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One of the major bottlenecks limiting farmers’ access to good quality seed for food crops in Uganda is the shortage of early generation seed (EGS - breeder and foundation) to produce sufficient quantities of certified and/or quality declared) to satisfy the needs of farmers. A national study was conducted between October 2015 to March 2016 to analyse pathways for promoting commercial and sustainable production and delivery of EGS. Five crops (hybrid maize, rice, beans, sesame and finger millet) were selected. The analysis provides real examples of potential business models that could scale in a commercially sustainable manner. For areas that are best suited to public sector investment, opportunities for public-private collaboration and increased efficiencies in the sector are outlined. Generalizable principles and recommendations to guide key stakeholders as they pursue policies, investments, and interventions are proposed.

The programme on Integrated Seed Sector Development in Ethiopia aims to strengthen the development of a vibrant, market-oriented and pluralistic seed sector in the country, where quality seed of superior varieties is available and affordable for a larger number of farmers, thereby contributing to food security and economic development in Ethiopia. The programme is a joint effort of Bahir Dar University, Haramaya University, Hawassa University, Mekelle University, Oromia Seed Enterprise, the Ethiopian Seed Association and Centre for Development Innovation of Wageningen UR. Partners include governmental organizations at federal, regional and local level, non-governmental organizations, development organizations, and seed businesses operating at different scales. The programme is funded by the Directorate General for International Cooperation through the Embassy of the Kingdom of the Netherlands in Addis Ababa.

This research investigates and describes the conservation and use of Plant Genetic Resources (PGRs), especially seeds through processes of commonisation. Seeds form an important element for sustaining human life (through food production) and social relations (by maintaining agricultural socialities). Therefore, conservation and management of PGRs in the form of seeds are essential for plant breeding, agricultural production and to meet the growing food demand of the increasing population. However, the changed use of PGRs through enclosures and appropriation of the Intellectual Property Rights creates underutilisation of these resources, risking their important societal role. Thus, this research aimed at analysing how the processes of commonisation of PGRs, especially seeds as biosocial commons emerge in the Indian context. The research applied an in-depth qualitative research approach using case study method. It focused on four distinct issues of disconnection, collective resistance, strategies of repossession and ability of stakeholders to provide insights broadly into the processes of commonisation of PGRs. Describing the different cases it also establishes whether and how opportunities for commonisation of PGRs as biosocial commons emerge within these contexts. The research analysed four cases where one case reflected on the intellectual commons produced through institutionalisation of PGRs and the other three cases reflected on the bottom-up perspective of commons produced through Non-Governmental Organisations (NGOs). The research through its first case, the Central Rice Research Institute (CRRI), a public ex situ genebank, describes the disconnection of PGRs, while through the second case reflects on the collective activity of resistance through management of community seed banks (CSBs) by the Deccan Development Society (DDS). The third and fourth cases involved small, local initiatives; Loka Samabaya Pratisthan (LSP) and Sambhav that fostered collective action for repossession through in situ seed banks. The research used various techniques, such as interviews with respondents, focus group discussions (FGDs) and participant observation for primary sources of data, with published and unpublished documents, reports and official websites as secondary sources. The second chapter of the thesis looks at the issue of disconnection and argues that storing seeds at genebanks disconnects the resources from their biosocial environment. Further, the evaluation of genetic traits within the stored seeds through the scientific intervention at the genebank creates the divide between the resources (seeds) and their informational content. Thus, this chapter concludes that disconnection of seeds from their biosocial environment leads to the creation of exclusive but positive intellectual commons. The third chapter of the thesis looks at the issue of collective resistance and argues that disconnection of the community from their local food system can generate resistance and collective activity among the community. This chapter finds that the resistance and collective activity further brought in the interaction between the resource and the stakeholders through informal social relations and seed networks. The fourth chapter of the thesis looks at the issue of strategies of repossession and argues that socio-political and ecological context play an important role in determining the strategy for repossession and commonisation of PGRs which further inhibits or facilitates the production of seeds as biosocial commons. The fifth chapter of the thesis analyses the ability of stakeholders and finds that apart from institutional rights other factors like the social relations, ideology, negotiations and social identity of a stakeholder determines their ability in accessing the conserved resources. The overall finding of the research suggests that the informal seed networks in the cases analysed stimulated in establishing the biosocial relations between the stakeholders and the resources. The biosocial relation further led seeds to function as biosocial commons. The research thus proposes that strengthening of these biosocial relations through informal seed networks can lead to the commonisation of the PGRs, especially seeds as biosocial commons in the Indian context.

The interest of research groups in desiccation tolerance (DT) has increased substantially over the last decades. The emergence of germinated orthodox seeds and resurrection plants as main research models has pushed the limits of our knowledge beyond boundaries. At the same time, new questions and new challenges were posed. The work presented in this thesis aims at shedding light on some of these questions, deepening our understanding of DT and providing relevant information to improve stress resistance in crops. Chapter 2 is a survey of the literature and discusses the ecological and evolutionary significance for seeds to be able to re-acquire DT after germination. This chapter also discusses recent progress in DT studies using developing and germinated seeds of the model plants Arabidopsis thaliana and Medicago truncatula. In Chapter 3 I used microarray data from a time series of DT re-acquisition, together with network analysis of gene expression, to gain temporal resolution and identify relevant genes involved in the re-acquisition of DT in germinated A. thaliana seeds by incubation in abscisic acid (ABA). Overall, genes related to protection, response to stresses, seed development and seed dormancy were up-regulated, whereas genes related to cell growth and photosynthesis were down-regulated with time. Genes that respond early to exogenous ABA were related to wax biosynthetic processes, lipid storage, seed development and response to ABA stimulus. Genes that respond late to exogenous ABA were related to syncytium formation and response to abiotic stimulus (mainly light stimulus). The robustness of the network was confirmed by the projection of sets of genes – related to the acquisition of DT, seed dormancy, drought responses of adult plants and re-induction of DT by polyethylene glycol – on this network. In Chapter 4 the relation between DT in germinated seeds and drought resistance in adult plants is analysed, using rice (Oryza sativa) as experimental model. Considering the predictions of a future with lower availability of fresh water, efforts to increase rice drought tolerance without reducing yield are increasingly important. The results presented in this chapter suggest that the intrinsic mechanisms of drought tolerance in adult plants are part of the mechanisms used by seeds to tolerate desiccation, but the molecular nature of these mechanisms remains elusive. Chapter 5 explores the relation between DT and longevity in germinated seeds of the Brazilian tree species Sesbania virgata as experimental model. DT and longevity are acquired by orthodox seeds during the maturation phase of development and lost upon germination. DT can be re-induced in germinated seeds by an osmotic and/or ABA treatment, but there is no information on how these treatments affect seed longevity. S. virgata seeds lose DT slowly upon radicle growth. The radicle appeared to be the most sensitive organ and the cotyledons the most resistant. The ability to produce lateral roots was key for whole seedling survival. An osmotic treatment improved DT in germinated S. virgata seeds, but not longevity. This implies that DT and seed longevity can be uncoupled. Xerophyta viscosa is one of the best studied resurrection species. Despite the fact that adult plants and mature seeds display DT, young X. viscosa seedlings are sensitive to fast drying. A treatment with ABA can induce DT early in shoots of these seedlings, but not in roots. Chapter 6 addresses the changes in the transcriptome and proteome of X. viscosa seedlings during induction of DT. A draft genome sequence of X. viscosa was used to improve transcript and protein identification and annotation. Differences in ABA signalling and the cross talk between ABA and ethylene were presented as determinant for shoot and root responses. Moreover, differences in the accumulation of late embryogenesis abundant proteins were also shown as being key for DT in shoots and roots. In Chapter 7, DT-transcriptomes of distantly related organisms are compared and surveyed for a core set of genes representing the signatures of critical adaptive DT mechanisms. A shortlist of 260 genes emerged, with a significant number of genes under the control of ABI3 and related to dormancy. The results reinforced the idea that core mechanisms and key regulators involved in DT developed early in the history of life and were carried forward by diverse species and life forms in a conserved manner and in conjunction with dormancy. In Chapter 8, the findings of this thesis are integrated, showing how they can contribute to future improvement of stress tolerance in crops. The ability of germinated seeds to re-acquire DT is discussed in combination with dormancy and longevity and related to seed survival under unfavourable environmental conditions. The relationship between drought- and desiccation tolerance and the role of ABA are presented briefly. Possible approaches to mine for new genes for crop improvement, such as searching for conserved genes and analysing new genome sequences, are addressed. Finally, a new perspective of the way to consider the evolution of DT is proposed.

The programme on Integrated Seed Sector Development in Ethiopia aims to strengthen the development of a vibrant, market-oriented and pluralistic seed sector in the country, where quality seed of superior varieties is available and affordable for a larger number of farmers, thereby contributing to food security and economic development in Ethiopia. The programme is a joint effort of Bahir Dar University, Haramaya University, Hawassa University, Mekelle University, Oromia Seed Enterprise, the Ethiopian Seed Association and Centre for Development Innovation of Wageningen UR. Partners include governmental organizations at federal, regional and local level, non-governmental organizations, development organizations, and seed businesses operating at different scales. The programme is funded by the Directorate General for International Cooperation through the Embassy of the Kingdom of the Netherlands in Addis Ababa.

The interest of research groups in desiccation tolerance (DT) has increased substantially over the last decades. The emergence of germinated orthodox seeds and resurrection plants as main research models has pushed the limits of our knowledge beyond boundaries. At the same time, new questions and new challenges were posed. The work presented in this thesis aims at shedding light on some of these questions, deepening our understanding of DT and providing relevant information to improve stress resistance in crops. Chapter 2 is a survey of the literature and discusses the ecological and evolutionary significance for seeds to be able to re-acquire DT after germination. This chapter also discusses recent progress in DT studies using developing and germinated seeds of the model plants Arabidopsis thaliana and Medicago truncatula. In Chapter 3 I used microarray data from a time series of DT re-acquisition, together with network analysis of gene expression, to gain temporal resolution and identify relevant genes involved in the re-acquisition of DT in germinated A. thaliana seeds by incubation in abscisic acid (ABA). Overall, genes related to protection, response to stresses, seed development and seed dormancy were up-regulated, whereas genes related to cell growth and photosynthesis were down-regulated with time. Genes that respond early to exogenous ABA were related to wax biosynthetic processes, lipid storage, seed development and response to ABA stimulus. Genes that respond late to exogenous ABA were related to syncytium formation and response to abiotic stimulus (mainly light stimulus). The robustness of the network was confirmed by the projection of sets of genes – related to the acquisition of DT, seed dormancy, drought responses of adult plants and re-induction of DT by polyethylene glycol – on this network. In Chapter 4 the relation between DT in germinated seeds and drought resistance in adult plants is analysed, using rice (Oryza sativa) as experimental model. Considering the predictions of a future with lower availability of fresh water, efforts to increase rice drought tolerance without reducing yield are increasingly important. The results presented in this chapter suggest that the intrinsic mechanisms of drought tolerance in adult plants are part of the mechanisms used by seeds to tolerate desiccation, but the molecular nature of these mechanisms remains elusive. Chapter 5 explores the relation between DT and longevity in germinated seeds of the Brazilian tree species Sesbania virgata as experimental model. DT and longevity are acquired by orthodox seeds during the maturation phase of development and lost upon germination. DT can be re-induced in germinated seeds by an osmotic and/or ABA treatment, but there is no information on how these treatments affect seed longevity. S. virgata seeds lose DT slowly upon radicle growth. The radicle appeared to be the most sensitive organ and the cotyledons the most resistant. The ability to produce lateral roots was key for whole seedling survival. An osmotic treatment improved DT in germinated S. virgata seeds, but not longevity. This implies that DT and seed longevity can be uncoupled. Xerophyta viscosa is one of the best studied resurrection species. Despite the fact that adult plants and mature seeds display DT, young X. viscosa seedlings are sensitive to fast drying. A treatment with ABA can induce DT early in shoots of these seedlings, but not in roots. Chapter 6 addresses the changes in the transcriptome and proteome of X. viscosa seedlings during induction of DT. A draft genome sequence of X. viscosa was used to improve transcript and protein identification and annotation. Differences in ABA signalling and the cross talk between ABA and ethylene were presented as determinant for shoot and root responses. Moreover, differences in the accumulation of late embryogenesis abundant proteins were also shown as being key for DT in shoots and roots. In Chapter 7, DT-transcriptomes of distantly related organisms are compared and surveyed for a core set of genes representing the signatures of critical adaptive DT mechanisms. A shortlist of 260 genes emerged, with a significant number of genes under the control of ABI3 and related to dormancy. The results reinforced the idea that core mechanisms and key regulators involved in DT developed early in the history of life and were carried forward by diverse species and life forms in a conserved manner and in conjunction with dormancy. In Chapter 8, the findings of this thesis are integrated, showing how they can contribute to future improvement of stress tolerance in crops. The ability of germinated seeds to re-acquire DT is discussed in combination with dormancy and longevity and related to seed survival under unfavourable environmental conditions. The relationship between drought- and desiccation tolerance and the role of ABA are presented briefly. Possible approaches to mine for new genes for crop improvement, such as searching for conserved genes and analysing new genome sequences, are addressed. Finally, a new perspective of the way to consider the evolution of DT is proposed.

One of the major bottlenecks limiting farmers’ access to good quality seed for food crops in Uganda is the shortage of early generation seed (EGS - breeder and foundation) to produce sufficient quantities of certified and/or quality declared) to satisfy the needs of farmers. A national study was conducted between October 2015 to March 2016 to analyse pathways for promoting commercial and sustainable production and delivery of EGS. Five crops (hybrid maize, rice, beans, sesame and finger millet) were selected. The analysis provides real examples of potential business models that could scale in a commercially sustainable manner. For areas that are best suited to public sector investment, opportunities for public-private collaboration and increased efficiencies in the sector are outlined. Generalizable principles and recommendations to guide key stakeholders as they pursue policies, investments, and interventions are proposed.

Physical treatments of seeds to eradicate pathogens have the risk of reducing vitality of the seeds. Seed lots may differ in sensitivity to these treatments, therefore factors influencing this sensitivity, including the physiological condition of the seeds, should be identified. In this study priming was used to mimic the effect of pre-harvest onset of germination on the sensitivity to these treatments. The effect of humidification, by incubation for one day at 100% RH, followed by drying back, on the sensitivity was also analysed. Primed, humidified and control seeds were subjected to hot water, aerated steam or electron beam treatments at various levels. Two seed lots from Daucus carota (carrot), two seed lots from Brassica oleracea (kohlrabi) and one seed lot from Allium cepa (onion) were used in the tests. Primed seeds from all seed lots were more sensitive to the aerated steam treatment and the B. oleracea and A. cepa seed lots were also more sensitive to the hot water treatment after priming. Priming had no effect on the sensitivity towards the electron beam treatment. Humidification had no effect on the sensitivity towards the physical seed treatments, with the exception of the humidified onion seeds, which were clearly more tolerant towards the aerated steam treatment. It is concluded that seed companies should be aware that the onset seed germination processes prior to harvest may increase the sensitivity of the seeds towards aerated steam and hot water treatments.

