Study of different antibiotic combinations for use in the elimination of Agrobacterium with kanamycin selection in carnation

The effect of several beta -lactam antibiotics on shoot regeneration, growt h and rooting of carnation (Dianthus caryophyllus L.), and their use in com bination with kanamycin in Agrobacterium-mediated genetic transformation st udies, 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-con taining medium. Cefotaxime inhibited root growth in both transgenic and non -transgenic shoots. Rooting of non-transgenic shoots was completely inhibit ed in all culture media containing kanamycin. The different antibiotics use d, alone or in combination, did not prevent the occurrence of false positiv e shoots, but it was possible to select transgenic shoots when rooting was induced in a kanamycin + ticarcillin-containing medium. Regenerated transfo rmed shoots were free of Agrobacterium after culturing in rooting medium, a s 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 kana mycin and timentin with or without carbenicillin, was very useful in the tr ansformation procedure, for the elimination of Agrobacterium in regenerated shoots before their transfer to greenhouse conditions and also in the sele ction of transgenic versus false-positive shoots.