Browsing by Author "O'Kennedy, Maretha M"
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Item The African perspective I: Using the new biosciences to support the African development agenda(Taylor & Francis Group publishers, 2016-09) Morris, EJ; Erasmus, C; O'Kennedy, Maretha MBiopharmaceutical proteins and vaccines are traditionally produced in bacteria, eggs, yeast and animal cell cultures and are well established industries. More recently, these molecules are produced in plants and became known as biopharming. Plant-based production systems have the advantages of eukaryotic protein processing properties, inherent safety due to a lack of adventitious agents, substantial cost reduction and facile scalability. The latter was demonstrated in two therapeutic and industrial enzyme technoeconomic case studies (Tusé et al., 2014). These advantages however are molecule/product-specific and depend on the relative cost-efficiency of alternative sources of the same product (biosimilars) or improved products (biobetters). Nevertheless, plant-based manufacturing is estimated to reduce the running costs (20-25%) and capital expenses (40%) compared to animal cell-based processes (Gleba and Giritch, 2012). A facility with the capacity of 1 ton of antibody per year would cost less than $50 million and operation costs for the first 3 years would amount to $45-60 million (Gleba and Giritch, 2012). Such a low financial entry barrier represents a very attractive opportunity for manufacturers in developing countries (Gleba and Giritch, 2012). In addition, the Defence Advanced Research Projects Agency (DARPA) recognises that plant-based transient systems overall represent the best manufacturing technologies when dealing with bioterrorism and pandemics. Biopharming is advancing fast and numerous reviews cover the latest developments concerning vaccines (Rybicki, 2014), antibodies (Whaley et al., 2014), purification strategies (Buyel and Fischer, 2014) and manufacturing platforms (Gleba and Giritch, 2012; Klimyuk et al., 2014).Item Biolistic mediated sorghum (Sorghum bicolor L. Moench) transformation via mannose and bialaphos based selection systems(Asian Network for Scientific Information, 2010) Grootboom, AW; Mkhonza, NL; O'Kennedy, Maretha M; Chakauya, E; Kunert, K; Chikwamba, Rachel KSorghum (Sorghum bicolor (L.) Moench) is recalcitrant to transformation and besides several reported cases of success, transformation is far from routine. The choice of the selectable markers is key factor influencing success and efficiency of transformation. In sorghum, concerns about flow of herbicide and antibiotic resistance gene into genetically related wild and weedy species have a direct bearing on the choice of suitable selectable markers in many tropical and subtropical regions. The authors studied the utility of bialaphos and phosphomannose isomerase selectable markers in microprojectile mediated transformation of P898012, a sorghum inbred line generally considered amenable to in vitro manipulation. Two plasmids containing bar gene, encoding bialaphos resistance, and manA gene, for phosphomannose isomerase, both under the control of maize ubiquitin promoter and nopaline synthase terminator were used to transform immature zygotic embryos (IZE) of sorghum via particle bombardment using a particle inflow gun. Transgene integration in putatively transgenic plants was confirmed by PCR and Southern blot analysis. The expression of the bar gene in transgenic plants was demonstrated by the BASTA leaf painting assay. Our results indicated a transformation efficiency of 0.11% and 75% escapes in the apparent bialaphos resistant plants. A transformation efficiency of 0.77% was observed on manA selection and all plants recovered contained the manA gene. Besides an improved transformation efficiency, mannose selection offers a more environmentally sound system as sugar metabolism is considered ecologically neutral should it inadvertently be transferred to wild sorghum speciesItem Biolistic-mediated transformation protocols for maize and pearl millet using pre-cultured immature zygotic embryos and embryogenic tissue(Springer, 2011-01) O'Kennedy, Maretha M; Stark, HC; Dube, NMaize (Zea mays L.) is the most important cereal food crop in sub-Saharan Africa and Latin