Wild rocket (Diplotaxis tenuifolia) has recently become a popular salad leaf in the UK due to its peppery taste. It is grown widely in Italy but is now being produced in Spring and Summer in England. It is part of the Brassicaceae family and thus has a high level of homology at the DNA level to other Brassica species and Arabidopsis. This project aims to produce late bolting genotypes of rocket to incorporate into commercial breeding programmes. Delayed bolting is important as current varieties flower at unpredictable times and often earlier than desired. This is a problem because when rocket flowers it becomes unsaleable. Ethyl methanesulphonate (EMS) was used to generate a mutant population of rocket. Late bolting lines were selected and whole genome sequencing was used to identify single nucleotide polymorphisms (SNPs) induced by the EMS which may be a cause of the late bolting phenotype. Six flowering pathway genes have been isolated from rocket and have been tested to see if they can functionally complement A. thaliana knockout lines in these genes. Further work was carried out to investigate how these genes were expressed over diurnal and developmental time courses to understand their function in the flowering time pathway in rocket. Together, these results show that most of the flowering pathway genes isolated from rocket are functional orthologues of those in Arabidopsis. The photoperiodic and vernalization requirements of rocket were investigated and it was found that rocket does not have a vernalization requirement and is a facultative long day plant. Targeted mutagenesis using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated system (cas9) was employed for the introduction of mutations into the FLOWERING LOCUS T (FT) gene in rocket and Arabidopsis. Many late flowering Arabidopsis lines were identified and sequencing revealed the successful manipulation of the FT gene. Two late bolting rocket lines were also identified. Overall, the project aims were achieved as late bolting rocket lines have been produced and will be incorporated into a rocket breeding programme.

Glyphosate is the world’s most used herbicide. There are currently 32 weedy species with resistant populations in 25 countries, although at present there are no reported cases of glyphosate resistance in the UK. As glyphosate use and selection pressure increases in the UK there is an excellent opportunity to investigate the potential for glyphosate resistance, and the evolutionary processes that may lead to resistance. The variability in standing genetic variation to herbicide susceptibility between weed populations can affect the amount of selection pressure and generations needed for resistance to evolve. If herbicide doses act within this standing genetic variation there may be a reduction in sensitivity due to a buildup of minor alleles related to reduced sensitivity. This thesis has investigated the glyphosate response of three UK weedy species, Alopecurus myosuroides (blackgrass), Anisantha sterilis (sterile brome), and Arabidopsis thaliana. Dose-response experiments showed significant variation in susceptibility between populations of all three species. Glasshouse selection experiments tested if glyphosate sensitivity could be further reduced under directional selection with below field rate doses, in Alopecurus myosuroides populations. Following selection, ten of eleven selected lines showed significantly different ED50 and ED90 values compared to unselected control lines, demonstrating that there is potential for selection of reduced glyphosate sensitivity, which may result in compromised field efficacy. Fitness cost experiments for two glyphosate-selected lines showed no major fitness costs associated with decreased glyphosate susceptibility both with and without wheat competition. Analysis of multi-parent advanced generation inter-cross Arabidopsis thaliana lines highlighted an area on chromosome 2 of the Arabidopsis thaliana genome that may be associated with variation in glyphosate susceptibility. These results are discussed in the context of the possibility of glyphosate resistance evolution in the UK.

Analysis of biological performance parameters of Aphis craccivora on groundnut variety ICG12991 in laboratory and field trials demonstrated that ICG12991 was resistant to the aphid vector of groundnut rosette diseases and that this resistance was stable over time and under high aphid pressure. Feeding experiments related slow population development and high aphid mortality on ICG 12991 to an inhibition of phloem feeding from the sieve elements. Consequently, virus transmission of all three agents of rosette disease was almost totally absent even under very high pressure of viruliferous aphids. Further investigation of the underlying mechanism of resistance in ICG 12991 showed that the resistance was induced by aphid probes rather than constitutive. The induced plant response was described as a hypersensitive response around the aphid feeding sites. Aphid-resistance and hypersensitivity were identified in at least 7 groundnut varieties and it is proposed that more varieties are likely to express these characteristics. The results led to the recommendation to evaluate groundnut varieties for aphid resistance by evaluating aphid population development. The identification of vector resistance and its efficacy in reducing virus spread of rosette disease in the field has opened the way for novel approaches for groundnut disease management.

