Korean Journal of Plant Resources (한국자원식물학회지)

The Plant Resources Society of Korea (한국자원식물학회)
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Efficient Transformation of Trifolium repens L. Using Acetosyringone

Acetosyringone을 이용한 효율적인 White Clover의 형질전환

  • Published : 1997.06.01
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Abstract

Transformants of White Clover(Trifolium repens L.) were efficiently produced from immature seed derived callus cocultivated with Agrobacterium twnefaciens LBA4404 harboring plant binary vector. pBI121, using acetosyringone. The mean frequencies of transformants on the two kanamycin-containing media were 16 to 19% when the immature seed-derived calli were infected with bacteria cultured in the presence of 100$\mu$M acetosyringone compared with 7% in media without acetosyringone. Transgenic white clover was subject to molecular analysis for integration into plant nuclear genome and expression of $\beta$-glucuronidase(GUS) gene. PCR and Northern blot analyses demonstrated that GUS gene was integrated into white clover nuclear genome and expressed into its mRNA. The expression of GUS gene into its protein was confirmed by spectrophotometric assay of GUS activity.

화이트 클로버의 배축, 잎, 미숙배 유래의 embryogenic callus에 식물 binary vector인 pBI121을 포함하는 A. tumefaciens LBA4404를 접종하여 효과적으로 화이트 클로버를 형질전환시켰다. A. tumefaciens를 이용한 화이트 클로버의 형질전환은 acetosyringone을 사용함으로써 품종간의 차이가 없이 배발생 캘러스에서 16-19%를 보였다. 재분화 식물체의 PCR 및 Northern 분서글 통하여 형질 전환된 화이트 클로버의 염색체내에 GUS 유전자가 안정되게 도입되었고 식물체내에서 mRAN로 발현됨을 확인하였다. 또한, GUS 유전자가 식물체내에서 단백질로 발현됨을 확인하기 위하여 형질 전환되어진 화이트 클로버부터 단백질을 추출하고 분광분석법에 의하여 GUS의 활성을 측정하였으며, 시료간에 약간의 차이는 있으나 유의적인 GUS 활성을 확인하였다.

Keywords

References

  1. J. Bacteriol. v.170 Ti plasmid-specified chemotaxis of Agrobacterium tumefaciens C58 super(1) toward vir-inducing phenolic compounds and soluble factors from monocotyledonous and dicotyledonous plants Ashby, A. M.;Watson, M. D.;Loake, G. J.;Shaw, C. H.
  2. J. Bacteriol. v.176 Glu-255 outside the predicted ChvE binding site in VirA is crucial for sugar enhancement of acetosyringone perception by Agrobacterium tumefaciens Banta, L. M.;Joerger, R. D.;Howtiz, V.R.;Campbell, A. M.;Binns, A. N.
  3. Anal. Biochem. v.72 A rapid and sensitive method for the quantatization of microgram quantities of protein utilizing the principle of protein-dye binding Bradford, M. M.
  4. J. Bacteriol. v.173 Controlled expression of the transcriptional activator gene virG in Agrobacterium tumefaciens by the Escherichia coli lac promoter Chen, C. Y.;Winans, S. C.
  5. Proc. Natl. Acad. Sci. USA v.71 A tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors Chilton, M. D.;Currier, T. C.;Farrand, S. K.;Bendich, A. J.;Gordon, M. P.;Nester, E. W.
  6. J. Mol. Biol. v.197 Characterization of Agrobacterium tumefaciens virulence proteins induced by the plant factor acetosyringone Engstroem, P.;Zambryski, P.;van-Montagu, M.;Stachel, S.
  7. Plant Cell Reports v.10 The use of the polymerase chain reaction in plant transformation studies Hamil, J. D.;Rounsley, S.;Spencer, A.;Rhodes, T. G.
  8. Plant J. v.6 Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA Hiei, Y.;Ohta, S.;Komari, T.;Kumashiro, T.
  9. Nature Biotech. v.14 High efficiency transformation of maize mediated by Agrobacterium tumefaciens Ishida, Y.;Saito, H.;Ohta, S.;Hiei, Y.;Komari, T.;Kumashiro, T.
  10. EMBO J. v.6 GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plant Jefferson, R. A.;Kavanagh, T. A.;Bevan, M. W.
  11. Anal. Biochem. v.163 Improved method for the isolation of RNA from plant tissues Logemann, J.;Schell, J.;Willmitzer, L.
  12. Ann. Bot. v.54 Direct somatic embryoid formation on immature embryos of Trifolium repens, T. pretense and Medicago sativa, and rapid clonal propagation of T. repens Maheswaran, G.;Williams, E. G.
  13. J. Plant Physiol. v.136 The promotion of Agrobacterium mediated transformation in Atropa belladona L. by acetosyringone Mathews, H.;Bharathan, N.;Litz, R. E.;Narayanan, K. R.;Rao, P. S.;Bhatia, C. R.
  14. Proc. Natl. Acad. Sci. USA v.87 A nontransformable Triticum monococcum monocotyledonous culture produces the potent Agrobacterium vir-inducing compound ethyl ferulate Messens, E.;Dekeyser, R.;Stachel, S. E.
  15. Physiol. Plant. v.15 A revised medium for rapid growth and bioassays with tobacco tissue cultures Murashige, T.;Skoog, F.
  16. Nucl. Acids. Res. v.8 Rapid isolation of high molecular weight DNA Murray, H. G.;Thompson, W. F.
  17. Physiol. Plant v.39 Callus and plantlet regeneration from cell culture of ladino clover and soybean Oswald, T. H.;Smith, A. E.;Phillips, D. V.
  18. Plant Cell Reports v.8 Transformation of Pea by Agrobacterium tumefaciens Puonti-Kaerlas, J.;Stable, P.;Erickson, R. J.
  19. Plant Mol. Biol. v.8 Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens Sheikholeslam, S. N.;Weeks, D. P.
  20. Proc. Natl. Acad. Sci. USA v.87 Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules and monosaccharides Shimoda, N.;Yamamoto, T.;Nagamine, J.;Usami, S.;Katayama, M.;Sakagami, Y.;Machida, Y.
  21. Mol. Gen. Genet. v.209 Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium Usami, S.;Morikawa, S.;Takebe, I.;Machida, Y.
  22. Plant Cell Reports v.13 Agrobacteriummediated transformation of white clover using direct shoot organogenesis Voisey, C. R.;White, D. W. R.;Dudas, B.;Appleby, R. D.;Ealing, P. M.;Scott, A. G.
  23. Biocontrol. Sci. Technol. v.4 Release of transgenic white clover plants expressing Bacillus thuringiensis genes: An ecological perspective Voisey, C. R.;White, D. W. R.;Wigley, P. J.;Chilcott, C. N.;McGregor, P. G.
  24. Plant Cell Reports v.12 Repetitive somatic embryogenesis and plant recovery in white clover WeissingerⅡ, A.;Parrott, W. A.
  25. Planta v.162 Plant regeneration from long-term suspension cultures of white clover White, D. W. R.
  26. Plant Mol. Biol. v.8 Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors White, D. W. R.;Greenwood, D.
  27. Plant Cell Reports v.13 Prolific direct plant regeneration from cotyledons of white clover White, D. W. R.;Voisey, C.