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International audience; Somatic embryogenesis (SE) has high potential for large-scale clonal propagation of conifers. Different types of bioreactor cultures have been tested for the conifer SE process where the temporary immersion bioreactors (TIBs) have proved to be useful across the different developmental steps of the SE process. In the present study the use of TIBs was tested for hybrid larch (Larix × eurolepis Henry). The results showed twofold increases in both fresh weight (FW) of pro-embryogenic masses (PEMs) and yield of cotyledonary embryos in the TIBs compared to solid medium in plates. For the germination phase, the highest number of roots per plant, the root length and height of plants were also obtained in the TIBs. The results show that the TIB system can be successfully used to support scale up of plant production in all steps of the SE process from proliferation to germination of hybrid larch (Larix × eurolepis Henry).

Aluminum (Al) is well known as a potent inhibitor of plant growth and development. It is notably present in soils in the soluble and bioavailable form Al3+ when the soil pH drops below 5. This situation is frequent, especially in softwood forests when litter decomposition is slow. In the present work, we studied the effects of Al3+ on the growth and development of Douglas fir plantlets. Somatic plantlets, regenerated via somatic embryogenesis, were grown in vitro on media supplemented with different concentrations of aluminum chloride (AlCl3): 0 &micro, M, 200 &micro, M, 500 &micro, M. and 1 mM. We show that a concentration of 500 &micro, M AlCl3 in medium significantly reduced root elongation (&minus, 21.8%), as well as stem growth (&minus, 14.6%). Also, a 25% reduction in dry mass of the plantlets was observed in presence of a concentration of 200 &micro, M of AlCl3. Histological analysis of root tissues revealed significant damage, especially in conducting vessels. In addition, mineral cation content of plantlets was disturbed under Al exposure. More particularly, the Mg and K contents of needles and the Ca content of stems and needles were significantly reduced in presence of a concentration of 500 &micro, M AlCl3 in the culture medium (&minus, 35.6%, &minus, 33.5%, &minus, 24%, and &minus, 34% respectively). However, all these damages appeared at relatively high Al concentrations when compared with other herbaceous species. This study shed light on the ability of Douglas fir in vitro plantlets to cope with the acid-driven toxicity of Al.

Douglas-fir is a conifer species of major economic importance worldwide, including Western Europe and New Zealand. Herein we describe some characterization and significant refinement of somatic embryogenesis in Douglas-fir, with focus on maturation. The most typical structures observed in the embryonal masses were large polyembryogenic centres (up to 800-1500 μm) with a broad meristem, creating a compact ce ll “package” with suspensor cells. Singulated somatic embryos composed of both a embryonal head (300-400 μm) and long, tightly arranged suspensor were also frequent. Embryo development was enhanced following embryonal mass dispersion on filter paper discs at low density (50-100 mg fresh mass). Moreover, increasing gellan gum concentration in maturation medium (up to 10 g L-1) improved both the quantity and quality of cotyledonary somatic embryos (SEs), which were subsequently able to germinate and develop into plantlets at high frequency. Embryogenic yield was highly variable among the seven embryogenic lines tested (27-1544 SE g-1 fresh mass). Interestingly secondary somatic embryogenesis could be induced from cotyledonary SEs of both low- and highly-productive lines with some useful practical outcomes: secondary lines from low-performance lines (30-39 478 SE g-1) displayed significantly higher embryogenic yield (148-1343 SE g-1). In our best conditions, the total protein content in cotyledonary SEs increased significantly with maturation 41 duration (up to 150 μg mg-1 fresh mass after 7 weeks) but remained below that of mature zygotic embryos (300 μg mg-1). The protein pattern was similar in both somatic and zygotic embryos, with major storage proteins identified as 7S-vicilin- and legumin-like proteins.

Key message This review highlights some of the discoveries and applications made possible by “omics” technologies over the last 10 years and provides perspectives for pioneering research to increase our understanding of tree biology. Context A decade after the first forest tree genome sequence was released into the public domain, the rapidly evolving genomics and bioinformatics toolbox has advanced our understanding of the structure, functioning, and evolution of forest tree genomes. Aims and methods This review highlights some of the discoveries and applications that “omics” technologies have made possible for forest trees over the past 10 years. Results In this review, we start by our current understanding of genome evolution and intricacies of gene regulation for reproduction, development, and responses to biotic and abiotic stresses. We then skim over advances in interactome analysis and epigenomics, the knowledge of the extent of genetic variation within and between species, revealing micro- and macro-evolutionary processes and species history, together with the complex architecture of quantitative traits. We finally end with applications in genetic resource conservation and breeding. Conclusion The knowledge gained through the use of these technologies has a huge potential impact for adapting forests to the main challenges they will have to face: changing demand from ecosystem services with potentially conflicting strategies in terms of conservation and use, as well as climate changes and associated threats. Genomics will undoubtedly play a major role over the next decade and beyond, not only to further understand the mechanisms underlying adaptation and evolution but also to develop and implement innovative management and policy actions to preserve the adaptability of natural forests and intensively managed plantations.

