ORCHID BIOTECHNOLOGY This page intentionally left blank ORCHID BIOTECHNOLOGY edited by Wen-Huei Chen National Inoversity of Kaohsiung, Taiwan Hong-Hwa Chen National Cheng Kung University, Taiwan World Scientific N E W J E R S E Y • L O N D O N • S I N G A P O R E • B E I J I N G • S H A N G H A I • H O N G K O N G • TA I P E I • C H E N N A I Published by World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE Library of Congress Cataloging-in-Publication Data Orchid biotechnology / editors, Hong-Hwa Chen, W.H. Chen. p. cm. Includes bibliographical references and index. ISBN-13: 978-981-270-619-5 (hardcover : alk. paper) ISBN-10: 981-270-619-4 (hardcover : alk. paper) 1. Orchids--Biotechnology. I. Chen, Hong-Hwa. II. Chen, W. H. (Wen Huei) QK495.O64O55 2007 635.9'344--dc22 2007016772 British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. Copyright © 2007 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher. For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher. Typeset by Stallion Press Email: enquiries@stallionpress.com Printed in Singapore. Foreword The Phalaenopsis is the national flower of Taiwan, first found and collected by the Japanese on Lanyu (Orchid Island) in 1897. After winning back-to-back championships in the International Orchid Exhibition in California in 1952 and 1953, Taiwan’s native Phalaenopsis has gained worldwide admiration and the pride of the Taiwanese people. With an optimal climate for growing the Phalaenopsis, Taiwan is well situated for commercialization of the flower, being in the northernmost area of Phalaenopsis’ natural germplasm. Today, the Phalaenopsis industry in Taiwan is well developed and has advanced to green house breeding and systematic production. Moreover, Taiwanese product varieties now account for more than 50% of the global Phalaenopsis market share. It is no surprise that the Phalaenopsis industry is viewed as an example of the most advanced knowledge-based agriculture in Taiwan. The future of the Phalaenopsis is exceptionally bright. On August 24, 2004, the New York Times reported that there is a $2 billion global market for orchids, with Phalaenopsis holding the leading share within that market. Phalaenopsis is also recognized as one of the most exciting and elegant indoor flowers by the American Orchid Society (AOS). And recently, Mr. Ed Matsui, owner of Matsui Nursery, the largest Phalaenopsis producer in the US, has estimated there will be a five-fold increase in the Phalaenopsis market within the next ten years. Furthermore, according to the December 2004 issue of FloraCulture International, the most influential floral magazine, the Phalaenopsis, a newly developing flower with 20% growth each year for the past five years, is the top seller among all pot flowers in The Netherlands and Japan. Moreover, with the advent of mass retail as a new distribution channel, demand has increased for mini-type, lowpriced product, and color variants, boosting flower sales in recent years. All these factors have led to the Phalaenopsis being selected as one of the top four most important export products for Taiwan by the Agriculture Product Competition Module (APCM), a group developed v vi ✦ Foreword by A-Turn Biotech Company, an advisor to the government for agriculture, to analyze and evaluate potential floral products for export. However, the value created by the Phalaenopsis is greater than that of just the plant itself; strategic alliances with other industries will provide the opportunity for extracting further value and greater margin from the flower. Phalaenopsis is recognized as a symbol of elegance amongst flowers. Properly managed, this rare property enables the attraction and development of many complementary products and industries such as gifts, arts, and home decoration. As Taiwan moves into the future, the establishment of acclimation and overseas sales points is one of the important steps in broadening the market of Phalaenopsis globally. Taiwan has a complete range of technologies from seedling acclimation to product vernalization. Through international strategic alliances enabling joint ventures and technology transfer, Taiwan will create higher profits through widening markets internationally for all parties involved. Currently, due to shipping costs and importation regulations, certain finished Phalaenopsis products are not able to reach the US markets. Thus, establishing a local acclimation facility for consumers is now the most cost effective way to market and distribute Phalaenopsis. Fortunately, the US government has recently begun accepting the importation of Phalaenopsis with moss as a supporting medium, which will provide a new international trading opportunity for Taiwan. There are many factors that contribute to Taiwan’s unique capability to take the greatest advantage of the Phalaenopsis phenomenon. As mentioned above, the subtropical climate makes Taiwan a near perfect environment for the production of Phalaenopsis. In addition, Taiwan is rich in orchid species, holding a worldwide leading position in new product development fueled by hundreds of professional and amateur breeders who have won gold medals in world competitions. Taiwan has maintained and will continue to maintain its competitive edge on research and development in the Phalaenopsis business. From a technical perspective, Taiwan’s Phalaenopsis industry has the strength to compete with any of the major floral countries. It is strongly supported by The National Science and Technology Program for Agricultural Biotechnology (NSTP.AB), a joint program of the National Science Council, Council of Agriculture and Academia Sinica. The NSTP.AB supports universities and research institutions in advancing the technology in genetic transforming, tissue culture, and production. Foreword ✦ vii Most of the chapters in this book have come about as a result of this National Project and I am honored to have served as the team leader since the initiation of the project in 1998. For many years, significant advances in the biotechnological research of Phalaenopsis have been made in the areas of thermo-tolerance, pathogen resistance, flowering control, flower color and virus diagnosis. This strength of research ability and experience should attract international cooperation on technological applications. Taiwan’s Phalaenopsis production system has evolved to a commercialized scale by through involvement of many breeders and companies. In addition, it has developed into a complete high tech system, i.e. from product selection, healthy seedling propagation, quarantine systems, and production automation, which form a well-set package in agriculture developments. As such, it is well situated for forming strategic alliances worldwide as a turn key project. In order to vertically integrate the orchid industry, the Council for Economic Planning and Development, has established and funded the Taiwan Orchid Plantation (TOP) in Tainan County in 2003. TOP was designed and developed by myself together with the management team of A-Turn Biotech. We believe that TOP will be the platform to strengthen the orchid industry in R&D, mass production, exhibition, trading and so forth, which will make Taiwan the largest orchid supplier in the world. In addition, the “TOP” name will provide a strong differentiating brand for Taiwan’s Phalaenopsis. Since there are many highly skilled breeders and technology developers on the island, TOP will also be acting as a platform for trading orchid varieties and technologies internationally. Taiwan has the capability to custom-make specific products for every specific market in the shortest possible period because of its rich breeding materials and skillful breeders. This will enhance its position as a global leader of Phalaenopsis suppliers. Technological development is the foundation of the industry; it takes tremendous time and investment to build up, but is absolutely essential. I am glad to see this book published and believe that the book will become the most useful and valuable technological resource for all orchid lovers worldwide. Dr. Irwin Y.E. Chu Founder A-Turn Biotech Co. This page intentionally left blank Preface The appreciation of orchid beauty has a long history in both western and eastern cultures. Over many years of development, the orchid has evolved such that it embraces not just the hobbyists’ market but a highly commercial market, thanks to advances in techniques such as breeding, micropropagation, industrial cultivation, etc. Today, orchid cut-flowers of Cymbidium, Dendrobium and Oncidium, and potted plants of Phalaenopsis are marketed globally. It is envisaged that growing tropical orchids for cut-flower production and potted plants will benefit from the recent advances in the crop science technology. However, for the orchid industry, producing an improved orchid through biotechnology is only the beginning. Taiwan has been the main driving force of the world’s Phalaenopsis breeding and plant production. The orchid research program was firstly supported 10 years ago by the Taiwan Sugar Corporation for the first three years, and currently has been one of the National Science-Tech Program for Agriculture Biotechnology (NSTP.AB) for more than six years. The budgets of the NSTP.AB are founded by National Science Council, Council of Agriculture and Academic Sinica, Taiwan. The contributors to the book include researchers from the Institute of Plant and Microbial Biology, Academia Sinica, National Taiwan University, National Tsing Hua University, National Cheng Kung University, National University of Kaohsiung, and National Pingtung University of Science & Technology. We collaborate with the growers of Taiwan Orchid Plantation, a government sponsored entity, in terms of research and training, in order to bring the Taiwan orchid industry to a new level of sophistication and profitability. This book is the first volume devoted exclusively to orchid biotechnology. It is extremely informative as it addresses many aspects of orchid biotechnology, including modern breeding (Chapters 1 and 2), in vitro morphogenesis (Chapter 3), somaclonal variation (Chapter 4), application of orchid mycorrhized fungi (Chapter 5), analysis of orchid genomes (Chapters 6–8) and functional genomics (Chapters 9–12), and ix x ✦ Preface genetic transformation (Chapter 13). It will be a valuable guide for readers such as research workers, graduate students, people interested in orchid biology and floriculturists. Its publication will be a milestone sets the foundation for the next level of orchid research. Wen-Huei Chen and Hong-Hwa Chen The Editors Contents v Foreword ix Preface xiii List of Contributors Chapter 1 Breeding and Development of New Varieties in Phalaenopsis Ching-Yan Tang and Wen-Huei Chen Chapter 2 Embryo Development of Orchids Yung-I Lee, Edward C Yeung and Mei-Chu Chung 23 Chapter 3 In vitro Morphogenesis and Micro-Propagation of Orchids Wei-Chin Chang 45 Chapter 4 Somaclonal Variation in Orchids Fure-Chyi Chen and Wen-Huei Chen 65 Chapter 5 The Screening of Orchid Mycorrhizal Fungi (OMF) and their Applications Doris C. N. Chang 77 Chapter 6 Analysis of the Orchid Genome Size Using Flow Cytometry Tsai-Yun Lin and Hsiao-Ching Lee 99 xi 1 xii ✦ Contents Chapter 7 The Cytogenetics of Phalaenopsis Orchids Yen-Yu Kao, Chih-Chung Lin, Chien-Hao Huang and Yi-Hsueh Li 115 Chapter 8 Analysis of the Chloroplast Genome of Phalaenopsis aphrodite Ching-Chun Chang, Hsien-Chia Lin and Wun-Hong Zeng 129 Chapter 9 Analysis of Expression of Phalaenopsis Floral ESTs Wen-Chieh Tsai, Yu-Yun Hsiao, Zhao-Jun Pan and Hong-Hwa Chen 145 Chapter 10 Orchid MADS-Box Genes Controlling Floral Morphogenesis Wen-Chieh Tsai, Chin-Wei Lin, Chang-Sheng Kuoh and Hong-Hwa Chen 163 Chapter 11 Pseudobulb-Specific Gene Expression of Oncidium Orchid at the Stage of Inflorescence Initiation Jun Tan, Heng-Long Wang and Kai-Wun Yeh 185 Chapter 12 Application of Virus-induced Gene Silencing Technology in Gene Functional Validation of Orchids Hsiang-Chia Lu, Hong-Hwa Chen and Hsin-Hung Yeh 211 Chapter 13 Genetic Transformation as a Tool for Improvement of Orchids Sanjaya and Ming-Tsair Chan 225 Index 255 List of Contributors Ming-Tsair Chan Agricultural Biotechnology Research Center Academia Sinica Taipei Taiwan Ching-Chun Chang Institute of Biotechnology National Cheng Kung University Tainan Taiwan Doris C. N. Chang Department of Horticulture National Taiwan University Taipei Taiwan Wei-Chin Chang Institute of Plant and Microbial Biology Academia Sinica Taipei Taiwan Fure-Chyi Chen Department of Plant Industry and Graduate Institute of Biotechnology National Pingtung University of Science & Technology Pingtung Taiwan xiii xiv ✦ List of Contributors Hong-Hwa Chen Department of Life Sciences National Cheng Kung University Tainan Taiwan Wen-Huei Chen Department of Life Sciences National University of Kaohsiung Kaohsiung Taiwan Mei-Chu Chung Institute of Plant and Microbial Biology Academia Sinica 115, Taipei Taiwan, ROC Yu-Yun Hsiao Department of Life Sciences National Cheng Kung University Tainan Taiwan Chien-Hao Huang Department of Botany National Taiwan University Taipei Taiwan Yen-Yu Kao Department of Botany National Taiwan University Taipei Taiwan Institute of Molecular and Cellular Biology National Taiwan University Taipei Taiwan List of Contributors ✦ xv Chang-Sheng Kuoh Department of Life Sciences National Cheng Kung University Tainan Taiwan Hsiao-Ching Lee Institute of Bioinformatics and Structural Biology and Department of Life Science National Tsing Hua University Hsinchu Taiwan Yung-I Lee Institute of Plant and Microbial Biology Academia Sinica 115, Taipei Taiwan, ROC Botany Department National Museum of Natural Science Taichung Taiwan Yi-Hsueh Li Department of Botany National Taiwan University Taipei Taiwan Chih-Chung Lin Department of Botany National Taiwan University Taipei Taiwan Chin-Wei Lin Department of Life Sciences National Cheng Kung University Tainan Taiwan xvi ✦ List of Contributors Hsien-Chia Lin Institute of Biotechnology National Cheng Kung University Tainan Taiwan Tsai-Yun Lin Institute of Bioinformatics and Structural Biology and Department of Life Science National Tsing Hua University Hsinchu Taiwan Hsiang-Chia Lu Department of Plant Pathology and Microbiology National Taiwan University Taipei Taiwan Zhao-Jun Pan Department of Life Sciences National Cheng Kung University Tainan Taiwan Sanjaya Agricultural Biotechnology Research Center Academia Sinica Taipei Taiwan Jun Tan College of Bioinformation Chongqing University of Post and Telecom Chongqing China List of Contributors ✦ xvii Ching-Yan Tang Department of Life Sciences National University of Kaohsiung Kaohsiung Taiwan Wen-Chieh Tsai Department of Biological Science and Technology Chung Hwa University of Medical Technology Tainan County Taiwan Heng-Long Wang Department of Life Science National Kaohsiung University Kaohsiung Taiwan Edward C. Yeung Department of Biological Sciences University of Calgary Calgary Alberta T2N 1N4 Canada Hsin-Hung Yeh Department of Plant Pathology and Microbiology National Taiwan University Taipei Taiwan Kai-Wun Yeh Institute of Plant Biology College of Life Science National Taiwan University Taipei Taiwan xviii ✦ List of Contributors Wun-Hong Zeng Institute of Biotechnology National Cheng Kung University Tainan Taiwan Chapter 1 Breeding and Development of New Varieties in Phalaenopsis Ching-Yan Tang† and Wen-Huei Chen*,† One of the most important strategies to keep Taiwan as the leading producer of Phalaenopsis in the world, is breeding and development of new varieties. Pedigree analysis of the 12 most popular white hybrids of Phalaenopsis indicated that the tetraploids of Phal. amabilis and the hybrid, Phal. Doris were used frequently as parents of these hybrids. Besides the standard big flower Phalaenopsis, development of novelty varieties, such as the Harlequins and the multi-floral types constitute the new trends in the Phalaenopsis breeding programs and markets in the last decade. The somaclonal mutants of Phal. Golden Peoker and the wild species, Phal. equestris played an important role in the development of these novelty varieties. Breeding for new varieties of Phalaenopsis is lengthy and time consuming. New techniques are needed to increase the breeding efficiency of crops having long life cycles. The recent development of molecular markers, such as restricted fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and DNA amplification fingerprinting (DAF) and their applications in Phalaenopsis breeding are discussed and evaluated in this chapter. 1.1 Introduction The 1980s was the decade that has divided the orchid business of Taiwan into two distinct phases. Before 1980, the cultivation of orchid *Corresponding author. † Department of Life Sciences, National University of Kaohsiung, Kaohsiung, Taiwan. 1 2 ✦ C.-Y. Tang and W.-H. Chen was considered a hobby. Most hobbyists were raising orchids in small scale with simple green-house facilities. With the rapid growth of economy, Phalaenopsis orchids became one of the most important commodity in the domestic as well as the international markets. Since 1988, Taiwan Sugar Corporation has started a comprehensive program to modernize Phalaenopsis production through intensive research effort, while more modern and well-equipped greenhouses1 were built to meet the demands of an expanding market which could not be met through the activities of orchid hobbyists. Moreover, Phalaenopsis breeding became more professional and was usually well designed as compared with the trial and error approach of the traditional breeding programs. Another change during this period was the product type in the export markets. Instead of cut flowers, the medium- and large-sized seedlings of selected hybrids became the major items for export. Consequently, Phalaenopsis growers in Taiwan had to equip themselves with modern greenhouses as well as facilities for mass production of hybrid seedlings which are normally derived from crosses of two high quality parental varieties. Seeds from the mature capsules are sown by in vitro method. Young seedlings developed in the test tube are transplanted into pots which are divided into four groups: small-, medium-, and large-sized seedlings and flowering plants, according to market demands.2 Progeny test is used to evaluate and select the potential hybrids at different stages of development. To save time, parental plants used for hybridization to produce hybrids are propagated by the mericlone method, while evaluation of the new hybrids is in progress. Therefore, at the final stage of selection of the hybrids, there will be enough parental stocks available for making crosses to produce a large amount of hybrid seedlings for the market.3 The Phalaenopsis varieties used for breeding are usually divided into two groups — the standard big flower group and the novelty group. The standard big flower group includes the white, pink as well as the varieties with stripes, being derivatives of the white Phal. amabilis and the pink Phal. schilleriana. The varieties of the novelty group are usually small flowers with special coloration; some have special fragrances, i.e. if Phal. violacea is involved in the pedigree. Other parental varieties in this group are Phal. amboinensis, Phal. venosa, etc. In recent years, Breeding and Development of New Varieties in Phalaenopsis ✦ 3 the pot varieties which have small but plentiful flowers have become a new market trend. Phal. equestris and Phal. stuartiana are the common parental varieties of this group. In general, the breeding programs are designed to improve the size and color of the flowers as well as other characteristics such as, longevity, stalk length, leaf shape, ease of cultivation, disease resistance and the number of viable seeds through the selection of parents for hybridization and so on. Through tremendous efforts in breeding, various types of Phalaenopsis varieties with attractive color and graceful appearance (Fig. 1.1) have been developed and the success of the development has made Taiwan one of the most important producers of Phalaenopsis in the world. The growing cycles of Phalaenopsis orchids are long, a cycle being 2–3 years. Using the traditional hybridization to transmit useful traits into the commercial varieties is a long process which takes years to achieve. In addition, some species of orchids are cross-incompatible, thereby limiting the work of variety improvement. Hence, new approaches and techniques are needed in order to produce superior Phalaenopsis varieties for the fast growing and highly competitive markets. This chapter discusses the recent developments in the breeding work of Phalaenopsis. Fig. 1.1. Phalaenopsis varieties showing various attractive colors and graceful appearance. 4 ✦ C.-Y. Tang and W.-H. Chen 1.2 Development of Phalaenopsis Varieties by Hybridization 1.2.1 White Phalaenopsis varieties The standard big white flower is the most important group of Phalaenopsis in the market (Fig. 1.2). Besides the large size, the breeding objectives of the white Phalaenopsis include long flower stalk, wellshaped flowers with long life span, etc. Taiwan is located at the northern border of the natural growth habitat of Phalaenopsis. A white flowered species, Phal. amabilis var. formosa was found native in Heng-Chung Peninsula, Taitung County and the Orchid Island off the coast of southern Taiwan.4 This native species had won several awards in different international orchid conferences as early as in the 1950s for the beauty of their multi-flowers. By using the technique of polyploidization, superior tetraploid varieties with short flower stalk, round-shaped petals and good quality flowers were developed. These varieties were well accepted by the Japanese market. The modern superior large white hybrids were developed through the hybridization of breeding stocks from different sources, including those from the local Phalaenopsis farms and many from foreign countries, such as Japan, the Netherlands and the United States. Based on these materials, large, well-shaped white-flowered Phalaenopsis Fig. 1.2. The appearance the standard big white variety “Phal. Taisuco Brinasu.” Breeding and Development of New Varieties in Phalaenopsis ✦ 5 hybrids with uniform morphology were developed. Through analysis of the pedigree of the 12 most popular white Taisuco Phalaenopsis hybrids in 1997/98, it was found that all of them were the offspring of Phal. amabilis, Phal. rimestadiana, Phal. aphrodite, Phal. schilleriana, Phal. stuartiana and Phal. sanderiana with the exception of the Phal. Taisuco white which was not related to Phal. stuartiana and Phal. sanderiana (Table 1.1). Among these wild species, Phal. amabilis, Phal. rimestadiana and Phal. aphrodite were the most important ancestors for the modern white commercial hybrids.5 The proportion of the genetic constitution contributed by Phal. amabilis, Phal. rimestadiana and Phal. aphrodite were 40.34%, 38.56%, and 16.41%, respectively. Based on this information, one can note that these hybrids were closely related in their genetic make-up. This narrow genetic background in Phalaenopsis white hybrids was difficult to avoid due to the demand for high uniformity of the hybrid seedlings by the market. That means genetic homogeneity of the parental stocks was required in order to produce uniform hybrid seedlings. However, one has to be aware that genetic depression may occur during the process of improvement. From the same analysis, it was found that these 12 Taisuco hybrids were originated from 17 ancestral hybrids (Table 1.2). However, the Table 1.1. Genetic Contribution of the Wild Species for the 12 Most Popular Commercial Hybridsa of the White Taisuco Phalaenopsis Wild Species Used in the Pedigree Phal. Phal. Phal. Phal. Phal. Phal. a amabilis rimestadiana aphrodite schilleriana stuartiana sanderianac Percentage of Genetic Contribution Mean of Percentage C.V.b 39.11–42.19 37.64–39.22 15.36–17.74 2.64–4.49 0–1.17 0–0.78 40.34 38.56 16.41 3.48 0.51 0.47 2.8 1.2 4.3 19.8 70.6 51.1 The name of the 12 hybrids are: Phal. Taisuco Kochdian, Phal. Taisuco Kaaladian, Phal. Taisuco Windian, Phal. Taisuco Bright, Phal. Taisuco Bridian, Phal. Taisuco Adian, Phal. Taisuco White, Phal. Taisuco Brinasu, Phal. Taisuco Silver, Phal. Taisuco Crane, Phal. Taisuco Swan, Phal. Taisuco Nasubula. b C.V. = coefficient of variation. c The commercial hybrid, Phal. Taisuco White, does not have the genetic contribution of Phal. stuartiana and Phal. sanderiana. 6 ✦ C.-Y. Tang and W.-H. Chen Table 1.2. Genetic Contribution of the Important Parental Hybrids in the Pedigree of the 12 Most Popular Commercial Hybridsa of the White Taisuco Phalaenopsis Name of Parental Hybrid Genetic Contribution (%) No. of Commercial Hybrids Group A Phal. Elisabethae Phal. Gilles Gratiot Phal. Katherine Siegwart 40.23 30.69 31.56 12 12 12 Group B Phal. Doris Phal. Doreen Phal. La Canada Phal. Winged Victory 50.47 17.36 12.18 19.37 12 9 9 12 Group C Phal. Grace Palm Phal. Thomas Tucker 23.57 12.26 12 9 Group D Phal. Elinor Shaffer Phal. Long Life Phal. Opaline Phal. Vallehigh 18.75 14.84 14.84 22.27 10 8 8 8 Group E Phal. Kochs Schneestern Phal. Meridian Phal. Mount Kaala Phal. Schone Von Unna 27.78 27.78 26.70 14.84 9 9 11 8 a The names of the 12 hybrids are the same as in Table 1.1. average genetic contribution for Phal. Doris to the current large white Phalaenopsis hybrids was about 50.47%, which was equally important to the direct parental hybrids. Through the genetic flow from the ancestors to the modern white Taisuco hybrids, it is observed that the superior clones of the large white Taisuco Phalaenopsis were developed firstly through the improvement of the genetic characters for Phal. Doris by chromosome doubling, resulting in a tetraploid with a larger genomic capacity to accumulate more additive alleles. Then it was Breeding and Development of New Varieties in Phalaenopsis ✦ 7 followed by backcrossing and hybridizing with its relatives to recombine and to accumulate desirable additive alleles for the flower size and other favorable traits. By this selection scheme, more than 30 Taisuco Phalaenopsis white hybrids were obtained and they won many awards throughout the world, including eight from the American Orchid Society. 1.2.2 Harlequin (novelty) varieties Development of the Harlequin varieties is a new trend in Phalaenopsis breeding which was developed in Taiwan in the last 12 years. The most distinguished characteristic of this new group of Phalaenopsis is the appearance of large blotches of coalesced spots with intense color against the light creamy white or other colors. The blotching appears to be unstable. It may vary in size, shape and location from flower to flower. It was also found that temperature may influence the expression of the blotches.6 With cooler temperatures, the color intensity of the Harlequin spot will increase. The breeding of the Harlequins began in Taiwan when the famous hybrid Phal. Golden Peoker “Brother” (Phal. Misty Green × Phal. Liu Tuen Shen, Reg. Brothers’s Orchid, 1983) was mericloned in the 1990s. The special feature of this variety is its creamy white flower with intense wine-colored spots. From the pedigree analysis, Phal. Golden Peoker was developed from 12 wild species through 11 generations.7 The genetic contribution in generating wine-colored spots is 25, 18.75, 12.5 and 6.25% from Phal. gigantea, Phal. leuddemanniana, Phal. amboinensis and Phal. faciata, respectively (Table 1.3). Besides the contribution of nicely spotted flowers in the particular group, these species also have a tendency to add other characters, such as leather-like texture, and flattened and round appearance of the flowers of the Harlequins. In the process of mericloning the Phal. Golden Peoker “Brother,” somaclonal mutants with different, remarkably, fused spots of Harlequin pattern on the sepals and petals have been found in different orchid farms in Taiwan. Among these mutants, three of them, namely “Ever-spring,” “Nan Cho” and “S.J.” were most famous and received AOS recognition and awards. The mericlones of these mutants also produced flowers that were dominated with this Harlequin pattern (Fig. 1.3). From the emergence of these clones, intensive breeding for the Harlequins began. They were used to hybridize high quality 8 ✦ C.-Y. Tang and W.-H. Chen Table 1.3. Peoker Genetic Contribution of the Wild Species to Phal. Golden No. of Times of Each Species Introduced into Each Generation Wild Species 1 2 P. gigantean P. luddemanniana P. rimestadiana P. amboinensis P. amabilis P. aphrodite P. faciata P. sumarana P. schilleriana P. stuartiana P. equestris P. sanderiana 3 4 1 1 5 6 7 8 9 10 11 1 4 19 18 9 2 3 1 13 12 9 13 4 5 2 1 1 1 2 1 1 2 1 1 % of Genetic Contribution 25.00 18.75 15.04 12.50 10.16 7.42 6.25 1.56 1.27 1.07 0.78 0.20 Data from RHS98 (1998). Fig. 1.3. Coalescence of red-brownish blotches on the flowers is the characteristic of Harlequin Phalaenopsis (Phal. Golden Peoker “A87–100”). parental varieties possessing different flower colors and to create novel cultivars of Harlequin flowers. Phal. Golden Peoker is an excellent parent. From 1992 to 2003, 423 hybrids developed from Phal. Golden Peoker were registered in Sander’s Breeding and Development of New Varieties in Phalaenopsis ✦ 9 list of orchid hybrids. Among them, 149 were the Harlequins. In addition to Phal. Golden Peoker, there were six important related varieties which were widely used in the breeding programs to create novel cultivars of Harlequins (Table 1.4). From 1994 to 2002, 58 Harlequin flowers won awards from the AOS. Among these varieties, Phal. Ever Spring Fairy “Tokai Silky Star” and Dtps. Chain Xen Diamond “Celebration” were so commanding that they received 90-points and won the much sought after FCC/AOS. Another variety, “Dtps. Ever Spring Prince” received seven AOS awards in 2001 and 2002, including two AMs and five HCCs. Good progress has been made to breed for Harlequin varieties since the development of Phal. Golden Peoker “ES,” a somaclonal variant. Due to the fascinating and unpredictable pattern of the Harlequin flowers, there remains a lot of room for the improvement of this group of novelty variety in the future. Table 1.4. Number of Registered Hybrids Derived from Harlequin Parents in the Breeding Programa Harlequin Variety Phal. Golden Peokerb Phal. Ever-spring King Phal. Ever-spring Light Phal. Ever-spring Prince Phal. Ching Her Prince Phal. Haur Jin Diamond Phal. Ho’s Fantastic Splash a Parent Phal. Misty Green × Phal. Liu Tuen-Shen Phal. Chih Shang’s Stripes × Phal. Golden Peoker Phal. Ever-spring Star × Phal. Golden Peoker Phal. Golden Peoker × Dtps. Taisuco Beauty Phal. Ever-spring King × Phal. Golden Peoker Phal. Golden Peoker × Phal. Ching her Buddha Phal. Ever-spring King × Phal. Ho’s French Fantasia No. of Hybrids Generation Registered 0 96 1 37 1 9 1 8 2 6 1 5 2 5 Data from Wildcatt Orchids Database (2003) and RHS98 (1998). No. of crosses including all clones of Phal. Golden Peoker which have “Brother”, “Everspring”, “Nan-Cho”, “S.J.” and “BL”, etc. b 10 ✦ C.-Y. Tang and W.-H. Chen 1.2.3 Potted varieties Before the 1980s, cut-flowers dominated the Phalaenopsis market. However, demands for potted Phalaenopsis varieties have increased tremendously in the last decade. Breeding for the potted-plant market is different from breeding for the cut-flower market. While the cutflower market emphasized floral traits, for the potted-plant market, vegetative traits are equally important. These traits include small plants with considerable number of flowers, shortened and multiple branching of the inflorescence, easy growing and flowering, etc. At the beginning, potted varieties available for the market were usually smaller version of the standard Phalaenopsis. Selections were made for more compact growth and flowering. As the demand of potted varieties increased, special effort was made to develop hybrids for this sector of the market. For this purpose, Phal. equestris is being used as the most important parent in producing hybrids for potted varieties.8 Phal. equestris is a native species in the Philippines and is one of the two indigenous Phalaenopsis species in Taiwan. Bearing either white or pink flowers are two common forms of Phal. equestris. Sequential flowering having flowers all around the inflorescence and short flower stalks are special characteristics of this species. In addition to Phal. equestris, Phal. stuartiana and Phal. schilleraiana are also important species used for breeding of potted varieties having heavily branching flowers. The first important hybrid in this line of breeding is Phal. Cassandra (Phal. equestris × Phal. stuartiana). Though it was made by Seden and was registered by Veitch in 1899,9 it was not heavily used in Phalaenopsis breeding until the 1960s. Numerous hybrids with multibranching which is expected as a major characteristic of multi-floral varieties, were developed by using Phal. Cassandra as one of the parents in their pedigree. More than 150 first generation hybrids were registered using Cassandra as one of the parents. Recently, Cassandra hybrid was remade by crossing the tetraploid form of Phal. equestris “Riverbend” and Phal. stuartiana. The resulting hybrid was a triploid hybrid which was sterile. The tetraploid form of the other parental varieties is needed in order to make use of the advantages of tetraploid breeding. Breeding for potted Phalaenopsis varieties is a new trend in the markets. One can expect to see more and better varieties in the near future. Breeding and Development of New Varieties in Phalaenopsis ✦ 11 1.3 Breeding Behavior and Inheritance 1.3.1 Inheritance of floral color Taiwan is one of the native habitats of Phal. amabilis and Phal. equestris which are used extensively in the development of Phalaenopsis hybrids.10 Phal. equestris which appeared naturally with pink or white flowers, produces branched inflorescences with a short juvenile period, and is naturally dwarf. It is an important parent for breeding the miniature type of plants which produce a large number of small flowers and are much easier to pack and to transport. It was also used to produce hybrids that had white petals and sepals with a red lip (semi-alba). Plants of Phal. equestris are highly variable in terms of morphology as well as in floral color. It can be divided into the following forms11: (1) Phal. equestris var. alba — a pure white form; no yellow pigments on the callus. (2) Phal. equestris var. aurea — white flowers with solid yellow lip. (3) Phal. equestris var. leucotante — flowers with white lips and yellow callus. (4) Phal. equestris var. rosea — flowers with even red petals and sepals; color of the mid-lobe of the lip varies from deep red to light red. (5) Phal. equestris var. leucaspis — small flowers with white edges on pink petals and sepals; mid-lobe of the lip is purple or orange in color with white or yellow callus. It was reported that several independent genes control the colors of the lip of Phal. equestris through the expression of both anthocyanins and carotenoids.12 By crossing between the white and red forms of Phal. equestris, Fu et al.11 reported that the pink floral color was controlled by a single dominant gene. This gene acts on the coloration of petals, sepals, the mid-lobe and the apex area of the side-lobe of the lip. Also, it is expressed as a brownish color through a pleiotrophic effect on the coloration of the floral stalk and the spot of the callus. Compared with the petals and sepals, the color inheritance of the lip is more complicated. Two dominant duplicated genes which are independent of the above mentioned red gene, control the yellow color of the base of the mid-lobe and side-lobe as well as the callus of the lip. 12 ✦ C.-Y. Tang and W.-H. Chen Since Phal. equestris is frequently used to cross with other hybrids, it has been found that the expression of these color-genes varies according to the different genetic backgrounds. For example, when a pink-flowered Phal. equestris was used to cross with various commercial hybrids of pink, yellow with magenta spots or semi-alba floral colors, the colors of the flower of the progenies were pink, orange with pink blush, lavender or white with pink splash, respectively. Retention of the pink color in the flowers of the progenies from crosses with different genetic backgrounds suggested that the inheritance of pink floral colors of Phal. equestris might be controlled by a dominant gene13 which was the same red gene as in the previous study. However, when the same Phal. equestris was used to cross with commercial hybrids of the white, orange or yellow varieties, flowers with pink lips and various degrees of pink blush were observed in the progenies. These results suggested the presence of two complementary genes, C and R which controlled the pink color in the flowers of Phal. equestris, similar to those in Cattleya.14 1.3.2 Influence of Phalaenopsis equestris parents on fertility To study the relationship between fertility and pollen or pod parents used in the crosses, four varieties (including two white and two pink forms) of Phal. equestris were used to cross with the commercial hybrids. Each form was used as either pollen or pod parents and vice versa for the commercial hybrids. A total of 147 crosses were made and the fertility of each cross was determined by measuring the viable seeds produced from each cross. The results showed that 50–57% of the crosses (Table 1.5) produced viable seeds if the white or pink forms of Phal. equestris were used as pollen parents to cross with the commercial hybrids. However, no viable seed was produced if Phal. equestris was used as pod parents, regardless of the floral color. The varieties of Phal. equestris used in this study were diploids while the majority of the other parents were tetraploid hybrids. That means failure of seed production was found when the tetraploid plants were used as pollen parents to cross with the diploid varieties. Therefore, in order to enhance the breeding efficiency, a breeder has to use the diploid varieties as pollen parents if the counterparts are tetraploid plants. Breeding and Development of New Varieties in Phalaenopsis ✦ 13 Table 1.5. Effect of Phalaenopsis equestris Parents on the Fertility Floral Color of Phal. equestris White White Pink Pink Fertility > 0a Phal. equestris Used as Pod or Pollen Parents Total No. of Crosses No. of Crosses % of Crosses Pollen parent Pod parent Pollen parent Pod parent 36 10 91 10 18 0 52 0 50 0 57 0 a The crosses with viable seeds were considered as fertility > 0. 1.4 Application of Molecular Markers in Phalaenopsis Breeding 1.4.1 Screening for red floral gene by RAPD markers A single dominant gene was found to control the red floral color (as against white color) in Phal. equestris.11 Due to the long life cycle of Phalaenopsis orchids, it is a time consuming procedure to identify the progenies carrying this gene after hybridization. Therefore, a rapid technique is needed for early detection of the presence of this gene in order to increase the efficiency of the breeding procedure. By using a stepwise screening method, Chen et al.15 identified a RAPD (random amplified polymorphic DNA) marker linked to the red floral gene in Phal. equestris. In this experiment, the leaf tissue of plants from the white and red parents, F1 and F2 progenies were subjected to RAPD analysis. In the first step, 920 primers were screened by RAPD analysis using the leaf-tissue from the white and red parents and a single F1 plant. One hundred and fifty (16.3%) of them were found to produce distinct DNA polymorphic bands in the red floral parent and F1 progeny. These were absent in the white floral parent (Table 1.6). In the second step, three F1 plants were used for the detection of polymorphic and homozygous bands that could distinguish the red floral parent and F1 progenies from the white floral parent. Homozygous trait could be confirmed if polymorphic bands were present in all three F1 plants. Among the 150 primers selected from the first step, 34 showed distinct polymorphic and homozygous DNA bands that could distinguish the red 14 ✦ C.-Y. Tang and W.-H. Chen Table 1.6. Numbers and Probabilities of Polymorphic Primers Detected by RAPD Analysis and PCR Reactions Required in the F1 and F2 Progenies of Phalaenopsis equestris from 920 Primers F1 Plants F2 Plants First Screening Second Screening Third Screening No. of polymorphic primers Probability of polymorphism (%) 150 16.3 34 3.7 1 0.1 No. of PCR reactions 2,760 750 3,604 flowered parent and the three F1 progenies from the white flowered parent. The third screening was made by using 34 primers for 106 individual plants (84 from red, 22 from white) randomly selected from the F2 population. The results showed that the primer “OPQ-10” (5′-TGTGCC-CGA-A-3′) generated a 380 bp DNA band (OPQ 10–380) that was linked to the red floral gene. Chi-square analysis indicated that the OPQ 10–380 marker and the red flower gene were two closely linked genes with a distance of 30.8 centiMorgan (cM) apart. With the use of this OPQ 10–380 marker, one can identify the presence of the redflower gene in the progenies of hybridization at any stage of the plant development. In association with the in situ hybridization technique, the molecular marker may potentially be used for the identification and gene mapping of the chromosome where the red flower gene is located. 1.4.2 Investigation of the parental and phylogenetic relationship by RFLP markers in chloroplast DNA Traditionally, morphological characteristics and cytological analysis were used for the classification of plant species as well as for the phylogenetic study. Recently, due to the fast development of biotechnology at the molecular level, molecular markers using restriction fragment length polymorphism (RELP) or random amplified polymorphic DNA (RAPD) were commonly used for various areas of plant sciences.16,17 Because of the specificity, consistency and precision of the performance of these molecular markers, these techniques became widely used to study the phylogenetic relationship of plant species or the parental relationship in the plant breeding programs. Breeding and Development of New Varieties in Phalaenopsis ✦ 15 In one of the studies, RFLP was used to analyze the mode of inheritance of chloroplasts in both interspecific hybrids of Phalaenopsis (Phal. amabilis × Phal. amboinensis; Phal. mannii × Phal. stuartiana) and intergeneric hybrids of Phalaenopsis equestris and Doritis pulcherrima.18 Chloroplast DNA digested with Dra I followed by hybridization with an rbcL probe revealed that Phal. amabilis, Phal. aphrodite and Phal. stuartiana had the same size 2.0-kb fragment while the Phal. mannii and Phal. amboinensis had a 2.3-kb fragment. The size of the fragment in Doritis pulcherrima was 3.5-kb. In the analysis of the interspecific reciprocal crosses between two Phalenopsis species or the intergeneric reciprocal crosses between Phal. equestris and Doritis pulcherrima, similar results were found, i.e. the sizes of the fragments shown in the F1 progenies were the same as that in the maternal parents (Table 1.7). Therefore, maternal inheritance of the cpDNA as revealed by the RFLP markers was clearly demonstrated in the reciprocal crosses between the interspecific hybrids and intergeneric hybrids. These results suggested that cpDNA can be used as a marker for the identification of the parentage and for phylogenetic studies of taxonomy. 1.4.3 Use of RAPD markers for phylogenetic study and variety identification By using the morphological characteristics of the petal and sepal, Sweet19 classified Phalaenopsis orchids into 45 species and 9 sections. All the species of Phalaenopsis have the same chromosome number (2n = 38) with chromosome sizes ranging from 1.5 to 3.5 µ m.20 They can be divided into large, medium and small chromosome groups according to their chromosome size.21 By using flow-cytometry, Lin et al.22 Table 1.7. Polymorphism as Shown by the RFLP Markers in the Parental Lines and the F1 Progenies of the Interspecific Reciprocal Crosses of Phalaenopsis Parents Fragment Size (kb) 2.3 2.0 a F1 Progenies Aa B A×B B×A +b − − + + − − + A and B represent Phal. amboinensis and Phal. amabilis, respectively. + and – represent presence or absence of polymorphism as shown by RFLP markers. b 16 ✦ C.-Y. Tang and W.-H. Chen studied the nuclear DNA content of 18 species of Phalaenopsis. The quantities of the nuclear DNA content ranged from 2.74–16.61 pg/2c. They were classified into eight groups according to the nuclear DNA content. This information is useful in terms of orchid classification as well as the phylogenetic relationship among species. In addition to the various approaches mentioned, RAPD analysis was also used for these purposes. Fu et al.23 studied the relationship of 16 wild species of Phalaenopsis using RAPD markers. They found that the similarity coefficient and the relative order were stabilized when 20 primers were used to generate 381 DNA bands for analysis. By using the results of this analysis, 16 wild species of Phalaenopsis could be classified into five groups (Table 1.8) according to the similarity Table 1.8. Comparison of the Classifications of 16 Wild Species of Phalaenopsis According to the Dendrogram Generated by RAPD,23 Morphological Characteristics19 and Chromosome Sizes20 Species Phal. Phal. Phal. Phal. Phal. Phal. Phal. Phal. Phal. Phal. Phal. Phal. Phal. Phal. Phal. Phal. a Group According to RAPD Dataa Section According to Sweet, 1980 Group According to Chromosome Size E E D D D D C C C B B A A A A A Amboinensis Phalaenopsis Polychilos Zebrinae Zebrinae Zebrinae Zebrinae Amboinensis Zebrinae Amboinensis Amboinensis Phalaenopsis Phalaenopsis Phalaenopsis Phalaenopsis Stauroglottis NAb NA Large Medium Small NA NA Medium Large Medium Medium Small Small Small Small Small micholitzii intermedia mannii lueddemanniana mariae pulchra sumatrana venosa violacea gigantea amboinensis schilleriana stuartiana amabilis aphrodite equestris Grouping according to Fu et al.23 NA = not available. b Breeding and Development of New Varieties in Phalaenopsis ✦ 17 coefficient and the relative order as shown by the dendrogram. The authors claimed that 11 out of 16 species studied were matched between the grouping methods based on morphological characteristics and use of molecular markers. Furthermore, Phal. amabilis and Phal. equestris were the most closely related species according to the RAPD data, but they were classified into two far-related sections based on morphology. Similarly, Phal. mannii and Phal. lueddemanniana were considered to be closely related according to the RAPD data which was different from the traditional taxonomic classification. If the cytogenetic evidence comes into the picture, one can find that the chromosome size of Phal. amabilis and Phal. equestris falls into to the small group, while those of the Phal. mannii and Phal. lueddemanniana falls into to the large group. It is more reasonable to put the varieties having similar chromosome size into the same group as shown by the RAPD data, instead of into different groups as in the traditional classification based morphological characteristics. In addition, based on comparison between the dendrogram generated by the RAPD analysis and chromosome sizes as shown by the study of the karyotype,20,21,24 it is noted that the tendency is for the Phalaenopsis chromosome size to probably evolve from large to small, and its origin seems to be polyphyletic. Cross-incompatibility is one of the problems needed to be solved in the Phalaenopsis breeding programs. Compatibility is usually correlated to the closeness of their phylogenetic relationship which is, on the other hand, related to the status of the chromosome (i.e. chromosome number and size) and the homology of the nuclear DNA. On the other hand, RAPD analysis as shown by the previous study provides a rapid method to understand the phylogenetic relationship of different species. If this relationship is correlated with compatibility among species, it becomes an useful reference for the choice of parents in the work of hybridization by the breeders. The morphological characteristics, and cytological and isozyme analysis were generally used in the identification of new species and cultivars. However, these methods are limited by the environmental effects and the diagnostic resolution. Recently, DNA amplification fingerprinting (DAF) has been shown to be an effective method in detecting polymorphism and thus is a powerful tool for species or cultivar identification.25 In a study, 20 random primers were used to 18 ✦ C.-Y. Tang and W.-H. Chen Table 1.9. DAF Patterns Generated by 20 Random Primers which could Distinguish among 5 Genera,a 5 Species in Phalaenopsis and 5 Clones in Phal. equestris Primer Genera Species Clones Primer Genera Species Clones OPF-1 OPF-2 OPF-3 OPF-4 OPF-5 OPF-6 OPF-7 OPF-8 OPF-9 OPF-10 −b + − − + + + + + + + + − + − − − − − + + − − + − − − − − − OPF-11 OPF-12 OPF-13 OPF-14 OPF-15 OPF-16 OPF-17 OPF-18 OPF-19 OPF-20 − + − − + − − − − − − − − − + + + + − − − − − − − − − − − + a 5 genera were: Phalaenopsis, Doritis, Cattleya, Dendrobium, Cymbidium; the 5 species of Phalaenopsis were: Phal. amabilis, Phal. amboinensis, Phal. mannii, Phal. violacea, Phal. equestris; 5 clones of Phal. equestris. b + and – represent presence or absence of polymorphism as shown by RAPD markers. analyze the DAF patterns among five genera, five species in the genus of Phalaenopsis and five clones in a species, Phalaenopsis equestris. Polymorphism was observed among them when a suitable primer was used in the PCR reaction. In this study, it was shown that 9, 8 and 3 primers produced considerable polymorphism which could distinguish among five genera, five species and five clones, respectively (Table 1.9). Distinguishable bands of DAF patterns among the clones with similar genetic background were obtained when a suitable primer was used. Therefore, DAF is a powerful and useful tool to generate a group of molecular markers which represent the identity of a new variety. It is one of the means by which one can use to protect the patent rights of the new varieties in Phalaenopsis as well as in other species. 1.5 Conclusion and Prospective The standard white Phalaenopsis is a successful “research and development” product from both the horticultural and industrial points of Breeding and Development of New Varieties in Phalaenopsis ✦ 19 view. Because of the development of the superior white Phalaenopsis varieties, not only did it lead to the opening of an international market for the Phalaenopsis business, but it also stimulated the modernization of the production facilities, technique and management for the orchid industry in Taiwan in the last two decades. In this review, one can find that the success of the standard white Phalaenopsis varieties was based on the discovery of the tetraploid from Phal. amabilis and the hybrid Phal. Doris. From the analysis of the 12 white TAISUCO varieties, these two tetrapoloids were involved in their pedigree one way or the other. This means the development of white Phalaenopsis is no longer at the diploid level; it is a kind of tetraploid breeding. Although there are only a few of tetraploid parental stocks, yet new and superior varieties of standard white Phalaenopsis varieties have been developed year after year. This indicates that the genetic heterogeneity of the tetraploid parents is broad enough to maintain the genetic variability for continuous selection. However, one cannot overlook the potential problem of genetic depression due to the narrow genetic background in these stocks. Exploration and development of new tetraploid breeding stocks with diverse genetic background is an urgent need in order to develop better white Phalaenopsis varieties as well as other types of moth orchid. Development of the novelty varieties, including Harlequin and multifloral Phalaenopsis is a new trend in the orchid business since the last decade. It opens the door to the exploitation of the use of the genetic diversity in various wild species of Phalaenopsis to create new types of varieties besides the standard moth orchids. This approach in Phalaenopsis breeding including various kinds of interspecific and intergeneric crosses will form more diverse and unusual types of Phalaenopsis that may be important in the future market. Because of the creation of novelty varieties, demands for Phalaenopsis orchids should continue to grow in the future. Phalaenopsis breeding is a lengthy and time consuming process due to the long life cycle. DNA markers associated with useful genes such as the red floral gene of Phal. equestris as reviewed in this chapter, will increase the breeding efficiency through identification of the desired offspring at the seedling stage. Use of the RFLP and RAPD markers to study the phylogenetic relationship of wild species or the parentage of breeding stocks will provide good information on the 20 ✦ C.-Y. Tang and W.-H. Chen genetic relationship among different species and cultivars. This kind of information is helpful for breeders to choose the parents for hybridization with more precision. The technique of DNA amplification fingerprinting (DAF) is useful to identify different varieties developed in a breeding program. With this method, a breeder can protect the patent rights of the Phalaenopsis hybrids produced. Looking into the future, there will be unlimited opportunities for the expansion of Phalaenopsis orchid in the international markets. However, in order to maintain the competitiveness of Taiwan in the Phalaenopsis business, development of new and superior varieties is the key to success. Besides the traditional hybridization technique, effort on the exploration of new sources of genetic diversity as well as the development in the biotechnology of Phalaenopsis orchids to increase the breeding efficiency and accelerate the development of novelty varieties should be emphasized, so as to maintain the leading role of Taiwan in the international orchid business. References 1. Huang TJ, Pan CL, Jean MH, Chen CF. (1996) Design and development of a greenhouse automatic environmental control system. Report of the Taiwan Sugar Research Institute 153:53–74. 2. Chen WH, Wang YT. (1996) Phalaenopsis orchid culture. Taiwan Sugar 43:11–16. 3. Chen WH, Chyou MS, Wu CC, et al. (1998) Breeding Phalaenopsis. Experimental Report of the Taiwan Sugar Research Institute 1997/1998:94–102. 4. Lin TP. (1977) Native Orchids in Taiwan. Vol. 2. Chong Tao Company, Chiayi, Taiwan. 5. Chen WH, Chen YH, Chyou MS, et al. (1999) Development of white Taisuco Phalaenopsis. In: Clark J, Elliott WM, Tingley G, Biro J (eds.), Proceedings of the 16th World Orchid Conference. Vancouver, Canada, pp. 272–278. 6. Fighetti C. (2004) Passing the torch. Phalaenopsis-J Int Phalaenopsis All, Winter 2004:20–31. 7. Chen WH, Chen TC, Wu WL. (2004) The influence of Phalaenopsis Golden Peoker “Brother” on Harlequins. Phalaenopsis-J Int Phalaenopsis All, Summer 2004:14–16. 8. Harper T. (1991). Mutliflora Phalaenopsis: The contribution of Phal. equestris in breeding multifloras. Amer Orch Soci Bull 60:106–114. Breeding and Development of New Varieties in Phalaenopsis ✦ 21 9. Griesbach RJ. (2002) Development of Phalaenopsis orchids for the massmarket. In: Janick J, Whipkey A (eds.), Trends in New Crops and New Uses. ASHS Press, Alexandra, VA. 10. Stubbings J. (2006) Development of white with colored lip Phalaenopsis. In: Hwang JH (ed.), Proceedings of Taiwan International Orchid Symposium, Taiwan Orchid Growers Association, Tainan, Taiwan, pp. 38–51. 11. Fu YM, Chen WH, Tsai WT et al. (1996) Studies on floral color heredity of Phalaenopsis equestris. Report of the Taiwan Sugar Research Institute 152:35–49. (In Chinese with English abstract). 12. Christense EA. (2001) Phalaenopsis: A Monograph. Timber Press, Inc., Portland, Oregon. 13. Chen WH, Tsai WT, Chyou MS, et al. (2000) The breeding behavior of Phalaenopsis equestris (Schauer) Rchb.f. Taiwan Sugar 47(1):11–14. 14. Lenz LW, Wimber DE. (1959) Hybridization and inheritance in orchids. In: Wither CL (ed.), The Orchids: A Scientific Survey. Ronald Press, New York, pp. 261–314. 15. Chen WH, Fu YM, Lin YS, Chen YH. (2001) Identification of RAPD markers linked to the red floral gene in Phalaenopsis equestris by a stepwise screening method. Taiwan Sugar 48(4):23–29. 16. Williams JGK, Kubelik AR, Livake KJ, et al. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl Acids Res 18:6532–6535. 17. Paran I, Kesseli R, Michelmore R. (1991) Identification of restriction fragment length polymorphism and random amplification polymorphic DNA markers linked to downy mildew resistance genes in lettuce using nearisogenic line. Genome 34:1021–1027. 18. Chang SB, Chen WH, Chen HH, et al. (2000) RFLP and inheritance patterns of chloroplast DNA in intergeneric hybrids of Phalaenopsis and Doritis. Bot Bull Acad Sin 41:219–223. 