Plant Cell Reports

, Volume 14, Issue 7, pp 450–454

Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker

  • Kazuhito Akama
  • Holger Puchta
  • Barbara Hohn
Article

Summary

We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.

Key words

Arabidopsis thaliana Transformation Phosphinothricin Bar Basta resistance 

Abbreviations

bar

bialaphos resistance gene

CIM

callus-inducing medium

DTNB

5,5′-dithiobis(2-nitrobenzoic acid)

GM

germination medium

HPT

hygromycin phosphotransferase

MS

Murashige and Skoog salts

NPTII

neomycin phosphotransferase II

PAT

phosphinothricin acetyltransferase

PPT

phosphinothricin

SIM

shoot-inducing medium

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References

  1. Aarts MGM, Dirkse WG, Stiekema WJ, Pereira A (1993) Nature 363:715–717Google Scholar
  2. Akama K, Shiraishi H, Ohta S, Nakamura K, Okada K, Shimura Y (1992) Plant Cell Rep 12:7–11Google Scholar
  3. Altmann T, Schmidt R, Willmitzer L (1990) In: Abstract of the 4th International Conference on Arabidopsis Research, Vienna, pp 26Google Scholar
  4. Bevan M (1984) Nucleic Acids Res 12:8711–8721Google Scholar
  5. Bradford MM (1976) Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  6. Chang S-S, Park S-K, Nam H-G (1990) In: Abstract of the 4th International Conference on Arabidopsis Research, Vienna, pp 28Google Scholar
  7. Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, Van Montagu M, Leemans J (1985) Nucleic Acids Res 13:4777–4788Google Scholar
  8. De Block M, Botterman J, Vandewiele M, Dockx J, Thoen C, Gossele Y, Movva NR, Thompson C, Van Montagu M, Leemans J (1987) EMBO J 6:2513–2518Google Scholar
  9. D'Halluin K, De Block M, Denecke J, Janssens J, Leemans J, Reynaerts A, Botterman J (1992) Methods Enzymol 216:415–426Google Scholar
  10. Dower WJ, Miller JF, Ragsdale CW (1988) Nucleic Acids Res 16:6127–6145PubMedGoogle Scholar
  11. Doyle JJ, Doyle JL (1990) Focus 12:13–15Google Scholar
  12. Feldmann KA, Marks MD (1987) Mol Gen Genet 208:1–9Google Scholar
  13. Feldmann KA (1991) Plant J 1:71–82Google Scholar
  14. Hrouda M, Paszkowski J (1994) Mol Gen Genet 243:106–111Google Scholar
  15. Kilby NJ, Leyser HMO, Furner IJ (1992) Plant Mol Biol 20:103–112Google Scholar
  16. Long D, Martin M, Sundberg E, Swinburne J, Puangsomlee P, Coupland G (1993) Proc Natl Acad Sci USA 90:10370–10374Google Scholar
  17. Lloyd AM, Barnason AR, Rogers SG, Byrne MC, Fraley RT, Horsch RB (1986) Science 234:464–466Google Scholar
  18. Meyerowitz EM (1989) Cell 56:263–269Google Scholar
  19. Murashige T, Skoog F (1962) Physiol Plant 15:473–497Google Scholar
  20. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  21. Sangwan RS, Bourgeois Y, Sangwan-Norreel BS (1991) Mol Gen Genet 230:475–485Google Scholar
  22. Schmidt R, Willmitzer L (1988) Plant Cell Rep 7:583–586Google Scholar
  23. Tachibana K, Kaneko K (1986) J Pestic Sci 11:297–304Google Scholar
  24. Tachibana K, Watanabe T, Sekizawa Y, Takematsu T (1986) J Pestic Sci 11:33–37Google Scholar
  25. Thompson CJ, Movva NR, Tizard R, Crameri R, Davies JE, Lauwereys M, Bottennan J (1987) EMBO J 6:2519–2523Google Scholar
  26. Valvekens D, Van Montagu M, Van Lijsebettens M (1988) Proc Natl Acad Sci USA 85:5536–5540Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Kazuhito Akama
    • 1
  • Holger Puchta
    • 1
  • Barbara Hohn
    • 1
  1. 1.Friedrich-Mieseher-InstitutBaselSwitzerland
  2. 2.Department of BiologyShimane UniversityMatsueJapan

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