Transgenic Research

, Volume 13, Issue 3, pp 271–287

A Novel Two T-DNA Binary Vector Allows Efficient Generation of Marker-free Transgenic Plants in Three Elite Cultivars of Rice (Oryza sativa L.)

  • Jean-Christophe Breitler
  • Donaldo Meynard
  • Jos Van Boxtel
  • Monique Royer
  • François Bonnot
  • Laurence Cambillau
  • Emmanuel Guiderdoni
Article

Abstract

A pilot binary vector was constructed to assess the potential of the 2 T-DNA system for generating selectable marker-free progeny plants in three elite rice cultivars (ZhongZuo321, Ariete and Khao Dawk Mali 105) known to exhibit contrasting amenabilities to transformation. The first T-DNA of the vector, delimited by Agrobacterium tumefaciens borders, contains the hygromycin phosphotransferase (hpt) selectable gene and the green fluorescent protein (gfp) reporter gene while the second T-DNA, delimited by Agrobacterium rhizogenes borders, bears the phosphinothricin acetyl transferase (bar) gene, featuring the gene of interest. 82–90% of the hygromycin-resistant primary transformants exhibited tolerance to ammonium glufosinate mediated by the bar gene suggesting very high co-transformation frequency in the three cultivars. All of the regenerated plants were analyzed by Southern blot which confirmed co-integration of the T-DNAs at frequencies consistent with those of co-expression and allowed determination of copy number for each gene as well as detection of two different vector backbone fragments extending between the two T-DNAs. Hygromycin susceptible, ammonium glufosinate tolerant phenotypes represented 14.4, 17.4 and 14.3% of the plants in T1 progenies of ZZ321, Ariete and KDML105 primary transformants, respectively. We developed a statistical model for deducing from the observed copy number of each T-DNA in T0 plants and phenotypic segregations in T1 progenies the most likely constitution and linkage of the T-DNA integration locus. Statistical analysis identified in 40 out of 42 lines a most likely linkage configuration theoretically allowing genetic separation of the two T-DNA types and out segregation of the T-DNA bearing the bar gene. Overall, though improvements of the technology would be beneficial, the 2 T-DNA system appeared to be a useful approach to generate selectable marker-free rice plants with a consistent frequency among cultivars.

