The Application of Biotechnology to Orchids

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The Application of Biotechnology to Orchids a

b

M. Musharof Hossain , Ravi Kant , Pham Thanh Van Jaime A. Teixeira da Silva

c d

e

f

, Budi Winarto , Songjun Zeng &

g

a

Department of Botany, University of Chittagong, Chittagong, Bangladesh

b

Department of Botany, Panjab University, Chandigargh, India

c

The United Graduate School of Agricultural Science, Ehime University, Matsuyama, Ehime, Japan d

Vietnam Academy of Agricultural Sciences, Institute of Agricultural Genetics, Pham Van Dong Street, Co Nhue, Tu Liem, Hanoi, Vietnam e

Tissue Culture Laboratory, Indonesian Ornamental Crops Research Institute, Sindanglaya, Pacet-Cianjur, West Java, Indonesia f

Key Laboratory of South China Agricultural Plant Genetics and Breeding, South China Botanical Garden, Chinese Academy of Sciences, China g

Bioresource Production, Department of Horticulture, Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Ikenobe, Kagawa, Japan Version of record first published: 12 Dec 2012.

To cite this article: M. Musharof Hossain , Ravi Kant , Pham Thanh Van , Budi Winarto , Songjun Zeng & Jaime A. Teixeira da Silva (2013): The Application of Biotechnology to Orchids, Critical Reviews in Plant Sciences, 32:2, 69-139 To link to this article: http://dx.doi.org/10.1080/07352689.2012.715984

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Critical Reviews in Plant Sciences, 32:69–139, 2013 C Taylor & Francis Group, LLC Copyright ISSN: 0735-2689 print / 1549-7836 online DOI: 10.1080/07352689.2012.715984

The Application of Biotechnology to Orchids M. Musharof Hossain,1 Ravi Kant,2 Pham Thanh Van,3,4 Budi Winarto,5 Songjun Zeng,6 and Jaime A. Teixeira da Silva7 1

Department of Botany, University of Chittagong, Chittagong, Bangladesh Department of Botany, Panjab University, Chandigargh, India 3 The United Graduate School of Agricultural Science, Ehime University, Matsuyama, Ehime, Japan 4 Vietnam Academy of Agricultural Sciences, Institute of Agricultural Genetics, Pham Van Dong Street, Co Nhue, Tu Liem, Hanoi, Vietnam 5 Tissue Culture Laboratory, Indonesian Ornamental Crops Research Institute, Sindanglaya, Pacet-Cianjur, West Java, Indonesia 6 Key Laboratory of South China Agricultural Plant Genetics and Breeding, South China Botanical Garden, Chinese Academy of Sciences, China 7 Bioresource Production, Department of Horticulture, Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Ikenobe, Kagawa, Japan

Downloaded by [South China Institute of Botany] at 16:39 12 December 2012

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Table of Contents I.

INTRODUCTION ............................................................................................................................................................................................... 70

II.

BIOTECHNOLOGY IN ORCHID PHYLOGENY

III.

ORCHID BREEDING AND BIOTECHNOLOGY-BASED BREEDING

IV.

ORCHID EMBRYOLOGY

V.

ORCHID TISSUE CULTURE, MICROPROPAGATION AND IN VITRO BIOTECHNOLOGY .............................. 82

VI.

GENETIC TRANSFORMATION OF ORCHIDS

VII.

BIOTECHNOLOGY IN ORCHID GERMPLASM CONSERVATION

VIII.

BIOTECHNOLOGICAL TOOLS TO DETECT SOMACLONAL VARIATION

IX.

BIOTECHNOLOGICAL TOOLS IN THE CONTROL OF ORCHID FLOWERING

X.

MYCORRHIZA AS A BIOTECHNOLOGICAL TOOL FOR IMPROVING ORCHID GROWTH

XI.

BIOTECHNOLOGY AND THE CONTROL OF ORCHID VIRUSES .................................................................................. 114

XII.

GENE SILENCING TECHNOLOGY IN GENE FUNCTIONAL VALIDATION OF ORCHIDS

............................................................................................................................ 70 ............................................................................... 79

............................................................................................................................................................................ 80

.............................................................................................................................. 93 ................................................................................... 94 ............................................................. 102 .................................................. 103 ...................... 108

........................... 119

Address correspondence to M. Musharof Hossain, Department of Botany, University of Chittagong, Chittagong 4331, Bangladesh. E-mail: [email protected]; Jaime A. Teixeira da Silva, Bioresource Production, Department of Horticulture, Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Ikenobe 761-0795, Kagawa, Japan. E-mail: [email protected]; and Songjun Zeng, Key Laboratory of South China Agricultural Plant Genetics and Breeding, South China Botanical Garden, Chinese Academy of Sciences, 510650, China. E-mail: [email protected]

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M. MUSHAROF HOSSAIN ET AL.

XIII.

GENES CONTROLLING FLORAL MORPHOGENESIS IN ORCHIDS: ORDER IN THE MADS-HOUSE . 119

XIV.

CYTOGENETICS OF ORCHIDS AND THE ROLE OF BIOTECHNOLOGY

XV.

MEDICINAL ORCHIDS AND PHARMACEUTICAL BIOTECHNOLOGY

XVI.

OTHER BIOTECHNOLOGICAL DEVELOPMENTS

............................................................... 120

................................................................... 122

................................................................................................................. 124

XVII. CONCLUSIONS ................................................................................................................................................................................................ 124 ACKNOWLEDGMENTS


REFERENCES

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................................................................................................................................................................................................................... 125

This review provides an informative and broad overview of orchid biotechnology, addressing several important aspects such as molecular systematics, modern breeding, in vitro morphogenesis, protoplast culture, flowering control, flower color, somaclonal variation, orchid mycorrhiza, pathogen resistance, virus diagnosis and production of virus-free plants, functional genomics, genetic transformation, conservation biotechnology and pharmaceutical biotechnology. This resource will provide valuable insight to researchers who are involved in orchid biology and floriculture, using biotechnology to advance research objectives. Producing an improved orchid through biotechnology for industrial purposes or to serve as a model plant for pure and applied sciences is well within reach and many of the current techniques and systems are already employed at the commercial production level. Keywords

I.

micropropagation, transgenics, in vitro flowering, taxonomy, conservation, Orchidaceae

INTRODUCTION The Orchidaceae is the largest, most highly evolved and most diverse family of flowering plants, and is comprised of 30,000–35,000 species belonging to 850 genera, accounting for almost 30% of monocotyledons or 10% of flowering plants (Dressler, 1993; Nikishina et al., 2001a; Lucksom, 2007). About 70% of orchids are epiphytic, comprising approximately twothirds of the world’s epiphytic flora (Gravendeel et al., 2004; Hsu et al., 2011). On the other hand, 25% orchids are terrestrial and the remaining 5% can be found on various supports (Atwood, 1986). While the majority of temperate orchids are terrestrial, tropical orchids are epiphytic or lithophytic. The specialized orchid floral morphology, structure, and physiological properties have always fascinated botanists, horticulturists, and evolutionary biologists, and the beauty and mystery surrounding these ornamental jewels have always captivated the attention of amateurs and scientists alike. The orchids form an extremely peculiar group of plants due to their highly specialized pollination system, small, thin and non-endospermic seeds, obligate requirement of mycorrhizal association during germination, diverse and cosmopolitan habitats, advanced evo-

lutionary trends, extraordinary mechanisms of adaptation and persistence in adverse environmental conditions (Rasmussen and Rasmussen, 1991; Arditti, 1992; Prutsch et al., 2000; Yam and Arditti, 2009; Phillips et al., 2009; Jiang et al., 2011; Hsu et al., 2011; Yagame et al., 2012). Nevertheless, rapid and dynamic progress in molecular biology and biotechnology have helped to produce new varieties with novel characteristics, such as multicolored flowers, disease and pest resistance, and have allowed the origin and evolutionary trends to be determined, clarified phylogenetic relationships, while the biophysiological activities of orchid alkaloids have allowed for the discovery of novel bioactive compounds. Moreover, due to the rising demand and popularity of orchids, it is now-a-days a multimillion dollar business, primarily as pot plants and cut flower stalks (Suda, 1995; Winkelmann et al., 2006). To meet the demand of orchids in the future, as well to conserve their biodiversity, the development and deployment of new technologies is very important for improving flower quality, resistance to biotic and abiotic stresses, and to rapidly mass propagation members of this phylum. More recently, orchids have become the center of attention of new areas of plant research, including genetic engineering, functional genomics, proteomics, and metabolomics (Hsiao et al., 2011a; Fu et al., 2011; Hsu et al., 2011; Chang et al., 2011), all of which require advanced transgenic strategies (Teixeira da Silva et al., 2011) to achieve their different goals. The successful application of these new approaches will help to further improve orchids and orchid products. In this review, a broad relation between several aspects of orchid research and biotechnology is discussed, and while it is impossible to cover the entire scope of literature that already exists, an attempt has been made to cover, in as much depth as possible, the most important factors that have defined orchid biotechnology, particularly over the past decade.

II.

BIOTECHNOLOGY IN ORCHID PHYLOGENY Traditionally, morphological and cytological characteristics are used to identify and classify the relatedness of orchid species


ORCHIDS AND BIOTECHNOLOGY

through phylogenetic studies, although these methods are limited by environmental effects and diagnostic resolution. Despite this, the rapid development of biotechnology at the molecular level has allowed orchid phylogeny at different taxonomic levels to be discerned and has also permitted the accurate evolutionary trends of the Orchidaceae to be traced out. The most commonly used molecular markers and techniques that have permitted these advances include restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter-simple sequence repeat (ISSR) markers, sequencing of particular genes such as internal transcribed spacer (ITS) of nuclear ribosomal DNA (nrDNA), ribulose 1,5-bisphosphate carboxylase large subunit (rbcL), plastid maturase K (matK), RNA polymerase-β subunit (rpoB), RNA polymerase-β subunit (rpoC1), NADH dehydrogenase-F subunit (ndhF), transfer RNA subunit-F (trnL-F), ATP synthase genes (atpF-atpH spacer) and transfer RNAs-photosystem II (trnH-psbA) intergenic spacers of the chloroplast genome (Table 1) (Geilly and Taberlet, 1994; Jarrell and Clegg, 1995; Neyland and Urbatsch, 1996; Fu et al., 1997; Cameron et al., 1999; Kores et al., 1997, 2000, 2001; Hedren et al., 2001; Been et al., 2002; Carlsward et al., 2006; Kishor et al., 2008; Bateman et al., 2009; Wang et al., 2009b; Ding et al., 2009; Hopper, 2009; Chen et al., 2010; Khew et al., 2011; Lu et al., 2011; Parveen et al., 2012). A DNA-based diagnostic assay such as RAPD can identify genotypes directly and can therefore help to mitigate incongruencies arising from cytological and morphological studies and has been extensively used for the identification of plants due to its rapidity, simplicity, economy and absence of prior genetic information and requirement of sophisticated equipment (Sheorey and Tiwari, 2011). For example, strap-leaved Vanda is very closely related to Euanthe from a cytological (karyotype) point of view (Tanaka and Kamemoto, 1961). Rudolf Schlechter in 1914 established it as a monotypic genus. Holttum (1953) referred to it as Euanthe sanderiana while Dressler (1981) referred to it as Vanda sanderiana, indicating that it was a synonym of Vanda and subsequently treating it as an invalid or doubtfully distinct ‘taxon’. Thus, the generic status of Euanthe remains open to debate. RAPD data allowed the similarity between Euanthe and Vanda to be confirmed and it has now been re-established as V. sanderiana (Lim et al., 1999). Similarly, based on RAPD analysis of terete leaves, Vanda teres and Vanda hookeriana were transferred to a separate genus, Papilionanthe. Goh et al. (2005), who studied 149 orchids, including 49 Phalaenopsis accessions, used RAPD analysis to conclude that species previously classified under the genera Doritis and Kingidium were closely related with Phalaenopsis species, and that P. doweryensis is a hybrid of P. gigantea and P. fuscatae due to its morphological and molecular resemblance to its parent plants. AFLP is also a valuable DNA-based technique allowing for the rapid analysis of genetic distances and providing information about multiple polymorphic anonymous loci, generally in the nuclear genome of a species and can be used to determine

71

genetic diversity in many plant species, particularly when there are few characterized molecular genetic markers (Hedren et al., 2001; Salas et al., 2007). Hedren et al. (2001) explored AFLP to elucidate details and support the concept of polyploid evolution in the Eurasian orchid genus Dactylorhiza which states that allotetraploid derivatives arose repeatedly as a result of hybridization between the two parental groups D. incarnata sensu lato (diploid marsh orchids) and the D. maculata group (spotted orchids). Morphologically distinct varieties within the D. incarnata group consisted of two clear subgroups, one containing autotetraploid D. maculata subsp. maculata and the other containing diploid D. maculata subsp. fuchsii. In addition, allotetraploids showed a high degree of additivity for the putative parental genomes, and relationships among them were partially correlated to morphologically based entities and to geographic distribution. The ITS of nrDNA is one of the most extensively applied molecular markers for angiosperm phylogenetic inference and genetic relatedness (Table 1) and is a proven useful source of characters for phylogenetic studies in many orchids (Pridgeon et al., 1997; Lau et al., 2001; Clements et al., 2002; Tsai et al., 2004a; van den Berg et al., 2005; Campbell et al., 2005; Chen et al., 2010; Sun et al., 2011; Takamiya et al., 2011; Parveen et al., 2012). ITS nrDNA sequences have been successfully used as a genetic marker for molecular authentication and identification of several medicinal orchids, including Dendrobium sp. and Paphiopedilum sp. (Cox et al., 1997; Cheng et al., 2004; Chung et al., 2006; Xu et al., 2006a; Parveen et al., 2012). Generally, ITS regions are a popular choice to infer phylogeny among closely related taxa and to identify species or strains (Lau et al., 2001; Cheng et al., 2004; Zeng et al., 2008; Chen et al., 2010), or to solve taxonomic ambiguities, particularly at the interspecific and intergeneric levels among angiosperms, derived from several merits such as biparental inheritance, universality, simplicity, intragenomic uniformity, intergenomic variability, low functional constraint and high copy number. Cox et al. (1997) produced nuclear ribosomal ITS nucleotide sequences for nearly 100 slipper orchids species (Paphiopedilum, Phragmipedium, Cypripedium, Selenipedium, Mexipedium) and used parsimony analysis to investigate their relationships. As for rbcL data, ITS sequences categorized Mexipedium as a sister to Phragmipedium. Relationships at the sectional level in Paphiopedilum are largely as described by Cribb (1987), although the division of Paphiopedilum into subgg. Brachypetalum and Paphiopedilum was not supported by Cox et al. (1997), whose study also showed that subg. Brachypetalum is paraphyletic to subg. Paphiopedilum. Phragmipedium species are divided into the same three major clades as in the taxonomic scheme of McCook (1989). Yuan et al. (2009a) studied the genetic relationship among 36 Dendrobium species in China including two different species, namely Epigeneium amplum and E. nakaharaei based on sequences analysis of the rDNA ITS region. The ITS sequences were unable to completely match the classification based on morphological characters with D. moulmeinense

72 Techniques applied

Remarks

Reference

185 taxa of Pleurothallidinae, including two Sequencing and analysis of Demonstrated phylogenetic relationships in Pleurothallidinae (Orchidaceae) Pridgeon et al., accessions each of Masdevallia internal transcribed spacers through combined analysis of nuclear and plastid DNA sequences. This is 2001 venezuelana, Brachionidium valerioi, and (ITS) of nrDNA, the plastid an extensive molecular phylogenetic study of subtribe Pleurothallidinae. Ophidion pleurothallopsis, as well as the matK, rbcL, trnL intron, and the In this analysis, authors were able to assess generic circumscriptions with outgroup taxa Dilomilis montana, trnL-F intergenic spacer hundreds of characters and produce trees for which internal support was Neocogniauxia hexaptera, Arpophyllum assessed rather than relying on a few characters selected a priori for giganteum, and Isochilus amparoanus reasons that are subjective and inconsistent. There was minimal (mostly Laeliinae) divergence among the various subgenera, sections, and even series of Pleurothallis, Masdevallia, and Dracula, blurring the distinctions among them and making them useful as categories only for identification purposes, which should not be misinterpreted as reflecting biological relationships. PCR-based AFLP elucidated details of polypoidy and supported the concept Hedren et al., D. incarnata vars. incarnata, D. ochroleuca, PCR-based AFLP analysis of polyploid evolution in the Eurasian orchid genus Dactylorhiza, i.e., that 2001 D. cruenta, D. borealis, and subspp. allotetraploid derivatives have arisen repeatedly as a result of coccinea and pulchella, D. incarnata s.l., hybridization between the two parental groups D. incarnata s.l. (sensu D. maculata subspp. fuchsii, D. maculata lato; diploid marsh orchids) and the D. maculata group (spotted orchids). subspp. maculata, and D. foliosa, D. Within the D. incarnata s.l. group, morphologically defined varieties were incarnata s.l., D. maculata/fuchsii, D. interdigitated. The D. maculata group consisted of two distinct subgroups, majalis subspp. majalis, lapponica, one containing autotetraploid D. maculata subsp. maculata and the other traunsteineri, praetermissa, purpurella, containing diploid D. maculata subsp. fuchsii. Allotetraploids showed a alpestris, D. majalis subspp. scotica, D. iberica, D. romana, D. sambucina and high degree of additivity for the putative parental genomes, and other Dactylorhiza spp. One sample each relationships among them were partly correlated to morphologically based of three genera was included, namely entities, but also to geographic distribution. Thus, allotetraploid taxa from Gymnadenia conopsea, Pseudorchis the British Isles clustered together, rather than with morphologically albida, and Coeloglossum viride, which is similar plants from other areas. closely related to Dactylorhiza Dendrobium tosaense, D. officinale, and Sequencing and analysis of partial This report describes the molecular identification of three medicinal orchids Cheng et al., D. moniliforme ribosomal DNA (rDNA) namely Dendrobium tosaense, D. officinale, and D. moniliforme. The 2004 rDNA regions (ITS1, 5.8S, and ITS2) of these three orchids were amplified by PCR. The length of the DNA fragments of D. tosaense, D. officinale, and D. moniliforme was 632 nt (nucleotides), 627 nt and 628 nt, respectively. Similarities in the rDNA region between the species pairs ranged from 91 to 95%. These results might be helpful in identifying other Dendrobium species.

Orchid taxa

TABLE 1 Molecular techniques applied to orchid phylogeny and evolution


73

(Continued on next page)

This study evaluated monopodial leaflessness, and provided molecular and Carlsward et al., Sequencing and analysis of ITS structural evidence to generate phylogenetic hypotheses for the Vandeae. nrDNA, the plastid matK (part 2006 Maximum parsimony analyses of these three DNA regions each supported of the exon encoding maturase two subtribes within monopodial Vandeae: Aeridinae and a combined K) and trnL-F (trnL intronþtrnL Angraecinae + Aerangidinae. Sequence data derived from the ITS regions 3 exon + trnL-trnF intergenic spacer) for all monopodial subtribes (Aeridinae, Aerangidinae, and Angraecinae) supported a monophyletic Vandeae. Adding structural characters to sequence data resulted in trees with more homoplasy, but gave fewer trees each with more well-supported clades than either data set alone. Two techniques for examining character evolution were compared: (1) mapping vegetative characters onto a molecular topology and (2) tracing vegetative characters onto a combined structural and molecular topology. In both cases, structural synapomorphies supporting monopodial Vandeae were nearly identical. Several important characters appear to be important in making the evolutionary transition to leaflessness: A change in leaf morphology (usually reduced to a nonphotosynthetic scale), monopodial growth habit, and aeration complexes for gas exchange in photosynthetic roots. This molecular phylogenetic work is a foundation for examining evolutionary questions within Vandeae and represents an integrated approach to answering the general question of parallelism that has occurred systematically and geographically in different lineages. Dactylorhiza majalis subsp. cordigera, D. AFLP fingerprints, nuclear Variation in nuclear AFLP fingerprints, nuclear isozymes, and hypervariable Hedren et al., baumanniana, D. smolikana, D. graeca, isozymes, and hypervariable plastid DNA loci were examined to describe speciation patterns and 2007 D. kalopissii, D. macedonica, D. pindica, plastid DNA loci, i.e. a portion species relationships in the Dactylorhiza incarnata/maculata polyploid D. pythagorae, and D. maculata subsp. of the trnT-trnL intergenic complex (marsh orchids) in Greece. Even though several endemic taxa saccifera. For AFLP analysis reference spacer, the trnL exon I, and the with restricted distribution have been described from this area, detailed material (D. majalis subsp. majalis and D. trnL intron relationships need to be understood in order to propose meaningful maculata subsp. fuchsia) from areas conservation priorities. Four independently derived allopolyploid lineages outside Greece was also included. which were poorly correlated with the prevailing taxonomy were identified. Three lineages were composed of populations restricted to small areas that may have been of recent origin from extant parental lineages. One lineage with a wide distribution in northern Greece was characterized by several unique plastid haplotypes that were phylogenetically related and evidently older. The D. incarnata/maculata polyploid complex in Greece showed high levels of genetic diversity at the polyploid level. This diversity had accumulated over a long period of time and may have included genetic variants originating from now extinct parental populations. This data set also indicated that the Balkans may have constituted an important refuge from which northern European Dactylorhiza were recruited after the Weichselian ice age.

193 species of monopodial Vandeae and four outgroup species from Polystachyinae were assessed.


74

Two commercially available Phalaenopsis species i.e., P. amabilis P. lindenii and 14 hybrids

Reciprocal crosses of Aerides vandarum and Vanda stangeana

Dendrobium officinale

354 species of the subtribe Maxillariinae

Orchid taxa

Remarks

Investigated phylogenetic relationships within 619 individuals representing ca. 354 species of subtribe Maxillariinae sensu using parsimony analyses of individual and combined DNA sequences data. They analyzed a combined matrix of ITS nrDNA, the plastid matK gene, rpoC1 gene and flanking trnK intron, and the plastid atpB-rbcL intergenic spacer. All the currently recognized minor genera of “core” Maxillariinae (Anthosiphon, Chrysocycnis, Cryptocentrum, Cyrtidiorchis, Mormolyca, Pityphyllum, and Trigonidium) are embedded within a polyphyletic Maxillaria. The results supported the recognition of a more restricted Maxillaria, of some previously published segregate genera (Brasiliorchis, Camaridium, Christensonella, Heterotaxis, Ornithidium, Sauvetrea), and of several novel clades at the generic level. These revised monophyletic generic concepts should minimize further nomenclatural changes, encourage monographic studies, and facilitate more focused analyses of character evolution within Maxillariinae. Sequencing and analysis of the The nrDNA ITS regions, chloroplast genes matK, rbcL and mitochondrial gene nad1 nrDNA ITS regions, chloroplast intron2 were used to survey eight populations of D. officinale, ultimately leading genes matK, rbcL and the to their molecular authentication. No differences were found in matK, rbcL and mitochondrial gene nad1 nad1 intron2 sequences between populations, except for 9 single nucleotide intron2 polymorphism (SNP) sites in nrDNA ITS regions. Two pairs of allele-specific PCR (AS-PCR) primers based on SNPs were designed to authenticate two genuine populations. A combined molecular protocol based on SNPs of rDNA ITS regions and AS-PCR is practical and effective and serves as a simple and convenient evaluation system for geoherbalism studies of D. officinale based on resource investigation and authentication research. Analysis of PCR-RAPD and In this study, an attempt was made to confirm hybridity of the intergeneric reciprocal sequencing of trnL-F crosses of A. vandarum and V. stangeana by developing RAPD markers using non-coding region of DNA extracted from in vitro-raised protocorms. Further, the trnL-F non-coding chloroplast DNA region of chloroplast DNA was employed for unequivocal identification of the reciprocal crosses by studying its inheritance pattern. The PCR-amplified product of the same region was subjected to RFLP to consolidate the finding. The hybridity of the reciprocal crosses could be established by employing PCR-RAPD at the protocorm stage although chloroplast DNA markers were more appropriate for identification of the reciprocal crosses. Such PCR-based molecular markers will also be useful to determine the genetic background of plant materials the breeders use to ensure greater success in achieving the specific aims of hybridization, rapidly and reliably. AFLP analysis of genomic DNA Using AFLP fingerprinting demonstrated the level of genetic variability in Phalaenopsis species and hybrids and estimated the correlation of genetic relatedness with the known pedigree in various hybrids. The results also demonstrated the usefulness of AFLP analysis in Phalaenopsis and its potential application in breeding and species conservation.

Sequencing and analysis of ITS nrDNA, the plastid matK gene, rpoC1 gene and flanking trnK intron, and the plastid atpB-rbcL intergenic spacer

Techniques applied

TABLE 1 Molecular techniques applied to orchid phylogeny and evolution (Continued)


Chang et al., 2009

Kishor et al., 2008

Ding et al., 2008

Whitten et al., 2007

Reference

75

Hundreds of orchid species available in Combined matK and trnL-F This paper briefly reviews the causes of recent taxonomic turmoil for Hopper, Australia primarily from the tribes plastid DNA sequences, ITS Australian orchids and outlines new research opportunities and 2009 Epidendroideae, Vanilloideae, Orchideae, nrDNA sequencing and analysis conservation implications arising from an improved understanding of their Diurideae, Cranichideae, molecular phylogenetics. The information traced in this study robustly Codonorchideae, among others supported molecular analyses of most clades now enabling rigorous comparative biological studies of orchids. Wang Dendrobium aduncum, D. hercoglossum, D. Analysis of inter-simple sequence Genetic diversity and phylogenetic relationships among 31 Dendrobium et al., chrysotoxum, D. lindleyi, D. thyrsiflorum, repeat (ISSR) marker DNA species were determined using ISSRs. In total, 2368 bands were amplified 2009 D. aphyllum, D. brymerianum, D. by 17 ISSR primers, resulting from 278 ISSR loci with 100% capillipes, D. chrysanthum, D. polymorphism at the genus level. All species were unequivocally crepidatum, D. crystallinum, D. distinguished based on ISSR fingerprinting. Species-specific ISSR devonianum, D. falconeri, D. gibsonii, D. markers were also identified in 9 out of 31 tested Dendrobium species. guangxiense, D. hancockii, D. Unweighted Pair-Group Mean Analysis (UPGMA) grouped the 31 heterocarpum, D. loddigesii, D. Dendrobium species into six clusters, indicating the polyphyletic nature of the genus with several well-supported lineages. The study demonstrated moniliforme, D. nobile, D. officinale, D. that ISSR was a very effective technique in determining genetic diversity pendulum, D. primulinum, D. dixanthum, in Dendrobium at the species level. Cluster analysis based on ISSR D. wardianum, D. longicornu, D. profiles clearly identified Dendrobium species and provided a molecular williamsonii, D. strongylanthum, D. diagnosis tool for the authentication of valuable medicinal plant species. stuposum, D. christyanum, and D. The ISSR markers reported in this study will facilitate an understanding of denneanum inter-species gene flow, genetic structure of species, genetic diversity and evolutionary relationships in the genus Dendrobium. 35 species of the Platanthera clade Analysis of nrITS DNA Molecular phylogenetics and morphological reassessment of the Bateman (Orchidaceae: Orchidinae) including Platanthera clade was described in this study. The ITS sequences were et al., Platanthera, Aceratorchis, Amerorchis, obtained from 86 accessions of 35 named taxa using Pseudorchis as the 2009 Chondradenia, Diphylax, Galearis, outgroup. By combining implicit assessment of morphological divergence Neolindleya, Piperia, Pseudorchis, and with explicit assessment of molecular divergence, 9 former genera could Tsaiorchis be rationalized into 4 revised genera by sinking the monotypic Amerorchis, together with Aceratorchis and Chondradenia, into Galearis, and by amalgamating Piperia, Diphylax and the monotypic Tsaiorchis into the former Platanthera section Platanthera. Further species sampling would allow this section to be sub-divided into at least three sections, suggesting that ITS sequence divergence characterizes most species other than the P. bifolia group. The floral differences that distinguished Piperia, Diphylax and Tsaiorchis from Platanthera, and Aceratorchis and Chondradenia from Galearis, reflect various forms of heterochrony. (Continued on next page)


76 Techniques applied

15 commercial Oncidiinae varieties Analysis of the complete including Oncidium Gower Ramsey chloroplast genome varieties (Lemon heart, Sunkiss, Sweet Sugar and Million Coins), five Beallara varieties (Eurostar, Morning Joy, Howard Dream, Sugar Sweet, and Smile Eri), two Odontoglossum varieties (Margarete Holm and Violetta von Holm), two Odontocidium varieties (Golden Gate, Wildcat ‘Garfield’), one Degarmoara variety (Flying High) and one Zelenkocidium variety (Little Angel)

123 species belonging to Laeliinae including Analysis of plastid trnL intron, the Scaphyglottis Alliance, the Brougtonia trnL-F spacer, matK gene and Alliance, the Encyclia Alliance, the trnK introns, upstream and Isabelia Alliance, Pleurothallidinae, dowstream from matK and Ponerinae, Bletiinae Chysiinae, Coeliinae, nuclear ITS rDNA Hagsatera, Arpophyllum, the Cattleya Alliance, Dinema / Nidema, etc.

Orchid taxa

Reference

This study assessed generic alliances of Laeliinae, a neotropical orchid van den Berg subtribe, based on molecular phylogenetic analysis of plastid trnL intron, et al., 2009 trnL-F spacer, matK gene, trnK introns upstream and dowstream from matK and nuclear ITS rDNA sequence data. Distant outgroups Earina valida and Polystachya galeata were chosen. The relationships between Laeliinae and outgroups were well supported. A combined tree followed the ITS trees more closely, but the levels of support obtained with maximum parsimony were low. Bayesian analysis recovered more well-supported nodes. The trees from combined maximum parsimony and Bayesian analysis allowed 8 generic alliances to be recognized within the Laeliinae, all of which showed trends in morphological characters but lacked unambiguous synapomorphies. By using combined plastid and nuclear DNA data in conjunction with mixed-models of Bayesian inference, it was possible to delimit smaller groups within the Laeliinae and to discuss general patterns of pollination and hybridization compatibility. Furthermore, these small groups can now be used for further detailed studies to explain morphological evolution and diversification patterns within the subtribe. Wu et al., 2010 The complete chloroplast genome of Oncidium Gower Ramsey was determined using PCR and Sanger-based ABI sequencing. The length of the Oncidium chloroplast genome was 146,484 bp. Genome structure, gene order and orientation were similar to Phalaenopsis, but differed from typical Poaceae, other monocots. The chloroplast-encoded NADH dehydrogenase (ndh) genes, except for ndhE, lacked apparent functions. Deletion and other types of mutations were also found in the ndh genes of 15 other Oncidiinae varieties, except for ndhE in some species. To identify the relationships between the 15 Oncidiinae hybrids, 8 regions of the O. Gower Ramsey chloroplast genome were amplified by PCR for phylogenetic analysis. A total of 7042 bp. derived from the 8 regions could identify the relationships at the species level, which were supported by high bootstrap values. One particular 1846 bp. region, derived from two PCR products (trnHGUG -psbA and trnFGAA-ndhJ) was adequate for correct phylogenetic placement of 13 of the 15 varieties. Thus, molecular markers of the complete chloroplast genome provide useful information for phylogenetic and evolutionary studies in Oncidium with applications for breeding and variety identification.

Remarks



77

Compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS)

Sequencing and analysis of bacterial artificial chromosome (BAC) end sequences (BESs)

Sequencing and analysis of nrDNA ITS regions

6600 plant samples belonging to 4800 species from 753 distinct genera of 193 families, including orchids

Phalaenopsis equestris

Dendrobium species from 21 samples of Dendrobii Herba collected from different herbal markets

In this study 7 candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, Chen et al., ycf5, ITS2, and ITS) from medicinal plant species were compared. The 2010 data suggests that the second internal transcribed spacer (ITS2) of nrDNA represents the most suitable region for DNA barcoding. Furthermore, the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera of 193 families was tested: the rate of successful identification with ITS2 was 92.7% at the species level. Therefore, the ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species and can serve as a novel universal barcode for the identification of a broader range of plant taxa, including orchids. Two BAC libraries of P. equestris (constructed using the BamHI and HindIII Hsu et al., 2011 restriction enzymes) were used to generate pair-end sequences from BAC clones. Simple sequence repeat (SSR) or microsatellite content, repeat element composition, GC content, and protein encoding regions were analyzed. The annotated BESs were an efficient and viable strategy for gaining insight into the sequence content and complexity of the Phalaenopsis genome, afforded by the construction of BAC libraries and end-sequencing of randomly selected BAC clones. Such BESs can be used as a primary scaffold for genome shotgun-sequence assembly and to generate comparative physical maps in genetic mapping and breeding. Takamiya et al., This study described identification of plant sources of Dendrobii Herba 2011 based on sequences of the ITS regions of nrDNA. The authors used an ITS1–5.8S-ITS2 sequence database of 196 Dendrobium species constructed earlier to identify plant sources of 21 Dendrobii Herba samples. They found that 13 Dendrobium species (D. catenatum, D. cucullatum, D. denudans, D. devonianum, D. eriiflorum, D. hancockii, D. linawianum, D. lituiflorum, D. loddigesii, D. polyanthum, D. primulinum, D. regium, and D. transparens) were possibly used as plant sources of Dendrobii Herba, and unidentified species allied to D. denudans, D. eriiflorum, D. gregulus, or D. hemimelanoglossum were also used. Therefore, ITS DNA sequencing may apply for identification of orchid species used in Dendrobii Herba available in medicinal orchid markets. (Continued on next page)


78 Remarks

Reference

AS-PCR, allele-specific PCR; BAC, bacterial artificial chromosome; BES, BAC end sequence; ISSR, inter-simple sequence repeat markers; ITS, internal transcribed spacers; NJ, neighbor joining; RAPD, random amplified polymorphic DNA; RFLP, restriction fragment length polymorphism; SSR, simple sequence repeat; UPGMA, Unweighted Pair-Group Mean Analysis.

RAPD-based technique

Techniques applied

This study reviewed the efficiency of RAPD-based DNA analyses for Sheorey and identification of herbs and herbal preparations. These analyses serve as a Tiwari, 2011 rapid, simple, efficient, reliable, economical and powerful technique that requires less sophisticated equipment. Moreover, without any prior information, these techniques can be successfully applied for phylogenetic analyses. 151 Cymbidium sinense cultivars Analysis of inter-simple sequence In this study, ISSR markers were used to assess the genetic diversity and Lu et al., 2011 repeat (ISSR) markers population structure of 151 C. sinense cultivars collected from China and Japan. These cultivars shared almost identical morphological characters with only a few differences, which can result in confusion during the assessment of genetic resources. The ISSR fingerprinting profiles for molecular identification of cultivars was effective. With neighbor joining (NJ) analysis, the 151 cultivars were clustered into 7 main groups which were approximately related to their geographical distribution. Population structure analysis revealed 6 subpopulations, generally consistent with NJ-clustering. The results of this study suggested that different provenance collection and in situ conversation are important for C. sinense conservation and genetic improvement. 100 slipper orchids species (incl. Sequencing and analysis of ITS sequences placed Mexipedium sister to Phragmipedium. Relationships Cox et al., 1997, Paphiopedilum, Phragmipedium, nrDNA ITS regions at the sectional level in Paphiopedilum are largely as described by Cribb 2011 Cypripedium, Selenipedium, Mexipedium) although the division of Paphiopedilum into subg. Brachypetalum and Paphiopedilum is not supported; subg. Brachypetalum is paraphyletic to subg. Paphiopedilum. Phragmipedium species are divided into the same three major clades as in the taxonomic scheme of McCook.

Medicinal herbs and herbal products, including orchids

Orchid taxa





diverging from the other 35 Dendrobium species. D. moulmeinense is not a well known species from Yunnan province (China) that had been included in the section Dendrobium by Tsi et al. (1999) based on morphological characteristics. Ding et al. (2003) used ITS-specific primers to authenticate the stems of Dendrobium officinale in China. Xu et al. (2006a) designed five species-specific primers pairs and used them for the rapid identification of five Dendrobium species using PCR. Takamiya et al. (2011) identified 13 Dendrobium species (D. catenatum, D. cucullatum, D. denudans, D. devonianum, D. eriiflorum, D. hancockii, D. linawianum, D. lituiflorum, D. loddigesii, D. polyanthum, D. primulinum, D. regium, and D. transparens) and an unidentified species allied to D. denudans, D. eriiflorum, D. gregulus, or D. hemimelanoglossum from 21 traditionally used herbal medicines (Dendrobii Herba) by using nrDNA ITS sequences. They also reported D. catenatum to be synonymous with D. officinale, one of the most important sources of Dendrobii Herba as defined by the Pharmacopoeia of The People’s Republic of China. matK and rbcL, the plastid trnL intron, the trnL-F spacer as well as the trnH-psbA intergenic spacer of the chloroplast genome are often used for molecular phylogenetic analysis of orchids (Table 1) (Hasebe et al., 1994; Hilu and Liang, 1997; Wilson, 2004; Kress and Erickson, 2007; Fazekas et al., 2008; Newmaster and Ragupathy, 2009; van den Berg et al., 2009; Parveen et al., 2012). Asahina et al. (2010) successfully distinguished five medicinal Dendrobium species, i.e. D. fimbriatum, D. moniliforme, D. nobile, D. pulchellum, and D. tosaense based on matK and rbcL sequencing. Lahaye et al. (2008) analysed the matK sequences of 1566 specimens of orchids representing 1084 species from Costa Rica, highlighting the relevance of this technique in species identification and in phylogenetic reconstruction. In addition to these molecular markers, several plastid DNA regions (atpF-atpH spacer, rpoB, rpoC1 gene, and psbK-psbI spacer) have also been used by the Consortium for the Barcode of Life (CBOL) in plant species identification (CBOL Plant Working Group, 2009) in which ‘DNA barcoding’ was successfully applied to identify orchids (Yao et al., 2009; Parveen et al., 2012). In 2009, the CBOL Plant Working Group recommended the two-loci combination of rbcL + matK as plant DNA barcodes, which are sequences that vary extensively between species but hardly at all within them (CBOL Plant Working Group, 2009). DNA barcoding can be applied to identify species and to provide taxonomic information that would allow their evolutionary relevance to be clarified (Chase et al., 2005; Vogler and Monaghan, 2007). Cameron et al. (1999) conducted cladistic parsimony analyses of rbcL nucleotide sequence data from 171 taxa representing nearly all tribes and subtribes of the Orchidaceae while Chase et al. (2003) provided a revised classification of the Orchidaceae based on molecular phylogenetics using combined analysis of atpB, rbcL, matK, PsaB, trnl-f , 26s rDNA and the nadl intron. Paphiopedilum species and their inter-species hybrids are morphologically difficult to differentiate, especially in their vegetative stage, thus,

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to determine the genetic relationships and proper identification of eight Paphiopedilum species and three inter-specific hybrids including P. druryi, P. hirsutissimum, P. fairrieanum, P. insigne, P. villosum, P. venustum, P. spicerianum, P. wardii, P. venustospicerianum, P venusto-insigne and P. pradhanii. Parveen et al. (2012) barcoded plastid genes including rbcL, rpoB, rpoC1, psbA-trnH spacer and the matK gene assisted in parallel by RAPD analysis and sequencing of ITS nrDNA. DNA amplification fingerprinting (DAF) is an effective method to detect polymorphism at the level of clones and cultivars and is thus a powerful tool for species or cultivar identification (Chen et al., 1995; Sheorey and Tiwari, 2011). Phalaenopsis clones were successfully identified by analyzing distinguishable bands from DAF patterns with a similar genetic background using a suitable primer sets (Tang and Chen, 2007). Molecular markers, particularly DNA barcoding and DAF, provide a robust means by which the patented rights of new orchid varieties or cultivars can be protected, although this possibility has not yet been applied.

III.

ORCHID BREEDING AND BIOTECHNOLOGY-BASED BREEDING Orchids have a weak reproductive barrier making them one of the most interesting plant groups for fabricating desirable hybrids. The Royal Horticultural Society registered, by the end of 2011, 250,000 cultivars and more than 106,000 attractive hybrids, both natural and man-made, with thousands of new hybrids being added each year (International Registration Authority for Orchid Hybrids, Royal Horticultural Society, UK; http://www.rhs.org.uk/Plants/RHS-Publications/Orchidhybrid-lists). This is the highest number of hybrids among all families in the plant kingdom. Many orchid hybrids are multigeneric. For example, the most popular orchid hybrid ‘Phalaenopsis Taisuco’ released in 1997/98 in Taiwan was the offspring of P. amabilis, P. rimestadiana, P. aphrodite, P. schilleriana, P. stuartiana and P. sanderiana and was developed when breeding stocks from different sources were hybridized, including those from local Phalaenopsis farms and many from foreign countries, including Japan, the Netherlands and the United States (Chen et al., 1999). Especially noteworthy is that some intergeneric hybrids are easy to be achieved successfully in Orchidaceae (Huang et al., 2011). Conventional breeding, which involves traditional hybridization to transmit useful traits into commercial varieties, and subsequent detection of novel varieties, is a laborious and time-consuming process that takes years to achieve, and in orchids this can translate to about 2–13 years (Teoh, 1986; Arditti, 1992; Kostenyuk et al., 1999; Kishor et al., 2006; Sim et al., 2007; Tang and Chen, 2007). In contrast, modern biotechnological approaches offer a promising alternative for the rapid detection and authentication of orchid germplasm. For example, the hybrid ‘Phalaenopsis Taisuco White’ is the progeny of P. amabilis, P. rimestadiana and P. aphrodite in which the proportion of the genetic constitution contributed by these three ancestors was 40.34, 38.56, and


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16.41%, respectively, which was only possible to discern when RAPD, RFLP, AFLP and DAF were used (Tang and Chen, 2007), emphasizing their prowess in the estimation of genetic constituent and variability among orchid species and hybrids (Chang et al., 2009) and to verify new and novel orchid varieties (Chen et al., 1995, Divakaran et al., 2006). The scope of varietal improvement is limited in orchids due to their notoriously slow growth and cross-incompatibility of some species. Therefore, new techniques that would allow the breeding efficiency of economically important crops that have long life cycles and cross-incompatibility, such as orchids, to be increased, are indispensable. RAPD, which is the simplest version of PCR with arbitrary primers used for detecting DNA variation and is popular due to its convenience, low cost and simplicity (Sun and Wong, 2001; Bairu et al., 2011), was used to find molecular differences to support physiological differences in Cymbidium (Teixeira da Silva et al., 2007a). AFLP, which is a PCR-based tool used in genetics research, DNA fingerprinting, and in genetic engineering, was developed in the early 1990s to overcome the limitations of reproducibility associated with RAPD and ingeniously combines the power of RFLP and the flexibility of PCR-based technology (Agarwal et al., 2008) to provide a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity. cDNA AFLP and three endonuclease AFLPs (TE-AFLPs), which are variations of AFLP, have been used to quantify differences in gene expression levels, detect transposable element mobility, and to determine parentage, relationships and variation among plants, including orchids (Weising et al., 2005; Hedren et al., 2007). It requires at least 3 to 10 years to confirm a successful orchid hybrid by conventional methods because the flower is the key characteristic to visually confirm hybrid progeny, and many orchids take several years to reach the flowering stage, although this can be overcome by inducing flowering in vitro within a compacted period of time (Sudhakaran et al., 2006). Molecular techniques can be used to easily detect the success of crossing soon after seedling development by in vitro tissue culture of the hybrid progeny. For example, RAPD markers allowed the hybridity of the reciprocal crosses of Aerides vandarum and Vanda stangeana, and Renanthera imschootiana and Vanda coerule to be determined as early as the immature seeds or embryos that had germinated in vitro (Kishor et al., 2008; Rajkumar and Sharma, 2009). By amplifying the trnL-F non-coding regions of chloroplast DNA (cpDNA) of the parent species and hybrids, the reciprocal crosses could be easily identified since maternal inheritance of cpDNA holds true for these intergeneric hybrids. PCR-based molecular markers have been used to determine hybridity early and for easy identification of reciprocal crosses in Vanilla planifolia, Renanthera imschootiana, Aerides vandarum, Vanda stangeana and V. coerulea (Besse et al., 2004; Divakaran et al., 2006; Kishor et al., 2008, Kishor and Sharma, 2009). Polyploidization allowed superior tetraploid hybrid varieties with large and multicolored flowers, short flower stalks, round-shaped petals and good quality flowers to be developed

in Taiwan, Japan, the Netherlands and the United States (Tang and Chen, 2007). Over the past few decades a number of multigeneric hybrids of diverse colors, shapes and sized flowers with a graceful appearance including Vanda, Dendrobium, Aranda, Cattleya, Darwinara, Knudsonara, Lindleyara, Mokara, have been well accepted by the world floriculture market (Fig. 1).

IV.

ORCHID EMBRYOLOGY The pattern of orchid embryo development is unique among flowering plants because of a) the lack of a clear pattern of histodifferentiation; b) the presence of a cuticle over the embryo proper; c) the absence of a cuticle in the suspensor cell wall; d) a suspensor having a transfer cell morphology; and e) accumulation of high levels of abscisic acid (ABA) in mature seeds of some terrestrial species (Lee et al., 2007). Thus, a comprehensive understanding of the structural and physiological changes during orchid embryo development and selection of the proper age of the developing embryos can facilitate the successful culture of embryos or immature seed, which is a useful in vitro micropropagation technique for orchids, especially for hybrids in which early embryo abortion takes place. The seeds of most angiosperms are characterized by the presence of an embryo which differentiates into the cotyledon(s), radicle, plumule and hypocotyl. However, in the Orchidaceae, embryo development is not as advanced as in other flowering plants (Kauth et al., 2008). In orchids, the embryo is poorly differentiated, and meristems and cotyledons are usually not present at the time of seed dispersal (Yeung et al., 1996). Seeds are minute in size, housed within a thin seed coat, consisting of about 100 cells that are arranged along a longitudinal axis according to size and the embryo contains only a small quantity of reserve material (Veyret, 1974; Leroux et al., 1997). A variety of embryo developmental patterns, especially regarding suspensor morphology, have been reported within the Orchidaceae (Swamy, 1949a, b). Megasporogenesis of Doritis pulcherrima and Phaius tankervilliae (Aiton) Bl. was observed under transmission electron microscope (Li et al., 2005; Wu et al., 2005), as were the growth paths of pollen tubes in the ovary of Phaius tankervilliae and the response of egg cell during the process of fertilization in Doritis pulcherrima Lindl (Li et al., 2006a, 2006b). The suspensor can be extremely reduced in size as in Lecanorchis japonica (Tohda, 1971) or it can become a haustoria-like organ with elaborate extensions as in Habenaria platyphylla (Swamy, 1946) or the suspensor cells may collapse in the mature embryo as they became dehydrated, as observed in Cymbidium sinense (Yeung et al., 1996). The suspensor can function in nutrient uptake and as a temporary site of food storage for the developing embryo (Lee et al., 2008). Embryo development is characterized by its irregular pattern of early divisions and its unique filamentous suspensor cells. Unfortunately, except for detailed embryological studies in Cymbidium bicolor by Swamy (1943), little information is available concerning ovule and embryo development in the Orchidaceae. Transient

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A

B

D

G

J

C

E

H

F

I

K

L

FIG. 1. The ultimate stage for testing the success of an in vitro protocol in orchids lies in the flowering stage. These photos represent flowering adult plants of species that were successfully propagated in vitro, and flowered in a greenhouse. (A) Phalaenopsis Balithi clone (E. 2122); (B) Phalaenopsis Barbara Moler × (Phalaenopsis Golden Buddha × Phalaenopsis Sara Lee Carlos); (C) Phalaenopsis plantlet after culture in vitro on New Phalaenopsis medium containing 0.5 mg/l BA; (D) Flowering plant derived from hybridization of Spathoglottis plicata × Spathoglottis ingham; (E) Flowering plant derived from hybridization of Spathoglottis Prime Rose × Spathoglottis vanoverberghii; (F) Paphiopedillum sangii; (G) Paphiopedillum Maudiae Black; (H) Indonesian Black orchid originated from Pagar Alam, Palembang (Coelogyne pandurata); (I) Dendrobium Burana Green; (J) Dendrobium Burana White; (K) Vanda Balithi accession Acc. No. 83; (L) Phalaenopsis Brother Girl. F-L were used as the mother plant in a hybridization orchid program. Unpublished photos (Budi Winarto) (color figure available online).


