Viral Infection and Propagation in Plant Tissue Culture

by FIONA STELLA SHADWICK

Thesis submitted for the degree of Doctor of Philosophy

School of Biotechnology and Biomolecular Sciences University of New South Wales January 2007

ACKNOWLEDGEMENTS

I wish to thank Professor Pauline Doran for her guidance, encouragement and generous support. I also wish to extend my gratitude to the staff of the school; in particular I would also like to thank Mr. Malcolm Noble, Dr. Russell Cail and Dr. David Chin for their technical advice and assistance. I also wish to thank Caleb Chung from his assistance with HPLC analysis and the assembly and operation of bioreactors. I wish to thank my husband and family for their patience and encouragement. For their companionship and generosity I thank my fellow laboratory members.

TABLE OF CONTENTS page

Table of Contents i

Abstract xiv

CHAPTER 1 – INTRODUCTION

1.1 Foreign Protein Expression in Whole Plants 1 1.1.1 Plants as expression systems 1 1.1.1.1 Expression systems 1 1.1.1.2 Plant-based expression systems 2 1.1.2 Heterologous protein production in whole plants 3 1.1.3 Approaches to increase protein yield 4 1.1.3.1 Promoter sequences 4 1.1.3.2 Protein targeting 6 1.1.3.3 Sequence optimisation 7 1.1.3.4 Alternate host plants 8 1.1.3.5 Plastid expression 8 1.1.3.6 Virus-based expression systems 9

1.2 Plant Tissue Culture 11 1.2.1 Undifferentiated plant tissue culture 11 1.2.2 Differentiated organ culture 12 1.2.2.1 Root cultures 12 Untransformed root cultures 13 Transformed “hairy root” cultures 13 1.2.2.2 Shoot cultures 16

1.3 Plant Tissue Culture as a Production System for Heterologous Proteins 17 1.3.1 Foreign protein production using plant tissue culture 19 1.3.2 Increasing protein expression and recovery 24 1.3.2.1 Increased protein expression 24 Promoter sequences 25 Protein targeting 26 Culture species and type 26

Alternate transformation 28 Bioprocess developments 29 1.3.2.2 Increased protein recovery 29

1.4 Plant Viruses as Vectors 32 1.4.1 Plant viruses 32 1.4.2 Plant-virus-based vectors 34 1.4.3 Vector design 36 1.4.4 Approaches for foreign protein expression 38 1.4.4.1 Fusions 38 1.4.4.2 Free proteins 40 Additional open reading frames 41 Expression of foreign proteins as part of the viral polyprotein 41

1.5 Foreign Protein Expression Using Plant Viral Vectors 42 1.5.1 Stability 50 1.5.2 Movement of plant viral vectors 51 1.5.3 Vector and foreign protein accumulation 53 1.5.3.1 Accumulation of viral vectors 54 1.5.3.2 Accumulation of foreign proteins 55 1.5.4 Safety 56

1.6 Tobacco Mosaic Virus 58 1.6.1 Virus structure 58 1.6.2 Genome organization and encoded product function 60 1.6.3 Early events in TMV infection 62 1.6.4 TMV replication and movement 63 1.6.4.1 Virus replication 63 1.6.4.2 Viral movement 64 Cell-to-cell movement 65 Long-distance movement 66

1.7 Tobacco Mosaic Virus as a Vector 67 1.7.1 30B vector 68 1.7.2 30B-GFPC3 70 1.7.3 Movement, host range and stability of 30B and 30B-GFPC3 70

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1.7.3.1 Vector stability 71 1.7.3.2 Host range and movement 71

1.8 TMV Infection of Plant Roots 72 1.8.1 TMV accumulation in roots from whole plants 72 1.8.2 TMV accumulation in cultured roots 74

1.9 Virus Infection of Cultured Plant Cells 75 1.9.1 Infection of cultured cells with virus 80 1.9.1.1 Initiation of infection in cultured cells 80 Intentional cell wall injury 80 Natural cell wall breaks 82 1.9.1.2 Culture characteristics 84 1.9.1.3 Culture age 85 1.9.1.4 Viral inoculum 86 1.9.2 Characteristics of viral accumulation in tissue culture 87 1.9.2.1 Kinetics of viral accumulation 87 Viral accumulation in plant cell protoplasts 88 Viral accumulation in plant suspension and callus cultures 89 1.9.2.2 Viral yield 94 1.9.2.3 Effect of viral infection on culture growth 96

1.10 Project Aims 97

CHAPTER 2 – MATERIALS AND METHODS

2.1 Cultures 98 2.1.1 Plant material 98 2.1.2 Viral material 98 2.1.3 Bacterial cultures 98

2.2 Media 99 2.2.1 Plant culture media 99 2.2.2 Bacterial culture media 100

2.3 Initiation and Maintenance of Plant Cultures 101 2.3.1 Surface sterilisation and germination of seeds 101 2.3.2 Maintenance of axenic plantlets 102

2.3.3 Hairy root cultures 102 2.3.3.1 Initiation of Nicotiana benthamiana hairy root cultures 102 2.3.3.2 Maintenance of hairy root cultures 104 2.3.4 Callus and suspension cultures 104 2.3.4.1 N. benthamiana callus and suspension cultures 104 2.3.4.2 N. tabacum callus and suspension cultures 105

2.4 Growth of Plants in Soil 106 2.4.1 N. tabacum plants 106 2.4.2 N. glutinosa plants 106 2.4.3 N. benthamiana plants 107

2.5 Production of TMV 107 2.5.1 Infection of N. tabacum plants with TMV 107 2.5.2 Purification of TMV 108 2.5.3 Concentration and purity of TMV preparations 110

2.6 Production of Transgenic Virus 111 2.6.1 Transformation of E. coli XL1-Blue with plasmid 30B-GFPC3 111 2.6.2 Production of in vitro transcript of 30B-GFPC3 111 2.6.3 Infection of plants with 30B-GFPC3 RNA transcript 112 2.6.4 Purification of TMV-GFPC3 113

2.7 Preliminary Investigations of TMV Accumulation in N. tabacum Suspension and Hairy Root Cultures 114 2.7.1 Growth of N. tabacum suspension and hairy root cultures 114 2.7.1.1 Growth of N. tabacum suspension cultures 114 2.7.1.2 Growth of N. tabacum hairy root cultures 114 2.7.2 Accumulation of TMV in N. tabacum suspension culture 115 2.7.3 Accumulation of TMV in N. tabacum hairy roots 116

2.8 Accumulation of TMV in N. benthamiana Suspension 116

2.9 Accumulation of TMV in N. benthamiana Hairy Roots 118 2.9.1 Selection of N. benthamiana hairy roots with suitable growth and viral accumulation characteristics 119 2.9.2 Time-course of TMV accumulation in N. benthamiana hairy roots 119

2.9.3 Effect of hairy root condition at the time of viral inoculation on TMV accumulation 120 2.9.3.1 Effect of root age 120 Variously aged roots in fresh medium 120 Variously aged roots in conditioned media 120 2.9.3.2 Effect of injuring hairy roots prior to viral infection 121 2.9.4 Characteristics of hairy root growth and viral accumulation in N. benthamiana hairy roots over 36 days 122 2.9.5 Effect of medium condition at the time of viral inoculation on TMV accumulation 122 2.9.5.1 Removal of TMV inoculum from cultures after an “inoculation phase” 122 2.9.5.2 Inoculation of hairy roots with virus in phosphate buffer 122 2.9.6 Alteration of viral inoculum concentration 123 2.9.7 Proportional scale-up in shake flasks 123

2.10 Accumulation of TMV in N. benthamiana Hairy Roots with an Established Viral Infection 126 2.10.1 Accumulation of TMV over three generations of culture 126 2.10.2 Effect of increasing the viral inoculum concentration used to initiate a primary viral infection in hairy roots on viral accumulation in a subsequent-generation hairy root culture with an established viral infection 127

2.11 Viral Stability in Medium 128 2.11.1 Viral stability in plant media and phosphate buffer 128 2.11.1.1 Virus stability in fresh media 128 2.11.1.2 Virus infectivity in fresh media 129 2.11.2 Virus characteristics in conditioned media 129 2.11.2.1 Virus stability in conditioned media 130 2.11.2.2 Virus infectivity in conditioned media 130

2.12 Short-Term Virus Association Experiments 131 2.12.1 Concentration of viral inoculum and viral association with hairy roots 131 2.12.2 Viral association with roots during proportional scale-up 131

2.13 Association of Deactivated TMV with Hairy Roots 132 2.13.1 Deactivation of TMV 132 2.13.2 Hairy roots and deactivated virus association 133

2.14 Accumulation of Genetically Modified Virus and GFP in Hairy Roots 134

2.15 Viral Distribution in Hairy Root Clumps in Shake Flasks 134 2.15.1 Distribution of virus in different concentric regions 135 2.15.2 Distribution of virus in radial segments 135

2.16 Accumulation of TMV in N. benthamiana Hairy Roots in a Stirred Bioreactor 136 2.16.1 Bioreactor configuration 136 2.16.2 Bioreactor culture methods 138

2.17 Analytical Procedures 141 2.17.1 Growth 141 2.17.2 Medium characteristics 141 2.17.2.1 Medium pH 141 2.17.2.2 Medium conductivity 142 2.17.2.3 Medium sugar concentration 142 2.17.3 Sample extraction for the analysis of virus, total protein and GFP 143 2.17.3.1 Sample extraction for the detection of virus in plant biomass using ELISA 143 2.17.3.2 Sample extraction for the detection of GFP in plant biomass and for virus detection using Western blot 144 2.17.3.3 Sample extraction for assessment of total soluble protein 145 2.17.4 Quantification and analysis of virus 145 2.17.4.1 Quantification of TMV using ELISA 145 2.17.4.2 Western blot detection of viral coat protein 149 2.17.4.3 Particle size analysis 151 2.17.4.4 Viral infectivity using local lesion assays 152 2.17.5 Detection of GFP 154 2.17.5.1 Detection of GFP using ELISA 154 2.17.5.2 Detection of GFP using Western blotting 156 2.17.5.3 Detection of GFP using fluorescence microscopy 157

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2.17.6 Quantification of virus using scanning electron microscopy 158 2.17.7 Total soluble protein 160

2.18 Statistical Analysis 161

CHAPTER 3 – RESULTS

3.1 Tobacco Mosaic Virus Accumulation in N. tabacum Plants 163

3.2 TMV Accumulation in N. tabacum Suspension and Hairy Root Cultures 164 3.2.1 Accumulation of TMV in N. tabacum suspension cultures 164 3.2.1.1 Growth of N. tabacum suspension cultures 164 3.2.1.2 Infection of N. tabacum suspension cultures with TMV 165 3.2.2 Accumulation of TMV in N. tabacum hairy roots 169 3.2.2.1 Growth of N. tabacum hairy roots 169 3.2.2.2 Infection of N. tabacum hairy root cultures with TMV 169

3.3 TMV Accumulation in N. benthamiana Suspension Cultures 174 3.3.1 Growth of TMV-infected N. benthamiana suspension cultures 174 3.3.2 Virus accumulation in TMV-infected N. benthamiana suspension cultures 176

3.4 Initiation and Selection of N. benthamiana Hairy Root Cultures 180 3.4.1 Initiation of N. benthamiana hairy roots 180 3.4.2 Infection of N. benthamiana hairy root clones with TMV 181 3.4.2.1 Growth of TMV-infected N. benthamiana hairy roots 181 3.4.2.2 TMV accumulation in N. benthamiana hairy roots 182

3.5 Summary of Preliminary Studies with N. tabacum and N. benthamiana Suspension and Hairy Root Cultures 184

3.6 Incubation of N. benthamiana Hairy Roots with Non-Infectious TMV 185 3.6.1 Deactivation of TMV 185 3.6.2 Incubation of N. benthamiana hairy root cultures with non-infectious TMV 187 3.6.2.1 Over 12 hours 188 3.6.2.2 Over 36 days 190

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3.7 Effect of Root Age on Viral Accumulation 193 3.7.1 Hairy roots in fresh medium 193 3.7.1.1 Effect of root age on hairy root culture growth 193 3.7.1.2 Viral accumulation in hairy root cultures initiated using variously-aged root inocula 195 3.7.2 Effect of infection of variously-aged hairy roots with TMV in conditioned medium on subsequent viral accumulation 197 3.7.2.1 Stability of TMV in conditioned media from variously-aged hairy root cultures 198 Protein concentration in conditioned medium 198 Stability of virus in conditioned plant media 199 Virus infectivity in conditioned media 200 3.7.2.2 Growth of variously-aged hairy roots in conditioned media 201 3.7.2.3 Viral accumulation when variously-aged hairy roots in conditioned media were infected with TMV 204 3.7.3 TMV accumulation in hairy root cultures initiated using 21-day N. benthamiana hairy roots and infected by co-incubation with TMV 208 3.7.3.1 Growth of N. benthamiana hairy roots – examined over a 52-day period 208 3.7.3.2 Virus accumulation in N. benthamiana hairy roots – examined over a 52-day period 210

3.8 Effect of Hairy Root Injury on Virus Accumulation 212 3.8.1 Effect of intentional root injury on subsequent root growth 212 3.8.2 Virus accumulation in hairy root cultures when root inocula were intentionally injured 215

3.9 Characteristics of Hairy Root Growth and Viral Accumulation in N. benthamiana Hairy Roots over 36 Days 217 3.9.1 Growth of N. benthamiana hairy roots 218 3.9.2 Medium characteristics 222 3.9.2.1 Sugar utilisation 222 3.9.2.2 Medium pH and conductivity 224

3.9.3 Virus accumulation in N. benthamiana hairy roots 225 3.9.3.1 Characteristics of virus accumulation in hairy root biomass 225 3.9.3.2 Virus in the biomass 228 3.9.3.3 Virus in the medium 231 3.9.3.4 Biomass-associated viral protein expressed as a percentage of total soluble protein 233 3.9.4 Viral infectivity and particle integrity in TMV-infected N. benthamiana hairy roots 235 3.9.4.1 Infectivity of virus in hairy root extracts 235 3.9.4.2 Particle length 237 3.9.5 Distribution of TMV in hairy roots 239 3.9.5.1 Distribution of virus in different concentric regions 240 3.9.5.2 Distribution of virus in radial segments 240

3.10 Medium Conditions at the Time of Viral Infection and Subsequent TMV Accumulation 243 3.10.1 Removal of TMV inoculum after an “inoculation phase” 243 3.10.1.1 Hairy root growth 243 3.10.1.2 Virus accumulation 243 3.10.2 Infection of N. benthamiana hairy roots with TMV in different “media” 249 3.10.2.1 Stability of TMV in different media 249 3.10.2.2 Infectivity of TMV in different media 250 3.10.2.3 Infection of hairy roots in phosphate buffer and subsequent medium exchange 251

3.11 Alteration of Inoculum Virus Concentration 259 3.11.1 Association of virus with hairy root biomass over 12 hours when the concentration of the viral inoculum was altered 259 3.11.2 Effect of altering the concentration of inoculum virus on hairy root growth and virus accumulation 261 3.11.2.1 Effect of inoculum virus concentration on hairy root growth 261 3.11.2.2 Accumulation of TMV in hairy root cultures when the inoculum virus concentration was altered 262

3.12 Proportional Scale-Up in Shake Flasks 270 3.12.1 Association of inoculum virus with hairy roots using scale-up conditions 270 3.12.2 Hairy root growth and viral accumulation during proportional scale-up in shake flasks 273 3.12.2.1 Hairy root growth 273 3.12.2.2 Virus accumulation 276

3.13 Accumulation of TMV in N. benthamiana Hairy Root Cultures with Established Infections 280 3.13.1 Accumulation of TMV in two generations of hairy root cultures with established TMV infections and in a hairy root culture with a primary TMV infection 280 3.13.1.1 Growth of hairy root cultures with established TMV infections 280 3.13.1.2 Viral accumulation in hairy root cultures with primary and established viral infections 282 Amount of virus in the biomass 282 Concentration of virus in the biomass 284 3.13.2 Effect of increasing the viral inoculum concentration used to initiate a primary viral infection in hairy roots on viral accumulation in a subsequent-generation hairy root culture with an established viral infection 290 3.13.2.1 Hairy root growth 291 3.13.2.2 Virus accumulation 293 Amount of accumulated virus 293 Concentration of accumulated virus 295

3.14 Infection of N. benthamiana Hairy Roots with TMV-GFPC3 in Shake Flasks 299 3.14.1 Production, concentration and infectivity of TMV-GFPC3 preparations 299 3.14.1.1 Production and concentration of TMV-GFPC3 preparations 299 3.14.1.2 Infectivity of TMV-GFPC3 purified from plants 301 3.14.1.3 Expression of GFP in plants infected with purified TMV-GFPC3 particles 301

3.14.2 Infection of N. benthamiana hairy roots with TMV-GFPC3 particles 304 3.14.2.1 Growth of N. benthamiana hairy roots inoculated with TMV-GFPC3 particles 304 3.14.2.2 Sugar utilisation by N. benthamiana hairy root cultures inoculated with TMV-GFPC3 305 3.14.2.3 TMV-GFPC3 accumulation in N. benthamiana hairy root cultures 307 3.14.2.4 Medium virus 311 3.14.2.5 Total amount of virus 312 3.14.2.6 GFP expression in hairy root cultures inoculated with TMV-GFPC3 312 3.14.3 Viral accumulation in N. benthamiana hairy root cultures inoculated with equal numbers of TMV-GFPC3, TMV and deactivated TMV particles 314

3.15 Infection of N. benthamiana Hairy Roots with TMV and TMV-GFPC3 in Bioreactors 319 3.15.1 Hairy root growth and sugar utilisation 319 3.15.2 Medium pH and conductivity 323 3.15.3 Accumulation of virus 325 3.15.3.1 Accumulation of virus in hairy root biomass 325 3.15.3.2 Viral accumulation in the medium 328 3.15.4 GFP expression in bioreactor grown hairy roots inoculated with TMV-GFPC3 330

CHAPTER 4 – DISCUSSION

4.1 Accumulation of Virus in Hairy Roots and Suspended Cells 331

4.2 Viral Accumulation in N. benthamiana Hairy Roots 332 4.2.1 Kinetics of TMV accumulation in N. benthamiana hairy roots 333 4.2.1.1 Inoculum virus in the medium 334 4.2.1.2 Association of inoculum virus with the biomass 336 4.2.1.3 Virus accumulation in the hairy root biomass 338 4.2.1.4 Virus accumulation in the culture medium 344 4.2.2 Hairy root growth and viral accumulation 345

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4.2.3 Variability in viral accumulation within cultures 346 4.2.4 Variability in viral accumulation between cultures 350 4.2.5 Particle length and infectivity 351 4.2.6 Virus yield 354 4.3 Modifications to Infection Procedures 359 4.3.1 Intentional root injury 359 4.3.2 Separation of the initial infection phase and culture growth 361 4.3.2.1 Medium exchange 361 4.3.2.2 Alternative infection media 362 4.3.3 Effect of hairy root pre-culture age on virus accumulation 365 4.3.4 Culture initiation in conditioned medium 367 4.3.5 Virus Inoculum concentration 370

4.4 Accumulation of TMV in N. benthamiana Hairy Root Cultures with Established Infections 376

4.5 Infection of N. benthamiana Hairy Roots with a Transgenic Plant Viral Vector 383

4.6 Infection Scale-up 390 4.6.1 Infection scale-up in shake flasks 390 4.6.2 Accumulation of virus in bioreactor-infected and -grown hairy roots 393 4.6.2.1 Hairy root growth 393 4.6.2.2 TMV accumulation 394 4.6.2.3 TMV-GFPC3 accumulation 397

4.7 Infection of N. benthamiana Suspended Cells with TMV 398

4.8 Virus infection of and accumulation in N. benthamiana suspension and hairy root cultures 402

CHAPTER 5 – CONCLUSIONS AND RECOMMENDATIONS

5.1 Conclusions 407

5.2 Recommendations 410

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Appendices

Appendix 1 Filter Sterilization of Purified TMV Preparations 412

Appendix 2 Extraction of TMV from Plant Biomass 413

Appendix 3 Growth of N. benthamiana hairy root cultures initiated using 14-day-old root inoculum 414

References 413

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ABSTRACT

The propagation of wild-type virus and a transgenic viral vector was examined in cultured plant cells to identify factors affecting viral infection of and accumulation in cultured cells and to determine if viral vectors could be used to facilitate the expression of heterologous proteins in vitro.

Tobacco mosaic virus (TMV) accumulation was examined in Nicotiana tabacum and

N. benthamiana suspension and hairy root cultures. TMV accumulation was superior in

N. benthamiana hairy roots. Hairy roots were infected by adding TMV to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by virus infection. TMV accumulated in actively growing root cultures. The distribution of virus within root mats from shake-flask grown cultures was non-uniform and the concentration of virus accumulated in replicate cultures was variable.

When N. benthamiana hairy roots were infected using 1.5 μg mL-1 TMV, the average maximum concentration of virus accumulated in the biomass was 1.6 ± 0.25 mg g-1 dry weight, or 15-fold lower than in TMV infected N. tabacum leaves. Virus coat protein accumulated to a level of (26 ± 10)% total soluble protein in the hairy roots which was not significantly different from that in N. tabacum leaves when the concentration of viral protein was expressed as a percentage of total soluble protein. Increasing the inoculum virus concentration resulted in increased virus accumulation in hairy roots, although virus accumulation per microgram of inoculum virus remained roughly constant. Virus accumulated in hairy roots to a concentration of 4.2 ± 0.60 mg g-1 dry weight when an inoculum of 9.0 μg mL-1 TMV was used. Virus accumulated to similar

xv concentrations in roots when the inoculum virus was retained in the medium for the duration of the culture or removed 16 hours after infection. The medium in which infection was performed affected the percentage of inoculated cultures that accumulated virus and the virus yield. Proportional scale-up of hairy root infection in shake flasks did not result in constant virus concentrations in the scaled cultures. TMV accumulation in bioreactor-infected and -grown hairy roots was poor.

N. benthamiana hairy roots were infected with a TMV-based vector (30B-GFPC3) that encoded Cycle 3 green fluorescent protein (GFP). TMV-GFPC3 was (260 ± 140)-fold less infectious than TMV as measured by local lesion assays. The patterns of TMV and

TMV-GFPC3 accumulation in hairy roots were similar, but the concentration of TMV-

GFPC3 in the biomass was approximately 65-fold lower than TMV when roots were inoculated with the same number of wild-type and transgenic viral particles.

Propagation of TMV-GFPC3 could not be confirmed using mass balance. GFP was not detected in the infected hairy roots or the culture medium.

Hairy roots represent a potentially viable culture-based system for the in vitro production of virus and virus products when field-grown agricultural systems do not adequately address issues of containment or product safety.

CHAPTER 1 – INTRODUCTION

1.1 Foreign Protein Expression in Whole Plants

Expression of heterologous proteins in plants has allowed the development of transgenic plants for research purposes. Crop plants that exhibit altered or additional characteristics such as disease and herbicide resistance, enhanced anti-microbial and insecticidal properties, improved nutritional value and improved production characteristics which benefit the producer and consumer have also been developed.

Transgenic plants are being utilised as expression systems for the production of heterologous proteins with industrial and pharmaceutical applications.

1.1.1 Plants as expression systems

1.1.1.1 Expression systems

The demand of the pharmaceutical industry for large quantities of therapeutic proteins, often of mammalian origin, has resulted in the development of expression systems for the production of proteins and peptides of varying complexity. The host organisms used range from bacteria to eukaryotic systems such as yeast, insect, mammalian and plant cell cultures, and transgenic animals and plants.

Bacterial expression systems allow relatively high levels of foreign protein expression at a low cost; however heterologous protein is often accumulated as insoluble inclusion bodies and only limited post-translational modifications are performed. In contrast, eukaryotic expression systems are able to glycosylate proteins and perform post- translational processing, although the nature of the protein modifications can vary depending on the eukaryotic system used, resulting in the production of recombinant

2 proteins that may not be identical to the native protein. The cost of protein production using different eukaryotic expression systems also varies widely. The complexity of a heterologous protein, its end use, the desired scale of production and the degree of similarity required between the transgenic and native proteins are factors considered when selecting an expression system.

1.1.1.2 Plant-based expression systems

Plant- and plant-cell-based expression systems can be utilised to produce active mammalian proteins. Post-translational protein modifications performed by plant cells are similar to those performed by mammalian cells and plant cells can facilitate the folding of multimeric proteins. The glycosylation sites on plant-produced mammalian proteins are the same as those on the native protein; however, processing of N-linked glycans in the plant cell secretory pathway results in the formation of a more diverse array of glycoforms than is produced in mammalian expression systems (Cabanes-

Macheteau et al., 1999; Gomord et al., 2004). Glycoprotein activity is retained in plant-produced mammalian proteins.

In addition to producing heterologous proteins similar to native mammalian proteins, plant-based production systems provide a number of production advantages over other expression systems, including product safety and, if agriculturally produced, ease of scale up. When compared with bacterial and other eukaryotic expression systems, the potential contamination of purified protein products with endotoxins, mycotoxins, and mammalian pathogens is significantly reduced by the production of heterologous proteins in plant-based systems. If proteins are expressed in whole plants, production levels can also be increased with relatively small capital investment by increasing the cropped area. The ability to cheaply scale production also reduces pressure on yield as

3 transgenic plants expressing low levels of protein can still be economically viable.

Equipment and processes developed for the food industry are often transferable for the production, harvest and primary processing of plant biomass or seeds containing foreign proteins. For some applications such as oral vaccination, the need for significant post-harvest processing and purification may be eliminated by expressing proteins designed for oral delivery in edible plants, further reducing processing costs.

1.1.2 Heterologous protein production in whole plants

Early attempts to express foreign proteins in whole plants focused on the expression of proteins from nuclear-transformed plants. Stable nuclear transformation of plant cells

(primarily callus or suspension cells) was achieved using Agrobacterium tumefaciens- mediated or particle bombardment (biolistic) transformation methods. Both methods resulted in the random insertion of the heterologous gene construct into the plant genomic DNA, often yielding unpredictable levels of protein expression due to gene position effects. Protein expression was generally under the control of the cauliflower mosaic virus (CaMV) 35S promoter, a strong constitutive promoter. Transformed plant cells containing heterologous gene constructs could be regenerated into whole plants.

However plant regeneration from individual transformed cells requires a significant time investment prior to protein analysis and quantification in regenerated plant tissue.

Early attempts to express foreign proteins in whole plants were often disappointing, with the levels of accumulated foreign protein generally, and often significantly, lower than 1% total soluble protein (TSP) (Cramer et al., 1999; Daniell et al., 2004). The levels of protein expression obtained were adequate to confer additional characteristics to whole plants but were generally not sufficient to make foreign protein expression and purification economically viable, or the use of unpurified plant material for oral

4 therapeutic applications practical. Low levels of foreign protein accumulation in plants have generally been attributed to a combination of low levels of protein expression and in vivo protein instability.

1.1.3 Approaches to increase protein yield

A variety of approaches have been examined to increase heterologous protein accumulation in whole plants. Major approaches include: using alternate promoter sequences to increase RNA transcription; targeting protein accumulation to sub-cellular locations that offer enhanced protein stability; optimising gene sequences to allow efficient transcription and translation; the use of alternate host plants that facilitate protein accumulation; and the use of alternate approaches to transformation that may allow higher levels of protein expression, including plastid transformation and transformation of plants with viral vectors.

1.1.3.1 Promoter sequences

Expression constructs contain a number of elements including promoter, 5′ untranslated region (5′-UTR) and terminator sequences that determine the level of foreign protein expression. Of these elements, the promoter sequence has been the most widely investigated in attempts to increase or more tightly control transgene expression.

The CaMV 35S promoter (Odell et al., 1985) and the enhanced CaMV 35S promoter

(Kay et al., 1987) are strong constitutive promoters commonly used to achieve heterologous protein expression in all tissues from dicotyledonous plants. Alternative constitutive promoters that have been associated with increased levels of heterologous protein expression have been developed (Ni et al., 1995); however CaMV 35S

5 promoters are still frequently used to control transcription. Promoter sequences that facilitate high levels of heterologous protein expression in dicotyledonous plants often require modification to work efficiently in monocotyledonous plants (Vain et al., 1996).

Specific promoter constructs that facilitate high levels of heterologous protein expression in monocotyledonous plants, such as the maize ubiquitin-1 (Ubi-1) promoter construct, have been developed (Christensen and Quail, 1996).

Tissue-specific promoters and inducible promoters have been utilised to obtain high levels of local protein expression and increased control over protein expression, or to achieve production advantages. Tissue-specific promoters allow the expression of heterologous proteins in particular plant tissues such as seeds, fruit, tubers or leaves, often with no or only low-level heterologous protein expression in other tissues (Russell and Fromm, 1997). Huang et al. (2002a) reported the expression of lysozyme in rice to a level of 45% TSP (0.6% brown rice dry weight) using the promoter and signal peptide from glutelin 1, a rice storage protein. ProdiGene Inc. (Texas, USA) commercially produce avidin and β-glucuronidase (GUS) in maize seed.

