The major objective of this study was to produce synthetic genotypes of Brassica juncea from its parental species <i> B. rapa </i> and <i> B. nigra </i> via somatic and sexual hybridization. As prerequisites for somatic hybridization experiments, methods were developed to improve the culture of mesophyll and hypocotyl protoplasts of <i> B. nigra </i> and <i> B. rapa </i>, to obtain reliable plant regeneration from mesophyll protoplast cultures of <i> B. nigra </i>, and to fuse protoplasts of <i> B. nigra </i> and <i> B. rapa </i>. A modified Kao's medium (1977), was found suitable for the culture of mesophyll protoplasts of <i> B. nigra </i> and <i> B. rapa </i>. At a density of approximately $110\sp5$ protoplasts/ml within a culture plate insert surrounded by culture medium, mesophyll protoplast cultures of <i> B. nigra </i> accessions R890, R1819, R3392 and U1218 and <i> B. rapa </i> cvs. R500 and Wong Bok formed colonies. Genotypic differences in cell division and colony formation were observed. Hypocotyl protoplasts of <i> B. nigra </i> and <i> B. rapa </i> were successfully isolated from 6 day-old seedlings cultured in a modified Kao's medium (1977). With <i> B. nigra </i> accession R890 and <i> B. rapa </i> cv. R500, cell division and colony formation were optimal when hypocotyl protoplasts were cultured at a density of 0.5 to $1.010\sp5$ protoplasts/ml within a culture plate insert surrounded by a nurse culture of 4 to 6 day-old mesophyll protoplasts of <i> B. nigra </i>. Plant regeneration was obtained from mesophyll protoplast-derived calli of <i> B. nigra </i> accession R890 originally cultured in inserts; a shoot regeneration frequency of 8.1% was obtained on a medium containing the salts and vitamins of medium K3 (Nagy and Maliga 1976) with 3 g/l sucrose, 18.2 g/l mannitol, 2 mg/l ZR, 0.1 mg/l NAA, 10 g/l agarose, pH 5.6. For somatic hybridizatian studies, methods were developed to select out parental protoplasts using iodoacetic acid and to efficiently fuse protoplasts on the bottom of a petri dish using PEG. Twenty-nine plants were recovered from fusion experiments between mesophyll protoplasts of <i> B. nigra </i> accession R890 and hypocotyl protoplasts of <i> B. rapa </i> cv. Tobin. The somatic hybrid plants resembled natural <i> B. juncea </i>, had $2n=36$ chromosomes and had pollen viabilities ranging from 30 to 45%. Twenty-one plants, derived from one callus colony, possessed the mitochondrial and chloroplast genomes of <i> B. rapa </i>, as found in natural <i> B. juncea </i>. Eight plants, derived from another callus, had a novel cytoplasmic combination consisting of the mitochondrial genome of <i> B. rapa </i> and the chloroplast genome of <i> B. nigra </i>. Synthetic genotypes of <i> B. juncea </i> were also produced from reciprocal sexual crosses between <i> B. rapa </i> and <i> B. nigra </i>. Seventy-eight interspecific hybrid plants from the cross <i> B. rapa </i> x <i> B. nigra </i> and six hybrid plants from the reciprocal cross were identified by their morphology, pollen viability and chromosome number. The colchicine-induced allotetraploids resembled natural <i> B. juncea </i> in morphology, had 18 bivalents at metaphase I, and had between 35 and 70% pollen viability.

Crop research has undergone a major transformation in North America and many other parts of the word. The introduction of biotechnology and Intellectual Property Rights (IPR) alter the nature of research products, which in turn changes the structure of the agricultural research industry from perfectly competitive to imperfectly competitive. The implications of these changes are not fully understood. The objective of this thesis is to develop a broader understanding of how biotechnology, changes in IPRs and the resulting changes in industry structure have affected private and public incentives for agricultural research. The specific goals include development of an analytical framework to examine the incentives for private R&D expenditure, and the spillovers between basic and applied research and between private and public firms. To achieve the objective of this study, a stochastic analytical model within an imperfect competitive framework was developed. Specifically, what is developed is a three-stage search/imperfect competition model characterized by two research firms developing and selling differentiated products to producers who are heterogeneous with respect to some attributes. Agricultural research is modeled with explicit recognition of the search process, which allows us to recognize research as a stochastic process with sporadic outcomes and to explicitly model the interaction between basic and applied research. The findings of this study are mainly in the form of propositions. It was shown that basic public research "crowds in" applied private research while applied public research "crowds out" applied private research. The current technology level and the cost of the experimentation negatively affect private investment, while the price of the final product positively affects the private investment. Moreover, it is concluded that, the greater the product heterogeneity, the higher the price charged with the same amount of R&D. Finally, it is shown that the increase in IPR's and the firm's market size has a positive effect on the private firm's amount of R&D investment. The econometric analysis, using data from the canola industry, provides empirical evidence to support the analytical framework and the proposition derived in this study. The study also draws a number of policy implications from the derived propositions.

