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Title: Frameworks for reprogramming early diverging land plants
Author: Pollak Williamson, Bernardo
ISNI:       0000 0004 7227 1424
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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Plant form is a product of emergent processes of cell division, patterning and morphogenesis. These fundamental processes remain poorly characterised in plants. However, engineering approaches can provide new tools and frameworks for the study and manipulation of plant development. This dissertation describes the development of engineering frameworks for reprogramming of the early diverging land plant Marchantia polymorpha (Marchantia). I describe the generation of genomic and transcriptomic datasets for Marchantia, which has provided the basis for the compilation of a gene-centric registry of DNA parts for engineering (MarpoDB). I describe the development of Loop assembly, an efficient and standardised DNA assembly system based on Type IIS restriction enzymes for recursive fabrication of DNA circuits with high efficiency. MarpoDB was used to mine new DNA parts compatible with Loop assembly which were used to generate plant transformation vectors for labelling of cellular features to study aspects of growth and development. I performed image analysis of genetic markers for segmentation and quantification of cellular properties in germinating gemmae. I implemented high-efficiency Cas9-mediated mutagenesis in Marchantia for use in functional molecular genetics studies. Furthermore, I produced inducible systems for expression of heterologous elements by transactivation which showed negligible levels of basal activity. It was possible to use this system for induction of gene expression in single cells. Finally, these new frameworks were applied to study the gametophytic meristem in Marchantia gemmae. I mapped the expression of several putative candidate homologues for higher plant meristem regulators, performed overexpression and loss-of-function studies for homologues of WUSCHEL, CLAVATA3 and SHOOT MERISTEMLESS. A strategy for misregulation of endogenous genes was developed using inducible transactivation, and was used with cellular markers for WUSCHEL and CLAVATA3 homologues in Marchantia.
Supervisor: Haseloff, Jim Sponsor: Becas Chile ; Cambridge Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Loop assembly ; Synthetic Biology ; Plant Synthetic Biology ; MarpoDB ; AND gate ; Cas9 ; Marchantia ; Marchantia polymorpha ; gemmae ; Marchantia meristem ; Wuschel ; Clavata ; Shootmeristemless ; Segmentation ; In planta cytometry ; Type IIS assembly ; Spore transcriptomics ; Transcriptional Units