America, and a key feed crop in Asia, whereas pearl millet [Pennisetum glaucum (L.) R. Br.] is a staple food that supplies a major proportion of calories and protein to large segments of the populations living in the semi-arid tropical regions of Africa and Asia. The limitations of biological gene transfer with Agrobacterium tumefaciens specifically related to recalcitrant cereal crops, led to the development of alternative methods of which high-velocity microprojectiles, biolistic genetic transfer is the most successful and also the most widely employed. Agrobacterium facilitated transformation is the method of choice especially for deregulation of commercial transgenic food crop products, but biolistic-mediated transformation are still valid for proof of concept and functional genomics applications. Biolistic-mediated transformation and the production of transgenic plantlets via somatic embryogenesis of two maize strains viz. Hi-II (a laboratory strain) and M37W (a South African elite white maize genotype) as well as a pearl millet strain (842B) are described in this chapter. The stages described include: 1) proliferation of immature zygotic embryos for biolistic-mediated transformation, 2) induction and maintenance of transgenic embryogenic tissue on selection medium; 3) maturation (both morphological and physiological) of transgenic somatic embryoids; and 4) germination of the somatic embryoids to transgenic putative primary events. Maize and pearl millet cultures were regenerated via somatic embryogenesis as they are bipolar structures that shoot and root simultaneously. The culture media described in this chapter rarely induced or regenerated plantlets via organogenesis.Item Co-suppression of synthesis of major x-kafirin sub-class together with y-kafirin-1 and y-kafirin-2 required for substantially improved protein digestibility in transgenic sorghum(Springer link, 2014-01) Grootboom, AW; Mkhonza, NL; Mbambo, Z; O'Kennedy, Maretha M; Da Silva, LS; Taylor, J; Taylor, JRN; Chikwamba, Rachel K; Mehlo, LCo-suppressing major kafirin sub-classes is fundamental to improved protein digestibility and nutritional value of sorghum. The improvement is linked to an irregularly invaginated phenotype of protein bodies.Item Dose immunogenicity study of a plant-produced influenza virus-like particle vaccine in layer hens(2022-06) Abolnik, C; Smith, Tanya; Wandrag, BDR; Murphy, M; Rautenbach, M; Olibile, O; O'Kennedy, Maretha MAvian influenza poses one of the largest known threats to global poultry production and human health, but effective poultry vaccines can reduce infections rates, production losses and prevent mortalities, and reduce viral shed to limit further disease spread. The antigenic match between a vaccine and the circulating field influenza A viruses (IAV) is a critical determinant of vaccine efficacy. Here, an Agrobacterium tumefaciens-mediated transient tobacco plant (Nicotiana benthamiana) system was used to rapidly update an H6 influenza subtype virus-like particle (VLP) vaccine expressing the hemagglutininn (HA) protein of South African H6N2 IAVs circulating in 2020. Specific pathogen free White Leghorn layer hens vaccinated twice with =125 hemagglutinating unit (HAU) doses elicited protective antibody responses associated with prevention of viral shedding, i.e. hemaglutination inhibition (HI) mean geometric titres (GMTs) of =7 log2, for at least four months before dropping to approximately 5–6 log2 for at least another two months. A single vaccination with a 250 HAU dose induced significantly higher HI GMTs compared lower or higher doses, and was thus the optimal dose for chickens. Use of an adjuvant was essential, as the plant-produced H6 HA VLP alone did not induce protective antibody responses. Plant-produced IAV VLPs enable differentiation between vaccinated and infected animals (DIVA principle), and with sucrose density gradient-purified yields of 20,000 doses per kg of plant material, this highly efficacious, safe and economical technology holds enormous potential for improving poultry health in lower and middle-income countries.Item Efficacy of a plant-produced clade 2.3.4.4 H5 influenza virus-like particle vaccine in layer hens(2022-06) Abolnik, C; O'Kennedy, Maretha M; Murphy, MA; Wandrag, DBROutbreaks