International audience; 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.

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.

Figure S3. Histology of non-embryogenic cells (NECs) clusters from 1ry and/or 2ry lines of genotypes TD17 and SD4. A / Histology of TD17 groups of loosely arranged NECs (arrow) in the vicinity of small somatic embryos (arrowhead); B / histology of TD17 compact NEC cluster accumulating phenolics (blue and brown cells, arrowheads) and starch (arrow); C / Lugol staining of TD17 NEC cluster showing starch (arrows) and phenolic compounds (small dark granules marked with arrowhead); D / TD17–1 NEC cluster with dividing cells (arrowheads); E /SD4–2 NEC cluster (arrow) within polyembryogenic center besides well-arranged embryonal heads (EH), note phenolic content (in dark blue-grey) of cells separating NEC from suspensor cells (in light blue); F / SD4–6 meristemoid-like NEC cluster; note phenolic content of cells between two meristemoid-like structures (in blue-grey). Scale bar: A, E = 200 μm; B, F = 100 μm; C, D = 50 μm. (DOCX 417 kb)

Figure S1. Macroscopic aspect of embryonal masses (EMs) from two Douglas-fir genotypes (SD4, TD17). These embryonal masses were obtained after 1ry somatic embryogenesis from zygotic embryo and two cycles (2ry and 3ry) of repetitive somatic embryogenesis from somatic embryos. Note the granular aspect of most embryonal masses indicating the occurrence of large polyembryogenic centers (arrowheads) and/or singulated early somatic embryos that are sometimes protruding from the embryonal masses surface. (DOCX 687 kb)

Figure S2. Structure of embryonal masses from primary and secondary lines of genotype TD17. A / TD17; B / TD17–1. Trypan blue staining of squashes of fresh EMs; m – meristem of polyembryogenic centers, s – suspensor. Scale bar = 200 μm. (DOCX 178 kb)

Table S3. Total protein content (mean ± SD, n = 4) in proliferating 2ry and 3ry embryogenic lines of Douglas-fir. (DOCX 45 kb)

Table S2. Total protein content (mean ± SD, n = 4) in proliferating 1ry and 2ry embryogenic lines of Douglas-fir. (DOCX 45 kb)

This paper describes the micropropagation of Douglas-fir via somatic embryogenesis using media and methods available in the public domain. Douglas-fir is a conifer species with high and increasing interest for forest industries in both New Zealand and France. Delivery of the best trees to the forest from breeding programmes is currently constrained by an inability to effectively and reliably multiply selections through vegetative propagation. Somatic embryogenesis coupled with cryopreservation is essential biotechnology tool in conifers for scaling up variety design and production. The aim of this work was therefore to develop protocols for the initiation and proliferation of Douglas-fir embryogenic cell lines based on the modifications of previously published techniques and media available in the public domain, especially successful methods developed for P. radiata at Scion. Three years of initiation experiments have resulted in a simple and efficient protocol. Disinfection of whole cones (instead of seeds) was sufficient to prevent contamination. Immature zygotic embryos were excised from megagametophytes and placed onto a modified Litvay medium (Plant Cell Rep 4(6):325–328, 1985***). At optimal development stages of zygotic embryos, the average initiation percentage of embryogenic tissue was 69.3% when our most effective protocol was used. Proliferation of initiated cell lines on a Glitz formulation was challenging, with a high percentage of cell lines composed of a mixture of both embryonal masses and callus. 2,4-D at a lower concentration reduced the number of such mixed lines. Repeated liquid suspension and subculture of the embryogenic parts of the tissue was an efficient means to increase the number of embryogenic cell lines with sustained proliferation. Maltose added to the proliferation medium in place of sucrose improved fresh mass gain and consistently increased early somatic embryo patterning and growth. A sample of proliferating cell lines was successfully cryopreserved, thawed and somatic embryos were matured and germinated from these lines. The method may be of practical interest and provide new opportunities to realize increased genetic gain in this species through the clonal-assisted deployment of the best genetic material in both New Zealand and France.

Table S1. A summary of macromorphological (EM colour and morphotype) and histo-cytological traits (occurrence of polyembryogenic centers (PECs), singulated SEs and NECs) of 1ry (SD4, TD17), 2ry (SD4–2, SD4–6, SD4–8; TD17–1) and 3ry (SD4–8-1, SD4–8-2, SD4–8-3) embryogenic lines of Douglas-fir. (DOCX 16 kb)

Table S4. Functional classification according to gene ontology (GO) of significant proteins identified after two cycles of repetitive somatic embryogenesis in Douglas-fir. (DOCX 72 kb)

International audience; 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.

