Despite the potential of kafirin (sorghum prolamin protein) films, up until now there has been no in-depth investigation on the plasticization of kafirin films, similar to that done on zein films. Since protein films tend to be very brittle, plasticization is a very important aspect. Cast films were produced from kafirin and plasticized with different combinations of plasticizers (glycerol (G), polyethylene glycol 400 (PEG) and lactic acid (LA)) according to a rotatable central composite statistical design. The effects of the different plasticizer combinations on the film properties (tensile-, Tg-, moisture and oxygen barrier properties), were investigated through a series of tests performed on the films. Plasticization of kafirin films was investigated further by determining the effect of an emulsifier, diacetyl tartaric ester of monoglyceride (DATEM) and an acidulant, glucono-_-lactone (GDL) on the films. To investigate the distribution and migration of the plasticizers in kafirin films, the films were studied by light microscopy. It was clear that G, PEG and LA together were necessary to plasticize kafirin films. G and PEG were found to be effective plasticizers, leading to a decrease in film strength and an increase in strain as the plasticizer amount increased. LA was, however, found to act rather as a solvent for kafirin during film casting, instead of acting as a plasticizer. An increase in plasticizer content also brought about a lowering in the Tg of the films, as well as an increase in film permeability to water vapour and oxygen. G and PEG were found to attract water from the atmosphere, which proved to be very influential on the properties of the films; the more plasticizer present in the film, the more moisture attracted, the greater the effect on the film properties. DATEM was not found to be a plasticizer for kafirin films. However, GDL did bring about changes in film properties, similar to G and PEG. It caused film strength to decrease and film strain to increase, but with less detrimental effects on the moisture barrier properties of kafirin films. Microscopy showed that the plasticizer migrated over time, apparently leading to plasticizer molecule coalescence, and the formation of plasticizer pools. The plasticizer combination of G, PEG and LA improved the qualities of kafirin films, reducing film brittleness, but it is not an ideal plasticizer combination due to the fact that it attracts water to the film and it is not stable over time. Moisture is another plasticizer to be taken into account, since it will be absorbed by the plasticizers in high relative humidity areas and will have an additional plasticizing effect on films properties. GDL proved to have potential as a kafirin film plasticizer as it affected the barrier properties of the films less. Further research is recommended into the plasticization mechanism of GDL. / Dissertation (MSc(Agric))--University of Pretoria, 2008. / Food Science / unrestricted

Whilst working on a Masters degree on alternative solvents and extractants for the sorghum prolamin protein, kafirin, the author serendipitously found an ethanol-free method of making kafirin microparticles in dilute organic acid. Further, on drying a suspension of kafirin microparticles in dilute organic acid, a clear, transparent film was found to be formed. Microparticles from zein, the maize prolamin protein, have shown potential for food and pharmaceutical applications. Kafirin is more hydrophobic and less digestible than zein so it was hypothesised that it may form microparticles with superior properties. However, the structural and functional characteristics of kafirin microparticles and films made from them needed to be known before any potential applications could be exploited. Kafirin microparticles were made by dissolution of kafirin in glacial acetic acid followed by precipitation on addition of water. They were characterized by Light microscopy (LM), Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) and were found to be mainly spherical, porous and between 1-10 ìm in diameter. The kafirin microparticles had very large internal surface area due to the presence of many smooth walled holes or vacuoles of variable sizes, probably caused by entrapment of air during microparticle formation. Increasing the final acetic acid concentration resulted in kafirin microparticles of increased size, with an increasing number of internal holes. At 40% acetic acid the spherical microparticle structures completely disappeared and were replaced by an open matrix which resembled an expanded foam. The kafirin microparticles were found to form very thin (<15 ìm) free standing films and coatings. A minimum concentration of organic acid (10.8 percent) is required to form a cohesive kafirin microparticle film relative to the concentration of protein (1 percent for acetic acid). Some functional properties, e.g. smooth film surface properties, low water vapour permeability (WVP) and low protein digestibility of these films are superior to those of similar conventionally cast kafirin films. With the aim of exploiting the porous nature of kafirin microparticles for encapsulation of nutrient additives, several factors were examined for their influence on retarding protein digestibility. Retardation of digestibility of kafirin microparticles would allow controlled release of the encapsulated agent in the stomach and gastrointestinal tract. The importance of disulphide cross-linking and sorghum condensed tannin protein interactions were confirmed as major causal factors of the poor protein digestibility of sorghum. Gamma-kafirin was found to bind the most condensed tannins compared to the a-and b- kafirins, probably due to its high proline content. As expected, the protein digestibility of kafirin-tannin complexes was much lower than unbound kafirins. This seems to slow the biodegradation of kafirin films made with bound tannins. The antioxidants, catechin and sorghum condensed tannins were encapsulated within kafirin microparticles and the antioxidant release profiles investigated under simulated gastric conditions. Over a period of four hours, catechin and condensed tannin encapsulated kafirin microparticles showed virtually no protein digestion but released approximately 70% and 50% respectively total antioxidant activity. The mechanism for the formation of kafirin microparticles and films formed from them seems to involve controlled aggregation of kafirin molecules. Models for the formation of both were proposed based on an analogy with protein body formation and the potential ability of -kafirin to undergo a structural inversion exposing either hydrophilic or hydrophobic ends depending on the prevailing conditions. Research into cross-linking by physical or chemical agents is needed before practical applications can be exploited. However, encapsulation of catechin and sorghum condensed tannins within kafirin microparticles seems to be an effective way to use the binding properties of polyphenols with protein to enhance potential health benefits by controlled release of antioxidant activity within the stomach and gastrointestinal tract. Copyright / Thesis (PhD)--University of Pretoria, 2009. / Food Science / unrestricted