This work centres on Cosmos atrosanguineus, a rare Mexican endemic self-incompatible species of Asteraceae that is now believed to be extinct in the wild. The two known wild C. atrosanguineus collections, made in made in the 19th century, localise the species to the pine-oak mountain forest ecological region in two areas of central Mexico. Its disappearance from the natural environment is attributed to habitat destruction by the copper mining industry and subsequent urbanisation, so that C. atrosanguineus is now known only as a cultivated species. European and American C. atrosanguineus populations are probably the progeny of seed collected in the 1860s by Benedict Roezl and brought to Europe for plant verification. Sir William Jackson Hooker (1785-1865), Director of the Royal Botanic Gardens, Kew initially identified the Roezl specimen as Cosmos diversifolius var. atrosanguineus, but it was classified as the distinct species, Cosmos atrosanguineus by Andreas Voss in 1894. Cosmos atrosanguineus is much prized in horticulture for its deep-red "atrosanguinate" blooms, which are deliciously chocolate scented, giving rise to its common name, "the Hot Chocolate Plant". From 1885 to 1942, C. atrosanguineus was sold as seed in England by Thompson and Morgan Seed Merchants, under the name of C. diversifolius atrosanguineus, but disappeared from the Thompson and Morgan seed catalogue thereafter. Cosmos atrosanguineus exhibits strong sporophytic self-incompatibility (SSI), does not set seed, and must be propagated vegetatively, a factor that has contributed to the proliferation of closely related or identical genotypes. Microsporogenesis within the species appears normal, with no discernible differences between microsporogenesis in C. atrosanguineus and two seed-producing Cosmos species. The C. atrosanguineus karyotype and chromosome associations at meiosis identify the species as an allotetraploid that produces viable gametes with ~30% pollen viability. Self- and cross-pollinations of C. atrosanguineus are strongly incompatible with an average of zero pollen grains germinating per stigma. In contrast, cross and self-pollinations in C. bipinnatus and C. sulphureus produced three compatibility groups; incompatible (-), compatible (+) and semi-compatible (±), and four categories of pollen-stigma interaction that putatively identify a gametophytic-sporophytic (G-S) incompatibility system in Cosmos. Dominance interactions of S-alleles are prevalent in the genus and pseudo-self-compatibility was observed in C. bipinnatus but not C. atrosanguineus. The Cosmos stigma is dry, papillate and becomes receptive only after stigmas become reflexed. Prior to this period, the stigmas do not respond to pollination and selfed bud pollinations and crossed bud pollinations are incompatible. Degenerate S-specific primers identified 41 S-domain-encoding sequences from Cosmos and four other genera of Asteraceae. These 41 Asteraceae sequences encoded S-domains related to Brassica S-domain proteins, but none identified as a putative self-incompatible molecule, indicating the control of SI in Cosmos is probably different to that in Brassica. Genetic fingerprinting studies (AFLP) of various European sources of C. atrosanguineus showed no genetic variation. In conclusion, Cosmos atrosanguineus is a strongly self-incompatible species with a reduced genome. It does not set seed because the gene pool of cultivated individuals has narrowed to a level where S-allele numbers are too few to produce cross compatible genotypes. For this to have occurred, the genetic diversity of plants at the Royal Botanic Gardens Kew and in commercial cultivation must have been reduced to genets that share one, but more probably both S-alleles, thus preventing germination of pollen and formation of seed.

The aim of this study was to evaluate the role of chitosan against Ganoderma pathogen of basal stem rot (BSR) disease under in vitro and in vivo conditions. Therefore, this study was conducted to investigate the effect of chitosan on (1) in vitro growth of Ganoderma sp. (2) production of lignin degrading enzymes (LDE) by Ganoderma sp. under in vitro cultivation system (3) disease severity and vegetative growth parameters on oil palm seedlings and (4) elicitation of plant defense response during the interaction between host-pathogen (oil palm-Ganoderma).

