@article {367,
	title = {Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics},
	journal = {TAG. Theoretical and Applied Genetics. Theoretische Und Angewandte Genetik},
	volume = {106},
	year = {2003},
	month = {2003/05//},
	pages = {1396 - 408},
	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 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.},
	keywords = {Caulimovirus, DNA, Bacterial, Genome, Plant, Genomics, Glucuronidase, Green Fluorescent Proteins, Luminescent Proteins, Oryza sativa, Promoter Regions, Genetic, Rhizobium radiobacter, Transformation, Genetic},
	isbn = {0040-5752},
	url = {http://www.ncbi.nlm.nih.gov/pubmed/12677401},
	author = {Christophe Sallaud and Donaldo Meynard and Boxtel,J. van and Gay,C. and Martine B{\`e}s and Brizard,J. P. and Larmande, Pierre and Ortega,D. and Raynal,M. and Portefaix,M. and Ouwerkerk,P. B F and Rueb,S. and Delseny,M. and Guiderdoni, Emmanuel}
}
