Effective protoplast isolation and transient transformation system for verification of gene function and editing efficiency in radish

Protoplasts serve as a versatile platform for genetic transformation and somatic hybridization. Radish ( Raphanus sativus L.) is an important root vegetable crop, and its taproot is derived mainly from the hypocotyl and main root. However, an efficient protoplast isolation and transient transformation system for radish, particularly for hypocotyl tissues, has not been well established. In this study, we developed an optimized, tissue-specific protocol for protoplast isolation and transformation from radish cotyledon and hypocotyl. Adding 0.4% polyvinylpyrrolidone (PVP) to the enzyme solution significantly improved protoplast yield and viability while reducing intracellular reactive oxygen species (ROS) accumulation. Under optimized conditions, cotyledon-derived protoplasts (CDPs) achieved a yield of 3.28 × 10 6 protoplasts g -1 fresh weight (FW) with 95.4% viability and 76.62% transformation efficiency, whereas hypocotyl-derived protoplasts (HDPs) reached 1.48 × 10 6 protoplasts g -1 FW with 94.3% viability and 69.33% transformation efficiency. The protocol was effective across radish genotypes and several Brassicaceae root crops. Furthermore, the resulting protoplasts supported subcellular localization and gene function assays, and rapid assessment of the editing efficiency of CRISPR/Cas9 vectors carrying different Cas9 promoters and single-guide RNAs. Overall, this system would provide a practical tool for gene function analysis and precision breeding in radish and related root vegetable crops.