In this thesis I examine how the scientific advisory system in England and Wales has responded to concerns about the risks of pesticide residues in food and demands for wider engagement in the formulation of advice. Specifically, I explore how the Advisory Committee on Pesticides (ACP) frames scientific uncertainties in risk assessment, and why some bodies outside and within government are critical of the ACP’s approach that is centred in the conventional single-chemical, high-dose-response paradigm of toxicology. Although some of these challenges date back to the early history of pesticide regulation in England and Wales, the emergence of scientific research employing different methods to assess the effects of chemical mixtures and chronic low-level exposure has stimulated new concerns about the risks posed by pesticide residues for human health. Using semi-structured interviews and documentary analysis, a key finding is that concerns about low-level exposure to chemical mixtures have been persistently bracketed in official advice as insufficient for changing current advice and regulation. Drawing from literature in science and technology studies, I account for this finding in three ways. First, it is perceived that change is unnecessary since established methods of pesticide risk assessment represent an exemplar for other domains. Secondly, evidence selection by the ACP and related committees is shaped by regulatory guidelines which aim to provide standardisation and quality assurance, but also constrain judgements about which risk assessment studies are considered admissible. Thirdly, fundamentally different notions are at play in terms of what constitutes legitimate expertise and who should embody it, leading to tensions within government as well as between the ACP and NGOs. These limit the impact of post-BSE attempts to make the role of scientific advice in policy-making more participatory and ‘evidence-based’, and the capacity to introduce new paradigms of chemical risk assessment in the pesticide advisory process.

In today's world of rapidly changing climates, ever-increasing human population growth and increased competition for space, the production of energy and high quality food to meet increasing demands presents an enormous challenge to the related industries. It has been predicted that global food production alone, must increase by 70% by 2050 to prevent worldwide starvation. The application of plant biotechnology offers a promising alternative over classical crop improvement techniques in the development of breeding programmes that could respond rapidly to future demands for high-yielding and nutritionally-enhanced crops. Adopting such strategies to underutilised legume crops represents a viable avenue for crop improvement, especially in developing countries with a high incidence of poverty and malnutrition, where local environmental conditions frequently prove too challenging for effective cultivation of commercial cultivars. The main objective of this thesis was to generate fundamental data regarding the adoption of tissue culture-based transformation strategies for the genetic improvement of two such crops, jicama [Pachyrhizus erosus (L.) Urban] and Bambara groundnut [Vigna subterranea (L.) Verdc.]. The work carried out on jicama investigated the regeneration potential of this plant in vitro as a basis for future biotechnological techniques. Preliminary tissue culture experiments identified explant types and plant growth regulator (PGR) matrices and concentrations that produced optimal regenerative responses in vitro. Histological analysis of regenerated tissue revealed that shoot formation occurred via somatic embryogenesis. Possible avenues for further optimisation of the explant-to-plant regeneration protocol, for downstream molecular genetic applications, were explored and discussed. For Bambara groundnut, this study initially focused on developing a novel explant-to-plant regeneration system, previously not reported for landraces DipC, Uniswa-Red and S 19-3, through the adoption and modification of techniques specific for distinct Bambara landraces. This micropropagation protocol employed whole zygotic embryos as starting explants, which formed the basis for subsequent transformation studies. Genetic transformation was attempted via biolistic- and Agrobacterium-mediated approaches, using transformation vectors pVDH65, pB1l21, harbouring the nptII selectable marker and uidA (gus) reporter genes, while pBIl21-LeB4-Ber e I additionally carried the methionine-rich ber el gene. T-DNA transfer in transformed explants was confirmed via histochemical GUS analyses, while peR assays identified putatively transformed regenerated shoots. Explant viability was significantly compromised following bombardment or inoculation, resulting in reduced shoot regeneration, and thus limited the production of transgenic plants. Improvements in transformation efficiency and regeneration frequency were made following protocol optimisation, and the potential for further improvements to efficiently produce well-developed, healthy transgenic shoots were explored and discussed. The results presented in this thesis describe the optimisation of innovative tissue culture and regeneration protocols, which form a fundamental prerequisite to future transformation-based experiments for jicama and Bambara groundnut. Additionally, previously unpublished data generated from transformation studies in Bambara groundnut has revealed its potential for genetic manipulation. These results have established the foundation of novel micropropagation and transformation systems with the potential to be further optimised to genetically enhance and exploit the enormous agronomic potential of these underutilised legume crops.