Doctor of Philosophy / Food Science Institute / Fadi Aramouni / Scott Bean / Zein, the storage protein of corn, has been shown to form a wheat-like dough; however the exact mechanism is unknown since zein lacks the large polymeric proteins found in wheat. To understand how zein forms a dough, different reagents were added during mixing of zein. Salts from the Hofmeister series were used to determine how hydrophobic interactions influence zein’s dough forming ability. In addition, urea, ethanol, and beta mercaptoethanol (β-ME) were also tested to evaluate the effects of protein denaturation and disulfide bonds on zein dough formation and bread quality. Kosmotropic salts had a negative effect on zein dough formation indicating that increasing hydrophobic interactions prevented dough formation. Surface hydrophobicity was found to decrease significantly (p < 0.05) when zein was exposed to 1M or 2M of the kosmotropic salts. Conversely, chaotropic salts had a slight positive effect on zein dough formation as did urea and ethanol. Interestingly, -ME had little effect on zein dough formation demonstrating that disulfide bonds played no role in zein dough development, and that large disulfide linked polymeric protein complexes were not present as found in wheat dough. Specific volumes of zein-starch bread increased as NaCl content in the bread formula decreased. Likewise, including 5% ethanol (v/v) in the bread formula was found to increase bread quality. Experiments were also conducted to compare the functionality of isolated sorghum proteins (kafirins) to commercially available zein produced during wet milling of corn. The effect of steeping, the first step in wet milling, on kafirin functionality was investigated. Sorghum flour was steeped for 0, 72, or 96 hours. Unsteeped sorghum flour was digested with Alcalase for 90 min at 50°C. After steeping or digestion with Alcalase, kafirins were isolated from the remaining material. Both groups of Kafirins had the ability to form a zein-like visco-elastic resin when mixed with warm water by hand. This is the first time that kafirin has been reported to form a visco-elastic resin using only water as a plasticizer.

