Norway spruce somatic embryogenesis benefits from proliferation of embryogenic tissues on filter discs and cold storage of cotyledonary embryos

Soil hydraulic properties are central for soil quality and affect forest productivity and the impacts of climate change on forests. The water retention characteristics (WRC) of mineral forest soils in Finland are not well known, and practical tools to predict them for hydrological, biogeochemical and forest models are lacking. We statistically analyzed mineral forest soils WRC from over 130 sites in Finland, focusing on the humus layer and main root zone (0–19 cm depth). We showed that mineral forest soils can be grouped into five WRC classes that are well predictable from soil bulk density, organic matter content and clay fraction. However, the majority of the forest soils are hydrologically rather similar. We found that neither topsoil maps nor any combination of open geospatial data were able to predict WRC. Thus, in the absence of site-specific soil data, parameterizing WRC as a function of forest site fertility type was proposed. We demonstrated the approach in soil moisture modeling at a small forest headwater catchment and showed that the soil moisture response to weather conditions is jointly affected by WRC, stand attributes and topography. We showed that drought risks are highest for dense mature forests at nutrient-poor, coarse-textured sites and lower for young stands on peatlands and lowland herb-rich sites with groundwater influence. The results improve hydrological predictions for Finnish forests, and the open dataset can contribute to the larger synthesis and development of boreal forest soil pedo-transfer functions

Maturation des embryons somatiques de meleze hybride (Larix x leptoeuropaea)

National audienc

Advances on somatic embryogenesis in forest trees

International audienc

L’embryogenèse somatique des conifères : une filière pour produire du matériel forestier de reboisement.

Parmi les techniques de culture in vitro, l’embryogenèse somatique est potentiellement la méthode de régénération la plus performante pour la propagation clonale d’arbres forestiers du fait de son taux de multiplication élevé. Chez les conifères, l’intérêt de cette technologie pour d’amélioration génétique est indéniable. La cryoconservation des cultures embryogènes (dans l’azote liquide à –196°C) laisse entrevoir la possibilité de disposer à volonté de matériel juvénile et la conservation des ressources génétiques. L’embryogenèse somatique représente aussi un outil de développement en foresterie. De fait, cette technologie est déjà utilisée à l’échelle industrielle pour les épicéas et différents pins au Canada, aux USA et en Nouvelle Zélande.Somatic embryogenesis is potentially recognised as the most efficient in vitro culture technique for clonal propagation of forest tree due to its high multiplication rate. In conifer species, this technology is very useful for breeding programmes. Embryogenic cultures can be stored in liquid nitrogen at -196ºC (cryopreservation) indefinitely without loss of juvenility. Somatic embryogenesis can be used also for large scale production of superior quality forest trees, as demonstrated at an industrial level in Canada, USA and New Zealand

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

• 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 $\times$ eurolepis, Larix $\times$ 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 $\mu$M of 2.4-dichlorophenoxyacetic acid and 2.3 $\mu$M 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$\cdot$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.Simplification et amélioration de l'embryogenèse somatique des mélèzes hybrides (Larix $\times$ eurolepis et Larix $\times$ marschlinsii). Perspectives pour l'amélioration. • 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 $\times$ eurolepis, Larix $\times$ 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 $\mu$M d'acide 2.4-dichlorophenoxyacetic et 2,3 $\mu$M 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$\cdot$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

Simple and efficient protocols for the initiation and proliferation of embryogenic tissue of Douglas-fir

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 programs is currently constrained by an inability to effectively and reliably multiply selections through vegetative propagation. Somatic embryogenesis coupled with cryopreservation are essential biotechnology tools 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 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 (Litvay et al. 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 realise increased genetic gain in this species through the clonal-assisted deployment of the best genetic material in both New Zealand and France

An improved method for somatic plantlet production in hybrid larch (Larix x leptoeuropaea) : Part 2. Control of germination and plantlet development

18 ref.International audienc

Influence of exogenous L-proline on embryogenic cultures of larch (Larix leptoeuropaea Dengler), sitka spruce (Picea sitchensis (Bong.) Carr.) and oak (Quercus robur L.) subjected to cold and salt stress

The effect of exogenous L-proline (1 mM, 10 mM, and 100 mM) on embryogenic cultures of larch, sitka spruce and oak subjected to environmental stresses was examined. Low temperature (4 °C) completely inhibited growth of the cultures and this was partially alleviated by the addition of proline. Our studies show that not only can cultures survive low temperatures, but are capable of active growth while the cold stress is still being applied. Growth was inversely related to [NaCl] with complete inhibition at 200 mM. Proline stimulated growth at all concentrations tested permitting growth with 200 mM NaCl even at low (1 mM) proline concentrations. Release of internal cellular potassium was inversely related to freezing temperature and this release was reduced by exogenous proline. These results for cultures of forest species are consistent with findings previously reported for deciduous herbaceous angiosperms and suggest that proline may have a role in protection of forest species from environmental stresses.Des cultures embryogènes de mélèze hybride, d'épicéa de sitka et de chêne ont été soumises à différentes conditions de culture et leur croissance étudiée en fonction de l'ajout de L-proline au milieu de culture (1 mM, 10 mM, et 100 mM). Si des températures basses (4 °C) inhibent totalement la croissance des cultures, celle-ci redevient partiellement normale en présence de proline. Nos résultats montrent que les cultures non seulement survivent à de basses températures mais sont aussi capables de croître activement au cours de la durée d'application du froid. De même, la croissance est inversement corrélée à la concentration en sel avec sa complète inhibition en présence de 200 mM de NaCl. L'addition de L-proline au milieu de culture (quelles que soient les concentrations testées) stimule la croissance des cultures, même en présence de 200 mM de NaCl. La libération de potassium intracellulaire est inversement corrélée à la température de congélation, libération qui est réduite en présence de proline exogène. Ces résultats, obtenus pour des cultures d'espèces forestières, sont en accord avec ceux précédemment rapportés pour des espèces herbacées. Ils suggèrent le rôle potentiel de la proline dans la protection de ces espèces forestières soumises à des stress abiotiques