Cranes are large elegant birds that occur on all continents of the world except for South America and Antarctica. Of the fifteen species of crane worldwide, three predominantly occur in southern Africa; the Wattled crane (Bugeranus carunculatus), the Blue crane (Anthropoides paradisea) and the Crowned crane (Balearica regulorum). Crane numbers throughout the world are diminishing, mostly because of the destruction of their habitat and illegal bird trading. Efforts are underway to prevent species extinction, legally and through the compilation of a studbook that contains descriptions of physical attributes, ownership, location and possible kinships of birds in captivity . This investigation, first of its kind, WdS undertaken to assess whether twelve published and unpublished microsatellite primers developed for the related Whooping crane and Red-Crowned crane could be used to fingerprint the southern African crane species using cost effective polyacrylamide gel electrophoresis. The results obtained were then used to determine the extent of genetic variation within species and distance between species. All primer sets amplified heterologous microsatellite loci in the three crane species, however, the unpublished primers produced poorly defined fingerprints even after extensive optimization. Of the twelve microsatellite loci investigated, the Blue crane and the Wattled crane revealed a high level of polymorphism. The Blue crane displayed 76% polymorphism and the Wattled crane 92%. In contrast, for the Crowned crane, that belongs to a different subfamily, Balearicinae, only 50% of the loci were polymorphic. The alleles displayed sizes similar to that of the species for which the primers were developed. Little variation in size, less than 10 bp, was noted for the different alleles of the polymorphic loci. The number of alleles, on the other hand, at each of the polymorphic loci was found to be low. The frequency of the most prevalent allele at most of the loci was generally reasonably high. These results therefore suggest that these primer sets are not suitable for individual identification and differentiation using polyacrylamide gel electrophoresis. Xll The observed heterozygosity of the three crane species was low; 12% in Blue crane; 7% in Crowned crane; and 13% in Wattled crane. Nei's identity further confirmed the high similarity between individuals; 66-100% for Blue crane; 55-100% for Crowned crane and 41-95% for Wattled crane. This low genetic variation is attributed to possible relatedness between birds supplied by aviculturists whom have a limited number of birds in captivity. A Hardy-Weinberg test for equilibrium revealed that most of the microsatellite loci displayed a deficiency of heterozygotes, while a few loci displayed an excess of heterozygotes. In general, the Hardy Weinberg test of equilibrium supported the notion that the individuals within each of the species might have been related. Differentiation between the three crane species ranged from 3-5%, with Blue and Wattled crane displaying a higher degree of genetic similarity when compared to the Crowned crane, known to be the oldest extant crane species. The limited allelic variation within the microsatellite loci tested, as well as the extensive genetic similarity between individuals suggests that a wide-ranging search for additional microsatellite loci that are more polymorphic and contain a larger number of alleles should be undertaken for the southern African crane species. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Ostrich (Struthio camelus) breeding is a well-developed industry in South Africa. However, successful genetic management has yet to be implemented. Parentage in colony breeding ostriches is unknown, where for a given offspring, a number of possible parents exist. Molecular markers have been extensively used in the livestock industry to resolve parentage issues and are only beginning to be utilized to address the issues of the ostrich industry. The aims of this investigation were to test known microsatellite markers developed for other ostrich subspecies in a South African Black ostrich population, and to further test these markers for their use in individual and parentage identification. DNA was extracted from venous blood obtained from two pair bred families and a colony of 97 individuals. Eleven polymorphic microsatellite markers were tested by PCR amplification of DNA samples followed by multiplexing on polyacrylamide gels to generate DNA fingerprints for each individual. Alleles were sized and quantified and used to create genotypes for each individual. Parentage analysis was performed using exclusion and likelihood methods. Pedigrees were constructed for the families by comparison of genotypes. Breeding statistics were calculated for the colony individuals. Three microsatellite markers did not amplify in this population