Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Development of herbicide tolerant soybean using Agrobacterium tumefaciens

아그로박테리움을 이용만 제초제 저항성 콩 개발

  • Lee, Ki-Jong (National Institute of Agricultural Biotechnology, Rural Development Administration) ;
  • Park, Hong-Jae (National Institute of Agricultural Biotechnology, Rural Development Administration) ;
  • Yi, Bu-Young (Department of Environmental Horticulture, The University of Seoul) ;
  • Lee, Kyeong-Ryeol (National Institute of Agricultural Biotechnology, Rural Development Administration) ;
  • Kim, Myung-Sik (National Institute of Agricultural Biotechnology, Rural Development Administration) ;
  • Woo, Hee-Jong (National Institute of Agricultural Biotechnology, Rural Development Administration) ;
  • Jin, Yong-Moon (National Institute of Agricultural Biotechnology, Rural Development Administration) ;
  • Kweon, Soon-Jong (National Institute of Agricultural Biotechnology, Rural Development Administration)
  • 이기종 (농촌진흥청 농업생명공학연구원) ;
  • 박홍채 (농촌진흥청 농업생명공학연구원) ;
  • 이부영 (서울시립대학교 환경원예학과) ;
  • 이경렬 (농촌진흥청 농업생명공학연구원) ;
  • 김명식 (농촌진흥청 농업생명공학연구원) ;
  • 우희종 (농촌진흥청 농업생명공학연구원) ;
  • 진용문 (농촌진흥청 농업생명공학연구원) ;
  • 권순종 (농촌진흥청 농업생명공학연구원)
  • Published : 2008.03.31
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Abstract

This study aims to establish the efficient soybean transformation system and develop soybean [Glycine max (L.) Merill] transformants using cotyledonary node explants. The cotyledonary node of soybean were co-cultivated with Agrobacterium tumefaciens strains (KYRT1, EHA105). These strains contain the binary vector pCAMBIA3301 which carries a herbicide-resistant far gene. Korean cultivars (Danbaekkong, Eunhakong) and foreign cultivars (Jack, Peking) were the most efficient in regenerating cotyledonary node. Therefore, they were chosen for the transformation. Results showed that the T-DNA transfer reached up to 60% and transformation efficiency reached up to 3% in the cotyledonary node explants from Jack cultivar, co-cultivated with EHA105 strain. Histochemical GUS evaluation showed that 12 individual lines, transformed with the 현 gene, have positive response. The transformed soybeans have been confirmed in the $T_0$ generation through phenotypic assay using herbicide $Basta^{(R)}$ and Southern blot analysis.

본 실험은 콩 [Glycine max (L.) Merill]의 자엽마디를 이용하여 효율적인 형질전환 시스템을 확립하고 그 방법을 통해 제초제 저항성 콩을 개발하기 위하여 수행되었다. 형질전환에 적합한 콩을 찾기 위하여 여러 가지 재배품종을 대상으로 재분화율을 조사한 결과 국내품종은 단백콩과 은하콩이 국외품종은 'Jack'과 'Peking'이 높은 재분화율을 나타내 콩의 형질전환에 적합한 품종으로 선발하였다. 콩의 자엽마디에 제초제 저항성 유전자 (bar)를 포함하고 있는 pCAMBIA3301 벡터로 형질전환한 아그로박테리움 (KYRT1, EHA105)을 접종하여 형질전환하였다. 콩의 자엽마디와 아그로박테리움을 공배양한 후 GUS발색 반응을 통해 절편체로의 T-DNA도입율은 최대 60%에 이르렀으나 형질전환율은 콩의 품종과 아그로박테리움의 종류에 따라 현저한 차이를 보였으며, 그 중에서 Jack품종의 자엽마디를 아그로박테리움 EHA105를 이용하여 형질전환하였을 때에 가장 높은 3%의 형질전환율을 나타내었다. 순화된 형질전환체 중 GUS발색 반응에서 양성반응을 보이는 식물체들을 PCR을 이용하여 검정하였으며, 제초제 (Basta(R))살포와 Southern분석을 통해 제초제 저항성 유전자가 도입되어 발현되는 형질전환체를 확인하였다. 현재 후대로의 유전양상과 도입된 유전자가 안정적으로 발현하는지의 여부를 실험중이다.

