“…Then, the tissue was rinsed three times with 0.6 M mannitol. The mycelia were incubated for 1.0 h in 15 mL of 10 mg/mL lywallzyme (Guangdong Institute of Microbiology, Guangdong, China) in 0.4 M mannitol at 30 • C. Optimized conditions include osmotic pressure stabilizers (mannitol, sorbitol, MgSO 4 , sucrose, or KCl), osmotic pressure stabilizer concentration (0.2, 0.4, 0.6, 0.8, and 1.0 mol/L), osmotic pressure stabilizer (10,15,20,25, and 30 mg/mL), reaction time (1, 2, 3, 4, and 5 h), and reaction temperature (22,26,30,34, and 38 • C). Protoplasts were separated by filtration through a 40 µm cell strainer, collected by centrifugation at 3500 g for 10 min at 4 • C, washed twice with 15 mL STC buffer (0.6 M sorbitol, 10 mM CaCl 2 , and 10 mM Tris-HCl pH 7.5), resuspended in STC buffer, and stored at 4 • C until use.…”