and one marker was found to be monomorphic in this population. Four of the microsatellite markers that successfully amplified produced anonymous amplification products suggesting a second annealing site in the genome sequence of Blacks. All loci displayed low observed heterozygosities indicative of little genetic variation in this population. For the colony sample, four individuals were not assigned either parent and one female did not contribute any offspring. On average females produced 4.86 ± 2.71 fertile eggs during the sampling period with a coefficient of variation of 55.86%. A total of 79.2% of individuals were assigned paternity and 88.3% were assigned maternity. A greater number of loci are required to improve the power of parentage analysis within breeding flocks incorporating all eggs laid. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

The face of animal breeding has changed significantly over the past few decades. Traditionally, the genetic improvement of both plant and animal species focussed on the selective breeding of individuals with superior phenotypes, with no precise knowledge of the genes controlling the traits under selection. Over the past few decades, however, advances in molecular genetics have led to the identification of genetic markers associated with genes controlling economically important traits, which has enabled breeders to enhance the genetic improvement of breeding stock through linkage disequilibrium marker-assisted selection. Since the integration of marker-assisted selection into breeding programmes has not been widely documented, it is important that breeders are able to evaluate the advantages and disadvantages of marker-assisted selection, in comparison to phenotypic selection, prior to the implementation of either selection strategy. Therefore, this investigation aimed to develop deterministic simulation models that could accurately demonstrate and compare the effects of phenotypic selection and marker-assisted selection, under the assumption of both additive gene action and complete dominance at the loci of interest. Six computer models were developed using Microsoft Excel, namely 'Random Mating,' 'Phenotypic Selection,' 'Marker-Assisted Selection,' 'Selection with Dominance,' 'Direct Selection' and 'Indirect selection.' The 'Random Mating' model was firstly used to determine the effects of linkage disequilibrium between two loci in a randomly mating population. The 'Phenotypic Selection' and 'Marker-Assisted Selection' models focused primarily on examining and comparing the response to these two selection strategies over five generations and their consequent effect on genetic variation in a population when the QTL of interest exhibited additive gene action. In contrast, the 'Selection with Dominance' model investigated the efficiency of phenotypic selection and marker-assisted selection under the assumption of complete dominance at the QTL under selection. Finally, the 'Direct Selection and 'Indirect Selection' models were developed in order to mimic the effects of marker assisted selection on two cattle populations utilizing both a direct and indirect marker respectively. The simulated results showed that, under the assumption of additive gene action, marker-assisted selection was more effective than phenotypic selection in increasing the population mean, when linkage disequilibrium was present between the marker locus and the QTL under selection and the QTL captured more than 80% of the trait variance. The response to both selection strategies was shown to decrease over five generations due to the decrease in genetic variation associated with selection. When the QTL under selection was assumed to display complete dominance, however, marker-assisted selection was markedly more effective than phenotypic selection, even when a minimal amount of linkage disequilibrium was present in the population and the QTL captured only 60% of the trait variance. The results obtained in this investigation were successful in simulating the theoretical expectations of markerassisted selection. The computer models developed in this investigation have potential applications in both the research and agricultural sectors. For example, the successful application of a model developed in this investigation to a practical situation that simulated markerassisted selection, was demonstrated using data from two Holstein cattle populations. Furthermore, the computer models that have been developed may be used in education for the enhancement of students understanding of abstract genetics concepts such as linkage disequilibrium and marker-assisted selection. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Eucalyptus macarthurii is one of the cold tolerant eucalypt species grown in South Africa for pulp and paper. However, little research has been done on this species’ growth performance. A study was therefore initiated to: i) analyse growth characteristics of Eucalyptus macarthurii at two sites and to calculate genetic parameters (genetic and phenotypic correlations, heritabilities and genetic gains), ii) develop a non-destructive near infrared calibration model to predict whole tree pulp yield of Eucalyptus macarthurii, and iii) screen a second generation Eucalyptus macarthurii breeding population, using the developed near infrared calibration model on core samples, to predict screened pulp yield and to rank and identify families with superior pulping properties. Eucalyptus macarthurii population growth data (diameter under bark, diameter over bark, bark thickness, bark stripping, height, basic wood density and stem form) were measured at Pinewoods and Vlakkloof sites and their respective genetic parameters calculated. Genotype by environment interaction was found in this population, indicating that different populations should perhaps be developed independently of each other for the two sites. Genetic and phenotypic correlations between diameter over bark and diameter under bark were, 0.96 and 0.98 for Pinewoods and 0.98 and 0.99 for Vlakkloof, respectively. These correlations indicated that selection of diameter over bark would lead to a positive indirect selection for diameter under bark. The heritability estimates also ranged from 0.03 to 0.23 at both sites, which indicated a reasonable response to selection. The predicted gains for all traits found at Pinewoods were higher than those at Vlakkloof for progeny trials E76/P1, except height for progeny trial E76/P2, which was 2.09m at Pinewoods site and 3.52m at Vlakkloof site which showed that, selection for taller trees will be more effective at Vlakkloof site. A preliminary study was undertaken from eleven second generation trees (2007 tree collection) to investigate if the radial strip core taken at breast height predicts the whole tree wood properties. Correlations found between laboratory Kraft pulping of whole tree wood discs and whole tree NIR spectra with that of the radial strip core NIR spectra were 0.9472 and 0.9506, respectively. These results confirmed that NIR spectra of the radial strip core at breast height predict the whole tree wood properties. A non-destructive near infrared calibration model using wood samples was obtained from Eucalyptus macarthurii felled trees. The wood samples were chipped into wood chips, pulped using Kraft pulping (reference method) and a sub-sample of wood chips of the same trees were ground into sawdust samples and analysed through near infrared spectroscopy for screened pulp yield. The screened pulp yield values obtained from both processes had a narrow screened pulp yield range of 40 to 48%. The Eucalyptus macarthurii screened pulp yield values obtained from both processes, as well as from values obtained from other eucalypt species, were subjected to Vision® Software for calibration and validation of the near infrared calibration model. The results indicated a strong calibration correlation coefficient of 94%, between Kraft pulping and near infrared spectroscopy with a validation coefficient of 89%. The strong correlation and validation coefficient indicated that a reliable non-destructive near infrared model to predict screened pulp yield was successfully developed. The successful development of the valid calibration model required a wider range of other eucalypts species, which improved the development of the model. The developed calibration model was applied to the second generation breeding population planted in KwaZulu-Natal and Mpumalanga provinces, using wood core samples obtained from standing trees for the prediction of screened pulp yield. The highest screened pulp yield achieved was 48%, which compared well to that found for Kraft pulping, which confirmed the success of the development of the calibration model. There was a wide scope of growth variation found amongst traits, which will be useful in selecting superior trees for the next generation. The development of the nondestructive near infrared calibration model was a success due to the strong correlation coefficients found between the screened pulp yields obtained from Kraft pulping and near infrared spectroscopy processes, which was achieved by the inclusion of other eucalypt species in the dataset. The calibration model can be used to select the top performing individual and family trees for the next generation based on screened pulp yield. Tree improvement trials can now be conserved for further breeding, without felling the trees for determination of pulping properties. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Leptin, a 16 (kilo Dalton) kDa hormone secreted predominantly by white adipocytes, regulates reproduction, energy intake and expenditure, and is involved in immune system function. Previous studies have identified associations between polymorphism E2FB in the leptin gene (lep) of cattle and milk quality and quantity, feed intake, and fat deposition in dairy and beef cattle though further studies have shown inconclusive results. Furthermore, indigenous South African cattle have not been involved in lep investigations or the applicability of the marker in South African beef grading systems. An investigation was conducted into the association of an SNP of a cytosine (C) to thymine (T) SNP (single nucleotide polymorphism) mutation in exon 2 of the bovine lep (leptin) gene with weight gain, body condition, carcass fat content and quality in a population of indigenous Nguni cattle (n = 70) as well as a population of exotic British Hereford cattle (n = 54). The Hereford population had higher T-allele frequencies and a lower P-value (P = 0.172) for the E2FB genotypes than the Nguni population (P = 0.958). The resulting E2FB lep genotypes CC, CT and TT did not show an association with the pre- and post-slaughter traits initial live weight (ILW), body condition score (BCS), slaughter live weight (SLW), carcass fat content (FAT), carcass conformation (CFN) or warm carcass mass (WCM) for either population though t-tests revealed an association with the CT genotype with increased ILW than TT and a significantly higher WG in the TT genotypes than the CT (P<0.05). Subsequently, differences in pre- and post-slaughter traits in both populations were largely attributable to breed differences. The Hereford population exhibited significantly higher WG, CFN, SLW, WCM and CCM (P<0.05) than the Nguni population. The Nguni displayed significantly higher ILW and BCS values when graded in terms of the commercial South African AAA feedlot system. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Thesis (MScAgric)--University of Stellenbosch, 1994 / 127 Leaves printed single pages, preliminary pages i-viii and numberd pages 1-118.Includes bibliography,tables and figures. / Date on t.p.: Dec. 1994. / ENGLISH ABSTRACT: Effects of pH,sucrose, boric acid and temperature on in vitro germination of pollen of Protea repens (L.) L. cv. 'Embers' were investigated in hanging-drop culture to establish optimum conditions for germination. Optimum values were found within ranges pH: 5 - 8, sucroseconcentration:0.4 - 0.7 M, boric acid concentration:50 - 500 mg.e-1 , and incubation temperature: 5 - 30°C. Storage temperature and humidity on pollen viability was studied in four Pro tea clones. Pollen was stored at a range of temperatures and relative humidities for up to one year and tested for ability to germinate in vitro. Pollen of P. repens cv. 'Sneyd', P. eximia cv. 'Fiery Duchess' andP. magnifica clone 'T 84 07 OS', stored in liquid nitrogen (-196°C) and in a freezer (-14° to -18°C), retained a germination percentage as high as that of fresh pollen regardless of humidity. The study showed that long-term storage of protea pollen is not feasible at temperatures above O°C. The correlations between the fluorochromatic reaction (FCR) and germinability were found to be low and nonsignificant. Fifteen month old cryopreserved 'Sneyd' pollen was shown to retain its ability to fertilize and set seed equal to that of fresh pollen. 'Sneyd', 'Fiery Duchess' and 'T 84 07 OS' pollen could be repeatedly thawed and frozen in liquid nitrogen before its germinability in vitro decreased. The morphology and size of Protea pollen was studied, using light and scanning electron microscopy. Polymorphic grains were observed in two interspecific hybrids. Very small differences in pollen grain size were recorded between clones/species. The male fertility of 25 interspecific Pro tea hybrids, based on in vitro pollen germinability, was investigated. The majority of hybrids were found to be sufficiently fertile to be used in a breeding programme. Pistil structure and pollen tube pathways were investigated in 'Sneyd' using light and scanning electron microscopy. The pistil had four distinct regions, consisting of the stigma, the vertebra-shaped upper style, the heart-shaped lower style, and the ovary. The pistil had a stylar canal along its entire length and this canal was also the route by which pollen tubes grew to the ovary. Very low numbers of pollen tubes reached the ovary. The breeding system of 'Sneyd' and 'Fiery Duchess' were determined from pollen tube and seed set data, after controlled hand-pollinations. Both clones were found to be fully selfcompatible. Very low percentages autogamous seed set were recorded. Interspecific crosses had a low success rate. An incompatibility reaction probably occurred on the stigma and/or in the upper style regions.The attainment of maximum stigma receptivity of two Protea cultivars was investigated by means of seed set experiments, pollen tube growth observations and measurement of the degree of opening and closing of the stigmatic groove. Both cultivars were found to be protandrous. The maximum stigmatic groove width of both cultivars never exceeded the pollen grain diameter. It was concluded that Protea spp. must be hand-pollinated two to six days after anthesis in order to obtain maximum seed set; while for the observations of pollen tubes in the ovary, inflorescences must not be harvested before seven days after pollination. / AFRIKAANSE OPSOMMING: Ten einde 'n optimale medium vir die in vitro-ontkieming van Protea-stuifmeel te ontwikkel, is die effek van pH, sukrose, boorsuur, en temperatuur op die in vitro-ontkieming van Protea repens (L.) L. cv. 