caused by Goose/Guangdong H5 highly pathogenic avian influenza (HPAI)-lineage viruses continue to occur in unprecedented numbers throughout Eurasia, the Middle East and Africa, causing billions of dollars in economic losses and the deaths or destruction of hundreds of millions of poultry, and pose a zoonotic threat. Here, a recombinant virus-like particle (VLP) displaying the hemagglutinin protein of a clade 2.3.4.4b H5N8 HPAI strain was produced in tobacco plants (Nicotiana benthamiana) and its immunogenicity with four commercial adjuvants was compared in layer hens. After two immunizations with 250 hemagglutinating unit doses, hens that received intramuscular injections of H5 VLPs formulated with Emulsigen D, Emulsigen P or Montanide ISA 71VG seroconverted with hemagglutination inhibition geometric mean titres (GMTs) of 7.3 log2 (± 1.17), 8 log2 (± 1.08) and 7.9 log2 (±1.07), respectively, but the GMT of hens inoculated by eye drop with VLPs plus Carbigen only reached 2.05 log2 (± 1.64). The H5 VLP plus Emulsigen-P vaccinated hens and a sham-vaccinated group were then challenged with a high dose of the homologous H5N8 HPAI virus. Vaccinated hens were completely protected and showed no clinical signs, whereas the sham-vaccinated birds all died within 3–4 days. The average oropharyngeal shedding in vaccinated hens was reduced by 3,487-fold and 472-fold on days 2 and 3 post challenge, respectively, whereas average cloacal shedding was reduced by 2,360,098-fold and 15,608-fold on days 2 and 3, respectively, compared to the sham-vaccinated controls. No virus was detected in the vaccinated hens after day 8 post challenge, and the plant-produced H5 VLP vaccine completely prevented H5N8 HPAI virus transmission to eggs. This highly efficacious, safe and non-toxic plant-produced H5 VLP vaccine with DIVA (differentiation of infected from vaccinated animals) capability could be rapidly produced with a yield of at least 85,000 doses per Kg of plant leaf material.Item Efficacy of a plant-produced virus-like particle vaccine in chickens challenged with Influenza a H6N2 virus(Wiley, 2019-07) Smith, Tanya; O'Kennedy, Maretha M; Wandrag, Daniel BR; Adeyemi, M; Abolnik, CThe efficacy, safety, speed, scalability, and cost-effectiveness of producing hemagglutinin-based virus-like particle (VLP) vaccines in plants is well-established for human influenza, but untested for the massive poultry influenza vaccine market that remains dominated by traditional egg-grown oil-emulsion inactivated whole virus vaccines. For optimal efficacy, a vaccine should be closely antigenically matched to the field strain, requiring that influenza A vaccines be updated regularly. In this study, an H6 subtype VLP transiently expressed in Nicotiana benthamiana was formulated into a vaccine and evaluated for efficacy in chickens against challenge with a heterologous H6N2 virus. A single dose of the plant-produced H6 VLP vaccine elicited an immune response comparable to two doses of a commercial inactivated H6N2 vaccine, with mean hemagglutination inhibition titers of 9.3 log2 and 8.8 log2, respectively. Compared to the non-vaccinated control, the H6 VLP vaccine significantly reduced the proportion of shedders and the magnitude of viral shedding by >100-fold in the oropharynx and >6-fold in the cloaca, and shortened oropharyngeal viral shedding by at least a week. Despite its potency, the cost of the antigenic mismatch between the inactivated H6N2 vaccine and challenge strain was evident not only in this vaccine’s failure to reduce viral shedding compared to the non-vaccinated group, but its apparent exacerbation of oropharyngeal viral shedding until 21 days post challenge. We estimate that a kilogram of plant leaf material can produce H6 VLP vaccines sufficient for between 5,000 and 30,000 chickens, depending on the effective dose and whether one or two immunizations are administered.Item Efficient in vitro plant regeneration from immature zygotic embryos of pearl millet [Pennisetum glaucum (L.) R. Br.] and Sorghum bicolor (L.) Moench(Springer-Verlag, 2001-07) Oldach, KH; Morgenstern, A; Rother, S; Girgi, M; O'Kennedy, Maretha M; Lorz, HThe authors report an in vitro culture system that provides reliable, highly efficient regeneration from immature embryos of pearl