Organophosphate hydrolase (OPH, EC 3.1.8.1) is a bacterial enzyme with a broad spectrum of potential substrates that include organophosphorus pesticides, herbicides, and chemical warfare agents. OPH has been expressed successfully in bacterial, fungal, and insect cell culture systems; however, none of these systems produces amounts of enzyme suitable for applications outside of the research laboratory. Therefore, a transgenic Zea mays L. (maize) system was developed to express OPH as an alternate to the current OPH expression systems. The bacterial gene encoding the OPH protein was optimized for transcriptional and translational expression in maize. The optimized gene was inserted into the maize genome under the control of embryo specific, endosperm specific, and constitutive plant promoters. Select transformants were analyzed for the expression of OPH. Expression was observed in the seeds of plants transformed with each of the three constructs with the highest expression observed with the embryo specific and constitutive promoter constructs. The highest OPH expressing lines of transgenic maize had expression levels higher than those reported for the E. coli expression system. OPH was purified from transgenic maize seed and analyzed for posttranslational modification and kinetic properties. OPH was observed to undergo a glycosylation event when expressed in maize that yielded at least two forms of OPH homogolous dimer. The glycosylated form of OPH bound tightly to the Concanavalin A sepharose and remained active after months of storage at room temperature. OPH activity was checked against a number of organophosphate herbicides. Enzymatic activity was observed against the herbicide Amiprophos-methyl and kinetic properties were measured. Enzymatic activity was also tested against the organophosphate Haloxon. Transgenic maize callus, leaf, and seed tissue could be screened for the presence of the optimized opd gene by enzymatic activity. Comparison of the growth of transgenic and control callus on media containing organophosphates showed that the transgenic callus was resistant to the herbicidal effects of haloxon. Transgenic plants expressing OPH were also resistant to the herbicide bensulide when compared to control plants. This indicates that OPH can be used as a screenable marker in plant systems and may be a potential scorable marker system as well.

Triticale (x Triticosecale Whitm.) is a cereal with favorable agronomic traits for a Canadian bioproduction platform crop. Appropriate tissue sampling times were determined and gene expression profiles were evaluated in five triticale seed tissues and eleven vegetative tissues using the Affymetrix Wheat GeneChip®. Genes that were expressed, not expressed, tissue-specific, tissue-enriched and developmentally regulated were identified. The percentage of probe sets on the wheat GeneChip with gene ontology annotations was improved from less than 3% to over 76% using homologous sequence identification and annotation transfer. This information was used to determine functions and processes over-represented within the identified gene lists and provide biological meaning to the results. Expression of candidate genes was further evaluated using qRT-PCR, RNA in situ hybridization and promoter characterization. This study has provided a comprehensive triticale gene expression atlas; knowledge regarding triticale development, gene function, expression and regulation; and tools enabling further triticale research and development. / xxiii, 425 leaves : col. ill. ; 29 cm

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2009. / Dichrostachys cinerea, Carpobrotus dimidiatus, Capparis tomentosa and Leonotis leonurus are all plants that are indigenous to South Africa. These plants are used in traditional medicine to treat various ailments. However, there is little or no scientific data to justify these traditional uses. Furthermore, it is difficult to reconcile traditional knowledge with scientific evidence because of the overwhelming targeting of signal-responsive systems by plant defensive compounds, multiple sites of action and the connectedness of the signaling pathways, which provide many cures and have pleiotropic effects. In order to evaluate the action spectrum of these plants, and validate its widespread use, this research evaluated the antibacterial, antioxidant, anti-inflammatory, anti-mosquito and immunomodulatory properties of these plants. Antimicrobial activity of the extract was determined by evaluating the bactericidal and fungicidal action using the agar disc diffusion assay. Anti-oxidative properties of the extracts were tested using the DPPH photometric assay. Anti-inflammatory properties were carried out using the 5-lipoxygenase assay. The larvicidal, repellency and insecticidal assay was determined against A.arabiensis. The safe use of these plant extracts was determined by evaluating toxicity, a brine shrimp lethality assay and an in vitro cell culture system using human myelogenous leukemia cell line. Potential carcinogenic activity was evaluated using the Ames Salmonella Mutagenecity assay. The immunomodulatory activity of the extracts on human peripheral blood mononuclear cells 6 was evaluated on freshly harvested lymphocytes using the MTT assay. Cytokine response was evaluated by measuring the secretion of interferon-gamma and interleukin-10. Elucidation of the B cells, T cells, activated T cells, CD 4+, CD 8+ and NK cells was performed by flow cytometry. The extracts showed anti-microbial activity against Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Salmonella typhimurium, Serratia marcescens, Bacillus cereus and Tricoderm sp. The highest activity was shown by methanolic and aqueous extracts of L. leonurus leaves followed by methanolic and aqueous extracts of D. cinerea. Extracts of C. tomentosa and D.cinerea demonstrated a higher degree of free radical scavenging than rutin, which was used as a standard indicating that these plants have strong antioxidant properties. None of the plants showed significant anti-inflammatory activity when compared to NDGA. In the anti-mosquito assays, the extracts showed strong repellency and insecticidal activity. L. leonurus extracts demonstrated the highest insecticidal and repellency activity against the mosquito, and was also found to cause ‗knockdown‘ and mortality. The extracts display no toxicity, cytotoxicity and mutagenicity. The immunological studies for immune modulation showed that the methanol extracts of these plants induce a Th1- predominant immune response because they significantly suppressed the secretion of IL-10 and augment IFN-γ production, which are hallmarks used to indicate a stimulation of the innate immune response. This study also provides new information, with respect to the potential use of these plants in producing a mosquito repellent and an immunostimulant.

