Establishment Of High Frequency Regeneration System And Transformation Of Eustoma Grandiflorum With Chitinase Gene

In the research, a lisianthus (Eustoma grandiflorum) cultivar "Double Mariachi Pink"was used. The research included establishment of regeneration system of lisianthus,construction of chitinase gene in plant expression vector and transformation of lisianthus with chitinase gene. The result of Km selection and PCR analysis showed the chitinase gene was transformed into "Double Mariachi Pink" .First, leaves of lisianthus were used to research the influence of different modes of incision and combinations of 6-BA((0,0.1,0.5 and 1.0mg/L) and NAA((0,0.01,0.05 and 0.1mg/L) to adventitious bud formation, and to test the sensitivity of adventitious bud formation with Kanamycin. The result showed that the leaves which had 4 incisions were not prominently different from those which had 2 incisions in the number of adventitious buds.The optimal buds differentiation medium was MS+6-BA1.0mg/L, in which the frequency of differentiation was 100%, and the number of adventitious buds was 25.4 in average. Adventitious buds proliferated on MS+6-BA 0.1mg/L+ NAA0.05mg/L.Plantlets rooted on 1/2MS +NAA0.1mg/L. High frequency regeneration system of "Double Mariachi Pink" was Established .And the lowest level of Kanamycin which could restrain the formation of adventitious buds was 25mg/L.Second,a pair of differential primers(P₁: 5'-tac tct aga atg aag aag aat agg atg atg 3'; P₂:5'cgc gaa tcc tca ctg aga ggt gac aag aag-3') was designed according to the sequence of P. vulgaris type I chitinase gene (M13968). The chitinase gene was separated and cloned into plant expression vector pBI121 and named pBI121-Chi. The pBI121-Chi was confirmed by restriction enzyme and PCR analysis. The results showed that the recombinant plasmid was constructed successfully. pBI121-Chi was transformed into Agrobacterium tumefacien LBA4404.At last, leaves were co-cultured with LBA4404 which contained pBI121-Chi to transform chitinase gene into the lisianthus. Some adventitious buds regenerated from leaves in medium with Kanamycin. Those were subcultured to select the transgenic plantlets in medium with Kanamycin.20 lines could grow in medium with Kanamycin. a pair of differential primers(P₃: 5'- aca gaa ctc gcc gta aag-3'; P4: 5'- atg aag gca tcg tag gtg tag aag c-3') was designed according to the sequence of chitinase gene(M13968) and pBI121(AF485783).A differential band was obtained by PCR method using P₃ and P₄ in 11 lines. The chitinase gene was transformed into"Double Mariachi Pink".In the research, the effect of different levels of GA₃(0,25,200 and 500 mg/L) to lisianthus plantlets was tested. The result showed that GA3 could promote the growth of lisianthus and prevent from fasciation...