Shoot regeneration from leaf explants of Withania coagulans (Stocks) Dunal and genetic stability evaluation of regenerates with RAPD and ISSR markers

• An efficient and cost effective in vitro regeneration protocol for a critically endangered medicinal plant, Withania coagulans (Stocks) Dunal was developed.
• Ex vitro rooting and simultaneous acclimatization of regenerates make protocol superior and reduce the time, energy and labor.
• The molecular marker (RAPD and ISSR) confirmed the reliability of regeneration system for genetic restoration program.
• The protocol developed would be useful for genetic restoration program, genetic transformation and improved phyto-pharming.

Withania coagulans (Stocks) Dunal (Solanaceae) is a critically endangered medicinal plant known for its multiple medicinal properties. We report here an efficient and high frequency plantlet regeneration system through direct organogenesis in W. coagulans using in vitro-derived leaves as explants. On Murashige and Skoog's (MS) medium containing 4.44 μM 6-benzylaminopurine (BAP) 73.7 ± 4.3% explants responded and produced 11.4 ± 0.9 shoot buds per explant. The regenerated shoot buds were elongated (6.7 ± 0.22 cm) on MS medium with 1.11 μM BAP and 0.57 μM indole-3-acetic acid (IAA). The elongated shoots were rooted both in vitro and ex vitro. The regenerates were acclimatized by slow and gradual exposure to different regimes of temperature and relative humidity. The leaf-regenerates were assessed for genetic stability using RAPD and ISSR molecular markers and confirmed true-to-type. The in vitro regeneration system developed would be useful for genetic restoration program as the overexploitation and reproductive failure forced W. coagulans towards the verge of complete extinction. The regeneration method would also be useful for genetic transformation for genetic improvement and improved phyto-pharming.