Preparation of protoplasts of the fungus Trametes hirsuta 072 and study of the effect of antioxidants on their formation and regeneration

Abstract

The consistent application of homogenization and enzymatic treatment is required to obtain protoplasts from the basidiomycete fungus Trametes hirsuta. The maximum yield of protoplasts (∼2.5 × 10⁷/mL) was achieved when mycelium in the exponential growth phase (60 h) was used. The maximum stability was observed in MES⁺ buffer during 4 h of incubation; in this case the titer reduction was 5–7%. Studies of the effect of antioxidants with different antioxidant capacities expressed in mmol equivalents of Trolox (ascorbate, 0.99; α-tocopherol, 1.0; β-carotene, 2.14; quercetin, 3.98) indicated that the yield of protoplasts was increased in the presence of β-carotene and quercetin by 18–24%. The studied antioxidants did not affect the protoplasts stability. The degree of regeneration of protoplasts correlated with the antioxidant capacity of the studied antioxidants and was maximal (0.4%) in the presence of β-carotene and quercetin; it was 0.1% in the presence of MES⁺. The rate of protoplast growth was two times higher in the presence of β-carotene and quercetin.