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Transformation of Filamentous Fungi by High-Voltage Electroporation

of transformants/g of DNA occurred. This low efficiency correlated with a decrease in the time constant probably caused by the high concentrations of OSC providing a higher resistance (data not shown). A sharp decrease in the transformation efficiency was observed when concentrations of OSC lower than 2.0 x 108/ml were used. The effect of plasmid DNA concentration on the number of transformants obtained by the electroporation of identical quantities of cells was also examined. The transformation efficiency increased with increasing concentrations (up to 40 g/ml) of plasmid DNA (data not shown).

To date, all protocols known for the chemical transformation of filamentous fungi are based on the utilization of PEG. Figure 3 shows the influence of different concentrations of PEG on the transformation efficiency. The use of 1.0% PEG in the electroporation medium made it possible to obtain an efficiency of 435 transformants/g of DNA. This efficiency is about four times greater than electroporation without PEG. A control pulse using only PEG in the same concentration did not yield any transformants. Recently, efficient transformation of Rhodococcus fascians (Desomer et al., 1990) and Bacillus thuringiensis (Mahillon et al., 1989) has been obtained by combining PEG and electroporation.

The decreased efficiency of transformation obtained by increasing the concentration of PEG above 1% did not correlate to the survival of the OSC (data not shown). Unexpectedly, the number of transformants was lower when concentrations of PEG below 1% were used than in the control treatment without PEG. This behavior was found to be reproducible in at least three independent experiments. Volumes smaller than
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