19. Sweet HR. (1980) The Genus Phalaenopsis. The Orchid Digest, Inc. USA. 20. Arends JC. (1970) Cytological observation on genome homology in eight interspecific hybrids of Phalaenopsis. Genetica 41:88–100. 21. Shindo K, Kamemoto H. (1963) Karyotype analysis of some species of Phalaenopsis. Cytologia 28:390–398. 22. Lin S, Lee HC, Chen WH, et al. (2001) Nuclear DNA contents of Phalaenopsis species and Doritis pulcherrima. J Amer Soc Hort Sci 126(2):195–199. 23. Fu YM, Chen WH, Tsai WT, et al. (1997) Phylogentic studies of taxonomy and evolution among wild species of Phalaenopsis by random amplified polymorphic DNA markers. Report of the Taiwan Sugar Research Institute 157:27–42. (In Chinese with English abstract). 22 ✦ C.-Y. Tang and W.-H. Chen 24. Sagawa Y. (1962) Cytological studies of the genus Phalaenopsis. Amer Orchid Bul 31:459–465. 25. Chen WH, Fu YM, Hsieh RM, et al. (1995) Application of DNA amplification fingerprinting in the breeding of Phalaenopsis orchid. In: Terzi M, et al. (eds.), Current Issues in Plant Molecular and Cellular Biology. Kluwer Academic, Netherlands, pp. 341–346. Chapter 2 Embryo Development of Orchids Yung-I Lee*,†,‡, Edward C Yeung§ and Mei-Chu Chung† The pattern of orchid embryo development is unique among flowering plants. The minute size of the embryos, lack of cotyledon, absence of an endosperm, the varied suspensor morphology, and the simple seed coat structure are some of the unique features of orchid seeds. This chapter summarizes the recent observations on the structural and physiological aspects of orchid embryo development using a few case histories, i.e. Cypripedium formosanum, Calanthe tricarinata and Phalaenopsis amabilis var. formosa. The unique features of orchid embryo development such as: 1) the lack of a clear histodifferentiation pattern; 2) presence of a cuticle over the embryo proper; 3) absence of a cuticle in the suspensor cell wall; 4) a suspensor having a transfer cell morphology; and 5) accumulation of high levels of ABA in mature seeds of some terrestrial species are discussed. A comprehensive understanding of the structural and physiological changes during the orchid embryo development will facilitate successful micropropagation of orchids. 2.1 Introduction Compared to a majority of flowering plants, the process leading to and the pattern of seed development in orchids are unique. In orchids, ovules are not present or poorly developed at the time of anthesis. *Corresponding author. † Institute of Plant and Microbial Biology, Academia Sinica, 115, Taipei, Taiwan, ROC. ‡ Botany Department, National Museum of Natural Science, Taichung, Taiwan. § Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada. 23 24 ✦ Y.-I. Lee, E.C. Yeung and M.-C. Chung A successful pollination event triggers ovule development within an ovary. Numerous pollen tubes penetrate into the ovary chamber. Once the ovules mature, double fertilization results in the formation of a zygote and a polar chalazal complex within the endosperm cavity of each fertilized ovule.1,2 The triggering of ovule development after a successful pollination event may ensure the survival of the parent plant since little energy is channeled to the unpollinated ovaries. The polarchalazal complex fails to develop into an endosperm. Hence, an endosperm is absent from mature orchid seeds. Numerous seeds are produced within a single capsule (Fig. 2.1A). The seeds, which are very small, contain a simple embryo with no distinct tissue differentiation (Fig. 2.1B).1,3–5 Seed morphology varies within the orchid family.3 The integuments develop into a thin seed coat with varied surface features.3,4 Under natural conditions, germination of the mature orchid seeds, especially those from the terrestrial orchids, is dependant upon the association of a mycorrhizal fungus. Since Knudson’s discovery,6 Fig. 2.1. The mature capsule and seed of Calanthe tricarinata. (A) Light micrograph showing a mature capsule of Calanthe tricarinata. There are numerous tiny seeds within a capsule. Scale bar = 15 mm. (B) Light micrograph showing a mature seed of Calanthe tricarinata. SC = seed coat; E = embryo. Scale bar = 150 µm. Embryo Development of Orchids ✦ 25 orchid seeds can also germinate successfully in a culture medium in the absence of a mycorrhizal fungus. This method is known as asymbiotic germination, and is a useful propagation technique for most orchids. Substantive information on orchid embryo development can be found in a number of reviews.3–5,7–9 In this chapter, we summarize recent observations on the structural and physiological aspects of orchid embryo development using several case histories as examples. Some of the unique characteristics of embryo development are discussed. 2.2 Embryo Development 2.2.1 Case histories of embryo development 2.2.1.1 Cypripedium formosanum Cypripedium formosanum is a terrestrial, native orchid species in Taiwan. At 60 days after pollination, fertilization has just taken place, and an elongated zygote (Fig. 2.2A) can readily be detected within the embryo sac in orchids grown in Mei-Fong farm (2100 m above sea level). Compared with other orchids (Table 2.1), the size of the embryo sac of Cypripedium formosanum is large (approximate 62 × 154 µm). In this species, the endosperm fails to develop. The polar-chalazal complex degenerates early in the endosperm cavity. The growing embryo gradually expands and fills the endosperm cavity. Judging from the location of the mitotic apparatus, the first division of the zygote is asymmetrical (Fig. 2.2A), giving rise to two daughter cells of different sizes and fates (Figs. 2.2B). The smaller terminal cell forms the embryo proper and the larger basal cell gives rise to a short-lived embryonic organ known as the suspensor. The suspensor of this species consists of a highly vacuolated, single cell. The two-celled embryo divides further, resulting in the formation of a four-celled embryo proper at 75 days after pollination (Fig. 2.2C). During globular embryo formation (90–105 days after pollination), cell divisions occur in the outermost as well as the inner layers of the embryo proper, resulting in an increase of embryo volume (Fig. 2.2D and E). Throughout proper embryo development, no further change occurs in the suspensor as cell division or swelling of the suspensor cannot be observed. The suspensor finally degenerates at the late globular stage (Fig. 2.2F). In contrast to the highly vacuolated nature of the suspensor 26 ✦ Y.-I. Lee, E.C. Yeung and M.-C. Chung Fig. 2.2. The embryo development of Cypripedium formosanum. (A) Light micrograph showing a longitudinal section of a zygote at 60 days after pollination. Before the first transverse division, the condensed chromosomes at metaphase (arrowhead) are located towards the chalazal end, while a large vacuole is located at the micropylar end of the cell. SG, starch grains. Scale bar = 30 µm. (B) Light micrograph showing a two-celled embryo. The first cell division of the zygote is unequal, resulting in the formation of a smaller terminal cell and a larger basal cell. Scale bar = 30 µm. (C) Light micrograph showing a five-celled embryo at 75 days after pollination. Scale bar = 30 µm. (D) Light micrograph showing a longitudinal section through an early globular embryo with a single-celled suspensor (S) at 90 days after pollination. The occurrence of periclinal division (arrowhead) within the embryo proper results in the formation of the inner tier of cells. Scale bar = 25 µm. (E) At 105 days after pollination, the globular embryo continues to develop by cell division (arrowhead) in the inner layer cell of the embryo proper. The suspensor cell (S) is highly vacuolated. Scale bar = 25 µm. (F) As the seed approaches maturity (150 days after pollination), large vacuoles begin to be replaced by small ones. Small protein bodies begin to appear within the cells of the embryo proper. The cells of both inner (IL) and outer (OL) layers of the seed coat become dehydrated and are gradually compressed into a thin layer. Scale bar = 45 µm. Embryo Development of Orchids ✦ 27 Table 2.1. The Characteristics of the Embryos of Three Orchid Species in the Case Histories Habitat Embryo sac size (X/Y-axis, µm)a Embryo size (X/Y-axis, µm)b Suspensor A gradient of cell size within embryo Cypripedium formosanum Calanthe tricarinata Phalaenopsis amabilis var. formosa Terrestrial 62 ± 7.3/154 ± 10.2 Terrestrial 31 ± 3.2/54 ± 4.6 Epiphytic 18 ± 1.8/30 ± 2.4 125 ± 11.4/ 228 ± 16.2 1-celled, not enlarged No 132 ± 8.5/ 184 ± 14.7 1-celled, enlarged 92 ± 10.1/ 250 ± 22.2 8-celled, tubular No Yes a, b X, Y ± standard deviation. At least 20 individual embryo sacs and mature embryos are measured. cell, the cytoplasm of the embryo proper cell is densely stained with amido black 10B — a protein stain. As the seed approaches maturity (150 days after pollination), the vacuoles begin to break down, and some small protein bodies begin to appear within the cells of the embryo proper (Fig. 2.2F). In this species, the seed coat is derived from the outer integument, which consists of two cell layers. The outer integumentary cells are highly vacuolated and contain chloroplasts with starch granules (Fig. 2.2A). A secondary wall is added to the seed coat cells as the seeds mature. The inner integument is composed of two layers of compact and vacuolated parenchyma cells (Fig. 2.2F). As the seed approaches maturity, the seed coat layers begin to desiccate and are compressed into a thin layer. The dehydrated inner integument is known as “carapace,” which is found in some terrestrial orchid species, such as Cephalanthera, Cypripedium and Epipactis. The carapace tightly envelops the embryo at seed maturity. It has been suggested that the carapace can play a role in seed dormancy.10,11 It is worthwhile to note that both the seed coat and carapace stained blue green with the TBO stain and react positively to the Nile red stain. The accumulation of cuticular material may increase the hydrophobic characteristic of the seed coat that impedes the uptake of water and nutrients during seed germination.12,13 28 ✦ Y.-I. Lee, E.C. Yeung and M.-C. Chung During the course of embryo development, the ABA level is low at the proembryo stage, then increases quickly through the late globular stages (120–150 days after pollination), and maintains a maximal level −1 (approximate 13.8 ng · mg of fresh weight) at seed maturity.14 2.2.1.2 Calanthe tricarinata In Calanthe tricarinata, the elongated zygote appears as a highly polarized cell at 70 days after pollination (Fig. 2.3A). Under the light microscope, the nucleus and most cytoplasm of the zygote are located toward the chalazal end, and a large vacuole is found near the micropylar end of the cell. The nucleolus is distinct at this stage. The endosperm of this species degenerates in the early stages of embryo development. After fertilization, the polar nuclei and one of the male nuclei forms the endosperm complex, and these nuclei eventually disintegrate (Fig. 2.3A and B). The first zygote division is unequal, producing a larger basal cell and a smaller terminal cell (Fig. 2.3B). Derivatives of the basal cell give rise to the suspensor, and the terminal cell gives rise to the embryo proper. Later, the terminal cell undergoes further anticlinal and periclinal divisions, resulting in the formation of a globular-shaped embryo proper (Fig. 2.3C and D). At 150 days after pollination, the protoderm has differentiated and the mitotic activity of embryo proper cells has ceased (Fig. 2.3E). During embryo development, the cytoplasm of the embryo proper cells remains dense. Starch granules are the first storage product to appear, and they tend to congregate around the nucleus of the cell. As the seed approaches maturity, the embryo proper is filled with protein and lipid bodies. At seed maturity, an embryo measures about six cells long and five to six cells wide (Fig. 2.3F). Since Calanthe and Phaius are relative genus, the pattern of suspensor development of Calanthe tricarinata is similar to that of Phaius tankervilliae as described by Ye et al.15 In Calanthe tricarinata, the suspensor consists of a large, single cell (Fig. 2.3E). During the course of the suspensor development, the suspensor cell begins to enlarge first, then elongates quickly by the process of vacuolation (Fig. 2.3C and D). Finally, the suspensor protrudes beyond the inner integument to make direct and contact with the seed coat cells. At seed maturity, the suspensor eventually degenerates and are compressed (Fig. 2.3F). The seed coat of this species is derived from the outer integument which has only two cell layers (Fig. 2.3F). The cell wall of the seed coat cells gives a greenish blue color when stained with the TBO stain, indicating the presence of polyphenols and lignin (Fig. 2.4A). The inner Embryo Development of Orchids ✦ 29 Fig. 2.3. The embryo development of Calanthe tricarinata. (A) Light micrograph showing a longitudinal section of a zygote, which is highly polarized with a chalazally located nucleus and a prominent vacuole occupying at the micropylar end. Scale bar = 8 µm. (B) Light micrograph showing a two-celled embryo. The first cell division of the zygote is unequal, resulting in the formation of a smaller terminal cell and a larger basal cell. In this species the endosperm fails to develop. The polar-chalazal complex (arrowhead) includes the chalazal nuclei, polar nuclei and one of the male nuclei. Scale bar = 10 µm. (C) Light micrograph showing a six-celled embryo. The cells of the embryo proper have condensed cytoplasm, while the suspensor cell (S) remains highly vacuolated. Scale bar = 8 µm. (D) Light micrograph showing an early globular embryo. The suspensor cell (S) begins to enlarge by vacuolation. Scale bar = 15 µm. (E) Light micrograph showing a globular embryo. At this stage, the mitotic activity of embryo proper cell has ceased. There are starch grains (arrowhead) scattered within the embryo proper cells. The cells are also with an abundant deposition of protein and lipid bodies. The suspensor (S) has enlarged and elongated toward the micropylar end of the seed. Both inner (IL) and outer (OL) layer of the seed coat become dehydrated and gradually are compressed into a thin layer. Scale bar = 80 µm. (F) A longitudinal section through a mature seed. As the seed approached maturity, there is a concurrent diminishing of starch granules within the cells. The lipid bodies (arrowhead) and protein bodies (arrow) of various sizes can be found within the cells of the embryo proper. The suspensor has degenerated at this stage. II, inner layer of the seed coat; OI, outer layer of the seed coat. Scale bar = 60 µm. 30 ✦ Y.-I. Lee, E.C. Yeung and M.-C. Chung Fig. 2.4. Nile red staining fluorescence micrograph of the embryos of Calanthe tricarinata. (A) Light micrograph showing a globular embryo with an enlarged and elongated suspensor (S) toward the micropylar end of the seed. IL, inner layer of the seed coat; OL, outer layer of the seed coat. Scale bar = 70 µm. (B) Nile red staining fluorescence pattern of a developing seed at the stage similar to that in A. The outermost layer of outer layer of the seed coat (OL) and the surface wall (SW) of the embryo proper react positively to the stain, while the inner layer of the seed coat (IL) does not fluoresce brightly. There is a thin layer cuticular substance covering the exposed portion of the suspensor (S). Scale bar = 50 µm. (C) Light micrograph showing a mature seed. The suspensor has degenerated at this stage, and the mature embryo is enveloped by the shriveled seed coat. IL, inner layer of the seed coat; OL, outer layer of the seed coat. Scale bar = 60 µm. (D) Nile red staining fluorescence pattern of a developing seed at the stage similar to that in C. The inner layer of the seed coat (IL) and the outermost layer of the outer layer of the seed coat (OL) reacted positively to the stain. Moreover, the surface wall (SW) of the embryo proper also fluoresced brightly. Scale bar = 50 µm. Embryo Development of Orchids ✦ 31 integument is composed of two layers of vacuolated parenchyma cells (Fig. 2.3B). The cell wall of the inner integument gives a purple color when stained with the TBO stain, suggesting a lack of a secondary wall. Nile red staining indicates the accumulation of cuticular substances in the outer layer of the seed coat, and the absence of cuticular substance in the inner layer of the seed coat (Fig. 2.4B and D). As the seed matures, the seed coat layers dehydrate and are compressed into thin layers (Fig. 2.4C). From determination of the endogenous ABA during the course of embryo development, it has been shown that relatively lower levels of −1 ABA (approximate 2 ng · mg of fresh weight) are found during early embryo development.13 Later, the ABA level continues to increase, and −1 is then maintained at a maximal level (approximate 11.6 ng · mg of fresh weight) at seed maturity. 2.2.1.3 Phalaenopsis amabilis var. formosa Phalaenopsis amabilis var. formosa is an epiphytic, native species of Taiwan. At 60 days after pollination, the zygote takes on an ovoid shape within the small embryo sac (Fig. 2.5A). The nucleus is located toward the chalazal end, and numerous tiny vacuoles are present in the cytoplasm of the zygote. Similar to the majority of orchids, the endosperm fails to develop in this species. Within the endosperm cavity, the endosperm complex eventually degenerates and is absorbed by the developing embryo (Fig. 2.5A and B). The first zygote division is asymmetrical, producing a larger basal cell and a smaller terminal cell (Fig. 2.5B). The basal cell of the two-celled embryo divides further, resulting in the formation of a three-celled embryo (Fig. 2.5C). The upper two cells of the three-celled embryo give rise to the embryo proper, and the larger basal cell becomes the suspensor initial cell. During the early stages of embryo proper formation, oblique cell divisions are commonly observed (Fig. 2.5F). Furthermore, the cells towards the chalazal end divide more frequently than the cells towards the micropylar end, forming two different sizes of cells within an ellipsoidal embryo: smaller cells in the anterior region and larger cells in the posterior end near the suspensor cells (Fig. 2.6B and C). During the early stages of embryo development, no distinct storage products are observed within the cytoplasm of the embryo proper (Fig. 2.6A). After the cells have ceased to divide, starch grains are the first storage product to appear within the embryo proper; the starch grains tend to congregate around the nucleus of the cells (Fig. 2.6B). As the seed approaches maturity, large vacuoles have broken 32 ✦ Y.-I. Lee, E.C. Yeung and M.-C. Chung Fig. 2.5. The early embryo development of Phalaenopsis amabilis var. formosa. (A) Light micrograph of a zygote (arrowhead) after fertilization. The zygote has a dense cytoplasm and a prominent nucleus. Scale bar = 20 µm. (B) The first cell division of the zygote is unequal, resulting in the formation of a smaller terminal cell and a larger basal cell. Scale bar = 20 µm. (C) Light micrograph showing a three-celled embryo. The basal cell towards the micropylar end is larger with a prominent nucleus. Scale bar = 20 µm. (D) Light micrograph showing a four-celled embryo. The two cells towards the chalazal end have a dense cytoplasm, whereas the other two cells towards the micropylar end continue to enlarge and elongate. Scale bar = 20 µm. (E) The basal cells toward the micropylar end continue to divide (arrowhead), and the daughter cells then differentiate into suspensor cells. In these dividing cells, vacuoles also become more prominent in the cytoplasm. Scale bar = 20 µm. (F) The oblique cell division occurs in the embryo proper (arrowhead), which signals the formation of the globular shaped embryo. At the same time, the suspensor cells (S) enlarge and elongate rapidly within the endosperm cavity towards both the micropylar and chalazal ends. Scale bar = 20 µm. Embryo Development of Orchids ✦ 33 Fig. 2.6. The later stages of embryo development of Phalaenopsis amabilis var formosa. (A) Light micrograph showing an early globular embryo. The suspensor cells (S) has elongated and surround the embryonic mass. In the Spurr’s resin section, it is worthwhile to note that several lipid droplets (arrowhead) appeared in the suspensor cells after TBO staining. Scale bar = 25 µm. (B) Light micrograph showing a globular embryo, which consists of two cell types: smaller cells in the anterior region and larger cells in the posterior end near the suspensor. The suspensor cells (S) begin to degenerate at this stage. In the embryo proper cells, starch grains (arrowhead) are abundant in the cytoplasm, and tend to congregate around the nucleus. Scale bar = 50 µm. (C) Light micrograph showing a longitudinal section through a mature seed. Several minute protein bodies (arrowhead) can be observed within the cells of embryo proper. In the historesin section, lipid is not preserved; the spaces between the protein bodies are occupied by storage lipid bodies. At maturity, the embryo is enclosed by the shriveled seed coat (SC). Scale bar = 50 µm. down and the starch grains have vanished. At the same time, lipid bodies and protein bodies start to accumulate within the cytoplasm. At seed maturity, protein and lipid bodies are the major storage products within the embryo proper (Fig. 2.6C). 34 ✦ Y.-I. Lee, E.C. Yeung and M.-C. Chung The embryo development of Phalaenopsis amabilis var. formosa is characterized by a vertical division in the basal cell of a three-celled embryo, resulting in the formation of a ⊥-shaped four-celled embryo (Fig. 2.5D). The two basal cells of the four-celled embryo divide two times and eventually give rise to a plate of eight suspensor cells (Fig. 2.5E). Soon after, the suspensor cells elongate rapidly by vacuolation, and some lipid droplets appear in the cytoplasm of the suspensor cell (Fig. 2.5F). Throughout embryo development, the tubular suspensor cells extend towards the micropylar and the chalazal ends, and surround the embryonic mass (Fig. 2.6A). As the seed approaches maturity, the suspensor cells shrivel, and the remnant of the suspensor could be observed in the mature seed (Fig. 2.6A and B). This pattern of embryo development occurs commonly in the genera of the Vandoid group, such as Phalaenopsis, Rhynchostylis and Vanda.16 The oblique division of the proembryo is characteristic of the Cymbidium type, suggesting that it is a programmed process that results in the formation of the suspensor structure17; in Phalaenopsis, the formation of a ⊥-shaped fourcelled embryo may be the trait of the Vandoid group that has the eight-celled filamentous suspensor. The mature seed coat is derived from the outer integument, which is two-three cells thick. The cells of the inner integument gradually collapse at the proembryo stage (Fig. 2.5F). The thickened radial walls of the outermost layer of the seed coat stain greenish blue with the TBO stain, indicating the presence of secondary walls (Fig. 2.7A). Autofluorescence of the seed coat indicates that lignin is present. Staining using Nile red indicates that cuticular materials are also present in the secondary walls (Fig. 2.7B). At maturity, the cells of seed coat become dehydrated and are compressed into a thin layer (Fig. 2.6C). 2.3 Integuments and the Seed Coat At the time of seed dispersal, the mature embryo of orchids is protected by a thin seed coat. The seed coat is derived from the maternal tissues — the integuments of the ovules. In Cymbidium sinense,17 the seed coat is derived from both the inner and outer integuments. The inner integument of some species degenerates in the early stages of embryo development, and the seed coat is derived from the outer integument, such as Calypso bulbosa18 and Phalaenopsis.19 Generally, the inner layers Embryo Development of Orchids ✦ 35 Fig. 2.7. Nile red staining fluorescence micrograph of the developing embryo of Phalaenopsis amabilis var. formosa. (Α) Light micrograph showing a cross section of a globular embryo of Phalaenopsis amabilis var. formosa. Secondary wall was present in the radial walls of the outer layer of the seed coat. E, embryo proper; OL, outer layer of the seed coat. Scale bar = 50 µm. (B) Nile red staining fluorescence micrograph of a developing seed at the stage similar to that in A. The radial walls of outer layer of the seed coat (OL) and the surface wall of the embryo proper react positively to the stain. E, embryo proper; OL, outer layer of the seed coat. Scale bar = 50 µm. of the seed coat is composed of two layers of vacuolated parenchyma cells (Figs. 2.2A and 2.3B). In Calypso bulbosa18 and Epidendrum ibaguense (Fig. 2.8), the inner layers of the seed coat that originated from the inner integument gives a strong positive protein staining during the ovule and early embryo developmental stages, suggesting that these cell layers may play a role as the nutrient supplier during these stages of development. In the outer layers of the seed coat, the cells are usually vacuolated. In many species, such as Cypripedium formosanum, starch grains can be found in the cells of the outer layers of the seed coat at the early stage of embryo development (Fig. 2.2A), and the starch grains disappear as the seed approaches maturity (Fig. 2.2F). As the seed matured, the seed coat cells dehydrate and are compressed into a thin layer, which envelopes the embryo. Although the seed coat of an orchid seed looks simple, the developmental pattern and the addition of secondary walls are diverse among the species. During seed development, cuticular materials, phenolic substances, and lignin may accumulate differently in the seeds coat. In Calanthe tricarinata (Fig. 2.4), Cymbidium sinense17 and Cypripedium formosanum,12 cuticular material as well as phenolic compounds are 36 ✦ Y.-I. Lee, E.C. Yeung and M.-C. Chung Fig. 2.8. Light micrograph showing a two-celled embryo of Epidendrum ibaguense. The inner integument cells gives a strong positive protein staining, Amido black 10B. Scale bar = 20 µm. present in the outermost layer of the seed coat, and persists through seed maturation. In Paphiopedilum delenatii, the cuticular material is present over the innermost walls of the inner layer of the seed coat that encloses the endosperm cavity at the early globular stage, while the outer layers lack the cuticular material.20 From previous reports, mature embryos of some terrestrial species are covered by a unique structure which is known as the “inner seed coat” or the “carapace.”21,22 The embryo with thick and complete “carapace” is hard to germinate, such as Cephalanthera,11,22 Cypripedium21 and Epipactis.22 Based on a series histological observation of developing seeds, the carapace is derived from the inner integument that envelops the embryo tightly as the seed matures.12 In Phalaenopsis amabilis var. formosa, an easy-togerminate epiphytic species, the presence of lignin and cuticular material are discontinuous, accumulating mainly in the radial walls of the outermost layer of the seed coat. This feature may allow water and nutrients access to the embryo for germination (Fig. 2.7A and B). In Cypripedium formosanum, Nile red staining indicates the accumulation Embryo Development of Orchids ✦ 37 of cuticular substance of the carapace.12 Ultrastructural observation also confirms the presence of an electron dense layer in the surface wall of the carapace (unpublished data). Seed germination of the terrestrial species is usually difficult. Treating mature seeds by calcium hypochlorite, sodium hypochlorite, or ultrasound may scarify and break the firm covering of the embryo, and thus improve seed germination.13,23–25 2.4 A General Discussion on Features of Orchid Embryo Development 2.4.1 The lack of a clearly histodifferentiation pattern One of the most striking features of embryo development is the lack of a clear histodifferentiation pattern (Figs. 2.2F and 2.3F). As illustrated in the above case histories, the embryo proper lacks defined tissues such as the apical meristems, cotyledon and primary tissue system. Although a protoderm is present, its organization is not as well defined as in other flowering plants such Brassica napus.17 In an easier and faster germinating species, Phalaenopsis amabilis var. formosa, a gradient of cell size is observed, with smaller cells occupying the future shoot pole (Fig. 2.6C). This difference in cell size may represent different developmental potential and may be a reflection of biological differentiation of cells within the embryo proper. Is the absence of a distinct histodifferentiation pattern due to the absent of the nutritive tissue, the endosperm? In flowering plants, the endosperm is an integral part of seed and embryo development. Abnormalities in endosperm development often result in embryo abortion.26 The failure of endosperm development may cause abnormal differentiation and starvation of the developing embryos.27,28 The endosperm is the food storage site for the developing embryo in most flowering plants, and the lack of an endosperm in orchids may have prevented further histodifferentiation of the globular embryo. Moreover, the absence of an endosperm results in an “empty” cavity. Since the seed coat of orchids is thin and without the nutritive endosperm tissue serving as buffer, the developing embryo may be subject to water stress. In Cypripedium formosanum14 and Calanthe tricarinata,13 the speedy increase of the ABA content is found as the seeds approach maturity. The rapid increase in the ABA content may be an indication of the unusually “dry” embryonic environment. It is well 38 ✦ Y.-I. Lee, E.C. Yeung and M.-C. Chung known that a majority of the developmental process cannot tolerate the lowering of water content and water stress. Mitotic divisions and DNA synthesis are both sensitive to water stress and increasing ABA levels.29,30 In maize, water deficit decreases the rate of endosperm cell division and inhibits DNA synthesis.31 If this is indeed the case, the mitotic activity becomes arrested early during the orchid embryo development. Hence, further histodifferentiation simply cannot occur. 2.4.2 Presence of a cuticle over the embryo proper All exposed plant surfaces have a cuticle. Rodkiewicz et al.32,33 clearly establish that the embryo proper in flowering plants has a distinct cuticle covering its surface, while a cuticle is absent from the suspensor. In orchid embryos, as shown in Section 2.2.2, a clearly defined cuticle is present over the entire embryo proper surface (Fig. 2.4B and D). In Cymbidium sinense,17 Paphiopedilum delenatii,20 and Phalaenopsis amabilis var. formosa,19 a cuticle can be detected as early as the globular stage of development. The formation of a cuticle at such as an early stage supports the notion that the embryonic environment may be “dry.” In plants, a cuticle functions to prevent water loss from the surface of the plant body. The cuticle covering the orchid embryo proper most likely serves to protect it from premature desiccation. The negative side of having a cuticle is that it may retard or impede nutrient absorption directly from the surface of the embryo proper. 2.4.3 The absence of a cuticle in the suspensor cell wall Histochemical studies using Nile red staining reveal that cuticular substances are absent from the suspensor cell wall.17,20 This observation clearly indicates structural and functional differences between the two parts of the embryo. In orchids as well as in a majority of flowering plants, the suspensor is usually embedded within the integumentary tissues and is not “exposed” (Figs. 2.3E and 2.6B). This may be the primary reason why a cuticle is absent from the suspensor. The consequence of not having a cuticle is important in facilitating nutrient uptake from the maternal tissues as the embryo is attached to the seed coat via the suspensor. The suspensor is a short-lived embryonic organ. Because of its precocious development during early embryogeny, together with its structural Embryo Development of Orchids ✦ 39 and physiological characteristics, the suspensor is believed to be essential to early embryo development.34 One of the important functions of the suspensor is to serve as a channel of nutrient uptake for the developing embryo as it connects the embryo proper to the maternal tissues.34 In the study of plant cell biology, the staining characteristics of a cell wall indirectly indicate the structure and function of the cell of interest. When suspensors are stained with toluidine blue O, the cell walls give a purple color (Fig. 2.4A). The walls stain negatively towards lipid stains such as Nile red and negatively toward the phloroglucinolHCl stain. These stains clearly indicate the cell wall is “primary” in nature and the absence of a cuticle and lignin in the wall (Fig. 2.4B). Since the primary cell wall allows for apoplastic movement of water and dissolved solutes, the suspensor cell wall can form an apoplastic continuum with cells of the maternal tissues, i.e. the seed coat. Albeit not necessary an efficient translocation process, the apoplastic continuum ensures nutrient flow directly from the maternal tissue to the embryo proper via the suspensor. 2.4.4 A suspensor having a “transfer cell morphology” It is well established that orchid suspensors have a varied morphology (Table 2.1); their nutritive function is usually inferred from their pattern of growth. At the light microscope level, orchid suspensor cells are usually highly vacuolated with no obvious structural specialization detected. The highly vacuolated cells together with the absence of structural specializations such as wall ingrowths suggest that the orchid suspensor may not have specialized functions similar to that reported in other flowering plants, such as the Phaseolus species.35,36 Ultrastructural studies concerning orchid suspensors are rare. As indicated above, in our recent ultrastructural studies, we noted that the Paphiopedilum delenatii suspensor indeed takes on a “transfer cell” morphology, indicating that the suspensor can play an important role in embryo development.20 The transfer cells function as the short-distance transporter for solutes across cells through wall ingrowths. In developing Vicia faba seeds, genes involved in plasma membrane sucrose transport (H+/sucrose symporter gene, sucrose binding protein gene, and H+-ATPase gene) are found to express in the wall ingrowths.37 Furthermore, H+/amino acid co-transporter genes are found to express predominantly in the transfer cells of pea seeds.38 These observations provide strong evidence 40 ✦ Y.-I. Lee, E.C. Yeung and M.-C. Chung that transfer cells play an important role in the uptake of the solutes. Our recent ultrastructural study clearly indicates that the suspensor of Paphiopedilum delenatii takes on a “transfer cell” morphology20; (Fig. 2.9A and B). Although there is no experimental data indicating that the orchid suspensor cells have similar biochemical functions as indicated above, having a transfer cell morphology strongly suggests that similar biochemical properties and functions are possible. In Paphiopedilum delenatii, wall ingrowths begin to appear when the embryo reaches the six-cell stage.20 The wall ingrowths are strategically located on the side walls abutting the maternal seed coat. The increase in the surface area of the suspensor cell supports the idea of enhancing nutrient uptake at this location. The early formation of wall ingrowths suggests that the ingrowths are important and have unique functions to play during the early stages of orchid embryo development. Additional ultrastructural Fig. 2.9. Electron micrographs of the suspensor development of Paphiopedilum delenatii embryo. (A) Electron micrograph of the micropylar end of the suspensor at the proembryo stage, showing the formation of the wall ingrowth (WI) along the basal wall. The cytoplasm is filled with the tubular SER system. D = dictyosomes; L = lipid body; M = mitochondrion; V = vacuole. Scale bar = 1 µm. (B) Electron micrograph showing the micropylar end of the fully developed suspensor at the globular stage. At this stage, the wall ingrowths (WI) are well developed; the rough ER (RER) begins to appear, which is in the form of long strands. The plastids (P), dictyosomes (D) and mitochondria (M) are plentiful in the cytoplasm. Scale bar = 1 µm. Embryo Development of Orchids ✦ 41 studies are needed to determine whether structural specializations are present in other orchid suspensor systems. 2.4.5 The accumulation of high levels of ABA in mature seeds of some terrestrial species ABA is essential in regulating seed embryonic maturation, dormancy and germination.39 In orchids, there are only a few reports on the endogenous ABA concentrations in developing seeds. In Epipactis helleborine, it has been shown that the endogenous ABA level in mature seeds is 14-fold greater than that in immature seeds.40 In recent studies on Calanthe tricarinata13 and Cypripedium formosanum,14 as the seeds approach maturity, there is a rapid increase in the ABA concentration (Fig. 2.4). In cereals, the ABA concentration is low in early developing seeds, reaches the highest concentration during mid-development, and then declines as the seed matures.41–43 In the developing seeds of Calanthe tricarinata and Cypripedium formosanum, the ABA concentration does not decrease, but continues to increase as the seeds approach maturity. Furthermore, in vitro germination decreases sharply as the seeds approach maturity; and this coincides with increasing ABA levels, and decreasing water content. These results suggest that ABA may regulate the processes of storage protein synthesis, acquisition of desiccation tolerance, and prevention of germination in orchid seeds. 2.5 Conclusion and Perspectives In Taiwan, a number of orchids, such as Cymbidium, Paphiopedilum, Phalaenopsis and Oncidium have become important floricultural crops. In addition, there are numerous native species with ornamental value. Asymbiotic germination is a useful and popular technique in obtaining seedlings for nursery culture, and to conserve our natural resources. Although most orchids can germinate successfully on a defined medium, asymbiotic seed germination is often intricate for some terrestrial orchids. A detailed knowledge of embryo development and its structure will facilitate our success in asymbiotic seed germination by selecting the proper age of developing seeds for in vitro germination. Our knowledge concerning the timing of the cessation of mitotic activity, appearance of the cuticle in the embryo, and presence 42 ✦ Y.-I. Lee, E.C. Yeung and M.-C. Chung or absence of specialization in cells of the embryo and surrounding maternal tissues will enable us to determine the most opportune time for the asymbiotic germination of immature seeds. The structure of the seed coat, thickness of the cuticle surrounding the embryo proper, cell number and presence or absence of a distinct cell size gradient also allow us to estimate the ease of symbiotic and asymbiotic germination of mature seeds. References 1. Arditti J. (1992) Fundamentals of Orchid Biology. John Wiley and Sons, Inc, New York. 2. Ye, XL, Yeung EC, Zee SY. (2002) Sperm movement during double fertilization of a flowering plant, Phaius tankervilliae. Planta 215:60–66. 3. Molvray M, Chase MW. (1999). Seed morphology. In: Pridgeon AM, Cribb PJ, Chase MW, Rasmussen FN (eds.), Genera Orchidacearum, Vol. 1. Oxford University Press, Oxford, UK, pp. 59–66. 4. Yam TW, Yeung EC, Ye XL, et al. (2002) Orchid embryos. In: Kull T, Arditti J (eds.), Orchid Biology: Reviews and Perspectives, 8th edn. Kluwer, Dordrecht, pp. 287–385. 5. Yam TW, Nair H, Hew CS, Arditti J. (2002) Orchid seeds and their germination: an historical account. In: Kull T, Arditti J (eds.). Orchid Biology: Reviews and Perspectives, 8th edn. Kluwer, Dordrecht, pp. 387–504. 6. Knudson L. (1922) Nonsymbiotic germination of orchid seeds. Bot Gaz 73:1–25. 7. Clements MA. (1999) Embryology. In: Pridgeon AM, Cribb PJ, Chase MW, Rasmussen FN (eds.), Genera Orchidacearum, Vol. 1. Oxford University Press, Oxford, UK, pp. 38–66. 8. Batygina TB, Bragina EA, Vasilyeva VE. (2003) The reproductive system and germination in orchids. Acta Biol Cracov Series Botanica 45:21–34. 9. Andronova EV. (2006) Embryogenesis in Orchidaceae. In: Batygina TB (ed.), Embryology of Flowering Plants, Vol. 2 Seed. Science Publishers, New Hampshire, pp. 355–363. 10. Rasmussen HN. (1995) Terrestrial Orchids from Seed to Mycotrophic Plants. Cambridge University Press, Cambridge. 11. Yamazaki J, Miyoshi K. (2006) In vitro asymbiotic germination of immature seed and formation of protocorm by Cephalanthera falcata (Orchidaceae). Ann Bot 98:1197–1206. Embryo Development of Orchids ✦ 43 12. Lee YI, Lee N, Yeung EC, Chung MC. (2005) Embryo development of Cypripedium formosanum in relation to seed germination in vitro. J Amer Soc Hort Sci 130:747–753. 13. Lee YI, Lu CF, Chung MC, et al. (2007) Developmental changes in endogenous abscisic acid concentrations and asymbiotic seed germination of a terrestrial orchid, Calanthe tricarinata Lindl. J Amer Soc Hort Sci 132:246–252. 14. Lee YI. (2003) Growth periodicity, changes of endogenous abscisic acid during embryogenesis, and in vitro propagation of Cypripedium formosanum Hay, Ph.D dissertation. National Taiwan University, Taipei, Taiwan, ROC. 15. Ye XL, Zee SY, Yeung EC. (1997) Suspensor development in the nun orchid, Phaius tankervilliae. Intern J Plant Sci 158:704–712. 16. Swamy BGL. (1949) Embryological studies in the Orchidaceae. II. Embryology. Am Midland Naturalist 41:202–232. 17. Yeung EC, Zee SY, Ye XL. (1996) Embryology of Cymbidium sinense: Embryo development. Ann Bot 78:105–110. 18. Yeung EC, Law SK. (1992) Embryology of Calypso bulbosa. II. Embryo development. Can J Bot 70:461–468. 19. Lee YI, Yeung EC, Lee N, Chung MC. (2007) Embryology of Phalaenopsis amabilis var. formosa: Embryo development. Botanical Study (submitted). 20. Lee YI, Yeung EC, Lee N, Chung MC. (2006) Embryo development in the lady’s slipper orchid, Paphiopedilum delenatii with emphases on the ultrastructure of the suspensor. Ann Bot 98:1311–1319. 21. Carlson MC. (1940) Formation of the seed of Cypripedium parviflorum. Bot Gaz 102:295–301. 22. Veyret Y. (1969) La structure des semences des orchidaceae et leur aptitude a la germination in vitro en cultures pures. Musee d’Histoire Naturelle de Paris, Travaux du Laboratoire La Jaysinia 3:89–98. 23. Van Waes JM, Debergh PC. (1986) Adaption of the tetrazolium method for testing the seed viability and scanning electron microscopy of some Western European orchids. Physiol Plant 66:435–442. 24. Van Waes JM, Debergh PC. (1986) In vitro germination of some Western European orchids. Physiol Plant 67:253–261. 25. Miyoshi K, Mii M. (1988) Ultrasonic treatment for enhancing seed germination of terrestrial orchid, Calanthe discolor, in asymbiotic culture. Scientia Hort 35:127–130. 26. White DWR, Williams E. (1976) Early seed development after crossing of Trifolium semipilosum and T. repens. NZ J Bot 74:161–168. 27. Lester RN, Kang JH. (1988) Embryo and endosperm function and failure in Solanum species and hybrids. Ann Bot 82:445–453. 44 ✦ Y.-I. Lee, E.C. Yeung and M.-C. Chung 28. Niroula RK, Bimb HP, Sah BP. (2006) Interspecific hybrids of buckwheat (Fagopyrum spp.) regenerated through embryo rescue. Scientific World 4:74–77. 29. Levi M, Brusa P, Chiatante D, Sparvoli E. (1993) Cell cycle reactivation in cultured pea embryo axes: Effect of abscisic acid. In Vitro Cell Dev Biol Plant 29P:47–50. 30. Liu Y, Bergervoet JHW, Ric De Vos CH, et al. (1994) Nuclear replication activities during imbibition of abscisic acid- and gibberellin-deficient tomato (Lycopersicon esculentum Mill.) seeds. Planta 194:368–373. 31. Setter TL, Flannigan BA. (2001) Water deficit inhibits cell division and expression of transcripts involved in cell proliferation and endoreduplication in maize endosperm. J Expt Bot 52:1401–1408. 32. Rodkiewicz B, Fyk B, Szczuka E. (1994) Chlorophyll and cutin in early embryogenesis in Capsella, Arabidopsis, and Stellaria investigated by fluorescence microscopy. Sex Plant Reprod 7:287–289. 33. Rodkiewicz B, Szczuka E. (2006) Cuticle of developing embryo. In: Batygina TB (ed.), Embryology of Flowering Plants, Vol. 2, Seed. Science Publishers, New Hampshire, pp. 373–374. 34. Yeung EC, Meink DW. (1993) Embryogenesis in angiosperms: Development of the suspensor. Plant Cell 5:1371–1381. 35. Yeung EC, Clutter ME. (1978) Embryogeny of Phaseolus coccineus: Growth and microanatomy. Protoplasma 94:19–40. 36. Yeung EC, Clutter ME. (1979) Embryology of Phaseolus coccineus: The ultrastructure and development of the suspensor. Can J Bot 57:120–136. 37. Harrington GN, Franceschi VR, Offler CE, et al. (1997) Cell specific expression of three genes involved in plasma membrane sucrose transport in developing Vicia faba seed. Protoplasma 197:160–173. 38. Tegeder M, Offler CE, Frommer WB, Patrick JW. (2000) Amino acid transporters are localized to transfer cells of developing pea seeds. Plant Physiol 122:319–325. 39. Bewley JD, Black M. (1985) Seeds: Physiology of the Development and Germination. Plenum Press, New York, USA. 40. Van der Kinderen G. (1987) Abscisic acid in terrestrial orchid seeds: A possible impact on their germination. Lindleyana 2:84–87. 41. Goldbach H, Michael G. (1976) Abscisic acid content of barley grains during ripening as affected by temperature and variety. Crop Sci 16:797–799. 42. Kawakami N, Miyake Y, Noda K. (1997) ABA insensitivity and low ABA levels during seed development of non-dormant wheat mutants. J Expt Bot 48:1415–1421. 43. King RW. (1976) Abscisic acid in developing wheat grains and its relationship to grain growth and maturation. Planta 132:43–51. Chapter 3 In vitro Morphogenesis and Micro-Propagation of Orchids Wei-Chin Chang* Plant biotechnology, especially in vitro regeneration and flowering, cell biology, DNA manipulation, and biochemical engineering, is reshaping orchid research in four major areas: 1) benefiting micropropagation and transgenic research with findings on the totipotency and regeneration ability through shoot-bud formation and somatic embryogenesis from callus, direct somatic embryogenesis from explants and thin-section cultures of leaves and roots, and even shoot-bud formation of suspension cells of several major commercial orchids; 2) active research into the dissection of genes responsible for controlling growth, meristem functioning, and flowering of orchids; 3) successful application of molecular genetics and plant transformation under laboratory conditions for protecting commercial orchids against biotic stress; 4) production of specialty biochemicals and pharmaceuticals. These are all good starts, but more devotion and support are needed for further research into both basic and practical aspects. This chapter will include findings on: 1) direct somatic embryogenesis of leaf explants of Oncidium, Phalaenopsis, and Dendrobium; 2) direct shootbud formation from leaf explants and regeneration from suspension cells of Paphiopedilum; 3) in vitro flowering of callus-derived somatic embryos and plantlets of Cymbidium, Dendrobium, and Phalaenopsis; and 4) disease-resistant Dendrobium and Oncidium through genetic transformation. For the challenges ahead in orchid biotechnology and its practical application, I strongly encourage integrating modern *Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan. 45 46 ✦ W.-C. Chang technologies with classical breeding for the future success of the commercial orchid industry. 3.1 Introduction This chapter is meant to be a personal view on the current advances in the micro-propagation of and transgenic approach to commercial orchids, not a review of all the current and past literature on the subject. I knew little about orchids, especially about their in vitro culture, 10 years ago. However, during the last 10 years, orchids have consumed my interest and my desire to learn more about exotic plants. When I finished my training at the University of California, Riverside, in 1972 and returned to Taipei to build a laboratory for working on in vitro morphogenesis at Academia Sinica, orchids were not the plants we chose as our early experimental material. Many colleagues in the academic community and the industry had worked on orchids for years and produced many publications on orchid tissue culture. Working on a variety of plants, we eventually focused on ginseng (Panax ginseng C. A Meyer), a popular herb among the Chinese community. We learned a lot about somatic embryogenesis and plant regeneration on ginseng callus culture1 and found a way to reduce its juvenility by inducing the callus-derived embryo, which flowered in a few months under a definite condition.2 Those experiences helped a lot in our later attempts at somatic embryogenesis and in vitro flowering in cultures of bamboo3,4 and orchids,5,6 the latter of which I will focus on in this chapter. 3.2 Cymbidium Cymbidium was the first orchid genus we learned to work on in vitro in the mid-1990s. Chen Chang, a Ph.D. student in my lab, tried to induce callus from various tissue explants of Cymbidium ensifolium var. misericors. The explants included roots, leaves, and pseudo-bulbs of 7- to 8-cm seedlings derived from seed-derived rhizomes and the rhizomes themselves. Chang eventually found that a medium of 1/2 In vitro Morphogenesis and Micro-Propagation of Orchids ✦ 47 Murashige and Skoog (MS) plus (2,4-dichlorophoxyacetic acid (2,4-D) (10 mg/l) and thioridazine TDZ (0.1 mg/l) was effective for callusing from explants of pseudo-bulbs and rhizomes. However, callusing at that time was a slow process: it took 12- to 18-months to obtain reliable callus for subculture. Those calli, sub-cultured on a 1/2 MS plus a smaller amount of 2,4-D (3.3 mg/l) and the same amount of TDZ (0.3 mg/l) demonstrated their totipotency through three different morphogenetic routes, including granular embryoid-like structures. Plants were regenerated from these calli through two morphogenetic routes: 1) rhizomes produced on basal medium plus 5 mg/l benzyaminopurine (BA), from which plantlets proliferated on basal medium plus 2 mg/l BA; and 2) embryoids formed on basal medium plus 5 mg/l BA or 1 mg/l TDZ, from which rhizomes developed on 1/10 basal medium plus coconut milk (150 ml/l), with lateral buds proliferating and eventually developing into plantlets on basal medium plus 2 mg/l BA (Fig. 3.1).5 We learned a lot about in vitro orchid culture from the early work on Cymbidium, especially the important role of TDZ in callusing and embryogenesis. Were those protocols useful for propagation of Cymbidium on a large scale? We demonstrated that one piece of rhizome produced seven shoot buds in 45 days cultured in agitated 1/2 MS liquid medium plus appropriate amounts of 2iP, TDZ, BA and naphthaleneacetic acid (NAA).7 Plantlets were then transferred onto with the same Gelritegelled basal medium supplemented with banana pulp for five months before being put into pots in the greenhouse. The scale was satisfactory for large-scale propagation, but still time consuming. Similar work on callusing from protocorm-like bodies (PLBs) of Cymbidium Twilight Moon “Day Light” was reported by Huan et al.8 The authors obtained embryogenic calli from longitudinally bisected segments of PLBs on modified Vacin and Went medium plus NAA or 2,4-D alone or in combination with TDZ within one month. The medium containing the combination of 0.1 mg/l NAA and 0.01 mg/l TDZ was optimal for callus formation and also good for callus subculture. Embryos/PLBs formed when callus was transferred to the basal medium devoid of hormonal additives. These callus-derived PLBs converted into normal plants with well-developed shoots and roots on the basal medium without hormones after about four months. Proper acclimatization in a green house gave with 100% survival rate and 48 ✦ W.-C. Chang Fig. 3.1. In vitro morphogenesis of Cymbidium ensifolium var. misericors. 3.1A Callus from lateral buds of rhizome explant (a); callus from a pseudobulb explant (b); callus form a root explant (c), callus from rhizome, after 3 subcultures (d). 3.1B Morphogenesis and structure of granular embryoids: longitudinal section of cluster of white granular embryoids (a); cross-section of white granular embryoids (b); secondary granular embryoids (arrows) proliferating from the surface of a granule (c). 3.1C Morphogenesis of subcultured rhizome calli: rhizomes (a); shoot buds (b), white granular embryoids (c). 3.1D Plantlet regeneration: granular embryoids developing into rhizomes (a); early germination stage of granular embryoids (b); rhizome-derived shoot buds (c); potted plantlets derived from granular embryoids (d). ( From Chang and Chang (1998). Plant Cell Rep 17:251–255.) demonstrated the efficiency of the method for micro-propagation of Cymbidium. We even found a repeatable way to encourage in vitro flowering of the sub-cultured callus-derived embryos and plantlets. More intensive In vitro Morphogenesis and Micro-Propagation of Orchids ✦ 49 studies on in vitro flowering led to a report on the role of cytokinins in flowering (Fig. 3.2).6 Among the eight cytokinins tested, only TDZ, 2iP, and BA induced de novo flowering of rhizomes. Callus-derived rhizomes produced flowers precociously within 100 days on 1/2 MS containing TDZ (33.3–10 µM) or 2iP (10–33 µM) combined with 1.5 µM NAA. The flowers we made bloomed for two weeks in vitro and were physically normal, with high pollen viability (Fig. 3.2). This was a promising result on the attempt to donate off-season pollen for other parents in terrestrial Cymbidium breeding programs. Fig. 3.2. In vitro flowering of Cymbidium ensifolium var. misericors. 3.2A Morphogenesis of callus-derived rhizomes in vitro: rhizome explant for in vitro flowering study (a); proliferated rhizomes on basal medium after 100 days of culture (b); shoot formed on basal medium supplemented with TDZ and NAA for 100 days (c); inflorescences formed on basal medium supplemented with BA and NAA after 100 days (d). 3.2B In vitro floral morphology of Cymbidium ensifolium var. misericors: undersized inflorescences derived from the apex and lateral buds of the rhizome (a); apex of young inflorescence that contains several floret primordia (b); erect raceme inflorescences (c); gravitropic panicle inflorescences (d), inflorescence clumps (e); normal flower with three sepals, 3 petals, and column (f ); column with anther and stigma (g); longitudinal section of a column showing anther stigma and pollen (h); germinated pollen (I). ( From Chang and Chang (2003). Plant Growth Reg 39:217–221.) 50 ✦ W.-C. Chang We admired a fine piece of work on in vitro flowering of Cymbidium published in 1999 by Kstenyuk et al.9 The authors used a combined treatment of cytokinins (BA), restricted nitrogen supply with phosphorus enrichment, and root excision to induce the shoots of 90-day-old rhizome-derived plantlets to flower. A few plants even produce fruit when self-pollinated. Chang et al.10 also checked the early morpghogenesis of seed germination of Cymbidium dayanum reichb. 3.3 Oncidium Oncidium were usually clonally propagated from the dormant buds on flower stalks. The method is quite effective and may not need other protocols to replace it on an industrial scale. Since 1999, we have released a series of reports on direct somatic embryogenesis for leaves and calli derived from a variety of vegetative and reproductive tissues. Segments taken from young leaves of Oncidium “Grower Ramsey” produced clusters of somatic embryos directly from epidermal and mesophyll cells of leaf tips and wound surfaces without any intervening callus within one month on culture with Gelrite-gelled 1/2 MS supplemented with low dosage (0.3–1 mg/l) TDZ (Fig. 3.3).11 Sub-cultures of these embryo clusters produced more embryos and subsequent plantlet formation on the same medium. Similar data were found with Oncidium “Sweet Sugar,”12 but requirements for cytokinin and medium composition for embryo formation were not exactly the same as in “Gower Ramsey.” The effects of different cytokinins and auxins and their combinations have been tested.13 Cytokinins played a promotive role on direct embryo formation, while auxins in general retarded the process. Embryo formation was significantly affected by explant position.14 Leaf tip segments had a significantly higher embryogenic response than other segments of leaves. Adaxial-side-up orientation significantly promoted embryogenesis as compared to abaxial-side-up orientation. The best response on direct embryo formation was obtained on modified 1/2 MS medium supplemented with 10–20 g/l sucrose, 170 mg/l NaH2PO4, and 0.5 g/l peptone. We also found that three ethylene synthesis inhibitors, 1-aminocyclopropane-1-carbocylic acid (ACC), silver nitrate, and cobalt chloride enhanced embryo In vitro Morphogenesis and Micro-Propagation of Orchids ✦ 51 Fig. 3.3. Direct somatic embryogenesis of Oncidium. 