A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Molecular and Cell Biology. Johannesburg, 2015. / With rising global food prices, growing populations, climate change and future demand for tuber crops for feed and potential energy source, cassava is well positioned to meet the needs of many countries in the SADC region, including South Africa. However a major constraint to cassava cultivation is cassava infecting begomoviruses (CBVs), including African cassava mosaic virus (ACMV) and South African cassava mosaic virus (SACMV). ACMV and SACMV belong to the family Geminiviridae, comprising of circular single-stranded bipartite. Symptoms associated with CBVs infection include yellow and/or green mosaic, leaf deformation, leaf curling and stunted plant growth. 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 to express hairpin RNA (hpRNA) silencing constructs against CBV. However cassava is recalcitrant and difficult to transform and regenerate. The aim of this study was to produce hpRNA/inverted repeat (IR) hpRNA constructs targeting ACMV AC1/4:AC2/3 open reading frames (ORF) and hpRNA targeting SACMV BC1 ORF to engineer hpRNA expressing transgenic cassava resistant to ACMV and SACMV. Furthermore, the approach was to stack two ACMV contiguous overlapping reading frames (AC1/4) and (AC2/3) in an attempt to improve resistance to CBV. However IR sequences are prone to unfavourable tight secondary structure formation known as cruciform structures. To circumvent this, one set of constructs (mutated sense-arm: mismatch constructs) were designed to contain sodium bisulfite deamination-induced mutations in the hairpin sense-arm making it less complementary to the antisense arm and therefore enhancing IR stability and cruciform junction formation. MM2hp (mismatch construct targeting ACMV AC1/4:AC2/3) and MM4hp (mismatch construct targeting SACMV BC1) were generated. The second construct set, non-mismatch: gateway, was designed based on the most currently used Gateway construct system. Gateway constructs contained an intron positioned between the IR fragments. MM6hp (non-mismatch construct targeting ACMV AC1/4:AC2/3) and MM6hp (non-mismatch construct targeting SACMV BC1) were generated. Similar to the deamination-induced mutations, the intron assisted with IR stability. ACMV- or SACMV-derived hpRNA constructs were transformed into model cassava cultivar cv.60444. Additionally, since few farmer-preferred cultivars or landraces have been transformed for resistance, South African high starch landrace T200 was also transformed with the hpRNA constructs. Agrobacterium-mediated transformation of friable embryogenic callus (FEC) was used and
plants regenerated. Several transgenic cv.60444 and T200 lines were regenerated. Cassava landraces are generally less amenable to transformation however were able to report 79 % and 76 % for model cv.60444 and landrace T200, respectively. T200 transformation efficiency reported in this study is 43% higher than previously reported. This is also the first report of South African cassava landrace T200 transformation with ACMV and SACMV-derived hpRNA constructs. Transgenic lines were selected and infected with ACMV and SACMV infectious virus clones. Lines were then monitored at 12, 32 and 67 days post infection (dpi) for symptom development, plant growth and SACMV and ACMV viral load. At 67 dpi, a more significant difference between transgenic lines and untransformed infected cv.60444 was observed. At 67 dpi, 69 % and 75% of ACMV AC1/4:AC2/3 and SACMV BC1 transgenic lines, respectively, showed lower symptoms and reduced viral load compared to control susceptible wild-type cv.60444, but comparable to virus-challenged non-transgenic tolerant landrace control TME3. Notably, a lack of correlation between viral load and symptoms was not always observed. Plant to plant variation was observed between individual transgenic lines generated from each construct (MM2hp; MM4hp; MM6hp and MM8hp) transformation events (A-MM2, A-MM4, C-MM6 and C-MM8). However, overall a positive correlation between symptoms and viral load was observed for virus challenge trials of transgenic lines generated from A-MM4, C-MM6 and C-MM8 transformation events, this overall positive correlation was observed at all 3 dpi (12, 32 and 67 dpi). A number of ACMV and SACMV tolerant transgenic lines were obtained for both mismatch and non-mismatch hpRNA expressing transgenic lines, where virus replication persisted, but symptoms were lower at 67 dpi compared to non-transgenic plants. CBV tolerance levels observed in transgenic lines expressing mismatch technology hpRNA was not significantly different to CBV tolerance levels observed in transgenic lines expressing non-mismatch hpRNA. Expression of ACMV and SACMV- derived constructs generated tolerant cassava lines, where tolerance is defined as plants displaying virus replication but lower to no symptoms. In addition to this, a recovery phenotype was observed in five MM2hp (ACMV AC1/4:AC2/4)- derived hp expressing transgenic lines at 365 dpi, where recovery is defined as no to mild symptoms after an initial period of symptoms, and a reduction in or no viral load. In five MM4hp (SACMV BC1)-derived hpRNA expressing transgenic lines, complete recovery was observed at 365 dpi; no symptoms and no detectable virus. From this study we propose that expression of CBV- derived hpRNA targeting ACMV AC1/4:AC2/4 and SACMV BC1 in CBV susceptible cv.60444
enhances cv.60444 ACMV and SACMV tolerance. Mismatch (mutated sense-arm) construct technology offered tolerance levels comparable to the more conventional and more expensive non-mismatch (Gateway) technology. We therefore also propose that the use of mismatch hpRNA technology in cassava genetic engineering can be used as an alternative approach to transgenic crop production. Promising transgenic lines, showing moderate SACMV and ACMV resistance, were identified and these will be used in further trials as they could be considered favourable to farmers.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/19289 |
| Date | January 2015 |
| Creators | Moralo, Maabo |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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