An Improved Transformation System for Molecular Biology Research in Peach Fruit

ABSTRACT Plant genetic engineering using Agrobacterium ‐mediated transformation or particle bombardment has enabled targeted trait introduction into many crops. Conventional transient transformation approaches, while offering rapid gene expression, are inherently short‐lived and insufficient for sustained functional studies. In this study, we establish an improved Agrobacterium tumefaciens (strain LBA4404)‐mediated transformation system for peach fruit by performing three consecutive infections at three developmental stages: fruit‐set, mid‐growth, and 2 weeks before harvest. Compared with conventional single‐time‐point transient transformation—which typically yields gene expression lasting only about one week—our protocol achieves prolonged and robust transformation effects, including stable overexpression of PpSNARE13 and efficient targeted mutagenesis of PpVOZ1 and PpVOZ2 persisting for up to 32 days. This repeated transformation strategy substantially extends the effective expression window and circumvents the lengthy selection procedures required for stable transformation. It enables efficient and reliable gene function screening directly in peach fruits, thereby offering a practical tool for postharvest biology and functional genomics in woody plants. This system can be widely applied to gene function studies in peach fruits and holds significant potential for advancing postharvest quality improvement and fruit preservation.