Ahighlye⁄cient polyethyleneglycol-mediated transformation method formushrooms

A highly efficient transformation system mediated by polyethylene glycol was developed for the cultivated mushroom Pleurotus ostreatus. Eighty to 180 integrative and stable-resistant colonies appeared per mg of DNA per 10 viable protoplasts in a transformation experiment with the hygromycin B phosphotransferase gene (hph), which is about 40–1800 times higher than that previously reported in P. ostreatus. One hundred to 150 transformants emitting green fluorescence were observed per mg of DNA per 10 viable protoplasts in a transformation with the green fluorescent protein gene, but green fluorescence disappeared 30 h after transformation, suggesting that the green fluorescent protein gene was only transiently expressed in P. ostreatus. Plasmid pAN7-1 was also transferred into two important cultivated mushrooms, Ganoderma lucidum and Lentinus edodes, and 120–150 and 85–100 transformants per mg of DNA per 10 viable protoplasts were obtained, respectively, which is seven to 38 times and 24–28 times greater than previously reported. These data indicate that this new polyethylene glycol-mediated transformation procedure is highly efficient for mushrooms, and could be a useful tool in mushroom improvement by gene engineering.