Advertisement

Theoretical and Applied Genetics

, Volume 109, Issue 4, pp 815–826 | Cite as

A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations

  • A. S. Afolabi
  • B. Worland
  • J. W. Snape
  • P. Vain
Original Paper

Abstract

Transgenic locus composition and T-DNA linkage configuration were assessed in a population of rice plants transformed using the dual-binary vector system pGreen (T-DNA containing the bar and gus genes)/pSoup (T-DNA containing the aphIV and gfp genes). Transgene structure, expression and inheritance were analysed in 62 independently transformed plant lines and in around 4,000 progeny plants. The plant lines exhibited a wide variety of transgenic locus number and composition. The most frequent form of integration was where both T-DNAs integrated at the same locus (56% of loci). When single-type T-DNA integration occurred (44% of loci), pGreen T-DNA was preferentially integrated. In around half of the plant lines (52%), the T-DNAs integrated at two independent loci or more. In these plants, both mixed and single-type T-DNA integration often occurred concurrently at different loci during the transformation process. Non-intact T-DNAs were present in 70–78% of the plant lines causing 14–21% of the loci to contain only the mid to right border part of a T-DNA. In 53–66% of the loci, T-DNA integrated with vector backbone sequences. Comparison of transgene presence and expression in progeny plants showed that segregation of the transgene phenotype was not a reliable indicator of either transgene inheritance or T-DNA linkage, as only 60–80% of the transgenic loci were detected by the expression study. Co-expression (28% of lines) and backbone transfer (53–66% of loci) were generally a greater limitation to the production of marker-free T1 plants expressing the gene of interest than co-transformation (71% of lines) and unlinked integration (44% of loci).

Keywords

Selectable Marker Gene Transgenic Locus Backbone Sequence aphIV Gene Dual Selection 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

We gratefully acknowledge The Rockefeller Foundation for its support. This document is an output from projects (Plant Sciences Research Programme R8031) funded by the UK Department for International Development (DFID) and administered by the Centre for Arid Zone Studies (CAZS) for the benefit of developing countries. The views expressed are not necessarily those of the DFID.

