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

Objective: To explore the optimal methods for the protoplast preparation and regeneration of Hirsutella sinensis by optimizing the limiting factors. Results: 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 20 days in the regeneration medium with 0.6 M mannitol and 1.5% agar. Conclusions: 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. [ABSTRACT FROM AUTHOR]