'Embers'-stuifmeel deur middel van die hangdruppel-metode ondersoek. Die volgende reekse van veranderlikes wat getoets is, is as optimaal gevind; pH: 5 - 8, sukrosekonsentrasie: 0.4 - 0.7 M, boorsuurkonsentrasie: 50 - 500 mg.e-1 en inkubasietemperatuur: 5 - 30°C. Die invloed van bergingstemperatuur en humiditeit op stuifmeel-Iewenskragtigheid is in vier Protea-klone ondersoek. Stuifmeel is gestoor by 'n reeks temperature en relatiewe humiditeite vir tot eenjaar, en vir in vitro-ontkiemingsvermoe getoets.' Stuifmeel van P. repens <?v. 'Sneyd', P. eximia cv. 'Fiery Duchess', en P. magnifica kloon'T 84 07 OS', in vloeibare stikstof (-196°C) en in 'n vrieskas (-14° tot - 18°C) geberg, het 'n ontkiemingspersentasie gelykstaande aan die van vars stuifmeel gehandhaaf, ongeag van die humiditeit. Hierdie studie het verder aangetoon dat langtermynberging van Protea-stuifmeel bokant O°C me die moeite werd is me. Die korrelasie tussen die fluorochromatiese reaksie (FCR) en ontkieming was laag en me betekemsvol me. 'Sneyd' -stuifmeel wat vir 15 maande in vloeibare stikstof gestoor is, het die bevrugtings- en saadsetvermoe gelykstaande aan vars stuifmeel behou. 'Sneyd', 'Fiery Duchess' en 'T 84 07 OS'-stuifmeel kon herhaaldelik in vloeibare stikstof gevries en ontdooi word voordat hul ontkiemingsvermoe afgeneem het. Die morfologie en grootte van Proteastuifmeel is deur middel van lig- en skandeerelektronmikroskopie bestudeer. Polimorfiese stuifmeelkorrels is in twee interspesie-hibriede waargeneem. Baie klein verskille in stuifmeelkorrelgroottes het tussen klone/spesies voorgekom. Die manlike vrugbaarheid van 25 Protea-interspesiehibriede, gebaseer op die in vitro-ontkiembaarheid, is ondersoek. Dit is gevind dat die meerderheid hibriede 'n voldoende graad van vrugbaarheid het om in 'n teelprogram te gebruik. Die stamperstruktuur en stuifmeelbuiswee in P. repens is deur middel van lig- en skandeer-elektronmikroskopie ondersoek. Die stamper bestaan uit vier kenmerkende gebiede, naamlik die stempel, die werwelvormige bo-styl, die hartvormige onderstyl, en die vrugbeginsel. Die stamper het 'n stylkanaal regdeur die totale lengte van die stamper, en hierdie kanaal is ook die weg waarvolgens stuifmeelbuise na die vrugbeginsel gegroei het. Min stuifmeelbuise het die vrugbeginsel bereik. Die teelsisteem van 'Sneyd' en 'Fiery Duchess' is deur middel van stuifmeelbuis- en saadsetdata na gekontroleerde handbestuiwings ondersoek. Beide kIone was ten volle selfverenigbaar. Die persentasie outogame saadset was baie laag. Interspesiekruisings het 'n baie lae sukses gehad. Dit is voorgestel dat die onverenigbaarheidsreaksie in die stempel en/of in die bopunt van die styl plaasvind. Die bereiking van maksimum stempelontvanklikheid van twee Protea-cultivars is deur middel van saadseteksperimente, stuifmeelbuisdata en waarnemings van die oop- en toemaak van die stempelgroef ondersoek. Beide cultivars was protandries. Die maksimum stempelgroefwydte het nooit die stuifmeelkorreldeursnee oorskry nie. Dit is afgelei dat Protea-spesies twee tot ses dae na antese handbestuif moet word vir optimale saadset. Vir die waarneming van stuifmeelbuise in die vrugbeginsel, moet bloeiwyses nie voor sewe dae na bestuiwing geoes word nie.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Despite numerous attempts involving a variety of target genes, the successful transgenic manipulation of sucrose accumulation in sugarcane remains elusive. It is becoming increasingly apparent that enhancing sucrose storage in the culm by molecular means may depend on the modification of the activity of a novel gene target. One possible approach to identify target genes playing crucial coarse regulatory roles in sucrose accumulation is to assess gene expression during the developmental transition of the culm from active growth to maturation. This study has resulted in the successful optimisation of a mRNA hybridisation technique to characterise the expression of 90 carbohydrate metabolism-related genes in three developmentally distinct regions of sugarcane culm. A further goal of this work was to extend the limited knowledge of the regulation of sucrose metabolism in sugarcane, as well as to complement existing data from physiological and biochemical studies. Three