Climate change, shrinking arable land, burgeoning population and malnutrition have made all aspects of crop improvement a critical issue. Of these, nutritional quality of crops is perhaps one of the most important aspects. Most cereals consumed in marginal agro-ecological zones of Africa, for example sorghum and maize are impoverished nutritionally. Given therefore the sole reliance on and the levels of consumption per day of such staples (up to 450 g/day), it is clear that most people cannot obtain the recommended daily allowance (RDA) for many nutrients including fibre, edible oil, protein, vitamins and mineral elements. In this thesis, the development of a sorghum mutant population using gamma irradiation and the subsequent employment of various analytical techniques to unravel multiple mutant traits with a significant positive impact on nutritional enhancement in sorghum is described. Protein analysis revealed a mutant designated SY accumulating (at the time) the highest ever reported amount of free lysine (21.6 g/100g) and other essential amino acids and that these changes were associated with induced protein polymorphisms. Adaptation of proton induced x-ray emission (PIXE) for the spatial profiling of the distribution of 9 elements in sorghum seed tissue allowed for the discovery of mutants with variations in the concentrations and distribution of these elements. The observed changes included enhanced or diminished accumulation of elements in preferential accumulation tissues and entire changes in cellular localisation. The locations within a cell and the quantities of an element are often critical determinants of bioavailability. The accumulation of multiple mutations affecting multiple nutritional traits in individual mutant sorghum clearly indicates the versatility of gamma irradiation induced mutations in addressing multiple nutritional challenges of sorghum. This desirable phenomenon was further demonstrated by electron microscopic analysis of starch granules and protein bodies across the mutants. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed changes in size, shape, ultra-structure and packed cell volumes of seed protein- and starch bodies. Induced mutation had a major effect on the protein body structure which in turn resulted in changes to protein digestibility. High digestibility mutants had a unique dense protein matrix with dark inclusions. However, improved protein quality traits were also associated with floury endosperm texture. Since endosperm texture is an important grain quality attribute and plays a major agronomic role, it is important to ensure that future work focuses on improving grain hardness. The mutants obtained in this study are therefore a valuable germplasm source for sorghum breeding and present real opportunities for addressing nutritional challenges of sorghum. / Ph.D. University of KwaZulu-Natal, Durban 2013.

In the breeding of grain sorghum (Sorghum bicolour L. Moench) towards improved nutrition and agronomic performance, new methodologies are required to increase genetic diversity and lower the inputs required to track and screen breeding populations. Near-infrared calibration models were developed by partial least squares (PLS) and test-set validation on 364 sorghum samples to predict crude protein and moisture content on whole-grain and milled flour samples. Models using milled flour spectra were more accurately predictive than those from whole grain spectra for all constituents (eg. Protein: R2 = 0.986 on flour vs R2 = 0.962 on whole grain). Discriminant calibrations were established to classify grain colour using partial least squares discriminant analysis (PLS-DA) based upon CIE L*a*b* reference values and visual ranking. Preliminary calibrations were developed for quantities of 18 amino acids, fat and apparent metabolisable energy (AME) on 40 samples using cross-validation, highlighting potential for reliable calibration for these parameters in sorghum. An investigation into the potential of 12C6+ heavy-ion beam mutagenesis of sorghum seed was undertaken by treatment at RIKEN Accelerator Research Facility (Saitama, Japan) and subsequent breeding at Ukulinga research farm and analysis at the Department of Plant Pathology, University of KwaZulu-Natal, Pietermaritzburg, South Africa. Dosage rates of 75, 100 and 150 Gy were compared in seven sorghum varieties to establish optimal dose treatments as determined by germination and survival rates, visible morphological changes and field data over two seasons of field trials. Crude protein variation within the M2 generation was analysed to compare dose rate effects. The need for higher dose rates was indicated by few quantified differences between treatments and control although good correlations between protein deviation and treatment dose rate were elucidated. Differences in varietal response suggest a need to optimize dose rate for specific varieties in future endeavours. In addition, all mutagenized populations were screened for crude protein content using near-infrared spectroscopy (NIRS). Significant differences in protein levels and standard deviations were observed between treated self-pollinated M2 generations and untreated control populations. Individual plants displaying significantly different protein levels were isolated. