Zhen-Zhu Su1, Meng-Di Dai2, Jia-Nan Zhu1, Yu-Lan Zeng1, Xuan-Jun Lu1, Xiao-Hong Liu1, Fu-Cheng Lin3. 1. Institute of Biotechnology, Zhejiang University, Hangzhou, 310058, China. 2. State Key Laboratory for Managing Biotic and Chemical Treats To the Quality and Safety of Agro-Products, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China. 3. State Key Laboratory for Managing Biotic and Chemical Treats To the Quality and Safety of Agro-Products, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China. fuchenglin@zju.edu.cn.
Abstract
OBJECTIVE: To investigate the protoplast preparation and transformation system of endophytic fungus Falciphora oryzae. RESULTS: F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9 M KCl solution and 10 mg mL-1 glucanase at 30 °C with shaking at 80 rpm for 2-3 h. When the protoplasts were plated on a regenerations-agar medium containing 1 M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired green fluorescent protein-expressing transformants by transforming the pKD6-GFP vector into protoplasts. Further, the GFP expression in fungal hyphae possessed good stability and intensity during symbiosis in rice roots. CONCLUSIONS: This study provided a protoplast transformation system of F. oryzae, creating opportunities for future genetic research in other endophytic fungi.
OBJECTIVE: To investigate the protoplast preparation and transformation system of endophytic fungus Falciphora oryzae. RESULTS: F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9 M KCl solution and 10 mg mL-1 glucanase at 30 °C with shaking at 80 rpm for 2-3 h. When the protoplasts were plated on a regenerations-agar medium containing 1 M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired green fluorescent protein-expressing transformants by transforming the pKD6-GFP vector into protoplasts. Further, the GFP expression in fungal hyphae possessed good stability and intensity during symbiosis in rice roots. CONCLUSIONS: This study provided a protoplast transformation system of F. oryzae, creating opportunities for future genetic research in other endophytic fungi.