millet [Pennisetum glaucum (L.) R. Br.] and sorghum [Sorghum bicolor (L.) Moench]. Immature embryos were isolated 10-20 days after pollination and cultured on various L3 media. The influence of different parameters during the callus induction phase was examined with respect to the regeneration rate: (1) the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and various cytokinins; (2) the addition of AgNO3; (3) the use of maltose or sucrose as a carbon source. Modifications in the phytohormones alone resulted in the regeneration of fertile sorghum plants at high efficiency. Significant increases in the regeneration rates of pearl millet genotypes were achieved by the combination of sucrose as a carbon source and silver nitrate as a potential ethylene inhibitor.Item Expression of a ß-1,3-glucanase from a biocontrol fungus in transgenic pearl millet(Elsevier Publishers, 2011-05) O'Kennedy, Maretha M; Crampton, BG; Lorito, M; Chakauya, E; Breese, WA; Burger, JT; Botha, FCSclerospora graminicola is an Oomycete (heterotrophic Stramenopiles), fungal-like obligate phytopathogen, the causal agent of downy mildew in pearl millet (Pennisetum glaucum [L.] R. Br.), and a major constraint in the production of this cereal crop. In this study a hydrolytic enzyme, ß-1,3-glucanase, from the biocontrol fungus Trichoderma atroviride (gluc78) was introduced into the genome of a pearl millet breeding line, 842B, by particle bombardment. Constructs were prepared containing the gluc78 gene, encoding the 78 kDa ß-1,3-glucanase protein, driven either by a constitutive ubiquitin promoter or the wound inducible potato proteinase inhibitor IIK gene promoter (pin2). The positive selectable marker gene, manA, encoding mannose-6-phosphate isomerase (phosphomannose isomerase) driven by the ubiquitin promoter, was used for co-transformation. Transgenic plants were obtained harbouring the manA selectable marker gene and the antifungal gene gluc78 driven either by the ubiquitin or pin2 promoter. Full constructs or minimal transgene expression cassettes containing the genes of interest were successfully introduced into the genome of pearl millet. Progeny of stably transformed plants, harbouring the gluc78 transgene which is driven by the pin2 promoter and followed by the rice Act1 intron sequences, were subjected to pathogenicity trials. Apart from one isolated event reducing incidence of S. graminicola infection by 58%, transgenic pearl millet showed no resistance to this phytopathogen. The event conferring decreased susceptibility to S. graminicola had high levels of the glucanase transcript especially in transgenic plants showing severe downy mildew infection.Item Harnessing sorghum and millet biotechnology for food and health(Elsevier Science Ltd, 2006-11) O'Kennedy, Maretha M; Grootboom, A; Shewry, PRThis review highlights recombinant DNA technology as a powerful tool to enhance the gene pools of sorghum and pearl millet crops regarded as jewels of Africa. Although important advances in the improvement of these species have been made by classical breeding and modern marker assisted selection, genetic manipulation and in vitro culture allows the gene pool to be broadened beyond that normally available for improvement by allowing the transfer of genes which control well-defined traits between species. The current state of sorghum and millet transformation technology is summarised and applications in the improvement of nutritional quality and the resistance to pathogens and pests for crops grown in Africa and Asia is discussed. Regulatory aspects including gene flow and future prospects are also discussedItem An investigation into robust spectral indices for leaf chlorophyll estimation(Elsevier, 2011-11) Main, Russell S; Cho, Moses A; Mathieu, Renaud SA; O'Kennedy, Maretha M; Ramoelo, Abel; Koch, SQuantifying photosynthetic activity at the regional scale can provide important information to resource managers, planners and global ecosystem modelling efforts. With increasing availability of both hyperspectral and narrow band multispectral remote sensing data, new users are faced with a plethora of options when choosing an optical index to relate to their chosen or canopy parameter. The literature base regarding optical indices (particularly chlorophyll