Vol. 9, Issue 3, Part H (2025)

Withania somnifera (L.) Dunal is a highly prized medicinal plant that has been valued for its extraordinary pharmaceutical potential since ancient times. Its demand has grown dramatically in both domestic and foreign markets as a result of scientific confirmation of its therapeutic efficacy against a wide range of diseases. Alternative methods to conventional cultivation are crucial to closing the significant gap between supply and demand. A practical method for creating large quantities of W. somnifera plants that are healthy, genetically stable, and phytochemically homogeneous is an in vitro propagation system. Both pre-clinical research and the herbal industry benefit from this approach. A significant obstacle in in vitro propagation, however, is maintaining a high rate of shoot multiplication in cytokinin-enriched MS medium for prolonged periods of time. In order to minimize the negative effects of cytokinin, the current study created a multi-step protocol that includes rotational subculturing of Withania shoots in auxin-cytokinin-enriched culture medium. Murashige and Skoog (MS) medium supplemented with different concentrations of BAP, IAA, 2,4-D, and IBA, as well as 0.8% agar and 3% sucrose, was used to start shoot cultures. Using BAP (5.0 mg/l) and IBA (3.0 mg/l) produced the best results, with the greatest number of shoots (56.71±24.91) after 60 days. Additionally, the highest shoot proliferation was obtained with a combination of BAP (5.0 mg/l) and IAA (3.0 mg/l); after 60 days, the average shoot length was 6.04±2.69 cm. Assuring the method's dependability, genetic homogeneity and the stability of pharmaceutically significant components in extended in vitro shoots were verified. Withania somnifera is becoming more and more in demand commercially, and this study provides a strong platform for its high-efficiency micropropagation.