3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Expanding the knowledge of genetically modified cassava through development of stacked traits for resistance and enhanced starch(2019) Walsh, Helen AnnCassava (Manihot esculenta Crantz) is ranked as the world's fourth most important food crop (FAO, 2016). Cassava mosaic disease (CMD), caused by 9 species of cassava mosaic geminiviruses including South African cassava mosaic virus, African cassava mosaic virus and East African cassava mosaic Cameroon virus (Brown et al, 2015), is one of the greatest hurdles to cassava cultivation in sub-Saharan Africa. The most promising method of improving cassava is through transgenic technology. The over-all aim of this project was the improvement of cassava through; improved disease resistance to South African cassava mosaic virus, and improved starch yield though the down-regulation of two genes involved in starch synthesis: plastidial Adenylate kinase and Uridine-5'-monophosphate synthase. In addition to these aims, another study conducted here investigated the off-target effects caused by a triple stacked construct targeting African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV) and South African cassava mosaic virus (SACMV) which was found to be toxic to cassava friable embryogenic callus (FECs). Previously, in order to increase CMD-susceptible cassava model cultivar 60444 resistance to SACMV, a RNA hairpin construct derived from the overlapping region of the replication associated protein (AC1)/ putative virus suppressor (AC4) of SACMV was used to transform cv.60444 (Taylor, 2009). This study reports on the screening of transformed lines for SACMV resistance. Three independent lines: O135, O13-8 and O12-2 showed tolerance to SACMV, with decreased symptom severity and viral loads compared to untransformed cv.60444 control. One line, O13-8, also showed a recovery phenotype associated with the resistant TME3 cultivar. This is the first time tolerance to SACMV has been reported in cassava cv.60444. The second aim of project was further elucidate the mechanism by which a triple stacked construct (pC-AES) targeting the AC1/AC4 overlap of ACMV, as well as the AC1/IR of SACMV and EACMV, induced 'off-target effects' in cv.60444, in order to improve virus target selection for future studies. This construct produced 'off-target effects' including high levels of mortality and very low transformation efficiency in FECs and regenerated lines also showed aberrant phenotypes including stunted growth and misshaped leaves. In order to confirm that the observed off-target effects were due to the stacked construct, possible siRNAs associated with the triple stacked construct were predicted bioinformatically, and siRNAs were compared to the cassava genome. Ten predicted gene targets which had partial homology to the predicted siRNA were identified, seven of which showed differential gene expression in FECs transformed with the pC-AES triple construct. The differentially regulated genes are involved in plant development, as well as defence response. These results show the limitations of PTGS, where the expression viral targets could interfere with the host biology. The third aim of this project was to increase starch yields through the down regulation of two genes plastidial Adenylate kinase (ADK) and Uridine-5'monophosphate (UMP) synthase, which are involved in starch synthesis. HairpinRNA constructs targeting ADK and UMP synthase, inserted into pCambia 1305.1 transformation vectors were used to transform cassava cv.60444 FECs. Ten independent UMPS lines and eight independent ADK lines were produced and were assessed over a 10 month period for tuber development. Two ADK lines, (1 and 5) and four UMPS lines (1, 2, 13 and 17) as well as control cv.60444 produced storage roots. Two of the UMPS lines storage roots (13 and 17) were significantly greater in size and weight than the untransformed regenerated control cv.60444 and three of the lines (1, 13 and 17) had a dry weight percentage mass higher than 25%, indicating high starch content. Analysis of the relative expression of UMP synthase in the 4 lines showed that there was a correlation between the increased storage root weight and the decreased expression of UMP synthase. The ADK lines did not produce any storage roots that were significantly greater than the control lines, although ADK 5 showed down-regulation of the ADK. Due to time constraints, no further testing of the starch content of these lines was performed, but this will be done in future studies. This is the first report of increased in storage root yields in cassava cv.60444 transformed with hp-RNA targeting UMP synthase. This