Abstract
An efficient and improved shoot regeneration technique for the micropropagation of Withania somnifera L. through in vitro culture of nodal segments with axillary buds using thidiazuron (TDZ) is developed. Murashige and Skoog (MS) medium containing TDZ (0.0 to 10.0 mu M) was effective in inducing shoot buds and maintaining high rates of shoot multiplication and subsequent elongation on hormone free MS medium. TDZ at a concentration of 0.5 mu M was found to be most effective in bud break and also produced maximum regeneration frequency (98%), number of shoots (23.8 +/- 0.33) with shoot length of (4.83 +/- 0.66 cm), after 4 weeks of culture. Such proliferating shoots when sub-cultured on MS media devoid of TDZ, showed the highest number (32.4 +/- 0.24) of shoots and shoot length (7.66 +/- 0.08 cm) at the end of fourth subculture passage. Among the different concentrations of Indole- 3-butyric acid (IBA) (50 to 500 mu M) tested, the maximum percentage of rooting 100% in vitro regenerated microshoots was achieved in soilrite when basal portion of the microshoots were treated with 200 mu M (IBA) for 15 min, which induced maximum roots (18.3 +/- 0.16) with root length of (7.63 +/- 0.08 cm) per shoot. The plantlets went through hardening phase in a growth chamber, prior to ex vitro transfer. Micropropagated plants grew well, attained maturity and flowered normally. The present regeneration process favoured large-scale multiplication and long-term in vitro conservation of W. somnifera.