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)--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 (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2006. / The young internodes of sugarcane are ideal targets for altering metabolism, through genetic manipulation, to potentially control known fungal diseases such as Smut or to increase sucrose yields in these regions that are currently being discarded. At present, no regulatory sequences that specifically drive transgene expression in young developing sugarcane tissues are available. The objective of this study was therefore to isolate and evaluate such a sequence. The promoter targeted for isolation in this study regulates the expression of UDP-glucose dehydrogenase (EC 1.1.1.22), an enzyme which catalyses the oxidation of UDP-glucose to UDP-glucuronic acid, a precursor for structural polysaccharides which are incorporated into the developing cell wall. A strong correlation between the expression of UDP-glucose dehydrogenase and a demand for structural polysaccharides in developing tissues could therefore be expected. The first part of this study addressed the general practicality of promoter isolation from sugarcane, a complex polyploid. A gene encoding UDP-glucose dehydrogenase was isolated from a sugarcane genomic library. The gene contains an open reading frame (ORF) of 1443 bp, encoding 480 amino acids and one large intron (973 bp), located in the 5’-UTR. The derived amino acid sequence showed 88 – 98% identity with UDP-glucose dehydrogenase from other plant species, and contained highly conserved amino acid motifs required for cofactor binding and catalytic activity. Southern blot analysis indicates a low copy number for UDP-glucose dehydrogenase in sugarcane. The possible expression of multiple gene copies or alleles of this gene was investigated through comparison of sequences amplified from cDNA prepared from different tissues. Although five Single Nucleotide Polymorphisms (SNP) and one small-scale insertion/deletion (INDEL) were identified in the aligned sequences, hundred percent identity of the derived amino acid sequences suggested the expression of different alleles of the same gene rather than expression of multiple copies. The finding that multiple alleles are expressed to provide the required level of a specific enzyme, rather than the increased expression of one dominant allele, is encouraging for sugarcane gene and promoter isolation. In the second part of the study the suitability of UDP-glucose dehydrogenase as a target for the isolation of a developmentally regulated promoter was investigated. The contribution of UDP glucose dehydrogenase to pentan synthesis, as well as the expression pattern and subcellular localisation of the enzyme in mature sugarcane plants was studied at the tissue and cellular level. Radiolabelling with positionally labelled glucose was used to investigate the relative contributions of glycolysis, the oxidative pentose phosphate pathway and pentan synthesis to glucose catabolism. Significantly (P=0.05) more radiolabel was released as CO2 from [6-14C]- glucose than [1-14C]-glucose in younger internodes 3, 4 and 5, demonstrating a significant contribution of UDP-glucose dehydrogenase to glucose oxidation in the younger internodes. In addition, there was significantly (P=0.05) more radiolabel in the cell wall (fiber) component when the tissue was labelled with [1-14C]-glucose rather than [6-14C]-glucose. This also demonstrates a selective decarboxylation of glucose in position 6 prior to incorporation into the cell wall and is consistent with a major role for UDP-glucose dehydrogenase in cell wall synthesis in the younger internodes. Expression analysis showed high levels of expression of both the UDP-glucose dehydrogenase transcript and protein in the leafroll, roots and young internodes. In situ hybridisation showed that the UDP-glucose dehydrogenase transcript is present in virtually all cell types in the sugarcane internode, while immunolocalisation showed that the abundance of the protein declined in all cell types as maturity increased. Results obtained confirmed that this enzyme plays an important role in the provision of hemicellulose precursors in most developing tissues of the sugarcane plant, indicating that UDP-glucose dehydrogenase was indeed a suitable target for promoter isolation. Lastly, the promoter region and first intron, located in the 5’-untranslated region (UTR) of this gene, were isolated and subsequently fused to the GUS reporter gene for transient expression analysis and plant transformation. Transient expression analysis showed that the presence of the intron was essential for strong GUS expression. Analysis of stably transformed transgenic sugarcane plants, evaluated in a green house trial, showed that the isolated promoter is able to drive GUS expression in a tissue specific manner under these conditions.

Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Please refer to fulltext for abstract / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Sugarcane is a crop that is farmed commercially due to the high amounts of sucrose that is stored within the mature internodes of the stem. Numerous studies have been done to understand sugar metabolism in this crop as well as to enhance sucrose yields. Until now sugarcane improvement strategies have been implemented through either breeding programs or transgenic manipulation. Public mistrust and regulatory hurdles, however, have made the commercialisation of transgenic crops difficult, expensive and timeconsuming. In this thesis two projects will address issues relating to the above. The first will address an effort to increase sucrose accumulation within the sugarcane culm. This was attempted via the expression of an Arabidopsis thaliana vacuolar pyrophosphatase (AtV-PPase) gene, linked to the maize ubiquitin promoter, in sugarcane callus. It was anticipated that increased activity of the tonoplast-bound AtV-PPase will result in increased sucrose accumulation in the vacuole. Transgenic sugarcane callus lines were tested for soluble sugar content which suggested no significant increase in sucrose content. However, this may change upon further assessment of sugarcane suspension cultures and glasshouse plants. The second project was concerned with the development of a novel sugarcane transformation technology that utilises only sugarcane sequences. This ‘cisgenic’ approach to sugarcane transformation will require a native sugarcane promoter, terminator, vector backbone and selection marker. It was attempted to first isolate a functional promoter as well as developing a selection system based on an endogenous selection marker. A promoter was amplified from sugarcane, using primers designed on a sorghum template, and its expression assessed using a GFP reporter gene. Unfortunately expression could not be confirmed in transgenic sugarcane callus. Currently, an alternative approach is followed by using short fragments of constitutively expressed genes to screen sugarcane Bacterial Artificial Chromosome (BAC) libraries to isolate their corresponding promoters. Lastly, it was attempted to develop a selection system for transgenic sugarcane based on resistance to the herbicide chlorosulfuron. A mutant acetolactate synthase (alsb) gene from tobacco, which has shown to confer resistance to the tobacco, was transformed into sugarcane callus. It was anticipated that this gene will confer chlorosulfuron resistance to transgenic sugarcane. If resistance is achieved, the corresponding sugarcane gene will be mutated via site-directed mutagenesis and checked if it also confers resistance to sugarcane. Results showed that although transgenic lines were generated, resistance development is still inconclusive. / AFRIKAANSE OPSOMMING: Suikerriet is ‘n kommersiële gewas wat verbou word as gevolg van die hoë hoeveelhede sukrose wat gestoor word in die volwasse tussenknope van die stam. Verskeie studies is al gedoen om suiker metabolisme in die gewas te ondersoek, sowel as om die sukrose opbrengs te verhoog. Huidige strategieë vir suikerriet verbetering word beywer deur middel van teel-programme of transgeniese manipulasie. Die kommersialiseëring van transgeniese gewasse word egter bemoeilik deur publieke wanpersepsies, sowel as regulatoriese uitdagings. Hierdie tesis beoog om boenoemde kwessies aan te spreek, deur middel van twee projekte. Die eerste projek poog om sukrose akkumulasie in sukerriet te verhoog. Dit was onderneem om die Arabidopsis thaliana vakuolere pirofosfatase (AtV-PPase) geen, wat verbind is met die mielie ubiquitien promoter, uit te druk in suikerriet kallus. Daar was verwag dat die verhoogde aktiwiteit van die tonoplast-gebonde AtV-PPase sal veroorsaak dat meer sukrose in die vakuool akkumuleer. Oplosbare suiker inhoud was getoets in transgeniese suikerriet kallus lyne, maar geen merkbare verhoging in sukrose inhoud was waargeneem nie. Hierdie mag egter verander met verdere ondersoeke in suikerriet suspensie-kulture en glashuis-plante. Die tweede projek het beywer om ‘n nuwe suikerriet transformasie tegnologie te ontwikkel, wat slegs van suikerriet genetiese materiaal gebruik maak. Hierdie ‘cisgeniese’ benadering tot suikerriet transformasie sal ‘n inheemse suikerriet promoter, terminator, vektor ruggraat en seleksie-merker, benodig. Dit was eers beoog om ‘n funksionele promoter te isoleer, sowel as om ‘n seleksie sisteem, gebasseer op ‘n inheemse seleksie merker, te ontwikkel. Deur gebruik te maak van primers wat op ‘n sorghum templaat gebasseer is, was ‘n