Thesis (Ph.D.)--Ohio University, March, 2007. / Title from PDF t.p. Includes bibliographical references.

Zugl.: Halle (Saale), Univ., Diss., 2008

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The goals of this project were firstly to develop the tissue preparation and in situ hybridisation protocols for sugarcane culm tissue, and secondly to use the developed techniques to examine the expression patterns of three invertase isoforms in sugarcane internodes of various developmental stages. Sugarcane invertases have been the focus of intense research for many years, yet almost nothing is known of their tissue-specific distribution. It was thought that by characterising their expression patterns using in situ hybridisation, more knowledge of their functions and involvement in sucrose accumulation would be gained. Although in situ hybridisation is now regularly used to study gene expression in plants, there is to date only a single publication describing its use on immature sugarcane tissue. Therefore this technique needed further development, and this was achieved by comparing different tissue preparation methods, as well as by systematically testing the various parameters pertaining to each method. The in situ hybridization technique was also developed by testing and comparing a number of key parameters. It was found that fixing whole mount tissue for 48 h preserved sugarcane tissue adequately. High hybridization temperatures and probe concentrations provided the best signal, and including pre-treatment with HCl and Pronase was essential in sensitizing the tissue to the probe. A less viscous detection buffer reduced both osmotic effects and time required for signal detection. In the second part of this study, the developed method was used to examine the expression patterns of the three invertase isoforms in young, maturing and mature internodes of sugarcane, and the results were complemented with Northern blot analysis. Transcript of all three isoforms was found to be present in the storage parenchyma and in the phloem tissue. Transcript levels of all three isoforms declined in maturing tissue, with soluble acid invertase declining sharply and dropping below detection in maturing and mature tissue. Transcript levels of cell wall invertase and neutral invertase declined only gradually, and appreciable levels of both were still present in mature tissue. Acid invertase is suggested to be mainly involved in internode elongation, while cell wall invertase would appear to play important roles in phloem unloading and turgor control. Neutral invertase is suggested to be involved in either sucrose cycling or maintenance of hexose pools, however the function of this enzyme remains unclear. This study has demonstrated the value of in situ hybridization, yet at the same time has shown its limitations, especially when more traditional biochemical techniques are not employed to complement the results. Although the precise functions of the invertase isoforms in sugarcane remain inconclusive, this study has opened up the way for tissuespecific promoter design and future in situ studies of sugarcane invertases / AFRIKAANSE OPSOMMING: Die doel van hierdie projek was tweeledig: eerstens om weefselvoorbereiding en in situhibridisasie- protokolle vir die stingelweefsel van suikerriet te ontwikkel; en tweedens om die ontwikkelde tegnieke te gebruik om die uitdrukkingspatrone van drie invertaseisovorme in die suikerriet-internodes van verskeie ontwikkelingstadia te ondersoek. Suikerriet-invertases is al vir jare lank die fokus van intense navorsing, maar baie min is bekend oor hulle weefselspesifieke verspreiding. Die idee was om meer kennis oor suikerriet-invertases se funksies en betrokkenheid by sukrose-akkumulasie te verkry deur in situ-hibridisasie te gebruik om hulle uitdrukkingspatrone te karakteriseer. Alhoewel in situ-hibridisasie deesdae gereeld gebruik word om geenuitdrukking in plante te bestudeer, is daar tot op datum slegs een publikasie wat die gebruik daarvan in onvolwasse suikerrietweefsel beskryf. Hierdie tegniek moes dus verder ontwikkel word, en dit is gedoen deur verskillende weefselvoorbereidingsmetodes te vergelyk en sistematies die verskillende parameters wat op elke metode van toepassing is te toets. Die in situ-hibridisasie-tegniek is ook ontwikkel deur die toetsing en vergelyking van 'n aantal sleutelparameters. Daar is gevind dat suikerrietweefsel