Doctor of Philosophy / Grain Science and Industry / Scott R. Bean / Hulya Dogan / The role of sorghum protein cross-linking into high M[subscript w] polymeric groups in grain hardness was investigated using a number of protein analytical techniques to study the protein composition (reduced and non-reduced) of isolated vitreous and floury endosperm. The relative molecular weight distributions of polymeric proteins within two of three differentially extracted fractions were determined by size exclusion chromatography (SEC). The proteins in vitreous endosperm showed more protein cross-linking and a larger M[subscript w] distribution than found in the floury endosperm. An improved method for fractionating sorghum proteins designed to obtain intact disulfide linked protein polymers was developed. Three protein fractions obtained by application of the method represented proportionally different protein polymer contents as evidenced by comparative SEC and provides an improved tool for polymeric protein content comparison and measurement. The improved method was applied to a highly diverse non-tannin wild-type sorghum sample set spanning a range of in-vitro protein digestibility (IVPD) values to determine polymers involved with and influencing IVPD. Grain traits other than cross-linked proteins were also investigated for significant relationships to IVPD. Three protein fractions (F1, F2, F3) containing intact protein polymers were obtained for analysis by SEC and RP-HPLC. Proteins represented by four of five individual SEC peaks from F3 were significantly negatively correlated to IVPD, with three of the correlated peaks being polymeric. A 2-dimensional (2-D) technique involving peak collection after size exclusion chromatography followed by reverse phase high performance liquid chromatography (SEC x RP-HPLC) of the collected peaks was applied to protein polymers previously determined to be correlated with IVPD. RP-HPLC chromatogram patterns unique to each collected SEC peak from three selectively extracted protein fractions allowed qualitative and quantitative comparisons of protein polymer components. A pair of early eluting peaks appearing in the [gamma]-kafirin region of 2nd-dimension RP-HPLC chromatograms from a protein fraction with the largest M[subscript w] distribution were significantly correlated to IVPD. The correlated peak of interest was collected and characterized using SDS-PAGE and was preliminarily identified as 27kDa [gamma]-kafirin. By combining techniques using differing selectivity’s (solvent based, molecular size based, hydrophobicity based), it was possible to disassemble and compare components of protein polymers significantly correlated to IVPD.

Microparticles (KEMs) made from the sorghum prolamin protein, kafirin, have internal vacuoles. Hence, they have potential as delivery vehicles for nutraceuticals. However, their physico-chemical properties need to be improved for this application. The influence of kafirin extracted from white tan-plant and red non-tannin sorghum types of 81% and 84% protein content respectively and the rate of water addition on the formation of KEMs from kafirin in acetic acid solution by coacervation on their morphology was investigated. A water flow rate of 1.4 and 0.7 ml/min during coacervation using 81% kafirin resulted in spherical-shaped KEMs between 1 and 10 μm in diameter and vacuoles up to 2 μm. KEMs made with 84% kafirin at a flow rate of 0.7 ml/min were large and oval-shaped with an average length and width of 43 and 21 μm respectively and numerous vacuoles up to 3 μm. At a flow rate of 1.4 ml/min, the KEMs were oval-shaped with larger vacuole sizes (5 μm), a length and width of 91 and 30 μm respectively. However, SDS-PAGE indicated that neither the source of kafirin, nor the conditions of microparticle preparation had an effect on KEMs protein molecular size.As the presence of phenolic compounds in the kafirins might have been responsible for the differences in KEMs morphology, the effect of sorghum-derived polyphenols (extracted from condensed-tannin and non-tannin black sorghum brans) on the physico-chemical properties of KEMs was then investigated using 81% kafirin. Aqueous condensed tannin (10.1 mg CE (catechin equivalent)/100 mg extract) and black non-tannin (4.6 mg CE/100 mg extract) extracts in varying concentrations, were substituted for the water used for coacervation. KEMs made with condensed tannin extracts were oval-shaped and much larger, than control KEMs ranging from 20 to 400 μm, with rough surfaces and enlarged vacuoles. The enlarged vacuoles were probably due to more air being trapped within the particles during formation. However, KEMs made from non-tannin phenolic extracts were smaller and spherical with average diameters up to 18 μm. Tannins are known to bind strongly to kafirin through hydrogen and hydrophobic bonds, which probably resulted in the larger microparticles. The KEMs made from condensed tannins also had high antioxidant capacities compared to KEMs made from non-tannin phenolic extracts, attributed to tannins being more potent antioxidants. Thus, condensed tannin extracts are the most beneficial as they contributed towards the antioxidant activity of the KEMs, resulting in the development of innovative KEMs with added antioxidant benefits and enlarged size. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Food Science / unrestricted

