Intraspecific hybridization of Aspergillus awamori var. kawachi by protoplast fusion.

Abstract

Protoplast fusion was studied to breed a koji-mold, Aspergillus awamori var. kawachi, used for making a Japanese spirit, shochu. Protoplasts were obtained from mycelia of two biochemical mutants using an enzyme system from Trichoderma viride consisting of mainly β-1, 3-glueanase and chitinase. Approximately 10-20% of these protoplasts regenerated on a solid culture medium with a soft agar overlay. When the two types of protoplasts were treated with a fusogen including 35% polyethylene glycol, they fused effectively and formed a heterokaryon. A strain obtained from the hetrokaryon using d-camphor showed high stability (98.8%) and numerical sporulation, and produced the same levels of amylase and citric acid as those by the original strain. From these results and the formation of recombinants, the new strain was assumed to be a heterozygous diploid. The appearance frequency of the diploid was 2.9 × 10-2. The existence of a parasexual cycle in this fungus was also discovered, using the protoplast fusion technique.