voldoende gepreserveer word deur die intakte gemonteerde weefsel vir 48 uur te fikseer. Hoë hibridisasietemperature en hoë peilerkonsentrasies het die beste sein gegee; die insluiting van voorbehandeling met HCl en Pronase was noodsaaklik om die weefsel meer gevoelig vir die peiler te maak. Osmotiese invloede en die tyd nodig vir seindeteksie is verminder deur die viskositeit van die buffer te verminder. In die tweede deel van die studie is die ontwikkelde metode gebruik om die uitdrukkingspatrone van die drie invertase-isovorme in jong, ontwikkelende en volwasse internodes te ondersoek en die resultate is deur 'n noordelike oordraganalise gekomplementeer. Transkripte van al drie isovorme is in die stoorparenchiem en floëemweefsel gevind. Transkripvlakke van al drie isovorme het afgeneem in ontwikkelende weefsel, met oplosbare suurinvertase wat skerp afgeneem en tot onder die deteksie-limiet gedaal het in ontwikkelende en volwasse weefsel. Transkripvlakke van selwandinvertase en neutrale invertase het slegs geleidelik afgeneem en merkbare vlakke van albei was teenwoording in ontwikkelende en volwasse weefsel. Daar word voorgestel dat suurinvertase hoofsaaklik betrokke is by internodeverlenging, terwyl selwandinvertase skynbaar 'n belangrike rol in floëem-ontlading en turgor-beheer speel. Daar word voorgestel dat neutrale invertase betrokke is óf by die sukrose-sirkulering óf by die onderhoud van heksose-poele; die funksie van hierdie ensiem is egter steeds nie duidelik nie. Hierdie studie het die waarde van in situ-hibridisasie gedemonstreer maar terselfdetyd ook die beperkinge daarvan uitgewys, veral as meer tradisionele biochemiese tegnieke nie gebruik word om die resultate aan te vul nie. Alhoewel daar onsekerheid is oor die presiese funksies van die invertase-isovorme in suikerriet, het die studie die weg gebaan vir weefselspesifieke promotorontwerp en toekomstige in situ-studies van suikerrietinvertases.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2006. / Fructose 2,6-bisphosphate (Fru 2,6-P2) is an important regulatory molecule in plant carbohydrate metabolism. There were three main objectives in this study. Firstly, to determine whether the recombinant rat 6-phosphofructo 2-kinase (6PF2K, EC 2.7.1.105) and fructose 2,6-bisphosphatase (FBPase2, EC 3.1.3.11) enzymes, which catalyse the synthesis and degradation of Fru 2,6-P2 respectively, showed any catalytic activity as fusion proteins. Secondly, to alter the levels of Fru 2,6-P2 in sugarcane, an important agricultural crop due to its ability to store large quantities of sucrose, by expressing the recombinant genes. Thirdly, to investigate whether sugar metabolism in photosynthetic- (leaves) and non-photosynthetic tissue (internodes) were subsequently influenced. Activity tests performed on the bacterially expressed glutathione-S-transferase (GST) fusion 6PF2K and FBPase2 enzymes showed that they were catalytically active. In addition antibodies were raised against the bacterially expressed proteins. Methods for extracting and measuring Fru 2,6-P2 from sugarcane tissues had to be optimised because it is known that the extraction efficiencies of Fru 2,6-P2 could vary significantly between different plant species and also within tissues from the same species. A chloroform/methanol extraction method was established that provided Fru 2,6-P2 recoveries of 93% and 85% from sugarcane leaves and internodes respectively. Diurnal changes in the levels of Fru 2,6-P2, sucrose and starch were measured and the results suggested a role for Fru 2,6-P2 in photosynthetic sucrose metabolism and in the partitioning of carbon between sucrose and starch in sugarcane leaves. Transgenic sugarcane plants expressing either a recombinant rat FBPase2 (ODe lines) or 6PF2K (OCe lines) were generated. The ODe lines contained decreased leaf Fru 2,6-P2 levels but increased internodal Fru 2,6-P2 levels compared to the control plants. Higher leaf sucrose and reducing sugars (glucose and fructose) were measured in the transgenic plants than the control plants. The transgenic lines contained decreased internodal sucrose and increased reducing sugars compared to the control plants. Opposite trends were observed for Fru 2,6-P2 and sucrose when leaves, internodes 3+4 or internodes 7+8 of the different plant lines were compared. In contrast, no consistent trends between Fru 2,6-P2 and sucrose were evident in the OCe transgenic lines.

