Transgenic Research

, Volume 27, Issue 6, pp 539–550 | Cite as

Coexpression of octopine and succinamopine Agrobacterium virulence genes to generate high quality transgenic events in maize by reducing vector backbone integration

  • Nagesh SardesaiEmail author
  • Stephen Foulk
  • Wei Chen
  • Huixia Wu
  • Emily Etchison
  • Manju Gupta
Original Paper


Agrobacterium-mediated transformation is a complex process that is widely utilized for generating transgenic plants. However, one of the major concerns of this process is the frequent presence of undesirable T-DNA vector backbone sequences in the transgenic plants. To mitigate this deficiency, a ternary strain of A. tumefaciens was modified to increase the precision of T-DNA border nicking such that the backbone transfer is minimized. This particular strain supplemented the native succinamopine VirD1/VirD2 of EHA105 with VirD1/VirD2 derived from an octopine source (pTi15955), the same source as the binary T-DNA borders tested here, residing on a ternary helper plasmid containing an extra copy of the succinamopine VirB/C/G operons and VirD1. Transformation of maize immature embryos was carried out with two different test constructs, pDAB101556 and pDAB111437, bearing the reporter YFP gene and insecticidal toxin Cry1Fa gene, respectively, contained in the VirD-supplemented and regular control ternary strains. Molecular analyses of ~ 700 transgenic events revealed a significant 2.6-fold decrease in events containing vector backbone sequences, from 35.7% with the control to 13.9% with the VirD-supplemented strain for pDAB101556 and from 24.9% with the control to 9.3% with the VirD-supplemented strain for pDAB111437, without compromising transformation efficiency. In addition, while the number of single copy events recovered was similar, there was a 24–26% increase in backbone-free events with the VirD-supplemented strain compared to the control strain. Thus, supplementing existing VirD1/VirD2 genes in Agrobacterium, to recognize diverse T-DNA borders, proved to be a useful tool to increase the number of high quality events in maize.


Agrobacterium-mediated transformation T-DNA borders Octopine virulence genes VirD1 VirD2 EHA105 



We would like to acknowledge Justin Komnick, Tatyana Minnicks and Heather Robinson for maize transformation. Authors are grateful to Cory Christensen and Otto Folkerts for their critical review of the manuscript, and to Rodrigo Sarria for his support.

Compliance with ethical standards

Conflict of interest

NS, SF, HW, and MG are the inventors on a US patent filing for the above listed work.

Supplementary material

11248_2018_97_MOESM1_ESM.pdf (301 kb)
Supplementary material 1 (PDF 301 kb)