Inducible promoters which allow transgene expression to be controlled externally by a variety of chemical (Padidam, 2003) and physical inducers (Cramer et al., 1999; Kim et al., 2003) have been developed for use in plants. Post-harvest production of heterologous proteins using a promoter induced by mechanical stress (shearing of leaves during harvest) has been demonstrated by CropTech Corp (Virginia, USA)

(Cramer et al., 1999; Twyman, 2004). Promoters that facilitate protein expression during specific plant developmental stages such as seed germination have also been developed and utilised to drive heterologous protein expression (Koivu, 2004).

1.1.3.2 Protein targeting

Heterologous protein accumulation in the cytosol of transgenic plant cells has generally been observed to be low or absent, while targeting of heterologous proteins to different sub-cellular compartments has been found to result in higher protein yields (Fecker et al., 1996; Schouten et al., 1996; Zimmermann et al., 1998). In an investigation of the effect of sub-cellular localisation on single-chain antibody (scFv) accumulation,

Schouten et al. (1996) reported that for a scFv that was undetectable in the cytosol, the addition of a signal peptide to direct the protein into the secretory pathway was associated with scFv accumulating to 0.01% TSP in the apoplast. Retention of the scFv in the endoplasmic reticulum (ER) by addition of the KDEL sequence resulted in further increases in scFv accumulation to 1.0% TSP.

Protein targeting is achieved by the addition of native plant (Fecker et al., 1996) or non-plant signals including mammalian signal peptides (Hiatt et al., 1989) to the heterologous protein, or by fusing heterologous proteins to host proteins with appropriate signal peptides (Boothe et al., 1997). Addition of a signal peptide allows the co-translational insertion of the heterologous protein into the ER. After entry into the ER, proteins can be targeted to different regions of the cell including oil bodies

(Boothe et al., 1997), vacuoles and protein storage vesicles (Murray et al., 2002;

Wright et al., 2001) or, if no additional targeting sequences are present, proteins will be secreted into the apoplast via the bulk transport pathway. Proteins can be retained in the ER by the addition of retention signals such as the tetrapeptide KDEL to the

C-terminal of the protein. Increases in yield observed when proteins were targeted to the ER have been attributed to enhanced protein stability due to correct protein folding and also the accumulation of proteins in regions of the plant cell that offer a more stable environment for protein storage than the cytosol (Farran et al., 2002; Hiatt et al., 1989)

7 rather than increased protein translation (Zimmermann et al., 1998). Passaging heterologous proteins through the ER is required for correct assembly, folding and glycosylation.

In addition to enhancing protein accumulation, sub-cellular protein targeting can reduce the effects of protein toxicity on plant cells (Murray et al., 2002) and can also simplify and reduce the cost of protein purification (Boothe et al., 1997; Borisjuk et al., 1999; van Rooijen and Moloney, 1995a, 1995b).

1.1.3.3 Sequence optimisation

Modification of coding and regulatory sequences of heterologous genes to eliminate sequences that may be deleterious to full-length transcription and transcript stability, such as AT-rich sequences that resemble plant mRNA processing sequences, potential polyadenylation signal sequences, and possible mRNA destabilising sequences, can result in increased levels of mRNA (Batard et al., 2000; Gleave et al., 1998; Perlak et al., 1991). Heterologous sequences can also be modified to take into account any disparity between codon bias between donor and host species. Rare codons, particularly if located towards the 5´ end of the mRNA transcript, can reduce the rate of protein translation and result in translation failure (Batard et al., 2000). Significant increases in both mRNA levels and heterologous protein production have been reported in plants transformed using sequence and codon-optimised heterologous genes; however significant increases in transcript stability and heterologous protein production have also been obtained when only the 5´ region of the heterologous gene has been sequence and codon optimised (Batard et al., 2000; Gleave et al., 1998; Perlak et al.,

1991).

1.1.3.4 Alternate host plants

Foreign proteins have been expressed in a range of host plants including leafy crops, cereals and legumes, oil crops and fruit and vegetable crops for the specific purpose of foreign protein production (Twyman, 2004). The protein content of different plants and plant tissues is variable and it has been assumed that the potential recombinant protein yield was dependant on the protein content of the plant or plant tissue in which the protein was expressed (Kusnadi et al., 1997). However, comparisons of scFv accumulation in a number of plants and tissues performed by Stoger et al. (2002) indicated that there was no correlation between the protein content of the host tissue and the heterologous protein yield. Although the effect of host plant on protein yield is unclear, careful host selection can result in total yield increases due to increased crop productivity. The choice of host plant has also been observed to affect heterologous protein stability (Khoudi et al., 1999) and protein characteristics such as glycan diversity (Gomord et al., 2004).

1.1.3.5 Plastid expression

The plastids of higher plants are able to be stably transformed and facilitate the expression of large amounts of heterologous protein (Svab et al., 1990). Multiple genes can be inserted into the plastid genome as an operon because polycistones are efficiently translated in chloroplasts (Daniell and Dhingra, 2002; De Cosa et al., 2001;

Staub and Maliga, 1995). Gene cassettes integrate stably into the plastid genome via homologous recombination so that the positional effects observed in plant nuclear transformation are not observed (Staub and Maliga, 1992). In stably transformed cell lines, genes inserted into the plastid genome can be present with a high copy number because individual plastids may contain up to 60 copies of the plastid genome and multiple plastids (50–60 for chloroplasts) are usually present within each cell (Bogorad,

2000). Proteins expressed in plastids can undergo processing such as disulfide bridge formation and folding; however protein glycosylation does not occur.

De Cosa et al. (2001) reported the accumulation of Bacillus thuringiensis Cry2Aa2 protein to 45.3% TSP in mature leaves and Molina et al. (2004) reported the accumulation of a canine parvovirus peptide fused with the cholera toxin B subunit to

31.1% TSP at full flowering using chloroplast expression; however heterologous protein yields of 5–15% TSP are more commonly observed (Daniell et al., 2004).

Heterologous protein expression has been reported in a range of plastids including chloroplasts, chromoplasts (Hibberd et al., 1998; Kumar et al., 2004; Ruf et al., 2001), amyloplasts (Hibberd et al., 1998) and proplastids (Kumar et al., 2004).

1.1.3.6 Virus-based expression systems

Virus-based vector systems utilise the ability of plant viruses to direct infected plant cells to synthesise large quantities of a limited range of viral proteins to produce heterologous proteins. Plant–virus vectors have been developed to facilitate transient and stable expression of heterologous proteins in plants.

Vectors designed to facilitate the transient expression of proteins in plants have been developed using a variety of viruses that primarily have single-stranded RNA (ssRNA) genomes. Heterologous proteins are co-expressed with viral proteins as the virus replicates and spreads throughout the plant. Expression of foreign proteins can be observed soon (1–3 days) after vector introduction into the plant and, provided the vector retains full viral movement capabilities, protein expression can be obtained throughout the biomass. The characteristics of transient protein expression from viral vectors are outlined in Sections 1.4 and 1.5. A variety of therapeutic proteins and

10 peptides have been expressed in plants using viral vectors. However heterologous protein yields have generally been disappointing with yields not significantly higher than those achievable using nuclear-transformed plants, although relatively high foreign protein yields (10% TSP) have been reported using a tobacco mosaic virus based vector

(Shivprasad et al., 1999).

Stable expression vectors have been developed using ssDNA geminiviruses (Meyer et al., 1992; Mor et al., 2003; Needham et al., 1998) and ssRNA viruses (Angell and

Baulcombe, 1997; Mallory et al., 2002; Mori et al., 2001). The characteristics of each vector system differ but in a number of successful systems (Mallory et al., 2002; Meyer et al., 1992; Mori et al., 2001; Needham et al., 1998) the viral cDNA construct containing the transgene has been randomly inserted into the plant chromosome using

T-DNA from the A. tumefaciens tumor-inducing (Ti) plasmid. The viral construct is stably inserted into the genome, but replicative elements (ssRNA or ssDNA) can be released from the genome and can replicate episomally to high copy number in the transformed cell.

By utilising inserted viral elements in stable transformation systems, it is hoped that the high-level protein expression characteristics of plant viruses can be obtained, but that problems of poor systemic movement (Section 1.5.2) and vector instability (Section

1.5.1) observed in transient viral expression systems can be overcome because replicative elements could potentially be released from the genome of every plant cell.

Episomal replication of the released viral vector may also minimise positional effects on foreign protein expression. Mallory et al. (2002) reported that, in plants stably transformed using two different potato virus X based vectors, GUS accumulated to approximately 3% total leaf protein.

1.2 Plant Tissue Culture

The in vitro culture of plants and plant cells covers a wide variety of techniques and culture types. Culture types demonstrated as suitable for the production of phytochemicals or heterologous proteins will be discussed in this section.

1.2.1 Undifferentiated plant tissue culture

Callus is de-differentiated or poorly differentiated plant tissue. Despite the essentially undifferentiated state of the cells, cell-to-cell contact is retained via plasmodesmata

(Spencer and Kimmins, 1969). Cell de-differentiation occurs under the influence of plant growth regulators which are provided in the medium to maintain culture morphology and promote rapid growth. Callus cultures may be maintained indefinitely but are generally considered to be genetically and biochemically unstable due to the high degree of somoclonal variation exhibited between cultures (Larkin and Scowcroft,

1981; Phillips et al., 1994).

Plant cell suspension cultures are formed by the dispersion of friable callus in liquid medium to form single cells or, more commonly, cell aggregates. Generally, aggregates are formed because dividing cells fail to separate after division (Kieran et al., 1997). The degree of aggregation within a culture depends on the cell line, culture age and also the cultivation conditions. Cells within aggregates retain cell-to-cell contact via plasmodesmata. Suspension cultures are genetically and biochemically unstable. Cultures can exhibit rapid growth with typical doubling times of 2–6 days (Scragg, 1992). While considerably slower than the growth of many microbial and mammalian cell cultures, this is considered rapid compared with other types of plant tissue culture. Bioreactors have been developed that allow the large-scale growth of plant suspension cultures.

Plant cell suspensions have been examined as a potential source of plant secondary metabolites for use in the pharmaceutical and food industries, as a source of plant- produced protein, and as a production system for foreign proteins (Section 1.3.1). Due to the instability of suspension cultures, periodic strain selection is frequently required to ensure that suitable growth characteristics and high product yields are maintained.

Despite considerable research, particularly into secondary metabolite production and the development of suitable bioreactors to permit large-scale production, only a limited number of products, including shikonin, ginseng, berberine and paclitaxel, have been produced commercially using plant suspension cultures.

1.2.2 Differentiated organ culture

Organ cultures comprised of isolated differentiated plant tissues such as roots and shoots can be produced. Directed differentiation of the cells occurs and is maintained by exposure to appropriate hormones. Genetically transformed organ cultures are transformed with genes of bacterial origin that direct and maintain culture differentiation without the addition of plant growth regulators to the medium.

Differentiated cultures are generally genetically and biochemically stable. In untransformed organ cultures, plant growth regulators must be added to the culture medium to maintain culture morphology. Organ cultures have a number of agricultural applications and have also been examined as potential sources of phytochemicals which are often poorly produced in undifferentiated suspension and callus cultures.

1.2.2.1 Root cultures

Root cultures contain differentiated root tissue that grows in vitro in the absence of other types of plant material such as shoots or undifferentiated callus. Anatomically, cultured roots resemble young roots from whole plants and secondary growth

13 characteristics are generally not observed (Butcher and Street, 1964; Payne, 1991;

White, 1936). Vascular tissue is observed in cultured roots (Butcher and Street, 1964;

White, 1936); however it does not function as in the roots of whole plants because transpiration-driven convection does not occur and other driving forces associated with the source and sink tissues in whole plants are also absent (Carvalho et al., 1997). Root growth occurs through cell division in the apical and lateral root meristems and by the enlargement of previously formed cells as roots elongate and thicken (Butcher and

Street, 1964; Payne, 1991).

Untransformed root cultures

Untransformed root cultures can be initiated by inoculating excised root tips into appropriate media. Alternatively, root cultures can be formed by differentiation of callus or suspension cells under the influence of auxin. Continued rapid growth of untransformed roots in culture generally requires the addition of auxin to the medium

(Butcher and Street, 1964), although prolonged growth of roots has been reported in medium without auxin (White, 1934a). Growth rates of cultured untransformed roots are often considered slow; however, with the appropriate addition of auxin, the growth rate of untransformed roots can be as rapid as that of corresponding suspension cultures

(Butcher and Street, 1964; Payne, 1991).

Transformed “hairy root” cultures

Transformed roots or hairy roots are initiated when susceptible host plants are infected with Agrobacterium rhizogenes, a gram-negative soil bacterium. Root induction by

A. rhizogenes infection is associated with the presence of a large (> 200 kb) root- inducing plasmid (Ri plasmid) that contains one or two T-DNA elements and a virulence (vir) region. Three types of Ri plasmid have been identified. These have

14 been designated as agropine, mannopine and cucumopine according to the opine that is encoded by the plasmid.

The agropine-type plasmid is the most virulent. Therefore, A. rhizogenes strains containing this plasmid are most frequently used for hairy root initiation. The agropine plasmid contains two T-DNA regions (White et al., 1985), designated left T-DNA

(TL-DNA) and right T-DNA (TR-DNA). When a wounded plant is infected with

A. rhizogenes, the T-DNA is excised from the Ri-plasmid and transferred into a plant cell. T-DNA regions integrate randomly into the plant genome (Chilton et al., 1982;

Willmitzer et al., 1982; Zambryski, 1988).

The TR-DNA region contains two genes that encode for synthesis of the auxin, indole-3-acetic acid (IAA), and genes that encode for opine (agropine) biosynthesis

(De Paolis et al., 1985; White et al., 1985). Opines are amino acid and sugar derivatives utilised by Agrobacterium species as a carbon source. The TL-DNA encodes oncogenes that direct transformed cells to differentiate into hairy roots in the presence of endogenous auxin (White et al., 1985). Cell transformation with TL-DNA is essential for hairy root induction (Cardarelli et al., 1987). Co-transformation with

TR-DNA is associated with increased virulence and expanded host range for hairy root initiation due to increased endogenous auxin levels (Cardarelli et al., 1987; White et al.,

1985). Cell transformation with TR-DNA alone can result in root initiation as a result of high endogenous auxin concentrations; however as the TL encoded oncogenes are not present, the initiated roots are not hairy roots (McInnes et al., 1989; Vilaine and Casse-

Dilbart, 1987). Mannopine and cucumopine strains have only one T-DNA region, which contains genes encoding for opine synthesis and root-inducing oncogenes. These

15 strains can initiate hairy root formation, but dependence on host plant auxins results in reduced virulence (Payne, 1991).

Neoplastic hairy roots can be excised from the infected tissue and maintain differentiated growth in plant growth regulator free medium. Hairy root cultures are characterised by a high degree of lateral branching and plagiotropic growth. The morphology of individual root clones (arising from a single transformed cell) is dependent on a variety of factors including the host plant (Carvalho et al., 1997), the

A. rhizogenes strain utilised for transformation, the number and location of integrated

T-DNA molecules in the host DNA (Payne, 1991), the ratio of integrated TL-DNA to

TR-DNA (Jouanin et al., 1987; Payne, 1991; White and Sinkar, 1987) and the conditions utilised for maintenance of the cultures (Carvalho et al. 1997; Kino-Oka et al., 1999). Hairy roots are generally considered to display greater genetic and biochemical stability than suspension cultures (Sevón et al., 1998).

Hairy roots can be propagated indefinitely in defined medium and often maintain their morphological integrity and stability in the absence of exogenous growth regulators, although addition of auxin to the medium of some hairy root cultures is associated with increased growth rates (Payne, 1991). Hairy roots cultured in liquid medium can exhibit rapid growth rates, which in some cases is comparable to that of suspension cultures (Payne, 1991; Sevón and Oksman-Caldentey, 2002).

Transformed hairy roots have been examined extensively for secondary metabolite production and biotransformation (Galun and Galun, 2001; Giri and Narasu, 2000;

Wysokinska and Chmiel, 1997). Hairy roots exhibit similar, although not always identical, biosynthetic profiles to parental plant roots and can also accumulate

16 secondary metabolites at levels similar to those observed in the roots of whole plants.

Foreign gene expression in hairy roots has been utilised to modify and enhance the biosynthetic capacities of some hairy root cultures (Sommer et al., 1999) and has allowed the accumulation of foreign proteins (Ehlers et al., 1991; Wongsamuth and

Doran, 1997). Plants regenerated from hairy roots also have horticultural applications due to phenotypic changes associated with oncogene expression (Christey, 2001).

Although hairy root cultures have significant industrial potential, they have yet to be utilised commercially for the production of plant-based products. The main limiting factor is the difficulty associated with designing large-scale bioreactor systems that provide a low-shear environment while maintaining adequate mixing and oxygen transfer. Reviews of the main reactor types examined for large-scale hairy root culture are provided by Giri and Narasu (2000), Kim et al. (2002) and Wysokinska and Chmiel

(1997).

1.2.2.2 Shoot cultures

Shoot cultures exhibit a proliferation of the aerial parts of a plant (leaves and stems) in vitro without the presence of roots or undifferentiated tissue (callus). Untransformed shoot cultures can be initiated from callus by modifying exogenous plant growth regulator concentrations to increase cytokinin levels or by exposing sterile explants to suitable combinations of plant growth regulators. Transformed shoot cultures can be initiated by infecting susceptible host plants with nopaline strains of A. tumefaciens or strains of A. tumefaciens with mutations in either of the auxin genes contained on the Ti plasmid. Shoots which develop as a result of cell transformation with T-DNA from the

Ti-plasmid are referred to as shooty teratomas and are capable of continued differentiated growth in the absence of exogenous plant growth regulators.

Shoot cultures are genetically and biochemically stable. Culture growth is often slower than that of corresponding suspension cultures; while culture doubling times of less than 3 days have been reported for some cultures (Subroto et al., 1996), significantly slower culture growth is often observed (Payne, 1991). Light is required for shoot greening and leaf development, and can also be required for the synthesis of some metabolites. Culture vitrification can occur if shoots are grown submerged in liquid medium.

Untransformed shoot cultures have been utilised for the micropropagation of horticultural plants. Additionally, both transformed and untransformed shoot cultures have been utilised for the study of biosynthetic pathways within plants and the production of aerially synthesised plant chemicals, particularly secondary metabolites.

However, the commercial utilisation of shoot cultures has been limited by the inability to scale production.

1.3 Plant Tissue Culture as a Production System for Heterologous

Proteins

Whole-plant-based expression systems have been demonstrated to be suitable for the production of heterologous proteins with regard to both protein integrity and activity.

In some situations, agricultural production has also been shown to be commercially competitive with other production systems. However for some therapeutic applications, agricultural production may not provide adequate assurance of product safety or quality, even when plant-produced proteins have appropriate biological activities. For example, heterologous proteins produced in field-grown plants may be subject to contamination with pesticides, herbicides and mycotoxins. Field-grown plants also

18 experience variable weather conditions, non-uniform soil composition, disease and insect infestation. Individually or in combination, these factors may result in inconsistent product yield and quality. The inability to control production conditions could mean that agriculturally produced whole-plant systems fail to comply with good manufacturing practice in many countries, particularly if the transgenic protein is to be used as a human therapeutic. Issues of environmental crop safety may also arise, particularly if heterologous proteins are toxic to soil microorganisms or to wild-life capable of consuming the plants.

When plant-derived products have suitable activity but contamination, inconsistent product yield and quality or regulatory issues prohibit the use of agricultural methods of production, large-scale plant tissue culture offers an alternative route for foreign protein production. This is particularly so if the volume of protein required is small.

Although large-scale plant tissue culture production of foreign proteins may be an alternative when plant-based production is deemed unsuitable, plant tissue culture is not economically competitive with whole plant systems. However plant tissue culture offers a number of advantages over the conventional animal cell culture methods currently applied to produce biopharmaceutical proteins. As plant cell culture media are relatively simple in composition and do not contain proteins, the cost of the process raw materials is reduced and product recovery from the medium is easier and cheaper compared with animal cell culture. In addition, as most plant pathogens are unable to infect humans, the risk of pathogenic infections being transferred from the culture via the product is also reduced.

1.3.1 Foreign protein production using plant tissue culture

Table 1.1 lists foreign proteins with human therapeutic and commercial applications that have been produced using plant tissue culture. Details of the expression systems, including culture type and species, promoter and signal sequence, and transformation method are presented with protein yields when reported. Protein production in protoplasts has not been reported as, to date, protoplast culture does not represent a viable production system for the large-scale production of heterologous proteins.

The production of foreign proteins using plant tissue culture has been examined at a research level only with the majority of investigations performed in shake flasks, although some small-scale bioreactor investigations have also been reported (Fischer et al., 1999b; Liu and Lee, 1999; Terashima et al., 1999a and 1999b; Trexler et al.,

2002; Verdelhan des Molles et al., 1999; Wongsamuth and Doran, 1997). As indicated by the protein accumulation levels in Table 1.1, low to moderate levels of foreign protein have been achieved in plant tissue culture systems. To take advantage of the cost benefits associated with recovering proteins from the culture medium rather than the biomass, expression systems that allow protein secretion into the medium have been developed. However, relative to production levels achieved using animal cell cultures, foreign protein concentrations achieved in plant cultures are typically low. In addition, the low growth rates of cultured plant cells result in long production cycles. Hellwig et al. (2004) estimated that expressed and recoverable protein levels from plant cell cultures were approximately one to two orders of magnitude below the threshold of economic feasibility.

Table 1.1 Expression of foreign proteins in plant tissue culture

Foreign protein Culture Plant Transformation method Promoter Signal sequence Production level Reference type species (maximum) Antibody, scFv, Suspension N. tabacum A. tumefaciens transformation CaMV 35S Tobacco 0.5 mg L-1 (e) Firek et al., against of leaf explants pathogenesis 5.0% of total medium 1993 phytochrome related protein protein (e) Antibody, scFv, Callus Oryza Microparticle bombardment of Maize Murine heavy- 0.45 μg g-1 fresh weight Torres et al., against sativa (rice) callus ubiquitin-1 and light-chain without KDEL (i) 1999 carcinoembryoni IgG 3.8 μg g-1 fresh weight c antigen with KDEL (i) Antibody, scFv, Suspension N. tabacum A. tumefaciens transformation CaMV 35S Sporamin 5 mg kg-1 fresh weight (i) Ramírez et against hepatitis of leaf explants 1 mg L-1 (e) al., 2000 B surface antigen Antibody, human Suspension N. tabacum A. tumefaciens transformation CaMV 35S Native human 8.0 mg L-1 (t) Yano et al., against hepatitis of suspension 0.2% TSP (t) 2004 B virus surface N. plumbaginfolia 16.2 mg L-1 (t) Yano et al., antigen calreticulin 0.6% TSP (t) 2004 Barley 7.5 mg L-1 (t) Yano et al., hordothionin 0.25% TSP (t) 2004 Antibody, heavy Suspension N. tabacum A. tumefaciens transformation CaMV 35S Native murine 150 μg L-1 (t) Wahl et al., chain, against of suspension 430 μg L-1 (t) with DMSO 1995 p-azophenyl- 170 μg L-1 (i) Magnuson et arsonate 10 μg L-1 (e) al., 1996 360 μg L-1 (e) with PVP 80 μg mL-1 (i) Liu and Lee, 300 μg mL-1 (e) 1999 Antibody, murine Suspension N. tabacum A. tumefaciens transformation Enhanced Murine 15 μg g-1 wet weight (i) Fischer et al., IgG -2b/κ, of leaf explants CaMV 35S 0.3% TSP (i) 1999a against tobacco 45 μg g-1 wet weight (i) mosaic virus with amino acids

Table 1.1 (continued)

Foreign protein Culture Plant Transformation method Promoter Signal sequence Production level Reference type species (maximum) Antibody, murine Suspension N. tabacum A. tumefaciens transformation CaMV 35S Murine Ig 1.2 mg g-1 dry weight (t) Sharp and -1 IgG1, against of leaf explant, regenerated 7.5 mg L (t) Doran, 2001a Steptococcus plants sexually crossed 3.6 mg L-1 (e) mutans surface 6.5% TSP (t) antigen 12% TSP (t) with PVP Hairy root 18 mg L-1 (t) Wongsamuth 1.8% TSP (i) and Doran, 3.2 mg L-1 (e) 1997 10.8 mg L-1 (e) with PVP 1.1 mg g-1 dry weight (t) Sharp and 7.0 mg L-1 (t) Doran, 2001a 1.4 mg L-1 (e) 3.0% TSP (t) 4.0% TSP (t) with PVP Shooty 0.28 mg g-1 dry weight (t) Sharp and teratoma 3.2 mg L-1 (t) Doran, 2001a α-antitrypsin, Suspension O. sativa Microparticle bombardment of Rice α- Rice α-amylase 85 mg L-1 (e) Terashima human suspension amylase, 5.7 mg g-1 dry weight (e) et al., 1999a inducible 51 mg L-1 (e) Trexler et al., 2002 Bryodin 1 Suspension N. tabacum Microparticle bombardment of CaMV 35S Extensin 30 mg L-1 (e) Francisco suspension et al., 1997 Cytochrome Hairy root Atropa A. rhizogenes transformation of CaMV 35S Not reported Not reported Banerjee P450 2E1, rabbit belladonna leaf explant et al., 2002 Erythropoietin, Suspension N. tabacum A. tumefaciens transformation Modified Native human 0.8 μg L-1 (t) Matsumoto human of suspension CaMV 35S erythropoietin 0.0026% TSP et al., 1995a

Table 1.1 (continued)

Foreign Culture Plant species Transformation method Promoter Signal sequence Production level Reference protein type (maximum) Granulocyte Suspension N. tabacum A. tumefaciens transformation Enhanced Native hG-CSF 105 μg L-1 (i) Hong et al., colony- of leaf explants CaMV 35S 2002 stimulating factor, human (hG-CSF) Granulocyte- Suspension N. tabacum A. tumefaciens transformation CaMV 35S Tobacco etch 150 μg L-1 (i) James et al., macrophage of callus virus 240 μg L-1 (e) 2000 colony- 0.5% total medium protein stimulating (e) factor, human A. tumefaciens transformation CaMV 35S Native 180 μg L-1 (e) Lee et al., (hGM-CSF) of leaf explants mammalian 783 μg L-1 (e) with gelatin 2002 270 μg L-1 (e) with PVP Kwon et al., 2003a O. sativa Microparticle bombardment of Rice α- Rice α-amylase 129 mg L-1 (e) Shin et al., callus amylase, 25% total medium protein 2003 inducible (e) Lycopersicum A. tumefaciens transformation Enhanced Native 45 μg L-1 (e) Kwon et al, esculentum of hypocotyls segments CaMV 35S hGM-CSF 2003c (tomato) Hepatitis B Suspension Glycine max Microparticle bombardment of Chimeric Not reported 1.7 mg g-1 dry Smith et al., surface antigen (soybean) suspension (ocs)3mas weight (i) 2002 20–22 mg L-1 (i) N. tabacum A. tumefaciens transformation Chimeric Not reported 0.31 mg g-1 dry weight (i) Smith et al., of suspension (ocs)3mas 2002

Table 1.1 (continued)

Foreign protein Culture Plant Transformation method Promoter Signal sequence Production level Reference type species (maximum) Hepatitis B Suspension N. tabacum A. tumefaciens transformation A. thaliana Not reported 1 μg g-1 fresh weight (i) Kumar et al., surface antigen of suspension ubiquitin 5 ng mL-1 (e) 2003 (continued) (ubq3) 2 μg g-1 fresh weight (i) with microsomal ER retention signal 10 ng mL-1 (e) with microsomal ER retention signal Interleukin-2, Suspension N. tabacum A. tumefaciens transformation CaMV 35S Natural 75 μg L-1 (i) Magnuson human of suspension mammalian 10 μg L-1 (e) et al., 1998 Interleukin-4, Suspension N. tabacum A. tumefaciens transformation CaMV 35S Natural 275 μg L-1 (i) Magnuson human of suspension mammalian 180 μg L-1 (e) et al., 1998 Interleukin-12, Suspension N. tabacum A. tumefaciens transformation Enhanced Native human 60 μg L-1 (i) Kwon et al., human of leaf explant, regenerated CaMV 35S interleukin-12 175 μg L-1 (e) 2003b plants sexually crossed subunit 700 μg L-1 (e) with gelatin Invertase, carrot Suspension N. tabacum A. tumefaciens transformation CaMV 35S Native carrot 1400 U L-1 (i) Verdelhan des of suspension invertase 40 U g-1 dry weight (i) Molles et al., 150 U L-1 (e) 1999 Lysozyme, Suspension O. sativa Microparticle bombardment of Rice Rice α-amylase 4% TSP Huang et al., human callus α-amylase, 2002b inducible Milk protein Suspension N. tabacum A. tumefaciens transformation CaMV 35S Native CD14 1.5 μg L-1 (e) Girard et al., sCD14, human of suspension 2004 Optimised 5 μg L-1 (e) Girard et al., tomato extension 2004 Ricin Suspension N. tabacum A. tumefaciens transformation CaMV 35S Not reported 25 ng L-1 (e) Sehnke and of leaf explants 0.05% TSP (e) Ferl, 1999 i = protein in the biomass; e = protein in the medium; t = total protein CaMV = cauliflower mosaic virus; DMSO = dimethylsulphoxide; PVP = polyvinylpyrrolidone; TSP = total soluble protein; ER = endoplasmic reticulum

23 24

Low levels of heterologous protein expression are observed in both agricultural- and plant-tissue-culture-based systems. Considerable research has been carried out into the molecular aspects of foreign protein production in whole plant expression systems to enhance the yield, quality and stability of the product and to facilitate protein separation and purification from the biomass (Section 1.1.3). Increasing the recoverable yield of foreign protein has also been a key research objective in plant tissue culture systems; however, little research has been undertaken to investigate specific issues associated with producing foreign proteins in plant tissue culture.