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accumulation of storage products such as starch grains in the suspensor cells has been observed during embryo development in a number of orchid species (Yeung and Law, 1992; Lee et al., 2007). Since suspensors in flowering plants play an important role during early development (Yeung and Meinke, 1993), careful analysis of embryo development may provide additional insight into the function of the suspensor (Yeung et al., 1996). During embryo development, cuticular materials can accumulate in different layers of the seed coat as reported for Cymbidium (Yeung et al., 1996), Cypripedium (Lee et al., 2005), Paphiopedilum (Lee et al., 2006), Calanthe (Lee et al., 2007), Phalaenopsis (Lee et al., 2008) and Oncidium (Mayer et al., 2011). Cuticular materials that accumulated usually formed a uniform layer enveloping the embryo proper (Lee et al., 2008). In some orchid species, for instance P. amabilis var. formosa, the cuticular materials form a discontinuous layer covering the embryo proper which might enable the embryo to access water and nutrients from the environment and involve fewer physical constraints on embryo growth and development, resulting in improved seed germination (Lee et al., 2008). On the other hand, in some terrestrial orchid species such as Cypripedium formosanum and Calanthe tricarinata, the accumulation of cuticular substances in the testa appears to be a tightly fitted cuticular coating which may be a physical barrier restricting embryo growth and a reason for low germination (Lee et al., 2005, 2007). It may also be related to protection of the embryo against anticipated desiccation since the seed has a thin integument and lacks an endosperm (Mayer et al., 2011) although, in orchids such as Spiranthes australis, which lack a structurally defined suspensor, further studies concerning nutrient acquisition by the developing embryo proper are needed (Clements, 1999; Lee et al., 2008). The ovule of orchids is anatropous and tenuinucellate, with one or two integuments and in most orchids the nucellus is represented only by the epidermis enveloping the megaspore mother cell. In certain species, namely Gymnadenia conopsea, Hetaeria nitida and Galeola septentrionalis, apart from the epidermal layer, the apical cells of the axial row also constitute the nucellus (Shamrov and Nikiticheva, 1992). In Paphiopedium insigne and Galeola septentrionalis, the nucellus in the chalazal portion of the ovule remains until fertilization while in Epipogium aphyllum the nucellus persists completely at the stage of the mature female gametophyte (Afzelius, 1954). In some species such as Gastrodia elata, Cypripedium parviflorum and Bulbophyllum neilgherrense the nucellus cells begin to degenerate during early embryogenesis (Shamrov and Nikiticheva, 1992). The archesporium is usually uni- to multicellular, and parietal cells are absent (Johri et al., 1992). Megaspore tetrads are linear or Tshaped and megaspore triads sometimes form instead of tetrads (cf . Batygina et al., 2003). In the Orchidoideae, embryo development follows the Polygonum or the Adoxa type (Johri et al., 1992; Mayer et al., 2011). The embryos consist of cells that are very similar to each other, specialized tissues are not evident and even mature embryos are not differentiated (Arditti, 1992). In Cypripedioideae, the female gametophyte develops according to

the Allium type. The development of a bisporic female gametophyte has been noted in primitive taxa of uni-stamenal orchids as well as in species from Neottiae and Epipogiae (Hagerup, 1947; Arekal and Karanth, 1981). In certain orchid species, both mono- and bisporic female gametophytes occur even in the same ovary (Andronova, 1988; Law and Yeung, 1993). Synergids are pear-shaped, with a filiform apparatus; antipodal cells are represented by nuclei or 1–3 cells; antipodal and polar nuclei fuse in some orchids (Calanthe), creating a polar-antipodal group (Batygina et al., 2003; Lee et al., 2007; Mayer et al., 2011). Polyembryony is typical for the Orchidaceae. The presence of several embryos in a single seed has been reported in certain tropical species, including Cymbidium bicolor, Eulophia epidendrrae, E. nuda, Zeuxine sulcata and Habenaria platyphylla (Arditti, 1992) although this feature rarely occurs in species growing in temperate zones such as Dactylorhiza baltica (Andronova, 1988) and Hammarbya paludosa (Bragina, 2001). Cleavage embryoidogeny is widespread among the orchids and can be observed at different stages of daughter sporophyte development (Calanthe veitchii, Habenaria platyphylla, Eulophia epidendraea, E. nuda, Geodorum densiflorum) (Batygina et al., 2003). Additional embryos can be created as a result of thorough budding, proliferation of the sexual embryo, nucellar, integumentary and monozygotic cleavage embryoidogeny, as well as gametophytic apomixis (Arditti, 1992; Batygina, 1998; Batygina et al., 2003). Pseudopolyembryogeny, i.e., the existence of two or more embryos in one seed, but not all of them derived from the same egg cells, also occurs in orchids such as Orchis morio, Gastrodia elata, Listeria ovata, Cyprepedium calceolus, among others (Arditti, 1992). Swamy (1943) described two variations of the development of monozygotic twins in orchid seed: (1) the zygote divides with abnormalities, forming a large number of cells; the cells located closer to the chalazal end grow simultaneously and give rise to numerous somatic embryos (embryoids); and (2) somatic embryos form from suspensor cells or from the body of an embryo itself; this is observed at late stages of daughter sporophyte development.

V.

ORCHID TISSUE CULTURE, MICROPROPAGATION AND IN VITRO BIOTECHNOLOGY Micropropagation or large-scale clonal propagation is the first major and widely accepted practical application of plant biotechnology that has gained the status of a multibillion-dollar industry throughout the world (Winkelmann et al., 2006; USDA 2009; Liao et al., 2011). According to a statistics from the United States Department of Agriculture, the US export value of orchid seedlings reached $270,000 in 2003 (Chan et al., 2005) and that of Phalaenopsis was approximately US $140 million in 2008 in the USA (USDA 2009). The sales volume of Phalaenopsis in the 2011 Chinese New Year of was about 43 million plants (China Flower and Gardening News, http://news.china-flower.com/paper/papernewsinfo.asp?n id= 223425; 2012-02-13).



The discovery of asymbiotic germination of orchid seeds by Lewis Knudson (1921) was the first practical procedure for in vitro propagation of any plant under axenic conditions. Without a doubt he made a quantum jump discovery by formulating a method for symbiotic germination of orchid seeds. His method was a significant conceptual and technological innovation that served as the basis of modern plant biotechnology and continues to remain the bulwark of plant tissue culture. The birth of orchid micropropagation was in 1960 when Morel first cultured Cymbidium shoot meristems, and since then, modern biotechnology has reshaped orchid research and revolutionized orchid industries. For example, in 2004, different laboratories in Germany produced more than 31 million Phalaenopsis plants in vitro (Winkelmann et al., 2006). The induction of totipotent callus, somatic embryogenesis from callus, direct somatic embryogenesis (protocorm-like body (PLB) induction; Teixeira da Silva and Tanaka 2006), direct regeneration through shoot-bud formation from different explants viz. leaf, nodal, flower stalk, root, rhizome and pseudobulb segments; thin-sections of shoot tips, leaves, protocorms and roots, and even shoot-bud or PLB formation from cell suspension cultures as well as scale-up of production through bioreactors of several major commercial orchids has benefited micropropagation and transgenic research. Good quality, long vase-life and attractive flower production is a major concern of the orchid industry and flower production depends on the genetic makeup of orchid hybrids and how well they are grown. To achieve maximum flower yield, proper agronomic practices must be understood and adopted. Equally important is the control of flowering to meet market demand (Hsiao et al., 2011b). The ability to control flowering in tropical orchids by physiological tools is crucial in the advance of orchid biotechnology (Sim et al., 2007). The thin cross section (TCS) and thin cell layer (TCL) culture systems are also promising and efficient techniques with regard to the total output of plantlets than other conventional in vitro methods for rapid regeneration of orchids. However, these culture systems have not yet been fully exploited for propagation of commercially important orchids except in a few orchid species including Cymbidium (Begum et al., 1994; Nayak et al., 2002; Teixeira da Silva and Tanaka 2006; Teixeira da Silva et al. 2005a, b, 2006a, b, 2007a, b; Hossain et al., 2008, 2009a, 2010), Aranda (Lakshmanan et al., 1995) Phaleanopsis, Rhynchostylis (Bui et al., 1999) Spathoglottis (Teng et al., 1997), and Dendrobium (Nayak et al., 2002). The advantage of the TCL system is to produce a high frequency of shoot regeneration and to reduce the time interval required. In an estimate it was shown that more than 80,000 plantlets could be produced from a single transverse TCL in a year as compared to the 11,000 plantlets produced by the conventional shoot tip method (Teixeira da Silva, 2003). Using the Plant Growth Correction Factor, it would now be easier to make direct comparisons between protocols that involve different sized explants, including TCLs and other explants (Teixeira da Silva and Dobránszki, 2011).

83

Somatic embryogenesis is another potential method to mass propagate orchids. A somatic embryo is widely considered to be of a single-cell origin and is advantageous in studies on spontaneous variation which originate during in vitro culture or experimentally induced by mutagens or transformation (Chen and Chang, 2006). However, somatic embryos have not been well-documented for orchids although histological and cytological analyses confirmed that PLBs are in fact equivalent to somatic embryos (Steward and Mapes, 1971; Begum et al., 1994; Chen et al., 1999; Teixeira da Silva and Tanaka, 2006). Spontaneous formation of organs that resemble protocorms are termed PLBs in orchids and their development is considered to be a peculiarity which could be developed in two ways, direct embryogenesis i.e., PLBs develop without intervening callus phase and indirect embryogenesis i.e., PLBs develop with an intermediary callus phase (Steward and Mapes, 1971; Chen et al., 1999; Begum et al., 1994; Huan et al., 2004; Huan and Tanaka, 2004a, b; Bhadra and Hossain, 2004; Teixeira da Silva et al., 2006a, 2007a; Liao et al., 2011; Ng and Saleh, 2011; Sujjaritthurakarn and Kanchanapoom, 2011). A number of earlier reports provided evidence for somatic embryogenesis in orchids, the most representative of which are summarized in Table 2. The morphogenetic response in orchids varies depending on the explants as well as on the endogenous hormone level (Chugh et al., 2009; Lu et al., 2001). However, the combinations, concentrations, and the ratio of plant growth regulators (PGRs) are critically important, Figure 2 (Teng et al., 1997; Huan et al., 2004). Many reports were published on organogenesis and somatic embryogenesis in orchids such as in Cymbidium ensifolium (Chang and Chang, 1998), Geodorum densiflorum (Bhadra and Hossain, 2003a), Cymbidium bicolor (Malabadi et al., 2008), Cymbidium hybrids (Teixeira da Silva et al., 2005b, 2006a, b, 2007a, b), Phalaenopsis (Ishii et al., 1998; Chen and Chang, 2006; Gow et al., 2008), Oncidium (Chen et al., 1999; Su et al., 2006), Dendrobium (Chung et al., 2007), Paphiopedilum (Hong et al., 2008; Long et al., 2010), Malaxis acuminata (Cheruvathur et al., 2010), Vanilla planifolia (Tan et al., 2011), Dendrobium nobile (Asghar et al., 2011), Nothodoritis zhejiangensis (Zeng et al., 2011), Phalaenopsis gigantea (Niknejad et al., 2011), Cymbidium bicolor (Mahendran and Bai, 2012), Renanthera (Wu et al., 2012), Cymbidium aloifolium and Cymbidium iridioides (Deb and Pongener, 2012), among others. Somatic embryogenesis is comparatively more efficient than orgaganogenesis, easy to carry out and can provide large numbers of embryos and plants for mass propagation within a short period of time. Tokuhara and Mii (2001, 2003) reported another efficient system for somatic embryogenesis from a cell suspension culture induced from shoot-tip explants of Phalaenopsis. Zhao et al. (2008) induced somatic embryos in Dendrobium candidum at a high frequency from bisected segments of protocorms. These systems would facilitate commercial micropropagation of orchids because they have very high regeneration potential compared to other methods. Moreover, embryogenic callus is considered to be an appropriate target material for genetic transformation

84

Shoot tips of flower stalk buds

Axillary buds

Foliar explants

Phalaenopsis and Doritaenopsis

Vanilla planifolia Jacks. ex Andrews

Acampe praemorsa (Roxb.) Blatter and McCann Phalaenopsis Richard Shaffer ‘Santa Cruz’

Oncidium Sweet Sugar

Oncidium Gower Ramsey Geodorum densiflorum (Lam.) Schltr.

Seed–derived rhizome Thin transverse sections of shoot tip

Cymbidium kanran Makino Aranda Deborah

Young leaf segments In vitro seed–derived rhizome sections Flower-stalk internodes

Protocorm-like bodies (PLBs)

Root segments

Explant used

Rhynchostylis retusa Bl.

Species

Type of response

1 2 MS

with TDZ (0.1–3 mg l−1) and NAA (0.1–3 mg l−1).

Organogenesis/ Embryogenesis

MS and 12 MS with TDZ (0.01–3.0 mgl−1), Embryogenesis 2,4–D (3.0 –30.0 mgl−1), NAA (0.5 mgl−1). MS, KC with BAP and NAA (0.5–20 μM Organogenesis each); IAA and IBA (0.5–5 μM each).

Solidified, semi–solidified and liquid MS and Organogenesis 1 −1 2 MS with BAP (1, 2, 5 or 10 mg1 ), NAA and IAA (0.5–1 mg1−1). MS with BA (1.0–10.0 mg1−1), KN (1.0–10.0 Organogenesis mg1−1), TDZ (0.01–2.0 mg1−1), NAA (0.5–5.0 mg1−1). Vacin and Went medium with 200 ml/l Embryogenesis coconut water and 40 g/l sucrose.

New Dogashima Medium (NDM, 1993) Embryogenesis containing 0.1 mg/1 NAA) and 1 mg/1 BAP.

MS with IAA, NAA, KN and BAP (1.0 Organogenesis/ each. Embryogenesis MS with BAP and NAA (0.1, 1.0, 10.0 mgl−1 Embryogenesis each). MS, KC, VW with BA (0 – 8.8 μM), NAA Embryogenesis (0.0–5.5 μM), AC (0.5 gl−1), CW (5–25%).

mgl−1)

Culture medium, PGRs and additives mgl−1).

Somatic embryos and shoot buds directly from wound surfaces or via proliferation of nodular masses in 3 mgl−1 TDZ in darkness. In light, the nodular masses produced green compact callus, and then somatic embryos, shoot buds and/or yellowish abnormal structures spontaneously. Supplementing 0.1 mgl−1 NAA enhanced embryo formation, but retarded proliferation of shoot buds and yellowish abnormal structures.

The frequency of direct shoot regeneration was highest in 1.0 mg1−1 TDZ which declined markedly at higher concentrations. Protocorm-like body (PLB) segments formed calli in Vacin and Went medium with sucrose. The optimal concentration of sucrose was 40 g/l. Medium containing 200 ml/l coconut water together with 40 g/l sucrose was effective for callus induction. Gellan gum was suitable than agar as a gelling agent for callus induction. The calli easily formed PLBs after being transferred to a medium without sucrose. 1 MS supplemented with 3.0 mgl−1 TDZ was efficient for 2 embryogenesis. MS with 2.0 μM NAA stimulated rhizome growth while BA at 5.0 μM favoured induction of multiple shoots.

Maximum number of PLBs induced in 10.0 mg1−1 BAP and 1.0 mg1–l NAA. Maximum number of PLBs (13.6/explant) produced in VW with 20% (v/v) CW. PLBs profusely proliferated in 2.75 NAA with 20% (v/v) CW in VW. These developed into plantlets on solid VW containing 10% (v/v) CW and 0.5 gl−1 AC. Green Protocorm-like Bodies (PLB) with high multiplication capacity were induced from shoot tips of flower stalk buds having 1 or 2 leaf primordia using New Dogashima Medium (NDM) containing 0.1 mg/l NAA and 1 mg/1 BAP. These PLB were subcultured on the same medium. More than 10,000 PLBs were obtained from a few buds on a single flower stalk within one year. After transfer onto NDM containing no plant growth regulator (PGR), the PLB developed into plantlets. The maximum number of direct multiple shoots were induced in solidified MS + 2.0 mg1−1 BA + 1.0 mg1−1 NAA.

Maximum number of PLBs observed in KN (1.0

Remarks

TABLE 2 Somatic embryogenesis (PLB production) and organogenesis in the Orchidaceae


Chen and Chang, 2000

Sheelavanthmath et al., 2000

Chen et al., 1999

Ishii et al., 1998

Nayak et al., 1997

George and Ravishankar, 1997

Tokuhara and Mii, 1993

Shimasaki and Uemoto, 1990 Lakshmanan et al., 1995

Vij et al., 1987

Reference

85

Rhizomes

Elongated stems

Flower-stalk internodes

Stem nodal explants

Cymbidium ensifoilum cv. Yuh Hwa Paphiopedilum hybrids

Epidendrum radicans Lindl.

Paphiopedilum philippinense hybrids (hybrid PH59 and PH60)

Modified MS with BAP (2.5–20 μM).

MS medium containing 45 gl−1 sucrose and BA (15 mgl−1) NAA (1 mgl−1). Hyponex medium (Kano, 1965; 6.5N – 4.5P – 19K 1 gl−1 + 20N – 20P – 20K 1 gl−1 + 1% potato homogenate)

MS with TDZ (0.45–4.54 μM), BA (0.44–44.38 μM), ZR (0.28–28.46 μM), KN (0.46–46.47 μM), sucrose (0, 10, 20, 30, 40 gl−1), NH4 NO3 (0, 165, 330, 495, 660, 825 mgl−1), KNO3 (0, 190, 380, 570, 760, 950 mgl−1). 1 MS with 2,4–D (4.52–45.25 μM), TDZ 2 (0.45–4.54 μM).

Modified MS with BA (0.13–444 μM), NAA (0.16–5.4 μM), TDZ (0.0014–0.14 μM), adenine (0.15–1.5 mM), CH (1.0–4.0 gl−1), CW (0.0–30% v/v), potato powder (10.0–30.0 gl−1) and banana powder (20.0–60.0 gl−1). Phalaenopsis [(Baby hat Shoot-tip explants New Dogashima Medium (NDM) containing 0.5 μM NAA, 4.4 μM BAP and excised from × Ann Jessica) 29.2–58.4 μM sucrose. flower stalk ×Equestris] buds

Flower stalk sections containing nodal buds

Phalaenopsis hybrid


Embryogenesis

(Continued on next page)

Tokuhara and Mii, Embryogenic calluses were induced from 73% of 2001 Phalaenopsis shoot-tip explants excised from flower stalk buds by culturing for 7 mo. on New Dogashima Medium (NDM) containing 0.5 μM NAA, 4.4 μM BAP and 29.2 μM sucrose. The sucrose concentration was increased to 58.4 μM 4 mo. after initiation of the callus culture. These calluses were successfully subcultured as cell suspension cultures in liquid NDM supplemented with 5.4 μM NAA and 58.4 μM sucrose. By simply reducing the sucrose concentration to 29.2 μM, the cells grew into plantlets through a developmental process similar to that of Phalaenopsis seedlings. Highest PLB induction was observed with 40 gl−1 sucrose + Chen et al., 2002 825 mgl−1 NH4 NO3 + 950 mgl−1 KNO3 while maximum number of PLBs with shoots was found at 10 gl−1 sucrose + 825 mgl−1 NH4 NO3 , + 950 mgl−1 KNO3 . The best hormone combinations were 4.44 μM BAP + 0.28 μM ZR, and only KN at 1.39 μM for proliferation of PLBs. A combination of 4.52 μM 2,4–D and 0.45 μM TDZ induced Chen et al., 2002 higher percentage of shoots in hybrid PH59 while in hybrid PH60, the highest shoot number was found in 4.52 μM 2,4-D. Embryogenesis

Organogenesis

Organogenesis

Protocorm-like bodies (PLBs) formed on leaf segments in Young et al., 2000 vitro were induced on MS medium containing 45 g/l sucrose and BA (15 mgl−1) NAA (1 mgl−1). A temporary immersion culture with charcoal filter attached was most suitable for PLB culture. About 18,000 PLBs were harvested from 20 g of inoculum (∼1000 PLB sections) in 2 l Hyponex medium after 8 weeks of incubation. Aeration in a bioreactor at 0.5 or 2.0 volume of air per volume of medium per min (vvm) yielded similar levels of biomass production. Hyponex medium was found to be suitable for conversion of PLBs into plantlets and 83% of PLBs transformed into plantlets on this medium. The feasibility of using PLBs for large-scale micropropagation was evaluated for scaled-up liquid cultures in bioreactors, rate of proliferation, and regeneration. The maximum number of shoot formation occurred in 2.5 μM Lu et al., 2001 BA Medium supplemented with 1.0 gl−1 CH, 15% CW, 10.0 gl−1 Huang et al., 2001 potato powder and 20.0 gl−1 banana powder was optimum for shoot multiplication.

Embryogenesis


86

Ipsea malabarica (Reichb. f.) J. D. Hook.

Rhizome segments

Calli originated from flower stalks 1 2 MS

Organogenesis with BA (2.22–6.66 μM), KN (2.32–6.96 μM), IBA (2.46–4.90 μM), IAA (2.85–5.71 μM), NAA (2.69–5.37 μM).

NP with potato extract (PE), corn extract (CE), Embryogenesis papaya extract (PAE) (25, 50, 100, 200 ml−1).

Organogenesis

High callus growth was found in 100 ml−1 PE or PAE and 200 ml−1 CE. Maximum PLBs regeneration from calli and subsequently plantlets was found in the medium fortified with 100 ml−1 CE. Highest multiple shoot induction and proliferation was observed in 6.97 μM KN.

Glucose (58.4 mM) was the best carbohydrate source for producing PLBs. Addition of 0.4 μM BA increased callus proliferation but completely inhibited PLB formation.

Embryogenesis

Phalaenopsis Snow Parade, P. Wedding Promenade and Doritaenopsis New Toyohashi Doritaenopsis sp.

Geodorum densiflorum (Lam.) Schltr.

MS, PM with NAA (0.5–2.0 mgl−1), BAP In vitro seed–derived (0.5–2.0 mgl−1), Pic (0.5–1.0 mgl−1), ZN rhizome (0.5–1.0 mgl−1), IAA and IBA (1.0 mgl−1 sections each). Shoot tip–derived NDM medium with NAA (0–0.5 μM), BAP suspension cells (0.4–4.4 μM), sucrose, glucose, fructose, sorbitol, maltose, lactose (29.2–58.4 mM each).

Modified KC with BAP (0.0, 1.0, 2.0, 3.0 and 4.0 mgl−1), NAA (0, 0.5, 1.0 and 2.0 mgl−1).

Shoot tip explants

Dendrobium fimbriatum Lindl. var. oculatum HK. f. Embryogenesis

Embryogenesis

Remarks

MS medium with BA (88.8μM) and NAA (5.4 μM). Hyponex medium (1 g1−l 6.5N-4.5P-19K + 20N-20P-20K + 2 g1−l peptone + 3% (w/v) potato homogenate + 0.05% activated 1 g1−l charcoal).

Type of response

Leaf segments Phalaenopsis Hybrids derived in vitro cv. ‘Taisuco Hatarot’, from flower Tinny Sunshine stalk nodes ‘Annie’, Taipei Gold ‘Golden Star’, and Tinny Galaxy ‘Annie’

Culture medium, PGRs and additives High induction of PLBs was established on MS medium supplemented with ZR at 14.0 mM for C. aloifolium while BA at 11.0 mM for D. nobile. Average number of PLBs per TCS explant was high in both the cases (28.2 in C. aloifolium, and 34.0 in D. nobile) though inhibitory effect was noticed with cytokinins above the optimal level. PLB development from TCS explants in both the species was enhanced by the use of suspension culture. High frequency of shoots (85% in C. aloifolium and 80% in D. nobile) regenerated of PLBs were rooted on MS medium containing 9.8 μM IBA. Regenerated plants were successfully acclimatized and eventually transferred to the field. MS medium with BA (88.8μM) and NAA (5.4 μM) produced an average of 10–13 protocorm-like bodies (PLBs) after 12 wk. PLB proliferation was achieved on a modified Hyponex medium (1 g1−l 6.5N-4.5P-19K + 20N-20P-20K + 2 g1−l peptone + 3% (w/v) potato homogenate + 0.05% activated 1 g1−l charcoal) and an optimal number of 13–18 PLBs developed from single PLB sections of different cultivars. Plantlet development was also achieved on a modified Hyponex medium. By repeated subculture of PLBs on a proliferation medium, and culturing them in the plantlet regeneration medium, plantlets could be produced continuously. Approximately 6 mo. were required from the initiation of culture to the production of plantlets for transplant to community pots Compact embryonic callus were induced in 0.5 mgl−1 NAA and 1.0 mgl−1 BAP. Following transfer to PGR-free medium the callus further proliferated along with PLBs production and well-rooted plantlets. The highest number of multiple shoots buds (4.50/explant) were induced in 2.0 mgl−1 BAP + 2.0 mgl−1 NAA.

Explant used Embryogenesis

Cymbidium aloifolium (L.) Sw. and Dendrobium nobile Lindl

MS medium with Zeatin Riboside (14.0 μM), Thin cross BA (11.0 μM) and IBA (9.8 μM) sections (TCSs) of protocormlike bodies

Species

TABLE 2 Somatic embryogenesis (PLB production) and organogenesis in the Orchidaceae (Continued)


Reference

Martin, 2003

Islam et al., 2003

Tokuhara and Mii, 2003

Bhadra and Hossain, 2003a

Roy and Banerjee, 2003

Park et al., 2002

Nayak et al., 2002

87

Half-PLBs, PLB TCLs

Leaf explants

Phalaenopsis amabilis var. formosa

Leaf tip explants (about 1 cm in length)

Nodal segments

modified MS medium supplemented with 0.1, 1 and 3 mg/dm−3 TDZ.

1 2 -strength

MS with BAP, TDZ, KN (0.5–5.0 μM each), NAA, IAA (0.5–1.0 μM each), CW (5, 10 and 15%). 1 MS with BAP (1.0–16.0 mgl−1), NAA 2 (0.10 mgl−1). 1 MS with 2,4-D (1.36–13.56 μM), IAA 2 (1.71–17.13 μM), IBA (1.48–14.70 μM), NAA (1.61–16.11 μM), 2iP (2.46–39.36 μM), BA (2.22–35.48 μM), Kn (2.32–37.16 μM), TDZ (2.27–36.32 μM), Zn (2.28–36.48 μM). VW + various concentrations of NAA, Kinetin, BA, and multiple media additives, including CW, AC

Protocorm and leaf sections

Cymbidium cv. Twilight Mon ‘Daylight’

Dendrobium candidum Wall. ex Lindl. Dendrobium cv. Chiengmai Pink

Phalaenopsis amabilis var. formosa Shimadzu Aerides crispum L.

MS with putrescine, spermidine, spermine (0.2, 0.4, 1.0 mM each), BAP (4.44 μM), NAA (5.38 μM). Modified 12 MS with NAA (0, 0.54, 5.37 μM, TDZ (0.45, 4.54, 13.62 μM).

Leaf explants

Paphiopedilum phiIippinense hybrids (hybrid PH59 and PH60) Dendrobium ‘Sonia’

Embryogenesis


Embryogenesis

Organogenesis

Embryogenesis

Embryogenesis

Embryogenesis

MS, KC, and Hyponex with BAP (0.1, 0.5, 1, Organogenesis 2 and 3 mgl−1), TDZ (0.1, 0.5, 1.0, 2.0 and 3.0 mgl−1), KN (0.1, 0.5, 1.0, 2.0 and 3.0 mgl−1), sucrose (1, 2, 3, 5 and 7%), and AC (0.0, 0.5, 1.0, 3.0 and 5.0 gl−1). Modified MS with TDZ (0.45–22.71 μM), Organogenesis 2,4-D (0.0–45.25 μM).

Shoot tip, fractionated PLB mass Protocorms

Shoot tip explants

Anoectochilus formosanus Hayata

Chung et al., 2005

Shiau et al., 2005

Sheelavanthmath et al., 2005

Teixeira da Silva Large variability in results depending on the medium and et al., 2005a,b, growth conditions and explant used. Light microscopy and 2006a,b, 2007a, b; scanning electron microscopy used to cofirm developmental Teixeira da Silva processes over time, flow cytometry to confirm ploidy and Tanaka, 2006 levels over the developmental period and RAPD analyses to confirm genetic stability. Teixeira da Silva coined the terms primary and secondary PLBs. Chen and Chang, Leaf explants of Phalaenopsis amabilis var. formosa formed 2006 clusters of somatic embryos directly from epidermal cells without an intervening callus within 20–30 d when cultured on 1/2-strength modified MS medium supplemented with 0.1, 1 and 3 mg/dm−3 TDZ. Repetitive production of embryos involved secondary embryogenesis could be obtained by culturing segments of embryogenic masses on TDZ-containing media. Plantlet conversion from embryos was successfully achieved on regulator-free growth medium. (Continued on next page)

Direct PLB formation was noticed. The maximum number of PLBs (49.1 and 22.0) were developed from protocorm and leaf sections in presence of 1.0 μM BAP. Highest shoot bud production was found in 2.0 mgl−1 BAP and 0.1 mgl−1 NAA and best rooting in 0.2 mgl−1 NAA. The best medium composition for direct embryo induction was 12 MS+18.16 μM TDZ where 33% of explants formed a mean number of 33.6 embryos/explant.

Direct embryo formation was reported. The best response was Chen and Chang, found in 12 MS modified with 13.62 μM TDZ. 2004

Ket et al., 2004 Hyponex medium supplemented with 1.0 mgl−1 BAP or 1.0–2.0 mgl−1 TDZ was most effective for induction of multiple shoots and shoot proliferation. Addition of AC (1.0 gl−1) with TDZ-containing medium promoted multiple shoot formation (11.1 shoots/explant). In hybrid PH59, 4.54 μM TDZ was effective for induction of Chen et al., 2004 high number of direct adventitious shoots/explant while 4.52 μM 2,4-D + 0.45 μM TDZ favoured shoot induction in hybrid PH60. Maximum number of PLBs formed in MS fortified with BAP Saiprasad et al., 2004 (4.44 μM) and 0.4 mM putrescine.


88

Dendrobium cv. Chiengmai Pink

Dendrobium phalaenopsis Lindl.

Shoot segments with meristematic auxillary buds Leaf tip segments of in vitro-grown plants

Cypripedium flavum P.F. Seedlings Hunt & Summerh

Phalaenopsis gigantea J. Protocorms with J. Smith trimmed and untrimmed bases Coelogyne stricta Pseudobulb (D.Don) Schltr. segments

Root tips and cut ends of stem and leaf segments

Axillary buds

Dendrobium Second Love (Dendrobium Peace × Dendrobium Awayuki Vanda coerulea Griff.

Explant used

In vitro derived foliar explants

Dendrobium hybrids Sonia 17 and 28

Species

MS with NAA (0, 0.1, 1 mgl−1) and TDZ (0, 0.3, 1, 3 mgl−1)

Harvais medium with BAP (2.22–4.44 μM), KN (2.32–4.65 μM), AC (0.3–0.6 gl−1), potato homogenate (0, 20 and 40 gl−1) Liquid and agar solidified MVW with Shrimp and fungal chitosan (0, 5, 10, 15, 20, 25 mgl−1)

Embryogenesis

Embryogenesis

Organogenesis

Organogenesis

Embryogenesis

XER medium (Ernst, 1994) with 0, 10, 15 or 20% (v/v) CW; and 0.0, 1.0, 2.0 or 2.5 gl−1AC MS with BAP and NAA (0.5–2.0 mgl−1 each)

Embryogenesis

MS basal medium supplemented with TDZ (0,1–3 mgl−l), and 2,4 D (3–10 mgl−l)

1 4

Organogenesis

Type of response Organogenesis/ Embryogenesis

Culture medium, PGRs and additives MS with BA (44.4 μM), KN (6.97 μM), 2 gl−l activated charcoal

VW medium supplemented with TDZ (0.45–1.8 μM)

1 2

Reference

Nge et al., 2006

Yan et al., 2006

Basker and Bai, 2006

Murdad et al., 2006

Lang and Hang, 2006

Ferreira et al., 2006

Martin and Madassery, 2006

Somatic embryos mostly developed from leaf surfaces near cut Chung et al., 2007 ends, and occasionally found on leaf tips. High frequency of embryogenesis obtained in light than in darkness. 5–25% of explants directly produced embryos after 60 d of culture, and 1 mgl−1 TDZ was the best treatment.

Growth of PLBs in liquid medium increased up to 15 times over solidified medium in chitosan oligomer, the optimal concentration being 15 mgl−1.

High shoot formation was found on 12 MS with BA (44.4 μM). Repeated-subculture of the shoots in the medium produced PLBs more than 200 in fifth subculture. High conversion of the PLBs to shoots was determined on 12 MS supplemented with KN (6.97 μM), easily rooted on 12 MS containing 2 gl−l activated charcoal, and successfully acclimatized ex vitro condition up to 80%. Shoots originated from the excised buds after 60 d of culture on VW medium supplemented with 0.45 μM TDZ. High shoot number up to 6.24 shoots per explant was determined on VW medium enriched with 1.8 μM TDZ. Embryogenic calli from root tips, cut ends of stem and leaf segments was easily determined on 14 MS basal medium supplemented with TDZ (0,1–3 mgl−l), and 2,4 D (3–10 mgl−l) for 4 weeks. Embryogenic callus was maintained by subculture on the same medium for callus induction and proliferated 2 times in 1 month. The survival rate of plantlets under in vitro condition was 90% after 2 months. The high regeneration capacity may be due to the callus origin from meristematic cells of root tips. It was also found that very different ability of somatic embryogenesis between cell lines in the same callus origin. Trimmed protocorms exhibited the highest PLB induction in 15% (v/v) CW and 2.5 gl−1 AC (4.24/protocorm) while untrimmed protocorms produced highest number (0.72/protocorm) in 20% (v/v) CW alone. BAP (1.0 mgl−1) + NAA (2.0 mgl−1) or BAP (2.0 mgl−1) individually induced maximum number of multiple shoots while maximum rooting was in NAA at 1.0 and 2.0 mgl−1. BAP (2.22 μM) and potato homogenate (20 gl−1) was the most effective for high frequency multiple shoot production.

Remarks



89

PLB tTCLs

In vitro PLBs

In vitro seedlings

Dendrobium candidum Wall. ex Lindl.

Dendrobium sp.

Cymbidium aloifolium (L.) Sw. Spiranthes acmella Murr.

medium with Sabri banana pulp (10%, w/v). KC medium with Sabri banana pulp (10%, w/v) MS with BAP (0–7.0 mgl−1)

1 2 MS

Embryogenesis

Embryogenesis

Embryogenesis

Organogenesis

Embryogenesis

Embryogenesis

Organogenesis

Embryogenesis New Dogashima medium (1/2 NDM) containing 0.44 M sorbitol, 0.06 M sucrose and 0.1 gl−l glutamine. NDM supplemented with 10 gl−l maltose and 0.3% (w/v) gellan gum

MS with different levels of NAA, 6-BA and KN alone or in combination

1 2

MS with BAP (0.5–4.0 mgl−1), KN (0.2–0.5 Nodal buds of in vivo and in vitro mgl−1), 2-ip (0.5–4.0 mgl−1), NAA (0.1–0.5 seedlings mgl−1), IAA (1.0 mgl−1), IBA (0.1 mgl−1). Phalaenopsis amabilis Leaf tip segments 12 MS with 2,4–D, IBA, IAA, NAA, 2iP, BA, Blume and P. Nebula KN, ZN, TDZ, GA3 , ancymidol, putrescine, spermine at different concentrations and combinations 1 −1 Dendrobium densiflorum Nodal segments 2 MS with BAP (0.1–10 mgl ), KN (0.1–2.0 −1 Lindl. mgl ), NAA (0.1–2.0 mgl−1), IAA (0.1–10.0 mgl−1), IBA (0.1–10.0 mgl−1), AC (1.0–5.0 gl−1) MS with NAA (0–10.8μM), BA (0–22.0 μM), Dendrobium candidum Longitudinally 2,4-D (0–13.8 μM), and Kn (0–13.8 μM) Wall. ex Lindl. bisected alone or in combination segments of protocorms

Protoplasts

Shoot-tip explants Knudson’s C medium with TDZ (2 μM), BAP Embryogenesis (2 and 8 μM), NAA (0.5–1 μM)

Phalaenopsis “Wataboushi”

Dendrobium chrysotoxum Lindl.

Frequency of callusing was best determined on Knudson’s C Roy et al., 2007 medium with 2 μM TDZ or BAP. Using a 3-month subculture period, a 69-fold increase in callus weight was achieved with 0.5 μM NAA, while as many as 133 PLBs could be obtained per 100 mg callus in the presence of 1 M NAA. For direct PLB formation, the optimum cytokinin dosage was dependent upon the type of cytokinin used. While TDZ was most effective at a concentration of 1 μM (15 PLBs per explant), for similar PLB yield the application of 8 μM BAP was essential. Shrestha et al., 2007 The highest frequency of microcolony formation (23%) was obtained when protoplasts were embedded in 1% Ca-alginate beads and subcultured every two weeks by replacing the surrounding liquid culture medium with a decrease in sorbitol concentration by 0.1 M. Colonies visible to the naked eyes were observed within 2 months of culture and the regenerated calluses were transferred onto hormone-free NDM supplemented with 10 gl−l maltose and 0.3% (w/v) gellan gum, on which PLBs were formed and proliferated profusely. The PLBs were regenerated into plantlets after changing the carbon source to maltose. High-frequency shoot regeneration occurred in 1.2 mgl−1 Zhao et al., 2007 NAA and 1.2 mgl−1 6-BA. Efficiency of shoot regeneration was related to the orientation and position of explants. With an upright orientation on medium, shoots were induced more efficiently. High number of 50 PLBs per explant and 17.5 shoots per Aktar et al., 2008 explants was easily recorded on 12 MS containing Sabri banana puld (10%, w/v) Hossain et al., 2008 High frequency PLBs produced from the base of seedlings. Maximum number of PLBs (35/seedling) at 5.0 mgl−1 BAP. Maximum number of adventitious shoots/explant (20 ± 0.47) Saritha and Naidu, 2008 produced in 3.0 mgl−1 BA and 1.0 mgl−1 IAA. Direct organogenesis confirmed through anatomical studies. Gow et al., 2008 Direct somatic embryogenesis occurred and highest embryogenic response was observed from cut ends of explants. 13.32 μM BA was the most effective in P. amabilis while 2iP at 4.92 μM was the best for P. Nebula. The highest number of PLBs induced in 5.0 mgl−1 BAP. PLBs Luo et al., 2008 proliferated further to form shoots in 2.0 mgl−1 BAP. Optimum rooting was found in 5.0 mgl−1 IBA with 5.0 gl−1 AC. Maximum callus induction was 82% on 12 MS with 8.8 μM Zhao et al., 2008 BA. PLB formation was achieved when callus was transferred onto PGR-free medium. PLBs developed into intact plantlets in 2.7 μM NAA. (Continued on next page)


90

Explant used

Internodal segments

Leaves and root tips for somatic embryo induction. Regenerated plantlets and somatic embryo for endoreduplication studies. Pseudobulb sections of 1 cm size

In vitro seedlings

Nodal explants

Malaxis acuminata D. Don.

Doritaenopsis ‘Happy Valentine’

Paphiopedilum callosum

Paphiopedilum rothschildianum

Lycaste armomatica

Leaf tip segments (about 1 cm in length)

Phalaenopsis amabilis and P. Nebula

Paphiopedilum Alma Seed–derived callus Gavaert

Species

Culture medium, PGRs and additives

Type of response

Embryogenesis

MS supplemented with different amounts (1, 2, 3, and 4 μM) of cytokinins (BA and KN) and 60 gl−1 banana homogenate or 60 gl−1 potato homogenate and 20% CW

MS and 12 MS with BAP, 2,4-D, TDZ at different concentrations

Embryogenesis

Organogenesis

MS medium augmented with 2 mgl−1 glycine, Organogenesis 100 mgl−1 myo-inositol, and cytokinins: KN, BAP, mT at 0, 0.46, 1.1, 2.2, 4.4, or 8.9 μM.

Modified Hyponex medium with 2gl−1 peptone, 3% (w/v) potato homogenate, 0.5% AC, and 30 gl−1 sucrose. 4 types of lighting conditions: red light, red + far-red light, red + blue light, and white light

MS medium with different concentrations and Organogenesis combinations of BA, KN, TDZ, NAA and various concentrations of different polyamines including spermine, spermidine, and putrescine

Embryogenesis

Embryogenesis/ MS with 2,4-D (22.60 μM), TDZ Organogenesis (4.54 μM), IAA (2.86, 5.71, 28.55 μM), NAA (2.69, 5.37, 26.85 μM), Kn (2.33, 4.65, 23.25 μM), Zn (2.28, 4.56, 22.80 μM)

MS with TDZ

1 2

Highest numbers of shoots/explant were achieved from basal sections cultured in media supplemented with TDZ at 4.4, 8.87 and 2.2 μM, forming on average 9.9, 8.6 and 7.3 shoots/explant, respectively. MS medium without PGRs allowed further development and rooting. 1 2 MS with BAP at 10 μM resulted in maximum number (2.30 ± 1.42) of shoots/explant. 12 MS without PGRs resulted in 100% root induction with an average of 3.70 ± 0.62 roots/explant and mean root length of 34.01 ± 4.87 mm. Propagation was achieved through in vitro formation of secondary PLBs from primary PLBs that developed from stem-derived callus. Highest number of secondary PLBs obtained on 12 MS with 4.0 μM kinetin (average of 4.1 PLBs/explant). Secondary PLBs continued to proliferate further and formed 9.5–12.1 new PLBs/secondary PLB after subculture onto PGR-free 12 MS with 60 gl−1 BH. Among the organic additives, 20% CW was the best for plantlet regeneration percentage from the PLBs.

Totipotent callus induced from seeds on 12 MS medium with 22.60 μM 2,4-D and 4.54 μM TDZ in darkness. The callus produced PLBs or shoot buds when transferred to 12 MS with 26.85 μM NAA. Kinetin at 4.65 μM was suitable for shoot multiplication. Explants cultured in darkness favoured direct embryogenesis. Embryogenic response was dependent on genotype, light regime and explant orientation. Explant placed adaxial-side-up on culture medium had higher embryogenic response than abaxial-side-up orientation. The cut end had highest embryogenic competence than other parts of the explant. The leaf basal segment had higher embryogenic competence than the leaf tip segment. Adventitious shoot buds were induced from internodal explants. The highest frequency of shoot induction (100%) and mean shoot number (14.6/explant) was observed with 3 mgl−1 TDZ, 0.5 mgl−1 NAA, and 0.4 mM spermidine. Highest frequency of rooting (96%) and mean number of roots/shoot (3.3) was observed on 4 mgl−1 IBA and 1.5 mgl−1 AC. Investigated the effects of light quality on endoreduplication and somatic embryo proliferation. Light combinations, i.e. red light and far-red light stimulates somatic embryogenesis and exert some control on degree of endoreduplication during cell division that can be applied to achieve a reduction in somaclonal variation for the purpose of mass proliferation and genetic improvement.

Remarks



Reference

Ng et al., 2011

Wattanawikkit et al., 2011

Mata-Rosas et al., 2010

Park et al., 2010

Cheruvathur et al., 2010

Gow et al., 2009

Hong et al., 2008

91

Phytotechnology medium (O753) Lateral shoots of Embryogenesis/ supplemented with 0.5, 1.0, 1.5, 2.0, 2.5 and about 8 cm Organogenesis 3.0 mgl−1 of BAP and Kin as well as CW at long, arising from the base of 50, 100, 150, 200, 250 and 300 mll−1 the stem of healthy plants maintained in a glasshouse MS with 4.43 μM BAP and 4.52 μM 2,4-D for the culture of Paphiopedilum Armeni White and with 44.39 μM BAP and 26.85 μM NAA for the culture of Paphiopedilum Deperle

Dendrobium nobile var. Emma White

Paphiopedilum Deperle and P. Armeni White

Cross-sectioned FBs; small (1.5–2 cm), medium (2.0–2.5 cm), and large (2.5–3 cm)

Trimmed protocorm cluster (TPC), nodal and shoot-tip explants

Organogenesis

P668 medium with different concentrations (5, Organogenesis 10, 12.5 and 15 μM) of various cytokinins including BA, zeatin, TDZ and mTR

Organogenesis

Embryogenesis

Ansellia africana Lindl.

MS medium with TDZ (0.5, 1.0, 2.0 and 3.0 mg l−1), NAA (0.5 and 1.0 mg l−1) and BAP (0.5, 1.0, 1.5 and 2.0 mgl−1) either individually or in combinations or with banana powder (30 gl−1) and coconut powder (30 gl−1)

MS medium containing 1 gl−1 casein hydrolysate, 2,4-D, NAA and BA at different combinations and concentrations

PLB segments (3 mm2 in size)

Vanilla planifolia

Coelogyne cristata Lindl.

