Study On Glyceollin Synthesis Induced By Various Kinds Of Oligosaccharides

In this study,soybean and black soybean were used as raw materials.As to soybean,soybean was firstly cultivated to form callus suspension cultures and cotyledons were prepared from soybean seed.Then alginate oligosaccharides?AOS?were employed to induce soybean callus suspension cultures and cotyledon tissues respectively,in which glyceollin synthesis was confirmed by UPLC-MS/MS.By comparing content of glyceollin produced by both materials treated with different concentration of AOS,suitable concentration of AOS was selected to induce both materials for studying mechanism underlying glyceollin synthesis.Optimization of glyceollin production from black soybean treated with xylooligosaccharides?XOS?using response surface methodology was carried out.The conclusion obtained from the present study was as follows:1.Study on the cultivating method of soybean callus and callus suspension cultures.By pretreatment of soybean with sodium hypochlorite including 1%active chlorine,well-grown aseptic seedling was acquired.Hypocotyl from aseptic seedling was transferred on the MS medium containing2,4-dichlorophenoxyacetic acid?2,4-D?and 6-benzylaminopurine?6-BA?with formation of white,compact and stiff callus,however,which wasn't available to grow to be callus suspension cultures.In addition,hypocotyl grown on the MS medium added only 2,4-D was induced to produce faint yellow,soft and incompact callus.Callus from hypocotyl under induction with 1.5 mg/L of 2,4-D achieved a maximum in fresh weight.So this callus was transferred into MS liquid medium containing 1.5 mg/L of2,4-D for cultivating soybean callus suspension cultures used as AOS-treated materials.2.Mechanism research of glyceollin synthesis from soybean callus suspension cultures and cotyledon tissue elicited with AOS.AOS application was exogenously executed to induce soybean callus suspension cultures and cotyledon tissue respectively.By soybean elicitation using AgNO₃,pure glyceollin was obtained.Confirmation of glyceollin synthesis in callus suspension cultures and cotyledon tissue was according to the retention time of pure glyceollin in the HPLC.Glyceollin accumulation in 2%AOS-treated callus suspension cultures was 8.2-fold higher than that obtained in the treated cotyledon tissue,so 2%AOS was used to investigate the change in metabolites content and expression of regulatory genes in the glyceollin synthesis pathway for both samples.The results showed uridine diphosphate glycosyltransferase?UGT?,3,9-dihydroxypterocarpan 6a-monooxygenase?CYP93A1?,glycinol 2-dimethylallyltransferase?G2DT?and glycinol 4-dimethylallyltransferase?G4DT?genes had a decisive effect on glyceollin synthesis.Moreover,as for glyceollin?,?,?,isomer?was prominent among the three isomers in elicited callus suspension cultures,whereas isomer?and?were dominant in the treated cotyledon tissue,revealing the mechanism involving glyceollin synthesis from both materials.3.Optimization of glyceollin synthesis from black soybean elicited with XOS.During the optimization of glyceollin production from XOS-elicited black soybean,the order of three factors affecting the glyceollin content was as follows:temperature>XOS concentration>induction time.Quadratic multiple regression model was acquired by fitting the actual content of glyceollin under different induction condition accompanied with a correlation coefficient of 0.9254,indicating this model could explain 92.54%of obtained glyceollin value.Optimized condition for glyceollin content was as follows:induction time 4.03 d,XOS concentration 4.08%and temperature 23.83?,and under this condition glyceollin accumulated by XOS-treated black soybean achieved 1.4118 mg/g dry weight.By analyzing the change of metabolites content and related regulatory genes in glyceollin synthetic route,effect of relevant genes regulation on metabolites was illuminated in this study,meanwhile UGT,CYP93A1,G2DT and G4DT genes showed a decisive influence on glyceollin accumulation and the proportion of isomer?,?and?.With the application of response surface methodology to optimize accumulation of glyceollin from black soybean elicited with XOS,glyceollin content was increased in exogenetic oligoses elicitor-treated varieties of beans and thus provided a basis for obtaining glyceollin with high efficiency.