The opportunities for vegetable seed sales in Ethiopia are derived from the size and type of the product market. The product market for vegetables in Ethiopia has been growing rapidly, both in terms of crop portfolio, as well as size.

The opportunities for vegetable seed sales in Ethiopia are derived from the size and type of the product market. The product market for vegetables in Ethiopia has been growing rapidly, both in terms of crop portfolio, as well as size.

One of the most important developmental processes in the life-cycle of higher plants is the transition from a seed to a plant and from a generative to a vegetative developmental program. The major hallmark or end-point of the transition from seed to plant is the onset of photosynthesis and the concomitant shift from a heterotrophic to an autotrophic organism. It is advantageous for a species to keep the period of seedling establishment as short as possible since young seedlings are highly sensitive to biotic and abiotic stresses. This implies that the extreme stress tolerance of seeds to i.e. desiccation is lost upon germination. If the regulatory principles of the seed-to-seedling transition are better understood it may become feasible to maintain the seed’s stress tolerance well into the seedling stage. Despite the profound impact of seedling performance on crop establishment and yield, the seed-to-seedling transition has hardly been studied at the molecular level. This thesis aims at deciphering and understanding the molecular processes that govern this transition in Arabidopsis thaliana. A high-resolution study of the molecular events that occur during these successive transitional stages may provide clues as to the regulatory principles that drive this transition. It may also yield information about the factors that determine the (in)ability to revert to a developmental mode and which features are critical for the maintenance and loss of desiccation tolerance and other stress responses. In Chapter 1 important processes such as abscisic acid and their regulation are described and it is discussed in what way the seed-to-seedling transition may have links to a trait such as desiccation tolerance. An overview is presented of the current knowledge of the seed-to-seedling phase transition and the existence of a temporal developmental block that can be manipulated by osmotic treatment, the carbon/nitrogen balance and by abscisic acid which results in the re-establishment of desiccation tolerance. Chapter 2 focuses on comprehensive gene regulation by a detailed transcriptional analysis across seven developmental stages of the seed-to-seedling transition. It describes the inference of a gene co-expression network and several transcriptional modules. I show that such an approach highlights important molecular processes during seedling development, which would not likely be derived from comparative transcript profiling. Moreover, I show that a putative key regulator in one of the transcriptional modules affects late seedling establishment. In Chapter 3 it is shown how this phase transition is expressed in the primary metabolite profiles in correlation with gene expression. A metabolite-metabolite correlation analysis suggested two profiles, which point at the metabolic preparation of seed germination and of vigorous seedling establishment. Furthermore, a linear correlation between metabolite contents and transcript abundance (Chapter 2) provides a global view of the transcriptional and metabolic changes during the seed-to-seedling transition. It creates new perspectives of the regulatory complexes underlying the seed-to-seedling transition. Chapter 4 describes the development of a novel method to re-establish desiccation tolerance during the seed-to-seedling transition without adverse effects such as those caused by an osmotic treatment with polyethylene glycol. By using this method, named ‘Mild Air Drying Treatment’ (MADT), I show that the re-establishment of desiccation tolerance is not linked to a reduced ability to accumulate ABA in the desiccation sensitive seeds (germinated seeds at root hair stage). I also present a genetic interaction study of ABSCISIC ACID INSENSITIVE (ABI) genes in their germination response to ABA, and their response to the re-establishment of desiccation tolerance using the MADT. The interaction between ABI3 and ABI4, and between ABI4 and ABI5 act synergistically in the re-establishment of DT, as well as in the germination response to ABA. In a more in depth study in Chapter 5 I carried out an extensive transcript analysis to infer possible mechanisms of the re-establishment of desiccation tolerance using the MADT protocol. Possible mechanisms underlying the re-establishment of desiccation tolerance were inferred by employing a time-series comparison of germinated desiccation tolerant and -sensitive seeds. Early-response genes of the re-establishment of desiccation tolerance may play a role in events that promote the initial protection to dehydration stress, whereas the late-response genes may play a role in events that help seed to respond to the changes in water dynamics. Moreover, using a gene co-expression network and transcriptional module I concluded that a crosstalk between ABA-dependent and ABA-independent transcription factors regulate the re-establishment of desiccation tolerance. In Chapter 6 I discuss how the results presented in this thesis contribute to our knowledge of the molecular basis of the seed-to-seedling transition and the re-establishment of desiccation tolerance during its phase changes. Finally, new possibilities for further research are discussed, as well as the further use of the data sets to delineate the mechanisms underlying the seed-to-seedling transition and desiccation tolerance. Possible applications of the results for crop improvement are addressed. Thus, the generation of genetically modified plants that produce seeds with a stress tolerance that extends well into seedling stage may be feasible.

Summary Seed is the basic and most critical input for seed propagated agricultural crops: seed quality and seedling vigour determine plant establishment, growth and development in both natural and agricultural ecosystems. Seed quality and seedling vigour are mainly determined by the interactions of the following three components: genetic background, physiological quality and the environmental conditions during seed set, seed ripening, storage, seed germination and early seedling development. In the past, many efforts have been made to improve seed germination and seedling vigour by optimizing physiological and environmental factors (non-genetic factors); however, the paradigm has shifted to investigate genetic factors and to use these to improve crop performance by plant breeding. The aim of this thesis is to unravel the genetics of seed germination and seedling vigour under different conditions in Brassica rapa, using a systems genetics approach. Studies in many crop species have reported that seed germination and seedling vigour traits are governed by many genes and are strongly affected by environmental conditions. As salinity stress is becoming one of the most important abiotic stresses affecting crop growth and yield, we studied the genetics of seed germination and seedling vigour under neutral and salt stress conditions. For a number of crops, it has been established that larger seed size and higher seed weight indicate more reserve food and contribute positively to seedling establishment. Therefore, our hypothesis for this thesis is that transcriptional regulation of genes during seed development determines the composition and content of seed reserves, and that these seed reserves play a major role in seed germination and seedling growth, especially at the heterotrophic stage under optimal and sub-optimal conditions. B. rapa is an extremely diverse Brassica species which includes, besides many diverse leafy vegetable types and turnips, also oilseed crops. Brassica seeds are of high economic importance for several reasons. They are the starting point of the life cycle of the crop, but also they are directly used as sources of vegetable oil or condiments. At present, B. napus is the most important source of vegetable oil worldwide, but B. rapa is often used for introgression breeding to broaden its narrow genetic base resulting in genetic improvements. Therefore, the acquired knowledge is also useful for the scientific community and plant breeders working in B. napus and other Brassica species. In Chapter 2 we evaluated the genetic diversity of a B. rapa core collection of 168 accessions representing different crop types and geographic origins. Using the Bayesian cluster analysis software STRUCTURE, we identified four subpopulations: subpopulation 1 with accessions of Indian origin, spring oil, yellow sarson and rapid cycling; subpopulation 2 consisting of several types from Asian origins: pak choi, winter oil, mizuna, mibuna, komasuna, turnip green, oil rape and Asian turnip; subpopulation 3, which included mainly accessions of Chinese cabbage and subpopulation 4 with mostly vegetable turnip, fodder turnip and brocoletto accessions from European origin. The geographical distribution of the accessions was very much congruent with genetic, metabolic and morphological diversity. This initial study was followed by association studies for secondary metabolites from the tocopherol and carotenoids pathways, using the population structure of these four subpopulations as a correction term to control for spurious marker-trait associations (Chapter 2). Additionally, we used a machine learning approach, Random Forest (RF) regression, to find marker-trait associations. We chose the RF approach as it can handle large numbers of variables (markers, metabolites, transcript abundance) in combination with relatively small sample sets of accessions, to show its perspectives for application to the increasing amounts of data available through the different ~omics technologies. In our analysis, the markers showing significant association with metabolites identified by the RF approach overlapped with markers obtained from association mapping. Those markers could potentially be used for marker-assisted selection (MAS) in breeding for these secondary metabolites in different morphotypes or sub-populations. Knowledge of genetic distance as evaluated in this chapter allowed the choice of parents to create a segregating population for QTL analyses by maximizing genetic variation between the parents. In Chapter 4, a doubled haploid (DH) population from a cross of genetically diverse morphotypes of B. rapa, an oil-type yellow sarson (YS143) and a vegetable pak choi (PC175) (Chapter 2), was used to evaluate the genetic basis of seed germination and seedling vigour traits under both non-stress and salt stress conditions. The yellow sarson parent had larger seed size and higher thousand-seed weight than the pak choi parent, and displayed earlier onset, higher uniformity in germination, faster germination and maximum germination, and higher root- and shoot- lengths and biomass under both non-stress and salt stress conditions. Positive correlations of thousand-seed weight with earliness, speed and uniformity of germination and maximum germination percentage, supports that larger seeds germinate earlier, faster, more uniformly and to a higher maximum germination percentage than smaller seeds. Thus, we conclude that yellow sarson had higher seed quality and seedling vigour than pak choi. However yellow sarson also contributed negative alleles to seed germination, as illustrated by its allele of the QTL at A05 which decreases the uniformity of seed germination. In addition we also observed that yellow sarson seedling growth was more affected by salt stress than pak choi. All traits were scored over the DH population, and this clearly showed transgressive variation for most traits. Eight QTL hotspots were identified for seed weight, seed germination, and root and shoot lengths. A QTL hotspot for seed germination on A02 co-located with a homologue of the FLOWERING LOCUS C (BrFLC2) genes and its cis-acting expression QTL (cis-eQTL). FLC2 (BrFLC2 in B. rapa) is an important repressor of flowering time in both A. thaliana and B. rapa and recently, FLC2 was reported for its pleiotropic effect on seed germination in A. thaliana. A QTL hotspot on A05 with salt stress specific QTL co-located with the FATTY ACID DESATURASE 2 (BrFAD2) gene and its cis-eQTL. Besides the role of FAD2 in fatty acid desaturation, the up-regulation of this gene was associated with enhanced seed germination and hypocotyl elongation under salinity in B. napus (BnFAD2) and A. thaliana (FAD2). We observed epistatic interactions between the QTL hotspots at the BrFLC2 and BrFAD2 loci, and between other QTL hotspots. Seed development is regulated by many dynamic metabolic processes controlled by complex networks of spatially and temporally expressed genes. Therefore, morphological characteristics and the transcriptional signatures of developing seeds from yellow- and brown/black-seeded genotypes were studied to get to know the timing of key metabolic processes, to explore the major transcriptional differences and to identify the optimum stage for a genetical genomics study for B. rapa seed traits (Chapter 3). This is the first study of genome-wide profiling of transcript abundance during seed development in B. rapa. Most transcriptional changes occurred between 25 and 35 days after pollination (between the bent-cotyledon stage and the stage when the embryo fully fills the seed), which is later than in the related species B. napus. A weighted gene co-expression network analysis (WGCNA) identified 47 gene modules with different co-expression patterns, of which 17 showed a genotype effect, 4 modules a time effect during seed development and 6 modules both genotype and time effects. Based on the number of genes in gene modules, the predominant variation in gene expression was according to developmental stages rather than morphotype differences. We identified 17 putative cis-regulatory elements (motifs) for four co-regulated gene clusters of genes related to lipid metabolism. The identification of key physiological events, major expression patterns, and putative cis-regulatory elements provides useful information to construct gene regulatory networks in B. rapa developing seeds and provides a starting point for a genetical genomics study of fatty acid composition and additional seed traits in Chapter 5. Since Brassica seeds are sources of vegetable oil, genetic studies of the gene regulatory mechanisms underlying lipid metabolism is of high importance, not only in relation to seed and seedling vigour, but also for Brassica oilseed breeding. In Chapter 5, an integrative approach of QTL mapping for fatty acids composition and for transcript abundance (eQTL) of genes related to lipid metabolism, together with gene co-expression networks was used to unravel the genetic regulation of seed fatty acid composition in the DH population of B. rapa. In this study, a confounding effect of flowering time variation was observed on fatty acid QTLs (metabolite level) at linkage group A02 and of seed colour variation on eQTLs (transcript level) at linkage group A09. At A02, fatty acid QTLs from 2009 seeds co-locate with the genetic position of a gene-targeted marker for BrFLC2, its cis-QTL, and a major flowering time QTL. Flowering time variation is very obvious in this DH population and the BrFLC2 gene at A02 (16.7 cM) is the major regulator of flowering time, with a non-functional allele in the yellow sarson parent. When QTL analysis was performed on seeds from 2011, from DH lines that flowered synchronously due to staggered sowing, this fatty acid QTL hotspot disappeared. The 2011 seed lot was used for further analysis combining fatty acid QTLs with eQTLs in this study. On A09, a large trans-eQTL hotspot was co-localized with a major seed colour QTL, in the region where the causal gene, the bHLH transcription factor BrTT8, was cloned. The role of this gene in seed colour development was functionally proven in B. rapa. As the yellow sarson and pak choi parents of this population have contrasting seed coat colour (Chapter 3) the DH lines segregated for seed colour. When seed colour variation was used as a co-variate in our statistical model, we could exclude its confounding effect on eQTL mapping. We compared the fatty acid QTL and eQTL results from the analyses before and after seed colour correction and later discuss the results from the analysis after correction. The distribution of major QTLs for fatty acids showed a relationship with the types of fatty acids: linkage group A03 contained major QTLs for monounsaturated fatty acids (MUFAs), A04 for saturated fatty acids (SFAs) and A05 for polyunsaturated fatty acids (PUFAs). Using a genetical genomics approach, eQTL hotspots were found at major fatty acid QTLs on A03, A04 and A05 and on A09. Finally, an eQTL-guided gene co-expression network of lipid metabolism related genes showed major hubs at the genes BrPLA2-ALPHA, BrWD-40, a number of seed storage protein genes and a transcription factor BrMD-2, suggesting essential roles of these genes in lipid metabolism. Several genes, such as BrFAE1, BrTAG1, BrFAD2, BrFAD5, BrFAD7, which were reported as important genes for fatty acid composition in seeds in other studies of related species, had relatively lower degrees of connection in the networks. However their cis-eQTLs co-localized with specific fatty acid QTLs, making them candidate genes for the observed variation. We hypothesize that these play a role in modifying fatty acid content or composition across genotypes, rather than playing essential roles in the pathway itself. These results suggest the need of a global study of lipid metabolism rather than a strict focus on the fatty acid biosynthesis pathway per se. This study gives a starting point for understanding the genetic regulation of lipid metabolism, by identification of a number of key regulatory genes, identified as major hub genes, and candidate genes for fatty acid QTLs. In the final chapter (Chapter 6) we summarize and critically discuss the relationships among phenotypic traits, metabolites and expression variation as well as the co-localization of QTLs from these different levels. In this thesis, we developed methodology to integrate transcriptomics and metabolomics data sets and to construct gene regulatory networks related to major fatty acids, and found a set of (possible) candidate genes involved in lipid metabolism. In the future, we recommend to integrate the genome-wide transcriptome data set with all major seed metabolites and phenotypic data on seed and seedling vigour to directly link all three components: transcriptome, metabolome and phenotypic traits, and ultimately expand the knowledge on the genetic regulation of seed metabolites, seed quality and seedling vigour in B. rapa to other Brassica species.

