Physical parameters affecting electrofusion of yeasts: ζ-potential on the surface of yeast protoplasts and osmotic pressure of the solution

Abstract

Yeast electrofusion is a relatively new technique which appears to offer some advantages over conventional chemical procedures. However, various parameters were found to affect fusion efficiency. The effects of both the osmotic pressure of the solution and the ζ-potential of yeast protoplasts on electrofusion were studied in detail in this study. The optimal osmotic pressure of the sorbitol solution for stability of protoplasts was about 0.9 Osm kg −1 because over 70% of protoplasts remained intact when an intensive pulse of 7 kV cm −1 of 100 μs duration was applied. On the other hand, the optimal pressure of sorbitol for the highest fusion efficiency was 0.6-0.7 Osm kg −1 when several pulses (5.5 kV cm −1, 25 μs, 1–10 charges) were applied. In the presence of divalent cations (especially Ca 2+ and Mg 2+) at a conductivity of 40–50 μs cm −1, a remarkable increase of fusion efficiency and frequent appearance of multimembrane-fused protoplasts were observed. Under the conditions established, fusion efficiency by electrofusion was 1:50-1:100 and approximately 10,000 times higher than that achieved by chemical methods such as the polyethylene glycol procedure. The acceleration of cell fusion by both divalent cations and change of conductivity was considered due to neutralization of anion residues of suger-protein complexes on the surface of the protoplasts because the ζ-potential of protoplasts was around 0 mV under the conditions used.