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Transformation of Filamentous Fungi by High-Voltage Electroporation

/sub>, 10 mM sodium phosphate, pH 5.8) containing Novozyme-234 (5 mg/ml) in a Petri dish; and, these plates were incubated at 28 C for 30 minutes with agitation in a rotary shaker at 150 rpm. The OSC generated with this treatment were then centrifuged in corex tubes at 4,000 x g for 5 minutes and the pellet was washed twice with 1.2 M sorbitol in water. After the last wash, the OSC were resuspended in 1.2 M sorbitol at the desired concentration.

Transformation by electroporation. Two micrograms of transforming DNA were mixed with 400 l of the OSC suspension and kept on ice. High voltage pulses were delivered to 400 l samples in 0.2 cm electrode gap cuvettes (Bio-Rad Laboratories) by using a Gene Pulser apparatus with the Pulse Controller (Bio-Rad Laboratories). Following delivery of the electrical pulse, OSC were mixed with 1.0 ml of Potato Dextrose Broth (PDB) plus 1.2 M sorbitol (PDBS), incubated for 10 minutes on ice, and then for 2 hours at 28 C. When PEG was used, the following modifications were made: before adding the transforming DNA, spheroplasts were centrifuged at 4,000 x g for 15 minutes; the pellet was resuspended in 400 l of 1.2 M sorbitol plus 1.0% PEG 6000 (Fluka); and, after electroporation, the OSC were mixed with 5.0 ml PDBS. After the incubation period of either treatment, aliquots were plated using an agar overlay on plates containing PDA plus 1.2M sorbitol and 100 g/ml of hygromycin B as previously described by Herrera-Estrella et al. (1990).

DNA preparation. DNA was isolated from T. harzianum mycelia grown in liquid cultures in PDB medium plus 20 g/ml of hygromycin B (Calbiochem) according to the method described by Raeder and Broda (1985).


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