Biochemical, physiological and molecular responses of Ricinus communis seeds and seedlings to different temperatures: a multi-omics approach by Paulo Roberto Ribeiro de Jesus The main objective of this thesis was to provide a detailed analysis of physiological, biochemical and molecular-genetic responses of Ricinus communis to temperature during seed germination and seedling establishment. In Chapter 2, I describe the assessment of 17 candidate reference genes across a diverse set of samples, including several tissues, various developmental stages and environmental conditions, encompassing seed germination and seedling growth in R. communis. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. Both GeNorm and Normfinder indicated that ACT, POB and PP2AA1 represent the optimal combination for normalization of gene expression data in inter-tissue studies. I also describe the optimal combination of reference genes for a subset of samples from root, endosperm and cotyledonary tissues. The selection of reference genes was validated by normalizing the expression levels of three target genes involved in energy metabolism with the identified optimal reference genes. This approach allowed me to identify stably expressed genes, and, thus, reference genes for use in RT-qPCR studies in seeds and seedlings of R. communis. In Chapter 3, a thermo-sensitive window is identified during seed germination in which high temperatures compromise subsequent seedling development. I assessed the biochemical and molecular requirements of R. communis germination for successful seedling establishment at varying temperatures. For that, I performed metabolite profiling (GC-TOF-MS) and measured transcript levels of key genes involved in several energy-generating pathways such as storage oil mobilization, β-oxidation of fatty acids and gluconeogenesis of seeds germinated at three different temperatures. Transient overexpression of genes encoding for malate synthase (MLS) and glycerol kinase (GK) resulted in higher starch levels in N. benthamiana leaves, which highlights the likely importance of these genes in energy-generating pathways for seedling establishment. Additionally, I showed that γ-aminobutyric acid (GABA), which is a stress-responsive metabolite, accumulated in response to the water content of the seeds during the initial phase of imbibition. In Chapter 4 I undertook a genomics approach using microarray analysis to determine transcriptome changes in three distinct developmental stages during seed germination at 20, 25 and 35ºC that could explain the thermo-sensitive window that is described in Chapter 3. Most of the differences in the R. communis transcriptome occurred between 6 hours of imbibition and the commencement of germination, i.e. radicle protrusion. This coincides with the thermo-sensitive window identified during seed germination in which high temperatures compromise seedling development. The transcriptome data was used to identify heat-stress responsive genes that might be involved in thermotolerance of R. communis during germination. Temperature had a major effect on genes involved in energy generating pathways, such as the Calvin-Benson-Bassham cycle, gluconeogenesis, and starch- and triacylglycerol degradation. Transcripts coding for ATP binding proteins, DNA binding proteins, RNA binding proteins, DNA-directed RNA polymerases I, II, and III, heat shock factor proteins, multiprotein-bridging factor proteins, and zinc finger proteins were also affected by temperature suggesting the whole transcriptional regulatory machinery was altered in response to temperature. Among the downregulated transcripts under high temperature, only three were shared by all three stages: an oxidation-related zinc finger 2, an F-box and wd40 domain protein, and a DNA binding protein/MYB-like transcription factor. Among the upregulated transcripts, nine were shared by all three stages: a BET1P/SFT1P-like protein, 14BB, a low-molecular-weight cysteine-rich protein LCR78, a WD-repeat protein, a GAST1 protein, an adenylate kinase 1/P-loop containing nucleoside triphosphate hydrolases superfamily protein, and four conserved hypothetical proteins. These genes constitute good candidates for further characterization of temperature-responsive genes in R. communis. In Chapter 5, I studied the genetic variation in the effect of temperature on growth of young R. communis seedlings and measured primary and secondary metabolites in roots and cotyledons of three R. communis genotypes, varying in stress tolerance. Seedling biomass was strongly affected by the temperature, with the lowest total biomass observed at 20ºC. The response in terms of biomass production for the genotype MPA11 was clearly different from the other two studied genotypes: genotype MPA11 produced heavier seedlings at all temperatures but the root biomass of this genotype decreased with increasing temperature, reaching the lowest value at 35ºC. In contrast, root biomass of genotypes MPB01 and IAC80 was not affected by temperature, suggesting that the roots of these genotypes are less sensitive to changes in temperature. A shift in carbon-nitrogen metabolism towards the accumulation of nitrogen-containing compounds seems to be the main biochemical response to support growth at higher temperatures. Carbohydrate content was reduced in response to increasing temperature in both roots and cotyledons, whereas amino acids accumulated to higher levels. The results in this chapter show that a specific balance between amino acids, carbohydrates and organic acids in the cotyledons and roots of genotype MPA11 seems to be an important trait for faster and more efficient growth of this genotype at higher temperatures. In Chapter 6, I decided to focus on the differential ability of genotypes MPA11 and IAC80 to sustain root biomass production at higher temperatures. Biomass allocation was assessed by measuring dry weight of roots, stems, and cotyledons of seedlings grown at three different temperatures. Seedlings grown at 25ºC and 35ºC showed greater biomass than seedlings grown at 20ºC. Cotyledon and stem dry weight increased for both genotypes with increasing temperature, whereas root biomass allocation showed a genotype-dependent behaviour. Genotype MPA11 showed a continuous increase in root dry weight with increasing temperature, while genotype IAC80 was not able to sustain further root growth at higher temperatures. Metabolite and gene expression profiles of genotype MPA11 demonstrated an increase in the levels of osmoprotectant molecules, such as galactinol and transcripts of genes encoding antioxidant enzymes and heat shock proteins, to a higher extent than in genotype IAC80. In Chapter 7 I raised the question whether carbohydrate accumulation in R. communis leaves, roots, and seeds, grown at low temperatures, as compared to higher temperatures, results from up-regulation of biosynthetic pathways, from down-regulation of catabolic pathways, or both. To answer this question, transcript levels were measured of genes encoding enzymes involved in starch biosynthesis, starch catabolism, and gluconeogenesis in leaves, roots, and seeds grown at 20ºC and 35ºC. Transcript levels of genes involved in starch catabolism were higher in leaves grown at 20ºC than at 35ºC, but up-regulation of genes involved in starch biosynthesis seems to compensate for this and, therefore, is the likely explanation for higher levels of starch in leaves grown at 20ºC. Higher levels of soluble carbohydrates in leaves grown at 20ºC may have been caused by a coordinated up-regulation of starch catabolism and gluconeogenesis pathways. In roots, starch catabolism and gluconeogenesis seem to be enhanced at elevated temperatures. Higher levels of starch in seeds germinated at low temperatures is associated with higher transcript levels of genes involved in starch biosynthesis. Similarly, higher transcript levels of RcPEPCK and RcFBPase are most likely causal for fructose and glucose accumulation in seeds germinated at 20ºC. This thesis provides important insights in the understanding of the plasticity of R. communis in response to temperature. The knowledge obtained may apply to other species as well. Additionally, based on the transcriptomics data, we selected several candidate genes that are potentially involved in, or required for, proper seed germination and seedling establishment under different temperatures, such as a number of transcription factors, a zinc finger protein, heat-shock proteins, malate synthase and glycerol kinase. Overexpressing Arabidopsis lines transformed with these R. communis genes, as well as Arabidopsis T-DNA lines, in which Arabidopsis homologs of these genes are knocked-out, are being generated for further phenotypical analysis. These overexpression and T-DNA lines should help us to understand the molecular requirements for vigorous seedling growth of R. communis under different environmental conditions. This work was performed at the Laboratory of Plant Physiology, Wageningen University. This research was financially supported by the Brazilian Government through the National Counsel of Technological and Scientific Development (CNPq grant number 200745/2011-5).

In this thesis I explore the ontological proposal of food sovereignty and I discuss the possibilities offered by studies like this one to the attempts of the social sciences to explain – in a symmetrical fashion - that develop between humans and other entities at the time of production, processing and consumption of food. In this effort I combine ethnography and history. I argue that in countries like Ecuador, food networks such as that of the lupine, Lupino mutabilis Sweet, since they do not establish ontological differences between nature and culture, promote the implementation of food sovereignty in practice, as long as agricultural and science and technology (S&T) policies enable the autonomous development of such networks. More specifically, food networks in the Andean highlands have functioned in a rhizomatic way, without establishing hierarchies between entities of different ontology: foods as goods or foods as gifts, society and nature, and have spread without discontinuities between town and country. This analysis enables me to show that these networks can promote food sovereignty, because in them is condensed an ontology distinct from that of modernity with regard to the cultivation, processing and consumption of food. Considering these findings I analyse the rationality of S&T policies and the current policies of the Ecuadorian State. I argue that such policies go against the logic of food networks. Food sovereignty is an achievable goal if Ecuadorian government policies contribute to the strengthening of food networks, creating new links so that they can sidestep the agribusiness model. The organisation of this thesis is unusual, as the object of study is a food network. This forced me to research and structure this dissertation in a particular way. So, I start with the ethnographic explanation of a food network. Here I analyse its operation, relationships and the paths it establishes. From this analysis it is possible to understand why S&T policies, specifically those related to plant breeding, created new social relations that affected the food networks of the highlands. I show here how the modern rationality on which agricultural policies are based inhibits the growth of food networks and works against food sovereignty. Then, from the analysis of the formation of the food sovereignty network, I examine the introduction of the food sovereignty proposal into the Ecuadorian Constitution and the changes made to the original proposal. I show how the translation of the Via Campesina proposal present in the constitution and the subsequent law is possible due to the intervention of actors linked with the big businesses of the food trade. All this enables me, finally, to discuss my contribution: the analysis of the ontological promise present in the food sovereignty proposal.

In this thesis I explore the ontological proposal of food sovereignty and I discuss the possibilities offered by studies like this one to the attempts of the social sciences to explain – in a symmetrical fashion - that develop between humans and other entities at the time of production, processing and consumption of food. In this effort I combine ethnography and history. I argue that in countries like Ecuador, food networks such as that of the lupine, Lupino mutabilis Sweet, since they do not establish ontological differences between nature and culture, promote the implementation of food sovereignty in practice, as long as agricultural and science and technology (S&T) policies enable the autonomous development of such networks. More specifically, food networks in the Andean highlands have functioned in a rhizomatic way, without establishing hierarchies between entities of different ontology: foods as goods or foods as gifts, society and nature, and have spread without discontinuities between town and country. This analysis enables me to show that these networks can promote food sovereignty, because in them is condensed an ontology distinct from that of modernity with regard to the cultivation, processing and consumption of food. Considering these findings I analyse the rationality of S&T policies and the current policies of the Ecuadorian State. I argue that such policies go against the logic of food networks. Food sovereignty is an achievable goal if Ecuadorian government policies contribute to the strengthening of food networks, creating new links so that they can sidestep the agribusiness model. The organisation of this thesis is unusual, as the object of study is a food network. This forced me to research and structure this dissertation in a particular way. So, I start with the ethnographic explanation of a food network. Here I analyse its operation, relationships and the paths it establishes. From this analysis it is possible to understand why S&T policies, specifically those related to plant breeding, created new social relations that affected the food networks of the highlands. I show here how the modern rationality on which agricultural policies are based inhibits the growth of food networks and works against food sovereignty. Then, from the analysis of the formation of the food sovereignty network, I examine the introduction of the food sovereignty proposal into the Ecuadorian Constitution and the changes made to the original proposal. I show how the translation of the Via Campesina proposal present in the constitution and the subsequent law is possible due to the intervention of actors linked with the big businesses of the food trade. All this enables me, finally, to discuss my contribution: the analysis of the ontological promise present in the food sovereignty proposal.

