Magnetofection of Tobacco Protoplasts: a Novel Mechanism for Plant Transformation

Magnetofection of tobacco protoplasts: a novel mechanism for plant transformation.

This thesis is presented for the degree of Bachelor of Science with

Honours (Hons) in Molecular biology

Alexander Christopher Stephen George

Western Australian State Agricultural Biotechnology Centre

School of Veterinary and Life Sciences

Murdoch University

October, 2018 i

Declaration Declaration

I declare that this thesis is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institution.

Abstract 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.

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Abstract 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.

Table of contents Table of contents

Title page i Declaration ii Abstract iii Table of contents v Acknowledgements viii

Chapter 1: Introduction 1 1.1 Transformation of plants 2 1.1.1 Conventional transformation systems 2 1.1.2 Novel nanoparticle-mediated transformation systems 3 1.2 Magnetofection 6 1.2.1 Mechanism of magnetofection 7 1.2.2 History of magnetofection 8 1.2.3 Magnetofection of plant cells and tissues 11 1.2.4 Factors affecting transformation efficiency of magnetofection 13 1.2.4.1 Static vs non-static magnetic fields 13 1.2.4.2 Length of magnetic field exposure 14 1.3 Protoplasts 15 1.3.1 Protoplast transient expression system 15 1.3.2 Mechanism of FDA staining in protoplasts 16 1.4 Reporter genes for transient expression assays 17 1.4.1 β-glucuronidase (GUS) 18 1.5 Research aims 20

Chapter 2: Materials & Methods 21 2.1 Transformation of competent E. coli cells & plasmid preparation 21 2.1.1 Heat-shock transformation and selection for transformants 22 2.1.2 Bacterial culture and preparation of glycerol stocks 22 2.1.3 Preparation of pCAMBIA1303 for leaf disc and protoplast transformation 22 2.1.4 Agarose gel electrophoresis 23 2.1.5 Confirmation of the identity of pCAMBIA1303 23 2.2 Plants and growth conditions 24 2.2.1 Cotton growth 24 2.2.2 Tobacco growth 25 2.3 Protoplast isolation 25

Table of contents

2.3.1 Leaf sterilisation 25 2.3.2 Digestion of leaf strips 26 2.3.3 Isolation of intact protoplasts 27 2.4 Plant transformation 27 2.4.1 Magnetofectin assembly and Magnetofection 28 2.4.2 Polyethylene glycol mediated transformation 29 2.4.3 Biolistic particle bombardment 29 2.5 Assessment of protoplast viability 30 2.5.1 Fluorescein diacetate staining of protoplasts 30 2.5.2 Image analysis 30 2.6 Assays for β-glucuronidase activity 31 2.6.1 GUS assay of transformed plant protoplasts - histochemical staining 31 2.6.2 GUS assay of transformed plant protoplasts - fluorescence assay 32 2.6.2.1 4-Methylumbelliferone standard curve preparation 32 2.6.3 GUS histochemical staining of leaves 33 2.7 Magnetofectin gel-retardation electrophoresis 33

Chapter 3: Results 35 3.1 Introduction 35 3.1.1 Confirmation of the identity of the pCAMBIA1303 binary vector 35 3.1.2 Functional expression of the pCAMIBA1303 binary vector 37 3.2 Optimisation of GUS assays to detect β-glucuronidase activity in protoplasts 39 3.2.1 Optimisation of the protoplast histochemical staining procedure 39 3.2.2 Calibration of the Titertek® Fluoroskan fluorimeter by generation of a 4-MU standard curve 41 3.2.3 Confirmation for the quantification of β-glucuronidase activity via 4-MU assay in GUS transgenic protoplasts 42 3.3 Tobacco protoplast isolation and detection of transient GUS expression 43 3.3.1 Optimisation of tobacco protoplast isolation 43 3.3.2 Confirmation for the detection of β-glucuronidase activity in transient GUS-expressing protoplasts 46 3.4 Optimisation of the conditions of magnetofection 47 3.4.1 Determination of the binding capacity of DNA to MNPs 48 3.4.2 Viability of control, magnetofected and PEG-mediated transformed protoplasts 49 3.5 Optimisation of magnetofection on tobacco protoplasts 52 3.5.1 Magnetofection of tobacco protoplasts 52 3.5.2 The effect of assembling magnetofectins in a larger volume of water on magnetofection 53 3.5.3 The effect of centrifugation of protoplasts before magnetofection 54

Table of contents Chapter 4: Discussion 57 4.1 Particle bombardment to confirm functional expression of pCAMBIA1303 57 4.2 Development of GUS assays to detect β-glucuronidase activity 58 4.3 Manipulation of protoplasts 59 4.3.1 Protoplast viability 59 4.3.2 Novel use of protoplasts for genome editing 61 4.4 Magnetofection as a novel transformation method in plants 62 4.5 Future studies 64 4.6 Conclusion 65 References 66

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Acknowledgements Acknowledgements

To my supervisors who have given me so much of their time and support through the year, I would like to express my deepest gratitude for all you have done. First, to Prof. Michael Jones, I would like to thank you for all your suggestions and corrections in the assembly of this manuscript and for teaching me how to not split infinitives… To Dr Sadia Iqbal, thank you for mentoring me throughout the year. In particular, your practical help in the laboratory was invaluable, your patience is truly astonishing. To Dr John Fosu-Nyarko, I can’t express just how much I appreciate what you have done for me, throughout the year you have gone out of your way and invested so much time and energy into mentoring me. I couldn’t have wished for a better supervisor.

To my family, I thank you for your understanding, encouragement and belief in me throughout my life, especially over the last four years. You have made me who I am today, I will leave it for you to decide if that is a good thing or not. A special thanks to you dad, your recent keen interest in molecular biology has spurred me on to continue down this path. I hope you to see you in the lab in the years to come.

To my cats, Luna & Stella, I thank you for keeping my lap warm while I typed up endless assignments and this manuscript over the years. While I appreciate your assistance typing on my keyboard, I feel I could go without.

Finally, to my better half, Tamsin. Your unwavering love and support has enabled me to achieve what I have. I lack the proficiency to express how deeply grateful I am for all you have done, I am forever in your debt. By the time you read this, I should have proposed to you, I hope you liked it and most importantly, I hope you said yes!

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1. Introduction

Chapter 1: Introduction

A growing population coupled with rapid climate change is a cause for major concern for the sustainability of global food production. Such threats make fulfilling the agricultural needs of the world a challenging task, and so improvements to all aspects of the agricultural industry - biological and technological - are being developed continuously. For approximately 10,000 years, farmers have selected plants with better traits. Initially this was not done systematically, but since the advent of Mendelian Genetics, directed plant breeding has been undertaken. This involves crossing plants with desirable traits which are selectively propagated by sexual reproduction, and has been practised widely to increase the yield and quality of crop plants (Gepts, 2002). This process takes a substantial amount of time as it requires crossing and selection through multiple generations. In addition, because of the reliance on sexual recombination for genetic inheritance and the issue of linkage drag, the transfer of unintended genes is unavoidable in conventional breeding (Jones et al., 2017).

Crop improvement via genetic engineering provides many benefits over conventional breeding, including a substantial reduction in the time required to produce improved crops. An additional important aspect is that specific, and in some cases, directed horizontal transfer of genes originating from the same, closely-related or unrelated organisms or species can be achieved. The latter point is critical, because it allows the improvement of traits which would not otherwise be impossible to achieve through conventional breeding if natural genetic variation is not present in the crop gene pool (Gepts, 2002). As a result, many genetically engineered crops are being developed with enhanced traits, which include increased yield, improved nutrient quality and increased resistance to abiotic stresses, diseases, insects and herbicides (Abdallah et al., 2015).

To produce genetically engineered crops, transformation systems are used to deliver biomolecules such as DNA into cells of the target plant. Despite the advances in genetic engineering, development of an efficient and genotype-independent transformation system for plants still remains a challenge.

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1.1 Transformation of plants

Horizontal gene transfer into plants can be achieved through the introduction of exogenous DNA with or without a gene vector, or more recently using forms of genome editing such as CRISPR/Cas9. There are a range of transformation systems which can be used to genetically modify crop plants, each with its own set of benefits and limitations. Discussed in detail below are specific applications and limitations of ‘conventional’ plant transformation systems, followed by the introduction of novel nanoparticle-mediated transformation systems whose development may overcome the constraints of conventional transformation systems.

1.1.1 Conventional transformation systems

Amongst the most established transformation systems for plants are the use of Agrobacterium tumefaciens as a gene vector, biolistic bombardment, electroporation and PEG-mediated transformation (Cunningham et al., 2018).

The most common method of Agrobacterium-mediated transformation is co-culture of leaf discs with Agrobacterium containing a Ti (tumour inducing) plasmid. In this procedure, Agrobacterium containing a modified Ti-plasmid is culture with leaf discs of the target plant, and plant cells at the cut surface that come into direct contact with Agrobacterium may be transformed with the T (transferred) -DNA portion of the Ti-plasmid. The major limitations of this system are: it can only be used to transform DNA, DNA integration into the genome is essentially at random sites, Agrobacterium has a narrow host-range, and it requires the laborious step of callus induction followed by plant regeneration, which is highly species- dependent, to regenerate transgenic plants. The importance of Agrobacterium-mediated transformation is that it provides a mechanism to transform plant cells despite the presence of the cell wall, which otherwise poses a major barrier to transformation (Cunningham et al., 2018).

Biolistic bombardment of DNA into plant cells was developed to overcome the block posed by the cell wall through mechanical force. The general principle of biolistic bombardment is to coat inert microcarriers such as tungsten or gold with DNA and accelerate the coated

1. Introduction particles using high pressure helium gas towards the plant tissue in a vacuum. The particles penetrate 1 -6 cell layers, and DNA can be released and integrated in the host cell genome, or be expressed transiently without integration. While this system can be applied to a wider range of plant tissues than Agrobacterium-mediated transformation, and can also be used deliver a wider variety of biomolecules, it is still genotype-dependent and, like Agrobacterium, requires callus induction and plant regeneration to produce transgenic plants (Birch, 1997).

Electroporation and PEG-mediated transformations are generally used to transform protoplasts, that is plant cells which lack a cell wall, which has been removed using wall degrading enzymes. Both of these systems work by disrupting the cell membrane to allow the entry of DNA, RNA or proteins. Protoplast systems overcome the need to find ways for such compounds to cross the cross the cell wall, however they come at the cost of the need for protoplast culture and regeneration, which adds additional regeneration steps prior to callus induction to produce transgenic plants (Cunningham et al., 2018). Additionally, isolation and efficient regeneration of protoplasts is labour-intensive and species-dependent (Birch, 1997).

In summary, these systems have been used successfully to generate genetically engineered plants, but no one transformation system can be applied to all agronomically important crops or tissue types, and typically they all require laborious regeneration protocols which are genotype-dependent.

1.1.2 Novel nanoparticle-mediated transformation systems

Nanoparticles (NPs) are of particular interest as biomolecule carriers for plant transformation because of their unique, and highly modifiable physical and chemical properties. NP cores can be composed of a wide variety of metals, semi-conductors and magnetic compounds, and can be coated with various polymeric ligands such as polyethylenimine (PEI) or polyethylene glycol (PEG), giving rise to the diverse physical and chemical properties (Thanh et al., 2010). As a results, NPs can be biofunctionalised with

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nucleic acids, proteins, phospholipids and carbohydrates, making them ideal biomolecule carriers.

More recently there has been increasing interest in using NP-mediated transformation systems to deliver biomolecules, particularly nucleic acids, into plant cells (Figure 1.1). A search of the literature showed that NP-mediated transformation systems were first developed to transfect animal cells in culture, and only recently has there been interest in their use in plant systems (Figure 1.1). While the disparity of published journal articles between plant and animal systems may be attributable in part to more research being done in animal cell culture systems, there is no doubt that the potential application of NP- mediated transformation has not been studied in detail in plant systems. A major reason for this is the presence of a cell wall. Generally, the cell wall was thought to have a size exclusion limit of 2-20nm, however, recent evidence suggests that NPs of at least 50nm and possibly larger are permeable to the cell wall (Schwab et al., 2016).

250 Plants 200 Animal

150

100

Number of published journal articles journal published Number of 0 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 Year

Figure 1.1: Number of unique published journal articles derived from using the search term “Nanoparticles” AND “plant” AND “transfection” OR “transformation” or, “Nanoparticles” AND “animal” AND “transfection” OR “transformation” in any field for the Web of Science and PubMed databases as of 12/10/2018.

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As NPs with novel properties and smaller sizes are being created, NP-mediated transformation systems may overcome the barrier to transformation imposed by the cell wall by virtue to their small size. In the first published use of carbon nanotubes as a potential delivery vehicle into plant cells, Liu et al. (2008), showed that single-walled carbon nanotubes (SWCNTs) with widths much less than 50nm conjugated to FITC could passively traverse the cell walls of Bright Yellow (BY-2) Nicotiana tabacum (tobacco) cells. They showed that SWCNTs-FITC treated BY-2 cells fluoresced significantly more than control cells incubated with only FITC or SWCNT. They also reported that SWCNTs did not affect the viability of the BY-2 cells, although in this case the evidence was not convincing, as they did not establish a base level of cell viability in the controls, or even provide statistical analysis of cell viability after treatment with SWCNTs. This aspect is important if the transformed cells were to be used to regenerate whole transgenic plants.

