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The structure and properties of a nascent pectin-xyloglucan complex in etiolated pea epicotyls were investigated. Membrane pellets were prepared and incubated with UDP-[14C] galactose, and were extracted with six different reagents: Tris-HC1 buffer (pH 7.4); 50mM EDTA/50mM PO4 (pH6.8) at 100°C; 50mM EDTA/59mM PO4 (pH6.8) at 25°C; phospholipase C with 100 ml of 0.1M Tris-HC1 pH7.4 at 25°C; trypsin at 25°C and 0.1% Triton X-100 at 25°C. The best extractant used to solubilise the pectin-complex from the pellets was 50nM EDTA/50mM PO4 pH 6.8 at 100°C. Aqueous solutions of pectic polyuronides tend to associate covalently into multichain aggregates. Because of the tendency of pectins to aggregate in solution, the effect of a number of eluents on the behaviour of the complex on gel filtration was studied. 10mM EDTA/10nM PO4/1M NaC1 (pH6.8) was chosen as the best eluent to minimise this aggregate formation. On gel filtration using Sepharose CL-2B with this eluent, the complex eluted with a Kav of around 0.8, corresponding to a molecular size approximately 200 kDa, as judged by dextran standards. In other solvents tested, aggregation appeared to occur. On Sepharose CL-4B and CL-6B columns using the same EDTA/Pi/NaC1 solvent, the apparent molecular size was significantly reduced by a xyloglucan-specific endoglucanase, confirming the presence of a xyloglucan rather that a glucan in the complex. Polygalacturonase caused a greater decrease in apparent molecular size, to 10-20 kDa while endo-1 4-b-galactanase converted the radioactivity to [14C] galactobiose and [14C] galactose. When the [14C] galactose-labelled complex was incubated in solution with 3MM paper, the radioactivity was almost completely absorbed onto the paper over a period of about 6 hours. This confirmed the presence of xyloglucan attached to pectin. A new model for the structure of the nascent pectin-xyloglucan complex is proposed.

Five experiments were conducted to investigate the effect of drought and high temperature stress on the growth and development of bambara groundnut (Vigna subterranea (L.) Verdc). Three glasshouse experiments were conducted at the University of Nottingham, Sutton Bonington Campus, UK, and two field experiments were conducted at the Botswana College of Agriculture, Gaborone, Botswana. In the glasshouse experiments, two landraces were grown, S19-3 (from hot, dry environment/ Namibia) and Uniswa Red (from cool, wet environment/ Swaziland) under two different temperatures, 33±5 oC and 23±5 oC. In the first experiment (2006), soil moisture was non-limiting. In the second experiment (2007) drought was imposed at pod filling stage (77 DAS). In the third experiment (2008), the same two landraces were grown under the same temperatures, but the drought was imposed at flowering (30 DAS). In the first field experiment, two landraces were grown under three sowing dates and two water regimes; rain fed and drought. The two landraces were Dip C (from hot, dry environment/ Botswana) and Uniswa Red. Drought was imposed approximately at pod filling (63 DAS). In the second field experiment, the same landraces were grown under the same sowing dates and water regimes with drought imposed at 30 DAS. Canopy development and growth were affected by temperature and water stress. In the glasshouse experiments, Uniswa Red always gave the highest leaf number at the high temperature and S19-3 had the lowest at the low temperature. Leaf number decreased with drought, it reached over 100 in the full irrigation treatment, and less than 100 in late season drought treatment and a maximum of 60 in the early season drought treatment. Crops grown under high temperature always had higher leaf area index and total dry matter. The highest yield (306 gm-2) was produced by S19-3 at 33°C in 2007 and the lowest (31.1 g m-2) by Uniswa Red at 33°C in 2008. Comparison of regressions showed no significant difference in water use efficiency (WUE) between treatments in 2007. However, there were significant differences in 2008 when S19-3 (1.80 g kg-1) had a greater WUE than Uniswa-Red (1.09 g kg-1) at the high temperature, but both landraces had similar WUE at the low temperature (S19-3 2.28 g kg-1, Uniswa Red 2.23 g kg-1). This indicates that, despite being from a hot, dry environment, S19-3 performs well at the low temperature, and this is supported by data from 2007 when S19-3 maintained the highest soil moisture content and the lowest evapotranspiration at the low temperature. For the field experiments, where the temperature decreased with delay in sowing, there was a reduction in development, growth and yield. The effect of sowing date on leaf number was significant in both field experiments. In the first field experiment, the four treatments mean of leaf number of leaves declined from 62 per plant in the first sowing date (D1) to 52 leaves per plant in the third sowing date (D3) and 46 leaves per plant in the fifth sowing date (D5) and it was 64, 52, and 37 for D1, D3, and D5 respectively in the second field experiment. WUE decreased with delay in sowing from average of 1.9 g kg-1 in D1 to average of 0.45 g kg-1 in D5. The landraces varied in their response to temperature and drought stress with respect to growth, development and resource capture and conversion. The landraces used different mechanisms to resist drought and temperature stress, that include high leaf water content, reduction in leaf area to reduce transpiration surface and avoidance through faster growth rate.

