Approximately 900bp of the Phaseolus vulgaris cytosolic glutamine synthetase gIn-a gene promoter has been cloned and the DNA sequence determined. The transcriptional start site was mapped using primer extension and RNAse protection techniques. The promoter fragment was fused to the E. coli uidA reporter gene encoding the B-glucuronidase (GUS) enzyme and introduced into transgenic Nicotiana tabacum (tobacco) and Lotus corniculatus. In transgenic tobacco, the gIn-a promoter directed uidA expression in the root-tip, in incipient lateral roots and in emerging lateral roots of all the transgenic plant lines studied. The promoter also directed uidA expression in the root vascular tissue and in the root hairs of a proportion of the lines studied. With the exception of root hairs, the promoter conferred a similar pattern of expression in transgenic L.corniculatus roots and expression of uidA was also observed in senescing mature nodules and possibly in incipient nodules. Expression of gigA was absent from young and mature transgenic nodules. Activity of the gln-a promoter was also associated with several tobacco flower structures including anthers, ovary placenta and vascular tissue, ova, pollen and the stigma and style. Several developing tobacco fruit structures including the placenta, vascular tissue, the developing seed cotyledon and the developing seed coat were also associated with gIn-a. promoter activity. The level of extractable leaf GUS activity was observed to increase between 2- and 5-fold 24 hours after mechanical wounding, relative to non-wounded transgenic leaf tissue. Although twenty four hours after wounding, uidA expression was not associated with those leaf tissues adjacent to the wound site, a higher proportion of wounded leaves showed uidA expression associated with the vascular tissue than in nonwounded leaves. The extractable leaf GUS activity of control cauliflower mosaic virus 358 promoter uidA transgenic plants were seen to decrease up to 50% 24 hours after mechanical wounding relative to non-wounded leaf tissue. Possible physiological functions for gln-a gene expression are discussed.

Potato virus Y (PVY) is considered to be the most serious viral pathogen that affects potato crops worldwide and can cause substantial yield losses. PVY exists as a complex of strains that can be distinguished on the basis of their biology, serology and genome analysis. In recent decades novel recombinant PVYN strains have emerged that can cause Potato Tuber Necrotic Ringspot Disease (PTNRD). It is therefore important to understand the potential threat to the Scottish seed potato industry. This molecular nature of PVY isolates in Scotland was established through the use of partial sequencing, revealing a predominance of isolates belonging to the molecular EU-NTN clade (ca 75%). Assessing the biological characteristics of selected isolates indicated that most isolates in Scotland belong to the biological PVYN type, however PVYE is also present. Molecular analysis of a PVYE isolate has shown that identifying the molecular determinants for vein necrosis production in tobacco is complex. Although it has not been reported from the field in Scotland, PTNRD initiation is possible with most PVYN isolates under optimal climatic conditions. Field trials suggest that PVYEU-NTN is more efficiently transmitted by aphids across a growing season than PVYNA-NTN and PVYO, with a higher than expected proportion of tubers infected with the PVYEU-NTN isolate. This suggests that once plants are inoculated with the virus, PVYEU-NTN isolates are more likely to infect progeny tubers. Taken together, the outcomes of this project should provide a better understanding as of PVY molecular nature in Scotland its pathogenicity and epidemiology with the view to understanding why PVYN variants have become an important threat for the seed potato industry both in Scotland and worldwide.

Cassava brown streak disease (CBSD) threatens cassava production in eastern and southern African countries. Diagnostic protocols currently available for the causal agents of CBSD, Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV), were unreliable but were urgently needed. In this study, sampling procedures and diagnostic protocols were developed for accurate and reliable detection of both CBSV and CBSUV. The cetyltrimethylammonium bromide (CTAB) method of RNA extraction was optimized for sample preparation from infected cassava plants and compared with the commercial kit RNeasy (Qiagen) for sensitivity and reproducibility. Results showed that both protocols were reliable but CTAB was more cost-effective and ideal for resource-poor laboratories. Mixed infections of cassava mosaic begomoviruses (CMBs) that cause cassava mosaic disease (CMD), CBSV and CBSUV have become more common with the recent spread of CBSD at mid-altitudes. A multiplex PCR for the simultaneous detection of viruses that cause both diseases, the first of its kind for cassava, was therefore developed to detect CBSV and CBSUV along with the three commonly occurring CMBs (African cassava mosaic virus (ACMV)), East African cassava mosaic virus (EACMV), and East African cassava mosaic virus-Uganda (EACMVUG) in eastern Africa. Similarly, a duplex PCR was developed for the simultaneous detection of CBSV and CBSUV, both viruses being detected in field-collected samples from Tanzania and Kenya. The genetic diversity of more than 40 CBSD isolates from Kenya, Tanzania, Uganda, and Mozambique was further examined by sequencing the coat protein (CP) gene and partial HAM1 gene sequences. The phylogenetic tree clustered the CBSD isolates into two groups reflecting the two virus species causing CBSD. In this study, various strategies were carried out for generating infectious clones of CBSV; gateway cloning, in vivo and in vitro transcription methods, and amplification of the viral genome in three fragments. Although 3 overlapping CBSV fragments were successfully cloned, the presence of an unexpected mutation at one of the cloning sites unfortunately did not allow reassembling of the fragments to construct the full-length cDNA.

