Protoplast Formation in Sclerotium rolfsii ATCC 201126 (poster)

J.I. FARIÑA; N.I. PEROTTI; O.E. MOLINA; L.I.C. FIGUEROA

Chicago

Congreso; 99th GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY; 1999

ASM

Sclerotium rolfsii ATCC 201126, a filamentous fungus isolated from environment, was selected on the basis of its ability to produce large amounts of the exopolysaccharide scleroglucan. The genetic characteristics of this organism have not been studied until present. A first approach for genetic studies such as electrophoretic karyotyping, transformation, and genetic manipulation involves a key step, the obtention of protoplasts. It is known that optimal conditions for protoplast release are often specific for each fungal species. In this case, we studied the influence of culture medium, osmotic stabilizers, biomass:enzyme ratio, blending of mycelium previous to protoplast formation, and pretreatment of mycelium with dithiothreitol (DTT). Using lysing enzymes from Trichoderma harzianum in a ratio of (1:1) biomass:enzyme, protoplast yields after 1 hour of incubation were affected by the culture medium composition, being the higher ones obtained in Czapek’s medium with ammonium as nitrogen source, for the same culture age. Among the osmotic stabilizers tested, 0.6 M MgSO4 led to the best results. When the mycelium was blended prior to the enzyme addition, a considerable decrease in protoplast formation was observed. The pretreatment with DTT did not show a noticeable effect on protoplast yields. Under the conditions selected, the microscopic observation revealed protoplasts with visible and large vacuoles in association with a slight granular appearence. A little amount of cellular debris was also observed, making the subsequent cleaning up procedures not tedious.