Studies On The Introduction Of Antisense ACS Gene And PEAS Gene To Banana Mediated By Argrobacterium Tumefaciens

In this experiment, a high-efficiency and systematic transgenic procedure mediated by Agrobacterium was developed in Musa spp. cv. Tianbaojiao (AAA group) . The plant transgenic system of thin-cross-section culture was optimized, which was further used for introducing antisense ACS gene to banana mediated by Agrobacterium tumefaciens, and selecting resistant buds and plant regeneration; the transgenic assays including transient expression of GUS, steadily expression of GUS and PCR assay of gus of the leaves of transgenic plants were conducted; and the subsequent studies on the introduction PEAS gene to banana were also carried out. The main result were described as follows:1.The plant regeneration system of thin-cross-section culture was optimizated by adjusting the factors such as explants, growth regulator types and concentrations, and AgNO₃ during bud induction from thin cross-sections of banana. The high rate of the bud induction was obtianed when the corm apical meristem was used as the explant and was placed directly on MS medium without plant growth regulators and the medium supplemented with 2 mg . L^-1 AgNO₃ was helpful for bud differentiation. It was good for transgenics to use the thin-cross-sections as the explants from the corm apical meristem of banana plantets which had been cultured on MS medium supplemented with 1.0 mg · L^-1 BA,0.1 mg · L^-1NAA 80mg · L^-1 Ad for 20 d . The thin cross-sections of banana were sensitive to kanamycin, and the concerntration of 100 mg· L^-1 for kanamycin was proved to be suitable for thin cross-sections of banana transgenic selection through further experiments.2.The introduction of antisense ACS gene to banana was conducted, and the optimized parameters used in Agrobacterium-mediatted transformation were obtained. The best transient expression of GUS was obtained under the conditions as follows: the thin cross-sections which were not precultured but were pretreated with 0.1 mol · L^-1 mannitol for 4 h; and the Agrobacterium suspension was with OD₆₀₀ value of 1.0 and supplemented with 100 g · L^-1 sucrose; the samples were infected for 10-15 mins, and then they were transferred onto the non-selective medium (pH 5.8) supplemented with 0.1 mg · L^-1 NAA and 1.0 mg · L^-1 BA for co-culture for 4 days at 26 ℃ in the dark.3. A whole technical system of banana resistant buds,transgenic plant regeneration and transgenic assay was established. After co-culture,the thin cross-sections were selected on MS medium supplemented with 100 mg · L^-1 kanamycin and 2 mg · L^-1 AgNO₃ ,which could be used to inhibit Agrobacterium contamination and be good for bud differentiation. At last,5 resistant bud lines were maintained for further studies. The PCR assays proved that the gus gene had integrated into the genome of the 5 resistant bud lines. About 30 transgenic plantlets regenerated. Among them plantlets regenerated...