INTRODUCTION Orchidaceae constitutes one of the largest and most diverse of all angiosperm families, with estimates of more than 25,000 species (Dress

Transcription

1 INTRODUCTION

2 INTRODUCTION Orchidaceae constitutes one of the largest and most diverse of all angiosperm families, with estimates of more than 25,000 species (Dressler, 1993; Cribb et al., 2003). Orchids comprise five sub-families and approximately 870 genera, and are considered almost ubiquitous, occurring on all vegetated continents and even some Antarctic islands (Dressler, 1981; Chase et al., 2003). Orchid distribution and abundance are distinctly skewed towards the tropics and vary between continents and within regions, following hotspots of species richness and high angiosperm endemism as described by Myers et al. (2000). Orchid-rich areas include the northern Andes of South America, Madagascar, Sumatra and Borneo for mostly epiphytic species, Indo-China for both epiphytic and terrestrial species, and South Western Australia as a centre of terrestrial orchid richness (Cribb et al., 2003; Swarts & Dixon, 2009). The spectacular diversification of orchids has been linked to the specific interaction between the orchid flower and pollinator (Cozzolino & Widmer, 2005), sequential and rapid interplay between drift and natural selection (Tremblay et al., 2005), the role of obligate orchidmycorrhizal interactions (Otero & Flanagan, 2006), and Crassulacean acid metabolism and epiphytism (Silvera et al., 2009). Orchids have adapted themselves to extremes of the environmental conditions producing thereby great variations in vegetative forms. According to their habitat, they can be broadly classified into following groups - terrestrial, epiphyte and lithophytes. Two-thirds of orchid species are epiphytes and lithophytes, with terrestrial species comprising the remaining third (Swarts & Dixon, 2009). The orchid plant structure is basically of two types (Chowdhery, 1998): Monopodial- such orchids do not have rhizomes and pseudobulbs and grow from a single vegetative apex. They sprout new growth by developing axillary shoots which grow into new plants. Examples: Vanda, Rhynchostylis. Sympodial- such orchids grow from a number of vegetative apices situated on the rhizome. These sometimes swell into reserve organs and are known as pseudobulbs. Examples: Dendrobium, Cymbidium. 1

3 The parts of an orchid flower (as described by Davies et al., 1988) are trimerous: three outer perianth segments or sepals, and three inner segments which correspond to petals. The inner segments comprise a pair of identical petals and a third, the lip or labellum the part of the lip nearest to its point of attachment to the rest of the flower is called the base and in many cases, this is drawn out behind the flower into a tube, called the spur where the nectar is stored. In orchids, male and female parts are fixed into a single structure called a column. The pollen grains are not separate but held together to form more or less solid masses called pollinia. The ovary is rather long and thin, with innumerable tiny ovules. The two stigmas capable of receiving pollen are found on the column in the very centre of flower, either below or immediately in front of the stamen. There is also a sterile third stigma in many orchids which has evolved into a curious structure called the rostellum. It prevents pollen spreading from a stamen onto a stigma of the same plant, which would cause self fertilization (Davies et al., 1988). India is considered as a rich orchid heritage and is estimated to have around 1,600 orchid species constituting almost 10% of the world orchid flora (Medhi & Chakrabarti, 2009). The main orchid rich belts in the country are North-Eastern India, Eastern and Western Himalayas and the Western Ghats. North-Eastern India, owing to its peculiar gradient and varied climatic conditions, contains largest group of temperate, sub-tropical orchids and has about 876 orchid species in 151 genera (Medhi & Chakrabarti, 2009). For the Chinese, orchids are a symbol of scholar unassuming, enduring and ascetic, it also stood as a symbol of love, beauty, grace, nobility and elegance in a woman (Chowdhery, 1998). Confucius ( BC) said acquaintance with good men was like entering a room full of fragrant orchids. The floral characteristics of orchids cover an exceptionally wide range of different shape, form, size, and colouration, surpassing flowers of all the other angiosperms (Thomas & Michael, 2007). The exquisite and bewitchingly beautiful flowers of various shapes, sizes and colours and also the medicinal virtues of some of the orchids have evoked considerable attention amongst the naturalists, the systematists and the herbalists (Singh, 2001). As a result, they are exceedingly valued in floriculture as species or hybrids for pot plant or cut flowers, and hold enormous promise. Orchids are well known for their beauty and use (Griesbach, 2003). Orchids have tremendous economic potential in cut flower industry, as 2

