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Sorghum [Sorghum bicolor (L.) Moench] is important both as a food and feed crop in Zimbabwe. Its yield losses can be up to 100% when the crop is heavily infested by witchweeds [Striga asiatica (L.) Kuntze]. Witchweed resistant cultivars offer the most practical control option under smallholder (SH) farmer conditions and could become part of a sustainable integrated control strategy. Development of S. asiatica resistant cultivars by conventional breeding is slow and has been hampered by the lack of efficient and reliable screening techniques in breeding programs. Molecular markers that are linked to witchweed resistance can expedite the development of resistant cultivars through adoption of appropriate markerassisted selection (MAS) strategies. The objectives of this investigation were to study the inheritance or low germination stimulant (lgs) production in cultivar SAR 29 and to identify molecular markers that are linked to this trait. Low germination stimulant production is one of the recognised mechanisms of witchweed resistance. A segregating F2 population derived from crosses between cultivars SV-1 (high germination stimulant producer, Striga-susceptible) and SAR 29 (low germination stimulant producer, Striga-resistant) was used for this purpose. Parental and F2 genotypes were screened for lgs production using the agar gel technique (AGT). Maximum germination distance (MGD) was used as the index ofresistance. Deoxyribonucleic acid was extracted from agar gel-screened F2s, and DNA bulks were createdfrom 16 resistant (MGD25 mm) progeny. Bulked segregantanalysis (BSA) was done using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers. Ninety-nine of the primers that were polymorphic between parent genotypes (10 SSR and 89 RAPD) were then used to screen a total of 77 segregating F2 progeny. Linkage analysis was performed using the computer software MAPMAKER 3.0b. Segregation ratios of high to low F2 stimulant producers did not differ significantly (P³0.05) from the expected ratio of 3:1. It was therefore deduced that a single recessive gene controlled lgs production in cultivar SAR 29. No molecular marker was found to be linked to the lgs locus. Instead, linkage analysis resulted in the construction of a molecular marker linkage mapconsisting of 45 markers that were distributed over 13 linkage groups (LGs). The other fifty-four loci, including the locus for lgs production, were completely unlinked and could not be assigned to any linkage group. The LGs consisted of 2-8 markers, identified at a LOD grouping threshold of 4.0. The map spanned a total distance of 494.5 cM, Haldane.

Cowpea aphid-borne mosaic virus (CABMV) is a potyvirus that infects cowpea causing significant yield reduction. However, there is no durable natural resistance to the virus within the crop and genetic engineering for virus resistance was not possible because of a lack of an efficient, reliable and reproducible cowpea transformation and regeneration protocol. Coat protein-mediated resistance to CABMV was evaluated in Nicotiana benthamiana, a model host for the virus. The CABMV coat protein gene from a Zimbabwean isolate of the virus was optimised for expression in plants under a CaMV 35S promoter and cloned into the Hind III site of the binary vector plasmid pBI121 to result in the plasmid pBI121-CPk. The plasmid pBI121-CPk was used in Agrobacterium-mediated transformation of N. benthamiana leaf sections following the co-cultivation method. Regenerated plants were analysed by PCR and Southern blot hybridisation. R1 seedlings were assayed for kanamycin resistance and for presence of the coat protein and challenged with CAMBV-infected sap. Lines showing delayed symptom development were identified but no line showing immunity was identified. Delayed symptom development is significant resistance since it affords protection to the plants during the crucial earlystages of development and exerts little evolutionary pressure on the virus to evolve new strategies.

No Abstract Available JASSA: Journal of Applied Science in Southern Africa Vol.8(2) 2002:65-75

Nicotiana benthamiana plants were engineered to express sequences of the helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic potyvirus (CABMV). The sensitivity of the transgenic plants to infection with parental and heterologous viruses was studied. The lines expressing HC-Pro showed enhanced symptoms after infection with the parental CABMV isolate and also after infection with a heterologous potyvirus, Potato virus Y (PVY) and a comovirus, Cowpea mosaic virus (CPMV). On the other hand, transgenic lines expressing nontranslatable HC-Pro or translatable HC-Pro with a deletion of the central domain showed wild type symptoms after infection with the parental CABMV isolate and heterologous viruses. These results showed that CABMV HC-Pro is a pathogenicity determinant that conditions enhanced sensitivity to virus infection in plants, and that the central domain of the protein is essential for this. The severe symptoms in CABMV-infected HC-Pro expressing lines were remarkably followed by brief recovery and subsequent re-establishment of infection, possibly indicating counteracting effects of HC-Pro expression and a host defense response. One of the HC-Pro expressing lines (h48) was found to contain low levels of transgenic HC-Pro RNA and to be resistant to CABMV and to recombinant CPMV expressing HC-Pro. This indicated that h48 was (partially) posttranscriptionally silenced for the HC-Pro transgene inspite of the established role of HC-Pro as a suppressor of posttranscriptional gene silencing. Line h48 was not resistant to PVY, but instead showed enhanced symptoms compared to nontransgenic plants. This may be due to relief of silencing of the HC-Pro transgene by HC-Pro expressed by PVY.

The helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic virus (CABMV) was expressed in Escherichia coli and used to obtain HC-Pro antiserum that was used as an analytical tool for HC-Pro studies. The antiserum was used in immunofluorescence assays to study the subcellular location of HC-Pro expressed with other viral proteins in cowpea protoplasts in a natural CABMV infection, or in protoplasts transfected with a transient expression construct expressing HC-Pro separately from other viral proteins under the control of the 35S promoter. In both cases the protein showed a diffuse cytoplasmic location. Similar localisation patterns were shown in live protoplasts when the transient expression system was used to express HC-Pro as a fusion with the green fluorescent protein as a reporter. In an alternative expression system, the HC-Pro coding region was subcloned in-frame between the movement protein and large coat protein genes of RNA2 of Cowpea mosaic virus (CPMV). Upon transfection of protoplasts with this construct, HC-Pro was expressed as part of the RNA2 encoded polyprotein from which it was fully processed. In this case, the protein localised in broad cytoplasmic patches reminiscent of the typical CPMV induced cytopathic structures in which CPMV replication occurs, suggesting an interaction of HC-Pro with CPMV proteins or host factors in these structures. Finally, recombinant CPMV expressing HC-Pro showed a strongly enhanced virulence on cowpea and Nicotiana benthamiana consistent with the role of HC-Pro as a pathogenicity determinant, a phenomenon now known to be linked to its role as a suppressor of host defense responses based on post-transcriptional gene silencing.

Can public policies and research institutions in African countries provide safe and useful genetically modified (GM) food crops? This is an urgent question, recognizing that advancing GM food crops can be difficult, affected by global debate, and various regulatory protocols. Reaching farmers has been achieved in several countries only for GM cotton for insect resistant while approvals for food and feed crops lag behind. To address this question, we identified and examined public research pipelines for GM crops in Egypt, Kenya, South Africa and Zimbabwe. Genetic transformation events are reported for 21 crops. Findings are presented for events nearing final stages of development, analysis of the crops, traits and genes involved, and details regarding biosafety. The paper concludes with a summary offering various policies, institutional and regulatory suggestions. Key Words: Africa, biosafety, biotechnology, genetic modification, public research African Journal of Biotechnology Vol.3(11) 2004: 564-571