The Genetic Transformation Of Fagopyrum Dibotrys By Ri Plasmid And Regulating The Synthesis Of Flavonoids In Hairy Root

Fagopyrum dibotrys,one of perennial herbs of the genus Polygonaceae in the Fagopgprum Mill family,is an important recource plant with rich nutrition and medicinal utilization values.The main bioactive components of Fagopyrum dibotrys is flavonoids.Because of the destroy of entironment and excessive exploitation in recent years,Fagopyrum dibotrys had been ranked into the second degree of national protected plants.Besides,cultured Fagopyrum dibotrys is low in the content of the active components and their medicinal quality is difficult to ensure.With the cesslessly increasing of the requirements of Fagopyrmn dibotrys,to exploit a new approach such as hairy root culture by application of biotechnology,for production of the active components,is of great significance to the protection of Fagopyrum dibotrys and the balance of ecology and the modcrnization of Chinese pharmacy.The experiment had established the genetic transformation systerm of Fagopyrum dibotrys by Agropyrum rhizogenes,and had primarily optimized the culture condition of genetic transformation hairy root,which had estsblished a definite foundation to the selection of metabolic mutant of Fagopyrum dibotrys and the study of genetic transformation,and which had also provided a perfect experiment systerm for the transformation and high efficient expression and functional mechanism of the key genes in flavonoids metabolism.The main results were following:1 Abtainment of callusCompared with other arrangements,tilting the seeds was beneficial to improve the percentage of germination and regeneration.The experiment showed that tilting the seeds could help to obtain more than 96%healthy seedlings in 7～12 days.Leaf blade and petiole from plants in vitro which had been subcultured 3～5 times,could be induced to form white loose callus,and the percentage of inducement was over 90%. 2 Establishment of genetic transformation systerm by Agropyrum rhizogenesThe different types of explants(leaf blade,petiole,callus)were infected by strain R1601 of Agropyrum rhizogenes,a highest frequency as 80%of root formation was achieved at the optimum transformation conditions.Leaf blade and petiole cultured in the medium MS+BA(2.0mg/L)+1BA (0.2mg/L)under the condition of 14h in light/10h in dark,could be induced to form callus in 10～15 days.White loose calli were dipped into R1601 bacteria solution with OD₆₀₀0.2～0.4,pH5.4～5.8,which had been actived by AS(100μmol/L)for 30 minutes,for 5～7 minutes.The infected calli were co-cultured on the mediurn 1/2MS+AS(100μmol/L)for 2～4 days in dark after dried by sterile paper carefully.When the co-culture was over,the calli were washed by sterile water 5～7 times,then disgermed in liquid MS containing Cef(500mg/L)for 30 minutes.After culture in the medium 1/2MS+cef(200 mg/L)+Km(20～30 mg/L).the calli were put into dark to induce hairy root.These roots were characterized by fast growth,frenquent branching and the ability to synthesize growth hormone as the roots of the intact plant.but the character of plagitropism were not marked.3 Detection of genetic transformation hairy rootDesigned primers according to the sequence of rolB gene and detected it by PCR.The DNA lade about 800bp was observed in transformation hairy root,same with positive control,but not in untransformed tissue,Those evidences showed that the genetic transformation systerm by Ri plasmid had been established successfully.4 Optimizing liquid culture condion of hairy rootThe hairy root liquid culture systerm was established from Fagopyrum dibotrys and the effects of different medium.different content of sucrose and different shaker revolution rates were studied.The results showed that the optirnum condition were N6,sucrose 30g/L,100rpm.The maximal content of flavonoids in hairy root was 7.84%(DW),and the multiplication of biomass was up to 206.34.