In two pot experiments using two different crop ryegrass (Lolium perenne) and two flax (Linum usitatissimum) varieties Viola and Elise, ryegrass decreased in the pool of heavy metals compared with bare soil using EDTA as extractant. NH4+ decreased the soil pH, increased EDTA-extractable Zn and increased the Zn uptake. Lime addition increased the pH and depressed Zn uptake. The pool of extractable EDTA was not changed by growing both of the flax varieties. Lime increased EDTA-extractable Cu and Pb significantly, but decreased the Zn, and pH increased in this order NH4+<NO3-<NH4++lime<NO3-+lime. The EDTA –extractable Cu decreased in the order NO3-+lime>NH4++lime>NH4+>NO3-. Ammonium decreased the pH more than other treatments. In agar using Bromocresol purple indicator NH4+ increased the pH in the rhizosphere of different plants. With two different initial pH treatments (7 and 3.2) the NH4_ decreased the pH in the rhizosphere at high initial pH 7 and maintained the low pH at initial pH 3.2 to 4 against the buffer capacity. At different initial pH 4, 5, 6, 7 and 8 the ammonium decreased the high pH and maintained the low pH, but NO3- had no effect on the pH. Ammonium increased the toxicity of Zn due to pH decreases. There was no effect of both nitrogen sources NH4+ or NO3- on rhizosphere pH when applied as a foliar application. These indicated that the NH4+ can decrease the pH in the rhizosphere of plants and could play an important role in manipulation of the rhizosphere bioavailability of heavy metals. Toxicity of the three metals is Cu>Pb>Zn in this order and the crops tolerance is following this order pea>flax>barley. An agar-Hoagland nutrient solution contaminated with two soils, sewage treated soil (SBS) and galena soil (G), was used with flax as a test crop. The ammonium treatment lowered the pH in both soils, but with galena treated greater than SBS soil, this is attributed to the buffering capacity of the SBS soil. Averaged over all the concentrations the NF4+ treatments resulted in higher Zn shoot content that NO3- treatment, while in Cu shoot content nitrate was more than ammonium. The transfer factor of lead with ammonium treatment was greater than nitrate treatments at the 0.1 and 0.25% galena and the transfer factor of the Zn and Pb more than Cu in all treatments. At high initial pH 8 and high concentration of Zn and Cu barley grew well and this is attributed to immobilisation of Zn and Cu compared with low pH 5 and 6.5 where the barley plant did not survive. Ammonium lowered the high pH 8 and caused lower biomass production of barley than nitrate.

This thesis describes the observations of field trials and analytical investigations of the residues of maleic hydrazide (1,2-dihydro-3,6-pyridazinedione) known as MH. MH is marketed in a formulation called `Fazor' and was introduced to the United Kingdom under PSPS (Pesticide Safety Precaution Scheme) in 1985. It is targeted at sprout suppression of ware tubers in store with the added benefits of increased marketable yield and reduced groundkeepers. Two field trials were conducted to investigate the sprout inhibition of MH and its effect on yield. Both field trials used a popular ware seed (c.v. Maris Piper) grown under typical growth conditions and treated with Fazor(5 kg/500 litres/hectare) under optimum conditions. Treated potatoes were observed to have typical characteristics of a break in apical dominance. Every eye on the tubers was active and formed spikelets that developed into growths similar in appearance to a small cauliflower head. It was also noticed that sprout suppression was not complete. A proportion of potatoes had inadequate suppression. Some showed no characteristics of MH, typical of an untreated potato. The inadequate sprout suppression was investigated by analysing the MH distribution and the comparison of sprout suppression. MH treated plots were found to have slightly lower yields than untreated plots. This reduction in yield was not shown to be statistically significant. Two methods to calculate the residues of MH were adapted. The first used the action of zinc in concentrated sodium hydroxide to produce hydrogen, which reduced and hydrolysed MH to hydrazine. The hydrazine was distilled and caught in an acid scrubber containing a colour reagent. The colour reagent and hydrazine combined to produce a coloured chromophore. The colour intensity was proportional to the amount of MH. This method was used to calculate the total MH. The second method calculated the amount of free MH (not metabolised or conjugated) by a methanol extraction, cleanup of the concentrate and analysis by high pressure liquid chromatography (HPLC). The total MH was roughly made up of three factions:- (1) Non-extractable MH (2) Methanol extractable MH (a) free MH (b) metabolised MH The uptake of MH over a four week period, after spraying Fazor, was assessed by harvesting at weekly intervals for four weeks and analysing the tubers by the above methods. The distribution of MH between individual tubers was investigated by calculating the MH in whole tubers. The distribution within individual tubers was calculated by analysing the skin, the outer flesh and the inner core. The distribution of MH between grades was calculated. The carryover of MH into processed foods was calculated by analysis of crisps and boiled potatoes from field treated plots. It was found that the MH accumulated in the potato tubers before one week and remained static up to four weeks. However, there was a fraction, a possible metabolite, that increased in concentration, whereas the free MH concentration decreased. Sprout suppression was found to be dependent on the total MH. Small tubers had lower concentrations than larger tubers. MH was found throughout the whole tuber with a slightly lower concentration in the core. MH was also found in fried and boiled potatoes and was identified as free MH. The metabolites of MH were investigated by applying radiolabelled MH to greenhouse grown potato plants. The potatoes were extracted by methanol and the concentrated extracts separated by thin layer chromatography (tlc). A metabolite was detected after exposing the plates to X-ray photographic plates. However, this metabolite was not successfully identified. This was due to the combination of low specific activity of the radiolabelled material, and the low quantity of metabolite produced. Hydrolytic enzymes were used to cleave possible conjugated metabolites in the potato juice concentrates. The action of a beta-D-glucosidase was found to increase the free MH. This would appear to suggest that a metabolite of MH is a conjugated product between MH and a glucose molecule. However, a conjugate could not be synthesized between uridine diphospho glucose (UDPG) and MH with soluble potato protein. There was no evidence to suggest that a metabolite is produced between MH and glutathione. It was suspected that MH would inhibit wound healing because it inhibits cell division. Cut potatoes treated with various concentrations of MH were examined histologically to assess cell division and suberin production. The rate of water loss from potato discs treated with MH was calculated. If the rate of water loss does not slow down because of chemical inhibition of the process, potatoes will lose weight and condition in store. No difference between control potatoes and MH treated potatoes was found at the beginning of a storage season. At the end of storage (four months), MH treated tubers had better wound healing than controls.