Factors regulating curd initiation in the summer cauliflower were examined with special attention given to responses to change in environment. Low temperature treatments of four weeks at 5°C accelerated curd initiation, reducing the number of leaves initiated before the curd. A reduction in vegetative growth was associated with earlier curd initiation. The extent of the low temperature effect was dependent on genotype, temperature, treatment duration and the plant's stage of development when low temperature treatments commenced. A distinct juvenile phase was observed when plants were incompetent to perceive chilling as a vernalization stimulus. Phase transition from the juvenile to the mature, competent form was associated with the initiation of a specific number of leaves. These were 14 and 18 leaves for cvs Perfection and White Fox respectively, leaf number being higher in the later season cultivar. The duration of phase change itself was short, lasting approximately two plastochrons. Increase in leaf initiation rate was associated with phase transition. Rate of leaf initiation increased with increasing temperature. Duration of the juvenile phase, measured chronologically, was therefore shorter at higher temperatures. Shoot dry weight was linearly related to leaf number in plants examined here; this characteristic would also be a stable marker for phase transition. Chilling imbibed seed proved ineffective in accelerating curd initiation. Reduction in total irradiance receipt delayed curd initiation in plants grown under warm conditions. Associated with this delay were reductions in rate of leaf initiation and stem dry weight increment. Photoperiods of 16 h following sub-optimal vernalization allowed faster curd initiation than photoperiods of 8 hand 24 h. A minimum stem dry weight at curd visibility was achieved under this regime. Reciprocal of leaf number subtending the curd, denoting the rate of progress towards curd initiation, was shown to be linearly related to temperature under controlled environment conditions. Curd initiation rate in . cv Perfection increased after treatment in the temperature range -1.2SoC to 5.5°C and decreased after treatment over the range 5.5°C to 23.5°C. Similarly, in cv White Fox, curd initiation rate increased following treatments at temperatures of -3.0°C to 8.6°C and declined over the range 8.6°C to 31.5°C. Linear regressions also adequately described the relationship of rate of curd appearance on temperature. Rate of curd appearance in cv Perfection increased following treatment at temperatures of -4.5°C to 12°C and declined over the range 12°C to 29.5°C. In cv White Fox rate of curd appearance was shown to increase following treatment over the range -3.5°C to 15.8°C and decline from 15.8°C to 28.3°C. The different cardinal temperatures from those established for curd initiation were probably the result of examining not one process but two: curd initiation and early curd growth. Their two distinct optimum temperatures would account for the observed parabolic trend. The relationship between leaf number subtending the curd and thermal time established under controlled environment conditions was extrapolated to predict curd initiation time in the field. Thermal times of vernalization for plants grown under field conditions showed close agreement with controlled environment figures at early transplantings, but not for late transplantings. This drift was probably due to irradiance receipt whereby increasing irradiance would partially SUbstitute for low temperature in accelerating curd initiation. Curd growth and morphology were clearly influenced by post-chilling temperature conditions. Linear functions adequately described regression of Loge curd diameter or curd weight on thermal time. Thermal requirement for a specified curd diameter could therefore be calculated. Increasing supply of mineral nitrogen accelerated curd initiation in unchilled plants. Curd initiation in chilled plants, however, was not affected by nitrogen applications. Increasing nitrogen increased both leaf area and dry weight. A minimum leaf dry weight below which curd initiation could not occur was indicated. Both larger module size for plant growth and increased nitrogen levels increased the number of leaves initiated during propagation. Increased nitrogen during propagation accelerated curd initiation in the field. Severe water stress also accelerated curd initiation and reduced leaf growth. Application of GA4+7 to unchilled plants accelerated curd initiation more. than applications of GAS. GA4+7 generally increased stem dry weight and decreased leaf dry weight. The hypothesis was proposed that GA4+7 accelerated curd initiation by redirecting assimilates to apical regions of the stem. Curd initiation under field conditions was also accelerated by GA4+7.

