Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Effect of cultivar and ascorbic acid on in vitro shoot regeneration and development of bombardment-mediated plastid transformation of tomato (Lycopersicon esculentum)

토마토 재분화 효율 향상 및 엽록체 형질전환 조건

  • Roh, Kyung-Hee (Department of Agricultural Bio-resources, National Academy of Agricultural Science, RDA) ;
  • Lee, Ki-Jong (Department of Agricultural Bio-resources, National Academy of Agricultural Science, RDA) ;
  • Park, Jong-Sug (Department of Agricultural Bio-resources, National Academy of Agricultural Science, RDA) ;
  • Kim, Jong-Bum (Department of Agricultural Bio-resources, National Academy of Agricultural Science, RDA) ;
  • Lee, Seung-Bum (Department of Agricultural Bio-resources, National Academy of Agricultural Science, RDA) ;
  • Suh, Seok-Cheol (Department of Agricultural Bio-resources, National Academy of Agricultural Science, RDA)
  • 노경희 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 이기종 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 박종석 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 김종범 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 이승범 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 서석철 (농촌진흥청 국립농업과학원 농업생명자원부)
  • Received : 2010.03.10
  • Accepted : 2010.03.18
  • Published : 2010.03.31
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Abstract

Eighteen cultivars of tomato were tested for their regeneration response. Lycopersicon esculentum cv. 2001-58 showed a very high frequency of regeneration (93%). We evaluated the effect of two compounds with known antioxidant activity (ascorbic acid and cystein). The use of ascorbic acid ($200\;-\;300\;{\mu}M/L$) has a positive effect on shoot regeneration. To develope a system for plastid transformation in tomato via homologous recombination, we constructed the tomato plastid expression vector (pKRT22-AG) harboring 2.2 kb flanking sequences cloned from intact plastid genome and gfp gene. To investigate the factors affecting the delivery system of the pKRT22-AG into chloroplast using bombardment, We assessed the optimal DNA concentration, gold particle volume and target distance. Expression of the GFP protein was observed within chloroplast on protoplast of cotyledon explant by confocal laser scanning microscopy, which indicates that the protocol developed in this study be useful for the production of plastid transgenic plants in tomato.

국립원예특작과학원에서 분양받은 토마토 18계통을 공시하여 재분화가 잘되는 적정 품종을 탐색한 결과, 계통번호 2001-58에서의 재분화율이 93%로 양호하였다. 또한 식물체로의 재분화 과정에서 보여 지는 갈변현상과 phenolic compound에 의한 식물조직의 괴사현상을 막기 위하여 항산화제인 ascorbic acid와 cystein을 단용 또는 혼용으로 첨가한 후 토마토 재분화에 미치는 영향을 살펴 본 결과, ascorbic acid $200{\sim}300\;{\mu}M/L$ 처리구에서 줄기형성율 및 생체중이 증가되는 현상을 관찰할 수 있었다. 토마토 엽록체 형질전환체 선발을 위해 spectinomycin의 적정 농도를 살펴본 결과, 재분화배지에 spectinomycin 20~25 mg/L 농도가 첨가되어진 처리구에서 재분화가 거의 이루어지지 않았다. 토마토 엽록체 형질전환을 위해 토마토 엽록체 게놈 일부를 분리하여 염기서열을 분석하여 담배와 비교 분석한 결과, homology가 매우 높음을 알 수 있었다. Homologous recombination에 의한 엽록체 형질전환이 되기 위해서 분리한 토마토 엽록체 게놈 일부를 border sequence로 이용하였고, transient assay를 위해 GFP 유전자가 포함된 토마토 엽록체 형질전환용 운반체 pKRT22-AG를 제작하였다. Bombardment을 한 후 원형질체를 나출하여 공초점 현미경하에서 관찰한 결과 엽록체 내에서만 GFP가 발현됨을 알 수 있었으며, DNA 농도 $1\;{\mu}g$, $0.6\;{\mu}m$ gold particle 1 mg, target distance 9 cm 조건이 가장 좋았다.

Keywords

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