Preparation and Regeneration of Didymella bryoniae Protoplasts

  Objective  To better understand the pathogenicity of Didymella bryoniae on melons, this study aimed to establish an efficient means of preparing and regenerating the fungal protoplasts.

  Method   Driselase at 20 g·L⁻¹ and a lysing enzyme at 8 g·L⁻¹ were combined and used for the protoplast preparations. A single-factor experiment was conducted to analyze the effects of age of mycelia, time and temperature of enzymatic digestion, rotational speed of culture vessel, type and concentration of osmotic stabilizer, and pH of medium on releasing of the protoplasts. Conditions for the subsequent protoplast regeneration in medium were also optimized.

  Result  The highest yield of protoplasts of 9.65×10⁷ cells·mL⁻¹ was achieved using the mycelia cultured for 36 h to be enzymatically digested in a solution containing NaCl 0.7 mol ·L⁻¹ as the osmotic stabilizer at 30 ℃ and pH 7.0 with 140 r·min⁻¹ constant rotation for 4 h. The protoplast regeneration rate could reach up to 22.53% in a 0.5% agar SR culture medium but declined rapidly in 10 h and then leveled off in storage.

  Conclusion  The protoplasmic preparation and regeneration methods were efficient and could materially aid the establishment and research on the genetic transformation of D. bryoniae.