Gametophyte and embryonic ontogeny: understanding the reproductive calendar of Cypripedium japonicum Thunb. (Cypripedoideae, Orchidaceae), a lady's slipper orchid endemic to East Asia.

BACKGROUND: The genus Cypripedium L. is one of the five genera of the subfamily Cypripedioideae, members of which are commonly known as lady's slipper orchids. Cypripedium japonicum is a perennial herb native to East Asia, specifically China, Japan, and Korea. Due to its limited distribution, the species is included in the Endangered category of the IUCN Red List. RESULTS: We investigated gametophyte development, including complete embryogenesis, in C. japonicum. The complete reproductive cycle is presented based on our observations. Anther development begins under the soil, and meiosis of pollen mother cells begins 3 weeks before anthesis, possibly during early April. The megaspore mother cells develop just after pollination in early May and mature in mid-late June. The pattern of embryo sac formation is bisporic, and there are six nuclei: three forming the egg apparatus, two polar nuclei, and an antipodal cell in the mature embryo sac. Triple fertilization results in the endosperm nucleus, which degenerates when the proembryo reaches the eight-to-sixteen-cell stage. CONCLUSION: Our overall comparisons of the features of gametophyte and embryo development in C. japonicum suggest that previous reports on the embryology of Cypripedium are not sufficient for characterization of the entire genus. Based on the available information, a reproductive calendar showing the key reproductive events leading to embryo formation has been prepared.

Chemical composition of cell walls in velamentous roots of epiphytic Orchidaceae.

The chemical composition of the cell walls strongly affects water permeability and storage in root tissues. Since epiphytic orchids live in a habitat with a highly fluctuating water supply, the root cell walls are functionally important. In the present study, we used histochemistry and immunocytochemistry techniques in order to determine the composition of the cell walls of root tissues of 18 epiphytic species belonging to seven subtribes across the Orchidaceae. The impregnation of lignin in the velamen cells reinforces its function as mechanical support and can facilitate apoplastic flow. Pectins, as well cellulose and lignins, are also essential for the stability and mechanical support of velamen cells. The exodermis and endodermis possess a suberinized lamella and often lignified walls that function as selective barriers to apoplastic flow. Various cortical parenchyma secondary wall thickenings, including phi, reticulated, and uniform, prevent the cortex from collapsing during periods of desiccation. The presence of highly methyl-esterified pectins in the cortical parenchyma facilitates the formation of gels, causing wall loosening and increased porosity, which contributes to water storage and solute transport between cells. Finally, cells with lipid or lignin impregnation in the cortical parenchyma could increase the water flow towards the stele.

Thirteen New Plastid Genomes from Mixotrophic and Autotrophic Species Provide Insights into Heterotrophy Evolution in Neottieae Orchids.

Mixotrophic species use both organic and mineral carbon sources. Some mixotrophic plants combine photosynthesis and a nutrition called mycoheterotrophy, where carbon is obtained from fungi forming mycorrhizal symbiosis with their roots. These species can lose photosynthetic abilities and evolve full mycoheterotrophy. Besides morphological changes, the latter transition is associated with a deep alteration of the plastid genome. Photosynthesis-related genes are lost first, followed by housekeeping genes, eventually resulting in a highly reduced genome. Whether relaxation of selective constraints already occurs for the plastid genome of mixotrophic species, which remain photosynthetic, is unclear. This is partly due to the difficulty of comparing plastid genomes of autotrophic, mixotrophic, and mycoheterotrophic species in a narrow phylogenetic framework. We address this question in the orchid tribe Neottieae, where this large assortment of nutrition types occurs. We sequenced 13 new plastid genomes, including 9 mixotrophic species and covering all 6 Neottieae genera. We investigated selective pressure on plastid genes in each nutrition type and conducted a phylogenetic inference of the group. Surprisingly, photosynthesis-related genes did not experience selection relaxation in mixotrophic species compared with autotrophic relatives. Conversely, we observed evidence for selection intensification for some plastid genes. Photosynthesis is thus still under purifying selection, maybe because of its role in fruit formation and thus reproductive success. Phylogenetic analysis resolved most relationships, but short branches at the base of the tree suggest an evolutionary radiation at the beginning of Neottieae history, which, we hypothesize, may be linked to mixotrophy emergence.

