Formation and Regeneration of Flammulina velutipes and Pleurotus ostreatus Protoplasts

Abstract

The preparation and regeneration of mycelial protoplasts from Flammulina velutipes and Pleurotus ostreatus were examined. With both F. velutipes and P. ostreatus, the age of mycelium remarkably affected the isolation of protoplasts. High yields of protoplasts (6.7×10⁷ cells/ml in F. velutipes and 3.8×10⁷ cells/ml in P. ostreatus)were obtained from young mycelium (2-3 days culture). Older mycelium(7-8 or 20-24 days culture) gave low yields. The components of a liquid medium showed a significant effect on the yield of protoplasts. In the case of both mycelium, the highest yield of protoplasts was obtained from the mycelium cultured on OS(Onion-soysauce)medium. The most suitable combination of commercial enzymes for digesting cell walls of mycelium was cellulase "ONOZUKA" RS (or R-10), Zymolyase 5000, chitinase and β- glucuronidase. In the case of both F. velutipes and P. ostreatus protoplasts, the development of hyphae was observed after 2 days on the solid media, but only after 5-10 days in liquid media. Three characteristic patterns could be observed for the regeneration of protoplasts. In the first, one or more germ tubes were directly produced from spherical protoplasts. Regeneration of this type occurred most frequently. In the second, some protoplasts formed a short chain of "yeast-like cells". In the third case, several modified "bud-like cells" were formed giving rise to germ tubes. The protoplasts cultured on the OS solid medium showed the highest regeneration frequency.