study contributes to improvement of cassava cv.60444, through the use of RNAi technology. Although several cassava lines have been developed which show increased resistance to CMG including ACMV and Sri Lankan cassava mosaic virus, this is the first report of tolerance to SACMV. Further, the improved storage root yield shown by UMPS lines increases the commercial value of cassava. These results could also contribute to further improvement in cassava, as the transgenes could be stacked to further improve yield. Further understanding of the production of 'offtargets' can also be used in improve transgene selection.Item Transcriptome analysis of cassava (Manihot esculenta Crantz) in response to mealybug (Phenacoccus manihoti) infestation(2018) Rauwane, Molemi EvelynCassava mealybug (Phenacoccus manihoti) is one of the most damaging pests of cassava (Manihot esculenta Crantz) globally. While biological control of mealybugs through the use of natural predators has been successfully practiced, breeding for resistant cultivars remains an important means of control. Understanding plant responses to insect herbivory, by determining and identifying differentially expressed genes (DEGs), is a vital step towards the understanding of molecular mechanisms of defence responses in plants and the development of resistant cultivars by gene editing. Mealybug isolates were collected from different cassava growing regions of South Africa and a detailed morphological and molecular characterization of the pests was undertaken. Morphological and molecular analysis confirmed the mealybug identity as Phenacoccus manihoti (Matile-Ferrero). The positively identified mealybug isolates were used to artificially infest three cassava genotypes with contrasting response to mealybug. The response of cassava genotypes AR23.1 (which has resistance to multiple diseases and to green mites), P40/1 (which has no known resistances) and 98/0581 (which has multiple pest resistances and resistance to cassava mosaic disease) was further investigated by analysing their transcriptome after mealybug infestation at 24 and 72 hours in comparison with non-infested mock tissues. A total of 301, 206 and 269 transcripts from AR23.1, P40/1 and 98/0581 were differentially expressed (log2 fold and P ≤ 0.05) per time point following mealybug infestation. In two genotypes (AR23.1 and 98/0581), mealybug infestation resulted in a higher number of down-regulated than up-regulated genes between mealybug-infested and mock-infested tissues, while in P40/1 between infested leaves at 24 hpi and 72 hpi, more genes were up-regulated than were down-regulated. There was an increase in the number of DEGs from 24 hpi to 72 hpi suggesting an early induction of defence responses during the mealybug infestation. Gene expression was further compared between AR23.1, P40/1 and 98/0581 to determine whether there were any genotype-specific expression patterns. Within the Gene Ontology (GO) functional classification, DEGs in the class Secondary Metabolic Processes were significantly enriched in AR23.1 in comparison with P40/1 and 98/0581. Additionally, DEGs in the class Regulation of Molecular Function were significantly enriched in P40/1, while DEGs in the classes Reproduction and Reproductive Processes, and Nutrient Reservoir, were enriched in the multi-pest resistant 98/0581 genotype compared to P40/1 and AR23.1. In the metabolic pathway analysis performed using the KEGG database, DEGs in the classes ABC Transporters and MAPK signalling pathways were present in AR23.1 and 98/0581; and not in P40/1. There were both up- and down-regulated genes that were mapped to the classes Plant-Pathogen Interactions, and Plant Hormone Signal Transduction pathways in genotypes AR23.1 and 98/0581; while in P40/1, there were only down-regulated genes classified under Plant-Pathogen Interactions pathway. The up-regulated DEGs associated with these pathways are suggested to be involved in defence in response to P. manihoti feeding since these stress-associated genes, including those related to transcription factors, phytohormones and secondary metabolism were significantly induced in the AR23.1 (resistant) and 98/0581 (tolerant) genotypes, and not in the P40/1 (susceptible) genotype. Stress-associated genes such as 2-oxogluterate / Fe (II)-dependent oxygenase superfamily protein, terpene synthase 21, heat shock family proteins and cytochrome P450 superfamily proteins were induced in AR23.1 and 98/0581 genotypes. The results revealed a significantly different response to mealybug infestation in the three genotypes studied, with genotypes AR23.1 and 98/0581 showing a higher proportion of differentially expressed