A toolkit for the rapid improvement of bioenergy crops: the cell wall fraction

With the realisation of the impending impact of climate change, the scientific community has been working towards generating viable renewable energy sources that do not contribute to the greenhouse effect. Bioethanol is one form of renewable energy that could replace the majority of transportation fuels, however, its production efficiency and commercial viability relies on optimisation of agricultural quality and yields, biomass conversion to sugars, and fermentation of these sugars into ethanol. The focus of this dissertation will be the optimisation of agricultural quality and yields of bioenergy crops. Most food crops have been domesticated for thousands of years providing selection pressure to increase harvest index (i.e. a high ratio of fruit/seed to vegetative biomass), among other traits. Bioenergy crops, however, are grown for their vegetative biomass more than fruit or seed yields, thus farmers seek a very different plant. To meet the demands of this growing industry, rapid improvements in agricultural yields of both soluble sugars and the cell wall fraction of bioenergy crops, driven by biomass increases, are required. Improvements through traditional breeding strategies, including screening of mutagenized populations, is limited by the availability of high-throughput methods for assessing phenotypes within large populations. Improvements through genetic modification are limited by a fundamental understanding of the biogenesis of vegetative biomass, the biosynthesis and composition of cell walls, and the accumulation of soluble sugars. Here I present a toolkit for the rapid improvement of bioenergy crops. A holistic high-throughput screening strategy was developed for the bioenergy crop, Sorghum bicolor, which rapidly quantifies biomass, cell wall composition and soluble sugar concentrations of stalks, and it was subsequently applied to a large mutagenesis population. Setaria viridis was established as a model C4 grass for the study of stalk development (the major source of bioenergy crop biomass). A developing internode was identified as an ideal experimental system for the study of the biogenesis of stalk tissue, the biosynthesis of cell walls and the import of soluble sugars for storage. RNA sequencing was performed in this system as a resource for gene discovery in relation to these three processes. To complement this resource for gene discovery a technique for 3D visualisation of proteins and tissue structure, PEA-CLARITY was developed and Raman confocal imaging was applied to S. viridis stems for in situ determination of cell wall composition. The high-throughput screening method will provide a tool to drive rapid improvements in commercial bioenergy crops and allow rapid assessment of phenotypes in a field setting. The use of S. viridis as a transformable model C4 grass, along with genetic resources for gene discovery and cutting edge imaging techniques, will allow rapid improvements in understanding of the processes that underpin bioenergy crop yields and will undoubtedly uncover opportunities for crop improvement through genetic manipulation. This toolkit will be integral for the improvement of agricultural yields from bioenergy crops and, therefore, the commercial viability of the biofuel industry.