%0 Journal Article %J TAG. Theoretical and Applied Genetics. Theoretische Und Angewandte Genetik %D 2003 %T Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics %A Christophe Sallaud %A Donaldo Meynard %A Boxtel,J. van %A Gay,C. %A Martine Bès %A Brizard,J. P. %A Larmande, Pierre %A Ortega,D. %A Raynal,M. %A Portefaix,M. %A Ouwerkerk,P. B F %A Rueb,S. %A Delseny,M. %A Guiderdoni, Emmanuel %K Caulimovirus %K DNA, Bacterial %K Genome, Plant %K Genomics %K Glucuronidase %K Green Fluorescent Proteins %K Luminescent Proteins %K Oryza sativa %K Promoter Regions, Genetic %K Rhizobium radiobacter %K Transformation, Genetic %N 8 %P 1396 - 408 %U http://www.ncbi.nlm.nih.gov/pubmed/12677401 %V 106 %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 beta-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 co-cultivated 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 T(0) plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T(1) 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. %8 2003/05// %@ 0040-5752