*Corresponding author: E-mail: gauchan@ku.edu.np. Journal of Advanced Laboratory Research in Biology E-ISSN: 0976-7614 Volume 5, Issue 3, July 2014 PP 41-45 https:/ / e-journal.sospublication.co.in Research Article Regenerative callus induction and biochemical analysis of Stevia rebaudiana Bertoni Dhurva P. Gauchan*, Ashna Dhakal, Nisha Sharma, Sabin Bhandari, Elina Maskey, Nayan Shrestha, Rachita Gautam, Sarala Giri and Sushma Gurung Department of Biotechnology, School of Science, Kathmandu University, Dhulikhel 45200, Nepal. Abstract: Stevia Leaves are the principal source of stevioside, which is estimated to be 100-300 times sweeter than table sugar. Stevioside has clinical significance as they are reported to maintain glucose levels in human blood. Owing to the difficulties in propagation of stevia through seeds and vegetative methods, callus culture has been an efficient alternative for generation of stevioside. The aim of this study is to develop an efficient and standardized protocol for maximum induction and multiplication of callus from a leaf. Callus culture was established from leaves in MS basal media fortified with various combinations (BAP, NAA, 2,4-D, KN, IBA) and concentrations of phytohormones. The best callusing (100%) was recorded in MS media supplemented with (2,4-D 1.0mg/l + NAA 1.0mg/l). The callus was harvested after 4 weeks and screened for the presence of various bioactive compounds. The qualitative results showed that the extracts of callus contained bioactive compounds like flavonoids, glycosides, phenol, tannins, sterols and saponins thereby making callus one of the sources for extraction of various secondary metabolites. Keywords: Stevia rebaudiana, callus, 2,4-D, phytochemical screening, plant growth regulators (PGR). 1. Introduction Stevia rebaudiana Bertoni is one of the 154 members of the genus Stevia (Pande & Khetmalas, 2012). It is a perennial herb and belongs to the family Asteraceae. It is a native of certain regions of South America, particularly Paraguay and Brazil (Anbazhagan et al., 2010). S. rebaudiana has its name after Dr. M.S. Bertoni who officially discovered Stevia during the early 20 th century (Das et al., 2006). It is otherwise popularly known as candy leaves, non-caloric sweet plant, Stevia, sugar substitute, sweet weed, honey leaf and sweet herb of Paraguay (Das et al., 2006; Anbazhagan et al., 2010). Stevia leaves contain a number of diterpene steviol glycosides (SGs) which are about 300 times sweeter than sucrose at their concentration of 4% (w/v) (Kinghora & Soejarto, 1986). Along with the steviol glycosides, Stevia leaf constituents include volatile oil components, sterols, triterpenes, flavonoids, coumarins, and non-glycosidic diterpenes (Talha et al., 2012). Particularly, leaves of stevia possess high protein, carbohydrate, and some active chemical. It has high Potassium, Calcium, Magnesium, Phosphorous, Sodium and Sulphur content in leaves and Cobalt, Copper, Iron, Manganese, Zinc, Selenium, Molybdenum are found in trace amounts. Vegetative propagation of stevia is limited by the low number of individuals that can be obtained simultaneously from a single plant. On the other hand, tissue cultured platelets offer excellent quality of foliage production in disease free condition and callus masses can sometimes yield the highest amount of secondary metabolites (Das et al., 2010). Thus, tissue culture is the only alternative for rapid mass propagation, conservation, and enhancement of the natural levels of valuable compounds of Stevia plants (Chan et al., 2005). Parts like leaves, nodes and shoot tips from Stevia can be used to raise plants in vitro (Naz et al., 2008). The objective of the present study was to develop an efficient protocol for the development of