Douglas-fir (Pseudotsuga menziesii (Mirb) Franco) is a native conifer from the Pacific North-West of the US and Canada, and is one of the most important timber species used in the world. Douglas-fir is highly productive in a range of climatic conditions and has valuable wood properties (quality) as well as a strong tolerance to diseases and insects. In Europe, Douglas-fir is a major species for reforestation with increasing demand for its wood. Adaptation of new varieties to climate change and associated stresses is one challenging question for ongoing breeding programs. Efficient selection and vegetative propagation of improved varieties are key issues to maintain productivity in plantation forestry (Lelu-Walter et al. 2013), however, as in many other conifers, early maturation is preventing clonal forestry through conventional multiplication methods in Douglas-fir (Bastien et al. 2013). Somatic embryogenesis from immature seeds, coupled with cryopreservation, is a promising retroactive clonal propagation system of selected trees. This technology has been developed for an increasing number of conifer species (Klimaszewska et al. 2016). Excluding patents, there are only a few published studies on Douglas-fir somatic embryogenesis (Durzan and Gupta 1987; Pullman et al. 2005, 2009). One recurrent problem is the sustainable multiplication of initiated embryogenic material, i.e., embryonal masses (EMs). Yellowish, non-embryogenic cells (NEC), which are interspersed with EM, is frequently observed during this process. In this work, we describe EM morphology and cytology (light and confocal microscopy) of different embryogenic lines. A suggested method to reduce the formation of NEC and sustain EM proliferation is presented.

International audience; Pinus pinaster (Ait.) somatic embryogenesis (SE) has been developed during the last decade, and its application in tree improvement programs is underway. Nevertheless, a few more or less important problems still exist, which have an impact on the efficiency of specific SE stages. One phenomenon, which had been observed in embryogenic tissue (embryonal mass, EM) initiated from immature seed, has been the loss of the ability to produce mature somatic embryos after the tissue had been cultured for several months. In an attempt to get insight into the differences between young cultures of EM (3-mo-old since the first subculture) of P. pinaster that produced mature somatic embryos and the same lines of significantly increased age (18-mo-old, aged EM) that stopped producing mature somatic embryos, we analyzed in both types of materials the levels of endogenous hormones, polyamines, the global DNA methylation, and associated methylation patterns. In addition, we included in the analysis secondary EM induced from mature somatic embryos. The analysis showed that the two tested genotypes displayed inconsistent hormonal and polyamine profiles in EM cultures of a similar phenotype and that it might be difficult to attribute one specific profile to a specific culture phenotype among genotypes. Experiments were also undertaken to determine if the global DNA methylation and/or the resulting methylation pattern could be manipulated by treatment of the cultures with a hypomethylating drug 5-azacytidine (5-azaC). An aged EM was exposed to different concentrations and durations of 5-azaC, and its response in culture was established by fresh mass increases and somatic embryo maturation potential. All of the analyses are new in maritime pine, and thus, they provide the first data on the biochemistry of EM in this species related to embryogenic potential.

chapitre en open access; Maritime pine is a major species in Europe, especially in France, Portugal and Spain. This species has long been subjected to advanced forestry and genetic breeding since the early sixties in France. However there are strong limitations to improvement progress, i.e. quite long generation time, high genetic load as well as high genetic redundancy within breeding population. Sudden and drastic socio-economic and environmental changes in recent years would need a significant paradigm shift in current breeding technology to deliver suitable tested tree varieties in plantation forestry, i.e. multi-varietal forestry targeting a wide range of end-products through various silvicultural regimes. Field comparison of vegetative propagules is a key towards individual selection and efficient capture of the best genetic stocks. Highly efficient clonal propagation technology is also required for scaling up production of improved varieties. Somatic embryogenesis is considered as the key technology to fulfil such requirements in maritime pine. The species is characterized by increased recalcitrance to vegetative propagation through conventional cuttings as trees are reaching their adult vegetative or reproductive phase. Somatic embryogenesis initiation from mature trees is still challenging in conifers. Therefore the approach developed in Maritime pine is the postponed propagation of tested trees combining somatic embryogenesis initiation from immature zygotic embryos and stable cryopreservation of juvenile embryogenic tissue. This review describes the most recent achievements and challenges towards efficient somatic embryogenesis as a key technology for multi-varietal forestry in Maritime pine.

chapitre en open access; International audience; Vegetative propagation of forest trees offers advantages to both tree breeders and the forest industry. In larch vegetative propagation has chronologically followed three major developments: (i) clonal propagation by cuttings; (ii) ‘bulk’ vegetative propagation by cuttings and (iii) somatic embryogenesis. The latter has potentially numerous applications such as the production of a large number of genetically improved plants and the amenability of embryogenic cultures to be stored in liquid nitrogen. In Larix sp. several improvements of the somatic embryogenesis protocol have been developed. Maturation conditions are now well enough refined to regenerate high quality somatic embryos that are highly similar to zygotic embryos in their anatomy, physiology and protein content. Among conifer species, somatic embryogenesis of Larix has become a model for its multiple uses; its integration in a breeding program is now undertaken for clonal propagation of improved material of hybrid larch Larix x eurolepis. Indeed, somatic embryogenesis assists breeding strategies by offering an alternative tool for at the same time accelerated production of plants for clonal testing and then for mass production; in addition, cryoconservation allows keeping material in a juvenile state and allows mass production of stored material at any time. This review describes the different methods of vegetative propagation of larch sp., in particular the advances in somatic embryogenesis and requirements for its integration into a breeding program.

