Skip to main content
SpringerLink
Log in

Characterisation of T-DNA loci and vector backbone sequences in transgenic wheat produced by Agrobacterium-mediated transformation

  • Original Paper
  • Published:
Molecular Breeding Aims and scope Submit manuscript

An Erratum to this article was published on 31 March 2007

Abstract

Detailed molecular characterisation of transgene loci is a requirement for gaining regulatory approval for environmental release of genetically modified crops. In cereals, it is generally accepted that Agrobacterium-mediated transformation generates cleaner transgene loci with lower copy number and fewer rearrangements than those generated by biolistics. However, in wheat there has been little detailed analysis of T-DNA insertions at genetic and molecular level. Wheat lines transformed using Agrobacterium tumefaciens with bar and gusA (GUS) genes were subjected to genetic and molecular analysis. Unlike previous studies of transgene loci in wheat, we used functional assays for PAT and GUS proteins, combined with PCR and Southern analysis to detect the presence, copy number, linkage and transmission of two transgenes inserted in the same T-DNA. Thirty-four independent transgenic lines were categorised into three types: type I events (38% of total) where the gusA and bar genes displayed complete genetic linkage, segregating together as a single functional locus at the expected ratio of 3:1; type II events (18%), which possessed two or more transgene loci each containing gusA and bar; and type III events (44%), containing an incomplete T-DNA in which either the gusA or bar gene was lost. Most lines in this last category had lost the bar gene situated near the left T-DNA border. Southern analysis indicated that 30% of all lines possessed a single T-DNA copy containing gusA and bar. However, when data on expression and molecular analysis are combined, only 23% of all lines have single copy T-DNAs in which both gene cassettes are functioning. We also report on the presence of plasmid backbone DNA sequence in transgene loci detected using primer pairs outside the left and right T-DNA borders and within the plasmid selectable marker (NptI) gene. Approximately two thirds of the lines contained some vector backbone DNA, more frequently adjacent to the left border. Taken together, these data imply unstable left border function causing premature T-strand termination or read-through into vector backbone. As far as we are aware, this is the first report revealing near border T-DNA truncation and vector backbone integration in wheat transgenic lines produced by Agrobacterium-mediated transformation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Afolabi AS, Worland B, Snape JW, Vain P (2004) A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations. Theor Appl Genet 109:815–826

    Article  PubMed  CAS  Google Scholar 

  • Barcelo P, Rasco-Gaunt S, Thorpe C, Lazzeri PA (2001) Transformation and gene expression. In: Shewry PR, Lazzeri PA, Edwards KJ (eds) Advances in botanical research incorporating advances in plant pathology. pp 59–126

  • Barro F, Cannell ME, Lazzeri PA, Barcelo P (1998) The influence of auxins on transformation of wheat and tritordeum and analysis of transgene integration patterns in transformants. Theor Appl Genet 97:684–695

    Article  CAS  Google Scholar 

  • Caplan AB, Vanmontagu M, Schell J (1985) Genetic-analysis of integration mediated by single T-DNA borders. J Bacteriol 161:655–664

    PubMed  CAS  Google Scholar 

  • Cheng M, Fry JE, Pang SZ, Zhou HP, Hironaka CM, Duncan DR, Conner TW, Wan YC (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980

    PubMed  CAS  Google Scholar 

  • Cheng M, Hu TC, Layton J, Liu CN, Fry JE (2003) Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat. In Vitro Cell Dev Biol-Plant 39:595–604

    Article  CAS  Google Scholar 

  • Cheng M, Lowe BA, Spencer TM, Ye XD, Armstrong CL (2004) Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cell Dev Biol-Plant 40:31–45

    Article  Google Scholar 

  • Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5:213–218

    Article  PubMed  CAS  Google Scholar 

  • Christou P (1992) Genetic-transformation of crop plants using microprojectile bombardment. Plant J 2:275–281

    Article  CAS  Google Scholar 

  • Cluster PD, Odell M, Metzlaff M, Flavell RB (1996) Details of T-DNA structural organization from a transgenic Petunia population exhibiting cosuppression. Plant Mol Biol 32:1197–1203

    Article  PubMed  CAS  Google Scholar 

  • De Buck S, De Wilde C, Van Montagu M, 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

    Article  Google Scholar 

  • Deblock M, Desonville A, Debrouwer D (1995) The selection mechanism of phosphinothricin is influenced by the metabolic status of the tissue. Planta 197:619–626