A system dynamics model is developed to study the technology adoption process (TAP) of modern agriculture input technology such as the biotechnologies. The work shows that the system dynamics approach is appropriate to integrate the different components considered in the TAP conceptual framework elaborated in this work. The conceptual framework illustrates the different system components found important in the literature, portfolio decision-making, learning, information gathering, uncertainties and economics perceptions and their involved relationships. / The model is first calibrated and validated using the case of soybeans adoption versus corn uses in Quebec from 1987 to 1998. Validation is performed through five tests, namely visual, statistical and sensitivity, modularity and extendibility are performed to show the relevancy of the approach. / The model is then applied to the case of four input biotechnology crops. Again three types of validation tests are carried out. Results show that the model predicted the shape of the curve for all application fields.

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 142-149). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 142-149). Also available on the Internet.

abstract: The HIV-1 pandemic continues to cause millions of new infections and AIDS-related deaths each year, and a majority of these occur in regions of the world with limited access to antiretroviral therapy. Therefore, an HIV-1 vaccine is still desperately needed. The most successful HIV-1 clinical trial to date used a non-replicating canarypox viral vector and protein boosting, yet its modest efficacy left room for improvement. Efforts to derive novel vectors which can be both safe and immunogenic, have spawned a new era of live, viral vectors. One such vaccinia virus vector, NYVAC-KC, was specifically designed to replicate in humans and had several immune modulators deleted to improve immunogenicity and reduce pathogenicity. Two NYVAC-KC vectors were generated: one expressing the Gag capsid, and one with deconstructed-gp41 (dgp41), which contains an important neutralizing antibody target, the membrane proximal external region (MPER). These vectors were combined with HIV-1 Gag/dgp41 virus-like particles (VLPs) produced in the tobacco-relative Nicotiana benthamiana. Different plant expression vectors were compared in an effort to improve yield. A Geminivirus-based vector was shown to increase the amount of MPER present in VLPs, thus potentially enhancing immunogenicity. Furthermore, these VLPs were shown to interact with the innate immune system through Toll-like receptor (TLR) signaling, which activated antigen presenting cells to induce a Th2-biased response in a TLR-dependent manner. Furthermore, expression of Gag and dgp41 in NYVAC-KC vectors resulted in activation of antiviral signaling pathways reliant on TBK1/IRF3, which necessitated the use of higher doses in mice to match the immunogenicity of wild-type viral vectors. VLPs and NYVAC-KC vectors were tested in mice, ultimately showing that the best antibody and Gag-specific T cell responses were generated when both components were administered simultaneously. Thus, plant-produced VLPs and poxvirus vectors represent a highly immunogenic HIV-1 vaccine candidate that warrants further study. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2016

abstract: Plants are a promising upcoming platform for production of vaccine components and other desirable pharmaceutical proteins that can only, at present, be made in living systems. The unique soil microbe Agrobacterium tumefaciens can transfer DNA to plants very efficiently, essentially turning plants into factories capable of producing virtually any gene. While genetically modified bacteria have historically been used for producing useful biopharmaceuticals like human insulin, plants can assemble much more complicated proteins, like human antibodies, that bacterial systems cannot. As plants do not harbor human pathogens, they are also safer alternatives than animal cell cultures. Additionally, plants can be grown very cheaply, in massive quantities. In my research, I have studied the genetic mechanisms that underlie gene expression, in order to improve plant-based biopharmaceutical production. To do this, inspiration was drawn from naturally-occurring gene regulatory mechanisms, especially those from plant viruses, which have evolved mechanisms to co-opt the plant cellular machinery to produce high levels of viral proteins. By testing, modifying, and combining genetic elements from diverse sources, an optimized expression system has been developed that allows very rapid production of vaccine components, monoclonal antibodies, and other biopharmaceuticals. To improve target gene expression while maintaining the health and function of the plants, I identified, studied, and modified 5’ untranslated regions, combined gene terminators, and a nuclear matrix attachment region. The replication mechanisms of a plant geminivirus were also studied, which lead to additional strategies to produce more toxic biopharmaceutical proteins. Finally, the mechanisms employed by a geminivirus to spread between cells were investigated. It was demonstrated that these movement mechanisms can be functionally transplanted into a separate genus of geminivirus, allowing modified virus-based gene expression vectors to be spread between neighboring plant cells. Additionally, my work helps shed light on the basic genetic mechanisms employed by all living organisms to control gene expression. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2017