ORCHID
BIOTECHNOLOGY
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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.
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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.
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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.
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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
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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
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3. Lin CS, Chen CT, Lin CC, Chang WC. (2003) A method for inflorescence
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13. Chen JT, Chang WC. (2001) Effects of auxins and cytokinins on direct
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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
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17. Chen JT, Chang WC. (2004) TIBA affects the induction of direct somatic
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19. Li SH, Kuoh CS, Chen YH, et al. (2005) Osmotic sucrose enhancement of
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20. Chen YQ, Piluek C. (1995) Effects of thidiazuron and N-6-beneyladaminopurine on shoot regeneration of Phalaenopsis. Plant Growth Reg
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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
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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
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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.
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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
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53. Yan N, Hu H, Huang Jl, et al. (2006) Micropropagation of Cypripedium
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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
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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.
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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.
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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
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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
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12. Ørgaard M, Heslop-Harrison JS. (1994) Relationships between species of
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13. Aggarwal RK, Brar DS, Khush GS. (1997) Two new genomes in the Oryza
complex identified on the basis of molecular divergence analysis using
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Ann Bot 87:387–395.
15. Flavell R. (1982) Sequence amplification, deletion and rearrangement:
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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
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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.
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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.
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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
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e
p
d
ce
a
k
s
p
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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
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o
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no
l ce su
ll io
ith
c la
ro
n
t
w
ce ract
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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.
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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.
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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.
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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.
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12. Burch-Smith TM, Anderson JC, Martin GB, Dinesh-Kumar SP. (2004)
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13. Robertson D. (2004) VIGS vectors for gene silencing: Many targets, many
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14. Brigneti G, Martin-Hernandez AM, Jin H, et al. (2004) Virus-induced gene
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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
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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
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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
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22. Peart JR, Cook G, Feys BJ, et al. (2002) An EDS1 orthologue is required for
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23. Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP. (2002) Tobacco Rar1, EDS1
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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
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26. Lu R, Malcuit I, Moffett P, et al. (2003) High throughput virus-induced
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27. Marathe R, Anandalakshmi R, Smith TH, et al. (2000) RNA viruses as
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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)
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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.
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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