Effects Of Exogenous Inducers On Flavonoid Accumulation In Ginkgo Biloba L.Tissue Culture

Ginkgo biloba L.is a perennial woody plants of the genus Ginkgo in the Ginkgo family.It is a relic plant in China.Ginkgo biloba L.has important medical value because leaves are rich in flavonoid,and widely used in the treatment of CCVD.However,the traditional method of extracting Flavonoid from leaves is greatly affected by the region,and over-exploitation will lead to resource shortage,which has certain limitations.Recent years,production of medicinal components by plant tissue culture is an important way to develop medicinal plants,which can maintain the stability of culture environment and screen the culture conditions to accumulation metabolites.In this study,a 10-15-year-old Ginkgo biloba L.tree on botanical garden of campus was used as material to induce callus.The leaves,stems and cotyledons were used as explants.Screening high-yield flavonoid cell lines and optimum culture conditions in solid culture period.Then in suspension culture period,MEJA and CTS were added to further increasing of flavonoid yield.The main conclusions are as follows:1.Callus induction of Ginkgo biloba L:Callus induction from leaves,stems and cotyledons of Ginkgo biloba L.The induction rate was cotyledon>leaf>stem segment,and the callus of leaves and cotyledons was loose,while the callus of stem segments was denser.2.Callus subculture of Ginkgo biloba L.:Through design orthogonal experiment,the best growth combination of Ginkgo biloba L.callus is:cotyledon callus+MS+3 mg/L NAA+1 mg/L KT+5 mg/L VC;the best anti-browning combination is:leaf callus+MS+1 mg/L NAA+1 mg/L KT+10 mg/L VC;the best flavonoid accumulation combination is:leaf callus+MS+2 mg/L NAA+1 mg/L KT+5 mg/L VC;Among the nine orthogonal treatment groups,the highest flavonoid yield group reached 6.32 mg/bottle.Use this callus for suspended cultured.And comprehensive browning index,Finally,MS+2 mg/L NAA+1 mg/L KT+10 mg/L VC medium combination was selected for subsequent experiments.3.Establishment of Ginkgo biloba L.suspension cell line:Under the basic conditions of 3 g/bottle of callus inoculation and 250 mL of culture bottle capacity,single factor experiments showed that Ginkgo biloba L.suspension cells have the highest flavonoid yield and can ensure a better growth state under the conditions of 80 mL liquid loading,110 r/min shaking speed,12 h/d illumination period and 15 d cultivation time.4.Effects of exogenous elicitors on cell growth and flavonoid accumulation:By adding different concentrations of elicitors MEJA and CTS to Ginkgo biloba L.suspension cell lines,it was found that MEJA treatment could significantly inhibit cell growth,but within a certain concentration range,the content of flavonoid can be increased and the final flavonoid yield can be increased either.The optimum concentration of MEJA was 50μmol/L,and the flavonoid yield could reach 369.26 mg in 15 days.CTS also inhibited cell growth,but the inhibition was not significant in the low concentration range.100 mg/L CTS could make the flavonoid yield of suspension cells reach 502.98 mg/L at 15 days,which is 1.66 times of the control.5.Effect of exogenous elicitors on cell enzymatic activity:Adding exogenous elicitors to Ginkgo biloba L.suspension cells.In a 15-day cycle,it was found that both MEJA and CTS could enhance the activities of flavonoid synthesis key enzymes(PAL)and antioxidant enzymes(SOD,POD,CAT)in suspension cell line.However,CTS was better than MEJA in improving PAL and CAT activity,and MEJA increased SOD and POD activity more significantly.