Yam (Dioscorea spp.) is an important staple food crop in Sub-Saharan Africa and is vegetatively-propagated. This had led to the accumulation of viruses decreasing yam production and hindering international movement of selected germplasm. This study was to determine the prevalence and diversity of yam badnaviruses, as well as determine if badnavirus sequences are also integrated in the genomes of West African yam breeding lines. DNAs were extracted from Nigerian yam leaf samples (177 breeding lines, 78 landraces), using an optimised CTAB-extraction method and then screened using degenerate badnavirus-specific PCR primers targeting a 579 bp RT-RNaseH region. All 255 yam samples (100%) tested badnavirus PCR-positive. Denaturation gradient gel electrophoresis (DGGE) analysis of these PCR products revealed 24 discrete bands in total. Sequence analysis of the bands confirmed they were typical of the genus Badnavirus and a nucleotide diversity of 1-37% in this partial RT-RNaseH region representative nine of badnavirus species group. To determine which sequences were from episomal infections, rolling circle amplification (RCA) was performed on samples, and three complete genome sequences of yam badnaviruses were amplified, cloned and sequenced. Two of these full viral genome sequences (7258 and 7538 bp) of D. rotundata origin represent new species in the genus Badnavirus and the third (7529 bp) from D. alata represented an isolate of Dioscorea bacilliform AL virus. The three new genomes shared nucleotide identities of 68.3-70.5% and demonstrated a typical size and organisation of yam badnaviruses. PCR-based assays were developed for the detection of the five yam badnavirus genomes, and for the detection of three putative badnavirus species groups (K08, K09 and U12) that contain integrated sequences. Southern hybridisation results using individual DGGE band partial RT-RNaseH sequences (NGb4_Dr, NGb5_Dr and NGb6_Dr), supported integration of badnavirus sequences in genomes of D. rotundata breeding lines. Fluorescent in situ hybridisation (FISH) results using badnavirus complete and partial cloned genome sequences as probes were inconclusive for the yam samples tested. The consequences of the integrated and episomal badnavirus sequences for yam improvement programmes in West Africa are discussed.

The research seeks to understand the virus-host plant interactions for cassava brown streak disease (CBSD) caused by two viruses, Cassava brown streak virus (CBSV) and Ugandan Cassava brown streak virus (UCBSV) of the genus Ipomovirus, family Potyviridae. The diversity of six CBSD isolates from the endemic (Kenya, Malawi, Mozambique and Tanzania) and the recently developed epidemic areas (Uganda) of the disease in eastern Africa was studied. Five cassava varieties differing in virus resistance levels; Albert, Columbian, Ebwanateraka, TMS60444 (all susceptible) and Kiroba (tolerant) were graft-inoculated with the UCBSV and CBSV isolates. Based on a number of parameters, the isolates can be grouped into two main categories; severe and milder forms. Transmission of viruses using non-vector modes confirmed that CBSV was sap transmissible from cassava to cassava. Graft-inoculation of infected scions onto CBSD-free cassava plants was the most efficient mode of transmission which resulted in 80 and 100% rate for UCBSV and CBSV respectively. The two virus isolates were not transmitted through contaminated tools and hands. The effect of host-tolerance on virus was investigated in a long-term experiment where three cassava varieties Albert, Kiroba and Kaleso (field-resistant to CBSD) were graft-inoculated with UCBSV and CBSV. The three cassava varieties showed differences in virus movement, symptom development, severity and relative virus titres. The mechanisms of resistance to CBSD were investigated by making cuttings, from various parts of the plants, and a greater number of disease-free plants were generated from cuttings made from Kaleso than Kiroba and Albert. The fecundity of B. tabaci and its ability to transmit the virus were determined and results indicated no significant differences in the ability of the three cassava varieties to support whitefly development. Finally, thermal and chemical treatments of tissue cultured plants were conducted and the combinations of both treatments produced the greatest number of disease-free plants in all three varieties; Kaleso (50%), Kiroba (44%) and Albert (35%). The information generated in this thesis has greatly improved our understanding of the interactions between the three biotic factors; the host, virus and vector in the CBSD-pathosystem, which would be highly useful in designing effective disease management strategies.