Organogenesis

Shoot tip, internode, leaf segment, auxillary bud and aerial roots Juvenile leaf and nodal segments

Vanilla planifolia Andr. or Vanilla fragrans Salisb

MS with BAP and IBA singly at different concentrations

Only axillary bud and shoot tip explants gave positive results. Neelannavar et al., Among different concentration of BAP, 1.0 mg1−1 produced 2011 highest number and better sized shoots. The maximum number of roots with good length within a short space of time was observed on 0.5 mgl−1 IBA-supplemented media. Juvenile leaf explants induced callus on 2.0 mgl−1 NAA and Tan et al., 2011 2.0 mgl−1 BA but no callus formed in the presence of any concentrations of 2,4-D and BA. The callus produced plantlets in 1.0 mgl−1 BA and 0.5 mgl−1 NAA. BAP in medium was distinctly better for shoot multiplication. Naing et al., 2011 The highest percentage of explants producing shoots, with a maximum average of 8.1/explant, was achieved on 1.0 mgl−1 NAA and 0.5 mgl−1 BA with CP. Shoots produced an average of 15 roots/explant on 12 MS with 2.0 mgl−1 IBA and BP. Ploidy analysis of regenerated plants using flow cytometry revealed the same ploidy level (diploid). Vasudevan et al., Nodal segments developed single shoots from axillary buds 2011 while shoot-tip explants simply elongated and grew, irrespective of PGR supplements. TPC explants proliferated new protocorms and shoot regeneration and highest number of adventitious shoots (6.94 ± 1.16) was observed with TDZ (12.5 μM). When TDZ and BA were joined, root differentiation and root growth were significantly lower. Asghar et al., 2011 Maximum number of shoots (4.33), as well as fresh and dry weights (752.5 and 52.99 mg) were obtained with 2 mgl−1 BAP. KN at 1.5 mgl−1 exhibited the highest shoot length (4.18 cm). Higher concentrations of BAP, KN (3.0 mgl−1) and CW (300 ml) resulted in yellowing, necrotic shoots and poor growth. IBA at 2 mgl−1 increased the rooting percentage (97.5%) number of roots (4.70) and root length (3.47 cm) more efficiently than NAA. Higher concentrations of IBA and NAA (3.0 mgl−1) showed poor rooting. In both species, only sections that contained the base tissue of Liao et al., 2011 FBs were able to produce shoots and plants. FB sections between 1.5 and 3.0 cm from Paphiopedilum Deperle were able to produce shoots, but only sections of FBs >2.5 cm from Paphiopedilum Armeni White were regenerable. Small bract at the FB base harbored a new miniature FB, which further harbored a primitive FB with dome-shaped meristem-like tissues that presumably led to plant induction. No callus formation was observed. (Continued on next page)


92 Culture medium, PGRs and additives

Type of response

Remarks

Leaf sections from NDM supplemented with cytokinins: BA, Direct and indirect The explants developed callus and PLBs within 6 weeks of TDZ, and KN, each at 0.01, 0.1, 0.5 and 1.0 in vitro young embryogenesis culture. Treatment with TDZ in combination with auxins mgl−1 alone and in combinations with plants was best for the induction of callus and PLBs. In vitro regeneration of PLBs was achieved by exposure to light and auxins: NAA, at 0.01, 0.1, 0.5 and 1.0 mgl−1 transferring to solid PGR-free NDM medium. Embryogenesis TDZ was a more effective inducer of PLBs and their MS liquid medium supplemented with Two-month-old proliferation than BA. The highest percentage of PLB different concen-trations of BA (4.4, 8.8, green induction (86%) and the highest number of PLBs 13.2, 17.6 or 22 μM) and TDZ (4.5, 9, 13.5, protocorms (3.6/protocorm) were observed after 9 weeks of culture in 18 or 22.5 μM) modified MS liquid medium supplemented with 18 μM TDZ. Young shoots VW or NDM with 2% sucrose and 15% CW Embryogenesis Most callus (79%) on NDM medium formed somatic embryos (9/callus cluster) within 3–5 months. Transfer to VW or NDM with 0.2% AC resulted in germination of somatic embryos. 1 Seed-derived Direct somatic Both epidermal and sub-epidermal cells were involved in the MS medium with BAP (0.5, 1.0, 2.0 and 2 protocorms embryogenesis formation of embryos. The proembryos developed into 3.0 mg l−1), NAA (0.5, 1.0, 2.0 and 3.0 mg globular stage and subsequently developed into protocorms. l−1) and 2,4-D (0.5, 1.0, 2.0 and 3.0 mg l−1) Complete plantlets were formed after 60 days of culture. 12 individually or in combinations, with or without 1.0 gl−1 peptone MS medium supplemented with 1.0 mg l−1 BAP and 2.0 mg l−1 2,4-D was best for high frequency induction of somatic embryos.

Explant used

Mahendran and Bai, 2012

Rittirat et al., 2011

Sujjaritthurakarn and Kanchanapoom, 2011

Niknejad et al., 2011

Reference

2,4-D, 2,4-dichlorophenoxyacetic acid; 2ip, N6-isopentenyladenine or 6-γ ,γ -dimethylallylaminopurine; AC, activated charcoal; BA, N6-benzyladenine; BAP, benzylaminopurine; CPPU, forchlofenuron–N-(2-chloro-4-pyridyl)-N -phenylurea; CW, coconut water; FB, flower bud; H3 medium, Hyponex medium; IAA, indole-3-acetic acid, IBA, indole-3-butyric acid; KC, Knudson medium; KN, kinetin; MS, Murashige and Skoog (1962) medium; mT, meta-topolin; mTR, meta-topolin riboside; NAA, α-naphthaleneacetic acid; NDM, New Dogashima medium (Tokuhara and Mii 1993); NP, New Phalaenopsis medium; P668, Phytotechnology Medium; Pic, picloram; PM, Phytamax medium; PGR, plant growth regulator; PLB, protocorm-like body; TCL, thin cell layer, TDZ, thidiazuron (N-phenyl-N’- 1,2,3-thiadiazol-5-urea); tTCL, transverse thin cell layer; VW, Vacin and Went (1949) medium; Zn, zeatin; ZR, zeatin-riboside.

Cymbidium bicolor Lindl.

Rhynchostylis gigantea var. Sagarik

Dwarf Dendrobium

Phalaenopsis gigantea

Species





(Belarmino and Mii, 2000; Teixeira da Silva et al., 2011). This ability also opens up the prospects of using biotechnological approaches for orchid improvement. Isolation of protoplasts in the Orchidaceae began in 1978 and successful isolation of protoplasts has been reported from Renantanda protocorms (Teo and Neumann, 1978a), Cattleya and Phalaenopsis leaves, Dendrobium and Paphiopedilum seedlings (Teo and Neumann, 1978b), and Vanilla planifolia leaf tissues (Price and Earle, 1984). To date, however, successful colony formation has only been reported in Dendrobium (Kuehnle and Nan, 1990; Kunasakdakul and Smitamana, 2003; Khentry et al., 2006; Tee et al., 2011) and Phalaenopsis (Sajise et al., 1990; Kobayashi et al., 1993; Ichihashi and Shigemura, 2002; Shrestha et al., 2007), isolation and fusion of protoplasts from mesophyll cells of Dendrobium Pompadour (Kanchanapoom et al., 2001). Protoplast culture is also a highly efficient method of regenerating plantlets and has great potential as a biotechnological tool for orchid improvement. Micropropagation by conventional methods is typically a labour-intensive and time-consuming means of clonal propagation. To overcome these limitations, a bioreactor system utilizing liquid culture medium could be employed. The application of bioreactors for plantlet production was first reported in 1981 for Begonia propagation (Takayama and Misawa, 1981). Since then, the technique has been applied to propagate a large number of plant species since the efficiency of plant propagation using bioreactors is quite high, particularly when automation and robotics are used (Takayama and Akita, 1998; Ziv, 2006). Since the liquid medium in the bioreactor allows for close contact with the tissue which stimulates and facilitates the uptake of nutrients and PGRs, this leads to better shoot and root growth. Continuous shaking promotes less expression of apical dominance which generally leads to induction and proliferation of numerous axillary buds (Mehrotra et al., 2007). In addition, the growth and multiplication rate of shoots is enhanced by forced aeration, since continuous shaking of medium provides ample oxygen supply to the tissue which ultimately leads to their faster growth (Takayama and Akita, 1994). Bioreactors, when used for orchid propagation have several benefits: large numbers of plantlets are easily produced, scaling up production is possible, inoculation or harvesting of cultures can be easily handled, saving labor costs and time. Although bioreactor-based micropropagation has been established in many economically important plants, in orchids only a limited number of experiments have been carried out (Ziv, 2005). The application of bioreactors for propagation of orchids was first reported in 2000 for Phalaenopsis propagation (Park et al., 2000; Young et al., 2000). Later on, continuous immersion culture (air-lift column and air-lift-balloon bioreactor), and temporary immersion cultures (with or without a charcoal filter attached) were used for the culture of Phalaenopsis PLB sections and an improved and automated low-cost bioreactor was established for Anoectochilus formosanus and Oncidium ‘Sugar Sweet’ (Paek et al., 2005, 2011; Yang et al., 2010).

93

VI. GENETIC TRANSFORMATION OF ORCHIDS Since orchids are primarily grown for their large, longlasting, and fascinating flowers, the improvement of quality attributes such as flower color, longevity, shape, architecture, biotic and abiotic stress tolerance, as well as the creation of novel variations, are important economic goals for floriculture biotechnologists around the world. Recent advances in genetic engineering and molecular biology, when considered in conjunction with genetic transformation, could help growers to meet the demand of the orchid industry in the new century (Teixeira da Silva et al., 2011). In the last two decades, significant developments in plant transformation technologies for the insertion of foreign DNA into plant genomes have been made, the most popular systems techniques for gene transformation being Agrobacterium tumefaciens-mediated transformation and microparticle bombardment (biolistics), with which transgenic orchid plants have been successfully developed. In addition, the pollen-tube pathway method (Zhou et al., 1983) bypasses the regeneration process and is a relatively simple procedure for transformation (Yang et al., 2009). The first reports of successful orchid transformation were in Vanda (Chia et al., 1990) and Dendrobium (Kuehnle and Sugii, 1992; Nan and Kuehnle, 1995) through particle bombardment and biolistic-transformed orchids were developed in a number of orchid species like Dendrobium hybrid (Yu et al., 1999), Brassia, Cattleya, Doritaenopsis (Knapp et al., 2000), Dendrobium phalaenopsis and D. nobile (Men et al., 2003a), Phalaenopsis hybrid (Mishiba et al., 2005), Dendrobium Sonia 17 (Tee and Maziah, 2005; Tee et al., 2011) Cymbidium (Yang et al., 1999; Chin et al., 2007; Teixeira da Silva and Tanaka, 2009b, 2011), Dendrobium (Chia et al., 2001; Tee et al., 2003; Tee and Maziah, 2005; Chai et al., 2007), Dendrobium Madame Thong-In and Dendrobium Chao Praya Smile (Chai et al., 2007), Dendrobium Jaquelyn Thomas (Suwanaketchanatit et al., 2007), Oncidium Sharry Baby (Li et al., 2005; Yee et al., 2008), among others (Teixeira da Silva et al., 2011). Hsieh and Huang (1995) reported that GUS and NPTII were transformed into Phalaenopsis via the pollen-tube pathway method.The first example of Agrobacterium-mediated transformation in orchids is credited to Nan et al. (1998) in Dendrobium. Thereafter, successful Agrobacterium-mediated transformation was reported in Dendrobium Madame Thong-In (Yu et al., 2001), Dendrobium nobile (Men et al., 2003b), Cymbidium (Chin et al., 2007), Phalaenopsis (Belarmino and Mii, 2000; Chai et al., 2002; Chan et al., 2005; Mishiba et al., 2005), Oncidium (Liau et al., 2003a, You et al., 2003), Phalaenopsis (Sjahril and Mii, 2006), Phalaenopsis violacea (Sreeramanan et al., 2009), Oncidium and Odontoglossum (Raffeiner et al., 2009), Vanda ‘Tokyo Blue’ (Shrestha et al., 2010), Phalaenopsis amabilis (Semiarti et al., 2007; Qin et al., 2011), Oncidium Gower Ramsey (Thiruvengadam et al., 2012), among other studies (Teixeira da Silva et al., 2011). Compared with the growing number of reports of genetically engineered crops in the literature, there are still relatively very few studies on genetically engineered orchids.


94


In most cases, only standardization of the technique was performed using the hpt gene (hygromycin phosphotransferase) as a selectable marker for hygromycin resistance or the nptII gene (neomycin phosphotransferase) for neomycin/kanamycin resistance and an intron containing the gus gene as a reporter gene (Chen et al., 2007; Chia et al., 1994; Kuehnle and Sugii, 1992; Yang et al., 1999; Knapp et al., 2000; Liau et al., 2003a; You et al., 2003; Yu et al., 2001), and in only a few cases has a desirable gene been inserted (Liau et al., 2003b; Chai et al., 2007; Raffeiner et al., 2009; Qin et al., 2011; Tee et al., 2011). In most studies the transformation efficiency was very low, therefore optimization of efficient DNA delivery, successful regeneration, selection of target gene and suitable selection systems need further improvement to optimize orchid transformation. Introduction of disease resistance traits such as antimicrobial peptides and viral coat proteins (CPs) into the genome of some orchids was achieved by genetic engineering (Liau et al., 2003b; Chan et al., 2005; Sjahril et al., 2006). Transgenic plants overexpressing those genes showed enhanced resistance to Erwinia carotovora and Cymbidium mosaic virus (CymMV). Chen et al. (2006) successfully cloned the Odontoglossum ringspot virus (ORSV) CP gene, green fluorescence protein (GFP) gene, and hygromycin resistance (Hygr) gene from ORSV-infected Epidendrum to Cymbidium niveo-marginatum through A. tumefaciens. Hence, a gene transfer approach may provide a powerful tool to improve disease resistance in orchid species and it may be possible to extend this approach to different orchid species (Chin et al., 2007). In most orchids, PLBs were used as target tissue for gene transformation since the origin of these PLBs are single somatic cells, they are easy to root, presumbly genetically uniform, and can be induced efficiently from various somatic tissues including young leaves, stem segments, or flower stalks, regardless of cultivar. Nowadays, routine gene transformation systems have been adapted in Oncidium, Dendrobium, Cymbidium and Phalaenopsis, with PLBs used as starting material. However, cell clumps/aggregates, TCL explants from PLBs, totipotent calli, protocorms and ovaries were also used as target explants for the transfer of alien genes into some orchids. A. tumefaciens was used by Qin et al. (2011) to develop transgenic Phalaenopsis amabilis incorporating a gene encoding rice cold-inducible lipid transfer protein (LTP) with high transformation success in whch all 470 transgenic plants, each derived from an independent PLB, showed a highly adaptive response to cold stress due to the increased accumulation of several compatible solutes such as total soluble sugars, proline, the antioxidant superoxide dismutase, and decreased accumulation of malondialdehyde. Most recently, Thiruvengadam et al. (2012) developed transgenic Oncidium Gower Ramsey by co-cultivating PLBs with A. tumefaciens harboring the Oncidium MADS box (OMADS1) gene. The transgenic plants with OMADS1 transgene flowered earlier and produced more flowers and pseudobulbs than nontransgenic plants and flower organ conversions were not observed in OMADS1 transgenic flowers. The production of transgenic orchids has advanced industrial applications and deepened the the-

oretical base of these model plants for a better understanding of physiological processes such as flowering (Hsiao et al., 2011b).

VII.

BIOTECHNOLOGY IN ORCHID GERMPLASM CONSERVATION The ruthless pillaging of wild orchid stocks by orchid lovers and collectors, over-exploitation for medicinal purposes, deforestation for urbanization, destruction of habitats by reclamation, shifting cultivation, killing of pollinators and unauthorized trade has led to a reduction in natural populations of many orchids (Hossain, 2009; Swarts and Dixon, 2009) leading many orchid species to become extinct and a large number of species to become rare or endangered (Yam et al., 2010). Ironically, orchids are fed to cows in north-eastern India with the belief that dendrobes enhance their milk yield and that cymbidiums improve their health (http://www.geocities.ws/ padmajaravihs/intro.html; http://english4u2.tripod.com/plants. html). Considering the present status of orchids, the family Orchidaceae as a whole was included in the CITES (Convention on International Trade in Endangered Species) Appendix II (Shefferson et al., 2005). Therefore, conservation of orchids is of utmost importance to meet future demand and for preserving this valuable group of ornamentals for humanity. There are a number of techniques devised to conserve orchids: seed bank development, slow growth conservation and cryopreservation of seed, meristem, tissue-cultured shoot primordia, immature seeds, somatic embryos, callus and other explants (Lurswijidjarus and Thammasiri, 2004; Hirano et al., 2009; Seaton et al., 2010; Yam et al., 2010; Khoddamzadeh et al., 2011) (Table 3). Cryopreservation is the most efficient technique that ensures the safe and cost-efficient long-term conservation of germplasm. It has a wide applicability for orchids both from temperate and tropical origin. Seeds, shoot meristems, protocorms, somatic embyos (i.e., PLBs) and cultured cells can be preserved in liquid nitrogen (N2 (l) ; –196◦ C) to create cryobanks for conserving rare and endangered species (Sakai et al., 2000; Gonzalez-Arnoa et al., 2008; Hirano et al., 2009; Khoddamzadeh et al., 2011). Such a low temperature inhibits metabolic processes and provides viability for an unlimited time period. Although cryopreservation is routinely employed for conservation of many economically important plants, there are only a limited number of experiments carried out in the Orchidaceae (Pritchard, 1984; Seaton and Hailes, 1989; Na and Kondo, 1996; Wang et al., 1998; Ishikawa et al., 1997; Popov et al., 2004; Hirano et al., 2005a, b; Hirano et al., 2009; Antony et al., 2011a, b; Khoddamzadeh et al., 2011; Hosomi et al., 2012; Huehne and Bhinja, 2012) (Table 3). For seeds to be successfully cryopreserved, seed moisture is an important factor because the presence of unbound water in seeds considerably reduces their germinability causing seeds to perish because of the formation of ice crystals in their cells during freezing in N2 (l) . Most researchers apply deep-freezing to seeds with a moisture content of < 13% while only scarce work has dealt with direct freezing of orchid seeds. In orchids, most

95

Explant used Tissue-cultured shoot primordia

Zygotic embryos

PLBs

Name of the species

Vanda pumila Hook.f.

Bletilla striata Rchb.f.

Darwinara ‘Pretty Girl’ and Brassocattleya Pastoral ‘Innocense’ BM/JOGA

Storage at 4 or 25◦ C in the dark with gradual desiccation

Cryopreservation by vitrification

Cryopreservation following ABA preculture and desiccation

Method applied

Reference

Na and Kondo, Tissue-cultured SP were precultured in B5 liquid media supplemented with 0.1, 1 .0 1996 and 10 mgl−l ABA for l-6 days to induce a high level of tolerance for cryopreservation. Following preculture, SPs were desiccated in a polycarbonate culture box with two holes on the lid designed especially for the desiccation R membrane filters (pore size experiment. The holes were covered with Milliseal 0.5 pm; Millipore, USA), which allowed ventilation under aseptic conditions. SPs were then placed on membrane rafts with filter paper in a polycarbonate culture box and kept in a desiccator with 40–45% relative humidity by adding silica gel. SPs precultured with 1.0 mgl−1 ABA showed the highest survival rate (65.0 ± 7.5%) while those precultured in B5 medium without ABA (control) did not withstand freezing. The cryopreserved SPs displayed no abnormalities in chromosome number or cell structure, and induced new meristematic tissues like those of non-cryopreserved controls. Pods were collected and stored at 10◦ C in a sealed metal case with dry silica gel and Ishikawa et al., seeds were cultured on solidified NDM for 10 days and germinated embryos were 1997 precultured on the same medium supplemented with 0.3 M sucrose for 3 days at 25◦ C in continuous dark. Embryos were then overlaid with a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at 25◦ C and finally dehydrated with highly concentrated PVS2 containing 30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethyl sulfoxide in 0.4 M sucrose solution (pH 5.8) for 3 h at 0◦ C prior to immersion into LN for 30 min. The cryotubes were then rapidly warmed in a water bath at 37◦ C and the embryos were washed with liquid ND medium supplemented with 1.2 M sucrose for 20 min and then plated on ND medium. Successfully vitrified and warmed embryos developed into normal plantlets. The rate of plant regeneration amounted to about 60%. PLBs ranging in size from 3.5 to 10 mm were gathered and placed on double layers of Kishi and Takagi, 1997 dry sterilized filter papers in plastic Petri dishes (diameter 9 cm, depth 3 cm) containing 1 g of PLBs and sealed with Parafilm for gradual desiccation. These PLBs were stored for 2, 4, 6, 8, 10 and 12 weeks in the dark at 4 or 25◦ C. Rapid desiccation of PLBs was carried out by keeping the Petri dishes, without the upper lid, under a sterile air stream in a laminar flow chamber at room temperature for 10, 20, 30, 60, 120 and 240 min. In case of preservation at 4◦ C, the regeneration frequency of preserved PLBs of both species remained almost constant during the initial 4 weeks, but decreased drastically after 6 weeks of preservation and finally all PLBs died. The regeneration frequency of the PLBs preserved at 25◦ C increased gradually until 6 weeks of preservation. When the preservation period increased to 12 weeks no conspicuous reduction of regeneration frequency was observed. (Continued on next page)

Method in brief

TABLE 3 Cryopreservation techniques applied to orchid conservation


96 Mature seeds

Explant used

Dendrobium Walter Oumae

Shoot tips

Calanthe vestita Lindl., var. Mature seeds rubro-oculata Paxt., Calanthe Gorey, Angraecum magdalenae Schltr. et Feddes, Trichopilia tortilis Lindl., Miltonia flavescens (Lindl.) Lindl.× Brassia Longissima (Reichb. f.) Nach, Encyclia cochleata (L.) Lemee, Eulophyella roempleriana Schltr., and Epigenium sp. Hybrid Bratonia orchid (Miltonia Seeds flavescens × Brassia longissima)

Angraecum magdalenae Schltr. et. Feddes, C. vestita Lindl. var. rubro-oculata Paxt., Calanthe Gorey, Trichopilia tortilis Lindl., Miltonia flavescens (Lindl.) Lindl.× Brassia longissima) (Reichb. f.) Nach, Encyclia cochleata (L.) Lemee

Name of the species

Method in brief

Reference

Direct immersion Seeds were frozen in plastic ampoules by rapid immersion into LN. More than Popova et al., 2003 in LN and stored 2000–3000 seeds were placed into a single ampoule. After one month of in a cryobank storage in LN in the cryobank, the ampoules were thawed in a water bath at 40◦ C within 40–60 s. Seeds were then sterilized with 5% calcium hypochlorite and sown on medium for germination. The germination rates of cryopreserved and control (nonfrozen) seeds did not differ and remained as high as 100%. ED Shoot tips were encapsulated in calcium-alginate before preculture on MVW Lurswijidjarusa agar medium supplemented with 0.3, 0.5 and 0.7 M sucrose for 2 d at 25 ± and Thammasiri, 2◦ C, 37 μmolm−2s−1, 16 h photoperiod. Encapsulated shoot tips were then 2004 dehydrated by incubation in a sterile air flow of a laminar air-flow cabinet for 0–10 h and immediately plunged into LN. After recovering from LN and rapid thawing in a waterbath (40 ± 2◦ C), the survival ratio and regrowth were measured by a TTC assay and a regrowth culture test, respectively. The highest survival ratio (16.18 mg living cells/100 mg total cells) and regrowth (13.33%) were obtained from encapsulated shoot tips that were previously precultured on 0.3 M sucrose agar medium for 2 d and sufficiently dehydrated for 6–8 h before storing in LN.

Seeds were frozen in plastic vials by direct immersion in LN. Frozen seeds were Nikishina et al., thawed by immersion of the vials for 60 s in water at 37◦ C. Before planting 2001a the seeds in nutrient medium, these were sterilized with 5% solution of calcium hypochlorite and washed in sterile distilled water. Both control and thawed seeds were grown in Petri dishes filled with modified KC medium in the dark or in the light (illuminance 2600 Lux, photoperiod, 16 h). Cryopreservation caused an increase in seed germination rate of all species except for Encyclia cochlerata when grown in the dark. There was no statistically significant difference between the protocorm growth rates in control and cryopreserved samples. The increase in the growth rate caused by rapid freezing and thawing can probably be explained by damage of the testa. Furthermore, cryopreservation of mature and sufficiently dry seeds of orchids caused little damage to the embryos. Nikishina et al., Low temperature Mature seeds were frozen in LN (–196◦ C) and preserved in a refrigerator for 7 days to 7 years. The viability of seeds was evaluated with the tetrazolium 2001b preservation and method, based on the reduction of a colourless solution of TTC to formazan, a cryopreservation red-coloured substance. The storage of seeds in refrigerator loss their viability up to 100% within 20 months of preservation while cryopreserved in LN had no negative effect on viability. The cryopreserved seeds showed normal growth and development of protocorms and juvenile plants. The feasibility of the conservation of mature seeds of some tropical orchids by freezing in LN was demonstrated, which offers opportunities for creating an orchid cryobank.

Direct immersion in LN

Method applied

TABLE 3 Cryopreservation techniques applied to orchid conservation (Continued)


97

Immature seeds

Mature and immature seeds

Protocorms and shoot buds

Bletilla striata Rchb. f.

Dactylorhiza fuchsia (Druce) Soo, D. maculata (L.) Soo, D. Baltica (Klinge) Orlova, D. incarnate (L.) Soo, Platanthera chlorantha (Cust.) Reichenb. and P. bifolia (L.) Rich.

Vanilla planifolia

Direct plunging into LN, Immature seeds were collected 2–4 MAP and three different cryogenic Hirano et al., 2005 vitrification, and procedures were applied: i) direct plunging into LN (seeds were vitrification with transferred to 2.0 ml cryotubes and the tubes directly plunged into LN), preculture ii) vitrification (seeds were transferred to 2.0 ml cryotubes containing 1.5 ml cryoprotective solution containing 2 M glycerol and 0.4 M sucrose in ND medium for 15 min at 25◦ C), and iii) vitrification with preculture (seeds were sown on 0.2% gellan gum-solidified ND medium supplemented with 0.3 M sucrose, and cultured at 25◦ C for 3 days under continuous illumination providing at an intensity of 62.0 μmolm−2s−1). The cryopreserved seeds by vitrification showed 78% germination, vitrification with preculture showed over 90% germination where direct plunging into LN showed only 32% germination. The seeds collected 3–4 MAP was appropriate for cryopreservation. Frozen in LN The procedure followed for cryopreservation is described above (Nikishina Nikishina et al., 2006 et al., 2001). Before sowing or freezing, freshly harvested seeds were stored in a refrigerator at 5◦ C for 2–3 months. The germinability of seeds was determined 60 days after thawing and sowing. Seed germinability after deep freezing was species-specific. In D. fuchsia and D. baltica, it did not substantially differ from control seeds, whereas in other species tested, the difference was substantial. The cause of decline in seed germinability was evidently related to the testa breakdown during rapid immersion into LN. The cryoconservation of seeds of six orchid species was successful, permitting a successful long-term storage of rare orchid seeds in a cryobank. Divakaran et al., Maintenance of shoots in An in vitro conservation protocol for Vanilla was established by 2006 slow growth medium encapsulating 3–5 mm in vitro regenerated shoot buds and protocorms in and conservation of 4% sodium alginate and stored at 22 ± 2◦ C in sterile water with a viability of > 80% up to 10 months. On the other hand, shoot cultures encapsulated remain fresh for > 1 year without subculture on a slow-growth medium, protocorms i.e. MS medium supplemented with 15 gl−1 each of sucrose and mannitol in sealed culture vessels at 22 ± 2◦ C. These cultures were maintained in vitro for more than 7 years with a single yearly subculture. Conserved material could be retrieved and multiplied normally in MS medium with 1.0 mgl−1 BA and 0.5 mgl−1 IBA with good growth and development of normal plants, suggesting that this slow growth and synseed conservation system can be utilized for conservation and exchange of Vanilla genetic resources. (Continued on next page)


98 Protocorms

Mature seeds

PLBs

Vanda coerulea Griff. ex Lindl.

Dendrobium candidum Wall. ex. Lindl.

Explant used

Cleiostoma arietinum (Rcbh.f.) Garay

Name of the species

Method in brief

Reference

ED

ManeerattanarunThis study reports a simple and reliable cyropreservation technique of protocorms by ED, groj et al., effects of dehydration time on water content, percentage of electrolyte leakage of TTC 2007 staining and survival rate of cryopreserved explants. The protocorms were immersed in a 3% (w/v) alginate solution prepared in NDM free of calcium, iron and plant growth regulators. Each protocorm was dropped into 100 mM CaCl2 solution and allowed to polymerize for 10 min. The resulting beads (3–5 mm in diameter) were washed with distilled water and precultured in semi-solid NDM enriched with 0.25 M sucrose for one week at 4◦ C in dark then transferred to liquid NDM enriched with 0.75 M sucrose for two days. The precultured encapsulated protocorms were then surface dried by placing on filter paper and desiccated in Petri dishes (30 protocorms/Petri dish containing 20 g of silica gel), and placed in a Desiccators for 0–6 h. The desiccated beads were transferred to 1.8-ml cryotubes (10 beads/tube) and directly immersed in LN for 1 h in the dark. Cryotubes were then warmed rapidly in a water bath at 28 ± 2◦ C for 2 min and the cryopreserved explants were cultured on agar-solidified NDM fortified with 2.0 mgl−1 BA, 2.0 mgl−1 NAA and 1% (w/v) sucrose (pH 5.4) for establishment. The viability of protocorms was determined by TTC test showed 77% viability and the survival of the cryopreserved protocorms in the establishment medium was 49%. Vitrification Seeds from 7-month-old mature pods were sufficiently dehydrated and approximately 8 mg Thammasiri and Soamkul, of seeds were placed in 2-ml cryotubes filled with 0.5 ml highly concentrated PVS2 2007 consisting of 30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethyl sulfoxide, prepared in modified VW liquid medium. Vitrification was carried out at 25 ± 2◦ C for 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 min, before plunging the cryotubes into LN where they were kept for 1 d. After 3 months of preservation, the seeds were cultured on VW agar medium to check for survival. The cultures were incubated at 25 ± 2◦ C under illumination of about 37 μmolm−2s−1 for 16 h. About 67% of cryopreserved seeds treated with PVS2 solution for 70 min were able to develop normal seedlings in vitro, while, without PVS2 treatment, there was no survival after LN storage. Cryopreservation PLBs 0.2–0.3 cm in diameter were excised and used for cryopreservation by EV. PLBs were Yin and Hong, 2009 by EV precultured in liquid MS medium containing 0.2 mgl−1 NAA, 0.5 mgl−1 BAP enriched with 0.75M sucrose, and grown under continuous light (36 μmolm−2s−1) at 25 ± 1◦ C for 5 days. PLBs were osmoprotected with a mixture of 2 M glycerol and 1M sucrose for 80 min at 25◦ C and dripped in a 0.5M CaCl2 solution containing 0.5M sucrose at 25 ± 1◦ C and left for 15 min to form Ca-alginate beads. The encapsulated PLBs were then dehydrated with a PVS2 solution consisting of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, and 15% (w/v) dimethyl sulfoxide in 0.5M sucrose, pH 5.8, for 150 min at 0◦ C. Encapsulated and dehydrated PLBs were plunged directly into LN for 1 h. Cryopreserved PLBs were then rapidly re-warmed in a water bath at 40◦ C for 3 min and then washed three times with MS medium containing 1.2 M sucrose at 10-min intervals. The cryopreserved PLBs developed normal shoots and roots without any morphological abnormalities. The survival rate of encapsulated-vitrified PLBs was > 85%.

Method applied



99

Mature seeds

Low temperature at 4◦ C and cryopreservation by vitrification

This study describes low temperature storage and cryopreservation by vitrification Hirano et al., of seeds. For low temperature preservation the seeds were placed in tubes and 2009 immediately sealed and transferred to 4◦ C for up to 6 months. For cryopreservation, freshly harvested seeds with or without preculture treatment were used. The seeds were sown for preculture treatment on 0.2% Gellan gum-solidified NDM with 0.3 M sucrose, and cultured at 25◦ C for 3 d under continuous illumination. Approximately 300–400 seeds, with or without preculture treatment, were transferred to 2.0-ml cryotubes, and then immersed in 1.5-ml cryoprotective solution consisting of 2 M glycerol and 0.4 M sucrose for 15 min at 25◦ C. The seeds which were dehydrated to 5% water content and preserved at 4◦ C, showed no decrease in viability and germinability after 3 months. After storage for 6 months, however, the seeds showed a drastic decrease in germinability (9%), even though survival rate of germinated seeds was high (69%). On the other hand, cryopreservation by vitrification was useful for long-term conservation of seeds. After 12 months of preservation the seeds showed equal rates of survival and germination and no apparent deterioration effect on germinability and survival rate was observed. Dendrobium Bobby Messina PLBs with a size ED PLBs were pretreated with 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 M sucrose and/or 0.0, 0.2, Antony et al., 2011 range of 1–2 0.4, 0.6, 0.8, 1.0, 1.2 M sorbitol supplemented in semi-solid 12 MS media at 24◦ C for 0–5 d under a 16-h photoperiod. These were encapsulated in 3% sodium and 3–4 mm alginate and 0.1M calcium chloride, both supplemented with liquid 12 MS media and 0.4 M sucrose. The resulting Ca-alginate beads were osmoprotected in 0.75M sucrose supplemented with liquid 12 MS media on an orbital shaker (110 rpm) at 24◦ C for 1 d under a 16-h photoperiod. The osmoprotected beads R -sealed culture jar containing 50 g of were dehydrated for 0–10 h in a Parafilm oven-sterilized silica gel followed by rapid freezing in LN. After thawing in a 40◦ C water bath for 90–120 s, the cryopreserved beads were cultured on growth recovery media containing semi-solid 12 MS media supplemented with 2% sucrose. Survival of the cryopreserved PLBs was assessed with the TTC test. The highest survival was observed in 3–4 mm size cryopreserved PLBs. Phalaenopsis bellina (Rchb.f.) PLBs Cryopreservation by ED Uniform sized PLBs measuring 3–5 mm in diameter were separated, blot dried, Khoddamzadeh Christenson mixed into a solution of 4% (w/v) sodium alginate in liquid 12 MS medium free of et al., 2011 calcium, plant growth regulators and iron and then dropped one by one into liquid 1 MS medium enriched with 75 mM CaCl2 ·2H2 O. The drops, each containing a 2 single PLB, were left in the CaCl2 ·2H2 O solution to polymerize for 30 min on a gyratory shaker (75 rpm). The resulting beads were recovered and washed three times with sterilized de-ionized water three times. Encapsulated PLBs were precultured in liquid 12 MS medium supplemented with 0 (control), 0.5, 0.75, 1.00 or 1.25 M sucrose for 0, 1, 3, 5 or 7 d with agitation (75 rpm). After preculture in sucrose, the PLBs were subjected to desiccation using silica gel and subsequently plunged into LN. The viability of encapsulated PLBs was assessed by TTC reduction. The PLBs precultured in 0.75 M sucrose-supplemented medium for 3 d followed by desiccation exhibited the highest viability (46.6%). (Continued on next page)

Phaius tankervilleae


100 Storage at 4◦ C, −20◦ C, and −70◦ C

ED and EV

PLBs

Mature seeds

Storage at −18 C

◦

Method applied

Seeds

Explant used

Reference

Cryopreservation of PLBs based on ED and EV was established. In both cryogenic Mohanty et al., procedures, PLBs were initially osmoprotected with a mixture of 0.4 M sucrose 2012 and 2 M glycerol, incorporated in the encapsulation matrix comprising 3% (w/v) sodium alginate and 0.1 M CaCl2 . Out of the two methods, EV resulted in higher survival (78.1%) and regrowth (75.9%) than ED (53.3 and 50.2%, respectively). Incorporation of 0.4 M sucrose and 2 M glycerol in the encapsulation matrix resulted in higher survival percentage after cryopreservation. EV method was the most appropriate way to cryopreserve PLBs. Regenerated plantlets showed normal morphology, similar to control plants. Five non-penetrating cryoprotectants, i.e. glucose (7.5% w/v), honey (105% w/v), Huehne and meso-inositol (5% w/v), mannitol (7.5% w/v) and skimmed milk (10% v/v) were Bhinja, 2012 used for the long-term cryopreservation of seeds of 12 orchid species. Around 35,000–50,000 seeds (100 mg) of each species were immersed with agitation in 100 ml of each cryoprotectant for 24 h at room temperature. The treated seeds were filtered through Myracloth (EMD Milllipore USA), and placed on sterile Whatman paper to absorb excess solution. The seeds were then placed on silica gel and air-dried in laminar air-flow cabinet for 10 h. These were then transferred to cryomicrotubes, sealed tightly and stored separately at 4◦ C, −20◦ C, and −70◦ C. The frozen seeds were rapidly thawed at 37◦ C in water bath for 2 min and directly overlaid on semi-solid VW medium and incubated at 25 ± 2◦ C under 60 μmolm−2s−1 light intensity for 18 hd−1. The germination percentage was tested every 6 months up to 5 years. The most effective cryoprotectant was 5% (w/v) meso-inositol, followed by mannitol and glucose (7.5% w/v each). The seeds of D. crystallinum, D. virgineum, and Vanda Miss Joaquim coated with meso-inositol and preserved at −70◦ C for one year showed 50.7, 31.5, and 8.6% germination, respectively. After further storage for 5 years the germination percentage remained stable at an average of 34.5, 17.4, and 4.1%, respectively and there was no effect on seedling development. The seeds stored at 4◦ C and −20◦ C lost viability within 1 and 2 years, respectively. Interestingly, the seeds of the other nine orchids could not survive under these storage conditions. Thus, a particular cryoprotectant, seed moisture, and other parameters should be standardized and improved for individual species.

Seeds were preserved (with or without pre-conditioning in 10% sucrose solution) at Hosomi et al., −18◦ C for 3 months. The stored seeds were germinated after 3 months; there was 2012 no loss of viability after storage.

Method in brief

B5 medium; ABA, abscisic acid; ED, encapsulation–dehydration; EV, encapsulation–vitrification; LN, liquid nitrogen; MAP, months after pollination; NDM, New Dogashima medium (Tokuhara and Mii 1993); PVS2 plant vitrification solution; SP, shoot primordium; TTC, 2,3,5-triphenyl-tetrazolium chloride; VW, Vacin and Went medium (Vacin and Went, 1949).

Dendrobium capillipes, D. cariniferum, D. chrysotoxum, D. compactum, D. cruentum, D. crystallinum, D. virginium, Grammatophyllum speciosum, Pholidota articulata and Vanda Miss Joaquim

Cattleya granulosa, C. hegeriana, C. intermedia, C. mossiae, C. purpurata, C. sanguiloba, C.tenuis, C. tigrina and C. walkeriana Dendrobium nobile

Name of the species





probably Pritchard (1984) was the first to apply cryopresevation through N2 (l) preservation of terrestrial and epiphytic orchid seeds with a moisture level below 11%, and seed germinability did not change after cryoconservation for most species examined. Ishikawa et al. (1997) successfully cryopreserved the zygotic embryos of Bletilla striata by vitrification. Na and Kondo (1996) described a cryopreservation protocol for preservation of tissue-cultured shoot primordia from shoot apices of cultured Vanda pumila protocorms following ABA preculture and desiccation. More specifically, after a three-day preculture in B5 liquid medium (Gamborg et al., 1968) supplemented with 1.0 mgl−1 ABA at 22◦ C under continuous lighting of 4000–6000 lux under halogen lamps, shoot primordia were desiccated from 40–45% relative humidity down to ∼24% relative water content and then cryopreserved in N2 (l) . The cryopreserved shoot primordia showed a survival rate of 65.0 ± 7.5% with no abnormalities in chromosome number or cell structure, similar to non-cryopreserved controls. Nikishina et al. (2001b) found that mature seeds of Calanthe Gorey, C. vestita, Angraecum magdalenae, Trichopilia tortilis, Miltonia flavescens×Brassia longissima, Encyclia cochleata, Eulophyella roempleriana, and Epigenium sp. stored in N2 (l) had no negative effect on the duration of germination, growth and development of protocorms or juvenile plants. The feasibility of the conservation of mature seeds by freezing in N2 (l) was demonstrated, which offers opportunities for creating an orchid cryobank. Hirano et al. (2005a) applied three cryogenic procedures: (1) direct plunging into N2 (l) , (2) vitrification, and (3) vitrification with preculture (3 days on New Dogashima medium (Tokuhara and Mii, 1993) supplemented with 0.3 M sucrose) for preserving immature seeds (2–4 months after pollination) of Bletilla striata. The survival rate after preservation was 81–92% and seedlings developed into normal plantlets in vitro. In Phaius tankervilleae, when the seeds were dehydrated to 5% water content and preserved at a low temperature (4◦ C), there was no decrease in viability or germinability after three months. After storage for six months, however, the seeds showed a drastic decrease (9%) in germinability (Hirano et al., 2009). Thus, drying and low temperature preservation is not reliable for long-term storage. Cryopreservation of P. tankervilleae seeds by the vitrification method showed no deterioration after 12 months of storage although this method is an efficient means of preserving orchid seeds (Hirano et al., 2009). Cryopreservation also offers other advantages, compared to other available storage approaches, such as the stability of phenotypic and genotypic characters, minimal storage space and maintenance requirements (cf . Khoddamzadeh et al., 2011). Classical cryopreservation procedures include the following successive steps: pregrowth of samples, cryoprotection, slow cooling (0.5–2.0◦ C/min) to a determined prefreezing temperature (usually around −40◦ C), rapid immersion of samples in N2 (l) , storage, rapid thawing and recovery which are generally complex since they require the use of sophisticated and expensive programmable freezers. Nowadays, improved and advanced cryopreservation pro-

101

cedures such as vitrification-based techniques are followed. To date, seven different vitrification-based techniques have been developed and applied: a) encapsulation–dehydration, b) a procedure actually termed vitrification, c) encapsulation–vitrification, d) dehydration, e) pregrowth, f) pregrowth–dehydration and g) droplet–vitrification (Engelmann, 2011). Vitrification involves the treatment of samples with cryoprotective substances, dehydration with highly concentrated vitrification solutions, rapid cooling and rewarming, removal of cryoprotectants and recovery. This procedure has been developed for meristem, cell suspensions and somatic embryos of numerous different species (Sakai and Engelmann, 2007; Sakai et al., 2008). In total, three cryopreservation techniques have been applied to orchid species, including desiccation (air-drying), vitrification and encapsulation-dehydration (Hirano et al., 2006; Tsai et al., 2009; Khoddamzadeh et al., 2011) (Table 3). This is because the desiccation technique involves direct dehydration of naked PLBs which are very sensitive to dehydration while the vitrification technique uses high concentrations of chemicals which can be toxic. However, it appears that the encapsulation-dehydration method may be the most suitable as it results in a high survival frequency after cryogenic storage. The encapsulation of explants in alginate beads for cryopreservation has some benefits compared to the use of nonencapsulated samples. The alginate beads provide enhanced protection of dried materials from mechanical and oxidative stress during storage and ease of handling of small samples during pre- and post-cryopreservation (Niino and Sakai, 1992; Maneerattanarungroj et al., 2007). Only a limited number of studies have reported on the cryopreservation of PLBs to date: Geodorum densiflorum (Datta et al., 1999), Doritaenopsis (Tsukazaki et al., 2000), Dendrobium Walter Oumae (Lurswijidjarus and Thammasiri, 2004), Dendrobium candidum (Yin and Hong, 2009), and Phalaenopsis bellina (Khoddamzadeh et al., 2011). Seeds of different orchids were also successfully preserved: Calanthe vestita, Calanthe Gorey, Angraecum, Trichopilia tortilis, Miltonia flavescens, Brassia longissima, Encyclia cochleata, Eulophyella roempleriana, and Epigenium (Nikishina et al., 2001b), Bratonia (Popova et al., 2003), Bletilla striata (Hirano et al., 2005a), Dactylorhiza fuchsia, D. maculata, D. baltica, D. incarnata, Platanthera bifolia and P. chlorantha (Nikishina et al., 2006), Vanda coerulea (Thammasiri and Soamkul, 2007) Phaius tankervilleae (Hirano et al., 2009), Cattleya granulosa, C. hegeriana, C. intermedia, C. mossiae, C. purpurata, C. sanguiloba, C.tenuis, C. tigrina and C. walkeriana (Hosomi et al., 2012), Dendrobium crystallinum, D. virgineum, and Vanda Miss Joaquim (Huehne and Bhinja, 2012). Orchid seeds are morphophysiologically different from other plants, to some extent limiting the application of cryopreservation and thus modification and proper adjustment of the techniques is required. Cryopreservation of vegetative tissue involves several stages, specifically the establishment of in vitro cultures, conditioning of these tissues, addition of an appropriate cryoprotectant, exposure of cultures to ultra-low


102


temperature, rewarming and regeneration of plant cells and tissues. Each stage plays an important role in determining the survival of tissue upon re-warming. Therefore, to develop a cryopreservation system for a species, including orchids, these stages need to be optimized (Khoddamzadeh et al., 2011). The best place to conserve plant germplasm and to promote biodiversity is in the wild, where a large number of species present in viable populations can persist in their natural habitats with their natural associated ecological interactions (McNaughton, 1989). Reintroduction of native species has become increasingly important in conservation worldwide for recovery of rare species and restoration purposes. However, some studies have reported the outcome of reintroduction efforts in plant species (Rout et al., 2009). In orchids, the reintroduced seedlings were usually from asymbiotic germination or tissue culture. Ramsay and Stewart (1998) reported re-establishment of lady’s slipper orchid (Cypripedium calceolus L.) in Britain in which 75% of reestablished seedlings survived. Seeni and Latha (2000) reported the ecorehabilitation of the endangered Blue Vanda (Vanda coerulea) and the survival rate was more than 70%. Zeng et al. (2012) successfully reintroduced Paphiopedilum wardii seedlings into habitats, including alien forest habitats. The highest survival rate (65%) occurred in the alien forest habitat of Ehuangzhang than on the habitat of Gaoligong Mountain, possibly because of more suitable irradiation, soil, water, nutrients, environmental humidity and other factors in the former.

VIII.

BIOTECHNOLOGICAL TOOLS TO DETECT SOMACLONAL VARIATION Somaclonal variation was first defined by Larkin and Scowcroft (1981) in CSIRO, Australia as variation originating in wheat cell and tissue cultures. Presently, the term is universally used for all forms of tissue culture-derived variants. However, other names such as protoclonal, gametoclonal and mericlonal variation are often used to describe variants from protoplast, gametic cells and meristem cultures, respectively (Chen et al., 1998). Some scientists added another aspect to the definition and required that somaclonal variation be heritable through a sexual cycle (Bairu et al., 2011). Unfortunately, it is not always possible to demonstrate heritability because of complex sexual incompatibilities, seedlessness, polyploidy or long generation cycles. The available evidence of somaclonal variation lends support to the notion that in vitro culture environments can be mutagenic due to physicochemical and biological agents that increase genetic variability and mutants and plants regenerated from these organ cultures, calli, protoplasts and via somatic embryogenesis sometimes exhibit phenotypic and DNA variations (e.g., Teixeira da Silva, 2004). The methods of propagation, genotype, nature of the tissue used as starting material, type and concentration of PGRs, number as well as duration of subcultures are other factors that can cause somaclonal variation.

Some of the resulting somaclones can possess desirable traits that remain stable and are heritable by progenies. In orchids, it generally occurs during proliferation of both shoots and PLBs and leads to morphological or physiological changes in the regenerants (e.g., Teixeira da Silva et al., 2006a, b, 2007). Many changes in ploidy may also arise, but these do not often express themselves as phenotypic variants (Fukai et al., 2002; Teixeira da Silva and Tanaka, 2006). Variants may be vegetative or reproductive, depending on the genetic background of the cultured orchid plants. The mechanism of these variations during tissue culture is still largely unknown in orchids. Since the generation time for orchids is relatively long, conventional cross-hybridization or selfing aimed at understanding the inheritance of somaclonal variation is difficult. Somaclonal variants can be detected using various techniques which are broadly categorized as morphological, physiological or biochemical and molecular detection techniques. Each of these has their peculiar strengths and limitations. Morphological characters have long been used to identify plant species, genera, and families. Variants can be easily detected based on characters such as differences in plant stature, leaf morphology and pigmentation abnormality (Israeli et al., 1991). However, morphological traits are often strongly influenced by environmental factors and may not reflect the true genetic composition of a plant (Mandal et al., 2001). In addition, morphological markers used for phenotypic characters are limited in number, often developmentally regulated and easily affected by environmental factors (Bairu et al., 2011). Relative to morphological detection, the use of physiological responses and/or biochemical tests for detecting variants is faster and can be carried out at juvenile stages to lower the possible economic losses. The response of plants to physiological factors such as PGRs and light can be used as a basis to differentiate between normal and variant somaclones (Peyvandi et al., 2009). For instance, gibberellic acid (GA3 ) regulates growth and influences various developmental processes such as stem elongation and enzyme induction. Therefore, disturbances in GA3 metabolism and level have been suggested as one of the possible indicators of somaclonal variation in higher plants (Sandoval et al., 1995). The use of molecular approaches such as AFLP, RFLP, methylation-sensitive restriction fragment-length polymorphism, isozyme electrophoretic patterns, candidate gene cloning by the use of degenerate primers and PCR based on conserved amino acid sequences of target genes, cDNA suppression subtractive hybridization (SSH), or flow cytometry have resulted in the successful study of epigenetic mutations, identification of expressed sequence tags (ESTs) from wild type and peloric flower buds of several orchids (Chen and Chen, 2007; Tsai et al. 2007a; Khoddamzadeh et al., 2010). In Phalaenopsis, Doritaenopsis and Oncidium, several candidate genes were cloned and transcript levels were compared (Tang and Chen, 2007). Some genes, such as a retroelements, may have been abnormally activated in the peloric mutants. DNA methylation status, activation of transposable elements or retrotransposons, and histone



modifications may also play a role in somaclonal variation in orchids. Somaclones help in the selection of valuable and novel genotypes as they show the inheritance of new phenotypes for disease resistance, altered flower color, peloric or semi-peloric flowers, stress tolerance and other traits. Some micropropagated plantlets may show vegetative abnormalities in the form of mosaic leaves, broad leaves, or distorted sickle-shaped leaves because of somaclonal variation, which is a concern for orchid growers because the variants may affect pot plant quality. The Golden Peoker ‘Brother’—somaclonal mutants of Phalaenopsis equestris with different, remarkable, fused spots of Harlequin pattern on the sepals and petals—were found in different orchid farms in Taiwan. A single dominant gene controls the red floral color (vs. white color) in P. equestris. However, as orchids have a long life cycle, one needs to wait for flowering to identify variant progenies carrying the mutant gene after hybridization, which is a time-consuming procedure. Therefore, a rapid technique is needed for early detection of the presence of mutant genes in order to increase the efficiency of the breeding procedure (Tang and Chen, 2007). The use of a stepwise screening method, such as RAPD, allows the presence of flower color-inducing gene(s) to be detected in the progenies of hybridization at any stage of plant development. In association with in situ hybridization, molecular markers such as RAPD may potentially be used for the identification and gene mapping of chromosomes where the flower color gene(s) are located (Khoddamzadeh et al., 2010).

IX.

BIOTECHNOLOGICAL TOOLS IN THE CONTROL OF ORCHID FLOWERING In vitro flowering is advantageous and has great potential in commercial application especially for species with a long juvenile period like orchids (Vaz and Kerbauy, 2008). Generally orchids take 3–13 years to produce a flowering plant from seeds, and this juvenile period can vary depending on environmental conditions and on the genetic set-up of the genus, species or hybrids (Chia et al., 1999; Bhadra and Hossain, 2003b; Ziv and Naor, 2006) thereby make breeding programs very slow (Duan and Yazawa, 1995a; Kostenyuk et al., 1999; Chang and Chang, 2003). Moreover, most of the orchids have definite flowering seasons. In vitro conditions significantly shortened the juvenile period and media components, while the level of PGRs, genetic set up of the species and culture conditions are strongly correlated with the initiation and development of floral organs from buds or callus tissue (Goh and Yang, 1978; Hew and Clifford, 1993; Blanchard and Runkle, 2008; Hsiao et al., 2011b). In vitro conditions can be manipulated and provide an alternative controlled system necessary to study flower induc-tion, as well as inflorescence and flower morphogenesis. In vitro flowering has always been of interest to orchidologists and horticulturists since it is highly desirable to formulate a system for precocious flowering, the so-called “in vitro bouquet” (Sudhakaran et al., 2006). Establishing a reliable in vitro protocol to induce early flowering in orchids is also

103

important for the study of molecular and genetic mechanisms of flower induction and for assisting orchid breeding programs. Orchid breeders can exploit the short flowering period to develop new hybrids much earlier then conventional methods. Mini orchids with flowers can also be sold as souvenirs and novelty gifts. The technique can also be applied to adjust commercial production of flowers and specific compounds from floral organs. For decades, scientists have tried to discover the inherent causes of flowering and many researchers pointed to a florigenic chemical stimulus (the florigen) that was photoperiodically induced in leaves and translocated to the bud, triggering off flowering. To date, identification of the florigen remains a mystery. Therefore, the direct role of internal factors remains obscure. Tissue culture is an important tool to study the controlled mechanisms involved in this complex physiological process and has been employed to induce flowering in vitro in several orchid species: Oncidium varicosum (Kerbauy, 1984), Doritis pulcherrima × Kingiella philippinensis and Phalaenopsis (Duan and Yazawa, 1994, 1995a, b), Cymbidium niveo-marginatum (Kostenyuk et al., 1999), Dendrobium huoshanase (Wen et al., 1999), Cymbidium ensifolium (Wang et al., 1981, 1988; Wang, 1988; Chang and Chang, 2003), Dendrobium candidum (Wang et al., 1995, 1997), Geodorum densiflorum (Bhadra and Hossain, 2003b), Dendrobium Madame Thong-In (Sim et al., 2007, 2008), Dendrobium Chao Praya Smile (Hee et al., 2007), Dendrobium Sonia 17 (Tee et al., 2008), Dendrobium nobile (Wang et al., 2009a), Friederick’s Dendrobium orchid (Te-Chato et al., 2009) Eulophia graminea (Chang et al., 2010), Psygmorchis pusilla (Vaz et al., 2004) and Phalaenopsis cornu-cervi, Dendrobium aphyllum (Hossain et al. unpublished data) (Table 4). Some reports indicated that flowering is accomplished by the combined synchronous effect of many endogenous substances, especially PGRs (Wang, 1988; Kostenyuk et al., 1999; Bhadra and Hossain, 2003b; Hsiao et al., 2011b). The proper manipulation of PGRs at appropriate doses to an appropriate explant can induce in vitro flowering, especially in orchids (Vaz and Kerbauy, 2008). However, different experimental conditions and types of orchids may have various effects. For Phalaenopsis, cytokinins (e.g., benzylaminopurine or BAP, equivalent to 6-benzyladenine, or BA) stimulate flowering, and auxin suppresses the effect of BAP; GA3 is not effective when applied alone but when added in combination with BAP seems to accelerate the effect of BAP slightly (Goh and Yang, 1978; Goh and Arditti, 1985; Hew and Clifford, 1993). For Dendrobium (Lee and Koay, 1986), Doritaenopsis and Phalaenopsis (Blanchard and Runkle, 2008), GA3 did not stimulate the effect of BAP. The encouragement of flowering by the application of BAP seems to suggest that cytokinins play a part in regulating inflorescence initiation of Doritaenopsis and Phalaenopsis, although the promotion of flowering depends on certain conditions (Blanchard and Runkle, 2008). Gibberellins are thought to play an essential role in the vegetative and reproductive development of plants (Tymoszuk et al., 1979; Cecich, 1985; Arditti, 1992).

104 Explant, culture condition and duration of flowering Culture medium

Major observations

Doritis pulcherrima In vitro plantlets VW medium and The in vitro plantlets induced flowering in the presence of BA × Kingiella Flowering occurred within 7 months Hyponex medium under certain nutritional conditions, including appropriate philippinensis of culture. Cultures were maintained supplemented sucrose content and nitrogen in the culture media. Use of KN, in 12-h photoperiod at 25 ± 2◦ C. with BAP, KN, 2iP or CW was ineffective for floral bud induction. No floral 2ip CW and buds formed in Hyponex medium, even with BA and sucrose. different N High N in the medium decreased or discouraged floral bud content formation while low N content improved the formation of floral buds. MS medium Floral buds were induced in 2.0 mgl−1 BAP and 0.5 mgl−1 Dendrobium Seed-derived protocorms NAA at a frequency of 27%. When protocorms were cultured supplemented candidum Wall. Flowering induced within 90 to in 0.5 mgl−1 ABA for 15 days and then transferred to 2.0 with BAP, NAA, ex Lindl. 150 days. Cultures were maintained mgl−1 BAP-supplemented medium the frequency of ABA at different in 12 h photoperiod at 1000–1500 ◦ flowering increased up to 84.0%. No flower buds were concentrations lux light at 25–27 C. observed when the protocorms were cultured on ABA-supplemented medium for long periods. Dendrobium Protocorms or shoots MS medium Plant hormones and polyamines played an important role in candidum Wall. Flowering induced within 3–6 supplemented flower initiation. The addition of spermidine, or BA, or the ex Lindl. months of culture. Cultures were with BAP, NAA, combination of NAA and BA in the culture medium induced maintained in a 12-h photoperiod at ABA at different protocorms or shoots to flower within 3–6 months with a 1000–1500 lux at 25–27◦ C. concentrations frequency of 31.6–45.8%. The flowering frequency was further increased to 82.8% on average by pre-treatment of protocorms in an ABA-containing medium followed by transfer onto medium with BA. The induction of precocious flowering depends on the developmental stage of the experimental materials. I need actual concentrations of hormones. Modified MS A combined treatment of cytokinin (BAP), restricted nitrogen Cymbidium niveo- Rhizomes (3–4 months old) and in medium supply with phosphorus enrichment, and root excision marginatum vitro plants 3–5 months old supplemented (pruning) resulted in flowering. When root excision and/or Mak. Cultures were maintained at with BAP and BAP were omitted from the combined treatments, flower 25–26◦ C, 50 mmol m–2 s−1, and a 16-h photoperiod. To stimulate restricted induction was significantly reduced. Root-excised plantlets short-day condition, an 8-h nitrogen supply cultured in medium containing BA with high P and low N photoperiod was employed at low with phosphorus content achieved about 2.5-fold higher number of in vitro (4–6◦ C) and high (30–32◦ C) enrichment flowers than cultures grown in 12 MS medium containing BA temperature. Nearly 100% of plants without a modified P/N ratio. flowered within 90 days.

Species

TABLE 4 In vitro flowering of orchids


Kostenyuk et al., 1999

Wang et al., 1997

Wang et al., 1995

Duan and Yazawa, 1994

Reference

105

Dendrobium Madame Thong-In

Dendrobium Chao Praya Smile

Geodorum densiflorum (Lam.) Schltr.

Cymbidium ensifolium var. misericors

1 Callus-derived rhizomes MS containing Precocious flowering occurred on a defined 12 MS medium Chang and 2 containing NAA with TDZ, 2iP or BA within 100 d of culture. Flowering occurred within 100 d NAA, TDZ, 2iP Chang, 2003 or BA at different Among eight cytokinins tested, TDZ at 3.3–10 μM or 2iP at of culture. The cultures were maintained for 100 d at 25 ± concentrations 10–33 μM combined with 1.5 μM NAA were the most effective combinations for achieving flower induction. TDZ had a stronger 1◦ C and exposed to artificial light (daylight fluorescent tubes inductive effect than BA on the flowering. FL-30D/29, 40 W) of 10 μmolm−2s−1 with a 16-h photoperiod. MS medium Two-three months old in vitro in vitro-grown seedlings flowered only on MS + 2.0 mgl−1 BAP + Bhadra and Hossain, 1.0 mgl−1 IAA and on MS + 2.5 mgl−1 BAP + 0.1% (w/v) AC supplemented grown seedlings having 3–4 2003 within 3–4 months of culture. BAP had a strong effect on in vitro with BAP, IAA leaves flowering. and AC at Flowering occurred within different 90–100 days. Cultures were concentrations maintained at 25 ± 2◦ C under a 16-h photoperiod of 1000 lux fluorescent light. in vitro-grown seedlings Modified KC BA at 11.1 μM induced the highest percent of flowering (45%) Hee et al., Flowering occurred within 60 d medium within 6 months from germination. The percentage of 2007 of culture. Cultures were supplemented inflorescence induction was increased to 72% in morphologically maintained at 25 ± 2◦ C and a with BAP at normal seedlings. Plantlets in culture produced both complete 16-h photoperiod of different and incomplete flowers. Pollen and female reproductive organs of 40 μmolm−2s−1 from daylight concentrations in vitro developed complete flowers were morphologically and fluorescent lamps. anatomically similar to flowers of field-grown plants. In addition, 65% of the pollen grains derived from in vitro developed flowers showed regular meiosis during microsporogenesis. Even so, a lower percentage of germination of the pollen grains derived from in vitro developed flowers could be self-pollinated and induced seed pods with viable seeds. CW promoted vegetative growth but did not support the formation Sim et al., in vitro seedlings with 2–3 leaflets Modified KC supplemented of flower buds. CW was essential to trigger the transition of the More than 60% of the flowers 2007 with BAP, CW shoot apical meristem from the vegetative to floral state and BA bloomed by the 6th week and and AC at enhanced inflorescence initiation and flower bud formation. 90% by the 9th week. Cultures different Inflorescence stalks became visible after 3 weeks of culture upon were incubated at 26 ± 2◦ C under a 16-h photoperiod of concentrations transfer to two-layered medium. By the 9th week, about 40–55% 35 μmolm−2s−1 from daylight of the protocorms produced inflorescence stalks. Both liquid and fluorescent lamps. Gelrite-solidified medium with 22.2 μM BA and 0.03% AC produced flowers. Healthy seedlings produced 100% flower buds, 75% of which resulted in abnormal flowers. (Continued on next page)


106

Friederick’s Dendrobium orchid

in vitro individual shoots 2–3 cm long consisted of 3–4 nodes After 3 months of culture floral buds were induced. Cultures were maintained at 26 ± 2◦ C under 1600 lux illumination for 14 h.

MS medium supplemented with PBZ at different concentrations

Three-leaf stage plantlets Modified 12 MS medium Cultures were kept at a supplemented temperature of 25 ± 2◦ C and 16-h photoperiod with irradiance with BAP and of 25 μmolm−2s−1. different N/P concentrations

Dendrobium Sonia 17

Modified KC

Culture medium

in vitro grown seedlings at different size cultures were incubated at 26 ± 2◦ C under a 16-h photoperiod of 35 μmolm−2s−1 from daylight fluorescent lamps

Explant, culture condition and duration of flowering

Dendrobium Madame Thong-In

Species

Major observations

Reference

Seedling growth stage and endogenous level of cytokinin was Sim et al., important for vegetative growth and flower-inductive response. 2008 Cytokinin content varied in different growth stages of seedlings. Seedlings 1.0–1.5 cm in size cultured in flowering-inductive medium [liquid KC medium containing 4.4 μM BA and 15% CW)] showed up to 200 and 133 pmol g−1 FW of iP and iPA, respectively. These levels were significantly higher than all other cytokinins analyzed in seedlings of the same stage and were about 80- to 150-fold higher than seedlings cultured in non-inductive medium. During the transitional (vegetative to reproductive) stage, the endogenous levels of iP (178 pmol g−1 FW) and iPA (63 pmol g−1 FW) were also significantly higher than cytokinins in the zeatin (Z) and dihydrozeatin (DZ) families in the same seedlings. This report provided strong evidence that cytokinins, especially the iP family, play an important role in early in vitro flowering in this orchid. Medium enriched with high P and low N was effective for inducing Tee et al., inflorescences while low P and high N content could only 2008 effectively promote shoot formation. In 52% of plantlets that formed an inflorescence in BA and high P and low N containing medium while only 20% of plantlets formed inflorescences in 1 MS medium containing BA without any modification of the P/N 2 ratio after 4 months. A repeatable method for in vitro inflorescence induction was found when the medium contained 20 μM BA. Different morphologies of in vitro flowers such as incomplete flower structures, abnormal and unresupinated in vitro flowers were observed. Without supplementation of PBZ in culture medium, no floral buds Te-chato et al., were induced. A low concentration of PBZ (0.025 mgl−1) promoted a small number of floral buds (10%). PBZ at 0.05 2009 mgl−1 gave the highest percentage of floral bud induction (29%) and highest concentration of PBZ (0.1 mgl−1) provided the lowest result (6.95%) of floral bud induction. Flowers obtained from all PBZ-containing media, i.e. at all concentrations of PBZ, showed normal morphology.

TABLE 4 In vitro flowering of orchids (Continued)


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Modified 12 MS Seedlings produced floral buds (33.3–34.8%) precociously on Wang et al., 2009 medium a defined MS medium containing paclobutrazol (PP333) at supplemented 0.5 mgl−1. In TDZ at 0.1 mgl−1 flowering occurred within 4 months of culture. The frequency of floral bud formation with paclobutrazol and increased to 95.6% by growing seedlings in 0.3 mgl−1 PP333 + 0.5 mgl−1 NAA followed by PP333 and TDZ. TDZ at different TDZ (0.05–0.1 mgl−1) and PP333 (0.5–1.0 mgl−1) were concentrations more effective for inducing floral buds of D. nobile plantlets than BA. Flowers that developed were deformed at 25◦ C but they developed fully when grown at a lower temperature regime (23◦ C/18◦ C, light/dark) for 45 days. 1 In vitro seed-derived rhizomes MS modified In vitro seed-derived rhizomes spontaneously sprouted flower Chang et al., 2010 4 All cultures were exposed to with coconut stems followed by flowering and subsequent fruiting to artificial light of 1000 lux with milk, potato complete a full life cycle. About 44–61% of rhizomes an average light/dark cycle of powder, peptone spontaneously produced flower stems from the apex. Among 16/8 h at 25 ± 1◦ C. and AC the flowers, 50% showed normal floral organs, 40% lacked both left and right sepals and 10% lacked the right petal only. 18% of the flowers showed autogamous mating and developed fruits. The seeds were harvested and sown further, showing a 59.6% average germination percentage. MS and VW In vitro flowering and fruit setting were mentioned but no Sotthikul et al., In vitro shoots medium specific reasons were provided the explain the phenomena. 2010 Cultures were maintained under fluorescent light at an intensity of 30 μmolm−2s−1, 16-h photoperiod. In vitro plants without roots VW medium The highest number of plants (38–43%) induced flower buds Rojanawong et al., Flowering occurred within supplemented 2006 after preculture in VW medium supplemented with 30 or 40–120 days. Cultures incubated with BA different 30–40 gl−1 sucrose before transfer onto standard or modified VW (i.e., MVW) medium supplemented with 66.6 μM of at 25 ± 2◦ C with a 16-h concentrations of photoperiod of BA for 40–120 days. BA and N concentration had major N supply 35–40 μmolm−2s−1 provided by effects on flower bud formation. cool white fluorescent lights.

In vitro seedlings Flowering was observed about 4–6 months after seeds were sown. The cultures were maintained in a culture room kept at 25◦ C with a 12-h photoperiod provided by white fluorescent light at 60 μmolm−2s−1.

2,4-D, 2,4-dichlorophenoxyacetic acid; 2ip, N6-isopentenyladenine or 6-γ ,γ -dimethylallylaminopurine; AC, activated charcoal; BA, N6-benzyladenine; BAP, benzylaminopurine; CPPU, forchlofenuron–N-(2-chloro-4-pyridyl)-N -phenylurea; CW, coconut water; H3 medium, Hyponex medium; IAA, indole-3-acetic acid, IBA, indole-3-butyric acid; 2ip, 6-(γ , γ -dimethylallylamino) purine (2ip); KC, Knudson medium; KN, kinetin; MS, Murashige and Skoog (1962) medium; mTR, meta-topolin riboside; NAA, a-naphthaleneacetic acid; NDM, New Dogashima medium (Tokuhara and Mii, 1993); NP, New Phalaenopsis medium; P668, Phytotechnology Medium; PM, Phytamax medium; PBZ, paciobutrazol; PGR, plant growth regulator; PLB, protocorm-like body; TCL, thin cell layer; TDZ, thidiazuron (N-Phenyl-N - 1,2,3-thiadiazol-5-ylurea); tTCL, transverse thin cell layer; VW, Vacin and Went (1949) medium; Zn, zeatin; ZR, zeatin-riboside.

Phalaenopsis Cygnus ‘Silky Moon’

Polystachya sp.

Eulophia graminea Lindl.

Dendrobium nobile Lindl.



108


Matsumoto (2006) reported that GA3 alone (2.5 mM) was effective in promoting inflorescence emergence in Miltoniopsis by increasing flower spike length and flower size (Sakai et al., 2000). Dewir et al. (2007) reported that GA3 was mandatory for a shift from the vegetative to the reproductive stage in Spathiphyllum (Araceae), an important tropical ornamental. However, Chen et al. (1997) reported that flowering in hybrid Phalaenopsis was clearly induced by GA3 at 1 μg/shoot at high temperature (30◦ C day/25◦ C night), and that flower primordial elongation, even though promoted by GA, was fully inhibited by BAP treatments while a combined treatment with BAP increased flower quality. In Cymbidium niveo-marginatum, a high concentration of GA3 (15 mgl−1) delayed flowering and decreased the percentage of plants exhibiting flower induction. However, it did not totally block flower induction (Kostenyuk et al., 1999). The blockage of flower development under high temperature can be rescued by applying GA3 exogenously, as observed in Phalaenopsis hybrida (Su et al., 2001). Dormant buds of Phalaenopsis contain a relatively high level of free ABA, whereas no detectable free or bound ABA was found in flowering shoots. A decrease in free ABA in buds may be associated with bud activation and the development of flowering shoots (Hsiao et al., 2011b). Recently, molecular techniques have been applied to study the mechanisms of flowering in orchids. As such, more then 70 genes have been cloned from seven orchid genera, some of which are related to flowering. The profile of gene expression during the transition to flowering has been established in Dendrobium spp. using an in vitro flowering system (Yu and Goh, 2000). For example, four color-related genes in Oncidium, including OgCHS, OgCHI, OgANS and OgDFR, have been identified (Chiou and Yeh, 2008). These genes are expressed during floral development. Among them, OgCHI and OgDFR showed especially low expression in yellow lip tissue but greater expression in the red part of Oncidium flowers (Hsiao et al., 2011b). A UDP-glucose anthocyanidin flavonoid glucosyltransferase (UFGT), which was isolated from P. equestris flower buds, showed high expression in red cultivars (Chen et al., 2011). Knockdown of the expression of PeUFGT3 by RNA interference resulted in various levels of fading color in Phalaenopsis, so PeUFGT3 may be associated with red color formation in Phalaenopsis. Chiou et al. (2008) identified OgCHRC and its promoter (Pchrc) in Oncidium that specifically expresses in flowers. Thiruvengadam et al. (2012) reported that overexpression of the Oncidium MADS box (OMADS1) gene promotes early flowering in transgenic Oncidium Gower Ramsey. Such tissue-specific promoters are key to tissue-targeted genetic transformation of orchids (Teixeira da Silva et al., 2011). Guo et al. (2006) cloned the pPI9 gene from Phalaenopsis but it only expressed in the reproductive organs, suggesting that it was probably involved in the regulation of floral morphogenesis. A more detailed review on the molecular basis underlying floral color and development in orchids has recently been reviewed by Hsiao et al. (2011b), and thus details will not be covered in our review.

X.

MYCORRHIZA AS A BIOTECHNOLOGICAL TOOL FOR IMPROVING ORCHID GROWTH Under natural and horticultural conditions, typical mutualistic associations are established among orchid roots and certain heterogeneous groups of fungi, the orchid mycorrhizae. More than a century ago, H. Wahrlich studied orchid mycorrhizae, describing the digestion of the fungus, establishing a fundamental fact that mycorrhiza are universal in the Orchidaceae. Subsequently, Noël Bernard perceptively observed these mycorrhiza and hypothesized that the role of these fungi in seed germination was in the form of a pervasive mutualistic symbiosis in which the fungus and host nutritionally depended on each other (Selosse et al., 2011). Initial groundwork of orchid mycorrhiza indicated Rhizoctonia to be the only fungal partner associated with orchids though, later on, a number of fungi were identified from orchids which showed resemblance in many aspects to Rhizoctonia; they were subsequently collectively termed Rhizoctonia-like fungi. More recently, many fungal symbionts were isolated from orchids and documented as belonging to the basidiomycete genera Ceratobasidium, Thanatephorus, Sebacina and Tulasnella. Selection of mycorrhizal roots, detection of colonized zones and thickness of cultured root sections is important for the isolation of mycorrhizal fungi. Root sections used by different researchers to isolate fungal endophytes were of varied thickness: 0.5 mm in Bletilla striata (Jonsson and Nylund, 1979), 0.5 cm in Campylocentrum fasciola, C. filiforme, Erythrodes plantaginea, Ionopsis satyrioides, I. utricularioides, Oeceoclades maculata, Oncidium altissimum, Psychilis monensis, and Tolumnia variegata (Otero et al., 2002), 2 mm in Eulophia flava, Goodyera procera, Habenaria dentata, Spiranthes hongkongensis (Shan et al., 2002), 3–5 mm in Vanilla planifolia (Porras-Alfaro and Bayman, 2007), 1 cm in Pterostylis nutans (Irwin et al., 2007); 2 cm in Cremastra appendiculata, Pleione bulbocodioides and P. yunnanensis (Zhu et al., 2008), 2 mm in Dendrobium officinale (Zhang et al., 2011), and 5 mm in Holcoglossum rupestre and H. flavcens (Tan et al., 2012). A single peloton or a clump of pelotons or colonized cells from the root cortex were also used to isolate fungal endophytes from Neuwiedia veratrifolia, Pleione spp., Caladenia spp., Dendrobium nobile, and D. chrysanthum (Kristiansen et al., 2001; Zhu et al., 2008; Wright et al., 2010; Chen et al., 2011). Different culture media have been used for growing fungal endophytes, including potato dextrose agar (PDA), cornmeal agar (CMA), oatmeal agar (OMA), water agar, malt extract agar, and bran agar but PDA was the most common (Warcup and Talbot, 1967; Jonsson and Nylund, 1979; Otero et al., 2002; Shan et al., 2002; Ma et al., 2003; Porras-Alfaro and Bayman, 2007; Zhang and Yang, 2008; Zhu et al., 2008; Nontachaiyapoom et al., 2010; Zhang et al., 2011; Wang et al., 2011). Interestingly, gelling agent, including the use of PDA, OMA and others, strongly affects the organogenic outcome of Cymbidium hybrids (Teixeira da Silva and Tanaka 2009a; Van et al., 2010). Some antibiotics such as streptomycin, tetracycline, chloramphenicol or penicillin at different concentrations (20–150 μg/ml) were used in the culture medium for controlling



bacterial growth, associated with mycorrhizal endophytes or in the surface of root sections (Otero et al., 2002; Zhu et al., 2008; Nontachaiyapoom et al., 2010; Wright et al., 2010; Chen et al., 2012). Different techniques were employed to isolate fungal endophytes. Warcup and Talbot (1967) selected the roots with external Rhizoctonia–like mycelium, washed them thoroughly in tap water and cut them into segments. The segments were teased apart in sterile water using two needles, releasing the pelotons into isolation plates. These were mixed with cooled, molten agar with 50 μg/ml streptomycin and 20 μg/ml tetracycline and poured into Petri dishes to obtain fungi growing from the pelotons. Currah et al. (1987) surface sterilized root segments with a 20% solution of household bleach for 1 min, rinsed twice in sterile distilled water, and decorticated with a sterile scalpel. Clumps of cells were removed from the inner cortex, macerated in a drop of sterile water, and plated in molten modified Melin–Norkran’s agar cooled to 55◦ C. The plates were then allowed to solidify and incubated in the dark at 18◦ C until hyphae were grown from the cortical cells into the media. Hyphal tips were transferred to PDA and serially transferred until pure cultures were obtained. Chang et al. (2007) developed a plastic bag cultivation method (PBCM) to grow Anoectochilus formosanus plants in plastic pots with fermented bark compost as the growth medium and covered with OPP transparent plastic bags to protect against water loss, disease and mite infection. Thereafter, plants with PBCM were placed under benches in greenhouses or trees, until 6–8 months later for harvesting. No more water or fertilizer was needed and minimum labor was required during cultivation. PBCM was a very effective and low-cost cultivation method for producing fungicide- and insecticide-free A. formosanus for medicinal use. Porras-Alfaro and Bayman (2007) isolated mycorrhizal fungi from Vanilla planifolia roots after removing velamen tissues and surface sterilization with ethanol R (52.6% sodium hypochlorite) for (70%) for 30 s, 50% Clorox 3 min and washed twice with distilled water and plated on PDA with penicillin and streptomycin (50 μg/ml each). A slightly different technique was applied by Zhang and Yang (2008) in Cremastra appendiculata and by Zhu et al. (2008) in C. appendiculata, Pleione bulbocodioides and P. yunnanensis. They peeled or scraped root hairs, epidermis, velamen tissue and other attachments in the roots and selected the roots with pelotons by microscopic examination. The roots were rinsed with sterilized distilled water five times then immersed in 10 ml of sterile distilled water with 150 μg/ml streptomycin sulfate and 150 μg/ml potassium Penicillin G and washed again with sterile distilled water. Roots with pelotons were cut into segments and cultured on PDA surface. Nontachaiyapoom et al. (2010) used 3% (v/v) H2 O2 and 70% (v/v) ethanol for 10 min for surface sterilization of mycorrhizal root and PDA medium containing 50 μg/ml oxytetracycline, 50 μg/ml streptomycin, and 50 μg/ml penicillin for culture of root sections. Zhang et al. (2011) rinsed healthy Dendrobium officinale roots under tap water, washed them in sterile de-ionized water, cut them into segments of about 1 cm in length and rewashed with sterile de-ionized water and surface

109

sterilized by 75% ethanol for 30 s, rinsed with sterile de-ionized water, soaked in 0.1% HgCl2 for 5 min and rinsed three times with sterile de-ionized water. Finally the roots were cut into 2-mm thick segments and cultured aseptically onto a PDA surface, incubated in the dark at 25◦ C until hyphae were visible. Pure cultures were obtained by transferring hyphae onto fresh PDA. A similar technique was followed by Ten et al. (2012) to isolate fungal endophytes from the roots of Holcoglossum rupestre and H. flavescens. They used PDA medium containing 50 μg/ml oxytetracycline, 50 μg/ml streptomycin, and 50 μg/ml penicillin. Traditionally, Rhizoctonia-like fungi are characterized based on cultural morphology, cytomorphology of the hyphae, monilioid cells, teleomorphs, affinities for hyphal anastomosis (rejoining of hyphae), number of nuclei in the cells and these features are studied by applying several staining methods such as Aniline blue or Trypan blue, HCl-Geimsa, Orcein, diamidino-2-phenylindole (DAPI), and Acridine orange (Shan et al., 2002; Ma et al., 2003). The hyphal diameter and dimensions of monilioid cells are generally measured by staining the hyphae with lactophenol triglycero-cotton blue or trypan blue or 0.5% (w/v) safranin and 3% KOH (1:1) solution. Culture growth rates are determined by inoculating uniform mycelium bits at the centre of PDA plates and measuring radial increments in colony size at definite time periods (Meyer et al., 1998; Shan et al., 2002; Nontachaiyapoom et al., 2010). The number of nuclei in hyphal cells is determined by fixing the mycelial mat in 2% formaldehyde for 2 min, rinsing it with distilled water for 1 min, followed by staining with DAPI or gold-antifade DAPI for 10 min, de-staining with distilled water for 2 min and observing under 50% glycerol (Shan et al., 2002). However, conventional approaches to identifying orchid mycorrhizal fungi have limitations as most of the fungal associates are mycelia sterilia. Consequently, broad vegetative criteria for identification have resulted in paraphyletic taxonomy with various unrelated fungi being grouped together, necessitating the application of molecular techniques for accurate identification (Sen et al., 1999; Otero et al., 2002; Shan et al., 2002; Dearnaley, 2007; Yagame et al., 2008). Sequencing of ITS regions is a common and powerful molecular technique for accurate identification of orchid mycorrhizal fungi (Kuninaga et al., 1997; Taylor and Bruns, 1997, 1999; Salazar et al., 2000; Gonzalez et al., 2001; Pope and Carter, 2001; Kristiansen et al., 2001; Sharon et al., 2008; Wright et al., 2010; Nontachaiyapoom et al., 2010; Wu et al., 2010; Zhang et al., 2011; Jacquemyn et al., 2011; Wang et al., 2011; Tan et al., 2012) (Table 5). This technique is performed by isolationg genomic or ribosomal DNA of fugal isolates, PCR amplification of ITS regions by specific primers such as ITS1 and ITS4, sequencing and then comparing sequences with the available data from GeneBank. The ITS region has several features that make it a strong candidate for a universal ‘barcode’ for fungal identification: it is easy to amplify due to high copy number, relatively few primer sets are needed

110 Ceratorrhiza cornigerum, C. globisporum, Rhizoctonia repens, R. solani, and Thanatephorus cucumeris

Mainly Rhizoctonia-like fungi (uni or bi-nucleate) related to Ceratobasidium spp., few Xylaria spp., Pestalotia spp., Colletotrichum spp., and many unidentified fungi

Eulophia flava, Goodyera procera, Habenaria dentata, and Spiranthes hongkongensis

Campylocentrum fasciola, C. filiforme, Erythrodes plantaginea, Ionopsis satyrioides, I. utricularioides, Oeceoclades maculata, Oncidium altissimum, Psychilis monensis, and Tolumnia variegata Cephalanthera austinae, Goodyera pubescens, Liparis lilifolia, Tipularia discolor Calanthe rubens, C. rosea, Cymbidium sinense, C. tracyanum, Goodyera procera, Ludisia discolor, Paphiopedilum concolor, P. exul, P. godefroyae, P. niveum and P. villosum Cypripedium calceolus, C. californicum, C. candidum, C. fasciculatum, C. guttatum, C. montanum, and C. parviflorum Epipogium roseum

Ionopsis utricularioides and Tolumnia Ceratobasidium spp. variegata Vanilla planifolia, V. poitaei, V. Ceratobasidium, Thanatephorus and Tulasnella phaentha, and V. aphylla

Ceratorhiza cerealis, C. goodyerae-repentis, C. pernacatena, C. ramicola, Ceratorhiza spp., Epulorhiza calendulina, E. repens, Rhizoctonia globularis, Sistotrema spp., Trichosporiella multisporum, Tulasnella spp. Waitea circinata and two Rhizoctonia spp. Fungi were mainly the members the family Tulasnellaceae. Rarely members of the Sebacinaceae, Ceratobasidiaceae, and an Ascomycetous genus Phialophora Psathyrella or Coprinus

Tulasnella

Tulasnella and Laccaria

Name of fungal isolate

Dactylorhiza majalis

Host plant

Reference

Athipunyakom et al., 2004

McCormick et al., 2004

Sequencing of ITS region of nuclear rDNA and phylogenetic analysis. Sequencing of ITS region of nuclear rDNA and phylogenetic analysis. Sequencing of ITS region of nuclear rDNA and part of the mitochondrial ribosomal large subunit (mtLSU) and phylogenetic analysis.

Yamato et al., 2005 Otero et al., 2004, 2007 Porras-Alfaro and Bayman, 2007

Sequencing and analysis of fungal ITS of nuclear Shefferson et al., large subunit and mitochondrial large subunit 2005 DNA and phylogenetic analysis.

Sequencing of ITS region of nuclear rDNA and part of the mitochondrial ribosomal large subunit (mtLSU) and phylogenetic analysis. Macroscopic features such as colony growth pattern, growth rate, color, sclerotia formation and nuclear number per cell.

Polymorphism analysis through sequencing of Kristiansen mitochondrial ribosomal large subunit (mtLSU) et al., 2001 DNA. Cultural and microscopic features of the fungal Shan et al., 2002 isolates such as colony color (young and mature, both front and reverse), growth rate, shape and diameter of monilioid cells, color of sclerotial masses, septal structure, teleomorph, diameter of vegetative hyphae, nuclei number per cell, hyphal branching, TEM, RAPD, RFLP and CAPS analysis of ITS rDNA. Sequencing of ITS region of nuclear rDNA and Otero et al., 2002 phylogenetic analysis.

Methods used for identification

TABLE 5 Key orchid fungal endophytes (i.e., orchid mycorrhizal fungi) and their methods of identification


111

35 species of fungi isolated from Cymbidium goeringii and C. faberi distributed in Tianmu Mountain, Zhingjiang Province, China showed that orchids and their mycorrhizal fungi had an obvious species-specificity relationship Dendrobium nobile Xylaria spp., Guignardia mangiferae, Clonostachys rosea, Trichoderma chlorosporum, Fusarium solani Dendrobium loddigesii Fusarium, Acremonium, Chaetomella, Cladosporium, Colletotrichum, Nigrospora, Pyrenochaeta, Sirodesmium, and Thielavia, etc. Changnienia amoena 18 fungal isolates identified Among them, 15 were Ascomycetes, and 3 belonged to Mortierella (Zygomycetes) and Tulasnella (Basidiomycetes) Hexalectris warnockii, H. grandiflora, A number of fungi belonging to Thelephoraceae, H. brevicaulis, H. revoluta, H. Russulaceae and Sebacinaceae families parviflora, H. arizonica, H. colemanii, H. nitida, and H. spicata Dendrobium officinale Mycena sp.

Rhizoctonia-like fungi, Fusarium spp. and Papulaspora spp.

Anacamptis pyramidalis, Orchis sancta, Ophrys fusca, and Serapias vomeracea subsp. orientalis

Cymbidium goeringii and C. faberi

Epulorhiza, Ceratorhiza, Ceratobasidium, Sistotrema, Thanatephorus and Tulasnella

A number of orchid species

Jiang et al., 2011

Kennedy et al., 2011

Sequencing of ITS region of nuclear rDNA and phylogenetic analysis.


Morphological, cultural and sporulation Zhang et al., characteristics were studied through light 2011 microscopy and SEM; and sequence analysis of the ITS nuclear rDNA regions. (Continued on next page)

Chen et al., 2010

Sequencing of 5.8S ITS regions rDNA and phylogenetic analysis for fungi.

SEM, sequencing of ITS region of nuclear rDNA Yuan et al., and phylogenetic analysis. 2009b

Taylor and McCormick, 2008 Cultural and morphological characteristics of the Gezgin and fungal isolates such as colony color, (both front Eltem, 2009 and reverse), growth rate, conidial morphology, hyphal morphology, presence or absence aerial mycelia, hyphal width, nuclei number per cell, hyphal branching, appearance of sclerotia, and monilioid cell morphology. Sequencing of ITS region of nuclear rDNA and Wu et al., 2009 phylogenetic analysis.



112

RAPD, random amplified polymorphic DNA; RFLP, restriction fragment length polymorphisms; CAPS, cleaved amplified polymorphic sequence; ITS, internal transcribed spacer; OMF, orchid mycorrhizal fungus; TEM, transmission electron microscopy; SEM, scanning electron microscopy.

Holcoglossum rupestre and H. flavescens

Platanthera minor

80 species of fungi belonging to 37 genera identified Among them Acremonium, Alternaria, Ampelomyces, Bionectria, Cladosporium, Colletotrichum, Fusarium, Verticillium and Xylaria were the dominant fungal endophytes. Yagame et al., Ceratobasidiaceae fungi Identified the fungal isolates by PCR 2012 amplification ITS of the fungal ribosomal DNA of hyphal coils extracted from P. minor roots. They tested for a 13C and 15N enrichment characteristic of mixotrophic plants. They also tested the ectomycorrhizal abilities of orchid symbionts using a new protocol of direct inoculation of hyphal coils onto roots of Pinus densiflora seedlings. 46 culturable fungal endophytes belonging to Identified the fungal isolates based on sequences Tan et al., 2012 four classes, i.e., Sordariomycetes (41.30%), analysis of the ITS nuclear rDNA region. Dothideomycetes (36.96%), Agaricomycetes (17.39%), Leotiomycetes (4.35%) were isolated. 36 strains were identified at the genus level. Among the fungal isolates, only two, i.e. Tulasnella calospora and Epulorhiza spp., were identified as OMF.

Dendrobium candidum, D. nobile, D. falconeri, D. loddigesii, D. primulinum, D. gratiosissimum, D. christyanum, D. hancockii, D. pendulum, and D. moniliforme

Reference

Identified based on spore and culture Chen et al., characteristics (colony shape, height and color 2011a of aerial hyphae, base color, growth rate, margin, surface texture, and depth of growth into medium). Sequence-based methods were also conducted through sequencing of ITS regions rDNA, the 5.8S and large subunit rRNA (nrLSU) and phylogenetic analysis. Identified the fungal isolates based on Chen et al., microscopic and culture characters and 2011b sequence analysis of the ITS nuclear rDNA region.

Methods used for identification

Rhizoctonia-like strains belonging to Tulasnellales, Sebacinales and Cantharellales In addition, species of Xylaria, Fusarium, Trichoderma, Colletotrichum, Pestalotiopsis, and Phomopsis were identified.

Name of fungal isolate

Dendrobium nobile and D. chrysantheum

Host plant

TABLE 5 Key orchid fungal endophytes (i.e., orchid mycorrhizal fungi) and their methods of identification (Continued)




as a result of the highly conserved small sub-unit (SSU) and large sub-unit (LSU) flanking regions, and it varies relatively little within species but dramatically between species, and is far better represented in GenBank than other loci in fungi (Jakucs et al., 2005; Taylor and McCormick, 2008; Zhang et al., 2011). Wu et al. (2009) reported 35 species of fungi isolated from Cymbidium goeringii and C. faberi distributed in Tianmu Mountain, Zhingjiang province, China and showed that these orchids and their mycorrhizal fungi have an obviously corresponding species-specificity ralationship by sequencing the ITS region of nuclear rDNA and phylogenetic analysis. Today, isolation of rDNA, amplification of specific DNA regions through direct PCR and sequencing or direct PCR amplification of DNA fragments from a single peloton of the mycorrhizal root using specific RFLP and RAPD primers are routinely used for characterization of orchid mycorrhiza (Sharon et al., 2008; Nontachaiyapoom et al., 2010; Wright et al., 2010). Seed germination, nutrition, ecological fitness and growth of numerous orchids, including Anoectochilus roxburghii, Arundina chinensis, Caladenia spp., Changnienia amoena, Cremastra appendiculata, Cymbidium ensifolium, C. lianpan, C. sinense, C. tracyanum, Dactylorhiza spp., Dendrobium brymerianum, D. candidum, D. chrysotoxum, D. densiflorum, D. loddigesii, D. nobile, D. primulinum, Epigeneium rotundatum, Erythrorchis ochobiensis, Gastrodia elata, Goodyera repens, Habenaria macroceratitis, Liparis viridiflora, Paphiopedilum armeniacum, Platanthera clavellata, P. integrilabia, P. bifolia, Pleione bulbolodiodes, Spathoglottis plicata, S. pubescens, Spiranthes hongkongensis, and Vanda amesiana have been promoted by inoculation with a range of orchid mycorrhizal fungi (OMF), including Ceratobasidium sp., Ceratorhiza sp., Epulorhiza sp., Mycena anoectochila, M. dendrobii, M. orchidicola, M. osmundicola, Rhizoctonia sp., and Tulasnella sp. (c.f. He et al., 2010). Seed germination and seedling development of some orchid species were strongly stimulated by Epulorhiza isolates and a Ceratorhiza isolate (Shan et al., 2002). The technique of burying seeds in “packets” either adjacent to or at varying distances from adult plants (i.e., in situ seed baiting method) in investigating in situ orchid seed germination and seedling development was found to be an effective means for orchid mycorrhizal fungal inoculum levels and specificity at the microhabitat level (Rasmussen and Whigham, 1993, 1998; Masuhara and Katsuya, 1994; Perkins and Mcgee, 1995; McKendrick et al., 2000). Recently, Wang et al. (2011) established a new in situ seed baiting method for Dendrobium officinale and D. nobile using seed packets to assess germination of orchid species within soil, providing a means of locating, collecting, and identifying specific fungi that are involved in the lifecycle of orchids in the wild and investigating their relationship with orchids under natural field conditions. This information will be critical to future plant production and reintroduction efforts aimed at the conservation of Dendrobium species in their natural habitats.

113

Hardening of tissue culture-raised plants is another important aspect of orchid mycorrhiza. Plantlets produced in vitro can not develop resistance against major and minor microbial pathogens. In addition, physiological and anatomical deficiencies of in vitro propagated plants make them incapable of surviving under field conditions (Pandey et al., 2000, 2002). As orchid seedlings can be infected with mycorrhizal endophytes during seed germination in nature and since they develop resistance to fungal infection at maturity and supply nutrition for their existence, there is a possibility of biological hardening for better growth and survival of asymbiotically raised orchid seedlings using appropriate OMF (Zhan and Zhou, 2004; Yamato and Iwase, 2008; Zhang et al., 2011). Since orchid plants permit the infection, colonization and co-existence of mycorrhizal fungi, it seems that during the process of root colonization, they initiate the synthesis and expression of defense mechanism-related secondary metabolites such as phytoalexins, isoflavonoids glyceollin, coumestrol, coumestin, isosojagol and other OMF-specific compounds that provide protection against biotic and abiotic stresses during the weaning phase, resulting in higher plant survival (Sahay and Varma, 2000; Hazarika, 2003). Many researchers are currently trying to find a way to promote the survival rate of transplantation through the symbiotic relationship of orchids with OMF (Ke et al., 2008; Zhang et al., 2011) while many studies have indicated that mycorrhizal fungi can effectively promote seed germination and seedling growth of many orchids (Guo et al., 2000; Chang and Chou, 2001; Kang et al., 2007; Jin et al., 2009; Chen et al., 2010; Dan et al., 2012). For example, growth of seedlings from Anoectochilus formosanus, Bletilla formosana, Cymbidium goeringii, Dendrobium amethystoglossum, D. Chao Praya Garnet, D. Snow Flake, D. tobaense, D. loddigesii, Doritis pulcherrima, Gastrodia elata, Haemaria discolor var. dawsoniana, Oncidium Gower Ramsey, Phalaenopsis spp., and other species from the genera Doritaenopsis, Phalaenopsis and Paphiopedium spp. was highly enhanced by specific Rhizoctonia strains (Chang and Chou, 2007; Chang et al., 2007; Chang, 2007, 2008; Ke et al., 2008; Wu et al., 2009, 2010; Chen et al., 2010; Zhang et al., 2011; Dan et al., 2012). Hossain et al. noted that Cymbidium aloifolium seedlings raised in vitro showed 100% survival in a greenhouse when inoculated with Ceratobasidium sp., an anamorphic Rhizoctonia-like OMF isolated from Rhynchostylis retusa (unpublished data). The survival rate of artificial cultivation of D. officinale transplanted from tissue culture could reach 80∼90% (Zhang et al., 2011). Resistance to bacterial and viral diseases and vegetative and reproductive growth was stimulated by OMF in a number of ornamental orchids, such as Doritaenopsis spp., Phalaenopsis spp., Cymbidium spp., Oncidium sp., and Phaphiopedium delenatii (Chang, 2007, 2008; Ke et al., 2008; Fang et al., 2008; Wu et al., 2011). Very recently, Dan et al. (2012) conducted extensive research on the effects of OMF on the growth of protocorms and Dendrobium candidum and D. nobile plantlets. Forty fungal endophytes isolated


114


from different orchid species were used for this study. Among the different OMF stains, six fungal stains (only 15%), including Mycena dendrobii (DC-13), Mycena anoectochila (AR-13), Epulorhiza sp. (AR-15), Epulorhiza sp. (DC-8), Gliocladium sp. (DN-3), and Cephalosporium sp. (AR-7), showed significant positive effects in terms of growth and height of orchid plantlets. The application of OMF may be performed both in vitro and ex vitro. In vitro inoculation may be achieved by placing the asymbiotically raised seedlings on moistened cotton wads overlying the fungal growth on PDA or other agar culture media such as 14 MS basal medium for one week and then transferring to pots outside. For the ex vitro application, in contrast, first the mycorrhizal inoculum is prepared by growing the fungi in potato dextrose broth for one week while the fresh fungal mycelium needs to be homogenized in distilled water or sterile 1 MS basal medium with a sterile homogenizer. Then the inocu4 lum is applied at the root surface before or after transplantation to pots containing sterilized potting mixture as support medium and kept under controlled environmental conditions for a few weeks before reintroduction to the natural habitat. Therefore, it is highly recommended that OMF be applied for the following purposes: a) to increase the survival rates of micropropagated plantlets or seedlings ex vitro; b) to enhance both vegetative and reproductive growth of orchid plants; c) to stimulate early flowering and enhance flower quality; and d) to reduce disease infection rates (Chang, 2006, 2007, 2008, Wu et al., 2011). Some studies indicated that orchid mycorrhizal fungi could secrete gibberellic acid (GA3 ), indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin (ZT) and zeatin riboside (ZR) and that these hormones have strong effects in improving the growth of medicinal orchids and seed germination and cell differentiation (Guo and Xu, 1990; Yang et al., 2008). Applying elicitors from pathogenic fungi has been an effective strategy for improving the productivity of useful secondary metabolites in medicinal orchids, such as D. officinale (Song and Guo, 2001; Chen et al., 2008) and C. goeringii (Zhao and Liu, 2008; Jin et al., 2009). Once the symbiotic relationship was established, the mycorrhizal fungi served as the dominant microorganism to release antagonistic substances, which are effective in preventing the invasion of other pathogens and indirectly enhanced the survival rate of seedlings and promoted the growth of seedlings (Chen et al., 2003).