transcripts compared with the susceptible genotype P40/1 during infestation with mealybugs. Putative candidate defence-related genes that were overexpressed in the AR23.1 genotype post-infestation will be useful in future functional studies towards the control of mealybugs. These results will further form the basis for more detailed future studies on the specific role of all the differentially expressed transcripts identified. The transcripts identified in this study will also contribute significantly to the cassava EST database, and can be used to improve the annotation of the cassava genome.Item Screening of cassava improved germplasm for potential resistance against cassava mosaic disease(2017) Mvududu, DonTafadzwa KudzanaiWith growing populations and climate change associated drought predicted for the future, cassava can provide one solution for food security and a source of starch for industrial use and biofuels in South Africa, and other countries in the SADC region. One of the severe constraints on cassava production is cassava mosaic disease (CMD) caused by cassava infecting begomoviruse species, including African cassava mosaic virus (ACMV), South African cassava mosaic virus (SACMV) and East African cassava mosaic virus (EACMV). Cassava begomoviruses (CBVs) are responsible for significant yield loss of the starchy tubers. Since no chemical control of virus diseases of plants is possible, one approach to develop virus resistance is via biotechnology, through genetic engineering (GE) of cassava with hairpin RNA (hpRNA) silencing constructs that express small interfering RNAs targeting CBVs and preventing severe disease development. The aim of this project was to subject previously transformed five CMM6 cassava lines (cv. 60444 transformed with a non-mismatched Africa cassava mosaic virus-[Nigeria:Ogorocco;1990] (ACMV-[NG:Ogo:90])-derived hpRNA construct, six AMM2 (cv. 60444 transformed with a mismatched ACMV-[NG:Ogo:90]-derived hpRNA construct), six CMM8 cassava lines (cv.60444 transformed with a non-mismatched SACMV BC1-derived hpRNA construct) and seven AMM4 cassava lines (cv.604444 transformed with a mismatched SACMV BC1-derived hpRNA construct) to reproducible trials, and evaluate for response to virus challenge. The ACMV-[NG:Ogo:90] hpRNAi constructs target 4 overlapping virus open reading frames (ORFs) (AC1 replication associated protein/AC4 and AC2 transcriptional/AC3 replication enhancer), while the SACMV hpRNAi constructs target the cell-to cell movement BC1 ORF. Non mismatched constructs consist of a transformation cassette that has an intron separating the sense and antisense arms of the viral transgene whilst mismatched constructs have the sense arm of the viral transgene treated with bisulfite to induce base mutation. This mutated sense arm is then separated from the non mutated antisense arm by a small spacer. Furthermore, a 229 bp inverted repeat hpRNA construct (DM-AES) was designed to target ACMV-[NG:Ogo:90] 117 nt putative promoter region (2714-49 nt), a 91 nt overlapping sequence (1530-1620 nt) between ACMV-[NG:Ogo:90] AC1 3’ end and AC2 5’ end (AC1 3’/AC2 5’-ter) as well as being efficient against SACMV and EACMV due to the inclusion of a 21 nt conserved sequence (1970-1990) of AC1/Rep shared between ACMV, EACMV and SACMV. Cassava landrace T200 friable embryogenic callus (FEC) were transformed with this construct. The selected transgenic lines were infected with either ACMV-[NG:Ogo:90] (CMM6 and AMM2 transgenic lines) or SACMV (CMM8 and AMM4 transgenic lines) by agro-inoculation and monitored at 14, 36 and 56, 180 and 365 days post infection (dpi) for symptom development, plant growth and viral load. From the ACMV trials 3 lines (CMM6-2, CMM6-6 and line AMM2-52) showed significantly lower symptom scores and lower viral load at 36, 56 and 365 dpi, compared with viral challenged untransgenic cv.60444. This phenotype is described as tolerance, not resistance, as despite ameleriorated symptoms virus replication persists at lower levels. From the SACMV infectivity trials even though all CMM8 and AMM4 transgenic lines had lower symptom severities and viral loads compared with infected untransformed cv.60444, the results were not highly significant (p˃ 0.05). From this study, tolerance or reduction of viral load and symptoms was attributed to the accumulation of transgene-derived siRNAs prior to infection. However there was no observable correlation between levels (semi-qauntitative northern blots) of siRNAs and tolerance or susceptible phenotypes. Tuber yield evaluation of the three tolerant lines (CMM6-2, CMM6-6 and line AMM2-52) showed that the tuber fresh and dry weight at 365 dpi was not affected by