KOREAN JOURNAL OF CROP SCIENCE (한국작물학회지)

The Korean Society of Crop Science (한국작물학회)
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Several Factors Affecting Transformation Efficiency of tall Fescue

톨페스큐의 효율적인 형질전환을 위한 몇 가지 요인의 영향

  • 김진수 (경상대학교 응용생명과학부) ;
  • 이상훈 (경상대학교 응용생명과학) ;
  • 이병현 (경상대학교 응용생명과학부)
  • Published : 2004.06.01
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Abstract

A system for the production of transgenic plants has been developed for tall fescue (Festuca arundinacea Schreb.) via Agrobacterium-mediated transformation of mature seed-derived embryogenic callus. Seed-derived calli were infected and co-cultured with Agrobacterium EHA101 carrying standard binary vector pIG121Hm encoding the hygromycin phosphotransferase (HPT), neomycin phosphotransferase II (NPTII) and intron-containing $\beta$-glucuronidase (intron-GUS) genes in the T-DNA region. The effects of several factors on transformation and the expression of the GUS gene were investigated. Inclusion of $200\mu\textrm{M}$ acetosyringone (AS) in inoculation and co-culture media lead to a increase in stable transformation efficiency. Transformation efficiency was increased when embryogenic calli were co-cultured for 5 days on the co-culture medium. The highest transformation efficiency was obtained when embryogenic calli were inoculated with Agyobacterium in the presence of 0.1% Tween20 and $200\mu\textrm{M}$ AS. Hygromycin resistant calli were developed into complete plants via somatic embryogenesis. GUS histochemical assay and Southern blot analysis of transgenic plants demonstrated that transgenes were successfully integrated into the genome of tall fescue.

우용유전자 도입을 통한 신품종 톨페스큐를 개발할 목적으로 Agrobacterium을 이용한 효율적인 형질전환 체계를 확립하였다 톨페스큐 성숙종자 유래의 캘러스를 standard binary vector인 pIG121Hm을 가지는 Agrobacterium EHA101을 이용하여 감염시킨 후 공동배양하여 형질전환시켰다. Agrobacterium을 이용한 형질전환에 있어서 중요한 인자로 작용하는 몇 가지 요인에 대한 톨페스큐 캘러스의 형질전환 효율을 GUS 유전자의 발현정도로 조사하였다. Agrobacterium 감염시에 접종배지와 공동배양배지에 $200\mu\textrm{M}$의 acetosyringone(AS)을 첨가해 주었을 때 형질전환 효율이 증가 되었으며, 공동배양기간을 5일까지 증가시켰을 때 형질전환효율이 증가되었다. 또한 Agrobacterium 감염시에 $200\mu\textrm{M}$의 AS와 0.l%의 Tween20을 동시에 첨가해 주었을 때 가장 높은 형질전환 효율을 나타내었다. 50 mg/L.의 hygromycin이 첨가된 선발배지에서 살아남은 캘러스로부터 정상적인 식물체가 재분화 되었으며 이들 형질전환체를 GUS 염색과 Southern blot 분석을 실시하여 본 결과 발현백터의 T-DNA 영역이 형질전환 식물체의 genome에 성공적으로 도입되었음을 확인할 수 있었다. 본 연구를 통하여 확립된 효율적인 형질전환 시스템은 분자육종을 통한 신품종 톨페스큐의 개발에 유용하게 이용될 수 있을 것이다.

Keywords

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