Thesis (Ph. D. (Plant Production)) -- University of Limpopo, 2015 / Wild watermelon (Cucumis africanus LF.) and wild cucumber (Cucumis myriocarpus Naude.) are known for their ethnomedicine, ethnopesticide, ethnonematicide and nutritional properties, along with nematode resistance. The two Cucumis species were successfully used as inter-generic seedling rootstocks for watermelon (Citrullus lanatus Thunb.) cultivars, where nematode-resistant genotypes are not available. Also, the two Cucumis species are hardy and resilient to inland South Africa conditions, where temperatures are predicted to increase by 6°C in the year 2030. Seeds in the Cucurbitaceae Family contain high concentration of cucurbitacins, which induce auto-allelopathy that inherently inhibits plant growth and germination. Poor germination and non-uniform stands as a result of seed dormancy are a major challenge in sexual propagation of wild Cucumis species for various potential industries. Generally, true-to-type, uniform and disease-free plants in plant production are asexually-generated through in vitro propagation techniques. This study was therefore, initiated to address seed dormancy and related challenges of sexual propagation in the two wild Cucumis species by determining whether: (1) seed dormancy in C. africanus and C. myiocarpus would be ameliorated to allow for in vitro sexual propagation to establish pathogen-free parent stock, (2) the testa in C. africanus and C. myiocarpus seeds would possess structures, which interfere with imbibition and movement of water to the endosperm, (3) all organs of C. africanus and C. myriocarpus would be suitable for in vitro propagation, (4) suitable potting medium for in vitro propagated plantlets of C. africanus and C. myriocarpus would be available for acclimatisation of plantlets and (5) in vitro-produced xxviii plantlets from nematode-resistant C. africanus and C. myriocarpus would retain their resistance to Meloidogyne incognita race 2 under greenhouse conditions. In vitro and ex vitro experiments were conducted to achieve the stated objectives, with treatments in the laboratory and the greenhouse being arranged in completely randomised and randomised complete block designs, respectively. Validity was primarily ensured through the use of factorial trials, while the reliability of data was ensured by using appropriate levels of statistical significance. Leaching alone in C. africanus improved germination, while in C. myriocarpus this treatment had no effect on germination. The optimum leaching time in leached-control seeds of C. africanus was achieved at 7.1 h, with a 25-day mean germination time (MGT) and 52% optimum germination percentage (GP). In the two Cucumis species, the combined effect of leaching seeds in running tapwater and physical scarification of seeds at the chalaza region escalated germination in both Cucumis species, suggesting that both chemical and physical seed dormancies were involved. In C. africanus, cucurbitacin B (C32H48O8) was deposited exogenously to the testa, whereas in C. myriocarpus cucurbitacin A [cucumin (C27H4009) and leptodermin (C27H3808)], was deposited endogenously to the testa. The optimum leaching time in leached-scarified (LS) seeds of C. africanus was achieved at 5.7 h, with at least 40-day MGT and 89% optimum GP. In contrast, in C. myriocarpus LS seeds had the optimum leaching time of 6.3 h, with at least 41 days MGT and 93% optimum GP. Field emission SEM confirmed that there were two “water-gaps”, one at the micropylar region (hilum end) and the other at chalaza region (abaxial end) of seeds in both Cucumis species. Five distinct testa layers in seeds of C. myriocarpus were observed, namely, (i) epidermis, (ii) hypodermis, (iii) sclerenchyma, (iv) aerenchyma xxix and (v) chlorenchyma. In contrast, C. africanus seeds did not have the hypodermis between the micropylar and chalaza regions, but was present around both regions, which may provide some explanation of sporadic germination in non-leached and non-scarified seeds in this Cucumis species. The most suitable plant propagules for in vitro mass propagation of the two Cucumis species were nodal and apical buds. The optimum PGRs for shoot regeneration using both propagules in C. africanus and C. myriocarpus were at 0.80 and 0.35 μM 6-benzyladeninepurine (BAP), respectively. In contrast, the largest number of roots was regenerated at 0.31 and 0.44 μM indole-3-butyric acid (IBA) for C. africanus and C. myriocarpus, respectively. In vitro-produced plantlets were successfully acclimatised to ex vitro conditions, with sand + compost potting medium being the most suitable growing medium for weaning both Cucumis species. The in vitro-produced plantlets retained their resistance to M. incognita race 2. In conclusion, seeds