TRAINING MANUAL ON POST-HARVEST PHYSIOLOGY OF FRUITS AND FLOWERS (27th January to '16th February, 2009) Sponsored by INDIAN COUNCIL OF AGRICULTURAL RESEARCH NEW DELHI ,, rl Division of Plant Physiology Indian Agricultural Research Institute New Delhi-110 012 TRAINING MANUAL ON Post-Harvest Physiology of Fruits and Flowers 27th January to 16th February, 2009 Compiled & Edited by Ajay Arora, V.P. Singh and Vijay Paul Course Director: Dr. Ajay Arora Course Coordinator: Dr. V.P. Singh Dr. Vijay Paul DIVISION OF PLANT PHYSIOLOGY INDIAN AGRICULTURAL RESEARCH INSTITUTE NEW DELHI-110 012 Citation: Ajay Arora, V.P. Singh and Vijay Paul (2009). Training Manual on Post­ harvest physiology of fruits and flowers. Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi, India. pp. 242 .. For Private Distribution only 2009 • " .. .. " ... .. ..• CONTENT Contributors Preface Programme schedule Participants i ii iii-v vi-vii Present and new trends in post-harvest research and technology J.D.S. Panwar and N.K. Prasad 1-10 Molecular strategies of extending cut-flower life and countering senescence/PCD Ajay Arora, Divya Choudhary and Gaurav Agarwal 11-20 Role of ROS and antioxidants in the regulation of leaf and flower senescence and fruit ripening R.K Sairam 21-31 1-Methylcyclopropene: a tool for managing ethylene and post-harvest losses in horticultural produce Vijay Paul and Rakesh Pandey 32-44 Physiological and biochemical changes in cut flowers V. P. Singh, Gaurav Agarwal and Divya Choudhary 45-57 Modern methods of potato storage Devendra Kumar 58-63 Harvesting techniques for fruits and vegetables D. V. K. Samuel 64-73 Ionizing radiations for improving quality and post harvest preservation of agricultural produce Bhupinder Singh 74-83 Biotechnology in horticultural gennplasm management Sandhya Gupta 84-88 Post harvest management of horticultural crops for domestic and export marketing R.K. Pal 89-93 Strategies for enhancing carotenoid levels in horticultural crops Ajay Arora, Divya Choudhary and Gaurav Agarwal 94-100 Fruit ripening: regulation and manipulation Vijay Paul and Rakesh Pandey 101-115 Physiological basis of color changes during fruit ripening Pramod Kumar, Madan Pal and Te} Pal Singh 116-125 .. 14. Electromagnetic energies for post harvest preservation of seeds Al'ljali Anand 122-125 15. Advances in packaging technology of fruits and vegetables ManishSrivastav and S. K. Jha 126-132 16. Importance of ethylene receptors in horticultural crops Gaurav Agarwal, Divya Choudhary and Ajay Arora 133-146 17. Role of plant hormones in flower senescence Vanita Jain and R.KSairam 147-155 18. Antioxidants in fruits and vegetables: a varietal profile Charanjeet Kaur 156-158 Biotechnology in the garden Ajay Arora, Gaurav Agarwal and Divya Choudhary 159-164 Monocarpic senescence and its regulation in plants Renu Khanna-Chopra and S. Srivalli 165 19. 20. -.... .... .... ... Experimentation/Practicals .... .... I. Oxidative stress and antioxidative defence system R.K. Sairam 166-179 2. Hydrolytic activities and softening in fruit ripening AtarSingh 180-184 3. Elemental analysis in plant tissue by atomic absorption spectroscopy AtarSingh 185-195 .. .. - 4. Regeneration protocol for gladiolus Divya Choudhary, Gaurav Agarwal and Ajay Arora 196-198 ..... 5. Tomato transformation and regeneration protocol Divya Choudhary, Gaurav Agarwal and Ajay Arora 199 .. 6. Estimation of ethylene by gas chromatography Sangeeta Khetarpal pnd Madan Pal 200-201 7. Development of protocol for regeneration of mungbean ( Vigna radiata (L.) Wilczek) Sangeeta Khetarpal and Madan Pal 202-203 8. A protocol on polarographic measurement of partial light reactions by oxygen electrode by oxygen electrode using N.K. Prasad 204-206 Chromatography D.V. Singh 207-211 9. ... "" ._ "" - .. ._ ... ... ._ ._ Membrane stability index -A simple technique for screening against drought and high temperature stress S.R. Kushwaha and R.K. Sairam 212 Measurement of chlorophylls content in plant tissues Te} Pal Singh 213-216 1-Methykyclopropene: technical bulletin for use on horticultural products Vijay Paul and Rakesh Pandey 217-221 Estimation of photosynthetic pigments and total carotenoids in fruit tissues Vijay Paul, Rakesh Pandey and Atar Singh 222-225 Estimation of lycopene pigment in fruits/plant tissues Vijay Paul, Rakesh Pandey and Atar Singh 226-228 · Gamma chamber (GC-5000) : An irradiation facility for crop improvement and post harvest preservation Bhupinder Singh 229-230 Isolation of plant DNA/RNA and preparation of cDNA Gaurav Agarwal, Divya Choudhary and Ajay Arora 231-234 Polymerase chain reaction Gaurav Agarwal, Divya Choudhary and Ajay Arora 235-236 Appendix 237-242 CONTRIBUTORS Gaurav Agarwal Division of Plant Physiology, IARI, New Delhi110 012 Madan Pal Division of Plant Physiology, IARI, New Delhi 110012 Anjali Anand Nuclear Research Laboratory, IARI, New Delhi 110012 Rakesh Pandey Division of Plant Physiology, IARI, New Delhi110012 Ajay Arora Division of Plant Physiology, IARI, New Delhi110 012 Renu Khanna-Chopra Stress Physiology & Biochemistry Laboratory, Water Technology Centre IARI, New Delhi- I 10012 J.D.S. Panwar Division of Plant Physiology IARI, New Delhi-110012 Divya Choudhary Division of Plant Physiology, IARI, New Delhi110 012 N.K. Prasad Division of Plant Physiology, IARI, New Delhi110012 Sandhya Gupta Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, New Delhi R.K Sairam Division of Plant Physiology, IARI, New Delhi] 10012 Vanita Jain Division of Plant Physiology, IARI, New Delhi] 10012 S. K. Jha Division of Post-harvest Technology !ARI, New Delhi-I JO 012 Charanjeet Kaur Division of Post Harvest technology, IARI, New Delhi-I I 0012 Sangeeta Khetarpal Division of Plant Physiology, IARI, New Delhi110012 Devendra Kumar Central Potato Research Institute Campus Modipuram, Meerut-2501 IO Pramod Kumar Division of Plant Physiology, IARI, New Delhi 110 012 S.R. Kushwaha Division of Plant Physiology, IARI, New Delhi110012 R.K. Pal Division of Post Harvest Technology, IARI, New Delhi- l JO 012 Vijay Paul Division of Plant Physiology, IARI, New Delhi110012 D.V. K. Samuel Division of Agricultural Engineering, IARI, New Delhi-I 10012 V. P. Singh Division of Plant Physiology, IARI, New Delhi110012 Bhupinder Singh Nuclear Research Laboratory, IARI , New Delhi] 10012 Atar Singh Division of Plant Physiology, IARI, New Delhi! 10012 D.V. Singh Division of Plant Physiology, IARI, New Delhi110012 Tej Pal Singh · Division of Plant Physiology, IARI, New Delhi! JOO 12 S. Srivalli Stress Physiology & Biochemistry Laboratory, Water Technology Centre IARI, New Delhi- 110012 Manish Srivastav Division of Fruits and Horticultural Technology, IARI, New Delhi-110 012 Preface India has a rich biodiversity of horticultural crops growing in its varied agro-cliipatic regions. Fruits, vegetables, flowers, ornamentals and spices are perishable in nature and their shelf life is limited, depending upon environmental and handling conditions. Due to poor-harvest management practices and lack for infrastructure facilities huge losses (20-40%) occur which cause an annual estimated loss of Rs. 23,000 crores. The subject matter of this winter school covers post harvest physiology, scientific handling, grading and storage methods, food irradiation, food biotechnology and food safety and quality assurance, floral dehydration and on-farm agro-processing/value addition techniques for employment generation. This course provides technical know how of the latest post-harvest management/ processing techniques which will have an impact on reduction of post harvest losses in perishable horticultural produce and will lead to sustainable development of our agrarian economy. The aim of thi's course is to create capacity that would enable safe handling of produce to process, package and transport to remunerative markets of as to minimize post harvest loss and improve net availability of quality saleable produce to consumers and net return to growers. Fruits and vegetables, as well as their processed products have become mainstream human dietary choices in recent days, primarily because of several epidemiological studies showing various health benefits associated with the consumption of fruits, vegetables, and their processed products. Fruits and vegetables share several common structural and nutritional properties and also characteristic differences due to differences in their biochemical composition. Fruits, in general, are attractive organs for vectors involved in seed dispersal, and thus have evolved features such as enhanced colour, attractive flavour, and taste. Consequently, the developmental and biochemical processes within a fruit are programmed to achieve this goal. By contrast to fruits and vegetables, the number of cultivated flowers is very small. The major flower crops include roses, carnations, aster, daisies and Chrysanthemum, snapdragons, Gladiolus, tulips, lilies, and Alstroemeria. In recent years, the cultivation of potted ornamentals has gained importance. Fruits and vegetables are nutritious, valuable foods full of flavour. However, in the low-income countries, poor care and handling of these crops frequently results in loss of quality, especially when not consumed immediately. In these countries, people are not sufficiently informed on how to make technical choices for better preservation of fruits and vegetables. Our goal is to bring together people using multi disciplinary approaches to the study of senescence and PCD. A systematic analysis of recycling processes and investigation of the role of organelles in senescence and cell death may well reveal exciting links between the different death processes. From a practical viewpoint, expanding our knowledge of the processes of plant senescence has many potential economic benefits. The timing and efficiency of senescence has an important role in determining the yield and pre-harvest quality of many cereal, forage and horticultural crops. In addition, a significant proportion of all fresh plant products for food or feed are lost, or suffer from reduced quality, due to plant senescence. Practical have been specifically designed to test the ripening and senescence mechanism in horticultural crops by physiological, .biochemical and molecular analyses. I sincerely thank my colleagues in the Division of Plant Physiology for providing the necessary protocols for this manual. I am also thankful to all other faculty members, students, technical officers and staff of the Division of Plant Physiology and other Departments for their help in conducting the training course. The financial help received from the Indian Council of Agricultural Research to organize this training course is gratefully acknowledged. 27th January 2009 \?A� f�trse-Director POST-HARVEST PHYSIOLOGY OF FRUITS AND FLOWERS Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India PRESENT AND NEW TRENDS IN POST-HARVEST RESEARCH AND TECHNOLOGY J.D.S. Panwar and N.K. Prasad Division of Plant Physiology Indian Agricultural Research Institute, New Delhi-110012 Fruits and vegetables are rich source of vitamins, minerals, dietary fibres and antioxidants. Many of them have medicinal value also. Medicinal nature of fruits is basically due to their antibacterial, anti-inflammatory, anti-carcinogenic and anti-oxidative properties. The awareness and improvement of the economic status globally has increased the demand of high quality, fresh and nutritious fruits. According to Indian Horticulture Database 2005, India has produced 45.2 million tonnes of fruits and 84.8 million tonnes of vegetables and occupies the second position (next to China) among the major fruit and vegetable producing countries (Table 1). Table 1. Major fruits and vegetables producing countries and production (million tonnes) Fruits Country China India Brazil USA Italy Spain Mexico Production 72.002 45.203 35.734 30.298 16.076 15.747 13.940 Vegetables Country Production China 250.341 India 84.815 USA 35.734 Turkey 21.777 Italy 15.723 Iran 14.194 Japan 13.115 Source: Indian Horticulture Database (2005) During the year 2004-2005, our country many countries and earned foreign exchange equivalent exported 0.5 million tonnes of fruits and vegetable to to 648 crores of Indian rupees (Table 2). Table 2. Export of horticultural produce (2004-2005) Items Total agricultural produce Flowers (cut flowers) Fruits and vegetables Processed fruits and vegetables Total horticultural produce Quality (Million tonnes) Value in Rs. (Crores) 3.335 2.060 0.503 0.374 7365.36 105.16 647.72 993.64 Share in total agricultural export (%) 1.43 8.79 0.13 0.885 1829.95 29.84 Source: Indian Horticulture Database (2005) 1 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India It must be mentioned that about 1/3rd of all the transporation. The estimated post-harvest losses of fruits suffer losses at various stages of storage and different fruits and vegetables in India are presented in Table 3. Table 3. Production of flowers, fruits and vegetables during 2003-04 and estimated post harvest losses Commodity Fruits Apple Banana Citrus Grapes Mango Papaya Sapota Production (Million tonnes) 1.348 13.304 1.137 1.248 12.733 2.147 0.80 Post-harvest Commodity loss (%) Vegetables Cabbage Okra Onion Peas Potato Tomato 14 11-14 8-31 27 17-37 40-100 NA Production (Million tonnes) Post-harvest loss (%) 5.392 3.420 4.201 2.061 23.161 7.617 10-40 NA 13-30 NA 8-40 13-16 Source: Database (2005) Commercial flowers constitute the most 2. profitable agro-industry now-a-days. Global trade for cut flowers and potted flowers amounts to US$ 40 billion, out of which cut flower’s share is about 60%. Non-climacteric fruits: The climacteric rise is respiration is completely lacking eg. pineapple and citrus fruits. Climacteric fruits generally produce higher amount of ethylene than non-climacteric fruits. Further, non-climacteric fruits do not respond to exogenous ethylene treatment for ripening. Climacteric fruits can ripen fully even if they are harvested at green mature (i) Poor storage; (ii) Poor transportation, and (iii) Fast stage. On the other hand, non-climacteric fruits can ripen fully only if they are allowed to remain attached senescence of flowers to the parent plants because the process of ripening CLIMACTERIC AND NON-CLIMACTERIC dose not occur very fast if they are detached from the FRUITS plant at green mature stage. Fruits, on the basis of their respiratory pattern The time and intensity of climacteric peak in during ripening, have been classified into two groups respiration (in climacteric fruits) can be delayed and (Kader and Barrett, 2003):lowered down by reducing the rate of respiration and India is very poor in export of cut flowers. The most serious problem before floriculture industry is the transportation because of short shelf life and vase life of fruits and flowers. Losses occur mainly due to- 1. Climacteric fruits: They show a sudden this way one can enhance the shelf life of fruits. burst/rise in respiration rate during ripening. The two major events during ripening of The rise in respiration is either simultaneous climacteric fruits areor it is just after the rise in the rate of ethylene Ethylene production; and production. Examples: mango, apple, papaya, guava, kiwi, tomato, peach, plum, banana, Rise in temperature pear, apricot, plum and avocado. 2 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ACC oxidase ACC + O2 C2H4 + HCN + CO2 + H2O HCN produced Metabolized (detoxified) B-cyanoalaline synthase B-cyanoalaline + H2S L-cysteine + HCN CN may inhibit cytochrome oxidase and triggers CN-resistant respiration. It has been reported that CN-resistant respiration rate becomes as high as 94% of the total respiration during ripening of tomato fruits. Malic enzyme : Malic acid : A marker enzyme of ripening Major organic acid in fruits During ripening process ME Malate CO2 + Pyruvate + NADPH + NADP TCA cycle 3 ETC and alternate respiration Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Table 4. Cyanide-sensitive and cyanide-resistant respiration (µmol O2/min/g dry weight) in tomato (var. Pusa Ruby) fruits during ripening Treatments Control 1 1.37 Days after harvest 4 6 8 1.54 2.40 1.94 SHAM (CN-sensitive decrease) 1.44 1.00 0.90 0.00 0.00 KCN (CN-insensitive increase) 0.00 0.56 1.44 1.92 1.65 KCN + SHAM 0.00 0.00 0.00 0.00 0.00 10 1.80 Alternate respiration (Cyanide-resistant respiration) during ripening in tomato Mature 61.04% of total respiration Half ripe 92.00% of total respiration Full ripe 93.75% of total respiration Source: Yenru et al. (1996) Malic enzyme is synthesized de novo. Treatment of fruits (mango) with AgNO3 and in vitro addition of AgNO3 completely inhibit ME activity (Srivastava et al., 1996). However, this chemical can not be recommended to be used commercially because it is poisonous in nature and good for academic purpose only. Rise in ME can be used as a marker of ripening stage Respiration: There is considerable amount of reducing equivalent production as follows: Oxidation PPP Glycolysis Malate Oxidation Kreb’s Cycle NADPH NADH NADPH NADH NADPH NADH Reducing equivalent Terminal oxidase NADH ATP(1) Flavoprotein UBQ ATP(2) Cyt. b. Cyt. C1 ATP(3) Cyt. C Cyt. a+a3 X O2 CN X O2 SHAM (Salicylic Hydroxamic Acid; 5 mM) In cytochrome oxidase system (ETC) 3 ATP are produced, where as 1 ATP is produced in cyanide resistant respiration. 2 ATP = energy is released to form heat and it causes the rise in temperature upto 100C Source: Kumar and Sinha (1992) 4 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India CN – Resistant Respiration –  Results in rising temperature; may be around 100C  Hydrolytic enzymes are favourably activated (iii) In addition, some hormones, such as cytokinin, benzyladenine and gibberellin are also used for delaying ripening of fruits. PHYSIOLOGICAL STUDIES ON RIPENING AND SENESCENCE It induces studies pertaining to: Ripening-associated gene; Ethylene-controlled gene-expression; Identification of senescence-related genes; Genes involved in protein degradation Genetic transformation of enzymes involved in fruit ripening; and PCD or Programmed Cell Death Molecular or Biotechnological Approach      The following morphological, physiological and biochemical changes occurs during ripening and senescence process. (i) Morphological changes  Change in colour  Loss in weight  Loss in moisture (ii) Physiological changes Ethylene production  Rise in respiration and  Rise in fruit temperature (iii) Biochemical changes Increase in anthocyanin content  Hydrolytic enzymes  Sugars; and  Oxidative stress We need to regulate ripening process in fruits and senescence in flowers. For that very purpose we generally make approaches which can be classified as(i) Physical Approach  Storage under modified atmosphere (CO2 : O2 ratio)  Storage under partial vacuum condition  Low temperature; and  Gamma-ray radiation (ii) Chemical Approach Some of the chemicals tried and tested include Ethanol  MCP  Ethylene  Polyamines (spermine, spermidine, putrescine)  Sulphur compounds (sulphosalicylic acid, thiourea and methionine)  Modified atmosphere storage Tremendous literature is available concerning with the extension of shelf-life of fruits by storing them at modified atmospheric conditions. Mango fruits (cv. Dashehari) stored at partial vacuum condition (-18” Hg) and normal temperature (250C) exhibited an extension of shelf-life for about 20 days without the pulp quality and taste being affected at all. Vacuum treated fruits revealed very low amount of ethylene evolution, which is a ripening associated hormone. The physiology behind may be attributed to declination of O2 concentration under vacuum condition. We know that ACC-oxidase oxidises ACC (1-amino cyclopropane-1-carboxylic acid) to form ethylene at a rate dependent upon O2 concentration (Kader, 1990). Similarly, acetaldehyde (1%) and N2 (97%) atmosphere at 150C storage also gave encouraging results. Free radical scavenging enzyme (SOD) activity was very low at 24 h after termination of treatments; however as storage period progressed, it increased and became about 10-fold on day-10th after harvest in case of all treatments. In control fruits, SOD activity did not show any conspicuous increase even on the 10th day following harvest when the fruits were full ripe. 5 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Fig. 1. Fig. 2. Superoxide dismutase activity (recorded on day-1 and day-10 following harvest) in mango fruits (cv. Dashehari) as influenced by acetaldehyde (1%), N2 (at 150C) and reduced pressure treatments Ethylene evolution (measured on day-10 following harvest) in mango fruits (cv. Dashehari) as influenced by acetaldehyde, N2 (at 150C) and reduced pressure treatments Source: Prasad et al. (1999) 6 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Scanty literature is available on the SOD activity during ripening of mango fruits. Zhang et al. (1998) reported that maintenance of SOD activity in the pericarp of mango fruits at a high level, retarded the increase in membrane permeability. Lower SOD activity in the pericarp of control fruits resulted in accelerated ripening. 1-MCP has a non-toxic mode of action and used at extremely low doses (usually parts per billion). Ethanol Table 6. Polygalacturonase activity (mg glucose eqv./g fr. wt./hr) and ethyelene evolution (nmoles/ g fr.wt./hr) during ripening of control and 1-MCP treated (1 ppm) tomato fruits (var. Desi) ---------------------------------------------------------------------------------------------------------------Days after Polygalacturonase Ethylene harvest activity evolution Control Treated Control Treated ---------------------------------------------------------------------------------------------------------------0 0 0 0.90 0 2 15.26 0 1.87 0 4 16.84 0 2.10 0 6 11.40 0 2.74 0 8 7.98 0 1.61 0 10 3.75 0 1.02 0 12 13.28 0.845 14 15.96 1.438 16 12.73 1.867 18 9.46 1.459 20 4.67 0.938 ---------------------------------------------------------------------------------------------------------------- In terms of its mode of actions, 1-MCP competitively binds to a metal in the ethylene receptors so it blocks the normal as well as autocatalytic (feed forward) system II of ethylene production (Blankenship and Dole, 2004). It has been shown that treating the tomato fruits with ethyl alcohol resulted in the extension of shelf life. It depends upon the quantity of ethanol and duration of treatment as shown in the table given below: Table 5. Effect of ethanol on shelf life of tomato fruits (var. Pusa Ruby) ---------------------------------------------------------------------------------------Ethanol Duration Days taken (ml/kg fruit) (hr) to ripe ---------------------------------------------------------------------------------------Control 8 1 ml 6 10 12 10 24 12 2 ml 6 12 12 16 24 18 4 ml 6 20 12 24 24 26 8 ml 24 32 ---------------------------------------------------------------------------------------Source: Mathur and Srivastava (2005) Source: Thakur (2000) The EPA (Environment Protection Agency, Gamma-ray treatment USA) has classified 1-MCP as a plant regulator Inhibition of de-greening by gamma radiation structurally related to the compounds present in plant. has been reported in mango (Paul and Srivastava, 1-MCP Unpublished). Gamma radiation (0.25 KGY) CH3-C maintained the green colour of the fruits but blackening 1- MCP (1-methyl cyclopropene) of skin was noticed. A six-day delay in ripening could (Mol. Wt. 54) be achieved in case of Alphanso mango fruits irradiated (C4H6) with an optimum dose of gamma radiation (25 Krads) under air or nitrogen. Radiation effect on the skin was HC CH2 more prominent interms of inhibition of chlorophyll disappearance and firmness of pulp (Dharkar et al., In 2003, it was approved for use in apples. 2006). More research is needed on radiation doses Today it is probably the most useful compound among and stage of fruit treatment to get a positive result. recently developed inhibitors of ethylene response. 7 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Polyamines, such on spermine, spermidine and putrescine have been reported to increase cumulative solution uptake and thus enhance the vase life in ethylene in- sensitive gladiolus cut flowers. FLOWER SENESCENCE To understand the mechanism of flower senescence, it is imperative to understand how to regulate- (i) Weight loss; (ii) Respiration, (iii) Ionleakage, (iv) Hydrolysing enzymes and (v) Oxidative stress. Table 7. Influence of polyamines on cumulative solution uptake, flower diameter and vase life of gladiolus (Var. Dhanwantri) Treatment Control (4% sucrose) 100 ppm spermine + 4% sucrose (1:1) 100 ppm spermidine + 4% sucrose (1:1) Cumulation solution uptake (ml/spike) 25.18 Vase life (days) 5.00 Flower diameter (cm) 7.85 52.17 9.60 11.56 42.13 7.70 10.90 Source: Singh et al. (2005) Equally encouraging results were obtained with some sulphur containing compounds such as 5sulphosalycylic acid, thiourea and methionine. The vase solution having 20-200 ppm 5-sulphosalicylic acid increased the vase life of Gladiolus grandiflora cut flowers by increasing the reactive oxygen species (ROS) scavenging activity of the enzyme lipoxygenase (Ezhilmathi et al., 2007). Free radicals Lipid peroxidation : Lipoxygenase activity membrane damage -lipic acid, a potent antioxidant used, in the vase solution (100 ppm along with 4% sucrose) increased SOD activity, thereby increasing the capacity to scavenge more free radicals. Moreover, -lipoic acid can reduce lipid peroxidation by decreasing lipoxygenase (LOX) activity which alleviate the membrane disintegration to some extent and consequently delay the senescence (Singh et al., Fig 3li. Effect of -lipoic acid on activity of SOD 2003). and LOX in gladiolus flowers Source: Singh and Jagadheesan (2003) 8 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India this increase shelf-life, but it also improves qualities of interest to processors. The tomato products are “thicker” (higher pectin to water ratio). This technique has been used in the well known “Flavr Savr” tomato (developed by Calgene, now owned by Monsanto). Programmed Cell Death PCD or Programmed Cell Death, is a well established mechanism in plants. It occurs during senescence of leaf and flowers. It also forms a part of plant’s adaptation to stresses, such as reactive oxygen. There occurs two overlapping mechanisms associated with the termination of a flower(i) Petals abscise before the majority of cells initiate a cell death program. (ii) Petals are more persistent and cell deterioration occur while the petals are still part of the flower (Arora, 2008b). One way of countering the effects of pathogen induced PCD is through the use of caspase inhibitors in the cut flower medium. Regulation of the genetic engineering in the US is governed by three agencies USDA (United State’s Department of Agriculture)  FDA (Food and Drug Administration); and  EPA (Environment Protection Agency) They look into the following aspects before commercially release of a GE fruit crop.  Food Allergy;  Food Toxicity and Antinutrients;  Crop Producing Pharmaceutical compounds; and  Antibiotic Resistance Post-harvest research is a very important field to enhance the shelf-life of fruits, vegetables and flowers. Various approaches have been adopted for the same such as physiological, biochemical and molecular approach. Use of different chemicals and genetic engineered plants need special care as they may affect the health of consumers and create allergy, toxicity and resistance to antibiotics. There is a need to combine the efforts of various agencies involved in horticultural research, genetic engineering of plants and environment protection including consumer’s health. MOLECULAR APPROACH It is well known that ethylene plays a regulatory role in fruit ripening. There are at least three methods employed to genetically – engineered reduction in ethylene (Nagata et al., 1995, Peggy et al., 2008 and Mamiko et al., 2006). (1) Decreased ACC-synthase ACC-synthase is an enzyme that is responsible for the next-to-the-last-step in the synthesis of ethylene in the fruit. Reducing the levels of ACC-synthase dramatically decreases ethylene production. Genetically engineering of a “minor-image” copy (or “antisense”) of the ACC-synhase gene into tomato, results in prevention of the production of both a backwards ACC enzyme and the normal “forward” version already in the plant, by a quirk of plant’s genetic editing machinery. This “anthisense” technique also has the advantage that no new protein is produced in the plant. These tomatoes are marketed in USA as “Endless Summer”. (2) Addition of ACC-deaminase A new gene is added to the tomato, from the soil bacteria Pseudomonas chlororaphis. This gene encodes an enzyme called ACC-deaminase, which causes the breakdown of one of the precursors of ethylene synthesis (ACC). The reduction in the levels of this precursor causes reduced ethylene production and delayed ripening. Monsanto developed this tomato, but it has not yet been marketed. (3) Addition of SAM-hydrolase Tomato, genetically engineered to produce SAM hydrolase (from the E. coli T3 bacteriophage) also breaks down one of the precursors of ethylene synthesis (SAM). This technology has been developed by Agritope Inc. for a variety of cherry tomato. Some tomatoes have been genetically engineered to alter one particular aspect of fruitripening–softening. The process of fruit softening is caused, in part, by the breakdown of pectins– compound which give support to the cell walls of fruits. Tomatoes have been engineered to have reduced levels of a pectin-breakdown enzyme, called Polygalacturonase (PG), using the same “antisense” technique applied to ACC-synthase. Not only does 9 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Nagata, M., Mori, H., Tabei, Y., Sata, T., Hirai, M. and Imaseki, H. (1995). Modification of tomato ripening by transformation with sense or antisense chimeric 1-aminocyclopropane-1carboxylate synthase genes. Acta Horticulturae, 1: 394-412. Peggy, G. and Lemaux, M. (2008). Genetically Engineered Plants and Foods : A Scientist’s Analysis of the Issue (Part-I). Ann. Rev. Pl. Biol., 59: 771-812. Prasad, N.K., Srivastava, G.C. and Pandey, M. (1999). Studies on mango fruit ripening with reference to superoxide dismutase and polygalacturonase enzyme activities under different storage conditions. J. Plant Biol., 26(2): 161-168. Singh, V.P. and Jegadheesan, A. (2003). Effect of lipoic acid on senescence in gladiolus flowers. Indian J. Plant Physiol. (Spl. Issue No. 1): 7279. Singh, V.P., Kiran, D. and Arora, A. (2005). Effect of spermine, spermidine and putrescine on the vase life and associated parameters in two gladiolus varieties. J. Orn. Hort., 8(3): 161-166. Srivastava, G.C., Zeng, Yanru, Pandey, M. and Prasad, N.K. (1996). Effect of silver nitrate on activity of malic enzyme during ripening in mango (Magnifera indica L.). Indian J. Expt. Biol., 34: 575-576. Thakur, A.K., Singh, A. and Pandey, M. (2000). Inhibition of respiration, ethylene synthesis and cell wall softening enzyme activities in tomato fruit during ripening by ethanol. Adv. Hort. Sci., 14: 176-184. Zeng, Yanru, Pandey, M., Prasad, N.K. and Srivastva, G.C. (1996). Hydrolysing enzymes and respiration during ripening of tomato (Cycopersicon esculentum) fruits. Current Sci., 70: 1017. Zhang, Z.Q., Hong, H.J., Li, X.P. and Ji, Z.L. (1997). Effects of intermittent warming on chilling injury and physiological and biochemical responses of mango fruits. Acta Hortic. Sin., 24: 329-332. References Arora, A. (2008b). “Programmed Cell Death During Plant Senescence”. In: Paliyath, G., Murr, D.P., Handa, A.K., Lurie, S. (eds.). Post harvest Biology and Technology of Fruits, Vegetables and Flowers. Blackwell Publishing, Iowa, USA. Pp. 86-124. Blankenship, S.M. and Dole, J.M. (2004). 1-methyl cyclopropene: A Review. Post harvest Bio. Technol., 28: 1-25. Dharkar, S.D., Savagaon, K.A., Sridangarajan, R.N. and Srinivasan, A. (2006). Irradiation of mangoes. 1. Radiation-induced Delay in Ripening of Alphanso Mangoes. J. Food Science, 31(6): 863-869. Ezhilmathi, K., Singh, V.P., Arora, A. and Sairam, R.K. (2007). Effect of 5-sulphosalicylic acid on antioxidant activity in relation to vase-life in gladiolus cut flowers. Plant Growth Regul., 51: 99-108. Kader, A. and Barrett, D.M. (2003). “Biology, Principles and Application in Processing Fruits”. In: Science and Technology Vol. I (Somogyi, J.P. et al., eds.). Technomic Publishing Co. Inc., Lancaster, Pennisylvania, USA Kader, A., Zagory, D. and Kerbel, E.L. (1990). Modified atmosphere packaging of fruits and vegetable. CRC Rev. Food Sci. Nutr., 20: 130. Kumar, S. and Sinha, S.K. (1992). Alternate respiration and heat production in ripening banana fruits (Musa paradisiana var. Mysore). J. Expt. Bot., 43: 1639-1642. Mamiko, Kitagawa; Nobutaka, Nakamura; Hiroyuki, Usida; Takeo, Shina; Hirotaka; ITO; Junichi, Yasuda; Takahiro, Inakuma; Yukio Ishiguro, Takafumi, Kasumi and Yashuhiro, ITO (2006). Ethylene biosynthesis regulation in tomato fruits from F1 hybrid of the ripening inhibitors (rin) mutant. Bioscience Biotechnology and Biochemistry, 70(7): 1769-1772. Mathur, K. and Srivastava, G.C. (2005). Effect of 1MCP on malic enzyme activity and ethylene production in mango during ripening. Indian J. Pl. Physiol., 10(2): 273-275. 10 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India MOLECULAR STRATEGIES OF EXTENDING CUT-FLOWER LIFE AND COUNTERING SENESCENCE/PCD Ajay Arora*, Divya Choudhary and Gaurav Agarwal Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110 012 *Email: romiarora@yahoo.com developmental continuum in the flower, preceded by tissue differentiation, growth and maturation of the petal, followed by growth and development of seeds, and co-coordinated by plant hormones. Cell-death processes are thought to be regulated by anti- and pro-death proteins, which may be expressed throughout the life of the flower, providing for the most part a highly regulated homeostatic balance. Future genetic analyses of floral senescence are likely to identify the proteins that function to maintain a nonsenescent ‘youthful’ state, and the ‘pro-senescence’ proteins which function to progress cell death. The past decade has seen increasingly rapid isolation and identification of senescence-associated genes from cut flower crops, with a somewhat slower movement towards understanding the function and significance of the gene products. Characterizing generic patterns of gene expression has identified common processes that are linked with the progression of flower senescence (e.g., ethylene signaling, proteolysis). This approach will also be useful in identifying the order of molecular changes associated with flower senescence, thereby enabling researchers to accurately study cause and effect. This chapter focuses on molecular and genetic research published within the last one decade Flower petals are ideal tissues for cell death that has increased our understanding of the processes studies as they are short lived, the tissue is relatively involved in or regulating flower senescence and its homogenous, chemical manipulation can be applied significance to the postharvest industry (Arora, 2008a). without substantial wounding (i.e., feeding through the Ethylene regulates the expression of senescence vascular tissue), and the process of flower senescence regulated genes has been shown to be a genetically programmed event. The plant hormone ethylene has been To date, most genetic analyses of floral senescence implicated in the regulation of both fruit ripening and have focused on changes that occur in mature flowers leaf and flower senescence (Abeles et al., 1992). A just prior to wilting or colour change. However, number of senescence regulated (SR) and ripening senescence of one floral organ (e.g., petal) is part of a related genes have been found to be up-regulated by All cut flowers are destined to die, and the challenge for postharvest researchers is to slow the processes controlling flower death to enable cut flowers to reach distant markets with a display life that will ensure their sale and display, and return custom of the buyers. A thorough understanding of the processes that lead to cell death of floral tissues is integral to achieving this goal. Postharvest performance of cut flowers is affected by the developmental stage of a flower at harvest, pro-senescence signals that originate from specific tissues within the flower (e.g., pollinationinduced petal senescence), and stress-related metabolism (in response to temperature, wounding, nutrient starvation). Cut flower stems are removed from a source of nutrients, undergo water restrictions and may be held at undesirable temperatures in the dark for days prior to sale. Plant hormones, membrane stability, water availability, cellular proteolysis and carbohydrate metabolism act in concert to determine the differential rate of senescence for each floral organ. Currently, flowers can be grouped into several categories based on postharvest technologies that can extend their vase life (e.g., sensitivity to ethylene, chilling sensitivity, leafy stems, multiple/single flowers per stem, woody stems, etc.). 11 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India not detectable or only moderately induced in young green leaves (Weaver et al., 1998). Immature tomato fruits and flowers also do not respond to exogenous ethylene with ripening or petal senescence. This ethylene treatment does not induce the expression of ripeningrelated genes in immature green fruit or SR genes in petals from flowers in the bud stage (Lawton et al., 1990). While some flowers like daylily and nonclimacteric fruits like strawberry are not regulated by ethylene, it is clear that ethylene plays a regulatory role in both senescence and fruit ripening through the transcriptional regulation of SR genes. the exogenous application of ethylene. Treatment of preclimacteric flowers with ethylene results in the induction of all the SR genes identified from carnation (Jones and Woodson, 1999). In tomato, the highest level of expression of pTOM genes in fruit was detected at the orange stage when ethylene production was highest and enhanced expression in leaves coincided with the first visible symptoms of leaf yellowing. Treatment with exogenous ethylene resulted in increased expression of pTOM genes in fruit and leaves, providing evidence that ethylene-controlled gene expression is involved in both fruit ripening and leaf senescence (Davies and Grierson, 1989). The observed differences in the timing of the response of various SR genes to external stresses and plant hormones indicate that some of the SR genes may respond directly to stress while others may be regulated by senescence that results from the stress or hormone application (Weaver et al., 1998). Further characterization of the response of SR genes to various stresses will help to identify those genes that are primarily responsive to senescence and are thus key regulators of senescence. There are many genes that are up regulated during senescence and involved in the activation and coordination of senescence, the down-regulation of genes that act as repressors of senescence may play an equally important role in regulating senescence. Currently most of the genes identified as down regulated during senescence are genes involved in photosynthesis (John et al., 1997). Transcript levels for the pea homologue of the defender against apoptotic death (dad) gene, a gene known to function as a repressor of programmed cell death (PCD) in Caenorhabditis elegans and mammals have been found to decrease during flower development (Orzaez and Granell, 1997), while the dad-1 cDNA from rice can rescue temperature sensitive dad-1 mutants of hamster from PCD. Yamada et al. (2004) isolated a homolog of the potential antiapoptotic gene (DAD1) from gladiolus petals as fullThe ability of plant organs to respond to length cDNA (GlDAD1), and investigated the exogenous ethylene appears to be developmentally relationship between its expression and the execution regulated as the enhanced expression of SAGs in processes of programmed cell death (PCD) in ethylene treated leaves is greatest in old leaves and senescing petals. RNA gel blotting showed that Never ripe (NR) tomatoes, which are insensitive to ethylene due to a mutation in the ethylene receptor, produce fruit in which ripening is inhibited, have flower petals that do not senesce and have leaves with delayed yellowing (Lanahan et al., 1994). In the fruit of NR tomatoes ripening-related transcripts accumulate too much lower levels than in wild-type fruit. Arabidopsis plants with a mutated ethylene receptor, etr1-1, also show delayed leaf senescence but, once initiated, the process of senescence and the level of SAG expression is similar to that detected in wild type leaves (Grbic and Bleecker, 1995). The treatment of tomato plants with the ethylene action inhibitor, silver thiosulfate, delays both fruit and leaf senescence and greatly reduces the expression of the mRNAs for pTOM31, pTOM36 and pTOM137 and to a lesser degree pTOM13, pTOM66 and pTOM75 in both fruit and leaves (Davies and Grierson, 1989). Treatment of carnation flowers with the ethylene action inhibitor, norbornadiene (NBD), delays the age-related accumulation of all SR genes except SR5 (Woodson et al., 1993). Treatment with NBD also reduces the basal levels of DCCP1 transcript in petals (Jones et al., 1995). These experiments indicate that while many SR genes are regulated by ethylene, they are also regulated by developmental or temporal cues. 12 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India the translation of this knowledge into strategies to counteract the detrimental effects of ethylene on ornamental products. Currently 1-Methyl cyclopropene (1-MCP) is entering the market worldwide to treat a variety of ornamental products and, genetically engineered ornamentals, with either a decreased ethylene production or perception and, Genes involved in ethylene biosynthesis and subsequent superior postharvest performance have been produced. Given the changing public and political perception opinions with regard to GMOs it is expected that the The biosynthesis and perception of the plant latter products will also soon enter the flower market. hormone ethylene are known to modulate specific components of leaf senescence, fruit ripening and flower Identification and classification of senescencesenescence (Arora, 2005). All three processes are also related genes known to be accompanied by increases in the synthesis Recent molecular studies have confirmed that of ethylene (Abeles et al., 1992) and therefore it is the processes of senescence and ripening are reasonable to assume that SR genes would include accompanied by changes in gene expression. Utilizing those involved in ethylene biosynthesis. Two enzymes, differential screening and subtractive hybridization 1-aminocyclopropane-l-carboxylate (ACC) synthase techniques a number of cDNAs that are upregulated and ACC oxidase have been identified as catalyzing during senescence have been cloned. Genes that rate-limiting steps in ethylene biosynthesis (Kende, exhibit enhanced expression during senescence have 1993). While no ACC synthase genes have specifically been cloned from the leaves of Arabidopsis, been isolated by differential screening of senescing or asparagus, Brassica napus, barley, maize, radish and ripening tissues, three SR clones have been identified tomato (Buchanan-Wollaston, 1997). Differential that encode ACC oxidase; these include pTOM13 from screening of senescence petal cDNA libraries and tomato; SR120 from carnation petals; and PCR-based differential display techniques have been pBANUU10 from banana. While pTOM13 is up- utilized to identify genes that are upregulated during regulated in leaves, flowers and fruits, SR120 is flower senescence of carnation and daylily flowers. specific. Upon the identification of additional ACC Most of the genes that have been identified as synthase and ACC oxidase genes, transcriptional up- senescence-related are expressed at basal levels in regulation has been reported during flower and leaf non-senescing tissues (green leaves and young flowers) senescence and fruit ripening in many species (Abeles and increase in abundance during senescence. A et al., 1992). While the ethylene biosynthetic pathway smaller number of SR genes are only detectable in is well established, components involved in ethylene senescing tissues and represent senescence-specific perception and signal transduction have only recently genes. An even smaller set of genes have been identified been identified. Initial studies on the expression of genes that have high levels of expression early in development, encoding the ethylene receptor report that specific decreased expression in young maturing tissue and receptor genes are up regulated during fruit ripening increased expression at the onset of senescence. Genes and senescence while others appear to be constitutively that fit within this class have only been identified in expressed in multiple tissues (Arora, 2005). vegetative tissues and represent genes that have a similar GlDAD1 expression in petals was drastically reduced, considerably before the first visible senescence symptom (petal wilting). A few days after downregulation GlDAD1 expression, DNA and nuclear fragmentation were observed, both specific for the execution phase of PCD but the function of the dad gene in plant senescence is still not very clear. During the last 10 years enormous progress role in multiple stages of development like germination has been made both in the understanding of the mode and senescence. of action of ethylene at the molecular level as well as in 13 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India napus and shows similar senescence-specified expression after the onset of leaf senescence. Weaver et al. (1998) showed the patterns of expression of a selected group of SAGs during agerelated senescence of Arabidopsis leaves. Most of these genes exhibit basal levels of expression in green non-senescing tissues. Within this broad classification genes are differentially regulated, with some increasing in abundance gradually as the leaf matures and others increasing more abruptly at various stages of leaf development. Only SAG12 and SAG13 show senescence-specific expression. Among the senescence-specific genes, SAG13 is detect before any visible signs of leaf senescence and as such may be responsible for initiation of the senescence process, while SAG12 is expressed after the leaf is visibly yellowing. While the majority of the research on SR gene expression has focused on vegetative tissues, there is significant evidence that petal senescence in some flowers is also a genetically programmed event that requires de novo protein synthesis and transcription of few genes (Woodson, 1994). In vitro translation of carnation petal mRNAs has revealed that the initiation of petal senescence is associated with increases in certain mRNAs. Differential screening of a cDNA library from senescing carnation petals has identified nine cDNAs that represent unique senescence-related mRNAs. A cysteine protease (DCCP1) and three ACC synthase (DCACS1, DCACS2 and DCACS3) cDNAs were identified from carnation petals using RT-PCR. Only SR139 and DCCP1 transcripts are detected in preclimacteric petals. Most of the SR genes are detected in petals at 5 days after harvest, corresponding to the first detectable ethylene production from the petals. Eight of the eleven SR genes from carnation are flowerspecific while low levels of SR139, SR123 and DCCP1 are detectable in leaves. Identifying the function of additional flower-specific SR genes will help to identify differences between the regulation of vegetative and floral senescence. Many of the genes that have been identified as senescence-related are identified from a particular plant organ and it is not known whether they are expressed in other senescing organs or during other developmental processes. The expression of a number of SAGs was investigated in roots, stems, flower buds and mature flowers of Arabidopsis. Expression of SAG12, SAG13, SAG25, SAG26 and SAG29 was not detected in any non-senescence tissues but was detected in both senescing flowers and leaves, indicating a common molecular regulation of senescing in vegetative and floral tissue. Some of the SAGs show low levels of expression in multiple tissues with upregulation in senescing leaves and flowers (SAG23) or up-regulation detected only in senescing leaves (SAG28 and SAG24). SAG27 shows strictly leaf senescence-specific expression (Quirino et al., 1999). While differential cDNA screening, differential display and cDNA subtraction have identified a number of senescence-related genes, the expression of most genes has not been investigated in flowers, leaves and fruits. The use of enhancer trap lines in Arabidopsis has resulted in the identification of over one hundred lines that have reporter gene expression in senescing but not in non-senescing tissues (He et al., 2001). This technology starts to reveal the complexity of the network of senescence-regulated pathways and will allow for the identification of many additional SR genes. The identification of senescence-specific promoter elements and the generation of mutants and transgenic plants will help us to better understand the regulation of SR genes during senescence. DNA micro-arrays Buchanan-Wollaston group have also identified a number of SR genes from Brassica napus utilizing both differential library screening and subtractive hybridization (Buchanan-Wollaston, 1997; BuchananWollaston and Ainsworth, 1997). It is not known whether any of these genes are up regulated during petal senescence, but similar to the Arabidopsis SAGs, they show differential patterns of expression during the development of the leaf. The homologue of the Arabidopsis (SAG12) gene has been cloned in B. 14 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India will allow temporal and spatial expression patterns to be determined for hundreds of genes involved in senescence. These technologies will lead to an increased understanding of the initiation and execution of senescence which will allow us to increase vase life and horticultural performance of ornamentals, increase yield in agronomic crops and decrease post-harvest losses of fruits and vegetables. suggests that the SAG12 protease might play a key role in the large-scale increases in protein degradation during senescence. The dismantling of the chloroplast, which contains greater than 50% of the leaf’s total protein, is a prominent process in leaf senescence. While many SR genes have been identified as proteases only one of these has been found to be localized to the Genes involved in protein degradation chloroplast (Erd1). Transcript levels of clp protease Decreases in total proteins during senescence have been reported to increase during leaf senescence result from increases in proteolytic enzyme activity and but protein levels were found to decline, suggesting decreases in protein synthesis. The degradation of that the clp protease does not play a primary role in proteins and remobilization of amino acids to the programmed disassembly of the chloroplast during developing tissues is the predominant metabolic senescence. process during senescence. Cysteine proteases are Similar to leaf senescence, protein degradation believed to be the main proteases involved in general has been demonstrated to be a major part of petal protein hydrolysis and recently a number of cysteine senescence and the remobilization of N to the protease have been identified from senescing leaves, developing ovary. A few of the SR cysteine proteases senescing flowers and ripening fruits (Arora, 2008a). have been shown to be up-regulated in both leaves Of those cysteine proteases identified from and petals (SAG12; DCCP1; Peth1; and GgCyP). senescing tissues, most share sequence homology with Large increases in proteolytic activity during the γ-oryzain from rice, a cysteine protease that has been senescence of the ephemeral flower, daylily have been implicated in the mobilization of reserve proteins during well documented and this proteolytic activity was seed germination. These include SAG2, See1, LSC7, correlated with increases in the expression of two SENU2 and SENU3. The expression patterns of these cysteine protease genes (Sen11 and Sen102) during five genes are similar, with low levels of expression in the senescence of tepals (Valpuesta et al., 1995). In young leaves and increased expression during contrast to the cysteine proteases from carnation and senescence (Buchanan-Wollaston, 1997). Both tomato petunia, transcripts are not detectable in young daylily cysteine protease, SenU2 and SenU3 and See1 from flowers (buds) and the level of transcript does not maize also show patterns of up-regulation during-seed increase in senescing leaves (Guerrero et al., 1998). germination, indicating that these proteases may play Both daylily cysteine proteases appear to be flower similar roles in protein degradation during germination senescence-specific. Arora and Singh (2004) studied and leaf senescence. While common to germination the changes in protein content and protease activity in and leaf senescence, the SENU2 and SENU3 the petals of ethylene-insensitive Gladiolus flowers, transcripts were not up regulated during fruit ripening. during development and senescence. There was a SAG12, which encodes a papain-like cysteine dramatic up-regulation in the expression of GgCyP at protease, is one of the few SR genes to the display the incipient senescent stage of flower development senescence-specific regulation. SAG12 mRNAs are indicating that this gene may encode an important not detectable in roots, stems, green leaves, or young enzyme for the proteolytic process in Gladiolus. The flowers, but increase in abundance in senescing petals gladiolus cysteine protease gene appears to be flower as well as leaves. This senescence specific expression senescence-specific. 15 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Regulation of senescence at the transcriptional, translational, and post-translational level Transcription factors and transcriptional regulators Senescence is regulated by levels of mRNA and proteins, and by activation of proteins. mRNA levels can be regulated by short interference RNA (siRNA) and micro-RNA (miRNA). A limiting factor can also be mRNA transport out of the nucleus. Proteins can be modified after translation. The eukaryotic translation initiation factor 5A (eIF-5A) is an example of post-translational modification. eIF-5A is apparently involved in translocation of specific mRNAs from the nucleus to the cytoplasm. During carnation petal senescence, the RNA abundance of eIF-5A increased, as well as the mRNA abundance of a gene involved in post-translational modification of eIF-5A, deoxyhypusine synthase. eIF-5A is unique among translation initiation factors because it contains hypusine [N-(4-amino-2-hydroxybutyl) lysine], which is formed post-translationally via the transfer and hydroxylation of a butylamino group to a specific lysine residue. These events activate eIF-5A. Deoxyhypusine synthase mediates the first step in the synthesis of hypusine in eIF-5A (Hopkins et al., 2007). Antisense suppression of deoxyhypusine synthase delayed senescence in Arabidopsis leaves and in carnation petals (Hopkins et al., 2007). These results are reminiscent of the delay of petal senescence by inhibitors of translation such as cycloheximide, showing the importance of de novo protein synthesis in senescence. This example illustrates the potential complexity of transcriptional, post-transcriptional, translational, and post-translational control of senescence. Transcription factors are usually classified according to specific functional amino acid sequences (domains). EIN3 and EIN3-like (EIL) transcription factors are crucial in the ethylene signal transduction chain. Thus far, four EIL genes have been isolated in carnation petals. When carnation flowers were treated with ethylene, the petal mRNA abundance of DcEIL1-2 and Dc-EIL4 slightly decreased, whereas the mRNA abundance of Dc-EIL3 drastically increased. Ethylene promoted and STS delayed the increase in Dc-EIL3 mRNA, and had no effect on Dc-EIL1. These data indicate that Dc-EIL3 is an important regulator of gene expression during carnation petal senescence. It might act as a master switch of ethylene induced gene expression (Hoeberichts et al. 2007). Targets of EIN/EIL transcription factors include genes with an ethylene-responsive element (ERE) in their promoter. Amongst these is a gene encoding the ethylene responsive transcription factor 2 (ERF2), which belong to the AP2 transcription factor family. In senescing daffodil petals, a gene (DAFSAG9) encoding an AP2 protein with very high homology to ERF2 was highly up-regulated. Aux/IAA genes are transcriptional suppressors, also often considered transcription factors. Aux/IAA is central in the regulation of auxin responses. In the absence of auxin, Aux/IAA inhibits all auxin-responsive genes. When auxin is present, it binds to its receptor, located on an F-protein in a nuclear-localized ubiquitin ligase. This ligase then targets Aux/IAA for degradation in the proteasome. Post-translational modifications also occur, for This results in the expression of auxin-responsive genes. example, in signal transduction (protein phosphorylation The protein sequence of the NAC in carnation by kinases), during autocatalytic activation of cysteine proteases in the vacuole, and prior to protein petals was highly homologous to that of NAC2 of degradation in proteasomes. Targeting for breakdown Arabidopsis. NAC factors (>100 in Arabidopsis) occurs through the transfer of several ubiquitin moieties have diverse functions, such as organ separation, but to a protein, by an ubiquitin ligase complex. a role for a NAC in senescence has not yet been Senescence, therefore, can be regulated at the level of described. AtNAC2 mRNA abundance was protein synthesis, protein activation, and protein increased after treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic-acid (ACC), by degradation. abscisic acid (ABA), and by an auxin. 16 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India postharvest performance. Many of the molecular mechanisms underlying senescence, and the respective genes involved in protein degradation, nucleic acid and chlorophyll breakdown, and lipid and nitrogen remobilization have been extensively covered in many reviews. An understanding of these mechanisms is vital to the use of molecular techniques to clone genes of interest to reverse, for example through antisense technology, the detrimental effects of senescence, ageing or PCD. Maternal inheritance of herbicide resistance via chloroplast engineering, or hyper expression of lethal insecticidal proteins (other than In senescing Iris petals, an up-regulated gene the Bt (Bacillus thuringiensis) gene product) provide encoded a plant homologue of the animal Grap2- and new genetic solutions to biocontrol of infectious agents cylininteracting protein (GCIP; van Doorn et al., 2003). in development of phytosanitary control. PCD in plants is well documented, and not GCIP is a helix–loop–helix leucine zipper family transcription factor that inhibits E2F1-mediated only is it synonymous with senescence (leaf and transcriptional activity The transcription factor E2F1 flower), but is also a fundamental part of a plant’s is a member of a family involved in cell cycle adaptation to stresses, such as reactive oxygen species. progression, differentiation, and cell death (Ma et al., The termination of a flower involves two overlapping mechanisms (Arora, 2008b), one being petals that 2007). In addition to the transcription factors abscise before the majority of their cells initiate a cell mentioned above, WRKY factors might also be death program, and where abscission may occur before regulators of petal senescence. The mRNA abundance or during the mobilization of food reserves to other of a gene encoding a RING domain ankyrin repeat parts of the plant. In the second, the petals are more protein, probably (part of) a ubiquitin ligase, drastically persistent, and cell deterioration and food remobilization increased during senescence in Mirabilis petals. occur while the petals are still part of the flower. One Silencing of the gene in Petunia petals delayed petal way of countering the effects of pathogen-induced wilting by a few days. The promoter of this gene had PCD is through the use of caspase inhibitors in the cut putative binding sites for bZip, HD-Zip, Myb, MADS, flower medium. Knotted1 is a homeobox gene found in apical meristems. Overexpression of this gene yielded phenotypes similar to those in which the cytokinin gene ipt is overexpressed. Knotted1 mRNA was not found in tobacco leaves undergoing senescence, but when knotted1 was overexpressed, driven by the promoter of a senescence specific cysteine protease (SAG12), it delayed leaf yellowing. This was associated with a 15-fold increase in cytokinin levels (Ori et al., 1999). These results suggest that the knotted1 found in carnation petals (Hoeberichts et al., 2007) is related to cytokinin metabolism. and also for WRKY (Xu et al., 2008). The data show that many transcriptional regulators become differentially expressed during petal senescence. However, these factors have as yet not been shown, for example by gene silencing or overexpression, to function as regulators of petal senescence. Molecular strategies Conventional breeding is still a practical form of increasing the number of flowering buds, extending the longevity of an inflorescence, and improving its Numerous ethylene-insensitive mutants, such as Arabidopsis thaliana etr1-1 or ein-2, or Never ripe tomato mutants exist. Flowers could be engineered to produce reduced levels of ethylene by introduction of an antisense ACC oxidase transgene, as occurs in tomatoes (FLAVR SAVR®), driven by a flower or senescence-specific promoter. Transgenic fruits containing ACC deaminase and antisense ACC synthase, ACC oxidase and polyphenol oxidase have been produced, the first three reducing ethylene production and slowing ripening, the last reducing browning of damaged tissue. 17 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Transgenic flowers had a much longer vase life than wild type but also showed problems related to a decreased resistance to fungal pathogens. Till now these products have not yet entered the market. The first attempt to block the function of the ethylene receptor was done by Hua et al. (1995) using a mutated Arabidopsis ers gene. Transgenic Arabidopsis plants showed strong tolerance to endogenous ethylene. Much more work has been done using the mutated dominant ethylene resistance gene etr1-1 from Arabidopsis. Early experiments with this gene were done in Petunia (Wilkinson et al., 1997). In these very thorough experiments a CaMV35S:etr1-1 construct was used, resulting in constitutive expression of the etr1-1 gene. The results showed that etr1-1 from Arabidopsis conferred ethylene insensitivity to the plants, but also that constitutive expression of the gene gave some additional effects that, though expected, are unacceptable from a grower’s point of view. When the endogenous cytokinin status is manipulated through transgenic intervention, a staygreen phenotype can be obtained, as occurred in the fusion of ipt, an Agrobacterium gene encoding a limiting step in cytokinin biosynthesis, to an Arabidopsis See (SAG12) promoter (Gan and Amasino, 1997). Greenness can also be altered (delay in leaf senescence) by down-regulating the production of a senescencepromoting hormone, as seen in tomato plants in which ethylene biosynthesis is inhibited by antisense suppression of the gene for ACC oxidase. Within flower species and cultivars there is great variability in ethylene sensitivity (of the flowers). This implies that breeding towards less sensitive flowers is possible. In fact, almost all modern carnation cultivars are much less sensitive to ethylene than the cultivar ‘White Sim’ that has been used over the years to study ethylene-induced senescence. In many flowers, breeding programs aiming at a better vase life may (unintentionally) target the ethylene biosynthesis and perception processes. Facilitated by the detailed knowledge of ethylene production and perception in plants, several attempts have been made to produce plants with prolonged flower life by genetic transformation. The first experiments in this line of work were based on ACC-oxidase (ACO), the last enzyme in the ethylene biosynthetic pathway. Savin et al. (1995) transformed carnation with a construct in which an antisense sequence of the carnation-ACO gene was placed under control of a constitutive promoter. This resulted in a few plants with dramatically reduced ethylene production during flower senescence, and with flower longevity of 8–9 days for cut flowers compared to 5 days for the non transformed flowers. No experiments were done with exposing these plants to exogenous ethylene, but there is no reason to think that the flowers would have a significantly better longevity under such conditions, as only the endogenous ethylene production and not the ethylene sensitivity is affected by ACO. Carnations with reduced ACC synthase activity using a co-suppression technique were produced at Florigene (transgenic carnations exhibit prolonged postharvest life). Another line of experiments have been carried out in Petunia by Chang et al. (2003). They used a construct (Psag12-IPT) containing a promoter from a senescence associated gene coupled to a cytokinin biosynthetic gene from A. tumefaciens to control the cytokinin production of senescing flowers. Several simultaneous effects were seen: the cytokinin level was enhanced, and this was accompanied by a delay in ethylene production and by enhanced ethylene tolerance of the flowers of the transgenic plants. In concert, these changes gave a dramatic effect: the flower longevity was prolonged with approximately 100% in non-pollinated flowers and approximately 450% in pollinated flowers. The report contains no information on other effects such as seed set or pollen viability. Conclusion and future perspectives There is little doubt that the molecular and genetic analyses of flower senescence made in the past 5 years have raised our awareness of the complex interactions that occur to regulate flower development and senescence. Gene technologies have enabled scientists to search for senescence-related genes in 18 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India plants often described as science models (e.g., Petunia, Arabidopsis), and then translate the data into other species to determine the functional significance of the expression of specific genes in specific tissues after harvest. Interactions between ethylene, cytokinin, sugars and various hydrolytic enzymes are now known to differentially mediate the progression of flower senescence. The individual importance of each signal appears to be species-specific and, in some instances, variety-specific, and varies differentially between floral organs. The challenge for postharvest scientists is to identify a hierarchy of regulators or a specific pattern of events that progresses senescence for certain groups of flower species. Subsequent categorization of cut flowers based on their metabolism and sensitivities will enable targeted application of appropriate postharvest technologies. References Abeles, F.B., Morgan, P.W. and Saltveit, Jr., M.E. (1992). Ethylene in Plant Biology. Academic Press, London. Arora, A. (2005). Ethylene receptors and molecular mechanism of ethylene sensitivity in plants. Current Science, 89: 1348-1361. Arora, A. (2008a). “Biochemistry of Flower Senescence”. In: Paliyath, G., Murr, D.P., Handa, A.K., Lurie, S. (eds.): Postharvest Biology and Technology of Fruits, Vegetables and Flowers. Blackwell Publishing, Iowa, USA, pp 51-85. Arora, A. (2008b). “Programmed Cell Death During Plant Senescence”. In: Paliyath, G., Murr, D.P., Handa, A.K., Lurie, S. (eds.): Postharvest Biology and Technology of Fruits, Vegetables and Flowers. Blackwell Publishing, Iowa, USA, pp 86-124. Arora, A. and Singh, V.P. (2004). Cysteine protease gene expression and proteolytic activity during floral development and senescence in ethyleneinsensitive gladiolus. Journal of Plant Biochemistry and Biotechnology, 13: 123126. Buchanan-Wollaston, V. (1997). The molecular biology of leaf senescence. J. Exp. Bot. 48:181-199. Buchanan-Wollaston, V. and Ainsworth, C. (1997). Leaf senescence in Brassica napus: cloning of senescence-related genes by subtractive hybridization. Plant Mol. Biol., 33: 821-834. Chang, H., Jones, M. L., Banowetz, G.M. and Clark, D.G. (2003). Overproduction of cytokinins in petunia flowers transformed with PSAG12-IPT delays corolla senescence and decreases sensitivity to ethylene. Plant Physiology, 132:2174–2183. Davies, K.M. and Grierson, D. (1989). Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene. Planta, 179: 73-80. Gan, S. and Amasino, R.M. (1997). Making sense of senescence. Plant Physiol., 113: 313-319. Grbic, V. and Bleecker, A.B. (1995). Ethylene regulates the timing of leaf senescence in Arabidopsis. Plant J., 8: 595-602. Guerrero, C., de la Calle, M., Reid, M.S. and Valpuesta, V. (1998). Analysis of the expression of two thiolprotease genes from daylily (Hemerocallis spp.) during flower senescence. Plant Mol. Biol., 36: 565-571. He, Y., Tang, W., Swain, J.D., Green, A.L., Jack, T.P. and Gan, S. (2001). Networking senescenceregulating pathways by using Arabidopsis enhancer trap lines. Plant Physiol., 126: 707716. Hoeberichts, F.A., van Doorn, W.G., Vorst, O., Hall, R.D. and van Wordragen, M.F. (2007). Sucrose prevents up regulation of senescence-associated genes in carnation petals. Journal of Experimental Botany, 58: 2873-2885. Hopkins, M., Taylor, C., Liu, Z., Ma, F., McNamara, L., Wang, T.W. and Thompson, J.E. (2007). Regulation and execution of molecular disassembly and catabolism during senescence. New Phytologist, 175: 201-214. Hua, J., Chang, C., Sun, Q. and Meyerowitz, E. M. (1995). Ethylene insensitivity conferred by Arabidopsis ERS gene. Science, 269:1712– 1714. John, I., Hackett, R., Cooper, W., Drake, R., Farrell, A. and Grierson, D. (1997). Cloning and 19 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India characterization of tomato leaf senescencerelated cDNAs. Plant Mol. Biol., 33: 641-651. Jones, M.L. and Woodson, W.R. (1999). Differential expression of three members of the laminocyclopropane-1-carboxylate synthase gene family in carnation. Plant Physiol., 119: 755-764. Jones, M.L., Larsen, P.B. and Woodson, W.R. (1995). Ethylene-regulated expression of a carnation cysteine proteinase during flower petal senescence. Plant Mol. Biol., 28: 505-512. Kende, H. (1993). Ethylene biosynthesis. Ann. Rev. Plant Physiol., 44: 283-307. Lanahan, M.B., Yen, H.C., Giovannoni, J.J. and Klee, H.I. (1994). The Never Ripe mutation blocks ethylene perception in tomato. Plant Cell, 6: 521-530. Lawton, K.A., Raghothama, K.G., Goldsbrough, P.B. and Woodson, W.R. (1990). Regulation of senescence-related gene expression in carnation flower petals by ethylene. Plant Physiol., 93: 1370-1375. Ma, W., Stafford, L.J., Li, D., Luo, J., Li, X., Ning, G. and Liu, M. (2007). GCIP/CCNDBP1, a helix-loop-helix protein, suppresses tumorigenesis. Journal of Cellular Biochemistry, 100: 1376-1386. Ori, N., Juarez, M.T., Jackson, D., Yamaguchi, J., Banowetz, G.M. and Hake, G.M. (1999). Leaf senescence is delayed in tobacco plants expressing the maize homeobox gene knotted1 under the control of a senescence-activated promoter. The Plant Cell, 11: 1073-1080. Orzaez, D. and Granell, A. (1997). The plant homologue of the defender against apoptotic death gene is down-regulated during senescence of flower petals. FEBS Letters, 404: 275-278. Quirino, B.F., Normanly, J. and Amasino, R.M. (1999). Diverse range of gene activity during Arabidopsis thaliana leaf senescence includes pathogen-independent induction of defenserelated genes. Plant Mol. Biol., 40: 267-278. Savin, K.W., Baudinette, S.C., Graham, M.W., Michael, M. Z., Nugent, G. D., Lu, C.Y., et al. (1995). Antisense ACC oxidase RNA delays carnation petal senescence. Hort Science, 30:970–972. Valpuesta, V., Lange, N.E., Guerrero, C. and Reid, M.S. (1995). Up-regulation of a cysteine protease accompanies the ethylene-insensitive senescence of daylily (Hemerocallis) flowers. Plant Mol. Biol., 28: 575-582. van Doorn, W.G., Balk, P.A., van Houwelingen, A.M., Hoeberichts, F.A., Hall, R.D., Vorst, O., van der Schoot, C. and van Wordragen, M.F. (2003). Gene expression during anthesis and senescence in Iris flowers. Plant Molecular Biology, 53: 845-863. Weaver, L.M., Gan, S., Quirino, B. and Amasino, R.M. (1998). A comparison of the expression patterns of several senescence-associated genes in response to stress and hormone treatment. Plant Mol. Biol., 37: 455-469. Wilkinson, J. Q., Lanahan, M. B., Clark, D. G., Bleecker, A. B., Chang, C., Meyerowitz, E. M. and Klee, H. J. (1997). A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous plants. Nature Biotechnology, 15:444–447. Woodson, W.R. (1994). Molecular biology of flower senescence in carnation. In Molecular and Cellular Aspects of Plant Reproduction (R.J. Scott and A.D. Stead, Eds.), pp. 225-267. Cambridge University Press, Cambridge, UK. Woodson, W.R., Brandt, A.S., Itzhaki, H., Maxon, J.M., Park, K.Y. and Wang, H. (1993). Regulation and function of flower senescencerelated genes. Acta Hort., 336: 41-46. Xu, X., Jiang, C. and Reid, M.S. (2008). Functional analysis of a putative ubiquitin ligase that is highly expressed during flower senescence. Journal of Experimental Botany (in press). Yamada, T., Takatsu, Y., Kasumi, M., Marubashi, W. and Ichimura, K. A. (2004). A homolog of the defender against apoptotic death gene (DAD1) in senescing gladiolus petals is down-regulated prior to the onset of programmed cell death. J Plant Physiol., 161:1281-1283. 20 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ROLE OF ROS AND ANTIOXIDANTS IN THE REGULATION OF LEAF AND FLOWER SENESCENCE AND FRUIT RIPENING R.K Sairam Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110012 Senescence is the age-dependent deterioration process at the cellular, tissue, organ, or organismal level, leading to death or the end of the life span. Flower senescence is organ level senescence but is often intimately associated with cellular or organismal death. Annual plants undergo flower senescence along with the organismal-level senescence when they reach the end of their temporal niche, as we observe at the grainfilling and maturation stage of the crop fields of soybean, corn, or rice. The connection between oxidative damage and aging, on one hand, and antioxidant defense mechanisms on the other, has been postulated in both animals and plants. A potentially decisive factor in determining the outcome of oxidative stress is the speed with which plants can activate their antioxidant reserves either by synthesizing new antioxidants or utilizing pre-existing pools. FLOWER SENESCENCE The flower is one of the most ephemeral of plant organ systems because it is specialized for the specific functions of pollen dispersal and reception, after which time many individual floral organs senesce while others develop further to form seeds and fruit. This transition in flower function involves the programmed senescence of the petals and sepals. Perianth senescence of some flowers occurs as part of a temporal program with the petals and sepals senescing strictly as a function of age. For example, daylily flowers senesce 12 ± 18 h after flower opening (Lukaszewski and Reid, 1989; Lay-Yee et al, 1992). The endogenous signals that regulate age-dependent petal senescence are completely uncharacterized, although the process is accompanied by the regulated expression of a suite of genes, some of which are functionally related to those associated with leaf senescence. Other flowers, such as petunia, gradually senesce over a period of days after flower opening, but this process is accelerated by pollination. In still other flowers perianth senescence is absolutely dependent on pollination and in these cases the external stimuli and endogenous signals that regulate programmed senescence have been examined in detail (O’Neill et al, 1993; O’Neill and Nadeau, 1997; O’Neill, 1997). OXIDATIVE STRESS AND ANTIOXIDANTS One of the paradoxes of life on this planet is that the molecule that sustains aerobic life, oxygen, is not only fundamentally essential for energy metabolism and respiration, but it has been implicated in many diseases and degenerative conditions (Marx, 1985). A common element in such diverse human disorders as ageing, arthritis, cancer, Lou Gehrig’s disease and many others is the involvement of partially reduced forms of oxygen. Our realization of the significance of oxygen in disorders and stress-induced dysfunctions in cultivated plants is recent due in no small part to the difficulty in detecting and tracing oxygen molecules, to the multitude of forms and intermediates that oxygen can assume, and to the extreme reactivity and rate of the chemical reactions involved. Atmospheric oxygen in its ground-state is distinctive among the gaseous elements because it is a biradical, or in other words it has two unpaired electrons. This feature makes oxygen paramagnetic; it also makes oxygen very unlikely to participate in reactions with organic molecules unless it is “activated”. The requirement for activation occurs because the two unpaired electrons in oxygen have parallel spins. According to Pauli’s exclusion principle, this precludes reactions with a divalent reductant, unless this reductant also has two unpaired electrons with 21 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India parallel spin opposite to that of the oxygen, which is a very rare occurrence. Hence, oxygen is usually nonreactive to organic molecules which have paired electrons with opposite spins. This spin restriction means that the most common mechanisms of oxygen reduction in biochemical reactions are those involving transfer of only a single electron (monovalent reduction). FREE ENERGY STATUS OF DIFFERENT OXYGEN SPECIES Superoxide radical Singlet state of oxygen Perhydroxy radical Hydrogen peroxide Triplet state Or ground state of oxygen Hydroxyl radical Activation of oxygen may occur by two different mechanisms: absorption of sufficient energy to reverse the spin on one of the unpaired electrons, or monovalent reduction. The biradical form of oxygen is in a triplet ground state because the electrons have parallel spins. If triplet oxygen absorbs sufficient energy to reverse the spin of one of its unpaired electrons, it will form the singlet state, in which the two electrons have opposite spins (Fig. 1). This activation overcomes the spin restriction and singlet oxygen can consequently participate in reactions involving the simultaneous transfer of two electrons (divalent reduction). Since paired electrons are common in organic molecules, singlet oxygen is much more reactive towards organic molecules than its triplet counterpart Water Fig. 1. The activation states of oxygen Hydrogen peroxide (H2O2) is noteworthy because it readily permeates membranes and it is therefore not compartmentalized in the cell. Numerous enzymes (peroxidases) use hydrogen peroxide as a substrate in oxidation reactions involving the synthesis of complex organic molecules. The well-known reactivity of hydrogen peroxide is not due to its reactivity per se, but requires the presence of a metal reductant to form the highly reactive hydroxyl radical, which is the strongest oxidizing agent known and reacts with organic molecules at diffusion-limited rates. Fenton described in the late nineteenth century (Fenton, 1894; 1899) the oxidising potential of hydrogen peroxide mixed with ferrous salts. Forty years later, Haber and Weiss (1934) identified the Hydroxyl radical (OH.) as the oxidizing species in these reactions: Fe3+ + OH. + OHFe2+ + H2O2 Nomenclature of the various forms of oxygen Given below in Figure 1 are some of the reactive forms of oxygen prevalent in biological systems. Superoxide radical: Superoxide (O2.-) can act as either an oxidant or a reductant; it can oxidize sulphur, 3+ .O2 + Fe2+ ascorbic acid or NADPH; it can reduce cytochrome Fe + O2 C and metal ions. In biological systems O2.- is generated Fe2+, Fe3+ at the site of PSI in chloroplastic electron transport .OH. + O2 + OHchain under conditions of high light and CO2 deficiency. H2O2 + O2 A dismutation reaction leading to the formation of Hydrogen peroxide is produced as the hydrogen peroxide and oxygen can occur dismutation product of O2.- in chloroplast, mitochondria spontaneously or is catalyzed by the enzyme superoxide and peroxysomes, and also a bye product of dismutase. photorespiration in peroxysomes. Hydrogen peroxide is scavenged by nonspecific peroxidase (POX), ascorbate peroxidase (APX), glutathione peroxidase (GPX) and catalase (CAT). SOD O2.- + 2H+ H2 O2 22 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India APX Exposure of plants to unfavourable monodehydroascorbic environmental conditions such as vicissitudes of Ascorbic acid + H2O2 temperature, high light intensity, water availability, air acid +H2O pollutants, salt-stress, and physiological conditions like POX aging/maturation and senescence can increase the RH2 + O2 RH + H2O2 production of reactive oxygen species such as singlet oxygen (1O2), superoxide radical (O2.-), hydrogen CAT peroxide (H2O2) and hydroxyl radical (OH.). Plants 2H2O2 2H2O + O2 possess both enzymic and non-enzymatic mechanisms Peroxidases are located in the apoplastic for scavenging of ROS. Superoxide radical is regularly region, where they function in the peroxidation of synthesized in the chloroplast (Elstner 1991) and phenolic compounds and synthesis of lignins. Ascorbate mitochondria (Rich and Bonner 1978), though some peroxidase is located in chloroplast, cytosol, and some quantity is also reported to be produced in microbodies reports have also suggested mitochondrial form of (Lindquist et al. 1991). APX. Catalases are also fund in mitochondria and The enzymic mechanisms are designated to chloroplast. minimize the concentration of O2.- and H2O2. The Reactivity of hydroxyl radical (OH.) enzymes overproduced so far include superoxide The extreme reactivity of hydroxyl radical dismutase (SOD), ascorbate peroxidase (APX), (OH.) is due to its addition of OH. to the organic glutathione reductase (GR) and glutathione-synthesizing molecule, or abstraction of a hydrogen ion (H+) from enzymes. Scavenging of O .- by SOD results in the 2 it. production of H2O2, which is removed by APX (Asada Addition reaction 1992) or Catalase (Scandalias 1992). However, both O2.- and H2O2 are not as toxic as the OH., which is formed by the combination of O2.- and H2O2 in the presence of trace amounts of Fe2+ and Fe3+ by the Haber–Weiss reaction (Fenton 1889, Haber and Weiss Subtraction reaction 1934). Hydroxyl radical can damage chlorophyll, protein, DNA, lipids and other important macromolecules (Frankel 1985, Farr and Kogama 1991, Imlay and Linn 1986), thus fatally affecting plant These reactive oxygen species (ROS) result metabolism and ultimately growth and development. in the degradation/inactivation of proteins, DNA/RNA, A schematic presentation of production and scavenging lipids and carbohydrates. A very well known example of O .- and H O and OH., and OH. mediated lipid 2 2 2 is that of lipid peroxidation by hydroxyl radical. peroxidation and glutathione peroxidase- mediated stabilization of lipids are presented in figure. 3. Increase in activities of SOD, APX, CAT and GR under drought, high temperature and salinity, and comparatively higher activity in tolerant wheat genotypes has also been reported by Sairam et al. (1998, 2000, 2001, 2005). Increase in activity of SOD, APX, GR, DHAR, CAT and POX in response to salinity stress as well as higher antioxidant activity in tolerant species/varieties have also been reported by various workers (Chen et al. Fig. 2. The peroxidation of linoleic acid 23 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India 1997, Gueta-Dahan et al. 1997, Gomez et al. 1999, Hernandez et al. 2000 ). Sairam and Srivastava (2002) reported omparatively higher Cu/Zn-SOD, Fe-SOD, APX and GR activity in chloroplastic fraction and Mn- Fig. 3. Generation and scavenging of superoxide radical and hydrogen peroxide, and hydroxyl radicalinduced lipid peroxidation and glutathione peroxidase-mediated lipid (fatty acid) stabilization. APX (ascorbate peroxidase), ASC (ascorbate), DHA (dehydro-ascorbate), DHAR (dehydro-ascorbate reductase), Fd (ferredoxin), GR (glutathione reductase), GSH (red glutathione), GSSG (oxi-glutathione), HO. (hydroxyl radical), LH (lipid), L., LOO., LOOH, (unstable lipid radicals and hydroperoxides); LOH (stable lipid -fatty acid), MDHA (monodehydro-ascorbate), MDHAR (mono dehydro-ascorbate reductase, NE (non-enzymatic reaction), PHGPX (phospholipid-hydroperoxide glutathione peroxidase), SOD (superoxide dismutase). ROLE OF ROS ANTIOXIDANTS IN LEAF senescence (Dhindsa et al. 1981, Thompson et al. 1987). Senescence in plants is generally associated SENESCENCE with decline in levels of phospholipids and galactolipids Organ senescence is an oxidative process that (Harwood et al. 1983) [13]. Free radical reactions involves degradation of the cellular and sub-cellular have been suggested to play an important role in the structures and macromolecules, and the mobilization degradation process of membrane polar lipids in of the products of degradation to other parts of the senescence (Grossman and Leshem 1978, Thompson plant. Susceptibility to oxidative stress depends on the et al. 1987, Lin, C.H. Kao 1998). Increased levels of overall balance between production of oxidants and TBARS indicating lipid peroxidation have also been antioxidant capability of the cell (del Rio et al. 1998). reported during senescence (Chia et al. 1981, Kunert Changes in membrane permeability and loss in ability and Ederer 1985). Antioxidant such as superoxide to retain solutes have been associated with fruit ripening dismutase, ascorbate peroxidase, glutathione (Sacher 1973) and senescence of green plant tissue reductase, peroxidase and catalase are involved in the (Ferguson and Simons 1973). Reactive oxygen species scavenging of reactive oxygen radicals (Bowler et al. such as superoxide radical, hydrogen peroxide and 1992, Scandalios 1993, Asada 1992, Foyer 1993). hydroxyl radical have a role in lipid peroxidation, There are reports suggesting both increase in the membranes damage and consequently in leaf activities of various antioxidants enzymes (Bueno and 24 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India del Rio 1992, del Rio et al.1992), as well as decrease petals. They suggest that oxidative stress and a in the activities of these enzymes (Dhindsa et al. 1981, concerted antioxidant response are associated with the initial stages of aging in Dendranthema morifolium Hurng and Kao 1994) during senescence. Prochazkova et al. (2001) studied leaf senescence petals. in maize and reported that leaf senescence in general and early senescence of early maturing cv. X 3342 was associated with higher oxidative stress and a decline in antioxidant activity towards maturity, and Mn-SOD has a major role in the scavenging of superoxide radicals during maize leaf senescence. Sairam et al. (2004) reported that SOD (Mn-SOD and Cu/Zn-SOD), APX and CAT follow a pattern similar to the pattern of Chl and Car contents in different leaves, i.e., in younger leaves the oxidative stress was minimum and the antioxidant demand was also less. Chl and Car contents increased to a peak in 7th - 9th leaves, which also showed the highest antioxidant enzyme activities. The decline in SOD, APX and CAT activity in subsequent older leaves was associated with a parallel decline in pigment contents, reflecting an enhanced senescence due to lower antioxidant activity. They further concluded that antioxidant enzymes such as SOD, APX and CAT play an important role in the regulation of senescence processes. ROS AND ANTIOXIDANTS DURING FLOWER SENESCENCE Bartoli et al. (1997) reported that aging of chrysanthemum petals was accompanied by a 9-fold increase in oxygen radical generation, and a 2-fold increase in the content of oxidized proteins. αTocopherol content increased at the onset of aging, and decreased as aging progressed. Total thiol content followed the a-tocopherol pattern during aging. Petals’ ascorbic acid content started to decline when the flowers were excised. Exposure of flowers showing browning of the petal, to 1 mM paraquat over 24 h did not cause increase in the activities of superoxide dismutase, glutathione reductase, and catalase but led to a 3-fold increase in oxygen radical generation, and to a 59% increase in hydroperoxide content in aged Ezhilmathi et al (2007) proposed that petal wilting in Gladiolus was associated with ROS induced lipid peroxidation, enhanced LOX activity, and decrease in ROS scavenging system in the form of SOD and CAT. Similar findings regarding the role of ROS in petal wilting of ethylene insensitive Gladiolus and ethylene sensitive carnation were reported by Yamane et al (1999). Celikel and Van Doorn (1995) also reported the interaction of ROS and antioxidant enzymes in the flower senescence in case of ethyleneinsensitive Daylily. Similar results have been reported in carnation petals (Sylvestre et al. 1989) and Daylily (Panavas and Rubinstein 1998), although in both cases the changes occur rather later in the progression of senescence. Catalase activity continuously decreased from harvest to the senescent stage. Panavas and Rubinstein (1998) also observed a steady decrease in CAT activity in Daylily from about 6 h before flower opening to the last stage. They further observed that when induced prematurely CAT activity decreases earlier. Kumar et al. (2007) studied the involvement of oxidative stress and the role of superoxide dismutases (SOD) in rose petal senescence with two popular rose cultivars, ‘Grandgala’ and ‘First Red’, from the seven whorls in each flower harvested at six different stages of development. The results revealed that rose petal senescence was associated with a higher production of O2 .– radicals (approx. 10-fold). A parallel increase in SOD activity was also noted in different petals whorls up to Stage-4, which declined thereafter. Different isoforms of SOD exhibited variable levels of activity, with Cu/Zn-SOD being the most active, followed by Mn-SOD, and Fe-SOD. A significant decline in SOD activity appeared to be associated with petal senescence. Kumar and Srivastava (2007) studied the changes in endogenous levels of various antioxidants from stage 1 to stage 6 in rose (Rosa hybrida L.) cv. First Red. Contents of ascorbic acid, dehydroascorbic acid, red.-glutathione, oxidized 25 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India However, Leverentz et al. (2002) using a combination of in vitro assays and chemical profiling of the lipid oxidation products generated studied the role of lipoxygenase (lox) in senescence of Alstroemeria peruviana flowers, and concluded that loss of membrane function was not related to lox activity or accumulation of lipid hydroperoxides per se and differs in these respects from other ethylene-insensitive floral tissues representing a novel pattern of flower senescence. glutathione, and activities of monodehydro-ascorbate reductase and dehydro-ascorbate reductase increased up to 4/5th stages and drastically declined at 6th stage. Kumar et al. (2008) further reported a continuous increase in hydrogen peroxide content from stage 1 to stage 6 in rose (Rosa hybrida L.) cv. Grand Gala and First Red. The activities of antioxidant enzymes viz., catalase, peroxidase and glutathione reductase increased up to 5th stage and declined drastically at the 6th stage, suggesting an association between decline in antioxidant status with increase in oxidative stress and consequently the senescence of the rose flower. Meir et al. (1994) reported that application of three water-soluble, antioxidant agents such as morin, kaempferol and glutathione (GSH), as a pre-treatment to various cut flowers and cuttings retarded significantly their leaf senescence and extended by a few days longevity of the individual flowers. Application of morin, kaempferol and GSH to cut rose flowers (Rosa hybrida, cv. ‘Jaguar’) improved significantly the appearance and opening of the petals, and extended their vase-life by 2 days. However, the response of rose flowers to antioxidants was found to be cultivardependent. Hossain et al. (2006) observed that petal senescence in gladiolus as a function of decline in membrane stability index was associated with increase in H2O2 content, which was highest at the complete wilting stage of the flower. SOD activity increased continuously till the last stage, while APX activity declined progressively and lowest was observed at the last stage. Though GR activity increased up to 50 % wilting stage but declined to the lowest level at the complete wilting stage of the flower. They concluded that a decline in H2O2 scavenging activity, in terms of APX and to some extent GR, was responsible for the flower wilting observed at the last stage. ROS AND ANTIOXIDANTS DURING FRUIT MATURITY AND RIPENING Fruit development and ripening are complex processes involving major changes in fruit metabolism. Fruit ripening has been described as an oxidative phenomenon, characterized by oxidative stress with chlorophyll and protein breakdown. Plant cells produce ROS, particularly superoxide and hydrogen peroxide, which have been implicated as a second messenger in many processes associated with plant growth and development. Rogiers et al. (1998) studied the extent of oxidative stress during ripening of saskatoon (Amelanchier alnifolia Nutt.) fruit. Lipid peroxidation during fruit development from the mature green to the fully ripe (purple) stage increased. Activities of superoxide dismutase (SOD) and catalase (CAT) fell about 4 and 18-fold, respectively, during development, indicating higher potential for the accumulation of cytotoxic H2O2. Peroxidase activity remained relatively low and constant from the mature green to the dark red stage of development, then increased towards the end of ripening as fruits turned purple. Lipoxygenase (LOX) activity increased 2.5-fold from the mature green to the fully ripe stage. Tissue prints showed LOX Kumar et al. (2008) reported a parallel to be present throughout fruit development and increase in lipoxygenase activity and lipid peroxidation, Western analysis revealed that the increase in activity as measured by TBARS contents and a continuous during ripening was due to increased synthesis of the decline in membrane stability index at progressive enzyme. Their results provide evidence that ripening stages of rose flower opening and development till of this climacteric fruit is accompanied by a substantial senescent stage. The result suggests a role of enzyme increase in free-radical-mediated peroxidation of lipoxygenase in inducing rose flower senescence. membrane lipids, probably as a direct consequence 26 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India of a progressive decline in the enzymatic systems responsible for catabolism of active oxygen species. The role of glutathione-mediated free-radical scavenging was also examined as a potential system for coping with this increased oxidative stress. Concentrations of reduced and oxidized glutathione (GSSG) increased 2- fold and GSSG increased as a percentage of total glutathione, reflecting the increase in oxidative status of fruits during ripening. Tissue prints of glutathione reductase (GRase) and transferase (GTase) showed these enzymes to be distributed throughout the pericarp at all stages of fruit development. GRase and GTase activities rose sharply during the later stages of fruit ripening, correlating well with substantial increases in the levels of both enzymes. Hence, the glutathione-mediated free-radical scavenging system was up-regulated towards the end of ripening, perhaps in response to the increasing oxidative stress resulting from the accumulation of lipid hydroperoxides from increased LOX activity, in conjunction with a decline in SOD/CAT activities. Mondal et al. (2004) reported that ripening of tomato fruits was accompanied by a progressive increase in oxidative/peroxidative stress. The cultivar with short shelf life had higher oxidative stress than the cultivar with longer shelf life. Increase in production of lipid hydroperoxides and other reactive oxygen species during development eventually induced higher activities of peroxidase, glutathione reductase, superoxide dismutase, catalase and ascorbate peroxidase, but not until the later stages of ripening. The activities of these enzymes were consistently low in the cultivar with short shelf life, suggesting that the reduced scavenging ability and associated increase in oxidative stress in the cultivar with short shelf life may be responsible for mediating many of the physicochemical changes that facilitate early ripening/softening of the fruits. higher than those in pericarp at all maturity stages, and the H2O2 content in the seed increased sharply after the pink stage. Higher activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) occurred in seeds than that in the pericarp. At the beginning of fruit ripening, an increase in CAT activity followed the accumulation of H2O2, but its activity declined quickly by the end of ripening. These patterns of enzyme activity suggest that the AsA-GSH cycle might play an important role in scavenging H2O2. The maximum malondialdehyde (MDA) content appeared in seeds earlier than that in the pericarp, the maximum being 1.5 fold of that in the pericarp. High concentration of ROS in the seed is closely related with fruit ripening, and H2O2 might be an important factor in triggering senescence. Bouvier et al (1998) tested the role of reactive oxygen species (ROS) as regulators of chromoplast carotenoid biosynthesis in vivo during maturity in Bell pepper (Capsicum annuum cv Yolo Wonder). Their results suggest that in vivo, transient, oxidative stress plays a key role in the induction of chromoplast carotenoid biosynthesis and in the transformation of chloroplasts into chromoplasts as evidenced by induction of expression of multiple carotenogenic genes mRNAS that give rise to capsanthin. Similarly, down-regulation of catalase by amitrole, so as to increase level of ROS, also induced the expression of carotenogenic gene mRNAs leading to the synthesis of capsanthin The specific activation of carotenogenic genes under was further supported by the fact that none of the ROS species used in induced the accumulation of capsidiol, a typical isoprenoid stress metabolite produced in elicited pepper cells. ROS AND ETHYLENE INTERACTION The accumulation of reactive oxygen species (ROS) is involved in regulating cell death. Hydrogen Shen et al. (2008) studied the changes in the peroxide and superoxide have emerged as the two levels of ROS and activities of antioxidant enzymes key ROS and recent studies have addressed their during fruit ripening and senescence in pericarp and sources and control of their production. ROS signals seed of tomato (Lycopersicon esculentum Mill.). interact directly or indirectly with several other signaling Superoxide (O2-.) production rate and hydrogen pathways, such as nitric oxide, and the stress hormones peroxide (H2O2) content in seed were significantly salicylic acid, jasmonic acid and ethylene. The 27 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Bouvier, F., Backhaus, R.A., and Bilal Camara, B. (1998). Induction and Control of Chromoplastspecific Carotenoid Genes by Oxidative Stress. J. Biol. Chem. 273: 30651–30659. interaction and balance of these pathways determines whether the cell lives or dies. Moeder et al. (2002) reported that ethylene synthesis and perception were required for active H2O2 production and cell death resulting in visible tissue damage. The results demonstrate a selective ozone response of ethylene biosynthetic genes and suggest a role for ethylene, in combination with the burst of H2O2 production, in regulating the spread of cell death. By using Arabidopsis mutant, radical-induced cell death1 (rcd1), in which ozone (O3) and extra-cellular superoxide (O2.