Improved Methods Of Transgenic And Viral Reagent Delivery For Genome Engineering In Plants

Abstract

Plant genetic improvement is a crucial lever to increase the productivity and sustainability of agricultural systems in the face of manifold economic and environmental challenges. The previous decade has witnessed burgeoning molecular technologies and genomic targets for directed modification, with the potential to underpin both incremental and radical genetic gains. The use of such technologies for plant breeding, however, depends on robust delivery and activity of molecular reagents into and within regenerable cells, in order to produce stably modified germplasm. This remains a significant barrier for the majority of plants. In this thesis, I present research at the intersection of reagent design and delivery for genome modification in plants. This work includes development of practical methods for the assembly and cloning of transformation constructs, guidance on transgene expression strategies to effect desired outcomes in planta, and a particular focus on the roles engineered plant viruses can play as augmenting or primary vectors to address specific challenges in transformation and gene editing. Three manuscripts constituting the primary chapters include a comprehensive historical review of viral vector development in plants; a methods paper advancing the design and assembly of transformation constructs; and a research paper describing an improved viral delivery system for site specific recombinases. In the conclusion, I consider perspectives for the broader field and prospects for future research.