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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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