-), induced cellular O2.- accumulation and transient spreading lesions it was shown that the cellular superoxide (O2.-) accumulation is ethylene dependent, occurs ahead of the expanding lesions before visible symptoms appear, and is required for lesion propagation. Exogenous ethylene increased O2.-dependent cell death, whereas impairment of ethylene perception by norbornadiene in rcd1or ethylene insensitivity in the ethylene- insensitive mutant ein2 and in the rcd1 ein2 double mutant blocked O 2.accumulation and lesion propagation (Overmyer et al. 2002). Bowler, C., Van Montague, M., Inze, D. (1992). Superoxide dismutase and stress tolerance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43: 83–116. Bueno, P., del Rio, L.A. (1992). Purification and properties of glyoxysomal cuperozinc superoxide dismutase from watermelon (Citrullus _ulgaris Scrad.). Plant Physiol. 98: 331–336. Celikel FG, Van Doorn WG (1995) Solute leakage, lipid peroxidation and protein degradation during the senescence of Iris petals. Physiol Plant 94:515–521. Chen, Y. W., Shao, G. H. and Chang, R. Z. (1997). The effect of salt stress on superoxide dismutase in various organelles of cotyledons of soybean seedlings. Acta Agron. Sin. 23: 214–219. Chia, L.S., Thompson, J.E., Dumbroff, E.B. (1981). Stimulation of leaf senescence on membranes by treatment with paraquat. Plant Physiol. 67: 415–420. In conclusion we can say that leaf and flower senescence and fruit ripening is accompanied by an del Rio, L.A., Pastori G.M., Palma, J.M., Sandalio, L.M., Sevilla, F., Corpas, F.J., Jimenez, A., increase in oxidative stress, which could be a direct Lopez-Huertas, E., Hernandez, J.A. (1998). result of decline in antioxidative potential of the tissue. The activated oxygen role of peroxisomes in ROS in turn degrade essential macro-molecules and senescence. Plant Physiol. 116: 1195–1200. thus aid in senescence and death. Recent studies have del Rio, L.A., Sandalio, L.M., Palma, J.M., Bueno, shown role of ethylene signaling in ROS generation in P., Corpas, F.J. (1992). Metabolism of oxygen relation to leaf necrosis/cell death. radicals in peroxisomes and cellular implications, Free. Radic. Biol. Med. 13: 557–580. References Asada K. (1992). Ascorbate peroxidase — A Dhindsa, R.A., Plumb-Dhindsa, P., Thorpe, T.A. (1981). Leaf senescence: correlated with hydrogen peroxide scavenging enzyme in plants. increased permeability and lipid peroxidation, Physiol. Plant. 55: 235–241. and decreased levels of superoxide dismutase Asada, K., Ascorbate peroxidase – a hydrogen and catalase. J. Exp. Bot. 126: 93–101. peroxide scavenging enzyme in plants. Physiol. Elstner, E. F. (1991). Mechanisms of oxygen activation Plant., 1992, 85, 235–241. in different compartments of plant cell. In Active Bartoli, C.G., Simontacchi, M., Montaldi, E.R. and Oxygen/Oxidative Stress and Plant Puntarulo, S. (1997). Oxidants and antioxidants Metabolism (eds Pell, E. J. and Stefen, K. L.). during aging of chrysanthemum petals. Plant Sci. American Society of Plant Physiology, Rockville, 129: 157-165 MD, pp. 13–25. 28 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India induction of antioxidant defences. Plant Cell Ezhilmathi, K., Singh, V. P., Arora, A. and Sairam, Biol. 23: 853–862. R. K. (2007). Effect of 5-sulfosalicylic acid on antioxidant activity in relation to vase life of Hurng, W.P., Kao, C.H. (1994). Lipid peroxidation Gladiolus cut flowers. Plant Growth Regul. 51: and antioxidative enzymes in senescing tobacco 99-108. leaves during post flooding. Plant Sci. 96: 41– 44. Farr, S. B. and Kogama, T. (1991). Oxidative stress response in Escherichia coli and Salmonella Imlay, J. A. and Linn, S. (1986) DNA damage and typhimurium. Microbiol. Rev. 55: 561–585 oxygen radical toxicity. Science, 240: 1302– 1309. Fenton, H. J. H. (1889). Oxidation of certain organic acids in the presence of ferrous salts. Proc. Jiménez, A., Hernandez, J.A., Pastori, G., del Rio, Chem. Soc. 25: 224. L.A., Sevilla, F. (1998). Role of ascorbateglutathione cycle of mitochondria and Ferguson, C.H.R., Simons, E.W. (1973). Membrane peroxisomes in the senescence of pea leaves. lipids in senescing green tissues. J. Exp. Bot. 24: Plant Physiol. 118: 1327–1335. 307–316. Foyer C.H. (1993). Ascorbic acid, in: R.C. Alscher, Kumar, N. and Srivastava, G.C. (2008). Antioxidant metabolism during rose (Rosa hybrida L.) petal J.L. Hess (Eds.), Antioxidants in Higher Plants, Senescence. J. Hort. Sci. Biotech. 83: 755– CRC Press, Boca Raton, , pp. 31–58. 759. Frankel, E. N. (1985). Chemistry of free radical and singlet oxidation of lipids. Prog. Lipid Res., 23: 197–221. Grossman, S., Leshem, Y. (1978). Lowering of endogenous lipoxygenase activity on Pisum sati_um foliage by cytokinins as related to senescence. Physiol. Plant. 43: 359–362. Kumar, N., Srivastava, G.C. and Dixit, K. (2007). Role of superoxide dismutases during petal senescence in rose (Rosa hybrida L.). J. Hort. Sci. .Biotech. 82: 673–678. Kumar, N., Srivastava, G.C. and Dixit, K. (2008). Role of Lipoxygenase During Flower Bud Opening in Roses (Rosa hybrida L.). Hort. Gueta-Dahan, Y., Yaniv, Z., Zilinskas, B. A. and BenEnviron. Biotechnol. 49:1-6. Hayyin, G. (1997). Salt and oxidative stress: Similar and specific response and their relation Kumar, N., Srivastava, G.C. and Dixit, K. (2008). Senescence in rose (Rosa hybrida L.): role of to salt tolerance in citrus. Planta 203: 460–469. the endogenous antioxidant System. J. Hort. Sci. Haber, F. and Weiss, J. (1934). The catalytic Biotech. 83: 125–131. decomposition of hydrogen peroxide by iron Kunert, K.J., Ederer M. (1985). Leaf aging and lipid peroxidation: the role of the antioxidants vitamin C and E., Physiol. Plant. 65: 85–88. Harwood, J.L., Jones, A.V.H.M., Thomas, H. (1982). Leaf senescence in a non-yellowing mutant of Leverentz, M.K., Wagstaff, C., Rogers, H.J., Stead, A.D. and Chanasut, U. (2002). Helena Festuca pratensis. Planta 156: 152–157. Silkowski, Brian Thomas, Heiko Weichert, Ivo Hernandez, J. A., Campillo, A., Jimenez, A., Alarcon, Feussner, and Gareth Griffiths Characterization J. J. and Sevilla, F. (1999). Response of of a Novel Lipoxygenase-Independent antioxidant systems and leaf water relations to Senescence Mechanism in Alstroemeria NaCl stress in pea plants. New Phytol. 141: peruviana Floral Tissue. Plant Physiol. 130: 241–251. 273–283, Hernandez, J. A., Jimerez, A., Mullineaux and Sevilla, P. F. (2000). Tolerance of pea (Pisum sativum) Lin, J.N., Kao, C.H. (1998). Effect of oxidative stress caused by hydrogen peroxide on senescence of to long term salt stress is associated with rice leaves. Bot. Bull. Acad. Sin. 39: 161–165. salts. Proc. R. Soc. London Ser. A, 147: 332– 334. 29 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Lindquist, Y., Branden, C. L., Mathews, F. S. and Prochazkova, D., Sairam, R.K., Srivastava, G.C. and Singh, D.V. (2001). Oxidative stress and Lederer, F., Spinach glycolate oxidase and yeast antioxidant activity as the basis of senescence in flavocytochrome b2 are structurally homologous maize leaves. Plant Sci. 161: 765-771. and evolutionarily related enzymes with distinctly different function and flavin mononucleotide Rich, P. R. and Bonner, W. D. Jr., The sites of binding. J. Biol. Chem., 1991, 266, 3198–3207. superoxide anion generation in higher plant mitochondria. Arch. Biochem. Biophys., 1978, Meir, S., Reuveni, Y., Rosenberger, I., Davidson, H., 188, 206–213. Philosoph-Hadas, S. (1994). Improvement of the postharvest keeping quality of cut flowers Rogiers, S.Y., Mohankumar, G. N. and Knowles, N. and cuttings by application of water-soluble R. (1998). Maturation and ripening of Fruit of antioxidants. Acta Hort. 368: 355-364. Amelanchier alnifolia Nutt. are Accompanied by increasing oxidative stress. Annl. Bot. 81: Moeder, W., Barry, C.S. , Tauriainen, A.A. , Betz, 203-211. C., Jaana Tuomainen, J., Utriainen, M., Grierson, D., Sandermann, H., Langebartels, C., and Sacher, J.A. (1973). Senescence and post harvest Kangasjarvi, J. (2002).Ethylene synthesis physiology. Annu. Rev. Plant Physiol. 24: 197– regulated by biphasic induction of 1224. aminocyclopropane-1-carboxylic acid synthase and1-aminocyclopropane-1-carboxylic acid Sairam, R. K. and Srivastava, G. C. (2002). Changes in antioxidant activity in sub-cellular fractions of oxidase genes is required for hydrogen peroxide tolerant and susceptible wheat genotypes in accumulation and cell death in ozone-exposed response to long-term salt stress. Plant Sci. 162, tomato. Plant Physiol. 130: 1918–1926. 897–904. Mondal, K., Sharma, N.S., Malhotra, S.P., Dhawan, K. and Singh, R. (2004). Antioxidant systems Sairam, R. K., Chandrasekhar, V. and Srivastava, G. C. (2001). Comparison of hexaploid and in ripening tomato fruits Biol. Plant. 48: 49-53. tetraploid wheat cultivars in their response to O’Neill, S.D. (1997). Pollination regulation of flower water stress. Biol. Plant. 44, 89–94. development. Ann. Rev. Plant Physiol. Mol. Sairam, R. K., Deshmukh, P. S. and Saxena, D. C. Biol. 48: 547 - 572 (1998). Role of antioxidant systems in wheat O’Neill, S.D. and Nadeau, J. (1997). Postpollination genotypes tolerance to water stress. Biol. Plant. flower development. Hortic. Rev. 19: 1-58 41, 384–389. O’Neill, S.D., Nadeau, J.A., Zhang, X.S., Bui, A.Q. Sairam, R. K., Deshmukh, P. S. and Shukla, D. S. and Halevy, A.H. (1993). Interorgan regulation (1997). Tolerance to drought and temperature of ethylene biosynthetic genes by pollination. stress in relation to increased antioxidant enzyme Plant Cell 5: 419 -432 activity in wheat. J. Agron. Crop Sci. 178, 171– 177. Overmyer, K., Tuominen, H., Kettunen, R., Betz, C., Langebartels, C., Sandermann, Jr., H., and Sairam, R. K., Srivastava, G. C. and Saxena, D. C. Kangasjärvi, J. (2000). Ozone-Sensitive (2000). Increased antioxidant activity under Arabidopsis rcd1Mutant Reveals Opposite elevated temperature: a mechanism of heat stress Roles for Ethylene and Jasmonate Signaling tolerance in wheat genotypes. Biol. Plant. 43, Pathways in Regulating Superoxide-Dependent 245–251. Cell Death Plant Cell 12: 1849–1862. Sairam, R.K., Singh, D.V. and Srivastava, G.C. (2003/ Panavas T, Rubinstein B (1998) Oxidative events 04). Changes in activities of antioxidant. enzymes during programmed cell death of Daylily in sunflower leaves of different age. Biol. Plant. (Hemerocallis hybrid) petals. Plant Sci 133:125– 47: 61-66. 13 30 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Scandalias, J. G. (1990). Response of plant antioxidant Sylvestre, I., Droillard, M.J., Bureau, J.M., Paulin, A. defence genes to environmental stress. Adv. (1989). Effects of the ethylene rise on the Genet. 28, 1–41. peroxidation of membrane lipids during senescence of cut Carnations. Plant Physiol. Scandalios, J.G. (1993). Oxygen stress and superoxide Biochem. 27: 407–413 dismutase. Plant Physiol. 101: 7–12. Shen, L., Ruan, Y., Liu, K. and Sheng, J. (2008). Metabolism of Reactive Oxygen Dynamic Changes during Tomato Fruit Ripening and Senescence. Acta Hort. 768: 517-523. Thompson, J.E., Legge, R.L., Barber, R.L. (1987). The role of free radicals in senescence and wounding. New Phytol. 105: 317–334. Yamane, K., Kawabata, S., Fujishige, N. (1999). Changes in activities of SOD, catalase and Stead, A., Van Doorn, W.G. (1994). Strategies of peroxidases during senescence of Gladiolus flower senescence: a review. In RJ Scott, AD florets. J. Jap. Soc. Hort. Sci. 68: 798–802 Stead eds, Molecular and Cellular Aspects of Plant reproduction. Cambridge University Press, Ye, Z., Rodríguez, R., Tran, A., Hoang, H., de los Santos, D., Brown, S., Vellanoweth R.L. Cambridge, pp 215–238 (2000). The developmental transition to Stead, A.D. (1992). Pollination-induced flower flowering repress ascorbate peroxidase activity senescence: a review. Plant Growth Regul. 11: and induces enzymatic lipid peroxidation in leaf 13-20 tissue in Arabidopsis thaliana. Plant Sci. 158: 115–127. 31 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India 1-METHYLCYCLOPROPENE: A TOOL FOR MANAGING ETHYLENE AND POST-HARVEST LOSSES IN HORTICULTURAL PRODUCE Vijay Paul* and Rakesh Pandey Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110 012 *E-mail: vijay_paul_iari@yahoo.com difference being that losses are not replaced in the postharvest environment. Hence, they suffer from detrimental changes after harvest. Fruit ripening and flower senescence are associated with a series of physiological and biochemical changes. These include an increase in hydrolytic enzymes, degradation of macromolecules, increased respiratory activity and a loss of cellular compartmentalization, utilization of energy reserves through respiration, changes in biochemical composition, textural changes, water loss, and increased ethylene production. During ripening/ senescence endogenous signals up regulate certain genes whose products show high homology to enzymes known to induce climacteric shift, aroma and flavour besides causing the degradation of proteins, RNA, lipids and chlorophyll etc. As a result of all the above said changes, both, qualitative and quantitative losses occur between harvest and consumptions. It is therefore desirable to minimize these losses by extensive understanding of underlying processes along with the elucidation of regulatory steps and mechanisms involved during post-harvest period. This is urgently needed, not only in view of providing the nutritional security to human beings but also, towards the sustained economic gain to the farmers. Post-harvest losses of fruits, vegetables and flowers are enormous in developing countries due to poor storage and transportation facilities. The available climatic diversity in India, which includes climates such as; tropical, sub-tropical and temperate, allows to grow wide range of fruits, vegetables and flowers. The importance of consumable commodities such as; fruits and vegetables can be judged from the fact that they also serve as functional food and nutraceuticals as they are source of energy, vitamins, minerals, dietary fibers, antioxidants and other phytochemicals. The tenet “Let food be thy medicine and medicine be thy food” as said by Hippocrates nearly 2,500 years ago is receiving renewed interest. Flowers, on the other hand, are highly perishable in nature. There are certain flowers like; rose and carnation where floral senescence is regulated in part by ethylene production. However, there is another big group of plants like; daylily, sandersonia, tulip, narcissus, Irish, hollandica, gladiolus etc. where flowers are insensitive to ethylene. If we increase the physiological level of this hormone the senescence is not hastened in these flowers or if we inhibit its production in flower or its action then senescence cannot be delayed. Loses in flowers are mainly due to the senescence of flower parts or/also the leaves of cut flowers. In general, it is estimated that post-harvest loss in India is up to 40 %. Thus, there is need to understand in depth the ripening and senescence process so that these processes can be regulated to save the fruits, vegetables and flowers from postharvest losses. The recent trends in food consumption habits show major emphasis on the consumption of fresh fruits and vegetables due to health concerns and increase in the purchasing power. Today the consumer demands good quality, fresh and nutritious fruits and vegetables. This has necessitated the development of suitable Harvest commodities are still living organs and technologies for enhancing the freshness and shelf life therefore they continue to respire and lose water as if of fruits and vegetables and preservation of food they were still attached to the parental plant, the only products. There is also little doubt that the molecular 32 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India and genetic analyses of flower senescence made in the past 5 years have raised our awareness of the complex interactions that occur to regulate flower development and senescence. Interactions between ethylene, cytokinin, sugars and various hydrolytic enzymes are now known to differentially mediate the progression of flower senescence. The individual importance of each signal appears to be speciesspecific and, in some instances, variety-specific, and varies differentially between floral organs. So, an important goal of the producers, stockiest, supplier, exporters and researchers is to minimize the losses at pre- and post-harvest stages. Approaches to delay ripening and senescence to minimize the post-harvest losses Approaches such as; controlled atmosphere, modified atmosphere and modified atmosphere package can retard the on going metabolic processes, respiration, transpiration, ethylene production and softening etc. Thereby the methods based on these approaches have become the established practices for extending the post-harvest life. However, the availability of such modified atmospheric conditions at reasonable price and at appropriate time is a practical problem in the developing and third world countries due to absence or lack of such infrastructure. As a result, a large quantity of produce undergoes spoilage. So, there is a need to develop simple and cost-effective methods of controlling fruit ripening and reducing the postharvested losses. Out of various other available approaches, approaches involving avoiding the exposure or minimizing the production or suppressing the perception/response of ethylene are very important. These approaches are of much relevance to the climacteric fruits because it is mainly the ethylene which is controlling the progress of ripening in these fruits. In this way, by following these approaches, effective postharvest management of edible commodities as well as ornamental products that are sensitive to ethylene can be achieved. Ethylene and 1-methylcyclopropene (1-MCP) Ethylene responses can be derived from either internal synthesis within plants and fruit (endogenous) or through exposure to external sources such as engine exhausts, heaters, fungi or ripening fruit (exogenous). Most common ways of blocking the perception/ response of ethylene are by the use of CO2 (higher concentrations), silver ion, 2, 5-norbornadiene, diazocyclopentadiene and trans-cyclooctene. In this direction, discovery of 1-MCP is the most important because it is an effective inhibitor of ethylene perception and thereby its response in plant system (Fig. 1). Chemically, 1-MCP (C4H6) is a cyclopropene with molecular mass of 54.09. It is volatile at STP. It inhibits the ethylene mediated ripening by binding to the ethylene receptors. The mode of action of 1-MCP is by its ability to binds to a metal in the ethylene receptors in a competitive way and thereby it stops further processes mediated by ethylene (Fig. 2). The presence of 1-MCP not only blocks the basal but also the auto-induced ethylene production (which is triggered production of ethylene in presence of ethylene itself due to its positive effect on the induction of ethylene biosynthetic enzymes i. e., ACC-synthase and ACC-oxidase enzymes (Fig 2). A breakthrough in application technology of 1-MCP resulted from the formulation of 1-MCP as a stable powder in which it is complexed with γ- cyclodextrin. As a result, 1-MCP can now be easily released as a gas when this powder formulation is dissolved in water. It is only after this that commercial products having 1-MCP as an active ingredient under the trade name of ‘EthylBloc’ (Floral Life Inc., USA) and ‘SmartFresh’ (AgroFresh Inc., USA) introduced. The ‘EthylBloc’ is aimed mainly for ornamental crops while the ‘SmartFresh’ is for edible commodities. In presence of 1-MCP, ethylene responses such as; fruit ripening and senescence of flowers, leaves and plant as a whole are found to be retarded. Beneficial effects of 1-MCP in delaying the senescence and enhancing the post-harvest life are demonstrated in number of fruits, vegetables, flowers, plants and bulbs as listed in Table 1. In this way, the usefulness of 1-MCP is very widespread. 33 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Table 1. Fruits, vegetables and ornamentals reported to show positive effect of 1-MCP treatment on their post-harvest aspects and shelf life. -------------------------------------------------------------------------------------Fruits Vegetables Ornamental products -------------------------------------------------------------------------------------Climacteric Broccoli Cut flowers -------------------------------------------------------------------------------------Apple Carrots Cymbidium orchid Apricot Lettuce Snapdragon Avocado Cucumber Delphinium Banana Geraldton waxflower Custard apple Lupine Kiwifruit Mango Flowering plants Nectarine Rose Papaya Kalanchoe Pear Campoanula carpatica Peach Lilium (oriental hybrid) Persimmon Begonia Plum Impatiens Tomato Poinsettia Non-climacteric Geranium Grapefruit Petunia Orange Catharanthus Pineapple Calceolaria Strawberry Schlumbergera Lime Nicotiana alata Mandarin Pepper Bulbs Watermelon Tulip -------------------------------------------------------------------------------------- CH3 1-Methylcyclopropene (1-MCP) Fig. 1: Molecular structure of 1-methylcyclopropene 1-MCP (a blocking agent of ethylene receptor). Methionine SAM-synthetase S-Aerosol methionine ACC-synthase 1-Aminocyclopropane -1-carboxylic acid ACC-oxidase P C-M Ethylene 1-MCP - e nleyht E detiade m dessreppussies nopsre er pin 1f oec nes Perception of ethylene by its binding to ethylene binding receptor Source: Watkins, C.B. and Miller, W.B. (2003) Response (Ripening and senescence etc.) 1-MCP is actively researched and has been approved for use in some fruits in USA. In 2003, it was approved for the use in apples. Today it is probably the most useful compound among recently developed inhibitors of ethylene response. It is relatively stable in dilute gas phase for several months. The EPA has classified 1-MCP as a plant regulator structurally related to the compounds present in the plant. It has been demonstrated that treatment with 1-MCP resulted in reduced accumulation of ACC-synthase as Fig. 2: Simplified biosynthetic and response pathway of ethylene in plants. 1-Methylcyclopropene (1-MCP) blocks the receptor of the ethylene and : means inhibition or thereby its response. suppression, : means blockage of response pathway in presence of 1-MCP. 34 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India well as ACC-oxidase transcripts and ethylene production. In terms of its mode of action, 1-MCP competitively binds to a metal in the ethylene receptors so it blocks the normal as well as autocatalytic (feed forward) system II of ethylene production. As a result of this, 1-MCP provides protection for a longer period of time than any other potential inhibitor compounds. 1-MCP has been reported to delay or reduce ethyleneinduced effects by suppressing the ripening and ripening-related changes in many fruits as described in detail in Table 2. Table 2: Some of the important fruits (climacteric and non-climacteric) where 1-methylcyclopropene (1-MCP) slowed down ripening and ripening related changes Fruits Climacteric Apple Appricot Effect of 1-MCP on ripening and ripening-related changes in different fruits Avocado Ripening: The 1-MCP concentrations required to saturate binding sites, and the extent and longevity of 1-MCP action, are influenced greatly by species, organ, tissue, and mode of ethylene biosynthesis induction, and temperature and length of exposure to this gas (Sisler and Serek 1997). Cultivar can affect product responses to 1-MCP (Abdi et al. 1998, Fan et al. 1999, Muller et al. 2000, Rupasinghe et al. 2000, Skog et al. 2001, Watkins et al. 2002). For fruit, these differences may be attributable to differences in maturity and ripening stages at harvest, as well as inherent rates of metabolism. In general, later harvested fruit are usually less responsive to 1-MCP, as might be expected for a compound that acts by inhibiting ethylene action. Effects of cultivar and harvest date could complicate commercial adoption of 1-MCP technology for fruit in some growing areas. Application of 1-MCP may be limited also for bananas that normally have mixed maturity of fruit within bunches at harvest (Harries et al. 2000). 1-MCP applications was found to be less effective in controlling banana ripening when applied at 2 oC compared with 20 oC (Macnish et al. 2003), and the authors suggested that 1-MCP binding is poorer at low temperature, perhaps due to conformational changes in a membrane-located ethylene receptor protein. Treatment at low temperature may result in relatively greater accumulation and/or non-specific binding of 1-MCP molecules in plant tissues. Banana Custard apple Guava Kiwifruit Mango Papaya Peach Pear Plum Tomato Reduction/delayed Reference Ethylene production, respiration,colour change, soft scald, internal browning, decay, softening Defilippi et al. (2005), Kondo et al. (2005), Lurie et al. (2005), Pechous et al. (2005), Tatsuki et al. (2007), Marin et al. (2008) Ethylene production, respiration, softening, decay, colour change, ripening, internal flesh browning, volatiles production Ethylene production, loss in green colour, respiration, ripening, softening, decay colour change, weight loss, chilling, injury, physiological disorders Ethylene production, respiration, ripening, chlorophyll loss, colour change, volatiles production, softening, loss of starch Softening Respiration rate, softening, colour change, ripening Ethylene production, respiration, softening colour change Botondi et al. (2003), Martino et al. (2006). Ethylene production, ripening, softening, colour change, volatiles production, aroma development Ethylene production, ripening, softening, de-greening Ethylene production, respiration, softening, ripening, browning, Ethylene production, respiration, softening, scald development Ethylene production, respiration, softening, colour change, aroma development, decay, browning, weight loss, Ethylene production, respiration, lycopene accumulation, softening chlorophyll loss, fruit abscission Non- climacteric Orange De-greening, rot incidence, abscission of fruit Pineapple Ethylene production, chilling injury, internal browning Strawberry Ethylene production, softening, colour change, decay Watermelon Water soaking, electrolyte leakage 35 Adkins et al. (2005), Woolf et al. (2005), Hershkovitz et al. (2005), Choi, et al. (2008) Jiang et al. (2004), Lohani et al. .(2004), Trivedi and Nath (2004), Gupta et al. (2006) Benassi et al. (2003) Bassetto et al. (2005), Bassetto et al. (2005), Singh and Pal (2008) Ding et al. (2003) Neves et al. (2003), Boquete et al. (2004), Koukounaras and Sfakiotakis (2007).. Lalel et al. (2003), Singh and Dwivedi (2008). Ergun and Huber (2004) Balbontínet et al. (2007), Manenoi et al. (2007) Liguori et al. (2004), Hayama, et al. (2008), Hayama et al. (2008) Mwaniki et al. (2005), Szczerbanik (2005), MacLean et al. (2007), Yamane et al. (2007), Calvo and Sozzi (2009) Menniti et al. (2004), Valero et al. (2004), Menniti et al. (2006), Khan and Singh (2007), Manganaris, et al. (2008) Opiyo and Ying (2005), Saltveit (2005), Guillén et al. (2006), Guillén et al. (2007), Choi and Huber (2008), Paul et al. (2009) Zhong et al. (2001) Selvarajan et al. (2001) Bower et al. (2003) Mao et al. (2004) Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India chilling injury (woolliness and internal browning) in 1MCP treated apricots, nectarines, peaches and plums (Dong et al. 2001, Dong et al. 2002, Fan et al. 2002). In orange, increased chilling injury caused by exogenous ethylene application was not prevented, but rather enhanced by 1-MCP application (Porat et al. 1999). Another important aspect of 1-MCP application on disorders has been highlighted by Flavour: Flavor is a composite of taste and odor, research on the effects of 1-MCP on apple fruit. The and volatile production can be greatly affected by observation showed that 1-MCP inhibits superficial ethylene. Therefore, decreased and/or altered volatile scald development in apple. production in 1-MCP compared with untreated fruits Diseases: Decay incidence can be increased by 1(Fan and Matheis 1999, Golding et al. 1999, Fan et MCP application in oranges and strawberries (Porat al. 2000, Rupasinghe et al. 2000), may impact product et al. 1999, Jiang et al. 2001), inhibited in apricots acceptability by consumers. 1-MCP decreased the (Dong et al. 2002), or unaffected in grapefruit (Mullins ripening-related increases of acetate and butyrate et al. 2000). Factors that influence the effects of 1esters, and decreases in alcohols and aldehydes (Pre- MCP on disease development are likely to be specific Aymard et al. 2003). Some flavor changes may be to the product and its interaction with a pathogen. unacceptable. Off-flavor development was increased Reduced ethylene sensitivity can be beneficial against by 1-MCP treatment (Porat et al. 1999), and higher some pathogens but deleterious to resistance against titratable acidity in treated apricots could result in other pathogens, e.g. in citrus, where mold and stem sourness [Fan et al. 2000]. The impact of 1-MCP on end rots are inhibited and enhanced by ethylene, acceptability by the consumer will not be understood respectively (Mullins et al. 2000). Small amounts of until 1-MCP products are available in the marketplace. endogenous ethylene may be necessary to maintain There appears to be no data on 1-MCP effects on basic levels of resistance to environmental and pathological stress, for example, by inducing PAL floral fragrance. Physiological disorders: In general, it might be activity and phenolics concentrations in the tissue (Jiang expected that the incidence of any disorder that is et al. 2001). Potential role of 1-MCP and plant associated with senescence, e.g. breakdown, might disease exists in the tulip bulb, where infection by the be decreased by 1-MCP treatment. Results with 1- fungus Fusarium is associated with the production of MCP also indicated that 1-MCP may reduce or large quantities of ethylene. The presence of this aggravate chilling injury depending on the commodity. ethylene is problematic to adjacent (but non-diseased) Chilling injury was decreased in 1-MCP treated fruit bulbs, as a number of ethylene-induced disorders can of apple (Rupasinghe et al. 2000, Watkins et al. occur, including flower abortion and gummosis 2002,), avocado (Pesis et al. 2002) and melon (Ben- (polysaccharide degradation and eruption). While 1-MCP clearly provides a powerful tool to manipulate senescence and ripening of horticultural products, adoption of commercial handling practices will need to take into account commodity type, cultivar, the stage of maturity or ripeness and temperature of treatment. Each of these factors may affect the appropriate 1-MCP concentration that should be applied. Amor et al. 1999), and the non-climacteric pineapple (Selvarajah et al. 2001). In the case of these climacteric fruit, reduced incidence of the particular disorder was associated with inhibited ethylene production. However, in the pineapple, maintenance of ethylene production may be involved in preventing development of chilling injury in such fruit. This possibility is highlighted by the greater incidence of Effect of 1-MCP on flower senescence Flowers (like fruits) are also categorized as being climacteric or non-climacteric. In climacteric or ethylene sensitive flowers such as carnations, Gypsophila and orchids, senescence is accompanied by a sudden, transient increase in ethylene production and respiration while treatment of non-senescent 36 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India flowers with ethylene rapidly induces petal senescence. In non-climacteric flowers such as gladiolous, tulip and iris, generally, no increases in ethylene production and respiration are a apparent during flower senescence, and exogenous ethylene has little or no effect on petal senescence. In these latter species, ethylene may, however, have severe effects on other plant parts such as bulbs or corms (Kamerbeek and De Munk 1976). Knowledge about ethylene sensitivity of flower species is necessary to predict the effects of e.g. mixed storage and transport of flowers with fruit species, to predict the usefulness of anti-ethylene treatments and to direct breeding programs towards better flower vase life. With respect to petal senescence, sensitivity to ethylene was found to be roughly determined at the plant family level. High sensitivity is found in e.g. Campanulaceae, Caryophyllaceae, Geraniaceae, Malvaceae, Orchidaceae, Primulaceae, Ranunculaceae and Rosaceae species; low sensitivity is found in Compositae and Iridacae species and in most of the Amarylliadaceae and Liliaceae species. Sensitivity of species within one plant family is generally comparable (Woltering and van Doorn 1988) The spectacular effect of 1-MCP has been well documented in a range of ornamental species (Table 3). The commercial producers of ornamental products in many countries are already benefiting from this new development. Table 3: List of flowers/plants where 1-MCP treatment was found to be beneficial in delaying senescence and enhancing shelf life or vase life. Ornamentals Alstroemeria, Dianthus caryophyllus, Antirrhinum majus Delphinium elatum, Catharanthus roseus, Easter lily (Lilum longiflorum), Impatiens, Petunia Campanula medium L. Carnation Chrysanthemum Daffodil (Narcissus pseudonarcissus) Ixora Orchid (Cymbidium, Phalaenopsis) Begonia x elatior Begonia x tuberhybrida Campanula carpatica Epipremnum pinnatum Geranium (Pelargonium x hortorum) Hibiscus Kalanchoe Lathyrus odoratus Oriental hybrid lilies (Lilium) Rosa hybrida Schlumbergera truncata Tulip References Serek et al. 1995c, Sisler and Serek, 1997, Skog et al. 2001b, Celikel et al. 2002 Skog et al. 2001a, Serek et al. 1995b Bosma and Dole 2002 Hassan and Gerzson 2002 Hassan and Gerzson 2002 Hunter et al. 2004 Michaeli et al. 2001 Heyes and Johnson 1998 Skog et al. 2001b Serek et al. 1994b Serek and Sisler 2001 Muller et al. 1997 Cameron and Reid 2001, Jones et al. 2001, Kadner and Druege 2004, Skog et al. 2001b Reid et al.2002, Serek et al. 1998 Skog et al. 2001b, Kebenei et al. 2003a, 2003b, Kebenei et al. 2003b Celikel et al. 2002, Han and Miller 2003 Serek et al. 1994b, Muller et al. 2000, Skog et al. 2001b Serek and Sisler 2001 de Wild et al. 2002 37 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India and commercial scale. The temperature control facilities are either costly or not available in recourse poor countries. It is therefore desirable to evaluate the effective dose and efficiency of 1-MCP at higher storage temperatures, which are prevalent in the subtropical and tropical regions. It would also be desirable to work out a dose of 1-MCP that could be effective for different stages and varieties of fruit stored at relatively higher temperatures. Safety concerns regarding 1-MCP The Environment Protection Agency (EPA) has classified 1-MCP as a plant regulator. It has considered 1-MCP as a reduced-risk product with safe profiles. As 1-MCP is applied at extremely low dose levels (usually in parts per billion (ppb) and therefore it has almost no measurable residues in food commodities. Further concentration of residues, if any in the treated commodity after few days of storage, is so low that it is found to be below the detection limits. The EPA, initially in April 1999, granted the use of 1-MCP in flower and then in 2003 approved its use in apples. The use of 1-MCP, at concentration up to 1.0 ppm, has been approved in USA for many fruits like; tomato, apple, plum, apricot, avocado, mango, papaya, peach, pear, persimmon, kiwifruit and nectarines. Registration of 1-MCP for its use had already been obtained by many countries such as; Argentina, Australia, Austria, Brazil, Canada, Chile, Costa Rica, France, Guatemala and Honduras, Israel, Mexico, The Netherlands, New Zealand, South Africa, Switzerland, Turkey, UK and US for number of commodities including; fruits, vegetables and flowers. The registration in several other countries, especially EU countries, for a range of horticultural products, is also in progress. Conclusions The enhancement in the shelf life for 1-MCP treated fruits was primarily because of suppression of ethylene action and its auto-induced production. 1MCP enhanced the shelf life but in a variety dependent manner. Small-scale trials have shown very encouraging results with 1-MCP. However, large-scale trials with a suitable dose of 1-MCP on different varieties, at different ripening stages/mixture of different ripening stages at higher storage temperatures of 25 and 30 0C need to be evaluated critically. Currently 1MCP is entering the market world-wide to treat a variety of ornamental products and genetically engineered ornamentals, with either a decreased ethylene production or perception and, thereby superior post-harvest performance. As per international standards (prescribed by EPA) the safety, clinical signs of systemic toxicity, acute toxicity and environmental profile of 1-MCP (which is a type of cyclopropene) in humans, animals and environment are extremely favourable (Environmental Protection Agency, 2002, Sisler et al. 2006). However, some naturally occurring cyclopropenes have been reported to inhibit fatty acid desaturation and gluconeogenesis in animals and possess carcinogenic and neurochemical activities (Pawlowski et al. 985, Salaun and Baird, 1995). Thereby, details and complete understanding of short and long-term effects of 1-MCP on animals, human beings and our environment were emphasized (Sisler et al. 2006). Work in the above suggested lines would be helpful in practically exploiting the use of 1-MCP at commercial levels especially in regions with higher ambient temperatures (sub-tropical and tropical parts of the world) for the effective storage of fruits and reducing the quantum of post-harvest losses. Some bottlenecks towards the commercialization of 1-MCP The bottlenecks towards commercialization of 1-MCP include its response being influenced by concentration, exposure duration, temperature (during treatment and storage), application method, type of fruit, variety and developmental stage or maturity of the commodity. This may be linked to the fact that, response to ethylene also depends on factors such as; type of produce, cultivars, maturity at the time of harvest, temperature and activity of other hormones. Most of the studies on fruit ripening have evaluated 1-MCP at temperatures of 15, 20 or 25 0 C. The delaying of ripening was found to be inversely related to the temperature during storage. Although, the findings with 1-MCP are positive but, studies are still required to determine the influence of its treatment on different cultivars and in different regions at large 38 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Bosma, T. and Dole, J.M. (2002). Postharvest handling of cut Campanula medium flowers. HortSci. 37: 954-958. Botondi, R., DeSantis, D., Bellincontro, A., Vizovitis, K. and Mencarelli, F. (2003). Influence of ethylene inhibition by 1-methylcyclopropene on apricot quality, volatile production, and glycosidase activity of low- and high-aroma varieties of apricots. J. Agricul. Food Chem. 51: 1189-1200. Bower, J., Holford, P., Latche, A. and Pech, J.C. (2002). Culture conditions and detachment of the fruit influence the effect of ethylene on the climacteric respiration of melon. Postharvest Biol. Technol. 26: 135-146. Calvo, G. and Sozzi, G.O. (2009). Effectiveness of 1MCP treatments on ‘Bartlett’ pears as influenced by the cooling method and the bin material. Postharvest Biol. Technol. 51: 49-55. Cameron, A.C. and Reid, M.S. (2001). 1-MCP blocks ethylene-induced petal abscission of Pelargonium peltatum but the effect is transient. Postharvest Biol. Technol. 22: 169-177. Celikel, F.G., Dodge, L.L. and Reid, M.S. (2002). Efficacy of 1-MCP (1-methylcyclopropene) and promalin for extending the postharvest life of oriental lilies (Lilium x ‘Mona Lisa’ and ‘Star Gazer’). Sci. Hort, 93: 149-155. Choi, S.T. and Huber, D.J. (2008). Influence of aqueous 1-methylcyclopropene concentration, immersion duration, and solution longevity on the postharvest ripening of breaker-turning tomato (Solanum lycopersicum L.) fruit. Postharvest Biol. Technol. 49: 147-154. Choi, S.T., Tsouvaltzis, P., Lim, C.I. and Huber D.J. (2008). Suppression of ripening and induction of asynchronous ripening in tomato and avocado fruits subjected to complete or partial exposure to aqueous solutions of 1-methylcyclopropene. Postharvest Biol. Technol. 48: 206-214. de Wild, H.P.J., Gude, H. and Pepplenbos, H.W. (2002). Carbon dioxide and ethylene interactions in tulip bulbs. Physiol. Plant. 114: 320-326. References Abdi, N., McGlasson, W.B., Holford, P., Williams, M. and Mizrahi, Y. (1998). Responses of climacteric and suppressed-climacteric plums to treatment with propylene and 1methylcyclopropene. Postharvest Biol. Technol. 14:29-39. Abeles, F.B., Morgan, P.W. and Saltveit, M.E. (1992). Ethylene in Plant Biology, Academic Press, San Diego, California, pp 414. Adkins, M.E., Hofman, P.J. Stubbings, B.A. and Macnish. A.J. (2005). Manipulating avocado fruit ripening with 1-methylcyclopropene. Postharvest Biol. Technol. 35: 33-42. Balbontín, C., Gaete-Eastman, C., Vergara, M., Herrera, R. and Moya-León, M. A. (2007). Treatment with 1-MCP and the role of ethylene in aroma development of mountain papaya fruit. Postharvest Biol. Technol. 43: 67-77. Bassetto, E., Jacomino, A.P., Pinheiro, A.L. and Kluge, R.A. (2005). Delay of ripening of ‘Pedro Sato’ guava with 1-methylcyclopropene. Postharvest Biol. Technol. 35: 303-308. Bassetto, E., Jacomino, A.P., Pinheiro, A.L. and Kluge, R.A. (2005). Delay of ripening of ‘Pedro Sato’ guava with 1-methylcyclopropene. Postharvest Biol. Technol. 35: 303-308. Ben-Amor, B., Latche, A., Bouzayen, M., Pech, J.C. and Romojaro, F. (1999). Inhibition of ethylene biosynthesis by antisense ACC oxidase RNA prevents chilling injury in Charentais cantaloupe melons. Plant Cell Environ. 22:1579-1586. Benassi, G., Correa, G.A.S.F., Kluge, R.A. and Jacomino, A.P. (2003). Shelf life of custard apple treated with 1-methylciclopropene - an antagonist to the ethylene action. Brazilian Archives Biol. Technol. 46: 115-119. Boquete, E.J., Trinchero, G.D., Fraschina, A.A., Vilella, F. and Sozzi, G.O. (2004). Ripening of ‘Hayward’ kiwifruit treated with 1methylcyclopropene after cold storage. Postharvest Biol. Technol. 32: 57-65. 39 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India DeEll, J.R., Murr, D.P., Porteous, M.D. and Rupasinghe, H.P.V. (2002). Influence of temperature and duration of 1methylcyclopropene (1-MCP) treatment on apple quality. Postharvest Biol. Technol. 24:349-353. Ding, J.G., Chen, K.S., Xu, W.P. and Xu, C.J. (2003). Regulation of 1-methylcyclopropene on the ripening and softening of postharvest kiwifruit. Acta Hort. Sinica. 30: 277-280. Dong, L., Lurie, S. and Zhou, H-W. (2002). Effect of 1-methylcyclopropene on ripening of ‘Canino’ apricots and ‘Royal Zee’ plums. Postharvest Biol.Technol. 24:135-145. Dong, L., Zhou, H-W., Sonego, L, Lers, A. and Lurie, S. (2001). Ethylene involvement in the cold storage disorder of ‘Flavortop’ nectarine. Postharvest Biol. Technol. 23:105-115. Environmental Protection Agency (2002). Federal Register, 67 (144) (pp. 48796-48800). July 28, 2002. Golding, J.B., Shearer, D., McGlasson, W.B. and Wyllie, S.G. (1999). Relationships between respiration, ethylene, and aroma production in ripening banana. J. Agric. Food Chem. 47:1646-1651. Guillén, F., Castillo, S., Zapata, P.J., MartínezRomero, D., Serrano, M. and Valero, D. (2007). Efficacy of 1-MCP treatment in tomato fruit: 1. Duration and concentration of 1-MCP treatment to gain an effective delay of postharvest ripening. Postharvest Biol. Technol. 43: 23-27. Guillén, F., Castillo, S., Zapata, P.J., MartínezRomero, D., Valero, D. and Serrano, M. (2006). Efficacy of 1-MCP treatment in tomato fruit: 2. Effect of cultivar and ripening stage at harvest. Postharvest Biol. Technol. 42: 235242. Gupta, S.M., Srivastava, S., Sane, A.P. and Nath, P. (2006). Differential expression of genes during banana fruit development, ripening and 1-MCP treatment: Presence of distinct fruit specific, ethylene induced and ethylene repressed expression. Postharvest Biol. Technol. 42: 1622. Han, S.S. and Miller, J.A. (2003). Role of ethylene in postharvest quality of cut oriental lily ‘Stargazer’. Plant Growth Regul. 40: 213-222. Harris, D.R., Seberry, J.A., Wills, R.B.H. and Spohr, L.J. (2000). Effect of fruit maturity on efficacy of 1-methylcyclopropene to delay the ripening of bananas. Postharvest Biol. Technol. 20:303-308. Hassan, F.A.S. and Gerzson, L. (2002). Effects of 1MCP (1-methylcyclopropene) on the vase life of chrysanthemum and carnation cut flowers. Int. J. Hort. Sci. 8: 29-32. Hayama, H. Tatsuki, M. and Nakamura, Y. (2008). Combined treatment of aminoethoxyvinylglycine (AVG) and 1-methylcyclopropene (1-MCP) reduces melting-flesh peach fruit softening. Postharvest Biol. Technol. 50: 228-230. Hershkovitz, V., Saguy, S.I. and Pesis, E. (2005). Postharvest application of 1-MCP to improve Ergun, M.and Huber, D.J. (2004). Suppression of ethylene perception extends shelf-life and quality of ‘Sunrise solo’ papaya fruit at both pre-ripe and ripe stages of development. Eur. J. Hort. Sci. 69: 184-192. Fan, X. and Mattheis, J.P. (1999). Impact of 1methylcyclopropene and methyl jasmonate on apple volatile production. J. Agric. Food Chem. 47:2847-2853. Fan, X., Argenta, L. and Mattheis, J.P. (2000). Inhibition of ethylene action by 1methylcyclopropene prolongs storage life of apricots. Postharvest Biol. Technol. 20:135142. Fan, X., Argenta, L. and Mattheis, J.P. (2002). Interactive effects of 1-MCP and temperature on ‘Elberta’ peach quality. HortSci. 37:134138. Fan, X., Blankenship, S.M. and Mattheis, J.P. (1999). 1-Methylcyclopropene inhibits apple ripening. J. Amer. Soc. HortSci. 124:690-695. 40 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India the quality of various avocado cultivars. Postharvest Biol. Technol. 37: 252-264. Heyes, J.A. and Johnson, J.W. (1998). 1Methylcyclopropene extends Cymbidium orchid vase life and prevents damaged pollinia from accelerating senescence. NZ J. Crop Hort. Sci. 26: 319-324. Iones, M.L., Kim, E.S. and Newman, S.E. (2001). Role of ethylene and 1-MCP in flower development and petal abscission in zonal geraniums. HorSci. 36: 1305-1309. Jiang, Y. and Joyce, D.C. (2000). Effects of 1methylcyclopropene alone and in combination with polyethylene bags on the postharvest life of mango fruit. Ann. Appl. Biol. 137:321-327. Jiang, Y., Joyce, D.C. and Terry, L.A. (2001). 1Methylcyclopropene treatment affects strawberry fruit decay. Postharvest Biol. Technol. 23:227-232. Jiang, Y.M., Joyce, D.C., Jiang, W.B. and Lu. W.J. (2004). Effects of chilling temperatures on ethylene binding by banana fruit. Plant Growth Regul. 43: 109-115. Kadner, R. and Druege, U. (2004). Role of ethylene action in ethylene production and poststorage leaf senescence and survival of pelargonium cuttings. Plant Growth Regul. 43: 187-196. Kamerbeek, G.A. and De Munk, W.J. (1976). A review of ethylene effects in bulbous plants. Sci. Hort. 4: 101-115. Kebenei, Z., Sisler, E.C., Winkelmann, T. and Serek, M. (2003a). Efficacy of inhibitors of ethylene perception in improvement of display life of Kalanchoe (K. blossfeldiana Poelln.) flowers. Postharvest Biol. Technol. 30: 169-176. Kebenei, Z., Sisler. E.C., Winkelmann, T. and Serek, M. (2003b). Effect of 1-octylcyclopropene and 1-methylcyclopropene on vase life of sweet pea (Lathyrus odoratus L.) flowers. J. Hort. Sci. Biotechnol. 78: 433-436. Khan, A.S. and Singh, Z. (2007). 1-MCP regulates ethylene biosynthesis and fruit softening during ripening of ‘Tegan Blue’ plum. Postharvest Biol. Technol. 43: 298-306. Kondo, S., Setha, S., Rudell, D.R., Buchanan, D.A. and Mattheis, J.P. (2005). Aroma volatile biosynthesis in apples affected by 1-MCP and methyl jasmonate. Postharvest Biol. Technol. 36: 61-68. Koukounaras, A. and Sfakiotakis, E. (2007). Effect of 1-MCP prestorage treatment on ethylene and CO2 production and quality of ‘Hayward’ kiwifruit during shelf-life after short, medium and long term cold storage. Postharvest Biol. Technol. 46: 174-180. Liguori, G., Weksler, A., Zutahi, Y., Lurie, S., I. and Kosto, I. (2004). Effect of 1methylcyclopropene on ripening of melting flesh peaches and nectarines. Postharvest Biol. Technol. 31:263-268. Lohani, S., Trivedi, P.K. and Nath, P. (2004). Changes in activities of cell wall hydrolases during ethylene-induced ripening in banana: effect of 1MCP, ABA and IAA. Postharvest Biol. Technol. 31: 119-126. Lurie, S., A. Lers, Z. Shacham, L. Sonego, S. Burd, and B. Whitaker. (2005). Expression of alphafarnesene synthase AFS1 and 3-hydroxy-3methylglutaryl-coenzyme a reductase HMG2 and HMG3 in relation to a-farnesene and conjugated trienols in ‘Granny Smith’ apples heat or 1-MCP treated to prevent superficial scald. J. Am. Soc. Hort. Sci. 130: 232-236. MacLean, D.D., Murr, D.P., DeEll, J.R., Mackay A.B. and Kupferman E.M. (2007). Inhibition of PAL, CHS, and ERS1 in ‘Red d’Anjou’ Pear (Pyrus communis L.) by 1-MCP. Postharvest Biol. Technol. 45: 46-55. Macnish, A.J., Joyce, D.C., Hofman, P.J., Simons, D.H. and Reid, M.S. (2000b). 1Methylcyclopropene treatment efficacy in preventing ethylene perception in banana fruit and grevillea and waxflower flowers. Aust. J. Exp. Agric. 40: 471-481. 41 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Macnish, A.J., Joyce, D.C., Hofman, P.J., Simons, D.H. and Reid, M.S. (2000). 1Methylcyclopropene treatment efficacy in preventing ethylene perception in banana fruit and grevillea and waxflower flowers. Aust. J. Exp. Agric. 40: 471-481. Manenoi, A., Bayogan, E.R.V., Thumdee, S. and Paull, R.E. (2007). Utility of 1-methylcyclopropene as a papaya postharvest treatment. Postharvest Biol. Technol. 44: 55-62. Manganaris, G.A., Crisosto, C.H., Bremer, V. and Holcroft, D. (2008). Novel 1methylcyclopropene immersion formulation extends shelf life of advanced maturity ‘Joanna Red’ plums (Prunus salicina Lindell). Postharvest Biol. Technol. 47: 429-433. Mao, L.C., Karakurt, Y. and Huber, D.J. (2004). Incidence of water-soaking and phospholipid catabolism in ripe watermelon (Citrullus lanatus) fruit: induction by ethylene and prophylactic effects of 1-methylcyclopropene. Postharvest Biol. Technol. 33:1-9. Marin, A.B., Colonna, A.E., Kudo, K., Kupferman, E. M. and Mattheis J. P. (2009). Measuring consumer response to ‘Gala’ apples treated with 1-methylcyclopropene (1-MCP). Postharvest Biol. Technol. 51: 73-79 Martino, G. De., Vizovitis, K., Botondi, R., Bellincontro, A. and Mencarelli, F. (2006). 1MCP controls ripening induced by impact injury on apricots by affecting SOD and POX activities. Postharvest Biol. Technol. 39: 3847. Menniti, A.M., Donati, I. and Gregori, R. (2006). Responses of 1-MCP application in plums stored under air and controlled atmospheres. Postharvest Biol. Technol. 39: 243-246. Menniti, A.M., Gregori, R. and Donati, I. (2004). 1Methylcyclopropene retards postharvest softening of plums. Postharvest Biol. Technol. 31: 269-275. Michaeli, R., Philosoph-Hadas, S., Riov, J., Shahak, Y., Ratner, K. and Meir, S. (2001). Chilling induced leaf abscission of Ixora coccinea plants. III. Enhancement by high light via increased oxidative processes. Physiol. Plant. 113: 338-345. Mir, N.A., Curell, E., Khan, N., Whitaker, M. and Beaudry, R.M. 2001. Harvest maturity, storage temperature, and 1-MCP application frequency alter firmness retention and chlorophyll fluorescence of ‘Redchief Delicious’ apples. Am. Soc. Hort. Sci.126: 618-624. Muller, R., Serek, M., Sisler, E.C. and Andersen, A.S. (1997). Poststorage quality and rooting ability of Epipremnum pinnatum cuttings after treatment with ethylene action inhibitors. J. Hort. Sci. 72: 445-452. Muller, R., Sisler, E.C. and Serek, M. (2000). Stress induced ethylene production, ethylene binding, and the response to the ethylene action inhibitor 1-MCP in miniature roses. Sci. Horti. 83:5159. Muller, R., Sisler, E.C.and Serek, M. (2000). Stress induced ethylene production, ethylene binding, and the response to the ethylene action inhibitor 1-MCP in miniature roses. Sci. Hort. 83: 5159. Mullins, E.D., McCollum, T.G. and McDonald, R.E. 2000. Consequences on ethylene metabolism of inactivating the ethylene receptor sites in diseased non-climacteric fruit. Postharvest Biol. Technol. 19: 155-164. Mwaniki, M. W., F. M. Mathooko, M. Matsuzaki, K. Hiwasa, A. Tateishi, K. Ushijima, A. Inaba and Y. Kubo. 2005. Expression characteristics of seven members of the β-galactosidase gene family in ‘La France’ pear (Pyrus communis L.) fruit during growth and their regulation by 1methylcyclopropene during postharvest ripening. Postharvest Biol. Technol. 36: 253–263. Neves, L.C., Corrent, A., Marini, L., Lucchetta, L., Zanuzzo, M.R., Goncalves, E.D., Zanatta, J., Cantillano, F.R. and Rombaldi, C.V. (2003). Atmosfera modificada e 1-metilciclopropeno na conservacao pos-colheita de kiwis cv. Bruno. 42 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India (Modified atmosphere and 1methylcyclopropene on postharvest storage of kiwis cv. Bruno.). Revista Brasileira de Fruticultura 25:390-393. Opiyo, A.M. and Ying, T.J. (2005). The effect of 1MCP treatment on the shelf life and quality of cherry tomato (Lycopersicon esculentum var. cerasiforme) fruit. Int. J. Food Sci. Technol. 40: 665-673. Pawlowski, N.E., Hendricks, J.D., Bailey, M.L., Nixon, J.E., and Bailey, G.S. (1985). Structural bioactivity relationship for tumour promotion by cyclopropenes. J. Agri. Food Chem. 33: 767770. Pechous, S.W., Watkins, C.B. and Whitaker, B.D. (2005). Expression of a-farnesene synthase gene AFS1 in relation to levels of a-farnesene and conjugated trienols in peel tissue of scaldsusceptible ‘Law Rome’ and scald-resistant ‘Idared’ apple fruit. Postharvest Biol. Technol. 35: 125-132. Pesis, E., Ackerman, M., Ben-Arie, R., Feygenberg, O., Feng, X., Apelbaum, A., Goren, R. and Prusky, D. (2002). Ethylene involvement in chilling injury symptoms of avocado during cold storage. Postharvest Biol. Technol. 24:171181. Porat, R., Weiss, B., Cohen, L., Daus, A., Goren, R. and Droby, S. (1999). Effects of ethylene and 1-methylcyclopropene on the postharvest qualities of ‘Shamouti’ oranges. Postharvest Biol. Technol. 15:155-163. Reid, M.S., Wollenweber, B., Serek, M. (2002). Carbon balance and ethylene in the postharvest life of flowering Hibiscus. Postharvest Biol. Technol., 25: 227-233. Rupasinghe, H.P.V., Murr, D.P., Paliyath, G. and Skog, L. (2000). Inhibitory effect of 1-MCP on ripening and superficial scald development in ‘McIntosh’ and ‘Delicious’ apples. J. Hort. Sci. Biotechnol. 75:271-276. Salaun, J. and Baird, M.S. (1995). Biologically active cyclopropanes and cyclopropenes. Curr. Medical Chem. 2: 511-542. Saltveit, M.E. (1999). Effect of ethylene on quality of fresh fruits and vegetables. Postharvest Biol. Technol. 15: 279-292. Saltveit, M.E. (2005). Aminoethoxyvinylglycine (AVG) reduces ethylene and protein biosynthesis in excised discs of mature-green tomato pericarp tissue. Postharvest Biol. Technol. 35: 183190. Selvarajah, S., Bauchot, A.D. and John, P.J. (2001). Internal browning in cold-stored pineapples is suppressed by a postharvest application of 1methylcyclopropene. Postharvest Biol. Technol. 23:167-170. Selvarajah, S., Bauchot, A.D. and John, P.J. (2001). Internal browning in cold-stored pineapples is suppressed by a postharvest application of 1methylcyclopropene. Postharvest Biol. Technol. 23: 167-170. Serek, M. and Reid, M.S. (2000). Ethylene and post harvest performance of potted kalanchoe. Postharvest Biol. Technol. 18: 43-48. Serek, M. and Sisler, E.C. (2001). Efficacy of inhibitors of ethylene binding in improvement of the postharvest characteristics of potted flowering plants. Postharvest Biol. Technol., 23: 161166. Serek, M., Prabucki, A., Sisler, E.C. and Andersen, A.S. (1998). Inhibitors of ethylene action affect final quality and rooting of cuttings before and after storage. HortSci. 33: 153-155. Serek, M., Woltering, E.J., Sisler, E.C., Frello, S. and Sriskandarajah, S. (2006). Controlling ethylene responses in flowers at the receptor level. Biotechnol. Advances 24: 368-381. Singh, R. and Dwivedi, U.N. (2008). Effect of Ethrel and 1-methylcyclopropene (1-MCP) on antioxidants in mango (Mangifera indica var. Dashehari) during fruit ripening. Food Chem. 111: 951-956. Singh, S.P. and Pal R.K. (2008). Response of climacteric-type guava (Psidium guajava L.) to postharvest treatment with 1-MCP. Postharvest Biol. Technol. 47: 307-314. 43 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Sisler, E.C. and Serek, M. (1997). Inhibitors of ethylene responses in plants at the receptor level: recent developments. Physiol. Plant. 100: 577582. Sisler, E.C. and Serek, M. 1997. Inhibitors of ethylene responses in plants at the receptor level: Recent developments. Physiol. Plant. 100:577-582. [4] Sisler, E.C., Grichko, V.P., and Serek, M. (2006). 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Influence of time from harvest to 1-MCP treatment on apple fruit quality and expression of genes for ethylene biosynthesis enzymes and ethylene receptors. Postharvest Biol. Technol. 43: 28-35. Trivedi, P.K. and P. Nath. (2004). MaExp1, an ethylene-induced expansin from ripening banana fruit. Plant Sci. 167: 1351-1358. Valero, D., Martinez-Romero, D., Valverde, J.M., Guillen, F. and Serrano, M. (2003). Quality improvement and extension of shelf life by 1methylcyclopropene in plum as affected by ripening stage at harvest. Innovative Food Sci. Emerging Technol. 4:339-348. Watkins, C.B. (2002). Ethylene synthesis, mode of action, consequences and control. In: Fruit Quality and its Biological Basis, Knee, M (ed.), CRC Press, Sheffield, Boca Raton, Florida. pp 180-224. Watkins, C.B. and Miller, W. B. (2003). Implications of 1-methylcyclopropene registration for use on horticultural products. In: Biology and Technology of the Plant Hormone Ethylene III, p385-390. Vendrell, M., Klee, H., Pech, J.C., Romojaro, F. (eds.) IOS Press, Amsterdam, Netherlands. Watkins, C.B., Nock, J.F. and Whitaker, B.D. (2000). Responses of early, mid and late season apple cultivars to postharvest application of 1methylcyclopropene (1-MCP) under air and controlled atmosphere storage conditions. Postharvest Biol. Technol. 19: 17-32. Woltering, E.J. and van Doorn, W.G. (1988). Role of ethylene in senescence of petals: morphological and taxonomical relationships. J. Exp. Bot. 39: 1605-1616. Woolf, A.B., C. Requejo-Tapia, K.A. Cox, R.C. Jackman, A. Gunson, M.L. Arpaia, and White. A. (2005). 1-MCP reduces physiological storage disorders of ‘Hass’ avocados. Postharvest Biol. Technol. 35: 43-60. Yamane, M., Abe, D., Yasui, S., Yokotani, N., Kimata, W., Ushijima, K., Nakano, R., Kubo, Y. and Inaba A. (2007). Differential expression of ethylene biosynthetic genes in climacteric and non-climacteric Chinese pear fruit. Postharvest Biol. Technol. 44: 220-227. Zhong, G.Y., Goren, R., Riov, J., Sisler, E.C. and Holland, D. (2001). Characterization of an ethylene-induced esterase gene isolated from Citrus sinensis by competitive hybridization. Physiol. Plant. 113: 267-274. 44 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India PHYSIOLOGICAL AND BIOCHEMICAL CHANGES IN CUT FLOWERS V. P. Singh, Gaurav Agarwal and Divya Choudhary Division of Plant Physiology, Indian Agricultural research Institute, New Delhi-110012 Email: drsinghvp@yahoo.com Flower is one of the most aesthetic creations of life. Higher plants have evolved to produce beautiful flowers for the propagation of species through sexual reproduction. In being so flowers carry huge commercial value as they are coveted by people from all walks of life to express their feelings. Throughout history, man has sought to produce plants for the beauty of flowers. Today, commercial floriculture is one of the most profitable agro-industries in the world and consumption of floricultural products is linked with gross domestic product (GDP). Global trade of flowers world wide is about worth $40 billion out of which share of cut flowers is 60%. Foreign exchange earning is more than Rs.330 Crores in 2008 (0.19 % of world trade, and expected to grow to 1000 Crores by 2010. Floriculture industry is growing @ 10-15% per annum but India has a growth potential of 25-30% per annum because of its diverse climate and availability of cheap labour. Not surprisingly, the longevity of many flowers is quite short, as their biological function of reproduction is transient in nature. Flowers are highly perishable and more than 30% post harvest quality loss has been reported. The post production quality of many ornamentals is influenced by both flowers and leaf senescence. Microbes and insects contribute to post harvest losses of quality in vegetables, fruits and flowers, but senescence exerts a very high toll and the keeping quality of flowers, vegetables and fruits is a function of the progress of senescence (Burton, 1982). High rate of metabolism in harvested flowers leads to rapid deterioration and loss of vase life and quality. Loss is very high in tropical regions with scarce facilities for refrigeration. Hence, the study of senescence is the underlying theme of most post harvest physiology research. Clearly an understanding of the physiology, biochemistry and molecular biology of senescence would have implications in the control of flower longevity. Senescence – is the ordered disassembly of cellular components in the senescing tissues and allows for the maximum recovery of nutrients from the senescing tissues for recycling to the parts of the plants that survive (Buchanan et al., 2003). Senescence, though a terminal developmental stage, can also be accelerated by an array of both abiotic and biotic factors, such as light, temperature nutrients and pathogens (Smart, 1991). It is a dynamic and closely regulated developmental process which involves highly coordinated changes in gene expression (Hensel et al., 1993). Patterns of floral senescence: Senescence is often dramatic as in monocarpic plants, where the completion of reproductive development culminates in to death of entire plant. In contrast, senescence in polycarpic plants is restricted to parts of the flower and fruits, while the plants continue to develop. Three general patterns of floral senescence based on differences in how flowers respond to ethylene are recognized. In ethylenedependent flowers, such as carnation, a rise in endogenous ethylene production triggers senescence (Woltering and van Doorn, 1988; van Doorn, 2001). If such flowers are treated with ethylene biosynthesis or action inhibitors, their life is extended substantially (Veen and van de Geijn, 1978; Fujino et al., 1980; Serek et al., 1994). Since ethylene is the endogenous senescence trigger, exogenous ethylene accelerates senescence (Woltering and van Doorn, 1988). In 45 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India to developing technical strategies for avoiding quality losses. External quality (appearance) is therefore extremely important to the consumer. Although consumer choice is driven by external quality, the postharvest life, or vase life, is key to convincing the consumer to re-purchase cut flowers. Postharvest physiology therefore has a double goal: to preserve external quality during the distribution chain and to extend the vase life of the flowers. ethylene independent flowers such as iris, there is little ethylene produced before or during senescence. Exogenous ethylene does not accelerate senescence and ethylene inhibitors do not elongate floral longevity (Woltering and van Doorn, 1988). Some flowers such as daffodil show an intermediate pattern of senescence. Without pollination, their senescence resembles that of the ethylene-independent flowers, in that there is little ethylene production and only a limited response to inhibition of ethylene biosynthesis and action. However, pollination results in an ethylene-dependent type of senescence, with an associated rise in endogenous ethylene production. Application of exogenous ethylene accelerates their senescence (Hunter et al., 2002). Factors affecting senescence of cut flowers: Senescence is influenced by pre-harvest, harvest and post-harvest factors. These factors could be summarized as follows: (a) Among the pre-harvest factors, it is the stage, development and nutritional status of the plant, which plays a primary role in regulating senescence. (b) Harvest factors include, time during the day of harvest, at which the flowers are harvested and disease are of the prominent factors. (c) Post-harvest factors include temperature at which the flowers are stored and gaseous atmosphere. Determinants of flower senescence: Senescence of flower petals is a complex process involving an increase of cell membrane permeability that results in wilting, pigment degradation, and ultimately, petal collapse (Jones and McConchie, 1995). Post harvest performance of cut flowers is affected by the developmental stage of a flower at harvest, pro-senescence signals that originate from specific tissues within the flower (e.g., pollinationinduced petal senescence), and stress-related metabolism (in response to temperature, wounding and nutrient starvation). Cut flower stems are removed from a source of nutrients, undergo water restrictions and may be held at undesirable temperatures in the dark for days prior to sale. Plant hormones, membrane stability, water availability, cellular proteolysis and carbohydrate metabolism act in concert to determine the differential rate of senescence for each floral organ. Temperature: Respiration is the fundamental metabolic process responsible for providing energy in all living cells. During postharvest life, current photo assimilates are not available reserved carbohydrates are used in respiration by cut flowers. Since, flowers are poikilotherms, the rate of respiration is dependent on temperature, increasing in an exponential fashion with increasing temperature. For cut flowers, the Q10 (the Physiology and biochemistry of cut flowers ratio of respiration at temperature T to that at The study of the postharvest physiology and temperature T-10) is usually at least 3, and may be as biochemistry of cut flowers has the aim of high as 9. This means that respiration (and deterioration) understanding the behaviour and disorders of harvested at 20 C is at least 9 times more rapid than at 0 C; flowers. The dramatic differences between the rapid cooling and maintenance of temperature is conditions in the growing environment and in the therefore key to maintaining the vase life of harvested storage facility dramatically affect the physiology of flowers. cut flowers. Understanding the biological mechanisms Temperature also promotes the senescence that are activated or inhibited during storage is essential 46 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India rate by increased ethylene production. Ethylene or free radicals generation in single flower was often found to be more in comparison to inflorescence. On the other hand lower temperature causes chilling injury and within a species, the sensitivity varied. The sensitiveness of single flower to high temperature or ethylene might be because once the flower open the senescence signal needs to be transported to the floret present on the apical side. In the early years of experimentation, it was shown that the fresh weight of the flowers decreased with time, due to which withering took place. Therefore, placing the cut ends in water resulted in overcoming the problem, in those, species where air blockage occurred. Cutting the stem about 2-3 cm resulted in restoration of continuous water supply, when supplied and unfurling of petals in rose took place. Thus the physical driving force, based on accumulation of osmotically active substances could be re-established Water relations at the initial stages but not once wilting symptoms When harvested spikes of gladiolus are kept begun. Interestingly, flowers often exhibit both opening in vase solution initially there is an increase in the fresh and closing of petals due to turgor changes, which often weight of spikes due to increased solution uptake as it was affected by water deficit. is required by spikes for opening of flowers. Then Prior to abscission, loss in solute and cell decline is attributed to high rate of respiration and also membrane leakage (Ezhilmathi et al., 2007; Singh, leakiness (Stead and Moore, 1983), inorganic ions, 2005, Singh et al., 2005).It has been shown that organic acids reduced anthocyanin content were negative water relation would lead to the wilting of reported. During the process of senescence, the rate flowers. By application of 13.3 Kpa pressure in of water flux through vessels, trachieds and fibers get Helianthus sps. wilting symptoms were often reduced and tylose formation was often shown to result manipulated by prolonging senescence. During the in reduced water and oxygen availability for the period of harvesting, without keeping in water, the respiration causing an imbalance in water relation of flowers often exhibit wilting symptoms, due to blocking flowers. This was further aggravated by increased loss of air or due to exuding the latex or mucilaginous in transpiration rate. material from cut end. Further, this often leads to the In cut flowers, Sonia or Frisco roses, when disease infestation causing senescence phenomenon held dry for 3 h and placed in water the stomata were to be set in at much earlier stage. ABA accumulation is found reopened and when held for 24 h, remarkable induced in leaves and petals by water stress, and may closure due to accumulation of ABA and its derivatives result from carotenoid degradation in leaves. Increased occurred (Aspinall, 1980). Increased day length to cut ABA concentrations may also trigger flower roses in vases, has a direct demonstration that, higher senescence in some species. water loss and loss in turgidity resulted due to Often, the flower buds were shown not fully open or sometimes total inhibition of flower opening occurred in roses, Gypsophila and Acacia. Whereas in tulips, Freesia and Iris flower bud opening due to exposure to air-interface was found to be not a problem. Opening of flower bud is related to the increased cell size, (which is due to expansion) although; to some extent cell division also takes place. Both theses processes are dependent upon cell water content, whereas cell differentiation generally does not take place in cut flowers and therefore differentiation is not directly dependent upon cell water content. disturbance in water relations followed by early senescence. Generally, the rate of transpiration has been shown to be dependent upon the water potential and dissolving glucose, fructose at 10 – 20 g/litre resulted in lowering the water potential, which decreased transpiration rate. In rose, an addition of sucrose was shown to decrease the rate of transpiration, which is largely attributed to the stomatal closure (Venkatarayappa et al., 1981). However, sucrose or addition of sugars often result in increased bacterial 47 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India properties. There is deterioration in membrane stability during flower senescence Singh and Jagadheesan, 2003; Singh et al., 2005; Singh, 2005; Singh et al., 2007). The first structural change often observed was invagination of tonoplast, resulting in release of various hydrolytic enzymes, followed by autolysis of cell. Often, acid hydrolases such as acid phosphatase, RNase and DNase were released. In those petals, which contain plastids, such as cucumber flowers, gradual disappearance of thylakoids followed by tubule presence with invaginations has been observed. Often, the single ribosome and vesiculation of endoplasmic reticulum were the changes at cell organelles level noticed during the early or middle part of senescence. During the course of senescence in flowers like roses there is an increase in pH. On the contrary, lowering pH induced senescence in Tradescantia, Ipomoea, and Fuchsia probably because of tonoplast breakdown, which was often associated with increased levels of organic acids. Several of the organic acids and enzymes which are pH dependent were found to increase in senescing tissues indicating that the membrane integrity has often been lost, which is rather a consequence than cause. The loss in membrane integrity was reported to be due to higher free radical production and consequently, there was a concomitant increase in the viscosity of the cell sap. Often the lost permeability was accompanied by a sharp increase in volume of the space of the tissue and this facilitated movement of water into the air. During the early period of loss in membrane integrity a dramatic decrease in petals pigments, amino acids, sugars, K and electrolyte occurred (Paulin 1986; Celikel and Van Doorn, 1995). At the time of wilting the leakage rates when measured were as high as 20 times is often regarded as death. growth which might promote the senescence in at early stages, which has often been countered by the addition of antimicrobial agents especially hydroquinoline citrate or sulphate in Chrysanthemum. The water relation of the tissues were also influenced by addition of inorganic ions such as Aluminium (Schnabl and Ziegler, 1974), through reduction in transpiration as a result of decreasing stomatal conductance in cut rose flowers. On contrary, wherein the larger surface area is present in inflorescences, the water losses when stored in dry condition were dramatic. With numerous small flowers in Astible, about 60% of the water is lost through the surface and, in Gladiolus, even flower opening is inhibited. Often during vase life, the rate of transpiration decreases, which is higher than the rate of water uptake, resulting in an imbalance with a decrease in water potential causing a turgor loss, increased plasticity and thus, the process of senescence starts. Proline, another important metabolite during stress is also found to increase in cut roses and addition of proline in aqueous solution was shown to be ineffective in delaying wilting and senescence (Tonecki et al., 1989). Thus many cut flowers when placed in water loose the water balance due to air blockage and or because of bacterial growth, without recycling the transpirational loss causing senescence. Due to cutting among the several effects, vacuolar occlusion in Zantedeschia aethiopia (Tjia and Funnell, 1986) takes place due to deposition of lignin, subarin and tannins (Van Doorn et al., 1989), deposition of gums in xylem vessels in roses (Lineberger and Steponkus, 1976), exudation of latex or other substances in Heliconia (Criley and Broschat, 1992) tyloses occurred in Syringa (Sytsema-Kalkman, 1991). Microbial growth causing vascular occlusions However, before the membrane integrity loss (Van Doorn et al., 1989) have been shown to reduce polar lipids and fatty acids decline in quantity in the water uptake, thereby resulting in turgor loss and ephemeral flowers (Suttle and Kende, 1980), whereas increased senescence rate. it started only after opening in longer lived carnations Changes in membrane properties (Mayak et al., 1985). This is mostly because of the Since the cell is enclosed within a membrane reduction in biosynthesis and increase in degradation it is logical to assume that regulation of the cell process. During the process of degradation, acyl metabolism is to a large extent by changes in membrane hydrolases, phospholipase A and D (Suttle and Kende, 48 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Involvement of free radicals in senescence had 1980; Borochov et al., 1982), non-specific lipase in carnation (Burgek et al., 1986), were higher resulting attracted considerable attention (Dhindsa et al., 1981; in the cleavage of fatty acids and production of lyso Lasham et al., 1986; Arora et al., 2002). In plant cell chloroplast, mitochondria and peroxisomes are compounds. oxygen species Since the phospholipid content of the petals intracellular generators of activated .such as H O , superoxide (O ) radicals, hydroxyl 2 2 2 membrane decline during senescence, its amount vis. . à-vis other membrane components also vary. Such radicals (OH ) and singlet oxygen ( O2) (del Rio and variations in ratios affect properties of membranes, such Donaldson, 1995; Lopez- Huertas et al., 1997). Free as fluidity and the temperature of phase transition of radicals are also produced because of air pollution, membrane’s lipids (Shinitzky, 1984). The molar ratio UV-irradiation and certain herbicides (Thompson et of sterol to phospholipids consequently increased with al., 1987) or xanthine/xanthidase oxidase reaction senescence, which was 2-6 times different in (Mayak et al., 1983). Some of these activated oxygen membranes from that of younger petals tissues, has species are highly reactive and in the absence of protective mechanisms, can cause damage to cell been supported by the findings of increased fluidity. structure and function (Halliwell and Gutterridge, 1989; Similar to membrane lipids, the proteins too Elstner, 1991). have been shown to be influenced causing fluidity during Activated oxygen species, such as O2. - or petal senescence. Among the different kinds of proteins, the specific content of thio groups, as well as H2O2 and their interaction products react with their total protein content were shown to decrease proteins, lipids and nucleic acids and disintegrate them significantly during carnation petal senescence. These (Elstner, 1982). Thus accumulation of these free changes were often associated with accessibility of radicals may initiate senescence. These activated. inactivate enzymes and in case of (OH ) hormones to their receptors (Shinitzky, 1984) and oxygen species ATPase activity, which were restored by 70% in and HO2 initiate lipid peroxidation (Asada and carnation petals. Amongst the hormones, the Takahashi, 1987). senescence promoting hormones, ethylene binding sites The change in the physical properties of were decreased more than 90% by age during the membrane is attributed to increased oxidation process, process of senescence is an indication that protein particularly double bond index reduction of fatty acids receptors have been lost in their functional integration. resulting a loss of the linolenic acid (Shinitzky, 1984), Whether, this was due to reduced affinity of the existing which perhaps is non-enzymatic since antioxidants receptors or due to decreased repair of the receptors increase the flower longevity (Paulin, 1986) and the or because of decreased de novo synthesis is not yet possible source of oxidants therefore could be free studied. radical ions. Not always the ethylene is the coordinating agent of senescence. Flower senescence was shown to proceed in certain ethylene-insensitive families without any ethylene induction like Liliaceae, Asteraceae and Umbelliferae. Reid and Wu (1992) reported that in tulip and Iris, petal senescence was initiated and progressed without the influence of ethylene. Lukazewski and Reid (1989) and Bieleski and Reid (1992) concluded that ethylene is not involved in the senescence of daylilies as application of STS has not delayed senescence. Thus, it may be concluded that a change in net decrease of phospholipid content, increased sterol to phospholipids molar ratio, decreased fluidity and membrane bound enzyme activity, probably because of free-radical production might lead to increased permeability facilitating smaller molecules to move out of the cell, finally leading to wilting and death. As a consequence of change in membrane physical properties, use of antioxidants, herbicides, sugars have been supplemented to enhance the vase life of flowers. 49 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India of protein (Woodson and Handa, 1987; Gao and Wu, 1990; Lay-Yee et al., 1992; Eason and Webster, 1995). The protein content is reduced due to little de novo synthesis and considerable protein degradation It is understood that free radicals attack amino acid residues of proteins causing conformational changes in proteins causing them to be recognized by specific proteases for degradation. Also a change in membrane lipid composition induces conformational changes in proteins, by providing an unfavorable environment deleteriously affecting the activity of key enzymes. (The proteins are excluded into liquid crystalline domain from gel phase liquid domain which progressively increases during senescence. Proteins trapped in the gel phase are degraded by proteases. During the process, several fold increase in ammonia has been observed in rose (Paulin, 1977), along with aspargine, glutamine and several other amino acids. This increase in ammonia was coincided with respiratory activity, similar to ethylene production. Formation of amides has often been considered as a mechanism of detoxification of excess ammonia. Carbohydrate metabolism Petal senescence is generally accompanied by a loss of dry matter due to hydrolysis of macromolecules such as starch, protein and nucleic acids and the redistribution of carbon and nitrogen compounds to other parts of the flower. Carbohydrate status of the flower petals is one of the factors, which ultimately determines their longevity. There is a sharp decline in the carbohydrate content during the final stage of flower development. This drop in level of macromolecules (starch, and cell polysaccharides) occurred with the onset of senescence. Since current photoassimilates are not available reserved carbohydrates are used in respiration leading to petal senescence. Supplying cut flowers with exogenous sugar maintains the pool of dry matter, respirable substrates in petals and extends longevity (Nichols, 1973; Mayak and Dilley, 1976). Decrease in reducing sugars with senescence was reported in daylily (Bieleski and Reid, 1992), rose (Sharma, 1981) and carnation (Halvey and Mayak, 1979). Often, invertase was shown to decrease (Woodson and Wang, 1987), with increasing age, which has been linked with de novo synthesis of invertase inhibitor (Halaba and Rudnicki, 1986), which makes the oxidation products of sucrose available for transport in carnation and Ipomea during wilting. Senescence is a degradative process, hence new proteins are synthesized (Wulster et al., 1982a, b), which was evidenced by the incorporation of radioactive amino acids into protein (Woodson and Handa, 1987). Simultaneously, decrease in the proteins which are involved in the synthesis process has been reduced (Woodson, 1987), clearly indicates that mRNA levels increased during the process. Polyadenylation of mRNA, however, has been found to be low in abundance because of change in geneexpression. Eason and De Vr’e (1995) also observed in Sandersonia that flower senescence required specific protein. Moreover, they found that the quantities of specific proteins present during flower development and senescence are regulated by sugars. Some of the important key enzymes, such as hydrolases, more specifically acid phosphatases, catalase and nucleasesRNase, DNase, esterase, were found to increase, which are active in acidic pH range, due to loss in membrane integration. However, whether these are synthesized de novo or increased affinity to substrate Sugars or carbohydrates increased the vase life of cut flowers by reducing the sensitivity to ethylene (Mayak and Dilley, 1976; Paulin, 1986). It has also been suggested that maintenance of osmotic pressure might be the reason for the delay in senescence. Paulin (1986) reported that exogenous application of sugars increases vase life by delaying proteolysis, promoting protein and amide synthesis, maintaining osmotic potential, delaying loss of membrane integrity and maintaining mitochondrial structure and function (Halvey and Mayak, 1979). Protein metabolism There is a decline in protein content during senescence. The senescence of both climacteric and nonclimacteric flowers has been associated with a loss 50 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Mayak, 1981). Along with it, there was a decrease in total protein content and increased RNase activity. However, in an attached system, the effects were reversible, probably because of closure of stomatal aperture (Halevy et al., 1974). Increased levels of internal ABA concentration in rose (Borochov et al., 1976) and carnation petals (Eze et al., 1986) often associated with petal senescence is mediated through ethylene action (Mayak and Dilley, 1976), since it could be delayed by CO2 treatment. Also in carnation increase ABA content could be prevented by pretreatment with silver thiosulphate (Nowak and Veen, 1982). By using external application and Hormones inducing internal level of ABA, it was found to be a While ethylene plays a pivotal role in flower possible candidate for hormonal trigger of senescence senescence and has been investigated in details, other of gladiolus flowers, as they did not respond to ethylene natural hormones have also been implicated in flower (Singh, 2007). senescence but their role in the process has not been conclusive. Role of IAA and GA in regulation of flower Ethylene senescence has not been clearly understood till date. Flower senescence is often associated with In carnation IAA was found to stimulate ethylene increased ethylene production. Halevy (1986) has production and consequently senescence (Wulster et suggested that flowers can be classified as climacteric al., 1982b) and ethylene production was dependent or non-climacteric based on the presence or absence on IAA concentration. Low level of 2,4-D to carnation of an increased rate of ethylene production. In promoted ethylene production and accelerated climacteric flowers, ethylene is regarded as a signal, senescence while higher level delayed the senescence mediating a sequence of events that eventually leads (Sacalis and Nichols, 1980). The senescence of to the organ death (Reid and Wu, 1992). Biosynthesis poinsettia flower was delayed by auxin due to of ethylene by petal tissues is under metabolic control. production of peroxidase and inhibition of IAA-oxidase The pathway for ethylene biosynthesis was elucidated and hydrogen peroxide during ageing (Gilbart and Sink, by Adams and Yang (1979) as methionine-S-adenosyl 1971). methionine (SAM)-1-amino-cyclopropane-1Cytokinins on the other hand when applied carboxilic acid (ACC) ethylene. In carnation flower exogenously delayed senescence very effectively petals, ACC was found to increase concomitant with (Kelly et al., 1985; Cook et al., 1985), which has the increase in ethylene biosynthesis (Bufler et al., been attributed to the limitation (Van-Staden et al., 1980). This indicated a possible regulatory role for 1987) or inhibition of ethylene climacteric (Mor et al., ACC synthase, the enzyme responsible for the 1983) or ACC (Mor et al., 1983) or inhibited conversion of SAM to ACC. Peiser (1986) reported biosynthesis of ethylene (Cook et al., 1985) or ACC synthase activity increases in climacteric carnation petals. sensitivity of petals to C H (Eisinger, 1977). due to change in pH had not been studied in details. Petal opening and senescence of cut gladiolus, Iris and Narcissus flowers were significantly inhibited by treatment with cycloheximide (CHI) and the vase life of gladiolus florets was doubled. These results indicate that de novo protein synthesis is required for bulb flower development, opening, petal wilting and senescence (Jones et al., 1994). Currently, molecular cloning techniques are being in use to isolate and characterize the gene specific to senescence phenomenon (Lawton et al., 1989; Woodson et al., 1989). 2 4 Abscisic acid (ABA) while applied exogenously accelerates petal senescence in cut carnation and flower petals treated with ABA were more sensitive to exogenous ethylene (Ronen and During senescence of carnation flowers there is a rise in ethylene production coinciding with the loss of petal turgor which is the first visible symptom of wilting (Trippi and Paulin, 1984; Whitehead, 1994). 51 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Ethylene production by these senescing climacteric flower petals is autocatalytic i.e. exposure to ethylene stimulates ethylene biosynthesis. In carnation, ethylene treatment results in a 90 to 100-fold increase in petal ACC, due to increased activity of ACC synthase (Mor et al., 1985). Since ethylene exposure stimulates its production in pre-climacteric carnation petals, it likely stimulates ACC oxidase activity in addition to ACC synthase as in the case of pre-climacteric fruits (Liu et al., 1985). The similar phenomenon occurs in flowers of Ipomea tricolor (Kende and Baumgartner, 1974) and Tradescantia (Suttle and Kende, 1978). senescence (Lovell et al., 1987). It was observed in carnation that petal senescence occur between 2-3 days after pollination, whereas in unpollinated ones in 6-7 days. The other parts of the flowers such as style, ovaries, receptacles etc have been shown to induce the synthesis of ethylene (Pech et al., 1987; Porat, 1994), however, not necessarily due to fertilization since, flowers, which have not pollinated with pollen tube still growing with in the style, have shown increased ethylene concentration suggesting that a transmissible factor is involved in senescence. Veen and Kwahhenbos (1982) reported that carnation flowers when treated with ethylene action inhibitor like silver thiosulphate (STS), did not exhibit the climacteric rise in ethylene production nor did they accumulate ACC. STS has spectacular effects in overcoming deleterious effects of ethylene in potted flowering plants (Cameron and Reid, 1981; Chang and Chen, 2001). As silver in STS is an environmental pollutant, its use for potted flowering plants has been restricted (Serek and Reid, 1993). A new gaseous binding competitor 1-methyl cycloporpene (1-MCP) was found to be effective usually at dose to the concentration at which STS caused phytotoxocity (Cameron and Reid, 1981). No phytotoxic symptoms of 1-MCP were observed over at 20 nl/l and at this minute concentration also, it provides as much protection of STS preventing ethylene induced bud and flower abscission, leaf abscission and flower senescence (Serek et al., 1994). The transmissible factor might be ACC (Whitehead et al., 1983), since pollens of many flowers enriched with ACC has been reported (Hoekstra and Wages, 1986), since application of AVG to stigmas inhibited senescence in petunia, but the action inhibitor nonbornadiene did not prevent ethylene biosynthesis or senescence. This proves that pollination results in the production and transmission of a senescence factor which does not initially require ethylene for its production but results in ethylene synthesis and senescence. Interestingly, wounds of stigma too had been shown to cause senescence (Lovell et al., 1987), which led to ethylene synthesis in the style (Hoekstra and Wages, 1986). Since petal senescence in carnation is accompanied by growth of the ovary, Nichols (1971) suggested former as a result of carbohydrate redistribution from petals, which was later, confirmed by 14C-sucrose study (Nichols and Ho, 1975a, b). Halevy et al., (1984) also observed simultaneous ovary growth and petal senescence in Cyclamen sp. as a result of carbohydrate redistribution. However, studies of Mor et al., (1980) indicated no appreciable increase in ovary dry weight after removal of petals. Rather removal of gynacium hastened the petal senescence (Sacalis and Lee, 1987), when flowers were left on plants or held in sucrose solution but interestingly not in water. ACC content and or ethylene production by gynacium have been implicated in initiation of petal senescence (Bufler et al., 1980). Serek et al., (1995) showed that symptoms such as electrolyte leakage and lipid fluidity were also reduced in petunia by 1-MCP. Sisler and Serek (1997) observed that a concentration as low as 0.5 nl/l of 1MCP was sufficient to protect carnation flowers for several days against ethylene. 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D. bacterial suspensions on vascular blockage in Stead (Eds), Cambridge University Press, ppstems of cut rose flowers. J. Appl. Bacteriol., 269-284. 71: 119-123. Plant., 60: 221-226. Van Doorn, W. G., Harkema, H. and Song, J. S. 1995. Whitehead, C. S., Fujino, D. W. and Reid, M. S. 1983. Identification of the ethylene precursor, 1-amino Water relations and senescence of cut Iris cyclopropane-1-carboxilic acid (ACC) in flowers. Post Harvest Biol. Tech., 5: 345-351. pollen. Scientia Hort., 21: 291-297. Van Doorn, W. G., Schurer, K. and De Witte, Y. 1989. Role of endogenous bacteria in vascular Woltering EJ, van Doorn WG. 1988. Role of ethylene and senescence of petals: morphological and blockage of cut flowers. J. Plant Physiol., 134: taxonomical relationships. Journal of 375-381. Expermental Botany. 39, 1605–1616. Van-Staden, J., Feathonby-Smith, B. C., Mayak, S., Spiegelstein, H. and Halevy, A. H. 1987. Woodson, W. R. 1987. Changes in protein and mRNA population during senescence of carnation petals. Cytokinin in cut carnation flowers. II Relationship Physiol. Plant., 71: 495-502. between endogenous ethylene and cytokinin levels in the petals. Plant Growth Regul., 5: Woodson, W. R. and Handa, A. K. 1987. Changes in 75-86. protein patterns and in vivo protein synthesis during pre senescence and senescence of Veen H, van de Geijn SC. 1978. Mobility and ionic Hibiscus petals. J. Plant Physiol., 128: 67form of silver as related to longevity of cut 75. carnations. Planta. 140: 93–96. Veen, H. and Kwahhenbos, A. A. M. 1982. The effects Woodson, W. R. and Wang, H. 1987. Invertase of carnation petals and changes in activity during of silver thiosulphate pretreatment on 1-amino petal growth. Physiol. Plant., 71: 224-228. cyclopropane-1-carboxylic acid content and action in cut carnation. Scientia Hort., 18: 277- Woodson, W. R., Lawton, K. A. and Goldsbrough, 286. P. B. 1989. Ethylene regulated gene expression during carnation petal senescence. Acta Hort. Venkatarayappa, T., Murr, D. P. and Tsujita, M. J. Sci., 261: 137-144. 1981. Effect of CO 2 and sucrose on the physiology of cut Samantha rose. J. Hort. Sci., 56: 21-25. 57 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India MODERN METHODS OF POTATO STORAGE Devendra Kumar Central Potato Research Institute Campus Modipuram, Meerut-250110 Potatoes contain 80% water and therefore, are semi-perishable in nature. The high water content makes handling and storage difficult. It has been estimated that under tropical and sub-tropical conditions, losses due to poor handling and storage can amount to 40-50%. Therefore, it is of utmost importance to minimize storage losses. It makes sense to minimize storage losses to increase the availability of potatoes because it costs less to store than to produce a given quantity of potatoes. while harvesting and therefore, curing is essential to heal the wounds. Suberization is the process by which wounds are healed in potatoes and the optimum conditions for suberization are a temperature of around 250C and relative humidity of 95%. Post harvest losses Post-harvest losses lead to reduction in the quantity as well as quality of potatoes. Quantitative losses are apparent and attempts are made to reduce these losses whereas qualitative losses are not apparent and their importance is underestimated. Since the qualitative losses can greatly reduce the value of potatoes, adequate attention should be paid to prevent loss in quality. Post-harvest losses result from physiological or pathological causes. Good post-harvest management begins at the production stage itself. The good quality of the tuber is greatly influenced by the cultural practices. Potatoes grown on improperly prepared fields are damaged while harvesting mechanically and have reduced storage quality. Conditions for high yield and for good storage characteristics may be contradictory. Further, adequate pest and disease management is essential for producing potatoes with good storage quality. Therefore, proper management of pre-storage factors that affect the keeping quality of potatoes is the first step in good storage management. Whatever may be the storage method, its success depends on the quality of the tubers entering storage. If the tubers entering storage are not sound, the tubers that come out of storage will also be of poor quality. Physiological losses: Natural respiration and evaporative loss of water are the main factors for these losses. The magnitude of physiological losses depends largely on the environmental conditions. Two important storage environmental factors that affect the storage behaviour of potatoes are temperature and relative humidity. (a) Effect of temperature: Physiological damage can occur from exposure to high or low temperatures both before and during storage. Overheating of tubers either due to direct exposure to sun light or during high temperature and non-refrigerated storage could lead to black heart symptoms. At high temperature storage, the high rate of respiration leads to larger oxygen requirement, which results in asphyxiation. The discolouration and breakdown of inner tissues of tubers is a result of asphyxiation. Tubers exposed to freezing temperatures also suffer internal damage. Tuber respiration during storage results in loss of dry matter. In addition, the heat generated during respiration called respiratory heat influences the storage temperatures Potatoes should be harvested in dry weather; irrigation should be stopped two weeks before dehaulming. Potatoes harvested under wet soil conditions must be dried before storage because even little moisture on the surface of tubers could lead to infection and rotting during storage. Only mature tubers should be stored. Immature tubers have poor keeping quality due to lower dry matter content and a weak skin. Harvesting is done 10-15 days after haulm cutting to facilitate proper skin curing. In spite of best efforts, cutting and bruising cannot be completely eliminated 58 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India and this affects the storage life of potatoes. Major weight loss during storage is due to evaporative loss of water. When the loss of water from tubers exceeds 10 percent, the market value of potatoes is greatly reduced because of shriveling. Freshly harvested immature potatoes loose more water than mature tubers because immature skin is more permeable to water. Sprouting results in increased water loss, as the surface of sprouts is 100 times more permeable to water vapour than the periderm of tuber per unit area and time. Sprouting also results in increased respiration leading to higher dry matter loss. (b) Effect of relative humidity: The relative humidity in the storage atmosphere affects the weight loss of tubers since water loss is directly proportional to the difference in water vapour pressure between the potatoes and the surrounding air. Generally a higher relative humidity of 90-95% is maintained in cold stores to minimize weight loss. However, the weight loss is higher under non-refrigerated storage conditions due to lower relative humidity in the atmosphere during the hot summer months. The relative humidity in the storage atmosphere also affects the rate of sprout growth and the form of sprouts. The degree of branching in sprouts is greater under lower humidity levels. Relative humidity also affects wound healing. Wound periderm formation is rapid at higher humidity. For example, at a storage temperature of 10 0C, wound healing is faster above 80% RH. Pathogenic losses: Post-harvest losses in potatoes caused by pathogens are greater than the losses due to physiological causes. Physical damage of tubers during harvesting and handling predispose tubes of attack by bacteria and fungi leading to quantitative loss. The more common storage diseases caused by fungi are late blight, dry rot and pink rot. The most severe bacterial disease that causes rotting is soft rot. When infection occurs in the field, rotting begins in the field and continues during storage, for example, with late blight, brown rot and pink rot. When infection occurs after harvesting, it is generally through mechanical injury as in the case of dry rot. High humidity and condensation of water on tuber surface can lead to infection by soft rot causing bacteria Erwinia Spp. Qualitative losses are caused by diseases such as common scab, powdery scab, black scurf and wart, which affect the appearance of the tuber and thus reduce the market value of potatoes. Among the insect pests, tuber moth causes maximum damage during storage and is common in potatoes stored under higher temperatures, as is the case with non-refrigerated storage. The larval damage results in weight loss and tuber moth infection greatly reduces market value of tubers. Storage methods About 90% of potatoes grown in India are harvested in February-March. Temperatures begin to rise at harvesting time and therefore, potatoes have to be stored during the hot summer months. The requirement for ware potatoes for 6-7 months during the year are met with stored potatoes. Pre-storage factors, viz. tuber maturity, proper skin curing, tuber health, variety etc. have significant implications on the ultimate quality of stored potatoes in all the methods of storage. Potato storage methods being practiced in India can be broadly divided in to two categories viz. refrigerated storage and non-refrigerated storage. Refrigerated Storage Storage at 2-4 oC Cold storage facility was developed primarily for the storage of seed potatoes, but they are also being used for the storage of table potatoes. About 93% of the cold storage capacity in the country is being used exclusively for the storage of potatoes. Seed potatoes are stored in cold stores which maintain temperature 2-4oC and 90-95% RH. At this temperature, sprouting does not take place, weight loss is less and seed maintains it proper vigour which is essential for taking subsequent crop. But 2-4oC is not the ideal storage temperature for table potatoes as at this temperature; they become sweet due to sugar accumulation and are not preferred for consumption. The sugar balance in potato tuber is given in table 1. Due to increased awareness, consumer preferences have changed and the requirements of potatoes with less sugar development are met through storage at elevated temperatures. 