Summary Seed is the basic and most critical input for seed propagated agricultural crops: seed quality and seedling vigour determine plant establishment, growth and development in both natural and agricultural ecosystems. Seed quality and seedling vigour are mainly determined by the interactions of the following three components: genetic background, physiological quality and the environmental conditions during seed set, seed ripening, storage, seed germination and early seedling development. In the past, many efforts have been made to improve seed germination and seedling vigour by optimizing physiological and environmental factors (non-genetic factors); however, the paradigm has shifted to investigate genetic factors and to use these to improve crop performance by plant breeding. The aim of this thesis is to unravel the genetics of seed germination and seedling vigour under different conditions in Brassica rapa, using a systems genetics approach. Studies in many crop species have reported that seed germination and seedling vigour traits are governed by many genes and are strongly affected by environmental conditions. As salinity stress is becoming one of the most important abiotic stresses affecting crop growth and yield, we studied the genetics of seed germination and seedling vigour under neutral and salt stress conditions. For a number of crops, it has been established that larger seed size and higher seed weight indicate more reserve food and contribute positively to seedling establishment. Therefore, our hypothesis for this thesis is that transcriptional regulation of genes during seed development determines the composition and content of seed reserves, and that these seed reserves play a major role in seed germination and seedling growth, especially at the heterotrophic stage under optimal and sub-optimal conditions. B. rapa is an extremely diverse Brassica species which includes, besides many diverse leafy vegetable types and turnips, also oilseed crops. Brassica seeds are of high economic importance for several reasons. They are the starting point of the life cycle of the crop, but also they are directly used as sources of vegetable oil or condiments. At present, B. napus is the most important source of vegetable oil worldwide, but B. rapa is often used for introgression breeding to broaden its narrow genetic base resulting in genetic improvements. Therefore, the acquired knowledge is also useful for the scientific community and plant breeders working in B. napus and other Brassica species. In Chapter 2 we evaluated the genetic diversity of a B. rapa core collection of 168 accessions representing different crop types and geographic origins. Using the Bayesian cluster analysis software STRUCTURE, we identified four subpopulations: subpopulation 1 with accessions of Indian origin, spring oil, yellow sarson and rapid cycling; subpopulation 2 consisting of several types from Asian origins: pak choi, winter oil, mizuna, mibuna, komasuna, turnip green, oil rape and Asian turnip; subpopulation 3, which included mainly accessions of Chinese cabbage and subpopulation 4 with mostly vegetable turnip, fodder turnip and brocoletto accessions from European origin. The geographical distribution of the accessions was very much congruent with genetic, metabolic and morphological diversity. This initial study was followed by association studies for secondary metabolites from the tocopherol and carotenoids pathways, using the population structure of these four subpopulations as a correction term to control for spurious marker-trait associations (Chapter 2). Additionally, we used a machine learning approach, Random Forest (RF) regression, to find marker-trait associations. We chose the RF approach as it can handle large numbers of variables (markers, metabolites, transcript abundance) in combination with relatively small sample sets of accessions, to show its perspectives for application to the increasing amounts of data available through the different ~omics technologies. In our analysis, the markers showing significant association with metabolites identified by the RF approach overlapped with markers obtained from association mapping. Those markers could potentially be used for marker-assisted selection (MAS) in breeding for these secondary metabolites in different morphotypes or sub-populations. Knowledge of genetic distance as evaluated in this chapter allowed the choice of parents to create a segregating population for QTL analyses by maximizing genetic variation between the parents. In Chapter 4, a doubled haploid (DH) population from a cross of genetically diverse morphotypes of B. rapa, an oil-type yellow sarson (YS143) and a vegetable pak choi (PC175) (Chapter 2), was used to evaluate the genetic basis of seed germination and seedling vigour traits under both non-stress and salt stress conditions. The yellow sarson parent had larger seed size and higher thousand-seed weight than the pak choi parent, and displayed earlier onset, higher uniformity in germination, faster germination and maximum germination, and higher root- and shoot- lengths and biomass under both non-stress and salt stress conditions. Positive correlations of thousand-seed weight with earliness, speed and uniformity of germination and maximum germination percentage, supports that larger seeds germinate earlier, faster, more uniformly and to a higher maximum germination percentage than smaller seeds. Thus, we conclude that yellow sarson had higher seed quality and seedling vigour than pak choi. However yellow sarson also contributed negative alleles to seed germination, as illustrated by its allele of the QTL at A05 which decreases the uniformity of seed germination. In addition we also observed that yellow sarson seedling growth was more affected by salt stress than pak choi. All traits were scored over the DH population, and this clearly showed transgressive variation for most traits. Eight QTL hotspots were identified for seed weight, seed germination, and root and shoot lengths. A QTL hotspot for seed germination on A02 co-located with a homologue of the FLOWERING LOCUS C (BrFLC2) genes and its cis-acting expression QTL (cis-eQTL). FLC2 (BrFLC2 in B. rapa) is an important repressor of flowering time in both A. thaliana and B. rapa and recently, FLC2 was reported for its pleiotropic effect on seed germination in A. thaliana. A QTL hotspot on A05 with salt stress specific QTL co-located with the FATTY ACID DESATURASE 2 (BrFAD2) gene and its cis-eQTL. Besides the role of FAD2 in fatty acid desaturation, the up-regulation of this gene was associated with enhanced seed germination and hypocotyl elongation under salinity in B. napus (BnFAD2) and A. thaliana (FAD2). We observed epistatic interactions between the QTL hotspots at the BrFLC2 and BrFAD2 loci, and between other QTL hotspots. Seed development is regulated by many dynamic metabolic processes controlled by complex networks of spatially and temporally expressed genes. Therefore, morphological characteristics and the transcriptional signatures of developing seeds from yellow- and brown/black-seeded genotypes were studied to get to know the timing of key metabolic processes, to explore the major transcriptional differences and to identify the optimum stage for a genetical genomics study for B. rapa seed traits (Chapter 3). This is the first study of genome-wide profiling of transcript abundance during seed development in B. rapa. Most transcriptional changes occurred between 25 and 35 days after pollination (between the bent-cotyledon stage and the stage when the embryo fully fills the seed), which is later than in the related species B. napus. A weighted gene co-expression network analysis (WGCNA) identified 47 gene modules with different co-expression patterns, of which 17 showed a genotype effect, 4 modules a time effect during seed development and 6 modules both genotype and time effects. Based on the number of genes in gene modules, the predominant variation in gene expression was according to developmental stages rather than morphotype differences. We identified 17 putative cis-regulatory elements (motifs) for four co-regulated gene clusters of genes related to lipid metabolism. The identification of key physiological events, major expression patterns, and putative cis-regulatory elements provides useful information to construct gene regulatory networks in B. rapa developing seeds and provides a starting point for a genetical genomics study of fatty acid composition and additional seed traits in Chapter 5. Since Brassica seeds are sources of vegetable oil, genetic studies of the gene regulatory mechanisms underlying lipid metabolism is of high importance, not only in relation to seed and seedling vigour, but also for Brassica oilseed breeding. In Chapter 5, an integrative approach of QTL mapping for fatty acids composition and for transcript abundance (eQTL) of genes related to lipid metabolism, together with gene co-expression networks was used to unravel the genetic regulation of seed fatty acid composition in the DH population of B. rapa. In this study, a confounding effect of flowering time variation was observed on fatty acid QTLs (metabolite level) at linkage group A02 and of seed colour variation on eQTLs (transcript level) at linkage group A09. At A02, fatty acid QTLs from 2009 seeds co-locate with the genetic position of a gene-targeted marker for BrFLC2, its cis-QTL, and a major flowering time QTL. Flowering time variation is very obvious in this DH population and the BrFLC2 gene at A02 (16.7 cM) is the major regulator of flowering time, with a non-functional allele in the yellow sarson parent. When QTL analysis was performed on seeds from 2011, from DH lines that flowered synchronously due to staggered sowing, this fatty acid QTL hotspot disappeared. The 2011 seed lot was used for further analysis combining fatty acid QTLs with eQTLs in this study. On A09, a large trans-eQTL hotspot was co-localized with a major seed colour QTL, in the region where the causal gene, the bHLH transcription factor BrTT8, was cloned. The role of this gene in seed colour development was functionally proven in B. rapa. As the yellow sarson and pak choi parents of this population have contrasting seed coat colour (Chapter 3) the DH lines segregated for seed colour. When seed colour variation was used as a co-variate in our statistical model, we could exclude its confounding effect on eQTL mapping. We compared the fatty acid QTL and eQTL results from the analyses before and after seed colour correction and later discuss the results from the analysis after correction. The distribution of major QTLs for fatty acids showed a relationship with the types of fatty acids: linkage group A03 contained major QTLs for monounsaturated fatty acids (MUFAs), A04 for saturated fatty acids (SFAs) and A05 for polyunsaturated fatty acids (PUFAs). Using a genetical genomics approach, eQTL hotspots were found at major fatty acid QTLs on A03, A04 and A05 and on A09. Finally, an eQTL-guided gene co-expression network of lipid metabolism related genes showed major hubs at the genes BrPLA2-ALPHA, BrWD-40, a number of seed storage protein genes and a transcription factor BrMD-2, suggesting essential roles of these genes in lipid metabolism. Several genes, such as BrFAE1, BrTAG1, BrFAD2, BrFAD5, BrFAD7, which were reported as important genes for fatty acid composition in seeds in other studies of related species, had relatively lower degrees of connection in the networks. However their cis-eQTLs co-localized with specific fatty acid QTLs, making them candidate genes for the observed variation. We hypothesize that these play a role in modifying fatty acid content or composition across genotypes, rather than playing essential roles in the pathway itself. These results suggest the need of a global study of lipid metabolism rather than a strict focus on the fatty acid biosynthesis pathway per se. This study gives a starting point for understanding the genetic regulation of lipid metabolism, by identification of a number of key regulatory genes, identified as major hub genes, and candidate genes for fatty acid QTLs. In the final chapter (Chapter 6) we summarize and critically discuss the relationships among phenotypic traits, metabolites and expression variation as well as the co-localization of QTLs from these different levels. In this thesis, we developed methodology to integrate transcriptomics and metabolomics data sets and to construct gene regulatory networks related to major fatty acids, and found a set of (possible) candidate genes involved in lipid metabolism. In the future, we recommend to integrate the genome-wide transcriptome data set with all major seed metabolites and phenotypic data on seed and seedling vigour to directly link all three components: transcriptome, metabolome and phenotypic traits, and ultimately expand the knowledge on the genetic regulation of seed metabolites, seed quality and seedling vigour in B. rapa to other Brassica species.

Keywords: Technography, Oryza glaberrima, Oryza sativa, farmer hybrids, sub-optimal agriculture, farmer adaptive management, plant genetic resources, peace and extreme (wartime) conditions, local seed channels, selection for robustness, Sierra Leone, West Africa. Abstract Mokuwa, G. A. (2015) Management of rice seed during insurgency: a case study in Sierra Leone. PhD Thesis, Wageningen University, 267 pp. In large parts of West Africa small scale farmers rely upon the cultivation of upland rice under low input conditions in a great diversity of micro-environments. It has been suggested that formal research should consider the context within which farmers address their food security issues. But these contexts need further clarification for poor and marginalized farm households facing many challenges, including dislocations associated with political and social unrest, and civil war. The research presented in this thesis builds on earlier findings concerning farmer management of rice genetic resources under farmer low-resource conditions. It starts with a regional focus, drawing on methods from the social and biological sciences, concerning the human, environmental and technical factors shaping the character and composition of rice varieties grown by small-scale farmers in coastal West Africa (seven countries from Senegal to Togo) and then focuses on specific in-depth field studies undertaken in Sierra Leone. Findings show that farmer rice genetic resources were persistently and enduringly adapted to local agro-ecologies via strong selection processes and local adaptation strategies, and that these adaptive processes were largely unaffected by the temporary contingencies of civil war. It is also shown that even under extreme (war-time) conditions success indicators in farmers’ local seed channels remain robust. Farmers continue to select and adapt their seed types to local contingencies, and war served as yet one more stimulus to further adaptation. This persistent human selective activity continues to make a significant contribution to the food security of poor and marginalized farm households in the region. The major finding of this thesis is that selection for robustness among varieties of the local staple, rice, helped to protect Sierra Leonean farmers against some of the worst effects of war-induced food insecurity. In this sense, therefore, war may have served to strengthen and prolong farmer preferences for robustness, but it was not the cause of this preference. The marked diversity farmers maintain in their rice varieties is understood to be part of a longer-term risk-spreading strategy that also facilitates successful and often serendipitous variety innovations. In a world facing major climatic changes this local capacity for seed selection and innovation ought to be a valued resource for technological change. The present study provides a starting point for thinking about the improved effectiveness of institutional innovation strategies for farmer participatory innovation activities.

One of the most important developmental processes in the life-cycle of higher plants is the transition from a seed to a plant and from a generative to a vegetative developmental program. The major hallmark or end-point of the transition from seed to plant is the onset of photosynthesis and the concomitant shift from a heterotrophic to an autotrophic organism. It is advantageous for a species to keep the period of seedling establishment as short as possible since young seedlings are highly sensitive to biotic and abiotic stresses. This implies that the extreme stress tolerance of seeds to i.e. desiccation is lost upon germination. If the regulatory principles of the seed-to-seedling transition are better understood it may become feasible to maintain the seed’s stress tolerance well into the seedling stage. Despite the profound impact of seedling performance on crop establishment and yield, the seed-to-seedling transition has hardly been studied at the molecular level. This thesis aims at deciphering and understanding the molecular processes that govern this transition in Arabidopsis thaliana. A high-resolution study of the molecular events that occur during these successive transitional stages may provide clues as to the regulatory principles that drive this transition. It may also yield information about the factors that determine the (in)ability to revert to a developmental mode and which features are critical for the maintenance and loss of desiccation tolerance and other stress responses. In Chapter 1 important processes such as abscisic acid and their regulation are described and it is discussed in what way the seed-to-seedling transition may have links to a trait such as desiccation tolerance. An overview is presented of the current knowledge of the seed-to-seedling phase transition and the existence of a temporal developmental block that can be manipulated by osmotic treatment, the carbon/nitrogen balance and by abscisic acid which results in the re-establishment of desiccation tolerance. Chapter 2 focuses on comprehensive gene regulation by a detailed transcriptional analysis across seven developmental stages of the seed-to-seedling transition. It describes the inference of a gene co-expression network and several transcriptional modules. I show that such an approach highlights important molecular processes during seedling development, which would not likely be derived from comparative transcript profiling. Moreover, I show that a putative key regulator in one of the transcriptional modules affects late seedling establishment. In Chapter 3 it is shown how this phase transition is expressed in the primary metabolite profiles in correlation with gene expression. A metabolite-metabolite correlation analysis suggested two profiles, which point at the metabolic preparation of seed germination and of vigorous seedling establishment. Furthermore, a linear correlation between metabolite contents and transcript abundance (Chapter 2) provides a global view of the transcriptional and metabolic changes during the seed-to-seedling transition. It creates new perspectives of the regulatory complexes underlying the seed-to-seedling transition. Chapter 4 describes the development of a novel method to re-establish desiccation tolerance during the seed-to-seedling transition without adverse effects such as those caused by an osmotic treatment with polyethylene glycol. By using this method, named ‘Mild Air Drying Treatment’ (MADT), I show that the re-establishment of desiccation tolerance is not linked to a reduced ability to accumulate ABA in the desiccation sensitive seeds (germinated seeds at root hair stage). I also present a genetic interaction study of ABSCISIC ACID INSENSITIVE (ABI) genes in their germination response to ABA, and their response to the re-establishment of desiccation tolerance using the MADT. The interaction between ABI3 and ABI4, and between ABI4 and ABI5 act synergistically in the re-establishment of DT, as well as in the germination response to ABA. In a more in depth study in Chapter 5 I carried out an extensive transcript analysis to infer possible mechanisms of the re-establishment of desiccation tolerance using the MADT protocol. Possible mechanisms underlying the re-establishment of desiccation tolerance were inferred by employing a time-series comparison of germinated desiccation tolerant and -sensitive seeds. Early-response genes of the re-establishment of desiccation tolerance may play a role in events that promote the initial protection to dehydration stress, whereas the late-response genes may play a role in events that help seed to respond to the changes in water dynamics. Moreover, using a gene co-expression network and transcriptional module I concluded that a crosstalk between ABA-dependent and ABA-independent transcription factors regulate the re-establishment of desiccation tolerance. In Chapter 6 I discuss how the results presented in this thesis contribute to our knowledge of the molecular basis of the seed-to-seedling transition and the re-establishment of desiccation tolerance during its phase changes. Finally, new possibilities for further research are discussed, as well as the further use of the data sets to delineate the mechanisms underlying the seed-to-seedling transition and desiccation tolerance. Possible applications of the results for crop improvement are addressed. Thus, the generation of genetically modified plants that produce seeds with a stress tolerance that extends well into seedling stage may be feasible.

The study presents an integrated assessment of Myanmar’s seed sector. The study includes information and analyses on regulatory environment for seed production and sales, a characterization of Myanmar’s seed sector with its various seed systems, a landscape of current seed sector interventions; an analysis of three seed value-chains and Myanmar’s seed farm system; as well as business opportunities for the private sector. The report concludes with a number of pathways for developing a vibrant seed sector in which quality seed of superior varieties can be accessed by farmers.

