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

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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.

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Author notes

  1. Jos Van Boxtel

    Present address: Department of Pomology, University of California, 1 Shields Drive, Davis, CA, 95616-8683, USA

Authors and Affiliations

  1. Cirad-Amis, UMR PIA1096, Biotrop and Crop Protection Programmes, TA40/03, Av. Agropolis, F-34398, Montpellier Cedex 05, France

    Jean-Christophe Breitler, Donaldo Meynard, Jos Van Boxtel, Monique Royer, Laurence Cambillau & Emmanuel Guiderdoni

  2. Cirad-Cp, TA 80/03, 34398, Montpellier Cedex 5, France

    François Bonnot

Authors
  1. Jean-Christophe Breitler
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  2. Donaldo Meynard
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  3. Jos Van Boxtel
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  4. Monique Royer
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  5. François Bonnot
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  6. Laurence Cambillau
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  7. Emmanuel Guiderdoni
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Correspondence to Jean-Christophe Breitler.

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Breitler, JC., Meynard, D., Van Boxtel, J. et al. A Novel Two T-DNA Binary Vector Allows Efficient Generation of Marker-free Transgenic Plants in Three Elite Cultivars of Rice (Oryza sativa L.). Transgenic Res 13, 271–287 (2004). https://doi.org/10.1023/B:TRAG.0000034626.22918.0a

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  • DOI: https://doi.org/10.1023/B:TRAG.0000034626.22918.0a

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