1.3.2 Increasing protein expression and recovery

The factors contributing to low foreign protein yields in plants and plant tissue cultures are not fully understood. Research aimed at increasing in vitro foreign protein yields have focused on two general areas. These are: (i) to increase the level of gene expression by cultured cells by altering transgenic constructs, methods of expression and culture systems, and (ii) to increase protein recovery by increasing the retention and stability of foreign proteins in the cultures and, in particular, the culture medium.

1.3.2.1 Increased protein expression

Compared to whole plants, there has been limited development of foreign protein expression systems designed specifically for use in plant tissue culture, with approaches found to be successful in whole plants often subsequently applied to cultured cells.

Some developments, such as the optimisation of coding sequences, are fully transferable to cultured plant cells; however other approaches have limited utility in cultured cells due to the different levels of tissue organisation and also differences in the desired characteristics of protein expression, such as secretion in cultured cells.

Promoter sequences

Transcription of foreign protein-encoding mRNA in cultured plant cells and organs generally occurs constitutively, under the control of the CaMV 35S promoter or the enhanced 35S promoter in cultures of dicotylenedonous origin, and the maize or

Arabidopsis ubiquitin promoters in cultures of monocotyledonous origin. The use of the constitutive chimeric ocs-mas promoter has also been reported in tobacco, soybean and carrot suspension cultures to control expression of the hepatitis B surface antigen

(Smith et al., 2002). In tobacco suspension cells, the ocs-mas promoter has been reported to direct higher levels of GUS expression than the CaMV 35S promoter

(Ni et al., 1995).

A range of inducible promoters have been developed and used in plant culture applications (David and Perrot-Rechemann, 2001; Hughes et al., 2002; Kurata et al.,

1998; Sommer et al., 1998; Suehara et al., 1996; Terashima et al., 1999a; Xiang et al.,

1996; Yoshida et al., 1995); however only the rice α-amylase promoter has been applied to the production of a pharmaceutical or commercial protein. The

α-amylase promoter controls the production of α-amylase isomerase, one of the most abundant proteins secreted from cultured cells after sucrose starvation. The use of this inducible promoter allows separation of cell growth and heterologous protein production by the use of two different media (Terashima et al., 1999b). Culture growth occurs in a sucrose-containing medium; when this medium is replaced by a sucrose-free production medium, frequently containing mannitol to maintain culture osmotic pressure, heterologous protein expression is induced. If appropriate signal peptides are used, the heterologous protein can be secreted into the culture medium, which, having been recently exchanged, is low in protease. The α-amylase promoter has been used for

expression of human α1-antitrypsin (Huang et al., 2001; Terashima et al., 1999a, 1999b;

Trexler et al., 2002), human granulocyte-macrophage colony-stimulating factor (hGM-

CSF) (Shin et al., 2003) and human lysozyme (Huang et al., 2002b) in Oryza sativa

(rice) suspension cultures.

Protein targeting

In whole plants, the addition of signal sequences to heterologous proteins to control sub-cellular localisation was observed to significantly affect protein accumulation

(Section 1.1.3.2). The effect of protein subcellular localisation on protein yield is similar in whole plants and cultured cells (Torres et al., 1999). In culture-based systems, despite lower potential yields, proteins are frequently targeted to accumulate in the apoplast from where they may be secreted into the medium to take advantage of the potential cost benefit of purifying heterologous proteins from the medium rather than the biomass. Purification of proteins from the medium can be facilitated by the addition of molecular tags to foreign proteins. The addition of a functional His6 tag to hGM-

CSF has been examined in tobacco suspension cultures (James et al., 2000) and allowed the specific removal of hGM-CSF from the culture medium using iminodiacetic acid metal affinity resin (James et al., 2002).

Culture species and type

In addition to altering expression constructs, altering host species and culture type have been observed to influence protein accumulation. As indicated in Table 1.1, most heterologous proteins have been produced in N. tabacum suspension cultures. A comparison of hepatitis B surface antigen production in suspensions derived from soybean, tobacco and carrot revealed considerable differences in foreign protein

27 accumulation in the cultures (Smith et al., 2002). Stable, high-level (65 μg g-1 fresh weight) expression was achieved in soybean, whereas maximum antigen accumulation was approximately 10-fold lower in N. tabacum suspensions. Lower accumulation in tobacco was attributed to a lower transgene copy number and strong down-regulation of antigen expression with successive sub-cultures of the suspension. Levels of antigen observed in carrot suspension cultures were approximately 2-fold lower than those in down-regulated tobacco suspensions. Rice has been used by several groups for the expression of heterologous proteins. Shin et al. (2003) reported that when using the rice α-amylase promoter inducible by starvation, hGM-CSF levels accumulated to 129 mg L-1 in the medium of rice suspension cultures. This was up to 1000-fold higher than the level observed when hGM-CSF was expressed in tobacco suspensions cultures using the CaMV 35S promoter sequence (James et al., 2000; Shin et al., 2003). Low protease activity in the medium of rice suspension cultures was suggested as a contributing factor (Shin et al., 2003). During the production phase, protease levels in the medium of rice suspension cultures were approximately one-quarter the levels observed in the medium of transgenic tobacco cultures.

Sharp and Doran (2001a) examined the accumulation of an antibody in tobacco suspensions, hairy roots and shooty teratomas. Antibody accumulation in shooty teratomas was low and the cultures were also morphologically unstable. Antibody accumulation was initially found not to differ significantly between suspension and hairy root cultures. However, over a period of one year, antibody titres in the suspensions decreased by 87%, while production levels in the hairy roots remained stable. Reductions in foreign protein expression were also observed in tobacco

28 suspensions expressing hepititis B surface antigen, but not in carrot suspensions (Smith et al., 2002), indicating that yield instability is not common to all suspension cultures.

Alternate transformation

Significant increases in foreign protein accumulation have been observed in whole plants using plastid transformation and transformation of plants using viral vectors.

Foreign protein expression has been reported in callus and suspension cultures containing transformed plastids (Daniell et al., 1990; Kumar et al., 2004). Plastid- transformed carrot suspensions expressing betaine aldehyde dehydrogenase (BADH), an enzyme which, when over-expressed, can enhance tolerance to salt stress, were able to survive and proliferate in the presence of high salt concentrations better than other cultures (Kumar et al., 2004). BADH activity in proplastid-containing callus was

53.1% of the activity observed in leaf chloroplasts. The application of plastid transformation for the production of therapeutic proteins may be limited as plastid- translated proteins are not glycosylated.

Expression systems involving viral vectors have been utilised for foreign protein production in cultured bacterial and animal cells; however, application of virus-based expression systems in cultured plant cells has been very limited and, in the case of non- integrative transient expression vectors, has not been reported. Mor et al. (2003) reported the development of a nuclear-integrating vector based on the genome of the bean yellow dwarf virus that was biolistically introduced into the genome of tobacco suspension cells. Green fluorescent protein (GFP) and GUS were expressed in the suspension cells with GUS accumulating up to 0.08% TSP. Palmer et al. (1999) stably transformed black Mexican sweetcorn suspension cells with an episomally replicating

29 viral vector based on maize streak virus. The selectable marker, bialaphos resistance gene, and a 500-base-pair maize intron were expressed in the suspension cells.

Bioprocess developments

Bioprocess developments have been associated with some increases in protein accumulation in plant tissue culture. Immobilisation of N. tabacum cells in alginate resulted in an increase in hGM-CSF production, although similar yield increases were not observed for human interleukin-4 production (Bodeutsch et al., 2001). Adequate aeration of plant tissue cultures is crucial for achieving maximum production of foreign proteins (Liu and Lee, 1999; Sharp and Doran, 2001a).

1.3.2.2 Increased protein recovery

In cultured transgenic plant cells, heterologous proteins are frequently secreted into the medium because protein purification from plant culture medium is potentially less expensive and complicated than protein recovery from the biomass. However, if high yields of heterologous proteins are to be recovered after secretion, foreign proteins must be retained stably in the medium prior to purification. The simplicity of plant culture medium is considered an advantage for foreign protein production. However, as a mixture of salts and sugar containing only a limited amount (and variety) of protein secreted from the plant cells themselves, plant culture medium provides an environment for secreted foreign proteins that is very different from the physiological conditions inside the cell. Tsoi and Doran (2002) observed significant differences between the extent of IgG1 antibody retention in sterile Murashige and Skoog (MS) and Gamborg’s

B5 plant culture media and in medium designed to support the growth of animal cells.

After 7 hours, approximately 80% and 70% of added antibody was retained in

Dulbecco’s minimal essential medium (DMEM) containing 10% fetal bovine serum and in serum-free Ex-Cell 302 medium, respectively. In contrast, in both plant culture media, less than 10% of the added antibody could be detected after only 2 hours, indicating that fresh, sterile plant culture media does not support the retention and stability of proteins in solution.

There have been many reports indicating that plant culture medium is not conducive to protein stability, and that the retention of secreted proteins in culture medium is poor

(James et al., 2000; LaCount et al., 1997; Lee et al., 2002; Magnuson et al., 1996;

Sharp and Doran, 2001a; Shin et al., 2003; Tsoi and Doran, 2002; Wahl et al., 1995).

The mechanisms responsible for protein loss from plant culture medium are not completely understood, however current indications are that multiple factors are involved. Processes that have been proposed include protein degradation (Kwon et al.,

2003a, 2003b; Sharp and Doran, 1999; Shin et al., 2003; Terashima et al., 1999b), protein instability due to defined or undefined conditions or components in the medium

(James et al., 2000; LaCount et al., 1997; Lee et al., 2002; Tsoi and Doran, 2002; Wahl et al., 1995), surface adsorption of proteins onto the culture vessels (Doran, 2006a;

Magnuson et al., 1996; Sharp and Doran, 2001a), and protein aggregation or insolubility (Sharp and Doran, 2001b).

Declining levels of foreign protein in tissue culture medium have been associated in some cases with an increase in extracellular protease activity (Kwon et al., 2003a,

2003b; Lee et al., 2002). Alternative culture techniques such as adjusting medium osmolarity to minimise cell disruption and protease release (Terashima et al., 1999b), the use of separate media for culture growth and production phases (Shin et al., 2003)

31 and the use of cultures such as rice which exhibit reduced extracellular protease activity compared with the commonly used tobacco (Shin et al., 2003) have been associated with increased heterologous protein production. The addition of protease inhibitors

(Sharp and Doran, 1999) and alternative protease substrates such as gelatin (Kwon et al., 2003b) to culture medium to reduce the protease degradation of heterologous proteins has also been examined.

Addition of protein stabilising agents such as polyvinylpyrrolidone (Kwon et al., 2003a;

LaCount et al., 1997; Magnuson et al., 1996; Sharp and Doran, 2001b; Wongsamuth and Doran, 1997), bovine serum albumin (James et al., 2000), gelatin (Kwon et al.,

2003b; Lee et al., 2002; Wongsamuth and Doran, 1997) and salt (NaCl) (James et al.,

2000) to culture medium has also been associated with increased heterologous protein retention. The mode of action of these additives is unclear: they may prevent protein aggregation, conformational changes in the proteins or the adsorption of proteins onto the internal surfaces of the culture vessels. Appropriate stabilising agents must be determined for each production system and protein. The addition of dimethylsulphoxide (Wahl et al., 1995), nitrate (Wongsamuth and Doran, 1997), or amino acids (Fischer et al., 1999a) to plant culture media have been reported in some cultures to result in increased product accumulation.

Improved bioreactor operating strategies also have the potential to enhance foreign protein recovery. Continuous and semi-continuous product recovery from plant culture medium has been used to remove active protein before it can be degraded or otherwise lost from the culture medium. Using a shake-flask-based affinity-chromatography bioreactor to simultaneously produce and purify antibodies from suspension cultures,

James et al. (2002) increased the total yield of secreted protein more than eight-fold. In similar experiments, hGM-CSF with a His6 tag (James et al., 2000) was harvested semi- continuously by recycling the medium through a metal affinity column (James et al.,

2002). Total hGM-CSF production was almost twice that obtained without product recovery.

Protein adsorption to culture vessels has been demonstrated by Doran (2006) to contribute to heterologous protein depletion from culture medium. Coating culture vessels with a protein-repellent polymer (Pluronic 127) was associated with significantly increased accumulation of an antibody secreted by hairy roots, relative to uncoated flasks. However, heterologous proteins were eventually lost from the culture medium, probably as a result of protease activity.

1.4 Plant Viruses as Vectors

1.4.1 Plant viruses

Plant viruses are genetically and morphologically diverse. The genomes of plant viruses can be single-stranded or double-stranded DNA or RNA, although a majority are single-stranded RNA (ssRNA) with positive polarity. The entire viral genome can be encoded on one molecule of nucleic acid (monopartite) or can be spread over a number (generally two or three) of separate molecules of nucleic acid (bipartite or tripartite). The viral genome is encapsidated in particles comprised of virally encoded coat proteins generally arranged in icosahedral or helical symmetry (Varma et al.,

2001).

Infection of plant cells is initiated by the entry of viral particles or the unpackaged viral genome. Animal and bacterial viruses utilise receptors to facilitate the entry of viral particles into cells; however, no such receptors have been identified for plant cells.

Virus infection of plant cells can only occur if the cell wall is mechanically breached, either by physical injury or by transmission vectors such as nematodes, arthropods, arachnids or zoosporis fungi, to allow the entry of viral particles. After entry into a suitable plant cell, viruses utilise plant replication machinery and virally encoded proteins to replicate. The replicated genome and virally encoded coat proteins self- assemble into viral particles and the particles may accumulate to high levels within the infected cells.

From the initial site of infection, assembled virus particles or viral nucleoprotein complexes can move through plasmodesmata and infect adjacent cells (cell-to-cell movement). Viral particles can also enter the plant vascular system and move in the phloem or, less frequently, the xylem (Leisner, 1999) from virally infected source material to uninfected sink material to initiate new sites of infection (long-distance movement). Via a combination of cell-to-cell and long-distance viral movement, plants can become systemically infected with virus.

Viral particles and viral genomic RNA can enter the cells of host and non-host plants, but virus will be able to replicate and move only in host plants. However, even host plants differ in the extent to which viral multiplication and movement are facilitated.

Host plants must support cell-to-cell and long-distance viral movement if they are to become systemically infected with virus. If a host is unable to facilitate long-distance

34 viral movement only local symptoms will develop, and if cell-to-cell movement is not facilitated infection will not move from the initially infected cells.

Different viruses can each infect a range of plants, generally at least 30 species (Gibbs and Harrision, 1976), although some viruses have significantly broader and narrower host ranges. Not all plants within the host range of a virus will be susceptible to infection to the same degree, and the level of viral accumulation, the movement of the virus and the severity of the symptoms associated with infection vary for individual virus:plant combinations. Virus infection of host plants is associated with the development of local and systemic symptoms, which depend on both the infecting virus and the host plant. Common symptoms include chlorosis, necrosis and changes in growth, including restricted growth and abnormal growth patterns.

1.4.2 Plant-virus-based vectors

Plant-virus-based transient expression vectors utilise characteristics of virus infection of plants, such as rapid viral replication, high levels of viral structural protein expression and systemic viral dissemination, to produce RNA or express foreign proteins in plants.

Virus-based vectors designed to facilitate transient protein expression in plants have generally utilised viruses that possess mono- or multipartite positive-sense ssRNA genomes. Using reverse genetics, these relatively small genomes can be readily manipulated into cDNA form. Infection of plants with vectors is most commonly achieved by mechanically inoculating leaves with naked RNA transcripts from the cDNA vectors or with viral particles formed by packaging the RNA transcripts in vitro.

Inoculation of plants directly with infectious cDNA vectors has also been reported

(Arazi et al., 2002; Ding et al., 1995) but has been infrequently utilised. Infiltration of plant leaves with A. tumefaciens containing plasmids based on viral vectors has also been demonstrated to be an efficient means of transforming plants (Liu and

Lomonossoff, 2002). If a vector is able to be packaged in planta using virally or host- encoded capsid proteins, it can be passaged through a host plant and the resulting viral particles used as inoculum for mechanical infection procedures.

RNA viruses replicate in the plant cytoplasm and, as a result, foreign protein expression from RNA-virus-based vectors is not influenced by the positional effects observed in stably transformed plants that can contribute to unpredictable protein yields.

If suitable vector and host plant combinations are used, cell-to-cell and long-distance movement of the vector from an initial site of infection can result in systemic viral infection and foreign protein expression throughout the plant. Foreign protein (GFP) expression has been observed one to three days after infection in inoculated leaves

(Casper and Holt, 1996; MacFarlane and Popovich, 2000; Takamatsu et al., 1987) and four days post-infection in un-inoculated leaves (MacFarlane and Popovich, 2000;

Casper and Holt, 1996). Systemic viral infection and associated foreign protein expression are often observed one to three weeks post-infection, although the timing and extent of vector dissemination are dependent on the vector and the host plant utilised. The ability of viral vectors to establish systemic infections allows relatively established plants with considerable biomass to be infected with vectors containing a gene of interest, resulting in the infection of large amounts of biomass and the rapid production of foreign protein. The rapid expression of foreign proteins is particularly

36 advantageous when the vector or expressed protein has a detrimental effect on plant growth.

The large number of plant viruses potentially available for use as vectors means that the range of plants amenable to transformation using viral vectors is very large. Plants which are difficult to transform using traditional stable transformation methods, such as legumes (Masuta et al., 2000) and crop cereals (Choi et al., 2000), can be readily transformed using vectors developed using viral pathogens of these plants. The varied host range exhibited by individual viruses also allows multiple host species to be transformed using the same vector construct.

1.4.3 Vector design

Viral vectors are derived from wild-type viral genomes that are modified to allow efficient foreign protein or RNA production. Several general approaches to viral vector design have been employed to obtain relatively stable, high-protein-expressing, easy-to- use and safe vectors. The two most frequently utilised approaches to vector design are to replace viral genes with the genes encoding foreign proteins (replacement vectors) or to insert the foreign-protein-encoding genes into the viral genome while retaining all the essential viral genes (full-function vectors).

In replacement vectors, viral genes that are not essential for replication, such as coat protein or movement protein genes or genes essential for virulence or insect transmission, are removed and replaced by genes encoding heterologous proteins

(MacFarlane and Popovich, 2000; Takamatsu et al., 1987). This approach to vector construction is advantageous when the vector genome size is limited by RNA

37 packaging constraints or when the wild-type virus genome contains genes that can be deleted without affecting virus multiplication or movement. However some virus functionality, such as systemic movement, can be lost, particularly if the genes required for viral movement or virion formation are removed. Vectors that do not retain all the essential genes for viral replication and movement can be fully functional in transgenic host plants that are stably expressing the required viral proteins (Gleba et al., 2004).

The genome packaging constraints of some viruses are relatively flexible allowing vectors with genomes significantly longer than the parental genome to be packaged.

Vectors based on viruses with flexible packaging constraints can contain genes encoding heterologous proteins in addition to all the genes required for full virus function. Even though vectors may contain all essential viral genes, full functionality may not be retained.

Non-native viral sequences can be utilised in vector construction if the nucleotide sequences or encoded proteins are functionally complementary to native sequences or viral proteins, resulting in the formation of chimeric vectors (Casper and Holt 1996;

Kumagai et al., 1993; Kumagai et al., 1995; Shivprasad et al., 1999; Yusibov et al.,

2002).

1.4.4 Approaches for foreign protein expression

Using viral vectors, heterologous proteins can be produced as fusions with viral structural proteins or as free cytosolic proteins.

1.4.4.1 Fusions

Heterologous proteins can be expressed as fusions with the viral coat protein, with the coat protein fusions retaining the ability to self-assemble into particles. Assembled viral particles containing peptide–viral protein fusions have been produced using alfalfa mosaic virus (AIMV), cowpea mosaic virus (CPMV), odontoglossum ringspot virus

(ORSV), plum pox potyvirus (PPV), potato leafroll virus (PLRV), potato virus X

(PVX), tobacco mosaic virus (TMV) and tomato bushy stunt virus (TBSV).

Fusion of foreign proteins or peptides with viral coat proteins can confer advantageous characteristics to expressed foreign proteins. The immunogenicity of epitopes can be enhanced by expression with a carrier protein (coat protein) and individual coat protein fusions or the assembled viral particles can function as epitope presentation systems

(Haynes et al., 1986; Jagadish et al., 1993; Johnson et al., 1997; Yusibov et al., 1997).

Additionally, self-assembly of coat protein fusions from multiple vectors can allow the formation of multivalent virus particles displaying multiple epitopes (Koprowski and

Yusibov, 2001). Heterologous protein stability can be enhanced in planta by fusion to viral coat proteins (Santa Cruz et al., 1996). Recombinant particles can also be readily purified from crude plant extracts (Beachy et al., 1996).

Viral coat proteins can accumulate to very high molar concentrations in plants. By expressing heterologous proteins as fusions with the coat protein, the heterologous

39 protein will be produced in the same molar ratio as the coat protein. Providing vector construction and the foreign protein insert do not significantly affect viral replication or movement, the coat protein fusion can accumulate to similar levels as the wild-type coat protein. However, although proteins may be produced at the same molar ratio as the viral coat protein, proteins expressed as coat protein fusions are often small and the absolute heterologous protein yield may be limited. Lim et al. (2002) produced a fusion between the coat protein of ORSV and the nocistatin (mNST) heptadecapeptide and, although the ORSV vector accumulated to 0.4 mg g-1 fresh leaf weight which was similar to the level of unmodified ORSV accumulation, as the peptide was equivalent to only 9.5% (w/w) of the coat protein-mNST fusion the cleaved nocistatin yield was only

13 µg mg-1 virus or 6.4 nmol mg-1 virus.

To allow particle assembly, foreign protein gene sequences must be positioned in the coat protein gene so that, when the protein is transcribed, the inserted protein is positioned on the ‘outer’ surface of the coat protein. This minimises the effect of the inserted protein on the protein–protein and protein–RNA interactions required for particle formation. Efficient viral particle formation is required for systemic viral movement and steric hindrance associated with coat protein fusions can limit particle assembly (Santa Cruz et al., 1996; Takamatsu et al., 1990) and vector movement.

Viruses differ with regard to the size of the peptide that can be inserted into the coat protein without particle formation being affected, and also the length of RNA that can be packaged within the viral particle. The capsid of TMV, a filamentous virus, can extend to accumulate additional RNA resulting from the insertion of foreign gene sequences into the genome; however, if peptides greater than 24–25 amino acids are

40 inserted into every coat protein molecule, TMV particles fail to self-assemble (Yusibov et al., 1997). CPMV, the most commonly utilised vector for epitope display, contains both large and small coat protein molecules in its capsid and can tolerate the fusion of larger peptides (30 amino acids) to one of the coat protein molecules (Porta et al., 1994,

2003). The size of RNA molecules able to be packaged into the CPMV capsid is also relatively flexible.

When particle assembly is limited by steric hindrance (Hamamoto et al., 1993), assembly can be achieved if a pool of coat protein molecules without fused peptides is provided. The resulting assembled particles contain both free and fused coat proteins.

Free coat proteins have been produced from an additional coat protein gene (Yusibov et al., 1997) by the use of leaky stop codons between the genes encoding the coat protein and heterologous protein (Hamamoto et al., 1993; Turpen et al., 1995), or by the insertion of cleavage sites between the coat protein and foreign peptide (Lim et al.,

2002; Santa Cruz et al., 1996; Smolenska et al., 1998). The reduced steric hindrance has allowed the assembly of viral particles from coat protein fusions displaying larger peptides and proteins, such as GFP (Santa Cruz et al., 1996), a scFv (Smolenska et al.,

1998), and angiotensin-I-converting enzyme inhibitor (Hamamoto et al., 1993).

1.4.4.2 Free proteins

Free heterologous proteins can be produced by introducing an additional open reading frame (ORF) into the vector under the control of a native viral or heterologous viral promoter, or as a cleavage product from viral polyproteins. Production of proteins as free molecules rather than as coat protein fusions can facilitate protein maturation and post-translational modification and, by the use of signal sequences, allows sub-cellular

41 targeting that can increase protein stability and simplify protein purification

(McCormick et al., 1999; Nemchinov et al., 2000).

Additional open reading frames

Free cytosolic proteins can be produced by inserting the ORF encoding the heterologous protein of interest into a vector under the control of an additional viral sub-genomic promoter. Increases in genomic RNA size can be large and, therefore, expression of proteins from separate ORFs requires the use of vectors derived from viruses that do not have strict RNA packaging requirements, such as TMV. Foreign protein production is not linked to the production of viral structural proteins.

Shivprasad et al. (1999) produced a chimeric TMV vector 30B that expressed foreign proteins from a separate ORF using the native TMV coat protein promoter to direct transcription of the foreign gene. The TMV coat protein promoter was used because it facilitates high levels of protein expression. The complete coat protein ORF of a related virus was included in the vector to allow particle assembly and long-distance viral movement. When Cycle 3 GFP was expressed using this vector, GFP RNA accumulated in the upper leaves of infected plants at levels twice those of the coat protein RNA. Cycle 3 GFP accumulated to 10% TSP. Expression of heterologous proteins from separate open reading frames has been demonstrated using vectors developed from AIMV, pea early-browning virus (PEBV), pepper ringspot virus

(PepRSV), PVX, TMV and tobacco rattle virus (TRV).

Expression of foreign proteins as part of the viral polyprotein

Some viruses (i.e. potyviruses and comviruses) translate structural and non-structural proteins as a polyprotein that, after translation, is self-processed by proteinase domains

42 to produce mature viral proteins. Expression of viral proteins as part of a viral polyprotein ensures that, while the transgene is maintained by the vector, the foreign protein will be produced at the same molar ratio as the viral proteins expressed from that polyprotein. Heterologous protein size is limited only by RNA packaging and relatively large proteins such as GUS (Guo et al., 1998; Arazi et al., 2001), GFP (Arazi et al., 2001; Gopinath et al., 2000; Verver et al., 1998) and interferon-alpha 2 (Arazi et al., 2001) have been expressed as part of viral polyproteins, although in some instances rates of vector accumulation (Guo et al., 1998) and the maximum concentration of accumulated vector (Arazi et al., 2001) were significantly lower than those observed in plants infected with wild-type virus or the vector without the foreign gene insert. Expression of heterologous proteins as part of a viral polyprotein has been demonstrated using vectors developed from PPV, clover yellow vein virus (CYVV),

CPMV, wheat streak mosaic virus (WSMV) and zucchini yellow mosaic virus

(ZYMV).

1.5 Foreign Protein Expression Using Plant Viral Vectors

Transient expression of foreign proteins in whole plants using viral vectors provides an alternative to stable transformation for the production of foreign proteins in plants. Use of viral vectors can potentially reduce the time required to produce useful quantities of heterologous proteins in plants, which may allow the production of individualised therapeutic proteins. For example, using a chimeric TMV vector, McCormick et al.

(1999) produced a scFv raised against an immunoglobin from mouse B cell lymphoma within 4 weeks of completing the molecular cloning. The time required to produce complex multi-subunit proteins in plants can also be reduced by infecting an individual plant with multiple vectors encoding different protein subunits. Verch et al. (1998)

43 produced a full-length monoclonal antibody by cloning the heavy- and light-chain antibody genes into separate TMV vectors, and infecting one plant with both vectors.

Expression of multiple heterologous proteins from one vector has also been demonstrated (Masuta et al., 2000). Scale-up times can be significantly reduced using viral vectors, as scale-up can be achieved by increasing the number of plants infected with the viral vector.

A range of heterologous proteins and peptides of varying complexity and size have been expressed in plants using viral vectors. In Table 1.2, expressed heterologous proteins, the viral vectors used to produce them and the vector and protein yields when reported are presented. High yields of heterologous proteins have been observed using viral transient expression vectors; however, as indicated in Table 1.2, reported yields are frequently relatively low.

Large Scale Biology Corporation (California, USA) have utilised TMV-based vectors and a specially developed host plant, N. benthamiana × N. excelsior, which is highly susceptible to viral infection and has advantageous agronomic characteristics (Gleba et al., 2004), to produce heterologous proteins commercially.