The study presents an integrated assessment of Myanmar’s seed sector. The study includes information and analyses on regulatory environment for seed production and sales, a characterization of Myanmar’s seed sector with its various seed systems, a landscape of current seed sector interventions; an analysis of three seed value-chains and Myanmar’s seed farm system; as well as business opportunities for the private sector. The report concludes with a number of pathways for developing a vibrant seed sector in which quality seed of superior varieties can be accessed by farmers.

The recommendations focus on the need to increase the availability of and access to quality seeds; improve quality, varietal sustainability and resilience of the seed system; build capacities of key actors to improve on their production and business skills; and address issues of enabling environment in seed security.

Biochemical, physiological and molecular responses of Ricinus communis seeds and seedlings to different temperatures: a multi-omics approach by Paulo Roberto Ribeiro de Jesus The main objective of this thesis was to provide a detailed analysis of physiological, biochemical and molecular-genetic responses of Ricinus communis to temperature during seed germination and seedling establishment. In Chapter 2, I describe the assessment of 17 candidate reference genes across a diverse set of samples, including several tissues, various developmental stages and environmental conditions, encompassing seed germination and seedling growth in R. communis. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. Both GeNorm and Normfinder indicated that ACT, POB and PP2AA1 represent the optimal combination for normalization of gene expression data in inter-tissue studies. I also describe the optimal combination of reference genes for a subset of samples from root, endosperm and cotyledonary tissues. The selection of reference genes was validated by normalizing the expression levels of three target genes involved in energy metabolism with the identified optimal reference genes. This approach allowed me to identify stably expressed genes, and, thus, reference genes for use in RT-qPCR studies in seeds and seedlings of R. communis. In Chapter 3, a thermo-sensitive window is identified during seed germination in which high temperatures compromise subsequent seedling development. I assessed the biochemical and molecular requirements of R. communis germination for successful seedling establishment at varying temperatures. For that, I performed metabolite profiling (GC-TOF-MS) and measured transcript levels of key genes involved in several energy-generating pathways such as storage oil mobilization, β-oxidation of fatty acids and gluconeogenesis of seeds germinated at three different temperatures. Transient overexpression of genes encoding for malate synthase (MLS) and glycerol kinase (GK) resulted in higher starch levels in N. benthamiana leaves, which highlights the likely importance of these genes in energy-generating pathways for seedling establishment. Additionally, I showed that γ-aminobutyric acid (GABA), which is a stress-responsive metabolite, accumulated in response to the water content of the seeds during the initial phase of imbibition. In Chapter 4 I undertook a genomics approach using microarray analysis to determine transcriptome changes in three distinct developmental stages during seed germination at 20, 25 and 35ºC that could explain the thermo-sensitive window that is described in Chapter 3. Most of the differences in the R. communis transcriptome occurred between 6 hours of imbibition and the commencement of germination, i.e. radicle protrusion. This coincides with the thermo-sensitive window identified during seed germination in which high temperatures compromise seedling development. The transcriptome data was used to identify heat-stress responsive genes that might be involved in thermotolerance of R. communis during germination. Temperature had a major effect on genes involved in energy generating pathways, such as the Calvin-Benson-Bassham cycle, gluconeogenesis, and starch- and triacylglycerol degradation. Transcripts coding for ATP binding proteins, DNA binding proteins, RNA binding proteins, DNA-directed RNA polymerases I, II, and III, heat shock factor proteins, multiprotein-bridging factor proteins, and zinc finger proteins were also affected by temperature suggesting the whole transcriptional regulatory machinery was altered in response to temperature. Among the downregulated transcripts under high temperature, only three were shared by all three stages: an oxidation-related zinc finger 2, an F-box and wd40 domain protein, and a DNA binding protein/MYB-like transcription factor. Among the upregulated transcripts, nine were shared by all three stages: a BET1P/SFT1P-like protein, 14BB, a low-molecular-weight cysteine-rich protein LCR78, a WD-repeat protein, a GAST1 protein, an adenylate kinase 1/P-loop containing nucleoside triphosphate hydrolases superfamily protein, and four conserved hypothetical proteins. These genes constitute good candidates for further characterization of temperature-responsive genes in R. communis. In Chapter 5, I studied the genetic variation in the effect of temperature on growth of young R. communis seedlings and measured primary and secondary metabolites in roots and cotyledons of three R. communis genotypes, varying in stress tolerance. Seedling biomass was strongly affected by the temperature, with the lowest total biomass observed at 20ºC. The response in terms of biomass production for the genotype MPA11 was clearly different from the other two studied genotypes: genotype MPA11 produced heavier seedlings at all temperatures but the root biomass of this genotype decreased with increasing temperature, reaching the lowest value at 35ºC. In contrast, root biomass of genotypes MPB01 and IAC80 was not affected by temperature, suggesting that the roots of these genotypes are less sensitive to changes in temperature. A shift in carbon-nitrogen metabolism towards the accumulation of nitrogen-containing compounds seems to be the main biochemical response to support growth at higher temperatures. Carbohydrate content was reduced in response to increasing temperature in both roots and cotyledons, whereas amino acids accumulated to higher levels. The results in this chapter show that a specific balance between amino acids, carbohydrates and organic acids in the cotyledons and roots of genotype MPA11 seems to be an important trait for faster and more efficient growth of this genotype at higher temperatures. In Chapter 6, I decided to focus on the differential ability of genotypes MPA11 and IAC80 to sustain root biomass production at higher temperatures. Biomass allocation was assessed by measuring dry weight of roots, stems, and cotyledons of seedlings grown at three different temperatures. Seedlings grown at 25ºC and 35ºC showed greater biomass than seedlings grown at 20ºC. Cotyledon and stem dry weight increased for both genotypes with increasing temperature, whereas root biomass allocation showed a genotype-dependent behaviour. Genotype MPA11 showed a continuous increase in root dry weight with increasing temperature, while genotype IAC80 was not able to sustain further root growth at higher temperatures. Metabolite and gene expression profiles of genotype MPA11 demonstrated an increase in the levels of osmoprotectant molecules, such as galactinol and transcripts of genes encoding antioxidant enzymes and heat shock proteins, to a higher extent than in genotype IAC80. In Chapter 7 I raised the question whether carbohydrate accumulation in R. communis leaves, roots, and seeds, grown at low temperatures, as compared to higher temperatures, results from up-regulation of biosynthetic pathways, from down-regulation of catabolic pathways, or both. To answer this question, transcript levels were measured of genes encoding enzymes involved in starch biosynthesis, starch catabolism, and gluconeogenesis in leaves, roots, and seeds grown at 20ºC and 35ºC. Transcript levels of genes involved in starch catabolism were higher in leaves grown at 20ºC than at 35ºC, but up-regulation of genes involved in starch biosynthesis seems to compensate for this and, therefore, is the likely explanation for higher levels of starch in leaves grown at 20ºC. Higher levels of soluble carbohydrates in leaves grown at 20ºC may have been caused by a coordinated up-regulation of starch catabolism and gluconeogenesis pathways. In roots, starch catabolism and gluconeogenesis seem to be enhanced at elevated temperatures. Higher levels of starch in seeds germinated at low temperatures is associated with higher transcript levels of genes involved in starch biosynthesis. Similarly, higher transcript levels of RcPEPCK and RcFBPase are most likely causal for fructose and glucose accumulation in seeds germinated at 20ºC. This thesis provides important insights in the understanding of the plasticity of R. communis in response to temperature. The knowledge obtained may apply to other species as well. Additionally, based on the transcriptomics data, we selected several candidate genes that are potentially involved in, or required for, proper seed germination and seedling establishment under different temperatures, such as a number of transcription factors, a zinc finger protein, heat-shock proteins, malate synthase and glycerol kinase. Overexpressing Arabidopsis lines transformed with these R. communis genes, as well as Arabidopsis T-DNA lines, in which Arabidopsis homologs of these genes are knocked-out, are being generated for further phenotypical analysis. These overexpression and T-DNA lines should help us to understand the molecular requirements for vigorous seedling growth of R. communis under different environmental conditions. This work was performed at the Laboratory of Plant Physiology, Wageningen University. This research was financially supported by the Brazilian Government through the National Counsel of Technological and Scientific Development (CNPq grant number 200745/2011-5).

This paper is intended as a basis for nature restoration activities using seeds of trees and (larger) shrubs native to Bonaire with the aim of reforestation. It describes the main seed biology issues relevant for species from this region, to facilitate decisions on time and stage of harvesting, safe storage, breaking dormancy and germinating seeds and planting the young trees in the field. The paper also emphasises that natural process of seedling establishment and succession must be observed in order to be successful. The choice of species and method of protection once planted in nature will prove essential. The paper ends with stressing that such a reforestation activity needs to be planned far ahead: seeds must be collected from tree species taking care of genetic diversity and their storage potential. Recalcitrant seeds (see paper) must be treated carefully and in a different way.

How different organisms survive in the absence or under very limited amounts of water is still an open question. The aim of the research presented in this thesis is to explore the molecular basis of desiccation tolerance in seeds. We investigated the possibilities of using germinated desiccation sensitive seeds which were rescued to become tolerant again as a system to study the mechanisms involved in surviving severe dehydration. Using this experimental approach in combination with technologies to assess which genes, proteins, metabolites and metabolic pathways are important in dry seeds, we obtained insights on the mechanism of survival in the dry state. The understanding of such mechanisms can aid to the design of crop improvement strategies, long-term storage of seeds, and dry storage of other biological materials such as blood cell, tissues and vaccines.

This paper is intended as a basis for nature restoration activities using seeds of trees and (larger) shrubs native to Bonaire with the aim of reforestation. It describes the main seed biology issues relevant for species from this region, to facilitate decisions on time and stage of harvesting, safe storage, breaking dormancy and germinating seeds and planting the young trees in the field. The paper also emphasises that natural process of seedling establishment and succession must be observed in order to be successful. The choice of species and method of protection once planted in nature will prove essential. The paper ends with stressing that such a reforestation activity needs to be planned far ahead: seeds must be collected from tree species taking care of genetic diversity and their storage potential. Recalcitrant seeds (see paper) must be treated carefully and in a different way.

How different organisms survive in the absence or under very limited amounts of water is still an open question. The aim of the research presented in this thesis is to explore the molecular basis of desiccation tolerance in seeds. We investigated the possibilities of using germinated desiccation sensitive seeds which were rescued to become tolerant again as a system to study the mechanisms involved in surviving severe dehydration. Using this experimental approach in combination with technologies to assess which genes, proteins, metabolites and metabolic pathways are important in dry seeds, we obtained insights on the mechanism of survival in the dry state. The understanding of such mechanisms can aid to the design of crop improvement strategies, long-term storage of seeds, and dry storage of other biological materials such as blood cell, tissues and vaccines.

The seed stage is an essential episode in the life cycle of higher plants. The environmental cues that seeds experience during their development are important components of their life history. The parental environment, from pre-fertilization until seed dispersal affects performance of the dry mature seed and, therefore, affects the life cycle of the next generation. The evolutionary response to environmental perturbations has resulted in genetic changes in order to increase the fitness of the population, which is called ‘adaptation’. The aims of this study were to increase our understanding of how environments regulate seed performance, both on the long term, i.e. through adaptation of seed performance traits to local conditions and on the short term, i.e. by acclimation of plants to different seed maturation environments.

The seed stage is an essential episode in the life cycle of higher plants. The environmental cues that seeds experience during their development are important components of their life history. The parental environment, from pre-fertilization until seed dispersal affects performance of the dry mature seed and, therefore, affects the life cycle of the next generation. The evolutionary response to environmental perturbations has resulted in genetic changes in order to increase the fitness of the population, which is called ‘adaptation’. The aims of this study were to increase our understanding of how environments regulate seed performance, both on the long term, i.e. through adaptation of seed performance traits to local conditions and on the short term, i.e. by acclimation of plants to different seed maturation environments.

The Netherlands has a long history of plant breeding which has resulted in a leading position in the world with respect to the sales of vegetable seeds. Nowadays high-tech methods are used for crop-production which demands high standards for the quality of the starting materials. While breeding has mainly focused on crop yield and disease resistance in the past, it now becomes equally important to create seeds that rapidly and uniformly germinate under a wide range of production environments. A better understanding of the molecular processes that are underlying seed quality is a crucial first step to enable targeted breeding. In this thesis we describe the results of new methods that were used to map the genetics of seed germination. For this research we have used the leading plant science model species Arabidopsis thaliana which has a short generation time and a fully sequenced genome. Further, the large scientific community working on this model species is providing a wealth of resources ranging from large collections of worldwide accessions, genetic mapping populations, mutants and knowledge about gene, protein and metabolite action. A disadvantage of using Arabidopsis is the small size of the seeds, which requires evaluation of the germination of individual seeds with the use of magnifying glasses. This problem has been solved by using image analysis to create an automated procedure to obtain detailed information for parameters such as rate, uniformity and maximum germination. This procedure, called ‘the Germinator’, is described in Chapter 2 and has been enthusiastically adopted by the seed community. Plants cannot walk away from the environment at which the seed is dispersed. To survive and to enable reproduction, plants adapt to the prevailing environment which results in considerable genetic variation. This ‘natural variation’ is a great resource to study the mechanisms of adaptation. In Chapter 3 we have used two distinct Arabidopsis accessions, one originating from Germany (Bayreuth) and the other from high altitude in the Pamiro-Alay Mountains in Tadjikistan (Shahdara). In contrast to the Bayreuth accession, the Shahdara accession is well adapted to survive harsh conditions and is known to be stress tolerant to a range of environments. A genetic mapping (recombinant inbred line; RIL) population, consisting of 165 lines, that was derived from these two accessions is therefore particularly suitable to locate the genomic regions with genetic differences that influence seed germination. Such genomic regions are commonly referred to as quantitative trait loci (QTL). With help of the Germinator system we were able to evaluate germination of this RIL population under many different conditions. This resulted in a description of the ‘genetic landscape of seed performance’ in which we identified many QTLs for Arabidopsis seed germination. QTL regions are often large and identification of the causal gene requires intensive follow up research. We therefore aimed for a high throughput analysis using modern ‘omics’ techniques to analyze differences in metabolite levels and gene expression between the lines. A method to classify and visualize the vast amount of data derived from such an approach is described in Chapter 4. The so called genetical ‘omics’ experiments are expensive and therefore often force researchers to limit their study to a single developmental stage or environment only. A novel generalized setup overcomes this limitation and was tested for metabolite level changes in Chapter 5. This setup offers a unique reduction of experimental load with minimal effect on statistical power and is of great potential in the field of system genetics. Four different developmental stages of seed germination were tested in the RIL population. This approach resulted in a large dataset for which efficient analytical procedures were lacking. Thus, Chapter 5 also includes a description of a newly developed statistical procedure to analyze this type of data. The same approach and material were used in Chapter 6 to evaluate the genetics of genome wide gene expression. Another approach to zoom in on the molecular mechanisms underlying seed performance is described in Chapter 7. Here, the genetic diversity was maximized by using 360 different Arabidopsis accessions which had been subjected to ultra-high density genotyping. In potential, such a genome wide association (GWA) study can provide high resolution mapping of genetic variation resulting in only a few candidate genes per association for the phenotype under study. Although we were able to replicate experiments over two years with a high level of heritability, no significant associations were found. This emphasizes the need to critically review the power of such an approach for traits that are expected to be determined by many small effect loci. Finally, closing in on the molecular mechanisms underlying the seed traits that we studied might be possible by a full integration of the datasets that were described in the different chapters. Two examples that show the potential and the complexity of such integration are described in the General Discussion (Chapter 8). Research focused on seed quality does not end here but has gained an impulse by the described new methods and hypotheses to continue on both the fundamental and applied level in the coming years.