4 medicines, food and perfumes. They are grown primarily as ornamentals and are valued as cut flowers not only because of their exotic beauty but also for their long shelf life. Orchids are marketed both as cut flowers and potted plants. The largest exporters of potted orchids are Taiwan, Thailand, UK, Italy, Japan, New Zealand and Brazil while United States is the largest importer of potted orchids. Orchids such as Cymbidium, Dendrobium, Oncidium and Phalaenopsis are marketed globally and the orchid industry has contributed substantially to the economy of many ASEAN (Association of the South East Asian Nations) countries (Hew, 1994; Laws, 1995). Orchids are currently the second most valuable potted crop in the United States with a total wholesale value of US$ 144 million in 2005 (U.S. Department of Agriculture, 2006). In 2005, 18 million potted orchids were sold at wholesale, with an average unit value of US$ At present, orchids are a million dollar industry in several countries such as Thailand, Australia, Singapore, Malaysia and several others. Thailand, which is the world s sixth largest exporter of cut flowers, earns US$ 30 million a year from orchid exports, and Singapore earns US$ 16 million a year (Reddy, 2008). However, India s annual flower production stands at around 1000 tonnes and its floriculture industry has a miniscule 0.01% share in the international market (Chugh et al., 2009). Besides their high ornamental values, orchids are of considerable importance in medicines as they have rich contents of alkaloids, glycerides and other useful phytochemicals (Gutierrez, 2010). Because of these properties, the plants are used for variety of folk medicines and cures by local people. Orchids are also collected for ethno botanical uses, for example the pseudobulbs of Dendrobium species are used in the Chinese medicinal plant trade while tuberous terrestrial orchids are collected in east sub-saharan Africa for the production of a cake called chikanda (Roberts & Dixon, 2008). Similarly, in Turkey, tubers of terrestrial orchids are used to make an extract known as salep that is used in the preparation of ice-cream. Malaxis acuminata is known for its therapeutic importance as its dried pseudobulbs are an important ingredient of Ashtavarga drugs used in the preparation of an ayurvedic medicine Chyavanprash (Kaur & Bhutani, 2010). Capsules of Vanilla planifolia are popularly used as the source of essence of vanilla. The therapeutic uses of orchids have been reviewed by Hossain (2011). 3

5 Many orchids can survive only in a specific undisturbed microclimate and are botanically very interesting for their floral complexity, free gene flow across specific barriers, minute seeds with undifferentiated embryos, suppressed endosperm formation, long-lived pollen tubes, and dependence on a suitable mycorrhizal association for germination of seeds. As a result of their complex ecological interaction with pollinators, mycorrhizal fungi and other plants and animals, orchids are often the first biological indicators of ecosystem decay (Thomas & Michael, 2007). Many decades may be needed to return to a level of ecological stability conducive to orchid persistence, because of the reliance of orchids on insect pollinators and mycorrhiza (Roberts & Dixon, 2008). Ecological specialization has not only contributed to the great species diversity in Orchidaceae, but has also resulted in the high level of threat to this family (Cribb et al., 2003). Anthropogenic threatening processes such as collection of wild orchids or land clearance directly limit or reduce the distribution and abundance of a species (Cribb et al., 2003; Koopowitz et al., 2003). The clearance of forests for development and agricultural purposes and unregulated collection of orchids for commercial and herbal uses have jeopardized the existence of the natural habitats and populations of a large number of orchids. The injudicious habitat destruction has brought 5-10% of the quarter of a million flowering plants under imminent danger of extinction. Fragmentation of habitats, removal of key species critical to the continued existence of ecosystems, increased susceptibility to fire threats and pollinator decline are also documented to result in drastic losses in orchid populations and diversity (Coates & Dixon, 2007).The high number of taxa, beautiful flowers, complicated structure, sophisticated pollination processes, delicate scents and difficult propagation of orchids led to high domestic and international demand for the last 40 years (Clemente, 2009). Wild specimens came under such pressure that fear over their conservation status led to listing the entire family in the Appendices of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). With nearly 12.5 % of the global vascular flora facing extinction, conservation of rare and threatened plants is of international concern (Walter & Gillet, 1998). Despite having a large number of orchid species and diversified agroclimatic conditions, the orchid industry has not been developed in India although it has a great 4