of C. africanus and C. myriocarpus are structurally and chemically different, with strong evidence of chemical and physical dormancies. Structurally, C. myriocarpus seeds consist of five layers, four lignified and one non-lignified, whereas those of C. africanus have four layers, three lignified and one non-lignified. Evidence suggested that in C. africanus seeds, allelochemicals were primarily deposited outside the testa, whereas in C. myriocarpus they were deposited within the testa. The identified seed dormancies could successfully be ameliorated through combining leaching and scarification in both Cucumis species. The developed in vitro propagation protocols accord the two Cucumis species the potential for use as future crops in the context of climate-smart agriculture and research. / Flemish Interuniversity Council (VLIR)

La qualité germinative (vigueur) des semences est un caractère agronomique majeur. Elle correspond à la capacité d'un lot de semences à germer de façon rapide et homogène dans une large gamme de conditions environnementales. Cette qualité germinative est notamment contrôlée par une interaction antagoniste entre deux phytohormones, l'acide abscissique (ABA) qui induit et maintient la dormance et les gibbérellines (GAs) qui stimulent la germination et la croissance de la plantule. La dormance, qui correspond à un blocage physiologique de la germination, est un paramètre non souhaitable d'un point de vue agronomique. Par conséquent la compréhension de la régulation hormonale sur la qualité des semences représente un intérêt fort pour la communauté scientifique mais aussi pour les acteurs de la filière "semences". De nombreuses études ont démontré l'existence d’une importante régulation de la synthèse des protéines au cours de l’imbibition des graines. Cette régulation traductionnelle contribuerait à la mise en place des programmes métaboliques différents en fonction de l’état physiologique des semences pour maintenir un état de dormance ou initier le processus de germination.Le travail réalisé dans le cadre de cette thèse s’est concentré à apporter des éléments nouveaux sur le rôle de l’ABA dans la détermination de la qualité physiologique des semences au cours du développement de la graine et au cours de la germination. L’impact de l’ABA a été particulièrement décortiqué à l’aide de mutant d’Arabidopsis thaliana présentant des teneurs en ABA endogènes contrastées. Par une approche multi-omique combinant des analyses transcriptomiques, protéomiques et métabolomiques, nous avons étudié les mécanismes moléculaires et biochimiques associées avec la mise en place de la qualité physiologique des semences en relation avec l’ABA. Nos résultats ont montré qu’au-delà du contenu en ABA, l’origine tissulaire de cette hormone dans les graines gouverne de nombreux réarrangements métaboliques qui participent au déterminisme de la profondeur de dormance et de la vigueur germinative. Il apparaît un lien entre l’ABA et l’activité traductionnelle, étroitement associé au métabolisme énergétique et à l’homéostasie RedOx.L’effet de l’ABA sur l’activité traductionnelle a été suivi par une adaptation des méthodes SILAC (stable-isotope labelled amino acids in cell culture) aux grains d’Arabidopsis. Cette technique a été utilisée pour décrire la dynamique du protéome dépendante du contenu en ABA des graines au cours de leur imbibition. Nos résultats montrent que cette approche originale permet d’enrichir les connaissances sur la biologie fondamentale des semences. En effet, nous avons montré que l’ABA est un régulateur clé de la synthèse protéique dans les graines et est un contributeur majeur dans la mise en place des différents programmes traductionnels. Cette approche a montré que l’ABA exerce un contrôle sur la traduction de plus de 400 ARNm au cours de l’imbibition des graines et ouvre de nouvelles pistes pour la compréhension de la régulation de la synthèse protéique chez les semences et chez les plantes. Ces données générées offrent un nouveau regard sur le processus germinatif et de sa régulation par l’ABA.Sur la base des données existantes au laboratoire et celles générées au cours de cette thèse, nous avons également développé une utilisation de bio-marqueurs pour l’évaluation de la qualité des semences et nous avons mis au point des traitements de semences innovants. Ces technologies ont été développé en accord avec les attentes des industriels de la filière « semences ». La récente obtention d’un financement pour ce projet de recherche appliquée démontre la complémentarité des recherches effectuées au sein du laboratoire avec les besoin des industriels de la filière « semences ». / Germination vigor is a main concern in agriculture. High seed vigor is defined as the capacity of a seed lot to germinate