Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Erica is a genus of about 860 species world wide, with 700 of these found in South Africa’s southwestern and southern Cape, making it by far the most speciose genus in the Cape Floristic Region. This poses a particular challenge in the construction of a molecular phylogeny of the genus. The choice of suitably variable gene regions is a crucial decision on which the successful phylogenetic reconstruction of this important genus is critically dependent. The aim of this project was therefore to determine which DNA regions, both chloroplast and nuclear, would be sufficiently variable to give adequate informative characters that may be useful at the species level phylogenetic reconstruction. A subset of 30 species, representing the range of morphological diversity and pollinator preference within Erica, was selected for study. For each of these species the variability in eight chloroplast regions (trnL-F, matK, trnS-G, rps12- rpl20, psbAtrnH, trnC-D, rps4-trnT and trnT-L) and the nuclear ITS region was investigated. The psbA-trnH, trnC-D, rps4-trnT and trnT-L chloroplast regions were found to be problematic to amplify and to possess too few Parsimony Informative Characters to be of use in phylogenetic reconstruction. Four of the chloroplast regions, trnS-G, trnL-F, matK and rpS12-rpL20 and the nuclear ITS region could be amplified and sequenced with success. The ITS region was found to be reasonably variable, with the chloroplast genes showing less variability. The DNA extraction method employed showed itself to be of critical importance in the success of the study. Two DNA extraction protocols, both modified from the original Doyle and Doyle (1987) method, were tested. The one included double the amount of β-mercaptoethanol and Polyvinylpyrrolidone (PVP) and the other included an extended phenol: chloroform: isoamylalcohol step. These variables, together with the effectiveness of these methods on fresh vs. silica dried plant samples, were investigated to determine which of the two would yield high quantities and qualities of DNA and result in the best method for the extraction of DNA from Erica species. / AFRIKAANSE OPSOMMING: Erica is ‘n genus van omtrent 860 spesies wêreldwyd, met 700 van hierdie spesies aanwesig in die suidwes en suid Kaap van Suid Afrika, wat dit by verre die mees spesieryke genus in die Kaapse Floristiese Streek maak. Dit stel ’n besondere uitdaging in die konstruksie van ’n molekulêre filogenie van die genus. Die keuse van geskikte variërende geen-areas is ‘n belangrike besluit waarvan die suksesvolle filogenetiese rekonstruksie van hierdie belangrike genus krities afhanklik sal wees. Die doel van hierdie projek was dus om te bepaal watter DNS areas, buide chloroplas en kern, genoegsaam varieer om voldoende informatiewe kenmerke te lewer om bruikbaar te wees in ’n spesie-vlak molekulêre rekonstruksie. ’n Subgroep van 30 spesies, wat die reeks van morfologiese diversiteit en bestuiwer voorkeure in Erica verteenwoordig, is dus vir die studie geselekteer. Vir elk van hierdie spesies is die variasie in agt chloroplast areas (trnL-F, matK, trnS-G, rps12- rpl20, psbA-trnH, trnC-D, rps4-trnT en trnT-L) en die kern ITS area ondersoek. Dit was problematies om die psbA-trnH, trnC-D, rps4-trnT en trnT-L chloroplast areas te amplifiseer, en daar is gevind dat hulle te min Parsimonie Informatiewe Kenmerke besig om bruikbaar te wees in filogenetiese rekonstruksie. Vier van die chloroplas areas, trnS-G, trnL-F, matK en rpS12-rpL20 en die kern ITS kon suksesvol geamplifiseer word en die basisvolgordes kon suksesvol bepaal word. Daar is gevind dat die ITS area redelik variërend is, terwyl chloroplas areas minder variasie getoon het. Die DNS ekstraksie metode wat gebruik is het die kritiese belang van die ekstraksie metode in die sukses van die studie bewys. Twee DNS protokolle, beide gemodifiseer van die oorspronklike Doyle en Doyle (1987) metode, is getoets. Die een het dubbel die hoeveelheid β-mercaptoetanol en Polyvinylpyrrolidone (PVP) bevat, en die het ’n uitgebruide fenol: chloroform: isoamylalkohol stap ingesluit. Hierdie veranderlikes, saam met die effektiwiteit van hierdie metodes op vars teenoor silika-gedroogde plant monsters, is ondersoek om vas te stel watter een van die twee die hoogste kwaliteit en kwantiteit DNS sou lewer en dus sal lei tot die beste DNS ekstraksie metode vir Erica spesies.

Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: The wild relatives are an important source of new genes for the genetic improvement of wheat. At Stellenbosch University the leaf and stripe rust resistance genes Lr54 and Yr37 were transferred from Aegilops kotschyi to chromosome 2DL of wheat. In an attempt to reduce the size of the whole-arm translocation on which the resistance genes occur, homoeologous pairing was induced between the wheat and corresponding Ae. kotschyi chromatin. The purpose of this study was to: (i) Evaluate the testcross progeny thus obtained; identify translocation recombinants that retained Lr54/Yr37 and to characterize these using molecular markers (ii) Test for the presence of genes for photoperiod insensitivity (Ppd) and reduced height (Rht) believed to be associated with the translocation (iii) Develop a SCAR marker for the most useful recombinant that could be recovered. Ten putative translocation recombinants were identified following the screening of 159 hemizygous testcross F1 plants with three microsatellite markers specific for chromosome arm 2DL. The recombinants were then characterized with another five microsatellite markers. Using the eight microsatellite markers the recombinants were ordered in two size categories with recombinant #74 being the shortest and having retained only proximal alien chromatin on 2DL. In addition to microsatellite markers, RAPDs, RGAs, AFLPs and SCAR markers were genetically mapped to the translocation and further resolved the recombinants into three size categories. In an attempt to find suitable markers linked to the shortest recombinant (#74) a polymorphic 410 bp AFLP fragment produced with the enzyme/selective nucleotide combination EcoRI – AAC/MseI – CAT, was converted into a dominant SCAR marker. In addition three microsatellite markers that mapped to recombinant #74 provided a useful recessive molecular marker system to detect Lr54/Yr37. Evaluation of the 10 recombinants with four 2DS-specific microsatellite markers revealed a large deletion of this chromosome arm in recombinant #74. This deletion may affect plant phenotypic characteristics and a strategy to replace the deleted region in recombinant #74 is proposed. To test for the presence of a gene for photoperiod insensitivity on the translocation, translocation-carriers plus controls were subjected to long and short day treatments, and the effect on time to flowering was studied. However, no evidence was found for the presence of such a gene. A height experiment to test for the presence of an Rht gene on the translocation confirmed its presence. This gene (designated H) appeared to be different from Rht8 on chromosome 2DS and was mapped on 2DL. While H does not occur in a chromosome region that corresponds with the location of Rht8, it does not rule out the possibility that they could be orthologous loci. Plant height data obtained for recombinant #74 suggested that H was lost through recombination in this particular recombinant. A greenhouse experiment suggested that the full-length translocation increased 100 kernel mass but had a detrimental effect on overall plant yield. Since a much shorter recombinant (#74) has been obtained, this will also have to be evaluated for associated effects. Such an evaluation needs to be done under commercial growing conditions and should involve the comparison of near-isogenic bulks with and without recombinant chromosome #74. The stripe rust resistance gene (Yr37) was mapped by screening hemizygous TF2 progeny of the 10 recombinants with Puccinia striiformis pathotype 6E22A+. Recombinant #74 retained both Lr54 and Yr37 and the two genes therefore occur towards the centromere. / AFRIKAANSE OPSOMMING: Wilde verwante spesies is ‘n belangrike bron van nuwe gene vir die genetiese verbetering