References

  1. Albert H, Dale EC, Lee E, Ow DW (1995) Site-specific integration of T-DNA into wild-type and mutant lox sites in the plant genome. Plant J 7:649–659CrossRefPubMedGoogle Scholar
  2. Bec S, Chen L, Ferriere NM, Legave T, Fauquet C, Guideroni E (1998) Comparative histology of microprojectile-mediated gene transfer to embryonic calli in japonica rice (Oryza sativa L.): influence of the structural organization of target tissues on genotype transformation ability. Plant Sci 138:177–190CrossRefGoogle Scholar
  3. Bhattacharyya M, Stemer BA, Dixon RA (1994) Reduced variation in transgene expression from a binary vector with selectable markers at the right and left T-DNA borders. Plant J 6:957–968Google Scholar
  4. Cluster PD, O’Dell M, Metzlaff M, Flavell RB (1996) Details of T-DNA structural organisation from a transgenic petunia population exhibiting co-suppression. Plant Mol Biol 32:1197–1203PubMedGoogle Scholar
  5. Cotsaftis O, Sallaud C, Breitler JC, Meynard D, Greco R, Pereira A, Guiderdoni E (2002) Transposon-mediated generation of T-DNA and marker free rice plants expressing a Bt endotoxin gene. Mol Breed 10:165–180CrossRefGoogle Scholar
  6. Dale EC, Ow DE (1990) Intra and intermolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase. Gene 91:79–85CrossRefPubMedGoogle Scholar
  7. Daley M, Knauf VC, Summerfelt KR, Turner JC (1998) Co-transformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker-free transgenic plants. Plant Cell Rep 19:489–496CrossRefGoogle Scholar
  8. De Block M, Debrouwer D (1991) Two T-DNAs co–transformed into Brassica napus by a double Agrobacterium infections are mainly integrated at the same locus. Theor Appl Genet 82:257–263Google Scholar
  9. De Framond AJ, Back EW, Chilton WS, Kayes L, Chilton M-D (1986) Two unlinked T-DNAs can transform the same tobacco plant cell and segregate in the F1 generation. Mol Gen Genet 202:125–131Google Scholar
  10. De Neve M, De Buck S, Jacobs A (1997) T-DNA integration patterns in co-transformed plant cells suggests that T-DNA repeats originate from co-integration of separate T-DNAs. Plant J 11:15–29PubMedGoogle Scholar
  11. Dong J, Kharb P, Teng W, Hall TC (2001) Characterization of rice transformed via an Agrobacterium-mediated inflorescence approach. Mol Breed 7:187–194CrossRefGoogle Scholar
  12. Ebinuma H, Sugita K, Matsunaga E, Endo S, Yamada K, Komamine A (2001) Systems for the removal of a selection marker and their combination with a positive marker. Plant Cell Rep 20:383–392CrossRefGoogle Scholar
  13. Goldsbrough AP, Lastrella CN, Yoder J (1993) Transposition mediated repositioning and subsequent elimination of marker genes from transgenic tomato. BioTechnology 11:1286–1292Google Scholar
  14. Hanson B, Engler D, Moy Y, Newman B (1999) A simple method to enrich an Agrobacterium-transformed population of plants containing only T-DNA sequences. Plant J 19:727–734CrossRefPubMedGoogle Scholar
  15. Hare P, Chua N-H (2002) Excision of selectable marker genes from transgenic plants. Nat Biotechnol 20:575–580CrossRefPubMedGoogle Scholar
  16. Hellens RP, Edwards A, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832Google Scholar
  17. Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries T-DNA. Plant J 6:271–282Google Scholar
  18. Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750PubMedGoogle Scholar
  19. James VA, Avart C, Worland B, Snape JW, Vain P (2002) The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants. Theor Appl Genet 104:553–561CrossRefGoogle Scholar
  20. James VA, Worland B, Snape JW, Vain P (2004) Development of a standard operating procedure (SOP) for the precise quantification of transgene expression levels in rice. Physiol Plant 120:650–656Google Scholar
  21. Jefferson RA, Kavanagh TA, Bevan MW (1987) β-glucuronidase as a sensitive and versatile fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  22. Jorgensen R, Snyder C, Jones JDG (1987) T-DNA is organized predominantly in inverted repeat structures in plants transformed with Agrobacterium tumefaciens C58 derivates. Mol Gen Genet 207:471–477Google Scholar
  23. Komari T, Hiei Y, Saito Y, Murai N, 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 10:165–174PubMedGoogle Scholar
  24. Koncz C, Nemeth K, Redei GP, Schell J (1994) Homology recognition during T-DNA integration into the plant genome. In: Paszkowski J (ed) Homologous recombination and gene silencing in plants. Kluwer, Dordrecht, pp 167–189Google Scholar
  25. Lu HJ, Zhou X-R, Gong Z-X, Upadhyaya NM (2001) Generation of selectable marker-free transgenic rice using double right-border (DRB) binary vectors. Aust J Plant Physiol 28:241–248CrossRefGoogle Scholar
  26. Matthew PR, Wang M-B, Waterhouse PM, Thornton S, Fieg SJ, Gubler F, Jacobsen JV (2001) Marker gene elimination from transgenic barley, using co-transformation with adjacent “twin T-DNAs” on a standard Agrobacterium transformatiom vector. Mol Breed 7:195–202CrossRefGoogle Scholar
  27. McCormac AC, Elliot MC, Chen DF (1999) pBeCKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates “clean-gene” facilities. Mol Gen Genet 261:226–235CrossRefPubMedGoogle Scholar
  28. McKnight TD, Lillies MT, Simpson RB (1987) Segregation of genes transferred to one plant cell from two Agrobacterium strains. Plant Mol Biol 8:439–445Google Scholar
  29. Miller M, Tagliani L, Wang N, Berka B, Bidney D (2002) High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system. Transgenic Res 11:381–396CrossRefPubMedGoogle Scholar
  30. Paszkowski J, Baur M, Bogucki A, Potrykus I (1988) Gene targeting in plants. EMBO J 7:4021–4026Google Scholar
  31. Sallaud C, Meynard D, Van Boxtel J, Gay C, Bes M, Brizard JP, Larmande P, Ortega D, Raynal M, Portefaix M, Ouwerkerk PBF, Rueb S, Delseny M, Guiderdoni E (2003) Highly efficient production and characterization of T-DNA plants for rice (Oriza sativa L.) functional genomics. Theor Appl Genet 106:1396–1408PubMedGoogle Scholar
  32. Srivastava V, Anderson OD, Ow DV (1999) Single-copy transgenic wheat generated through the resolution of complex integration patterns. Proc Natl Acad Sci USA 96:11117–11121PubMedGoogle Scholar
  33. Terada R, Urawa H, Inagaki Y, Tsugane K, Lida S (2002) Efficient gene targeting by homologous recombination in rice. Nat Biotechnol 20:1030–1034CrossRefPubMedGoogle Scholar
  34. Vain P, James VA, Worland B, Snape JW (2002) Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment. Theor Appl Genet 105:878–889CrossRefGoogle Scholar
  35. Vain P, Afolabi A, Worland B, Snape JW (2003) Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup. Theor Appl Genet 107:210–217CrossRefPubMedGoogle Scholar
  36. Xing A, Zhang Z, Sato S, Staswick P, Clement T (2000) The use of two T-DNA binary system to derive marker-free transgenic soybeans. In Vitro Cell Dev Plant 36:456–463Google Scholar
  37. Yoder JI, Goldsbrough AP (1994) Transformation systems for generating marker-free transgenic plants. BioTechnology 12:263–267Google Scholar
  38. Zubko E, Scutt C, Meyer P (2000) Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes. Nat Biotechnol 18:442–445CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • A. S. Afolabi
    • 1
  • B. Worland
    • 1
  • J. W. Snape
    • 1
  • P. Vain
    • 1
  1. 1.John Innes CentreNorwich Research ParkNorwichUK

Personalised recommendations