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Magnetofection of tobacco protoplasts: a novel mechanism for plant transformation

George, Alexander Christopher Stephen (2018) Magnetofection of tobacco protoplasts: a novel mechanism for plant transformation. Honours thesis, Murdoch University.

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Abstract

To help meet the challenges of climate change and a growing global population, it has never been more critical that the yields of agricultural crops are increased: new breeding technologies such as genetic engineering can play a major role in achieving increased food production. Despite several decades of study there are still limitations to plant transformation technologies. These include transformation methods based on vector-mediated introduction of gene constructs using Agrobacterium, or direct gene transfer methods such as particle bombardment or transformation of protoplasts using polyethylene glycol or other methods to disturb cell membranes. Such limitations have slowed applications to generate genetically engineered plants with improved properties.

One potentially new approach to transform cells is based on nanoparticle-mediated transformation. The use of nanoparticles as gene vectors may overcome some current limitations when applied to plant systems. The small size and novel properties of nanoparticles can be exploited since the physicochemical properties of nanoparticles can be modified to suit different applications, so that they can deliver a diverse range of biomolecules. One such novel system is termed ‘magnetofection’, where superparamagnetic iron oxide nanoparticles coated with biomolecules (magnetofectins) are directed towards target cells by application of a strong external magnetic field. Magnetofection has been studied for almost two decades in animal systems, but to date there are only two reports of magnetofection being applied to plant cells.

This study was a ‘proof-of-concept’ to determine whether magnetofection could be used as a transformation system for tobacco mesophyll protoplasts, including optimisation of conditions, and to study the effects of magnetofection treatments on the viability of protoplasts.

In initial experiments, a transgenic line of cotton was obtained which expressed the reporter gene β-glucuronidase (GUS), and systems were developed to assay GUS expression both by histochemical staining and fluorescence. A GUS expression plasmid (pCAMBIA1303) was obtained for experimental work and its identity checked.

pCAMBIA1303 was bound via electrostatic interactions to PEI derivatised 100nm superparamagnetic iron oxide nanoparticles. After application of a powerful external magnetic field, the magnetofectins were attracted towards protoplasts, and transient gene expression of GUS was demonstrated 48 hours post transfection. Of the tested complexes, the transformation efficiency was highest when magnetofectins were assembled with 1:5 and 1:10 MNP:DNA weight ratios. It was shown that the density of MNPs affected the viability of magnetofected protoplasts, and that increasing MNP density significantly reduced protoplast viability. An optimal density of MNPs (1.0μg/mL) was found that resulted in transient GUS expression and did not affect protoplast viability.

This work provides a starting point for further development of magnetofection as a novel plant transformation system, the advancement of which may broaden the scope of plant molecular biology and genetic engineering.

Item Type: Thesis (Honours)
Murdoch Affiliation: School of Veterinary and Life Sciences
United Nations SDGs: Goal 2: Zero Hunger
Supervisor(s): Jones, Michael, Fosu-Nyarko, John and Iqbal, Sadia
URI: http://researchrepository.murdoch.edu.au/id/eprint/49707
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