Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 200-215). Also available on the Internet.

Thesis (PhD)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: Barry J. Jacobsen. Includes bibliographical references.

The propagation of wild-type virus and a transgenic viral vector was examined in cultured plant cells to identify factors affecting viral infection of and accumulation in cultured cells and to determine if viral vectors could be used to facilitate the expression of heterologous proteins in vitro. Tobacco mosaic virus (TMV) accumulation was examined in Nicotiana tabacum and N. benthamiana suspension and hairy root cultures. TMV accumulation was superior in N. benthamiana hairy roots. Hairy roots were infected by adding TMV to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by virus infection. The distribution of virus within root mats from shake-flask grown cultures was non-uniform and the concentration of virus accumulated in replicate cultures was variable. When N. benthamiana hairy roots were infected using 1.5 μg mL-1 TMV, the average maximum concentration of virus accumulated in the biomass was 1.6 ?? 0.25 mg g-1 dry weight, or 15-fold lower than in TMV infected N. tabacum leaves. Virus coat protein accumulated to a level of (26 ?? 10)% total soluble protein in the hairy roots. Inoculum virus concentration and the medium in which infection was performed affected the virus yield and the percentage of inoculated cultures that accumulated virus. When cultures were inoculated using 9.0 μg mL-1 TMV, virus accumulated in the biomass to 4.2 ?? 0.60 mg g-1 dry weight. Proportional scale-up of hairy root infection in shake flasks did not result in constant virus concentrations in the scaled cultures. TMV accumulation in bioreactor-infected and -grown hairy roots was poor. N. benthamiana hairy roots were infected with a TMV-based vector (30B-GFPC3) that encoded Cycle 3 green fluorescent protein (GFP). TMV-GFPC3 was (260 ?? 140)-fold less infectious than TMV as measured by local lesion assays. Propagation of TMV-GFPC3 could not be confirmed using mass balance. GFP was not detected in the infected hairy roots or the culture medium. Hairy roots represent a potentially viable culture-based system for the in vitro production of virus and virus products when field-grown agricultural systems do not adequately address issues of containment or product safety.

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 200-215). Also available on the Internet.

Thesis (Ph. D.)--University of Kentucky, 2004. / Title from document title page (viewed on June 22, 2006). Document formatted into pages; contains viii, 116 p. : ill. (some col.). Includes abstract and vita. Includes bibliographical references (p. 103-114).

Viruses are ubiquitous in natural growth environments and cause severe losses to crop yields, globally. Approximately 30% of plant viruses described to date are grouped within the family Potyviridae, making it one of the largest plant virus families. Furthermore, certain potyvirus species can cause devastating diseases in several agriculturally and economically important crops. Hence, gaining insight into potyvirus resistance and recovery mechanisms in plants is an important research focus. This thesis firstly explores how environmental cues can modulate the activity of a central form of viral defence, namely RNA silencing. Specifically, high temperatures and low light intensities were found to increase the efficacy of viral RNA silencing in Arabidopsis, resulting in recovery from infection by Turnip Mosaic Virus. The biological context and potential for agricultural exploitation of these phenomena are discussed. Secondly, this thesis explores the ability to engineer resistance alleles using the latest genome editing techniques. Specifically, resistance to Turnip Mosaic Virus was successfully engineered in Arabidopsis by CRISPR/Cas9-induced deletion of a known susceptibility factor eIF(iso)4E. Biotechnological methods to implement this proof of concept research in crop species were also investigated.