rapidly, uniformly and in a wide range of environmental conditions. Seed quality is controlled by a dynamic balance between two antagonistic hormones, abscisic acid (ABA), which induces and maintains dormancy and gibberellins (GAs), which stimulate seed germination and seedling establishment. Seed dormancy corresponds to a block to the completion of germination and is an undesirable characteristic from an agronomic point of view. Thus, investigation of seed quality toward a better understanding of hormonal regulation is of fundamental concern for scientific community and seed industry.Recent studies have highlighted the intensive regulation of protein synthesis during seed germination. Translational regulation would govern the implementation of different metabolic programs during seed imbibition in order to maintain seed dormancy or to initiate the germination process. In this thesis, we explore the role of ABA in the control of germination quality during seed development and seed germination, using Arabidopsis thaliana mutant displaying contrasted ABA content.By combined “omic” approaches, we have highlighted the impact of ABA level on metabolic rearrangements during seed maturation. Our results showed that ABA origin in the seeds governs many metabolic rearrangements controlling dormancy depth and germination vigor. In addition, the present work suggests an intimate linkage between translational activity and ABA content, in association with energetic pathways and redox homeostasis.The impact of ABA on proteome turnover during seed germination was studied by adapting a metabolic labeling of neosynthesized proteins based on SILAC methods (stable isotope labelled amino acids in cell culture) to Arabidopsis seeds. Our results suggest that ABA is a key regulator of protein synthesis and modulates metabolic changes during seed imbibition. Indeed, this novel approach has highlighted that ABA controls the translation of more than 400 mRNAs during seed imbibition. This work provides an original perspective on the contribution of ABA and mRNA translation in seed germination and provides a valuable basis for further investigation of translational regulation in seeds and in plants.Based on existing data and those generated during this thesis, we also developed innovative seed treatments and new biomarkers for seed quality assessment. Recent funding for a maturation program dedicated to improve these biotechnologies demonstrates that our research meets the needs of seed industry.

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Organisms are often adapted to their local environment, but the role of early life stages in adaptive differentiation among populations remains poorly known. The aim of my thesis was to investigate the contribution of early life stages to the magnitude and genetic basis of local adaptation, and to identify the underlying adaptive traits. For this, I used two natural populations of the annual plant Arabidopsis thaliana from Italy and Sweden, and a Recombinant Inbred Line (RIL) population derived from a cross between these populations. By combining greenhouse and field experiments, Quantitative Trait Loci (QTL) mapping, and path analysis, I examined (1) the genetic basis of seed dormancy, (2) the contribution of differential seedling establishment to local adaptation, (3) among-year variation in selection during seedling establishment, (4) direct and indirect effects of seed dormancy and timing of germination on fitness, and (5) the adaptive value of the seed bank. I found that both the level and the genetic basis of seed dormancy were affected by the maternal environment. One major-effect QTL was identified in all maternal environments, which overlaps with the dormancy gene DELAY OF GERMINATION 1 (DOG1). Selection through seedling establishment success contributed strongly to local adaptation and genetic tradeoffs, and varied among years. Variation in seedling establishment and overall fitness among RILs could be explained by genetically based differences in seed dormancy and timing of germination. Seed dormancy affected fitness throughout the life cycle, by affecting the proportion of germinated seeds, and indirectly via effects on timing of germination, plant size and flowering time. My results suggest that a considerable portion of A. thaliana seeds enter the seed bank. I found genetic differences in dormancy cycling behaviour between the two populations, which could contribute to local adaptation. The value of a seed bank should be higher at the Swedish study site than at the Italian study site due to lower rate of seed mortality in the soil. Overall, the results of this thesis demonstrate that early life stages contribute strongly to both the magnitude and the genetics of local adaptation.

