Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Transformation of Bottle Gourd Rootstock (Lagenaria siceraria Standl.) using GFP gene

GFP유전자를 이용한 대목용 박 형질전환

  • 임미영 ((주)농우바이오 생명공학연구소) ;
  • 박상미 ((주)농우바이오 생명공학연구소) ;
  • 권정희 ((주)농우바이오 생명공학연구소) ;
  • 한상렬 ((주)농우바이오 생명공학연구소) ;
  • 신윤섭 ((주)농우바이오 생명공학연구소) ;
  • 한증술 (농촌진흥청 원예연구소) ;
  • 한지학 ((주)농우바이오 생명공학연구소)
  • Published : 2006.03.01
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Abstract

Bottle gourd (Lagenaria siceraria Standl.) has been used as a rootstock for the watermelon cultivation because of better growth ability at low temperature and avoidance from contamination of the soil disease. Since the genetic source for the elite rootstock is limited in nature, the genetic engineering method is inevitable to develop new lines especially to obtain the functionally important or multi-disease resistant bottle gourd. Recently, our lab has set up a successful system to transform the bottle gourd. in order to monitor the transformation process, GFP gene is used. Cotyledons of the inbred line 9005, 9006 and G5 were used to induce the shoot under the selection media with MS + 30 g/L sucrose + 3.0 mg/L BAP + 100 mg/L kanamycin + 500 mg/L cefotaxime + 0.5 mg/L $AgNO_3$, pH 5.8. The shoot was developed from the cut side of the explants after 3 weeks on the selection media. The shoot was incubated in the rooting media with 1/2 MS + 30 g/L sucrose + 0.1 mg/L IAA + 50 mg/L kanamycin + 500 mg/L cefotaxime, pH 5.8 and moved to pot for acclimation. Although the shoot development rate was depended on the genotype, the G5 was the best line to be transformed. Monitoring GFP expression from the young shoot under microscope could make the selection much easier to distinguish the transformed shoot from the non-transformed shoots.

박과 (Cucurbitaceae) 작물에서 박 (Lagenaria siceraria Standl.)은 식용뿐만 아니라 주로 저온 신장성의 확보와 토양전염성 병해를 회피하기 위한 수단으로서 수박의 대목으로 많이 사용된다. 유전공학 방법을 이용한 토양병 내성이 증대된 신품종 개발이 필요한 요즘 박 형질전환체 개발의 효과적인 시스템 확립을 위하여 식물형질전환 연구에 매우 효율적으로 이용되는 마커인 GFP 유전자를 도입하였다. 실험재료로 이용된 박 계통은 9005, 9006 및 G5였으며, 신초 유기를 위한 선발 배지는 3.0 mg/L_ BAP + 100 mg/L kanamycin +500 mg/L cefotaxime + 0.5 mg/L $AgNO_3$, pH 5.8이었다 박 선발배지에 치상된 자엽 절편체는 3주 정도 지나면 절단면 즉 치상 부위에서 작은 돌기들이 발생하였고, 갈수록 절편체는 갈변하여 고사하였다. 선발된 개체에서는 신초가 발생하였으며 선발 후 7주가 경과하면 pot로 이식하여 발근된 완전한 식물체를 얻을 수 있었다. 본 실험결과 박 형질전환 시 genotype에 따른 신초 발생률에 상당한 차이를 보였는데 계통 9005의 경우 신초 발생율이 0.0%이였으며 계통 G5의 경우 4.1%이였다. 형질전환으로 발생된 신초는 GFP가 발현되지 않는 것과 GFP가 발현되는 것 두 가지 양상을 볼 수 있었다. PCR 및 Southern blot 분석을 한 결과, 9006과 G5 두 genotype에서 형질전환 신초에서 GFT 유전자를 가지고 있는 것으로 확인되었으며 GFP 유전자가 도입된 박 형질전환체를 얻을 수 있었다. 따라서 GFP 유전자는 박 형질전환시 유식물체에서 형질전환체를 쉽게 선발할 수 있는 유용한 마커로 이용 될 수 있다.

Keywords

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