Mechanistic Study of Electrofusion of Plant Protoplasts

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1. A new electrofusion method using dielectrophoretic focusing was developed. Using this method electrofusion of barley mesophyll protoplasts was studied as function of applied pulse and ac fields. Application of ac field after induction of fusion by pulse electric field caused immediately transient extension of the fusing pair of protoplasts toward both electrodes. The extension increased with increasing strength of pulse or ac field but this phenomenon was not observed when the time taken from the fusion induction to application of the ac field was longer than about 10 s. These results suggest that the properties of the plasma membrane being in the fusible or fusing state are very different from those before the fusion induction and that the life time of the fusible membrane state is about 10 s. Quantitative analysis of this phenomenon will provide a useful approach for the study of the plasma membrane of the fusing protoplasts.2. The surface charge density of the hetro-fused protoplast, which was produced by electrofusion from protoplasts of barley mesophyll and Rauwolflia sperpentina cultured cells, was studied by cell electrophoresis. The results indicated that the surface charge density of the fused protoplasts is essentially determined by the surface charge density and size of each parent protoplast.3. The methodology was advanced for the evaluation of membrane potentials of plant protoplasts using a fluorescent probe, diS-C_3-(5). The quantitative relationship between the fluorescent quenching and membrane potential was derived and was verified experimentally for protoplast isolated from Lithospermum erythrorhizon cultured cell (MA line). This method is being applied to study the effects of Ca , La ions calmoduline antagonists (these chemicals were reported to influence the electrofusion processes) on the membrane potential of the protoplasts.