In 2009, a sweet potato begomovirus (sweepovirus) was detected for the first time in Uganda. An isolate was sequenced, providing the first full sequence of a sweepovirus from mainland Africa which differed from other sweepoviruses by at least 13%, discriminating this isolate as a new species, ‘Sweet potato leaf curl Uganda virus’ (SPLCUV). SPLCUV was quite common in cultivars (cvs) Ejumula, New Kawogo and 318L having uneven distribution in infected plants and reversion to healthy occurred, especially in cv New Kawogo. SPLCUV was observed not to be synergised by Sweet potato chlorotic stunt virus (SPCSV), apparently making it the first report of a sweet potato virus not synergised by SPCSV. Besides SPLCUV, a ‘mild’ SPCSV strain that induced purpling symptoms and 50% yield reduction similar to wild type SPCSV when infecting alone was identified from Busia district, Uganda. ‘Mild’ SPCSV was never observed to be co-infected with Sweet potato feathery mottle virus (SPFMV) in farmers’ fields. Experimentally, ‘mild’ SPCSV induced mild symptoms in Ipomoea setosa and sweet potato plants and SPFMV titre was greater in co-infections of SPFMV and wild type SPCSV than in co-infections of SPFMV and ‘mild’ SPCSV. Both RNase3 (accession No. HE575406) and p22 (accession No. HE575409) genes on RNA1 of ‘mild’ SPCSV compared closely to those reported previously. Instead, RNA1 region appears to be expressed less in the ‘mild’ SPCSV infection than in the wild type SPCSV infection though RNA2 continues to be more expressed in the ‘mild’ SPCSV infection than in the wild type SPCSV infection. Recovery from SPVD symptoms and reversion from SPFMV were observed in cv Kampala White co-infected with ‘mild’ SPCSV and SPFMV. Reversion from SPFMV single infections occured in several landraces with higher rates observed in shoots of resistant than susceptible cultivars. Overall, cv NASPOT 11 was the fastest to revert while cv Beauregard was the slowest.

Wheat grain yield is strongly related to nitrogen (N) fertiliser input, a major cost factor and potential environmental pollutant. Much of the grain N requirement is met by N remobilisation from the canopy. Unfortunately, a consequence is canopy senescence and decreased photosynthetic capacity, reducing carbon available for grain-filling. One approach to achieve both higher N use efficiency and grain yield would be to extend the duration of photosynthesis using delayed leaf senescence “stay-green” phenotypes. Three stay-green and two fast-senescing EMS mutants of wheat (cv. Paragon) were characterised. A fast-senescing line, a stay-green line and the wild-type were grown to characterise the interaction between senescence and N availability. Stay-green line SG3 was able to allocate similar proportions of N to the grain under N-limiting and N-sufficient conditions. The accelerated senescence of line FS2 reduced grain yield and N allocation to the grain. Candidate regulatory genes of leaf senescence genes were characterised by correlating their expression with leaf senescence by screening wheat genotypes with varied senescence characteristics in the field. Among the genes were members of the WRKY and NAC transcription factor families that have been related to senescence. Overexpression of the NAC gene resulted in a stay-green phenotype and increased grain N concentrations, but had no effects on shoot biomass or grain yield. Expression of a WRKY-RNAi construct did not reduce WRKY mRNA levels, but led to accelerated leaf senescence and increases in plant height, the number of fertile tillers and grain yield. These results show that the relationships between senescence, nitrogen remobilisation and grain yield are complex and not easily manipulated. The phenotypes and genes identified could contribute to wheat improvement.

Cut flower production in Colombia has grown dramatically over the past forty years; meanwhile in the last decade consumption in the UK has almost doubled. Gender plays a central role in the industry since women provide the main labour force for production and are the main buyers and recipients of cut flowers. Conditions in the sector are characterised by widespread violations of labour rights and there is considerable academic and activist research in this area. However, the emphasis is on material factors with little attention paid to a cultural analysis of production. Moreover, analyses of production tend to ignore aspects of consumption in the North. Cut flowers have a double life, as commodities and as gifts. They are material things that are shaped, made, cultivated and are replete with meanings; they are 'cultural' in the full sense of the word. This idea is central to the thesis, which combines a cultural and material analysis of interview material and secondary material gathered in Colombia and the UK. I examine the social relations of cut flower production and consumption and the prospects for cut flower campaigns to secure better living standards and working conditions for Colombian workers. The research shows that little is known among ethical consumers in the UK about the characteristics of cut flower production. I argue that the positive meanings ascribed to flowers as gifts (love, sympathy etc. ) obscure the processes under which the flowers are produced. In production, I show that actors have differing understandings of the cycles organising the labour process, depending on their position in the production hierarchy. Discourses around the 'backwardness' of workers are used by owners and managers to exclude workers from decisionmaking. These discourses are also used to co-opt aspects of the ethical trade language to implement a 'home-grown' programme, Florverde, one that I argue serves the interests of cut flower entrepreneurs and not of labour. The thesis concludes by considering the prospects for ethical cut flowers from Colombia.

