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International Journal of Recent Scientific Research Vol. 4, Issue, 7, pp.979 983, July, 2013 ISSN: 0976-3031

International Journal of Recent Scientific Research

REVIEWARTICLE ROLE OF TISSUE CULTURE IN MASS MULTIPLICATION AND CONSERVATION OF TEMPERATE HIMALAYAN ORCHIDS: AREVIEW
Burhan M Padder, Zahoor A Kaloo,Seema Singh and Gowhar A Shapoo
Department of Botany University of Kashmir Hazratbal Srinagar India, 190006 ARTICLE INFO
Article History:
Received 15th, June, 2013 Received in revised form 27th, June, 2013 Accepted 12th, July, 2013 Published online 30th July, 2013

ABSTRACT
Orchids are beautiful, mysterious and slow growing perennial plants exhibiting an incredible range of diversity in size, shape and colour of their flowers. They are known for longer lasting and bewitchingly beautiful flowers which fetch a very high price in the international market. Orchids are commercially grown worldwide for their cut flower and as potted plants with 8% share in floriculture trade and have the potential to alter the economic landscape of a country. It is considered outstanding in the ornamentals due to its diverse colours, shapes, forms and long lasting blooms. Orchids are valued for their medicinal properties as well. Many orchid plants are dwindling from their natural habitat due to over harvesting, destruction of habitat, population growth and urbanization; this has rendered Orchids as one of the most threatened groups of plants. Many species are considered endangered; therefore the management of orchid plant resources has become a matter of urgency. Tissue culture technology is potent and has opened extensive areas of research for biodiversity conservation. Tissue culture protocols have been developed for many plants, which includes endangered, rare and threatened plant species. Studies on invitro propagation and subsequent ex vitro cultivation of rare and endangered terrestrial orchid species could also be important for species reintroduction programs. This review article emphasizes work done in micro propagation and conservation strategies of temperate orchids therefore suggesting that more steps need to be taken for large scale propagation of these plants which are highly threatened and are at the front-line of extinction, with more species under threat globally than any other plant family and even the most modest of impacts are likely to add to the perilous conservation situation of orchids. Copy Right, IJRSR, 2013, Academic Journals. All rights reserved.

Key words: Orchids, micropropagation, terrestrial, Kashmir Himalaya

INTRODUCTION
Orchidaceae is a unique and one of the few largest families having fascinating and colourful flowers. They have a wide ecological range of distribution and their growth is closely correlated with environmental conditions (Aravindhan et al. 2011 Research in the last decades has provided recordings of numerous new species from the Iberian Peninsula (Bernardos et al. 2004) and the Balkan Peninsula (Petrova and Venkova, 2006; 2008).World over, presently 24,500 species are reported in Orchidaceae (Dressler 2005). In India, the family is represented by 177 genera with 1195 species (Singh et al. 2001) of which 400 species are endemic (Misra 2007).The temperate Himalaya includes the North Eastern Hill states, Uttrakhand, Parts of Himachal Pradesh and Jammu and Kashmir. The North Eastern region is considered as the most important biodiversity hot spot of the Indian subcontinent. This region has high humidity and low temperature accompanied by good rainfall that makes this entire region of the country a hot spot of Orchids also. This region has about 876 orchid species in 151 genera which constitute nearly 70% of total orchid flora of our country (Jain 1985). In Uttrakhand 72 genera with 237 species of Orchids are reported. Of these 127 are terrestrial, 99 epiphytic and 11 saprophytic. 17 species

are medicinally important, 12 taxa are recorded in Red Data Book of Indian plants (Jalal 2007, Jalal 2008). More than seventy six Orchid species representing different habits (terrestrial, epiphytic, lithophytic) occur in Himachal Pradesh state, many of which are of Ornamental and medicinal importance (Verma et al. 2009, Vij et al. 2011).In Indian state of Jammu and Kashmir nearly 47 species have been listed, out of which one species is endemic (Akther et al. 2011).However, as per the present surveys carried out by our team on diversity of Orchid species in Kashmir Himalaya, the species which have been reported so far include Cephalenthera longifolia; Epipactis helleborine, Listeria ovata and Oreorchis micrantha in coniferous forests of Dachigam National Park (1600m asl), Daksum (2600m asl) etc. Goodyera repens along the dense shady places of coniferous forests of Daksum. Spiranthes sinensis lancea was reported from the semi aquatic habit in the areas of the Manasbal Lake,Dactylorhiza hatagirea from the marshy areas of Kargil at an altitude of 3500m. On the other hand, orchids are one of the most threatened groups of plants. Many species are considered endangered (Salazar 1996, Morales 2000). The genus Epipactis (Orchidaceae) includes both highly selfing and highly outcrossing taxa. The outcrossing species are geographically

* Corresponding author: Burhan M Padder Department of Botany University of Kashmir Hazratbal Srinagar India, 190006