Phytoextraction of Cd is a growing biotechnology although we currently know few Cd hyperaccumulators, i.e., plant species able to accumulate at least 0.1 mg Cd g(-1) dry weight in aerial organs. Owing their deep root system and high biomass, trees are more and more preferred to herbaceous species for phytoextraction. Assuming that conifers could be relevant models under cold climates, we investigated cadmium tolerance of the hybrid larch Larix × eurolepis Henry (Larix decidua × Larix kaempferi) and the efficiency of this species to store this metal. In vitro grown larches were chosen in order to reduce time of exposure and to more rapidly evaluate their potential efficiency to accumulate Cd. One-month-old plantlets were exposed for 2 and 4 weeks to 250 and 500 μM Cd. Results showed that they tolerated a 4-week exposure to 250 μM Cd, whereas the content of photosynthetic pigment strongly dropped in plantlets growing in the presence of 500 μM Cd. In the presence of 250 μM Cd, shoot growth slightly decreased but photosynthetic pigment and total soluble carbohydrate contents were not modified and no lipid peroxidation was detected. In addition, these plantlets accumulated proline, particularly in shoots (two to three times more than control). In roots, Cd concentration in the intracellular fraction was always higher than in the cell wall fraction contrary to shoots where Cd concentration in the cell wall fraction increased with time and Cd concentration in the medium. In shoots, Cd concentration was lower than in roots with a ratio of 0.2 after 4 weeks of exposure but stayed around 0.2 mg g(-1) dry weight, thus a value higher than the threshold requested for Cd hyperaccumulators. Hybrid larch would thus be a relevant candidate for field test of Cd phytoextraction.

Genome-wide profiling (transcriptomics, proteomics, metabolomics) is providing unprecedented opportunities to unravel the complexity of coordinated gene expression during embryo development in trees, especially conifer species harboring "giga-genome." This knowledge should be critical for the efficient delivery of improved varieties through seeds and/or somatic embryos in fluctuating markets and to cope with climate change. We reviewed "omics" as well as targeted gene expression studies during both somatic and zygotic embryo development in conifers and tentatively puzzled over the critical processes and genes involved at the specific developmental and transition stages. Current limitations to the interpretation of these large datasets are going to be lifted through the ongoing development of comprehensive genome resources in conifers. Nevertheless omics already confirmed that master regulators (e.g., transcription and epigenetic factors) play central roles. As in model angiosperms, the molecular regulation from early to late embryogenesis may mainly arise from spatiotemporal modulation of auxin-, gibberellin-, and abscisic acid-mediated responses. Omics also showed the potential for the development of tools to assess the progress of embryo development or to build genotype-independent, predictive models of embryogenesis-specific characteristics.

This review compiles research results published over the last 14 years on conifer somatic embryogenesis (SE). Emphasis is placed on the newest findings that affect the response of seed embryos (typical explants) and shoot primordia (rare explants) to the induction of SE and long-term culture of early somatic embryos. Much research in recent years has focused on maturation of somatic embryos, with respect to both yield and quality, as an important stage for the production of a large number of vigorous somatic seedlings. Attempts to scale up somatic embryo production numbers and handling have resulted in a few bioreactor designs, the utility of which may prove beneficial for an industrial application. A few simplified cryopreservation methods for embryonal masses (EM) were developed as a means to ensure cost-efficient long-term storage of genotypes during clonal field testing. Finally, recent long-term studies on the growth of somatic trees in the field, including seed production yield and comparison of seed parameters produced by somatic versus seed-derived trees, are described.

Cold, salt and frost are important environmental stresses in forest trees and may significantly reduce productivity. Elevated levels of proline are associated with these stresses and may help alleviate their effects. Transgenic hybrid larch Larix leptoeuropaea has been produced expressing a Vigna aconitifolia gene for pyrroline 5-carboxylate synthase, the rate-limiting step in proline synthesis. Embryogenic masses of hybrid larch were co-cultivated with Agrobacterium tumefaciens harbouring a binary vector expressing the gene. The integration of the gene into the plant genome was confirmed by Southern blot and by proline content analysis. There was an approximately 30-fold increase in proline level in transgenic tissue compared to non-transformed controls. The transgenic tissue lines were significantly more resistant to cold, salt, and freezing stresses and grew under conditions (200mM NaCl or 4 °C) that completely inhibited the growth of control cell lines. Our results indicated that introduction of proline over-accumulation into forest trees might be an effective strategy for ameliorating the effects of environmental stresses.

