Browsing by Author "Masoabi, Malira"
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- ItemBiotechnological approaches for sugarcane enhancement – Drought tolerance and Sucrose accumulation(Stellenbosch : Stellenbosch University, 2016-12) Masoabi, Malira; Van der Vyver, Christell; Lloyd, James Richard; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.ENGLISH ABSTRACT: The application of biotechnological approaches can supplement traditional crop breeding strategies to combat the effects of adverse environmental conditions on plant production in order to bridge the gap between supply and demand worldwide. In Southern Africa adverse environmental conditions, such as drought, are a serious agronomic problem that threatens crop productivity, food security and eventually negatively impact the economy of the region. Urgent attention to overcome drought stress is therefore vital to eradicate or minimise its effects on crop productivity. Furthermore, the use of fossil fuels as the primary source of energy worldwide has led to demonstrable detrimental effects to the environment and its sustainability. Efforts have been made to seek other cost effective energy sources that counteract the disadvantages of fossil fuels towards the environment. In this regard, research has unravelled the potential of feedstock such as maize and sugarcane as fuel sources and tremendous advancements have been made to explore the possibility of using both sugarcane and maize for biofuel production. Sugarcane is an economically important crop that can be exploited both as food source and for production of biofuels. This study aimed to apply biotechnological methods to: firstly, produce drought tolerant sugarcane plants from three drought sensitive cultivars of South African origin using mutation breeding; secondly, manipulate the myo-inositol oxidation pathway (MIOP) in the model NCo310 sugarcane cultivar to alter sucrose and possibly cell wall composition which would enhance the plants viability for second-generation biofuel production. To accomplish this, optimal in vitro growth conditions for callus obtained from NCo376, N19 and N40 sugarcane cultivars were first established by testing different auxin concentrations for in vitro callus induction. NCo376, N19 and N40 callus grew optimally on 1, 0.5 and 2 mg/L 2,4-D, respectively. The NCo310 model cultivar was furthermore exposed to different ethylmethanesulfonate (EMS) concentrations to induce random mutations without compromising in vitro plant regeneration abilities of the model cultivar. Subsequently an optimal EMS concentration of 16 mM was selected and used for all further mutagenesis experiments across all cultivars. Additionally, calli from the NCo310 cultivar were exposed to polyethylene glycol (PEG) and mannitol at different concentrations to identify a suitable osmoticum, which simulates a drought stress environment in vitro. PEG at a 20% (w/v) concentration was determined as the most suitable osmoticum to induce negative osmotic pressure and allow selection of drought tolerant sugarcane plantlets in vitro. Sugarcane cultivars included in this study were consequently exposed to 16 mM EMS and in vitro selected on 20% PEG, which resulted in the survival of 7, 18 and 19 plantlets of NCo376, N19 and NCo310, respectively. These plantlets were multiplied to prepare for future ex vitro drought pot trials. NCo310 in vitro selected lines were subjected to ex vitro preliminary and follow-up pot trials to confirm drought tolerance. In the preliminary drought trials, single biological clones of selected lines were exposed to water deficit conditions. Of the 19 plantlets selected in vitro, only five outlived the wildtype NCo310 control plants. Plants were re-watered three days after plant to monitor possible plant recovery. Only one line, 310EP 4.1A, recovered from the drought stress and formed new shoots upon re-watering. To confirm these preliminary results, follow-up drought pot trials were executed using three biological repeats of each line together with NCo310 wildtype controls. . Preliminary results from this study indicate that biotechnological approaches, such as mutation breeding and in vitro selection for osmotic pressure, can potentially be successfully applied to develop crops to combat the negative effects of abiotic stresses, such as drought. Nucleotide sugars are activated monosaccharides that act as glycosyl donors in reactions that contribute to the production of cell wall biopolymers in plants. Cell wall components might play a role in biofuel potential of crops and are synthesised by various pathways. The nucleotide 5’-diphospho-sugar (NDP-sugar) interconversion pathway uses the sequential action of kinases and NDP-sugar phosphorylases to produce uridine 5’-diphosphate glucuronic acid (UDP-glucuronic acid), a precursor for cell wall sugars and lignocellulosic polymers. The NDP-sugar interconversion pathway is composed of the sugar nucleotide oxidation pathway (SNOP) and the myo-inositol oxidation pathway (MIOP). In this study one of the MIOP enzymes isolated from Arabidopsis thaliana, the glucuronokinase enzyme was overexpressed in NCo310 sugarcane, to evaluate changes in sucrose content. Morphological differences were observed between transgenic lines and the NCo310 wildtype, no significant sucrose content or glucoronokinase enzymatic activity differences were detected in leaf material isolated from these lines. Inconclusive results obtained from this study, together with the lack of published literature on the production of cell wall components and still remaining knowledge gaps regarding sucrose metabolism by the NDP-sugar interconversion pathways in sugarcane, demonstrate the need for further research into this particular field of cell wall biosynthesis and sucrose metabolism.