Comparative survey of secretory structures and floral anatomy of Cohniella cepula and Cohniella jonesiana (Orchidaceae: Oncidiinae). New evidences of nectaries and osmophores in the genus.

The morpho-anatomical structure of nectaries, osmophores, and elaiophores, and the anatomical and micromorphological features of floral pieces of Cohniella cepula Hoffmans. and Cohniella jonesiana Rchb.f. were comparatively analyzed. In both species, bracteal and sepal nectaries are structured, i.e., they present a secretory epidermis, secretory parenchyma, and vascular bundles. Nectar secretion is released through stomata. The anatomical and micromorphological traits are similar in both nectaries, which can be detected only if the nectar drops are secreted. Considering the location of these nectaries, the secreted nectar would not be a reward to pollinators. Osmophores are located at the base of both callus and laterals lobes, and consist of a layer of secretory epidermis composed of quadrangular cells and papillae. Elaiophores are found on the callus of the labellum and are of the epithelial type. The anatomical features of floral pieces are similar in both species. The anatomical analysis of sepals and petals showed a few differences, which could be of potential taxonomic value. Our results contribute valuable and novel information for the knowledge of these species and the genus, which will be useful in future taxonomic evaluations.

Comparative anatomical properties of some Epidendroideae and Orchidoideae species distributed in NE Turkey.

In this research, anatomical, leaf micromorphological features of the samples belonging to 25 taxa (Anacamptis Rich., Cephalanthera Rich., Dactylorhiza Necker ex Nevski, Gymnadenia R.Br., Himantoglossum Spreng., Limodorum Boehm., Ophrys L., Orchis L., Platanthera Rich., Serapias L., Spiranthes Rich. and Steveniella Schltr.) spread in the Karadeniz Region have been evaluated comparatively. In anatomical studies, the transverse section from root, stem and leaf, and surface section from leaves of plants were examined. In addition, micromorphological properties of leaf were determined by electron microscopy. Morphometrical analyses were carried out using the anatomical and leaf micromophological characters of each taxa. The data matrices were obtained by examining the results of at least three samples collected from different localities. The data were evaluated using Statistical Package for the Social Sciences (SPSS) and PAleontological STatistics (PAST) statistical programs with PCA, linear discriminant analysis (LDA), and unweighted pair group method with arithmetic mean analysis. Anatomical characteristics of plants such as root epidermis cell length, cortex diameter and pith cell diameter, leaf upper epidermis length-width and bulliform cell length-width were determined to be important characteristics. It was concluded that these characters are especially important in grouping at the genus level.

Floral nectary and osmophore of Epipactis helleborine (L.) Crantz (Orchidaceae).

The analysis of flowers collected at different stages of anthesis provides strong evidence to conclude that the shell-shaped hypochile and the knobs of epichile form a nectary. The scent comes from the aromatic constituents of nectar and the epichile tissue and the apices of all tepals (osmophores). The comparison between pollinated and unpollinated flowers revealed that the anthesis of unpollinated flowers lasted up to the 16th day. The nectariferous secretory cells formed single-layered epidermis and several layers of underlying parenchyma built by small, isodiametric cells with thin walls and dense cytoplasm, relatively large nuclei, supplied by collateral vascular bundles. During the floral lifespan, the residues of secreted material were higher on the hypochile cells. The lipoid-carbohydrate material and lipid globules in the cell walls and in the cytoplasm were localised. The abundance of starch grains was observed at the beginning of anthesis and their gradual reduction during the flower lifespan. At the end of anthesis in unpollinated flowers, the lipoid-carbohydrate-phenolic materials have been demonstrated. The phenolic material was the same as in plastoglobuli. The features such as irregular plasmalemma, the secretory vesicles that fuse with it, fully developed dictyosomes, numerous profiles of ER indicate vesicle-mediated process of secretion. The substances could be transported by vesicles to the periplasmic space via granulocrine secretion and then to the external surface. Both micro-channels and slightly developed periplasmic space were visible in the hypochile epidermis. This is the first time for anatomical survey of secretory tissue in pollinated and unpollinated flowers of E. helleborine.

Floral features of two species of Bulbophyllum section Lepidorhiza Schltr.: B. levanae Ames and B. nymphopolitanum Kraenzl. (Bulbophyllinae Schltr., Orchidaceae).

