Thesis (M.S.)--Bowling Green State University, 2009. / Document formatted into pages; contains viii, 38 p. : ill. Includes bibliographical references.

Thesis (M.Sc.)(Plant Biotechnology))--University of Pretoria, 2002. / Summaries in Afrikaans and English. Includes bibliographical references.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Zymomonas mobilis is a non-pathogenic, facultatively-anaerobic Gram-negative bacterium, which has historically been used for the fermentation of alcoholic beverages in many tropical/sub-tropical countries. Due to its excellent ethanol-producing capabilities, significant effort has been undertaken over recent years to utilize it for industrial ‘bioethanol’ production. Its physiological and metabolic properties indicate that it may also be an excellent organism for the bio-production of many different types of organic molecules. Consequently, the aim of my thesis was to develop new molecular methodologies that would enable Z. mobilis to be ‘engineered’ for use in future ‘bioproduction’ endeavours. In the first part of my study, I analyzed the native (cryptic) plasmids present within a variety of Z. mobilis strains, including two poorly-studied Z. mobilis strains: NCIMB 11163 and NCIMB 8227. Several plasmid libraries containing restriction-digested fragments of Z. mobilis cryptic plasmid DNA were prepared, and their inserts were sequenced. This enabled the complete DNA sequences of three small (non-integrating, double-stranded DNA) cryptic plasmids to be determined: pZMO1A and pZMO7 from NCIMB 11163, and pZMO1B from NCIMB 8227. Their DNA sequences were analyzed using bioinformatic approaches, to identify open reading frames, and regions of DNA that were putatively involved in transcription or DNA replication. In the second part of this thesis, the minimally-replicating region from plasmid pZMO7 was used to construct a series of Escherichia coli-Z. mobilis shuttle vectors. These vectors were found to be stable within several Z. mobilis strains for over 60 generations without antibiotic selective pressure. A reliable and reproducible method based on quantitative real time PCR (Q-RT-PCR) was developed to accurately determine the copy number of cryptic plasmids and shuttle-vectors present in Z. mobilis cultures. The pZMO7-based shuttle vectors exhibited good compatibility with cryptic plasmids as well as the widely-used pZM2-based shuttle vectors. Genes encoding glutathione S-transferase (GST) as well as green and red fluorescent protein (GFP and RFP) reporters were cloned into various shuttle vector constructs; placing them under the control of endogenous (Ppdc) or exogenous (Plac and Ptac) promoters. Promoter strength was evaluated by quantifying the reporter gene expression. The plasmid-based expression of GFP and RFP was visualized within planktonic and biofilm cultures using confocal laser scanning microscopy (CLSM). Shuttle vector-based GST pull-down experiments were used to study intracellular protein-protein binding interactions. In the third part of my thesis, I explored the potential use of Z. mobilis for the bioproduction of isoprenoid (terpenoid) compounds. Five predicted sesquiterpene synthases (terpene cyclases) of unknown function from the dimorphic fungus Penicillium marneffei, and several terpene cyclases from several other bacteria, fungi and plants were initially functionally-analyzed in E. coli. Several cyclase genes were cloned into E. coli-Z. mobilis shuttle vectors for expression trials within Z. mobilis cells. In summary, this thesis describes the development of a variety of novel methodologies and genetic ‘tools’ that may be used to express heterologous genes within Z. mobilis cells. These will be invaluable for future studies concerned with exploring the biology and industrial applications of this ‘microbial cell factory’. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Tobacco is an important crop in South Africa. The genetic basis of tobacco breeding is very narrow and cultivars are closely related. The production of new tobacco hybrids with novel characteristics through classical breeding techniques is difficult. Genetic engineering could assist plant breeders to introduce new herbicide, disease and pest resistance traits into existing proven cultivars. Plant genetic engineering has not previously been applied to the improvement of South African commercial tobacco cultivars. Agrobacterium-mediated leaf disc transformation was used to create transgenic tobacco plants from South African commercial tobacco cultivars TL33, J6 and 20/19. The cultivar samsun was also used to create transgenic plants. The Agrobacterium tumefaciens helper strain C58C1 (pGV2260) containing the binary vector pJIT119 was used to carry out the transformation. As well as the leaf disc transformation method, other methods of obtaining transgenic tobacco plants were explored. These methods included the use of Agrobacterium-mediated transformation of tobacco cell cultures and direct DNA-mediated transformation of tobacco protoplasts. The vector pJIT119 