promotor geisoleer vanuit suikerriet; die uitdrukking hiervan is bepaal deur gebruik te maak van ‘n GFP verklikker geen. Ongelukkig kon uitdrukking nie bevestig word in transgeniese suikerriet kallus nie. Tans word suikerriet Kunsmatige Bakterieële Chromosoom (KBC) biblioteke geskandeer, deur gebruik te maak van geen-fragmente van globaal-uitgedrukte gene, om ooreenstemmende suikerriet promoters te isoleer. Die tweede deel van die cisgeniese projek het beoog om ‘n seleksie sisteem vir transgeniese suikerriet te ontwikkel, wat gebasseer is op weerstand teen die plantdoder chlorosulfuron. Suikerriet kallus was getranformeer met ‘n mutante tabak geen – asektolaktaat sintase (alsb) – wat chlorosulfuron weerstand in tabak meebring. Daar was verwag dat die geen chlorosulfuron weerstand aan transgeniese suikerriet sou oordra. Indien weerstand ontwikkel, sal die ooreenstemende suikerriet geen deur gerigte mutagenese gemuteer word; dan sal dit kan bepaal word of weerstand ook oorgedra word aan suikerriet. Daar is bevind dat alhoewel transgeniese lyne gegenereer is, daar steeds nie ‘n konklusiewe bevestiging van weerstand ontwikkeling is nie.

The main aim of the work presented in this dissertation was to explore the possibility to genetically manipulate the sucrose storing crops, sugarcane and sweet sorghum, to convert their sucrose reserves into higher-value alternatives. For the purpose of this study we focussed on fructans as alternative sucrose-based high-value carbohydrates, since these fructose polymers are of significant commercial interest. To investigate the technical feasibility of transforming sugarcane and sweet sorghum to produce this novel carbohydrate, we proposed to transfer the fructosyltransferase genes from Cynara scolymus into these plants by means of particle bombardment. In order to apply this technology to sweet sorghum, an in vitro culture system suitable for transformation had to be established. For this purpose an extensive screening process with different combinations of variables were conducted. Though the relationships between these variables proved to be complex, it was concluded that immature zygotic embryos could be used to initiate a genotype-independent totipotent regeneration system with a 65% callus induction rate, provided that initiation takes place during summer. Stable transformation and regeneration of these calli were however not successful and will have to be optimised to allow future applications. By introducing fructosyltransferase genes into sugarcane, we succeeded in transforming sugarcane into a crop that produces a variety of fructans of the inulintype. Low molecular weight (LMW) inulins were found to accumulate in the mature internodes of 42% of the transgenic sugarcane plants expressing the sucrose:sucrose 1-fructosyltransferase (1-SST) gene, and in 77% of the plants that incorporated both 1-SST and fructan:fructan 1-fructosyltransferase (1-FFT), while only 8% of these plants accumulated high molecular weight (HMW) inulins. Our results demonstrated that sugarcane could be manipulated to synthesise and accumulate fructans without the induction of phenotypical irregularities. Inulins with a degree of polymerisation up to 60 were found in sugarcane storage tissue. In these HMW inulin-producing plants, up to 78% of the endogenous sucrose in the mature sugarcane culm was converted to inulin. This enabled inulin accumulation up to 165.3 mg g-1 fresh weight (FW), which is comparable to that found in native plants. These transgenic sugarcane plants, therefore exhibit great potential as a future industrial inulin source. Fructan production was detected in all the sugarcane plant tissue tested, predominantly as 1-kestose. In contrast with the fact that fructan accumulation in leaves did not affect the endogenous sucrose concentrations in these organs, the sucrose content of mature internodes that accumulated high levels of 1-kestose was severely reduced. However, increases in total sugar content, in some instances up to 63% higher than control plants, were observed. This phenomenon was investigated with the use of radio-labelled-isotopes. An increase in the allocation of incoming carbon towards sucrose storage, resulting in higher carbon partitioning into both 1- kestose and sucrose, were detected in the culms of transgenic compared to control lines. This modification therefore established an extra carbohydrate sink in the vacuoles that affected photosynthate partitioning and increased total soluble sugar content. The data suggests that sucrose sensing is the main regulatory mechanism responsible for adapting carbon flow in the cells to maintain sucrose concentration.