Magister Scientiae - MSc (Biotechnology)
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Item 3,3'-diindolylmethane improves drought tolerance of Zea mays through enhancing antioxidant activity(University of the Western Cape, 2018) Basson, Gerhard Leroy; Ludidi, Ndiko; Keyster, Marshall; Gokul, ArunMaize is one of the most abundantly produced cereals and contributes to about 40% of the global cereal production. This figure will have to increase in order to feed the ever-growing human population. One of the major environmental constraints that impact maize production is drought. Plants use antioxidant defences to cope with drought stress. Understanding and improving these defence mechanisms will be important to improve overall drought tolerance. A previous study done by Gokul and authors in 2016 showed that 3,3’-diindolylmethane (DIM) improves both seed germination and seedling shoot growth in Brassica napus. Plants belonging to the Brassicaceae family have the metabolic machinery to synthesize glucosinolates such as DIM, which play vital roles in physiological and stress responses. These responses have not been investigated in plants such as maize, which lack the machinery to produce DIM. Therefore, this study investigated the effects of exogenously applied DIM on the physiological and biochemical responses of maize under drought stress. Physiological parameters such as relative water content, chlorophyll content and lipid peroxidation, were determined in order to understand how drought and DIM , as separate or combined treatments, affected the plants. Additionally, proline accumulation was also assessed because free proline plays a role as an osmoprotectant during stress. The accumulation of ROS, namely hydrogen peroxide, was measured using spectrophotometric assays to determine how the above treatments affect ROS accumulation in maize. As a result of changes in the ROS content in due to the treatments, it would only be natural to investigate the changes in antioxidants as well. Given that hydrogen peroxide was the ROS to be measured, we therefore investigated the antioxidant enzymatic activities responsible for hydrogen peroxide scavenging. Therefore, changes in Ascorbate peroxidase (APX) and catalase (CAT) were assessed. An improved drought response was observed in maize plants treated with DIM as these plants had better ability to maintain their water status than when no DIM was applied. This is indicated by water-deprived plants treated with DIM having a higher RWC than water-deprived plant without DIM.Item A comparative ancestry analysis of Y-chromosome DNA haplogroups using high resolution melting(University of the Western Cape, 2018) Michelle Burrows, Adria; Eugenia D'Amato, MariaThe objective of this study is to deduce paternal ancestry using ancestry informative single nucleotide polymorphisms (SNPs) by means of High Resolution Melting (HRM). This was completed by producing a multiplex system that was designed in a hierarchical manner according to the YSNP tree. This project mainly focused on African ancestry and was used to infer paternal ancestral lineages on the Johannesburg Coloured population. South Africa has a diverse population that has ancestral history from across the globe. The South African Coloured population is the most admixed population as it is derived from at least five different population groups: these being Khoisan, Bantu, Europeans, Indians and Southeast Asians. There have been studies done on the Western Cape/ Cape Town Coloured populations before but this study focused on the Johannesburg Coloured population. The first step was to design the multiplex system. This was done by using inhouse SNPs. A total of seven multiplexes were designed and optimised, each consisting of two, three or four different SNPs respectively. A total of 143 saliva and buccal samples were collected from male Johannesburg Coloureds. DNA was extracted from the saliva samples using an optimised organic method. DNA was extracted from the buccal samples using an optimised salting out method. DNA was successfully extracted from 77 of the male samples. A total of 69 samples were screened using Multiplex 1; of the 69 samples 56 samples were successfully screened to infer the paternal lineage of the samples. The results show that the most frequent haplogroup of the Johannesburg male samples was haplogroup CF (39%). The second most frequent haplogroup was haplogroup DE (38%). Under further analysis of haplogroup DE it was seen that 37% of those samples were derived for the haplogroup E1b1b.Item Actinobacterial and archaeal diversity in lake Magadi, Kenya(University of the Western Cape, 2013) Halimat, Olubukola Ibrahim; Cowan, D. A.Microorganisms of the class Actinobacteria and domain Archaea are interesting