Expanding the Development of Engineered Genetic Incompatibility in Three Plant Systems

Abstract

The rapid advancement of agricultural synthetic biology necessitates robust biocontainment strategies to prevent the ecological escape of transformative traits. Current methods, such as physical isolation and sterility-based systems, are insufficient due to environmental instability and biological leakage. This dissertation investigates Engineered Genetic Incompatibility (EGI) as a mechanism for synthetic speciation to enforce durable reproductive isolation in plants. EGI utilizes a programmable transcriptional activator to drive lethal overexpression of essential genes in hybrid offspring, while maintaining fertility within the engineered population through a refractory guide target site. This research addresses the biological hurdles of implementing EGI, across three plant systems of increasing complexity. First, using the model organism Arabidopsis thaliana, this work optimizes EGI components to overcome incomplete lethality observed in previous prototypes. By shifting the lethal target from the developmental regulator WUSCHEL to the metabolic gene ELO1, and integrating intron-mediated enhancement with the Rpl23 promoter, containment efficacy was improved to 93% lethality in hybrid lines. Second, these components were translated to the oilseed cover crop pennycress (Thlaspi arvense) to evaluate EGI in an agronomic context. This compared protoplast, viral, and stable transformation methodologies, revealing critical bottlenecks in guide RNA toxicity and the transferability of tools from model systems. Finally, this dissertation addresses transformation recalcitrance in perennial ryegrass (Lolium perenne), optimizing protocols to facilitate the future incorporation of EGI into the ecologically critical grass family. Collectively, this work demonstrates that while barriers to absolute lethality remain, EGI represents a distinct maturation in biocontainment technology. By engineering bidirectional incompatibility that mimics natural speciation, this strategy offers a pathway to secure the biosafety of next-generation crops while preserving germplasm integrity.