Molecular Breeding

, Volume 10, Issue 3, pp 165–180 | Cite as

Transposon-mediated generation of T-DNA- and marker-free rice plants expressing a Bt endotoxin gene

  • Olivier Cotsaftis
  • Christophe Sallaud
  • Jean Christophe Breitler
  • Donaldo Meynard
  • Rafaella Greco
  • Andy Pereira
  • Emmanuel Guiderdoni


Transposon-mediated repositioning of transgenes is an attractive strategy to generate plants that are free of selectable markers and T-DNA inserts. By using a minimal number of transformation events a large number of transgene insertions in the genome can be obtained so as to benefit from position effects in the genome that can contribute to higher levels of expression. We constructed a Bacillus thuringiensis synthetic cry1B gene expressed under control of the maize ubiquitin promoter between minimal terminal inverted repeats of the maize Ac-Ds transposon system, which was cloned in the 5' untranslated sequence of a gfp gene used as an excision marker. The T-DNA also harboured the Ac transposase gene driven by the CaMV 35S promoter and the hph gene conferring resistance to the antibiotic hygromycin. Sixty-eight independent rice (Oryza sativa L.) transformants were regenerated and molecularly analysed revealing excision and reinsertion of the Ds-cry1B element in 37% and 25% respectively of the transformation events. Five independent transformants harbouring 2–4 reinserted Ds-Cry1B copies were analysed in the T1 progeny, revealing 0.2 to 1.4 new transpositions per plant. Out segregation of the cry1B gene from the T-DNA insertion site was observed in 17 T1 plants, representing 10 independent repositioning events without selection. Western analysis of leaf protein extracts of these plants revealed detectable Cry1B in all the plants indicating efficient expression of the transgene reinsertions. Stability of position and expression of the cry1B transgene was further confirmed in T2 progeny of T-DNA-free T1 plants. New T-DNA-free repositioning events were also identified in T2 progenies of T1 plants heterozygous for the T-DNA. Furthermore, preliminary whole plant bioassay of T-DNA-free lines challenged with striped stem borer larvae suggested that they are protected against SSB attacks. These results indicate that transposon mediated relocation of the gene of interest is a powerful method for generating T-DNA integration site-free transgenic plants and exploiting favourable position effects in the plant genome.

Ac/Ds Bacillus thuringiensis Insect resistance Repositioning Selectable marker free Transgenic rice 