Article en open access; Background and Aims In conifers, mature somatic embryos and zygotic embryos appear to resemble one another physiologically and morphologically. However, phenotypes of cloned conifer embryos can be strongly influenced by a number of in vitro factors and in some instances clonal variation can exceed that found in nature. This study examines whether zygotic embryos that develop within light-opaque cones differ from somatic embryos developing in dark/light conditions in vitro. Embryogenesis in larch is well understood both in situ and in vitro and thus provides a suitable system for addressing this question.Methods Features of somatic and zygotic embryos of hybrid larch, Larix × marschlinsii, were quantified, including cotyledon numbers, protein concentration and phenol chemistry. Somatic embryos were placed either in light or darkness for the entire maturation period. Embryos at different developmental stages were embedded and sectioned for histological analysis.Key Results Light, and to a lesser degree abscisic acid (ABA), influenced accumulation of protein and phenolic compounds in somatic and zygotic embryos. Dark-grown mature somatic embryos had more protein (91·77 ± 11·26 µg protein mg–1 f.wt) than either dark-grown zygotic embryos (62·40 ± 5·58) or light-grown somatic embryos (58·15 ± 10·02). Zygotic embryos never accumulated phenolic compounds at any stage, whereas somatic embryos stored phenolic compounds in the embryonal root caps and suspensors. Light induced the production of quercetrin (261·13 ± 9·2 µg g–1 d.wt) in somatic embryos. Mature zygotic embryos that were removed from seeds and placed on medium in light rapidly accumulated phenolics in the embryonal root cap and hypocotyl. Delaying germination with ABA delayed phenolic compound accumulation, restricting it to the embryonal root cap.Conclusions In larch embryos, light has a negative effect on protein accumulation, but a positive effect on phenol accumulation. Light did not affect morphogenesis, e.g. cotyledon number. Somatic embryos produced different amounts of phenolics, such as quercetrin, depending on light conditions. The greatest difference was seen in the embryonal root cap in all embryo types and conditions.

Chapitre 8; Genome-wide profiling (transcriptomics, proteomics, metabolomics) is providing unprecedented opportunities to unravel the complexity of coordinated gene expression during embryo development in trees, especially conifer species harbouring “giga-genome”. This knowledge should be critical for the efficient delivery of improved varieties through seeds and/or somatic embryos in fluctuating markets and to cope with climate change. We reviewed “omics” as well as targeted gene expression studies during both somatic and zygotic embryo development in conifers and tentatively puzzled over the critical processes and genes involved at the specific developmental and transition stages. Current limitations to the interpretation of these large datasets are going to be lifted through the ongoing development of comprehensive genome resources in conifers. Nevertheless omics already confirmed that master regulators (e.g. transcription and epigenetic factors) play central roles. As in model angiosperms, the molecular regulation from early to late embryogenesis may mainly arise from spatio-temporal modulation of auxin-, gibberellin- and abscisic acid-mediated responses. Omics also showed the potential for the development of tools to assess the progress of embryo development or to build genotype-independent, predictive models of embryogenesis-specific characteristics.

Chapitre 7; This review compiles research results published over the last 14 years on conifer somatic embryogenesis (SE). Emphasis is placed on the newest findings that affect the response of seed embryos (typical explants) and shoot primordia (rare explants) to the induction of SE and long-term culture of early somatic embryos. Much research in recent years has focused on maturation of somatic embryos, with respect to both yield and quality, as an important stage for the production of a large number of vigorous somatic seedlings. Attempts to scale up somatic embryo production numbers and handling have resulted in a few bioreactor designs, the utility of which may prove beneficial for an industrial application. A few simplified cryopreservation methods for embryonal masses (EM) were developed as a means to ensure cost-efficient long-term storage of genotypes during clonal field testing. Finally, recent long-term studies on the growth of somatic trees in the field, including seed production yield and comparison of seed parameters produced by somatic versus seed-derived trees, are described.

International audience; Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

International audience

International audience; 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.

Article fédérateur entre toutes les équipes Européennes travaillant sur le pin maritime. Assemblage du transcriptome des données venant de France (INRA Orléans + INRA Pierroton) + Espagne + Portugal = projet Sustainpine; International audience; Maritime pine (Pinus pinasterAit.) is a widely distributed conifer species in Southwestern Europe and one of the most advanced models for conifer research. In the current work, comprehensive characterization of the maritime pine transcriptome was performed using a combination of two different next-generation sequencing platforms, 454 and Illumina. De novo assembly of the transcriptome provided a catalogue of 26 020 unique transcripts in maritime pine trees and a collection of 9641 full-length cDNAs. Quality of the transcriptome assembly was validated by RT-PCR amplification of selected transcripts for structural and regulatory genes. Transcription factors and enzyme-encoding transcripts were annotated. Furthermore, the available sequencing data permitted the identification of polymorphisms and the establishment of robust single nucleotide polymorphism (SNP) and simple-sequence repeat (SSR) databases for genotyping applications and integration of translational genomics in maritime pine breeding programmes. All our data are freely available at SustainpineDB, the P. pinaster expressional database. Results reported here on the maritime pine transcriptome represent a valuable resource for future basic and applied studies on this ecological and economically important pine species.