59 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Table 1. Sugar balance (mg/hr/kg) in potato tuber during storage Biochemical activity Starch? Sugars Respiration Sugars ? Starch Sugar Balance 0 32.0 2.3 1.7 28.0 Storage temperature (oC) 3 6 10 32.6 33.6 35.8 2.8 3.5 4.5 20.8 25.8 31.3 9.0 4.3 0.0 15 39.3 6.5 32.5 0.3 20 44.0 9.5 34.5 0.0 storage technology has been tried by several cold stores in the country during the last 4-6 years with encouraging results. This technology needs further improvement for successful storage of potatoes meant either for ware purpose or for processing use for 7-8 months. In this direction efforts are being made to identify a safe chemical, preferably originated from plant sources, which work as sprout suppressant. Storage at 10-12oC Potatoes meant for table use and processing are stored at 10-12oC, in most of the countries. When potatoes are stored at this temperature, accumulation of reducing sugars is minimum and therefore they do not become sweet. Besides, the chips produced from these potatoes are light in colour. However, at this storage temperature, potatoes sprout. Therefore, it is necessary to treat the stored potatoes with a sprout suppressant like CIPC (isopropyl N- (3-chlorophenyl) carbamate). CIPC is available in liquid formulation and is applied in the form of a fog using a fogging machine. It is applied @ 35 ml (50 % a.i.) per tonne of potatoes. CIPC treated potatoes are safe for consumption 30 days after treatment as the CIPC residue is within the acceptable limit of 25 mg CIPC/kg of potatoes. This Non-refrigerated Storage: On-farm storage Though the refrigerated storage is essential for long-term storage but for short-term storage of 3-4 months non-refrigerated methods can be used profitably. Traditionally, farmers in India have been storing potatoes in cool dry rooms, heaps and pits. Potato Heap 60 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Heaps are made by heaping potatoes in the shade of a tree to a maximum height of 1m and covering them with 1 foot thick wheat /rice straw or sugarcane trash. Generally 5-30 tonnes of potatoes are stored in heaps. Pits are also dug under the tree shade and these are of two types, viz. Katcha and Pukka. Usually 1040 tonnes of potatoes are stored in pits. The pit method is very popular in the Malwa region of Madhya Pradesh. Katcha Pit Traditional storage methods are in use in states of Assam, Bihar, UP, MP, Gujrat Maharashtra and Karnatka and these have several advantages. They are cheap and no investment is needed on storage structure. Furthermore, the materials required are locally available. The temperature in heaps and pits vary from 23-32oC and the RH: 60-96%. The higher temperatures prevailing in heaps and pits prevent accumulation of reducing sugars and therefore these potatoes are preferred for table use as well as for processing. The reducing sugar content normally does not exceed 150mg/100 g tuber fresh weight and the chips are generally acceptable (score <3). The only disadvantage of traditional methods is that storage losses are high due to rotting. Improving the efficiency of these storage methods can reduce storage losses. Experiments carried out by CPRI at three locations viz. Jalandhar, Modipuram and Patna have shown that it is possible to store potatoes on-farm for 3-4 months with acceptable storage losses. Potatoes of Kufri Jyoti can be stored in heaps for 105 days with total loss of 10.9% at Jalandhar, for 90 days with total loss of 7.1% at Modipuram and for 80 days with total loss of 27% at Patna. On-farm storage in heaps is recommended Pukka Pit for short term for ware use and also for processing purposes. The preferred specifications for various methods of on-farm storage are given in table-2. Table 2. Preferred specifications and functional details for methods of on-farm storage ---------------------------------------------------------------------------------------a. Heap  3m Bottom width  Heap height-1m  Length as per requirement  Protective cover thickness-15m  Preference in shaded places like orchards b. Katcha Pit  Ideal location: shaded area  Dimensions : 6 x 4.5 x 4.5 m (L x B x W)  Lower the temperature by 10-12 oC than ambient  Increase inside RH by 20%  Potatoes remain good for table & processing purpose  Safe duration 80-90 days  A thatched roof over the pit increase cooling efficiency 61 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India c.         Pucca Pit Preferred location : shaded area Diameter 3.5 x 4.5 m (diameter x depth) Pit is lined with bricks Lower the temperature by 12oC Increase inside RH by 25% Potatoes suitable for table and processing 80 to 90 days is safe duration Thatched roof to protect from untimely rains and to improve cooling efficiency ---------------------------------------------------------------------------------------Evaporative Cooled Store (ECS) ECS-CPRI model The insulated potato store (capacity: 20 tonnes) is equipped with passive evaporative cooling which does not require any other source of energy for cooloing. These ECS were evaluated at Jalandhar and Modipuram and it was found that the daily maximum temperatures in the ECS remains 6-13 oC lower than the ambient during March-June. RH remains high at 72-98 %. Due to moderate temperature and high RH the weight loss in potato tubers is significantly reduced. ECS-CIP model This store is consisting of a thatched hut having the roof made up of straw and bamboos supported with a suitable wooden beam. On floor 12 cm wide and 24cm deep alternate channels, half filled with sand are provided to retain water for gradual and regular cooling. To heap potatoes round bamboo mats are placed over these channels. A door, invariably on 62 northern side is provided with “L” shaped small chamber having similar roof to prevent direct entry of hot air. A self rotating wind turbine of aluminum, connected with PVC pipe (diameter: 10 cm) is fixed to exhaust the foul air from the store. The channels are watered daily to keep the sand wet for necessary cooling during the storage period. Table 3. Preferred specifications and functional details for Evaporative Cooled Store (ECS) ---------------------------------------------------------------------------------------a. ECS-CPRI model  Pucca cemented structure (9.5 x 4.7 x 4.3 m3 ) with GI roof  Approximate cost Rs. 1.0 Lakh  Capacity 200 q  Maintain 11-15oC lower temperature than ambient.  Maintain 70-85% RH  Hold potatoes in good conditions for 80-90 days b. ECS-CIP model  Katcha structure with thatched roof  Dimensions: 6.5 x 4.5 x 2.3 m3 for 100q capacity  Maintain 11-15oC lower temperature than ambient  RH ranges 70-90%  Roof require replacement after every 3rd year  Very good for 80-90 days on-farm storage ---------------------------------------------------------------------------------------- Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Ware potatoes can be stored in good condition up to the beginning of June when the price in the market is much higher than the prices at the time of harvest. During storage the reducing sugar decrease and potatoes are not as sweet as the cold stored potatoes. Potatoes stored in ECS fetch premium price as they are suitable for ware consumption as well as for processing. The preferred specifications for Evaporative Cooled Stores are given in table-3. The storage life as well as tuber quality can be further improved by using sprout inhibitor like CIPC. This technology is suitable for 80-90 days in regions where the temperatures are high and humidity is low during the storage period. Suggested reading Burton, W.G. 1989. The potato, 2nd Edn. Longman, Essex. 742 pp. Ezekiel, R., P.S. Dahiya and G.S. Shekhawat, 2002. Traditional methods of potato storage in the Malwa region of Madhya Pradesh. Technical Bulletin No. 57, C.P.R.I. Shimla, 39pp. Burton, W.G., A van Es and K. J. Hartmans. 1992. The physics and physiology of storage. Talburt, W.F. and O.Smith. 1987. Potato processing, 4th Edn. Van Nostrand Reinhold, New York. 796 pp. In: The potato crop, 2nd Ed. (P.M. Harris, Ed.) pp. 608-727. Chapman and Hall, London. Kaul, H.N. and Ashiv Mehta. 1999. Storage of potatoes in India. Technical Bulletin No.47, CPRI, Shimla. 29 pp. Kumar, Devendra; Jagpal Singh and P.C. Pandey. 1998. Storability evaluation of selected TPS lines under passive evaporative cooling. J. Indian Potato Assoc. 25 (3&4): 91-94. Sukumaran, N.P. and S.C.Verma. 1993. Storage and processing. In: Advances in horticulture. Vol.7Potato (K.L.Chadha and J.S.Grewal, Eds.), pp. 701-732, Malhotra Publishing House, New Delhi. 63 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India HARVESTING TECHNIQUES FOR FRUITS AND VEGETABLES D.V. K. Samuel Division of Agricultural Engineering, Indian Agricultural Research Institute, New Delhi-110012 Care in harvesting and handling is necessary to preserve subsequent quality of horticultural produce. Maintenance of the garden fresh quality, which to-day’s consumers demand requires best possible protection at each step of the complex marketing structure. Rough handling at the farm directly affects market quality. Bruises and injuries later show up as brown and black patches making the commodities unattractive. Some physiological disorders are attributed to rough handling. Injuries to the peel or surface of the produce during harvesting and handling serves as avenues for microorganism and lead to rotting. Moreover, respiration is increased markedly by the damage and storage life is shortened. Lack of knowledge in harvesting and handling will result in a substantial waste of horticultural produce. Whole selling of perishable horticultural produce involves receipt, unloading and warehousing of van or rail shipment and subsequent distribution of the products to retail stores. However, depending on the type of commodity being handled, some or all of the following operations may be undertaken. (Fig.1) Harvesting Care in harvesting and handling is necessary to preserve subsequent quality. As far as practicable fruits and vegetables should be harvested during cooler part of the day and they should be immediately removed to a shady place. Harvesting immediately after rain should be avoided as it creates conditions most favourable for multiplication of decay causing pathogens. In India most fruits and vegetables are harvested manually. Some vegetables like potato, onion require special post harvest operation like ‘curing’ before marketing / storage. Thus harvesting at the proper stage of maturity and careful handling of the produce will help in maintaining better post harvest quality and reduce losses. Harvesting methods There are various methods which are used to remove fruits from the parent plants or from the trees. The factors, which should be taken into consideration, are the perishability of the fruit, the economy of labor and market requirements. The following methods are used generally to harvest the fruits. Receiving of product after harvest Sorting / grading Hand/Manual harvesting This method is frequently carried out in traditional ways. For soft fruits and vegetables such as strawberries, raspberries and tomatoes, which are borne on low growing plants, harvesting is done by simply removing them from the plant and putting them into a suitable container. Fruits, which are borne on trees, such as apples, mangoes and citrus fruits are more difficult to harvest. Traditionally the harvester would carry a ladder and use that to reach the fruit. This is very time consuming. Picking plate-forms which can be raised and lowered, are used by the harvester and is to be moved around the orchard from tree to tree. Cleaning / washing etc. Application of protectants (if any) / special treatments viz. vapour heat treatment Packaging Post packaging treatment (fumigation, cooling etc.) Dispatch Fig. 1. Unit Operations of Fruits and Vegetables 64 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Mechanical harvesting proportion of the leaves. Very little fruit required for the fresh market is harvested by machines because the likely damage could result in rapid deterioration in quality during the marketing chain. The fruits required for processing may be harvested mechanically, but it is usually important to process it very soon, otherwise it can deteriorate. Oranges for juice extraction may be removed from the trees by powerful wind machines being dragged through the orchard followed by a device for collecting the oranges from the ground. Tree shakers can also be used which are attached to the tree trunk and violently shake the tree to dislodge the fruit. In order to reduce the difficulty of dislodging the fruit from the tree, chemical sprays are used in certain fruits for the formation of natural abscission layer on the fruit stalk and should be applied a few days prior to harvesting. In India, generally manual harvesting of the fruits is followed, except for the introduction of mango harvesters developed at the Konkan Krishi Vidyapeeth, Dapoli, Maharashtra, IIHR, Bangalore and at the central institute of Subtropical Horticulture, Lucknow. Some more research institutes and universities have also tested their harvesters successfully(Table 1). These harvesters harvest the fruit with 1 to 2 cm long pedicel. This prevents sap bleeding and reduces the latent infections, thereby increasing shelf life of the fruits by 2 to 4 days. The harvesting capacity is 274 to 714 fruits per hour and cost of harvesting is Rs. 40 to 130 per tonnes. Proper maturity of the fruit and their subsequent harvesting is important in horticultural industry. These factors ultimately decides the quality, marketability and Grapes and soft fruits for processing such as consumer acceptability. Every fruit has separate Black Currants may be harvested by tractor mounted maturity indices as well as harvesting method, which machines with combing fingers which are run up the should be followed to minimize the post-harvest losses stems, pulling of the fruit bunches as well as a high and to prolong their shelf life. Table 1: Comparison of Different Harvesting Tools Region Method of Harvesting Karnataka Local Harvester IIHR Harvester GKVK Harvester Traditional Harvester IARI, N. Delhi Harvester Pusa Harvester CIHNP, Lucknow Harvester KKV, Dapoli HOPCOMS, Dapoli APAU, Bapatala i) Adjustable height type ii) Fixed height type U. P. / Delhi Region Maharastra Region Andhra Region Total fruit harvested / hour Numbers kg 584 77.1 714 376 41.0 330 38.2 353 40.5 396 41.7 396 45.3 714 - Local Harvester Gujrat Region Dharti Model RTTC model Positioner 65 312 274 15.9 13.9 165 360 8.5 62.0 Remarks Good performance Good performance Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India  Gujrat Agricultural University, Junagarh Bidhan Chandra Krishi Vishwavidhyalya, The Institutes / Universities where work is /  Kalyani was undertaken on development of mango harvesters CIHNP, Lucknow  Indian Institute of Horticultural Research,  HOPCOMS, Dapoli Banglore Some of these mango harvesting gadgets  University of Agricultural Sciences, GKVK, developed by organizations and performance data of Banglore  Indian Agricultural Research Institute, New Delhi a few of them is presented in the Table 1 and Fig.2. Apart from these Institutes, some other Public & private  Konkan Krishi Vidhyapeeth, Dapoli  Andhra Pradesh Agricultural University, Institutions are also engaged in the development of mango harvester. Bapatala Harvesting tools for mangoes Traditional Mango Harvester, Karnataka Dapoli Mango Harvesters - I IIHR Mango Harvester Dapoli Mango Harvesters - II UAS (GKVK) Harvester Dev Agro, Banglore Harvester ` Traditional Harvester (A.P.) IARI Mango Harvester Fig. 2: Mango Harvesters from Different Regions of India 66 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India and in the green houses. To overcome this problem, a manually operated tomato harvesting tool made from 5.0 mm diameter steel rod, has been developed (Fig.4). The hand held gadget has twelve picking fingers spaced 10.0 mm apart. The tomatoes get detached from the plant with a slight pulling action of the gadget. Harvested tomatoes fall and get collected in the nylon wire net attached to the gadget. Harvesting trials were conducted in the departmental Multi Span Saw Tooth Greenhouse facility on three tomato varieties namely, Navin, Rakshita and Avinash-2.The results were compared with manual (with bare hands) harvesting employing farm labourers. The analysis of the data reveals that the manual (bare hands) harvesting rate for three varieties ranged between 22.0 Kg/hr to 30.0 Kg/hr, whereas harvesting with the developed gadget resulted in harvesting rates ranging between 54.0 Kg/ hr to 59.0 Kg/hr (Table 2 & Fig. 5). This shows that harvesting rate was close to one kg of tomatoes per minute which is 2 – 2.7 times more than tomato harvested manually with bare hands. The gadget is light in weight and simple in construction and costs less than Rs. 100/- (US $2.00) to fabricate. “Pusa” Mango Harvester developed at IARI A mango harvesting tool was designed and fabricated to harvest mangoes from the tree by pulling action. The main consideration was given to the angle of pull which will require minimum force to detach the mango from the tree branches. Most of the manually operated mango harvesters developed earlier have their cutting edges, handle bar and mounting pole aligned in straight line; whereas it was felt during trials that consideration must be given to the optimum angle of pull for effortless cutting of mangoes. Hence, the new design (above) has a 35° angle of pull which is expected to result in right cutting angle for mangoes and more stability to the tools when attempting to harvest mangoes from toll trees. (Fig.3) The harvesting tools were fabricated out of 2 mm M. S. rod and shaped to provide an protruding head to overcome the problem in the movement of mango harvester in the tree canopy. It has a triangular front shape and is wide enough to hold a number of mangoes. The performance evaluation of the developed harvester was done in this season. The results show that the harvester can harvest 353 fruits in one hour time which is very much comparable to some of the best tools developed to harvest mangoes in recent times. 50 mm 180 mm 35° Straight Fingers 35° 25 mm ø Fig. 3: Pusa Mango harvester developed at IARI Development of Tomato Harvester Traditionally, tomatoes are harvested with bare hands by the farmers in the most part of the world. This not only involves drudgery but also results in low harvesting rates both for the crop grown in open fields li Curved Fingers Fig. 4: Tomato Harvesters 67 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Table 2: Comparative Performance of Developed Harvester with Manual Plucking (Tomatoes) ----------------------------------------------------------------------------------------Variety Harvesting Rate, kg/min Curved Straight Manual Fingers Finger Harvester Harvester ----------------------------------------------------------------------------------------Navin 0.984 0.980 0.363 Rakshita 0.850 0.840 0.438 Avinash – 2 0.905 0.750 0.503 ----------------------------------------------------------------------------------------Manual Straight Finger Harvester Curved Fingers Harvester 1 0.9 0.8 Harvesting Rate, Kg/min 0.7 0.6 horticultural produce. Some of the important cleaning, sorting and grading techniques for horticultural produce are discussed in the subsequent paragraphs. Development of a Fruit and Vegetable Cleaning Machine A manual fruit & vegetable cleaning machine is designed to remove impurities from the surface of the produce (Fig.6). A hollow cylinder with one side open for input and the other side for delivery is used for holding the fruits and vegetables. This cylinder is mounted on a stainless steel shaft which moves on the bearings which are resting on the frame. The machine can be operated by a handle provided on one end. Preliminary studies have shown that the machine can remove the impurities like dust, chaff and particles of soil sticking to the product. Efforts are being made to convert this machine to power operated one. 0.5 0.4 0.3 0.2 0.1 0 Navin Rakshita Variety Avinash - 2 Fig. 5. Comparative Performance of Developed Harvester with Manual Plucking of Tomatoes Cleaning, Sorting and Grading techniques for Fruits and vegetables Cleaning, sorting and grading of fresh horticultural produce is one of the most important activity after harvesting. Care in handling is necessary to preserve subsequent quality of horticultural produce. Maintenance of the garden fresh quality, which to-day’s consumers demand requires best possible protection at each step of the complex marketing structure. Rough handling at the farm directly affects market quality. Injuries to the peel or surface of the produce during harvesting and handling serve as avenues for microorganism and lead to rotting. Moreover, respiration is increased markedly by the damage and storage life is shortened. Lack of knowledge in harvesting and handling techniques will result in a substantial waste of 68 Fig. 6. Fruit and Vegetable Washing Machine Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India A rotary vegetable washing machine (drum washer) was developed and fabricated at PAU Ludhiana. Portable, one hp. electric motor operated machine was designed for washing 4-5 quintals per hour of potato/carrots. 62 cm dia stainless steel washing drum with 6.0 mm holes was provided with electronic device to regulate precisely rotational speed of drum upto 100 rpm. Proper arrangements for feeding water into machine and draining out dirty water and silt were provided. Rotating parts and moving belts were covered with guard for safety of operator. All the parts coming in contact with vegetable were made from food grade stainless steel. The electronic circuit provides regulation in speed control and timer for automatic stopping of machine at desired periods. The machine has been evaluated for washing carrots, potatoes and spinach. The machine can successfully wash 3.5 qph of carrots, 3.5 qph potatoes and 1 qph spinach. The cost of 1 hp electric motor operated machine is Rs.25,OOO/- and the cost of washing carrots and potatoes is Rs.2/qtl and spinach Rs.7/qtl respectively. The microbial load reduction by washing spinach, potatoes and carrot in this machine was evaluated by washing efficiency ranged from 96.599.8%. Sorting Sorting and grading are the important steps to protect the value of the produce in market. Sorting the produce before packing eliminates low quality produce and thereby reducing packing costs of high quality produce. As sorting also takes care of mis-shaped, decayed, diseased and injured fruits, the losses during post-packing operations are also reduced. The objective of sorting is to remove over ripe or damaged fruits, which are likely to decay before reaching the consumers, often damage the good fruit in contact with it and to remove abnormal coloured or deformed fruits that lower the standard of the whole lot. Such fruits may sometimes be sold locally at low price or used for processing. efficient marketing system as a well designed programme on grading and standardization to bring about an overall improvement not only in the marketing system but also in raising the quality of produce. Grading is a critically important process because produce presentation, an aspect of quality is often judged on the basis of uniformity. Uniformity is important as it presents a standard product for handling and marketing. Fruits when graded fetch suitable returns commensurate with quality. Grading or sorting should be limited to doing what is necessary; split, puncture, deformed fruits and incipient rots must be removed. Off grade fruit should be removed from the packing house line as early as possible, so that dump rate can be proportionally increased to replace defective fruit with sound fruit that will return to profit. Grades are based on soundness, firmness, size, weight, colour, shape, maturity, freedom from foreign matter, diseases, insect damage and mechanical injury etc. Manual Sorting/grading/sizing Hand sizing is useful for small-scale operation. Some times sorting, grading and packing are integrated in such a way that a single person or three persons on a single table undertake all the three operations. The incoming produce is placed on sorting table (Fig.7), the workers pick up the undesirable fruit and the second worker grades the produce for size or colour, and the third one pack the produce. This method is inadequate for the separation of citrus fruits into uniform size groups. The first simple approach can be merely providing grids of stretched wires of increasing spacing in which each fruit is graded by size. Sorting / grading tables of various size and shapes are used for different commodities depending upon the type of the commodity and size of the operation. Here, care must be exercised while dumping the produce to avoid mechanical injury. The provision of a foam pad at dumping table can be helpful. Grading Systematic grading is a pre-requisite for 69 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Mechanical grading for apples in HP, J & K and UP, citrus in Maharashtra and Punjab (Kinnow) etc. is being undertaken in packing centers installed with mechanical graders. Grading and sorting of kinnow over the mechanized line is undertaken at 5 waxing and grading units installed by Punjab Agro in various kinnow estates of the state, where kinnow fruits are graded and sorted on custom basis. The size grader can be adjusted according to size as per demand. The mechanical grading of other fruits in India due to the smaller farm size and the high costs of the Fig. 7. Sorting Bench equipment is still is in its infancy. However, with Partly mechanical and partly manual system of Governments or Cooperative’s initiative, some grading sorting/grading facilities where farmers can get their produce graded In the partly mechanical and partly manual on custom basis like kinnow waxing and grading system of sorting grading, the fruit is continuously stations, can prove to be a new beginning for the aspect moving on a conveyor belt and workers stand along of fruit/vegetable handling which has not been given the length of the belt at various locations pick us the the attention it deserved since long. undesirable produce. The speed of the conveyor belt A simple and low cost fixed roller belt-type is adjusted according to the capacity of the workers, vegetable grader was developed at IARI, New Delhi. volume of the undesirable produce etc. The details of the prototype vegetable grader are shown in Fig.8. The machine consists of the endless belts, a feed hopper and a frame. The two endless fixed roller belts having rectangular openings of sizes 40 cm x 5 cm and 40 cm x 2.5 cm move one over the other with same speed and in same direction with gentle shaking. The shaking helps in changing the orientation of products and thus increases the grading efficiency. These belts are made from 5 cm wide flat canvas belt and 2 cm diameter PVC pipes. Both the ends of these pipe rollers are fixed on the canvas belt with M.S. bolts and nuts at a desired spacing. The ungraded material from feed hopper is fed to one end of the upper belt. The product of size larger than 50 mm is carried over by this roller belt to the other end of the belt where it is discharged into the bags attached with the holder. The product, smaller than 50 mm size fall on the lower belt which has 25mm openings. This belt retains the product of size between 25mm and 50 mm and allows the product of less than 25mm to fall through the gap between PVC rollers of lower belt. This graded material is collected and bagged. The medium size product is carried away by the lower belt, Mechanical grading/sorting The rotary cylinder sizer that has hollow cylinder which rotates with electric motor fitted with these is used for size grading of produce. Each cylinder has holes large enough to let commodity drop in on the line, the first cylinder is with smallest diameter hole and the diameter progressively enlarges to the last cylinder in the assembly. The angle of the cylinder is tilted to guide the fruits to the exit point and the fruit falls into collecting bin through a slanting tray. This system works well with round commodities. In certain sizing lines, a conveyor belt is installed with a wide variety of sizing chains and belts. These chains and belts may have square, round, rectangular or hexagonal openings. For regular shaped fruits like citrus, central longitudinal belt and roll seizers are of many types or variations such as transverse roll and drop roll are used. The different sizes drop is guided through the canvas distribution belts and is transported to the packaging stations. 70 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India discharged and collected at the end of the lower belt. Thus, this machine separates the products into three grades of desired size. The belts of different spacings of PVC rollers can be used for sizing the products. Seven M.S. rollers are provided for supporting and moving these belts. Wooden fenders are provided on both sides to avoid spilling of products over the sides. namely A, B, C & D. Number and weight of tomatoes which fall under each of these categories was recorded before starting the experiments. For conducting the experiments, tomatoes were filled into the feed hopper and the machine was started. The feed conveyer lifts the tomatoes and drops it on the moving belt conveyer from where the tomatoes move and passing through their opening of relevant size, drop in to the respective collection buckets. After completing the experimental run, the collection buckets are taken for analysis.Data is collected on the tomatoes which passed through different openings and is analyzed for calculating the efficiency of the machine. The capacity of the machine works out to be 500 kg/hr (approx) for tomatoes with a grading efficiency between 80 – 85 percent. Optical methods of grading In several crops such as peaches, apples, peppers and tomato etc. colour is of significance Fig. 8. Fruit and Vegetable Grader bearing on the quality of the produce. Optical methods Fruit and Vegetable Grader for Tomato & Mango such as colour grading, spectrophotometric techniques, A fruit and vegetable grader has been designed delayed light emission and Minolta or Hunter Colour and fabricated in the Agricultural Engineering Division, meters of grading have been developed and are IARI, New Delhi. The fruit grader is designed to available for commercial use. separate on the basis of size difference. The machine Colour grading has a feeding hopper, a feed conveyer and a sizing In the colour grading, a beam of light is put on table comprising of a moving belt. A taper opening is provided on one side of moving belt. The material is the surface of the fruit. This light is reflected back and lifted from the hopper below & is carried and dropped the light so reflected is received by one or more (if the on the moving belt. The material in single layer travels grading for more than one colour is to be undertaken) parallel to the belt and along the taper wooden roller photoelectric cells. A reflector of the colour of the and is separated depending on its size. The graded commodity (red tomatoes should have red reflector) material in three sizes is then collected in the plastic placed immediately behind the commodity shows its colour. When a red fruit is put on the system for sorting, crates for further use (Fig. 9). the photoelectric cell would not find any difference Trials were conducted on the developed grader between red tomato fruit and red reflector. But in case with tomatoes. Assorted tomatoes of various sizes were of a green or yellow tomato, due to the difference in first counted and weighed on a platform balance. Only colour of fruit and reflector, the photoelectric cell would healthy tomatoes were selected for the trials. The receive different reflective signal. This signal is passed sample heap was first analysed manually and size and over to the computer attached to it. The computer weight of tomatoes which fall in to one category were analyses the colour data and sends message for the counted. For example, the outlets were divided commodity to be dropped in the bin assigned to that according to their opening sizes in to 4 categories particular colour such as red, yellow, green etc. The 71 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Outlets Moving Belt Conveyer Guide Feed Conveyer with hopper Electric Motor Fig. 9. Fruit and Vegetable Grader for Tomato & Mango c* which describes the level of colour intensity on the scale of 0 to 60. A camera installed in the grading/ sorting line can scan the commodity and sends the image to the computer, which control the drop zone of the produce after analyzing the colour data. grading of the crops by mechanized system with colour sensors is common practice outside India in crops where specific colour requirements are absolute. This system can also be extended to detect the surface defects or blemishes. A considerable research data have been generated and published on the advanced methods of This is another technique, which is used for grading/sorting but significant amount of the produce fruits like mandarin, tomato, and papaya to grade is still graded using eye and hand. Some commodities maturity based on chlorophyll content. A fruit is such as grapes and bananas are in bunch form making subjected to bright light and later the fruit is shifted to these crops unsuitable for grading by these methods. darkness while sensor measures the amount of light References emitted. The difference in colour ripeness and damage Arora, M abd Sehgal, V. K. (2002). Performance can be detected by this method. evaluation of power operated vegetable washing Colour meters machine. Proc. – National Workshop on Post Hunter and Minolta system of colour analysis Harvest Management of Horticultural Produce are based on three components of colour. These are organized by National Productivity Council, value, hue and chroma. Value is denoted by L * and Chandigarh. pp 215-303 gives the value of light to darkness. Hue is represented Javrae, G. S., Kumargaud, V., Venkatesh, B. S., by a* which measures the range of colour between Mandhar, S. C. and Shankar, A. N. (1995), green and red over a scale of -60 to +60. Similarly, Comparative evaluation of mango harvesting b* represents the range of colour between yellow and techniques. Proc. Of conference “Phala blue in the range of -60 to +60. Chroma is denoted by Samaskarna – 95” at Banglore. pp 48 - 51 Delayed light emission 72 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Prasad, S. S., Rao, K. V., Venkantaramaiah, K and Samuel, D. V. K. (2002), Annual Report of NATP Sukumaran C. R. (1995). Improved manual project on “Development, Evaluation and operated fruit harvester. Proc. Of conference Adoption of Equipment and Gadgets for “Phala Samaskarna – 95” at Banglore. pp 29 – Harvesting, Grading, Packaging and Transport 34 of Selected Fruits and Vegetables”. Randhawa, J. S., Bala, J. S. and Kamboj, J. S. (2002). Singh, A and Jha, S. K. (2001). Cleaning, grading, Grading and Sorting of Horticultural Produce in drying and handling of vegetables in “Post Harvest handling of fruits and vegetables” “Mechanization of vegetable crops” (Ed: (Ed: Sandhu and Bal), Punjab Agricultural Srivastave et. al.), Division of Agricultural University, Lushiana, pp 29-38 Engineering, IARI, New Delhi, pp 229-241 Ranganna, B., Babu, C. K. and Subramanya, S. Singhal O. P. and Singhal, D. V. K. (2002). Post (2001), Research Digest (1991 – 2000), AICRP harvest handling of fruits and vegetables in in on “Post Harvest Technology Scheme”, UAS, “Post Harvest handling of fruits and vegetables” Banglore. pp 74 (Ed: Sandhu and Bal), Punjab Agricultural University, Lushiana, pp 5-10 73 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India IONIZING RADIATIONS FOR IMPROVING QUALITY AND POST HARVEST PRESERVATION OF AGRICULTURAL PRODUCE Bhupinder Singh Nuclear Research Laboratory, Indian Agricultural Research Institute, New Delhi radiation is obtained through the use of radioisotopes, generally Cobalt- 60. Cesium-137 is the only other gamma emitting radionuclide suitable for industrial processing of materials. The irradiation is measured in the SI unit known as “Gray” (Gy). 1 Gy dose of irradiation is equal to 1 Joule of energy absorbed per kg of food material. In irradiation processing of samples, the doses are generally measured in Kilo gray (1KGy = 1000 Gy). The source of gamma radiation in use at the Nuclear Research Laboratory, Indian Agricultural Research Institute, New Delhi is Co- 60 Gamma Chamber, Model: GC 5000, supplied by BRIT, Mumbai. Radiation can be defined as the energy emitted by a body in the form of rapidly spreading waves or particles as it moves from a higher energy level to a lower energy level. The most common use of the word “radiation” refers to ionizing radiation. The other class of radiation is the non-ionizing type that have enough energy to move atoms in a molecule around or cause them to vibrate, but not enough to remove electrons. Examples of this kind of radiation are sound waves, visible light, and microwaves. A radioactive material is a material that emits ionizing radiation of any of the three principal types i.e., alpha, beta and gamma radiation, which have enough energy to displace electrons and cause ionization in matter. The penetrating power of the above particles governs the amount of damage the radiation can cause. Among different types of ionizing radiations, gamma rays are most widely studied for their applications in the field of agriculture, medicine and industry. RADIATION TECHNOLOGY Radiation processing of food involves controlled application of energy from ionizing radiations such as gamma rays, electrons and X-rays for food preservation. Irradiation works by disrupting the biological processes that lead to decay. In its interaction with water and other molecules that make up food and living organisms, the radiation energy is absorbed by the molecules it contacts. The interaction of radiation and radiolytic products of water with DNA cause biological changes at call/tissue or organ level. Gamma rays were first discovered by Paul Villard, a French chemist and physicist, in 1900, while studying Uranium. Villard recognized them as being different from x rays because the gamma rays had a much greater penetrating depth. They were emitted from radioactive substances and were not affected by electric or magnetic fields. However, these came to be called gamma rays by another scientist, Ernest Rutherford in 1914. Gamma rays are generally characterized as electromagnetic radiation having the highest frequency and energy and also the shortest wavelength (below about 10 Pico meter). Gamma rays consist of high energy photons with energies above about 100 keV. Due to their high energy content, gamma rays can cause serious damage when absorbed by living cells, in high amounts. Materials such as lead, concrete are the best shielding materials. The gamma Ionizing radiations provide an effective alternative to fumigants, which are being phased out owing to their adverse effects on the environment and human health. Further, the exposure of food material to radiation has strong advantages over conventional methods of preservation viz., cold storage, fumigation, salting and drying because it does not lead to loss of flavour, odour, texture or quality. Irradiation is the exposure of food products to ionizing radiation at levels: to destroy micro-organisms, insects, and parasites that cause disease and spoilage. Radiation processing technology 74 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India can be used for disinfestation of food grains and pulses, inhibition of sprouting in bulbs and tubers, extending shelf-life under recommended conditions of storage, ensuring microbiological safety and in overcoming quarantine barriers to international trade. The technology can also be used for hygienization and sterilization of non-food farm products including cutflowers, pet food, cattle feed, aqua feed, ayurvedic herbs and medicines and packaging materials. The process is approved by international bodies like WHO, FAO, IAEA and Codex Alimentarius Commission. More than 40 countries have approved the process for over 100 items of food. India, the world’s second largest producer of fruit and vegetable, produces a wide range of horticultural crops such as fruits, vegetables, roots, tropical tuber crops, medicinal and aromatic plants, spices and plantation crops like coconut, cashew, cocoa etc. It produces 160 million tons of horticultural produces per year out of which more than 85 million tons are vegetables and 50 million tons are fruits. The processing of these crops is just 2 percent in India where as in other developed countries, more than 50 per cent of the total horticultural produce is processed. The economic value and health benefits to humans of the food saved from spoilage would more than cover the cost for irradiation. BIOLOGICAL EFFECTS OF GAMMA IRRADIATION had a higher dry matter, protein and RNA content. Investigations pertaining to the effect of gibberellin and gamma irradiation on wheat showed gibberellin aided stimulation in growth of irradiated plants mainly through an increase in cell expansion, cell division or both. In photosynthetic organisms, carotenoids play a vital role in the photosynthetic reaction center (Ananthaswamy et al; 1985). Gamma irradiation of three centimeter apical leaf tips excised from 7 day old seedlings of wheat showed no apparent affect on chlorophyll content. A comparison of amino acid profile of wheat, maize, chickpea, and mung bean seeds irradiated at 0.5, 1.0, 2.5 and 5.0 KGy indicated that sulphur containing amino acids like cysteine and methionine were radiation labile, particularly in the legumes. Gamma rays accelerate the softening of fruits, caused by the breakdown of middle lamella in cell wall. They also influence the plastid development and function, such as starch sugar inter-conversion. Gamma rays penetrate through the cells. Investigations on the effect of gamma rays on growth and cellular contents of soluble carbohydrates, protein and nucleic acids in sunflower revealed a significant increase in protein, carbohydrate and DNA but a significant reduction in RNA content of irradiated plants. Gamma treated plants showed significant increase in P, K, cellulose and total nitrogen. Studies on the irradiated wheat starch revealed that irradiated plants constituents are more susceptible to enzyme actions, compared to their non- irradiated controls. Maltose was found to be the chief radiolytic breakdown product of starch at high dose of 1 Mrad. Various gamma irradiation doses applied on the wheat plants showed a significant increase in micronutrients (Cu, Mn, Zn, and Fe) concentration. Reducing sugar levels were increased in irradiated wheat at doses in the range 20 to 200 Krad. Both á and â amylases retain their activities in irradiated wheat, but the sensitivities of starch to amylolysis is increased with an increase in dose of irradiation. Biological effect of gamma rays is based on the interaction with atoms or molecules in the cell, particularly water to produce free radicals, which can damage different important compounds of the plant cell. Plant types developed from seeds irradiated gamma rays, at a dose rate of 4 Gray and dose intensity of 2 Gy/min, had higher vigour productivity and the photosynthetic pigments. Chl-a, Chl-b and carotenoid concentration in irradiated plants were high as compared to non irradiated control. Gamma irradiation in the range up to 30 KGy produced a decrease in wheat starch concentration. Large gamma doses increase the quantity of reducing sugars but decrease POTENTIAL OF RADIATION TECHNOLOGY the starch content. In a separate study, seedlings The major technological benefits that can be produced from wheat grains irradiated at 800 Krad 75 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India achieved by radiation processing of food at different doses include (Table 1). Fruits and vegetables are essential dietary sources of vitamins and antioxidants. Since more than Rs.70000 crores worth of fruits and vegetables are produced annually, they are also commercially important. Hence, wastage of these commodities has to be reduced to a minimum without compromising on the quality of the produce. Research has proved beyond doubt that it is possible to extend the shelf life of fresh fruits at the ambient as well as refrigerated temperatures through the judicious application of ionizing radiations like gamma rays. Table 1: Applications of radiation processing ------------------------------------------------------------------------------------Low dose applications (Less than 1 kGy)  Inhibition of sprouting in potato and onion  Insect disinfestation in dry commodities  Destruction of parasites in meat and fish Medium dose applications (1-10 kGy)  Elimination of spoilage microbes in fresh fruits, meat and fish  Elimination of food pathogens in meat and fish  Hygienization of spices and herbs High dose applications (above 10 kGy)  Sterilization of food for special requirements  Shelf-stable foods without refrigeration ------------------------------------------------------------------------------------Gray is the unit of radiation absorbed dose = 1Joule/kg. The old unit of dose is rad (1Gy = 100 rad) DOSE EFFECT RELATIONSHIP On the basis of dose requirements these benefits could be classified in to low dose, medium dose, and high dose applications (Table 1). The dosages vary with varying purposes like: Sprout inhibition in tuber, bulb, and root vegetables; inhibition of growth in asparagus and mushroom (0.05-0.15 kGy); Insect dis-infestation (0.15-0.75 kGy); Delayed ripening of some tropical fruits viz., banana, mango, and papaya (0.25-0.50 kGy); Control of postharvest diseases (>1.75 kGy) etc. Accelerated softening; development of offflavors were, however, reported in some commodities at 1-3 kGy followed by excessive softening; abnormal ripening; incidence of some physiological disorders; impaired flavor at >3 kGy dosages. Most of the advantages or effects are realized due to highly penetrating nature of the ionizing radiations. Irradiation is a cold process and can be used to disinfest, disinfect, pasteurize and sterilize food items without causing perceptible changes in quality of food. Unlike chemical fumigants, irradiation does not leave any harmful toxic residues in food and is more effective. It is highly efficient and can be used to treat pre-packed commodities. It can be applied only to those foods that have been established by experimentation to benefit from the technology. It cannot inactivate many viruses or The primary, tangible action brought about by enzymes in food. Therefore, for certain processes it these rays are: (a) disinfestations of fruit flies/insects; may need to be coupled with mild heat treatment such (b) inactivation or reduction of spoilage as blanching to get rid of these enzymes and viruses. microorganisms; (c) delaying ripening and /or Irradiation produces very little chemical senescence. The hormetic effect of irradiation are also changes in food. None of the chemical changes found realized by some of the breeders and which has resulted to occur in food have been found to be harmful. It has in the development of a number of mutants (2252) in a been found that there are no unique radiolytic products wide range of crops including 552 mutants in formed and free radicals in the system disappear ornamental crops. An attempt has been made here to depending on the nature of the commodity and its postprovide an overview of potentials and innumerable irradiation storage and treatment. The radiolytic applications of ionizing radiation in general and gamma products and free radicals produced are identical to irradiation in particular to improve fruit and vegetable those present in foods subjected to treatment such as crops as such, their post harvest shelf life and quality. cooking, frying and canning. Highly sensitive scientific 76 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India tests carried out during the past 30 years have failed to detect any harmful chemical products in radiation processed foods. In fact, the chemical differences between radiation processed foods and non-irradiated foods are too small to be detected easily. There is no evidence to suggest that free radicals or radiolytic products affect the safety of radiation- processed food. This has even been confirmed by many long term multigeneration studies in which laboratory animals were fed irradiated products exposed up to a dose of 45 kGy. In comparison to other food processing and preservation methods the nutritional value is least affected by irradiation. Extensive scientific studies have shown that irradiation has very little effect on the main or macronutrients such as proteins, carbohydrates, fats and minerals. Vitamins show varied sensitivity to food processing methods including irradiation. For example, vitamin C and B1 (thiamine) are equally sensitive to irradiation as well as to heat processing. Vitamin A, E, C, K and B1 in foods are relatively sensitive to radiation, while riboflavin, niacin, and vitamin D are much more stable. The change induced by irradiation on nutrients depends on a number of factors such as the dose of radiation, type of food, and packaging conditions. Very little change in vitamin content is observed in food exposed to doses up to 1 kGy. processed food as part of their ration on the various space flights from Apollo to space shuttle. As in many other food processing procedures only fruits and vegetables of good hygienic quality should be irradiated. It is very important that foods intended for processing are of good quality and handled and prepared according to good manufacturing practice (GMP) established by national and international standards. Like any other food treatment irradiation cannot reverse the spoilage process and make bad food good. A spoilt but irradiated food is unfit for consumption. APPROVAL OF RADIATION PROCESSING OF FOOD IN INDIA Government of India in 1994 amended Prevention of Food Adulteration Act (1954) Rules and approved irradiation of onion, potato and spices for domestic market. Additional items were approved in April 1998 and in May 2001 (Table 2). In 2004, Ministry of Agriculture, Government of India, amended plant protection and quarantine regulations to include irradiation as a quarantine measure. Laws and regulations enacted under the Atomic Energy Act enforced by the Atomic Energy Regulatory Board, an independent body, govern operations of irradiators used to process non-food products, such as medical IS RADIATED COMMODITY SAFE FOR supplies as well as food. Many medical product CONSUMPTION irradiators are operating in India and around the world. Animal feeding studies have been the most The plants that must be approved by the government time consuming and expensive feature of before construction and operation, and are subject to wholesomeness testing of irradiated foods. None of regular inspection, safety audits, and other reviews to the short- or long-term feeding studies and also the ensure that they are safely and properly operated. Only detailed mutagenicity testing studies on animals, as well those foods approved under the Prevention of Food as trials on human volunteers, has not revealed any Adulteration Act rules can be irradiated and sold in adverse effect of consumption of irradiated diet. In domestic market. the early 1980s, eight feeding studies using several APPLICATIONS OF GAMMA RADIATION IN radiation processed food items, including wheat, were AGRICULTURE conducted in USA and China with human volunteers, Research and development in the field of More than 400 volunteers consumed radiation processed food under controlled conditions for 7-15 radiation processing of food in India had its beginning weeks. Results showed no significant differences in the late 1950’s at the Bhabha Atomic Research between the control and the test groups. In fact, Centre (BARC), Mumbai. Irradiation is a cold process astronauts and cosmonauts have been taking radiation and dose not significantly increase the temperature or 77 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India change the physical characteristics of most foods. Process parameters in terms of commodity, specification, optimal radiation dose, packaging and post irradiation storage and handling have been standardized for a large number of food items of both plant and animal origin, particularly those of economic importance to India. Food is irradiated to provide the same benefits as when it is processed by heat, refrigeration, freezing or treated with chemicals to destroy insects, fungi or bacteria that cause food to spoil or cause human diseases. GAMMA IRRADIATION IN POST HARVEST STORAGE OF AGRICULTURAL PRODUCE Vegetables seed crops and fruits are essential dietary sources of nutrients, proteins, vitamins and antioxidants. Further, since more than Rs.40000 crores worth of perishable produce are generated annually, they are also commercially important. Hence, wastage of this commodity has to be reduced to a minimum, while at the same time maintaining the quality. It has been conclusively shown that it is possible to extend the shelf life of fresh fruits at the ambient as well as refrigerated temperatures through the judicious application of ionizing radiations like gamma rays. The primary, tangible action brought about by these rays are: (a) disinfestations of fruit flies/insects; (b) inactivation or reduction of spoilage microorganisms; (c) delaying ripening and /or senescence. Amount of ionizing energy used for a specific purpose determines the resultant benefits. A very low dose of irradiation inhibits microbial growth. Gamma irradiation is thus useful in controlling insects, pests in stored rice, wheat flour, pulse, gram, dry fruits, nuts, spice powders, dry fish etc. At higher doses, irradiation pasteurizes or retards spoilage of meat, poultry, and fishery products by killing bacteria responsible for spoilage of these foods and also ensures food safety by destroying food borne pathogenic bacteria such as Salmonella, Listeria, Staphylococcus, E. coli and parasitic organisms such as protozoa, roundworms, tapeworms etc. At still higher doses, irradiation can improve quality and microbial safely of spices and dried herbs. The provision of safe and nutritionally adequate diets for the world’s population is increasingly becoming a major challenge. In spite of great efforts at the national and international levels, progress in combating food borne diseases has largely been offset by other global trends, including longer food distribution networks and many basic changes in the way food is produced, transported, processed, prepared and consumed. The scenario can be turned to achieve economic advantage through adoption of radiation technology. Gamma rays are widely used to generating mutant population with desirable traits in crop breeding programme in several species. More than 2500 radiomutant varieties of different crops have been released worldwide. Besides, variety development, gamma irradiation is also useful to alter morphological, physiological and biochemical traits to for commercial gains. Gamma irradiation was used to achieve chemical modification of starch to develop starches with different physical characteristics to suit varied commercial applications. These rays are also useful to improve germination of hardy seeds and to induce/ improve tolerance of crop species to abiotic stresses. Irradiation can also be used to increase yield of plants. Some of the above reflected gamma irradiation linked affects could be manifested at molecular level. IONIZATION IRRADIATION IN FRUITS Effect on crop response and produce shelf life and quality Since fruits generally contain very high moisture content, the high - energy gamma rays generate copious amounts of free radicals in the fruit immediately following radiation, the amount of free radicals formed depending upon the intensity of the radiation dose. The free radicals in turn bring about the breakage of the genetic material (DNA) of the insects and spoilage organisms, thus destroying them. But after a short period (2-3 days) the free radicals get scavenged off or converted into harmless molecules. What preserves the fruit thereafter, and helps extend its shelf-life? 78 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Table 2. Items of food permitted for irradiation under Indian Prevention of Food Adulteration Act (PFA) Rules S.No 1 2 3 4 5 6 7 8 9 10 11 12 13 Name of food Onion Potato Shallots (small onion) garlic, ginger Rice Semolina (Sooji or Rawa), Wheat atta and Maida Pulses Dried sea-food Raisins, figs and dried dates Mango Meat and meat products including chicken Fresh sea-food Frozen sea-food Spices Dose of irradiation (kGy) Purpose Min 0.03 0.06 0.03 Max 0.09 0.15 0.15 0.25 0.25 1.0 1.0 Insect disinfestation Insect disinfestation 0.25 0.25 0.25 0.25 1.0 1.0 0.75 0.75 2.5 4.0 1.0 4.0 6.0 3.0 6.0 14.0 Insect disinfestation Insect disinfestation Insect disinfestation Shelf-life extension and quarantine treatment Shelf-life extension and pathogen control Shelf-life extension Microbial pathogen control Microbial decontamination Biochemical processes that are related to the biosynthesis of substances to the pathogens get triggered by the radiation. These are mainly phenolics. One enzyme whose activity becomes important is phenylalanine ammonia lyase (PAL). The activity of this enzyme correlates well with dose of radiation. Low activity of the enzyme (which correlates with low dose of radiation) and high activity of the enzyme (which correlates with high dose of radiation) are both detrimental for fruit preservation. Sprout inhibition Sprout inhibition Sprout inhibition has been observed in the treatments. However, irradiation of mangoes (var. Amrapali) with 0.5 kGy was found optimum to enhance the shelf-life of the fruits up to 6 days with minimum degree of blackening. Infect US export market has opened its door to Indian mangoes only if it is irradiated to suffice the US quarantine requirement. Senescence in radiationtreated non-climacteric fruits is perhaps delayed by inhibition of/decrease in the activity of the amylolytic and proteolytic enzymes. However, detailed Gamma radiation itself delays ripening by investigationis desired to understand the mechanisms decreasing the activity of the cell wall enzyme involved. pectinmethyl esterase (PME) and the activity of ACCThe quality parameters that are looked for in oxidase involved in ethylene synthesis. Activities of some fruits usually are: (a) soluble solids; (b) titratable acidity; other enzymes like polygalactosidase, cellulase and 1- (c) appearance; and (d) organoleptic and sensory aminocyclopropane-1-carboxylate oxidase are also evaluation. All case studies reviewed showed that negatively affected. Mangoes (var. Amrapali), irradiated gamma radiation does not affect the quality parameters (0.5, 0.75 and 1.0 kGy) just after the harvest, indicated adversely. Health-promoting compounds like (a) that the softening of the fruits was delayed up to 6 flavanones; (b) limonin; (c) carotenoids; (d) lycopene days of irradiation in all the treatments as compared to and (e) Vitamin C either increase or remain unchanged the un-irradiated control but some degree of blackening as a result of gamma radiation. Positive benefits in 79 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India kGy of gamma radiation and stored at room temp. (20-30oC), did not show any insect infestation even after 2 months of storage. Chickpea (var. Pusa-362, Pusa-256, Pusa-1053), Lentil (cv. L-4147, Pea (cv. DMR-7), irradiated with different doses of gamma radiation (within 2 months of the harvest) and stored for 1year along with other infested pulse samples, indicated that the recommended dose (1.0 kGy) of gamma radiation is most effective in controlling the growth of infestation in pre-packed pulses stored over a long period. Mung seeds (cv. Asha), irradiated with a dose of 1.0 kGy were found free of natural infestation even after 3 years of storage. biomolecules may not have any practical significance if irradiation used makes the fruit unmarketable. Sensory qualities such as appearance and flavour are retained in the irradiated fruits. Polyamines, that are known to be scavengers of free radicals, interact with phospholipids to stabilize the bilayer surface and retard membrane deterioration in radiation treated fruits. This stimulates the activity of lignifying enzymes and suppresses the loss of water from the fruit. Overall benefits of irradiation in terms of preservation, quality and value addition in fruits are immense and we must develop ways and means to take their advantage. Studies done at SRI, New Delhi on gamma radiation processing of various varieties of apples for shelf life enhancement have yielded favorable results. Irradiated apple were found to retain most of its physico-chemical properties such as fruit firmness, acidity, total soluble solids reducing sugar and sensory properties even up to 7-8 months of storage at 2-4°C at an optimized dose of 0.25 kGy. Whereas unirradiated (Control) samples started decaying even after 5 months of cold storage at 2 - 4°C. A pilot study on radiation processing of apples for consumer acceptability involving various orchard owners of apple growing regions of Himachal Pradesh is underway. The outcome of this study may open a new horizon in setting up gamma irradiation facilities at suitable locations in the Himachal Pradesh, which will be of immense benefit to the orchardists and farmers. However, there is a need to work out the risk and success factors of the radiation processing units before hand. Three different varieties of chickpea and 2 varieties of mung bean, exposed to gamma radiation doses of 0.2, 0.4, 0.6, 1.0 and 1.6 kGy, exhibited that with the increase in gamma radiation dose, free amino acid content showed a decreasing trend, while the content of reducing sugars tended to increase up to the dose of 1.0 kGy. Studies on the content of free amino acids and reducing sugar in different varieties of pea and lentil exhibited reduction in the content of reducing sugar and free amino acid in the radiated (1.0 k Gy) as compared with the un-radiated ones in all the cases.During long post harvest storage periods (3-1 year) the seed hardness decreased with increasing irradiation dosage (0.5 to 5.0KGy) in three Mungbean genotypes (Pusa Visal, IPM-99-125 and K851) while Pusa 9531 maintained the hardness. Its consequences for insect damage will be worked out. Seed protein content also declined and followed a pattern as observed for hardness index across mungbean genotypes. Production of free radicals upon irradiation was monitored in terms of NBT reduction and was found to increase with dose while reverse occurred with time. The cooking quality of radiated chickpea and mung seeds was enhanced as seen by reduction in cooking time when compared with the control. The taste and softness of the cooked material, however, remained unchanged. Similar results were recorded with other irradiated pulses also. IONIZATION IRRADIATION IN VEGETABLES Effect on crop response and produce shelf life and quality Gamma irradiation, of the seeds of 2 var. of mung, with 0.05, 0.1, 0.5 and 1.0 kGy dose of gamma radiation indicated that insect (weevil) infestation could be contained in both the varieties even up to 3 months of radiation. Freshly harvested pea (var. DMR-11, DMR-7, HEP-4); lentil (var. E-4147) and chickpea Chickpea cv. P-362, irradiated with 1.0 kGy (var. Pusa-256, Pusa-362, Pusa1053), exposed to 1.0 of gamma radiation, did not show any change in milling 80 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India quality over the non-radiated samples. The loss of about 25 per cent material in gamma irradiated seeds was similar to that of the un-irradiated seeds. Obviously, gamma radiation did not have any adverse effect on milling quality. Losses could be reduced considerably if large amounts of the seeds are milled in a large commercial mill. Germination of gamma-radiated chickpea seeds did not indicate any difference due to radiation treatment. Low dose of radiation (0.2 kGy), on the other hand, considerably enhanced the germination as compared to control. In mung, however, a low dose of gamma radiation seems to reverse to some extent the damage caused by accelerated ageing of mung seeds and it may be used to enhance the germination characteristics of stored seeds. Soybean seeds (cv. PK-1189), treated with gamma radiation and then sown in the field, gave enhanced yield at the dose of 0.1 kGy. Weight of 1000 seeds was also considerably enhanced over control with the dose of 0.2 kGy. In another study the response of chickpea lines Pusa 362 (desi) and Pusa -1108 (Kabuli) to irradiation (0.01, 0.03, 0.05 and 0.1kGy) was investigated in terms of growth and nodulation characteristic. Kabuli type produced more plant-root and shoot mass, nodule number, mass than desi type. Irradiation inhibited not only the growth but also the nodule characteristics per plant and weight of a single nodule. The inhibition was however, less pronounced for the desi chickpea type. Studies on the effect of 0, 0.05, 0.10, 0.20 and 0.40 kGy on morphological, physiological traits of tomato (cv. Pusa Ruby) exhibited complete inhibition of germination at 0.4 kGy while germination was 2530% higher at 0.05 kGy over un-irradiated control. Total plant biomass was highest at low dosage of irradiation (0.05 kGy) but decreased by more than 50% of control at 0.01 and 0.02 kGy. A similar pattern of variation was observed for other traits like leaf area weight and number and weight of fruits (3 times higher over control). Freshly harvested tomatoes (var. DT39) irradiated with different doses of gamma radiation showed enhanced shelf-life. A dose of 1 kGy was found to optimum in increasing its storability up to 2 weeks with minimum change in its toughness and without any rotting or weight loss. The fruits can be stored up to 4 weeks, however, with significantly less weight loss and rotting than the un-irradiated control. Gamma irradiation with 0.1 and 0.5 kGy gamma rays was effective in suppressing sprout growth of potatoes during storage at 8,12 and 16ºC, it however, resulted in increased weight loss, rotting and accumulation of sugars, which led to browning of chips. There is a need to focus to create genetically modified potato plants where cold and irradiation induced negative effects could be minimized in potato tubers, by more studies on dose-effect relationships. Recent research findings by our group reveal that gamma irradiation can be potentially used as a non-chemical method for degradation of pesticide residues which often accompany vegetables and fruits to our kitchen. IONIZING RADIATION FOR DEVELOPING IMPROVED PLANT TYPES Ionizing radiations can be used to develop mutant lines in specific crops in the following manner and purpose, (i) Induction of mutation for broadening of genetic base of crop (ii) In vitro culture in combination with induction of mutation and, (iii) Anther culture technique with induction of mutation. The object can be irradiated in two ways: (a) With an aid of a powerful source of a short-duration gamma rays for short duration radiation. (b) A much weaker radiation but operating continuously (gamma field). However, while irradiating the tissue, the dosage must be varied depending on the plant species whose seeds/organs are to be irradiated. Plant must be irradiated heavily enough to ensure as many inherited changes as possible but without seriously affecting the germination, growth and fertility of plant directly emerging from the irradiated seeds or vegetative organs (critical radiation dose: dosage which strong enough to assure many mutation not yet so strong as to kill plants. Normally low physiological doses are used for favorably altering a desired trait. At physiological doses, changes may not necessarily occur at the gene level but only the rate of 81 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India certain metabolic processes and reactions are hastened application in improving quality and yield of vegetable that result in enhanced expression of a trait or (Table 3) and fruit (mangoes, banana, pomegranate production of higher amount of new or existing etc) crops. phytocompound. In sexually produced crops, the Work on irradiation effects of gamma on shelf probability of producing desirable mutations and life of cut flowers has not yielded encouraging results genetic variability is theoretically higher in irradiated However, hormetic influence of lower doses of gamma seeds and very young seedling in self than cross at 10 and 15 Gy have been successfully used to achieve pollinated crops. In asexually produced crops, it has improvement in growth and flowering attributes of been much easier and quicker to obtain variant plant chrysanthemum and promising mutants like Brick Red types using ionizing radiations. For this, specific mutant, Fluted mutant, Cup shaped mutant have been location of the mutation event (segmental chimera) developed at IARI. For boosting flower industry and becomes important. The mutant must be in improving consumer interest, it is important to create meristematic tissue that will reproduce faithfully through variability which can be effectively done using gamma cutting or other vegetative means bud, scion, cutting, irradiation. tuber, bulbs. Ionizing radiations have found immense Table 3. Mutants developed using ionization radiation with altered quality attributes Species Lycopersicon esculentum Mutagen Gamma rays, fast neutrons Capsicum annum Gamma rays Phaseolus vulgaris Solanum tuberosum X-rays Gamma rays isotopes Gamma rays Ipomoea batatas and TECHNOLOGY DEMONSTRATION UNITS Changed quality High dry matter and sugar, uniform ripening Higher capsicin content, amino acid composition of seeds From yellow to green podded radio White skinned, fleet eyes from deep eyes. Change in tuber colour, variation in sugar content kCi of cobalt-60. In the year 2004 more than 300 tons of agricultural commodities were processed. These included onion for sprout inhibition and raisins, mango, and spices for disinfestation. It is expected that the technology demonstration units would serve as forerunners for setting up radiation processing plants by entrepreneurs both in private and co-operative sectors. Already, a number of entrepreneurs have signed MoUs with BARC/BRIT for setting up radiation processing units. The department of Atomic Energy has set up two technology demonstration units in India. The Radiation Processing Plant at Vashi, Navi Mumbai, is a 30 ton per day capacity unit capable of delivering medium and high doses. The plant is being operated by the Board of Radiation & Isotope Technology (BRIT) since January 2000 and is currently loaded with 430 kCi of cobalt-60. It is processing more than 1000 tons of spices and other materials annually. Another unit KRUSHAK (Krushi Utpadan Sanrakshan Kendra) at Lasalgaon near Nashik, is primarily a low dose irradiator capable of delivering doses less than 1 kGy to agricultural commodities. The unit is a 10 ton per hour capacity plant built and operated by BARC. It is currently loaded with 30 GAMMA IRRADIATION FACILITY AT THE INSTITUTE Gamma irradiation facility is available at the Nuclear Research Laboratory of this Institute and caters to the students, trainees, researchers, industries 82 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India and farmers from all around the Delhi region The facility available with the laboratory is Gamma Chamber 5000 (12000 Ci, 5 lit volume) and contains 60Co as a radiation source. The laboratory is continuously focusing its research to develop new applications of gamma besides optimization of dose requirements for post harvest preservation of different agricultural crops. The present list of users of this facility is given below: ICAR Institutes  IARI, Delhi  CPRI, Shimla  CRRI, Cuttak  NRC Spices  CSSRI, Karnal State and Agriculture Universities  CSKHP Krishi Vishwavidyalaya, Palampur  PAU, Ludhiana  State Agricultural University, Jammu  Rajasthan Agri-College, Udaipur  AMU, Aligarh  Allahabad Agricultural University, Allahabad  Kisan PG college, Gaziabad  Rajasthan Agricultural University, Bikaner  SGPG College, Meerut  Central Agricultural university, Imphal  Agri-Science Centre, Mathura Private Companies  ITC R and D Centre, Hyderabad  MAHYCO Seed Ltd, Medak, Andhra Pradesh  Shakti Seeds, Hyderabad The vast variety of samples irradiated at our center for varied applications are listed below: Crop/commodity Rose, Chrysanthemum, Jasmin, Carnations Kinnow seeds Jatropha oil Purpose/Activity development of mutants for variability for colour and size Deltamithrine, monocrotophos Induction of seedless ness in Kinnow fruits Induction of useful modification in fatty acids of Jatropha oil to suit biodiesel properties Pesticide degradation potential of radiation Garden Pea Sorghum, Wheat, Rice Pea seeds Mango, Guava Tobacco,Eucalyptus, Babul Potato, Onion Mungbean, Soybean, Chickpea Tissue cultured material In vivo and In vitro mutagenesis for inducing Fusarium wilt resistant Mutational breeding for quality For identifying biotic stress resistant mutants To enhance shelf life and storability Development of better flavour and low lignin Inhibition of Sprouting, chipping quality Post harvest storage For better efficiency of regeneration. COMMERCIAL PROSPECTS Radiation processing of food and agricultural commodities can be undertaken both for export and domestic markets. The technology can be used to overcome quarantine barriers for the export of fruits and vegetables as well as for cut flowers. The estimated cost of setting up a commercial radiation processing facility is in the range of Rs.5-7 crore. Many variables affect food costs, and one of them is cost of processing. Any processing will add to the cost of food. But processing also brings many hidden benefits to consumers in terms of increased availability, storage life, distribution, and improved hygiene of food. It can have a stabilizing effect on market price of commodities by reducing storage losses resulting in increased availability of produce. These factors should therefore be taken in to account while considering the economics. Generally speaking, irradiation costs may range from Rs.0.25 to 0.50 / kg for a low dose application in vegetables and fruits to Rs.1-3/kg for high dose applications such as treatment of spices for microbial decontamination. The costs could be brought down in a multipurpose facility treating a variety of products round the year. 83 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India BIOTECHNOLOGY IN HORTICULTURAL GERMPLASM MANAGEMENT Sandhya Gupta Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, New Delhi E-mail: sandhya@nbpgr.ernet.in, sandhya_gupta87@yahoo.com  The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds  The regeneration of whole plants from plant cells that have been genetically modified  The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens  The production of plants from seeds that may have very low chances of germinating and growing  To clean particular plant of viral and other infections and to quickly multiply these plants as ‘cleaned stock’ for horticulture and agriculture  In vitro techniques are widely used for micropropagation and germplasm storage Plant tissue culture techniques can be applied in various ways that can be divided conveniently into five broad areas, namely: a) Cell behavior b) Plant modification and improvement c) Pathogen-free plants and germplasm storage d) Clonal propagation e) Product formation In vitro collecting Introduction A wide range of biotechnological techniques are being used for efficient management of plant genetic resources viz., tissue culture, cryopreservation, biochemical and molecular genetic marker analysis, immunological diagnosis etc. These techniques are being used for various activities related to management of germplasm such as collecting, characterization, disease indexing and elimination, propagation, conservation, and exchange or distribution. The various techniques related to each of these activities are briefly discussed in this chapter. Plant tissue culture The last three decades have seen a very rapid rise in the number of plant scientists using the technique of organ, tissue and cell culture for management of plant genetic resources. The term ‘plant tissue culture’ broadly refers to the in vitro cultivation of plants, seeds, plant part (tissue, organ, embryos, single cell, protoplasts etc.) on nutrient media under aseptic conditions. Tissue culture techniques are becoming increasing popular as alternative means of plant vegetative propagation. Plant tissue culture involves asexual methods of propagation and its primary goal is crop improvement. The success of many in vitro selection and genetic manipulation techniques in higher plants depends on the success of in vitro plant regeneration. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation that include:  The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits  To quickly produce mature plants in absence of long juvenile phase In vitro techniques have potential for their use in germplasm collecting from the field in nearly aseptic conditions and their subsequent transportation to the tissue culture laboratory for further culture establishment and multiplication. The in vitro collecting has advantages under such circumstances where either the mature seeds are not available at the time of collecting mission, or they are short-lived and bulky, or plants are damages due to disease infestation or 84 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India grazing. The in vitro collecting techniques have been c) Somatic embryogenesis is the formation of a developed for few species (e.g. cacao, coconut and bipolar structure containing both shoot and root cotton), but only been routinely in use for collecting meristem either directly from the explant, from the coconut germplasm. callus and cell culture induced from the explant. Disease indexing and elimination Micropropagation Clonal propagation in vitro is called micropropagation. The word ‘clone’ was first used by Webber for cultivated plants that were propagated vegetatively. Suitable explants from vascular plants including angiosperms, gymnosperms and pteridophytes can be cultured in vitro and induced to from adventitious buds, shoots, embryoids or whole plants. Murashige (1974) outlined three major stages involved in micropropagation.  In vitro grown tissues are normally devoid of any contamination such as fungi and bacteria and other disease causing organisms except viruses and viriods. Meristem-culture techniques alone or in combination with thermotherapy help eliminating virus from various vegetatively propagated crops. For indexing the viruses in plant tissue various techniques such as infectivity test or grafting on indicator plants, Enzyme-Linked Immunosorbent Assay (ELISA), Immunosorbent Electron Microscopy (ISEM) and Polymerase Chain Reaction (PCR) etc. are used. Stage 1: Selection of suitable explants, their sterilization and transfer to nutrient media for establishment, i.e. initiation of a sterile culture of Exchange or distribution the explant. In vitro techniques help multinational exchange  Stage 2: Proliferation or multiplication of shoots of germplasm. Tissue culture-raised plant materials are pest- and pathogen-free thus facilitate distribution of from the explants on media. germplasm with minimum risks of introducing any pests  Stage 3: Transfer of shoots to a rooting media of quarantine importance. In vitro exchange is the safest followed later by plating into soil. and most efficient method for the movement of Plant regeneration can be achieved by culturing germplasm across the quarantine boundaries. The in tissue sections either lacking a preformed meristem or vitro plant material is mostly distributed in the form of from callus and cell cultures. Micropropagation or plant well established proliferating shoot cultures that are regeneration can be grouped into the following easy to multiply for research or for further distribution. Besides, other in vitro explants that may be used for categories: a) Enhanced release of axillary bud proliferation, exchange purposes include shoot apices (sometimes i.e. by multiplication through growth and encapsulated in alginate beads), nodal cuttings, zygotic/ proliferation of existing meristems. It can be somatic embryos, callus, cell suspension, storage through apical shoots excised form the parent organs (e.g. micro rhizome, micro tubers, bulblets) etc. plant (meristem and shoot tip culture) and by Characterization and evaluation multiplication of existing meristems within axillary Characterization and evaluation of germplasm shoots, which proliferate on explants after removal from the parent plant (single node and collection is very important part of germplasm management. Characterization of germplasm can be axillary bud culture). b) Organogenesis is the formation of individual achieved through various techniques such as organ such as shoots and roots either directly morphological, cytological, biochemical and molecular on the explant where a performed meristem is genetic analysis, alone or in combination one another. lacking from callus and cell culture induced from Characterization and evaluation of conserved germplasm is extremely important for its efficient the explants. utilization. 85 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India crop in vitro genebank at NBPGR (Gupta et al., 2002; In the past two decades the significant Gupta and Mandal, 2003; NBPGR, 2008) with the advances made in the plant tissue culture technology conservation period of ranging from 4 to 12 months have paved way to adopt in vitro conservation methods or more (Gupta, 2004). as a complementary strategy to the field genebank (ii) Storage using in vitro slow growth: conservation. Use of in vitro techniques can overcome Conventional method of in vitro short- to the problems many problems associated with the medium-term storage is using slow growth techniques vegetatively propagated crop species. Various in vitro with the aim to extend subculture intervals. Slow growth storage techniques have been applied to a wide range methods are used to reduce the growth rate of plant of fruit species (Ashmore, 1997). An essential pre- tissues, in order to prolong the subculture interval requisite for in vitro conservation is availability of without causing any damage to the tissue. Strategies micropropagation protocol, and that has been for slow growth need to be developed for each species developed for a number of fruit species. Various and even for different genotypes within species as methods are now being routinely followed for the short- responses to culture conditions are quite variable to medium-term conservation of vegetatively (Lynch, 1999). The various methods to achieve slow propagated species, whereas cryopreservation offers growth include one or a combination of the following potential for their long-term conservation. The main approaches: aim in developing an efficient in vitro conservation Reduction in temperature and/or light protocol is to reduce frequent subculturing. There are 1. Effect of enclosures three major in vitro conservation strategies: (i) storage 2. 3. Minimal growth media using in vitro normal growth, (ii) storage using in vitro Inclusion of growth retardants in medium slow growth, and (iii) storage using cryopreservation. 4. 5. Inclusion of osmotica in medium (i) Storage using in vitro normal growth 6. Effect of gelling agents in the medium Modification of gaseous environment Method has been found very useful for slow 7. 8. Encapsulation and desiccation growing, stable cultures for short- to medium- term storage. Cultures are readily available for multiplication (iii) Storage using cryopreservation: and distribution. Besides, it saves inputs on low Cryopreservation refers to the storage of temperature facility especially in tropical region. biological material at ultra-low temperature (-196°C) However, cultures maintained under normal growing in liquid nitrogen (LN). It is the only available method conditions involve frequent subculturing, which is at present for the conservation of germplasm on longlabour intensive, costly and poses risks of losses due term basis. Theoretically, all cellular and metabolic to contamination or error. Studies done at National activities are stopped at this temperature. Thus, the Bureau of Plant Genetic Resources (NBPGR) plant tissue can be stored without alteration for laboratory for the in vitro conservation of various unlimited period of time. Another advantage is that the species showed that after initial fast growth for 3-4 plant material is stored in a small volume, with minimum months cultures entered into stationary phase and were day to day handling and very limited maintenance. It is sustained on limited nutrients available to them for the potentially the most appropriate strategy for long-term next 6-8 months. Using that strategy more than 2000 conservation of in vitro cultures (Benson, 1999; Reed, accessions including 600 accessions of temperate and 2008). Currently, cryopreservation is in routine use tropical fruit species (apple, pear, strawberry, for a wide range of genotypes in several horticultural blackberry, raspberry, blueberry, kiwifruit, mulberry, crops (Ashmore, 1997) using shoot apex, somatic bael, banana and grapes) are being maintained at multi- embryo and dormant vegetative bud as explants etc. Conservation 86 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India germplasm. The application of new biotechnological In the classical cryopreservation techniques the approaches can increase the efficiency of management plant tissue is treated with cryoprotectants and cooled of plant genetic resources in various ways. slowly (0.1-4°C/min.), down to about -40°C followed References by rapid cooling in liquid nitrogen followed by rapid Ashmore S.E. (1997) Status Report on the thawing. This method is performed under Development and Application of In Vitro programmable freezing equipment. This technique is Techniques for the Conservation and Use of used by the National Clonal Germplasm Repository Plant Genetic Resources. International Plant (NCGR) at Corvallis in the USA for routine storage Genetic Resources Institute, Rome, Italy, 67 p. of apices of Pyrus and Rubus (Reed et al., 2000). Benson E.E. (ed.) (1999) Plant Conservation Biology. New cryopreservation techniques: Taylor and Francis, London, UK. New cryopreservation techniques are based de Langhe E.A.L. (1984) The role of in vitro on vitrification-based procedure. These are simple and techniques in germplasm conservation. In: J.H.W. relatively inexpensive methods suitable for routine use Holden and J.T. Williams (eds.), Crop Genetic for plant germplasm conservation (Benson, 1999; Resources: Conservation and Evaluation. Engelmann, 2000; Reed, 2008), involve George Allen and Unwin, London, UK, pp. 163encapsulation-dehydration, vitrification, encapsulation179. vitrification, desiccation and droplet freezing Englemamm F. and Takagi H. (eds.) (2000) techniques. The technique of encapsulationCryopreservation of Tropical Plant Germplasm. dehydration has been applied to apices of several Current Research Progress and Application. genotypes of pear, apple, mulberry, blackberry and JIRCAS, Tsukuba, Japan/IPGRI, Rome, Italy. raspberry (Niino and Sakai, 1992; Gupta and Reed, 2006; Reed et al., 2008; Gupta et al., 2006). The Gupta S. (2004) In vitro conservation of temperate and minor fruit crop germplasm at NBPGR in vitrification techniques have been developed for apple, India. In Vitro Cell Dev. Biol. Plant 40: 418pear (Niino and Sakai, 1992) and mulberry (Niino et 419. al., 1992). Gupta S. and Mandal B.B. (2003) In vitro methods Monitoring of genetic stability for PGR conservation. In: Chaudhury et al. (eds) Monitoring genetic stability of in vitro In Vitro Conservation and Cryopreservation of conserved germplasm is very important because plant Tropical Fruit Species. IPGRI/NBPGR, New tissue may have got somaclonal variation. Efficient in Delhi, pp. 71-80. vitro conservation technique should ensure the Gupta S. and Reed B.M. (2006) Cryopreservation of maintenance of genetic stability of stored germplasm. Shoot Tips of Blackberry and Raspberry by Somaclonal variation can be assessed using Encapsulation-Dehydration and Vitrification. morphological, cytological, biochemical and molecular CryoLetters. 27 (1): 29-42. genetic marker techniques. Use of combination of techniques is recommended for the accurate Gupta S., Mandal B.B. and Gautam P.L. (2002) In vitro and cryorepository of NBPGR. In: Kumar assessment of genetic stability of conserved germplasm. et al. (eds.) Plant Biotechnology for Sustainable It is evident from the literature that the Hill Agriculture. DARL, Pithoragarh, Uttaranchal biotechnology plays an important role in managing the India, pp. 20-25. plant germplasm. Different biotechnological tools are being used for the various activities related to the Gupta S., Kumar M. and Saxena S. (2006) In vitro conservation and cryopreservation of mulberry Classical cryopreservation techniques: 87 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India germplasm. In: Meshram S.U., Shinde G.B., Niino T., Sakai A., Enomoto S. and Kato S. (1992) Shanware A.S., Meshram J.S. and Ingle A.O. Cryopreservation of in vitro grown shoot tips (eds) 2nd Global Biotech Congress 2006 ADof mulberry by vitrification. CryoLetters 13: Memoir Journal. Published by Chairman 303-312. Organizing Committee, Global Sustainable 2nd Reed B.M. (ed.) (2008) Plant Cryopreservation: A Biotech Congress 2006 AD, Nagpur. p 192. Practical Guide. Springer, New york, USA. Lynch P.T. (1999) Tissue culture techniques in in vitro 503P. plant conservation. In: E.E. Benson (ed.), Plant Reed B.M., J. DeNoma and Y. Chang (2000) Conservation Biotechnology. Taylor and Application of cryopreservation protocols at a Francis, London, UK, pp. 41-62. clonal genebank. In: F. Engelmann and H. Takai Murashige T. (1974) Plant propagation through tissue cultures. Ann. Rev. Plant Physiol. 25: 135-166. (eds.), Cryopreservation of Tropical Plant Germplasm. Current Research Progress and Application. Japan International Research Centre for Agricultural Sciences, Japan/ International Plant Genetic Resources Institute, Rome, Italy, pp. 246-249. NBPGR (2008) Annual Report of NBPGR 20072008, NBPGR, New Dehi, 154 p. Niino T. and Sakai A. (1992) Cryopreservation of alginate-coated in vitro grown shoot tips of apple, pear and mulberry. Plant Sci. 87: 199206. Reed B.M., Hummer K.E., Gupta S. and Chang Y. (2008) Medium and Long-Term Storage of Rubus Germplasm. Acta Hort. 777: 91-98. 88 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India POST HARVEST MANAGEMENT OF HORTICULTURAL CROPS FOR DOMESTIC AND EXPORT MARKETING R.K. Pal Division of Post Harvest Technology, Indian Agriclutural Research Institute, New Delhi- 110 012 e-mail: rkpal@iari.res.in, rkrishnapal@rediffmail.com    Root and shoot growth, Seed germination and Fiber development. Temperature surrounding the produce is the single most important factor responsible for maintaining quality of fruits and vegetables after harvest. Storage in refrigerated atmosphere retards the aging due to ripening, softening and color changes. The undesirable metabolic changes and respiratory heat production are also reduced by refrigeration. Temperature control throughout the period between the harvest and Appropriate Post Harvest Management utilization has been found to be the most important (PHM) of horticultural crops fruits and vegetables in factor in maintaining the product quality. their fresh form prolongs their usefulness and in some Loss of water from produce is often associated cases improves their quality. It also checks market glut, with a loss of quality, as visual changes such as wilting, helps in orderly marketing, increases financial gain to shriveling and textural changes. Any method of the producers and preserves quality of produce for increasing the relative humidity of the storage much longer time. Horticultural produce like fruits, environment will slow down the rate of water loss. vegetables and cut flowers are living, respiring tissues The best method of increasing relative humidity is to separated from the parent plant. Therefore, the aim of reduce temperature. Another method is to add PHM is to control various physiological processes viz. moisture to the air around the commodity as mists or respiration, transpiration and other metabolic activities sprays, or by wetting the storeroom floor. Composition to keep produce in maximum usable form. of air inside the storage room or package also plays a Proper control of pre and post harvest crucial role in extending the marketability of fresh fruits diseases, regulation of post harvest physiology, storage and vegetables. In general elevated levels of CO2 and atmosphere, application of chemical treatments, reduced levels of O2 in the storage room or package irradiation and refrigeration of produce may prolong coupled with low temperature are very useful in long term storage of apples, pears with premium quality the storage life of horticultural produce. attributes. The deterioration of fresh horticultural produce Several physiological changes contribute to a after harvest occurs mainly through great extent in limiting storage life of fruits and  Loss of moisture,  Loss in quality due to physiological disorders, vegetables. Sprouting in onion, ginger, garlic and potatoes is one of the most serious causes of  Disease and pest attack,  Loss of nutrients like carbohydrates, vitamins, deterioration. Rooting is initiated by a condition of Ever since the civilization of mankind, efforts have been directed towards accumulating and storing foods when they are in plenty in order to meet needs during the days of scarcity. In case of food grains not much problem was faced due to nature’s noble way of reducing the moisture level as the grains mature. However, in case of fruits and vegetables, long-term storage in their fresh form was not possible (until development of modern methods) primarily due to their high degree of perishability owing to high moisture content of these commodities at the time of harvest. 