Transfer Bt Cry1Iα Gene Into Cauliflower (Brassica Oleracea Var.Botrytis) By Agrobacterium-Mediated Transformation

Cauliflower (Brassica oleracea Var.botrytis), one of the most important vegeTab. crops, is planted all over the world. Nowadays, it is also a main vegeTab. in China. But insect-pests have caused severe damage. Among them green worm (Pieris rapae L.) and diamondback moth (Plutella xylostella L.) are the primary pests. In China,diamondback moth is one of the most destructive pests and its damage has risen rapidly especially for cruciferous vegeTab.s. Because the pests are easy to induce resistance to insecticide, it is the best way to obtain the cultivars of insect-resistance in pest control.Bt cry1Ia gene has been cloned from Bacillus thuringiensis strains Btc008 and has no homology with cry1A-type which is commonly used in pest control. Cry1Ia protein have relatively broad insecticidal controlling spectrum, strong insecticidal activity for corn borer, diamondback moth. Insecticidal activity of Cry1Ia protein is similar to Cry1Ab and Cry1AC, but didn't have cross-resistance with both of them.The cotyledon petioles and hypocotyls of cauliflower(Brassica oleracea Var.botrytis))were transformed with Agrobacterium tumefaciens harboring CaMV35S-1Ia-NOS expression vector containing CaMV35S promoter,nptâ…¡marker gene and the insecticidal protein gene Bt cry1Ia gene. In this study the effects of influencing factors on genetic transformation frequency of cauliflower were investigated. The results showed: Both the hypocotyls and cotyledon of RuiXue were perfect explants for transformation; The hormone was an important factor on the regeneration of adventitious buds, we could obtain high regeneration frequency by supplementing 6-BA 2 mg/L and NAA 0.05 mg/L on the MS medium; Adding 0.lmg/L IAA to MS medium had good effect on rooting period and root quality of regenerate shoots; Infectious activity of Agrobacterium tumefaciens EHA105 was superior to LBA4404; Acetosyringone could promote the regeneration of adventitious buds in the transformation; It was no obvious difference to add silver nitrate on transformation frequency of cauliflower; The result showed that 10mg/L kanamycin would completely inhibit green shoot regeneration from explants of cauliflower, so it was available to selecting transgenic plants; The carbenicillin concentration was decided to use with 300mg/L; The experiment detected that the optimized infection time was 8 min for cotyledon and 15 min for hypocotyls.Through successive selection in the period of shoot and root induction under kanamycin selecting pressure,42 kanamycin resistant regenerated plants were obtained,18 of them were positive resulted by PCR detection which demostrated that cry1Ia gene was integrated into the genome. Moreover, RT-PCR and Western blot analysis demonstrated that cry1Ia gene was expressed in RNA and protein levels in 14 plants. The transformation plants were given insect feeding test with larvae of diamondback moth.The results showed the mortality of insect larvae was accounted for 99.6%,and the growth of the survival larvae were seriously inhibited.