Establishment of protoplasts isolation and transient transformation system for kiwifruit

Abstract

Protoplast isolation and transient transformation have proven to be valuable tools for studying gene function in plants. However, these techniques are rarely utilized in most woody plants due to the lack of an efficient protoplast isolation system. In this study, we successfully established a protocol for isolating and purifying protoplasts from kiwifruit callus. First, loose kiwifruit callus was induced using young true leaves as explants on MS medium containing 0.5 mg·L−1 zeatin and 0.5 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D), followed by propagation on medium supplemented with 1.0 mg·L−1 2,4-D + 0.5 mg·L−1 6-benzylaminopurine + 0.5 mg·L−1 1-naphthlcetic acid. Subsequently, the callus subcultured at 15 d was digested with an enzyme solution consisting of 2.0 % Cellulase R-10, 0.5 % Macerozyme R-10, and 0.7 M mannitol for 7 h in the dark to obtain protoplasts at a yield of approximately 2.8 × 106 protoplasts·g−1FW with viability reaching up to 95.2 %. Furthermore, we optimized the conditions for transient transformation in kiwifruit protoplasts, achieving an approximately 43.5 % transformation efficiency using a concentration of PEG4000 at 40 %. By employing this method, we successfully verified the subcellular localization of AcMYB6l-GFP fusion protein. In conclusion, we have successfully established an efficient protocol for protoplast isolation and transient transformation in kiwifruit, laying a foundation for future research on gene function and molecular breeding in Actinidia.