6 potential (Chugh et al., 2009). Lack of organized efforts, market information and postharvest technology are the major constraints in India (Ahuja et al., 2001). Hence, it is imperative to design an efficient strategy not only to save these beautiful members of the plant kingdom but also to harness the economic potential by scientific and judicious management. This calls for a need to standardize commercial-scale micropropagation techniques for production of quality planting material of important, rare, endangered, threatened as well as exotic hybrid orchids keeping the developing countries in mind (Chugh et al., 2009). The major bottleneck in plant tissue culture technology is the survival of plants in the green house and field conditions. Therefore, it is necessary to devise a methodology for in vitro hardening of tissue culture raised plants for successful establishment. Conventional propagation through seed is less desirable especially for horticultural uses due to the long juvenile period before flowering (Decruse et al., 2003). The regeneration of plantlets from mature tissue is important since the flowering stage is reached after several years of vegetative growth from protocorms (Le et al., 1999). Since orchids are out breeders, their propagation using seeds is undesirable as heterozygous plants are produced and their vegetative propagation is extremely slow, the commercial application of tissue culture techniques for rapid clonal propagation of some hybrids is of great importance. In vitro micropropagation technique has many unique advantages over the conventional propagation methods such as rapid multiplication of valuable genotypes, expedited release of improved varieties, fast production, germplasm conservation and facilitating their easy international exchange (Kannan, 2009). In vitro techniques are persuasive tools for plant breeders in all the fields of promoting the performance of agriculture, horticulture and floriculture plant yield. The major benefit of clonal propagation is that the plantlets produced are identical to their parents (clones). This is of great advantage to the cut flower industry in production of uniform blossoms during predictable period to meet the market demands. Tissue culture techniques have been extensively exploited not only for rapid and large scale propagation of orchids but also for their ex situ conservation. Tissue culture techniques are of great interest for multiplication and storage of plant germplasm (Engelmann, 2011). Tissue culture systems allow propagating plant material with high multiplication rates in an aseptic environment. Virus-free plants can be obtained through meristem culture, thus ensuring 5

