Transformation of Botrytis cinerea with the hygromycin B resistance gene, hph

A transformation method has been developed for the phytopathogenic fungus Botrytis cinerea. Protoplasts were transformed with pAN7-1 plasmid carrying the Escherichia coli hygromycin phosphotransferase gene (hph), conferring hygromycin B resistance, downstream from an Aspergillus nidulans promoter. Molecular analysis, showed that transformation resulted in an integration of the plasmid into different regions of the B. cinerea genome and occurred through non-homologous recombination. The frequency was 2-10 transformants per micrograms of DNA. Transformants expressed phosphotransferase activity confirming that the hph gene conferred the hygromycin-resistance phenotype. All transformants analysed so far proved to be stable after several subcultures without any selective pressure.