XI.

BIOTECHNOLOGY AND THE CONTROL OF ORCHID VIRUSES Different biotechnological assays have been successfully applied for the diagnosis of orchid viruses, their control measures, production and certification of virus-free plants. Orchids are affected by more than 50 viruses. Cymbidium mosaic potexvirus (CymMV) and Odontoglossum ringspot tobamovirus (ORSV) are the most prevalent and economically important viruses infecting orchids worldwide (Wong et al., 1994; Ryu and Park, 1995a, b; Ajjikuttira et al., 2002; Zheng et al., 2008a; He et al.,

2011; Mohd-Hairul et al., 2011; Yoon et al., 2011, Zheng et al., 2011). Viral diseases are a serious threat to the orchid industry as they reduce plant vigour, decrease flowering capacity and lower flower quality, all of which significantly reduce their commercial value (Mackenzie et al., 1998; Mohd-Hairul et al., 2011). Many orchid viruses are highly contagious and are transmitted mechanically through infected sap on cutting tools and potting media when plants are pruned or divided for repotting and some viruses even retain their infectivity for long periods. In general, once viral diseases occur, there is no way of getting rid of them, short of destroying the infected plants. A number of sophisticated biotechnological approaches are now routinely used for detecting and identifying orchid viruses including bioassays, electron microscopy, reverse transcription-polymerase chain reaction (RT-PCR), hybridization and molecular beacon, enzyme-linked immunosorbent assay (ELISA), quartz crystal microbalance (QCM), immunosensors, immunocapillary zone electrophoresis (I-CZE), immunocapture-PCR (IC-PCR), liquid chromatography/mass spectrometry (LC/MS), matrix-assisted laser desorptionio-nization (MALDI) and digoxigenin (DIG)labelled cRNA probes (Lim et al., 1993; Wong and Chang, 1993; Ryu and Park, 1995a, b; Barry et al., 1996; Hu and Wong, 1998; Seoh et al., 1998; Eun and Wong, 1999, 2000; Tan et al., 2000; Eun et al., 2002; Lee and Chang, 2006; Sherpa et al. 2006, 2007; Liu et al., 2009; Mohd-Hairul et al., 2011; Padmavathi et al., 2011; Yoon et al., 2011; Zheng et al., 2011) (Tables 6 and 7). In many cases, a simple diagnostic test is not sufficient for diagnosis (Elliot et al., 1996). In such instances, it is necessary to apply more than one technique to identify viruses and to confirm whether the plant is infected with a virus or whether symptoms are due to other causes (Hu et al., 1993; Elliot et al., 1996; Eun and Wong, 1999). Among these detection methods some are very sensitive and efficient such as ELISA. Both direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) are cost-effective and can satisfactorily detect different viruses (Hull, 2002). For a detection method to be sensitive depends on the properties of viral proteins, and preparation of an efficient antibody is important for its specificity and sensitivity. Viral CP is responsible for the immune reaction against virus particles. DAS-ELISA is a widely used serological technique for virus indexing and offers an ideal diagnostic technique for routine mass-indexing programs such as virus-free certification, breeding, plant quarantine, and germplasm screening because it is relatively sensitive and cheap although it may give unreliable results, especially with potyviruses (Arunasalam and Pearson, 1989; Mackenzie et al., 1998; Mohd-Hairul et al., 2011). In addition, ELISA is particularly suitable for dealing with large amounts of samples in surveys or indexing. PCR has been shown to be very useful for detecting and characterizing plant pathogens and it is more sensitive than direct probing or serological techniques (Hadidi and Podleckis, 1995; Randles et al., 1996). RT-PCR using specific primers enables small numbers of viral RNA molecules to be detected in nucleic acid

115

BYMV CymMV and ORSV

Calanthe

Oncidium

Molecular beacons

This rapid and specific technique is applicable to the orchid industry, which routinely carries out virus indexing and screening for virus-resistant cultivars. The use of molecular beacon approach can be extended to the detection of multiple plant viruses in various crops. (Continued on next page)

Matsumoto et al., 1999 Eun and Wong, 2000

Mackenzie et al., 1998

Multi-RIPA

EM, Modified RT-PCR Modified RT-PCR improved the speed, sensitivity and (“salt wash/One specificity of the method. Application of multi-techniques Tube/RT-PCR”), gene helped in successful detection of viruses. sequencing and analysis EM, ELISA Both techniques were efficient for identification of this virus.

RIPA is very easy and convenient, enabled rapid, simultaneous Tanaka et al., diagnosis of virus diseases in the field without specialized 1997 equipment and gave satisfactory results. This technique can show results of diagnosis in a short time (about 5 min per sample) with high sensitivity.

EM

References

Aranthera Bulbophyllum, Calanthe, CymMV and Cattleya, Cymbidium, ORSV Gromatophyllum, Phalaenopsis, Oncidium and Vanda Arachnostylis, Ascocenda, Arachnis, CymMV and Cymbidium, Christeara, Calanthe, ORSV Cattleya, Cymbidiella, Dendrobium, Doritis, Gramanthophylum, Kagawara, Lowana, Mokara, Oncidium, Paphiopedilum, Phalaenopsis, Renanthera, Rhynchostylis, Thunia, Vanda, Vandopsis, and Vascostylis Representative orchid species CerMV belonging to 33 genera

Efficiency/sensitivity of the technique

It is more sensitive than ELISA. Tissue prints derived from Chia and different parts of the plants could show the distribution of Chan, 1992 viruses within plants. On the basis of its sensitivity and ease of use, this method may find wide application in the detection and localization of viruses in orchids. EM has moderate efficiency and it is comparatively easy in Inouye and virus detection. It helps in detecting the shape of viruses Gara, 1996 which is not possible by other serological techniques.

Techniques applied for detection Tissue-print hybridization assay

Virus name

Dendrohium, Cattleya and Oncidium CymMV and ORSV

Species

TABLE 6 Some common methods used for the detection of orchid viruses


116 Virus name CymMV and ORSV

CymMV CymMV and ORSV

CymMV and ORSV

CymMV and ORSV Cal MMV

CymMV

Species

Oncidium

Cattleya

Oncidium

Cymbidium Sw.

Phalaenopsis

Cymbidium pendulum and C. tigrinum

Phaius tankervilliae

Efficiency/sensitivity of the technique

LC-and/or MALDI-mass These techniques are capable of providing molecular mass spectrometry information on biological samples with high speed, accuracy and sensitivity and can be applied to all viruses with known CP molecular weights. LC/MS and MALDI allow automated analyses of multiple samples with simple preparation steps; both techniques are ideal for rapid identification of viruses from a large number of samples. GPAT It is a very rapid, economical, and accurate technique for virus detection in a number of plant species of different groups. QCM-based DNA QCM is a sensitive mass-measuring device. The QCM DNA biosensors biosensors method is efficient, highly sensitive, capable of detecting target viral RNAs in virus-infected plants. It is a more economical means of plant virus detection than other techniques. EM, DAS-ELISA DAS-ELISA is a sensitive serological technique currently used in routine diagnosis of plant virus infection. However, it is laborious and less sensitive than other approaches. It also may give unreliable results, especially with potyviruses. Multiplex RT-PCR The detection methods based on RT-PCR are highly sensitive, reliable and rapid for detecting small amounts of viral templates. ELISA, RT-PCR and Application of more than one technique is obviously Northern blot analysis trustworthy but the cost of detection increases and gene sequencing proportionally. Sometimes a single technique is not enough for accurate detection of viruses. Northern blotting is also a reliable technique. The sequencing of a gene is very helpful to compare the similarity and dissimilarity among the viruses infecting different orchids. ELISA, RT-PCR and Genome analysis of the CymMV isolates from different gene sequencing, EM regions shared good similarity, determined by sequencing and phylogenetic analysis.

Techniques applied for detection

TABLE 6 Some common methods used for the detection of orchid viruses (Continued)


Sherpa et al., 2007

Singh et al., 2007

Lee and Chang, 2006

Navalinskienë et al., 2005

Han et al., 2001 Eun et al., 2002

Tan et al., 2000

References

117

CymMV and ORSV

Phalaenopsis spp.

Using these techniques it is possible to identify the cytoplasmic types of OFV in orchids. Using these techniques is possible to identify CyMV and ORSV in orchids.

Using these techniques it was possible to identify distinct isolates of CymMV co-infecting Dendrobium and determination of phylogenetically distinct CymMV strains. Successful identification of CyMV and ORSV.

As above

Successful identification of a new virus for Phalaenopsis and determination of phylogenetically related viruses.

It is now considered as a reliable and shortcut method for detection of viruses and phylogenetic analysis. Using this technique it is possible to determine the subgroups of viruses displaying few differences due to regional and species differences. RT-PCR, DAS-ELISA, Using these techniques it was shown that the CP gene Molecular cloning and sequences are highly conserved among ORSV isolates, sequence analysis irrespective to geographic location. Thus, only CP gene sequencing and analysis may give accurate results for respective viruses.

Complete nucleotide sequencing

DAS –ELISA and RT-PCR

RFLP, dot-blot hybridization, PCR, gene sequencing RT-PCR, I-ELISA, Immunoblotting, bacterially expressed recombinant CP gene sequencing TEM, PTA-ELISA

EM, Immunoblotting, I-ELISA, Molecular cloning and sequence analysis As above

Yoon et al., 2011

He et al., 2011

Kubo et al., 2009 Liu et al., 2009

Lee and Chang, 2008

Zheng et al., 2008b Vaughan et al., 2008

Zheng et al., 2008a

BYMV, Bean yellow mosaic virus; CaCV-Ph, Capsicum chlorosis virus-Phalaenopsis; Cal MMV, Calanthe mild mosaic virus; CerMV, Ceratobium mosaic potyvirus; CP, coat protein; CymMV, Cymbidium mosaic potexvirus; GPAT, gelatin particle agglutination test; I-ELISA, indirect enzyme-linked immunosorbent assay; ORSV, Odontoglossum ringspot tobamovirus; OFV, Orchid fleck virus; PhCSV, Phalaenopsis chlorotic spot virus; MALDI, matrix-assisted laser desorption-ionization; PTA-ELISA, plate-trapped antigen – ELISA; QCM, quartz crystal microbalance-based DNA biosensors; RIPA, rapid immunofilter paper assay; RT-PCR, reverse transcription-polymerase chain reaction.

Cymbidium virescens, other ORSV Cymbidium spp., Dendrobium spp., and Oncidium spp

Miltonia spp., Cattleya spp., OFV Jumellea spp., and Coelogyne sp. 14 orchids. Arachnis spp., Cattleya CymMV and spp., 2 Cymbidium hybrid, C. ORSV sinense, C. goeringii, C. kanran, C. faberi, C. ensifolium, C. ensifolium var. rubrig emmum, Dendrobium spp., Grammatophyllum speciosum, Oncidium spp., Phalaenopsis spp. Vanilla fragrans CymMV

CymMV

PhCSV

CaCV-Ph

Dendrobium spp.

Phalaenopsis spp.


118


TABLE 7 A comparison of some common methods of plant virus detection.


Method∗

Real-time Nature of analysis? Sensitivity probe

Relative cost per sample analyzed

ELISA

No

2.5 ng

Antibody

Low

Tissue-print hybridization Multi-RIPA

Yes

20 pg

Nucleic acid

High

Yes

1.5 ng

Antibody

Medium

DIG-labeled cRNA probes RT-PCR

No

100 pg

Nucleic acid

Medium

No

100 fg

Nucleic acid

Medium

I-CZE

Yes

10 fg

Antibody

Medium

Molecular beacons QCM-based DNA biosensors Immunoblotting

Yes

0.5 ng

Nucleic acid

High

Yes

1 ng

Nucleic acid

High

No

1–5 ng

Antibody

Medium

Speed of analysis 4 h for 96 samples 24–72 h for 96 sample 5 min per sample 4 h for 1 sample 3.5 h for 96 samples 10 min for 1 sample 1 h for 96 samples 40 min for 1 sample 12–36 h for 96 sample

Ability to detect specific genes

References

No

Hill et al., 1981

No

Chia et al., 1992

No

Tanaka et al., 1997 Hu and Wong, 1998 Seoh et al., 1998

Yes Yes No

No

Eun and Wong, 1999 Eun and Wong, 2000 Eun et al., 2002

No

Zheng et al., 2008

Yes

I-CZE, immuno-capillary zone electrophoresis; DIG, digoxigenin; RT-PCR, reverse transcription-polymerase chain reaction; ELISA, enzymelinked immunosorbent assay.

mixtures. There are now many published reports of the use of RT-PCR with degenerate primers that produce specific DNA fragments from all species of a group, genus or even family of viruses, including potyviruses (Gibbs and Mackenzie, 1997; Langeveld et al., 1991; Mohd-Hairul et al., 2011; Yoon et al., 2011), carlaviruses (Badge et al., 1996), closteroviruses (Karasev et al., 1994; Tian et al., 1996), tospoviruses (Mumford et al., 1996; Zheng et al., 2011) and geminiviruses (Deng et al., 1994). Dot/slot blot hybridization and molecular beacons are also a proven efficient technique for virus indexing and screening for virus-resistant cultivars (Eun and Wong, 2000; Vaughan et al., 2008) (Tables 6 and 7). Molecular beacons, which are applicable to the orchid industry, are single-stranded nucleic acid molecules with a stem-loop conformation which are routinely applied to virus indexing and screening for virus-resistant cultivars. This technology was successfully applied to detect CymMV and ORSV in Oncidium (Eun and Wong, 2000). In addition, several companies such as Mukoyama Orchids in Japan, Forsite Diagnostics in Great Britain and Agdia, Inc., in the United States have recently developed some easy and rapid techniques for diagnosis of orchid viruses that can be performed in the field and provide results within 5 to 10 min (Batchman, 2008; Hossain et al., 2009b; www.agdia.com; www.forsitediagnostics.com). Tissue culture techniques such as seed propagation and meristem culture have been used for production of virus-free plants (Morel, 1960; Freitas and Rezende, 1998). Cybularz-Urban and

Hanus-Fajerska (2006) investigated the chemotherapeutic effect of PGRs in eradicating a mixed infection of CyMV and ORSV from Cattleya schönbrunnensis × C. leopoldii gutata. They reported that cytokinins, specifically N6-benzyladenine (BA), kinetin (KN) and zeatin (ZN) at different concentrations in MS medium could effectively eliminate CyMV from proliferating Cattleya mericlones with mericlone induction in 3.2 mg/l KN and further propagation in the presence of 0.2 mg/l ZN surprisingly eliminating CyMV. Another PGR combination namely induction on 5.0 mg/l BA then transfer to 0.5 mg/l ZN also effectively eliminated CyMV. Interestingly, ORSV infection persisted in all treatments. Chemotherapy using specific antiviral compounds has been used in orchid tissue culture media for eliminating viruses. Yap et al. (1999) used ribavirin (virazole) against CymMV in tissue cultured Oncidium Goldiana plantlets in which 10 mg/l ribavirin for 30 days was sufficient to eradicate CymMV from 43% of meristem-raised plantlets compared to 31% eradication for plantlets derived from callus tissue treated with 20 mg/l for 60 days. Chemotherapy using dithiouracil or ribavirin was also suitable for detecting CymMV-negative tissue (Kuehnle, 1996). Freitas and Rezende (1998) obtained 83–100% ORSV-free orchids by using meristem culture in association with chemotherapy by virazole at various concentrations. Development of transgenic orchids is possible by incorporation the CP gene for virus resistance into a number of orchids (reviewed in Teixeira da Silva et al., 2011). For example,



Phalaenopsis expressing heterologous CymMV CP can effectively confer resistance to CymMV (Liao et al., 2004) and the Odontoglossum ringspot virus (ORSV) CP gene was cloned from ORSV-infected Epidendrum to Cymbidium niveomarginatum through A. tumefaciens for the same purposes (Chen et al., 2006). Development of Phalaenopsis orchids with resistance to both viral and bacterial phytopathogens has also been possible by applying gene stacking. For this purpose, PLBs were first transformed with a CP cDNA of CymMV to confer protection against CymMV infection via particle bombardment then re-transformed with sweet pepper ferredoxin-like protein cDNA (Pflp) by A. tumefaciens which expressed dual (viral and bacterial) disease resistant traits (Chan et al., 2005). Harikrishna et al. (2010) developed a novel method for the isolation of viral CP-specific peptides using superparamagnetic beads. This resulted in the identification of a peptide with specificity for CymMV and several other peptides which were able to detect both CymMV and ORSV. They also reported that the use of double-stranded RNA (dsRNA) produced in E. coli could reduce CymMV infection and/or replication, although the same effect could be produced by pathogen-derived re-sistance via plant transformation. The use of bacterially pro-duced dsRNA encoding viral genes offers several advantages as a method for use in plant protection because it is relatively in-expensive and faster than producing transgenic plant varieties expressing the same RNA. XII.

GENE SILENCING TECHNOLOGY IN GENE FUNCTIONAL VALIDATION OF ORCHIDS The Orchidaceae has diverse, specialized pollination and ecological strategies that provide a rich subject for investigating evolutionary relationships and developmental biology (e.g., Singer et al., 2007). However, the study of these non-model organisms may be hindered by challenges such as their large genome size, low transformation efficiency, long regeneration period, and long life cycle. In fact, orchids are the opposite of model plants in every trait studied by geneticists and molecular biologists. To overcome these obstacles, Lu and his collaborators in Taiwan first developed vectors with the use of a symptomless CymMV, which infects most orchids and established the strategy of virus-induced gene silencing (VIGS) (Lu et al., 2007). By this method, they became successful in the functional analysis of plant genes involved in loss-of-function. The success of their strategies was verified by functional validation of floral identity gene(s) in tetraploid Phalaenopsis, which has a long life cycle. Functional analysis of orchid genes by VIGS has advantages over other approaches, such as transforming plants with antisense and/or sense genes that can produce dsRNA of target genes (Hsiao et al., 2011b). 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 and prevent suppression of some essential genes required for seed maturation, a common problem with mutagenesis screening. The

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application of functional genomics has been made possible by knocking down PeUFGT3 via VIGS in a Doritaenopsis hybrid derived from a cross of Doritaenopsis ‘I-Hsin Lucky Girl’ × Dtps. ‘I-Hsin Song’. The PeUFGT3-suppressed Doritaenopsis hybrids exhibited various levels of fading flower color, which was well correlated with the extent of reduced PeUFGT3 transcriptional activity (Chen et al., 2011). Therefore, VIGS offers an opportunity for functional analysis of genes in plants with long life cycle, such as orchids. XIII.

GENES CONTROLLING FLORAL MORPHOGENESIS IN ORCHIDS: ORDER IN THE MADS-HOUSE The versatility and specialization in orchid floral morphology, structure, and physiological properties have fascinated botanists for centuries. Reproductive development in the flowering shoot of orchids begins with the transition of the dormant meristem from producing vegetative structures to producing inflorescence branches, floral bracts, and finally flowers (Hsiao et al., 2011b). Orchids represent 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. 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. Since 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 (Tsai et al., 2007b; Fernando 2010; Chen et al., 2011). In floral studies, MADS-box genes contribute to the “ABCDE model” of floral organ identity. According to this model, the organ identity in each whorl is determined by a unique combination of three activities by floral organ identity genes, A, B, C, D and E. Generally it is believe that, the expression of ‘A’ gene alone specifies sepal formation. The combination of ‘BC’ genes offers stamen formation; the expression of the C function alone determines the development of carpels. The functions of ‘A’ and C are mutually repressive, D-class genes specify ovules while ‘E’ gene functioned in developing vegetative leaves rather than sepals, petals, stamens, or carpels (Theissen et al., 2000; Tsai et al., 2008a, b; Chang et al., 2010, 2011; Xu et al., 2010; Fernando, 2010). Some recent experimental information on MADS-box genes indicated the specific function of these genes. For instance, two A-class MADS-box genes (ORAP11 and ORAP13) identified from Phelaenopsiss hybrida cv. ‘Formosa rose’ (Chen et al., 2007), one gene (OMADS10) from Oncidium Gower Ramsey (Chang et al., 2009) and four genes (DOMADS2, DthyrFL1, DthyrFL2 and DthyrFL3) from Dendrobium (Yu and Goh, 2000;


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Skipper et al., 2005), seven floral organ identity genes from Dendrobium crumenatum i.e. DcOAP2 (D. cru-menatum AP2-like gene), DcOAP3A, DcOAP3B, DcOPI, DcOAG1, DcOAG2 and DcOSEP1 (Xu and Kumar, 2010) are involved in the transition of flowering and floral organ development through expression and over expression patterns in transgenic plants. Several members of B-class genes (APETALA3 or AP3 and PISTILLATA or AP1-like genes), isolated from P. equestris and O. Gower Ramsey, specify the identity of petals in whorl 2 and stamens in whorl 3 (Theissen et al., 2000; Hsu and Yang, 2002; Tsai et al., 2004b, 2005; Chang et al., 2009, 2011). In addition, two AP3- and one PI-like genes have also been identified from D. crumenatum (Xu et al., 2006b). Expression and protein-protein interaction studies suggest that the specific combination of duplicate gene expression and protein-protein interactions are responsible for the development of different floral organs (Tsai et al., 2008a, b; Chang et al., 2009, 2011). However, the proposed models derived from studies of Phalaenopsis and Oncidium to explain lip development are diverse. From a study on Phalaenopsis, PeMADS4 (clade AP3A2 or 4) was suggested to play a critical role in determining lip development because its transcripts are specifically detected in lip and in peloric mutant lip-like petals (Tsai et al., 2004b, 2008a). Very recently, B-class genes were collected from 11 species in four orchid subfamilies except for Apostasioideae to give a picture of floral organ development controlled by B-class genes (Pan et al., 2011). After analyzing the expression patterns of B-class genes by PCR-based methods and in situ hybridization, the “Homeotic Orchid Tepal (HOT) Model” was proposed to illustrate the regulation of perianth morphogenesis in orchids (Hsiao et al., 2011b). In this model, PI and AP3B clades determine the formation of sepals. The combination of PI and both AP3A1 and AP3B clades controls lateral petal formation at the inflorescence stage, whereas PI and AP3A1, AP3A2 clade genes and AGL6-like genes contribute to lip morphogenesis in the floral bud stage (Pan et al., 2011). The HOT model is somewhat similar to the “orchid code” (MondragónPalomino and Theissen, 2008) but more precisely points out the effect of PeMADS4 (AP3A2 clade) in determining lip morphogenesis at a relevant later floral development stage (Hsiao et al., 2011b). Interestingly, only PI-like genes i.e. PeMADS6, DcOPI and OMADS8 reported in P. equestris, D. crumenatum and O. Gower Ramsey, respectively were expressed in all floral organs (Tsai et al., 2005; Xu et al., 2006b; Chang et al., 2010, 2011). However, OMADS8 is also expressed in vegetative roots and leaves, whereas both PeMADS6 and DcOPI are not expressed in vegetative tissues. In addition, PeMADS6 expressed in the undeveloped ovary suggests that the expression of PeMADS6 in the ovary has an inhibitory effect on the development of the ovary (Tsai et al., 2005). Thus, it is evident that the expression patterns of B-class genes in orchid floral organs fit nicely into the “modified ABC model.” Further investigation of the B-class genes in Apostadioideae will undoubtedly shed light on the mystery of the great diversity of orchid flowers (Hsiao et al., 2011b). The reasoning behind the models proposed to

explain orchid floral development were derived from comparisons of expression patterns of B-class genes between wild type and peloric flowers in orchids, the latter being actinomorphic mutants with lip-like petals (Tsai et al., 2004b). The C-class genes specify stamen and carpel development and function in meristem determination because C-class mutants are indeterminate in whorl 4 and form a new C-class mutant flower instead of carpels (Hsiao et al., 2011b). More recently, one C-class gene and one D-class gene were isolated from O. Gower Ramsey, and two C-class genes were isolated from Cymbidium ensifolium (Hsu et al., 2010; Wang et al., 2011). The expression pattern of a D-class gene, OMADS2, is similar to that of OMADS4, suggesting that there is a close relationship in regulating the formation of the stigmatic cavity and ovary in O. Gower Ramsey (Hsu et al., 2010). Two E-class MADSbox genes and two AGL6-like genes identified from O. Gower Ramsey are expressed in the apical meristem and in the lip and carpel of the flower but not in vegetative tissues (Hsu et al., 2003; Chang et al., 2009). Four E-class genes (OM1, DOMADS1, DOMADS3 and DcOSEP1) have been identified from Dendrobium (Lu et al., 1993; Yu and Goh, 2000; Xu et al., 2006b) and two E-class genes from P. equestris whose expression patterns overlaps with that of ABCD genes in orchids suggests that higherorder MADS-box complexes are involved in all floral organ development in orchids, similar to Arabidopsis E-class genes (Hsiao et al., 2011b). However, whether orchid E-class genes play a role in floral determinacy still needs further investigation. Further investigation of these gene functions in orchids will provide useful information in understanding the evolutionary roles of E-class MADS-box genes in orchid flowering and/or flower development. These findings are in line with current thoughts on how major evolutionary changes in the genetic basis of organ identity were established by gene duplication and the separation of functions (Hsiao et al., 2011b). However, we cannot be sure that all the genes within each family have been isolated in orchids and recently established orchid EST databases provide plentiful gene information while integrated bioinformatic tools offer opportunities for finding A, B, C, D and E-function paralogous genes or additional genes related to flower development, allowing scientists to better understand the molecular and genetic mechanisms of floral control in orchids (Fu et al., 2011). A more detailed review on the genetic basis underlying floral color and development in orchids has recently been reviewed by Hsiao et al. (2011b).

XIV.

CYTOGENETICS OF ORCHIDS AND THE ROLE OF BIOTECHNOLOGY Only an estimated 90% of angiosperms (Joubès and Chevalier, 2000; Lim and Loh, 2003). Endopolyploidy has been reported in many plant tissue types, especially those with cells of large size and high specificity and for orchids in particular, it has been observed in leaves (Jones and Kuehnle, 1998), root tips (Jones and Kuehnle, 1998; Teixeira da Silva and Tanaka, 2006), the perianth (Mishiba and Mii, 2000), growing embryos (Alvarez, 1968; Nagl, 1972), PLBs (Teixeira da Silva and Tanaka, 2006), and several parts of Phalaenopsis hybrids, Cymbidium hybrids and Paphiopedilum delenatii flowers (Teixeira da Silva, unpublished data). Endopolyploidization increases, often in response to harsh environments, including the in vitro environment, in response to radical treatments such as—at least for Cymbidium and Phalaenopsis hybrids—high light intensity, super-CO2 enrichment (10,000 ppm), antibiotics, strong PGRs such as thidiazuron (TDZ) or high concentrations of more moderate PGRs such as BA, heat, excessively long tissue culture periods, infrequent sub-cultures and liquid medium (Teixeira da Silva and Tanaka, 2006; Teixeira da Silva, unpublished data). Endopolyploidization in these cases is not resticted to a whole organ and several tissues within a single organ can have radically contrasting states of endoplyploidization, e.g., root or shoot tip vs root base, PLB outer vs inner layers, petal vs lobe (Teixeira da Silva and Tanaka, 2006), although, for Cymbidium hybrids at least, the level never exceeds 2n = 16. Chromosme counts provide indispensable information on genetic discontinuities within and among species and they contribute to our understanding of phylogenetic relationships at all taxonomic levels and play an important role in plant breeding programmes (Barnardos et al., 2006; Lee et al., 2011). Polyploid induction plays a significant role in the hybridization and improvement of orchids, which can produced new orchid varieties and help to restore fertility by doubling the number of chromosomes and creating allotetraploids (Ranney, 2006; Miguel and Leonhardt, 2011). Several authors have reported the successful use of colchicine to convert diploid to tetraploid forms in Dendrobium (Nakasone and Kamemoto, 1961; Nakasone,

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1962; Sanguthai et al., 1973; Li et al., 2004; Zheng et al., 2009), Cymbidium (Menninger, 1963; Yin et al., 2010; Wang et al., 2011), Oncidium (Cui et al., 2010), Vanda (Nakasone and Kamemoto, 1961; Nakasone, 1962; Sanguthai and Sagawa, 1973), Phalaenopsis (Griesbach, 1981, 1985; Chen et al., 2009; Cui et al., 2010), Paphiopedilum (Watrous and Wimber, 1988), Pleione maculate (Cheng et al., 2010), Habenaria and Calanthe (Tahara and Kato, 1987), and Cattleya (DeMello e Silva et al., 2000). Miguel and Leonhardt (2011) studied polyploid induction of Dendrobium, Epidendrum, Odontioda and Phalaenopsis using oryzalin. Meiotic chromosome associations in interspecific hybrids have long been used to indicate genome homology between plant species, but the degree of chromosome pairing is not the only criterion for evaluating species relationships. Additionally, analysis of chromosome pairing is difficult, because a) many plants produce very few flowers, hindering collection of sufficient microsporocytes at the right stages for analysis; b) microsporocytes are enclosed in a thick callose wall, which hampers stain penetration; and c) meiotic chromosomes cannot be spread well due to clumping and stickiness. Nowadays, three molecular techniques, namely fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH), and southern hybridization of genomic DNA are being used in the cytogenetics of orchids and have proved to be effective tools for genome research. FISH is useful for localization of specific DNA sequences on chromosomes, while GISH for total genomic DNA of a species (Jiang and Gill, 1994; Kao et al., 2001, 2007; Begum et al., 2009). GISH, a modified FISH method, uses total genomic DNA as a probe usually with excess unlabelled DNA from another species as blocking agent for better differentiation (Lee et al., 2011). Southern hybridization of genomic DNA reveals the amount and distribution of repetitive DNA sequences in genomes, and is, therefore, also a useful tool to achieving an understanding of genome differentiation between species. The karyotypes, genome organization and localization of tandemly repetitive sequences [(microsatellites or SSRs] on the chromosomes of some representative orchid species were studied in the past few years and their outcome clearly demonstrate that these techniques are effective and more accurate than conventional methods (Kao et al., 2001, 2007; Begum et al., 2009). The variability in basic chromosome number, chromosome size and ploidy level has been very useful in studies of cytotaxonomy and karyotype evolution (Felix and Guerra, 2005). The identification of basic chromosome number in members of the Orchidaceae is fundamental to an understanding of the ploidy level of its members and the relationships between them. Cytotaxonomic analyses of a number of groups of orchids has supported classical taxonomic divisions, such as in Cymbidium (Seth and Cribb, 1984), where the two sub-genera are separated by different basic numbers, as well as in the subtribe Aerangidinae, which has a distinct basic number within the tribe Vandeae (Jones, 1966). Phylogenetic analyses within the subfamily Cypripedioideae (Cox et al., 1998) and the subtribes Orchidinae (Pridgeon et al.,


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1997) and Lycastinae (Ryan et al., 2000), for example, have also received strong support from studies that demonstrated variation in chromosome numbers. Orchidaceae is a diverse family in terms of ploidy i.e. chromosome number. Differences exist among genera, species and hybrids (Arditti, 1992). For example, 136 Cattleya plants examined in a study showed 5 pentaploids (100 chromosomes), 63 tetraploids (78–82 chromosomes), 39 triploids (59–62 chromosomes), 18 diploids (40–42 chromosomes) and 11 aneuploids (63–77 chromosomes) (Arditti, 1992). The chromosome numbers of 41 mainly terrestrial Brazilian orchid species belonging to 11 genera (subfamilies Cypripedioideae, Spiranthoideae, Orchidoideae, and Vanilloideae) were determined (Felix and Guerra, 2005). The variation in chromosomes was high: Spiranthoideae (2n = 28, 36, 46, 48 and 92), Orchidoideae (2n = 42, 44, ca. 48, ca. 80, 84, and ca. 168), and in the subfamily Spiranthoideae, some species of Prescottia (subtribe Prescottiinae) and some genera of Spiranthinae showed a bimodal karyotype with one distinctively large pair of chromosomes. The analysis of chromosome numbers of the genera in subfamilies revealed the predominance of the polyploid series 7, 14, 21, 28, 42 with a dysploid variation of ±1 in each ploidy level, suggesting that the basic chromosome number of terrestrial orchids is x1 = 7 for the subfamilies Spiranthoideae and Orchidoideae, as well as other Epidendroid orchids, and that the majority of the genera are composed of palaeopolyploids.

XV.

MEDICINAL ORCHIDS AND PHARMACEUTICAL BIOTECHNOLOGY Orchids have been used as a source of medicine for millennia to treat different diseases and ailments. Orchids have been used in Chinese medicine for thousands of years. One of the first records of Chinese medicine, The Book of Herbs (reportedly published in 500 A.D) included the use of dendrobiums. Dendrobium (D.officinale, D. nobile, D. chrysotoxum, D. fimbriatum, among others), Gastrodia elata and Bletilla striata have been officially listed in the Chinese Pharmacopoeia for a long time (State Pharmacopeia Committee of China, 2010). Besides, many orchidaceous preparations are used as emetic, purgative, aphrodisiac, vermifuge, cooling agent, bronchodilator, sex stimulator and contraceptive agents. Some of the preparations have supposedly miraculous curative properties but rare scientific evidence is available, although this is the primary requirement for clinical implementation. Several biotechnological approaches have been adopted for studying orchid phytochemicals such as high-performance liquid chromatography (HPLC), HPLC coupled with diode array detection (DAD), mass spectrometry (MS), HPLC-MS, HPLC-UV, NMR, among others. HPLC is the most widely used method for the analysis of pharmaceuticals and medicinal herbs. HPLC-DAD and MS have grown into the most powerful analytical techniques for the detection and determination of biologically active components in natural products. HPLC-MS methods provide qualitative information of

the targeted compounds present in complex matrices and from which compound identity may be inferred with a high degree of certainty. Using these techniques, several novel compounds and drugs, both with a phytochemical and pharmacological interest, have been detected in orchids (Hossain, 2009, 2011). Linking indigenous knowledge to modern biotechnological tools is providing a new arena for drug discovery for much more effective and potent alternatives to contemporary synthetic medicines, using ginseng as a model system (Yan, 2012). Orchids, like other plants, produce a large number of bioactive phytochemicals inlcuding alkaloids, flavonoids, carotenoids, anthocyanins and sterols. Only a few of them have been investigated for their biological function while others remain unknown. Among them, alkaloids and flavonoids are the most important for their biological properties. To date, more than 2000 orchid species have been screened for their alkaloid and/or flavonoid content and more than 100 bioactive alkaloids have been isolated from orchids (Gutierrez, 2010; Chinsamy et al., 2011; Hossain, 2011). From a pharmaceutical point of view, the most important orchid alkaloids are Dendrobine, Moscatilin, Moscatin, Nobilin-D, Nobilin-E, Nobilonine, Nobilone, Dendrocandin-A,B; Loroglossin, Crepidatin, Crepidine, Crepidamine, Gastrodin, Dendrochrysanene, Dendrophenol, Chrysotoxin, Tristin, Chrysotoxene, Denfigenin, Defuscin, Amoenumin, Rotundatin, Cumulatin, Gigantol, CymbinodinA, Cypripedine, Dactylorhins A-E, Coumarin, Confusarin, Chrysotobibenzyl, Dendrocrepine, Shihunidine, Dendrophenol, Dendroxime, Dendrine, Dendroxine, Dedramine, Nudol, Malaxin, Melianin, 6-hydroxynobilonine, Phenanthrenes, Dihydrophenanthrene, Erianthridin, Ephemeranthoquionone, Shihunine, Catachin, Coumarin, Saponin, Callosuminin, although many more have been identified (Majumder and Sen, 1987; Majumder and Chatterjee, 1989; Majumder and Pal, 1992; Yamaki and Honda, 1996; Honda and Yamaki, 2000; Krohn et al., 2001; Yu et al., 2005; Yang et al., 2006; Zhang et al., 2007; Li et al., 2008; Xu et al., 2009; Gutiérrez, 2010; Lalitharani et al., 2011; Majumder et al., 2011). Among the different orchid flavonoids and flavonols the medicinally important ones are scutellarein 6-methyl ether, dihydroquercetin, apigenin 7-O-glucoside, apigenin-7neohesperidoside quercetin-7-O-β-D-[6 -O-(transferuloyl)]glucopyranoside, 8-C-p-hydroxybenzylquercetin, isorhamnetin-7-O-β-D-[glucopyranoside, quercetin, chrysin, homoeriodictyol, homoisoflavanone, fimbriol A, plicatol B and glycoside apigenin-6-O-β-d-glucopyranosil-3-O-α-l-rhamnopyranoside, inter alia (Wu et al., 2006; Gutiérrez, 2010; Gutierrez et al., 2011). Although orchids have long been used and continue to be used as medicine in all parts of the world, pharmacological and toxicological studies of orchid phytochemicals on humans—which are required for clinical implementations—are rare (Kuo et al., 2009; Gutierrez et al., 2011). Studies that were conducted on animals including mice, rabbits and guinea pigs created optimism that to create life-saving and health-improving



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FIG. 2. In vitro development to ex vitro acclimatization of Renanthera imschootiana. (A) Flowering plant; (B) White protocorms from asymbiotic seed germination in darkness on half-strength MS medium containing 10% coconut water; (C) PLB proliferation on half-strength MS medium containing 1.0 mg l−1 N6-benzylaminopurine, 1.0 mg l−1 NAA and 10% coconut water under light conditions; (D) PLB differentiation into shoots and roots on half-strength MS medium containing 0.2 mg l−1 N6-benzylaminopurine, 1.0 mg l−1 NAA and 10% coconut water under light conditions; (E) Rooting and seedling growth on half-strength MS medium containing 1.0 mg l−1 NAA, 100 g l−1 banana homogenate and 10.5 g l−1 activated charcoal under light conditions; (F) Establishment of in vitro seedlings in the field after six months acclimatization on Chilean sphagnum moss. Unpublished photos (Songjun Zeng) (color figure available online).

phytochemicals like taxol, vinblastine or quinine. Khouri et al. (2006) conducted an experiment to determine the effect the bulb extract of Orchis anatolica on male fertility; their results suggested that the ingredients of this plant could be used to create a safe drug that increases male sexual potential. Wang et al. (2006) noted that polysaccharides isolated from Bletilla striata stimulated the proliferation of human umbilical vascular endothelial cells and has been widely used in Eastern Asian countries to treat alimentary canal mucosal damage, ulcer, bleeding, bruises, and burns and with polysaccharide content, such as glucomannan. A study performed in Mexico on mice evaluated two complex organic chemicals from the orchid Scaphyglottis

livida as a potential relaxant of heart contractions caused by the excitatory hormone noradrenaline. The results showed that organic compounds, the stilbenoids, inhibited aortic contractions provoked by noradrenaline, and caused vasodilation, relaxation and widening of blood vessels in the body, although the implications of these chemicals for use in human models may be promising in cardiology, pending further examination (Saleem, 2007). Vanda tessellata has a long history of use by the native people of India for its antiinflammatory properties, although Suresh Kumar et al. (2000) discovered its role as a potent aphrodisiac and fertility booster in mice, similar to Orchis maculata (Ageel et al., 1994), lending to the possibility of extrapolating

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FIG. 3. The application of permanent magnetic fields (MFs) in orchid research. (A) Phalaenopsis Gallant Beau ‘George Vazquez’ PLB proliferation in liquid R culture system under heterotrophic culture treated by different MF intensities: 0.1; 0.15; 0.2 Tesla (T), N or S pole. (B) Hybrid medium in “Miracle Pack” R culture system under photoautotrophic culture conditions: 25 ± 1◦ C, Cymbidium Music Hour ‘Maria’ shoot development in liquid medium in “Miracle Pack” 16-h photoperiod, light intensity (45 μmol m−2 s−1; Plant Lux, Toshiba Co., Japan), CO2 enrichment (3,000 μmol mol−1 24 h−1), treated by different MF intensities: 0.1; 0.15; 0.2 T N or S pole. Unpublished photos (Pham Thanh Van) (color figure available online).

results to humans. Moscatilin has anti-cancer effects (Ho and Chen, 2003) but also showed antiproliferative effects against various types of cancers, including those from choriocarcinoma (cancer of germ cells), lung cancers, and stomach cancers by arresting the cell cycle at G2 -M, with an increase of cells at the sub-G1 phase (Chen et al., 2008). It also induced the phosphorylation of JNK1/2 (c-Jun NH2 -Terminal Protein Kinase), which significantly inhibited the activation of caspase-9 and caspase3 through moscatilin-induced apoptosis signaling. Similar to moscatilin, a number of orchid biochemicals have shown the ability of treating different diseases and ailments. Eulophia epidendraea is a terrestrial orchid traditionally used for treating tumours, abscesses, wounds and other ailments (Maridass et al., 2008), with recent antidiarrhoeal activity of the ethanolic-tuber extract, which included alkaloids, saponins, flavonoids, sterols and/or terpenes and sugars (Maridass, 2011). Many bioactive constituents of Dendrobium species have been identified. The macromolecules included lectins, the enzymes chalcone synthase, sucrose synthase, and cytokinin oxidase, and polysaccharides, the latter displaying immunomodulatory and hepatoprotective activities. Alkaloids exhibit antioxidant, anticancer, and neuroprotective activities. Other compounds manifest antioxidant, anticancer, and immunomodulatory (Ng et al., 2012). Gastrodia elata has been widely used to treat headaches, migraines, dizziness, epilepsy, rheumatism, neuralgia, paralysis, inflammatory diseases, and other disorders. The components in tall gastrodia tuber include gastrodin (ρ-hydroxymethylphenylβ-D-Glucopyranoside), ρ-hydroxybenzyl alcohol, vanillyl alcohol, parishin, gastrodioside, p-hydroxybenzal- dehyde, vanillin, and β-sitosterol (Liu et al., 2002; Lee et al., 2006a).

XVI. OTHER BIOTECHNOLOGICAL DEVELOPMENTS A number of problems have been encountered during orchid propagation, conservation, and modification for commercial interest, but some promising successes have been achieved. Permanent magnetic fields (MFs) have been used to manipulate orchid organogenesis and development in vitro, in Phalaenopsis.

The MFs of 0.1 Tesla–S pole, applied to either PLBs or plantlets, could affect root and shoot growth parameters, PLB proliferation and photosynthetic parameters (Tanaka et al., 2010; Van et al., 2011a, b; Fig. 3). In vitro germination of orchid seeds was successful on low cost and easy available coir fibres as an ecofriendly substitute for costly gelling agents (agar, gelatin, etc.) in culture media (Aggarwal and Nirmala, 2012). Enhancement of seedling growth and resistance to diseases by co-culturing seedlings with OMF is another successful method already applied in the commercial production of orchids (Wu et al., 2010, 2011). Priming abiotic factors (gelling agent, media pH, media additives and volume, solid versus liquid versus membrane raft culture) for mass PLB induction (Teixeira da Silva et al., 2006), mass-scale seedling production without damaging any plant parts (Hossain et al., 2008), studies on the effects of photon flux density on the morphology, photosynthesis and growth of orchid seedlings during post-micropropagation acclimatization (Jeon et al., 2005), dual phase culture (multiplication of PLBs in liquid medium and plantlet regeneration in agar-gelled medium) for reducing the time required for mass propagation (Hossain et al., 2010), development of multiple regeneration R and dipathways, rooting in shoots on rockwool in the Vitron rect transfer to the greenhouse, without special acclimatization procedures (Teixeira da Silva and Tanaka, 2006) are other landmark achievements that have, in recent years, revolutionized orchid propagation and biotechnology.

XVII. CONCLUSIONS Some 2500 years ago Confucius wrote “Lan (orchid) that grows in the deep forests never withholds its fragrances even when no one appreciates it.” Incredible diversity, a sky-scraping demand for cut flowers, the drive towards making orchids model plants in understanding flower structure and development, a high content of alkaloids and glycosides, and dozens of as yet unexplored avenues of biotechnological research make research on orchids, without a doubt, full of potential. The application of advanced biotechnological approaches for studying orchid


biology may conclude many controversies and resolve clashes in taxonomic disaccord. The efforts by many scientists to use a plethora of biotechnological tools will lead to a promising understanding of the biological, physiological, molecular and genetic mechanisms of orchids in years to come.


ACKNOWLEDGMENTS The authors thank the assistance of Kasumi Shima (GSB) for assistance with the figure plates. Sincere thanks to Prof. Masahiro Mii (Chiba University) for feed-back and constructiuve comments on orchid transformation and to Prof. Michio Tanaka (Kagawa University) for constructive feed-back on tissue-culture related aspects of orchid biotechnology.