This work has been conducted under Research Infrastructure Concerted Action “Treebreedex” (http://treebreedex.eu, contract RICA-CT-2006-026076).; Vegetative propagation of forest trees offers advantages to both tree breeders and the forest industry. This review will describe benefits, type of vegetative propagation, and its integration into breeding programmes. Of all of the different methods for vegetative propagation, only rooted cuttings and somatic embryogenesis (and the combined use of both) offer any practical methods for large-scale commercial use. However, it is very difficult to fully appreciate the overall level of activity of the research and application of somatic embryogenesis of forest trees. Publications and reports only highlight a small fraction of the ongoing work. To this end, a survey was conducted across Europe (under EU Research Infrastructure Concerted Action “Treebreedex”) to document the species involved, the state-of-the-art of somatic embryogenesis, its stage of development and its application in tree improvement programmes and to commercial forestry. The results of this survey are presented and discussed. In addition, this review presents the challenges (biological, economic, public acceptance and regulatory) and their relationships to European forestry. Finally, a strategy to promote the use of this technology is proposed.

Scots pine (Pinus sylvestris L.) is an economically very important and scientifically well investigated tree species of the northern hemisphere occupying large areas with different climate condition in Eurasia. It has been in the focus of tree improvement since many decades. Breeding programmes were initiated with selection of plus trees, establishment of seed orchards and progeny testing in nearly all countries where this species is of economic importance. Today these programmes are developed very differently. There are countries with advanced programmes for improvement of Scots pine and others were such programmes were stopped in the last years. In this chapter the situation of breeding of Scots pine is described for different European countries. Short overviews are given about the distribution of Scots pine, its genetic diversity, the wood production and the state of forest reproductive material of this species in Europe.

FCBA et l'INRA ont engagé des recherches il y a maintenant près de 20 ans pour développer une méthode de multiplication végétative performante du pin maritime. Ses applications dans le programme d'amélioration génétique seraient multiples, depuis la gestion durable des ressources génétiques jusqu'à la sélection plus efficace des meilleures variétés et leur déploiement facilité dans les plantations. Suite aux développements pionniers chez l'épicéa auxquels FCBA a contribué (années 80, Afocel) et comme chez la plupart des autres conifères, c'est le processus d'embryogenèse somatique à partir de graines immatures couplé à la cryoconservation des embryons somatiques obtenus qui offre actuellement les meilleures perspectives pratiques. Des progrès importants ont été obtenus pour la maîtrise de cette technologie, particulièrement depuis la mise en place en 2004 (et continue depuis) d'une collaboration spécifique sur ce thème entre les équipes " Biotechnologie & Sylviculture Avancée " de FCBA et "Amélioration, Génétique et Physiologie Forestières " de l'INRA. Nous faisons ici un bilan des avancées majeures à différentes étapes de l'embryogenèse somatique jusqu'à la mise en place d'essais au champ en cours d'évaluation. Nous portons également un regard sur les verrous techniques et les contraintes socio-économiques qu'il faudrait lever pour amener cette technologie prometteuse à franchir les portes de l'application pratique.

European larch (Larix decidua) is a native species from the Alps and from several mountainous ranges in Central Europe. Its early introduction outside its native range, especially in lowlands western and northern Europe has been problematic for ecological (phenology) and sanitary (canker) reasons due to the lack of knowledge of its genetic variability. As a substitute, Japanese larch (Larix kaempferi) and then hybrids between European and Japanese larches have been preferred in these regions together with Siberian larches in northern Scandinavia. Results from intensive provenances testing (as early as in the 1940s) have been the corner stone on which breeding programmes have developed both at the intra- (mostly in Central and Eastern Europe) and inter-specific (mostly in North-Western Europe) levels. Inter-specific hybridization is probably the most emblematic and promising breeding strategy for larch, taking benefit of both species trait complementarity, hybrid vigour (heterosis) and hybrid homeostasis. While seed orchards are now the main source for dissemination of genetic progress for European larch, mass-production of hybrid larch remains problematic either through sexual (hybridization seed orchard) or asexual (vegetative propagation by cutting) ways and still impedes large deployment of hybrids in Europe. While progress is expected from current research on this topic (coupling of somatic embryogenesis with bulk vegetative propagation by cutting, 2nd generation hybridization orchard), other hot issues include decoding of heterosis, evaluation of genetic variability for the most original larch wood properties (heartwood formation and durability), adaptation to climate, etc. Joined efforts at European level are expected for genetic resource conservation, breeding and deployment, and tackling of several scientific and technical bottlenecks.