Aspects of the biology, epidemiology and control of Rhizoctonia solani from potato were investigated using a range of laboratory and field-based experiments. In vitro experiments revealed nutritional factors including a range of carbon sources, and inorganic and organic nitrogen did not affect significantly mycelial growth or sclerotial germination. Carbon and nitrogen sources including cellobiose, glucose, glycerol and potassium nitrate significantly increased sclerotial biomass production in vitro. Mycelial growth, sclerotial production and germination occurred over a temperature range of 10-30oC, with an optimum of 25oC for both AG 2-1 and AG 3 isolates. Mycelial growth and sclerotial germination occurred at pH 4-9 with an optimum of pH 5.6, whereas sclerotial production occurred between pH 4-6 for AG 2-1 isolates and pH 4-8 for AG 3 isolates. Mycelial growth, sclerotial biomass production and germination declined with decreasing osmotic, matric and soil water potential, with mycelial growth prevented between -3.5 MPa and -4.0MPa on osmotically adjusted media, at -2.0 MPa on metrically adjusted media and -6.3 MPa in soil. Sclerotial production ceased prior to the limits for mycelial growth and germination for all isolates, between -1.5 MPa and -3.5 MPa on osmotically adjusted media and -1.5 MPa on metrically adjusted media. AG 3 isolates produced significantly more well-formed sclerotia during all in vitro experiments compared to the loosely constructed sclerotia produced by AG 2-1 isolates. A pathogenicity bioassay, coupled with staining and microscopic examination of stem tissues, showed all AGs formed infection cushions as a prerequisite to infection, with clear differences in the extent of infection cushion formation and subsequent stem lesion severity. AG 2-1 produced small, infrequent infection cushions, causing stem lesions only 1-2 mm in length which did not increase in size or severity after initial formation.