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

Barley net blotch (BNB) caused by Pyrenophora teres, and Septoria tritici blotch (STB) caused by Mycosphaerella graminicola, are destructive cereal diseases worldwide on barley and wheat respectively. Due to the lack of highly resistant cultivars, both diseases are widely controlled using fungicides. Systemic, site-specific modern fungicides have played an essential role in disease management in cereals. Triazole-based fungicides, which inhibit the C14 demethylation step in fungal ergosterol biosynthesis, known as demethylation inhibitors (DMIs) and strobilurins, known as quinine outside inhibitors (QoIs), which interfere with energy production in the fungal cell, by blocking electron transfer at site of quinone oxidation in the cytochrome bc1 complex, are two major site-specific systemic groups of fungicides, currently used to control cereal diseases. Multiple, consecutive and extensive use of these fungicides has led to the emergence of fungicide resistance in these plant pathogens. The existence of G143A and F129L mutations has been found to be associated with resistance of many plant pathogens to QoIs. However, in P. teres only F129L was found to confer insensitivity. The presence of an intron in several fungi (including rusts and P. teres) determines that it is impossible for the G143A mutation to survive and thus be selected for. Alterations in CYP51 gene in plant pathogens has also been found to be one of the major mechanisms resulting in reduced sensitivity towards DMIs. The aim of this research was to investigate the impact of the F129L mutation in isolates of P. teres, and mutations in the CYP51 gene in M. graminicola isolates on the activity of QoI and DMI fungicides respectively. Results revealed a high frequency of the F129L mutation within recent UK P. teres isolates. Furthermore, the common change (G143A) in cytochrome b was not found in P. teres strains. The results also showed a lack of any fitness penalty associated with the mutation. Bioassay tests indicated that inhibition of net blotch by QoIs was variable. Single QoI fungicides such as pyraclostrobin and picoxystrobin were found to be highly inhibitory whilst the efficacy of other QoIs was less pronounced. It has been found that efficacy of QoI fungicides varied amongst a population of isolates with the F129L mutation. This might suggest that some QoIs were compromised by the F129L mutation to some degree. However, the results obtained were in agreement with previous reports that the F129L mutation in the cytochrome b gene generates lower levels of resistance and was not as serious as that posed by the G143A mutation in other plant pathogens. In addition, fungicide mixtures, comprising QoIs and DMIs or the novel SDHI formulations, were found to have great efficacy in net blotch disease management. Sequence results of CYP51 gene fragment indicated existence of 15 alterations in recent UK and German isolates of M. graminicola. Some of these mutations, such as Y137F, were found to be rare whilst the I381V mutation was found to be increasing with time. However, investigations indicated a lack of phenotypic fitness penalties associated with these alterations. Apical germ tube growth measurement was found an effective method to assess in vitro activity of DMI fungicides against M. graminicola isolates. Based on bioassay studies, six categories within M. graminicola isolates were detected, showing different sensitivities to azole fungicides. In general, genotypes characterised S, R3+ and R4 were sensitive to most azole fungicides. The R3+ variant, however, showed less sensitivity to tebuconazole and prochloraz. In in vitro studies, the R5 variants, exhibited sensitivity to many DMIs but were less sensitive to prochloraz. This supporting the results obtained from in planta assays, where this genotype was found to be sensitive to tebuconazole but less sensitive to prochloraz. On the other hand, genotypes characterised R6a, R7 and R8, containing I381V mutation, were resistant to tebuconazole but sensitive to prochloraz. The latter variant, however, were more sensitive to prochloraz. It can be suggested from results obtained in this study that CYP51 alterations were differentially selected by different members of the azole class of fungicides. Q-PCR was also used to evaluate in planta fungicide activity on both diseases. The method indicated similar pattern to that observed in visual assessments. Detection of medium to high correlation values between both assessments confirmed the validity of q-PCR assessment. This suggests that q-PCR assays may serve as an alternative method for accurate assessment of the fungicide effects on cereal diseases. The method can be a valuable tool to evaluate disease occurrence in pathogens with a long latent period, such as M. graminicola, as q-PCR could readily detect the pathogen during the asymptomatic latent period.