7 the production of disease free stocks and simplifying quarantine procedures for the international exchange of germplasm (Engelmann, 2011). The miniaturization of explants allows reducing space requirements and consequently labour costs. Vegetative propagation of orchids in vitro is mainly through protocorm-like bodies (PLBs) induced from various explants either directly or via an intermediary callus phase (Lakshmanan, 1995a). Protocorm like-bodies (PLBs) are organs that resemble protocorm and produce plantlets like the protocorm does (Teixeira da Silva et al., 2006a). They are also referred to as shoot buds and the terms are used interchangeably. The cells of PLBs are highly meristematic and thus can be used to enhance proliferation and production of plantlets. Apart from that the cells of protocorms are highly meristematic in nature, thus can be used to enhance proliferation and simultaneous production of orchid plantlets (Teixeira da Silva et al., 2005a). The term protocorm was first introduced by Treub (1890) in lieu of the previously used embryonic tuber (tubercule embryonaire) to describe a tuberidiform structure with hairs on the lower part, which is formed during germination of club moss embryos. The apical part of the protocorm, consisting of smaller cells, is the shoot apex. The basal part, functioning as a storage organ, consists of larger parenchymatous cells. Vegetative reproduction of specimens at protocorm stage is hardly ever observed in nature (Tatarenko & Vakhrameeva, 1998), whereas in culture in vitro protocorms are employed to obtain the maximum number of regenerants (Batygina & Shevtsova, 1985; Shevtsova et al., 1986). There are two ways by which protocorms are differentiated in vitro, through the formation of a number of shoot apices, with further production of adventitious roots and the formation of numerous secondary protocorms from epidermal cells of a single protocorm (Batygina et al., 2003). Protocorms can possess several centers of meristematic activity, but usually they develop only one shoot. Because of this attribute, protocorms obtained from different plant parts are being frequently used by many workers as explants for mass propagation of many rare orchid species like, transverse stem thin cell layers of Rhynchostylis gigantea (Le et al., 1999), leaf explants of Renanthera imschootiana (Seeni & Latha, 1992), shoot-tips and leaves of V. coerulea (Seeni & Latha, 2000), shoot-tips of Cymbidium hybrid (Teixeira da Silva et al., 2006a,b), seeds of Geodorum densiflorum (Sheelavantmath et al., 2000), Zygopetalum 6

8 intermedium (Nagaraju & Mani, 2005) Malaxis khasiana (Deb & Temjensangba, 2006), Cymbidium giganteum (Hossain et al., 2009), etc. Nagaraju and Parthasarathy (1999) used seed-derived protocorms as explants and BAP, NAA and kinetin as growth regulators for micropropagation. Corrie and Tandon (1993) reported use of encapsulated protocorms for propagation. Above all, the main advantages of direct PLB formation without an intervening callus phase is that it saves time to regenerate uniform plantlets and are economically viable. The development of protocorms is slow in vitro especially in many economically important hybrid orchids (Arditti & Ernst, 1993) and this makes commercial tissue culture expensive and time consuming. In this perspective, propagation of orchids by using TCLs can prove to be useful. The thin cell layer (TCL) system is a simplified system that requires only a small amount of plant material and provides a good system for the study of fundamental and applied aspects of regeneration and transformation. TCL technique employs various explants of small size from different plant organs excised either longitudinally (ltcl, containing one tissue type) or transversely (ttcl, containing small number of cells from different tissue types) (Tran Thanh Van, 1980). By manipulation of culture conditions desired morphogenic responses can be obtained from a very small explant (consisting of a few layers of cells) and this makes the thin cell layer technique efficient for mass propagation of many economically important plant species. This technique has also been employed for controlling various morphogenic responses of an explant in a repeatable and controlled manner (Nhut et al., 2003). In orchids, this technique has been successfully employed in Aranda Deborah from shoot tip (Lakshmanan et al., 1995), Phalaenopsis from young leaf lamina (Tran Thanh Van, 1974), Rhynchostyli sgigantea (Le et al., 1999), Dendrobium moschatum (Kanjilal et al., 1999), Phalaenopsis hybrid from stem (Guha, 2007), Cymbidium Sleeping Nymph using PLBs (Vyas et al., 2010) for regeneration. Chugh et al. (2009) have mentioned in their review that thin cross sections of actively growing parts such as shoots, leaves, inflorescence stalks and developing PLBs have been used for plantlet regeneration in some orchids. Regeneration of PLBs from thin sections has also been reported in Cymbidium hybrids (Begum et al., 1994; Teixeira da Silva et al., 2006a, b) and Cymbidium aloifolium and Dendrobium nobile (Nayak et al., 2002). 7

