In vitro shoot multiplication using meta-Topolin and leaf-based regeneration of a withaferin A rich accession of Withania somnifera (L.) Dunal

Withania somnifera (L.) Dunal is a popular medicinal herb used in traditional Ayurvedic medicines. The pharmacological properties of this plant are largely due to the presence of withanolides such as withanone, withanolide A, withanolide D and withaferin A. Among these, withaferin A has gained a lot of attention because of its anti-cancer activity against a number of cancer cell lines. The current study deals with the selection of an elite accession of W. somnifera with high withaferin A content and the establishment of aseptic cultures under in vitro conditions for clonal mass propagation. Here, a novel cytokinin meta-Topolin (mT) was used for the first time in W. somnifera for shoot proliferation. mT (2.5 mu M) was recorded as an efficient cytokinin for successful shoot multiplication and their growth. Further, an effective and reproducible regeneration protocol using leaf explant of W. somnifera has been developed for in vitro mass propagation and genetic transformation studies. The best shoot regeneration frequency and the maximum number of shoot buds were recorded in MS medium amended with BAP (10.0 mu M). Among various auxins such as IAA, IBA, and NAA, IAA (10.0 mu M) was proved to be optimal for both shoot and root growth. The regenerated plants were transferred to the greenhouse for their hardening and a high rate of survival (80 %) was recorded. The genetic uniformity of in vitro regenerated shoots was verified with RAPD, ISSR, and SCoT markers.

Automated summary

This study successfully selected an elite accession of Withania somnifera with high withaferin A content and utilized meta-Topolin (mT) as a novel cytokinin for aseptic cultures, achieving clonal mass propagation of shoots. Additionally, an effective regeneration protocol using leaf explant of W. somnifera for in vitro mass propagation and genetic transformation studies was developed. The genetic uniformity of the in vitro regenerated shoots was confirmed using RAPD, ISSR, and SCoT markers.