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A Tool for Monitoring Trichoderma harzianum: I. Transformation with the GUS Gene by Protoplast Technology. C. Thrane, Plant Pathology Section, Institute of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark; M. Lübeck(2), H. Green(3), Y. Degefu(4), S. Allerup(5), U. Thrane(6), and D. Funck Jensen(7). (2)(3)(4)(7)Plant Pathology Section, Institute of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark; (4)Current address: Plant Breeding Section, Department of Plant Biology, University of Helsinki, FIN-00710 Helsinki, Finland; (5)Plant Physiology and Anatomy Section, Institute of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark; (6)Department of Biotechnology, Technical University of Denmark, DK-2800 Lyngby, Denmark. Phytopathology 85:1428-1435. Accepted for publication 28 August 1995. Copyright 1995 The American Phytopathological Society. DOI: 10.1094/Phyto-85-1428.

To obtain a genetically marked strain of Trichoderma harzianum that can be used as a tool for studies of population dynamics, T. harzianum was cotransformed with the Escherichia coli uidA β-glucuronidase (GUS) gene and the hygromycin B (hygB) gene as the selective marker. To improve the efficiency of conditions used for the transformation, the isolation, reversion, and germination of mycelial protoplasts were studied by scanning electron microscopy (SEM). Hexamethyldisilzane was useful for preparing protoplasts from suspensions for SEM. It was essential to obtain a transformant that phenotypically resembled the wild-type. After mitotic stabilization of the transformants by single-spore isolations, three transformants were compared to the wild-type by measurement of spore germination and mycelial growth rates, identification of 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 wild-type. Furthermore, transformant T3a remained genetically stable during nonselective cultivation on plates and in sterile peat-bran.

Additional keywords: reporter gene.