Coffee is an important beverage product in many parts of the world. During the production and processing of coffee the prevailing environmental factors and rate of drying can have a profound influence on colonisation by mycotoxigenic fungi and contamination with ochratoxin A (OTA). In Kuwait, coffee beans are imported from various parts of the world. The objectives of this project were to (a) to examine the diversity of mycotoxigenic fungi found in green and roasted coffee beans bought in the Kuwaiti market from different source countries and identify the dominant fungal populations, (b) to examine the ecology and ochratoxin A production by the ochratoxigenic strains and species isolated, (c) determine the effect of caffeine concentrations in vitro on growth and OTA production by strains from the Aspergillus section Circumdati and Section Niger groups, (d) evaluate the impact of interacting climate change factors (water activity (aw) x temperature x elevated CO2) on growth and OTA production in vitro and in situ, (e) determine the effect of aw and temperature interactions on ecology of strains of two new species, A. aculeatinus and A. sclerotiicarbonarius, isolated from coffee beans and (f) evaluate the efficacy of gaseous ozone (O3) for controlling OTA producing fungi and control of contamination in coffee beans after treatment and after storage. Cont/d.

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Cacao (Theobroma cacao L.) is widely farmed throughout the humid tropics where it is an important crop to smallholder farmers. Climate change projections suggest increased temperatures and altered rainfall patterns in these regions. The effects of two key climate change variables; elevated carbon dioxide (CO2) concentrations and water deficit were investigated on the reproductive development of six genotypes of cacao (CL19/10, ICS 1, IMC 47, Pound 7/B, SCA 6, and SPEC 54/1). Genotypes showed variability in their response to treatments; however general observations were also made. At elevated CO2 (700ppm) the mean length of pollen tubes decreased and the degree of fluctuation in flowering intensity increased over time without any overall reduction in flower production. Final pod size and maximum rate of growth increased in response to elevated CO2 in the second year of study. Additionally in the second year there was an increase in husk weight and thickness, and individual bean weight. There was no increase in bean number or shell percentage. Total cocoa butter content and percentage unsaturated fat was lower under elevated CO2 along with reductions in stearic, oleic and linoleic fatty acid content; however these responses varied between genotypes and sampling period. Final woody biomass increased under elevated CO2 whereas leaf biomass was unaffected. Water deficit stress had no observed effect on pollen performance, flowering behaviour, or cocoa butter content and composition. Final pod size and rate of growth was reduced under water stress in the first year of study only. The reduced pod size was reflected in reduced husk and bean weights. Final wood and leaf biomass were both reduced in response to the water stress treatment. Across all parameters measured, no evidence of interaction between elevated CO2 and water deficit stress was observed. Overall, these results demonstrate the potential for yield improvement in cacao through breeding in preparation for future climate scenarios.

‘Molecular farming’, the use of transgenic plants to produce biopharmaceutical proteins is emerging as a new biotechnological paradigm. Transgenic plants offer several advantages over conventional microbial and mammalian cell host technologies. In particular, transplastomic plants, with transformed plastid genomes, are capable of massive expression of foreign proteins and represent a promising platform for biopharmaceutical synthesis. The main theme of this PhD thesis is the investigation of in vitro regeneration of tobacco (Nicotiana tabacum) shoots from callus tissue in temporary immersion (TI) culture for heterologous biopharmaceutical synthesis. There is special emphasis on subunit vaccine expression in transplastomic tobacco, in which foreign protein accumulation is correlated with chloroplast number and development during the organogenesis process. Studies using transplastomic N. tabacum expressing TetC (tetanus toxin fragment C) investigated the influence of several culture parameters on biomass regeneration and recombinant protein expression. The parameters investigated include medium nitrogen source ratio, sucrose concentration and hydrodynamics. These studies highlight the sensitivity of transplastomic protein yields to the culture microenvironment, and provide a starting point for further optimisation. Further studies demonstrated the feasibility of TI culture for biosynthesis of proteolytically-unstable transplastomic subunit vaccines, p24 (HIV antigen) and VP6 (rotavirus antigen). TI culture is also demonstrated as a means for nuclear expression of functional Guy’s 13 monoclonal antibody. Finally, the use of TI culture as the basis of novel technological innovations is investigated. This includes the demonstration of transplastomic protein expression in a prototype large-scale mechanical temporary immersion bioreactor. Encapsulation of callus aggregates in an alginate matrix for long-term germplasm preservation was trialled, prior to temporary immersion regeneration. Overall, this work presents a novel in vitro propagation method for the contained large-scale biosynthesis of biopharmaceutical proteins, as a potential alternative to conventional plant propagation platforms based on agricultural cultivation or cell suspension culture.

