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

Sallaud C., Meynard D., Van Boxtel J., Gay C., Bes M., Brizard J.P., Larmande P., Ortega D., Raynal M., Portefaix M., Ouwerkerk P.B.F., Rueb S., Delseny M., Guiderdoni E.. 2003. Theoretical and Applied Genetics, 106 : p. 1396-1408.

DOI: 10.1007/s00122-002-1184-x

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 : oryza sativa; génie génétique; gène; adn; mutation; mutagène; méthode d'amélioration génétique; transfert de gène

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