Effects Of Basal Media And Phytoormones On Accumulation Of Flavonoids And Carotenoids Of Embryogenic Calli In Dimocarpus Longan Lour.

Longan?Dimocarpus longan Lour.?,a plant belongs to Sapindaceae Euphoria,is a characteristic fruit in south China.It is rich of flavonoids,carotenoids and some other wholesome ingredients,and therefore it is thought to be of high medicinal value.The longan embryogenic callus?EC?is a nice modle for the study of somatic embryogenesis and secondary metabolism for its advantages of high speed proliferation,fast growth and high regeneration frequency.In this study,longan 'Honghezi' EC was used to investigate the effects of basic medium,phytohormone and incubation time on the proliferation of EC,flavonoids and carotenoids accumulation and flavonoids and carotenoids metabolism related genes.The results generated here would provide basis for the understanding and exploration of the flavonoids and carotenoids in longan EC.Results were listed as follows:1.Effects of Different Basic Media on the Proliferation and Flavonoidss Contents and yield of Longan ECBy using single factor test,the effects of 4 different basic media,i.e.MS,1/2MS,1/4MS and B5,on the proliferation of EC,the content of flavonoidss and carotenoidss in longan EC were investigated.Results showed that the impact of basic medium on longan EC proliferation from high to low in order is:MS>1/2 MS>B5>1/4MS.And the highest proliferation rate?17.48?was obtained when using MS as basic medium.The impact of basic medium on flavonoids content from high to low in order is also:MS>1/2 MS>B5>1/4MS.The highest is 5.06 mg/g.Results generated in this part indicated that MS medium is the best medium for EC proliferation and flavonoids accumulation,while 1/4 MS was the worst.2 Effects of Phytohormones on Proliferation,Flavonoidss and Carotenoids Content and Yield of Longan ECOrthogonal test was used for the study of the influences of phytohormones on proliferation,flavonoidss and carotenoids content and yield of Longan EC.Results showed that the impact of phytohormones on longan EC proliferation from high to low in order is:2,4-D>AgNO₃>KT.Treatment 5?MS + 2,4-D 1.0 mg/L+ KT 0.50 mg/L + AgNO₃ 7.5 mg/L?was found to be the best cultivation condition for longan EC proliferation,followed by treatment 6?MS + 2,4-D 1.0 mg/L+ KT 0.75 mg/L + AgNO₃ 2.5 mg/L?.The proliferation rate for the two treatments was 24.50 and 23.82,respectively.Treatment 7?MS +2,4-D 1.5 mg/L+ KT 0.25 mg/L + AgNO₃ 7.5 mg/L?and treatment 9?MS + 2,4-D 1.5 mg/L+ KT 0.75 mg/L + AgNO₃ 5.0 mg/L?were found to the not very good for EC proliferation,and the proliferation ratio under the two treatments was respectively 10.38 and 9.85.The highest flavonoids content?4.24 mg/g?was obtained under the treatment 8?MS + 2,4-D 1.5 mg/L+ KT 0.50 mg/L + AgNO₃ 2.5 mg/L?,while the lowest content?1.85 mg/g?was obtained under the treatment 7?MS + 2,4-D 1.5 mg/L+ KT 0.25 mg/L + AgNO₃ 7.5 mg/L?.The impact of phytohormones on flavonoids content from high to low in order is:2,4-D>KT>AgNO₃.And the best treatment for the production of longan flavonoids is treatment 5?MS + 2,4-D 1.0 mg/L+ KT 0.50 mg/L+ AgNO₃ 7.5 mg/L?.The highest carotenoids content?25.75 ?g/g?was obtained under the treatment 1?MS + 2,4-D 0.5 mg/L+ KT 0.25 mg/L + AgNO₃ 2.5 mg/L?,while the lowest content?9.5 ?g/g?was obtained under the treatment 4?MS + 2,4-D 1.0 mg/L+ KT 0.25 mg/L + AgNO₃ 5.0 mg/L?.The impact of phytohormones on carotenoids content from high to low in order is:AgNO₃>2,4-D>KT.And the best production system of longan carotenoids is treatment 1?MS + 2,4-D 0.5 mg/L+ KT 0.25 mg/L + AgNO₃ 2.5 mg/L?.By synthesizing various influence factors,the best treatment for longan EC proliferation and production of flavonoids and carotenoids is treatment 1?MS + 2,4-D 0.5 mg/L + KT 0.25 mg/L+ AgNO₃ 2.5 mg/L?.3.Effects of Cultivation Time on Proliferation,Flavonoidss and Carotenoids Content and Yield of Longan ECAccording to the result of part 2,single factor test was performed to investigate the influence of cultivation time on the proliferation,flavonoidss and carotenoids content and yield of Longan EC.Results showed that the impact of cultivation time on longan EC proliferation from high to low in order is:10d>15d>20d>7d.The impact of cultivation time on flavonoids content from high to low in order is:10 d>15d>7d>20 d,and the flavonoids content at 10 d is high up to 4.2 mg/g.The impact of cultivation time on flavonoids yield from high to low in order is:10 d>15 d>7 d>20 d.The impact of cultivation time on carotenoids content from high to low in order is:7 d>15 d>20 d>10 d,and the flavonoids content at 7 d is high up to 25.12 ?g/g.The impact of cultivation time on carotenoids yield from high to low in order is:7 d>10 d>15 d>20 d.In summary,10 d cultivation is the best for EC proliferation and flavonoids and carotenoids accumulation.4 Effects of Medium,Phytohormones and Cultivation Time on Flavonoidss and Carotenoids Related genes in Longan ECQuantitative Real-time PCR?qRT-PCR?was performed to evaluate the influences of medium,phytohormones and cultivation time on flavonoidss metabolism related genes?CHS,CHI,F3H and DFR?and carotenoids related genes?GGPS,PSY,ZDS and PDS?in Longan EC by using FSD,EF-1? and EIF-4a as the internal reference genes.QRT-PCR result showed that the expression levels of CHS,CHI and F3H in different medium from high to low in order were:1/2MS>MS>1/4MS>BS,while the order for DFR is:B5>MS>1/2MS>1/4MS.The highest expression levels of GGPS,PSY,ZDS and PDS in longan EC were all found when it is cultured in 1/2MS.The effects of different phytohormones treatments on the expression of CHS,CHI,DFR and F3H varied a lot.The highest expression level of CHS was found under Treatment 8?MS + 2,4-D 1.5 mg/L+ KT 0.50 mg/L + AgNO₃ 2.5 mg/L?,and the highest expression levels of CHI and DFR were found under Treatment 6?MS + 2,4-D 1.0 mg/L+ KT 0.75 mg/L + AgNO₃ 2.5 mg/L?.For F3H,however,the highest expression level was found under Treatment 9?MS + 2,4-D 1.5 mg/L+ KT 0.75 mg/L + AgNO₃ 5.0 mg/L?.Interestingly,the highest expression levels of all the carotenoids metabolism related genes were all found under treatment 9?MS + 2,4-D 1.5 mg/L+ KT 0.75 mg/L +AgNO₃ 5.0 mg/L?.Notably,highest expression levels of all the flavonoids metabolism related genes and carotenoids metabolism related genes were found when cultivated for 10 d.