A Pipeline for Exogenous Gene Expression and Biomass Analysis in Plants

Biological pretreatment of biomass is typically required for the production of liquid biofuels, which are viable alternatives to traditional fossil fuels. While traditional thermomechanical pretreatments are effective at helping to deconstruct lignocellulose, they are often expensive and tend to generate environmentally dangerous waste materials. An alternative to traditional pretreatment strategies is biological pretreatment, which has focused on lignocellulose-degrading organisms, such as white-rot fungi. While direct pretreatment of biomass with white-rot fungi has shown great promise, directly expressing functional white-rot fungi genes in plants (i.e. in planta expression) has the potential to be even more efficient because the biomass itself would produce the deconstruction enzymes, which would decrease the amount of steps toward biofuel production. This thesis details an efficient pipeline for cloning and expressing genes in planta followed by protocols for assessing changes to carbohydrates and lignin chemistry. An In-Fusion cloning system was developed for Agrobacterium-based transformation into Arabidopsis thaliana using green fluorescent protein as a representative gene. Modified acid hydrolysis and thioacidolysis procedures were used to explore cellulose and lignin chemistry on pretreated (i.e. non-traditional) biomass. Overall, this research generated a procedural work-flow for the rapid cloning and transformation of exogenous genes into Arabidopsis, followed by reliable carbohydrate analysis of the resulting biomass. This pipeline is expected to be used in future studies examining the expression of enzymes involved in biomass deconstruction from white-rot fungi into commercially-relevant plant biomass