Research objectives were to study dormancy/sprout control in potato and sweetpotato, and to identify novel sprout control strategies. Consistent with sprout stimulation by gibberellins (GAs) in potato, 10 mM GA3 enhanced sprout length and number in sweetpotato roots and sprout growth was decreased by 20 ml/L piccolo (GA synthesis inhibitor). Continuous application of 10 ppm ethylene or greater prevented sprouting in sweetpotato roots over 4 weeks storage at 25° C. Sprout growth was also inhibited by 1000 ppm aminoethoxyvinylglycine (AVG) (ethylene synthesis inhibitor) or 625 ppb 1-methylcyclopropene (1-MCP) (ethylene antagonist). Continuous ethylene treatment or single 1-MCP treatment could be a practical sprout control method for sweetpotatoes stored at >15° C. 5 ppm or greater ethylene increased root respiration rates, but this effect was reduced by 1-MCP or AVG. Sugar content in ethylene treated roots +/- 1-MCP or AVG were lower than untreated roots, with lowest levels in roots treated with ethylene alone. Hormonal control of dormancy/sprout growth was studied in excised buds from potato tubers transformed to over-express a bacteria gene encoding 1-deoxy-D-xylulose 5 phosphate synthase (DXS), which exhibit arrested buds, Five weeks post-harvest over-expressing lines, DXS1 and DXS2, showed greater sprout growth compared to wild type when treated with 1 mM GA3 or tZR in 2009, whereas DXS1 showed less sprout growth 4 weeks post-harvest in 2007. There was no difference in DXS1 and DXS2 behaviour 4 months after harvest in 2007. The different behaviour of DXS1 over seasons and with time from harvest underlines how tuber state can change with maturity. No consistent differences were found in chemical profile of peel among potato tubers of accessions with a range of dormancy characteristics. Although 1, 4-Dimethylnaphthalene has been identified as a natural sprout suppressant, it could not be detected in any accession even though measurements were sensitive to below 100 ppb.

A three-year study on the practice of ratooning of sorghum was conducted in Eastern Kenya (1999 to 2002), with emphasis on the stem borer (Chilo partellus) and covered kernel smut (Sporisorium sorghi Ehrenberg Link). Ratooning is the practice of stimulating tillering by cutting the old straw after harvest (Doggett, 1988). A six season on-station experiment in Machakos District showed the practice of ratooning short duration sorghum increased the reliability and yields in comparison to a direct sown with yield ranges of 1630-1778kg/ha and 0-148kg/ha, respectively. The higher number of heads and stems per unit area meant the ratooned crop had higher level of stem borers per unit area than the direct sown crop and when infected with covered kernel smut was a greater source of inoculum. Unlike the incidence of CKS, the number of stem borers had little correlation with the numbers in the previous season’s crop; there was no upward trend to the number of stem borers per stem during the experiment suggesting factors other than the presence of a sorghum crop have a stronger influence on the population. Yield loss was an interaction between cultivar*incidence of stem borers * stage of infestation * rain quantity and distribution, but rain was the most important factor. An on-station trial in Kitui (2001-2) found the ratooned crop outperformed the direct sown crop in yield by a factor of three and non-cutting of stems produced a similar yield to cutting back stems after harvest. The different ‘ratoon’ methods did not significantly affect the incidence of covered kernel smut or level of stem borers. On-farm trials in Mwingi District (2000-2) showed that short duration sorghum ratooned outperformed direct sown sorghum. The timing of the cutting back of the stems had an effect on plant survival and yield; cutting back stems at harvest produced higher yields than cutting the stems at the on-set of rains, however cutting the stems at the onset of rains increased plant survival when the stems were dry at harvest. A decision tree was produced outlining the decisions a farmer needs to make when deciding whether or not to practice ratooning. Four factors were identified as important for varieties to perform well under the practice of ratooning: drought tolerance, stem strength, non-senescence and the ability to produce tillers during growth stage 3.