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Transformation of Filamentous Fungi by High-Voltage Electroporation

Contributed by G.H. Goldman, R. Geremia, M. Van Montagu, and A. Herrera-Estrella, Laboratorium voor Genetica, Rijksuniversiteit Gent, B-9000 Gent, Belgium


Introduction
Virtually all fungal transformation protocols call for the addition of a high concentration of polyethylene glycol (PEG) following the initial period of exposure to DNA. However, electroporation could also be useful for introducing genes into filamentous fungi. Recently, electroporation has become a valuable technique for transferring nucleic acids into both eukaryotic and prokaryotic cells (Miller et al., 1988; Fster and Neumann, 1989). In this report, we show the use of high voltage-mediated transformation as an efficient method for genetic transformation of Trichoderma harzianum with plasmid DNA.


Materials and Methods
Strains and plasmid. T. harzianum strain IMI206040 was used as the transformation host. The plasmid used was pAN7-1, a derivative of pUC19 containing the E. coli hygromycin B phosphotransferase gene as a dominant selectable marker, and the gpd promoter and trpC terminator signals from Aspergillus nidulans (Punt et al., 1987). Plasmid DNA was purified from E. coli MC1061 by standard procedures (Maniatis et al., 1982) and dialyzed against distilled water prior to use.

Electro-competent cells. Osmotically sensitive cells (OSC) were prepared according to Laurila et al. (1985) with the following modifications: cellophane sheets placed on Potato Dextrose Agar (PDA) plates were inoculated with 5 x 106 spores/ml and incubated for 21 hours at 28 C; the germinative tubes from five cellophane sheets were suspended in 15 ml of buffer (1.2 M MgSO4<
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