Study On The Transformation Of AOC Transcriptionally Fused With Ipt To S. Guianensis Cv. Reyan No.5

In this study, AOC and ipt in transcriptional fusion has been transformed to Stylosanthes guianensis cv. Reyan No.5 mediating by Agrobacterium LBA 4404; which AOC gene is related with salt resistance, and ipt gene is the key gene in the pathway of cytokinin synthesis. The researches included:researches on the conditions of tissue culture and gene transformation of S. guianensis cv. Reyan No.5, such as:the best explants for gene transformation; the best callus inducible medium of S. guianensis cv. Reyan No.5; antibiotic concentration selection for gene transformation; suitable Agrobacterium strains and concertration for gene transformation; the effects of Agrobacterium concentration on the efficiency of transformation; the effects of submersion time of cotyledon with Agrobacterium on the efficiency of transformation; the effects of co-cultivation temperature on the efficiency of transformation; the effects of AS concentration on efficiency of transformation; the effects of co-cultivation time of cotyledon with Agrobacterium on the efficiency of transformation. About 500 pVKH-AOC-ipt independent lines of transformants that survived in the selection medium with hygromycine have been obtained. Parts of these lines have been furtherly identified by PCR and RT-PCR. The results as the following:1、The best callus induction medium of S. guianensis cv. Reyan No.5 consists of MS+4 mg/L 6-BA+2 mg/L NAA+30 g/L sucrose+8.0 g/L Agar powder, pH 6.0. The composition for bud differentiation medium is MS+0.4 mg/L 6-BA+0.1 mg/L NAA+30 g/L sucrose+ 8.0 g/L Agar powder, pH 6.0. The root inducible medium is the MS solid medium or 1/2MS solid medium, pH 6.0. The best explant is the cotyledon.2、Using the improved system of plant regeneration, and several factors that influence genetic transformation of S. guianensis cv. Reyan No.5 mediated by Agrobacterium have been studied. The results showed:LBA4404 Agrobacterium could more functionally infected S. guianensis cv. Reyan No.5 than GV3101. The optimum conditions for gene transformation were:OD600 of Agrobacterium concentration was 0.6; the submersion time of cotyledon with Agrobacterium was 10～15 mins, and co-culture temperature was 25℃for 60 h. Acetosyringone (AS) had no influence on transformation efficiency of S. guianensis cv. Reyan No.5.3、Researches on the conditions for plant transplantation from tissue conditions to soil have been done. The results showed that before transplantition, the seedlings in the medium with the cover open for a short-term could increase survival rate in soil. After transplantition, keeping the plants enough moisture, and letting them to be in shaded conditions are also necessary for the transplants to be survival.4、Using the improved gene transformation system, AOC-ipt has been successfully transformed to the genome of of S. guianensis cv. Reyan No.5. Appromixately 500 independent transfomant lines have been selected in the selection medium with hygromycine. Furtherly, parts of these transformants have been identified by PCR and RT-PCR, and 14 independent transgenic lines that could be transcripted to mRNA from genome have been obtained. These transgenic plants have been successfully transferred to soil, and grow well.