Isolation and regeneration of protoplasts fromPenicillium digitatum

AbstractThe effects of some factors on the isolation of protoplasts fromPenicillium digitatumwere studied, including the appropriate material (young mycelia and generating spores), the concentrations of enzyme and osmotic pressure stabilizers, reaction time and reaction temperature. Results demonstrated that germinating spores were an ideal material resource for the isolation ofP. digitatumprotoplasts. Highest yield and quality ofP. digitatumprotoplasts were obtained by shaking germinating spores suspended in a solution of 10 mg/ml Lywallzyme™ dissolved in 0.7 M NaCl as osmotic pressure stabilizer at 80 rev/min and 30°C for 3.0–3.5 h. The regeneration rate of the isolated protoplasts was as high as 24.9% on double-layer Czapek medium containing 0.7 M NaCl. Additionally, observation of the protoplast release pattern showed that the protoplasts ofP. digitatumwere released primarily from the hyphal apex and occasionally from the subapical or original sites of germinating tubes. The protoplasts ofP. digitatumwere regenerated in a direct manner of either yeast-like cell development or mycelium formation.