Made available in DSpace on 2016-04-28T14:56:18Z (GMT). No. of bitstreams: 1 2005- Juliana Muller Freire.pdf: 826620 bytes, checksum: f7f3878211e26134428516a4db9f4737 (MD5) Previous issue date: 2005-03-15 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The aim of this work was to estimate the level and distribution of genetic variation within and among five Schizolobium parahyba (guapuruvu) populations and to evaluate the morphometric traits and dormancy of seeds of two populations of these species. The collect was carried out at coastal and mountain regions in the south of Rio de Janeiro. RAPD (Random Amplified Polymorphic DNA) markers were used in order to estimate the similarity of the genotypes and to indicate the genetic distance among individuals and populations. The following morphometric traits of seeds was assessed: width, lenght, thickness and weight. Variation in dormancy level was tested in seeds of two populations comparing the performance of scratching and not scratching seeds. Germination porcentage, germination and emergency speed index, normal seedling porcentage and death porcentage was assessed and compared among individual plant and populations by using ANOVA test. The polymorphic loco was 97%, as expected for a wide distributed specie. Of the total genetic diversity, 89% was atributtable to differences within populations and 11% to differences among populations, indicating a low amount of populations differentiation. The estimative of gene movement among populations revealed a high value (3,18 migrant per generation). Correlations among genetic and geographic distance were not found, indicating the lack of spatial structure of the specie. All morphometric traits differed significantly among individual within each of the population. The individuals plants from the mountain region presented the highest seed size. The major morphometric variation ocurred within population. The seed lenght responded with 60.87% of the total morphometric variation, followed by width (26,58%) and thickness (11,193%). Four groups were created by cluster analysis, with no origin relationship. Dormancy level differed significantly among and within populations and the highest dormancy level was presented by the coastal population. The germination speed index and death porcentage expressed better the dormancy levels than the other variables, differing significantly in the most of individuals trees. Seeds not scratched from mountain population presented better performance than the coastal one. Morphometrical and germination traits were not correlated. There were a high death percentage, specially for the scratched seeds. The role of biotics and abiotics factors in the selection of germination behavior are discussed based in the results and field observations. / Os objetivos deste estudo foram estimar o n?vel e a distribui??o da varia??o gen?tica entre e dentro de cinco popula??es de Schizolobium parahyba (guapuruvu), analisar a diversidade morfom?trica e a dorm?ncia de sementes considerando duas popula??es desta esp?cie. As popula??es estudadas est?o localizadas na regi?o litor?nea e serrana do sul do Estado do Rio de Janeiro. A an?lise gen?tica foi realizada utilizando-se marcadores RAPD (Random Amplified Polymorphic DNA) que resultou na estimativa de similaridade dos gen?tipos, indicando a dist?ncia gen?tica entre indiv?duos e entre as popula??es. A an?lise morfom?trica foi realizada em sementes colhidas em duas popula??es, tendo sido avaliadas: largura, comprimento, espessura e peso das sementes. No estudo de germina??o foi avaliado o n?vel de dorm?ncia das sementes, comparando matrizes de duas popula??es. Foram calculados a porcentagem de germina??o, ?ndice de Velocidade de Germina??o (IVG), n?mero de pl?ntulas normais e anormais, ?ndice de Velocidade de Emerg?ncia (IVE) e mortalidade. A diferen?a entre tratamentos e locais foi feita atrav?s da ANOVA. A propor??o de locos polim?rficos foi de 97%, valor considerado compat?vel para uma esp?cie de ampla distribui??o geogr?fica. Da varia??o gen?tica total observada, 89% ocorreu dentro das popula??es e 11% entre as popula??es, indicando um baixo n?vel de diferencia??o entre as popula??es. A estimativa de fluxo g?nico entre popula??es (NM) foi alto, alcan?ando 3,18 migrantes por gera??o. N?o foi encontrada correla??o entre dist?ncia gen?tica e geogr?fica (r=0,036), evidenciando a falta de estrutura??o espacial da esp?cie. A diversidade morfom?trica indicou diferen?a significativa entre matrizes de uma mesma regi?o para todas as vari?veis morfom?tricas analisadas. As matrizes de Miguel Pereira apresentaram maior tamanho de semente do que as de Paraty, sendo esta diferen?a significativa para todas as vari?veis, exceto comprimento. A maior varia??o no tamanho da semente ocorreu a n?vel intra-populacional, sendo significativo para todas as vari?veis. O comprimento respondeu com 60.87% da varia??o total do tamanho da semente, seguido da largura (26,58%) e da espessura (11.193%). A an?lise de agrupamento permitiu a forma??o de quatro grupos, n?o sendo poss?vel associ?