Physiological And Molecular Mechanisms Of Somatic Embryogenic Potential Of Korean Pine Cell Lines

Somatic embryogenesis is currently one of the most promising methods for conifer reproduction in commercial use and can rapidly produce large quantities of high-value seedlings that meet the needs of plantations.This plays an important role in tree improvement,establishment of commercial forests,and increased forest productivity,sustainability and consistency.Although many conifer species have successfully obtained regenerated plants through somatic embryogenesis,there are still some important problems to be solved.In conifers,serious genotype-dependent and embryogenic loss problems were found during somatic embryogenesis,which seriously affected the cultivation and propagation of elite tree species and limited the commercial development of this technology.Therefore,the analysis of the differences and key driving factors of different somatic embryogenic cell lines in the process of somatic embryogenesis plays a crucial role in dissecting the molecular genetic network of somatic embryogenesis expression.In this study,the cell lines of Korean pine with different somatic embryogenic abilities were used as research objects.After synchronization treatment,the somatic embryogenesis ability of different cell lines was identified,and morphological observation and physiological measurement were carried out,in order to screen out the morphological indicators and biochemical markers of somatic embryogenesis ability cell lines.Therefore,in this study,the cell lines of Korean pine with different somatic embryogenic abilities were used as research materials.Morphological indicators and biochemical markers of somatic embryogenic cell lines were screened.To investigate the morphological and physiological changes of the responsive somatic embryogenesis cell line（R-EC）,the blocked somatic embryogenesis cell line（B-EC）and the loss of somatic embryogenic cell line（L-EC）during somatic embryogenesis.Transcriptomic and metabolomic studies were performed simultaneously on R-EC,B-EC and L-EC.To comprehensively explore the dynamic transcriptome profiles,gene regulation and metabolic pathway patterns involved in somatic embryogenesis of cell lines with differential somatic embryogenesis.Analyzing the key factors that determine the expression of totipotency in somatic cells lays the foundation for revealing the molecular genetic network of totipotency expression in plant cells.The main findings are as follows:（1）The embryogenic callus suspension culture system of Korean pine was successfully established,and 40 mg·m L-1 embryogenic callus was transferred to 20 m L liquid medium in a200 m L flask,cultured at 100 rpm for 14 d to obtain the maximum proliferation（FW=1.96g,DW=0.132g）.Embryogenic callus propagated in liquid medium had the ability to mature after transfer to somatic embryo maturation medium and the yield of somatic embryos was higher than that of semi-solid medium.（2）Established using the number of primordial mass III（PEMIII）and early somatic embryos（E-SE）as morphological indicators for screening early high somatic embryogenic cell lines,while low levels of starch content,indole acetic acid（IAA）,abscisic acid（ABA）content and putrescine:spermine（Put:Spm）as physiological markers.It was also found that with the long-term proliferation of embryogenic callus,the somatic embryogenic ability gradually decreased,and the somatic embryogenic ability was completely lost after 12 months of proliferation.During this period,starch content gradually decreased（1.3-2.6-fold）and IAA content in most cell lines showed an upward trend.（3）During proliferation,there was no significant difference in fresh weight between R-EC and B-EC,but both were significantly higher than L-EC.There was no significant difference in the amount of PEMIII in R-EC and L-EC,both were higher than in B-EC,while the amount of E-SE in R-EC was significantly higher than that in L-EC and B-EC.The storage substances in R-EC were significantly higher than that in B-EC and L-EC,and the content of storage substances in L-EC was the lowest,and the antioxidant enzyme activity in R-EC was significantly higher than that in B-EC and L-EC at the later stage of proliferation culture.The antioxidant enzyme activities in EC and B-EC were significantly higher than those in L-EC,and it was also found that the contents of IAA and Put were significantly accumulated in L-EC compared with R-EC and B-EC.