Molecular Mechanism Of Dormancy Release Of American Ginseng Seeds After Removing The Endosperm

American ginseng（Panax quinquefollium L.）is a world-famous traditional medicine with important medicinal value.However,the deep dormancy of seeds brings severe challenges to artificial cultivation.Although the existing research methods shorten the dormancy period of seeds,the work is cumbersome and the cost is high,and the mechanism of embryo dormancy release is still poorly understood.The germination rate,soluble sugar content,amylase activity,G6PDH activity,hormone content and ginsenoside content of American ginseng seeds during physiological after-ripening process were determined.At the same time,in order to verify the existence of germination inhibitors in endosperm,the endosperm of American ginseng seeds was removed to investigate the germination of embryo.The contents of hormones and ginsenosides after removing the seed embryos were determined by HPLC-MS,and the transcriptome sequencing of American ginseng seed embryos was carried out by illumination technology.The gene expression during seed embryo germination was analyzed in order to explore the important genes closely related to seed embryo dormancy and germination,and speculate the dormancy release mechanism of American ginseng seed,so as to shorten its dormancy period and accelerate germination.The main results are as follows:1.During the physiological after-ripening process,the germination rate of American ginseng gradually increased at the seven stages,in the seventh period,the germination rate was 91%;the soluble sugar content gradually increased,from the first stage to the seventh stage,the soluble sugar content in endosperm increased 1.1 times;the activity of G6PDH and amylase increased gradually,and the activity of G6PDH and amylase increased 3.3 times and 0.9 times respectively in the seventh stage than in the first stage;the content of ABA in embryo and endosperm decreased gradually with the release of dormancy,at the seventh stage,ABA content in embryo and endosperm decreased by 71%and 94%,respectively;the content of ginsenosides in seed embryo increased gradually,the most significant change was 20（R）-Rh₁,which increased by 13.58 times during the whole process,and only three ginsenosides in endosperm changed significantly,among which Ro increased by 1.88 times.2.The germination rate of intact seeds of American ginseng on filter paper and culture medium was0%,and the germination rate of embryo in vitro was 100%and they all grew radicle.The fresh weight,seedling height and radicle length of in vitro embryos grown on MS medium were significantly（P<0.05）higher than that of in vitro embryos grown on filter paper or pure agar.Pure agar culture resulted in significantly（P<0.05）higher fresh weight and radicle length compared with filter paper culture in American ginseng.3.After embryo germination,the content of ABA in the experimental group decreased by 96%,the content of IAM and GA19 decreased by 66.67%and 66.06%respectively,while the content of IPYA and TZR increased by 17.9 and 7.9 times respectively.After embryos germination,the type of ginsenosides increased from 24 to 35（including iso）.The most significant increase was PPD type ginsenosides,which increased by 5,followed by PPT type ginsenosides,which increased by 4,while OA type ginsenosides decreased by 1.Rb₁ of PPD type ginsenoside increased 131.56 times,followed by Rg₁ of PPT type,increased 118.83 times.OA-glc-glura-xyl-iso-b of OA type was the only ginsenoside whose content decreased after embryos germination.4.Transcriptomic analysis showed that a total of 8207 differentially expressed genes were obtained by transcriptome analysis,of which 1972 were down-regulated and 6235 were up-regulated.One differentially expressed gene related to ABA degradation was detected,which was enriched in carotenoid biosynthesis pathway,eight differentially expressed genes related to ginsenoside synthesis were detected,which were mainly enriched in the pathways of terpenoid backbone biosynthesis,sesquiterpenoid and triterpenoid biosynthesis and carbohydrate transport and metabolism,and they were up-regulated.Molecular docking predicted that UGT85A24 was the most likely to catalyze the glycosylation of PPD ginsenoside and CYP71D313 was the most likely to catalyze the hydroxylation of PPD ginsenoside.qRT-PCR results were consistent with transcriptomic results,which proved that transcriptomic results were true and reliable.