encodes the uidA gene for the β-glucuronidase (GUS) enzyme, the nptl/ gene for neomycin phosphotransferase (NPTII) and the sul I gene for the dihydropteroate enzyme conferring asulam resistance. The presence and expression of these three foreign genes uidA , npt/l and sul I from pJIT119 in transgenic tobacco plants was confirmed by a variety of experimental approaches, including the culture of transgenic plants on medium containing kanamycin or asulam, the GUS histochemical assay, the neomycin phosphotransferase assay, DNA dot-blot analysis, in situ hybridization, computerized image analysis, polymerase chain reaction and progeny analysis. A detailed analysis of individual transgenic plants is necessary in order to select those plants which express the foreign genes maximally. Only these plants would be given to plant breeders for field trial assessment. A high level of foreign gene inactivation was observed in transgenic tobacco plants obtained from the Agrobacterium-mediated leaf disc transformation method. Approximately 20% of the original transgenic plants were discarded as "escapes" as they contained a defective npt/l gene. The remaining kanamycin resistant plants, however, had inactive copies of either the sul I or the uidA gene, or both. The use of in situ hybridization and the polymerase chain reaction (PCR) helped to explain the foreign gene inactivation. The lack of foreign gene expression in individual transgenic plants was not due to the physical loss of entire foreign genes, DNA methylation or the position effect. The lack of expression was due to possible T-DNA rearrangements or deletions which disabled certain genes carried on the T-DNA. Transcription and translation of these foreign genes occurred, but the final uidA and sul I gene products (β-glucuronidase and dihydropteroate synthase, respectively) were possibly defective and did not confer GUS activity or asulam resistance on the transgenic plants The tissue specific activity of the uidA gene under the control of the cauliflower mosaic virus (CaMV) 358 promoter was studied. In the vegetative structures of transgenic tobacco plants, the uidA gene activity was located within the cells surrounding the vascular traces and within the glandular hairs. The effects of stress on 358 promoter activity was also investigated. Chemical and nutrient stess in vitro did not have a significant effect to decrease uidA gene expression under 358 promoter control. Foreign gene expression (uidA) under CaMV 358 promoter control may be enhanced by in vitro stress. Oxygen stress (anaerobic culture under waterlogged conditions) induced uidA expression in areas of the plant which usually did not show usual tissue specific patterns of uidA expression. The stage of differentiation in tissue culture when compared to the mature hardened off transgenic plant, also had an effect on the amount of uidA gene expression. Mature hardened off plants expressed less GUS activity than immature in vitro plants. The tissue specific pattern of foreign gene expression under CaMV 358 promoter direction was conserved in the reproductive structures of transgenic tobacco plants. In floral organs, the pattern of uidA gene expression was essentially the same as that found in vegetative tissues. In all floral organs examined, uidA expression was found associated with the vascular system and within the glandular hairs. The uidA gene with a CaMV 358 promoter was not expressed in pollen. Because of the ease of transformation of tobacco, it is possible that genes for pharmaceutically valuable proteins and peptides could be expressed in tobacco, for agricultural scale fine chemical production("pharming"). This could be of economic advantage for the survival of tobacco as a commercial agricultural crop in the future when tobacco smoking is no longer popular. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1994.

Lactic acid bacteria (LAB), a group of generally recognized as safe (GRAS) organisms that metabolize sugars into primarily lactic acid, have traditionally been used for the fermentation and preservation of various foods and beverages. There is increasing interest in the genetic manipulation of LAB to improve existing characteristics or introduce novel, industrially pertinent phenotypes. However, because these bacteria have food-related applications, their genetic modification requires the use of food-grade genetic engineering tools. LAB plasmids, self-replicating extrachromosomal DNA molecules, can be used to derive food-grade cloning vectors. The rationale of this research was to develop a food-grade cloning vector using a lactobacilli cryptic plasmid and to investigate its cloning and expression properties. The main objectives were to (i) screen Lactobacillus spp. for plasmids, (ii) isolate and characterize a plasmid, and (iii) use the plasmid replicon to construct a cloning vector and express heterologous genes in various hosts. This is the first step in the development of a new family of food-grade cloning vectors for the genetic modification of lactobacilli.

