Establishment Of Genetic Transformation System For Lily And Transformation With Antisense ACC Synthase Gene

Delaying postharvest senescence of cut flowers, it has become one of the main topics for twenty years' study. Many resarches have showed that controlling the biosynthesis of ethylene is the effective method of extending cut flower longevity.By using Lilium 'Siberia', Lilium 'Sorbonne', and Lilium Longiflorum as the materials, plant regeneration and the sensibility to several antibiotics were conducted. GUS gene and anti-ACC synthase gene were introduced into Lilium 'Siberia' with Agrobacterium tumefaciens-mediated genetic transformation. All of these results indicated that efficient and stable Agrobacterium-mediated genetic transformation system of lily had been established.Among three varieties of lily, the results showed that regeneration efficiency varied depending on genotype. The regeneration ability of Lilium 'Siberia' was the highest, then Lilium 'Sorbonne' and Lilium Longiflorum. 0.2,0.5mg/L 6-BA and 0.5～2.0mg/L 2, 4-D or 0.1, 0.2mg/L NAA combination could induce varieties regeration with high frequency. Callus and squama of lily were sensitive to antibiotics. 50～100mg/L kanamycin or 7.5～10mg/L hygromycin concentration can restrain regeneration of material.Agrobacterium tumefaciens-mediated genetic transformation protocol was developed for Lilium 'Siberia'. The results showed that calli explants were more sensitive to Agrobacterium infection, induction with AS greatly improved the transformation effects. The infection effect of Agrobacterium strain LBA4404 was better than GV3101. In addition, preculture days, concentration of Agrobacterium, infection time, as well as the time of co-cultivation had effects on the transformation efficiency. The optimum concentration of Agrobacterium using affection is about OD₆₀₀=0.4, infection time could be 15 min, co-culture period was 2～3 days, the highest transformation frequency could be obtained under this condition. Resistant plants could be obtained after transformtion and selective regeneration. GUS staining, PCR analysis confirmed that foreign gene had been integrated into the Lilium 'Siberia' genome with transformation frequency of 5% - 8%.Anti-ACC synthase gene had been transferred into Lilium 'Siberia'. Eventurally nine kanamycin-resistant culture lines were obtained. Stable transformation was verified via PCR analysis. Two of the culture lines were PCR-positive. PCR analysis preliminarily indicated...