Thesis (MSc.)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The detrimental effect of soil salinity on crop production is a growmg problem worldwide (Tanji, 1990b). The degree to which plants can tolerate high concentrations of salt in their rooting medium is under genetic control with different genetic and physiological mechanisms contributing to salt tolerance at different developmental stages (Epstein & Rains, 1987). Only limited variation exists for salt tolerance in the cultivated cereals. This has prompted attempts to select tolerant progeny following hybridisation of cultivated species and wild, salt-tolerant species. Thinopyrum distichum, an indigenous wheatgrass that is naturally adapted to saline environments (McGuire & Dvorak, 1981), was crossed with triticale (x Triticosecale) in an attempt to transfer its salt tolerance and other hardiness characteristics (Marais & Marais, 1998). The aims of this study were to (i) identify Thinopyrum chromosomes carrying genes for salt tolerance and to identify molecular markers for these chromosomes, (ii) identify a number of diverse monosomic and disomie addition plants. Bulked segregant analysis (BSA), in combination with AFLP, RAPD and DAF marker analysis was implemented to screen for polymorphisms associated with salt tolerance. Five putative AFLP markers and two RAPD markers were detected using bulks composed of salt tolerant plants and bulks composed of salt sensitive plants. The distribution of the markers in these bulks suggests that more than one Thinopyrum chromosome carry genes for salt tolerance. Salt tolerant monosomic and disomie addition plants were characterised for AFLP, RAPD and DAF polymorphisms in an attempt to find markers associated with the chromosome(s) conditioning salt tolerance. One salt tolerant monosomic and one disomie addition plant was identified. One AFLP and two RAPD markers were identified for the Thinopyrum chromosome( s) present in the monosomic addition plant, while three AFLP and three RAPD markers were identified for the disomie addition plant. An attempt was also made to identify diverse chromosome addition plants having complete or near complete triticale genomes plus an additional random Thinopyrum chromosome. Plants with 2n = 43 /44 were identified and characterised for molecular markers (AFLP and RAPD). Cluster analysis was used to group the putative monosomic or disomie addition plants according to the specific Thinopyrum chromosomes they retained. Seventeen AFLP and RAPD markers could be used to group the 24 putative addition plants into six broadly similar groups with different additional Thinopyrum chromosomes. While the members of each group are likely to carry the same additional Thinopyrum chromosomes, this may not necessarily be the case as the interpretation of the marker results is complicated by heterogeneity among plants with regard to the triticale background chromosomes they possess. It is also likely that chromosome translocations occurred during backerossing which may further complicate data. Nonetheless, it is now possible to select disomie addition plants from each group that are likely to represent different Thinopyrum chromosomes. The data will also be useful in future attempts to find further addition plants carrying the remaining Thinopyrum chromosomes. / AFRIKAANSE OPSOMMING: Die skadelike effek van grond versouting op gewasproduksie neem wêreldwyd toe (Tanji, 1990b). Die mate waartoe plante hoë konsentrasies sout in die wortelstelsel kan hanteer is onder genetiese beheer en verskillende genetiese en fisiologiese meganismes dra by tot die soutverdraagsaamheid tydens verskillende ontwikkelingstadia (Epstein & Rains, 1987). Slegs beperkte variasie bestaan vir soutverdraagsaamheid in verboude grane. Dit het aanleiding gegee tot pogings om soutverdraagsame nageslag te selekteer na hibridisasie van verboude spesies en wilde, soutverdraagsame spesies. Thinopyrum distichum, 'n inheemse koringgras, wat aangepas is by brak omgewings (McGuire & Dvorak, 1981), is met korog (x Triticosecale) gekruis in 'n poging om die gene vir soutverdraagsaamheid en ander gehardheidseienskappe oor te dra (Marais & Marais, 1998). Die oogmerke van hierdie studie was om (i) Thinopyrum chromosome te identifiseer wat gene bevat vir soutverdraagsaamheid en molekulêre merkers te vind vir hierdie chromosome, (ii) 'n aantal diverse monosomiese en disomiese addisieplante te identifiseer. Bulksegregaatanalise (BSA), gekombineer met AFLP-, RAPD- en DAF-merkeranalise, is gebruik om polimorfismes geassosieerd met soutverdraagsaamheid op te spoor. Vyf moontlike AFLPmerkers en twee RAPD-merkers is geïdentifiseer met gebruik van bulks bestaande uit soutverdraagsame plante en bulks bestaande uit soutgevoelige plante. Die verspreiding van die merkers in soutverdraagsame bulks dui daarop dat meer as een Thinopyrum chromosoom bydra tot soutverdraagsaamheid. Soutverdraagsame, monosomiese en disomiese addisieplante is gekarakteriseer vir AFLP- en RAPD-polimorfismes in 'n verdere poging om merkers te vind vir chromosome betrokke by soutverdraagsaamheid. Een soutverdraagsame monosomiese en een disomiese addisieplant is geïdentifiseer. Een AFLP- en twee RAPD-merkers is geïdentifiseer vir die Thinopyrum chromosoom(e) teenwoordig in die monosomiese addisieplant, terwyl drie AFLP- en drie RAPDmerkers geïdentifiseer is vir die disomiese addisieplant. 'n Poging is ook gemaak om diverse addisieplante te identifiseer met 'n volledige koroggenoom plus 'n addisionele Thinopyrum chromosoom. Plante met 2n = 43 / 44 is geïdentifiseer en gekarakteriseer met molekulêre merkers (AFLP en RAPD). Tros-analise is gebruik om die vermoedelik monosomiese of disomiese addisieplante te groepeer volgens die spesifieke Thinopyrum chromosome wat hulle behou het. Sewentien AFLP- en RAPD-merkers is gebruik om die 24 vermoedelike addisieplante in 6 groepe met verskillende Thinopyrum chromosome te groepeer. Alhoewel dit voorkom of die verskillende plante in 'n groep dieselfde addisionele Thinopyrum chromosoom het, is dit nie noodwendig die geval nie aangesien die interpretasie van die merkers bemoeilik word deur die heterogeniteit tussen die plante wat betref die agtergrond korogchromosome wat hulle besit. Dit is ook moontlik dat chromosoom herrangskikkings plaasgevind het gedurende die terugkruisings, wat die data verder kan bemoeilik. Nietemin, dit is nou moontlik om disomiese addisies te selekteer uit elke groep wat moontlik verskillende Thinopyrum chromosome bevat. Die data kan ook gebruik word om in die toekoms verdere addisieplante te identifiseer wat die oorblywende Thinopyrum chromosome bevat.

Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Much has been written about the effects of soil salinity on plant growth. Its devastating effects have already been reported 2000 years BC. In the 21· century an alarming 80 million hectares of cultivated land area are affected by salt (Munns, 2002a) and represent a growing threat to agriculture. Salt tolerance is a complex trait moderately expressed in only a few plant genotypes (Ruiz, 2001). An attempt to transfer salt tolerance genes from the wild grass, Thinopyrum distichum, to triticale and éommon wheat was initiated by Marais and Marais (2003). A study of Th. distichum x rye hybrids enabled the authors to identify chromosomes 2Jld , 3Jld , 4Jld and SJld as being involved in the determination of salt tolerance. Indirect (yet unconfirmed) evidence suggested that 7Jld might also have a role. A programme aiming to transfer regions of the critical chromosomes to homoeologous triticale chromosomes, which relies heavily on the use of molecular markers, was launched. While an RFLP marker is available for each of the Thinopyrum chromosomes, these are not suited for the screening of large numbers of segregates. This study therefore represents an attempt to convert the RFLP markers into less time consuming and cost-effective SCAR markers. The published DNA sequences of the RFLP probes in question were used as templates to design PCR primers. The PCR reactions were optimised using DNA of Th. distichum, rye and their FI hybrid. When Thinopyrum specific amplification products were obtained, the primers were also tested on a panel of genotypes with and without the target chromosomes. Seemingly polymorphic bands were confirmed by Southern blotting and hybridisation with the corresponding RFLP probes. The primers were also tested on a panel of genotypes that included 'Rex' triticale to ensure that they would also detect a difference in a triticale genetic background during transfer. Polymorphic bands were then isolated and sequenced to further refine the markers. In certain eases, sequences of the same fragment amplified in triticale ('Rex') and Thinopyrum were aligned in an attempt to design more specific markers. Using this approach, it was possible to develop chromosome specific SCARs for Thinopyrum chromosomes 3Jld and 7J2 d . Three and one set(s) of PCR markers, respectively, have been developed and can be used to unequivocally detect the Thinopyrum chromosomes involved in salt tolerance against a triticale background. A SCAR marker was also found for chromosome 6J. Thus, an attempt was made to convert thirteen RFLP probes to SCAR markers. Only three were successfully converted. The main reason for the low success rate is the syntenic relationships between the genomes of the different cereals that made it an arduous- task to find discriminating primer sets. Based on the results obtained, an adapted procedure is suggested for future attempts to develop chromosome specific markers utilizing published sequence information that was obtained for a different species. / AFRIKAANSE OPSOMMING: Baie is al geskryf oor die uitwerking van grond versouting op plantproduksie. Die vernietigende gevolge van versouting is alreeds 2000 jaar VC gerapporteer. In die 21* eeu is 'n geraamde 80 miljoen hektaar (Munns, 2002a) bewerkte land-area sout-geaffekteerd. Die ontstellende verwikkelinge verteenwoordig 'n groeiende bedreiging vir die landbou. Soutverdraagsaamheid is 'n komplekse kenmerk en slegs enkele plantgenotipes met matige verdraagsaamheid kon nog ontwikkel word (Ruiz, 2001). 'n Poging om soutverdraagsaamheidsgene vanaf die wilde gras, Thinopyrum distichum, na triticale en gewone koring oor te dra, is deur Marais en Marais (2003) geïnisieer. 'n Studie van Th. distichum x rog hibriede het die skrywers in staat gestelom chromosome (2Jld, 3Jld, 4Jld en SJld) wat bydra to soutverdraagsaamheid te identifiseer. Indirekte (maar onbevestigde) aanduidings is gevind dat 7J1dook' n rol mag speel. 'n Program is daarna geloods om segmente van chromosome na homoeoloë triticale chromosome oor te dra, 'n onderneming wat swaar steun op die gebruik van molekulêre merkers. Alhoewel daar'n RFLP merker beskikbaar is vir elk van die Thinopyrum chromosome, is hierdie merkers nie geskik vir die sifting van groot getalle segregate nie. Hierdie studie verteenwoordig 'n poging om die RFLP merkers om te skakel na 'n minder tydrowende en meer koste-effektiewe SCAR merkers. Die gepubliseerde DNS-volgordes van die betrokke RFLP peilers is as templaat gebruik om PKR inleiers te ontwerp. Die PKR reaksies is geoptimiseer deur gebruik te maak van DNS van Th. distichum. rog en hulle FI hibried. In gevalle waar Thinopyrum spesifieke amplifikasie produkte verkry is, is die inleiers ook getoets op 'n paneel van genotipes met en sonder die teikenchromosoom. Skynbare