Influence of Additives on Enhanced in Vitro Shoot Multiplication of Stevia rebaudiana (Bert.)—An Important Anti Diabetic Medicinal Plant


The present study was designed to develop an efficient protocol for micro propagation of S. rebaudiana from nodal explants and study the influence of additives on enhancement of shoot proliferation. A two-step protocol has been standardized in which, first step comprising growth hormones concentration is optimized and it was found that MS medium supplemented with 2.0 mg/l BAP + 0.5 mg/l Kin + 0.1 mg/l NAA turned out to be the best treatment for shoot induction. In the second step, the best treatment for shoot induction was fortified with different growth additives for further shoot proliferation. Among the different types of additives used, casein hydrolysate at 0.05% (w/v) was found to be most effective, resulted with maximum of 15.0 shoots. 90% regeneration frequency and shoot length of 6.0 cm were recorded per explant. Thus, the procedure described is a quick and reliable method which could be applied for efficient large scale propagation, genetic transformation assays and secondary metabolite production of Stevia.

Share and Cite:

T. Sridhar and C. Aswath, "Influence of Additives on Enhanced in Vitro Shoot Multiplication of Stevia rebaudiana (Bert.)—An Important Anti Diabetic Medicinal Plant," American Journal of Plant Sciences, Vol. 5 No. 1, 2014, pp. 192-199. doi: 10.4236/ajps.2014.51025.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] D. D. Soejarto, C. M. Compadre, P. J. Medon, S. K. Kamath and A, D. Kinghorn, “Potential Sweetening Agents of Plant Origin, II. Field Search for Sweet Tasting Stevia species,” Economic Botany, Vol. 37, No. 1, 1983, pp. 71-79.
[2] M. Bridel and R. Lavielle, “The Sweet Principle of Kaahe-e (Stevia rebaudiana),” Pharmaceutical Chemistry Journal, Vol. 14, 1931, pp. 99-154.
[3] A. S. Richman, M. Gizen, Al N. Starratt, Z. Y. Yang and J. E. Brandle, “Diterpene Synthesis in Stevia rebaudiana: Recruitment and Up-Regulation of Key Enzymes from the Gibberellin Biosynthetic Pathway,” The Plant Journal, Vol. 19, No. 4, 1999, pp. 411-421.
[4] D. Preethi, T. M. Sridhar and C. V. Naidu, “Effect of Bavistin and Silver Thiosulphate on in Vitro Plant Regeneration of Stevia rebaudiana,” Journal of Phytology, Vol. 3, 2011, pp. 74-77.
[5] M. D. Tofazzal Islam, “Stevia rebaudiana News: Is There Any Safe and Natural Alternative to Sugar? From This,” Financial Express, 2006, pp. 8-26.
[6] V. Yodyingyuad and S. Bunyawong, “Effects of Stevioside on Growth and Reproduction,” Human Reproduction, Vol. 6, 1991, pp. 158-165.
[7] D. Preethi, P. Shanmukanad, S. Hemadri Reddy, S. P. Jeevan Kumar, P. Josthna and C. V. Naidu, “In Vitro Plant Regeneration of Stevia rebaudiana,” Journal of Tropical Medicinal Plants, Vol. 9, No. 1, 2008, pp. 71-76.
[8] P. B. Jeppesen, S. Gregersen and S. E. Rolfsen, “Antihyperglycemic and Blood Pressure-Reducing Effects of Stevioside in the Diabetic Goto-Kakizaki Rat,” Metabolism, Vol. 52, No. 3, 2003, pp. 372-378.
[9] R. Abudula, P. B. Jeppesen, D. S. E. Rolfsen, J. Z. Xiao and K. Hermansen, “Rebaudioside A Potently Stimulates Insulin Secretion from Isolated Mouse Isolates: Studies on the Dose-Glucose and Calcium-Dependency,” Metabolism, Vol. 53, No.10, 2004, pp. 1378-1381.
[10] K. Yasukawa, S. Kitanaka and S. Seo, “Inhibitory Effect of Stevioside on Tumor Promotion by 12-O-Tetradecanoylphorbol-13-Acetate in Two Stage Carcinogenesis in Mouse Skin,” Biological and Pharmaceutical Bulletin, Vol. 25, No. 11, 2002, pp. 1488-1490.
[11] S. Nakamura and Y. Tamura, “Variation in the Main Glycosides of Stevia,” Japanese Journal of Tropical Agriculture, Vol. 29, 1985, pp. 109-116.
[12] S. Jayakumar and R. R. Lingam, “Influence of Additives on Enhanced In Vitro Shoot Multiplication of Orthosiphon aristatus (Blume) Miq,” Notulae Scientia Biologicae, Vol. 5,No. 3, 2013, pp. 338-345.
[13] S. Neelam and K. P. S. Chandel, “Effects of Ascorbic Acid on Axillary Shoot Induction in Tylophora indica (Burm. F.) Merr,” Plant Cell Tissue and Organ Culture, Vol. 29, No. 2, 1992, pp. 109-113.
[14] A. Vasudevan, N. Selvaraj, A. Ganapathi, S. Kasthurirengan, V. R. Anbbazhagan and M. Manickavasagam, “Glutamine: A Suitable Nitrogen Source for Enhanced Shoot Multiplication in Cucumis sativus,” Biologia Plantarum, Vol. 48, No. 1, 2004, pp. 125-128.
[15] T. Murashige and F. A. Skoog, “Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures,” Physiology Plant, Vol. 15, No. 3, 1962, pp. 473-497.
