Abstract: This study aimed to explore the protoplast preparation and regeneration conditions of Tricholoma matsutake by orthogonal experiment. The results showed that there were four release modes of T. matsutake protoplasts: release from top，release from the side，hyphal fragment end release and release in situ. The optimal protoplast preparation condition was selecting mycelium cultured in liquid medium for 2 d，for enzymolysis in mixed enzyme solution containing 1% cellulose， 1% snailase and 1% lywallzyme at 34℃ for 2 h，with 06 mol·L-1 KCl as osmotic stabilizer，and the obtained preparation rate reached 275 ×107protoplasts·mL-1. The optimal regeneration condition was using mycelium cultured in liquid medium for 2 d，for enzymolysis in enzyme solution containing 1% lywallzyme and 1% snailase at 30℃ for 4 h，with 06 mol·L-1 MgSO4·7H2O as osmotic stabilizer，and the regeneration rate reached 416%．

Key words: Tricholoma matsutake, protoplast, release modes, orthogonal experiment design, regeneration rate