van koring. By die Universiteit van Stellenbosch is die blaar-roes en streep-roes weerstandsgene Lr54 en Yr37 vanaf Aegilops kotschyi na chromosoom 2DL van koring oorgedra. ‘n Poging is vervolgens aangewend om die vol-armtranslokasie waarop die weerstandsgene voorkom te verklein deur homoeoloë paring tussen die koring en ooreenstemmende Ae. kotschyi chromatien te induseer. Die doelstelling van hierdie studie was daarom as volg: (a) Evaluering van die verkreë toetskruis-nageslag asook die identifisering en karakterisering van translokasie rekombinante wat Lr54/Yr37 behou het. (b) Toetsing vir fotoperiode onsensitiwiteits- (Ppd) en verkorte plant-hoogte (Rht) gene wat moontlik op die translokasie kon voorkom. (c) Die ontwikkeling van ‘n volgorde-spesifieke polimerase kettingreaksie (PKR) vir die mees bruikbare rekombinant. Tien translokasie rekombinante is geïdentifiseer nadat 159 hemisigotiese toetskruis F1-plante met drie mikrosatelliet-merkers, spesifiek vir chromosoom-arm 2DL, ge-evalueer is. Die rekombinante is hierna met vyf verdere mikrosatellietmerkers getoets. Die data van die agt mikrosatelliet-loci het die rekombinante in twee grootte-kategorieë geplaas waarvan rekombinant #74 die kortste was met slegs die proksimale gedeelte van 2DL wat uit vreemde chromatien bestaan. Behalwe mikrosatellite-merkers is toevallig-geamplifiseerde polimorfiese DNS (RAPD), weerstandsgeen-analoog (RGA), geamplifiseerde volgordelengte polimorfisme (AFLP) en volgorde-gekarakteriseerde geamplifiseerde-streke (SCAR) merkers ook geneties op die translokasie gekarteer. Data van die addisionele merkers het dit moontlik gemaak om die rekombinante in drie grootte-kategorieë te skei. Pogings om ‘n merker vir die kortse rekombinant (#74) te vind, het gelei tot die omskakeling van ‘n 410 bp polimorfiese AFLP-fragment (geproduseer met die ensiem/selektiewenukleotied kombinasie EcoRI - AAC/MseI - CAT), na ‘n dominante, volgordespesifieke PKR-merker. Hierbenewens kan drie mikrosatelliet-merkers wat op rekombinant #74 karteer as resessiewe merkers vir die identifisering van Lr54/Yr37 gebruik word. Die evaluering van die 10 rekombinante met vier chromosoom 2DSspesifieke mikrosatelliet-merkers het ‘n groot delesie van chromosoom-arm 2DS in rekombinant #74 uitgewys. Die delesie mag plant fenotipiese kenmerke beïnvloed en daarom is ‘n strategie vir die vervanging daarvan in rekombinant #74 voorgestel. Ten einde te toets of ‘n geen vir fotoperiode-onsensitiwiteit op die translokaie voorkom is translokasie-draers en kontroles aan lang- en kortdag-behandelings onderwerp en is die effek hiervan op dae-tot-blom gemeet. Geen bewyse vir so ‘n geen kon gevind word nie. ‘n Hoogte-eksperiment om te toets vir die teenwoordigheid van ‘n Rht-geen op die translokasie, het bevestig dat so ‘n geen wel voorkom. Die geen (voorgestelde simbool H) is gekarteer op 2DL en verskil oënskynlik van Rht8 op chromosoom 2DS. Die verskillende chromosoom-ligging van H en Rht8 skakel egter nie die moontlikheid dat hulle ortoloë loci mag wees uit nie. Plant-hoogte data vir rekombinant #74 het daarop gedui dat H nie meer in hierdie rekombinant voorkom nie. Data van ‘n glashuis-eksperiment het daarop gedui dat die vollengte-translokasie 100-korrel-massa verhoog maar dat dit plant-opbrengs verlaag. Aangesien ‘n aansienlike korter rekombinant (#74) verkry is, sal dit ook vir gekoppelde effekte getoets moet word. So ‘n evaluering moet egter onder kommersiële toestande gedoen word met gebruik van naby isogeniese-lyne met en sonder rekombinante chromosoom #74. Die streep-roes weerstandgeen (Yr37) is gekarteer deur hemisigotiese TF2- nageslag van die 10 rekombinante te toets vir weerstand teen Puccinia striiformis patotipe 6E22A+. Rekombinant #74 het beide Lr54 en Yr37 behou en die twee gene karteer dus naby die sentromeer.

