Marie-Anne Lelu-Walter

Simplified and improved somatic embryogenesis for clonal propagation of Pinus pinaster (Ait.)

In this study, several improvements and simplifications of SE protocols in Pinus pinaster (Ait.), a species of economic importance in the regions of Western Europe, are described. These improvements pertained to all stages of SE including high initiation frequencies in eight control pollinated seed families, relatively high somatic embryo maturation yield when cells were coated with particles of activated charcoal and a rapid production of plants directly in a shade house. The SE initiation frequency from isolated zygotic embryos was high (up to 100%) and plants were produced from 11 embryogenic lines representing all crosses. Based on these results, the estimated number of somatic embryos required to produce 1,000 plants varied from slightly more than the required number of plants to more than double this number depending on the line. Such an estimate is critical in developing plant production strategy when a number of embryogenic lines are considered for production of clonal plants.

Initiation of somatic embryogenesis in Pinus banksiana, P. strobus, P. pinaster, and P. sylvestris at three laboratories in Canada and France

During 2002–2004, three laboratories in Canada and France collaborated to improve initiation of somatic embryogenesis (SE) in jack pine (Pinus banksiana Lamb.), eastern white pine (P. strobus L.), maritime pine (P. pinaster Ait.), and Scots pine (P.␣sylvestris L.), giving particular attention to the effects of (1) N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) versus various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA), (2) differences in basal nutrient media, i.e., macro- and microelements, and (3) gelling agent concentration. The work was carried out separately at␣each laboratory, but the details of media compositions were shared and tested on their respective species. Results indicate that the developmental stage of the zygotic embryo (ZE) and genotype effects had a large influence on SE initiation, and that genetic effects were consistent over time. Different species responded differently to PGR types and concentration, basal nutrient media, trace elements, and their combinations. Currently, our best initiation rates based on a selected group of genotypes, optimal development stage of ZE, and medium are 3.9% for jack pine, 54.6% for eastern white pine, 76.2% for maritime pine, and 19.7% for Scots pine.

Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction

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.)

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.

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.

Overproduction of proline in transgenic hybrid larch (Larix x leptoeuropaea (Dengler)) cultures renders them tolerant to cold, salt and frost

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.

Simplified and improved somatic embryogenesis of hybrid larches (Larix × eurolepis and Larix × marschlinsii). Perspectives for breeding

Abstract• Development of clonal propagation method, such as somatic embryogenesis, has numerous applications such as mass-production of genetically improved plants and the amenability of embryogenic cultures to cryogenic storage. Since the 90’s, researchers at INRA have engaged in research on somatic embryogenesis in Larix species (Larix × eurolepis, Larix × marschlinsii).• The aim of this work was to improve and to simplify all steps of somatic embryogenesis and to apply this protocol to the new hybrid variety REVE-VERT.• The somatic embryogenesis initiation frequency from immature zygotic embryos was high (65%) on a medium with reduced plant growth regulator concentrations (2.2 μM of 2.4-dichlorophenoxyacetic acid and 2.3 μM of 6-benzyladenine). Simplified cryopreservation method (no need of programmable freezer) of the embryonal masses resulted in 100% recovery of cryopreserved lines. Maturation of a large number of somatic embryos was greatly improved when embryonal masses were dispersed on filter paper placed on medium containing high concentration of gellan gum (8 g·L−1). Under these conditions, 94% of the lines matured somatic embryos that developed into plantlets. Clearly ageing and cryopreservation did not reduce embryogenic potential of embryonal masses.• Requirements for the effective integration of somatic embryogenesis into the larch breeding programme are discussed.Résumé• Le développement de méthode de multiplication clonale, telle l’embryogenèse somatique, a de nombreuses applications comme la production en masse de plants génétiquement améliorés et la disponibilité des cultures embryogènes en cryoconservation. Depuis les années 1990, l’INRA a engagé des recherches en embryogenèse somatique des mélèzes (Larix × eurolepis, Larix × marschlinsii).• L’objectif du travail a été de simplifier et d’améliorer chaque étape de l’embryogenèse somatique, et d’appliquer ce protocole à la nouvelle variété de mélèze hybride REVE-VERT.• L’initiation de l’embryogenèse somatique à partir d’embryons zygotiques immatures est obtenue à des fréquences élevées (65 %) avec un milieu de culture contenant des concentrations hormonales réduites (2,2 μM d’acide 2.4-dichlorophenoxyacetic et 2,3 μM de 6-benzyladenine). Une méthode simplifiée de congélation (sans l’utilisation d’un congélateur programmable) des masses embryogènes permet la survie de 100 % des lignées congelées. La maturation d’un grand nombre d’embryons somatiques a été fortement améliorée lorsque les masses embryogènes ont été étalées sur filtre papier placé sur milieu de culture à forte concentration en gelrite (8 g·L−1). Sous ces conditions, 94 % des lignées embryogènes régénèrent des embryons somatiques matures qui se développent en plantes. Clairement l’âge et la cryoconservation des lignées embryogènes ne réduisent pas leur potentiel embryogène.• Les besoins pour une intégration efficace de l’embryogenèse somatique au programme d’amélioration du mélèze, sont discutés.

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.

Advances in Conifer Somatic Embryogenesis Since Year 2000

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.

Larches (Larix sp.)

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.