Studies On Agrobacterium-Mediated Transformation Of Carnation With ACC Oxidase Gene

As one of the major contributors and a commercial leader to the cut-flower market, carnation (Dianthus caryophyllus L.) is an important target for the breeding of new cultivars with novel characteristics. In conventional method of prolonging vase life of carnation cut flower, the substance containing Ag+ was used and this led to pollution of environment. In this study, ACC oxidase (AGO) gene genetic DNA was cloned from carnation cultivar 'America' genome. Four T-DNA structures of plant expression vectors of AGO gene were constructed and integrated into carnation cultivars 'Master', 'Mabel', 'Yellow Star', 'Opera' and 'Aicardi'. The vase lifeof transgenic cut flower was prolonged.1 Cloning of ACC oxidase gene of carnation and four constructions of its plant expression vectorsA pair of primers was designed according to the 1-aminocyclopropone-l-carboxylic acid (ACC) oxidase gene sequence of carnation, and the primers were used to amplify the genomic DNA fragment of 1.2 kb by polymerase chain reaction (PCR) by taking genome DNA from carnation cutivar 'America' leaves as template. The PCR product was cloned into pMD18-T vector. Sequencing indicated that the cloned ACC oxidase gene consisted of 3 exons interrupted by 2 introns. Totally four T-DNA structures of plant expression binary vectors were constructed. Those were sense, antisense, direct repeated of sense and antisense.2 Establishment of efficient regeneration system from leaf of 5 carnation cultivarsThe regeneration system of carnation was established with the method of direct adventitious shoots inducing. The adventitious shoots were induced from leaf explants based on MS basal medium supplemented with 1 .0mg/L BA and 0.3mg/L NAA and its differentiation frequencies were between 38.6% and 61.8%. The age of leaf affects its differentiation frequency and AgNO3 in adventitious medium inhibits adventitious shoot inducing. The regenerated plants were rooted on MS medium containing 0.1mg/L NAA.3 Genetic transformation of carnationTaken the young leaf as explants, the Agrobacterium rwme/ac/ens-mediated genetic transformation system of carnation was established. A. tumefaciens strain LBA4404 harboring pMOGMON containing four T-DNA structures of ACO gene were used totransform 5 carnation cultivars. Leaf explants were pre-cultured on shoot-inducing medium for 2 days, then immersed in Agrobacteriwn suspension for 8-12 min. The co-cultivation was carried out on medium containing 1 00 mol/L acetosyringone (AS) for3 days. After that the transformants were obtained by transferring explants to selection medium supplemented with 4-5 mg/L hygromycin (Hyg) and 400mg/L cefotaxime (Cef). Some factors that affect the transformation frequency were compared. The regeneration frequencies on selection medium were between 4.3% and 11.2%.4 Identification of transgenic plantsIn four methods of DNA extraction, the modified SDS method was the best. 48.3 percent transformed plants rooted on rooting medium containing 4mg/L Hyg. And 24.5 percent of the PCR amplification of HPT gene was positive. Southern blot analysis demonstrated that 33 transformed plants, which was 14.2 percent of resistant rooting plants, were positive, and foreign gene were integrated into plant genome. 3 of the 33 transformed plants have two copies of foreign gene and the rest of transformed plants have single copy of foreign gene.5 Comparison of vase life and ethylene production of transgenic plant cut flowers with different T-DNA structuresVase life and ethylene production of 17 transgenic lines of 3 cultivars were analyzed. Vase life and ethylene production were different in transgenic lines with different T-DNA structures.Vase life of 5 direct repeat transgenic lines of 'Master' cultivar was all prolonged by about 6 days, and antisense direct repeat transgene produced only a negligible amount of ethylene during cut flower natural senescence. 3 lines vase life of 7 single transgenic lines of 'Master' cultivar did not change compared with control. This suggested that direct repeat transgene ind...