Thesis (PhD)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Tuberculosis (TB) has been declared a global health emergency by the World Health Organisation, and consequently there is an urgency to develop improved methods of diagnosis and treatment. Despite the current TB epidemic, the disease can be treated effectively using isoniazid (INH) in combination with other antibiotics. However, INH is inactivated in the body by certain drug metabolising enzymes, which may reduce the efficacy of TB treatment. The activity of these drug metabolising enzymes, called NAT, are in turn reduced by nucleotide changes (SNPs) in the gene. These genetic variants (alleles) have been correlated with the rapid- (FA), intermediate- (IA), and slow acetylation (SA) enzymatic activity, and one is therefore able to investigate potential phenotypic effects via genotypic analyses. We investigated these genetic changes in the NAT1 and NAT2 genes in individuals from the local Coloured community (SAC) since this group has one of the highest TB incidences in the country. NAT2 is primarily responsible for the inactivation of INH, whilst NAT1 metabolises para-aminosalicyclic acid (PAS) which is used in the treatment of drug resistant TB. The NAT2 results indicated that the NAT2 alleles were not equally represented in three local ethnic groups studied, and subsequently the rapid, intermediate and slow acetylation activity reflected these differences. However, the relative frequency of these variants in the SAC and Caucasian groups were relatively low. These differences require further investigation to determine their overall relevance to the NAT2 activity differences between groups. In the case of the NAT1 analysis we also observed differences in the relative frequency of various NAT1 alleles between Caucasian and SAC individuals. However, many of these NAT1 SNPs and alleles have not as yet been characterised, so effects of these variants are currently unknown. Interestingly, the NAT1*4 and NAT1*10 alleles were the most prevalent NAT1 alleles in both Caucasians and SAC. The NAT1*4 allele exhibits the rapid NAT1 activity, whilst the activity of the NAT1*10 allele is currently subject to ongoing debate. In this respect, the analysis of NAT1 continues to be a topic for ongoing research. These results, observed for the NAT genes, underscore the importance of doing genetic analyses in local ethnic groups, since these differences may vary significantly between the groups. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) is deur die Wêreldgesondheidsorganisasie (WGO) tot 'n globale gesondheidsnood verklaar en derhalwe is dit noodsaaklik dat nuwe, verbeterde diagnostiese metodes ontwikkel word, wat tot meer effektiewe behandeling kan lei. Ten spyte van die huidige TB-epidemie, kan die siekte doeltreffend behandel word deur middel van isoniasied (INH), in kombinasie te met ander antibiotika. INH kan egter geïnaktiveer word deur sekere ensieme in die liggaam, met die gevolg dat INH nie meer effektief is nie in die behandeling van TB. Die aktiwiteit van hierdie ensiem, die sogenaamde NAT2 (Arielamien N-asetieltransferase 2) ensiem, word op sy beurt beïnvloed deur sekere nukleotied veranderings (SNPs) in die geen. Hierdie genetiese veranderings gekorreleer met ensiemaktiwiteitsveranderings (geklassifiseer as vinnig (FA) Intermediêr (IA) en stadig (SA)), wat mens in staat stel om potensiële fenotipiese effekte te ondersoek deur middel van genotipiese analise. Ons het hierdie genetiese veranderings ondersoek in die NAT1 en NAT2 gene in individue van die Kleurling-gemeenskap (SAC) omdat díe bevolkingsgroep die hoogste voorkoms van TB in die land het. NAT2 is primêr verantwoordelik vir die inaktivering van INH, terwyl NAT1 para-amienosalisilaat (PAS) inaktiveer, wat gebruik word in die behandeling van midel-weerstandige TB. Die NAT2 resultate dui daarop dat die allele van die NAT2 geen nie eweredig verteenwoordig wasin die drie etniese groepe nie en derhalwe word die vinnige (FA), intermediêre (IA) en stadige (SA) ensiemaktiwiteite deur hierdie verskille weerspieël. Hoewel die teenwoordigheid van hierdie variante relatief laag was in die SAC en Koukasiër gemeenskappe, is verdere studies nodig om die omvang van hierdie verskille te bepaal ten onsigte van NAT2 aktiwiteit tussen groepe. In die geval van die NAT1 analise het ons verskille waargeneem in die voorkoms van verskeie NAT1 allele tussen Koukasiese en SAC individue. Baie van hierdie NAT1 SNPs is egter nog nie gekarakteriseer nie, en derhalwe is die effek van hierdie NAT1 variante onbekend. Die NAT1*4 en NAT1*10 allele was die prominentste NAT1 alleel in beide Koukasiërs en SAC. Die NAT1*4 is betrokke by vinnige NAT1 aktiwiteit, terwyl die effek van die NAT1*10 alleel nog onderhewig is aan aktiefwe debat. In hierdie verband, is die studie van NAT1 steeds 'n onderwerp vir toekomstige navorsing. Hierdie resultate, wat vir die NAT gene waargeneem is, beklemtoon die belangrikheid van verdere genetiese analises in plaaslike etniese groepe, aangesien hierdie verskille beduidend kan wees tussen die verskillende groepe.

