A TOOL FOR MONITORING TRICHODERMA-HARZIANUM .1. TRANSFORMATION WITH THE GUS GENE BY PROTOPLAST TECHNOLOGY

To obtain a genetically marked strain of Trichoderma harzianum that ca n be used as a tool for studies of population dynamics, T. harzianum w as cotransformed with the Escherichia coli uidA beta-glucuronidase (GU S) gene and the hygromycin B (hygB) gene as the selective marker. To i mprove the efficiency of conditions used for the transformation, the i solation, reversion, and germination of mycelial protoplasts were stud ied by scanning electron microscopy (SEM). Hexamethyldisilzane was use ful for preparing protoplasts from suspensions for SEM. It was essenti al to obtain a transformant that phenotypically resembled the wild-typ e. After mitotic stabilization of the transformants by single-spore is olations, three transformants were compared to the wild-type by measur ement of spore germination and mycelial growth rates, identification o f secondary metabolite profiles, and studies of extracellular proteins . One transformant, T3c, was dissimilar to the wild-type in most tests , whereas transformant T3a was physiologically very similar to the wil d-type. Furthermore, transformant T3a remained genetically stable duri ng nonselective cultivation on plates and in sterile peat-bran.