한국초지조사료학회지 (Journal of The Korean Society of Grassland and Forage Science)

  • 제33권2호
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  • Pages.100-104
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  • 2013
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  • 2287-5824(pISSN)
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  • 2287-5832(eISSN)
한국초지조사료학회 (The Korean Society of Grassland and Forage Science)
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포장생육 잔디 포복경을 이용한 잔디 형질전환에 있어서 살균방법의 영향

Effect of Surface Sterilization Method on Agrobacterium-mediated Transformation of Field-grown Zoysiagrass Stolon

  • 안나영 (경상대학교 응용생명과학부(BK21), 농업생명과학연구원) ;
  • 알람 이프테칼 (경상대학교 응용생명과학부(BK21), 농업생명과학연구원) ;
  • 김용구 (경상대학교 응용생명과학부(BK21), 농업생명과학연구원) ;
  • 배은지 (국립산림과학원 남부산림자원연구소) ;
  • 이광수 (국립산림과학원 남부산림자원연구소) ;
  • 이병현 (경상대학교 응용생명과학부(BK21), 농업생명과학연구원)
  • Ahn, Na-Young (Division of Applied Life Science (BK21 program), IALS, Gyeongsang National University) ;
  • Alam, Iftekhar (Division of Applied Life Science (BK21 program), IALS, Gyeongsang National University) ;
  • Kim, Yong-Goo (Division of Applied Life Science (BK21 program), IALS, Gyeongsang National University) ;
  • Bae, Eun-Ji (Southern Forest Research Center, Korea Forest Research Institute) ;
  • Lee, Kwang-Soo (Southern Forest Research Center, Korea Forest Research Institute) ;
  • Lee, Byung-Hyun (Division of Applied Life Science (BK21 program), IALS, Gyeongsang National University)
  • 투고 : 2013.03.04
  • 심사 : 2013.04.05
  • 발행 : 2013.06.30
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초록

포장에서 생육된 잔디 포복경을 이용한 Agrobacterium 형질전환에 있어서 큰 제한인자였던 곰팡이 오염을 제거할 목적으로 포복경 조직에 대한 새로운 살균법을 개발하고자 하였다. 여러 가지 살균방법 중에서 30% NaOCl로 15분간 처리한 다음 0.1% $HgCl_2$로 25분간 처리 했을 때 포복경 절편체의 생존율이 높았으며, 0.1% $HgCl_2$로 처리시 800 mbar의 진공처리를 5분간 실시했을 때 가장 효과적이었다. 또한 Agrobacterium과 공동배양 시 2.5 mg/l의 amphotericin B를 첨가해 준 배지에서 배양했을 때 가장 높은 생존율을 나타내었다. AmB의 처리는 Agrobacterium의 생장에 영향을 미치지 않았다. 또한 살균된 포복경으로 부터 신초의 재분화에도 영향을 미치지 않았으며 곰팡이 오염만을 효율적으로 억제하는 것으로 나타났다. 이러한 결과는 포장에서 대량으로 생육시킨 잔디 포복경을 이용한 Agrobacterium 형질전환 시 그 효율을 증가시키는데 큰 기여를 할 것으로 추측된다.

Zoysiagrass (Zoysia japonica Steud.) is an important forage and turfgrass that spreads by stolons and rhizomes. Zoysiagrass stolon can be used directly for Agrobacterium-mediated genetic transformation by exploiting the potential of direct shoot formation. However, surface sterilization of field-grown stolons is difficult and remains to be explored. We developed an effective surface sterilization and culture method using the stolon explant for infection with Agrobacterium tumefaciens. Among various treatments, sequential disinfection in 30% bleach for 15 min followed by 0.1% mercuric chloride for 25 min resulted in the highest number of clean stolons. The efficacy of mercuric chloride was increased under vacuum conditions by incubating at 800 mbar for 5 min. The inclusion of 2.5 mg/l amphotericin B further prevents fungal growth in in vitro cultures. This protocol would speed up the development of transgenic plants by utilizing field-grown stolon nodes.

키워드

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