Ce travail est basé sur le constat du manque de méthodes permettant l'analyse intégrée des processus contrôlant le développement végétatif d'Arabidopsis thaliana dans les études phénotypiques multi-échelles. Un phénotypage préliminaire de la croissance foliaire de 91 génotypes a permis de sélectionner 3 mutants et des variables d'intérêt pour une étude plus poussée du développement de la pousse. Un pipeline de méthodes d'analyses combinant techniques d'analyse d'images et modèles statistiques a été développé pour intégrer les mesures faites à l'échelle de la feuille et de la pousse. Des modèles multi-phasiques à changements de régime semi-markovien ont été estimés pour chaque génotype permettant une caractérisation plus pertinente des mutants. Ces modèles ont validé l'hypothèse selon laquelle le développement de la rosette peut être découpé en une suite de phases de développement, pouvant varier selon les génotypes. Ils ont aussi mis en évidence le rôle structurant de la variable «trichome abaxial», bien que les phases de développement ne puissent être entièrement expliquées par ce trait. Un 2nd pipeline d'analyses combinant une méthode semi-automatique de segmentation d'images de l'épiderme foliaire et l'analyse des surfaces de cellules par un modèle de mélange de lois gamma à paramètres liés par une loi d'échelle a été développé. Ce modèle nous a permis d'estimer la loi du nombre de cycles d'endoréduplication. Nous avons mis en évidence que cette loi dépendait du rang de la feuille.Le cadre d'analyses multi-échelles développé et testé durant cette thèse devrait être assez générique pour être appliqué à d'autres espèces végétales dans diverses conditions environnementales. / This study is based on the observation of a lack of methods enabling the integrated analysis of the processes controlling the vegetative development in Arabidopsis thaliana during multi-scale phenotypic studies. A preliminary leaf growth phenotyping of 91 genotypes enabled to select 3 mutants and different variables of interest for a more in depth analysis of the shoot development.We developed a pipeline of analysis methods combining image analysis techniques and statistical models to integrate the measurements made at the leaf and shoot scales. Semi-Markov switching models were built for each genotype, allowing a more thorough characterization of the studied mutants. These models validated the hypothesis that the rosette can be structured into successive developmental phases that could change depending on the genotype. They also highlighted the structuring role of the ‘abaxial trichomes' variable, although the developmental phases cannot be explained entirely by this trait. We developed a second pipeline of analysis methods combining a semi-automatic method for segmenting leaf epidermis images, and the analysis of the obtained cell areas using a gamma mixture model whose parameters of gamma components are tied by a scaling rule. This model allowed us to estimate the mean number of endocycles. We highlighted that this mean number of endocycles was function of the leaf rank.The multi-scale pipeline of analysis methods that we developed and tested during this PhD should be sufficiently generic to be applied to other plant species in various environmental conditions.

Le contrôle moléculaire du patterning au cours des processus développementaux est aujourd’hui bien décrit chez les organismes multicellulaires. A l’inverse, la contribution de la croissance dans l’émergence des patterns reste peu explorée, et est souvent réduite à un rôle passif. Au cours de cette thèse, j’ai étudié cette question en utilisant le méristème apical caulinaire (MAC) d’Arabidopsis comme modèle. Le méristème est un groupe de cellules en divisions situé à l’extrémité de toutes les tiges et les branches et qui génère tous les organes aériens de la plante selon un patron stéréotypé, aussi appelé phyllotaxie. Dans une première partie, j’ai étudié comment la croissance de la tige pouvait influencer le patron phyllotactique. Plus précisément, en découplant dépôt de la cellulose dans la paroi et l’orientation des microtubules, j’ai montré que le patron de phyllotaxie devenait bimodal en raison de l’induction d’une torsion lors de la croissance de la tige. Dans une seconde partie, j’ai analysé le lien entre forme du MAC et expression génétique. En particulier, j’ai pu corréler l’expression d’un gène maître : SHOOTMERISTEM LESS (STM) au degré de courbure dans le MAC. De plus, en utilisant des approches de micromécaniques, j’ai aussi pu montrer que l’expression de STM pouvait être induite par le patron de contraintes localement généré par la courbure. Pour finir, j’ai aussi étudié comment la taille du méristème influence la robustesse du pattern de phyllotaxie sur la tige en modulant la fréquence d’initiation des organes. L’ensemble de ce travail met ainsi en avant le rôle de la croissance dans le patterning, notamment via des mécanismes de rétrocontrôles géométriques et mécaniques. / The molecular mechanisms behind the emergence of patterns during developmental processes have been well described in multicellular organisms. However, the contribution of growth in patterning is still poorly understood; growth is often seen as a passive output of the activity of the patterning signals. In this PhD, I have studied the relation between growth and patterning using the shoot apical meristem of Arabidopsis as a model system. The meristem is a group of dividing cells located at the tip of every stems and branches that generates all the aerial organs of the plant following a typical spatio-temporal pattern also called phyllotaxis. In a first part, the influence of post-meristematic growth on phyllotaxis was assessed. More precisely, by uncoupling cellulose deposition from the orientation of the microtubule array, I showed that the resulting stem torsion induces the emergence of a new and robust bimodal phyllotactic pattern. In a second part, the relation between meristem shape and gene expression was analyzed. More precisely, I correlated the expression of a master regulatory gene: SHOOT MERISTEMLESS (STM) to tissue curvature in the boundary domain that separates the emerging organ from the meristem. Furthermore, I showed that STM expression can be induced by micromechanical perturbations thus suggesting that shape-derived mechanical stresses in the meristem boundary contribute to STM expression. Finally, I also studied how meristem size can influence the robustness of the pattern of phyllotaxis along the stem through a modulation of the frequency of organ initiation. Altogether, this work highlights the important contribution of growth in patterning, notably thanks to the existence of geometrical and mechanical feedbacks.

Le maintien de la croissance foliaire en situation de déficit hydrique résulte du maintien de l´absorption racinaire et de la production de biomasse au niveau foliaire. Pour optimiser les deux processus, la plante ajuste la croissance de ses organes, et la répartition de la biomasse produite, entre les différents organes (root/shoot ratio) ou au sein de chaque organe (surface foliaire spécifique, longueur racinaire spécifique). Les principaux objectifs de ce travail de thèse étaient (i) d´évaluer l´impact des modifications de répartition de biomasse sur le maintien de la croissance foliaire en situation de déficit hydrique, (ii) de relier la réponse de la croissance d´un génotype aux caractéristiques de son habitat d´origine, and (iii) d´identifier les régions du génome responsables de la variation des croissances foliaires et racinaires en situation de déficit hydrique. Différent types de populations d´Arabidopsis thaliana ont été utilisés, une population de lignées recombinantes, ainsi que différents groupes d´accessions collectées dans des environnements naturels contrastés. Une analyse des relations allométriques entre les variables foliaires et racinaires en conditions de culture optimales puis en situation de déficit hydrique a permis de mettre en évidence le rôle clé de la surface foliaire spécifique dans l´amélioration de la tolérance au déficit hydrique. Une caractérisation détaillée du climat des régions dans lesquelles les accessions avaient été collectées a permis de faire le lien entre la tolérance accrue de certains génotypes et la faible balance climatique dans laquelle ils évoluaient. Enfin, en utilisant ces génotypes, une analyse de génétique quantitative (combinant recherche de QTL et génétique d´association) a été menée. Les régions génomiques controllant les croissances foliaires et racinaires étaient très liées, en particulier en situation hydrique optimale, mais le calcul