Combination of H2O2 and Ca2+ Regulates Regeneration of Successively Subcultured Somatic Embryos in Pinus massoniana

A major barrier to the large-scale production of plantlets using somatic embryogenesis technology in Pinus massoniana is recalcitrance to continue somatic embryo proliferation and germination, resulting from degeneration of successively transferred somatic embryos. However, the mechanism underlying reduced regeneration capacity of somatic embryos from long-term subculture remains unexplored. Thus, an integrative analysis for molecular and physiological mechanisms on degeneration of somatic embryos during long-term subculture was performed to identify key factors involved in regulating somatic embryo proliferation and germination. Regeneration ability of somatic embryos was distinct between 10 and 30 transfer cycles (S10, S30). In comparison to S10 somatic embryos, proliferation coefficient and germination rate was respectively reduced by 90.9% and 86.1% for S30 somatic embryos. Transcriptome analysis indicated that a total of 841 differentially expressed genes (DEGs) were identified, consisting of 826 upregulated and 15 downregulated genes (S30 vs S10). By exploring the biological metabolic pathways associated with the DEGs, signal transduction related to redox was one of the top five most significantly enriched pathways. In addition, almost all of DEGs involved in the pathway were upregulated. With an application of 1.0 mM antioxidant against H2O2, glutathione (GSH), in the culture media, increases in both proliferation coefficient and germination rate were investigated for S30 somatic embryos, which indicated that the oxidization might be a possible cause of the declined regeneration for somatic embryos after long-term subculture. Further evidence revealed the physiological mechanism involved in somatic embryo response to oxidization by which H2O2 regulated Ca2+ signal by increasing Ca2+ influx and decreasing Ca2+ efflux, resulting in cytosolic Ca2+ accumulation. In addition, we also observed that somatic embryo regeneration was able to be furtherly enhanced for those cultured on media containing low Ca2+ concentration. Our findings suggest that improved regeneration of somatic embryos from long-term subculture gets involved in H2O2 and Ca2+. We have developed information demonstrating that regulation of Ca2+ level and oxidation environment in vitro can enhance proliferation and germination of somatic embryos in P. massoniana.