Expanding the knowledge of genetically modified cassava through development of stacked traits for resistance and enhanced starch
Date
2019
Authors
Walsh, Helen Ann
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Abstract
Cassava (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.
Description
A thesis submitted in fulfilment of the requirement for the degree Doctor of Philosophy in Molecular and Cell Biology to the Faculty of Science, University of the Witwatersrand, Johannesburg, 2019
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Citation
Walsh, Helen Ann. (2019). Expanding the knowledge of genetically modified cassava through development of stacked traits for resistance and enhanced starch. University of the Witwatersrand, https://hdl.handle.net/10539/29292