Key words: Bénin, genetic quality, growth dynamics, innovation, institutions, multistakeholders process, oil palm, perennials, physiological quality, pot size, seed quality, seed systems, smallholders, social learning. The seed supply system used by smallholder farmers is characterised by many dysfunctions. For perennials, including oil palm, there is a knowledge gap regarding these dysfunctions. In this thesis, we used oil palm as a case to analyse the performance of seed systems. We conducted social surveys, sampled farmers’ oil palm plots, ran field experiments and documented the social learning process of stakeholders involved. To identify major constraints in the seed system, we conducted a diagnostic study using farmers’ perspective. Jointly with stakeholders the following major constraints were identified (in order of importance): poor geographic distribution of official nurseries, poor genetic quality of palms in smallholder plots, high costs of hybrid seedlings, and poor seedling care in nurseries leading to poor physiological quality. The poor care was specifically mentioned in the eastern part of the study area. We investigated the reliability of genetic quality of seedlings supplied to smallholder farmers as one of the constraints that emerged from the diagnostic study. Main drivers of reliability in genetic quality over the past decades were analysed. Using event ecology approach, we document the historical events that may have affected the oil palm seed system. Proportions of hybrid palms varied with seedling supply source, farmers’ geographic position, seedling purchase price and year of planting. Socio-institutional mechanisms associated with observed variation in smallholder plantations were national policy change, local arrangements for seedling supply to smallholder farmers, and farmers’ personal characteristics. Local arrangements improved genetic quality in villages located far away from official supply sources. Villages where local seedling supply initiatives withdrew showed reduced genetic quality with farmers having fewer tenera in their fields than before. Membership of farmers’ organisation correlated positively with proportion of tenera. Farmer’s use of informal intermediaries showed negative effects on genetic quality they received. To evaluate the efficacy of on-going nursery management practices on the seedling phenotype at planting we conducted joint learning experiments. In two full 3 × 4 × 3 factorial experiments, bag size (small, medium, large), type of soil substrate ("forest" soil, household waste substrate, arable soil, and arable soil with animal manure) and fertiliser supply (no fertilisation, split dose every 15 days, and full dose every 30 days) were tested for their effects on seedling phenotype. Bag size proved the main factor determining oil palm seedling phenotype in both the 2011 and 2012 experiment. Although large-sized bags produced largest seedlings, medium-sized bags filled with a mixture of arable soil and animal manure without any fertiliser supply sustained seedling growth well and seemed the best balance between physiological quality and production cost. Growth variables were highly correlated. Height and root-collar diameter constituted good proxies to estimate seedling biomass differences between objects in a non-destructive way. To gain insight into temporal patterns of effects of bag size, substrate, fertiliser supply, and their interactions on seedling growth, we analysed the dynamics of oil palm seedling growth using monthly observations. Overall, in both experiments, bag size explained the largest proportion of experimental error and started to deviate earlier than substrate, fertiliser supply or their two and three way interactions. Curve fitting showed different growth models for height, root-collar diameter and number of leaves. The analysis of growth rates showed that (relative and absolute) growth rates were mainly affected by bag size in both years with larger F-values than for substrate, fertiliser supply, and interactions. Experimental findings indicated that pot size matters and cannot be compensated by fertiliser addition and should therefore be considered carefully for tree seedling production in nurseries. We analysed the joint experiment as a multi-stakeholder process and contributed to understand how the way of organising social learning affects stakeholders’ ownership of process outcomes. Stakeholders’ perceptions of seedling quality and their appreciation of treatment performance varied with the use they make of planting material. While farmers, as end-users, put forward seedling vigour when describing quality seedlings, nursery holders underlined production costs and reported that seedling quality is a compromise with production costs. Field observations further to the joint experiment indicated changes in practices among nursery holders, research, and farmers. The level of stakeholders’ involvement increased their participation, ownership of the learning process, and could lead to sustainable practices. The research approach developed in this thesis to analyse seed systems performance contributed to the methodology for seed systems analysis. It also contributed to knowledge of dysfunctions of seed systems for perennials, tree nursery seedling production and social learning processes.

The seed business plays a crucial role at the basis of the food supply chain, and companies which are active in plant breeding and production and sales of seeds are embedded in a competitive environment. They continuously face challenges to develop higher yielding varieties with better or new qualities, optimized for sustainable production under a wide variety of growing conditions. To meet those challenges innovation through R&D is extremely important. For this study, I chose the vegetable breeding industry as the subject of this research and focused on two similar business sectors in two different parts of the world, China and the Netherlands.

The seed business plays a crucial role at the basis of the food supply chain, and companies which are active in plant breeding and production and sales of seeds are embedded in a competitive environment. They continuously face challenges to develop higher yielding varieties with better or new qualities, optimized for sustainable production under a wide variety of growing conditions. To meet those challenges innovation through R&D is extremely important. For this study, I chose the vegetable breeding industry as the subject of this research and focused on two similar business sectors in two different parts of the world, China and the Netherlands. Dit onderzoek werd geïnitieerd door de cruciale rol die de internationale zaadindustrie speelt aan de basis van de voedselproductieketen, waardoor de economische effecten van innovatie groot kunnen zijn. Bedrijven die actief zijn in de veredeling van gewassen en in de productie en verkoop van zaden voeren hun activiteiten uit in een zeer competitieve omgeving. Ze hebben te maken met voortdurend veranderende uitdagingingen zoals het leveren van een bijdrage aan de voedselzekerheid, de noodzaak om nieuwe rassen te ontwikkelen die een hoge opbrengst en hoge kwaliteit producten leveren en die gebruikt kunnen worden voor een duurzame productie. Om die uitdagingen aan te kunnen gaan is innovatie in R&D van groot belang. Voor dit onderzoek hebben we gekozen voor innovatie in de groenteveredeling in twee identieke sectoren, maar in twee verschillende delen van de wereld, China en Nederland.

Key words: Bénin, genetic quality, growth dynamics, innovation, institutions, multistakeholders process, oil palm, perennials, physiological quality, pot size, seed quality, seed systems, smallholders, social learning. The seed supply system used by smallholder farmers is characterised by many dysfunctions. For perennials, including oil palm, there is a knowledge gap regarding these dysfunctions. In this thesis, we used oil palm as a case to analyse the performance of seed systems. We conducted social surveys, sampled farmers’ oil palm plots, ran field experiments and documented the social learning process of stakeholders involved. To identify major constraints in the seed system, we conducted a diagnostic study using farmers’ perspective. Jointly with stakeholders the following major constraints were identified (in order of importance): poor geographic distribution of official nurseries, poor genetic quality of palms in smallholder plots, high costs of hybrid seedlings, and poor seedling care in nurseries leading to poor physiological quality. The poor care was specifically mentioned in the eastern part of the study area. We investigated the reliability of genetic quality of seedlings supplied to smallholder farmers as one of the constraints that emerged from the diagnostic study. Main drivers of reliability in genetic quality over the past decades were analysed. Using event ecology approach, we document the historical events that may have affected the oil palm seed system. Proportions of hybrid palms varied with seedling supply source, farmers’ geographic position, seedling purchase price and year of planting. Socio-institutional mechanisms associated with observed variation in smallholder plantations were national policy change, local arrangements for seedling supply to smallholder farmers, and farmers’ personal characteristics. Local arrangements improved genetic quality in villages located far away from official supply sources. Villages where local seedling supply initiatives withdrew showed reduced genetic quality with farmers having fewer tenera in their fields than before. Membership of farmers’ organisation correlated positively with proportion of tenera. Farmer’s use of informal intermediaries showed negative effects on genetic quality they received. To evaluate the efficacy of on-going nursery management practices on the seedling phenotype at planting we conducted joint learning experiments. In two full 3 × 4 × 3 factorial experiments, bag size (small, medium, large), type of soil substrate ("forest" soil, household waste substrate, arable soil, and arable soil with animal manure) and fertiliser supply (no fertilisation, split dose every 15 days, and full dose every 30 days) were tested for their effects on seedling phenotype. Bag size proved the main factor determining oil palm seedling phenotype in both the 2011 and 2012 experiment. Although large-sized bags produced largest seedlings, medium-sized bags filled with a mixture of arable soil and animal manure without any fertiliser supply sustained seedling growth well and seemed the best balance between physiological quality and production cost. Growth variables were highly correlated. Height and root-collar diameter constituted good proxies to estimate seedling biomass differences between objects in a non-destructive way. To gain insight into temporal patterns of effects of bag size, substrate, fertiliser supply, and their interactions on seedling growth, we analysed the dynamics of oil palm seedling growth using monthly observations. Overall, in both experiments, bag size explained the largest proportion of experimental error and started to deviate earlier than substrate, fertiliser supply or their two and three way interactions. Curve fitting showed different growth models for height, root-collar diameter and number of leaves. The analysis of growth rates showed that (relative and absolute) growth rates were mainly affected by bag size in both years with larger F-values than for substrate, fertiliser supply, and interactions. Experimental findings indicated that pot size matters and cannot be compensated by fertiliser addition and should therefore be considered carefully for tree seedling production in nurseries. We analysed the joint experiment as a multi-stakeholder process and contributed to understand how the way of organising social learning affects stakeholders’ ownership of process outcomes. Stakeholders’ perceptions of seedling quality and their appreciation of treatment performance varied with the use they make of planting material. While farmers, as end-users, put forward seedling vigour when describing quality seedlings, nursery holders underlined production costs and reported that seedling quality is a compromise with production costs. Field observations further to the joint experiment indicated changes in practices among nursery holders, research, and farmers. The level of stakeholders’ involvement increased their participation, ownership of the learning process, and could lead to sustainable practices. The research approach developed in this thesis to analyse seed systems performance contributed to the methodology for seed systems analysis. It also contributed to knowledge of dysfunctions of seed systems for perennials, tree nursery seedling production and social learning processes.

The Netherlands has a long history of plant breeding which has resulted in a leading position in the world with respect to the sales of vegetable seeds. Nowadays high-tech methods are used for crop-production which demands high standards for the quality of the starting materials. While breeding has mainly focused on crop yield and disease resistance in the past, it now becomes equally important to create seeds that rapidly and uniformly germinate under a wide range of production environments. A better understanding of the molecular processes that are underlying seed quality is a crucial first step to enable targeted breeding. In this thesis we describe the results of new methods that were used to map the genetics of seed germination. For this research we have used the leading plant science model species Arabidopsis thaliana which has a short generation time and a fully sequenced genome. Further, the large scientific community working on this model species is providing a wealth of resources ranging from large collections of worldwide accessions, genetic mapping populations, mutants and knowledge about gene, protein and metabolite action. A disadvantage of using Arabidopsis is the small size of the seeds, which requires evaluation of the germination of individual seeds with the use of magnifying glasses. This problem has been solved by using image analysis to create an automated procedure to obtain detailed information for parameters such as rate, uniformity and maximum germination. This procedure, called ‘the Germinator’, is described in Chapter 2 and has been enthusiastically adopted by the seed community. Plants cannot walk away from the environment at which the seed is dispersed. To survive and to enable reproduction, plants adapt to the prevailing environment which results in considerable genetic variation. This ‘natural variation’ is a great resource to study the mechanisms of adaptation. In Chapter 3 we have used two distinct Arabidopsis accessions, one originating from Germany (Bayreuth) and the other from high altitude in the Pamiro-Alay Mountains in Tadjikistan (Shahdara). In contrast to the Bayreuth accession, the Shahdara accession is well adapted to survive harsh conditions and is known to be stress tolerant to a range of environments. A genetic mapping (recombinant inbred line; RIL) population, consisting of 165 lines, that was derived from these two accessions is therefore particularly suitable to locate the genomic regions with genetic differences that influence seed germination. Such genomic regions are commonly referred to as quantitative trait loci (QTL). With help of the Germinator system we were able to evaluate germination of this RIL population under many different conditions. This resulted in a description of the ‘genetic landscape of seed performance’ in which we identified many QTLs for Arabidopsis seed germination. QTL regions are often large and identification of the causal gene requires intensive follow up research. We therefore aimed for a high throughput analysis using modern ‘omics’ techniques to analyze differences in metabolite levels and gene expression between the lines. A method to classify and visualize the vast amount of data derived from such an approach is described in Chapter 4. The so called genetical ‘omics’ experiments are expensive and therefore often force researchers to limit their study to a single developmental stage or environment only. A novel generalized setup overcomes this limitation and was tested for metabolite level changes in Chapter 5. This setup offers a unique reduction of experimental load with minimal effect on statistical power and is of great potential in the field of system genetics. Four different developmental stages of seed germination were tested in the RIL population. This approach resulted in a large dataset for which efficient analytical procedures were lacking. Thus, Chapter 5 also includes a description of a newly developed statistical procedure to analyze this type of data. The same approach and material were used in Chapter 6 to evaluate the genetics of genome wide gene expression. Another approach to zoom in on the molecular mechanisms underlying seed performance is described in Chapter 7. Here, the genetic diversity was maximized by using 360 different Arabidopsis accessions which had been subjected to ultra-high density genotyping. In potential, such a genome wide association (GWA) study can provide high resolution mapping of genetic variation resulting in only a few candidate genes per association for the phenotype under study. Although we were able to replicate experiments over two years with a high level of heritability, no significant associations were found. This emphasizes the need to critically review the power of such an approach for traits that are expected to be determined by many small effect loci. Finally, closing in on the molecular mechanisms underlying the seed traits that we studied might be possible by a full integration of the datasets that were described in the different chapters. Two examples that show the potential and the complexity of such integration are described in the General Discussion (Chapter 8). Research focused on seed quality does not end here but has gained an impulse by the described new methods and hypotheses to continue on both the fundamental and applied level in the coming years.