polimorfiese bande is bevestig deur 'n 'Southern' klad te maak en te hibridiseer met die tersaaklike RFLP peiler. Die inleiers is ook getoets op 'n paneel van genotipes waarby 'Rex' triticale ingesluit was om te verseker dat dit ook verskille in 'n triticale genetiese agtergrond opspoor (nodig tydens oordrag). Polimorfiese bande is verder verfyn. Dit is geïsoleer en die DNS-volgorde daarvan is bepaal. Tn sekere gevalle is ooreenstemmende fragmente geamplifiseer in triticale ('Rex') en Thinopyrum. Die volgordes is dan bepaal en met mekaar vergelyk in 'n poging om meer spesifieke merkers te ontwerp. Met die gebruik van hierdie benadering was dit moontlik om chromosoom-spesifieke SCAR-merkers vir die Thinopyrum chromosome 3Jld en 7J2d te ontwikkel. Drie en een stel(le) PKR merkers is onderskeidelik ontwikkel en kan gebruik word om ondubbelsinnig te bepaal of die betrokke Thinopyrum chromosoom segregeer in 'n triticale kruising. 'n SCAR merker is ook gevind vir chromosoom 6J. Dus, daar is probeer om dertien RFLP peilers na SCAR merkers om te skakel. Slegs drie van die pogings was suksesvol. Die hoofrede vir die lae sukseskoers is die hoë graad van sintenie tussen die genome van die verskillende grane wat dit 'n moeilike taak gemaak het om diskriminerende inleierstelle te ontwerp. Op grond van die resultate word 'n ietwat gewysigde prosedure vir die toekomstige pogings om chromosoom-spesifieke merkers te ontwerp met gebruik van gepubliseerde volgorde inligting vanaf' n ander spesie, voorgestel.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The deterioration of harvested sugarcane as a result of bacterial growth causes major losses of sucrose and a build-up of exopolysaccharides (EPS). Polysaccharides present during production increase the massecuite viscosity, which negatively influences evaporation and crystallisation. In this study 38 culturable EPSproducing bacteria were isolated from milled sugarcane. Analysis of the EPS showed the ubiquitous presence of glucose, however, 14 polysaccharides also contained mannose, fructose or galactose. In vitro treatment using Chaetomium erraticum dextranase to evaluate is effectiveness indicated that 37 of the EPS were hydrolysed to some extent. There were 21 polysaccharides that were only partially digested. The capacity of the isolates to produce EPS on different sugars indicated a correlation between sucrose and polysaccharide formation in 37 isolates. The results indicate there are more species involved in EPS production than previously thought as well as the presence of non-dextran polysaccharides. / AFRIKAANSE OPSOMMING: Bakteriële groei veroorsaak ‘n afname in gehalte, sukrose en ‘n verhoging in die hoeveelheid van eksternepolisakkeriede (EPS). Die verhoogde konsentrasie van polysakkariede gedurende die verwerkingsprosses veroorsaak ‘n verhoging in “massecuite” viskositeit. Hierdie verskynsel het ‘n nadelige uitwerking op die verdamping en kristalvorming van die produk. In gemaalde skuikerriet was 38 groeibare EPS-produserende bakterieë geisoleer. Die geanaliseerde EPS van hierdie bogenoemde bakterieë was daar in almal glukose teenwoordig. In 14 van hulle was mannose, fruktose en galaktose ook gevind. Die in vitro effektiwieteit van Chaetomium erraticum dekstranase op die EPS het gewys dat 37 het tot ‘n mate gehidroliseer maar 21 was net gedeeltelik verteer. As gevolg van die bo-genoemde resultate was daar gevind dat sukrose was ‘n noodsaaklike subtraat vir EPS produksie in die geisoleerde bakterieë. In hierdie studie was bevestig ‘n groter verskiedenheid EPS-produserende bakterieë gevind was en dat hulle assosiasie aan sukierriet prossering meer kompleks is as wat vooreen gedink was.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Metagenomic studies that make use of next-generation sequencing (NGS) generate large amounts of sequence data, representing the genomes of multiple organisms of which no prior knowledge is necessarily available. In this study, a metagenomic NGS approach was used to detect multiple novel mycoviral sequences in grapevine phloem tissue. Individual sequencing libraries of doublestranded RNA (dsRNA) from two grapevine leafroll diseased (GLD) and three shiraz diseased (SD) vines were sequenced using an Illumina HiScanSQ instrument. Over 3.2 million reads were generated from each of the samples and these reads were trimmed and filtered for quality before being de novo assembled into longer contigs. The assembled contigs were subjected to BLAST (Basic Local Alignment Search Tool) analyses against the NCBI (National Centre for Biotechnology Information) database and classified according to database sequences with which they had the highest identity. Twenty-six putative mycovirus species were identified, belonging to the families Chrysoviridae, Endornaviridae, Narnaviridae, Partitiviridae and