Thesis (PhD)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative organism of tuberculosis (TB), is a major cause for mortality and morbidity world-wide with a death toll only second to HIV among infectious diseases. Drug resistance is widespread and cases of multiple drug resistant TB (MDR-TB) and extensively drug resistant TB (XDR-TB) have emerged in several countries. Drug treatment is problematic and new drugs are not developed rapidly enough to offset the rapid drug resistance mutation rate of M. tuberculosis. Simple and effective diagnostics are required to contain the spread of the disease as current routine diagnostics are not fulfilling this role. Additionally, current rapid TB diagnostics are out of reach to resource poor settings due to infrastructure, cost and skill requirements. Novel TB diagnostics are thus required that meet these requirements. Mycobacteriophages are phages that infect mycobacteria and could offer a viable and cost effective alternative rapid TB diagnostics. In this study, an affinity-tagged fluorescent reporter mycobacteriophage is described, which was engineered to act as a TB diagnostic. Its performance proved favourable and superior to current existing mycobacteriophage-based TB diagnostics. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die organisme verantwoordelik vir tuberkulose (TB), is `n groot bron van mortaliteit en morbiditeit wêreldwyd en slegs HIV is verantwoordelik vir groter getalle sterftes as gevolg van n aansteeklike siekte. Middelweerstandigheid is algemeen en gevalle van meervoudigemiddelweerstandige tuberkulose (MDR-TB) en uiters weerstandige tuberkulose (XDR-TB) kom in verskeie lande voor. Antibiotika behandeling is problematies en nuwe anti-TB middels word nie vinnig genoeg ontwikkel om die antibiotika weerstandigheid mutasie spoed van M. tuberculosis te bekamp nie. Doeltreffende diagnostiese toetse word benodig om die verspreiding van die siekte te beheer en bestaande roetine diagnostiese toetse voldoen tans nie aan hierdie vereiste nie. Behalwe hiervoor, is huidige vinnige TB diagnostiese toetse buite bereik van arm instansies weens vereistes aan infrastruktuur, meegaande kostes en werknemervaardigheid. Nuwe TB diagnostiese toetse is dus nodig om aan hierdie vereistes te voldoen. Mikobacteriofaage is fage wat mikobacteria infekteer en kan moontlik 'n lewensvatbare en koste-effektiewe alternatief bied vir vinnige TB diagnostiese toetse. In hierdie studie word 'n affiniteitgekoppelde fluoreserende rapporteringsmikobakteriofaag beskryf wat ontwerp is om op te tree as `n nuwe vinnige TB diagnostiese toets. Die werking hiervan vertoon gunstige en beter resultate as die huidige, mikobacteriofaaggebaseerde TB-diagnostiese toetse.