Bambara groundnut (Vigna subterranea (L.) Verdc) is an indigenous legume that is still cultivated in subsistence agricultural systems in sub-Saharan Africa, despite the lack of any major research effort until recently. The crop is cultivated from local landraces as there are no true varieties of the species bred for specific traits. The variable and hostile climates in the region mean that annual yields of most rainfed crops including bambara groundnut are far below their agronomic or genetic potential. The lack of quantitative information on the eco-physiological responses of the crop to various abiotic factors has resulted in poor decision making on crop management practices especially in relation to sowing date and the selection of appropriate landraces for different locations. Modelling of bambara groundnut was initiated previously but there is still insufficient understanding of how growth and developmental processes can be simulated under abiotic stress and different photoperiods. The aim of this study was to develop a crop simulation model for bambara groundnut to predict growth, development and yield under drought, heat and cold stress and different daylengths. The present model (BAMGRO) is an adaptation of the established CROPGRO and previous bambara groundnut models; BAMnut and BAMFOOD project model. It uses climate data, landrace specific parameters and soil characteristics and runs on a daily time-step to determine the canopy development, biomass production and yield of a landrace in a specific environment. The parameters of the model have been determined with glasshouses data (TCRU, University of Nottingham) and published information. BAMGRO is capable of describing differences between landraces, and the influence of drought, temperature and photoperiod using a simplified approach. The present modelling approaches with BAMGRO model provide useful predictive information on canopy development, biomass production and yield formation of bambara groundnut landraces under contrasting environments. Two contrasting landraces; Uniswa Red (Swaziland) and S19-3 (Namibia) were used in the present study to evaluate the growth and yield performances under drought, heat and cold stress. BAMGRO has been primarily validated against independent data sets of two years glasshouse for two contrasting landraces; Uniswa Red and S19-3 grown under two temperatures (23 ± 5 0C, 33 ± 5 0C) with drought. Further, it was validated for field data in Botswana with two sowing dates (January 18, February 1) during the 2007 season and for Swaziland for three landraces; Uniswa Red, DipC, OM1. The model achieves a good fit between observed and predicted data for LAI (Nash and Sutcliffe (N-S), 0.78-0.98; Mean Absolute Error, ± 0.14-0.57) for tested four landraces. Pod yield simulation was correlated well with measured values especially for Uniswa Red and S19-3 (N-S 0.73-0.87; Mean Absolute Error ± 16 g m-2) while it was poor for DipC and OM1 (N-S, 0.46-0.50; Mean Absolute Error, ± 15.6-17.7 g m-2). Further, the comparison of simulated and measured data of TDM reported lower correlation compared to LAI and yield. (N-S, 0.59-0.79; Mean Absolute Error ± 48-100 g m-2) indicating overall underestimation. The performance of the BAMGRO-soil water module was tested by validating the available soil moisture and results indicating that it over estimated for upper layers while deeper layers showed lower prediction. The possible reasons for the discrepancies in measured and simulated data are differences in quality and quantity of solar radiation in UK summer and Semi-arid Africa, intra-landrace variability and poor calibration of soil water module. Four potential applications of BAMGRO and three future developments are presented in this thesis.