Since the early 1990s, the concept of substantial equivalence has been a guiding principle of the Canadian Food Inspection Agency and Health Canada's regulatory approach toward products of plant biotechnology destined for the food and livestock feed markets. To assess substantial equivalence in terms of chemical composition, genetically modified (GM) plants are compared to conventional counterparts at the level of macro- and micro-nutrients, allergens and toxicants. Such targeted comparative analyses are limited in their scope and their capacity to detect unintended changes in chemical composition. There is a need to develop more effective testing protocols to improve the substantial equivalence assessment of GM crops. The objective of this thesis was to explore high-density oligoarrays as tools to assess substantial equivalence of Roundup Ready(TM) soybean. Three conventional and two GM soybean varieties were selected according to the similarity of their performance in field trials. Total RNA was extracted from first trifoliate leaves harvested from soybean plants grown in a controlled environment until the V2 stage. To annotate the 37 776 soybean probesets present on the multi-organism Soybean Affymetrix GeneChip(TM), consensus sequences were aligned with TIGR Soybean Gene Index tentative consensus sequences using BLASTN. After redefining the chip description file to exclude non-soybean probesets, the effects of three different normalization methods (Robust Multichip Average (RMA), Microarray Analysis Suite (MAS 5.0) and Model-Based Expression Index) were compared and Significance Analysis of Microarrays (SAM for R-Bioconductor) was applied to detect differential gene expression between conventional and GM soybean varieties. Eleven candidate genes were selected for further studies.

Regeneration and shoot multiplication of common bean (Phaseolus vulgaris L. 'ICA Pijao') from half-cotyledonary nodes was achieved on modified Murashige and Skoog (1962) basal medium amended with 5 $ mu$M 6-benzylaminopurine. Histological studies confirmed the adventitious origin of the regenerated buds. Shoots were rooted ex vitro and developed into morphologically normal plants compared with seed-grown controls. The relative susceptibility of bean tissues to infection by a collection of wild-type Agrobacterium strains was tested. Positive transformation events were evaluated based on morphological and biochemical changes observed following Agrobacterium infection. The A. tumefaciens strain C58 was particularly virulent on greenhouse-grown plants, in vitro-derived stem sections, half-cotyledonary nodes and seedlings. A sensitive and rapid method was developed to detect opines using thin layer chromatography. Transient $ beta$-glucuronidase (GUS) gene expression was detected in 'ICA Pijao' bean buds regenerated from half-cotyledonary nodes following Agrobacterium-mediated gene transfer with the binary vector pGV1040 or p35SGUSINT. Four out of eight putative transformants contained the chimeric GUSINT gene following polymerase chain reaction (PCR) analysis. This was confirmed by Southern analysis of blotted PCR gels. However, there was no stable integration of the GUSINT gene as none of the R1 progeny showed an amplified GUSINT fragment with PCR.

Plants have evolved numerous strategies for the adaptation to drought. Although many investigations reported on the potential value of proline accumulation during environmental stress, it is still unknown whether or not a constitutive higher level of proline accumulation enhances plant tolerance. Thus, it was investigated if underproduction and overproduction of proline will influence the susceptibility to drought stress in soybean plants. This was made possible with the transformation of soybean plants with an L-Δ¹-pyrroline-5-carboxylate reductase (P5CR) gene. First, an Agrobacterium-mediated vacuum infiltration transformation system, using partially germinating Carnia 2233 soybean seed, was established through the assessment of several conditions that can affect transformation efficiency with the use of β-glucuronidase reporter genes. Transformation was confirmed with PCR and Southern blot analysis and results indicated that stable transgenic soybean plants were obtained within one generation with a transformation rate of± 30%. This technique was used in the transformation of Carnia 2233 soybean seed with the P5CR gene in the antisense orientation under the control of an inducible heat shock gene promoter (IHSP). It was confirmed that the P5CR-IHSP gene construct was integrated into the soybean cells and was conserved over three generations. Physiological screening of the antisense P5CR transgenic plants in the greenhouse proved that, with activation of the promoter, an under-expression of the P5CR gene and subsequent inhibition of the accumulation of proline were experienced during drought and osmotic stress. The decline of the viability of the transgenics with prolonged drought stress, as monitored with a woodenbox screening test, is an indication that proline is needed for survival of soybean plants under drought stress conditions. The transgenic plants demonstrated a sensitive reaction in contrast to the control plants that displayed a tolerant reaction to osmotic stress in a TTC assay. The underexpression of the P5CR gene resulted in a decline protein synthesis due to proline shortage as was observed with the evaluation of the efficiency of protein synthesis. All these results suggest that a decrease in the proline level due to the antisense P5CR gene, yielded plants that are more osmotic and drought stress sensitive. Subsequently, the soybean cultivar Ibis was successfully transformed with the P5CR-IHSP construct in the sense and antisense