A number of characteristics of viral vectors that impact on their utility for foreign protein production are presented below, including vector stability, movement, protein accumulation and safety.

Table 1.2 Examples of vectors expressing heterologous proteins

Virus Protein expression Protein/peptide Yield References strategy Vector/coat Foreign protein protein fusion Alfalfa mosaic virus Fusion with coat protein Respiratory syncytial virus epitope 0.8 mg g-1 fresh leaf 80 μg g-1 fresh leaf Belanger et al., 2000 Rabies virus epitope Modelska et al., 1998 Chimeric peptide containing rabies 0.4 ± 0.07 mg g-1 Yusibov et al., 2002 virus glycoprotein (G protein) and fresh leaf nucleoprotein (N protein) epitopes Free protein from an Green fluorescent protein Sanchez-Navarro additional sub-genomic et al., 2001 promoter Clover yellow vein Free protein from a Green fluorescent protein 46 μg g-1 fresh Masutu et al., 2000 virus polyprotein weight Green fluorescent protein:soybean Masutu et al., 2000 glutamine synthase as a fusion and free proteins Cowpea mosaic virus Fusion with coat protein Canine parvovirus epitopes Nicholas et al., 2002 Foot and mouth disease virus VP1 Porta et al., 1994 epitope Foot and mouth disease virus VP1 Usha et al., 1993 epitope Human immunodeficiency virus-1 1–2 mg g-1 fresh leaf McLain et al., 1995 glycoprotein 41 epitope Human rhinovirus 14 epitope 1.2–1.5 mg g-1 fresh Porta et al., 1994 tissue Mink enteritis virus VP2 capsid protein 1–1.2 mg g-1 leaf Dalsgaard et al., 1997 epitope fresh weight Pseudomonas aeruginosa Protein F Gilleland et al., 2000 epitope Coat protein fusion with Green fluorescent protein 1–2% TPS Gopinath et al., 2000 a protease cleavage site

Table 1.2 (continued)

Virus Protein expression Protein/peptide Yield References strategy Vector/coat Foreign protein protein fusion Cowpea mosaic virus Free protein from a Green fluorescent protein Verver et al., 1998 (continued) polyprotein Odontoglossum Fusion with coat protein Mouse nocistatin peptide 0.4 mg g-1 fresh 6.4 nmol mg-1 virus Lim et al., 2002 ringspot virus with factor Xa cleavage weight 13 μg mg-1 virus linker 3 μmoles kg-1 leaf fresh weight Pea early-browning Free protein from an Green fluorescent protein MacFarlane and virus additional sub-genomic Popovich, 2000 promoter Pepper ringspot virus Free protein from an Green fluorescent protein MacFarlane and additional sub-genomic Popovich, 2000 promoter Plum pox virus Fusion with coat protein Canine parvovirus VP2 capsid protein Fernández-Fernández epitope et al., 1998 Free protein from a β-Glucuronidase Guo et al., 1998 polyprotein Potato leafroll virus Fusion protein with the Green fluorescent protein Nurkiyanova et al., coat protein readthrough 2000 protein P5 Potato virus X Free protein from an Antibody, scFv 210 μg g-1 fresh 258 ng g-1 fresh Roggero et al., 2001 additional sub-genomic weight inoculated weight inoculated promoter leaves leaves 367 μg g-1 fresh 265 ng g-1 fresh weight systemically weight systemically infected leaves infected leaves Blue fluorescent protein Divéki et al., 2002 scFv against PVX coat protein epitope Hendy et al., 1999

Table 1.2 (continued)

Virus Protein expression Protein/peptide Yield References strategy Vector/coat Foreign protein protein fusion Potato virus X Free protein from an Diabody against PVX coat protein Hendy et al., 1999 (continued) additional sub-genomic epitope promoter (continued) Enhanced green fluorescent protein Lawrence and Novak, 2001 Bacillus thuringiensis Cry1AC toxin Lawrence and Novak, 2001 Wound induced proteinase inhibitor, 0.1–0.2% TSP of Lawrence and Novak, poplar leaf 2001 Fusion with coat protein Green fluorescent protein Santa Cruz et al., 1996 with a FMDV 2A Antibody, scFv against diuron 100–250 μg g-1 fresh Smolenska et al., 1998 peptide leaf Free protein from an Green fluorescent protein Toth et al., 2001 internal ribosome entry site (IRES) Tobacco mosaic virus Free protein from an Antibody, heavy chain, against Verch et al., 1998 additional sub-genomic colorectal cancer promoter Antibody, light chain, against colorectal Verch et al., 1998 cancer Antibody, scFv of immunoglobin from 25–800 μg mL-1 McCormick et al., mouse B cell lymphoma interstitial fluid 2003 Foot and mouth disease virus protein 50–150 μg g-1 fresh Wigdorovitz et al., (VP1) leaf 1999 Bet v 1 Birch pollen allergen 250 μg g-1 fresh leaf Krebitz et al., 2000 2.5% TSP Bovine herpes virus-1 glycoprotein D 20 μg g-1 fresh leaf Pérez Filgueira et al., 2003

Table 1.2 (continued)

Virus Protein expression Protein/peptide Yield References strategy Vector/coat Foreign protein protein fusion Tobacco mosaic virus Free protein from an Hepatitis C virus hypervariable region 1 0.07–0.9% TSP Nemchinov et al., (continued) additional sub-genomic fused with cholera toxin subunit B 6–80 μg g-1 fresh 2000 promoter leaf (continued) Human immunodeficiency virus-1 Yusibov et al., 1997 epitope fused with alfalfa mosaic virus coat protein Green fluorescent protein Casper and Holt, 1996 Green fluorescent protein 5% leaf TSP Shivprasad et al., 1999 α-Galactosidase 2% TSP Yusibov et al., 1999 Cycle 3 green fluorescent protein 10% leaf TSP Shivprasad et al., 1999 Cytokines Up to 1% TSP Yusibov et al., 1999 Mal d 2 apple allergen 240 μg g-1 fresh leaf Krebitz et al., 2003 2.4% TSP Single chain version of follicle Dirnberger et al., 2001 stimulating hormone, bovine Rabies virus epitope fused with alfalfa Yusibov et al., 1997 mosaic virus coat protein α-Trichosanthin Up to 2% TSP Kumagai et al., 1993 Free protein from a Rabies peptide (g24) fused with AIMV 50 ± 12 μg g-1 fresh Yusibov et al., 2002 sub-genomic promoter coat protein leaf (no native coat protein) Fusion with coat protein Foot and moth disease virus epitopes 5 mg g-1 leaf 0.3–0.4 mg g-1 leaf Wu et al., 2003 fresh weight Pseudomonas aeruginosa outer Staczek et al., 2000 membrane protein epitope

Table 1.2 (continued)

Virus Protein expression Protein/peptide Yield References strategy Vector/coat Foreign protein protein fusion Tobacco mosaic virus Fusion with coat protein Pseudomonas aeruginosa Protein F Gilleland et al., 2000 (continued) (continued) epitope Coat protein fusion with Angiotensin-I-converting enzyme 100 μg g-1 fresh 7.0 μg g-1 leaf fresh Hamamoto et al., 1993 a modified amber stop inhibitor peptide weight weight codon readthrough Coat protein fusion with Angiotensin-I-converting enzyme 1.2 mg g-1 fresh Sugiyama et al., 1995 a modified amber stop inhibitor peptide weight codon readthrough (vector without insert: 1.8 mg g-1 fresh weight) HIV-1 envelope protein epitope 1.6 mg g-1 fresh Sugiyama et al., 1995 weight (vector without insert: 1.8 mg g-1 fresh weight) Influenza virus hemagglutin epitope (8 2.0 mg g-1 fresh Sugiyama et al., 1995 amino acids) weight Influenza virus hemagglutin epitope 0.8 mg g-1 fresh Sugiyama et al., 1995 (18 amino acids) weight Plasmodium yoelii epitope 3 μM kg-1 leaf fresh Turpen et al., 1995 weight Plasmodium vivax epitope 20 μM kg-1 leaf Turpen et al., 1995 fresh weight Fusion with coat protein Mouse nocistatin peptide 0.8 mg g-1 fresh Lim et al., 2002 with a factor Xa weight cleavage linker Tobacco rattle virus Free protein from Green fluorescent protein MacFarlane and additional subgenomic Popovich, 2000 promoter Galanthus nivalis agglutinin (GNA) 10 μg g-1 root fresh MacFarlane and (snowdrop lectin) weight Popovich, 2000

Table 1.2 (continued)

Virus Protein expression Protein/peptide Yield References strategy Vector/coat Foreign protein protein fusion Tomato bushy stunt Fusion with a truncated Human immunodeficiency virus (HIV) 0.9 mg g-1 fresh leaf Joelson et al, 1997 virus coat protein type 1 glycoprotein 120 epitope tissue HIV nucleoprotein (p24) 5% leaf TSP Zhang et al., 2000 Wheat streak mosaic Free protein from a β-Glucuronidase Choi et al., 2000 virus polyprotein Neomycin phosphotransferase (NPT II) 300 ng mg-1 soluble Choi et al., 2000 protein (leaf) 14 μg g-1 fresh weight (leaf) 507 ng mg-1 soluble protein (root) Zucchini yellow Free protein from a Interferon-alpha 2 430000 IU g-1 fresh Arazi et al., 2001 mosaic virus polyprotein weight 2 μg active protein g-1 fresh weight β-Glucuronidase Arazi et al., 2001 Green fluorescent protein Arazi et al., 2001 Gelonium anti-HIV protein Arazi et al., 2002 Momordica anti-HIV protein Arazi et al., 2002

49 50

1.5.1 Stability

The positive-strand RNA genomes of plant viruses utilised for the construction of transient expression vectors are replicated using RNA-dependent RNA polymerases.

These polymerases facilitate rapid replication and high-level accumulation of viral genomes, but generally have low fidelity so that replication is error-prone. It was originally considered that the low fidelity of RNA polymerases would prevent the use of RNA viruses as protein expression vectors because mutations would be introduced into the inserted foreign sequences. However, Kearney et al. (1993) demonstrated that low-fidelity RNA polymerase was not a practical limitation to the use of TMV as a transient expression vector because mutations accumulated at a low rate in a foreign gene sequence inserted into a TMV-based vector.

RNA-based viral vectors can exhibit sufficient stability to allow the transient expression of foreign proteins in plants. However, over time, the function of the inserted gene can be lost either as a result of the accumulation of mutations in the inserted gene

(Wu et al., 2003; Joelson et al., 1997) or the loss of all or significant portions of the inserted gene as a result of recombination within the vector (López-Moya et al., 2000;

Rabindran and Dawson, 2001). Vectors with deletions in the sequences encoding foreign proteins have been observed to have a competitive advantage over complete vectors (Guo et al., 1998; Porta et al., 1994, 2003). After successive viral transfers, as selection pressures in favour of complete vectors are usually not present, vectors with deleted sequences become the dominant species in infected plants.

Significant differences in stability have been observed between different RNA-based vectors. Vector design has been shown to significantly affect stability and, in

51 particular, the tendency of vectors to lose inserted sequences because of recombination

(Dawson et al., 1989; Porta et al., 1994; Rabindran and Dawson, 2001). The foreign gene sequence inserted into the vector also affects stability, with some sequences deleted more rapidly than others (Arazi et al., 2001; Choi et al., 2000; López-Moya et al., 2000). The size of the foreign gene insert (Arazi et al., 2001), and the insert sequence (Choi et al., 2000) have been associated with vector instability.

Vector stability is generally determined by examining the vector genome length using reverse transcription-polymerase chain reaction (RT-PCR) and monitoring the ability of the vector to facilitate the expression of foreign protein during successive passages

(every two to four weeks) through a host plant. Reductions in foreign protein expression and vector truncation have been reported for some vectors in the second generation of infected plants (Guo et al., 1998; Rabindran and Dawson, 2001), although many vectors exhibit moderate stability (Joelson et al., 1997; Lim et al., 2002; Porta et al., 1994; Wu et al., 2003), allowing successive generations of virus to be used as inocula for plant infection. Porta et al. (1994) reported that after 10 serial passages through host plants, CPMV-based vectors in which antigenic peptides were fused to the small coat protein were detectable using RT-PCR.

1.5.2 Movement of plant viral vectors

The ability of plant viral vectors to accumulate and facilitate foreign protein accumulation in both initially infected tissue and non-inoculated tissue is a desirable characteristic of virus-mediated protein expression. To achieve systemic protein expression, vectors must retain cell-to-cell and long-distance movement capabilities.

For full movement, sufficient levels of movement and coat proteins must be available.

Some protein expression vectors, even if peptide epitopes are displayed on the coat protein, retain rates of cell-to-cell movement (Turpen et al., 1995) and long-distance movement (Fernández-Fernández et al., 1998; Turpen et al., 1995) similar to those of the wild-type virus. However, delayed systemic protein expression is frequently observed. Vector design can influence the extent and speed of movement (Hamamoto et al., 1993; Toth et al., 2002), as can the foreign gene sequence inserted into the vector

(Porta et al., 2003).

The size of a foreign gene insert and the nature of the insert can influence vector dissemination throughout the plant. Many vectors without foreign gene inserts establish systemic infections more rapidly than the same vectors with foreign gene inserts (Toth et al., 2002). The insertion of large gene sequences such as those encoding GFP, GUS and scFv into some vectors is associated with delays in systemic infection and, in some host plants, the complete loss of systemic vector movement

(Choi et al., 2000; Hendy et al., 1999; Shivprasad et al., 1999; Toth et al., 2002). A wheat streak mosaic virus vector without a foreign gene insert and the same wheat streak mosaic virus vector expressing neomycin phosphotransferase (29-kDa), both retained systemic movement in wheat, barley, oat and maize plants; however, when the larger GUS gene was inserted into the vector, systemic movement was retained only in wheat and barley and only cell-to-cell movement was observed in oats (Choi et al.,

2000). A similar loss of systemic movement and protein expression in some host plants was observed for a chimeric TMV-based vector, 30B, when the gene encoding GFP was inserted into the vector (Shivprasad et al., 1999) (Section 1.7.1). Toth et al. (2002) proposed that in vectors with large foreign gene inserts, the increased genetic load resulted in reduced expression of the movement protein required for cell-to-cell

53 movement of vectors, which also limited systemic vector movement. Gene shuffling of the TMV movement protein of the 30B vector expressing GFP resulted in increased rates of cell-to-cell movement in N. benthamiana and N. tabacum cv. Xanthi, the development of systemic infections in N. tabacum cv. Xanthi, and more rapid development of systemic infections in N. benthamiana (Toth et al., 2002).

The nature of some sequences rather than the insert size appears to affect the movement and symptomology of some vectors (Bendahmane et al., 1999; Hendy et al., 1999;

Porta et al., 2003). For example Porta et al. (2003) and Bendahmane et al. (1999) noted that the isoelectric point of peptides fused to the viral coat protein influenced the interaction of virus with the host plants, resulting in altered virus movement and plant symptomology.

1.5.3 Vector and foreign protein accumulation

Plant viral vectors have been investigated for foreign protein expression because of the large amounts of viral proteins often detected in infected plant material. The efficient expression of foreign proteins using viral vectors is dependant on high levels of viral multiplication, systemic vector movement and vector stability. The concentration of viral proteins expressed by an individual virus in a defined host has generally been considered to represent the upper limit for vector-mediated foreign protein expression.

Moderately high levels of protein expression have been achieved using some vectors

(Gopinath et al., 2000; Shivprasad et al., 1999; Wu et al., 2003; Yusibov et al., 1999;

Zhang et al., 2000); however levels of foreign protein accumulation (Table 1.2) are generally lower than those observed for wild-type viral structural proteins in the same host. Despite only moderate levels of protein expression, the potential reduction in

54 development time, the enhanced characteristics of coat protein fusions and the ability to scale-up production have continued to make the use of viral vectors for heterologous protein production attractive.

Heterologous proteins are expressed from plant viral vectors as free proteins or as coat protein fusions. When heterologous proteins are expressed as fusions with the viral coat protein, yields are generally reported as vector yields. When proteins are expressed as free proteins, in addition to the heterologous protein yield, the vector yield may also be reported because comparisons of vector yield with the wild-type virus yield provides an indication of vector efficiency, and because inconsistent vector:protein levels may be indicative of vector or heterologous protein instability (Guo et al., 1998).

1.5.3.1 Accumulation of viral vectors

The alteration of virus sequences required for vector construction can significantly affect the ability of the vector to accumulate in host plants (Choi et al., 2000).

Comparison of accumulation of wild-type virus, vectors without foreign gene inserts and the same vectors expressing foreign proteins indicates whether vector design alone, or the insertion of foreign protein genes, influences the accumulation. The ability of vectors to accumulate to high levels is particularly important when foreign proteins are expressed as fusions to the viral coat protein or are translated as part of a viral polyprotein, as foreign protein levels are directly related to host protein levels.

The accumulation of vector viral particles containing sequences encoding for foreign proteins is frequently the same or not significantly lower than the accumulation of the wild-type virus (Fernández- Fernández et al., 1998; Joelson et al., 1997; Lim et al.,

2002; Porta et al., 1996; Wu et al., 2003), or not significantly lower than the accumulation of the vector without the foreign gene insert (Choi et al., 2001).

However, as with vector movement, it has been observed that the size (Arazi et al.,

2001; Choi et al., 2001) and nature (Porta et al., 2003) of the foreign gene insert can affect vector accumulation.

1.5.3.2 Accumulation of foreign proteins

The ability of vectors to facilitate foreign protein expression, even if the host plant becomes systemically infected with virus, can be highly variable.

Foreign protein gene instability and the instability of the expressed foreign proteins negatively affect foreign protein accumulation. Vector instability ( Section 1.5.1) can result in the rapid deletion of foreign gene sequences so that, although the vector may accumulate to high levels within the plant biomass, heterologous protein yields will be comparatively low. Ideally, vectors should be sufficiently stable to allow high levels of foreign protein production prior to the loss of foreign protein genes; however the sometimes extended period between plant infection and the establishment of systemic infection can result in reduced efficiency of foreign protein expression due to an increasing proportion of the vector population accumulating mutations in or deletions of the foreign gene insert.

Foreign protein instability in plant cells is largely independent of the protein expression system and dependent on the host plant. Turnover of heterologous proteins expressed from viral vectors has been reported in whole plants (Arazi et al., 2001). As in stably transformed whole plants, signal sequences can be utilised to target protein

56 accumulation to different regions within the cell (Hendy et al., 1999; McCormick et al.,

1999; Nemchinov et al., 2000) (Section 1.1.3.2); this can reduce protein degradation and simplify purification. However, signal sequences have been infrequently used in viral expression system. Protein stability can be enhanced by expressing proteins as fusions with the viral capsid protein. Assembled viral particles are highly stable and foreign proteins assembled into viral particles have enhanced stability (Fernández-

Fernández et al., 1998).

Heterologous protein expression within the plant biomass can be non-uniform (Arazi et al., 2001; Choi et al., 2000). Some viral vectors preferentially accumulate in particular plant organs such as leaves and roots and viral accumulation can also be highly variable within individual tissues, resulting in patchy expression of foreign proteins (Arazi et al., 2001; Krebitz et al., 2000; Lawrence and Novak, 2001;

MacFarlane and Popovich, 2000). The maximum local protein concentration is often significantly different from the average (whole plant) expression levels.

1.5.4 Safety

The potential risks associated with heterologous protein expression in plants using transient expression vectors has two main components: (i) the purity and quality of the product, and (ii) the effect of the vector on the environment.

The safety of proteins expressed using plant viral vectors is similar to the safety of protein products produced using stably transformed whole plants. Plant viral vectors provide the mRNA, but the mRNA is translated and the proteins are processed by the host plant and therefore the characteristics of the protein will be identical to plant-

57 expressed proteins produced by nuclear-transformed plants (Pogue et al., 2002). The risks associated with the contamination of heterologous proteins with plant, bacterial and fungal toxins are similar when proteins are expressed using plant viral vectors or transgenic plants. However the purification of viral vector-encoded heterologous proteins requires the removal of potentially large amounts of viral RNA and protein

(Pogue et al., 2002). Plant viruses are not pathogenic to humans.

Foreign gene dissemination into the environment resulting from vector escape is potentially greater when heterologous proteins are expressed using viral vectors than when proteins are expressed in stably transformed plants. Vector escape independent of foreign gene dissemination also poses a threat to the environment. Vector escape could result in foreign protein production in non-target plants and also negative growth effects associated with viral infection. However, the risk of vector escape is generally considered to be low. Transgenic viruses have been demonstrated to be relatively easy to contain in field trials (Pogue et al., 2002; Turpen, 1999) and retain activity in the soil for only short periods (Della Cioppa and Grill, 1996), further reducing the risk of transmission. Vector instability also limits the likelihood of long-term foreign protein expression in non-target plants. Depending on the vector used, the deletion of inserts may result in the formation of viruses very similar to the parental wild-type virus or, if chimeric vectors were used, hybrid viruses. Viruses formed by vector recombination may produce milder symptoms and be less competitive than wild-type viruses (Pogue et al., 2002; Rabindran and Dawson, 2001). The potential economic impact of vector escape could still be significant and should be avoided. Vectors can be designed to reduce the likelihood of transmission to non-target plants. When using whole-virus vectors, which encode all the proteins required for virus multiplication, it is preferable

58 that they are derived from viruses such as TMV that have no known transmission agents, or that genes encoding proteins or motifs essential for virus transmission by animal vectors are removed from the vector construct (Arazi et al., 2001; Fernández-

Fernández et al., 1998; MacFarlane and Popovich, 2000). The use of vectors in which proteins essential for the establishment of infection, such as the movement protein, are encoded by host plants can prevent virus spread to non-host plants (Yusibov et al.,

2002).

1.6 Tobacco Mosaic Virus

Tobacco mosaic virus (TMV) is a member of the alpha-like virus supergroup and the type member of the tobamovirus group of viruses. Vectors based on TMV and other closely related tobamoviruses have been used extensively for foreign protein production.

1.6.1 Virus structure

The TMV virion is a filamentous and generally straight particle that self-assembles from viral genomic RNA and 17.5-kDa coat proteins (Figure 1.1). The coat protein molecules are arranged in an α-helical pattern around the viral genomic RNA (Watson,

1954) and each coat protein sub-unit is bound to three nucleotides (Namba et al., 1989).

Particle assembly commences from an origin positioned at the 3′ end of the genome, between nucleotides 5420 and 5546, and occurs bi-directionally using different mechanisms (Butler, 1999; Jonard et al., 1977; Zimmern, 1977). Forty-nine coat protein sub-units are required to form three turns of the helix and, if the full-length

RNA genome (6.395 kb) is packaged, the particle will contain 131 turns of the helix

(approximately 2130 coat protein subunits) (Namba et al., 1989). The assembled

59 particle is 300 nm (3000 Å) in length and 18 nm (180 Å) in diameter. A hole of radius

2 nm is located down the axis of the particle and the viral genomic RNA is located within the particle at a radius of 4 nm (Butler, 1999). The TMV virion is composed of

95% protein, 5% RNA and a small amount of calcium. TMV genomic RNA can be packaged into viral particles using the coat protein of other closely related viruses from the tobamovirus group.

Assembled TMV particles are stable, remain infectious for extended periods and are relatively resistant to chemical degradation (Chapman, 1998; Dijkstra and de Jager,

1998; Zaitlin, 2000).

A. B.

Figure 1.1 (A) Electron micrograph of a TMV virion (ICTVdB – The Universal virus database, version 4. http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB) and (B) a schematic of a TMV viral particle showing the helical arrangement of coat protein molecules and the genomic RNA (Locke, 1974).

1.6.2 Genome organization and encoded product function

Tobacco mosaic virus (common strain) has a single-stranded linear positive-sense RNA genome. The genome sequence is known (Goelet et al., 1982). The genome contains four ORFs and 5′ and 3′ untranslated regions. A schematic representation of the genome organization of TMV is shown in Figure 1.2.

Figure 1.2 Schematic diagram representing the organisation of the TMV genome, sub- genomic RNA molecules and translated proteins (Dawson, 1992).

A 5′ terminal cap structure (m7GpppG) is attached to the first nucleotide of the viral genome (Zimmern, 1975). The cap structure is followed by a 68 nucleotide guanine-deficient leader sequence (Ω) that acts as a translational enhancer

(Okada, 1999; Richards et al., 1978).

The 5′ ORF is translated directly from the genomic RNA and encodes two proteins.

The major translational product of the 5′ ORF genomic RNA is a 126-kDa protein.

Readthrough of an amber codon that terminates translation of the 126-kDA product results in the translation of a 183-kDa protein (Pelham, 1978). The 183-kDa readthrough protein comprises 10% (numerically) of the translated protein from the 5′

ORF (Dawson, 1992; Pelham, 1978). The 126-kDa and 183-kDa proteins associate with at least one host protein, possibly a 56-kDa protein (Osman and Buck, 1997), to form an endoplasmic-reticulum-bound RNA-dependant RNA polymerase that is required for replication of the RNA genome.

The remaining virus-encoded proteins are translated from subgenomic RNA derived from the full-length minus strand of the genomic RNA (Bruening et al., 1976; Buck,

1996, 1999; Hunter et al., 1976). An ORF encoding a 30-kDa protein overlaps the last five codons of the 5′ ORF coding for the183-kDa protein (Goelet et al., 1982). The

30-kDa protein functions as a movement protein facilitating the movement of virus from infected cells to adjacent uninfected cells (Deom et al., 1987). The origin of assembly (Section 1.6.1) is located within the cistron encoding the 30-kDa protein

(Zimmern, 1977). The 3′ proximal ORF is initiated two nucleotides after the ORF encoding the 30-kDa protein terminates (Goelet et al., 1982). This ORF encodes the only structural protein produced by TMV, a 17.5-kDa coat protein that stabilises the

RNA genome via capsid formation and facilitates the rapid systemic dissemination of viral particles via the phloem (Dawson, 1992).

The existence of an additional ORF located in the read through region of the 183-kDa protein that encodes a 54-kDa protein product of unknown function has been postulated

(Sulzinski et al., 1985); however this product has not been detected in vivo.

The terminal 3′ untranslated region (204 nucleotides) of the TMV genome contains three consecutive pseudoknot structures at the 5′ end, and a tRNA-like structure that accepts histidine at the 3′ end. The pseudoknotted region interacts with the cap structure to facilitate RNA translation (Gallie, 1996).

1.6.3 Early events in TMV infection

The early events in TMV infection of plants, including the attachment, entry and uncoating of viral particles, are poorly defined. TMV is mechanically transmitted and virus transmission requires direct contact between the plant and TMV-contaminated surfaces such as tools or soil. TMV has no true vectors; however mechanical transmission can be facilitated by feeding insects. Infection of whole plants with either viral particles or infectious RNA requires the plant (most frequently leaves) to be wounded. Protoplasts isolated from plant leaves can be infected by TMV RNA or particles; however infection occurs only if the protoplasts are exposed to polycationic substances such as poly-L-ornithine or cell-fusing agents such as polyethylene glycol, or if the protoplasts are electroporated in the presence of the virus (Mühlbach, 1982;

Nishiguchi et al., 1986; Sander and Mertes, 1984). The requirement for plant wounding prior to infection, the failure to locate membrane-associated cell receptors that facilitate viral movement across the cell membrane, and the inability of protoplasts to become infected without electroporation or the addition of substances that enhance viral uptake, have been taken to indicate that wounding allows the direct deposition of TMV into the cell cytoplasm (Shaw, 1999). Subsequent viral replication and dissemination throughout the plant does not require viral particles to cross the cell plasma membrane.

Alternative mechanisms of TMV entry into plant cells have been proposed but not confirmed (reviewed in Shaw, 1999).

After particle entry to the cell, viral RNA must be released from the viral particle to allow translation of the viral genes and genome replication. Purified viral particles are generally stable in vitro but disassemble rapidly in vivo to allow translation and replication. Disassembly of the viral particle occurs bi-directionally (Wu and Shaw,

1996, 1997), with disassembly occurring rapidly in the 5′ to 3′ direction, and more slowly from the 3′ to 5′ direction. The region around the origin of assembly is the last to be uncoated.

1.6.4 TMV replication and movement

Mechanical transmission of TMV results in virus entry into the cells of host plants that are able to support viral replication and movement and non-host plants in which the virus cannot replicate and move. The ability of TMV to replicate in plant cells and to move throughout the plant is dependent on the interaction of viral and host plant proteins.

1.6.4.1 Virus replication

RNA replication, synthesis of viral proteins, formation of movement complexes and virus encapsidation occur in the cytoplasm of infected cells, probably in inclusions called viroplasms or ‘X bodies’ (Buck, 1999; Heinlein et al., 1998; Saito et al., 1987).

Viroplasms consist of replicase complexes associated with the endoplasmic reticulum, ribosomes and aggregated tubules (Buck, 1996). During the course of infection viroplasms enlarge to form X-bodies (Szécsi et al., 1999).

