Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Development of Antibiotics Marker-free Potato Having Resistance Against Two Herbicides

두 가지 제초제에 대하여 저항성을 가지는 항생제 마커-프리 형질전환 감자 육성

  • Fang, Yi-Lan (Department of Plant Biotechnology, Division of Biotechnology, School of Bioscience & Biotechnology, Kangwon National University) ;
  • Kim, Jin-Seog (Chemical Biotechnology Research Center, Korea Research Institute of Chemical Technology (KRICT)) ;
  • Gong, Su (Department of Plant Biotechnology, Division of Biotechnology, School of Bioscience & Biotechnology, Kangwon National University) ;
  • Mo, Hwang-Suk (Department of Plant Biotechnology, Division of Biotechnology, School of Bioscience & Biotechnology, Kangwon National University) ;
  • Min, Seok-Ki (Chemical Biotechnology Research Center, Korea Research Institute of Chemical Technology (KRICT)) ;
  • Kwon, Suk-Yoon (Plant Genome Research Center, KRIBB) ;
  • Li, Kui-Hua (Department of Plant Biotechnology, Division of Biotechnology, School of Bioscience & Biotechnology, Kangwon National University) ;
  • Lim, Hak-Tae (Department of Plant Biotechnology, Division of Biotechnology, School of Bioscience & Biotechnology, Kangwon National University)
  • Published : 2007.09.29
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Abstract

This study was conducted to develop an antibiotics marker-free potato (Solanum tuberosum L., cv. Taedong valley) plant having resistance against two herbicides. Agrobacterium tumefaciens strain EHA105, harboring a binary vector plasmid pCAMBIA3300 containing bar gene under the control of a promoter CaMV35S and linked CP4-EPSPS genes driven by CaMV35S promoter, was used in the current study. The leaf segments of newly bred potato variety (cv. Taedong Valley) was co-cultured with Agrobacterium. Then, the regenerated individual shoots were excised and transferred to potato multiplication medium supplemented with 0.5 mg/L phosphinothricin. The shoots were rooted in MS medium without hormone and obtained putative transgenic plant E3-6. Integration of target genes into the E3-6 plant and their expression was confirmed by PCR, Southern analysis, and ELISA test. The tissue necrosis test on young leaf blade and shikimic acid accumulation test using the tissue of E3-6 plant were conducted to investigate the resistance to glufosinate-ammonium and glyphosate, respectively. The transgenic plants (E3-6) simultaneously showed a high resistance to both herbicides. The same results were surely obtained also in the whole plants foliar-treated with alone or mixture of two herbicides, glufosinate-ammonium and glyphosate.

본 연구에서는 제초제 저항성 bar 유전자 및 CP4-EPSPS 유전자를 포함하는 발현벡터로 형질전환되고 항생제 마커 유전자를 포함하지 않는 제초제 복합 저항성 감자 식물체를 육성하고자 실험하였다. Bar 유전자를 포함하는 pCAMBIA3300에 CaMV35S 프로모터에 의해 조절되는 CP4-EPSPS 유전자를 도입하여 식물체용 발현 운반체를 제작하고, 이를 Agrobacterium tumafaciens EHA105에 도입하였다. 태동밸리 잎 절편체를 Agrobacterium과 공동배양한 다음, phosphinothricin 0.5 mg/L이 첨가된 배지에서 선발하고 호르몬 무처리 MS발근시켜 형질전환체 (E3-6)를 얻었다. PCR, Southern 분석, 효소면역반응 분석 등을 통해 두 가지 유전자가 도입되었으며 이들이 정상적으로 발현됨이 확인되었다. E3-6 식물체는 glufosinate-ammonium의 어린 식물체 잎 도포처리, glyphosate 용액에 치상한 식물체 조직에서의 shikimate 축적 여부 조사를 통하여 조사한 결과, 두 제초제에 대해 저항성을 나타내었다. 또한 형질전환감자의 전식물체에 대해 glyphosate와 glufosinate-ammonium 각각의 용액 또는 이들의 혼합물을 처리한 후 제초활성 반응을 조사한 결과, E3-6 형질전환 감자는 두 제초제를 각각 단독으로 처리할 때나 혼합하여 동시 처리할 때에도 동일한 저항성이 나타남을 확인하였다.

Keywords

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