Transformation Mediated By Agrobacterium Tumefaciens And The Introduction Of Ipt Gene Into Chrysunthemum

Chrysanthemum, one of the traditional ornamental flowers in China, is one of the four cut flowers in the world, making 23% of the total commercial production. New cultivars were traditionally breeded by using bud mutation. It takes long period of time and is poor-oriented. Genetic engineering provides an alternative. It can be used effectively to modify a specific character while shorten the breeding time. Foreign gene(s) have been engineered to obtain novel ornamental cultivars with particular color, fragnance and morphology. After regeneration experiment, two cultivars,cut flower Shanchengzhiguang and Japanese xiaohuang, were selected as target materials for genetic transformation in this paper. SAG-IPT prolong the senescence of leaves.Armed with SAG-IPT, transgenic cut flowers with storage resistant, longer shelf life and longer flower period may be obtained.1. Regeneration system was established with young leaves and shoots as explants.iYoung leaves and shoots were both pre-cultured on MSI (MSO containing l.Omg/L 2,4-D and 0.1mg/L6-BA ) for 3-6days, transferred to MS2 (MSO supplemented with 0.5mg/L6-BA and O.lmg/LNAA) andMSS (MSO supplemented with 3.0mg/L6-BA and O.lmg/LNAA) respectively to induce bud initiation directly. With young leaves, Light of Moutain City and Japanese xiaohuang were regenerated with frequency amounting to 100% while Baixue^ GuanghuK Dongqing and Baixiufang were regenerated with much lower frequency which is hard to meet the needs of genetic transformation. With shoots, Japanese xiaohuang and Light of Moutain City were regenerated with frequency amounting to 92% and 70% respectively while Baixue^ Guanghuk Dongqing and Baixiufang with the same low frequency. Regenerated bus were rooted on 1/2 strength MS medium for 7-10days,and rooting frequency amounted to 100%.2. Transformation system was established with young leaves as target material. Young leaves were excised from sterile seedlings of Light of Moutain City and Japanese xiaohuang, cut into 4x4mm, dipped into Agrobaterium suspention (ODeoo adjusted to 0.5) for 30min. Parted dry on the filter paper, transferred to MSI medium and co-cultured for 3 days in darkness. After co-cultivation , all the explants were washed in liquid MSO medium containing 500mg/L Cef and 500mg/L Carb for 2 hours. Washed with sterile water for 5 times and patted dry on the filter paper again. Subsequently, the explants were transferred to MS2 medium supplemented with 50mg/L Cef for 3 days delay selection and the transferred to MS2 medium supllemented with 50mg/L Cef and 5mg/L Km for selection culture. After 30 days .green buds were rooted on 1/2 strength MSO medium containing lOmg/L Km. Plantlet with normal roots were transferred to field for further analysis. 3. Confirmation of transformant.Integration of SAG-IPT into chrysanthemum genome was confirmed in two thirds of the putative tansgenic plantlet be PCR amplification. Difference of horticultural character between transgenic and wild-type plants was observed.