Sorghum (Sorghum bicolor L. Moench) ranks fifth worldwide in production among cereals. It is a major staple food for millions in Africa and Asia, and a major livestock feed grain in developed countries. However, the sorghum grain is poor in lysine content, limiting its value as food and feed. In this study, I hypothesize that reduction of some of the major storage proteins that are inherently poor in lysine through in vitro manipulation will result in the enhanced expression of proteins with a better lysine profile and, thus, increased overall grain lysine content. Sorghum genotypes were screened for in vitro amenability and a sorghum genotype-tissue culture medium combination that yielded the highest somatic embryo callus formation and regeneration potential, was identified. This resulted in the establishment of a sorghum biolistic transformation method with a transformation efficiency of 3.36%, the highest reported to date. Using genetic engineering tools, the enhancement of the nutritional quality of grain sorghum was achieved by increasing the seed lysine content. An RNAi co-suppression strategy was employed and resulted in 45.23 and 77.55% increase in whole seed and endosperm lysine increase, respectively. The co-suppression RNAi constructs targeted the endosperm specific suppression of three lysine-poor storage proteins, namely ä-kaf-2, ã-kaf-1 and -2, and an enzyme that catalyzes seed lysine degradation, lysine keto-gluterate reductase (LKR). Seven independent transgenic events displayed successful transgene integration for both the selectable marker gene and the target constructs. However, the Southern blot hybridization analysis revealed two transgenic events that displayed transgene re-arrangement at the 5’promoter end, thus resulting in a lack of suppression of target proteins. Variations in target proteins co-suppression was observed with Western blot analysis and RT-PCR for both the target kafirins and LKR suppression, and no lysine improvement was observed where no kafirin suppression occurred. The transgenic co-suppression of the target kafirins resulted in the endosperm structural change from a hard, corneous endosperm to a soft, floury endosperm, consistent with ã-zein suppression in the Opaque-2 maize mutant. / Thesis (PhD)--University of Pretoria, 2010. / Plant Science / unrestricted

Master of Science / Department of Grain Science and Industry / Rebecca Miller / Sorghum is a vital cereal crop grown in many regions around the world. Tolerance to harsh climates and low moisture conditions are unique traits making sorghum an economical choice in an era of global water scarcity. In recent years, sorghum has gained greater recognition as a gluten-free grain and is a safe alternative for individuals suffering from gluten sensitivities or celiac disease. Still, the lack of gluten proteins does not allow sorghum to form a viscoelastic dough. In this study reducing agents were added to improve functional properties of sorghum kafirins for bread baking. Study objectives were to determine the effect of reducing agents on protein body structure of sorghum kafirins, investigate the influence on the sorghum batter consistency, and evaluate the effects on the physical characteristics of sorghum bread. Protein analysis, accomplished using RP-HPLC, showed reducing agents, L-cysteine and sodium metabisulfite, reduced protein structure; increasing RP-HPLC total peak area up to 747% and 681%, respectively. Batter consistency was obtained using a RVA. Treatments of L-cysteine (2.5% fwb) expressed increased RVA peak viscosity and decreased final viscosity. Samples treated with sodium metabisulfite (500 ppm fwb) had increased peak viscosity, holding strength and final viscosity. Yeast activity of batter treated with ≥3000 ppm (fwb) sodium metabisulfite caused volume loss of 95% yet at 500 ppm (fwb) sodium metabisulfite did not have an effect. Batter with 2.5% (fwb) L-cysteine experienced reduced yeast activity after 20 min. Sorghum bread characteristics were altered. Loaf volume and crumb grain characteristics of bread produced using sodium metabisulfite (500 ppm) were equal to that of the control, while initial texture and staling were improved. The addition of L-cysteine (2.5% fwb) to breads lowered loaf volume but produced softer initial crumb texture and improved in-vitro protein digestibility by 18.8%.