89 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India visible symptoms of diseases, insect damage and mechanical injury. Deformed fruits and those unmarketable fruits with splits, punctures and rotting along with foreign matter like plant debris, soil or stones are removed during sorting. Even after the produce is sorted and graded in the farm, there may be a further selection before it is packed for quality and size. elevated humidity, which may result in rapid decay, shriveling, and exhaustion of food reserves in roots and tubers. Solanine, a toxic alkaloid is found in green tissues of potatoes, which is a result of exposure of potatoes to light during storage. Green beans and sweet corn may get tough during prolonged storage due to development of spongy tissue. Similarly, a large number of biochemical changes are associated with the changes in colour, texture, flavour and other sensory quality during storage. Wounding stimulates respiration and developmental changes that promotes ethylene liberation and leads to undesirable ripening. Ethylene also stimulates respiration and stress-induced ethylene may have many physiological effects on commodities. Further, the degree of microbial infection at the time of harvest i.e. the primary inoculum load plays a significant role in determining the storage life. There exists an inverse relationship between the primary inoculum load of commodities at harvest and the storage life. Packaging The primary goal of packaging is assembling the produce in convenient units for marketing and distribution. Selection of packaging material depends on (a) influence and demands of different chains right from farm to transportation, wholesale and retail marketing (b) method of packaging (c) contamination or migration of package components to produce e.g. odour, chemicals etc. (d) ease of availability (e) ease of handling and (f) the value for money. The fundamental essence in ideal packaging should envisage: communication, legal information (e.g. weight, Therefore, an integrated approach towards size, contents, use etc.), bar coding, appeal, split post harvest management with appropriate backward second decision making, reliability, consistent quality Linkage at the farm level will go a long way in quality (i.e. sample pack and the consignment pack should assurance of harvested horticultural produce during be the same), in-time delivery and value for money. subsequent storage and marketing (Fig.1) Modified atmosphere packaging (MAP) Harvesting and picking methods Modified atmosphere packaging consisting of Harvesting deals with removal of fruits from reduced O and elevated CO concentration compared 2 2 their parent plant. There are certain guiding principles to air are created either by rapidly flushing the headto be followed before selection of fruits or vegetables space of the package with the desired gas mixture or for harvest. Generally maturity is assessed by subjective by allowing the produce to respire inside the package evaluation of commodities e.g. sight, smell, touch, so that an equilibrium is slowly attained. Extensive morphological changes and resonance. However, continued use of MAP in preservation of horticultural combination of both subjective evaluation and objective produce is anticipated for the future. One of the newest measurement of appropriate parameter (TSS, acidity, trends in MAP is the shrink-wrapping of individual sugar, fat/oil, texture, specific gravity, colour, rate of produce items. Shrink-wrapping has been used respiration, internal ethylene evolution etc) with respect successfully to package apples, mangoes and a variety to particular crop may be more significant for predicting of tropical fruits. Shrink-wrapping with an engineered optimum maturity. plastic wrap can reduce shrinkage, protect the produce Sorting and grading: from disease, reduce mechanical damage and provide Fruits and vegetables show considerable a good surface for stick-on labels. variations in quality due to genetic, environmental and Pre-cooling agronomic factors. Grades are based on size, weight, Pre-cooling is the process of “rapid” removal of field colour, shape, firmness, cleanliness, maturity, and 90 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India heat/respiratory heat usually practiced for fresh fruits, vegetables and flowers immediately after harvest, before shipment, before storage or before processing depending on the commodity. This is the first step of good temperature management. The primary advantages of pre-cooling are: (a) Inhibition of the growth of decay causing organism, (b) restriction of the enzyme activity, (c) reduction of water loss, (d) reduction in rate of respiration and C2H4 liberation and (e) rapid wound healing. The production and action of ethylene from harvested fruits, vegetables and flowers are temperature dependent. Harvested produce kept at 250 C with 30% RH shows a tendency of 36 times more water loss as compared to that stored at 00C with 90% RH. Hence pre-cooling serves as an essential practice in any successful cool chain management of horticultural produce. retention of essential vitamins in fruits and vegetables; uniform ripening of tomatoes and banana even in peak summer months. It is very effective in storing kinnowmandarin up to one month with prime quality attributes. The commercial size (6-8 MT) chamber could be successfully utilized for onion storage during rainy season after withholding the water supply. Controlled atmosphere storage: Atmosphere at ambient conditions comprises of 78.08% N2, 20.98% O2 and 0.03% CO2 under normal conditions. Any deviation from this normal atmosphere composition e.g. elevated level of CO2 reduced level of O2, N2 or any other combination is known as ‘Modified Atmosphere’. When this deviated normal atmosphere is precisely kept under control then it is termed as “Controlled Atmosphere”. This control can be done in package (Controlled There are basically four methods used for Atmosphere Packaging) or in the storage chamber horticultural commodities. These are (i) Room/air CA-storage. Similar is the case for Modified cooling. (ii) Water/ hydro cooling (iii) Forced air- Atmosphere Storage (MA-storage) and Modified cooling (iv) Vacuum cooling and (v) Package icing. Atmosphere Packaging (MAP). Generally, O2 below On-farm storage systems 8% and CO2 above 1% are used in CA-storage. Pusa Zero Energy Cool Chamber is an on-farm Atmospheric modification is a supplementary practice cuboidal storage chamber developed at IARI, New to temperature management in preserving quality and Delhi works on the principle of evaporative cooling. It safety of fresh fruits, vegetables, ornamentals and their can be constructed easily anywhere with locally products throughout postharvest handling available materials like brick, sand, bamboo, khaskhas Essentiality of CA/MA technology should be or straw, gunny bags etc. and its operation needs a justified only if (a) the commodities are having high steady source of water. It consists of a double-walled market value (b) it significantly enhances storage life bricks having cavity in between that is filled up with (c) it retains significantly better quality (d) it fetches fine riverbed sand on all four sides. The bricks are better price compared to conventional cool stored porous enough to allow seepage of water. The water produce. Retardation of ripening, reduction in decay, seeped through the walls and sand matrix gets prevention of specific disorders and maintenance of evaporated, and consequently, reduce the temperature product texture are some of the potential advantages of the cool chamber. Water seeping through inner wall of CA/MA storage. provides necessary moisture in the enclosure, and Beneficial Effects of CA (in optimum commodity consequently, increases the humidity. specific composition): The Major benefit of Pusa zero energy cool chamber Retardation of senescence (including ripening) could be derived for extension of growing period of and associated biochemical and physiological changes button mushrooms with 24% higher yield then i.e. slowing down rates of respiration, ethylene conventional growing; orderly marketing of potatoes; production, softening, and compositional changes. quality assurance through better appeal and high 91 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India  Reduction of sensitivity to ethylene action at O2 levels < 8% and/or CO2 levels > 1%.  Alleviation of certain physiological disorders such as chilling injury of avocado and some storage disorders, including scald of apples.  CA can have a direct or indirect effect on postharvest pathogens (bacteria and fungi) and consequently decay incidence and severity. For example, CO2 at 10 to 15% significantly inhibit development of Botrytis rot on strawberries, cherries, and other perishables.  Low O2 (< 1%) and/or elevated CO2 (40 to 60%) can be a useful tool for insect control during storage of dried products from fruits, vegetables, flowers, nuts and grains. Detrimental Effects of CA (above or below optimum composition for the commodity):  Initiation and/or aggravation of certain physiological disorders such as internal browning in apples and pears, brown stain of lettuce, and chilling injury of some commodities.  Irregular ripening of fruits, such as banana, mango, pear, and tomato can result from exposure to O2 levels below 2% and/or CO2 levels above 5%.  Development of off-flavors and off-odors at very low O2 concentrations and very high CO2 (as a result of anaerobic respiration and fermentative metabolism)  Increased susceptibility to decay when the fruit is physiologically injured by too-low O2 or toohigh CO2 concentrations. Ethylene in post harvest management of horticultural produce: these peaks of ethylene production and respiratory activity. Threshold limit for ethylene action in various fruits varies from the type of commodity, but in most cases it ranges between 0.1-1.0 ppm. The duration of exposure may vary from 12 h to 72 h for initiation of ripening but full ripening may take several days. The effectiveness of ethylene in achieving proper and more uniform ripening depends on three important factors. viz (i) stage of maturity (ii) temperature and RH in the ripening room and (iii) concentration and duration of exposure to ethylene. In general, the ripening of fruits occur at 18-250C, 90-95% RH with 10-100 ppm ethylene and 24-72 h exposure depending on the commodity. Ethylene can be applied through ethylene generator using ethanol or by use of ethrel/ ethephon (2-chloroethyl phosphonic acid). One must remember the fact that ripening of fruits can be initiated by many other hydrocarbons viz. acetylene, propylene etc. But the efficacy of ethylene is considered to be 100 times more than acetylene. The use of calcium carbide for fruit ripening is banned in India vide Rule 44 AA of the Prevention of Food Adulteration Act. Storage disorders and diseases: Nature of post harvest deterioration of horticultural crops can be classified into two major groups viz. (i) Disorders and (ii) Diseases. The type of spoilage, which can usually be identified by microscopic examination or by various specialized techniques are termed as “disease” whereas the types of spoilage that needs the history of the consignment for confirmation are usually grouped as “disorders”. Prompt cooling and maintenance of low temperatures reduces the result of injuries of affecting all these processed. All fruits that ripen in response to ethylene show a characteristic rise in respiratory rate before the ripening phase, called a “climacteric”. Some fruits such as apples, bananas, avocados, figs, mangoes, peaches, pears, persimmons, plums and tomatoes show a sharp peak in ethylene production just before the respiratory burst, and these are called climacteric fruits. On the other hand non-climacteric fruits, such as grapes, strawberries, cherries and citrus fruits, do not show Post harvest diseases: Different types of micro-organisms can cause spoilage in storage houses. However, bacteria and fungi are most common. Bacteria are generally propagated by direct contact between perishable goods or with contaminated surfaces, or else with the water used during treatment before or after storage. Some of these bacteria can be pathogenic for human as well, such as 92 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Listeria monocytogenes, which is sometimes found on certain vegetables. Vegetables are mostly spoiled by bacteria due to their high pH (4.5-7.0) whereas many fruits particularly the acidic fruits (pH < 4.5) inhibits the growth of bacteria but encourage fungal spoilage. A relative humidity less than 94 to 95 % is considered low enough for growth of bacteria. Majority of the soft rots causing bacteria have their optimum temperature for growth around 30oC. Control of Quiescent infection in fruits: i) Strict sanitation in pack house to reduce conidial rot ii) Avoidance of injury iii) Post harvest dips with systemic fungicides a) Benlate @ 100 ppm b) Benomyl @ 100 –400 ppm c) Thiabendazole @ 200 ppm iv) Pre-cooling or cool chain management INTEGRATED POST HARVEST MANAGEMENT Harvesting Processing Contract growing Fresh Marketing Cattle Feed Packing station Others Processed products. Canned, frozen, Dried, Pulps, Beverages, Ketchups, Sauces etc. Sorting, grading Primary processing e.g. trimming, removal of undesirable parts Wastes Wastes: Seeds, peels, Pomace etc. Value added products e.g. Food colours, enzymes, essences etc. Cull/damaged Value added processed products Pre-treatments Waxing, antisprouting fungicide treatment etc. Bulk packaging Unit packaging Palletization Pre-cooling Storage for future marketing Transport for Retail marketing 93 Animal feed Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India STRATEGIES FOR ENHANCING CAROTENOID LEVELS IN HORTICULTURAL CROPS Ajay Arora*, Divya Choudhary and Gaurav Agarwal Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110 012 *Email: romiarora@yahoo.com fruit production and provides livelihood security to thousands of people. The crop is largely grown by small scale and subsistence farmers. With a total production of 14.2 million tones grown on 4.7 million hectares, India is the largest producer of banana in the world, contributing to about 22% to the global production. Concerted efforts through research and development during the past decade have resulted in an appreciable increase in production and productivity of banana in the country. Productivity has increased from 20 tones/ha in 1991 to 34 tonnes/ha in 2000. Productivity reaches 65 tonnes/ha in some states such as Maharashtra. A National Research Centre under the Indian Council of Agricultural Research is working exclusively of banana research. The Centre maintains a rich germplasm bank containing 690 accessions. Due to varied agro-climatic conditions a large number of varieties of banana are being cultivated in various parts of the country. Though more than 20 varieties are being cultivated commercially ‘Dwarf Cavendish’ forms the mainstay of the Indian banana industry due to its high yield, market acceptability, short crop cycle and high economic returns per unit area. The other commercial varieties of banana grown are ‘Poovan’, ‘Njali poovan’ and ‘Rastali’. Short distances are by road whereas the longer ones are by rail but no specialized vans or wagons exist for transporting bananas, post-harvest losses are high. Although there is no standard for grading and sorting, size of the bunch and external fruit appearance determine the quality of the produce. Despite being the largest producer of banana in the world, the Indian share in the export market is negligible. Only next to food security, nutritional security is an important national goal. In India, 63% children (under 5) are malnourished, highest in the world excluding only Nepal and Bangladesh and 33% infants are borne with low birth weight. This is due to inadequate intake of essential nutrients like nutritious proteins and vitamins. According to a survey conducted by National Nutrition Monitoring Bureau (NNMB), major nutritional deficiency among children is protein energy malnutrition (PEM) and vitamin deficiency. The NNMB data revealed that there is a higher degree of vitamin deficiency than PEM. Therefore, we are interested in a long-term goal of making available convenient and cost-effective methods of vitamin fortification through transgenic fruits and vegetables, including specific anti-oxidants. Vitamin A deficiency is considered a priority among global health problems, since it can be related to increased mortality among children and women, particularly in developing countries. The underlying cause of vitamin A deficiency is a lack of vitamin A in the diet. Food sources of vitamin A include animal foods rich in vitamin A (retinol) and plant foods containing provitamin A carotenoids, such as β-carotene, the carotenoid contributing most to vitamin A status. Previous foodbased strategies for decreasing vitamin A deficiency, such as horticultural programs and nutrition education, have focused on the production and promotion of vitamin A-rich foods, including eggs; milk; liver; darkgreen leafy vegetables; orange and yellow fruits and vegetables such as papaya, mango, pumpkin, squash, carrot, and orange-fleshed sweet potatoes; red palm oil; etc. Bananas are an important food for many Banana is one of the most important fruit crops of India, which contributes about 37 per cent of total people in the world. Thus, banana cultivars rich in 94 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India provitamin A carotenoids may offer a potential food source for alleviating vitamin A deficiency, particularly in developing countries. Many factors are associated with the presently known food sources of vitamin A that limit their effectiveness in improving vitaminA status. Acceptable carotenoid-rich banana cultivars have been identified in Micronesia, and some carotenoid-rich bananas have been identified by our group in IARI, New Delhi. Bananas are ideal foods for young children and families for many regions of the world, because of their sweetness, texture, portion size, familiarity, availability, convenience, versatility, and cost. Foods containing high levels of carotenoids have been shown to protect against chronic disease, including certain cancers, cardiovascular disease, and diabetes. Because the colouration of the edible flesh of the banana appears to be a good indicator of likely carotenoid content, it may be possible to develop a simple method for selecting carotenoid-rich banana cultivars in the community. Research is needed on the identification of carotenoid-rich cultivars, targeting those areas of the world where bananas are a major staple food; investigating factors affecting production, consumption, and acceptability; and determining the impact that carotenoid-rich bananas may have on improving vitamin A status. Based on these results, interventions should be undertaken for initiating or increasing homestead and commercial production. In photosynthetic tissues, carotenoids are synthesized within chloroplasts and function as photoprotectants. In non-green plant tissues (such as banana fruit), carotenoids accumulate in chromoplasts. A significant increase in total carotenoids occurs during fruit ripening, which is primarily due to the accumulation of two carotenes. The biosynthesis of these carotenoids is best understood in tomato. Phytoene, the first carotenoid in the pathway, is synthesized from two molecules of geranyl geranyl diphosphate by phytoene synthase (PSY). Subsequently, phytoene is converted to Lycopene and beta-carotene. In this pathway, the first step catalyzed by phytoene synthase is rate limiting and the level of PSY protein is the main factor determining lycopene and beta-carotene production in ripening fruits. Thus, we ultimately aim to introgress the phytoene synthase gene encoding PSY protein, and other related genes, which are specific to ripening fruits from high carotenoid containing species to cultivated elite low carotenoid containing cultivars to increase various kinds of carotenoids in fruits. Genetic stocks are available for use in analyzing genetic variation in carotenoid biosynthesis and accumulation. It is thus possible to assess the relationship between variations in carotenoid content, particularly beta-carotene, and expression of the carotenoid biosynthesis pathway genes, which in turn will help identify key genes regulating carotenoid accumulation. Thus, we would like to study expression analysis of carotenoid biosynthesis pathway genes in banana by utilizing introgression lines (ILs), mutants and wild species by cloning. Impact on global health Given some of the issues associated with the production and consumption of foods that are the current focus of many nutrition programs to alleviate vitamin A deficiency and the lack of success of many of these programs, we suggest that consideration be given to the promotion of carotenoid-rich banana cultivars in communities where they could be readily available and culturally acceptable as human food. Bananas: A major staple food Bananas, a major global food staple is the fourth most important food in the world, after rice, wheat, and maize. The term “banana” as used here includes plantain. Plantains are just types of bananas that are commonly more starchy at ripeness. Bananas are eaten as ripe raw fruit or cooked as a staple food, green or ripe. Plantains are usually eaten cooked. However, the use of the terms differs among countries, indicating that care is needed in communications about bananas and interpretations of studies on “banana” and global “banana” production data. Bananas and plantains have both been classified as fruits in global Food and Agriculture Organization (FAO) reports and FAO Food Balance Sheets, although they are both 95 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India small children. This calculation is based on the assumption that the conversion of dietary β-carotene to vitamin A in the rice would be at the standard ratios (12:1 or 6:1). However, the rice matrix is known to be very digestible. Thus, the bioavailability may be much higher. The β-carotene bioavailability in banana may be higher than the 12:1 or 6:1 ratio, because the banana matrix is almost totally digestible. However, this has not yet been demonstrated. Virtually all people do not choose their food for its vitamin A content but eat what they like and what is most available. People often do not associate the concept of nutrition and diet with illness or good growth; some believe that illness is contracted because of supernatural or other events, making it more difficult to effect dietary change on the basis of a healthier diet. eaten as cooked staple foods. This underestimates their use as staple foods and presents a particular challenge in the interpretation of global data on the production and consumption of these foods. Vitamin A deficiency is common in areas where bananas are grown as a staple food crop. A shift from low-carotenoid to high-carotenoid banana cultivars would lead to increased vitamin A content of the diet and thus possibly lead to improved vitamin A status in those areas. The Micronesian high-carotenoid bananas are well liked for their good taste, and a number of them have been eaten for many years, indicating high acceptability [34] (see also the section on the Karat banana). McLaren suggested that for understanding the cause of vitamin A deficiency, investigation of the staple food eaten is important, particularly since riceeating communities are prone to vitamin A deficiency disorders. Bouis argued that the strategy of improving the vitamin and mineral content of staple foods has several advantages: little behavioral change on the part of the people is required, and large amounts of these foods are consistently eaten on a daily basis by all family members. There has been considerable success in the introduction of orange-fleshed sweet potatoes in parts of Africa, providing an example of a successful shift from a low carotenoid to a high-carotenoid staple food cultivar. Other potential health benefits Epidemiological studies suggest that carotenoid-rich food protects against chronic diseases, including certain cancers, cardiovascular disease, diabetes, some inflammatory diseases, and age-related macular degeneration. Consumption of carotenoid-rich bananas should help protect against those diseases, which are growing problems throughout the world. Increased consumption of carotenoid-rich foods may be more likely if they are promoted for protection against both vitamin A deficiency disorders and chronic Work is also being carried out on plant- diseases, since chronic diseases are more visible in breeding strategies to increase the nutrient density of the community. β-carotene, iron, and zinc in wheat, rice, maize, Bananas are also a good source of energy, cassava, and the common bean. Golden rice is rice vitamin C, potassium, and fiber and therefore provide that has been genetically modified to increase its β- a variety of important nutrients for good health. carotene content. No modified rice normally contains no provitamin A carotenoids and is the staple food Other positive factors related to bananas as a eaten by most societies that have the worst vitamin A crop and a food deficiency. Bananas are a crop that is easy to grow in a tropical climate. They do not require replanting or seed Golden rice was developed as a solution to vitamin A deficiency, but there are questions about the purchase, and they generally bear fruit throughout the acceptability of rice with a different colour. Some year, providing ongoing availability, unlike some other analyses suggest that golden rice is relatively low in crops. Although there is no “perfect banana cultivar,” carotenoid content and could meet only 6% to 12% when pest and disease resistance, yield, taste, and of the estimated vitamin A requirements for infants and nutritional content are taken into consideration cultivars 96 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India can be selected with the most positive characteristics that are suitable for the particular environment. Fungal diseases (e.g., black Sigatoka) threaten global banana production, in particular that of the Cavendish cultivar, but some cultivars, including Karat, have been found to show resistance to these diseases, emphasizing the need to maintain a diversity of banana cultivars. Obtaining planting material may be difficult for rare banana cultivars, but banana tissue culture offers many advantages to ensure that cultivars are maintained free from dangers of the environment, pests, or disease. Tissue culture can provide planting material as pathogen-tested plantlets to be sent all over the world. items to produce beta-carotene. Monsanto produced rapeseed and mustard rich in beta-carotene and, more recently, scientists funded by the Rockefeller Foundation produced a strain of rice - golden rice genetically modified to produce beta-carotene. This may well herald an important strategy for controlling vitamin A deficiency, particularly because rice is the dietary staple of many of the most-deficient populations. Some hurdles need to be surmounted before golden rice or its variants can have an effect. The strains must be able to grow under the varied conditions in countries with vitamin A-deficient populations. The yield and the cost must be attractive to the farmer (or benefit from public sector subsidization). The organoleptic qualities of the rice must be acceptable to the target population (women and children). The beta-carotene needs to be bioavailable, the degree dependent on its concentration in the rice, the matrices to which it is bound, the effect of traditional cooking methods and the amount consumed. Although genetically modified rice could go a long way toward controlling vitamin A deficiency, it will never completely solve the problem. Many deficient populations do not consume rice, and even within traditional riceconsuming countries, some high-risk groups will not be able to afford it. Young children are fed bananas in many cultures because of their sweet taste and soft texture and because children seem almost universally to like them. Bananas have great versatility and are prepared in a variety of recipes, including baby food preparations, puddings, pancakes, and breads. In the Pacific, bananas are mixed with taro or yam, along with coconut cream, and baked in several recipes. Because they can be eaten raw, bananas provide a convenient food for busy mothers and a ready-to-eat, hygienically packed food for the child. Bananas can be considered a healthful “fast food.” Even when ripe bananas are cooked as a staple food, the cooking time is relatively short and they can be prepared without milling or grinding into flour, as must be done with Strategies cereal products. Before embarking on an experimental The colouration of the edible portion of the programme to enhance nutritionally important banana fruit appears to be a good indicator of its carotenoids in crop plants several pre-requisites should carotenoid content. Five grades of colour have been be considered. Confronting these issues at an early identified: white, creamy, yellow, yellow-orange, and conceptual stage will enable achievable aims and orange. Thus, banana fruit colouration might be used objectives to be devised as well as placing proof of in the community to select banana cultivars with the concept approaches on sound foundations for greatest potential for health benefits. However, further subsequent exploitation. One of the first questions to work is needed to verify that colour does consistently be considered is what are the disease states to be reflect the carotenoid content in different banana addressed by dietary intake? Secondly, is there sound cultivars. experimental evidence supporting the health benefits In an attempt to overcome some of the claimed? For example, vitamin A deficiency is a clear obstacles facing conventional fortification, scientists contributor to childhood blindness in Southeast Asia; have begun to genetically modify traditional dietary while strong epidemiological evidence indicates that 97 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India breeding has resulted in a wide range of varieties with different carotenoid profiles many of which await full nutritional exploitation. Pepper (Capsicum) varieties also exist with a diverse range of carotenoid profiles. Recently a variety of cauliflower (Brassica oleracea var. botrytis) with an intense orange-coloured curd, contrasting the white/cream of the wild type has been reported. high dietary intake of lycopene (in the form of tomato products) reduces the onset of prostate cancer in western societies. These two examples also show how geographical and social-economic factors must be taken into account when devising an effective strategy, which can be tailored to enhance a specific carotenoid. However, the health benefits of carotenoid mixtures and their synergy with other phytochemicals in fruits and vegetables must not be overlooked. These factors also have a bearing on the choice of crop. Ideally, the crop plant should be amenable to conventional breeding approaches and/or genetic manipulation, contain an endogenous carotenoid pathway and a known basal profile of carotenoids. It is advantageous, but not essential, for the crop plant to be a staple dietary consistent. Alternatively, production of carotenoids in non-food crops followed by introduction into the food chain as supplements may represent a means of supplying the consumer with desired product, whilst alleviating concerns over consumption of genetically modified (GM) foods. Once the particular carotenoid(s) and crop plants have been targeted there are two strategies available for the enhancement of carotenoids in plants: (1), conventional plant breeding/ varietal differences and (2), genetic engineering (often termed metabolic engineering or genetic manipulation). Genetic engineering of carotenoid biosynthesis in crop plants The advantages of genetic engineering over conventional breeding methods include the ability to transfer gene(s) in a faster and targeted manner. In addition to the transfer of genes and cDNAs from the same species, modern recombinant technologies facilitate the introduction of genetic material from diverse planta and unrelated species such as microorganisms. The main disadvantage of secondgeneration GM crops at present resides with the lack of consumer acceptance of novel foods within the market place. A good starting point prior to performing plant transformations is to determine the profile and levels of carotenoids present in the target crop and/or tissue of interest and compare these with amount of carotenoid required. The latter may be extrapolated from RDAs or related to the amount consumed in an average meal. Such data will help define the engineering approach required. For example, if more of the end product carotenoid is required then a strategy aimed at increasing flux through the pathway is appropriate (e.g. quantitative engineering of the pathway). Typically, amplification of the ‘rate-limiting’ enzyme or more correctly the enzyme with the highest flux control coefficient is the principal target for manipulation. Alternatively, it may be desirable to change the composition of carotenoids or create a new carotenoid pathway in the tissue of interest (e.g. qualitative engineering). Another approach that has been successful, but is difficult to design rationally, is pleiotrophic engineering. In this instance the carotenoid content is altered as a result of manipulations to another pathway or biological process from which crosstalk with carotenoid formation (often normally silent) Conventional plant breeding Over the past 50 years modern plant breeding has successfully increased productivity by pursuing higher yields and better adaptation to biotic and abiotic stress. However, breeding programmes aimed at improving health-promoting phytochemicals (such as carotenoids) have been largely overlooked. Tomato is a case where the effect of genotypic variation on carotenoid content has been studied. Of the nine related species of the genus Lycopersicon six have green coloured fruits with a carotenoid and chlorophyll profile similar to that found in leaf tissue. The two species L. cheesmanni and L. pimpinellifolium yield ripe fruit containing no chlorophyll but b-carotene and lycopene, respectively. These species are the phylogenetically closest to L. esculentum, which has with ripe fruit containing both lycopene and b-carotene. Tomato 98 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India operates. Metabolite/precursor pool sizes, enzyme activities and location, gene expression profiles, carotenoid catabolism, interaction with other isoprenoid pathways and regulatory mechanisms are needed to influence the choice and combination of genes and promoters necessary to manipulate the pathway. Most of the carotenoid biosynthetic genes have now been isolated and characterized from bacteria, fungi, algae and higher plants. Several genes encoding retinoic acid (vitamin A) have also been isolated from animals. Only one gene, fibrillin has been isolated that is associated with carotenoid sequestration and no regulatory genes influencing carotenoid formation have been isolated from plants. A comprehensive selection of biosynthetic genes or cDNAs with diverse homologies but functional identity is advantages to prevent genetic effects such as cosuppression (sense suppression and/or gene silencing). If bacterial or fungal genes are used targeting to the plastid is achieved by creating a chimeric gene, containing a leader sequence fused to the 50 end of the transgene. The choice of promoter is very important, as its properties control the dynamics, level and tissue specificity of expression of gene of interest. From a commercial viewpoint, it is important to be aware of intellectual and tangible property (IP/TP) rights. Being aware of ownership and material transfer agreements (MTA) can save time in the long-term and impact on downstream commercial development. Since the first successful plant transformation in 1988 there has been reports of transformation systems for virtually all crop plants. However, it is important to ensure that the system is applicable to varieties within a genus and will create stable lines. Finally, molecular, biochemical and nutritional methodologies must be in place not only to assist in plant regeneration but direct further rational design of metabolic engineering, assess nutritional potential and implement safety requirements. Future prospects The application of molecular genetics to the biotechnological exploitation of carotenoid formation has facilitated rapid advancements in our understanding of carotenoid biosynthesis and its manipulation in higher plants. The fundamental reaction sequences involved in the biosynthesis of carotenoids are now known and all encoding genes, with the exception of the ahydroxylase, have now been isolated and identified. Quantitative and qualitative manipulations of the pathway have been reported. Despite these significant advances, however, some aspects of carotenoid formation and manipulation in higher plants remain poorly understood. In order to make further progress and attain a state of expertise that will alter the carotenoid content of crop plants at a level that impact on the prevention of human disease states, research must proceed concurrently in fundamental and applied directions. Diverse scientific disciplines as well as emerging technologies need to be used. The manipulation of carotenoid biosynthesis has facilitated our understanding of the pathway and its regulation. It is important to maintain the generation and full characterization of varieties (either transgenic, natural mutant or from biodiversity) in which carotenoid levels are altered. Typically studies have tended to focus on a specific step in the biosynthetic pathway, either at a gene or enzyme level. Such studies remain valuable, but there is a growing need to perform a systems approach to the pathway by determining changes occurring within the whole pathway in response to perturbations arising in response to biological processes and/or manipulation. The advances in targeted metabolite profiling, global metabolomics and mathematical assignment of fluxes will greatly assist in furthering our appreciation of the pathway and its relationship with cellular metabolism and function. Hopefully, identification of limiting substrates and cellular compartments for accumulation will occur. At the level of gene expression transcriptome analysis will provide a valuable insight into regulatory aspects of the pathway and target possible sites for pleiotropic engineering, whilst proteomic studies can assess changes of the protein components that result from alterations to carotenoid content in higher plants. In addition, protein in biosynthetic complexes could be identified providing an insight into the mechanisms of metabolite channeling, a regulatory process that appears to limit the effectiveness of metabolic 99 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India engineering. A common feature of many studies attempting to manipulate carotenoid biosynthesis by amplifying a single enzyme, judged to rate-limiting, has been the relative ineffectiveness of the approach, in part due to the ability of other pathway components to compensate for the fluctuations. These findings have led to suggestions for multiple gene manipulations or the use of transcription factors to facilitate the coordinate expression of the pathway. Such an approach has been used successfully with flavonoid formation. To date, no transcription factor influencing carotenoid formation has been identified, but hopefully techniques such as activation tagging and the Arabidopsis ORF knockout collection will enable putative transcription factors influencing carotenoid formation to be identified. In addition, comparative bioinformatics studies on promoter regions of carotenoid genes may also elucidate common binding motifs involved in carotenoid formation. Finally, the consumer perception of GM crops means that it is important to show conclusively that alterations to carotenoid content in plants have no adverse effects and represent a substantial equivalent. With regard to the perceived health benefits of dietary carotenoids, there are many epidemiological and human supplementation studies underway that should clarify the rather confusing evidence that has been published to date. Similarly, investigations on the relative benefits of pure carotenoid supplements compared to high carotenoid foods are in progress. In all of these cases, extrapolations from animal studies to humans need to be viewed cautiously. Finally, post genomic technologies will allow studiers of human biomarkers that will help to elucidate the precise modes of action of these compounds. References Arora, A, Choudhary, D., Agarwal, G. and Singh, V.P. (2008). Compositional variation in β-carotene content, carbohydrate and antioxidant enzymes in selected banana cultivars. International Journal of Food Science & Technology 43(11): 1913-1921. Arora, A., Sairam, R.K. & Srivastava, G.C. (2002). Oxidative stress and antioxidative system in plants. Current Science, 82, 1227-1238. Bouis, H.E. (2000). Improving human nutrition through agriculture: the role of international agricultural research. Conference summary and recommendations. Food Nutrition Bulletin, 21, 550–67. Cooper, D.A. (2004). Carotenoids in health and disease: recent scientific evolutions, research recommendations and the consumer. Journal of Nutrition 134, 221-224. Fraser, P.D. & Bramley, P.M. (2004). The biosynthesis and nutritional uses of carotenoids. Progress in Lipid Research, 43, 228-265. Romer, S., Fraser, P.D., Kiano, J.W., Shipton, C.A., Misawa, N., Schuch, W. & Bramley, P.M. (2000). Elevation of the provitamin A content of transgenic tomato plants. Nature Biotechnology, 18, 666-669. Stahl, W. & Sies, H. (2005). Bioactivity and protective effects of natural carotenoids. Biochemica et Biophysica Acta, 1740, 101-107. 100 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India FRUIT RIPENING: REGULATION AND MANIPULATION Vijay Paul* and Rakesh Pandey Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110 012 *E-mail: vijay_paul_iari@yahoo.com Fruits have always been an attractive food for humans as they are rich source of energy, vitamins, minerals, antioxidants and dietary fiber etc. Fruits also serve as a protective ingredient in our food due to their health benefits and medicinal properties. Consumption of fruits helps us in protecting from various ailments such as; xerophthalmia, macular degradation, chronic disease states, different types of cancers, cardiovascular diseases and other age-related problems. Medicinal nature of fruits is basically due to their antibacterial, anti-inflammatory, anti-mutagenic, anticarcinogenic and anti-oxidative properties. The discovery of large number of health promoting phytochemicals in fruits has helped in creating awareness about the versatile beneficial effect of consuming fresh as well as processed fruits. The awareness and improvement in the economic status of the people has increased the demand of high quality, fresh and nutritious fruits. The supply, however, remained restricted because the fruits suffer from quantitative and qualitative losses not only between harvest and consumption but even prior to their harvest as well. Therefore, an important goal of the producers, stockiest, supplier, exporters and researchers is to minimize the losses at pre- and post-harvest stages. A fruit is basically a developed ovary and its main biological function is to facilitate seed dispersal. Some important vegetables like; tomato, cucumber, squash, pepper or brinjal are examples of fruits. So, with seed maturation the fruit undergoes many changes and becomes attractive to the agents of seed-dispersal. Some of these important changes include; increase in sweetness, softening, loss of tartness/acidity, breakdown of chlorophyll, synthesis of new pigments and production of highly distinctive flavour and aromas. The sum total of these changes in the fruit is called as fruit ripening. The physiological and developmental processes play central role in determining the postharvest aspects of fruits. In this generalized article, attempt has been made to describe and discuss importance of ripening, regulatory aspects of fruit ripening and how physiological inputs (basic as well as applied) can contribute for better post-harvest management in minimizing the quantitative and qualitative losses in fruits. Fruit production and their post-harvest losses There is gradual increase in the area under cultivation of fruits and vegetables in India (Table 1). As a result of this there is significant increase in the production of fruits and vegetables. Country wise production data of fruits and vegetables indicate that India is the second largest producer after China and it contributes 10 % of total world production of fruits and vegetables (Table 2). Table 1: Change in the cultivation area (000’ hectares) under fruits and vegetables in India (1990-2004) ----------------------------------------------------------------------------------------------------------Commodities 1990-95 1995-2000 2000-04 1990-04 Fruits 483 512 1095 2090 Vegetables 258 915 506 1163 ----------------------------------------------------------------------------------------------------------Table 2: Major producers of fruits and vegetables ----------------------------------------------------------------------------------------------------------Country Production (mt) Percentage of 2003 during 2003 world production ----------------------------------------------------------------------------------------------------------China 483 37 India 128 10 USA 66 5 Brazil 42 3 Turkey 37 3 Itlay 31 2 Spain 29 2 Iran 24 2 Mexico 24 2 Egypt 22 2 ----------------------------------------------------------------------------------------------------------Source: FAO (2004). Statistical Yearbook; FAO, Rome (2005). 101 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Current and projected export of fruits and vegetables (raw and processed) by India is presented in Table 3. Providing the required per capital availability of fruits and vegetables to growing population of second largest populated country of the world is a challenge before us. And this is besides the exploitation of enormous potential of our produce in earning the foreign exchange through the export of these commodities. Current production and projected requirement of fruits and vegetables for India is presented in Table 4. One way to attain the projected goal is by gradually increasing the production itself. But, keeping in view the problems like; availability of cultivable land, degradation of soil, scarcity of water, environmental pollution and climate change – continuous increase in the production will be the most challenging task before the plant scientists. Another available option for sustaining the production status is by lowering the post- harvest losses to a minimum level. In this way, our aims of preserving the natural resources and to stop their over-exploitation can also be partly achieved. Table 3: Current and projected export of fruits and vegetables (raw and processed) by India. Values are in rupees (crores) Commodity Fresh Fruits Fresh Vegetables Processed Fruits and Vegetables 1995-96 2005-06 229.9 301.2 491.6 1120.7 919.8 1093.2 Table 4: Current production (2006-07) and projected requirement (2011-12) of fruits and vegetables. Values are in million tonnes -----------------------------------------------------------------------------------Commodity Production Projected requirements (2006-07) (2011-12) -----------------------------------------------------------------------------------Fruits 59 81 Vegetables 116 185 -----------------------------------------------------------------------------------It is important to note that about 1/3rd of all the fruits produced worldwide are never consumed by humans due to losses at various stages and the losses are generally more in developing countries in comparison with developed countries especially when compared between production and retail sites (Table Percent increase over 1995-96 387.4 205.4 122.4 2011-12 (Projected value) 1804.9 1481.4 1760.7 5). The estimated post-harvest losses of different fruits in developing countries are presented in Table 6. Table 6: Estimated post-harvest losses of fruits in developing countries -----------------------------------------------------------------------------------Fruit Post-harvest losses (%) -----------------------------------------------------------------------------------Apple 14 Banana 11-14 Mango 17-37 Papaya 40-100 Tomato 13-16 Citrus 8-31 Grapes 27 -----------------------------------------------------------------------------------Source: Verma and Joshi (2000). Table 5: Comparative estimate of post-harvest losses of fruits produced in developed and developing countries Location 1. From production to retails sites 2. At retail, food service and consumer sites 3. Cumulative total Developed countries Developing countries Range % Mean % Range % Mean % 2-23 12 5-50 22 5-30 20 2-20 10 7-53 32 7-70 32 Source: Kader (2005). 102 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Such heavy losses therefore provide us an opportunity to look for the aspects associated with the factors influencing the post harvest losses and how we can reduce such losses. First and foremost requirement to achieve this is the holistic understanding of physiological and molecular basis of fruit ripening and senescence along with complete deciphering of mechanisms associate with such processes. Factors affecting quantity and quality of fruits Harvest commodities are still living organs. They continue to respire and lose water as if they were still attached to the parental plant, the only difference being that losses are not replaced in the post-harvest environment. They therefore suffer detrimental changes after harvest. These changes include the utilization of energy reserves through respiration, changes in biochemical composition, textural changes, water loss, and increased ethylene production. Being a living commodity, the quantitative as well as qualitative aspects of fruit are mainly governed by the physiological status of the fruit itself. This status is critical and also of central importance in determining the final quantity as well as the quality at a given time and under prevailing conditions. The physiological status is directly or indirectly influenced by extent of diseases, pests’ infestation and mechanical injuries along with the factors influencing the pre-harvest, harvest and post-harvest aspects of a fruit. Therefore, the understanding of post-harvest physiology of fruit is fundamental in deciding different approaches for controlling the losses. Climacteric and non-climacteric fruits Fruits, in general, show two distinctive respiratory patterns during ripening and on this basis fruits are categorized into climacteric and nonclimacteric groups (Table 7). Climacteric fruits show a dramatic increase in the rate of respiration during ripening and it is referred as climacteric rise. The rise in respiration is either simultaneous or it is just followed after the rise in the rate of ethylene production. The time and intensity of this climacteric peak can be delayed and lowered down, respectively by reducing the rate of respiration and in this way one can enhance the shelf life of fruits. Climacteric fruits can be ripen fully even if they are harvested at green mature stage from the plant. The non-climacteric fruits, on the other hand, can ripen fully only if they are allowed to remain attached to the parent plant because the process of ripening does not occur very fast if they were detached from the plant at green mature stage. Further, non-climacteric fruits do not respond to exogenous ethylene treatment for ripening, except the response of degradation of chlorophyll in citrus fruits and pineapples. The production of ethylene by fruits vary substantially from <0.1 to >100 ml kg-1 h-1. Climacteric fruits, generally, produce a higher amount of ethylene than nonclimacteric fruits. Overall losses are reported to be more severe in climacteric fruits in comparison to nonclimacteric fruits and therefore much of the efforts to minimize the losses and associated physiological studies were directed towards climacteric fruits. Differences in the levels of ethylene between the climacteric and non-climacteric fruits and also among the fruits with in each group are presented in Table 8. Table 7: Classification of fruits based on presence or absence of sharp respiratory peak (climacteric rise) during ripening Climacteric Apple, Mango, Papaya, Guava, Kivi, Tomato, Cherimoya, Banana, Pear, Apricot, Peach, Plum, Avocado, Plantain Non-climacteric Citrus fruits (orange, grapefruit, lemon etc.), Berries (cherry, strawberry, blueberry etc.), Pineapple, Fig, Lychee, Melon, Loquat, Pomegranate, Cucumber, Tamarillo Source: Salunkhe et al. (1991); Kader and Barrett (2003). 103 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Table 8: Endogenous ethylene concentration of regulated programme). Role of some of the important enzymes and their physiological consequence are being selected fruits ------------------------------------------------------------------------------------ presented in Table 10. Climacteric fruit µl/l Non-climacteric µl/l Table 9: Important activities and changes occur during fruit ------------------------------------------------------------------------------------ the ripening of fleshy fruits -----------------------------------------------------------------------------------Apple 25-2500 Lemon 0.1-0.2  Seed maturation Pear 70-80 Lime 0.3-2.0 Peach 1-21 Orange 0.1-0.3  Colour changes Avocado 29-74 Pineapple 0.2-0.4  Rate of respiration and ethylene production Mango 0.05-3.0  Tissue permeability and cellular Passion fruit 466-530 compartmentalization Plum 0.2-0.3 Softening ------------------------------------------------------------------------------------  Source: Burg and Burg (1962).  Carbohydrate composition  Levels of organic acids Climacteric fruit ripening Quantitative and qualitative pattern of proteins Some of the important changes occurring   Development of wax on skin during the course of climacteric fruit ripening are Production of volatiles including flavour volatiles presented in Table 9. Many physiological, biochemical   Abscission and developmental changes (as listed in Table 9) occur during ripening through a coordinated and genetically -----------------------------------------------------------------------------------Source: Pratt (1975). Table 10: Important enzymes and key reactions associated with ripening of fruits Enzyme Polygalacturonase Pectin esterase Chlorophyllase Cellulase and hemicellulases Amylases Polyphenol oxidase, catalase, peroxidase Reaction Hydrolysis of glycosidic bonds between adjacent poly galacturonic acid residues in pectin Hydrolysis of ester bonds of galacturonans in pectin Cleavage of phytol ring from chlorophyll Hydrolysis of cell wall Result Tissue softening Hydrolysis of amylase and amylopectin Oxidation of phenolics Loss of texture and increase in sweetness due to production of sugars Formation of precursors to coloured and polymers leading to undesirable browning Hydrolytic rancidity Production of off-flavour and offodours Loss of nutritional value and increase or decrease in digestibility Loss of nutritional quality Formation of hydrogen peroxide Liberation of phosphates Lipase Lipoxygenase Hydrolysis of lipids Oxidation of lipids Proteases Hydrolysis of proteins Ascorbic acid oxidase Glucose oxidase Phytase Oxidation of ascorbic acid Oxidation of glucose Hydrolysis of phytic acid 104 Tissue firming Loss of green colour Loss of texture Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Ethylene is one of the main regulators of climacteric fruit ripening. Ethylene is supposed to start the cascade of events leading to many interactive signaling and metabolic pathways for the progress of ripening in climacteric fruits [see Fig. 1 (a-b) and its details]. ethylene-signaling pathway. Activation of the EIN3/EIL family of transcription factors induces a transcriptional cascade to establish ethylene responses. b Ethylene receptors. The family is divided into two subfamilies (I and II) based on phylogenetic analysis and the presence of conserved sequences (H, N, G, F, and G) in the histidine kinase domain (gray rectangle). The ethylene-binding domain (input) consists of three transmembrane domains (shaded bars). Subfamily II receptors have an additional putative signal sequence (black bar) preceding the transmembrane domains. All five members of the ethylene receptor family have a GAF domain (black diamond) of unknown function. The receiver domain is indicated by an oval. Conserved histidine (H) and aspartate (D) phosphorylation sites are indicated Source: Etheridge et al. 2006. System 1 and system 2 of ethylene production Two systems of ethylene production have been defined in plants. System I functions during normal growth and development and during stress responses, whereas system 2 operates during floral senescence and fruit ripening. System 1 is autoinhibitory, such that exogenous ethylene inhibits synthesis, and inhibitors of ethylene action can stimulate ethylene production (Fig. 2). In contrast, system 2 is stimulated by ethylene and is therefore autocatalytic, and inhibitors of ethylene action inhibit ethylene production (McMurchie and Others, 1972). Fig. 1 (a-b): Ethylene signal transduction. In the absence of ethylene, the receptors activate CTR1, a negative regulator that suppresses downstream signaling. CTR1 is related to the MAPKKKs, but it is unclear whether CTR1 functions as the first step in a MAP kinase cascade. Downstream of CTR1, EIN3 levels are reduced by proteasome-mediated degradation, involving action of the E3 complex components EBF1 and EBF2. Perception of ethylene results in the inactivation of CTR1 and prevents EIN3 degradation, thus activating the Fig. 2: Differential expression of ACC synthase (ACS) and ACC oxidase (ACO) genes associated with system 1 and system 2 ethylene synthesis during fruit development and ripening in tomato. Autoinhibition of ethylene synthesis during system 1 ethylene production is mediated by a reduction in LeACS1A and 6 (genes of ACS) expression. Autocatalytic ethylene synthesis at the onset of fruit ripening is mediated through ethylene-stimulated expression of LeACS2 and 4 (genes of ACS) and LeACO1 and 4 (genes of ACO) (see text for details). Source: Barry and Giovannoni (2007) 105 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ACC synthase (ACS) and ACC oxidase (ACO) enzymes encoded by multigene families in higher plants, with tomato possessing at least nine ACS (LEACS1A, LEACS1B, and LEACS2-8) and five ACO (LEACO1-5) genes (Barry et al. 1996, Nakatsuka et al. 1998, Oetiker et al. 1997, Vander-Hoeven et al. 2002, Zarem-binski and Theologis 1994). Expression analysis has revealed that at least four ACS (LEACS1A, LEACS2, LEACS4, LEACS6) and three ACO (LEACO1, LEACO3, LEACO4) genes are differentially expressed in tomato fruit (Barry et al. 1996, 2000, Nakatsuka et al. 1998, Rottmann et al. 1991). LEACOl, LEACO3, and LEACO4 are expressed at low levels in green fruit that are in a system 1 mode of ethylene synthesis, but the transcripts of each increase at the onset of ripening as the fruit transition to system 2 ethylene production and response. During ripening, LEACOI and LEACO4 are sustained in expression, whereas the increase in LEACO3 expression is transient (Barry et al. 1996, Nakatsuka et al. 1998). In the case of LEACO1 and LEACO4, ripening-related increases in transcript abundance are largely blocked by treatment with ethylene response inhibitor (1-MCP), indicating that these genes are positively regulated by ethylene. The regulation of ACS gene expression during fruit ripening has been investigated using a combination of ethylene and inhibitor studies together with expression analysis in various ripening mutants (Barry et al. 2000, Nakatsuka et al. 1998). LEACS6 is expressed in wild-type green fruit but rapidly declines at the onset of ripening during the transition to system 2 ethylene synthesis. In contrast, LEACS6 transcripts persist throughout development and ripening in the ripening inhibitor (rin) mutant (Barry et al. 2000). LEACS6 is responsible for low-level ethylene production in preclimacteric fruit (Barry et al. 2000, Nakatsuka et al. 1998). LEACS1A gene may be important in regulating ethylene synthesis during the transition from system 1 to system 2 ethylene synthesis (Barry et al. 2000). LEACS4 is not expressed in green fruit but is induced at the onset of ripening. LEACS2 expression is also induced at the onset of ripening; this induction requires ethylene. Therefore, it seems likely that LEACS1A and LEACS4 are responsible for initiating system 2 ethylene synthesis and that this is maintained by a combination of LEACS2 and LEACS4. Interaction of ethylene with its environment and within the plant system It is important to know that ethylene not only interact with plant but also with its immediate environment. Further, there are many factors and basically it is the combination of these factors which decides the final response of ethylene for a given plant and its parts such as fruit (Fig. 3). Fig. 3: Factors governing plant responses to ethylene. Adapted and modified from: Saltveit (1999). Approaches to regulate the fruit ripening I. Controlled ripening and colour development Regulated and controlled ripening is of great importance in providing the properly ripened fruits as per demand. Climacteric fruits such as; banana and mangoes are harvested well before they are fully ripe to avoid any mechanical injury to the fruit. The fruits are ripened under controlled conditions of temperature, relative humidity and ethylene gas, which facilitate uniform development of colour, texture and flavour for consumption or processing. Ethylene is the most active ripening agent. However, some other sources causing effect equivalent to that of ethylene are also being used. For example, acetylene, generated by mixing water with calcium carbide salt, is also used as ripening agent. However, 106 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Table 11: Typical conditions for controlled post-harvest ripening and colour development by using ethylene and storage temperature of some important fruits. Relative humidity is normally maintained at 85-90% Fruits Avocado Banana Honeydew-melon Kiwifruit Mango Orange Tomato Pear Persimmon Ethylene concentration (ppm) 10-100 100-150 100-150 10-100 100-150 1-10 100-150 10 10 Temperature (0C) 15-18 15-18 20-25 0-20 20-22 20-22 20-25 15-18 18-21 Time (h) 12-48 24 18-24 12-24 12-24 24-72 24-48 24 24 Application for inducing Ripening Ripening Ripening Ripening Ripening Degreening Colour development Ripening Ripening Source: Kitinoja and Kader (1995). it is 100 times less effective compared to ethylene. The typical ripening conditions used for some important fruits are presented in Table 11. Ethephon is used commercially for stimulation of ripening in many fruits including tomato and banana. The major commercial application of ethephon for ripening of fruits is for processing tomatoes. It is important to mention here that ethephon treatment to tomato plants was shown to have adverse effects on the germinability of seeds so, treatment should not be given to the tomatoes destined solely for the seed production. II. Minimizing losses in fruits Response to ethylene depend on several factors such as; type of produce, cultivars, maturity at the time of harvest, temperature and activity of other hormones as already explained in Fig. 3. Quantitative losses (production, reduction in weight and decay due to physiological or biotic factors) and qualitative deteriorations (pre-harvest, reduction in colour, overall appearance, nutritional status, flavour, aroma and texture) can occur any where along the whole chain starting from field, pre-harvest, during harvesting, after harvest, packaging, storage, transportation, distribution, reaching up to the consumer and till it is being finally consumed. In this way, preserving the quantity as well as quality over the entire chain is an onerous task. Important approaches to minimize the losses in fruits are being summarized (Fig. 4) and the suitability of these treatment(s) or approach(es) depends on the type of fruit and the purpose for which the fruit will be used. Manipulating the level, perception and response of ethylene: Ethylene production rates by fresh fruits and the rate of ripening can be reduced if fruits are storage at low temperature, at reduced oxygen level (less than 8%), at elevated carbon dioxide concentration (above 1%) and by avoiding any type of stress (such as; fruit injury diseases incidence, water stress). There are three approaches to regulate ethylene mediated responses. These are by controlling the 1. Exposure 2. Perception and 3. Response of ethylene. Tissue exposure can be avoided by excluding ethylene from the surrounding environment by scrubbing and proper ventilation. Reduction in the levels of ethylene can be achieved by 1. Forcing air through filters of activated charcoal (brominated), 2. Treatment with silver thiosulfate, potassium permanganate (KMnO4) or purafil and 3. Oxidation of ethylene by UV light. KMnO4 is the most accepted ethylene remover used commercially. It oxidizes ethylene into ethylene glycol and it is often incorporated into different carrier materials such as; activated alumina and silica gel. It is also applied in sachets, tubes and blankets during storage and transportation of fresh fruits. 107 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Approaches to minimize the losses in fruits Crop improvement Breeding and transgenics - Traditional breeding - Making use of mutants - Genetic engineering/ transgenic production Pre-harvest Genetic - Selection of suitable variety Climatic - Suitable soil and environmental conditions Cultural - Optimum flowering and fruit setting - Use of hormones and growth regulators to manipulate growth and development - Proper cultural practices - Optimum nutrition - Suitable maturity index - Suitable harvesting method - Optimum harvesting time Post-harvest Post harvest handling - Sorting, grading, curing - Pre-treatments - Pre-cooling - Heating etc. Coating and waxing Storage - Controlled atmosphere - Modified atmosphere - Low temperature - Hypobaric - Modified humidity Avoiding/reducing exposure to ethylene - Ventilation - Use of ethylene absorbers - O3 mediated oxidation of ethylene - UV light for oxidation of ethylene - Low-pressure storage Irradiation Packaging Transportation Controlled ripening Other methods - High intensity pulsed electric field - Oscillating magnetic field - High intensity pulse light - High hydrostatic pressure - Ultrasound - Ohmic heating - Light energy (UV) - Encapsulation Decontamination Preservation Processing and value addition Fig. 4: Available approaches for minimize the losses in fruits. Compiled from: Yahia et al. (2004), Mishra and Gamage (2007). 108 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Damaged or diseased fruits produce more ethylene and this further stimulates deteriorative effects. So, removal of damaged and diseased fruits helps in maintaining lower levels of ethylene. Inhibiting the biosynthesis of ethylene within the plant is also an important way to suppress or minimize the exposure of tissue or fruit to ethylene. Based on this approach, genetically modified fruits with reduced activity of ACC-synthase and ACC-oxidase and over expression of SAM-decarboxylase, SAM-hydrolase, ACCdeaminase and ACC-N-malonyl transferase resulted in reduced biosynthesis of ethylene (Fig. 5). Reduced perception of ethylene has been achieved by the use of CO2 (higher concentrations), silver ions, 1-methylcyclopropene (1-MCP), reduction in temperature and using ethylene-insensitive cultivars. Genetic engineering techniques can also play vital role here by reducing the sensitivity or creating insensitivity towards ethylene by targeting the ethylene receptors (Etr 1, ETR 2, ERS 1, ERS 2 and EIN 4) (Fig. 5). 1MCP (which binds to ethylene receptor) is actively researched and has been approved for use in some fruits in USA. 1-MCP has a non-toxic mode of action and used at extremely low doses (usually in parts per billion) and has almost no measurable residues in food commodities. 1-MCP has been reported to delay or reduce ethylene-induced effects by suppressing the ripening and ripening-related changes in many fruits as compiled by Blankenship and Dole (2004), Watkins and Miller (2005) and Watkins (2006). L-Methionine SAM-synthetase SAM-decarboxylae * SAM ACC-synthase * SAM-hydrolase * ACC-deaminase * ACC ACC-oxidase * deSAM 5-MTA α-Ketobutyrate + NH3 ACC-N-malonyl transferase * N-Malonyl-ACC Ethylene Reducing sensitivity or creating insensitivity to ethylene * ETR1, ETR2, ETR3, ERS1, ERS2, EIN4, RAN1 Ethylene binding / receptor Hindering proper signal transduction * CTR1, EIN2 Signal transduction Hindering the transcription and translational processes * EIN3, ERFs, EILs Transcription and translation Altering the expression/ activity of ethylene responsive genes/ enzymes/ proteins * PG, PME, Exp1, Cel1, Cel2 etc. Response (Ripening) Fig. 5: Ethylene biosynthesis, perception and its response. The symbol denotes the sites which have been targeted through genetic engineering to suppress/ inhibit the production, sensitivity or response/action of ethylene. SAM: S-Adenosyl-L-methionine, ACC: 1Aminocyclopropane-1-carboxylic acid, 5-MTA: 5’Methyl thioadenosine and deSAM: decarboxylated SAM. expression of ethylene responsive genes coding for Ethylene response by the plant can also be enzymes/proteins associated with the process of reduced by reduction in temperature, controlled or ripening (PG, PME, Exp1, Cel1, Cel2 etc., as detailed modified atmosphere storage, using chemicals to inhibit in Table 12 and 13) as shown in Fig. 5. biosynthesis of some enzymes and proteins and by Dip or spray treatments to control fruit ripening: making use of genetic engineering techniques (Table Various plant growth regulators are being used at 12). Molecular studies have also shown that after the various stages of production and post-harvest handing perception, the response related changes can be for delaying ripening and colour alteration. These hindered by targeting the components of signal chemicals can be applied as dip or spray. Some transduction (CTR and EIN 2) and transcription commonly used chemicals and respective effects on factors (EIN3, ERFs and EILs) besides following the some fruits are presented in Table 12. antisense or co-suppression approaches to reduce the 109 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Table 12: Selected work where the ethylene response was altered using genetic engineering techniques targeting the ethylene biosynthesis, perception or other aspects related with fruit softening Target gene/enzyme/protein (A) Altered ethylene biosynthesis Bacteriphage SAM hydrolase Apple ACS/ACO Fruit crop Tomato Apple P. chloraphis ACC deaminase Melon ACO Tomato Melon (B) Altered ethylene perception Tomato ETR4 Tomato Effect Reduced ethylene production Reduced ethylene production, firmer fruits with an increased shelf life Delayed fruit ripening Reduced ethylene production, rind stayed greener during ripening, increased concentration of sucrose and citric acid Reference Good et al. (1994) Dandekari et al. (2004) Klee et al. (1991) Flores et al. (2001) Loss of flowers and early fruit ripening, more sensitive to ethylene Fruits ripen normally Tieman et al. (2000) Smith et al. (1988), Giovannoni et al. (1989), Sheehy et al. (1998), Tieman et al. (1992) Tomato NR (C) Altered fruit softening Tomato PG (polygalacturonase) Tomato Hackett et al. (2000) Tomato Suppression of PG mRNA and protein accumulation, no effect on fruit softening Tomato PME (pectin methylesterase) Tomato Tomato TBG4 (beta-galactosidase) Strawberry PL (Pectate lyase) Tomato expansin (Exp1) antisense Tomato PG x Tomato Exp1 Tomato Suppression of PME mRNA and protein accumulation, no effect on fruit ripening and softening Fruit firmness higher than control fruit Strawberry Significantly firmer fruits Tomato Reduced fruit softening during ripening Jimenez-Bermudez et al. (2002) Brummell et al. (1999b) Tomato Significantly firmer fruits, juice more viscous Powell et al. (2003) Smith et al. (2002) Table 12: Chemicals used for delaying ripening in fruits Chemical Cytokinin, kinetin Benzyladenine Benzylaminopunine Gibberellin Effect Fruit used Delays chlorophyll degradation and senescence Cucumber Delays chlorophyll degradation and senescence Cherry Delays chlorophyll degradation and senescence Sweet cherry Retards maturation, ripening, delay chlorophyll Tomato, kiwi banana, degradation, increase peel firmness, delay citrus accumulation of carotenoids Maleic hydrazide and its Delays ripening Mango, tomato, sapota analogs Delay in the onset of ripening Grape Synthetic auxin and benzothiazole-2-oxyacetic acid (BTOA) IAA Suppression in the activities of major cell wall Banana hydrolasis Source : Modified after Salunkhe et al. (1991). 110 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Genetic transformation of enzymes involves in fruit ripening (influencing activities other than plant hormones): Specialized enzymes catalyzing the changes in fruit occurring during ripening/ senescence were also targeted. By switching off or over expressing genes encoding these enzymes one can achieve either acceleration or inhibition of the process of ripening without affecting any other aspects. For example, it is possible to delay tissue softening by removing genes responsible for cell-wall degradation where as colour changes and carbohydrate metabolism proceed at a normal rate. Likewise, colour changes can be accelerated for better fruit colour without affecting tissue softening. List of enzymes/proteins being targeted to alter selected changes affecting softening, colour and flavour of fruit by genetic engineering is presented in Table 13. Table 13: List of enzymes/proteins being targeted to alter the selected changes associated with ripening by genetic engineering Target (enzyme/protein) Action Result Affecting softening Polygalacturonase (PG) Suppression Decrease in softening Polygalacturonase inhibitors Expression Firmness Pectin methylesterase (PME) Suppression Firmness and slower softening Cellulase (Cel1, Cel2) Suppression Difficult abscission Expansins (Exp) Suppression Nil or positive effect on softening Alcohol dehydrogenase Expression Increase in volatiles and flavour Invertase Expression Increase in flavour and quality Taumatin II gene Expression Increase in sweet proteins Dihydroflavonol 4-reductase Expression Enhanced anthocyanin so colour Affecting fruit colour and flavour Role of plant hormones other then ethylene in application of gibberellins and cytokinins were shown fruit ripening to delay ripening of several fruit species. However, no Auxins: Auxins in general show inhibitory consistent relationship between those growth regulators effect on ripening. Auxins have been reported to act and fruit ripening and senescence has been found. opposite to ethylene response in several processes in Abscisic acid: The abscisic acid (ABA) a dose-dependent manner, but its role during fruit enhanced ripening of both climacteric as well as nongrowth and development sometimes extends during the climacteric fruits. ABA was reported to exert its effect early phase of ripening and therefore it is suspected to by increasing sensitivity of the fruit towards ethylene. be related to the sensitivity of ethylene response. Auxin In all the study where ABA was detected, it was found (especially the IAA) is the main hormone controlling to present substantially in the unripe fruit and so it could ripening in non-climacteric fruit with strawberry fruit play the interactive role with other factors/hormones as a model system. Specific role of auxins in regulating promoting the ripening. As increase in ABA was found the genes involved in cell wall loosening during fruit across the climacteric and non-climacteric categories growth but not during ripening was also reported. of fruits and therefore, ABA may be a dominant Gibberellins and cytokinins: Exogenous ripening promoter in non-climacteric fruits where as auxins have been reported to play negative role. 111 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Polyamines: In general, polyamines influence early fruit development and ripening. They act as antisenescence agents, causing retarded fruit colour change, increased fruit firmness, delayed ethylene and respiration rise, induce mechanical stress resistance and also reduced chilling symptoms. Polyamines are metabolites that utilize a common precursor of ethylene biosynthesis i.e., S-adinosylmethionine. It was believed that polyamine biosynthesis would negatively affect ethylene biosynthesis due to competition for a common substrate and therefore inhibit the ripening process. It was found that exogenous application of putrescine, spermidine and spermine might inhibit ACC-synthase activity in tomato and avocado. However, when endogenous polyamine biosynthesis was enhanced in tomato it did not significantly affect the ripening process. Brassinosteroids: Brassinosteroids (BRs) are a group of plant polyhydroxysterols that have been identified as a class of phytohormones that play diverse roles in plant growth and development. For tomato fruit, exogenous BR application (to tomato pericarp discs) leads to elevated levels of lycopene, lowered chlorophyll levels, decreased ascorbic acid and increased carbohydrate content (brought about by the increased level of an extracellular invertase). Tomato mutant with deficiency in brassinosteroid display delay in ripening severely reduced levels of most carbohydrates with lower dry mass, acid invertase and fruit yield. Methyl jasmonate: Its application found to accelerate ripening (mango) enhanced the flavour volatile production and softening (mango). Post harvest methyl jasmonate application increased firmness and reduced chilling injury in papaya, avocado, grapefruits and bell peppers. Its application in tomato and apple found to increase level of -carotene besides promoting chlorophyll degradation, ethylene production. In tomato, endogenous concentrations of jasmonates increases at the onset of fruit ripening and exogenous jasmonate application stimulates ethylene production and colour change. From the detailed studies carried out in apple and tomato it was concluded that jasmonates are involved in early steps in the modulation of climacteric fruit ripening. There appears to be interaction between jasmonate and ethylene. Convergence of ethylene and jasmonate pathways is at transcriptional activation level i.e., ERF1 (it encodes a transcriptional factor that regulate the expression of pathogen response genes). The expression of this transcriptional factor can be obtained by both i.e. jasmonate and ethylene. It is interesting to note that both signaling pathways (jasmonate and ethylene) are required simultaneously to activate ERF1 expression because mutations that block either of them prevent ERF1 induction by both in hormones either alone or in combination. Conclusions It is well known and also proved experimentally that the consumption of fruits just after their harvest at an appropriate stage is the most appropriate, nutritious and healthy way of consuming the fruits. However, most of the time and for the majority of the people it is simply impossible. The imbalance in the of demand and supply of fresh fruits become more compounded in view of the differences in the requirements of climatic conditions for the growth of different fruits. Further, there is also a wide variation in the ideal conditions for the storage of different fruits. Today the demand of fresh fruits is increasing not only for the locally produced fruits but also for the fruits produced in the different states, countries and even in the different continents. Now, in view of widely known health benefits of fruits their pre and post-harvest management are critical. Approaches to be followed for minimizing the losses at one or different steps starting from production to consumption must be cost-effective, non-toxic and suitable for a given location. Many approaches have been described and discussed in this article with especial emphasis on physiological aspects and background that are being exploited for managing the enormous losses in fruits. In recent time, much understanding at molecular level has been gained and as a result, genetic engineering approaches are being followed to alter the traits and physiological and biochemical events associated with the process of fruit ripening and success has also been 112 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India achieved. The tremendous progress made in the area of gene manipulation allows us to modify ripening of practically all climacteric fruits. However, for most of the fruit, which are tree or woody species, introduction of transgenic cultivars is not economically sound. The future of genetically engineered fruits with delayed ripening depends more on economics than on technology. Consequently, despite significant advancement in cloning ripening-related genes and development of transformation method, no revolutionary change can be expected through this field at least in the next several years. One example to substantiate the above statement is of apple. The existing technologies enable storage of apple for up to one year without a significant loss of their quality and such storage facilities are ample in most of the apple producing countries. So, experimenting the apple crop through genetic engineering for delaying ripening and developing such transgenic would be costly but without any practical relevance. In this way, it would be appropriate that the approach or approaches to be followed for minimizing the losses should be based on location, the fruit and its overall importance, market and the aspects contributing towards the preference of the consumers. Ideally, it is the combination of different approaches/methods that can easily be integrated in to the existing chain of fruit starting from its production in the field and till it finally reaches to the consumer would be the best for minimizing the losses or reducing them at least to an acceptable levels. So, development and/or adoption of appropriate postharvest management practice/s in terms of utilization, storage, processing etc. will not only reduce wastage of most of perishable fruits and vegetables but also help in managing gluts and price destabilization which adversely affect the farmers. References Barry, C.S., Llop-Tous, M.I. and Grierson, D. (2000). The regulation of 1-aminocyclopropane-1carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato. Plant Physiol. 123: 979– 986. Barry, C.S. and Giovannoni, J.J. (2007). Ethylene and fruit ripening. J. Plant Growth Regul. 26: 143159. Blankenship, S.M. and Dole, J.M. (2004). 1Methylcyclopropene: a review. Postharvest Biol. Technol. 28: 1-25. Burg, S.P. and Burg, E.A. (1962). The role of ethylene in fruit ripening. Plant Physiol. 37: 179-189. Ciardi, J. and Klee, H. (2001). Regulation of ethylene mediated responses at he level of the receptor. An. Bot. 88: 813-822. Dandekari, A.M., Teo, G., Defilippi, B.G., Uratsu, S.L., Passey, A.J., Kader, A.A., Stow, J.R., Colgan, R.J. and James, D.J. (2004). Effect of down regulation of ethylene biosynthesis on fruit flavor complex in apple fruit. Transgenic Res. 13: 373-384. Etheridge, N., Hall, B.P. and Schaller, G.E. (2006). Progress report: ethylene signaling and responses. Planta 223: 387-391. FAO (2004). Statistical Yearbook - 2004. FAO, Rome (2005). Flores, F., Martinez-Madrid, M.C., Sanchez-Hidalgo, F.J. and Romojaro, F. (2001). Diferential rind and pulp ripening of transgenic antisense ACC oxidase melon. Plant Physiol. Biochem. 39: 3743. Giovannoni, J.J., Della Penna, D., Bennett, A.B. and Fischer, R.L. (1989). Expression of a chimeric poly-galacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening. Plant Cell 1: 53-63. Barry, C.S., Blume, B, Bouzayen, M., Cooper, W., Hamilton. A.J. and Grierson, D. (1996). Differential expression of the 1- Good, X, Kellogg, J.A., Wagoner, W., Langoff, D., aminocyclopropane-1-carboxylate oxidase gene Matsumura, W. and Bestwick, R.K. (1994). family of tomato. Plant J. 9: 525–535. Reduced ethylene synthesis by transgenic tomatoes expression S-adenosylmethionine hydrolase. Plant Mol. Biol. 26: 781-790. 113 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Hackett, R.M., Ho, C., Lin, Z., Foote, H.C.C., Fray, R.G. and Grierson, D. (2000). Antisense inhibition of the Nr gene restores normal ripening to the tomato Never-ripe mutant, consistent with the ethylene receptor inhibition model. Plant Physiol. 124: 1079-1085. Nakano, R., Kubo, Y. and Inaba, A. (1998). Differential expression and internal feed back regulation of 1-aminocyclopropane-1carboxylate synthase, 1-aminocyclopropane-1carboxylate oxidase and ethylene receptor genes in tomato fruit during development and ripening. Plant Physiol. 118: 1295-1305. Hamilton, A.J., Lycett, G.W. and Grierson, D. (1990). Antisense gene that inhibits synthesis of the Oetiker, J.H., Olson, D.C., Shiu, O.Y. and Yang, S.F. hormone ethylene in transgenic plants. Nature (1997). Differential induction of seven 1346: 284-287. aminocyclopropane-1-carboxylate synthase genes by elicitor in suspension cultures of tomato Jimenez-Bermudez, S., Redondo-Nevado, J., Munoz(Lycopersicon esculentum). Plant Mol. Biol. Blanco, J., Caballero, J.L., Lopez-Aranda, J.M., 34: 275-286. Valpuesta, V., Pliego-Alfaro, F., Quesada, M.A. and Mercado, J.A. (2002). Manipulation of Powell, A.L., Kalamaki, M.S., Kurien, P.A., Gurrieri, strawberry fruit softening by antisense expression S. and Bennett, A.B. (2003). Simultaneous of a pectate lyase gene. Plant Physiol. 128: 751transgenic suppression of LePG and LeExp1 759. influences fruit texture and juice viscosity in a fresh market tomato variety. J. Agric. Food Chem. Kader, A.A. (2005). Future research needs in post51: 7450-7455. harvest biology and technology of fruits. Acta Hort. 485: 209-213. Pratt, H.K. (1975). The role of ethylene in fruit ripening. Facteurs et regulation de la maturation des fruits. Kader, A.A. and Barrett, D.M. (2003). Biology, Centre National de La Recherche Scientifique: principles and application. In: Processing Fruits: Pairs, France, Pp. 153-160. Science and Technology, Vol I (Somogyi, L.P. et al., Eds.). Technomic Publishing Co. Inc., Rottmann, W.H., Peter, G.F., Oeller, P.W., Keller, J.A., Lancaster, Pennsylvania, USA. Shen, N.F., Nagy, B.P., Taylor, L.P., Campbell, A.D. and Theologies, A. (1991). 1 Kitinoja, L. and Kader, A.A. (1995). Small Scale PostAminocyclopropane-1-carboxylate synthase in Harvest Handling Practices: A Manual for tomato is encoded by a multigene family whose Horticultural Crops, (3rd ed). University of transcription is induced during fruit and floral California, Davis, USA. senescence. J. Mol. Biol. 222: 937-962. Klee, H.J., Hayford, M.B., Kretzmer, K.A., Barry, G.F. and Kishmore, G.M. (1991). Control of Saltveit, M.E. (1999). Effect of ethylene on quality of fresh fruits and vegetables. Postharvest Biol. ethylene synthesis by expression of a bacterial Technol. 15: 279-292. enzyme in transgenic tomato plants. Plant Cell Salunkhe, D.K., Bolin, H.R. and Reddy, H.R. (1991). 3: 1187-1193. Storage, Processing and Nutritional Quality of McMurchie, E.J., McGlasson, W.B. and Eaks, I.L. Fruits and Vegetables. VI. 1, Fresh Fruits and (1972). Treatment of fruit with propylene gives Vegetables, CRC Press, Boca Raton. information about biogenesis of ethylene. Nature, Sheehy, R.E., Kramer, M. and Hiatt, W.R. (1988). Reduction of polygalacturonase activity in tomato Mishra, V.K. and Gamage, T.V. (2007). Post-harvest fruit by antisense RNA. Proc. Natl. Acad. Sci. handling and treatments of fruits and vegetables USA. 85: 8805-8809. In: Handbook of Food Preservation (2nd ed.). (Rahman, M.S. Edi.). CRC Press, Taylor and, Smith, C.J.S., Watson, C.P., Ray, J., Bird, C.R., Morris, P.C., Schuch, W. and Grierson, D. Francis Group, Boca Raton, Pp. 49-72. (1988). Antisense RNA inhibition of Nakatsuka, A., Murachi, S., Okunishi, H., Shiomi, S., 237: 235-236. 114 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India polygalacturonase gen expression in transgenic Watkins, C.B. (2006). The use of 1methylcyclopropene (1-MCP) on fruits and tomatoes. Nature 334: 724-726. vegetables. Biotechnol. Advances 24: 389-409. Tieman, D.M., Harriiman, R.W., Ramamohan, G. and Handa, A.K. (1992). An antisense pectin Watkins, C.B. and Miller, W.M. (2005). A summary of physiological processes or disorders in fruits, methylesterase gene alters pectin chemistry and vegetables and ornamental products that are soluble solids in tomato fruit. Plant Cell 4: 667delayed or decreased, increased or unaffected 679. by application of 1-methylcyclopropene (1Tieman, D.V., Taylor, M.G., Ciardi, J.A., Klee, H.J. MCP): http://www.hort.cornell.edu/ mcp/ (2000). The tomato ethylene receptors NR and ethylene.pdf. LeETR4 are negative regulators of ethylene response and exhibit functional compensation Yahia, E.M., Barry-Ryan, C. and Dris, R. (2004). Treatments and techniques to minimize the within a multigene family. Proc. Natl. Acad Sci. postharvest losses of perishable food crops. In: USA 97: 5663-5668. Production Practices and Quality Assessment of Van-der-Hoeven, R., Ronning, C., Giovannoni, J., Food Crops, Vol. 4, Post-harvest Treatment and Martin, G. and Tanksley, S. (2002). Deductions Technology (Dris, R. and Jain, S.M. Eds.). about the number, organization, and evolution of Kluwer Academic Publishers. The Netherlands, genes in the tomato genome based on analysis of Pp. 95-133. a large expressed sequence tag collection and selective genomic sequencing. Plant Cell. 14: 1441-1456. Verma, L.R. and Joshi, V.K. (2000). Post-Harvest Technology of Fruits and Vegetables. Indus Publishing Co., New Delhi, India, Pp. 3-4. Zarembinski, T.I. and Theologis, A. (1994). Ethylene biosynthesis and action: A case of conservation. Plant Mol. Biol. 26: 1579-15970. 115 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India PHYSIOLOGICAL BASIS OF COLOR CHANGES DURING FRUIT RIPENING Pramod Kumar, Madan Pal and Tej Pal Singh Division of Plant Physiology, Indian Institute of Agricultural Research, New Delhi 110 012 Color is a cosmetic indicator of fruit and vegetable quality. It is major factor for appearance of fruit and vegetable, which also encompasses gloss, lesions, and other attributes detected by visual evaluation of the product. In many cases, it serves as an index of physiological maturity, ripeness. Regardless, color and other appearance factor are the primary means of evaluating fruit or vegetable quality and in consumer’s purchase decision. Plant pigments provide the chemical basis for fruit and vegetable’s color. Color changes are important criteria for visual evaluation of advance of senescence, especially in fruits (Goldschmidt, 1980). Thus, an understanding of the physiology of changes in color during fruit ripening and its role in consumer acceptability are critical to maintain or enhance fruit & vegetable quality. Fruit refers to a structure developing from ovary and often other surrounding appendages of the flower. In other words fruit may be defined as the end product of matured ovary. It should be noted that many vegetables including tomatoes, peas, beans, cucumbers, squash, peppers, egg plant and okra are botanically fruits. Fruit ripening is one of the most dramatic events for color changes in harvested fruit. The fruit grows to achieve full size, and that there is a diversity of fruit types among the flowering plants. The green and immature fruit is full size and to cause it to ripen various events must now happen so that it is attractive and rewarding for an animal to carry it off for dispersing the seeds. There are two types of fruits: climacteric and non-climacteric. Climacteric include for example tomatoes, peaches etc. They are capable of generating ethylene, the hormone required for ripening even when detached from the mother plant. Nonclimacteric for example peppers, citrus etc. Commercial maturity is only obtained on the plant (Table 1). Climacteric fruits are autonomous from the ripening point of view and changes in taste, aroma, color and texture are associated with a transitory respiratory peak and closely related to autocatalytic ethylene production. Fruit ripening is commonly observed by a ripening signal i.e. a burst of ethylene production. Ethylene is a simple hydrocarbon gas (H2C=CH2) that ripening fruits make and shed into the atmosphere. Sometimes a wound or infection of bacteria or fungi on the fruit cause rapid ethylene production. Thus picking a fruit sometimes produces the signal it to ripen. This ethylene signal causes developmental changes that result in fruit ripening. Various new enzymes are synthesized because of the ethylene signal. These enzymes include hydrolases to help break down chemicals inside the fruits, amylases to accelerate hydrolysis of starch into sugar, pectinases to catalyze degradation of pectin (the glue between cells), and so on. Ethylene apparently “turns on” the genes that are then transcribed and translated to make these enzymes. The enzymes then catalyze reactions to alter the characteristics of the fruit. The actions of the enzymes cause the ripening responses. Chlorophyll is broken down and sometimes new pigments are 116 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Table 1: Climacteric and non-climacteric fruits Non-climacteric Bell pepper Blackberries Blueberries Cacao Cashew apple Cherry Cucumber Eggplant Grape Grapefruit Lemon Lime Loquat Lychee Climacteric Olives Orange Pineapple Pomegranate Pumpkin Raspberries Strawberries Summer squash Tart cherries Tree tomato Apple Apricot Avocado Banana Breadfruit Cherimoya Feijoa Fig Guanábana Guava Jackfruit Kiwifruit Mamey Mango Melons Nectarine Papaya Passion fruit Peach Pear Persimmon Plantain Plum Quince Sapodilla Sapote Tomato Watermelon Source: Wills, et al., 1982; Kader, 1985 made which causes the fruit skin changes color been shown to decline with the advance of fruit ripening from green to red, yellow, or blue (Goldschmidt, or senescence. Carotenoids are lost at a much lower 1980). During ripening acids are broken down and rate than chlorophyll. taste of fruit changes from sour to neutral. The degradation of starch by amylase produces sugar. This reduces the mealy (floury) quality and increases juiciness (by osmosis). The breakdown of pectin is catalyzed by pectinase that result in a softer fruit. At an extreme, pectin losses may make a fruit “pithy”. Enzymes also break down large organic molecules into smaller ones that can be volatile (evaporate into the Chlorophyll degradation in banana air) and we can detect as an aroma. PIGMENT CHANGES DURING FRUIT RIPENING Fruit ripening is usually associated with changes in color of fruit due to changes in composition of chlorophylls, carotenoids and anthocyanins pigments, which, helps in the visual evaluation of maturity and ripening. During the fruit ripening, there is the visible loss of green color with time due to disappearance of chlorophyll. In apple 75 percent chlorophyll is degraded during ripening. The chlorophyll a/b ratio has 117 Chlorophyll degradation in citrus Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India In maturing citrus fruit the first step in chlorophyll catabolism is conversion of chlorophyll to chlorophyllide in a reaction catalyzed by chlorophyllase (Amir-Shapira et al., 1987). The other product of chlorophyllase action is phytol, which usually accumulates in the lipid globules of gerontoplasts, mostly, in the form of esters. An enzyme called dechelatase is required to remove Mg from chlorophyllide, yielding pheophorbide. chlorophyllide and pheophorbide retain their intact porphyrin ring structures at green (Figure 1). Figure 1: Topographical model of the Chl breakdown pathway of higher plants and chemical structures of Chl and of Chl catabolites. Putative (enzymatic) reactions are indicated with a question mark. Pyrrole rings (A–D), methine bridges (α-δ), and relevant carbon atoms are labeled in Chl (top left). R0 = CH3, Chl a; R0 = CHO, Chl A critical step in the degradation path way opens the ring to generate a colorless straight-chain tetrapyrrole. Two enzymes responsible for this are, Pheophorbide a oxidase (PaO) and red color catabolite (RCC) reductase. The PaO reaction requires O2 and involves Fe, which operates in a redox cycle driven by reduced ferredoxin. PaO uses pheophorbide a but not pheophorbide b as a substrate, so chlorophyll b must be converted to chlorophyllide a pheophorbide a before it can be catabolozed. The bright red bilin compound produced by the PaO reaction is similar to pigments excreted by certain single-celled algae when they are starved of nitrogen or are transferred to heterotrophic conditions. Figure 2: Subcellular compartmentation of the pheophorbide a pathway of chlorophyll catabolism in leaf mesophyll cells. X represents a hypothetical part of the catabolic system thought to be responsible for dismantling the thylakoid pigment-protein complexes and the transport of the resulting chlorophyll molecules to the envelope membrane. (Adopted from Buchnan, et al., 2000). 118 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India This catabolite (RCC) does not accumulate in plants but is immediately metabolized further by RCL reductase, which catalyzes the ferredoxin dependent reduction of a double bond in pyrrole sytem of RCC to produce an almost colorless tetraapyrrole with 4 strong blue fluorescence (flourescent chlorophyll. catabolite; FCC). Studies of the sub cellular location of the enzymes of chlorophyll catabolism have established that chlorophyllase, PaO, and probably Mg dechelatase activities are associated with the gerontoplast envelope, whereas RCC reductase is a soluble plastid protein (Figure. 2). Chlorophyll is thought to be conveyed from the thylakoid membrane to the envelope by a carrier protein. carotenoids. Fruits like mango, tomato, bell pepper and papaya and carrot synthesize β carotene. Tomato and pink flesh guava accumulate lycopene. Genes for three of key enzymes have been cloned from several plant sources. FCC export from the gerontoplast is ATP dependent the final destination of catabolites is the vacuole, which they enter through ATP-binding casette (ABC) transporters. Inside the vacuole, FCC may be conjugated-malonyl and β- glucosyl derivatives have been identified-or otherwise modified. The end products of these modifications are various nonfluorescent catabolites (NCCs), which vary greatly in number and type, depending on the species. Chlorophyll catabolism unmasks carotenoids during fruit ripening: In fruit, chlorophyll loss is accompanied by decreases in carotenoids. Fruits also become brightly’ colored during ripening. In such cases, the loss of chlorophyll unmasked derlying carotenoids as in banana which provide a yellow or orange background against which new pigment accumulate. New carotenoids are synthesized by the isoprenoid pathway (Figure 3). Some of the citrus fruits Figure 3 (a-b):Outline of carotenoid biosynthesis. Sequential are reported to contain more than 100 types of addition of isopentenyl diphosphate (IPP) generates Degree of changes in color and ripening in tomato geranylgeranyl diphosphate (GGPP) by way of several intermediates shown here. Two GGPP molecules combine head to head to make the symmetrical C40 phytoene, which in turn is desaturated to yield lycopene, the bright red pigment of many ripe fruit such as tomatoes. Xanthophylls are the products of subsequent cyclization and oxidation reactions. Color changes in ripening are associated with increased activity of enzymes in the pathway, particularly GGPP synthase, phytoene synthase, and phytoene desaturase. DMAPP, dimethylallyl diphosphate. 