[16] P. Larkin and W. Scowcraft, “Somaclonal Variation—A Novel Source of Variability from Cell Culture for Plant Improvement,” Theoretical and Applied Genetics, Vol. 60, No. 4, 1981, pp. 197-214.
[17] L. W. Leng and C. Lai-Keng, “Plant Regeneration from Stem Nodal Segments of Orthosiphon stamineus Benth. A Medicinal Plant with Diuretic Activity,” In Vitro Cellular & Developmental Biology—Plant, Vol. 40, No. 1, 2004, pp. 115-118.
[18] D. Misick, D. Grubisic and R. Konjevic, “Micro Propagation of Salvia brachyodon through Nodal Explants,” Biologia Plantarum, Vol. 50, No. 3, 2006, pp. 473-476.
[19] M. E. Hembrom, K. P. Martin, S. K. Patchathundikandi and J. Madassery, “Rapid in Vitro Production of True-to-Type Plants of Pogostemon heyneaus through Dedifferentiated Axillary Buds,” In Vitro Cellular & Developmental Biology—Plant, Vol. 42, No. 3, 2006, pp. 283-286.
[20] H. Cao, J. Yang, Z. S. Peng, C. Y. Kang, D. C. Chen, Z. C. Gong and X. Tan, “Micro Propagation of Penthorium chinense through Axillary Bud,” In Vitro Cellular & Developmental Biology—Plant, Vol. 12, 2007, pp. 33-38.
[21] M. S. Rao and S. D. Purohit, “In Vitro Shoot Bud Differentiation and Plantlet Regeneration in Celastrus paniculates Wild,” Biologia Plantarum, Vol. 50, No. 4, 2006, pp. 501-506.
[22] T. M. Sridhar and C. V. Naidu, “High Frequency Plant Regeneration, in Vitro Flowering of Solanum nigrum (L.) —An Important Antiulcer Medicinal Plant,” Journal of Phytology, Vol. 3, No. 2, 2011, pp. 85-93.
[23] K. Sanatombi and G. J. Sharma, “In Vitro Propagation of Capsicum chinense Jacq,” Biologia Plantarum, Vol. 52, No. 3, 2008, pp. 517-520.
[24] D. Negi and S. Saxena, “Micro Propagation of Bambusa balcooa Roxb. through Axillary Shoot Proliferation,” In Vitro Cellular & Developmental Biology—Plant, Vol. 47, No. 5, 2011, pp. 604-610.
[25] E. F. George and P. D. Sherrington, “Plant Propagation by Tissue Culture,” Handbook and Dictionary of Commercial Laboratories, Exgenetics Ltd., England, 1984.
[26] J. Patnaik and B. K. Debata, “Micro Propagation of Hemidesmus indicus (L.) R. Br. through Axillary Bud Culture,” Plant Cell Reports, Vol. 15, No. 6, 1996, pp. 427-430.
[27] N. Komalavalli and M. V. Rao, “In Vitro Micro Propagation of Gymnema sylvestre—A Multipurpose Medicinal Plant,” Plant Cell, Tissue and Organ Culture, Vol. 61, No. 2, 2000, pp. 239-245.
[28] E. F. George and G. J. De Klerk, “The Components of Plant Tissue Culture Media I: Macro and Micro Nutrients,” Plant Propagation by Tissue Culture, 3rd Edition, Springer-Verlag, Dordretch, Vol. 1, 1984, pp. 65-113.
[29] F. Parabia, B. Gami, L. L. Kothari, J. S. S. Mohan and M. H. Parabia, “Effect of Plant Growth Regulators on in Vitro Morphogenesis of Leptadenia reticulata Retz. from Nodal Explant,” Current Science, Vol. 92, 2007, pp. 1290-1293.
[30] S. Saxena and V. Dhawan, “Large-Scale Production of Anogeissus pendula and Anogeissus latifolia by Micro Propagation,” In Vitro Cellular & Developmental Biology—Plant, Vol. 37, No. 5, 2001, pp. 586-591.
[31] N. Walia, A. Kour and S. B. Babbar, “An Efficient, in Vitro Cyclic Production of Shoots from Adult Trees of Crataeva nurvala Buch. Ham,” Plant Cell Reports, Vol. 26, No. 3, 2007, pp. 277-284.
[32] S. H. Reddy, M. Chakravarthi, K. N. Chandrashekara and C. V. Naidu, “Influence of Bavistin and Silver Thiosulphate on in Vitro Regeneration of Asclepias curassavica (L.) Using Nodal Explants,” American Journal of Plant Sciences, Vol. 3, 2012, pp. 941-946.
[33] Z. Molnar, E. Virag and V. Ordog, “Natural Substances in Tissue Culture Media of Higher Plants,” Acta Biologica Szegediensis, Vol. 55, No. 1, 2011, pp. 123-127.
[34] W. Tefera and S. Wannakrairoj, “Micro Propagation of Karwan,” Science Asia, Vol. 30, 2004, pp. 9-15.
[35] T. S. Rangan, “Clonal Propagation, Cell Culture and Somatic Cell Genetics of Plants, Laboratory Procedures and Their Applications,” Academic Pres, Inc., Orland, Vol. 1, 1984, pp. 68-73.
[36] L. Dix and J. Van Staden, “Auxin and Gibberellin Like Substances in Coconut Milk and Malt Extract,” Plant Cell, Tissue and Organ Culture, Vol. 1, 1982, pp. 239-245.
[37] M. C. S. Gras and M. C. Calvo, “Micro Propagation of Lavandula latifolia through Nodal Bud Culture of Mature Plants,” Plant Cell, Tissue and Organ Culture, Vol. 45, No. 3, 1996, pp. 259-261.
[38] K. Vinod, G. Parvatam and A. R. Gokare, “AgNO3-A Potential Regulator of Ethylene Activity and Plant Growth Modulator,” Electronic Journal of Biotechnology, Vol. 12, No. 2, 2009, pp. 1-15.

Copyright © 2022 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.