Two representatives of section Lepidorhiza, previously sometimes considered conspecific, Bulbophyllum levanae and Bulbophyllum nymphopolitanum, demonstrated both similarities and differences in floral features. There were significant differences in the length of sepals and micromorphological features of the labellum. In both species, osmophores are located on the extended apices of sepals and possibly on petals. An abundance of proteins in tepals is probably associated with the unpleasant scent of the flowers, whereas the thin wax layers on the epidermis are probably involved in the maintenance of the brilliance of floral tepals, which strongly attracts flies. In all tepals of both species, we noted the presence of dihydroxyphenolic globules in the cytoplasm after staining with FeCl3. Comparison with ultrastructure results revealed that they were associated with plastids containing plastoglobuli. The most remarkable feature was the presence of a prominent periplasmic space in the epidermal cells of both investigated species. Furthermore, in the labellum of B. levanae, the cuticle contained microchannels. The combination of periplasmic space and microchannels has not previously been recorded.

Cell-specific expression of plant nutrient transporter genes in orchid mycorrhizae.

Orchid mycorrhizal protocorms and roots are heterogeneous structures composed of different plant cell-types, where cells colonized by intracellular fungal coils (the pelotons) are close to non-colonized plant cells. Moreover, the fungal coils undergo rapid turnover inside the colonized cells, so that plant cells containing coils at different developmental stages can be observed in the same tissue section. Here, we have investigated by laser microdissection (LMD) the localization of specific plant gene transcripts in different cell-type populations collected from mycorrhizal protocorms and roots of the Mediterranean orchid Serapias vomeracea colonized by Tulasnella calospora. RNAs extracted from the different cell-type populations have been used to study plant gene expression, focusing on genes potentially involved in N uptake and transport and previously identified as up-regulated in symbiotic protocorms. Results clearly showed that some plant N transporters are differentially expressed in cells containing fungal coils at different developmental stages, as well as in non-colonized cells, and allowed the identification of new functional markers associated to coil-containing cells.

Development of polyspermic zygote and possible contribution of polyspermy to polyploid formation in angiosperms.

Fertilization is a general feature of eukaryotic uni- and multicellular organisms to restore a diploid genome from female and male gamete haploid genomes. In angiosperms, polyploidization is a common phenomenon, and polyploidy would have played a major role in the long-term diversification and evolutionary success of plants. As for the mechanism of formation of autotetraploid plants, the triploid-bridge pathway, crossing between triploid and diploid plants, is considered as a major pathway. For the emergence of triploid plants, fusion of an unreduced gamete with a reduced gamete is generally accepted. In addition, the possibility of polyspermy has been proposed for maize, wheat and some orchids, although it has been regarded as an uncommon mechanism of triploid formation. One of the reasons why polyspermy is regarded as uncommon is because it is difficult to reproduce the polyspermy situation in zygotes and to analyze the developmental profiles of polyspermic triploid zygotes. Recently, polyspermic rice zygotes were successfully produced by electric fusion of an egg cell with two sperm cells, and their developmental profiles were monitored. Two sperm nuclei and an egg nucleus fused into a zygotic nucleus in the polyspermic zygote, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle. The two-celled proembryos further developed and regenerated into triploid plants. These suggest that polyspermic plant zygotes have the potential to form triploid embryos, and that polyspermy in angiosperms might be a pathway for the formation of triploid plants.

Distinct chromatin environment associated with phosphorylated H3S10 histone during pollen mitosis I in orchids.

Pollen developmental pathway in plants involving synchronized transferal of cellular divisions from meiosis (microsporogenesis) to mitosis (pollen mitosis I/II) eventually offers a unique "meiosis-mitosis shift" at pollen mitosis I. Since the cell type (haploid microspore) and fate of pollen mitosis I differ from typical mitosis (in meristem cells), it is immensely important to analyze the chromosomal distribution of phosphorylated H3S10 histone during atypical pollen mitosis I to comprehend the role of histone phosphorylation in pollen development. We investigated the chromosomal phosphorylation of H3S10 histone during pollen mitosis I in orchids using immunostaining technique. The chromosomal distribution of H3S10ph during pollen mitosis I revealed differential pattern than that of typical mitosis in plants, however, eventually following the similar trends of mitosis in animals where H3S10 phosphorylation begins in the pericentromeric regions first, later extending to the whole chromosomes, and finally declining at anaphase/early cytokinesis (differentiation of vegetative and generative cells). The study suggests that the chromosomal distribution of H3S10ph during cell division is not universal and can be altered between different cell types encoded for diverse cellular processes. During pollen development, phosphorylation of histone might play a critical role in chromosome condensation events throughout pollen mitosis I in plants.