the viral presence. These are promising lines for larger greenhouse and field trials. A comparison between the two different constructs showed that the two tolerant CMM6 lines-2 and 6 appeared to perform better (viral load) compared with AMM2 tolerant line-52 with regards to levels of viral amplification. The mismatched construct in AMM4 lines and the nonmismatched construct in CMM8 lines induced the same viral and symptom severity score (sss) reduction. Transformation of T200 FECs with the DM-AES construct was unsuccessful due to the age (more than six months old) of the FECs. FECs are more likely to lose their regeneration and totipotent nature with age. We therefore propose the use of fresh T200 FECs in future transformation studies to test the DM-AES construct.Item The role of small RNAs in susceptibility and tolerance to cassava mosaic disease(2016) Rogans, Sarah JaneCassava (Manihot esculenta, Crantz) is considered to be an important food security crop consumed by over a billion peoples globally, many who subsist on it. Cassava mosaic disease (CMD) is one of the main biotic and economically important constraints to cassava cultivation in sub-Saharan Africa. Geminiviruses are the casual agents of CMD and cause disease to many staple food and cash crops of great economic importance worldwide. There are currently 11 species of Begomoviruses that belong to the Geminiviridae family. South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite (DNA A and DNA B components) begomovirus belonging to the family Geminiviridae, and is one of the causal agents of cassava mosaic disease (CMD) endemic to southern Africa. Various strategies to control CMD are currently being investigated, one of which is cis-genics, which involves manipulation of endogenous host genes to combat viral pathogens. In order to achieve this, it is imperative to elucidate molecular mechanisms involved in host-virus interactions. Endogenous small RNAs (sRNAs), including microRNAs (miRNAs), have been found associated with gene regulatory mechanisms in response to virus infection. Amongst the non-coding host sRNAs targeting viruses are small interfering RNAs (siRNAs) associated with posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS), which are involved in the host RNA silencing pathway. The RNA silencing pathway is a highly conserved basal immunity pathway involved in host defence against plant viruses. The aim of this study was to identify siRNAs and miRNAs associated with gene regulatory mechanism in response to SACMV infection and to determine if they a play a role in the susceptible or recovery phenotype observed in SACMV tolerant cassava landrace TME3 or T200, respectively. Furthermore, virus-derived siRNA (vsRNA) populations targeting the DNA A and B components of SACMV were also investigated. MicroRNAs (miRNAs) are an important class of endogenous non-coding single-stranded small RNAs (21-24 nt in length), which serve as post-transcriptional negative regulators of gene expression in plants. Despite the economic importance of Manihot esculenta Crantz (cassava) only 153 putative cassava miRNAs (from multiple germplasm) are available to date in miRBase (V.21). Therefore, both conserved and novel miRNAs needed to be identified in cassava before we could determine what association they had with SACMV infection. In this part of the study, mature sequences of all known plant miRNAs were used as a query for homologous searches against cassava EST and GSS databases, and additional identification of novel and conserved miRNAs were gleaned from next generation sequencing (NGS) of two cassava landraces (T200 from southern Africa and TME3 from West Africa) at three different growth stages post explant transplantation and acclimatization. EST and GSS derived data revealed 259 and 32 conserved miRNAs in cassava, and one of the miRNA families (miR2118) from previous studies has not been reported in cassava. NGS data collectively displayed expression of 289 conserved miRNAs in leaf tissue, of which 230 had not been reported previously. Of the 289 conserved miRNAs identified in T200 and TME3, 208 were isomiRs. Thirty-nine novel cassava-specific miRNAs of low abundance, belonging to 29 families, were identified. Thirty-eight (98.6%) of the putative new miRNAs identified by NGS have not been previously reported in cassava. Several miRNA targets were identified in T200 and TME3, highlighting differential temporal miRNA expression between the two cassava landraces. This study contributes to the expanding knowledge base of the micronome of this important crop. MicroRNAs play a crucial role in stress response in plants, including biotic stress caused by viral