Studies On Genetic Transformation Of Embryogenic Suspension Cells In Atropa Belladonna L.

In this paper, the establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation system were bulit in Atropa belladonna L., by screening the level of various factors which affected the transformation frequence. PCR identification showed that the foreign genes has been successfully integrated into the genomic DNA of A.belladonna. This study will been explore the conditions of Agrobacterium-mediated genetic transformation used embryogenic suspension cells for infection, and analyze the role of pmt genes in Tropane alkaloid (TAs) biosynthetic pathway of A.belladonna. It would be an efficient and new approach to improving the biosynthesis of TAs in A.belladonna. The main findings were as follows:1.Petioles, stems and leaves as materials, the optimal medium for callus induction in A.belladonna was studied. The results revealed that Y1-3(MS+2.0mg.L-16-BA+0.2mg.L-1NAA), Y2-3(MS+2.0mg.L-12,4-D), Y3-4(MS+3.0mg-L-116-BA+0.1mg.L/-1NAA) was the optimal medium for callus development to leaves, petioles and stems, respectively. Compared with callus growth condition of different explant, leaves was optimal explant, and the calli grew fast and had a high differentiation rate.2. Embryogenic callus could be obtained directly on the UMO sold medium with the combination of2,4-D and KT. The primary callus ultimately yielded embryonic callus after subculture2to3months in medium (MS+2.0mg-L-12,4-D). Callus became light yellow, loose, granular, finely divided and relatively friable. So, leaves was the best sources for embryonic cell suspension culture system.3. Plant regeneration and suspension embryogenic cell lines in A.belladonna were obtained. Culture condition of suspension medium was optimized by orthogonal test? that media was MS+2.0mg.L-12,4-D+0.25mg.L-1KT,0.5mL cultures,50mL medium each200mL erlenmeyer flask for10d. Embryogenic cell suspension culture lines was established after2to3months, and contained dense embryogenic cell mass. Cell growed and breeded rapidly, their sizes were similar, their colors were whity-yellow. Embryogenic callus could produce somatic embryos and production rates was above40.9%after subculture of5cycles, and germinated into regeneration plants on MS solid medium readily and directly, and germination rate was84.36%. The survival rate of transplanted plantlets of regeneration was above95%after habituation.4. Several factors effecting Agrobacterium-mediated gene transformation of embryogenic cell suspensions in A.belladonna were studied by using the transient expression of gus as criteria. The study showed that the genetic transformation efficiency could be effectively enhanced with A.tumefaciens concentration (OD600=0.5～0.6), incubation for20min, and100μmol-L-1AS co-culture for2days.5. Resistant embryogenic callis and hairy roots were induced and screened for resistance to Hygromycin B. Carbenicillin concentration of400mg.L-1could be effectively inhibit Agrobacterium after co-culture, and Hygr concentration gradient from low to high (10,15mg-L-1) was used to select resistant embryogenic callus in A.belladonna. Total of21lines of resistant hairy roots were obtained, but PCR identification showed that pmt as foreign gene had been integrated into genome of the7Hygr-resistant hairy root clones. It was feasible to use Agrobacterium-mediated method to introduce foreign gene into A.belladonna cells by embryogenic suspension cultures.6. Compared with non-transgenic hair root cultures, HPLC results confirmed that contents of total TAs and scopolamine had been improved in five transgenic hair root lines in A.belladonna. Moreover, total TAs, scopolamine, hyoscyamine of contents increased to3.3,3.7,2.6fold in optimal transgenic line compared with that of wild type control line, respectively.