    CAS  Google Scholar 

  • Durrenberger F, Crameri A, Hohn B, Koukolikovanicola Z (1989) Covalently Bound Vird2 Protein of Agrobacterium-Tumefaciens Protects the T-DNA from Exonucleolytic Degradation. Proc Natl Acad Sci U S A 86:9154–9158

    Article  PubMed  CAS  Google Scholar 

  • Frame BR, Shou HX, Chikwamba RK, Zhang ZY, Xiang CB, Fonger TM, Pegg SEK, Li BC, Nettleton DS, Pei DQ, Wang K (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol 129:13–22

    Article  PubMed  CAS  Google Scholar 

  • Fu D, St. Amand PC, Xiao Y, Muthukrishnan S, Lang GH (2006) Characterization of T-DNA integration in creeping bentgrass. Plant Sci 170:225–237

    Article  CAS  Google Scholar 

  • Hellens R, Mullineaux P, Klee H (2000a) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446–451

    Article  CAS  Google Scholar 

  • Hellens RP, Edwards EA, Leyland NR, Bean S and Mullineaux PM (2000b) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832

    Article  CAS  Google Scholar 

  • 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 of the T-DNA. Plant J 6:271–282

    Article  PubMed  CAS  Google Scholar 

  • Hu T, Metz S, Chay C, Zhou HP, Biest N, Chen G, Cheng M, Feng X, Radionenko M, Lu F, Fry J (2003) Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Reports 21:1010–1019

    Article  PubMed  CAS  Google Scholar 

  • 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 14:745–750

    Article  PubMed  CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) Beta-Glucuronidase (Gus) as a Sensitive and Versatile Gene Fusion Marker in Plants. J Cell Biochem 57–57

  • Jones HD (2005) Wheat transformation: current technology and applications to grain development and composition. J Cereal Sci 41:137–147

    Article  CAS  Google Scholar 

  • Jones HD, Doherty A, Wu H (2005) Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods 1:5

    Article  PubMed  Google Scholar 

  • Khanna HK, Daggard GE (2003) Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium. Plant Cell Reports 21:429–436

    PubMed  CAS  Google Scholar 

  • Kim SR, Lee J, Jun SH, Park S, Kang HG, Kwon S, An G (2003) Transgene structures in T-DNA-inserted rice plants. Plant Mol Biol 52: 761–773

    Article  PubMed  CAS  Google Scholar 

  • Kononov ME, Bassuner B, Gelvin SB (1997) Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J 11:945–957

    Article  PubMed  CAS  Google Scholar 

  • Kuraya Y, Ohta S, Fukuda M, Hiei Y, Murai N, Hamada K, Ueki J, Imaseki H and Komari T (2004) Suppression of transfer of non-T-DNA ‘vector backbone’ sequences by multiple left border repeats in vectors for transformation of higher plants mediated by Agrobacterium tumefaciens. Mol Breed 14:309–320

    Article  Google Scholar 

  • Makarevitch I, Svitashev SK and Somers DA (2003) Complete sequence analysis of transgene loci from plants transformed via microprojectile bombardment. Plant Mol Biol 52:421–432

    Article  PubMed  CAS  Google Scholar 

  • Martineau B, Voelker TA, Sanders RA (1994) On Defining T-DNA. Plant Cell 6:1032–1033

    Article  PubMed  Google Scholar 

  • Mehlo L, Mazithulela G, Twyman RM, Boulton MI, Davies JW and Christou P (2000) Structural analysis of transgene rearrangements and effects on expression in transgenic maize plants generated by particle bombardment. Maydica 45:277–287

    Google Scholar 

  • Miranda A, Janssen G, Hodges L, Peralta EG, Ream W (1992) Agrobacterium-Tumefaciens Transfers Extremely Long T-Dnas by a Unidirectional Mechanism. J Bacteriol 174:2288–2297

    PubMed  CAS  Google Scholar 

  • Ooms G, Bakker A, Molendijk L, Wullems GJ, Gordon MP, Nester EW, Schilperoort RA (1982) T-DNA Organization in Homogeneous and Heterogeneous Octopine-Type Crown Gall Tissues of Nicotiana-Tabacum. Cell 30:589–597