International Journal of Recent Scientific Research, Vol. 4, Issue, 7, pp. 979 - 983, July, 2013 widespread and morphologically very variable (Richards 1982). Contrary to this, the self-fertilizing species, which are more geographically restricted, tend to show very little morphological variation within populations, although they show some variation among populations. Moreover, the outcrossing taxa usually have larger and less pendulous flowers than the autogamous taxa (Proctor and Yeo 1973, Richards 1982). Dactylorhiza hatagirea known for its traditional medicinal values is dwindling rapidly from nature, which is alarming need for its conservation (Giri and Tamta, 2010).Orchids are cultivated for beautiful flowers, although they are known for their economic importance in cut flower industry and less for their medicinal value. Some like Dendrobium crumenative, Eulophia campestris, Orchis latifolia, Vanda roxburghii and Vanda tessellata have been documented for their medicinal value.Phytochemically some orchids have been reported to contain alkaloids, triterpenoids, flavonoids and stilbenoids. Ashtavarga (group of eight medicinal plants) is vital part of Ayurvedic formulations like Chyvanprasha and four plants viz, Riddhi, Vriddhi, Jivaka and Rishbhaka have been discussed as possible members of family Orchidaceae. Recently there has been tremendous progress in medicinal plants research; however orchids have not been exploited fully for their medicinal application (Singh and Duggal 2009). Tubers of Dactylorhizahatagirea which is commonly known as Hatajari in Uttarakhand; Salampanja in Kashmiryield a high quality Salep used for its aphrodisiac properties in traditional medicine in different part of Himalaya. It is considered as a nutritive and restorative tonic and also as an alternative source of Salep used very commonly in Europe. The annual consumption of salep obtained from the species in India is about 7.38 tonnes (valued at about Rs 50 lakhs) most of it imported from the other countries (Vij et al. 1992). All orchid species have the capacity for vegetative propagation. It manifests itself at various stages of ontogenesis and establishes a system of reproductive reliability (Batygina and Vasilyeva, 1980; Batygina and Shevtsova, 1985; Shevtsova et al., 1986). There are several patterns of vegetative reproduction of orchids varying between species possessing different life forms (Tatarenko and Vakhrameeva, 1998). In some groups of orchids the capacity for vegetative propagation is obvious even at the protocorm stage (Ziegenspeck, 1936; Batygina and Vasilyeva, 1983a,b; Andronova, 1988; Vinogradova, 1996). Protocorms can possess several centres of meristematic activity, but usually they develop only one shoot. Vegetative reproduction of specimens at protocorm stage is hardly ever observed in nature (Tatarenko and Vakhrameeva, 1998), whereas in culture in vitro protocorms are employed to obtain the greatest number of regenerants (Batygina and Shevtsova, 1985; Shevtsova et al., 1986). The period of time necessary for development of individual plants before flowering of different terrestrial orchids is extremely long. On average a period of 12 years is necessary for development of Cypripedium species from seed germination until flowering (Curtis 1943; Kull1999). The major constraint in conventional propagation of Orchids is slow clonal multiplication and in provision of insufficient clones within a short time frame (Martin and Madassary, 2006). The use of in vitro culture technique in orchid propagation allows obtaining flowering plants within 4 to 5 years of culture or even after 2 to 3 years (Stoutamire 1974; Rasmussen 1995).Orchids are known for their ability to overcome consolidated genetic barriers, and thus to generate intra- and intergeneric hybrids (Bonati et al. 2006). Hence, in vitro propagation is used for rapid mass multiplication of these valuable plants, as these techniques are universally applicable in large-scale multiplication of plants but at the same time delivery of hardened small micropropagated plants to growers and market also requires extra care (Ahloowalia et al. 2002). Invitro seed germination leading to the development of protocormshas been an efficient technique for micropropagation of temperate orchids including Cymbidium elegans Lindl. Using BAP1mgl-1(Pant and Pradhan 2010); Cypripedium calceolus L., Dactylorhiza macualata L. Soo, Epipactis helleborine L. Crantz, Gooderya repens L. R. Br. and Gymnadenia conopsea L. Br. on MS+NAA+BA 1.0mgl1( Pindel and Pindel 2004); Cypripedium formosanum (Lee and Lee 2003); Cypripedium candidum at BA and 2ip upto 0.8mgl-1(Depauw et al. 1995); Dactylorhiza fuschii at Knudson C and half strength MS(Jakobsone 2008); Liparis loselli half strength MS (Zoltan et al. 2005); Grammatophyllum speciosumhalf strength MS+ Chitosan 25mgl-1(Sopalum et al. 2010); Geodorum densiflorum Lam. Schltr. at 2.0 mgl1 +NAA+2.0mgL-1 BAP, MS and Phytamax Media(Bhadra and Hussain 2003), Knudson 1946, Cymbidium Scott and Arditti 1959; Dactylorhiza fuschii Druce Soo and Platenthera bifolia L. Rich. Fast medium+ 1.0mgl-1 BA(Nikishina et al. 2007). During the present study a number of temperate Orchid species were subjected to in vitro studies. The results reported so far include asymbiotic seed germination of Epipactis royleana, Dactylorhiza hatagirea and callus development from leaf explants in Epipactis helleborine. An efficient method of propagation in Dactylorhiza hatagirea has been developed via protocorm regeneration by culturing immature seeds from unripe fruits on half strength Murashige and Skoog medium supplemented with BAP 2.5mgl-1 and activated charcoal (AC) 5gl-1. Epipactis royleana seeds germinated on MS medium supplemented with BAP 2.5mgl-1. While evaluating the regeneration competence of different explants in Epipactis helleborine, the leaf explants used produced Callus on BAP (2.5mgl-1) supplemented MS medium. The Callus was acholorophyllous and compact in nature. Cytokinins have proved to be the most effective growth regulators affecting invitro seed germination of temperate terrestrial orchids. Among these BA and 2ip enhanced germination whereas kinetin had little effect. It is also reported that lower concentrations of the above two hormones are more effective than kinetin at higher concentrations including the reports of Arditti 1979; Arditti and Ernest1984; Anderson 1990; Blonstein et al. 1991; Borse and Van Staden 1989, Depauw et al. 1995; Harvais 1982, Henrich et al. 1981;; Shimasaki and Uemoto1990; Van Waes and Debergh 1986; and Vander Krieken et al. 1990. Orchids require specific mycorrhizal association especially during the seed germination stage for uptake of nutrients. With an objective to isolate the endophytic mycorrhiza from the natural species and applying it as a biofertilizer by inoculating into the invitro cultured seedlings of rare, endangered and commercially viable orchids for successful establishment in the field with better survival rate (Saha and Rao 2006). The optimum temperature for seedling growth in vitro is usually around 20-25C (Rasmussen, 1995), but some species