Work described in this thesis was designed to address a number of environmental issues relating to the use of chlorpropham (CIPC) as a sprout suppressant in potato stores. Studies considered the behaviour of CIPC during the application process and storage and also the potential for it to be released into the environment. All commercial studies were carried out in box stores, rather than bulk stores. A survey of potato growers was carried out to provide up-to-date industry information on store management practice and to determine the extent of chlorpropham use in the UK. Results confirmed that the vast majority of crop held in the UK for both pre-packing and processing is treated with chlorpropham. In most cases, several applications are necessary to maintain sprout control throughout the season, which highlights the inefficiency of the application process. Thermal fog application (the industry standard) is known to be inefficient, and to result in uneven distribution of chlorpropham around the store. This can lead to unacceptably high chemical residues in crop at certain locations, and poor sprout control in places that do not receive the correct dose. The imminent introduction of a Maximum Residue Level (MRL) for chlorpropham means that store managers must be able to predict with confidence the amount of chemical reaching each tuber. Analysis of crop from commercial stores found chemical levels ranging from 0-50 mg kg-1 following conventional application. Washing significantly reduced these very high levels in most cases. Improvements in chemical distribution (and a lowering of the highest chemical levels) were seen when the movement of air and fog around the store were manipulated using fans or by restricting air flow using polythene sheeting. A method for the collection and analysis of air samples was developed and used to quantify CIPC in samples of air from treated stores. Vapour concentrations were found to be of the order of mg l-1 (parts per billion), and to increase linearly with air temperature. 3-chloroaniline (a metabolite of CIPC) was also identified in the air samples, suggesting significant breakdown of the CIPC molecule may occur during chemical application or storage. The mechanism of breakdown was not identified. Contaminated fabrics within the store are believed to provide a reservoir of chemical that can readily volatilise and be found in the vapour phase. The presence of chlorpropham in the air has implications for crop contamination and the extent of chemical loss from the store. Samples of effluent from potato washing plants were collected and analysed on several occasions. The CIPC concentration to liquid effluent (after removal of all suspended material) was found to range from several mg/l (parts per million) in untreated samples to <0.01 mg l-1 following filtration and digestion.

No description available.

In the model plant Arabidopsis thaliana, the circadian clock is believed to be composed of a number of coupled transcriptional negative feedback loops. The LATE ELONGATED HYPOCOTYL (LHY) gene is thought to form part of at least two of these transcriptional feedback loops, as well as playing a role in the perception of light signals by the clock. To better understand how multiple transcriptional feedback loops might be integrated in the transcriptional regulation of LHY, we have performed an analysis of the cis-regulation of this gene. Through deletion analysis of reporter gene constructs, we have identified a 957 basepair region of the LHY promoter which contains sufficient sequence to direct the characteristic expression profile of LHY. Furthermore, we provide evidence that at least two circadian signals converge on this region. Electrophoretic mobility shift assays identified four classes of candidate cis-elements within the LHY promoter including a poly-CTT tract, an AAAAA motif, a candidate MYB-binding site and a G-box motif. Through mutational analysis of these elements, we have been able to determine aspects of their in vivo regulatory function. We report that a G-box motif and the previously uncharacterized AAAAA element are implicated in the regulation of LHY transcription by light signals. In etiolated seedlings, the region of the LHY promoter containing the MYB-binding site motif and multiple copies of the poly-CTT motif mediates regulation of LHY by both light-responsive and circadian signals.

Post harvest discolouration in lettuce is an increasingly important problem due to the shift in the market for prepacked processed salads. Variation in post harvest discolouration was recorded in a lettuce diversity set of 28 accessions representative of the lettuce genepool. The parents of the WHRI lettuce mapping population, Saladin and Iceberg were included in the diversity set. They showed significantly different responses for discolouration and the difference between them was representative of a major part of the variation seen in the diversity set. F7 RILs derived from a cross between Saladin and Iceberg were suitable for genetic analysis of post harvest discolouration. As a precursor to the genetic analysis, a good quality linkage map based on the F7 Sal x Ice population was generated. Significant genetic variation in the post harvest response was demonstrated for these RILs. Twenty-one significant QTL were identified for post harvest discolouration traits, and the markers linked to the QTL can be used for marker assisted selection. Significant but weak correlations were recorded between discolouration and important agronomic traits, however as these were not highly correlated this means that post harvest discolouration and agronomic traits can generally be independently selected for by breeders without having to compromise on other traits. Research was also initiated to understand the metabolic changes underlying the phenotype change. Significant variation in levels of metabolites related to post harvest discolouration including phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and total phenolic content (TPC) was observed in RILs with extreme phenotypes. The differences in metabolite levels were significantly correlated with the discolouration phenotypes. Work was also initiated to identify candidate genes associated with the biosynthetic pathway responsible for discolouration (phenylpropanoid pathway) in an attempt to identify genes underlying QTL. Twenty-three genes have been placed on the Sal x Ice map using comparative genomic approaches. Some of these co-locate within the region of a discolouration QTL and are therefore candidate genes for the QTL effect. Mutants with altered post harvest discolouration phenotypes were also confirmed in this study; however the point of mutation could not be identified although it is thought to be downstream of PPO. This indicates that a desired phenotype with reduced levels of post harvest discolouration can be achieved by two approaches to breeding; using natural and induced variation. This study has provided the tools and knowledge to do this.