infection. Viruses however can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of miRNAs. This study aimed to understand the regulation of miRNAs in tolerant (TME3) and susceptible (T200) cassava landraces infected with SACMV. Next-generation sequencing was used for analysing small RNA libraries from infected and mock-inoculated cassava leaf tissue collected at 12, 32 and 67 dpi (days post-inoculation). The total number of differentially expressed miRNAs (normalized against mock-inoculated samples) across all three time points was 204 and 209 miRNAs, in TME3 and T200 infected plants, respectively, but the patterns of log2fold changes in miRNA families over the course of infection differed between the two landraces. A high number were significantly altered at 32 dpi when T200 and TME3 plants showed severe symptoms. Notably, in T200 69% and 28 (100%) of miRNA families were upregulated at 12 and 32 dpi, respectively. In contrast, TME3 showed an early pre-symptomatic response at 12 dpi where a high number (87%) of miRNAs showed a significant log2fold downregulation. Endogenous targets were predicted in the cassava genome for many of the identified miRNA families including transcription factors, disease resistance (R)-genes and transposable elements. Interestingly, some of the miRNA families (miR162, miR168 and miR403) that were significantly affected in both T200 and TME3 upon SACMV infection were shown to target proteins (DCL1, AGO1 and AGO2) that play important roles in the RNA silencing pathway. From results, we suggest that the early (12 dpi) miRNA response to SACMV in TME3 appears to involve PTGS-associated AGO1, DCL2 and a cohort of R genes belonging to the miR395 family which may prime the plant for tolerance and recovery downstream, while in T200, SACMV suppresses AGO1, AGO2 (at 32 and 67 dpi), and DCL2 (32 dpi) mediated RNA silencing, leading to severe persistent disease symptoms. This study provides insights into miRNA-mediated SACMV cassava interactions and may provide novel targets for control strategies aimed at developing CMD-resistance cassava varieties Endogenous small RNAs (sRNAs) associated with gene regulatory mechanisms respond to virus infection, and virus-derived small interfering RNAs (vsRNAs) have been implicated in recovery or symptom remission in some geminivirus-host interactions. Transcriptional gene silencing (TGS) (24 nt vsRNAs) and post transcriptional gene silencing (PTGS) (21-23 nt vsRNAs) have been associated with geminivirus intergenic (IR) and coding regions, respectively. In this Illumina deep sequencing study, we compared for the first time, the small RNA response to South African cassava mosaic virus (SACMV) of cassava landrace TME3 which shows a recovery and tolerant phenotype, and T200, a highly susceptible landrace. Interestingly, different patterns in the percentage of SACMV-induced normalized total endogenous sRNA reads were observed between T200 and TME3. Notably, in T200 there was a significant increase in 21 nt sRNAs during the early pre-symptomatic response (12 dpi) to SACMV compared to mock, while in TME3, the 22 nt size class increased significantly at 32 dpi. While vsRNAs of 21 to 24 nt size classes covered the entire SACMV DNA- A and DNA-B genome components in T200 and TME3, vsRNA population counts were significantly lower at 32 (symptomatic stage) and 67 dpi in tolerant TME3 compared with T200 (non-recovery). It is suggested that the high accumulation of primary vsRNAs, which correlated with high virus titres and severe symptoms in susceptible T200, may be due to failure to target SACMV-derived mRNA. In contrast, in TME3 low vsRNA counts may represent efficient PTGS of viral mRNA, leading to a depletion/sequestration of vsRNA populations, supporting a role for PTGS in tolerance/recovery in TME3. Notably, in TME3 at recovery (67 dpi) the percentage (expressed as a percentage of total vsRNA counts) of redundant and non-redundant (unique) 24 nt vsRNAs increased significantly. Since methylation of the SACMV genome was not detected by bisulfite sequencing, and vsRNA counts targeting the IR (where the promoters reside) were very low in both the tolerant or susceptible landraces, we conclude that 24 nt vsRNA-mediated RNA directed genome methylation does not play a central role in disease phenotype in these landraces, notwithstanding recognition for a possible role in histone modification in TME3. This work represents an important step toward understanding variable roles of sRNAs in different cassava genotype-geminivirus interactions. Also, by comparing the differences between a tolerant and susceptible host the aim is to achieve better understanding of the effect of pathogens