FEATURES OF EMBRYONIC DEVELOPMENT OF DIENIA OPHRYDIS (ORCHIDACEAE).

On example of Dienia ophrydis (J. Köenig) Seidenf (Orchidaceae), we have described a new type of embryogenesis of orchids ­ Dienia-type, which is differs from Liparis-type learned earlier in the tribe Malaxideae. Embryogenesis of Dienia-type is characterized by 1) the development of a single-celled suspensor formed by cb-derivative, 2) linear arrangement of germ cells in the tetrad stage, 3) the special structure of the embryo in the stages of tetrads and octants (l, lR, m, ci, cb), and 4) the absence of ci and cb cell division. The convergent similarity of embryogenesis of Dienia- and Caryophyllaceae-types is proposed. A number of specific for D. ophrydis structures of embryo sac and embryo, including «petassum¼, «fitting¼ and «suspensor coat¼ are described for the first time. Petassum represents remains of the cell walls of pollen tube, and perhaps of filamentous apparatus of synergids, plugging the micropyle side of the fertilized embryo sac. The only cell of suspensor has a specific appendix («fitting¼), that connects it with the embryo itself. There is «suspensor coat¼ which surrounds the only suspensor cell, including «fitting¼, but does not extend to the basal cells of the embryo itself.

Phylogenetic Analysis of a 'Jewel Orchid' Genus Goodyera (Orchidaceae) Based on DNA Sequence Data from Nuclear and Plastid Regions.

A molecular phylogeny of Asiatic species of Goodyera (Orchidaceae, Cranichideae, Goodyerinae) based on the nuclear ribosomal internal transcribed spacer (ITS) region and two chloroplast loci (matK and trnL-F) was presented. Thirty-five species represented by 132 samples of Goodyera were analyzed, along with other 27 genera/48 species, using Pterostylis longifolia and Chloraea gaudichaudii as outgroups. Bayesian inference, maximum parsimony and maximum likelihood methods were used to reveal the intrageneric relationships of Goodyera and its intergeneric relationships to related genera. The results indicate that: 1) Goodyera is not monophyletic; 2) Goodyera could be divided into four sections, viz., Goodyera, Otosepalum, Reticulum and a new section; 3) sect. Reticulum can be further divided into two subsections, viz., Reticulum and Foliosum, whereas sect. Goodyera can in turn be divided into subsections Goodyera and a new subsection.

In vitro propagation of Paphiopedilum orchids.

Paphiopedilum is one of the most popular and rare orchid genera. Members of the genus are sold and exhibited as pot plants and cut flowers. Wild populations of Paphiopedilum are under the threat of extinction due to over-collection and loss of suitable habitats. A reduction in their commercial value through large-scale propagation in vitro is an option to reduce pressure from illegal collection, to attempt to meet commercial needs and to re-establish threatened species back into the wild. Although they are commercially propagated via asymbiotic seed germination, Paphiopedilum are considered to be difficult to propagate in vitro, especially by plant regeneration from tissue culture. This review aims to cover the most important aspects and to provide an up-to-date research progress on in vitro propagation of Paphiopedilum and to emphasize the importance of further improving tissue culture protocols for ex vitro-derived explants.

LONG-TERM CONSERVATION OF PROTOCORMS OF Brassavola nodosa (L) LIND. (ORCHIDACEAE): EFFECT OF ABA AND A RANGE OF CRYOCONSERVATION TECHNIQUES.

