Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Perilla transformation using selection markers containing antibiotics and basta

항생제와 제초제 이중 선발 마커를 이용한 들깨 형질전환

  • Kim, Kyung-Hwan (Department of Agricultural Bio-resources, National Academy of Agricultural Science, Rural Development Administration) ;
  • Lee, Jung-Eun (Department of Agricultural Bio-resources, National Academy of Agricultural Science, Rural Development Administration) ;
  • Ha, Sun-Hwa (Department of Agricultural Bio-resources, National Academy of Agricultural Science, Rural Development Administration) ;
  • Hahn, Bum-Soo (Department of Agricultural Bio-resources, National Academy of Agricultural Science, Rural Development Administration) ;
  • Park, Jong-Sug (Department of Agricultural Bio-resources, National Academy of Agricultural Science, Rural Development Administration) ;
  • Lee, Myung-Hee (Department of Functional Crop Science, National Institute of Crop Science, Rural Development Administration) ;
  • Jung, Chan-Sik (Department of Functional Crop Science, National Institute of Crop Science, Rural Development Administration) ;
  • Kim, Yong-Hwan (Department of Agricultural Bio-resources, National Academy of Agricultural Science, Rural Development Administration)
  • 김경환 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 이정은 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 하선화 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 한범수 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 박종석 (농촌진흥청 국립농업과학원 농업생명자원부) ;
  • 이명희 (농촌진흥청 국립식량과학원 기능성작물부) ;
  • 정찬식 (농촌진흥청 국립식량과학원 기능성작물부) ;
  • 김용환 (농촌진흥청 국립농업과학원 농업생명자원부)
  • Published : 2008.12.31
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Abstract

A modified method of Agrobacterium-mediated perilla transformation was developed using two selection markers of an antibiotics (either hpt or nptll) and an herbicidal (bar) gene. Perilla hypocotyl explants were cocultured with Agrobacterium tumefaciens EHA 105 strain harboring plasmid vector (either pMOG6-Bar or pCK-Bar) for three days, respectively. Primary shoots were selected with antibiotics of hygromycin (15 mg/L) or kanamycin (125 mg/L) and regenerated shoots were further selected with herbicide phosphinothricin (ppt,1.2 mg/L) to obtain authentic transformants. Roots were induced for the regenerated shoots on the MS medium without hormone and 80 putative transgenic plants were obtained. Transgene integration into perilla genome was confirmed by Southern blot and their expression was analyzed by Northern blot. T1 perilla seeds drived from To plants were tested 0.3% basta spray for identification of stable gene delivery to next generation.

선발마커로 두 종류의 항생제 (hpt와 nptII)와 제초제(bar) 유전자를 사용하여 아그로박테리움을 이용한 수정된 들깨 형질전환방법을 개발하였다. 들깨 배축 절편을 pMOG6-Bar 운반체 혹은 pCK-Bar 운반체를 가진 아그로박테리움 EHA 105와 각각 3일간 공동배양 하였다. 1차로 형성된 신초는 하이그로마이신(15mg/L)이나 카나마이신(125 mg/L)을 사용하여 선발하였고 재분화 된 신초들은 확실한 형질전환체를 얻기 위해서 포스피노트리신(1.2 mg/L)에서 한번 더 선발하였다. 뿌리는 호르몬이 없는 MS배지에서 재분화 신초로 부터 유도하였으며 80개의 재분화개체를 획득하였다. 들깨 지놈으로 형질전환유전자의 삽입은 서던 블럿으로 확인하였고 유전자의 발현은 노던 블럿으로 분석하였다. To 식물체로부터 유도된 T1들깨 종자들은 후대로의 안정된 유전자 전이를 확인하기 위하여 0.3% 바스타 제초제 살포로 확인하였다.

Keywords

References

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