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

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dc.contributor.author Makwarela, Murunwa
dc.date.accessioned 2006-11-13T12:08:37Z
dc.date.available 2006-11-13T12:08:37Z
dc.date.issued 2006-11-13T12:08:37Z
dc.identifier.uri http://hdl.handle.net/10539/1640
dc.description Faculty of Science School of Molecular and Cell Biology 9714718d MMakwarela@arc.agric.za en
dc.description.abstract Cassava (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 en
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dc.language.iso en en
dc.subject Cassava en
dc.subject Genetic en
dc.subject Engineering en
dc.subject CMD en
dc.subject SACMV en
dc.title 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 en
dc.type Thesis en


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