119 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Geranylgeranyl diphosphate (GGPP) synthase makes the basic C20 unit from which all carotenoids are constructed. Phytoene synthase condences two molecules of GGPP in to the C 40 carotenoids phytoene. Phytoene desaturase (PDS) activity leads to formation of ζ – carotene, which is desaturated further to generate lycopene, the red pigment of tomato, bell pepper, and similar fruits. The activities of GGPP synthase and PDS increase markedly during ripening of pepper, and the expression of phytoene synthase in tomato is enhanced by treatment with the ripening hormone ethylene (Bramley, 2002). condensed tannins accumulate, DFR is subsequently down-regulated and then is strongly up-regulated again as fruit color develops. The pigments of grapes are anthocyanins (red, blue, purple and black) modified by the attachment of a molecule of glucose. Five anthocyanins – cyaniding, peonodin, petunidin and malvidin- make up the basic parts of grapes. Among vegetables, anthocyanins are responsible for red (radish, red cabbage), purple (eggplant). Betalins provide the distinct red and yellow coloration associated with beets. Carotenoids synthesized in the plastid of ripening fruits are concentrated in structures variously described as fibrils, crystals or globules, which become increasingly numerous as chloroplasts redifferentiate into chromoplasts. Fibrillins, specific protein associated with the carotenoid bodies of ripening fruit tissues, have also been detected on the surface of plastoglobuli, the lipid droplets that accumulate in the plastids of mango fruits. CHRC, a cDNA encoding a carotenoidassociated protein with marked sequence similarity to fibrillin, has been cloned from several plant species. These proteins may have a role in storing lipids, transporting newly synthesized or modified carotenoids, or stabilizing plastid structure in fruits, leaves, and other colored organs. Primary metabolism Calvin cycle Shikimate Phenylalanine Secondary metabolism Lignins Flavones Phenylalanine ammonia lyase Cinnamate Coumarins Chalcone synthase Chalcones Flavones Flavnoids Phenylpropanoid metabolism alteration during fruit ripening: Flavanones Dihydroflavonols Dihydro -flavonol reductase Phenylpropanoid pathways are complex, branched metabolic sequences with several control points (Figure 4). Phenylalanine ammonia lyase (PAL) is a key early enzyme in phenylpropanoid metabolism. Isoflavonoids Isoflavans Isoflavones Coumestans Pterocarpan (DFR) Flavan-3,4- diols Condensed tannins Anthocyanins Many of the striking red, purple, and yellow pigments of vegetables and fruits are anthocyanins, betacyanins, and flavanoids- water-soluble phenylpropanoid derivatives that accumulate in cell vacuoles. The pigments of ripe strawberry fruit likewise include anthocyanins and proanthocyanins. Expression of the branch-point enzyme dihydroflavonol 4reductase (DFR) varies during the development of strawberry fruit. Active in early development as Figure 4: Phenylpropanoid metabolism. Phenylalanine, a product of the shikimate pathway enter secondary metabolism by way of the reaction catalyzed by phenylalanine ammonia lyase (PAL). Among the phenylpropanoid, anthocyanins are responsible for some of the vibrant pigments of ripening fruits and senescing leaves. Anthocyanin production depend dihydroflavonol 4-reductase (DFR), a developmentally regulated enzyme that is active during the ripening of fruit like strawberry. 120 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Before enhancing the color of a fruit or vegetable, it is important to understand the role color plays in the quality of the product. To date, unfortunately, an ideal color standard does not exist for most fruits and vegetables. Though, for some crops, charts have been developed to aid in color grading. A rational starting point would be to understand the consumer preference. Since, color is the first characteristic a consumer observes in evaluating the fruits and vegetables. Physiological basis of changes in color may provide some potential for genetic manipulation of specific fruits and vegetables for better color. The most promising work to date has been started to under stand the genetics of carotenoids biosynthesis in tomato fruits (Hamilton et al., 1995; Bramley, 2002). Progress has also been made in understanding the genetics of anthocyanin accumulation in flowers (Quattrocchio et al., 1993) but, in context to genetic manipulation the studies are inadequate for the improvement of fruits and vegetables, particularly with respect to anthocyanins (Francis, 1989). Further, in rush to manipulate genes for improved quality, the importance of preventing losses due to to post harvest deterioration of fruits and vegetables must not be underestimated. Quality management must be emphasized to deliver a fruit or vegetable with color, flavour and texture to meet consumer expectations. Suggested Readings Buchnan, B.B., Gruissem, W. and R. L. Jones (2000). Biochemistry and Molecular Biology of Plants. Publisher I.K. International Pvt. Ltd., New Delhi pp. 1057-1062. Francis, F.J. (1989). Food colorant: Anthocyanins. Crit. Rev. Foo Sci. Nutr. 28: 273-314. Goldscmidt, E.E. (1980). Pigment changes associated with fruit maturation and their control. In: K.V. Thiman, ed. Senescence in Plants. Boca Raton.PL:CRC Press, pp 207-218. Grieson, D., and A. Kader (1986). Fruit ripening and quality. In: Atherton J. Ruddich J., eds.. The tomato crop: a scientific basis for the improvement. London: Chapman and Hall. 241280. Hortensteiner, S. (2006). Chlorophyll degradation during senescence. Annu. Rev. Plant Biol.., 57:55-77. Hamilton, A.J., R.G. Fray and D. Grieson. (1995). Sense and antisense inactivation of fruit ripening genes in tomato. Curr. Tropics Microbiol. Immunol., 197:77-89. Mazza, G. and E. Miniati (1993). Anthocyanins in fruits, vegetables, and grain. CRC Press, Boca Raton, Fl. Ranjan, R., S.P. Bohra and M.J. Asija (2001). Plant Senescence: Physiological, Biochemical and Molecular Aspects. In: Studies in Plant Physiology Series No.1. Publisher, Agribiose (India). Amir-Shapira, D., E.E. Goldschmidt and A. Altman (1987). Chlorophyll catabolism in senescing plant tissues: in vivo breakdown intermediates suggest different degradative pathways for Citrus fruit and parsley leaves. Proc. Acad. Sc. USA Quattrocchio, F., J.F. Wing, H.T.C. Leppen, J.N.M. 84:1901- 905. Mol and R.E. Koes. (1993). Regulatory genes Bartz, J.A. and J.K. Brecht (2003). Post harvest controlling anthocyaninis pigmentation are Physiology and Pathology of vegetables. Inc. functionally conserved among plant species and Mercel Dekker, Inc., New York. have distinct set of target genes. Plant Cell, 5:1497-1512. Bramley, P.M. (2002). Regulation of carotenoids formation during tomato fruit ripening and development. J. Exp. Bot., 53: 2107-2113. 121 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ELECTROMAGNETIC ENERGIES FOR POST HARVEST PRESERVATION OF SEEDS Anjali Anand Nuclear Research Laboratory, Indian Agricultural Research Institute, New Delhi 110012 Crop yields can be maximized by establishment of an adequate and uniform plant population for which good quality seed is a pre requisite. What the plant will be and what results we will get depend upon the quality of the seed. The gains from other agronomic inputs are drastically reduced if the seed is of poor quality resulting in a poor stand. Seed quality encompasses several important attributes of seed that present planting performance. Although all seed quality components are important, the single most recognized and accepted index is germination. is widely practised due to low cost, fast speed in processing and ease of use. However, concerns have been raised about the health hazards of chemical pesticides and its environmental pollution leading to research on the ionizing radiations, controlled atmosphere, cold treatment, conventional hot air, and radiofrequency and microwave dielectric heating for preservation of seeds, grains and other agricultural produce. Deterioration of seeds by insects and mites is a serious problem, particularly in warm and humid climates. Storage fungi cause more seed spoilage even in situations where insects and rodents can be effectively controlled. They can tolerate low levels of moisture and can survive well in stored seeds. Storage fungi not only reduce seed germination but also cause discolouration of seed, production of mycotoxins, heating and caking of seed and decay in final stages of deterioration. internal damage caused by heat over long exposure times including peel browning, pitting and poor colour development. All these methods are accompanied by certain unavoidable drawbacks therefore quest has been on for newer methods of food preservation with least change in sensory qualities. The potential of other alternative techniques like exposure of seeds to certain electromagnetic radiation for enhancing germination and post harvest protection of seeds from insects and pathogens has been explored. Radiofrequency (RF) and microwave (MW) heat treatments have been proposed to reach the same level of insect mortality in a shorter time than chemical treatments. Nelson (1996) summarized research on the susceptibility of various stored grain insect species to RF and MW treatments. Ionising radiation like gamma rays, high energy electrons and X rays are used to sterilize, kill or prevent The potential energy of self-preservation in emergence of insect pests in food. Due to the high seeds differs at different stages of development. During initial investment to establish irradiation facilities and the harvest collection, seeds also contain different disposal of radioactive wastes this technology has not energy levels, and not all planted seeds actually grow been widely favoured or accepted. into plants. Maintenance of seed quality in storage from Controlled atmosphere (O below 1% and time of production until it is sown is imperative to assure CO above 20%) and chilled aeration2 have been used 2 its planting value. Prolonged storage of seed can lead to control stored product insects in grains. Forced hot to gradual loss of vigor and finally loss of viability due air is used as an alternative treatment to the chemical to attack by insects, mites, fungi. fumigation. This method is limited due to external and Various methods of post harvest seed storage and food preservation have been practised since time immemorial. Post harvest preservation of seeds and grains is achieved with fumigation with methyl bromide or contact treatment with an appropriate pesticide. This 122 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India The Federal Commissions allocated five frequencies of RF and MW for industry, scientific and medical applications i.e., 13.56, 27.12 and 40.68 MHz for RF, 915 and 2450 MHz for MWs. The short time exposure to RFs and MWs makes it possible to design continuous treatment of large quantities of products in a short period of time. This reduces the labour cost, use of space and results in less mechanical damage. These processes are safe to operators and have little impact on the environment. Rincker et. al. (1957) observed that the germination of hard seed alfalfa increased by RF treatment, compared to hot air oven method and infra red treatment. Nelson and Walker (1961) determined the maximum level of treatment tolerated by wheat seed at different moisture contents, without affecting seed germination. Stone et al. (1973) worked on saw ginned cotton seeds and found that due to RF treatment the field emergence capability of seeds increased from 50-76%. biochemical terms. The structure and processes of various organisms may be modified under the influence of electromagnetic fields. The influence of geomagnetic field on the growth of plants was established by Louis Pastuer in 1860 as he discovered that earth’s magnetic field had a stimulating effect on fermentation process. The first patent for stimulation of plant growth using magnetically treated seeds was given to Dr. Albert Roy Davies in 1950. Besides other physical treatments like electrical and laser rays that help in increasing the vigour of the seeds and consequently the development of the plant, magnetic treatment is also one of the physical techniques that may be used as a presowing treatment to stimulate plant growth. This technique helps in enhancing germination rate, thus decreasing seed rate per hectare and also fulfilling the requirement of being an environmentally friendly technology. Post harvest loss of seed viability may be overcome to some extent by magnetic treatment of the seeds. Evidence for the biostimulation of seeds on pre sowing exposure to magnetic field dates back to the work of Pittman (1962) when he showed that winter wheat matures 4 to 6 days earlier when seeded in rows oriented north to south than in east and west. He further showed in 1965 that speed of germination and seedling growth of corn and beans were positively affected by pre germination exposure to magnetic field. Boe and Salunkhe (1963) observed that tomatoes placed in magnetic field ripened faster than controls. Bhatnagar and Deb (1978) studied the effect of pre-germination exposure of wheat seeds to magnetic field. Magnetically treated seeds at a field of 500 to 3000 Oersted showed higher respiratory quotient to alpha amylase activity as compared to control seeds. Gubbels et al. (1982) observed that seed lots of flax (Linum usitatissimum L.), buckwheat (Fagopyrum esculentum Moench.), sunflower (Helianthus annuus L.) and field pea (Pisum sativum L.) exposed to a magnetic field produced earlier and more vigorous Living organisms have electrical and magnetic seedlings growth in some seed lots and increased the properties but it is difficult to present them in yield of sunflower. Saktheeswari and Subramanyam (1989) reported that there was an increase in the Nelson (1976) exposed seed lots of alfalfa, red clover and ladino clover to MW and it was concluded that there was a substantial increase in germination. Ponomarev et al.(1996) studied the effect of electromagnetic radiation of the microwave range on the germination of cereals (winter and spring wheat, spring barley, oats) and noticed increased germination in all the tested seeds, the optimal stimulating effect being reported at 20 min exposure. Aladjadjiyan (2002) measured the effect of microwave radiation on the germination and germinating energy of seeds of Gledistichia triacanthos and Robinia pseudoacacia and reported that MW irradiation increases the germination energy and germination proportionally to the output power. Subbarao (1979) evaluated the effect of frequency and power of microwaves on germination percentage of seeds. He observed that lower power time constant treatments increased the percentage germination of all varieties. Most of the seeds responded well with 9.535 GHz. 123 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India number of parenchymatous cells in the root and leaf of paddy, the root hairs were more in number and increased cell division in roots. Exposure of the seeds to pulsed magnetic field with their germination tip in east and south was more beneficial than other orientation. Alexander and Doijode (1995) found that onion and rice seeds exposed to weak electromagnetic field significantly increased germination and seedling vigor. Phirke (1996) reported that a magnetic field strength of 0.1 Tesla and exposure time of 25 minutes was optimum to increase yields of soybean and cotton by 46% and 32 % respectively. Harchand et al. (2002) reported that field trial (100 gauss) with 40h exposure indicated an increase in plant height, seed weight per spike and yield of wheat. Gallard and Pazur (2005) explained the magnetic response on the basis of two hypothesis; a) radical pair mechanism- This consists of modulation of singlet- triplet interconversion rates of a radical pair by weak magnetic fields b) ion cyclotron resonance- this is based on the fact that ions should circulate in a plane perpendicular to an external magnetic field with their Larmor frequencies which can interfere with an alternating electromagnetic field. Seeds of chickpea, maize and sunflower were exposed to static magnetic field of varying strength and exposure time in our laboratory. Magnetic field enhanced seed performance in terms of germination, speed of germination, seedling length and seedling dry weight significantly compared to unexposed control. Root characteristics of the plants showed dramatic increase in root length, root surface area and root volume. CONTROL CONTROL 500 Gauss 1000Gauss 2000Gauss Fig. 2. Effect of magnetic field on root growth of one month old maize plants Concern about health hazards of chemical pesticides in 1950s through 1970s stimulated studies on the use of RF and MW for controlling stored grain and other stored product insects. Differences among various stored grain insect species in their susceptibility to RF dielectric heating exposures have been noted when they were treated in common host grains under similar conditions (Nelson et al. 1966; Nelson, 1973). Anglade et al. (1979) found differences between developmental stages within species of insects exposed to RF. In general, the adult stages were more susceptible to RF treatment than immature stages. Larvae of cadelle were more susceptible to 39 MHz exposures than the adult of this species (Nelson et al. 1966). The electric field intensity to which insects are subjected depends on geometric and spatial factors as well as the dielectric properties of the insects and their host medium. Host medium particle size in relation to insect dimensions is expected to influence lethal exposure levels. RF frequencies between 10 and 90 MHz have achieved control of insects treated in grain and grain products by exposures that raised the grain temperature to about 60 to 65 oC . Orset and Raghavan (1996) observed wheat seed infection by Fusarium graminearum lowered seed germination and quality of the harvest. They optimized the RF treatment for which the fungus mortality is maximized while conserving 70 to 80% of the germination quality of the seeds. TREATED Fig. 1. Effect of magnetic field (2000 Gauss, 1h) on growth of one month old maize plants The burgeoning concern about environmental hazard due to use of traditional methods for the control of pests in seeds and grains makes it pertinent to 124 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India address the problem through alternative physical Nelson, S.O. (1966). Electromagnetic and sonic energy for insect control. Trans. of the ASEA, methods. Electromagnetic radiation provide a promise 9: 398-403 in this direction as it reduces the labour cost, use of space, mechanical damage and is environment friendly. Nelson, S.O. (1973). Insect control studies with It will reduce/prevent post harvest and storage losses microwaves and other radiofrequency energy. in the commodities by cutting down insect infestation. Bull. Entomol.Soc. Am., 19:157-163. References Nelson, S.O. (1996). Review and assessment of radio-frequency and microwave energy for stored Aladjadjiyan, A. (2002). Influence of microwave grain insect control. Trans. of the ASEA, 39: irradiation on some vitality indices and 1475-1484. electroconductivity of ornamental perennial crops. J. Central European Agriculture, 3: 271-276. Nelson, S.O. and Walker, E.R. (1961). Effects of radio-frequency electrical seed treatment. Alexander, M.P. and Doijode, S.D. (1995). Agricultural Engineering, 42: 688-691. Electromagnetic field, a novel tool to increase germination and seedling vigor of conserved onion Nelson, S.O., Ballard, L.A.T., Stetson, L.E., (Allium cepa L.) and rice (Oryza sativa L.) seeds Buchwald, T. (1976). Trans. of the ASEA, 19: with low viability. Plant Genetic Resources 369-371. Newsletter No. 104: 1-5. Orset, V. and Raghavan, V. (1996). Radiofrequency Anglade, P., Cangardel, H. and Lessard, F.F. (1979). treatment of seed quality wheat infected with Application des O.E.M. de haute frequence et Fusarium graminareum CSAE/ SCGR des micro-ondes a la desinsectisation des den Processing Papers. rees stockees. In Proceedings of Microwave Power Symp. 1979 Digest (XIV Symp. Int. sur Phirke, P.S., Kubde, A.B. and Umbarkar, S.P. (1996). The influence of magnetic field on plant growth. les Applications Energetiques des MicroSeed Sci and Technol. 24: 375-392. ondes), 67-69. Monaco, 11-15 June. Boe, A. and Salunke, D.K. (1963). Effects of magnetic Pittman, U. J. (1962). Growth reaction and magnetotropism in roots of winter wheat. Can. J. field on tomato ripening, Nature. 199: 91. Plant Sci. 42: 430-436. Bhatnagar, D. and Deb, A.R. (1978). Some effects of pregermination exposure of wheat seeds to Ponomarev, L.I. (1996). In: Aladjadjiyan, A. (2002). Influence of microwave irradiation on some magnetic field. II. Effects on some physiological vitality indices and electroconductivity of process. Seed Research, 6: 14 -22. ornamental perennial crops. J. Central Gallard, P. and Pazur, A. (2005). Magnetoreception European Agriculture, 3: 271-276. in plants. J. Plant Res. 118: 371-389. Rincker, C.M. (1957). Bull. No. 352, Wyoming Agril. Gubbels, G. H. (1982). Seedling growth and yield Expt. Station. response of flax, buckwheat, sunflower and field pea after preseedling magnetic treatment. Stone, R.B. and Chiristiansen, Nelson, S.O., Webb, J.C., Goodwonge, J.L., Stetson,L.E. (1973). Canadian Journal of Plant Science 62: 61-64. Crop Sci., 13(2) : 159-161. Harchand, K.S., Narula, V., Raj, D. and Singh, G. (20020. Effect of magnetic fields on germination, Subbarao, Y.V. (1979). Effect of microwaves on the seed germination and plant growth. M.Sc Thesis, vigor and seed yield of wheat. Seed Res. 30: 289Delhi College of Engg., New Delhi. 293. 125 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ADVANCES IN PACKAGING TECHNOLOGY OF FRUITS AND VEGETABLES Manish Srivastav* and S. K. Jha** *Division of Fruits and Horticultural Technology, ** Division of Post-harvest Technology Indian Agricultural Research Institute, New Delhi-110 012 to understand a product characteristics or biology of India is fortunate to have suitable agro-climatic the produce. The following important product conditions for the production of various fruits and characteristics are to be considered. vegetables and hold second position in the world in a. Respiration terms of production. Despite having such a huge Fresh horticultural produce respire even after production in the country, our share in the world trade harvesting. During this process oxygen is used and CO is very less. This clearly indicates that the quality of is released. The rate of deterioration is proportional to2 produce in terms of appearance, colour, size, weight, the high respiration rate. Rapid respiration result in fast etc. are not up to the level of international standards. ripening of the produce. If the availability of oxygen is Moreover, there has been inadequacy for post-harvest restricted, it results in changes in chemical reactions handling facilities, transportation and improper an breakdown of cells, Production of small quantity of packaging for the horticultural produce. In fruits and alcohol, off flavor and colour and finally bring about vegetables post-harvest physical and qualitative losses the decay or spoilage of the fresh produce. are very high. The post harvest losses are estimated to be 25-30% of the value of the produce depending b. Moisture upon the perishability of the product. Fruits and vegetables have very high moisture Packaging fresh fruits and vegetables is one content of 75 to 95%. Under ambient conditions, loss of the important steps in the long and complicated of moisture causes rapid drying of the product causing journey from grower to consumer. Packaging play a wilting, shriveling and loss of rigidity. Loss in moisture vital role not only for food preservation and protection results in rapid weight loss during storage and during storage, handling, transportation and distribution transportation. but has assumed a multifunctional role by serving as a c. Micro-organism symbol of value addition, and assurance of quality and The surface of fruits and vegetables might get quantity/ number and also a conveyor of convenience and thus an instrument or tool for marketing. In recent bruise or injury during handling and transportation. The years, the importance of packaging has been increased MO yeast, bacteria and mould invade through this injury to a great extent. This is mainly due to increase of and cause internal decay. consumer awareness and willingness to pay for hygienic d. Changes in colour, texture and flavour and value added package. During normal ripening alterations takes place Product characteristics in colour, texture and odour and flavor of the fresh Horticultural products are highly perishable in produce and hence the produce should reach the nature and are very easily affected by climatic consumer at proper ripening stage. Introduction conditions, distribution hazards and microbial decay. e. Temperature In order to develop a suitable package t is important The process of respiration is dependent on 126 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India temperature. Therefore, it is necessary to slow down the reaction by storing the fruits under refrigeration. Very low temperature causes chilling injuries which damage the delicate tissues of fresh fruits and vegetable and must be avoided. In order to restrict the damages of fresh fruits due to the effect of Temperature the fresh fruits are required to be subjected into pre-cooling prior to packaging. The main objective of pre-cooling is to slow down the enzymatic and respiratory activity, minimize the susceptibility of MOs and to reduce the water loss and ethylene production. Different methods used for pre-cooling the fresh fruits and vegetables are room cooling for 3-5 days, hydro-cooling by means of drenching the fruits at low temperature less than 10C and forced air cooling where air is drawn through the packages by creating a pressure difference across the packages. f. Volatiles Some fresh produce releases volatile compound such as ethylene during ripening. If these volatile are not allowed to escape and unacceptable odour develop and the produce ripens rapidly. The production rate of ethylene depends on variety maturity stage temp oxygen level and CO2 levels of fresh produce. g. Ventilation Since the horticultural products respire even after harvesting, the package should be provided with the ventilation hole during the transportation. Cold air is constantly circulated through the container to remove the heat transmitted the cooling transmitted. Packaging requirements Considering the product characteristics of fresh fruit and vegetables the different packaging material and packages are selected. To meet the requirement of fresh fruit and vegetables. The important requirements are as follows: 1. Protection against bruising and physical injuries. 2. Protection against microbial contamination and deterioration. 3. Provide ventilation for respiration and exchange of gases. 4. Protection against moisture loss/ weight loss. 5. Control the ethylene concentration in the package. Characteristics of packaging material a. To contain produce  As an efficient handling unit, easy to handle by one person.  As a marketable unit, with the same weight and contents. b. To protect produce against  Rough handling during loading, unloading and transport- regid crates.  Pressure during stacking  Moisture or water loss with consequent weight and appearance loss.  Heat: air flow through crates or boxes via ventilation holes.  Fumigation possible through ventilation holes. c. To communicate  Identification:” a label with country of origin, volume, type or variety of product, etc. printed on it.  Marketing, advertising- recognizable trade name and trademark. d. To market product  Proper packaging will lead to reduce injuries and subsequently to improve its appearance.  Standard units of a certain produce will increase speed and efficiency of marketing.  With reduced cost of transport and handling, stacking and combining of packages into layers units pallets are possible. A more efficient use of space and reduced losses will lower the marketing costs.  Labels and slots facilitates inspection. Types of packaging materials Different types of packages are used throughout world, many of which have been carefully evaluated with respect to produce and market system, while other types have been often been adopted for 127 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India general use without thorough evaluation. Changes to improve such packages are still required. Some different types of packages includes: 1. Sacks: Flexible, made of plastic or jute a. Bags: small size sack b. Nets: sacks made of open mesh c. Wooden crates d. Carton of fiber board boxes. e. Plastic crates f. Pallet boxes and shipping containers. g. Baskets: made of woven strips of leaves, bamboo, plastic etc. Sacks and nets wire-bound crates are used extensively for snap beans, sweet corns and several other commodities that require hydro-cooling. Wire-bound crates are sturdy, rigid and have very high stacking strength that is essentially unaffected by water. Wooden crates and lugs Wooden crates, once extensively used for apples, stone fruits and potatoes have been almost totally replaced by other types of containers. The relative expense of the container, a greater concern for tare weight, and advances in material handling have reduced their use to a few specialty items, such as expensive tropical fruit. The 15, 20 and 25 pound The material used for sacks and nets may be wooden lugs still used for bunch grapes and some woven natural fiber (jute, kenaf, sisal, cotton), woven specialty crops are being gradually replaced with less synthetic (Polypropylene, polyethylene), knitted natural costly alternatives. fabrics (cotton), knitted synthetic (polyethylene) or Wooden baskets and hampers non-woven synthetic (propylene). Wire-reinforced wood veneer baskets and Wood pallets hampers of different sizes were once used for a wide Literally form the base on which most fresh variety of crops from strawberries to sweet potatoes. produce is delivered to the consumer. Pallets were first They are durable and may be nested for efficient used during world War –II as an efficient way to move transport when empty. However, cost, disposal goods. Depending on the size of produce package, a problem, and difficulty inefficient palletilization have single pallet may carry from 20 to over 100 individual severely limited their use to mostly local growers packages. Because these packages are often loosely markets where they may reuse many items. stacked to allow air circulation, or are bulging and Corrugated fibreboard difficult to stack evenly, they must be secured (unitized) Corrugated fiberboard (often mistakenly called to prevent shifting during handling and transit. cardboard or pasteboard) manufactured in many Pallet bins different styles and weights. Because of its relatively Substantial wooden pallet bins of milled lumber low cost and versatility, it is the dominant produce or plywood are primarily used to move produce from container material and will probably remain so in the field or orchard to the packaging house. Depending near future. The strength and serviceab8lity of on the application, capacities may range from 12 to corrugated fiber board have been improving in recent more than 50 bushels. Although the height may vary, years. the length and width is generally the same as a standard Most corrugated fiber board is made for three pallet (48 inches x 40 inches). More efficient double- or more layers of paperboard manufactured by the wide pallet bins (48 x 80 inches) are becoming more kraft process. In the recent years, large double-wall common in some produce operations. or even triple wall corrugated fiber board containers have increasingly been used as one way pallet bins to Although alternatives are available, wooden ship bulk produce to processes and retailers. Cabbage, melons, potatoes, pumplkins and citrus fruits have all Wire-bound crates 128 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India been shipped successfully in these containers. The Shrink wrap containers cost per pound of produce is as little as One of the newest trends in produce one fourth of traditional size containers. Some bulk packaging is the shrink wrapping of individual produce containers ay be collapsed and re-used. items. Shrink wrapping has been used successfully to package potatoes, sweet potaoes, apples, onions, Pulp containers Containers made from recycled paper pulp sweet corn, cucumbers and a variety of tropical fruits. and a starch binder are mainly used for small consumers Shrink wrapping with a n engineered plastic wrap can packages of fresh produce. Pulp containers are reduce shrinkage, protect the produce from diseases, available in a large variety of shapes and sizes and are reduce mechanical damage and provide a good surface relatively inexpensive in standard size. Pulp containers for stick-on labels. can absorb surface moisture from the product, which is a benefit for small fruit and berries that are easily harmed by water. Pulp containers are also bidegradable, made from recycled materials, and recyclable. Rigid plastic packages predominant material for fruit and vegetable consumer packaging. Besides the very low material costs, automated bagging machines further reduces packing costs. Film bags are clear, allowing for inspection of the content, and readily accept high quality graphics. Plastic films are available in a wide range of thickness and grades and may be engineered to control the environmental gases inside bags. The film material ‘breathes” at a rate necessary to maintain the correct mix of oxygen, carbon di-oxide, and water vapour inside bags. Since each produced items has its own unique requirement for environmental gases, modified atmosphere packaging material must be specially engineered for each items. Modification of packaging atmosphere from normal atmosphere (78% nitrogen, 21 % Oxygen) is widely used as effective modern packaging technologies which can significantly extend shelf life and improve quality level of most food products. The modification of internal package atmosphere may take place at the level of total pressure and/or partial pressures of component gas components. When the total pressure inside the package becomes lower than that of outside environment as a consequence of a more or less evacuation of the internal atmosphere, it is commonly referred to as vacuum packaging. Packages with a top and bottom that are heat formed from one or two pieces of plastic are known as clamshells. Clamshells are gaining popularity because they are inexpensive, versatile, provide excellent protection to the produce, and present a very Paper ad mesh bags Consumers packs of potatoes and onions are pleasing consumer package. Clamshells are most often about the only produce items now packed in paper used with consumers packs of high value produce items bags. The more sturdy mesh bags has much wider use. like small fruits, berries, mushrooms, etc., or items that In addition to potatoes and onions, cabbage, turnip, are easily damaged by crushing. citrus fruits, and some specialty items are packaged in Recent developments in food packaging mesh bags. Sweet corn may still be packed in mesh techniques bags in some market. In addition to its low cost, mesh  Vacuum packaging has the advantages of uninhibited airflow. Good  Active and intelligent packaging ventilation is particularly beneficial to onions.  Modified atmosphere packaging  Aseptic packaging Plastic bags Plastic bags (polyethylene film) are the Vacuum packaging Active packaging Fresh foods just after harvest are still active biological 129 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India systems. The atmosphere inside a package constantly changes as gases and moisture are produced during metabolic processes. The type of packaging used will also influence the atmosphere around the food because some plastics have poor barrier properties to gases and moisture. system that is capable of carrying out intelligent functions (such as detecting, sensing, recording, tracing, communicating, and applying scientific logic) to facilitate decision making in order to extend shelf life, enhance safety, improve quality, provide information and warn about possible problems. The metabolism of fresh food continues to use Table 1. Packaging systems and their application up oxygen in the headspace of a package and increases Active packaging system Application Most food classes the carbon dioxide concentration. At the same time Oxygen scavenging Carbon di-orxide production Most food affected by moulds water is produced and the humidity in the headspace Water vapour removal Dried and mould-sensitive foods of the package builds up. This encourages the growth Ethylene removal Horticultural Produce of spoilage microorganisms and damages the fruit and Ethanol release Baked foods (where permitted) vegetable tissue. Intelligent packaging can play an important role Many food plants produce ethylene as part of their normal metabolic cycle. This simple organic in facilitating the flow of both materials and information compound triggers ripening and aging. This explains in the food supply chain cycle. An intelligent packaging why fruit such as bananas and avocados ripen quickly system consists of 4 components viz. smart package when kept in the presence of ripe or damaged fruits in devices, data layers, data processing and information a container and broccoli turns yellow even when kept highway. The smart package devices are largely responsible for giving birth to the concept of intelligent in the refrigerator. packaging since they impart the package with a new Extensive trials have shown that each fresh ability to acquire, store and transfer data. The data food has its own optimal gas composition and humidity layers, data processll1g and information highway are level for maximizing its shelf life. Active packaging offers collectively referred as decision support system. promise in this area; it is difficult with conventional packaging to optimize the composition of the Controlled and modified atmosphere storage headspace in a package. Controlled atmosphere usually indicates monitoring and control of gaseous composition. This Active packaging systems is the case with bulk stores for fruit and sometimes Active packaging, employs a packaging with transport containers. It is not practical with small material that interacts with the internal gas environment packages in the distribution system. to extend the shelf-life of a food. Such new The term modified atmosphere is used when technologies continuously modify the gas environment (and may interact with the surface of the food) by the composition of the storage atmosphere is not closely removing gases from or adding gases to the heads pace Table 2. Smart package devices and their functions inside a package. The table below sets out some areas Functions of atmosphere control in which active packaging is Category TTI (Time temperature Food quality monitoring being successfully used. integrator or indicator) Intelligent packaging Leak or gas indicator It is defined as the packaging that monitors the conditions of packaged foods to give information about the quality of the packaged food during the transport and storage. It is also defined as a packaging Freshness indicator Bar code Electronic identification tags 130 Food quality monitoring, temperature evidence Food quality monitoring Tracking, logistic control Antitheft, brand-protection, tracking, logistic control, information flow, tamper-evidence Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India controlled. The initial atmosphere is intentionally  adjusted to give a gas mix as close as possible to that which will optimize the shelf life. Subsequent movement of gases and moisture into and out of the package is controlled only by the ability of the packaging film to act as a barrier. Modified atmosphere packaging Concept of food packaging - the entire  dynamic interaction between food, packaging material and ambient atmosphere has to be considered. Design and manufacture of packaging materials:  a multistep process.  involve careful and numerous considerations to successfully engineer the final package with all the required properties. Properties to be considered in relation to food distribution may include gas or water vapour permeability, mechanical properties, sealing capability, thermoforming properties, resistance (towards water, grease, acid, UV, light, etc.), machinability (on the packaging line), transparency, anti fogging capacity, printability, availability and, of course cost. As a rule, packaging materials with oxygen permeability lower than 100 cm3/m2 24 h 101.3 kPa are used in gas packaging. In the literature, on the other hand, very few reported investigations are available on the significance of oxygen permeability between 0-100 cm3/m2 24h 101.3 kPa for quality gas-packed products. Vegetables and fruits differ from other foodstuffs in that they continue to respire even when placed in a modified atmosphere. Due to the respiration, there is a danger that CO2 will increase to levels injurious to the packed commodities. On the other hand, respiration consumes oxygen and there is a danger of anaerobiosis. A number of special packaging materials intended for vegetables and fruits have been developed such as smart films, microporous film and micro-perforated films. Mechanical strength The gas barrier properties needed In choosing packaging materials for food one has also pay attention to how resistant to mechanical stresses (e.g. puncture), humidity and temperature (frozen or chilled) the material needs to be. If a material is of poor mechanical strength, the mechanical stresses, humidity and low temperature during storage transport and handling can damage the package and cause leakage.  Integrity of sealing Packaging materials requirements  In most gas packaging applications, excluding vegetables and fruits, it is desirable to maintain the atmosphere initially injected into the package for as long a period as possible. Some of the polymers currently used include PE, PETP, metallised PETP, PP, PS, PVC, PVDC, PA, EVA, and EVOH. The adequate integrity of the seal is important in order to maintain the correct atmosphere in the package. Type of package The type of package to be used, rigid or semiThese polymers are normally used as laminated rigid, lidded tray or flexible film pouch has to be taken or co-extruded multilayer materials in order to into consideration when choosing packaging materials. have the barrier properties required. The inner Fogging layer is usually polyethylene or its co-polymer In order to improve the appearance of the which forms the food contact and heat seal medium; polyethylene or ethylene vinyl acetate packages in retail outlets, the polyethylene in the alone are not suitable for gas packaging because packaging laminates can be specially treated to prevent condensation of water, which fogs the package and of their high gas permeability. prevents the consumer examining the product. 131 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Microwaveability of packaging materials is a factor that should also be considered in gas packaging, particularly in the case of ready-to-eat food products. For instance, the low melting point of PVC makes the PVC-LOPE-laminate or co-extrusion film much used as a base web material in deep - draw machines unsuitable for microwave oven heating. considerably at increased humidity levels. This phenomenon is also seen with conventional polymers. The gas permeability of high gas barrier materials, such as nylon and ethylvinyl alcohol, is likewise affected by increasing humidity. Gas barriers based on PLA and PHA are not expected to be dependent on humidity. Biodegradability and recyclability Aseptic packaging  These factors are new trends in packaging business.  A major challenge for the materials manufacture is the natural hydrophilic behaviour of many biobased polymers as a lot of food applications demand materials that are resistant to moist conditions. Heat is the most important means for food preservation. A typical thermal process consists of heating, holding and cooling of a food product. Thermal processes may be c1assifed based on whether the inactivation of microorganisms occurs before or after the food is filled inside the container. The former includes hot filling and aseptic packaging, in which a heat processed food is filled and packed in a clean environment. In aseptic packaging, the food and the package are sterilized (by heating and subsequent cooling) before the filling operation and the recontamination of the heat processed foods is prohibited completely by maintaining the environment and containers aseptic. Thermal and mechanical properties  Most bio-based polymer materials perform in a similar fashion to conventional polymers.  The mechanical properties in terms of modulus and stiffness are not very different compared to conventional polymers. References Compostability    Burg, S.P. (2004). Postharvest Physiology and The compostability of the materials is highly Hypobaric Storage of Fresh Produce. dependent of the materials e.g. the first step of composting is often a hydrolysis or wetting of the CABI Publishing, Wallingford, Oxfordshire, England. material. Kader, A.A. and Watkins, C.B. (2000). Modified atmosphere packaging - toward and beyond. In general, the oxygen permeability and the Hart. Technology, 10, 483-486. permeability of other gases of a specific material are closely interrelated. This relation is also Lee, D.S., Yam, K.L. and Piergiovanni, L. (2008). observed for bio-based materials. However, for Food Packaging Science and Technology. CRC some bio-based materials, e.g. PLA and starch, Press. the permeability of carbon dioxide compared to oxygen is much higher than for conventional Robertson, G.L. (2006). Food Packaging: Principles and Practices. CRC Press. plastics. As many of these bio-based materials are hydrophilic, their gas barrier properties are dependent on the humidity conditions for the measurements and the gas permeability of hydrophilic bio-based materials may increase 132 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India IMPORTANCE OF ETHYLENE RECEPTORS IN HORTICULTURAL CROPS Gaurav Agarwal, Divya Choudhary and Ajay Arora Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110 012 (Rottman et al. 1991; Woodson, 1994; Tang et al. Just like fruits, flowers are categorized as being 1994). Current status of molecular studies on ethylene climacteric or nonclimacteric. In climacteric flowers such as carnations, Gypsophila, and orchids, signal transduction provides potential points to be taken senescence is accompanied by a sudden, transient care of by the use of genetic engineering to alter increase in ethylene production and respiration, while ethylene sensitivity for crop improvement. Considering treatment of nonsenescent flowers with ethylene rapidly the pathway there are a few strategies and potential induces petal senescence. In nonclimacteric flowers points. Expressing any receptor gene in the sense such as gladiolus, tulip, and iris, generally, no increases direction would increase the receptor protein in the in ethylene production and respiration are apparent transgenic plant leading to reduced sensitivity to during flower senescence, and exogenous ethylene ethylene as a larger volume of ethylene is needed to application has little or no effect on petal senescence bind with the increased number of receptors which (Serek et al., 2006). Several studies to date suggest will not be available to inactivate the receptors and that abscission and senescence of flowers may be CTR1, consequently leaving the receptors in active triggered by the perception of endogenous ethylene mode henceforth rejecting the activation of EIN2. This by ethylene receptors. Abscission is a typical ethylene approach was used by Ezura et al., in developing response induced through ethylene receptors and is transgenic plant expressing the sense gene of melon influenced by mutations in ethylene receptors (Patterson Cm-ERS1. However; the number of transgenic plants and Bleecker, 2004). Therefore, investigations of with sense gene was lower than the plants with ethylene receptors and associated signal transduction antisense gene. The other approach is to express the pathways are essential for understanding of ethylene gene in antisense direction which will enhance sensitivity to ethylene because the transgenic plants will require a perception in flowers. Cross species transfer of mutated ethylene small volume of ethylene for rejecting the down regulation of EIN2, compared with wild type plants. receptor genes The transgenic generated using the same Cm-ERS1 There are many examples of post harvest receptor gene this time showed phenotype identical to disorders of flowers, fruits and vegetables caused by etr1 mutant, antagonistic to previous results i.e. with ethylene. For example lettuce and cucumbers must be enhanced sensitivity. physically separated from ethylene producing crops After elucidation and characterization of like tomatoes. Wilting of flower is caused by the death Arabidopsis ethylene response mutants and of cells resulting from increased membrane permeability, activation of reactive oxygen species and decreased establishment of ethylene signal transduction expression of protective enzymes (Rubinstein, 2000). components (Bleecker and Kende, 2000; Stepanova These changes are the repercussion of ethylene effect and Ecker, 2000) attempts have been made by several and upregulation of ethylene biosynthesis genes are groups to isolate and characterize homologus genes noticed prior to senescence of many flower species from different crops in order to asses the degree of Ethylene perception in flowers 133 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India variability and conservation of genes involved in the cascade compared to Arabidopsis. This would open new horizons for cross species transfer of receptor genes. Mutated version of ethylene receptor genes have been put to good use to covey resistance to ethylene in transgenic floral species. Bleecker et al. (1998) showed that etr1-1 is a dominant mutation that confers ethylene insensitivity in other plants. A few fruitful examples putting in use the above mentioned work is described below. Along with the transgenic approaches using different signaling molecules including receptors and downstream intermediates to delay senescence, the regulation of all these signaling intermediates are also discussed in depth in different flowers individually. Understanding the roles of the various ethylene receptors in flower development will be very handy in selectively blocking the process in flower development that economically detrimental and will improve the display life using molecular and genetic engineering approaches Rose ethylene or ACC in Arabidopsis (Chao et al., 1997), tomato (Tieman et al., 2001), tobacco (Kosugi and Ohashi, 2000; Rieu et al., 2003), or miniature potted roses (Müller et al., 2003).Rh-EIN3-1 and Rh-EIN32 showed constitutive expression in cut rose , a result consistent with previous reports. Moving a step further, Muller et al. (2000) demonstrated the cultivar difference in ethylene receptor levels during flower development. Also demonstrated the ethylene treatment mediated flower sensitivity to ethylene by RhETR1 regulation. Though the exact mechanism by which ethylene sensitivity is regulated by ethylene receptor dynamism is still not understood fully. An effort has been made to unravel the mechanism, upto a certain extent by receptor expression analysis in rose. Higher expression of RhETR1 was observed in cultivar ‘Bronze’ (having shorter flower life) compared to ‘Vanilla’(long lasting) alongwith the maximal expression of RhETR1 at the bud and young flower stage in bronze and vanilla cultivar respectively. As far as the RhETR3 expression levels are concerned, in ‘Bronze’ the transcript level increased as the senescence approaches however, in ‘Vanilla’ flowers it was constitutively expressed at very low levels. In case of RhETR2, the constitutive expression was observed during senescence although the transcript level was different for the two cultivars. Early expression of RhETR1, even before the ethylene production, alongwith the increase in RhETR3 expression in senescencing flowers of ‘Bronze’ prompts that ethylene response system in rose is mediated via overlapped expression of the multiple receptors. This also explains the reason for the multiplicity of ethylene receptors to some extent. Thus, it can be concluded that RhETR1 and RhETR3 are rate limiting for ethylene perception and determinant for flower longevity. These observations also justify Lashbrook et al. (1998) theory that increase in ethylene sensitivity is manifested by increase in receptor abundance. In a nutshell the negative regulation model can be defined as an inverse relation between the level of ethylene receptor expression and sensitivity to ethylene or the active state of ethylene receptors and CTR1 genes in the presence of ethylene and inhibition of EIN3 and other downstream signaling molecules in case of ethylene sensitive plants undergoing senescence process. Thus under normal physiological conditions, Rosa hybrida (ethylene sensitive) is expected to show decrease in expression of ethylene receptors and CTR1 undergoing senescence. However, study on rose tells a different story, CTR1, CTR2 (CTR1 homolog) were constitutively expressed during senescence and increased in response to exogenous ethylene. On the contrary, RhEIN3 was constitutively expressed throughout the process under the influence of both exogenous ethylene and ABA (Muller et al. 2000b; Muller et al. 2002a). Also, RhETR3 (one of the four Nan Ma and coworkers (2006) unearthed the ethylene receptors in rose) expression increased in senescing flowers. EIN3 is a positive regulator in the ethylene’s regulatory role in flower opening thus ethylene signalling pathway. However, there is no understanding the effect of ethylene on flower preevidence to indicate that EIN3 is up-regulated by pollination opening, and identifying key regulatory 134 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India components in ethylene biosynthesis and signaling pathways in cut roses. Although ethylene hastens the process of flower opening, 1-MCP (1methylcyclopropene), an ethylene action inhibitor, impeded it. Ethylene promoted ethylene production in petals, but 1-MCP did not inhibit ethylene biosynthesis and decrease in ethylene production was not observed. Thus, confirming that ethylene regulates flower opening of roses through signaling pathway and not through biosynthetic pathway. It was also established that continuous ethylene perception is required for flowers to open without the feedback regulation of ethylene biosynthesis, in accordance with Wang and Woodson’s (1989) theory of flower opening. Out of seven signaling component genes studied in rose, including three ethylene receptors (Rh-ETR1, RhETR3, and Rh-ETR5), two CTRs (Rh-CTR1 and RhCTR2), and two transcription factors (Rh-EIN3-1 and Rh-EIN3-2), transcripts of Rh-ETR5, Rh-EIN3-1, and Rh-EIN3-2 were accumulated in a constitutive manner and had no or little response to ethylene or 1-MCP, while transcript levels of Rh-ETR1 and Rh-CTR1 were substantially elevated by ethylene, and those of RhETR3 and Rh-CTR2 were greatly enhanced by ethylene. 1-MCP reduced all the four genes to levels much less than those in control flowers. These results show that ethylene triggers physiological responses related to flower opening in cut rose cv. Samantha, and that continued ethylene perception results in flower opening. Ethylene may regulate flower opening mainly through expression of two ethylene receptor genes (RhETR1 and Rh-ETR3) and two CTR (Rh-CTR1 and Rh-CTR2) genes (Ma et al. 2006). Carnation Ethylene plays a crucial role in the senescence of carnation flowers. During natural- and pollinationinduced senescence of the flowers, ethylene is first produced in the gynoecium (pistil) and the evolved ethylene acts on petals and induces the expression of genes for ACC (1-aminocyclopropane-1-carboxylate) synthase, ACC oxidase and cysteine proteinase, resulting in the autocatalytic ethylene production from the petals and wilting of the petals (ten Have and Woltering, 1997; Jones and Woodson, 1999; Shibuya et al., 2000). Also, exogenous ethylene applied to carnation flowers, which have not yet started ethylene production, induces autocatalytic ethylene production in petals, resulting in wilting of the petals (Borochov and Woodson, 1989; Wang and Woodson, 1989; ten Have and Woltering, 1997; Shibuya et al., 2000). Thus, Carnation (Dianthus caryophyllus L.) flowers are considered as an excellent model system for the study of ethylene perception and signaling. Till date three genes of ethylene receptor family have been isolated in carnation namely DC-ERS1 (Charng et al.1997 ), DC-ERS2 (Shibuya et al. 1998 ) and partial sequence of DC-ETR1 (Nagata et al. 2000).The expression analysis reveled that only DCERS2 and DC-ETR1 are involved in flower opening but in a tissue specific manner as the mRNAs of both the genes were present in abundance in petals at the full opening stage and level of both the mRNAs, DCERS2 in particular declined as the senescence progresses. However in ovaries the level of DC-ERS2 increased slightly and that of DC-ETR1 remained constant and in style the expression was constant during opening and senescence (Shibuya et al. 2002). Flowers when treated with 1, 1-dimetly-4(phenylsulfonyl)-semicarbazide (DPSS), known to block ethylene production in flowers (Onoue et al.2000) resulted in decreased expression of the genes in petals independently of ethylene production. In presence of exogenous ethylene the expression stayed the same as it was under the influence of increase in endogenous ethylene production. The decrease in DCERS2 mRNA in petals with the progress of senescence goes in accordance with the negative regulation model of ethylene signaling but further work needs to be done at the protein level to confirm this. (Shibuya et al. 2002). Downstream most of the signaling pathway lies EIN3-like protein (DC-EIL1) was identified in carnation by Waki et al. (2001) and demonstrated the expression of the same. DC-EIL1 levels were decreased in petals during natural senescence. In ovaries marginal increase in DC-EIL1 expression was reported as the senescence approaches, which otherwise was almost constant in styles and ovaries 135 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India even during senescence. Exogenous ethylene and ABA treatment showed increased expression initially followed by a decline as the senescence progresses. However, small amount of RNA accumulated on ABA and ethylene treatment. Since the exogenous application of ethylene or ABA leads to declined DC-EIL1 mRNA level and also the increased ethylene production during natural senescence demonstrated the same results. This leads to the conclusion that EIN3 level decreases with the increase in ethylene production, contradicting the findings of Chao et al. (1997) and Kosugi and Ohashi (2000) stating that EIN3 and TEIL mRNA levels in Arabidopsis and Tobacco respectively are not affected by ethylene. Thus, indicating that there could be other senescence factors apart from ethylene resulting in decline of DC-EIL1 expression. Also, abiding by the finding that EIN3 and its homologues acts as positive regulator of ethylene response (Chao et al. 1997), the DC-EIL1 level should be expected to increase or remain unchanged in the presence of ethylene. However, the declined DC-EIL1 mRNA levels recommends the decrease in ethylene response of petal tissues, thus mitigating the ethylene response in senescing carnation petals in response to exogenous and natural ethylene produced, contradicting the predicted enhanced ethylene response in the tissues due to DC-ERS2 and DC-ETR1 expression in carnation (Shibuya et al. 2002). Since the expression analysis of a limited number of putative ethylene receptors in carnations, DC-ERS2, DC-ETR1 and a downstream signalling DC-EIL1 gene throws a few probable mechanisms which the flowers can follow to combat senescence. A robust explanation for changes in mRNA accumulation is still needed (Shibuya et al. 2002). In addition, ethylene binding does not change throughout petal development suggesting that other steps in ethylene signalling may control ethylene responsiveness (Brown et al., 1986). Overexpression of several EIN3/EIL members confers constitutive ethylene responses without significant changes in transcript levels. These findings led to the hypothesis that EIN3 may be regulated at the protein level (Wang et al., 2002). Same has been confirmed and discussed recently by (Guo and Ecker, 2003; Potuschak et al., 2003; Yanagisawa et al., 2003). Apart from DC-EIL1 three more EIN3-like (EIL) genes DC-EIL1/2 (AY728191), DC-EIL3 (AY728192), DC-EIL4 (AY728193) were isolated, cloned and analysed for their expression on vegetative and flower tissues (petals, ovaries and styles) during growth and development and natural and ethylene induced senescence in carnation (Dianthus caryophyllus) by Iordachescu and Verlinden (2005) to get a better insight into ethylene responsiveness during flower development and senescence leading to ethylene climacteric. The DC-EILs compared to other EILs from other organisms show high similarity to EIN3. DC-EIL3 mRNA levels in flower petals and style increased both under normal flower development and exogenous ethylene application. Similar pattern has also been observed for some senescence related genes (Lawton et al., 1989; Jones et al., 1995; Verlinden et al., 2002). SR8 and SR12 (containing ethylene responsive elements in the promoter region (Verlinden et al., 2002)), the two senescence related gene transcripts accumulated in carnation petal flower senescence at the onset ethylene climacteric (Lawton et al., 1989) during natural senescence process and similar results were observed on exogenous ethylene exposure of pre-senescent tissues. Also the EIN3 like proteins have shown to interact with ethylene responsive elements (EREs) (Solano et al. 1998). These observations suggest that DC-EIL3 may be playing a role in regulating SR8 and SR12 gene expression, therefore regulating senescence (Iordachescu and Verlinden 2005). However, dip in DC-EIL3 expression was reported on wounding the plant suggesting the suppression of onset of senescence in leaves by decreasing the tissue sensitivity to ethylene or activation of some defense mechanisms similar observations of temporary decline in DC-EIL3 mRNA status in ovaries during senescence when higher ethylene production is reported in the flower (negative correlation of ethylene production and DC-EIL3 mRNA accumulation). Thus, showing the dynamism of regulation of DC-EIL3 in different parts (Photosynthetic, ovaries and non photosynthetic) of the flower. Another evidence of dynamism of DC-EIL3 was supported by the 136 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India unexpected maintained increase in its mRNA level in sucrose treated ovaries of the flower. Although it has been well established that sucrose and other sugars are known to extend the vase life of flowers (Van Droon 2004), it is sucrose which is particularly involved in the decrease of ethylene responsiveness in carnation petals (Verlinden and Garcia, 2004), raising the expected possibility of delayed increase in DC-EIL3 mRNA. Also the elevated levels of DC-EIL1/2 and DC-EIL4 mRNA in flowers treated with sucrose were in contradiction with depleted ethylene responsiveness, prompting towards tissue dependent and complex ethylene signaling. disease resistance and other ethylene mediated activities to continue in other parts of the plant. Symptoms like petal in rolling phenotype, hallmark of ethylene-dependent carnation flower senescence was not observed in transgenic carnation flowers. Instead, the petals remained firm throughout the senescence process and gradually became brown on the edges (loosing colour) and died from rotting (Bovy et al. 1999). Transgenic carnation cut flowers had three times the vase life of non transformed flowers and lasted upto 16 days, which is longer than flowers treated with ethylene inhibitors of ethylene biosynthesis or ethylene antagonists (Bovy et al, 1999; Baudinette et al., 2000). Exploiting the fact that ethylene production shoots up post pollination in carnation flowers at different intervals of time (Jones Woodson 1997) an attempt was made to interpretate the regulation of DCEINs with the ethylene production in flower.NBD (an ethylene inhibitor) treated pollinated showed higher accumulation of DC-EIL1/2 and DC-EIL4 transcripts (at 12 and 48 hours after pollination) which was expected to show lower DC-EILs mRNA levels. DCEIL3 transcript levels showed subtle differences between NBD non treated flowers and only pollinated flowers. Taking all these observations into account it can be said that EIN3 like genes and DC-EIL3 gene in particular is involved in significant regulation by ethylene in initiating and sustaining of the senescence process in carnation petals (Iordachescu and Verlinden 2005). Chrysenthamum Transgenic carnation plants were obtained by using etr1-1 allele driven either by constitutive CaMV 35S or flower specific petunia FBP1 promoter (Bovy et. al, 1999), CMB2 (carnation MADS box) containing promoter (Baudinette et al. 2000); MADS domain proteins are important in fruit ripening (Vrebalov et al. 2002). Lower transformation efficiency along with diminished disease resistance and lower ethylene sensitivity (Hoffman et al. 1999; Ohtsubo et al. 1999) was observed using constitutive promoter compared to flower specific promoters, which took care of both the above stated drawbacks (Angenent et al. 1993; Bovy et al. 1999) and allowed In the cut flowers of “Seiko-no-makoto” (ethylene-sensitive cultivar) chrysanthemum (Dendranthema grandifiorum (Ramat.) Kitamura), DG-ERS1 mRNA was present in a large amount in the petals on day 0 (at the full-opening stage of flower) and its levels decreased markedly with the lapse of time in air or in response to a 12-h exposure to ethylene, although these were not evident in “Iwa-nohakusen” chrysanthemum (ethylene-insensitive cultivar). DG-ERS1 mRNA was present in a large amount in nonsenescent flower tissues of an ethylenesensitive “Seiko-no-makoto” cultivar, but its rnRNA level was very low in an ethylene-insensitive “Iwa-nohakusen” cultivar. The observed difference between the two cultivars is probably related to the variation in sensitivity to ethylene between them, and suggest that the DG-ERS1 gene and its resultant DG-ERS1 protein may be involved in the perception of ethylene signal in flower and leaf tissues of the cut chrysanthemum, especially those of “Seiko-no-makoto” (Narumi et al., 2005a). Delphinium Delphinium is another ethylene sensitive ornamental crop composing five petaloid sepals, with the posterior sepal forming the spur, four petals into two unequal pairs, termed as “bee” by gardeners, numerous stamens in eight spiral series, and 3-5 pistils. Delphinium flowers show a climacteric-like rise in 137 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ethylene production before abscission of sepals and sepals are abscised by exposure to exogenous ethylene (Ichimura et al., 2000).It was also reported by the same group that carbohydrate accumulation is responsible for reduced ethylene sensitivity. However, at the molecular level not much is explored in relation with abscission of florets in this particular crop. Two ethylene receptor genes in delphinium have been isolated namely Dl-ERS1 type1 (AB055429) and DlERS1 type2 (AB055430) differing by three amino acids (Kuroda et al, 2003). Recently two more cDNA encoding ethylene receptors Dl-ERS1-3 and Dl-ERS2 were isolated from Delphinium flowers by Tanase and Ichimura (2006). However, the most common ethylene receptor ETR1 seems to be missing from delphinium (Kuroda et al. 2003). The Flower parts showed higher Dl-ERS1-3 transcript levels than leaves and stems whereas Dl-ERS2 transcript levels in leaves and flowers were equal but greater than that of stem. These observations suggest differential regulation of receptor genes (Dl-ERS1-3 and Dl-ERS2) and their involvement in ethylene signaling of delphinium flower senescence. Exogenous ethylene treatment revealed distinct effects on the receptor transcripts in different parts. In sepals Dl-ERS1-3 and Dl-ERS2 transcript levels increased but unchanged in gynoecia and receptacle. This increase in transcript level in sepals was not observed during flower senescence, may be because of lower level ethylene production in flower than exogenous ethylene. Thus, in accordance with negative regulation of ethylene signaling model (Hua and Mayerowitz, 1998) it was proposed that increased Dl-ERS1-3 and Dl-ERS2 in sepals might be contributing in allaying sepal senescence due to ethylene production by gynoecia and receptacles. Abiding by the finding in A. thaliana that ethylene receptors are not specifically expressed in the abscission zone (Hua et al. 1998), Dl-ERS1-3 and Dl-ERS2 are not expected to determine the ethylene response of the abscission zone and ethylene induced sepal abscission is controlled both by ethylene signaling components and Dl-ERS1-3 and Dl-ERS2 proteins (Tanase and Ichimura,2006). Shibuya and coworkers (2002) work on carnation threw light upon behavior of ETR1 and ERS2 gene in response to endogenous and exogenous ethylene but how these receptors behave in response to ethylene produced by abscission of flower after stem excision needed to be answered. Above mentioned case studies (rose and carnation) and the examples indicating upregulation of ERS type ethylene receptors: NR in tomato fruit ripening (Lashbrook et al. 1998 and Wilkinson et al. 1995), Nt-ERS1 in developing tobacco (Terajima et al. 2001), RP-ERS1 in R.palustris leaves (Virezen et al. 1997), derives enough evidences in favour of the expected rise in the levels of Dl-ERS1 with the due course of endogenous ethylene evolution. The correlation between abscission of florets, ethylene evolution and Dl-ERS1 expression after cutting the stems was elucidated by the finding that maximum ethylene evolution and Dl-ERS1 transcript level was observed at the time of maximal floret abscission. Kuroda et al. (2003) also concluded that Dl-ESR1 expression is more or less proportional to the endogenous ethylene production implying that ethylene evolved by the florets is perceived by elevated levels of Dl-ERS1 leading to flower senescence. In a nut shell, can be said that abscission and flower senescence in delphinium is ERS1 mediated considering that ETR1 has not yet been reported in this flower (Kuroda et al. 2003). Petunia Two forms of the etr1-1 were engineered for the transformation of tomato and petunia. First form used the vector pMON11063 which contained CaMV35S promoter driving expression of the Arabidopsis etr1-1 cDNA. The other one pMON26601 contain figwort mosaic virus (FMV) 35S promoter and nopaline synthase gene at 3’ end flanking a chimeric etr1-1/Nr cDNA. This construct carried a fusion protein with the N-terminal hydrophobic domains of etr1-1, including the cys-65 to tyr mutation and the C- terminal histidine kinase 138 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India domain of wild type (WT) NR. By exchanging the Nterminus of the endogenous tomato ethylene receptor with a mutant Arabidopsis protein, they alter the binding activity dimerization of the NR such that potentially different phenotypes might occur. The transgenic generated due to pMON26601 transfer containing chimeric protein are more efficient than the other and also showed ethylene insensitivity indicating that response regulator domain is not necessary for the etr1 mutants to confer ethylene insensitivity. Tomato and petunia plants transformed with the Arabidopsis mutated dominant ethylene insensitive etr1-1 cDNA showed that cross species transfer of an ethylene insensitivity phenotype is possible (Wilkinson et al., 1997). The creation of ethylene insensitive tomato and petunia plants via the introduction of dominant Arabidopsis ethylene receptor alleles depicts the functional conservation for this component of ethylene signaling (Giovannoni, 2004). This strategy of developing plants without ethylene perception however, does not lead to a normal development of plant. Therefore in an attempt later to improvise the previous work Cobb et al. (2002) transformed petunia with etr1-1 under the influence of floral specific promoters FBP1 (floral binding protein) and AP3 (involved in floral organ development) resulted in increased vase life of upto 5 times compared to control flowers. receptor genes attaining the desired objective. The delay in senescence observed in transgenic etr1-1 flowers was longer than in flowers pretreated with chemicals that inhibit either ethylene biosynthesis (amino-oxyacetic acid) or the ethylene response (silver thiosulfate) and blocked ethylene biosynthesis by ACO1 gene co-suppression (Bovy et al.) . The possible reason for this difference might be that ACO1 co-suppressed flowers are still sensitive to basal levels of ethylene, either produced endogenously or by flowers and fruits in the vicinity, whereas etr1-1 transformed flowers are not. This observation is also of great importance to non-climacteric flowers and fruits that can be damaged by ethylene produced in the surrounding. However, the disease resistance was compromised as the plant became more susceptible to fungal disease. Earlier, Clark et al. (1999) reported reduced adventitious root formation in ethyleneinsensitive transgenic petunia and concluded that ethylene plays an important role in response of roots to environmental stimuli. Shibuya et al. (2004) figured out the central role of signaling molecule EIN2 in petunia plant development. Transgenic petunia plants with reduced PhEIN2 expression were produced using co suppression (expressing PhEIN2 sense RNA) and RNA-interference (PhEIN2-RNAi) approach. Out of 68 lines produced two homozygous lines, EIN2s-182 Mutated version of BOERS, an Ethylene and EINr-12, exhibiting the greatest flower longevity Response Sensor (ERS) gene of broccoli, Brassica after ethylene treatment and pollination in T1 generation oleracea, boers (obtained by removing an EcoRI were picked from PhEIN-sense and PhEIN2-RNAi cutting site with a silent mutation at Gly-521 and lines, respectively. RNAi mediated gene silencing was introducing a point at Ile-62, replacing isoleucine with more efficient than co suppressed lines. These phenylalanine) (Chen et al. 1998) conferred ethylene transgenic plants were compared to wild-type Petunia insensitivity (because of non functional sensor domain, x hybrida cv Mitchell Diploid (MD) and two ethylene not able to bind ethylene) when transformed into insensitive petunia plants transformed with Atetr1 petunia x hybrida Hort. Vilm.-Andr. Transformed namely etr-44568 (Wilkinson et al., 1997) and etrpetunia flowers retained pigmentation longer and 56(Clevenger, 2000). showed longer life span irrespective of either its storage Geranium in water or exposure to exogenous ethylene. These Two ethylene receptors, PhETR1 and observations were consistent with those of Bovy et PhETR2 are deduced till date, though there is scope al. (1999) and Wilkinson et al. (1997), stating yet another fruitful example of cross species transfer of for more receptors to be decoded (Dervinis et al. 139 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India 2000). They have also been characterized for their distribution and temporal and spatial regulation in geranium (Pelargonium X hortorum L.H. Bailey). Because of the rapidity and nature of their response to ethylene geranium florets are considered for the expression analysis of ETR1 homologue in response to pollination and exogenous ethylene during flower petal abscission. The rapid nature of ethylene response was figured out by Clark et al. (1997) by demonstrating that during the receptive stage of pollination, geranium florets were observed to undergo complete abscission even in the turgid petals when treated with very low concentrations of exogenous ethylene (Clark et al., 1997). Self pollination of geranium flower causes rapid burst of ethylene production by the gynoecium within 1 hour, which leads to petal abscission in 2-4 hr. Enhanced sensitivity of geranium florets to exogenous ethylene at the pollination receptive stage was also reported by Evensen et al. (1991). Older florets of Pelargonium x domesticum were observed to abscise quickly compared to younger florets when exposed to minimal levels of exogenous ethylene. In contrast with orchid (O’Neill et al, 1993) and carnation (Van Altvorst and Bovy, 1995) ethylene produced triggers its own biosynthesis (autocatalytic activity), geranium flowers are not known to display this autocatalytic feature (Clark et al. 1997). PhETR expression analysis revealed that it was expressed least in roots and maximum in pistils. The assessment of PhETR expression at the site of pollination induced ethylene production i.e. in pistil and the abscission zone site i.e. receptacles showed that PhETR1 and PhETR2 transcript levels were unaltered before and after ethylene treatment suggesting that these genes are not ethylene inducible in petal abscission. Also, the pollinated and non-pollinated pistils and receptacles PhETR expression levels were consistenet with these results. This prompts either at a post transcriptional level or another member of the PhETR family (which needs to be figured out), might be controlling or regulating ethylene induced petal abscission in geranium. Considering these observations, hypothesis put forth by Dervinis et al. (2000) can not be ruled out, which states that the control of ethylene sensitivity in geranium florets may not be determined by the amount of receptor protein present justifying the repression of ethylene signal transduction pathway by the receptors unless ethylene binds the receptors. Abundant expression of ethylene receptor prior to ethylene production might be a factor responsible for allowing non-autocatalytic plants like geranium to respond quickly to the ethylene generated after pollination (Dervinis et al. 2000). These observations raise a possibility of overlapping functions and genetic redundancy adopted by PhETR genes. Coriander Delaying senescence in coriander using a dominant negative mutant ERS1 of Arabidopsis (Aters1) is yet another attempt of heterologus expression. This work was a crucial breakthrough as not only it established the transformation protocol for coriander but also the plant resources beneficial for mankind remained in the plant for longer duration and resource like fatty acid (petroselinic acid) can be exploited for prolonged period. Coriander seeds are known to contain many fatty acids like petroselinic acid (18:16cis), the principal fatty acid of the seed oil of most Umbelliferae, Araliaceae, and Garryaceae species. This fatty acid composes as much as 85% of the total fatty acid of Umbelliferae seeds but is virtually absent from leaves and other tissues of these plants (Klieman et al. 1982; Dutta et al. 1991; Ellenbracht et al. 1980) The structure of petroselinic acid differs from that of oleic acid (18:1A9cis), a common plant fatty acid, by the position of its double bond. Because of the unsaturation at carbon 6, petroselinic acid is of potential industrial significance. Through chemical cleavage at its double bond, petroselinic acid can be used as a precursor of lauric acid (12:0). Petroselinic acid in humans can be absorbed but not synthesized by mammals, can be useful in clinical investigation of fat absorption by monitoring the ingested petroselinic acid in blood lipids (Weber et al. 1997). Agrobacterium mediated transformation of ERS1 gene harbored by binary vector pCGN1547 was performed. The cloned cDNA expression analysis of ACO and ERS1 homologs in both transgenic and wild type plants 140 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India revealed subtle downregulation of ACO in transgenic plants compared to wild type. The marginal downregulation of ACO could be due to the failure of feedback control mechanism of ethylene synthesis by heterologus dominant negative receptor. Thus, the delay in senescence in transgenic plant is primarily due to the effect of dominant negative ERS1 and not due to reduction of ethylene biosynthesis. Also ERS1 homolog in coriander was not altered significantly indicating no cosuppression by the transgene to confer its function (Wang and Kumar 2004). Gladiolus Two ethylene receptor paralogous genes, GgERS1 a and GgERS1 b, have been isolated from gladiolus (Gladiolus fandiflora hort cv. Traveler), an ethylene-insensitive flower (Arora et al., 2006). The cDNA sequence indicated that both genes have almost exact similar sequence except that GgERS1 blacks 636 nucleotides, including first (H) and second (N) in the histidine kinase motifs, present in GgERS1 a. The analysis of the genomic DNA sequences (4, 776-bp nucleotide designated as GgERS1 long DNA and 3,956-bp nucleotide designated as GgERS1 short DNA) revealed that both sequences were identical except that GgERS1 short DNA was devoid of an 820-bp long nucleotide segment in the first intron of GgERS1 long DNA. These data suggested that each of the GgERS1 genes was generated by duplication and splicing from different genomic DNA. The GgERS1b mRNA level de- creased in petals during flower development, whereas the expression of GgERS1a mRNA was constitutive, however, with a high accumulation level, suggesting that high expression level of GgERS1a conferred the ethylene insensitive nature in petals of gladiolus. On the other hand, the sensitivity to ethylene might be regulated by GgERS1b expression. Nemesia Recently, flower longevity in transgenic plants of an ethylene-sensitive ornamental plant, Nemesia strumosa, was established by introducing the mutated melon ethylene receptor gene Cm-ETR1/H69A (Cui et al., 2004; Takada et al., 2005). Based on the mutation in Arabidopsis err1-1, the mis-sense mutation His-69 to Ala (H69 A) was introduced into Cm-ETR1 to create the mutant gene Cm-ETRI/H69A. The CmETR1/H69A expression inhibited the ethylene response during the senescence of Nemesia flowers, resulting in longer shelf life. This technique can be useful in delaying flower senescence in heterologous plants. Ethylene perception in fruits and vegetables Tomato has emerged as the most useful model to date, due to its commercial importance. ease of genetic manipulation, rapid life cycle, year-round nonseasonal greenhouse fruit production, wellcharacterized single gene-ripening mutants such as never ripe (nr), nonripening (nor), ripening inhibitor (rin), and green ripe (gr) and the availability of detailed genetic maps, EST collections, microarray chips, and full-length cDNA collections (Alexander and Grierson, 2002; Barry et al., 2005; Barry and Giovannoni, 2006; Klee, 2006).While much is known about regulation of ethylene synthesis in tomato, less is known about its perception and signal transduction. Never ripe (Nr) is a semidominant ethylene receptor mutant, has a mutation in ethylene binding domain of the NR receptor, unable to bind ethylene and the first to be identified in tomato (Wilkinson et al., 1995).In tomato, there are six ethylene receptors (LeETR1, LeETR2, LeETR4, LeETR5, LeETR6; Klee and Tieman, 2002) and NR. LeETR1, LeETR2, LeETR4 and LeETR5 were cloned by homology to the Arabidopsis ETR1, ETR2 and tomato NR genes. The predicted structures of the tomato receptor family are very similar to those in Arabidopsis. The tomato receptors are quite divergent, exhibiting less than 50% identity in primary sequence at the extremes. Three receptors have a potential extra amino terminal membrane-spanning domain. Only one, NR, lacks the receiver domain. Three (LeETR4–6) are missing one or more conserved HK domains, thus resembling the Subfamily II Arabidopsis receptors. Despite the extensive structural differences between them, all are receptors, as defined by their ability to bind ethylene (F. Rodriguez, A. Bleecker, and H. Klee, unpublished data).NR and 141 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India LeETR4 expression increases during fruit ripening (Payton et al., 1996; Lashbrook et al., 1998; Tieman and Klee, 1999) whereas LeETR1 and LeETR2 are constitutively expressed in all tissues throughout development (Lashbrook et al., 1998) with LeETR1 expressed at about 5-fold higher level than LeETR2. In contrast, expression patterns of the other four genes are highly regulated. For example, during fruit development, ovaries express high levels of NR mRNA at anthesis. The level then drops approximately 10fold until the onset of ripening whereupon it rises approximately 20-fold. Functional compensation for reduced expression of one member of a gene family by increased expression of another member of the same family has been reported in several mammalian multigene families like retinoic acid receptor, retinoblastoma and connexin gene families (Berard et al. 1997; Mulligan et al. 1998; Minkoff et al. 1999). Are ethylene receptors in tomato negatively regulated? It has already been established that Arabidopsis is best known example to demonstrate ethylene perception and signal transduction via receptor inhibition mechanism (Hua and Meyerowitz, 1988). However, the same can not be said with authenticity in case of tomato until Tieman et al. (2000) came up with the results which answered a few questions. Even if we presume that ethylene signaling in tomato is negatively regulated and is in accordance with Arabidopsis receptor inhibition model than one question which needs to be answered is why the NR gene expression increases with the ripening process and thus with ethylene evolution . This was explained by Hackett et al. They used antisense inhibition of a mutant tomato ethylene receptor, Nr gene to show that the mutant tomato restored the normal ripening suggesting that the mutant Nr receptor is unable to bind ethylene and prevents its inactivation, therefore suppressing the ethylene responses. The transgenic plants developed using antisense Nr produced three different phenotypes depending upon the transgene dosage in the progeny plant. Progeny with two copies of homozygous transgene produced wild-type fruit but gave intermediate fruit type if they were hemizygous for both copies, in case of single transgene inheritance in a homozygous state fetched fruits with intermediate (between Nr and wild type) phenotype, whereas hemizygotes produced Nr-type fruit. This finding suggests that a threshold level of mutant receptor is needed to suppress the ethylene response pathway and prevent normal ripening. However, antisense inhibition of the wild type NR gene showed no effect on ripening, raising a question about normal function of this gene. Probable reason of the functional NR gene might be to achieve wild type levels of LeETR4 expression. The Nr mutants are known to affect ethylene responses in tissues other than ripe fruit (Lanahan et al., 1994), including seedlings. Nr seedlings were devoid of ethylene triple response (shortened, swollen hypocotyls, exaggerated apical hook and shortened root) when germinated in the dark on media with ethylene precursor ACC. Similar results were observed in case of homozygous NR antisense transgenic and Nr seedlings when given similar treatment. These data suggests that although downregulation of the mutant Nr gene was sufficient to alleviate its effect on fruit ripening in the transgenic raised, insensitivity of seedlings to ethylene was not altered. Results indicate that Wild type NR gene product is not required for normal ripening and antisense inhibition of the mutant Nr gene products restores normal ripening provides enough evidence in support of receptor inhibition model and negative regulation of ethylene signaling in tomato. The tomato has been renamed Solanum lycopersicum (formerly Lycopersicon esculentum), and this had led to the renaming of its genes. Other than tomato fruit, the ethylene receptors have been isolated in several climacteric and nonclimacteric fruits, and exhibits different expression patterns during ripening. The melon fruit is second to tomato fruit when it comes to research work carried on ethylene perception. Melon fruit is an ideal fruit for these studies due to the fact that its development has three distinct stages: phase I, II, and III; the flesh, embryo, placenta, and seeds are well ordered; the fruit 142 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India development can be clearly divided into ethyleneinsensitive and ethylene-sensitive stage, and the developing fruit has a lower sensitivity to ethylene than does the ripening fruit (Gillaspy et al., 1993; Takahashi et al., 2002). In muskmelon, Cm-ERS1 mRNA increased slightly in the pericarp of fruit during ripening, followed by a marked increase of Cm-ETR1 mRNA, which paralleled climacteric ethylene production. The increase of Cm-ERS1 mRNA at a low concentration of ethylene before the increase of Cm-ETR1 mRNA and ethylene production indicates that Cm-ERS1 may be sensitive to a much lower concentration of ethylene, while Cm-ETR1 may be involved in the response at a high concentration of ethylene (Sato-Nara et al., 1999). Studies carried out to examine the temporal and spatial expression pattern of Cm-ERS1 protein, during fruit development, revealed that a posttranscriptional regulation of Cm-ERS1 expression affects stage- and tissue-specific accumulation of the protein (Takahashi et al., 2002). The melon receptor CmERS1 was localized at the endoplasmic reticulum and its topology indicates that there are three membrane-spanning domains, with its N-terminus facing the luminal space and the large C-terminal portion being located on the cytosolic side of the ER membrane (Fig. 6.3) (Ma et al., 2006a). The melon subfamily II ethylene receptor, Cm-ETR2 mRNA, exhibits earlier accumulation compared to Cm-ETR1 during ripening, and its transcript accumulation increased during melon ripening, and declined in parallel with a reduction in ethylene production. Furthermore, the Cm-ETR2 mRNA was induced by ethylene treatment and inhibited by 1-MCP (Owino et al., unpublished results). The expression of two ethylene receptor genes in passion fruit (Passifiora edulis), PeETR1 and PeERS1, did not change significantly during ripening. However, the levels of PeETR1 and PeERS1 mRNA were much higher in arils than in seeds (Mita et al., 1998). High levels of PeERS2 mRNA were detected only in the arils of ripe purple fruit. Although the expression of PeERS2 mRNA was enhanced during ripening, a markedly high level of PeERS2 mRNA was detected only in the arils of ripened purple passion fruit, and no ripening-regulated expression was apparent in seeds. Exposure of mature green fruit to ethylene increased the levels of PeERS2 mRNA, suggesting that PeERS2 might playa role in repressing ethylene responses at later developmental stages after fruit ripening has been completed (Mita et al., 2002). Ethylene perception has also been described to be involved in apple fruitlet abscission and early development (Cin et al., 2005). The apple (Malus domestica) MdETR1 and MdERS1 gene expression patterns were tissue specific, with MdETR1 transcripts being abundant in the peduncle, abscission zone, and seed than in the cortex of early developing fruits (nonabscising fruitlets), even though the expression always remained at a steady-state level. The MdERS1 transcripts increased throughout shedding in all tissues of abscising fruitlets, indicating a possible role for this ethylene receptor in abscission. An increase in ethylene evolution and/or sensitivity at the abscission zone level would regulate the expression of genes encoding specific cell wall hydrolases, leading to abscission zone cell separation and to fruitlet shedding. The avocado (Persea americana) PA-ERS1 mRNA increased gradually from the day of harvest, and did not change significantly until the climacteric peak when it was hyperinduced. 1-MCP however suppressed the accumulation of PA-ERS1 to basal levels suggesting that the stimulated induction of PAERS1 at the climacteric peak maybe a mechanism by the avocado fruit to dissipate the high levels autocatalytic ethylene (Owino et al., 2002). In peach (Prunus persica), the expression of Pp-ETR1 appeared to be constitutive and ethylene independent during fruit development and ripening, while Pp-ERS1 transcripts increased during fruit ripening and its expression appeared to be upregulated by propylene treatment (Rasori et al., 2002). Application of the ethylene antagonist, 1-MCP, delayed fruit ripening, ethylene evolution, and concurrently downregulated Pp-ERS1, while Pp-ETR1 transcription was unaffected. 1-MCP action was rapidly abolished after moving fruits to air, when a rapid stimulation of 143 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ethylene evolution and a concurrent increase of PpERS1 mRNAs were observed. Cold treatment of late-season pear (Pyrus communis cv. Passe-Crassane) fruit leads to a gradual increase in ethylene production and a commensurate increase in ethylene receptor mRNA expression (ElSharkawy et al., 2003). The Pc-ETR1 a mRNA accumulation was upregulated by cold and during ripening, whereas Pc-ERS1 a and Pc-ETR5 were less affected by cold treatment, but all increased during postcold treatment, ethylene-dependent ripening. A sharp peak of Pc-ETR1 a and Pc-ERS1 a mRNA accumulation was observed during ripening in the earlyseason pear cultivars, in contrast to the gradual increase seen in late-season pear cultivar, Passe-Crassane (PC). A more pronounced difference between earlyseason cultivars and late-season cultivar PC was seen in the behavior of Pc-ETR5 transcript accumulation. Transcript levels for Pc-ETR5 diminish sharply before and during the ethylene climacteric and ripening of earlyseason pear fruit, whereas in late-season cultivar they increase sharply. This suggests that a decrease in the expression of a negative regulator could result in an increase in ethylene sensitivity early in the ripening phase of early fruit development. However, given the potential for redundancy in the ethylene receptor family, it remains to be determined whether reduced levels of Pc-ETR5 affect the overall ethylene sensitivity of earlyseason pear fruit. Three ethylene receptors-DkERS1, DkETR1, and DkETR2-have been isolated and their expression determined during ripening of persimmon (Diospyros kaki) fruit (Pang et al., 2006). The DkETR1 mRNA is constitutively expressed during all stages of fruit ripening and is ethylene-independent. Conversely, DkERS1 and DkETR1 mRNA levels correlated with ethylene production during fruit development and ripening and were induced by ethylene. The DkERS1 protein decreased gradually prior to fruit maturation and reached its lowest level at the ripening stage when ripening-related ethylene was produced, suggesting the involvement of DkERS1 in ethylene perception during fruit ripening. In contrast to the great deal of information available regarding the ethylene receptors in climacteric fruits, much less is known about nonclimacteric fruits. At present, no single growth regulator appears to play a positive role analogous to the role played by ethylene in the ripening of climacteric fruits. Nonclimacteric fruits are also able to synthesize ethylene, and in some cases, it has been shown that ethylene can hasten the postharvest deterioration. However, in spite of many efforts, no results have been obtained that can demonstrate a clear relation between ethylene and the ripening of these fruits. Three ethylene receptors were isolated and expression patterns detennined in strawberry (Fragaria ananassa) fruits (Trainotti et al., 2005). The FaEtr1 mRNA was low in flowers, but showed an increase in the small green fruits and a subsequent decrease in the large green fruits that was followed by a steep increment, which continued throughout the ripening phase. The FaErs1 mRNA was very high in flowers but steadily decreased to reach a minimum in the large green fruits. Afterward, it increased again till the ripening was completed. On the other hand, F’aEtr2 mRNA increased about threefold to reach a maximum in the white fruits. Afterward, although a slight decrease was observed, the transcript amount remained high in the red fruits at well over twice that of the small green fruits. The FaEtr1 and FaEtr2 genes were more responsive to ethylene in the white fruits, while FaErs1 was highly responsive to ethylene at the red stage. This study suggested that ethylene receptors might have a physiological role in the ripening of nonclimacteric strawberries. Even though citrus (Citrus sinensis) fruits are nonclimacteric, exogenous ethylene is able to stimulate ripening by accelerating respiration and inducing pigment changes of peel, chlorophyll degradation, as well as carotenoid biosynthesis. In young “Valencia” fruitlets, CsERS1 expression was detected in fruits on tree, immediately after harvest, and was further induced in the subsequent days (Katz et al., 2004). The CsERS1 expression was slightly induced by ethylene treatment and reduced by 1-MCP treatment in young 144 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India elements and their expression during ripening in fruitlets. The CsETR1 expression was constitutive in pears with/without cold requirement. J. Exp. Bot., young fruitlets, but was not affected by detachment 54: 1615-1625. from the tree and was ethylene-independent. In mature fruit, the expression of both CsERS1 and CsETR1 Gillaspy, G., Ben-David, H., and Gruissem, W. (1993). Fruit: a developmental perspective. Plant Cell, 5: genes was constant and was not affected either by 11439-1451. MCP or propylene treatments. The differences in the Hua J and Meyerowitz EM. (1998) Ethylene responses expression of CsERS1 between young fruitlets and are negatively regulated by a insensitive3 (EIN3) mature fruit suggest that CsERS1 may modulate the homolog. Nucleic Acids Research 28, 960–967 differential sensitivity to ethylene in fruitlets versus Iordachescu, M. and Verlinden, S. (2005). mature citrus fruit Transcriptional regulation of three EIN3-like genes of carnation (Dianthus caryophyllus L. cv. Suggestive reading Improved White Sim) during flower development and upon wounding, pollination, and ethylene Alexander, L. and Grierson, D. (2002). Ethylene exposure. Journal of Experimental Botany, biosynthesis and action in tomato: a model for 56:2011–2018. climacteric fruit ripening. J. Exp. Bot., 53: 20392055. Katz, E., Lagunes, P.M., Riov, J., Weiss, D., and Goldschmidt, E.E. (2004). Molecular and Arora, A., Watanabe, S., Ma, B., Takada, K., and Ezura, physiological evidence suggests the existence of H. (2006). 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Overexpression of a mutated melon ethylene receptor gene Cm-ETR1/H69A confers reduced Mita, S., Kawamura, S., and Asai, T. (2002). Regulation of the expression of a putative ethylene receptor, ethylene sensitivity in a heterologous plant, Nemesia PeERS2, during the development of passion fruit strumosa. Plant Sci, 167:253–258. (Passiflora edulis). Physiol. Plant, 114: 271-280. El-Sharkawy, I., Jones, B., Li, Z., Lelievre, J., Pech, J.C., and Latche, A. (2003). Isolation and Mita, S., Kawamura, S., Yamawaki, K., Nakamura, K., and Hyodo, H. (1998). Differential expression of characterization of four ethylene perception genes involved in the biosynthesis and perception 145 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India of ethylene during ripening of passion fruit (Passiflora edulis Sims). Plant Cell Physiol., 39: 1209-1217. Müller R, Lind-Iversen S, Stummann BM, Serek M. (2000a) Expression of genes for ethylene biosynthetic enzymes and an ethylene receptor in senescing flowers of miniature potted roses. Journal of Horticulture Science and Biotechnology 75:12–18. Müller R, Owen CA, Xue Z-T, Welander M, Stummann BM. (2002) Characterization of two CTR-like protein kinases in Rosa hybrida and their expression during flower senescence and in response to ethylene. Journal of Experimental Botany 53:1223–1225. Müller R, Stummann BM, Serek M. (2000b) Characterization of an ethylene receptor family with differential expression in rose (Rosa hybrida L.) flowers. Plant Cell Reports 19:1232–1239. Narumi, T., Aida, R., Ohrniya, A., and Satoh, S. (2005a). Transformation of chrysanthemum with mutated ethylene receptor genes: mDG-ERS1 transgenes conferring reduced ethylene sensitivity and characterization of the transformants. Postharvest Biol. Technol., 37: 101-110. Owino, W.O., Nakano, R., Kubo, Y., and Inaba, A. (2002). Differential regulation of genes encoding ethylene biosynthesis enzymes and ethylene response sensor ortholog during ripening and in response to wounding in avocados. J. Am. Soc. Hort. Sci., 127: 520-527. Pang, J.H., Ma, B., Sun, H.J., Ortiz, G.I., Imanishi, S., Sugaya, S., Gemma, H. and Ezura, H. (2006). Identification and characterization of ethylene receptor homologs expressed during fruit development and ripening in persimmon (Diospyros kaki Thumb.). Postharvest Biol. Technol., 44: 195-203. Patterson, S.E. and Bleecker, A.B. (2004). Ethylenedependent and -independent processes associated with floral organ abscission in Arabidopsis. Plant Physiol., 134: 194-203. Rasori, A., Ruperti, B., Bonghi, C., Tonutti, P., and Ramina, A. (2002). Characterization of two putative ethylene receptor genes expressed during peach fruit development and abscission. J. Exp. Bot., 53: 2333-2339. Rubinstein, B. (2000). Regulation of cell death in flower petals. Plant Mol. Biol., 44: 303-318. Serek, M., Woltering, E.J., Sisler, E.C., Frello, S., and Sriskandarajah, S. (2006). Controlling ethylene responses in flowers at the receptor level. Biotechnol. Adv., 24: 368-381. Shibuya, K., Barry, K.G., Ciardi, J.A., Loucas, H.M., Underwood, B.A., Nourizadeh, S., Ecker, J.R., Klee, H.J. and Clark, D.G. (2004). The central role of PhEIN2 in ethylene responses throughout plant development in petunia. Plant Physiology, 136:2900–2912. Tahashi, H., Kobayashi, T., Sato-Nara, K., Tomita, K.O., and Ezura, H. (2002). Detection of ethylene receptor protein Cm-ERSI during fruit development in melon (Cucumis melo L.). J. Exp. Bot., 53: 415-422. Tan, H., Liu, X., Ma, N. Xue, J., Lu, W.L., Bai, J., and Gao, J. (2006). Ethylene-influenced flower opening and expression of genes encoding Etrs, Ctrs, and Ein3s in two cut rose cultivars. Postharvest Biol. Technol., 40: 97-105. Tang X and Woodson WR. (1996) Temporal and spatial expression of 1-aminocyclopropane-1-carboxylate oxidase mRNA following pollination of immature and mature petunia flowers. Plant Physiology 112:503–511. Tieman, D., and Klee, H. (1999). Differential expression of two novel members of the tomato ethylenereceptor family. Plant Physiol. 