Study of different antibiotic combinations for use in the elimination of Agrobacterium with kanamycin selection in carnation |
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Authors: | Estopà Montserrat Marfà V Melé E Messeguer J |
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Affiliation: | (1) Departament de Genètica Vegetal, Centre de Cabrils, IRTA, Carretera de Cabrils s/n, Cabrils, 08348 Barcelona, Spain |
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Abstract: | The effect of several β-lactam antibiotics on shoot regeneration, growth and rooting of carnation (Dianthus caryophyllus L.), and their use in combination with kanamycin in Agrobacterium-mediated genetic transformation studies, was determined. Carbenicillin, cefotaxime and ticarcillin increased the regeneration
rate when added alone in non-inoculated explants; whereas, with inoculated explants, this effect was only observed in ticarcillin-containing
medium. Cefotaxime inhibited root growth in both transgenic and non-transgenic shoots. Rooting of non-transgenic shoots was
completely inhibited in all culture media containing kanamycin. The different antibiotics used, alone or in combination, did
not prevent the occurrence of false positive shoots, but it was possible to select transgenic shoots when rooting was induced
in a kanamycin + ticarcillin-containing medium. Regenerated transformed shoots were free of Agrobacterium after culturing in rooting medium, as was proven by the PCR analysis for the nptI gene, the antibiogram and the culture of tissue pieces of transgenic shoots on LB broth. The use of kanamycin and timentin
with or without carbenicillin, was very useful in the transformation procedure, for the elimination of Agrobacterium in regenerated shoots before their transfer to greenhouse conditions and also in the selection of transgenic versus false-positive
shoots.
This revised version was published online in June 2006 with corrections to the Cover Date. |
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Keywords: | carbenicillin cefotaxime Dianthus caryophyllus rooting ticarcillin transgenic plant selection |
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