Abstract
Somatic embryogenesis techniques have been developed for most coniferous species, but only using very juvenile material. To extend the techniques’ scope, better integrated understanding of the key biological, physiological and molecular characteristics of embryogenic state is required. Therefore, embryonal masses (EMs) and non-embryogenic calli (NECs) have been compared during proliferation at multiple levels. EMs and NECs originating from a single somatic embryo (isogenic lines) of each of three unrelated genotypes were used in the analyses, which included comparison of the lines’ anatomy by transmission light microscopy, transcriptomes by RNAseq Illumina sequencing, proteomes by free-gel analysis, contents of endogenous phytohormones (indole-3-acetic acid, cytokinins and ABA) by LC-MS analysis, and soluble sugar contents by HPLC. EMs were characterized by upregulation (relative to levels in NECs) of transcripts, proteins, transcription factors and active cytokinins associated with cell differentiation accompanied by histological, carbohydrate content and genetic markers of cell division. In contrast, NECs were characterized by upregulation (relative to levels in EMs) of transcripts, proteins and products associated with responses to stimuli (ABA, degradation forms of cytokinins, phenols), oxidative stress (reactive oxygen species) and carbohydrate storage (starch). Sub-Network Enrichment Analyses that highlighted functions and interactions of transcripts and proteins that significantly differed between EMs and NECs corroborated these findings. The study shows the utility of a novel approach involving integrated multi-scale transcriptomic, proteomic, biochemical, histological and anatomical analyses to obtain insights into molecular events associated with embryogenesis and more specifically to the embryogenic state of cell in Douglas-fir.
Highlights
Douglas-fir [Pseudotsuga menziesii (Mirb) Franco] is a conifer native to the Pacific North-West of the United States and Canada, and one of the most important timber species globally
embryogenic line (EM) and non-embryogenic callus (NEC) cultures derived from all three Douglas-fir genotypes (SD4-8, TD15-1, and TD17-1) fundamentally differed in color, morphology, cell arrangement and levels of secondary metabolites
Similar differences in sugar partitioning between embryogenic and NEC have been previously described in alfalfa (Martin et al, 2000)
Summary
Douglas-fir [Pseudotsuga menziesii (Mirb) Franco] is a conifer native to the Pacific North-West of the United States and Canada, and one of the most important timber species globally. In Europe, it is frequently used for reforestation, partly to meet increasing demand for its wood, which has outstanding mechanical properties and durability. Vegetative propagation could provide a flexible, fast and efficient way to produce enough uniform genetically improved material for dissemination (Lelu-Walter et al, 2013). Somatic embryogenesis from immature seeds coupled with cryopreservation is a promising retroactive approach for clonal propagation of selected trees (Klimaszewska et al, 2016). Despite several published studies on somatic embryogenesis in Douglas-fir (Durzan and Gupta, 1987; Pullman et al, 2009; Lelu-Walter et al, 2018; Reeves et al, 2018), and several relevant patents (Reeves et al, 2018 references therein), further information is required to realize its full potential
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