Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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GUS Gene expression and plant regeneration via somatic embryogenesis in cucumber (Cucumis sativus L.)

오이에서 체세포배 발생을 통한 GUS유전자의 발현 및 식물체 재생

  • Kim, Hyun-A (Department of Medicinal Plant Resources, Nambu University) ;
  • Lee, Boo-Youn (Department of Medicinal Plant Resources, Nambu University) ;
  • Jeon, Jin-Jung (Department of Medicinal Plant Resources, Nambu University) ;
  • Choi, Dong-Woog (Department of Biological Education, Jeonnam National University) ;
  • Choi, Pil-Son (Department of Medicinal Plant Resources, Nambu University) ;
  • Utomo, Setyo Dwi (Department of Crop Science, College of Agriculture, University of Lampung) ;
  • Lee, Jae-Hyoek (Department of Medicinal Plant Resources, Nambu University) ;
  • Kang, Tong-Ho (Department of Medicinal Plant Resources, Nambu University) ;
  • Lee, Young-Jin (Department of Medicinal Plant Resources, Nambu University)
  • 김현아 (남부대학교 한방제약개발학과) ;
  • 이부연 (남부대학교 한방제약개발학과) ;
  • 전진중 (남부대학교 한방제약개발학과) ;
  • 최동욱 (전남대학교 생물교육학과) ;
  • 최필선 (남부대학교 한방제약개발학과) ;
  • 세이토우토모 (람풍대학교 농과대학 작물학과) ;
  • 이재혁 (남부대학교 한방제약개발학과) ;
  • 강동호 (남부대학교 한방제약개발학과) ;
  • 이영진 (남부대학교 한방제약개발학과)
  • Published : 2008.12.31
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Abstract

One of the limitation for Agrobacterium-mediated transformation via organogenesis from cotyledon explants routinely in cucumber is the production of chimeric plants. To overcome the limitation, Agrobacterium-mediated transformation system via somatic embryogenesis from hypocotyl explants of cucumber (c.v., Eunsung) on the selection medium with paromomycin as antibiotics was developed. The hypocotyl explants were inoculated with Agrobacterium tumefaciens strain EHA101 carrying binary vector pPTN290; then were subsequently cultured on the following media: co-cultivation medium for 2 days, selection medium for $5{\times}14$ days, and regeneration medium. The T-DNA of the vector (pPTN290) carried two cassettes, Ubi promoter-gus gene as reporter and 35S promoter-nptll gene conferring resistance to paromomycin as selectable agent. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to paromomycin indicated by the growth of putative transgenic calli on selection medium amended with 100mg/L paromomycin, and GUS gene expression. Forty eight clones (5.2%) with GUS gene expressed of 56 callus clones with resistance to paromomycin were independently obtained from 928 explants inoculated. Of 48 clones, transgenic plants were only regenerated from 5 clones (0.5%) at low frequency. The histochemical GUS assay in the transgenic seeds ($T_1$) also revealed that the gus gene was successfully integrated and segregated into each genome of transgenic cucumber.

Agrobacterium공동배양법으로 오이의 기관발생을 통한 형질전환에서 가장 문제점 중 하나는 chimeric 형질전환체의 발생빈도이다. 이러한 문제점을 극복하기 위하여 항생제로서 paromomycin이 첨가된 선발배지에서 "은성" 품종의 배축절편으로부터 체세포배발생을 통한 형질전환시스템을 개발하였다. 배축절편을 pPPTN290발현벡터가 도입된 Agrobacterium 균주 (EHA101)에 30분간 접종한 후 2일간 공동배양 하였고, 선발배지에서 2주 간격으로 5회 계대 배양하면서 항생제 저항성 캘러스 선발, 체세포배발생 및 식물체를 유도하였다. pPPTN290발현벡터의 T-DNA는 reporter유전자로서 Ubi 프로모터에 의해 gus유전자가 발현조절 되도록 그리고 항생제로서 paromomycin에 저항성을 갖는 nptII유전자가 35S 프로모터에 의해 발현되도록 제조하였다. 안정적 형질전환과 빈도는 캘러스의 paromomycin항생제 저항성과 GUS유전자의 발현 여부에 의해 조사하였다. Agrobacterium과 공동배양한 928개의 배축절편에서 paromomycin에 저항성을 갖는 56개의 캘러스 클론을 얻었고, 이중 48개 캘러스 클론 (5.2%)에서 GUS유전자가 안정적으로 발현되고 있음을 확인하였다. 48개의 캘러스 클론중에서 오직 5개의 캘러스 클론으로부터 식물체를 얻어 낮은 빈도 (0.5%)를 나타냈다. 수확한 $T_1$종자에서 GUS양성반응은 gus유전자가 오이 게놈에 안정적으로 도입 및 발현되고 있음을 확인하였다.

Keywords

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