Replication of TMV genomic RNA requires the synthesis of a negative-strand RNA using the positive-strand genomic RNA as a template. Positive-strand progeny

64 genomic RNA is then synthesised using the negative-strand RNA as a template.

Virus-encoded replicase proteins (126-kDa and 183-kDa) and host-encoded proteins are required for RNA replication (Osman and Buck, 1997). Failure of host proteins to interact with the viral replicase proteins is a determinant of viral host range (Dawson,

1992). The full-length negative-strand RNA is also used as a template for synthesis of the sub-genomic mRNAs.

Translation of 126-kDa and 183-kDa proteins that comprise the viral component of the replicase complex occurs directly from the positive-strand genomic RNA early in the infection cycle (Agranovsky and Morozov, 1999; Siegel et al., 1978). The remaining viral proteins, the 30-kDa movement protein and the 17.5-kDa coat protein, are translated from sub-genomic RNA molecules. The movement protein is expressed transiently early in the infection cycle at relatively low levels (Joshi et al., 1983; Lehto et al., 1990a; Watanabe et al., 1984). The coat protein is translated in large amounts late in the replication cycle (Siegel et al., 1978). The timing of translation from sub-genomic RNA is probably controlled by the sub-genomic promoter sequence

(Dawson, 1992; Lehto et al., 1990b).

1.6.4.2 Viral movement

TMV is disseminated from the point (or points) of infection throughout the plant using two transport mechanisms, cell-to-cell movement and long-distance movement.

Efficient viral replication, cell-to-cell movement and long-distance movement are required for systemic infection (Hilf and Dawson, 1993)

Cell-to-cell movement

The cell-to-cell movement of TMV is an active process that requires the specific interaction of the viral movement protein and the plasmodesmata. Within the viroplast, the 30-kDa movement protein binds to newly synthesised genomic RNA in a non-sequence-specific manner (Citovsky et al., 1990, 1992). This inhibits translation of the RNA (Karpova et al., 1997) and removes most of the RNA secondary structure resulting in the formation of a nucleoprotein with a diameter of approximately

2.0–2.5 nm (Citovsky et al., 1992). It has been proposed that the nucleoprotein complexes are transported from the viroplast towards the plasmodesmata by microtubules and actin microfilaments (Boyko et al., 2000; Carrington et al., 1996;

Citovsky, 1999; Heinlien et al., 1995, 1998; McLean et al., 1995).

The size exclusion limit of plasmodesmata within plant tissue is approximately 0.73 nm

(Wolf et al., 1989) and only relatively small molecules (< 1 kD) are able to pass through (Oparka and Roberts, 2001). The movement protein, either in a free form or as part of the nucleoprotein complex, interacts with host proteins resulting in a transient increase in the size-exclusion limit of the plasmodesmata from 0.73 nm to 2.4–3.1 nm

(Wolf et al., 1989). The nucleoprotein complex can be transported, probably by an active process, through plasmodesmata with increased size-exclusion limits (Citovsky et al., 1999; Karpova et al., 1997). During passage through the plasmodesmata, the nucleoprotein complex composed of the viral genome and the movement protein is destabilised, which allows translation and replication of the viral RNA in the newly infected cell (Karpova et al., 1997). The increase in plasmodesmata size required for cell-to-cell movement occurs in cells at the leading edge of infection only (Oparka et al., 1997). The movement of viruses via cell-to-cell movement occurs relatively

66 slowly. TMV cell-to-cell movement in N. benthamiana, a permissive host, occurs at an approximate rate of 25 µm h-1, which is equivalent to the infection of one cell every two hours (Cheng et al., 2000).

Movement protein–host interactions can define viral host range. Absence of cell-to-cell movement can result in the failure of infection to spread from the initially infected cells

(subliminal infection) (Sulzinski and Zaitlin, 1982), and inefficient movement can limit the establishment of systemic viral infections.

Long-distance movement

In permissive hosts, if the viral infection is developing in a source tissue, the expansion of the viral infection via cell-to-cell movement allows virus to enter the plant vasculature (Cheng et al., 2000). Sieve elements within the phloem transport water, inorganic nutrients and photoassimilates from source tissues to sink tissues. TMV can move passively with the flow of photoassimilates from source tissue, through the sieve elements and to the sites of photoassimilate utilisation (sink tissue). Sink tissues include young leaves and roots. In the sink tissue, cells associated with major veins can become infected with TMV (Cheng et al., 2000). Long-distance transport allows the rapid (cm h-1) (Nelson and van Bel, 1998) dissemination of viral particles from the point of infection to distant regions of the plant.

Efficient long-distance movement of TMV requires that the virus be assembled into particles. Viruses with mutations in the coat protein gene or the origin of assembly are generally impaired with regard to long-distance movement (Dawson et al., 1988;

Leisner, 1999; Saito et al., 1990). The function of coat protein in long-distance

67 movement is not known, although it may be required for efficient phloem invasion, transit or exit or may protect the nucleic acid during viral transport through the phloem

(Leisner, 1999).

1.7 Tobacco Mosaic Virus as a Vector

TMV naturally has a large host range that potentially allows the expression of foreign proteins in a wide variety of plants, including highly productive crop plants such as

N. tabacum. TMV has been reported to accumulate in inoculated leaves of

N. tabacum L. ‘Havana 38’ to levels greater than 60 mg g-1 dry weight and in systemically infected leaves to 23.5 mg g-1 dry weight (Copeman et al., 1969). TMV coat protein accounts for 10–40% of the protein content of systemically infected tobacco leaves (Beachy et al., 1996). TMV invades and replicates in almost all plant tissues including the leaves, stems and roots; however root and shoot apices and reproductive cells remain uninfected (Harrison and Wilson, 1999).

TMV-based vectors have been utilised extensively for the transient expression of foreign genes in plants (Table 1.2). The well defined genomic and structural organisation of TMV has allowed the design of a variety of vectors for the expression of foreign proteins as free cytosolic proteins (Donson et al., 1991; Kumagai et al.,

1993; Shivprasad et al., 1999; Takamatsu et al., 1987) and coat protein fusions

(Bendahmane et al., 1999; Hamamoto et al., 1993; Lim et al., 2002; Turpen et al.,

1995; Yusibov et al., 1997; Wu et al., 2003). Vector design is not limited by

RNA- packaging restraints because the rod-shaped particles extend to accommodate the longer vector RNA. Particle assembly can be limited by the display of peptides longer than 24–25 amino acids (Koprowski and Yusibov, 2001) on the coat protein due to

68 steric hindrance, although if a pool of unmodified coat protein is available, longer peptides can be displayed (Hamamoto et al., 1993; Turpen et al., 1995).

The risk of foreign gene dissemination from TMV vectors is considered to be low as infection control methodologies for TMV are well established. TMV has no transmission vectors and TMV viral vectors are relatively unstable with added sequences rapidly lost (Turpen, 1999; Della-Cioppa and Grill, 1996).

Large Scale Biology Corporation have designed and commercially utilised TMV-based vectors that allow the expression of free cytosolic proteins and coat protein fusions with pharmaceutical applications (www.lsbc.com). Production facilities allow field-, glasshouse- and growth-room-grown biomass to be processed.

1.7.1 30B vector

30B is a TMV-based hybrid vector developed by Shivprasad et al. (1999) to allow transient gene expression in plants. Foreign proteins expressed using the 30B vector are produced as free cytosolic proteins. A schematic diagram of the genetic organisation of the 30B vector is shown in Figure 1.3. The vector contains the

5′ untranslated region (UTR), the 5′ ORF encoding the 126-kDa and 183-kDa replicase proteins and the ORF encoding the 30-kDa movement protein from TMV variant U1

(vulgare). The TMV U1 coat protein ORF and 3′ UTR are replaced with heterologous sequences from tobacco mild green mosaic virus (TMGMV) variant U5. The 3′ UTR of TMGMV variant U5 is longer (357 nucleotides) (Shivprasad et al., 1999) than the

3′ UTR of TMV variant U1 (204 nucleotides) (Okada, 1999). The 3′ UTR of TMGMV contains six psuedoknots preceding the tRNA-like structure.

Figure 1.3 Schematic diagrams of the genomic organisation of TMV and the 30B and 30B–GFPC3 hybrid vectors (Shivprasad et al., 1999).

The complete TMV variant U1 coat protein sub-genomic mRNA promoter and PacI,

AgeI, PmeI and EhoI restriction endonuclease sites are positioned 3′ of the movement protein ORF (Shivprasad et al., 1999). The multiple cloning site (MCS) allows the insertion of a foreign ORF under the control of the TMV U1 coat protein sub-genomic promoter. Three pseuodknots from the 3′ UTR of TMV variant U1 were inserted 3′ of the MCS and 5′ of the heterologous coat protein ORF because proximity of internal genes to the pseudoknot of the 3′ UTR is associated with increased gene expression

(Shivprasad et al., 1999; Culver et al., 1993).

The 30B vector has been made available for research purposes and has been used to express a variety of reporter genes and pharmaceutical proteins including GFP and

Cycle 3 GFP (Shivprasad et al., 1999), hepatitis C virus epitope fused to the B subunit of cholera toxin (Nemchinov et al., 2000), bovine herpes virus type 1 glycoprotein D

(Pérez Filgueira et al., 2003), apple allergen Mal d 2 (Krebitz et al., 2003) and the heavy and light chains of the monoclonal antibody CO17-1A directed against colorectal cancer cloned into separate vectors (Verch et al., 1998).

1.7.2 30B-GFPC3

Cycle 3 GFP is utilised as a reporter gene in the 30B vector to allow movement of the vector and the stability of the inserted gene to be examined. The cycle 3 GFP gene is a synthetic gene with codon usage optimised for expression in E. coli and also containing mutations introduced using DNA shuffling (Crameri et al., 1996). Modification of the wild-type GFP gene to form the cycle 3 GFP gene was associated with the introduction of 25 silent mutations and three mis-sense mutations into the nucleotide sequence. The three amino acid substitutions in the cycle 3 GFP protein promote native protein folding rather than protein aggregation (Crameri et al., 1996). The emission and excitation maxima of Cycle 3 GFP are unchanged from those of wild-type GFP; however Cycle 3

GFP exhibits significantly higher fluorescence at equivalent protein levels, possibly due to reduced protein aggregation.

When expressed in N. benthamiana plants from the 30B vector encoding Cycle 3 GFP

(30B-GFPC3), GFP accumulated in upper systemically infected leaves to more than

10% of TSP (Shivprasad et al., 1999).

1.7.3 Movement, host range and stability of 30B and 30B-GFPC3

Characteristics of the 30B vector–host interactions are often examined using the

30B-GFPC3 vector. The expression of Cycle 3 GFP allows the movement and stability of the vector to be monitored and the effect of increased genetic load on vector characteristics to be examined.

1.7.3.1 Vector stability

The stability of the Cycle 3 GFP gene in the 30B vector has been assessed by passaging the vector through N. benthamiana plants. Instability of the inserted foreign gene sequence (Cycle 3 GFP) was detected by a loss of GFP expression in vector-containing leaves. Rabindran and Dawson (2001) reported that N. benthamiana plants infected with the 30B-GFP and 30B-GFPC3 vectors exhibited a lack of GFP expression after two or three serial passages of the virus, when the virus was passaged every 2 weeks.

The GFP gene was deleted from the 30B vector by recombination between similar sequences in the TMVU1-and TMGMVU5-encoded portions of the vector (Rabindran and Dawson, 2001). The hybrid viruses produced by recombination exhibited delayed symptom development and milder symptoms than wild-type TMV. The hybrid viruses were generally out-competed by TMV.

1.7.3.2 Host range and movement

The 30B vector without a foreign gene insert systemically infects N. tabacum and

N. benthamiana plants, two permissive hosts for TMV. 30B-GFPC3 was able to systemically infect and express Cycle 3 GFP in N. benthamiana; however in

N. tabacum, Cycle 3 GFP expression was limited to leaves inoculated with 30B-GFPC3

(Rabindran and Dawson, 2001; Shivprasad et al., 1999). The 30B-GFPC3 vector

72 exhibited reduced host range and limited systemic invasion compared with 30B and

TMV; this was attributed to the increased genetic load of the vector compared with

TMV (Toth et al., 2002). Increased genetic load can limit the rate of systemic infection due to reductions in the rate of viral replication, cell-to-cell movement and long-distance movement of the vector. The inability of TMV-GFPC3 to systemically infect N. tabacum is probably an effect of reduced cell-to-cell movement, which limits virus entry into the vasculature. By altering the sequence of the TMVU1 movement protein in 30B-GFPC3 by gene shuffling, Toth et al. (2002) were able to obtain a vector that exhibited increased rates of cell-to-cell movement compared to the original

30B-GFPC3 vector and could also systemically infect N. tabacum.

1.8 TMV Infection of Plant Roots

1.8.1 TMV accumulation in roots from whole plants

TMV replicates and accumulates within the roots of susceptible host plants. Using

TMV labelled with GFP (30B-GFP) it has been observed that, although TMV accumulated to high concentrations within plant roots, accumulation was patchy

(MacFarlane and Popovich, 2000). Valentine et al. (2002) have examined the progression of TMV infection in the roots of N. benthamiana seedlings. When virus entered the plant roots via the phloem, cells within the vascular tissue became infected.

The infection progressed both longitudinally along the root vascular cylinder and also via cell-to-cell movement into the root cortex, epidermis and root hairs. The development of infection in established lateral roots paralleled the infection pattern in primary roots, except that a zone of approximately 0.5 mm behind the root tip remained free of replicating virus. Lateral-root primordia forming within a TMV-infected root become heavily infected with virus but, as the lateral roots began to elongate, viral

73 replication was inhibited. Inhibition was initiated in the lateral root meristem and progressed up the lateral root but did not prevent viral replication in the primary root.

The inhibition of replication occurred because of a gene-silencing-like mechanism that originated in the lateral meristem. Suppression of replication can be overcome by co-infecting roots with a virus such as TRV that suppresses gene silencing (Valentine et al., 2002).

Plants are able to control the accumulation of virus in infected cells using a defensive mechanism called post-transcriptional gene silencing (PTGS). Transcription of the viral genome is unaffected, but as a result of sequence specific RNA degradation the viral mRNA and proteins fail to accumulate. In virus infected plant cells PTGS is triggered by the presence of double-stranded RNA (dsRNA) (Waterhouse et al., 1998), which forms as a result of virus replication. The dsRNA is cleaved into small interfering RNA molecules (siRNA) 21–25 nucleotides in length by a host encoded type III RNase-like enzyme complex referred to as DICER (Galun, 2005). The siRNA are separated into ssRNA fragments and recruited into a multi-protein RNA-induced silencing complex (RISC). The RISC facilitates the sequence specific degradation of viral mRNA. Silencing signals are able to move throughout the infected plant using similar mechanisms to those employed by viruses to move cell-to-cell and long distance

(Section 1.6.4.2). This results in the induction of local and systemic viral silencing

(Ruiz-Medrano et al., 2004). PTGS can limit the concentration of virus and heterologous proteins expressed from viral vectors, and in some cases infection can be almost completely suppressed (Marthe et al., 2000).

A number of plant viruses including potato virus Y, PVX, tobacco etch virus, TRV and

TBSV encode RNA suppressor proteins that can suppress gene silencing, improving the movement and accumulation of the infecting virus. Expression of suppressor proteins from native viral hosts or as plant transgenes can improve the accumulation of other infecting viruses and heterologous proteins if accumulation is limited by PTGS (Pruss et al., 1997; Anandalakshmi et al., 1998).

1.8.2 TMV accumulation in cultured roots

TMV-infected root cultures have been established using excised roots from systemically infected plants (White, 1934b) and by mechanically inoculating cultured roots with virus (White, 1934b; Kassanis et al., 1958). When infected root cultures were initiated using roots obtained from systemically infected plant material, infections were maintained over successive subcultures (White, 1934b), but when cultured roots were mechanically inoculated with virus, although the inoculated roots became infected, White (1934b) was unable to detect virus in new root tips after subculture.

Kassanis et al. (1958), although initiating infections in five root cultures, were only able to maintain infection in one root-tip culture. The progression of virus infection in mechanically inoculated roots has not been determined, but Kassanis et al. (1958) suggested that the failure to maintain infection after subculture was due to the slow progression of viral infection in cultured roots.

1.9 Virus Infection of Cultured Plant Cells

Virus infection of suspension and callus cultures was investigated extensively from the late 1950s to the mid-1970s in an attempt to develop a system that could be readily controlled and manipulated, in which plant:virus interactions such as viral replication, cell-to-cell movement, cell responses to infection and plant resistance mechanisms could be examined. Viral infection of cultured cells and subsequent viral multiplication were obtained; however their use was limited by the inability to initiate synchronous viral infection and thus synchronous viral replication in callus and suspension cultures

(White et al., 1977), and the relatively low levels of viral infection and accumulation that were generally observed. For a majority of research applications protoplasts, which could be synchronously infected, became the system of choice in plant virology and the use of callus and suspension cultures was predominantly limited to the examination of multi-cell characteristics such as plant resistance to viral infection.

Virus-based transient expression vectors have been utilised to obtain heterologous protein expression in cultured mammalian and insect cells and also in bacteria. Virus infection of animal cells and bacteria is receptor-mediated. RNA-virus-based transient expression vectors have been developed for use in whole plants, however they have not been utilised to express heterologous proteins in cultured plant cells or tissues

(Section 1.2). Efficient foreign protein production in cultured plant cells using virus- based transient expression vectors would require the cells to become readily infected with virus and, once infected, to accumulate virus at relatively high concentrations.

Viral-vector-mediated heterologous protein production has been obtained in transiently infected plant protoplasts (Guo et al., 1998; Hamamoto et al., 1993; Shivprasad et al.,

1999; Zhang et al., 2000); however protoplasts do not represent a practical large-scale

76 production system, and infection methodologies developed for use in protoplasts are not directly transferable to cultured plant cells and tissues.

Viral infections have been initiated in plant callus, suspension and root cultures. Virus- infected cultures can be obtained by initiating cultures from virus-infected plant material (Berlin et al., 1985; Hansen and Hildebrandt, 1966; Kassanis, 1957; Mühlbach and Sänger, 1981; Murakishi and Carlson, 1982; Raychaudhuri and Mishra, 1965;

Reinert, 1966; Toyoda et al., 1989; White, 1934b) or virus-free cell cultures can be infected with virus. In Table 1.3, viruses that have been demonstrated to infect and accumulate in cultured cells are listed with the plant species and culture types that have been infected. The infection methods are also provided. Cell cultures initiated from plant material already infected with virus are not listed.

Virus infection and multiplication in cultured cells has been investigated using a variety of virus:plant cell combinations (see Table 1.3). However a majority of investigations have been performed using TMV and either callus or suspension culture initiated using a range of N. tabacum cultivars that exhibit systemic or hypersensitive responses to

TMV infection. TMV infection has also been investigated in cell cultures from other

Nicotiana and non-Nicotiana species.

Although a considerable number of investigations have examined the infection of cell cultures with virus and the subsequent multiplication and dissemination of virus in vitro, reported results were frequently variable and inconsistent. The variability in results obtained may reflect the specific nature of host cell:virus interactions, the

Table 1.3 Virus Infection of cultured plant cells

Virus Plant species Tissue type Infection method References used for inoculation Aster yellow virus Daucus carota L. (carrot) Callus Virus transferred to callus using an insect vector Mitsuhashi and (six-spotted leafhopper Macrosteles fascifrons) Maramorosch, 1964 Clover yellow mosaic Trifolium ambiguum × Callus Callus dispersed in liquid medium and then Jones et al., 1981 virus T. hybhridum inoculated with virus using vibratory T. hybridum L. inoculation (alsike clover) T. repens L. (white clover) Clover yellow vein T. hybridum L. Callus Callus dispersed in liquid medium and then Jones et al., 1981 virus (alsike clover) inoculated with virus using vibratory T. repens L. inoculation (white clover) Potato spindle tuber Solanum tuberosum L. Protoplasts Suspension regenerated from protoplasts Mühlbach and Sänger, 1981 viroid inoculated with the viroid Southern bean mosaic Glycine max L. Callus and Callus and suspension cells inoculated with Wu and Murakishi, 1978 virus ‘Harosoy 63’ (soybean) suspension virus using agitation (120 rpm) on a rotary shaker Callus and Callus and suspension cells inoculated with Wu and Murakishi, 1978, suspension virus using vibratory inoculation 1979; White et al., 1977 Phaseolus valgaris L. Callus and Callus and suspension cells inoculated with Wu and Murakishi, 1978 ‘Prince’ (bean) suspension virus using agitation (120 rpm) on a rotary shaker Callus and suspension cells inoculated with virus using vibratory inoculation Tobacco mosaic virus Daucus carota L. Suspension Suspended cells inoculated with virus using Warren and Hill, 1989 agitation (150 rpm) on a rotary shaker

Table 1.3 (continued)

Virus Plant species Tissue type Infection method References used for inoculation Tobacco mosaic virus Lycopersicom esculentum Cultured Cultured roots wounded repeatedly with fine White, 1934b (continued) var. ‘Bonny Best’ roots pins in the presence of juice from crushed TMV-infected roots L. esculentum L. var. Excised root Root tips rubbed with a micro-spatula dipped in Kassanis et al., 1958 ‘Kondine Red’ tips Celite and virus N. glutinosa Callus Callus inoculated with virus using vibratory Beach and Murakishi, 1970, inoculation and inoculated cells grown on agar 1971 N. rustica Callus Callus inoculated with virus using vibratory Beach and Murakishi, 1970 inoculation and inoculated cells grown on agar N. suaveolens Callus Callus inoculated with virus using vibratory Beach and Murakishi, 1970 inoculation and inoculated cells grown on agar N. tabacum cv. Callus Callus inoculated with virus using vibratory Beachy and Murakishi, 1970 ‘Ambalema’ inoculation and inoculated cells grown on agar N. tabacum cv. Callus and Callus and suspension cells inoculated with Beachy and Murakishi, 1970, ‘NN Burley’ suspension virus using vibratory inoculation and inoculated 1971 cells grown on agar N. tabacum L. cv. Callus Callus inoculated with virus using vibratory Beachy and Murakishi, 1970; ‘Havana 38’ inoculation and inoculated cells grown on agar White et al., 1977 Suspension Suspension inoculated with virus using Murakishi et al., 1970, 1971; vibratory inoculation and inoculated cells grown Pelcher et al., 1972 on agar N. tabacum cv. Callus Callus inoculated with virus using vibratory Beachy and Murakishi, 1970 ‘Maryland Mammoth’ inoculation and inoculated cells grown on agar N. tabacum L. cv. Suspension Suspension cells microinjected with virus Halliwell and Gazaway, ‘Samsun’ 1975; Russell and Halliwell, 1974

Table 1.3 (continued)

Virus Plant species Tissue type Infection method References used for inoculation Tobacco mosaic virus N. tabacum L. cv. Callus and Callus and suspensions cells inoculated with Beachy and Murakishi, 1970, (continued) ‘NN Samsun’ suspension virus using vibratory inoculation and inoculated 1971 cells grown on agar Suspension Suspension cells microinjected with virus Nims et al., 1967; Russell and Halliwell, 1974 N. tabacum cv. Suspension Suspended cells inoculated with virus using Thomas and Warren, 1994 ‘White Burley’ agitation (150 rpm) on a rotary shaker Callus Callus inoculated with virus using vibratory Beachy and Murakishi, 1970 inoculation and inoculated cells grown on agar N. tabacum cv. ‘Xanthi-nc’ Suspension Suspension inoculated with virus using Beachy and Murakishi, 1971, vibratory inoculation and inoculated cells grown 1973, 1976 on agar N. tabacum × N. glutinosa Suspension Suspension inoculated using vibratory Wu et al., 1960 inoculation and inoculated cells grown in liquid medium N. tabacum Callus Callus inoculated by pouring virus over callus Kassanis et al, 1958 Callus soaked in virus Callus inoculated by pricking with a needle dipped in virus Callus inoculated by rubbing with a micro-spatula dipped in Celite and virus Tobacco mosaic virus L. esculentum var. Callus Callus inoculated with viral RNA using Murakishi, 1968 RNA ‘Bonny Best’ vibratory inoculation and inoculated cells grown in liquid medium Tobacco necrosis virus N. tabacum Callus Callus inoculated with virus using parasitic Kassanis and MacFarlane, fungi, Olpidium brassicae 1964

79 80 variability in cell culture parameters and characteristics, and the range of methods used to examine and assess the developing viral infections.

1.9.1 Infection of cultured cells with virus

1.9.1.1 Initiation of infection in cultured cells

Plant cells without cell walls (protoplasts) can be readily and synchronously infected with virus as the deposition of virus into the cytoplasm necessary for infection requires virus to cross the cell membrane only. This can be achieved by inducing transitory breaks in the cell membrane or by treating the protoplasts and/or virus to increase the frequency of virus:cell membrane interactions or membrane fusions (Sander and

Mertes, 1984). Callus and suspension cultures (Section 1.2.1.1) are composed of cells that retain functional cell walls and, as with whole plants, viral particles or RNA must cross both the cell wall and membrane to initiate infection. The cell walls of callus and suspension cells can be intentionally breached permitting viral entry; however virus can also enter cells through naturally occurring breakages in the cell wall.

Intentional cell wall injury

Virus infection of callus and suspension cultures and cultured roots can be achieved by intentionally injuring cells. Kassanis et al. (1958) infected callus cultures with TMV by pricking cultures with a needle that had been dipped in virus and by rubbing callus with a spatula that had been dipped in a mixture of virus and abrasive. Similar methods were also used to infect cultured plant roots with TMV (Kassanis et al., 1958; White, 1934b).

Microinjection of virus directly into cells (Russell and Halliwell, 1972, 1974; Nims et al., 1966, 1967) has been utilised to initiate viral infection in chains of callus and suspension cells. Virus can also be transmitted to cultured cells using transmission

81 vectors that physically injure cells. Mitsuhashi and Maramorosch (1964) utilised six- spotted leaf hoppers (Macrosteles fascifrons) to infect carrot callus with aster yellow virus during feeding and Kassanis and MacFarlane (1964) utilised the obligately parasitic fungus, Olpidium brassicae, to transmit tobacco necrosis virus to tobacco callus cultures.

Inoculation methods that result in the mechanical injury of cells within a culture are generally thought to result in the initial injury and subsequent viral infection of only a small proportion of the cell population (Warren et al., 1992). Callus and suspension cultures lack the vascular tissue that facilitates the rapid dissemination of virus in whole plants and virus dissemination in cultures occurs primarily via cell-to-cell movement

(Section 1.6.4.2). Cell-to-cell movement of virus occurs slowly (Kassanis et al, 1958;

Russell and Halliwell, 1974) and virus is unable to spread to cells that do not share protoplasmic connections with infected cells. If mechanical inoculation methods result in only a small proportion of the cell population initially becoming infected, levels of overall viral accumulation within a culture will probably be low as the infection does not spread extensively.

Low levels of viral infection and subsequent viral accumulation, and difficulties associated with the application of mechanical inoculation techniques to large-scale cultures, have limited the adoption of culture inoculation methods dependent on intentional cell injury.

Natural cell wall breaks

Kassanis et al. (1958) observed that when virus was poured onto callus, cultures that had not been mechanically injured could become infected with virus. Callus and suspension cells retain plasmodesmata (Spencer and Kimmins, 1969) and the rupture of plasmodesmata between cells exposes gaps in the cell wall through which the virus can enter and initiate infection. In callus, plasmodesmata may rupture as a result of varying rates of cell division within the cultures and, in suspension cultures, the dissociation of cell aggregates results in the rupture of plasmodesmata (Murakishi et al., 1971; Warren,

1992).

The efficiency of culture infection through ruptured protoplasmic connections is influenced by the number of ruptured plasmodesmata and the concentration of the viral inoculum applied (Murakishi, 1968; Murakishi et al., 1971; Wu and Murakishi, 1978).

Established suspension cultures in liquid medium can be infected by co-incubating cells with virus without the application of additional agitation above that required for culture mixing (100–120 rpm on an orbital shaker) (Warren and Hill, 1989; Wu et al., 1960;

Wu and Murakishi, 1978). The infection efficiency achieved when inoculum virus and cells are co-incubated can be highly variable (Kassanis, 1967; Kassanis et al., 1958).

For some host:virus combinations, acceptable levels of infection can be obtained by co-incubation without special mechanical treatment (Warren and Hill, 1989; Wu and

Murakishi., 1978).

Infection efficiency can be increased by vortexing established suspension cultures or callus in liquid medium in the presence of inoculum virus (Murakishi et al., 1971).

Vortexing the cultures is thought to result in the dispersion of cell clumps into smaller

83 aggregates and single cells, rupturing protoplasmic connections and possibly causing additional cell injuries, through which viral particles can enter (Murakishi et al., 1971;

Wu and Murakishi, 1978). Because cell-to-cell virus movement through cultures occurs relatively slowly, increasing the number of cells within a culture that are initially infected can result in an increase in the proportion of cells that become infected with virus and the overall viral yield. The potential infection sites exposed as cells dissociate appear to be only temporarily available for viral particle and RNA entry; therefore, cultures must either be vortexed in the presence of the virus, or the virus must be added immediately after vortexing (Murakishi, 1968).