3.3A In vitro morphogenesis of leaf explants on 1/2 MS basal medium plus TDZ: embyogenic nodular mass protruding from the wound surfaces (a); embryogenic nodular mass on epidermal cell layers of leaf explant (b); cluster of embryos on tip of leaf explant (c); small globular embryo formed form the mesophyll cells under the leaf surface (d); small PLBs eventually formed from embryos (e); cluster of 4 young PLBs from embryos (f ); PLBs developed on the surface of a piece of leaf explant (g); tip of a leaf explant bearing a cluster of embryos. 3.3B Morphogenesis of embryos and PLBs: cross section of a leaf explant showing embryonic cell of epidermal layer with densely stained cytoplasm and nuclei (a); a young embryo with approximately 10 cells protruding from the epidermal layer of a leaf explant (b); a developing embryo with more embryonic cells (c); well-developed embryo or young PLB protruding into the adjacent surface (d). ( From Chen et al. (1999) Plant Cell Rep 19:143–149.) formation.15 Gibberellic acid (GA3), cycocel, and paclobutrazol, in general, retarded embryo formation, while ancymidol in low concentration had a promotive effect.16 Also, 2,3,4-triiodobenzoic (TIBA) had a promotive effect on embryo formation.17 We also reported that calli derived from Oncidium roots, leaves, and stems all showed their embryogenic ability for plant regeneration 52 ✦ W.-C. Chang via embryo formation,18 and auxins and cytokinins played a role in embryogenesis similar to direct embryogenesis. Such embryogenesis from a variety of tissue explants of Oncidium is easy to induce in large quantity. The usefulness of direct embryogenesis for genetic transformation was clearly demonstrated in a recent report on transformation attempt in Oncidium. Seed-derived PLBs were bombarded with psSPFLP containing genes encoding a sweet pepper ferredoxin-like protein (pflp), hygromycin phosphotransferase, and b-glucuronidase (GUS) driven by the cauliflower mosaicvirus (CaMV) 35S promoter. PCR analysis confirmed the success of the transformation (Li et al.19). 3.4 Phalaenopsis Ten years ago, the use of flower stalks20 and cytokinin-induced nodes21 became popular for mass propagation of Phalaenopsis. In addition, PLB proliferation in liquid medium for mass propagation has been reported.22 A paper on Phalaenopsis regeneration from callus culture via somatic embryogenesis has generated much attention.23 The authors believed that their histological observations of the early young stage of PLBs regenerated from the primary callus are somatic embryos, with no variation in flowering plants regenerated through somatic embryogenesis. However, lacking were data and descriptions on the subculture of the primary callus culture. Chen et al.24 reported a reliable protocol for plant regeneration from subcultured callus of Phalaenopsis nebula. The authors still used the term “PLB” for the early structure of the regeneration (Fig. 3.4). Successive mass culture of PLBs in suspension culture for mass propagation is promising. Doritaenopsis callus cells can be subcultured in suspension culture, and the suspension cells can be cryopreserved.25 Young et al.26 demonstrated mass multiplication of PLBs in a bioreactor system and subsequent plant regeneration. Tokuhara and Mii27 obtained embryogenic callus and cell suspensions from shoot tips of flower stalk buds and later found that carbohydrate sources played a crucial role in somatic embryogenesis in cell suspension cultures.28 The thin-cell layer method has been an efficient means for plant regeneration of orchids. A thin-section culture system for rapid regeneration In vitro Morphogenesis and Micro-Propagation of Orchids ✦ 53 Fig. 3.4. Plant regeneration for callus culture of Phalaenopsis Nebula: protocorm-like body (PLB)-derived sub-culturable callus (a); embryo-like structures on the surface of callus (b); longitudinal section of callus showing a pro-embryolike structure formed on the surface of the callus (c); PLBs formed on subcultured green callus (d); cluster of PLBs derived from green calli (e); regenerated plantlets in vitro (f ); potted regenerants grown in sphagnum moss in the greenhouse (g); 76 chromosomes of a metaphase cell form a rot-tip squash of a regenerant (h); root-tip squash showing chromosomes of the donor plant. ( From Chen et al. (2000) In Vitro/Plant 46:420–423.) 54 ✦ W.-C. Chang of the monopodal orchid hybrid Aranda has been developed by Lakshmanan et al.29 Le et al.30 obtained high-frequency shoot regeneration from Rhynchostylis gigantean using thin cell layers of leaves. Park et al.31 developed an efficient and rapid method of PLB regeneration of Doritaenopsis that utilizes thin sections obtained from leaves as explants. Root tips also produced PLBs on a defined medium supplemented with TDZ.32 Direct somatic embryogenesis occurs in a culture of Phalaenopsis. A simple protocol for regenerating a Phalaenopsis cultivar through direct somatic embryogenesis on leaf explants in a defined medium plus TDZ (Fig. 3.5) was established.33,34 Repetitive production of embryos involving secondary embryogenesis could be obtained by culturing segments of embryogenic mass on TDZ-containing medium, and plant conversion from embryos was successfully achieved on regulator-free growth medium. Basically, the plantlet obtained through direct somatic embryogenesis originates from a single cell origin. Therefore, the method is suitable for regenerating transgenic plants and could avoid the formation of chimera. 3.5 Dendrobium Highlights among the Dendrobium literature can be categorized into three groups: shoot-bud proliferation, regeneration from callus, and direct somatic embryogenesis. Nayak35 developed a TDZ-induced high-frequency shoot proliferation of two Dendrobium spp. High-frequency proliferation from thin cross-sections of PLBs of Dendrobium nobile was achieved in a defined medium.36 Plant regeneration of D. fimbriatum from shoot-tip-derived callus has been documented.37 Encapsulated PLBs has been attempted for long-term storage of the seed-derived PLBs.38 Tricontanol (TRIA), a long 30-carbon primary alcohol, also a naturally occurring plant growth promoter, has been used for promoting shoot proliferation from cultured shoot tips of D. nobile.39 Several medicinally important orchids have been intensively studied in Taiwan over the last decade. Asymbiotic germination of immature seeds, plant development, and ex vitro establishment of plants of Shi-hu (D. tosaense) has been studied in detail by Tsay’s group.40 Nodal In vitro Morphogenesis and Micro-Propagation of Orchids ✦ 55 Fig. 3.5. Direct somatic embryogenesis for leaf explants of Phalaenopsis “Little Steve” and subsequent plant regeneration. 3.5A Somatic embryos formed directly from the leaf surface after 30 d of culture (a); embryos formed directly from the adaxial surface and the wounding of a leaf explant (b); Scanning electron microscopy (SEM) photograph of somatic pre-embryos formed from the leaf surface (c); SEM photograph of leaf-derived embryos (d); cluster of leaf-derived embryos forming shoots (e); regenerated plantlets (f ). 3.5B Histology of direct somatic embryogenesis from leaf explants. Embryogenic cells originating from the leaf epidermal cell layer (a); a globular embryo contains densely stained and smaller embryonic cells than leaf cells (b); a developing embryo (c); a mature embryo, so-called protocorm, consisting of a shoot apical meristem and root meristem, and meristematic cells, formed from the basal region, were the origin of secondary embryogenesis (d). ( From Kuo et al. (2005). In Vitro/Plant 41:453–456.) segments have been used for mass propagation of D. candidum, a Chinese medicinal plant, through lateral bud proliferation.41 Direct organogenesis and direct somatic embryogenesis in Dendrobium spp. in vitro have been documented recently. Cytokinins were found causing direct somatic embryo formation on leaf explants of Dendrobium chiengmai Pink (Fig. 3.6).42,43 Direct shoot formation from 56 ✦ W.-C. Chang Fig. 3.6. Direct somatic embryogenesis from leaf explants of Dendrobium “Chiengmai Pink.” Somatic embryos directly formed from leaf tip after 30 d of culture (a); embryo formed on the cut end (b); leaf-derived embryos formed shoots (c); regenerants (d); scanning electron microscopy (SEM) photograph of embryos were originating from leaf epidermal cells (e); an embryo in globular shape (f ); secondary somatic embryos formed from the sheath leaf of a primary somatic embryos (g); secondary embryos in globular shape (h). ( From Chung et al. (2005). In Vitro/Plant 41:765–769.) In vitro Morphogenesis and Micro-Propagation of Orchids ✦ 57 foliar explants and PLBs has been developed for in vitro propagation of two Dendrobium hybrids. Lin’s group44 demonstrated a successive transformation on a dendrobium to resist cymbidium mosaic virus. 3.6 Paphiopedilum and Cypripedium Not much research has been documented in the past 10 years on lady’s slippers. Efficient protocols for seed germination of slippers for propagation are still urgently needed. Regeneration from cultured explants is still considered difficult. Totipotent calli of a Paphiopedilum hybrid were induced from seed-derived protocorms on a defined medium containing 2,4-D and TDZ (Fig. 3.7).45 Huang et al.46 released a mericloning protocol enabling shoot multiplication and rooting of Paphiopedilum in a single medium. Nodal explants were useful for mass shoot bud proliferation on two Paphiopedilum hybrids (Fig. 3.7).47 Direct multiple shoot bud proliferation for leaf explants of two Paphiopedilum hybrids clearly demonstrated that a practical mass propagation protocol can be developed from the morphogenesis.48 In the case of Cypripedium, five papers on improving seed germination have been published in the last decade. Cold temperature pre-treatment in seeds at 4°C is crucial for seed germination of C. candidum.49 A fungus isolated from the roots of C. macranthos var rebunense induced symbiotic germination. Cold treatment of seeds at 4°C prior to fungal inoculation was required for symbiotic germination. Changing the timing of inoculation of the fungus to the seeds greatly improved germination frequency.50 Totipotent callus of C. formosanum, an endangered slipper orchid species, was induced for seed-derived protocorm segments.51 Protocorms were regenerated from the callus on a medium supplemented with BA only and, eventually, developed into plants. The age of seed capsule is crucial for good germination in C. formosanum.52 Yan et al.53 recently demonstrated an efficient way to mass-propagate C. flavum through multiple shoots of seedlings derived from mature seeds. The authors demonstrated that the efficiency in plantlets from the multiple shoots was much higher than that through PLBs derived from seeds. 58 ✦ W.-C. Chang Fig. 3.7. Plant regeneration of Paphiopedilum in vitro. 3.7A Plant regeneration via shoot-bud formation from seed-derived subculturable callus ( From Lin et al. (2000). Plant Cell Tiss Org Cult 62:21–25.) 3.7B Plant regeneration via direct shoot-bud formation from nodal tissue of dwarf stem (Chen et al. (2002). In Vitro/Plant 38:595–597.) 3.7C Plant regeneration through direct shoot-bud formation from leaf explants ( From Chen et al. (2004). Plant Cell Tiss Org Cult 76:11–15.) In vitro Morphogenesis and Micro-Propagation of Orchids ✦ 59 3.7 Epidendrum Sympodium orchid Epidendrum radicans was successively propagated via multiple-shoot proliferation from stem explants.54 3.8 Outlook: How Basic Research can Help the Orchid Industry Any orchid biotechnology research should be relevant to the needs of orchid growers in any country and the benefits of the research transmitted to consumers at an affordable price. For the challenges ahead in orchid biotechnology and its practical application, I strongly encourage integrating modern technologies with classical breeding and physiological studies for the future success of the orchid industry. The physiological basics of crop yield have been dealt with in great detail for most agricultural crops. Of course, orchids are no exception. In starting an orchid business or research, an important consideration is to ensure a steady supply of plant materials. Obtaining plant materials through conventional vegetative propagation methods is slow and costly. Today, the supply of uniform clonal planting materials comes, mainly from in vitro culture. The demand for micropropagated orchids also explains the recent, rapid increase in the number of commercial orchid laboratories and companies operating in Asia. Clonal propagation of orchids, involving batch tissue culture, has been the mainstay throughout the world since 1960. Many problems are associated with the in vitro method and the batch tissue culture approach, as you can see in the presentations today. For example, batch culture is essentially a closed system, and in vitro conditions change with time and may not be optimal for cell growth. To optimize cell growth, all the factors must be maintained at optimal conditions. However, in batch culture, this condition is only possible by frequent subculturing. Subculturing involves considerable time and effort and will certainly increase production costs. Thus, improved cultural methodology is essentially based on a better understanding of basic physiology. 60 ✦ W.-C. Chang Furthermore, orchid seedlings grown in flasks are first transferred to a community pot, then to thumb pots, and then to a larger typical commercial pot. The duration of each transfer is about 3- to 6-months. Surprisingly, few scientific studies have investigated the growth and survival rate of plantlets during and after transfer from culture flasks to community pots in the greenhouse. In fact, high plantlet mortality rates have often been showed with some orchids. The hardening or acclimatization of plants in flasks and community pots certainly deserves more research. More than two years are required for the orchid plantlets to reach the flowering stage. Orchids, particularly those with an epiphytic origin, are notoriously slow-growing plants. In their natural habitat, epiphytes usually meet with a greater degree of environmental stress, such as the supply of water and minerals. An understanding of how these orchids cope physiologically with environmental stress is important in order to improve their cultivation. Flowering production is a major concern of any orchid industry. Flowering production depends on the genetic make-up of the orchid hybrids and how well they are grown. To achieve maximal flower yield, proper agronomic practices must be understood. Equally important is the control of flowering to meet market demand. The ability to control flowering in tropical orchids by physiological tools is indeed crucial. The importance of proper post-harvest handling of cut-flowers has often been overlooked in the orchid cut-flower industry. The lack of proper post-harvest management in the industry is attributed to the paucity of information on the post-harvest physiology of orchid flowers. New findings on the formation of direct embryos and shoot-buds for various tissue explants and in vitro flowering of regenerated embryos and plantlets on several orchids may address only a few aspects of the basic studies on orchids. The new knowledge may someday help to support the research I have mentioned previously. In the Chinese literature, Lan (which means orchid in Chinese), is often personified as a man of virtue who strives for self-discipline, champions principles, and does not succumb to poverty and distress. Confucius wrote some 2500 years ago the following: “Lan that grows in deep forests never withholds its fragrances even when no one appreciates it.” As a biotechnology researcher of orchids, I would like to see more insightful researches on such unique plants in the years to come. In vitro Morphogenesis and Micro-Propagation of Orchids ✦ 61 References 1. Chang WC, Hsing YE. (1980) In vitro flowering of embryoids derived from mature root callus of ginseng (Panax ginseng). Nature 284:341–342. 2. Chang WC, Hsing YE. (1980) Plant regeneration through somatic embryogenesis in root-derived callus of ginseng (Panax ginseng C. A. Meyer). Theor Appl Genet 57:133–135. 3. Lin CS, Chen CT, Lin CC, Chang WC. (2003) A method for inflorescence proliferation. Plant Cell Rep 21:838–843. 4. Lin CS, Lin CC, Chang WC. (2004) Effect of thidiazuron on vegetative tissue-derived somatic embryogenesis and flowering of Bambusa edulis. Plant Cell Tiss Org Cult 76:75–82. 5. Chang C, Chang WC. (1998) Plant regeneration from callus culture of Cymbidium ensifolium var. misericors. Plant Cell Rep 17:251–255. 6. Chang C, Chang WC. (2003) Cytokinins promotion of flowering in Cymbidium ensifolium var. misericors in vitro. Plant Growth Reg 39:217–221. 7. Chang C, Chang WC. (2000) Effect of thidiazuron on bud development of Cymbidium sinense Willd in vitro. Plant Growth Regul 30:171–175. 8. Huan LVT, Takamura T, Tanaka M. (2004) Callus formation and plant regeneration from callus through somatic embryo structures in Cymbidium orchid. Plant Sci 166:1443–1449. 9. Kstenyuk I, So IS. (1999) Induction of early flowering in Cymbidium niveo-narginatum (Mark) in vitro. Plant Cell Rep 19:1–5. 10. Chang C, Chen YC, Yen HF. (2005) Protocorm or rhizome? The morphology of seed germination in Cymbidium dayanum reichb. Bot Bull Academia Sinica 46:71–74. 11. Chen JT, Chang C, Chang WC. (1999) Direct somatic embryogenesis on leaf explants on Oncidium Grower Ramsey and subsequent plant regeneration. Plant Cell Rep 19:144–149. 12. Chen JT, Chang WC. (2000). Plant regeneration via embryo and shoot bude formation from flower-stalk explants of Oncidium Sweet Sugar. Plant Cell Tiss Org Cult 62:95–100. 13. Chen JT, Chang WC. (2001) Effects of auxins and cytokinins on direct somatic embryogenesis on leaf explants of Oncidium Grower Ramsey. Plant Growth Reg 34:229–232. 14. Chen JT, Chang WC. (2002) Effects of tissue culture conditions and explant characteristics on direct somatic embryogenesis on Oncidium Grower Ramsey. Plant Cell Tiss Org Cult 69:41–44. 15. Chen JT, Chang WC. (2003) 1-aminocyclopropane-1-carboxylic ancid enhanced direct somatic embryogenesis from Oncidium leaf cultures. Biol Plant 46:455–458. 62 ✦ W.-C. Chang 16. Chen JT, Chang WC. (2003) Effects of GA3, ancymidol, cycocel and paclobutrazol on direct somatic embryogenesis of Oncidium in vitro. Plant Cell Tiss Org Cult 72:105–108. 17. Chen JT, Chang WC. (2004) TIBA affects the induction of direct somatic embryogenesis from leaf explants of Oncidium. Plant Cell Tiss Org Cult 79:315–320. 18. Chen JT, Chang WC. (2000) Efficient plant regeneration through somatic embryogenesis form callus cultures of Oncidium (Orchidaceae). Plant Sci 160:87–93. 19. Li SH, Kuoh CS, Chen YH, et al. (2005) Osmotic sucrose enhancement of single-cell embryogenesis and transformation efficiency in Oncidium. Plant Cell Tiss Org Cult 81:183–192. 20. Chen YQ, Piluek C. (1995) Effects of thidiazuron and N-6-beneyladaminopurine on shoot regeneration of Phalaenopsis. Plant Growth Reg 16:99–101. 21. Duan DX, Chen H, Yazawa S. (1996) In vitro propagation of Phalaenopsis via culture of cytokinin-induced nodes. J Plant Growth Reg 15:133–137. 22. Park YS, Kakuta S, Kano A, Okabe M. (1996) Efficient propagation of protocorm-like bodies of Phalaenopsis in liquid medium. Plant Cell Tiss Org Cult 45:79–85. 23. Ishii Y, Takamura T, Goi M, Tanaka M. (1998) Callus induction and somatic embryogenesis of Phalaenopsis. Plant Cell Rep 17:446–450. 24. Chen YC, Chang C, Chang WC. (2000) A reliable protocol for plant regeneration form callus culture of Phalaenopsis. In Vitro/Plant 36:420–423. 25. Tukazaki H, Mii M, Tokuuhara K, Ishikawa K. (2000) Cryopreservation of diritaenopsis suspension culture by vitrification. Plant Cell Rep 19:160–164. 26. Young PS, Murthy HN, Yoeup PK. (2000) Mass multiplication of protocorm-like bodies using bioreactor system and subsequent plant regeneration in Phalaenopsis. Plant Cell Tiss Org Cult 63:67–72. 27. Tokuara K, Mii M. (2001) Induction of embryogenic callus and cell suspension culture from shoot tips excised from flower stalk buds of Phalaenopsis (Orchidaceae). In Vitro/Plant 37:457–461. 28. Tokuara K, Mii M. (2003) Highly-efficient somatic embryogenesis from cell suspension cultures of Phalaenopsis orchids by adjusting carbohydrate sources. In Vitro/Plant 39:635–639. 29. Lakshmanan P, Loh CS, Goh CJ. (1995) An in vitro method for rapid regeneration of manopodial orchid hybrid Aranda Deborah using thin section culture. Plant Cell Rep 14:510–514. 30. Le BV, Phuong NTH, Hong LTA, et al. (1999) High frequency shoot regeneration from Rhynchostylis gigantean (Orchidaceae) using thin cell layers. Plant Growth Reg 28:178–185. In vitro Morphogenesis and Micro-Propagation of Orchids ✦ 63 31. Park SY, Yeung EC, Chakrabarty D, Paek KY. (2002) An efficient direct induction of protocorm-like bodies form leaf subepidermal cells of Doritaenpsis hybrid using thin-section culture. Plant Cell Rep 21:46–51. 32. Park SY, Murthy HN, Peak KY. (2003) Protocorm-like body induction and subsequent plant regeneration from root tip cultures of Doritaenopsis. Plant Sci 164:919–923. 33. Ku HL, Chen JT, Chang WC. (2005) Efficient plant regeneration through direct somatic embryogenesis from leaf explants of Phalaenopsis “little Steve.” In Vitro/Plant 41:453–456. 34. Chen JT, Chang WC. (2006) Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis anabilis. Biol Plant 50:169–173. 35. Nayak NR, Rath SP, Patnaik S. (1997) In vitro propagation of three epiphytic orchids Cymbidium aloifolium (L.) Sw., Dendrobium apyllum (Roxb.) fishch. and Dendrobium moschatum (Buch-Ham) Sw. Through thidiazuroninduced high frequency shoot proliferation. Sci Hort 71:243–250. 36. Nayak NR, Sahoo S, Patnaik S, Rath SP. (2002) Establishment of thin cross section (TCS) culture method for rapid micropropagation of Cymbidium aloifolium (L.) Sw. and Dendrobium nobile Lindl. (Orchidaceae). Sci Hort 94:107–116. 37. Roy J, Banerjee N. (2003) Induction of callus and plant regeneration form shoot-tip explants of Dendrobium fimbriatum Lindl. Var. oculatum HK.f. Sci Hort 97:333–340. 38. Saiprasad GVS, Polisetty R. (2003) Propagation of three orchid genera using encapsulated protocorm-like bodies. In Vitro/Plant 39:42–48. 39. Malabadi RB, Mulgund GS, Kallappa N. (2005) Micropropagation of Dendribium nobile form shot tip sections. J Plant Physiol 162:473–478. 40. Lo SF, Nalawade SM, Kuo CL, et al. (2004) Asymbiotic germination of immature seeds, plantlet development and ex vitro establishment of plants of Dendrobium tosaense makino — A medicinally important orchid. In Vitro/Plant 40:528–535. 41. Shiau YH, Malawade SM, Hsia CN, et al. (2005) In vitro propagation of the Chinese medicinal plant, Dendrobium candidum Wall, ex Lindl., from axenic nodal segments. In Vitro/Plant 41:666–670. 42. Chung HH, Chen JT, Chang WC. (2005) Cytokinins induce direct somatic embryogenesis of Dendrobiu chiengmai Pink and subsequent plant regeneration. In Vitro/Plant 45:765–769. 43. Chung HH, Chen JT, Chang WC. Cytokinins induce direct somatic embryogenesis from leaf explants of Dendrobiu chiengmai Pink. Biol Plant (in press). 44. Chang C, Chen YC, Hsu YH, et al. (2005) Transgenic resistance to Cymbidium mosaic virus in Dendrobium expressing the viral capsid protein gene. Transgenic Res 14:41–46. 64 ✦ W.-C. Chang 45. Li YH, Chang C, Chang WC. (2000) Regeneration from callus culture of Paphiopedilum hybrid. Plant Cell Tiss Org Cult 62:21–25. 46. Huang LC, Lin CJ, Kuo CI, et al. (2001) Paphiopedilum cloning in vitro. Sci Horti 91:111–121. 47. Chen TY, Chen JT, Chang WC. (2002) Multiple shoot formation and plant regeneration from stem nodal explants of Paphiopedilum orchids. In Vitro/ Plant 38:595–597. 48. Chen TY, Chen JT, Chang WC. (2004) Plant regeneration through direct shoot bud formation from leaf cultures of Paphiopedilum orchids. Plant Cell Tiss Org Cult 76:11–15. 49. Shimura H, Koda Y. (2004) Micropropagation of Cypripedium macranthos var. rebunense through protocorm-like bodies derived from mature seeds. Plant Cell Tiss Org Cult 78:273–276. 50. Shimura H, Koda Y. (2005) Enhanced symbiotic seed germination of Cypripedium macranthos var. rebunense following inoculation after cold treatment. Physiol Plant 123:281–287. 51. Lee YI, Lee N. (2003) Plant regeneration from protocorm-derived callus of Cypripedium formosanum. In Vitro/Plant 39:475–479. 52. Lee YI, Lee N, Yeung EC, Chung MC. (2005) Embryo development of Cypripedium formosanum in relation to seed germination in vitro. J Amer Soc Horti Sci 130:747–753. 53. Yan N, Hu H, Huang Jl, et al. (2006) Micropropagation of Cypripedium flavum through multiple shoots of seedlings derived from mature seeds. Plant Cell Tiss Org Cult 84:113–117. 54. Chen LR, Chen JT, Chang WC. (2002) Efficient production of protocormlike bodies and plant regeneration from flower stalk explants of the sympodial orchid Epidendrum Radicans. In Vitro/Plant 38:441–445. Chapter 4 Somaclonal Variation in Orchids Fure-Chyi Chen*,† and Wen-Huei Chen‡ Mass production of orchids is achieved through micropropagation of axillary buds of flower stalks or shoot meristem culture. Somaclonal variation occurs during proliferation of both shoots and protocormlike bodies (PLBs) and leads to morphological or physiological changes in the finished potted plants. We observed both vegetative and reproductive variants, depending on the cultivar or genetic background, among tissue-cultured orchid plants. The use of molecular approaches such as random amplified polymorphic DNA (RAPD), cDNA-RAPD and cDNA suppression subtractive hybridization has resulted in the successful identification of expressed sequence tags (ESTs) from wildtype and peloric flower buds of several Phalaenopsis and Doritaenopsis hybrids. Several candidate genes were cloned and transcript levels compared in these plants. The results suggested that some genes, such as a retroelement, could have been abnormally activated in the peloric mutants. We discuss other investigations, such as DNA methylation status, which might also play a role in somaclonal variations in orchids. 4.1 Introduction Somaclonal variation refers to the variation seen in plants regenerated from tissue culture of any plant tissue. Earlier work on *Corresponding author. † Department of Plant Industry and Graduate Institute of Biotechnology, National Pingtung University of Science & Technology, Pingtung, Taiwan. ‡ Department of Life Sciences, National University of Kaohsiung, Kaohsiung, Taiwan. 65 66 ✦ F.-C. Chen and W.-H. Chen somaclones aimed at selecting for breeding valuable genotypes such as variant protoclones inheriting new phenotypes1,2 or with altered ploidy levels.3,4 For other crops, interest was in selecting somaclonal variants with disease resistance, low-temperature tolerance and other traits;5–7 (for detailed reviews of somaclonal variation, see Refs. 1, 7, 8). Orchids are grown worldwide for their commercial value.9 In Taiwan, Phalaenopsis, Oncidium and several other orchid genera have been developed quickly in response to market demand. A novel orchid variety selected from a seedling population results from crosshybridization of two parents with desirable traits. The selected plant is then propagated from either axillary buds of flower stalks or shoot meristem culture in vitro. Such mass propagation can result in several hundred thousands of plantlets with uniform growth and flowering time. Some micropropagated plantlets of certain hybrids may appear abnormal during the vegetative or reproductive stages because of somaclonal variation, which is a concern for orchid growers because the variants may affect pot plant quality. The mechanism of these variations during tissue culture is still largely unknown. Regarding somaclonal variation of other crop plants, the past two decades have seen investigations into DNA methylation status, activation of transposable elements or retrotransposons, and histone modifications.10–16 This chapter covers the methods of tissue culture, somaclonal variation, and molecular approaches to understanding the mechanisms of somaclonal epigenetic changes in exploring novel strategies for lowering the somaclonal variation rate during tissue culture of orchids. 4.2 Orchid Tissue Culture and Plantlet Production To evaluate horticultural traits or performance of a hybrid of two desirable cultivars, plants of a small population of the same clones derived from tissue culture must be grown in potting medium. For tissue culture, first an elite orchid plant from the hybrid combination is selected, usually during the flowering stages, then an axillary bud is excised from a pseudobulb axil or flower-stalk node for use as starting explant materials. The materials are surface sterilized with diluted sodium hypochloride Somaclonal Variation in Orchids ✦ 67 containing surfactant and washed with sterile water several times. The explants are then cultured on a suitable nutrient medium, such as Hyponex-based medium supplemented with cytokinin and auxin,17 and plantlets are potted. Oncidium and Phalaenopsis orchids have been successfully propagated by culture of flower-stalk nodes.17,18 Generally, two methods of micropropagation are adopted by the industry, one through multiplication of adventitious buds and the other through the induction and subsequent proliferation of protocorm-like bodies (PLBs). PLBs have been successfully induced from in vitro-grown etiolated leaves of Phalaenopsis aphrodite.19 4.3 Somaclonal Variation in Orchids Orchid mutants can be derived from variations during tissue culture or from seedling mutations. Since these days mericloned plants are preferred for the flower market,20 one can look for orchid plants with abnormal morphology during visits to orchid nurseries. We grew such plants in the greenhouse for at least two flowering seasons to further observe their stability. From our observations, the mutations could be divided into two categories: those affecting vegetative growth and those rendering reproductive abnormalities, such as peloric or semipeloric flowers on which the lateral petals are completely or partially converted into labellum (lip)-like structures.21 We obtained several vegetative mutations, including overabundant leaves, multiple axillary shoots, distorted sickle-shaped leaves, and polyploidy (Fig. 4.1). In reproductive mutants, peloric and semipeloric flowers have been observed in mericloned plants of many Phalaenopsis and/or Doritaenopsis hybrids, with semipeloric flowers being more common (Fig. 4.2). Since Doritaenopsis hybrids are derived from cross-hybridization of Phalaenopsis and Doritis genotypes, their progenies tend to be more unstable genetically than those of Phalaenopsis hybrids because of variation in chromosome configuration or genome size.21,22 Mutations affecting internode length were reported in tissue culture-derived plants of Phalaenopsis and Doritaenopsis orchids.23 Peloric flower mutations vary depending on the degree of severe conversion of petals into lip-like protrusions or narrowed petals. In some 68 ✦ F.-C. Chen and W.-H. Chen A B C D E F Fig. 4.1. Vegetative mutants in orchids. (A) Carotenoid accumulation in leaves and inflorescence of a Phalaenopsis aphrodite; (B) Sickle-shaped leaves in a P. Hwafeng Redjewel mutant plant; (C), A Doritaenopsis Purple Gem mutant with an overabundance of leaves; (D) A Dtps. mutant with flower-like structures on the leaves; (E), A hybrid of Dtps. Sogo Pride mutant with multiple axillary shoots; (F) Normal plants of Phalaenopsis mericlones. A B C D E F Fig. 4.2. Reproductive mutants in orchids. (A) Mutation with petaloid column; (B) A semi-peloric flower with labellum-like lateral petals; (C) Sepal mutation with protrusion on the upper epidermal tissues of lateral sepals; (D) Mutation possessing anther-like structures in the distal part of lateral petals; (E) Yellow peloric flowers; (F) Anthocyanin pattern mutation in a Doritaenopsis hybrid, wild-type on the left and color-stripe mutant on the right. Somaclonal Variation in Orchids ✦ 69 severe cases, the lateral petals are changed completely into lip structures, and the plants lack pollinia. Another class of mutations involves serrated petals and labellum. The remaining plant tissues are not affected. The serrated petals are visible only during the flowering stages. The cause of this mutation is not clear. A serrated-petal mutation in Arabidopsis was caused by altered sterol composition and ecotopic endoreduplication in petal tips.24 Methods successful in Arabidopsis study could be used to investigate whether endoreduplication caused the serrated petals of our Phalaenopsis mutants and to clone the candidate gene for sterol biosynthesis. 4.4 Strategy for Studying Somaclonal Variation Since the generation time for orchids is relatively long, conventional cross-hybridization or selfing to learn the inheritance of somaclonal variation is difficult. Unconventional molecular approaches to study epigenetic mutations in other organisms have involved suppression subtractive hybridization (SSH);25,26 differential display;27,28 molecular markers such as random amplified polymorphic DNA (RAPD);29–33 amplified fragment-length polymorphism (AFLP);34,35 methylationsensitive restriction FLP (RFLP);13 and candidate gene cloning by use of degenerate primers and PCR based on conserved amino acid sequences of target genes. Flow cytometry has been used to study endopolyploidy of embryos or PLBs in Vanda and Doritaenopsis orchids during in vitro germination, with ploidy levels enhanced by treatment with the auxins N-acetyl aspartate or 2,4-dichlorophenoxyacetic acid.36,37 4.4.1 Molecular markers Somaclones derived from in vitro culture of flower-stalk nodes of the Phalaenopsis cultivar True Lady were studied by use of RAPD markers and isozyme electrophoretic patterns. With the use of 38 selected random primers for PCR amplification of genomic DNAs, polymorphism was revealed in somaclones with deformed flower shapes,30 and with two isozymes, viz., aspartate aminotransferase and phosphoglucomutase, wild types were differentiated from variants.30 cDNA-RAPD analysis revealed differences in mRNA expression in both the wild type and peloric and semipeloric variants in P. Little Mary.26 70 ✦ F.-C. Chen and W.-H. Chen 4.4.2 cDNA suppression subtractive hybridization Differentially expressed genes have been effectively studied by use of cDNA SSH25 and modified SSH methods such as SSH followed by negative subtraction chain (NSC.)38 Both SSH or NSC techniques can enrich target sequences at different efficiency rates, but background signals may be a concern. We adopted SSH to compare transcripts enriched in young flower buds of both the wild-type and peloric Phalaenopsis and Doritaenopsis hybrids.26 One problem encountered with SSH of Phalaenopsis orchids is that often the plants were infected by viruses such as cymbidium mosaic virus (CymMV) and odontoglossum ringspot virus (ORSV), which may interfere with the effectiveness of the SSH approach. In one case, approximately 28% of the ESTs from the peloric mutants were due to CymMV sequences.26 A similar result has been reported for P. Hsiang Fei with use of cDNA-AFLP.39 To confirm the expression level of selected ESTs resulting from our SSH analysis, we used real-time RT-PCR to compare the transcripts in both the wild-type and peloric and semipeloric flower buds. CymMV RNAdependent RNA polymerase, a retroelement, and an auxin-regulated dual-specificity cytosolic kinase were preferentially upregulated in the peloric flower buds (Fig. 4.3) and to a lesser extent in semipeloric buds.26 A TCP protein, reported to be responsible for the symmetric development of plant organs,40 was slightly up-regulated in peloric flower buds.26 Obtaining virus-free orchid plants will help in better adopting SSH for future analysis of transcript profiling in the somaclonal mutants. 4.4.3 Methylation-specific restriction PCR assays DNA hypomethylation, which may lead to abnormal activation of certain genes, has been related to epigenetic changes in diseased animal tissues.41 Methylation-sensitive restriction enzymes and quantitative PCR have been widely used to investigate methylation status in the promoter regions of target genes.42,43 Mutation in histone H3 methylation may also contribute to DNA methylation changes.44,45 McrBC, a restriction enzyme that preferentially digests tracts of DNA flanked by methylcytosine residues, has been used to reveal genome-wide reduction in DNA methylation in the mantled phenotypes of regenerated oil palm.16 The use of these techniques in investigating somaclonal variation in orchids may be profitable. Somaclonal Variation in Orchids ✦ 71 Fig. 4.3. Transcript profiles in wild-type, peloric and semi-peloric flower buds of Phalaenopsis Little Mary by real-time RT-PCR analyses. (A) a TGA1a-like protein; (B) a protein kinase; (C) CymMV RNA-dependent RNA polymerase; (D) a proline iminopeptidase; (E) a retroelement; (F) an unknown protein; (G) auxin-regulated dual specificity cytosolic kinase; (H) cyclophilin-like protein; (I) a transcriptional factor PCF6 or TCP family protein. (Chen et al., 2005; Courtesy of Cell Research.) 4.4.4 Cloning of candidate genes Peloric flowers show a change from bilateral to radial symmetry; however, an attempt to clone the TCP homolog of cycloidea,46 based on conserved amino acid sequences by RT-PCR, was fruitless (Chen FC, unpublished result). Thus, the conserved amino acid sequences of Phalaenopsis orchids may differ from that of other higher plants. A candidate gene of the knotted1-like homeobox gene family,47 which may have roles in the patterning of plant body plans, has been cloned by the design of degenerate primers following alignment of several protein sequences. The knotted1like homeobox genes may play a role in the formation of the lobe in the 72 ✦ F.-C. Chen and W.-H. Chen labellum of Phalaenopsis flowers. Four B-class Phalaenopsis DEF-like MADS-box genes were detected in floral organs; the expression of PeMADS4 was in the lips and column of the wild type, and it extended to the lip-like petals in the peloric mutant.48 4.5 Conclusions We have obtained many somaclonal variants of Phalaenopsis and Oncidium orchids. Molecular and morphological studies have revealed abnormalities in the vegetative and reproductive tissues. Orchid viruses exist widely in orchid plants and may hinder some molecular analyses. Several candidate genes were preferentially expressed in the peloric mutants. The wide spectrum of somaclonal variation may contribute to our further understanding of the molecular basis of epigenetic changes at the DNA or chromatin levels. Recently, we cloned DNA methyltransferase from Phalaenopsis. Our preliminary study indicated its downregulation in peloric mutants. Further characterization of this gene should offer an opportunity to elucidate its function in the DNA methylation status of orchid genomes. 4.6 Perspectives Because epigenetic changes often occur in orchid tissue culture, the question is whether the phenomenon actually disturbs the developmental pattern of an orchid flower. Several approaches to investigation are available and await confirmation. Genetic transformation of candidate genes to reveal their function in orchids requires efficient tissue culture and regeneration, such as induction of PLBs from cultures of etiolated leaf segments.19 The candidate genes can also be used to transform corresponding mutants of Arabidposis or rice to complement their defect. One can also use RNA interference to block the candidate genes in planta to abolish their morphological competency. Acknowledgments The work described in this chapter was funded by the Council of Agriculture, Taiwan, awarded to the first author. We thank the graduate students and research assistants in both authors’ labs for assistance and support. Somaclonal Variation in Orchids ✦ 73 References 1. Bidney DL, Shepard JF. (2004) Phenotypic variation in plants regenerated from protoplasts: the potato system. Biotechnol Bioeng 23:2691–2701. 2. Ramulu KS, Dijkhuis P, Roest S. 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(2002) Methylation-sensitive RFLPs: Characterisation of two oil palm markers showing somaclonal variation-associated polymorphism. Theor Appl Genet 104:1263–1269. 14. Jaligot E, Beule T, Baurens FC, et al. (2004) Search for methylation-sensitive amplification polymorphisms associated with the mantled variant phenotype in oil palm (Elaeis guineensis Jacq). Genome 47:224–228. 74 ✦ F.-C. Chen and W.-H. Chen 15. Koukalova B, Fojtova M, Lim KY, et al. (2005) Dedifferentiation of tobacco cells is associated with ribosomal RNA gene hypomethylation, increased transcription, and chromatin alterations. Plant Physiol 139:275–286. 16. Kubis SE, Castilho AM, Vershinin AV, Heslop-Harrison JS. (2003) Retroelements, transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation. Plant Mol Biol 52:69–79. 17. Chen FC, Hsu CC. (2002) Techniques of orchid tissue culture. In: Proceedings of the Current Situation of Floricultural Industry and Future Perspectives Taiwan Floricultural Association, Taipei, pp. 26–36. 18. Chen FC, Chen TC. (1998) Effect of salt strength and organic additives on the in vitro growth of protocorm-like-bodies and plantlets of Oncidium Grower Ramsey. J Chin Soc Hortic Sci 44:403–412. 19. Hsu CC, Chen FC. (2003) Plant regeneration from protocorm-like bodies induced in etiolated leaves of Phalaenopsis aphrodite Rchb. f. J Chinese Soc Hort Sci 49:335–342. 20. Shen T-M, Hsu ST, Chen JJ, Chen FC. (2006) Research and development of Phalaenopsis industry in Taiwan. In: Proceedings of the 2006 Taiwan Intl Orchid Symp, Taiwan Orchid Growers Association, Tainan, Taiwan, pp. 7–13. 21. Chen FC, Chen YH, Lee WL, et al. (2004) Orchid mutants from tissue culture — implication of gene expression to orchid development. In: Proceedings of the 8th Asia Pacific Orchid Conference, Taiwan Orchid Growers Association, Tainan, Taiwan, pp. 314–328. 22. Lin S, Lee HC, Chen WH, et al. (2001) Nuclear DNA contents of Phalaenopsis sp. and Doritis pulcherrima. J Amer Soc Hort Sci 126:195–199. 23. Tokuhara K, Mii M. (1998) Somaclonal variations in flower and inflorescence axis in micropropagated plants through flower stalk bud culture of Phalaenopsis and Doritaenopsis. Plant Biotech (Japan) 15:23–28. 24. Hase Y, Fujioka S, Yoshida S, et al. (2005) Ectopic endoreduplication caused by sterol alteration results in serrated petals in Arabidopsis. J Exp Bot 56:1263–1268. 25. Diatchenko L, Lau YF, Campbell AP, Chenchik A. (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 93:6025–6030. 26. Chen, YH, YJ Tsai, JZ Huang, FC Chen. (2005) Transcription analysis of peloric mutants of Phalaenopsis orchids derived from tissue culture. Cell Res 15:639–657. 27. Liang P, Pardee AB. (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–971. 28. Tregear JW, Morcillo F, Richaud F, et al. (2002) Characterization of a defensin gene expressed in oil palm inflorescences: induction during tissue Somaclonal Variation in Orchids ✦ 75 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. culture and possible association with epigenetic somaclonal variation events. J Exp Bot 53:1387–1396. Purwantoro A, Supaibulwatana K, Mii M, Koba T. (1999) Cytological and RAPD (random amplified polymorphic DNA) analysis of somaclonal variation in Easter lily (Lilium longiflorum Thinb). Plant Biotechnol (Jpn) 16:247–250. Chen WH, Chen TM, Fu YM, et al. (1998) Studies on somaclonal variation in Phalaenopsis. Plant Cell Rep 18:7–13. Martins M, Sarmento D, Oliveira MM. (2004) Genetic stability of micropropagated almond plantlets, as assessed by RAPD and ISSR markers. Plant Cell Rep 23:492–496. Palombi MA, Lombardo B, Caboni E. (2007) In vitro regeneration of wild pear (Pyrus pyraster Burgsd) clones tolerant to Fe-chlorosis and somaclonal variation analysis by RAPD markers. Plant Cell Rep 26:489–496. [Epub ahead of print] 10.1007/s00299-006-0256-9 Rival A, Bertrand L, Beulé T, et al. (1998) Suitability of RAPD analysis for the detection of somaclonal variants in oil palm (Elaeis guineensis Jacq.). Plant Breed 117:73–76. Chen J, Henny RJ, Devanand PS, Chao CT. (2006) AFLP analysis of nephthytis (Syngonium podophyllum Schott) selected from somaclonal variants. Plant Cell Rep 24:743–749. Matthes M, Singh R, Cheah S-C, Karp A. (2001) Variation in oil palm (Elaeis guineensis Jacq.) tissue culture-derived regenerants revealed by AFLPs with methylation-sensitive enzymes. Theor Appl Genet 102:971–979. Lim WL, Loh CS. (2003) Endopolyploidy in Vanda Miss Joaquim (Orchidaceae). New Phytol 159:279–287. Mishiba K-I, Okamoto T, Mii M. (2001) Increasing ploidy level in cell suspension cultures of Doritaenopsis by exogenous application of 2,4-dichlorophenoxyacetic acid. Physiol Plant 112:142–148. Li L, Techel D, Gretz N, Hildebrandt A. (2005) A novel transcriptome subtraction method for the detection of differentially expressed genes in highly complex eukaryotes. Nucleic Acids Res 33:e136. Chen H-H, Tsai W-C, Lin S, Chen W-H. (2001) cDNA-AFLP analysis of differential gene expression in the flower buds of Phalaenopsis HSING FEI cv. H. F. and its somaclonal variant. Plant and Animal Genome IX Conference, p. 323. Cubas P, Lauter N, Doebley J, Coen E. (1999b) The TCP domain: a motif found in proteins regulating plant growth and development. Plant J 18:215–222. Ortmann CA, Burchert A, Hölzle K, et al. (2005) Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region. Nucleic Acids Res 33:6895–6905. 76 ✦ F.-C. Chen and W.-H. Chen 42. Germann S, Juul-Jensen T, Letarnec B, Gaudin V. (2006) DamID, a new tool for studying plant chromatin profiling in vivo, and its use to identify putative LHP1 target loci. Plant J 48:153–163. 43. Ott T, Nelsen-Salz B, Doring HP. (1992) PCR-aided genomic sequencing of 5’ subterminal sequences of the maize transposable element Activator (Ac) in transgenic tobacco plants. Plant J 2:705–711. 44. Jackson JP, Lindroth AM, Cao X, Jacobsen SE. (2002) Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416:556–560. 45. Johnson L, Cao X, Jacobsen S. (2002) Interplay between two epigenetic marks. DNA methylation and histone H3 lysine 9 methylation. Curr Biol 12:1360–1367. 46. Cubas P, Vincent C, Coen E. (1999a) An epigenetic mutation responsible for natural variation in floral symmetry. Nature 401:157–161. 47. Serikawa KA, Martinez-Laborda A, Zambryski P. (1996) Three knotted1like homeobox genes in Arabidopsis. Plant Mol Biol 32:673–683. 48. Tsai WH, Kuoh CS, Chuang MH, et al. (2004) Four DEF-like MADS box genes displayed distinct floral morphogenic roles in Phalaenopsis orchid. Plant Cell Physiol 45:831–844. Chapter 5 The Screening of Orchid Mycorrhizal Fungi (OMF) and their Applications Doris C. N. Chang* Wild-grown and greenhouse-cultivated orchid roots and the associated fungi were isolated on 1/6 PDA. The fungi isolated from various Taiwan orchid roots were Acremonium spp.; Alternaria spp.; Cylindrocarpon spp.; Fusarium spp.; mycelia sterile; Penecillum spp.; Rhizoctonia spp.; and Trichoderma spp. Those isolates that showed no pathogenecity to the major crops of Taiwan: mungbean, cucumber, radish and rice seedlings were used for inoculation of orchid mycorrhizal fungi. Only those proving beneficial in the growth of orchids were considered as orchid mycorrhizal fungi (OMF). Trichoderma and Rhizoctonia were the most frequent fungi isolates from the Taiwan wild-grown orchid roots. Based on 10 years of inoculation tests, R01, R02, and R04 are shown to be the three isolates with OMF of commercial value. Among them, R01 and R02 are binucleate, while R04 is a multinucleate Rhizoctonia sp. Up till now the identification of R01 and R02 has been uncertain. However, it is known that R04 is Rhizoctonia solani, in the AG6, which has no pathogenic effects on orchids. Seed germinations were enhanced by R02 for Anoectochilus formosanus Hayata and R01 for Haemaria discolor var. dawsoniana. Growth of seedlings was also highly enhanced by the inoculation of Rhizoctonia OMF isolates for three medicinal uses of orchids, such as A. formosanus (R02, R04), H. discolor (R01), and Dendrobium spp. Higher acid and alkaline phosphatases and superoxide dismutase activities, together with *Department of Horticulture, National Taiwan University, Taipei, Taiwan. 77 78 ✦ D.C.N. Chang flavonoids, phenolic compounds, polysaccharides, ascorbic acids, and phosphate contents were all higher in mycorrhizal tissues than the non-mycorrhizal control of A. formosanus. Vegetative and reproductive growth was stimulated by OMF for ornamental orchids, such as Doritaenopsis spp. (R02, R01, R04), Phalaenopsis spp. (R01, R02, R04), and Phaphiopedium delenatii (R02, R04). Oat meal agar should replace MS or Kyoto agar medium if OMF is inoculated in vitro. The physiological effects of OMF and their potential applications on commercially cultivated orchids are presented. It was highly recommended that the OMF be applied as follows: 1) to increase the survival rates of micropropagated plantlets or seedlings ex vitro; 2) to enhance both vegetative and reproductive growth of orchid plants; 3) to result in earlier flowering and enhanced flower quality; and 4) to reduce disease infection rates. 5.1 Introductions It is well-established that orchids have fungi growing in their roots. Cells of the root cortex are occupied, to a greater or lesser extent, by clusters of fungal hyphae, and these occur under both natural and horticultural conditions. The orchid-fungus relationship is one of many known forms of mycorrhiza (the term means fungus-root), a common form of symbiosis between fungi and plants.1 Many orchid mycorrhizal fungi belong to the genera Rhizoctonia, Epulorrhiza, or Ceratorhiza.2 Bernard3 and Burgeff 4–7 studied seed germination of orchids and recognized that, in general, and particularly under natural conditions, successful germination was virtually impossible in the absence of a fungus. This implies that germination is not successful unless it is succeeded by growth. In the absence of infection, the growth rate is often negligible regardless of the availability of nutrients. Consequently, a symbiotic method for seed germination was developed, which involved culture tubes inoculated with both seeds and fungi.7–13 This procedure was difficult for the commercial grower. It was also characterized by a low success rate.1,3 Hadley1 wrote a chapter called “Orchid mycorrhiza” in the book Orchid Biology Reviews and Perspectives, II,14 an excellent volume on orchid mycorrhizal researches from 1960- to the 1980s. In contrast to symbiotic germination, the non-symbiotic method of seed germination offered obvious The Screening of Orchid Mycorrhizal Fungi (OMF) and their Applications ✦ 79 attractions in orchid horticulture.1 The success of orchid seed germination was achieved by Knudson15,16 on a medium containing only sugars and mineral salts, and then the non-symbiotic methods are successfully used by many growers.1 Since then, the non-symbiotic seed germination methods have been very well established in the orchid industry. Now, the non-symbiotic method is applied to orchid seed germination and growth of orchid plants by almost all of the commercial orchid growers. Because the orchid industry became very important in Taiwan in the late 20th century, the author decided to study vesicular arbuscular mycorrhiza since many years ago. Therefore, orchid mycorrhiza is included in my research programs. In Taiwan, it was reported by three orchid mycorrhizal research groups that Rhizoctonia spp. could enhance the growth of orchids, including Bletilla formosana (Hayata) Schltr. by Su,17 Oncidium spp. by Chu18 and Dendrobium spp. by Lin.11 This review covers most of the orchid mycorrhizal researches conducted in my laboratory at the National Taiwan University which were aimed at the following: 1) To find out whether the orchid plants grown in green houses are associated with fungi; 2) To test the growth effects of inoculation with orchid mycorrhizal fungi (OMF) in the roots of greenhouse grown orchids; 3) To determine if those inoculated i.e. the mycorrhizal orchid plants were healthier than the non-mycorrhizal control plants; 4) To observe the flowering responses of the mycorrhizal and non-mycorrhizal orchid plants. 5) To understand the growth and developmental response of Anoectochilus formosanus Hayata, Dendrobium spp., Haemaria discolor var. dawsoniana, Phalaenopsis spp. Dendrobium spp., and slipper orchids (Paphiopedium spp.) to the inoculation of OMF. Hopefully, the importance of OMF could be recognized and reconsidered by the commercial and amateur orchid growers. 5.2 The Isolation, Purification, and Culturing of Orchid Mycorrhizal Fungi (OMF) 5.2.1 Isolation, purification, and culturing of OMF Wild-grown and greenhouse-cultivated orchid roots are collected and pasteurized using sodium hypochloride solution, then cultured on a 1/6 PDA 80 ✦ D.C.N. Chang A B C D Fig. 5.1. Orchid mycorrhizal fungi were isolated from wild grown (A), or greenhouse cultivated (B), orchid roots (C) on 1/6 PDA (D). (potato dextrose agar) medium at 20–30°C (Fig. 5.1). Single hypha is isolated for the pure culture of single fungus. The fungi isolated from the various Taiwan orchid roots are Acremonium spp.; Alternaria spp.; Cylindrocarpon spp.; Fusarium spp.; mycelia sterile; Penecillum spp.; Rhizoctonia spp.; and Trichoderma spp. (Table 5.1). Trichoderma and Rhizoctonia are most frequently isolated from the wild-grown orchid roots. The results showed that greenhouse grown orchids could become mycorrhizal without artificial inoculation. It was reported that the majority of the fungi associated with orchids are regarded as members of the form-genus Rhizoctonia.1 Thus, the Rhizoctonia spp. was isolated (Fig. 5.2). Note the presence of septa in the hyphae, the angle of the branching hyphae (90° or 45°), and the monilioid cells (Fig. 5.2). The isolation results showed that the orchid roots usually harbors more than one fungus. The unifying feature of most isolates of fungi from orchid roots remains their vegetative form, i.e. the features that enables them to be recognized as the Rhizoctonia species. At present, the classification of Rhizoctonia spp. is based on cytomorphology of hyphae, morphology of cultures, morphology of teleomorphs, affinities for hyphal anastomosis (rejoining of hyphae), the number of nuclei in the cells, and more recently, DNA The Screening of Orchid Mycorrhizal Fungi (OMF) and their Applications ✦ 81 Table 5.1. Fungi Isolated from Various Kinds of Healthy Orchid Roots in Taiwan Fungus Name Plant Host Acremonium spp. Alternaria spp. Cylindrocarpon spp. Fusarium spp. Bletilla, Cleisostoma & Dendrobium spp. Dendrobium spp. Dendrobium spp. Anoectochilus, Cleisostoma, Dendrobium & Phalaenopsis spp. Bulbophyllum, Dendrobium & Paphiopedilum spp. Anoectochilus, Bletilla, Bulbophyllum, Cleisostoma, Dendrobium, Haemaria & Phalaenopsis spp. Anoectochilus, Bletilla, Dendrobium, Haemaria, Spiranthes, Paphiopedilum & Phalaenopsis spp. Anoectochilus, Paphiopedilum & Phalaenopsis spp. mycelia sterile Penicillium spp. Rhizoctonia spp. Trichoderma spp. c S C s Fig. 5.2. The hyphae with septum (s) and constriction (c) at the base of branching (left), and there were monilioid cells appearing in later stages (right). base sequence homology. Rhizoctonia is divided into species with binucleate vegetative hyphal cells and those with multinucleate hyphal cells. Several effective staining methods have been developed, such as Aniline blue or Trypan blue, HCl-Geimsa, Orcein, DAPI, and Acridine orange or Safranin O.19 82 ✦ D.C.N. Chang 5.2.2 Pathogenecity analysis of the isolated fungi The isolates of purified fungi are cultured on PDA; about 1 mm2 of hyphae are injected under the roots of each seedling, such as cabbage, cucumber, mungbean and rice, which are the major crops in Taiwan (Fig. 5.3). Only those isolates with no harmful symptoms on all the four crop seedlings are chosen as the potential orchid mycorrhizal fungi (POMF). Those isolates of Rhizoctonia spp. which show beneficial effects for the growth of orchids are screened as OMF inocula and are named R01–R09. Based on 10 years of inoculation tests, R01, R02, and R04 are shown to be the three isolates with OMF of commercial value. Among them, R01 and R02 are binucleate, while R04 is a multinucleate Rhizoctonia spp. Up until now, the identification of R01 and R02 has been uncertain. However, it is known that R04 is Rhizoctonia solani, in the AG6,20 which could enhance seed germination of orchids and was non-pathogenic to the major crops, such as mungbean, cucumber, cabbage, and rice seedlings. R04 R04 R04 R02 R02 R02 R01 R01 R01 NM NM NM Fig. 5.3. The inoculation of isolated orchid root fungi for pathogenisity test on cucumber, mungbean and rice, respectively. The Screening of Orchid Mycorrhizal Fungi (OMF) and their Applications ✦ 83 5.2.3 The application of orchid mycorrhiza 5.2.3.1. Anoectochilus formosanus Hayata and Haemaria discolor var. Dawsoniana (A) Seed germination of Anoectochilus formosanus Hayata (Fig. 5.4) For in vitro germination, Hyponex # 3 agar medium (containing 3 g of Hyponex # 3, 3 g tryptone, 30 g sucrose and 8 g agar (modified from Kano, 1968) was used as a non-mycorrhizal (NM) control and oat meal agar (2.5 g oat meal in 1 L of water, with 8 g of agar, pH 5.7) was used for both mycorrhizal seedlings. For non-symbiotic germination of H. discolor, WM agar medium (1 L containing NaNO3, 0.3 g, KH2PO4, 0.2 g, MgSO4.7H2O, 0.1 g, MgSO4.7H2O, 0.1 g, KCl, 0.1 g, malt extract, 0.1 g, agar, 11.5 g) was used. Results showed that the germination rates were about the same for both treatments.9,25 However, the growth of seedlings was faster for the symbiotic germination than for the nonsymbiotic one (Fig. 5.5). Jewel’s orchids Anoectochilus formosanus Hayata. Haemaria discolor var. dawsoniana Fig. 5.4. Anoectochilus formosanus was called as “The King of Medicine” by aborigines in Taiwan, used mainly for lung and liver protection, and cancer prevention. H. discolor was used for lung protection. 