Viral vector-mediated CRISPR-Cas9 gene editing for fundamental and applied plant research

Abstract

Agricultural systems face significant challenges in achieving sustainable and productive growth in light of increasing global population and climate change. In the past decade, advances in genome editing and sequencing technologies have offered promising opportunities for improving various aspects of plant productivity and sustainability. However, while major crops such as maize and rice have been the primary focus of research in this area, regional staple crops also referred to as “underutilized”, “minor” or “orphan” crops have been largely neglected. This has resulted in significant yield gaps for these crops, which could be closed through the application of gene editing techniques. The main challenge in using gene editing technologies for plant improvement is the inefficient delivery of gene editing reagents into plant cells and the subsequent regeneration of an edited plant. This is especially challenging for orphan crops, which often lack reference genomes and efficient plant transformation methods.This thesis comprises three manuscripts and a concluding chapter that address challenges, opportunities, and potential solution in utilizing advanced plant biotechnology tools for fundamental and applied plant research, with a particular focus on orphan crops. The first manuscript reviews biotechnological approaches that have been attempted in Eragrostis tef, a cereal orphan crop, emphasizing the potential of gene editing for crop improvement. In the second manuscript, we demonstrated the use of plant viral vectors to deliver editing reagents into the model plant Nicotiana benthamiana, aiming to regulate stomatal development for functional characterization and enhanced drought tolerance. In the third manuscript, we extended this technique to the orphan fruit crop P. grisea (groundcherry), demonstrating the introduction of domestication trait focused on boosting fruit quality and production. The final chapter concludes the thesis covering the available plant transformation and delivery methods and their potential application in rapid improvement of orphan crops including monocots such as tef. Ultimately, this thesis demonstrates the viability of gene editing technologies to enhance orphan crops and prompts the need for further research aimed at improving the current bottleneck of delivering editing reagents to a wide array of crops thereby supporting global food security and resilient agricultural systems.