siRNA-mediated silencing of mgfp5-ER in GFP-expressing protoplasts

RNA-mediated gene silencing is a highly conserved cellular mechanism induced by the intracellular presence of double-stranded RNA (dsRNA). Small interfering RNA duplexes (siRNA) are produced after enzymatic cleavage of dsRNA and induce the degradation of homologous RNA. siRNA-mediated silencing appears to be an RNA based system for counteraction of virus infections in plants and it could be used in specific silencing of genes for genomic studies involving gene characterization. This thesis explores the use of multiple siRNAs for silencing of the mgfp5-ER gene in protoplasts expressing the Green Fluorescent Protein (GFP). To investigate this, hairy roots expressing GFP were produced by Agrobacterium-mediated transformation. Nicotiana tabacum cv. Petit Havana SR1 (tobacco), Solanum tuberosum cv. Iwa (potato), rapid-cycling Brassica oleracea, and B. campestris var. pekinensis cv. Wong Bok (Chinese cabbage) explants were co-cultivated with Agrobacterium strain A4T. Hairy roots were produced in tobacco and Chinese cabbage; the efficiency of transformation was 33% and 7% respectively. The hairy roots were selected on the basis of their uniform expression of GFP and their characteristic hairy root phenotype. PCR was used to confirm the presence of the GFP gene. Additionally, seeds were obtained from rapid-cycling B. oleracea plants stably expressing GFP. Chinese cabbage hairy roots and hypocotyls from etiolated rapid-cycling B. oleracea seedlings were subsequently used as source material for protoplast isolation. Yields of 3-5x10⁵ protoplasts per gram of Chinese cabbage hairy roots and a mean yield of 1.2x10⁶ protoplasts per gram of etiolated hypocotyls were obtained. A mixture of multiple siRNA was produced to target silencing of GFP in the protoplasts by transfection. Delivery of the siRNA was confirmed by observing the fate of control Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA labelled with Cy3 fluorescent dye. As performed in this study, transfection of siRNA using a combination of polyethylene glycol (PEG) and Lipofectamine™ 2000 was not successful in delivering the siRNA into the protoplasts. Image analysis was used to measure GFP levels in this experiment. Successful transfection was achieved using Lipofectamine™ 2000 alone. Control Cy3-labeled GAPDH siRNA was observed in the protoplasts transfected, which confirmed transfection. Approximately 30% of the protoplasts showed red-fluorescent spots consistent with fluorescence of the dye. However, Lipofectamine™ 2000 was toxic and protoplast viability was reduced to approximately 50% when measured 48 h after transfection. Silencing of GFP was measured by qualitative RT-PCR. Preliminary studies were carried out to partly optimise qualitative RT-PCR analysis for confirmation of GFP silencing. Much work needs to be done before gene silencing in protoplasts using transfection of siRNA becomes efficient but this study has set the basis for future research on transfection of siRNA into protoplasts to reduce gene expression