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The optimum conditions of electric stimulation for electrofusion of protoplasts of petunia, carrot and soybean, and the effects of calcium, magnesium, protease, trypsin, triton X-100, concanavalin A, dimethyl sulfoxide(DMSO), glycerol monooleate and spermine on fusion frequency and/or viability of petunia protoplast were investigated. The optimum frequencies(Hz)-amplitudes(V/㎝) of AC pulse for protoplast pearl-chain formation were 10 kHz-20 V/㎝ and 1 MHz-60 V/㎝ for petunia, 100 kHz-40 V/㎝ and 1. MHz-40∼60 V/㎝ for carrot, and 1 MHz-40∼80 V/㎝ for soybean, respectively. The optimum condition of DC pulse treatment at the 1 MHz-60 V/㎝-15 sec treatment of AC for electrofusion of petunia protoplasts was 2.5 kV/㎝-40 sec, and under this condition the fusion frequency and viability of protoplasts were 45 % and 10 %, respectively, Both of the protoplasts of carrot and soybean were not fused under the AC and DC conditions. tested in this experiment. The electrofusion of petunia protoplasts was stimulated by calcium, and the fusion frequency and the viability of the protoplasts were 43 % and 11 %, respectively at the calcium concentration of 140 mM. Although fusion frequency was not affected by magnesium only, magnesium stimulated fusion frequency in the presence of calcium, and the viability and fusion frequency of petunia protoplasts were 45 % and 13 %, respectively, at 140 mM of magnesium-140 mM of calcium. The relative fusion frequencies of petunia protoplasts to the controls were increased by 2.4, 2.1, 1.6, 1.4, 1.8, 1.5 and 2.2 folds, respectively, by the treatments of protease, trypsin, triton X-100, concanavalin A, DMSO, glycerol monooleate, and spermine. The viabilities of petunia protoplasts were decreased by these substances.
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