Ashwagandha (Withania somnifera L. Dunal) is a medicinally important plant with withanolides as its major bioactive compounds, abundant in the roots and leaves. We examined the influence of plant growth regulators (PGRs) on direct organogenesis, adventitious root development, withanolide biosynthetic pathway gene expression, withanolide contents, and metabolites during vegetative and reproductive growth phases under in vitro and ex vitro conditions. The highest shooting responses were observed with 6-benzylaminopurine (BAP) (2.0 mg L^-1) + Kinetin (KIN) (1.5 mg L^-1) supplementation. Furthermore, BAP (2.0 mg L^-1) + KIN (1.5 mg L^-1) + gibberellic acid (GA₃) (0.5 mg L^-1) exhibited better elongation responses with in vitro flowering. Half-strength MS medium with indole-3-butyric acid (IBA) (1.5 mg L^-1) exhibited the highest rooting responses and IBA (1.0 mg L^-1) with highest fruits, and overall biomass. Higher contents of withaferin A (WFA) [∼8.2 mg g^-1 dry weight (DW)] were detected in the reproductive phase, whereas substantially lower WFA contents (∼1.10 mg g^-1 DW) were detected in the vegetative phase. Cycloartenol synthase (CAS) (P = 0.0025), sterol methyltransferase (SMT) (P = 0.0059), and 1-deoxy-D-xylulose-5-phosphate reductase (DXR) (P = 0.0375) genes resulted in a significant fold change in expression during the reproductive phase. The liquid chromatography-mass spectrometry (LC-MS) analysis revealed metabolites that were common (177) and distinct in reproductive (218) and vegetative (167) phases. Adventitious roots cultured using varying concentrations of indole-3-acetic acid (IAA) (0.5 mg L^-1) + IBA (1.0 mg L^-1) + GA₃ (0.2 mg L^-1) exhibited the highest biomass, and IAA (0.5 mg L^-1) + IBA (1.0 mg L^-1) exhibited the highest withanolides content. Overall, our findings demonstrate the peculiarity of withanolide biosynthesis during distinct growth phases, which is relevant for the large-scale production of withanolides.

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