Tobacco mesophyll protoplasts were transfected with tobacco mosaic virus (TMV) RNA in an electric field using a newly devised chamber constructed with gold-coated glass panel electrodes. Up to 95 9/oo of protoplasts subjected to several DC pulses (50 Ixs, 550 to 800 V/cm) while suspended in 0.5 i-mannitol containing 10 ~tg/ml TMV RNA became infected. The treatment did not affect the viability of the protoplasts or their stability during 40 h of incubation. The transfection of animal cells with

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... oreign nucleic acids mediated by an electric field was first reported by Neumann et al. (1982) , Recently, the method, which we term electrotransfection, has also been used with plant protoplasts. For instance, Ti plasmid DNA (Langridge et al., 1985) , the Tn 9 chloramphenicol acetyltransferase gene linked to the nopaline synthase promoter or to the cauliflower mosaic virus 35S promoter (Fromm et al., 1985) , and the Tn 5 aminoglycoside phosphotransferase gene linked to the cauliflower mosaic virus 19S promoter have been transfected into carrot or tobacco protoplasts in an electric field. Similar treatment of tobacco protoplasts resulted in 75~ transfection with tobacco mosaic virus (TMV) RNA and 46 9/o transfection with cucumber mosaic virus RNA (Nishiguchi et al., 1986) . However, the conditions used for these transfections, such as salt concentrations and DC pulse voltages, differ considerably. Electrolyte concentration affects the conductivity, which itself affects the DC pulse voltage required fo.r the transfection. High DC pulse voltages and long pulse durations as used in the published work quoted above are known to cause the disruption of protoplasts (Zimmermann & Scheurich, 1981; Zimmermann et al., 1981) and Nishiguchi et al. (1986) found that approximately 30 to 40~ of protoplasts survived the DC pulse used for the transfection. One proposed mechanism of transfection is a reversible breakdown of the membranes of protoplasts (Neumann et al., 1982) , similar to that during fusion as proposed by Zimmermann et al. (1981). Highly efficient (up to nearly 100~) fusion has been achieved with protoplasts of several species of plants using a DC pulse of low enough voltage to cause little damage (Zimmermann & Scheurich, 1981 ; Zimmermann et al., 1981 ; Bates et al., 1983; Zachrisson & Bornman, 1984). Moreover, low concentrations (100 ~tM) of Ca 2÷ were reported to promote the electro-fusion of protoplasts (Zimmermann, 1982; Watts & King, 1984) . On the other hand, for transfection, Mg 2÷ has been used at high concentration (9 to 15 mM) and reported to be essential (Langridge et al., 1985) . We have therefore tried to find suitable conditions for transfection in which almost all protoplasts survive, by a combination of decreasing the pulse voltage and controlling the ionic conditions in the transfection medium. Therefore, we have examined the effects on the efficiency of electro-transfection of different RNA concentrations, protoplasts concentrations, pulse voltages, pulse durations, pulse times and concentrations of Ca 2+ and Mg 2+ using tobacco mesophyll protoplasts and TMV RNA. We also describe a newly devised chamber for conducting transfection experiments. 0000-7079 © 1986 SGM