Keywords

References

  1. Cho JY (2001) Dry field farming. Hyangmunsa, Seoul
  2. Cho MA, Choi DW, Liu JR, Clement T, Choi PS (2004) Development of transgenic soybean using Agrobacterium tumefaciens. Kor J Plant Biotechnol 31: 255-259 https://doi.org/10.5010/JPB.2004.31.4.255
  3. Donaldson PA, Simmonds DH (2000) Susceptibility to Agrobacterium tumefaciens and cotyledonary node transformation in short-season soybean. Plant Cell Rep 19: 478-484 https://doi.org/10.1007/s002990050759
  4. Hinchee MAW, Connor-Ward DV, Newell CA, McDonnell RE, Sato SJ, Gasser CS, Fischhoff DA, Re DB, Fraley RT, Horsch RB (1988) Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Bio/Technol 6: 915-922 https://doi.org/10.1038/nbt0888-915
  5. Jefferson RA, Kavanagh TA Bevan MW (1987) GUS fusion: $\beta$-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901-3907
  6. Ko TS, Lee SM, Krasnyanski S, Korban SS (2003) Two critical factors are required for efficient transformation of multiple soybean cultivars: Agrobacterium strain and orientation of immature cotyledonary explant. Theor Appl Genet 107: 439-447 https://doi.org/10.1007/s00122-003-1264-6
  7. Meurer CA, Dinkins RD, Collins GB (1998) Factors affecting soybean cotyledonary node transformation. Plant Cell Rep 18: 180-186 https://doi.org/10.1007/s002990050553
  8. Olhoft PM, Somers DA (2001) L-Cysteine increases Agrobacteriummediated T-DNA delivery into soybean cotyledonarynode cells. Plant Cell Rep 20: 706-711 https://doi.org/10.1007/s002990100379
  9. Olhoft, PM, Flagel LE, Donovan CM, Somers DA (2003) Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method Planta. 216: 723-735
  10. Paz MM, Martinez JC, Kalvig AB, Fonger TM, Wang K (2005) Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Rep 25: 206-213 https://doi.org/10.1007/s00299-005-0048-7
  11. Rie T, Ko S (1990) Expression of CaMV35S-GUS gene in transgenic rice plants. Mole. Genetics Genomics 220: 389-392
  12. Sambrook J, Russell DW (2001) Molecular cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  13. Sandermann H (2006) Plant biotechnology: ecological case on herbicide resistance. Trends in Plant Science 11: 324-328 https://doi.org/10.1016/j.tplants.2006.05.004
  14. Santarem ER, Trick HN, Essig JS, Finer JI (1998) Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: Optimization of transient expression. Plant Cell Rep 17: 752-759 https://doi.org/10.1007/s002990050478
  15. Stewart CN, Adang MJ, All JN, Boerma HR, Cardineau G, Tucker D, Parrott WA (1996) Genetic transformation, recovery, and characterization of fertile soybean transgenic for a synthetic Bacillus thuringiensis crylAc gene. Plant Physiol 112: 121-129 https://doi.org/10.1104/pp.112.1.121
  16. Torisky RS, Kovacs L, Avdiushko S, Newman JD, Hunt AG, Collins GB (1997) Development of a binary vector system for plant transformation based on the supervirulent Agrobacterium tumefaciens strain Chry 5. Plant Cell Rep 17: 102-108 https://doi.org/10.1007/s002990050360
  17. Trick HN, Finer JJ (1997) SAAT: Sonication-assisted Agrobacterium-mediated transformation. Transgenic Res 6: 329-337 https://doi.org/10.1023/A:1018470930944
  18. Trick HN, Finer JJ (1998) Sonication-assisted Agrobacteriummediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue. Plant Cell Rep 17: 482-488 https://doi.org/10.1007/s002990050429
  19. Yan B, Reddy MSS, Collins GB, Dinkins RD (2000) Agrobacterium tumefaciens-mediated transformation of soybean [Glycine max (L.) Merrill] using immature zygotic cotyledon explants. Plant Cell Rep 19: 1090-1097 https://doi.org/10.1007/s002990000236
  20. Zeng P, Vadnais DA, Zhang Z, Polacco JC (2004) Refined glufosinate selection in Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill]. Plant Cell Rep 22: 478-482 https://doi.org/10.1007/s00299-003-0712-8

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