Studies On The Regulation System Of Secondary Metabolism Of Resveratrol Callus In Polygonum Cuspidatum

Polygonum Cuspidatum is a kind of herbage belongs to polygonaceae.It is a Chinese traditional medicine.The mostly activity component is Resveratrol in Polygonum Cuspidatum. Resveratrol is consider of a promising natural chemical anticarcinogen.In order to achieve the sustainable use of Polygonum Cuspidatum resources,regard obtaining high-load resveratrol as goal,this research set up Polygonum callus clones.From the callus of light,temperature culture conditions,precursors of phenylalanine added,as well as fungal elicitor,Salicylic acid and UV-induced,we Carried out systematic research.Through to inducing the analysis of training physiological biochemistry of Polygonum cuspidatum callus in the course to change,have canvassed the measure of adjusting and controlling to the biosynthesis influence of resveratrol in the callus,have set up the technology system of improving the resveratrol content of Polygonum cuspidatum.And draw the following main conclusion:1.Through studying the impact on callus culture from explant and medium,understand that regards stem as explant,its callus grows faster than the blade,and the concentration of resveratrol among them is 5 times in the blade;The callus on MS culture medium is the most in proliferation,fresh heavy growth rate of its section of stem and blade is higher than N6 and B5 culture medium extremely notably,up to 141.0%and 73.8%separately,but the alcohol content of the white black false hellebore is slightly lower than B5 culture medium,but the concentration of resveratrol is slightly lower than B5 culture medium.So regard MS as basic culture medium,stem as explant induce appear callus relatively high,grow vigorously,maintain long time,the concentration of resveratrol is high,superior to other treatment.2.Passing analyzes the relation between the callus grows and the concentration of resveratrol,it is obvious the callus grows in the fastest period,while the concentration of resveratrol reach the most high too,so Polygonum callus harvest should be selected in the callus Organization of rapid growth when it reached saturation period,the entire culture can be completed in the same medium.3.Orthogonal experimental study the impact of solvent kind,time and temperature on the extraction rate of resveratrol,the variance analysis show that the most important factor is the extraction temperature,secondly the extraction time,and it is minimum to show the impact on drawing extraction rate of the resveratrol of solvents.Comprehensive all,using the ethyl acetate extract 4℃under 48 h is the best results.4.The callus inducing out the stem section,locate in dark mere(0 lx),weak light(1340-1560 separately Lx),strong light(3110-3310 Train under lx) three conditions,the result reveals,the biomass of the callus increases with enhancement of illumination intensity,the strong light trains the biomass of the lower callus to be up to 53.682 g/LFW.But the resveratrol content and illumination intensity of the resveratrol do not become positive correlation,only train weakly,the content of resveratrol is higher than other two to deal with notably in the callus,it is 140.074μg/ gDW.So the Polygonum cuspidatum callus is sensitive to the light,by only training the result to be best weakly.25℃trains one section of calluses growth speed of stem under the condition to be suitable,the callus relatively appears early,resveratrol has the highest content,is superior to other treatment in the callus.5.Feed Polygonum Callus on the different concentrations of phenylalanine precursor substances, the study found that 10～30 mg/L concentrations of phenylalanine can promote callus proliferation of biomass,20 mg/L deal Callus proliferation of the largest biomass.Richards equation with simulated callus biomass growth cycle in a process of dynamic accumulation,found no precursor substances in the circumstances,can get steady and reliable estimates,phenylalanine in a certain extent,the regulation of callus increased.By the rate of growth parameters(K),we can see that with the increase in the concentration of phenylalanine,callus growth rate accelerated significantly higher than the control,higher than the 16.5 times.The resveratrol also with the content and phenylalanine concentration increases,30mg/L deal with the maximum concentration of resveratrol,fresh,dried samples were 18.025μg / g and 241.489μg/g,controls more than 16.8%and 5.7%.6.Precursor substances