（4）During the maturation of R-EC,B-EC and L-EC somatic embryos,the existence of early somatic embryos could be observed in R-EC after 7 days of culture.After 7 days,the early somatic embryos gradually developed and formed complete somatic embryos.However,early somatic embryos were observed in L-EC and B-EC at 7 d and 14 d,respectively,but with the extension of somatic embryo maturation and culture time,early somatic embryos could not further develop to the late stage of early somatic embryos and were gradually aborted.At the later stage of somatic embryo development,the ABA content in R-EC was significantly higher than that in B-EC and L-EC.The levels of starch,catalase（CAT）,superoxide dismutase（SOD）,catalase（POD）,hydrogen peroxide（H₂O₂）,adenosine triphosphate（ATP）,Put and IAA in B-EC were significantly higher than R-EC and L-EC.Soluble protein and reduced coenzyme II（NADPH）in R-EC and L-EC were significantly higher than B-EC.（5）In R-EC and L-EC,2566 differentially expressed genes（DEGs）were identified and assigned to 122 KEGG pathways.In R-EC and B-EC,13,768 DEGs were assigned and assigned to 137 KEGG pathways,and in B-EC and L-EC,13,900 DEGs were assigned and assigned to 137 KEGG pathways.23,319 DEGs in R-EC and R-SE were assigned to 139KEGG pathways.The most representative pathways are phenylpropane biosynthesis,plant hormone signal transduction,flavonoid biosynthesis,and starch and sucrose metabolism.We found the most DEGs annotated to peroxidase in phenylpropane synthesis.During plant hormone signal transduction,AUX1 and AUX/IAA family genes were significantly up-regulated in L-EC,and TIR1,ARF and CH3 family genes were significantly up-regulated in R-EC.Most ABA-related genes were highly expressed in R-SE,and most ethylene signaling differentially expressed genes were highly expressed in R-SE and B-SE.Most enzymes were significantly up-regulated in L-ECs during starch and sucrose metabolism.In addition,the expression level of most of the DEGs involved in the regulation of SOD was higher in L-EC than in R-EC and B-EC,while most of the DEGs involved in CAT and glutathione peroxidation during the somatic embryo maturation culture of R-EC.The expression of DEGs of GPX was significantly up-regulated,and the expression of most of the DEGs involved in the regulation of GPX was significantly increased during the maturation and culture of B-EC somatic embryos.（6）During the somatic embryogenesis of Pinus chinensis,a total of 1113 metabolites were detected.Among them,the phytochemical properties of R-SE are quite different from other tissues.High accumulation of flavonoids,terpenoids,lignans,and quinones was observed in R-SE.Flavonoid biosynthesis and phenylpropanoid biosynthesis pathways were identified as key metabolic pathways affecting somatic embryogenesis potential by combined transcriptomic and metabolomic analysis.In flavonoid biosynthesis,phloretin,prunin,avucatechin,naringenin,epicatechin,catechin,epiaphlocatechin,chalcone 2’O-glucoside and dihydromyricetin were significantly up-regulated in R-EC,naringenin,chalcone,vitexin,apigenin and gallocatechin were significantly up-regulated in B-EC,and phloridzin was significantly up-regulated in L-EC.In addition,among R-SE,B-SE and L-SE,14 metabolites were significantly up-regulated in R-SE,7 metabolites were significantly up-regulated in B-SE,and no metabolites were significantly up-regulated in L-SE.In phenylpropane biosynthesis,8metabolites were up-regulated in R-SE,7 metabolites were up-regulated in B-SE,and 14metabolites were up-regulated in L-SE.In the comparison of R-SE,B-SE and L-SE,1metabolite was up-regulated in R-SE,4 metabolites were up-regulated in B-SE,and 1metabolite was up-regulated in L-SE,among which the differential metabolites included scopolamine,caffeic acid,ferulic acid and coniferin.In conclusion,this study obtained the morphological and physiological markers of some high somatic embryogenic cell lines by comparing the differences between different somatic embryogenic cell lines.It was also found that early somatic embryo abortion,changes in endogenous auxin content and redox reactions were closely related to the loss of somatic embryogenesis.These findings not only have important theoretical significance for in-depth understanding of the mechanism of regulating somatic embryogenesis during somatic embryogenesis,but also provide theoretical basis for accelerating the application of biotechnology in large-scale artificial breeding.