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Studies On Gene Transformation Influential Factors Of Medicago. Sativa By Agrobacterium Tumefaciens With SacB Gene

Posted on:2007-09-19Degree:MasterType:Thesis
Country:ChinaCandidate:M M YuFull Text:PDF
GTID:2143360185481343Subject:Forest cultivation
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Saline soil is widespread in China. The development of saline soil is of significance with the increasing population and decreasing arable land. Cultivated alfalfa(Medicago sativa) perennial legume crop is wildly planted in the world. The development and application of gene engineering creates a new path for alfalfa resistant breeding. In this research, a protocol for SacB gene transformation of alfalfa by Agrobacterium tumefaciens mediated has been developed.The mainly conclusions are as follows,1. 300mg/L Cef could beused to inhibit the Agrobacterium strain LBA4404 after transformation availably;2. The results showed that induction of callus from alfalfa was completely restrained by 100mg/L Kanamycin, the suitable selection pressure should be 100mg/L Kanamycin;3. The different gene types showed different results. In this experiment, the hypocotyls from hetian had highest calli induction frequence.4. The hypocotyls had the highest Kanr calli induction frequence under together cultivation time for 3days, but it showed that together cultivation was not necessary during the cultibation followed.5. Agrobacterium concentration, inoculation time, advance culture time and together culture time were associated. The best combination contains Agrobacterium concentration whose OD600 was 0.3~0.5, The explants from alfalfa were inoculated for 10~15 minutes, advance culture time and together culture.6. The inoculated explants were incubated on MS medium supplemented with 2mg/L 2,4-D,0.5mg/L KT, 30g/L sugar and 7g/L agar ,then the explants were transferred into MS medium supplemented with 2mg/L 2,4-D,0.5mg/L KT, 30g/L sugar and 7g/L agar and 300mg/L Cef to induce callus and somatic embryogenesis and subcultured onto MS medium supplemented with 1.0mg/L BA,0.3mg/L NAA, 30g/L sugar, 7g/L agar,50mg/L Kan and 300mg/L Cef to promete further embryos development. Transgenetic plantlets regenerated from somatic embryos on medium mentioned above after several subcultruing. Transgenic plantlets could root on 1/2 MS medium supplemented with 1.0mg/L IBA,10g/L sugar,8g/L agar and 80mg/L Kan.
Keywords/Search Tags:Medicago sativa, Gene Transformation, SacB gene, Protocol by Agrobacterium tumefaciens mediated
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