Intraspecific hybridization of Trichoderma reesei by protoplast fusion

Protoplasts obtained from mycelia of a single auxotrophic mutant of Trichoderma reesei QM 9414 were fused with those of T. reesei QM 9136 in the presence of 0.5 M glycine-NaOH buffer, pH 7.5, containing 0.05 M CaCl₂ · 2H₂O and 35% polyethylene glycol 4,000. The regeneration frequency of these protoplasts was 8.9–12.0% on a solid culture medium with soft agar overlay. The fused protoplasts successfully formed heterokaryons showing 3.33% of the fusion frequency. A heterozygous diploid was obtained from conidia of the heterokaryon by treatment with 0.1% d-camphor. The diploid showed a 1.9 fold DNA content per conidial nucleus compared to T. reesei QM 9414. The frequency of diploid formation was about 1.9 × 10⁻⁴ per conidium. Cellulase activities, such as filter paper degrading and CM-cellulose and Avicel saccharifying activities, and the xylanase activity of the diploid showed intermediate values between those of T. reesei QM 9414 and T. reesei QM 9136. However, the β-glucosidase, β-1,3-glucanase and chitinase activities of the diploid increased to levels equal to on above those of T. reesei QM 9414 and T. reesei QM 9136. The existence of a parasexual cycle of T. reesei and the possibility of its application to enhanced enzyme productivity were confirmed using the protoplast fusion technique.