120, 165–17 Trainotti, L., Pavanello, A., and Casadoro, G. (2005). Different ethylene receptors show an increased expression during the ripening of strawberries: does such an increment imply a role for ethylene in the ripening of these non-climacteric fruits? J. Exp. Bot., 56: 2037-2046. Wilkinson, J. Q., Lanahan, M. B., Clark, D. G., Bleecker, A. B., Chang, C., Meyerowitz, E. M. and Klee, H. J. (1997). A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous plants. Nature Biotechnology, 15:444–447. Woodson, W.R. (1994). Molecular biology of flower senescence in carnation. In Molecular and Cellular Aspects of Plant Reproduction (R.J. Scott and A.D. Stead, Eds.), pp. 225-267. Cambridge University Press, Cambridge, UK. Yamamoto, K., Komatsu, Y., Yokoo, Y., and Furukawa, T. (1994). Delaying flower opening of cut roses by cis- propenylphosphonic acid. J. Jpn. Soc. Hort. Sci., 63: 159-166. 146 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ROLE OF PLANT HORMONES IN FLOWER SENESCENCE Vanita Jain and R.K Sairam Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110012 Senescence is the age-dependent deterioration process at the cellular, tissue, organ, or organismal level, leading to death or the end of the life span (48). Flower senescence is organ level senescence but is often intimately associated with cellular or organismal death. Annual plants undergo flower senescence along with the organismal-level senescence when they reach the end of their temporal niche, as we observe at the grain-filling and maturation stage of the crop fields of soybean, corn, or rice. Flower senescence The flower is one of the most ephemeral of plant organ systems because it is specialized for the specific functions of pollen dispersal and reception, after which time many individual floral organs senesce while others develop further to form seeds and fruit. This transition in flower function involves the programmed senescence of the petals and sepals. Perianth senescence of some flowers occurs as part of a temporal program with the petals and sepals senescing strictly as a function of age. For example, daylily flowers senesce 12 ± 18 h after flower opening (Lukaszewski and Reid, 1989; Lay-Yee et al, 1992). The endogenous signals that regulate age-dependent petal senescence are completely uncharacterized, although the process is accompanied by the regulated expression of a suite of genes, some of which are functionally related to those associated with leaf senescence. Other flowers, such as petunia, gradually senesce over a period of days after flower opening, but this process is accelerated by pollination. In still other flowers perianth senescence is absolutely dependent on pollination and in these cases the external stimuli and endogenous signals that regulate programmed senescence have been examined in detail (O’Neill et al, 1993; O’Neill and Nadeau, 1997; O’Neill, 1997). Endogenous signals regulating pollination induced flower senescence In flowers whose senescence is pollinationdependent or pollination-accelerated, including petunia, carnation, cyclamen and orchids, senescence and the pollination event is accompanied by a sudden and rapid increase in endogenous ethylene production (Nichols, 1966; Bufler et al, 1980; Halevy et al, 1984; Nichols et al, 1983; Whitehead et al, 1983; Hall and Forsyth, 1967; Porat et al, 1994). Indeed, it has been known for over 30 years that senescence of certain orchid flowers was accompanied by ethylene evolution and that senescence could be induced by application of exogenous ethylene (Akamine, 1963). Most importantly, the effects of exogenous ethylene could be mimicked by pollination, which indicated that ethylene played a central role in signaling the onset of programmed flower senescence. Because the stigma is the initial site of perception of pollination, the initial pollination signal must be transduced and translocated to promote senescence processes in the distal organs of the flower, such as the perianth. A detailed characterization of transduction of the pollination signal in orchid flowers indicates that the initial pollination event promotes the synthesis of the immediate precursor of ethylene, aminocyclopropane-carboxylic acid (ACC), in the stigma by induction of expression of an ACC synthase gene. ACC produced in the stigma may be oxidized directly to ethylene or potentially translocated to distal floral organs where it serves as substrate for ACC oxidase, the enzyme responsible for its conversion to ethylene. This model suggests that inter-organ transduction of the pollination signal occurs by translocation of a hormone precursor, but that the endogenous signal initiating programmed senescence of flower organs is ethylene. This conclusion is supported by tomato mutants defective in ethylene 147 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India perception in which the flowers do not senesce (Lanahan et al, 1994). In this regard, the regulation of flower senescence differs fundamentally from the regulation of leaf senescence, where ethylene appears to only modify the primary signals that underlie the senescence process. In this chapter, however, we will discuss role of plant hormones other than ethylene on flower senescence. Gibberellins (GAs) The role of GAs in petal growth has been demonstrated in many plants. Analysis of endogenous GAs in young petunia anthers and corolla indicated the presence of biologically active GA1 and GA4. In Arabidopsis nap gene is a good candidate to play a role in the regulation of the transition to the second phase of corolla development, which involves rapid petal expansion (Sablowski and Meyerowitz 1998). Since, Arabidopsis petal elongation requires GA (Koornneef and Van der Veen 1980), it is possible that nap gene expression is indeed induced by the hormone. Weiss (2000) reported that GAs controls pigmentation only in those cases where pigment accumulation is directly tied to the petal cell expansion. This effect of GA3 is not exclusive to the anthocyanin biosynthetic genes, but it also regulate some others gene on the corolla, such as gibberellin-induced gene gip, (Ben-Nissan and Weiss 1996) expressed in corollas during cell elongation, is specifically induced by GA3. Although the function of GIP protein is still unknown, Kening (1985) found a sharp increase in GA concentrations in Gaillardia petals with fast growth of corolla, followed by a decrease at later stage, which indicates that even at low concentration, GA may induce an entire developmental program, probably via the activation of master regulatory genes. These factors may induce genes from various pathways. A MYBtype regulatory gene, myb-92 was cloned from petunia corolla and its expression was found to be induced by GA. Although myb-92 may be a master regulator in the GA-signal cascade, its target DNA sequences are still unknown (Mur 1995). Sabehat and Zieslin (1994) showed that GAs might play a role in preserving membrane integrity and controlling the cell membrane permeability of rose petals. Observations from the work of Kuiper et al. (1991) suggests GAs can change the capacity for sugar uptake in rose petals and seems to stimulate active sucrose uptake. Results of Ganelevin and Zieslin (2002) indicate that sepals may be a source of GA during flower bud development. Removal of sepals reduced fresh and dry weights, as well as length of bud and the peduncle. External application of GA3 completely restored the length of the peduncle. Besides this, formation of star-shaped abnormality in rose flowers exposed to external ethylene was completely prevented by applying GA3 after the petals were excised. This supports the role of GA in reducing the sensitivity of rose flower to ethylene. GAs modified the climacteric ethylene rise in a manner that consists with the extension of longevity (Saks et al. 1992). Abscisic acid (ABA) As early as 1972, Mayak and Halvey showed that endogenous concentration of ABA increased in rose petals as the flower senesced. Moreover, higher ABA concentrations were found in short lived cultivars compare to long-lived cultivars (Halvey and Mayak 1975). These results indicate that ABA participates in the endogenous regulation of senescence processes in rose flower (Halvey and Mayak 1981). Borochov et al. (1976) reported that ABA accelerates senescence of cut roses by promoting petal growth and respiration, thus decreasing carbohydrate concentration in the petals and triggering the chain of metabolic processes leading to ageing. During the vase life of a rose flower ABA concentration decline during the first 3 days, followed by a steady state at a low level, and finally a sharp increase in the late senescence (Le Page-Degivry et al. 1991). Additionally, a direct relationship between petal ABA concentration and flower longevity was observed, the higher the ABA concentration at harvest, the shorter the subsequent vase life (Muller et al. 1999a). This correlation was verified for roses of different genetic origin as well as for the same cultivars grown under different cultural conditions. Increasing 148 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India evidence suggests that ABA is a natural regulator of petal senescence in flowers (Hunter et al. 2004). The hormone is present in higher amounts in naturally senescing petals (Wie et al. 2003), and in petals senescing pre-maturely in response to water stress (Panavas et al. 1998a, b), or an alteration in light quality (Garello et al. 1995). The senescence caused by ABA is mediated through ethylene in many cultivars of roses as exogenously applied ABA does not cause senescence when pre-treated with inhibitors of ethylene action (Muller et al. 1999b). In daylilies, the ABA concentration of petals increased before the increase in activities of hydrolytic enzymes (Panavas et al. 1998a, b), whereas in roses, ABA concentration increases comparatively late in the petals, 2 days after the surge in ethylene production (Mayak and Halvey 1972). However, in daffodil ABA accumulated in sepals in a biphasic manner. It initially increased as the flowers opened and then later as they senesced (Hunter et al. 2004). The role of this early increase in ABA is not clear. The ability of exogenous ABA to cause senescence could be inhibited by treatment of the flower with GA3 (Hunter et al. 2004). It is likely that the significance of ABA in floral senescence will only become clear when ABA biosynthesis or action can be specifically inhibited in floral tissue. Kumar et al. (2008) observed several fold increase in ABA concentration during the later stages of rose flower senescence, which was found to be associated with a drastic reduction of flower water potential and water uptake. During the later stages of senescence higher ABA concentration coincides with the elevated concentration of ethylene production. ABA and ethylene both stimulate senescence and are suggested to interact during flower senescence under water limitations. Menard and Dansereau (1995) observed an interaction between N-supply, ABA levels and vase life of cut rose flowers. Longevity was greater when plants were previously grown on a medium supplemented with low nitrogen than when supply was higher, as the high pre-harvest nitrogen induced higher and early rise in ABA, resulted in shorter vase life. Cytokinins Cytokinins are known to defer leaf senescence (Richmond and Lang 1957) and improve the keeping qualities of cut carnation (Heide and Oydvin, 1969, Maclsan and Dedolph 1962) and rose flowers (Mayak and Halevy. 1970). MacLsan and Dedolph (1962) reported a decrease in the respiration rate of cytokinintreated flowers and proposed that cytokinins increase flower longevity as a result of this reduction in respiration. However, Heide and Oydvin (1969) found only small and inconsistent effects of cytokinins on the respiration rate of cut carnation flowers. They concluded that processes other than respiration mediate the cytokinin retardation of senescence. Mayak and Halevy (1974) have shown that kinetin increases net water uptake of expanding rose petals and delays wilting of petals especially when flowers are subjected to heat (28 ºC) and low relative humidity (40-50%). Kinetin had no effect on protein content of rose petals under these stressful conditions, but kinetin retarded the increase in RNase activity normally seen in rose flowers at the onset of senescence. Kinetin is proposed to increase rose flower longevity by improving water balance and delaying senescence processes. Eisinger (1977) suggested that kinetin may be extending the life span of cut carnation flowers because it is replacing the natural cytokinins, which are normally supplied to the flower from the parent plant. The fact that kinetin, a synthetic cytokinin, increases the life span of cut flowers, especially those with short stems (3 cm), indicates that the natural antisenescence factor in carnation flowers may be endogenous cytokinins. This is further supported by the fact that increased stem length provides no significant additive effect with kinetin-treated flowers. The increased longevity of cytokinin-treated flowers might be the result of many different physiological effects of the hormone on the flower tissues. They may operate by maintaining membrane permeabilities (Kende and Baumgartner (1974), water balance (Mayak and Halevy 1974), and/ or protein and nucleic acid metabolism (Osborne 149 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India 1962). The presence of optimal concentrations of cytokinins may also increase longevity of flowers by reducing their ethylene production and responsiveness. Research with rose flowers suggests that cytokinins play a role in natural senescence. Rose flowers show a peak in natural cytokinins levels as the flower opens, but decline as the flower ages on the parent plant (Mayak et al.1972). Also, higher natural cytokinin levels were found in more long lived rose varieties (Muller et al. 1999b). When flowers are detached from the parent plant, the natural cytokinin source (the roots) would be lost and internal levels would decline (Weiss and Vaadis 1965). This decline in endogenous cytokinin levels could serve as a trigger for senescence initiated by increased ethylene production and responsiveness by the cut flowers. Intact flowers might also use this trigger since their endogenous cytokinin levels decline with age (Mayak et al.1972). In nature, pollination often serves as the trigger for increased ethylene production (Buag and Dukan 1967), and as a result of this ethylene exposure, carnation flowers begin the process of senescence 2 to 4 days after pollination (Nichols 1966). Using a different tissue system, Kende and Hanson (1976) reported that BA delayed ethylene production by isolated rib segments of Ipomoea tricolor (morning-glory) flowers and the rolling up response of these rib segments. Thus, the morning glory flowers show the same kind of delay of ethylene production as do carnation flowers in response to cytokinin treatment. The reduced responsiveness to exogenous ethylene seen with kinetin treated carnations was not seen in the leaf rolling up response of their rib segments with BA treatment. Cytokinins slow down the process of senescence possibly by their ability to promote the transport, accumulation and retention of metabolites in tissues and organs; besides protecting membranes against degradation (Beckman and Ingram, 1994). Applied cytokinins have been shown to slow down the aging process in rose and carnation petals (Borochov and Woodson, 1989; Van Staden et al., 1990). Gulzar et al. (2005) reported that treatment of partially open flowers with BAP or Kinetin were the effective treatments in delaying senescence, maintaining flower quality and thereby prolonging longevity of isolated flowers of Hemerocallis fulva. In these treatments lesser ion leakage, besides higher fresh mass and water content was recorded suggesting maintenance of membrane integrity. These treatments also maintain the respiratory pool of sugars in the perianth tissues, besides being effective in slowing down protein degradation with flower opening and senescence. Serek and Andersen (1995) also reported that higher concentration of benzyl adenine (0.8 mM) was the best in increasing the longevity of post harvest Rosa hybrida L. ‘Victory Parade’ flowers, and concluded that a higher concentration than 0.8 mM give additional improvement of the measured parameters. Sankhla et al. (2003) reported the effect of synthetic cytokinins thidiazuron (TDZ) on Cut Phlox Inflorescences. Treatment with TDZ greatly reduced the shedding of flowers and, induced opening of a significant number of additional flower buds during vase life. TDZ also counteracted the flower abscissionaccelerating and leaf-senescence promoting effect of abscisic acid. These results indicate that TDZ may prove highly useful in improving post-harvest performance of phlox cut flower heads. Chang et al. (2003) transformed petunia flowers over-expressing ipt, a cytokinin biosynthetic gene from Agrobacterium tumefaciens. The transformed lines have elevated cytokinin content and extended flower longevity. Floral senescence in these lines was delayed 6 to 10 d relative to wild-type (WT) flowers. Ipt transcripts increased in abundance after pollination and were accompanied by increased cytokinin accumulation. Though endogenous ethylene production was induced by pollination in both WT and IPT corollas, but this increase was delayed in IPT flowers. Flowers from IPT plants were less sensitive to exogenous ethylene and required longer treatment times to induce endogenous ethylene production, corolla senescence, and up-regulation of the senescence-related Cys protease phcp1. 150 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Polyamines Polyamines, ubiquitous in plants and known to be associated with cell division and growth, have been shown to act as anti-senescence agents in a number of tissues (Altman 1982, Galston and KaurSawhney 1987, Kaur-Sawhney et al. 1978, 1982a, 1982b, Muhitch et al.1983, Popovic et al.1979, Roberts et al.1984, Shih et al.1982, Srivastava et al.1983). The most commonly known of these polycationic, bioregulatory compounds are putrescine, spermidine and spermine. The diamine putrescine is produced from the amino acids, arginine and ornithine, and gives rise to the triamine spermidine and tetramine spermine, through the addition of aminopropyl groups from decarboxylated SAM (Adiga and Prasad 1985). Wang and Baker (1980) demonstrated that polyamines applied with a preservative solution increased the longevity of carnation flowers. However, a similar experiment indicated that ethylene production was unchanged by increases in the levels of endogenous polyamines within the floral tissues. Inhibition of polyamine production, however, did result in increased ethylene level and reduction in flower longevity. Upfold and Van Staden (1991) showed that, in open flowers, putrescine was present in the petals at harvest. It increased in the petals, and then decreased to undetectable levels at day 9 when the flowers had senesced. As the ovary started to show senescenceinduced development, which coincided with petal senescence and inrolling (day 6), putrescine increased and reached a high level at day 9. The further reported that no detectable endogenous levels of polyamines were recorded in young flower buds, which could reflect extensive polyamine metabolism and utilization or could be due to the fact that these compounds are only produced during later stages of flower development. Flower senescence was also delayed by the application of low levels of putrescine and spermine, indicating that when these compounds are available at higher levels at the early stages of lower development, beneficial effects are derived with respect to petal senescence. Burtin et al (1991) reported that increased accumulation of polyamine conjugates occurs in parallel to enhanced activity of ornithine decarboxylase (ODC), which is responsible for PA synthesis, at the time of flowering initiation. This coincides with translocation of conjugates from leaves to the young floral tissue (Havelange et al 1996). They may act as storage forms of PAs, from which the free bases may be released during growth or these conjugates may be transported as and when required (Martin-Tanguy 1997). The involvement of PAs in floral induction has been documented by spraying the compounds directly on the plants. In Iris hollandica buds, Spd has been suggested to be a marker for floral induction. In Douglas fir, Pseudotsuga menziesii, putrescine was the dominant PA in vegetative buds, while Spd predominated in floral buds (Kakkar and Rai 1993, Kumar et al. 1997). Singh et al. (2005a) reported that polyamines like spermine, spermidine and putrescine at 100 ppm, applied alone or in combination with sucrose in vase solution were effective in extending vase life of gladiolus. Among the three, 100 ppm spermine along with sucrose in vase solution was also able to keep fresh weight of gladiolus spikes higher for longer duration in comparison to other treatments due to higher uptake of holding solution. Aesthetic look of cut spikes in terms of floret diameter, turgidity and freshness was also better in this treatment. They concluded that increase in vase life by 100 ppm spermine or spermidine + 2 % sucrose in vase solution was due to an increase in protein content and activities of antioxidant enzymes viz., superoxide dismutase, ascorbate peroxidase, glutathione reductase and Catalase. References Adiga RR and Prasad GL (1985) Biosynthesis and regulation of polyamines in higher plants . Plant Growth Regul 3 : 205-226 Akamine, E.K. (1963). Ethylene production in fading Vanda orchid blossoms. Science 140: 1217 1218 151 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Altman A (1982) Retardation of radish leaf senescence Galston AW and Kaur-Sawhney R (1987) Polyamines by polyamines . Physiol Plant 54 : 189-193 and senescence in plants . In : Med. 5: 39-49. Beckman, K.B. and Ingram, D.S. (1994). The Ganelevin, R., Zieslin, N. (2002). Contribution of inhibition of the hypersensitive response of sepals and gibberellin treatments to growth and carnation flowers. VIII. Uptake, transport and development of rose (Rosa hybrida) flowers. Plant metabolism of benzyladenine and the effect of Growth Regul. 37:225–261 benzyleadenine derivatives on flower longevity. Garello, G., Menard, C., Dan Seraeu, B., Le PageJ. Plant Physiol. 135: 703 -707. Degivry, M.T. (1995). The influence of light quality Ben-Nissan, G., Weiss, D. (1996). 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Regulation of ABA AW (1982a) Relation of polyamine synthesis and levels in senescing petals of rose flowers. J. Plant titer to ageing and senescence in oat leaves . I . Growth Regul. 10: 67–72 Plant Physiol 69: 405-410 Kaur-Sawhney R, Shih, LM, Flores HE and Galston Lukaszewski, T.A. and Reid, M.S. (1989). Bulb-type flower senescence. Acta Hort. 261: 59-62 AW (1982b) Relation of polyamine synthesis and titer to ageing and senescence in oat leaves . II. Maclsan, D.C., Dedolph, R.R. (1962). Effects of N’Plant Physiol 69 : 411-415 benzylaminopurine on postharvest respiration of Chrysanthemum morifolium and Dianthus Kende, H., Baumgartner, B. (1974). Regulation of caryophyllus. Bot. Gaz. 124: 20-21 aging in flowers of Ipomoea tricolor by ethylene. Planta 116: 279-289 Martin-Tanguy, J. (1997). Conjugated polyamines and reproductive development: Biochemical, Kende, H., Hanson, A.D. (1976). Relationship between ethylene evolution and senescence in molecular and physiological approaches. Physiol. morning-glory flower tissue. Plant Physiol. 57: Plant. 100 675–688 523-527 Mayak, S., Halvey, A.H. (1970). Cytokinin activity in Kening, R.E. (1985). Gaillardia. In: Halevy AH (ed). rose petals and its relation to senescence. Plant Handbook of flowering, vol 5. CRC, Press, Boca Physiol. 46: 497-499 Raton, pp 117–126 Mayak, S., Halvey, A.H. (1972). Interrelationship of Koornneef, M., Van der Veen, J.M. (1980). Induction ethylene and abscisic acid in the control of rose petal senescence. Plant Physiol. 50: 341–346 and analysis of gibberellin sensitive mutant in Arabiodopsis thaliana (L.) Heynh. Theo. Appl. Mayak, S., Halvey, A.H. (1974). The action of kinetin Gene. 58: 257–263 in improving the water balance and delaying senescence processes of cut rose flowers. Plant Kuiper, D., Van Reenen, H.S., Ribot, S.A. (1991). Physiol. 32: 330-336 Effect of gibberellic acid on sugar transport in to petals of ‘Madelon’ rose flowers during bud Mayak, S., Halvey, A.H. Katz, M. (1972). Correlative opening. Acta Hort. 298: 93–95 changes in phytohormones in relation to senescence processes in rose petals. Physiol. Kumar, A., Altabella, T., Taylor, M.A. and Tiburcio, Plant. 27: 1-4 A. F. (1997). Recent advances in polyamine research. Trends Plant Sci. 2:124–130 Menard, C. and Dansereau, B. (1995). Influence of nitrogen supply on ABA levels and flower Kumar, N., Srivastava, G.C., Dixit, K. (2008). senescence in Rosa Hybrida cv. Royalty. Acta Hormonal regulation of flower senescence in roses Hort. 424: 151-155. 153 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India spermine during stress-induced senescence of Muhitch MJ, Edwards LA and Fletcher JS (1983) barley leaves . Plant Physiol 64: 721-726 Influence of domancy and polyamines on senescence of plant suspension cultures. Plant Cell Porat, R., Borochov, A., Halevy, A.H. and O’Neill, Resp . 2 : 82-84 S.D. (1994). Pollination-induced senescence of Phalaenopsis petals ± The wilting process, Muller, R., Andersen, A.S., Serek, M. (1999b). ethylene production and sensitivity to ethylene. Differences in display life of miniature potted Plant Growth Regl. 15: 129 - 136 roses. Scientia. Hort. 76: 59–71 Muller, R., Stummann, B.M., Andersen, A.S., Serek, Richmond, A., Lang, A. (1957). Effect of kinetin on protein content and survival of detached Xanthium M. (1999). Involvement of ABA in postharvest leaves. Science 125: 650-651 life of miniature potted roses. Plant Growth Regul. 29: 143–150 Roberts DR, Walker MA, Thompson JE and Dumbroff EB (1984) The effects of inhibition of Nichols, R. (1966). Ethylene production during polyamine and ethylene biosynthesis on senescence of flowers. J. Hort. Sci. 41: 279-290 senescence, ethylene production and polyamine Nichols, R., Bufler, G., Mor, Y., Fujino, D.W. and Reid, levels in cut carnation flowers . Plant Cell Physiol M.S. (1983). Changes in ethylene production and 25 : 315-322 1-aminocyclopropane-1-carboxylic acid content of pollinated carnation flowers. J. Plant Growth Sabehat, A., Zieslin, N. (1994). GA3 effects on postRegl. 2: 1 - 8 harvest alterations in cell membranes of rose (Rosa × Hybrida) petals. J. Plant Physiol. O’Neill, S.D. (1997). Pollination regulation of flower 144:513–517 development. Ann. Rev. Plant Physiol. Mol. Biol. 48: 547 - 572 Sablowski, W.M., Meyerowitz, E.M .(1998). A homology of NO APICAL MERISTEM is an O’Neill, S.D. and Nadeau, J. (1997). Postpollination immediate target of the floral homeotic genes flower development. Hortic. Rev. 19: 1-58 APETLA3/PISILLATA. Cell 92:93–103. O’Neill, S.D., Nadeau, J.A., Zhang, X.S., Bui, A.Q. and Halevy, A.H. (1993). Interorgan regulation Saks, Y., Van Staden, J., Smith, M.T. (1992). Effect of gibberellic acid on carnation flower of ethylene biosynthetic genes by pollination. Plant senescence: evidence that the delay of carnation Cell 5: 419 -432 flower senescence by gibberellic acid depends Osborne, D. (1962). Effects of kinetin on protein and on the stage of flower development. Plant Growth nudeic acid metabolism in Xanthium leaves during Regul. 11:45–51 senescence. Plant Physiol. 37: 595-602 Sankhla, N., Mackay, W.A. and Davis, T.D. (2003). Panavas, T., Reid, P.D., Rubinstein, B. (1998b). Reduction of flower abscission and leaf Programmed cell death of daylily petals: activity senescence in cut Phlox inflorescences by of wall based enzymes and effects of heat shock. thidiazuron. Acta Hort. 628: 837-841 Plant Physiol. Biochem. 36: 379–388 Serek, M. and Andersen, A.S. (1995). The possibility Panavas, T., Walker, E.L., Rubinstein, B. (1998a). for improvement of the longevity of intact flowers. Possible involvement of abscisic acid in Acta Hort. 404: 48-57 senescence of daylily petals. J. Exp. Bot. 49: Shih, LM, Kaur-Sawhney R, Fuhrer J, Samanta S 1987–1997 and Galston AW (1982) Effects of exogenous Popovic RB, Kyle DJ, Cohen AS and Zalik S (1979) 1,3-diaminopropane and spermidine on Stabilization of the thylakoidmembrane by senescence of oat leaves . Plant Physiol 70:15921596 154 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Singh VP (2005) Influence of 8-hydroxy Quinoline (8HQ) and sucrose pulsing on membrane stability and post harvest quality of gladiolus cut spikes. J ornamental Hort. 8: 243-248. Wang CY and Baker JE (1980) Extending vase life of carnation with amino-oxyacetic acid, polyamines, EDU and CCCP. Hort Sci 15 : 805806 Srivastava SK, Vashi DJ and Naik BJ (1983) Control Weiss, C., Vaadis, Y. (1965). Kinetin activity in root apices of sunflower plants. Life Sci. 4: 1323of senescence by polyamines and guanidines in 1326 young and mature barley leaves . Phytochem 22 : 2151-2154 Weiss, D. (2000). Regulation of flower pigmentation and growth: multiple signaling pathways control Upfold SJ and Van Staden J (1990) Cytokinins in cut anthocyanin synthesis in expanding petals. carnation flowers: VII. The effect of zeatin and Physiol. Plant. 110:152–157 dihydrozeatin derivatives on flower longevity. Plant Growth Regul 9: 77–81. Whitehead, C.S., Fujino, D.W. and Reid, M.S. (1983). Identification of the ethylene precursor, Van Staden, J. Bayley, A.D., upfold, S.J. and Drewes, 1-aminocyclopropane-1-carboxylic acid (ACC) F.E. 1990. Cytokininis in Cut carnation flowers. VIII. Uptake, transport and metabolism of in pollen. Scienti. Hort. 21: 291 – 297 benzyladenine and the effect of benzyleadenine Wie, Z., Zhang, H., Gu, Z.P., Zhang, J.J. (2003). Cause derivatives on flower longevity. J. Plant Physiol. of senescence of nine sorts of flowers. Acta Bot 135: 703 -707. Sinica 33: 429–436 155 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ANTIOXIDANTS IN FRUITS AND VEGETABLES: A VARIETAL PROFILE Charanjeet Kaur Division of Post Harvest technology, Indian Agricultural Research Institute, New Delhi-110012 Food research in the last decade has been the most spectacular and intriguing one. The adage of Hippocrates “let thy food be your medicine and medicine be food” has become the credo of modern dietians and nutritionists. Research has proven the truth of his words raising nutrition science to status equal to that of a medicine. Discovery of antioxidant phytochemicals and their promising health promoting effects have paved the way to a new revolution what is termed as a food revolution. A revolution promising a age of good health. A continuous development in understanding relationship between the food genotype and diet related diseases has initiated an exciting era for nutritional sciences. Pleothera of review articles on enormous potentiality of these antioxidants have geared up researchers in area of food processing to explore their foods with a new perspective. Epidemiological studies and successive scientific investigations have transformed foods into functional foods and neutraceuticals as important tools in promoting health and reducing health care tools. The role of traditional antioxidants such as ascorbic acid and carotenoids has now taken aback seat. Phenolic and flavonoids are emerging as ideal candidates for assuming the status vitamins and guardians of health. Food producers and processors increasing interest in developing nutraceutical is fuelled by the increasing interest of consumers in understanding the relationship between diet and health. In this context, it is relevant to know which health promoting compounds are present in raw materials their concentration and total antioxidant effects which governs their health promoting effects. In this context it is of paramount importance to examine varieties, cultivars for their antioxidant content and antioxidant activity for exploring ideal genotypes high in antioxidants. Researchers unanimously agree that their exists wide variation that exists among genotypes of a specific crop. This offers a great deal of opportunity for enhancing the levels of antioxidants and their antiproliferative and chemo protective properties through genetic manipulation. Higher levels of antioxidants in advanced breeding lines and many wild types as reported by many workers shows that there is great opportunity to increase the overall antioxidant status of crops by conventional means. Such a information could be incorporated into the breeding programmes for breeding antioxidant rich high quality varieties. Utilization of such varieties for processing into functional foods will add additional value to these delicious fruits and vegetables. They can further be used for isolation of bioactive principles for development of neutraceuticals. Berries Berry fruits are an important source of vitamin C apart from bioactive non-nutrient such as phenolic compounds and flavonols.. They have been shown to be effective in inhibiting oxidation of human low-density lipoproteins, which may have potential health effects In fact Vaccinium sp (Ericaceae) which includes low bush, high berries, blue berry, berry cranberry and ligonberry are richest sources of anthocyanins, flavonoids, pro-anthocyanidin and other phenolics which are stable than other significant constituents such as vitamin C. Anthocyanins have been shown to strong antioxidants with free radical scavenging properties attributed to phenolic hydroxy groups attached to ring structures. They offer protection against free radical damage and low-density lipoprotein oxidation, platelet aggregation, and endothelium-dependent vasodilation of arteries. Cranberries and blue berries designated to be number of one antioxidant fruits are reported to contain proanthocyanidin that are useful in treating urinary tract infections. Various pharmaceutical products prepared from Vaccinium species have been 156 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India accounting for high antioxidant activity. Importance of polyphenolics, particularly in apple flesh has already been overemphasized Vitamin-C was responsible for less than 0.4% of the AOX indicating the contribution of phenols. In a recent study, Monroe variety had In an elaborate study, with 87 cultivars of the highest content of flavonoids, quercetin glycosides Vaccinium, a wide variation has been observed ( 4.44mg/100g), which accounted for the highest AOX between varieties. Cultivars Elliot, ornablue, friendship, (40.39VCEAC). Apple peels are an exceptional good source and Burlington also had appreciably high ORAC values. Considerable common ancestry in these high ORAC of phenolic compounds While flesh contains catechins, clones, cv. Burlington and Rancocas has been reported procyanidin, phloridin, phloetus, glycosides, caffeic a due to presence of Rubel as a parent. Both total procyanidin, caffeic a chlorogenic acid, the peels, phenolics and anthocyanin exhibited a four-fold possess additional flavonoids quercetin along with their difference between low and high values. Since the compounds. Peels of Ida red and Rome Beauty apple majority of antioxidants are concentrated in skin and are reported to have the highest total phenolics and seeds, the expression of antioxidant activity as ORAC/ flavonoid content and correspondingly AOA. cm2 seems to be more meaningful as breeders are Plums interested in high antioxidant concentration per unit area Fresh plums are rich source of phenolic of skin. Vaccinium pallidum was found to represent compounds, many of them concentrated in the skin. a valuable source of genes for achieving AOX. In another study of twenty clones of low and high bush, High antioxidant activity of plums has been mainly due despite environmental factors, all low bush blue berries to their high content of polyphenols. Skin contains clones had higher levels of anthocyanin, total phenols about five times more phenolics per unit than the pulp. and ORAC as compared to high bush berries. Although Neochlorogenic acid (3-O-caffeoylquinic acid) was a environmental factors can significantly influence predominant polyphenol in the fresh or dried plum and synthesis of compounds total phenols and anthocyanin quercetin 3-rutinoside, the major flavonol in plums. In responsible for high ORAC, however, authors are of addition, all the cultivars except yellow plums the opinion that species or genotypes still remain to be commonly contain anthocyanins such a s cyaniding 3dominant factor. Cranberries (V. macrocarpon Aiton) glucoside and cyanidin3-rutinoside exocarp also among Vaccinium genus represents another potential contains significant quality of rutin (2.2 mg/g d.w.) and catechins (0.74 mg/g d.wt.). Apart from polyphenols source of anthocyanins and total polyphenols. fresh plums are rich source of sorbitol and dietary fibre Strawberry fruits have been found to have high which makes them ideal for laxative. Plums had lower AOX as indicated by ORAC assay than plums, orange, ORAC value (9.5 uM Te/g), second to strawberries. red grapes, and apple. Cultivar Earliglow, kent have Royal Garnet, Beltsville, Elite B 70197, cacak and been found to be promising cultivar of strawberry French damson genotype plum had total highest phenol with highest antiproliferative activity. content of 534.8 mg/100 g among the 16 fruits analysed Apples including apple. primarily due to their antioxidant action Blue berries are one of richest sources of antioxidant phytochemicals encountered with AOX as high as 45.9 umol TE/g and total phenolic content (5 g/kg), further emphasising the link with in vitro AOA and phenolic content. Consumption of apples has been linked to the prevention of chronic disease, lung cancer) risk of thermobiotic risk. Apples are significant part of diet in Netherlands, Finland and United States . The red colour of apple peels is due to presence of cyanidin 3galactoside . Phenolics are one of the major constituents Broccoli Broccoli is a potential vegetable high in antioxidants, is known to contain diverse antioxidants including carotenoids, tocopherol, ascorbic acid and flavonoids. The antioxidant AOX varied 10 fold from variety to variety. This was apparently explained due 157 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India to influences of genotype, location, and harvest conditions on biosynthesis of the natural secondary metabolites accounting for differences in AOA. MA 191, Packman, Ev6-1, Brigadier, Peto37, Vi158, Majestic and Peto 315 were found to be promising varities high in antioxidants. Capsicum Capsicum is a versatile vegetable rich in array of phytochemicals, however variable at green and red stage. Green cultivars of TAES Hidalgo and red cultivars of Rio grande gold are characterized as high carotene containing varieties. Among spiced pungent paprika cultivar has been thoroughly investigated for its bioactive content and antioxidant activity. Hungarian cultivars km-622, K801, semi-determinate 7/92 had high total carotene content thus characterized as best for its bioantioxidant potency. Bibor and Napfeny were recognized as potential cultivars with Fruits of ‘Ancho’ type had the most β –cryptoxanthin and β—carotene while fruits of ‘Red cherry’ and ‘bell captain’ had the highest capsanthin and lutein respectively . Mature fruits of ‘Tabasca ’ and ‘Cayenne ’ pepper types had high capsanthin whereas immature fruits of ‘Inferno’ and ‘Petocas’ had high flavonoid quercetin. Antioxidant activity was however high in ‘Petocasbella’ Onion Onion (Allium cepa ) among the vegetables is the richest source of flavonoids in human diet. Currently flavonoids are being considered as gold mines due to their potential role in preventing a wide range of degenerative physiological processes, including cancer, cardiovascular, diabetes, osteoporosis. Generally red and strong flavoured onions have higher total phenolics, flavonoid and high antioxidant activity , than white and mild flavoured onions. Quercetin is the major flavonoid in onions although minor amounts of kaempferol and myrecitin are also present. Shallots and strong flavoured western yellow, New York bold and northern red to be potential genotypes of united states having high total flavonoids and AOX. g . Kaempferol and myrecetin content was higher in Vidalia onion variety DPS 1032. Potential onion varieties rich in antioxidant have been identified by several workers. Tomato Tomato among vegetables tomato cultivars have been the subject of thorough investigation of its high and varied antioxidant content. Overwhelming epidemiological evidence that lycopene rich diets are protective against some cancer and cardiovascular diseases has led to the evaluation of wide genotypes of tomato for their antioxidant characterization. While the earlier focus of the research was on targeting the varieties with high lycopene content and high total soluble solids, the recent attention has been on phenolics and flavonoids. Tomatoes are rich in chlorogenic acids, flavonoids such as quercetin, kaempferol, naringenin and rutin. Cherry tomatoes containing high levels of antioxidants than the normal types of tomatoes have the subject of investigation by many workers. Cherry Siracursa and cherry Ragusa were found to potential Spanish varieties of having higher total carotene(13.19 mg/100g) and high antioxidant activity ( 0.55 Trolox or ascorbic acid/100 g respectively. Italian varieties of tomato however seem to dominate the tomato varities grown around the world. Genotypes, growing conditions and biotic and abiotic stresses seem to play an important role in affecting metabolism of antioxidant components and antioxidant capacity .Researchers agree that no clear advantage can be accrued from one single genotype, since a single genotype may not contain all potential antioxidants in their high doses. A genotype may contain high concentration of one bioactive compound whereas may be deficit in the other. Such variation offers a base for improving cultivars with health benefits. And strengthen the notion that health benefits can be realized by taking a mixture of antioxidants from a diet containing mixture of potential gentoypes. This also offers a great deal of challenge for the breeders to develop varieties and optimize agronomic practices to Red onion varieties are potential source of maintain a optimum balance of these health benefits quercetin content ranging from 99.7 – 118.7 mg/100 to produce varieties with high antioxidant capacity. 158 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India BIOTECHNOLOGY IN THE GARDEN Ajay Arora*, Gaurav Agarwal and Divya Choudhary Senior Scientist, Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-12 *Email: romiarora@yahoo.com The application of biotechnology has extended beyond the production of field crops and medicines. Biotechnology plays an important role in the fruit and ornamental market. From tissue culture to ‘molecular breeding’, biotechnology offers several valuable tools to those in the horticulture industry. At home a green thumb can grow a beautiful garden or flowerbed and accent the house with greenery. These plants can be nurtured from seeds or purchased ready to plant or pre-potted. Many plants offered for retail have origins in the biotech lab. Tissue culture is widely used for mass production, especially for plants, which reproduce asexually such as ferns and orchids. Plant tissue culture involves the cloning (genetically identical copies) of plants under laboratory conditions. Plants with desirable characteristics are identified and a small piece of the plant from an actively growing tissue is removed, sterilized and placed on a nutrient medium. The small piece of tissue is known as the meristem. The nutrient medium is designed to induce rapid shoot multiplication. This results in several clones of the original plant being produced over a short period of time. Once multiplication of the clones has taken place, the micro-plants are rooted and grown in a greenhouse. This process is known as micropropagation and is similar to taking clippings of a favorite plant at home. Tissue cultured plants provide many benefits such as uniform orchard development, uniform berry quality, rapid introduction of new cultivars, year round production, and vigorous high quality plants that are free of disease. What can be done when a plant does not have the desired characteristic? Genetic modification can be used to transfer this new trait into the plant. This may be done through traditional breeding or by using genetic engineering. Improvements to flowers – whether in shape, colour, vase life, disease resistance, or other characteristics – have historically been achieved by cross breeding existing varieties. This can be a long process, often 10 to 12 years, and may only produce a small change or improvement. For example, of the five leading cut flower species – rose, gerbera, lily, chrysanthemums, and carnation – none are easy to breed for blue flowers. This is because none contain the enzyme pathways to produce those pigments (delphinidin). Transgenic flowers can have colours in the blue to mauve range. Genetic modification technology can bridge the genetic gaps and rapidly deliver new traits to flower species. With biotechnology, the time to commercialize a new colour variety may be only 4 to 5 years. For many years, the quest for the “holy grail” in horticulture, the blue rose, never attained its ultimate goal. However, many breeders have anticipated this achievement because the commercial names of more than 100 cultivars refer to the term of blue, bleu (in French), or blau (in German). In fact, on the basis of Royal Horticultural Society (RHS) colour codes, and colorimetric measurements for some, the colour palette of these “blue” varieties covers the red-purple (RHS sheets 70-74), purple (sheets 75-79), purple-violet (sheets 80-82), and violet (sheets 83-88) groups and, exceptionally, the violet-blue group (sheets 89-98). The roses corresponding to the first two colour groups display a wide range of colour “intensities”, from light to dark coupled to dull to vivid colours, that is, from purplish-pink to purplish-red, whereas those in the second group are generally restricted to light and dull colours, such as lavender or mauve. On the basis of objective colour measurement, their basic tonalities correspond to hue angles on the CIELAB colour wheel approximately between 355 and 345* for the first 159 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India group and between 345 and 325* for the second one. The colour gap between these tonalities and really blue ones is impressive because blue colours cover the ~275-240* portion of the colour wheel. In the RHS colour chart, sheets 99-110 represent the blue group, and there is no rose cultivar having a colour code in this area. Recently, a newly introduced shrub rose cultivar was reported to represent “an important colour break”, and its colour was described as a striking “iridescent purple” that in overcast conditions looks remarkably blue. Its original name, cv. Frantasia, was finally changed to cv. Rhapsody in Blue when the rose was introduced into the commercial market. It also has won several awards. The availability of this plant material in quantities represented an interesting opportunity to investigate the physical and chemical basis of this apparently unusual colour in roses. “Cyanic” colours (red to purple and up to blue shades) are currently based on the presence of water-soluble anthocyanin pigments, the colour expression of which depends on multiple factors in the vacuolar compartment: the nature of the basic pigment itself; vacuolar pH effects; and the presence and relative concentration of other phenolic molecules named copigments. This study was undertaken to determine the factors responsible for the “blue” colour of the cv. Rhapsody in Blue rose variety. Blue roses and black orchids were among the first wonders promised by horticultural biotechnology. However, difficulty in achieving stable plant transformation and problems expressing heterologous genes has shown that genetic modification of ornamental crops is far more complicated than originally anticipated. Despite biotechnology’s tremendous success in improving traditional crop plants are still few and far between. Now thanks to recent advances in plant transformation technology and a better understanding of plant metabolic pathways, horticultural biotechnology’s time may have finally arrived. At the vanguard into ornamental plant biotechnology was Australia’s Florigene. In the early ‘90s, this company created a buzz with its wellpublicized attempt to create a blue rose. Species such as rose, tulip, and carnation are not naturally blue as they lack the “enzymatic machinery” to synthesize blue coloured pigments. Therefore, researchers at Florigene cloned two enzymes from petunia, flavonoid 3’5’ hydroxylase (F3’5’H) and dihydroflavonol reductase (DFR), that are responsible for producing the blue pigment, delphinidin, found in the vacuole of petunia cells. In an effort to generate a blue rose, researchers at Florigene transferred these two metabolic genes into rose’s plants. Despite the difficulties often encountered with ornamental transformation, stable transgenic roses were successfully regenerated. However, when the blooms finally opened, they were anything but blue. The problem is that the delphinidin pigments acts very much like litmus paper in the alkaline vacuolar environment of the petunia, delphinidin is blue, but in the acidic environment of the rose vacuole, it is pink. Similar experiments performed by Florigene with carnation, which has a more alkaline vacuolar environment, were more successful and the company has since released two transgenic varieties, Moonglow and Moon shadow. Nevertheless, even these blue flowers appear more mauve than truly blue. Now, a report by deVetten et al. (1999) in the Proceeding of the National Academy of Sciences (PNAS) has reawakened interest in the possibility of creating a blue rose. Production of blue pigments in flower requires the 3’, 5’-hydroxylation of the purple anthocyanin precursors. As mentioned above, two of the enzymes for “blue genes” required for this process, F3’5’H and DFR, have already been characterized. In the PNAS paper, deVetten and colleagues report the discovery of third blue genes, difF that is required for blue pigment synthesis in petunia. This novel gene is required for the formation of 3’, 5’ substituted anthocyanins. After cloning and sequencing the difF gene, they found that it encoded a b(5) cytochrome that was only expressed in flowers. By creating difF gene knockouts through transposons mutagenesis, they 160 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India were able to demonstrate that floral tissues lacking difF activity displayed a 60% reduction in delphinidin accumulation compared to wild type. This was due to the fact that in difF-plants, F3’5’H activity in flowers is reduced by as much as 20-fold. These findings therefore demonstrate the importance of the difF gene product in the synthesis of blue flower pigment. the brand Moondust, according to the firm. (The Japan Times: July 1, 2004) The authors suggest that by introducing this new blue gene into roses and carnations, along with the two previously isolated blue genes, it may now be possible to create truly blue flowers, although this has yet to be substantially evaluated. Whereas it may now Scientists’ efforts to create the world’s first be possible to generate much higher levels of blue rose have come up. Distillery and beverage delphinidin in transgenic roses and carnations with this manufacturer Suntory Ltd., Japan, said it has developed approach, the effect of low pH on delphinidin colour the world’s first blue roses with Australian firm Florigene still need to be addressed. To accomplish this, it will Ltd. Suntory officials said researchers extracted the be necessary to either modify vacuolar pH, which gene that produces blue pigment in pansies and would be an extremely ambitious undertaking, possibly with many unforeseen side effects, or to introduce other activated it inside the roses. enzymes to engineer blue pigments that are less pH sensitive. Either way, as promising as these results are, it is still not clear whether the production of a truly blue rose is any closer to reality. Recently, scientists have found a way to produce a blue rose; something that rose growers and breeders had spent years on experimenting with grafts and crossbreeding in unsuccessful attempts. The chance discovery was made by two biochemists conducting research into drugs for cancer and Alzheimer’s in a Tennessee medical laboratory. They came across a liver enzyme into a bacterium, the bacterium turned blue. They then moved the gene into plants and produced a blue rose (Hindustan Times, 23rd May, A model shows off the world’s first blue roses- 2004). long thought impossible to create-during a media Lasting Beauty preview in Tokyo. There are already “bluish” roses in the market, but these flowers were created through crossbreeding and cannot be called true blue, according to Suntory. The gene of the enzyme that produces the blue pigment, delphinidin, is not found in rose petals to begin with. Thanks to biotechnology, the petals of Suntory’s blue roses contain nearly 100 percent of the blue pigment, it said. Suntory and Florigene, which is 98.5 percent Suntory-owned, bred a blue carnation using the same basic technology in 1995. The carnations were marketed in Japan, North America and Australia under In addition to their Blue Gene Technology, Florigene has also developed Long Vase Life (LVL) Gene Technology. Premature inrolling, what most call wilting, of the petals is a serious problem for carnations and can begin just a few days after cutting. Wilting occurs because the plant starts to produce ethylene – a natural plant hormone that triggers the aging process and leads to petal wilting. To slow this process and provide customers will longer lasting flowers, growers often chemically treat carnations with a solution of silver thiosulfate or similar chemicals. These eliminate the 161 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India plant’s sensitivity to ethylene and thereby slow down significant market opportunity down this path, as wilting. commercial sweet potato plants with black leaves are Florigene scientists have been able to extend already huge sellers as ornamental bedding plants. the vase life of carnations while avoiding the use of For more information: chemicals. The LVL technology achieves the same The Florigene website: http://www.florigene.com result by suppressing or ‘turning off’ the particular gene http://www.agwest.sk.ca/infosource/inf_oct01.pdf responsible for producing ethylene, the ACS gene. http://www.accessexcellence.org/LC/ST/ Carnations incorporating this technology are close to st2bgplant.html market and will be available in the near future. Forever Flowers and Fruits: When the tomato or banana fruits turn mushy or if those carnation flowers in the vase droop quickly, blame it on ethylene. This gaseous hormone elicits a cascade of developmental responses in plants resulting in fruit ripening and flower senescence. Biotechnologists have sought to extend the shelf life of fruits and flowers by silencing the genes involved in ethylene biosynthesis. However, such transgenic fruits and flowers still respond to ethylene produced by other plants and begin to decay just like non-transgenic plants. A recent report describes a new solution to this problem that entails the use of a hormone receptor gene from Arabidopsis, which confers ethylene insensitivity. Tomato fruits ripened very slowly on plants engineered with this gene, while petunia flowers from transgenic plants remained fresh longer than their The Moonshade carnation nontransgenic counterparts. The dominant mutant etr1Florigene is not the only group using 1 gene, cloned from Arabidopsis by Elliot Meyerowitz biotechnology to develop colours in flowers. The first and colleagues at CalTech, encodes a protein that alters application of modern biotechnology in flowers was the perception of ethylene by plant cells and thus makes the creation of an orange petunia developed by the plant unresponsive to the hormone. introducing a pigment-producing gene from corn. A team led by Harry Klee, who initiated the Another example, eustomas, very similar to roses in appearance, is being bioengineered by a group in New work while at Ceregen Technology (Monsanto Zealand to be redder and become a less-expensive Company) and continued it at the University of Florida, alternative to roses. Eustomas have been created in introduced this gene into tomato and petunia using sky blue, but the red remain a better seller. In addition, Agrobacterium vectors. Transgenic tomato plants eustomas can be cut and shipped dry, reducing exposed to ethylene exhibited a dramatically delayed fruit ripening and senescence compared with those on distribution costs. untransformed plants. Harvested tomato fruits retained Flowers are not the only targets for new their original golden yellow colour even when stored colours. Plant pigments can also be expressed in their for 100 days while the regular tomato fruits soon leaves. It is possible then that plants can be produced “turned red, became soft and started to rot”. Similarly, with purple, red, black, or blue leaves. There may be petunia flowers with the ‘ethylene- insensitive’ gene 162 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India senesced slowly and remained longer on the plant. When exposed to ethylene, the transgenic flowers stayed fresh for nine days in the vase while the untransformed flowers wilted within just three days. Arora, A. (2008). “Getting more colourful flowers by painting with genes”. Indian Horticulture, July-August: Vol. 53 (4): 21-23. Arora, A. (2008). “Programmed Cell Death During According to the researchers, the ethylene Plant Senescence”. In: Paliyath, G., Murr, D.P., insensitive gene from Arabidopsis may have to be Handa, A.K., Lurie, S. (eds.): Postharvest weakened by molecular alterations to ensure its broad Biology and Technology of Fruits, Vegetables and application, because fruits and vegetables eventually Flowers. Blackwell Publishing, Iowa, USA, pp must respond to ethylene for ripening to proceed. The 86-124. use of appropriate promoters may also permit targeted Arora, A. and Ezura, H. (2003) Isolation, molecular ripening. Monsanto scientists anticipate that the characterization and regulation of cysteine immediate beneficiary of their finding will be the protease gene in Gladiolus grandiflora. floriculture business, a multibillion-dollar industry Molecular and Cellular Proteomics. 2 (9): 746. worldwide. Arora, A. and Singh, V.P. (2004). Cysteine protease Many chemicals that affect ethylene synthesis gene expression and proteolytic activity during or its action, which are currently in use to extend the floral development and senescence in ethyleneshelf life of flowers, are being banned because of insensitive gladiolus. Journal of Plant environmental concerns. The floriculture industry thus Biochemistry and Biotechnology, 13 (2): 123may gain substantially from the use of the ‘ethylene126. insensitive’ gene by making their colourful blooms last longer either on plants or in vases. Arabidopsis may Arora, A. and Singh, V.P. (2006). Polyols regulate the flower senescence by delaying programmed cell never be considered pretty enough to be taken seriously death in Gladiolus. Journal of Plant by nurserymen but the Nature Biotechnology study Biochemistry and Biotechnology 15 (2): 139clearly underscores one of the potential pay-offs to 142. agriculture from the investment in research on this humble weed. Arora, A. and Singh, V.P. (2007). RNA interference: a novel approach for gene silencing. In: Recent Selected References advances in Plant Sciences. Edited by Setia, Arora, A. (2005). Ethylene receptors and molecular R.C., Setia, N., Thind, S. K. and Nayyar, H., mechanism of ethylene sensitivity in plants. Punjab Agricultural University, Ludhiana. Current Science 89 (8): 25th Oct. 2005: 1348Arora, A., Sairam, R.K. and Srivastava, G.C. (2002). 1361. Oxidative stress and antioxidative system in Arora, A. (2008). “Biochemistry of Flower plants. Current Science 82 (10): 1227-1238. Senescence”. In: Paliyath, G., Murr, D.P., Handa, A.K., Lurie, S. (eds.): Postharvest Biology and Arora, A., Singh, V. P., Sindhu, S. S., Rao, D. N. and Voleti, S. R. (2007). Oxidative stress mechanisms Technology of Fruits, Vegetables and Flowers. during flower senescence. Plant Stress 1(2), 157Blackwell Publishing, Iowa, USA, pp 51-85. 172, Global Science Books, Ltd., London, UK. Arora, A. (2008). “Biotechnological approaches for improving post harvest life of fruits, vegetables Arora, A., Singh, V.P., Sindhu, S.S., Rao, D.N. and Voleti, S.R. (2007). “Oxidative stress and flowers”. Article in Delhi Agri-Horticultural mechanisms during flower senescence”. In: Society (DAHS) Annual Magazine during DAHS Floriculture, ornamental and plant biotechnology: Annual Pusa Horticultural Show. 163 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Advances and Topical Issues. (Ed. Jaime A. Teixeira da Silva), Global Science Books, Ltd., London, UK. antioxidant activity in relation to vase life of Gladiolus cut flowers. Plant Growth Regulation, Volume 51, Number 2 / February, 2007, 99-108 Arora, A., Watanabe, S., Ma, B., Takada, K. and Ezura, H. (2006) A novel ethylene receptor Singh, V.P., Kiran, D. and Arora, A. (2005). ). Effect of spermine, spermidine and putrescine on the homolog gene isolated from ethylene-insensitive vase life and associated parameters in two flowers of gladiolus (Gladiolus grandiflora Gladiolus varieties. J. Orn. Hort. 8 (3): 161-166. hort.). Biochemical and Biophysical Research Communications Dec 22: 351(3): 739-44. Epub Singh, V.P., Kiran, D. and Arora, A. (2005). Alleviation 2006 Oct 30. of antioxidants activity in gladiolus flowers during senescence by spermine and spermidine. J. Orn. Ezhilmathi, K., Singh, V.P. Arora, A. and Sairam, R.K. Hort. 8 (3): 167-172. (2007). Effect of 5-sulfosalicylic acid on 164 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India MONOCARPIC SENESCENCE AND ITS REGULATION IN PLANTS Renu Khanna-Chopra and S. Srivalli Stress Physiology & Biochemistry Laboratory, Water Technology Centre Indian Agricultural Research Institute, New Delhi – 110012, Email: renu.chopra3@gmail.com Senescence is a genetically controlled system of nutrient relocation involving a co-ordinated action at the cellular, tissue, organ and the whole plant level leading to cell death. Leaf senescence is an integrated response to developmental age and also various internal factors and environmental signals. The environmental signals that influence leaf senescence include biotic and abiotic stresses such as drought stress. In monocarpic plants, the developing reproductive sink (grain/pod) often governs the senescence of the whole plant, and removal of reproductive sink usually delays senescence whereas drought stress hastens the process of senescence. Proteases are responsible for protein degradation leading to nitrogen mobilization during monocarpic senescence. Our studies in cowpea showed that cysteine proteases played a predominant role in the degradation of the large subunit of Rubisco protein during both monocarpic and drought induced senescence. Vacuoles were found to be the major storehouse of these cysteine proteases. The enhanced monocarpic senescence as a result of drought stress was correlated with the increased hydrolytic activities of endopeptidases where new forms of papain family cysteine proteases of molecular weights 69 kDa, 60 kDa and 48 kDa were induced whereas a single 48 kDa protease was observed under monocarpic senescence which increased nine days after flowering. The removal of reproductive sink led to lowered proteolytic activities and lower levels of reactive oxygen species (ROS). This in turn led to slower nitrogen mobilization from the leaves of these plants. Thus, it was observed that reproductive sink drives the nitrogen mobilization through increased ROS levels. Key issues in the senescence process still need to be elucidated, including the integration mechanism of various senescence-affecting signals. 165 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India OXIDATIVE STRESS AND ANTIOXIDATIVE DEFENCE SYSTEM R.K. Sairam Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110012 biologists as oxygen free radicals which is a misnomer because in chemistry a free radical is defined as an Activation of oxygen atom or molecule with an unpaired electron. It is more One of the paradoxes of life on this planet is appropriate to refer to the intermediate reduction that the molecule that sustains aerobic life, oxygen, is products of oxygen as activated not as free radicals not only fundamentally essential for energy metabolism because triplet oxygen (ground state) is a radical and and respiration, but it has been implicated in many hydrogen peroxide is not. diseases and degenerative conditions (Marx, 1985). Superoxide Hydroperoxy A common element in such diverse human disorders Singlet oxygen radical as ageing, arthritis, cancer, Lou Gehrig’s disease and many others is the involvement of partially reduced Hydrogen forms of oxygen. peroxide Triplet 1. Generation of oxidative stress The biradical form of oxygen is in a triplet ground state because the electrons have parallel spins. Activation of oxygen may occur by two different mechanisms: absorption of sufficient energy to reverse the spin on one of the unpaired electrons, or monovalent reduction. If triplet oxygen absorbs sufficient energy to reverse the spin of one of its unpaired electrons, it will form the singlet state, in which the two electrons have opposite spins. This activation overcomes the spin restriction and singlet oxygen can consequently participate in reactions involving the simultaneous transfer of two electrons (divalent reduction). Since paired electrons are common in organic molecules, singlet oxygen is much more reactive towards organic molecules than its triplet counterpart. oxygen Hydroxy radical Water The activation states of oxygen. Hydrogen peroxide is noteworthy because it readily permeates membranes and it is therefore not compartmentalised in the cell. The well-known reactivity of hydrogen peroxide is not due to its reactivity per se, but requires the presence of a metal reductant to form the highly reactive hydroxyl radical which is the strongest oxidizing agent known and reacts with organic molecules at diffusion-limited rates. Fenton described in the late nineteenth century (Fenton, The second mechanism of activation is by the 1894; 1899) the oxidising potential of hydrogen stepwise monovalent reduction of oxygen to form peroxide mixed with ferrous salts. Forty years later, superoxide (O 2.-), hydrogen peroxide (H 2O 2), Haber and Weiss (1934) identified the hydroxyl radical hydroxyl radical (OH.) and finally water. The first step as the oxidising species in these reactions: in the reduction of oxygen forming superoxide is In biological systems the availability of ferrous endothermic but subsequent reductions are exothermic. ions limits the rate of reaction, but the recycling of iron The univalent reduction of superoxide from the ferric to the ferrous form by a reducing agent produces hydrogen peroxide which is not a free radical can maintain an ongoing Fenton reaction leading to because all of its electrons are paired. Very often the the generation of hydroxyl radicals. One suitable reduction products of oxygen are referred to by 166 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India reducing agent is superoxide which participates in the Mitochondria overall reaction. Most oxygen is consumed by the cytochrome oxidase enzyme in the mitochondrial electron transport system, and involves the sequential transfer of four electrons to oxygen, releasing water. Plant mitochondria have an additional site of oxygen reduction at the alternative oxidase, distinguished from Therefore, in the presence of trace amounts cytochrome oxidase by its resistance to cyanide. of iron, the reaction of superoxide and hydrogen However, neither of these sites produce significant peroxide will form the destructive hydroxyl radical and quantities of superoxide (Rich and Bonner, 1978). initiate the oxidation of organic substrates. Metals other than iron, such as copper, may also participate in these electron transfer reactions by cycling between oxidised and reduced states. Sites of activated oxygen production As indicated above, there are two forms of activated oxygen that are formed by distinctly different mechanisms. The reduction of oxygen to form superoxide, hydrogen peroxide and hydroxyl radicals is the principle mechanism of oxygen activation in most Schematic representation of the electron biological systems. However in photosynthetic plants, transport system in the mitochondrial membrane the formation of singlet oxygen by the photosystems showing a possible site of superoxide production has importance. by reduced ubiquinones. Chloroplasts However, isolated mitochondria produce As described by Elstner (1991), there are at H O and O .- in the presence of NADH (Loschen et 2 2 2 least four sites within the chloroplast that can activate al., 1973; 1974). Antimycin A, which blocks electron oxygen). PSI can reduce oxygen by the Mehler reaction flow after ubiquinone (Fig. 7) enhances oxygen which is an important mechanism of oxygen activation reduction. Presumably other conditions which also in the chloroplast. The reducing side of PSI is thought increase the reduction of ubiquinone favour reduction to contribute significantly to the monovalent reduction of oxygen in the ubiquinone Ä cytochrome b region of of oxygen under conditions where NADP is limiting. the chain (Rich and Bonner, 1978). The various Fe-S This would occur, for example, if the Calvin cycle did proteins and NADH dehydrogenase have also been not oxidise NADPH as rapidly as PSI supplied implicated as possible sites of superoxide and hydrogen electrons. peroxide formation (Turrens et al., 1982). Endoplasmic Reticulum Various oxidative processes, including oxidation, hydroxylations, dealkylations, deaminations, dehalogenation and desaturation, occur on the smooth endoplasmic reticulum. Mixed function oxygenases that contain a heme moiety add an oxygen atom into an organic substrate using NAD(P)H as the electron Schematic representation of the electron transport system donor. The generalised reaction catalysed by in the thylakoid membrane showing three possible sites of cytochrome P450 is: activated oxygen production. 167 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India The best characterised cytochrome P450 in plants is cinnamate-4-hydroxylase which functions in flavonoid and lignin biosynthesis, but other mixed function oxidases function in other biochemical pathways including gibberellin and sterol biosynthesis. Activation of oxygen by these systems is an essential prerequisite to oxygen addition reactions in the synthesis of these “complex” metabolites. Schematic representation of the cytochrome P450 electron transport system on the endoplasmic reticulum showing one possible site of superoxide production. Superoxide is produced by microsomal NAD(P)H dependent electron transport involving cytochrome P450 (Winston and Cederbaum, 1983). One possible site at which this may occur is shown in figure. After the univalent reduction of the substrate (RH) and the addition of triplet oxygen to form the complex P450 - RHOO the complex may decompose to P450-RH and release superoxide. Microbodies Peroxisomes and glyoxysomes are organelles with a single membrane that compartmentalises enzymes involved in the ß-oxidation of fatty acids, and the glyoxylic acid cycle including glycolate oxidase, catalase and various peroxidases. Glycolate oxidase produces H2O2 in a two electron transfer from glycolate to oxygen (Lindqvist et al., 1991). Xanthine oxidase, urate oxidase and NADH oxidase generate superoxide as a consequence of the oxidation of their substrates. The xanthine oxidase reaction is often used in vitro as a source of superoxide producing one mole of superoxide during the conversion of xanthine to uric acid (Fridovich, 1970). Plasma Membranes A superoxide-generating NAD(P)H oxidase activity has been clearly identified in plasmalemma enriched fractions (Vianello and Macri, 1991). These flavoproteins may produce superoxide by the redox cycling of certain quinones or nitrogenous compounds. In the root, NAD(P)H oxidase reduces Fe3+ to Fe2+ converting it to a form that can be transported. Dysfunction of this root enzyme will produce superoxide (Cakmak and Marschner, 1988). An auxinactivated NADH oxidase has been associated with acidification of the cell wall and auxin-stimulated cell elongation (Morré et al., 1988). 1.2. Measurement of Oxidative Stress 1.2.1 Superoxide radicles (O2..-) The spectrophotometric assay of total superoxide radical content in the fresh tissue is based on the principle of formation of blue coloured formazone by nitroblue tetrazolium chloride with superoxide radicals (O2.-) in the absence of/or by inhibiting total superoxide dismutase (SOD) activity, as described by Chaitanya and Naithani (1994). Requirements Test tubes, test tube stand, micro-pipettes (20200 µl, 100-1000 µl and 5 ml), refrigerated centrifuge, visible spectrophotometer and “chemicals” as listed in reagents below. Reagents 1. Methionine (200 mM): 2. Nitroblue tetrazolium chloride (NBT) (2.25 mM): 3. EDTA (3.0 mM): 4. Sodium carbonate (1.5 M): 5. Grinding media: (0.2 M phosphate buffer, pH 7.2, containing 1 mM diethyl dithio carbamate) Sol A: Potassium dihydrogen phosphate 100 mM was prepared in 500 ml with double distilled water. Sol B: Di-potassium hydrogen phosphate 100 mM was prepared in 500 ml with double distilled water. 168 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Mix 28 ml of sol. A + 72 ml of sol. B and 0.017 g sodium diethyl dithio carbamate, and final pH is adjusted with the help of pH meter. Extraction Weighed amount of plant tissue is homogenized in pre-cooled phosphate buffer (0.2 M, pH 7.2) containing 1 mM diethyl dithio carbamate (to inhibit SOD). The homogenate is centrifuged at 5000 rpm for 5 min. Supernatant is used immediately for the estimation of superoxide radical. Assay of O2.In the supernatant superoxide anion (O2.-) was measured by its capacity to reduce nitroblue tetrazolim and formation of blue coloured formazone, whose absorption is measured at 540 nm. Reaction mixture 250 µl plant extract 100 µl NBT (2.25mM) 50µl Na2CO3 (1.5 M) 100 µl EDTA (3.0mM) 200 µl L-methionine (200 mM) 2.30 ml water Incubate at 30 oC for 10-15 min. Superoxide radical (O2.-) content is expressed as A540 min-1 g-1 dry weight. Reference Chaitanya, K.S.K. and Naithani, S.C. (1994). Role of superoxide, lipid peroxidation and superoxide dismutase in membrane perturbation during loss of viability in seeds of Shorea robusta Gaertn.f. New Phytol. 126: 623-627. 1.2.2. Hydrogen Peroxide Hydrogen peroxide (H2O2) is a reactive oxygen species formed in various components of plant cell, such as glyoxisomes during -oxidation of fatty acids, peroxisomes during photo-respiratory glycolate oxidation and in chloroplast, mitochondria and cytosol stroma during dismutation of superoxide radical. Principle Hydrogen peroxide is estimated by forming titanium-hydro peroxide complex (Rao et al. 1996). Hydrogen peroxide forms a light yellow coloured titanium-hydro-peroxide complex with titanium reagent. H2O2 + Ti5+ Titanium-hydro peroxide complex (yellow colour) This yellow complex absorbs at 415 nm. Requirements Test tubes, test tube stand, micro-pipettes (20200 µl, 100-1000 µl and 5 ml), refrigerated centrifuge, visible spectrophotometer and “chemicals” as listed in reagents below. Reagents Titanium reagent: One-gram titanium dioxide and 10 g potassium sulphate are digested in 150 ml conc. sulphuric acid over a hot plate for 4 h. The digested mixture is diluted to 500-600 ml and stirred with a magnetic stirrer cum heater at 70 – 80 oC till a clear transparent solution is obtained, diluted to 1.5 liter and store in dark brown bottle (Teranishi et al. 1974). Acetone: Analytical grade reagent Ammonium hydroxide/liquid ammonia:Analytical grade reagent Procedure: One g leaf material was grinded to fine powder with the help of liquid nitrogen in cold room with 10 o C temperature, followed by addition of 10 ml cooled acetone in Mixture is filtered with Whatman No. 1 filter paper followed by the addition of 4 ml titanium reagent and 5 ml ammonium solution to precipitate the titaniumhydro peroxide complex. Reaction mixture is centrifuged at 10 000 g for 10 min in a refrigerated centrifuge Precipitate is dissolved in 10 ml 2 M H2SO4 and than recentrifuged. Supernatant is read at 415 nm against reagent blank in UV-visible spectrophotometer. Concentration of hydrogen peroxide is computed by referring to a standard curve made from known concentrations of hydrogen peroxide. Standard curve of hydrogen peroxide Standard curve of hydrogen peroxide is prepared by taking a range of concentrations of H2O2, from 0.1 to 1.0 µmol. To this 10 ml of cold acetone is added, followed by various steps as in case of sample and a curve is drawn by plotting concentrations against respective absorbance. 169 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India References Rao, M.V., Paliyath, G., Ormrod, D.P., Murr, D.P. and Watkins, C.B. (1997). Influence of salicylic acid on H2O2 production, oxidative stress and H 2O2 metabolizing enzymes. Plant Physiol., 115: 137-149. Teranishi, Y., Tanaka, A., Osumi, M. and Fukui, S. (1974). Catalase activity of hydrocarbon utilizing candida yeast. Agric. Biol. Chem., 38: 1213-1216. 1.2.3. Superoxide generation-NADPH oxidase NADPH oxidase is a membrane linked enzyme, found in plasma membrane, endoplasmic reticulum and microsomal fraction. It catalysis the oxidation of NADPH resulting in generation of superoxide. NADPH-oxidase NADPH H+ + 2O2 O2.- + NADP + H2O Requirements Test tubes, test tube stand, micro-pipettes (20200 µl, 100-1000 µl and 5 ml), refrigerated centrifuge, UV-visible spectrophotometer and “chemicals” as listed in reagents below. Reagents 1. Tris-HCL buffer 150 mM, (pH 8.6): Tris (hydroxyl methyl) amino methane and pH of the solution is adjusted with 0.15 N HCl to 8.6 and final volume is made to 200ml by double distilled water. 2. Sucrose (750 mM): 3. Nitroblue tetrazolim chloride (NBT) (3 mM):. Store in dark bottle. 4. Diphenylene iodonium chloride (DPI) (0.3 mM):. Store in dark bottle. 5. Grinding media (pH 7.5, 50 mM Tris-HCL, 250mM sucrose, 3 mm Na 2EDTA, 10 mM ascorbic acid and 5 mM diethyl dithiocarbamate): Tris 12.223 g + sucrose 17.115 g + EDTA 0.223 g + ascorbic acid 0.352 g + diethyl dithio carbamate sodium salt 0.225 g were dissolved in appox. 150 ml water and pH was adjusted to 7.5 by pH meter and final volume is made to 200 ml with double distilled water. 6. Resuspension buffer (5 mM K-phosphate buffer, pH 7.8, 250 mM sucrose and 3 mM KCl): (A) KH2PO4 0.136 g and (B) K2HPO4 0.174 g are separately dissolve in 100 ml distilled water to get 10 mM solution of each. Solutions A and B are dissolved in the ratio of 15:85 ratio and to this solution 17.115 g sucrose and 0.089 g KCl are added and pH is adjusted to 7.5 with the help of a pH meter, and volume is made to 200 ml by double distilled water. Preparation of crude extract (membrane + microsomal and cytosolic) Fresh leaf tissue is cut in to pieces and ground in pre-chilled mortar and pestle with 5 times its weight in ice cold extraction buffer consisting of 50 mM TrisHCL, pH 7.5, 250mM sucrose, 3 mm Na2EDTA, 10 mM ascorbic acid and 5 mM diethyl dithiocarbamate. Homogenate is filtered through 4 layers of nylon/cheese cloth and centrifuged at 10000g for 10 min. The crude microsomal + plasma membrane (PM) fraction was isolated by further centrifugation of supernatant at 65000g for 10 min. The pallet is resuspended in resuspension buffer made up of 5 mM K-phosphate buffer, pH 7.8, 250 mM sucrose and 3 mM KCl. NADPH oxidase assay NADPH dependent O2.- generating activity in crude extract and mixed microsomal + PM fraction is determined the rate of SOD-inhibitable (by adding 5 mM diethyl dithiocarbamate in extraction buffer) reduction of NBT using NADPH as electron donor as per the method of Quartacci et al. (2001). The 3 ml reaction mixture consisted of: 50 mM Tris-HCL buffer, pH 8.6 (1 ml of 150 mM buffer) 250 mM sucrose (1 ml of 750 mM sucrose) 0.1 mM NBT (0.1 ml of 3 mM NBT) 50-100 µg protein (as per protein concentration) Water to make up volume ( 3 ml) [10µM DPI (optional to inhibit NADPH O 2.- dependent generation)] (0.1 ml of 0.3 mM DPI) After 1 min pre-incubation the reaction is started by the addition of 0.1 mM NADPH (0.1 ml of 3 mM NADPH) and the absorbance change at 530 nm were measured for 5 min. Rates of O2.- generation were calculated using an extinction coefficient of 12.8 mM-1 cm-1. 170 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Activity = OD x 3 12.8 * total vol. of extract Wt. of plant sample x vol. of enzyme used x time in min. = µmol O 2 .- g -1 fr. wt. min -1 Activity can also be expressed on dry weight and protein basis. Reference Quartacci, M.F., Cosi, E. and Navari-Izzo, F. (2001). Lipids and NADPH dependent superoxide production in plasma membrane vesicles from roots of wheat grown under copper deficiency or excess. J. Exp. Bot. 52 (354): 77-84. 1.2.4. Lipid peroxidation Lipid peroxidation is oxidative degradation of lipid-fatty acids by reactive oxygen species. The level of lipid peroxidation is measured in terms of thiobarbituric acid reactive substances (TBARS) content (Heath and Packer, 1968). Principle Lipids peroxidation products like malondialdehyde, fatty acid-hydro-peroxides reacts with thiobarbituric acid and forms a red colour complex, known in general as thiobarbituric acid reactive substances, which is taken as the measure of in vivo lipid peroxidation in plant tissue. This red coloured complex absorbs at 532 nm. Requirements Test tubes, test tube stand, micro-pipettes (20200 µl, 100-1000 µl and 5 ml), refrigerated centrifuge, visible spectrophotometer and “chemicals” as listed in reagents below. Reagents Trichloro-acetic acid (TCA) (0.1 %): Trichloro-acetic acid solution is prepared by dissolving 0.1 g TCA in water and than the volume is made to 100 ml. Thiobarbituric acid reagent: 0.5% thiobarbituric acid (TBA) in 20% TCA was prepared by first preparing 20 % TCA (20 g TCA in 100 ml). 0.5 g TBA was dissolved in 20 % TCA and than volume was made to 100 ml by 20 % TCA. Procedure: Extraction Leaf sample (0.5 g) is homogenized in 10 ml 0.1 % trichloro-acetic acid (TCA). The homogenate is centrifuged at 15 000 g for 15 min. Supernatent is used for the estimation of TBARS contents. Assay To 1.0 ml aliquot of the supernatant 4.0 ml of 0.5% thiobarbituric acid (TBA) in 20% TCA is added. The mixture is heated at 95 C for 30 min in the laboratory electric oven and than cooled in an ice bath. After cooling the aliqut is centrifuged at 10 000 g for 10 min The absorbance of the clear supernatant is recorded at 532 nm. Values of non-specific absorption recorded at 600 nm are subtracted from the values recorded at 532 nm. The TBARS content is calculated according to its extinction coefficient  = 155 mM-1 cm-1. References Heath, R.L. and Packer, L. (1968). Photoperoxidation in isolated chloroplast. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys., 125: 189-198. 1.3. Antioxidant Metabolites 1.3.1.Ascorbic Acid Total ascorbic acid by dinitrophenylhydrazine reagent method Ascorbic acid is an important chemical antioxidant, which is responsible for the non-enzymatic scavenging of superoxide radical and hydrogen peroxide, regeneration of -tocopherol in chloroplast and in enzymatic scavenging of H2O2 in association with ascorbate peroxidase. Its estimation is based on the formation of pink coloured complex due to the reduction of dinitrophenylhydrazine by ascorbic acid to phenyl hydrazone in acidic medium (Mukherjee and Choudhuri 1983). Requirements Test tubes, test tube stand, micro-pipettes (20-200 µl, 100-1000 µl and 5 ml), refrigerated centrifuge, visible spectrophotometer and “chemicals” as listed in reagents below. Reagents: Trichloroacetic acid (6 %): Six grams of trichloroacetic acid is dissolved in distilled water and volume made up to 100 ml with distilled water. Dinitrophenylhydrazine (2 %): Two gram 171 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India dinitrophenylhydrazine is dissolved in distilled water made acidic by adding few drops of analytical grade concentrated hydrochloric acid, and than volume is made up to 100 ml. Thiourea (10 % in 70 % ethanol): Ten gram thiourea is dissolved in 70 % ethanol and than volume is made up to 100 ml. Extraction Fresh leaf sample (0.5 g) or preserved in liquid nitrogen is extracted with 10 ml of 6 % trichloroacetic acid. Homogenate is centrifuged at 5000 g at 4 0C temperature in a refrigerated centrifuge. Supernatant is used for estimation of ascorbic acid. Assay Mix 4 ml of the extract with 2 ml of 2 % dinitrophenylhydrazine (in acidic medium) followed by the addition of 1 drop of 10 % thiourea (in 70 % ethanol). The mixture is heated for 15 min in a boiling water bath (100 oC). After cooling to room temperature, 5 ml of 80 % (v/v) H2SO4 is added to the mixture at 0 oC (in an ice bath). The absorbance is recorded at 530 nm. Quantification The concentration of ascorbic acid in the sample is calculated from a standard curve plotted by taking known concentration of ascorbic acid (25 - 250 nmol) and following the steps as mentioned for samples. References Mukherjee, S.P. and Choudhari, M.A. (1983). Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in vigna seedlings. Physiol. Plant., 58: 166170. Dipyridyl-ferric chloride reagents methods Reagents Metaphosphoric acid 5% Trichloro acetic acid (TCA) 10 % Orthophosphoric acid 44% Phosphate buffeer 150 mM, pH 7.4 containing 5 mM EDTA 10 mM dithiotrietol (DTT) Dipyridyl 4 % in 70 % ethanol Ferric chloride (FeCl3) 0.3% (w/v) N-ethylmaleimide 0.5 % Extraction Leaf samples were prepared for ascorbic acid (AsA) and dehydroascorbic acid (DAsA) by homogenising 1 g fresh leaf material in 10 ml of cold 5% metaphosphoric acid.. The homogenate is centrifuged at 22000 g for 15 min at 4 oC, and the supernatent is collected for analysis of AsA and DAsA. Assay AsA, DAsA and total ascorbate (AsA + DAsA) are analyzed as per Law et al. (1983). Total ascorbate was estimated after reduction of DAsA to AsA by DTT, and the concentration of DAsA was determined from the difference between the total ascorbate and AsA. The reaction mixture for total ascorbate contained; Plant extract (aliquot) 0.3 ml Phosphate buffer 150 mM, pH 7.4 (+5 mM EDTA) 0.75 ml DTT 10mM 0.15 ml Incubate at room temperature for 10 min, and add; N-ethylmaleimide 0.5 % 0.15 ml (to remove excess DTT) AsA is determinde in a similar reaction mixture except 0.3 ml water is added rather than DTT and Nethylmaleimide. Plant extract (aliquot) 0.3 ml Phosphate buffer 150 mM, 0.75 ml pH 7.4 (+5 mM EDTA) Water 0.3 ml Colour development Colour is developed in both reaction mixtures by addition of the followings: TCA 10 % 0.6 ml Ortho-phosphoric acid 44 % 0.6 ml Dipyridyl 4 % in 70 % ethanol 0.6 ml FeCl3 0.3% 0.3 ml After vortex mixing the reaction mixture is incubated at 40 oC for 40 min. Absorbance is read at 525 nm. 172 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Concentration is quatified by reffering to a standard curve of ascorbic acid in the range of 0 100 µg ml-1. References Law, M.Y., Charles, S.A. and Halliwell, B.(1983). Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplast. Biochem. J. 210: 899-903. Gossett, O.W., Millhollon, E.P. and Lucas, M.C. (1994). Antioxidant response to NaCl stress in salt tolerant and salt sensitive cultivars of cotton. Crop Sci. 34: 706-714. 1.3.2. Glutathione(Total, reduced and oxidized) Glutathione is an important antioxidant. It is a tripeptide of glutamic acid, cycteine and glycine. Its oxidation-reduction reaction is due to the presence of sulfhydryl (-SH) group in cysteine residue and its oxidation to disulfide (-S-S-) bond, and consequently it exist in reduced (GSH) form and oxidized form (GSSG). GSH can function as an antioxidant in many ways. It can react chemically with singlet oxygen, superoxide and hydroxyl radicals and therefore function directly as a free radical scavenger. GSH may stabilise membrane structure by removing acyl peroxides formed by lipid peroxidation reactions. GSH is the reducing agent that recycles ascorbic acid from its oxidised to its reduced form by the enzyme dehydroascorbate reductase. GSH can also reduce dehydroascorbate by a non-enzymatic mechanism at pH > 7 and at GSH concentrations greater than 1 mM. This may be a significant pathway in chloroplasts whose stromal pH in the light is about 8 and GSH concentrations may be as high as 5 mM. Reagents Metaphosphoric acid 5% Phosphate buffer, 0.5 M, pH 7.5 Phosphate buffer 0.1 M, pH 7.5 2-Vinylpyridine NADPH 6.0 mM EDTA 150 mM 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) 18 mM Glutatione reductase (GR) , dilute the appropriate quantity in phosphate buffer, 0.1 M, pH 7.5 to get 3 units per 1.0 ml Extraction Leaf samples were prepared for reduced glutathione (GSH) and oxidized glutathione (GSSG) by homogenising 1 g fresh leaf material in 10 ml of cold 5% metaphosphoric acid. The homogenate is centrifuged at 22000 g for 15 min at 4 oC, and the supernatent is collected for analysis of AsA and DAsA. Assay GSH and GSSG are assayed accoding to the methods of Griffith (1985) and Smith (1985). For total glutathione (GSH + GSSG) 1 ml aliquot of the supernatent is neutralized with 1.5 ml of 0.5 M phosphate buffer (pH 7.5), followed by addition of 50µl of water. For GSSG assay another 1 ml of the aliquot is neutralized with 1.5 ml of 0.5 M phosphate buffer (pH 7.5), followed by addition of 50 µl of 2-vinylpyridine, to mask the GSH, and the contents of the tube are.mixed/vortexed until an emulsion formed. The tube is then incubated for 60 min at room temperature. The 3 ml reaction mixture for glutathione content contained the followings: 0.2 mM NADPH 100 mM phosphate buffer, pH 7.5 5 mM EDTA 0.6 mM DTNB 3 units of GR Water 0.1 ml of 6.0 mM 0.6 ml of 0.5 M) 0.1 ml of 150 mM 0.1 ml of 18 mM 1 ml 1 ml Reaction is started by adding 0.1 ml of extract sample as described above. The reaction rate is monitored by measuring the change in absorbance at 412 nm for 1 min. The concentration of glutathione is quantified by reffering to a standard curve based on GSH in the range of 0 to 50 µM ml-1. Reference Griffith, O.W. (1985). Glutathione and glutathione disulfide. In: Bergmeyer, H.U. (Ed.). Methods of Enzymatic Analysis. Weinheim, Verlagsgesellschaft mbH, pp. 521-529. Smith, I.K. (1985). Stimulation of glutathione synthesis in photorespiring plants by catalase inhibitors. Plant Physiol. 79: 1044-1047. 173 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India 1.3.3. Carotenoids Carotenoids are C 40 isoprenoids (tetraterpenes) that are located in the plastids of both photosynthetic and non-photosynthetic plant tissues. In chloroplasts, the carotenoids functions as accessory pigments in light harvesting, but a more important role is their ability to detoxify various forms of activated oxygen and triplet chlorophyll that are produced as a result of excitation of the photosynthetic complexes by light. There are two types of carotenoids. The main protective role of ß-carotene in photosynthetic tissue may be through its direct quenching of triplet chlorophyll, which would prevent the generation of singlet oxygen, and therefore completely avoid oxidative stress. 3 1 Chl* + 1ß-car Chl + 3ß-car* 3 1 ß-car* ß-car + heat The carotenoids act as a competitive inhibitor for the formation of singlet oxygen, and this is aided considerably by their proximity to chlorophyll in the light harvesting complex. This method of protection is especially critical as light intensity increases above saturating levels. The xanthophyll cycle involves the reversible conversion of the xanthophylls between two forms, violaxanthin and zeaxanthin. A de-epoxidase enzyme catalyses the de-epoxidation of violaxanthin to zeaxanthin in the presence of excess light, and an epoxidase catalyses the reverse reaction in darkness or low light. Zeaxanthin, therefore accumulates under light intensities that exceed photosynthetic capacity. The de-epoxidase has a low pH optimum (5.1), whereas the epoxidase has a high pH optimum (7.5). Reduced ascorbate serves as the electron donor for the de-epoxidase, whereas NADPH supplies reducing equivalents for the epoxidase. Structure of two common carotenoids found in plants, ß-carotene and zeaxanthinin. The ß-carotenes are hydrocarbons, while the xanthophylls are carotene derivatives that contain one or more oxygen atoms. The carotenoids can exist in a ground state or in one of two excited states after the absorption of light energy. In terms of its antioxidant properties carotenoids can protect the photosystems in one of four ways: (i) by reacting with lipid peroxidation products to terminate chain reactions, (ii) by scavenging singlet oxygen and dissipating the energy as heat, (iii) by reacting with triplet or excited chlorophyll molecules to prevent formation of singlet oxygen, (iv) or by the The Xanthophyll cycle for the cycling of dissipation of excess excitation energy through the violaxanthin and zeaxanthin. xanthophyll cycle. Carotenoids may also augment aReagents tocopherol in scavenging peroxy radicals. Acetone 80 % Or Dimethyl sulfoxide (DMSO) 3 1 Chl* + O2 O2 + 1Chl Assay 1 O2 + 1ß-car O2 + 3ß-car* Total carotenoids (ß-carotene + xanthophyll) 3 1 estimation require simualtaneous estimation of ß-car* ß-car + heat chlorophyll-a and chlorophyll-b. The assay is based on the various equations for the determination of 174 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India pigments based on their absorption in organic solvents at various wavelength as described by Arnon (1949) and Lichtenthaler and Wellburn (1983). The leaf material 500 mg is grounded in 80 % acetone, filtered through whatman No 1 filter paper and filterate is collected in a 50 ml volumetric flask. Volume is made to 50 ml with 80 % acetone. Alternatively 50 mg leaf material is placed in 10 ml of DMSO in test tubesas suggested by Hiscox and Israelstam (1979), and thereafter the test tubes are placed in a constant temperature incubator at 65 o C for 4 h. After the completion of period the tubes are cooled to room temperature. The aborbance of extracts for both the sovents is recorded at 470, 645 and 663 nm against 80 % acetone/DMSO blank. The chlorophyll-a, chlorophyll-b and total carotenoid are calculated as per the equations given below: Chl-a = 12.7 A663 - 2.69 A645 Chl-b = 22.9 A645 - 4.68 A663 acetone) References Arnon, D.I. (1949). Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24: 1-15. Hiscox, J.D. and Israelstam, G.F. (1979). A method for extraction of chloroplast from leaf tissue without maceration. Canadian J. Bot. 57: 1332-1334. Lichtenthaler, H.K. and Wellburn, W.R. (1983). Determination of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans. 11: 591-592. 1.4. Antioxidant Enzymes 1.4.1. Superoxide dismutase(SOD) Principle SOD catalyze the dismutation of superoxide radical (O2.-) to hydrogen peroxide (H2O2). Arnon (1949) (80 % O2.- + 2H+ SOD H2O2 + O2 The assay is based on the formation of blue coloured formazone by nitro-blue tetrazolium and O2.Wellburn radical, which absorbs at 560 nm, and the enzyme (80 % (SOD) decreases this absorbance due to reduction in .The values obtained for chlorophyll-a, the formation of O2 radical by the enzyme (Dhindsa chlorophyll-b and total carotenoid are in µg ml-1 of et al., 1981). The blank, without enzyme, consequently gives highest absobance, which decreases with increase leaf extract. Results obtained with both DMSO and 80 % in enzyme activity. acetone using Arnon (1949)’s equation do not differ Requirements Test tubes, test tube stand, micro-pipettes (20for chlorophyll content. Therefore we recommened that DMSO extraction can also be ued for carotenoids 200 µl, 100-1000 µl and 5 ml), refrigerated centrifuge, estimation using the equations of Lichtenthaler and visible spectrophotometer and “chemicals” as listed in reagents below. Wellburn (1983). Comparison of chlorophyll concentration in Reagents: tissues extracted by grinding with 80 % 1. Methionine (200 mM) acetone and incubation-without grinding in 2. Nitroblue tetrazolium chloride (NBT) (2.25 mM) 3. EDTA (3.0 mM) DMSO (Data from Hiscox and Israelstam 1979) -1 4. Riboflavin (60 µM) Sr. Plant Species Chlorophyll contents (mg g fresh No. weight) 5. Sodium carbonate (1.5 M) Extraction in Extraction in DMSO 6. Phosphate buffer (100 mM, pH 7.8): acetone 1 Pisum sativum 2.6 2.6 (15 min)* Sol A: Potassium dihydrogen phosphate 6.80 g was 2 Citrus lemonii 2.3 2.2 (120 min) 3 Pinus sylvestris 1.4 1.4 (360 min) dissolved in water and the volume was made upto Figure in parenthesis indicate time of incubation of tissue in DMSO 500 ml with double distilled water. Chl-a = 12.21 A663 2.81 Lichtenthaler and Chl-b = 20.13 A645 - 5.03 A663 (1983) Total carotenoids = [1000 A470 - (3.27 Chl-a + 104 Chl-b)]/229 acetone) A645 175 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Sol B: Di-potassium hydrogen phosphate 8.71 g was dissolved in water and the volume was made up to 500 ml with double distilled water. Mix 8.5 ml of sol. A and 91.5 ml of sol. B and final pH was adjusted with the help of pH meter. 7. Grinding media: (0.1 M phosphate buffer, pH 7.5, containing 0.5 mM EDTA) in case of SOD, CAT, GR and POX: EDTA 0.0186 g is dissolved in phosphate buffer 0.1 M, pH 7.5 (made by mixing 16 ml of sol. A and 84 ml of sol. B and final pH is adjusted with the help of pH meter) and volume is made to 100 ml with the buffer. Preparation of enzyme extract Enzyme extract for superoxide dismutase, ascorbate peroxidase, peroxidase and catalase was prepared by first freezing the weighed amount of leaf samples (1 g) in liquid nitrogen to prevent proteolytic activity followed by grinding with 10 ml extraction buffer (0.1 M phosphate buffer, pH 7.5, containing 0.5 mM EDTA in case of SOD, CAT, GR and POX, and 0.5 mM EDTA and 1 mM ascorbic acid in case of APOX). Brie was passed through 4 layers of cheesecloth and filtrate was centrifuged for 20 min at 15 000 g and the supernatant was used as enzyme. Enzyme assay Superoxide dismutase activity was estimated by recording the decrease in optical density of formazone made by superoxide radical and nitro-blue tetrazolium dye by the enzyme (Dhindsa et al., 1981). Three ml of the reaction mixture contained 13.33 mM methionine (0.2 ml of 200mM) 75 µM nitroblue tetrazolium chloride (NBT) (0.1 ml of 2.25 mM) 0.1 mM EDTA (0.1 ml of 3mM) 50 mM phosphate buffer (pH 7.8) (1.5 ml of 100 mM) 50 mM sodium carbonate (0.1 ml of 1.5M) 0.05 to 0.1 ml enzyme 0.9 to 0.95 ml of water (to make a final volume of 3.0 ml) To distinguish SOD isoforms viz., Cu/Zn-SOD, FeSOD and Mn-SOD, the sensitivity of Cu/Zn-SOD to cyanide (3 mM), and Cu/Zn-SOD and Fe-SOD to hydrogen peroxide (5 mM) were used, whereas MnSOD is unaffected (Yu and Rengel, 1999). Complete reaction mixture plus KCN 3 mM (0.1 ml of 90 mM solution) was used to inhibit Cu/ZnSOD Complete reaction mixture plus 3 mM KCN (0.1 ml of 90 mM solution) and 5 mM H2O2 (0.1 ml of 150 mM solution) were used to inhibit both Cu/ZnSOD and Fe-SOD activities. Separate controls (lacking enzymes) were used for total SOD and inhibitor studies. The absorbency was recorded at 560 nm, and one unit of enzyme activity was taken as that amount of enzyme, which reduced the absorbency reading to 50 % in comparison with tubes lacking enzyme. Control - Sample unit (of enzyme) = Control/2 References Dhindsa, R.A., Plumb-Dhindsa, P. and Thorpe, T.A. (1981). Leaf senescence: Correlated with increased permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot., 126: 93-101. Yu, Q. and Rengel, Z. (1999). Drought and salinity differentially influence activities of superoxide dismutase in narrow-leafed lupins. Plant Sci., 142; 1-11. 1.4.2. Ascorbate Peroxidase (APX) Principal Ascorbate peroxidase (APX) reduces hydrogen peroxide to water with the help of ascorbic acid, which is oxidized to mono-dehydroascorbic acid, which is spontaneously metabolized to dehydroascorbate. Reaction was started by adding 2 M riboflavin (0.1 ml) and placing the tubes under two 15 W fluorescent lamps for 15 min. A complete reaction mixture with out enzyme, which gave the maximal colour, served as control. Switching off the light and putting the tubes into dark stopped the reaction. A nonirradiated complete reaction mixture served as a blank. 176 H2O2 + APX Ascorbic acid Mono dehydroAscorbic acid Dehydro-ascorbic acid H2O Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India The assay is based on the decrease in absorbance of ascorbic acid at 290 nm, due to oxidation of ascorbic acid to mono-dehydroascorbic acid and dehydroascorbic acid (Nakano and Asada, 1981). Requirements Test tubes, test tube stand, micro-pipettes (20200 µl, 100-1000 µl and 5 ml), refrigerated centrifuge, UV-visible spectrophotometer and “chemicals” as listed in reagents below. Reagents: 1. Ascorbic acid (3.0 mM): 2. EDTA (3.0 mM 3. Hydrogen peroxide (3.0 mM): 4. Phosphate buffer (100 mM, pH 7.0): Sol A: Potassium dihydrogen phosphate 6.80 g was dissolved in water and the volume was made upto 500 ml with double distilled water. Sol B:Di-potassium hydrogen phosphate 8.71 g was dissolved in water and the volume was made upto 500 ml with double distilled water. Mix 39 ml of sol. A and 61 ml of sol. B, and final pH was adjusted with the help of pH meter. 7. Grinding media: (0.1 M phosphate buffer, pH 7.5, containing 0.5 mM EDTA and 1 mM ascorbic acid): EDTA 0.0186 g and ascorbic acid 0.0176 g are dissolved in phosphate buffer 0.1 M, pH 7.5 (made by mixing 16 ml of sol. A and 84 ml of sol. B and final pH is adjusted with the help of pH meter) and volume is made to 100 ml with the buffer. Preparation of enzyme extract As in case of SOD, except the extraction buffer contain 1 mM ascorbic acid (0.0176 g in 100 ml buffer) in addition to other ingredients. Enzyme assay: Ascorbate peroxidase was assayed by recording the decrease in optical density due to ascorbic acid at 290 nm (Nakano and Asada, 1981). The 3 ml reaction mixture contained: 50 mM potassium phosphate buffer (pH 7.0) (1.5 ml of 100 mM) 0.5 mM ascorbic acid (0.5 ml of 3.0 mM) 0.1 mM EDTA (0.1 ml of 3.0 mM) 0.1 mM H2O2 (0.1ml of 3.0 mM) 0.1 ml enzyme Water 0.7 ml (to make a final volume of 3.0 ml). The reaction was started with the addition of 0.2 ml of hydrogen peroxide. Decrease in absorbency for a period of 30 sec. is measured at 290 nm in an UV-visible spectrophotometer. The initial and final contents of ascorbic acid are calculated by comparing with a standard curve drawn with known concentrations of ascorbic acid. Enzyme activity is calculated as concentration of ascorbic acid oxidized (initial reading – final reading = quantity of ascorbic acid oxidized) per min. per mg protein. References Chen, G.X. and Asada, K. (1989). Ascorbate peroxidase in tea leaves: occurance of two isozymes and the differences in their enzymatic and molecular properties. Plant Cell Physiol. 30 (7): 987-998. Nakano, Y.and Asada, K. (1981). Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant Cell Physiol., 22: 867-880. 1.4.3. Glutathione reductase (GR) Glutathione reductase catalyze the reduction of oxidized glutathione (glutathione disulphide) (GSSG) to reduced glutathione (GSH) by using NADPH as reductant. C y s te in e G l y c in e G lu ta m ic a c id G lu ta m ic a c id C=O C=O NH NH GR C H -C H 2-S -S -C H 2-C H C=O C=O N AD PH NH NH C O O H -C H 2 C O O H -C H 2 G SSG 2 G lu ta m ic a c id C =O NH H S -C H 2-C H + NADP C =O NH C O O H -C H 2 GSH The assay of the enzyme is based on the formation of a red coloured complex by reduced glutathione with 5,5-dithiobis-2-nitrobenzoic acid (DTNB), which absorbs at 412 nm (Smith et al. 1988). Requirements Test tubes, test tube stand, micro-pipettes (20200 µl, 100-1000 µl and 5 ml), refrigerated centrifuge, UV-visible spectrophotometer and “chemicals” as listed in reagents below. 177 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Reagents 1. 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) (3.0 mM) in phosphate buffer (10 mM, pH 7.5) 2. Oxidized glutathione (GSSG) (20 mM) 3. NADPH (2.0 mM): 4. Phosphate buffer (200 mM, pH 7.5): Sol A: Potassium dihydrogen phosphate 6.80 g was dissolved in water and the volume was made upto 500 ml with double distilled water. Sol B: Dipotassium hydrogen phosphate 8.71 g was dissolved in water and the volume was made upto 500 ml with double distilled water. Mix 15 ml of sol. A and 85 ml of sol. B and final pH was adjusted with the help of pH meter. Preparation of enzyme extract As in case of SOD Enzyme assay Glutathione reductase was assayed as per the method of Smith et al. (1988). The reaction mixture contained: 66.67 mM potassium phosphate buffer (pH 7.5) and 0.33 mM EDTA (1 ml of 0.2 M buffer containing 1 mM EDTA) 0.5 mM DTNB in 0.01 M potassium phosphate buffer (pH 7.5) (0.5 ml of 3.0 mM) 66.67 µM NADPH (0.1 ml of 2.0 mM) 666.67µM GSSG (0.1 ml of 20 mM) 0.1 ml enzyme extract Distilled water to make up a final volume of 3.0 ml Reaction was started by adding 0.1 ml of 20.0 mM GSSG (oxidized glutathione). The increase in absorbance at 412 nm is recorded spectro-photometrically. The activity is expressed as total absorbance at 412 nm (A412) per mg protein per min. References Smith, I.K., Vierheller, T.L., Thorne, C.A. (1988). Assay of glutathione reductase in crude tissue homogenates using 5, 5'-dithiobis (2-nitrobenzoic acid). Anal. Biochem., 175: 408-413. 1.4.4.Catalase (CAT) Principal Catalase catalyze the reduction of hydrogen peroxide to water and molecular oxygen. Compared to ascorbate peroxidase it is considered a less efficient system of H2O2 scavenging due to its higher Km value for H2O2 than APX. It is also localized in mitochondria and peroxisomes, and absent in chloroplast, one of the important site of H2O2 generation. 2 H2 O2 Catalase 2 H2 O + O2 Catalase assay is based on the absorbance of H2O2 at 240 nm in UV-range. A decrease in the absorbance is recorded over a time period as described by Aebi (1984). Requirements Test tubes, test tube stand, micro-pipettes (20200 µl, 100-1000 µl and 5 ml), refrigerated centrifuge, UV-visible spectrophotometer and “chemicals” as listed in reagents below. Reagents 1. Hydrogen peroxide (75 mM): Dissolve 775 µl of 30 % H2O2 in double distilled water and make up the volume to 100 ml. 2. Phosphate buffer (100 mM, pH 7.0): Sol A: Potassium dihydrogen phosphate 6.80 g is dissolved in water and the volume was made upto 500 ml with double distilled water. Sol B: Dipotassium hydrogen phosphate 8.71 g is dissolved in water and the volume was made upto 500 ml with double distilled water. Mix 39 ml of sol. A and 61 ml of sol. B and adjust final pH is adjusted with the help of pH meter. Preparation of enzyme extract As in case of SOD Enzyme assay The 3.0 ml reaction mixture consisted of the following: 1. Potassium phosphate buffer 50 mM (1.5 ml of 100mM buffer, pH 7.0) 2. Hydrogen peroxide 12.5 mM (0.5 ml of 75 mM H2O2) 3. Enzyme 50µl 4. Water to make up the volume to 3.0 ml Adding H2O2 started reaction and decrease in absorbance atb 240 nm was recorded for 1 min. Enzyme activity was computed by calculating the amount of H2O2 decomposed. The initial and final 178 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India contents of hydrogen peroxide are calculated by comparing with a standard curve drawn with known concentrations of hydrogen peroxide. Enzyme activity is calculated as concentration of hydrogen peroxide reduced (initial reading – final reading = quantity of hydrogen peroxide reduced) per min. per mg protein. Reference Aebi, H. (1984). Catalase in vitro. Meth Enzymol., 105: 121-126. 1.4.5. Peroxidase (POX) Peroxidase, also referred as non-specific peroxidase or guaiacol-peroxidase catalyse the reduction of hydrogen peroxide with a concurrent oxidation of a substrate. In plant cell it is mostly located in cell wall and is involved in the oxidation of phenolic compounds towards the synthesis of lignins. Peroxidase RH2 + H2O2 2 H2O + R Peroxidase activity is assayed as increase in optical density due to the oxidation of guaiacol to tetraguaiacol (Castillo et al. 1984). The 3 ml reaction mixture contained 16 mM guaiacol, 2 mM H2O2, 50 mM phosphate buffer (pH 6.1) and 0.1 ml enzyme extract diluted 10 times. Requirements Test tubes, test tube stand, micro-pipettes (20200 µl, 100-1000 µl and 5 ml), refrigerated centrifuge, UV-visible spectrophotometer and “chemicals” as listed in reagents below. Reagents 1.Phosphate buffer (100 mM, pH 6.1) Sol A: Potassium dihydrogen phosphate 6.80 g dissolved in water and the volume made up to 500 ml with double distilled water. Sol B: Dipotassium hydrogen phosphate 8.71 g dissolved in water and the volume made up to 500 ml with double distilled water. Mix 85 ml of sol. A and 15 ml of sol. B and final pH is adjusted with the help of pH meter. 2. Hydrogen peroxide (12 mM): Dissolve 124 µl of 30 % H2O2 in double distilled water and make up the volume to 100 ml. 3. Guaiacol (96 mM): Dissolve 1075 µl of analytical grade guaiacol in distilled water and make the volume to 100 ml. Preparation of enzyme extract As in case of SOD Reaction mixture Phosphate buffer (50 m M, pH 6.1) Guaiacol (16 mM) H2O2 (2 mM) Enzyme Water 1.0 ml of 100 mM 0.5 ml of 96 mM 0.5 ml of 12 mM 0.1 ml 0.4 ml, to make final volume of 3.0 ml. Absorbance due to the formation of tetraguaiacol was recorded at 470 nm and enzyme activity was calculated as per extinction coefficient of its oxidation product, tetra-guaiacol =26.6 mM-1 cm-1. Enzyme activity is expressed as µmol tetraguaiacol formed per min per g fr. wt or per mg protein References Castillo, F.I., Penel, I. and Greppin, H. (1984). Peroxidase release induced by ozone in Sedum album leaves. Plant Physiol. 74 846-851. MISCELLANEOUS CALCULATION OF ENZYME ACTIVITY = [*units of substrate/product (µmol or nmol) g-1 fresh wt. min-1] *Units of substrate used or product formed can be calculated either by referring to a standard curve prepared from known concentration or by multiplying with its extinction coefficient (), which has been reported in literature. Activity = *units of substrate/products x vol. of total enzyme extract = 1 x vol. of enzym e used x 1 x x fr. wt. of 60 x Tim e of sam ple incubation If the above value is = ‘X’ Specific activity is calculated by dividing the activity (‘X’) by protein content (mg protein g-1 fr. wt.). Specific activity = [*units of substrate/product (µmol or nmol) mg-1 protein min-1] = X/mg protein g-1 (fr. wt.) Calculation of molarity of liquid chemicals/ compounds M o la rity = 1000 x sp . g r. M o l. w t. x x % 100 Sp. gr. = specific gravity (g/ml) of the chemical/compound. Mol. wt. = molecular weight of the chemical/compound. % = % availability of the chemical/compound in the solution. 179 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India HYDROLYTIC ACTIVITIES AND SOFTENING IN FRUIT RIPENING Atar Singh Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110012 Plant cell wall : Role in fruit ripening/senescence Plant cell walls consist of a complex mixture of polysaccharides and other polymens that are secreted by the cell and are assembeled into an organize network linked together by a mixture of covalent and non covalent bonds. Plant cell wall also contain structural proteins, enzymes, phenolics polymens, and other material that modify the wall’s physical and chemical characteristics, sometimes in seconds. Much of the carbon i.e. assimilated in photosynthesis ends up as polysaccharides in the wall. During the specific phase of development, these polymens may be hydrolyzed into their constituent sugars (cell wall may be massively degraded, such as occur in fruit ripening or in the endosperm of germinating seeds) which may be scavenged by the cell and used to make new polymens (eg. Wall polysaccharides of the endosperm or cotyledons function primarily as food reserve). Oligosaccharide component of the cell wall, act as important signaling molecules during cell differentiation and during the recognition of pathogens and symbionts. Messenger RNAs for expensin have recently been found to be highly and selectively expressed in ripening tomato fruits, suggesting that they play important role in wall disassembly (Rose et al., 1997). Similarly softening fruits express high levels of pectin methyl esterase, which hydrolyse the methyl ester from pectins. This hydrolysis makes the pectin more susceptible to subsequent hydrolysis by pectinases and related enzymes. The presence of these enzymes in cell wall indicate that walls are capable of significant modification during development. Softening is normally accompanied by increase in soluble uronoides (Huber, 1983). Cell wall hydrolydrolyzing enzymes responsible for wall softening also includes endopolygalacturonase, pectin methyl esterase, cellulase, xylanase, -galactosidase etc. (Paull and Chen, 1983). During ripening of tomato fruits, several polysacchaside degrading enzymes are known to increase activity, including cellulase (Dickinson and McCollum, 1964), pectinesterase and polygalacturonase (PG) (Hobson, 1963), Grierson et Enzymes mediate wall hydrolysis and degradation al., 1981). Degradation of the walls can result in the Hemicelluloses and pectins, the structural component of the cell wall, may be modified by a production of biologically active fragments, called variety of enzymes that are found naturally in the cell oligosaccharins, that may be involved in natural wall. Softening of fruits is thought to be the result of developmental responses and in defense responses. disassembly of the wall (Fischer and Bennett, 1991). Some of the reported physiological and developmental Glucanases and related enzymes may hydrolyse the effects of oligosaccharins include stimulation of backbone of hemicelluloses xylosidases and related phytoalexin synthesis, oxidative brusts, ethylene enzymes may remove the side branches from the synthesis, membrane depolarization, changes in backbone of xyloglucans. Transglycosylases may cut cytoplasmic calcium, induced synthesis of pathogen and join hemicelluloses together. Such enzymatic related proteins such as chitinase, and glucanase, other changes may alter the physical properties of the cell systemic and local “Wound” signals and alterations in wall, for example, by changing viscosity of the matrix the growth and morphogenesis of isolated tissue or by altring the tendency of the hemicelluloses to stick samples (John et al., 1997). The best scidied examples are oligosaccharide elicitors produced during invasion to cellulose. 180 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India of pathogen. For example, the fungus phytophthora secretes an endopolygalacturonase (a type of pectinase) during its attack on plant tissues. As this enzyme degrades the pectin component of the plant cell wall, it produces pectin oligogalacturonans that elicit multiple defense responses by the plant cell. The oligogalacturonans that are 10 to 13 residues long are most active in these responses. Plant cell wall also contain -D-glucanase that attacks the -D-glucan i.e. specific of fungal cell wall. When this enzyme attacks the fungal cell wall, it releases the glucan oligomens with potent elicitor activity. The wall component serve in this case as part of the sensitive system for the detection of pathogen invasion. Oligosaccharins may also function during the normal function of cell growth and development/ differentiation. For example, a nona saccharide (an oligosaccharide cantaining nine sugar residues) derived from xyloglucan has been found to inhibit growth permotion by the auxin 2,4-D (optimal concentration 10-9M). Structure of common hemicelluloses A. Galacturonic acid oligomers i) (1→ 4) -D-Gal A – [(1 → 4) –-D-Gal A] – (1→4) – -D-Gal A B. Three oligosaccharins from xyloglucan i) Xyl Xyl Xyl Glc Glc Glc Glc Fuc Gal Gal Xyl Xyl Xyl Glc Glc Glc ii) iii) Xyl Glc (C) Glc Glc Glc Oligosaccharins from fungal cell wall -D-Glc-(1→ 6)–-D-Glc – (1 → 6)–-D-Glc–(1→6)–-D-Glc–(1→6)–Glucitol 3 3 This xyloglucan oligosaccharins may act as a feedback inhibitor of growth eg. When auxin induced breakdown of xyloglucan is maximal, it may prevent excessive weakening of the cell wall. Related xyloglucan oligomers have also been reported to influence organogenesis in tissue cultures and may play a wider role in cell differentiation (Greelman and Mullet, 1997). Polygalacturonase (PG) (EC. 32.1.15): Role in cell wall hydrolysis Polygalacturonase plays the most important role in softening of tomatoes. The enzyme is absent from the unripe fruits and is synthesize de-nova during ripening. It has been found in the cell wall fraction of homogenates in various isozyme forms (Ali Brady, 1982). There are three isozymes forms which are structurally and immunologically related. PGI is the first to appear at the on set of ripening, but in ripe fruit it constitutes only a major percentage (%) of the total. Isozyme 2A and 2B appears 1-2 days after isozyme1 and constitute the majority of the PG protein present in the ripe fruits (Brady et al., 1985). Zeng Yanru et al. (1996) reported an increase in PG and PME activity as the ripening proceeds and reached the peak on 4th day after harvest. The first detectable dissolution of middle lamella occurs very early in ripening when PG-1 is present. Later stages of disorganization may be due to the action of PG2. It has been seen that Never-ripe fruit soften more slowly and only PG-1 activity was detected in it (Crookes and Grierson et al., 1983) PG antisense fruits showed an initial inhibition of pectic degradation and no changing in softening (Smith et al., 1988, Giovannoni, 1993). However, differences in fruit softness was measured in fully ripe transgenic fruit (Grierson and Schuch, 1993) which questions PGs role in softening. Flavr SavrTM , the PG antisense tomato started coming to USA market during 1994. It is characterized by higher viscosity and soluble solids which offer them greater potential in processing industry (Grierson and Schuch, 1993).   -D-Glc -D-Glc Pectic substances of fruit tissues change during 181 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India the ripening period. An overall loss of polymerized Extraction of crude enzyme: galacturonates has sometimes been observed, the large (5 g of fruit tissue (mango/tomato) + effect is an increase in solubility which may result from (10 ml of 0.2 M sodium acetate buffer (ph 6.0) an increase in methyl estrification of the + polygalacturonates or from a decrease in chain length. (A pinch of Na2S2O4) The increased polygalacturonase (PG) activity during + ripening of a number of fruits suggests that (A pinch of polyvinyl pyrrolidone (PVP) depolymerization commonly contributes to increased solubility. mortar under chilled condition (at The enzyme PG (EC 3.2.1.15) catalyze the Grind in pestle and 0 4 C) in ice cool bath hydrolytic cleavage of -1,4 galacturonan linkage in polygalacturonic acid polysaccharide of pectin. Filter through 4 layer of cheese cloth and squeezed in centrifuge tube Assay of polygalacturonase (PG): Principle The enzyme is assayed as the amount of Centrifuge at 15000g for 20 min. At 40C reducing sugars formed calorimetrically using (Please counter balance the tube before run) polygalacturonic acid as the substrate. (The standard curve may be drawn using -D glacturonate or Dglucose as std.) Collect supernatant (enzyme extract) and make up Requirements volume (eg 20 ml or 50 ml) Grinding buffer (Used for both PG and PME assay) i.) Instruments : Centrifuge : Spectrophotometer Assay of polygalacturonase (PG): : Water baths (0.2 ml of assay mixture + 2 ml enzyme extract) ii) Chemicals/solution a) 0.2 M sodium acetate buffer (pH 6.0) 27.2 g of CH3COONa.3H2O dissolved in distilled Incubate in a warm water both at 370C for 2 hr. H 2O adjusted pH 6.0 with acetic acid diluted to 1000 ml with distilled H2O b) 0.4% sodium acetate (CH 3COONa3.H2O) Add 1 ml of 5% phenol solution to 0.05 ml of above buffer (pH 3.8) incubated enzyme extract + 5 ml of 96% H2SO4 and allowed to react for 15 min.) (0.4 g) of CH3COONa.3H2O dissolved in distl. (The blank contain 0.05 ml dist. H2O instead of H2O adjusted pH 3.8 with glacial acetic enzyme extrude) acid diluted to 100 ml with distl. H2O. c) Assay mixture: 0.45% (w/v) pectin and 0.1% casein in 0.4% sodium acetate buffer (pH 3.8) [Add 5 ml distl. H2O and mixed evenly (vortex) and (0.45 g) of pectin and 0.1 g of casein dissolved in cool to room temperature (RT)] 0.4% sodium acetate buffer solution (pH 3.8) d) 5% phenol solution: 5g of distilled phenol dissolved in distl. H2O diluted to 100 ml (Read optical density (OD) at 490 nm on with distl. H2O stored in brown glass spectrophotometer) bottle. 182 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Calculation Assay of PME PG activity (mg of glucose equivalent released/ (10 ml of 0.3 mM NaCl containing 0.5% pectin (pH g fr.wt.hr) = 288.07 x OD 8.0) are measured for a original pH value with pH meter) Pectin methyl esterase (PME) (EC.3.1.1.11) Pectin methyl esterase (PME) catalyzes the demethoxylation of carboxyl groups from the galacturonosyl residues of pectin. This enzyme is widely (Added 3 ml of enzyme extract gradually. The reacts flask is constantly stirred) distributed in plants. Pectin degradation play important role in the plant disease, fruit ripening, nutrition and food product stability. Pectin which is composed of galacturonic acid and galacturonic acid methyl esters (After 5 min. the pH of the mixture is brought to the original pH value (Stp I) with 0.1 M-NaOH are hydrolyzed by pectin methyl esterase as shown below: PME Pectin – COOCH3 + H2O Pectin – COO– + H+ + CH3OH (Volume of 0.1 M-NaOH consumed noted down) Either the methanol or the increase in free carboxyle group may be measured. While the former is cumbersome, measurement of the later is easily done. Calculation: PME activity (units equivalents/min/g/ fr.wt.) = 0.13 x vol of 0.1 M NaOH consumed References Pectin methyl esterase activity Requirements i) Instruments : pH meter : Titrimeter ii) Chemicals/solution needed a) Assay mixture: 0.3 mM NaCl (pH 8.0) containing 0.5% pectin 0.0035 g of NaCl dissolved in dist. H2O + 1 g of pectin added to the solution Ali, Z.M. and Brady, C.J. (1982). Purification and characterization of the poly galacturonases of the tomato fruits. Aust. J. Plant Physiol., 9: 155169. Brady, C.J. McGlasson, W.B., Pearson, J.A., Meldrum, S.K. and Kopeliovitch, E. (1985). Interaction between the amount and molecular forms of polygalcturonase, calcium and firmness in tomato fruits. J. Amer. Soc. Hort. Sci., 115(2): 254-258. Dissolved by magnetic starring over might (if not dissolved filter through Whatman No. 1 filter paper) Crookes, P.R. and Grierson, D. (1983). Ultra structure of tomato fruit ripening and the role of polygalacturonase isozymes in cell wall degradation. Plant Physiol., 71: 1088-1093. Adjusted the pH of the solution to 8.0 with 1.0 M NaOH Dickinson, D.B. and McCollum, D.P. (1964). Cellulose in tomato fruit. Nature, 203: 525-527. Dilute to 200 ml with distl. H2O Fischer, R.L. and Bennett, A.B. (1991). Role of the cell wall hydrolase in fruit ripening. Annu. Rev. Plant Physiol. Plant Mol. Biol., 42: 675-703. b) c) 0.1 M NaOH : NaOH (4 g/lit.) Enzyme extraction : Same as PG activity Grierson, D. and Schuch, W. (1993). Control of ripening. Philosophical Transactions of the Royal Society of London, 342: 241-250. 183 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Grierson, D., Tucker, G.A. and Roberston, N.G. (Carica papaya L.) during fruit ripening. Plant (1981). The molecular biology of ripening. In: Physiol., 72: 382-385. Biochemistry of Fruit and Vegetables. Pp. 179- Rose, J.K.C., Lec, H.H. and Bennett, A.B. (1997). 191 ed. (Friend, J.) Academic Press, London. Expression of a divergent expansion gene in fruit Hagerman, A.E. and Anstin, P.J. (1986). J. Agric Food specific and ripening regulated. Proc. Natl. Acad. Chem., 34(3): 440-444. Sci. USA, 94: 5955-5960. Hobson, G.E. (1963). Pectinesterase in normal and Rouse, A.H. and Atkins, C.D. (1955). Pectin methyl abnormal tomato fruit. Biochem. J., 92: 324-333. esterase and pectin in commercial citrus fruits as determined by methods used at the citrus Huber, D.J. (1983). The role of cell wall hydrolases in experimental station. Florida Agric. Exp. fruit softening. Hort. Rev., 5: 169-219. Station Bulletin, 570: 1-9. John, M., Rohrig, H., Schmidt, J., Walden, R. and Schell, J. (1997). Cell signaling by Smith, C.J.S., Watson, C.F., Ray, J., Bird, C.R., Morris, P.C., Schuch, W. and Grierson, D. oligosaccharides. Trends Plant Sci., 2: 111-115. (1988). Antisonse RNA inhibition of Lazan, H., Ali, Z.M. and Sani, H.A. (1990). Effect of polygalacturonase gene expression in transgenic vapour guard on polygalacturonase activity and tomatoes. Nature, 334: 724-726. variation in ripening of papaya fruit with tissue depth and heat treatment. Physiol. Plantarum, Zeng, Yanru, Pandey, M., Prasad, N.K. and Srivastava, G.C. (1996). Hydrolyzing enzymes 77: 93-98. and respiration during ripening of tomato Paull, R.E. and Chen, N.J. (1983). Post harvest (Lycopersicon esculentum). Fruits Curr. Sci., variation in cell wall degrading enzymes of papaya 70(11): 1017-1018. 184 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ELEMENTAL ANALYSIS IN PLANT TISSUE BY ATOMIC ABSORPTION SPECTROSCOPY Atar Singh Division of Plant Physiology, IARI, New Delhi-110012 Sampling and method of digestion for elemental analysis Although it is possible to assay just about any plant part, or even the whole plant itself but the biological significance of such analysis depends on the availability of interpretive data for the plant part collected. Therefore, the primary objective should be to obtain that the plant part for which the assay result is comparing with non interpretive values. Prior to elemental analysis, possible error can occur at various stages such as Sample collection, Initial handling, Transportation, Decontamination, drying and Particle size reduction. Thus possible source of error must be known to the investigator for accurate analysis and following precautions should be taken at the time of sampling andvarious stages of sample preparation for analysis. Collection of plant sample: The important components for plant tissue collection are to define The plant part at specific location of plant. Specific sampling time or plant growth stage. Number of the plant part taken per plants. Number of plants selected for sampling. Avoidance criteria for collecting appropriate sample is also crucial. Plants to be avoided are ones that i) Have been damaged mechanically or by insects. ii) Have been under long climatic or nutritional stress. iii) Are infested with disease. iv) Are covered with dust or soil or foliar applied spray materials unless these extraneous substances can be removed (decontamination procedures) v) Are border row plants or shaded leaves contain dead plant tissue. In general the nutritional status of the plant is better reflected in mineral content of the leaves than that of other plant part. Thus leaves are usually used for plant analysis. For some species and for certain mineral nutrients, nutrient content in dry matter may differ considerably between leaf blades and petioles, sometime the petioles are a more suitable indicator of nutritional status. In fruit trees, fruit analysis themselves is the better indicator, especially for Ca, B in relation to fruit quality and storage properties. Under certain climatic conditions, draught stress during seed filling in particular, in legumes seed content of Zn appears to be more sensitive parameter for Zn fertilizer supply as, for example, foliar analysis. For statistical analysis it is always advisable to collect the required number of plants in order to make the composite samples, which will ensure the sufficient replication. Initial handling and transportation: Fresh plant tissue is perishable and therefore, must be kept cool and dry atmosphere prior to delivery to lab. It is best to transport plant tissue in clean paper or cloth bags, not on airtight containers or plastic bags. If possible the tissue will be air dried prior to shipment, particularly if tissue is succulent or the time in transit will be greater than 24 hr. Integrity of the sample is also crucial to ensure the accurate assay results. Therefore, care should be taken during collection of sample. The sample should not be altered chemically or by extraneous material as a result of contact with sampling tools or container. Decontamination of sample: Plant tissue i.e, covered with dust, soil particles or coated with that element of interest in the plant analysis require decontamination prior to drying of sample. Only fully turgid plant tissue can be subjected 185 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India to a decontamination procedure. Decontamination by washing must be done either in the field or later in the lab and keep the sample in cool and moist atmosphere. To remove most of the extraneous materials washing of the fresh plant tissue can be done in 0.1% to 0.3% P- free detergent solution followed by rinse by pure water. Dipping of tissue for 15 seconds in 0.1% teepol and 0.1M-HCl followed by rinsing by pure water can also be followed. Normally decontamination (washing) is not recommended unless absolutely necessary. Reason being, elements like Fe, Al, Mn, Zn, Si and Cu are reduced by washing. Washing cause significant reduction in Fe, Mn and Zn and moderate reduction in Cu content. Washing in the citrus leaves resulted reduction in  Fe, from 186 to 6  Mn from 182 to 94  Zn from 123 to 68 and  Cu from 5.6 to 5.1 mg/ Kg. In some instances, the decontamination procedure itself may add the elements to the tissue or it may leach elements such as K, Cl, B and NO3 during the washing process. Thus mechanical wiping or brushing may also be followed to remove the dust etc. if required. Contamination on rough or pubescent plant tissues surfaces are difficult to remove, posing serious problem, as washing may not be able to remove the contaminants effectively. Unfortunately, there is no satisfactory alternative decontamination procedure so far. Drying: Elemental concentration is totally based on dry weight of collected plant tissue will affect its elemental composition. Decaying of plant tissue causes significant reduction in dry weight of plant tissue, in addition some elements; particularly N and S may be lost by volatilization. Fresh plant tissue is best dried at 80 ºC in an oven. Temperature below 80 ºC may not be sufficient to remove all the moisture while above 80 ºC can cause thermal decomposition. Thus, once dried the sample must be stored in a moisture free atmosphere. Plant tissues high in soluble sugar are not easily oven dried and therefore moisture removal is best done by vacuum oven drying or by fridge drying. Grinding: Particle size reduction can be done by various ways;  Mechanical grinding/crushing.  Cutting action using Wiley or hammer Mill.  By abrasion in Cyclonic Mill or  By crushing in ball Mill. In most Mill particle of contact surfaces will be added to the sample such asCu and Zn addition from brass fittings even Fe when fittings, cuttings and crushing surfaces are made of steel. Fe contamination is best reduced by hand cutting or by crushing in agate ball Mill. The finer the ground powder, more homogeneous the sample will be. The ground powder become more sensitive to thermal decomposition at temperature above 80 ºC, therefore this temperature limit should not be exceed prior to weighing for analysis. For long-term storage, ground tissue must be kept in airtight containers and stored at low temperature (below 10 ºC) in dark environment. Digestion of the plant sample: For nutrient other then N the plant material can be digested in a diacid mixture or tri acid mixture or dry ashed and dissolved in acid. The diacid digestion is used for the determination of P, K, Ca, Mg, S, Fe, Mn, and Cu. It must be followed for the determination of Ca and Mg. Diacid digestion normally recommended for the plant analysis. Triacid digestion recommended only for K, P to be estimated. S cannot be estimated from triacid extract. Wet digestion is normally not used for the estimation of B and Mo. Diacid digestion: It is carried out using 9:4 mixture of HNO3: HClO4, if the sample is high in fat/oils, predigestion using 25 ml HNO3/g sample is recommended to avoid explosion. Procedure for Diacid digestion: One gram of ground plant material is place in 100ml volumetric flask. To this 10 ml acid mixture is added and mixed the content by swirling the flask. The flask is placed on low heat hot plate in digestion 186 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India chamber. Then the flask is heated at higher temperature until the production of red NO2 fumes ceases. The content are further evaporated until the volume is reduced to about 3 to 5 ml but not dryness. The completion of digestion is confirmed when the liquid become colourless. After cooling the flask, add 20 ml of glass distilled water and made the volume upto the mark. The solution is filtered through whatman no.42 filter paper. The filtered solution is used for the determination of P, K, Ca, Mg, S, Fe, Mn, Zn and Cu. Triacid digestion: It is carried out using the mixture of HNO3: H2SO4: HClO4, in the ratio of 9:4:1. The sample digestion is carried out as described under Diacid digestion. Dry ashing: Organic matter destruction can also be done by high temperature oxidation. The critical requirements are the - Nature of the ashing vessel - Placement in the muffle furnace - Ashing temperature and time The vessel used for ashing range from silica to platinum crucible or dishes. The sample size can very from 0.5 to 2.0g depending of expected concentration of elements to be determined. The temperature used for ashing ranges from 475 to 600 ºC and time varies from 4 to 12 hr depending on sample weight and sample type. The ash residue is usually dissolved in a HNO3 or HCl solution and diluted to a specific volume with deionized water. Procedure: To carry out ashing, 1g of dried, ground plant material is placed in silica crucible. The crucible is placed in a cool muffle furnace and ashed at 550 ºC for 5 hr. The ash is then cooled and dissolved in 20% HCl, warming the solution if necessary to dissolve the residue. The crucible should be placed 2cm away from the walls and bottom of furnace to avoid localized over heating. The solution is filter through acid washed filter paper into a 50 ml volumetric flask. The filter paper is washed and the solution is diluted with demonized water and mixed well. Dry ashing can be used for the determination of Na, K, Ca, Mg, Cu, Fe, Mn, Zn, B and Mo in plant tissue. It is preferred technique particularly for the estimation of B and Mo. Dry ashing provide good precision and is an easy, rapid method requiring minimal analyst attention. It is also free from reagent contamination. Main disadvantage of this process is that it cannot be used for the element such as N, P and S, which are volatile at ashing temperature. Atomic absorption spectroscopy: The first observation of an atomic absorption spectra was made by Wollaston who in 1802, described dark line in solar system but the technique was introduced for analytical purposes by Walsh and by Alkemade and Milatz (1955) under the designation of atomic absorption Spectroscopy. This involves the absorption of radiation energy (usually UV and Visible region) by neutral atoms in gaseous state. In atomic absorption spectroscopy the sample is converted into atomic vapour and then absorption of these vapours is measured at a selected λ which is characteristic of individual element. All the analytical applications of atomic absorption involve spraying a solution of the sample into the flame. Thus technique is also called Absorption flame photometry. Principle of Atomic absorption spectroscopy: The principle is Similar basically to those considered for absorption of UV and visible radiations by solution, but the equipment and the spectra are quite different. In atomic absorption analysis the element being determined must be reduced to the atomic state, vaporized and imposed in the beam of radiation from the source. This process is frequently accomplished by drawing the solution of the sample, as a fine mist into a suitable flame. Thus the flame serves the function analogous to that of Cell/Cuvette and solution in conventional spectroscopy. The absorption spectrum of an element in its gaseous, atomic form consists of a series of well defined, extremely narrow lines arising by the electronic transitions of outer most electrons. In case of metals most of these transitions correspond to the transitions that occur in the UV and visible regions. 187 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Why elemental analysis? Some elements particularly metals, play an important role in biological systems whether as simple cofactor as coenzymes, the central atom on biological macromolecules such as iron (Fe) in hemoglobin or magnesium (Mg) in Chl or toxic substances that affect the metabolism (eg. Cd is very toxic and poisonous and its traces may cause adverse changes of human kidneys. It is also toxic to certain varieties of fishes if present in the concentration more than 200ppb in water) or sodium (Na) and potassium (K) in biological fluids (blood serum, cerebro spinal fluid) and urine. Therefore, use of atomic absorption spectroscopy enables to obtained the data that are important in the understanding of biological role of these elements. In a spectrum of an element of most obvious λs at which absorption or emission is observed are associated with the transitions where minimal energy change occurs eg. 3s-3p transition in Na atom that gives rise the emission of yellow light, referred as Dline transition. When the electronic transition occurred in an atom they are limited by the availability of empty orbital or levels. A orbital or level could not be overfilled without contravening the Pauli’s principle. Adequate tissue levels of element that may be required by plants: In order for energy changes to be minimal, transition tend to occur between the levels close together in energy terms. These limitations of energy and occupancy of levels means that observed absorption or emission are absolutely the characteristics of the element concerned. Measurement of atomic absorption: Analytical methods based on atomic absorption are highly sensitive because atomic absorption lines are extremely narrow and transition energies are unique for each element. But limited line width creates the measurement problem, not encountered in solution absorption. If the band width is narrow w.r.t. the width of absorption peak, a linear relationship between absorbance and concentration is expected inspite of the fact that Beer’s law is only applicable for monochromatic radiation. In contrast to rather broad bands shown in the absorption spectra of compositions, atoms absorb at sharply defined λs . Consequently, an extremely small amount of compound exciting light is used and the radiant energies corresponding to these λs presenting in the exciting beam is quite insufficient for practical applications. Moreover, a band of radiation ie., as narrow as peak width of an atomic absorption line (0.02Element Conc. In dry matter Relative no. of atoms (%) with respect to Mo. 0.5A0) cannot be obtained by ordinary ________________________________________________________________________ monochromator. Thus, if a continuous source of Obtained from H2O and CO2 radiation is used with the monochromator, only a H 6 60,000,000 C 45 40,000,000 fraction of emerging radiation is of the λ ie., absorbed O 45 30,000,000 Obtained from the soil macronutrient and the relative changes in the intensity of emerging N 1.5 1,000,000 band is small in comparison to the changes takes place K 1.0 250,000 Ca 0.5 125,000 in the radiation corresponding to the absorption peak. Mg 0.2 80,000 These cause two main difficulties. P 0.2 60,000 S Si Micro nutrients 0.1 0.1 ppm 30,000 30,000 Relative no. of atoms with respect to Mo. i) Beer, law will not be applicable under these conditions. ii) The sensitivity of the method decreases. Cl 100 3,000 Fe 100 2,000 It is therefore, necessary to use the exciting B 20 2,000 Mn 50 1,000 beam that contain the high intensity of light of the Na 10 400 required λs ( Hollow cathode lamp is used )because Zn 20 300 Cu 6 100 the particular λ is specific for the element and unable Ni 0.1 2 Mo 0.1 1 to excite any other element. ________________________________________________________________________ 188 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Reference Modulated Power supply Monochromator PMT HCL Sample Automizer Amplifier D2 Lamp PC r pe p o Ch Data conversion Fig. Schematic of Double beam AAS 4139 Fuels and Oxidant • • used for elements, which produce refractory oxides and require somewhat high temperature for decomposition. Oxygen –acetylene or N2O –acetylene may be employed for elements, which form the stable oxides (eg. Al, Be and rare earths). -Natural gas-Hydrogen, Propane, Butane and *Acetylene *Acetylene most important and most widely used flame. Oxidants: -Air -Air rich oxygen and Nitrous oxide (N 2O) Fuels: Flame Approx Temp ºC Element for which suitable ________________________________________________________________________ Air-coal gas Air-propane 1800 ºC 1900 ºC Air-acetylene (lean) Air-acetylene (rich) N2O/ acetylene 2300 ºC 2300 ºC 2955 ºC Cu, Zn, Pb, Cd, alkali metals Same+ volatile elements and noble metals Alkaline earths Sn, Ba, Cr, etc. Al, V, Ti, Ta, Be, Se, etc Consumption : Air –acetylene flame 1-4lit/min N2O-acetylene flame 4-9lit/min 10-20lit/min **N2O flame ** Where the room temperature lowers than 20 ºC, internal freezing of the gas may be experienced. To prevent this, an IR heat lamp can be directed on the regulator. Low temperature flame (Natural gas) produce satisfactory results for elements which are redialy converted into atomic state ( eg. Cu, Zn, Pb, and Cd). High temperature flame (Air-acetylene mixture) Burners: Two types of burners- Made up of corrosion resistant material. • Stainless Steel - For air-acetylene flame • Titanium - N2O –acetylene flame 189 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Burners i) Total consumption burner P0 P From source To monochromator Aspired sample Ai r Fuel gas ii) Premix burner Sampl e From source To monochromator Fue l Sampl e Bafles for mixing Drai n Interference: Chemical Interference: An absorption peak of an element is free from interference by other elements can be obtained by using the good monochromator. Moreover chemical reactions taking place in the flame give rise interferences which arise as a result of interactions and chemical competitions. These interactions and competitions affect the number of atoms present in the light path under specified conditions. However, it is not possible to predict these effects theoretically. They can be determined experimentally only. The formation of ground state atoms can be inhibited by two general forms of chemical interference. i) Incomplete dissociation of compounds ii) Ionization i) Incomplete dissociation of compounds: The most common form of this type of interference is the formation of refractory compounds as calcium phosphate and potassium fluorotantalate. Such interferent forms the compounds which are not completely dissociated at flame temperature and therefore prevent the formation of neutral ground state atoms. With non flame atomization problem is similar eg. Graphite furnace, it is carbon rather than oxygen that forms the refractory compounds. Amos (1972) has successfully dissociated the carbides of Al and Si thermally. Carbide formation can be prevented by coating the graphite surface with pyrolytic graphite or vitreous carbon which is highly impervious (impermeable) form of element. But the coating has been found to decrease the penetration of the sample in the porous graphite. The interference can be overcome by using following means. a) use of higher temperature flame - N2O – acetylene flame (Frequently supply the sufficient thermal energy to cause the complete dissociation of compound which are insufficiently dissociated in cooler flames eg. Ca interference on Al can overcome by 190 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India dissociating the calcium aluminate found in the cooler flames) b) Extraction the analyte element: It is sometime possible to extract the analyte element in the organic medium eg. Potassium (K) interference on tantalum (Ta) can be avoided by extracting the Ta as the fluoride complex into methylisobutyl-ketone, thus preventing the refractory potassium fluorotantalate. c) Extraction the interferent: Extraction of interferent in organic solvent is also possible from the solution. A highly specific and quantitative extraction is not always necessary eg. In the determination of trace metals in iron ores the excess Fe can be extracted in isobutyl- acetate as chloride complex. This allows trace analysis to be carried out in the aqueous solution. d) Use of releasing agent: The formation of refractory compounds can be prevented by adding the excess of another element which will combine preferencelly with the interferent in the presence of analyte eg. In the determination of Ca; Lanthanum (La) or Strontium (Sr) nitrate can be added to the solution containing phosphorous. This will allow the determination of Ca in an acetylene flame without interference due to the formation of calcium phosphate. Some elements form the stable compounds with some other constituents of the sample eg. the determination of Sr in the presence of Al or Si can be carried out only by adding the lanthanum (La) salts. La preferancelly binds the Al or Si, leaving the Sr free. Some elements like Ti, Al, and V oxidized in the flame forming compounds that stand with the temperature of air-acetylene flame (refractory compounds). Therefore, their molecules rather than the atoms are present in the flame. ii) Ionization: High temperature flame such as acetylene and N2O-acetylene may cause the appreciable ionization of the analyte element. Alkali metals and alkaline rare earth metals are more susceptible to ionization than the transistor elements. To control the ionization of analyte it is necessary to add the suitable cation having an ionization potential lower than that of analyte. eg. Additions of Na, K and Cs at concentration between 2000 and 5000 μg/ ml create excess electrons in the flame and effectively suppress ionization of analyte. Table. Ionization potential and % ionization of different elements in air–acetylene and N 2Oacetylene flame at their corresponding concentrations. ---------------------------------------------------------------------------------------------------------------Metal Ionization Conc. % ionization Potential (µg/ ml) Air-acetylene N 2O (eV) acetylene ---------------------------------------------------------------------------------------------------------------Al 6.0 100 10 Ba 5.2 30 0 88 Be 9.3 2 0 Ca 6.1 5 3 43 Cs 3.9 K 4.3 Mg 7.6 2 0 6 Na 5.1 Sr 5.7 5 3 84 Tb 6.2 15 20 ---------------------------------------------------------------------------------------------------------------- It should be noted that the degree of ionization will vary with the concentration of the analyte element. When using a more concentrated solution (when operating on alternative) ionization may not a serious problem. Cation Interferance: The absorption of one cation is affected by the presence of another cation eg. In the determination of Mg the presence of Al causes low results. It is due to the formation of Al-Mg compound (heat stable) which reduces the concentration of Mg in the flame. In the determination of Ca, the interference is caused by Al, Be and Mg, however this type of interaction is rare. Anion Interference: The height of the absorption peak for a metal 191 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India is affected by the type and concentration of the anion • present in the sample solution. In many cases anion interference can be avoided by adding the complex formining agent (chelator) such as EDTA to both the standard and sample. In this manner formation of atoms • takes place from the decomposition of complex, instead of different compounds into the flame. Analytical variables: The most important analytical variables are i) Rate and flow of the oxidant and fuel gases. ii) Position of the beam w.r.t. the flame. iii) Nature of fuel and oxidant. iv) Nature of anion present. v) Rate of introduction of sample Among all these factors the last one is of the special value. If the rate is too low, the number of atomic particles is small, while the higher rate of introduction causes an inordinate consumption of the flame energy by the evaporation process and very small amount of energy is thus available for the formation of atoms.Viscosity of the solvent and the presence of combustible organic solvents also influence the atomic absorption peak eg. Two solution of same concentration of metal but varying amount of the other extraneous materials may give the divergent readings. Advantages: • Minute amount of an element can be determined in the presence of high concentration of other element. • The method is more sensitive than emission flame photometry. • It is highly specific . • It is rapid and require small amount of material. Disadvantages and limitations: • The main disadvantage of this method is the need of separate lamp source for each element to be determined. To avoid this difficulty a continuous source of light is used with a very high resolution of monochromator, or alternatively to produce a line source by introducing a flame. Element such as Al, Ti, W, Mo, V, Si. etc. can not be detected when a flame is used to produce atomic state. This is due to the fact these element give rise the oxides in the flame. The use of this technique is limited to metals only. With nonmetals which have their resonance line in the vacuum ultra violet, difficulties arise from the strong absorption of light by the oxygen in the light path and from the flame gases themselves. • If aqueous solutions are used the prominent anion affects the signal to a noticeable degree. Application: Agriculture: Soil extract, plant material and fertilizers have been analyzed for Ca,Cu, Fe, K, Na,Mg, Mo,Mg, Mn, Sr. and Zn oil analysis: crude oil, feed stock and lubricating oil have been analyzed for Ag, Ba, Ca, Cr, Cu, Fe,Na, Ni and Pb Natural water and trade effluents: Metallic Fe and contaminants (Cd, Cr, Cu, Fe, Pb, Ni, and Zn) Medical: -Na and K in biological fluids. They have depressive effect of phosphate and proteins in blood serum, cerebrospinal fluid and urine is overcome by adding the single diluents in each case - Fe in blood. - Cd and Zn in the urine of industrial workers. - Toxic level of Pb and Hg. Metallurgy and general inorganic analysis: -Light alloys -In Al, residual and alloying concentration of Mg, Cu, Zn, Pb, Mn, Ni, etc are easily determined -In iron and steel Co, Cr,Mg, Mn, Pb, and Zn. -Non ferrous alloys analyzed for Ag, Bi, Cd, Co, Cr, Mg, Mn, Mo, Ni, Pb, and Sb. -in noble metals Zn, Ag, Au, Pd, and Pt -Residue containing noble metal for Ag, Co, Cu, Fe, Ni, Pd, and Zn Flame Emission Spectroscopy or flame Photometry: Basis for flame emission spectroscopy: When small amount of Na introduce in the flame of 192 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Bunsen burner a characteristic yellow light flame is emitted and the brightness of the flame varies with the amount of Na or other metal introduced. Emission of such characteristic radiation by Na or other metal and correlation of the emission intensity with the concentration of the element form the bases of flame photometry, which is actually a part, in broader sphere, of emission spectroscopy. Principle: The principle of flame emission spectroscopy is same as flame atomic absorption spectroscopy but the main difference is that the former depends upon the particles which are electronically excited in the medium while the later is based on the behavior of the particles that exist in ground state in the flame. Flame and flame characteristics: The most important characteristics of the flame in flame emission spectroscopy are i) to convert the constituents of liquid sample into vapor state ii) to decompose these constituents into atoms or simple molecules. iii) to electronically excite a fraction of the resulting atomic or molecular species. Flame temperature: Between 1000 º - 3000 ºC, Mixture of coal gas air don’t give very hot flames due to the presence of nitrogen -----------------------------------------------------------------------------------Fuel Temp ºC In Oxygen In air -----------------------------------------------------------------------------------Methane 2700 2000 Propane 2800 1925 Butane 2900 1900 Hydrogen 2780 2100 Acetylene 3050 2000 *Cyanogen 4580 -----------------------------------------------------------------------------------*Highest energy flame reported is produced by combustion of cyanogens gas in oxygen. The spectra of cyanogen gas is nearly arc like and permit the determination of elements with high excitation energy but because of its toxicity and other disadvantages it is not likely to be used widely. CO + NO 2 C 2 N 2 + O2 Variation in emission intensity in the flame: The intensity of emission line varies in different parts of the flame, and hence the adjustable burner mounts are desirable eg. The line of various elements such as Mo, Ti, W, V, and Re etc are either absent or very weak in the oxygen gas flame while appear in unusual strength in the spectrum of a rich acetylene oxygen flame when the analyte is dissolved in hydrocarbon solvent. Fuel rich flames provide an environment more favorable for the existence of free atoms of those elements having strong predictions to form stable monoxide molecules in ordinary flame. The spectrum of the inner cone and reaction zone of hydrocarbon flames another rich source of lines. Lines are primarily from elements of high excitation potential and ionization potential; they include Sb, As, Bi, Hg, B, Pt, Tl, Sn and Zn from oxygen- acetylene- ketone flame or oxygen-hydrogen –naphtha flames Metallic Spectra in flames: In flame photometry both line and band spectra are useful for analysis, but the presence of other metals causes background luminosity. This is due to the fact that the excitable cation will give some radiation over a wide spectral region, even at the considerable distance from its discrete lines. The background effect can best be eliminated by the application of baseline technique, if the spectra observed with double beam spectrophotometer. In general lowest energy resonance lines of atoms have been employed because they have the sufficient energy and these characteristics of flame arise directly from low excitation energy involved. Flame spectra are of great importance in quantitative analysis since it permit the use of lower 193 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India dispersion monochromator or filters and reduces the influence from overlapping spectra. Nearly 1/3rd elements including rare earths and alkali metals have been determined by using band spectra in flame photometry. Excitation of metal oxides gives the band spectra The use of organic solvents such as alcohols, ketones and esters alone or mixed with water increase the sensitivity of flame when spray the sample in flame. When an aerosol is uniformly delivered into the flame, the following sequence of events take place in rapid succession. • The water or other solvent is vaporized leaving the minute particles of salts. • The dry salt is vaporized at high temperature of flame then a part of vapour or a gaseous molecules are progressively dissociated to neutral atoms. Some of the free metal atoms react with other radicals or atoms present in the flame gas or introduce concomitantly with the test element. • The vapor of the neutral atoms of metal or the molecules containing the metal atoms, are then excited by thermal energy of the flame or by chemiexcitation mechanism. Ionization and excitation of ionized atoms may takes place to some extant. • From the excited state of the atoms or molecules or ions a reversion occur to the ground state. This process takes place partly by impacts with other species and partly by emission of characteristics radiation. • Although most of the lines emitted in the flame are from neutral atoms, but in high temperature flames, emission lines arising from singly ionized atoms specially incase of alkaline earth metals have also been observed. Flame Background: The spectrum associated with the flame of a given fuel depends mainly on the condition of the flame, specially the fuel oxygen ratio and flame temperature. The hydrogen flame gives the best combination of signal to background. Prominent OH band structure takes place between 280 and 295 mμ and 306 and 320mμ and 340 and 348mμ. A broad emission due to the spectrum of water occurs between 800 and 1250mμ. Spectrum of water occurs as a product of combustion. The acetylene flame shows principally OH band spectrum and the continuum of dissociating CO molecules. Pressure regulators and flow meter: • In order to achieve the steady emission readings, it is necessary that the gas pressure and gas flow must be maintained constant during the use of flame photometer. • Double diaphragm pressure regulators- A 10lb / in2 gauge for the fuel and - 30lb / in2 gauge for oxygen or air supply followed by a rotameter should be installed in the line from the gas cylinder to burner. Analytical errors: Analytical errors arise when the component present in the sample as impurity. The magnitude depends upon the following factors. i) Quality of the monochromator. ii) The temperature of the source. iii) Concentration ratio between the contaminants and the element sought. Chemical interference: Chemical interference occurs when a species in the flame react with the atoms, as a result emission is decreased. Naturally anions present in the solution will interfere. Anions have the strong dispersion effect on the intensities of a number of cation lines. eg. The intensities of the alkaline earths, may decrease even to 5% or more by the presence of phosphate, oxilate, sulphate and aluminate eg. When increasing amount of suphate is added to the solution containing Ca Cl2, the line intensity of Ca decreases progressively but after some time a point is reached where the addition of more sulphate has no effect on the line intensity. 194 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Chemical interference may be reduced to greater extent by adding organic solvent, complex forming agent or both to the solution. If the amount of interfering anion is greater, then pass the solution through - anion exchange resin in the chloride or nitrate form precipitating agent can also be used releasing agent can be employed. These agents firmly combine with interfering anion or displace it by forming stable complex with cation eg. Phosphate interfere in the determination of Ca. It can be eliminated by adding Zirconium or Lanthanum. This agent strongly bonded with phosphate and leaves the Ca available for excitation. Radiation Interference: When the emission line of the element is very close to the element under study the monochromator is unable to distinguish between the line of analyte element and interfering element. To ovecome this problem a identical amount of interfering element is added to the solution of calibration curve or it should be removed. Excitation Interference: In case of alkali metals due to low ionization energy cause this type of interference. The presence of another metal causes the ionization and increase the electron concentration which reverse the equilibrium of the following equation. M M+ + eConsequently more atoms of the metal to be determined are present and the line intensity is increased. Flame Temperature: When the flame temperature is too low either no lines or very weak lines are obtained. Low temperature is insufficient to cause Dissociation of salt. To effect vaporization* To excite the atoms of metal and - Too high temperature is also deteriorative effects; hence for good precision, the temperature of the flame must be appropriate. (*The energy is sufficient to vaporize only metal of high volatility. The vaporization decompose the salts but the rate at which the decomposition occurs is low for some salts and high for others.) Application: • Elements commonly determined by flame photometry areAl, Ba, B, Ca, Ce, Cr, Cu, Fe, Pb, Li, Mg, Mn, K, Ru, Na, and Sr. • Elements easily determined but more or less neglected – Sb, Ag, Bi, Cd, Co, Ga, Id, La, Ni, Pd, rare earths (except Cs), rhodium, ruthenium, scandium, Ag, Tl, Th, Tin and Yttrium. • Elements with distinctive but less sensitive flame spectra- Be, Ge, Au, Mo, Hg, Nb, Rb, Si, Ti, and W • Elements determined by indirect meansBr, Cl, F, I, P and Si. Br, Cl, and F can however, be determined by their metallic halide spectra. References: • Gieseking et al., (1935). Ind. Eng. Chem. A.E. 7:185. • Toth et al., (1948). Soil Sci. 66: 459. • Parks et al., (1943). Anal. Chem.15: 527. • Piper (1944). Soil and plant analysis (New york: Interscience, Publishers, INC). • Methods Mannual of Atomic Absorption Spectrometer (Eletrnic corpration of India Ltd.) • Soil chemical analysis by Jackson (1958). Printice-Hall Englewood Cliffs, NJ. • Instrumental method s of chemical analysis by BK Sharma (2001). Goel Publishing House Meerut 195 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India REGENERATION PROTOCOL FOR GLADIOLUS Divya Choudhary, Gaurav Agarwal and Ajay Arora* Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110 012 Procedure for micropropagation of Gladiolus grandiflora, an economically important horticultural crop has been developed using asceptically grown cormel’s slices as explants. Higher proliferation and multiplication of callus were obtained on Murashige and Skoog basal medium supplemented with 2.0 mg/ l NAA and 1.0 mg/l BAP. Regenerable callus were formed from the basal region of in vitro cultured cormel slices which took approximately three weeks for callus induction after explants were placed on callus induction medium. For shoot induction one gram (Fresh Weight) of callus were placed on shoot induction media containing MS basal salt and 0.20 mg/l BAP. Shoot induction takes place after one month of callus inoculation on shoot induction media. Introduction The exuberance of colorful spikes of gladiolus is a delight to any floret bouquet. It is an all time favorite for the flower lovers in flower shows and cultivate worldwide as commercial horticultural crop. The species and varieties of it are numerous since the genus of gladiolus includes 180 species with more than 10,000 cultivars (1). Every year the number of varieties is rising through hybridization, which can prolong the vase life in introducing new colors in spikes, in floret arrangement on the spikes and to extend the flowering period (2). Due to large commercial worldwide trade and vegetative propagation by corms and cormels are affected by a large number of viruses. Gladiolus highly susceptible crop that may suffer considerable losses if control measures are not taken, the infection by soilborne fungus, Fusarium oxysporumf. sp. gladioli causing severe loss and damage are a major bottleneck to its mass propagation The main viruses that effect gladiolus and cause a severe financial loss every year for the farmers are bean yellow mosaic virus (BYMV) (3-7), cucumber mosaic virus (CMV) (8-16) , tomato ringspot nepovirus1, tomatoblack ring nepovirus, tomato spotted wilt tospovirus, tobacco mosaic tobamovirus and tobacco ringspot nepo virus, arabis mosaic nepo virus and strawberry latent ringspot nepo virus, tobacco streak ilarvirus (17-22) in different parts of the world. Biotechnogly offers a potentials for significant advances for improvement of ornamental species. Novel techniques such as genetic engineering are contributing increasingly to the improvement of horticultural crops especially with the introduction of new desirable traits. Target traits include flower texture, pigment, insect resistance, virus resistance and prolong flower vase life. But for these an efficient regeneration protocol for Gladiolus is essential. Here we tried different combination of plant hormones with standard MS media to figure out a media which can provide maximum regeneration from the explant. Materials and Methods Corms of gladiolus grandiflora var snowprincess were collected from Division of Horticulture, I.A.R.I, New Delhi. For cormel formation corms were inoculated in autoclaved sand with essential moisture, after 4-5 weeks small cormel comes out from corms. These cormels were selected for further culturing. Before inoculation in media the cormels were surface sterilized with continuous stream of running tap water for 30 minutes, washed with tween 20 for 15 minutes and then with 0.1% antiseptic savlon 5% (v/ v) for 10 minutes, these corms are now subjected to 0.1% HgCl2 treatment for 10 minutes in a laminar air flow cabinet and washed for three times with autoclaved double distilled water to remove any traces of HgCl2 followed by 70% ethanol treatment for 10 minutes then washed with double distilled water for three times. The cormels blotted dry on autoclaved sterile filter paper for 5-10 minutes. MS containing standard salts and vitamins, 3% sucrose and 0.7% (w/ 196 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India v) agar was used with different combination of auxins and cytokinin. The pH of the medium was adjusted 5.8 before adding agar. The medium was autoclaved at 1.1 Kg/cm2 (15 psi) for 20 minutes at 121oC. The small cormel were sliced approximately 1-2 cm with sharp sterilized scalpel and placed on media (20 slices on each plate of media), cultures were incubated at 10,000 lux with cool fluorescent light and temperature 0 at 24±2 C. For shoot induction one gram (approximately) of callus were placed on shoot induction media containing standard MS media with 0.2 mg/l BAP. Shoot induction takes place after one month of callus inoculation on shoot induction media. Results Number of cormel's slices undergoes callus formation Plant regeneration occurred from callus of gladiolus derived from either inflorescence stalks (23), cormel stem tips (24) or suspension cells (25). Plants Regenerable callus were formed from basal have been regenerated from callus of gladiolus by many region of in vitro cultured cormel slices approximately researchers (23, 24, 26, 27, 28, 29, 30, 31, 32, 33). 3 weeks after the explant placed on callus induction During this experiment total 8 types of media medium. Different (8) callus induction medium had tried containing MS Basal salts, 3% (w/v) sucrose, 0.7% were utilized containing auxin and cytokinin in different agar and the following in mg/l glycine 2.0, thiamine proportion. All the media gave significant result but 1.0, pyridoxine 0.5, nicotinic acid 0.5, mesoinositol the most significant result was given by media number 100.0 and two plant hormones NAA and BAP, 3 which contain NAA 2mg/l and BAP 1 mg/l here 17 out of 20 inoculated cormel’s slices undergoes callus individually and in combination as shown in table 1. formation and gave maximum shoot regeneration after Table 1. shows different combination of plant transferring to the shoot regeneration media. Media hormones with standard MS medium. number 4 and 8 gave intermediate result , in media -----------------------------------------------------------------------------------number 4 NAA (0.5 mg/l) act as callus promoting plant Media number NAA (mg/l) BAP (mg/l) ------------------------------------------------------------------------------------ hormone but in media number 8 BAP (0.2 mg/l) promoted callusing clearly indicated that both hormones 1 4 2 in combination could give best result. Media number 2 2 1 3 containing both plant hormones clearly proved this 3 0.5 hypothesis. 4 0.1 5 0.1 0.2 Our observation also gave the conclusion that 6 0.5 auxin and cytokinin at higher concentration for example 7 0.2 medium number 2 ( NAA 4 mg/l and BAP 2mg/l ) and 8 ------------------------------------------------------------------------------------ lower concentration medium number 8 ( 0.2 mg/l BAP) and medium number 1 (MS Basal) gave almost same Regeneration efficiency of different media rate of callusing. Recent investigation (1) on plant 20 regeneration in gladiolus through cormel slices also 18 16 explained the role of NAA and BAP in callus formation. 14 Medium containing less concentration of NAA with 12 10 or without BAP gave least regeneration efficiency. 8 6 4 2 0 Conclusion Media Media Media Media Media Media Media Media 1 2 3 4 5 6 7 8 Media Fig. 1. Shows regeneration efficiency of different media The developed protocol highlights the usefulness of Auxin and Cytokinin for enhancing the callus formation that can be exploited for mass propagation of virus free gladiolus cultivar which can be used for introducing desirable character into the 197 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India plant through genetic engineering and transformation Lee TC, Francki RIB, Hatta T, Plant Dis Rep, 63 (1979) 345-8. approach. Park I S, Kim K W, Jung K H & Chang M, Korean References J Plant Pathol, 14 (1998) 83–91. Bajaj YPS, Sidhu MMS & Gill APS, Biotechnology in agriculture and forestry, 20 (1992) 135-143. Raj SK, Saxena S, Hallan V, & Singh B P, Biochem Mol Biol Int, 4 (1998) 89–95. Bajaj YPS, Sidhu MMS & Gill APS, Sci.Hortic 18 Remotti PC & Loffler HJM, Plant Cell Tissue Organ (1982) 269-275. Cult, 42 (1995) 171-178. Bellardi M G & Vicchi V , Sementi Elette, 41 (1995) Remotti PC, Plant Sci, 107 (1995) 205-214. 19–21. Bellardi MG, Canova A & Gelli C, Phytopathol Mediterr 25 (1986) 85-91. Rosner A, Stein A & Levy S , ibid, 121 (1992) 269– 276. Bellardi MG, Vicchi V & Gelli C, Phytopathol Mediterr 26 (1987) 73-80. Rosner A, Stein A, Levy S & Liliau-kipnis H, J. Virol. Methods, 47 (1994) 227–235. Bing A & Johnson G V, Cornell Ornamental Research Simonsen J & Hildebrandt AC, Can J Bot 49 (1971) 473-492. Lab, Farmingdale NY, USA, (1973) 286–291. Bozarth RF,Corbett MK, Plant Dis Rep, 42 (1957) 217-21. Sinha P & Roy SK, Plant Tiss. Cult, 12 (2002) 139145. Francki R I B, Randles J W & Chambers T C, Abstr. Phytopathol, 54 (1964) 892–893. Srivastava K M, Raizada R K & Singh B P, Indian J. Plant Pathol, 1 (1983) 83–88. Fukumoto F, Yto Y & Tochihara H, Ann Phytopathol Stefaniak B, Plant Cell Rep, 13 (1994) 386-389. Soc Jap, 48 (1982) 68-71. Stein A, Levy S & Loebenstein G, Acta Hortic, 234 (1988) 371–378. Hort Kaminska M, Zesz. Probl. Postepow Nauk Roln, 182 (1976) 157–164. Stein A, Loebenstein G & Koenig R, Plant Dis. Rep, 63 (1979) 185–188. Kaminska M, Zeszyty Problemowe Postepow Nauk Rolniczych 214 (1978) 109-17 Takamatsu S, Tsuchiya T & Makara K, The Bulletin of the Faculty of Bioresources, Mie University, Kamo K, Chen J & Lawson R, In Vitro Cell Dev Tsu, Japan, 13 (1994) 1–6. Biol 26 (1990) 425-430. Kamo K, In Vitro Cell Dev Biol, 30 (1994) 26-31. Kamo K, In Vitro Cell Dev Biol, 31 (1995) 113115. Kim J S, Choi G S & Lee K H, Research Report of Rural Development Administration, Crop Protection 34 (1992) 18–27. Vunsh R, Rosner A & Stein A, Ann. Appl. Biol, 117 (1990) 561–569. Ziv M, Halevy AH & Shilo R, Ann Bot 34 (1970) 671-676. Kim KW, Choi JB & Kwon KY, J.Korean Soc. Hortic.Sci, 29 (1988) 312-318. Kumar A, Sood A, Palni LMS & Gupta AK, Plant Cell Tiss. Org. Cult, 57 (1999) 105-112. 198 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India TOMATO TRANSFORMATION AND REGENERATION PROTOCOL Divya Choudhary, Gaurav Agarwal and Ajay Arora Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110012 1. Seed germination of tomato Wash the tomato seeds with running tap water three times  Add tween-20 or liquid detergent and shake vigorously for 10 minutes. Wash seed three times with double distilled water  For surface sterilization of seeds treat the seeds with 0.1% HgCl2 for 5 minutes in laminar air flow hood and wash the seeds with autoclaved double distilled water three times to remove traces of HgCl2  Add 70% ethanol and shake the seeds for 5 minutes and wash three times with autoclaved double distilled water  Inoculate these seeds in half strength MS media for seed germination (with the help of flame sterilized forceps)  Keep these seeds for 15-20 days in tissue culture room  After 15-20 days cotyledonary leaves comes out (three leaves)  (2) Precallusing Cut cotyledonary leaves into small pieces and place on regeneration Media (MS + 2 mg/l BAP + 0.1 mg/l IAA)  (3) Tomato transformation After precallusing for 2 days on regeneration media these explants can be used for transformation (a) Co-cultivation Take single positive colony of Agrobacterium (containing the gene to be transformed) and inoculate in 25 ml of LB media containing appropriate antibiotics grow the culture at 200 rpm for 24 hours at 280C  Transfer the grow culture in 30 ml centrifuge tube and centrifuge it at 6000 rpm at 40C for 10-15 min  Dissolve the pellet in half strength MS liquid media (0.5-1 ml)  Take 25 ml of half strength MS in fresh autoclaved Petri plate and transfer the explants into the Petri plate  Now add 80-100 µl of grown Agrobacterium culture  Shake it gently (leave it for 12-15 min)  Drain the medium and blot dry the explants  (b) Regeneration Transfer the explants into the regeneration medium (MS + 2 mg/l BAP + 0.1 mg/l IAA)  Cover the plates with aluminum foil or brown bag and place in tissue culture room for 2 days  After 2 days transfer the explants in selection media (MS + 2 mg/l BAP + 0.1 mg/l IAA + appropriate antibiotics)  Within 7-8 days callusing starts  After 25-30 days transfer in shooting media (MS + 2 mg/l BAP + antibiotics)  After shooting transfer the explants into elongation media or rooting media (MS + 0.1 mg/l IAA + antibiotics) for 20-25 days  (c) Hardening After rooting transfer the plants in half strength MS liquid media for 2 days  Transfer the plants in autoclaved distilled water for 1-2 days  Transfer the plants in autoclaved artificial soil pots  Covered the plants with plastic bags (to maintain humidity)  After 5-7 days transfer the plants in green house and remove the plastic bags 199 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ESTIMATION OF ETHYLENE BY GAS CHROMATOGRAPHY Sangeeta Khetarpal and Madan Pal Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110012 Plants use ethylene (C2H4, also called ethene) as a hormone. It is a very small, simple molecule that exists as a gas at biological temperatures. Different kinds of plants use ethylene differently; which includes the promotion of fruit development and ripening, release of buds from dormancy in springtime, stimulation of leaf and fruit abscission (dropping), causing some plants to become female, stimulation of leaf senescence, induction of flowering, etc. Ethylene can cause significant economic losses for florists, markets, suppliers, and growers. Researchers have come up with several ways to inhibit ethylene, including inhibiting ethylene synthesis and inhibiting ethylene perception. Inhibiting ethylene synthesis is less effective for reducing post-harvest losses since ethylene from other sources can still have an effect. Ethylene production is regulated by a variety of developmental and environmental factors. During the life of the plant, ethylene production is induced during certain stages of growth such as germination, ripening of fruits, abscission of leaves, and senescence of flowers. Ethylene production can also be induced by a variety of external aspects such as mechanical wounding, environmental stresses, and certain chemicals including auxin and other regulators. Since the ethylene production occurs in small quantities as gaseous molecule in plant tissues, a very precise technique for its estimation is needed. Gas chromatography technique has been very successful to estimate the production of ethylene and widely used by researchers as well as in industries. This chapter explains the methodology of ethylene detection in plant tissues using gas chromatography. Principle Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction (mobile phase). Those substances distributed preferentially in the moving phase pass through the chromatographic system faster than those that are distributed preferentially in the stationary phase. As a consequence the substances are eluted from the column in inverse order of their distribution coefficients with respect to the stationary phase. Ethylene estimation by gas chromatography is based on the method provided by Wilson (1986). Requirements Assay vials/culture tubes with serum stoppers, compressed standard ethylene gas cylinder, gas tight syringes (1, 2 and 5 ml), incubator and gas chromatograph. Gas chromatograph conditions Column : Porapak N 80/100 mesh column Detector : Flame ionization detector (FID) Carrier gas : Nitrogen with a flow rate of 20 ml/min. Convenient rates of hydrogen and air are also used in this detector as a fuel. Column/oven temp. : 60 0C Detector temp. : 110 0C Injector temp. : 110 0C Generally the injector and detector temperatures are kept 50 0 C above column temperature. Procedure The culture tubes/assay vials containing plant samples are sealed with rubber serum stoppers and incubated for 24h before taking observations. For each assay, 2 ml of gas sample is with drawn from each tube/assay vial and injected into gas chromatograph. 1 ml standard ethylene is also injected in a similar manner. 200 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Fresh weight of the sample is measured and volume of air space above the sample is also measured after the measurement of the gas. Rate of ethylene production is calculated using the following formula and expressed as nmoles of ethylene evolved g-1 FW h-1 as per the formula given below: Ethylene (nmoles g-1 = fr.wt. h-1) nmoles of sample Air space Attenuation volume (ml) of sample 60 1 x x x x Gas sample Attenuation Incubation Fresh injected (ml) of standard time (min.) wt. (g.) Whereas, Concentration of standard in nmoles nmoles of sample = x area of sample Unit area of standard Calculation of ethylene concentration (C) in standard ethylene gas Standard ethylene gas is supplied as compressed gas in air light cylinders. The volume of ethylene in gas mixture is mentioned on the cylinder and can be 105 vpm or 1050 vpm (volume per million), that is, 106 ml (1 kl) of gas mixture will have 105 or 1050 ml of ethylene. To calculate ethylene concentration let us consider a standard ethylene cylinder with a volume of 105 vpm. The volume of 105 x 103/22,246 nmoles represents the concentration of standard ethylene in 1 ml of gas mixture. Precautions 1. Do not switch on the gas chromatograph without the carrier gas flow. 2. If proper resolution of the peaks is not there, wait for sometime and find out the reasons for it. May be the flame has extinguished or carrier gas, hydrogen or air has exhausted. Then inject the sample again. 3. In case of power failure, continue the flow of carrier gas and after resumption of power, allow the instrument to run for sometime to remove the sample already injected in the column. 4. Strictly avoid introduction of moisture in the column. 5. To activate the column, allow it to condition at 200-2100C for 4-5 h to get good resolution. 6. While switching off the instrument, first switch off injector, detector and oven but continue the carrier gas flow till the oven temperature reaches 400C. 10 6 m l o f gas m ixture co ntains = 105 m l o f ethylene gas 105 i.e. 1 m l o f gas m ixture co nta ins = m l o f ethylene gas 10 6 According to Avagadro ’s law 22,400 m l o f any gas at NT P = 1 mo le T herefore, 22,246 m l o f ethylene gas at N TP = 1 mo le 1 or 1 m l ethylene gas at N T P = mo le s 22,246 105 or 105 m l o f ethylene gas at N TP = 10 6 10 6 x 22,246 105 x 106 = mo les 6 10 6 x 22,246 µmoles (1 mole = 10 µmoles) REFERENCES 105 = µmoles 22,246 105 x 103 = nmoles (1 µmol = 10 3 nmoles) Wilson K (1986). Chromatographic Techniques in A biologist’s Guide to Principles and Techniques of Practical Biochemistry, K. Wilson and K.H. Goulding (eds.), 3rd edn., Arnold, London. 22,246 201 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India DEVELOPMENT OF PROTOCOL FOR REGENERATION OF MUNGBEAN (Vigna radiata (L.) Wilczek) Sangeeta Khetarpal and Madan Pal Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110012 Mungbean (Vigna radiata (L.) Wilczek) is an important grain-legume crop and a good source of dietary protein. Development of resistance to diseases (yellow mosaic virus and powdery mildew) and insect pests (white fly, jassids, pod borers etc.) are major problems in this crop. Besides, there is a need to improve grain yield, quality and content of grain protein. However, conventional breeding as a means of genetic improvement for yield increase is often limited by narrow genetic variability within species. Recent advances in biotechnology have offered an opportunity to develop new germplasm. The development of such technology largely depends on an efficient in vitro regeneration protocol which should also be suitable for genetic transformation. However, genetic improvement of this crop through biotechnological methods has not yet been achieved mainly due to its recalcitrance in culture. of B5 medium was used, while for shoot bud induction, full strength of B5 media supplemented with BAP (Table 1) was used. More details of the media used for various plant components and other requirements of regeneration are mentioned in Table 1 & 2. For establishing the regenerated plantlets one fourth concentration of Hoagland’s solution (Table 3) was used. Growth and development of regenerated plantlets are shown in plates A-D. Table 1. Detail of mungbean plant and media used for regeneration Variety Explant Germination medium Shoot bud induction medium Shoot bud development Pusa 105 Cotyledonary node ¼ B5 medium supplemented with 10-5 M BAP B5 basal medium supplemented with 10 µM BAP (kept for 15 days) Two subcultures at the interval of 15 days (Total duration 30 days) MS medium supplemented with 1 µM IAA ¼ Hoagland solution, pH 5.5 Pots containing sterile vermiculite At present, in mungbean, a few protocols of regeneration are available i.e., through shoot meristem Rooting medium (Mathews, 1987; Gulati and Jaiwal, 1992), cotyledon (Chandra and Pal, 1995; Gulati and Jaiwal, 1990) and Plantlet establishment cotyledonary node explants (Gulati and Jaiwal, 1994). Transfer of plantlets A major limitation of these protocols is their genotype specificity and low efficiency of regeneration. Often Table 2. Formulations of B and MS media 5 the regeneration protocol is influenced by variables (constituents in mg l-1) such as genotype, explant type, physical environmental Constituents B5 MS medium composition. A. Inorganic salts i) Macro nutrients Understanding the various physiological and NH4NO3 1650.00 biochemical factors affecting regeneration has been (NH4)2SO4 134.00 KNO3 2500.00 1900.00 useful in developing a regeneration protocol adaptable CaCl2.2H2O 150.00 440.00 to wide range of genotypes. Keeping in view, the MgSO4.7H2O 250.00 370.00 present protocol was developed in our laboratory using KH2PO4 170.00 cotyledonary node explant of mungbean variety Pusa NaH2PO4.H2O 150.00 105. For germination of seeds, one fourth concentration 202 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India B. C. ii) Micro nutrients MnSO4.H2O ZnSO4.7H2O H3BO3 KI Na2MoO4.2H2O CuSO4.5H2O CoCl2.6H2O iii) Iron source FeSO4.7H2O Na2EDTA.2H 2O Organic compounds Glycine Nicotinic acid Pyridoxine HCl Thiamine HCl Meso-inositol Carbohydrate source Sucrose 10.00 2.00 3.00 0.75 0.25 0.025 0.025 16.90 8.60 6.20 0.83 0.25 0.025 0.025 27.80 37.30 27.80 37.30 1.00 1.00 10.00 100.00 2.00 0.50 0.50 0.10 100.00 20,000 30,000 A B C D Table 3. Composition of Hoagland and Arnon’s solution Salt KNO3 Ca(NO3)2.4H2O NaH2PO4.2H2O MgSO4.7H2O Fe-chelate MnCl2.4H2O H3BO3 ZnSO4.7H2O CuSO4.5H2O H2MoO4.H2O mg l-1 606.60 944.80 115.00 493.00 10.00 1.78 2.84 0.23 0.08 0.02 References Chandra, M. and Pal, A. (1995). Differential response of the two cotyledons of Vigna radiata in vitro. Plant Cell Rep., 15: 248-253. Gulati, A. and Jaiwal, P.K. (1990). Culture conditions effecting plant regeneration from cotyledon of Vigna radiata (L.) Wilczek. Plant Cell Tissue Org. Cult., 23: 1-7. Gulati, A. and Jaiwal, P.K. (1992). In vitro induction of multiple shoots and plant regeneration from Plates (A-D): Regeneration from cotyledonary node shoot tips of mungbean (Vigna radiata (L.) explants Wilczek). Plant Cell Tissue Org. Cult., 29: 199205. A. Shoot bud induction in 4d old cotyledonary Gulati, A. and Jaiwal, P.K.(1994). Plant regeneration node at 15 days after inoculation from cotyledonary node explants of mungbean B. In vitro root initiation in regenerated shoots (Vigna radiata (L.) Wilczek). Plant Cell Rep., C. Plantlet establishment in Hoagland solution 13: 523-527. D. Established plantlet from pot containing Mathews, H. (1987). Morphogenetic responses from vermiculite in vitro cultured seedling explants in mungbean (Vigna radiata (L.) Wilczek). Plant Cell Tissue Org. Cult., 11: 233-240. 203 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India A PROTOCOL ON POLAROGRAPHIC MEASUREMENT OF PARTIAL LIGHT REACTIONS BY OXYGEN ELECTRODE BY OXYGEN ELECTRODE USING N.K. Prasad Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110 012 Oxygen Electrode is the most widely used instrument for the assay of photosynthetic electron transport via PSI and PSII. M/s Hansatech, UK provides with two models of oxygen electrode viz. DW2 (model, used for the measurement of photosynthesis and respiration at the organelle level i.e. using isolated chloroplasts and mitochondria respectively) and LD2 (model, used for the measurement of the same processes but in the detached leaves). Four electrons are generated at the anode which is used to reduce a molecule of O2 at the cathode. The O2 tension at the cathode then drops to zero and this acts as a sink. O2 diffuses towards it to make up the deficit. 4 Ag 4 Ag+ + 4e– 4 Ag+ 4Cl– 4 Ag Cl The DW2 model is being described here along with the working principle and method of measurement of photosynthetic partial light reactions. Construction of Oxygen Electrode and its Principle DW2 model is a “Clark Type” of oxygen electrode. It consists of a platinum cathode in conjunction with a silver anode; both immersed in a semi-saturated solution of KCl and separated from the test solution by a membrane. The membrane is generally made of “Teflon”, measures 12 µm in thickness and is oxygen permeable. Oxygen diffuses from the test solution in the reaction vessel through the membrane into electrode compartment. The reduction of oxygen at the cathode gives rise to a current. The current flows though the Fig. 1. The Hansatech DW2 Oxygen Electrode Unit circuit which is completed by the KCl bridge. This current is proportional to oxygen-tension in the solution Calibration of Oxygen Electrode using Sodium provided a voltage of 0.5 to 0.8 V is applied across Dithionite the electrodes. The calibration technique is based on the fact Electrochemical reactions that occur at the that fully air-saturated water al 250C temperature and cathode are: atmospheric pressure yields 240 nmoles of oxygen/ H2O2 O2 + 2e– + 2H+ ml, if stirred continuously at a constant speed. H2O2 + 2e– + 2H+ 2H2O 204 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Steps of Calibration Drop 1 ml of air-saturated distilled water into the reaction vessel  Screw the plunger upto the upper level of water. Then put a cap on the plunger. (There should be no air bubble under the plunger).  Switch the magnetic stirrer and the recorder ‘ON’  Note down the distance traveled by the recorder pen on the chart paper (That much distance denotes the full saturation value of O2 i.e. 240 n moles)  Stop the stirrer  Add a pinch of sodium dithionite (Na2 S2 O3) into the reaction vessel through the hole in the plunger  The recorded comes to zero  Clean the reaction vessel thoroughly (There should be no residue of Na2 S2 O3 while sampling) Measurement of Partial Light Reactions Preparation of grinding medium It consists of 20 mM Tricine-NaOH buffer (pH 8.5), 0.35 M sorbitol, 0.1 M potassium chloride, 5 mM magnesium chloride, 1 mM Na-EDTA, 5 mM sodium ascorbate, 1 mM sodium azide (freshly prepared) and 1% PVP-4000 in a total volume of 500 ml. Suspension Medium The suspension medium is the same as grinding medium except that it does not contain sodium ascorbic acid. Isolation of thylakoids Take freshly cut leaves  Wash thoroughly with water  Blot them dry  Cut fine segments (using a sharp razor blade)  Take 1 g of leaf pieces  Grind in a pre-chilled pestle with mortar (use 10 ml of “Grinding Medium”)  Filter through 6-8-layered muslin cloth  Centrifuge the extract at 4000x g for 4 minutes at 04 0C  Discard the supernatant  Suspend the pellets in 10 ml of NaCl solution (10 mM)  Centrifuge at 5000x g for 5 minutes at 0-40C  Discard the supernatant  Wash the pellets (containing thylakoids) twice with the “Suspension Medium”  Re-suspend it into “Suspension Medium” (1-0 ml volume) Estimation of Chlorophyll in the Thylakoids Chlorophyll content in the thylakoids may be estimated following Arnon’s (1949) method. 0.1 ml of thylakoid suspension in dissolved in 10 ml of 80% acetone and after grinding in a chilled pestle-motar for about thirty seconds, it is filtered through a Whatman40 filter paper. Optical density of chlorophyll solution is recorded using a spectrophotometer at 645, 663 and 710 nm wavelengths. Chlorophyll content (µg/ml) is calculated using the formula given below (Mohanty and Boyer, 1976). Total chlorophyll (a+b) = (OD645 – OD710) 20.2 + (OD663 – OD710) 8.02 205 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Volume of thylakoid suspension equivalent to Photosystem-I (PSI) 20-50 µg of chlorophyll is used each time for the Electron transport through PSI is measured measurement of light reactions. using electron donor, reduced DCPIP in the presence Chloroplast Assay of methyl viologen. Assay medium (2.5 ml) contains 5 mM phosphate buffer (pH 6.8), 2 mM NH4Cl, 0.5 Photosystem-II (PS-II) mM sodium azide (prepared daily), 5 mM sodium The assay medium consists of 5 mM MgSO4, ascorbate, 15 µM DCPIP, 0.1 mM DCMU, 2 mM 2 mM NH4Cl, 0.5 mM sodium azide (freshly MV and 20 µg chlorophyll equivalent thylakoid prepared), 15 µM DCPIP and 20 µg chlorophyll suspension. While illuminating the reaction mixture, O2 equivalent thylakoid suspension in a final volume of uptake takes place by the solution, so that the recorder 2.5 ml. The assay mixture is irradiated with saturating registers a fall unlike PS-II. The negative deflection light (1000 µE m-2 s-1) for two minutes. The recorder indicates PSI activity. goes uphill showing oxygen evolution. Whole Chain Electron Transport The rate of electron absorption by DCPIP is For the estimation of whole chain electron equivalent to the PSII function. transport (excluding ferrodoxin), the assay medium consists of 5 mM phosphate buffer (pH 6.8), 100 mM + – 4H + 4e + O2 2H2 KCl, 1 mM MgSO4, 5 mM NH4Cl, 2 mM sodium azide (prepared fish), 50 µM methyl viologen and 20 2DCPIP + 4H+ + 4e– 2 DCPIPH2 µg chlorophyll equivalent thylakoid suspension in a final volume of 2.5 ml. The stoichiometry is-four electrons 2H2 + 2DCPIP 2 DCPIPH2 + O2 transported per O2 molecule consumed. Referencesli Aron, D.I. (1949). Copper enzymes in isolated chloroplasts: Polyphenoloxidases in Beta vulgaris. Plant Physiol., 24: 1-15. Coombs, J., Hall, D.O., Long, S.P. and Scurlock, J.M.O. (1987). Techniques in Bioproductivity and Photosynthesis. Pergamon Press Ltd. Pp. 62-93. D.W L.W. Oxygen Electrodes : Operator’s Manual (1992). M/s Hansatech Ltd., Hardwick Industrial Estate, King’s Lynn, Norfolk, UK. Henley, W.J., Levavasseur, G., Franklin, L.A., Osmond, C.B. and Ramus, J. (1991). Photoacclimation and photoinhibition in Ulva rotundata as influenced by nitrogen availability, Planta, 184: 235-243. Mohanty, P. and Boyer, J.S. (1976). Chlorophyll response to low leaf water potentials. IV : Fig. 2. Schematic ‘Z’ scheme of electron transport Quantum yield is reduced. Plant Physiol., 22: pinponing sites of action of inhibitors 395-430. (artificial electron acceptors) 206 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India CHROMATOGRAPHY D.V. Singh Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110012 water, Acetic acid, petroleum ether, butanol, chloroform, benzene, phenol, ammonia, hydrate of organic and inorganic solvents etc. More than 2 solvent can be used at a time in different proportion as require for separations. For example for GA isolation from Earlier a Russian Scientist, Mechaet Iswett, plant extraction are used Isopropanol: Amonium coined this technique in 1906, while he was trying hydroxides: water in ratio of 8:1:1. Basically all chromatographic system consist separation of pigments using ETHER as solvent. of two, phases. Chromoato = colour and graphy = The technique for separation, purification, identification of substances in a mixture, in which separatoin is brought about by different movement of individual substance through a process medium under the influence of moving solvents. diagrammatic presentation thus the name of this 1. Stationary phase: Medium on which various component are adsorbed, may be solid, gel, liquid technique was initiated as chromatography because or solid/liquid mixture and remains immobilized earlier the technique was used to separate colour or stable. components (Pigments of chlorophyll etc). Now-aday this technique is used to separate, identify and purity 2. Mobile phase: Carries the mixture of substances the coloured substances as well colour less substances over stationary phase, which may be liquid or also. The advantage of this technique is to separate gaseous, which flow over/through the stationary and identify the substances in a very small quality upto phase. ng and picogram. Modes of chromatography By this technique separation of pigments, There are three modes of chromatography on sugars,, proteins amino acids, organic acids, phenolic acids drugs etc. can be separated quantitatively. The the basis of stationary and mobile phases: resolution/separation of substances from a mixture is 1. Column chromatography: The stationary phase is packed into glass or metal column. due to different adsorption capacities of substances. 2. Thin layer chromatography: The stationary Principle of chromatography phase thinly coated on glass, plastic or foils plates. The principle behind the chromatography is that the mixture of substances (under similar conditions), when passing through a material of different phase are adsorbed in different rates. 3. Paper chromatography: The cellulose fibbers of a paper sheet support the stationary phase. All above three modes of chromatography has its own specific advantages and can be use according to our decided work. The medium, which carries the mixture of substances over another phase is called as moving phase or mobile phase and medium on which various 1. Column chromatography component are adsorbed are called as stationary Most of the chromatography is carried out phase. using the column mode such as: Some important solvents/resolving fluids are 1. Adsorption chromatography: Adsorption 207 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India equilibrium between a stationary solid phase and 3. Paper chromatography mobile liquid phase. The separation of substances of a mixture on a sheet of filter paper (generally Whatman No.1) is the typical example of paper chromatography. The filter paper has numerous cellulose fibers and each fiber together with their associated fibres constitute minute 3. Gas liquid chromatography: Partition between cells which accommodate a certain percentage of a stationary liquid and a mobile gaseous phase. moisture. The moisture present in these cells acts as stationary phase. 