directions in order to test the reproducibility of the transformation process and to assessed the link between the biochemical traits involved in the drought stress mechanism. Three different experiments were conducted: a mild heat and drought stress on "To" transgenic plants exploring changes in chlorophyll fluorescence transients, a mild heat stress on "T1" transgenic plants comparing proline accumulation and chlorophyll fluorescence transients and a severe drought and heat stress on the "T1" transgenic plants comparing proline accumulation NADP⁺synthesis and chlorophyll fluorescence transients. Chlorophyll fluorescence transients were successfully used as a screening method for transgenic soybean plants during this study. The sense transgenics responded to the mild stresses with a significant decrease in their electron transport, trapping and absorption compared to the antisense plants that displayed significant increases in electron transport and trapping. During the severe stress, the antisense transgenics experienced total photoinhibition indicated by the enormous loss of electron transport but the sense plants had the ability to overcome the stress as is revealed in the increase in the electron transport. It was demonstrated that although proline accumulation yielded no significant differences during the mild heat stress, the sense plants accumulated substantially more proline than the control and antisense plants during the severe heat and drought stress. It was demonstrated that proline plays an important role in the plant's response to a drought stress as well as in the recovery phase after drought, as the sense plants also had the ability to reduce the accumulated proline during the recovery period in contrast to the antisense transgenics that experienced protein degradation. The transgenics responded to a period of heat and drought stress with a reduction in NADP⁺ levels in the antisense plants and increasing levels in the sense plants. The sense plants were able to fully recover after the stress period, thus adaptation to drought may depend on different mechanisms, including the capacity to maintain high levels of proline and to regenerate them through the "reduction" of NADP⁺. It was possible to alter the drought tolerance of Ibis by transformation with antisense and sense P5CR gene constructs, which resulted in respectively more sensitive and more tolerant Ibis plants. It can be concluded that over-expression of P5CR during a drought stress resulted in higher proline levels, better photosynthetic efficiency, higher NADP⁺ production and thus a more drought tolerant plant. This study gave additional proof that a constitutively higher level of proline accumulation enhances drought tolerance in soybean. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2000.

The use of genetic markers that are linked to specific traits in sugarcane has the potential to increase the efficiency of the selection of improved varieties. Conventionally, markers are identified by analysing the segregation of potential markers and traits in the progeny of single crosses. However, this approach is not practical for sugarcane breeding programmes where replicated, well characterized progenies do not exist. The objective of this project was to investigate the potential of using commercial varieties for identifying markers associated with some of the important traits in sugarcane. This approach would be far more effective than dealing with single progenies since the traits of commercial varieties have already been characterized. The DNA of fifty commercial varieties of sugarcane was amplified by RAPD PCR using forty-one arbitrary decamer primers. Analysis of the resulting banding profiles, obtained by agarose gel electrophoresis, yielded fifty-four reliable polymorphic fragments. Two approaches were used to identify putative markers linked to the traits of resistance to eldana, sugarcane mosaic virus, and smut: (1) a correlation approach which attempted to identify whether the presence of any polymorphisms could be used to imply the existence of a particular phenotypic state, and (2) multiple regression analysis, in order to determine whether polymorphisms could be used to predict the performance of the varieties for each of the traits. Both approaches appeared to identify associations between polymorphisms and the traits, although multiple regression analysis yielded the most informative results and was able to assign statistical values to the associations. Using multiple regression, the best predictive model was obtained for sugarcane mosaic virus resistance. This model consisted of four polymorphisms and had an r² of 0.40l. By dividing the resistance ratings into three groups (resistant, intermediate and susceptible), 52% of the varieties were correctly classified and only 2% of the varieties were predicted in opposite groups (i .e. predicted susceptible when actually resistant, and vice versa). The predictive model for eldana resistance consisted offour polymorphisms and had an r² of 0.347. This model classified 30% of the varieties in the correct group of three while none of the varieties were predicted in opposite groups. The predictive model for smut resistance consisted of three polymorphisms and had an r² of 0.316. This model classified 30% of the varieties in the correct group of three while 2% of the varieties were predicted in opposite groups. Further analysis of sugarcane varieties using additional polyrnorphisrns has the potential to identify markers linked to important traits. These markers could be used for marker-assisted selection to increase the efficiency of selecting for improved sugarcane genotypes for commercial release. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.