The possibilities of commercialization of Indian orchids: application of tissue culture techniques

SoP, Vij Orchid Laboratory. Department a/Botany, Panjab Un iverslIy. Chandigarh- 160 ON. India.

The orchids represent a group of botanically interesting and commercially significan;. ;:Li":': which have outnumbered and outsmarted their counterparts by evolving ingenl\::~ higher levels of specializations, They comprise nearly 20,000 species in 800 genera, culminate one of the evolutionary lines of monocots, and are still in an active state of speciation due to poorly developed barriers of reproductive isolation. Suppressed development of endosperm, microscopic seeds with undifferentiated embryos, and dependence upon a variety offactors including specific pollinators and fungi for continued reproduction in nature are some of their distinguishing features. The orchids possess eXlfPmely beautiful, intricately fabricated, highly colourful, and long-lasting flowers of myriad sizes and have significantly ','ontributed to the development of international trade in cut-flowers and pot-plants. Possibilities of producing new and novel varieties through successful wide matings have added new dimensions to the significance of these plants in the trade~ they rank among the top ten sought after plants for cut-t1ower production, Many of them are rich in phytochemical contents as well and are extensively used in local medicines for their curative and aphrodisiac properties. Thailand, Singapore, Malaysia, Taiwan, Korea, and Sri Lanka are the major asian players in the orchid trade with Japan and US.A as the biggest expol1 destinations.

India with a vast geographic expanse and varied climatic regions is rich in plant genetic resources. TheiJrchids have naturalized here in great profusion~ they are represented by nearly 1200 species (Sathish Kumar, Manila!, 1994). The rich diversity of Indian orchids includes several with proven ornamental and/or therapeutic values. Many of them pa11icularly in the genera Aerides. Arachnis. Calanthe, Cymbidium, Cypripedillm. Den,''',billm, Paphiopedilul11. Phalaellopsis, Pleiolle. Renanthera. Rhynchostylis, Vanda etc. are source of incalculable aesthuic pleasure because of their floral excellence. Besides adding to the prized collections of almost all the famed botanical gardens, the world over, they have been extensively used to progenate internationally acclaimed hybrids with heavier texture, exotic colour combinations, and long keeping qualities of flowers (Bose, Bhattacharjee, 1980). Unfortunately, the orchid genetic resource remained unexploited in

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A. Altman et al. (eds,), Plant Biotechnology and In Vitro Biology in the 21st Century, 729-736. © 1999 Kluwer Academic Publishers.

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the country due to different national priorities and lack of awareness about its commercial significance. The scenario has now changed with the increased level of awareness about these plants and changed national policies which favour orchid based floriculture, but we are faced with conservation related problems. A sizeable number ofIndian orchids have succumbed to unregulated collection and habitat destruction pressures; their natural populations are declining rapidly. While the Government ofIndia has already banned the expo:. Jf orchids collected in wild and has taken steps to protect orchid rich habitats, the efforts are not as yet commensurate with the dimensions of the problem. The orchids are still being collected stealthily from their natural and foster homes and shall continue to be collected as long as they enjoy an economic status and demand. Mass propagation using conventional and tissue culture techniques thus seems to be the only strategy to commercialize orchids and save their natural populations from collection pressures.

The orchids are primarily sexual but they reproduce a great deal through vegetative means as welL Their vegetative propagation through conventional means i.e. keikis, back-bulbs, division of shoots, etc., is rather slow. Even their natural regeneration through seeds is limited. Only 0.2-0.3% seeds germinate because they are poorly organized (the development of endosperm is suppressed and that of embryo remains arrested at the 'globular'tpre­heart-shaped' stage), lack ail appropriate metabolic machinery to utilize their own lipidaceous food reserves, and require a suitable mycorrhizal association for germination in nature. Th~ fungus is believed to provide the necessary stimulus by aiding carbohydrate/auxin and/ or vitalnin transport during germination (Hayes, 1969). The tissue culture techniques have, on the other hand, opened new possibilities in conservation and commercialization of orchids. Round the year propagation of genetically uniform, disease-free, fast maturing and high yielding plants is now within the realms of reality. The technique exploits the regenerative potential of plants much more effectively than the conventional methods of vegetative reproduction. and can easily satisty the large scale requirements for genetically identical planting materials. Moreover, the micropropagated plants grow more vigorously due to their better health status. Even to keep pace with the ever changing consumer preferences, the possibility of using tissue culture techniques for mass multiplication of new and better developed plant varieties (through desired matings and/or genetic engineering) is tremendous. It is also possible to enlarge the genetic base by inducing somaclonal variations. Incidentally, the orchids represent the first floricultural crop successfully propagated through tissue culture techniques. The immature embryos and shoot meristems are amongst the most commonly used explants for in vitro propagation of orchids. The embryo culture helps in enbrging the genetic diversity and shoot meristems in maintaining genetic purity.