A more recent report showed that SWCNTs and multi-walled carbon nanotubes (MWCNTs) can be used for passive transfection of cells in Eruca sativa (arugula), Triticum aestivum (wheat), and tobacco leaves by infiltrating the SWCNTs or MWCNTs coated with DNA encoding the green fluorescent protein (GFP) onto small punctures made with a razor (Demirer et al., 2018). The single-walled or MWCNT/DNA complexes had a length of 12- 23nm, theoretically small enough to permeate the cell wall. They provided convincing evidence of transient GFP expression in leaves transformed with DNA coated onto SWCNTs and MWCNTs, through quantitative confocal imaging three days post transfection. They also confirmed transient expression with Reverse Transcription and quantitative PCR, showing that GFP mRNA was only present in leaves three days post treatment with SWCNTs and MWCNTs and not in untreated controls.

Two papers have recently suggested that mesoporous silica nanoparticles (MSNs) have the capacity to transfect plant cells with plasmid DNA (Chang et al., 2013; Torney et al., 2007). In the first paper, Torney et al. (2007) used MSNs of ~100nm coated with plasmid DNA encoding a GFP gene and incubated them with tobacco protoplasts, after which the protoplasts were evaluated for GFP expression after 36 hours via confocal microscopy. While they did provide images suggesting that GFP was expressed in treated protoplasts, no images of true controls, that is, protoplasts not treated with MSNs or treated with MSNs without plasmid DNA were provided, making it difficult to establish the veracity of their

1. Introduction

results. In the same paper, they attempted to show that MSNs can be used to induce transient expression of GFP in MSN-bombarded immature embryos of maize, however, again they did not provide adequate controls and therefore it cannot be concluded that GFP had been expressed. In the second paper Chang et al. (2013), showed that 50nm MSNs could mediate transient expression of the mCherry protein in Arabidopsis thaliana root cells when they transferred and incubated 20 day old seedlings in media containing a mCherry plasmid construct coated-MSNs for 48 hours. In this case appropriate controls were provided and convincing evidence was shown that the DNA construct-coated MSNs induced transient expression of mCherry in transfected root cells. As with the two publications on carbon nanotubes, it has been shown that passive uptake of the nanoparticle vehicles through plant cell walls is possible.

NP-mediated transformation systems have the potential to overcome many of the limitations of the conventional transformation systems discussed above (Cunningham et al., 2018). Firstly, narrow host ranges and laborious regeneration protocols may be avoided through the transformation of embryonic and pollen tissue, which could be simplified by efficient transformation through the cell wall. Finally, random DNA integration could be avoided by delivery of endonucleases such as CRISPR/Cas9 ribonucleoproteins (RNPs), facilitated by the diverse biomolecule carrying capability of NPs.

1.2 Magnetofection

A new approach to plant transfection using NPs was published recently (Zhao et al., 2017) . It makes use of magnetic nanoparticles (MNPs) and powerful magnets to attract MNPs to the surface or through cells such that they can release coated DNA constructs into the target. This technique has been termed ‘Magnetofection’ and is the focus of the research presented in this thesis. Magnetofection is a simple and efficient transformation mechanism in animal cell cultures for transformation of target cells with the application of the external magnetic field. The most commonly used magnetofection system comprises superparamagnetic iron oxide nanoparticles coated with the cationic polymer, polyethylenimine (PEI), which binds negatively charged nucleic acids.

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1.2.1 Mechanism of magnetofection

The fundamental unit of delivery in magnetofection is the ‘magnetofectin’. A magnetofectin is comprised of an MNP coated with a cationic polymer associated with the desired cargo (DNA, RNA, virus). The magnetofectin complex is assembled by electrostatic self-assembly or salt-induced colloid aggregation. To facilitate integration of nucleic acids or proteins, magnetofectins are added to cell culture or tissues in an appropriate medium followed by application of the external magnetic field. The purpose of the magnetic field is to increase the rate and likelihood that magnetofectins will associate with or penetrate the surface of target cells. The consensus in the animal literature is that magnetofectins are then internalised by cells via endocytosis, however, it is has been suggested that some magnetofectins can directly penetrate target cell membranes (Mondalek et al., 2006; Muthana et al., 2008). Once inside cells, magnetofectins either escape the endosomal pathway or prevent endolysosome formation due to the proton sponge effect (Ma, Zhang et al. 2011). This is thought to occur as a result of the attraction of hydrogen and chloride ions into the endosome by the cationic polymer (conjugated to the MNP), resulting in a change of osmotic pressure causing the endosome to burst. However, the exact mechanism of the proton sponge effect is heavily debated (Benjaminsen et al., 2013). In the cytoplasm, the magnetofectin then dissociates, so that the nucleic acid/viral cargo is seperated from the MNP; at present the mechanism for this process is not understood (Ma et al., 2011). The delivered cargo is then free to express once inside the cytoplasm, in the case of plasmid DNA or DNA viruses, the DNA first enters the nucleus, where it may integrate, prior to expression. A simplified schematic of magnetofection is shown in Figure 1.2.

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Figure 1.2: Schematic diagram showing the process of magnetofection. Adapted from (www.ozbiosciences.com)

1.2.2 History of magnetofection

A search of the literature using the term “Magnetofection” in the Web of Science and PubMed databases returned 334 and 160 hits, respectively, of which 335 were unique articles. The distribution of this search by publication year is shown in Figure 1.3. A further search with “Gene therapy” or “Cancer” as a key word in titles, abstract or keywords of the literature returned 55% of the articles, whereas “Human” or “Animal” yielded 23% of the articles, but “Plant” or “Agriculture” resulted in just two hits, 0.6%, with only one of these articles being directly related to magnetofection of plant cells.

Most publications related to magnetofection orginate from Germany, which contributed 25% of the articles, with 19% from the USA, 18% from China, and just 2% from Australian scientists/authors.

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The most highly cited of the papers was the second to be published in which Scherer et al. (2002), showed that magnetofectins carrying plasmid DNA, adeno- and retro-viral vectors can be used to transfect human (K562) and murine (NIH3T3 & CHO) cell lines. Specifically, they showed that the application of a magnetic field increased gene expression by up to 500-fold in NIH3T3 cell lines compared to controls without a magnetic field. Additionally, magnetofection was performed in vivo on Wistar rats, where magnetofectins associated with DNA encoding the β-glucuronidase (GUS) gene, was injected directly into the surgically exposed ileum after which a magnetic block was placed directly below the site of injection for twenty minutes. Transfection was confirmed through histochemical staining, which showed that transfection was highly localised to the site of magnetic field application compared with controls where no GUS expression was observed. Magnetofection has been largely limited to medical research in animal or human cell lines with some in vivo applications for animals. A common theme in the literature is the potential applications of magnetofection in gene therapy to treat cancer. For example, in the past five years, the top three cited works have all involved magnetofection of murine melanoma or human cancer cells lines. (Govindarajan et al., 2013; Huang et al., 2015; Prosen et al., 2013).

In 2001 the first paper related to retroviral nucleic acid delivery enhanced by a magnetic field was published, though the term magnetofection was not used in the paper (Hughes et al., 2001). The authors showed that retroviral vectors bound to paramagnetic particles of 1µm diameter could be used to transfect HeLa cells, and that expression in cells exposed to magnetofectins was highly localised to cells that were exposed to a magnetic field. They suggested that magnetic field-dependent localisation of retroviral infection may be beneficial for in vivo administration of target tissues and organs. In the following three years, ten more journal articles were published in the field of magnetofection (Figure 1.3). The first of these was by Scherer et al. (2002), who showed that magnetofection can also be used to transfect or be used as a carrier for non-viral and multiple viral vectors. Huth et al. (2004) and Ma et al. (2011), went on to described the possible mechanism of magnetofection, showing the entry and intracellular trafficking of magnetofectins, although much remains to be elucidated on this topic. In more recent years, magnetofection has been used to transform an ever-increasing diversity of human and animal cell types with the end

1. Introduction goal of gene therapy being a common theme (Huang et al., 2015; Pereyra et al., 2016; Prosen et al., 2016; Przybylski et al., 2017; Shalaby et al., 2016).

Magnetofection of animal cells is not generally superior to other traditional transformation methods, such as electroporation, sonoporation, hydroporation and ballistic methods (Plank et al., 2011). However, the advantages include localisation of delivery and transfection, low cost, simplicity of use and enhanced transformation efficiency in some cell lines (Plank et al., 2011). Nevertheless, the number of published journal articles per year in this field peaked in 2012, and is perhaps declining as shown in (Figure 1.3).

Number of published journal articles journal published Number of

Year

Figure 1.3: Number of unique published journal articles derived from using the search term “Magnetofection” in all fields for the Web of Science and PubMed databases as of 28/05/2018.

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1.2.3 Magnetofection of plant cells and tissues

Although magnetofection has not yet been ground-breaking for transformation in animal systems, there are features which are promising for application to plant systems. In particular, if this strategy can be used to overcome the barrier of the plant cell wall, then magnetofection may provide some advantages over conventional plant transformation systems.

To date, only one published journal article describes the application of magnetofection of plant material. This work by Zhao et al. (2017), was innovative in that it showed the successful use of magnetofection to introduce plasmid DNA coated superparamagnetic iron oxide nanoparticles (168nm diameter), conjugated with PEI, into pollen grains of cotton, pumpkin, zucchini, capsicum and lily. In one experiment, a vector containing the GUS gene was magnetofected and the expression of GUS in the pollen, as determined by histochemical staining, was species-dependent, with transfection efficiencies of 38%, 65%, 53%, 66% and 90% for the cotton, pumpkin, zucchini, capsicum and lily respectively. The authors did not suggest a reason for these differences in transformation efficiency, however, they speculated that magnetofectins gain entry to the pollen cells via apertures in the pollen wall. A possible reason for the differences in transformation efficiency could be variations in the morphological structure, specifically aperture size. The advantage of transforming pollen is that laborious tissue culture procedures involved in the regeneration of transformed cells are not required. Instead, transformed pollen can be used directly for fertilisation to produce transgenic seed. While transformation of pollen via magnetofection and the generation of transgenic lines was accomplished by Zhao et al. (2017), at least two backcrosses were required to produce pure transgenic lines and so ultimately it may not significantly reduce the amount of time required to produce stable transgenic plants. The authors found no significant difference in the viability of magnetofected pollen compared to control pollen, suggesting that magnetofection has no deleterious effect on the viability of magnetofected pollen.

While the research by Zhao et al. (2017) is the only published work directly related to iron oxide NP- based magnetofection of plant cells, an earlier paper by Hao et al. (2013) reported magnetic field enhanced transfection of magnetic gold coated nanoparticles (GNPs) into

1. Introduction canola root cells with and without a cell wall. This is not considered magnetofection due to the use of GNPs as opposed to superparamagnetic iron oxide NPs, however, the principle of the transformation system is very similar. In that research, GNPs of 25nm diameter conjugated with polyethylene glycol (PEG) and associated with either Fluorescein isothiocyanate (FITC) or plasmid DNA encoding the GUS gene were used for transfection. GNP/PEG/plasmid DNA complex delivery was enhanced by exposure to a rare metal Neodymium magnet for 24 hours. Exposure to a magnetic field was typically limited to 30 minutes in magnetofection experiments, so the longer exposure may have been excessive as any enhancement of the interaction between the GNPs and the cell suspension would occur shortly after exposure to the magnetic field. Using Fluorescence-Activated Cell Sorting (FACS), an extremely high delivery efficiency of ~99% for FITC in canola protoplasts was reported. Although the expression efficiency of GUS was not reported, the activity of β- glucuronidase was confirmed by histochemical staining with 5-bromo-4-chloro-3-indoyl β-D- glucuronide (X-gluc), showing convincing evidence of transient GUS expression. They reported a minimal reduction (~1-3%) in the viability of cells undergoing magnetic enhanced delivery compared to control cells. GNPs offer many traits that make them particularly desirable for transfection; they are readily functionalised, easily synthesised, biologically inert, and have been shown to be non-cytotoxic in many mammalian cell lines (Ghosh et al., 2008), perhaps making them more desirable for transfection than iron oxide NPs used for magnetofection.

The little to no effect that magnetic-enhanced transfection of NPs have on the viability of transformed tissues is a promising feature of this transformation system. As for many cell types, such as protoplasts, transformation is most efficient in cell populations with high viability (Miao et al., 2007). A transfection system which has such a mild effect on viability may promote regeneration of plants which are otherwise recalcitrant to transformation. Pollen transfection by magnetofection may lead to creating a genotype-independent transformation system for plants, which would be highly desirable for plant biotechnology and genetic manipulation.

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1.2.4 Factors affecting transformation efficiency of magnetofection

Since the advent of magnetofection as a transformation system, many factors that impact the transformation efficiency of magnetofection have been explored, the most well explored of which are, the use of non-static magnetic fields as opposed to static magnetic fields, and the length of time of magnetic field exposure. These aspects are discussed below.

