Synthetic biology for engineering Arabidopsis thaliana protoplasts
File(s)
Author(s)
Kocaoglan, Elif Gediz
Type
Thesis
Abstract
For over 10,000 years, humans have domesticated plants, but plant synthetic biology has ushered in a new era by facilitating the engineering of development. Plant development shows remarkable plasticity, enabling plants to adapt to the ever-changing environment. This plasticity even extends to the cell fates, for example, in protoplast regeneration. Upon removal of the cell wall, fully differentiated plant cells can revert to a stem-cell-like state, giving rise to protoplasts capable of regenerating cells, tissues, and even whole plants. Engineering this process has great potential for creating transgenic plants for agriculture and new cell culture lines for sustainable bioproduction. However, engineering protoplast regeneration requires tools that offer precise spatiotemporal control over gene expression, ensuring stable expression levels without mutations across generations. Efforts to identify the target genes regulating protoplast regeneration are equally crucial. Yet, protoplasts currently lack both the genetic tools and knowledge of target genes. To address these gaps, I aimed to establish Arabidopsis thaliana protoplasts as a new synthetic biology chassis. This involved characterising basic DNA parts which revealed combinatorial interactions in gene expression. With RNA folding analysis, I proposed physical interactions among the parts as one of the mechanisms underlying these interactions. Additionally, I have re-designed vectors with enhanced stability for assembling DNA constructs. These constructs have been used to create stable A. thaliana marker lines as stage-specific reporters of protoplast regeneration. Fluorescent proteins were screened for strongest signals after a protoplast transformation protocol was optimised. Furthermore, I also established a platform for regenerating protoplasts into microcallus. Plant growth and cell wall digestion conditions were shown to be crucial in this process. The tools and platforms developed in this study will facilitate precision engineering in plant synthetic biology. Additionally, they will enhance our understanding of molecular pathways underlying protoplast regeneration and further biotechnology driven by protoplasts.
Version
Open Access
Date Issued
2024-04
Date Awarded
2024-08
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Nakayama, Naomi
Sponsor
Imperial College London
Publisher Department
Bioengineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)