REFERENCES Afzelius, K. 1954. Embryo sac development in Epipogium aphyllum. Svensk Botanisk Tidskrift Utgifven af Svenska Botaniska Föreningen 48: 513–520. Agarwal, M., Shrivastava, N., and Padh, H. 2008. Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Rep. 27: 617–631. Ageel, A. M., Islam, M. W., Ginawi, O. T., and Al-Yahya, M. A. 1994. Evaluation of the aphrodisiac activities of Litsea chinensis (Lauraceae) and Orchis maculata (Orchidaceae) extracts in rats. Phytother Res. 8: 103–105. Aggarwal, S., and Nirmala, C. 2012. Utilization of coir fibres as an eco-friendly substitute for costly gelling agents for in vitro orchid seed germination. Sci. Hortic. 133: 89–92. Ajjikuttira, P. A., Ho, C. L. L., Woon, M. H., Ryu, K .H., Chang, C. A., Loh, C. S., and Wong, S. M. 2002. Genetic variability in the coat protein genes of two orchid viruses: Cymbidium mosaic virus and Odontoglossum ringspot virus. Arch. Virol. 147: 1943–1954. Aktar, S., Nasiruddin, K. M., and Hossain, K. 2008. Effects of different media and organic additives interaction on in vitro regeneration of Dendrobium orchid. J. Agric. Rural Dev. 6: 69–74. Alvarez, M. R. 1968. Quantitative changes in nuclear DNA accompanying postgermination embryonic development in Vanda (Orchidaceae). Amer. J. Bot. 55: 1036–1041. Andronova, E. V. 1988. Embryogenesis and postseminal development of orchids (on example of Dactylorhiza baltica, D. incarnata, Thunia alba, Bletilla striata). Ph.D. dissertation, Komarov Botanical Institute, Leningrad, Russia (in Russian). Antony, J. J. J., Keng, C. L., Rathinam, X., Marimuthu, S., and Subramaniam, S. 2011a. Effect of preculture and PVS2 incubation conditions followed by histological analysis in cryopreserved PLBs of Dendrobium Bobby Messina. Aus. J. Crop Sci. 5: 1557–1564. Antony, J. J. J., Sinniah, U. R., Keng, C. L., Pobathy, R., Khoddamzadeh, A. A., and Subramaniam, S. 2011b. Selected potential encapsulation-dehydration parameters on Dendrobium Bobby Messina protocorm-like bodies using TTC analysis. Aus. J. Crop Sci. 5: 1817–1822. Arditti, J. 1992. Fundamentals of Orchid Biology. John Wiley and Sons, New York. Arekal, G. D., and Karanth, K. A. 1981. The embryology of Epipogium roseum (Orchidaceae). Plant Syst. Evol. 138: 1–7. Arunasalam, G., and Pearson, M. N. 1989. ELISA detection of Odontoglossum ringspot virus in mature plants and protocorms of Cymbidium orchids: Potential solutions to problems of sample preparation time and low virus concentration. J. Phytopathol. 126: 160–166. Asahina, H., Shinozaki, J., Masuda, K., Morimitsu, Y., and Satake, M. 2010. Identification of medicinal Dendrobium species by phylogenetic analyses using matK and rbcL sequences. J. Nat. Med. 64, 133–138.

125

Asghar, S., Ahmad, T., Hafiz, I. A. and Yaseen, M. 2011. In vitro propagation of orchid (Dendrobium nobile) var. Emma White. Afr. J. Biotechnol. 10 (16): 3097–3103. Athipunyakom, P., Manoch, L., and Piluek, C. 2004. Isolation and identification of mycorrhizal fungi from eleven terrestrial orchids. Kasetsart J. Nat. Sci. 38: 216–228. Atwood, J. T. 1986. The size of Orchidaceae and the systematic distribution of epiphytic orchids. Selbyana 9, 16. Badge, J., Brunt, A., Carson, R., Dagless, E., Karamagioli, M., Phillips, S., Seal, S., Turner, R., and Foster, G. D. 1996. A carlavirus-specific PCR primer and partial nucleotide sequence provides further evidence for the recognition of Cowpea mild mottle virus as a whitefly transmitted carlavirus. Eur. J. Plant Pathol. 102: 305–310. Bairu, M. W., Aremu, A. O., and van Staden, J. 2011. Somaclonal variation in plants: causes and detection methods. Plant Growth Regul. 63: 147–173. Bajaj, Y. P. S. 1990. Somaclonal variation - origin, induction, cryopreservation, and implications in plant breeding. In: Biotechnology in Agriculture and Forestry II. Somaclonal Variation in Crop Improvement, pp. 3–35. Bajaj, Y. P. S., Ed. Springer, Berlin. Barnerdos S., Barriuso, M. G., Leon-Arencibia, M. C., Reyes-Betancort, A., Gonzalez-Gonzalez, R., Padron, M., and Amich, F. 2006. A cytotaxonomic study of three endemic orchids of the Canary Islands. Ann. Bot. Fannici. 43: 161–166. Barry, K., Hu, J. S., Kuehnle, A. R., and Sugii, N. 1996. Sequence analysis and detection using immunocapture-PCR of Cymbidium mosaic virus and Odontoglossum ringspot virus in Hawaiian orchids. J. Phytopathol. 144: 179–186. Basker, S., and Bai, V. N., 2006, Micropropagation of Coelogyne stricta (D. Don) Schltr. via pseudobulb segment culture. Tropical Subtropical Agroecosys. 6: 31–35. Batchman, L. 2008. Orchid Ailments: Detecting virus in orchids. Orchids, pp. 242–243. www.aos.org; Agdia, Inc. (Web site: www.agdia.com; www.forsitediagnostics.com) Bateman, R. M., James, K. E., Luo, Y. B., Lauri, R. K., Fulcher, T., Cribb, P. J., and Chase, M. K. W. 2009. Molecular phylogenetics and morphological reappraisal of the Platanthera clade (Orchidaceae: Orchidinae) prompts expansion of the generic limits of Galearis and Platanthera. Ann. Bot. 104: 431–445. Batygina, T. B. 1998. Certain aspects of the reproductive system in orchids. Byulleten’ Botanicheskogo Sada I. S. Kosenko 7: 155–157 (in Russian). Batygina, T. B., Bragina, E. A., and Vasilyeva, V. E. 2003. The reproductive system and germination in orchids. Acta Biol. Cracov. Ser. Bot. 45 (2): 21–34. Been, C. G., Na, A. S., Kim, J. B., and Kim, H. Y. 2002. Random amplified polymorphic DNA (RAPD) for genetic analysis of Phalaenopsis species. J. Korean Hort. Sci. 43: 387–391. Begum, A. A., Tamaki, M., and Kako, S. 1994. Formation of protocorm-like bodies (PLBs) and shoot development through in vitro culture of outer tissue of Cymbidium PLB. J. Jpn. Soc. Hort. Sci. 63 (3): 663–673. Begum, R., Alam, S. S., Menzel, G., and Schmidt, T. 2009. Comparative molecular cytogenetics of major repetitive sequence families of three Dendrobium species (Orchidaceae) from Bangladesh. Ann. Bot. 104: 863–872. Belarmino, M. M., and Mii, M. 2000. Agrobacterium-mediated genetic transformation of a Phalaenopsis orchid. Plant Cell Rep. 19: 435–442. Bernardos, S., Garc´ıa-Barriuso, M., León-Arencibia, M. C., Reyes-Betancort, A., González-González, R., Padrón, M., and Amich, F. 2006. A cytotaxonomic study of three endemic orchids of the Canary Islands. Ann. Bot. Fennici 43: 161–166. Besse, P., da Silva, D., Bory, S., Grisoni, M., Le Bellec, F., and Duval, M. F. 2004. RAPD genetic diversity in cultivated vanilla: Vanilla planifolia, and relationships with V. tahitensis and V. pompona. Plant Sci. 167: 379–385. Bhadra, S. K., and Hossain, M. M. 2003a. In vitro germination and micropropagation of Geodorum densiflorum (Lam.) Schltr., an endangered orchid species. Plant Tissue Cult. 13: 165–171.


126


Bhadra, S .K., and Hossain, M. M., 2003b. In vitro flowering in Geodorum densiflorum (Lam.) Schltr. J. Orchid Soc. India 17: 63–66. Bhadra, S. K., and Hossain, M. M. 2004. Induction of embryogenesis and direct organogenesis in Micropera pallida Lindl., an epiphytic orchid of Bangladesh. J. Orchid Soc. India 18: 5–9. Biswas, B. K., and Sharma, A. K. 1979. Some indications of evolution in the genus Paphiopedilum (Orchidaceae). Evol. Bot. Biostratigr. 1979, 195–201. Blanchard, M. G., and Runkle, E. S. 2008. Benzyladenine promotes flowering in Doritaenopsis and Phalaenopsis orchids. J. Plant Growth Regul. 27: 141–150. Bragina, E. A. 2001. Morphogenesis of ovule and seed in Hammarbya paludosa (L.) Kuntze (Orchidaceae). Abstracts of the 10th International Conference on Plant Embryology, 5–8 September 2001, 138. Nitra, Slovak Republic. Bui, V. L., Phuong, N. T. H., Hong, L. T. A., and Van, K. T. T. 1999. High frequency shoot regeneration from Rhynchostylis gigantean (Orchidaceae) using thin cell layer. Plant Growth Regul. 28: 179–185. Cameron, K. M., Chase, M. W., Whitten, M. W., Kores, P. J., Jarrell, D. C., Albert, V. A., Yukawa, T., Hills, H. G., and Goldman, D. H. 1999. A phylogenetic analysis of Orchidaceae: evidence from rbcL nucleotide sequences. Amer. J. Bot. 86: 208–224. Campbell, C. S., Wright, W. A., Cox, M., Vining T. F., Major C. S., and Arsenault, M. P. 2005. Nuclear ribosomal DNA internal transcribed spacer 1 (ITS1) in Picea (Pinaceae): sequence divergence and structure. Mol. Phylogenet. Evol. 35: 165–185. Carlsward, B. S., Whitten, W. M., Williams, N. H., and Bytebier, B. 2006. Molecular phylogenetics of Vandeae (Orchidaceae) and the evolution of leaflessness. Amer. J. Bot. 93: 770–786. CBOL Plant Working Group, 2009. A DNA barcode for land plants. Proc. Natl. Acad. Sci. USA 106: 12794–12797. Cecich, R. A. 1985. White spruce (Picea glauca) flowering in response to spray application of gibberellin A. Can. J. For. Res. 15: 170–174. Chai, D., Lee, S. M., Ng, J. H., and Yu, H. 2007. L-Methionine sulfoximine as a novel selection agent for genetic transformation of orchids. J. Biotech. 131: 466–472. Chai, M. L., Xu, C. J., Senthil, K. K., Kim, J. Y., and Kim, D. H. 2002. Stable transformation of protocorm-like bodies in Phalaenopsis orchid mediated by Agrobacterium tumefaciens. Sci. Hortic. 96: 213–224. Chan, Y. L., Lin, K. H., Sanjaya, Liao, L. J., Chen, W. H., and Chan, M. T. 2005. Gene stacking in Phalaenopsis orchid enhances dual tolerance to pathogen attack. Transgenic Res. 14: 279–288. Chang, D. C. N., Chou, L. C., and Lee, G. C. 2007. New cultivation methods for Anoectochilus formosanus Hayata. Orchid Sci. Biotech. 1: 55–60. Chang, Y. K., Veilleux, R. E., and Iqbal, M. J. 2009. Analysis of genetic variability among Phalaenopsis species and hybrids using amplified fragment length polymorphism. J. Amer. Soc. Hort. Sci. 134: 58–66. Chang, C., and Chang, W. C. 1998. Plant regeneration from callus of Cymbidium ensifolium var ‘Misericors’. Plant Cell Rep. 17: 251–255. Chang, C., and Chang, W. C. 2003. Cytokinins promotion of flowering in Cymbidium ensifolium var. misericos in vitro. Plant Growth Regul. 39: 217–221. Chang, C., Hu, W. H., Chen, Y. C., Su, Y. L., and Chiu, Y. T. 2010. In vitro flowering and mating system of Eulophia graminea Lindl. Bot. Stud. 51: 357–362. Chang, D. C. N. 2006. Research and application of orchid mycorrhizal fungi in Taiwan. Internatl. Hortl. Cong. (IHC) August 13–19, 2006, Seoul, Korea. Chang, D. C. N. 2007. The Screening of Orchid Mycorrhizal Fungi (OMF) and their Applications. In: Orchid Biotechnology, Chen, W. H., Chen, H. H. (Eds.), World Scientific, Hong Kong. pp. 77–98. Chang, D. C. N. 2008. Research and application of orchid mycorrhiza in Taiwan. Acta Hortic. (ISHS) 766: 299–306. Chang, D. C. N., and Chou, L. C. 2001. Seed germination of Haemaria discolor var. dawsoniana and the use of mycorrhizae. Symbiosis 30: 29–40. Chang, D. C. N., and Chou, L. C. 2007. Growth response, enzyme activities, and component changes as influenced by Rhizoctonia orchid mycorrhiza Anoectochilus formosanus Hayata. Bot. Stud. 48: 445–451.

Chang, Y. Y., Chiu, Y. F., Wu, J. W., and Yang, C. H. 2009. Four orchid (Oncidium Gower Ramsey) AP1/AGL9-like MADS box genes show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis thaliana. Plant Cell Physiol. 50: 1425–1438. Chang, Y. Y., Chu, Y. W., Chen, C. W., Leu, W. M., and Yang, C. H. 2011. Characterization of Oncidium ‘Gower Ramsey’ transcriptomes using 454 GS-FLX pyrosequencing and their application to the identification of genes associated with flowering time. Plant Cell Physiol. 52: 1532–1545. Chang, Y. Y., Kao, N. H., Li, J. Y., Hsu, W. H., Liang, Y. L., and Wu, J. W. 2010. Characterization of the possible roles for B functional MADS box genes in regulation of perianth formation in orchid (Oncidium Gower Ramsey). Plant Physiol. 152: 837–853. Chase, M. W., Cameron, K. M., Barrett, R.L., and Freudenstein, J. V. 2003. DNA data and Orchidaceae systematics: a new phylogenetic classification. In: Orchid Conservation, pp. 69–89. Dixon, K. W., Kell, S. P., Barrett, R. L., and Cribb, P. J., Eds. Natural History Publications, Kota Kinabalu. Chase, M. W., Salamin, N., Wilkinson, M., Dunwell, J. M., Kesanakurthi, R. P., Haidar, N., and Savolainen, V. 2005. Land plants and DNA barcodes: shortterm and long-term goals. Philos. Trans. R. Soc. London Bull. Biol. Sci. 360: 1889–1895. Chatterji, A. K. 1966. Polysomaty in orchids. Naturwissenschaften 19: 509–510. Chen, D., Guo, B., Hexige, S., Zhang, T., Shen, D., and Ming, F. 2007. SQUAlike genes in the orchid Phalaenopsis are expressed in both vegetative and reproductive tissues. Planta 226: 369–380. Chen, F. C., and Chen, W. H. 2007. Somaclonal variation in orchids. In: Orchid Biotechnology, pp. 65–76. Chen, W. H. and Chen, H. H., Eds. World Scientific, Hong Kong. Chen, J., Hu, K. X., Hou, X. Q., Guo, S. X. 2011. Endophytic fungi assemblages from 10 Dendrobium medicinal plants (Orchidaceae). World J. Microbiol. Biotechnol. 27: 1009–1016. Chen, J., Wang, H., and Guo, S. X. 2012. Isolation and identification of endophytic and mycorrhizal fungi from seeds and roots of Dendrobium (Orchidaceae). Mycorrhiza 22: 297–307. Chen, J. T., and Chang, W. C. 2006. Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis. Biol. Plant. 50: 169–173. Chen, J. T., and Chang, W. C. 2000. Plant regeneration via embryo and shoot bud formation from flower-stalk explants of Oncidium Sweet Sugar. Plant Cell, Tissue Organ Cult. 62: 95–100. Chen, J. T., Chang, C., and Chang, W. C. 1999. Direct somatic embryogenesis on leaf explants of Oncidium Gower Ramsey and subsequent plant regeneration. Plant Cell Rep. 19: 143–149. Chen, J. T., and Chang, W. C. 2002. Effects of tissue culture conditions and explant characteristics on direct somatic embryogenesis in Oncidium ‘Gower Ramsey’. Plant Cell, Tissue Organ Cult. 69: 41–44. Chen, J. T., and Chang, W. C. 2004. Induction of repetitive embryogenesis from seed-derived protocorms of Phalaenopsis amabilis var. formosa Shimadzu. In Vitro Cell Dev. Biol.—Plant 40: 290–293. Chen, J. T., and Chang, W. C. 2006. Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis. Biol. Plant. 50: 169–173. Chen, L., Kawai, H., Oku, T., Takahashi, C., and Niimi, Y. 2006. Introduction of Odontoglossum ringspot virus coat protein gene into Cymbidium niveomarginatum mediated by Agrobacterium tumefaciens to produce transgenic plants. J. Japan. Soc. Hort. Sci. 75: 249–255. Chen, R. R., Lin, X. G., and Shi, Y. Q. 2003. Research advances of orchid mycorrhizae. Chin. J. Applied Env. Biol. 9 (1): 97–101. Chen, S., Yao, H., Han, J., Liu, C., Song, J., Shi, L., Zhu, Y., Ma, X., Gao, T., Pang, X., Luo, K., Li, Y., Li, X., Jia, X., Lin, Y., and Leon, C. 2010. Validation of ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS ONE 5: e8613. Chen, T. H., Pan, S. L., Guh, J. H., Liao, C. H., Huang, D. Y., Chen, C. C., and Teng, C. M. 2008. Moscatilin induces apoptosis in human colorectal cancer cells: a crucial role of C-Jun NH2 -terminal protein kinase activation


ORCHIDS AND BIOTECHNOLOGY caused by tubulin depolymerization and DNA damage. Clin. Cancer Res. 14: 4250. Chen, T. Y., Chen, J. T., and Chang, W. C. 2002. Multiple shoot formation and plant regeneration from stem nodal explants of Paphiopedilum orchids. In Vitro Cell Dev. Biol.—Plant 38: 595–597. Chen, T. Y., Chen, J. T., and Chang, W. C. 2004. Plant regeneration through direct shoot bud formation from leaf cultures of Paphiopedilum orchids. Plant Cell Tissue Organ Cult. 76: 11–15. Chen, W. H., Chen, T. M., Fu, Y. M., Hsieh, R. M., and Chen, W. S. 1998. Studies on somaclonal variation in Phalaenopsis. Plant Cell Rep. 18: 7–13. Chen, W. H., Chen, Y. H., Chyou, M. S., Fu, Y. M., Lin, Y. S., and Hsiau, P. T. W. 1999. Development of white Taisuco Phalaenopsis. In: Proceedings of the 16th World Orchid Conference, pp. 272–278. Clark, J., Elliott, W. M., Tingley, G., and Biro, J., Eds. Vancouver, Canada. Chen, W. H., Fu, Y. M., and Hsieh, R. M. 1995. Application of DNA amplification fingerprinting in the breeding of Phalaenopsis orchid. In: Current Issues in Plant Molecular and Cellular Biology, pp. 341–346. Terzi, M. et al., Eds. Kluwer Academic, Netherlands. Chen, W. H., Hsu, C. Y., Cheng, H. Y., Chang, H., Chen, H. H., and Ger, M. J. 2011. Down regulation of putative UDP-glucose: flavonoid 3-Oglucosyltransferase gene alters flower coloring in Phalaenopsis. Plant Cell Rep. 30: 1007–1017. Chen, W. H., Tang, C. Y., and Kao, Y. L. 2009. Ploidy doubling by in vitro culture of excised protocorms or protocorm-like bodies in Phalaenopsis species. Plant Cell Tiss. Organ Cult. 98: 229–238. Chen, W. S., Chang, H. Y., Chen, W. H, and Lin, Y. S. 1997. Gibberellic acid and cytokinin affect Phalaenopsis flower morphology at high temperature. HortScience 32: 1069–1073. Chen, X. M., Dong, H. L., Hu, K. X., Sun, Z. R., Chen, J., and Guo, S. X. 2010. Diversity and antimicrobial and plant-growth-promoting activities of endophytic fungi in Dendrobium loddigesii Rolfe. J. Plant Growth Regul. 29: 28–337. Chen, X. M., Guo, S. X., and Meng, Z. X. 2008. Effects of the fungal elicitors on the growth of Dendrobium candidum protocorms. Chin. Trad. Herb Drugs 39 (3): 423–426. Chen, Z. L., Duan, J., Zeng, S. J., Liang, C. Y., and Ye, X. L. 2007. Agrobacterium-mediated transformation of Dendrobium orchid by targeting protocorms. Acta Sci. Nat. Uni. Sunyatseni 46 (1): 86–90. Cheng, K. T., Lo, S. F., Lee, C. Y., Chen, C. C., and Tsay, H. S. 2004. The rDNA sequence analysis of three Dendrobium species. J. Food Drug Anal. 12: 367–369. Cheng, Q., Tang, Y. M., Zhang, W., Hu, X. L., Wei, X. Q., and Na, H. Y. 2010. Study of polyploid induction of Pleione maculate lindl. with colchicine. J. Sichun Uvi. (Nat. Sci. Edi.) 47 (3): 635–638. Cheruvathur, M. K., Abraham, J., Mani, B., and Thomas, T. D. 2010. Adventitious shoot induction from cultured internodal explants of Malaxis acuminata D. Don, a valuable terrestrial medicinal orchid. Plant Cell, Tiss. Organ Cult. 101: 163–170. Chia, T. F., Arditti, J., Segeren, M. I., and Hew, C. S. 1999. Review: In vitro flowering in orchids. Lindleyana 14: 60–76. Chia, T. F., Chan, Y. S., and Chua, N. H. 1992. Detection and localization of viruses in orchids by tissue-print hybridization. Plant Pathol. 41: 355–361. Chia, T. F., Chan, Y. S., and Chua, N. H. 1990. Genetic engineering of tolerance to Cymbidium Mosaic Virus. In: Proceedings of the 13th World Orchid Conference, pp. 284. Kernohan, J., Bonham, N., Bonham, D., and Cobb, L., Eds.. World Orchid Conference Trust, Auckland, New Zealand. Chia, T. F., Chan, Y. S., and Chua, N. H. 1994. The firefly luciferase gene as a non-invasive reporter for Dendrobium transformation. Plant J. 6: 441–446. Chia, T. F., Lim, A. Y. H., Luan, Y., and Ng, I. 2001. Transgenic Dendrobium (Orchid). In: Biotechnology in Agriculture and Forestry (Vol. 48) Transgenic Crops III, pp. 95–106. Bajaj, Y. P. S., Ed. Springer, Berlin, Chin, D. P., Mishiba, K. I., and Mii, M. 2007. Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium. Plant Cell Rep. 26: 735–743.

127

Chinsamy, M., Finnie, J. F., and van Staden, J. 2011. The ethnobotany of South African medicinal orchids. South Afr. J. Bot. 77: 2–9. Chiou, C. Y., Wu K., and Yeh, K. W. 2008. Characterization and promoter activity of chromoplast specific carotenoid associated gene (CHRC) from Oncidium Gower Ramsey. Biotechnol. Lett. 30: 1861–1866. Chiou, C. Y., and Yeh, K. W. 2008. Differential expression of MYB gene (OgMYB1) determines color patterning in floral tissue of Oncidium Gower Ramsey. Plant Mol. Biol. 66: 379–388. Chugh, S., Guha, S., and Rao, I. U. 2009. Micropropagation of orchids: A review on the potential of different explants. Sci. Hort. 122: 507–520. Chung, H. H., Chen, J. T., and Chang, W. C. 2005. Cytokinins induce direct somatic embryogenesis of Dendrobium Chiengmai Pink and subsequent plant regeneration. In Vitro Cell. Dev. Biol.—Plant 41: 765–769. Chung, S. Y., Choi, S. H., Kim, M. J., Yoon, K. E., Lee, G. P., Lee, J. S., and Ryu, K. H. 2006. Genetic relationship and differentiation of Paphiopedilum and Phragmepedium based on RAPD analysis. Sci. Hortic. 109: 153–159. Chung, H. H., Chen, J. T., and Chang, W. C. 2007. Plant regeneration through direct somatic embryogenesis from leaf explants of Dendrobium. Biol. Plant. 51: 346–350. Clements, M. A. 1999. Embryology. In: Genera Orchidacearum Vol. 1: General Introduction, Apostasioideae, Cypripedioideae, pp. 38–58. Pridgeon, A. M., Cribb, P. J., Chase, M. W., and Rasmussen, F. N., Eds.. Oxford University Press, Oxford. Clements, M. A., Jones, D. L., Sharma, I. K., Nightingale, M. E., Garratt, M. J., Fitzgerald, K. J., and Mackenzie, A. M. 2002. Phylogenetic systematics of the Diurideae (Orchidaceae) based on the ITS and 5.8S coding region of nuclear ribosomal DNA. Lindleyana 17: 135–171. Cox, A. V., Pridgeon, A. M., Albert, V. A., and Chase, M. W. 1997. Phylogenetics of the slipper orchids (Cypripedioideae Orchidaceae): nuclear rDNA ITS sequences. J. Plant Syst. Evol. 208: 197–223. Cox, A. V., Abdelnour, D. G., Bennett, M. D., and Leitch, I. J. 1998. Genome size and karyotype evolution in the slipper orchids (Cypripedioideae: Orchidaceae). Amer. J. Bot. 85: 681–687. Cribb, P. J. 1987. The genus Paphiopedilum. Collingridge, London. Cui, G. R., Zhang Z. X., Zhang, C. Y., Hu, N. B., Sui, Y. H., and Li, J. Q. 2010. Polyploid induction and identification of Oncidium. Pratac. Sci. 19 (1): 184–190. Cui, G. R., Zhang, Z. X., Hu, N. B., Zhang, C. Y., and Hou, X. L. 2010. Tetraploid of Phalaenopsis induction via colchicine treatment from protocorm-like bodies in liquid culture. J. Zhejiang Univ. (Agri. & Life Sci.) 36 (1): 49–55. Currah, R. S., Sigler, L., and Hamilton, S. 1987. New records and taxa of fungi from the mycorrhizae of terrestrial orchids of Alberta. Can. J. Bot. 65: 2473–2482. Cybularz-Urban, T., and Hanus-Fajerska, E. 2006. Therapeutic effect of cytokinin sequence application on virus-infected Cattleya tissue cultures. Acta Biol. Cracov. Ser. Bot. 48 (2): 27–32. Dan, Y., Meng, Z. X., and Guo, S. X. 2012. Effects of forty strains of Orchidaceae mycorrhizal fungi on growth of protocorms and plantlets of Dendrobium candidum and D. nobile. Afr. J. Microbiol. Res. 6: 34–39. Datta, B. K., Kanjilal, B., and Sarker, D. D. 1999. Artificial seed technology: development of a protocol in Geodorum densiflorum (Lam) Schltr: an endangered orchid. Curr. Sci. 74: 1142–1145. Daviña, J. R., Grabiele, M., Cerutti, J. C., Hojsgaard, D. H., Almada, R. D., Insaurralde, I. S., and Honfi, A. I. 2009. Chromosome studies in Orchidaceae from Argentina. Genet. Mol. Biol. 32: 811–821. Dearnaley, J. D. W. 2007. Further advances in orchid mycorrhizal research. Mycorrhiza 17: 475–486. De Mello e Silva, M., Callegari, J., and Zanettini, B. 2000. Induction and identification of polyploids in Cattleya intermedia Lindl. (Orchidaceae) by in vitro techniques. Ciência. Rural 30: 105–111. Deb, C. R., and Pongener, A., 2012. Studies on the in vitro regenerative competence of aerial roots of two horticultural important Cymbidium species. J. Plant Biochem. Biotechnol. 21(2): 235–241.


128


Deng, D., McGrath, P. F., Robinson, D. J., and Harrison, B. D. 1994. Detection and differentiation of whitefly-transmitted geminiviruses in plants and vector insects by the polymerase chain reaction with degenerate primers. Ann. Appl. Biol. 125: 327–336. Dewir, Y. H., Chakrabarty, D., Ali, M. B., Singh, N., Hahn, E. J., and Paek, K. Y. 2007. Influence of GA3 , sucrose and solid medium/bioreactor culture on in vitro flowering of Spathiphyllum and association of glutathione metabolism. Plant Cell, Tiss. Organ Cult. 90: 225–235. Ding, G., Li, X., Ding, X., and Qian, L. 2009. Genetic diversity across natural populations of Dendrobium officinale, the endangered medicinal herb endemic to China, revealed by ISSR and RAPD markers. Russ. J. Genetics 45: 327–334. Ding, G., Xu, G., Zhang, W., Lu, S., Li, X., Gu, S., and Ding, X. Y. 2008. Preliminary geoherbalism study of Dendrobium officinale food by DNA molecular markers. Eur. Food Res. Technol. 227: 1283–1286. Ding, X., Wang, Z., Zhou, K. Xu, L., Xu, H., and Wang, Y. 2003. Allele-specific primers for diagnostic PCR authentication of Dendrobium officinale. Planta Medica 69: 587–588. Divakaran, M., Babu, K. N., Ravindran, P. N., and Peter, K. V. 2006. Interspecific hybridization in Vanilla and molecular characterization of hybrids and selfed progenies using RAPD and AFLP markers. Sci. Hortic. 108: 414–422. Dong, F., Liu, H. X., Jin, H., and Luo, Y. B. 2008. Symbiosis between fungi and hybrid Cymbidium and its mycorrhizal microstructure. For. Stud. China 10: 41–44. Dressler, R. L. 1981. The Orchids: Natural History and Classification. Harvard University Press, Cambridge Dressler, R. L. 1993. Phylogeny and Classification of the Orchid Family. Dioscorides Press, Portland, Oregon. Duan, J. X., and Yazawa, S. 1994. Induction of floral development xDoriell a Tiny (Doritis pulcherrima x Kingiella philippinensis). Sci. Hortic. 59: 253–264. Duan, J. X., and Yazawa, S. 1995a. Floral induction and development in Phalaenopsis in vitro. Plant Cell, Tiss. Organ Cult. 43: 71–74. Duan, J. X., and Yazawa, S. 1995b. Induction of precocious flowering and seed formation of x Doriella Tiny (Doritis pulcherrima x Kingiella philippinensis) in vitro and in vivo. Acta Hortic. 397: 103–110. Duval, M. F., Bory, S., Andrzejewski, S., Grisoni, M., Besse, P., and Causse, S. 2006. Diversite genetique des vanilliers dans leurs zones de dispersion secondaire. In: Les ressources génétiques: des ressources partagées: 6ème Colloque national BRG, La Rochelle, 2–4 October 2006, pp 181–196. BRG, Paris. Elliot, M. S., Zettler, F. W., Zimmerman, M. T., Barnett, O. W., and LeGrande, M. D. 1996. Problems with interpretation of serological assays in a virus survey of orchid species from Puerto Rico, Ecuador and Florida. Plant Dis. 80: 1160–1164. Engelmann, F. 2011. Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell. Dev. Biol.—Plant 47: 5–16. Ernst, R. 1994. Effects of thidiazuron on in vitro propagation of Phalaenopsis and Doritaenopsis (Orchidaceae). Plant Cell, Tiss. Organ Cult. 39: 273–275. Eun, A. J. C., Huang, L., Chew, F. T., Li, S. F. Y., and Wong, S. M. 2002. Detection of two orchid viruses using quartz crystal microbalance (QCM) immunosensors. J. Virol. Methods 99: 71–79. Eun, A. J. C., and Wong, S. M. 1999. Detection of Cymbidium mosaic potexvirus and Odontoglossum ringspot tobamovirus using immuno-capillary zone electrophoresis. Phytopathology 89: 522–528. Eun, A.J.C., and Wong, S. M. 2000. Molecular beacons: a new approach to plant virus detection. Phytopathology 90: 269–275. Fang, D., Liu, H-X., Jin, H., and Luo, Y-B. 2008. Symbiosis between fungi and the hybrid Cymbidium and its mycorrhizal microstructures. For. Studies China 10: 41–44. Fazekas, A. J., Burgess, K. S., Kesanakurti, P. R., Graham, S. W., Newmaster, S. G., Husband, B. C., Percy, D. M., Hajibabaei, M., and Barrett, S. C. H. 2008. Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well. PLoS ONE 3, e2802.

Felix, L. P., and Guerra, M. 2000. Cytogenetics and cytotaxonomy of some Brazilian species of Cymbidioid orchids. Gen. Mol. Biol. 23 (4): 957–978. Felix, L. P., and Guerra, M. 2005. Basic chromosome numbers of terrestrial orchids. Plant Syst. Evol. 254: 131–148. Fernando, D. D. 2010. Plant development and evolution. Intl. J. Plant. Dev. Biol. 4 (Special Issue 1): 1–121. Ferreira, W. D., Kerbauy, G. B., and Costa, A. P. P. 2006. Micropropagation and genetic stability of a Dendrobium hybrid (Orchidaceae). In Vitro Cell. Dev. Biol.—Plant 42: 568–571. Freitas, A. J., and Rezende, A. M. 1998. Odontoglossum ringspot virus-free Cymbidium obtained through meristem tip chemotherapy. Fitopatol. Bras. 23: 158–160. Fu, S. F., Lin, C. W., Kao, T. W., Huang, D. D., and Huang, H. J. 2011. PaPTP1, a gene encoding protein tyrosine phosphatase from orchid, Phalaenopsis amabilis, is regulated during floral development and induced by wounding. Plant Mol. Biol. Rep. 29: 106–116. Fu, Y. M., Chen, W. H., Tsai, W. T., Lin, Y. S., Chyou, M. S., and Chen, Y. H. 1997. Phylogenetic studies of taxonomy and evolution among wild species of Phalaenopsis by random amplified DNA markers. Rep. Taiwan Sugar Res. Inst. 157: 27–42. Fukai, S., Hasegawa, A., and Goi, M. 2002. Polysomaty in Cymbidium. HortScience 37: 1088–1091. Gamborg, O. L., Miller, R. A., and Ojima, K. 1968. Nutrient requirement suspension cultures of soybean root cells. Exp. Cell Res. 50: 151–158. Geilly, L., and Taberlet, P. 1994. The use of chloroplast DNA to resolve plant phylogenies: noncoding versus rbcL sequences. Mol. Biol. Evol. 11: 769–777. George, P. S., and Ravishankar, G. A. 1997. In vitro multiplication of Vanilla planifolia using axillary bud explants. Plant Cell Rep. 16: 490–494. Gezgin, Y., and Eltem, R. 2009. Diversity of endophytic fungi from various Aegean and Mediterranean orchids (Saleps). Turk. J. Bot. 33: 439–445. Gibbs, A., and Mackenzie, A. 1997. A primer pair for amplifying part of the genome of all potyvirids by RT-PCR. J. Virol. Methods 63: 9–16. Goh, C. J., and Arditti, J. 1985. Handbook of Flowering. CRC Press, Boca Raton, Florida. Goh, C. J., and Yang, A. L. 1978. Effects of growth-regulators and decapitation on flowering of Dendrobium orchid hybrids. Plant Sci. Lett. 12: 287–292. Goh, M. W. K., Kumar, P. P., Lim, S. H., and Tan, H. T. W. 2005. Random amplified polymorphic DNA analysis of the moth orchids, Phalaenopsis (Epidendroideae: Orchidaceae). Euphytica 141: 11–22. Goldman, D. H., Jansen, R. K., van den Berg, C., Leitch, I. J., Fay, M. F., and Chase, M. W. 2004. Molecular and cytological examination of Calopogon (Orchidaceae, Epidendroideae): circumscription, phylogeny, polyploidy and possible hybrid speciation. Am. J. Bot. 91 (5): 707–723. Gonzalez, D., Carling, D. E., Kuninaga, S., Vilgalys, R., and Cubeta, M. 2001. Ribosomal DNA systematics of Ceratobasidium and Thanatephorus with Rhizoctonia anamorphs. Mycologia 93: 1138–1150. Gonzalez-Arnoa, M. T., Panta, A., Roca, W. M., Escobor, R. H., and Engelmann, F. 2008. Development and large-scale application of cryopreservation technique of shoot and somatic embryo cultures of tropical crops. Plant Cell, Tiss. Organ Cult. 92: 1–13. Gow, W. P., Chen, J. T., and Chang, W. C. 2008. Influence of growth regulators on direct embryo formation from leaf explants of Phalaenopsis orchids. Acta Physiol. Plant. 30: 507–512. Gow, W. P., Chen, J. T., and Chang, W. C. 2009. Effects of genotype, light regime, explant position and orientation on direct somatic embryogenesis from leaf explants of Phalaenopsis orchids. Acta Physiol. Plant. 31: 363–369. Gravendeel, B., Smithson, A., Slik, F. J., and Schuiteman, A. 2004. Epiphytism and pollinator specialization: drivers for orchid diversity? Philos. Trans. R. Soc. Lond. B. Biol. Sci. 359: 1523–1535. Griesbach, R. J. 1981. Colchicine-induced polypoloidy in Phalaenopsis orchids. Plant Cell, Tissue Organ Cult. 1: 103–107. Griesbach, R. J. 1985. Polyploidy in Phalaenopsis orchid improvement. J. Hered. 76: 74–75.


ORCHIDS AND BIOTECHNOLOGY Guo, S. X., Cao, W. Q., and Gao, W. W, 2000. Isolation and biological activity of mycorrhizal fungi from Dendrobium candidum and D. nobile. China J. Chin. Mat. Med. 25: 338–341. Guo, B., Chen, D. H., Dai, W., Wei, X., and Ming, F. 2006. Cloning and expression analysis of a Phalaenopsis flowering-related gene pPI9. J. Fudan Uni. (Nat. Sci) 45 (3): 277–282. Guo, S. X., and Xu, J. T. 1990. Determination of primary metabolic products of fungi promoting seed germination of Gastrodia elata Bl. and other orchidaceae medicinal plants. China J. Chin. Mat. Med. 15 (6): 12–14. Gutierrez, R. M. P. 2010. Orchids: A review of uses in traditional medicine, its phytochemistry and pharmacology. J. Med. Plants Res. 4: 592–638. Gutierrez, R. M. P., Sosa, I. A., Vadillo, C. H., and Victoria, T. C. 2011. Effect of flavonoids from Prosthechea michuacana on carbon tetrachloride induced acute hepatotoxicity in mice. Pharm. Biol. 49: 1121–1127. Hadidi, A., Levy, L., and Podleckis, E. V. 1995. Polymerase chain reaction technology in plant pathology. In: Molecular Methods in Plant Pathology, pp. 167–187. Singh, R. P., and Singh, U. S., Eds. CRC Press, Boca Raton. Hagerup, O. 1947. The spontaneous formation of haploid, polyploidy and aneuploid embryos in some orchids. Biologiske Meddelelser, Kongelige Danske Videnskabernes Selskab 20: 1–22. Han, J. H., Jeong, H. W., and La, Y. J. 2001. Use of gelatin particle agglutination test for the detection of Cymbidium mosaic virus in Cattleya plants. Plant Pathol. J. 17: 325–328. Handa, T., Iwahori, M., Kano, Y., Shinkai, S., Sugiyama, N., and Sakiyama, R. 1998. Utilization of molecular markers for ornamental plants. J. Jpn. Soc. Hortic. Sci. 67: 1197–1199. Harikrishna, J. A., Miin, D. O. J., Lian, A. S. A., Dzulkurnain, A., Othman, R. Y., and Hoon, L. S. 2010. Biotechnological approaches to protection against Cymbidium mosaic virus. AsPac J. Mol. Biol. Biotechnol. 18: 181–184. Hasebe, M., Omori, T., Nakazawa, M., Sano, T., Kato, M., and Iwatsuki, K. 1994. rbcL gene sequences provide evidence for the evolutionary lineages of leptosporangiate ferns. Proc. Natl. Acad. Sci. USA 91, 5730–5734. Hazarika, B. N. 2003. Acclimatization of tissue-cultured plants. Curr. Sci. 85: 1704–1712. He, X. H., Duan, Y. H., Chen, Y. L., and Xu, M. G. 2010. A 60-year journey of mycorrhizal research in China: Past, present and future directions. Sci. China Life Sci. 53: 1374–1398. He, Z., Jiang, D., Liu, A., Sang, L., Li, W., and Li, S. 2011. The complete sequence of Cymbidium mosaic virus from Vanilla fragrans in Hainan, China. Virus Genes 42: 440–443. Hedren, M., Fay, M. F., and Chase, M. W. 2001. Amplified fragment length polymorphisms (AFLP) reveal details of polyploid evolution in Dactylorhiza (Orchidaceae). Amer. J. Bot. 88: 1868–1880. Hedrén, M., Nordström, S., Hovmalm, H. A. P., Pedersen, H.Æ., and Hansson, S. 2007. Patterns of polyploid evolution in Greek marsh orchids (Dactylorhiza; Orchidaceae) as revealed by allozymes, AFLPs, and plastid DNA data. Amer. J. Bot. 94: 1205–1218. Hee, K. H., Loh, C. S., and Yeoh, H. H. 2007. Early in vitro flowering and seed production in culture in Dendrobium Chao Praya Smile (Orchidaceae). Plant Cell Rep. 26: 2055–2062. Hew, C. S., and Clifford, P. E. 1993. Plant-growth regulators and the orchid cut-flower industry. Plant Growth Regul. 13: 231–239. Hill, J. H., Bryant, G. R., and Durand, D. P. 1981. Detection of plant virus by using purified IgG in ELISA. J. Virol. Methods 3: 27–35. Hilu, K., and Liang, H. 1997. The matK gene: sequence variation and application in plant systematics. Amer. J. Bot. 84: 830–839. Hirano, T., Godo, T., Mii, M., and Ishikawa, K. 2005a. Cryopreservation of immature seeds of Bletilla striata by vitrification. Plant Cell Rep. 23: 534–539. Hirano, T., Godo, T., Miyoshi, K., Ishikawa, K., Ishikawa, M., and Mii, M. 2009. Cryopreservation and low-temperature storage of seeds of Phaius tankervilleae. Plant Biotech. Rep. 3: 103–109. Hirano, T., Ishikawa, K., and Mii, M. 2005b. Cryopreservation of immature seeds of Ponerorchis graminifolia var. suzukiana by vitrification. CryoLetters 26: 139–146.

129

Hirano, T., Ishikawa, K., and Mii, M. 2006. Advances in orchid cryopreservation. In: Teixeira da Silva, J.A. (Ed.) Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues (Vol. II). Global Science Books, Ltd., Isleworth, UK, pp. 410–414. Ho, C. K., and Chen, C. C. 2003. Moscatilin from the orchid Dendrobrium loddigesii is a potential anticancer agent. Cancer Investigation 21: 729–736. Holttum, R. E. 1953. A Revised Flora of Malaya; an illustrated systematic account of the Malayan Flora, including commonly cultivated plants. Government Printing Office, Singapore. Honda, C., and Yamaki, M. 2000. Phenanthrenes from Dendrobium plicatile. Phytochemistry 53: 987–990. Hong, P. I., Chen, J. T., and Chang, W. C. 2008. Plant regeneration via protocormlike body formation and shoot multiplication from seed-derived callus of a Maudiae type slipper orchid. Acta Physiol. Plant. 30: 755–759. Hopper, S. D. 2009. Taxonomic turmoil down-under: recent developments in Australian orchid systematics. Ann. Bot. 104: 447–455. Hosomi, S. T., Custódio, C. C., Seaton, P. T., Marks, T. R., and Neto, N. B. M. 2012. Improved assessment of viability and germination of Cattleya (Orchidaceae) seeds following storage. In Vitro Cell. Dev. Biol.—Plant 48: 127–136. Hossain, M. M. 2009. Traditional therapeutic uses of some indigenous orchids of Bangladesh. Medicinal Aromatic Plant Sci. Biotechnol. 3: 100–106. Hossain, M. M. 2011. Therapeutic orchids: Traditional uses and recent advances—An overview. Fitoterapia 82: 102–140. Hossain, M. M., Khan, S. N., Sharma, M., Pathak, P., Hallan, V., and Zaidi, A. A. 2009b. Cymbidium Mosaic Potexvirus and Odontoglossum Ringspot Tobamovirus—A major threat to the orchid industry. J. Orchid Soc. India 23: 103–107. Hossain, M. M., Sharma, M., and Pathak, P. 2008. In vitro mass propagation of an economically important orchid, Cymbidium aloifolium (L.) Sw. J. Orchid Soc. India 22: 91–95. Hossain, M. M., Sharma, M., and Pathak, P. 2009a. Cost effective protocol for in vitro mass propagation of Cymbidium aloifolium (L.) Sw.—a medicinally important orchid. Eng. Life Sci. 9: 1–10. Hossain, M. M., Sharma, M., Teixeira da Silva, J. A., and Pathak, P. 2010. Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl. Sci. Hortic. 123: 479–487. Hsiao, Y. Y., Chen, Y. W., Huang, S. C., Pan, Z. J., Fu, C. H., Chen, W. H., Tsai, W. C., and Chen, H. H. 2011a. Gene discovery using next-generation pyrosequencing to develop ESTs for Phalaenopsis orchids. BMC Genomics 12: 360. Hsiao, Y. Y., Pan, Z. J., Hsu, C. C., Yang, Y. P., Hsu, Y. C., Chuang, Y. C., Shih, H. H., Chen, W. H., Tsai, W. C., and Chen, H. H. 2011b. Research on orchid biology and biotechnology. Plant Cell Physiol. 52 (9): 1467–1486. Hsieh, Y. H., and Huang, P. L. 1995. Studies on genetic transformation of Phalaenopsis via pollen tube pathway. J. Chinese Soc. Hort. Sci. 41 (4): 309–324. Hsu, C. C., Chung, Y. L., Chen, T. C., Lee, Y. L., Kuo, Y. T., Tsai, W. C., Hsiao, Y. Y., Chen, Y. W., Wu, W. L., and Chen, H. H. 2011. An overview of the Phalaenopsis orchid genome through BAC end sequence analysis. BMC Plant Biol. 11: 3. Hsu, H. F., Hsieh, W. P., Chen, M. K., Chang, Y. Y., and Yang, C. H. 2010. C/D class MADS box genes from two monocots, orchid (Oncidium Gower Ramsey) and lily (Lilium longiflorum), exhibit different effects on floral transition and formation in Arabidopsis thaliana. Plant Cell Physiol. 51: 1029–1045. Hsu, H. F., Huang, C. H., Chou, L. T., and Yang, C. H. 2003. Ectopic expression of an orchid (Oncidium Gower Ramsey) AGL6-like gene promotes flowering by activating flowering time genes in Arabidopsis thaliana. Plant Cell Physiol. 44: 783–794. Hsu, H. F., Yang, C. H. 2002. An orchid (Oncidium Gower Ramsey) AP3-like MADS gene regulates floral formation and initiation. Plant Cell Physiol. 43: 1198–1209.