BACKGROUND: Populations of Brassavola nodosa have been severely affected by habitat destruction and illegal collecting, and as with the majority of orchid species, it is critical to take action to guarantee their continued survival. OBJECTIVE: The present study aimed to establish protocols for the long-term conservation of protocorms of species. MATERIALS AND METHODS: Four different cryogenic techniques were compared: encapsulation-dehydration (ED), encapsulation-vitrification (EV), encapsulation-dehydration-vitrification (EDV) and vitrification. RESULTS: Preculture of protocorms with ABA was a critical factor in obtaining high percentages of regrowth. With vitrification, 100% regrowth was achieved in five treatments, mainly when protocorms were dehydrated with PVS2 for 120 min. 100% regrowth was also obtained with EDV, where the protocorms were precultured with ABA 5 mg/l for 3 days and incubated with PVS2 for 60 min. With the ED, regrowth of 72% was achieved with the preculture of protocorms with ABA 5 mg/l for the three times of incubation used (3, 6 and 9 days). In the case of EV, 92% regrowth, was recorded when protocorms were precultured for 9 days with ABA 3 mg/l and incubated with PVS2 for 90 min. CONCLUSION: Although regrowth of protocorms was obtained with all the techniques used, the vitrification technique is preferred since it requires less labour and is less costly.

Biogeography of the Phalaenopsis amabilis species complex inferred from nuclear and plastid DNAs.

BACKGROUND: Phalaenopsis is one of the important commercial orchids in the world. Members of the P. amabilis species complex represent invaluable germplasm for the breeding program. However, the phylogeny of the P. amabilis species complex is still uncertain. The Phalaenopsis amabilis species complex (Orchidaceae) consists of subspecies amabilis, moluccana, and rosenstromii of P. amabilis, as well as P. aphrodite ssp. aphrodite, P. ap. ssp. formosana, and P. sanderiana. The aims of this study were to reconstruct the phylogeny and biogeographcial patterns of the species complex using Neighbor Joining (NJ), Maxinum Parsimony (MP), Bayesian Evolutionary Analysis Sampling Trees (BEAST) and Reconstruct Ancestral State in Phylogenies (RASP) analyses based on sequences of internal transcribed spacers 1 and 2 from the nuclear ribosomal DNA and the trnH-psbA spacer from the plastid DNA. RESULTS: A pattern of vicariance, dispersal, and vicariance + dispersal among disjunctly distributed taxa was uncovered based on RASP analysis. Although two subspecies of P. aphrodite could not be differentiated from each other in dispersal state, they were distinct from P. amabilis and P. sanderiana. Within P. amabilis, three subspecies were separated phylogenetically, in agreement with the vicariance or vicariance + dispersal scenario, with geographic subdivision along Huxley's, Wallace's and Lydekker's Lines. Molecular dating revealed such subdivisions among taxa of P. amabilis complex dating back to the late Pleistocene. Population-dynamic analyses using a Bayesian skyline plot suggested that the species complex experienced an in situ range expansion and population concentration during the late Last Glacial Maximum (LGM). CONCLUSIONS: Taxa of the P. amabilis complex with disjunct distributions were differentiated due to vicariance or vicariance + dispersal, with events likely occurring in the late Pleistocene. Demographic growth associated with the climatic oscillations in the Würm glacial period followed the species splits. Nevertheless, a subsequent population slowdown occurred in the late LGM due to extinction of regional populations. The reduction of suitable habitats resulted in geographic fragmenttation of the remaining taxa.

Differential protein expression in Phalaenopsis under low temperature.

A comparative proteomic analysis was carried out to explore the molecular mechanisms of responses to cold stress in Phalaenopsis after treated by low temperature (13/8 °C day/night) for 15 days. Differentially expressed proteins were examined using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF/MS). Among 85 differentially expressed proteins, 73 distinct proteins were identified. Comparative analysis revealed that the identified proteins mainly participate in photosynthesis, protein synthesis, folding and degradation, respiration, defense response, amino acid metabolism, energy pathway, cytoskeleton, transcription regulation, signal transduction, and seed storage protein, while the functional classification of the remaining four proteins was not determined. These data suggested that the proteins might work cooperatively to establish a new homeostasis under cold stress; 37 % of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology, and 56 % of them were predicted to be located in the chloroplasts, implying that the cold stress tolerance of Phalaenopsis was achieved, at least partly, by regulation of chloroplast function. Moreover, the protein destination control, which was mediated by chaperones and proteases, plays an important role in tolerance to cold stress.

Orchids (Oncidium and Phalaenopsis).