SeedlEmbryo Culture Ever since Knudson (] 922) demonstrated the possibility of bypassing the fungal requirement of orchid seeds during germination in vitro, asymbiotic seed germination has been accepted as an important procedure for propagating orchids (Arditti et aL, 1982). The orchid seeds can also germinate prior to reaching maturity. The technique of culturing immature seeds

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is variously referred to as ovule/embryo/green pod/green fruit culture technique (Sagawa, 1963). It IS easy to follow. ensures better germination frequency, and favours rapid production of virus-free seedlings. Its additional utility in recovering the progenies of desired matings, propagating rare and endangered species, and cloning apomictic (obligate) genotypes is also well documented (Vij, 1992). The asymbiotic germination potential of seeds, representing different developmental stages, has been positively tested in several commercially viable and/or threatened Indian taxa (cf Vij, 1995). In orchids, very young ovules do not form suitable explants hcause the embryo sac development is a post -pollination phenomenon and fertilization a pre-requisite for obtaining seedlings. However, as the ovules can be used for raising cultures immediately after fertilization, the importance of information on time interval between pollination and fertilization has often been stressed (Valmayor, Sagawa, 1967). Though such studies are yet to be initiated in the Indian orchids, the time (in wks) after pollination when the ovules can be successfully germinated seems to vary with species (Vij, 1995). Eulophia hormllsjii embryos, procured between 8 and 16 wks after pollination, germinate readily but their germination frequency declines sharply with further passage of time; only a few of them respond when obtained later than 16 wks of pollination. Likewise in Satyrillm nepalense. Nephalaphyllum cord~fo1illm. Phaills rankervi!!ieae. and cymbidiums, the germination frequency shows a sharp decline when the embryos are collected 3-4 wks prior to fruit dehiscence; the mature embryos either fail to germinate or germinate very poorly. Since they can be induced to germinate by treating with cold temperature, 20% sucrose, and/or KCl, it appears that dormancy rather than the non-"i~bility factors are responsible for their poor response in vitro. The impaired germination of the mature seeds has also been attributed to the quality of their food reserves. Harvais (1974) reported that the food reserves comprise starch in the immature and lipids in the mature seeds of Corallorhiza maC/tlata, and suggested that conversion of starch and other simpler carbohydrates into lipids, during seed maturation may be a common feature of orchids. The inability of the mature seeds to germinate, with ease, was correlated with lack of an appropriate metabolic machinery (glyoxysome bodies) to utilize their own lipid reserves. Raghavan (1976) recorded changes in the enzyme complements at different stages of seed maturation in orchids but the critical stage at which they acquire dormancy is yet to be identified. Moreover, as the dormant (mature) and metabolically active (immature) seeds are morphologically more or less indistinct, Mitra (1986) stressed the importance of information on histochemical and biochemical features for selecting the fruits for the right type of immature ovules (seeds). In this connection, the ablility of embryos from fruits which develop prominent ridges along the valves and cease to grow in diameter to respond better (Vij, 1995) may be useful in selecting the right stage offruits for embryo culture.

Meristem Culture Resident meristems: Since the orchids are outbreeders and generate a great deal of heterozygosity in their progenies, their propagation through embryo culture appears to be a disadvantageous proposition in cut-flower industry where pure lines of desired genotypes are preferred.

