Studies On The Transformation Of Antisense ACS Gene Mediated By Agrobacterium Tumefaciens In Oncidium

In this experiment, a high-efficiency and systematic transgenic procedure mediated by Agrobacterium tumefaciens with antisense ACS gene was developed from protocorm-like bodies (PLBs) in Oncidium. The main results were described as follows:1. Establishment of the plant regeneration system of OncidiumThe studies on the efficiently transgenic establishment of the regeneration from leaves and roots were carried out. The results showed that the rate of PLBs induced from leaves cultured on the 1/2 MS medium supplemented with 0.2 mg/L 6-BA and 1.0 mg/L NAA was 72.6%, which was higher than the roots cultured on the same medium. Therefore, the leaf was suitable as the material for inducing the PLBs. The proliferation coefficient was high when the PLBs cultured on the MS medium supplemented with 2.5 mg/L 6-BA and 0.5 mg/L NAA or 2.0 mg/L 6-BA and 0.5 mg/L NAA, and the 1/2 MS medium with 0.8 mg/L 6-BA and 0.2 mg/L NAA was suitable for the the PLBs differentiation. The 1/2 MS medium supplemented with 0.5 mg/L NAA and 100 g/L apple juice was suitable for the test tube plantlet growth and rooting. The aquatic weed was the best transplantation substrate for cultivation of Oncidium.2. Establishment and optimization of the acceptor system of protocorm-like bodiesIn the research of genetic transformation in Oncidium, the PLBs should be tiny and granular when taken as the acceptor, because it was difficult to obtain non-chimaeric transgenic plantlets if the larger PLBs were used as the acceptor. The acceptor system was established and optimized by adjusting factors such as different sorts and concentrations of plant growth regulators, the basal medium, the pH value, the type and concentration of sugars, light conditions, antibiotics, etc. The results showed that the 1/2 MS medium adding 6-BA 2.5 mg/L and NAA 0.5 mg/L was suitable for establishing the acceptor system, and the best culturing condition was with 30.0 g/L sugar, pH 5.4 , 5.8 g/L agar, 1 500 lx light intensity and the 20~25℃temperature. The PLBs were very sensitive to kanamycin, and PLBs of Oncidium could be killed with kanamycin concentration as low as 25.0 mg/L, and the kanamycin concentration of 50.0 mg/L was proved to restrict rooting of non-transgenic plantlets. Therefore, 25.0 mg/L kanamycin and 50.0 mg/L kanamycin were as the effective concentrations of selection pressure for selecting the acceptor system and the rooting transgenic plantlets, respectively.3. The introduction of antisense ACS gene to Oncidium mediated by Agrobacterium tumefaciens The best transient expression of GUS was obtained under the conditions as follows: taking the PLBs cultured on the solid culture for 30 days as the materials, and cutting the upper part of the PLBs, then being pretreated with 0.25 mol/L mannitol for 5 h; and with 0.4 OD600 value of Agrobacterium suspension containing 30.0 g/L sugar, the samples being infected for 35 mins, then being transferred onto the 1/2 MS medium (pH 5.4) supplemented with 2.5 mg/L 6-BA and 0.5 mg/L NAA for co-cultivation for 4 days at 25℃in the dark..4. The selection of Oncidium resistant PLBs and resistant plants and transplant of the transgenic plantsA whole technical system of the selection of Oncidium resistant PLBs and resistant plants were developed and the transgenic plants were be transplanted. The indirect method of selecting resistant PLBs was as follows: after co-culture, the PLBs were cultured on the 1/2 MS medium supplemented with 2.5 mg/L 6-BA and 0.5 mg/L NAA and 50.0 mg/L Cef. for 30 days, then selected on the 1/2 MS medium supplemented with 2.5 mg/L 6-BA and 0.5 mg/L NAA and 25.0 mg/L Km. for 3 months with the gradual enhancement of Km. concentrations, and further transferred on the 1/2 MS medium supplemented with 0.8 mg/L 6-BA and 0.2 mg/L NAA for differentiation, and finally rooted on the 1/2 MS medium supplemented with 0.5 mg/L NAA , 100 g/L apple juice, 50.0 mg/L Km. and 50.0 mg/L Cef. . There was no significant difference between the non-transgenic plants and transgenic plants on the biological characteristics according the preliminary observation. The transgenic plantlets were survival after transplanting for 2 months.5. The detection of Oncidium resistant plantletsIn the experiment 121 plantlets differentiated from resistant PLBs were obtianed, among them 23 plantlets rooted cultured with Km., and at last, 9 plantlets detected by GUS histochemical assay and PCR assays preliminarily were proved that the gus gene and ACS gene were integrated into the genome of these resistant plantlets.As a whole, in this experiment the efficient regeneration of plantlets in Oncidium was established; the acceptor of the light yellow granular PLBs was obtained; the stable genetic transformation system of antisense ACS gene mediated by Agrobacterium tumefaciens to PLBs in Oncidium was established; the method and the selecting time of the transgenic PLBs were determined; the technique system of the high transformation rate by enhancing sugar concentration without AS was founded. These results would be of great importance for the new method of the prolonging life of cut flowers and the technique of the transgenic research in Oncidium.