The Study On Preservation Of Germplasm Resources In Vitro And Anthocyanins In The Fruit Of Aronia Melanocarpa

Aronia melanocarpa is a deciduous shrub that belongs to Rosaceae.The tree is small,with leaves that change color seasonally and dark blue-purple fruit,which is great ornamental value in landscaping.The fruit of Aronia melanocarpa is rich in anthocyanins and other biologically active substances,and has extremely high medicinal value.Aronia melanocarpa is a multifunctional tree species with ornamental,ecological,edible and medicinal purposes.The research of Aronia melanocarpa has important theoretical and practical significance.There are abundant resources and many high-quality varieties and the content of anthocyanins in the fruit in the Northeast region is higher than that of other regions.Therefore,the excellent local germplasm resource ‘Fukangyuan No.1’ with high content of anthocyanins was preserved in vitro.This method is used for long-term retention,in order to introduce more widely,cultivate varieties with high anthocyanin content in the future,and obtain more anthocyanins more conveniently.This paper mainly did the following research: The high-content anthocyanin resource of Aronia melanocarpa was preserved by the method of delayed growth in vitro preservation,so as to prolong the subculture preservation time.The anthocyanins in fruit were extracted by ultrasonic wave and the extraction process was optimized,more crude extracts of which were obtained by technical means.The crude extracts of anthocyanin were separated and purified by high speed countercurrent chromatography.The anthocyanins were analyzed and identified by UV-visible spectroscopy,high performance liquid chromatography and high performance liquid chromatography-mass spectrometry.Finally,the antioxidant activity and antibacterial activity of anthocyanin monomers were studied,and the results were as follows:1.A method for in vitro culture of Aronia melanocarpa was established and optimized.‘Fukangyuan No.1’ test-tube seedlings were used as materials,the effects of different concentrations of paclobutrazol,mannitol and sucrose on the preservation of ‘Fukangyuan No.1’test-tube seedlings were investigated.The results showed that all three can effectively limit growth.Orthogonal test of three levels of factors,the optimal formula is: MS medium,6 mg/m L PP333,10 g/L mannitol,40 g/L sucrose.‘Fukangyuan No.1’ was carried out in the optimal medium.The test-tube seedlings can survive as long as 6-7 months,and the survival rate can reach 67.4%.2.The extraction process of fruit anthocyanins was optimized by response surface methodology.The study showed that four factors(ethanol volume fraction,ultrasonic power,ultrasonic time,and liquid-to-solid ratio)all had an effect on extraction of anthocyanins from fruits.On the basis of a single factor,a response surface model with four factors and three levels was established.The model fitting showed that the influence of each factor on the extraction volume was in the following order: ultrasonic power(B)> ethanol volume fraction(A)>ultrasonic time(C)>liquid-material ratio(D);the optimal extraction process parameters are:ethanol volume fraction 56 %,ultrasonic power 125 W,ultrasonic time 4 min,liquid-material ratio 20:1 m L/g,the maximum extraction amount of anthocyanins is 6.489 mg/g.3.Three anthocyanin monomers of Aronia melanocarpa were isolated and identified.High-speed countercurrent chromatography was used to separate and purify anthocyanin monomers.The research results showed that: using n-butanol: methyl tert-butyl ether:acetonitrile: water: trifluoroacetic acid=2: 2: 1: 5: 0.01 as the solvent system of HSCCC,under the conditions of rotating speed of 900 rpm and detection wavelength of 510 nm,three components were separated and identified by UV-visible spectroscopy,high performance liquid chromatography,and high performance liquid chromatography-mass spectrometry.The components were preliminarily determined as cyanidin-3-O-arabinoside,cyanidin-3-O-glucoside and delphinidin-3-O-glucoside.4.The antioxidant and antibacterial activities of anthocyanin monomers of Aronia melanocarpa were tested.The antioxidant activity of three anthocyanin monomers and crude extracts was measured in vitro,and the antibacterial activity was determined by Oxford cup method.The results showed that: three anthocyanin monomers can effectively scavenge free radicals;The extract has inhibitory effects on four common bacteria including Staphylococcus aureus,Bacillus subtilis,Escherichia coli and Salmonella Among them,delphinidin-3-O-glucoside has the best inhibitory effect,and the minimum inhibitory concentration is 0.313 mg/m L.In conclusion,Aronia melanocarpa is a multi-functional tree species with ornamental,ecological,edible and medicinal functions.‘Fukangyuan No.1’ is a high-quality variety with high content of anthocyanins,and its preservation can better obtain more anthocyanins.By optimizing the extraction and purification process of anthocyanins in fruits,the content of anthocyanins can be increased,the purity can be improved,and the antioxidant and antibacterial activities can be enhanced.