The life cycle of most plants starts, and ends, at the seed stage. In most species mature seeds are shed and dispersed on the ground. At this stage of its life cycle the seed may be dormant and will, by definition, not germinate under favourable conditions (Bewley, 1997). Seasonal dormancy cycling is a characteristic found in plant seeds. Being able to cycle in and out of dormancy allows the seed to survive decades or even centuries, allowing germination to be spread over time, but only when optimal conditions are available, not only for germination but especially for seedling establishment. In this thesis we have attempted to further elucidate the mechanisms behind dormancy, germination and dormancy cycling. Sisymbrium officinale seeds need nitrate and light to start germination (Chapter 2, 3, 4, 6). Nitrate acts in part by reducing the abscisic acid (ABA) levels (a plant hormone that elevates dormancy levels). The action of light and nitrate can also be reached by applying gibberellins (GAs) to the seeds (Chapter 2, 3, 4, 6). GAs are capable of inducing enzymes that hydrolyze the ensdosperm walls (Debeaujon and Koornneef, 2000; Chen and Bradford, 2000; Nonogaki et al., 2000; Manz et al., 2005) In this way GAs could be involved in lowering the physical restrictions imposed by the resistance of the seed coat and the endosperm. On the other hand, GAs may also increase the embryo growth potential. For successful survival of the dormant seed, metabolic activity is reduced to avoid rapid depletion of reserves. The metabolic activity of the seed was measured using electron paramagnetic resonance (EPR), with TEMPONE as a spin probe, and the respiratory activity was measured with the Q2-test (Chapter 2).We showed that primary dormancy was accompanied by hardly any metabolic or respiratory activity, and this increased considerably when dormancy was broken by nitrate. However, when the light pulse was not given and the seeds had become secondary dormant the metabolic activity slowed down. Regulation of dormancy is tightly linked with abiotic stress factors from the environment. The regulation and survival of the seed under stress conditions is largely dependent on the composition of the cytoplasm. We tested this by EPR, using carboxyl-proxyl (CP) spin probe (Chapter 4). The primary dormant and sub-dormant seeds possessed a higher viscosity than the germinating seeds. The viscosity of secondary dormant seeds appeared intermediate; however, the ease at which the vitrified water melted was similar to that of primary dormant seeds. As a result of the differences in viscosity, the temperature of vitrified water melting differed between the different dormancy states. The changes in cytoplasmic viscosity and vitrified water melting may be linked to changes in metabolism and the content of high molecular weight compounds. As membranes are the primary target for temperature perception, they are often implicated in regulating dormancy. Therefore, Hilhorst (1998) put forward a hypothesis in which changes in responsiveness to dormancy breaking factors like nitrate and light was a function of cellular membrane fluidity. In Chapter 3 we indeed showed that dormancy is a function of membrane fluidity. Primary dormant seeds of Sisymbrium officinale appeared to have very rigid membranes, whereas breaking dormancy increased membrane fluidity considerably. However, when sub-dormant seeds became secondary dormant membrane fluidity decreased again, but not to the rigidity seen in primary dormant seeds. One of the most common ways in which cells control membrane fluidity is by homeoviscous adaptation with the help of desaturases. Desaturase involvement in changes in membrane fluidity due to changes in dormancy was tested in Chapter 3 (using Sisymbrium officinale) and Chapter 5 (using Arabidopsis thaliana). Here we found that although desaturase activity may change the membrane fluidity or influence the germination/dormancy phenotype, the two are not linked, unless the effects of these enzymes are very local within the seed. Finally, in Chapter 7, we presented a new model in which a membrane anchored dormancy related protein/transcription factor is activated by changes in membrane fluidity. The activated form is transported to the nucleus, where it starts the germination process, which includes changes in metabolism and mobilization of storage reserves.

The life cycle of most plants starts, and ends, at the seed stage. In most species mature seeds are shed and dispersed on the ground. At this stage of its life cycle the seed may be dormant and will, by definition, not germinate under favourable conditions (Bewley, 1997). Seasonal dormancy cycling is a characteristic found in plant seeds. Being able to cycle in and out of dormancy allows the seed to survive decades or even centuries, allowing germination to be spread over time, but only when optimal conditions are available, not only for germination but especially for seedling establishment. In this thesis we have attempted to further elucidate the mechanisms behind dormancy, germination and dormancy cycling. Sisymbrium officinale seeds need nitrate and light to start germination (Chapter 2, 3, 4, 6). Nitrate acts in part by reducing the abscisic acid (ABA) levels (a plant hormone that elevates dormancy levels). The action of light and nitrate can also be reached by applying gibberellins (GAs) to the seeds (Chapter 2, 3, 4, 6). GAs are capable of inducing enzymes that hydrolyze the ensdosperm walls (Debeaujon and Koornneef, 2000; Chen and Bradford, 2000; Nonogaki et al., 2000; Manz et al., 2005) In this way GAs could be involved in lowering the physical restrictions imposed by the resistance of the seed coat and the endosperm. On the other hand, GAs may also increase the embryo growth potential. For successful survival of the dormant seed, metabolic activity is reduced to avoid rapid depletion of reserves. The metabolic activity of the seed was measured using electron paramagnetic resonance (EPR), with TEMPONE as a spin probe, and the respiratory activity was measured with the Q2-test (Chapter 2).We showed that primary dormancy was accompanied by hardly any metabolic or respiratory activity, and this increased considerably when dormancy was broken by nitrate. However, when the light pulse was not given and the seeds had become secondary dormant the metabolic activity slowed down. Regulation of dormancy is tightly linked with abiotic stress factors from the environment. The regulation and survival of the seed under stress conditions is largely dependent on the composition of the cytoplasm. We tested this by EPR, using carboxyl-proxyl (CP) spin probe (Chapter 4). The primary dormant and sub-dormant seeds possessed a higher viscosity than the germinating seeds. The viscosity of secondary dormant seeds appeared intermediate; however, the ease at which the vitrified water melted was similar to that of primary dormant seeds. As a result of the differences in viscosity, the temperature of vitrified water melting differed between the different dormancy states. The changes in cytoplasmic viscosity and vitrified water melting may be linked to changes in metabolism and the content of high molecular weight compounds. As membranes are the primary target for temperature perception, they are often implicated in regulating dormancy. Therefore, Hilhorst (1998) put forward a hypothesis in which changes in responsiveness to dormancy breaking factors like nitrate and light was a function of cellular membrane fluidity. In Chapter 3 we indeed showed that dormancy is a function of membrane fluidity. Primary dormant seeds of Sisymbrium officinale appeared to have very rigid membranes, whereas breaking dormancy increased membrane fluidity considerably. However, when sub-dormant seeds became secondary dormant membrane fluidity decreased again, but not to the rigidity seen in primary dormant seeds. One of the most common ways in which cells control membrane fluidity is by homeoviscous adaptation with the help of desaturases. Desaturase involvement in changes in membrane fluidity due to changes in dormancy was tested in Chapter 3 (using Sisymbrium officinale) and Chapter 5 (using Arabidopsis thaliana). Here we found that although desaturase activity may change the membrane fluidity or influence the germination/dormancy phenotype, the two are not linked, unless the effects of these enzymes are very local within the seed. Finally, in Chapter 7, we presented a new model in which a membrane anchored dormancy related protein/transcription factor is activated by changes in membrane fluidity. The activated form is transported to the nucleus, where it starts the germination process, which includes changes in metabolism and mobilization of storage reserves.

This thesis focuses on the ethnobotany, phylogeny and taxonomy of the genus Gloriosa L. over its distributional range. Some Gloriosa species are known to have economic and commercial value, but the genus is also well known for its complex alpha taxonomy. An appropriate taxonomy for this group is of great importance because it includes widely used species as traditional medicine, horticultural plants and sources of industrial and pharmaceutical chemical colchicine. The seeds and tubers of G. superba are valued as a commercial source of colchicine. The genus Gloriosa has considerable horticultural appeal because of the conspicuous inflorescence of its members and the ease with which taxa are propagated, introduced into new areas and hybridise in cultivation. G. carsonii, G. modesta, G. simplex and G. superba have been taken into cultivation as ornamental plants in several countries, including native countries of these species.

This thesis focuses on the ethnobotany, phylogeny and taxonomy of the genus Gloriosa L. over its distributional range. Some Gloriosa species are known to have economic and commercial value, but the genus is also well known for its complex alpha taxonomy. An appropriate taxonomy for this group is of great importance because it includes widely used species as traditional medicine, horticultural plants and sources of industrial and pharmaceutical chemical colchicine. The seeds and tubers of G. superba are valued as a commercial source of colchicine. The genus Gloriosa has considerable horticultural appeal because of the conspicuous inflorescence of its members and the ease with which taxa are propagated, introduced into new areas and hybridise in cultivation. G. carsonii, G. modesta, G. simplex and G. superba have been taken into cultivation as ornamental plants in several countries, including native countries of these species.

In mei 2012 is een verkenning uitgevoerd van de voorziening van groentezaden in Suriname. Zaden voor de groenteteelt in Suriname kunnen lokaal worden geproduceerd of de zaden worden door bedrijven of particulieren geïmporteerd. De eigen zaadproductie is kleinschalig en voor eigen gebruik. Het betreft vooral groentegewassen die van belang zijn voor de export van groenten. Contacten met buitenlandse zaadbedrijven voor de import van groentezaad door agrarische handelsbedrijven komen veelal tot stand via internet. Telers en andere particulieren brengen van buitenlandse reizen zaden mee voor de teelt in Suriname. Er bestaat geen systematische import en toetsing van alle potentieel voor Suriname geschikte groenterassen. Er worden aanbevelingen gedaan voor het professionaliseren van de Surinaamse zaadgoedsector.

Rangelands in Africa provide important forage resources for herbivores; particularly perennial grasses provide grazing for domestic and wild herbivores. However, semi-arid African rangelands experience severe vegetation and soil degradation due to heavy grazing, causing negative impacts on the ecosystems, livestock production and livelihoods of the people. Semi-arid African rangelands can be described by a state-and-transition models, often with three stable states, the first one being a state with ample herbaceous cover, perennial grasses and scattered trees, the second one as a state with a poor cover of annual grasses, absence of perennial grasses, and the third state with a high proportion of bare soil and/or often bush encroached. This thesis aims to fill important information gaps concerning soil seed banks that play in the recovery and possible restoration of degraded semi-arid African rangelands with particular emphasis on Ethiopia. This was done through investigating the mechanisms of how heavy grazing affects the soil seeds bank dynamics so as to understand stable states and transition processes of aboveground vegetation. In this thesis, aboveground vegetation and soil seed bank dynamics were studied under heavy and light grazing pressures in a field and experimental conditions. Results show that heavy grazing resulted in the disappearance of perennial grasses, a reduction in herbaceous species diversity and their plant abundance, standing biomass and basal cover, as well as a decrease in the soil nutrient conditions. The soil seed banks was correlated to differences in grazing pressure, with a greater seedling density under light grazing compared with heavy grazing. Immediately after seed dispersal, the seedling density increased over the first first three months until the eight months of soil sampling, and decreased thereafter. Under light grazing, perennial grass species dominated, whereas annual species were abundant at the heavily grazed sites, indicating that perennial grasses, with good fodder value, are replaced by annual species in the soil seed banks due to heavy grazing. With increasing soil depth, the seedling density and its species richness declined. Moreover, the seeds of perennial grasses were less abundant in the soil seed banks under heavy grazing. The similarity in species composition between the soil seed banks and aboveground vegetation was low under heavy grazing. Results also show that annual grasses had a lower germination and mortality, and higher viability, leading to a longevity of 62% over the 120 days burial in the soil, which was high compared to the 28% for perennials. Moreover, most perennial grasses germinate rapidly after initial seed dispersal at the first rains early in the year, whereas annual grasses show a linear germination pattern over time, indicating that perennial grasses have different survival strategy in semi-arid Africa. As a result, annual species are expected to dominate the soil seed banks, whereas most perennial grass species do not form persistent soil seed banks.The mean mortality from the seedling stage to adult plants in grass species was 65%, and the seed–to–seedling stage was found the most critical transitional stage for grass survival on these rangelands, suggesting that exclusion from grazing and trampling in the early germination stage is important to facilitate the transition from seedling to established plants. Depletion of perennial grass seeds in the soil due to heavy grazing coupled with high seedling mortality leads to a strong decrease in perennial grasses both in the soil seed banks, as well as in the aboveground vegetation. I found that the positive relationship between plant cover and differences in soil seed bank dynamics, i.e., seed density, seed germination rate and longevity, trigger the transition from perennial grasses to annuals and from annual plant cover to bare soil under heavy grazing. I hypothesize that the restoration of perennial grasses from the soil seed banks in heavily grazed areas in semi-arid African rangelands cannot be successful without an extraneous source of perennial grass seeds and without protecting the young plant’s regrowth from trampling and grazing. Therefore, the persistence of species and maintenance of biodiversity in semi-arid rangelands depends mainly on the recruitment of seedlings from annual species, and on vegetative reproduction of perennial grasses and woody species. These findings have important implications for the management, conservation and restoration of semi-arid African rangelands. Keywords:Africa; Aboveground vegetation; Ethiopia; Pastoral system; Rangeland; Restoration; Soil seed bank; Savanna; Semi-arid ecosystem; Vegetation and soil degradation

Rangelands in Africa provide important forage resources for herbivores; particularly perennial grasses provide grazing for domestic and wild herbivores. However, semi-arid African rangelands experience severe vegetation and soil degradation due to heavy grazing, causing negative impacts on the ecosystems, livestock production and livelihoods of the people. Semi-arid African rangelands can be described by a state-and-transition models, often with three stable states, the first one being a state with ample herbaceous cover, perennial grasses and scattered trees, the second one as a state with a poor cover of annual grasses, absence of perennial grasses, and the third state with a high proportion of bare soil and/or often bush encroached. This thesis aims to fill important information gaps concerning soil seed banks that play in the recovery and possible restoration of degraded semi-arid African rangelands with particular emphasis on Ethiopia. This was done through investigating the mechanisms of how heavy grazing affects the soil seeds bank dynamics so as to understand stable states and transition processes of aboveground vegetation. In this thesis, aboveground vegetation and soil seed bank dynamics were studied under heavy and light grazing pressures in a field and experimental conditions. Results show that heavy grazing resulted in the disappearance of perennial grasses, a reduction in herbaceous species diversity and their plant abundance, standing biomass and basal cover, as well as a decrease in the soil nutrient conditions. The soil seed banks was correlated to differences in grazing pressure, with a greater seedling density under light grazing compared with heavy grazing. Immediately after seed dispersal, the seedling density increased over the first first three months until the eight months of soil sampling, and decreased thereafter. Under light grazing, perennial grass species dominated, whereas annual species were abundant at the heavily grazed sites, indicating that perennial grasses, with good fodder value, are replaced by annual species in the soil seed banks due to heavy grazing. With increasing soil depth, the seedling density and its species richness declined. Moreover, the seeds of perennial grasses were less abundant in the soil seed banks under heavy grazing. The similarity in species composition between the soil seed banks and aboveground vegetation was low under heavy grazing. Results also show that annual grasses had a lower germination and mortality, and higher viability, leading to a longevity of 62% over the 120 days burial in the soil, which was high compared to the 28% for perennials. Moreover, most perennial grasses germinate rapidly after initial seed dispersal at the first rains early in the year, whereas annual grasses show a linear germination pattern over time, indicating that perennial grasses have different survival strategy in semi-arid Africa. As a result, annual species are expected to dominate the soil seed banks, whereas most perennial grass species do not form persistent soil seed banks.The mean mortality from the seedling stage to adult plants in grass species was 65%, and the seed–to–seedling stage was found the most critical transitional stage for grass survival on these rangelands, suggesting that exclusion from grazing and trampling in the early germination stage is important to facilitate the transition from seedling to established plants. Depletion of perennial grass seeds in the soil due to heavy grazing coupled with high seedling mortality leads to a strong decrease in perennial grasses both in the soil seed banks, as well as in the aboveground vegetation. I found that the positive relationship between plant cover and differences in soil seed bank dynamics, i.e., seed density, seed germination rate and longevity, trigger the transition from perennial grasses to annuals and from annual plant cover to bare soil under heavy grazing. I hypothesize that the restoration of perennial grasses from the soil seed banks in heavily grazed areas in semi-arid African rangelands cannot be successful without an extraneous source of perennial grass seeds and without protecting the young plant’s regrowth from trampling and grazing. Therefore, the persistence of species and maintenance of biodiversity in semi-arid rangelands depends mainly on the recruitment of seedlings from annual species, and on vegetative reproduction of perennial grasses and woody species. These findings have important implications for the management, conservation and restoration of semi-arid African rangelands. Keywords:Africa; Aboveground vegetation; Ethiopia; Pastoral system; Rangeland; Restoration; Soil seed bank; Savanna; Semi-arid ecosystem; Vegetation and soil degradation

