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Molecular characterization of a new cassava geminivirus (ACMV-SA) in South Africa

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dc.contributor.author Essiedu EKO en
dc.date.accessioned 2016-09-22T11:18:14Z
dc.date.available 2016-09-22T11:18:14Z
dc.date.submitted 1995 en
dc.identifier.uri http://hdl.handle.net/20.500.11892/109545
dc.description.abstract Cassava (Manihot esculenta Crantz) is a perennial woody shrub grown mainly in Latin America, Africa and Asia for its starchy tuberous roots. The cassava crop provides a lot of calories for both animal and human consumption as well as starch for industrial use. Because of its broad adaptability to a variety of soil and climatic conditions (such as drought tolerance and ability to grow on depleted and marginal soil), the crop is very important to the agroeconomy of several tropical countries. The main threat to the crop's survival is the Cassava Mosaic Disease (CMD) caused by the African Cassava Mosaic Virus (ACMV). This virus is a bipartite geminivirus.<br><br> Recently a new strain of ACMV (i.e. ACMV-SA) has been observed to infect cassava crops in Kwazulu-Natal and Eastern Transvaal in South Africa. Initial studies of a 793 bp Polymerase chain reaction ( PCR) generated fragment sequence of the DNA-A component of the virus reveals that it shares a higher sequence homology with a monopartite TYLCV than with its other bipartite ACMV counterparts. This unique ACMV-strain is a cause for concern which needs to be further investigated so that an effective approach can be taken to combat its devastating influence on cassava in South Africa.<br><br> In this project further characterization of DNA-A of ACMV-SA was pursued so that- (1) antisense-ACI ORF (putative polymerase) expression could be developed in genetically engineered virus-resistant cassava; (2) the new virus could be compared with other similar geminiviruses. The characterization was done by: (1) Partially sequencing a 607bp ACI PCR-generated fragment of ACMV-SA cloned into Psil siteof pBluescript SK(-) and comparing the sequence with other geminiviruses; (2) constructing a restriction map of the DNA-A of ACMV-SA cloned into pBluescript KS so as to identify convenient restriction sites for future subcloning experiments. For the Agrobacterium transformation studies/in vitro plantlets, cal- lus and suspension cell cultures for cassava and tobacco were maintained. Protoplast isolation from cell suspension cultures was carried out for purposes of inoculating them with DNA-A of ACMV-SA. An attempt was made to clone the 607bp ACI fragment of ACMV-SA into the plant transformation vectors pBIN19 and pBI121.<br><br> In this study, 371bp of the 607bp ACI fragment were sequenced. Sequence comparisons with ACI regions of other geminiviruses indicated that ACI ACMV-SA shared the highest sequence homology with ACMV-Nig (from Nigeria, i.e 80%). However, it also showed a reasonably high sequence homology with To- mato Yellow Leaf Curl Virus (TYLCV)-isolates from Italy (75% with TYLCV from Sardinia and 73% with TYLCV from Sicily) as well as 78% sequence homology with Tomato Leaf Curl Virus (ToLCV) from Australia.<br><br> The restriction map of DNA-A of ACMV-SA was established but a convenient restriction site for subcloning the whole of AC 1 putative open reading frame (ORF) of the DNA-A genome was not observed. However, using Pstl and EcoRl a large part of it could be subcloned. Cloning of the 607bp ACI fragment of ACMV-SAinto plant transformation vectors pBIN19 and pBI121 was not achieved. The transformation control experiments did work, hence the reason for cloning failure may be due to the ligation step.<br><br> Healthy plantlets of Nigerian cassava cultivars TMS87100609, TMS63397 and tobacco (Nicotiana tabacum) with developed roots, stems and leaves were successfully propagated from their nodal explants. Calli from immature leaves of thecassava and tobacco cultivars were successfully induced on Murashige and Shoog (MS) medium containing 8mg/l 2,4-D. Cassava calli were creamy in appearance and tobacco calli were greenish in colour. It took 3 weeks to induce the cassava calli while in tobacco 2 weeks was sufficient to obtain callus. Cell suspension cultures of the cassava and tobacco cultivars were initiated using friable calli as inocula. The suspension cells of both cassava and tobacco cultivars were subcultured two weeks after initiation of the primary cultures. Suspension cells counts showed that the viable cell density range of 0.5 - 2.0 x 10<sup>6</sup> cells per ml was attained after 14 days, after which subculturing took place.<br><br> Protoplasts were isolated from tobacco and cassava suspension cultures after 5h and 8h of incubation respectively in 2% cellulase Onozuka RIO and 0.1% pectolyase Y23. en
dc.language English en
dc.title Molecular characterization of a new cassava geminivirus (ACMV-SA) in South Africa en
dc.type Masters degree en
dc.description.degree MSc en


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