1.2.4.1 Static vs non-static magnetic fields

The earliest reported use of a non-static magnetic field for magnetofection was by Kamau et al. (2006). In this study, 293T, HeLa, Cos7 and synoviocyte cells were magnetofected with either a static or a pulsating magnetic field, with a sinus type wave applied perpendicular to the culture plate. For all cell types it was shown that exposure to a static magnetic field resulted in higher transformation efficiency then for a pulsating magnetic field, when both were applied for five minutes. They go on to show that the applying a static field for five minutes followed by a pulsating magnetic field for five minutes, resulted in a higher transformation efficiency then for only static or pulsed magnetic fields for five minutes. The authors claim that this shows that the application of the pulsed magnetic field increases transformation efficiency, however, this is an inappropriate conclusion as they do not have controls for 10 minutes of only static or pulsating magnetic field exposure. The observed increase in transformation efficiency that they showed could be attributable solely to additional exposure time to the magnetic field, and not the type of magnetic field. Two years later, McBain et al. (2008), applied an oscillating magnetic field to add a lateral motion to the MNPs as they were pulled towards human lung epithelial cells (NCI-H292 ). It was shown that transformation efficiency was significantly higher in oscillating magnetic fields compared to static fields under some amplitudes and frequencies. However, this was not consistent across all amplitudes and frequencies tested. From their results, it is seen that the highest reported transformation efficiency occurred under a static magnetic field, perhaps indicating that transformation efficiency is more dependent on the amplitude of the magnetic field as opposed to the frequency. Vainauska et al. (2012), constructed a magnet system that applied a complex dynamic gradient magnetic field, where magnets were oriented in a checker board pattern and rotated in a circular motion. The authors

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reported significantly enhanced transformation of PC3 cells compared to magnetofection with a static magnetic field. The results were convincing as appropriate controls were shown. An explanation for this enhanced transformation efficiency was later offered by Kozlov et al. (2015), who showed in a computer model that in a 1 x 1 x 1m3 cuvette, approximately 30 - 40% of the basal area is inaccessible to MNPs under the effect of a static magnetic field. Overall, it appears as if the application of a non-static magnetic field can enhance transformation efficiency, however the enhancement is dependent on the nature of the non-static field.

1.2.4.2 Length of magnetic field exposure

Another factor of magnetofection that has been shown to influence transformation efficiency is the time of exposure to the magnetic field. Plank et al. (2003) showed that under a static magnetic field, maximum transfection levels were achieved at ten minutes of magnetic field exposure in NIH 3T3 cells, and that increasing the length of incubation out to four hours did not result in a significant difference in transformation efficiency. A study by Gersting et al. (2004), on immortalised bronchial (16HBR14o) cells, showed that transgene expression increased to a maximum after 15 minutes of magnetofection in a static magnetic field, after which transgene expression levels remained constant for treatments up to four hours, supporting the findings of Plank et al. (2003). Kamau et al. (2006), showed that in cells magnetofected with a vector coding for GFP, that increasing the time of magnetic field exposure from five to 20 minutes resulted in over double the transformation efficiency in HeLa and Cos7 cells for 50nm MNPs. Conversely, in magnetofected PC3 cells it was shown that increased time exposure to a magnetic field resulted in decreased transformation efficiency, with a maximum transformation efficiency of ~65% at five minutes and a minimum of ~40% at 20 minutes of exposure (Vainauska et al., 2012). In this case the magnetic field used was dynamic as opposed to static, which may explain why these findings were not consistent with the other work. In summary, it appears that 10 – 15 minutes of magnetic field exposure is required to achieve maximum transformation efficiency, in general longer incubations do not appear to have any negative impact on transformation.

1. Introduction

1.3 Protoplasts

Protoplasts are cells from which cell walls have been removed. Almost 60 years ago, Cocking (1960), described the isolation of protoplasts from plants for the first time. It was achieved by enzymatic digestion of root tips of tomato seedling using a fungal cellulase: there are now many protocols for the isolation of protoplasts derived from a many plant tissues and species. Successful transformation of protoplasts was achieved for the first time by introducing RNA into tobacco mesophyll protoplasts (Aoki et al., 1969). After the establishment of efficient protoplast transformation methods such as PEG and electroporation, and the development of sensitive reporter gene assays such as those for GUS and green fluorescent protein (GFP), the use of protoplasts in transient expression systems became an important tool in plant molecular and cell biology (Sheen, 2001).

1.3.1 Protoplast transient expression system

Following the enzymatic removal of cell walls, plant protoplasts retain many of the physiological properties of intact plants (Sheen, 2001). A diverse range of biomolecules including DNA, RNA and proteins can all be delivered into protoplast via methods such as PEG-mediated transformation, electroporation and microinjection while maintaining some degree of viability (Yoo et al., 2007). As a result, plant protoplasts are ideal to study transient gene expression systems, and have been used to analyse expression of chimeric reporter genes, to describe cis-acting elements and trans-acting factors related to gene regulation and signal transduction (Abel et al., 1994).

There are, however, limitations to the use of protoplast transient expression systems. For example, processes that are dependent on the cell wall, plasmodesmata or intracellular interactions cannot be studied using protoplasts (Sheen, 2001). A drawback of using protoplast transient expression systems is the demanding and practical expertise required to use protoplast systems. It can be difficult to generate intact protoplasts (Chen et al., 2006; Sheen, 2001; Yoo et al., 2007), and healthy plant starting material, optimisation of the amount and conditions of enzymatic digestion and meticulous handling procedures are needed for efficient transformation (Miao et al., 2007). As a result, protoplast viability in

1. Introduction

transformation experiments is often reported in the literature (Abel et al., 1994; Hao et al., 2013).

1.3.2 Mechanism of FDA staining in protoplasts

There are a range of methods to determine the viability of protoplasts, the most commonly used method is fluorescein diacetate (FDA) staining. The principle of FDA staining is that the membrane-permeable and non-fluorescent compound FDA is added to protoplasts whose viability is to be determined. FDA enters the cell where it is hydrolysed by endogenous plant esterases into the membrane-impermeable, fluorescent compound, fluorescein. Fluorescein only accumulates in cells with intact cell membranes, where it exhibits a bright green fluorescence. In cells with damaged cell membranes, fluorescein leaks into the surrounding medium and there is no accumulation of fluorescence. In this method, cells exhibiting fluorescence are deemed viable, while cells that do not are not intact or viable (Figure 1.4).

1. Introduction

Fluorescein diacetate (FDA)

H2O

Dead cell Viable cell

Fluorescein

Hydrolysis by plant esterases

Fluorescein

Figure 1.4: Schematic diagram showing the hydrolysis of non-fluorescent, membrane permeable fluorescein diacetate to the fluorescent, membrane impermeable compound fluorescein via endogenous plant esterases. Fluorescein is retained in cells with intact cell membranes. 1.4 Reporter genes for transient expression assays

Reporter genes are nucleic acid sequences which encode proteins whose expression in appropriate media and conditions is readily detectable and in some cases, quantifiable. An ideal reporter gene for plants would have no endogenous activity, is highly sensitive and quantifiable, could be assayed non-destructively, and could be localised to sub-cellular compartments or target tissues (Jefferson et al., 1987). No individual reporter gene meets all these criteria, therefore, reporters such as Chloramphenicol Acetyl Transferase (CAT), GUS, GFP and Luciferase (LUC) may be used in combination for gene expression analyses. Methods for detecting expression of these markers range from histochemical staining,

1. Introduction

fluorometric and colorimetric assays, to spectrophotometry and immunoassays and PCR methods (Ziemienowicz, 2001).

1.4.1 β-glucuronidase (GUS)

The GUS gene encodes β-glucuronidase, a hydrolase that catalyses the cleavage of many β- glucuronides such as 5-bromo-4-chloro-3-indoyl β-D-glucuronide (X-gluc) and 4- methylumbelliferyl-β -D-glucuronide (MUG). The products of such reactions are relatively easily detectable.

Jefferson et al. (1987) developed the GUS reporter system during Jefferson’s Ph.D studies. This was done by generating gene fusion systems for analyses of gene products which are present in cells in moderate to low abundance. The GUS reporter system works well in plants as the expression of GUS is non-deleterious and there is little or no endogenous β- glucuronidase activity in higher plants (Jefferson et al., 1987). Additionally, the system is relatively cheap, highly sensitive, quantifiable, and the activity can be targeted and even localised to sub-cellular compartments (Jefferson et al., 1987). Unfortunately, the assay system is also destructive, and so tracking expression in real-time is difficult.

Histochemical and fluorometric GUS assays were used to detect β-glucuronidase activity in this study. The histochemical stain requires minimal effort and is simple to perform, however, it is not easily quantifiable and is relatively insensitive. The opposite is true for GUS fluorescence assays (Ziemienowicz, 2001).

The histochemical substrate X-gluc is used in the histochemical assay, X-gluc is cleaved by β- glucuronidase to produce glucuronic acid and chlorodibromoindigo, which then oxidises to form the dark blue insoluble dimer dichlorodibromoindigo (ClBr-Indigo) (Figure 1.5). The ClBr-Indigo is easily visualised under visible light and its presence indicates the activity of β- glucuronidase.

1. Introduction

Β-glucuronidase (GUS) Glucuronic acid

5-bromo-4-chloro-3- Chlorodibromoindigo indoyl β-D-glucuronide (X-gluc) Oxidation dimerization

Dichlorodibromoindigo (blue coloured)

Figure 1.5: Schematic diagram showing the cleavage of X-gluc by β-glucuronidase to produce the insoluble blue product dichlorodibromoindigo. Adapted from Karcher (2002).

When the GUS fluorometric assay is used to quantify the activity of β-glucuronidase in plant tissues or cells, the fluorometric substrate MUG is cleaved by β-glucuronidase to produce the fluorescent compound 4-methyl-umbelliferone (4-MU) which can be detected using a fluorimeter with an excitation wavelength of 365 nm and emission wavelength of 455 nm (Figure 1.6).

Β-glucuronidase (GUS) 4-methyl-umbelliferone (4-MU) 4-methylumbelliferyl- β -D-glucuronide (MUG) Glucuronic acid

Figure 1.6: Schematic diagram showing the cleavage of MUG by β-glucuronidase to produce the fluorescent product 4-MU. Adapted from Wu et al. (1998).

1. Introduction

1.5 Research aims

The first report of successful use of magnetofection for plant transformation was published recently by Zhao et al. (2017). The significance of this report is that, if repeatable, it could reduce the bottleneck of tissue culture procedures in plant transformation. Therefore, the overall aim of this project was to examine this new technology and determine as a proof-of- concept study whether magnetofection can be used to transfect tobacco mesophyll protoplasts. If successful, then in future studies at Murdoch University, magnetofection could be applied to plant material such as pollen or embryonic tissues to reduce the time needed to generate transgenic plants, and possibly allow transfection of species recalcitrant in tissue culture.

The specific aims of this project were:

1. To determine the effect of magnetofection treatments on the viability of tobacco mesophyll protoplasts. 2. To optimise the magnetofection system in tobacco mesophyll protoplasts. 3. To establish that magnetofection can enhance transfection and expression of reporter genes such as GUS in tobacco mesophyll protoplasts.

2. Materials & Methods

Chapter 2: Materials & Methods

2.1 Transformation of competent E. coli cells & plasmid preparation

Chemically competent Escherichia coli strain JM 109 was used for all plasmid transformations following the method outlined by the E. coli Competent Cells: Single-Use Promega protocol.

The binary vector pCAMBIA1303 (Figure 2.1), which has the kanamycin gene for bacterial selection and a GUS/GFP gene fusion driven by the 35S Cauliflower mosaic virus promoter was used for biolistic bombardment of tobacco leaf discs, as well as magnetofection and PEG-mediated transformation of tobacco protoplasts.

Figure 2.1: Map of the pCAMBIA1303 binary vector (from http://www.snapgene.com/resources/plasmid_files/plant_vectors/pCAMBIA1303).

2. Materials & Methods

2.1.1 Heat-shock transformation and selection for transformants

Competent cells (50µL) stored at -80°C in 1.5mL microcentrifuge tubes were thawed on ice and approximately 50ng of pCAMBIA1303 was added and gently mixed. The mixture was incubated on ice for 30 minutes. The cells were then heat-shocked in a 42°C water bath for 45 seconds and returned to ice for a further 2 minutes. Subsequently, 500µL of LB broth (10g/L Bacto®-tryptone, 5g/L Bacto®-yeast extract, 5g/L NaCl, pH 7.5) was then added to heat-shocked cells and placed on a shaker at 220 rpm for a minimum of 90 minutes at 37°C.

Antibiotic selection for transformants was then carried out by spreading 100µL of heat- shocked cells on kanamycin selective plates (10g/L Bacto®-tryptone, 5g/L Bacto®-yeast extract, 5g/L NaCl, 15g/L agar, 25mg/L Kanamycin monosulphate-A, pH 7.5) and incubating at 37°C for 16-24 hours.

2.1.2 Bacterial culture and preparation of glycerol stocks

Individual colonies from the kanamycin selective plates were inoculated in 8mL of LB broth supplemented with 25mg/L kanamycin monosulphate-A in 20mL McCartney bottles and incubated overnight on a shaker at 220 rpm at 37°C.

Glycerol stocks of pCAMBIA1303 transformed cells were prepared by mixing an equal volume of transformed culture and 50% glycerol. The glycerol stocks were stored at -20°C for two months at a time or -80°C indefinitely. Following the preparation of glycerol stocks, all future cultures were produced by inoculation with 5μL of pCAMBIA1303 glycerol stock as described above.

