Role of Activated Charcoal and Polyvinylpyrrolidone (PVP) in Enhancing Callus Quality in Recalcitrant Plants

Recalcitrance in plant tissue culture remains a major constraint to efficient callus induction, regeneration, and downstream biotechnological applications, particularly in phenolic-rich and woody plant species. Excessive phenolic exudation, oxidative stress, and accumulation of inhibitory metabolites often compromise callus quality, leading to tissue browning, necrosis, and poor regenerative competence. This study evaluated the roles of activated charcoal and polyvinylpyrrolidone as media additives for improving callus quality in recalcitrant plant species. Using a factorial experimental design, the individual and combined effects of these additives on callus induction, growth, morphology, and physiological stability were systematically assessed. Activated charcoal enhanced callus induction frequency and biomass accumulation by adsorbing inhibitory metabolites and stabilizing the culture medium during prolonged culture. Polyvinylpyrrolidone effectively mitigated oxidative stress during early culture stages, reducing phenolic oxidation and improving callus color, friability, and viability. Notably, the combined application of activated charcoal and polyvinylpyrrolidone produced synergistic effects, resulting in the highest callus quality, lowest browning indices, and improved survival across subculture cycles. Enhanced callus quality translated into improved regeneration potential, with positive implications for somatic embryogenesis, organogenesis, and genetic transformation efficiency. The findings demonstrate that integrated use of adsorptive and antioxidant additives offers a robust strategy for overcoming in vitro recalcitrance and provide a practical framework for optimizing tissue culture protocols for difficult-to-culture plant species.