from a biotechnological perspective owing to their metabolic attributes as producers of secondary metabolites and resilience under harsh environmental conditions respectively. Lake Magadi is a soda lake well studied in terms of its geology and limnology. Research attention has also been drawn to the microbial populations which thrive in this unique habitat but currently there are no reports on the assessment of its microflora using molecular methods. This study aimed to assess the actinobacterial and archaeal communities within Lake Magadi, Kenya a hypersaline –highly alkaline habitat using metagenomic methods as a preliminary study to identify potential candidates for exploitative biology Samples from two sites dubbed Lake Magadi station 2 (LM2) and Lake Magadi salt pan 4 (LMS4) within the Lake Magadi were analyzed using the 16S rRNA gene as a phylogenetic marker. Cluster analysis of taxon-specific 16S rDNA PCR-DGGE profiles revealed moderately heterogeneous actinobacterial and archaeal populations across the sample sites under investigation which is probably a reflection of the differences in abiotic conditions at the study sites. This observation was also confirmed from the multi-dimensional scaling (MDS) plot. PCR-based clonal libraries of actinobacterial and archaeal communities of both study sites retrieved a total of thirty-two clones (twenty actinobacterial and twelve archaeal) were sequenced. Analysis of the sequences revealed cultured and uncultured signatures of microorganisms typical of hypersaline and or highly alkaline niches. A few (3) sequences presented novelty (<96%) in identities with any previously identified organism. It was concluded that the species dominance at site LMS4 [situated within the salt flats of Lake Magadi and site for exploration of trona and its mineralized extensions (nacholite and gayllusite)] is likely to be dictated by anthropogenic stress since most of the microbial signals associated with the study site are typical of saline and or alkaline environmental samples exposed to especially mining but also agricultural and waste management practices. Isolation studies also revealed previously identified strains peculiar to hypersaline brines and sediments. The strains retrieved were affiliated to the taxonomically diverse genus Bacillus and Halomonas sp. The true applications and potential opportunities these isolates have for biotechnology have been well documented. Observations made from the culture dependent and culture independent methods suggests strongly that study site LMS4 is subjected to environmental conditions more severe than at site LM2. This study is a guide for future studies as it provides primary information on the haloalkaliphilic representatives of the actinobacteria phylum and domain Archaea within the soda lake environment. It can serve as a pedestal for investigation into the molecular machinery that supports the haloalkaliphilic lifestyles of inhabiting microorganisms and consequently give leads as to how they can be commercially exploited.Item Actinobacterial diversity of the Ethiopian Rift Valley lakes(University of the Western Cape, 2011) Du Plessis, Gerda; Cowan, D.A.; Tuffin, MarlaThe class Actinobacteria consists of a heterogeneous group of filamentous, Gram-positive bacteria that colonise most terrestrial and aquatic environments. The industrial and biotechnological importance of the secondary metabolites produced by members of this class has propelled it into the forefront of metagenomics studies. The Ethiopian Rift Valley lakes are characterized by several physical extremes, making it a polyextremophilic environment and a possible untapped source of novel actinobacterial species. The aims of the current study were to identify and compare the eubacterial diversity between three geographically divided soda lakes within the ERV focusing on the actinobacterial subpopulation. This was done by means of a culture-dependent (classical culturing) and culture-independent (DGGE and ARDRA) approach. The results indicate that the eubacterial 16S rRNA gene libraries were similar in composition with a predominance of α-Proteobacteria and Firmicutes in all three lakes. Conversely, the actinobacterial 16S rRNA gene libraries were significantly different and could be used to distinguish between sites. The actinobacterial OTUs detected belonged to both the Rubrobacterales and Actinomycetales orders with members of the genus Arthrobacter being found in all three lakes. Geochemical properties were significantly different between the lakes, although more than one property attributed to the variance between community compositions. The diversity detected in the culture-based study differed significantly and all isolates belonged to the genus Streptomyces. Two novel