Research Journal of Recent Sciences ______ ______________________________ ______ ____ ___ ISSN 2277 - 2502 Vol. 4 ( I YS C - 201 5 ), 1 0 - 13 (201 5 ) Res. J . Recent . Sci. International Science Congress Association 10 Impact of temperature and pH Variation on in - vitro Protocorm formation of Vanda tessellata (Roxb.) Hook. ex G.Don an endangered medicinal Orchid Bindiya Sachin Department of Botany, Sarojini Naidu Govt. Girls Post Graduate (Autonomus) College Shivaji Nagar , Bhopal, MP, INDIA Available online at: www.isca.in , www.isca.me Received 18 th June 201 5 , revised 22 nd August 20 1 5 , accepted 5 th September 20 1 5 Abstract The present research work is carried out to study the In - vitro protocorm formation of an endangered medicinal orchid Vanda tessellata at different temperature(15°C, 20°C, 25°C, 30°C) and pH (4.5, 5.5, 6.5,7.5) . Immature seeds obtained from green pods successfully shown protocorm for mation on basal media MS without various combinations of growth hormones. Highest protocorm formation ( 94±0.17%) were observed at temperature 20°C and pH 5.5 , whereas the medium which is adjusted at temperature 15°C, 25°C, 30°C and pH 4.5, 6.5, 7.5 show ed poor protocorm formation. Keywords : Vanda tessellata , seed germination, protocorm, In - vitro . Introduction ORCHIDS, the most beautiful flowers in God's creation, comprise a unique group of plants. Orchidaceae were reported that has belonging a lot of species grew in the world, consisting of 35,000 species and 800 genera 1 . Orchids exhibit an incredible range of diversity in size, shape and colour of their flowers. Among the various orchids, Vanda tessellata (Roxb.) Hook. ex G.Don is one of the most eth no - botanically important taxa. It is an endangered medicinal plant belongs to the family orchidaceae. It is an epiphytic orchid, 30 - 60 cm high, with leafy stem. Capsules 7.5 - 9 cm long, narrowly clavate - oblong with acute ribs. Paste of its leaves is used as application in fevers. It is used in the treatment of arthritis and rheumatism. The root is used as antidote against scorpion sting and remedy for bronchitis 2 . As the seeds do not have sufficient reserve food material (lacks endosperm) to take care of the growth of embryo during germination they have to depend on some external source for nutrients so as to make their undifferentiated embryo to develop into a protocorm. However Vanda tessellata in natural habitats are quickly diminishing due to heavy defore station, urbanization, utilization of land for agriculture and over - exploitation of agro - resources for commercial purposes. To save this diverse orchid species from extinction, in vitro culture techniques have been utilized to propagate them 3 . In the prese nt study, an attempt is made to have a mass clonal propagation of Vanda tessellata an endangered species within a short span of time; the aim is to study the effect of temperature and pH on protocorm formation in MS media. Material and Method s The immatur e pods of Vanda tessellata where collected from the forest around Madhya Pradesh. Firstly the dry petals attached to the green pods were removed, then the pods were washed properly by using running tap water for (30min), and was further treated with an ant ifungal agent (Bivastin) for 1 hour and with detergent for 10 min. The pods were surface sterilized by soaking in 0.1% mercuric chloride ( H g C l 2 ) solution for 25 - 30 min in laminar air flow thereafter by t hrow washing in distilled water . Then the pods was di pped quickly in 70% alcohol and flamed over a spirit lamp. Each pods were then transferred to a sterile petri plate. Then the pods were cut longitudinally into two equal halves using sterile scalpel, and the seeds together with cottony fibers in between we re scooped out. After careful separation of the seeds from the fibers, the seeds were transferred onto MS 4 . One set of each seed culture was maintained at temperature (15°C, 20°C, 25°C, 30°C) and pH (4.5, 5.5, 6.5,7.5). The Basal medium was amended with 3 % (w/v) sucrose. The temperature was adjusted from 15°C to 30°C and pH was adjusted from 4.5 to 7.5, prior to adding 0.8% agar. The media was autoclaved at a temperature of 125°C at pressure of 15 psi for 15 - 20min in 100ml conical flasks 5 . All such opera tion was done within a Laminar Air Flow Cabinet. The culture bottles were incubated in culture room at 25 ± 20°C under 16 hrs. photoperiod of approximately 2,500 flux light intensity from cool fluorescent tubes. Result s and Discussion The response of prot ocorm formation was noted by observing the colour change and shape of the seed. During germination, embryos were seen to emerge from the seed coat as yellow to creamy structure. The cultur e attained sperule shape (Figur e - 2(a) and soon after it starts proto corm formation within 60 to 120 day (Fig ure - 2b). The onset of protocorm development by immature seeds at different temperature and pH where recorded periodically after the day of initial inoculation (table - 1). Highest protocorm formation ( 94±0.17% ) was ac hieved in MS medium adjusted at temperature 20°C and pH 5.5. In our present study MS media adjusted at temperature 20°C and pH Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( I YS C - 201 5 ), 1 0 - 13 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 11 5.5 is