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Nanoelectroporation of single cell plants https://doi.org/10.17863/CAM.126604 Swan, Gemma Haseloff, Jim Fruk, Ljiljana 0000000321045817 There has been significant interest in the development of recombinant proteins as novel catalysts for green chemistry, diagnostics for integration into biosensors and therapeutic agents such as nanobodies. However, the application of these reagents is hindered by the lack of production capacity. Plants such as Marchantia polymorpha, offer a promising, chassis for the scalable bioproduction of biologics. The prospects for distributed and low-cost production offer the potential to support rapid responses to emerging diseases and tackle global health inequity. This thesis aims to support the use of M. polymorpha as a model plant and potential bioproduction chassis for the expression of high value biologics, through the use of chloroplast expression. One of the major bottle necks to scale is low chloroplast transformation rates. This work investigates the adoption of nanotechnology and novel delivery mechanisms to plant transformation, in order to deliver cargo into M. polymorpha sporelings. Nutrient film technique hydroponic units were explored as a method for optimising and scaling up both the sexual and asexual propagation of M. polymorpha. Growth conditions were explored and optimised for vegetative growth and spore production. The 3D printed hydroponic units also feature embedded low-cost sensors for real time, remote monitoring offering a low-cost, flexible platform that has the potential to be expanded to deliver an automated growth platform. A method was developed to trigger arrested spore development in M. polymorpha sporelings, yielding a near uniform population of single cells that were significantly larger than their light grown counterparts, forming an attractive target for genetic transformation. In further studies, the single cell sporelings were proven to be metabolically active, through FDA staining for cell viability, which also revealed starch granule accumulation in chloroplasts of the arrested sporelings. Software simulations with COMSOL were used to probe physical conditions for nanoelectroporation and the enhanced field effect generated by nanopores. The simulations informed the design of a novel poration mechanism, that utilised the double enhancement effect seen by nanopores with gold nanoparticles. A low-cost 3D printed nanoelectroporation device was constructed based on simulation results. Optimal device settings for achieving high poration efficiency rates whilst maintaining cell viability were established by assessing the nanoelectroporation of arrested sporelings. Optimised device parameters achieved a 95% poration efficiency while maintaining 95% cell viability. Finally, a nanoparticle candidate was identified for the loading and retention of DNA, to be used in conjunction with the nanoelectroporation device. While this thesis focuses on the establishment of a novel delivery mechanism in M. polymorpha, it also lays the groundwork for wider research and future application of nanoelectroporation in plant systems. University of Cambridge 2025-07-21 Thesis Doctoral Doctor of Philosophy (PhD) eng NanoDTC, Cambridge Display Technology (LTD) https://www.repository.cam.ac.uk/handle/1810/397474 embargo 2027-02-09 https://www.repository.cam.ac.uk/bitstreams/9d59366d-0ab6-4895-a59a-4713d6d5b7a1/download ffe025cdf36b9afc80e8ffe3581f76a9 https://www.repository.cam.ac.uk/bitstreams/1799468a-0571-4aa9-b800-ab4f8df964a3/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved bioproduction chloroplast engineering COMSOL simulations delivery mechanisms marchantia polymorpha nanoelectroporation