Factors Affecting Electrofusion of Plant Protoplasts

식물 Protoplast의 전기자극 융합에 관여하는 인자

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/cm) of AC Pulse for protoplast pearl-chain formation were 10 kHz-20 V/cm and 1 MHz-60 V/cm for petunia, 100 kHz-40 V/cm and $1\;MHz-40{\sim}60\;V/cm$ for carrot, and $1\;MHz-40{\sim}80\;V/cm$ for soybean, respectively. The optimum condition of DC pulse treatment at the 1 MHz-60 V/cm-15sec treatment of AC for electrofusion of petunia protoplasts was 2.5 kV/cm-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.

Petunia, 당근 및 대두의 Protoplast를 전기자극법으로 융합시킬 때의 적정 전기자극 조건과, petunia protoplast의 융합율 그리고 또는 viability에 대한 calcium, magnesium, pretense, trypsin, triton X-100, concanavalin A, dimethyl sulfoxide(DMSO), glycerol monooleate 그리고 spermine의 영향을 조사하였다. Protoplast pearl chain 형성을 위한 교류의 주파수와 전압은, Petunia의 경우 10kHz-20 V/cm와 1 MHz-60 V/cm, 당근의 경우에 100 kHz-40 V/cm와 $1\;MHz-40{\sim}80\;V/cm$, 그리고 대두의 경우에 $1\;MHz-40{\sim}80\;V/cm$가 양호하였다. Protoplast융합을 위한 최적 직류 처리조건은, petunia의 경우에 교류를 1MHz, 60 V/cm (15 sec) 처리한 후 직류를 2.5 kV/cm, $40\;{\mu}sec$ 처리했을 때였는데, 이 때 Protoplast의 생존율 과 융합율은 각각 45 %와 10 %였다. 당근과 대두의 경우는 처리시간에 관계없이 직류 2.5 V/cm 이하에서는 융합이 일어나지 않았고, 2.5 V/cm 이상에서는 protoplast가 파괴되었다. Calcium은 전기자극융합을 촉진시켰는데, 최적 농도는 140 mM이었고 이 때 protoplast의 생존율과 융합율은 43 %와 11 %였다. Magnesium은 융합촉진효과가 없었으나 calcium이 존재할 때는 융합을 촉진하였는데, calcium 140 mM일 때 최적 magnesium농도는 140mM이었고, 이때의 생존을과 융합율은 각각 45%와 13%였다. 융합배지에 protease, trypsin, triton X-100, concanavalin A, DMSO, glycerol monooleate, spermine을 처리했을 때 융합율은 대조구(11.2 %)에 비해 각각 2.4, 2.1, 1.6, 1.4, 1.8, 1.5, 2.2배로 증가하였고, 생존율은 각각 37 %, 35 %, 38 %, 41 %, 31 %, 37 %, 42 %로 대조구(45.2 %)에 비해 모두 저하되었다.