Two-stage culture procedure using thidiazuron for efficient micropropagation of Stevia rebaudiana, an anti-diabetic medicinal herb

Abstract

Stevia rebaudiana Bertoni, member of Asteraceae family, has bio-active compounds stevioside and rebaudioside which taste about 300 times sweeter than sucrose. It regulates blood sugar, prevents hypertension and tooth decay as well as used in treatment of skin disorders having high medicinal values, and hence there is a need for generating the plant on large scale. We have developed an efficient micropropagation protocol on half strength Murashige and Skoog (MS) media, using two-stage culture procedures. Varying concentrations of cytokinins, i.e., benzylaminopurine, kinetin and thidiazuron (TDZ) were supplemented in the nutrient media to observe their effects on shoot development. All the cytokinins promoted shoot formation, however, best response was observed in the TDZ (0.5 mg/l). The shoots from selected induction medium were sub-cultured on the multiplication media. The media containing 0.01 mg/l TDZ produced maximum number of shoot (11.00 ± 0.40) with longer shoots (7.17 ± 0.16) and highest number of leaves (61.00 ± 1.29). Rooting response was best observed in one-fourth strength on MS media supplemented with indole-3-butyric acid (1.0 mg/l) and activated charcoal (50 mg/l) with (11.00 ± 0.40) number of roots. The plantlets thus obtained were hardened and transferred to the pots with soil and sand mixture, where the survival rate was 80 % after 2 months. Quantitative analysis of stevioside content in leaves of in vivo mother plant and in vitro plantlets was carried out by high performance liquid chromatography. A remarkable increase in stevioside content was noticed in the in vitro-raised plants as compared to in vivo grown plants. The protocol reported here might be useful in genetic improvement and high stevioside production.