9 The control of growth and development in plants is by the simultaneous interactions of different plant hormonesacting synergistically or antagonistically rather than to the effect of a single hormone (Gaspar et al., 2003). Hormones can influence each other s biosynthesis so that the effects produced by one hormone may infact be mediated by others (Taiz & Zeiger, 2006). Different cell types respond differentially to various signals and hormonal controls act in a developmental and tissue-dependent manner. Orchids require auxins and/or cytokinins for neoformation of PLBs and plantlet development. The type and concentrations of growth regulators play an important role during in vitro multiplication of many orchid species (Arditti & Ernst, 1993). Cytokinins occur as free molecules in plants but are also found in the t-rnas of the cytoplasm and chloroplast (George et al., 2008). They have been implicated in many aspects of plant development including cell division and differentiation (Miller et al., 1955; Beyl, 2011). It plays a central role in cell cycle and other developmental programs such as shoot initiation and growth, apical dominance and senescence (D Agostino & Kieber, 1999). Cytokinins are classified into two major groups: synthetic phenyl urea derivatives, such as 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron; TDZ) and N-(2-chloro-4-pyridyl)-N9-phenylurea (CPPU), and adenine derivatives such as kinetin (Kn) and 6-benzylaminopurine (BAP) (Nandi et al., 1989). The development of agonists and antagonists of a particular physiological effect is useful in studying the mode of action of biologically active compounds. Cytokinin antagonists are of potential value in studies of cytokine action and other physiological studies of growth in various systems including plants and tissues (Skoog et al., 1973). In this study, 8-azaadenine and 8-azaguanine have been used to study the effects on regeneration from ttcl in Cymbidium Lunalvin Atlas. Auxins are very widely used in plant tissue culture and usually form an integral part of nutrient media. Auxins promote, mainly in combination with cytokinins, the growth of calli, cell suspensions and organs and also regulate the direction of morphogenesis (Machakova et al., 2008). At the cellular level, auxins control basic processes such as cell division and cell elongation. Since they are capable of initiating cell division, they are involved in the formation of meristems giving rise to either unorganized tissue, or defined organs (Machakova et al., 2008). In organized tissues, auxins are involved in 8

10 the establishment and maintenance of polarity and in whole plants, their most marked effect is maintenance of apical dominance and mediation of tropisms (Friml, 2003). Several compounds can act as auxin transport inhibitors including NPA (1-Nnaphthylphthalamic acid) and TIBA (2,3,5-triiodobenzoic acid). Auxin transport inhibitors are usually included in tissue culture medium to correct the cytokinin:auxin ratio. These were reported to promote or modify morphogenesis by negating the effects of exogenous or endogenous auxins (Singh & Syamal, 2000). Gibberellic acid regulates many processes in plant development, including leaf initiation and outgrowth and morphogenesis (Fleet & Sun, 2005) and has been found to promote cell division and elongation in the sub-apical zone of the shoots (George, 1993). The physiological effect of GA 3 and positive effect on plant growth is wellknown as it increases the concentrations of soluble carbohydrates during seed germination and plant growth (Bialecka & Kępczyński, 2007). Polyamines include diamine (putrescine), triamine (spermidine) and tetraamine (spermine) and are aliphatic compounds distributed widely from bacteria to higher plants (Smith, 1985). Polyamines have been implicated in the regulation ofplant growth as well as in various developmental processes such as cell division, cell and tissue differentiation, organogenesis and embryogenesis (Galston & Sawhney, 1990). At the cellular level, polyamines are organic cations, interacting with the macromolecules that possess anionic groups such as DNA, RNA, lipids, proteins, thereby influencing DNA conformation, gene expression and protein synthesis and modulating enzyme activity (Ge et al., 2006). Polyamines interact with phytohormones, act as plant growth regulators or hormonal secondary messengers, and as reserve of carbon and nitrogen in culturing tissues (Couee et al., 2004). Silver nitrate has been shown to be effective in improving somatic embryogenesis and plant regeneration in a number of crop species (Williams et al., 1990; Zhang et al., 2001; Chen & Chang, 2002; Cogbill et al., 2010). Silver nitrate has proved to be a very potent inhibitor of ethylene action and is widely used in plant tissue culture. A few properties of silver nitrate such as easy availability, solubility in water, specificity and stability makes it very useful for various applications in exploiting plant growth regulation and morphogenesis in vivo and in vitro (Kumar et al., 2009). 9

