| Picea balfourian is an evergreen conifer, which is distributed mostly in the southwest andnorthern margin species of Picea grow on the Tibetan plateau. Due to the high quality of thewood of this specie as well as their fast growth, their plantations are regularly selected as majorspecies for afforestation. However, it reproduces primarily sexually, its seedlings initially growslowly, as well as it sets seed late. So research on somatic embryogenesis of Picea balfourianwould lay the foundation for the scarcity of seedlings. To understand the molecularmechanisms regulating early somatic embryogenesis in Picea balfouriana, we used the newlydeveloped RNA-seq technology to investigate the transcriptome. We compared the expressedgenes of embryogenic and non-embryogenic tissues from three Picea balfouriana genotypes.In addition, RNA-Seq, iTRAQ and GS-MS were combined to clarify the mechanism under theembryogenic ability affected by6-BAP. Finally, the content of endogenous hormones andpolyamines, and the activities of antioxidase were investigated. The mean results are asfollows:Zygotic embryos with different developmental stages of eight genotypes from Piceabalfouriana were used as explants to explore the effects of developmental stages, basal media,as well as different kinds and levels of plant growth regulators on callus and somatic embryosinduction. The results showed that the developmental stage2is most suitable for the inductionof embryogenic tissue and somatic embryos. The best initiation medium was1/2LMmedium+10μM2,4-D+5μM6-BAP+3.8μM ABA or20μM AgNO3, with the highest initiationrate40%; The best maturation medium was1/2LM medium+61μM ABA+0.4%active charcoal,and every100mg embryogenic tissues could produced124somatic embryos. Optimal mediumfor germination was1/4LM medium+0.125%active charcoal+50μM ascorbic acid (24.83%).This study indicated that both of ABA and AgNO3could increase the initiation rate ofembryogenic tissue by10%at least. A total of54978834reads were obtained for embryogenic and non-embryogenic tissuesof Picea balfouriana, and for pro-embryos and cotyledon embryos of Picea likiangensis. Usingthe National Center for Biotechnology Information database annotation, we obtained68070identified unigenes. In total,18897(27.8%) unigenes were classified into Gene Ontology, and15439(22.7%) unigenes were found in121KEGG categories. These genes includedrepresentatives from almost all functional categories. Using Solexa,3280differentiallyexpressed genes (P <0.05) were detected by comparative analysis of the expression profiles inembryogenic and non-embryogenic tissues, including1371significantly up-regulated genesand1909significantly down-regulated genes. In addition, SERK and arabinogalactan proteins,may could act as molecular makers in the early stage of somatic embryogenesis as previouslyreported.A total of51375unigenes and2617proteins were obtained after quality filtering. Therewere2770transcripts for proteins found among these unigenes. GO analysis of the mostregulated unigenes and proteins showed that they were involved in cell and binding activityand were enriched in ribosome and glutathione metabolism pathways. Ribosomal proteins,glutathione S-transferase proteins, germin-like proteins and calmodulin-independent proteinkinases were up-regulated in the embryogenic tissues with the highest embryogenic ability(treated with3.6μM6-BAP), which was validated via qRT-PCR analysis, and these proteinsmight serve as molecular markers of embryogenic ability.A total of309compounds were obtained, among which123metabolite identities could beassigned to KEGG. The levels of35metabolites were significantly different among the threetreatments, and32differed between2.5μM and5μM treatments with lower embryogeniccapacities. A total of17metabolites appeared only once among the three comparisons. Thecombination of S-plot and Loading plot showed that mostly up-regulated metabolites wereamino acids, whereas primary carbohydrates (especially sugars) were down-regulated insamples with higher embryogenic ability. These results suggested that6-BAP was likely toinfluence the embryogenic competence by nitrogen metabolism, leading amino acids, such as the amount of aspartate, isoleucine, and leucine, increase in tissues with higher embryogenicability. Furthermore, the results speculated that6-BAP may affect the amount of tryptophan,which leading level of Indole-3-acetic acid (IAA) changed and embryogenic ability waschanged finally. Asparagines, tryptophan and serine could be metabolites makers foridentification of embrogenic ability.The contents of endogenous hormonals (IAA, ZR, ABA and GA3) in embryogenic tissuesof three genotypes were all lower than those in non-embryogenic tissues. However, theactivities of SOD and POD and contents of polyamines (Put, Spd and Spm) in embryogenictissues of three genotypes were all higher than those in non-embryogenic tissues. The contentof IAA and the activity of SOD were highest in tissue treated by3.6μM6-BAP, whereas, thecontent of ZR and the activity of POD were lowest. The contents of IAA and ZR decreased tothe lowest, but GA3and ABA increased to the highest when compared with2.5μM and5μMtreatments. The contents of polyamines in tissues treated by3.6μM6-BAP were higher thanother two treatments, especially after transferred to somatic embryos initiation medium.In conclusion, complicated biochemical and molecular regulation mechanism wereinvolved in the process of Picea balfourian somatic embryogenesis, which was the result of theintereaction of genes, proteins and metabolites. This study not only lain a good foundation forobtaining and maintenance tissues with higher embryonic ability, but also provided substantialbasis for solving the further application of the technology of somatic embryogenesis inconifers. |
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