indices) is wide ranging and extensive, however it is without much consensus regarding robust indices. The wider spectral community could benefit from studies that apply a variety of published indices to differing sets of species data. The consistency and robustness of 73 published chlorophyll spectral indices have been assessed, using leaf level hyperspectral data collected from three crop species and a variety of savanna tree species. Linear regression between total leaf chlorophyll content and bootstrapping were used to determine the leafpredictive capabilities of the various indices. The indices were then ranked based on the prediction error (the average root mean square error (RMSE)) derived from the bootstrapping process involving 1000 iterative resampling with replacement. The results show two red-edge derivative based indices (red-edge position via linear extrapolation index and the modified red-edge inflection point index) as the most consistent and robust, and that the majority of the top performing indices (in spite of species variability) were simple ratio or normalised difference indices that are based on off-chlorophyll absorption centre wavebands (690–730 nm).Item Pearl millet transformation system using the positive selectable marker gene phosphomannose isomerase(Springer-Verlag, 2004-04) O'Kennedy, Maretha M; Burger, JT; Botha, FCFertile transgenic pearl millet plants expressing a phosphomannose isomerase (PMI) transgene under control of the maize ubiquitin constitutive promoter were obtained using the transformation system described here. Proliferating immature zygotic embryos were used as target tissue for bombardment using a particle inflow gun. Different culture and selection strategies were assessed in order to obtain an optimised mannose selection protocol. Stable integration of the manA gene into the genome of pearl millet was confirmed by PCR and Southern blot analysis. Stable integration of the manA transgene into the genome of pearl millet was demonstrated in T-1 and T-2 progeny of two independent transformation events with no more than four to ten copies of the transgene. Similar to results obtained from previous studies with maize and wheat, the manA gene was shown to be a superior selectable marker gene for improving transformation efficiencies when compared to antibiotic or herbicide selectable marker genes.Item Physiological responses of selected African sorghum landraces to progressive water stress and re-watering(Elsevier, 2016-03) Devnarain, N; Crampton, BG; Chikwamba, Rachel K; Becker, JVW; O'Kennedy, Maretha MSorghum is particularly drought tolerant compared with other cereal crops and is favoured for subsistence farming in water scarce regions of the world. This study was conducted to identify South African sorghum landraces with superior drought tolerance compared with a drought-tolerant breeding line (P898012). Seedlings of 14 South African sorghum landrace accessions were initially screened for drought tolerance by assessing percentage leaf water content (LWC) during progressive water deficit. Four landraces (designated LR5, LR6, LR35, and LR36) recorded higher LWC than P898012. These were subsequently evaluated with P898012 during the reproductive growth stage, for their physiological responses to mild (4 days) and severe (6 days) water stress treatments and a moderate re-watered treatment on day 7. Plant height, soil moisture, and LWC were measured during harvests. Chlorophyll, carotenoid, and proline contents were quantified. All five genotypes maintained LWC above 80% during mild and severe stress treatments. For LR35 and LR36, LWC were recorded within 8% less in comparison to their well-watered controls following the moderate re-watered treatment. Significantly higher chlorophyll and carotenoid contents were recorded for both LR6 and LR35 in comparison to P898012 during severe stress. When LWC was reduced in LR36 (to 73.68%) and LR35 (to 73.51%), their proline content significantly increased by 14- and 16-fold, respectively. In this study, we have identified four previously uncharacterised sorghum genotypes exhibiting drought tolerance and described their physiological responses during water deficit and moderate re-watering. Aside from their application to breeding, these landraces