de variables utilisant la croissance de la plante comme cofacteur a permis d´identifier des régions spécifiques de la croissance racinaire, dont une a été confirmée en utilisant des lignées quasi isogéniques. En situation de contrainte hydrique, les déterminants génétiques des croissances foliaires et racinaires étaient moins liés, et plusieurs régions très fortement associées spécifiquement aux variations de croissance racinaire ou foliaire ont été détectées. Des régions associées au maintien de la croissance foliaire en situation de déficit hydrique ont pu être mises en évidence, et la précision des études de génétique d´association a permis de réveler la présence de gènes d´intéret dans ces régions. / Growth maintenance under water deficit mainly results from the maintenance of water uptake at the root level,and assimilates production by leaves. To optimize both processes, plant need to adjust organ growth and biomassallocation patterns between roots and shoots (root/shoot ratio), but also within the organs, through specific leaf areaand specific root length variations. The main objectives of this study were (i) to evaluate the impact of growth andbiomass allocation patterns modifications on growth maintenance under drought conditions, (ii) to rely the genotypicresponses to water deficit conditions and the climatic features of the natural environment in which they evolved, and(iii) to identify the key genetic regions responsible for shoot and root growth variation in response to water deficitconditions. We used different sets of genotypes, a population of recombinant inbred lines, and different sets ofaccessions of Arabidopsis thaliana, collected in a wide range of environments. An analysis of the allometricrelationships between shoot and root growth related variables under both well watered and water deficit conditionsallowed to highlight the importance of specific leaf area plasticity to maintain plant growth under water deficit. Adetailed climatic characterization of the natural habitats of the accessions studied, combined to the evaluation ofgrowth response to water deficit in these accessions allowed connecting low climatic water balance to better toleranceto water deficit conditions in specific regions, suggesting that this climatic feature could have shaped the evolution ofgenotypes in certain regions. Finally, using these two sets of genotypes, joint linkage and linkage disequilibriumanalysis were performed on growth related traits under well watered and water deficit conditions. Some genetic regionsinvolved in the control of root and shoot related traits were strongly coupled, especially in well watered experiments,but we managed to identify root specific regions using calculated variables that takes global plant growth as a cofactor.Under water deficit, the regions controlling root and shoot growth were less associated, and very strong QTL weredetected, specifically associated to one or the other part. Genomic regions associated to growth response to waterdeficit were also detected, and the accuracy of association mapping enabled to identify target genes that could be playa role in growth maintenance under drought.

Made available in DSpace on 2016-01-26T18:56:20Z (GMT). No. of bitstreams: 1 Disertacao.pdf: 273120 bytes, checksum: 0281bd7efcba57034bce3538d8a8412d (MD5) Previous issue date: 2009-10-30 / The main objective of this work was to verify the effect of substitution of phosphate by its related compound phosphite in the in vitro growth of sweet potato nodal segments. These segments from a previous culture were incub ated in MS media with the following combinations of phosphate and phosphite T (100% / 0%); T1 (87.5% /12.5%); T2 (75% / 25%); T3 (50% / 50%) and T4 (0% / 100%) respectively, in a factorial arrangement of 5x5, with 5 treatments (T, T1, T2, T3 and.T4) and five evaluation periods (7, 14, 21, 28 and 35 days). The design was completely random with three replicates of two explants per period. The evaluations were done weekly, by counting the number of shoots, length and dry weight of shoot and root. The increment in the phosphite concentration, decreased all atributes, as growth velocity, lenght and dry weight, meaning that this salt could not be used in substitution to phosphate in sweet potato tissue culture. / O objetivo deste trabalho foi avaliar o efeito de fosfito no crescimento in vitro das plântulas de batata-doce em substituição parcial ao fosfato. Segmentos nodais, provenientes de uma subcultura, foram inoculados em meio MS e com as seguintes combinações entre fosfato e fosfito: T (100% e 0%); T1 (87,5% e 12,5%); T2 (75% e 25%); T3 (50% e 50%) e T4 (0% e 100%) respectivamente. Adotou-se esquema fatorial 5x5, com 5 tratamentos (T, T1, T2, T3 e T4) e 5 períodos de avaliação (7, 14, 21, 28 e 35 dias). O delineamento experimental foi inteiramente casualizado, com três repetições por período de avaliação, sendo cada parcela formada por dois explantes. As análíses foram realizadas semanalmente e avaliaram-se o número de brotos originados; o comprimento e a massa seca de parte aérea e de raízes. Verificou-se que aumento da concentração de fosfito em substituição a fosfato inibiu a velocidade de crescimento, comprimento da parte aérea e de raízes das plântulas de batata-doce, indicando que o fosfito não deve ser utilizado como nutriente na cultura de tecidos de batata doce.

