Intraspecific Hybridization Between Color Mutants of Trichoderma harzianum by Protoplast Fusion

Abstract

Protoplasts of two color mutants of Trichoderma harzianwn derived from ultraviolet treatment were prepared by using lytic enzyme system (10 mg Zymolvase. 150 mg Uzukizyme, 10 mg Chitinase and 150 mg Funcelase in 3 ml water, pH 5.0) containing 0.6 M NaCl as an osmotic stabilizer. Fusion between protoplasts nt two color mutants was observed in the solution containing 0.05 M glycine-NaOH buffer, pH 7.5, 0.05 M CaCl₂^. 2H₂O and 35% polyethylene glycol ( MW. 6,000). The fused protoplasts were successfully detected at the rate of 2% as green colonies developed on a hypersonic solid culture medium (complete medium) overlaid with hypertonic soft medium (MM with 0.5% agar). Production of well defined white and yellow sectors on tear surface of minimal medium supplemented with 0.1% (W/V) d-camphor represented the heterokaryotic usants. Green conidia were constantly obtained after transferring the green part from green-sectored colonies onto MM agar. This indicated that protoplast fusants were heterozygous diploid.