Protoplast formation, regeneration and transformation from the taxol-producing fungus Ozonium sp.

The effects of some factors on protoplast isolation and regeneration from taxol-producing fungus Ozonium sp. BT2 were investigated, including the enzymolysis time and temperature, the osmotic pressure stabilizer, mycelial incubation time, the culture medium, the culture methods and preprocessing. The mycelia were digested by enzyme combination of 1.5% lywallzyme, 0.5% snailase, 1.5% cellulase and 1.0% lysozyme for 3 h at 30°C, and a high yield of protoplasts (1.31 × 108/g moist mycelia) were obtained. The protoplasts were purified, and then regenerated by different culture methods. The results showed that regeneration frequency was not different. The protoplasts could regenerate on both PDA and Czapek medium with 0.6 M sucrose, 0.6 M sodium chloride and 0.6 M mannitol, respectively. The regeneration rate was about 2.56% under 25°C on Czapek medium using 0.6 M sodium chloride as osmotic pressure stabilizer. Furthermore, transformants were obtained by transforming the protoplasts with plasmid pAN7-1 carrying hygromycin B phosphotransferase gene (hph) conferring hygromycin resistance. This study provides the foundation to develop an engineered strain of taxol-producing fungus by protoplast mutagenesis, fusion and genetic transformation. Key words: Endophytic fungus, enzyme combination, regeneration, protoplast, transformation, Ozonium sp.