strains were characterized by means of phylogenetic (16S rRNA gene sequence), physiological, morphological and biochemical analyses. Both novel isolates were capable of growing under "extreme" conditions- pH 12, 10% NaCl and 45°C. Partial enzyme characterization revealed that both strains produced xylanase enzymes that were active at pH 6.5 and 8.5 with an increase in activity up to 45°C. The results obtained revealed a previously undetected diversity of actinobacteria in the Ethiopian Rift Valley with a potentially novel subpopulation adapted to haloalkaline conditions. The low 16S rRNA sequence similarity of a substantial proportion of the libraries suggests that culture-based isolation may play a vital role in deciphering the community fingerprint.Item An investigation of the zinc binding characteristics of the RING finger domain from the human RBBP6 protein using heteronuclear NMR spectroscopy.(University of the Western Cape, 2007) Mulaudzi, Takalani; Pugh, David J.R.; Magister Scientiae - MSc; Faculty of ScienceRetinoblastoma binding prot ein 6 (RBBP6) is a 250 kDa human splicing-associated protein that is also known to interact with tumour suppresso r proteins p53 and pRb and to mediate ubiquitination of p53 via its intera ction with Hdm2. RBBP6 is highly up regulated in oesophageal cancer, and has been shown to be a promising target for immunotherapy against the disease. RBBP6 is also known to play a role in mRNA splicing, cell cycle control and apoptosis.Item Analysis of ammonia-oxidizing bacteria associated with the roots of Proteaceae plant species in soils of Fynbos ecosystem(University of the Western Cape, 2005) Cowan, D. A.Molecular methods were used to investigate the microbial diversity and community structure of ammonia-oxidizing bacteria (AOB) associated with the roots of the Proteaceae plant family. The identification of ammonia oxidizing bacteria in this ecosystem is of particular interest since Proteaceae are adapted to acidic, low nutrient (e.g. nitrogen) soils. The ammonia monooxygenase operon was used as a molecular marker to identify ammonia-oxidizing bacteria associated with the proteoid roots of the three Proteaceae members and compared to non-plant associated soil. PCR amplification using primer sets targeting the ammonia monooxygenase gene (amoA subunits) were used to construct a clone library. Sequence diversity was determined by RFLP analysis of amoA to identify major groups of AOB of the ~-subclass of Proteobacteria in total community DNA, and DNA sequencing and phylogenetic analysis were also applied. DGGE analysis was performed to determine the community structure and distribution of ammonia-oxidizing bacteria in plant-associated and non-plant associated soils. The AOB genotypic diversity was similar in the plant-associated samples and non-plant associated soil. All AOB phylotypes belonged to Nitrosospira species and clustered with Nitrosospira cluster 3. The abundance of the amoA was quantified to be approximately 4.2 x 107 copies/g of dry soil, using a real-time PCR assay. These data suggest that the Nitrosospira species are the dominant phylotypes in that environment. This investigation provides new insights into the relationships between plants and ammonia-oxidizing bacteria in natural Fynbos ecosystems.Item Analysis of ammonia-oxidizing bacteria associated with the roots of Proteaceae plant species in soils of Fynbos ecosystem(University of the Western Cape, 2005) Lako, Joseph; Cowan, Don A.; Dept. of Biotechnology; Faculty of ScienceThe major objective of this study was to investigate soil ammonia-oxidizing bacterial diversity and composition associated with plant roots of Proteaceae plants and to compare it with non-plant associated soil.Item Analysis of the proteome of red and green leaf phenotypes of 'Bon Rouge' pear trees \trus communrls L. by 2-dimensional gel electrophoresis(University of the Western Cape, 2007) Sehata, Majimi James; du Preez, Marlene; Ndimba, Bongani; Rees, JasperThe 'Bon Rouge' pear is a red pear cultivar derived from a rare, spontaneous bud mutation which occurred on the green pear 'William's Bon Chretien' (Bartlett). 'Bon Rouge' pear cultivar was observed to be reverting back to its original phenotypic green colour. To study the cellular changes occurring within the cells both the mutated red phenotype and the wild type green phenotype of 'Bon Rouge' were investigated using proteomics approach. The proteins from the tissues of interest were precipitated in the presence of l0o/o TCA and solubilized in urea/thiourea lysis buffer. The proteins were separated onl2Yo SDS-PAGE and2D PAGE in order to compare their protein expression profiles. The results from SDS-PAGE and 2D PAGE profiles show an elevated level of 53 kDa protein in green pears which was either absent or less expressed in red phenotype and this protein appears to be the only differentiating factor between the red and green 'Bon Rouge' phenotypes. This protein was subsequently identified by the MALDI-TOFMS to be large subunit of RuBisCO. Expression proteomics is a preferred method due to its reproducibility and it has a potential to provide direct identification of cultivars related proteins. Additionally, proteomics approaches could help the fruit crop gtowers andbreeders, to select and determine types of different cultivars at various growth stages.Item Ancient Genes in Cancer Gene Expression?