High Yield Preparation of Lentinus edodes ("Shiitake") Protoplasts with Regeneration Capacity and Mating Type Stability

Abstract

An efficient method for the liberation and regeneration of L. edodes protoplasts was developed using an enzyme mixture of Cellulase Onozuka RS and chitinase, pH 4.6. Homogeneous young mycelia as the protoplast material were quickly cultured in a liquid medium with 'wood components' after inoculation of cut fragments of hyphae. The preparation of more than 6 × 10⁷ protoplasts/ml/4hr and regeneration of more than 15% were attained. The dikaryons formed through the mating of monokaryotic strains of protoplast origin could develop complete fruit bodies on small scale sawdust cultivation (30g medium) in 50-60 days. After protoplasting or fusion treatment with polyethyleneglycol, the mating phenotypes were sufficiently well preserved.