co-transformation segregation analysis selectable marker gene transgenic rice 

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References

  1. Bouchez D and Tourneur J (1991) Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes. Plasmid 25: 27–39.CrossRefPubMedGoogle Scholar
  2. Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Annal Biochem 72: 248–254.CrossRefGoogle Scholar
  3. Chen L, Zhang S, Beachy RN and Fauquet CM (1998) A protocol for consistent, large scale production of fertile transgenic rice plants. Plant Cell Rep 18: 25–31.Google Scholar
  4. Christensen A and 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.CrossRefPubMedGoogle Scholar
  5. Corneille S, Lutz K, Svab Z and Maliga P (2001) Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox-site-specific recombination system. Plant J 27: 171–178.CrossRefPubMedGoogle Scholar
  6. Cotsaftis O, Sallaud C, Breitler JC, Meynard D, Greco R, Pereira A, et al. (2002) Transposon-mediated generation of marker free rice plants containing a Bt endotoxin gene conferring insect resistance. Mol Breeding 10: 165–180.CrossRefGoogle Scholar
  7. Dale EC and Ow DW (1994) Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad USA 88: 10558–10562.Google Scholar
  8. Daley M, Knauf VC, Summerfelt KR and 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 17: 489–496.CrossRefGoogle Scholar
  9. De Block M and Debrouwer D (1991) Two T-DNA's co-transformed into Brassica napus by a double Agrobacterium tumefaciens infection are mainly integrated at the same locus. Theor Appl Genet 82: 257–263.Google Scholar
  10. De Framond AJ, Back EW, Chilton WS, Kayes L and 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: 125–131.Google Scholar
  11. De Neve M, De Buck S, Jacobs A, Van Montagu M and Depicker A (1997) T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs. Plant J 11: 15–29.PubMedGoogle Scholar
  12. De Pater BS van der Mark F Rueb S Katagiri F Chua NH Schilperoort RA, et al. (1992) The promoter of the rice gos2 is active in various different monocot tissues and binds rice nuclear factor ASF-1. Plant J 2: 837–844.CrossRefPubMedGoogle Scholar
  13. De Vetten N, Wolters A-M, Raemakers K, van der Meer I, Stege R, Heeres E, et al. (2003) A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop. Nat Biotech 21: 439–442.CrossRefGoogle Scholar
  14. Ebinuma H, Sugita K, Matsunage E, Endo S, Yamada K and Komamine A (2001) Systems for the removal of a selection marker and their combination with a positive marker. Plant Cell Rep 20: 383–392.Google Scholar
  15. Feller W (1968) An Introduction to Probability Theory and Its Applications. New York, John Wiley & Sons.Google Scholar
  16. Gilbertson L, Ekena J, House I, Huang S, Krieger E, Luethy M, et al. (2003) Novel T-DNA vector designs to facilitate the production of transgenic marker genes and vector backbone. In: 7th International Congress of Plant Molecular Biology, 23–28 June, Barcelona, Spain, 438 p.Google Scholar
  17. Gleave AP, Mitra DS, Mudge SR and Morris BA (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.CrossRefPubMedGoogle Scholar
  18. Goldsbrough AP, Lastrella CN and Yoder JI (1993) Transposition mediated re-positioning and subsequent elimination of marker gene from transgenic tomato. Biotechnology 11: 1286–1292.Google Scholar
  19. Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG et al. (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2: 603–618.CrossRefPubMedGoogle Scholar
  20. Hansen G and Wright MS (1999) Recent advances in the transformation of plants. Trends Plants Sci 4: 226–231.Google Scholar
  21. Hare PD and Chua N-H (2002) Excision of selectable marker genes from transgenic plants. Nat Biotech 20: 575–580.CrossRefGoogle Scholar
  22. Heim R, Cubitt AB and Tsien RY (1995) Improved green fluoresence. Nature 373: 663–664.CrossRefPubMedGoogle Scholar
  23. Hohn B, Levy AA and Puchta H (2001) Elimination of selection markers from transgenics plants. Curr Opin Biotech 12: 139–143.PubMedGoogle Scholar
  24. Hoisington D (1992) Laboratory Protocols. CIMMYT, Mexico DF.Google Scholar
  25. Hood EE, Gelvin SB, Melchers LS and Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2: 208–218.CrossRefGoogle Scholar
  26. Horsch RB and Klee HJ (1986) Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens: role of T-DNAs borders in the transfer process. Proc Natl Acad Sci USA 83: 4428–4432.Google Scholar
  27. Kim SR, Lee J, Jun SH, Park S, Kang HG, Kwon S et al. (2003) Transgene structures in T-DNA-inserted rice plants. Plant Mol Biol 52: 761–73.PubMedGoogle Scholar
  28. 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.CrossRefPubMedGoogle Scholar
  29. Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.CrossRefPubMedGoogle Scholar
  30. Lu H, Zhou X, Gong Z and Upadhyaya N (2001) Generation of selectable marker-free transgenic rice using a double rigth-border. Aust J Plant Physiol 28: 241–248.Google Scholar
  31. Matsuoka M, Kano-Murakami Y, Tanaka Y, Ozeki Y and Yamamoto N (1988) Classification and nucleotide sequence of cDNA encoding the small subunit of ribulose-1,5-biphosphate carboxylase from rice. Plant Cell Physiol 29: 1015–1022.Google Scholar
  32. Matthews P, Wang MB, Waterhouse P, Thornton S, Fieg S, Gubler F et al. (2001) Marker gene elimination from transgenic barley, using co-transformation with adjacent ‘twin T-DNAs’ on a standard Agrobacterium transformation vector. Mol Breeding 7: 195–202.CrossRefGoogle Scholar
  33. McCormac AC, Fowler MR, Chen DF and Elliott MC (2001) Efficient co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separation. Transgenic Res 10: 143–155.CrossRefPubMedGoogle Scholar
  34. McKnight TD, Lillis MT and Simpson RB (1987) Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Plant Mol Biol 8: 439–445.CrossRefGoogle Scholar
  35. Miller M, Tagliani L, Wang N, Berka B, Bidney D and Zhao ZY (2002) High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system. Transgenic Res 11: 381–396.CrossRefPubMedGoogle Scholar
  36. Poirier Y, Ventre G and Nawrath C (2000) High-frequency linkage of co-expression T-DNA in transgenic Arabidopsis thaliana transformed by vacuum-infiltration of Agrobacterium tumefaciens. Theor Appl Genet 100: 487–493.CrossRefGoogle Scholar
  37. Puchta H (2000) Removing selectable marker genes: taking the shortcut. Trends Plant Sci 5: 273–274.CrossRefPubMedGoogle Scholar
  38. Sallaud C, Meynard D, van Boxtel J, Gay C, Bés M, Brizard J-P, et al. (2003) Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theor Appl Genet 106: 1396–1408.PubMedGoogle Scholar
  39. Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual. 2nd édn, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  40. Speulman E, Metz PL, Van Arkel G, Lintel Hekkert BT, Stiekema WJ and Pereira A (1999) A two-component enhancer-inhibitor transposon mutagenesis system for functional analysis of the Arabidopsis genome. Plant Cell 11: 1853–1866.CrossRefPubMedGoogle Scholar
  41. 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.CrossRefGoogle Scholar
  42. Vain P, Afolabi AS, Worland B and Snape JW (2003) Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup. Theor Appl Genet 107: 210–217.CrossRefPubMedGoogle Scholar
  43. Vaucheret H, Beclin C, Elmayan T, Feuerbach F, Godon C, Morel JB, et al. (1998) Transgene-induced gene silencing in plants. Plant J 16: 651–659.CrossRefPubMedGoogle Scholar
  44. Wang MB, Upadhyaya NM, Brettell RI and Waterhouse PM (1997) Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens. J Genet Breed 51: 325–334.Google Scholar
  45. Xing A, Zhang Z, Sato S, Staswick P and Clemente T (2000) The use of the two T-DNA binary system to derive marker-free transgenic soybeans. In Vitro Cell Dev Biol 36: 456–463.Google Scholar
  46. Yin Z and Wang GL (2000) Evidence of multiple complex patterns of T-DNA integration into the rice genome. Theor Appl Genet 100: 461–470.CrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Jean-Christophe Breitler
    • 1
  • Donaldo Meynard
    • 1
  • Jos Van Boxtel
    • 1
  • Monique Royer
    • 1
  • François Bonnot
    • 3
  • Laurence Cambillau
    • 1
  • Emmanuel Guiderdoni
    • 1
  1. 1.Cirad-AmisUMR PIA1096, Biotrop and Crop Protection ProgrammesMontpellier Cedex 05France
  2. 2.Department of PomologyUniversity of CaliforniaDavisUSA
  3. 3.Cirad-CpMontpellier Cedex 5France

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