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  1. Bohorova N., Frutos R., Royer M., Estanol P., Pacheco M., Rascon Q. et al. 2001. Novel synthetic Bacillus thuringiensis cry1B gene and the cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize. Theor Appl. Genet. 103: 817–826.Google Scholar
  2. Bradford M. 1976. A rapid and sensitive method for the quantifi-cation of microgram quantities of protein utilizing the principle of protein-dye binding. Annal. Biochem. 72: 248–254.Google Scholar
  3. Chen L., Zhang S., Beachy R.N. and Fauquet C.M. 1998. A protocol for consistent, large scale production of fertile transgenic rice plants. Plant Cell Rep. 18: 25–31.Google Scholar
  4. Chin H.G., Choe M.S., Lee S.H., Park S.H., Park S.H., Koo J.C. et al. 1999. Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system. Plant J. 19: 615–623.Google Scholar
  5. Chiu W., Niwa Y., Zeng W., Hirano T., Kobayashi H. and Sheen J. 1996. Engineered GFP as a vital reporter in plants. Curr. Biol. 6: 325–330.Google Scholar
  6. Christensen A. and Quail P.H. 1996. Ubiquitin promoter-based vectors for high level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 5: 213–218.Google Scholar
  7. Dale E.C. and Ow D.W. 1991. Gene transfer with subsequent removal of the selection gene from the host genome. Proc. Natl Acad. Sci U.S.A 88: 10558–10562.Google Scholar
  8. Daley M., Knauf V.C., Summerfelt K.R. and Turner J.C. 1998. Cotransformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing markerfree plants. Plant Cell Rep. 17: 489–496.Google Scholar
  9. De Block M. and Debrouwer D. 1991. Two T-DNA's co-transformed in Brassica napus by a double Agrobacterium infection are mainly integrated at the same locus. Theor Appl. Genet. 82: 257–262.Google Scholar
  10. De Buck S., De Wilde C., Van Montagu M. and Depicker A. 2000. T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation. Mol. Breed. 6: 459–468.Google Scholar
  11. Enoki H., Izawa T., Kawahara M., Komatsu M., Koh S., Kyozuka J. et al. 1999. Ac as a tool for the functional genomics of rice. Plant J. 19: 605–613.Google Scholar
  12. Gleave A.P., Mitra D.S., Mudge S.R. and Morris B.A. 1999. Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene. Plant Mol. Biol. 40: 223–235.Google Scholar
  13. Goldsbrough A.P., Lastrella C.N. and Yoder J.I. 1993. Transposition mediated re-positioning and subsequent elimination of marker gene from transgenic tomato. Bio/Technology 11: 1286–1292.Google Scholar
  14. Greco R., Ouwerkerk P.B.F., Sallaud C., Kohli A., Colombo L., Puigdomenech P. et al. 2001a. Transposon Insertional Mutagenesis in rice. Plant Physiol. 125: 1175–1177.Google Scholar
  15. Greco R., Ouwerkerk P.B.F., Taal A.J.C., Favalli C., Beguiristain T., Puigdomenech P. et al. 2001b. Early and multiple Ac transpositions in rice generated by an adjacent strong enhancer. Plant Mol. Biol. 46: 215–227.Google Scholar
  16. Hansen G. and Wright M.S. 1999. Recent advances in the transformation of plants. Trends Plant Sci. 4: 226–231.Google Scholar
  17. Hohn B., Levy A.A. and Puchta H. 2001. Elimination of selection markers from transgenic plants. Curr. Opin. In Biotech 12: 139–143.Google Scholar
  18. Kohli A., Xiong J., Greco R., Christou P. and Pereira A. 2001. Tagged transcriptome display (TTD) in indica rice using Ac transposon. Mol. Genet. Genomics 266: 1–11.Google Scholar
  19. Komari T., Hiei Y., Saito Y., Murai N. and Kumashiro T. 1996. Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J. 10: 165–174.Google Scholar
  20. Koprek T., Rangel S., Mc elroy D., Louwerse J.D., Williams Carrier R.E. and Lemaux P.G. 2001. Transposon-mediated single copy gene delivery leads to increased transgene expression stability in barley. Plant Physiol. 125: 1354–1362.Google Scholar
  21. Hoisington D. 1992. Laboratory Protocols. CIMMYT, Mexico DF.Google Scholar
  22. Hood E.E., Gelvin S.B., Melchers L.S. and Hoekema A. 1993. New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res. 2: 208–218.Google Scholar
  23. Izawa T., Miyazaki C., Yamamoto M., Terada R., Iida S. and Shimamoto K. 1991. Introduction and transposition of the maize transposable element Ac in rice (Oryza sativa L.). Mol. Gen. Genet. 227: 391–396.Google Scholar
  24. Izawa T., Ohnishi T., Nakano T., Ishida H., Enoki H., Hashimoto K. et al. 1997. Transposon tagging in rice. Plant Mol. Biol. 35: 219–229.Google Scholar
  25. Laemmli U. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.Google Scholar
  26. Mc Knight T.D., Lillis M.T. and Simpson R.B. 1987. Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Plant Mol. Biol. 8: 439–445.Google Scholar
  27. Mao L., Wood T.C., Yu Y., Budiman M.A., Tomkins J., Woo S.S. et al. 2000. Rice transposable elements: A survey of 73,000 sequence-tagged-connectors. Genome Res. 10: 982–990.Google Scholar
  28. Nakagawa Y., Machida C., Machida Y. and Toriyama K. 2000. Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants. Plant Cell Physiol. 41: 733–742.Google Scholar
  29. Sambrook J., Fritsch E.F. and Maniatis T. 1989. Molecular cloning: a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.Google Scholar
  30. Shimamoto K., Myazaki C., Hashimoto H., Izawa T., Itoh K., Terada R. et al. 1993. Trans-activation and stable integration of the maize transposable element Ds cotransfected with the Ac transposase gene in transgenic rice plants. Mol. Gen. Genet. 239: 354–360.Google Scholar
  31. Sijmons P.C., Dekker B.M., Scrammeijer N., Verwoerd T.C., van den Eck and Hoekema A. 1990. Production of correctly processed human serum albumin in plants. Bio/Technology 8: 217–221.Google Scholar
  32. Smith N., Kilpatrick J.B. and Whitelam G.C. 2001. Superfluous transgene integration in plants. Crit. Rev. Plant Sci. 20: 215–249.Google Scholar
  33. Speulman E., Metz P.L., van Arkel G., Lintel Hekkert B.T., Stiekema W.J. and Pereira A. 1999. A two-component Enhancer/ inhibitor transposon mutagenesis system for functional analysis of the Arabidopsis genome. Plant Cell 11: 1853–1866.Google Scholar
  34. Sugita K., Matsunaga E. and Ebinuma H. 1999. Effective selection system for generating marker-free transgenic plants independent of sexual crossing. Plant Cell Rep. 18: 941–947.Google Scholar
  35. Tyagi A.K. and Mohanty A. 2000. Rice transformation for crop improvement and functional genomics. Plant Sci. 158: 1–18.Google Scholar
  36. Tu J., Zhang G., Datta K., Xu C., He Y., Zhang Q. et al. 2001. Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis endotoxin. Nat. Biotech. 18: 1101–1104.Google Scholar
  37. Wang M.B., Upadhyaya N.M., Brettell R.I.S. and Waterhouse P.M. 1997. Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens. J. Genet. Breed. 51: 325–334.Google Scholar
  38. Yin Z. and Wang G.L. 2000. Evidence of multiple complex patterns of T-DNA integration into the rice genome. Theor. Appl. Genet. 100: 461–470.Google Scholar
  39. Yoder J.I. and Goldsbrough A.P. 1994. Transformation systems for generating marker-free transgenic plants. Bio/Technology 12: 263–267.Google Scholar
  40. Zubko E., Scutt C. and Meyer P. 2000. Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes. Nat. Biotech. 18: 442–445.Google Scholar
  41. Zuo J., Niu Q.W., Moller S.G. and Chua M.H. 2001. Chemicalregulated, site specific DNA excision in transgenic plants. Nat. Biotech. 19: 157–161.Google Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Olivier Cotsaftis
    • 1
  • Christophe Sallaud
    • 1
  • Jean Christophe Breitler
    • 1
  • Donaldo Meynard
    • 1
  • Rafaella Greco
    • 2
  • Andy Pereira
    • 2
  • Emmanuel Guiderdoni
    • 1
  1. 1.Biotrop ProgrammeCirad-AmisMontpellierFrance
  2. 2.Plant Research InternationalWageningenThe Netherlands

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