Totiviridae. Two of the identified mycoviruses, namely grapevine-associated chrysovirus (GaCV) and grapevine-associated mycovirus 1 (GaMV-1) have previously been identified in grapevine while the rest appeared to be novel mycoviruses not present in the NCBI database. Primers were designed from the de novo assembled mycoviral sequences and used to screen the grapevine dsRNA used for sequencing as well as endophytic fungi isolated from the five sample vines. Only two mycoviruses, related to sclerotinia sclerotiorum partitivirus S and chalara elegans endornavirus 1 (CeEV-1), could be detected in grapevine dsRNA and in fungus isolates. In order to validate the presence of mycoviruses in grapevine phloem tissue, two additional sequencing runs, using an Illumina HiScanSQ and an Applied Biosystems (ABI) SOLiD 5500xl instrument respectively, were performed. These runs generated more and higher quality sequence data than the first sequencing run. Twenty-two of the putative mycoviral sequences initially detected were detected in the subsequent sequence datasets, as well as an additional 29 species not identified in the first HiScanSQ sequence datasets. The samples harboured diverse mycovirus populations, with as many as 19 putative species identified in a single vine. This indicates that the complete virome of diseased grapevines will include a high number of mycoviruses. Additionally, the complete genome of a novel endornavirus, for which we propose the name grapevine endophyte endornavirus (GEEV), was assembled from one of the second HiScanSQ sequence datasets. This is the first complete genome of a mycovirus detected in grapevine. Grapevine endophyte endornavirus has the highest sequence similarity to CeEV-1 and is the same virus that was previously detected in fungus isolates using the mycovirus primers. The virus was detected in two fungus isolates, namely Stemphylium sp. and Aureobasidium pullulans, which is of interest since mycoviruses are not known to be naturally associated with two distinctly different fungus genera. Mycoviral sequence data generated in this study can be used to further investigate the diversity and the effect of mycoviruses in grapevine. / AFRIKAANSE OPSOMMING: Metagenomiese studies, wat gebruik maak van volgende-generasie volgordebepalingstegnologie, het die vermoë om die genetiese samestelling van veelvoudige onbekende organismes te bepaal deurdat dit groot hoeveelhede data genereer. Die bogenoemde tegniek was in hierdie studie aangewend om aantal nuwe mikovirusse in die floëem weefsel van wingerd te identifiseer. Dubbelstring-RNS was gesuiwer vanuit twee druiwestokke met rolbladsiekte en drie met shirazsiekte en Illumina HiScanSQ instrument is gebruik om meer as 3.2 miljoen volgorde fragmente te genereer van elk van die monsters. Lae-kwaliteit volgordes was verwyder en die oorblywende kort volgorde fragmente was saamgestel om langer konstrukte te vorm wat met behulp van BLAST soektogte teen die NCBI databasis geïdentifiseer kon word. Ses-en-twintig mikovirus spesies, wat aan die families Chrysoviridae, Endornaviridae, Narnaviridae, Partitiviridae en Totiviridae behoort, was geïdentifiseer. Twee van die geïdentifiseerde mikovirusse, naamlik grapevine-associated chrysovirus (GaCV) en grapevine-associated mycovirus 1 (GaMV-1), was voorheen al in wingerd gekry terwyl die res nuwe mikovirusse is wat tans nie in die NCBI databasis voorkom nie. Inleiers was ontwerp vanaf die saamgestelde mikovirus basisvolgordes en gebruik om wingerd dubbelstring-RNS sowel as swamme wat vanuit die wingerd geïsoleer is te toets vir die teenwoordigheid van hierdie mikovirusse. Slegs twee mikovirusse, wat onderskeidelik verwant is aan sclerotinia sclerotiorum partitivirus S en chalara elegans endornavirus 1 (CeEV-1), kon deur middel van die inleiers in wingerd en swam isolate geïdentifiseer word. Twee addisionele volgordebepalingsreaksies, wat gebruik gemaak het van die Illumina HiScanSQ en ABI SOLiD 5500xl volgordebepalingsplatforms, was gebruik om die teenwoordigheid van mikovirusse in wingerd te bevestig. Groter hoeveelheid volgorde fragmente was geprodusser wat ook van hoër gehalte was as dié van die eerste volgordebepalingsreaksie. Twee-en-twintig mikovirus spesies kon weer geïdentifiseer word, sowel as 29 spesies wat nie in die eerste HiScanSQ basisvolgorde datastelle gevind was nie. Die wingerdstokke wat in hierdie studie ondersoek was, het hoë diversiteit van mikovirusse bevat aangesien daar tot 19 mikovirus spesies in enkele wingerdstok geïdentifiseer was. Dit is aanduiding dat volledige virus profiele van siek wingerdstokke aantal mikovirusse sal insluit. Die vollengte genoomvolgorde van voorheen onbekende endornavirus was saamgestel vanuit een van die tweede HiScanSQ volgorde datastelle. Dit is die eerste mikovirus wat in wingerd gevind word waarvan die