Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics

Sallaud Christophe, Meynard Donaldo, Van Boxtel Jos, Gay Céline, Bes Martine, Brizard Jean-Paul, Larmande Pierre, Ortega D., Raynal Monique, Portefaix Murielle, Ouwerkerk Pieter B.F., Rueb S., Delseny Michel, Guiderdoni Emmanuel. 2003. Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theoretical and Applied Genetics, 106 : pp. 1396-1408.

Journal article ; Article de revue à facteur d'impact
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Abstract : We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice (Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance (hpt), green fluorescent protein (gfp) and [bêta]-glucuronidase (gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75-95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per cocultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30-40% of the T0 plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T1 progenies showed that 30-50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed. (Résumé d'auteur)

Mots-clés Agrovoc : Oryza sativa, Génie génétique, Gène, ADN, Mutation, Mutagène, Méthode d'amélioration génétique, Transfert de gène

Classification Agris : F30 - Plant genetics and breeding

Auteurs et affiliations

  • Sallaud Christophe, CIRAD-AMIS-BIOTROP (FRA)
  • Meynard Donaldo, CIRAD-AMIS-BIOTROP (FRA)
  • Van Boxtel Jos, CIRAD-AMIS-BIOTROP (FRA)
  • Brizard Jean-Paul, IRD (FRA)
  • Larmande Pierre, CIRAD-AMIS-BIOTROP (FRA)
  • Ortega D., CNRS (FRA)
  • Raynal Monique, CNRS (FRA)
  • Portefaix Murielle, INRA (FRA)
  • Ouwerkerk Pieter B.F., Universiteit Leiden (NLD)
  • Rueb S., Universiteit Leiden (NLD)
  • Delseny Michel, CNRS (FRA)
  • Guiderdoni Emmanuel, CIRAD-AMIS-BIOTROP (FRA)

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