A vibratory method of cell dispersal and viral inoculation was reported by Murakishi et al. (1970). Using this method, cell aggregates (0.2–0.3 g fresh cells) were dispersed in a small volume (3 mL) of medium using a vortex mixer, prior to the addition of a large amount of virus (83 μg mL-1) and a further cycle of vortex dispersion. The vibratory method of cell dispersal and inoculation resulted in a large proportion of the cells (up to 94% seven days after inoculation) becoming visibly infected with virus

(Murakishi et al., 1970) and a significant increase in virus titre compared with cultures inoculated using gentle agitation (Murakishi et al., 1971). Vibratory dispersion and inoculation were adopted widely for the inoculation of suspension and callus cultures with virus in applications where the cell injury caused by vortexing cultures was acceptable (Beachy and Murakishi, 1970, 1971; Jones et al., 1981; Murakishi 1968;

Murakishi et al., 1970, 1971; Pelcher et al., 1972; Wu and Murakishi 1978, 1979).

1.9.1.2 Culture characteristics

The efficiency of culture infection using vibrational cell dispersion and the subsequent accumulation of virus are sensitive to the physical characteristics of cell cultures, both prior to and after inoculation. Whether a cell culture has been grown on agar (callus) or in liquid medium (suspension) prior to inoculation by vibrational cell dispersion can affect infection efficiency. Virus accumulation was observed to be lower in cultures inoculated by vortexing callus in the presence of virus than in cultures similarly inoculated but with cells obtained from suspension cultures (Murakishi et al., 1971;

Wu and Murakishi, 1978). The dissociation of callus probably results in the rupture of a larger number of plasmodemata than the dissociation of suspensions cultures; however Murakishi et al. (1971) proposed that the hydrophobic nature of callus limited medium and virus contact with the dissociated cells. Relatively newly initiated suspension cultures (two subcultures) have been demonstrated to be more efficiently infected with virus than suspension cultures maintained over a long period (Beachy and

Murakishi, 1973).

The amount of virus that can accumulate in an infected cell appears to be limited

(Huber et al, 1981; Sander and Mertes, 1984), and high viral yields within cultures are generally observed when virus accumulates in a high percentage of cells.

Dissemination of virus throughout cultures from initial sites of infection is affected by culture characteristics, particularly friability. After vortex treatment, higher viral yields were observed in cultures containing a large proportion of the cells as aggregates

(0.5–2.0 mm diameter) compared with highly friable cultures where vortexing resulted in a large proportion of the cell population existing as single cells (Beachy and

Murakishi, 1973). This finding was attributed to the ability of virus to spread readily to

85 adjacent cells within aggregates via cell-to-cell movement. In highly friable cultures, although initial levels of infection may be high, subsequent infection of uninfected cells or aggregates that do not share protoplasmic connections with infected cells requires that new infections be initiated by virus in the medium. This is thought to occur at a reduced rate compared with viral spread via cell-to-cell movement.

Incubation conditions after cultures are inoculated with virus have also been demonstrated to significantly affect viral accumulation. Murakishi et al. (1971) observed that enhanced viral yields were obtained when vortex-inoculated cells were incubated on solid rather than in liquid medium. This was attributed to increased viral entry through broken plasmodesmata as the inoculum virus dried onto the surface of the cells incubated on solid medium. However, in contrast, Wu and Murakishi (1978) found that incubation of virus-infected cultures in liquid medium resulted in more rapid viral accumulation than in the same cultures incubated on agar, possibly because the rapid cell proliferation in suspension culture was more conducive for viral replication.

1.9.1.3 Culture age

The age of the cultures (i.e. the time after subculture) and the growth phase of the cells when a culture is inoculated with virus have been found to affect viral infection and subsequent accumulation. Warren and Hill (1989) examined viral accumulation in suspension cultures initiated using variously aged carrot suspension cultures that were inoculated by co-incubation with virus. Suspended cells from cultures in lag, early exponential, late exponential and late stationary phases were transferred to fresh medium containing TMV and the accumulation of virus was monitored. It was found that, as the age of the cells at the time of virus inoculation increased, there was a

86 progressive reduction in the amount of viral antigen (coat protein) accumulated in cultures at equivalent stages of the growth cycle.

Wu et al. (1959, 1960) observed a different relationship between culture age and viral accumulation when variously aged suspension cultures were inoculated with virus without being transferred to fresh medium. Viral accumulation was assessed 7 days after culture infection with local lesion assays used to measure viral infectivity. There was considerable variability in infectious virus levels in different cultures; however it was observed that cultures containing predominantly senescent cells (32 and 40 days after subculture) when inoculated with virus accumulated higher levels of infectious virus than cultures inoculated with virus 3 days after subculture when a majority of cells were still actively dividing.

The considerable differences observed in these studies may reflect the differences in experimental methods utilised. The results observed by Warren and Hill (1989) primarily reflect the susceptibility of variously aged cultures to infection whereas the results obtained by Wu et al. (1959, 1960) reflect a combination of the susceptibility of variously aged cells in conditioned media to infection and subsequent replication and accumulation of virus in cells at different stages of the growth cycle.

1.9.1.4 Viral inoculum

The concentration of the inoculum virus affects the initial infection levels and subsequent viral accumulation. Murakishi et al. (1971) reported that use of a

12 µg mL-1 TMV inoculum resulted in lower viral accumulation than an inoculum of

83 µg mL-1 TMV. A similar concentration effect was demonstrated when TMV RNA

87 was used as inoculum (Murakishi, 1968). An inoculum of 60 µg mL-1 RNA resulted in superior viral accumulation compared with 15 and 30 µg mL-1 RNA, although levels similar to those for 60 µg mL-1 RNA were achieved using 160 µg mL-1 RNA. The entry of one viral particle into a cell can result in infection, but the entry of more than one particle is generally required. Halliwell and Gazaway (1975) determined that an infectious load of 620 TMV particles per cell was required to achieve 100% infection of microinjected cells. Increasing the inoculum above the level required to achieve the infection of all susceptible cells within a culture does not increase the amount of virus accumulated within a culture, possibly because there is an upper limit to virus accumulation in individual infected plant cells (Huber et al., 1981; Sanders and Mertes,

1984).

The infectivity of virus particles and viral RNA decreases rapidly after addition to medium containing plant cells (Murakishi, 1968; Murakishi et al., 1971)

1.9.2 Characteristics of viral accumulation in tissue culture

1.9.2.1 Kinetics of viral accumulation

The kinetics of viral accumulation in cultured cells was determined by monitoring the accumulation of assembled virus particles, viral components (RNA and coat proteins) or infectious virus in cel extracts. The concentration of viral particles and viral components in cell extracts were determined using specific Enzyme Linked

Immunosorbent Assays (Sander and Mertes, 1984; Thomas and Warren, 1994; Warren and Hill, 1989) or by monitoring the incorporation of radiolabels into viral RNA and coat proteins (Beachy and Murakishi, 1973; Pelcher et al., 1972). The concentration of infectious virus was determined using local lesion bioassays (Murakishi et al., 1971;

Thomas and Warren, 1994; White et al., 1977). In local lesion assays the concentration of infectious virus in a sample is assessed by counting the number of discrete lesions formed on the leaves from hypersensitive host plants after inoculation with a virus containing extract. Infectious virus concentrations determined using local lesion assays are relative because leaf cells differ in susceptibility to infection, and the ability of infectious virus to initiate infections also fluctuates (Dijkstra and de Jager, 1998).

Viral accumulation in plant cell protoplasts

The kinetics of viral accumulation in isolated virus infected cells has been investigated using synchronously infected plant cell protoplasts. The pattern of viral accumulation reported when tobacco protoplasts were inoculated with TMV particles is shown in

Figure 1.3 and described below. Immediately after the addition of TMV to protoplasts in a suitable inoculation buffer, virus adsorption to protoplasts resulted in a small increase in the concentration of infectious virus associated with the protoplasts. This was followed by a decrease in the concentration of infectious virus associated with the protoplasts as viral particles that had entered the plant protoplasts uncoated (eclipse)

(Takebe and Otsuki, 1969). Soon after infection viral RNA began to accumulate exponentially in protoplasts, but RNA encapsidation was delayed by approximately 4 to

5 hours (Harrision and Mayo, 1983) and the exponential accumulation of viral particles was not observed until approximately 6 hours post-inoculation (Takabe, 1975). Viral

RNA continued to accumulate exponentially until approximately 10 hours-post inoculation (Harrision and Mayo, 1983) and exponential particle accumulation continued until approximately 12 hours post-inoculation (Takebe, 1975). RNA synthesis and particle assembly continued at a reduced rate until approximately 72 hours post-infection (Takebe, 1975). Protoplast lysis resulted in the release of small

89 amounts of virus into the medium (Takabe, 1975; Takebe and Otsuki, 1969). The kinetics of viral accumulation are similar in protoplasts derived from plants that displayed systemic and hypersensitive responses to viral infection (Otsuki et al., 1972).

30 lesions 10

log 20

0 020406080 Time after inoculation

Figure 1.3 Growth curve of TMV in tobacco protoplasts determined by local lesion formation as described by Takebe (1975).

Viral accumulation in plant suspension and callus cultures

Two distinct patterns of viral accumulation have been reported to occur in batch grown callus and suspension cultures derived from susceptible host plants. The pattern observed was determined by whether the infected culture was initiated from a plant that displayed a systemic or a hypersensitive response to the infecting virus. The growth kinetics of virus infected cultures was generally not reported.

In callus and suspension cultures initiated from permissive host plants that exhibited systemic responses to viral (TMV and SBMV) infection, one accumulation peak was observed when virus accumulation was monitored using local lesion assays (Murakishi

90 et al., 1971; White et al., 1977) and radio-label incorporation into viral RNA and viral particles (Pelcher et al., 1972; Wu and Murakishi, 1978). The kinetics of viral accumulation in N. tabacum L. cv. Havana 38 suspension cultures infected with TMV using the vibratory inoculation technique are shown in Figure 1.4 and described below.

A. B.

120 60 60

100 50 50

80 40 40

60 30 30 local lesins 10 40 20 20 log Viral RNA concentration 20

Virus particle concentration 10 10

0 0 0 0 40 80 120 160 0 40Time 80 after 120 infection 160 200 240 Time after infection

Figure 1.4 Growth curve of TMV in a suspension culture of a systemic host monitored by (A) local lesion formation (Murakishi et al., 1971) and (B) the accumulation of viral particles (▬▬) and viral RNA (▪▪▪▪▪) (Pelcher et al., 1972).

Shortly after the inoculation of suspension cells with TMV, when viral accumulation was monitored using local lesion assays, an initial increase in the concentration of infectious virus attributed to inoculum virus adsorption to cells was followed by a decrease in viral titre that was possibly associated with the uncoating of the viral RNA and the initiation of replication (Murakishi et al., 1971). A minimum concentration of infectious virus was observed at approximately 10 hours post-infection. When virus accumulation was monitored using radio-label incorporation into viral RNA and coat

91 protein (particles), only low levels of incorporation were observed during the first 24 hours after culture infection and labelling (Pelcher et al., 1972).

The initial lag in accumulation was followed by a short period of rapid viral accumulation (until 48–60 hours post-infection) observed as a logarithmic increase in the concentration of infectious virus (Murakishi et al., 1971; White et al., 1977) and a nearly linear increase in the concentration of labelled virus particles (Pelcher et al.,

1972). After the early rapid phase of virus accumulation, the concentration of viral particles (Pelcher et al., 1972) and infectious virus (Murakishi et al., 1971; White et al.

1977) continued to increase but at reduced rates compared with the previous phase.

Pelcher et al. (1972) attributed the phase of rapid viral synthesis primarily to the replication and accumulation of virus in cells with primary viral infections, and in surrounding cells in individual aggregates that had become infected as a result of cell- to-cell movement of virus. Pelcher et al. (1972) also suggested that the division of virus infected cells could result in further accumulation of virus. Maximum concentrations of infectious virus and assembled virus were observed at varying times post-infection (4–8 days) and were maintained in the cultures for a short period before the concentration declined (Murakishi et al., 1971; Pelcher et al., 1972).

The concentration of infectious virus in the medium of suspension cultures increased throughout the culture period and was highest when the concentration of infectious virus in the biomass was highest (Murakishi et al., 1971). The maximum amount of infectious virus in the medium was equivalent to only 0.15% of the amount of virus accumulated in the biomass.

In suspension and callus cultures initiated from host plants exhibiting hypersensitive responses to viral infection two distinct cycles of viral synthesis and accumulation were observed. These cycles were observed when viral accumulation was monitored by lesion formation, particle accumulation and viral RNA synthesis (Beachy and

Murakishi, 1973; Sander and Mertes, 1984). The pattern of viral accumulation in

N. tabacum cv. Xanthi-nc, a hypersensitive host, infected with TMV is shown in Figure

1.5 and described below.

A. B.

60 60

50 50

40 40

30 30

20 20 Concentration of RNA Concentration of virus

10 10

0 0 0 40 80 120 160 0 40 80 120 160 Time post infection Time post infection

Figure 1.5 Growth curve of TMV in a suspension culture of a hypersensitive host determined by (A) concentration of viral particles and (B) concentration of viral RNA (Beachy and Murakishi, 1973).

When monitoring radio-labelled viral RNA and particle accumulation in TMV-infected

N. tabacum cv. Xanthi-nc suspension cultures, Beachy and Murakishi (1973) observed an initial cycle of rapid viral synthesis that commenced 12–24 hours-post-inoculation and lasted until 60–72 or 72–80 hours post-inoculation. The initial cycle of rapid virus synthesis was followed by a sharp decline in the virus content of the cultures, and then

93 by a second cycle of rapid viral synthesis. A similar response was reported by Sander and Mertes (1984) also using N. tabacum cv. Xanthi-nc suspension cultures infected with TMV. In this case an initial replication cycle was followed by a marked decrease in the concentration of infectious virus in cultures and the addition of fresh medium to the culture was associated with a second cycle of virus synthesis. In both cultures, the first cycle of infection was attributed to the replication of virus in cells with primary infection and the second cycle to the multiplication of virus in neighbouring cells. The two cycles of accumulation were distinct because of the necrosis of tissue infected during the initial (primary) infection stage.

A medium-induced alteration of viral accumulation patterns in callus and suspension cultures initiated from a plant that exhibited systemic responses to viral infection

(N. tabacum cv. White Burley) has been reported when a medium that suppressed embryogenesis was used (Thomas and Warren, 1990, 1994). Virus accumulation patterns observed by Thomas and Warren (1994) are shown in Figure 1.6 when virus accumulation was monitored using ELISA for viral coat protein and by assessing the concentration of infectious virus using local lesion assays.

Rather than the expected single accumulation peak, two peaks of viral coat protein accumulation were observed in the TMV-infected N. tabacum cv. White Burley suspension cultures. The first peak in coat protein accumulation (Days 0–2) was associated with a corresponding increase in the infectious virus titre. Both the concentration of coat protien and infectious virus decreased at the onset of mitosis. The second cycle of coat protein accumulation commenced as mitosis ceased, however it was not associated with an increase in infectious virus titre. Examination of viral particle length throughout the culture period showed that the proportion of viral

300 100

250 80

200 60 300 nm

150 ≥ fresh weight)

-1 40

100 length g Viral coat protein protein coat Viral μ (

20 Local lesions per half leaf 50 Percentage of viral particles with

0 0 0 5 10 15 Time (days)

Figure 1.6 Characteristics of TMV accumulation in N. tabacum cv. White Burley suspension as described by Thomas and Warren (1994). (▬▬) Concentration of viral antigen; (▬▬) lesions per half leaf (infectivity); (▬▬) percentage of viral particles of full length or longer.

particles that were full length or longer had decreased from 81% when the investigation commenced to only 26% at the commencement of the second phase of antigen accumulation and 7% when cultures were terminated. Thomas and Warren (1994) observed that disrupted viral particles gave a higher titre in their ELISA than undisrupted virus and therefore suggested that the second cycle of antigen accumulation was due primarily to particle disruption rather than viral multiplication.

1.9.2.2 Viral yield

Individual cells from callus cultures and protoplasts have been reported to accumulate virus at the same order of magnitude as cells from infected leaves (Takebe, 1975).

Murakishi et al. (1971) also reported that the yield of TMV in callus, vortex inoculated

95 in the presence of a high concentration of virus and subsequently grown on solidified medium, was equivalent to the viral yield in TMV infected plant leaves. However, more commonly the average concentration of accumulated virus in cell cultures is lower than in virus-infected leaves (Kassanis, 1957, 1967). Low levels of viral accumulation have been reported in callus and suspension cultures inoculated directly with virus and also in cultures initiated from systemically infected plant material. In a callus culture of crown gall origin, initiated from tobacco that was systemically infected with TMV,

Kassanis (1957) estimated that the viral yield was one twentieth to one thirtieth of that seen in systemically infected leaves. The low virus yield was considered to reflect the less active protein metabolism of cultured cells (Kassanis, 1967).

The low levels of viral accumulation frequently observed in cultured cells have been attributed to either generally low levels of viral accumulation across a cell population in which a large proportion of cells were infected (Hansen and Hildebrandt, 1966) or to a small proportion of cells within infected cultures accumulating virus with the majority of the cells remaining uninfected (Kassanis, 1967). It is probable that both factors contribute to low virus yields in different cultures.

When low levels of viral accumulation is attributed to a low proportion of cells within a culture accumulating virus, increases in viral titre can be obtained by the use of inoculation methods that increase the proportion of cells that become infected with virus. The use of inoculation methods such as vortex inoculation in the presence of high concentrations of inoculum virus that increase the level of primary infection, have been reported to result in increased concentrations of accumulated virus in cultures

(Murakishi et al., 1971). As new virus infections occur more readily via cell-to-cell

96 movement of virus from infected to connected uninfected cells than the initiation of new primary infections by virus in the medium, selection of suspension cultures with a tendency to form large cell aggregates through which virus can move via cell-to-cell movement has also been associated with increased levels of virus accumulation

(Beachy and Murakishi, 1973). Pelcher et al. (1972) observed that in cell aggregates containing virus infected cells, nearly all the cells contained viral inclusions.

A variety of modifications to plant culture medium and incubation conditions, and the associated alterations in the characteristics of cultures, appear to increase viral accumulation without necessarily directly increasing the percentage of cells infected with virus. Limiting medium nitrogen (Hill et al., 1990) and the addition of some pyridine and purine bases to culture medium (Wu et al., 1960) have been associated with increased viral accumulation in infected cells. The type and concentration of auxin added to culture medium can also effect viral accumulation (Hill et al., 1990; Warren et al., 1992; Warren and Hill 1989). Viral accumulation rates are also sensitive to incubation temperature (Mühlbach and Sänger, 1981), however optimum rates of viral multiplication are frequently observed at temperatures that also facilitate rapid plant cell growth. Green pigmented photosynthesising cell cultures have also been observed to accumulate more virus than non-pigmented cultures (Murakishi et al., 1971).

1.9.2.3 Effect of viral infection on culture growth

The effect of viral infection on culture growth is dependant on the virus: host combination. Virus infection of plant cells results in significant intracellular changes.

Premature cell death and necrotic lesion formation has been reported in virus infected cells from hypersensitive hosts (Beachy and Murakishi, 1970, 1971), although cells

97 from systemic hosts were not subject to premature cell death (Russell and Halliwell,

1974). The effect of viral infection on the overall growth of susceptible cultures is generally reported be slight (Thomas and Warren, 1994; Reinert, 1966). Culture discolouration without growth inhibition has been reported to occur in some virus- infected cultures as they aged (Wu et al., 1959; Thomas and Warren, 1994).

1.10 Project Aims

The primary objective of this project was to examine the characteristics of tobacco mosaic virus accumulation in plant suspension and hairy root cultures to determine if the plant viral vectors could facilitate transient heterologous protein expression in cultured plant cells. Specific objectives were:

1. to select a plant cell or tissue culture in which TMV was observed to accumulate

2. to examine the general characteristics of TMV accumulation in the selected

culture in shake flasks

3. to investigate the effect of inoculation methods and culture parameters on viral

accumulation

4. to examine culture scale-up to a 2-L bioreactor

5. to determine if transient heterologous protein expression could be achieved in a

plant cell or organ culture using a transgenic plant viral vector and the infection

parameters elucidated using TMV

CHAPTER 2 – MATERIALS AND METHODS

2.1 Cultures

2.1.1 Plant material

Seeds of Nicotiana tabacum var. Hicks were provided by Mr. Geoffrey McDonald,

School of Biological, Earth and Environmental Sciences, University of New South

Wales. Seeds of Nicotiana benthamiana were provided by Mr. Mervyn Rettke,

Australian Tropical Crops Genetic Resource Centre, Queensland Department of

Primary Industries, Biloela, Queensland. Seeds of Nicotiana glutinosa were provided by Ms. Sally Dillon, Australian Tropical Crops Genetic Resource Centre, Queensland

Department of Primary Industries, Biloela, Queensland. N. tabacum hairy roots were provided by Dr. Janet Sharp, School of Biotechnology and Biomolecular Sciences,

University of New South Wales. Initiation procedure for N. tabacum hairy roots are detailed in Sharp and Doran (2001b).

2.1.2 Viral material

Tobacco mosaic virus (TMV) was provided by Dr. Peter Waterhouse, CSIRO Division of Plant Industry, Canberra. Plasmid 30B-GFPC3, used to produce TMV-GFPC3 virus

(Section 1.7.2), was provided by Dr. Shailaja Rabindran, Department of Plant

Pathology, University of Florida, USA.

2.1.3 Bacterial cultures

Agrobacterium rhizogenes strain A4 was provided by Professor Alan Kerr, Waite

Agricultural Institute, Adelaide, Australia. A. rhizogenes strain ATTC 15835 was provided by Professor J. D. Hamill and Professor V. Krishnopillai, Department of

Genetics and Developmental Biology, Monash University, Australia. Escherichia coli

XL1-Blue {recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F΄proAB laclqZΔM15

Tn10 (Tetr)]} was obtained from Stratagene, USA.

2.2 Media

2.2.1 Plant culture media

Gamborg’s B5 basal medium with minimal organics (Sigma-Aldrich, USA) supplemented with 30.0 g L-1 sucrose (Ajax Finechem, Australia) was used as the growth medium for N. tabacum and N. benthamiana hairy roots. The medium pH was adjusted to 5.8 prior to sterilisation using 0.1 M potassium hydroxide (Asia Pacific

Specialty Chemicals, Australia). The above medium supplemented with 0.8% (w/v) agar (Bacto Laboratories, Australia) was used for the maintenance of hairy root cultures and in vitro axenic N. tabacum plants.

Murashige and Skoog (MS) medium (ICN Biomedicals, USA) supplemented with

30.0 g L-1 sucrose, 5.0 mg L-1 α-naphthaleneacetic acid (NAA: Sigma-Aldrich),

0.25 mg L-1 kinetin (Sigma-Aldrich) and 0.8% (w/v) agar was used for the maintenance of N. tabacum callus cultures. The medium pH was adjusted to 5.8 prior to sterilisation using 0.1 M potassium hydroxide. The above medium without the addition of agar was used for the maintenance of N. tabacum suspension cultures.

MS medium supplemented with 30.0 g L-1 sucrose, 0.1 mg L-1 kinetin, 0.2 mg L-1

2,4-dichlorophenoxyacetic acid (2,4-D: Sigma-Aldrich) and 0.8% (w/v) agar was used for the maintenance of N. benthamiana callus cultures. The medium pH was adjusted to

5.8 using 0.1 M potassium hydroxide prior to agar addition and sterilisation. The above

100 medium without the addition of agar was used for the maintenance of N. benthamiana suspension cultures.

Axenic in vitro cultures of N. benthamiana plantlets were maintained initially on MS medium supplemented with 30.0 g L-1 sucrose and 0.8% (w/v) agar. The medium pH was adjusted to 5.8 using 0.1 M potassium hydroxide prior to agar addition and sterilisation. This medium was later supplemented with 0.01 mg L-1 NAA to promote rooting of explants.

All plant culture media were prepared using Milli-Q water (Millipore Corporation,

USA) and were heat sterilised at 121ºC and 15 psi for 20 minutes.

When culture medium in the text is described as Gamborg’s B5 medium, this refers to liquid Gamborg’s B5 basal medium with minimal organics supplemented with

30.0 g L-1 sucrose. Similarly when medium is described as MS medium, this refers to liquid MS medium supplemented with 30.0 g L-1 sucrose. Where medium compositions are altered, the modifications are specifically mentioned in the text.

When medium is referred to as solidified, the medium has been supplemented with

0.8% (w/v) agar as a gelling agent.

2.2.2 Bacterial culture media

A. rhizogenes strains A4 and 15834 were maintained on yeast mannitol medium. This medium contains 0.5 g L-1 di-potassium hydrogen orthophosphate (Asia Pacific

Specialty Chemicals), 0.2 g L-1 magnesium sulphate (Ajax Finechem), 0.1 g L-1 sodium chloride (Asia Pacific Specialty Chemicals), 10.0 g L-1 mannitol (Sigma-Aldrich),

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0.4 g L-1 yeast extract (Oxoid, England) and 15.0 g L-1 agar. Medium pH was adjusted to 7.0 prior to agar addition and sterilisation. Agar was omitted for the preparation of liquid medium.

E. coli XL1-Blue containing the plasmid 30B-GFPC3 was maintained on yeast tryptone medium supplemented with 100 mg L-1 ampicillin (Sigma-Aldrich). Yeast tryptone medium contains 5.0 g L-1 tryptone (Oxoid), 2.5 g L-1 yeast extract, 5.0 g L-1 sodium chloride and 15.0 g L-1 agar. The medium pH was adjusted to 7.0 prior to agar addition and sterilisation. Agar was omitted in the preparation of liquid medium.

All media were prepared using Milli-Q water and heat sterilised at 121ºC and 15 psi for

20 minutes.

2.3 Initiation and Maintenance of Plant Cultures

2.3.1 Surface sterilisation and germination of seeds

Prior to surface sterilisation, seeds were placed in folds of Whatman No. 1 filter paper

(Whatman International, England) for ease of handling. Seeds in the filter paper folds were rinsed briefly in 70% ethanol (Asia Pacific Specialty Chemicals) prior to immersion for 20 minutes in a solution containing commercial bleach (White King:

SaraLee Household and Bodycare, Australia ) diluted to contain 1% active chlorine and

0.05% (w/v) Tween 20 (polyoxyethyenesorbitan monolaurate) (Sigma-Aldrich). After immersion in bleach, seeds were rinsed six times in heat-sterilised Milli-Q water.

Sterilised seeds were placed on MS medium solidified with 0.8% (w/v) agar or, in the case of N. benthamiana seeds used for the initiation of axenic plantlet cultures, seeds

102 were placed on half-strength MS medium supplemented with 10.0 g L-1 sucrose and

0.8% (w/v) agar. Seeds were incubated at 25ºC in the dark until germination, at which time the cultures were transferred to continuous fluorescent light (Cool White: Osram,

Indonesia) conditions (650 lux).

2.3.2 Maintenance of axenic plantlets

Axenic N. benthamiana plantlets were maintained using a single-node method of clonal propagation, in which sections of plantlet stem containing at least one node were inserted upright into solidified medium. Initial axenic explants were obtained from in vitro germinated surface-sterilised seeds. The explants were initially maintained using MS medium solidified with 0.8% (w/v) agar, however this medium was later supplemented with 0.01 mg L-1 NAA to promote rooting of explants. Cultures were maintained at 25ºC under continuous fluorescent light (Cool White: 650 lux).

Axenic N. tabacum plantlets were maintained using single-node clonal propagation.

Explants were placed on Gamborg’s B5 medium solidified with 0.8% (w/v) agar.

Cultures were maintained at 25ºC under continuous fluorescent light (Cool White:

650 lux).

2.3.3 Hairy root cultures

2.3.3.1 Initiation of N. benthamiana hairy root cultures

Stem and leaf explants and rooted stem sections from in vitro grown N. benthamiana plantlets were used for the initiation of hairy roots. A. rhizogenes strains A4 and 15834 were inoculated into liquid yeast mannitol medium from solid medium, and incubated at 25ºC on an orbital shaker operated at 100 rpm for 48 hours. An aliquot of the

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48-hour culture was used to inoculate fresh yeast mannitol medium. The inoculated medium was incubated under the above conditions for 48 hours prior to hairy root initiation.

A. rhizogenes was applied using a syringe and 25-gauge needle (Becton Dickson

Medical Products, Singapore) to freshly-cut surfaces of rooted stems and stem explants, and wounds on leaves created by puncturing or scratching the leaves with the needle.

Excess bacterial suspension was removed from the explants using the syringe. The explants were inserted into solidified MS medium in a manner that minimised contact between the cut or wounded surfaces to which bacteria had been applied and the agar.

Explants to which bacteria had been applied were incubated at 25ºC in the dark.