Reverse genetics, defined as ectopic candidate gene expression or silencing, has become an indispensable tool for functional dissection of traits of interest in forest trees. In maritime pine as in other conifers, long generation time and long life span, high genetic loads as well as high genetic redundancy are major obstacles to perform standard genetic approaches including association genetics. Validating marker associations with specific properties before transferring into breeding selection models is still challenging. Stable Agrobacterium-mediated genetic transformation of maritime pine was first reported by Trontin et al. (2002) and developed in both France (FCBA, INRA) and Portugal (IBET) with sufficient refinement (reviewed in Trontin et al. 2007) to envisage practical application in reverse genetics as an attractive alternative to association studies. As a result, the technology has been recently implemented in French (GenoQB, 2006-2009) and transnational (Sustainpine, 2010-2013) or European initiatives (ProCoGen 2012-2016). Somatic embryogenesis (SE) was revealed as a key tissue culture system for achieving genetic transformation, easy cryopreservation of transgenic tissue and efficient transgenic plant regeneration in maritime pine. Much consideration is given in France, Portugal and Spain to apply SE as a clonal propagation system for maritime pine improvement and deployment strategies in multivarietal forestry (MVF, Park YS, reviewed in Klimaszewska et al. 2007). By using this technology it is expected that we will obtain greater genetic gain, high flexibility in deployment of tested embryogenic varieties and easy balancing of genetic gain and diversity in plantations. Both SE and Agrobacterium-mediated transformation methods developed at FCBA, INRA or IBET are being successfully transferred to different partners through running the Sustainpine project (http://www.scbi.uma.es/sustainpine/).

International audience

An integrated physiological and proteomic approach was used to investigate the effects of high gellan gum concentration in the medium during maturation of somatic embryos (SE) of hybrid larch, by comparing embryos incubated in media with a high gellan gum concentration (8 g l(-1) ) and the standard concentration (4 g l(-1) ) after 1, 3, 6 and 8 weeks of maturation. Because of the reduced availability of water in the 8 g l(-1) medium, the cultured embryos had a lower osmotic water potential (Ψπ) and water contents, but higher dry weights (DWs), at 8 weeks compared with embryos cultured on the standard medium. The high gellan gum concentration induced a desiccation that is characteristic in zygotic embryo maturation. Total soluble proteins were extracted from SE with trichloroacetic acid (TCA)-acetone after 1 and 8 weeks of maturation on media with 4 and 8 g l(-1) of gellan gum, and separated by two-dimensional gel electrophoresis (2-DE) at pH 4-7. More than 1100 proteins were reproducibly detected on each gel. At 1 and 8 weeks respectively, the abundances of 62 and 49 spots detected in analyses of embryos matured at the two gellan gum concentrations, significantly differed. Among 62 significantly differing spots at 1 week of maturation, the corresponding proteins of 56 were reliably identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), and were found to be mainly involved in 'carbohydrate metabolism', 'genetic information processing' or 'environmental information processing' according to kegg taxonomy. Both physiological parameters and the proteins identified suggested that the embryos were stressed when they were cultured on 4 g l(-1) of gellan gum.

International audience; Methods for genetic transformation of maritime pine embryonal masses (EM) using Agrobacterium tumefaciens (At) have been independently established at 2 laboratories (FCBA and INRA) in France. The two procedures appeared roughly similar but technical differences were noticed during the decontamination and washing of co-cultivated EM, the subsequent reactivation culture step and the selection phase of transgenic lines. One major difference was the basal formulations of media used for At-mediated transformation development at FCBA (mDCR, modified Gupta and Durzan medium) and INRA (mLV, modified Litvay medium). Joined experiments were carried out in order to get insights on factors affecting the transformation rate based on a comparison of both procedures (one model genotype, C58pMP90 At strain, modified pCambia1301 binary vector harbouring the uidA-intron, hpt and bar genes). Our ultimate goal is to foster the transgenic approach as an efficient tool for establishing direct linkages between genes and phenotypes. We showed that the efficiency of the selected transformation procedures is tightly correlated with the basal medium formulation. Using mLV medium, currently recognized in both laboratories as a superior mineral base for maritime pine somatic embryogenesis, transformation yields were higher using the INRA procedure following either hygromycin or phosphinothricin selection. In contrast full advantage of the FCBA procedure could only be raised by using mDCR basal medium instead of mLV. In conjunction with strong phosphinothricin selection (1 mg l-1) the mDCR-based FCBA procedure was revealed as the best option to transform one model genotype of maritime pine (89.6 ± 18.4 phosphinothricin-resistant lines per g EM).

International audience; Development of clonal propagation method, such as somatic embryogenesis, has numerous applications such as massproduction of genetically improved plants. Since the 90’s, researchers at INRA have engaged research on SE in Larix species (Larix eurolepis, Larix marschlinsii), resulting in a controlled protocol (Lelu and Label 1994). If the positive effect of high gellan gum concentration on somatic embryo maturation is well-known in pine species (Lelu-Walter et al., 2006, 2008), but had not been previously demonstrated with hybrid larches. Maturation of somatic embryos was greatly improved when embryonal masses were placed on medium supplemented with 0.2M sucrose, 60μM ABA containing high concentration of gellan gum (8gl-1). Increasing the gellan gum concentration improved both the quantity and quality of the somatic embryos by reducing EM proliferation and giving rise to well-shaped somatic embryos which subsequently germinated and developed into plantlets. Somatic embryos maturated in presence of high gellan gum, presented reduced moisture content, increase of their dry weight that could resulted from an increase of their protein content (soluble and total part). Gels 2D were realised at 8 weeks, following by LC-MS/MS analysis to identify proteins. In conclusion, hybrid larch SE should influence breeding strategies by offering an alternative tool for accelerated production of large quantities of plants for clonal tests. The improved protocol was applied for the propagation of the new hybrid variety REVE-VERT. Integration of SE into the breeding programme is now undertaken (Lelu-Walter and Pâques 2009).

