T-DNA as a potential insertional mutagen in rice
2003
Sallaud, C. | Meynard, D. | Brizard, J.P. | Bès, M. | Gay, C. | Raynal, M. | Bourgeois, E. | Hoge, H. | Delseny, M. | Guiderdoni, E.
We investigated the potential of Agrobacterium tumefaciens - mediated transformation for producing a genomewide library of T-DNA rice plants individually characterized by their flanking sequence taf (FST). A japonica rice callus transformation procedure - which relies both on high frequency (75% and 96% in Taipei 309 and Nipponbare, respectively) of cocultured calli-yielding resistant cell lines and independent generation of multiple (2 to 30) resistant cell lines per cocultured callus - was optimized. Potential efficiencies were as high as 5 and 7 independent transformants per cocultured callus in cultivars Taipei 309 and Nipponbare. We further studied the integration of the T-DNA in more than 200 transgenic plants. Around 35% of the T0 plants were found to harbor one copy of the T-DNA in both populations. In Taipei 309, 90% of these plants did not have integrated plasmid backbone sequences and 95% segregated the hygromycin resistance trait according to a 3:1 ratio in their T1 progenies. In multiple-copy plants, 31% of the plants had integrated plasmid backbone sequences and 52% and 10% segregated according to a 3:1 and 15:1 ratio, respectively. Using an efficient polymerase chain reaction walking method, 82 (58%) DNA regions adjacent to the left border of T-DNA inserts (FSTs) have been isolated and sequenced. Results of homology search indicated that eight known genes had been tagged with the T-DNA.
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