84 ✦ D.C.N. Chang NM +M A A +M B B +M NM C D Fig. 5.5. In vitro (A, C & D) and in vivo germinations (B) of Anoectochilus formosanas Hayata (A, B) and Haemaria discolor var. dawsoniana (C & D) as affected by orchid mycorrhizal fungi (A, +M; B & D) (Chang, 2006). NM: non-mycorrhiza; +M: mycorrhiza. (B) Seedling growth of both A. formosanus and H. discolor in vivo and in vitro The growth of both seedlings of A. formosanus9,20–24 and H. discolor 8 (Fig. 5.6; Refs. 8, 9, 21, 25) was promoted by inoculation of OMF. In most cases, better combinations of plant-OMF are as follows: A. formosanus — R02 or R04; B. H. discolor — R01 or R02. These results were useful for the production of orchids for both medicinal and ornamental uses. (C) Component changes of A. formosanus The activities of acid and alkaline phosphatases and SOD (superoxide dismutase), contents of polysaccharides, polyphenols, flavonoids, ascorbic The Screening of Orchid Mycorrhizal Fungi (OMF) and their Applications ✦ 85 acid, and phosphate were markedly higher in the mycorrhizal tissues of A. formosanus than the non-mycorrhizal controls (NMC) (Ref. 9; Table 1). Thus, the mycorrhizal A. formosanus should be more effective in their medicinal function. Currently, wild-grown A. formosanus could sell for about four times the price compared with greenhouse-cultivated plants. Actually, most of the wild-grown A. formosanus plants were mycorrhizal. The growth of mycorrhizal H. discolor var. dawsoniana seedlings was almost doubled that of the NMC (Fig. 5.6). 5.2.3.2. The application of orchid mycorrhizal fungi: Phalaenopsis spp. (A) Vegetative growth Seed germination of many orchids could be greatly enhanced by the presence of OMF.1,6,12,25–27 Plants of Phalaenopsis spp. of various sizes were tested (Fig. 5.7). The growth of most of the Phalaenopsis spp. and Doritaenopsis spp. cultivars could be enhanced after their transplantation out of glass containers and inoculation with Rhizoctonia spp. of OMF. 28 A NM R02 R04 C B D NM R02 NM R04 R02 Fig. 5.6. In vitro and ex vitro seedling growth of Anoectochilus formosanas Hayata (A & B) and Haemaria discolor var. dawsoniana (C & D) in oat meal agar (A & C) and commercial growth media in pots (B & D) (Chang, 2006). NM: non-mycorrhiza; R02, R04: mycorrhiza. 86 ✦ D.C.N. Chang Young plants Middle-sized plants Adult plants Fig. 5.7. In Taiwan, commercially available small, medium and large plants of Phalaenopsis orchids are grown in 2.5-, 3.5- and 4.5-inch pots, respectively. NM R01 R02 R04 NM R02 R04 Fig. 5.8. Growth of Phalaenopsis amabilis and Doritaenopsis sp. ex vitro after the inoculation of orchid mycorrhizal fungi for 4 months (Chang, 2006). Good combinations of host and orchid mycorrhizal fungi are as follows: Phalaenopsis amabilis: R01, R04. Doritaenopsis sp.: R02, R04. The vegetative growth of Phalaenopsis spp. could be enhanced by the inoculation of Rhizoctonia spp. of OMF (Fig. 5.8); thus, leaf numbers, leaf span, leaf length, and leaf width were enhanced.21 The chlorophyll contents in some Phalaenopsis spp. were greatly increased by the presence of OMF (Fig. 5.9). However, the increase of chlorophyll contents was not consistent for all of the orchids. (B) Reproductive growth The reproductive growth (Fig. 5.10) of many Phalaenopsis spp. cultivars was enhanced by the presence of OMF, including earlier flowering, The Screening of Orchid Mycorrhizal Fungi (OMF) and their Applications ✦ 87 42 41 Chlorophyll content 40 39 38 37 36 35 34 33 0 CK R01 R02 R04 FL01 G04 Fig. 5.9. Chlorophyll contents in the leaf of Dtps. Luchia Davis × Dtps. Taisuco Firebird as influenced by the inoculation of orchid mycorrhizal fungi. CK: non-mycorrhizal control; R01, R02, R04, FL01, G04: mycorrhizal plants. Phalaenopsis sp. Dtps . Ho s Happy Auckland Song Phal. White hybrid Phal. amabilis Fig. 5.10. Dtps. Sogo Manager Flowers of tested Phalaenopsis spp. 88 ✦ D.C.N. Chang NM R02 Fig. 5.11. Flower color of Doritaenopsis Minho Princess was brighter, as influenced by the inoculation of orchid mycorrhizal fungi (R02), than the nonmycorrhizal control (NM). increase in flower number, bigger flower diameter, etc.21 Lan28 reported better plant growth and a greater percentage of orchid blooming through inoculation with OMF. OMF also resulted in brighter orchid flowers (Fig. 5.11). Thus, it was concluded that both the quantity and quality of orchid flowers were enhanced by OMF. If, after inoculation with OMF, GA3 was applied to the Phalaenopsis amabilis, a much higher flowering rate under greenhouse conditions could result, without need for the temperature to be lowered.29 However, if after inoculation with OMF, GA3 or BA (benzyl adenine), is applied, a greater flower stalk emergence rate could result.21 Thus, all the results indicated that inoculation with OMF could significantly promote quality and quantity of Phalaenopsis spp. (C) Disease infection Much less infection was found in the greenhouses of mycorrhizal orchids. In our greenhouse, only about 1% of infection was found for all the mycorrhizal and non-mycorrhizal orchids. In contrast, in the greenhouses of commercial orchid growers, the infection rate was 3% or greater. The Screening of Orchid Mycorrhizal Fungi (OMF) and their Applications ✦ 89 One commercial orchid grower in southern Taiwan observed that inoculation of Phalaenopsis spp. with Rhizoctonia spp. OMF resulted in virus-free orchid plants in a greenhouse, while the non-mycorrhizal orchid plants were all infected with CymMV. Cultivation of virus-free orchid plants is an important goal for most orchid growers. Therefore, research on the ability of OMF to enhance orchid plants to resist infection by CymMV is being conducted and is in progress in our laboratory. (D) Seasonal infection of OMF In Taiwan, the percentage of infection of OMF was 100% within one month of inoculation with the three Rhizoctonia spp. isolated in Taiwan (R01, R02 and R04), i.e. during spring, summer, and fall. Only in winter season was the percentage of the infection of OMF slightly lower. It was found that the rates of infection of KC 1111 (white flower of Phalaenopsis spp.) by the various Rhizoctonia spp. varied; some were fast (such as R02, R04, and R19) and some were slow (R01 and R15). 5.2.3.3. Application of orchid mycorrhizal fungi: Dendrobium spp. and Slipper orchids The vegetative growth of Dendrobium spp. of orchids — for medicinal uses — could be promoted by inoculation with OMF, such as Rhizoctonia spp. and Cylindricarpon spp. (Fig. 5.12).30,31 The growth of slipper orchids could also be promoted by Rhizoctonia spp. of OMF.32,33 After inoculation of OMF (R02 and R04) for six months, leaf length, fresh weight, and survival rates were increased.33 5.3 Observations of Orchid Mycorrhizae by Various Microscopies The orchid mycorrhizae were observed using light microscopy, SEM microscopy, or confocal microscopy. Aniline blue (0.05%) in lactoglycerin was the stain used in light microscopy.34 SEM samples were prepared by fixing with 2% glutaraldehyde, dehydrating in acetone series, then critical-point drying with liquid carbon dioxide, and finally coated with gold (modified from Ref. 35. Fresh roots of Jewels orchid (Haemaria sp.) inoculated with OMF were sectioned using Lancer vibratome series 1000 to obtain sections 30–50 µm in thickness; these were stained with 90 ✦ D.C.N. Chang A NM B +M C Fig. 5.12. Growth of Dendrobium sp. (A), Oncidium sp. (B), (Chu, 2000) and Phaphiopedium sp. (C) as affected by orchid mycorrhizal fungi. (A) NM: non-mycorrhiza; +M: mycorrhiza (B) CK: non-mycorrhiza; DC, GD, BF2: mycorrhiza (C) NM: non-mycorrhiza; TR3, TR2, TR1, R04: mycorrhiza 0.05% aniline blue in lactoglycerin for 3 to 4 h (modified from Ref. 34) and were mounted on slides. The stained sections were observed via Leica TCS SP2 laser scanning confocal microscope under 488 and 543 nm, and photos were taken using a computer. The results of light microscopy showed that the hyphae of OMF usually infected root hairs or root epidermal cells in orchids (Fig. 5.13) and to form peletons in the cortex of the roots. In germinating seeds, the fungal hyphae penetrate via the base end of the seed. The hyphae enter the cells and coil into structures called peletons. Germination of the seeds into a portocorm follows. The cells eventually digest the peletons.2 SEM revealed the three-dimensional structure of the peletons in the cortex cells (Fig. 5.13). Confocal microscopy showed that in the hyphae infection processes,9 the hyphae passed mainly through intercellular spaces of the root epidermal cells and the few layers underneath, then entered the cortex cells intracellularly to form peletons. According to Burgeff 6 and Hadley,1 there were two types of OMF infection, i.e. a) Tolypophagy: showing one layer of host cells adjacent to the epidermis and two layers of digestion cells; and b) Ptyophagy: showing two layers of passage cells and the phagocyte layer with hyphae The Screening of Orchid Mycorrhizal Fungi (OMF) and their Applications ✦ 91 Fig. 5.13. Hyphae (H) of orchid mycorrhizal fungi (OMF) could infect root hair (RH) or orchid root epidermal cells (A) and formed peletons (P) in cortex cells (B) as stained by aniline blue and revealed by light microscopy. Also, SEM observation of non-mycorrhizal (C) and mycorrhizal (D) orchid roots showed numerous peletons (P) formed in the mycorrhizal cortex cells, which was a tolypophagy type of infection. liberating the cytoplasm, which is absorbed into spherical ptysomes. For the infection of three Rhizoctonia spp. in several orchid roots (such as Anoectochilus sp., Haemaria sp., Dendrobium sp., Phalaenopsis spp., and Paphiophilium sp.), only tolypophagy type was observed. 5.4 Identification of OMF by Hyphal Anastomosis Group or RAPD and SSR Markers Hyphal fusion (anastomosis): Isolates of Rhizoctonia are assigned to the anastomosis groups by pairing the isolates with “tester” strains and observing the hyphae for fusion. There are three types of hyphal fusion: perfect fusion, imperfect fusion, and contact fusion.36 Carling et al.37 reported that of 23 selected mycorrhizal isolates of Rhizoctonia solani from an orchid, 20 were members of AG-12 and the rest were members of AG-6. It is known that our R04 is Rhizoctonia solani, which is in the AG6;20 AG12 also could enhance seed germination of orchids.38 The pathogenic Rhizoctonia solani belong to AG 1, 2, 3, 4, 5, 7, and 8.38–41 Rhizoctonia spp. of OMF with multinucleate, were first identified as Rhizoctonia solani. Thus, five R. solani isolates (R04–7, and R09) 92 ✦ D.C.N. Chang from orchids were compared with a total of 19 ATCC test isolates for classifying the anastomosis groups. They were then compared with ATCC 76129 and 76130, two AG-6 isolates, for further grouping. RAPDs with nine primers (OPD-01, 05–08, 10–12 and 14) and four primers (Y 17, 18, 19 and 27) were used for SSR analysis of OMF. Amplification of ribosomal DNA, sequencing and data analysis were carried out by a commercial company in Taiwan.20 PCR reactions were based on the analysis techniques recommended by Rafalaski and Tingey.42 The results of nine OMF calculated from RAPD and SSR were given by Lee.20 Multinucleate Rhizoctonia solani (R04, R05, R06, R07 and R09) all belong to AG-6, which has not shown any pathogenic effects on various kinds of orchids since they were isolated in 1997. 5.5 Specificity of OMF and Hosts The orchid mycorrhizal fungi (OMF) showed some degree of specificity in its effect on seed germination of orchids (Fig. 5.5). Both binucleate and multi-nucleate Rhizoctonia spp. could promote seed germination and growth of orchids. However, usually for seed germination, the binucleate species is more effective. That is why a better combination of plant-OMF should be tested for practical use. 5.6 Important Features for the Successful Application of OMF In Vitro Inoculation of the roots of orchids with Rhizoctonia spp. showed that the growth of many kinds of orchids could be thus enhanced (Fig. 5.5, 5.6, 5.8, 5.12 and Table 5.3). Rhizoctonia spp. produced a higher germination rate than the multi-nucleated ones.9 It is very important that, in vitro, the growth media for the inoculation be changed to oat meal agar (OMA). If an MS medium or a Hyponex medium for the micropropagation of orchid seedlings or plantlets is used, the growth of OMF would be very fast and is thus harmful to the plantlets or seedlings. Usually these seedlings or plantlets will be covered by the hyphae of OMF and will eventually die (Fig. 5.14). Also the in vitro inoculation of OMF was often contaminated by other microorganisms. Therefore, inoculation The Screening of Orchid Mycorrhizal Fungi (OMF) and their Applications ✦ 93 Table 5.2. Mycorrhizal A. formosana Contained Significantly Higher Enzyme Activities or Component Contents than Non-mycorrhizal Control (modified from Refs. 9 and 21) Plant Part Leaf Stem Root Enzyme Activity Component Contents superoxide dismutase — acid & alkaline phosphatases ascorbic acid, flavonoid, phosphate, polyphenol and polysaccharide polyphenol and polysaccharide ascorbic acid, polyphenol, and polysaccharide Fig. 5.14. Mass hyphae of orchid mycorrhizal fungus covered and resulted in the death of seedling in MS medium. with OMF in vitro is currently not recommended for mass production of mycorrhizal orchid plants in commercial tissue cultural companies. There was some degree of plant-OMF specificity for the useful infection of orchid plants by OMF. For example, some Dendrobium sp. showed better growth enhancement by infection of R01, while some other Dendrobium sp. needed R04 for growth.30 Therefore, proper combination tests for orchid-OMF should be conducted before mass inoculation of the orchid plants. 94 ✦ D.C.N. Chang Table 5.3. Combinations of Orchid Hosts and Rhizoctonia spp. which could result in significant differences in growth of orchids (Chang, 2006) Host Cultivar Anoectochilus formosanas Haemaria discolor Dendrobium spp. Doritaenopsis sp. Ho’s Happy Auckland ‘Song’ Ho’s Happy Auckland ‘Song’ Paphiopedium delenatii Phalaenopsis spp. P. amabilis P. Sogo Manager P. Sogo Manager Orchid Mycorrhizal Fungi (OMF) Vegetative Growth R02, R04 + R01, R02 R01, R04 + + R02 + R02, R01, R04 Reproductive Growth + R02, R04 + R02 R01, R02, R04 R04, R02 + + + + 5.7 Physiological Effects of Orchid Mycorrhizal Fungi and Their Possible Applications (a) To enhance symbiotic seed germination of orchids in vitro and in vivo. (b) To increase the survival rates of the micropropagated plantlets or seedlings in vivo. Specificity of OMF-hosts should be considered. (c) To stimulate nutrient absorption and translocation. (d) To enhance both vegetative and reproductive growth of orchid plants. (e) To result in earlier flowering and increase in flower quality. (f ) May help to supply plant hormones (GAs, CK). (g) To increase chlorophyll contents (photosynthesis might be related). (h) To increase enzymatic activities (acid and alkaline phosphatases, SOD), and to promote antioxidant abilities by increasing amounts of ascorbic acids, flavonoids, polyphenols, and polysaccharides in Anoctochilus formosana Hayata. (i) To reduce the infection rate and hence less agrichemicals are required. The Screening of Orchid Mycorrhizal Fungi (OMF) and their Applications ✦ 95 ( j) To combine the use of proper plant growth substances and cool temperature to enhance the production of more flowers as well as to increase flower quality and quantity. (k) To promote the growth and flowering ability of plants after bareroot shippings. (l) May be helpful for the restoration of orchids in the wilderness. Among them, items (b), (d), (e), (i), and ( j) were more readily achievable. Better combinations of orchid cultivars and OMF, which could result in significant differences in growth of orchids, are listed in Table 5.2. It is concluded that various orchid hosts had some degree of specificity for orchid mycorrhizal fungi. Therefore, before large scale applications, tests for proper combinations of orchid-OMF should be conducted. The enhancement of disease resistance of OMF might be the most unique feature that could not be easily replaced by other cultural practices. References 1. Hadley G. (1982) Orchid Mycorrhiza. In: Arditti J (ed.), Orchid Biology: Reviews and Perspective II. Cornell University Press, Ithaca, New York, pp. 83–118. 2. Ian St, G. (2006) Orchids and fungi. New Zealand Native Orchid Group. Orchid Journal 66. 3. Benard N. (1909) L’évolution dans la symbiose. Ann Sci Nat Bot 9:1–196. 4. Burgeff H. (1936) Samenkeimung der Orchideen. Fischer, Jena. 5. Burgeff H. (1943) Problematik der Mycorrhiza. Nautrwiss 31:558–567. 6. Burgeff H. (1959) Mycorrhiza of orchids. In: Withner CL (ed.), The Orchids: A Scientific Survey. Ronald Press, New York, pp. 361–395. 7. Arditti J. (1967) Factors affecting the germination of orchid seeds. Bot Rev 31:1–97. 8. Chou LC. (1997) Mass propagation of Haemaria discolor var. dowsoniana and the use of Rhizoctonia spp., Master’s thesis. Department of Horticulture, National Taiwan University. 9. Chou LC. (2004) The mycorrhizal physiology and cultivation of Anoectochilus formosanus Hayata, Haemaria discolor var. dawsoniana and their F1 hybrids, Ph.D. dissertation. Institute of Horticulture, National Taiwan University. 10. Deutsch G. (2001) In vitro-propagation of Nigritella (Orchidaceae-Orchideae) from seeds with the help of mycorrhizal fungi. Phyton 41:111–128. 96 ✦ D.C.N. Chang 11. Lin CF. (2002) Effects of orchid mycorrhizal fungi on seed germination and the seedling growth of Dendrobium, Master’s thesis. Department of Horticulture, National Chung Hsing University. 12. Sharma J, Zettler LW, Sambeek JWV, et al. (2003) Symbiotic seed germination and mycorrhizae of federally threatened Platanthera praeclara (Orchidaceae). Am Midl Nat 149:104–120. 13. Takahashi K, Ogiwara I, Hakoda N. (2001) Inoculation of Habenaria (Pecteilis) radiata with two isolates of orchid mycorrhizal fungi: Germination and growth response to separate, simultaneous, and consecutive inoculation. Lindleyana 16:102–108. 14. Arditti J. (1982) Orchid Biology Reviews and Perspectives, II. Cornell Univ. Press. 15. Knudson L. (1922) Nonsymbiotic germination of orchid seeds. Bot Gaz 73:1–25. 16. Knudson L. (1924) Further observations on nonsymbiotic germination of orchid seeds. Bot Gaz 77:212–219. 17. Su MC. (1995) Preliminary study of the symbiosis Bletilla formosana in Taiwan, Master’s thesis. Department of Forestry, National Taiwan University. 18. Chu JN. (2000) Isolation and inoculation of orchid mycorrhizal fungi and their effects on the seedling growth of Oncidium Goldiana × Onc. Guiena Gold, Master’s thesis. Institute of Tropical Agriculture & International Cooperation. 19. Sneh B, Burpee L, Ogoshli A. (1991) Identification of Rhizoctonia species. APS Press, Minnesota, USA. 20. Lee GC. (2001) Identification and improvement of production techniques for Anoectochilus formosanus Hayata and orchid mycorrhizal fungi, Ph.D. dissertation. Department of Horticulture, National Taiwan University. 21. Chang DCN. (2006) Research and application of orchid mycorrhizal fungi in Taiwan. Internatl Hortl Cong (IHC) 2006, Aug. 13–19, Seoul, Korea. 22. Li HMC. (2001) Physiology of orchid mycorrhizal fungi (Rhizoctonia spp.), inoculum production and their effects on the growth of Anoectochilus formosanus Hayata, Master’s thesis. Department of Horticulture, National Taiwan University. 23. Sheu CL. (1994) The effect of mycorrhizae on the growth of Carrot plantlet derived from somatic embryo and Anoectochilus formosanus Hayata tissue culture plantlet, Master’s thesis. Department of Horticulture, National Taiwan University. 24. Tsai CY. (1997) Effects of Rhizoctonia spp, and temperature on the growth of Anoectochilus formosanus Hayata plantlets, Master’s thesis. Department of Horticulture, National Taiwan University. 25. Chang DCN, Chou LC. (2001) Seed germination of Haemaria discolor var. dawsoniana and the use of mycorrhizae. Symbiosis 30:29–40. The Screening of Orchid Mycorrhizal Fungi (OMF) and their Applications ✦ 97 26. Uetake Y, Farquhar ML, Peterson RL. (1997) Changes in microtubules arrays in symbiotic orchid protocorms during fungal colonization and senescence. New Phytol 135:701–709. 27. Zettler LW, Hofer CJ. (1998) Propagation of the little club-spur orchid (Platanthera clavellata) by symbiotic seed germination and its ecological implications. Environ Exp Bot 39:189–195. 28. Lan IC. (2001) Effects of orchid mycorrhizal fungi on the growth and development of Phalaenopsis and Paphiopedium spp., Master’s thesis. Department of Horticulture, National Taiwan University. 29. Wang YF. (2005) Effects of orchid mycorrhizal fungi and plant growth substances on the growth and flowering of Phalaenopsis spp., Master’s thesis. Department of Horticulture, National Taiwan University. 30. Kang CW. (2004) Effects of orchid mycorrhizal fungi and plant growth substances on the growth and development of medical Dendrobium spp., Master’s thesis. Department of Horticulture, National Taiwan University. 31. Tseng CJ. (2001) Isolation, identification and culture of Dendribium mycorrhizal fungi and their effects on the growth of Dendrobium spp., Master’s thesis. Department of Horticulture, National Taiwan University. 32. Tsai LC. (2003) Effects of orchid mycorrhizal fungi and plant growth substances on the growth and development of Paphiopedilum spp., Master’s thesis. Department of Horticulture, National Taiwan University. 33. Chen CC. (2005) Effects of orchid mycorrhizal fungi with plant growth substances on the growth and development of Paphiopedilum spp., Master’s thesis. Department of Horticulture, National Taiwan University. 34. Koske RE, Gemma JN. (1989) A modified procedure staining roots to detect VA mycorrhizas. Mycol Res 92:486–488. 35. Porter KR, Kelly D, Andrews PM. (1972) The preparation of cultured cells and soft tissues for scanning electron microscopy. Proc Fifth Ann Stereoscan Colloq (1972). Kent Cambridge Corp., 19 pp. 36. Matsumoto T, Yamamoto W, Hirane S. (1932) Physiology and parasitology of the fungi generally referred to as Hypochnus sasakii Shirai. I. Differentiation of the strains by means of hyphal fusion and culture in differential media. J Soc Trop Agric 4:370–388. 37. Carling DE, Pope EJ, Brainard KA, Carter DA. (1999) Characterization of mycorrhizal isolates of Rhizoctonia solani from an orchid, including AG-12, a new anastomosis group. Phytopathology 89:942–946. 38. Pope J, Carter DA. (2006) Phylogenetic placement and host specificity of mycorrhizal isolates belonging to AG-6 and AG-12 in the Rhizoctonia solani species complex. Mycologia 93:712–719. 39. Harikrishnan R, Yang XB. (2002) Effects of herbicides on root rot and damping-off caused by Rhizoctonia solani in glyphosate-tolerant soybean. Plant Dis 86:1369–1373. 98 ✦ D.C.N. Chang 40. Priyatmojo A, Escopalao VE, Tangonan NG, et al. (2001) Characterization of a new subgroup of Rhizoctonia solani anastomosis group 1 (AG-1-ID), causal agent of a necrotic leaf spot on coffee. Phytopathology 91:1054–1061. 41. Stepniewska-Jarosz S, Manka M, Asiegbu FO. (2006) Studies on anastomosis group of isolates causing disease in two forest nurseries in Poland. Pathology 36:97–109. 42. Rafalski JA, Tingey SV. (1993) Genetic diagnostics in plant bredding: RAPDs, microsatellites and machines. TIG 9:275–280. Chapter 6 Analysis of the Orchid Genome Size Using Flow Cytometry Tsai-Yun Lin*,† and Hsiao-Ching Lee† Flow cytometry is an efficient and reliable method for the estimation of nuclear DNA contents. Nuclei suspension from orchid plant is often contaminated with a high level of crystalline calcium oxalate that blocks the fluidics system of the flow cytometer. A simple and highly efficient protocol enables isolation of intact nuclei from recalcitrant plant tissues containing high levels of polysaccharides, calcium oxalate crystals, and other metabolites. This method was applied to determine the genome sizes of Phalaenopsis sp. using Pisum sativum Minerva Maple as the standard. The occurrence of endoreduplication in orchids during organ development and the influences of environmental perturbations are also discussed. 6.1 Introduction Interspecific hybridization and chromosome doubling are the techniques often applied to produce new cultivars of orchids.1 A better understanding of the karyotypes and DNA contents of orchid will aid in the development of new cultivars of high quality. Intraspecific variations in genome size occur due to environmental influences, common chromosome polymorphism, and spontaneous aberration. However, the DNA content per genome is fairly constant, both between cells of an *Corresponding author. † Institute of Bioinformatics and Structural Biology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan. 99 100 ✦ T.-Y. Lin and H.-C. Lee individual and between different individuals of the same species.2,3 An accurate determination of genome size provides basic information for breeders and molecular geneticists. Interspecific comparison of nuclear DNA amounts is also useful in cytotaxonomic and evolutionary studies. The classical methods for the estimation of genome sizes are based on the determination of the phosphate content in the DNA backbone of total DNA isolated from a defined number of cells or on re-association kinetics of high molecular weight genomic DNA. More recent techniques employ DNA-specific fluorescent dyes in flow cytometry analysis. Cell polyploidy is often due to endoreduplication, when one or several rounds of DNA synthesis occur in the absence of mitosis or cytokinesis, resulting in an increase in the genomic DNA content.4 Endoreduplication is an important component of organ development and is often related to plant cell size. The number of endoreduplication cycles is tissue-specific and is characteristic of the stage of development.5 6.2 Flow Cytometric Methods 6.2.1 Flow cytometers Flow cytometry has proven to be an efficient and reliable method for analyzing plant genomes. The principle of flow cytometry is that single particles suspended within a stream of liquid are interrogated individually in a very short time as they pass through a light source focused at a very small region. The optical signals generated are mostly spectral bands of light in the visible spectrum, which represent the detection of various chemical or biological components, mostly fluorescence.6 6.2.2 Applications of flow cytometry DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathways, and estimation of absolute DNA amount or genome size.7 Plant scientists have used flow cytometry to determine the nuclear DNA content of a wide variety of species, such as Arabidopsis8; cucumber9; tomato10; orchids11,12; several succulents13; and endosperm in maize.14 In addition, Analysis of the Orchid Genome Size Using Flow Cytometry ✦ 101 flow cytometry has been used to analyze the cell cycle, sort protoplasts and chromosomes, and examine various cellular parameters.15 6.3 Estimation of Orchid Genome Size 6.3.1 Preparation of orchid nuclei suspensions There is no general procedure that works with all plants, although several protocols have been described for nuclei isolation.16–18 Most protocols use young tissues to avoid the high concentration of starch, polysaccharides, calcium oxalate crystals, and other metabolites found in old tissues. Orchid plant nuclei suspension often contains a high level of crystalline calcium oxalate that blocks the fluidics system of the flow cytometer. To avoid this problem, a cotton column was designed and polyvinyl pyrrolidone-40 (PVP-40) was added to the buffer to remove phenolic impurities and cytoplasmic compounds from plant nuclei, making the suspension suitable for flow cytometry. This simple and highly efficient protocol enables the isolation of intact nuclei from plant tissues containing high levels of polysaccharides, calcium oxalate crystals, and other metabolites.19 6.3.1.1 Solution and reagents • MgSO4 buffer: 10 mM MgSO4.7H2O, 50 mM KCl, 5 mM HEPES [N-2-hydroxyethylpip erazine-N′-2-ethanesulfonic acid], pH 8.0. • Extraction buffer A: MgSO4 buffer with 1% polyvinyl pyrrolidone40, 6.5 mM dithiothreitol, 0.25% Triton X-100; stored at 4°C. • Extraction buffer B: MgSO4 buffer with 6.5 mM dithiothreitol, 0.25% Triton X-100, 0.2 mg ml−1 propidium iodide (Calbiochem, La Jolla, Calif.), 1.25 µ g ml−1 RNase (DNase-free); prepared on ice just prior to use. 6.3.1.2 Column for nuclei filtration To make the column for nuclei filtration, cosmetic cotton made of 100% cotton is cut into 5 × 1-cm pieces moistened with MgSO4 buffer and loosely rolled into a cylindrical shape. The cotton ball is gently inserted into the middle of a 5 ml pipette tip (Fig. 6.1). The cotton ball filters out 102 ✦ T.-Y. Lin and H.-C. Lee Fig. 6.1. The cotton column for filtration of plant nuclei. Cosmetic cotton is cut into 5 × 1-cm pieces, wetted with MgSO4 buffer and rolled into a cylindrical shape loosely, then inserted into a 5 ml pipette tip to form a filtration column.19 (Photo from Lee et al., courtesy of the ISPMB abd PMBR.) the particles on the basis of differences in physical structure of the particles, and the position of the cotton ball affects the efficiency of filtration and yield of nuclei. Users may shift the position of the cotton ball up or down to find the optimal pore size for different plant materials. Prior to use, the column must be rinsed with 10 ml or more of MgSO4 buffer solution to remove floating cotton fibers. 6.3.1.3 Nuclei isolation While on ice, 50 to 300 mg of fresh tissue is sliced into thin strips of less than 0.5 mm with a sharp razor blade in a glass petri dish containing 1 ml of ice-cold extraction buffer A. Two ml of ice-cold extraction buffer A is added to the chopped tissue and gently shaken for 1 min on a shaker or by hand. The nuclei extract is filtered into a 15-ml Falcon tube through the cotton column. The homogenate is transferred to the cotton column and nuclei are eluted with a 7 ml ice-cold extraction buffer A. The homogenate is centrifuged at 120 g for 10 min at 4°C. The supernatant is then carefully decanted and the pellet gently suspended in 400 µ l of extraction buffer B, which is then incubated in the dark at 37°C for 15 min before flow cytometry analysis. 6.3.1.4 Flow cytometry analysis Flow cytometry analysis lacks the intrinsic ability to deal with cell aggregates or other large particles, thus requiring the isolation of intact nuclei. Isolating intact nuclei from plant tissues is not easy, especially when young tissues are unavailable. Fully developed, mature organs are usually heavily loaded with polysaccharides, calcium oxalate crystals, and Analysis of the Orchid Genome Size Using Flow Cytometry ✦ 103 Fig. 6.2. Effect of the cotton column on plant nuclei isolation confirmed by microscopy. Samples prepared from mature leaves of Phalaenopsis aphrodite subsp. formosana and stained with carmine showing nuclei were examined under a microscope (Axioskop, Zeiss, Germany) with a hemacytometer. (a) The suspension without filtration contains a large amount of calcium oxalate crystals (needleshaped) that clog the tube of flow cytometer. (b) The suspension filtered through a 30-µm nylon mesh still contains many calcium oxalate crystals and other residues of cells. (c) The suspension filtered through the cotton column significantly reduced contaminants.19 (Photo from Lee et al., courtesy of the ISPMB and PMBR.) other metabolites that decrease the purity of intact nuclei. These contaminants, such as calcium oxalate crystals, have smaller diameters than the nuclei and are difficult to remove by the small pore size (30–50 µ m) nylon mesh generally used in other protocols.16–18 The cotton column forms meshed network to retard the irregular-shaped contaminants, and the addition of PVP-40 to the buffer removes phenolic impurities. Microscopy confirmed the purity of the isolated nuclei (Fig. 6.2). The disadvantage of this protocol is a 50% decrease in nuclei yield. To overcome this problem, the amount of tissue used needs to be increased for nuclei extraction. This cotton column is particularly effective on recalcitrant plant tissues and allows precise measurement using flow cytometry. 6.3.2 Fluorescent staining of nuclear DNA Propidium iodide (PI) and 4′-6-diamidino-2-phenylindole (DAPI) are commonly used fluorochromes. The choice of fluorochromes is primarily determined by the excitation source available. PI is excited by visible light with an absorbancy maximum at 490 nm, while DAPI is excited by UV light at 350 nm.20 However, the two dyes have quite different stain reactions. PI intercalates between base pairs of double-stranded DNA 104 ✦ T.-Y. Lin and H.-C. Lee and RNA with little or no base specification,21 while DAPI is a nonintercalating stain that binds preferentially and in a complex manner to A-T base regions.22 Highly significant differences were detected in plant nuclear DNA contents obtained using DAPI and PI and this cast doubt on the reliability of base preference fluorochromes.23 PI-based flow cytometry produced results consistent with those based on Feulgen microspectrophotometry; DAPI was suggested not to be used as the sole basis for DNA content comparisons.20 PI was recommended as the fluorochrome of choice for flow cytometric determination of plant DNA content. DAPI should be used only if the estimated DNA value is corroborated by using a second stain that has no bias for AT- or GC-rich sequences within genomes.20 6.3.3 DNA reference standards Using proper reference standards for flow cytometric determination of DNA contents is important. Most of the published DNA values for plants have been calibrated against chicken erythrocytes. However, this may result in significant error in estimated DNA contents, as there is considerable reported variations in DNA content among chicken lines.24 Price et al.25 suggested that for technical reasons a plant standard is better for estimating the DNA content of plants. Five species are recommended by Johnston et al.20 as an initial set of international standards for plant DNA content determinations: Sorghum bicolor cv. Pioneer 8695 (2C = 1.74 pg); P. sativum cv. Minerva Maple (2C = 9.56 pg); Hordeum vulgare cv. Sultan (2C = 11.12 pg); Vicia faba (2C = 26.66 pg); and Allium cepa cv. Ailsa Craig (2C = 33.55 pg). It is recommended that one of the reference standards be chosen for determination of DNA content of plant genome. 6.4 Nuclei DNA Contents of Orchids 6.4.1 Nuclei DNA contents of Phalaenopsis sp. Chromosome numbers for the orchids are usually examined and stained by basic fuchsin and acetic orcein. There is a 6.07-fold variation in genome size within 18 Phalaenopsis species, ranging from 2.74 pg/2C for P. sanderiana to 16.61 pg/2C for P. parishii (Table 6.1).11 Analysis of the Orchid Genome Size Using Flow Cytometry ✦ 105 Table 6.1. Nuclear DNA Contents of Phalaenopsis sp. Determined by Flow Cytometry of Propidium Iodide Attained Nuclei using P. sativum Minerva Maple as the Standard11 Genus Speciesa TSRI Cloneb Mean (pg/2C) aphrodite Rchb.f.c sanderiana Rchb.f. stuartiana Rchb.f. equestris (Schauer) Rchb.f. P. amboinensis J. J. Smith P. amboinensis J. J. Smith P. gigantea J. J. Smith P. micholitzii Rolfe P. venosa Shim & Fowl. P. fasciata Rchb.f. P. lueddemanniana Rchb.f. P. modesta J. J. Smith P. mariae Burb. ex Warn. & Wms. P. pulchra (Rchb. f.) Sweet P. sumatrana Korth. & Rchb.f. P. bellina (Rchb. f.) Cristensond P. cornu-cervi (Breda) Bl & Rchb.f. P. mannii Rchb.f. P. mannii Rchb.f. P. parishii Rchb.f. W01–38 W36–01 W40–04 W09–51 2.80 2.74 3.13 3.37 W02–07 14.36 W02–08 14.50 S82–314 5.28 W27–05 W42–01 6.49 9.52 W10–01 W23–02 6.56 6.49 W28–06 W26 5.15 6.48 W33–04 6.37 W41–03 6.62 S82–429 15.03 W08–01 6.44 W25–06 W25–07 W32 13.50 13.61 16.61 Sectiona Phalaenopsis Phalaenopsis P. P. P. Stauroglottis P. Amboinenses Zebrinae Polychilos Parishianae a Classification according to Sweet (1980). Source of plant material: Taiwan Sugar Research Institute (TSRI). c Chromosome number are 2n = 2x = 38. d Nomenclature according to Christenson and Whitten (1995). b 106 ✦ T.-Y. Lin and H.-C. Lee Mean DNA content (pg/2C) 8 6 4 2 0 Diploidy Triploidy Tetraploidy Fig. 6.3. Nuclear DNA contents of diploidy, triploidy, and tetraploidy of P. aphrodite determined by flow cytometry of propidium iodide stained nuclei. The DNA contents (mean ± SE) of diploidy, triploidy, and tetraploidy of P. aphrodite are 2.8 ± 0.06, 4.52 ± 0.16 and 5.89 ± 0.10 pg/2C, respectively.11 Nuclei extracted from P. sativum Minerva Maple were used as the standard. Chromosome numbers of different TSRI clones of P. aphrodite were confirmed to be 2n = 2x = 38 for W01-38; 2n = 3x = 57 for W01-41; and 2n = 4x = 76 for W01-22. The nuclear DNA contents of diploidy, triploidy and tetraploidy of P. aphrodite had a ratio near 2:3:4, corresponding to their somatic chromosome numbers, 38, 57 and 76 (Fig. 6.3). The linear relationship between nuclear DNA contents and chromosome numbers of these three clones indicated the accuracy of flow cytometry. 6.4.2 Intraspecific variations in genome size No variation in DNA content was found between the two different P. amboinesis clone, W02–07 (14.36 pg/2C) and W02–08 (14.50 pg/2C) or between the two different P. mannii clones, W25–07 (13.50 pg/2C) and W25–06 (13.61 pg/2C) (Table 6.1). Our DNA 2C-values of 3.37 pg/2C for P. equestris and 6.49 pg/2C for P. lueddemanniana are smaller than that previously reported (5.53 pg/2C for P. equestris and 8.65 pg/2C for P. lueddemanniana).26 These differences could have resulted from intraspecific variations.11 Analysis of the Orchid Genome Size Using Flow Cytometry ✦ 107 6.5 Endoreduplication Occurrence in Orchid 6.5.1 Phenomenon of endoreduplication Cell polyploidy is often due to endoreduplication, when one or several rounds of DNA synthesis occur in the absence of mitosis or cytokinesis, resulting in an increase in the genomic DNA content.4,27–29 Endoreduplication is common in animal and plant cells, especially in those undergoing differentiation and expansion.30–32 Endoreduplication in the endosperm and cotyledons of developing seeds is well documented, but it also occurs in many tissues throughout the plant. An increase in ploidy may be part of the differentiation of a single cell, as it is in Arabidopsis trichomes,33–35 and may also be related to cell size.36 Cell size in yeast increases with increasing ploidy.37 In plants, increases in ploidy level are related to increases in nuclear volume and cell size. Cells that have undergone endoreduplication are larger than comparable cells that have not.5,38 Endoreduplication is widespread in orchid plants. Sturdier flowers and better forms may accompany an increase in ploidy in orchids. Although chromosome doubling is a technique for breeders to obtain new cultivars, little is known regarding the mechanism of endoreduplication in orchids. Use of flow cytometry in determining polyploidy is fast and makes determining polyploidy easier. 6.5.2 Occurrence of endoreduplication during organ development The frequency of nuclei at different ploidy levels differs in various organs and is correlated with development. Older tissues often possess nuclei of higher ploidy levels than younger ones within the same plant. For example, in Cucumis sativus, ploidy levels were described as a continuous process during the developmental stages from seed to flowering plants.9 The distribution of nuclei at different ploidy levels also changes with the developmental stages as determined by flow cytometry. The number of endoreduplication cycles appears to be controlled by developmental signals.27 Nevertheless, direct evidence for the link between development and the ploidy level in various organs and tissues of plant still remains to be determined.4 In P. aphrodite and P. equestris, endoreduplication stopped once the flower was fully expanded,12,39 and ploidy levels stabilized, similar to 108 ✦ T.-Y. Lin and H.-C. Lee leaves in Arabidopsis,8 cucumber,9 and several succulents.13 Floral buds and opened flowers in an inflorescence had different frequencies of endoreduplication. The distribution of endopolyploidy also varied among the different parts of a flower (Fig. 6.4). The proportion of endoploidy in cells increased with the increasing size of a floral bud. A similar pattern of polysomaty is found in the fully opened flowers. Our results showed that the younger floral buds have higher proportions of 2C cells. To describe the relationship between endoreduplication and cell growth, a model was designed combining a logistic growth model with an endoreduplication model of Schweizer et al.14 Our results indicate that cells with higher C values had lower transition rates and less potential for further endoreduplication, and when cessation of endoreduplication occurred, the flower fresh weight also stopped increasing. The average cell fresh weight was positively correlated to the average C value, suggesting that endoreduplication in Phalaenopsis is a contributing factor to cell growth.39 The Arabidopsis plant-specific cyclin-dependent kinase CDKB1;1 and transcription factor E2Fa-DPa control the balance between mitotic division and endoreduplication.40 Plants which overexpressed a dominant negative allele of CDKB1;1 were found to undergo enhanced endoreduplication, demonstrating that the CDKB1;1 activity is required to inhibit the endocycle. In E2Fa-DPa–over expressing plants, DNA replication is strongly activated. Plant organ cell number and cell size might be controlled by modulation of genes involved in cyclin D-CDK inhibitor interaction. Plant CDK inhibitors, ICK1/KRP1 can move between cells and can inhibit entry into mitosis.41 6.5.3 Endoreduplication influenced by environmental perturbations Environmental perturbations can influence endoreduplication. For example, both DNA endoreduplication and mitosis in maize endosperm cells were significantly correlated with decreased fresh weight and nuclei number after exposure to 35°C for 4 to 6 days.42 In Arabidopsis, reductions in light intensity and soil water content reduces the extent of endoreduplication.42 The hypocotyls cells go through several cycles of endoreduplication, and the process is differentially regulated in the light and in the dark.44 Activated phytochrome specifically inhibits a third endoreduplication cycle in developing hypocotyls cells. The 16C Analysis of the Orchid Genome Size Using Flow Cytometry ✦ 109 TS97u, 2n=3x TS97v, 2n=4x lateral petals & sepals lateral petals & sepals 80 80 60 60 40 40 20 20 0 0 F1 F2 B1 B2 B3 B4 B5 B6 F1 F2 B1 lower labellum B2 B3 B4 B5 B6 lower labellum 80 80 60 60 40 40 20 20 0 0 F1 F2 B1 B2 B3 B4 B5 F1 B6 F2 B1 B2 upper labellum B3 B4 B5 B6 upper labellum 80 80 60 60 40 40 20 20 0 0 F1 F2 B1 B2 B3 B4 B5 F1 B6 F2 B1 B2 gynostemium B3 B4 B5 B6 gynostemium 80 80 60 60 40 40 20 20 0 0 F1 F2 B1 B2 B3 B4 B5 F1 B6 F2 B1 B2 B3 ovary B4 B5 B6 B5 B6 ovary 80 80 60 60 40 40 20 20 0 0 F1 F2 B1 B2 B3 B4 B5 B6 F1 F2 B1 B2 B3 B4 Fig. 6.4. Frequencies of cells with various C-values in different parts of flower. The flowers of TS97u (2n = 3x) and TS97v (2n = 4x) in a florescence were numbered from the bottom to the top, with the oldest flower as number one. The ploidy distribution of two inflorescences was recorded. The fully opened flowers had a similar pattern of polysomaty (
: 2C, : 4C, ▲: 8C, : 16C). 110 ✦ T.-Y. Lin and H.-C. Lee nuclei were present in the hypocotyls of phytochrome ( phy) A mutants, but not in that of wild type Arabidopsis plants under continuous far red light.45 The salt treatment (150 mM NaCl) induced chromosome endoreduplication during the differentiation of cells in the root cortex of Sorghum bicolor cv. 610 plants, but not in the cells of the root vascular cylinder or the leaves. In another S. bicolor genotype DK 34-Alabama, noncompetent for salt adaptation, the same salt treatment did not induce chromosome endoreduplication in the root cortex cells. Endopolyploidy may be considered as a part of the adaptive response of S. bicolor competent genotypes to salinity.46 The number of endoreduplication cycles appears to be under the control of developmental and environmental signals. Acknowledgments We thank AH Marhkart for comments on the manuscript and the National Science Council, Republic of China for grant NSC 92-2317-B007-001 and Council of Agriculture, Republic of China for grants 94AS5.2.1-ST-a1 and 95AS-6.2.1-ST-a1. References 1. Arditti J. (1997) Fundamentals of Orchid Biology. Wiley, New York. 2. Greilhuber J. (1998) Intraspecific variation in genome size: A critical reassessment. Ann Bot 82(Supp. A):27–35. 3. Greilhuber J, Obermayer R. (1997) Genome size and maturity group in Glycine max (soybean). Heredity 78:547–551. 4. Joubès J, Chevalier C. (2000) Endoreduplication in higher plants. Plant Mol Biol 43:735–745. 5. Kudo N, Kimura Y. (2002) Nuclear DNA endoreduplication during petal development in cabbage: Relationship between ploidy levels and cell size. J Exp Bot 53:1017–1023. 6. Gary W, Gary LB. (2004) Encyclopedia of Biomaterials and Biomedical Engineering. Marcel Dekker, New York. 7. Dolezel J, Bartos J. (2005) Plant DNA flow cytometry and estimation of nuclear genome size. Ann Bot 95:99–110. 8. Galbraith DW, Harkins KR, Knapp S. (1991) Systemic endopolyploidy in Arabidopsis thaliana. Plant Physiol 96:985–989. Analysis of the Orchid Genome Size Using Flow Cytometry ✦ 111 9. Gilissen LJW, van Staveren MJ, Creemers-Molenaar J, Verhoeven HA. (1993) Development of polysomaty in seedlings and plants of Cucumis sativus L. Plant Sci 91:171–179. 10. Smulders MJM, Rus-Kortekaas W, Gilissen LJW. (1994) Development of polysomaty during differentiation in diploid and tetraploid tomato (Lycopersicon esculentum) plants. Plant Sci 97:53–60. 11. Lin S, Lee HC, Chen WH, et al. (2001) Nuclear DNA contents of Phalaenopsis sp. and Doritis pulcherrima. J Amer Soc Hort Sci 126:195–199. 12. Yang M, Loh CS. (2004) Systemic endopolyploidy in Spathoglottis plicata (Orchidaceae) development. BMC Cell Biol 5:33. 13. de Rocher EJ, Harkins KR, Galbraith DW, Bohnert HJ. (1990) Developmentally regulated systemic endopolyploidy in succulents with small genomes. Science 250:99–101. 14. Schweizer L, Yerk-Davis GL, Phillips RL, et al. (1995) Dynamics of maize endosperm development and DNA endoreduplication. Proc Natl Acad Sci USA 92:7070–7074. 15. Brown SC, Bergounioux C. (1989) Plant flow cytometry. In: Yen A (ed.), Flow Cytometry: Advanced Research and Clinical Applications, Vol. 2. CRC Press, Boca Raton, Florida, pp. 195–220. 16. Galbraith DW, Harkins KR, Maddox JM, et al. (1983) Rapid flow cytophotometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051. 17. Arumuganathan K, Earle ED. (1991) Estimation of nuclear DNA content of plants by flow cytometry. Plant Mol Bio Rep 9:229–233. 18. Ulrich I, Ulrich W. (1991) High-resolution flow cytometry of nuclear DNA in higher plants. Protoplasma 165:212–215. 19. Lee HC, Lin TY. (2005) Isolation of plant nuclei suitable for flow cytometry from recalcitrant tissue using a filtration column. Plant Mol Biol Rep 23:53–58. 20. Johnston JS, Bennett MD, Rayburn AL, et al. (1999) Reference standards for determination of DNA content of plant nuclei. Amer J Bot 86:609–613. 21. Properi E, Giangare MC, Bottiroli G. (1991) Nuclease induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide. Cytometry 12:323–329. 22. Godelle B, Cartier D, Marie D, et al. (1993) Heterochromatin study demonstrating the non-linearity of fluorometry useful for calculating genomic base composition. Cytometry 14:618–626. 23. Dolezel J, Sgorbati S, Lucretti S. (1992) Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiol Plantarum 85:625–631. 24. Bennett MD, Leitch I. (1995) Nuclear DNA amounts in angiosperms. Ann Bot 76:113–176. 112 ✦ T.-Y. Lin and H.-C. Lee 25. Price HJ, Bachmann K, Chambers KL, Riggs J. (1980) Detection of intraspecific variation in nuclear DNA content in Microseris douglasii. Bot Gaz 141:195–198. 26. Jones WE, Kuehnle AR, Arumuganathan K. (1998) Nuclear DNA content of 26 orchid (Orchidaceae) genera with emphasis on Dendrobium. Ann Bot 82:189–194. 27. Traas J, Hülskamp M, Gendreau E, Höfte H. (1998) Endoreduplication and development: Rule without dividing? Curr Opin Plant Biol 1:498–503. 28. Grafi G, Larkins BA. (1995) Endoreduplication in maize endosperm: Involvement of M phase-promoting factor inhibition and induction of S phase-related kinases. Science 269:1262–1264. 29. Grafi G. (1998) Cell cycle regulation of DNA replication: The endoreduplication perspective. Exp Cell Res 244:372–378. 30. Nagl W. (1976) DNA endoreduplication and polyteny understood as evolutionary strategies. Nature 261:614–615. 31. Brodsky WY, Uryvaeva LV. (1977) Cell polyploidy: Its relation to tissue growth and function. Int Rev Cytol 50:275–332. 32. Larkins BA, Dilkes BP, Dante RA, et al. (2001) Investigating the hows and whys of DNA endoreduplication. J Exp Bot 52:183–192. 33. Hülskamp M, Schnittger A, Folkers U. (1999) Pattern formation and cell differentiation: Trichomes in Arabidopsis as a genetic model system. Int Rev Cytol 186:147–178. 34. Szymanski DB, Lloyd AM, Marks MD. (2000) Progress in the molecular genetic analysis of trichome initiation and morphogenesis in Arabidopsis. Trends Plant Sci 5:214–219. 35. Folker U, Berger J, Hülskamp M. (1997) Cell morphogenesis of trichomes in Arabidopsis: Differential control of primary and secondary branching by branch initiation regulators and cell growth. Development 124:3779–3786. 36. Kondorosi E, Roudier F, Gendreau E. (2000) Plant cell-size control: Growing by ploidy? Curr Opin Plant Biol 3:488–492. 37. Galitski T, Saldanha AJ, Styles CA, et al. (1999) Ploidy regulation of gene expression. Science 285:251–254. 38. Mizukami Y. (2001) A matter of size: Development control of organ size in plants. Curr Opin Plant Biol 4:533–539. 39. Lee HC, Chiou DW, Chen WH, et al. (2004) Dynamics of cell growth and endoreduplication during orchid flower development. Plant Sci 166:659–667. 40. Boudolf V, Vlieghe K, Beemster GTS, et al. (2004) The plant-specific cyclindependent kinase CDKB1;1 and transcription factor E2Fa-DPa control the balance of mitotically dividing and endoreduplicating cells in Arabidopsis. Plant Cell 16:2683–2692. Analysis of the Orchid Genome Size Using Flow Cytometry ✦ 113 41. Weinl C, Marquardt S, Kuijt SJ, et al. (2005) Novel functions of plant cyclin-dependent kinase inhibitors, ICK1/KRP1, can act non-cellautonomously and inhibit entry into mitosis. Plant Cell 17:1704–1722. 42. Engelen-Eigles G, Jones RJ, Phillips RL. (2000) DNA endoreduplication in maize endosperm cells: The effect of exposure to short-term high temperature. Plant Cell Environ 23:657–663. 43. Cookson SJ, Radziejwoski A, Granier C. (2006) Cell and leaf size plasticity in Arabidopsis: What is the role of endoreduplication? Plant Cell Environ 29:1273–1283. 44. Gendreau E, Traas J, Desnos T, et al. (1997) Cellular basis of hypocotyl growth in Arabidopsis thaliana. Plant Physiol 114:295–305. 45. Gendreau E, Hofte H, Grandjean O, et al. (1998) Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl. Plant J 13:221–230. 46. Ceccarelli M, Santantonio E, Marmottini F, et al. (2006) Chromosome endoreduplication as a factor of salt adaptation in Sorghum bicolor. Protoplasma 227:113–118. This page intentionally left blank Chapter 7 The Cytogenetics of Phalaenopsis Orchids Yen-Yu Kao*,†,‡, Chih-Chung Lin†, Chien-Hao Huang† and Yi-Hsueh Li† All Phalaenopsis species have the same chromosome number (2n = 2x = 38), but their karyotypes and genome sizes vary markedly. The variation is positively correlated with the amount of constitutive heterochromatin in the genome. Genomic in situ and Southern hybridizations indicate that species with large genomes contain more repetitive sequences; however, species with small genomes also have their own specific repetitive sequences. One family of tandem repeats named Pvr I consisting of 7-bp repeat units was isolated and characterized. Chromosomal locations of these repeats coincide with heterochromatic blocks. 7.1 Introduction The genus Phalaenopsis (Orchidaceae) represents an important source for floricultural commodity. Many species in this genus and interspecific and intergeneric hybrids are of high value due to their graceful and long-lasting flowers. Phalaenopsis plants are slow-growing perennials *Corresponding author. † Department of Botany, National Taiwan University, Taipei 106, Taiwan. ‡ Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan. 115 116 ✦ Y.-Y. Kao et al. that take two to three years to reach maturity. In addition, each plant produces very few roots and flowers, making cytogenetic analysis difficult. According to Sweet,1 the genus Phalaenopsis is composed of approximately 47 species that are grouped into nine sections, while Christenson2 recognizes 63 species in the genus and divides them into five subgenera and eight sections. These species are widely distributed, ranging from the Himalayas of northern India, through Southeast Asia to northern Australia.1 Previous studies showed that all Phalaenopsis species possess the same chromosome number (2n = 2x = 38),3,4 but their karyotypes differ markedly.5,6 Arends7 studied meiotic chromosome associations at metaphase I of eight interspecific hybrids. His results indicated that hybrids between species both with large chromosomes (e.g. P. amboinensis × P. mannii) and between species both with small chromosomes (e.g. P. amabilis × P. stuartiana) had complete bivalent formation, whereas hybrids between one species with large and the other species with small chromosomes (e.g. P. mannii × P. equestris) showed low frequencies of bivalents. Two molecular techniques, fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH), have been developed and shown as effective tools for genome research. FISH is useful for localization of specific DNA sequences on chromosomes,8 while GISH, in which total genomic DNA from one species is the probe, can discriminate parental chromosomes in interspecific or intergeneric hybrids.9,10,11 In addition, genomic Southern hybridization reveals the amount and distribution of repetitive DNA sequences in genomes, and is therefore also a useful tool to achieve an understanding of genome differentiation between species.12,13 The karyotypes, genome organization and localization of tandemly repetitive sequences on chromosomes of some representative Phalaenopsis species were studied by us in the past few years. The results from these studies are summarized below. 7.2 Karyotypes Karyotypes of nine Phalaenopsis species were analyzed based on Feulgen-stained somatic metaphase chromosomes prepared from root tips.14 Consistent with previous studies,4,6 all the species are diploids with a chromosome number of 2n = 38, but their chromosomes vary The Cytogenetics of Phalaenopsis Orchids ✦ 117 in size and centromere position. The chromosomes of five species, P. aphrodite, P. stuartiana, P. equestris, P. cornu-cervi and P. lueddemanniana, are small and uniform in size (1–2.5 µ m) and are all metacentric or submetacentric (Fig. 7.1A–B; Table 7.1). However, two species, P. venosa and P. amboinensis have strongly asymmetrical and bimodal karyotypes. They possess small, medium and large chromosomes, most of them being subtelocentric or acrocentric (Fig. 7.1C). These two species differ in the number of small chromosomes: P. venosa has 12 small chromosomes whereas P. amboinensis has 8 (Table 7.1). The karyotype of Fig. 7.1. Feulgen-stained somatic metaphase chromosomes. (A) Phalaenopsis stuartiana; (B) P. cornu-cervi; (C) P. venosa; (D) P. violacea; (E) P. mannii. Scale bar = 10 µm. Taxona Chromosome Size (µm)c TSRIb Accession Number 2n < 2.0 W1 W40 38 38 38 38 0 0 0 0 ND 23.70 ± 4.51 2.80 ± 0.06 3.13 ± 0.07 1.87 ± 0.24 1.77 ± 0.42 W9 38 38 0 0 22.29 ± 4.04 3.37 ± 0.05 2.74 ± 0.33 W8 W25 38 38 34 0 4 6 0 32 35.91 ± 5.75 86.06 ± 19.06 6.44 ± 0.16 13.50 ± 0.12 2.44 ± 0.47 11.32 ± 1.08 W2 W42 38 38 8 12 4 4 26 22 ND 79.76 ± 24.98 14.36 ± 0.19 9.52 ± 0.27 14.17 ± 1.08 13.25 ± 0.88 W23 W43 38 38 28 6 10 6 0 26 44.58 ± 3.48 87.84 ± 8.32 6.49 ± 0.22 15.03 ± 0.21 5.51 ± 0.46 27.17 ± 2.03 2.0–2.5 > 2.5 TCVc (µ m3) Nuclear DNA Contentd (pg 2C−1) CHc (% Nuclear Area) Phalaenopsis Sect. Phalaenopsis P. aphrodite Rchb.f. P. stuartiana Rchb.f. Sect. Stauroglottis P. equestris (schauer) Rchb.f. Sect. Polychilos P. cornu-cervi Rchb.f. P. mannii Rchb.f. Sect. Amboinenses P. amboinensis J. J. Smith P. venosa Shim & Fowl Sect. Zebrinae P. lueddemanniana Rchb.f. P. violacea Witte (Bornean form) a Classification of Sweet.1 Taiwan Sugar Research Institute. c Kao et al.14 d Lin et al.17 ND: not determined. b 118 ✦ Y.