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Aflatoxins are toxic, and highly carcinogenic secondary metabolites of Aspergillus flavus and Aspergillus parasiticus. They pose a serious health hazard to humans and animals that consume contaminated grain. Recently, the National Maize Breeding Program at National Crop Resources Research Institute (NaCRRI) incorporated breeding for resistance to A. flavus infection and aflatoxin accumulation in its initiative to improve maize grain quality in Uganda. The breeding strategy had three major components: (i) identifying locally adapted, elite germplasm with resistance to A. flavus and reduced aflatoxin accumulation, (ii) improving locally adapted, elite germplasm for host resistance, and (iii) formation of new populations for pedigree breeding. To identify sources of resistance, we first compared effectiveness of media plating and media free techniques for assessment of kernel infection rate (KIR) on various germplasm. We generated 144 three-way test crosses and screened them together with their parental inbred lines and 4 single cross testers for host resistance to A. flavus and reduced aflatoxin accumulation. Using line by tester analysis, we identified 5 resistant inbred lines and 7 hybrids. We used the resistant inbred lines to make crosses with new sources of resistance from International Center for Maize and Wheat Improvement (CIMMYT) and International Institute for Tropical Agriculture (IITA) in diallel design. To strengthen this work, we conducted a survey to assess farmers’ knowledge on A. flavus and aflatoxins and their management practices. Results obtained provided us with a foundation for development and deployment of new germplasm with resistance to A. flavus and aflatoxin accumulation for improved grain quality for both domestic consumption and export.