Thesis(Ph.D.(Botany)) --University of Limpopo,2019 / Genetic transformation offers great opportunities for rapidly introducing, selecting or inducing desired characteristics in various leguminous plants for breeding purposes. But, this technique remains aloof for soybean improvement due to challenges such as genotype specificity, inefficient regeneration protocols and the rapid loss of viability in seeds required to develop explants. However, the rate of seed deterioration and its influence on in-vitro plant genetic transformation differs according to the age, storage duration and moisture content of the seeds used. The moisture status of the seeds is usually high during harvesting and deterioration (loss of viability) starts to occur when seeds are stored under ambient conditions for long periods. This seed deterioration also results in a phenomenon called “recalcitrance”, which is predominantly realised in soybean. In the present study, selected soybean genotypes were analysed for: (i) the efficiency of germination using seeds stored for 0, 3, 6 and 9-months under ambient conditions (ii) the effect of seed storage on in-vitro multiple shoot induction, (iii) the competency of the selected soybean genotypes on callus induction and Agrobacterium-mediated genetic transformation and (iv) the evaluation of protein profiles of the genotypes following co-cultivation of cotyledonary node explants with A. tumefaciens. The results obtained in this study showed that, seed stored for more than 3-months had reduced rates of germination, seedling development and in-vitro shoot multiplication. In particular, seed stored for 9-months showed a significant drop in seed germination, and less than 50% overall seed germination (Dundee-42%, LS678- 49%, TGx140-2F-44% and TGx1835-10E-48%) except for LS677 and Peking with 52 and 55%, respectively. The efficiency of multiple shoot induction also decreased with the prolonged seed storage, with all genotypes recording overall decline from about 96% to 40% regeneration efficiency over this period. The mean number of induced shoots decreased from more than 10.5 to 4.2 shoots per explant, for each genotype. The results obtained clearly indicated that efficient in-vitro shoot induction depended largely on seed storage duration, viability and significantly differed according to genotype. Following the evaluation for callus induction and Agrobacterium-mediated genetic transformation frequencies, the results indicated that the responses were genotype specific. This trend was consecutively observed in all soybean cultivars used (LS677, LS678, Dundee, Peking, TGx1740-2F and TGx1835-10E). Furthermore, the responses of the genotypes were also dependent on the culture media composition,especially, plant growth regulators and antibiotics. Amongst the cultivars used, Peking demonstrated the highest callus induction capacity (more than 70%) on MS-A and the mean number of shoots induced (1.65) using cotyledonary explants co-cultivated with Agrobacterium. This was followed by LS677 (1.42 shoots), LS678 (1.40 shoots), Dundee (1.30 shoots), TGx1835-10E (0.80 shoots) and TGx1740-2F (0.75 shoots), respectively. These genotypes also demonstrated low yields of proteins, extracted using a TCA buffer, and separated by means of two-dimensional polyacrylamide gel electrophoresis. The one-dimensional and two-dimensional profiles of proteins extracted from explants infected with Agrobacterium differed significantly to those expressed without co-cultivation of cotyledonary nodes with bacteria. These observations suggested that, the infection and co-cultivation of explants with Agrobacterium may have caused the expression of new proteins. Newly expressed proteins could also be found to either promote or inhibit transgene integration and expression on the cotyledonary node explants transformed with Agrobacterium tumefaciens for trait improvement. This study has clearly demonstrated that soybean production is confronted with a myriad of stress factors, including seed storage and quality problems due to unfavourable storage duration and weather conditions, amongst others. Thus, soybean seeds used for germination, callus induction, multiple shoot induction and genetic improvement should be harvested at R8 stage after reaching physiological maturity (with 20-35% seed moisture content) to avoid any mechanical damage, shattering or loss of seed viability. / National Research Foundation

Le caroubier est considéré comme l'un des arbres fruitiers et forestiers qui présente le plus grand potentiel de valorisation puisque toutes les parties de cette plante sont utilisables dans plusieurs applications industriels. Le caroubier est cultivé dans plusieurs régions du Liban mais peu d'études sont disponibles sur les voies de valorisation et sur les propriétés fonctionnelles et structurales des graines et des gousses. Ainsi, dans le cadre d'une démarche qui vise à promouvoir et contribuer à une meilleure valorisation et gestion de cette ressource renouvelable, nous avons entrepris dans cette thèse des travaux consacrés à la caractérisation et à l'évaluation de la valeur nutritive et fonctionnelle des gousses de caroube issues de plusieurs régions libanaise. Ces travaux ont porté notamment sur la caractérisation morphologique et physico-chimique des gousses et sur la purification des gommes extraits des graines. Ces gommes ont fait l'objet d'une étude approfondie portant sur leur comportement rhéologique et la relation entre ce comportement, la variété et le lieu géographique. Nous avons aussi déterminé les isothermes de sorption de ces gommes pour prédire les conditions idéales de leur conservation. Les résultats obtenus à l'issue de cette étude ont démontré clairement la présence des corrélations morphologie, composition chimique et coordonnées géographiques de différents cultivars étudiés. De même, les études structurales et rhéologiques ont montré des différences significatives entre les gommes purifiées. Cette différence semble être liée à une variation du rapport