2.1.3 Preparation of pCAMBIA1303 for leaf disc and protoplast transformation

Plasmid DNA was isolated and purified using the Wizard® Plus SV Minipreps DNA Purification System as per the manufacturer’s protocol, with the following modifications. All centrifugation steps were carried out at 14680 rpm in a 5424 table top centrifuge (Eppendorf) at room temperature. Elution of plasmid DNA was carried out with 100μL of

2. Materials & Methods

nuclease free water warmed to 55°C, which was allowed to sit in the Spin Column for 10 minutes prior to centrifugation. This method was performed routinely with 10 replicates concurrently, typically yielding approximately 250μg of pCAMBIA1303 plasmid DNA in 1mL of nuclease-free water.

Plasmid DNA, resuspended in nuclease-free water, was quantified using a NanoDrop Onec spectrophotometer (Thermo Scientific) by measuring 1μL of DNA and using the nuclease- free water as a blank.

To obtain high DNA concentrations of 1μg/μL for PEG-mediated transformation of protoplasts, pCAMBIA1303 isolated as described above was concentrated using a RVC 2-18 CDplus benchtop mini concentrator (Christ) at 1300rpm and 25°C. The length of vacuum concentration depended on the starting concentration and volume of DNA. Mostly, 120 minutes was sufficient to concentrate 1mL of DNA at 250ng/μL to approximately 1μg/μL.

2.1.4 Agarose gel electrophoresis

All agarose gel electrophoresis used 1% (w/v) agarose run at 70V for 1-2 hours. The agarose gel and running buffer were prepared with TAE (0.04M Tris-acetate, 0.002M EDTA [pH 8.0]). SYBR safe DNA gel stain (Invitrogen Pty. Ltd, Australia) was used for staining DNA. Gel imaging was performed with a Biovision+1000 gel imager (Fisher Biotec). For all gels, 5µL of a 1kb DNA ladder (Axygen) was used as a marker.

2.1.5 Confirmation of the identity of pCAMBIA1303

pCAMBIA1303 was digested with the restriction endonucleases EcoRI and BsrGI in NEBuffer 2.1 (New England Biolabs) as shown in Table 2.1. The reaction mixtures were incubated at 37°C for 3 hours and then run on an agarose gel for 1 hour.

2. Materials & Methods

Table 2.1: Reaction setup for restriction digestion of the pCAMBIA1303 binary vector.

Components Restriction digest (μL) Control reaction (μL)

Restriction enzymes:

EcoRI (20,000U/mL) 0.5 0

BsrGI (20,000U/mL) 0.5 0

10x NEBuffer 2.1 5 5 Cresol Red 15 15 pCAMBIA1303 x (1μg) x (1μg)

Nuclease free water to 50 50

* x is variable based on the concentration of DNA used.

2.2 Plants and growth conditions

Seeds of tobacco (Nicotiana tabacum) and cotton (Gossypium hirsutum), both Sicot 71 and GUS4 transgenic cotton driven by the 35S promoter (here on referred to as 35SGUS4 cotton) were kindly provided by Dr Stephen Milroy (Murdoch University) and Dr Danny Llewellyn (Chief Research Scientist, Cotton Biotechnology, CSIRO Agriculture & Food) respectively. Tobacco leaf discs were used for biolistic bombardment, and leaf mesophyll protoplasts were used for PEG-mediated transformation and magnetofection. 35SGUS4 cotton leaf discs and leaf mesophyll protoplasts were used as positive controls for β- glucuronidase activity and Sicot 71 cotton leaf mesophyll protoplasts were used as a negative control for β-glucuronidase activity. GUS transgenic tobacco lines were not available for the project.

2.2.1 Cotton growth

For each 10L pot, five cotton seeds were sowed into thoroughly wetted soil containing

(0.375g/L CaCO3, 0.5g/L CaMg(CO3)2, 2g/L Growers blue, 2g/L Osmocote® Native Gardens) at a depth of ~2cm. The plants were kept in an environment-controlled chamber with a 14 hour light/10 hour dark photoperiod at 28°C.

2. Materials & Methods

Periodically, cotton plants were sprayed with Yates Rose Gun (active ingredients – 0.1g/L tau-fluvalinate, 0.05g/L myclobutanil) for control of thrips and fungal growth on the soil surface.

2.2.2 Tobacco growth

Tobacco seeds were sprinkled onto the surface of thoroughly wetted soil and allowed to germinate into seedlings for approximately three weeks. Individual seedlings were then re- potted into 3L pots for further growth. At all times tobacco plants were maintained in an environment-controlled chamber with a 16 hour light/8 hour dark photoperiod at 23°C or 14 hour light/10 hour dark photoperiod at 28°C. Two different environment-control chambers were used because of limited space, however, tobacco appeared to grow well in both conditions.

2.3 Protoplast isolation

The method of isolating sterile leaf mesophyll protoplasts used in the research involved three distinct steps. Firstly, the surface of leaves were sterilised by exposure to ethanol and bleach. Secondly, sterile leaves were finely sliced to maximise their exposed surface area and were digested with enzymes that release leaf mesophyll protoplasts by degrading structures adjoining adjacent cells and the cell wall. Lastly, intact protoplasts were isolated from non-digested leaf debris and damaged protoplasts. The details of these methods are described below.

Sterility was required as the goal is to perform transient assays for β-glucuronidase activity after PEG-mediated transformation and magnetofection; bacterial contamination could have resulted in false positive results.

2.3.1 Leaf sterilisation

Approximately three-week old leaves were detached from tobacco and cotton plants. From tobacco leaves, the midrib was removed. Leaves were washed under running water for one minute and transferred to a sterile 500mL beaker in a laminar flow cabinet. Leaves were

2. Materials & Methods

washed with 250mL of 70% ethanol for 30 seconds with continuous swirling, after which the ethanol was subsequently decanted. The leaves were then washed with 250mL of 1.8% sodium hypochlorite supplemented with three drops of tween-20 for one minute with continuous swirling. The solution was decanted and the leaves were washed a total of five times with 250mL autoclaved sterile water for one minute per wash with continuous swirling, decanting between each wash. The leaves were kept in sterile water under a laminar flow until processed further.

2.3.2 Digestion of leaf strips

The sterilised leaves were transferred to a sterile plastic Petri dish and the abaxial side of the leaf was abraded with a sterile scalpel blade. They were then sliced as thinly as possible with a sterile scalpel blade and were immediately transferred into sterile Murashige and Skoog solution supplemented with sucrose (4.4g/L MS, 3% sucrose, pH 5.6) until all leaf strips were cut.

For tobacco, an optimisation was performed using two leaf digestion solutions, approximately 1g of leaf strips were transferred to 20mL filter-sterilised tobacco leaf

digestion (1.5% cellulase, 0.3% macerozyme, 0.5M mannitol, 6.5mM CaCl2 · 2H2O, 750μM

KNO3, 60μM MgSO4 · 7H2O, 50μM KH2PO4, pH 5.6), or (1% cellulase R-10, 0.25%

macerozyme R-10, 0.5M mannitol, 8mM CaCl2, 5mM MES [pH 5.7]) in a 50mL sterile plastic falcon tube. The solution was lightly rocked up and down on a rocker for three or five hours in the dark (using tubes wrapped with aluminium foil) at room temperature respectively. The latter leaf digestion solution was ultimately used for the isolation of protoplasts used in all down-stream experiments. Optimisations of these leaf digestion solutions are described in the results, section 3.3.1.

For cotton, filter sterilised cotton leaf digestion solution (1.5% cellulase, 0.3% macerozyme,

0.5M mannitol, 6.5mM CaCl2 · 2H2O, 750μM KNO3, 60μM MgSO4 · 7H2O, 50μM KH2PO4, pH 5.6) was used and leaf strips were digested for four hours.

2. Materials & Methods

Prior to filter sterilisation, digestion solutions were passed through Whatman filter paper to remove any unwanted proteases present in the cellulase R-10 and macerozyme R-10 (Yakult Pharmaceutical) products.

2.3.3 Isolation of intact protoplasts

Following digestion of leaf strips, the digestion suspension was diluted with an equal volume of W5 solution (154mM NaCl, 125mM CaCl2, 5mM KCl, 2mM MES [pH 5.7]) and incubated at room temperature for two minutes. The suspension was then passed through a sterile 50 micron stainless steel sieve to remove leaf strips, the sieve was pre-wetted with W5 solution. The sieved suspension was then divided evenly between four 10mL sterile plastic centrifuge tubes and centrifuged at 50 x g, using the minimum acceleration and deceleration parameters of the centrifuge, for five minutes at 25°C. The supernatant was discarded and isolated protoplasts were resuspended in 4mL of W5 solution per tube, following which protoplasts were consolidated into two tubes. The protoplasts were then centrifuged as before and then resuspended in 4mL of W5 solution per tube. Each tube of protoplasts were slowly and carefully layered on top of 4mL of 21% sucrose cushion in a 10mL sterile plastic centrifuge tube, being careful not to disrupt the interphase of the two solutions. Following, protoplasts were centrifuged as before. Intact protoplasts, those that were floating at the interphase, were transferred to a 10mL sterile plastic centrifuge tube via slow and careful pipetting using 1mL sterile pipette tips with the pointed tip cut back. Protoplasts were resuspended in 4mL of W5 solution and were stored at 4°C in the dark for 30 minutes prior to down-stream experiments.

A 10μL aliquot of protoplasts was used for quantification using a haemocytometer (BLAUBRAND 0.0025mm2 Improved Neubauer counting chamber).

2.4 Plant transformation

Three methods of plant transformation were used in the research. Both magnetofection and PEG-mediated transformation were performed on tobacco protoplasts, magnetofection as a

2. Materials & Methods

novel method of introducing DNA into protoplasts, and PEG-mediated transformation as a control to gauge the effectiveness of magnetofection. As a guide to ensure the plasmid DNA was intact and will indeed express β-glucuronidase when introduced into plant cells, particle bombardment of pCAMBIA1303 into tobacco leaf discs was performed and the expression of β-glucuronidase was assessed after 48 hours via histochemical staining. The details of the transformation methods are described below.

2.4.1 Magnetofectin assembly and Magnetofection

Superparamagnetic iron oxide nanoparticles (100nm diameter) coated with PEI (PolyMAG- 1000; Chemicell, Berlin, Germany) were supplied at a concentration of 1µg/µL. In total, six magnetofectin preparations were constructed, the magnetofectins were prepared in three MNP/DNA ratios of 1:5, 1:10 and 1:20 (w/w), each with two different amounts of MNPs, 0.5µg and 1.0µg, corresponding to densities of 1.0µg MNPs/mL and 2.0µg MNPs/mL post transformation, which were mixed with pCAMBIA1303 at a concentration of approximately 1µg/µL. Magnetofectins were then diluted up to 150μL with sterile water and the MNP/DNA mix was incubated for 30 minutes at room temperature to allow for magnetofectin binding via electrostatic interactions. Assembled magnetofectins were used immediately for transfection.

Isolated intact protoplasts stored at 4°C for 30 minutes were resuspended in W1 solution at a concentration of 2.86x105 protoplasts/mL. An aliquot of 350µL of intact protoplasts in W1 solution were used per magnetofection event. Aliquots were transferred to glass vials (1.5cm diameter x 5.0cm height) which were placed directly on top of the 24 well-format magnetic plate (MagnetoFACTOR-24 plate, Chemicell, Berlin, Germany). Assembled magnetofectins were pipetted dropwise onto the protoplast cell suspension, distributing magnetofectins as uniformly as possible. After magnetofectins were added, protoplasts were in a final volume of 500µL at a concentration of 2x105 cells/mL. The transfected protoplasts were incubated in the dark at room temperature for 30 minutes on the magnetic plate, after which the magnetic plate was removed. The glass vials, with protoplasts and magnetofectins were kept in the dark in a culture room at 23.5°C for 48 hours prior to β-glucuronidase expression analysis.

2. Materials & Methods

2.4.2 Polyethylene glycol mediated transformation

PEG-mediated transformation of protoplasts was carried out as described by Yoo et al. (2007) with the following adaptations. The number of protoplasts transformed per event was increased by five times the stated amount, to a total of 1x105 protoplasts per event. The amount of DNA, and volumes of solutions used were increased correspondingly. Protoplasts were incubated with PEG solution (40% (w/vol) PEG4000 Fluka, 0.2M mannitol,

100mM CaCl2) for 10 or 15 minutes. Post transformation, protoplasts were resuspended in 0.5mL of W1 solution in glass vials. The glass vials, with protoplasts were kept in the dark in a culture room at 23.5°C for 48 hours prior to β-glucuronidase expression analysis.

2.4.3 Biolistic particle bombardment

Coating of DNA on microcarriers was similar to the method described by (Fosu-Nyarko, 2005). Microcarriers were prepared by resuspending 3mg tungsten micro-particles of 0.7µm diameter (Tungsten M-10, Biorad) in 50μL of 50% (v/v) glycerol. The microcarriers were vigorously vortexed during which the following were added in quick succession: 10μL of

pCAMBIA1303 (1μg/μL), 50µL of 2.5M CaCl2 · 2H2O and, 20µL of 0.1M spermidine. Vortexing was carried out for an additional 3 minutes then the mixture was allowed to settle for 15 minutes. The micro-particles were then centrifuged for 5 seconds in a micro centrifuge. Excess supernatant was discarded until the final volume of the solution was approximately 50µL. A helium-driven particle inflow gun (CSIRO, Australia) was used for particle bombardment with a pressure of 2000kPa. For each shot, 10μL aliquots of the micro-particle suspension were used, these aliquots were loaded onto a plastic mesh. Tobacco leaf discs of 2.5cm diameter were placed on a Petri dish containing CCLM media at a distance of 14cm away from the filter assembly at the bottom of the vacuum chamber. A protective mesh with pore size 0.25mm2 was placed 2cm above the leaf disc. The vacuum chamber was sealed and a negative pressure of 50kPa was applied. Tobacco leaf discs were bombarded once, twice or three times. Subsequently leaf discs were removed from the assembly, the Petri dishes were sealed with parafilm and placed in a culture room with a 16 hour light/8 hour dark photoperiod at 23.5°C for 48 hours prior to β-glucuronidase expression analysis. 29

2. Materials & Methods

2.5 Assessment of protoplast viability

FDA staining was used to assess the viability of isolated and transfected protoplasts 48 hours post transformation. Protoplasts exhibiting fluorescein induced fluorochromasia are deemed viable.