on host sRNAome, an area that is deserving of me attention in plant systems. The expectation is that these findings presented in the PhD will contribute to the long-term goals of devising new methods of disease control against SACMV and understanding the complex interconnected mechanisms involved in virus-host interactome.Item Epidemiology of Cassava mosaic disease and molecular characterization of Cassava mosaic viruses and their associated whitefly (Bemisia Tabaci) vector in South Africa(2008-06-19T12:16:54Z) Mabasa, Kenneth GazaCassava mosaic disease (CMD) is caused by whitefly-transmitted geminiviruses and is a major constraint to cassava production in Africa. Field surveys were conducted in three (Bushbuckridge, Mariti and Tonga) cassava growing areas of Limpopo and Mpumalanga provinces in South Africa during two seasons (2004/2005 and 2005/2006). Results showed that a higher percentage (27.1%) of CMD infection was due to the use of infected planting materials compared to whitefly borne-infections (10.4%). Disease symptoms were generally mild. There was no change in disease incidence over the survey period. Molecular characterization of cassava mosaic geminiviruses (CMG’s), using differential primer PCR, restriction fragment length polymorphisms (RFLP’s), phylogenetic and recombination analysis and screening for satellite DNA’s. Differential primer PCR and RFLP’s showed that African cassava mosaic virus (ACMV) was the most prevalent virus in South Africa and that mixed infections were a common occurrence. Phylogenetic analysis and RFLP’s showed the presence of a ‘new’ strain of ACMV in South Africa. EACMV isolates from this study showed more frequent recombination compared to ACMV isolates. None of the samples tested positive for satellite DNA’s. Phylogenetic analysis of Bemisia tabaci using the mitochondrial cytochrome oxidase gene sequences revealed a ‘new’ sister clade of B. tabaci that is closely related to the previously identified southern African clade and the presence of the Q biotype that groups with Q biotypes of North African/Mediterranean origin. Good cultural practices, introduction of resistant cultivars and continuous monitoring are required to reduce the impact of CMD in South Africa.Item Engineering virus resistant transgenic cassava: the design of long hairpin RNA constructs against South African cassava mosaic virus(2008-03-19T06:20:56Z) Harmse, JohanABSTRACT Cassava is currently the second most important source of carbohydrates on the African continent. In the last two decades, cassava crops have been severely affected by outbreaks of cassava mosaic disease (CMD). South African cassava mosaic virus (SACMV) has been associated with CMD outbreaks in the Mpumalanga province. Advances in post-transcriptional gene silencing (PTGS) technology have provided promising new strategies for the engineering of virus resistance in plants. Inverted repeat (IR) constructs are currently the most potent inducers of PTGS, however, these constructs are inherently unstable. The purpose of this study was to develop IR constructs with an improved stability for the efficient induction of PTGS in plants. Two mismatched inverted repeat constructs, one targeting the SACMV BC1 open reading frame, the other targeting the Maize streak virus (MSV) AC1 open reading frame, were successfully created. Sodium bisulfite was used to deaminate cytosine residues on the sense arm of the constructs. The resulting number of GT mismatches was seemingly sufficient to stabilize the linear conformation of the IR constructs, as they were efficiently propagated by E.coli DH5!, and subsequently behaved like linear DNA molecules. Furthermore, it was found that the number of mismatches on the BC1 construct (17.5%) was ideal, as the subsequent stability of the predicted RNA hairpin was not affected. Due to the higher number of mismatches on the AC1 construct (23.5%), it was found that the loop region of the RNA hairpin was marginally destabilized. Despite this, long stretches of stable dsRNA were still produced from the AC1 IR construct, and is likely to induce PTGS. Interestingly, it was observed that the mismatched IR constructs, although still replicated in E.coli, were marginally destabilized in Agrobacterium. Therefore, it was deduced that the stability of a mismatched IR construct may be influenced by the particular intracellular environment of an organism. Due to the recalcitrance of cassava to transformation, a model plant system, Nicotiana benthamiana, was used to screen constructs for toxicity, stability, and efficiency of PTGS induction. Agrobacteriummediated transformation and regeneration of N. benthamiana was optimized, and 86% transformation