Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Transgenic (TG) sorghum genotypes with altered kafirin synthesis were developed by the Africa Biofortified Sorghum Project, employing recombinant DNA technology, with the aim of improving the protein nutritional quality of the grain. In this project, the effects of altered kafirin synthesis on kafirin polymerisation, protein quality, protein body structure and endosperm texture in different TG lines were investigated. The first generation of TG lines were in a type II low-tannin sorghum background. Altered synthesis of different major kafirin sub-classes (α-, γ- and σ-kafirin) was targeted. Some TG lines had improved lysine content (3.17 g/100 g protein) and moderate (55%) to high (74%) cooked in vitro protein digestibility, compared to the parent (2.05 g/100 g protein; 47.4%, respectively). This is of significance as tannins reduce protein digestibility, by complexing with the proline-rich kafirins. Transmission electron microscopy revealed that the improved protein quality traits were associated with floury endosperm texture and irregular protein body structure. Irregular protein bodies were 2-3 ìm in diameter, with few to numerous invaginations, compared to normal protein bodies. The high digestibility TG line also had a unique dense protein matrix, with occasional thick dark-staining inclusions. It appears that reduced kafirin synthesis, specifically γ-kafirin, has a major effect on the protein body structure, which in turn results in changes in protein digestibility and endosperm structure. To further improve the protein quality and poor endosperm texture of the first generation of TG lines, improved non-tannin sorghums were transformed to suppress kafirin synthesis, or they were back-crossed into TG lines with improved protein quality. Co-suppression of the α-, γ- and σ-kafirin sub-classes and removal of the tannin trait, resulted in TG with high cooked protein digestibility (±80%), improved Amino Acid Score (0.8) and Protein Digestibility Corrected Amino Acid Score (0.7) compared to the non-TG null controls (±50%, 0.4 and 0.2, respectively). However, these high-protein quality lines still had a floury endosperm texture. They also had irregular shaped protein body structure, as described previously. When fewer kafirin sub-classes were suppressed (only γ- and σ-kafirin) the endosperm was corneous with normal protein body structure, but the improvement in cooked protein digestibility was less. Apparently, co-suppression of several kafirin sub-classes is required to obtain high-protein quality sorghum, but this seems to result in floury-type grain endosperm. Further work conducted on the high digestible TG line revealed that the proportion of kafirin-1, extracted with 60% tert-butanol alone, was greatly increased. However, the total amount of kafirin remained unchanged. Also, the kafirin was much less polymerised by disulphide bonding, and there was evidence of compensatory synthesis of other kafirin proteins. Hence, the mechanism for the increased protein digestibility of TG lines is probably related to their lower levels of disulphide-bonded kafirins, allowing better access of proteases. This work appears to confirm that disulphide bond formation in kafirin is responsible for the reduced protein digestibility of cooked sorghum. Since grain hardness is an important grain quality attribute, playing a major agronomic role, in sorghum processing and in the end-use quality of sorghum-based foods, further research should focus on transforming sorghum to have both improved protein nutritional quality and good grain endosperm texture. / Thesis (PhD)--University of Pretoria, 2012. / Food Science / unrestricted

This experimental work investigated the development and testing of a controlled release system for methionine. Methionine is one of the limiting amino acids for the milk production in dairy cows. The quantities of methionine which reach the small intestine are affected by the bacteria in the rumen which utilize methionine. A controlled release system which will offer a protective barrier for methionine may ensure that the methionine reaches the small intestine in sufficient quantities. The work involved the development of a coating around methionine crystals, which would act as a barrier, protecting it from the rumen conditions. Zein and kafirin proteins from maize and sorghum, respectively, were used as the principal coating components for the controlled release system. Two different approaches were used in the development of the controlled release system. First, the zein and kafirin proteins were tested for their ability to act as barriers for the controlled release of methionine, and second, zein and kafirin microparticles were used as the controlled release agents. Relatively successful, laboratory-scale methods were developed for coating the methionine with the proteins and the microparticles. Protein coatings were made by addition of methionine crystals to acid-dissolved proteins which led to the formation of a protein/methionine matrix. For coating the methionine with microparticles, glacial acetic acid was used to fuse microparticles around the methionine crystals. Dissolution assays were performed to test the release of methionine from the coatings under simulated rumen conditions. Both the zein and kafirin and microparticle coatings exhibited a barrier effect for methionine. The barrier effects of these coatings were influenced by several factors. Increasing the proportion of the coating agents led to improved barrier properties. However, this only occurred until a certain proportion of coating agent was present (50%), after which the barrier properties no longer increased. Heat treatment of the coatings also increased the barrier properties of the coatings. This may be due to the formation of disulphide cross-links being formed during the application of heat. When a simple extrusion method was used to form the coatings, the barrier properties also improved in comparison to those coatings which were not formed using extrusion. When producing the microparticles, it was found that only the laboratory extracted kafirin preparation with 85% (db) protein formed microparticles. It was hypothesized that microparticle formation might be related to the purity of the protein preparations. Scanning electron microscopy of the coatings after the dissolution tests and pepsin digestion revealed pores on the surface of the coating. These were probably where the methionine leached from the coating into the dissolution medium. The protein coatings did act as partial barriers, extending the release of methionine. From the release curves of methionine from the coatings, it could be seen that a sustained release of methionine occurred over a period of time, rather than a controlled release of methionine at a certain time. The aim of the application was thus only partially achieved as a complete protective barrier for methionine was not obtained from the protein coatings. No significant difference between the barrier properties of the coatings prepared from the proteins themselves and the microparticles were found. However, when based on equal protein purity the kafirin protein coatings showed the most effective barrier properties. Further research regarding kafirin coatings as a controlled release agent is recommended based on the results of the above named calculation. This research would entail investigating various coating technologies and methods. / Dissertation (MSc)--University of Pretoria, 2011. / Food Science / unrestricted

