Membrane permeability changes and ultrastructural abnormalities observed during protoplast fusion

A comparative study between polyethylene glycol (PEG) fused and electrofuse d 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 ha d decreased viability, respiratory O-2 consumption was lower, and PEG marke dly decreased O-2 evolution in the light, the rate of CO2 fixation, protein and chlorophyll levels. Membrane damage was present in PEG treated protopl asts, as evident by the large amount of efflux of intracellular potassium ( K) (>70 %) and CO2 fixation products (>40 %). The best electrofusion protoc ol (achieved by trial and error) produced 25-30% fusion products, and highe r protoplast viability than PEG. Protoplast membrane damage due to electric currents was lower, and respiratory O-2 consumption, O-2 evolution and CO2 fixation in the light were dose to, and in some cases higher than in fresh ly isolated (control) protoplasts. Protein and chlorophyll content of elect rofused protoplasts were close to controls, and the loss of intracellular K was now about 10 %. Electron micrographs of isolated protoplasts showed co nsiderable accumulation of osmiophilic deposits near membranes, and especia lly at membrane fusion sites. PEG mediated fusion of protoplasts caused gre ater protoplast distortion and membrane disruption compared to electrofusio n, bur PEG fusion caused increased vesicle formation near membranes in clos e contact. Only PEG treatment induced a high percentage (40 %) of inter-org anelle fusion even within intact protoplasts, but these were predominantly only part fusion of the outer membranes.