-Y. Kao et al. Table 7.1. Somatic Metaphase Chromosomes, Total Chromosome Volume (TCV), Nuclear DNA Content and Constitutive Heterochromatin (CH) of Nine Phalaenopsis Species The Cytogenetics of Phalaenopsis Orchids ✦ 119 P. violacea is also bimodal, with12 chromosomes being small or medium and 26 chromosomes, large (Table 7.1). The 16 largest chromosomes are metacentric or submetacentric, while the remaining 10 large chromosomes are subtelocentric or acrocentric (Fig. 7.1D). P. mannii has comparatively more symmetrical karyotype, with 6 chromosomes being medium and 32, large (Table 7.1). Most of its chromosomes are metacentric or submetacentric (Fig. 7.1E). The variation in genome size between related species is attributed to the difference in the amount of heterochromatin.15,16 In order to understand the causes of karyotype variation in Phalaenopsis species, the amount of constitutive heterochromatin (CH) in the cell cycle from interphase was estimated and compared (Fig. 7.2; Table 7.1). The value, expressed as a percentage of the total area of CH in the interphase Fig. 7.2. Feulgen-stained interphase nuclei. (A) Phalaenopsis aphrodite; (B) P. lueddemanniana; (C) P. amboinensis; (D) P. violacea. Scale bar = 10 µm. 120 ✦ Y.-Y. Kao et al. nucleus, showed a range of a 15-fold variation, ranging from 1.77% in P. stuartiana to 27.17% in P. violacea (Table 7.1). The amount of CH is positively correlated with the nuclear DNA content as reported by Lin et al.17 (Table 7.1). Therefore, we suggested that differential accumulation of CH may be a major cause for karyotype variation in Phalaenopsis orchids. The distribution of CH at late prophase/early metaphase chromosomes indicates that large chromosomes generally contain more CH than small ones, and this is true for chromosomes both within and between species (Fig. 7.3). In P. aphrodite, P. stuartiana, P. equestris, Fig. 7.3. Feulgen-stained late prophase/early metaphase chromosomes. (A) Phalaenopsis aphrodite; (B) P. cornu-cervi; (C) P. venosa. (D) P. violacea. Scale bar = 10 µm. The Cytogenetics of Phalaenopsis Orchids ✦ 121 P. cornu-cervi and P. lueddemanniana, all species containing small chromosomes, CH mainly clusters around centromeres (Fig. 7.3A–B). In P. venosa, P. amboinensis and P. violacea, both ends of large metacentric/submetacentric chromosomes and the ends of the long arms of subtelocentric/acrocentric chromosomes possess large blocks of CH, whereas small chromosomes have little or no CH (Fig. 7.3C–D). The heterochromatin blocks in P. mannii are smaller in size compared with those in the species with bimodal karyotypes, and the distribution of CH is not restricted to chromosome ends. To understand the base composition of CH, root-tip cells were stained with the fluorochrome 4′-6-diamidino-2-phenyl-indole (DAPI). The result showed that DAPI-positive bands coincide with CH regions as revealed by Feulgen staining. Since DAPI binds preferentially to AT-rich regions,18 the result suggests that the DNA of CH in Phalaenopsis orchids is rich in AT base pairs. 7.3 Genome Organization and Relationships Revealed by GISH Meiotic chromosome associations in interspecific hybrids have long been used to indicate genome homology between plant species,19 but the degree of chromosome pairing is not the only criterion for evaluating species relationships. Additionally, analysis of chromosome pairing in Phalaenopsis is difficult because: 1) each plant produces very few flowers, hindering collection of sufficient microsporocytes at the right stages for analysis; 2) microsporocytes are enclosed in a thick callose wall, which hampers stain penetration; and 3) meiotic chromosomes cannot be spread well due to clumping and stickiness. GISH has been shown to be a powerful tool for identifying genome constitution,20,21 and therefore, may be a method complementing analysis of meiotic chromosome pairing. In order to understand the relationships of Phalaenopsis species with various sizes of genomes, we examined seven interspecific hybrids of Phalaenopsis by GISH.22 The results are listed in Table 7.2. In hybrid P. aphrodite × P. sanderiana, in which both parents contained small genomes, the parental chromosome sets could not be distinguished even in the presence of 100-fold blocking DNA (Fig. 7.4A–B). A similar result was observed in hybrid P. mannii × P. violacea, in which both parents possessed large genomes (Fig. 7.4C–D). Normal chromosome pairing in hybrids with 122 ✦ Y.-Y. Kao et al. Table 7.2. Hybrids The Results from GISH in Seven Phalaenopsis Interspecific Hybrid Hybrid between species with small genomes: P. aphrodite × P. sanderiana Hybrid between species with large genomes: P. mannii × P. violacea P. mannii × P. violacea Hybrids between species with small and large genomes: P. amboinensis × P. stuartiana P. amboinensis × P. stuartiana P. violacea × P. equestris P. violacea × P. equestris P. mannii × P. stuartiana P. mannii × P. stuartiana P. mannii × P. aphrodite P. mannii × P. aphrodite Hybrid between species with medium and large genomes: P. mannii × P. lueddemanniana 2n Probe DNA Differentiation of Parental Chromosomesa 38 P. sanderiana − 38 38 P. violacea P. mannii − − 38 38 38 38 38 38 38 38 P. P. P. P. P. P. P. P. + + + + + + + + 38 P. mannii amboinensis stuartiana violacea equestris mannii stuartiana mannii aphrodite ± a: No differentiation; ±: slight differentiation; +: clear differentiation. similar sizes of genomes was reported by Arends.7 A comparison of our GISH results with those of Arends supports the suggestion that species with similar genome sizes have close phylogenetic relationships. In hybrids P. mannii × P. stuartiana, P. mannii × P. aphrodite, P. amboinensis × P. stuartiana and P. violacea × P. equestris, the genomes of P. mannii, P. amboinensis and P. violacea are much larger than those of P. stuartiana, P. aphrodite and P. equestris,14,17 and the parental species of these hybrids belong to different sections.1,2 In general, results obtained from these hybrids were similar (Table 7.2). When genomic DNAs from species with large genomes were used as probes, the parental chromosome sets could be distinguished in all The Cytogenetics of Phalaenopsis Orchids ✦ 123 Fig. 7.4. Genomic in situ hybridization in Phalaenopsis interspecific hybrids. Somatic metaphase chromosomes prepared from the hybrids were probed with FITC-labeled genomic DNA from one parent in the presence or absence of unlabeled (blocking) DNA from the other parent. Chromosomes were counterstained with propidium iodide. (A–B) Hybrid P. aphrodite × P. sanderiana chromosomes probed with P. sanderiana DNA in the absence (A) and presence of 100× blocking DNA (B); (C–D), Hybrid P. mannii × P. violacea chromosomes probed with P. mannii DNA in the absence (C) and presence of 100× blocking DNA (D); (E), Hybrid P. mannii × P. stuartiana chromosomes probed with P. mannii DNA in the absence of blocking DNA; (F–G) Hybrid P. mannii × P. stuartiana chromosomes probed with P. stuartiana DNA in the absence (F) and presence of 50× blocking DNA (G); (H–I), Hybrid P. mannii × P. lueddemanniana chromosomes probed with P. mannii DNA in the absence (H) and presence of 50× blocking DNA (I). Scale bar = 10 µm. 124 ✦ Y.-Y. Kao et al. hybrids without the application of blocking DNA (Fig. 7.4E). The strength and distribution of hybridization signals in these hybrids indicate that species with large genomes contain more repetitive sequences than species with small genomes and confirm distant relationships between the two parental species based on morphological characters.1,2 The results are consistent with Arend’s findings that hybrids between species with dissimilar sizes of genomes showed poor chromosome pairing.7 When total DNA from species with small genomes was used as the probe in the absence of blocking DNA, strong signals dispersed on chromosomes of the species with small genomes (Fig. 7.4F). After addition of 50× unlabeled blocking DNA from species with large genomes, hybridization signals were restricted to centromeric regions of small chromosomes (Fig. 7.4G). These results suggest that species with small genomes have their own specific sequences at or around centromeres. In hybrid P. mannii × P. lueddemanniana, nuclear DNA contents of these two parents differ approximately twofold; addition of 50-fold blocking DNA was necessary to distinguish the parental chromosomes (Fig. 7.4H–I). This observation indicates that P. mannii and P. lueddemanniana share a high degree of sequence homology and, therefore, have a close phylogenetic relationship. This is consistent with the cytological work of Arends,7 who found complete bivalent formation at the first meiotic division in hybrid P. lueddemanniana × P. mannii. 7.4 Repetitive Sequences 7.4.1 (GA)n microsatellites Microsatellites, also called simple sequence repeats (SSRs), are tandem repeated arrays of short core sequence. One microsatellite, (GA)11, has been mapped physically to seven Phalaenopsis species and Doritis pulcherrima.23 (GA)11 signals are dispersed on the chromosomes of P. amboinensis and P. violacea, but clustered in the centromeric regions of P. stuartiana, P. equestris, P. aphrodite, P. mannii, P. lueddemanniana and D. pulcherrima chromosomes (Fig. 7.5A–B). Signals were much stronger in P. stuartiana and P. aphrodite than in other species. In GISH experiment of hybrids between species with small and large The Cytogenetics of Phalaenopsis Orchids ✦ 125 Fig. 7.5. Fluorescence in situ hybridization of (GA)11 microsatellite (A–B) and Pvr-93 sequences (C–D) on chromosomes of Phalaenopsis species. The probe was labeled with digoxigenin and detected with anti-digoxigenin-fluorescein. Chromosomes were counterstained with propidium iodide. (A) Phalaenopsis stuartiana; (B) P. equestris; (C) P. venosa; (D) P. mannii. Scale bar = 10 µ m. genomes mentioned above, when total DNA from species with small genomes was used as the probe in the presence of 50× blocking DNA, hybridization signals were restricted to the centromeric regions of the small chromosomes (Fig. 7.4G). From these, we suspect that GISH signals in the centromeric regions of P. stuartiana, P. aphrodite, and P. equestris chromosomes were from hybridization of (GA)n microsatellite sequences. 126 ✦ Y.-Y. Kao et al. 7.4.2 Pvr I tandem repeats Genomic Southern hybridization was used to investigate the amount and distribution of repetitive DNA sequences in Phalaenopsis.24 The hybridization patterns in P. violacea and P. mannii were similar when the total genomic DNA from P. violacea was the probe. This result suggests that the kind and amount of repetitive sequences in these two species are similar. This may explain the difficulty of discriminating the chromosomes of these two species by GISH (Fig. 7.4C–D; Table 7.2). In addition, the probe from P. violacea hybridized strongly to DNAs from species with large genomes, but weakly to DNAs from species with small genomes. However, when total genomic DNA from P. aphrodite was used as a probe, an opposite result was obtained, i.e. species with small genomes showed stronger signals than did species with large genomes. These results are consistent with the observations of GISH that species with large genomes contain more repetitive sequences and species with small genomes have their own specific sequences. One family of tandemly repeated sequences, Pvr I, was isolated from P. violacea.24 Twelve clones of this family were sequenced. Sequences of these clones are not found in the EMBL/Genbank database. All sequences are AT-rich at the 5′- and 3′-ends and consist of 7-bp repeat units organized in tandems, ranging from 13 to 22 copies. The consensus sequence of these repeats is GTAAGCC. Physical mapping of one sequence of this family, Pvr-93, was preformed by FISH on mitotic metaphase chromosomes prepared from the root tips of Phalaenopsis species and the related species Doritis pulcherrima. The results revealed that in species with large genomes, including P. violacea, P. mannii, P. amboinensis, P. venosa and D. pulcherrima, Pvr-93 sequences were located in the constitutive heterochromatin regions of large chromosomes corresponding to DAPIpositive bands (Fig. 7.5C–D). No hybridization signals were observed on the small chromosomes of P. aphrodite and P. equestris. These results indicate that Pvr I sequences are species-specific and reside in AT-rich heterochromatic blocks of large chromosomes. 7.5 Conclusion The nine representative Phalaenopsis species studied by us have the same chromosome number (2n = 2x = 38), but their karyotypes differ The Cytogenetics of Phalaenopsis Orchids ✦ 127 markedly. We suggest that differential accumulation of CH is a major cause for karyotype variation. Our results from FISH, GISH and genomic Southern hybridization demonstrate that the Phalaenopsis species possess different kinds and amounts of repetitive sequences. A more detailed understanding of genome organization and relationships in Phalaenopsis will result from comparative studies of chromosome pairing at meiosis, isolation and characterization of species-specific sequences. Acknowledgment We are grateful to Dr. Chi-Chang Chen for his critical reading of this manuscript. References 1. Sweet HR. (1980) The Genus Phalaenopsis. Day Printing Corporation, California. 2. Christenson EA. (2001) Phalaenopsis: A Monograph. Timber Press, Portland, Oregon. 3. Sagawa Y. (1962) Cytological studies of the genus Phalaenopsis. Amer Orchid Soc Bull 31:459–465. 4. Sagawa Y, Shoji T. (1968) Chromosome numbers in Phalaenopsis and Doritis. Bul Pacific Orchid Soc 26:9–13. 5. Woodard JW. (1951) Some chromosome numbers in Phalaenopsis. Amer Orchid Soc Bull 20:356–358. 6. Shindo K, Kamemoto H. (1963) Karyotype analysis of some species of Phalaenopsis. Cytologia 28:390–398. 7. Arends JC. (1970) Cytological observations on genome homology in eight interspecific hybrids of Phalaenopsis. Genetica 41:88–100. 8. Jiang J, Gill BS. (1994) Nonisotopic in situ hybridization and plant genome mapping: the first 10 years. Genome 37:717–725. 9. Schwarzacher T, Leitch AR, Bennett MD, Heslop-Harrison JS. (1989) In situ localization of parental genomes in a wide hybrid. Ann Bot 64:315–324. 10. Ørgaard M, Heslop-Harrison JS. (1994) Investigations of genome relationships between Leymus, Psathyrostachys, and Hordeum inferred by genomic DNA: DNA in situ hybridization. Ann Bot 73:195–203. 11. D’Hont A, Paget-Goy A, Escoute J, Carreel F. (2000) The interspecific genome structure of cultivated banana, Musa spp. revealed by genomic DNA in situ hybridization. Theor Appl Genet 100:177–183. 128 ✦ Y.-Y. Kao et al. 12. Ørgaard M, Heslop-Harrison JS. (1994) Relationships between species of Leymus, Psathyrostachys, and Hordeum (Poaceae, Triticeae) inferred from Southern hybridization of genomic and cloned DNA probes. Plant Syst Evol 189:217–223. 13. Aggarwal RK, Brar DS, Khush GS. (1997) Two new genomes in the Oryza complex identified on the basis of molecular divergence analysis using total genomic DNA hybridization. Mol Gen Genet 254:1–12. 14. Kao YY, Chang SB, Lin TY, et al. (2001) Differential accumulation of heterochromatin as a cause for karyotype variation in Phalaenopsis orchids. Ann Bot 87:387–395. 15. Flavell R. (1982) Sequence amplification, deletion and rearrangement: Major sources of variation during species divergence. In: Dover FA, Flavell RB (eds.), Genome Evolution. Academic Press, London, pp. 301–323. 16. Kubis S, Schmidt T, Heslop-Harrison JS. (1998) Repetitive DNA elements as a major component of plant genomes. Ann Bot 82 (Suppl. A):45–55. 17. Lin S, Lee HC, Chen WH, et al. (2001) Nuclear DNA contents of Phalaenopsis species and Doritis pulcherrima. J Amer Soc Hort Sci 126:195–199. 18. Sumner AT. (1990) Chromosome Banding. Unwin Hyman, London. 19. Singh RJ. (2003) Plant Cytogenetics. 2nd Edition. CRC Press, Boca Raton, Florida, pp. 277–306. 20. Bennett ST, Kenton AY, Bennett MD. (1992) Genomic in situ hybridization reveals the allopolyploid nature of Milium montianum (Gramineae). Chromosoma 101:420–424. 21. Fukui K, Shishido R, Kinoshita T. (1997) Identification of the rice D-genome chromosomes by genomic in situ hybridization. Theor Appl Genet 95: 1239–1245. 22. Lin CC, Chen YH, Chen WH, et al. (2005) Genome organization and relationships of Phalaenopsis orchids inferred from genomic in situ hybridization. Bot Bull Acad Sin 46:339–345. 23. Li YH. (2000) Investigation on the physical distribution of microsatellites in Phalaenopsis orchids by fluorescence in situ hybridization. Master thesis, Department of Botany, National Taiwan University, Taipei, Taiwan. 24. Huang CH. (1999) Isolation and characterization of two repetitive DNA sequences in Phalaenopsis orchids. Master thesis, Department of Botany, National Taiwan University, Taipei, Taiwan. Chapter 8 Analysis of the Chloroplast Genome of Phalaenopsis aphrodite Ching-Chun Chang*,†, Hsien-Chia Lin† and Wun-Hong Zeng† The complete nucleotide sequence of the chloroplast genome of the Taiwan moth orchid (Phalaenopsis aphrodite subsp. formosana) was determined. The circular, double-stranded DNA of 148,964 bp comprises a pair of inverted repeats of 25,732 bp, which are separated by a small single copy (SSC) and a large single copy (LSC) region of 11,543 and 85,957 bp, respectively. The genome contains 76 protein coding genes, four ribosomal RNA genes, 30 tRNA genes, and 24 putative open reading frames (ORFs). Seventeen genes are intron-containing, including 6 tRNA and 11 protein coding genes. Unlike other chloroplast genomes of photosynthetic angiosperms, which have a complete set of genes in the 11 subunits of NADH dehydrogenase, the chloroplast genome of Phalaenopsis completely lacks the ndhA, ndhH, and ndhF genes. The other eight ndh genes have various degrees of nucleotide insertion/deletion, and they are all frameshifted. This loss results in the SSC region in Phalaenopsis being the shortest among known photosynthetic angiosperms. 8.1 Introduction Chloroplasts, in which photosynthesis takes place, have distinct functional genomes. They were once free-living cyanobacteria and even now contain preserved remnants of eubacterial genomes,1 but with some of their genes *Corresponding author. † Institute of Biotechnology, National Cheng Kung University, Tainan, Taiwan. 129 130 ✦ C.-C. Chang, H.-C. Lin and W.-H. Zeng having been transferred to the nucleus of the cell during the course of evolution.2,3 Since the complete plastid genome sequences of tobacco and liverwort were reported in 1986,4,5 over 30 complete sequences representing the major lineages of vascular plants have been made available in the genomic database of NCBI (http://www.ncbi.nlm.nih.gov/genomes/ ORGANELLES/plastids_tax.html). The DNA molecules in the chloroplast genome of photosynthetic plants are circular and range in size from 116 to 204 kb. With few exceptions, such as in conifers6 and Medicago (accession number NC_003119, unpublished), most of the genomes that have been examined have a large (5–76 kb) inverted repeat (IR) segment, which accounts for the variation in length of the genomes.2,7,6 Two segments of the IRs are divided by a large single copy (LSC) and a small single copy (SSC) region.2,7 Among vascular plants, chloroplast genome contains 110–130 genes, and most of these genes code for proteins mostly involved in photosynthesis or gene expression, with the remainder being transfer RNA or ribosomal RNA genes.8 Furthermore, the polycistronic transcription units of the known chloroplast genomes are also highly conserved.7 Although the plastid genomes of vascular plants are conserved in the overall structure,7 comparative genomic studies have recently revealed many evolutionary hotspots and single nucleotide polymorphism that are useful for phylogenetic studies below the family and subspecies level.9–12 Furthermore, genome-wide analysis of transcripts in the chloroplast has revealed that plants have undergone dramatic changes in both the levels and patterns of editing, from hornworts (1.2%) and ferns (0.38%) to seed plants (less than 0.05%).13,14 In addition to phylogenetic studies, complete genome sequences provide useful information for designing expression cassettes for chloroplast genetic manipulation and for elucidating functional genomics.15–17 With approximately 30,000 species, orchids are one of the largest and most diverse families of flowering plants.18 The mode of chloroplasts DNA transmission in orchids is uniparentally inherited from female ancestors.19,20 Based on molecular markers from plastid genes, the phylogenetic relationship has been investigated at the family and subfamily level within Orchidaceae.21,22 As a part of a larger effort to preserve the native species and to utilize its chloroplast genomes for various biotechnological purposes, we sequenced the complete chloroplast genome of Phalaenopsis aphrodite subsp. formosana. It is anticipated that knowledge of the chloroplast genome of this species will be useful for commercial breeding and for phylogenetic classification within Orchidaceae as well as for genome-based analysis in angiosperm evolution. Analysis of the Chloroplast Genome of Phalaenopsis aphrodite ✦ 131 8.2 Phalaenopsis Chloroplast DNA Extraction, Genome Sequencing, and Gene Annotation Leaves of Phalaenopsis aphrodite subsp. formosana were obtained from seedlings at the four-leaf stage of development. Intact chloroplasts were fractionated with step percoll (40–80%) gradient.23 The chloroplast DNA was further isolated according to a CTAB-based protocol.24 Chloroplast DNA was randomly fragmented into 2 to 3 kb pieces through hydroshear with GeneMachine (Genomic Solutions Inc., Michigan, USA), and then cloned into the pBluescriptSK vector. After transformation, positive shotgun clones were propagated on microtiter plates. Plasmid DNAs were isolated and used as templates for sequence analysis. The sequencing reaction was performed using the BigDye terminator cycle sequencing kit (Applied Biosystems, USA), according to the protocol recommended by the manufacturer. The DNA sequencer used was an Applied Biosystems ABI 3700. The sequence data from both ends of each shotgun clone were accumulated, trimmed, aligned, and assembled using Phred-Phrap programs (Phil Green, University of Washington, Seattle, USA). The sequence data were accumulated to 1,728,000 nucleotides, which is 11.6X coverage for the chloroplast genome of P. aphrodite subsp. formosana. Database searches were conducted with the BLAST algorithm provided by the National Center for Biotechnology Information (NCBI). The tRNAscan program was used to assign the tRNA genes. 8.3 Phalaenopsis Chloroplast Genome Properties 8.3.1 Overall genome structure The complete circular chloroplast DNA of Phalaenopsis aphrodite subsp. formosana (the Taiwan moth orchid) is 148,964 bp (accession number AY916449) long and can be separated into two regions by a pair of inverted repeat regions (IR) of 25,732 bp (Fig. 8.1). The large single copy region (LSC) and the small single copy region (SSC) are 85,957 bp and 11,543 bp long, respectively. The overall A+T content of the chloroplast genome of P. aphrodite subsp. formosana is 63.4%, which is close to the A+T percentages of rice (61%), maize (61.5%), wheat (61.7%), wild rice (61%), and sugarcane (61.6%). The A+T contents in the LSC and SSC region are higher, being 66.1% and 71.9%, respectively, than in the IR regions (56.8%). The gene order in the Phalaenopsis chloroplast 132 ✦ C.-C. Chang, H.-C. Lin and W.-H. Zeng Fig. 8.1. Gene map of Phalaenopsis aphrodite subsp. formosana chloroplast genome.30 Genes shown inside circle are transcribed clockwise; those outside are transcribed counterclockwise. Genes belonging to different functional groups are color coded. Asterisks indicate genes containing introns. Analysis of the Chloroplast Genome of Phalaenopsis aphrodite ✦ 133 genome is more similar to that of the known dicots than to the grasses, as in the latter, gene rearrangements caused by inversion are extensive in the LSC region.9,25–28 8.3.2 Gene content, intron, and codon usage Table 1 presents the content of genes in the Phalaenopsis chloroplast DNA. A total of 110 genes (not including genes duplicated in the IR) were identified as being scattered around the chloroplast genome. They include 76 protein coding genes, four ribosomal RNA genes, and 30 transfer RNA genes. Among them, 17 are intron-containing genes, including 6 tRNA and 11 protein coding genes (Table 2). Three of the latter genes, i.e. clpP, rps12, and ycf3, contain two introns. In the five grasses, there are 16 introncontaining genes, and the clpP and rpoC1 genes have lost introns.9,25–28 The rps12 gene is a divided gene whose 5′-end exon is located in the LSC region, but whose second and third exons are in the IR regions. It appears that a trans-splicing mechanism is required between exon I and exon II to yield mature rps12 transcripts. The intron of the trnL-UAA gene contains minisatellite repeat elements in the marsh orchid, Orchis palustris,29 but in P. aphrodite subsp. formosana it does not. In addition, 24 ORFs were identified with a threshold of 225 bp. An ORF, orf91, located in the complementary strand of the rrn23 gene, was reported for the first time.30 Its amino acid sequence is 81% identical to a hypothetical protein (NCBI accession no. ZP_00203428) encoded in the genome of Anabaena variabilis. We also observed the orf91 transcripts in the total RNAs of P. aphrodite and tobacco by RT-PCR assay and RNA filter hybridization,30 which demonstrated that orf91 was actually transcribed in the chloroplasts. However, further studies are required to determine if the orf91 transcripts are translated and to characterize the function of the translated protein. The codon usage in Phalaenopsis chloroplast genome and the anticodons of 30 tRNA species are summarized in Table 3. The codon usage determined from 67 protein coding genes, is strongly biased toward A or T due to the high A-T content at the third codon positions. For example, degenerated codons at the third position exhibit usage frequencies of 65% to 81% for both A or T. The frequencies of the stop codon usage for UAA, UAG, and UGA are 48%, 27%, and 25%, respectively. These are also strongly biased toward A or T at both the second and third positions. The strong AT bias of codon usage in Phalaenopsis is consistent List of Gene Contents in Phalaenopsis Chloroplast Genome (Total 110 Genes) Group of Genes RNA genes rRNA genes tRNA genes Protein coding genes Ribosomal proteins Small subunit Large subunit Transcription/translation apparatus DNA dependent RNA polymerase Translation initiation factor Photosynthesis Subunits of Photosystem I Subunits of Photosystem II Subunits of Cytochrome b6/f complex Subunits of ATP synthase Subunits of NADH-dehydrogenase Large subunit of RubisCO rrn4.5,b rrn5,b rrn16,b rrn23b trnA (ugc),a,b trnC (gca), trnD (guc), trnE (uuc), trnF (gaa), trnG (gcc), trnG (ucc),a trnH (gug),b trnI (cau), trnI (gau),a,b trnK (uuu),a trnL (caa),b trnL (uag), trnL (uaa),a trnM (cau),b trnfM (cau), trnN (guu),b trnP (ugg), trnQ (uug), trnR (ucu), trnR (acg),b trnS (gcu), trnS (uga), trnS (gga), trnT (ggu), trnT (ugu), trnV (gac),b trnV (uac),a trnW (cca), trnY (gua) rps2, rps3, rps4, rps7,b rps8, rps11, rps12,a,b rps14, rps15, rps16,a rps18, rps19b rpl2,a,b,c rpl14, rpl16,a rpl20, rpl22, rpl23,b rpl32, rpl33, rpl36 rpoA, rpoB, rpoC1,a rpoC2 infA psaA, psaB, psaC, psaI, psaJ, ycf3,a ycf4 psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ petA, petB,a petD,a petG, petL, petN atpA, atpB, atpE, atpF,a atpH, atpI ψ -ndhB,a,b ψ -ndhC, ψ -ndhD, ψ -ndhE, ψ -ndhG, ψ -ndhI, ψ -ndhJ, ψ -ndhK rbcL (Continued ) 134 ✦ C.-C. Chang, H.-C. Lin and W.-H. Zeng Table 8.1. Table 8.1. (Continued ) Miscellaneous proteins Conserved proteins Open reading frames a accD, clpP,a matK, cemA, ccsA ycf1, ycf 2b ORF114 (175d) ORF80 (1754d) ORF103 (51817d) ORF88 (61993e) ORF79 (70125d) ORF99 (70354e) ORF90 (93146d)b ORF81B (97939d)b ORF81A (102460d)b ORF115 (102606d)b ORF91 (109119d)b ORF131 (110120d)b Intron containing gene. Two copies due to inverted repeat. c Initiation codon created by RNA editing. d ORF translated clockwise. e ORF translated counterclockwise. The position of start codon of putative ORF is given in parenthesis. b ORF98 (22677d) ORF87C (64106d) ORF81C (74208e) ORF79 (99557d)b ORF86A (104928d)b ORF77B (112628e) ORF87B (23929d) ORF85 (69399e) ORF86B (86687d)b ORF77A (100484d)b ORF170 (105185d)b ORF87A (122213d) Analysis of the Chloroplast Genome of Phalaenopsis aphrodite ✦ 135 Group of Genes 136 ✦ C.-C. Chang, H.-C. Lin and W.-H. Zeng Table 8.2. Gene atpF clpP ndhB petB petD rpl2 rpl16 rpoC1 rps12* rps16 trnA-ugc trnG-ucc trnI-gau trnK-uuu trnL-uaa trnV-uac ycf3 The Splitting Genes on Phalaenopsis Chloroplast Genome Location Exon I Intron I Exon II LSC LSC IR LSC LSC IR LSC LSC IR LSC IR LSC IR LSC LSC LSC LSC 145 71 733 6 8 391 9 453 114 245 38 23 37 37 35 39 124 986 981 532 726 865 664 1251 758 — 914 802 687 944 2896 717 582 731 410 292 763 642 484 431 399 1608 232 40 35 48 35 35 50 35 230 Intron II Exon III 689 252 547 26 750 153 *The rps12 gene is divided. The 5′-rps12 locates on the LSC region, while the 3′-rps12 locates on the IR region. The number indicates the length of the intron or exon in the base pairs. with the results published elsewhere concerning the chloroplast genome of many higher plants.5,11,25,27 8.3.3 Loss of ndh genes Ndh genes that encode the subunits of the NADH dehydrogenase complex are involved in the cyclic electron flow of photosystem I and chlororespiration in tobacco.15 All 11 subunits of ndh genes are present in the chloroplast genomes of photosynthetic angiosperms so far sequenced. In contrast, in the chloroplast genomes of black pine, the ndhA, ndhG, ndhF, and ndhJ genes were completely absent, and sequences of the other seven ndh genes were truncated as obvious pseudogenes.31 In Phalaenopsis, the ndhA, ndhF, and ndhH genes are completely absent from the chloroplast genome, and the other remaining eight ndh genes are frameshifted. The ndhB, ndhC, and ndhJ genes have small region of nucleotide insertion/deletion, and the ndhD, ndhE, ndhG, ndhI, and Table 8.3. F F L L 612 331 557 386 trnF-GAA CUU CUC CUA CUG L L L L 363 113 226 119 AUU I AUC I AUA I AUG M UCU UCC UCA UCG S S S S 398 trnS-GGA 215 254 trnS-UGA 100 UAU UAC UAA UAG Y 475 trnY-GUA Y 116 — 32 — 18 UGU UGC UGA UGG C 147 trnC-GCA C 48 — 17 trnW-CCA W 308 trnL-UAG CCU CCC CCA CCG P 287 trnP-UGG P 167 P 200 P 68 CAU CAC CAA CAG H 355 trnH-GUG H 95 Q 501 trnQ-UUG Q 158 CGU CGC CGA CGG R 272 trnR-ACG R 60 R 250 R 64 727 trnI-GAU 294 423 trnI-CAU 395 trnM-CAU ACU ACC ACA ACG T 379 trnT-GGU T 152 T 263 trnT-UGU T 98 AAU AAC AAA AAG N N K K 612 trnN-GUU 165 663 trnK-UUU 243 AGU S 289 trnS-CCU AGC S 62 AGA R 331 trnR-UCU AGG R 119 GCU GCC GCA GCG A 473 trnA-UGC A 138 A 312 A 90 GAU GAC GAA GAG D D E E 570 trnD-GUC 124 701 trnE-UUC 228 GGU GGC GGA GGG trnL-UAA trnL-CAA trnfM-CAU GUU GUC GUA GUG V V V V 369 129 357 155 trnV-GAC trnV-UAC G G G G 433 trnG-GCC 122 490 trnG-UCC 207 Analysis of the Chloroplast Genome of Phalaenopsis aphrodite ✦ 137 UUU UUC UUA UUG Codon Usage of the Phalaenopsis Chloroplast Genome 138 ✦ C.-C. Chang, H.-C. Lin and W.-H. Zeng ndhK genes are truncated with large deletion of 26%, 60%, 45%, 46%, and 32% of the sequences, respectively, as compared with that of tobacco. Probably, all the resident ndh genes were pseudogenes. The loss of functional ndh genes explains well why P. aphrodite subsp. formosana has the smallest SSC region among the sequenced monocots. Although the majority of the ndh genes have been lost from the chloroplast genome of Pinus and Phalaenopsis, the ndhF gene was present in all vascular plant divisions,32 suggesting that the lost ndhF and other ndh genes in the plastid genome of Phalaenopsis might have been transferred to the nuclear genome. Indeed, we were able to detect the presence of ndhA, ndhF, and ndhH gene fragments by RT-PCR (unpublished data). Further studies will unravel the details of the lost ndh genes in the plastid of P. aphrodite. 8.3.4 RNA editing RNA editing plays an important role in the regulation of gene expression in chloroplasts. The plastid rpl2 gene of Phalaenopsis has an ACG codon rather than an ATG codon at the translation initiation site (positions 88242–88244) as was observed in maize.33 The ACG rather than an ATG was also found at the initiation codon of the ndhD genes (positions 115315–115317), as in tobacco and spinach.34 Furthermore, frameshifts were observed in the ndh genes, suggesting RNA editing is required to restore their gene function. Our RT-PCR assays indicated that RNA editing occurs in the orchid because a C to U conversion was verified in the initiation codon of rpl2 transcripts.30 However, a C to U conversion was not apparent in the ndhD transcripts, which further indicates that ndhD is a pseudogene. Furthermore, we did not detect RNA editing in the ndhC, ndhD, ndhJ, and ndhK transcripts for repairing the internal stop codon,30 further implying that the eight variously truncated and frameshifted ndh genes are probably pseudogenes. Currently, we have been analyzing the transcripts of 76 protein coding genes from P. aphrodite subsp. formosana to extensively study the pattern of RNA editing in chloroplast. 8.3.5 IR contraction and expansion The border between the inverted repeat region and the single copy region usually shifts among the various plastome species.11,26,35 Using tobacco as a representative of dicots, a comparison of the exact IR-border positions and their adjacent genes among tobacco, orchid, maize, rice, and wheat is shown in Fig. 8.2. This comparison further demonstrates Analysis of the Chloroplast Genome of Phalaenopsis aphrodite ✦ 139 Fig. 8.2. Comparison of border positions of LSC/IR and SSC/IR regions and gene order within two LSC/IR regions among five chloroplast genomes.30 Various lengths of ycf1 or ndhH pseudogenes are created in tobacco, rice and wheat at the IRb/SSC border. 29 bp of ndhF pseudogene is created in maize at the SSC/IRa border. 31 bp of rpl22 pseudogene is created in Phalaenopsis aphrodite subsp. formosana at the IRa/LSC border. Both rps19 and trnH genes are located within IR region in P. aphrodite subsp. formosana, maize, rice, wheat; whereas in tobacco they are located in LSC. The rps15 gene is located in SSC in P. aphrodite subsp. formosana and tobacco, whereas it is present in the IR regions in the chloroplast genomes of five grasses. The ycf1 and ycf2 genes are lost in five grasses, while ndhA, ndhF, ndhH genes are lost in P. aphrodite subsp. formosana. Black triangles indicate loss of genes. that the border positions vary among the chloroplast genomes of angiosperms. As illustrated in Fig. 8.2, the IRb/LSC border in P. aphrodite subsp. formosana is located within the coding region of rpl22, while in maize, rice, and wheat it is located up-stream of the noncoding 140 ✦ C.-C. Chang, H.-C. Lin and W.-H. Zeng region of rpl22. The IRa/LSC borders in P. aphrodite subsp. formosana, maize, rice, and wheat are located on the downstream of the non-coding region of psbA. The IRa/LSC and IRb/LSC borders of tobacco, however, are located downstream of the noncoding region of trnH-GUG and upstream of the non-coding region of rps19, respectively. The IRb/LSC borders in many other dicot plants, such as Panax,11 Spinacia,36 Oenothera,37 Arabidopsis,38 Lotus,39 and Atropa,40 are located within the coding region of rps19, and as a result, give rise to the rps19 pseudogenes in IRa. In monocots, the rps19 and trnH genes appear to have been completely shifted into the IR region, which may represent a major structural difference between the chloroplast genomes of monocots and dicots. In P. aphrodite subsp. formosana, the IRa/SSC border (Fig. 8.2) is located upstream of the noncoding region of ycf1, while in tobacco the border is in the coding region of ycf1, with the result that the 996 bp of ycf1 pseudogene in IRb is created. In grasses, however, the IRa/SSC border is either within the initiation codon of the ndhH gene as in maize or shifted into the central region of the ndhH gene as in rice and wheat, resulting in the creation of the 163 bp and 207 bp of the ndhH pseudogene at the IRb/SSC borders in rice and wheat, respectively. The IRb/SSC border resides downstream of the noncoding region of rpl32 in P. aphrodite subsp. formosana and downstream of the coding region of the ndhF gene in tobacco, rice and wheat, but in maize it is shifted into the ndhF coding region, thereby creating the 29 bp of ndhF pseudogene in IRa. The intramolecular recombination of short direct repeat sequences probably is responsible for the shift of the IR/SSC or IR/LSC borders during evolution.11,26 The expansions and contractions of the IR region contribute to the variation in length of the chloroplast genome among plant lineages.11 The IR regions of the grass family (including maize, rice, wild rice, wheat and sugarcane) are shorter than those in P. aphrodite subsp. formosana and tobacco, mainly because their ycf2 were lost. The smaller sizes in the SSC regions of the six known monocots are due to loss of the ycf1 genes in the grass family and the deletion of several ndh genes from P. aphrodite subsp. formosana. In P. aphrodite subsp. formosana and in dicots, the rps15 genes are located in the SSC regions, while in the grass family the gene is in the IR regions. Likely, a duplication of the rps15 gene followed by an inversion occurred in the grass lineage during evolution. Analysis of the Chloroplast Genome of Phalaenopsis aphrodite ✦ 141 8.4 Conclusion and Prospective The complete nucleotide sequence of the chloroplast genome of the Taiwan moth orchid (Phalaenopsis aphrodite subsp. formosana) was determined. The genome contains 76 protein coding genes, four ribosomal RNA genes, 30 tRNA genes, and 24 open reading frames (ORFs). The availability of complete chloroplast genomic sequence of Phalaenopsis aphrodite subsp. formosana will be very useful in designing molecular markers both for commercial breeding and for phylogenetic classification within Orchidaceae. Furthermore, the plastid genomic information provides the fundamentals to study gene expression and regulation in the chloroplasts of Phalaenopsis orchid. In addition, since the lack of complete chloroplast genome sequences is still one of the major limitations for extending chloroplast genetic engineering technology to useful crops, a complete genomic sequence of Phalaenopsis chloroplast will provide valuable information. This information can be used for the construction of chloroplast expression vector for site-specific integration of foreign genes through homologous recombination in orchid plastid transformation in the near future. Acknowledgments This work was financially supported by grants (NSC92-2317-B006-004) to C.-C. Chang from the National Science Council of Taiwan. References 1. Martin W, Stoebe B, Goremykin V, et al. (1998) Gene transfer to the nucleus and the evolution of chloroplasts. Nature 393:162–165. 2. Palmer JD. (1991) Plastid chromosome: Structure and evolution. In: Vasil IK, Bogorad L (eds.), Cell Culture and Somatic Cell Genetics in Plants, Vol. 7A, The Molecular Biology of Plastids. Academic Press, San Diego, pp. 5–53. 3. Martin W, Rujan T, Richly E, et al. (2002) Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc Natl Acad Sci USA 99:12246–12251. 4. Ohyama K, Fukuzawa H, Kohchi T, et al. (1986) Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322:572–574. 142 ✦ C.-C. Chang, H.-C. Lin and W.-H. Zeng 5. Shinozaki K, Ohme M, Tanaka M, et al. (1986) The complete nucleotide sequence of tobacco chloroplast genome: Its gene organization and expression. EMBO J 5:2043–2049. 6. Raubeson LA, Jansen RK. (2005) Chloroplast genomes of plants. In: Herry RJ (ed.), Plant Diversity and Evolution: Genotypic and Phenotypic Variation in Higher Plants. CABI Publishing, Cambridge, pp. 45–68. 7. Sugiura M. (1995) The chloroplast genome. Essays Biochem 30:49–57. 8. Jansen RK, Raubeson LA, et al. (2005). Methods for obtaining and analyzing whole chloroplast genome sequences. Methods Enzymol 395:348–384. 9. Asano T, Tsudzuki T, Takahashi S, et al. (2004) Complete nucleotide sequence of the sugarcane (Saccharum officinarum) chloroplast genome: A comparative analysis of four monocot chloroplast genomes. DNA Res 11:93–99. 10. Calsa T, Carraro DM, Benatti MR, et al. (2004) Structural features and transcript-editing analysis of sugarcane (Saccharum officinarum L.) chloroplast genome. Curr Genet 46:366–373. 11. Kim KJ, Lee HL. (2004) Complete chloroplast genome sequences from Korean ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants. DNA Res 11:247–261. 12. Tang J, Xia H, Cao M, et al. (2004) A comparison of rice chloroplast genomes. Plant Physiology 135:412–420. 13. Kugita M, Yamamoto Y, Fujikawa T, et al. (2003) RNA editing in hornwort chloroplasts makes more than half the genes functional. Nucleic Acids Res 31:2417–2423. 14. Wolf PG, Rowe CA, Hasebe M. (2004) High levels of RNA editing in a vascular plant chloroplast genome: Analysis of transcripts from the fern Adiantum capillus-veneris. Gene 339:89–97. 15. Burrows PA, Sazanov LA, Svab Z, et al. (1998) Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes. EMBO J 17:868–876. 16. Drescher A, Ruf S, Calsa T, et al. (2000) The two largest chloroplast genome-encoded open reading frames of higher plants are essential genes. Plant J 22:97–104. 17. Daniell H, Khan MS, Allison L. (2002) Milestones in chloroplast genetic engineering: An environmentally friendly era in biotechnology. Trends Plant Sci 7:84–91. 18. Atwood JT. (1986) The size of the Orchidaceae and the systematic distribution of epiphytic orchids. Selbyana 9:171–186. 19. Chang SB, Chen WH, Chen HH, et al. (2000) RFLP and inheritance patterns of chloroplast DNA in intergeneric hybrids of Phalaenopsis and Doritis. Botany Bulletin of Academia Sinica 41:219–223. Analysis of the Chloroplast Genome of Phalaenopsis aphrodite ✦ 143 20. Cafasso D, Widmer A, et al. (2005) Chloroplast DNA inheritance in the orchid Anacamptis palustris using single-seed polymerase chain reaction. J Hered 96:66–70. 21. Neyland R, Urbatsch LE. (1996) The ndhF chloroplast gene detected in all vascular plant divisions. Planta 200:273–277. 22. Cameron KM. (2004) Utility of plastid psaB gene sequences for investigating intrafamilial relationships within Orchidaceae. Mol Phylogenet Evol 31:1157–1180. 23. Robinson SP, Downton WJ. (1984) Potassium, sodium, and chloride content of isolated intact chloroplasts in relation to ionic compartmentation in leaves. Arch Biochem Biophys 228:197–206. 24. Stewart CN Jr, Via LE. (1993) A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Biotechniques 14:748–750. 25. Hiratsuka J, Shimada H, Whittier R, et al. (1989) The complete sequence of the rice (Oryza sativa) chloroplast genome: Intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol Gen Genet 217:185–194. 26. Maier RM, Neckermann K, Igloi GL, Kossel H. (1995) Complete sequence of the maize chloroplast genome: Gene content, hotspots of divergence and fine tuning of genetic information by transcript editing. J Mol Biol 251:614–628. 27. Ogihara Y, Isono K, Kojima T, et al. (2002) Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA. Mol Genet Gen 266:740–746. 28. Shahid Masood M, Nishikawa T, Fukuoka S, et al. (2004) The complete nucleotide sequence of wild rice (Oryza nivara) chloroplast genome: First genome wide comparative sequence analysis of wild and cultivated rice. Gene 340:133–139. 29. Cafasso D, Pellegrino G, Musacchio A, et al. (2001) Characterization of a minisatellite repeat locus in the chloroplast genome of Orchis palustris (Orchidaceae). Curr Genet 39:394–398. 30. Chang CC, Lin HC, et al. (2006) The chloroplast genome of Phalaenopsis aphrodite (Orchidaceae): Comparative analysis of evolutionary rate with that of grasses and its phylogenetic implications. Mol Biol Evol 23:279–291. 31. Wakasugi T, Tsudzuki J, Ito S, et al. (1994) Loss of all ndh genes as determined by sequencing the entire chloroplast genome of the black pine Pinus thunbergii. Proc Natl Acad Sci USA 91:9794–9798. 32. Neyland R, Urbatsch LE. (1996) Phylogeny of subfamily Epidendroideae (Orchidaceae) inferred from ndhF chloroplast gene sequences. Amn J Bot 83:1195–1206. 144 ✦ C.-C. Chang, H.-C. Lin and W.-H. Zeng 33. Hoch B, Maier RM, Appel K, et al. (1991) Editing of a chloroplast mRNA by creation of an initiation codon. Nature 353:178–180. 34. Neckermann K, Zeltz P, Igloi GL, et al. (1994) The role of RNA editing in conservation of start codons in chloroplast genomes. Gene 146:177–182. 35. Goulding SE, Olmstead RG, Morden CW, Wolfe KH. (1996) Ebb and flow of the chloroplast inverted repeat. Mol Gen Genet 252:195–206. 36. Schmitz-Linneweber C, Maier RM, Alcaraz JP, et al. (2001) The plastid chromosome of spinach (Spinacia oleracea): Complete nucleotide sequence and gene organization. Plant Mol Biol 45:307–315. 37. Hupfer H, Swiatek M, Hornung S, et al. (2000) Complete nucleotide sequence of the Oenothera elata plastid chromosome, representing plastome I of the five distinguishable euoenothera plastomes. Mol Gen Genet 263:581–585. 38. Sato S, Nakamura Y, Kaneko T, et al. (1999) Complete structure of the chloroplast genome of Arabidopsis thaliana. DNA Res 6:283–290. 39. Kato T, Kaneko T, Sato S, et al. (2000) Complete structure of the chloroplast genome of a legume, Lotus Japonicus. DNA Res 7:323–330. 40. Schmitz-Linneweber C, Regel R, Du TG, et al. (2002) The plastid chromosome of Atropa belladonna and its comparison with that of Nicotiana tabacum: The role of RNA editing in generating divergence in the process of plant speciation. Mol Biol Evol 19:1602–1612. Chapter 9 Analysis of Expression of Phalaenopsis Floral ESTs Wen-Chieh Tsai†, Yu-Yun Hsiao‡, Zhao-Jun Pan‡ and Hong-Hwa Chen*,‡ Orchids have great diversity of specialized pollination and ecological strategies and provide a rich setting for studying evolutionary relationships and molecular biology. The sophisticated orchid flower morphology offers an opportunity to discover new variant genes and different levels of complexity in the morphogenesis of flowers. To obtain plentiful gene information from orchid reproductive organs, we constructed a cDNA library of mature flower buds of Phalaenopsis equestris, a native diploid species of Phalaenopsis in Taiwan. A total of 5,593 expressed sequence tags (ESTs) from randomly selected clones were identified and characterized. Cluster analysis enabled the identification of a unigene set of 3,688 sequences. The abundance of transcripts with predicted cellular roles were functionally characterized by using the BLASTX matches to known proteins. Comparison of the relative EST frequencies based on functional categories among floral tissues of five species including P. equestris, Acorus Americanus, Asparagus officinalis, Oryza sativa, and Arabidopsis thaliana was performed. The most highly transcribed genes in Phalaenopsis floral buds are those coding for RNA-dependent RNA polymerase of Cymbidium mosaic virus, followed by heat shock protein genes. A total 217 putative transcription factor related ESTs were identified. C3H and trihelix *Corresponding author. † Department of Biological Science and Technology, Chung Hwa University of Medical Technology, Tainan County, Taiwan. ‡ Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan. 145 146 ✦ W.-C. Tsai et al. families occupied 25% of the transcribed transcription factor genes, indicating that the profile of the transcription factors in orchid flower buds is polarized. The extensive analysis of the genes in floral organs adds to the growing repertoire of known plant genes and may also reveal unique features of the reproductive organs of orchids. 9.1 Introduction 9.1.1 Arabidopsis and rice structure genomics Access to a complete, finished genome for any organism provides the basis for large-scale exploration of biology. With respect to plants, Arabidopsis has secured the historical record of being the first plant genome to be sequenced (Arabidopsis Genome Initiative, 2000),1 with rice (Oryza sativa) coming in second.2,3 The Arabidopsis genome is essentially complete with the exception of a few gaps, primarily at the centromeres. The rice genome was completed in December 2004 by a public effort of the International Rice Genome Sequencing Project (IRGSP; http://rgp.dna.affrc.go.jp/IRGSP/). The value of both organisms as model species for plant biology is further supported by the availability of not one genome sequence, but multiple genome sequences. For Arabidopsis, the public consortium sequenced to the draft level the heavily utilized Columbia accession, while a private company, Cereon, sequenced the second most utilized accession, Landsberg erecta (Ler).4 For rice, there have been four genome sequencing efforts, three being focused on the Nipponbare cultivar from the temperate subspecies japonica2,5,6 and one on the 93–11 variety from the tropical subspecies indica.3 In addition to the nuclear genomes, both the chloroplast and mitochondrial genomes of Arabidopsis and rice are publicly available.7–9 Both Arabidopsis and rice genome sequencing was preceded by expressed sequence tag (EST) sequencing, as this provides not only an inexpensive sampling method for the expressed fraction of a genome, but also a quantitative profile of the expression levels in specific tissues. ESTs also have utilities, as the cDNA clones themselves are valuable reagents for functional genomic studies. Currently, there are 326,202 Arabidopsis and 299,998 rice ESTs in the dbEST division of GenBank (http://www.ncbi.nlm.nih.gov/dbEST/ dbESTsummary.html). Analysis of Expression of Phalaenopsis Floral ESTs ✦ 147 9.1.2 The significance of Orchidaceae for plant science The family of Orchidaceae has an estimated 17,000 to 35,000 species, making it among the largest families of flowering plants.10 The species are known for their diversity of both specialized pollination and ecological strategies and provide a rich subject for investigating evolutionary relationships and molecular biology. The versatility and specialization in orchid floral morphology, structure, and physiological properties have fascinated botanists and collectors for centuries. Their most astonishing evolution is seen in their reproductive biology. Like most angiosperm flowers, orchids have two whorls of perianth segments. The outer whorl consists of three sepals. Three petals make up the inner whorl, one of them (the labellum or lip) being highly evolved with different size, form, color, and general appearance from the other two. In some genera, such as Paphiopedilum and Phragmipedium, the labellum has even evolved into a pouch. In all genera, the male and female reproductive organs are fused to form a gynostemium. Modifications of the perianth, androecium, and gynoecium can represent a basis for a variety of floral morphology. The highly sophisticated flower organization offers the opportunity to discover new variant genes and different levels of complexity in the morphogenetic networks.11,12 9.2 Expressed Sequence Tags 9.2.1 Significance of EST in genome research Single-run partial sequencing of randomly selected clones is a widely used tool in genome research. Expressed sequence tags (ESTs) have played significant roles in accelerating gene discovery, including gene family expansion,13,14 large-scale expression analysis,15–17 and elucidating phylogenetic relationships.17 Libraries of cDNAs are routinely prepared and contain tens of thousands of clones, representing a variety of specific tissue types and a snapshot of gene expression during defined developmental stages and following specific biotic and abiotic challenges. Recent developments in high volume biotechnology combined with advanced DNA sequencing technology have made it feasible to perform large-scale EST sequencing projects. 148 ✦ W.-C. Tsai et al. The concept of using cDNAs as a route to expedite gene discovery was first demonstrated in the early 1980s.19 In 1990, Sydney Brenner proposed that an obvious method for characterizing the “important” part of the human genome would involve looking at messengers from the expressed genes, thus advocating the application of highthroughput methods for transcriptome sampling.20 Mark Adams first used the term EST in relation to gene discovery and the human genome project in 1991.21 Currently there are near 25 million ESTs in the National Center for Biotechnology Information (NCBI) public collection-dbEST database (http://www.ncbi.nlm.nih.gov/dbEST/ index.html), representing a wide taxonomic variety of fungi, plants, and animals. With many large-scale EST sequencing projects in progress and new projects being initiated, the number of ESTs in the public domain will likely increase substantially, providing additional opportunities for intra- and inter-specific expression comparisons on a genomics scale. Expressed sequence tags are created by partially sequencing randomly isolated gene transcripts that have been converted into cDNA. EST sequencing initially favored the 5′-end of directionally cloned cDNAs because the 5′-sequences are likely to contain more protein coding sequence than the 3′-ends, which often contain significant untranslated regions (UTRs). Currently, the 3′-end of the cDNA clone is often preferred because it is likely to offer more unique sequence (in many cases, the UTR) and can be used to distinguish between gene paralogues. EST sequencing strategies in which both ends of the cDNA are sequenced are also becoming prevalent. In the absence of complete genome sequences, the cDNA (and its EST) remains the only link back to the genome. However, cDNA sequencing remains one of the more accessible and widely used methods for sampling the actively transcribed portion of the gene space. The preparation of cDNA libraries depends on the underlying mRNA population of a cell, tissue, or organism. The genomic structure of the host plant is, therefore, largely inconsequential. EST data have been directly applied for gene discovery,22,23 evaluation of the genome-wide gene content and structure,24 as well as in silico comparative expression analysis between different plant tissues.16,17 Moreover, ESTs can be a valuable resource for highthroughput expression analysis via the cDNA-array technology.25–28 Recent studies have revealed that mRNA transcripts can also Analysis of Expression of Phalaenopsis Floral ESTs ✦ 149 contain repeat motifs, and the abundance of ESTs available makes this an attractive potential source of microsatellite markers.29 ESTderived SSRs have been developed for many plants, such as Triticum aestivum L.,30 Hordeum vulgare L.,31 Gossypium L.,32 and Medicago truncatula.33 9.2.2 Application of full-length cDNAs (FL-cDNA) to functional genomics Most of the EST projects are based on cDNA libraries in which most of the inserts are not full-length. ESTs are useful for making a catalogue of expressed genes, but not for further study of gene function. The only viable alternatives to EST sequencing that address the attributes of incomplete sequence coverage and nucleotide quality are the full-length cDNA sequences. Consequently, genome-scale collections of the full-length cDNAs of expressed genes are both essential for gene identification and annotation of complex genomes and provide a valuable resource for experimentalists interested in gene function. In addition, full-length cDNAs are useful resources for transgenic analysis, such as overexpression, antisense suppression, double-stranded RNA interference (dsRNAi), and biochemical analysis to study the function of the encoded proteins.34 Thus, there has been a large effort to produce annotated EST and full-length cDNA collections for mammals and plants.35–37 Full-length cDNA sequences are obtained by Cap-Trapper or other technologies that take advantage of the property of the cap structure, and sequencing clones for both 5′- and 3′-end.38–44 Such a strategy yields many individual ESTs that can be assembled into a single contig and greatly improves identification of the complete exon structures of eukaryotic genes.45 9.2.3 Bioinformatics of plant EST collections It has been shown previously that EST databases are valid and reliable sources of gene expression data.15–17 With the rapid expansion of available EST data, opportunities for digital gene expression analysis will continue to expand. As a result of advances in computational molecular biology and biostatistics, it is possible to mine and analyze large-scale EST datasets efficiently and exhaustively.15–17 150 ✦ W.