Caroline Teyssier

In search of markers for somatic embryo maturation in hybrid larch (Larix × eurolepis): global DNA methylation and proteomic analyses

A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level: from 45.8% mC for undifferentiated somatic embryos (1-week proliferation) to 61.5% mC for immature somatic embryos (1-week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8-weeks maturation). The presence of 5-azacytidine (hypo-methylating agent) or hydroxyurea (hyper-methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin- and vicilin-like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two-dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper- and hypo-treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos.

Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses.

Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.

Somatic Embryogenesis for More Effective Breeding and Deployment of Improved Varieties in Pinus spp.: Bottlenecks and Recent Advances

Global transition towards a bioeconomy sets new demands for wood supply (bioenergy, biomaterials, biochemicals, etc.), and the forestry sector is also expected to help mitigate climate change by increasing carbon fixation. For increased biomass production, the use of improved, genetically superior materials becomes a necessity, and vegetative propagation of elite genotypes provides a potential delivery mechanism for this. Vegetative propagation through somatic embryogenesis alone or in combination with rooted cuttings obtained from somatic young trees can facilitate both tree breeding (greater selection accuracy and gains, breeding archives of donor material for making crosses after selection) and the implementation of deployment strategies for improved reforestation materials. To achieve these goals, progress in the efficiency of pine somatic embryogenesis biotechnology has been made for a few commercial pine species, and a better understanding has been gained of the molecular mechanisms underpinning somatic and zygotic embryo development.

Micromorphology, structural and ultrastructural changes during somatic embryogenesis of a Tunisian oat variety (Avena sativa L. var ‘Meliane’)

To better understand micromorphological and structural changes, histological sections provide additional insight into cellular process and developmental pathways occurring in oat somatic embryogenesis. Environmental scanning electron microscopy (ESEM) and transmission electron microscopy (TEM) were also used to follow the ultrastructural modifications during this system. Histological observations allowed following the events leading to the development of mature somatic embryos. The scheme includes the following steps: cell reactivation, the first organized cell division in diads, triads, tetrads as well as octant stages, the observation of an extracellular matrix (ECM) as a fibrillar material that bounded the surface of individualized proembryos. The transition from proembryo stage to an early globular somatic embryo was noted, where the embryogenic cortex is surrounded by the protoderm. The late globular stage was marked by bipolarity. The early and late transitional stages, the coleoptilar, mature and germinated stages were also described. The ESEM allowed us to follow some rearrangements, related to the morphology and surfaces involved in somatic embryos development. These events are proembryo formation, transition from proembryo to globular stage, marked by protoderm formation, scutellum and coleoptile development and finally somatic embryos germination. The TEM showed that embryogenic cells were very rich in organelles; mitochondria, rough endoplasmic reticulum, Golgi apparatus and ribosomes. Cells of proembryos, globular and late somatic embryos showed more vacuoles and differentiated organelles. The ECM was also detected by TEM as fibrillar material coating the cell walls. These results on structural and ultrastructural changes provided new insights and findings on oat somatic embryogenesis.