In Vitro Shoot Multiplication of Stevia and Assessment of Stevioside Content and Genetic Fidelity of the Regenerants

Abstract

A reliable protocol for micropropagation was developed for an anti-diabetic medicinal plant species, Stevia rebaudiana. In the present investigation the highest percentage of response was shown by nodal segment (98.00 ± 1.22) and both MS full and half strength performed well and were almost equally effective. Callus along with multiple shoots was obtained from nodal segments of S. rebaudiana on half strength Murashige and Skoog (MS) media supplemented with various concentrations of benzyl amino purine (BAP) and indole-3-butyric acid (IBA) (0.2, 0.5, 1.0, 1.5 mg l−1 each). The in vitro shoots produced along with callus were re-cultured on one-fourth strength MS media supplemented with kinetin (Kn) (0.5 mg l−1), IBA (1.0 mg l−1), activated charcoal (50 mg l−1), polyvinylpolypyrrolidone (100 mg l−1) and gibberellic acid (1 mg l−1) to obtain optimum shoot regeneration. Shoot multiplication and simultaneous rooting of S. rebaudiana was observed. The results obtained showed multiple shooting (18–20) with shoot length (6.5–7.2 cm), number of leaves (42–50), number of roots (12–14) and root length (5.8–6.2 cm), after 4 weeks of re-culturing of callus. Among different auxins and cytokinins used for secondary callus formation in half strength MS media. 2, 4-D (0.5 mg l−1) showed maximum growth index (266.384), after 8 weeks of callus culture. In vitro raised plantlets were transferred for hardening in plastic pots containing garden soil and perlite (1:1). A total of 80 % survival rate was observed. Quantitative analysis of stevioside content of in vitro and field grown plants was carried out by HPLC which showed increase of about 2 % stevioside content in the regenerants as compared to field grown plants. The genetic fidelity of micropropagated plantlets was confirmed by ISSR analysis confirming that in vitro plants obtained from callus showed genetic variation during the period of culturing, whereas those from nodal segments did not. Overall, this system results in mass multiplication of S. rebaudiana with simultaneous production of callus within short period. The high stevioside content in micropropagated plants may be of immense use to the pharmaceutical industry.