    Article  PubMed  CAS  Google Scholar 

  • Peralta EG, Hellmiss R, Ream W (1986) Overdrive, a T-DNA Transmission Enhancer on the a-Tumefaciens Tumor-Inducing Plasmid. Embo J 5:1137–1142

    PubMed  CAS  Google Scholar 

  • Ramanathan V, Veluthambi K (1995) Transfer of Non-T-DNA Portions of the Agrobacterium-Tumefaciens Ti Plasmid pTiA6 from the Left Terminus of TL-DNA. Plant Mol Biol 28:1149–1154

    Article  PubMed  CAS  Google Scholar 

  • Rasco-Gaunt S, Riley A, Lazzeri P, Barcelo P (1999) A facile method for screening for phosphinothricin (PPT)-resistant transgenic wheats. Mol Breed 5:255–262

    Article  Google Scholar 

  • Rooke L, Steele SH, Barcelo P, Shewry PR, Lazzeri PA (2003) Transgene inheritance, segregation and expression in bread wheat. Euphytica 129:301–309

    Article  CAS  Google Scholar 

  • 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 and Guiderdoni E (2003) Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. TAG Theoretical and Applied Genetics 106:1396–1408

    CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press

  • Sawada H, Ieki H, Matsuda I (1995) Pcr detection of Ti and Ri plasmids from phytopathogenic agrobacterium strains. Appl Environ Microbiol 61:828–831

    PubMed  CAS  Google Scholar 

  • Shewry PR, Jones HD (2005) Transgenic wheat: where do we stand after the first 12 years? Ann Appl Biol 147:1–14

    Article  CAS  Google Scholar 

  • Shou HX, Frame BR, Whitham SA and Wang K (2004) Assessment of transgenic maize events produced by particle bombardment or Agrobacteriummediated transformation. Mol Breed 13:201–208

    Article  CAS  Google Scholar 

  • Stacey J and Isaac P (1994) Isolation of DNA from plants. In: Isaac P (eds) Methods in molecular biology – protocols for nucleic acid analysis by nonradioactive probes. Totowa NJ, Humana Press Inc., pp 9–15

    Google Scholar 

  • Svitashev SK, Pawlowski WP, Makarevitch I, Plank DW, Somers DA (2002) Complex transgene locus structures implicate multiple mechanisms for plant transgene rearrangement. Plant J 32:433–445

    Article  PubMed  CAS  Google Scholar 

  • Tingay S, McElroy D, Kalla R, Fieg S, Wang MB, Thornton S and Brettell R (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J 11:1369–1376

    Article  CAS  Google Scholar 

  • Trifonova A, Madsen S, Olesen A (2001) Agrobacterium-mediated transgene delivery and integration into barley under a range of in vitro culture conditions. Plant Sci 161:871–880

    Article  CAS  Google Scholar 

  • van der Graaff E, den Dulk-Ras A, Hooykaas PJJ (1996) Deviating T-DNA transfer from Agrobacterium tumefaciens to plants. Plant Mol Biol 31:677–681

    Article  PubMed  Google Scholar 

  • Vasil IK (2005) The story of transgenic cereals: The challenge, the debate, and the solution – A historical perspective. In Vitro Cell Dev Biol-Plant 41:577–583

    Article  Google Scholar 

  • Wu H, Sparks C, Amoah B, Jones HD (2003) Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Reports 21:659–668

    PubMed  CAS  Google Scholar 

  • Yin Z, Wang GL (2000) Evidence of multiple complex patterns of T-DNA integration into the rice genome. Theor Appl Genet 100:461–470

    Article  CAS  Google Scholar 

  • Zambryski P (1988) Basic processes underlying Agrobacterium-mediated DNA transfer to plant-cells. Annu Rev Genet 22:1–30

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. CPI Division, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK

    Huixia Wu, Caroline A. Sparks & Huw D. Jones

Authors
  1. Huixia Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Caroline A. Sparks
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huw D. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Huw D. Jones.

Additional information

An erratum to this article is available at http://dx.doi.org/10.1007/s11032-007-9090-1.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, H., Sparks, C.A. & Jones, H.D. Characterisation of T-DNA loci and vector backbone sequences in transgenic wheat produced by Agrobacterium-mediated transformation. Mol Breeding 18, 195–208 (2006). https://doi.org/10.1007/s11032-006-9027-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11032-006-9027-0

Keywords

Search

Navigation