A laboratory process exists for the extraction of kafirin protein from sorghum grain in order to form kafirin encapsulating microparticles. This laboratory process extracts approximately 2 g of protein and takes in excess of 60 hours from start to finish. A scaled-up extraction process based on the current laboratory process, consisting of a 100 L extraction vessel, was established in order to extract large volumes of kafirin protein from sorghum grain. Approximately 2.5 kg of kafirin protein, which contained approximately 80 % protein after defatting, was extracted from red sorghum grain. This blended kafirin protein, which was the product of combining 9 batches done on the up-scaled process, was needed in order to obtain a consistent base raw material for further experimentation. The blended kafirin was used to investigate the formation of kafirin encapsulating microparticles. This was achieved by means of the solvent phase separation technique with acetic acid as the solvent phase. A series of experiments, selected from a partial factorial design, were used to screen how the formation of microparticles was affected by various parameters. The parameters investigated were solvent to protein ratio, stirring speed, water addition rate and number of water droplets. The morphology of the various microparticles produced was analysed by means of light microscopy, FTIR and particle size analysis, and the different formed microparticles characterised. From the screening partial factorial experimental design, it was determined that the acetic acid concentration was crucial for the formation of microparticles. Microparticles did not form at a low mass ratio (2.3) of glacial acetic acid solvent to protein. Water addition rate and stirring rate also affected microparticle formation while the number of water droplets was insignificant. Therefore, using a high solvent to protein mass ratio (6.8), additional refined partial factorial experiments were conducted. These experiments focused on the effect of water addition rate and stirring speed on the final kafirin microparticle size. Ultimately, a polynomial model was developed to predict the final kafirin microparticle size using only the water addition rate and stirring speed as inputs. The model had an R2 value of 0.986 and was found to relatively accurate during validation. The model also identified that three distinct regions existed within the workspace: _ A region containing large particles due to protein mass agglomeration and crosslinking, which occurs at low stirring speeds (< 400 rpm) and high water addition rates (> 5 mL/min) _ A region where only small individual microparticles exist, which occurs at high stirring speeds (< 800 rpm) and low water addition rates (> 2 mL/min) _ A region where moderate particles existed as uniform agglomerates of the microparticles, which occurs at moderate stirring speeds (+- 600 rpm) and moderate water addition rates (+- 3.5 mL/min) Ultimately these kafirin microparticles, prepared from protein extracted in an up scaled process, were used to form qualitative microparticle films. The microparticle films were made without plasticiser and without dewatering the microparticles. Furthermore these films were made from microparticles in the regions identified in the model. This qualitative film formation showed that agglomerated microparticles can form films. This could be beneficial for the feasibility of a commercialised process for kafirin microparticle films since the production time would be shorter and less energy intensive. / Dissertation (MEng)--University of Pretoria, 2016. / Chemical Engineering / MEng / Unrestricted

Doctor of Philosophy / Department of Biochemistry / Gerald R. Reeck / Prolamins are the alcohol soluble storage proteins found in the endosperm of seeds from cereals and related grasses. The physical and biochemical properties of prolamins vary between species; and due to their relative abundance can greatly affect the properties and healthfulness of foods from those sources. In this work I investigate peptides from the high molecular weight glutenin of wheat, which is linked to dough elasticity and finished product quality. Using 2D NMR I determined the three-dimensional structure for the repeat peptide Ac- GQQPGQG-Am, which makes up ~50% of the 700 residue central domain. The structure was found to be a flexible β-hairpin with a type II β-turn across residues QPGQ. The NMR structure was later compared to 33 proteins with known three-dimensional structure carrying the exact sequence (backbone RMSD=0.802Å). This finding provides useful insight into the structure of high molecular weight glutenin and the molecular nature gluten elasticity. Alternatively, I studied the kafirin storage prolamins from sorghum, which do not have important physical properties, but are poorly digestible by humans and livestock. Improving digestibility of sorghum could significantly impact human health and nutrition in countries where sorghum is a dietary staple. In this work I devised a unique protocol to isolate kafirins under both non-reducing and reducing conditions. I studied kafirin extracts using SDS-PAGE, HPLC and MALDI-TOF MS, then purified β-kafirin, for the first ever characterization of this single protein. Past studies implicate β-kafirin as a source of poor digestibility due to extensive intermolecular disulfide cross-linking. Contrary to this claim I found more than 50% of β-kafirin was extractable without reducing agents. I used chymotrypsin to digest pure β-kafirin and map 10 cysteine residues to 5 intra-molecular disulfide bonds. Precise pairings have yet to be determined although the protein is largely intact after 12 hours of digestion. This work challenges us to think about sorghum protein body formation and the mechanism that leads to disulfide cross-linking during seed desiccation at maturity.

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.

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.