galactose/mannose et de la masse molaire observée selon les variétés. Par ailleurs, les études des isothermes de sorption ont permis de fournir des informations complémentaires sur leurs hygroscopicités et par conséquence sur les conditions idéales de leur conservation / The carob plant is considered as one of the fruit and forester tree that has the greatest potential of valorization since all the parts of this plant can be used in several food and cosmetic applications. The carob is cultivated in several areas of lebanon but few studies are available on the ways of valorization and the functional and structural properties of the different fractions of this tree (seeds and pods). Thus, in view to develop a strategy aims to promote and contribute to a better valorization and management of this renewable resource, we undertook in this thesis of work devoted to the characterization and the evaluation of nutritive and functional properties of the carob pods resulting from several lebanese areas. This work focused particularly on the morphological and physicochemical characterization of the pods and the purified fraction of gum. These gums were the subject of a thorough study relating to their rheological behavior and the relation between this behavior, the variety and the geographical localization of the studied varieties. We also determined the isotherms of sorption of these gums to predict the ideal conditions of their conservation. The obtained results clearly showed the presence of the correlations between morphology, chemical composition and geographical localization of the different studied cultivars. In the same way, the structural and rheological studies showed significant differences between purified gums. This difference seems to be related to a variation of the galactose /mannose ratio and molar mass according to the varieties. These interesting rheological properties open the carob gums a new niches of valorization with added values. In addition, the studies of the isotherms of sorption provided additional information on their hygroscopicities and by consequence on the ideal conditions of their conservation

Le changement climatique entraîne un changement profond des régimes thermiques qui a des conséquences importantes sur la croissance des plantes. Toutefois, les effets des températures extrêmes ont été peu étudiés chez espèces fourragères pérennes, et ne sont pas intégrés dans les modèles actuels de prairie. Or ces modèles ont un rôle central à jouer dans le développement de stratégies d'adaptation au changement climatique. Dans ce contexte, cette thèse a cherché à approfondir la compréhension de la réponse de la luzerne et de la fétuque élevée à une large gamme de températures et à améliorer la prise en compte de ces réponses dans le modèle STICS. Un volet de travail expérimental en conditions contrôlées n'a pas permis de mettre en évidence de différences de réponses entre génotypes d'origine contrastées (tempéré/Méditerranéen) pour différents processus de croissance et de développement des deux espèces. En revanche, il est apparu des différences significatives entre les réponses normalisées de différents processus ; l'allongement des tiges en particulier se distinguant nettement des autres processus chez la luzerne. La réponse de la photosynthèse à la température a montré une forte acclimatation à la température de croissance, aussi bien pour des génotypes d'origine tempérée que méditerranéenne. Ces derniers sont apparus plus sensibles aux températures élevées du fait d'une désactivation de la RUBISCO. La modification des formalismes de réponse à la température de la croissance et du développement dans STICS (fonction non linéaire, pas de temps) a eu un fort impact sur le cumul de temps thermique pour des scénarios climatiques « futurs ». Toutefois, du fait de l'effet majoritaire d'autres stress conjoints durant les périodes de stress thermique et d'une rapide saturation du rayonnement intercepté, l'impact sur les projections de production fourragère s'est avéré limité pour les conditions testées. Au total ces résultats suggèrent que les productions fourragères pourraient être moins exposées au stress thermique que les cultures annuelles. / Due to climate change, the global average temperature is expected to rise with marked impacts on crop growth. However, extreme temperature's impacts on perennial herbaceous species have received much less attention to date than other crops and aren’t currently implemented in grassland models. Yet, grassland models have a major role to play in defining adaptation options to global warming’s impacts on agriculture. In this context, our aims were to characterise the responses to temperature of two major perennial forage species over a large range of growth temperature and to improve the temperature response formalisms in the STICS model. First, experiment conducted in growth chambers indicated that the responses of normalized developmental rates did not differ between genotypes from contrasting thermal areas (Mediterranean/temperate) within each species. On the other hand, the responses to temperature of normalised rates differed significantly between several of the physiological processes studied; in particular stem elongation rate differed from other developmental rates in alfalfa. Irrespective of cultivar origin, a remarkable acclimation of photosynthesis to growth temperature was observed. Mediterranean genotypes displayed a greater sensitivity of the maximum rate of Rubisco carboxylation to elevated temperatures. Modifications of temperature formalism in the STICS crop model (implementation of nonlinear response, hourly/daily step of response) had a significant impact on thermal time accumulation and crop development for future climate scenarios. However, due to larger effects of water stress and to saturation of radiation interception, impacts on projections of grassland dry matter production were limited. This result suggests that grassland production could be less exposed to thermal stress than annual crops.