Made available in DSpace on 2016-07-18T17:51:05Z (GMT). No. of bitstreams: 1 Disertacao.pdf: 273120 bytes, checksum: 0281bd7efcba57034bce3538d8a8412d (MD5) Previous issue date: 2009-10-30 / The main objective of this work was to verify the effect of substitution of phosphate by its related compound phosphite in the in vitro growth of sweet potato nodal segments. These segments from a previous culture were incub ated in MS media with the following combinations of phosphate and phosphite T (100% / 0%); T1 (87.5% /12.5%); T2 (75% / 25%); T3 (50% / 50%) and T4 (0% / 100%) respectively, in a factorial arrangement of 5x5, with 5 treatments (T, T1, T2, T3 and.T4) and five evaluation periods (7, 14, 21, 28 and 35 days). The design was completely random with three replicates of two explants per period. The evaluations were done weekly, by counting the number of shoots, length and dry weight of shoot and root. The increment in the phosphite concentration, decreased all atributes, as growth velocity, lenght and dry weight, meaning that this salt could not be used in substitution to phosphate in sweet potato tissue culture. / O objetivo deste trabalho foi avaliar o efeito de fosfito no crescimento in vitro das plântulas de batata-doce em substituição parcial ao fosfato. Segmentos nodais, provenientes de uma subcultura, foram inoculados em meio MS e com as seguintes combinações entre fosfato e fosfito: T (100% e 0%); T1 (87,5% e 12,5%); T2 (75% e 25%); T3 (50% e 50%) e T4 (0% e 100%) respectivamente. Adotou-se esquema fatorial 5x5, com 5 tratamentos (T, T1, T2, T3 e T4) e 5 períodos de avaliação (7, 14, 21, 28 e 35 dias). O delineamento experimental foi inteiramente casualizado, com três repetições por período de avaliação, sendo cada parcela formada por dois explantes. As análíses foram realizadas semanalmente e avaliaram-se o número de brotos originados; o comprimento e a massa seca de parte aérea e de raízes. Verificou-se que aumento da concentração de fosfito em substituição a fosfato inibiu a velocidade de crescimento, comprimento da parte aérea e de raízes das plântulas de batata-doce, indicando que o fosfito não deve ser utilizado como nutriente na cultura de tecidos de batata doce.

Upland cotton (Gossypium hirsutum L.) is the world's most prominent fiber crop. Cotton transformation is labor intensive and time consuming, taking 12 to 18 months for rooted T0 plants. One rate limiting step is the necessary production of somatic embryos. In other recalcitrant species, ectopic expression of three genes were shown to promote somatic embryogenesis: WUSCHEL (WUS), SHOOT MERISTEMLESS (STM), and BABY BOOM (BBM). WUS is responsible for maintaining stem-cell fate in shoot and floral meristems. STM is needed to establish and maintain shoot meristems. STM and WUS have similar functions but work in different pathways; overexpression of both together converts somatic cells to meristematic and embryogenic fate. BBM encodes an AP2/ERF transcription factor that is expressed during embryogenesis and ectopic expression of BBM reprograms vegetative tissues to embryonic growth. In prior studies, these genes were constitutively expressed, and cultures did not progress beyond embryogenesis because the embryogenic signal was not turned off. In our study, we set out to use these genes to increase the efficiency of cotton transformation and decrease the time it takes to regenerate a plant. A disarmed cotton leaf crumple virus (dCLCrV) vector delivers WUS, STM, or BBM into cotton tissue cultures through Agrobacterium tumefaciens infection. We propose that virus delivery of embryo-inducing genes is a better approach for transformation because A) inserts more than 800 nucleotides are unstable, and will spontaneously inactivate, B) virus DNA can migrate through plasmodesmata to cells around the infected cell, creating a gradient of embryonic potential, C) the virus DNA does not pass through the germ line and the seed will not contain virus. We propose this method of inducing embryogenesis will facilitate the stable transformation of cotton and will be beneficial to the cotton industry. Ectopic expression of AtBBM, AtSTM, and AtWUS GrWUS:meGFP from a constitutive CaMV 35S promoter produced plants with phenotypes similar to those described in previous studies overexpressing AtBBM, indicating that the AtBBM gene was functional. The cotton cotyledon infiltration of the pART27 constructs showed transformed cells in Coker 312 by GFP localization in the nucleus. Although GFP was detected, no visible embryos appeared from the cotyledon. Cotyledons infiltrated with Agrobacterium harboring overexpression vectors withered and aborted after ~2 weeks. The virus-based vector in tissue culture failed to increase transformation efficiency, resulting in no embryos. The combination of hormone concentration showed no contribution to increasing the transformation efficiency.

