Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA |
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Authors: | M Lorito C K Hayes A Di Pietro G E Harman |
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Institution: | (1) Department of Horticultural Sciences and Plant Pathology, Cornell University, 14456 Geneva, NY, USA;(2) Present address: Istituto di Patologia Vegetale, Università degli Studi di Napoli and Istituendo Centro CNR di Studio delle Tecniche di Lotta Biologica, I-80055 Portici (Napoli), Italy;(3) Present address: Departmento de Genetica, Universidad de Córdoba, E-14071 Córdoba, Spain |
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Abstract: | Biolistic (biological ballistic) and protoplast-mediated procedures were compared as methods for transforming strains of Gliocladium virens and Trichoderma harzianum. For biolistic transformation, conidia were bombarded using a helium-driven biolistic device to accelerate M5 tungsten particles coated with plasmid or genomic DNA. DNA from either source contained a bacterial hygromycin B resistance gene (hygB) as a dominant selectable marker. The same sources of DNA were also used to transform protoplasts using a standard polyethylene glycol-CaCl2 protoplast fusion protocol. Hygromycin B-resistant (HygBR) transformants were recovered from all strains, methods, and DNA sources except for genomic DNA used with the protoplast method. The biolistic procedure was technically simpler, and increased transformation frequency and genetic stability in the progeny as compared with the protoplast-mediated transformation. Southern analysis of homokaryotic HygBR progenies showed that the transforming sequences were integrated into the genome of the recipient strains, and apparently were methylated. This is the first study presenting detailed results on biolistic transformation of a filamentous fungus. |
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Keywords: | Fungal transformation Particle gun Biolistic gene transfer Biocontrol Biological control Methylation |
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