L’amélioration génétique de la luzerne (Medicago sativa), une légumineuse fourragère autotétraploïde, pourrait bénéficier de l’allele mining. Cette méthode consiste à rechercher, dans la diversité naturelle, des allèles ayant potentiellement un effet sur le phénotype. Pour évaluer l’intérêt de cette stratégie qui exploite la diversité naturelle, cinq gènes impliqués dans des caractères sélectionnés ont été retenus : CAD1 et CCoaOMT (digestibilité), CONSTANS-like (rendement fourrager), NHX1 (tolérance à la salinité) et WXP1 (tolérance à la sécheresse). La diversité de ces gènes a été étudiée en séquençant 387 génotypes cultivés et 20 génotypes sauvages. L’analyse des données confirme la présence d’un goulot d’étranglement durant la domestication et la sélection de la luzerne. CONSTANS-like et WXP1 révèlent de nombreux variants alors que CAD1, CCoaOMT et NHX1 sont très peu variables. Des variants ayant un effet potentiel sur le phénotype ont été identifiés dans les zones de la séquence protéique qui sont conservées au sein des Faboideae. L’impact sur le phénotype de deux variants du gène CONSTANS-like a été étudié : Constans-634, à l’origine d’un codon stop prématuré et Constans-4111, situé dans une région conservée du gène. Pour cela des croisements ont été réalisés afin d’obtenir une descendance avec toutes les doses possibles (AAAA, AAAB, AABB, ABBB and BBBB) pour chaque mutation étudiée. Des marqueurs KASPar ont été développés afin de déterminer les doses de mutations chez les descendants. Aucun génotype homozygote muté pour Constans-634 n’a été détecté parmi les 1505 descendants. Ce variant a induit une floraison plus précoce de trois jours pour les génotypes portant trois doses d’allèle muté. Le variant Constans-4111 a induit un effet additif sur la hauteur de tige. Les génotypes homozygotes de type sauvage étaient en moyenne 11,8 cm plus petits que les génotypes homozygotes portant trois ou quatre doses du variant. L’intégration de la stratégie allele mining dans les schémas de sélection des espèces végétales autotétraploïdes hétérozygotes a été discutée. / Lucerne (Medicago sativa) is an autotetraploid forage legume, whose breeding could beneficiate from allele mining. This strategy is based on the natural diversity and consists in seeking alleles with a potential effect on the phenotype. The interest of this approach was evaluated by studying five genes of agronomic interest: CAD1 and CCoaOMT (digestibility), CONSTANS-like (forage yield), NHX1 (salt tolerance) and WXP1 (drought tolerance). The diversity of these five genes was evaluated by sequencing 387 genotypes of cultivated accessions and 20 genotypes of wild accessions. The results confirmed a bottleneck during lucerne domestication and selection. CONSTANS-like and WXP1 were very variable whereas CAD1, CCoaOMT and NHX1 contained very few variants. Variants with a potential strong impact on the phenotype were identified in conserved parts of protein sequence within the Faboideae. The impact on phenotype was studied for two mutations of the CONSTANS-like gene: constans-634, causing a premature stop codon and constans-4111, located in a conserved region of the gene. Genotypes carrying one to three doses of the mutations (AAAB, AABB and ABBB) were polycrossed in order to obtain offsprings with every allele combination (AAAA, AAAB, AABB, ABBB and BBBB). KASPar markers were developed to determine the mutation doses in offspring progeny. No homozygous genotype was found for constans-634 in the 1505 offspring progeny. This mutation induced a premature flowering of three days for the genotypes carrying three doses of the mutation. The mutation constans-4111 induced an additive effect on stem height and the homozygous genotypes without the variant where on average 11.8 cm shorter than homozygous genotypes carrying three or four doses of the variant. The application of allele mining strategy in plant schemes of heterozygous autotetraploid species was discussed.