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.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Worldwide, the rust diseases cause significant annual wheat yield losses (Wallwork 1992; Chrispeels & Sadava 1994). The utilization of host plant resistance to reduce such losses is of great importance particularly because biological control avoids the negative environmental impact of agricultural chemicals (Dedryver et al. 1996). The wild relatives of wheat are a ready source of genes for resistance to disease and insect pests. A large degree of gene synteny still exists among wheat and its wild relatives (Newbury & Paterson 2003). It is therefore possible to transfer a chromosome segment containing useful genes to a homologous region in the recipient genome without serious disruption of genetic information. Special cytogenetic techniques are employed to transfer genes from the wild relatives to the wheat genomes (Knott 1989). Unfortunately the transfer of useful genes may be accompanied by the simultaneous transfer of undesirable genes or redundant species chromatin which has to be mapped and removed (Feuillet et al. 2007). DNA markers are extremely useful for the characterisation and shortening of introgressed regions containing genes of interest (Ranade et al. 2001), and may also be used for marker aided selection of the resistance when the genes are employed commercially. Eight wheat lines containing translocations/introgressions of wild species-derived resistance genes were developed by the Department of Genetics (SU). These lines are presently being characterized and mapped and attempts are also being made to shorten the respective translocations. This study aimed to find DNA markers for the various translocations and to convert these into more reliable SCAR markers that can be used in continued attempts to characterize and improve the respective resistance sources. A total of 260 RAPD and 21 RGAP primers were used to screen the eight translocations and, with the exception of Lr19, it was possible to identify polymorpic bands associated with each translocation. However, it was not possible to convert all of these into more reliable SCAR markers. The primary reason for this was the low repeatability of most of the bands. Certain marker fragments turned out to be repeatable but could not be converted successfully. Some of the latter can, however, be used directly (in RAPD or RGAP reactions) as markers. The Lr19 translocation used in the study (Lr19-149-299) is a significantly reduced version of the original translocation and failure to identify polymorphisms associated with it can probably be ascribed to its small size. The following numbers of markers (direct and converted into SCARs) were Worldwide, the rust diseases cause significant annual wheat yield losses (Wallwork 1992; Chrispeels & Sadava 1994). The utilization of host plant resistance to reduce such losses is of great importance particularly because biological control avoids the negative environmental impact of agricultural chemicals (Dedryver et al. 1996). The wild relatives of wheat are a ready source of genes for resistance to disease and insect pests. A large degree of gene synteny still exists among wheat and its wild relatives (Newbury & Paterson 2003). It is therefore possible to transfer a chromosome segment containing useful genes to a homologous region in the recipient genome without serious disruption of genetic information. Special cytogenetic techniques are employed to transfer genes from the wild relatives to the wheat genomes (Knott 1989). Unfortunately the transfer of useful genes may be accompanied by the simultaneous transfer of undesirable genes or redundant species chromatin which has to be mapped and removed (Feuillet et al. 2007). DNA markers are extremely useful for the characterisation and shortening of introgressed regions containing genes of interest (Ranade et al. 2001), and may also be used for marker aided selection of the resistance when the genes are employed commercially. Eight wheat lines containing translocations/introgressions of wild species-derived resistance genes were developed by the Department of Genetics (SU). These lines are presently being characterized and mapped and attempts are also being made to shorten the respective translocations. This study aimed to find DNA markers for the various translocations and to convert these into more reliable SCAR markers that can be used in continued attempts to characterize and improve the respective resistance sources. A total of 260 RAPD and 21 RGAP primers were used to screen the eight translocations and, with the exception of Lr19, it was possible to identify polymorpic bands associated with each translocation. However, it was not possible to convert all of these into more reliable SCAR markers. The primary reason for this was the low repeatability of most of the bands. Certain marker fragments turned out to be repeatable but could not be converted successfully. Some of the latter can, however, be used directly (in RAPD or RGAP reactions) as markers. The Lr19 translocation used in the study (Lr19-149-299) is a significantly reduced version of the original translocation and failure to identify polymorphisms associated with it can probably be ascribed to its small size. The following numbers of markers (direct and converted into SCARs) were v identified: S8-introgression (Triticum dicoccoides) = one RAPD and two SCARs; S13-translocation (Aegilops speltoides) = four RAPDs, three RGAPs and five SCARs; S15-translocation (Ae. peregrina) = one RAPD and two SCARs; S20-translocation (Ae. neglecta) = two RAPDs, two RGAPs and one SCAR. The markers are already being employed in current projects aiming to map and shorten these translocations. Some of the markers can be combined in multiplex reactions for more effective mass screening. No repeatable markers could be identified for the four remaining translocations (S12 from Ae. sharonensis; S14 from Ae. kotschyi; Smac from Ae. biuncialis and Lr19-149-299 from Thinopyrum ponticum).