Quantification of physical and cyto-physiological conditions for the electrofusion of Saccharomyces cerevisiae.

Various conditions for obtaining hybrids of the auxotrophic mutants SH1509 and SH1512 of Saccharomyces cerevisiae by electrofusion were investigated. An AC field of 400 Vp/cm and a DC field of 2 square pulses (7 kV/cm; 60 microsec each) at an interval of 0.5 sec were effective. Treatment with 0.2 (SH1509) or 1.0 mg/ml (SH1512) Zymolyase for 1 or 1.5 hr was essential. As to the molarity of the osmotic stabilizer (sorbitol), the hybrid yield peaked at 0.6 M. The presence of CaCl2 (up to 0.4 mM) or 0.1 mM CaCl2 with 0.1 mM MgCl2 enhanced the yield. The temperature of the spheroplast suspension during pulsations also affected the yield, the most suitable temperature being 28 degrees C.