Enhancement of protoplast preparation and regeneration of Hirsutella sinensis based on process optimization.

Abstract

To explore the optimal methods for the protoplast preparation and regeneration of Hirsutella sinensis by optimizing the limiting factors. During the treatment of enzymatic protoplast preparation, mycelium cultured for 7 days was the optimal start material. The maximum protoplast preparation rate of 4.3×107 protoplasts/g fresh weight (FW) was obtained after 0.5 h treatment of 1 mg/ml mixed lytic enzymes in KH2PO4-K2HPO4 buffer (pH 5.5) with 0.6 M KCl at 18°C. As for the protoplast regeneration, the maximum protoplast regeneration rate reached 12.32% through 5×103 protoplasts mL-1 cultivated for 20days in the regeneration medium with 0.6M mannitol and 1.5% agar. The preparation and regeneration of H. sinensis protoplasts was firstly established based on process optimization and it provided a foundation for the study of H. sinensis mutagenesis.