| As ethylene plays a main role in growth, development and senescence of plants, it becomeresearch focus in order to prevent fruit setting and prolong vase life of cut flowers. Inconventional method of preservation the ethylene inhibitor and antagonist was used and thisled to pollution of environment. Biotechnology is a good way to improve metabolize andstorable characteristic of cut flowers. In this study, ACC oxidase (1-aminocyclopropone-1-carboxylic acid oxidase, ACO ) gene fragments DNA was cloned from carnation(Dianthus caryophyllus L.) cultivars 'Master','Tasman' and 'Isabelle'. The hpRNA structureof plant expression vector of ACO gene fragment was constructed and integrated intocarnation(Dianthus caryophyllus L.) cultivars in hope of prolong vase life of carnation cutflowers. In addition, optimal concentrations of 6-BA and NAA and their combinations weretested with quadratic regressive orthogonal design for their effect on shoot regeneration ofcarnation to use in integration.1. Cloning of ACO gene fragments of carnation and construct its RNAi expression vectorA pair of primers was designed according to ACO gene sequence of carnation, and theprimers were used to amplify the genomic DNA fragment by polymerase chain reaction (PCR)by taking genome DNA from carnation cutivar 'Master', 'Tasman' and 'Isabelle' leaves astemplate. It was found that the fragment was similar to the ACO gene with 98.9% homology. Itwas used as template to amplify a smaller fragment (536bp). These two fragments were ligatedin opposite directions and inserted into plant expression vector pCAMBIA1301 to producehpRNAi vector pCAM-ACOi. PCR and restriction enzyme analysis confirmed correctness ofthe vector construction.2. Es Tab.lishment of regeneration system of carnationOptimal concentrations of 6-BA and NAA were tested with quadratic regressiveorthogonal design for their effect on shoot regeneration of Dianthus Caryophyllus L.. Itshowed that the best combination of 6-BA and NAA for the shoot regeneration was 6-BA1.21mg·L-1 and NAA 0.35mg·L-1, and the propagation coefficient reached 3.65. When 6-BAconcentration was in a range of 1.20～1.79mg·L-1 and NAA concentration in 0.15～0.63mg·L-1,the propagation coefficient more then 2 with the probability of 95%.To testify the reliability ofresult 'Master', 'Tasman' and 'Isabelle' was cultivated in the MS media containing 6-BA1.2mg·L-1 and NAA 0.3mg·L-1. The propagation coefficient reached 3.75, 3.57, and 3.26 respectively.3. Genetic transformation of carnationTaken the young leaf as explants, the Agrobacterium tumefaciens-mediated genetictransformation system of carnation was esTab.lished. Agrobacterium strain GV3101 harboringpCAMBIA1301 which contained double ACO fragments was used to transform 3 carnationcultivars. Carnation was sensitive to hyg (hygromycin).The concentrations of hyg for leaveregeneration was 4～5 mg·L-1. The bacteria-free seedling was cultivated on MS media withoutany plant hormone for 30 days. Put leaf explants on shoot-inducing media 2 days forpre-culturing, then immersed into Agrobacterium suspension (OD600=0.6～0.8) for 8～12 min.The co-cultivation was carried out on medium containing 100μmol·L-1 AS (acetosyringone) for3 days. After that the transformants were obtained by transferring explants to selectionmedium.27 hyg resistant transformants were selected.
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