Hairy roots that formed at the site of wounding and bacterial application were excised when one to two centimeters in length. Hairy roots originating from the same wound site were assumed to originate from one transformation event. Hairy roots developing from different wounds were assumed to originate from different transformation events and are referred to as separate clones. Excised roots were placed in 50 mL Gamborg’s

B5 medium supplemented with 200 µg mL-1 Claforan (cefotaxime) (Hoechst Marion

Roussel, England) in 250-mL Erlenmeyer flasks. Hairy root cultures were incubated in the dark at 25ºC on an orbital shaker operated at 100 rpm. Roots were subcultured

(0.2 g fresh root material into fresh medium) every three to four weeks and maintained in medium containing antibiotic for at least nine weeks post-excision (i.e. three consecutive subcultures).

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2.3.3.2 Maintenance of hairy root cultures

N. benthamiana hairy roots and N. tabacum hairy roots (Section 2.1.1) were maintained using Gamborg’s B5 medium. N. benthamiana hairy roots were subcultured every 21 days and N. tabacum hairy roots every 14 days by transferring 0.2 g fresh root material to 50 mL fresh medium in 250-mL Erlenmeyer flasks. Cultures were incubated in the dark at 25ºC on an orbital shaker operated at 100 rpm.

Hairy root cultures were placed on Gamborg’s B5 medium solidified with agar for long-term culture maintenance with subculturing every 2 months.

2.3.4 Callus and suspension cultures

2.3.4.1 N. benthamiana callus and suspension cultures

N. benthamiana callus was initiated using two media, MS medium and MS basal salts

(ICN Biomedicals) supplemented with 0.5 mg L-1 pyridoxine monohydrochloride

(Sigma- Aldrich), 0.5 mg L-1 nicotinic acid (Sigma-Aldrich), 100 mg L-1 myo-inositol

(Sigma-Aldrich), 0.4 mg L-1 thiamine HCl (Sigma-Aldrich) and 30.0 g L-1 sucrose. The media were supplemented with kinetin at a concentration of 0.05 mg L-1,

0.1 mg L-1 or 0.2 mg L-1 and 2,4-D at a concentration of 0.1 mg L-1 , 0.2 mg L-1 or

0.4 mg L-1. The media were solidified using 0.8% (w/v) agar. Stem, root, petiole, leaf and flower bud explants from in vitro grown N. benthamiana plantlets were placed on the growth-regulator containing media and incubated at 25ºC in the dark. Resulting callus was subcultured every 4 weeks.

A friable callus that was pale in colour was selected for suspension culture initiation.

The selected callus was initiated from leaf material on MS medium supplemented with

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0.1 mg L-1 kinetin and 0.2 mg L-1 2,4-D. Suspension cultures were initiated by placing a small amount of the callus (approximately 1 cm3) into liquid MS medium containing the above growth regulator concentrations and incubating on an orbital shaker operated at 100 rpm in the dark at 25ºC to allow cell dissociation. Suspension cultures were maintained by transferring a 20% (v/v) inoculum to fresh medium (10 mL suspension to

40 mL fresh medium in a 250-mL Erlenmeyer flask) every 10 days.

2.3.4.2 N. tabacum callus and suspension cultures

N. tabacum callus was initiated using MS medium supplemented with NAA and kinetin. The media were supplemented with NAA at a concentration of 0.1 mg L-1,

1.0 mg L-1 or 2.0 mg L-1 and kinetin at a concentration of 0.25 mg L-1 or 0.5 mg L-1.

The media were solidified using 0.8% (w/v) agar. Stem, root and leaf explants from in vitro grown N. tabacum plantlets were placed on the media and incubated at 25ºC in the dark. Resulting callus was subcultured every 4 weeks.

A friable callus that was pale in colour was selected for suspension culture initiation.

The selected callus was initiated from leaf material on MS medium supplemented with

5.0 mg L-1 NAA and 0.25 mg L-1 kinetin. Suspension cultures were initiated by placing a small amount of the callus (approximately 1 cm3) into liquid MS medium containing the above growth regulator concentrations and incubating on an orbital shaker operated at 100 rpm in the dark at 25ºC to allow cell dissociation. Suspension cultures were maintained by transferring a 20% (v/v) inoculum to fresh medium (10 mL suspension to

40 mL fresh medium in a 250-mL Erlenmeyer flask) every 7 days.

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2.4 Growth of Plants in Soil

2.4.1 N. tabacum plants

Plants of N. tabacum var. Hicks were grown in a glasshouse for use in TMV production. The plants were grown from seed in a potting mix containing a 1:1:1 ratio of river sand, coconut peat and an organic garden mix comprising 50% black soil,

20% course sand and 30% organics (Australian Native Landscape Company, Australia).

The potting mix was supplemented with dolomite, lime, blood and bone, ammonium nitrate, trace elements, superphosphate and gypsum. The plants were grown at ambient temperatures, were watered using a drip irrigation system, and were fertilised using

Thrive All Purpose Soluble Fertiliser (Yates, Australia) according to the manufacturer’s instructions every 7 days. Plants were repotted to larger pots as required.

2.4.2 N. glutinosa plants

N. glutinosa plants for use in local lesion assays were grown in potting mix under artificial light at 25ºC. Seeds were surface sterilised (Section 2.3.1) and germinated on solidified MS medium in Petri dishes. The seedlings were transplanted to potting mix in sealed plastic containers when they reached the four-leaf stage. The potting mix,

Yates Thrive Premium (Yates), was heat sterilised to kill any insects, viruses and seeds in the mix prior to use. The seedlings were grown under continuous fluorescent light

(Cool White: 650 lux) and fertilised every 7 days with Thrive All Purpose Soluble

Fertiliser according to the manufacturer’s directions. Seedlings were transferred to larger uncovered containers as required.

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2.4.3 N. benthamiana plants

N. benthamiana seeds were surface sterilised (Section 2.3.1) and germinated on solidified MS medium in Petri dishes. After 7 days, seedlings were transplanted to potting mix in sealed plastic containers. The potting mix, Yates Thrive Premium, was heat sterilised to kill any insects, viruses and seeds in the mix prior to use. The seedlings were grown under continuous fluorescent light (Cool White: 650 lux) for

2 days prior to transfer to a glasshouse where they were transplanted into larger uncovered pots. Seedlings were watered using a drip irrigation system and fertilised every 7 days with Thrive All Purpose Soluble Fertiliser according to the manufacturer’s directions.

2.5 Production of TMV

2.5.1 Infection of N. tabacum plants with TMV

Glasshouse-grown N. tabacum plants were inoculated with TMV when approximately

30 cm tall. The uppermost fully-unfurled leaf was rubbed lightly using 500 mesh carborundum (Naxos Products, Sydney) and a small amount of purified TMV

(Section 2.1.2) in 0.01 M sodium phosphate buffer at pH 7.4 was applied to the leaf and spread over the abraded surface. The leaf surface was rinsed with sterile water to remove excess carborundum and virus. Leaves showing systematic infection were harvested 28 days post-inoculation.

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2.5.2 Purification of TMV

TMV was purified from N. tabacum leaf material using a method provided by the

Commonwealth Myrological Institute Association of Applied Biologists: Description of

Plant Viruses, description 156 (www.dpvweb.net/dpv/showdpv.php?dpvno=156) with minor modification.

All glassware and phosphate buffers were heat sterilised and sterile disposable plastic ware was used for centrifugation. The homogenisation buffer used was 0.05 M sodium phosphate buffer at pH 7.4 (Sambrook et al., 1989b), which was prepared by combining

34.2 mL of 1.0 M di-sodium phosphate (Asia Pacific Specialty Chemicals) with

15.8 mL of 1.0 M monosodium phosphate (Asia Pacific Specialty Chemicals), making up to 1 litre and adding 0.1% (v/v) thioglycolic acid (Sigma-Aldrich).

Infected leaf material was frozen overnight at –20ºC. Frozen leaf material was homogenised using a cold mortar and pestle with 4.0 mL of sodium phosphate buffer per gram of biomass. The homogenate was squeezed through pre-boiled, sterile gauze.

Butan-1-ol (APS Ajax Finechem, Australia) was added drop-wise to the collected extract to give 9.3 mL butanol per 100 mL extract. The plant extract/butanol mix was incubated at room temperature on an orbital shaker for 45 minutes. The extract was clarified by centrifuging at 10000 × g at 15ºC for 30 minutes. Polyethylene glycol

(PEG) of average molecular weight 6000 (Fluka Chemie, Switzerland) was added to the supernatant to give 4.0 g PEG per 100 mL of supernatant, and mixed until dissolved.

The mixture was incubated at room temperature for 30 minutes and then centrifuged at

15ºC for 15 minutes at 10000 × g to sediment the precipitated virus. The resulting pellet was re-suspended in a volume of 0.01 M sodium phosphate buffer at pH 7.4

109 equivalent to 20 mL per 100 mL of initial leaf extract. Sodium phosphate buffer

(0.01 M) at pH 7.4 (Sambrook et al., 1989b) was prepared by combining 7.74 mL of

1.0 M di-sodium phosphate and 2.26 mL of 1.0 M monosodium phosphate and making up to 1 litre. The solution was clarified by centrifugation at 10000 × g at 15ºC for

15 minutes to remove insoluble non-viral material.

Further purification was achieved using a second PEG treatment. PEG and sodium chloride were each added at a concentration of 4.0 g per 100 mL of supernatant and mixed until dissolved. The virus was precipitated by centrifugation at 10000 × g at

15ºC for 15 minutes, and the pellet re-suspended in 2 mL of 0.01 M sodium phosphate buffer per 100 mL of initial leaf homogenate. The solution was clarified by centrifugation at 10000 × g at 15ºC for 5 minutes to remove insoluble non-viral material.

The purified virus was frozen at –20°C in aliquots and defrosted as required.

Prior to use as an inoculum for cultures, the virus was diluted with sterile

0.01 M sodium phosphate buffer to the required concentration and filtered through a

0.2-µm Minisart filter (Sartorius, Germany). Filtration of virus preparations did not significantly affect virus concentration (Appendix 1).

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2.5.3 Concentration and purity of TMV preparations

The concentration and purity of the TMV preparation were assessed by UV adsorption using the method outlined by Dijkstra and de Jager (1998).

Purified viral preparations were diluted in 0.01 M sodium phosphate buffer. A scan between the wavelengths of 225 nm and 350 nm was performed using a UV–visible recording spectrophotometer UV160 (Shimadzu, Japan), and an absorbance curve with absorbance expressed as a function of wavelength was obtained.

From the known A260/280 of purified TMV, 1.19, the purity of the preparation was assessed. Using the reported extinction coefficient for purified TMV, the concentration

(mg mL-1) of the virus was determined using the following formula:

E 260 c = 0.1% (2.1) E1 cm, 260nm

-1 where c is the concentration of the virus preparation (mg mL ), E260 is the extinction

0.1% (adsorption) of the unknown TMV preparation at 260 nm and E 1 cm, 260 nm is the extinction coefficient of a 1.0 mg mL-1 (0.1%) suspension of TMV at 260 nm at an

0.1% optical path length of 1 cm. For TMV, E 1 cm, 260 nm is 3.0.

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2.6 Production of Transgenic Virus

2.6.1 Transformation of E. coli XL1-Blue with plasmid 30B-GFPC3

E. coli strain XL1-Blue was prepared to make the cells electroporation-competent and transformed with the plasmid 30B-GFPC3 (Section 2.1.2) by electroporation according to the method of Sambrook et al. (1989a) for the transformation of E. coli. Preparation of competent XL1-Blue cells and electroporation was performed by Ms. Ellen De Leon,

School of Biotechnology and Biomolecular Sciences, University of New South Wales.

Transformants were selected by growth on solidified yeast tryptone medium containing

100 mg L-1 ampicillin. Glycerol stocks were prepared from transformants to allow long-term storage of E. coli XL1-Blue containing the plasmid 30B-GFPC3. The stocks were prepared by suspending bacterial colonies grown on solid yeast tryptone medium containing 100 mg L-1 ampicillin in yeast tryptone medium containing 10% (v/v) glycerol (Asia Pacific Specialty Chemicals). The glycerol stocks were frozen and stored at –70ºC.

2.6.2 Production of in vitro transcript of 30B-GFPC3

In vitro transcription was performed with the assistance of Mr. Luke Selth, CSIRO

Plant Industry Horticulture Unit, Adelaide.

Overnight cultures of E. coli XL1-Blue transformed with 30B-GFPC3 were prepared in yeast tryptone medium containing 100 mg L-1 ampicillin. The cultures were incubated at 37ºC with shaking.

Plasmids were purified by alkaline extraction using a protocol provided by the

International Maize and Wheat Improvement Centre (CIMMYT, Centro Internacional

112 de Mejoramiento de Maíz y Trigo: www.cimmyt.cgiar.org/abc/protocols/plasmidminipreps.pdf) based on the method by

Birnboim and Doly (1979). Purified plasmid was linearised with Kpn I or Pst I

(Promega, USA), using 2 units of enzyme per 1.0 µg of plasmid DNA at 37ºC for

2 hours. The linearised plasmid was purified using the QUIquick® PCR purification kit

(Qiagen, The Netherlands) according to the manufacturer’s directions.

RNA transcript was produced from the template using a mMESSAGE mMACHINE®

High Yield Capped RNA Transcription Kit (Ambion, USA). The manufacturer’s directions were followed with the following modifications: 1–5 µg of linearised plasmid was used as template DNA, an additional 1 µL of 30 mM guanosine 5’-triphosphate

(GTP) was added to the reaction mix and the reaction time was extended to 2 hours.

2.6.3 Infection of plants with 30B-GFPC3 RNA transcript

Infection of plants with RNA transcript was performed with the assistance of Mr. Luke

Selth, CSIRO Plant Industry Horticulture Unit, Adelaide.

Leaves on glasshouse-grown Nicotiana clevelandii plants of height approximately 5 cm

(provided by Mr. Luke Selth) were dusted with 500 mesh carborundum. Six µL of

RNA transcript (Section 2.6.2) was placed onto each leaf and gently rubbed over the surface with a gloved finger until the leaf surface was lightly abraded. The leaves were rinsed with milli-Q water to remove excess carborundum and transcript. Three leaves on each plant were inoculated with transcript.

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Plant leaves and stems were harvested 23 days after inoculation with RNA transcript and the plant material was frozen at –20ºC. Prior to plant harvest, selected leaves were exposed to ultraviolet irradiation to confirm that GFP had been expressed in the plant material. Plants were harvested and frozen by Mr. Luke Selth.

2.6.4 Purification of TMV-GFPC3

TMV-GFPC3 was purified from frozen plant material (Section 2.6.3) using the method used for purification of TMV as described in Section 2.5.2. Virus was purified from

RNA inoculated leaves, upper un-inoculated leaves and stems.

Infectivity of the purified TMV-GFPC3 was examined using local lesion assays

(Section 2.17.4.4). The ability of the purified TMV-GFPC3 preparation to facilitate the production of Cycle 3 GFP in inoculated plants was examined by infecting

N. benthamiana plantlets (Section 2.4.3) at the four leaf stage with purified

TMV-GFPC3 and crude N. clevelandii leaf extracts according to the method for infection of N. tabacum plants with TMV (Section 2.5.1)

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2.7 Preliminary Investigations of TMV Accumulation in N. tabacum

Suspension and Hairy Root Cultures

Preliminary investigations into the accumulation of virus in tissue culture were performed using N. tabacum suspension and hairy root cultures in shake flasks.

2.7.1 Growth of N. tabacum suspension and hairy root cultures

2.7.1.1 Growth of N. tabacum suspension cultures

Shake-flask investigations with N. tabacum suspension cultures were performed using

250-mL Erlenmeyer flasks containing a final volume of 50 mL. Ten mL of a mid-exponential-phase (Day 8) N. tabacum suspension culture was added to 40 mL MS medium containing 5.0 mg L-1 NAA and 0.25 mg L-1 kinetin. The flasks were incubated with shaking on an orbital shaker operating at 100 rpm in the dark at 25ºC. Suspension growth was examined over a 22-day period by periodically harvesting triplicate flasks.

2.7.1.2 Growth of N. tabacum hairy root cultures

Shake-flask investigations with N. tabacum hairy root cultures were performed using

50 mL Gamborg’s B5 medium in 250-mL Erlenmeyer flasks. Hairy roots (0.2 g fresh weight) from 14-day cultures were placed in the medium and incubated with shaking on an orbital shaker operating at 100 rpm in the dark at 25ºC. Hairy root growth was examined over a 36-day period by periodically harvesting triplicate flasks.

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2.7.2 Accumulation of TMV in N. tabacum suspension culture

Shake-flask investigations with N. tabacum suspension cultures and TMV were performed using 250-mL Erlenmeyer flasks containing a final volume of 50 mL.

Ten mL of a mid-exponential-phase (Day 8) N. tabacum suspension culture was added to 40 mL MS medium containing 5.0 mg L-1 NAA and 0.25 mg L-1 kinetin. The flasks were incubated with shaking on an orbital shaker operating at 100 rpm in the dark at

25ºC for 1 hour. TMV (75 µg TMV to give a concentration of 1.5 µg mL-1 TMV) or an equivalent volume of 0.01 M sodium phosphate buffer at pH 7.4 was added to the flasks. The cultures were vortexed (Janke & Kunkel VF2, IKA®-Labortechnik) for

30 seconds and then incubated on an orbital shaker operated at 100 rpm in the dark at

25ºC.

After 2 hours, viral inoculum was removed from half of the suspension cultures using medium exchange. The suspended cells and virus-containing medium were transferred to centrifuge tubes, and the cells allowed to settle to the base of the tubes. The virus- containing medium was removed and the cells were rinsed by re-suspending in

10 mL of fresh medium. The cells were again allowed to settle and the medium removed. Rinsed suspension cells were placed in new 250-mL Erlenmeyer flasks and the volume was made up to 50 mL with fresh medium. Cultures were returned to the orbital shaker operated at 100 rpm in the dark at 25°C.

Triplicate cultures were harvested 0, 5, 10, 14 and 19 days post virus inoculation for cell dry weight analysis and quantification of virus. Day 0 cultures were harvested

8 hours after virus addition to cultures. Fresh biomass samples taken to allow quantification of virus were frozen in liquid nitrogen prior to viral extraction.

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2.7.3 Accumulation of TMV in N. tabacum hairy roots

Shake-flask investigations with N. tabacum hairy root cultures and TMV were performed using 50 mL Gamborg’s B5 medium in 250-mL Erlenmeyer flasks. Hairy roots (0.2 g fresh weight) from 14-day cultures were placed in the medium and

1.5 µg mL-1 TMV (75 µg) or an equivalent volume of 0.01 M sodium phosphate buffer at pH 7.4 was added. Cultures were vortexed for 30 seconds and then incubated on an orbital shaker operated at 100 rpm in the dark at 25ºC.

After 4 hours, viral inoculum was removed from half of the hairy root cultures using medium exchange. Hairy roots were separated from the medium by vacuum filtration using a Buchner funnel and sterile Whatman No. 1 filter paper. Hairy roots on the filter paper were rinsed with fresh Gamborg’s B5 medium. Rinsed hairy roots were removed from the filter paper and placed in 50 mL fresh Gamborg’s B5 medium in 250-mL

Erlenmeyer flasks. Cultures were returned to the orbital shaker operated at 100 rpm in the dark at 25ºC.

Triplicate cultures were harvested 0, 5, 14 and 22 days post viral-inoculation and quadruplicate cultures were harvested 32 days post viral-inoculation for cell dry weight analysis and quantification of virus. Day 0 cultures were harvested 7 hours after virus addition to cultures. Fresh biomass samples taken to allow quantification of virus were frozen in liquid nitrogen prior to viral extraction.

2.8 Accumulation of TMV in N. benthamiana Suspension

In general hairy root investigations (N. tabacum and N. benthamiana), 0.2 g fresh weight hairy roots (approximately 0.011 g dry weight) was inoculated into 50 mL

117 medium and 1.5 µg mL-1 TMV was added, resulting in a total viral inoculum of 75 µg

TMV. The relatively low biomass:medium ratio used for hairy root cultures

(approximately 0.011 g dry weight in 50 mL) was not considered desirable for use with

N. benthamiana suspension cultures as this would require the addition of only a 4%

(v/v) inoculum from a mid-exponential-phase (Day 11) suspension culture, rather than the usual 20% inoculum which is conducive for suspension culture growth. As a compromise, a 14% inoculum (approximate dry weight of 0.033 g in 50 mL) from a

Day 11 suspension culture was selected for inoculation of N. benthamiana suspension cultures. The total culture volume used was 50 mL (43 mL fresh medium and 7 mL suspension culture) in 250-mL Erlenmeyer flasks. The same viral inoculum:dry biomass ratio was applied in the suspension and hairy root cultures (6820 µg TMV per gram dry weight biomass). Accordingly an inoculum TMV concentration of 4.5 µg mL-1 was used to inoculate the N. benthamiana suspension cultures.

Seven mL of Day 11 suspension culture was added to 43 mL of MS medium containing

30 g L-1 sucrose, 0.1 mg L-1 kinetin and 0.2 mg L-1 2,4-D in 250-mL Erlenmeyer flasks.

TMV was diluted with 0.01 M sodium phosphate buffer, pH 7.4, and added to the cultures to give a viral inoculum of 4.5 µg mL-1. An equivalent volume of 0.01 M sodium phosphate buffer was added to control cultures without virus. Cultures were incubated on an orbital shaker operated at 100 rpm in the dark at 25ºC. Cell growth and viral accumulation were examined over a 36-day period. Day 0 cultures were harvested

3.5 hours after virus addition. Quadruplicate flasks were harvested periodically for cultures inoculated with TMV; triplicate flasks were harvested for control cultures without virus. Fresh biomass samples taken to allow virus quantification were frozen in liquid nitrogen prior to viral extraction.

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2.9 Accumulation of TMV in N. benthamiana Hairy Roots

Viral accumulation in N. benthamiana hairy root cultures was examined using shake-flask time course studies.

All experiments examining viral accumulation in N. benthamiana hairy roots were performed using the following conditions unless otherwise stated. Cultures were grown in 250-mL Erlenmeyer flasks containing 50 mL of Gamborg’s B5 medium. The flasks were incubated in the dark at 25ºC on an orbital shaker operated at 100 rpm. Roots used as inoculum (0.2 g fresh weight) were from 21-day cultures, and were separated from the medium by vacuum filtration using a Buchner funnel and Whatman No. 1 filter paper. Virus was added to the hairy roots at the same time as root inoculation.

Virus was diluted in heat-sterilised 0.01 M sodium phosphate buffer at pH 7.4 and filtered using 0.2-µm syringe filters (Sartorius). In all experiments, an equivalent volume of 0.01 M sodium phosphate buffer was added to control flasks without virus.

Quadruplicate flasks were harvested for each measurement when cultures were inoculated with TMV; triplicate flasks were harvested for control cultures without virus. Fresh biomass samples were taken to allow virus and total soluble protein quantification and frozen in liquid nitrogen prior to processing. Fresh biomass samples taken to allow virus quantification accounted for at least 6% of the fresh biomass from advanced cultures (from Day 18) and up to 50% of the fresh biomass from less mature culture.

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2.9.1 Selection of N. benthamiana hairy roots with suitable growth and viral accumulation characteristics

Hairy root growth and TMV accumulation were examined in six N. benthamiana hairy root clones (Section 2.3.3.1) using shake-flask experiments. N. tabacum hairy root cultures were also included in the experiments to allow comparisons between the two species. Of the six N. benthamiana clones examined, one was initiated using

A. rhizogenes 15834 from a stem explant, and five were initiated using A. rhizogenes

A4, four from stem explants, and one from a leaf explant.

N. tabacum hairy roots (0.2 g fresh weight) from 14-day-old cultures and

N. benthamiana hairy roots (0.2 g fresh weight) from 14-day-old cultures were placed in 50 mL Gamborg’s B5 medium. TMV (1.5 µg mL-1) was added to the medium immediately after root addition. Triplicate cultures were harvested 0 and 5 days post- inoculation and quadruplicate cultures were harvested 14 days post-inoculation to monitor hairy root growth and viral accumulation. Day 0 cultures were harvested

4 hours after virus addition to cultures.

Based on the results of this experiment, a N. benthamiana hairy root clone initiated using A. rhizogenes A4 from a leaf explant was selected and used for all further examinations of viral accumulation in N. benthamiana hairy roots.

2.9.2 Time-course of TMV accumulation in N. benthamiana hairy roots

N. benthamiana hairy roots were inoculated with 1.5 µg mL-1 TMV. Hairy root growth and viral accumulation in treated and control cultures were examined over a 52-day period, by periodically harvesting quadruplicate or triplicate flasks.

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2.9.3 Effect of hairy root condition at the time of viral inoculation on TMV accumulation

2.9.3.1 Effect of root age

Variously aged roots in fresh medium

N. benthamiana hairy root cultures, initiated using root inocula from 6-, 10-, 14- and

21-day-old shake-flask cultures, were inoculated with 1.5 µg mL-1 TMV. Hairy root growth and viral accumulation were monitored over a 25-day period. The variously aged hairy-root pre-cultures were initiated using inoculum roots obtained from 14-day- old cultures (Appendix 3).

Variously aged roots in conditioned media

The feasibility of initiating TMV infection in established hairy root cultures by direct addition of virus to the cultures without medium exchange was investigated by infecting hairy root cultures in conditioned media.

N. benthamiana hairy root inocula from 6-, 10-, 14-, and 21-day-old shake-flask cultures were placed in 50 mL of conditioned medium collected from several root cultures of corresponding age. The cultures were inoculated with 1.5 µg mL-1 TMV.

Hairy root growth and viral accumulation were monitored over a 25-day period. The variously aged hairy-root pre-cultures were initiated using inoculum roots obtained from 14-day-old cultures (Appendix 3).

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2.9.3.2 Effect of injuring hairy roots prior to viral infection

The effect of intentional root injury prior to viral inoculation was examined. In these experiments carborundum and sandpaper were used as abrasives. The injury inflicted in the treatments was additional to the injury that inevitably occurs during the division of hairy root mats to form the 0.2 g hairy root inocula (subculture trauma).

For the control cultures without specific root injury (subculture trauma),

N. benthamiana hairy roots were inoculated with 1.5 µg mL-1 TMV by direct addition of the virus to the medium. This infection method can be considered as inoculation by simple co-incubation with virus.

For the cultures treated with carborundum, 0.03 g carborundum (500 mesh) was added to the medium before inoculation with N. benthamiana hairy roots. After hairy root addition, the flasks were vortexed on maximum speed for 20 seconds. After vortexing,

1.5 µg mL-1 TMV was added directly to the medium.

For the cultures treated with sandpaper, N. benthamiana hairy roots were drawn firmly over sandpaper (T421 Waterproof B07 P1000 sandpaper, Norton, Australia) five times to ensure that most of the roots came into contact with the abrasive surface. TMV

(37.5 µg) was added directly onto the injured roots and incubated for 1 hour prior to placing the roots in medium. A further 37.5 µg TMV was added to the medium after inoculum root addition to give a total viral inoculum concentration of 1.5 µg mL-1.

Hairy root growth and viral accumulation were examined in the control and treated flasks over a 36-day culture period.

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2.9.4 Characteristics of hairy root growth and viral accumulation in

N. benthamiana hairy roots over 36 days

N. benthamiana hairy roots were inoculated with 1.5 µg mL-1 TMV. Growth, medium characteristics and the characteristics of viral accumulation in TMV-infected and control cultures were examined over a 37-day period by periodically harvesting quadruplicate or triplicate flasks.

2.9.5 Effect of medium condition at the time of viral inoculation on TMV accumulation

2.9.5.1 Removal of TMV inoculum from cultures after an “inoculation phase”

N. benthamiana hairy roots were inoculated with 1.5 µg mL-1 TMV. After 16 hours of incubation the roots were separated from the medium by filtering the cultures through

Whatman No. 1 filter paper on a Buchner funnel using vacuum filtration, and the roots were rinsed with 3.0 mL fresh Gamborg’s B5 medium to remove residual medium and virus. The roots were removed from the filter paper using tweezers and placed into

50 mL of fresh Gamborg’s B5 medium. Hairy root growth and viral accumulation examined over a further 35 days. The results were compared with those from control cultures without inoculum TMV removal.

2.9.5.2 Inoculation of hairy roots with virus in phosphate buffer

N. benthamiana hairy roots were inoculated into either 50 mL of Gamborg’s B5 medium or 50 mL of 0.01 M sodium phosphate buffer at pH 7.4. TMV was added to the cultures at a concentration of 1.5 µg mL-1. After 23 hours incubation the roots were separated from the liquid by filtering the cultures through Whatman No. 1 filter paper on a Buchner funnel using vacuum filtration, and the roots rinsed with 2.0 mL of fresh

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Gamborg’s B5 medium. The roots were removed from the filter paper using tweezers and placed into 50 mL of Gamborg’s B5 medium. Hairy root growth and viral accumulation were examined over a further 35 days.

