Abstract
Isolation and culture conditions of protoplasts from spores and hyphae of Pyricularia oryzae were studied. The combination of zymolyase and β-glucuronidase and that of driselase and cellulase were effective for the isolation of viable protoplasts from spores and hyphae of P. oryzae, respectively. A majority of protoplasts thus obtained had the potential to germinate and reverse. Protoplasts germinated in two different types. One is direct reversion to hyphae from regenerated mother cells (hyphal type germination) and another is formation of budded cells (budding type germination). The germination type of protoplasts was significantly influenced by pH and presence of agar in the medium. In Czapek-yeast extract liquid medium (CzYLM), the frequency of hyphal type germination was maximum at pH7.0. About 70% of hyphal protoplasts and about 50% of spore protoplasts germinated in direct reversion to hyphae in CzYLM at pH7.0. In contrast, the frequency of budding type germination was minimum at pH7. Presence of agar in the medium increased the frequency of hyphal type germination. The intensity of fluorescence by a calcofluor white of regenerated mother cells and budded cells increased with increase of pH of incubation medium while that of reversional hyphae was not influenced by pH. Thus we suggest that cell wall regeneration process was one of the factors involved in determination of germination type. This is the first report on the successful isolation of abundant viable protoplasts from spores of P. oryzae.