4. Ion exchange chromatography: Ion exchange It is the partition of the substances between equilibrium between an ion exchange resin the moisture among the fibers (stationary phase) and stationary phase and a mobile electrolyte phase. the solvent (mobile phase) following over the cells, 5. Exclusion chromatography: Equilibrium which actually brings about the separation. between a liquid phase inside and outside a The cellulose of chromatography paper acts porous structure or molecular sieve. as supporting matrix for the stationary phase. The paper 6. Affinity chromatography: Equilibrium between may be of different running characteristics eg. Slow a macromolecule and a micro molecule for which medium or fast medium and others may be acid washed it has a high biological specificity hence called as to remove the traces of impurities. The paper for affinity chromatography. reverse phase chromatography must be prepared 2. Thin layer chromatography (TLC) immediately before use. A uniform layer is prepared of any suitable RF value: adsorbent like silica gel, CaSO4 etc for a stationary The identification of given compound may be phase. This technique is simple) quick and allows a made on the basis of the retention factor (R ) value, F large number of samples to be studied concurrently. It which the distance moved by solutes relative to the can be used for analytical and preparative. purposes. distance moved by the solvent front. For analytical separation the layer is 0.25 mm thick is Distance move by the solutes from origin prepared and for preparative purposes it can be of RF = upto 5 mm thick. Distance move by the solvent from origin Although the movement of compound on TLC This value is a constant for a particular may be characterized by RF value (as in paper compound under standard conditions and closely reflect chromatography) but such values are not so reliable the distribution coefficient for that compound. as those in paper chromatography. HPLC The amount of compounds present in a given Originally, HPLC was referred to as high pressure spot may be determined in a number of ways. liquid chromatography but now-a-days it is preferred Quantification may be achieved by use of radio as high performance liquid chromatography since it is chromatogram scanning or by densitometry. Precision better description of characteristics of HPLC. densitometry is commercially available, which measure the ultraviolet or visible absorption of the compounds. Components of HPLC system The amount of compound in a solution can be then 1. Solvent reservoir determined by standard methods. It consist the solvent, which may be one or more as required in our experiments. 2. Partition chromatography: Partition equilibrium between stationary liquid (semi liquid) and mobile liquid phase. It is also known counter current chromatography. 208 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Fig. 1. Diagrammatic representation of the HPLC components Solvents (Mobile phase) 2. Choice of mobile phase to be use depends on the type of separation to be achieved. Separation of the substances may be made with a single or two or more solvents mixed in a fixed proportion. Alternatively a gradient elution system may be used, where the composition of solvent is continuously changed by using a suitable gradient programmer. Degassing may be carried out in several ways: All solvents must be specially purified as HPLC grade otherwise a trace of impurities can affect the column and may interfere with detection system. Before introduce the solvents into HPLC the following points should keep in mind. 1. To avoid some impurities the solvents have to pass or filter separately by a filtration kit having 1-5 µm micro filter prior to introduce into the pump. It is also essential that all solvents are degassed before use otherwise any gas bubbles in the solvent can alter the column resolution and continuous monitoring of the column effluent. 1. By warming the solvent (at 400C) 2. By stirring it vigorously with magnetic stirrer. 3. Subjecting it to a vacuum. 4. By Ultra sonic vibration 5. By budding helium gas through the solvent. 2. Pumping system The pumping system one of the most important features of HPLC system. The following specification must be in pumping system. 1. 209 There should be a higher resistance to solvent Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India flow due to the narrow column packed with small 1. particle. Gently pouring slurry of stationery phase (adsorbent resin or gel) in column. 2. It should capable of constant and reproducible 2. mobile phase flow over a range of about 0.1 to 10.0 ml/min. Gently tapped to ensure that no air bubbles are for append and the packing settled evenly. 3. Can operate at pressure of 4000 Psi or greater. 4. Capable with wide range of solvents. Some important examples of HPLC packing materials / stationary phases and their applications The 2nd point is most essential because Retention Time (RT) is directly related to the flow rate. (Normally flow rate are 2 ml/min. and upto 2000-3000 Psi pump presume. Material for stationary phase Silica Alumina Commercial Name Corsil Pellicular Pellumina Silica Alumina Partisil Microporous Micropack A1 Application Sterioids and drugs, vitamins, pesticides polar herbicides, plant pigments, triglyeerids Alkaloids, glycosides, drugs, plant growth regulators etc. There are different types of pumps are available which operate on the principle of constant pressure or All the column packing materials can be constant displacement. The choice of solvent depends divided in to 3 types. upon the experiment’s aims 1. Microporus, where micropores ramify through the 3. Sample injection loop: particle (5-1 0 µm). A metal loop of small volume (5-10 micro 2. Pellicular (superficially porous) supports where litres) which can be filled with the sample before porous particles coated on to an inert solid core introduce into HPLC. To use this loop there is no need such as glass bead of about 40 µm in diameter. to stop the pumping system and no chances of error in 3. Bonded phase, where stationary phase is handling the samples. chemically bonded into an inert support. 4. Column: Stationary phase depends upon the form of The column used for HPLC are- generally chromatography. Most stationary phases are available made of stainless steel and are manufactured so that in a range of size and shape so selection of material is they can bear pressure upto 5.5 x 107 Pa or 8000 Psi. very important because they influence the flow rate Straight atmosphere pression column are generally 20 and resolution characteristics. The longer the particle, to 50 cm in length and 1 to 4 mm diameter. faster the flow rate. Porous plugs of stainless steel or Teflon are The particle size commonly expressed by mesh used in the ends of the column to retain the packing size which is a measure of the opening per inch in a material. The things must be homogenous to ensure sieve hence the longer the mash size, smaller the uniform flow of solvent through the column. It is also particle. 100- 120 mash is most commonly for routine important in some separation such as liquid partitioning use, while 200- 400 mash is used for high resolution and ion exchange that column temperature is work. thermostatically controlled during the analysis. 5. Detector system Guard columns are short (2-10 cm) of the same Since the quantity of material applied to the diameter packing with similar material are necessary column is frequently very small so sensitivity of the to prevent the main column. detector system should be sufficiently high and stable. Most commonly detectors are of variable wavelength, Packing of columns (Ultravoilet to visible) spectrophotometer, a fluorimeter, Most critical factors all successful separation a refractive index monitor and an electrochemical by any form of column/or packing a columns. detector. 210 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India 6. Filtration kits a. Sample filtration kit Sample filtration kit (Having nylon membrane filter 13 mm in diameter having 02-0.45 micron pores) b. Solvent filtration kit Solvent filtration kit (Having nylon membrane filter 47 mm diameter having 0.1-0.2 micron pores). Others Sample syringes (5-10 µl or required as per experiments requirement. Applications of HPLC: 1. Separation of polar compounds (using reverse phase column) such as drugs and their metabolites, vitamins, polyphenols and steroids 2. To purify the biological molecule. 3. Widely use in clinical and pharmaceutical work such as serum. Urine directly to column 4 Separation of highly polar compounds such as amino acids, organic acids act. 5. Separation glycopeptides and proteins. The separation of proteins has given rise to the technique of FPLC (fast protein liquid chromatography on reverse phase Extraction of samples Distilled water 2 ml with 1% acetic acid (v/v) was added in the round bottom flasks, which were then sonicated thoroughly. Therefore, the contents of the flasks were transferred to the sample vials. Finally flasks were rinsed twice with 2 ml distilled water having 1 % acetic acid (v/v) and their contents were transferred to respective vials. The volume of solution in the vial was finally made to 5 ml with distilled water containing 1% acetic acid (v/v). 1. Always use a micro syringe designed to withstand high pressure (For ABA extraction) 2. Sample should be injected through a septurn I an injection loop Operational instructions for HPLC Chart speed : 5-1 cm/min Flow rate : 1-2 ml/min Detector (wavelength) : 265 nm UV radiation Column : C18 reverse phase of 5 µ posticle size Calibration curve : Standard ABA solution in 95% ethanol (5-10 ppm) Loading of samples: The correct application of a sample into a HPLC column is most important factor in achieving the successful result so following instruction should be in mind. Each leaf sample/plant sample was transferred in 100 ml conical flask and the sufficient amount of 3. Using loop injector is always better acetone (acetone contained 1% acetic acid) was Calculation added to dip the material properly. Then the flask was Peak area of sample x amount of material in sample kept overnight at 40C. The extract was filtered in round Content of materials = bottom flask through Whatman No. 4 filter paper and Peak area of standard x fresh weight of the sample filter paper was washed thoroughly with acetone. Suggestive readings The residue was again dipped in acetone and A Biologist Guide to Principle and Techniques of kept again over night for second extraction. Next Practical Biochemistry. Edited by Keith Wilson morning, the extract was filtered in the round bottom and Kenneth H. Goulding flask in the same way. The residue was then crushed with the help of mortar and pastel and kept in acetone Organic Trace Analysis by Liquid chromatography by James F. Lawrence. for overnight again. Finally, the extract was filtered in the round bottom flask in the same way as described Zevaart, et al. (1980). Change in the level of abscisic earlier. The acetone from the extract was evaporated acid and its metabolites in exided leaf blades of with the help of rotary evaporator at 400C. The round Xanthium strumarium during and after water bottom flasks containing residue kept in dark and cool stress. Plant Physiol., 66: 672-678. place for the analysis of ABA. 211 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India MEMBRANE STABILITY INDEX – A SIMPLE TECHNIQUE FOR SCREENING AGAINST DROUGHT AND HIGH TEMPERATURE STRESS S.R. Kushwaha and R.K. Sairam Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi - 110012 Abiotic stresses induce profound changes in cellular membranes. When the stress level is high, it affects the membrane function and leakage of cellular ions and other molecules. The change in permeability of membranes or injury can be readily quantified by measuring cellular electrolyte leakage from affected leaf tissue into an aqueous medium. Moisture and high temperature stress results in loss in membrane permeability resulting in ion/solute leakage. This could possibly be due to a shift in the ratio of unsaturated to saturated fatty acids in the plasma membrane and/or could be due to stress induced reactive oxygen species (ROS) production and consequent lipid peroxidation. In the Division of Plant Physiology, MSI have been shown good relationship amongst stress (drought, high temperature and salinity) in chickpea and wheat under field conditions. Membrane stability index (MSI) has positively and significantly correlated with biomass and grain yield under late planting conditions in wheat. The relationship between MSI and biomass and seed yield was linear and higher yield was obtained in those genotypes which have higher stability index. In chickpea similar trend was observed under late sown rainfed conditions. At Karnal under salinity conditions twenty chickpea genotypes were screened for salinity tolerant (Deshmukh and Kushwaha (2002). On the basis of MSI grouping of genotypes could be possible which could be utilized by plan breeders in identification of suitable parents in breeding programme. Sairam et al (2005) also found close association between MSI under stress environment and also found close relation with Induced ROS production, lipid peroxidation and membrane stability index. Membrane stability index can be estimated by Deshmukh et al. (1991). Membrane stability index a very simple technique and can be used for screening large number of genotypes/breeding material for heat and drought resistance. Leaf membrane stability index (MSI) is determined by recording the electrical conductivity of leaf lichgates in double distilled water at 40 and 100o. Leaf samples (0.1g) were cut into disces pf uniform size and taken in test tubes containing 10 ml of double distilled water in two sets. One set was kept at 40o for 30 minutes and another set at 100oC in boiling water bath for 15 minutes and their respective electric conductivities C1 and C2 were measured by Conductivity meter. Membrane stability index = (1-(C1/C2))*100 References Deshmukh, P.S. Sairam, R.K. and Shukla, D.S. (1991). Measurement of Ion-leakage as a screening techniques for drought resistance in wheat genotypes. Indian J. Plant Physiol. 34(1): 89-91. Deshmukh, P.S. and Kushwaha, S.R. (2002). Variability in membrane injury index in chickpea genotypes. Indian J. Plant Physiol., 7(3): 285287. 212 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India MEASUREMENT OF CHLOROPHYLLS CONTENT IN PLANT TISSUES Tej Pal Singh Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110012 The chlorophylls are the pigments that give plants their characteristics green colour. They constitute about 4% of chloroplast dry mass. In plants (except in red algae and cyanobacteria), chlorophyll ‘b’ is present about one third of the content of chlorophyll ‘a’. Further, chlorophyll ‘a’ is a constituent of the photosynthetic reaction centres and thus we can consider it as the essential photosynthetic pigment. Light absorption pattern for chlorophyll ‘a’ and chlorophyll ‘b’ is shown in Fig. 1. I. Destructive method Principle Chlorophylls and carotenoids are extracted from leaf (or shoot) material and their concentrations determined spectrophotometrically. By use of simultaneous equations it is possible to determine the concentration of chlorophyll ‘a’, ‘b’ and total chlorophyll. Method of extraction A wide range of solvents were used for extraction of pigments such as; 80% acetone, acetonemethanol-water (80:15:5), dimethylsulphoxide (DMSO), DMSO-90% acetone, N, Ndimethylformamide (DMF), dichlormethane-methanol (9:1), cold methanol, chloroform and other organic solvents and other less used combinations. For most of the plant samples extraction with 80% acetone and DMSO appears to give acceptable results. Procedure Fig. 1. The absorption spectra of chlorophyll ‘a’ and chlorophyll ‘b’ Chlorophyll ‘a’ shows two major absorption bands: one in the red, with a maximum around 660 nm and other in the blue to violet region, with maximum around 435 nm. chlorophyll ‘b’ also shows two major absorption bands: one in the red-orange of the visible spectrum, with a maximum at 650 nm and the other one in the blue range. Chlorophyll ‘a’ is found in all photosynthetic eukaryotes and the other secondary chlorophyll (b, c, d or e) may have an evolutionary significance with a restricted presence in some groups of organism. Two most acceptable methods i.e. A: Acetone method (Arnon, 1949) and B: DMSO (Hiscox and Israelstam, 1979) are described here. A: Acetone method Materials required: Plant material (leaf/shoot), distilled water, 80% acetone, mortar and pestle, funnels, Whatman filter papers grade 1, volumetric flasks, measuring cylinders, pipettes, Sepectronic 20 etc. Procedure: Prepare 80% acetone. Weight 250 mg of fresh leaf material (avoid mid ribs). Ground the pieces of plant material in pestle and mortar using 5 ml of 80% acetone. Filter the homogenate in 25 ml of Both destructive and non-destructive method volumetric flask (25 ml) by using Whatman paper grade is being described here for actual as well as relative 1. Wash out the homogenate 3-4 times with 5 ml of estimation of photosynthetic pigments.. 80% acetone each time. Make the final volume of 213 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India V -1 Chlorophyll ‘a’ (mg g fw)= [(12.7 x A663) – (2.69 x A645)] x 1000 x W V Chlorophyll ‘b’ (mg g-1 fw)= [(22.9 x A645) – (4.68 x A663)] x 1000 x W V -1 Total chlorophyll ‘a’ + ‘b’ (mg g fw)= [(8.02 x A663) + (20.20 x A645)] x 1000 x W Where, A = Absorbance at given wavelength; V = Final volume of 80% acetone in ml and W = Weight of plant tissue in grams filterate to 25 ml. Record the absorbance of filterate at two different wavelengths (663 and 645 nm) using spectrophotometer by keeping 80% acetone as blank. Place the material in test tube and add 20 ml of DMSO in tube. Cover the tubes with aluminium foil (to avoid photooxidation of pigments) and keep in oven at 650C for 5 hours. Record the absorbance of chlorophylls containing solution at 663 and 645 nm. Calculate the chlorophyll ‘a’, ‘b’ and total by using the same formulas mentioned above as given by Arnon (1949). Use DMSO solution as blank. Express the results in terms of mg g-1 fw. The amount of chlorophyll ‘a’, ‘b’ and total are determined using the following formulas given by Arnon (1949) based on the work of Mac Kinney (1941) who provided the values of extraction Merits of DMSO method over acetone method coefficients. Precautions  It is highly recommended to extract as soon as possible after collection of samples.  For later use, extracts must be stored in deep freeze for up to five months with only minor charges. 1. No maceration or grinding of plant tissue is required. This makes this method simple and rapid. 2. Chances of loosing any chlorophyll due to filtration step are totally eliminated. 3. Chlorophyll extracted by DMSO remains stable for number of days and hence spectrometric analysis need not to be carried our immediately.  Because of light influence on the pigments, extraction should be carried out in subdued light II. Non-destructive method at low temperatures.  Ensure maximum extraction of chlorophyll pigments by minimum of 3-4 washings of homogenate with 80% acetone.  If not stored in deep freeze, the absorbance of the filtrate must be taken as early as possible after chlorophyll extraction as pigment in not stable for a long time. B: DMSO method Materials required: DMSO, test tubes, aluminium foil, oven etc. Procedure: Weight accurately 100 mg of finely chopped fresh leaf sample (avoid the mid rib portion). Chlorophyll meter or SPAD meter The chlorophyll meter is a simple, portable diagnostic tool that measures the greenness or relative chlorophyll content of leaves (Inada, 1963, 1985), Meter readings are given in Minolta Company defined SPAD (soil plant analysis development) values that indicate relative chlorophyll contents. There is a strong linear relationship between SPAD values and weight based leaf N concentration (Nw), but this relationship varied with crop growth stage and/or variety (Takebe and Yoneyama, 1989; Turner and lund, 1994), mostly because of leaf thickness or specific leaf weight (Peng et al., 1993). The confounding effect of leaf thickness 214 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India can be eliminated if foliar N concentration is expressed 4. on a leaf area basis. Leaf area based N concentration (Na) has a unique linear relationship with SPAD values 5. of rice plants at all growth stages (Peng et al., 1995). The linear relationship between Na and SP AD values has led to the adaptation of the SPAD meter to 6. assess crop N status and to determine the plant’s need for additional N fertilizer (Balasubramanian et al., 1999). SP AD readings indicate that plant N status and the amount of N to be applied are determined by 7. the physiological N requirement of crops at different 8. growth stages. Factors affecting SP AD values 9. 1. Radiation differences between seasons. 2. Plant density 3. Varietal groups 4. Nutrient status other than N in soil and plant 5. Biotic and abiotic stresses that include leaf discoloration. You must shield the leaf from direct sun light by having your back to the sun. Leaves should be at the 1.5 “Y leaf” stage. First fully expended leaf with the next emerging leaf approximately half way up its length. Take readings between the leaf margin and the mid rib (avoid the mid rib or only partially reading a leaf, i.e. edge, these gives false readings) Close until hear “BEEP”. To check a reading, take another reading on the sample leaf. Readings from the same leaf should be very similar. Push “A VERAGE” to automatically calculate the average for readings taken. 10. Clear data by pressing “ALL CLEAR DATA”. Note: You can remove leaves from the field for reading, but they need to be wrapped in a paper towel and put in an ice chest to avoid wilting. Advantage of using SPAD meter over conventional methods of using SP AD meter over Chlorophyll has distinct optical absorbency conventional methods of chlorophyll estimation Laboratory methods for determination of characteristics. Strong absorbency bands are presents in the blue and red but not in the green or infrared chlorophyll content are both time consuming and bands, hence the green appearance of a leaf. By destructive to the sample. Typically, a sample must be measuring the amount of energy absorbed in the red detached, ground up in a solvent and then assayed in band, an estimate of the amount of chlorophyll present a spectrophotometer. A sample can be measured only in the tissue is possible. Measurements in the infrared once, precluding the monitoring of trends in chlorophyll band show absorbencies due to cellular structure content over the growing cycle. The SP AD meter materials. By using this infrared band to quantify bulk provides non-destructive measurements of relative leaf absorbency, factors such as leaf thickness can be chlorophyll content without the need to detach the leaf. taken into account in the chlorophyll content index References value. Aron, D.I. (1949). Copper enzymes in isolated Calibration: There is a disk provided to check the chloroplasts. Polyphenol oxidases in Beta accuracy of the meter. vulgaris. Plant Physiol., 24: 1-14. Taking measurements Balasubramanian, V., Morales, A.C., Cruz, R.T. and Principle 1. Switch “ON” 2. Close for calibration until hear “BEEP”. 3. Leaves should not have free water. Abdulrachman, S.(1999). On farm adaptation of knowledge-intensive nitrogenmanagement technologies for rice systems. Nutr. Cycl. Agroeco., 53: 93-101. 215 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Hiscox, J.D. and Israelstam, G.F. (1979). A method for extraction of chlorophyll from leaf tissue without maceration. Can. J. Bot., 57: 13321334. Peng, S., Garcia, F.C., Laza, R.C. and Cassman, K.G. (1993). Adjustment for specific leaf weight improves chlorophyll meter’s estimation of rice leaf nitrogen concentration. Agron.J., 85: 987990. Inada, K. (1963). Studies on a method for determining deepness of green colour and chlorophyll content Peng, S., Laza, R.C., Garcia, F.C. and Cassman, K.G. of intact crop leaves and its practical application. (1995). Chlorophyll meter estimates leaf area 1. Principle for estimating the deepness of green based N concentration of rice. Commun. Soil colour and chlorophyll content of whole leaves. Sci. Plant Anal., 26: 927-935. Proc. Crop Sci. Soc. Jpn., 32: 157-162. Takebe, M. and Yoneyama, T. (1989). Measurement Inada, K. (1985). Spectral ratio of reflectance for estimating chlorophyll content of leaf. Jpn. J. Crop Sci., 54: 157-162. of leaf colour scores and its implication to nitrogen nutrition of rice plants. Jpn. Agric. Res. Q., 23: 86-93. Mac Kinney, G. (1941). Absorption of light by chlorophyll solutions. J. Biol. Chem., 144: 315323. Turner, F.T. and Jund, M.F. (1994). Assessing the nitrogen requirements to rice crops with a chlorophyll meter method. Aust. J. Exp.Agric., 34: 1001-1005. 216 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India 1-METHYLCYCLOPROPENE: TECHNICAL BULLETIN FOR USE ON HORTICULTURAL PRODUCTS Vijay Paul* and Rakesh Pandey Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110 012 *: E-mail: vijay_paul_iari@yahoo.com Ethylene regulates a number of developmental processes and stress responses, including leaf abscission, fruit ripening, organ senescence, seed germination, seedling growth, development of physiological disorders, sprouting, and pathogen responses (Abeles et al. 1992, Saltveit 1999). While the molecular details of the many diverse ethylene responses are still not well understood, it is now known that the signals for these responses are mediated by ethylene receptor proteins located on cell membranes. It has been postulated that ethylene initiates signaling that allows a series of down stream events to occur resulting in the typical ethylene response. Because of the diverse effects of ethylene on a wide range of plant species, many of which are harmful, it would be highly beneficial to manage the effect of ethylene during the post-harvest life of fruits, vegetables and flowers. Over the years many techniques have been developed to regulate the physiological effects of ethylene. Controlled atmosphere (CA) developed in the early sixties is still used extensively for controlling post-harvest ethylene production and respiration in many fruits and vegetables during their storage. Later on, modified atmosphere (MA) films are being developed to regulate gas exchange at the package level and they are effective in extending the shelf-life of fresh cut produce. Aminoethoxyvinylglycine (AVG), an ethylene biosynthesis inhibitor, has been introduced commercially as ReTain to reduce internal ethylene production resulting in delayed ripening especially in apples. However, AVG is less effective in post-harvest applications because it does not control the effects of ethylene from external sources. Many types of ethylene scrubbers were therefore developed over the years, however, none have been highly successful. While, application of these techniques is important for horticultural industries but because of the limitations research interest focused towards the improvement in control of ethylene physiology, whether by transgenic or chemical approaches (Watkins 2002). So, there is a need to develop simple and cost-effective methods of controlling fruit ripening and reducing the postharvested losses. Out of various other available approaches, approaches involving avoiding the exposure or minimizing the production or suppressing the perception/response of ethylene are very important. Development of 1-methylcyclopropene (1-MCP) In the mid-nineties, Prof. E. C. Sisler at North Carolina State University discovered that some cyclopropenes counteract the effects of ethylene of which 1-MCP holds the most commercial promise (Grichko et al. 2006). Prof. Sisler has shown that 1MCP binds to the ethylene receptor (Sisler and Serek 1997). Chemically, 1-MCP (C4H6) is a cyclopropene with molecular mass of 54.09. It is volatile at STP. The presence of 1-MCP not only blocks the basal but also the autoinduced ethylene production (which is triggered production of ethylene in presence of ethylene itself due to its positive effect on the induction of ethylene biosynthetic enzymes i. e., ACC-synthase and ACC-oxidase enzymes. So, 1-MCP has the potential to be the most efficient and convenient approach for managing the diverse ethylene responses in fruits, vegetables or flowers (Grichko et al. 2006). 1-MCP is being developed by AgroFresh Inc., a wholly owned subsidiary Rohm and Haas Company, for use in post-harvest fruit, vegetables and flowers world wide. 1-MCP is currently formulated as SmartFresh, powder for post-harvest use in fruits and vegetables and EthylBloc for use in flowers. 217 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Acute toxicity, mutagenicity and product chemistry  studies conducted on the SmartFresh formulation indicate a favorable toxicology profile. In addition, 1MCP has a non-toxic mode of action, is applied at extremely low ppb dose levels and has no expectation  of measurable residues in food commodities. The EPA has classified 1-MCP as a biopesticide, which qualifies it for an expedited review. EPA registration was granted in flowers in April, 1999, and the registration for post-harvest use on fruits and vegetable is done in 2002 (EPA, 2002). The development of 1-MCP as a complexed formulation with -cyclodextrin to form a white stable powder that releases 1-MCP when dissolved in water, marketed as EthylBloc TM (Floralife, Inc.) and SmartFreshTM (AgroFresh, Inc., Rohm and Haas) for use on ornamental and edible horticultural products respectively, has lead to a surge of research and commercial interest around the world. Rohm and Haas Company purchased the worldwide rights to 1-MCP and licensed back to Floralife the commercial rights to ornamentals in the USA and Canada. All other global use rights, both ornamental and food, are retained by AgroFresh Inc. 1-MCP has a non-toxic mode of action, negligible residue and is active at very low concentrations. The US Environmental Protection Agency has classified 1-MCP a plant growth regulator. SmartFresh is currently formulated as a 3.3 % a.i. powder that liberates the active ingredient, 1methylcyclopropene, when added to water. There is also a 0.14 % a.i. powder, which is usually preferred for small-scale research trials since it is easier to weigh and handle. Harvested fruits and vegetables must be exposed to the active ingredient in its gaseous form in enclosed areas such as storage rooms, coolers, shipping containers or trailers. These enclosed areas should be fairly air tight, as excessive leakage will reduce SmartFresh effectiveness. This product is not intended for use outdoors or in other non-enclosed areas.  For research purposes small containers should be used such as drums, plastic chambers, plastic bags of at least 3 mil (63 m) thickness, drying chambers, etc.  For commercial use, plant material has to be treated in enclosed areas such as tightly built greenhouses, rooms, shipping boxes/containers, etc. The treatment areas should be as much as possible gas-tight for preventing gas leakage, which will reduce effectiveness of 1-MCP.  In the large rooms/treatment areas it is recommended an establishment of an internal air circulation system during the treatment (without bringing outside air).  In most of the cases the plant material, once treated, does not need re-treatment, however retreatment is not harmful and can even be beneficial. Some species of ornamental plants would likely to get benefited from additional or even continuous treatment, especially with flowers in different stages of development on the same plant, which are continuously developing new ethylene binding sites (Serek and Sisler, 2005). Directions for use: Generation, application and quantification of 1-MCP  SmartFresh can be applied to fruits and vegetables immediately after harvest, upon entering storage, in transit or at the distribution center. To realize maximum benefit for ripening control, fruit should be treated after the ripening process  has been initiated (green ripe) but before the climacteric peak has occurred. And to get maximum benefit in controlling senescence, produce should be treated immediately after harvest. 218 It has been documented that only very small concentrations of 1-MCP, in the range of µl L-1, are sufficient for preventing ethylene responses in ornamental crops and cut flowers (Serek et al. 1994, 1995a, b, c). However, for commercial use, the recommended concentration for Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India EthylBloc® and SmartFreshTM is in the range of 100 to 500 µl L-1, some 1000 fold higher, probably because of a high possibility for leakage of 1MCP.  warm temperature conditions (55 to 75 F, 13 to 24 C). Longer treatment periods of >12 hours may be required for applications at lower temperatures (below 55 F, 13 C). Irrespective of the treatment temperature, the release 1-MCP from the SmartFresh powder is most efficient when the solution is stirred or aerated. SmartFresh is formulated as a powder that, when added to water, releases the active ingredient 1-methylcyclopropene.  SmartFreshTM has been available for only a short  period of time and no published data are available. However, it apparently has faster and more complete release characteristics, especially at lower temperatures, than EthylBlocTM.  Table 1 gives the quantity of SmartFresh powder needed per cubic meter (and cubic feet) treatment chamber volume to achieve target application rates in ppb of active ingredient. For example, treating a volume of one m3 with 1.6 g of 0.14 % SmartFresh formulation will result in an approximate concentration of 1000 ppb 1MCP a.i. vol/vol in air.     1-MCP, quantified by gas chromatography (GC) with calibration against butane, 1-butene, isobutylene, or ethylene), is applied by injecting known quantities of 1-MCP into air-tight containers. This method continues to be used, despite the availability of the commercial product (Jiang and Joyce 2000, Pesis et al. 2002). Table 1. Application rate of SmartFresh (0.14 % a.i.) product in ppb v/v and water volume (ratio of SmartFresh :water = 1:16) needed to release the 1-MCP. The 1-MCP has either been released directly in the treatment container, (DeEll et al. 2002, Dong et al. 2002, Watkins et al. 2002), or generated separately and aliquots injected into the container (Mir et al. 2001). 1-MCP Application Rate (ppb a.i. vol/vol) 10 30 50 100 300 500 1000 SmartFresh per m3 mg product 16 48 80 160 480 800 1600 ml water 1 1 2 3 8 17 25 SmartFresh  per ft3 mg product 0.45 1.36 2.27 4.53 13.6 22.7 45.3 ml water 1 1 1 1 1 1 1 The final 1-MCP concentration has sometimes been quantified by GC (Mir et al. 2001), but often the final concentration has been assumed from the known weights of EthylBlocTM in the container (Dong et al. 2002, Watkins et al. Recommendations for different commodities 2002). SmartFresh has demonstrated excellent The recommended temperature during the activity for delaying the onset of ripening in fruits, treatment is based on several scientific reports as vegetables, cut flowers and potted plants. Almost all well as on practical trials, which for ornamental fruits and vegetables are ethylene sensitive to a varying crops is not lower than 13 0C. Treatments at lower degree. Many, especially those of the climacteric class, temperature require a substantial increase of the also generate large amounts of ethylene. Consequently, concentration of 1-MCP. Both EthylBloc® and it is expected that most fruits and vegetables would SmartFreshTM were qualified for review in USA. benefit from SmartFresh treatments. Table 2 1-MCP is more effective when fruits and contains a list of fruits and vegetables along with their vegetables are exposed for at least 4 hours under relative degree of ethylene production and sensitivity and the suggested 1-MCP application range. 219 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Table 2. Level of ethylene production, sensitivity and Recommended personal protective equipment suggested 1-MCP application rate range for fruits and Gloves-Polyvinyl chloride-coated glove or other vegetables. chemical resistant rubber coated glove. Commodity Pome Fruit Apple Asian Pear Pear (Anjou) Pear (Bosc, Bartlett) Stone Fruit Apricot Peach Plum & Prune Berries Raspberry Strawberry Leafy Vegetables Endive leafy greens Spinach Lettuce (iceberg) Celery Parsley Cut Vegetables Broccoli Brussel Sprouts Cabbage, Chinese Cabbage (white) Cabbage (green, red, savory) Cauliflower Vegetables (Other) Beans (snap or green) Cucumbers Onion (dry) Onion (green) Peas Potato Squash (summer) Tomato (breaker) Melons Cantaloupe Crenshaw melon Honeydew Melon Tropical Fruit Banana (green) Banana (yellow) Mango Olive Kiwifruit Papaya Avocado Passion fruit Ethylene sensitivity Suggested 1MCPapplication range (ppb a.i.) Eye protection -Protective eye wear goggles for particles and organic gases, tightly fit to the face. High High High High 600 – 1000 300 – 1000 100 – 1000 100 – 1000 Respirator -Face mask with a multi gas/vapor cartridge High High High 300 – 1000 300 – 1000 300 – 1000 Low Low 30 – 100 30 – 100 Medium High High High Medium High 500 - 1000 500 - 1000 500 - 1000 500 - 1000 500 - 1000 500 - 1000 High High High High High 500 - 1000 500 - 1000 500 - 1000 500 - 1000 500 - 1000 High 500 - 1000 Medium 500 - 1000 High Low Medium Medium Medium Medium High 500 - 1000 500 - 1000 500 - 1000 500 - 1000 500 - 1000 500 - 1000 300 - 500 Medium High High 300 - 500 300 - 500 300 – 500 High High High Medium High High High High 30 – 100 300 – 1000 100 – 500 100 – 500 100 – 500 100 – 500 100 – 500 100 – 500 Safe handling procedures for working with 1-MCP or its commercial formulation Do not handle material near food, feed or drinking water. While weighing and mixing, wear gloves, eye protection and keep spectators away. When water is added to the SmartFreshTM formulation, the vapor will be released. Avoid breathing the vapor and prevent it from contacting your face and eyes. For many small research trials, the SmartFreshTM vapor is released in a chamber remote from the user, but if there is any chance of contact with the vapor, a respirator must be worn. A respirator must be worn for treatments in walk-in rooms, and the room must be vented for 15 minutes before entering after the treatment. Gloves should be removed and replaced immediately if there is any indication of degradation or chemical breakthrough. Rinse and remove gloves immediately after use. Wash hands with soap and water. If exposed to material, remove all contaminated clothing promptly. Wash all exposed skin areas with soap and water immediately after exposure. First Aid Measures, if required to be taken along with the consultation of Doctor. Thoroughly launder clothing before reuse. Do not take clothing home to be laundered. If spilled, appropriate protective equipment must be worn when handling a spill of this material. Keep spectators away. Avoid breathing dust. Transfer spilled material to suitable containers for recovery or disposal. Keep dust to a minimum. Keep spills and cleaning runoff out of municipal sewers and open bodies of water. 220 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Serek, M. and Sisler, E.C. (2005). Impact of 1-MCP on post harvest quality of ornamentals. APEC Abeles, F.B., Morgan, P.W. and Saltveit, M.E. (1992). Symposium on Quality Management of Ethylene in Plant Biology, Academic Press, San Postharvest Systems Proceedings, pp. 121-128. Diego, California, pp 414. References DeEll, J.R., Murr, D.P., Porteous, M.D. and Rupasinghe, H.P.V. (2002). Influence of temperature and duration of 1methylcyclopropene (1-MCP) treatment on apple quality. Postharvest Biol. Technol. 24:349-353. Serek, M., Sisler, E.C. and Reid, M.S. (1994). Novel gaseous ethylene binding inhibitor prevents the effects in potted flowering plants. J. Am. Soc., Hort. Sci. 119: 1230-1233. Serek, M., Sisler, E.C., Tirosh, T. and Mayak, S. (1995a). 1-Methyleyclopropene prevents bud, flower, and leaf abscission of Geraldton Dong, L., Lurie, S. and Zhou, H-W. (2002). Effect of waxflower. HortSci. 30: 310. 1-methylcyclopropene on ripening of ‘Canino’ apricots and ‘Royal Zee’ plums. Postharvest Serek, M., Tamari, G., Sisler, E.C. and Borochov, A. Biol.Technol. 24:135-145. (1995b). Inhibition of ethylene induced cellular senescence symptoms of ethylene action. Environmental Protection Agency (2002). Federal Physiol. Plant. 94: 229-232. Register, 67(144) (pp. 48796-48800). July 28, 2002. Grichko, V., Serek, M., Watkins, C.B. and Yang, S.F. (2006). Father of 1-MCP (Editorial). Biotechnol. Advances 24: 355-356. Serek, M., Sisler, E.C. and Reid, M.S. (1995c). Effects of 1-MCP on the vase life and ethylene response of cut flowers. Plant Growth Regul. 16: 93-97. Jiang, Y. and Joyce D.C. (2000). Effects of 1- Serek, M., Woltering, E.J., Sisler, E.C., Frello, S. and Sriskandarajah, S. (2006). Controlling ethylene methylcyclopropene alone and in combination responses in flowers at the receptor level. with polyethylene bags on the postharvest life of Biotechnol. Advances 24: 368-381. mango fruit. Ann. Appl. Biol. 137:321-327. Mir, N.A., Curell, E., Khan, N., Whitaker, M. and Sisler, E.C. and Serek, M. (1997). Inhibitors of ethylene responses in plants at the receptor level: Beaudry, R.M. (2001). Harvest maturity, storage Recent developments. Physiol. Plant. 100: temperature, and 1-MCP application frequency 577-582. alter firmness retention and chlorophyll fluorescence of ‘Redchief Delicious’ apples. Am. Watkins, C.B. (2002). Ethylene synthesis, mode of action, consequences and control. In: Fruit Soc. Hort. Sci.126: 618-624. Quality and its Biological Basis, Knee, M (ed.), Pesis, E., Ackerman, M., Ben-Arie, R., Feygenberg, CRC Press, Sheffield, Boca Raton, Florida. Pp O., Feng, X., Apelbaum, A., Goren, R. and 180-224. Prusky, D. (2002). Ethylene involvement in chilling injury symptoms of avocado during cold Watkins, C.B., Nock, J.F. and Whitaker, B.D. (2000). Responses of early, mid and late season apple storage. Postharvest Biol. Technol. 24:171cultivars to postharvest application of 1181. methylcyclopropene (1-MCP) under air and Saltveit, M.E. (1999). Effect of ethylene on quality of controlled atmosphere storage conditions. fresh fruits and vegetables. Postharvest Biol. Postharvest Biol. Technol.19: 17-32. Technol. 15: 279-292. 221 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ESTIMATION OF PHOTOSYNTHETIC PIGMENTS AND TOTAL CAROTENOIDS IN FRUIT TISSUES Vijay Paul*, Rakesh Pandey and Atar Singh Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110 012 *: E-mail: vijay_paul_iari@yahoo.com Plant pigments (chlorophylls and carotenoids etc.) are of tremendous significance in the biosphere. They are essential not only for photosynthesis but also for plant and animal survival through their photosynthetic and nutritional functions (Davies, 2004). Pigments are very important component for any given fruit as they are not only associated with the nutritional aspects but also linked to the appearance and ripening status of a fruit. From epidemiological studies, clinical trials and experiments on animals as well as in vitro studies, this protective effect has been mainly attributed to carotenoids. Moreover, carotenoids are a major class of compounds providing precursors to essential vitamins and antioxidants. Because tomato is the second-most important vegetable in the world after potato this horticultural crop is the predominant source of carotenoids. From a total of around 40 carotenoids found in the human diet, only 25 are found in human blood due to selective uptake by the digestive tract. Of that number, 9-20 are derived from fresh and processed tomato; major ones being lycopene, -and -carotene, lutein, zeaxanthin and -cryptoxanthin. Once again, -carotene, a potent dietary precursor of vitamin A (Olson 1989), accounts for around 7 % of tomato carotenoid content (Nguyen and Schwartz 1999). All these factors influence the consumers’ acceptability and preference. Further, information regarding different plant pigments pertaining to their temporal dynamics and spatial variations can form an applied perspective to a wide range of scientific investigations and environmental/agricultural management endeavors. Pigment assays assist us to monitor the temporal dynamics of pigments at the individual scale (leaf, fruit) or at group level (crop, canopy). Faster, non-destructive measurements of leaf chlorophyll concentration can be obtained using handheld field instruments such as the SPAD (Markwell et al. 1995). However, there is still need to measure the actual levels of chlorophyll ‘a’, chlorophyll ‘b’, total chlorophyll and carotenoids etc.. It is important to note that even today plant physiological aspects/processes highlighted by temporal and/or spatial changes in the pigments remained under exploited at basic as well as at applied levels. Information on pigment status not only provides us quantity of pigments but also the composition, content and their degradation (during plant tissue growth, stress conditions, maturation and senescence) under a given condition and/or over a period of time. The dynamics of plant pigments in fact relate strongly to the physiological status of plants, leaves or plant parts (such as; fruits). In this article we are presenting the methodology for the estimation of chlorophyll and total carotenoids especially for the fruits along with the improvements that has taken place with respect to the wavelengths at which such estimations to be actually done for the accurate quantification of these pigments in plant tissues including fruits. Principle Chlorophylls and carotenoids are extracted from plant parts and their concentrations are determined spectrophotometrically. By use of simultaneous equations it is possible to determine the concentration of chlorophylls ‘a’ and ‘b’, total chlorophyll and to estimate total carotenoids from the same extract. Materials required Plant material (leaf/shoot/ fruit), distilled water, 222 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India 80 % acetone, mortar and pestle, funnels, Whatman filter papers grade 1, volumetric flasks, measuring cylinders, pipettes, colorimeter or spectrophotometer etc. are determined using the following formulas given by Arnon (1949) based on the work of Mac Kinney (1941) who provided the values of extraction coefficients. Procedure Chlorophyll ‘a’ = [(12.7 x A 663 ) – (2.69 x A 645 )] x –––––––––– (mg g -1 fresh weight) 1000 x W Prepare 80 % acetone. Weigh 250 mg of fresh leaf material (avoid mid ribs) or 0.5 to 1.0g of fruit part, mostly the pericarp tissue is being taken although the part taken is dependent on the objectivity of the experiment. V Chlorophyll ‘b’ = [(22.9 x A 645 ) – (4.68 x A 663 )] x –––––––––– -1 (mg g fresh weight) 1000 x W V Total chlorophyll ‘a’ + ‘b’ = [(8.02 x A 663 ) + (20.2 x A 645 )] x –––––––––– -1 1000 x W (mg g fresh weight) Where, A = Absorbance at given wavelength, V = Final volume of 80 % acetone in ml and W = Weight of plant tissue in grams. Estimation of total carotenoids and improvements in the quantification of chlorophyll The above extract (i. e., in 80 % acetone) can also be used for the quantification of total carotenoids. A. Method given by Kirk and Allen (1965) and this was also used by Venkatarayappa et al. (1984) gives For fruits or leaves: Grind the pieces initially only the relative OD or absorption values. in liquid nitrogen in pestle and mortar so that tissue Carotenoids = [A480 + (0.114 x A663) – (0.638 x A645)] powder or a fine paste can be obtained. To this paste A add 5 ml of 80 % acetone and grind again. Wash out the content present in the pestle and mortar in centrifuge B. As per the method given by Lichtenthaler (1987) tube of 50 ml capacity by giving wash with 3 ml of 80 we can calculate not only total carotenoids but also % acetone at least three times. Put the centrifuge tubes the chlorophyll ‘a’, ‘b’ and total chlorophyll by using on shaker (200 rpm) for 2 hours. Centrifuge it and following calculations. 1000 x A470 - (1.82 x Ca) - (85.02 x Cb) decant the supernatant in another tube/vial. Add 3 or Total carotenoids = 5 ml of 80 % acetone in the centrifuge tube containing (µg/ml) 198 the pallet. Put the tube again for shaking (250 rpm) for 3 to 4. Repeat the process of centrifugation and shaking Where, A = Absorbance at given wavelength at least 1 to 2 times more for complete extraction of Ca is chlorophyll ‘a’ = (µg/ml) = (12.25 x A 663.2) - (2.79 x A 646.8) pigments especially the carotenoids. Now make the Cb is chlorophyll ‘b’ = (µg/ml) = (21.50 x A 646.8) - (5.10 x A ) volume of repeatedly collected supernatant to 25 ml 663.2 with 80 % acetone. We can also find out total chlorophyll by the formulae: Estimation of chlorophyll ‘a’, ‘b’ and total (µg/ml) = (7.15 x A 663.2) + (18.71 x A 646.8) chlorophyll -1 Record the absorbance of filterate/supernatant For converting µg/ml into µg g fresh weight: at two different wavelengths (663 and 645 nm) using µg g-1 fresh weight (fw) = µg/ml x Final volume made spectrophotometer by keeping 80 % acetone as blank. Weight of tissue taken in g For leaves: Grind the pieces of plant material in pestle and mortar using 5 ml of 80 % acetone. Filter the homogenate in 25 ml of volumetric flask (25 ml) by using Whatman paper grade 1. Wash out the homogenate 3-4 times with 5 ml of 80 % acetone each time. Grind the tissue once again with minimum quantity of 80 % acetone if required as it helps in complete extraction of plant pigments. Make the final volume of filterate to 25 ml. The amount of chlorophyll ‘a’, ‘b’ and total 223 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India C. The above calculations were examined further in For converting µg/ml into µg g-1 fresh weight: 80 % acetone extracting medium and still finer µg g-1 fw = µg/ml x Final volume made improvements were suggested by Wellburn (1994): Weight of tissue taken in g Total carotenoids = 1000x A470 - (3.27 x Ca) – (104 x Cb) (µg/ml) 198 Precautions  It is highly recommenced to extract the pigments soon after the collection of samples. Where, A = Absorbance at given wavelength Ca is chlorophyll ‘a’ = (µg/ml) = (12.21 x A 663) - (2.81 x A 646)  Because of light influence on the pigments, extraction should be carried out in subdued light Cb is chlorophyll ‘b’ = (µg/ml) = (20.13 x A 646) - (5.03 x A 663) at low temperatures preferably 5 0C and if We can also find out Total chlorophyll by Ca + Cb appropriate facilities not available then For converting µg/ml into µg g-1 fresh weight: temperature should not be more than 20-22 0C. -1 µg g fw = µg/ml x Final volume made  Ensure maximum extraction of chlorophyll Weight of tissue taken in g pigments by minimum of 3-4 washings of homogenate with extracting medium. D. Specifically for the leaves of grasses and cereals  If not stored in deep freeze, the absorbance must be taken as early as possible after the extraction following formulae were reported by Price and Hendry as pigments especially the carotenoids are not (1991) and Hendry and Price (1993) for the estimation stable for a long time. of total carotenoids: Total carotenoids = 1000 x A480 - (0.114 x A 663) – (0.638 x A 645) x 5.45 (µg/ml) 112.5 References Arnon, D.I. (1949). Copper enzyme in isolated Where, A = Absorbance at given wavelength chloroplasts polyphenoloxidase in Beta vulgaris. Please see above for converting µg/ml into µg g-1 fresh Plant Physiol. 24: 1-15. weight Davies, K.M. (ed.) (2004). Plant pigments and their manipulation. Annual Plant Reviews, Vol. 14. Estimations using pure acetone Oxford, UK: Blackwell, Publishing. It is not always 80 % acetone that is being Hendry, G.A.F. and Price, A.H. (1993). Stress used as extracting medium but pure acetone can also indicators: chlorophylls and carotenoids. In be used. In pure acetone extract we can calculate Methods in Comparative Plant Ecology. A chlorophyll ‘a’, ‘b’, total chlorophyll and total Laboratory Manual (Hendry, G.A.F. and Grime, carotenoids by using the equations given by J.P. eds.), Chapman and Hall, London, pp. 48Lichtenthaler (1987) 152. Kirk, J.T.O. and Allen, R.L. (1965). Dependence of Total carotenoids = 1000 x A470 - (1.90 x Ca) – (63.14 x Cb) chloroplast pigment synthesis of protein synthesis (µg/ml) 214 effect of actidione. Biochem. Biophys. Res. Commun. 21: 523-530. Where, A = Absorbance at given wavelength Lichtenthaler, H.K. (1987). Chlorophylls and Ca is chlorophyll ‘a’ = (µg/ml) = (11.24 x A 661.6) - (2.04 x A 644.8) carotenoids: pigments of photosynthetic membranes. Meth. Enzym. 148: 350-382. Cb is chlorophyll ‘b’ = (µg/ml) = (20.13 x A 644.8) - (4.19 x A 661.8) We can also find out Ca + Cb i. e., total chlorophyll (µg/ml) = (7.05 MacKinney, G. (1941). Absorption of light by chlorophyll solutions. J. Biol. Chem. 144: 315x A 661.6) + (18.09 x A 644.8) 323. 224 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Markwell, J., Osterman, J.C. and Mitchell, J.L. contribution to drought damage in nine native (1995). Calibration of the Minolta SPAD-502 grasses and three cereals. Plant Cell Environ. leaf chlorophyll meter. Photosynthesis Res. 46: 14: 477-484. 467-472. Venkatarayappa, T., Fetcher, R.A. and Thompson, Nguyen, M.L., Schwartz, S.J. (1999). Lycopene: J.E. (1984). Retardation and reversal of chemical and biological properties. Food senescence in bean leaves by benzyladenine and Technol. 53: 38-45. decapitation. Plant Cell Physiol., 25: 407-418. Olson, J.A. (1989). Provitamin A function of Wellburn, A.R. (1994). The spectral determination of carotenoids: the conversion of beta-carotene into chlorophyll a and b, as well as total carotenoids, vitamin A. J. Nutr. 119: 105-108. using various solvents with spectrophotometers Price, A.H. and Hendry, A.F. (1991). Iron catalysed of different resolution. J. Plant Physiol. 144: oxygen radical formation and its possible 307-313. 225 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ESTIMATION OF LYCOPENE PIGMENT IN FRUITS/PLANT TISSUES Vijay Paul*, Rakesh Pandey and Atar Singh Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110 012 *: E-mail: vijay_paul_iari@yahoo.com It is widely accepted that a healthy diet is an important factor in preventing chronic diseases such as cancer, cardio-vascular and neuro-degenerative diseases, and in improving energy balance and weight management. In the literature, studies have shown strong inverse correlations between fruit (especially the tomato) consumption and the risk of certain types of cancer, cardiovascular diseases and age-related macular degeneration (Gianetti et al. 2002, Giovannucci 2002, Giovannucci et al. 2002, Khachik et al. 2002, Muller et al. 2002, Sesso et al. 2003, 2004, Stahl and Sies 2005). From epidemiological studies, clinical trials and experiments on animals as well as in vitro studies, this protective effect has been mainly attributed to pro-vitamin A and other carotenoids. Moreover, carotenoids are a major class of com- pounds providing precursors to essential vitamins and antioxidants. Moastofi et al. 2003, Cano et al. 2003). There are also variations in the levels of lycopene due to the effect of variety (Tonucci et al. 1995, Abushita et al. 2000) or maturity status of the tomato fruit (Chiesa et al. 1998, Arias et al. 2000). Differences in colour and nutritional quality were attributes for attached and detached fruits (Hobson 1989, Giovanelli et al. 2001, Wills and Ku 2002). During storage of harvested tomato fruits, the optimum temperature for colour development was 18-24 0C. At 30 0C or above 30 0 C lycopene was not formed and ripening was also delayed (Goodwin and Jamikorn 1952, Laval-Martin et al. 1975, Timdall 1983). Tomato fruit is the predominant source of carotenoids. From a total of around 40 carotenoids found in the human diet, only 25 are found in human blood due to selective uptake by the digestive tract. Of that number, 9-20 are derived from fresh and processed tomato; major ones being Iycopene, -and -carotene, lutein, zeaxanthin and -cryptoxanthin. Lycopene, which constitutes about 80-90% of the total carotenoid content of red- ripe tomatoes (Shi and Maguer 2000) is the most efficient antioxidant among carotenoids through its quenching activity of singlet oxygen and scavenging of peroxyl radicals (Mortensen and Skibsted 1997, Sies and Stahl 1998). Principle In this article we are presenting the methodology for the estimation of lycopene pigment in fruits. Although the method is being described for tomato fruit but it is valid for any other fruit or plant parts. Pigment is being extracted from plant parts and its concentration is determined spectrophotometrically. Method This method is modification of procedures described earlier by Sadler et al. 1990, Fish et al. 2002 and Davis 2003. Weigh 0.5 to 1.0g of fruit part, mostly the pericarp tissue is being taken although the part taken can vary with the objectivity of the experiment. Grind the pieces of fruit tissue initially in liquid nitrogen using pestle and mortar so that tissue powder or a fine paste can be obtained. To this paste Lycopene is the major membrane bound add 5 ml of 80 % acetone and grind again. Wash out (inside the chromoplast) antioxidant in mature tomato the content present in the pestle and mortar in centrifuge fruit. Lycopene content increased gradually during the tube of 50 ml capacity by giving wash with 3 ml of 80 course of ripening of tomato fruit under attached as % acetone at least three times. Put the centrifuge tubes well as detached conditions (Chiesa et al. 1998, on shaker (200 rpm) for 2 to 3 hours. Centrifuge it 226 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India and decant the supernatant in another tube/vial. Add 3 or 5 ml of 80 % acetone in the centrifuge tube containing the pallet. Put the tube again for shaking (250 rpm) for 4 hours. Repeat the process of centrifugation and shaking at least 2 more times more for complete extraction of pigment. Now make the volume of repeatedly collected supernatant to 25 ml with 80 % acetone. Now take 10 ml of extract and into it add 10 ml of hexane. In this mixture add 1 ml of chilled water. Shake the content gently for 10 minutes and then allow it to stand still for 1 to 2 hours. Now take out the upper phase containing the lycopene in hexane for the estimation of lycopene. Precautions  It is highly recommenced to extract the pigment soon after the collection of samples.  Because of light influence on the pigments, extraction should be carried out in subdued light at low temperatures.  If not stored in deep freeze, the absorbance of the filtrate must be taken as early as possible after chlorophyll extraction as pigment in not stable for a long time. References Abushita, A.A., Daood, H.G. and Biacs, P.A. (2000). Record the absorbance at 503 nm using Change in carotenoids and antioxidant vitamins spectrophotometer by keeping hexane as blank. Now in tomato as a function of varietal and calculate the lycopene content using the following technological factors. J Agric. Food Chem. equation which is primarily based on the molecular 48:2075-2081. extinction coefficient of lycopene i.e., 17.2 x 104 mole Arias, R., Lee, T.C., Logendra. L. James, H. (2000). cm-1 as reported by Beerh and Siddappa (1959): Correlation of lycopene measured by HPLC Lycopene (µg/ml) = 3.12 x A503 with L*, a*, b* colour readings of a hydroponic tomato and the relationship of maturity with colour of lycopene content. J Agric Food Where, A = Absorbance at given wavelength Chem. 48: 1697-1702. For converting µg/ml into µg g-1 fresh weight: Beerh, O.P. and Siddappa, G.S. (1959). A rapid spectrophotometric method for the detection and µg g-1 fresh weight (fw) = µg/ml x Final volume made estimation of adulterants in tomato ketchup. Weight of tissue taken in g Food Technol., 13: 414-418. The above said method can also be followed Cano, A., Acosta, M. and Arnao, M.B. (2003). Hydrophilic and lipophilic antioxidant activity using pure acetone in place of 80 % acetone. changes during on-vine ripening of tomato Modifications and suggested improvements (Lycopersicon esculentum Mill.). Postharvest Biol Technol. 28: 59-65.  Some workers have used hexane and acetone mixture (v/v) in the ratio of 3:2 or 1:2 for the Chiesa, A., Sackmann, V.M. and Fraschina, A. extraction of the lycopene pigment. (1998). Acidity and pigment changes in tomato (Lycopersicon esculentum Mill.) fruit ripening.  Still some other prefer to use hexane, acetone Acta Hort. 484: 487-490. and ethanol mixture (v/v) in the ratio of (2:1:1) for the extraction of this pigment. Davis, A., Fish, W.W. and Perkins-Veazie, P. (2003). A rapid spectrophotometric method for  0.05 to 0.1 % (w/v) butylated hydroxyl toluene analyzing lycopene content in tomato and tomato (BHT) can be added in the extracting medium to products. Postharvest Biol. and Technol. 28: reduce the oxidative damage to the pigment. 425-430 227 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Fish, W.W., Perkins-Veazie, P. and Collins, J.K. methylcyclopropene on ripening of green house (2002). A quantitative assay for lycopene that tomatoes at three storage temperatures. utilizes reduced volumes of organic solvents. J. Postharvest Biol. Technol. 27: 285-292. Food Comp. Analysis 15: 309-317. Muller, K., Carpenter, K.L., Challis, I.R, Skepper, Gianetti, J., Pedrinelli, R., Petrucci, R., Lazzerini, G., J.N. and Arends, M.J. (2002). Carotenodis De Caterina M., Bellomo, G. and De Caterina, induce apoptosis in the T-lymphoblast cell line R. (2002). Inverse association between carotid Jurkat E6.1. Free Radic. Res. 36: 791-802. intima media thickness and the antioxidant Sadler, G., Davis, J. and Deyman, D. (1990). Rapid lycopene in atherosclerosis. Am. Heart J. 143: extraction of lycopene and b-carotene from 467-474. reconstituted tomato paste and pink grapefruit Giovanelli, G., Lavelli, V., Peri, C. and Nobili, S. homogenate. J. Food Sci. 55: 1460-1465. (2001). Variation in antioxidant components of Sesso, H.D., Buring, J.E., Norkus, E.P. and Gaziano, tomato during vine and postharvest ripening. J. J.M. (2004). Plasma lycopene, other Sci. Food Agric. 79: 1583-1588. carotenoids, and retinol and the risk of Giovannucci, E. (2002). Lycopene and prostate cancer cardiovascular disease in women. Am. J. Clin. risk. Methodological considerations in the Nutr. 79: 47-53. epidemiologic literature. Pure Appl. Chem. 74: Sesso, H.D., Liu, S., Gaziano, J.M. and Buring, J.E. 1427-1434. (2003). Dietary lycopene, tomato based food Giovannucci, E., Rimm, E., Liu, Y., Stampfer, M. and products and cardiovascular disease in women. Willett, W. (2002). A prospective study of J. Nutr. 133: 2336-2341. tomato products, lycopene, and prostate cancer Shi, J., Le and Maguer, M. (2000). Lycopene in risk. J. Natl. Cancer Inst. 94: 391-398. tomatoes: chemical and physical properties Goodwin, T.W. and Jamikorn, M, (1952). Biosynthesis affected by food processing. Crit. Rev. of carotenes in ripening tomatoes. Nature 170: Biotechnol. 20: 293-334. 104-105. Sies, H. and Stahl, W. (1998). Lycopene antioxidant Hobson, G.E. (1989). Manipulating the ripening of tomato fruit low and high technology. Acta Hort. 258:593-600. and biological effects and its bioavailability in the human. Proc. Soc. Exp. Biol. Med. 218: 121124. Khachik, F., Carvalho, L., Bernstein, P.S., Muir, G.J., Stahl, W. and Sies, H. (2005). Bioactivity and Zhao, D.Y. and Katz, N.B. (2002). Chemistry, protective effects of natural carotenoids. Rev. distribution, and metabolism of tomato Biochem. Biophys. Acta. 1740: 101-107. carotenoids and their impact on human health. Timdall, H.D. (1983). Vegetables in the tropics. Mac Exp. Biol. Med. 227: 845-851. Millian Press, London. Laval-Martin, D., Quennemet, J. and Meneger, R. Tonucci LH, Holden JM, Beecher GR, Khachik F, (1975). Pigment evolution in Lycopersicon Davis CS, Mulokozi G 1995. Carotenoid esculentum fruits during growth and content of thermally processed tomato-based development. Phytochem. 14: 2357-2362. food products. J. Agric. Food Chem. 43: 579586. Mortensen, A. and Skibsted, L.H. (1997). Importance of carotenoid structure in radical scavenging Wills RBH, Ku, VVV 2002. Use of 1-MCP to extend reactions. J. Agric. Food Chem. 45: 121-124. the time to ripen the green tomatoes and postharvest life of ripe tomatoes. Postharvest Mostofi, Y., Toivonen, P.M.A., Lessant, H., Babaler, Bio. Technol. 26: 85-90. M. and Lu, C. (2003). Effects of 1228 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India GAMMA CHAMBER (GC-5000) : AN IRRADIATION FACILITY FOR CROP IMPROVEMENT AND POST HARVEST PRESERVATION Bhupinder Singh Nuclear Research Laboratory, Indian Agricultural Research Institute, New Delhi-110012 Nuclear Research Laboratory, apart from research efforts, is also providing gamma irradiation services to the researchers, trainees and students from all around the Delhi region. The facility available with the laboratory i.e., 5 litre capacity Gamma radiation chamber containing a 60Co source of 12000 Ci (GC5000), is good enough for the scientific community and the farmers. Efforts are on to develop new applications of gamma and to optimize dose requirement of different crops for improving the post harvest shelf life and quality of the agricultural produce. Gamma Chamber Radiation field is provided by a set of stationary cobalt-60 sources placed in a cylindrical cage. The sources are doubly encapsulated in corrosion resistant stainless steel pencils and are tested in accordance with international standards. A mechanism for rotating/stirring samples during irradiation is also incorporated. The lead shield provided around the source is adequate to keep the external radiation field well within permisible limits. Features of interest  Gamma Chamber 5000 is a compact self shielded cobalt-60 gamma irradiator providing an irradiation volume of approximately 5000cc. The material for irradiation is placed in an irradiation  chamber located in the vertical drawer inside the lead flask. This drawer can be moved up and down with the help of a system of motorised drive which enables precise positioning of the irradiation chamber at the centre of the radiation field. Safe and self-shielded: The shielding provided is adequate to limit the radiation field on the external surface of the unit, well within the permissible levels. No additional shielding is required for its installation and use. Automatic control of irradiation time: Builtin timer provides accurate control of irradiation time from 6 seconds onwards. The unit can also be operated manually. Solid state programmable controls have been provided. In the event of power failure battery backup displays the programmes.  Manual control of irradiation temperature: It is possible to irradiate samples at low or high temperature by circulating liquid nitrogen or hot air. These can be introduced through the service sleeves provided in the vertical drawer. The irradiation temperature is sensed by a thermocouple and displayed on the panel.  Remote operation: An additional table top control panel is provided for remote operation in addition to the normal one provided on the unit.  Dose uniformity: Stationary source pencils, symmetrically placed in a cylindrical cage ensure good uniformity of radiation field in the sample chamber. In addition a mechanism is also A picture of Gamma Chamber (GC-5000) 229 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India   provided for rotating/stirring samples during irradiation. A Facility for All code No.SC/TR-1, 1986 of Atomic Energy Regulatory Board of INDIA. For more details you can contact The gamma irradiation facility at the Easy loading and unloading of samples: Laboratory, IARI is available to users at a very nominal Sample chamber extends to a convenient height cost (Rs 400 per dose per chamber volume for ICAR for easy loading and unloading of samples. and SAUs, Rs 800 for State universities and others Safety assurance: The design of Gamma institutions, Rs 1600 for Industry). The number of Chamber conforms to American National samples irradiated for gamma has been growing year Standards, ANSI-N433.1-1977 for safe design after year, roughly in excess of 1000 machine runs a and use of self-contained dry source storage year. It is only apt to conclude that the technology has gamma irradiators(Category I). It also meets the the support of the researchers and a greater role to requirements of type B(U) package for safety in play in changing certain facets of agriculture through transport of radioactive materials as per AERB applications that are just waiting to be discovered. Applications Dr Bhupinder Singh bsingh@iari.res.in, phone: 011-25842139 Gamma Chamber is a versatile equipment for research studies in many fields such as:  Radiobiology  Preservation of tissue grafts  Mutation breeding  Food preservation  Sterile male insect technique  Biological and genetic effects of radiations  Radiation chemistry  Radiation effects on materials  Radiation sterilization  Modification of properties of materials Control Irradiated (0.5 kGy) Gamma irradiation delays ripening in Mango Gamma Chamber can also be used in many other research applications which require irradiation of materials with ionizing radiations to varying doses. Accessories Radio mutant developed using gamma irradiation 1. Attenuators to bring down the radiation dose in the sample chamber by 25% or 50% - are available on request. 2. Dosimeters are available in sets of 100 ampules of 2 ml. The Kilo Gray Gamma Dosimeter system to read out the dose delivered, is also available. It is essential for calibration purpose. 230 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India ISOLATION OF PLANT DNA Gaurav Agarwal, Divya Choudhary and Ajay Arora Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110012 The isolation of high molecular weight DNA that is suitable for digestion with restriction endonuclease is an essential requirement for confirming transgene interaction through polymerase chain reaction or by southern blot hybridization. The plant tissues are notoriously difficult material for DNA isolation due to the presence of various secondary plant products. A protocol that works with one plant group may fail with others. Consequently, a number of DNA isolation methods have been developed for different target plant groups. Two methods developed originally by Dellaporta et al. (1983) and Murray and Thompason (1980) respectively, are being commonly used with certain modifications for isolating high molecular weight DNA from small amounts of tissue. In our hands a modified Murray and Thompson protocol, as described below has worked equally well in a number of plant genera including rice, wheat, chickpea, Nicotiana and Brassica. Protocol (i) DNA isolation 1. Grind 0.5 g of leaf material in liquid nitrogen to fine powder using pre-chilled mortar and pestle. 2. Transfer the powder to a 15 ml polypropylene centrifuge tube containing 5 ml of pre-warmed extraction buffer. Use spatula to disperse the material completely. 3. Incubate samples at 600C for 30 min with occasional mixing by gentle swirling. 4. Add 3 ml chloroform isoamyl alcohol and mix by inversion to emulsify. 5. Spin at 15000 rpm for 10 min at room temperature. 6. Remove the aqueous phase with a wide-bore pipette, transfer to a clean tube, add 2/3 volume of isopropanol and mix by quick gentle inversion. 7. Spool DNA using a bent Pasteure pipette and transfer to another tube. Alternatively, if the DNA appears flocculent, centrifuge at 5000 rpm for 2 min and gently pour off the supernatant. 8. Wash the DNA pellet in 70% ethanol (5-10 ml) for 20 min. 9. Dry the pellet and dissolve in 500 µl TE buffer. (ii) Purification of DNA Major contaminants in crude DNA preparation are RNA, protein and polysaccharides. Inclusion of CTAB in DNA extraction buffer helps elimination of polysaccharides from DNA preparations to a large extent. The RNA is removed by treating the sample with RNase. Proteins including RNase can be removed by treatment with proteinase K. Extraction with phenol: chloroform following RNase treatment is also employed for eliminating RNA and most of proteins. Given below is the protocol of DNA purification routinely being used in our laboratory. 1. Dissolve 10 mg RNase in 1 ml of 10 mM TrisHCL (pH 8.0) and 15 mM NaCl in a 1.5 ml microfuge tube. 2. Heat the tube in boiling water bath at 1000C for 15 min to denature contaminating DNase. 3. Cool slowly at room temperature. 4. Add RNase @ 50 µg/ml to DNA sample and incubate at 370C for one hour. 5. Add equal volume of phenol: chloroform (1:1) and mix. 6. Spin at 11000 rpm for 2 min at room temperature, take out the aqueous phase and transfer to a fresh microfuge tube. 7. Extract twice with equal volume of chloroform: isoamyl alcohol (24:1), centrifuge and take out the aqueous phase. 8. Add 0.1 volume of 3 M sodium acetate (pH 4.8) and mix properly. 9. Add 2.5 times absolute alcohol, mix by quick gentle inversion to precipitate the DNA. 231 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India 10. Pellet the DNA by centrifugation at 10000 rpm for 5 min in a microfuge. 11. Decant supernatant carefully, wash the pellet with 70% cold ethanol, air dry and dissolve DNA in 50-100 µl TE buffer. (iii) Quantitation of DNA Reliable measurement of DNA concentration is important for many applications in molecular biology including complete digestion of DNA by restriction enzymes and amplification of target DNA by polymerase chain reaction. DNA quantitation is generally carried out by spectrophotometric measurements or by agarose gel analysis. (a) Spectrophotometric measurement 1. Take 1 ml TE buffer in a cuvette and calibrate the spectrophotometer at 260 nm as well as 280 nm wavelength. 2. Add 2 to 5 µl of DNA, mix properly and record the optical density (OD) at both 260 and 280 nm. 3. Estimate the DNA concentration employing the following formula: (OD)260 x 50 x dilution factor Amount of DNA (µg/µl) = 1000 4. Judge the quality of DNA from the ratio of the OD values recorded at 260 and 280 nm. The A260 / A280 ratio around 1.9 (1.85-1.95) indicates best quality of DNA. (b) Gel analysis 1. Cast agarose gel (0.7%) in 0.5 x TBE (TrisBorate-EDTA) buffer. 2. Load 2-5 µl of DNA sample 3. Load a known amount of uncut  phage DNA as control in the adjacent well. 4. Run the gel at 50 V for 1 h. 5. Stain the gel with Ethidium bromide solution (5 µg/ml) for 10 min, wash with distilled water and visualize under UV light. 6. Judge the DNA quality. Presence of a single compact band at the corresponding position to  phage DNA indicates high molecular weight of the isolated DNA. 7. Estimate the quantity of DNA in the sample by its comparison with the control either by eye judgment or by densitometric measurements. Technical tips 1. The plant sample should not be subjected to frequent freezing and thawing during grinding. 2. The DNA pellet should not be over dried. A hard, over dried DNA pellet takes very long time to dissolve completely. 3. DNA should not be forced into solution. It might cause shearing. 4. DNA from tissues of cereal crop, particularly wheat, must not be dissolved in TE buffer. Instead, only 10 mM Tris-HCL, pH 8.0 has to be used since EDTA is known to activate DNase enzymes which cause extensive shearing of the DNA. 5. One OD (A260 unit) corresponds to 50 µg/ml of double stranded DNA, while in case of single stranded DNA, RNA and oligonucleotides it is 33, 40 and 20-30 µg/ml, respectively. 6. Only A260 reading between 0.1 and 1.0 are reliable enough for accurate DNA quantitation. 7. Low-salt, alkaline buffer should be used as a solvent for spectrophotometric measurement of DNA in order to achieve reproducible A260 values and A260/A280 ratio. Requirements  Extraction buffer: 2% (2 w/v) CTAB, 100 mM Tris HCL, pH 8.0, 1.4 M NaCl, 20 mM EDTA, 0.2% mercaptoethanol.  TE buffer: 10 mM Tris, 1 mM EDTA, pH 8.0  Isopropanol  Ethanol  Chloroform  Isoamyl alcohol  RNase  Agarose  Ethidium bromide References Dellaporta, S.L., Wood, J. and Hicks, J.B. (1983). A plant DNA minipreparation: Version II. Plant Molecular Biology Report, 1: 19-21. Murray, M.G. and Thompson, W.F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acid Research, 8: 4321-4325. 232 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India TRIzol method for RNA isolation transfer 50-100 mg of frozen tissue in a 15 ml tube with 1 ml TRIzol (GIBCO BRL) homogenize for 60 sec in the polytron add 200 ml chloroform mix by inverting the tube for 15 sec incubate for 3 min at room temperature centrifuge at 12.000 g for 15 min transfer the aqueous phase into a fresh Eppi tube add 500 ml isopropanol centrifuge at max. 12.000 g for 10 min in the cold room wash the pellet with 500 ml 70 % ethanol centrifuge at max. 7.500 g for 5 min in the cold room dry the pellet on air for 10 min disolve the pellet in 50-100 ml DEPC-H2O incubate for 10 min at 60° C analyse 1 or 2 ml on a MOPS gel: disolve 1-3 mg RNA in 11 ml denaturation buffer (50 % deionized formamide, 2.2 M formaldehyde, MOPS buffer, pH 7.0, 6.6 % glycerol, 0.5 % bromphenol) add 1 ml ethidium bromide (1mg/ml) and denaturate at 65° C for 15 min load a 1 % agarose gel in MOPS buffer plus 5 % formaldehyde run the gel at 25 V for 15 h Protocol for cDNA synthesis Reagents 10x buffer RT dNTP Mix (5 mM each dNTP) OligodT primer (10 µM) RNase inhibitor (10 units/µl) Omniscript Reverse Transcriptase RNase free water Template RNA Procedure: 1. Template RNA solution was thawed on ice. Primer solution (not supplied), 10x Buffer RT, dNTP Mix, and RNase-free water were thawed at room temperature and were stored on ice immediately after thawing. 2. RNase inhibitor was diluted to a final concentration of 10 unit/µl in ice cold 1x Buffer RT was mixed carefully by vortexing, and centrifuged briefly to collect residual liquid from the sides of the tubes. 3. A fresh master mix was prepared on ice, was mixed thoroughly and carefully by vortexing for an more than 5 sec, centrifuged briefly to collect residual liquid from the walls of tube, and stored on ice. 4. Add template RNA to the individual tubes containing the master mix, Mix thoroughly and carefully by vortexing for not more than 5 sec. Centrifuged briefly to collect residual liquid from the walls of the tube. 5. Incubate for 60 min at 370 C. 6. stored at -200 C till used. Reaction mixture was prepared as follows: 10x buffer RT 2.0 µl dNTP Mix(5 mM each) 2.0 µl Oligo dT primer (10 µM) 2.0 µl RNase Inhibitor (10units/ µl) 1.0 µl Omniscript Reverse Transcriptase 1.0 µl RNase Free water variable Template RNA variable Total 20 µl Polymerase Chain Reaction (PCR): Material: 1. Taq DNA polymerase (5 units / µl) 2. cDNA. 3. dNTPs (2.5 mM). 4. 10x buffer 233 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India 3. Loading Dye 5. Primers. Forward primers 4. EtBr solution Reverse primers Reagents Preparation: 6. RNase free water. 1. 1X TAE- 250ml of 50 X TAE was prepared by adding 60.5 g Tris base,14.275ml of acetic acid, 25ml of 0.5M EDTA to minimum amount of distilled water.finally volume was make up to 250ml.Was further diluted to get 1X TAE. 2. 1% Agarose Gel-1g of Agarose(Sigma,molecular biology grade) was added to 100ml of1X TAE.Heated it in microwave for 1 min at 30 seconds interval.Mixture was cooled under tap water and 2ìl of EtBr was added,swirled lightly,then the gel mixture was poured into the gel caster with preadjusted comb. 7. Thermal Cycler. Protocol: 1. Prepare reaction mix as follows: 10x PCR buffer 5.0 µl 2.5 mM dNTPs 4.0 µl 10 µM primer 1 2.0 µl 10 µM primer 2 2.0 µl cDNA from section I 2.0 µl H2 O 34.5 µl Taq DNA polymerase (5U/µl) 0.5 µl PROTOCOL Total 50 µl 1. 1% Agarose Gel was prepared in 1X TAE buffer. 2. Sample mix was prepared by mixing 6ìl of loading dye with sample DNA. 3. Sample was loaded onto the gel and electrophoresis was carried out at 70V for 3 hrs. 4. The gel was stained with EtBr solution and viewed under U.V light. 2. Was mixed gently and spun down. 3. PCR reaction was started: First denaturation: 940 C 15 min PCR Amplification 30 cycles for 940 C 1 min 50-60 0 C 1 min 720 C 1 min/Kb Final extension: 4. 720 C 10 min 40 C Storage The tube were stored electrophoresis. at -20 0 C till Agarose Gel Electrophoresis: Reagents: 1. 1% Agarose Gel(Sigma Molecular Biology Grade) 2. 1X TAE Buffer 234 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India POLYMERASE CHAIN REACTION Gaurav Agarwal, Divya Choudhary and Ajay Arora Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110012 Polymerase chain reaction (PCR) is a technique used to selectively amplify in vitro a specific segment of the total genomic DNA a billion-fold (Saiki et al., 1985; Mullis et al., 1986). The most essential requirement of PCR is the availability of a pair of short (typically 20-25 nt) oligonucleotides called primers having sequence complementarity to either end of the target DNA segment (called template DNA) to be synthesized in large amount. The PCR involves three basic steps which constitute a single cycle: (i) denaturation of the target DNA at 92-94 0C, (ii) annealing of the primers to the template DNA at 55600C and (iii) primer extension by addition of nucleotides to the 3’ end of the primers at 720C by the enzyme DNA polymerase. As the number of PCR cycle increases, the amount of target DNA synthesized increases exponentially. Availability of thermostable DNA polymerase like Taq (from the bacteria Thermus aquaticus) has facilitated automation of the PCR (Saiki et al., 1988). 3. Prepare two negative control tubes. Add DNA from the untransformed control plant to one tube and no DNA to another tube containing all other PCR components. 4. Carry out the reaction in a thermocycler for 30 cycles with the following specifications: Cycle 1 : Cycle 2-29 : Cycle 30 : In a sterile 0.2 ml thin-wall PCR tube, add the following components of the reaction and mix in the order as given below: ---------------------------------------------------------------------------------------------------------------Sterile water 18 µl 10x buffer 2.5 µl dNTPs 0.5 µl (200 µM) each Primers 1 µl (300 ng) each Enzyme 1 unit Template DNA 2 µl (100 ng) Sterile water To a final volume of 25 µl ---------------------------------------------------------------------------------------------------------------2. Prepare a positive control. Add the vector DNA carrying the gene construct to the reaction mix instead of target genomic DNA. 4 min 1 min 2 min 1 min 1 min 2 min 1 min 1 min 7 min 5. Prepare 1.5% agarose gel in 0.5 x TBE buffer. 6. Add 2.5 µl gel loading dye to each tube containing the amplified DNA. 7. Load the samples and carry out electrophoresis at 50V for 3h. 8. Stain the gel with ethidium bromide solution (0.5 µg/ml). 9. View the gel under UV light. Compare the band position of the amplified DNA from plants with that in the positive control. Protocol 1. Denaturation (940C) Primer annealing (550C) Primer extension (720C) Denaturation (940C) Primer annealing (550C) Primer extension (720C) Denaturation (940C) Primer annealing (550C) Primer extension (720C) 10. Photograph the gel. Precautions 1. Put on a fresh pair of gloves prior to starting the work. 2. Use fresh sterile pipette tips, PCR tubes and buffers. 3. Do not use the PCR reagents and solutions for other purposes. 235 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India 4. Add all components of PCR reaction before adding the template DNA. integration (and also of the gene of interest) into the nuclear genome. Keep the level of EDTA in template DNA solution  Deoxynucleotide triphosphates (10 mM) as low as possible to prevent chelating of Mg++.  Taq DNA polymerase buffer (10X) 6. Do not use excess of DNA polymerase; use (100 mM Tris-HCl, pH9, 15 mM MgCl , 500 mM 2 dNTPs at saturating concentrations (200 µM for KCl and 0.1% gelatin) each dNTPs).  Agarose 7. Start the PCR cycling process within 30 min after addition of MgCl2 to minimize the formation of  Ethidium bromide (10 mg/ml)  Gel loading dye (6X) primer dimer. (0.25% bromophenol blue, 0.25% Xylene cyanol Technical tips FF, 15% Ficoll Type 400 in water) 1. One unit of Taq DNA polymerase is the amount 5. of enzyme which incorporates 10 nmoles of total  TBE buffer (5X) deoxyribonucleotide triphosphates into acid(0.45 M Tris-borate, 0.01M EDTA) precipitable DNA within 30 min at 740C.  DNA size standard. 2. One microgram of 1000 bp DNA fragment References corresponds to 1.52 picomoles. The relation between nanogram and picomole quantities of Mullis, K.B., Faloona, F., Scharf, S., Saiki, R., Horn, G. and Erlich, H. (1986). Specific enzymatic primer is depicted by the following equation: amplification of DNA in vitro: The Polymerase Nanogram of oligonucleotide primer = chain reaction. Cold Spring Harbor Symp. = picomole of the primer x number of nucleotides x 0.33 Quart. Biol., 51: 263. 3. Primer annealing temperature depends on its Tm value which is calculated from the following Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N. (1985). equation: Enzymatic amplification of -globin genomic Tm (0C) = 4 (G+C) + 2 (A + T) sequences and restriction site analysis for diagnosis Where, A,T,G and C stand for the number of of sickle cell anaemia. Science, 230: 1350. corresponding nucleotides in the primer. Primer Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., annealing may fail at a temperature much higher than Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, Tm, whereas annealing temperature much below Tm H.A. (1988). Primer directed enzymatic may lead to non-specific amplification. amplification of DNA with a thermostable DNA Requirements polymerase. Science, 239: 487.  Taq DNA polymerase (3 units/µl)  Oligonucleotide primers (300 ng/µl) which flank the nptII gene. Note: The nptII gene is used as a selectable marker gene in raising transgenic plants and gets integrated into the nuclear genome of the recipient species along with the gene of interest. Therefore, by using the nptII primers, the nptII gene is amplified to confirm its 236 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Appendix Good Laboratory Practices i. ii. iii. iv. v. vi. vii. viii. ix. x. xi. Working with RNA: Precautions Use a pipettor for all the solution transfers. No mouth pipetting RNA is more susceptible to degradation than DNA, due to the ability of the 2' hydroxyl groups adjacent to the phosphodiester linkages in RNA to Periodically, clean out refrigerators and act as intramolecular nucleophiles in both base- and deep-freeze. enzyme-catalyzed hydrolysis. Whereas Avoid smoking, eating and drinking in the deoxyribonucleases (DNases) require metal ions for laboratory. activity and can therefore be inactivated with chelating Do not reuse plasticware that has been used agents (e.g. EDTA), many ribonucleases (RNases) for PCR, recombinant DNA work and plant bypass the need for metal ions by taking advantage of transformation work. the 2' hydroxyl group as a reactive species. Sterilize all the plasticware before discarding RNase A is single-strand specific it. endoribonuclease that is resistant to metal chelating Use gloves and shields. Any radiation is harmful agents and can survive prolonged boiling or for biological systems. Hard beta from 32p is autoclaving. However, RNase A-type enzymes rely on active site histidine residues for catalytic activity very penetrating. and consequently, these enzymes can be inactivated Use glass or plastic goggles while viewing in by the histidine-specific alkylating agent diethyl UV light. Turn UV on for just the minimum pyrocarbonate (DEPC). required time. UV is damaging to skin and eye. Sources of RNase contamination Use gloves while handling Ethidium bromide It is possible to reintroduce RNases during the (EtBr). EtBr is highly carcinogenic. Where possible, avoid exposure of nucleic acids to course of experimentation via aqueous solutions or ultraviolet light and also visible light where from the environment at large. Autoclaving a solution ethidium bromide is present. This is most will kill contaminating bacteria, but RNases liberated important at intermediate steps in synthetic from the dead bacteria will still be active. Additionally, procedures. Avoid contact with hand and skin. ungloved fingers can introduce bacteria into solutions resulting in RNase contamination. Fortunately, the use Wash hands thoroughly with soap before of DEPC to remove ribonuclease activity from leaving the laboratory and before taking an) aqueous solutions, coupled with few laboratory prefood etc. cautions, is generally sufficient to prevent RNase Use gloves and sterile tubes, tips etc. for the contamination. success of experiment. Fingers contain Laboratory precautions nucleases, and dust contain microorganisms, Always wear gloves when working with RNA. heavy metals etc. all of which degrade nucleic It is a good idea to maintain a separate area for RNA acids. work that has its own lab equipment. This is especially Avoid contact with skin or inhalation of important if your work requires the use of RNase A chemicals like acrylamide (neurotoxic), phenol (e.g. plasmid preps). Sterile, disposable plasticware (corrosive), chloroform, lysozyme etc. 237 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India can safely be considered RNase-free and should be used when possible. Metal spatulas can quickly be decontaminated by holding in a burner flame for several seconds. Contaminating RNases can be inactivated by baking glassware at 1800C or higher for several hours. Alternatively, glassware can be soaked in freshly prepared 0.1% (v/v) DE PC in water or ethanol for 1 hour, drained and autoclaved (necessary to destroy any unreacted DEPC which can otherwise react with other proteins and RNA). DEPC will destroy polycarbonate or polystyrene materials (e.g. electrophoresis tanks), which should instead be decontaminated by soaking in 3% hydrogen peroxide for 10 minutes. Remove perodixe by extensively rinsing with DEPC-treated and autoclaved water prior to use. DNA Data Average weight of DNA base pair (sodium salt) = 650 daltons MW of a double-stranded DNA molecule = (# of base pair)’ (650 daltons/base pair) Moles of ends of a double-stranded DNA molecule = 2’ (grams of DNA) / (MW in daltons) Moles of ends generated by restriction endonuclease cleavage: a) Circular DNA molecule: 2 x (moles of DNA) x (number of sites) b) linear DNA molecule: 2 x (moles of DNA) x (number of sites) + 2 x (moles of DNA) 1.0 kb DNA-= coding capacity for 333 amino acids = 37,000 dalton protein 10,000 dalton protein = 270 bp DNA 50,000 dalton protein = 1.35 kb DNA Linear DNA µg/ml Moles/ml Molecules/ml Conc. Conc. 5’ ends 1.0 A260 DNA = 50 µg/ml 1.6x10-12 9.8x1011 1.6 nM 3.2 nM 1.0A260pBR 322 DNA = 50 µg/ml 1.8x10-11 1.1x1013 18.0 nM 36.0 nM 1.0A260 linker (8 MER) = 50 µg/ml 9.8x10-9 5.9x1015 10.0 µM 20.0 µM Common Conversion Metric Prefixes 1 Ci 1 mCi 1 µCi Isotope Data T = tera = 1012 Particle G = giga = 109 Isotope Emited M = mega = 106 14 C  5,730 years 3 H  12.3 years k = kilo = 10 3 m = milli = 10 -3 µ = micro = 10 n = nano = 10 -6 -9 Half life I 60 years 32 P 14.3 years 35 87.4 years 125 S p = pico = 10-12 f = femto = 10-15 a = atto = 10-18 238 = 1,000 mCi = 1,000 µCi = 2.2x106 disintegrations/ minute 1 Becquerel = 1 disintegration/second 1µCi = 3.70 x 104 Becquerels 1 Becquerel = 2.75 x 10-5 µCi Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India Recommended Acrylamide Gel Percentages for Resolution of Linear DNA ------------------------------------------------------------------------------------------------------1 A260 unit of double-stranded DNA = 50 µg/ml Gel percentage Protein size range 1 A260 unit of single-stranded DNA = 33 µg/ml ------------------------------------------------------------------------------------------------------1 A260 unit of single-stranded RNA= 40 µg/ml 8% 40–200 kDa 10% 21–100 kDa DNA Molar Conversions 10–40 kDa 1 µg of 1,000-bp DNA = 1.52 pmol (3.03 pmol ends) 12% ------------------------------------------------------------------------------------------------------1 pmol of 1,000-pb DNA 0.66 µg Sepectrophotometric Conversion Lambda Markers (size in kb) ------------------------------------------------------------------------------------------------------EcoRI Hind III Eco RI + HindIII 12.226 23.130 21.227 7.421 9.416 5.148 5.804 6.557 4.973 DNA/Protein Conversion 4.361 4.268 1 kb of DNA = 333 amino acids = 37 kDa of protein 5.643 4.878 2.322 3.530 3.530 2.027 2.027 Gel Electrophoresis 0.564 1.904 0.125 1.584 Recommended Agarose Gel Percentages for 1.375 Resolution of Linear DNA 0.974 ------------------------------------------------------------------------------------------------------0.831 Gel percentage DNA size range 0.564 ------------------------------------------------------------------------------------------------------0.125 0.5% 1,000–30,000 bp ------------------------------------------------------------------------------------------------------0.7% 800–12,000 bp 1.0% 500–1,0000 bp 1.2% 400–7,000 bp 1.5% 200–3,000 bp 2.0% 50–2,000 bp ------------------------------------------------------------------------------------------------------Recommended Polyacrylamide Gel Percentages for Resolution of Linear DNA ------------------------------------------------------------------------------------------------------Gel percentage Protein size range ------------------------------------------------------------------------------------------------------3.5% 100–1,000 pb 5.0% 75–500 pb 8.0% 50–400 pb 12.0% 35–250 pb 15.0% 20–150 pb 20.0% 5–100 pb ------------------------------------------------------------------------------------------------------Protein Molar Conversions 100 pmol of 100-kDa protein = 10 µg 100 pmol of 100-kDa protein = 5 µg 100 pmol of 100-kDa protein = 1 µg 239 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India pH AND BUFFER Before discussing the concept of pH, it is necessary to have an idea of acid and base. ACID AND BASE According to Bronsted concept, a proton donor is denoted as an acid, and a proton acceptor as a base, Acid proton + Conjugate base incomplete. The concentration of free H+ and OHdepends on the value of their dissociation constant: Weak acid, HCOOH Weak base, C6H5NH2+H+ H+ + HCOOC6H5NH3+ IONISATION OF WATER Water molecules have a tendency to undergo reversible ionization to yield a hydrogen ion and a hydroxyl ion H2O H+ + OH- Amino acid possess both acid and base function. When a crystalline amino acid is dissolved in The degree of ionization of water at equilibrium water, it exists in solution as the dipolar ion, or Zwitter is small. The equilibrium constant of reversible ionization ion, which can act either as an acid or a base. of water is given by H H [H+] {OH] R C COO R C COO + H K = ———————— NH NH [H2O] - - + + 3 3 H R C + NH 3 H COO - +H + R C The concentration of water for all purpose is constant, so we can write COO - NH 3 The carboxyl ion can also act as a base and accept a proton and is called a conjugate base. Similarly, protonated amino group can donate a proton and act as an acid and is called a conjugate acid. The charge molecule in a reaction is known as a conjugate acid or a base, while uncharged substance as an acid or a base. STRONG ACIDS OR BASES These compound are completely ionized in solution, so that the concentration of free H+ or OH- is the same as the concentration of acid or base Strong acid, HCl H+ + ClStrong base, NaOH Na+ + OHWEAK ACIDS OR BASES The dissociation of these compounds is Kw = [H+][OH-] At 25oC, the ionic product of water is 10-14. Since [H2O] is constant, Kw is also constant. Therefore, [H+][OH-] = 10-14 Whenever, concentration of H+ ions is greater than 1x 10-7, the concentration of OH- must become less than 1x10-7 and vice versa. In case of solution of hydrochloric acid, the concentration of H+ will be very high, the OH- concentration will be low. CONCEPT OF PH In 1909 Sorenson introduced the term pH as a convenient way of expressing hydrogen ions by means of logarithmic function and is defined as, the negative logarithm of hydrogen ion concentration. 240 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India 1 pH = log ——— = -log [H+] [H+] The corresponding term for hydroxyl is defined as pOH = -log [OH-] The equation for Kw can be written as -log Kw = pH + pOH = 14 The proton added combines instantly with CH3COO- present in the buffer mixture to form the undissociated weak acid. Consequently, resulting pH change is much less than would occur if the conjugate base is present. The buffering capacity is directly proportional to the concentration of the buffer component. The concentration of buffer reference is the sum of the Thus the sum oh pH and pOH is 14 and the concentration of the weak acid and conjugate base. two compounds are related reciprocally. Neutrality Henderson and Hasselbalch equation is important in prevails at pH = pOH = 7 various buffers and for determining the concentration of conjugate base and acid for the preparation of the BUFFERS buffers. Buffer solution is the one that resists changes in pH when small amounts of acids or base are added. References: A buffer solution consists of a weak acid and its 1. Lehninger, A.L., Nelson, L.D. and Cox, M.M. conjugate base. Principles of Biochemistry, Worth Publisher Inc. The quantitative relationship among pH, the N. Y. (1993). buffering action of a mixture of a weak acid with its 2. Stryer, L. Biochemistry, Freeman and Co. N. Y. conjugate base and the pK of the weak acid is given (1995) by the Henderson-Hasselbalch equation. 3. Plummer, D.T. An Introduction to Practical [A-] Biochemistry. Tata McGraw Hill Publishing PH = pK = log——— Company Ltd. (1987). [HA] When (A-) = [HA], then pH = pK and under PREPARATION OF BUFFERS such conditions the system has a maximum buffering action since addition or removal of protons is absorbed 1. Phosphate buffer (0.1 M) in the transition between A- and HA. For preparation of phosphate buffer use either On addition of alkali to a mixture of acetic acid (a) or (b) (a) A: 0.2 M solution of monobasic potassium and potassium acetate, following reaction occurs: phosphate [27.2 g KH2PO4 (Mol Wt 136) in OH = CH3COOH CH3COO + H2O 1000 ml water] B: 0.2M solution of dibasic potassium phosphate As OH- is added to the weak electrolyte acetic [34.8 g K2HPO4 (Mol Wt 174) in 1000 ml water] acid, which passes through a transition from acid to a (b) A: 0.2 M solution of monobasic potassium conjugate base or OH ion reacts with proton furnished phosphate [27.6 g NaH2PO4.H2O (Mol Wt 138) with dissociation of the weak acid and forms H2O and or 31.2 g NaH2PO4.2H2O (Mol Wt 156) in 1000 on further addition of alkali there is further dissociation ml water] of available CH3COOH to furnish additional protons B: 0.2M solution of dibasic potassium phosphate [35.6 g Na2HPO4.2H2O (Mol Wt 178) or 53.6 and thus keep the hydrogen concentration or pH unchanged. When acid is added the following reaction g Na2HPO4.7H2O (Mol Wt 268) or 71.6 g occurs. Na2HPO4.12H2O (Mol Wt358) in 1000 ml H+ + CH3COOHCH3COOH water] 241 Training Manual on “Post-Harvest Physiology of Fruits and flowers” 27th January to 16th February, 2009, IARI, New Delhi, India X ml of solution A + Y ml of solution B diluted to 200 ml X 92.5 92.4 90.0 87.7 85.0 81.5 77.5 73.5 68.5 62.5 56.5 51.0 45.0 39.0 33.0 28.0 23.0 19.0 16.0 13.0 10.5 8.5 7.0 5.3 Y 6.5 8.6 10.0 12.3 15.0 18.5 22.5 26.5 31.5 37.5 43.5 49.0 55.0 61.0 67.0 72.0 77.0 81.0 84.0 87.0 89.5 91.5 93.0 94.5 pH 5.7 5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 C2H3O2Na. 3H2O (Mol Wt 136) in 1000 ml water) X ml of solution A + Y ml of solution B diluted to 100 ml X Y pH 46.3 3.7 3.6 44.0 6.0 3.8 41.0 9.0 4.0 36.8 13.2 4.2 30.5 19.5 4.4 25.5 24.5 4.6 20.0 30.0 4.8 14.8 35.2 5.0 10.5 39.5 5.2 8.8 41.2 5.4 4.8 45.2 5.6 4. Glycine-HCl buffer (0.05M) A: 0.2 M solution of glycine [15.01 g (Mol wt 75) in 1000 ml water] B: 0.2 M HCl 50 ml of solution A + X ml of solution B diluted to 200 ml -----------------------------------------------------------------------------------X pH -----------------------------------------------------------------------------------5.0 3.6 6.4 3.4 8.2 3.2 11.2 3.0 2. Tris-HCl buffer (0.05 M) 16.8 2.8 A: 0.2 M Tris (hydroxyl methyl) amino methane 24.2 2.6 [Mol Wt 121 (24.2 g) in 1000 ml water] 32.4 2.4 B: 0.2 M HCl 44.0 2.2 50 ml of solution A + X ml of solution B diluted to 200 ml -----------------------------------------------------------------------------------X pH 5. Citrate buffer (0.05M) 5.0 9.0 A: 0.1 M solution of citric acid monohydrate [21.1 8.1 8.8 g (Mol Wt 210) in 1000 ml water] 12.2 8.6 B: 0.1 M solution of sodium citrate [29.41 g 16.5 8.4 Na .2H2O (Mol Wt 294) in 1000 ml], use of salt 21.9 8.2 3 with 5 H2O is not recommended. 26.8 8.0 X Y pH 32.5 7.8 38.4 7.6 46.5 3.5 3.0 41.5 7.4 43.7 6.3 3.2 44.2 7.2 40.0 10.0 3.4 37.0 13.0 3.6 3. Acetate buffer (0.1 M) 35.0 15.0 3.8 A: 0.2 M solution of glacial acetic acid [Mol Wt 60 33.0 17.0 4.0 (11.51 ml in 1000 ml water)] B: 0.2 M solution of sodium acetate [16.4 g of 31.5 18.5 4.2 C2H3O2 Na (Mol Wt 82) or 27.2 g of 28.0 22.0 4.4 242