Repetitive somatic embryogenesis induced cytological and proteomic changes in embryogenic lines of Pseudotsuga menziesii [Mirb.]

Abstract

BackgroundTo explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2ry) and tertiary (3ry) lines from cotyledonary somatic embryos (SEs) of two Douglas-fir genotypes: SD4 and TD17. The 2ry lines exhibited significantly higher embryogenic potential (SE yields) than the 1ry lines initiated from zygotic embryos (SD4, 2155 vs 477; TD17, 240 vs 29 g− 1 f.w.). Moreover, we observed similar differences in yield between 2ry and 3ry lines of SD4 (2400 vs 3921 g− 1 f.w.). To elucidate reasons for differences in embryogenic potential induced by repetitive somatic embryogenesis we then compared 2ry vs 1ry and 2ry vs 3ry lines at histo-cytological (using LC-MS/MS) and proteomic levels.ResultsRepetitive somatic embryogenesis dramatically improved the proliferating lines’ cellular organization (genotype SD4’s most strongly). Frequencies of singulated, bipolar SEs and compact polyembryogenic centers with elongated suspensors and apparently cleavable embryonal heads increased in 2ry and (even more) 3ry lines. Among 2300–2500 identified proteins, 162 and 228 were classified significantly differentially expressed between 2ry vs 1ry and 3ry vs 2ry lines, respectively, with special emphasis on “Proteolysis” and “Catabolic process” Gene Ontology categories. Strikingly, most of the significant proteins (> 70%) were down-regulated in 2ry relative to 1ry lines, but up-regulated in 3ry relative to 2ry lines, revealing a down-up pattern of expression. GO category enrichment analyses highlighted the opposite adjustments of global protein patterns, particularly for processes involved in chitin catabolism, lignin and L-phenylalanine metabolism, phenylpropanoid biosynthesis, oxidation-reduction, and response to karrikin. Sub-Network Enrichment Analyses highlighted interactions between significant proteins and both plant growth regulators and secondary metabolites after first (especially jasmonic acid, flavonoids) and second (especially salicylic acid, abscisic acid, lignin) embryogenesis cycles. Protein networks established after each induction affected the same “Plant development” and “Defense response” biological processes, but most strongly after the third cycle, which could explain the top embryogenic performance of 3ry lines.ConclusionsThis first report of cellular and molecular changes after repetitive somatic embryogenesis in conifers shows that each cycle enhanced the structure and singularization of EMs through modulation of growth regulator pathways, thereby improving the lines’ embryogenic status.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1337-y) contains supplementary material, which is available to authorized users.

New Approaches to Optimize Somatic Embryogenesis in Maritime Pine

“…Embryogenic cultures of gymnosperms proliferate as EMs that typically have a whitish to translucent appearance and may have a granular to spiky (SK) morphotype due to early embryos protruding at their surface (see Gautier et al, 2018 and references therein). Modifications in water availability, either by solute-imposed water stress or by physical restriction, will impact the development of embryogenic tissue (Klimaszewska et al, 2000).…”

“…In different Picea glauca x engelmanni genotypes the ability of embryogenic tissue to utilize ABA from the medium may reflect their maturation capability (Kong and von Aderkas, 2007). The switch from EMs proliferation to somatic embryo development and maturation has recently been investigated by both transcriptome and proteome profiling (Morel et al, 2014a; Plomion et al, 2016; Trontin et al, 2016; Klubicová et al, 2017; Gautier et al, 2018). In maritime pine, yield and quality of mature somatic embryos is strongly dependent on the embryogenic line (Lelu-Walter et al, 2016; Cano et al, 2018).…”

“…To date, knowledge on the molecular factors initiating and controlling SE are scarce (Uddenberg et al, 2011; Lara-Chavez et al, 2012; Morel et al, 2014a; Rupps et al, 2016; Gautier et al, 2018). However, studies on some of the genes associated with conifer embryogenesis as LEAFY COTYLEDON ( LEC1 ), WUSCHEL-related HOMEOBOX2 ( WOX2 ) and SOMATIC EMBRYOGENESIS RECEPTOR-like KINASE ( SERK ) demonstrated that the expression of these genes varied along embryogenesis and are differentially expressed in embryogenic and non-embryogenic cultures in some species (Yang and Zhang, 2010; Klimaszewska et al, 2016).…”

Identification and Characterization of Phosphoproteins in Somatic Embryogenesis Acquisition during Oil Palm Tissue Culture

“…The ubiquitin protein ligase plays a major role as a robust marker of correct embryo development in Pinus pinaste [26] and the somatic embryogenic lines of Pseudotsuga menziesii [Mirb.] [27]. In our study, the sister chromatid cohesion protein PDS5 homolog A isoform X2 was specifically expressed in the globular, torpedo and plantlet stages that regulate chromosome segregation during mitosis and meiosis and was also found to play a role in DNA repair.…”

Somatic Embryogenesis and Plant Regeneration From Primordial Shoot Explants of Picea abies (L.) H. Karst. Somatic Trees

“…Despite similar embryo productivity, i.e., around 120 embryos per g FW in the original EM lines (derived from the zygotic embryos), the EM sublines of two responsive Norway spruce genotypes grew very differently after initiation. This may be due to differentially expressed proteins like in secondary and tertiary lines of Douglas-fir ( Gautier et al, 2018 ). The EM sublines of 11Pa3794 had much lower embryo productivity than sublines of 11Pa4066, possibly due to the untimely subculture, or other factors.…”