Abstract: The conditions for protoplast preparation and regeneration from the mycelia of Inonotus obliquus were investigated. The optimal amounts of four enzymes were explored by orthogonal array design and other conditions were studied by single factor method. The results showed that the mycelium harvesting after 7-day culture, adding 5 mg/mL cellulase, 15 mg/mL snailase, 15 mg/mL lywallzyme and 15 mg/mL drislease, using 0.6 mol/L MgSO4 as the osmotic stabilizer, digestion at 35 ℃ for 5.5 h were found to be best for the preparation of protoplasts. The optimal conditions for protoplast regeneration of Inonotus obliquus were CYM or GYM as the medium, 0.6 mol/L mannitol as the osmotic stabilizer using and digestion time of 3.5 h. No significant difference in protoplast regeneration rate was observed between CYM and GYM media. The highest protoplast regeneration rate was 0.13%.

Key words: Inonotus obliquus, protoplast, orthogonal test