Redox-linked hormetic responses of Withania coagulans (Stocks) Dunal to Mn, Zn, and Fe supplementation in vitro

Withania coagulans (Stocks) Dunal is a “Near-Threatened” medicinal herb valued for its bioactive withanolides, yet its natural regeneration remains poor. This study evaluated the responses of in vitro cultured shoot tip and nodal explants to graded supplementation of Mn, Zn, and Fe in the culture medium (1x, 5x, 10x, 20x, 30x, and 50x Murashige and Skoog levels) to assess their effects on morphogenesis, antioxidant activity, and micronutrient accumulation. It was hypothesised that moderate micronutrient enrichment would enhance growth and antioxidant activity, whereas higher levels would inhibit development. Moderate supplementation, particularly at 10x–20x levels, significantly improved regeneration efficiency (up to 95%), shoot elongation (up to 5.8 cm), chlorophyll content and biomass accumulation, while excessive exposure (30x–50x) resulted in pronounced growth inhibition. Antioxidant enzymes displayed distinct micronutrient-specific activation patterns, with Mn maximised superoxide dismutase (SOD) and guaiacol peroxidase (GPX) activities, Zn enhanced catalase and GPX, and Fe strongly induced catalase, (SOD), and polyphenol oxidase (PPO) at optimal concentrations before declining at higher levels. Micronutrient uptake increased with external supplementation, following the order Zn > Mn > Fe, and nodal explants consistently exhibited greater tolerance than shoot tips. This study provides the first integrated Mn–Zn–Fe response profile for W. coagulans, offering a physiological basis for nutrient-optimised micropropagation, stress management, and ex situ conservation.