-los ? regi?o de origem. A dist?ncia morfom?trica e gen?tica aparentemente n?o apresentaram correla??o. O n?vel de dorm?ncia das sementes variou entre e dentro de popula??es. Paraty apresentou maior diferen?a entre os tratamentos (sementes escarificadas e n?o escarificadas), evidenciando seu maior grau de dorm?ncia. Os par?metros que melhor expressaram a dorm?ncia foram o ?ndice de Velocidade de Germina??o e a taxa de mortalidade. As sementes n?o escarificadas de Miguel Pereira se mostraram superiores as de Paraty em praticamente todos os par?metros germinativos. N?o houve correla??o entre as vari?veis morfom?tricas e germinativas. Houve alta mortalidade das sementes por fungos, principalmente em rela??o ?s sementes escarificadas. A influ?ncia dos fatores bi?ticos e abi?ticos (precipita??o) no comportamento germinativo s?o discutidos com base nos resultados obtidos e observa??es de campo.

A large number of weed seeds in the soil persist because of seed dormancy, and depletion of the seed bank through manipulation of seed dormancy has been suggested as one of the goals in weed control. This study was designed to investigate some of the factors which control dormancy and germination in Amaranthus retroflexus seeds. Germination studies were conducted at different temperatures, and either in continuous white light or in the dark. Higher temperatures increased germination and, although light interacted with temperature, its effect on germination varied with the temperature. In an attempt to determine changes in dormancy during dry storage, two lots of seeds were stored dry at different temperatures. Loss in dormancy increased with an increase in storage temperature and duration, but the time required for maximum germination varied according to the seedlot. Seeds germinated to higher percentages at high temperatures, but storage at higher temperatures and for prolonged duration resulted in seeds gaining the ability to germinate at lower temperatures. Changes in dormancy under field conditions were also examined. Seeds were buried at different depths and for different durations and they all lost viability with time, but this loss was greater in surface-sown and shallowly buried seeds. Dormancy was broken during cold periods and induced as warmer periods progressed. The effects of chemical stimulants on dormancy and germination were investigated. The response of seeds to ethephon and nitrate were assessed at different temperatures either at continuous white light or in the dark. Germination increased with the concentration of the chemicals, and a greater response was observed at lower temperatures. The response to light varied depending on temperature / Master of Science (Hons)

Studies of seed dynamics and seedling establishment at the community level in savanna ecosystems are scant, particularly in the tropical savannas of Central Brazil. The Brazilian savannas (cerrado) have the largest diversity of plant species among Neotropical savannas. Cerrado vegetation exhibits consistent changes in tree density and tree size along shallow topographic gradients. Vegetation types differ from closed savannas with high tree density, and a nearly continuous canopy cover in the uppermost portions, to open savannas with scattered short trees in the low portions of the topographic gradients. Whether or not dynamics of seeds and seedlings of woody species are consistent with variations in tree density and tree canopy cover across the cerrado landscape has not been determined, but could potentially influence such spatial patterns of tree density and canopy cover. The main objective of this study was to evaluate seed dynamics (i.e., seed rain, soil seed banks, and seed characteristics) and seedling establishment of woody species in three major cerrado vegetation types (closed, intermediate and open savannas) which differ in tree canopy cover and tree height. These vegetation types are located along topographic gradients of approximately 30 m in elevation and 1000 m in length. I conducted field work from June 2005 to August 2008 at the Instituto Brasileiro de Geografia e Estatística (IBGE) reserve, a