Membrane Permeability Changes and Ultrastructural Abnormalities Observed During Protoplast Fusion

A comparative study between polyethylene glycol (PEG) fused and electrofused protoplasts of pea has shown that the chemical fusion method was inferior, and PEG fusion products of about 15 % were possible, but over 50% of the initial protoplast mix was lost or damaged. The PEG treated protoplasts had decreased viability, respiratory O2 consumption was lower, and PEG markedly decreased O2 evolution in the light, the rate of CO2 fixation, protein and chlorophyll levels. Membrane damage was present in PEG treated protoplasts, as evident by the large amount of efflux of intracellular potassium (K) (>70%) and CO2 fixation products (>40%). The best electro fusion protocol (achieved by trial and error) produced 25—30% fusion products, and higher protoplast viability than PEG. Protoplast membrane damage due to electric currents was lower, and respiratory O2 consumption, O2 evolution and CO2 fixation in the light were close to, and in some cases higher than in freshly isolated (control) protoplasts. Protein and chlorophyll content of electrofused protoplasts were close to controls, and the loss of intracellular K was now about 10%. Electron micrographs of isolated protoplasts showed considerable accumulation of osmiophilic deposits near membranes, and especially at membrane fusion sites. PEG mediated fusion of protoplasts caused greater protoplast distortion and membrane disruption compared to electrofusion, but PEG fusion caused increased vesicle formation near membranes in close contact. Only PEG treatment induced a high percentage (40%) of inter-organelle fusion even within intact protoplasts, but these were predominantly only part fusion of the outer membranes.