| The petals of Hemerocallis middendorfii Trautv.et Mey.were used as test materials to develop a best extraction method,detection condition and identification method of carotenoids in petals based on UPCC-MS.And the method was applied to the analysis of qualification and quantification of carotenoids in petals of H.middendorfii and H.‘Pardon Me’ which were the representatives of yellow and red petals.At the same time,the hybridization(H.middendorfii × H.‘Pardon Me’)was proceeding methodically.Superior line were selected from hybrid progeny to study the differences of components and content of carotenoids between parents and hybrid progeny.The whole experiment process was carried out to explore the genesis of flower color and provide a theoretical support for hybridization breeding work of the genus Hemerocallis.The results are as follows.1.Five extraction reagents,two shaking methods and two saponification methods were set up to optimize the extraction method of carotenoids and the extraction effect of different combinations were compared.Three factors have the significant effect on the results.In the selection of extraction reagent,reagent B acetone: n-hexane(3:5/V:V)and D acetic ester: nhexane(1:1/V:V)obtained the highest content of carotenoids in total,which is 56.37 mg/g and 53.92 mg/g,respectively.In the selection of shaking method,extracting method used by shaking incubator obtained a total amount of 56.43 mg/g,which is higher than another.In the selection of saponification method,the total amount of carotenoids extracted from saponification at normal temperature for 16 h was higher,for 53.73 mg/g.In the interaction of three factors,the interaction between extract reagent and shaking method was significant.The combination of reagent B acetone: n-hexane(3:5/V:V)and useness of shaking incubator obtained the highest content in total,for 67.32 mg/g.According to the sensitivity of carotenoids to high temperature,the best optimal extraction method was determined as follows: B acetone: hexane(3:5/V:V)as extraction reagent,extracted by shaking incubator and saponification for 16 h at room temperature.2.The samples of carotenoids were separated and detected by UPCC-MS method.The results of two kinds of mobile phase modifier and three kinds of elution gradient were compared.The modifier contains methanol and ethanol,elution gradient contains degree elution and two kinds of gradient elution.Degree elution was set as 20% B,elution time 10 min.Gradient elution 1 was set as 0 min,5% B;5 min,25% B;6 min,30% B;8 min,5% B;10 min,5% B.Gradient elution 2 was set as 0 min,5% B;5 min,20% B;7 min,20% B;8 min,5% B;10 min,5% B.The optimum detection conditions were determined by comparing the number of chromatography peaks and their separation degrees.The best condition was finally set as follows: ethanol used as the mobile phase modifier and gradient elution 2.Under this condition,the chromatography peaks of isomers were separated greatly.3.According to extraction,separation and determination of carotenoids in petals of H.middendorfii and H.‘Pardon Me’,the results showed that there was a big difference in components and content of carotenoids between yellow and red petals.In carotenoids varieties,petals of H.middendorfii contain 16 kinds of carotenoids,while 18 varieties in H.‘Pardon Me’.Zeaxanthin isomers were detected in the former petal which were not contained in the latter one.And violaxanthin,all-trans-neoxanthin and 5,6-epoxy-β-carotene detected in the latter petal were not found in the former one.As for the content,petals of H.middendorfii contain 126.38mg/g carotenoids in total,while in H.‘Pardon Me’ was 115.36mg/g.The carotenoids variety of highest content was all-trans-β-carotene in both species,while the variety of lowest content was dihydrolutein in the former petal and dihydrolutein and lutein in the latter one.4.Through the hybridization test of H.middendorfii and H.‘Pardon Me’,114 seedlings were taken.The color performance of the hybrid progeny petals was from yellow to red,and the majority showed orange.Two superior line NO.46 and NO.84,were selected from hybrid progeny,which showed uniform and stable color and representative performance.The results showed that the components and contents of carotenoids were different between the two superior line and their parents,as well as two parts of the same flower.In the respect of variety,new variety of carotenoids different with their parents were not found.The number of variety is the same as or less than that of parents.The number of carotenoid variety of NO.46 is the lowest,just for 14 varieties.And the variety in the same plant flowers is similar.In the respect of content,the total amount of carotenoids in hybrid progeny is less than that of their parents,and the content is 43.55、62.35、80.34、69.01 mg/g respectively.As for NO.84,the variety of highest content is the same as that of their parents,means all-trans-β-carotene.And the variety of lowest content were dihydrolutein in NO.84 and lutein isomer(tR=3.50)in NO.46. |
PDF Full Text Request