A novel approach for direct shoot regeneration, anatomical characterization, and withanolides content in micropropagated plants of Withania somnifera (L.) Dunal—an important medicinal plant

Withania somnifera (Ashwagandha) is a valuable therapeutic plant used in many Ayurvedic drugs marketed for health restoration. However, its commercial production faces challenges due to poor seed viability, low germination rates, and the absence of standardized propagation methods. Therefore, this investigation aimed to establish a fast and highly efficient method for direct shoot regeneration in W. somnifera by using the apical meristem perforation technique, which is a novel approach. Initially, rapid seed germination was standardized by soaking the seeds in distilled water for varying durations (0 to 48 h). The highest rates of shoot initiation (90.40 ± 0.51%) and mean number of direct shoot regenerations (16.20 ± 0.50 per explant) were achieved on MS basal medium fortified with meta-topolin (mT) at 3.0 mg L⁻¹. For optimal root development, shoots (3 to 4 cm) were cultured on half-strength Murashige and Skoog (MS) medium with 3.0 mg L⁻¹ indole-3-butyric acid (IBA). Subsequently, well-rooted seedlings (90% survival rate) were acclimatized under glasshouse conditions in pots filled with a mixture of sandy loam and vermicompost (4:1). Anatomical examination of leaves and stems from micropropagated and acclimatized glasshouse plants revealed well-developed cuticles, differentiated mesophylls, and vascular bundles. Furthermore, the accumulations of withaferin A (1.26 ± 0.12 mg g⁻¹), withanone (1.39 ± 0.18 mg g⁻¹), and withanoside V (0.11 ± 0.04 mg g⁻¹) in greenhouse-acclimated plant leaves were 1.88, 2.28, and 2.30 times higher, respectively, than in tissue culture samples (0.52 ± 0.07 mg g⁻¹, 0.73 ± 0.02 mg g⁻¹, and 0.05 ± 0.02 mg g⁻¹). In conclusion, the described procedure offered an effective and straightforward method for large-scale production of W. somnifera plants for commercial metabolites.