(University of the Western Cape, 2004) Panji, Sumir; Hide, WinstonBacksround: The Cancer/testis (CT) antigens are a division of germ cell specific genes not expressed in somatic cells, exceptions being placental cells and 20Vo - 4OVo of cancer types. The aptitude of CT antigens to elicit humoral immune responses, their restricted expression profile, absence of major histocompatability complex expression in male germline cells have contributed to the emergent attraction of CT antigens as ideal, prospective cancer vaccination candidates. Motivation: Presently there are M CT gene families containing a total of 97 gene products and isoforms. Due to the promulgation in sensitivity and specificity of rapid serological immunodetection assays e.g. serial analysis of recombinant cDNA expression libraries (SEREX), the magnitude of novel CT genes and gene families will increase. Hence, characteization of this unique subset of CT genes is fundamental to our erudition of this rapidly emerging novel subset of genes. Obiectives: The sequencing of the human genome provides a useful biological framework for the categoization and systematization of rapidly accumulating biological information. A genomic approach was used to ascertain the locations of the CT genes in the human genome and determine if the genomic locations of the CT genes is nonrandom. An in-silico expression study was conducted for the CT genes with the aim of establishing if CT gene expression is restricted to the testis. A portion of the human genome housing the largest proportion of the CT genes was selected for analysis in order to determine if the surrounding genomic architecture influences CT gene expression. A comparative genomics approach was used in determining if the CT genes are "ancient genes.Item Antibacterial effects of biogenic silver nanoparticles synthesized using manihot esculenta (cassava) leaf aqueous extract(University of the Western Cape, 2023) Oliver, Toni Charlize Alexia; Madiehe, AbramAntimicrobial resistance (AMR) is a growing global health concern that poses a serious threat to the health of humans, animals, and plants. The major public health problem of AMR is primarily caused by the incorrect use of antibiotics, which is further compounded by the reduced novel antibiotics discovery rate. AMR bacteria cause infections that are difficult to treat, resulting in prolonged hospital stays and increased healthcare costs. Moreover, AMR is associated with a high risk of morbidity and mortality. Current therapeutic strategies for AMR infections are often inefficacious, associated with side effects, and may further exacerbate AMR. Therefore, there is an urgent need to develop alternative strategies to treat AMR bacterial infections.Item The antimicrobial activity of zinc oxide nanoparticles synthesized with plant extracts against Staphylococcus aureus(University of the Western Cape, 2019) Kriek, Gisela; Antunes, EdithNanoparticles using a green synthesis production method is of increasing interest for biomedical applications. Zinc oxide is currently used in medicinal and cosmetic formulations, and zinc oxide nanoparticles have potential in biomedical applications. Antibiotic-resistant bacterial infections are of growing concern globally, and novel antimicrobial agents are drastically needed. In this work, zinc oxide nanoparticles were synthesized using Calendula officinalis flowers aqueous extract and zinc nitrate hexahydrate using a single-pot green synthesis approach. The synthesized particles were calcined and characterised using UV-Vis spectrophotometry, Dynamic Light Scattering (DLS), X-Ray Diffraction (XRD), Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Thermogravimetric Analysis (TGA), Fourier-Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance Imaging (NMR). Quasi-spherical, hexagonal phase zinc oxide nanoparticles with core size <10nm according to TEM and average size of ~53nm according to XRD were successfully synthesized. The particles tended towards agglomeration. Antioxidant assays were run to determine the reducing power, antioxidant activity, and presence of polyphenols in the final products. The final