| Dimocarpus longan Lour.is a fruit tree unique to tropical and subtropical areas.It possesses rare characteristics which make it a highly valued fruit in China.Longan fruit is rich in flavonoids,polyphenols and carotenoids and other nutrients components.It can be used as medicine and as a food crop.Longan embryogenic cultures have fast growth and multiplication factors,regeneration ability,and many other advantages.Longan embryogenic callus can be used as good source of secondary metabolite production.Additional studies have shown that the accumulation of plant secondary metabolites are often affected by stress adversity as well as other external environment influences.In view of this,longan 'honghezi' embryogenic cultures were used as the research material to study the effects of temperature and SA on flavonoids and carotenoids accumulation,at the same time,the cell morphology and the activities of cell protective enzymes as well as the flavonoid-related and carotenoid-related biosynthetic genes were studied.The purpose of the this study was to provide material basis and theoretical guidance for commercial production of longan secondary metabolites such as flavonoids and carotenoids.The main results were as follows:1 The effects of culture time and culture temperature as well as the concentration of SA on the accumulation of flavonoids and carotenoids in longan embryogenic callusUsing longan 'honghezi' embryogenic callus as the experimental material,the effect of culture time,temperature and the concentration of SA on the accumulation of flavonoids and carotenoids were studied.The results were as follows:the best culture time for the accumulation of the flavonoids and carotenoids in longan embryogenic callus was 30 d;the best constant culture temperature for the accumulation of flavonoids and carotenoids in longan embryogenic callus was 30/30 ?,the best fluctuation culture temperature for the accumulation of the flavonoids in longan embryogenic callus was 35/20 ?,the best fluctuation culture temperature for the accumulation of carotenoids in longan embryogenic callus was 30/20 ?;the best concentrations of SA in medium for the accumulation of flavonoids in longan embryogenic callus were 0.50 and 1.00 and 1.50 and 2.00 mg·L-1,the best concentrations of SA in medium for the accumulation of carotenoids in longan embryogenic callus was 2.50 mg·L-1.2 The effects of culture temperature and addition time of SA on the accumulation of flavonoids and carotenoids in longan embryogenic suspension cellsUsing longan 'honghezi' embryogenic suspension cells as the experimental material,based on the result of the effect of temperature and SA on the accumulation of flavonoids and carotenoids in longan embryogenic callus,the culture temperature and the addition time of SA on the accumulation of flavonoids and carotenoids were studied as well as the effects of the cooperation of temperature and SA on the accumulation of flavonoids and carotenoids were studies.The results were as follows:the best time for 2.00 mg·L-1 SA added to the medium for the accumulation of the flavonoids and carotenoids in longan suspension cells was on the 2nd day;when constant culture temperature was 30/30 ?and with the 2nd day added 2.00 mg·L-1 SA that the accumulation of flavonoids and carotenoids were significantly positive compared with constant culture temperature 30/30 ? and constant culture temperature 25/25 ?.3 The effects of culture time and temperature as well as the concentration of SA on the propagation of longan embryogenic callusUsing longan 'honghezi' embryogenic callus as the experimental material,the effect of culture time and the culture temperature as well as the concentration of SA on the propagation of longan embryogenic callus were respectively studied.The results were as follows:it was on 35/35 ? constant culture condition that the proliferation rate was the highest;it was on 35/20 ? fluctuation culture condition that the proliferation rate was the highest;the concentration in medium of the SA were 2.00 mg·L-1 and 1.50 mg·L-1 that the proliferation rate was the highest.4 The effect of temperature and SA on the activities of SOD and CAT as well as POD cell protective enzymes in longan embryogenic callusLongan 'honghezi' embryogenic callus were used as materials to studied variations in activities of SOD and CAT as well as POD cell protective enzymes under different temperatures and SA treatments on the 30th day.The results were as follows:under the conditions of constant temperature culture,the activities of SOD and POD enzyme both were 25/25 ?<20/20 ?<30/30 ?<35/35 ?,The activities of CAT enzyme were 20/20 ?<25/25 ?<30/30 ?<35/35 ?;under the conditions of varying temperatures,the activity of SOD and POD as well as CAT enzymes were all the same 20/20 ?<25/25 ?<30/20 ?