Cette thèse étudie les phénomènes de concentration préférentielle et de sédimentation de particules inertielles transportées dans un écoulement turbulent. Pour cela, des expériences ont été menées en soufflerie dans une turbulence engendrée en aval d’une grille active et ensemencée avec des gouttelettes d'eau. La concentration préférentielle se manifeste par la ségrégation spatiale des particules qui bien qu’initialement ensemencée de façon homogène, tendent à se regrouper en amas, laissant en déplétion d’autres zones de l’écoulement. Un effort particulier a été consacré à séparer les mécanismes liés à l’inertie des particules, à la turbulence et aux effets collectifs impactant la formation des amas et modifiant la vitesse de sédimentation des particules. Quatre principaux paramètres non-dimensionnels ont été variés afin d’établir le rôle spécifique de chacun d’entre eux sur les processus de concentration préférentielle et de sédimentation : le nombre de Rouse $Ro $, représentant le rapport de la vitesse de sédimentation des particules à la vitesse fluctuante de l’écoulement; le nombre de Stokes $St$, quantifiant l'inertie des particules comme le rapport entre le temps de réponse des particules et le temps dissipatif de l’écoulement; le nombre de Reynolds $ RE_lambda$ représentant le degré de turbulence et enfin la fraction volumique de la phase dispersée $phi_v$.Deux techniques expérimentales (suivi Lagrangien des particules et interférométrie à phase Doppler) ont été utilisées pour l'acquisition des données et pour le diagnostic de la concentration préférentielle et de la sédimentation des gouttelettes dispersées. Le suivi Lagrangien de particules a été réalisé par visualisation à haute vitesse cadence des gouttelettes dispersées dans une nappe de laser. Cela donne accès aux statistiques simultanées de la distribution spatiale des particules et de leur vitesse. La niveau de clustering a été quantifié à l’aide de tessélation de Voronoï. Nous établissons des lois d’échelles quantitatives caractérisant la dépendance du degré de clustering et de la géométrie des amas en fonction des paramètres de l’étude ($St$, $Re_lambda$ et $ phi_v$. Ces lois d’échelles indiquent une forte influence de $Re_lambda$ et de $phi_v$, mais un faible effet de $St$. Ce résultat est cohérent avec un rôle dominant du mécanisme « sweep-stick » comme origine de la concentration préférentielle, tel que proposé par Vassilicos. En outre, l'analyse conditionnelle des vitesses de sédimentation des particules en fonction de leur appartenance ou non à des amas montre que les zones à fortes concentration tendent à sédimenter plus rapidement que les zones peu concentrées, suggérant un possible rôle des effets collectifs dans l’augmentation de la vitesse de chute. Les mesures par interférométrie de phase Doppler ont ensuite permis d’analyser plus en détail les statistiques de vitesse et de concentration de particules conditionnées à la taille des particules. Ces mesures montrent une augmentation de la vitesse de sédimentation pour les particules de petits diamètres, en accord avec des études précédentes. En revanche, la sédimentation est ralentie pour les particules de plus grand diamètre. Ceci indique une subtile intrication de plusieurs mécanismes possibles affectant la sédimentation turbulente de particules. / This PhD thesis investigates the phenomena of preferential concentration and settling of sub-Kolmogorov inertial particles transported in a turbulent flow. To this end, experiments have been carried out in active-grid-generated turbulence in a wind-tunnel, seeded with water droplets. Preferential concentration manifests itself as the emergence of spatial segregation of the particles, which where initially homogeneously seeded in the carrier flow, leading to clusters and voids. A particular effort has been put in disentangling the roles of particles inertia, of turbulence and of collective effects on the emergence of clustering and the modification of settling velocity