This chapter describes an efficient and reproducible method for large-scale propagation of Oncidium and Phalaenopsis protocorm-like bodies (PLBs) using floral stalk sections and seeds, respectively. The propagated PLBs can be used for Agrobacterium-mediated transformation. An advanced transformation system for Oncidium and Phalaenopsis orchids has been established. This protocol demonstrates that the time during which the PLBs are cocultivated with Agrobacterium is the key to promoting transformation efficiency. Modified DNA and RNA extraction methods are also provided to diminish polysaccharide contamination and to improve the quality for further molecular analysis.

Physical wounding and ethylene stimulated embryogenic stem cell proliferation and plantlet regeneration in protocorm-like bodies of Phalaenopsis orchids.

Phalaenopsis orchids have been regenerated by inducing protocorm-like bodies (PLBs) from etiolated leaf sections. However, the physiological and molecular mechanisms of secondary PLB development and subsequent proliferation have not been explored. Bisectionally cutting primary PLBs resulted in more secondary PLBs at 5 weeks, suggesting an embryogenic stem cell property imposed by wounding of primary PLB tissues. The ethylene precursors ethephon and 1-aminocyclopropanecarboxylic acid and the ethylene perception inhibitor silver nitrate increased PLB formation, while aminoethoxyvinylglycine decreased PLB formation. Ethylene content in wounded PLB explants increased over culture time in media containing ethylene precursors or inhibitors. mRNA levels of PhACS2, PhACS3, and PhACO were increased by ethephon and decreased by ethylene inhibitors. Expression of genes in the ethylene signaling pathway was enhanced following ethylene-precursor treatment and was mitigated by ethylene inhibitors during PLB proliferation. Transcription of PhETR and PhEIN3, as well as PhERS, PhCTR, and PhGTP, was significantly increased 12 h after ethylene treatment. Ethylene and physical wounding stimulated secondary PLB formation in Phalaenopsis, probably through ethylene biosynthesis and signal transduction.

Cold response in Phalaenopsis aphrodite and characterization of PaCBF1 and PaICE1.

Phalaenopsis is a winter-blooming orchid genus commonly cultivated in tropical Asian countries. Because orchids are one of the most economically important flower crops in Taiwan, it is crucial to understand their response to cold and other abiotic stresses. The present study focused on gene regulation of P. aphrodite in response to abiotic stress, mainly cold. Our results demonstrate that P. aphrodite is sensitive to low temperatures, especially in its reproductive stage. We found that after exposure to 4°C, plants in the vegetative stage maintained better membrane integrity and photosynthetic capacity than in the flowering stage. At the molecular level, C-repeat binding factor1 (PaCBF1) and its putative target gene dehydrin1 (PaDHN1) mRNAs were induced by cold, whereas inducer of CBF expression1 (PaICE1) mRNA was constitutively expressed. PaICE1 transactivated MYC motifs in the PaCBF1 promoter, indicating that up-regulation of PaCBF1 may be mediated by the binding of PaICE1 to MYC motifs. Overexpression of PaCBF1 in transgenic Arabidopsis induced AtCOR6.6 and RD29a without cold stimulus and maintained better membrane integrity after cold stress. Herein, we present evidence that cold induction of PaCBF1 transcripts in P. aphrodite may be transactivated by PaICE1 and consequently protect plants from cold damage through up-regulation of cold-regulated (COR) genes, such as DHN. To our knowledge, this study is the first report of the isolation and characterization of CBF, DHN and ICE genes in the Orchidaceae family.

In vitro regeneration of Coelogyne nervosa A.Rich. and Eria pseudoclavicaulis Blatt., threatened orchids of Western Ghats, India.

The seeds of C. nervosa and E. pseudoclavicaulis were germinated asymbiotically on Knudson C (KC) and Schenk and Hildebrandt basal medium (SH). Growth regulators such as 2,4-Dichlorophenoxyacetic acid (2,4-D) individually and in combinations with benzyladenine (BA) and kinetin were used for callus induction from the protocorm like bodies. Coelogyne nervosa showed maximum (90%) callus induction in Knudson C medium supplemented with 2,4-D (2.26 microM) and Eria pseudoclavicaulis showed 60% callus induction in Schenk and Hildebrandt medium supplemented with 2,4-D (2.26 microM). Calli developed a route of production of protocorm-like bodies and eventually developed into plantlets on transfer to growth regulator free half strength basal medium. The well rooted plants were hardened successfully in the potting mixture containing coconut husk, charcoal, and brick pieces in the ratio 2:1:1.