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Cassava protoplast transformation using Agrobacterium tumefaciens

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dc.contributor.author McDonnell SL 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/20.500.11892/109554
dc.description.abstract The cassava (Manihot esculenta Crantz) root crop is one of the most important staple foods of the lowland tropics. The crop is the main food source for approximately five hundred million people, in over sixty countries worldwide. Cassava is mainly grown by peasant, subsistence farmers, but the crop also has the potential of being a lucrative cash crop. In South Africa, the cassava perennial shrub is the ideal alternative or seasonal rotational crop to both sugarcane and maize, due to its ability to grow in sandy, infertile soils in areas that have a low annual rainfall. The aim of this project was to determine a mechanism whereby foreign genetic material could be stably integrated into the cassava genome, and maintained in the plant line through successive plant generations. Transformation of genetic material encoding agronomically superior characteristics was therefore noted to be a rapid and preGise alternative to conventional cassava breeding programs. In this study the selectable genetic marker, β-glucuronidase (GUS), was successfully transformed into cassava protoplasts, isolated from leaf mesophyll cells. The bacterium-mediated transformation procedure made use of Agrobacterium tumefaciens' natural ability to stably insert a portion of plasmid DNA into the plant genome. Cassava plantlets were successfully cultured in MS growth medium, in vitro. Cassava protoplast isolation and resuspension in Thomas medium was then performed, and the protoplast developmental stages observed. Cell wall resynthesis was observed 4 to 5 days after culture initiation, and the first cellular division was observed 16 days after cell culture. The protoplasts underwent a series of cellular divisions to form microcalli within one month of culture. The triparental mating system was performed using A.tumefaciens strain LBA4404. The binary vectors pBI121 and pBIN19, housing the reporter genes β-glucuronidase (GUS) and β-galactosidase (l/acZ) respectively, were successfully transformed into individual bacterial colonies. Agrobacterium-mediated transformation was performed by the addition of LBA4404pBI121 to the protoplast digestion medium. Protoplast transformation took place at the stage of cell wall digestion, during protoplast isolation. Following Agrobacterium co-culture, the bacteria were removed from the protoplast cultures by separation in a mannitol concentration gradient, and the addition of various concentrations of carbenicillin and cefotaxime. The protoplasts treated with 50mg/l of the antibiotics yielded the highest GUS activity reaction rates. The success of protoplast transformation was determined using the pNPG GUS spectrophotometric assay. Transient GUS expression was detected two days after the transformation procedure, with a maximum GUS reaction rate of 0.0251 nanomoles p-nitrophenol/min/20μl protoplast cell extract analyzed. One week after bacterial infection, the assay was repeated. Residual GUS activity showed that GUS transformation had been successful. The highest reaction rate observed one week after culture was 0.00913 nanomoles p- nitrophenol/min/20μl protoplast cell extract. Cassava protoplast isolation, transformation, and GUS enzymatic activity detection was therefore accomplished within only four days. Twenty days after bacterial infection, viable microcalli were observed in the transformed protoplast cultures. Confirmation of the transformation results observed using the spectrophotometric assay could not be obtained using either electrophoretic or colorimetric GUS assays. en
dc.language English en
dc.title Cassava protoplast transformation using Agrobacterium tumefaciens en
dc.type Masters degree en
dc.description.degree MSc en


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