Physiological Analysis And Key Gene Screening Of Exogenous Ascorbic Acid Regulating Embryogenic Cell Proliferation Of Pinus Koraiensis

Pinus koraiensis is a kind of plant belonging to pinaceae,which has important economic and ecological value.In the previous studies,the system of Korean pine somatic embryogenesis and plant regeneration has been basically established.However,due to different reproductive materials and culture conditions,there are significant differences in the process of Korean pine somatic embryogenesis and proliferation,which has become an obstacle to its large-scale propagation.Ascorbic acid（ASA）is an important antioxidant in plants.Many studies have proved that exogenous ASA can promote the process of plant somatic embryogenesis.In this study,three Korean pine embryogenic cell lines with different proliferation ability were used as materials.By adding different concentrations of ASA,the effects of exogenous ASA on the proliferation rate of Korean pine embryogenic callus,the content of key substances and related enzyme activities in endogenous Ascorbic acid-Glutathione（ASA-GSH）cycle and reactive oxygen species（ROS）metabolism,the endogenous hormone level and the relative expression of related genes in the process of proliferation were studied.The optimum concentration of exogenous ASA treatment and related physiological and molecular mechanisms were determined.The main conclusions are as follows:（1）Exogenous ASA significantly affected the proliferation of embryogenic callus of different cell lines of Korean pine,showing a trend of low concentration promoting and high concentration inhibiting.Among them,20 mg/L and 50 mg/L ASA had the best promoting effect,and the proliferation rate was 372.5%-480%（both significantly higher than other concentrations）,while 200 mg/L ASA significantly inhibited the proliferation（the proliferation rate was 137.5%-155%）.（2）The addition of exogenous ASA decreased the content of endogenous H₂O₂ in Korean pine embryonic cells,promoted the ASA-GSH cycle,increased the contents of endogenous ASA and GSH,as well as the activities of APX and GR enzymes;The contents of DHA and GSSG were reduced,and the values of ASA/DHA and GSH/GSSG were increased,so that the cells were in a reducing environment,among which the treatment effect of 20 mg/L or50 mg/L ASA was the most significant.At this time,the endogenous H₂O₂ content of the three cell lines decreased by 36.0%,59.2%and 69.4%respectively compared with the control,the content of ASA increased by 167%,770%and 905%respectively,and the content of GSH increased by 185%,206%and 188%respectively.（3）The addition of exogenous ASA changed the endogenous hormones in embryonic cells of Korean pine.Among the embrygenic cells of Korean pine treated with exogenous ASA at different concentrations for the same time,the content of endogenous IAA was the lowest after50 mg/L ASA treatment,which decreased by 2.1%compared with the control on the 14th day;After 200 mg/L treatment,the contents of endogenous IAA and ZR were the highest,increased by 8.2%and 12.4%respectively,and the content of ABA was the lowest,decreased by 33.4%compared with the control.In the embryogenic cells of Korean pine treated with the same concentration for different times,the contents of endogenous IAA and ABA decreased and ZR increased with the increase of days;（4）The addition of exogenous ASA changed the expression of genes related to the proliferation of embryonic cells of Korean pine.Among them,50 mg/L exogenous ASA treatment increased the expression of genes related to ASA metabolism and decreased the expression of genes related to ROS metabolism and auxin signal transduction;After 200 mg/L exogenous ASA treatment,the expression of genes related to ROS metabolism and auxin signal transduction was increased,and the expression of genes related to ASA metabolism was reduced.The relative expression of genes obtained by qRT-PCR was consistent with the results of various physiological indexes.In conclusion,the concentration of exogenous ASA that is most conducive to the proliferation of embryogenic callus of Korean pine is determined to be 50 mg/L from both physiological and molecular aspects.The physiological mechanism of exogenous ASA on the proliferation of Korean pine embryogenic cells was obtained,that is,exogenous ASA promoted the endogenous ASA-GSH cycle,increased ASA/DHA and GSH/GSSG,put the cell in a reducing environment,reduced the level of endogenous IAA,and finally promoted the proliferation of Korean pine embrygeonic cells by reducing the content of endogenous H₂O₂,increasing the content of ASA,GSH and APX,GR activities.It provides a basis for the follow-up study of genetic transformation,and optimizes the efficient large-scale propagation technology of somatic embryogenesis pathway of Korean pine.