Two APETALA2 domain transcription factors were characterized first in angiosperms, and, recently, in several gymnosperms. These proteins are involved in several processes, from flowering to embryogenesis in Arabidopsis thaliana. We extrapolated this result to hybrid larch (Larixxmarschlinsii Coaz) resulting from a cross between European (Larix decidua) and Japanese (Larix kaempferi) larches. Somatic embryogenesis is well described and controlled for this Pinaceae. We characterized two-AP2 domain genes: LmAP2L1 and LmAP2L2. Phylogenetic analysis confirmed that LmAP2L1 and LmAP2L2 were orthologous to Norway spruce PaAP2L1 and PaAP2L2 and that L1 forms appeared to be specific to Pinaceae. RT-PCR analysis showed that larch APETALA2 was differentially expressed during late somatic embryogenesis and during the first steps of germination. Whereas LmAP2L2 was constitutively expressed during this process, LmAP2L1 expression appeared only during late somatic embryogenesis, when embryos were able to germinate. Further, LmAP2L1 appeared to be the preferentially expressed form during embryo germination. Thus, LmAP2L1 seems to be a valuable molecular marker for hybrid larch late somatic embryogenesis and could play a role during post-embryonic development.

Several factors affecting somatic embryogenesis (SE) in Pinus sylvestris from self- and cross-pollinated seed families were studied with the aim of producing large quantities of clonal plants. Somatic embryogenesis initiation from zygotic embryos was improved on a medium with lower than standard concentrations of 2,4-dichlorophenoxyacetic acid (2.2 vs. 9.5 mu M) and 6-benzyladenine (2.2 vs. 4.5 mu M). On this medium, initiation rates of four controlled crosses, including one self-cross, varied from 3% to 25%. Among the maturation factors tested, the concentration of abscisic acid (ABA 80, 120 mu M) had no significant effect on the production of mature somatic embryos when the medium contained 0.1 M sucrose. When sucrose concentration was 0.2 M, however, 1.4 times more mature somatic embryos were produced on medium with 80 mu M compared with 120 mu M ABA. Under our best maturation conditions, mature somatic embryos accumulated amounts of storage proteins that were similar to the amounts in mature zygotic embryos. Activated charcoal exerted a beneficial effect on mature somatic embryo production of 24-week-old cultures; there was no evidence of such an effect in 8-week-old cultures. Thirty-seven embryogenic lines from a self-cross and an out-cross were chosen for clonal plant production. Highly embryogenic lines produced mature somatic embryos that were more likely to convert to plants than those from less embryogenic lines. After 4 months of growth in a shade house, plantlet survival rates exceeded 70% for 31 lines out of 35. This report describes an improved method for accelerated production of large quantities of Scots pine for clonal tests.

International audience; Somatic embryogenesis (SE) in conifers is at the base of nearly all biotechnology products that potentially might be developed including transgenic trees. SE is a powerful tool for clonal propagation that has opened avenues for deployment of superior clonally replicated planting stock in forest plantations. Although the SE process can be managed for high efficiency there are still several problems that require better understanding of underlying causes. One phenomenon, which occurs after the long-term culture of embryogenic tissue, particularly of pines, is the loss of ability to produce mature somatic embryos and consequently plants. Such unproductive cultures must be replaced, which is both expensive and undesirable. In an attempt to get an insight into the differences between young, primary lines of embryonal mass of maritime pine that produced somatic embryos and the same lines of significantly increased age that stopped producing somatic embryos, we analyzed in both types of material the levels of endogenous hormones and their metabolites, polyamines, global DNA methylation and DNA methylation patterns. DNA methylation plays an important role in regulation of gene expression and may cause epigenetic change leading to suppressed embryogenic potential. To determine if the DNA methylation could be altered, we exposed the cultures to different concentrations of hypomethylating agents such as 5-aza-2'- deoxycytidine, 5-aza-cytidine and zebularine and tested the embryogenic potential of a line after the treatment. For comparison, we induced secondary SE from a few mature somatic embryos of the same lines and carried out the same as above analyzes. Secondary SE has been implicated in restoring/enhancing the embryogenic potential of a given line and therefore theoretically the tissue was expected to be biologically similar to the primary young line. Our results indicated that embryogenic cultures of different ages varied with respect to the polyamines levels and DNA methylation pattern but not with respect to the hormone concentration. Treatment of the cultures with the hypomethylating agents resulted in different methylation patterns and somatic embryo production ability.