Genetic engineering of sugarcane for increased sucrose and consumer acceptance

[摘要] ENGLISH ABSTRACT: Sugarcane is a crop that is farmed commercially due to the high amounts of sucrose thatis stored within the mature internodes of the stem. Numerous studies have been done tounderstand sugar metabolism in this crop as well as to enhance sucrose yields. Until nowsugarcane improvement strategies have been implemented through either breedingprograms 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 addressan effort to increase sucrose accumulation within the sugarcane culm. This was attemptedvia the expression of an Arabidopsis thaliana vacuolar pyrophosphatase (AtV-PPase)gene, linked to the maize ubiquitin promoter, in sugarcane callus. It was anticipated thatincreased activity of the tonoplast-bound AtV-PPase will result in increased sucroseaccumulation in the vacuole. Transgenic sugarcane callus lines were tested for solublesugar content which suggested no significant increase in sucrose content. However, thismay change upon further assessment of sugarcane suspension cultures and glasshouseplants.The second project was concerned with the development of a novel sugarcanetransformation 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 endogenousselection marker.A promoter was amplified from sugarcane, using primers designed on a sorghumtemplate, and its expression assessed using a GFP reporter gene. Unfortunatelyexpression could not be confirmed in transgenic sugarcane callus. Currently, an alternativeapproach is followed by using short fragments of constitutively expressed genes to screensugarcane Bacterial Artificial Chromosome (BAC) libraries to isolate their correspondingpromoters.Lastly, it was attempted to develop a selection system for transgenic sugarcane based onresistance to the herbicide chlorosulfuron. A mutant acetolactate synthase (alsb) genefrom tobacco, which has shown to confer resistance to the tobacco, was transformed intosugarcane callus. It was anticipated that this gene will confer chlorosulfuron resistance totransgenic sugarcane. If resistance is achieved, the corresponding sugarcane gene will bemutated via site-directed mutagenesis and checked if it also confers resistance tosugarcane. Results showed that although transgenic lines were generated, resistancedevelopment is still inconclusive.

[发布机构] Stellenbosch University