As a plant operating the C3 photosynthetic pathway and commonly grown under tropical conditions of high light intensity and temperature, rice (Oryza sativa) displays high levels of photorespiration, to the detriment of photosynthetic efficiency. For this reason it is thought that improvements to net photosynthesis via an increased photosynthetic efficiency could provide significant gains in terms of grain yield. There is great interest in 1. Introducing CO2 concentrating mechanisms into C3 crop plants such as the C4 photosynthetic pathway in order to facilitate enhanced photosynthetic efficiency. This requires an understanding of C3 and C4 leaf development and establishing whether there is sufficient plasticity in the rice genome to produce plants with C4-like properties. 2. Improving existing C3 photosynthesis by means of increasing leaf thickness, vein density and investigation of the impacts of mesophyll cell size. It is in this context that a forward screen of approximately 100 mutant lines of the indica rice variety IR64 was developed at Nottingham to search for relevant changes in leaf morphology. Mutant seed produced using chemical mutagenesis (diepoxybutane and ethylmethanesulfonate) and irradiation (gamma and fast neutron) was supplied by the International Rice Research Institute (IRRI) in the Philippines. A rapid low resolution screen was devised using light microscopy of fresh, untreated hand cut leaf sections of plants at the leaf six stage. Seven mutant lines were identified as showing altered leaf morphologies and were termed alm mutants. alm1, alm5 and alm6 displayed a reduced interveinal distance between neighbouring veins, a common feature of C4 plants with Kranz anatomy, whilst alm1 and alm5 also displayed a reduction in the size of minor veins. alm3 and alm4 produced significantly thicker leaves than wild type plants, whilst the leaves of alm7 were significantly thinner. A detailed anatomical characterisation of leaf structure revealed that alm3, alm4 and alm5 plants all displayed a significant reduction in the size of mesophyll cells and that for all the mutant lines, the distance between veins was strongly correlated with mesophyll cell size rather than the number of mesophyll cells spanning the interveinal regions. Physiological properties of the alm lines were investigated using infra-red gas analysis (IRGA) measurements of gas exchange and chlorophyll fluorescence. It was shown that none of the mutant lines displayed an increase in photosynthetic capacity when compared to wild type plants, even in lines which were shown to possess what was thought to be a favourable leaf anatomy, quite possibly a result of widespread effects of the mutation process. The alm1 line was shown to display interesting physiological responses, with almost no transpiration and a severely reduced photosynthetic capacity, yet functioning stomata and an unimpaired stomatal conductance. In conclusion, the future success of photosynthetic improvement in rice will rely on the screen of much larger numbers of mutant lines of rice and C4 plants in order to identify the genes determining key conserved morphological features such as interveinal cell number, cell size and the degree to which rice mesophyll cells are lobed.