mRNA populations derived from the different culm regions were assayed and their hybridisation intensities to the immobilised gene sequences statistically evaluated. The relative mRNA transcript abundance of 74 genes from three differing regions of culm maturity was documented. Genes exhibiting high relative expression in the culm included aldolase, hexokinase, cellulase, alcohol dehydrogenase and soluble acid invertase. Several genes (15) were demonstrated to have significantly different expression levels in the culm regions assessed. These included UDP-glucose pyrophosphorylase and UDP-glucose dehydrogenase, which were down-regulated between immature and mature internodes. Conversely, sucrose phosphate synthase, sucrose synthase and neutral invertase exhibited up-regulation in maturing internodal tissue. A variety of sugar transporters were also found to be up-regulated in mature culm, indicating a possible control point of flux into mature stem sink tissues. Combined with knowledge of the levels of key metabolites and metabolic intermediates this gene expression data will contribute to identifying key control points of sucrose accumulation in sugarcane and assist in the identification of gene targets for future manipulation by transgenic approaches. / AFRIKAANSE OPSOMMING: Ondanks verskeie pogings, waartydens verskeie gene geteiken is, is daar nog weinig sukses behaal om sukrose-akkumulering te verhoog. Toenemend wil dit voorkom asof suksesvolle genetiese manipulering van sukroseberging in die stingel van die verandering van ‘n nuwe geen afhanklik sal wees. Een van die moontlike benaderings wat gevolg kan word om potensiële teiken gene wat ‘n belangrike rol in die beheer van sukrose-opberging speel te identifiseer, is om geen uitdrukkingspatrone in die stingel tydens die omskakeling van aktiewe groei tot volwassenheid te karakteriseer. In hierdie studie is ‘n metode gebaseer op die hibridisering van mRNA geoptimiseer en suksesvol aangewend om die uitdrukkingspatrone van 90 verskillende geselekteerde gene, wat vir sleutelensieme in die beheer van koolhidraatmetabolisme kodeer, te bestudeer. Die doel met die ondersoek was om die beperkte kennis oor die regulering van koolhidraatmetabolisme uit te brei en om die bestaande inligting afgelei van fisiologiese en biochemiese-studies aan te vul. Drie verskillende mRNApopulasies, verkry uit verskillende dele van die stingel, is ontleed deur verskillende peilers te gebruik. Die gegewens is statisties ontleed en dit het afleidings oor die verandering in uitdrukking van hierdie gene moontlik gemaak. Die relatiewe konsentrasies van 74 verskillende gene is gedokumenteer. Gene wat sterk uitgedruk word het aldolase, heksokinase, sellulase, alkoholdehidrogenase en ongebonde suurinvertase ingesluit. Die uitdrukkingspatrone van 15 gene het tussen die verkillende weefsels gevarieer. Gene waarvan die uitdrukking tydens die oorgang na volwassenheid verlaag sluit in UDP-glukose pirofosforilase en UDP-glukose dehidrogenase en waarvan die uitdrukking verhoog sukrosefosfaatsintase, sukrosesintase en neutrale invertase in. Die uitdrukking van verskeie suikertransporter gene verhoog tydens volwassewording. Hierdie inligting te same met die huidige kennis oor heersende metabolietvlakke sal bydrae tot die identifisering van geenteikens vir toekomstige genetiese manupulering.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination. / AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies. Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde. Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie materiaal word meestal geënt op onderstokmateriaal waarvan die virus status onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n noodsaaklike beheermeganisme vir virus-infeksie. Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut konstruk bevat. ‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3 infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat v getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het. Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante wat teenwoordig is in Suid-Afrika nie. Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus weerstandbiedendheids studies. Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Nitric oxide (NO) has previously been shown to play a vital role in plants that are undergoing oxidative stress arising from abiotic stress. To better understand the role of NO on the antioxidative pathway, the effect of NO on Superoxide Dismutase (SOD) activity was studied during salt stress on soybean nodules. The enzymatic activity of specific MnSOD and FeSOD isoforms increased upon 1 week of exposure of nodules to NO or salt stress, the activity of CuZnSOD isoforms however increased in response to salt stress only. Furthermore, 4 putative FeSOD and MnSOD transcripts were identified and shown to increase in response to NO and salt stress. The promoter sequences of these NO-responsive putative SOD genes were analysed alongside a cystatin (AtCYS-1) which is also NO-inducible. Putative NO-responsive cis-acting elements as well as abiotic stress-responsive cis-acting elements were studied amongst these promoter sequences. The MYCL element and the AtMYB4 binding site were found to occur in all four NO-inducible SOD promoter sequences as well as in the AtCYS-1 promoter sequence. This suggests that NO acts via MYCL and/or AtMYB4 to up-regulate specific FeSODs and MnSODs, causing an increase in the activity of these SOD isoforms, thus reducing oxidative stress and cell death in soybean nodules. Furthermore, NO may also be up-regulating cystatins to inhibit cysteine proteases, thus preventing the onset of programmed cell death (PCD) and subsequently reducing salt stress-induced cell death. / AFRIKAANSE OPSOMMING: Geen opsomming

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Inorganic Phosphate (Pi) is one of the key nutrients required by all living organisms on earth. This nutrient is of vital importance to higher plants but it is not readily available for uptake from the soil, implying constant stress on plants. During photosynthetic dark and light reactions, phosphate is a prerequisite for all reactions to occur and to ensure plant survival. This statement implies that a careful homeostatic control of this nutrient is necessary in order to maintain a balanced carbon flow in all sub-cellular plant compartments. Phosphate limitation is a threat to plant survival and one way of addressing this nutritional hurdle is by feeding plants with fertilizer. This method of crop development and general plant maintenance by humans has a devastating effect on the environment, as phosphate causes eutrophication and various other consequences which are detrimental to animal life. Plants, however, are naturally equipped with Pi transporters which are activated conditionally depending on the external Pi availability. These transporters are present in most sub-cellular compartments and some of them have been identified and characterised, while others remain to be a prediction. If these transporters are characterised accordingly it might eventually mean that the use of fertilizers may no longer be necessary. In order to contribute to successful Pi-efficient crop development, a clearer understanding of P-dynamics in the soil and its recycling ability inside the plant itself is necessary. During this study it was attempted to characterise a putative high affinity Pi transporter, PHT1;5, from Arabidopsis thaliana via a Escherichia coli and yeast heterologous expression system and its Km value predicted in order to verify/hypothesise whether it is a high or low affinity transporter. This transporter is expressed in leaves and could be a promising tool for future carbon partitioning studies during phosphate limitation. / AFRIKAANSE OPSOMMING: Anorganiese fosfaat (Pi) word beskou as een van die belangrikste nutriente benodig vir alle lewe op aarde. Dit vervul ‘n hoof rol in talle noodsaaklike prosesse in hoër plante en is veral ‘n voorvereiste vir fotosintetiese reaksies om plaas te vind. In ‘n plant se natuurlike omgewing is anorganiese fosfaat nie geredelik bekskikbaar in grond nie en dus word daar vermoed dat plante onder konstante fosfaat stres gevind word. Omdat fosfaat so ‘n belangrike rol speel tydens fotosintese is dit noodsaaklik dat daar ‘n balans op sellulêre vlak gehandhaaf word, veral wat die verspreiding van koolhidrate tussen die verskillende kompartemente van die sel betref. Plante se oorlewing word bedreig deur ‘n tekort aan fosfaat in die omgewing en die enigste onmiddelike oplossing daarvoor is deur die toediening van bemestingstowwe. Hierdie metode van landery ontwikkeling en algemene instandhouding van plante deur die mensdom het ’n baie negatiewe effek op die omgewing. ‘n Oormaat fosfaat lei tot eutrifikasie en het verkeie ander negatiewe nagevolge wat dodelik is vir die dierelewe. Plante beskik ook oor natuurlike interne fosfaat transporters om hierdie tekort te oorkom. Hierdie transporters word op grond van eksterne fosfaat beskikbaarheid ge-aktiveer of ge-deaktifeer. Die transporters is teenwoordig in meeste sub-sellulêre kompartemente en sommige is al ge-identifiseer en gekarakteriseer, terwyl ander slegs ‘n voorspelling bly. Gedurende hierdie studie was ‘n poging aangewend om ‘n anorganiese fosfaat transporter van Arabidopsis thaliana, PHT1;5, te karakteriseer met behulp van mikro-organismes soos Escherichia coli en gis. Die poging het ingesluit om ‘n Km waarde vir hierdie transporter te voorspel en sodoende ‘n hipotese daar te stel van of dit hoë of lae affiniteit het vir fosfaat. Die transporter word groot en deels aangetref in blare en kan dus dien as ‘n belowende apparaat vir toekomstige koolhidraat uitruiling studies gedurende fosfaat tekort.