FUSION OF PROTOPLAST FROM ANTAGONISTIC TRICHODERMA HARZIANUM STRAINS

Protoplasts from T. harzianum isolates and from their auxotrophic mutants obtained after N-methyl-N'-nitro-N-nitrosoguanidine treatment could be produced efficiently by incubating the mycelium in a crude cell wall lytic enzyme solution containing NovoZyme 234 + Lytic enzyme No.2 from Cytophaga and with 0.7 M NaCl as osmotic stabilizer. In average 10⁷ to 10⁸ protoplasts could be obtained. Between 50–70 % of these protoplasts regenerated to osmotically stable cells, when embedded in a soft agar overlay on a suitable medium.

Protoplast fusion was carried out by treating a mixture of approximately equal numbers of protoplasts from complementary auxotrophs with a 30 % (w/v) polyethylene glycol (M.W. 6 000) solution in 0.05 M CaCl₂, 0.6 M sucrose and 0.05 M glycine, pH 8.0. This suspension was embedded, without further treatment, in a soft agar overlay of selective minimal medium. Fused protoplasts of complementary auxotrophic strains could be selected as heterokaryons on selective medium. They produced prototrophic mycelium and on sporulation conidia of both the constitutive parents were produced. In order to stimulate nuclear fusion, the heterokaryotic mycelium was treated with d-camphor or exposed to UV, but heterozygous diploids could as yet not be isolated. However, when heterokaryons were transferred and subcultivated on MM or MM supplemented with 1.5 % (w/v) Oxgall, colonies of different morphological types were observed.

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