While mutagenesis breeding has been widely applied in major crops, its implementation in tropical horticultural species remains limited. In this study, we established a pingyangmycin-induced mutagenesis system using in vitro –cultured embryogenic callus of litchi. Treatment with gradient concentrations of pingyangmycin (0–40 mg/L) revealed an inverse relationship between chemical dosage and cell viability, with somatic embryogenesis completely inhibited at 40 mg/L. Whole-genome resequencing of t