11 Natural additives are complex undefined supplements from plants which form a natural source of minerals, sugars, vitamins, amino acids and plant growth substances (Vyas, 2010). Addition of organic additives to an orchid culture medium is a simple, practical, beneficial, and convenient means to improve culture media used for commercial production (Ichihashi & Islam, 1999). Coconut water (CW) was first used in plant tissue cultures by Van Overbeek et al. in 1941 for the development of embryos of Datura stramonium and then with the passage of time it was found that coconut water could be used to initiate and maintain tissue cultures of several plants (Nasib et al., 2008). Coconut water is composed of many amino acids, nitrogenous compounds, inorganic elements, organic acids, sugars and their alcohols, vitamins, growth substances (cytokinins and auxins) and many other unknown components (George, 1993). Beneficial effects of organic additives, such as coconutwater and/or banana homogenate and/or potato homogenateadded to medium on seedling growth have been reported in many orchid species like Aranda Deborah (Goh & Wong, 1990), Vanda coerulea (Seeni & Latha, 2000), V. spathulata (Decruse et al., 2003), Dendrobium tosaense (Lo et al., 2004), D. lituiflorum (Vyas et al., 2009), Cymbidium Sleeping Nymph (Vyas et al., 2010). Orchid seeds are minute and have no endosperm, and in nature they must be symbiotic with some kinds of fungi in order to germinate. As an adaptation to wind dispersal, orchid seeds are minute varying in size from 150 to 6,000 µm (Molvray & Kores, 1995). In spite of a very large number of seeds produced, only % germinate in nature (Singh, 1992). The seeds contain a small embryo and lack enzymes to metabolize polysaccharides, but utilize lipids as a major nutrient source. The embryo also lacks enzymes to convert lipids to soluble sugars (Manning & Van Staden, 1987). The technique of asymbiotic seed germination by in vitro culture was first introduced by Knudson (1946). Since then, in vitro propagation protocols have been established for many orchid species, and a number of media, salts, and plant-growth regulators have been employed for several orchid species (Arditti, 1993). Since many native orchids are rare, threatened, or endangered, it is important to establish in vitro propagation protocols for its conservation. Although conservation is still a primary goal of native orchid, a recent purpose is to provide plants to the consumer to alleviate collection 10

12 pressure (Kauth, 2005). Hardy terrestrial orchids are adapted to variable habitats (Stoutamire, 1983) making the plants suitable for a wide range of garden conditions. Lack of knowledge and interest by the consumer and industry, difficulties in propagation methods, and a long maturation process has limited the market for native orchids (Kauth, 2005). Recent advances in the application of tissue culture have lead to an expanding market; however, small hobby growers make up the majority of the market, and availability of numerous native orchid genera is relatively small. Orchids are inherently slow growing plants and consequently, their clonal propagation is also relatively slow (Jheng et al., 2006). The development of an efficient plant regeneration protocol through PLB formation is advantageous in accelerating plant production, reducing labor costs and producing more uniform plants. Hybridization in orchids is a common means for producing new and improved material, including new flower colors, color patterns, flower size and number, and other additional characteristics of commercial value (Vendrame et al., 2007). Hybridization introduces a new dimension in floriculture industry with constant production of better breeds. Over 100,000 commercial hybrids are registered worldwide to date, being grown as cut flowers and potted plants. The great popularity of hybrid orchids is due to a variety of reasons like superior quality, ease of cultivation, free-blooming habit, incredible array of shapes, blend of colours and longer shelf-life (Kishor et al., 2006). Although development of new hybrid orchid is a tedious work that calls for great patience, synthesis of better hybrid orchids will reduce the threatening pressure on their wild parents and would be helpful for conservation (Kishor et al., 2006). Orchid conservation has become a major issue because of indiscriminate loss of native habitats. The ultimate success of in vitro propagation on commercial scale depends on the ability to transfer plants out of culture on a large scale, at a low cost and with high survival rates (Hazarika, 2006). A substantial number of micropropagated plants do not survive transfer from in vitro conditions to greenhouse or field environment (Hazarika, 2003). The greenhouse and field have substantially lower relative humidity, higher light level and septic environment that are stressful to micropropagated plants compared to in vitro conditions. Hence, a slow and gradual ex vitro transfer process is necessitated during the period in which a sustained acclimatization is effected to correct the physiological 11

