Browsing by Author "Rosmarin, Michael"
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- ItemEnhancing drought and osmotic stress tolerance by overexpressing α-acetolactate decarboxylase and acetoin 2,3-butanediol dehydrogenase in planta(Stellenbosch : Stellenbosch University, 2020-03) Rosmarin, Michael; Hills, Paul N.; Van der Vyver, Christell; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).ENGLISH ABSTRACT: The effect of certain rhizosphere colonising bacteria on plant growth has been extensively exploited in agriculture since the green revolution of 1950. The bacteria symbiotically colonise the rhizosphere, utilising exudates from the plant roots, while providing the plant with a number of beneficial actions such as increasing the nutrient availability to roots. These plant growth-promoting rhizobacteria (PGPR) have been well documented and researched since the green revolution began, however, many of the exact bacterial mechanisms responsible for plant growth promotion remain unknown. Previous research, aiming to elucidate such mechanisms, discovered that bacterial volatile organic compounds (VOCs) such as acetoin and 2,3-butanediol were able to increase growth of Arabidopsis thaliana. Subsequent research identified the genes responsible for the production of acetoin and 2,3-butanediol as a-acetolactate decarboxylase (ALDC) and 2,3-butanediol dehydrogenase (BDH1) respectively. These genes were previously successfully transformed into and expressed within Arabidopsis plants within our research group, leading to increased growth and disease tolerance. In this study, Arabidopsis thaliana plants were transformed with the ALDC and BDH1 genes using an Agrobacterium-mediated floral dip method in order to confirm the above-mentioned research, and to allow for the detection of the volatiles in the transgenic plants. However, due to a 240 base pair deletion within the ALDC gene, discovered in the T2 generation, further research could not be performed using Arabidopsis. Sugarcane (Saccharum officianarum) was transformed with the ALDC and BDH1 genes using a particle bombardment approach. Transgenic sugarcane plants were successfully genotyped, sequenced and assessed for transgene expression. The transgenic sugarcane was tested for increased growth, under both in vitro and ex vitro conditions, as well as for drought tolerance via an ex vitro pot trial. No significant differences were observed for the growth of the transgenic sugarcane in vitro compared to the untransformed control plants. The limited availability of transgenic material lead to difficulties in selecting plantlets that were of uniform size and root development for in vitro trials. This led to high variance in the data and inconsistent results within each transformed line. Larger quantities of transgenic material would have alleviated this issue by allowing for selection of plantlets at a uniform developmental stage. Neither were overall significant differences observed between the transformed and untransformed lines within the drought trial. Inconsistent conditions within the growth room where the drought trial was performed led to inconsistent drought pressures applied to the plants. In addition, a temperature spike during the trial led to the rapid onset of drought shock rather than the intended slower onset of drought stress. Untransformed sugarcane was also exposed to synthetic acetoin in vitro, with no significant differences in growth observed after the allowed growth period. In general, this study was inconclusive. However, various aspects of the research were identified which could lead to more conclusive and consistent results. In addition, a method for directly confirming the production of VOCs in planta still needs to be established.