product possessed antioxidant activity, polyphenols and had reducing power, confirming the constituents from Calendula officinalis playing a role in the reduction of the zinc ions and capping of the zinc oxide nanoparticles. Minimum inhibitory concentration of the samples by tube dilution was conducted to determine potential of the samples as antimicrobial agent against methicillin-resistant Staphylococcus aureus, using Clindamycin as control. Results of antimicrobial studies indicate the need for investigation of MIC using a variety of concentrations of the samples, and a variety of solvents, in order to determine exact MIC values. Further work is needed to improve the methodology to obtain particles that have a narrow size distribution, that are well dispersed, and are of higher concentration to be considered for antimicrobial use in the biomedical field.Item Antimicrobial, anticancer and catalytic activities of green synthesized Avocado seed extract-gold nanoparticles(University of the Western Cape, 2019) Ngungeni, Yonela; Madiehe, Abram; Dube, AdmireNature through billions of years of trial and error has produced an immeasurable amount of natural systems like plants, birds and animals. The intelligence of nature is hidden in these natural systems and researchers are turning towards “Nature’s intelligence” to find inspiration and advance novelty in the development of nanomaterials. Gold nanoparticles (AuNPs) have unique optical, electronic and physicochemical features which has gained them popularity and widespread exploitation in various applications. The conventional methods used for AuNPs synthesis employs toxic chemicals which makes these NPs unsafe for biomedical applications. Hence, there is a search for new, ‘green’ and more cost effective methods for AuNPs synthesis. Plant extracts are regarded as a highly desirable system for nanoparticle synthesis due to their tremendous capability to produce a wide range of phytochemicals that can act as reducing agents. The main goal of this study was to synthesize AuNPs in a cost effective manner without the use of toxic chemicals in the synthesis process. Avocado seeds which are an agricultural waste by-product were used for the biosynthesis of AuNPs. The study reports on the synthesis optimization, characterization and activities of the biogenic AuNPs. The avocado seed extract mediated - AuNPs (AvoSE-AuNPs) were optimized by varying reaction parameters and characterized by UV-visible, Dynamic Light Scattering (DLS) and High Resolution Transmission Electron Microscopy (HRTEM), Zetasizer and Fourier Transform Infrared Spectroscopy (FTIR). The formation of AvoSE-AuNPs had an absorption maximum at 534 nm. HRTEM and DLS confirmed that the NPs were polydispersed and present in different shapes. The presence of phytochemical constituents on the AvoSE-AuNPs were confirmed by FTIR. Their potential antibacterial activity was tested on bacterial strains known to exhibit resistance to a number of current antibiotics. The catalytic activity of AvoSE-AuNPs was also assessed as a means to contribute to the development of new methods aimed at alleviating organic pollutants such as nitrophenols in the environment. The AvoSE-AuNPs demonstrated excellent catalytic activity in the reduction of 4-NP by NaBH4 as shown by the rapid decrease in the nitrophenolate absorption band at 400 nm and the appearance of new absorption band at 298 nm, revealing the formation of the 4-aminophenol. Furthermore, the rate constants calculated demonstrated that the reaction occurs faster in the presence AvoSEAuNPs. The AvoSE-AuNPs showed low significant cytotoxicity. Cell cycle analysis was conducted to further investigate the apparent exhibited toxicity of the AvoSE-AuNPs. The results showed that in both cell lines treated with AvoSE-AuNPs and AvoSE there was a ii | P a g e disruption in the regulation of cell cycle. Cell cycle analysis helped improve understanding of the low cytotoxicity observed by the MTT assay results. The results presented in this study clearly demonstrate the feasibility of using AvoSE for the synthesis of AuNPs. This study demonstrated that AvoSE mediated AuNPs synthesis is a greener alternative as it abides by the green chemistry principles. Furthermore, the study outcomes contributed to minimizing environmental pollution by finding use for agricultural waste and thus ultimately adding value to the field.Item The assessment of the bactericidal effect of green synthesized silver nanoparticles against a panel of infectious microorganisms(University of the Western Cape, 2016) Mokone, Mmola; Beukes, D. R.; Antunes, E. M.; Meyer, M.The emergence of multiple drug resistant microorganisms poses a major threat to human life. These microorganisms have made the currently used antibiotics