2.5.1 Fluorescein diacetate staining of protoplasts

A 5mg/mL stock of FDA (Sigma-Aldrich) was first prepared in acetone and a working solution was then prepared by diluting 20mL of the stock up to 1mL with 0.45M mannitol. For the protoplast staining, an equal volume of protoplast suspension was mixed with the working solution as a single drop on a glass microscope slide. The mixture was incubated for five minutes to allow for fluorescein accumulation within cells, and to allow protoplasts to settle by gravity.

FDA-stained protoplasts were viewed via fluorescence microscopy using an Olympus BX51 photomicroscope, 24, and 48 hours post-transformation. FDA-stained protoplasts were visualised with an excitation/emission wavelength of 460-490/520nm at which they appeared green. Chloroplast autofluorescence of the protoplast suspension was visualised with an excitation/emission wavelength of 510-550/590nm, which appeared red.

2.5.2 Image analysis

Using the DPmanager software, FDA-stained and chloroplast autofluorescence images were merged. The merged image was assessed in the Image J software package by manually counting cells of the different colours; dead protoplasts appeared red in the merged image, while all other protoplasts on the spectrum from orange to green were considered viable. In total, three fields of view at 100 x magnification were counted per treatment to determine the mean viability.

2. Materials & Methods

Viability of protoplasts was determined as follows:

Number of dead protoplasts % = 100 100 Total number of protoplasts 𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑙𝑙𝑖𝑖𝑖𝑖𝑖𝑖 − ∗

2.6 Assays for β-glucuronidase activity

Magnetofected and PEG-mediated transformed tobacco protoplasts were tested for β- glucuronidase activity via histochemical staining and fluorescence assay methods 48 hours post transformation. These methods were also applied to Sicot 71 and 35SGUS4 cotton protoplasts immediately post isolation.

Both freshly-removed and particle bombarded tobacco leaves were tested for β- glucuronidase activity via leaf histochemical stain immediately, or 48 hours post transformation respectively. These methods were also applied to Sicot 71 and 35SGUS4 cotton leaves immediately post isolation.

2.6.1 GUS assay of transformed plant protoplasts - histochemical staining

For histochemical staining of 35SGUS4 and Sicot 71cotton protoplast, an optimisation was performed using two staining solutions. Protoplasts were allowed to settle by gravity for 30 minutes post isolation. Taking care not to disturb protoplasts, as much W1 solution as possible was carefully removed from the protoplast suspension by pipetting, and 1x105 protoplasts were resuspended in 500µL of ‘GUS histochemical staining solution #1’ (2mM X- gluc, 4.3g/L MS, 0.3M mannitol, 10mM EDTA, 0.5mM K-ferrocyanide, 0.5mM K-ferricyanide, 50mM K-phosphate buffer [pH6.5]), or ‘GUS histochemical staining solution #2’ (2mM X- gluc, 0.4M mannitol, 124mM CaCl2, 50mM Tris HCL [pH 7.0]). Protoplasts were then incubated at 37°C in the dark for 24 hours. A droplet of protoplasts was mounted for microscopy and visualised using an Olympus IMT-2 inverted microscope 24 hours post staining. A protoplast testing positive for β-glucuronidase activity appears blue in the visible light spectrum.

2. Materials & Methods

For magnetofected or PEG-mediated transformed protoplasts, histochemical staining was performed as previously described with the following changes: Histochemical staining was performed 48 hours post-transformation, and protoplasts were only stained with ‘GUS histochemical staining solution #2’.

2.6.2 GUS assay of transformed plant protoplasts - fluorescence assay

When Β-glucuronidase activity was determined in magnetofected or PEG-mediated transformed protoplasts via the 4-methylumbelliferone b-D-galactopyranoside (MUG) fluorescence assay, the method described by Yoo et al. (2007) was used with adaptations appropriate for a 96-well format. To do this, magnetofected, PEG-treated or control protoplasts were centrifuged at 50g for 5 min using a centrifuge with a swinging rotor. The protoplasts were resuspended in 50μL of freshly prepared protoplast lysis buffer (1mM DTT,

2mM Na2EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 25mM Tris-phosphate [pH 7.8]). Protoplast were then vigorously vortexed for 10 seconds. The protoplast lysis buffer solution

was then added to 500μL of MUG substrate mix (1mM MUG, 2mM MgCl2, 10mM Tris-HCl [pH 8.0]) pre-warmed to 37°C in a 1 .5mL microcentrifuge tube, and the solution was then incubated at 37°C for 48 hours. Periodically, the reaction was stopped by adding 27.5μL aliquots of the protoplast lysis buffer/MUG substrate mix to 222.5μL of Stop buffer (0.2M

Na2CO3) in wells of a 96-well plate. The time intervals chosen were as follows: 5, 30, 60, 90, 120, 180 minutes, 24, and 48 hours after starting the assay. Fluorescence was measured with a 96-well format Titertek Fluoroskan® fluorometer (Titertek, Germany) at an excitation/emission wavelength of 355/480nm.

2.6.2.1 4-Methylumbelliferone standard curve preparation

A 1mM stock of 4-Methylumbelliferone (4-MU; Sigma-Aldrich) was prepared by first dissolving 1.76mg of 4-MU in 100μL Dimethyl sulfoxide (Ajax Finechem) and diluting up to 10mL with milliQ water. A serial dilution of 4-MU was prepared from this stock, each time diluting with milliQ water such that the following sub-stocks were prepared: 50μM, 25μM, 10μM, 7.5 μM, 5μM, 2.5μM, 1nM, 750nM, 500nM, 250nM, and 100nM. A 25μL aliquot of

2. Materials & Methods

these sub-stocks were placed into wells of a 96-well plate, in duplicate, and were diluted up to 250μL with 0.2M Stop buffer, such that the final concentration of 4-MU was: 5μM, 2.5μM, 1μM, 750nM, 500nM, 250nM, 100nM, 75nM, 50nM, 25nM, and 10nM. A blank consisting of 225μL of Stop buffer and 25 μL of milliQ water was also prepared in duplicate. Fluorescence intensity (FI) of the dilution series was measured at an excitation/emission wavelength of 355/480nm with a Titertek Fluoroskan. The standard curve was produced by subtracting the average FI value of the blank from the average FI value of the dilution series and plotting pmoles 4-MU vs FI using Microsoft Excel.

2.6.3 GUS histochemical staining of leaves

Freshly removed tobacco, Sicot 71, and 35SGUS4 cotton leaves were sliced into 2-3 pieces and covered with 2mL of leaf GUS histochemical staining solution (1mM X-gluc, 50mM

Na2HPO4, 0.1% (v/v) Triton X-100) in a 10mL plastic tube. The set-up was incubated at 37°C in the dark for 24 hours. Leaves were then transferred to 8mL of 70% ethanol and incubated at 37°C on a 220rpm shaker for one week for chloroplast decolouring. For tobacco leaves it was necessary to change the 70% ethanol twice over this period to attain complete decolouring.

Β-glucuronidase activity was tested for in particle-bombarded tobacco leaf discs 48 hours post transformation. The leaf discs were covered with 2mL GUS leaf histochemical staining solution and were incubated at 37°C in the dark for 48 hours. The same chloroplast decolouring process was applied as described above. Non-bombarded tobacco leaf discs and 35SGUS4 cotton leaf discs were also histochemical stained, being used as negative and positive controls for β-glucuronidase activity respectively.

2.7 Magnetofectin gel-retardation electrophoresis

To determine the optimal binding ratio of MNPs to DNA, a series of magnetofectin complexes were assembled as described previously in section 2.4.1. Each sample contained 2μg of linearised pCAMBIA1303 in DNA/MNP mass ratios of: 1:1, 1:0.5, 1:0.3, 1:0.25, 1:0.2,

2. Materials & Methods

1:0.125, 1:0.1, 1:0.05, 1:0.025, and 1:0.016, magnetofectins were assembled in a total of 25µL of nuclease free water. Assembled magnetofectins and control linear pCAMBIA1303 were mixed with 7.5µL of cresol red and loaded onto an agarose gel for 2 hours.

The pCAMBIA1303 binary vector was linearised with HindIII (New England Biolabs) as shown in Table 2.2. The linear product was purified with the Wizard® SV Gel & PCR Clean-Up System according to the manufacturer’s protocol.

Table 2.2: Reaction set-up for linearisation of the pCAMBIA1303 binary vector. Components Restriction digest (μL)

Restriction enzyme:

HindIII (20,000U/mL) 8

10x NEBuffer 2.1 6 Plasmid DNA x (50μg)

Nuclease free water to 60

* x is variable on the concentration of DNA used.

3. Results

Chapter 3: Results

3.1 Introduction

The results of a series of experiments to determine whether a novel transfection technique, magnetofection, could be applied to plant cells, are presented. The research involved checking the identity of the binary vector pCAMBIA1303 containing the GUS reporter gene, and ensuring that it was expressed in plant cells. As a positive control, transgenic cotton plants expressing GUS were obtained to check that GUS could be detected. This was followed by developing protoplast systems for transfection, and assaying expression of the reporter gene after transfection using histochemical and fluorescence assays. Having developed the assay systems, the potential of magnetofection for protoplast transfection was examined and its effectiveness compared to a conventional transfection technique using PEG-mediated transformation.

3.1.1 Confirmation of the identity of the pCAMBIA1303 binary vector

It was important to confirm the identity of the pCAMBIA1303 binary vector before proceeding with any transformation experiments. To show that the pCAMBIA1303 binary vector used in all following experiments contained a functional GUS gene and could be expressed in plant cells to produce β-glucuronidase, the following tests were done. First, the identity of pCAMBIA1303 was confirmed by digestion of the plasmid with restriction endonucleases. The products were separated by electrophoresis using agarose gel, and the resulting bands where compared to those expected by in silico digestion. Second, it was shown that pCAMBIA1303 can be functionally expressed in tobacco cells by histochemically staining of particle-bombarded tobacco leaves for β-glucuronidase activity.

The identity of the pCAMBIA1303 binary vector was assessed by digestion with the restriction endonucleases EcoRI and BsrGI (Figure 3.1A). In silico digestion of pCAMBIA1303 with EcoRI and BsrGI resulted in three fragments of size 9985 bp, 1950 bp, and 426 bp. The 1950 bp fragment contained the CaMV 35S promoter and the 5’ end of the GUS/GFP gene fusion, whereas the 426bp fragment contained a downstream fragment of the GUS/GFP gene fusion. The results of restriction digestion showed that all and only the expected

3. Results

fragments were present, confirming the identity of the pCAMBIA1303 binary vector (Figure 3.1A).

Additionally, the binary vector pCAMBIA1303 was linearised by digestion with the restriction endonuclease HindIII that cuts at a single position on the vector. The results show that linearisation of the pCAMBIA1303 binary vector was successful, with the generation of a single band corresponding to the size of the pCAMBIA1303 binary vector, 12,362 bp (Figure 3.1B). The linear fragment of pCAMBIA1303 appears larger than the circular plasmid as the supercoiled circular plasmid migrates faster in the agarose gel in virtue to its condensed state.

A M 1 2 B M 1 2 12,362 10,000 10,000 9,985

2,000 1,950

500 426

Figure 3.1: (A) Restriction digestion analysis of the pCAMBIA1303 binary vector with EcoRI and BsrGI. Lane M: Axygen 1kb DNA ladder. Lane 1: Undigested pCAMBIA1303. Lane 2: pCAMBIA1303 digested with EcoRI and BsrGI. (B) Restriction digestion analysis of the pCAMBIA1303 binary vector with HindIII. Lane M: Axygen 1kb DNA ladder. Lane 1: Undigested pCAMBIA1303. Lane 2: pCAMBIA1303 digested with HindIII. Numbers on the right of the gels represent expected band sizes from the in silico digestion.

3. Results

3.1.2 Functional expression of the pCAMIBA1303 binary vector

To determine if the GUS gene contained in pCAMBIA1303 could be transiently expressed in tobacco cells, tobacco leaf discs were bombarded using a particle inflow gun with 0.6mg of pCAMBIA1303-coated tungsten microcarriers per shot. The bombarded tissue was then histochemically stained for β-glucuronidase activity after allowing 48 hours for transient expression. This experiment was replicated twice, all observations were consistent between the replicates. A 35SGUS4 cotton leaf disc was used as a positive control, to show that cells expressing GUS are stained blue by the histochemical stain (Figure 3.2A). The results showed that β-glucuronidase activity was only detectable in tobacco leaf discs which had been bombarded with microcarriers coated with pCAMBIA1303 (Figure 3.2D-F). Tobacco leaf discs which were untreated or bombarded without pCAMBIA1303 showed no evidence of β-glucuronidase activity (Figure 3.2B-C). Bombardment of tobacco leaf discs with one or two shots of 0.6mg pCAMBIA1303-coated tungsten microcarriers resulted in an average of 1.5 and 3.5 blue spots per leaf disc respectively, showing that multiple bombardments resulted in additional transformation events (Figure 3.2E-F). It is clear from the results that the GUS gene encoded by pCAMBIA1303 can be transiently expressed in tobacco cells.