volledige genoomvolgorde bepaal is en ons stel die naam grapevine endophyte endornavirus (GEEV) voor vir hierdie virus. Grapevine endophyte endornavirus is die naaste verwant aan CeEV-1 en is dieselfde virus wat voorheen in wingerd dubbelstring-RNS en swam isolate gevind was deur middel van die mikovirus inleiers. Swam isolate waarin GEEV gevind is, was geïdentifiseer as Stemphylium sp. en Aureobasidium pullulans. Dit is van belang dat GEEV in twee swam isolate gevind is wat aan verskillende genusse behoort aangesien hierdie verskynsel nog nie voorheen in die natuur gevind is nie. Mikovirus nukleiensuurvolgordes wat in hierdie studie bepaal was kan gebruik word in toekomstige studies om die verskeidenheid en impak van mikovirusse in wingerd verder te ondersoek. / National Research Foundation (NRF) / Stellenbosch University

Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Flocculation can be defined as the asexual aggregation of yeast cells in a liquid environment. This aggregation of cells, also referred to as "floc formation", will in most cases lead to rapid settling or sedimentation. However, in so-called top-fermenting yeast strains, the floes can move to the surface of the liquid growth substrate to form a thin layer, called a "velum", that has been compared to other microbial biofilms. The factors that trigger flocculation can be divided into two groups, physical/chemical (e.g. sugar content, the presence of inorganic salts, organic solvents, ethanol concentration, pH, agitation etc.) and genetic factors (genes that encode for proteins that are either directly or indirectly involved in flocculation). In top-fermenting yeast strains, several physical and chemical factors that trigger the process have been described, including ethanol concentration, the presence of organic solvents, the absence of molecular oxygen and the presence of inorganic salts (Ca2+ and Mg2+). These factors appear to affect the cell hydrophobicity and the cell surface charge. As for genetic factors, no specific genes have thus far been associated with flocculation in top fermenting yeast strains. In bottom-fermenting yeast strains, the physical and chemical factors that affect the process are similar to the ones described for top-fermenting yeast strains, but include, more specifically, the concentration of hexoses in the media (mannose or glucose), which may inhibit the process. Indeed, flocculation in bottom-fermenting yeast strains has been divided into the NewFlo type (inhibited by both mannose and glucose) and the Fl01 type (inhibited by mannose) on the basis of the inhibitory effect of specific sugars. Various genes have been associated with the flocculation of bottom-fermenting yeast strains. Through genetic analysis, the genes have been categorised into dominant genes, semidominant genes and recessive genes. In order to better understand the role of some of the proteins responsible for flocculation in S. cerevisiae, and to create strains whose flocculation properties would correspond to those wanted in the wine and beer industries, three of the dominant flocculation genes, FL01, FL05 and FL011, were placed under the control of the promoters of the stationary phase-induced genes, ADH2 and HSP30. This was achieved by replacing the native promoters of the flocculation genes with the heterologous promoters through homologous recombination. The laboratory strain FY23, which is nonflocculent due to the absence of the transcription factor that is required for flocculation, F108p,was used as a model system. Some of the transformed strains showed high flocculation, especially when the genes were placed under control of the ADH2 promoter. In addition to this, the strains carrying a modified FL011 gene showed increased adhesion to solid agar media and were able to invade the growth substrate. These strains also showed an increased velum-forming ability when grown in media containing only non-fermentable carbon sources. / AFRIKAANSE OPSOMMING: Flokkulasie kan gedefinieër word as die ongeslagtelike aggregasie van gisselle in 'n vloeibare medium. Hierdie aggregasie van selle, kan ook na verwys word as flok formasie, en in meeste gevalle lei dit tot In vinnige sedimentering. In oppervlak-fermenterende giste, beweeg die flokke na die oppervlakte van die vloeibare medium om sodoende 'n flor -lagie te vorm. Hierdie verskynsel was ook al gevind in ander organismes. Verskeie faktore is verantwoordelik vir die effektiwiteit van flokkuklasie. Hierdie faktore kan in twee groepe verdeel word, nl. fisiese en chemiese faktore (byv. suikerkonsentrasie, die teenwoordigheid van anorganiese soute, organiese oplossings, etanol konsentrasie, pH, ens.) en genetiese faktore (gene wat kodeer vir die proteïene wat of direk of indirek betrokke is by flokkulasie). In oppervlak-fermenterende giste is daar al heelwat