Some decades ago it became clear that formal agricultural research institutions - and hence formal interventions in agriculture - were somehow missing their targets for African farmers, mainly because their proposed solutions, and the ways these solutions were developed and introduced to African farming communities, did not match the realities of peasant life. It was recommended that the formal research should consider the wider contexts within which farmers operated to formulate better solutions. These solutions are essential for low-resource farmers facing many (socio-economic and cultural) constraints and having to cope with uncertainties (climate change, market variations, soil degradation, political and social unrest). The research presented in this thesis analyses the functioning of West African rice seed systems with regards to this recommendation. It starts with a regional focus (seven West African coastal countries) and then focuses on specific in-depth field studies undertaken in Guinea (with some comparison from neighbouring Sierra Leone). The study is based on an interdisciplinary approach combining methods from social and natural sciences. Findings show that despite efforts from governments, policy makers and formal agricultural research, the informal seed system still predominates, largely because it is the seed system closest to low-resource farmers. The objective of replacing the informal seed system by a formal seed system exclusively promoting improved varieties is a distant prospect. The research shows that local varieties are, to a large extent, superior to improved varieties in the sub-optimal conditions facing most farmers. It is also shown that even when improved varieties suit farmers’ conditions they are often channeled through inappropriate institutional arrangements that block access by low-resource farmers. Formal seed projects often lack follow-up to sustain actions. Innovations are lost between research planning, donor requirements to demonstrate adoption and the realities of peasant coping strategies. It is argued that success indicators in the formal seed system need to be redefined based on a clear conceptual divide between variety dissemination and bulk seed supply. The formal seed system merges these two activities whereas the informal seed system pursues a different path and addresses different procedural constraints. We suggest seed projects should concentrate on variety dissemination and leave bulk seed supply to local seed dealers. The thesis demonstrates that local dealers are effective and more closely in tune with farmer needs. The major finding of this thesis is that the informal seed system is closer to farmers, and works well, because it reflects (and is integrated with) local ideas about food security and social solidarity. This social dimension is missing in the formal system, designed and funded by experts who neither live by planting rice nor share in the local sets of assumptions about social reciprocity and obligation. Guinea may be undermining its long-term food security if it continues to seek to replace a social seed system with one driven solely by abstract ideas of economic rationality. The better option, supported by the weight of evidence in this thesis, is to seek complementarity and synergy between the two systems. Keywords: Oryza sativa, Oryza glaberrima, food security, formal seed system, informal seed system, varietal diversity, sub-optimal agriculture, small-scale farmers, farmers’ practices, Guinea, Sierra Leone, West Africa.

Key words: farmer seed production school, farm-saved seed, formal seed sector, impact assessment, improved practice, local practice, rice (Oryza sativa), seed production, seed quality, VietnamRice (Oryza sativa) is the most important food and cash crop of Vietnam. It is cultivated in all provinces of the country since ancient times. Farm-saved seed is the most important seed source covering more than 80% of the farmers’ seed needs. However, farmers not always use the best techniques of producing and selecting seeds. Inadequate seed quality is an important yield limiting factor in rice production.To improve the farmers’ capacity to produce, process, store and use good rice seed, the farmer seed production school (FSPS) training programme was conducted in seven provinces of Vietnam during the period 2003−2007. The study reported in this thesis took place in four out of those seven provinces, i.e. Nam Dinh, Nghe An, Binh Dinh and Dong Thap. The objective was to assess to what extent farmers’ knowledge in seed production practices and seed quality management had increased and whether that knowledge increase was reflected in an increase in potential rice yields and profits, and in diffusion of retained practices after training to other farmers in communities.A long seed production training programme with the farmer field school approach was combined with field demonstrations including plots with either local practices or improved practices which were conducted in each FSPS. We recorded and analysed data on on-farm demonstrations at 429 FSPSs and on ex-ante and ex-post tests of knowledge at the FSPSs. Moreover, we carried out a survey among 240 rural households.Results of the study indicate that some rice varieties were better adopted in the farming systems than other varieties: well adopted ones were KD18 in both Nam Dinh and Nghe An province and OM1490, Ai32 and MO2718 in Binh Dinh and Dong Thap. With local practices in the farm-saved seed system of the transplanted rice crop, farmers used old seedlings, planted many seedlings per hill, planted too many or too few plants per unit area and applied unbalanced quantities of fertilizers; for the directly sown crop farmers used high seed rates in the traditional system. Rice yields showed larger differences between local practices and improved practices in the dry season than in the wet season all across Vietnam. With improved practices at the FSPSs, rice yields were 8.5% higher in the wet season and 13.6% higher in the dry season; additional profits associated with the improved practice in both the dry and wet seasons averaged 212 US$ ha-1. The majority of the FSPS-farmers moved from food production to seed production, reduced seed rates by about 50%, and used high quality seed to produce seeds with much better quality. More important is that the FSPS-farmers diffused improved practices (79%) and shared good seeds (57% of respondents) with other farmers in their communities to help other rice growers to improve their productivity. A large proportion of non-FSPS farmers learned and applied improved practices for rice production through neighbouring FSPS-farmers within the community. Besides, evaluation in acquired knowledge during training showed that FSPS-farmers with lower scores (

Key words: farmer seed production school, farm-saved seed, formal seed sector, impact assessment, improved practice, local practice, rice (Oryza sativa), seed production, seed quality, VietnamRice (Oryza sativa) is the most important food and cash crop of Vietnam. It is cultivated in all provinces of the country since ancient times. Farm-saved seed is the most important seed source covering more than 80% of the farmers’ seed needs. However, farmers not always use the best techniques of producing and selecting seeds. Inadequate seed quality is an important yield limiting factor in rice production.To improve the farmers’ capacity to produce, process, store and use good rice seed, the farmer seed production school (FSPS) training programme was conducted in seven provinces of Vietnam during the period 2003−2007. The study reported in this thesis took place in four out of those seven provinces, i.e. Nam Dinh, Nghe An, Binh Dinh and Dong Thap. The objective was to assess to what extent farmers’ knowledge in seed production practices and seed quality management had increased and whether that knowledge increase was reflected in an increase in potential rice yields and profits, and in diffusion of retained practices after training to other farmers in communities.A long seed production training programme with the farmer field school approach was combined with field demonstrations including plots with either local practices or improved practices which were conducted in each FSPS. We recorded and analysed data on on-farm demonstrations at 429 FSPSs and on ex-ante and ex-post tests of knowledge at the FSPSs. Moreover, we carried out a survey among 240 rural households.Results of the study indicate that some rice varieties were better adopted in the farming systems than other varieties: well adopted ones were KD18 in both Nam Dinh and Nghe An province and OM1490, Ai32 and MO2718 in Binh Dinh and Dong Thap. With local practices in the farm-saved seed system of the transplanted rice crop, farmers used old seedlings, planted many seedlings per hill, planted too many or too few plants per unit area and applied unbalanced quantities of fertilizers; for the directly sown crop farmers used high seed rates in the traditional system. Rice yields showed larger differences between local practices and improved practices in the dry season than in the wet season all across Vietnam. With improved practices at the FSPSs, rice yields were 8.5% higher in the wet season and 13.6% higher in the dry season; additional profits associated with the improved practice in both the dry and wet seasons averaged 212 US$ ha-1. The majority of the FSPS-farmers moved from food production to seed production, reduced seed rates by about 50%, and used high quality seed to produce seeds with much better quality. More important is that the FSPS-farmers diffused improved practices (79%) and shared good seeds (57% of respondents) with other farmers in their communities to help other rice growers to improve their productivity. A large proportion of non-FSPS farmers learned and applied improved practices for rice production through neighbouring FSPS-farmers within the community. Besides, evaluation in acquired knowledge during training showed that FSPS-farmers with lower scores (

Brassica rapa is an important crop with a variety of forms, and a wide distribution in the world. It is used as oil seed and vegetable crop and a valuable source of diverse health-promoting metabolites. It also can serve as a model for genetic and molecular analysis in the Brassica genus, to which all rapes, kales and cabbages belong, as it has the smallest genome size and some genotypes with a rapid life cycle. Insertional mutagenesis using heterologous maize transposons has been a valuable tool for the identification and isolation of genes in Arabidopsis. Transposon-based activation tagging systems use a construct with constitutive enhancer elements that can cause transcriptional activation of flanking plant genes, which can result in dominant mutant phenotypes and subsequent isolation of the genes involved. Chapter 2 describes the action of an En/I activation tagging construct in B. rapa through Agrobacterium rhizogenes–mediated hairy root transformation. Successful transformation of this construct to B. rapa ssp. by A. tumefaciens was not achieved, probably due to the combination of an inefficient plant transformation and regeneration system, the length of the construct and most importantly the presence of the SU1 gene in the construct that appears to inhibit the regeneration of transformed shoots. As an alternative to the insertional mutagenesis approach to identify genetic loci that impact traits, there is a genetic approach based on quantitative trait locus (QTL) analysis. Segregating populations are needed to map QTLs for traits of interest. Chapter 3 describes the analysis of an F2 population derived from a cross between two distinct, but early flowering and self compatible, B. rapa genotypes, L58 and R-o-18. Amplified fragment length polymorphism (AFLP) markers together with simple sequence repeat (SSR) markers were used to genotype this F2 population and anchor the linkage map to the reference genetic map of B. rapa. Highly significant QTLs associated with the production of adventitious roots and the transformation competence to A. rhizogenes were detected, which will allow the selection of lines that are more efficient in transformation experiments. The analysis detected a strong QTL associated with seed coat color as well as QTLs for various morphological traits. To fix the recombination events as much as possible and to obtain an “immortal” mapping population, a recombinant inbred line (RIL) population was developed from this F2 population. Chapter 4 describes development of this RIL population, for which a genetic linkage map was constructed using the Illumina® BeadXpressTM genotyping platform of Keygene NV and additional SSR markers. Analysis revealed an additional QTL for seed coat colour as well QTL for pod shattering, carpel number, cuticular wax and seed vivipary. Chapter 5 describes the detection of QTLs related to primary and secondary metabolites in this RIL population. The two parental lines show clear differences in metabolite profile, which allowed the finding of QTLs for glucosinolates, phenylpropanoids, glucose, glutamate and amino acids after analysis with H1- NMR. HPLC analysis of tocopherols revealed four QTLs controlling the levels of this important antioxidant. The information on the genetic control of health related compounds indicates the potential to improve nutritional quality in classical crop breeding programs.

Brassica rapa is an important crop with a variety of forms, and a wide distribution in the world. It is used as oil seed and vegetable crop and a valuable source of diverse health-promoting metabolites. It also can serve as a model for genetic and molecular analysis in the Brassica genus, to which all rapes, kales and cabbages belong, as it has the smallest genome size and some genotypes with a rapid life cycle. Insertional mutagenesis using heterologous maize transposons has been a valuable tool for the identification and isolation of genes in Arabidopsis. Transposon-based activation tagging systems use a construct with constitutive enhancer elements that can cause transcriptional activation of flanking plant genes, which can result in dominant mutant phenotypes and subsequent isolation of the genes involved. Chapter 2 describes the action of an En/I activation tagging construct in B. rapa through Agrobacterium rhizogenes–mediated hairy root transformation. Successful transformation of this construct to B. rapa ssp. by A. tumefaciens was not achieved, probably due to the combination of an inefficient plant transformation and regeneration system, the length of the construct and most importantly the presence of the SU1 gene in the construct that appears to inhibit the regeneration of transformed shoots. As an alternative to the insertional mutagenesis approach to identify genetic loci that impact traits, there is a genetic approach based on quantitative trait locus (QTL) analysis. Segregating populations are needed to map QTLs for traits of interest. Chapter 3 describes the analysis of an F2 population derived from a cross between two distinct, but early flowering and self compatible, B. rapa genotypes, L58 and R-o-18. Amplified fragment length polymorphism (AFLP) markers together with simple sequence repeat (SSR) markers were used to genotype this F2 population and anchor the linkage map to the reference genetic map of B. rapa. Highly significant QTLs associated with the production of adventitious roots and the transformation competence to A. rhizogenes were detected, which will allow the selection of lines that are more efficient in transformation experiments. The analysis detected a strong QTL associated with seed coat color as well as QTLs for various morphological traits. To fix the recombination events as much as possible and to obtain an “immortal” mapping population, a recombinant inbred line (RIL) population was developed from this F2 population. Chapter 4 describes development of this RIL population, for which a genetic linkage map was constructed using the Illumina® BeadXpressTM genotyping platform of Keygene NV and additional SSR markers. Analysis revealed an additional QTL for seed coat colour as well QTL for pod shattering, carpel number, cuticular wax and seed vivipary. Chapter 5 describes the detection of QTLs related to primary and secondary metabolites in this RIL population. The two parental lines show clear differences in metabolite profile, which allowed the finding of QTLs for glucosinolates, phenylpropanoids, glucose, glutamate and amino acids after analysis with H1- NMR. HPLC analysis of tocopherols revealed four QTLs controlling the levels of this important antioxidant. The information on the genetic control of health related compounds indicates the potential to improve nutritional quality in classical crop breeding programs.