ineffective and therefore continue to thrive. Therefore, there is a need for development of new, broad-secptrum antibiotics which is effective against almost every infectious microorganism. These antibiotics should ensure high effectiveness against the infectious pathogens while it is less detrimental to human health. Thus the search is channelled in nanoscience and nanotechnology in order to develop antibiotics that can kill infectious microorganisms effectively and hindering the development of drug resistance by these microorganisms. Nanoscience is the study of properties of a material when reduced to it smallest size (below 100 nm). It is a newly developing field of science which includes chemistry, physics and biology and has made it easy to synthesise nanomaterials for applications in many sectors of life including in medicine. The synthesis of nanoparticles can be achieved by physical and chemical methods. However, these methods are energy and capital intensive. Additionally, chemical method of synthesis uses chemicals that may be toxic which restrict the use of resultant nanoparticles in medicine. Therefore, there is a need for the use of eco-friendly methods of nanoparticle synthesis. The synthesis of silver and gold nanoparticles in this study was carried out by a green synthesis method, at room temperature, using an aqueous extract from the endemic brown alga Sargassum incisifolium. For comparison, commercially available brown algal fucoidans were also used to synthesise these nanoparticle, in addition to conventional methods of synthesis. The formation of nanoparticles was followed by the use of UV-Vis spectrophotometry. The characterization of the nanoparticles was done by TEM, XRD, DLZ and FT-IR. The rate of nanoparticle formation varied with specific reducing agent used. The faster reaction rate was recorded with S. incisifolium aqueous extracts pretreated with organic solvents while extracts obtained without this pretreatment produced slightly slower reaction rates. However, the commercially available fucoidans were less effective and required elevated temperatures for nanoparticle formation. Sodium borohydride reduction of silver nitrate was faster than the biological methods while the reduction of auric chloride by the S. incisifolium extracts and sodium citrate proceeded at similar rates. The nanoparticles synthesised with the help of the untreated aqueous extract were bigger than those synthesised from pre-treated extracts with both giving irregular shaped of nanoparticles. Also the nanoparticles formed from commercially available fucoidans were not of the same size, with bigger sizes recorded with Macrocystis fucoidan and smaller sizes with Fucus fucoidan. The shapes of nanoparticles from these fucoidans were spherical. From the conventional method, the nanoparticle sizes were smaller compared to the green synthesised nanoparticles and were predominantly spherical. The silver nanoparticles synthesised from the Sargassum aqueous extracts showed excellent antimicrobial activity against five pathogenic microorganisms including A. baumannii, K. pneumoniae, E. faecalis, S. aureus, and C. albicans. The gold nanoparticles were much less effective. To adequately assess the antimicrobial activities of the nanoparticles, it is or paramount importance to also asses their cytotoxicity activity. Three cell lines were used in this study namely, MCF-7, HT-29 and MCF-12a. The silver nanoparticles were found to be toxic to HT-29 and MCF-7 cell lines, exhibiting sligtly less toxicity against MCF-12a cells. The gold nanoparticles showed lower toxicity but a similar trend was observed.Item An assessment of the dietary intake of pregnant women in the West Coast / Winelands region, Western Cape Province: relation to low birth weight(University of the Western Cape, 2008) Jaffer, Sharmilah; Swart, E.C.; Jackso, D.; Faculty of Community and Health SciencesThis secondary analysis aimed to develop dietary scores to assess the dietary intake of pregnant women in the West Coast/ Winelands region and determine the association with LBW. Further to determine the association between the dietary scores and maternal socioeconomic and socio-demographic characteristics and maternal smoking and/or alcohol consumption during pregnancy.Item Association between antioxidant activities and drought responses of two contrasting sugar beet genotypes(University of the Western Cape, 2017) Majola, Anelisa; Ludidi, NdikoSugar beet (Beta vulgaris L), which belongs to the Amaranthaceae family is a biennial crop widely used as a source of domestic sugar in the European countries, United States, China and Japan. However, in South Africa, sugar cane is the sole source of natural sugar and therefore sugar beet is a potential source of