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


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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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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