Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of Gaeumannomyces tritici
Mei Wang | Jie Zhang | Lanying Wang | Lirong Han | Xing Zhang | Juntao Feng
Take-all, caused by Gaeumannomyces tritici, is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, G. tritici 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&mdash:temperature, enzyme lysis time, and concentration of the lysing enzyme&mdash: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&ndash:54 transformants/&mu:g DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of G. tritici. An optimised PEG-mediated protoplast transformation system for G. tritici was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of G. tritici and could be used to transform other fungi.
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