An optimized regeneration protocol for chili peppers (<i>Capsicum annuum</i> L.) through genotype-specific explant and growth regulator combinations

Efficient regeneration of pepper (<italic>Capsicum annuum</italic> L.) is essential for genetic transformation and improving agronomic traits. However, the recalcitrant nature of pepper to <italic>in vitro</italic> manipulation has led to relatively low regeneration efficiencies. This study presents an optimized regeneration protocol for the Zunla-1 variety, focusing on genotype, explant type, and growth regulator combinations. Cotyledon and hypocotyl explants showed superior callus induction with 1 mg/L IAA or 1 nM CaREF1 peptide compared to 2,4-D treatments, while root explants exhibited limited callus formation. Shoot regeneration was most effective with 5 mg/L AgNO₃ and 1 nM CaREF1 for cotyledon explants, and shoot elongation was enhanced with GA₃ (0.5 mg/L) and CaREF1 (1 nM) in Zunla-1. Rooting efficiency was improved with IBA combined with CaREF1, yielding higher rooting percentages than IBA or IAA alone. Supplementation with CaREF1 peptide increased the overall ratio of regenerated plants from 27.2% to 55.0%. Histological analysis showed improved cellular organization in explants treated with CaREF1, indicating active cell division and differentiation. This study provides an optimized protocol for efficient regeneration of Zunla-1, highlighting the role of CaREF1 in overcoming regeneration recalcitrance.