The biological degradation of lignocellulose into fermentable sugars for the production of liquid transportation fuels is feasible and sustainable, but equires a variety of enzymes working in synergy as lignocellulose is a complex and recalcitrant substrate. The cellulosome is a multi-enzyme complex (MEC) with a variety of cellulolytic and hemicellulolytic enzymes that appears to facilitate an enhanced synergy and efficiency, as compared to free enzymes, for the degradation of recalcitrant substrates such as lignocellulose and plant cell walls. Most of the studies on cellulosomes have focused on a few organisms; C. thermocellum, C. cellulovorans and C. cellulolyticum, and there is only limited knowledge vailable on similar complexes in other organisms. Some MECs have been identified in aerobic bacteria such as Bacillus circulans and Paenibacillus curdlanolyticus, but the nature of these MECs have not been fully elucidated. This study investigated the cellulolytic and emi-cellulolytic system of Bacillus licheniformis SVD1 with specific reference to the presence of a MEC, which has never been reported in the literature for B. licheniformis. A MEC of approximately 2,000 kDa in size, based on size exclusion chromatography using Sepharose 4B, was purified from a culture of B. licheniformis. When investigating the presence of enzyme activity in the total crude fraction as well as the MEC of a birchwood xylan culture, B. licheniformis was found to display a variety of enzyme activities on a range of substrates, although xylanases were by far the predominant enzyme activity present in both the crude and MEC fractions. Based on zymogram analysis there were three CMCases, seven xylanases, three mannanases and two pectinases in the crude fraction, while the MEC had two CMCases, seven xylanases, two mannanases and one pectinase. The pectinases in the crude could be identified as a pectin methyl esterase and a lyase, while the methyl esterase was absent in the MEC. Seventeen protein species could be detected in the MEC but only nine of these displayed activity on the substrates tested. The possible presence of a β-xylosidase in the crude fraction was deduced from thin layer chromatography (TLC) which demonstrated the production of xylose by the crude fraction. It was furthermore established that B. licheniformis SVD1 was able to regulate levels of enzyme expression based on the substrate the organism was cultured on. It was found that complexed xylanase activity had a pH optimum of between pH 6.0 and 7.0 and a temperature optimum of 55oC. Complexed xylanase activity was found to be slightly inhibited by CaCl2 and inhibited to a greater extent by EDTA. Complexed xylanase activity was further shown to be activated in the presence of xylose and xylobiose, both compounds which are products of enzymatic degradation. Ethanol was found to inhibit complexed xylanase activity. The kinetic parameters for complexed xylanase activity were measured and the Km value was calculated as 2.84 mg/ml while the maximal velocity (Vmax) was calculated as 0.146 U (μmol/min/ml). Binding studies, transmission electron microscopy (TEM) and a bioinformatic analysis was conducted to investigate whether the MEC in B. licheniformis SVD1 was a putative cellulosome. The MEC was found to be unable to bind to Avicel, but was able to bind to insoluble birchwood xylan, indicating the absence of a CBM3a domain common to cellulosomal scaffoldin proteins. TEM micrographs revealed the presence of cell surface structures on cells of B. licheniformis SVD1 cultured on cellobiose and birchwood xylan. However, it could not be established whether these cell surface structures could be ascribed to the presence of the MECs on the cell surface. Bioinformatic analysis was conducted on the available genome sequence of a different strain of B. licheniformis, namely DSM 13 and ATCC 14580. No sequence homology was found with cohesin and dockerin sequences from various cellulosomal species, indicating that these strains most likely do not encode for a cellulosome. This study described and characterised a MEC that was a functional enzyme complex and did not appear to be a mere aggregation of proteins. It displayed a variety of hemi-cellulolytic activities and the available evidence suggests that it is not a cellulosome, but should rather be termed a xylanosome. Further investigation should be carried out to determine the structural basis of this MEC.

