Electrofusion and preparation of transgenic plant by direct insert of marker gene

Marker gene의 직접삽입에 의한 transgenic plant의 제조 및 전기융합

The conditions required for plant transformation through the electroporation system and for the electrofusion of the prtoplasts were investigated for geranium (Pelargonium zonale hybrids). The optimum condition for electroporation was 1.77 kV/cm for $40\;{\mu}sec$ under which 70% of the protoplasts were viable and 58% of the viable protoplasts were stained with methylene blue. The pBin19 DNA plasmid used as a carrier vector was isolated from E.coli $DH5{\alpha}$ strain, purified, identified by the electrophoresis on agarose gel and electroporated into the protoplasts. The KM8 liquid medium gave better cell division than any other media. One MHz of AC frequency with 40 V/cm of amplitude for 15 sec followed by 0.5 kV/cm of DC amplitude for $60\;{\mu}sec$ was most efficient for the electrofusion of protoplasts.

제라니움(Pelargonium zenale hybrids)의 원형질체에 외래유전자를 전기충격에 의해 직접도입시키는 조건을 methylene blue 염색법을 이용하여 조사하였다. 그 결과 1.77 kV/cm의 직류전압을 $40{\mu}\;sec$ 동안 가해 주는 것이 제일 효과적이었으며 이때의 원형질체 생존율은 70% 염색율은 58%이었다. 정제한 pBin19 plasmid를 전기충격법으로 원형질체에 삽입시킨 뒤 kanamycin이 포함된 배지에서 배양하였으며 원형질체의 세포분열은 KM8P 액체배지에서 제일 높았다. 윈형질체의 최적 전기융합 조건은 교류 주파수 1 MHz를 40 V/cm에서 15 sec 동안 가한 후 직류전압 0.5 kV/cm를 $60{\mu}\;sec$ 동안 처리해 준 결과 이때의 융합율과 생존율은 각각 13%, 81%이었다.