3. Results

A B

C D

E F

Figure 3.2: Transient GUS expression in tobacco leaf discs bombarded once or twice with 0.6mg of pCAMBIA1303 coated tungsten microcarriers. 48 hours post transformation, bombarded leaf discs were histochemically stained with X-gluc, and subsequently destained with 70% ethanol. (A) Un-bombarded, stained 35SGUS4 cotton (positive control). (B) Un- bombarded, stained tobacco (negative control). (C) Stained tobacco bombarded once without. pCAMBIA1303. (D) Stained tobacco bombarded once with pCAMBIA1303. (E) Stained tobacco bombarded twice with pCAMBIA1303. (F) 2.5x magnification of the image in (E).

3. Results

3.2 Optimisation of GUS assays to detect β-glucuronidase activity in protoplasts

To confirm the functional expression of GUS in protoplasts, two different assays, an X-gluc histochemical stain and a 4-MU fluorescence assay, were optimised to detect of β- glucuronidase activity in isolated cotton protoplasts which either express GUS (35SGUS4) or do not (Sicot 71). For histochemical staining, two different stains were tested, with the procedure providing the clearest evidence of β-glucuronidase activity being used for further experiments. In order to perform the 4-MU fluorescence assay, the fluorometer was first calibrated by generating a standard curve of 4-MU fluorescence, after which the 4-MU fluorescence assay was used to quantify β-glucuronidase activity.

3.2.1 Optimisation of the protoplast histochemical staining procedure

Two different protoplast histochemical staining procedures described in the literature, outlined in section 2.6.1, were tested for their effectiveness in indicating β-glucuronidase activity in cotton protoplasts, that were either known to have β-glucuronidase activity (35SGUS4) or that did not (Sicot 71).

The results showed that both histochemical stains were positive for β-glucuronidase activity in GUS-transgenic protoplasts, as evidenced by the blue staining of the protoplasts (Figure 3.3B,F). Blue stained protoplasts were not observed in non-GUS transgenic protoplasts (Figure 3.3A,E). The differentiation between protoplasts with or without β-glucuronidase activity was much clearer in ‘GUS histochemical staining solution #2’, both in protoplasts under brightfield and in the histochemical staining solution (Figure 3.3), therefore method #2 was used in all further experiments.

3. Results

A B

40µm 40µm C D

E F

80µm 80µm G H

Figure 3.3: A comparison of the two X-gluc histochemical staining procedures used for cotton protoplasts to indicate β-glucuronidase activity. Staining was done for 24 hours. (A-D) ‘GUS histochemical staining solution #1’. (E-H) ‘GUS histochemical staining solution #2’. (A,E) Sicot 71 protoplasts. (B,F) 35SGUS4 protoplasts. (C,G) Histochemical staining solution of stained Sicot 71 protoplasts. (D,H) Histochemical staining solution of stained 35SGUS4 protoplasts.

3. Results

3.2.2 Calibration of the Titertek® Fluoroskan fluorimeter by generation of a 4- MU standard curve

Before conducting a 4-MU fluorescence assay to quantify β-glucuronidase activity in protoplasts, it was necessary to calibrate the fluorimeter so that any measured fluorescence values could be attributed to a known concentration of the fluorophore 4-MU, the fluorescent product from enzymatic cleavage of MUG by β-glucuronidase. Serial dilutions of 4-MU were set up and their corresponding Fluorescence Intensity (FI) values were determined as described in section 2.6.2.1. A standard curve was produced by plotting the measured FI values against the known amount of 4-MU in the samples. The results show that the FI of 4-MU is linear between 0 and 1250pmoles (Figure 3.4). With this standard curve, FI values of any appropriately blanked solution can be attributed to an amount of 4- MU derived from the linear model, y = 0.1713x, where y = FI and x = amount of 4-MU in pmoles. The smaller graph embedded below shows a magnified view of the standard curve for 4-MU amounts between 0 – 200pmole (Figure 3.4).

4-MU (pmoles)

Figure 3.4: Standard curve of 4-MU fluorescence measured at excitation/emission wavelengths of 355/480nm. Amounts ranging from 0-200pmole of 4-MU are embedded in the graph. Error bars represent the standard deviation of FI means of two replicates.

3. Results

3.2.3 Confirmation for the quantification of β-glucuronidase activity via 4-MU assay in GUS transgenic protoplasts

To show that β-glucuronidase activity was detectable in protoplasts via 4-MU fluorescence, a time-course 4-MU fluorescence assay was performed on protoplast lysate derived from cotton protoplasts that had β-glucuronidase activity (35SGUS4), and those that do not (Sicot 71). The results showed that β-glucuronidase activity was detectable in 35SGUS4 protoplast lysate and was not detectable in the Sicot 71 protoplast lysate, the difference in β- glucuronidase activity between the two cultivars was statistically significant at 180 minutes, p-value < 0.05 (Figure 3.5). Time points beyond 180 minutes have been omitted as the measured FI of 35SGUS4 protoplast lysate was beyond the range of the 4-MU standard curve.

Figure 3.5: β-glucuronidase activity of GUS transgenic (35SGUS4) or non-transgenic (Sicot 71) cotton protoplasts at regular time intervals. Error bars represent the standard deviation of means of β-glucuronidase activity for two replicates. Statistical significance was assessed with an α = 0.05 for a two-way t-test between the β-glucuronidase activity of transgenic and non-transgenic protoplast lysate at 180 minutes.

3. Results

3.3 Tobacco protoplast isolation and detection of transient GUS expression

Because successful transfection of protoplasts depends on the quality of the protoplasts isolated, it was important to optimise the isolation of tobacco protoplasts so that enough high-quality protoplasts could be generated routinely.

Subsequently, the GUS assays which had been shown to detect β-glucuronidase activity in GUS-transgenic protoplasts (Figure 3.3, 3.5), were used to assess β-glucuronidase activity in transient GUS-expressing protoplasts. Transient GUS-expressing tobacco protoplasts were obtained using PEG-mediated transformation.

3.3.1 Optimisation of tobacco protoplast isolation

Two different enzyme digestion solutions, described in the literature were followed, with some optimisations, to develop a protoplast isolation methodology that yielded enough high-quality tobacco protoplasts. The composition of each enzyme digestion solution is described in section 2.3.2. Using the first of the two methods, based on the protocol of Nicolia et al. (2015), tobacco protoplasts were obtained successfully. Optimisation of the protocol was undertaken first by addition of the enzyme pectolyase (which catalyzes the eliminative cleavage of a-(1- 4)-D galacturonan methyl ester to give oligosaccharides with 4- deoxy-6-O-methyl-a-D-galact-4-enuronosyl groups at their non-reducing ends - it contain two types of pectinase, endopolygalacturonase, endo-pectin lyase, Sigma Aldrich). This enzyme preparation is often used in plant protoplast isolation protocols. From a visual inspection of protoplasts by brightfield microscopy it was determined that the addition of pectolyase resulted in the release of more protoplasts, but that this was accompanied by reduction in their quality, as evident by breaking and distortions of the spherical shape of some isolated protoplasts (Table 3.1).

A second protoplast isolation protocol was tried, as described by Kosicki et al. (2018). The quality of protoplasts derived was much improved, as determined by brightfield microscopy. An optimisation of the time that leaf strips were incubated in the enzyme digestion solution

3. Results was undertaken, showing that five hours of incubation resulted in release of more protoplasts without noticeable decline in quality compared to four hours incubation (Table 3.1). This protoplast isolation protocol was used for all further experiments.

3. Results

Table 3.1: A summary of the different enzyme digestion protocols used for optimising the isolation of tobacco protoplasts.

Method

1 2 3 4

Isolation protocol (Nicolia et al., 2015) (Nicolia et al., 2015) (Kosicki, Tomberg et al. 2018) (Kosicki, Tomberg et al. 2018) (modified) (modified)

Cellulase (%) 1.5 1.5 1 1

Macerozyme (%) 0.3 0.3 0.25 0.25

Pectolyase (%) -* 0.5* - -

Mannitol (M) 0.5 0.5 0.5 0.5

CaCl2 (mM) 6.5 6.5 8 8 pH 5.6 5.6 5.7 5.7

Digestion time (hours) 3 3 4** 5**

Appearance of tobacco protoplasts post isolation.

* A change in % pectolyase between otherwise identical protocols. ** A change in digestion time between otherwise identical protocols.

3. Results 3.3.2 Confirmation for the detection of β-glucuronidase activity in transient GUS-expressing protoplasts

To show that the two developed GUS assays, histochemical staining and 4-MU fluorescence, could be used to detect β-glucuronidase activity in transient GUS- expressing tobacco protoplasts, PEG-mediated transformation was performed on isolated tobacco protoplasts, followed by GUS assays 48 hours post transformation. The results show that the 4-MU fluorescence assay could be used to quantify β- glucuronidase activity in transiently expressing tobacco protoplasts, indicated by rising β-glucuronidase activity in only the protoplasts treated with PEG and pCAMBIA1303 (Figure 3.6A). Additionally, the 4-MU fluorescence assay showed that tobacco protoplasts transformed with 40% PEG solution for 10 minutes yielded greater levels of β-glucuronidase activity compared to those treated for 15 minutes, and that this difference was statistically significant at 24 hours, p-value < 0.05 (Figure 3.6A). Consequently, a 10-minute PEG-treatment was used for all subsequent PEG- transformations. Β-glucuronidase activity was not detected in untransformed protoplasts or protoplasts treated with DNA but without PEG (Figure 3.6A).

The β-glucuronidase activity in transient GUS expressing tobacco protoplasts was much lower than in the stable GUS expressing 35SGUS4 cotton protoplasts, consequently, it was necessary to extend the 4-MU assay out to at least 24 hours to be confident that there were real differences in β-glucuronidase activity.

The histochemical staining procedure showed no indication of β-glucuronidase activity in any PEG-mediated transformed protoplasts, evidenced by the lack of protoplasts stained blue (Figure 3.6B), despite that β-glucuronidase activity was quantifiable via 4- MU assay in the same protoplasts. This difference in results is probably because of low levels of expression, and the relative insensitivity of the histochemical staining procedure compared to the fluorescent assay. As a result, the 4-MU fluorescence assay was used as the primary tool to confirm β-glucuronidase activity in all magnetofection experiments.

3. Results

B i ii

Figure 3.6: (A) β-glucuronidase activity of tobacco protoplasts transformed with pCAMBIA1303 and 40% PEG solution for 10 or 15 minutes. Samples were blanked against mock PEG-mediated transformed tobacco protoplasts. Error bars represent the standard deviation of means of β-glucuronidase activity for two replicates. Statistical significance was assessed with an α = 0.05 for a two-way t-test between the β- glucuronidase activity of PEG treatments at 24 hours. (B) Assay for transient β- glucuronidase activity in tobacco protoplasts via histochemical staining. (i) Mock PEG- mediated transformed protoplasts. (ii) PEG-mediated transformed protoplasts.

3.4 Optimisation of the conditions of magnetofection

Because magnetofection has not been used to transform protoplasts before, it was important to optimise conditions that may be appropriate for successful magnetofection. These conditions included, the ratio of MNP:DNA to use in

3. Results magnetofectin assembly, and the viability of protoplasts when magnetofected with various densities of MNPs.

First, the binding capacity of DNA to MNPs was determined, both to indicate appropriate MNP:DNA ratios for magnetofectin assembly and to provide evidence to support why particular magnetofection treatments resulted in successful or unsuccessful transient β-glucuronidase activity. Second, the viability of protoplasts magnetofected with 1.0, 2.0 and 4.0μg MNP/mL/105 cells was determined by FDA staining.

3.4.1 Determination of the binding capacity of DNA to MNPs

To determine the binding capacity of DNA to MNPs, magnetofectins with varying mass ratios of MNPs to DNA were established as described in section 2.7. The results of the gel-retardation electrophoresis assays showed that at MNP:DNA mass ratios of 1:1 – 1:10 almost all available DNA was complexed with MNPs, indicated by both the absence of a DNA band at the expected size of pCAMBIA1303, 12,362 bp, and the presence of DNA in the wells of most of these lanes (Figure 3.7 Lanes 2-9). At MNP:DNA ratios of 1:20 – 1:60, the presence of bands at the expected size of the linearised pCAMBIA1303, shown by the control, indicated that some DNA was unbound to MNPs and was free to migrate through the gel as normal (Figure 3.7 Lanes 1, 10-12). Despite that the same amount of DNA was used for each preparation, DNA was hardly visible in the lanes that contained the most MNPs (Figure 3.7 Lanes 2-4). It is possible that the MNPs either blocked the luminescence of DNA on the gel, or prevented the binding of SYBR safe to DNA, resulting in a decreased luminescence intensity of DNA when more MNPs were present. This experiment was repeated twice, and observations were consistent in both the replicates.