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Water availability is predicted to be reduced and temperature to rise in the global climate change context. Future climate conditions may thus represent a serious risk for coffee cultivation especially in less favorable environment. Agroforestry has been postulated as a promising strategy to adapting to climate changes. Shade tree may minimize radiation and temperature near the soil surface and reduce soil evaporation. Shade tree may enhance infiltration, reduce runoff and increase rainfall water use efficiency by taking up water from deep soil layers. However, shade tree may reduce the water that reaches the soil by rainfall interception. Trees may consume additional water and can establish a competitive relationship depending on tree species characteristics, soil water availability, site conditions and management. In this experiment water dynamics and use was monitored in a mature agroforestry experiment where coffee shaded by a mixture of Tabebuia rosea and Simarouba glauca is compared to full sun coffee over 2012 and 2013. The water balance was obtained by two independent approaches: 1) measuring directly all components of water balance (trees and coffee transpiration; soil evaporation; rainfall interception); and 2) measuring changes in the soil water stock through Time Domain Reflectometers (TDR) probes. Agroforestry (AFS) showed greater transpiration and lower soil surface evaporation compared to full sun (FS). Shade tree did not represent a serious constraint for coffee water use during most of the period of the experiment. Coffee water consumption represented 75% of the total transpiration in agroforestry while Tabebuia rosea transpired 17% and Simarouba glauca 8%. Complementarity was demonstrated by root niche differentiation between coffee and Simarouba glauca that seemed to be more suitable as coffee shade tree compared to Tabebuia rosea. We also demonstrated high competition between coffee and shade tree when an atypical very dry season occurred. Transpiration was stabilized although the high evaporative demand and coffee leaf water potential reached its lowest value in AFS which suggested high level of coffee water stress. Adaptation strategies for coping with climate change using shade trees need to be devised taking into account this quantified information.

The tomato lat52 gene encodes an essential cysteine-rich protein required for normal pollen tube growth which is preferentially transcribed in the vegetative cell during pollen maturation. The identity, organisation and role of cis-regulatory elements which control the development and cell-specific expression of lat52 were investigated in detail. A series of 5' promoter deletion mutants fused to the E. coli -glucoronidase gene uidA (gus) were stably introduced into tobacco. Detailed analysis of the accumulation of GUS activity during anther development demonstrated that lat52 promoter activity during early to mid-pollen maturation was regulated specifically by the region -492 to -101.;Transgenic studies and transient expression analysis demonstrated that the major cis-regulatory elements responsible for high-level pollen-specific transcriptional activity were located between -492 and -42. Fusion of the region -492 to -52 to a minimal CaMV35S promoter enhanced reporter gene expression in a pollen-specific manner. This gain of function approach was used to show that the previously defined sequence motifs PBII and PBIII did not contribute to pollen-specific transcription in a germinating pollen. Analysis of the region -100 to -52 led to the identification of two novel co-dependent cis-regulatory elements within the minimal pollen-specific activator unit -71 to -52.;In addition to multiple upstream cis-regulatory elements which control the level and specificity of lat52 transcription, the 5' untranslated region (5'-UTR) conferred a dramatically increased translational yield to heterologous transcripts in a pollen-specific and highly developmentally regulated manner during the final stages of pollen maturation in transgenic tobacco. Taken together, these data suggest that lat52 expression is developmentally regulated in a tri-phasic manner during tobacco pollen development.

This work investigates differences in the survey methodologies of the monitoring programmes of the United Nations Office on Drugs and Crime (UNODC) and the US Government that lead to discrepancies in quantitative information about poppy cultivation. The aim of the research is to improve annual estimates of opium production. Scientific trials conducted for the UK Government (2006–2009) revealed differences between the two surveys that could account for the inconsistency in results. These related to the image interpretation of poppy from very high resolution satellite imagery, the mapping of the total area of agriculture and stratification using full coverage medium resolution imagery. MODIS time-series profiles of Normalised Difference Vegetation Index (NDVI), used to monitor Afghanistan’s agricultural system, revealed significant variation in the agriculture area between years caused by land management practices and expansion into new areas. Image interpretation of crops was investigated as a source of bias within the sample using increasing levels of generalisation in sample interpretations. Automatic segmentation and object-based classification were tested as methods to improve consistency. Generalisation was found to bias final estimates of poppy up to 14%. Segments were consistent with manual field delineations but object-based classification caused a systematic labelling error. The findings show differences in survey estimates based on interpretation keys and the resolution of imagery, which is compounded in areas of marginal agriculture or years with poor crop establishment. Stratified and unstratified poppy cultivation estimates were made using buffered and unbuffered agricultural masks at resolutions of 20, 30 and 60 m, resampled from SPOT-5 10 m data. The number of strata (1, 4, 8, 13, 23, 40) and sample fraction (0.2 to 2%) used in the estimate were also investigated. Decreasing the resolution of the imagery and buffering increased unstratified estimates. Stratified estimates were more robust to changes in sample size and distribution. The mapping of the agricultural area explained differences in cultivation figures of the opium monitoring programmes in Afghanistan. Supporting methods for yield estimation for opium poppy were investigated at field sites in the UK in 2004, 2005 and 2010. Good empirical relationships were found between NDVI and the yield indicators of mature capsule volume and dry capsule yield. The results suggested a generalised relationship across all sampled fields and years (R2 >0.70) during the 3–4 week period including poppy flowering. The application of this approach in Afghanistan was investigated using VHR satellite imagery and yield data from the UNODC’s annual survey. Initial results indicated the potential of improved yield estimates using a smaller and targeted collection of ground observations as an alternative to random sampling. The recommendations for poppy cultivation surveys are: the use of image-based stratification for improved precision and reducing differences in the agricultural mask, and use of automatic segmentation for improved consistency in field delineation of poppy crops. The findings have wider implications for improved confidence in statistical estimates from remote sensing methodologies.

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