Establishment of a PEG-mediated protoplast transformation system for the orchid mycorrhizal fungus Mycena purpureofusca.

Mycena purpureofusca is an essential symbiotic fungus associated with Gastrodia elata and Dendrobium officinale, two economically important medicinal and edible orchids in China. However, the lack of an efficient and stable genetic transformation system has limited research on orchid-fungal symbiosis and constrained the development of improved fungal strains. In this study, we established an efficient PEG-mediated protoplast transformation system for M. purpureofusca using the pCAMBIA1303 vector. Key parameters affecting protoplast preparation were evaluated by step-wise single-factor experiments followed by orthogonal optimization, including fungal age, osmotic stabilizer, lywallzyme concentration, digestion temperature, and digestion time. The highest protoplast yield was obtained from 24-h-old mycelia digested with 2.5% lywallzyme in 0.8 M KCl at 24°C for 4 h, resulting in 8.90 × 106 protoplasts/mL. Protoplast regeneration efficiency reached 3.54% on SGAY medium. Seven hygromycin-resistant isolates remained stable after three rounds of selective subculture, and five were confirmed by diagnostic PCR, corresponding to a stable transformation frequency of 4.3% based on the total regenerants obtained on non-selective regeneration plates. This transformation system provides a useful genetic tool for functional studies in M. purpureofusca and for future investigations of orchid-fungal symbiosis.