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International Journal of Recent Scientific Research, Vol. 4, Issue, 7, pp. 979 - 983, July, 2013 develop better at cooler temperatures. Periods of lower temperatures resembling natural conditions should occur to induce dormancy periods (Malmgren, 1989; 1993). Thermo periodicity with chilling temperatures should also be utilized in vitro for breaking bud dormancy (Riley, 1983; Malmgren, 1988; Anderson 1990; Rasmussen, 1995). Most investigators suggest vernalization for 3 months at 5C. For Dactylorhiza majalis bud dormancy can be broken in 8 weeks if the seedlings are sufficiently large (longer than 5-8 mm) when the cold treatment is begun (Rasmussen, 1995). Orchids are one of the most fascinating groups of ornamental plants, and numerous novel cultivars have been produced by interspecific, as well as intergeneric, hybridization of plants with exotic and elegant flowers. However, many orchid species are endangered as a consequence of environmental disruption, succession of natural habitats, and overexploitation for horticultural purposes. In situconservation by preservation and enhancement of dwindling populations of endangered orchid species is very difficult because of the relatively slow growth of orchids and low germination rates which requires symbiotic relationships with mycorrhizal fungi in natural habitats (Bernard 1903; Warcup 1973; Rasmussen 1995). Thus, in recent years, the maintenance of living collections has been considered to be an important aspect of conservation (Sharrock 2007; Somiya 2007). India has a rich biological diversity due to its varied climatic, altitudinal variations and ecological habitats. There have been increasing rates of threats of depletion to these biological resources due to immense biotic and abiotic stresses. Indiscriminate collection of plants for their medicinal, ornamental, perfumery uses, etc. and habitat loss and degradation are potential causes of threats. Conventionally, there are two methods of conservation: in situ and ex situ conservation, both are complementary to each other. Ex situ conservation via in vitro propagation also acts as a viable alternative for increase and conservation of populations of existing bioresources in the wild and to meet the commercial requirements (Kapaia, et al. 2010). The prime importance of in vitro propagation of rare, critically endangered, endangered and vulnerable plants would be to generate a large number of planting materials from a single explant without destroying the mother plant and subsequently their restoration in the natural habitat, thus conserving the biodiversity. The significance of an efficient in vitro protocol would be to obtain maximum number of plantlets in minimum period of time with proper rooting along with acclimatization in the field. The different regeneration systems which have been developed need to be field tested and the field data is collected so that the complete technology packages could be ready for commercialization and transfer to the user agencies Indian Himalayan State of Jammu and KashmirPleione5(1):1 - 9. Anderson, A.B. 1990. Asymbiotic germination of seeds of some North American orchids, In: North American native terrestrial orchid propagation and production, ed. Sawyers, C.E., p. 7580. Andronova E.V. 1988. Embryogenesis and post seminal development of orchids (on example of Dactylorhiza baltica, D. incarnata, Thunia alba,Bletilla striata) Ph.D dissertation, Komarov Botanical Institute, Leningrad, Russia (in Russian). Aravindhavan V., Sathiyadas K., Rajendran A. and Thomus Binu 2011. 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