informasie beskikbaar omtrent fisies en chemiese faktore se effekte op flokkulasie. Van die faktore waarvan heelwat informasie beskikbaar is sluit in, etanol konsentrasie, die teenwoordigheid van organiese oplossings, die afwesigheid van molekulêre suurstof en die teenwoordigheid van anorganiese soute (Ca2+ en Mg2+). Hierdie faktore toon 'n effek of hidrofobisiteit en elektriese lading op die seloppervlakte. Geen genetiese faktore kon tot dusver gekoppel word aan flokkulasie in oppervlak-fermenterende giste nie. Benede-oppervlak fermenterende giste se fisies en chemiese faktore wat effektiwiteit van flokkulasie beïnvloed is dieselfde as die van oppervlak-fermenterende giste, maar sluit in meer spesifiek, die konsentrasie van heksoses in die media (nl. mannose en glukose), wat 'n inhiberende effek het op flokkulasie. Die benede-oppervlak fermenterende giste se flokkulasie kan in twee segmente verdeel word nl. die NewFlo tipe (word geïnhibeer deur die teenwoordigheid van mannose en glukose) en die Flo1-tipe (word geïnhibeer deur slegs die teenwoordigheid van mannose). Verskeie gene was ook al geidentifiseer wat die effektiwiteit van flokkulasie beïnvloed in benede-oppervlak fermenterende giste. Hierdie gene kan in drie kategorieë opverdeel word, nl dominante-, semi-dominante- en ressessiewe flokkulerende gene. Ten orde 'n beter begrip te kry rondom die proteïene verantwoordelik vir die meeste effektiwiteit ten opsigte van flokkulasie in S. cerevisiae, asook om giste te manipuleer om spesifieke flokkulasie eienskappe te toon volgens die belange van die wyn en bierindustrieë, was drie dominante flokkulerende gene, nl. FL01, FL05, en FL011, onder regulering van stationêre fase-geïnduseerde promotors, PADH2 en PHSP30, geplaas. Dit was verkry deur die vervanging van die wilde tipe promotors van die drie gene met die stationêre fase-geïnduseerde promotors deur middel van homoloë rekombinasie. Die laboratorium gisras, FY23, wat 'n nie-flokkulerende gisras is vanweë die afwesigheid van 'n transkripsionele faktor, Flo8p, wat verantwoordelik is vir die aktivering van belangrike gene in flokkulasie, was gebruik as 'n wilde tipe ras. Sommige van die transformante het In hoë mate van flokkulasie getoon, veral wanneer onder die regulering van die PADH2. Tesame met laasgenoemde verskynsel, was daar gevind dat FL011-transformante 'n verhoging in hul vermoeë het om te bind aan die agar en ook om die agar te penetreer. Laasgenoemde gisrasse het ook die vermoë getoon om 'n flor-lagie te vorm bo-op die oppervlakte van die medium, maar slegs wanneer dit in niefermenteerbare koolstofbronbevattende media opgegroei word.

Thesis (MPhil)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: The aim and function of this paper is to provide a balanced account of how the media, international and South African, have dealt with the issue of genetically modified organisms (GMOs). A selection of interviews, presentations, articles, transcripts and published reports forms the background of this interpretation, and offers insight into the history of the technology, the major role players, the legislation required and implemented, the question of environmental accountability, and the power of the media's influence. It addresses aspects of the causal relationship between the media and public understanding, and the subsequent power of the consumer as manifested by the perception of risk. The central theme of genetic engineering conjures up a variety of meanings and applications, and the plethora of available information is evaluated in an attempt to develop informed understanding for reporters covering the many dimensions of this development within the arena of science and technology. / AFRIKAANSE OPSOMMING: Die doel van hierdie verhandeling is om 'n ewewigtige oorsig te verstrek van hoe die media - Suid-Afrikaans sowel as internasionaal - die kwessie van geneties gemodifiseerde organismes gehanteer het. 'n Seleksie onderhoude, aanbiedinge, artikels, transkripsies, en gepubliseerde verslae vorm die basis van hierdie interpretasie, en verskaf 'n insig in die geskiedenis van die tegnologie, die belangrike rolspelers, nodige en géimplementeerde wetgewing, die vraag van omgewingstoerekenbaarheid, en die mag van die media se invloed. Dit spreek aspekte aan van die kousale verwantskap tussen die media en begrip deur die algemene publiek, en die daaropvolgende mag van die verbruiker, soos dit duidelik word in hulle insig in en begrip van die risiko-faktor. Die sentrale tema van genetiese modifisering bring te voorskyn 'n verskeidenheid betekenisse en aanwendings; en 'n oorsig van die massa beskikbare inligting word hier aangebied in 'n poging om aan verslaggewers ingeligte begrip aan te bied van die veelsydige omvang van die ontwikkeling van genetiese modifisering in die gebied van wetenskap en tegnologie.