Temporal variations in biochemical attributes and stress responses in Ashwagandha (Withania somnifera L. Dunal) during in vitro propagation

Ashwagandha ( Withania somnifera L. Dunal) is a medicinally important plant with high demand in Ayurveda and other indigenous medicinal systems. Conventional propagation through seeds is inadequate to meet pharmaceutical demand due to poor germination, pathogen susceptibility, and genetic heterogeneity. Micropropagation provides a reliable alternative to overcome these limitations. In the present study, efficient protocols were developed for both direct and indirect regeneration. Shoot tip explants regenerated directly on MS medium supplemented with 5.0 mg/L BAP, while callus cultures induced with 0.5 mg/L 2,4-D supported indirect regeneration. Complete plantlets were established on MS medium containing 2.0 mg/L kinetin (KIN) and 0.1 mg/L IAA. Prolonged in vitro culture induced morphological, biochemical, and genetic variations among regenerants. Total protein and flavonoid contents were elevated during early subcultures, whereas H₂O₂ levels progressively declined with successive passages. Activities of ROS-scavenging enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX) increased significantly. Regenerated plants exhibited distinct morphological leaf variations, further supported by genetic fidelity analysis, which confirmed divergence from greenhouse-grown counterparts. To evaluate stress responses, transcript levels of oxidative and abiotic stress-related genes (SOD, CAT, MYB, and HSP70) were quantified. Among these, HSP70 showed a pronounced upregulation (6.93-fold) in regenerants. Moreover, withanolide A accumulation under heat stress was significantly enhanced, highlighting tissue culture-induced metabolic shifts. Overall, this study establishes tissue culture-induced biochemical reprogramming, stress-gene upregulation, and secondary metabolite enhancement. These findings emphasize the need for monitoring physiological and genetic stability during in vitro culture of medicinal plants.