M.B.A. / The debate over plant biotechnology and genetic engineering (GE) is surrounded with controversy. On the one side of the debate, phrases such as `Frankenfood' and `terminator seed' have been used to describe food and seed resulting from plant biotechnology. On the other hand, Agricultural Scientists see biotechnology and genetic engineering as a solution to keep feeding and clothing the increasing world population with static or reducing world resources. Many farmers in developing countries eke out a living based on the production techniques that are becoming increasingly unaffordable because of increasing inputs cost and are no longer producing enough output to provide adequate access to food. Plant biotechnology holds the potential for increasing the productivity of agriculture in developing countries. New crop varieties are developed which are resistant to insects and are tolerant to certain non-selective herbicides. All these plant biotechnology developments are said to help reduce the cost of inputs, protects yields and reduce environmental contamination with toxic insect sprays. For many commercial farmers, cotton production is the only possibility they have to produce a cash crop but spraying to control insect pests is required several times during the season resulting in high input costs. An alternative to spraying is to introduce a built-in defense into the plant itself to combat insect damage. The introduction of the insecticidal gene from the bacterium Bacillus thuringiensis (Bt) into the cotton plant trough biotechnology, has secured a built-in defense against the most common insect family in cotton namely the lepidopteran caterpillar. Cotton engineered with such genes was introduced into commercial production for the first time in 1996 with over 1.8 million acres planted with transgenic cotton in the US. Following early trials, Bt-cotton has since the 1998/99 growing season been commercialized in South Africa. Since the introduction of genetically engineered (GE) crops, US farmers have rapidly adopted most of them. Analyses by USDA's Economic Research Services (ERS) and others indicate economic benefits to many farmers adopting first-generation GE crops. This research study was carried out to determine to what extend the South African cotton growers embraced this kind of technology through usage thereof and if they benefited as much as other cotton farmers world-wide. The specific research objectives for this study were as follows: An evaluation of the adoption rates and benefits derived from plant biotechnology on a worldwide base through literature. An evaluation of the acceptance and penetration of Bt-cotton amongst the South African commercial cotton growers. To determine the benefits of Bt-cotton to the South African commercial cotton growers. To determine to what extend the South African cotton growers benefited economically through the use of the Bt-cotton trait. To identify segmental differences in the adoption of the Bt-cotton trait amongst commercial cotton growers in South Africa. The main findings of this research indicate that almost three quarters of the cotton hectares surveyed were planted with the Bt-cotton trait and that almost ninety percent of cotton growers surveyed, adopted the Bt-cotton trait in the 2001/2002 season. Comparing the benefits derived from Bt-cotton in South Africa to those in other countries, it can be concluded that the South African commercial cotton growers had equal results. Most of the cotton growers in South Africa experienced yield increases with a substantial reduction in the use of insecticides. This resulted in higher gross margins from Bt-cotton compared to the non-Bt cotton or commercial cotton varieties. The South African commercial cotton grower has benefited agronomically, economically and environmentally through the adoption of the Bt-cotton trait. Future introductions of plant biotechnology traits such as herbicide tolerance will further enhance the production potential of cotton.

No description available.

Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2006. / The main aim of the work presented in this thesis was to elucidate the apparent role of pyrophosphate fructose 6-phosphate 1-phosphotransferase (PFP) in sucrose accumulation in sugarcane. PFP activity in sugarcane internodal tissue is inversely correlated to the sucrose content and positively to the water-insoluble component across varieties which differ in their capacities to accumulate sucrose. This apparent well defined and important role of PFP seems to stand in contrast to the ambiguity regarding PFP’s role in the general literature as well as the results of various transgenic studies where neither the downregulation nor the over-expression of PFP activity had a major influence on the phenotype of transgenic potato and tobacco plants. Based on this it was therefore thought that either the kinetic properties of sugarcane PFP is significantly different than that of other plant PFPs or that PFP’s role in sucrose accumulating tissues is different from that in starch accumulating tissues. In the first part of the study sugarcane PFP was therefore purified and its molecular and kinetic properties were determined. It consisted of two subunits which aggregated in dimeric, tetrameric and octameric forms depending on the presence of Fru 2,6-P2. Both the glycolytic and gluconeogenic reactions had broad pH optima and the kinetic parameters for all the substrates were comparable to that of other plant PFPs. The conclusion was therefore that sugarcane PFP’s molecular and kinetic characteristics do not differ significantly from that of other plant PFPs. The only direct way to confirm if PFP is involved in sucrose accumulation in sugarcane is to alter its levels in the same genetic background through genetic engineering. This was therefore the second focus of this study. PFP activity was successfully down-regulated in sugarcane. The transgenic plants showed no visible phenotype under greenhouse and field conditions and sucrose concentrations in their immature internodes were significantly increased. PFP activity was inversely correlated with sucrose content in the immature internodes of the transgenic lines. Both the immature and mature internodes of the transgenic plants had significantly higher fibre contents. This study suggests that PFP plays a significant role in glycolytic carbon flux in immature, metabolically active sugarcane internodal tissues. The data presented here confirm that PFP can indeed have an influence on the rate of glycolysis and carbon partitioning in these tissues. It also implies that there are no differences between the functions of PFP in starch and sucrose storing tissues and it supports the hypothesis that PFP provides additional glycolytic capacity to PFK at times of high metabolic flux in biosynthetically active tissue. This work will serve as a basis to refine future genetic manipulation strategies and could make a valuable contribution to the productivity of South African sugarcane varieties.