130


Hu, J. S., Ferreira, S., Wang, M., and Xu, M. Q. 1993. Detection of Cymbidium mosaic virus, Odontoglossum ringspot virus, Tomato spotted wilt virus, and Potyviruses infecting orchids in Hawaii. Plant Dis. 77: 464–468. Hu, W. W., and Wong, S. M. 1998. The use of DIG-labelled cRNA probes for the detection of Cymbidium mosaic potexvirus (CymMV) and Odontoglossum ringspot tobamovirus (ORSV) in orchids. J. Virol. Methods 70: 193–199. Huan L. V. T., and Tanaka, M. 2004b. Callus induction from protocorm-like body segments and plant regeneration in Cymbidium (Orchidaceae). J. Hortic. Sci. Biotechnol. 79: 406–410. Huan, L. V. T., Takamura, T., and Tanaka, M. 2004. Callus formation and plant regeneration from callus through somatic embryo structures in Cymbidium orchid. Plant Sci. 166: 1443–1449. Huan, L. V. T., and Tanaka, M. 2004a. Effects of red and blue light emitting diodes on callus induction, callus proliferation, and protocorm-like body formation from callus in Cymbidium orchid. Environ. Control Biol. 42: 57–64. Huang, L. C., Lin, C. J., Kuo, C. I., Huang, B. L., and Murashige, T. 2001. Paphiopedilum cloning in vitro. Sci. Hortic. 91: 111–121. Huang, W. T., Zeng S. J., Wu K. L., Zhang, J. X., and Duan, J. 2012. Research progress on cross breeding of Phalaenopsis. J. Trop. Subtrop. Bot. 20 (1): 14–25. Huehne, P. S., and Bhinja, K. 2012. Application of cryoprotectants to improve low temperature storage survival of orchid seeds. Sci. Hortic. 135: 186–193. Hull, R. 2002. Matthews’ Plant Virology (4th ed.). Academic Press, San Diego. Ichihashi, S., and Shigemura, S. 2002. Phalaenopsis callus and protoplast culture. Proc. 17th World Orchid Conf., Malaysia. pp. 257–261. Inouye, N., and Gara, I. W. 1996. Detection and identification of viruses of orchids in Indonesia. Bull. Res. Inst. Bioresour. Okayama Univ. 4: 109–118. Irwin, M. J., Bougoure, J. J., and Dearnaley, J. D. W. 2007. Pterostylis nutans (Orchidaceae) has a specific association with two Ceratobasidium root-associated fungi across its range in eastern Australia. Mycoscience 48: 231–239. Ishii, Y., Takamura, T., Goi, M., and Tanaka, M. 1998. Callus induction and somatic embryogenesis of Phalaenopsis. Plant Cell Rep. 17: 446–450. Ishikawa, K., Harata, K., Mii, M., Sakai, A., Yoshimatsu, K., and Shimomura, K., 1997. Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification. Plant Cell Rep. 16: 754–757. Islam, M. O., Rahman, A. R. M. M., Matsui, S., and Prodhan, A. K. M. A. 2003. effects of complex organic extracts on callus growth and PLB regeneration through embryogenesis in the Doritaenopsis orchid. JARQ 37: 229–235. Israeli, Y., Reuveni, O., and Lahav, E. 1991. Qualitative aspects of somaclonal variations in banana propagated by in vitro techniques. Sci. Hortic. 48: 71–88. Jacquemyn, H., Brys, R., Cammue, B. P. A., Honnay, O., and Lievens, B. 2011. Mycorrhizal associations and reproductive isolation in three closely related Orchis species. Ann. Bot. 107: 347–356. Jakucs, E., Kovács, G. M., Agerer, R., Romsics, C., and Er˝os-Honti, Z. 2005. Morphological-anatomical characterization and molecular identification of Tomentella stuposa ectomycorrhizae and related anatomotypes. Mycorrhiza 15: 247–258. Jarrell, D. C., vClegg, M. T. 1995. Systematic implications of the chloroplastencoded matK gene on the tribe Vandeae (Orchidaceae). Amer. J. Bot. 82: 137. Jeon, M. W., Ali, M. B., Hahn, E. J., and Paek, K. Y. 2005. Effects of photon flux density on the morphology, photosynthesis and growth of a CAM orchid, Doritaenopsis during post-micropropagation acclimatization. Plant Growth Regul. 45: 139–147. Jiang, J., and Gill, B. S. 1994. Nonisotopic in situ hybridization and plant genome mapping: the first 10 years. Genome 37: 717–725. Jiang, W., Yang, G, Zhang, C., and Fu, C. 2011. Species composition and molecular analysis of symbiotic fungi in roots of Changnienia amoena (Orchidaceae). Afr. J. Microbiol. Res. 5: 222–228. Jin, H., Wu, J. R., Chen, X., and Han, S. F. 2006. Effects of mycorrhizal fungi on the growth and mineral absorption of Cymbidium goeringii. Northern Hortic. 6: 90–92.

Jin, H., Xu, Z. X., Chen, J. H., Han, S.F., Ge, S., and Luo, Y. B. 2009. Interaction between tissue-cultured seedlings of Dendrobium officinale and mycorrhizal fungus (Epulorhiza sp.) during symbiotic culture. Chin. J. Plant Ecol. 33: 433–441. Johri, B. M., Ambegaokar, K. B., and Srivastava, P. S. 1992. Comparative embryology of angiosperms. Springer Verlag, Berlin. Jones, W. E., and Kuehnle, A. R. 1998. Ploidy identification using flow cytometry in tissues of Dendrobium species and cultivars. Lindleyana 13: 11–18. Jonsson, L., and Nylund, J. E. 1979. Favolaschia dybowkyana (Singer) Singer (Aphyllophorales), a new orchid mycorrhizal fungus from tropical Africa. New Phytol. 83: 121–128. Joubès, J., and Chevalier, C. 2000. Endoreduplication in higher plants. Plant Mol. Biol. 43: 735–745. Kanchanapooma, K., Jantarob, S., and Rakchad, D. 2001. Isolation and fusion of protoplasts from mesophyll cells of Dendrobium Pompadour. Sci. Asia 27: 29–34. Kang, Z .H., Han, S. F., and Han, Z. M. 2007. Effects of orchidaceous Rhizoctonias on the growth of Dendrobium candidum. J. Nanjing For. Univ. (Nat. Sci.) 31: 49–52. Kano, K. 1965. Studies on the media for orchid seed germination. Mem. Fac. Agric. Kagawa Univ. 20: 1–68. Kao, Y. Y., Chang, S. B., Lin, T. Y., Hsieh, C. H., Chen, Y. H., Chen, W. H., and Chen, C. C. 2001. Differential accumulation of heterochromatin as a cause for karyotype variation in Phalaenopsis orchids. Ann. Bot. 87: 387–395. Kao, Y. Y., Lin, C. C., Huang, C. H., and Li, Y. H. 2007. The cytogenetics of Phalaenopsis orchids. In: Orchid Biotechnology, pp. 115–128. Chen, W. H. and Chen, H. H., Eds., World Scientific, Hong Kong. Karasev, A. V., Nikolaeva, O. V., Koonin, E. V., Gumpf, D. J., and Garnsey, S. M. 1994. Screening of the closterovirus genome by degenerate primer-mediated polymerase chain reaction. J. Gen. Virol. 75: 1415–1422. Kauth, P. J., Dutra, D., Johnson, T. R., Stewart, S. L., Kane, M. E., and Vendrame, W. 2008. Techniques and applications of in vitro orchid seed germination. In: Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues (1st Ed., Vol. V), Chapter 45, pp. 375–391. Teixeira da Silva, J. A., Ed. Global Science Books, Ltd., Isleworth, UK. Ke, H. L., Song, X. Q., Luo, Y. B., Zhu, G. P., and Ling, X. B. 2008. Seedling cultivation of Doritis pulcherrima Lindl. with mycorrhizal fungi. Acta Hortic. Sin. 35: 571–576. Kennedy, A. H., Taylor, D. L., and Watson, L. E. 2011. Mycorrhizal specificity in the fully mycoheterotrophic Hexalectris Raf. (Orchidaceae: Epidendroideae). Mol. Ecol. 20: 1303–1316. Kerbauy, B. B. 1984. In vitro flowering of Oncidium varicosum mericlones (Orchidaceae). Plant Sci. Lett. 35: 73–75. Ket, N.V., Hahn, E. J., Park, S. Y., Chakrabarty, D., and Paek, K. Y. 2004. Micropropagation of an endangered orchid Anoectochilus formosanus. Biol. Plant. 48: 339–344. Khentry, Y., Paradornuvat, A., Tantiwiwat, S., Phansiri, S., and Thaveechai, N. 2006. Protoplast isolation and culture of Dendrobium Sonia “Bom 17”. Kasetsart J. (Nat. Sci.) 40: 361–369. Khew, G. S., and Chia, T. F. 2011. Parentage determination of Vanda Miss Joaquim (Orchidaceae) through two chloroplast genes rbcL and matK. AoB Plants plr018. Khoddamzadeh, A. A., Sinniah, U. R., Kadir, M. A., Kadzimin, S. B., Maziah, M., and Sreeramanan, S. 2010. Detection of somaclonal variation by random amplified polymorphic DNA analysis during micropropagation of Phalaenopsis bellina (Rchb. f.) Christenson. Afr. J. Biotechnol. 4: 6632–6639. Khoddamzadeh, A. A., Sinniah, U. R., Lynch, P., Kadir, M. A., Kadzimin, S. B., Mahmood, M., and Sreeramanan, S. 2011. Cryopreservation of protocorm-like bodies (PLBs) of Phalaenopsis bellina (Rchb.f.) Christenson by encapsulation-dehydration. Plant Cell Tiss, Organ Cult. 107: 471–481. Khouri, N. A., Nawasreh, M., Al-hussain, S. M., and Alkofahi, A. S. 2006. Effects of orchids (Orchis anatolica) on reproductive function and fertility in adult male mice. Reprod. Med. Biol. 5: 269–276.


ORCHIDS AND BIOTECHNOLOGY Kishi, F., and Takagi, K. 1997. Efficient method for the preservation and regeneration of orchid protocorm-like bodies. Sci. Hortic. 68: 149–156. Kishor, R., Devi, H. S., Jeyaram, K., and Singh, M. R. K. 2008. Molecular characterization of reciprocal crosses of Aerides vandarum and Vanda stangeana (Orchidaceae) at the protocorm stage. Plant Biotechnol. Rep. 2: 145–152. Kishor, R., Khan, P. S. S. V., and Sharma, G. J. 2006. Hybridization and in vitro culture of Ascocenda ‘Kangla’. Sci Hortic. 108: 66–73. Kishor, R., and Sharma, G. J. 2009. Intergeneric hybrid of two rare and endangered orchids, Renanthera imschootiana Rolfe and Vanda coerulea Griff. ex L. (Orchidaceae): Synthesis and characterization. Euphytica 165: 247–256. Knapp, J. E., Kausch, A. P., and Chandlee, J. M. 2000. Transformation of three genera of orchid using the bar gene as a selectable marker. Plant Cell Rep. 19: 893–898. Knudson L. 1921. La germinación no simbiótica de las semillas de orquideas. Bol. Real. Soc. Española Hist. Nat. 21: 250–260. Kobayashi, S., Kameya, T., and Ichihashi, S. 1993. Plant regeneration from protoplasts derived from callus of Phalaenopsis. Plant Tiss. Cult. Lett. 10: 267–270. Kores, P. J., Cameron, K. M., Molvray, M., and Chase, M. W. 1997. The phylogenetic relationships of Orchidoideae and Spiranthoideae (Orchidaceae) as inferred from rbcL plastid sequences. Lindleyana 12: 1–11. Kores, P. J., Molvray, M., Weston, P. H., Hopper, S. D., Brown, A. P., Cameron, K. M., and Chase, M. W. 2001. A phylogenetic analysis of Diurideae (Orchidaceae) based on plastid DNA sequence data. Amer. J. Bot. 88: 1903–1914. Kores, P. J., Weston, P., Molvray, M., and Chase, M. W. 2000. Phylogenetic relationships within Diurideae: inferences from plastid matK DNA sequences. In: Monocots: Systematics and Evolution. Wilson, K. L., and Morrison, D. A., Eds.. CSIRO Publishing, Collingwood, Victoria, Australia. Kostenyuk, I., Oh, B. J., and So, I. S. 1999. Induction of early flowering in Cymbidium niveo-marginatum Mak in vitro. Plant Cell Rep. 19: 1–5. Kress, W. J., and Erickson, D. L. 2007. A two-locus global DNA barcode for land plants: the coding rbcL gene complements the noncoding trnH-psbA spacer region. PLoS ONE 2, e508. Kristiansen, K. A., Taylor, D. L., Kjøller, R., Rasmussen, H. N., and Rosendah, S. 2001. Identification of mycorrhizal fungi from single pelotons of Dactylorhiza majalis (Orchidaceae) using single-strand conformation polymorphism and mitochondrial ribosomal large subunit DNA sequences. Mol. Ecol. 10: 2089–2093. Krohn, K., Loock, U., Paavilainen, K., Hausen, B. M., Schmalle, H. W., and Kiesele, H. 2001. Synthesis and electrochemistry of annoquinone-A, cypripedin methyl ether, denbinobin and related 1,4-phenanthrenequinones. Arkivoc 1: 88–130. Kubo, K. S., Freitas-Astúa, J., Machado M. A., Kitajima, E. W. 2009. Orchid fleck symptoms may be caused naturally by two different viruses transmitted by Brevipalpus. J. Gen. Plant Pathol. 75: 250–255. Kuehnle, A. R. 1996. Towards control and eradication of virus in Dendrobium by hybridization, tissue culture, and genetic engineering. J. Orchid Soc. India. 10: 43–51. Kuehnle, A. R., and Nan, G. L. 1990. Factors influencing the isolation and culture of protoplasts from Hawaiian Dendrobium cultivars. In: Bonham, D.G., Kernohan, J. (Eds.) Proceedings of the 13th World Orchid Conference, Auckland, New Zealand. pp. 259–262. Kuehnle, A. R., and Sugii, N. 1992. Transformation of Dendrobium orchid using particle bombardment of protocorms. Plant Cell Rep. 11: 484–488. Kunasakdakul, K., and Smitamana, P. 2003. Dendrobium Pratum Red protoplast. Thai J. Agric. Sci. 36: 1–8. Kuninaga, S., Natsuaki, T., Takeuchi, T., and Yokosawa, R. 1997. Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani. Curr. Gen. 32: 237–243. Kuo, C. T., Chen, B. C., Yu, C. C., Weng, C. M., Hsu, M. J., Chen, C. C., Chen, M. C., Teng, C. M., Pan, S. L., Bien, M. Y., Shih, C. H., and Lin, C. H. 2009. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells. J. Biomed. Sci. 16: 43.

131

Lahaye, R., van der Bank, M., Bogarin, D., Warner, J., Pupulin, F., Gigot, G., Maurin, O., Duthoit, S., Barraclough, T. G., and Savolainen, V. 2008. DNA barcoding the floras of biodiversity hotspots. Proc. Natl. Acad. Sci. USA 105: 2923–2928. Lakshmanan, P., Loh, C. S., and Goh, C. J. 1995. An in vitro method for rapid regeneration of a monopodial orchid hybrid Aranda Deborah using thin section culture. Plant Cell Rep. 14: 510–514. Lalitharani, S., Mohan, V. R., and Maruthupandian, A. 2011. Pharmacognostic investigations on Bulbophyllum albidum (Wight) Hook. f. Intl. J. PharmTech Res. 3: 556–562. Lang, N. T., and Hang, N. T. 2006. Using biotechnological approaches for Vanda orchid improvement. Omonrice 14: 140–143. Langeveld, S. A., Dore, J. M., Memelink, J., Derks, A. F. L. M., van der Vlugt, C. I. M., Asjes, C. J., and Bol, J. F. 1991. Identification of Potyviruses using the polymerase chain reaction with degenerate primers. J. Gen. Virol. 72: 1531–1541. Larkin, P., and Scowcroft, W. 1981. Somaclonal variation - a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60: 197–214. Lau, D. T. W., Shaw, P. C., Wang, J., and But, P. P. H. 2001. Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA. Planta Med. 67: 456–460. Law, S. K., and Yeung, E. C. 1993. Embryology of Cypripedium passerium (Orchidaceae): ovule development. Lindleyana 8: 139–147. Lee, J. Y., Jang, Y. W., Kang, H. S., Moon, H., Sim, S. S., and Kim, C. J. 2006a. Anti-inflammatory action of phenolic compounds from Gastrodia elata root. Arch. Pharm. Res. 29 (10): 849–858. Lee, S. C., and Chang, Y. C. 2006. Multiplex RT-PCR detection of two orchid viruses with an internal control of plant nad5 mRNA. Plant Pathol. Bull. 15: 187–196. Lee S. C., and Chang, Y. C. 2008. Performances and application of antisera produced by recombinant capsid proteins of Cymbidium mosaic virus and Odontoglossum ringspot virus. Eur. J. Plant Pathol. 122: 297–306. Lee, S. K., and Koay, S. H. 1986. Effects of benzyladenine and gibberellic acid on the flowering of Dendrobium Louisae. In: Proceedings of 5th Asian Orchid Congress, pp. 87–88. Singapore. Lee, Y. I., Chang, F. C., and Chung, M. C. 2011. Chromosome paring affinities in interspecific hybrids reflect phylogenetic distances among lady’s slipper orchids (Paphiopedilum). Ann. Bot. 108: 113–121. Lee, Y. I., Lee, N., Yeung, E. C., and Chung, M. C. 2005. Embryo development of Cypripedium formosanum in relation to seed germination in vitro. J. Am. Soc. Hortic. Sci. 130: 747–753. Lee, Y. I., Yeung, E. C., and Chung, M. C. 2007. Embryo development of orchids In: Orchid Biotechnology, pp. 23–44. Chen, W. H., and Chen, H. H., Eds. World Scientific, Hong Kong. Lee, Y. I., Yeung, E. C., Lee, N., and Chung, M. C. 2006. Embryo development in the lady’s slipper orchid, Paphiopedilum delenatii with emphases on the ultrastructure of the suspensor. Ann. Bot. 98: 1311–1319. Lee, Y. I., Yeung, E. C., Lee, N., and Chung, M. C. 2008. Embryology of Phalaenopsis amabilis var. formosa: embryo development. Bot. Stud. 49: 139–146. Lee, Y. I., Yeung, E. C., Lee, N., Lur, C. F., and Chung, M. C. 2007. Changes in endogenous abscisic acid levels and asymbiotic seed germination of a terrestrial orchid, Calanthe tricarinata Lindl. J. Am. Soc. Hortic. Sci. 132: 246–252. Leroux, G., Barabi, D., and Vieth, J. 1997. Morphogenesis of the protocorm of Cypripedium acaule (Orchidaceae). Plant Syst. Evol. 205: 53–72. Li D.M., Xia, K.F., Ye, X. L., and Liang, C. H. 2005. Ultrastructural observations on megasporogenesis in Phaius tankervilliae (Aiton) Bl. Acta Sci. Nat. Uni. Sunyatseni 44 (2): 209–212. Li D. M., Wu, C. H., Ye, X. L., and Liang, C. H. 2006a. The response of egg cell during the process of fertilization in Doritis pulcherrima Lindl. Acta Sci. Nat. Uni. Sunyatseni 45 (6): 93–96.


132


Li D. M., Ye, X. L., Liang, C. H., and Hu, X. Y. 2006b. Growth paths of pollen tubes in ovary of Phaius tankervilliae (Aiton) Bl. (Orchidaceae). J. Trop. Subtrop. Bot. 14 (2): 130–133. Li, H., Zheng, S. X., Li, Z. L., and Yu, C. X. 2004. Prelimonary study on polyploid of Dendyobium devonianum. Chin. Agric. Sci. Bull. 20 (4): 198–199. Li, S. H., Kuoh, C. S., Chen, Y. H., Chen, H. H., and Chen, W. H. 2005. Osmotic sucrose enhancement of single-cell embryogenesis and transformation efficiency in Oncidium. Plant Cell, Tiss. Organ Cult. 81: 183–192. Li, Y., Wang, C. L., Guo, S. X., Yang, J. S., and Xiao, P. G. 2008. Two new compounds from Dendrobium candidum. Chem. Pharm. Bull. 56: 1477–1479. Liao, L. J., Pan, I. C., Chan, Y. L., Hsu, Y. H., Chen, W. H., and Chan, M. T. 2004. Transgene silencing in Phalaenopsis expressing the coat protein of Cymbidium Mosaic Virus is a manifestation of RNA-mediated resistance. Mol. Breed. 13: 229–242. Liao, Y. J., Tsai, Y. C., Sun, Y. W., Lin, R. S., and Wu, F. S. 2011. In vitro shoot induction and plant regeneration from flower buds in Paphiopedilum orchids. In Vitro Cell. Dev. Biol.—Plant 47: 702–709. Liau, C. H., Lu, J. C., Prasad, V., Hsiao, H., You, S. J., Lee, J., Yang, N. S., Huang, H. E., Feng, T. Y., Chen, W. H., and Chan, M. T. 2003b. The sweet pepper ferredoxin-like protein (pflp) conferred resistance against soft rot disease in Oncidium orchid. Transgenic Res. 12: 329–336. Liau, C. H., You, S. J., Prasad, V., Hsiao, H. H., Lu, J. C., Yang, N. S., and Chan, M. T. 2003a. Agrobacterium tumefaciens-mediated transformation of an Oncidium orchid. Plant Cell Rep. 21: 993–998. Lim, S. H., Teng, P. C. P., Lee, Y. H., and Goh, C. J. 1999. RAPD analysis of some species in the genus Vanda (Orchidaceae). Ann. Bot. 83: 193–196. Lim, S. T., Wong, S. M., Yeong, C. Y., Lee, S. C., and Goh, C. J. 1993. Rapid detection of Cymbidium mosaic virus by the polymerase chain reaction (PCR). J. Virol. Methods 41: 37–46. Liu, A. C., Liu, C., Zhao Y., and Xu, X. C. 2009. Detection of two orchid viruses using ELISA. Acta Agric. Zhejiangensis 21 (2): 91–95. Liu, C. L. Liu, M. C., and Zhu, R. L. 2002. Determination of gastrodin, ρ-hydroxybenzyl alcohol, vanillyl alcohol, ρ-hydroxylbenzaldehyde and vanillin in tall gastrodia tuber by high-performance liquid chromatography. Chromatographia 55: 317–320. Lim, W. L., and Loh, C. S. 2003. Endopolyploidy in Vanda Miss Joaquim (Orchidaceae). New Phytol. 159, 279–287. Long, B., Niemiera, A. X., Cheng, Z. Y., and Long, C. L. 2010. In vitro propagation of four threatened Paphiopedilum species (Orchidaceae). Plant Cell Tiss. Organ Cult. 101: 151–162. Lu, H. C., Chen, H. H., and Yeh, H. H. 2007. Application of virus-induced gene silencing technology in gene functional validation of orchids. In: Chen, W. H., Chen, H. H. (Eds.) Orchid Biotechnology, World Scientific, Hong Kong. pp. 211–223. Lu, I. L., Sutter, E., and Burger, D. 2001. Relationships between benzyladenine uptake, endogenous free IAA levels and peroxidase activities during upright shoot induction of Cymbidium ensifoilum cv. Yuh Hwa rhizomes in vitro. Plant Growth Regul. 35: 161–170. Lu, J., Hu, X., Liu, J., and Wang, H. 2011. Genetic diversity and population structure of 151 Cymbidium sinense cultivars. J. Hortic. Forestry 3: 104–114. Lu, Z. X., Wu, M., Loh, C. S., Yeong, C. Y., and Goh, C. J. 1993. Nucleotide sequence of a flower-specific MADS box cDNA clone from orchid. Plant Mol. Biol. 23: 901–904. Lucksom, S. Z. 2007. The orchids of Sikkim and North-east Himalaya. Author Publishers and Distributors, Gangtok, East Sikkim, Assam, India. Luo, J. P., Wang, Y., Zha, X. Q., and Huang, L. 2008. Micropropagation of Dendrobium densiflorum Lindl. ex Wall. through protocorm-like bodies: effects of plant growth regulators and lanthanoids. Plant Cell, Tiss. Organ Cult. 93: 333–340 Lurswijidjarus, W., and Thammasiri, K. 2004. Cryopreservation of shoot tips of Dendrobium Walter Oumae by encapsulation/dehydration. Sci. Asia 30: 293–299.

Ma, M., Tan, T. K., and Wong, S. M. 2003. Identification and molecular phylogeny of Epulorhiza isolates from tropical orchids. Mycol. Res. 107: 1041–1049. Masuhara, G., and Katsuya, K. 1994. In situ and in vitro specificity betwee Rhizoctonia spp. & Spiranthes sinensis (Persoon.) Ames. Vaamoena (M. Beiberstein) Hara (Orchidaceae). New Phytol. 127: 711–718. Mackenzie, A. M., Nolan, M., Wei, K. J., Clements, M. A., Gowanlock, D., Wallace, B. J., and Gibbs A. J. 1998. Ceratobium mosaic potyvirus: another virus from orchids. Arch. Virol. 143: 903–914. Mahendran, G., and Bai, V. N. 2012. Direct somatic embryogenesis and plant regeneration from seed derived protocorms of Cymbidium bicolor Lindl. Sci. Hortic. 135: 40–44. Majumder, P. L., and Chatterjee, S. 1989. Crepaditin, a bibenyl derivative from the orchid Dendrobium crepidatum. Phytochemistry 28: 1986–1988. Majumder, P. L., Guha, B. N. S., and Ray, P. N. S. 2011. Four new stilbenoids from the orchids Coelogyne ochracea and Coelogyne cristata. J. Indian Chemical Soc. 88: 1293–1304. Majumder, P. L., and Pal, S. 1992. Rotundatin, a new 9,10-dihydrophenanthrene derivative from Dendrobium rotunatum. Phytochemistry 31: 3225–3228. Majumder, P. L., Sen, R.C. 1987. Moscatilin, a bibenzyl derivative from the orchid Dendrobium moscatum. Phytochemistry 26: 2121–2124. Martin, K. P., and Madassery, J. 2006 Rapid in vitro propagation of Dendrobium hybrids through direct shoot formation from foliar explants, and protocormlike bodies. Sci. Hortic. 108: 95–99. Nakasone, H. Y. 1962. Chromosome doubling in orchids with colchicine. Hawaii Orchid J. 4: 46–50. Nakasone, H. Y., and Kamemoto, H. 1961. Artificial induction of polyploidy in orchids by the use of colchicine. Hawaii Agric. Exp. Sta. Bull. 42: 3–27. Malabadi, R. B., Teixeira da Silva, J. A., Nataraja, K., and Mulgund, G. S. 2008. Shoot tip transverse thin cell layers and 24-epibrassinolide in the micropropagation of Cymbidium bicolor Lindl. Floriculture Ornamental Biotechnol. 2: 44–48. Mandal, A., Maiti, A., Chowdhury, B., and Elanchezhian, R. 2001. Isoenzyme markers in varietal identification of banana. In Vitro Cell. Dev. Biol.—Plant 37: 599–604. Maneerattanarungroj, P., Bunnag, S., and Monthatong, M. 2007. In vitro conservation of Cleiostoma areitinum (Rcbh.f.) Garay, rare Thai orchid species by encapsulation- dehydration method. Asian J. Plant Sci. 6: 1235– 1240. Maridass, M. 2011. Anti diarrhoeal activity of rare orchid Eulophia epidendraea (Retz.) Fisher. Nat. Pharm. Tech. 1: 5–10. Maridass, M., Raju, G., and Ghanthikumar, S. 2008. Tissue—regenerative responses on tuber extracts of Eulophia epidendraea (Retz.) Fischer in wistar rat. Pharmacology Online 3: 631–636. Martin, K. P. 2003. Clonal propagation, encapsulation and reintroduction of Ipsea malabarica (Reichb. f.) J. D. Hook., an endangered orchid. In Vitro Cell. Dev. Biol.—Plant 39: 322–326. Mata-Rosas, M., Baltazar-Garc´ıa. R .J., Moon, P., Hietz, P., and LunaMonterrojo, V. E. 2010. In vitro regeneration of Lycaste aromatica (Graham ex Hook) Lindl. (Orchidaceae) from pseudobulb sections. Plant Biotechnol. Rep. 4: 157–163. Matsumoto, J. I., Maeda, T., and Inouye, N. 1999. Some properties of bean yellow mosaic virus isolated from Calanthe sp. (Orchidacese) in Japan. Bull. Res. Inst. Bioresour. Okayama Univ. 6: 43–51. Matsumoto, T. K. 2006. Gibberellic acid and benzyladenine promote early flowering and vegetative growth of Miltoniopsis orchid hybrids. HortScience 41, 131–135. Mayer, J. L. S., Carmello-Guerreirob, S. M., and Appezzato-da-Glória, B. 2011. Anatomical development of the pericarp and seed of Oncidium flexuosum Sims (Orchidaceae). Flora 206: 601–609. McCook, L. 1989. Systematics of Phragmipedium (Cypripedioideae: Orchidaceae). PhD thesis, Cornell University, Ithaca, New York McCormick, M. K., Whigham, D. F., and O’Neill, J. 2004. Mycorrhizal diversity in photosynthetic terrestrial orchids. New Phytol. 163: 425–438.


ORCHIDS AND BIOTECHNOLOGY McNaughton, S. J. 1989. Ecosystems and conservation in the twenty-first century. In: Conservation for the Twenty-first Century, pp. 109–120. Western, D., and Pearl, M., Eds. Oxford University Press, New York. Mehrotra, S., Goel, M. K., Kukreja, A. K., and Mishra, B. N. 2007. Efficiency of liquid culture systems over conventional micropropagation: A progress towards commercialization. Afr. J. Biotechnol. 6: 1484–1492. McKendrick, S. L, Leake, J. R., Taylor, D. L., and Read, D. J. 2000. Symbiotgermination and development of myco-heterotrophic plants nature: ontogeny of Corallorhiza trifida and characterisation of its mycorrhizal fungi. New Phytol. 145: 523–537. Men, S., Ming, X., Liu, R., Wei, C., and Li, Y. 2003b. Agrobacterium-mediated transformation of a Dendrobium orchid. Plant Cell, Tiss. Organ Cult. 75: 63–71. Men, S., Ming, X., Wang, Y., Liu, R., Wei, C., and Li, Y., 2003a. Genetic transformation of two species of orchid by biolistic bombardment. Plant Cell Rep. 21: 592–598. Menninger, E. D. 1963. Diary of a colchicine-induced tetraploid Cymbidium. Amer. Orchid Soc. Bull. 32: 885–887. Meyer, L., Wehner, F. C., Nel, L. H., and Carling, D. E. 1998. Characterization of the crater disease strain of Rhizoctonia solani. Phytopathology 88: 366–371. Miguel, T. P., and Leonhardt, K. W. 2011. In vitro polyploid induction of orchids using oryzalin. Sci. Hort. 130: 314–319. Mishiba, K., Chin, D. P., and Mii, M. 2005. Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination. Plant Cell Rep. 24: 297–303. Mishiba, K., and Mii, M. 2000. Polysomaty analysis in diploid and tetraploid Portulaca grandiflora. Plant Science 156: 213–219. Mohanty, P., Das, M. C., Kumaria, S., and Tandon, P. 2012. High-efficiency cryopreservation of the medicinal orchid Dendrobium nobile Lindl. Plant Cell Tiss. Organ Cult. 109(2): 297–305. Mohd-Hairul, A. R., Namasivayam, P., Abdullah, J. O., and Lian, G. E. C. 2011. Screening, isolation, and molecular characterization of putative fragrance-related transcripts from Vanda Mimi Palmer. Acta Physiol. Plant. 33: 1651–1660. Mondragón-Palomino, M., and Theissen, G. 2008. MADS about the evolution of orchid flowers. Trends Plant Sci. 13: 51–59. Morel, G. 1960. Producing virus free Cymbidium. Amer. Orchid Soc. Bull. 29: 459–497. Mumford, R. A., Barker, I., and Wood, K. R. 1996. An improved method for the detection of tospoviruses using the polymerase chain reaction. J. Virol. Methods 57: 109–115. Murashige, T., and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tabacco tissue cultures. Physiol. Plant. 15: 473–497. Murdad, R., Hwa K. S., Seng, C. K., Latip, M. A., Aziz, Z. A., and Ripin, R. 2006. High frequency multiplication of Phalaenopsis gigantean using trimmed bases protocorms technique. Sci. Hortic. 111: 73–79. Na, H. Y., and Kondo, K. 1996. Cryopreservation of tissue-cultured shoot primordia from shoot apices of cultured protocorms in Vanda pumila following ABA preculture and desiccation. Plant Sci. 118: 195–201. Nagl, W. 1972. Evidence of DNA amplification in orchid Cymbidium in vitro. Cytobios 5: 145–154. Naing, A. H., Chung, J. D., Park, I. S., and Lim, K. B. 2011. Efficient plant regeneration of the endangered medicinal orchid, Coelogyne cristata using protocorm-like bodies. Acta Physiol. Plant. 33: 659–666. Nair, R. R., and Ravindran, P. N. 1994. Somatic association of chromosomes and other mitotic abnormalities in Vanilla planifolia (Andrews). Caryologia 47: 65–73. Nan, G. L., and Kuehnle, A. R. 1995. Factors affecting gene delivery by particle bombardment of Dendrobium orchids. In Vitro Cell. Dev. Biol.—Plant 31: 131–136. Nan, G. L., Kuehnle, A. R., and Kado, C. I. 1998. Transgenic Dendrobium orchid through Agrobacterium-mediated transformation. Malay Orchid Rev. 32: 93–96.

133

Navalinskienë, M., Raugalas, J., and Samuitienë, M. 2005. Viral diseases of flower plants 16. Identification of viruses affecting orchids (Cymbidium Sw.). Biologia 2: 29–34. Nayak, N. R., Patnaik, S., and Rath, S. P. 1997. Direct shoot regeneration from foliar explants of an epiphytic orchid, Acampe praemorsa (Roxb.) Blatter and McCann. Plant Cell Rep. 16: 583–586. Nayak, N. R., Sahoo, S., Patnaik, S., and Rath, S. P. 2002. Establishment of thin cross section (TCS) culture method for rapid micropropagation of Cymbidium aloifolium (L.) Sw. and Dendrobium nobile Lindl. (Orchidaceae). Sci. Hortic. 94: 107–116. Neelannavar, V. S., Biradar, M. S., Kumar, A., and Shivamurthy, D. 2011. In vitro propagation studies in Vanilla (Vanilla planifolia Andr.). Plant Arch. 11: 377–378. Newmaster, S. G., and Ragupathy, S. 2009. Testing plant barcoding in a sister species complex of pantropical Acacia (Mimosoideae, Fabaceae). Mol. Ecol. Res. 9: 172–180. Neyland, R., and Urbatsch, L. 1996. Evolution in the number and position of fertile anthers in Orchidaceae inferred from ndhF chloroplast gene sequences. Lindleyana 11: 47–53. Ng, C. Y., and Saleh, N. M. 2011. In vitro propagation of Paphiopedilum orchid through formation of protocorm-like bodies. Plant Cell, Tiss. Organ Cult. 105: 193–202. Ng, T. B., Liu J, Wong J. H., Ye X., Wing Sze, S. C., Tong Y., and Zhang, K.Y. 2012. Review of research on Dendrobium, a prized folk medicine. Appl. Microbiol. Biotechnol. 93: 1795–1803. Nge, K. L., New, N., Chandrkrachang, S., and Stevens, W. F. 2006. Chitosan as a growth stimulator in orchid tissue culture. Plant Sci. 170: 1185–1190. Niino, T., and Sakai, A. 1992. Cryopreservation of alginate-coated in vitro grown shoot tips of apple, pear and mulberry. Plant Sci. 87: 199–206. Nikishina, T. V., Popov, A. S., Kolomeitseva, G. L., and Golovkin, B. N. 2001a. Cryopreservation of seeds of some tropical orchids. Doklady Biochemistry and Biophysics 378, 231–233. Translated from Doklady Akademii Nauk 378: 555–557. Nikishina, T. V., Popov, A. S., Kolomeitseva, G. L., and Golovkin, B. N. 2001b. Effect of cryoconservation on seed germination of rare tropical orchids. Russian J. Plant Physiol. 48: 810–815. Nikishina, T. V., Vakhrameeva, M. G., Varlygina, T. I., Pavlov, V. N., Shirokov, A. I., Burov, A. V., Popovich, E. A., and Popov, A. S. 2006. Cryoconservation of seeds of rare Russian orchids. Doklady Biol. Sci. 408: 214–216. Niknejad, A., Kadir, M. A., and Kadzimin, S. B. 2011. In vitro plant regeneration from protocorms-like bodies (PLBs) and callus of Phalaenopsis gigantea (Epidendroideae: Orchidaceae). Afr. J. Biotechnol. 10: 11808–11816. Nontachaiyapoom, S., Sasirat, S., and Manoch, L. 2010. Isolation and identification of Rhizoctonia-like fungi from roots of three orchid genera, Paphiopedilum, Dendrobium and Cymbidium, collected in Chiang Rai and Chiang Mai provinces of Thailand. Mycorrhiza 20: 459–471. Otero, J. T., Ackerman, J. D., and Bayman, P. 2004. Differences in mycorrhizal preferences between two tropical orchids. Mol. Ecol. 13: 2393–2404. Otero, J. T., Flanagan, N. S., Herre, E. A., Ackerman, J. D., and Bayman, P. 2007. Widespread mycorrhizal specificity correlates to mycorrhizal function in the neotropical, epiphytic orchid Ionopsis utricularioides (Orchidaceae). Amer. J. Bot. 94: 1944–1950. Otero, J. T., Ackerman, J. D., and Bayman, P. 2002. Diversity and host specificity of endophytic Rhizoctonia-like fungi from tropical orchids. Amer. J. Bot. 89: 1852–1858. Padmavathi, M., Srinivas, K. P., Reddy, C. V. S., Ramesh, B., Navodayam, K., Krishnaprasadji, J., Babu, P. R., and Sreenivasulu, P. 2011. Identification of a new potyvirus associated with chlorotic vein banding disease of Spathiphyllum spp., in Andhra Pradesh, India. Plant Pathol. J. 27: 33–36. Paek, K. Y., Chakrabarty, D., and Hahn, E. J. 2005. Application of bioreactor systems for large scale production of horticultural and medicinal plants. Plant Cell, Tiss. Organ Cult. 81: 287–300.


134


Paek, K. Y., Hahn, E. J., and Park, S. Y. 2011. Micropropagation of Phalaenopsis orchids via protocorms and protocorm-like bodies. Methods Mol. Biol. 710: 293–306. Pan, Z. J., Cheng, C. C., Tsai, W. C., Chung, M. C., Chen, W. H., Hu, J. M., and Chen, H. H. 2011. The duplicated B-class MADS-Box genes display dualistic characters in orchid floral organ identity and growth. Plant Cell Physiol. 52: 1515–1531. Pandey, A., Bag, N., Chandra, B., and Palni, L.M.S. 2002. Biological Hardening: A promising technology for tissue culture industry. In: Role of Plant Tissue Culture in Biodiversity Conservation and Development, pp. 565–577. Nandi, S. K., Palni, L. M. S., and Kumar, A., Eds. Gyanodaya Prakashan, Nainital, India. Pandey, A., Palni, L. M. S., and Bag, N. 2000. Biological hardening of tissue culture raised tea plants. Biotechnol. Lett. 22: 1087–1091. Park, S. Y., Murthy, H. N., and Paek, K. Y. 2000. Mass multiplication of protocorm-like bodies using bioreactor system and subsequent plant regeneration in Phalaenopsis. Plant Cell, Tiss. Organ Cult. 63: 67–72. Park, S.Y., Murthy, H. N., and Paek, K. Y. 2002. Rapid propagation of Phalaenopsis from floral stalk-derived leaves. In Vitro Cell. Dev. Biol.—Plant 38: 168–172. Park, S. Y., Yeung, E. C., and Paek, K. Y. 2010. Endoreduplication in Phalaenopsis is affected by light quality from light-emitting diodes during somatic embryogenesis. Plant Biotechnol. Rep. 4: 303–309. Parveen, I., Singh, H. K., Raghuvanshi, S., Pradhan, U. C., and Babbar, S. B. 2012. DNA barcoding of endangered Indian Paphiopedilum species. Mol. Ecol. Res. 12: 82–90. Perkins, A. J., and McGee, P. A. 1995. Distribution of the orchid mycorrhizfungus Rhizoctonia solani, in relation to its host, Pterostylacuminata, in the field. Aust. J. Bot. 43: 565–575 Peyvandi, M., Noormohammadi, Z., Banihashemi, O., Farahani, F., Majd, A., Hosseini-Mazinani, M., and Sheidai, M. 2009. Molecular analysis of genetic stability in long-term micropropagated shoots of Olea europaea L. (cv. Dezful). Asian J. Plant Sci. 8: 146–152. Phillips, R. D., Faast, R., Bower, C. C., Brown, G. R., and Peakall, R. 2009. Implications of pollination by food and sexual deception for pollinator specificity, fruit set, population genetics and conservation of Caladenia (Orchidaceae). Aus. J. Bot. 57: 287–306. Pope, E. J., and Carter, D. A. 2001. Phylogenetic placement and host specificity of mycorrhizal isolates AG-6 and AG-12 in the Rhizoctonia solani species complex. Mycologia 93: 712–719. Popov, A. S., Popova, E. V., Nikishina, T. V., and Kolomeytseva, G. L. 2004. The development of juvenile plants of the hybrid orchid Bratonia after seed cryopreservation. CryoLetters 25: 205–212. Popova, E. V., Nikishina, T. V., Kolomeitseva, G. L., and Popov, A. S. 2003. The effect of seed cryopreservation on the development of protocorms by the hybrid orchid Bratonia. Russian J. Plant Physiol. 50: 672–677. Porras-Alfaro, A., and Bayman, P. 2007. Mycorrhizal fungi of Vanilla: diversity, specificity and effects on seed germination and plant growth. Mycologia 99: 510–525. Price, G. R., and Earle, E. D. 1984. Sources of orchid protoplasts for fusion experiments. Amer. Orchid. Soc. Bull. 53: 1035–1043. Pridgeon, A. M., Bateman, R. M., Cox, A. V., Hapeman, J. R., and Chase, M. W. 1997. Phylogenetics of subtribe Orchidinae (Orchidoideae, Orchidaceae) based on nuclear ITS sequences. 1. Intergeneric relationships and polyphyly of Orchis sensu lato. Lindleyana 12: 89–109. Pritchard, H. W. 1984. Liquid nitrogen preservation of terrestrial and epiphytic orchid seed. CryoLetters 5: 295–300. Prutsch, J., Schardt, A., and Schill, R. 2000. Adaptations of an orchid seed to water uptake and storage. Plant Syst. Evol. 220: 69–75. Qin, X., Liu, Y., Mao, S., Li, T., Wu, H., Chu, C., and Wang, Y. 2011. Genetic transformation of lipid transfer protein encoding gene in Phalaenopsis amabilis to enhance cold resistance. Euphytica 177: 33–43. Raffeiner, B., Serek, M., and Winkelmann, T. 2009. Agrobacterium tumefaciensmediated transformation of Oncidium and Odontoglossum orchid species with the ethylene receptor mutant gene etr1–1. Plant Cell Tiss. Organ Cult. 98: 125–134.

Rajkumar, K., and Sharma, G. J. 2009. Intergeneric hybrid of two rare and endangered orchids, Renanthera imschootiana Rolfe and Vanda coerulea Griff. ex L. (Orchidaceae): Synthesis and characterization. Euphytica 165: 247–256. Randles, J. W., Hodgson, R. A. J., and Wefels, E. 1996. The rapid and sensitive detection of plant pathogens by molecular methods. Aus. Plant Pathol. 25: 71–85. Ramsay, M. M., and Stewart, J. 1998. Re-establishment of the lady’s slipper orchid (Cypripedium calceolus L.) in Britain. Bot. J. Linn. Soc. 126 (1): 173–181. Ranney, T. G. 2006. Polyploidy: from evolution to new plant development. Proc. Int. Plant Propagators Soc. 56: 137–142. Rasmussen, H. N., and Rasmussen, F. N. 1991. Climactic and seasonal regulation of seed plant establishment in Dactylorhiza majalis inferred from symbiotic experiments in vitro. Lindleyana 6: 221–227. Rittirat, S., Te-chato, S., Kerdsuwan, N., and Kongruk, S. 2011. Micropropagation of Chang Daeng (Rhynchostylis gigantea var. Sagarik) by embryogenic callus. Songkalakarin J. Sci. Technol. 33: 659–663. Rojanawong, T., Thepsithar, C., and Thongpukdee, A. 2006. Micropropagation of Phalanopsis cygnus ‘Silky Moon’ from leaf segments. In: Proceedings of the 32nd Congress on Science and Technology of Thailand, Bangkok, Thailand, October 10–12, 2006. Rout, T. M., Hauser, C. E., and Possingham, P. 2009. Optimal adaptive management for translocation of a threatened species. Ecol. Appl. 19: 515–526. Roy, J., and Banerjee, N. 2003. Inducton of callus and plant regeneration from shoot-tip explant of Dendrobium fimbriatum Lindl. var. oculatum Hk. f. Sci. Hortic. 97: 333–340. Roy, J., Naha, S., Majumdar, M., and Banerjee, N. 2007. Direct and callusmediated protocorm-like body induction from shoot-tips of Dendrobium chrysotoxum Lindl. (Orchidaceae). Plant Cell, Tiss Organ Cult. 90: 31–39. Ryan, A., Whitten, W. M., Johnson, M. A. T., and Chase, M. W. 2000. A phylogenetic assessment of Lycaste and Anguloa (Orchidaceae: Maxillarieae). Lindleyana 15: 33–45. Ryu, K. H., and Park, W. M. 1995a. Occurrence of Cymbidium mosaic virus and Odontoglossum ringspot virus in Korea. Plant Dis. 79: 321–326. Ryu, K. H., and Park, W. M. 1995b. Rapid detection and identification of Odontoglossum ringspot virus by polymerase chain reaction amplification. FEMS Microbiol. Lett. 133: 265–269. Sahay, N. S., and Varma, A. 2000. A biological approach towards increasing the rates of survival of micropropagated plants. Curr. Sci. 78: 126–129. Saiprasad, G. V. S., Raghuveer, P., Khetarpal, S., and Chandra, R. 2004. Effect of various polyamines on production of protocorm-like bodies in orchid—Dendrobium ‘Sonia’. Sci. Hortic. 100: 161–168. Sajise, J. U., Valmayor, H. L., and Sagawa, Y. 1990. Some problems in isolation and culture of orchid protoplasts. Proceedings of the Nagoya International Orchid Show (NIOS), Japan. pp. 84–89. Sakai, A., and Engelmann, F. 2007. Vitrification, encapsulation–vitrification and droplet–vitrification: a review. CryoLetters 28: 151–172. Sakai, A., Hirai, D., and Niino, T. 2008. Development of PVS-based vitrification and encapsulation–vitrification protocols. In: Reed, B.M. (Ed.) Plant Cryopreservation: A Practical Guide. Springer, Berlin. pp. 33–58. Sakai, A., Matsumoto, T., Hirai, D., and Niino, T. 2000. Newly developed encapsulation-dehydration protocol for plant cryopreservation. CryoLetters 21: 53–62. Sakai, W. S., Adams, C., and Braun, G. 2000. Pseudo-bulb injected growth regulators as aids for year around production of Hawaiian Dendrobium orchid cut flowers. Acta Hort. (ISHS) 541: 215–220. Salas, M. R., Ranilla, J. B., and Espinoza, J. R. 2007. Genetic relationships of Phragmipedium species (Orchidaceae) using amplified fragment length polymorphism (AFLP) analysis. Lankesteriana 7: 493–496. Salazar, O., Julian, M. C., Yacumachi, M., and Rubio, V. 2000. Phylogenetic grouping of cultural types of Rhizoctonia solani AG 2–2 based on ribosomal ITS sequences. Mycologia 92: 505–509. Saleem, U. 2007. Investigating the medicinal properties of orchids. Columbia Sci. Rev. 4: 22–23.


