A study one isopentenyl transferase gene by using Agrobacterium tumefaciens to increase the longevity of carnation (Dianthus caryophyllus L.)
Keywords:Agrobacterium tumefaciens, carnation, hpt, gusA, ipt, GUS, mediated transformation, PCR, promoter SAG12, TDZ
In vitro leaves of carnation (cut-off flower) variety CCF cut into small pieces about 1x1cm in size were precultured for 5 days and were co-cultivated with the Agrobacterium tumefaciens strain LBA 4404 harbouring the plasmid pVDH1396 contained the β-glucuronida gene (gusA), hygromycin phosphotransferase gene (hpt) under the control of cauliflower Mosaic virus 35S promoters, terminators, and an isopentenyl transferase (ipt) gene from Agrobacterium tumefaciens, driven by the SAG12 promoter from senescence associated gene 12 of Arabidopsis thaliana and its terminator. After 5 days of co-cultivation, explants were transferred onto the MS selection medium containing 1.0mg/l TDZ and 0.1mg/l NAA, 5mg/l hygromycin, 500 mg/l cefotaxime. Some putative shoots were tested for GUS assay. Putative shoots were transferred on MS medium 3 containing 0.5 mg/l IBA and 10 mg/l hygromycin. PCR analyses on hygromycin resistant shoots generated via Agrobacterium tumefaciens transformation to confirm the presence of the, ipt, hpt and gusA genes with expected bands 615bp, 508bp and 365bp. Analysis of putative carnation transformants to confirm that the foreign genes are inserted into the carnation genome by Southern blot analyses will be done in the future.