efficiency was achieved when using leaf disk explants. It was found that the addition of an ethylene scrubber, potassium permanganate, substantially increased the rate of regeneration by reducing the frequency of hyperhydritic plants. Transgene iv integration was confirmed by PCR amplification of the hptII gene in the T-DNA region. Transgene expression was confirmed by screening for GUS and GFP reporter genes. No toxic responses to the transgene have been observed thus far. Studies are currently underway to confirm the stability of the mismatched IR constructs in N. benthamiana. PAGE Northern blotting is being done, as the detection of siRNAs derived from the transgene will confirm that constructs are functional. In addition, infectivity assays are underway to determine the efficacy of BC1 knockdown by a stably integrated construct. Due to the enhanced stability of mismatched IR constructs, they may be an appealing alternative to currently available intron-spliced, or exact matched hairpin systems.Item Optimisation of regeneration systems for a range of Cassava (Manihot Esculenta Crantz) cultivars suitable for growth in South Africa and transformation with SACMV N-REP gene(2006-11-13T12:08:37Z) Makwarela, MurunwaCassava (Manihot esculenta Crantz) is a vegetatively propagated root crop used as a staple throughout the tropics and subtropics. It is the fourth most important and cheapest staple food crop after rice, wheat and maize in developing countries, providing food for over 600 million people. However, its production is severely limited by a wide variety of viral and bacterial diseases, especially Cassava Mosaic Disease (CMD) which is caused by several geminivirus species including, South African cassava mosaic virus (SACMV), African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), Indian cassava mosaic virus (ICMV) and the Ugandan recombinant virus (UgV). In South Africa (SA), there has recently been an enormous upsurge of interest in cassava for industrial applications such as the manufacture of starch, animal feeds, and in its potential as a food security crop for marginalised farmers. However, due to serious losses in cassava yields by begomoviruses, such as SACMV, there is an urgent need for the development of appropriate systems that allows for transformation and regeneration of virus-resistant transgenic cassava cultivars suitable for diverse needs and growth requirements in different geographical areas in southern Africa. The potential application of cassava tuber disks as an alternative system to leaf tissue for transformation and regeneration was investigated. Furthermore, the antibiotic, carbenicillin, was tested as a possible shoot inducing factor. Disks from freshly-harvested cassava tubers were cultured on 25 different sets of MS supplemented with zeatin (0.01-5 mgl-1) and indole-3-acetic acid (0.01-5 mgl-1). Carbenicillin at 500 μgl-1 was included in each treatment as a potential viii organogenesis inducing factor. The results observed after 21 days in culture indicated that non-embryogenic friable callus formed readily on MS medium supplemented with MS vitamins, 30 gl-1 sucrose, 0.01 mgl-1 indole-3-acetic acid (IAA), 0.01 mgl-1 zeatin (ZEA), 500 μgml-1 carbenicillin and 0.8% agar, pH 5.8. Shoots or somatic embryos were never formed and only adventitious roots developed at a frequency of 60% on shoot induction medium supplemented with 2μM copper sulphate (CuSO4), 1 mgl-1 6-benzylaminopurine (BAP) and 0.5 mg-1 indole-3-butyric acid (IBA). The current study also investigated infection of cassava and tobacco by the SA begomovirus species SACMV, dimer A and B using the particle inflow gun. Full-length head-to-tail dimers of DNA-A and DNA-B of SACMV were constructed by digestion with SalI or EcoRI, respectively. The DNA-coated particles were used to shoot 3-week-old cassava plantlets (cv. TMS60444) at a pressure of 1500 psi using the Bio-Rad biolistic device. Thirty-day-old N. benthamiana seedlings were also inoculated in the same manner. In both cases young tender uppermost leaves were targeted (five plants inoculated and another 5 as control). Disease symptoms were recorded daily on the first emerging leaves. Cassava plantlets and tobacco seedlings showed infection by visibility of symptoms. On the other hand, control plantlets that were not inoculated were symptomless. Symptoms appeared 7 dpi in tobacco whereas mosaic symptoms became visible 14 dpi in cassava. The pre-requisite for any cassava transformation program that proposes to develop improved plants is the availability of a reliable regeneration system. Presently many laboratories that prioritize cassava research are able to reliably