Faculty of Science School of Molecular and Cell Biology 9714718d MMakwarela@arc.agric.za / 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

Student Number : 0413249D - MSc research report - Faculty of Science / Cassava is one of the most important staple crops in the world and is consumed by over 700 million people around the globe and is a profitable product commercially due to the high starch content of its tubers. One of the future aims is to produce cassava that is high yielding, resistant to cassava mosaic geminiviruses (CMGs) and high in starch content. To be able to achieve commercially attractive cassava varieties, research need to be carried out to investigate the virus resistance status of different cassava cultivars, which can later be used in the future breeding programme. In South Africa, cassava is used for commercial starch manufacturing purposes, as a cash crop and a food source by small-scale farmers. Cassava Mosaic Disease (CMD) is having a negative impact on yield of the crop globally and therefore dropping profitability of cassava on a commercial scale. The aims of this research were to propagate thirteen cassava cultivars and then to test them for virus susceptibility or resistance. Eleven cassava cultivars received from the International Institute of Tropical Agriculture (IITA) were tested for resistance or susceptibility against South African cassava mosaic virus (SACMV). Two local, commercial cultivars T200 and T400, were tested for East African cassava mosaic virus (EACMV) and African cassava mosaic virus (ACMV) resistance. Cassava cultivars were successfully propagated in vitro and thereafter transferred into soil and acclimatized to adapt to environmental conditions. When the plantlets were three weeks old, the plantlets were infected with cassava mosaic viruses. Plants were infected with SACMV via Agrobacterium-mediated transfer and infectious EACMV and ACMV monomers were used to biolistically bombard the plantlets. Resistance/susceptibility results of seven of the thirteen cultivars were obtained with SACMV, these cultivars being T200 (susceptible), T400 (susceptible), TME3 (highly resistant), I30572 (susceptible), I420251 (highly susceptible), I60506 (susceptible) and TMS60444 (susceptible). Due to destruction by fungal gnats eating the roots of the plants, acclimatization of the remaining six cultivars was not possible. Also, due to the nature of the biolistic equipment, infection of the cultivars with EACMV and ACMV was not achieved as the plantlets were not robust enough to survive the pressure.