  1. Abdal-Aziz SA, Pliego-Alfaro F, Quesada MA, Mercado JA (2006) Evidence of frequent integration of non-T-DNA vector backbone sequences in transgenic strawberry plant. J Biosci Bioeng 101:508–510CrossRefGoogle Scholar
  2. Ainley M, Armstrong K, Belmar S, Folkerts O, Hopkins N, Menke MA, Pareddy D, Petolino JF, Smith K, Woosley A (2004) Regulatory sequences for transgenic plants. US Patent 6699984Google Scholar
  3. Atmakuri K, Cascales E, Burton OT, Banta LM, Christie PJ (2007) Agrobacterium ParA/MinD-like VirC1 spatially coordinates early conjugative DNA transfer reactions. EMBO J 26:2540–2551CrossRefGoogle Scholar
  4. Beringer J, Chen W, Garton R, Sardesai N, Wang P-H, Zhou N, Gupta M, Wu H (2017) Comparison of the impact of viral and plant-derived promoters regulating selectable marker gene on maize transformation and transgene expression. Plant Cell Rep 36:519–528CrossRefGoogle Scholar
  5. Christensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18:675–689CrossRefGoogle Scholar
  6. Cowen NM, Armstrong K, Smith KA (2007) Use of regulatory sequences in transgenic plants. US Patent 7179902Google Scholar
  7. 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 6:459–468CrossRefGoogle Scholar
  8. De Framond AJ, Barton KA, Chilton MD (1983) Mini-Ti: a new vector strategy for plant genetic engineering. Biotechnology 1:262–269Google Scholar
  9. Dürrenberger F, Crameri A, Hohn B, Koukolíková-Nicola Z (1989) Covalently bound VirD2 protein of Agrobacterium tumefaciens protect the T-DNA from exonucleolytic degradation. Proc Natl Acad Sci 86:9154–9158CrossRefGoogle Scholar
  10. Eamens AL, Blanchard CL, Dennis ES, Upadhyaya NM (2004) A bidirectional gene trap construct suitable for T-DNA and Ds-mediated insertional mutagenesis in rice (Oryza sativa L.). Plant Biotechnol J 2:367–380CrossRefGoogle Scholar
  11. Fronzes R, Christie PJ, Waksman G (2009) The structural biology of type IV secretion systems. Nat Rev Microbiol 7:703–714CrossRefGoogle Scholar
  12. Gelvin SB (2010) Plant proteins involved in Agrobacterium-mediated genetic transformation. Annu Rev Phytopathol 48:45–68CrossRefGoogle Scholar
  13. Hanson B, Engler D, Moy Y, Newman B, Ralston E, Gutterson N (1999) A simple method to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences. Plant J 19:727–734CrossRefGoogle Scholar
  14. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180CrossRefGoogle Scholar
  15. Howard EA, Winsor BA, De Vos G, Zambryski P (1989) Activation of the T-DNA transfer process in Agrobacterium result in the generation of a T-strand-protein complex: tight association of VirD2 with the 5′ ends of T-strands. Proc Natl Acad Sci 86:4017–4021CrossRefGoogle Scholar
  16. Iglesias VA, Moscone EA, Papp I, Neuhuber F, Michalowski S, Phelan T, Spiker S, Matzke M, Matzke AJM (1997) Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco. Plant Cell 9:1251–1264CrossRefGoogle Scholar
  17. Jen GC, Chilton M-D (1986) The right border region of pTiT37 T-DNA is intrinsically more active than the left border region in promoting T-DNA transformation. Proc Natl Acad Sci 83:3895–3899CrossRefGoogle Scholar
  18. Jin S, Komari T, Gordon MP, Nester EW (1987) Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J Bacteriol 169:4417–4425CrossRefGoogle Scholar
  19. Kohli A, Leech M, Vain P, Laurie DA, Christou P (1998) Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots. Proc Natl Acad Sci 95:7203–7208CrossRefGoogle Scholar
  20. Komari T (1990) Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542. Plant Cell Rep 9:303–306CrossRefGoogle Scholar
  21. Kondrak M, van der Meer IM, Banfalvi Z (2006) Generation of marker- and backbone-free transgenic potatoes by site-specific recombination and a bi-functional marker gene in a non-regular one-border Agrobacterium transformation vector. Transgenic Res 15:729–737CrossRefGoogle Scholar
