Development of a simplified and efficient method for genetic transformation of Gymnoascus reesii

The identification of complex genetic determinants that control the ability of a fungus to produce bioactive compounds enhances molecular breeding strategies. Through a series of optimizations, we developed a system for the genetic transformation of Gymnoascus reesii that can produce nematicidal activity against the root-knot nematode Meloidogyne incoginta. The optimal conditions for protoplast preparation and generation are as follows: mycelia growth time in CM medium of 42 h, Driselase as the cell wall-degrading enzyme, a ratio of 1.5 mL enzyme solution (20 mg/mL Driselase) per gram mycelia, a treatment time of 4 h at 28°C, and 0.7 M NaCl buffer. With the above conditions, the yield of G. reesiiprotoplasts reached 1.84 × 107 protoplasts per mL with a regeneration rate of up to 21.17% on solid regeneration medium (SR medium). The pKNTG vector with green fluorescent protein (GFP) was transformed into the protoplasts by polyethylene glycol (PEG), and 20 positive transformants were obtained. The results provide a basic technique for molecular breeding of G. reesii strains that has improved nematicidal activity.   Key words: Gymnoascus reesii, nematicidal activity, genetic transformation.