are valuable resources to elucidate genetic mechanisms that enable drought tolerance in South African sorghum.Item Protective efficacy of a plant-produced beta variant rSARS-CoV-2 VLP vaccine in golden Syrian hamsters(2024-02) Lemmer, Yolandy; Chapman, R; Abolnik, C; Smith, Tanja; Schäfer, G; Hermanus, T; Du Preez, Ilse; Sepotokele, Kamogelo M; Roth, Robyn; Truyts, Alma; O'Kennedy, Maretha MIn the quest for heightened protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, we engineered a prototype vaccine utilizing the plant expression system of Nicotiana benthamiana, to produce a recombinant SARS-CoV-2 virus-like particle (VLP) vaccine presenting the S-protein from the Beta (B.1.351) variant of concern (VOC). This innovative vaccine, formulated with either a squalene oil-in-water emulsion or a synthetic CpG oligodeoxynucleotide adjuvant, demonstrated efficacy in a golden Syrian Hamster challenge model. The Beta VLP vaccine induced a robust humoral immune response, with serum exhibiting neutralization not only against SARS-CoV-2 Beta but also cross-neutralizing Delta and Omicron pseudoviruses. Protective efficacy was demonstrated, evidenced by reduced viral RNA copies and mitigated weight loss and lung damage compared to controls. This compelling data instills confidence in the creation of a versatile platform for the local manufacturing of potential pan-sarbecovirus vaccines, against evolving viral threats.Item Protective immunity of plant-produced African horse sickness virus serotype 5 chimaeric virus-like particles (VLPs) and viral protein 2 (VP2) vaccines in IFNAR-/- mice(2022-08) O'Kennedy, Maretha M; Coetzee, P; Koekemoer, O; Du Plessis, L; Lourens, CW; Kwezi, Lusisizwe; Du Preez, Ilse; Mamputha, Sipho; Rutkowska, Daria A; Lemmer, YolandyNext generation vaccines have the capability to contribute to and revolutionise the veterinary vaccine industry. African horse sickness (AHS) is caused by an arbovirus infection and is characterised by respiratory distress and/or cardiovascular failure and is lethal to horses. Mandatory annual vaccination in endemic areas curtails disease occurrence and severity. However, development of a next generation AHSV vaccine, which is both safe and efficacious, has been an objective globally for years. In this study, both AHSV serotype 5 chimaeric virus-like particles (VLPs) and soluble viral protein 2 (VP2) were successfully produced in Nicotiana benthamiana ΔXT/FT plants, partially purified and validated by gel electrophoresis, transmission electron microscopy and liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing before vaccine formulation. IFNAR-/- mice vaccinated with the adjuvanted VLPs or VP2 antigens in a 10 µg prime-boost regime resulted in high titres of antibodies confirmed by both serum neutralising tests (SNTs) and enzyme-linked immunosorbent assays (ELISA). Although previous studies reported high titres of antibodies in horses when vaccinated with plant-produced AHS homogenous VLPs, this is the first study demonstrating the protective efficacy of both AHSV serotype 5 chimaeric VLPs and soluble AHSV-5 VP2 as vaccine candidates. Complementary to this, coating ELISA plates with the soluble VP2 has the potential to underpin serotype-specific serological assays.Item Safety and immunogenicity of plant-produced African horse sickness virus-like particles in horses(BioMed Central, 2018-10) Dennis, SJ; O'Kennedy, Maretha M; Rutkowska, Daria A; Tsekoa, Tsepo L; Lourens, Carina W; Hitzeroth, II; Meyers, AE; Rybicki, EPAfrican horse sickness (AHS) is caused by multiple serotypes of the dsRNA AHSV and is a major scourge of domestic equids in Africa. While there are well established commercial live attenuated vaccines produced in South Africa, risks associated with these have encouraged attempts to develop new and safer recombinant vaccines. Previously, we reported on the immunogenicity of a plant-produced AHS serotype 5 virus-like particle (VLP) vaccine, which stimulated high titres of AHS serotype 5-specific neutralizing antibodies in guinea pigs. Here, we report a similar response to the vaccine in horses. This is the first report demonstrating the safety and immunogenicity of plant-produced AHS VLPs in horses.Item Sorghum biotechnology for food and