Effects Of Light And ABA On Callus Growth And Carotenoid Content Of Longan And The Mechanism Of DlPIFs

Longan(Dimocarpus longan Lour.)is a plant of the genus Dipterocarpus in the family Apocynaceae.Longan pulp,nucleus,shell and other parts are rich in secondary metabolism substances,especially flavonoids and carotenoids.In this experiment,the embryogenic callus(EC)of the embryonic callus was treated with different light qualities and ABA concentrations,the carotenoid content of the embryonic callus was determined and the expression of DlPIFs and DlPSY was analyzed.Thus,the effects of light quality and ABA on the carotenoid content in the embryonic callus of the longan and the mechanism of action of the PIF genes were studied.The main results of this study were as follows:1.Effects of light quality on the proliferation of embryogenic callusLongan EC was treated with different light qualities(blue,red,yellow,white and green,with darkness as a control,light intensity of about 1100 Lux),the results showed: In dark,white and green light conditions,longan EC grows optimally;Under blue light conditions,the growth state is better,but the color is slightly browned;Under red and yellow light treatment,cell clumps are coarsely granulated,the color browning is severe,and the growth state is not good.The proliferation coefficient of longan EC was the largest and the proliferation coefficient was significantly different when the incubation time was 20 d under different light qualities.The influence ranking of different light qualities on the embryonic callus proliferation of the longan was red light > green light > yellow light > dark > blue light >white light.Combined with the appearance morphology of longan EC,it can be concluded that green light is the most favorable photoglychote condition for longan embryonic callus culture.2.Effect of ABA on proliferation of embryonic callus in the longanWith different concentrations of ABA treatment(0,0.02,0.04,0.08,0.1,0.2,0.4,0.8 μmol/L),the growth morphology of longan EC was different.At ABA concentrations of 0 and 0.02 μmol/L,the growth state of longan EC was optimal;The ABA concentration of 0.04 was good for growth,but the color is browned;At ABA concentrations of 0.1,0.2,and0.4 μmol/L,the longan EC grows well but the cell clumps are coarser and the color was slightly browned;At ABA concentrations of 0.08 μmol/L,the cell mass particles were coarse,water-stained,and the growth condition was not good;When the ABA concentration is 0.8 μmol/L,the cell mass was coarse,appeared severe browning,and the growth state was poor.The proliferation coefficients of the embryonic callus of the longan under different light conditions were significantly different.The proliferation factor of longan embryonic callus was greatest at Day 20.At the incubation time of 20 days,the proliferation effects of different concentrations of ABA on longan EC were arranged as: 0.1 > 0.8 >0.04 > 0.08 > 0.2 > 0.02 μmol/L.3.Response surface test employed to optimize the process of ethanol extraction of carotenoids from longan callusIn order to achieve the efficient and green safe extraction of carotenoids in longan callus,the ethanol extraction process was optimized by the response surface method on the basis of previous research,and the extraction scheme was upgraded.The test conditions are: material-liquid ratio 1:20 g/m L,the ethanol concentration 71 %,the extraction temperature 88.5 °C,the extraction time 15 min,the extraction time 15 min,and the supernatant centrifugation for 10 min,and the supernatant as carotenoid extract,and the absorbance value determined at 445 nm using a UV spectrophotometer.This method is not only efficient,but also green and safe.4.Light quality and ABA affect the content of carotenoids in the embryonic callus of the longanThe experimental results showed that the effects of different light qualities on the carotenoid content in longan EC were blue light > dark >white light > green light > yellow light > red light,and blue light had a significant role in promoting the content of carotenoids in callus.Under different ABA concentrations,the content of carotenoids in longan EC was sorted as follows: 0.2 > 0.1 > 0.08 > 0.8 > 0.4 > 0.02 > 0.04>0μmol/L.Further experiments showed that under blue light conditions,when the ABA concentration was 0.1 μmol/L,the content of carotenoids in the embryonic callus of the longan was the highest,and it was higher than that of the blue light or the ABA treatment alone.5.Identification and expression analysis of longan PIFs familyIn this study,the DlPIFs was identified and analyzed based on the longan genome.Eight members of the PIF family with complete domains were identified from Longan.Bioinformatics was discovered,and each member contained many elements of cis-acting such as light and hormones in the promoter region.Transcriptome gene expression data analysis and q RT-PCR results show that DlPIFs members are closed related to the growth and development process of different tissues and organs of the longan and play an indispensable and important function in it;DlPIFs members also played important roles in the response to abiotic stresses such as high temperature,low temperature,drought and exogenous hormones.6.Effects of light quality and ABA on PSY expression of PIFs and carotenoid synthesis-related genes in longan calluses.The expression of DlPIFs and correlation analysis under different light qualities treatments showed that DlPIF1-2,DlPIF3,DlPIF4,DlPIF5,DlPIF6,DlPIF7 and DlPIF8 were all negatively correlated with DlPSY,and DlPIF4 was significantly negatively correlated with DlPSY.Under the ABA treatment at different concentrations,DlPIF1-1,DlPIF1-2,DlPIF4 and DlPIF6 were inversely correlated with DlPSY,and both DlPIF4 and DlPIF6 were significantly negatively correlated with DlPSY,indicating that DlPIF4 was closely related to the regulation of DlPSY.