ORCHIDS AND BIOTECHNOLOGY Sandoval, J., Kerbellec, F., Côte, F., and Doumas, P. 1995. Distribution of endogenous gibberellins in dwarf and giant off-types banana (Musa AAA cv. ‘Grand Nain’) plants from in vitro propagation. Plant Growth Regul. 17: 219–224. Sanguthai, O., Sanguthai, S., and Kamemoto, H. 1973. Chromosome doubling of a Dendrobium hybrid with colchicine in meristem culture. Hawaii Orchid J. 2: 12–16. Sanguthai, S., and Sagawa, Y. 1973. Induction of polyploidy in Vanda by colchicine treatment. Hawaii Orchid J. 2: 7–19. Saritha, K. V., and Naidu, C. V. 2008. Direct shoot regeneration from leaf explants of Spilanthes acmella. Biol. Plant. 52: 334–338. Seaton, P. T., and Hailes, N.S.J. 1989. Effect of temperature and moisture content on the viability of Cattleya aurantiaca seed. In: Modern Methods in Orchid Conservation: The Role of Physiology, Ecology and Management, pp. 17–29. Pritchard, H. W., Ed. Cambridge University Press, Cambridge. Seaton, P. T., Hu, H., Perner, H., and Pritchard, H. W. 2010. Ex situ conservation of orchids in a warming world. Bot. Rev. 76: 193–203. Selosse, M. A., Boullard, B., and Richardson, D. 2011. Noël Bernard (1874–1911): orchids to symbiosis in a dozen years, one century ago. Symbiosis 54: 61–68. Semiarti, E., Indrianto, A., Purwantoro, A., Isminingsih, S., Suseno, N., Ishikawa, T., Yoshioka, Y., Machida, Y., and Machida, C. 2007. Agrobacterium-mediated transformation of the wild orchid species Phalaenopsis amabilis. Plant Biotechnol. 24: 265–272. Sen, R., Hietala, A. M., and Zelmer, C. D. 1999. Common anastomosis and internal transcribed spacer RFLP groupings in binucleate Rhizoctonia isolates representing root endophytes of Pinus sylvestris, Ceratorhiza spp. from orchid mycorrhizas and a phytopathogenic anastomosis group. New Phytol. 144: 331–341. Seoh, M. L., Wong, S. M., and Zhang, L. 1998. Simultaneous TD/RT-PCR detection of Cymbidium mosaic potexvirus and Odontoglossum ringspot tobamovirus with a single pair of primers. J. Virol. Methods 72: 197–204. Seth, C. J., and Cribb, P. J. 1984. A reassessment of the sectional limits in the genus Cymbidium Swartz. In: Orchids Biology—Reviews and Perspectives, III, pp. 283–321. Arditti, J., Ed. Cornell University Press, London. Shamrov, I. I., and Nikiticheva, Z. I. 1992. Morphogenesis of ovule and seed in Gymnadenia conopsea (Orchidaceae): structural and histochemical investigation. Botanicheskii Zhurnal USSR 77: 45–60 (in Russian). Shan, X.C.E., Liew, C. Y., Weatherhead, M. A., and Hodgkiss, I. J. 2002. Characterization and taxonomic placement of Rhizoctonia-like endophytes from orchid roots. Mycologia 94: 230–239. Sharma, A. K., and Chatterji, A. K. 1966. Cytological studies on orchids with respect to their evolution and affinities. Nucleus 9: 177–203. Sharon, M., Kuninaga, S., Hyakumachi, M., Naito, S., and Sneh, B. 2008. Classification of Rhizoctonia spp. using rDNA-ITS sequence analysis supports the genetic basis of the classical anastomosis grouping. Mycoscience 49: 93–114. Sheelavanthmath, S. S., Murthy, H. N., Hema, B. P., Hahn, E. J., and Paek, K. Y. 2005. High frequency of protocorm like bodies (PLBs) induction and plant regeneration from protocorms and leaf sections of Aerides crispum. Sci. Hortic. 106: 395–401. Sheelavanthmath, S. S., Murthy, H. N., Pyati, A. N., Kumar, H. G. A., and Ravishankar, B. V. 2000. In vitro propagation of the endangered orchid, Geodorum densiflorum (Lam.) Schltr. through rhizome section culture. Plant Cell, Tiss. Organ Cult. 60: 151–154. Shefferson, R. P., L Weiß, M., Kull, T., and Taylor, L. 2005. High specificity generally characterizes mycorrhizal association in rare lady’s slipper orchids, genus Cypripedium. Mol. Ecol. 14: 613–626. Sheorey, R. R., and Tiwari, A. 2011. Random amplified polymorphic DNA (RAPD) for identification of herbal materials and medicines – A review. J. Scientific Indust. Res. 70: 319–326. Sherpa, A. R., Bag, T. K., Hallan, V., and Zaidi, A. A. 2006. Detection of Odontoglossum ringspot virus in orchids from Sikkim, India. Aust. Plant Pathol. 35: 69–71.

135

Sherpa, A. R., Hallan, V., Pathak P., and Zaidi, A. A. 2007. Complete nucleotide sequence analysis of Cymbidium mosaic virus Indian isolate: further evidence for natural recombination among potexviruses. J. Biosci. 32: 1–7. Shiau, Y. J., Nalawade, S. M., Hsai, C. N., and Tsay, H. S. 2005. Propagation of Haemaria discolor via in vitro seed germination. Biol. Plant. 49: 341–346. Shimasaki, K., and Uemoto, S. 1990. Micropropagation of a terrestrial Cymbidium species using rhizomes developed from seeds and pseudobulbs. Plant Cell, Tiss. Organ Cult. 22: 237–244. Shrestha, B. R., Chin, D.P., Tokuhara, K., and Mii, M. 2010. Agrobacteriummediated transformation of Vanda using protocorm-like bodies. AsPac J. Mol. Biol. Biotechnol. 18: 225–228. Shrestha, B. R., Tokuhara, K., and Mii, M. 2007. Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis. Plant Cell Rep. 26: 719–725. Sim, G. E., Goh, C. J., and Loh, C. S. 2008. Induction of in vitro flowering in Dendrobium Madame Thong-In (Orchidaceae) seedlings is associated with increase in endogenous N6-(2-isopentenyl)-adenine (iP) and N6-(2isopentenyl)-adenosine (iPA) levels. Plant Cell Rep. 27: 1281–1289. Sim, G. E., Loh, C. S., and Goh, C. J. 2007. High frequency early in vitro flowering of Dendrobium Madame Thong-In (Orchidaceae). Plant Cell Rep. 26: 383–393. Singer, R. B., Breier, T. B., Flach, A., and Farias-Singer, R. 2007. The pollination mechanism of Habenaria pleiophylla Hoehne & Schlechter (Orchidaceae: Orchidinae). Func. Ecosystems Communities 1: 10–14. Singh, M. K., Sherpa, A. R., Hallan, V., and Zaidi, A. A. 2007. A potyvirus in Cymbidium spp. in northern India. Aus. Plant Dis. Notes 2: 11–13. Sjahril, R., Chin, D. P., Khan, R. S., Yamamura, S., Nakamura, I., Amemiya, Y., and Mii, M. 2006. Transgenic Phalaenopsis plants with resistance to Erwinia carotovora produced by introducing wasabi defensin gene using Agrobacterium method. Plant Biotech. 23: 191–194. Sjahril, R., and Mii, M. 2006. High-efficiency Agrobacterium-mediated transformation of Phalaenopsis using meropenem, a novel antibiotic to eliminate Agrobacterium. J. Hortic. Sci. Biotech. 81: 458–464. Skipper, M., Pedersen, K. B., Johansen, L. B., Frederiksen, S., Irish, V. F., and Johansen, B. B. 2005. Identification and quantification of expression levels of three FRUITFULL-like MADS-box genes from the orchid Dendrobium thyrsiflorum (Reichb. f.). Plant Sci. 169: 579–586. Song, J. Y., and Guo, S. X. 2001. Effects of fungus on the growth of Dendrobium candidum and D. nobile in vitro culture. Acta Academic Med. Sinicae 23 (6): 547–551. Sotthikul, C., Choomporn, P., Kammuen, S., and Suwanthada, C. 2010. Effects of some cytokinins, auxins and medium constituents on in vitro propaga-tion of Polystachya sp. AsPac J. Mol. Biol. Biotechnol. 18: 111–114. State Pharmacopeia Committee of China. 2010. Pharmacopoeia of The People’s Republic of China Vol. I, Beijing. Chinese Medical Science and Technology Press. Sreeramanan, S., Vinod, B., Ranjetta, P., and Rathinam, X. 2009. Chemotaxis movement and attachment of Agrobacterium tumefaciens to Phalaenopsis violacea orchid tissues: an assessment of early factors influencing the efficiency of gene transfer. J. Biosci. 20: 43–54. Steward, F. C., and Mapes, M. O. 1971. Morphogenesis in aseptic cell culture of Cymbidium. Bot. Gaz. 132: 65–70. Su, W. R., Chen, W. S., Koshioka, M., Mander, L. N., and Hung, L. S. 2001. Changes in gibberellin levels in the flowering shoot of Phalaenopsis hybrida under high temperature conditions when flower development is blocked. Plant Physiol. Biotech. 39: 45–50. Su, Y. J., Chen, J. T., and Chang, W. C. 2006. Efficient and repetitive production of leaf-derived somatic embryos of Oncidium. Biol. Plant. 50: 107–110. Sudhakaran, S., Teixeira da Silva, J. A., and Sreeramanan, S. 2006. Test tube bouquets—in vitro flowering. In: Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues (1st Ed., Vol II), Chapter 45, pp. 336–346. Teixeira da Silva, J. A., Ed. Global Science Books, Ltd., Isleworth, UK.


136


Sujjaritthurakarn, P., and Kanchanapoom, K. 2011. Efficient direct protocormlike bodies induction of dwarf Dendrobium using thidiazuron. Not. Sci. Biol. 3: 88–92. Sun, M., and Wong, K. C. 2001. Genetic structure of three orchid species with contrasting breeding systems using RAPD and allozyme markers. Amer. J. Bot. 88: 2180–2188. Sun, Y. W., Liao, Y. J., Hung, Y. S, Chang, J. C., and Sung, J. M. 2011. Development of ITS sequence based SCAR markers for discrimination of Paphiopedilum armeniacum, Paphiopedilum micranthum, Paphiopedilum delenatii and their hybrids. Sci. Hortic. 127: 405–410 Suresh Kumar, P. K., Subramoniam, A., and Pushpangadan, P. 2000. Aphrodisiac activity of Vanda tessellata (Roxb) hook; Ex Don extract in male mice. Indian J Pharmacol. 32: 300–304. Suwanaketchanatit, C., Piluek, J., Peyachoknagul, S., and Huehne, P. S. 2007. High efficiency of stable genetic transformation in Dendrobium via microprojectile bombardment. Biol. Plant. 51: 720–727. Swamy, B. G. L. 1943. Female gametophyte and embryogeny in Cymbidium bicolor Lindl. Proc. Indian Acad. Sci. Section B 15: 194–201. Swamy, B. L. G. 1946. Embryology of Habenaria. Proc. Indian Acad. Sci. Section B 12: 413–426. Swamy, B. C. L. 1949a. Embryological studies in the Orchidaceae II. Embryogenie. Amer. Midl. Nat. 41: 202–232. Swamy, B. C. L. 1949b. Embryological studies in the Orchidaceae I. Gametophytes. Amer. Midl. Nat. 41: 184–201. Swarts, N. D, and Dixon, K. W. 2009. Terrestrial orchid conservation in the age of extinction. Ann. Bot. 104: 543–556. Tahara, M., and Kato, M. 1987. Polyploidy and hybridization in Habenaria and Calanthe. In: Proceedings of the World Orchid Hiroshima Symposium, pp. 79–82. Takamiya, T., Wongsawad, P., Tajima, N., Shioda, N., Lu, J. F., Wen, C. L., Wu, J. B., Handa, T., Iijima, H., Kitanaka, S., and Yukawa, T. 2011. Identification of Dendrobium species used for herbal medicines based on ribosomal DNA internal transcribed spacer sequence. Biol. Pharm. Bull. 34: 779–782. Takayama, S., and Akita, M. 1994. The types bioreactors used for shoots and embryos. Plant Cell, Tiss. Organ Cult. 39: 147–156. Takayama, S., and Akita, M. 1998. Bioreactor techniques for large-scale culture of plant propagules. Adv. Hort. Sci. 12: 93–100. Takayama, S., and Misawa, M. 1981. Mass propagation of Begonia x Hiemalis plantlets by shake culture. Plant Cell Physiol. 22: 461–467. Tan, B. C., Chin, C. F., and Alderson, P. 2011. Optimisation of plantlet regeneration from leaf and nodal derived callus of Vanilla planifolia Andrews. Plant Cell, Tiss. Organ Cult. 105: 457–463. Tan, S. W., Wong, S. M., and Kini, R. M. 2000. Rapid simultaneous detection of two orchid viruses using LC and/or MALDI-mass spectrometry. J. Virol. Methods 85: 93–99. Tan, X. M., Chen, X. M., Wang, C. L., Jin, X. H, Cui, J. L., Chen, J., Guo, S. X., and Zhao, L. F. 2012. Isolation and identification of endophytic fungi in roots of nine Holcoglossum plants (Orchidaceae) collected from Yunnan, Guangxi, and Hainan Provinces of China. Curr. Microbiol. 64: 140–147. Tanaka, M., Van P. T., Teixeira da Silva, J. A., and Ham, L. H. 2010. Novel magnetic field system: application to micropropagation of horticultural plants. Biotechnol. Biotechnol. Equipment 24: 2160–2163. Tanaka, R., and Kamemoto, H. 1961. Meiotic chromosome behaviour in some intergeneric hybrids of the Vanda alliance. Amer. J. Bot. 48: 573–582. Tanaka, S., Nishii, H., Ito, S., Kameya-Iwaki, M., and Sommartya, P. 1997. Detection of Cymbidium mosaic potexvirus and Odontoglossum ringspot tobamovirus from Thai orchids by rapid immunofilter paper assay. Plant Dis. 81: 167–170. Tang, C. Y., and Chen, W. H. 2007. Breeding and development of new varieties in Phalaenopsis. In: Orchid Biotechnology, pp. 1–22. Chen, W. H., and Chen, H. H., Eds. World Scientific, Hong Kong.

Taylor, D. L., and McCormick, M. K. 2008. Internal transcribed spacer primers and sequences for improved characterization of basidiomycetous orchid mycorrhizas. New Phytol. 177: 1020–1033. Taylor, L. D., and Bruns, T. D. 1997. Independent, specialized invasion of ectomycorrhizal mutualism by two nonphotosynthetic orchids. Proc. Nat. Acad. Sci. USA 94: 4510–4515. Te-chato, S., Nujeen, P., and Muangsorn, S. 2009. Paclobutrazol enhance budbreak and flowering of Friederick’s Dendrobium orchid in vitro. J. Agric. Technol. 5: 157–165. Tee, C. S., Lee, P. S., Kiong, A. L. P., and Mahmood, M. 2011. Optimisation of protoplast isolation protocols using in vitro leaves of Dendrobium crumenatum (pigeon orchid). Afr. J. Agric. Res. 5: 2685–2693. Tee, C. S., Marziah, M., Tan, C. S., and Abdullah, M. P. 2003. Evaluation of different promoters driving the GFP reporter gene and selected target tissues for particle bombardment of Dendrobium Sonia 17. Plant Cell Rep. 21: 452–458. Tee, C. S., and Maziah, M. 2005. Optimization of biolistic bombardment for Dendrobium Sonia 17 calluses using GFP and GUS as the reporter system. Plant Cell, Tiss. Organ Cult. 80: 77–89. Tee, C. S., Maziah, M., and Tan, C. S. 2008. Induction of in vitro flowering in the orchid Dendrobium Sonia 17. Biol. Plant. 52: 723–726. Tee, C. S., Maziah, M., Tan, C. S., and Abdullah, M. P. 2011. Selection of cotransformed Dendrobium Sonia 17 using hygromycin and green fluorescent protein. Biol. Plant. 55: 572–576. Teixeira da Silva, J. A. 2003. Thin Cell Layer technology in ornamental plant micropropagation and biotechnology. Afr. J. Biotechnol. 2: 683–691. Teixeira da Silva, J. A. 2004. Chrysanthemum: ex vitro to in vitro to ex vitro. Acta Hortic. 616: 443–447. Teixeira da Silva, J. A., Chan, M. T., Sanjaya, Chai, M. L., and Tanaka, M. 2006a. Priming abiotic factors for optimal hybrid Cymbidium (Orchidaceae) PLB and callus induction, plantlet formation, and their subsequent cytogenetic stability analysis. Sci. Hortic. 109: 368–378. Teixeira da Silva, J. A., Chin, D. P., Van, P. T., and Mii, M. 2011. Transgenic orchids. Sci. Hortic. 130 (4): 673–680. Teixeira da Silva, J. A., and Dobránszki, J. 2011. The plant Growth Correction Factor. I. The hypothetical and philosophical basis. Intl. J. Plant Dev. Biol. 5: 73–74. Teixeira da Silva, J. A., Giang, D. T. T., Chan, M. T., Sanjaya, Norikane, A., Chai, M. L., Chico-Ru´ız, J., Penna, S., Granström, T., and Tanaka, M. 2007a. The influence of different carbon sources, photohetero-, photoautoand photomixotrophic conditions on protocorm-like body organogenesis and callus formation in thin cell layer culture of hybrid Cymbidium (Orchidaceae). Orchid Sci. Biotechnol. 1: 15–23. Teixeira da Silva, J. A., Giang, D. T. T., and Tanaka, M. 2005a. In vitro acclimatization of banana and Cymbidium. Intl. J. Bot. 1: 41–49. Teixeira da Silva, J. A., Norikane, A., and Tanaka, M. 2007b. Cymbidium: successful in vitro growth and subsequent acclimatization. Acta Hort. 748: 207–214. Teixeira da Silva, J. A., Singh, N., and Tanaka, M. 2006b. Priming biotic factors for optimal protocorm-like body and callus induction in hybrid Cymbidium (Orchidaceae), and assessment of cytogenetic stability in regenerated plantlets. Plant Cell Tiss. Organ Cult. 84: 135–144. Teixeira da Silva, J. A., and Tanaka, M. 2006. Multiple regeneration pathways via thin cell layers in hybrid Cymbidium (Orchidaceae). J. Plant Growth Regul. 25: 203–210. Teixeira da Silva, J. A., Tanaka, M. 2009a. Impact of gelling agent and alternative medium additives on hybrid Cymbidium protocorm-like body and callus formation. Floriculture Ornamental Biotechnol. 3: 56–58. Teixeira da Silva, J. A., and Tanaka, M. 2009b. Optimization of particle bombardment conditions for hybrid Cymbidium. Transgenic Plant J. 3 (Special Issue 1): 119–122. Teixeira da Silva, J. A., and Tanaka, M. 2011. Optimization of particle bombardment conditions for hybrid Cymbidium: Part II. Transgenic Plant J. 5 (1): 78–82.


ORCHIDS AND BIOTECHNOLOGY Teixeira da Silva, J. A., Yam, T., Fukai, S., Nayak, N., and Tanaka, M. 2005b. Establishment of optimum nutrient media for in vitro propagation of Cymbidium Sw. (Orchidaceae) using protocorm-like body segments. Prop. Ornam. Plants 5: 129–136. Teng, W. L., Nicholson, L., and Teng, M. C. 1997. Micropropagation of Spathoglottis plicata. Plant Cell Rep. 16: 831–835. Teo, C. K. H., and Neumann, K. H. 1978a. The culture of protoplasts isolated from Renantanda Rosalind Cheok (J). Orchid Rev. 86: 156–158. Teo, C. K. H., and Neumann, K. H. 1978b. The isolation and hybridization of protoplasts from orchids. Orchid Rev. 86: 186–189. Teoh, S. B. 1986. A breeding program for the development of seed propagated uniform hybrid allopolyploid Aranda cultivars for cut flowers. Malay Orchid Rev. 20: 42–45. Thammasiri, K., and Soamkul, L. 2007. Cryopreservation of Vanda coerulea Griff. ex Lindl. seeds by vitrification. Sci. Asia 33: 223–227. Than, M. M. M., Pal, A., and Jha, S. 2011. Chromosome number and modal karyotype in a polysomatic endangered orchid, Bulbophyllum auricomum Lindl., the royal flower of Myanmar. Plant Syst. Evol. 294: 167–175. Theissen, G., Becker, A., Di-Rosa, A., Kanno, A., Kim, J. T., and Munster, T. 2000. A short history of MADS-box genes in plants. Plant Mol. Biol. 42: 115–149. Thiruvengadam, M., Chung, I. M., and Yang, C. H. 2012. Overexpression of Oncidium MADS box (OMADS1) gene promotes early flowering in transgenic orchid (Oncidium Gower Ramsey). Acta Physiol. Plant. 34(4): 1295–1302. Tian, T., Klaasen, V. A., Soong, J., Wisler, G., Duffus, J. E., and Falk, B. W. 1996. Generation of cDNAs specific to lettuce infectious yellows closterovirus and other whitefly-transmitted viruses by RT-PCR and degenerate oligonucleotide primers corresponding to the closterovirus gene encoding the heat shock protein. Phytopathology 86: 1167–1173. Tohda, H. 1971. Development of the embryo of Lecanorchis japonica. Sci. Rep. Tohoku Uni. Ser. IV (Biol.) 35: 245–251. Tokuhara, K., and Mii, M. 1993. Micropropagation of Phalaenopsis and Doritaenopsis by culturing shoot tips of flower stalk buds. Plant Cell Rep. 13: 7–11. Tokuhara, K., and Mii, M. 2001. Induction of embryogenic callus and cell suspension culture from shoot tips excised from flower stalk buds in Phalaenopsis (Orchidaceae). In Vitro Cell. Dev. Biol. – Plant 37: 457–461. Tokuhara, K., and Mii, M. 2003. Highly-efficient somatic embryogenesis from cell suspension cultures of Phalaenopsis orchids by adjusting carbohydrate sources. In Vitro Cell. Dev. Biol. – Plant 39: 635–639. Tsai, C. C., Peng, C. I., Huang, S. C., Huang, P. L., and Chou, C. H. 2004a. Determination of the genetic relationship of Dendrobium species (Orchidaceae) in Taiwan based on the sequence of the internal transcribed spacer of ribosomal DNA. Sci. Hortic. 101: 315–325. Tsai, S. F., Yeh, S. D., Chan, C. F., and Liaw, S. I. 2009. High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines. Plant Cell, Tiss. Organ Cult. 98: 157–164. Tsai, W. C., Chuang, M. H., Kuoh, C. S., Chen, W. H., and Chen, H. H. 2004b. Four DEF-like MADS box genes displayed distinct floral morphogenetic roles in Phalaenopsis orchid. Plant Cell Physiol. 45: 831–844. Tsai, W. C., Hsiao Y. Y., Pan Z. J., and Chen, H. H. 2007a. Analysis of expression of Phalaenopsis floral ESTs. In: Chen, W. H., Chen, H. H., (Eds.) Orchid Biotechnology, World Scientific, Hong Kong. pp. 145–161. Tsai, W. C., Hsiao, Y. Y., Pan, Z. J., Hsu, C. C., Yang, Y. P., and Chen, W. H. 2008a. Molecular biology of orchid flower - with emphasis on Phalaenopsis. Adv. Bot. Res. 47: 99–145. Tsai, W. C., Lee, P. F., Chen, H. I., Hsiao, Y. Y., Wei, W. J., and Pan, Z. J. 2005. PeMADS6, a GLOBOSA/PISTILLATA-like gene in Phalaenopsis equestris involved in petaloid formation, and correlated with flower longevity and ovary development. Plant Cell Physiol. 46: 1125–1139. Tsai, W. C., Lin, C. W., Kuoh, C. S., and Chen, H. H. 2007b. Orchid MADSbox genes controlling floral morphogenesis. In: Orchid Biotechnology, pp. 163–183. Chen, W. H., and Chen, H. H., Eds. World Scientific, Hong Kong.

137

Tsai, W. C., Pan, Z. J., Hsiao, Y. Y., Jeng, M. F., Wu, T. F., and Chen, W. H. 2008b. Interactions of B-class complex proteins involved in tepal development in Phalaenopsis orchid. Plant Cell Physiol. 49: 814–824. Tsi, Z. H., Chen, S. C., Luo, Y. B., and Zhu, G. H. 1999. Orchidaceae (3). In: Angiospermae, Monocotyledoneae, Flora Reipublicae Popularis Sinica, pp. 19–33. Tsi, Z. H., Ed. Science Press, Beijing. Tsukazaki, H., Mii, M., Tokuhara, K., and Ishikawa, K. 2000. Cryopreservation of Doritaenopsis suspension culture by vitrification. Plant Cell Rep. 19: 1160–1164. Tymoszuk, J., Saniewski, M., and Rudnicki, R. M. 1979. The physiology of Hyacinth bulbs. 15. The effect of gibberellic acid and silver nitrate on dormancy release and growth. Sci. Hortic. 11: 95–100. USDA., 2009. Floriculture crops 2008 summary. National Agricultural Statistics Service, Washington, DC. Vacin, E., and Went, F. W. 1949. Some pH changes in nutrient solutions. Bot. Gaz. 110: 605–613. van den Berg, C., Goldman, D. H., Freudenstein, J. V., Pridgeon, A. M., Cameron, K. M., and Chase, M. W. 2005. An overview of the phylogenetic relationships within Epidendroideae inferred from multiple DNA regions and recircumscription of Epidendreae and Arethuseae (Orchidaceae). Amer. J. Bot. 92, 613–624. van den Berg, C., Higgins, W. E., Dressler, R. L., Whitten, W. M., Soto-Arenas, M. A., and Chase, M. W. 2009. A phylogenetic study of Laeliinae (Orchidaceae) based on combined nuclear and plastid DNA sequences. Ann. Bot. 104, 417–430. Van, P. T., Tanaka, M., and Teixeira da Silva, J. A. 2010. Gelling agent affects hybrid Cymbidium plantlet growth. Floriculture Ornamental Biotechnol. 4 (Special Issue 1): 45–47. Van, P. T., Teixeira da Silva, J. A., Ham, L .H., and Tanaka, M. 2011a. The effects of permanent magnetic fields on Phalaenopsis plantlet development. J. Hortic. Sci. Biotech. 86: 473–478. Van, P. T., Teixeira da Silva, J. A., and Tanaka, M. 2011b. Study on the effects of permanent magnetic fields on the proliferation Phalaenopsis protocorm-like bodies using liquid medium. Sci. Hortic. 128: 479–484. Vasudevan, R., and van Staden, J. 2011. Cytokinin and explant types influence in vitro plant regeneration of Leopard orchid (Ansellia africana Lindl.). Plant Cell, Tiss. Organ Cult. 107: 123–129. Vaughan, S. P., Grisoni, M., Kumagai, M. H., and Kuehnle, A. R. 2008. Characterization of Hawaiian isolates of Cymbidium mosaic virus (CymMV) coinfecting Dendrobium orchid. Arch. Virol. 153: 1185–1189. Vaz, A. P. A., and Kerbauy, G. B. 2008. In vitro precocious orchid flowering: a strategy for basic research and commercial approaches. In: Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues (1st Ed., Vol. V), Chapter 45, pp. 421–426. Teixeira da Silva, J. A., Ed. Global Science Books, Ltd., Isleworth, UK. Vaz, A. P. A., Figueiredo-Ribeiro, R. C. L., and Kerbauy, G. B. 2004. Photoperiod and temperature effects on in vitro growth and flowering of P. pusilla an epiphytic orchid. Plant Physiol. Biochem. 42: 411–415 Veyret, Y. 1974. Development of the embryo and the young seedling stages of orchids. In: The Orchids: Scientific Studies, pp. 223–265. Withner, C. L., Ed. John Wiley & Sons, New York. Vij, S. P., Pathak, P., and Sharma, M. 1987. On the regeneration potential of Rhynchostylis retusa: A study in vitro. J. Orchid Soc. India 1: 71–74. Vogler, A. P., and Monaghan, M. T. 2007. Recent advances in DNA taxonomy. J. Zool. Syst. Evol. Res. 45: 1–10. Wang, C., Sun, J., Luo, Y., Xue, W., Diao, H., Dong, L., Chen, J., and Zhang, J. 2006. A polysaccharide isolated from the medicinal herb Bletilla striata induces endothelial cells proliferation and vascular endothelial growth factor expression in vitro. Biotechnol. Lett. 28: 539–543. Wang, G. Y., Liu, P., Xu, Z. H., and Chua, N. H. 1995. Effect of ABA on the in vitro production of flower buds of Dendrobium candidum Wall. Ex Lindl. Acta Bot. Sin. 37: 374–378. Wang, G. Y., Xu, Z. H., Chia, T. F., and Chua, N. H. 1997. In vitro flowering of Dendrobium candidum. Sci. China (Ser. C) 40: 35–42.


138


Wang, H., Fang, H., Wang, Y., Duan, L., and Guo, S. 2011a. In situ seed baiting techniques in Dendrobium officinale Kimuraet Migo and Dendrobium nobile Lindl.: the endangered Chinese endemic Dendrobium (Orchidaceae). World J. Microbiol. Biotechnol. 27: 2051–2059. Wang, H. Z., Feng, S. G., Lu, J. J., Shi, N. N., and Liu, J. J. 2009b. Phylogenetic study and molecular identification of 31 Dendrobium species using intersimple sequence repeat (ISSR) markers. Sci. Hortic. 122: 440–447. Wang, J. H., Ge, J. G., Liu, F., Bian, H. W., and Huang, C. N. 1998. Cryopreservation of seeds and protocorms of Dendrobium candidum. CryoLetters 19: 123–128. Wang, S. Y., Lee, P. F., Lee, Y. I., Hsiao, Y. Y., Chen, Y. Y., and Pan, Z. J. 2011b. Duplicated C-class MADS-box genes reveal distinct roles in gynostemium development in Cymbidium ensifolium (Orchidaceae). Plant Cell Physiol. 52: 563–577. Wang, X. 1988. Tissue culture of Cymbidium: plant and flower induction in vitro. Lindleyana 3: 184–189. Wang, X., Chen, J. C., Liu, G. Y., Gu, M. X., and Boa, C. H. 1981. Clonal propagation of orchids by means of tissue culture. Acta Phytophysiol. Sin. 7: 203–207. Wang, X., Zhang, J. Y., Lian, K., Gong, S. F., and Jin, Y. R. 1988. Studies on Cymbidium ensifolium Susin clonal propagation and flower bud differentiation by means of tissue culture. Acta Hortic. Sin. 15: 205–208. Wang, Z. H., Wang, L., and Ye, Q. S. 2009. High frequency early flowering from in vitro seedlings of Dendrobium nobile. Sci. Hortic. 122: 328–331. Warcup, J. H., and Talbot, P.H.B. 1967. Perfect states of Rhizoctonias associated with orchids. New Phytol. 66: 631–641. Wang, M. G., Zeng, R. Z., Xie, L., Gao, X. H., and Zhang, Z. S. 2011. In vitro polyploid induction and its identification in Cymbidium sinense. Chin. Agric. Sci. Bull. 27 (2): 132–136. Watrous, S. B., and Wimber, D. E. 1988. Artificial induction of polyploidy in Paphiopedilum. Lindleyana 3: 177–183. Wattanawikkit, P., Bunn, E., Chayanarit, K., and Tantiwiwat, S. 2011. Effect of cytokinins (BAP and TDZ) and auxin (2,4-D) on growth and development of Paphiopedilum callosum. Kasetsart J. (Nat. Sci.) 45, 12–19. Weising, K., Nybom, H., Wolff, K., and Kahl, G. 2005. DNA Fingerprinting in Plants: Principles, Methods, and Applications. CRC Press, New York. Wen, Y. F., Lu, R. L., and Xie, Z. L. 1999. Rapid propagation and induction of floral buds of Dendrobium huoshanase. Plant Physiol. 35: 296–297. Whitten, W. M., Blanco, M. A., Williams, N. H., Koehler, S., Carnevali, G., Singer, R. B., Endara, L., and Neubig, K. M. 2007. Molecular phylogenetics of Maxillaria and related genera (Orchidaceae: Cymbidieae) based on combined molecular data sets. Amer. J. Bot. 94: 1860–1889. Wilson, C. A. 2004. Phylogeny of Iris based on chloroplast matK gene and trnK intron sequence data. Mol. Phylogenet. Evol. 33: 402–412. Winkelmann, T., Geier, T., and Preil, W. 2006. Commercial in vitro plant production in Germany in 1985–2004. Plant Cell Tiss. Organ Cult. 86: 319–327. Wong, S. M., and Chng, C. G. 1993. Evaluation of sample processing methods in detecting Cymbidium mosaic virus in orchids using indirect ELISA. Intl. J. Tropical Plant Dis. 11: 139–145. Wong, S. M., Chng, C. G., Lee, Y. H., Tan, K., and Zettler, F. W. 1994. Incidence of Cymbidium mosaic and Odontoglossum ringspot viruses and their significance in orchid cultivation in Singapore. Crop Prot. 13: 235–239. Wright, M. M., Cross, R., Cousens, R. D., May, T. W., and McLean, C. B. 2010. Taxonomic and functional characterization of fungi from the Sebacina vermifera complex from common and rare orchids in the genus Caladenia. Mycorrhiza 20: 375–390. Wu, B, He, S., and Pan, Y. J. 2006. New dihydrodibenzoxepins from Bulbophyllum kwangtungense. Planta Medica 72: 1244–1247. Wu, C. H., Li, D. M., Liang, C. H., and Ye, X. L. 2005. Ultrastructural observations on megasporogenesis in Doritis pulcherrima (Orchidaceae). J. Trop. Subtrop. Bot. 13 (1): 45–48. Wu, F. H., Chan, M. T., Liao, D. C., Hsu, C. T., Lee, Y. W., Daniell, H., Duvall, M. R., and Lin, C. S. 2010. RComplete chloroplast genome of Oncidium

Gower Ramsey and evaluation of molecular markers for identification and breeding in Oncidiinae. BMC Plant Biol. 10: 68. Wu, J., Ma, H., Lu, M., Han, S., Zhu, Y., Jin, H., Liang, J., Liu, L., and Xu, J. 2010. Rhizoctonia fungi enhance the growth of the endangered orchid Cymbidium goeringii. Botany 88: 20–29. Wu, J. Y., Hu, T., Yang, S. Z., Liu, L., Wang, Q., Wang, T., and Li, L. B. 2009. rDNA ITS analysis and preliminary study in the specificity for the symbiotic mycorrhizal fungi of Cymbidium goeringii and C. faberi. Ecol. Sci. 28 (2): 134–138. Wu, K. L., Zeng, S. J., Teixeira da Silva, J. A., Chen, Z. L., Zhang, J. X., Yang, Y. S., and Duan, J. 2012. Efficient regeneration of Renanthera Tom Thumb ‘Qilin’ from leaf explants. Sci. Hortic. 135 (1): 194–201. Wu, P. H., Huang, D. D., and Chang, D. C. N., 2011. Mycorrhizal symbiosis enhances Phalaenopsis orchid’s growth and resistance to Erwinia chrysanthemi. Afr. J. Biotechnol. 10, 10095–10100. Xu, H., Wang, Z., Ding, X., Zhou, K., and Xu, L. 2006a. Differentiation of Dendrobium species used as “Huangcao Shihu” by rDNA ITS sequence analysis. Planta Medica 72: 89–92. Xu, J. T., and Guo, S. X. 1991. The relationship of nutrient between Gastrodia elata -Mycena osmundicola and Armillaria mella and its application in the cultivation. J. Med. Res. 1: 31–32. Xu, J., Yu, H., Qing, C., Zhang, Y., Liu, Y., and Chen, Y. 2009. Two new biphenanthrenes with cytotoxic activity from Bulbophyllum odoratissimum. Fitoterapia 80: 381–384. Xu, Y., Teo, L. L., Zhou, J., Kumar, P. P., and Yu, H. 2006b. Floral organ identity genes in the orchid Dendrobium crumenatum. Plant J. 46: 54–68. Xu, Y., Yu, H., and Kumar, P. P. 2010. Characterization of floral organ identity genes of the orchid Dendrobium crumenatum. AsPac J. Mol. Biol. Biotechnol. 18: 185–187. Yagame, T., Fukiharu, T., Yamato, M., Suzuki, A., and Iwase, K. 2008. Identification of a mycorrhizal fungus in Epipogium roseum (Orchidaceae) from morphological characteristics of basidiomata. Mycoscience 49: 147–151. Yagame, T., Orihara, T., Selosse, M. A., Yamato, M., and Iwase, K. 2012. Mixotrophy of Platanthera minor, an orchid associated with ectomycorrhizaforming Ceratobasidiaceae fungi. New Phytol. 193: 178–187. Yam, T. W., Chua, J., Tay, F., and Ang, P. 2010. Conservation of the native orchids through seedling culture and reintroduction – a Singapore experience. Bot. Rev. 76: 263–274. Yam, T. W., and Arditti, J. 2009. History of orchid propagation: a mirror of the history of biotechnology. Plant Biotechnol. Rep. 3: 1–56. Yamaki, M., and Honda, C. 1996. The stilbenoids from Dendrobium plicatile. Phytochemistry 43: 207–208. Yamato, M., and Iwase, K. 2008. Introduction of asymbiotically propagated seedlings of Cephalanthera falcata (Orchidaceae) into natural habitat and investigation of colonized mycorrhizal fungi. Ecol. Res. 23: 329–337. Yamato, M., Yagame, T., Suzuki, A., and Iwase, K. 2005. Isolation and identification of mycorrhizal fungi associating with an achlorophyllous plant, Epipogium roseum (Orchidaceae). Mycoscience 46: 73–77. Yan, J. 2012. Ginseng research in the era of systems biology. Intl. J. Biomedical Pharma. Sci. 6 (Special Issue 1): 1–124. Yan, N., Hu, H., Huang, J. L., Xu, K., Wang, H., and Zhou, Z. K. 2006. Micropropagation of Cypripedium flavum through multiple shoots of seedlings derived from mature seeds. Plant Cell, Tiss. Organ Cult. 84: 113–117. Yang A. F., Su, L. J., and An, L. J. 2009. Ovary-drip transformation: a simple method for directly generating vector- and marker-free transgenic maize (Zea mays L.) with a linear GFP cassette transformation. Planta 229: 793–801. Yang, J., Lee, H. J., Shin, D. H., Oh, S. K., Seon, J. H., Paek, K. Y., and Han, K. H. 1999. Genetic transformation of Cymbidium orchid by particle bombardment. Plant Cell Rep. 18: 978–984. Yang, J. F., Piao, X. C., Sun, D., and Lian, M. L. 2010. Production of protocormlike bodies with bioreactor and regeneration in vitro of Oncidium ‘Sugar Sweet’. Sci. Hortic. 125: 712–717. Yang, L., Qin, L. H., Bligh, S. W., Bashall, A., Zhang, C. F., Zhang, M., Wang, Z. T., and Xu, L. S. 2006. A new phenanthrene with a spirolactone from


ORCHIDS AND BIOTECHNOLOGY Dendrobium chrysanthum and its anti-inflammatory activities. Bioorg. Med. Chem. 14: 3496–3501. Yang, Y. L., Lai P. F., and Jiang, S. P. 2008. Research development in Dendrobium officinale. J. Shandong Univ TCM 32 (1): 82–85. Yao, H., Song, J. Y., Ma, X. Y., Liu, C., Li, Y., Xu, H. X., Han, J. P., Duan, L. S., and Chen, S. L. 2009. Identification of Dendrobium species by a candidate DNA barcode sequence: the chloroplast psbA-trnH intergenic region. Planta Medica 75: 667–669. Yap, M. L., Lam, L.T.C., Yik, C. P., Kueh, J., Lee, S. M., and Loh, M. 1999. Eradication of viruses from commercial orchids. Singapore J. Prim. Indust. 27: 11–19. Yee, N. C., Abdullah, J. O., Mahmood, M., and Basiron, N. 2008. Co-transfer of gfp, CHS and hptII genes into Oncidium Sharry Baby PLB using the biolistic gun. Afr. J. Biotechnol. 7: 2605–2617. Yeung, E C., Zee, S. Y., and Ye, X. L. 1996. Embryology of Cymbidium sinense: Embryo development. Ann. Bot. 78: 105–110. Yeung, E. C., and Meinke, D. W. 1993. Embryogenesis in angiosperms: development of the suspensor. Plant Cell 5: 1371–1381. Yeung, E. C., Zee, S. Y., and Ye, X. L. 1996. Embryology of Cymbidium sinense: Embryo development. Ann. Bot. 78: 105–110. Yin, C. C., Zhang, Y., Zhang, J. H., Chen, Y. Y., and Wang, G. D. 2010. Tetraploid induction by colchicine and indidentication in Cymbidium interspecific hybrids. Acta Agric. Nucl. Sin. 24 (3): 518–521. Yin, M. H., and Hong, S. R. 2009. Cryopreservation of Dendrobium candidum wall. ex Lindl. protocorm-like bodies by encapsulation vitrification. Plant Cell Tiss. Organ Cult. 98: 179–185. Yoon, J. Y., Chung, B. N., and Choi, S. K. 2011. High sequence conservation among Odotoglossum ringspot virus isolates from orchids. Virus Genes 42: 261–267. You, S. J., Liau, C. H., Huang, H. E., Feng, T. Y., Venkatesh, P., Hsiao, H. H., Lu, J. C., and Chan, M. T. 2003. Sweet pepper ferredoxin-like protein (pflp) gene as a novel selection marker for orchid transformation. Planta 217: 60–65. Young, P. S., Murthy, H. N., and Paek, K. Y. 2000. Mass multiplication of protocorm like bodies using bioreactor system and subsequent plant regeneration in Phalanopsis. Plant Cell Tiss. Organ Cult. 63: 67–72. Yu, H., and Goh, C. J. 2000. Identification and characterization of three orchid MADS-box genes of the AP1/AGL9 subfamily during floral transition. Plant Physiol. 123: 1325–1336. Yu, H., Yang, S. H., and Goh, C. J. 2001. Agrobacterium-mediated transformation of a Dendrobium orchid with the class 1 knox gene DOH1. Plant Cell Rep. 20: 301–305. Yu, S. J., Kim, J. R., Lee, C. K., Han, J. E., Lee, J. H., Kim, H. S., Hong, J. H., and Kang, S. G. 2005. Gastrodia elata blume and an active component, phydroxybenzyl alcohol reduces focal ischemic brain injury through antioxidant related gene expressions. Biol. Pharm. Bull. 28: 1016–1020. Yu, Z., Chen, M., Lin, N., Lu, H., Ming, X., Zheng, H., Qu, L. J., and Chen, Z. 1999. Recovery of transgenic orchid plants with hygromycin selection by particle bombardment to protocorms. Plant Cell, Tiss. Organ Cult. 58: 87–92. Yuan, Z. L., Chen, Y. C., and Yang, Y. 2009. Diverse non-mycorrhizal fungal endophytes inhabiting an epiphytic, medicinal orchid (Dendrobium nobile): estimation and characterization. World J. Microbiol. Biotechnol. 25: 295–303. Yuan, Z. Q., Zhang, J. Y., and Liu, T. 2009a. Phylogenetic relationship of China Dendrobium species based on the sequence of the internal transcribed spacer of ribosomal DNA. Biol. Plant. 53: 155–158.

139

Zeng, J., Zhang, L., Fan, X., Zhang, H. Q., Yang, R. W., and Zhou, Y. H. 2008. Phylogenetic analysis of Kengyilia species based on nuclear ribosomal DNA internal transcribed spacer sequences. Biol. Plant. 52: 231–236. Zeng, S. J., Chen, Z. L., Wu, K. L., Bai, C. K., Zhang, J. X., Teixeira da Silva, J. A., and Duan, J. 2011. Asymbiotic seed germination, induction of calli and protocorm-like bodies, and in vitro seedling development of the rare and endangered Nothodoritis zhejiangensis Chinese orchid. HortScience 46: 460–465. Zeng, S. J., Wu, K. L., Teixeira da Silva, J. A., Zhang, J. X., Chen, Z. L., Xia, N. H., and Duan, J. 2012. Asymbiotic seed germination, seedling development and reintroduction of Papiopedilum wardii Sumerh., an endangered terrestrial orchid. Sci. Hortic. 138: 198–209. Zhang, D. C., and Zhou, L. C. 2004. Effects of orchidaceous Rhizoctonia spp. on the seedlings of several orchids. Plant Seedling 6: 30–41. Zhang, L., Chen, J., Lv, Y., Gao, C., and Guo, S. 2012. Mycena sp., a mycorrhizal fungus of the orchid Dendrobium officinale. Mycol. Progress 11(2): 395–401. Zhang, M. S., and Yang, Y. H. 2008. Endophyte extracts in the improvement of Cremastra appendiculata (D.Don.) Makino (Orchidaceae) in vitro tissue culture and micropropagation. In: Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues (1st Ed., Vol. V), Chapter 45, pp. 433–437. Teixeira da Silva, J. A., Ed. Global Science Books, Ltd., Isleworth, UK. Zhang, X., Xu, J. K., Wang, J., Li, W.N., Kurihara, H., Kitanaka, S., and Yao, X. S. 2007. Bioactive bibenzyl derivatives and fluorenones from Dendrobium nobile. J. Nat. Prod. 70: 24–28. Zhao, J. N., and Liu, H. X. 2008. Effects of fungal elicitors on the protocorm of Cymbidium eburneum. Ecol. Sci. 27 (2): 134–137. Zhao, P., Wang, W., Feng, F. S., Wu, F., Yang, Z. Q., and Wang, W. J. 2007. High-frequency shoot regeneration through transverse thin cell layer culture in Dendrobium candidum Wall ex Lindl. Plant Cell, Tiss. Organ Cult. 90: 131–139. Zhao, P., Wu, F., Feng, F. S., and Wang, W. J. 2008. Protocorm-like body (PLB) formation and plant regeneration from the callus culture of Dendrobium candidum Wall ex Lindl. In Vitro Cell. Dev. Biol. – Plant 44: 178–185. Zheng B. Q., Zhang Y., Wang, Y., Li, Z. J., Zhu, X. T., and Lu, C. Y. 2009. Polyploid induction of Nobile-type Dendrobium. Acta Hortic. Sin. 36 (9): 1381–1384. Zheng Y. X., Chen, C. C., Yang, C. J., Yeh, S. D., and Jan, F. J. 2008b. Identification and characterization of a tospovirus causing chlorotic ringspots on Phalaenopsis orchids. Eur. J. Plant Pathol. 120: 199–209. Zheng, Y. X., Chen, C. C., Chen, Y. K., and Jan, F. J. 2008a. Identification and characterization of a potyvirus causing chlorotic spots on Phalaenopsis orchids. Eur. J. Plant Pathol. 121: 87–95. Zheng, Y. X., Chen, C. C., and Jan, F. J. 2011. Complete nucleotide sequence of capsicum chlorosis virus isolated from Phalaenopsis orchid and the prediction of the unexplored genetic information of tospoviruses. Arch. Virol. 156: 421–432. Zhou, G. Y., Weng, J., Zeng, Y., Huang, J., Qian, S., and Liu, G. 1983. Introduction of exogenous DNA into cotton embryos. Methods Enzymol. 101: 433–481 Zhu, G. S., Yu, Z. N., Gui, Y., and Liu, Z. Y. 2008. A novel technique for isolating orchid mycorrhizal fungi. Fungal Div. 33: 123–137. Ziv, M. 2005. Simple bioreactors for mass propagation of plants. Plant Cell, Tiss. Organ Cult. 81: 277–285. Ziv, M., and Naor, V. 2006. Flowering of geophytes in vitro. Propag. Ornam. Plants 6: 3–16.