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.