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Transformation of Filamentous Fungi by High-Voltage Electroporation


Electroporation of germinative tubes or mycelia from T. harzianum did not yield transformants, so osmotically sensitive cells were tested. The effect of incubation time of the germinative tubes with the cell wall-degrading enzymes on the electro-transformation efficiency was tested. Intervals of 15, 30, and 45 minutes were used. An incubation period shorter than 30 minutes did not yield any transformants while a 45 minute incubation yielded 50% less transformants than did a 30 minute incubation time (data not shown). To optimize conditions, different combinations of voltages and capacitances were chosen, while holding the parallel resistor at 200 ohms and holding the concentration of OSC at 2.0 x 108 per ml (data not shown). The electroporation medium consisted of distilled water with 1.2 M sorbitol as an osmotic protectant. These preliminary results indicated that transformation could be obtained at 2.0 kV/cm with a capacitance of 25 F. Figure 1A shows that this was a good approximation since the maximum yield of transformants was found at 2.8 kV/cm.

The effect of the parallel resistor (and thus the time constant, τ=RxC) on electroporation efficiency was also examined (Figure 1B). The best yield was obtained when the pulse was delivered using a field strength of 2.0 kV/cm, a parallel resistance of 800 ohms and a capacitance of 25 F. Using 2.0 kV/cm with 25 F, the survival of the OSC decreased sharply at 100 ohms reaching 20% survival, and continued to decrease slowly from 200 ohms to 800 ohms. At 800 ohms, the survival was about 10.0%. From these initial experiments, the best electrical conditions found for the electroporation of T. harzianum were a field strength of 2.8 k
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