-C. Tsai et al. Bioinformatics-based sequence resources have been developed that address the quality, redundancy, and partial nature of EST sequences. The first crucial step in adding value to EST sequences involves clustering and assembling the ESTs into a more manageably sized dataset of better quality clustered sequences. Sequences are aggressively trimmed of vector and polylinker remnants before a fast clustering method places the ESTs into buckets of similar sequences. A final assembly step places the clustered sequences into logical contigs and singletons.46,47 The clustered sequences are typically longer than any individual EST and are of a higher quality. These cluster consensus and singleton sequences form the core sequence data within several plant specific EST derived databases. Most of these sequence databases have added further value to the sequences by attaching additional annotation to the sequences and by providing methods to select specific sequences or groups of sequences that satisfy specific criteria. The most valuable annotations and methods are those that assign a tentative function and allow retrieval and identification of sequences on the basis of tissue or challenge specificity. 9.2.4 Reasons for applying ESTs to study Phalaenopsis equestris Based on morphological characteristics, the genus Phalaenopsis comprises approximately 45 species that are grouped into nine sections.10 They have broad geographical distribution as well as commercial value as floricultural commodities. Two native species of Phalaenopsis, P. equestris and P. aphrodite var. formosa48 have been reported in Taiwan. Few molecular studies of Phalaenopsis orchids’ reproductive biology exist because of their long life cycles and inefficient transformation systems. With the use of flow cytometry, nuclear DNA contents of P. equestris were estimated to be 1.6 × 109 bp (2n = 2x = 38), about fourfold that of the rice genome.49,50 The expansion of plant genomes has mainly been the result of multiplications of retrotransposon repeat sequences.51 Thus, expressed sequence tags (ESTs) avoid the highly repetitive DNA that make up the bulk of most plant genomes and offer both a reasonable cost and rapid generation of data that can be exploited for gene discovery and comparative genomics. Analysis of Expression of Phalaenopsis Floral ESTs ✦ 151 9.3 Characterization and Significance of Phalaenopsis Floral EST 9.3.1 Significance of ESTs shared no significant similarity to any other protein sequences in public databases To provide considerable sequence information from orchid, we characterized the 5,593 floral bud ESTs reading from the 5′-end. These ESTs were assembled and generated by the PHRAP program. Among them, 2,637 ESTs were assembled into 732 contigs. These contigs and 2,956 singletons represented up to 3,688 different genes. The results reflected 34.1% (1 − number of unigenes/number of ESTs) redundancy rate of the library. The unigene set was used for similarity searches and annotation using BLAST algorithm. Putative functions of the cDNAs were assigned after applying a stringency of BLASTX scores >50. More than 75% of the database-matched unigenes were found to match proteins from plants (Table 9.1). By contrast, 8.32% of the unigenes represented genes that had been previously found in non-plant organisms, such as E. coli, Drosophila, fungi, mice, and humans. Furthermore, about 15% of the unigenes revealed no hits, indicating that these sequences may have specific roles in orchids. Although the deposited sequences in public databases are increasing exponentially year by year, recent EST analysis in non-model plants always showed a substantial portion of expressed genes that are species specific.52–54 Defining the functional identities of these unidentified genes might advance the understanding of the differences among plant species. Table 9.1. Source Overview of the Unigenes BlastX Analysisa No. of Unigenes Percentage of Total ESTs (%) Plant Non plant No hits found 2,833 307 548 76.82 8.32 14.86 Total 3,688 100.000 a Adapted from Tsai et al., Plant Science 170 (2006): 426–432. 152 ✦ W.-C. Tsai et al. 9.3.2 Significance of highest expressing level for transcripts of the Phalaenopsis flower buds The sequences in the Phalaenopsis flower bud dbEST were further characterized by the functional category of the plant genes.55 Details of the gene species included in each category are given in Fig. 9.1. A significant proportion of ESTs (15%) exhibited similarity to genes that encode enzymes of primary and secondary metabolism. In addition, the Phalaenopsis flower bud cDNA library contains transcripts encoding proteins involved in subcellular organization (6%), transcription (5%), signal transduction related genes (4%), protein fate (4%) and cell cycle (3%), tags to transport (3%) and cell rescue (3%), as well as genes involved in proteins with binding function (3%), protein synthesis (3%), interaction with the cellular environment (1%), transposable element 25% 20% 15% 10% 5% 0% sm gy oli ner ing on b a e s i is et e s p t e s a te t m oc ri th f or n pr ansc sy n tein n sp ctio nce A tr in ro tra d u le nt en t s DN ro te p lar rans viru n meop m ent on n n d p llu l t d ro el em ti io o n an ce g na an nv i d ev e el n iza ntiat izati atio o n in le s c l l z i si ns e ar e cy ab rga re ca li ct ote in n ll os ar o iffe r lo loca fu n e pr ro te no w un d ffe llu l e p d ce a k s p d e n lul e u ue ng ag d n fo e, c tra cel typ cell tiss ind i sto r ifie u hits c u ith f ll b b ss es n w o r no l ce su ll io ith c la ro n t w ce ract u n n o i c e te ot in pr P. equestris A. americanus A. officinalis O. sativa A. thaliana Fig. 9.1. Classification of the BLAST results of the floral ESTs from P. equestris, A. americanus, A. officinalis, O. sativa and A. thaliana. Relative frequencies of ESTs assigned to predicted functions were similar across these species. (Adapted from Tsai et al., Plant Science 170 (2006): 426–432.) Analysis of Expression of Phalaenopsis Floral ESTs ✦ 153 Table 9.2. Highly Abundant Transcripts Detected in the P. equestris c Flower Bud dbEST No. of ESTsa Percentageb (%) Cymbidium mosaic virus Dictyostelium discoideum Oryza sativa Oryza sativa Arabidopsis thaliana Aerides japonica 111 1.98 73 1.40 48 45 44 36 0.86 0.80 0.79 0.64 Musa acuminata 30 0.54 Nicotiana glauca Cymbidium hybrid 27 24 0.48 0.43 Dendrobium crumenatum 20 0.36 Putative Function Organism RNA-dependent RNA polymerase heat shock cognate protein Hsc70-1 beta-1,3-glucanase 40S ribosomal protein S5 lipid transfer protein phospholipid transfer protein metallothionein-like protein lipid transfer protein mannose-binding lectin precursor S-adenosyl-L-methionine synthetase a Number of clones assigned to the related gene function. Abundant genes/total number *100. c Adapted from Tsai et al., Plant Scinece 170 (2006): 426–432. b (1%), cellular organization (1%), energy production (1%), and development (1%). Another 30% showed similarity to sequences of unknown proteins (24%) and unclassified functions (6%). These ESTs that have been obtained from a non-normalized cDNA library could be used to reveal global gene expression patterns as deduced from transcript abundance. The highly expressed transcripts of the Phalaenopsis flower buds are listed in Table 9.2. The highest expressing level for transcripts of the Phalaenopsis flower buds was homologous to RNA-dependent RNA polymerase (1.98%) of Cymbidium mosaic virus. Although no virus infection symptoms were observed, these transcripts existed significantly in the floral buds, indicating that experimental materials used here were infected with the virus. Correlated with the highly expressed virus RNA-dependent RNA polymerase transcripts, ESTs related to β-1,3-glucanase were also comparably detected (0.86%). Considerable evidence suggested that β-1,3-glucanase is induced 154 ✦ W.-C. Tsai et al. during response of plants to viral, bacterial, and fungal pathogens.56 In the top 10 highly abundant ESTs, three different kinds of ESTs related to lipid transfer proteins occupied at least 1.91% of the total ESTs. These lipid transfer protein related genes, including lipid transfer protein per se, its precursor, and phospholipid transfer proteins play roles in defense and pollen tube adhesion in plants.57–59 ESTs homologous to heat shock cognate protein Hsc70-1 genes also presented a significant expression level (1.4%). Hsp70s function as molecular chaperones. In addition to heat stress, these proteins can also be induced by other environmental stresses, including cold, drought, or salinity, as well as during various developmental processes, such as embryogenesis, germination, and fruit development.60–62 Dissection of these gene functions might be a useful direction for further study of orchid floral development. 9.3.3 Significance of similar expression patterns in floral tissues of flowering plants For comparison of floral transcriptome among different plant species, plentiful EST resources deposited at public databases were downloaded to the local server for in silico comparative analysis. In particular, ESTs of floral tissue were chosen from Arabidopsis thaliana, the model plant of eudicots, Oryza sativa, the first monocot whose genome was completely sequenced (International Rice Genome Sequencing Project, 2005);63 Acorus americanus, the basal monocot which provides support as a sister to all other monocots, and Asparagus officinalis, an economically important non-grain monocot with the highly derived floral structures found in the grasses and grass relatives.64 The number of unigene sets were 2,707, 3,934, 4,548, and 4,267 from assembled floral ESTs of Arabidopsis, rice, A. americanus, and A. officinalis, respectively, and functionally characterized and compared to that of P. equestris. The results showed that the relative frequencies of ESTs assigned to predicted functions were very similar across these species (Fig. 9.1). Interestingly, transcripts related to cell type differentiation, tissue localization, and storage protein were barely detected in the floral tissues of these five species (Fig. 9.1). These findings suggested that the basic floral transcriptomes were parallel among the flowering plants from basal monocots to monocots and to eudicots. Analysis of Expression of Phalaenopsis Floral ESTs ✦ 155 9.3.4 Significance of transcription factor expression profiles in the transcriptome of Phalaenopsis flower buds Because transcription factors control the expression of a genome and play important roles in all aspects of a higher plant’s life cycle, we characterized the transcription factor associated ESTs from the transcriptome of P. equestris flower bud through the use of Arabidopsis transcription factor sequences as queries.65 In the orchid flower bud dbEST, 217 ESTs encoding putative transcription factors were identified, occupying 4% (217/5593) of flower bud transcriptome. The most abundantly expressed transcription factor gene families, C3H and trihelix, accounted for a full 25% (54/217) of the overall transcription factor expression (Fig. 9.2). In addition, bHLH (9%), C2H2 (8%) and WRKY (6%) families were found in turn (Fig. 9.2). These five families occupied approximately 50% of the expressed transcription factors. However, BBR-BPC, CCAAT-DR1, CCAAT-HAP2, CPP, GRF, SBP, VOZ-9, and WHIRLY families were not detected. The results suggested that expression of transcription factors in mature orchid flower buds is highly regulated. The C3H family has been reported to be involved in Arabidopsis embryogenesis66 and response to abscisic acid in Craterostigma plantagineum.67 To our knowledge, documents describing the relationship between C3H 30 27 27 No. of clones 25 19 20 17 14 15 10 9 9 7 7 5 5 1 2 1 2 0 11 10 3 2 1 2 1 0 0 1 2 0 8 6 5 4 3 1 1 2 2 0 3 2 0 0 0 A BI 3 A Alfi VP1 P2 n -E -lik RE e B A P A R BB RR F R- -B B BH PC LH bZ C2 IP C2 -C BZ C2 O-l R C2 ike C C2 2 -D C2 C2 of -Y -Ga A ta BB C2 Y H 2 CC CA C3 CC AA M H TA T A CC A -D T R CCAA -HA 1 A T-H P2 A A T- P H 3 A P5 E2 CPP FD P G EIL 2li G ke e G BP RA G S RF H B M M HSF Y A B- D re S la t M ed Y N B or AC ph a RE n M SB P T Tr CP ih el i TU x V W OZ B H -9 IR W LY RK Y 0 Fig. 9.2. Number of ESTs related to transcription factors appearing in each transcription factor family. A total of 1,583 putative Arabidopsis transcription factors were searched against P. equestris flower bud dbEST, and then the target ESTs were classified into corresponding transcription factor families. Each bar represents the number of EST clones in one family. (Adapted from Tsai et al., Plant Science 170 (2006): 426–432.) 156 ✦ W.-C. Tsai et al. and floral development have not been proposed. In addition to involvement in light signaling, the trihelix family also regulates perianth architecture in Arabidopsis flowers.68,69 Understanding these gene functions will advance our understanding of orchid floral development. However, there are eight transcription factor families, including BBR-BPC, CCAATDR1, CCAAT-HAP2, CPP, GRF, SBP, VOZ-9, and WHIRLY, which were not discovered in our floral bud dbEST. A CPP family gene, TSO1, modulates cytokinesis and cell expansion in Arabidopsis flowers.70 The SBP family controls the flowering time, leaf organogenesis, and pollen sac development and is associated with floral development in silver birch.71–73 These two families of transcription factors might either be rarely expressed genes or might not appear in stage IV flower buds so that EST sampling would not detect them. While no literature concerning the functions of the other six transcription factor families exists, the coincidence of absence of expression of these six families of transcription factors in orchid flower dbEST and the lack of functions in floral development for these transcription factors may explain the precise regulation of transcription factor expression in orchid flowers. 9.4 Conclusions Large-scale EST sequencing provides a gateway into the genome of organisms owing to the massive information buried in the genome-scale expression data. So far, less than 150 orchid protein sequences have been deposited in public databases (http://www.ncbi.nlm.nih.gov). The random and partial sequencing of orchid cDNA has been a highly rewarding program, particularly because genomic information is still lacking in orchid. The data presented here represents a significant contribution to the publicly accessible expressed sequences for the Orchidaceae family. Application of this knowledge through the common language of nucleotide sequences will provide many candidate genes involved in orchid floral development for further characterization. References 1. Arabidopsis Genome Initiative. (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815. 2. Goff SA, Ricke D, Lan TH, et al. (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296:92–100. Analysis of Expression of Phalaenopsis Floral ESTs ✦ 157 3. Yu J, Hu S, Wang J, et al. 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(1999) Molecular characterization of the Arabidopsis SBP-box genes. Gene 237:91–104. Lannenpaa M, Janonen I, Holtta-Vuori M, et al. (2004) A new SBP-box gene BpSPL1 in silver birch (Betula pendula). Physiol Plant 120:491–500. Unte US, Sorensen AM, Pesaresi P, et al. (2003) SPL8, an SBP-box gene that affects pollen sac development in Arabidopsis. Plant Cell 15:1009–1019. This page intentionally left blank Chapter 10 Orchid MADS-Box Genes Controlling Floral Morphogenesis Wen-Chieh Tsai†, Chin-Wei Lin‡, Chang-Sheng Kuoh‡ and Hong-Hwa Chen*,‡ Orchids are known for both their floral diversity and ecological strategies. The versatility and specialization in orchid floral morphology, structure, and physiological properties have fascinated botanists for centuries. In floral studies, MADS-box genes contributing to the now-famous “ABCDE model” of floral organ identity control have dominated conceptual thinking. The sophisticated orchid floral organization offers an opportunity to discover new variant genes and levels of complexity different from the ABCDE model. Recently, several remarkable researches involving orchid MADS-box genes have revealed the important roles of these genes in orchid floral development. Knowledge about MADSbox genes encoding ABCDE functions in orchids will provide insights into the highly evolved floral morphogenetic networks of orchids. 10.1 Introduction With more than 270,000 known species, the angiosperms are by far the most diverse and widespread group of plants. The ancestry of angiosperms is still uncertain. The fossil records showed that they appeared at the *Corresponding author. † Department of Biological Science and Technology, Chung Hwa University of Medical Technology, Tainan County, Taiwan. ‡ Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan. 163 164 ✦ W.-C. Tsai et al. Fig. 10.1. Phylogeny of angiosperms. Eudicots Monocots Magnollids Star anise and relatives Water lilies Amborella early Cretaceous period, about 130 million years ago. By the end of the Cretaceous period, 65 million years ago, the angiosperms had radiated and become the dominant plants on earth, as they are today. The origin and diversification of angiosperms, what Charles Darwin characterized as “an abominable mystery,” has been the subject of much speculation over the last 100 years.1–3 The rapid explosion in diversity that followed their origin in the early Cretaceous period may be linked to modularity within a new structure, the flower.4 The flower is the defining reproductive adaptation of angiosperms and is the predominant source of characters for angiosperm taxonomy and phylogeny reconstruction.5 Over the past decades, the codification of rigorous methods of phylogenetic analysis, the emergence of molecular techniques, and a renewed interest in developmental pathways during the growth of plant organs have improved the understanding of the relations among angiosperms.6–9 The angiosperms consist of some small relic basal clades (basal angiosperms), magnollids, and two main clades, monocots and eudicots (Fig. 10.1). The basal angiosperms and magnollids share some primitive traits, such as a typical spiral rather than whorled Orchid MADS-Box Genes Controlling Floral Morphogenesis ✦ 165 arrangement of flower organs.10,11 The monocots show extreme variation in floral form, including bilaterally symmetric (zygomorphic) flowers with elaborately modified perianth parts. The organization of flower parts is slightly less variable in the core eudicots. The MADS-box-containing transcriptional regulators have been the focus of floral organ specification, development, and evolutionary studies in plants.12–14 In the well-known “ABC model,” the organ identity in each whorl is determined by a unique combination of three activities by floral organ identity genes, A, B, and C.12 In any one of the four flower whorls, the expression of A alone specifies sepal formation. The combination of AB determines the development of petals, and the combination of BC offers stamen formation. The expression of the C function alone determines the development of carpels. The functions of A and C are mutually repressive.15 The ABC genes were cloned from a wide range of species, and the model has been used to explain floral organ development in plants.15–21 From studies of Petunia, the ABC model was later extended to include D-class genes, which specify ovules.22 An important recent discovery was that another set of MADS-box genes, SEPALLATA1, 2, and 3, function redundantly to specify petals, stamens, and carpels as well as floral determinacy.23 Recently, SEP4 has been defined. sep1 sep2 sep3 sep4 quadruple mutants develop vegetative leaves rather than sepals, petals, stamens, or carpels.24 SEPALLATA function or E function has led to a revision of the ABC model into the “ABCDE model.”25,26 The diversification of MADS-box genes during evolution has been proposed to be a major driving force for floral diversity in land plant architecture.15,27 Containing more than 20,000 species, the Orchidaceae, of class Liliopsida and order Asparagales, is one of the largest angiosperm families. Associated with this enormous size is extraordinary floral diversity. Orchids are extremely rich in species, and speciation rates are presumed to be exceptionally high.28 Although this spectacular diversification is often thought to be linked to the intimate and sometimes bizarre interaction of many species with their pollinators,29 we still face the challenge of explaining how these mechanisms work and why they have evolved. According to the classic view, the orchid flower is composed of five whorls of three segments, each including two perianth whorls, two staminal whorls, and one carpel whorl (Fig. 10.2A).30 This formation conforms to the general flower structure of many other monocotyledonous 166 ✦ W.-C. Tsai et al. (A) (B) Fig. 10.2. Flowers from wild type (A) and peloric mutant (B) of Phalaenopsis equestris. Bar = 1 cm. families. Orchidaceae represents an unusually coherent group among monocots, possessing several reliable floral morphological synapomorphies, including the presence of a gynostemium, or column, fused by the style and at least part of the androecium, a highly evolved petal called the labellum, and resupination caused by 180o torsion of the pedicel.31 Within the monocots, only well-known crop species, such as rice and maize have been studied thoroughly, but the highly reduced flowers make these plants unsuitable for general floral development studies. All expected whorls in the flowers are present in orchids, and the highly sophisticated flower organization offers an opportunity to discover new variant genes and different levels of complexity within morphogenetic networks. Therefore, the Orchidaceae can be used to validate the ABC model in the monocots and study how MADS-box genes are involved in defining the different, highly specialized structures in orchid flowers. 10.2 Phalaenopsis Orchid Floral Ontogeny Floral ontogenetic studies provide an essential tool for research into systematics and phylogenetics as well as developmental regulation. Kurzweil and Kocyan32 reviewed research on species that have been studied for orchid floral ontogeny and included data on the total number of genera and species in Dressler’s subfamilies of Orchidaceae and the number of species whose floral ontogeny was studied, for a total of 128 genera and 211 species in five subfamilies examined. The authors Orchid MADS-Box Genes Controlling Floral Morphogenesis ✦ 167 concluded that the sequence of floral organ initiation on the floral apex and the timing are identical in all monandrous species studied, including Phalaenopsis.32 Our recent studies of floral ontogeny in Phalaenopsis equestris, a native species in Taiwan, could strengthen the evidence for monandrous orchid floral ontogeny (Fig. 10.3). The floral primordium of this species emerges on the flanks of the inflorescence meristem and then enlarges (Fig. 10.3A and 3B). The first organs initiated are the lateral sepals located at the adaxial side (Fig. 10.3C). The primordium of the lip soon follows (Fig. 10.3D). Then the lateral petal primodia emerge (Fig. 10.3E). The last observed tepal is the dorsal sepal located at the abaxial side (Fig. 10.3F). Subsequently, the first organ of the gynostemium A B C FP FP D LS IM IM E F LS L G DS LS DS LP LP LS LS AC L L I DS LP LP LS L H FS LP LS LP PO PO R LS Fig. 10.3. Transverse sections of developing P. equestris flowers showing floral ontogeny. (A) and (B) Initiation of floral primodium. (C)–(F) Differentiation of floral perianth. (G) Initiation of gynostemium. (H) and (I) Late developmental stage of gynostemium. FP: floral primodium, IM: inflorescence meristem, LS: lateral sepal, L: lip, LP: lateral petal, DS: dorsal sepal, FS: fertile stamen, AC: anther cap, PO: pollinium, R: rostellum. 168 ✦ W.-C. Tsai et al. to be initiated is the fertile stamen (Fig. 10.3G). Then, the carpel apices become visible in the central part of the receptacle. The anther cap and the rostellum derived from the median stigma lobe develop later (Fig. 10.3H and 3I). This process of floral development in P. equestris is nearly parallel to that of the other monandrous orchids. The detailed knowledge gained from orchid floral ontogeny will provide a solid foundation for the study of morphogenetic functions of orchid floral organ identity genes. 10.3 A-Class Genes in Orchids To date, almost all cloned orchid MADS-box genes involved in floral development were from Epidendroideae, the largest orchid subfamily with many more genera and species than all of the other four subfamilies together (Table 10.1). So far, four A-class genes were identified from Dendrobium. One of them, DOMADS2, was isolated from Dendrobium grex Madame Thong-In.33 The other three genes were cloned from Dendrobium thyrsiflorum and named DthyrFL1, DthyrFL2, and DthyrFL3.34 The APETALA1/FRUITFULL (AP1/FUL) MADS-box genes lineage within the eudicots was recognized as two clades (the euAP1 and euFUL clades), while the non-core eudicots and monocots have sequences similar only to euFUL genes.35 Sequence analysis showed that these genes contain the C-terminal FUL-like motif LPPWML of monocot FUL-like proteins, but this motif is not present in the sequence of DthyrFL3.34 Phylogenetic analysis showed that DthyrFL1 and DOMADS2 are orthologous genes (Fig. 10.4A). The existence of DthyrFL2 and DthyrFL3 represents a recent duplication event in D. thyrsiflorum (Fig. 10.4A).34 DOMADS2 is expressed in both the shoot apical meristem and the emerging floral primordium throughout the process of floral transition and later in the column of mature flowers.33 The expression pattern of DOMADS2, from the shoot apical meristem and increasing in later stages of floral development, suggests that DOMADS2 is one of the earliest regulatory genes during the transition of flowering. DthyrFL genes are expressed during inflorescence development and also in developing ovules.34 These A-class genes in orchid may be involved in floral meristem identity and column and ovule development. Unlike its homolog AP1 in Arabidopsis, DOMADS2 may not be associated with the development of the first Table 10.1. Floral MADS-box Genes and their Expression in Orchidaceae Expression Location Gene A DOMADS2 Dendrobium grex Madame Thong-In DthyrFL1 Dendrobium thyrsiflorum DthyrFL2 Dendrobium thyrsiflorum DthyrFL3 Dendrobium thyrsiflorum B (paleoAP3) OMADS3 PeMADS2 PeMADS3 PeMADS4 PeMADS5 DcOAP3A DcOAP3B Species Oncidium Gower Ramsey Phalaenopsis equestris Phalaenopsis equestris Phalaenopsis equestris Phalaenopsis equestris Dendrobium crumenatum Dendrobium crumenatum Sepal Petal Labellum Column Pedicel/ Ovary Root Leaf Shoot Ovule Analysis −a − − +b − − − + NDc Id, IIe ND ND ND ND ND − − ND + IIIf ND ND ND ND ND − − ND + III ND ND ND ND ND − − ND + III + + + + ND ND + ND ND IVg + + − − − − − − ND I − + + − − − − − ND I − − + + − − − − ND I + + + − − − − − ND I + + + + + ND + ND ND II, IV − + + + + ND − ND ND IV (Continued ) Orchid MADS-Box Genes Controlling Floral Morphogenesis ✦ 169 Class (Continued ) Expression Location Class Gene Species B PeMADS6 Phalaenopsis equestris Dendrobium crumenatum Orchis italica (PI) DcOPI ORCPI C PeMADS1 PhalAG1 DthyrAG1 DcOAG1 D PhalAG2 DthyrAG2 DcOAG2 Phalaenopsis equestris Phalaenopsis Hatsuyuki Dendrobium thyrsiflorum Dendrobium crumenatum Phalaenopsis Hatsuyuki Dendrobium thyrsiflorum Dendrobium crumenatum Sepal Petal Labellum Column Pedicel/ Ovary Root Leaf Shoot Ovule Analysis + + + + + − − − ND I, II + + + + + ND − ND ND II, IV ND ND ND ND ND ND ND ND ND − − − + + − − − ND I, IV − − − + + ND ND ND + II, IV − − − + ND ND ND ND + II, III + + + + + ND − ND ND II, IV − − − + + ND ND ND + II, IV − − − + ND ND ND ND + II, III − − − + + ND − ND ND II, IV (Continued ) 170 ✦ W.-C. Tsai et al. Table 10.1. Table 10.1. (Continued ) Class Gene E OM1 Aranda Deborah DOMADS1 Dendrobium grex Madame Thong-In DOMADS3 Dendrobium grex Madame Thong-In DcOSEP1 Dendrobium crumenatum a Species Transcripts of gene not detected. Transcripts of gene detected. c Detection of transcripts has not been performed. d Transcripts detected by northern blotting. e Transcripts detected by in situ hybridization. f Transcripts are detected by real-time RT-PCR. g Transcripts detected by RT-PCR. b Sepal Petal Labellum Column Pedicel/ Ovary Root Leaf Shoot Ovule Analysis + + + + + + − + ND + ND − ND − ND − ND ND I I, II − − − − + − − − − I, II + + + + + ND − ND ND IV Orchid MADS-Box Genes Controlling Floral Morphogenesis ✦ 171 Expression Location 172 ✦ W.-C. Tsai et al. E class PI B class AP3 A class (A) Fig. 10.4. Phylogenetic relationship of MADS-box genes of ABCDE class. (A) Phylogenetic analysis of A-, B- and E-class genes. (B) Phylogenetic analysis of C- and D-class genes. Orchid MADS-box genes are highlighted by open boxes. Orchid MADS-Box Genes Controlling Floral Morphogenesis ✦ 173 C class D class (B) Fig. 10.4. (Continued) 174 ✦ W.-C. Tsai et al. two whorls. However, whether the DthyrFL genes associate with perianth formation is not clear. In addition, a MADS-box gene OMADS1 in Oncidium is also involved in floral initiation and formation and belongs to the AGL6 subfamily rather than the A-class genes.36 Transgenic Arabidopsis and tobacco overexpressing OMADS1 showed significantly reduced plant size, flowering extremely early, and losing inflorescence indeterminacy.36 10.4 B-Class Genes in Orchids Both the developmental and the biochemical aspects of B-class genes required to specify the identity of petals in whorl 2 and stamens in whorl 3 appear to be conserved in many core eudicots.15,16 The B-class genes in the monocots rice and maize are similar in function to the core eudicots B-class genes.19,20,37,38 The orchid flowers have a petaloid perianth arrangement that could be explained by a “modified ABC model” in that the expression of the B-class genes has expanded to whorl 1.39 In addition, the orchid flowers display an elaborated labellum that is a highly modified petal. Because the function of A-class genes is poorly defined in angiosperms, studies of petal development and evolution have generally focused on B-class genes.40 The extraordinary floral diversity in orchids may be associated with the evolution of B-class genes. The molecular functions of B-class genes have been studied in more detail than that of any other floral homeotic genes in orchids. So far, various numbers of APETALA3 (AP3)-like and PISTILLATA (PI)-like genes have been isolated from several orchids. These include 1 AP3-like OMADS3 from Oncidium Gower Ramsey; 4 AP3- and 1 PI-like genes from P. equestris; and 2 AP3- and 1 PI-like genes from Dendrobium crumenatum.41–43 All these AP3-like genes are members of the paleoAP3 lineage (Fig. 10.4A). The paleoAP3 genes identified from orchids were subdivided into two subclades. One subclade contains OMADS3, PeMADS5, DcOAP3A, and PeMADS2 and the other OcOAP3B, PeMADS3, and PeMADS4 (Fig. 10.4A). This division suggests that the ancestor of Orchidaceae might have two paleoAP3-like genes, and further gene duplication has at least taken place in the AP3 clade in the monocots. Interestingly, both OMADS3 and PeMADS5 do not show a paleoAP3 motif, which suggests Orchid MADS-Box Genes Controlling Floral Morphogenesis ✦ 175 that they are orthologous genes. Although they share similar expression patterns in orchid floral organs, PeMADS5 is not expressed in vegetative tissues, but OMADS3 can be detected in leaves.41,42 Phylogenetic analysis also showed that DcOAP3A and PeMADS2 are orthologous genes (Fig. 10.4A). However, they possess different expression patterns. Similar to OMADS3, DcOAP3A is ubiquitously expressed in all floral organs and leaves, while PeMADS2 is predominantly expressed in sepals and petals.41–43 Recently, we discovered at least three paleoAP3 genes displaying distinct expression patterns in Oncidium floral organs (our unpublished data). In addition, we also noticed that the expression profile of OMADS3 examined by Hsu and Yang41 was indeed composed of the expression patterns from 2 paleoAP3 genes of Oncidium (our unpublished data). A specialized paleoAP3 gene, PeMADS4, discovered in P. equestris, is specifically expressed in the labellum and column, which suggests that its function is associated with the development of these organs. Gene duplication is important for generating new genes during evolution44 and, therefore, may lead to the generation of new organs. In orchids, duplication of paleoAP3-like genes, followed by diversification and specialization of PeMADS4-like genes probably is concomitant with the appearance of a new floral organ, the labellum. Overexpression of paleoAP3 genes from Oncidium, Dendrobium, and Phalaenopsis under the control of the cauliflower mosaic virus 35S promoter has been examined in Arabidopsis41,43 (our unpublished data). Consistently, these results showed that the flower morphology of transgenic Arabidopsis plants overexpressing the orchid paleoAP3 genes is indistinguishable from that of wild-type plants. Dominantnegative mutation strategy was further used to investigate the functions of OMADS3 and DcOAP3A.43 By doing this, the OMADS3 was shown to have a function similar to that of the A functional gene in regulating flower formation as well as floral initiation, while DcOAP3A had a putative B function.41,43 However, these results could not reflect the real roles the genes may play during orchid floral development. Peloric flowers that are actinomorphic mutants with lip-like petals are widely found in natural populations of species from Veronicaceae, Gesneriaceae, Labiatae, and Orchidaceae.45 With the high frequency of orchid peloric mutants derived from micropropagation (Fig. 10.2B), we were able to infer the individual roles the diversified paleoAP3 genes play in orchids by comparing the expression patterns of the 4 paleoAP3 176 ✦ W.-C. Tsai et al. genes (PeMADS2, PeMADS3, PeMADS4 and PeMADS5) in wild-type Phalaenopsis floral organs and peloric mutants.42 First, both PeMADS2 and PeMADS5 were expressed in sepals of wild-type plants, but only PeMADS2 transcript was detected in the sepals of peloric mutants whose morphology is not affected. This result suggests that PeMADS5 is dispensable, while PeMADS2 is crucial for sepal development. Second, the expression of all four PeMADS genes, except PeMADS4, was detected in wild-type petals. However, the expression of PeMADS5 was not noted in the lip-like petals of peloric mutant. This result suggests that PeMADS5 is associated with petal development. Third, the expression of PeMADS4 was concentrated in the lips and columns in wild-type plants and is extended to the lip-like petals in the peloric mutant. Because PeMADS4 was detected as well in the lip-like petal of the peloric mutant, it might be required for labellum identity. Fourth, PeMADS3 showed similar expression patterns in the wild-type plant and the peloric mutant, which implies its important function in inner perianth whorl morphogenesis. So far, only one PI-like gene has been found in D. crumenatum and P. equestris: DcOPI and PeMADS6, respectively.43,46 Southern blot hybridization results supported that the Phalaenopsis orchid genome contains only one copy of the PI-like gene.46 Both genes are expressed in all the floral organs, except that PeMADS6 is not detected in the pollinia of P. equestris.43,46 In addition, PeMADS6 is expressed in the undeveloped ovary.46 Tsai et al.46 suggested that the expression of PeMADS6 in the ovary has an inhibitory effect on the development of the ovary, and auxin acts as the candidate signal to regulate the repression of PeMADS6 expression in the ovary. Furthermore, PeMADS6 is not differentially expressed in wild-type and peloric floral organs, which suggests that PeMADS6 is not responsible for the altered phenotype of the peloric mutant. Overexpression of DcOPI or PeMADS6 in Arabidopsis demonstrated that both genes share the angiosperm PI function.43,46 Further evidence came from the complementation of the pi-1 phenotype in Arabidopsis by overexpressing DcOPI and showed that DcOPI is able to substitute PI in Arabidopsis,46 while PeMADS6 could not complement the pi-4 mutant (our unpublished data). In conclusion, the expression patterns of B-class genes in orchid floral organs nicely fits the “modified ABC model” in that their expression has expanded to whorl 1 in plants possessing nearly identical morphology Orchid MADS-Box Genes Controlling Floral Morphogenesis ✦ 177 of sepals and petals.39 paleoAP3 genes are highly duplicated in the Epidendroideae genome. Diversification and fixation of both these gene sequences and expression profiles might cause subfunctionalization and even neofunctionalization. The driving force of the specialized labellum and diversified orchid flowers may be linked to the fast evolution rate of paleoAP3 genes. Studies of the B-class genes from other orchid subfamilies, such as Apostasioideae, Cypripedioideae, Spiranthoideae, Orchidoideae, and even more members of Epidendroideae,47 will provide profound knowledge to resolve the mystery of orchid floral development and diversification. 10.5 C- and D-Class Genes in Orchids A gynostemium or column, comprising stamen filaments adnate to a syncarpous style, is normally regarded as a structure peculiar to orchids.47 The development of the column, which involves whorls 3 and 4, would be one of the most interesting subjects for elucidating the evolution of C-class genes. In most orchid flowers, ovary and ovule development is precisely and completely triggered by pollination and, therefore, orchids offer a unique opportunity to study D-class genes involved in ovule development. Recently, one C- and one D-class gene were isolated independently from three orchid species: Phalaenopsis Hatsuyuki (PhalAG1, PhalAG2),48 D. thyrsiflorum (DthyrAG1, DthyrAG2),49 and D. crumenatum (DcOAG1, DcOAG2).43 PhalAG1, DthyrAG1, DcOAG1 were classified in the C lineage of AG-like genes, with PhalAG2, DthyrAG2, DcOAG2 classified in the D lineage (Fig. 10.4B). PhalAG1, PhalAG2, DthyrAG1, and DthyrAG2 share similar spatial expression patterns in the column, ovary and developing ovules, despite the fact that these four genes belong to different lineages.48,49 One possible explanation is that C- and D-class genes in orchid would act redundantly with each other in floral and ovule development. Although PhalAG1 is expressed in all the floral organs at their initiation, its expression quickly decreases and is detected only in the column and ovary of mature flowers, which is also true for DthyrAG1.48,49 We also identified a C-class gene, PeMADS1, from P. equestris, whose expression patterns were consistent with those of PhalAG1 and DthyrAG1 (our unpublished data). However, DcOAG1 is expressed in all mature floral organs, and DcOAG2 is expressed in only the anther cap and column of D. crumenatum.43 The unusual expression 178 ✦ W.-C. Tsai et al. patterns of DcOAG1 in monocots evoke that its regulatory mechanism is independently evolved in D. crumenatum as in some basal angiosperms, such as Illicium and Persea,50 but the function of DcOAG1 and Arabidopsis AG is conserved, as seen by the phenotypic similarity between transgenic Arabidopsis expressing either 35S::DcOAG1 or 35S::AG.43 The molecular mechanism of morphogenesis of orchid gynostemium is still enigmatic, but mutation of C-class genes in orchids could possibly provide the opportunity to shed light on the mystery. 10.6 E-Class Genes in Orchids E-class genes are required for floral organ identity in all four orchid floral organs as well as for floral determinacy.24,51 They have been shown to form ternary complexes with A- and B-class proteins, by a yeast three-hybrid system, and can mediate the interactions between B- and C-class proteins in higher-order complexes.52 The first E-class gene found, OM1, was isolated from the supposed bigeneric hybrid Aranda Deborah.53 The other three E-class genes, DOMADS1, DOMADS3, DcOSEP1, were identified from Dendrobium. Two were cloned from D. grex Madame Thong-In and the third from D. crumenatum.33,43 Phylogenetic analysis showed that OM1 was clustered with DOMADS1 and DcOSEP1, with DOMADS3 separated at a distance from the other three E-class genes (Fig. 10.4A). DOMADS1 RNA is uniformly expressed in both the inflorescence meristem and floral primordium and later exists in all of the floral organs.33 The expression pattern of DOMADS1 in mature flowers coincides with that of its counterpart DcOSEP1 in D. crumenatum and their orthologs in Arabidopsis.23,33,43 However, OM1 is expressed in mature flowers and not in young developing inflorescence or young floral buds. In mature flowers, it is expressed only in petals and weakly in sepals, but not in the column.53 Spatiotemporal expression differences imply that functional diversification among these genes closely relates to phylogeny. The onset of DOMADS3 transcription occurs in the early shoot apical meristem at the stage before the differentiation of the first flower primordium and later can only be detected in the pedicels.33 DOMADS3 may function as a regulatory factor not only in early floral transition, but also in the development of the pedicel. The fact that the expression of E-class genes overlaps with that of ABC genes in orchids suggests that the higher-order MADS complexes are involved in orchid floral development. Recently, one line of evidence Orchid MADS-Box Genes Controlling Floral Morphogenesis ✦ 179 that MADS proteins form higher-order complexes comes from the formation of DcOAP3A-DcOPI-DcOSEP1 and DcOAP3B-DcOPI-DcOSEP1 detected by yeast three-hybrid experiments.43 10.7 Perspective Owing to the large genome size, the long life cycle and the inefficient transformation system of orchids, few studies of orchid biology exist. Recently, the genetic architecture of the sophisticated floral organization of orchids has begun to be investigated. Given that orchids represent one of the most successful and diverse plant families worldwide, the development of genomic resources is imperative.54 Thanks to advanced progress in genomics and bioinformatics, plentiful gene information and integrated bioinformatics tools are ready to be used for studying orchid biology.55–57 The effort of many scientists will promise to lead to a better understanding of the molecular and genetic mechanisms of orchid floral control in the years to come. Acknowledgments We thank Wen-Huei Chen (Department of Life Sciences, National Science Council, National University of Kaohsiung, Kaohsiung, Taiwan) for leading us into the field of orchids; Chang-Sheng Kuoh (Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan) for sharing his knowledge of orchid floral ontogeny; and Tung-Hua David Ho (Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan) and Michel Delseny (Laboratory of Plant Genome and Development, University of Perpignan, France) for helpful discussions. Grant funding from both the National Science Council and Council of Agriculture in Taiwan supports the current research in our labs. References 1. Cronquist A. (1988) The Evolution and Classification of Flowering Plants, 2nd ed. New York Botanical Garden, Bronx, NY. 2. Darwin C. (1903) The Different Forms of Flowers on Plants of the Same Species. D. Appleton, New York. 180 ✦ W.-C. Tsai et al. 3. Doyle JA. 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(2006) Expression analysis of the ESTs derived from the flower buds of Phalaenopsis equestris. Plant Sci 170:426–432. This page intentionally left blank Chapter 11 Pseudobulb-Specific Gene Expression of Oncidium Orchid at the Stage of Inflorescence Initiation Jun Tan†, Heng-Long Wang‡ and Kai-Wun Yeh*,§ Oncidium pseudobulb is a critical organ in the developmental stage. It is a sink for nutrition, water, and mineral storage organ during the vegetative growth stage. The growth and development determine Oncidium plant growth cycle transforming from the vegetative into the reproductive stage. Therefore, the genes activating in the pseudobulb of beforeinflorescence stage attract much research interest. In this chapter, we present a subtractive EST data bank of EST genes, which are actively expressing in the pseudobulb tissues before inflorescence initiation. In total, 74.8% of 636 unique gene parts were annotated on the database of the NCBI GenBank. Largely, all EST was classified into carbohydrate metabolism involved in mannan, pectin, and starch bosynthesis, transportation, stress-related, and regulatory function. Most of the genes that were differentially expressed are involved in early flowering development, carbohydrate metabolism, and stress-response physiology. The efficient pseudobulb-specific EST-library represented an explicit transcriptome profile before the flowering stage. It is a valuable data bank for molecular biology study in orchid. *Corresponding author. † College of Bioinformation, Chongqing University of Post and Telecom, Chongqing, China. Department of Life Science, National Kaohsiung University, Kaohsiung, Taiwan. § Institute of Plant Biology, College of Life Science, National Taiwan University, Taipei, Taiwan. ‡ 185 186 ✦ J. Tan, H.-L. Wang and K.-W. Yeh 11.1 Introduction Orchids (Orchidaceae, L.) are the largest family of plants and are predominantly found in the tropics and subtropics, especially in the mountains of the tropical Americas and Southeast Asia. The number of orchid species may exceed 30,000 and constitute almost 30% of monocots or 10% of flowering plants (Royal Botanic Gardens, Kew, 2003). The genus Oncidium has become more and more commercially important in the market for cut flowers and houseplants.1 Consequently, efforts to improve the economic traits of this ornamental plant are immense at present. After the method of efficient plant regeneration through somatic embryogenesis from Oncidium orchid callus,2 a routine procedure of transformation with Agrobacterium tumefaciens was also established.1 Sweet pepper ferredoxin-like protein ( pflp) was used as a novel selection marker for orchid transformation.3 At the same time the selection marker was demonstrated to confer resistance against soft rot disease in Oncidium orchid.4 Other results showed that the function of its AP3-like MADS gene in regulating floral formation and initiation.5 Recently, a sucrose-phosphate synthase gene highly expressed in Oncidium flowers and leaves was cloned.6 At the base of the second upper leaf, Oncidium orchid has an enlarged bulb-like stem, termed a pseudobulb, which is important for the storage and maintenance of moisture, mineral nutrition, and carbohydrates during both auxiliary bud and flower development.7 It is clear that current and former pseudobulbs are connected,8 and the carbohydrate pool in the current pseudobulb varied strikingly during inflorescence development.9 From initiation to the end of inflorescence development, galactonic acid 1,4-lactone, mannan, and hexoses including glucose, fructose, and galactose gradually decreased in the current pseudobulb, but sucrose and mannose contents almost always remained low. During the period before flowering, there was dramatic accumulation followed by degradation of starch.10 Here, subtractive ESTs were generated by subtracting Rsa I-digested cDNAs of the upper leaf tissue from those of the pseudobulb. The highly expressed and tissue-specific genes, such as peroxidase, sodium/ dicarboxylate cotransporter, mannose binding lectin, senescence, or resistance associated proteins and other genes were thought of as having specific functions in the pseudobulb. Redundancies of these ESTs also revealed some interesting information on their expressed gene family members. The sequence dataset was not on a large scale, but did Pseudobulb-Specific Gene Expression of Oncidium Orchid ✦ 187 cover most of the genes involved in the metabolisms of Oncidium pseudobulb, mainly carbohydrates, transportation, stress-related, cell cycle, and regulatory function. All of them were related to the water, nutrition, and energy support of the pseudobulb during inflorescence development. Our research on pseudobulb- specific gene expression is beneficial for the further functional determination of these genes and to identify the physiological linkage with floral time and qualities. 11.2 Overall Distribution of Subtractive ESTs from Pseudobulb of Oncidium Gower Ramsy In total, 1248 clones were sequenced. 1088 clones were readable and had an average length of 1031 bp. The distribution of their length is shown in Fig. 11.1. After the pGEM-T easy vector sequence was removed by cross match from them, 1080 inserts with a length more than 100 bp were accepted as subtractive ESTs for further study. Their length was 430 bp on average and in the range of 100 to 1200 bp, mainly from 200 to 800 bp (Fig. 11.1A). After phrap, 1080 subtractive ESTs were assembled into 149 contigs and 543 singletons. Furthermore, they were aligned into 51 clusters and 585 singles by blastn. There were 69 contigs in the 585 singles, so 516 real singles had only one subtractive EST, 48.3% of the total ESTs. The number of ESTs in clusters, also called cluster size, reflected the abundance of mRNAs. Including singles with one EST, the distribution of cluster size was indicated in Fig. 11.1B, and clusters were divided Fig. 11.1. Alignment and annotation of subtractive ESTs. (A) Length distribution of readable sequences and subtractive ESTs. (B) Prevalence distribution of subtractive EST cluster size. (From Tan et al., Biotech Letts (2005) 27: 1517–1528. The authors wish to thank Biotechnology Letters for providing the figures and tables cited in the text.) 188 ✦ J. Tan, H.-L. Wang and K.-W. Yeh into six classes. There were 95 clusters containing 2–4 ESTs, constituting 22.3% of the total accepted ESTs (241 of 1080 subtractive ESTs) and 79.1% of the total clusters (95 of 120 clusters). There were 12 clusters containing 5–7 ESTs, constituting 6.5% of the total accepted ESTs (70 of 1080 subtractive ESTs) and 10.0% of the total clusters (12 of 120 clusters). These clusters represented lipid transfer protein, DP-E2F-related protein, senescence-associated protein, mannosebinding lectin, sucrose synthase, peroxidase, GDSL-motif lipase/ hydrolase, pollen allergen, and two unknown protein genes. One cluster had no significant similarity to any protein sequence in the GenBank nr database. There were five clusters containing 8–10 ESTs, constituting 4.1% of the total accepted ESTs (44 of 1080 subtractive ESTs) and 4.1% of the total clusters (5 of 120 clusters). These clusters represented mannosebinding lectin, BURP domain protein, peroxidase, chitinase, and 19 genes. One cluster had no significant similarity to any protein sequence in the GenBank nr database. There were another five clusters containing 11–13 ESTs constituting 5.5% of the total accepted ESTs, 59 of 1080 subtractive ESTs. These clusters represent short-chain dehydrogenase/ reductase and two unkonwn genes (Arabidopsis T23E23.17, T18N14.110). Two clusters had no significant similarity to any protein sequence in the GenBank nr database. Three clusters were regarded as the most abundant transcripts. They constituted only 2.5% of the total clusters (3 of 120 clusters), but 12.7% of the total subtractive ESTs (137 of 1080 subtractive ESTs). Two of them were peroxidase genes, and one was a sodium/dicarboxylate cotransporter gene. More details and the possible functions of these genes are shown and discussed in Table 11.1. Comparatively, mannose binding lectin genes were expressed predominately in normal pseudobulb (data not shown). 11.3 Annotation and Classification of the Subtractive ESTs A total of 636 clusters and singles including 1080 subtractive ESTs were submitted to blastX for homologous searching with the nr database in GenBank. 439 clusters and singles composed of 808 ESTs (484 contigs and singletons) were identified. A total of 309 of them comprising 614 ESTs Pseudobulb-Specific Gene Expression of Oncidium Orchid ✦ 189 Table 11.1. Assembled Clusters that Contain more than 4 ESTs Gene Annotation peroxidase (EC 1.11.1.7) sodium/dicarboxylate cotransporter peroxidase (EC 1.11.1.7) 2, cationic T23E23.17 similar to Arabidopsis thaliana T18N14.110 short-chain dehydrogenase/ reductase No hits found mannose-binding lectin BURP domain protein No hits found peroxidase (EC 1.11.1.7) glycosyl hydrolase family 19 (chitinase) lipid transfer protein isoform 4 unknown DP-E2F-related protein 1 unknown protein senescence-associated protein mannose-binding lectin Reference Organism GI Number E Value ESTs Gossypium irsutum Arabidopsis thaliana Glycine max 7433087 2.10E-47 92 15238130 5.10E-43 24 7433098 6.10E-26 21 9369404 7.00E-11 12 13486662 7.10E-29 12 15224306 1.10E-76 11 Cymbidium hybrid Vigna unguiculata 2144226 2.10E-51 11 10 7106540 6.00E-12 9 Gossypium irsutum Arabidopsis thaliana Vitis vinifera 7433087 2.00E-17 9 8 15228911 1.10E-37 8 28194086 3.00E-16 7 21553375 1.10E-22 7 22331664 1.10E-48 7 28393189 7.10E-70 6 13359451 3.10E-42 6 2144226 2.10E-21 6 Arabidopsis thaliana Oryza sativa Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Pisum sativum Cymbidium hybrid (Continued) 190 ✦ J. Tan, H.-L. Wang and K.-W. Yeh Table 11.1. Gene Annotation sucrose synthase peroxidase (EC 1.11.1.7) 2, cationic GDSL-motif lipase/ hydrolase protein pollen allergen-like protein No hits found peroxidase (Continued) Reference Organism GI Number E Value ESTs Oncidium Glycine max 22347630 7433098 1.10E-95 1.10E-90 6 5 Arabidopsis thaliana Arabidopsis thaliana 15228189 3.10E-90 5 21593946 6.10E-20 5 Glycine max 5002234 7.00E-18 5 5 (From Tan et al., Biotech Letts (2005) 27: 1517–1528. The authors wish to thank Biotechnology Letters for providing the figures and tables cited in the text.) were annotated with a gene name and could be analyzed further. From Fig. 11.1A, it is obvious the length of subtractive ESTs had a linear relationship with the number of ESTs that could be annotated. The longer the sequence, the greater the chance of being annotated and vice versa. Using the GO classification system, only 155 ESTs (25.2% of 614 annotated ESTs) were matched, protein sequences with special GI numbers which had been previously classified on three different GO trees (cell component, molecular function, and cellular process) (Fig. 11.2). This represented three different points of view of classified genes. There were 104 classified ESTs belonging to cellular components, 29 ESTs belonging to extracellular components, and 19 ESTs to unlocalized components. According to the molecular function tree, the majority (85 ESTs) were classified as enzymes, 44 ESTs as binding proteins, and 20 as transporters. A total of 130 ESTs were involved in physiological processes, and 42 were involved in cellular processes. 11.4 Northern Blot Check of the Subtractive Efficiency of the Subtractive ESTs Dataset From the ESTs clones, 16 sequences, including eight abundant transcripts, were selected as probes for Northern blot (Fig. 11.3). Sodium/dicarboxylate Pseudobulb-Specific Gene Expression of Oncidium Orchid ✦ 191 Fig. 11.2. Classification of subtractive ESTs with 3 GO trees. (From Tan et al., Biotech Letts (2005) 27: 1517–1528. The authors wish to thank Biotechnology Letters for providing the figures and tables cited in the text.) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 sodium/dicarboxylate cotransporter [Arabidopsis thaliana] mannose-binding lectin [Cymbidium hybrid] 1 mannose-binding lectin [Cymbidium hybrid] 2 BURP domain protein [Oryza sativa] peroxidase [Gossypium irsutum] sucrose synthase [Oncidium] GDP-mannose pyrophosphorylase [Oryza sativa] mannose-6-phosephate isomerase [Arabidopsis thaliana] proline-rich-like protease inhibitor [Asparagus officinalis] glycine_rich RNA binding protein [Oryza sativa] AP2 domain transcription factor [Arabidopsis thaliana] leucine_rich receptor-related protein kinase [Arabidopsis thaliana] Na+/H+ antiporter isoform 2 [Lycopersicon esculentum] Invertase [Zea Mays] granule-bound starch synthase [Pisum sativum] pectate lyase [Arabidopsis thaliana] Fig. 11.3. Northern blot check of 10 subtractive ESTs. PB, pseudobulb; L, leaf. 1–8 were subtractive ESTs with redundancy, and 9–16 were without redundancy. (From Tan et al., Biotech Letts (2005) 27: 1517–1528. The authors wish to thank Biotechnology Letters for providing the figures and tables cited in the text.) 192 ✦ J. Tan, H.-L. Wang and K.-W. Yeh cotransporter, BURP domain protein (dehydration-responsive protein RD22), peroxidase, mannose-6-phosephate isomerase, proline-rich-like protease inhibitor, Na+/H+ antiporter isoform 2, invertase, and pectate lyase were highly expressed in the pseudobulbs at the initiation of inflorescence, but they exhibited almost no expression in its upper leaf. The expression patterns of mannose binding lectin, sucrose synthase, GDPmannose pyrophosphorylase, and granule-bound starch synthase were similar. Glycine-rich RNA binding protein and leucine-rich receptorrelated protein kinase genes were highly expressed in the pseudobulb and slightly in leaves at the same time. Only AP2 domain transcription factor gene displayed the opposite quality, the expression level in pseudobulbs was high, but that in leaves seemed higher. Obviously, abundant ESTs had more specific expression profiles. Significantly, the results indicated that subtractive ESTs dataset showed pseudobulb-specific genes expressed at the initiation of the inflorescence. Thus, these Northern data demonstrated that the EST subtraction was very precise and reliable. 