Microparticles produced from kafirin, the sorghum grain prolamin protein, by molecular selfassembly using coacervation with acetic acid solvent are vacuolated. They have shown considerable potential for encapsulation of antioxidants and for preparation of high quality free-standing bioplastic films. However, the functional quality of these kafirin microstructures needs to be improved to exploit their potential application, particularly as biomaterials. Wet heat, transglutaminase and glutaraldehyde treatments were used to modify the physical structure and chemical properties of the kafirin microstructures. Heat treatment (50–96°C) increased microparticle average size by up to four-fold to ≈20 μm, probably due to disulphide cross-linking of kafirin proteins. The vacuoles within these microparticles enlarged up to >10-fold, probably due to greater expansion of air within the microparticles with higher temperature, as the vacuoles are probably footprints of air bubbles. As with heat treatment, glutaraldehyde (10–30%) treatment resulted in oval microparticles, up to about four-fold larger than the control, probably due to covalent glutaraldehyde-polypeptide linkage. Transglutaminase (0.1–0.6%) treatment had only slight effect on the size and shape of microparticles, probably because kafirin has very low lysine content, inhibiting transglutaminase-catalysed cross-linking through ε-(-glutamyl)-lysine bonding. Surface morphology using atomic force microscopy indicated that the microparticles apparently comprised coalesced nanostructures. With heat and transglutaminase treatments, the microparticles seemed to be composed of round nanostructures that coalesced into random irregular shapes, indicative of non-linear protein aggregation. In contrast, with glutaraldehyde treatment, the nanostructures were spindle-shaped and had a unidirectional orientation, probably due to linear alignment of the nanostructures controlled by glutaraldehyde-polypeptide linkage. Thin (<50 μm) films prepared from kafirin microparticles and conventional cast kafirin films were compared in terms of their water stability and other related properties. Films cast from microparticles were more water-stable compared to conventional kafirin films, probably because the large vacuoles within the kafirin microparticles may have enhanced protein solubility in the casting solution, thereby improving the film matrix cohesion. The films prepared from microparticles treated with glutaraldehyde were more water-stable compared to the control, despite the loss of plasticizer, probably due to formation of the covalent glutaraldehyde-polypeptide linkages. The potential of modified kafirin microparticles to bind bone morphogenetic protein-2 (BMP- 2) was investigated. Compared to a collagen standard, the BMP-2 binding capacity of control, heat-treated, transglutaminase-treated and glutaraldehyde-treated kafirin microparticles were 7%, 18%, 34% and 22% higher, respectively, probably mainly due to the vacuoles within the microparticles creating greater binding surface area. The safety, biodegradability and effectiveness of kafirin microparticle film and kafirin microparticle film-BMP-2 system in inducing bone growth were determined by a subcutaneous bioassay using a rat model. Kafirin microparticle film and kafirin microparticle film-BMP-2 system was non-irritant to the animals, probably because kafirin is non-allergenic. The kafirin microparticle film implants showed signs of some degradation but a large proportion of these implants was still intact by Day 28 post implantation, probably because of the low susceptibility of kafirin to mammalian proteolytic enzymes. Kafirin microparticle film-BMP-2 system did not induce bone growth, probably mainly due to low BMP-2 dosage and short study duration. Modification of kafirin microparticles by wet heat or glutaraldehyde treatment both result in increased size of the microparticles with similar gross structure. However, it is apparent that with both treatments the proteins within the pre-formed kafirin microparticles undergo some form of further assisted-assembly through different mechanisms. It seems that heat-induced disulphide cross-linking reinforces a layer around the nanostructures, probably rich in γ- kafirin polypeptides, that stabilizes the structure of the nanostructures. In contrast, glutaraldehyde-treatment appears to destabilize this structure-stabilizing layer through formation of γ-kafirin polypeptide-glutaraldehyde covalent bonding. This probably offsets the balance of attractive and repulsive forces between the different kafirin subclasses within the nanostructures, thereby resulting in collapsed nanostructures and linear realignment. A deeper understanding of the mechanism of kafirin self-assembly will be important for further development of kafirin microstructures for different applications. / Thesis (PhD)--University of Pretoria, 2012. / Food Science / unrestricted

Although work on the prolamins of pearl millet (Pennisetum glaucum) has revealed partial amino acid sequences for several alcohol-soluble storage proteins (Marcellino et al. 2002) the genes encoding them have not yet been isolated. We constructed a cDNA library from developing P. glaucum seed tissue and screened it using maize zein gene probes to isolate several α-prolamin-like gene sequences. The proteins encoded by these genes generally fall into two size classes: 20.6kD and 27.1kD, which we call the 21kD and 27kD pennisetins. Both proteins are similar in composition and sequence to α-prolamins from maize, sorghum and Coix. Protein bodies that appear as occlusions within the rough ER of P. glaucum endosperm cells are also very similar in size and shape to maize and sorghum protein bodies. The SDS-PAGE gel of the alcohol soluble protein fraction shows two distinct bands in the region corresponding to the 19kD and 22kD of maize α-zein. Both classes of pennisetins appear to be more similar to the 19kD α-zein of maize than to the 22-kD α-zein judging from sequence homology and maize antibody binding. Phylogenetic reconstruction suggests that P. glaucum may have branched from maize prior to the gene duplication which created the 19kD and 22kD α-zein families.