A novel two T-DNA binary vector allows efficient generation of marker-free transgenic plants in three elite cultivars of rice (Oryza sativa L.)

Breitler Jean-Christophe, Meynard Donaldo, Van Boxtel Jos, Royer Monique, Bonnot François, Cambillau Laurence, Guiderdoni Emmanuel. 2004. 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 Research, 13 (3) : 271-287.

Résumé : 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.

Mots-clés Agrovoc : Oryza sativa, plante transgénique, gène, expression des gènes, marqueur génétique, adn, transformation génétique

Classification Agris : F30 - Génétique et amélioration des plantes

Auteurs et affiliations

• Breitler Jean-Christophe, CIRAD-AMIS-BIOTROP (FRA)
• Meynard Donaldo, CIRAD-AMIS-BIOTROP (FRA)
• Van Boxtel Jos, CIRAD-AMIS-UMR PIA (FRA)
• Royer Monique, CIRAD-AMIS-PROTECTION DES CULTURES (FRA)
• Bonnot François, CIRAD-CP-DIR (FRA)
• Cambillau Laurence, CIRAD-AMIS-UMR PIA (FRA)
• Guiderdoni Emmanuel, CIRAD-AMIS-BIOTROP (FRA)

Source : Cirad - Agritrop (https://agritrop.cirad.fr/521633/)

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