11.5 Alignment of Subtractive ESTs with Known Sucrose Synthase and Peroxidase Genes Sucrose synthase was the only known Oncidium gene as a reference sequence for ESTs annotation. Using blastn, three EST contigs and three singletons were aligned with 2520 bp, a complete CDS. They were aligned on four different regions of the gene and resulted in three clusters and one single (Table 11.3). This is because RsaI digested a complete cDNA into several pieces. It also hinted that perhaps two gene family members were expressed at that time. Obviously, this example helped explain why several clusters or singles had the same annotation (Fig. 11.4A). Another explanation may lie in the large gene family. All ESTs annotated by the peroxidase gene were aligned with a reference CDS of Gossypium irsutum. There were nine overlapped sequences, including eight contigs and one singleton, in the middle of the genes (Fig. 11.4B). Their homologous sequences were 205 pb. Based on their single nucleotide polymorphism, a phylogenetic tree was constructed with clustal W (Fig. 11.5). It showed three main groups on the tree. In Table 11.1, five annotations refer to peroxidase genes. Two annotations were sequence Pseudobulb-Specific Gene Expression of Oncidium Orchid ✦ 193 Fig. 11.4. Statement of subtractive ESTs with same annotations. (A) Subtractive ESTs were aligned with a known sucrose gene of Oncidium. (B) Subtractive ESTs were aligned with a known peroxidase gene of Gossypium irsutum. (From Tan et al., Biotech Letts (2005) 27: 1517–1528. The authors wish to thank Biotechnology Letters for providing the figures and tables cited in the text.) Fig. 11.5. Phylogenetic analysis of peroxidase genes expressed in pseudobulb according to sequences of overlapped 120 bp of subtractive EST contigs. (From Tan et al., Biotech Letts (2005) 27: 1517–1528. The authors wish to thank Biotechnology Letters for providing the figures and tables cited in the text.) consensuses belonging to the front and rear part of the gene, respectively. The other three annotations came from sequences aligned in the middle of the gene. That is to say, the peroxidase gene family was expressed in the pseudobulb, and it could be divided into three primary subfamilies. 194 ✦ J. Tan, H.-L. Wang and K.-W. Yeh 11.6 Subtractive ESTs Relevant to Inflorescence, Carbohydrate Metabolisms, Transportation, Stress, Cell Cycle, and Regulation According to the annotations and references about their functions, the subtractive ESTs were gathered and analyzed manually. One cluster and seven singles (10 ESTs in total) were related to specific flower genes (Table 11.2). Abnormal inflorescence meristem 1 (AIM1) could affect inflorescence and floral development in Arabidopsis.11 MADS box protein genes were expressed in different organs and mainly during floral development. DOMADS2 was expressed throughout the process of flower development and responded to osmotic changes and darkness.12 Shaggy-like kinase was flower-specific and responsible for osmotic changes and darkness13 inflorescence development dependent genes. The others were related to four kinds of genes within the synthesis pathway of flower pigment. Chalcone synthase14 and chalcone-flavanone isomerase15 were at the upper stream of the pathway. More chalconeflavanone isomerase gene seemed to be expressed in the pseudobulb at Table 11.2. Selected Examples of ESTs for Genes with Known or Putative Functions Related to Inflorescence Gene Annotation abnormal inflorescence meristem 1 (AIM1) chalcone sythase C2 chalcone-flavanone isomerase cytochrom P450 cytochrome P450 71D2 dihydroflavonol reductase MADS box protein (DOMADS2) shaggy-like kinase Reference Organism GI Number E Value ESTs Arabidopsis thaliana Zea mays Arabidopsis thaliana Arabidopsis thaliana Catharanthus roseus Arabidopsis thaliana Dendrobium grex Ricinus communis 15235527 2.00E-36 1 116380 18414838 6.00E-15 3.00E-29 1 3 21595357 2.00E-34 1 28261339 2.00E-30 1 18390863 1.00E-16 1 6467974 5.00E-24 1 1877397 8.00E-99 1 (From Tan et al., Biotech Letts (2005) 27: 1517–1528. The authors wish to thank Biotechnology Letters for providing the figures and tables cited in the text.) Pseudobulb-Specific Gene Expression of Oncidium Orchid ✦ 195 the initiation of inflorescence. Two ESTs annotated with cytochrome P450 genes could also be annotated as flavanone 3′-hydroxylase or flavanone 3′-5′-hydroxylase. They catalyzed dihydrokaempferol into dihydroquercetin or dihydromyrlcetin.16 Incorporated with UDP-glucose transferase, dihydroflavonol reductase was a downstream gene of the pathway and took part in the synthesis of anthocyanins.16 We found 61 subtractive ESTs annotated with genes involved in the metabolism of saccharides, including mannose, glucose, fructose, galactose, sucrose, starch, pectin, and cellulose (Table 11.3). Based on this information, we could draw a draft of carbohydrate pathways to explain what happened in pseudobulbs at the initiation of inflorescence development (data not shown). A total of 91 ESTs were thought to have probable relationships with transportation (Table 11.4). Most of the proteins the genes encoded were localized in the kinds of membranes or on cell matrixes that help material transportation. Sodium/dicarboxylate cotransporter and GDSL-motif lipase/hydrolase were most abundant. Sodium/dicarboxylate cotransporter was a single copy gene in Arabidopsis and localized on vacuole membrane to transfer malate into vacuole.17 GDSL-motif lipase/hydrolase was a lipolytic enzyme which might be related to a secretion mechanism.18 ADP-ribosylation factor played a critical role in intracellular trafficking and maintenance of endoplasmic reticulum morphology in Arabidopsis.19 Gamma-adaptin is involved in Golgi-endosome traffic, including the recruitment of accessory proteins, gammasynergin, and Rabaptin-5.20 Golgi-localized protein (GRIP) could maintain normal Golgi morphology and function.21 C2 domain-containing protein was found in a large variety of membrane trafficking and signal transduction proteins and most of their biological roles have not been identified.22 Including transporters in membranes, others interacting with cell scaffolds were also expressed, such as dynamin-like protein, F-actin capping protein, kinesin, and villin. We identified 186 ESTs as possible stress-related genes (Table 11.5). Amazingly, 131 ESTs were peroxidase genes. In Arabidopsis, they are a large gene family composed of 78 members with different expression profiles in different organs.23 Based on EST alignment, expressed peroxidase genes in pseudobulbs belong to a large family too. The others were genes induced by different biotic and abiotic stresses, including wounding, drought, and pathogens. Among them, AP2 domain transcription factor could be induced by cold, dehydration, and ABA stress,24 196 ✦ J. Tan, H.-L. Wang and K.-W. Yeh Table 11.3. Selected Examples of ESTs for Genes Related to Carbohydrate Metabolisms Gene Annotation ADP-glucose pyrophosphorylase aldose 1-epimerase alpha 1,4-glucan phosphorylase L isozyme alpha-galactosidase beta-1,3 glucanase beta-fructofuranosidase 1 beta-galactosidase beta-galactosidase beta-mannosidase cinnamyl alcohol dehydrogenase dTDP-glucose 4-6-dehydratase epimerase/dehydratase glucosyltransferase glycogenin glucosyltransferase (EC 2.4.1.186) glycogenin glucosyltransferase (EC 2.4.1.186) glycosyl hydrolase 1 granule-bound starch synthase mannose-6-phosphate isomerase mannose-6-phosphate isomerase Reference Organism GI Number E Value ESTs Arabidopsis thaliana Arabidopsis thaliana Oryza sativa 30699056 4.00E-66 2 15242099 3.00E-65 2 13195430 1.00E-43 1 Arabidopsis thaliana Oryza sativa Zea mays Oryza sativa Oryza sativa Lycopersicon esculentum Populus balsamifera Arabidopsis thaliana Oryza sativa Arabidopsis thaliana Oryza sativa 11264291 3.00E-33 1 20161490 1352468 20514290 18461259 17226270 7.00E-39 2.00E-31 7.00E-63 2.00E-49 1.00E-103 1 1 1 1 1 9998899 3.00E-38 1 21594350 1.00E-16 1 20042976 25408401 1.00E-56 2.00E-26 3 1 5441877 5.00E-84 1 Oryza sativa 5441877 7.00E-93 1 Arabidopsis thaliana Pisum sativum 15220627 6.00E-36 1 15626365 4.00E-86 1 Arabidopsis thaliana Oryza sativa 15232927 8.00E-50 1 11275529 6.00E-45 1 (Continued ) Pseudobulb-Specific Gene Expression of Oncidium Orchid ✦ 197 Table 11.3. Gene Annotation mannosyltransferase NAD-dependent epimerase/dehydratase nucleoside-diphosphatesugar pyrophosphorylase N-acetylglucosaminephosphate mutase pectate lyase pectin esterase pectinesterase 1 phosphoglucomutase, cytoplasmic phosphoglucose isomerase phosphomannomutase polygalacturonase polygalacturonase ripening-related protein starch phosphorylase sucrose synthase sucrose synthase sucrose synthase sucrose synthase triosephosphate isomerase, cytosolic (TIM) (Continued ) Reference Organism GI Number E Value ESTs Arabidopsis thaliana Arabidopsis thaliana Oryza sativa 22326970 4.00E-15 1 15231926 2.00E-63 1 29893646 6.00E-68 4 Arabidopsis thaliana Arabidopsis thaliana Oryza sativa Lycopersicon esculentum Solanum tuberosum Dioscorea septemloba Arabidopsis thaliana Pisum sativum Arabidopsis thaliana Vitis vinifera Ipomoea batatas Oncidium Oncidium Oncidium Oncidium Petunia x hybrida 30686654 8.00E-38 1 10177179 4.00E-23 1 20161185 6174913 1.00E-12 1.00E-13 2 4 12585316 2.00E-84 1 2351056 2.00E-80 1 15225896 2.00E-16 2 13958032 18412253 3.00E-57 2.00E-22 3 1 7406669 12658431 22347630 22347630 22347630 22347630 1351279 4.00E-79 2.00E-31 1.00E-96 2.00E-27 1.00E-38 4.00E-30 4.00E-27 3 1 6 2 2 1 1 (From Tan et al., Biotech Letts (2005) 27: 1517–1528. The authors wish to thank Biotechnology Letters for providing the figures and tables cited in the text.) and was involved in the regulation of low-temperature responsive genes in barley.25 A total of 23 ESTs were found to have functions in the cell cycle (Table 11.6). That is to say, the cells in pseudobulbs kept growing actively and differentiating at this stage. DP-E2F-related protein 1 and 198 ✦ J. Tan, H.-L. Wang and K.-W. Yeh Table 11.4. Selected Examples of ESTs for Genes Related to Transportation Gene Annotation Acyl-CoA-binding protein ADP-ribosylation factor ADP-ribosylation factor C2 domain-containing protein C2 domain-containing protein C2 domain-containing protein dynamin like protein 2a F-actin capping protein, alpha subunit gamma-adaptin 1 GDSL-like lipase/ acylhydrolase GDSL-like lipase/ acylhydrolase GDSL-motif lipase/ hydrolase GDSL-motif lipase/ hydrolase GDSL-motif lipase/ hydrolase GDSL-motif lipase/ hydrolase GDSL-motif lipase/ hydrolase golgi-localized protein (GRIP) high mobility group protein 2 kinesin kinesin-related protein lipid transfer protein isoform 4 Reference Organism GI Number E Value ESTs Panax ginseng Glycine max Oryza sativa Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Oryza sativa 19352190 4324967 18844784 15239959 3.00E-19 2.00E-52 2.00E-25 2.00E-36 2 2 1 1 15223764 5.00E-15 1 15217968 5.00E-37 1 19032337 2.00E-44 1 23617186 2.00E-12 1 Oryza sativa Oryza sativa 19386749 29837765 1.00E-30 1.00E-26 1 4 Oryza sativa 29837765 9.00E-34 2 Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Oryza sativa 15228189 3.00E-91 5 21593518 2.00E-25 3 15221260 6.00E-54 2 18416824 2.00E-25 1 15224201 4.00E-12 1 22093862 2.00E-18 1 Arabidopsis thaliana Daucus carota Arabidopsis thaliana Vitis vinifera 15231065 7.00E-31 1 15186760 22327641 3.00E-30 3.00E-54 2 3 28194086 3.00E-16 7 (Continued ) Pseudobulb-Specific Gene Expression of Oncidium Orchid ✦ 199 Table 11.4. Gene Annotation membrane bound O-acyl transferase (MBOAT) membrane bound O-acyl transferase (MBOAT) mitochondrial carrier protein myosin heavy chain permease peroxisomal targeting signal type 1 receptor PEX14 protein plasma membrane intrinsic protein Rer1A protein (AtRer1A) Sec31p secretory carrier membrane protein signal peptidase sodium/dicarboxylate cotransporter sodium/dicarboxylate cotransporter sodium-dicarboxylate cotransporter vesicle transport v-SNARE protein villin 1 (VLN1) (Continued ) Reference Organism GI Number E Value ESTs Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Oryza sativa Arabidopsis thaliana Arabidopsis thaliana Oryza sativa 22329514 9.00E-24 1 22329514 1.00E-75 1 15240756 5.00E-39 1 18402909 5.00E-17 1 27545049 15241175 2.00E-17 3.00E-15 3 1 30697742 3.00E-15 1 22831004 2.00E-44 4 Oryza sativa Oryza sativa Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Oryza sativa 10945247 22831279 15222550 2.00E-37 4.00E-63 1.00E-30 2 1 1 15240934 7.00E-56 1 15238130 5.00E-44 24 15238130 2.00E-39 3 21536650 2.00E-35 1 19571103 8.00E-55 1 Arabidopsis thaliana 26451417 2.00E-22 1 (From Tan et al., Biotech Letts (2005) 27: 1517–1528. The authors wish to thank Biotechnology Letters for providing the figures and tables cited in the text.) homobox genes were most abundant among this group. The E2F/DP protein family controls cell cycle progression by acting predominantly as an activator or repressor of transcription.26 Arabidopsis had more than 180 potential E2F target genes with various functions: cell cycle, transcription, stress and defense, or signaling.27 Homeobox 20 had a 200 ✦ J. Tan, H.-L. Wang and K.-W. Yeh Table 11.5. Selected Examples of ESTs for Known or Putative Stressrelated Genes Gene Annotation acid phosphatase AP2 domain transcription factor AP2 domain transcription factor beta-Nacetylhexosaminidase biostress-resistancerelated protein bZIP DNA-binding protein chloroplastic light-induced, drought-induced stress protein choline monooxygenase dehydration-induced protein DHHC-type zinc finger domain-containing protein disease resistance protein disease resistance protein (NBS-LRR class) extensin farnesyltranstransferase glyceraldehyde 3-phosphate dehydrogenase, cytosolic glycosyl hydrolase family 19 (chitinase) heat shock protein Reference Organism GI Number E Value ESTs Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Triticum aestivum Capsicum chinense Solanum tuberosum 22330531 1.00E-35 1 21593696 2.00E-18 1 21593696 5.00E-58 1 21537026 4.00E-50 1 29409364 1.00E-61 1 4457221 3.00E-27 1 22261807 4.00E-40 1 Suaeda liaotungensis Arabidopsis thaliana Arabidopsis thaliana 21217447 6.00E-19 1 18411430 2.00E-68 1 18409331 2.00E-34 1 Arabidopsis thaliana Arabidopsis thaliana Populus nigra Oryza sativa Magnolia quinquepeta 15232373 9.00E-26 3 15231860 4.00E-18 1 7484770 20160508 120669 6.00E-42 7.00E-11 2.00E-99 4 1 2 Arabidopsis thaliana Arabidopsis thaliana 15228911 1.00E-38 8 15225377 3.00E-20 1 (Continued ) Pseudobulb-Specific Gene Expression of Oncidium Orchid ✦ 201 Table 11.5. Gene Annotation heat shock protein heat shock protein cognate 70 heat shock protein hsc70-3 (hsc70.3) late embryogenesis abundant protein leucine rich repeat protein major intrinsic protein (MIP) Na+/H+ antiporter 2 nodulin PDR-like ABC transporter peroxidase peroxidase (EC 1.11.1.7) peroxidase (EC 1.11.1.7) peroxidase (EC 1.11.1.7) 2, cationic peroxidase (EC 1.11.1.7) 2, cationic phosphoethanolamine methyltransferase plastid-lipid associated protein PAP/fibrillin proline rich protein 3 proline-rich protein APG isolog proline-rich-like protein senescence-associated protein senescence-associated protein wound-induced protein (Continued ) Reference Organism GI Number E Value ESTs Arabidopsis thaliana Oryza sativa 15225377 1.00E-40 1 29124135 2.00E-42 1 Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Lycopersicon esculentum Oryza sativa Oryza sativa Glycine max Gossypium irsutum Gossypium irsutum Glycine max 15232682 7.00E-29 1 15224810 2.00E-16 1 30686169 1.00E-37 1 15236485 1.00E-81 2 15982206 4.00E-17 1 11072005 27368827 5002234 7433087 9.00E-31 4.00E-35 7.00E-18 2.00E-48 1 1 5 92 7433087 2.00E-17 8 7433098 6.00E-27 21 Glycine max 7433098 1.00E-91 5 Oryza sativa 22535531 8.00E-13 1 Arabidopsis thaliana Cicer arietinum Cicer arietinum 18403751 2.00E-35 1 21615411 10638955 5.00E-75 4.00E-16 1 1 Asparagus officinalis Pisum sativum 1531756 2.00E-29 1 13359451 3.00E-43 6 18398417 2.00E-20 1 15234987 2.00E-15 3 Arabidopsis thaliana Arabidopsis thaliana (From Tan et al., Biotech Letts (2005) 27: 1517–1528. The authors wish to thank Biotechnology Letters for providing the figures and tables cited in the text.) 202 ✦ J. Tan, H.-L. Wang and K.-W. Yeh Table 11.6. Selected Examples of ESTs for Genes Related to Cell Cycle Gene Annotation 26S proteasome non-ATPase, regulatory subunit 6 3-hydroxy-3methylglutarylcoenzyme A reductase 3 (HMG3.3) AUX1-like permease auxin efflux carrier protein biotin carboxyl carrier protein subunit cyclic nucleotide-regulated ion channel (CNGC9) cysteine proteinase AALP cysteine proteinase mir3 (EC 3.4.22.) DP-E2F-related protein 1 histone deacetylase 2 isoform b homeobox 20 homeobox protein knotted-1 2 (KNAP2) homeobox-leucine zipper protein ATHB-13 Homeotic protein knotted-1 (TKN1) nucleolysin Reference Organism GI Number E Value ESTs Oryza sativa 20978545 8.00E-79 1 Solanum tuberosum 11133016 5.00E-32 1 Arabidopsis thaliana Arabidopsis thaliana Glycine max 5881784 2.00E-24 1 15239215 3.00E-50 1 12006165 2.00E-29 1 Arabidopsis thaliana Arabidopsis thaliana Zea mays 15234769 8.00E-34 1 23397070 6.00E-30 1 7435806 5.00E-47 1 Arabidopsis thaliana Zea mays 22331664 1.00E-49 7 7716948 6.00E-19 1 Nicotiana tabacum Malus x domestica Arabidopsis thaliana Lycopersicon esculentum Oryza sativa 4589882 1.00E-49 1 6016217 8.00E-46 3 15222452 9.00E-38 1 3023974 9.00E-16 1 4680340 1.00E-15 1 (From Tan et al., Biotech Letts (2005) 27: 1517–1528. The authors wish to thank Biotechnology Letters for providing the figures and tables cited in the text.) common motif and took part in xylem cell differentiation.28 Homeoboxleucine zipper protein ATHB-13 was a transcription factor. It could specify the cell fate and body plan in early embryogenesis.29 A total of 59 ESTs were annotated with known or putative regulatory functions (Table 11.7). It seemed that the regulation of the genes Pseudobulb-Specific Gene Expression of Oncidium Orchid ✦ 203 Table 11.7. Selected Examples of ESTs for Known or Putative Regulatory Functions Gene Annotation adapter protein SPIKE1 adenine phosphoribosyltransferase form 2 amidase amidase BURP domain protein c-myc binding protein cupin domain-containing protein DEAD/DEAH box helicase DEAD/DEAH box helicase DnaJ protein DnaJ protein homolog 2 DNAJ-like protein elongation factor 1-alpha GAMYB-binding protein GF14 protein glycine-rich RNA-binding protein HD-Zip transcription factor Athb-14 helicase homeobox-leucine zipper protein ATHB-13 MuDR mudrA-like protein phosphoprotein phosphatase (EC 3.1.3.16) Reference Organism GI Number E Value ESTs Oryza sativa Oryza sativa 24899400 29826070 4.00E-56 8.00E-75 1 1 Arabidopsis thaliana Oryza sativa Vigna unguiculata Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Salix gilgiana Allium porrum Oryza sativa Elaeis oleifera Hordeum vulgare Fritillaria agrestis Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Oryza sativa Arabidopsis thaliana 8163875 7.00E-31 1 18542894 7106540 1.00E-12 6.00E-12 1 9 22325671 2.00E-12 1 15226403 3.00E-29 2 15222526 5.00E-40 1 15219185 1.00E-26 1 11277163 1169382 29367357 18419676 27948448 1.00E-106 4.00E-26 8.00E-33 8.00E-40 7.00E-51 2 1 1 1 1 2921512 2.00E-66 1 21553602 7.00E-21 1 15226808 1.00E-96 1 18395518 4.00E-30 1 15222452 9.00E-38 1 5441874 25513447 4.00E-31 2.00E-94 1 1 (Continued ) 204 ✦ J. Tan, H.-L. Wang and K.-W. Yeh Table 11.7. Gene Annotation probable protein disulfide-isomerase Ras-related protein Rab11C receptor-like kinase RHG4 receptor-like protein kinase receptor-like protein kinase (EC 2.7.1) receptor-related protein kinase RNA recognition motif (RRM)-containing protein RNA-binding protein RNA-binding protein serine/threonine kinase serine/threonine protein kinase serine/threonine protein kinase (EC 2.7.1) serine/threonine-specific protein kinase SNF2 domain/helicase domain-containing protein sphingosine kinase transcription factor LIM transcription factor X1 transducin/WD-40 repeat protein transfactor (Continued ) Reference Organism GI Number E Value ESTs Nicotiana tabacum Nicotiana tabacum Glycine max 7489183 1.00E-88 1 3024503 5.00E-31 1 21239384 2.00E-17 1 Arabidopsis thaliana Oryza sativa 7487253 6.00E-41 1 7434420 3.00E-18 1 Arabidopsis thaliana Arabidopsis thaliana 15240720 6.00E-45 1 22328805 8.00E-18 1 Oryza sativa 18087662 Mesembryanthe- 1076251 mum crystallinum Arabidopsis 25387051 thaliana Nicotiana 3811293 tabacum Avena sativa 7489361 2.00E-37 4.00E-26 2 1 2.00E-51 1 3.00E-19 1 5.00E-37 1 Arabidopsis thaliana Arabidopsis thaliana 25751318 2.00E-18 1 15226870 4.00E-42 1 Oryza sativa Nicotiana tabacum Oryza sativa Arabidopsis thaliana Arabidopsis thaliana 13786462 18565124 3.00E-71 5.00E-46 1 2 6650526 30682603 2.00E-28 3.00E-21 1 1 6223653 2.00E-34 1 (Continued ) Pseudobulb-Specific Gene Expression of Oncidium Orchid ✦ 205 Table 11.7. Gene Annotation translational activator translational activator WD-40 repeat protein zinc finger (C3HC4-type RING finger) protein zinc finger protein zinc finger protein 5 (ZFP5) zinc-finger protein Lsd1 (Continued ) Reference Organism Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Pisum sativum Arabidopsis thaliana Arabidopsis thaliana GI Number E Value ESTs 25404492 2.00E-18 1 15217742 5.00E-42 1 30685408 5.00E-46 1 15233298 9.00E-28 1 11288368 21592423 3.00E-77 5.00E-12 1 1 30685085 1.00E-22 1 (From Tan et al., Biotech Letts (2005) 27: 1517–1528. The authors wish to thank Biotechnology Letters for providing the figures and tables cited in the text.) involved in the active material and energy metabolism in the pseudobulb was very complex. 11.7 Discussion In Oncidium plant, the pseudobulb is a critical organ regulating plant growth. In general, when the pseudobulb grows healthily and accumulates abundant nutrition, the embedded young bud develops into inflorescence, a so-called reproductive state. However, the bud might develop into a young adventitious shoot and begin a new vegetative state. Therefore, the pseudobulb plays a functional role in regulating Oncidium’s switch between the reproductive state and the vegetative state. In the previous study, we analyzed the carbohydrate composition and amino acid pool in the pseudobulb prior to and during inflorescence development.10 Alternation of nutrition in the pseudobulb during floral development was also investigated.10 To our knowledge, mannan is the principal storage polysaccharide synthesized in the pseudobulb before inflorescence initiation. It is degraded into mannose and de novo synthesized into starch during inflorescence development. Eventually, starch is utilized while the flower is forming.10 Therefore, active metabolism related to carbohydrate and other types of metabolism may affect 206 ✦ J. Tan, H.-L. Wang and K.-W. Yeh floral development. However, the genes involved in these active metabolic steps are still unknown. Indubitably, subtractive EST data have more redundancy than EST data. Including the inevitable consequence of the nonuniform abundance of mRNA from different genes with dramatically various numbers in the cDNA pool, the bias must be further enhanced by hybridization, PCR amplification twice, ligation, and transformation. To this point, this is also the main problem in EST sequencing based on the cDNA library. It could perhaps be resolved by subtracting newly found redundant sequences from templates during stepwise sequencing. EST sequencing only from the 5′-ends of cDNAs is a rapid, economical, and amenable strategy.6 However, for subtractive EST sequencing, not only is Rsa I digestion infeasible, it also causes shorter pieces of ESTs. Furthermore, it seemed the middle parts of genes have more chances to be sequenced than the ends (Fig. 11.4). Thus, a large-scale analysis of gene expression related to physiological responses of the Oncidium pseudobulb, particularly during the early floral stage, has not been reported. Therefore, the expressed gene catalogue presented here will provide the basic information to investigate the molecular genetics basis of the Oncidium pseudobulb’s early flowering stage by transcriptome profiling. In this small-scale subtractive EST, a conclusive picture of the cellular processes of stress-response (Table 11.5), carbohydrate metabolism (Table 11.3), and transportation (Table 11.4) was obtained. Also, the RNA gel-blot expression data showed some evidence that this EST-set is indeed enriched with such genes (Fig. 11.3), indicating the high efficiency of the cDNA subtraction strategy. Based on the types of genes represented, the mechanism of inflorescence initiation in the early flowering stage of the Oncidium pseudobulb relies on precise integration of developmental processes with the carbohydrate metabolism-related response. The pseudobulb at the stage of inflorescence initiation seems like a ripening fruit for the coming flower development. First, it is in growing, including cell enlargement and differentiation. Especially, several genes involved in pectin degradation are expressing actively (Table 11.3). This may be a physiological marker for the pseudobulb, which is going to mature.30 From the Northern results of genes related to mechanisms of saccharides at different stages of inflorescence, the ripening of Pseudobulb-Specific Gene Expression of Oncidium Orchid ✦ 207 pseudobulb is just in the initiation of inflorescence development (data not shown). At the same time, transportation is very active, and storage seems to be in preparation. There are two main directions of transportation: extracellular and intracellular, in which vacuoles play a predominant role. For extracellular transportation, saccharides and proteins are transferred to cell wall for synthesis. Intracellular transportation involves mainly the transfer of low molecular saccharides into vacuoles to maintain osmosis and to prepare for future storage functions. Pseudobulb mannan is very rich and in degradation at the initiation of inflorescence. Plant mannose binding lectin is believed to play a role in the recognition of high-mannose type glycans of foreign microorganisms or plant predators.31 However, mannose is grasped with mannose binding lectin in the pseudobulb, for it is harmful to plant cells.32 Then the mannose can be further catalyzed into fructose and glucose. Starch is also synthesized at this stage, but almost no starch is stored in cells compared with the starch granules seen before blossoming (data not shown). Starch is perhaps also degraded into sucrose and other monohexoses for cell growth and differentiation. At the bottom side of the pseudobulb, the little flower bud is also in differentiation and growing at the same time. Usually the bud there develops into inflorescence, but it can sometimes turn into a vegetative reproductive bud. This is a hint that the pseudobulb offers signals leading to inflorescence development unless some accident occurs. The genes involved in this function are perhaps flower-specific along with some transcriptional factors. It is no wonder so many genes are related to regulation because the pseudobulb is a very active organ leading to inflorescence at this stage. Making their functions clear is important and will require further research. In summary, the subtractive EST approach is an efficient tool to provide an overview of gene expression profiles in the metabolically active tissue of the Oncidium pseudobulb. The EST data provide us with an insight into a wide range of genes. These genes represent the physiological status in the pseudobulb during the early inflorescence development. The abundant genes, e.g. peroxidase and sodium/dicarboxylate cotransporter, shown in the profile revealed some especially unexpected facts. These will someday make it possible to exploit flowering-related mechanisms for the benefit of mankind. 208 ✦ J. Tan, H.-L. Wang and K.-W. Yeh Acknowledgment This work was financially supported by National Science Council, ROC under Grant NSC 91-2317-B-002-041 to Professor K. W. Yeh. References 1. Liau CH, You SJ, Prasad V, et al. (2003a) Agrobacterium tumefaciensmediated transformation of an Oncidium orchid. Plant Cell Rep 21: 993–998. 2. Chen J, Chang W. (2000) Efficient plant regeneration through somatic embryogenesis from callus cultures of Oncidium (Orchidaceae). Plant Sci 160:87–93. 3. You SJ, Liau CH, Huang HE, et al. (2003) Sweet pepper ferredoxin-like protein (pflp) gene as a novel selection marker for orchid transformation. Planta 217:60–65. 4. Liau CH, Lu JC, Prasad V, et al. (2003b) The sweet pepper ferredoxin-like protein (pflp) conferred resistance against soft rot disease in Oncidium orchid. Transgenic Res 12:329–336. 5. Hsu HF, Yang CH. (2002) An orchid (Oncidium Gower Ramsey) AP3-like MADS gene regulates floral formation and initiation. Plant Cell Physiol 43:1198–1209. 6. Li CR, Zhang XB, Hew CS. (2003) Cloning of a sucrose-phosphate synthase gene highly expressed in flowers from the tropical epiphytic orchid Oncidium Goldiana. J Exp Bot 21:89–91. 7. Hew CS, Yang JWH. (1994) Growth and photosynthesis of Oncidium Goldiana. J Hort Sci 69:809–819. 8. Wadasinghe G, Hew CS. (1995) The importance of back shoots as a source of photo-assimilates for growth and flower production in Dendrodium cv. Jashika pink (Orchidaceae). J Hort Sci 70:207–214. 9. Hew CS, Ng CKY. (1996) Changes in mineral and carbohydrate content in pseudobulbs of the C3 epiphytic orchid hybrid Oncidium Goldiana at different growth stages. Lindleyana 11:125–134. 10. Wang HL, Chung JD, Yeh KW. (2003) Changes of carbohydrate and free amino acid pools in current pseudobulbs of Oncidium Gower Ramsey during inflorescence development. J Agric Associ China 4:476–488. 11. Richmond TA, Bleecker AB. (1999) A defect in beta-oxidation causes abnormal inflorescence development in Arabidopsis. Plant Cell 11:1911–1924. 12. Yu H, Goh CJ. (2000) Identification and characterization of three orchid MADS-box genes of the AP1/AGL9 subfamily during floral transition. Plant Physiol 123:1325–1336. Pseudobulb-Specific Gene Expression of Oncidium Orchid ✦ 209 13. Charrier B, Champion A, Henry Y, Kreis M. (2002) Expression profiling of the whole Arabidopsis shaggy-like kinase multigene family by real-time reverse transcriptase-polymerase chain reaction. Plant Physiol 130:577–590. 14. Wade HK, Bibikova TN, Valentine WJ, Jenkins GI. (2001) Interactions within a network of phytochrome, cryptochrome and UV-B phototransduction pathways regulate chalcone synthase gene expression in Arabidopsis leaf tissue. Plant J 25:675–685. 15. van Tunen AJ, Koes RE, Spelt CE, et al. (1988) Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: Coordinate, light-regulated and differential expression of flavonoid genes. EMBO J 7:1257–1263. 16. de Vetten N, ter Horst J, van Schaik HP, et al. (1999) A cytochrome b5 is required for full activity of flavonoid 3′, 5′-hydroxylase, a cytochrome P450 involved in the formation of blue flower colors. Proc Natl Acad Sci USA 96:778–783. 17. Emmerlich V, Linka N, Reinhold T, et al. (2003) The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier. Proc Natl Acad Sci USA 100:11122–11126. 18. Wilhelm S, Tommassen J, Jaeger KE. (1999) A novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa. J Bacteriol 181:6977–6986. 19. Lee MH, Min MK, Lee YJ, et al. (2002) ADP-ribosylation factor 1 of Arabidopsis plays a critical role in intracellular trafficking and maintenance of endoplasmic reticulum morphology in Arabidopsis. Plant Physiol 129:1507–1520. 20. Nogi T, Shiba Y, Kawasaki M, et al. (2002) Structural basis for the accessory protein recruitment by the gamma-adaptin ear domain. Nat Struct Biol 9:527–531. 21. Ungar D, Oka T, Brittle EE, et al. (2002) Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function. J Cell Biol 157:405–415. 22. Ochoa WF, Corbalan-Garcia S, Eritja R, et al. (2002) Additional binding sites for anionic phospholipids and calcium ions in the crystal structures of complexes of the C2 domain of protein kinase calpha. J Mol Biol 320:277–291. 23. Tognolli M, Penel C, Greppin H, Simon P. (2002) Analysis and expression of the class III peroxidase large gene family in Arabidopsis thaliana. Gene 288:129–138. 24. Shen YG, Zhang WK, He SJ, et al. (2003) An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold, dehydration and ABA stress. Theor Appl Genet 106:923–930. 25. Xue GP. (2003) The DNA-binding activity of an AP2 transcriptional activator HvCBF2 involved in regulation of low-temperature responsive genes in barley is modulated by temperature. Plant J 33:373–383. 210 ✦ J. Tan, H.-L. Wang and K.-W. Yeh 26. Cayirlioglu P, Ward WO, Silver Key SC, Duronio RJ. (2003) Transcriptional repressor functions of Drosophila E2F1 and E2F2 cooperate to inhibit genomic DNA synthesis in ovarian follicle cells. Mol Cell Biol 23:2123–2134. 27. Ramirez-Parra E, Frundt C, Gutierrez C. (2003) A genome-wide identification of E2F-regulated genes in Arabidopsis. Plant J 33:801–811. 28. Hertzberg M, Olsson O. (1998) Molecular characterisation of a novel plant homeobox gene expressed in the maturing xylem zone of Populus tremula x tremuloides. Plant J 16:285–295. 29. Yang JY, Chung MC, Tu CY, Leu WM. (2002) OSTF1: A HD-GL2 family homeobox gene is developmentally regulated during early embryogenesis in rice. Plant Cell Physiol 43:628–638. 30. Medina-Escobar N, Cardenas J, Moyano E, et al. (1997) Cloning, molecular characterization and expression pattern of a strawberry ripening-specific cDNA with sequence homology to pectate lyase from higher plants. Plant Mol Biol 34:867–877. 31. Chai Y, Pang Y, Liao Z, et al. (2003) Molecular cloning and characterization of a mannose-binding lectin gene from Crinum asiaticum. J Plant Physiol 160:913–920. 32. Stein JC, Hansen G. (1999) Mannose induces an endonuclease responsible for DNA laddening in plant cells. Plant Physiol 121:71–80. 33. Tan J, Wang HL, Yeh KW. (2005) Analysis of organ-specific, expressed genes in Oncidium orchid by substractive expressed sequence tags library. Biotech Lett 27:1517–1528. Chapter 12 Application of Virus-induced Gene Silencing Technology in Gene Functional Validation of Orchids Hsiang-Chia Lu†, Hong-Hwa Chen‡ and Hsin-Hung Yeh*,† The largest family of angiosperms, Orchidaceae, has diverse, specialized pollination and ecological strategies and provides a rich subject for investigating evolutionary relationships and developmental biology. However, the study of these non-model organisms may be hindered by challenges, such as their large genome size, low transformation efficiency, long regeneration time, and long life-cycle. To overcome these obstacles, we first developed vectors with the use of a symptomless Cymbidium mosaic virus, which infects most orchids, then combined simple physiological controls and virus-induced gene silencing (VIGS) for validation of gene function in orchids. The success of our strategies was verified by our functional validation of floral identity gene(s) in the tetraploid Phalaenopsis orchids, which have an unusually long life-cycle (two years from sowing to flowering). We could knock down the RNA level of either a specific Phalaenopsis floral identity gene or a family of genes congruently in two months. Functional analysis of orchid genes could become easier and profit from the VIGS approach. *Corresponding author. † Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan. ‡ Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan. 211 212 ✦ H.-C. Lu, H.-H. Chen, and H.-H. Yeh 12.1 Introduction Orchids, plants with elegant and beautiful flowers, have attracted humans for centuries. Known for their diversity, they form the largest family of angiosperms, Orchidaceae.1 Orchids have evolved specialized and astonishing pollination and ecological strategies for survival. Therefore, they are a rich source for investigating evolutionary relationships and developmental biology. Although evolutionary developmental biology (Evo-Devo) has become an important topic in every field of biology today,2–4 the study of orchids has been hindered by several challenges, including their large genome size, long life-cycle, and low transformation efficiency. In fact, orchids are the opposite of model plants in every trait studied by geneticists and molecular biologists. For example, the most widely grown orchid, Phalaenopsis spp., has a large genome size, ranging from 1 × 109 to 6 × 109 bp/1C,5,6 and several important commercial cultivars are multiploid. The life-cycle of Phalaenopsis spp. naturally takes about 2 to 3 years from the vegetation to the reproductive phases. Therefore, applications of commonly used genetic and molecular approaches to the study of orchids is difficult. However, the development of several new research techniques has led to increased investigation of model plants and numerous exciting findings. From these developments, we can select suitable tools to solve the problems faced in the study of orchids. 12.2 Approaches Used for Gene Functional Analysis Forward and reverse genetics is commonly used in the elucidation of the function of a gene or genes. Forward genetics tries to identify the genetic source of a trait of interest, beginning with random mutagensis of the whole genome to follow the selection of the trait and subsequently identifying the mutated gene or genes related to the trait. Reverse genetics, however, aims to disrupt the expression of genes to search for a phenotype and usually begins with cloned DNA sequences, with or without prior knowledge about the DNA. In plants, disrupting the expression of a gene of interest is difficult owing to low homologous recombination frequencies. Alternatively, plants transformed with antisense and/or sense genes that can produce dsRNA from an Application of Virus-Induced Gene Silencing Technology ✦ 213 inverted-repeat of a target gene may disrupt the RNA or block the translation of the gene.7 Forward genetics is not practical for functional analysis of plants, such as orchids because of their long life-cycle and lack of high-resolution genetic maps. Reverse genetics to find genes is problematic as well because of difficulties in plant transformation and long regeneration time, especially for genes involved in reproductive stages, the last stages of the life-cycle and so important to orchid study. In addition to genetic transformation, a recently developed approach to functional analysis of plant genes involves loss-of-function mediated by virus-induced gene silencing (VIGS). Since the early success of VIGS with Tobacco mosaic virus (TMV)- and Potato virus X (PVX)-based vectors in silencing the phytoene desaturase gene (PDS) in Nicotiana benthamiana,8,9 the use of virus vectors to induce plant gene silencing has been efficient and reliable in many systems, such as tomato (Lycopersicum esculentum), Arabidopsis spp., and N. benthamiana, some Solanum species; a legume species, Pisum sativum; soybean (Glycin spp.); and Papaver somniferum.10–18 Among the monocots, only a Barley strip mosaic virus-derived vector has been developed for VIGS.19,20 VIGS has seen success in diverse fields of plant science. For example, Tobacco rattle virus (TRV)-based VIGS vectors have been applied to the study of floral development,21 plant resistance against pathogens,22–24 and fruit ripening.25 A PVX-based vector has been applied in high throughput analysis to screen 4,992 cDNAs derived from N. benthamiana. Discovered were genes involved in signal transduction pathways of Pto-mediated resistance to a pathogenic bacteria, Pseudomomas syringae.26 VIGS has advantages over other approaches, such as transforming plants with antisense and/or sense genes that can produce dsRNA of target genes. The technique can be induced within a month after inoculation in every plant tested so far. In addition, it can be applied at different stages of plant development, as long as viruses can infect the plant tissues. Therefore, it prevents suppression of some essential genes required for seed maturation, a common problem with mutagenesis screening. For the detailed mechanism and advantages of VIGS, see Refs. 11, 12, 27 and 28. Therefore, VIGS offers an opportunity for functional analysis of genes in plants with long life-cycle, such as orchids. 214 ✦ H.-C. Lu, H.-H. Chen, and H.-H. Yeh 12.3 Viral Vectors Suitable for Gene Functional Analysis of Orchids Among the currently constructed viral vectors, only TRV can infect orchids.29 However, despite the successful application of the TRV vector in the systems described above, its application in orchids has not been reported.10–18 Since viral isolates may differ in their biological nature in terms of symptoms and host range, we do not know if the currently available TRV vectors are suitable for orchid gene functional analysis. For application of VIGS, the desired viral vectors must not induce symptoms in plants, so as not to complicate the functional interpretation of the gene of interest. The currently available TRV vectors cause no visible symptoms in tested plants. However, whether the TRV vectors can infect orchids and induce symptoms or not remains to be resolved. Another important issue is biological safety of the vectors. In countries, such as Taiwan, infection with TRV has not been reported in any plants. Therefore, application of TRV vectors must not violate local biosafety legislation and/or potential release of the viruses outside the tested plants. Alternatively, orchid viruses belonging to the same genus of the currently successful VIGS vector are potential resources for developing VIGS vectors. Examples are the successful PVX vector and Cymbidium mosaic virus (CymMV), belonging to Potexvirus, and TMV and Odontoglossum ringspot tobamovirus (ORSV), belonging to Tobamovirus. Recently, we screened and selected a symptomless CymMV, one of the most prevalent orchid viruses, to develop a VIGS vector (Fig. 12.1).30 The vector could induce gene silencing by knocking down the RNA level of PDS in leaves and that of a specific floral identity gene or, congruently, floral identity family genes in Phalaenopsis in less than two months. The following describes our novel use of VIGS in functional analysis of floral identity genes in Phalaenopsis. 12.4 Applicability of Vigs in the Study of Gene Function in Leaves of Orchids To monitor VIGS efficacy, we inserted a Phalaenopsis amabilis var. formosa PDS fragment into our CymMV vector (pCymmV-pro60) and inoculated P. amabilis var. formosa with the vector. Real-time RT-PCR was Application of Virus-Induced Gene Silencing Technology ✦ 215 Fig. 12.1. Schematic representation of CymMV cDNA infectious clone and derivative vectors. Schematic representation of CymMV infectious clone (A), and derivative vectors (B–D). Rectangles represent ORFs encoded by CymMV genomic RNA. RNA-dependent RNA polymerase (RDRP), triple gene block 1, 2, and 3, capsid protein (CP) are indicated. The T3 promoter immediately adjacent to the most 5′-end and a poly(A) tail (25 adenosine) at the most 3′-end are indicated. The SpeI restriction enzyme digestion site is indicated by a red arrow. The numbers below the red lines indicate the selected region of CP subgenomic promoter, and plus or minus corresponds to the upstream and downstream CP translation start codon, respectively. The light green line indicates the duplicated CP subgenomic promoter. Head to head arrows on (C) indicate that the selected 150 bp of PeMADS6 was cloned as an inverted repeat. The direct arrow on (D) indicates that the PeMADS6 conserved region was cloned directly. used to monitor the knockdown of PDS in leaves beginning at three weeks after inoculation (approximately three weeks are needed for the CymMV to establish systemic infection). The RNA level of PDS was gradually reduced to 54% in plants at eight weeks postinoculation, the level increasing thereafter; thus, gene-silencing efficacy was reduced after eight weeks. Our results showed that the CymMV vector can induce gene silencing in leaves of orchids and are consistent with a previous report indicating that VIGS efficacy is progressively reduced over time.21 However, the result also revealed the limitation of the application of VIGS in leaves of Phalaenopsis orchids. PDS is a visual marker commonly used for VIGS because the bleaching phenotype is easily 216 ✦ H.-C. Lu, H.-H. Chen, and H.-H. Yeh observed,8,9,21 but only on newly emerging leaves, not fully expanded leaves. Because Phalaenopsis is a Crassulacean acid metabolism (CAM) plant with very slow growth rate (more than six months to generate a new leaf ), no new leaves were able to generate before the VIGS efficacy was reduced. Therefore, no visible phenotype was observed on the plants silenced with PDS. Although the CymMV vector could induce gene silencing in the leaves of orchids, a visible phenotype could not be easily observed on those leaves. However, since gene silencing was induced in leaves, the transcriptome was changed, and an invisible phenotype might be measured or analyzed by biochemical or molecular biology approaches. 12.5 Strategies for Functional Validation of Genes Involved in the Reproductive Stage of Orchids For functional validation of orchid genes, the most intriguing and challenging studies are those analyzing genes involved in the reproductive stages, intriguing because of the diversity of orchid flowers and challenging because in orchids, the transition from vegetative to reproductive stages usually takes about 2 to 3 years. In addition, orchids usually bloom only once a year, which limits the time for conducting experiments. To overcome such obstacles in the study of orchids, we first learned from commercial orchid growers. Commercial orchid growers usually keep their plants under low temperature controls (25°C/day and 20°C/night) with appropriate humidity and fertilization to induce stalks for flowering.31 With this process, orchid growers have flowers to sell year-round and we have more flowers for study. To test whether the CymMV vector (pCymMV-pro60) could induce gene silencing in all floral organs, we selected a B-class MADS-box family gene, PeMADS6, which is transcribed in all flower organs.32 Since MADS-box family genes are less conserved in their 3′-terminus, we selected a stretch of 150 nucleotides (nts) in this region and inserted it to the CymMV vector. We directly inoculated the CymMV vector containing the 150 nts of PeMADS6 [pCymMV-pro60-PeMADS6IR (Fig. 12.1B)] into emerging stalks with six nodes (about 8 cm) of P. amabilis var. formosa. Approximately six weeks later, the flowers blossomed. Compared with mock-inoculated plants, the plants inoculated Application of Virus-Induced Gene Silencing Technology ✦ 217 with pCymMV-pro60-PeMADS6IR showed reduced PeMADS6 RNA levels in sepals, petals, lips, and columns to 63 ± 2%, 33 ± 3%, 23 ± 5% and 33 ± 2%, respectively, as measured by real-time RT-PCR.30 However, the plants inoculated with the pCymMV-pro60 vector showed a PeMADS6 RNA level similar to that of mock-inoculated plants. We also analyzed the RNA level of PeMADS1 and PeMADS3, which belong to C- and B-class-like MADS-box genes, in the plants inoculated with pCymMV-pro60-PeMADS6IR. No obvious transcriptional changes in PeMADS1 and PeMADS3 were detected among the plants inoculated with the buffer, pCymMV-pro60, or pCymMV-pro60-PeMADS6IR. Thus, only PeMADS6 was silenced in pCymMV-pro60-PeMADS6IRinoculated plants. To confirm that the reduced expression of PeMADS6 was due to the presence of RNAi mediated by VIGS, we purified small-molecular-weight RNA from inoculated plants and performed Northern blot hybridization, with the CymMV CP gene or PeMADS6 used as probes. CymMV could induce 21-nt siRNA, because the CymMV CP probe could detect 21-nt siRNA in both CymMV-pro60- and pCymMV-pro60-PeMADS6IRinoculated plants. However, the PeMADS6 21-nt siRNA was detected only in plants inoculated with pCymMV-pro60-PeMADS6IR (the PeMADS6 region used for probes is different from that inserted in pCymMV-pro60-PeMADS6). No siRNA was detected in the mock-inoculated plants using of both probes. Thus, the generation of PeMADS6 siRNA is specific, and the reduction of the PeMADS6 RNA level is through the gene-silencing mechanism.30 However, we observed no visible morphologic changes in PeMADS6silenced plants, perhaps because the knockdown level induced by VIGS was enough to induce flower morphologic change. The expression of MADS-box genes is dose-dependent, and such genes may require complete silencing to produce a phenotype.33–35 12.6 Simultaneous Knockdown of MADS-Box Family Genes Family genes with redundant functions are not easily targeted by genetic knockout assay. In addition, a vector that can easily induce a visible phenotype in orchids during VIGS will be desirable for further research. Therefore, we inserted a 500-bp DNA fragment of a PeMADS6 218 ✦ H.-C. Lu, H.-H. Chen, and H.-H. Yeh conserved region of the MADS-box family of genes into pCymMV-pro60 [pCymMV-pro60-PeMADS6 (Fig. 12.1C)]. We expected that several MADS-box family genes would be affected, with consequent prominent morphologic changes. In plants inoculated with pCymMV-pro60PeMADS6, the flower blossomed, but streaks or patches of greenish tissue appeared in the sepals, petals, and lips (Fig. 12.2). Interestingly, some of the inoculated plants initially produced flower buds on the lower stalks, but the buds could not blossom (Fig. 12.2A). The flower buds on the upper stalk could blossom to some extent, but streaks or patches of greenish tissue were observed in the sepals, petals, and lips. We dissected some initial flower buds that turned yellow (an indication that these buds would eventually abort) and found fully formed sepals, petals, lips, and columns within the buds and morphology similar to that of green buds of healthy plants. These results suggest that the reduced transcript level of MADS-box family genes still allowed the flower to develop normally at first, but not enough for the flower to further develop and blossom. Real-time RT-PCR revealed that PeMADS6 and two randomly selected C- and B-class-like MADS-box genes, PeMADS1 and PeMADS3, were all silenced in plants inoculated with pCymMVpro60-PeMADS6. Especially, for the three analyzed genes, more than 85% of the transcript level was reduced in the initial flower buds.30 In addition, the transcript level of all the analyzed genes was similar, and no phenotype described above was observed in either the mock- or pCymMV-pro60-inoculated plants. The MADS-box family gene-silenced plants were unable to blossom or showed a greenish structure in the sepals and lips. This result was consistent with the prediction of the well-known ABC model, which suggests that A-, B-, and C-class-like MADS-box genes act cooperatively to control floral identity.36,37 We performed experiments in two different orchid varieties, P. amabilis and P. Sogo Musadium and produced similar results. Both varieties are tetraploid and, in general, loss-of-function assays are not easy to perform in plants with a multiploid genome or when the genes of interest have multiple copies. With loss-of-function assays, such as T-DNA insertion or transposon tagging, simultaneously targeting all genes with functional redundancy is difficult. In contrast, VIGS can knock down the RNA level after RNA transcription, regardless of the RNAs transcribed from genes in different genome locations and, thus can silence all genes simultaneously. Application of Virus-Induced Gene Silencing Technology ✦ 219 Fig. 12.2. Phenotype on MADS-box gene-silenced plants P. Sogo Musadium infected with pCymMV-pro60 (B, D) and pCymMV-CP60- PeMADS6 (A, C, E). The arrow on (A) indicates an aborted flower bud. Below this node, flower buds were all aborted, and flower buds produced beyond this node blossomed normally. The arrows on (C) indicate the greenish streaks in the sepal and petals. The arrows on (E) indicate the greenish patches in the lip. 220 ✦ H.-C. Lu, H.-H. Chen, and H.-H. Yeh 12.7 Perspective We demonstrated that our newly constructed CymMV-based vector is suitable for analyzing genes involved in floral morphogenesis of Phalaenopsis spp. As CymMV has a wide host range among species belonging to Orchidaceae, including Phalaenopsis, Cymbidium, Cattleya, Dendrobium, Epidendrum, Laelia, Laeliocattleya, Oncidium, Zygopetalum, Vanilla, and Vinda,38,39 the developed vectors will contribute well to functional genomic studies of orchids. With the advent of genomics and bioinformatics, the past few years have seen great strides in the amount of gene information available and development of tools for their analysis of orchids.40–42 Here, we have demonstrated that VIGS can be used for the functional validation of genes in orchids, difficult plants to study by the traditional genetic approaches because of their large genome size, low transformation efficiency, long regeneration time, and long life-cycle. The study of orchids will flourish and profit from approaches such as VIGS. Acknowledgments We thank Dr. Wen-Hui Chen (Department of Life Sciences, National Science Council, National University of Kaohsiung, Kaohsiung, Taiwan) for orchid variety consulting and Dr. Hong-Ji Su for helping to collect virus isolates. We also thank the National Science Council and Council of Agriculture in Taiwan for supporting the research grants in HHY’s laboratory. References 1. Dressler RL. (1993) Phylogeny and Classification of the Orchid Family. Dioscorides Press, Portland, Oregon. 2. Pennisi E. (2004) RNAi takes Evo-devo world by storm. Science 304:384. 3. Babbitt C, Giorgianni M, Price A. (2002) Evo-devo comes into focus. BioEssay 24:677–679. 4. Kramer EM, Hall JC. (2005) Evolutionary dynamics of genes controlling floral development. Curr Opin Plant Bio 8:13–18. 5. Chen WH, Kao YY, Lin TY, et al. (2001) Genomic study of Phalaenopsis orchid. Proceedings of APOC7, Nagoya, Japan. Application of Virus-Induced Gene Silencing Technology ✦ 221 6. Lin S, Lee HC, Chen WH, et al. (2001) Nuclear DNA contents of Phalaenopsis sp. and Doritis pulcherrima. J Am Soc Hortic Sci 126:195–199. 7. Kusaba M. (2004) RNA interference in crop plants. Curr Opin Biotechnol 15:139–143. 8. Kumagai MH, Donson J, della-Cioppa G, et al. (1995) Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proc Natl Acad Sci USA 92:1679–1683. 9. Ruiz MT, Voinnet O, Baulcombe DC. (1998) Initiation and maintenance of virus-induced gene silencing. Plant Cell 10:937–946. 10. Wang MB, Waterhouse PM. (2002) Application of gene silencing in plants. Curr Opin Plant Biol 5:146–150. 11. Benedito VA, Visser PB, Angenent GC, Krens FA. (2004) The potential of virus-induced gene silencing for speeding up functional characterization of plant genes. Genet Mol Res 3:323–341. 12. Burch-Smith TM, Anderson JC, Martin GB, Dinesh-Kumar SP. (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39:734–746. 13. Robertson D. (2004) VIGS vectors for gene silencing: Many targets, many tools. Annu Rev Plant Biol 55:495–519. 14. Brigneti G, Martin-Hernandez AM, Jin H, et al. (2004) Virus-induced gene silencing in Solanum species. Plant J 39:264–272. 15. Constantin GD, Krath BN, MacFarlane SA, et al. (2004) Virus-induced gene silencing as a tool for functional genomics in a legume species. Plant J 40:622–631. 16. Fofana IB, Sangare A, Collier R, et al. (2004) A geminivirus-induced gene silencing system for gene function validation in cassava. Plant Mol Biol 56:613–624. 17. Hileman LC, Drea S, Martino G, et al. (2005) Virus-induced gene silencing is an effective tool for assaying gene function in the basal eudicot species Papaver somniferum (opium poppy). Plant J 44:334–341. 18. Zhang C, Ghabrial SA. (2006) Development of Bean pod mottle virus-based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean. Virology 344:401–411. 19. Holzberg S, Brosio P, Gross C, Pogue GP. (2002) Barley stripe mosaic virusinduced gene silencing in a monocot plant. Plant J 30:315–327. 20. Scofield SR, Huang L, Brandt AS, Gill BS. (2005) Development of a virusinduced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiol 138:2165–2173. 21. Ratcliff F, Martin-Hernandez AM, Baulcombe DC. (2001) Technical Advance. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J 25:237–245. 222 ✦ H.-C. Lu, H.-H. Chen, and H.-H. Yeh 22. Peart JR, Cook G, Feys BJ, et al. (2002) An EDS1 orthologue is required for N-mediated resistance against tobacco mosaic virus. Plant J 29:569–579. 23. Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP. (2002) Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30:415–429. 24. Sharma PC, Ito A, Shimizu T, et al. (2003) Virus-induced silencing of WIPK and SIPK genes reduces resistance to a bacterial pathogen, but has no effect on the INF1-induced hypersensitive response (HR) in Nicotiana benthamiana. Mol Gen Genomics 269:583–591. 25. Fu DQ, Zhu BZ, Zhu HL, et al. (2005) Virus-induced gene silencing in tomato fruit. Plant J 43:299–308. 26. Lu R, Malcuit I, Moffett P, et al. (2003) High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance. The EMBO J 22:5690–5699. 27. Marathe R, Anandalakshmi R, Smith TH, et al. (2000) RNA viruses as inducers, suppressors and targets of post-transcriptional gene silencing. Plant Mol Biol 43:295–306. 28. Burch-Smith TM, Schiff M, Liu Y, Dinesh-Kumar SP. (2006) Efficient virus induced gene silencing in Arabidopsis thaliana. Plant Physiol 142:21–27. 