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Analysis of genetically transformed coffee plants (Coffea canephora Pierre) for resistance to coffee leaf miner: bioassays, molecular and immunological analyses Proceedings

by Dufour, M; Philippe, R; Fenouillet, C; Carasco Lacombe, C; Gruchy, D; Jourdan, I; Leroy, T; Association Scientifique Internationale du Cafe, París (Francia); 19. International Scientific Colloquium on Coffee Trieste (Italia) 14-18 May 2001.
Publisher: Trieste (Italia) ASIC 2001Description: 1 disco compacto.ISBN: 290012189.Subject(s): COFFEA | COFFEA CANEPHORA | LEUCOPTERA | INSECTOS DAÑINOS | COMPOSICION QUIMICA | CAFE | EXPRESION GENICA | GENES | INSECTOS DAÑINOS | GENETICA MOLECULAR | RESISTENCIA A LAS PLAGAS | ENFERMEDADES DE LAS PLANTAS | CONTENIDO PROTEICO | ADN | PLANTAS TRANSGENICAS | COFFEA | COFFEA CANEPHORA | LEUCOPTERA | CHEMICAL COMPOSITION | COFFEE | GENE EXPRESSION | GENES | MOLECULAR GENETICS | PEST RESISTANCE | PLANT DISEASES | PROTEIN CONTENT | DNA | TRANSGENIC PLANTS | COFFEA | COFFEA CANEPHORA | LEUCOPTERA | COMPOSITION CHIMIQUE | CAFE | EXPRESSION DES GENES | GENE | GENETIQUE MOLECULAIRE | RESISTANCE AUX ORGANISMES NUISIBLES | MALADIE DES PLANTES | TENEUR EN PROTEINES | ADN | PLANTE TRANSGENIQUESummary: Sixty transgenic coffee plants (C. canephora) were obtained with genetic constructs containing a Bacillus thuringiensis gene efficient against Lepidopterae (cryIAc). Their molecular analyses (Southern blots and PCR) have been performed including analysis of T-DNA integration quality, and estimation of copy number. These plants were also submitted to bioassays and to studies of toxin occurrences by immunoassays (Western blotting). For the bioassays, coffee plants were put in the presence of adult Tanzania leaf miners (Leucoptera caffeina) for 24 hours and checked 2 weeks later. In order to perform biochemical analysis, proteins were extracted from the same plants, concentrated, and checked for protein content. After migration and transfer to nitrocellulose membranes, Cry1A(c) protein was identified using a polyclonal rabbit antiserum. Most of the plants harbour single copy integration of complete T-DNA. Transgene expression varies among bioassays and this variation is not always detected through the Western blots. Hypothesis about the kind of antibodies for future experiments will be discussed, as well as the integration of new genetic constructs, harbouring tissue-specific promoters.
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Sixty transgenic coffee plants (C. canephora) were obtained with genetic constructs containing a Bacillus thuringiensis gene efficient against Lepidopterae (cryIAc). Their molecular analyses (Southern blots and PCR) have been performed including analysis of T-DNA integration quality, and estimation of copy number. These plants were also submitted to bioassays and to studies of toxin occurrences by immunoassays (Western blotting). For the bioassays, coffee plants were put in the presence of adult Tanzania leaf miners (Leucoptera caffeina) for 24 hours and checked 2 weeks later. In order to perform biochemical analysis, proteins were extracted from the same plants, concentrated, and checked for protein content. After migration and transfer to nitrocellulose membranes, Cry1A(c) protein was identified using a polyclonal rabbit antiserum. Most of the plants harbour single copy integration of complete T-DNA. Transgene expression varies among bioassays and this variation is not always detected through the Western blots. Hypothesis about the kind of antibodies for future experiments will be discussed, as well as the integration of new genetic constructs, harbouring tissue-specific promoters.

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