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 AJPS  Vol.4 No.1 , January 2013
TDZ-Induced High Frequency Plant Regeneration through Direct Shoot Organogenesis in Stevia rebaudiana Bertoni: An Important Medicinal Plant and a Natural Sweetener
Abstract: An efficient high frequency plant regeneration protocol through direct organogenesis was developed for Sevia rebaudiana Bert. Nodal segments containing axillary buds were used as an explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 1.00 to 9.00 μM. Maximum multiple shoots (96%) were obtained in MS medium supplemented with 1.0 μM TDZ with an average of 60 shoots per culture, having an average shoot length of 6.0 cm. The best in vitro root induction (89%) was achieved on half strength MS medium without any growth regulator with an average of 24 roots per culture and root length of7 cm. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 95% in the indoor grow room. High-performance liquid chromatography was used to assess the stability in chemical profile and quantification of stevioside and rebaudioside A content of in vitro propagated S. rebaudiana plants and compared with their mother plant at the peak vegetative stage. Our results show no significant differences (p < 0.05) between the mother and in vitro propagated plants. Furthermore, fully developed in vitro propagated S. rebaudiana plants were also compared with mother plant for their gas and water vapour exchange characteristics and leaf anatomy. The results show that in vitro propagated and hardened plants of S. rebaudiana are morphologically as well as functionally comparable to each other and to their mother plant.
Cite this paper: H. Lata, S. Chandra, Y. Wang, V. Raman and I. Khan, "TDZ-Induced High Frequency Plant Regeneration through Direct Shoot Organogenesis in Stevia rebaudiana Bertoni: An Important Medicinal Plant and a Natural Sweetener," American Journal of Plant Sciences, Vol. 4 No. 1, 2013, pp. 117-128. doi: 10.4236/ajps.2013.41016.
References

[1]   S. Bhosle, “Commercial Cultivation of Stevia rebaudiana,” Agrobios Newsletter, Vol. 3, No. 2, 2004, pp. 43-45.

[2]   D. D. Soejarto, A. D. Kinghorn and N. R. Fransworth, “Potential Sweetening Agents of Plant Origin,” Journal of Natural Products, Vol. 45, No. 5, 1982, pp. 590-599. doi:10.1021/np50023a013

[3]   M. B. Ahmed, M. Salahin, R. Karim, M. A. Razvy, M. M, Hannan, R. Sultana, M. Hossain and R. Islam, “An Efficient Method for in Vitro Clonal Propagation of a Newly Introduced Sweetener Plant (Stevia rebaudiana) in Bangladesh,” American-Eurasian Journal of Scientific Research, Vol. 2, No. 2, 2007, pp. 121-125.

[4]   N. Bondarev, O. Reshetnyak and A. Nosov. “Features of Development of Stevia rebaudiana Bertoni Shoots Cultivated in the Roller Bottle System and Their Production of Steviol Glycosides,” Planta Medica, Vol. 68, No. 8, 2002, pp. 759-762. doi:10.1055/s-2002-33809

[5]   H. Miyagawa and N. Fujioka, “Studies on The Tissue Culture of Stevia rebaudiana and Its Components: II. Induction of Shoot Primordia,” Planta Medica, Vol. 52, No. 4, 1986, pp. 321-323. doi:10.1055/s-2007-969165

[6]   M. Anbazhagan, M. Kalpana, R. Rajendran, V. Natrajan and D. Dhanavel, “In Vitro Production of Stevia rebaudiana Bertoni,” Emirates Journal of Food and Agriculture, Vol. 22, No. 3, 2010, pp. 216-222.

[7]   M. Sakaguchi and T. Kan, “Japanese Researches on Stevia rebaudiana,” Ci Cult, Vol. 34, 1982, pp. 235-248.

[8]   M. Debnath, “Clonal Propagation and Antimicrobial Activity of an Endemic Medicinal Plant Stevia rebaudiana,” Journal of Medicinal Plants Research, Vol. 2, No. 2, 2008, pp. 45-51.

[9]   P. K. Mishra, R. Singh, U. Kumar and V. Prakash, “Stevia rebaudiana—A Magical Sweetener,” Global Journal of Biotechnology and Biochemstry, Vol. 5, No. 1, 2010, pp. 62-74.

[10]   O. V. Kornilova and E. A. Kalashnikova, “Clonal Micropropagation of Stevia (Stevia rebaudiana),” Cercetari Agrton Moldova, Vol. 30, 1997, pp. 80-85.

[11]   S. Verma, K. Yadav and N. Singh, “Optimization of the Protocols for Surface Sterilization, Regeneration and Acclimatization of Stevia rebaudiana Bertoni,” American-Eurasian Journal Agriculture and Environment Science, Vol. 11, No. 2, 2011, pp. 221-227.