bioethanol since it would not be in direct competition with a food crop for sugar production. Thus, sugar beet can be utilized as a renewable source of energy, reducing the need and use of fossil fuels (such as petrol) that produce greenhouse gases and consequently cause global warming and climate change. Nonetheless, drought stress is one of the major factors limiting the growth of sugar beet, resulting in sugar beet yield reductions worldwide.Item AtNOGC1 protein bioelectrode for the determination of stress signalling molecules - Nitric Oxide (NO), Carbon Monoxide (CO) and Calcium ion (Ca2+)(University of the Western Cape, 2018) Tshivhidzo, Tsumbedzo Tertius; Mulaudzi-Masuku, Takalani; Iwuoha, Emmanuel; Ajayi, FanelwaIt has been estimated that the world population will reach about 10 billion by the year 2050 and in order to accommodate the increased demand of food, the world agricultural production needs to rise by 70 % in the year 2030. However, the realisation of the goal in food production is hindered by limited arable land caused by urbanisation, salinisation, desertification and environmental degradation. Furthermore, abiotic and biotic stresses affect plant growth and development, which lead to major crop losses. The long term goal of this study is to improve food security by producing genetically engineered agricultural crops that will be tolerant to diverse stresses. This research aims at developing stress tolerant crops through the determination of important signalling molecules and second messengers, such as nitric oxide (NO), carbon monoxide (CO) and calcium ion (Ca2+), which can bind to plant proteins such as AtNOGC1 in order to induce stress tolerance in plants.Item Bacteriophage diversity in haloalkaline environments(University of the Western Cape, 2013) Nemavhulani, Shonisani; Tuffin, MarlaThere are limited reports on virus population in haloalkaline environments; therefore the aim of this study was to investigate the genetic diversity and biology of bacteriophage communities in these environments. Bacteria were isolated to be used as phage hosts. One bacterium from Lake Magadi and four bacteria from Lake Shala were successfully isolated from sediment samples. A further two Lake Shala bacterial hosts from the IMBM culture collection were also used to isolate bacteriophages. Bacterial isolates were identified to be most closely related to Bacillius halodurans, Halomonas axialensis, Virgibacillus salarius, Bacillus licheniformis, Halomonas venusta, Bacillus pseudofirmus and Paracoccus aminovorans. Bacteriophages were screened using all bacteria against sediment samples from both Lake Shala and Lake Magadi. One phage was identified from Lake Magadi sediments (MGBH1) and two phages from Lake Shala sediments (SHBH1 and SHPA). TEM analysis showed that these phages belong to three different dsDNA phage families; Siphoviridae (MGBH1), Myoviridae (SHBH1) and Podoviridae (SHPA). All phages showed different genome sizes on agarose gel. Due to the small genome size, phage SHPA was chosen for further investigation. Partial, genome sequence analysis showed homology to both bacterial and phage proteins. A further investigation of phage diversity in this environment is essential using metagenomic approaches to understand these unique communities.Item Barley anther culture: determining the optimal pre-treatment for green plant regeneration(2013) Horn, Marizanne; du Preez, MarleneDoubled Haploid (DH) Technology is an important tool for plant breeding and biotechnological applications as it accelerates the breeding cycle of plants by shortening the time required to attain homozygosity. Anther culture has become one of the most frequent and well-established methods for the induction of haploid embryogenesis and regeneration in barley. Anther culture is easily reproduced and workable for a wide range of genotypes. The aim of this study was to determine the optimal pre-treatment for barley anther culture. Three pre-treatments, 0.3 M Mannitol, 0.7 M Mannitol and a cold treatment with a moist cloth (CMC), were studied. The results suggest that CMC is the optimal pre-treatment to use for green plant regeneration. Anthers treated with CMC showed a higher response percentage than that of 0.7 M Mannitol and 0.3 M Mannitol. CMC also induced a significantly higher callus formation and green plant regeneration frequencies than 0.7 M Mannitol and 0.3 M Mannitol. Further research has to be conducted to further optimize green plant yields per treatment as well as reduce the number of albinos regenerated through barley anther culture.Item Bioactivity and genome guided isolation of a novel antimicrobial protein from Thalassomonas viridans(University of the Western Cape, 2019) Adams, Shanice Raquel; Trindade, MarlaThe continued