Selection Of Lily Transformation Varieties With The Blue Genes And A Study Of Genetic Transformation

Recently, the successful commercialization of transgenic violet carnations and roses has attracted people's attention in blue flower colour genetic engineering, and it has been extensively studied by more researchers. Being the same as roses and carnations, lilies lack violet to blue ?ower varieties. This is attributed to their lack of the flavonoid 3',5'-hydoxylase (F3'5'H), which is a key enzyme for delphinidin biosynthesis.It has been widely demonstrated that the main factor controlling the blue flower colour formation is blue genes, while other factors such as the presence of co-pigments and the vacuolar pH also affect flower color. So firstly 10 lilium cultivars whose flower colour ranging from pink to mauve were selected, and their flavonoid composition were analyzed and the pH of their petal juice were measured in order to select hosts of genetic transformation that would be suitable for the exclusive accumulation of delphinidin and the resulting color changes toward blue. Then the efficient acceptor system for genetic transformation of L tenuifolium OT'Robina'was constructed using thin cell layer technology. Finally blue genes such as phalaenopsis F3'5'H gene,petunia DifF gene and hyacinth DFR gene were transformed through Agrobacterium-mediated transformation for the attempt of acquiring blue lilium. Several achievements were obtained as follows:1. Lilium tenuifolium oriental x tumpet'Robina'and Lilium tenuifolium oriental'Bernini'are selected as the suitable hosts of blue genes genetic transformation for their higher vacuolar pH, higher flavonol content and lower cyanidin content.2. The construction of acceptor system for genetic transformation of L tenuifolium OT'Robina'using thin cell layer technology(1)It was found that 2,4-D combined with 6-BA and NAA combined with TDZ in various concentration was not able to induce somatic embryogenesis;(2)The suitable medium for bulblet regeneration from field-derived bulb scale tTCLs is MS+1.0 mg l^-1 NAA+0.1 mg l^-1 TDZ+30 g l^-1sucrose+8 g l^-1 agar;(3)The suitable medium for shoot regeneration from invitro-derived bulb scale tTCLs is MS+0.3 mg l^-16-BA+0.8 mg l^-1 2,4-D+30 g l^-1sucrose+8 g l^-1 agar;(4)The pretreatment of tTCLs with liquid MS basal medium containing 0.8 mg l^-12,4-D in combination with 0.3 mg l^-1 6-BA for 18 h and liquid MS basal medium containing 4.8 mg l^-1 2,4-D alone for 6 h significantly promoted the formation of shoots, and the shoot regeneration rate was 68.61%(30.83% in control cultures)and 80.82% (49.52% in control cultures), respectively;(5)The regenerated shoots were transferred to half-strength MS medium containing 0.5 mg l^-1 IBA and 0.1 % AC, and the rooting rate was up to 100% with an average number of 3.17 roots per shoot after 2 months. The rooted plantlets were successfully transplanted to soil with 74.29% of survival rate.3. Agrobacterium-mediated genetic tranformation of L tenuifolium OT'Robina'(1)Field bulb scale tTCLs were not able to be used as the acceptor for genetic tranfoemation, while in vitro bulb scale tTCLs were the suitable acceptor;(2)Hygromycin concentration for selection of in vitro bulb scale tTCLs was determined to be 20 mg l^-1;(3)Two kinds of plant expression vectors were used for the genetic transformation: 40 resistant-hygromycin shoots were obtained while the objective genes were phalaenopsis F3'5'H gene and petunia DifF gene,60 resistant-hygromycin shoots were obtained while the objective genes were phalaenopsis F3'5'H gene hyacinth DFR gene.