Enhancing callus induction of pomegranate (Punica granatum L.) leaf explants

Pomegranate (Punica granatum L.) is a nutritionally and medicinally valuable woody plant with considerable potential for commercial propagation. However, its in vitro regeneration is often constrained by challenges such as microbial contamination, phenolic browning, and low callus induction efficiency. The present study aimed to optimize key tissue culture parameters—namely surface sterilization treatments, subculturing frequency, antioxidant supplementation, and plant growth regulator (PGR) combinations—for improved in vitro culture response of P. granatum leaf explants. Leaf explants were subjected to varying sterilization protocols involving sodium hypochlorite, Bavistin, and Tween20. The most effective treatment (10% NaOCl for 15 min + 0.2% Bavistin + 0.1% Tween20) achieved 84% survival and the lowest contamination rate (17.71%). Phenolic browning was mitigated through frequent subculturing, with four transfers per week completely eliminating browning. Antioxidant supplementation, particularly the combination of polyvinylpyrrolidone (PVP) and citric acid, significantly reduced browning (20%) and enhanced callus quality (score 4.1/5). Callus induction was highest (95%) on MS medium supplemented with 1.5 mg/L BAP and 0.5 mg/L NAA, producing friable, light-green callus with high morphogenic potential. These findings underscore the importance of integrating optimized sterilization protocols, antioxidant strategies, and tailored PGR combinations to improve the tissue culture response of P. granatum. The established protocol provides a reliable foundation for further regeneration, genetic transformation, or secondary metabolite production studies in this species.