A highly efficient polyethylene glycol-mediated transformation method for mushrooms

Abstract

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 μg of DNA per 10 7 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 μg of DNA per 107 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 μg of DNA per 107 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. © 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved.