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Abstract
For most plant species, transformation has historically been a technically challenging process, with numerous bottlenecks throughout the pipeline. There is an increased demand for genetically engineered crops that possess a stack of multiple traits for yield protection, improved nutrition, improved shelf stability, and more, and current technologies are not considered to be up to the task. The shift in engineering complexity has come with a new set of challenges: how to stack traits, how to deliver traits, and how to do so in both a time and resource efficient manner.
There have been several cloning methods proposed for stacking transgenes in bacteria prior to transforming a plant. Of these, GoldenGate-based cloning systems have seen recent widespread adoption due to their modular nature that lends itself to rapid vector construction, but there remains a need for further development to better accommodate cloning needs. In chapter two, an expansion of the GreenGate cloning kit, called MultiGreen, is proposed to address the major shortcomings of GreenGate. With MultiGreen, multi-gene plant transformation vectors can be produced in a time and resource efficient manner using backward compatible parts from GreenGate, while strictly adhering to the original kit’s modular assembly structure.
Agrobacterium-mediated transformation is the most popular method for delivering recombinant DNA to plants, yet strains of laboratory Agrobacterium have remained largely unchanged since their inception. While the popular strains are effective for a range of host plants, these are largely of wild type genotype, aside from being rendered non-oncogenic. Chapter three describes a suite of new Agrobacterium strains produced through a simplified CRISPR-mediated base editing workflow. Existing workflows for mutant generation through base-editing have several bottlenecks, slowing the process and increasing the barrier to adopting base-editors as mutant generation tools. Chapter three addresses these inefficiencies through a series of visually marked single component base-editors, and an associated Geneious Prime plugin for automated target filtering. The simplified workflow was validated by editing various strains for thymidine auxotrophy and recA deficiency, thus, expanding the available options for plant transformation.
All the tools produced in this dissertation will be made available to better serve the plant transformation community.
There have been several cloning methods proposed for stacking transgenes in bacteria prior to transforming a plant. Of these, GoldenGate-based cloning systems have seen recent widespread adoption due to their modular nature that lends itself to rapid vector construction, but there remains a need for further development to better accommodate cloning needs. In chapter two, an expansion of the GreenGate cloning kit, called MultiGreen, is proposed to address the major shortcomings of GreenGate. With MultiGreen, multi-gene plant transformation vectors can be produced in a time and resource efficient manner using backward compatible parts from GreenGate, while strictly adhering to the original kit’s modular assembly structure.
Agrobacterium-mediated transformation is the most popular method for delivering recombinant DNA to plants, yet strains of laboratory Agrobacterium have remained largely unchanged since their inception. While the popular strains are effective for a range of host plants, these are largely of wild type genotype, aside from being rendered non-oncogenic. Chapter three describes a suite of new Agrobacterium strains produced through a simplified CRISPR-mediated base editing workflow. Existing workflows for mutant generation through base-editing have several bottlenecks, slowing the process and increasing the barrier to adopting base-editors as mutant generation tools. Chapter three addresses these inefficiencies through a series of visually marked single component base-editors, and an associated Geneious Prime plugin for automated target filtering. The simplified workflow was validated by editing various strains for thymidine auxotrophy and recA deficiency, thus, expanding the available options for plant transformation.
All the tools produced in this dissertation will be made available to better serve the plant transformation community.