PROTOPLAST ISOLATION AND REGENERATION AND NUCLEAR STAINING OF MYCOPARASITIC GLIOCLADIUM SPECIES
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Protoplast isolation and regeneration from 24 h germinating conidia of Gliocladium catenulatum, G. roseum and G. virens have been optimized. The number of nuclei per cell of four cell types of the Gliocladium spp. was determined. The optimal enzyme combination for protoplast production contained chitinase, lyticase and cellulase onuzaku in 0.5 M mannitol osmoticum. The quantity of protoplasts produced was dependent on the duration of enzymatic digestion, the concentration of germinating condidia and the species of Gliocladium being assayed. A maximum of 11 to 55% of the protoplasts of wild-type Gliocladium spp. and of two double amino acid autoxotrophic mutants of G. roseum (both Met- Leu-) regenerated to mature, conidiating colonies depending on the regeneration medium utilized and the strain assayed. Protoplasts of G. roseum and G. catenulatum were approximately 4% multinucleate, 56% uninucleate and 40% anucleate. Protoplasts of G. virens were approximately 81% multinucleate, 6% uninucleate and 13% anucleate. Regenerating protoplasts and germinating conidia of G. catenulatum and G. roseum were predominantly uninucleate; those of G. virens were predominantly multinucleate. Conidia of all three Gliocladium spp. were predominantly uninucleate. 1988.