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Sorghum (Sorghum bicolor) is an important cereal crop in sub-Saharan Africa. The parasitic weed Striga hermonthica is a major biotic constraint to sorghum production. A novel technology where planting seeds are coated with herbicide to kill Striga that attach to the roots of the host has been shown to be effective in protecting the cereal crop from Striga damage. However, the host plant must have herbicide tolerance. This technology has not been tested in sorghum because there are no herbicide tolerant sorghum varieties available in Kenya and is, therefore, unavailable for subsistence farmers. One of the ways in which genetic variation can be enhanced and herbicide resistance developed is through chemical mutagenesis with ethyl methane sulfonate (EMS). The objectives of this project, therefore, were to: 1) identify sorghum production constraints through farmer PRA in order to determine breeding priorities.in two Striga endemic districts in western Kenya; 2) develop an EMS mutagenesis protocol for sorghum and to enhance the genetic variability of the crop using chemical mutagenesis; 3) evaluate EMS-derived sorghum mutants for improved agronomic performance; 4) develop acetolactate synthase (ALS) herbicide resistance in sorghum and to characterize the mode of inheritance of the trait; 5) determine the effect of herbicide coating of seed of herbicide tolerant sorghum on Striga infestation. In order to determine breeding priorities and constraints in sorghum production and the likelihood of adoption of herbicide seed coating technology, a survey involving 213 farmers was conducted in two Striga endemic rural districts of Nyanza province in Kenya. Results indicated that local landraces like Ochuti, and Nyakabala were grown by more farmers (> 60%) than the improved varieties like Seredo and Serena (48%). Popularity of the landraces was linked to Striga tolerance, resistance to drought, bird damage and storage pests, yield stability and high satiety value. Major constraints to sorghum production were drought, Striga weed, storage pests, bird damage and poverty among the rural farmers. Important characteristics farmers wanted in new varieties were Striga and drought resistance, earliness, resistance to bird and weevil damage and good taste. Striga infestations in sorghum fields were > 70%. Cultural Striga control options were considered inadequate while inorganic fertilization and chemical control were considered effective but unaffordable. Farmers’ willingness to pay a premium of over 30% for a Striga solution gave indication that herbicide seed coating if effective could be adopted by farmers. As a prerequisite to development of herbicide resistance, a comparative study was carried out to determine optimum conditions for mutagenesis and to induce genetic variation in the sorghum. Two sorghum varieties were mutagenized using varying concentrations (0.1 to 1.5% v/v) of EMS and two exposure times (6h and 12h). In laboratory and greenhouse experiments, severe reduction of sorghum root and shoot lengths indicated effective mutagenesis. The LD50 based on shoot length reduction was 0.35% and 0.4% EMS for 6h for Seredo and Kari/mtama-1, respectively. The highest mutation frequency based on chlorophyll abnormalities was 56% for 0.3% EMS for 6h. In the M2 generation, phenotypic variances for panicle characteristics were increased on treatment with EMS. However, significant effects of exposure time and variety indicated the necessity of genotype optimization for some traits. In order to determine the significance of mutation breeding in sorghum, 78 mutant lines derived from EMS mutagenesis, their wild type progenitor (Seredo) and two local checks (Kari/mtama-1 and Serena) were evaluated for agronomic performance in two locations in Kenya. There were significant (P = 0.05) effects among entries for grain yield, 1000-seed weight and visual scores for height uniformity, head exertion, head architecture and overall desirability. The highest yielding entry-mutant line “SB2M13” had a yield of 160% and 152% relative to the wild type (Seredo) and the best check Kari/mtama-1, respectively. Mutant line “tag27” had the highest 1000-seed weight which was 133% relative to the wild type. Seven mutant lines were rated superior to the wild type for panicle characteristics, head exertion and overall desirability. However, the majority of mutants were inferior to the wild type for most characteristics. Superior mutant lines may be developed into direct mutant varieties after multi-location trials or used as breeding material for sorghum improvement. In order to develop acetolactate synthase (ALS) herbicide resistance in sorghum, over 50,000 seeds of Seredo were mutagenized with 0.3% EMS. Over four million M2 plants were screened using 20g ha-1 of the ALS herbicide, sulfosulfuron. Five mutants (hb46 hb12, hb462, hb56 and hb8) survived the herbicide treatment and were confirmed to be tolerant. Mutant lines displayed differential herbicide tolerance, and the general order of tolerance after spray or seed coat application was hb46 > hb12 > hb462 ~ hb56 > hb8. The LD50 values for herbicide application as a spray, or seed coat, showed mutant lines to be up to 20 and 170 fold, respectively, more resistant than the wild type. Chi square analysis of data from herbicide screening of F2 generation of mutant X wild type crosses indicated no difference from the Mendelian segregation of 1:2:1 indicating the herbicide tolerance was inherited as a single semi-dominant gene. Mutant X mutant crosses did not show allelism indicating that the tolerance in all five mutants could be a result of the same gene mutation. To determine effect of herbicide seed coating on Striga infestation, the five herbicide tolerant mutant lines, hb46, hb12, hb462, hb56 and hb8 and the wild type progenitor Seredo were coated with varying concentrations (0.5-1.5% g ha-1) of sulfosulfuron and planted in a Striga endemic field. There were significant (P=0.05) effects of herbicide concentration on Striga density, Striga flowering and seed set, and sorghum plant stand and biomass. All treatments with herbicide coated on sorghum seeds had lower Striga emergence. Coating sorghum seed with 1g ha-1 sulfosulfuron reduced Striga infestation, Striga flowering and Striga seed set by 47%, 52% and 77%, respectively, and was considered the most effective rate as it did not result in sorghum biomass reduction. Mutants displayed differential herbicide tolerance and Striga resistance. Combining seed coating with high herbicide tolerance and inherent Striga resistance would be most effective for Striga control. Overall, the study showed that EMS mutagenesis is effective in inducing variation in sorghum for several traits including herbicide resistance. The mutants developed in this study will be important for sorghum breeding and for protection of sorghum against the Striga weed. / Thesis (Ph.D.) - University of KwaZulu-Natal, Pietermaritzburg, 2009.