  22. 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 11:945–957CrossRefGoogle Scholar
  23. Kuraya Y, Ohta S, Fukuda M, Hiei Y, Murai N, Hamada K, Ueki J, Imaseki H, 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 14:309–320CrossRefGoogle Scholar
  24. Martineau B, Voelker TA, Sanders RA (1994) On defining T-DNA. Plant Cell 6:1032–1033CrossRefGoogle Scholar
  25. Merlo DJ, Russell SM, Retallack DM, Woosley AT, Meade T, Narva KE (2017) Method of increasing plant transformation frequency using modified strains of Agrobacteria. US Patent 9617551Google Scholar
  26. Nester EW (2015) Agrobacterium: nature’s genetic engineer. Front Plant Sci 5:730CrossRefGoogle Scholar
  27. Olszewski N, Tzafrir I, Somers DA, Lockhart B, Torbert KA (2002) Sugarcane bacilliform virus promoter. US Patent 6489462Google Scholar
  28. Oltmanns H, Frame B, Lee L-Y, Johnson S, Li B, Wang K, Gelvin SB (2010) Generation of backbone-free, low transgene copy plants by launching T-DNA from the Agrobacterium chromosome. Plant Physiol 152:1158–1166CrossRefGoogle Scholar
  29. 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 30:589–597CrossRefGoogle Scholar
  30. Peralta EG, Ream LW (1985) T-DNA border sequences required for crown gall tumorigenesis. Proc Natl Acad Sci 82:5112–5116CrossRefGoogle Scholar
  31. Peralta EG, Hellmiss R, Ream W (1986) Overdrive, a T-DNA transmission enhancer on the A. tumefaciens tumor-inducing plasmid. EMBO J 5:1137–1142CrossRefGoogle Scholar
  32. Podevin N, De Buck S, De Wilde C, Depicker A (2006) Insights into recognition of the T-DNA border repeats as termination sites for T-strand synthesis by Agrobacterium tumefaciens. Transgenic Res 15:557–571CrossRefGoogle Scholar
  33. 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 28:1149–1154CrossRefGoogle Scholar
  34. Richael CM, Kalyaeva M, Chretien RC, Yan H, Adimulam S, Stivison A, Weeks JT, Rommens CM (2008) Cytokinin vectors mediate marker-free and backbone-free plant transformation. Transgenic Res 17:905–917CrossRefGoogle Scholar
  35. Scheiffele P, Pansegrau W, Lanka E (1995) Initiation of Agrobacterium tumefaciens T-DNA processing. Purified proteins VirD1 and VirD2 catalyze site- and strand-specific cleavage of superhelical T-border DNA in vitro. J Biol Chem 270:1269–1276CrossRefGoogle Scholar
  36. Shagin DA, Barsova EV, Yanushevich YG, Fradkov AF, Lukyanov KA, Labas YA, Semenova TN, Ugalde JA, Meyers A, Nunez JM, Widder EA, Lukyanov SA, Matz MV (2004) GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Mol Biol Evol 21:841–850CrossRefGoogle Scholar
  37. Shou H, Frame BR, Whitham SA, Wang K (2004) Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Mol Breed 13:201–208CrossRefGoogle Scholar
  38. Smith N, Kilpatrick JB, Whitelam GC (2001) Superfluous transgene integration in plants. Crit Rev Plant Sci 20:215–249CrossRefGoogle Scholar
  39. Stachel SE, Zambryski P (1986) VirA and virG control the plant-induced activation of the T-DNA transfer process of Agrobacterium tumefaciens. Cell 46:325–333CrossRefGoogle Scholar
  40. Stachel SE, Timmerman B, Zambryski P (1986) Generation of single-stranded T-DNA molecules during the initial stages of T-DNA transfer from Agrobacterium tumefaciens to plant cells. Nature 322:706–712CrossRefGoogle Scholar
  41. Stachel SE, Timmerman B, Zambryski P (1987) Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirements for 5′ virD gene products. EMBO J 6:857–863CrossRefGoogle Scholar
  42. Steck TR, Lin T-S, Kado CI (1990) VirD2 gene product from the nopaline plasmid pTiC58 has at least two activities required for virulence. Nuc Acids Res 18:6953–6958CrossRefGoogle Scholar
  43. Stuive MH, Ponstein AS, Ohl SA, Goddijn OJM, Simons LH, Dekker BMM, Hoekstra S, Tigelaar H, Elzinga N (2006) Plasmids for plant transformation and method for using the same. US Patent 7029908Google Scholar
