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Generation of enhancer trap lines of rice (Oryza sativa L.) expressing GAL4 and GFP in specific cell types [W04-6]

Johnson A., Tester M., Hibberd J., Gay C., Sallaud C., Guiderdoni E.. 2003. In : 7th International Congress of Plant Molecular Biology, ISPMB 2003, Barcelona (Spain), June 23-28. Book of abstracts. Barcelone : ISPMB Office, p. 466-466. International Congress of Plant Molecular Biology. 7, 2003-06-23/2003-06-28, Barcelone (Espagne).

We are generating a set of 10,000 enhancer trap lines of rice (Oryza sativa L., cv. Nipponbare) expressing an exogenous transcription factor (gal4) and green fluorescent protein (gfp) in specific cells or tissues. In addition to gene discovery resulting from visualisation of GFP activity, this system will provide for the first time a powerful tool for the targeted expression of transgenes in an important monocotyledonous crop. An enhancer trap cassette containing gal4, as well as gfp placed downstream of the upstream activation sequence (UAS) for GAL4, was mobilized into two supervirulent strains of Agrobacterium tumefaciens and embryogenic calli were transformed by co-cultivation with the bacteria. Screening at the callus stage using a UV light stereomicroscope indicated GFP activity in 22.9% of developing, hygromycin-resistant cell lines (n=11,645). GFP activity was also detected in vegetative and floral organs (10.6% and 5.0%, respectively) of adult primary transformants (n=2,082) with specific organs and cell types marked. In flowers, for example, GFP expression specific to lodicules, lemma and palea, trichomes, stomata, ovary, filaments and pollen were detected. Continued screening for GFP activity in shoots and roots of T1 seedlings confirmed the stable transmission of shoot expression patterns from the T0 to T1 generation. as well as enabled screening of the root for novel expression patterns. Confocal microscopy has been used to characterise the GFP-expressing cell types in selected enhancer trap lines, particulary those that may be used for future targeted expression of transgenes. To date, more than 8,000 primary transformants harbouring this enhancer trap cassette have been generated and a database documenting the expression patterns is being prepared. This work is funded by Génoplante, a national genomics initiative based in France, and by the Biotechnology and Biological Sciences Research Council (UK). (Texte intégral)

Mots-clés : oryza sativa; génie génétique

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