Electrofusion of Yeast Protoplasts

Electrofusion is an important method in genetics and biotechnology. The first morphological ( 1 ) and genetical ( 2 ) evidence for yeast electrofusion was presented at the 5th Bioelectrochemical Symposium held at Weimar in 1979. Protoplasts from auxotrophic yeast strains were fused in a macrochamber (volume > 0.2 mL) by single-capacitor discharge pulses with cell-membrane contact facilitated by PEG ( 3 – 10 ; see also Note 6 ). Early experiments tested the viability of several yeast strains with field strength E > 5 kV/cm (Table 1 ). It could be demonstrated further ( 11 ) that cells from the stationary growth phase are much more resistant to killing by electric fields ≤20 kV/cm than cells from the exponential phase ( see Note 2 ). Knowing the specific threshold of a strain, a lower E value should be applied for the expansion of statistical small hydrophobic pores in the lipid bilayer to larger hydrophilic pores as the first prerequisite for fusion. The driving force for this electroporation process is the enhancement of the transmembrane voltage Δφ _m from its normal value of about 100 to about 1000 mV ( 12 ), depending on the protoplast radius r according to the general relation: