In Vitro Shoot Regeneration of Stevia rebaudiana through Callus and Nodal Segments

Stevia rebaudiana Bertoni of Asteraceae family, commonly known as ‘madhu patra’, is a valuable medicinal herb having anti-diabetic properties. Due to the presence of secondary metabolite, stevioside, it is highly used as low calorie sweetener and has importance in the nutraceutical market. Nodal segments of Stevia rebaudiana were cultured in Gamborg's (B₅) medium supplemented with BAP, Kn and IBA alone and in combination (0.2, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mg/l). Almost in all the cultures, initiation of shoot primordia was observed 4–8 days after the inoculation. Overall, from the economical point of view, B₅ media with 0.2 mg/l BAP was the best producing multiple shoot (2.00±0.00), shoot length (3.87±0.37) and number of leaves (33.50±1.50), after 30 days of inoculation. The culture after 60 days showed rooting response without any rooting hormone. The sub-culturing after 30 days in 0.5 mg/l BAP showed the appearance of callus from leaves. Leaf derived primary callus when transferred in fresh B5 media with 0.2 mg/l BAP resulted in shoot and root formation (organogenesis). Moreover, 88.20 g/l fresh weight of 8 weeks old secondary callus was obtained in 0.5 mg/l 2,4-D. Regenerated plantlets were hardened in perlite and showed survival rate of 80% after 2 months in the field. This micropropagation protocol could be effectively used for mass production of Stevia rebaudiana in vitro, besides for further investigation of medicinally active constituents of the plant through callus production.