Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of .

Take-all, caused by , is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors-temperature, enzyme lysis time, and concentration of the lysing enzyme-were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 10⁷ protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46⁻54 transformants/μg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of . An optimised PEG-mediated protoplast transformation system for was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of and could be used to transform other fungi.