Abstract: The aim of this research is to obtain more active and larger amount of protoplasts of Embellisia fungal endophyte from Oxytropis glabra, investigate the optimal conditions of protoplast preparation and regeneration, and lay foundation for setting up later exogenous gene transformation system. The protoplasts are made by enzymes, and the influences on protoplast yield regarding different combined enzymes and their concentration, pH, temperatures, osmotic solutions, enzymolysis time and hyphal culture time were discussed. The protoplasts regenerated on TB3 medium were analyzed for understanding the impacts of different enzymolysis time on the regeneration. The protoplast concentration reached to 4.42×10⁵ /mL when medium mass was hydrolyzed by enzymes consisting of 2.5%（W/V）lysing enzymes, 2%（W/V）cellulose and 3%（W/V）helicase, the culture time was 10 days, the enzymolysis temperature was set to 30℃, the pH was 5.8, the 1.2 mol/L MgSO₄ solution was selected as osmotic stabilizer and the cultures were incubated in a flat shaker（80 r/min）for 8 h. The protoplast regeneration rate reached to 46.7% when enzymolysis time was 6 h.

Key words: Oxytropis glabra, fungal endophytes, protoplast, preparation and regeneration