This study considered a novel approach to selecting isolates of Hyphomycete fungi as mycoinsecticides for biological control of aphids in arable crops in the UK. The approach was designed to select isolates which were compatible with both the biotic and abiotic environment. Aphis fabae was chosen as a representative target aphid for bioassays, based on results of preliminary experiments. Eighteen isolates of fungi were screened at a single concentration of 1x 10⁸ conidia ml⁻¹ against apterous adult A. fabae, which were incubated at 23°C. Spray applications were made using an electrostatic rotary atomiser in both laboratory and field experiments. Isolates that originated from aphid hosts were most pathogenic to A. fabae. Four isolates were selected for further studies; ARSEF 2879 (Beauveria bassiana), HRI 1.72 (Verticillium lecanii), Mycotrol strain GHA (B. bassiana) and Z11 (Paecilomyces fumosoroseus). Isolate HRI 1.72 was most virulent to A. fabae in dose-response assays compared to other isolates; at concentrations of 1x 105 conidia ml⁻¹ and above, mortality of aphids due to infection by HRI 1.72 was 100%. Isolates of P. fumosoroseus and V. lecanii were able to grow and germinate better in vitro at low temperatures (10 & 15°C), than isolates of B. bassiana and Metarhizium anisopliae. Aphis fabae and Myzus persicae, inoculated with isolate HRI 1.72 and incubated at 10°C, succumbed to infection after a significantly shorter period of time compared to other isolates. Rhopalosiphum padi was most resistant to infection by the four isolates compared to five other species of aphid. Aphis fabae, Acyrthosiphon pisum, Sitobion avenae, Metopolophium dirhodum, R. padi and M. persicae were most susceptible to infection by isolate HRI 1.72. Aphids infected with isolates of V. lecanii often had fungal sporulation on their legs and died attached to leaves on which they were feeding. The isolates Mycotrol strain GHA and ARSEF 2879 were pathogenic to the 7-spot ladybird Coccinella septempunctata and the generalist parasitoid Praon volucre. The isolates HRI 1.72 and Z11 had very little impact on these natural enemies tested. When aphids of A. fabae were co-inoculated with isolates of Hyphomycete fungi and Erynia neoaphidis, most individuals succumbed to infection with E. neoaphidis. A significant number of aphids died within 24 hours of inoculation and showed no signs of external sporulation. The potential interactions between these natural enemies in the biocontrol of aphids are discussed. The spatial and temporal distribution of aphids and their natural enemies, in field bean and wheat crops, was determined in two field seasons (1997 & 1998). Aphids sampled from the field, after application of Hyphomycete fungi in 1998, mostly succumbed to infection with E. neoaphidis. Epizootics of E. neoaphidis were recorded in both years. Greater numbers of healthy laboratory aphids succumbed to infection with Hyphomycete fungi when they were bioassayed on leaves sampled immediately following spraying (51 - 100%) compared to 24 hours later (8 - 65%). Microclimate recordings showed humidity in both crops was generally >90% overnight and differences were as great as 15% between the top and bottom of crop canopies. Temperature differences were as great as 5-7°C between individual sensors. The implications of using a biorational approach as part of the development of Hyphomycete fungi as mycoinsecticides for the control of aphids is discussed.

Many of the wild and some cultivated species of potato are true diploids and are therefore more amenable for genetic studies than the majority of tetraploid cultivars. However, the use of these diploid Solanum species is complicated by almost universal self-incompatibility (SI). In Solanum, SI is gametophytic and pistil specificity is controlled by a polymorphic ribonuclease (S-RNase), as found in other members of the Solanaceae. The genetic engineering of self-compatible (SC) diploid potato lines would benefit potato breeding in general and allow inbred lines to be established for the first time. This would facilitate genetic analyses including that of complex traits such as drought resistance or yield. The aim of this thesis is to down regulate the expression of S-RNases in diploid potatoes using the RNAi technique and established procedures for Agrobacterium-mediated transformation. This approach to engineering self-compatibility has already been successfully demonstrated in SI Petunia inflata (Lee et al., 1994) and other species of the Solanaceae. To date just a handful of S-RNase sequences are available for potato species. The characterization of S-RNases in targeted diploid Solanum species was an initial requirement for our approach. To develop the tools, S-alleles have initially been characterized in both Petunia inflata and P. hybrida cv Mitchell both phenotypically (by pollination tests using a diallel cross) and/or genotypically (by RT-PCR). This approach was then transferred to three diploid potato species, specifically accessions of Solanum stenotomum, Solanum phureja and Solanum okadae. These wild species are