29. Korotieieva HV, Polishchuk VP. (2004) Viruses of orchids of the natural flora in Ukraine. Mikrobiol Z 66:74–80. 30. Lu HC, Chen HH, Tsai WC, et al. (2006) Strategies for functional validation of genes involved in reproductive stages of orchids. (Accepted) 31. Gorden B. (1998) Phalaenopsis flower induction (or, how to make them bloom). Am Orchid Soc Bull 9:908–910. 32. Tsai WC, Lee PF, Chen HI, et al. (2005) PeMADS6, a GLOBOSA/PISTILLATA-like gene in Phalaenopsis equestris involved in petaloid formation, and correlated with flower longevity and ovary development. Plant Cell Physiol 46:1125–1139. 33. Scortecci KC, Michaels SD, Amasino RM. (2001) Identification of a MADSbox gene, FLOWERING LOCUS M, that represses flowering. Plant J 26:229–236. 34. Yu H, Xu Y, Tan EL, Kumar PP. (2002) AGAMOUS-LIKE 24, a dosagedependent mediator of the flowering signals. Proc Natl Acad Sci USA 99:16336–16341. 35. Zachgo S, Silva Ede A, Motte P, et al. (1995) Functional analysis of the Antirrhinum floral homeotic DEFICIENS gene in vivo and in vitro by using a temperature-sensitive mutant. Development 121:2861–2875. 36. Bowman JL, Smyth DR, Meyerowitz EM. (1991) Genetic interactions among floral homeotic genes of Arabidopsis. Development 112:1–20. 37. Coen ES, Meyerowitz EM. (1991) The war of the whorls: Genetic interactions controlling flower development. Nature 353:31–37. Application of Virus-Induced Gene Silencing Technology ✦ 223 38. Zettler FW, KO NJ, Wisler GC, et al. (1990) Viruses of orchids and their control. Plant Dis 74:621–626. 39. Hu SJ, Ferreira S, Wang M, Xu MQ. (1993) Detection of Cymbidium mosaic virus, Odontoglossum ringspot virus, Tomato spotted wilt virus, and Potyviruses infecting Orchids in Hawaii. Plant Dis 77:464–468. 40. Tsai WC, Hsiao YY, Lee SH, et al. (2006) Expression analysis of the ESTs derived from the flower buds of Phalaenopsis equestris. Plant Sci 170: 426–432. 41. Wang HC, Kuo HC, Chen HH, et al. (2005) KSPF: Using gene sequence patterns and data mining for biological knowledge management. Expert Syst Appl 28:537–545. 42. Hsiao YY, Tsai WC, Kuoh CS, et al. (2006) Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis pathway. BMC Plant Biol. (Accepted) This page intentionally left blank Chapter 13 Genetic Transformation as a Tool for Improvement of Orchids Sanjaya† and Ming-Tsair Chan*,† Orchids are primarily grown for their large, long-lasting, and fascinating flowers; thus, the improvement of quality attributes such as flower color, longevity, shape, architecture, biotic and abiotic stress tolerance, and creation of novel variations are important economic goals for floriculturists across the world. Recent advances in genetic engineering and molecular biology techniques augmented with gene transformation could help growers to meet the demand of the orchid industry in the new century. Presented is an overview of Agrobacterium-mediated and particle bombardment or direct gene transformation in orchids and the essential factors involved in the technology systems. Available methods for the transfer of genes could greatly simplify traditional breeding procedures and overcome some of the inherent genetic problems, which otherwise would not be achievable through conventional methods. Indeed, more recently, orchids have been the subject of new areas of research, including functional genomics, proteomics, and metabolomics. The successful application of these new approaches to improve traits requires a reliable and reproducible transformation technique. The development and remarkable achievements of biotechnology in orchids during the past decade are reviewed, as are potential areas of research for the improvement of orchids. *Corresponding author. † Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan. 225 226 ✦ Sanjaya and M.-T. Chan 13.1 Introduction Orchids are the largest group of flowering plants and comprise approximately 750 genera, more than 25,000 identified species, and 120,000 hybrids. They represent the upper class in floriculture as cut flowers and potted plants worldwide.1,2 According to the 2005 floriculture crops survey of the United States Department of Agriculture, orchid sales increased by approximately 12% over the previous year and currently account for the second highest sales in potted flowering plants (behind poinsettias), with a wholesale value of nearly US$144 million. The demand for orchids is increasing tremendously; in 2005, more than 18 million orchids were sold in the United States alone (http://www. orchidweb.org). In response to ever-increasing consumer demand, the creation of new varieties with unique flower patterns, colors, shapes, fragrance, and resistance against pests and diseases is highly desirable.1 Recently, Taiwan has emerged as a leader in the breeding and export of orchids. According to statistics from the Bureau of Foreign Trade in Taiwan, sales in 2003 accounted for approximately US$20 million. As a token of encouragement, the United States gave Taiwan permission to export Phalaenopsis grown in approved media to US soil in 2004. Currently, approximately 200 hectares of land in Taiwan are devoted to cultivating orchids, with most orchid farms being located in Yunlin, Chiayi, and Tainan counties of southern Taiwan.2 To boost the floriculture industry in Taiwan, the government has invested nearly US$25 million in infrastructure development across the country.3 Traditionally, classical breeding has been used to introduce new traits for creating new hybrids in many orchid species. However, the technique is tedious and selected offspring are maintained throughout vegetative propagation to ensure genetic identity. Genetic improvement in orchids by sexual hybridization is hampered because of long juvenile periods and reproductive cycles. In addition, the available gene pool for new traits in orchids is limited because of the genetic background of parents. Nevertheless, rapid and dynamic progress in molecular biology and biotechnology has allowed for producing new orchid varieties with novel characteristics, such as flower pigmentation as well as disease and pest resistance. Sustaining both supply and demand of orchids in the future will depend more heavily on the development and deployment of new technologies, including genetic engineering.2,4,5 Genetic Transformation as a Tool for Improvement of Orchids ✦ 227 Gene transformation is an essential tool, both for the experimental investigation of gene function and the improvement of orchids, either by enhancing existing traits or introducing new genes.5 Introducing and expressing foreign genes stably into the orchid genome by different means (bacteria/viruses), with Agrobacterium-mediation or microprojectile bombardment, is now possible, and many aspects of plant physiology and biochemistry that cannot be addressed easily by any other experimental means can be investigated by the analysis of gene function and regulation in transgenic orchids. 13.2 Foreign DNA Transfer Methods In the last century, traditional plant breeding techniques have been used to increase many agronomic traits and the commercial value of orchids. The main drawback of traditional plant breeding is that it relies on the use of germplasm of the same or closely related species, which is sometimes a serious limiting factor.5 In addition, progress is time-consuming and relies on the extensive use of natural resources. Gene transformation is an important tool in biotechnology and is based on the introduction of DNA into totipotent plant cells, followed by the regeneration of such cells into whole fertile plants. Early developments arising from transgenic techniques in plants in the mid-1970s produced tomatoes, corn, soybean, and canola with modified traits. Since the 1980s, these techniques have matured, become more target-specific, and generated transgenic plants in most major agricultural crops. Particularly, the last two decades have seen significant developments in plant transformation technologies for the insertion of foreign DNA into plant genomes.6 Among available techniques for gene transformation, the most popular systems are Agrobacterium tumefaciensmediated transformation and microparticle bombardment (biolistics), with which transgenic plants have been successfully established in a variety of plant species such as maize, rice, wheat, and barley. Compared with the growing number of reports of genetically engineered crops in the literature, very few studies have demonstrated engineered plants for orchids. The optimization of efficient DNA delivery, successful regeneration, and suitable selection system are, therefore, prerequisites for most orchid transformation systems. 228 ✦ Sanjaya and M.-T. Chan The three major plant transformation systems routinely employed in orchid transformation — Agrobacterium tumefaciens-mediated transformation, microprojectile bombardment, and direct gene transfer into protoplasts — have been used in the transfer of orchids, but only in a few species compared with agronomic plants.7–11 So, research into improving existing or establishing new transformation techniques for orchids is essential. 13.3 Prerequisites for DNA Transfer into Orchids 13.3.1 Explant type and regeneration capacity A reliable gene transformation system depends on the regeneration capacity of putatively transformed tissues so that various potential genes and their functions can be explored. In most orchids, protocormlike bodies (PLBs) serve as target tissue for gene transformation: as the origin of these PLBs are single somatic cells, they are easy to root, presumed to be genetically uniform, and can be induced efficiently from various somatic tissues including young leaves, stem segments, or flower stalks, regardless of cultivar. Routine gene transformation systems have been developed in Oncidium, Dendrobium, and Phalaenopsis, with PLBs used as starting material.10,12–15 However, Belarmino and Mii7 used cell clumps/aggregates in Phalaenopsis transformation. Yu et al.13 regenerated Dendrobium transgenic plants by using thin-section explants from PLBs, whereas calli or PLBs were used as target explants in Dendrobium phalaenopsis and D. nobile transformation.10 Several media compositions have been standardized for orchid cultivars for successful regeneration of PLBs and subsequent regeneration of putatively transformed plants. However, the nutritional requirement in orchid transformation depends on the species or variety. For example, in Oncidium transformation, a G10 medium supplemented with 4.3 g/L MS salts, 1 g/L tryptone, 20 g/L sucrose, 1 g/L charcoal, 65 g/L potato tubers, and 3 g/L phytagel (pH 5.4) is routinely used.12 T2 medium (3.5 g Hyponex No. 1 [N:P:K 6:6.5:19; Hyponex Co. Ltd., Japan], 1 g tryptone, 0.1 g citric acid, 20 g sucrose, 1 g charcoal, 20 g sweet potato, 25 g banana and 3 g phytagel, pH 5.5) was successful in Phalaenopsis transformation.1,14,15 Growth and morphogenesis in vitro are said to be Genetic Transformation as a Tool for Improvement of Orchids ✦ 229 controlled by the interaction and balance between growth regulators in the medium and growth substances produced endogenously by cultured cells.16 The use of auxin and cytokinin concentration is unresolved in orchid transformation. 13.3.2 Selection marker gene The proportion of totipotent cells that become transformed is low compared with untransformed cells. Most foreign genes introduced into the orchid genome do not confer a phenotype that can be conveniently used for selective propagation of transformed cells. For this reason, a selectable marker gene is introduced at the same time as the nonselectable foreign DNA to enable the survival of transformed cells in the presence of a particular chemical, the selective agent, which is toxic to nontransformed cells. Currently, more than 50 selection systems have been reported, but only a few are frequently used in plant transformation.17,18 Examples are those that used a gene encoding neomycin phosphotransferase II (NPT II), hygromycin phosphotransferase II (HPT II), and phosphinothricin acetyltransferase (bar) to develop the first generation of transgenic crops, regardless of tissue systems,17,19 in order to confer antibiotic resistance with the gene of interest. The most commonly used antibiotics are hygromycin and kanamycin. Resistance to these antibiotics is conferred by insertion and expression of a gene encoding hygromycin phosphotransferase II (HPT II) under the control of Cauliflower mosaic virus (CaMV) 35S promoter. In direct DNA transfer methods, the selectable marker and nonselected transgene(s) may be linked on the same cointegrate vector or may be introduced on separate vectors (cotransformation). Both strategies are suitable because exogenous DNA, whether homogeneous or a mixture of different plasmids, predominantly integrates at a single locus. Experiments in our laboratory involving the pCAMBIA (Center for the Application of Molecular Biology of International Agriculture, Black Mountain, Australia) binary vector series showed the most consistent results in Oncidium and Phalaenopsis transformation.8,14,15 Screenable markers or reporter genes are widely used, particularly when transformation procedures are being optimized. The most commonly used gene in higher plants is gusA, which encodes β -glucuronidase (GUS) (Tables 13.1 and 13.2). Expression and activity of this enzyme are easily Agrobacterium-Mediated Transformation in Orchids Species/Variety Target Tissue Agrobacterium Selection Reporter/ Transformation Efficiency Strain Marker Gene Foreign Gene and Remarks Dendrobium (UH800, UH44 and Thai hybrid M61) Phalaenopsis (True Lady A76-13, Brother Mirage A79-69, Asian Elegance B79-11 and Taisuco Kaaladian F80-13) Phalaenopsis (Doritaenopsis Coral Fantasy X Phalaenopsis) (Baby Hat X Ann Jessica) PLBs LBA4301 vir Transversely bisected PLBs EHA105 (pMT1) NPT II Intron-GUS Cell clumps LBA4404 (pTOK233) and EHA101 (pIG121Hm) NPT II and HPT II Intron-Gus PLBs LBA4404 NPT II Orchid DOH1 antisense gene Dendrobium (Dendrobium Somasak X Dendrobium Suzie Wong) Reference No. Presence of coniferyl alcohol in PLBs acts as vir gene inducer 100% GUS gene expression in Asian Elegance B79-11 and Taisuco Kaaladian F80-13, 50–80% in True Lady A76-13 and lowest in Brother Mirage A79-69 22 More than 100 hygromycinresistant plantlets produced and transformation was confirmed by histochemical GUS assay, PCR and Southern hybridization analysis Expression of DOH1 antisense gene caused abnormal shoot development and presence of transgene in transgenic plants confirmed by genomic PCR and Southern hybridization 7 23 8 (Continued ) 230 ✦ Sanjaya and M.-T. Chan Table 13.1. Table 13.1. (Continued ) Agrobacterium Selection Reporter/ Transformation Efficiency Strain Marker Gene Foreign Gene and Remarks Target Tissue Phalaenopsis (T0, T5, T10 and Hikaru) Intact and transversely bisected PLBs LBA4404 NPT II Intron-GUS Dendrobium nobile PLBs AGL1 and EHA105 HPT II Intron-GUS Oncidium (Sherry Baby cultivar OM8) PLBs EHA105 and LBA4404 HPT II Intron-GUS Transgenic plants derived from T0 and Hikaru showed 70% GUS expression and 10% of transformation efficiency was observed in Hikaru PLBs 18% transformation efficiency obtained with 73 stably transformed lines and transgenes confirmed by Southern blot and GUS histochemical assay Among 1000 inoculated PLBs, 108 putatively transformed PLBs were proliferated on hygromycin selection medium (10%). A total of 28 independent transgenic orchid plants were obtained, from which six transgenic lines were confirmed by Southern, Northern, and Western blot analyses Reference No. 24 9 12 (Continued ) Genetic Transformation as a Tool for Improvement of Orchids ✦ 231 Species/Variety (Continued ) Species/Variety Target Tissue Agrobacterium Selection Reporter/ Transformation Efficiency Strain Marker Gene Foreign Gene and Remarks Oncidium (Sherry Baby cultivar OM8) PLBs EHA105 HPT II Oncidium (Sherry Baby cultivar OM8) PLBs EHA105 HPT II pflp, GFP and Demonstrated pflp as selection Intron GUS marker gene and Erwinia carotovora as selection agent. Out of 32 independent transgenic lines, 9 were randomly selected and confirmed by Southern and Northern blot analyses pflp, GFP and Among 17 independent Intron GUS transgenic orchid lines, 6 (GUS) positive were randomly selected and confirmed by Southern, Northern, and Western blot analyses. Transgenic plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen Reference No. 25 26 (Continued ) 232 ✦ Sanjaya and M.-T. Chan Table 13.1. Table 13.1. (Continued ) Agrobacterium Selection Reporter/ Transformation Efficiency Strain Marker Gene Foreign Gene and Remarks Target Tissue Phalaenopsis (TS97K) PLBs EHA105 transformed with CymMV cDNA (gene stacking) HPT II Phalaenopsis (S122-2 × S153 and S153 × S119-4) Protocorms from germinating seeds EHA101 (pIG121Hm) HPT II Phalaenopsis (Wataboushi ‘#6.13) Embryonic cell suspension culture EHA101 (pEKH-WT) NPT II and HPT II pflp, GFP and Transgene integration Intron GUS confirmed by Southern and Northern blot analysis for both CP and pflp genes. Transgenic lines exhibited enhanced dual disease resistance to CymMV and E. carotovora Intron-GUS A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca.12,500 mature seeds 6 months after infection with Agrobacterium and integration of HPT II gene confirmed by Southern blot analysis Rice wasabi Integration and expression of defensine defensin gene was confirmed by PCR, Southern, and Western blot analyses. Most transgenic plants showed strong resistance to E. carotovora Reference No. 15 11 27 Genetic Transformation as a Tool for Improvement of Orchids ✦ 233 Species/Variety Particle Bombardment/Direct Gene Transformation in Orchids Selection Marker Gene Reporter/ Foreign Gene Species/Variety Target Tissue Dendrobium (Dendrobium × Jaquelyn Thomas hybrids) Dendrobium (White angel) Protocoms NPT II Calli cultured in liquid medium NPT II Phalaenopsis (Danse × Happy Valentine) PLBs NPT II and bar Intron-GUS Dendrobium (hybrid ‘MiHua’) Protocorms HPT II Intron-GUS Cymbidium PLBs NPT II Intreon-GUS Papaya ringspot virus (PRV) coat protein (CP) gene Luciferase Transformation Efficiency and Remarks Obtained 13 kanamycin resistant plants and confirmed foreign gene (NPT II) by genomic PCR and Southern blot analyses Transformed tissues expressing luciferase were detected, allowed to regenerated into complete plant on selection medium and integration of transgene confirmed by Southern and Northern blot analyses About 7 bialophos-resistant plantlets were obtained from 622 bombarded PLBs and only one showed GUS expression 15 hygromycin-resistant lines recovered on selection medium and integration of foreign genes was confirmed by GUS histochemical assay and Southern blot analysis About 85% of GUS positive shoots were obtained and further transgenic nature confirmed by genomic PCR and Southern blot analyses of the PCR product Reference No. 37 29 39 13 40 (Continued) 234 ✦ Sanjaya and M.-T. Chan Table 13.2. Table 13.2. Selection Marker Gene (Continued ) Reporter/ Foreign Gene Target Tissue Brassia, Cattleya and Doritaenopsis PLBs bar Dendrobium phalaenopsis Banyan Pink and D. nobile Calli HPT II Intron-GUS Phalaenopsis TS444 [(New Eagle × Pinlong Cinderella) × Dtps. Taisuco Red] Petels — Flavonoid-3′,5′hydroxylase gene from Phalaenopsis Phalaenopsis “TS340” (P. Taisuco Kochdiam × P. Taisuco Kaaladian) PLBs HPT II CymMV coat protein (CP) cDNA Transformation Efficiency and Remarks Selection of putative transformants was accomplished by using bialaphos. The presence of bar gene was confirmed by PCR, Southern, and Northern blot analyses About 12% (D. Phalaenopsis) and 2% (D. nobile) transformation efficiency was achieved and integration of foreign gene confirmed by Southern and Northern blot analyses Transient transformation achieved by particle bombardment and the transgenic petals changed from pink to magenta, demonstrated the role of flavonoid-3′,5′-hydroxylase gene in anthocyanin pigment synthesis Among 13 transgenic plants confirmed by Southern blot analysis, most transgenic lines showed CymMV protection. Nuclear run-on and small interfering RNA (siRNAs) analyses showed that CymMV resistance was RNAmediated through a posttranscriptional gene silencing mechanism (PTGS) in the silenced transgenic orchid plants. Reference No. 20 10 43 14 (Continued ) Genetic Transformation as a Tool for Improvement of Orchids ✦ 235 Species/Variety Selection Marker Gene (Continued ) Reporter/ Foreign Gene Species/Variety Target Tissue Dendrobium (Hickam Deb) Protocorms HPT II CymMV coat protein (CP) cDNA Phalaenopsis (TS97K) PLBs HPT II CymMV coat protein (CP) cDNA Oncidium (Sharry Baby “OM8”) PLBs (pretreated with sucrose) HPT II pflp Transformation Efficiency and Remarks Presence of the transgene confirmed by PCR, Southern, Northern, and Western blot analyses. Transgenic plants harboring the CymMV CP gene expressed a very low level of virus accumulation 4 months postinoculation with CymMV PLBs transformed with CymMV coat protein cDNA (CP) were then retransformed with sweet pepper ferredoxin-like protein cDNA ( pflp) by Agrobacterium tumefaciens, to enable expression of dual (viral and bacterial) disease resistant traits. Double transformants confirmed by molecular analysis and exhibited enhanced dual disease resistance Sucrose pretreatment enhanced the regeneration of PLBs, single-cell embryogenesis, and transformation efficiency Reference No. 42 14 41 236 ✦ Sanjaya and M.-T. Chan Table 13.2. Genetic Transformation as a Tool for Improvement of Orchids ✦ 237 detectable by use of both histochemical and fluorigenic substrates. This reporter gene was successfully used in the transformation of different orchids.10,11 In some cases, the herbicide resistance (bar) gene has also been useful in the selection of transgenic orchids.20 13.4 General Protocol for Agrobacterium-Mediated Transformation in Orchids The landmark discovery of A. tumefaciens21 provided a natural gene transfer mechanism to introduce and express DNA stably in different plant species, regardless of their origin. Several hybrid orchids, which are difficult for A. tumefaciens infection, have been transformed by direct gene gun or microparticle bombardment. However, gene transfer techniques have been established only in a few orchids.7,10,11,13 In general, Agrobacterium-mediated transformation in orchids was achieved by cocultivating a virulent A. tumefaciens strain containing a recombinant Ti plasmid with PLBs/cell clumps, from which complete plants were regenerated. Although Agrobacterium-mediated transformation needs to be optimized for different species, the Agrobacterium infection can be promoted under conditions that induce virulence, such as use of acetosyringone (AS) or a-hydroxyacetosyringone, acidic pH, and appropriate incubation temperature. After coculture for 2 to 3 days, PLBs are transferred to a medium containing selective agents to eliminate nontransformed plant cells, antibiotics to kill the Agrobacterium, and hormones to induce shoot and root growth. After a few months, shoots develop from transformed PLBs. These can be removed and transferred to a rooting medium, and then shifted to soil. Most current protocols for the Agrobacterium-mediated transformation of orchids involve variations in the starting material. In Phalaenopsis, using PLBs as target tissue, we have developed a reliable and reproducible transformation protocol (Fig. 13.1). 13.4.1 Recent advances in Agrobacterium-mediated transformation Several plant phenolic compounds, including AS, coumaryl alcohol, and sinapyl alcohol, are known as vir gene inducers; but in orchids, their 238 ✦ Sanjaya and M.-T. Chan Regenerate of PLBs from leaf segments Transfer PLBs (45-day-old) to fresh T2 medium supplemented with 200 µM AS and continue in dark for 1 h Infect with Agrobacterium for 1 h in dark Cocultivate blot-dried PLBs on T2 solid medium supplemented with 200 µM AS and 5% glucose at 26˚C in dark for 3 days Wash infected PLBs in MS liquid medium supplemented with 200 mg/L timentin Transfer blot-dried PLBs on to T2 medium supplemented with 200 mg/L timetin, incubate at 26˚C with 16 h light/8 h dark photoperiod for 4 weeks at a light intensity of 100 to 200 µE/m2/s. Separate newly differentiated PLBs from the original explant, subculture on T2 medium supplemented with 200 mg/L timentin and the optimal concentration of antibiotics for selection of putative transformants Transfer actively growing PLBs on T2 medium supplemented with the optimal concentration of antibiotics for 2nd selection to establish transgenic lines After 6–8 weeks, transfer survival and actively growing putatively transformed PLBs on fresh T2 medium for shoot elongation Transfer to pots containing sphagnum moss and acclimatize under greenhouse conditions Transfer well-rooted plants for hardening and carry out molecular analysis Fig. 13.1. Different stages of A. tumefaciens-based transformation in Phalaenopsis. Genetic Transformation as a Tool for Improvement of Orchids ✦ 239 presence and characteristics were not well understood until Nan et al.22 identified these compounds in high amounts in light-grown Dendrobium PLBs. The authors demonstrated activation of the Agrobacterium vir gene in the presence of these phenolic compounds, and revealed the possibility of Agrobacterium-mediated transformation in orchids. These observations come as a great boon to orchid biologists to initiate gene transformation studies in various commercial varieties. The feasibility of using Agrobacterium-mediated transformation system in orchid transformation was not explored until Hsieh et al.23 first reported the transformation of different varieties of Phalaenopsis with NPT II and GUS genes using PLBs as target tissue (Table 13.1). Subsequently, other authors reported success with Agrobacteriummediated transformation in orchids. Belarmino and Mii7 reported Phalaenopsis transformation with a GUS gene and confirmed the transgenic nature of developed plants by histochemical GUS assay, PCR analysis, and Southern hybridization. Use of this transformation system successfully produced more than 100 hygromycinresistant plantlets within 7 months following infection of cell aggregates. Dendrobium was transformed with DOH1 by use of thin-section explants from PLBs.13 Chai et al.24 evaluated two types of explants, intact and transversely bisected PLBs, in Phalaenopsis transformation, and observed that transversely bisected PLBs responded better than intact ones in terms of efficiency of multiplication and transformation. A high efficiency of transformation (18%) in Dendrobium was reported, with 73 stably transformed lines accomplished in the presence of 100 µ M AS and hygromycin as the selection marker.10 Liau et al.12 first reported a transformation method suitable for improving Oncidium by using PLBs derived from protocorms as target tissue. Among 1000 inoculated PLBs, 108 putatively transformed PLBs were proliferated on hygromycin selection medium. A total of 28 independent transgenic orchid plants were obtained, from which 6 transgenic GUS-positive lines were randomly selected and confirmed by Southern, Northern, and Western blot analyses. This successfully demonstrated the integration and expression of foreign DNA in the Oncidium genome. Soonafter, the same group transformed sweet pepper ferredoxin-like protein (pflp) cDNA, with a HPT II and GUS coding sequence, into PLBs of Oncidium, and demonstrated the applicability of the pflp gene as an antibiotic-free selection marker in Oncidium25; intriguingly, transgenic plants showed 240 ✦ Sanjaya and M.-T. Chan enhanced resistance to Erwinia carotovora, which causes soft rot disease, even when the entire plant was challenged with the pathogen.26 Recently, Chan et al.15 used “gene stacking” in Phalaenopsis by double transformation events. Phalaenopsis PLBs originally transformed with CymMV coat protein cDNA (CP) were then transformed with pflp cDNA by A. tumefaciens infection to enable the expression of dual (viral and bacterial) disease-resistant traits. The immature protocorms developed from Phalaenopsis seeds were also demonstrated to be a potential starting source for Agrobacterium-mediated transformation.11 However, this protocol may not have broader application because most of the commercial hybrids are sterile in nature. Interestingly, 5 day-old embryogenic suspension cells of Phalaenopsis were used as starting material in rice wasabi defensin gene transformation, and the authors claimed that most of the transgenic plants exhibited enhanced resistance to E. carotovora.27 13.5 General Conditions for A. TumefaciensMediated Transformation 13.5.1 Effect of feeder cells/acetosyringone on transformation efficiency Acetosyringone (AS), a phenolic compound produced in metabolically active plant cells or released by wounded cells, induces the expression of the vir genes on the Ti plasmid of A. tumefaciens, which in turn facilitates T-DNA transfer into plant cells.28 Although orchid cells are capable of producing certain phenolic compounds at a low level, such production is not sufficient for high and consistent transformation events.22 The feasibility of AS in orchid transformation was demonstrated by Hsieh et al.23 in Phalaenopsis transformation; the authors added 100 µ M AS and regeneration medium to the Agrobacterium culture prior to infection to enhance transformation events. In concordance with the above observations, Chia et al.29 and Mishiba et al.11 used 100 µ M AS during the infection and cocultivation stages in Phalaenopsis transformation. In Dendrobium, Men et al.10 observed higher GUS gene expression when PLBs were cocultivated with 100 µ M AS-activated A. tumefaciens for 2 to 3 days. Interestingly, Chan et al.15 reported the Genetic Transformation as a Tool for Improvement of Orchids ✦ 241 use of 200 µ M AS and 5% glucose during cocultivation and found enhanced transformation efficiency. Belarmino and Mii7 infected explants with 200 µ M AS-treated Agrobacterium and included 500 µ M AS in cocultivation medium. In Oncidium transformation, the use of 1 mL of tightly packed 3-day-old freshly subcultured tobacco suspension cells on G10 medium supplemented with 200 µ M AS as a cocultivation medium and dark incubation for 3 days at 26°C significantly triggered the A. tumefaciens vir genes, which in turn resulted in high transformation efficiency. Furthermore, these results were consistent and reproducible.12 However, in the absence of AS, only a few transgenic plants were obtained. The use of feeder cells from species such as tobacco petunia, and tomato as a phenolic source to enhance the transformation rate still needs to be optimized for various commercial orchids. 13.5.2 Agrobacterium strain and density The choice of Agrobacterium strain greatly influences the transformation success in orchids. A wide range of A. tumefaciens strains used include LBA4301,22 EHA105 (pMT1),23 LBA4404 (pTOK233),8,24 EHA101 (pIG121Hm),11 and EHA101 (pEKH-WT).27 However, others have advocated that the success of transformation efficiency is not influenced by the Agrobacterium strain alone.7,30 In Dendrobium, Men et al.10 examined two Agrobacterium strains, AGL1 and EHA105, and observed a high level of GUS staining in AGL1-infected PLBs. Simultaneously, Liau et al.26 using EHA105 and LBA4404, found no difference in transformation efficiency. However, the shoot formation rate of EHA105-transformed PLBs was higher than that of LBA4404treated PLBs. In addition, after hygromycin selection, the antibiotics used (timentin/cefatoxime) did not always control the overgrowth of LBA4404 and resulted in a low rate of shoot formation. Therefore, in general, the tissue culture conditions and choice of bacteriostatic substances employed to suppress the overgrowth of Agrobacterium are important factors in orchid transformation. Although the bacterial density was not thoroughly examined in most of the transformation studies, most authors used diluted Agrobacterium at an A600 of 0.2–1.0 for improved transformation efficiency. Thus, Agrobacterium density is also a key factor for successful transformation.11,23–26 242 ✦ Sanjaya and M.-T. Chan 13.5.3 Effect of selection agents In general, Agrobacterium-mediated transformation in orchids is preceded by infection, 2 to 3 days of cocultivation, and selection of putative transformants on selection medium supplemented with antibiotics and bacteriostatic substances to cull untransformed tissues and A. tumefaciens. The ideal antibiotic for inhibiting Agrobacterium growth should be highly effective, inexpensive, not have a negative effect on plant growth and regeneration, and be stable in culture.31 The selective effects of different antibiotics in plant transformation have been reported for several species.32 In orchid transformation, most authors used different concentrations of cefotaxime/carbenicillin as antibiotics for suppressing Agrobacterium growth after cocultivation.7–9,23,24,27 Mishiba et al.11 used meropenem ( β -lactam antibiotics) to remove excess Agrobacterium from PLBs. In our lab, we standardized a two-step transformation procedure in Oncidium: after infection, PLBs were cocultivated for 3 days; then, infected PLBs were transferred to a medium supplemented with low concentrations of timentin/cefatoxime to suppress A. tumefaciens growth and incubated at 26°C for a 16-h photoperiod at a light intensity of 100–200 µ Em−2s−1 for a month. No selective antibiotic had been incorporated in the medium to select putatively transformed PLBs because the long-term transformation process might enhance transfer of foreign DNA from A. tumefaciens into actively growing PLBs. Subsequently, A. tumefaciens was completely avoided with high concentrations of timentin/cefatoxime and a selective agent was used to eliminate untransformed PLBs. We achieved approximately 10% transformation efficiency, with 108 antibiotic-resistant independent PLBs proliferated from 1000 infected PLBs.12 13.6 Microprojectile/Particle Bombardment or Direct Gene Transformation A novel method for plant transformation was introduced by John Sanford and colleagues in 1987. The authors showed that projectile bombardment, with several modifications, was an efficient method for stable integration of foreign genes into the plant genome.33 Subsequently, a gunpowder-based device was refined for a system based Genetic Transformation as a Tool for Improvement of Orchids ✦ 243 on high-pressure blasts of helium;34 this is the only commercially available particle gun, which is marketed by Bio-Rad. Particle bombardment is a simple technique: a plasmid DNA is prepared by standard methods and precipitated onto tungsten or gold particles with use of CaCl2, spermidine, and PEG to protect the DNA during precipitation; and then the particles are washed and suspended in ethanol before being fired against a retaining screen that allows the microprojectiles through to strike the target tissue. Particle bombardment is widely used because it circumvents two major limitations of the Agrobacterium system. First, any hybrid orchid species and cell type can be transformed by this method because DNA delivery is controlled entirely by physical rather than biological parameters. The range of species transformable by particle bombardment is, therefore, restricted only by the competence of cells for regeneration. The technique is independent of genotype and, consequently, useful for the transformation of elite cultivars. However, careful optimization is required to tailor the method for different varieties or cell types and to achieve the highest efficiency transformation with the least cell damage. Important parameters include acceleration method, particle velocity (controlled by the discharge voltage and/or gas pressure), particle size, and use of different materials (tungsten, gold).35 Second, particle bombardment allows the stable and heritable introduction of many different genes at once with the use of different plasmids, as these tend to concatemerize to form one DNA cluster that integrates at a single locus. Remarkably, the transgene silencing, instability, and rearrangements are more evident with particle bombardment than the Agrobacterium method of gene transformation.36 13.6.1 Recent advances in particle bombardment in orchids Pioneering gene transformation experiments augmented with particle bombardment were successfully used to produce transgenic orchids with a marker/reporter gene in Dendrobium,10,13,28,37 Phalaenopsis,39 Cymbidium,40 Brassia, Cattleya, and Doritaenopsis20 (Table 13.2). Osmotic pretreatment of Oncidium PLBs on high sucrose medium before bombardment and transformation have enhanced single-cell embryogenesis and transformation efficiency.41 This situation can also be achieved by partial drying or the addition of osmoticum (mannitol 244 ✦ Sanjaya and M.-T. Chan Table 13.3. Other Direct Gene Transformation Methods in Orchids Species/Variety Target Tissue Dendrobium Seeds Dendrobium Dendrobium Phalaenopsis Ovary Protocorms Ovary Transformation Method Imbibition in solution containing foreign DNA Pollen-tube pathway Electro injection Pollen-tube pathway Reference No. 44 44 44 38 and/or sorbitol) to the culture medium. In general, stable transformation by any direct DNA transfer method occurs at a much lower frequency than transient transformation. The direct gene transformation has also been successfully used in developing transgenic orchids resistant to Cymbidium mosaic virus in Phalaenopsis14,15 and Dendrobium.42 Interestingly, Chan et al.15 demonstrated the usefulness of direct gene transformation in introducing multiple genes into the Phalaenopsis genome. Su and Hsu43 reported transient transformation of Phalaenopsis petals with the putative cytochrome P450 gene. In addition, other direct gene transformation methods, including seed imbibition and pollen tube mediation, have been reported in Dendrobium and Phalaenopsis38,44 (Table 13.3). However, these methods are not in frequent use because of low transformation efficiency. 13.7 Selection of Putative Transformants and Molecular Analysis Selection of the transformed cells and subsequent regeneration of plants is the most important requirement in orchid transformation. The most commonly used antibiotics for selection of transformed orchid PLBs are hygromycin and kanamycin. Resistance to these antibiotics is conferred by insertion and expression of HPT II or NPT II under the control of a CaMV35S promoter.7,12,13,23,24,37 Knapp et al.20 demonstrated the usefulness of bar as a selection marker in Brassia, Cattleya, and Doritaenopsis transformation. Our laboratory experiments involving Genetic Transformation as a Tool for Improvement of Orchids ✦ 245 pCAMBIA binary vector series showed the most consistent results in Phalaenopsis and Oncidium transformation. The screenable markers or reporter genes are widely used, particularly when transformation procedures are being optimized. The most commonly used reporter gene in higher plants is GUS; the expression and activity of this enzyme are easily detectable by the use of both histochemical and fluorigenic substrates. This reporter gene was used successfully in the transformation of other orchids, including Cymbidium,37 Dendrobium,10 and Phalaenopsis.11 Furthermore, the available molecular tools such as PCR, Northern, Southern, and Western blot analyses have allowed for confirming transgene integration and expression and have clarified the outcome of such transformation experiments. Because orchids have a long flowering stage, the molecular analysis of T1 progeny is time-consuming. Alternatively, T0 transgenic lines can be thoroughly diagnosed for foreign gene integration by Southern, Northern, and Western blot analyses.12 Southern and Northern blot analysis was used to confirm the transformation, copy number, and transcriptional activity of transgenes in putatively transformed orchids. 13.8 Expression of Genes with Potential Commercial Application The main drawback of traditional plant breeding is that it relies on the use of germplasm of the same or closely related species, and the progress is time-consuming. The 21st century will likely pose a new set of challenges for orchid growers, as increased demand for cut flowers and potted plants will require quick production practices. Sustaining both supply and demand in the future will depend more heavily on the development and deployment of a range of new technologies, including biotechnology. 13.8.1 Marker-free transgenic orchids To obtain maximum putatively transformed plants in each transformation event, effective selectable markers, antibiotics, or herbicideresistant genes have been used to simplify detection procedures. The existing selection systems can be divided into two groups: conventional 246 ✦ Sanjaya and M.-T. Chan selection systems, the largest group, which rely on an antibiotic or a herbicide as the selective agent detoxified by a selective gene;45 and positive selection systems, in which the selective agent is converted into a simple compound by the selective gene product and transformed cells have metabolic or developmental advantages.46,47 In most of the orchid transformation protocols, the available methods for screening transgenic plants are based on antibiotics or herbicide-resistant marker genes such as NPT II, HPT II, and bar on media containing the selective agent.7,8,20 However, the use of these selection marker genes is a matter of concern because of their possible toxicity or allergenicity to humans and other organisms. In addition, the presence of such resistance genes and their proteins is undesirable in transgenics. Furthermore, these selection systems are time-consuming, expensive, and labor-intensive, therefore, the development of alternative or new selection marker genes and improved selection procedures is essential for the orchid industry. Sweet pepper ferredoxin-like protein ( pflp), isolated from pepper, was successfully cloned and reported to have antimicrobial activity against Pseudomonas syringae by delaying the hypersensitive response through the release of a hairpin proteinaceous elicitor.48 We developed a novel selection procedure for orchid transformation by introducing pflp as a selection marker into the Oncidium genome via A. tumefaciens infection and particle bombardment, and putatively transformed plants were screened by the use of the natural bacterial pathogen E. carotovora as a selection agent (Fig. 13.2). We were able to obtain approximately 32 independent transgenic lines without the use of an antibiotic selection agent.25 Although the selection efficiency with pflp or hygromycin does not differ much, the time required for selecting transgenic plants by use of pflp is shorter. Therefore, to minimize the chances for somaclonal variation caused by long-term tissue culture selection procedures and to enhance the rooting ability of transgenic plants, always a serious problem in the antibiotic selection system, the use of pflp is preferable. Furthermore, the pflp system can be used to introduce economically important genes into any other orchid. Recently, Chan et al.15 successfully demonstrated the practicality of the pflp system as a nonantibiotic selection system in Phalaenopsis transformation. Plants that are not natural hosts of E. carotovora, used as transformation materials, do not conform to the pflp selection system. However, the deployment of this system may substantially decrease concerns about the negative effect on Genetic Transformation as a Tool for Improvement of Orchids ✦ 247 Fig. 13.2. Schematic representation of the development of marker-free transgenic orchids with pflp used as a marker gene and Erwinia carotovora (ECC) as a selection agent. biodiversity with the use of antibiotics or herbicide-resistance marker genes and their presence in transgenic plants. 13.8.2 Disease-resistant genes The efficient control of phytopathogens affecting orchid crops represents one of the major challenges in the floriculture industry worldwide. Several orchid species are highly susceptible to different pathogens, among them the devastating disease soft rot caused by E. carotovora, and this genus is considered to be the natural host. Advances in technology have allowed for introducing a novel gene(s) singly or in combination into the orchid genome for the dual purposes of transformation and disease resistance. For example, the overexpression of pflp in rice confers resistance against Xanthomonas oryzae.49 We introduced an 248 ✦ Sanjaya and M.-T. Chan antimicrobial gene ( pflp) into the Oncidium genome via A. tumefaciens infection. Application of bacteria on the leaves of transgenic and wildtype plants showed characteristic symptoms of water-soak regions followed by maceration, more evident in wild-type plants than in transgenic plants; resistance scoring at the whole plant level also revealed that pflp confers enhanced resistance to E. carotovora.26 Viral diseases in orchids, especially those caused by Odontoglossum ringspot tobamovirus (ORSV) and Cymbidium mosaic virus (CymMV), are a major concern among breeders because of their catastrophic effects on plant yield and market value. Liao et al.14 successfully created transgenic Phalaenopsis orchid plants transformed with CymMV coat protein cDNA (CP), which conferred protection against CymMV infection. To enhance the resistance of orchids to both viral and bacterial phytopathogens, Chan et al.15 used the “gene-stacking” phenomena in Phalaenopsis by double transformation events: Phalaenopsis PLBs, originally transformed with CP, were then transformed with pflp cDNA by the use of A. tumefaciens to enable the expression of dual (viral and bacterial) disease-resistant traits. Disease-resistant assays revealed transgenic plants with enhanced resistance to CymMV and E. carotovora infection. Recently, Chang et al.39 created transgenic Dendrobium plants harboring the CymMV CP gene, which expressed a very low level of virus accumulation 4 months after inoculation with CymMV. Large numbers of toxic compounds exist in higher plants to ensure various physical and biochemical defense barriers against pests and pathogens. Among these, defensin proteins form a well-defined group of low-molecular-weight cystein-rich polypeptides whose toxic effect on fungi and bacteria have been studied in model systems and are known to be expressed either by pathogen attack or elicitors. Recently, Sjahril et al.27 successfully introduced the wasabi defensin gene into Phalaenopsis and recorded enhanced tolerance to E. carotovora. Similar strategies can be extended to other commercially important orchids for single- or multiple-disease resistance. 13.9 Conclusions and Future Prospects As more genes are being isolated and understood in model systems and crop plants, more options are becoming available for the application of genetic modification in orchid improvement. However, such advances have not been fully realized in orchids, probably because most hybrid Genetic Transformation as a Tool for Improvement of Orchids ✦ 249 orchids are recalcitrant to tissue culture and transformation. So, research to increase transformation efficiency, develop new marker genes, introduce new genes alone or in combination by multigene transformation (gene stacking), and improve cryopreservation techniques would be extremely useful for orchid improvement. Recent studies of the entire chloroplast genome sequencing (cpDNA) of Phalaenopsis are novel examples of the successful utilization of cutting-edge technology in orchids, and the information generated can be successfully utilized in chloroplast transformation in Phalaenopsis and other orchids.50 Functional genomics studies of Oncidium and Dendrobium flower formation and initiation are promising and useful in terms of industrial as well as academic interests.51–53 In addition, the information generated by Wu et al.54 on Dendrobium MYB genes during flower development could be useful in exploring new genes involved in early/late flower development, subsequent functional analysis, and innovation of novel flower-specific promoters that will certainly benefit orchid improvement. Pathway engineering, involving the simultaneous introduction and concerted expression of multiple transgenes, will be featured prominently in future strategies for orchid transformation. Finally, the outputs from current genomics, proteomics, and metabolomics programs can be fully exploited with sophisticated methods to introduce large pieces of DNA into the orchid genome. Acknowledgments Updates are not intended to be comprehensive reviews, and the authors apologize to those colleagues whose works could not be cited because of the restricted number of references. The authors thank S.J. Yu, I.C. Pan, and C.H. Liau for graphics. This work was supported by grants from Academia Sinica as well as the National Science and Technology Program for Agricultural Biotechnology of the Republic of China. References 1. Chan MT, Chan YL, Sanjaya. (2006) Orchids (Cymbidium spp., Oncidium and Phalaenopsis). In: Kang Wang (ed.), Methods in Molecular Biology, Vol. 344: Agrobacterium Protocols. 2/e, Vol. II, Humana Press Inc., Totowa, USA, pp. 331–337. 250 ✦ Sanjaya and M.-T. Chan 2. Chan MT, Sanjaya, Teixeira da Silva JA. (2006) Oncidium tissue culture, transgenics and biotechnology. 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Plant Mol Biol 51:959–972. This page intentionally left blank Index coat protein (CP) 234, 240, 248 codon usage 133, 137 constitutive heterochromatin 115, 118, 119, 126 cross-incompatibility 17 cuticular material 27, 34, 35, 36 Cymbidium 18, 34, 35, 38, 41, 45–50, 57, 70, 145, 153, 189, 191, 211, 214, 220, 234, 243–245, 248 Cymbidium mosaic virus 57, 70, 145, 153, 211, 214, 244, 248 ABCDE model 163, 165 acetosyringone 237, 240 A-class genes 168, 174 AFLP 69, 70 agrobacterium-mediated transformation 230, 237, 239, 240, 242 Anoectochilus 77, 79, 81, 83–85, 91, 94 B-class genes 174, 176, 177 bioinformatics 149, 150, 179, 220 biotechnology 14, 20, 45, 59, 60, 131, 147, 148, 225–227, 245, 249 breeding 1–4, 7, 9–14, 17, 19, 20, 46, 49, 59, 66, 130, 141, 225–227, 245 DAF 1, 17, 18, 20 D-class genes 165, 172, 177 Dendrobium 18, 45, 54–57, 77, 79, 81, 89–91, 93, 94, 153, 168–171, 174, 175, 178, 194, 220, 228, 230, 231, 234–236, 239–241, 243–245, 248, 249 developmental biology 211, 212 disease resistance 3, 66, 95, 200, 233, 236, 247, 248 DNA methylation 65, 66, 70, 72 Doritaenopsis 52, 54, 65, 67–70, 78, 85, 86, 88, 94, 230, 235, 243, 244 dsRNA 149, 212, 213 carbohydrate metabolism 185, 194, 196, 206 C-class genes 177, 178 cDNA 65, 69, 70, 145–149, 151–153, 156, 186, 192, 206, 213, 215, 233, 235, 236, 239, 240, 248 cDNA library 145, 152, 153, 206 cDNA suppression subtractive hybridization 65, 70 cDNA-RAPD 65, 69 centromere 117, 121, 124, 146 chloroplast 14, 15, 27, 129–134, 136–141, 146, 200, 249 chloroplast DNA 14, 15, 131, 133 chloroplast DNA extraction 131 chloroplast genome structure 129–134, 136–141, 249 E-class genes 172, 178 embryo proper 23, 25–33, 35, 37–39, 42 embryology 25, 34, 40 endoreduplication 69, 99, 100, 107, 108, 110 Epidendroideae 168, 177 255 256 ✦ Index Erwinia carotovora 240, 247 expressed sequence tag (EST) 65, 70, 145–156, 185–207 fertility 12, 13 Feulgen staining 121 floral color 11–13 floral morphogenetic networks 163 floral morphology 49, 147, 163 floral ontogeny 166–168, 179 floral organ identity genes 165, 168 floral-identity gene 211, 214 flow cytometry 15, 69, 99–107, 150 flowering 2, 10, 23, 37–39, 45, 46, 48–50, 52, 60, 66, 67, 69, 78, 79, 86, 88, 94, 95, 107, 130, 147, 154, 156, 168, 174, 185, 186, 206, 207, 211, 216, 226, 245 fluorescence in situ hybridization 116, 125 full-length EST database 148, 149 functional genomics 130, 149, 225, 249 gene annotation 131, 189, 190, 194, 196–205 gene contents 134 gene functional analysis 212, 214 gene map 14, 132 gene silencing 211, 213–217, 235, 243 gene stacking 240, 248, 249 genetic engineering 141, 225, 226 genome organization 116, 121, 127 genome relationship 121, 122, 124–127 genome size 67, 99, 100, 101, 104, 106, 115, 119, 122, 179, 211, 212, 220 genomic in situ hybridization 116, 123 genomic Southern hybridization 116, 126, 127 genomics 130, 146, 148–150, 179, 220, 225, 249 growth enhancement 93 gynostemium 109, 147, 166, 167, 177, 178 Haemaria 94 Harlequin 77, 79, 81, 83–85, 89, 91, 7–9, 19 in vitro flowering 45, 46, 48–50, 60 inflorescence 10, 11, 49, 68, 108, 109, 167, 168, 174, 178, 185–187, 192, 194, 195, 205–207 integument tissues 24, 27, 28, 31, 34–36, 38 interspecific hybrid 15, 99, 116, 121–123 inverted repeat region (IR) 130, 131, 133, 136, 138, 139, 140, 146, 155, 156, 216, 217 inverted-repeat 213 IR contraction 138, 140 IR expansion 138, 140 karyotypes 126 99, 115, 116, 117, 121, labellum 67–69, 72, 109, 147, 166, 169–171, 174–177 large single copy region (LSC) 129–131, 133, 136, 139, 140 MADS-box genes 72, 163, 165, 166, 168, 169, 172, 217, 218 mannan 185, 186, 205, 207 mannose-binding lectin 186, 188, 189, 191, 192, 207 microprojectile/particle bombardment 225, 227, 228, 234, 235, 237, 242, 243, 246 micropropagation 23, 45, 65, 67, 92, 175 Index ✦ 257 microsatellite 124, 125, 149 molecular markers 13, 14, 17, 18, 69, 130, 141 NCBI GenBank 126, 146, 185, 188, 190 ndh genes 129, 136, 138, 140 nonantibiotic selection marker 186, 229, 239, 244, 246 novelty varieties 7, 9, 19, 20 nuclear DNA content 16, 99, 100, 104, 105, 106, 118, 120, 124, 150 Oncidium 41, 45, 50–52, 66, 67, 72, 79, 90, 169, 174, 175, 185–187, 190–193, 197, 205–207, 220, 228, 229, 231, 232, 236, 239, 241–243, 245, 246, 248, 249 orchid 2–4, 7, 9, 13, 15, 16, 19, 20, 23–25, 27, 31, 34, 35, 37–41, 45–47, 52, 54, 57, 59, 60, 65–72, 77–95, 99–101, 104, 107, 115, 120, 121, 129–131, 133, 138, 141, 145–147, 150, 151, 154–156, 163, 165–169, 172, 174–179, 185, 186, 211–218, 220, 225–232, 234, 235, 237 Orchidaceae 115, 130, 141, 147, 156, 165, 166, 169, 174, 175, 186, 211, 212, 220 organogenesis 55, 156 organ-specific 165 ornamental crops 41, 78, 84, 186 Paphiopedilum 36, 38–41, 45, 57, 58, 81, 147 PDS 213–216 pectin, 185, 195, 197, 206 peroxidase 186, 188–193, 195, 201, 207 peloric mutants 65, 70, 72, 166, 175, 176 pepper ferredoxin-like protein ( pflp) 52, 186, 232, 233, 236, 239, 240, 246–248 Phal. amabilis 2, 4, 5, 11, 15, 16, 17, 18, 19, 87 Phal. Cassandra 10 Phal. Doris 6, 19 Phal. equestris 3, 10–13, 15–19 Phal. Golden Peoker 7–9 Phalaenopsis 2–8, 10–20, 23, 27, 31–38, 41, 45, 52–55, 65–72, 78, 79, 81, 85–89, 91, 94, 99, 103–105, 108, 115–127, 129–134, 136–139, 141, 145, 150–153, 155, 166, 167, 169, 170, 175–177, 211, 212, 214–216, 220, 226, 228–231, 233–240, 243–246, 248, 249 Phalaenopsis amboinensis J. J. Smith 105, 118 Phalaenopsis aphrodite rchb. f. 105 Phalaenopsis aphrodite subsp. Formosana 103, 129–133, 138–141 Phalaenopsis bellina (Rchb. f.) Cristenson 105 Phalaenopsis cornu-cervi (Breda) Bl & Rchb. f. 105 Phalaenopsis equestris 12–15, 18, 145, 150, 166, 167 Phalaenopsis equestris (Schauer) Rchb. f. 105, 118 Phalaenopsis fasciata Rchb. f. 105 Phalaenopsis gigantea J. J. Smith 105 Phalaenopsis lueddemanniana Rchb. f. 105, 118 Phalaenopsis mannii Rchb. f. 105, 118 Phalaenopsis mariae Burb. ex Warn. & Wms. 105 Phalaenopsis micholitzii Rolfe 105 Phalaenopsis modesta J. J. Smith 105 Phalaenopsis parishii Rchb. f. 105 Phalaenopsis pulchra (Rchb. f.) Sweet 105 258 ✦ Index Phalaenopsis sanderiana Rchb. f. 105 Phalaenopsis stuartiana Rchb. f. 105, 118 Phalaenopsis sumatrana Korth. & Rchb. f. 105 Phalaenopsis venosa Shim & Fowl 105, 118 phylogenetic 14–17, 19, 122, 124, 130, 141, 147, 164, 166, 168, 172, 175, 178, 192, 193 plastid/chloroplast transformation 141, 249 ploidy 66, 67, 69, 100, 106–110 postharvest improvement 60 potted varieties 10 propidium iodide 4′-6-diamidino-2phenylindole 103 protocorm-like bodies (PLBs) 47, 51–54, 57, 65, 67, 69, 72, 228, 230–244, 248 pseudobulb 48, 66, 185–188, 191–195, 197, 205–207 RAPD 1, 13–19, 65, 69, 91, 92 Real-time RT-PCR 70, 71, 171, 214, 217, 218 reproductive growth 78, 86, 94 reproductive stages 66, 213, 216 RFLP 1, 14, 15, 19, 69 Rhizoctonia 77–82, 85, 86, 89, 91, 92, 94 rhizome 46–50 RNA editing 135, 138 RNAi 149, 217 RT-PCR 70, 71, 133, 138, 171, 214, 217, 218 seed coat 42 selection marker 186, 229, 239, 244, 246 shoot-bud formation 45, 58 single copy region 138 singlet 150, 151, 187, 192 small single copy region (SSC) 129–131, 138–140 soft rot disease 186, 240 somatic embryogenesis 45, 46, 50–52, 54–56, 186 storage products 31, 33 subtractive cDNA library 206 suspensor 23, 25–34, 38–41 Taiwan moth orchid 129, 131, 141 tandem repeats 115, 126 tetraploid 4, 6, 10, 12, 19, 106, 211, 218 thioridazine 47 tissue culture 46, 59, 65–67, 72, 241, 246, 249 total chromosome volume 118 transcription factor 108, 145, 146, 155, 156, 191, 192, 202, 204 transcription profile 71, 146, 155, 185, 206 transcriptome 148, 154, 155, 185, 206, 216 transformation efficiency 211, 212, 220, 230–236, 240–244, 249 transplanting 2, 85 Trichoderma 77, 80, 81 unigene 145, 151, 154 vegetative growth 67, 85, 86, 89, 185 viral vectors 214 23, 24, 26–31, 33–38, 40, zygote 24, 25, 26, 28, 29, 31, 32