An efficient callus protoplasts isolation and PEG-mediated transient expression system for subcellular localization in kiwifruit

Abstract

Abstract Protoplast isolation and transient gene expression have been served as valuable tools for gene function study in plants, however, they are rarely used in most woody plants due to the lack of efficient protoplast isolation system. In the present study, the protoplast isolation and purification system of kiwifruit callus was established after condition optimization. First, the loose kiwifruit callus were obtained using young true leaves as explants, inducing on MS medium containing 0.5 mg/L zeatin and 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and propagating on medium supplemented with 1.0 mg/L 2,4-D + 0.5 mg/L 6-benzylaminopurine + 0.5 mg/L 1-naphthlcetic acid. Then, the callus subcultured at 15 d were digested with enzyme solution containing 2.0% Cellulase R-10, 0.5% Macerozyme R-10, and 0.7 M mannitol for 7 h in the dark to obtain protoplasts, reaching the yield of 2.8 × 106 protoplasts·g−1FW and the viability up to 87%. In addition, transient transformation condition in kiwifruit protoplast were optimized, approximately 40% transfection efficiency was obtained with 40% PEG4000 and 15 min transfection duration. By this way, the subcellular localization of AcMYB6l-GFP fusion proteins was verified. Taken together, we developed an efficient protocol for protoplast isolation and transient transfection in kiwifruit, laying a foundation for future research on gene function and molecular breeding in Actindia.