found suitable over the temperature 15°C, 25°C, 30°C and pH 4.5, 6.5, 7.5 for protocorm formation ( figure - 1). Similar res ults were observed when the seeds of orchid Cattleya mossiae and Lealia purpurea germinated well when adjusted at 20°C 6 . In the earlier studies, D.nobile germinated better within a pH range 4.0 - 5.0 7 . The present investigation suggest the specificities of t emperature and pH requirements during In - vitro protocorm formation of orchid. Conclusion From the above findings, it may be concluded that MS medium adjusted at temperature 20°C and pH 5.5 is best for protocorm formation of Vanda tessellata orchid in co mparison to the temperature adjusted at, 15°C, 25°C, 30°C and pH adjusted at 4.5,6.5,7.5 . Hence all this data there by suggests that the temperature and pH plays a very important role in In - vitro protocorm formation of Vanda tessellata a medicinally impor tant endangered orchid. Table - 1 Protocorm formation of Vanda tessellate (Roxb.) Hook.ex G. D on at different temperature and pH %Protocorm formation in mean ± SE MS media Different Temp. Different pH 30Days 60Days 90Days 120Days 15°C 4.5 0.0±0.0 0.0±0. 0 0.0±0.0 30±0.12 20°C 5.5 0.0 ± 0.27 75±0.20 94±0.17 80.0 ±0.10 25°C 6.5 0.0±0.0 20±0.18 0.0±0.0 0.0±0.0 30°C 7.5 0.0±0.0 0.0±0.0 0.0±0.0 1.0±0.19 0 10 20 30 40 50 60 70 80 90 100 30 Days 60 Days 90 Days 120 Days P E R C E N T A G E DAYS temperature pH Figure - 1 Protocorm formed at different temperature and pH. Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( I YS C - 201 5 ), 1 0 - 13 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 12 Figure - 2 (a)PLB’S formed in MS media adjusted at temperature 20°C and pH 5.5 in 90 days, (b) PLB’S formed after 120 days in MS media adjuste d at temperature20°C and pH 5.5 References 1. Singh M.K., Sherpa A.R., Hallan V and Zaidi A.A. , Autralasian Plant pathology , 2, 11 - 13 (2007) 2. N.S. Chauhan, Medicinal and Aromatic Plants of Himachal Pradesh, Indus Publishing Company, New Delhi, (1999) 3. Fay M.F., Conservation of rare and endangered plants using in vitro methods, In vitro Cell Dev., Biol. — Plant; 28, 1 – 4 (1992) 4. Mura shige I. and Skoog F., A revised medium for rapid growth and bioassys with tobacco tissue culture, Plant Physiol, 15 , 473 - 497 (1962) 5. Bindiya Prakash, Shagufta Khan, Ritu Thakur Bais (2012) Effect of different media on In vitro Seed Germination and Protoco rm Formation of Vanda tessellata (Roxb.) Hook. Ex. G an endangered medicinal orchid , Researcher , 4(12) (2012) http://www.sciencepub.net/researcher. 6. Bernard, La germination des orchidees , Compt. Rend. Acad. Sci. Paris, 137, 483 - 485 ( 1903) 7. Ito I., Germinatio n of seed from immature pod and subsequent growth of seedling in Dendrobium nobile Lindl . Sci. Reports Saikyo Univ. Agr., 7, 35 - 42 (1955) 8. Arditti J., Factors affecting the germination of orchid seeds, Botanical Review, 33, 1 - 97 (1967) 9. Bindiya P., Ritu T.B. , Prathibha S., Shagufta K., Effect of different pH on In vitro Seed Germination of Vanda tessellata (Roxb.) Hook.Ex.G an endangered medicinal orchid , Advances in Life Science and Technology , 8, ( 2013) 10. Guay A.T., Spark R.F., Bansal S., Cunningham G.R., Goo dman N.F., Nankin H.R., Petak S.M. and Perez J.B., American Association of Clinical Endocrinologists Male Sexual Dysfunction Task , Force. Endocr Pract. Jan - Feb; 9(1), 77 - 95 (2003) 11. Ichihashi S and M. Yamashita, Studies on the media for orchid seed germinati on. I. The effects of balances inside each cation and anion group for the germination and seedling development of Bletilla striata seeds , J. Jap. Soc. Hort. Sci., 45, 407 - 413 (1977) 12. Knudson C., A new nutrient solution for germination of orchid seed, Amer Orchid Soc Bull, 15, 214 - 217 (1946) 13. Mead J.W and C. Bulard, Vitamins and nitrogen requirement of orchis laxiflora Lamk, New Phytol. , 83, 129 - 36 (1979) 14. Mitra, G.C, Studies on seed, shoot tip and stem disc of an orchid grown in aseptic culture, Indian J.Exp. Biol ., 9, 79 - 85 (1971) 15. Pierik R.L.M., In vitro Culture of Higher Plants Martinus Higoff Publ , Netherlands, 344, (1987) 16. Prasad R.N. and G.C. Mitra, Nutrient requirement for germination of seed and development of protocorm and seedling of Cymbidium in aseptic culture, Indian J. Exp. Biol ., 13, 123 - 26 (1975) 17. Quednow K.G., Beitrage Zue Frage der aufahme geloster Kohleenstoff Verbin dungen durch Orchideen und andere Pflanzen, Bot. Arch., 30, 51 - 108 (1930) 18. Raghvan V. and J.G. Torrey, Inorganic nitrogen nutrition o f the embryos of the orchid Cattleya, Am. J. Bot ., 51, 264 - 74 (1964) a b Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 4 ( I YS C - 201 5 ), 1 0 - 13 (201 5 ) Res. J. Recent. Sci. International Science Congress Association 13 19. Sinha S.K., Singh L.S. and Hegde S.N., In - vitro multiplication of Aerides rosea Loddiges ex. through asymbiotic seed germination, 16, 38 - 44 (1998) 20. Straus M.S. and D.M. Reisinger, Effect o f NAA on seed germination, Am.Orchid Soc. Bull., 45, 722 - 23 (1976) 21. Vacin E and Went F., Some pH changes in nutrient solution, Bot Gardens Conserv. News, 11, 605 - 613 (1949) 22. Yakubu M.T, Akanji M.A and Oladiji A.T., Male sexual dysfunction and methods used in assessing medicinal plants with aphrodisiac potentials, Pharmacognosy Reviews, 1(1), 39 - 46 (2007)