field experimental station located 35 Km south of Brasilia, Brazil (15˚ 56' S, 47˚ 63' W, altitude 1100 m). I established permanent plots in each of the three vegetation types to assess seed rain, seed limitation, seed predation, and seed removal. I also quantified density and composition of the soil seed bank in each vegetation type and studied seed characteristics (i.e., kind of dormancy, moisture content, longevity) of 14 common cerrado woody species. In the field, I quantified establishment of woody seedlings in fire-protected savannas as well as in accidentally burned and frequently burned savannas. In the greenhouse I performed experiments to evaluate the effect of litter cover and light level on seedling emergence of 9 common cerrado woody species. Finally, I evaluated the effect of pulses of heat on seed germination of 5 common cerrado woody species under controlled conditions. The overall results of this study show that regardless of tree canopy cover, cerrado vegetation types are limited in seeds and seedlings of woody species, but these limitations are higher in open than in closed savannas. Most woody species do not form persistent soil seed banks along cerrado vegetation types because their seeds are short-lived, predated and quickly removed from the ground. In addition, dispersal season and dormancy appear to control timing of germination at the onset of the rainy season to ensure survival of seedlings in the field. In the absence of fire, establishment of woody seedlings is higher in closed than in open savannas because canopy cover mitigates the stressful environmental conditions of open savannas and thus facilitates establishment of woody seedlings. As a result, closed savannas are likely to maintain higher density of woody elements over time than open savannas. In burned savannas, however, species composition of woody and herbaceous seedlings is significantly affected because establishment of seedlings of woody and herbaceous species decreases but vegetative reproduction increases. This dissertation study indicates that interactions of early life-history stages of woody species with environmental and disturbance factors appear to play a more significant role in maintaining woody cover variations along cerrado topographic gradients than previously recognized.

Seed heteromorphism related to chasmogamous (CH) and cleistogamous (CL) seeds can have different ecological significance in species with different functional characteristics, competitive ability and palatability. Punagrass [<i>Achnatherum brachychaetum </i> (Godr.) Barkworth], an invasive, perennial grass is common in grasslands of Argentina and it is an aggressive weed in other parts of the world. <i>Flechilla grande</i> [<i>Nassella clarazii</i> (Ball) Barkworth] is a palatable perennial grass associated with the dominant "climax" vegetation in grasslands of Argentina. Seeds of the two grasses were collected from grasslands of Argentina, and growth chamber and greenhouse experiments were conducted to determine: 1) germination, dormancy breaking, and mass of CH and CL seeds of the two species 2) effects of contrasting range condition on germination and seed mass of punagrass, 3) the relative fitness of plants from CH and CL seeds, 4) if contrasting range condition affect fitness of CH plants of punagrass, and 5) the effect of maternal nutrient environments on CH and CL seeds and on fitness in the two species. Small CH seeds of high dispersal potential were less dormant than large CL seeds of low dispersal potential in punagrass. CH and CL seeds of flechilla grande had similar mass, germination, and response to dehulling. CL seed size and CL seed production of punagrass increased with good range condition. Increasing the maternal, nutrient environment enhanced germination of CH seeds, CL seed size, growth rate, development, biomass and seed production more in punagrass than flechilla grande. Under low nutrient conditions, flechilla grande produced a few large CH seeds. CH progeny of punagrass grew fast and developed rapidly as compared to CL progeny, which in turn produced many CH seeds. CH and CL seeds of flechilla grande had similar contribution to the fitness of adult plants. In both species, the ecological significance of having seed heteromorphism is that sibling competition is probably reduced by having more diverse offspring. Heavy grazing of competitive species such as flechilla grande may favours species as punagrass with many small CH seeds, high potential for colonization as well as large CL seeds for persistence in the seed bank and seedling competition.