Predicted climate change and increasing global populations suggest that water will become an increasingly scarce and valuable commodity. Breeding plants which produce equivalent yields with reduced water input is therefore vital to sustain and increase crop production in the future. The phytohormone, abscisic acid (ABA), is important in controlling plant responses to water stress. It may be possible to improve water use efficiency (WUE) by genetically modifying tomato and other species to maintain elevated levels of ABA under optimal (unstressed) conditions, thereby manipulating an intrinsic signalling mechanism which is known to mediate drought-induced alterations of stomatal behaviour. ABA is synthesised via the oxidative cleavage of C40 epoxycarotenoid precursors, a reaction catalysed by the key enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). Pure breeding transgenic tomato lines constitutively over-expressing LeNCED1, known as sp5 and sp12, both have elevated ABA concentrations, which reduce stomatal conductance under optimal (unstressed) growth conditions, thus conserving soil water during periods when corresponding wild type (WT) control plants were inefficient in its use. Under well-watered conditions, whole plant transpiration efficiency (TEp) was significantly greater in both 'high ABA' lines (sp12 and sp5) than in WT plants. The over-expression of LeNCED1 was combined with over-expression of a gene (LeBCH2) encoding β-carotene hydroxylase (BCH), an enzyme acting earlier in the ABA biosynthetic pathway. These 'double transgene' lines (G28 and G29) consistently exhibited further improvements in ABA accumulation and TEp relative to corresponding 'single transgene' parental lines. Lines G28 and G29 respectively exhibited 37 and 54 % improvements in TEp relative to WT controls. When evaluated as a potential 'high ABA' rootstock, it was found that the 'double transgene' G29 line did not provide a sufficiently strong root-sourced signal to affect the stomatal behaviour of scions. To increase ABA biosynthesis in the roots further, a programme designed to combine the over-expression of three ABA biosynthetic genes (LeNCED1; LeBCH2; LePSY1) was initiated with the objective of obtaining a rootstock which produced sufficient ABA to affect stomatal behaviour when grafted onto WT scions. Unfortunately, there was insufficient time to complete this work by the end of the period reported in this thesis, although the programme is ongoing.

Turnip yellows virus (TuYV) is the most common and important virus infecting oilseed rape (Brassica napus) in the UK. It causes reductions in growth and seed yield in oilseed rape. Between 2007 and 2010, the prevalence of TuYV in oilseed rape crops in Lincolnshire, Warwickshire and Yorkshire was determined; incidences of infection ranged from 0 and 100%. The highest levels of infection were detected in Lincolnshire and the lowest in Yorkshire. Highest incidences were recorded during 2009-10 and the lowest in 2008-9. Incidences of TuYV were closely related to the flight activities Myzus persicae vector. Most fields showed slightly aggregated pattern of infection during autumn but spring sampling revealed more random patterns. Phylogenetic analysis of both nucleotide and amino acid sequences of the P0 and P3 genes of TuYY revealed three and two genetic groups of TuYV respectively, infecting oilseed rape in Lincolnshire, Warwickshire and Yorkshire. The P0 gene was more variable than the P3 gene and both were under purifying selection. TuYV populations in the three regions were highly structured with limited gene flow between them. Analysis of molecular variance (AMOVA) indicated 96- 97% of the observed variation was due to the variation between isolates within fields. Three RT-PCR assays were developed to differentiate the three genotypes. They successfully detected and discriminated isolates of the two major genotypes from oilseed rape in Lincolnshire. Twenty seven accessions of a B. napus Diversity Fixed Foundation Set (DFFS) screened for resistance against TuYV infections varied in their susceptibility to the virus. An accession Yudal had partial resistance to some but not all the isolates of the two major genetic groups tested. TuYV caused yield losses of up to 44.7% in a glasshouse experiment. A major QTL for the partial TuYV resistance was detected on chromosome C4 (N14), explaining up to 50.5% of the observed resistance.

The aim of the work presented in this thesis was to assess the genetic variation present within the wild tomato species Solanum pennellii that can be adapted to improve the texture of the domesticated tomato species. Using a population of S. pennellii introgression lines, 23 significant QTL supporting intervals were identified. Nine of the QTL were significant in two growing seasons. Three QTL were identified for pericarp firmness. Lines containing the firmness QTL F-Sp 2.1 were used to create BC1 populations. Sensory analyses were used to correlate the instrumental texture measurements to those perceived during mastication. Repeated texture measurements were conducted on lines representing similar chromosomal regions from another wild tomato species Solanum habrochaites introgression line population. To better understand the genetic basis of one of the firmness QTL identified, the Syngenta tomato Affymetrix GeneChip was used to quantify the differential expression of S. pennellii genes within the QTL-introgression line through development in comparison to the recurrent parent S. lycopersicum L. cv M82. The microarray analyses were extended to the ripening mutants Cnr, nor and rin. Differential gene expression between the ripening mutants and the wild type Ailsa Craig were compared through development. Candidate genes for the firmness QTL and fruit development were nominated.