In vitro plant regeneration from epicotyl explant of Withania somnifera (L.) Dunal

Abstract

Studies were carried out to investigate the regeneration and rapid multiplication of Withania somnifera (L.) in vitro. Direct and indirect regeneration protocols for multiple shoots development from epicotyl explants of 50 to 60 days old seedlings were established. The shoots were initiated directly from epicotyl explant on 6-benzyl amino purine (BAP: 2.0 mg/L) along with indole-3-acetic acid (IAA: 0.2 mg/L), and the maximum of 15.5 ± 0.90 shoots/explant were achieved by subsequent subcultures at 4 weeks interval in the same medium. Calli (98.3%) were produced from epicotyl explant on 2,4-dichlorophenoxy acetic acid (2,4-D: 2.0 mg/L) along with kinetin (Kn: 0.6 mg/L), and shoots were initiated from calli on BAP (1.0 mg/L) along with adenine sulphate (AdS: 20.0 mg/L). Proliferation of shoots was achieved by subsequent subcultures at 4 weeks interval in the same medium. The maximum value of 25.3 ± 1.81 shoots/explant was achieved in the second subculture of indirect regeneration. Murashige and Skoog (MS) medium along with gibberellic acid (GA3) at 1.0 mg/L produced maximum 73.3 and 95.5% of shoot elongation in direct and indirect regenerated shoots, respectively. On the other hand, MS medium with indole-3-butyric acid (IBA) at 0.8 mg/L induced maximum 86.7% and 90.0% of rooting from elongated shoots of direct and indirect regeneration, respectively. The rooted plants were transferred to small cups filled with sterilized mixture containing soil, sand and vermiculite (1:2:1, v/v/v) for hardening. About 90% of plants survived in the hardening process, and then the plants were established successfully in the experimental field. This protocol yielded a higher number of shoots within a short period. Consequently, the protocol developed in this study offers a simple and improved in vitro method to regenerate W. somnifera.   Key words: Withania somnifera, epicotyls, tissue culture, shoot elongation, root induction.