Preparation,Regeneration and Transformation of Aspergillus niger Protoplasts

To establish protoplast-mediated genetic transformation system of Aspergillus niger,the conditions for its protoplast preparation and regeneration of the mycelium incubation time,various enzymes and osmotic stabilizers were examined.The results showed that the mycelia incubated for 4 days were most suitable for protoplast release.1 mol/L sorbitol was the most effective osmotic stabilizer and the mixture solution of 1 % snailase/1% cellulose/0.1% lysing enzyme was most favorable for protoplast preparation.The suitable incubation temperature and time for the release of protoplast was 30℃ and 2.5～3 h.For the regeneration of the protoplast prepared freshly,the TZ medium containing 0.6 mol/L MgSO_(4)was the best.PEG and CaCl_(2) mediated protoplast transformation of A.niger with pBC-Hygro was performed and 4~5 stable transformats per microgram DNA were obtained.