Optimization of Phytohormone Combinations for Enhanced Callus Induction in Medicinal Plant Explants

Plant tissue culture is a cornerstone of medicinal plant biotechnology, with callus induction serving as a critical prerequisite for applications such as micropropagation, genetic transformation, and in vitro production of bioactive secondary metabolites. Despite its widespread use, callus induction in medicinal plants remains highly variable and often irreproducible due to strong explant- and genotype-dependent responses and an overreliance on empirically derived phytohormone recipes. The present study addresses this challenge by systematically optimizing auxin–cytokinin combinations for efficient and high-quality callus induction using an interaction-based experimental framework. Multiple explant types (leaf, nodal segment, internode, petiole, and root) were evaluated under controlled in vitro conditions, followed by preliminary hormone screening and advanced optimization using Design of Experiments (DoE) and Response Surface Methodology (RSM). The results demonstrated significant non-linear interactions between auxin and cytokinin concentrations, revealing narrow, explant-specific optimal regions that maximized callus induction frequency and biomass accumulation while minimizing oxidative browning and tissue necrosis. Balanced auxin–cytokinin ratios consistently outperformed high-auxin or empirically selected treatments, producing friable, viable, and morphogenically competent callus. Validation experiments confirmed the predictive accuracy, robustness, and reproducibility of the optimized hormone combinations across independent culture batches. Overall, this study establishes a robust, transferable, and data-driven optimization strategy for callus induction in medicinal plants, advancing reproducibility in tissue culture protocols and supporting sustainable propagation, conservation, and biotechnological exploitation of medicinal plant resources.