phenylalanine can induce phenylalanine pyrolysis of enzyme activity,PAL activity of the show along with the added phenylalanine concentrations higher increase.To deal with all the activity of PAL's basically the same trend,both in the development of the first 7 d maximum,pre-show culture,phenylalanine concentration promoted an increase in PAL enzyme activity increased,for the synthesis of resveratrol the substrate to provide adequate and promote the synthesis of resveratrol.Therefore,add the appropriate amount of precursor substances to delay the Polygonum callus cell senescence,increased resistance to the callus.7.Through Aspergillus niger,Penicillium,brewer's yeast and the Botrytis cinerea extracts for the induction of sub callus induction carried out and found niger-induced synthesis of resveratrol on the most significant role in the promotion,improved control 50.2%that of Polygonum-type fungi induced selective.At the same time,in order to study found that 100 mg/ L of niger-induced best, 128.9%higher than the control group.When elicitor was added at different times,it was to different influence on callus and content of resveratrol.The content of resveratro was highest at the beginning,but the callus on the accumulation of biomass is a disincentive and in the first 14d and 21d join the sub-induced callus on the accumulation of biomass did not affect.In the process of induction training,the content of resveratro changes in the law of the analysis.It found that the cultured content of resveratro reached highest in the first 6d and the content was 227μg/g,but after the first 6d the content of resveratro started to decline.As a result,by a comprehensive analysis,we receive a best program: Of fungi induced callus.On the 14 day of Polygonum callus was inoculated, elicitor(100mg/L)from Aspergillus niger was added into the medium,the introduction of elicitor from Aspergillus niger the content of resveratrol in Polygonum callus was 2.25 times higher than that of the control.8.Aspergillus Niger inducer separation results show that Aspergillus niger-induced extract the main wall is a fungal polysaccharide induced a callus in the biological synthesis of resveratrol,and the composition of protein in callus inhibit the synthesis of resveratrol.9.Through fungal elicitor of Polygonum callus in PAL(PAL),superoxide dismutase(SOD), peroxidase(POD) and catalase(CAT) activity,Endogenous hormone changes in the ABA,as well as CAT and POD isozyme analysis found that the fungal elicitor stimulated the first Culture in 1d to 4d endogenous ABA have a lot of content,showed a sharp upward trend in the first 4d peak, PAL activity increased rapidly,reaching a peak in the first 4d;at the same time in training before the 3d,SOD and POD activity decreased,CAT in the first 2d appeared for the first peak;when the first train to 5d to 6d,a great deal of difference between Control and induced,SOD activity with POD induced processing suddenly dropped sharply,then a sharp upward trend,while the control is at a fairly high level;CAT at the 5th day of the second peak;POD isozyme have with the emergence of a new enzyme;and CAT MB is no significant change.10.Filtrating the adding the concentration of salicylic acid,adding time and screening time,By adding salicylic acid of 7mg/L by the last 14d,induced 6d of the best,we can make resveratrol nearly 1 time higher than the control group,but the effects induced by Aspergillus niger as elicitor. A.Niger elicitor and salicylic acid,phenylalanine in different combinations,the combination of resveratrol synthesis have a certain role,but with a separate add-in comparison A,niger induced change little,induced by A..niger,salicylic acid and phenylalanine is the most effective combination of three types of material.11.By exposuring UV(30W) under the department 20cm at per 15 min,stopped 5 min and repeated exposure to 6 times,the content of resveratro can not raise in 2h exposure processing.12.Set up the technology system to improve the resveratrol of Polygonum cuspidatum,regard stem section of Polygonum cuspidatum as explant,establishing callus clones on MS十6-BA 1.0十KT 0.2～0.5十NAA 0.2(mg/L) culture medium,Switching adds crude extract of dark Aspergillus 100 mg/L as Niger inducer on the 14th day after taking the place of on liquid culture medium,feeds 20 mg/L phenylalanine and minim salicylic acid at the same time,trains in weak mere 13001x,25℃.On the 6th day after adding,we can improve by more than 3 times of resveratrol in Polygonum cuspidatum callus.