[12]   A. Das, S. Gantait and N. Mandal, “Micropropogation of an Elite Medicinal Plant (Stevia rebaudiana Bertoni),” International Journal of Agriculture Research, Vol. 6, No. 1, 2011, pp. 40-48. doi:10.3923/ijar.2011.40.48

[13]   K. A. Malik and P. K. Saxena, “Thidiazuron Induces High Frequency Shoot Regeneration in Intact Seedlings of Pea (Pisum sativum) and Lentil (Lens culinaris),” Australian Journal of Plant Physiology, Vol. 19, No. 6, 1992, pp. 731-740. doi:10.1071/PP9920731

[14]   B. Vinocur, T. Carmi, T. Altman and M. Ziv, “Enhanced Bud Regeneration in Aspen (Populus tremula L.) Roots Cultured in Liquid Media,” Plant Cell Report, Vol. 19, No. 12, 2000, pp. 1146-1154. doi:10.1007/s002990000243

[15]   S. Y. Park, H. N. Murth and K. Y. Paek, “Protocorm-Like Body Induction and Subsequent Plant Regeneration from Root Tip Cultures of Doritaenopsis,” Plant Science, Vol. 164, No. 6, 2003, pp. 919-923. doi:10.1016/S0168-9452(03)00019-0

[16]   G. Sujatha and R. B. D. Kumari, “High-Frequency Shoot Multiplication in Artemisia (Vulgaris L.) Using Thidiazuron,” Journal of Plant Biotechnology Report, Vol. 1, No. 3, 2007, pp. 149-154. doi:10.1007/s11816-007-0028-1

[17]   H. Lata, S. Chandra, I. Khan and M. A. ElSohly, “Thidiazuron Induced High Frequency Direct Shoot Organogenesis of Cannabis Sativa L.,” In vitro Cellular and Developmental Biology-Plant, Vol. 45, No. 1, 2009, pp. 12- 19. doi:10.1007/s11627-008-9167-5

[18]   S. D. Singh and G. P. Rao, “Stevia: The Herbal Sugar of 21st Century,” Sugar Technology, Vol. 7, No. 1, 2005, pp. 17-24. doi:10.1007/BF02942413

[19]   R. Upton, A. Graff, G. Jolliffe, R. Langer and E. Williamson, “American Herbal Pharmacopoeia: Botanical Pharmacognosy-Microscopic Characterization of Botanzcal Medicines,” CRC Press, New York, 2011. doi:10.1201/b10413

[20]   T. D. Thomas and J. T. Puthur, “Thidiazuron Induced High Frequency Shoot Organogenesis in Callus from Kigelia pinnata L.,” Botanical Bulltein of Academic Sinica, Vol. 45, No. 4, 2004, pp. 307-313.

[21]   M. P. A. Jones, Z. Yi, S. J. Murch and P. K. Saxena, “Thidiazuron-Induced Regeneration of Echinacea purpurea L.: Micropropagation in Solid and Liquid Culture Systems,” Plant Cell Reports, Vol. 26, No. 1, 2007, pp. 13-19. doi:10.1007/s00299-006-0209-3

[22]   S. Parveen and A. Shahzad, “Thiadiazuron Induced High Frequency Shoot Regeneration in Cassia sophera L. via cotyledonary Node Explants,” Journal of Plant Physiology and Molecular Biology, Vol. 16, No. 2, 2010, pp. 201-206. doi:10.1007/s12298-010-0022-x

[23]   S. C. Capelle, D. W. S. Mok, S. C. Kirchner and M. C. Mok, “Effects of Thidiazuron on Cytokinin Autonomy and the Metabolism of N6-(Y2-Isopentyl) [8-14c] Adenosine in Callus Tissues of Phaseolus lunatus L.,” Journal of Plant Physiology, Vol. 73, No. 3, 1983, pp. 796-802. doi:10.1104/pp.73.3.796

[24]   C. Magioli, A. P. M. Rocha, D. E. de Oliveira and E. Mansur, “Efficient Shoot Organogenesis of Eggplant (Solanum melongena L.) Induced by Thidiazuron,” Plant Cell Report, Vol. 17, No. 8, 1998, pp. 661-663. doi:10.1007/s002990050461

[25]   J. Mithila, J. C. Hall, J. M. R. Victor and P. K. Saxena, “Thidiazuron Induces Shoot Organogenesis at Low Concentrations and Somatic Embryogenesis at High Concentrations on Leaf and Petiole Explants of African Violet (Saintpaulia ionantha Wendl.),” Plant Cell Reports, Vol. 21, No. 5, 2003, pp. 408-414.

[26]   J. C. Bespalhokk, L. G. E. Vicira and J. M. Hashimoto, “Embryogenic Callus Formation and Histological Studies from Stevia rebaudiana Bert. Floret Explants,” Revista Brasileira de Fisiologia Vegetal, Vol. 5,1997, pp. 51-53.

[27]   C. Visser, J. K. Qureshi, R. Gill and P. K. Saxena, “Morphoregulatory Role of Thidiazuron: Substitution of Auxin-Cytokinin Requirement of Somatic Embryogenesis in Hypocotyl Cultures of Geranium,” Journal of Plant Physiology, Vol. 99, No. 4, 1992, pp. 1704-1707. doi:10.1104/pp.99.4.1704

[28]   B. N. S. Murthy, S. J. Murch, S. KrishnaRaj and P. K. Saxena, “Thidiazuron: A Potent Regulator of in Vitro Plant Morphogenesis,” In Vitro Cellular Developmental Biology Plant, Vol. 34, No. 4, 1998, pp. 267-275. doi:10.1007/BF02822732

[29]   R. Radhakrishnan, A. Ramachandran and B. D. R. Kumari, “Rooting and shooting: Dual function of Thidiazuron in Vitro Regeneration of Soybean (Glycine max. L.),” Acta Physiologiae Plantarum, Vol. 31, No. 6, 2009, pp. 1213-1217. doi:10.1007/s11738-009-0356-6

[30]   N. Bag, L. M. S. Palni, S. Chandra and S. K. Nandi, “Somatic Embryogenesis in ‘Maggar’ Bamboo (Dendrocalamus hamiltonii) and Field Performance of Regenerated Plants,” Current Science, Vol. 102, No. 9, 2012, pp. 1279-1287.

 
 
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