13 anomalies and deficiencies. Tissue culture conditions that promote rapid growth and multiplication of shoot often result in the formation of structurally and physiologically abnormal plants. They are often characterized by poor photosynthetic efficiency, malfunctioning of stomata and a marked decrease in epicuticular wax. High sucrose and salt containing media often employed for raising cultures and poor light conditions seems to restrict photosynthetic efficiency of leafy shoots. Although such plantlets may appear normal, they are unlikely to be actively photosynthesizing because of the exogenous supply of sucrose, which does not necessitate the normal development of photosynthetic apparatus. Therefore, in the present study, SEM analyses were performed to examine the changes in the morphology of the leaf surfaces of plantlets in vitro and after transfer to greenhouse in all the three genera. Photosynthetic pigments were also estimated to ascertain the normal development of photosynthetic apparatus of the in vitro raised plantlets. In the present study, the candidate has worked on three orchid genera Cymbidium Lunalvin Atlas, a hybrid and two native orchid genera: Thunia venosa and Vanda testacea. Cymbidium constitutes 50% of the micropropagated temperate commercially important plants and its hybrids are in high demand in the cut flower industry. In vitro propagation of orchid hybrids like that of Cymbidium is important as most of them do not set seeds because of the absence of the natural pollinating insect. Even after seeds are formed, it may take many years for the orchid to grow and eventually flower and only then the success of the breeder can be ascertained (Vyas, 2010). Approximately 70% of orchid species have an epiphytic growth habit (Royal Botanic Gardens, Kew, 2003). Vanda testacea is an alkaloid rich epiphytic species of vandaceous orchids and is widely known for its medicinal properties. It is also a potential anti-cancerous drug (Chauhan, 1990). Many vandaceous orchids are also important in hybridization programmes for the cut flower industry (Goh & Kavaljian, 1989). The genus Thunia comprises about six terrestrial species that are native to India, Burma and South-East Asia. Thunia venosa, commonly called as the veined orchid is useful for breeding programmes. In the present study, to establish rapid regeneration protocols for in vitro mass propagation of these three economically important orchid genera by rapid development. 12

14 The specific objectives of the present research study are as follows: Study the effects of different plant growth regulators on the multiplication and development of protocorm-like bodies of Cymbidium Lunalvin Atlas. To use thin cell layer culture technique for regeneration of PLBs of Cymbidium hybrids using different plant growth regulators such as cytokinins, auxins, gibberellic acid, putrescine, AgNO 3 anti-cytokinins, auxin transport inhibitors and natural additives. To develop a regeneration protocol for native species - Thunia venosa Rolfe and Vanda testacea (Lindl.) Reichb. F. using different plant growth regulators. To understand the localization of starch and ultrastuctural details during the initial period of regeneration of PLBs from ttcl by histological techniques and transmission electron microscopy. To study the changes in the growth and development of the transferred plantlets to greenhouse by analyzing the changes in leaf surface morphology by scanning electron microscopy, estimation of photosynthetic pigments and anatomical changes occurring during acclimatization to assess formation of healthy and normal plants. 13