Optimization Of Synchronization Of Somatic Embryogenesis And Transcriptome Analysis Of Larix Olgensis

Larix ssp.is an important forest tree species.Efficient and stable larch somatic embryo regeneration system can make excellent varieties reproduce quickly,and also lay a foundation for physiological,biochemical and genetic transformation studies.Analysis of transcriptomes at different developmental stages of somatic embryos is helpful to understand the mechanism of embryonic development.The immature zygote embryos of Larix olgensis collected on June 15 and June 25 were used as explants to induce embryogenic callus,and the embryogenic callus induction rate was about 30%.The components of the callus induction medium are: S medium + 2,4-D1.0mg/L + 6-BA 0.5mg/L + KT 0.3mg/L,sucrose 20g/L,agar 3g/L,p H 5.8.?6.0.Comparing the differences between embryogenic callus subcultured on different subculture media,it was found that Under the conditions of more Available water and higher plant growth regulator(2,4-D,6-BA)concentration,the ESM(embryogenic suspensor mass)are mostly at the PEM(proembryogenic mass)I or PEMII.However,under the conditions of less available water and lower plant growth regulator concentration,the ESM are mostly at PEMIII.Successively use 4 subculture mediums to subculture the induced embryogenic callus,gradually reduce the concentration of auxin and cytokinin and reduce the concentration of available water,,so that the ESM can develop to PEM III,with similar size and synchronized development,is conducive to optimizing the generation of somatic embryos.In turn,three maturation media A,B,and C were used to induce maturation of somatic embryos.Mature medium A is S medium + PEG4000 100 g/L + ABA 15 mg/L,sucrose 45 g/L liquid medium;The maturation media B and C are maturation media A supplemented with 2 g/L and 10 g/L agar,respectively.Compared with the single maturation medium,the maturation schemes of the three mediums optimized the formation of somatic embryos.The maturation time of mature somatic embryos in this method was about 32 days,the synchronization rate was 78%,and the proportion of normal morphological embryos was 83%.The number of somatic embryos in normal morphology was 223/g embryogenic callus,and the germination rate of somatic embryos was 47.2%Samples were taken at different stages of somatic embryo development(PEM,early embryogeny,late embryogeny)to analyze gene expression levels.Functional annotation found that among the differentially expressed genes of cotyledonary embryos and PEM,44 late embryogenesis abundant proteins genes,16 storage proteins and precursor genes,13 ABA-related genes and 3 larch growth regulating factor genes expression levels were up-regulated.GO enrichment analysis and KEGG enrichment analysis of differentially expressed genes showed that the pathways with high levels of differentially expressed genes are: flavonoid biosynthesis,plant hormone signaling pathway,phenylpropanoid biosynthesis,MAPK signaling pathway,diterpenoid biosynthesis and fatty acid degradation.,etc.