health(2006-02) Mehlo, L; Grootboom, M; O'Kennedy, Maretha M; Okole, BThe goal of this project is to develop transgenic sorghum varieties that will deliver essential amino acids (lysine, threonine and tryptophan), vitamins A and E, iron and zinc - all of which are deficient in sorghum - to African populations in the arid and semi-arid tropics. The end-product is referred to as African Biofortified Sorghum (ABS). Taking cognisance of the limitations of classic breeding in the area of nutrition in fortifying sorghum with these essential amino acids, vitamins and minerals, the strategy will rely on genetic engineeringItem Stable transformation of Hi-II maize using the particle inflow gun(Bureau Scientific Publ, 1998-04) O'Kennedy, Maretha M; Burger, JT; Watson, TGFocuses on the transformation of the embryogenic type II callus, initiated from cultured immature zygotic embryos of the maize line Hi-II, by microprojectile bombardment using a particle inflow gun. Use of the plasmid pAHC25; Tissue culture and plant growth conditions; Optimization of bombardment conditions by transient expression; Southern blot analysis.Item Transcriptomic analysis of a Sorghum bicolor landrace identifies a role for beta-alanine betaine biosynthesis in drought tolerance(2019-12) Devnarain, A; Crampton, BG; Olivier, N; Van der Westhuyzen, Christiaan W; Becker, JVW; O'Kennedy, Maretha MSorghum is indigenous to Africa and a remarkably drought tolerant cereal crop. In this study, the genetic response mechanisms involved in sorghum's tolerance to progressive water deficit and re-watering were investigated in a South African landrace (designated LR6), using cDNA microarrays comprising 35,899 transcript probes. Significant differential expression of 902 transcripts, including 128 transcripts with currently unknown functions, was altered in response to progressive water stress and re-watering. The modulated sorghum genes had homology to proteins involved in growth, regulation, and protection. Gene Ontology (GO) analysis identified significant enrichment of 26 genes involved in the ‘response to abiotic stimulus’ GO category during severe stress. The expression of two genes associated with beta(b)-alanine betaine biosynthesis was validated with quantitative RT-PCR. Importantly, the detection of b-alanine betaine in sorghum leaf extracts using NMR spectroscopy, and the significant increase in relative abundance during severe stress supports the microarray and qRT-PCR findings, thereby highlighting a role for b-alanine betaine biosynthesis in drought tolerance of sorghum. In future, crop improvement initiatives that consider metabolic engineering of the b-alanine betaine biosynthesis pathway should be explored.Item Transformation of elite white maize using the particle inflow gun and detailed analysis of a low-copy integration event(Springer-Verlag, 2001-12) O'Kennedy, Maretha M; Burger, JT; Berger, DKElite white maize lines W506 and M37W were transformed with a selectable marker gene (bar) and a reporter gene (uidA) or the polygalacturonase-inhibiting protein (pgip) gene after bombardment of cultured immature zygotic embryos using the particle inflow gun. Successful transformation with this device did not require a narrow range of parameters, since transformants were obtained from a wide range of treatments, namely pre-culture of the embryos for 4-6 days, bombardment at helium pressures of 700-900 kPa, selection-free culture for 2-4 days after bombardment and selection on medium containing bialaphos at 0.5-2 mg l(-1). However, bombardments with helium pressures below 700 kPa yielded no transformants. The culture of immature zygotic embryos of selected elite white maize lines on medium containing 2 mg l(-1) 2,4-dichlorophenoxyacetic acid and 20 MM L-proline proved to be most successful for the production of regenerable embryogenic calli and for the selection of putative transgenic calli on bialaphos-containing medium after transformation. Transgenic plants were obtained from four independent transformation events as confirmed by Southern blot analysis. Transmission of the bar and uidA genes to the T-4 progeny of one of these transformation events was demonstrated by Southern blot analysis and by transgene expression. In this event, the transgenes bar and uidA were inserted in tandem.