IN VITRO MICROPROPAGATION OF DENDROBIUM HERBACEUM LINDL. FROM LEAF EXPLANTS Sr. Sagaya Mary.B and Divakar K.M.

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1 ISSN X 1 International Journal of Advance Research, IJOAR.org Volume 3, Issue 10, October 2015, Online: ISSN X IN VITRO MICROPROPAGATION OF DENDROBIUM HERBACEUM LINDL. FROM LEAF EXPLANTS Sr. Sagaya Mary.B and Divakar K.M. Plant Tissue Culture Division, Department of Botany, St. Joseph s Post-Graduate Studies and Research Centre Langford Road, Bangalore ABSTRACT Dendrobium herbaceaum Lindl the white coloured flowers of this sympodial orchid usually blooms in the months of January-May. This bushy leafless epiphyte with its inflorescence having innumerable flowers sitting high on trees being the highlight helping in identification of this species. The typical size of the flower is less than a centimetre in width and when observed closely the greenish lip or labellum is seen.tender Leaf explants of Dendrobium herbaceaum plant were inoculated on various medium VW, B5, MS and KC for In vitro micro propagation. B5 medium produced numerous shoot lets. Small shoot lets were transferred to shoot elongation medium supplemented with 2 mg BAP, 1mg NAA, for plantlets and 2 mg BAP, 1mg IAA, for rooting. Rooted plantlets were transferred for hardening showed per cent of plantlets success-fully established in their field. Keywords: Dendrobium herbaceum Lindl. VW, B5, MS, KC, NAA, IAA, BAP, AC & CM. ABBREVIATIONS: VW - Vacin and Went medium, B5 - Gamborg B5 medium, MS- Murashige and Skoog medium, KC Knudson C, NAA Naphthalene Acetic Acid, IAA Indole Acetic Acid, BAP Benzyl Amino Purine, AC Activated Charcoal & CM Coconut Milk.

2 ISSN X 2 INTRODUCTION: Dendrobium herbaceum Lindl. Orchids are herbaceous plants that are classified on their name in the family, the Orchidaceae. In many Asian countries, orchids are used as traditional drugs since time immemorial. The roots, tubers, stems, leaves or the whole plants have been used as medicines known to possess antibiotic, ant malarial, rejuvenating and many other properties. The medicinal importance of orchids was known in India since Vedic period. The genus dendrobium is the second largest group among the orchids plant in India and exhibit diverse shapes, colour and morphological characters. Earlier in vitro propagation of Dendrobium herbaceum was attempted through organogenesis from different explants such as leaves, shoot tips, axillary buds and callus (Rout 2002, Saatheesh Kumar and Seeni 2003, Satheesh Kumar and bhavanandam 1988) and somatic embryo (das and rout 2002).The present study was aimed at developing a simple, rapid and an efficient protocol of plant regeneration from leaf explants of Dendrobium herbaceum. In order to meet the demand of this species an in vitro system has been investigated. Dendrobium herbaceum plants are medicinal and its roots are used for Skin diseases, Fresh roots are burnt and 10 g of the resultant ash is mixed with 10 ml mustard oil and applied on the affected portion 2 to 3 times daily for several days till it disappears. (Mishra, 1956, 1990; Saxena, 1970) In Dendrobium herbaceum usually the flowering lasts several weeks. Normally once the flowering season is complete and rains set in, the same plant can be seen full of new leaves and shoots. Typical habitat to find this species is mid to lower elevations of the Southern Western Ghats. It also observed in other parts of India as well. Even though this species flowers profusely, flowers being very small may be the reason behind this species being not very popular in cultivation. This hardy species once established will start flowering regularly every year or so. When it is not in bloom, with its numerous attractive canes and leaves it is wonderful orchid to grow and conserve. Materials and Methods: The healthy plants of Dendrobium herbaceum were collected from somavarpet / coorg, from the natural environment in perforated, clean, polythene bags. Care was taken to ensure to retain the mother plant intact in its natural epiphytic territory. And raised in pots containing soil and farm yard manure (1:1) in green house in the department of botany, St. Joseph s Post-Graduate and Research Centre, Bangalore. Surface sterilization using leaf culture Leaf explants were surface sterilized by cleaning thoroughly under running tap water and washed with a solution of Tepol (Two drops in 100 ml water) for 5 min, and again washed with sterile distilled water. The cleaned explants were finally treated with Hg cl2 (0.1%) for 3 min under aseptic conditions and washed six times with sterile distilled water to remove traces of Hg cl2. After surface sterilization, leaf explants were trimmed and inoculated on B5 basal medium supplemented with different concentrations and combinations of 2mg BAP+1 mg NAA shoot induction. Fully developed plantlets transferred to B5 basal medium with plant growth