important sources of new traits studied by The James Hutton Institute (formally the Scottish Crop Research Institute). The approach taken to amplify partialS-RNase sequences from pistil RNA was RT-PCR using a degenerate primer. PCR products were cloned using a TA vector (Invitrogen) and sequenced. For two alleles full length sequences were obtained by 5'RACE. Database searches with these sequences, revealed sixteen S-RNases several of which are novel. Phylogenetic analYSis was carried out with the cloned S-RNases together with selected published S-RNase and S-like RNase sequences of solanaceous species. The S-RNases revealed extensive trans-generic evolution and are clearly distinct from and distantly related to S-like RNases. For two alleles (501 and 502), S-RNase gene expression profiling was performed to check the developmental expression of the S-RNase gene, tissue-specific expression and also test whether these S-RNases (e.g. Sor and S02-RNases) are expressed at a similar level. Wide variation in S-RNase gene expression levels have been reported in the literature. An RNAi construct has been designed to down-regulate two specific 5-RNases in an 501/502 heterozygote of S. okadae. To increase the chance of Silencing, the RNAi construct has been designed to use a chimeric 5-RNase gene involving the 5' end of the SorRNase and the 3' end of the S02-RNase. The correct chimeric S-RNase construct (SOl/S02-RNase) has now been identified and inserted into an RNAi vector (pHelisgate8) using Gateway® technology. This RNAi construct (pHG8-S01/S02) is now a valuable resource for use in S-RNase gene silencing in potato leading to the development of self-compatible diploid potato lines and ultimately the development of the first inbred lines of S. okadae.

Bambara groundnut is an indigenous African legume grown mainly in sub-Saharan Africa; it is an important source of protein to the rural majority. There are no established varieties and subsistence farmers grow locally adapted landraces which are generally low yielding. Bambara groundnut is a predominantly self-pollinating crop and is expected to exist as non-identical inbred lines, although the previous lack of co-dominant markers has prevented a formal assessment of heterozygosity within bambara groundnut genotypes. A total set of 75 microsatellites that were characterised in this study were used to investigate the genetic diversity of a set of 24 bambara groundnut landraces, to provide an evaluation of the markers for polymorphism and provide a link with DArT marker data that were previously analysed. Sixty eight microsatellites were identified that were found to be consistent and reproducible, from which a set of markers were selected and used for genetic variability studies of bambara groundnut, to compare the use of molecular markers with morphological markers, and to investigate using SSR markers in pure line selection. The genetic diversity of bambara groundnut was assessed based on morphological characters for two seasons; in a glasshouse experiment at the University of Nottingham, Sutton Bonington Campus, UK and in a field experiment that was conducted at the Botswana College of Agriculture (Notwane farm), Gaborone in a randomised block design with three replicates. The landraces were characterised for 24 quantitative and 13 qualitative characters. The results indicated considerable variation for quantitative characters, while significant morphological differences were also recorded for most characters. Multivariate data analysis was conducted using principal component analysis, cluster analysis and heritability estimates were developed. The low cost, simplicity and agricultural relevance of morphological characterisation makes it an important tool in germplasm genetic variation studies. Thirty four lines from field experiments were investigated for genetic diversity based on 20 microsatellites. The expected heterozygosity (He) had an average of 1 in agreement with the fact that bambara groundnut is predominantly self-pollinating. Both cluster analysis and principle component analysis (PCoA) grouped landraces based mainly on their areas of origin. A thorough molecular analysis of genetic and morphological variation in bambara groundnut was conducted to investigate the relationship between the two assessment techniques. This comparison will assist in breeders making informed decisions as to which approach is best to use in germplasm characterisation and plant breeding and how best to apply such knowledge in practical situations. DNA markers could then aid with the selection of germplasm for breeding, quality control within breeding programmes and, potentially, direct selection via Marker Assisted Selection (MAS). Euclidean distance estimates for morphological data and (Nei’s 1972) genetic distance estimates for SSR data were strongly correlated (r = 0.7; P < 0.001) in the agronomy bay and (r = 0.6; P< 0.001) in the controlled growth room. These results suggest the two approaches are generating the same pattern of genetic diversity, and as such can be used as a surrogate for each other.