Protoplast culture and transformation technology offer a novel approach for developing new plant varieties. However, efficient protocols for isolating and transforming watermelon protoplasts have remained elusive. This study aimed to establish a robust protocol for high‐yield protoplast isolation and transient transformation. The objective was to establish a high‐yield, high‐viability mesophyll protoplast isolation system and to achieve transient gene transformation and expression. The results demonstrated that the highest yield (17.25 × 10 6 protoplasts per gram fresh weight) was obtained from true leaves digested for 16 h with an enzyme mixture containing 2.0% cellulase, 0.6% macerozyme, and 1.5% pectinase, followed by purification via sedimentation. Viability assessed by fluorescein diacetate (FDA) staining reached 83%. The recombinant vector pCAMBIA‐Super1300‐ ClBBM ‐GFP, harboring GFP as a reporter gene, was successfully constructed. Optimal transient transformation efficiency (64.9%) was achieved using 40% PEG6000 for 30 min. Subcellular localization analysis revealed that the ClBBM protein was predominantly localized to the nucleus. This study successfully established efficient isolation and transformation system, provides a crucial foundation for gene function analysis, genome editing and the development of new varieties in watermelon.
Xie et al. (2026) studied this question.