3 ISSN X 3 regulators for root induction. For shoot elongation, small shoots were transferred to B5 basal medium supplemented with 2 mg BAP, 1mg IAA after 25 days; the cultures containing the rooted plantlets were transferred to the greenhouse at per cent humidity. In vitro rooting In vitro rooting was successful with B5 medium supplemented with 2 mg BAP, 1mg IAA, 50 ml CM and 500 mg AC. Ex vitro rooting The basal ends of healthy shoots from the shoot multiplication medium were dipped in an auxin solution, 10 ml of IAA (made in distilled water) then planted in small pots containing solrite (potting mix) sprayed with bavistin to avoid fungal infection. In vitro rooted plants in the pot trays containing potting mixture maintained under mist chamber and covered with perforated plastic cups. Hardening Well grown shoots were directly transferred to small pots containing soil, sand and solrite (mixture of pearlite and peat moss) and were kept in the green house. Successfully established plantlets were subsequently transferred to field condition. OBSERVATIONS: 1.Dendrobium herbaceum in coorg 2.dendrobium herbaceum 3.Dendrobium herbaceum flowers 4. dendrobium herbaceum with fruits 5. Leaf culture 6. Plantlet formation

4 ISSN X 4 7. Leaf Sub culturing 8. In- vitro Rooting 9. Ex-vitro Rooting 10.Hardening 11. Hardening in Green House 12.Transfer to Green House RESULTS AND DISCUSSION MS, B5 and KC media was used (Table 1) Media used Media composition Average plantlet formation (percentage) 3 VW 2 mg BAP +1.5 mg NAA + 50 ml CM 1 mg BAP mg NAA + 50 ml CM 2.5mg BAP + 2 mg NAA + 50 ml CM 40% 30% 2 1 Series1 0% B 5 1mg BAP mg IAA + 50ml CM 1.5mg BAP + 0.5mg IAA+50ml CM 2mg BAP+ 1mg IAA + 50 ml CM 30% 40%

5 ISSN X 5 MS 2 mg BAP + 1 mg NAA + 50 ml CM 1.5 mg BAP mg NAA + 50 ml CM 1 mg BAP +1.5 mg NAA + 50 ml CM 30% 40% Media used Media composition Average plantlet formation (percentage) B5 1mg BAP mg NAA + 50ml CM+500 mg AC 1.5mg BAP mg NAA+50ml CM +500 MG AC 2 mg BAP + 1mg NAA + 50 ml CM+ 500 mg AC 40% 60% 100% 0% Series1

6 ISSN X 6 KC - For the In vitro Rooting (Table 3) Med ia Used Media Composition Results The average rooting (percentage) B5 1mg BAP mg IAA + 50ml CM+500 mg AC 1.5mg BAP + 0.5mg IAA+50ml CM +500 MG AC 2 mg BAP + 1mg IAA + 50 ml CM+ 500 mg AC 80% 85% 95% % Series1 Conclusion: The results showed the ability of the leaf explants to produce higher number of shoot lets without intervening callus phase, where all the plantlets were uniform in height and growth. This study establishes a simple, rapid, high frequency microprobagation method for Dendrobium herbaceum from leaf explants and also this present system of normal root culture would be beneficial for the sustainable utilization of this rare endemic orchid for its bioactive ingredients, thereby providing an alternative method rather than destroying whole plants that are not under cultivation. From these studies it can be concluded that B5 medium suitable for Dendrobium herbaceum Lindl. This study also revealed that a low concentration of 2 mg BAP + 1 mg NAA + 50 ml CM was found to be more suitable for plantlets and multiple plantlets. B5 medium supplemented with Basal B5 Medium + 2 mg BAP+ 1mg IAA+ 50 ml CM +500 AC giving highest percentage was found to be suitable for In vitro Rooting.