3. Results From this result, it was expected that an MNP:DNA ratio of 1:10 would have the greatest transformation potential as it would enable the most DNA to be introduced to magnetofected cells. Magnetofectins with MNP:DNA ratios of 1:5, 1:10 and 1:20 were chosen for further experiments as a result.

M 1 2 3 4 5 6 7 8 9 10 11 12

12,362 10,000

1:1 1:2 1:3 1:4 1:5 1:6 1:8 1:10 1:20 1:40 1:60 MNP:DNA mass ratios Figure 3.7: Gel-retardation electrophoresis of magnetofectins with varying mass ratios of MNP:DNA. Each lane contained 2μg of linearised pCAMBIA1303. Lane M: Axygen 1kb DNA ladder. Lane 1: Linearised pCAMBIA1303. Lanes 2-12: Magnetofectin complexes with MNP:DNA mass ratios of 1:1, 1:2, 1:3, 1:4, 1:5, 1:8, 1:10, 1:20, 1:40, 1:60 respectively. The number on the right of the image represents the band size of the linearised pCAMBIA1303 binary vector.

3.4.2 Viability of control, magnetofected and PEG-mediated transformed protoplasts

The viability of protoplasts magnetofected with three different densities of MNPs, 1.0, 2.0, and 4.0μg MNP/mL/105 cells, was assessed by FDA staining 48 hours post transformation. It was found that as the density of MNPs increased, the viability of protoplasts decreased. The viability of protoplasts magnetofected with 1.0μg MNP/mL (80%) was significantly different from those with 2.0μg MNP/mL (38%), p-value < 0.05, and those magnetofected with 2.0μg MNP/mL was significantly different from those with 4.0μg MNP/mL (19%), p-value < 0.05 (Figure 3.8A). To compare the viability of magnetofected protoplasts with that of conventional protoplast transformation methods, the viability of PEG-mediated transformed protoplasts was assessed. It was found that the viability of protoplasts magnetofected with 1.0μg MNP/mL (80%) was not significantly different from PEG-mediated transformed protoplasts (75%), p-value < 0.05 (Figure 3.8A). As a control, the same number of protoplasts (105) used for PEG and magnetofection treatments was kept in W1 solution, and the viability was 49

3. Results determined as for the other treatments. The viability of the control protoplasts (84%), was not significantly different from the 1.0μg MNP/mL treatment (80%), p-value < 0.05, while the viability of control protoplasts was significantly different from PEG treatment (75%), p-value > 0.05 (Figure 3.8A).

Representative images of FDA stained protoplasts which were used to determine viability are shown, and these demonstrate clearly that protoplasts magnetofected with an increasing density of MNPs were less viable (Figure 3.8B). As a consequence of these results, magnetofection treatments with MNP densities of 1.0 and 2.0μg MNP/mL were chosen for subsequent magnetofection experiments.

3. Results

A B i

ii iii

iv v

Figure 3.8: (A) A comparison of the effects of different transformation treatments on the viability of tobacco protoplasts 48 hours post transformation as determined by FDA staining. Control: Untransformed protoplasts. PEG: Protoplasts transformed in 40% PEG solution for 10 minutes. 1.0μg MNP/mL – 4.0μg MNP/mL: Protoplasts magnetofected with 1.0μg MNP/mL/105 cells, 2.0μg MNP/mL/105 cells, and 4.0μg MNP/mL/105 cells respectively. Error bars represent the standard deviation of % viability as determined by n = 3 replicates. Lower case letters above the bars represent statistical significance based on α = 0.05 for two-sample t-tests. (B) Merged images showing FDA induced fluorochromasia and chloroplast autofluorescence of tobacco protoplasts that were: (i) Untreated. (ii) PEG-mediated transformed. (iii) Magnetofected with 1.0μg MNP/mL/105 cells. (iv) Magnetofected with 2.0μg MNP/mL/105 cells. (v) Magnetofected with 4.0μg MNP/mL/105 cells.

3. Results 3.5 Optimisation of magnetofection on tobacco protoplasts

Β-glucuronidase activity was measured in magnetofected tobacco protoplasts and the conditions of magnetofection were sequentially optimised. The initial starting conditions for magnetofection of tobacco protoplasts were based on the previous assessments of DNA binding capacity to MNPs and protoplast viability in section 3.4. After each magnetofection experiment, alterations of the previous methodology were made in an attempt to increase the level of transient GUS expression in the following experiment.

3.5.1 Magnetofection of tobacco protoplasts

To determine if magnetofection of tobacco protoplasts with pCAMBIA1303 resulted in transient GUS expression, various magnetofection treatments were conducted followed by assessment of β-glucuronidase activity via 4-MU fluorescence assay 48 hours post transformation. The only difference between magnetofection treatments were the conditions of magnetofectin assembly. In all, six different magnetofection treatments of protoplasts were used: Three different MNP:DNA ratios of magnetofectins (1:5, 1:10 and 1:20), each with two different densities of MNPs, 1.0μg MNP/mL and 2.0μg MNP/mL. For each individual treatment, the MNPs/DNA were incubated in 33.3µL of water during magnetofectin assembly. PEG-mediated transformed protoplasts were used as a positive control for transient levels of β- glucuronidase expression, the protoplasts used for PEG and magnetofection were isolated from the same tissue. The results showed that there was no significant difference between the mean β-glucuronidase activity of all magnetofection treatments and that of untransformed protoplasts, p-value > 0.05, indicating that magnetofection did not induce transient GUS expression.

3. Results

Figure 3.9: β-glucuronidase activity in tobacco protoplasts transformed via magnetofection with three different ratios of MNP:DNA, 1:5, 1:10, and 1:20, each with two different densities of MNPs, 0.5µg MNP/mL or 1.0μg MNP/mL. Each treatment was performed on 10,000 protoplasts. Magnetofectins were assembled in 33.3µL of water per treatment. Error bars represent the standard deviation of means of β- glucuronidase activity for two replicates. Statistical significance was assessed with an α = 0.05 for an ANOVA test between all magnetofection groups for β-glucuronidase activity at 48 hours.

3.5.2 The effect of assembling magnetofectins in a larger volume of water on magnetofection

During magnetofectin assembly, it was observed that at MNP:DNA ratios of 1:20, agglutination of magnetofectin complexes could be seen with the naked eye. It was suspected that clumping of magnetofectins would hinder successful transformation, therefore, another magnetofection trial was established in which the volume of water that magnetofectins were assembled in was increased to 150μL per treatment, to reduce possible magnetofectin agglutination. All other aspects of the methodology were unchanged.

The results of the 4-MU fluorescence assay show that the magnetofection treatment where magnetofectins were assembled with a 1:10 MNP:DNA ratio at 1.0μg MNP/mL with 150μL of water did have β-glucuronidase activity, and that this activity was

3. Results significantly different from when the same conditions were used but with magnetofectins assembled in 33.3μL of water at 48 hours, p-value < 0.05 (Figure 3.10). None of the other treatments showed any indication of transient GUS expression, as no significant difference between the mean β-glucuronidase activity of these treatments were found compared to the untransformed control, p-value > 0.05.Results of magnetofection treatments performed where magnetofectins were assembled with 1.0μg of MNP have been left out to reduce visual clutter on the graph, however, none of those treatments showed any significant β-glucuronidase activity compared to the untransformed control, p-value > 0.05.

Figure 3.10: β-glucuronidase activity in tobacco protoplasts transformed via magnetofection with three different ratios of MNP:DNA, 1:5, 1:10, and 1:20, with a MNP density of 1.0µg MNP/mL. Each treatment was performed on 10,000 protoplasts. Magnetofectins were either assembled with 33.3µL or 150µL of water per treatment. Error bars represent the standard deviation of means of β-glucuronidase activity for two replicates. Statistical significance was assessed with an α = 0.05 for a two-way t- test between the ‘MNP:DNA 1:10, 1.0µg MNP/mL, 33μL’ and ‘MNP:DNA 1:10, 0.5µg MNP/mL, 150μL' treatments for β-glucuronidase activity at 48 hours.

3.5.3 The effect of centrifugation of protoplasts before magnetofection

In an attempt to further optimise magnetofection, protoplasts were centrifuged prior to magnetofection. The purpose was to increase the number of protoplasts that were available for interaction with magnetofectins by ensuring all protoplasts sedimented to the bottom of the transfection container. Protoplasts centrifuged before

3. Results magnetofection and treated with 1.0μg MNP/mL with ratios of 1:5 and 1:10 MNP:DNA showed significantly different β-glucuronidase activity compared to untreated controls, p-value < 0.05, and were not significantly different from each other, p-value > 0.05. The β-glucuronidase activity of the centrifuged protoplasts treated with 1:5 MNP:DNA was significantly different from the equivalent treatment that was not centrifuged, p-value < 0.05, while the activity of protoplasts treated with 1:10 MNP:DNA, with or without centrifuging, were not significantly different from each other, p-value < 0.05 (Figure 3.11). β-glucuronidase activity for centrifuged protoplasts treated with 1:20 MNP:DNA was significantly different from the untreated control, p- value < 0.05, however was significantly less than the equivalent 1:5 and 1:10 treatments, p-value < 0.05.

Figure 3.11: β-glucuronidase activity in tobacco protoplasts transformed via magnetofection with three different ratios of MNP:DNA, 1:5, 1:10, and 1:20, with a MNP density of 1.0µg MNP/mL. Magnetofectins were assembled with 150µL of water per treatment. Each treatment was performed on 10,000 protoplasts, where protoplasts either were or were not centrifuged prior to magnetofection. Error bars represent the standard deviation of means of β-glucuronidase activity for two replicates. Statistical significance was assessed with an α = 0.05 for a two-way t-test between the equivalent treatments that were either centrifuged or not for β- glucuronidase activity at 48 hours.

3. Results At this stage, β-glucuronidase activity had been detected in protoplasts magnetofected with 1.0μg MNP/mL at 1:5, 1:10 and 1:20 ratios of MNP:DNA, but was not detectable at MNP densities of 2.0μg MNP/mL.

The results obtained in these first ‘proof-of-concept’ experiments to determine the effectiveness of magnetofection as a method of transfection of plant protoplasts are discussed in Chapter 4.

4. Discussion Chapter 4: Discussion

The major aim of this study was to determine whether magnetofection could be used as a novel plant transformation system. In the time available, specific aims included a proof-of-concept study that magnetofection could be used to transform plant protoplasts, including optimisation of conditions and to study the effects of magnetofection treatments on the viability of protoplasts. To achieve these aims, magnetofection of tobacco mesophyll protoplasts was carried out with MNPs to which were bound a binary vector encoding the reporter gene GUS, and assays were carried out to confirm transient expression by measuring β-glucuronidase activity. FDA staining was also done to assess the viability of protoplasts after various treatments. The results showed that protoplasts magnetofected with MNP:DNA ratios of 1:5, 1:10, and 1:20 showed transient GUS expression 48 hours after introducing magnetofectins, and the effect of magnetofection on the viability of these protoplasts was insignificant at densities of 1.0µg MNP/mL.

4.1 Particle bombardment to confirm functional expression of pCAMBIA1303

To confirm that pCAMBIA1303 could be functionally expressed in tobacco cells, particle bombardment of leaf tissue was carried out with tungsten microcarriers. The results unequivocally show that GUS was transiently expressed in bombarded tissue, confirming the functionality of the expression vector (Figure 3.2). While transient expression did occur, the number of transgenic events that were observed were quite low, with a maximum of four transgenic events per leaf, as indicated by blue histochemically stained spots. Using an almost identical methodology, Fosu-Nyarko (2005) showed that particle bombardment of wheat leaf tissue with a plasmid containing the GFP reporter resulted in a maximum of 16 fluorescent spots over a surface area of ~ 4cm2. An explanation for this greater level of transient expression is that the negative pressure attained in the vacuum chamber by Fosu-Nyarko (2005) was -95 to -100kPA, much higher than the -50kPA achieved in this study. Unfortunately, this was the maximum negative pressure attainable, as there was a

4. Discussion small break in the seal of the chamber door, nevertheless, transient GUS expression was clearly evident.

4.2 Development of GUS assays to detect β-glucuronidase activity

In this study, two assays were used to detect β-glucuronidase activity in protoplasts, a histochemical stain and a fluorescence assay. To confirm that both methods could be used to detect GUS, mesophyll protoplasts were isolated from GUS transgenic and non-transgenic cotton leaves and both assays were applied immediately post isolation.

For histochemical staining, two methods described in the literature were tested. The first, ‘GUS histochemical staining solution #1’, was described by Jefferson et al. (1987) and the second, ‘GUS histochemical staining solution #2’, by Mathur et al. (1995). The main difference between the two staining solutions were the pH of the solutions, 6.5 and 7.0, and the osmoticum, 0.3M and 0.4M mannitol, respectively. The results showed that the latter staining procedure produced clearer evidence of β- glucuronidase in GUS transgenic protoplasts, evidenced by an intense blue staining (Figure 3.3). The optimum pH of β-glucuronidase activity is reported to be 6.5-7.5 (Alwen et al., 1992), and so, it is possible that the pH of ‘GUS histochemical staining solution #2’ may have been more optimal for the enzymatic cleavage of X-gluc. In addition, the higher osmoticum used may have reduced protoplast viability, causing release of cellular β-glucuronidase into the X-gluc solution.