  44. Vain P, Harvey A, Worland B, Ross S, Snape JW, Lonsdale D (2004) The effect of additional virulence genes on transformation efficiency, transgene integration and expression in rice plants using the pGreen/pSoup dual binary vector system. Transgenic Res 13:593–603CrossRefGoogle Scholar
  45. van der Graaff E, den Dulk-Ras A, Hooykaas PJJ (1996) Deviating T-DNA transfer from Agrobacterium tumefaciens to plants. Plant Mol Biol 31:677–681CrossRefGoogle Scholar
  46. Wang K, Herrera-Estrella L, Van Montagu M, Zambryski P (1984) Right 25 bp terminus sequence of the nopaline T-DNA is essential for and determines direction of DNA transfer from Agrobacterium to the plant genome. Cell 38:455–462CrossRefGoogle Scholar
  47. Wang K, Genetello C, Van Montagu M, Zambryski PC (1987) Sequence context of the T-DNA border repeat element determines its relative activity during T-DNA transfer to plant cells. Mol Gen Genet 210:338–346CrossRefGoogle Scholar
  48. Wang K, Herrera-Estrella A, Van Montagu M (1990) Overexpression of virD1 and virD2 genes in Agrobacterium tumefaciens enhances T-complex formation and plant transformation. J Bacteriol 172:4432–4440CrossRefGoogle Scholar
  49. Wenck A, Czako M, Kanevski I, Marton L (1997) Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation. Plant Mol Biol 34:913–922CrossRefGoogle Scholar
  50. Wenck AR, Quinn M, Whetten RW, Pullman G, Sederoff R (1999) High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda). Plant Mol Biol 39:407–416CrossRefGoogle Scholar
  51. Winans SC, Ebert PR, Stachel SE, Gordon MP, Nester EW (1986) A gene essential for Agrobacterium virulence is homologous to a family of positive regulatory loci. Proc Natl Acad Sci 83:8278–8282CrossRefGoogle Scholar
  52. Woosley A, Worden S (2015) Increased protein expression in plants. US Patent application US 2015/0203857Google Scholar
  53. Wu H, Sparks CA, Jones HD (2006) Characterisation of T-DNA loci and vector backbone sequences in transgenic wheat produced by Agrobacterium-mediated transformation. Mol Breed 18:195–208CrossRefGoogle Scholar
  54. Yadav NS, Vanderleyden J, Bennett DR, Barnes WM, Chilton M-D (1982) Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc Natl Acad Sci 79:6322–6326CrossRefGoogle Scholar
  55. Yanofsky MF, Porter SG, Young C, Albright LM, Gordon MP, Nester EW (1986) The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Cell 47:471–477CrossRefGoogle Scholar
  56. Ye X, Williams EJ, Shen J, Esser JA, Nichols AM, Petersen MW, Gilbertson LA (2008) Plant development inhibitory genes in binary vector backbone improve quality event efficiency in soybean transformation. Transgenic Res 17:827–838CrossRefGoogle Scholar
  57. Ye X, Williams EJ, Shen J, Johnson S, Lowe B, Radke S, Strickland S, Esser JA, Petersen MW, Gilbertson LA (2011) Enhanced production of single copy backbone-free transgenic plants in multiple crop species using binary vectors with a pRi replication origin in Agrobacterium tumefaciens. Transgenic Res 20:773–786CrossRefGoogle Scholar
  58. Yin Z, Wang G-L (2000) Evidence of multiple complex patterns of T-DNA integration into the rice genome. Theor Appl Genet 100:461–470CrossRefGoogle Scholar
  59. Zambryski PC, Depicker A, Kruger K, Goodman HM (1982) Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. J Mol Appl Genet 1:361–370PubMedGoogle Scholar
  60. Zhi L, TeRonde S, Meyer S, Arling ML, Register JC III, Zhao Z-Y, Jones TJ, Anand A (2015) Effect of Agrobacterium strain and plasmid copy number on transformation frequency, event quality and usable event quality in an elite maize cultivar. Plant Cell Rep 34:745–754CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Dow AgroSciences LLCIndianapolisUSA

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