7 ISSN X 7 Reference list of important books, journals and reports referred. 1. Ambasta SP. (Eds.). The Useful Plants of India. Councile of Scientific & Industrial Research, New Delhi, Anonymous. The Wealth of India Raw Materials. Council of Scientific & Industrial Research, New Delhi, Bondya SL, Khanna KK, Singh KP. Ethno medicinal uses of leafy vegetation from the folk-lore of Achanakmar-Amarkantak Biosphere Reserve (Madhya Pradesh and Chhattishgarh). Ethnobotany 2006; 18: Bondya SL, Singh KP, Khanna KK. Exploitation of ethnomedicinal plants and their marketing status in Achanakmar-Amarkantak Biosphere Reserve. Journal of Tropical Forestry 2009; 25 (I & II): Bulpitt CJ. The uses and misuses of orchids in medicine, Q J Med 2005; 98: Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants. Council of Scientific & Industrial Research, New Delhi, Dubey PC, Khanna KK, Sikarwar RLS, Tiwari AP. Threat Assessment of Plant Diversity in Amarkamtak Area. 2007; In: Joshi, K.C. & A.K. Mandal (Eds): Research Needs for Achanakmar-Amarkantak Biosphere Reserve. 9. Jain SK. Medicinal Plants. National Book Trust, India, New Delhi, Jain SK, Mudgal V. Handbook of Ethnobotany. Bishen Singh Mahendra Pal Singh, Dehradun, Kapale R. Ethno medicinal Plants used by Baiga Tribals in Amarkantak Meikal forest of Madhya Pradesh (India). Bulletin of Environment, Pharmacology & Life Sciences 2012; 1(4): Kirtikar KR, Basu BD. Indian Medicinal Plants. Vol. 1-4 (2nd Ed.). Bishen Singh Mahendra Pal Singh, Dehra Dun and Periodical Experts, New Delhi, Kumar R, Suman NR, Dash SS. Traditional Uses of Plants by Tribal of Amarkantak Region, Madhya Pradesh. Indian Journal of Traditional Knowledge 2004; 3(4): Mishra R. The vegetation of Amarkantak. Bulletin Botanical Society, University of Saugar, 1956; 8: Mishra OP. Addition to the flora of Amarkantak (M.P.). Journal of Economic and Taxonomic Botany 1990; 14: Sahu PK.Traditional knowledge and indigenous medicine of the tribal of Biosphere Reserve, Central India. International Journal of Pharmacy & Life Sciences 2010; 1(8):

8 ISSN X Satyavati GV, Gupta AK, Tandon N. Medicinal Plants of India. Indian Council of Medical Research, New Delhi, Saxena HO. The flora of Amarkantak (Madhya Pradesh). Bulletin Botanical Survey of India. 1970; 12 (1-4): Shukla AN, Singh KP. Diversity of woody plants in Achanakmar-Amarkantak Biosphere Reserve of Central India. Indian Journal of Forestry 2007; 31(2): Shukla AN, Singh KP, Kumar A. Ethno veterinary uses of plants from Achanakmar- Amarkantak Biosphere Reserve of Madhya Pradesh and Chhattisgarh. Journal of Non-timber Forest Product 2007; 14: Shukla AN, Singh KP. Contribution to the flora of Achanakmar-Amarkantak Biosphere Reserve, Central India. Indian Forester 2012; 138(1): Sikarwar RLS. Some Unrecorded Ethno medicinal Plants from Amarkantak Plateau of Madhya Pradesh. Tribal Research and Development Institute, Bhopal, Ingh NP, Khanna KK, Mudgal V, Dixit RD. Flora of Madhya Pradesh, Vol.-3.Botanical Survey of India, Calcutta, Singh S, Dixit RD, Sahu TR. Ethno medicinal uses of Pteridophytes of Amarkantak, Madhya Pradesh. Indian Journal of Traditional Knowledge 2005; 4(4): Singh KP, Shukla AN, Singh JS. Floristic diversity and taxonomic profile of the vegetation of Achanakmar-Amarkantak, Biosphere reserve, Central India. Journal of the Bombay Natural History Society 2010; 107 (2):