Bambara groundnut presents a great adaptation potential for mitigating climate change and as a potential alternative crop for the future in its production regions and beyond. Experiments were conducted at the Field Research Centre of the Crops for the Future and the Screenhouse of the University of Nottingham Malaysia Campus to determine the growth variables, yield and nitrogen fixation of Bambara groundnut landraces at different rates of Christmas Island Rock Phosphate and rice biochar in the study area. The treatments consisted of three Bambara groundnut landraces [Ex-Sokoto, Kaaro and NN-1, for which two (Ex-Sokoto and Kaaro) were maintained after the first experiment], two biochar levels (0 and 10 t ha-1) and four levels of rock phosphate (0, 20, 40 and 60 kg P2O5 ha-1, the equivalent of 0, 555.6, 1111.1 and 1666.7 kg ha-1 Christmas Island Rock Phosphate). The treatments in experiment 1 and 3 were laid out in a randomized complete block design (RCBD) replicated three times, whereas experiment 2 was laid out in a completely randomized design (CRD) replicated three times. For all the experiments, data were collected on growth, yield and nitrogen fixation of the crop. The results obtained revealed grain yield and N fixed ranging from 703-2256 kg ha-1 and 32-81 kg ha-1, respectively in experiment 1, 13.2-18.0 g plant-1 and 587-894 mg plant-1, respectively in experiment 2 and 891-1220 kg ha-1 and 101.6-103.4 kg ha-1, respectively in experiment 3, which are comparable to the yields obtained in other regions. Hence, Bambara groundnut can adapt to the tropical acid soil and the growing conditions in the study area. The two field experiments confirm Ex-Sokoto landrace to perform better in the study area in terms of yield than Kaaro landrace. Growing Bambara groundnut and returning the residues (haulm + shells) back to the soil resulted in net addition of N to the soil in the range of 1 to 25.6 kg ha-1, 150-261 mg plant-1 and 63.3-74.1 kg ha-1 observed in experiment 1, 2 and 3, respectively. Application of biochar increased the yield and nitrogen fixation of the crop in the study area. However, the effect of rock phosphate application on the yield and N fixation of the crop was only observed in the screenhouse studies and was optimum at the application rate of 40 kg P2O5 ha-1 (0.56 g kg-1 soil). Thus, Ex-Sokoto landrace could be recommended for grain yield in the study area. Application of biochar at 10 t ha-1 alone was optimum for increased yield of the crop. The implication of the study is that farmers in the study area could apply 10 t ha-1 of biochar on Ex-Sokoto landrace to enhance the yield of the crop.

A stable mixed microbial community able to utilise picolinic acid as the sole carbon and energy source was isolated from soil by continuous- flow culture enrichment. The community contained six different organisms three of which were capable of growth on picolinate in pure culture. These three primary utilisers were Pseudomonas aeruginosa, Alcaligenes faecalis and a second Alcaligenes sp. The three secondary organisms were Bacillus licheniformis, a Rhodococcus sp and a Corynebacterium sp. of the C.aquaticum type. These secondary organisms were presumed to be growing on the secondary metabolites of the primary utilisers but contributing something to the mixed culture due to its stability. The community was adapted to degrade a chlorinated analogue of picolinate, namely 3,6-dichloropicolinate, and the kinetics of enzyme attack on picolinate, in the presence and absence of the chlorinated compound was studied. At low growth rates, that would represent nature more accurately, there was a decrease in 36-DCPA concentration of approximately 15% indicating some microbial degradation. Oxygen uptake by the community in the presence of various substrates was studied and, generally, it was found that 36DCPA inhibited, to some extent, the uptake by the microbial community.