and in investigating the interplay between clustering and settling. Four main non-dimensional parameters have been varied to establish the role of each in the clustering process and on the settling of the particles: the Rouse number $Ro$, representing the ratio of the settling velocity of the particles to the fluctuating velocity of the fluid ; the Stokes number $St$ , quantifying particle inertia as the ratio of the particle response time to the flow dissipative time scale ; the Reynolds number $Re_lambda$ representing the degree of turbulence and the volume fraction $phi_v$ representing the concentration of the particles in the two-phase flow.Two experimental techniques (Lagrangian Particle Tracking and Phase Doppler Interferometry) are used to acquire data and diagnose the clustering and settling properties of the dispersed droplets.2D-Lagrangian Particle Tracking has been performed using high-speed visualization of the dispersed droplets in a laser sheet. This gives access to simultaneous statistics of particles spatial distribution and velocity. Clustering has been quantified using Voronoï tessellation and quantitative scalings on the dependency of clustering intensity and clusters dimensions on $St$, $Re_lambda$ and $phi_v$ are found. They show a strong influence of $Re_lambda$ and volume fraction $phi_v$ but a weak effect of $St$. This finding is consistent with a leading role of the “sweep-stick” mechanism in the clustering process, as proposed by Vassilicos. Furthermore, conditional analysis of the velocities of particles within clusters and voids has been performed showing that clusters tend to settle faster than voids, pointing to the role of collective effects in the enhancement of settling.Phase Doppler Interferometry has then been used to further analyse velocity statistics, and particle concentration field conditioned on particle diameter. Enhancement of the settling velocity for small diameters is observed, in agreement with previous studies. On the contrary, for larger particles settling velocity is found to be hindered. This indicates a subtle intrication of several possible mechanisms affecting the settling, including preferential sweeping, loitering and collective effects.

No description available.

Among the major phytohormones, the cytokinin exhibits unique features for its ability to positively affect the developmental status of plastids. Even early on in its research, cytokinins were known to promote plastid differentiation and to reduce the loss of chlorophyll in detached leaves. Since the discovery of the components of cytokinin perception and primary signaling, the genes involved in photosynthesis and plastid differentiation have been identified as those directly targeted by type-B response regulators. Furthermore, cytokinins are known to modulate versatile cellular processes such as promoting the division and differentiation of cells and, in concert with auxin, initiating the de novo formation of shoot apical meristem (SAM) in tissue cultures. Yet how cytokinins precisely participate in such diverse cellular phenomena, and how the associated cellular processes are coordinated as a whole, remains unclear. A plausible presumption that would account for the coordinated gene expression is the tight and reciprocal communication between the nucleus and plastid. The fact that cytokinins affect plastid developmental status via gene expression in both the nucleus and plastid is interpreted here to suggest that cytokinin functions as an initiator of anterograde (nucleus-to-plastid) signaling. Based on this viewpoint, we first summarize the physiological relevance of cytokinins to the coordination of plastid differentiation with de novo shoot organogenesis in tissue culture systems. Next, the role of endogenous cytokinins in influencing plastid differentiation within the SAM of intact plants is discussed. Finally, a presumed plastid-derived signal in response to cytokinins for coupled nuclear gene expression is proposed.