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Morel (1960) demonstrated the possibility of using excised shoot-meristems for regenerating complete plants of Cymbidium, in vItro, and Wimber (1963) formulated, described, and published a procedure for the purpose. The technique of resident meristem (shoot-tip, axillary bud) culture has opened new vistas in orchid micropropagation (cf Arditti, Ernst, 1993). Upto 200,000 plants can be regenerated, within a year, from a single resident meristem The technique has, however, a limited utility in monopodial taxa as it requires the sacrifice ofthc nntire new grovvth or the only growing point and endangers the survival of the mother plant

Adventitiolls meristems: Several efforts have been made to develop an equally effective multiplication system by inducing de novo formation of adventitious meristems in organs whose excision is not detrimental to the survival of mother plant. The proliferative potential of explants from leaf (Chaturvedi, Sharma, 1986; Mathews, Rao, 1985; Seeni, 1988; Seeni, Latha, 1992; Vij et ai, 1984, 1986a, 1988a; Vij, Pathak, 1988, 1990), root (Chaturvedi, Sharma, 1986; Sood, Vij, 1986; Vij, et ai, 1987; Vij, Pathak, 1988b; Vij, 1993, 1994) and flower stalks (Singh, Prakash, 1984; Kaur, Vij, 1995; Vij, et ai, 1986b; Vij et ai, 1997) has been positively tested in some Indian orchids. The source, genetic constitution, and physiological age of the explants are, however, some of the important factors for regeneration. The juvenile tissues from greenhouse grown plants respond better than the mature ones from plants grown outdoors. The proliferative loci, in generaL get activated in the dermal cells and soon deve!, ':) into somatic embryos (Proto corm Like Bodies: PLBs). Direct or callus mediated development, multiplication, and di11erentiation of the PLBs is influenced by the chemical stimulus in the nutrient pool (Vij, Pathak, 1990; Seeni, Latha, 1992). Most orchid tissues require a treatment with plant growth regulators for growth and development This requirement, however, varies with the species and source of explant. The use ofNAA and either of BAP and KN, in the medium, yields a rich crop of PLBs in Luisia trichorhiza, Satyriul11 nepafense, vanda cristata, and V testacea leaf segment culture (Vij, 1995). Similarly, a synergistic action ofKN and IAN NAA, in peptone enriched medium, favours enhanced production ofPLBs in Rhynchostylis retllsa cultures. Yeast extract is obligatory for regeneration in Aerides l11ultiflorum, Papilionanthe teres, and Satyrium nepalense foliar cultures and peptone in those of randa. However, as the juvenile leaves can proliferate all along and the relatively older ones only in their basal regions (Vij et ai, 1986) and the tip region regenerates infrequently when excised and more frequently in an entire leaf(Mathews, Rao, 1985), it was suggested that the inherent (latent!) meristematic potential in orchid lea'les is perhaps controlled by some factors emanating from the leaf base and it diminishes with ,';,dturity (Vij, Pathak, 1990).

In the root explants, the effect of an exogenous supply of plant growth regulators is species specific and it varies during initiation, multiplication, and differentiation of cultures. It is possible to induce etiolated growth in the explants or produce plantlets therefrom (through direct or callus mediated development of either of PLBs and shoot buds) by varying the

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quality of the growth adjunct(s) in the medium. Incidentally, as the explants fromjuvenile roots (with poorly developed root- caps) regenerate with ease, and the ones from mature roots with well developed root-caps faii to do so, it appears that the root-cap plays an important role in controlling the regenerative potential of roots. In this connection, it is worthwhile to mention that root-cap is an active site of IAA accumulation and the transformation of a root meristem into a shoot meristem is positively influenced by the endo­and/or exo-genous level of the auxin; its supra-optimal level maintains root meristem and sub-optimal level brings about a switch in the activities of the meristematic cells with the result that the quiescent centre and other derivative cells are induced to develop into a shoot meristem (Philip, Nainar, 1988).

The advantages ofleaf and root segment culture are apparent for more than one reason; they are easy to obtain, easier to disinfect, and their excision does not endanger the mother plant. Moreover, as the regeneration occurs in the dermal cells which are cytologically more stable, mass production of genetically uniform plants from these is a distinct possibility. A two-step technique involving culture initiation from flower stalk cuttings and subsequent use offoliar explants is expected to yield better results for cloning desired genotypes.

The release of brownish exudates (phenolics!) by the explants into medium is a serious problem in orchid cultures as these significantly impair the growth of the germinating entities or regenerants. We have successfully alleviated the harmful effect of these exudates by using activated charcoal (AC) in the medium. According to Yam et al. (1989), AC favours better health of the cultures because of its ability to adsorb exudates/growth inhibitors, enhance medium aeration and, absorb light and provide enhanced quantum of energy per unit plant material.