Pest risk assessment is an essential yet problematic stage in pest risk analysis (PRA) that concerns the likelihood and consequences of pest introduction. The aim of this study was to develop methodologies for risk assessment and to explore different approaches that could lead to the development of new methods for practical PRA in line with the requirement of "scientific justification" by World Trade Organisation and Food and Agriculture Organisation of the United Nations. Current international practices were discussed and research reviewed on qualitative and quantitative approaches to risk assessment. It was proposed that risk assessment be divided into two steps: Pest risk identification (PRI) and pest risk evaluation (PRE). Mind Mapping was a valuable tool for PRI that reduced ambiguity and increased transparency. Approaches to PRE were proposed that facilitated the scoring and weighting of risk factors, and the subsequent combining of risk scores. Several methods were developed to incorporate weighting into PRA, which included subjectively assigned weighting and Delphi technique-derived weighting. Metrics for combining risk scores into an overall risk value were also explored, compared and evaluated. Correlation and interaction between risk factors were analysed, which revealed that some risk factors were highly correlated and some were relatively independent, which meant there was some information redundancy, and therefore simplification of risk assessment was possible. Cluster analysis was applied to risk factor scores and different clusters of risk factors were identified: some more appropriate for preliminary assessment; some for determining the level of risk; and some could be eliminated. A method to apply Principal Components Analysis (PCA) to derive weighting for individual risk factors was developed. PCA could be applied to historical data of pest introductions, previous PRA cases, or expert opinion. Genetic algorithms implemented in the software BEAGLE, were applied to PRA data. The rules obtained could distinguish high-risk situations with high accuracy, which was useful in predicting the risk of an organism by using a simplified set of conditions. The results showed that weightings and rules differed for different taxonomic groups. Therefore it was implausible to develop a generic scheme in this way. However, it may be possible to develop patterns based on taxonomy. The results of applying several different techniques all suggested that by grouping risk factors for different purposes, risk assessment could be simplified without compromising rigor, because a) some factors were redundant; b) some factors are more important than others; and c) high risk situation could be predicted with a few key factors.

The objective of this study is to develop a versatile vector system for the delivery and expression of transgenes in the chloroplast genome of N. benthamiana. The successful advent of such a system would vastly streamline the construction process of chloroplast transformation vectors for the expression of recombinant proteins, such as vaccine candidates, in the chloroplasts of N. benthamiana. Transgenes targeted to the chloroplasts of higher plants are expected to be expressed at considerably higher levels as compared to nuclear expression, resulting in more significant accumulation of recombinant proteins. In this study, a 2-part chloroplast transformation vector system was developed and two new GFP vector prototypes, pEXPR-G and pEXPR-UG were generated for preliminary evaluation of functionality. The aadA and GFP expression cassettes of pEXPR-G and pEXPR-UG were evaluated in E. coli prior to actual delivery into N. benthamiana via particle bombardment. Particle bombardment parameters were optimised with particular emphasis on minimising excessive damage to the target tissue in order to facilitate the recovery of antibiotic resistant shoots and calli following transformation. To further evaluate the versatility of the developed system for the expression of vaccine antigens, recombinant vectors, pEXPR-HA and pEXPR-NA were constructed for the delivery of hemagglutinin (HA) and neuraminidase (NA) genes of avian influenza strain H5N1 into the chloroplast genome of N. benthamiana. Experimental results indicated that pEXPR-G and pEXPR-UG were fundamentally functional in E. coli and both the aadA and GFP expression cassettes were active, allowing the bacteria to withstand 500mg/l spectinomycin and express the transgene of interest at the protein level. Similar results were also observed in transplastomic N. benthamiana transformed with pEXPR-UG and pEXPR-NA. In essence, the developed 2-part chloroplast transformation vector system was found to be highly versatile and could be conveniently applied for the construction of transformation vectors for the delivery and expression of HA and NA in the chloroplast of N. benthamiana.

Postharvest tools have long been developed for minimising postharvest stresses and preserving fruit quality. However, increasing evidence points towards the role of abiotic stress in improving the bioactivity and nutraceutical value of fruits. This study was an investigation into the effects of postharvest stresses on the bioactive content of the tropical fruits carambola (Averrhoa carambola), dragonfruit (Hylocerues sp.) and mangosteen (Garcinia mangostana). The fruits were subjected to stresses induced by cold storage at 6 °C along with exposure to four different levels of one of the stress hormones salicylic acid (0, 0.1, 1, 2 and 5 mM) and methyl jasmonate (0, 0.01, 0.1, 0.2 and 0.5 mM). Physicochemical responses of all three fruits to the postharvest stresses along with changes in the antioxidant activity were assessed throughout the shelf life of the fruits. The goal was to characterise physiological and biochemical associated stress responses of the tropical fruits, and to evaluate the potential of using these stress responses for enhancing fruit quality.