<35/20 ?;with the increase of SA concentration in the medium,the activities of SOD and CAT as well as POD cell protective enzymes in longan embryogenic callus increased at first and then decreased,the activity of SOD enzyme was the highest when the concentration of SA in medium was 1.50 mg·L-1;the activity of POD enzyme was the highest when the concentration of SA in medium was 1.00 mg·L-1;the activity of CAT enzyme was the highest when the concentration of SA in medium were 1.00 and 1.50 mg·L-1.5 The effects of temperature and SA on the flavonoid-related and carotenoid-related biosynthetic genes in longan embryogenic callus and correlation analysisLongan 'honghezi' embryogenic callus were used as materials,to study the effects of different temperature and the concentration of SA on the expression of flavonoid-related and carotenoid-related biosynthetic genes on the 30th day as well as the effects of the best condition of temperature and SA concentration on the expression of flavonoid-related and carotenoid-related biosynthetic genes on the 10th,the 15th,the 20th,the 25th and the 30th day.The results were as follows:under the different temperature and the concentration of SA treatments on the 30th day,The correlation of CHI gene expression and DFR gene expression were both significantly positive with the content of flavonoids.The correlation of GGPS gene expression and PSY gene expression were both significantly positive with the content of carotenoids.Under best temperature treatment(30/30 ?),The correlation of CHIgene expression,CHS gene expression,F3H gene expression and DFR gene expression were positive in a pair wise manner under embryogenic callus growth for 20th and 25th;under best temperature treatment(30/30 ?),for carotenoid-related gene,the correlation of GGPS gene expression,PSY gene expression,PDS gene expression and ZDS gene expression were also positive in a pair wise manner under the process of embryogenic callus growth.Under the best SA treatment(1.50 mg·L-1),the correlation of CHS gene expression and CHI gene expression,F3H gene expression and DFR gene expression,GGPS gene expression and PSY gene expression as well as PDS gene expression and ZDS gene expression were always positive in pair wise manner under the process of embryogenic callus growth for all cases.6 The effects of temperature and SA on the flavonoids and carotenoids biosynthetic genes in longan embryogenic suspension cells and correlation analysisLongan 'honghezi' embryogenic suspension cells were used as materials,to study the effects of temperature with SA on the expression of flavonoid-related and carotenoid-related biosynthetic genes on the 10th,the 15th,the 20th,the 25th and the 30th day as well as the effects of the best condition of temperature and SA concentration on the expression of flavonoid-related and carotenoid-related biosynthetic genes on the 10th,the 15th,the 20th,the 25th and the 30th day.The results were as follows:under the cooperation of temperature and SA treatments,the significant rise of flavonoids was related to the stability of CHI,CHS,F3H,DFR gene high expression under the process of embryogenic suspension cells growth;under the cooperation of temperature and SA treatments,the correlation between CHI gene expression and CHS gene expression,F3H gene expression and DFR gene expression were respectively negative under embryogenic suspension cells growth on the 8th and the 10th day.Under the cooperation of temperature and SA treatments,the significant rise of carotenoids content on the 10th day of embryogenic suspension cells culture was related to the high expression of GGPS,PSY,PDS and ZDS gene under the process of embryogenic suspension cells growth on the 2nd and the 4th day.Under the cooperation of temperature and SA treatments,the correlation between PSY gene expression,PDS gene expression and ZDS gene expression were positive in a pair wise manner under embryogenic suspension cells growth on the 2nd,the 4th and the 6th day;under the cooperation of temperature and SA treatments,the correlation between GGPS gene expression and PSY gene expression were always high positive under the process of embryogenic suspension cells growth.The futher research about the accumulation of flavonoids and carotenoids on emberyogenic cultures,the production work of longan flavonoids and carotenoids can be more normative.By establish a scientific and efficient factory production process can improve the enterprise production efficiency and competivite power.It has important significance to the excavatoion of the traditional Chinese medicine resources and the modernization as well as the internationalization of traditional Chinese medicine.The published of longan genome database provides a new opportunity to futher research the longan flavonoids and carotenoids. |
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