The same cotton protoplasts were used to test a fluorometric assay for the detection of the fluorophore 4-MU. The only fluorometer that was available with the required filter set to detect 4-MU was limited to detection in a 96-well format. Therefore, a 4- MU assay was designed to use this format: a protocol described by Yoo et al. (2007), for a 24-well format was modified for this study. It was shown that β-glucuronidase activity was quantifiable for GUS transgenic cotton protoplasts (Figure 3.5).

Subsequently, both GUS assays were tested for their ability to detect β-glucuronidase activity in transiently expressing tobacco protoplasts that were transformed with pCAMBIA1303 via PEG-mediated transformation. The results showed that transient

4. Discussion expression of GUS could be detected in the 4-MU fluorescence assay (Figure 3.6A), but was not detectable using the histochemical stain (Figure 3.6B). An explanation for this is that the fluorometric assay is much more sensitive to GUS activity than the histochemical stain (Jefferson et al., 1987). This does not fully explain these results, however the successful histochemical staining of protoplasts transformed via PEG- mediated transformation with GUS constructs has been reported in the literature (Abel et al., 1994; Dhir et al., 1998; Gallie et al., 1989; Hao et al., 2013). Therefore, a more likely explanation for the results obtained here is that the transfection levels achieved was relatively low, and usually more protoplasts were used for analysis in previous studies. A reflection of the level of expression was that the time that the 4-MU fluorescence assay was run for had to be increased to 24 – 48 hours, to obtain clear differentiation of expression in transformants and controls, longer than the three hours of the original protocol (Yoo et al., 2007). This may have been due to relatively low amounts of 4-MU in each well as a consequence of downscaling the system from a 24 to a 96 well format. A recommendation to overcome this issue would be to increase the number of protoplasts tested, which should increase the β-glucuronidase activity, and hence increase the sensitivity of the assay. Nevertheless this, the assay did clearly indicated β-glucuronidase activity in transformed tobacco protoplasts (Figure 3.6A).

4.3 Manipulation of protoplasts

4.3.1 Protoplast viability

It has been established that protoplast transformation efficiency depends very much on the viability of protoplasts (Miao et al., 2007). Hence, it was necessary to ensure high quality protoplasts were isolated and that MNP densities used for magnetofection did not affect the viability of protoplasts. The results from the optimisation of tobacco mesophyll protoplast isolation experiments showed that high quality protoplasts could be prepared (Table 3.1). The optimum procedure was used routinely and the quality of protoplasts isolated was consistent between experiments. As determined in later experiments, the viability of these protoplasts was determined to be 84%, 48-hours

4. Discussion post isolation. This viability is similar to those reported for canola and carrot protoplasts (~85%) 24 hours post isolation (Hao et al., 2013). In some systems, e.g. Arabidopsis protoplast viability can be up to 95%, however this value was obtained immediately post isolation (Sheen, 2001).

To determine MNP densities that would have negligible effect on protoplast viability, three different densities of MNPs were assessed. The results clearly show that viability of magnetofected protoplasts decreased as the density of MNP increased (Figure 3.8). This trend is consistent with reports by Arsianti et al. (2010), who showed the inverse relationship between viability of magnetofected baby hamster kidney cells (BHK21) and MNP dose. Transformation efficiencies were maximised when magnetofection treatments that resulted in a decrease of 10-15% viability compared to untreated controls (95% viability) were used. They determined that the effect on viability caused by MNPs was directly attributable to cell membrane damage, as opposed to cell cytotoxicity. In this case, the viability was determined by propidium iodide (PI) staining: PI exclusively stains the nucleus of cells with damaged cell membranes, as PI is impermeable to the cell membrane. This is consistent with findings in this study when tobacco protoplast viability was determined by FDA staining. The latter only results in fluorescence in cells with intact cell membranes (Figure 1.4). In a similar study, both the survival and transformation efficiency of magnetofected Escherichia coli decreased as the density of MNPs increased (Chen et al., 2006). Transformation efficiency was highest when a density of 960µg/mL MNPs was used, over 1000 times more than used in this study. A likely explanation for this is that the effect of magnetofection on cell viability is cell type-dependent, and bacterial cells are protected by cell walls, unlike plant protoplasts.

To date, there is no report on the effect of varying MNP densities on the viability of magnetofected plant cells – making this research the first to analyse such an important aspect, especially in cases where magnetofected cells would be needed as explants for regeneration of whole transgenic plants. The only study on plant tissues used cotton pollen grains transfected with 1µg/mL of MNPs, a statistically insignificant decline in the viability of magnetofected pollen compared to untreated control was reported (Zhao et al., 2017). Similarly, the same density of MNPs in this study also showed a statistically insignificant decline in viability (Figure 3.8). Hao et al. (2013) reported that

4. Discussion magnetically-enhanced delivery of 3.7µg/mL of GNPs did not affect the viability of canola and carrot protoplasts. Although the density of GNPs used was higher than that of the MNPs used in this study, a possible explanation for the difference in their effects on protoplasts could be the smaller average diameter of the GNPs, 25nm, compared to the 100nm iron NPs used in this study. In summary, it is concluded that a high density of MNPs causes a decrease in cell viability, and this finding is consistent with results in the literature. Overall, the nature of the relationship between magnetofection and viability may also depend on a variety of other factors, including: target cell type, the magnetic field strength used, cell concentration, and properties of the MNPs such as the diameter and composition.

4.3.2 Novel use of protoplasts for genome editing

Protoplasts can be used as tools to study transient gene expression of introduced expression constructs, for genetic and biochemical analyses, and in longer term studies to regenerate genetically modified plants. Because the global landscape of GMO regulations is changing to adjust to new technologies, genetically edited plants that are produced using ‘DNA-free’ systems with SDN1 (Site-directed nuclease type-one: mutations of only a few bases induced by endogenous cellular DNA repair mechanisms) may be exempt from being defined as GMOs, and so are of particular interest in agriculture. The use of CRISPR/Cas9, in the form of combined Cas9 enzyme and guide RNA RNPs, in which no DNA is present, is one such way to generate SDN1 mutations. Protoplasts are well-suited for genetic modification with CRISPR/Cas9 RNPs because high levels of transformation efficiency can be achieved with the system. This makes high throughput genetic screening of regenerants practicable for identifying SDN1 gene edits, without the need for screening using a selectable marker. For example, Woo et al. (2015) recently showed that Arabidopsis, tobacco, rice, and lettuce protoplasts transformed via PEG with CRISPR/Cas9 RNPs could be regenerated into whole plants, with a targeted mutagenesis frequency of up to 46%.

In animal systems, magnetofection has been shown to generate the same levels of transformation with up to 1000 times less DNA compared to conventional methods (Plank et al., 2011). If this characteristic was applicable to RNPs, then magnetofection 61

4. Discussion of RNPs bound to MNPs, which are then used to edit genomes using a protoplast system, could well reduce the cost of transformation considerably, as RNPs are currently expensive to buy and make.

4.4 Magnetofection as a novel transformation method in plants

To date, magnetic-enhanced delivery of nanoparticles has only been applied twice to plant systems, of which only one fits the strict definition of magnetofection. The first was the reported magnetic-enhanced delivery of GNPs with plasmid DNA into walled and root protoplasts from canola and carrot (Hao et al., 2013). The second reported the introduction of magnetofectins coated with plasmid into pollen grains of cotton, pumpkin, zucchini, capsicum and lily. In the case of cotton, magnetofection was followed by pollination with the transformed pollen, which led to the generation of transgenic plants (Zhao et al., 2017).

The demonstration of GUS expression in tobacco mesophyll protoplasts following magnetofection supports the use of this solanaceous plant in many aspects of plant research as a model. Tobacco has many properties that make it ideal as a model, such as: well established transformation protocols, the generation of thousands of seed per plant, and its close relatedness to agronomically important solanaceous crops such as potato and tomato. Mesophyll protoplasts were used for two reasons: first, without a cell wall there is less of a barrier to transformation, and second, transient analysis systems for protoplasts are established (Yoo et al., 2007).

The results presented here show, for the first time, that magnetofection can be applied successfully to tobacco mesophyll protoplasts: and this was confirmed by analysis of transient expression of the GUS reporter gene. In a preliminary magnetofection set up which used 1:5, 1:10, and 1:20 ratio of MNP:DNA, no β- glucuronidase activity was observed in protoplasts when they were assembled in a lower volume of water (Figure 3.9). For the 1:20 MNP:DNA ratio, agglutination of magnetofectins could be seen. This was thought to hinder successful transformation, either because large clumps of magnetofectins would not be able to traverse the cell membrane, or perhaps that magnetically accelerated agglutinations of magnetofectins might cause more damage to protoplast cell membranes. 62

4. Discussion The binding capacity of positively charged MNPs and negatively charged DNA enables the formation of both individual magnetofectin complexes, and agglutinations of magnetofectins, the nature of this mechanism is exploited in MNP agglutination assays (Mezger et al., 2015). To minimise the latter, magnetofectins were then assembled in an increased volume of water, in order to reduce the likelihood of MNP/DNA agglutination interactions. This resulted in successful magnetofection at the ratio of 1:10 MNP:DNA (Figure 3.10). Research by Chen et al. (2006), indirectly supports these findings in magnetofected E. coli, where the efficiency of transformation increased as the ratio of MNP:DNA was decreased, when the MNP density was kept constant. This result suggested that excess DNA may have contributed to the agglutination of the MNPs thereby reducing the number of cells interacting with the magnetofectins and subsequent transformation. To provide direct evidence to support this hypothesis, it would be necessary to track the entry of magnetofectins into cells, which can be done with fluorescently labelled MNPs or by transmission electron microscopy. Centrifuging protoplasts prior to introduction of magnetofectins resulted in a significant increase in transient GUS expression with MNP:DNA ratios of 1:5 and 1:20, but not for 1:10 (Figure 3.10). The centrifugation step may have made more protoplasts accessible for magnetofectin interaction by ensuring protoplasts were at the bottom of the transfection vessel.

As a transformation system, magnetofection has many properties that may make it appear well-suited for treatments to introduce external compounds such as DNA, RNA, viral vectors, and proteins, followed by regeneration of transgenic plants. Because MNPs can be loaded with a diverse range of biomolecules, and treatments can be optimised to not affect cell viability (MNP density of 1.0µg/mL, which was confirmed in this study) (Figure 3.8), and they can under certain circumstances penetrate plant cell walls, such as for pollen (Zhao et al., 2017), there is a wide variety of potential applications of magnetofection in plant transformation. Success in this area would have the major advantage of avoiding laborious tissue culture protocols. Other potential, but as of yet unexplored in planta systems to which magnetofection could be applied are, magnetofection of ovary cells or apical meristem cells. It is possible that magnetofection of these tissues would enable the generation of transgenic plants

4. Discussion for species/varieties which are typically recalcitrant to culture, such as cereals, which are of enormous importance in agriculture.

A possible application of magnetofection is to be applied directly to cells in leaf tissues. A major disadvantage of particle bombardment of leaf tissue is the potential for incorporation of high copy number gene inserts in transformed cells, whereas for Agrobacterium transformation, the main limitations are its relatively narrow host range and the strict use of DNA as a vector (Cunningham et al., 2018). Magnetofection may offer a scenario of the ‘best of both worlds’, by introducing low/single copy numbers of gene constructs, due to the low amount of DNA required for transformation, or by enabling the use of DNA-free (RNP) transformation systems, and being applicable to a wide-range of species. Further study is clearly warranted to determine the full potential of magnetofection.

4.5 Future studies

Further optimisation of the magnetofection procedure described in this research could result in a higher transformation efficiency. Due to time constraints, it was not possible to thoroughly assess all the factors that may have increased the efficiency of magnetofection of protoplasts. These factors included:

1. Concentration of protoplasts – when increased, it may enable greater interaction of magnetofectins with protoplasts. 2. Strength of the magnetic field gradient – stronger field gradients may allow magnetofectins to penetrate directly through the cell membrane. 3. The use of dynamic magnetic fields – allowing for greater interactions between magnetofectins and protoplasts. This has been shown in some cases to increase transformation efficiency in animal systems (Vainauska et al., 2012). 4. Length of magnetic field exposure – directly altering the time that magnetofectins are attracted towards target cells. 5. Change of transformation buffers – such buffers may alter the permeability of the cell membrane/wall to magnetofectins.

4. Discussion 6. Type of polymer functionalisation – different polymers confer various physicochemical properties to the MNP, such as charge, size and biomolecule binding capacity. 7. MNP size – smaller MNPs may be able to penetrate cell walls, while larger particles may penetrate directly through cell membranes more easily because of greater physical momentum.

Such optimisations will broaden the application of magnetofection to tissue types such as leaves, pollen, ovaries and meristems and ensure that MNPs can introduce biomolecules other than DNA, such as CRISPR/Cas9 RNPs, which may lead to the development of a transformation system which is much improved over conventional systems.

4.6 Conclusion

The research conducted shows for the first time that magnetofection can be used to induce transient expression of DNA in tobacco mesophyll protoplasts. High density (2- 4µg/mL) of MNPs can drastically reduce the viability of protoplasts, however, an optimised density (1.0µg/mL) of MNPs was used to induce transient GUS expression which was are not detrimental to protoplast viability. Overall, this work provides a starting point for future development of magnetofection as a plant transformation system. Further optimisation of the procedure for different tissue types can widen the scope of plant molecular biology.

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