ABSTRACT An efficient repeatable protocol has been developed for rapid clonal multiplication of an endemic, perennial herb Stevia rebaudiana using juvenile axillary buds from in vivo grown seedlings. MS basal medium supplemented with BAP (8.88 μM) and NAA (5.37 μM) was found to be the most suitable medium for initiation of shoots with 92% response. The repeated subculture resulted in high-frequency shoot multiplication (9.82 ± 1.93) at the average rate of 3-fold per subculture. The axenic multiple shoots were rooted ex-vitro on horticultural grate soilrite mix-peat:perlite:vermiculate (1:1:1 v/v) with half strength MS salts fortified with NAA (5.37 μM). The complete plantlets were successfully established in soil with 86% survival frequency. The statistical analysis of the data pertaining to multiple shoots and root formation revealed significant difference between and within the treatments.
Cite this paper
Rangappa, K. and Aind, D. (2013) High frequency multiplication of shoots using axillary buds for production of elite lines of Stevia rebaudiana. Advances in Bioscience and Biotechnology, 4, 781-784. doi: 10.4236/abb.2013.47102.
 Brandle, J. E. and Rosa, N. (1992) Heritability for yield, leaf: stem ratio and stevioside content estimated from a landrace cultivar of Stevia rebaudiana. Canadian Journal of Plant Science, 72, 1263-1266. doi:10.4141/cjps92-159
 Anbazhagan M., Kalpana, M., Rajendran, R., Natarajan, V. and Dhanavel, D. (2010) In vitro production of Stevia rebaudiana Bertoni. Emirates Journal of Food and Agriculture, 22, 216-222.
 Ishima, N. and Katayama, O. (1986) Sensory evaluation of stevioside as a sweetener. Reports of National Food Research Institute, 31, 80-85.
 Tanaka, O. (1982) Steviol-glycosides: New natural sweeteners. TrAC Trends in Analytical Chemistry, 1, 246-248.
 Carneiro, J.W.P., Muniz, A.S. and Guedes, T.A. (1997) Green house bedding plant production of Stevia rebaudiana (Bert.). Canadian Journal of Plant Science, 77, 473474. doi:10.4141/P96-166
 Debnath, M. (2008) Clonal propagation and antimicrobial activity of an endemic medicinal plant Stevia rebaudiana. Journal of Medicinal Plants Research, 2, 45-51.
 Taware, A.S., Harke, S.N., Mukadam, D.S., Chavan, A.M. and Taware, S.D. (2010) Effect of different extracts of callus and plantlets of Stevia rebaudiana (Bertoni) on seed germination of some agricultural crops. African Journal of Biotechnology, 9, 6675-6683.
 Bhojwani, S.S. and Razdan, M.K. (2004) Plant tissue culture: Theory and practice. Revised Edition, Elsevier Publication, Amsterdam.
 Sugimura, Y., Adachi, T., Kotani, E. and Furusawa, T. (1998) Shoot bud formation and plantlet regeneration from the basal tissues of mulberry leaves. The Journal of Sericultural Science of Japan, 67, 421-424.
 George, E.F. and Sherrington, P.D. (1984) Plant propagation by tissue culture. Exegetics Ltd., Eversley, Basingstoke, 39-71.
 Sairkar, P., Chandravanshi, M.K., Shukla, N.P., Mehrotra N.N. (2009) Mass production of an economically important medicinal plant Stevia rebaudiana using in vitro propagation techniques. Journal of Medicinal Plants Research, 3, 266-270.
 Ahmed, M.B., Salahin, M., Karim, R., Razvy, R.A., Hannan, M.M., Sultana, R., Hossain, M. and Islam, R. (2007) An efficient method for in vitro clonal propagation of a newly introduced sweetener plant (Stevia rebaudiana Bertoni.) in Bangladesh. American-Eurasian Journal of Scientific Research, 2, 121-125.
 Salim, M.U., Shaheed, M.H.C., Mouoztaba, M.M.K., Belal, M.U., Ahmed, R. and Baten, M.A. (2006) In vitro propagation of Stevia rebaudiana Bert in Bangladesh. African Journal of Biotechnology, 5, 1238-1240.
 Yukiyoshi, T., Shigeharu, N., Hiroshi, F. and Mamoru, T. (1984) Clonal propagation of Stevia rebaudiana Bertoni by stem-tip culture. Plant Cell Reports, 3, 183-185.
 Giridhar, P., Sowmya, K.S., Ramakrishna, A. and Ravishankar, G.A. (2010) Rapid clonal propagation and stevioside profiles of Stevia rebaudiana Bertoni. International Journal of Plant Developmental Biology, 4, 47-52.
 Das, K., Dang, R. and Rajasekharan, P.E. (2006) Establishment and maintenance of callus of Stevia rebaudiana Bertoni under aseptic environment. Natural Product Radiation, 5, 373-376.
 Das, A, Gantait, S. and Mandal, N. (2011) Micropropagation of elite medicinal plant: Stevia rebaudiana Bert. International Journal of Agricultural Research, 6, 40-48.
 Sivaram, L. and Mukundan, U. (2003) In vitro culture studies on Stevia rebaudiana. In Vitro Cellular & Developmental Biology—Plant, 39, 520-523.
 George, E.F. (1996) Plant propagation by tissue culture part 2: In practice. 2nd Edition, Exegetics Ltd., Westbury.
 Rafiq, M., Dahoti M.U., Mangrios, S.M., Naqvi, H.A. and Qarshi, I.A. (2007) In vitro clonal propagation and biochemical analysis of field established Stevia rebaudiana Bertoni. Pakistan Journal of Botany, 39, 2467-2474.
 Thiyagarajan, M. and Venkatachalam, P. (2012) Large scale in vitro propagation of Stevia rebaudiana (bert) for commercial application: Pharmaceutically important and anti-diabetic medicinal herb. Industrial Crops and Products, 37, 111-117. doi:10.1016/j.indcrop.2011.10.037
 Hossain, M.A., Kabir, A.H.M, Jahan, T.A. and Hasan, M.N. (2008) Micropropagation of Stevia. International Journal of Sustainable Crop Production, 3, 1-9.
 Steven, M.S., Gail, B.M. and Christopher, W.W.B. (1992) Stevioside biosynthesis by callus, root, shoot and rootedshoot cultures in vitro. Plant Cell Tissue and Organ Culture, 28, 151-157.