Precocious flowering Free gene-flow across the taxonomic limits favours the development of new genotypes in orchids; their evaluation and subsequent selection for floricultural purposes is, however, a time consuming process because these plants take several years to reach maturity. The recent possibilities of inducing precocius flowers in vitro, however, offer tremendous opportunities to the breeder to select suitable clones from a hybrid progeny for commercial purposes within a short period of time. We have successfully induced in vitro florigenesis in Dendrobiul11 denlldans, Dendrobiul11 Snow Fire, Vanda coerulea , Eulophia dabia, Spiranthes sinensis. and Zeuxine stratelll11atica cultures. While a cytokinin (BAPIKN) treatment proved useful for flower induction in most of these taxa, a treatment with IAA was obligatory for the purpose in Dend. denudans. In vanda, the in vitro induced floral buds failed to open into complete flowers despite repeated subcultures as is also true in Phalaenopsis and Dendrobium cultures; frequent subcultures were required for normal development of flo.al buds in Doriella cultures (Duan, Yazawa, 1994).

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Culture transplant and transport Incidentally, regeneration in the explants, proliferation of the regenerants, differentiation of the plantlets (complete with roots and shoots) and their transplantation to soil/field are the 4 important stages in micropropagation process. The micropropagants have poorly developed cuticle, stomatal apparatus, photosynthetic ability, and conducting tissues, and they fail to withstand direct exposure to harsher climates outside the m vitro regimes until or unless they are properly acclimatized prior to transplanting. The procedure, known as hardening, helps cutting down their mortality rate, during lab to land transfers, by correcting the above mentioned morpho-physiological aberrations. Storage and long distance transport of the micropropagants are among other problematic aspects. The possibility of overcoming these probkillS by preparing somatic/artificial 'seeds' or beads (encapsulation ofpropagules in a nutritive gel) is being increasingly realized. Such 'seeds' have so far been prepared in nearly 20 orchid species by encapsulating their meristems/embryoids in nutrient supplemented calcium alginate gel. The quality of the beads varies with the concentration of calcium chloride and sodium alginate in the medium; they are best formed by using 2.5% sodium alginate and 100 mM calcium chloride with a complexation period of 30-40 min (Vij et al.,1992). They can germinate readily on a variety of substrata including agar, sand, vermiculite, etc. and retain their viability for about 4 months when stored at 4°C. The synthetic 'seeds' are easy to handle and ensure economy of space, medium, and time during storage, lab to land transfer, and long distance transport of tissue culture raised genotypes. The nutritive gel, in them, acts like an artificial endosperm and provides necessary growth stimulus to the enclosed embryoids/meristems besides saving them from mechanical injury. Since the encapsulated propagules invariably proliferate during germination, the synthetic 'seeds' represent a novel system for exploiting the inherent polyembryonate potential of orcilids.

Conclusions The international trade in floriculture is developing rapidly and the prevalent 'orchid mania', the world over augurs well for orchid based floriculture. India is a rich repository of orchids (1,200 species). Many an Indian species have been used to progenate internationally acclaimed hybrids; they are even known for their curative and aphrodisiac properties. Unfortunately, till very recently, the orchid based floriculture could not pick up in the country due primarily to different national priorities, conservation related problems, and lack of appropriate planting materials. Application of tissue culture techniques has, however, opened up new possibilities to conserve and commercialize Indian orchids. The immature embryos and shoot meristems are amongst the most commonly used explants for in vitro propagation of orchids; large scale propagation of elite clones and disease-free propagules, and development of new and novel varieties are within the realm of reality. Embryo culture helps in enlarging genetic diversity and shoot meristems in maintaining genetic purity. Since the shoot meristem culture requir~s the sacrifice of entire new growth or the mother plant itself, the possibility of using leaves (whole/segments/dermal pee1.,), roots, and flower stalks as alternate but equally effective donor tissues has been positivley tested. Their efficacy, however, varies with the

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species, maturity level of the donor tissues, and chemical stimulus in the nutrient pool. Since orchid plants raised form seeds require several years to reach maturity, the technique of inducing precocious flowering in the proto corms provides the breeder with an opportunity for early selection of desirable hybrid genotypes for commercial propagation. Proper devc;opment of orchid based floriculture, however, calls constant efforts to improve the quality of planting material through desired matings and by inducing somaclonal variations and to develop cost effective reproducible micropropagtion protocols for commercially viable genotypes particularly the hardy ones with a wider ecological amplitude

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