emergence of bacterial resistance to the antibiotics currently employed to treat several diseases has added to the urgency to discover and develop novel antibiotics. It is well established that natural products have been the source of the most effective antibiotics that are currently being used to treat infectious diseases and they remain a major source for drug production. Natural products derived from marine microorganisms have received much attention in recent years due to their applications in human health. One of the biggest bottlenecks in the drug discovery pipeline is the rediscovery of known compounds. Hence, dereplication strategies such as genome sequencing, genome mining and LCMS/MS among others, are essential for unlocking novel chemistry as it directs compound discovery away from previously described compounds. In this study, the genome of a marine microorganism, Thalassomonas viridans XOM25T was mined and its antimicrobial activity was assessed against a range of microorganisms. Genome sequencing data revealed that T. viridans is a novel bacterium with an average nucleotide identity of 81% to its closest relative T. actiniarum. Furthermore, genome mining data revealed that 20% of the genome was committed to secondary metabolisms and that the pathways were highly novel at a sequence level. To our knowledge, this species has not previously been exploited for its antimicrobial activity. Hence, the aim of this study was to screen for bioactivity and identify the biosynthetic gene/s responsible for the observed bioactivity in T. viridans using a bioassay-and-genome- guided isolation approach to assess the bioactive agent. The bioassay-guided fractionation approach coupled to LCMS/MS led to the identification of a novel antimicrobial protein, TVP1. Bioinformatic analyses showed that TVP1 is a novel antimicrobial protein that is found in the tail region of a prophage in the T. viridans genome. Phage-derived proteins have previously been shown to induce larval settlement in some marine invertebrates. Since the mechanism of action of TVP1 remains unknown, it remains a speculation whether it may offer a similar function. More research is required to determine the biotechnological application and the role of TVP1 in its host and natural environment.Item A biochemical and proteomic analysis of sugargraze sorghum under hyperosmotic stress(University of the Western Cape, 2015) Nxele, Xolisa; Ndimba, Bongani K.; Klein, AshwilSugargraze is a moderately drought tolerant sweet sorghum hybrid which is ideal for grazing, winter stand over and pit silage. A major advantage that Sugargraze has over other forages is its very high sugar content which improves feed quality thus increasing palatability and results in significantly reduced feed wastage. This study explored the influence of hyperosmotic stress on plant development, ROS accumulation, antioxidant capacity and the extent of cell death. Heat shock protein (Hsp70) expression immunoblotting assays were used to demonstrate whether the various treatment conditions induced stress within natural physiological parameters for the experimental material. This was coupled with the separation, visualization and identification of abundant proteins in Sugargraze leaves in response to hyperosmotic stress using two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MS). The results showed that hyperosmotic stress significantly influences plant development by reducing plant biomass and increasing the levels of ROS accumulation, proline content and subsequently reducing total chlorophyll content. An over accumulation of ROS in the form of hydrogen peroxide and lipid peroxidation was observed in the stressed plants which was supported by the extent of cell death. Although an increase in antioxidant enzyme activity (in the form of total enzymatic activity or individual isoform activity) in response to hyperosmotic stress was observed, this increase was not sufficient to counter the deleterious effects caused by the stress conditions hence the decrease in plant biomass and increase in cell death. Western blotting analysis of Sugargraze leaf tissues using Hsp70 antibodies showed that hyperosmotic stress induced Hsp70 expression to levels significantly higher than observed for the control plants. A total of thirteen CBB stained spots were selected for mass spectrometric identification, owing to their good resolution and abundance levels, and of these, nine were positively identified. Identified proteins were divided into functional categories including both known and novel/putative stress responsive proteins. Molecular and physiological functions of some of the proteins of interest identified will be subjected to further investigation via bioinformatic and molecular biology approaches.