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Transformation of Filamentous Fungi by High-Voltage Electroporation

V/cm with a capacitance of 25 F and a parallel resistance of 800 ohms. T. harzianum OSC subjected to these conditions in the absence of the pAN7-1 plasmid did not produce any hygromycin-resistant colonies.

Total cellular DNA of several independent transformants was isolated. Undigested DNA, as well as DNA digested with EcoRV, BamHI, HindIII, and EcoRI was subjected to agarose gel electrophoresis and analyzed by Southern blot using pAN7-1 as a probe. Wild-type T. harzianum contained no sequences hybridizing to the vector. Figure 2 shows the pattern of hybridization for three transformants. The pAN7-1 plasmid is about 6.5 kb in size, and it has no EcoRV sites, one site each for BamHI and HindIII, and two sites for EcoRI (which yields two fragments of about 3.9 and 2.6 kb). The digestions showed the restriction patterns expected from the restriction map of pAN7-1. Each transformant most likely contains tandem repeats of the vector. Hybridization of undigested DNA occurred only in the high molecular weight genomic band (UC), suggesting that pAN7-1 integrated into the genome and did not replicate autonomously.

The effect of the number of electro-competent cells on the transformation efficiency has already been observed in bacteria, mammalian cells, and plant protoplasts (Fromm et al., 1985; Dower et al., 1988; Miller et al., 1988; Shigekawa and Dower, 1988). We investigated the effect of the number of electro-competent T. harzianum cells on the transformation efficiency. By increasing the concentration of OSC to about 1.0 x 109/ml, we were able to increase the transformation efficiency 2.3-fold. When a concentration of 2.5 x 109 OSC/ml was used, a sharp decrease in the number
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