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 : 1396-1408.

Résumé : 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.

Mots-clés Agrovoc : Oryza sativa, génie génétique, gène, adn, mutation, mutagène, méthode d'amélioration génétique, transfert de gène

Classification Agris : F30 - Génétique et amélioration des plantes

Auteurs et affiliations

• Sallaud Christophe, CIRAD-AMIS-BIOTROP (FRA)
• Meynard Donaldo, CIRAD-AMIS-BIOTROP (FRA)
• Van Boxtel Jos, CIRAD-AMIS-BIOTROP (FRA)
• Gay Céline, CIRAD-AMIS-BIOTROP (FRA)
• Bes Martine, 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)

Source : Cirad - Agritrop (https://agritrop.cirad.fr/514145/)

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