Introduction Of Ipt Gene Into Petunia Mediated By Agrobacterium Tumefaciens

The quality of many ornamentals is influenced by both flower and leaf senescence and the cytokinins have been shown to delay leaf senescence. We have transformed petunia discs mediated by Agrobacterium tumefaciens , which contain the isopentenyl transferase gene(ipt) with a senescence-specific promoter(sag12). The ipt(isopentenyl transferase) is the rate limiting enzyme in cytokinin biosynthesis. We hope the over-expressing ipt would increase the cytokinin and delay leaf senescence.1. Establishment of regeneration systemThe sterile young leaves of petunia were cultured on MS1(MSO containing 2.5mg/L ZT) to induce bud initiation directly. The regenerating frequency amounted 100%. Regenerated buds were rooted on 1/2 strength MS medium and the rooting frequency amounted to 100%.2. Choice of selection agent concentrationThe leaves of petunia sterile seedlings were cultured on MSI (including different concentration of Km). The result showed that the callus could not be induced when the concentration of Km was 50mg/L.3. Establishment of transformation systemYoung leaves were excised from sterile seedlings of petunia, cut into 5 X 5mm2 , dipped into Agrobaterium (OD600 adjusted to 0.2) for 15min. Patted dry on the filter paper, transferred to MSI medium and co-cultured for 3~4 days in darkness. After co-cultivation, all the explants were washed in liquid MSO medium containing 500mg/L Cef for 2 hours. Washed with sterile water for 5 times and patted dry on the filter paper again. Subsequently,the explants were transferred to MSI medium supplemented with 200mg/L Cef for 3~6 days delay selection and then transferred to MS 1 medium supplemented with 200mg/L Cef and 50mg/L Km for selection culture. The resistant shoots were rooted on 1/2 strength medium containing 10mg/L Km and then transferred the plantlets with normal roots to field for further analysis.4. The PCR identification of transformed plantsThe DNA of Agrobaterium plasmid and transformed plants growed on selected medium were distilled and were amplified by PCR. The result of electrophoresis was that eight of twenty-six transfotmed plants had the same line as plasmid. It showed that the target gene had inserted in the geonome of plants.5. Phenotypes of transgenic petuniaTransgenic plants were shorter, reduced leaf width, increased lateral branching, had an underdeveloped root system and slower growing. Some resistant shoots had much axillary buds and couldn't root. In later growing, transgenic plants have been shown to have delayed leaf and flower senescence.