Environmental pollution of heavy metals has become an increasing problem. Environmental risk assessments can be conducted to investigate and determine the potential risk of polluted terrestrial environments. Traditionally, risk assessments are based on total soil metal concentrations on a dry weight basis. Assessments based on total concentrations do not account for metal bioavailability. The bioavailable fraction of the metal is that available for metabolic uptake over a biological membrane, and it is largely controlled by the physiochemical characteristics of the soil solution. For soil-dwelling organisms the most important physiochemical parameters governing copper toxicity are pH and dissolved organic carbon. To incorporate these parameters into risk assessments mechanistic chemical equilibrium models can be used, such as biotic ligand models. These have previously been applied to mainly aquatic environments and only in recent years they have been expanded to the terrestrial realm. The overall aim of this thesis was to test the applicability of a leachate-based biotic ligand model, which takes pH dependency into account, for ecotoxicological risk assessment of soil-dwelling organisms. Toxicity data with associated soil solution pH for seven soil-dwelling organisms and microbially mediated soil processes were obtained from the Swedish Geotechnical Institute. Physiochemical soil characteristics of three Swedish field sampled soils amended with biochar were also obtained from the Swedish Geotechnical Institute. The toxicity data were used to derive two key parameters for calibration of the soil biotic ligand model through linear regression analysis i.e., the pH dependency and the species-specific intrinsic sensitivity. The calibrated biotic ligand models were applied to the field soils and species sensitivity distributions were derived for each soil to calculate hazardous metal concentrations. A simplified risk assessment of the soils was performed based on the results of the leachate-based biotic ligand models and on measured total concentrations on a dry weight basis.  As expected, the results of the regression analysis showed a strong pH dependency between toxicity effect concentrations and pH. For all included test organisms, the copper toxicity effect concentration decreased as the pH of the soil solution increased. Although Cu2+ toxicity increased with increasing pH, the considered organisms showed individual and varying pH-dependencies especially at pH 3-4 and pH 7-8. Further, the results showed that the risk assessment based on the Swedish EPA method, which use total metal concentrations on a dry weight basis, yielded different results than risk assessment based on the leachate-based biotic ligand models. The soils that had been amended with biochar contained lower total Cu concentrations on a dry weight basis compared to those which had not been amended. Consequently, total Cu concentrations exceeded the guideline value for sensitive land-use only in the non-amended soils. Total Cu concentrations exceeded the guideline value for less sensitive land-use in all field soils. Similar to total Cu concentrations on a dry weight basis, the total dissolved Cu concentrations also decreased with added biochar. The same trend could not be seen for Cu2+ in CaCl2 leachates. DOC in the leachates decreased with added biochar, suggesting that biochar sorbs DOC. A majority of the total dissolved Cu was bound to DOC and only a small fraction left as free ions.  The lower DOC concentrations led to higher Cu2+ concentrations in the leachate. Consequently, two of the biochar amended soils had Cu2+ concentrations exceeding the calculated HC50 (protection level for LSL). The predicted toxic effect concentrations ranged from 0.001 μg/L for the most sensitive organism Tomato shoot (L. esculentum) to 3.53 μg/L for the least sensitive organism Soil induced respiration (SIR). The most sensitive field soil had the highest measured pH and had been amended with 6% biochar, the two least sensitive field soils had the lowest measured pH and had been amended with 3 and 6% biochar respectively. The risk assessment based on the soil-BLM approach yielded different, but not less conservative, results compared to the traditional risk assessment based on total concentrations on a dry weight basis. The expected result was for the BLM-based risk assessment to be less conservative as it takes the site-specific bioavailability into account. The leachate-based soil-BLM seems to be sensitive to changes and variations of the input parameters in the speciation. To improve the robustness of the model, and accuracy of risk assessments, additional organisms should be included in the SSDs and speciation should be performed on soils with a wider range of pH. The potential of leachate-based BLMs for risk assessment has been demonstrated. The results invite to further v development of leachate-based soil-BLMs and has the potential to increase the knowledge of the chemistry and toxicology of copper in soils as well as the effects and behaviour of biochar as a metal sorbent.