Studies On Agrobacterium-Mediated Transformation Of Lilium Asiatic With ACO IhpRNAi Vector

As one of the major contributors and a commercial leader to cut-flower market, lily is widely favored by people for the characters of the big flower, varied flower color, graceful posture, and strong fragrance. With the limit of common hybridization and negative effection of chemical on fresh cut flowers, biotechlonogy development provided new concepts, new methods and new tools to overcome the shortcomings of traditional breeding and the use of the preservative chemical toxicity.In this study, we established the acceptor system of Agrobacterium-mediated transformation of lilium elite. Then by using the bulb leaf and scale as the gene transformation acceptor, the foreign gene ACO which cloned from lily was built as a dsRNA-mediated gene silencing vector and transformed by Agrobacterium EHA105. The purpose of this study is to achieve transgenic lily of ACC Oxidase RNAi gene and provide some beneficial foundation in lily in the future. The results were as follows:1. Establishment of efficient scale regeneration system from Asiatic hybrid lily"elite"The adventitious shoots(62.5%) were induced from scales explants based on MS basal medium supplemented with 2.0 mg·L-1 BA and 0.2 mg·L-1 NAA.2. Establishment of High-efficiency acceptor system of direct differentiation for the gene transformation of lilium"Elite"Taken the young leaf and scale as explants, the Agrobacterium tumefaciens-mediated genetic transformation system of lily was established. The medium suitable for the leaves of the test-tube plantlets to differentiate adventitious bud was MS+TDZ 1.0 mg·L-1+NAA 0.2 mg·L-1; The medium suitable for the scales leaves of the test-tube plantlets to differentiate bud was MS+6-BA 2.0 mg·L-1+2,4-D 0.1 mg·L-1; The concentrations of kanamycin were 80 mg·L-1 for the leaf and 115 mg·L-1 for the scales.3. Genetic transformation of Asiatic hybrid lilyAgrobacterium tumefaciens strain EHA105 was used. The ACC Oxidase (ACO) gene, which cloned from lily, was built as dsRNA-mediated gene silencing vector and transformed by Agrobacterium EHA105. Leaf and scale explants were pre-cultured on shoot-inducing medium for 3 days, then immersed in Agrobacterium suspension for 8-15 min. The co-cultivation was carried out on medium containing 25 mg·L-1 acetosyringone (AS) for medium supplemented with 80 mg·L-1or 115 mg·L-1 kanamycin and 300 mg·L-1 carbimycin (Carb). Some factors that affect the transformation frequency were compared. The regeneration frequencies on selection medium were 90.2% and 100% respectively.4. Identification of transgenic plantsIn this study, 200 kanamycin resistant plantlets were obtained, 50kanamycin resistant plantlets were at random selected for PCR and got the 7 plants strip. It proved that foreign gene had been integrated into these 7 plants genome.