PURPOSE:To obtain easily mononuclear hyphae capable of breeding by crossing without forming spores, by treating dinuclear hyphae of basidiomycetes with an enzyme to form protoplasts and regenerating the protoplasts. CONSTITUTION:Dinuclear hyphae of basidiomycetes, e.g. SHIITAKE mushroom or Lyophyllum decastes Sing., obtained by carrying out the slant culture are inoculated into a complete culture medium, cultivated by the stationary culture method and then centrifuged to collect the dinuclear hyphae. A cell wall lytic enzyme, e.g. cellulase, is then reacted with the resultant dinuclear phae to dissolve the cell wall of the dinuclear hyphae and form protoplasts, which are then collected by using a nylon mesh. The collected protoplasts are then cultivated in a minimum culture medium containing agarose by the plate culture method to form colonies. Fungi are then collected from the colonies deficient in clumps and cultivated to give the aimed mononuclear hyphae capable of breeding by crossing.