2.9.6 Alteration of viral inoculum concentration

In order to allow the effect of viral inoculum concentration on subsequent virus accumulation in N. benthamiana hairy roots to be examined, the concentration of viral inoculum was altered by changing the total amount of virus that was added to the medium.

N. benthamiana hairy roots were placed in 250-mL Erlenmeyer flasks containing

50 mL Gamborg’s B5 medium. TMV was added to the flasks at a concentration of

0.75 µg mL-1, 1.5 µg mL-1, 3.0 µg mL-1, 6.0 µg mL-1 or 9.0 µg mL-1. Each viral inoculum was diluted in 0.01 M sodium phosphate buffer, pH 7.4, such that the total inoculum added per culture was contained within a buffer volume of 54.7 µL. Control flasks were inoculated with 54.7 µL of 0.01 M sodium phosphate buffer, pH 7.4. Hairy root growth and viral accumulation were monitored over a 36-day period.

2.9.7 Proportional scale-up in shake flasks

The scale-up of viral inoculation in hairy roots was examined in shake flasks by keeping the ratio of root weight:amount of virus:medium volume constant while altering the volume of medium. As the medium volume was increased, the flask size was also altered. The medium volumes and flask sizes were selected with the aim of ensuring that gas–liquid oxygen mass transfer rates in the cultures were the same as that seen when 50 mL medium is used in a 250-mL Erlenmeyer flask. This was determined

124 according to the relationship provided by Henzler and Schedel (1991) for gas–liquid mass transfer in shake flasks:

1 8 1 8 a ⎛ v ⎞ 3 ⎛ D3 ⎞ 9 ⎛ D ⎞ 2 ⎛ v 2 ⎞ 27 ⎛ n 2 eD ⎞ k a⎜ ⎟ = B⎜ ⎟ ⋅ ⎜ F ⎟ ⋅ ⎜ ⎟ ⋅ ⎜ ⎟ (2.2) L ⎜ 2 ⎟ ⎜ ⎟ ⎜ 3 ⎟ ⎜ ⎟ ⎝ g ⎠ ⎝ VF ⎠ ⎝ v ⎠ ⎝ D g ⎠ ⎝ g ⎠

-1 where kL a is the gas–liquid oxygen mass transfer coefficient (s ), v is the kinematic viscosity of the liquid (m2s-1), g is the acceleration due to gravity (ms-2), B is a constant,

3 D is the widest diameter of the flask (m), VF is the volume of liquid in the flask (m ),

2 -1 DF is the diffusion coefficient of oxygen in the liquid (m s ), n is the speed of shaking

(s-1), e is the shaker eccentricity (m) and a is a specific phase interface exponent.

A number of these factors ( v , g, DF , n and e ) remain constant when only the flask and liquid volumes are changed. Therefore, the above equation can be simplified to:

8 8 3 9 ⎛ D ⎞ ⎛ 1 ⎞ 27 a k a ∝ ⎜ ⎟ ⋅ ⋅ D (2.3) L ⎜ ⎟ ⎜ 3 ⎟ () ⎝ VF ⎠ ⎝ D ⎠

If the value of a is taken at 0.5, as for water/sodium sulfite and glucose/sodium sulfite solutions (Henzler and Schedel, 1991), Equation (2.3) becomes:

−8 2.03 9 kL a ∝ D ⋅ VF (2.4)

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Using this relationship, the liquid volumes VF required in Erlenmeyer flasks of different

size were calculated to give the same kL a as obtained with 50 mL of liquid in 250-mL flasks. The results for 100-mL, 500-mL and 1000-mL flasks are listed in Table 2.1.

Once the liquid volumes VF were determined, the mass of hairy root inoculum and the

amount of virus added to the cultures were scaled in proportion to VF , based on the use of 0.2 g fresh weight hairy roots, 75 µg of virus and 50 mL medium. These results are also listed in Table 2.1.

The designated root weights were added to flasks containing the appropriate medium volumes, and the corresponding virus inocula or an equivalent volume of sodium phosphate buffer was added. Hairy root growth and viral accumulation were examined over a 36-day period.

Table 2.1 Flask sizes, medium volumes, root inocula and viral inocula used in the investigation of scale-up

Erlenmeyer Maximum Medium Hairy root Viral flask nominal flask base volume VF fresh weight inoculum (µg) volume (mL) diameter, D (mL) (g) (cm)

100 6.40 27 0.11 41.3

250 8.45 50 0.20 75

500 10.5 82 0.32 120

1000 13.0 134 0.54 201

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2.10 Accumulation of TMV in N. benthamiana Hairy Roots with an

Established Viral Infection

Viral accumulation in hairy root cultures with an established (systemic) viral infection was examined and compared to the viral yield and pattern of viral accumulation in hairy root cultures with a primary viral infection. The primary viral infection was initiated in hairy roots by co-incubating virus and freshly subcultured roots for 21 days. The roots with primary viral infections were transferred to fresh medium to initiate hairy root cultures with established viral infections.

2.10.1 Accumulation of TMV over three generations of culture

Primary TMV infection of N. benthamiana hairy roots was established by co-incubating 0.2 g fresh weight of roots with TMV (1.5 µg mL-1) in 50 mL medium.

Hairy root growth and viral accumulation in the biomass were examined over a 36-day period. Hairy root cultures infected by co-incubating hairy root inoculum with the inoculum virus in the culture medium are referred to as having a primary viral infection.

A first-generation root culture with an established viral infection was initiated by inoculating 0.2 g fresh weight of hairy roots from cultures with a primary TMV infection into 50 mL medium. Hairy roots with a primary TMV infection were used as the root inoculum 21 days after the cultures had been infected with TMV by co-incubation. Hairy root growth and viral accumulation in the first generation of cultures with an established viral infection were examined over a 36-day period.

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A second-generation hairy root culture with an established viral infection was initiated using roots from the first-generation hairy root culture with an established viral infection as an inoculum. The culture was established by adding 0.2 g fresh weight of roots from the first-generation culture with an established infection to 50 mL of medium. Hairy roots with first-generation established viral infections were used as the root inoculum 21 days after culture initiation. Hairy root growth and viral accumulation were examined over a 36-day period.

Inoculum roots used to initiate each generation of culture with an established infection were obtained from four replicate pre-cultures. At each sample time, cultures initiated using inoculum roots from each of the four pre-cultures were harvested.

2.10.2 Effect of increasing the viral inoculum concentration used to initiate a primary viral infection in hairy roots on viral accumulation in a subsequent-generation hairy root culture with an established viral infection

Primary TMV infection of N. benthamiana hairy roots was established by co-incubating

0.2 g fresh weight of roots with 9.0 μg mL-1 TMV in 50 mL of medium. Root growth and viral accumulation were examined over a 36-day period.

A first-generation hairy root culture with an established viral infection was initiated by inoculating 0.2 g fresh weight of hairy roots from the above cultures with primary viral infections into 50 mL medium. Hairy roots with a primary TMV infection were used as the root inoculum 21 days after the cultures had been infected with TMV by co-incubation. Inoculum roots used to initiate cultures with an established infection

128 were obtained from four replicate pre-cultures. At each sample time, cultures initiated using inoculum roots from each of the four pre-cultures were harvested. Hairy root growth and viral accumulation were examined in the cultures with established viral infections over a 36-day period.

The results for these experiments were compared with those obtained using a viral inoculum of 1.5 µg mL-1 as described in Section 2.10.1.

2.11 Viral Stability in Medium

2.11.1 Virus characteristics in plant media and phosphate buffer

2.11.1.1 Virus stability in fresh media

The stability of TMV in fresh plant culture media (Gamborg’s B5 medium and MS medium) and 0.01 M sodium phosphate buffer, pH 7.4, was examined over a 29-hour period. Fifty mL of sterile plant medium or sodium phosphate buffer was added to 250- mL Erlenmeyer flasks post-sterilisation to ensure the liquid volume was not reduced during sterilisation. Flasks were prepared in triplicate. Virus was added at a concentration of 1.5 µg mL-1 to the media and buffer that had been pre-warmed to 25ºC.

After viral addition, the flasks were incubated in the dark at 25ºC on an orbital shaker operated at 100 rpm. Samples were taken periodically, frozen immediately using liquid nitrogen, and then stored at –20ºC until assayed. Samples were assayed using Enzyme

Linked Immunosorbent Assay (ELISA) to measure virus concentration in the media

(Section 2.17.4.1).

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2.11.1.2 Virus infectivity in fresh media

The determination of virus infectivity using local lesion assays (Section 2.17.4.4) required a TMV concentration higher than 1.5 g mL-1, the concentration added to media for general investigations. To allow the infectivity of TMV in different medium to be determined, the above investigation (Section 2.11.1.1) was repeated using a higher concentration of TMV.

Twenty-five mL of sterile B5 medium, MS medium or 0.01 M sodium phosphate buffer, pH 7.4, was added to 250-mL Erlenmeyer flasks post-sterilisation to ensure the liquid volume was not reduced during sterilisation. Flasks were prepared in triplicate.

Virus was added at a concentration of 11.25 µg mL-1 to the media and buffer that had been pre-warmed to 25ºC. After viral addition, flasks were incubated in the dark at

25ºC on an orbital shaker operated at 100 rpm. Samples were taken 0.06, 10.5 and

29 hours after virus addition, frozen immediately using liquid nitrogen, and then stored at –20ºC until assayed. Infectivity of virus in the media and buffer was examined using local lesion assays.

2.11.2 Virus characteristics in conditioned media

The stability of virus in conditioned media was examined to help assess whether viral inoculation of root cultures could occur directly into conditioned plant media

(Section 2.9.3.1). The stability and infectivity of virus in the conditioned media were examined over a 12-hour and 27-hour period, respectively.

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2.11.2.1 Virus stability in conditioned media

Virus-free conditioned media were obtained from N. benthamiana hairy root cultures

6, 10, 14 and 21 days post-culture-initiation, where cultures were initiated by the addition of 0.2 g fresh weight of N. benthamiana hairy roots into 50 mL of medium.

The conditioned media were separated from the hairy roots by filtering the cultures through sterile Whatman No. 1 filter paper using a Buchner funnel. Medium from replicate cultures (between 5 and 8 cultures depending on the medium volume remaining in the cultures) was pooled and 50 mL of the pooled medium was added to each of three 250-mL Erlenmeyer flasks. Protein concentrations in the conditioned media were determined using the method described in Section 2.17.7. Virus was added to each of the media (pre-warmed to 25ºC) at a concentration of 1.5 µg mL-1 and incubated in the dark at 25ºC on an orbital shaker operated at 100 rpm. Fresh

Gamborg’s B5 medium was also inoculated with virus as a control. One-mL samples were removed periodically over a 12 hour period, with the first sample taken as close to the time of virus addition as possible (less than 5 minutes after virus addition). Samples were frozen in liquid nitrogen and stored at –20ºC until analysed for virus concentration using ELISA (Section 2.17.4.1).

2.11.2.2 Virus infectivity in conditioned media

As described in Section 2.11.1.2 the above investigation (Section 2.11.2.1) was repeated using a higher concentration of TMV. Fifteen mL of conditioned medium was added to each of three sterile 250-mL Erlenmeyer flasks. Virus was added at a concentration of 11.25 µg mL-1 and incubated in the dark at 25ºC on an orbital shaker operated at 100 rpm. Samples taken periodically over a 27-hour period were assayed for infectivity using local lesion assays (Section 2.17.4.4).

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2.12 Short-Term Virus Association Experiments

Short-term experiments were performed to allow analysis of viral association with hairy roots. The aim of these experiments was to determine if there was any relationship between the concentration of viral inoculum and the association of virus to roots, or between inoculum virus association with hairy roots and long-term viral accumulation in the root cultures.

2.12.1 Concentration of viral inoculum and viral association with hairy roots

N. benthamiana hairy roots were inoculated into 50 mL medium and incubated for approximately 16 hours. TMV was added to the cultures to achieve viral concentrations of 0.75 µg mL-1, 1.5 µg mL-1, 3.0 µg mL-1, 6.0 µg mL-1 and 9.0 µg mL-1. In negative control flasks, an equivalent volume of 0.01 M sodium phosphate buffer, pH 7.4, was added instead of virus. Viral association with hairy roots was timed from the point of virus addition to the cultures. Quadruplicate flasks were harvested periodically over a

12 hour period after virus addition. Roots were separated from the medium using a

Buchner funnel and the roots were gently rinsed with Milli-Q water. Hairy roots from each flask were divided and either used for determination of biomass dry weight or frozen in liquid nitrogen for later processing and analysis of virus concentration using

ELISA (Section 2.17.4.1).

2.12.2 Viral association with roots during proportional scale-up

The concentration of inoculum virus associated with hairy roots was examined using scale-up parameters to determine the effect of proportional scale-up of root weight, amount of virus and medium volume on the association of inoculum virus with

132 inoculum roots. The flask sizes, medium volumes, root inocula and viral inocula used in the investigation were the same as those described in Section 2.9.7 and listed in

Table 2.1.

The designated N. benthamiana hairy root weights were added to flasks containing the appropriate medium volumes and incubated for approximately 16 hours. Appropriate amounts of TMV were added to cultures to give a medium virus concentration of

1.5 µg mL-1. In negative control flasks, an equivalent volume of 0.01 M sodium phosphate buffer, pH 7.4, was added instead of virus. Virus association with the hairy roots was timed from the point of virus addition to the cultures. Quadruplicate flasks were harvested periodically over a 12 hour period after virus addition. Roots were separated from the medium using a Buchner funnel and the roots were gently rinsed with Milli-Q water. Hairy roots from each flask were divided and used for either determination of biomass dry weight or frozen in liquid nitrogen for later processing and analysis of virus concentration using ELISA (Section 2.17.4.1).

2.13 Association of Deactivated TMV with Hairy Roots

2.13.1 Deactivation of TMV

TMV was deactivated by exposure to ultraviolet (UV) irradiation to render it non- infectious without significantly altering the virus particle structure or the ability of specific antibodies to interact with viral coat protein.

TMV was diluted in 0.067 M sodium phosphate buffer, pH 7.0, so that the final virus concentration was 0.652 mg mL-1. Diluted TMV (12 mL) was added to

100-mm-diameter glass Petri dishes. The depth of virus preparation prior to

133 evaporation was approximately 1.5 mm. The virus preparation was exposed to ultraviolet radiation in a laminar flow cabinet (Airpure Laminar Flow Cabinet: Email

Westinghouse, Australia) approximately 34 cm from the lamp. A minimum of 400 mW m-2 UV radiation (254 nm) was generated by the lamp used. The Petri dishes were rocked to promote mixing of the viral preparations and UV inactivation. Viral preparations were exposed to UV radiation for 0.5, 1, 2, 4, 6, 8, 10 and 12 hours.

The volume of the viral preparations was made up to 12 mL after UV exposure and infectivity of virus in the preparations was determined using local lesion assays

(Section 2.17.4.4). The viral concentration in irradiated preparations was determined spectrophotometrically (Section 2.5.3) and using ELISA (Section 2.17.4.1) to determine if irradiation reduced the binding of anti-TMV antibody to TMV. The ratio of absorbance of the viral preparations at 260 nm and 280 nm (A 260/280) was examined to determine if RNA and protein ratios were altered.

2.13.2 Hairy roots and deactivated virus association

N. benthamiana hairy roots were inoculated into 50 mL medium and incubated for approximately 16 hours. Deactivated TMV (6–8 hours UV exposure) was added to the cultures to achieve a viral concentration of 1.5 µg mL-1. In negative control flasks, an equivalent volume of 0.067 M sodium phosphate buffer, pH 7.0, was added instead of virus. Viral association with hairy roots was timed from the point of virus addition to the cultures. Quadruplicate flasks were harvested periodically over a 36-day period, with samples taken frequently within the first 12 hours after virus addition. Hairy roots were separated from the medium using a Buchner funnel and the roots were gently rinsed with Milli-Q water. Hairy roots from each flask were divided and used for either

134 determination of biomass dry weight or frozen in liquid nitrogen for later processing and analysis of viral association with the biomass. Medium samples were taken prior to root separation from the medium to allow medium virus concentration to be determined.

2.14 Accumulation of Genetically Modified Virus and GFP in Hairy Roots

N. benthamiana hairy roots were inoculated into 50 mL medium in 250-mL Erlenmeyer flasks. Using the viral quantification methods described in Section 2.17.6, 75 µg TMV was found to be equivalent to approximately 1.98 × 1011 TMV particles. Accordingly, to provide an equivalent number concentration of TMV-GFPC3 particles to the cultures

1.98 × 1011 TMV-GFPC3 particles (Section 2.6) were added to each flask. Hairy root cultures were also inoculated with 3.97× 1011 TMV-GFPC3 particles. TMV-GFPC3 particles were diluted in 0.01 M sodium phosphate buffer, pH 7.4, and filtered through a 0.2-µm Minisart filter (Sartorius) prior to addition to the cultures. Control cultures were inoculated with an equivalent volume of 0.01 M sodium phosphate buffer, pH 7.4.

Cultures were incubated in the dark at 25ºC on an orbital shaker operated at 100 rpm.

Hairy root growth, viral accumulation and GFP accumulation were monitored over a

36-day period.

2.15 Viral Distribution in Hairy Root Clumps in Shake Flasks

The viral distribution in hairy roots grown in Erlenmeyer flasks was examined to determine if TMV was distributed evenly throughout the root mass. N. benthamiana hairy roots (0.2g fresh weight) from 21-day-old cultures were placed in 50 mL

Gamborg’s B5 medium in 250-mL Erlenmeyer flasks. TMV (1.5 µg mL-1) was added to the medium immediately after hairy root addition. The cultures were incubated in

135 the dark at 25ºC on an orbital shaker operated at 100 rpm. N. benthamiana hairy roots grown in Erlenmeyer flasks with shaking form a circular root mat. The root mat can be removed from the flask without being significantly disrupted.

2.15.1 Distribution of virus in different concentric regions

Circular root mats from five TMV-infected N. benthamiana hairy root cultures grown in 250-mL Erlenmeyer flasks were divided into three concentric regions. The regions were an inner core region that contained older roots including roots that comprised the initial root inoculum, a middle concentric region, and an outer concentric region that predominantly contained young roots. Samples from each concentric region were analysed for viral accumulation to determine if virus was distributed evenly across the different regions. Virus concentrations were determined in the hairy roots using approximately 16–42% of the biomass from each concentric region. The hairy roots were examined 27 days post-inoculation.

2.15.2 Distribution of virus in radial segments

Circular root mats from three TMV-infected N. benthamiana hairy roots grown in

250-mL Erlenmeyer flasks were divided into nine radial segments of mass 0.6–1.3 g fresh weight each. Each segment contained old roots from the centre of the root clump as well as younger roots from the outer edge of the clump. Eight segments were analysed for virus accumulation and one was used to determine biomass dry weight.

The hairy roots were examined 34 days post-inoculation.

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2.16 Accumulation of TMV in N. benthamiana Hairy Roots in a Stirred

Bioreactor

2.16.1 Bioreactor configuration

Hairy roots were grown in 2-L bioreactors (Quickfit, UK) fitted with stainless steel headplates (LH Fermentations, USA). Duplicate bioreactors were used to culture non- infected and TMV-infected N. benthamiana hairy roots simultaneously, while

TMV-GFPC3-infected hairy roots were grown in a single bioreactor.

The bioreactors were operated with an initial medium volume of 2.0 L. Fish-tank air pumps (Air pump 301: Rena, France) were used to provide air to the bioreactors. The air was humidified to minimise medium evaporation using a 500-mL Quickfit Dreschel

Bottle with a stopper incorporating a glass sparger. The flow rate of the air was regulated using a gas rotameter (Platon, Duff and Macintosh, Australia) to supply an air flow rate of 230 mL min-1. The air was filter-sterilised using a Midisart 2000

0.2-µm polytetrafluoroethylene (PTFE) filter (Sartorius) and entered the vessel through a horizontal sintered stainless steel sparger (length 25 mm and diameter 6 mm) with

2-µm pore size (Mott Pacific, Australia) located approximately two centimeters above the base of the vessel. Dissolved oxygen tension was monitored using an Ingold polargraphic electrode (Ingold, Switzerland) connected to an oxygen amplifier (Type

170% aire, Ingold). The probe was calibrated in Gamborg’s B5 medium using nitrogen gas (BOC Gases, Australia), and air. Exit gas was passed through a condenser operated at 4ºC to minimise liquid loss from the bioreactor, and then through 0.2-µm PTFE filters (Sartorius) and a glass-fibre-filled depth filter arranged in series. The filters were exchanged every 10 days, to ensure that gas exiting the vessel was not limited by filter blockage.

137

The temperature of the bioreactors was controlled using a combination of cooling water and a heating lamp. Each bioreactor was cooled by circulating water at 15ºC through a thin-walled stainless steel tube within the vessel. A custom made temperature probe placed inside a stainless steel sleeve in the bioreactor was connected to an on-off proportional band controller (RKC Instrument Inc., Japan). The controller was set to switch on a heating lamp (Siccatherm 250: Osram, Germany) situated approximately

20 cm from the bioreactor when the temperature inside the Vessel fell below 23ºC. The bioreactor was covered with black cardboard to exclude light from the cultures. Mixing in the bioreactors was achieved using a Teflon-covered 5.5-cm-long magnetic stirrer- bar and magnetic stirrer (B211: J. Bibby Scientific, USA).

A flat-bottomed cylindrical stainless steel mesh basket with a diameter of 2.2 cm and length 18 cm was used to contain the hairy root inoculum. The basket was suspended from a stainless steel tube (1.6 cm internal diameter) inserted through the central port on the headplate so that its base was 3 cm from the base of the bioreactor and directly above the sparger. The top of the basket extended above the surface of the medium in the bioreactor.

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2.16.2 Bioreactor culture methods

The bioreactor and medium (2.0 L) were sterilised separately. Gamborg’s B5 medium

(1.2 L) was added initially to the bioreactor to allow the equilibration and calibration of the dissolved oxygen probe. After calibration of the probe, the remainder of the medium, the hairy roots and any viral inoculum used were added. Medium and roots were added using a 500-mL bottom-side-opening inoculation flask connected with wide-bore silicone tubing (1.3 cm diameter) to a stainless steel tube inserted through the centre of the headplate. N. benthamiana hairy roots (8.0 g fresh weight) were separated into small clumps and mixed with a large proportion of the remaining medium in the inoculation flask, and inoculated into the cylindrical mesh basket in the bioreactor. The remaining volume of medium was used to wash any trapped roots from the silicone and stainless steel tubing into the basket. If virus was to be added to the bioreactor, it was mixed with the medium used to wash the roots from the tubing. TMV was added to the bioreactor to give a viral concentration of 1.5 µg mL-1. TMV-GFPC3 was added so that

7.94 ¯ 1012 particles were inoculated into the bioreactor to give a particle number concentration equivalent to 1.5 µg mL-1 TMV.

Medium samples were taken from the bioreactors using a sampling port every 3 days for 30 days. The total sugar concentration in the medium and the medium viral concentration, where applicable, were determined. Medium conductivity and pH were also monitored.

The cultures were terminated on Day 31, and the harvested roots and medium were separated. Root samples were taken for analysis of virus accumulation in different regions of the root mass, and the remainder was dried. A schematic of the cylindrical

139 root mass indicating the position of root samples within the biomass is shown in

Figure 2.1. Triplicate radial samples of thickness 0.5 cm were taken from roots at the top of the root mass adjacent to the headspace (top), roots growing near the base of the vessel (base) and also roots halfway (mid-height) up the vessel height. The mid-height radial root samples were divided into three sections, allowing analysis of viral concentrations in roots adjacent to the mesh basket, mid-radius and adjacent to the bioreactor wall (each sample encompassing a radial length of 2 cm). The viral concentration in roots in the mesh basket was also analysed, using samples taken from the base, top and mid-height of the basket.

Bioreactor investigations were performed in duplicate for non-infected hairy roots and hairy roots infected with TMV. Only one bioreactor investigation was performed when roots were infected with TMV-GFPC3 due to the limited availability of viral inoculum.

140

A. B.

Top

Mid-height b.

Base

Figure 2.1 Schematic of the cylindrical root mass of bioreactor grown hairy roots indicating sampling regions. (A) Cylindrical root mass indicating regions from which samples were taken for analysis of viral accumulation. (B) Position of hairy root samples obtained from within the mesh basket (●). (C) Schematic of transverse root sections (0.5 cm thickness) through the cylindrical root mass from near the top, mid- height and base of the root mass. Triplicate rectangular sections (▬) taken from different regions of the transverse sections, containing hairy roots from near the mesh basket and close to the bioreactor wall were analysed for viral accumulation. (D) Schematic of a rectangular sample from the mid-height of the root mass, indicating the division of the triplicate root samples into 2 cm sections from (a) adjacent to the mesh basket, (b) mid radius and (c) adjacent to the bioreactor wall.

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2.17 Analytical Procedures

2.17.1 Growth

The fresh weight of hairy roots and suspended cells was determined by separating the medium from the biomass using suction filtration through dried pre-weighed Whatman

No. 1 filter paper. The biomass and wet filter paper were separated and the total biomass weighed to obtain the biomass fresh weight. A known proportion of the fresh biomass was placed on the pre-weighed filter paper and dried in an oven for 2 days at

50ºC. The dry paper and biomass were cooled in a desiccator. The cooled dry samples and the filter paper were weighed together. The net dry sample weight was taken to be the difference between the combined weight of the dried plant biomass and filter paper, and the weight of the pre-weighed dry filter paper. From the net dry weight, the total biomass dry weight was determined taking into account the proportion of the biomass sampled.

The biomass dry weight of tissue from whole plants was also determined using the above method.

2.17.2 Medium characteristics

2.17.2.1 Medium pH

The pH of medium samples was determined after suspended cells and hairy roots were removed from the medium. pH was measured using a double-junction pH electrode

(Cole-Parmer, USA) and a pH meter with temperature compensation (Activon,

Australia).

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2.17.2.2 Medium conductivity

Medium conductivity was monitored using a CDM 80 conductivity meter (Radiometer,

France) with a type CDC114 probe (Radiometer) referenced at 25ºC. Conductivity of the medium was measured after suspended cells and hairy roots were removed from the medium.

2.17.2.3 Medium sugar concentration

Medium samples from the bioreactors and selected shake-flask hairy root cultures were analysed for sucrose, glucose and fructose by High Performance Liquid

Chromatography (HPLC) (Waters, USA). Medium samples were frozen at –20ºC until analysed. Prior to analysis, the samples were thawed and filtered (0.45-µm GHP

Acrodisk: Gelman, USA) to remove particulate matter. Samples were injected at ambient temperature into a 250 mm × 4.6 mm I.D. Econosil NH2 5-µm column

(Alltech, USA). The mobile phase was 79% acetonitrile (Asia Pacific Specialty

Chemicals) and 21% Milli-Q water. The flow rate was 1.6 mL min-1, the sample injection volume was 7.0 µL and the run time was 26 minutes. Each sample was analysed in duplicate. Data were collected and the peaks integrated using a Delta 5

Chromatography Data System (Digital Solutions, Australia). Standard solutions containing 30.0 g L-1, 15.0 g L-1, 5.0 g L-1 and 1.0 g L-1 of sucrose, D (-) glucose

(Asia Pacific Specialty Chemicals) and D (-) fructose (Sigma-Aldrich) were used to obtain standard curves.

The concentrations of sucrose, glucose and fructose in the medium samples were used to calculate total sugar concentrations as sucrose equivalents, i.e.

143

[Total sugar] = [Sucrose] + 342 ([Glucose] + [Fructose]) (2.5) 360

where [] denotes concentration in units of g L-1, to take into account the molecule of water incorporated during hydrolysis of sucrose (Sharp and Doran, 1990).

2.17.3 Sample extraction for the analysis of virus, total protein and GFP

2.17.3.1 Sample extraction for the detection of virus in plant biomass using ELISA

The composition of the viral extraction buffer was 1.3 g L-1 sodium sulphite (BDH,

Australia), 0.2 g L-1 potassium chloride (Asia Pacific Specialty Chemicals),

20.0 g L -1 polyvinylpyrrolidone average molecular weight 40 000 (Sigma-Aldrich),

2.0 g L-1 bovine albumin (BSA) (Sigma-Aldrich) and 20.0 g L -1 Tween 20. The buffer components were added to sodium phosphate buffered saline (PBS), which consisted of

8.0 g L-1 sodium chloride, 1.15 g L-1 di-sodium phosphate, 0.2 g L-1 monopotassium phosphate (Asia Pacific Specialty Chemicals) and 0.05% (w/v) Tween 20. The pH of the extraction buffer was adjusted to pH 7.4 using potassium hydroxide.

Suspension cells, hairy roots or whole plant material was frozen in liquid nitrogen prior to virus extraction. Frozen material was ground in a cold mortar and pestle in the presence of a small amount of 50–150-mesh acid-purified sand (BDH) and cold viral extraction buffer, using 1.0 mL buffer per 0.1 g of plant biomass. The extract was centrifuged in a refrigerated centrifuge (4ºC) at 10 000 × g for 20 minutes. The supernatant was collected and frozen using liquid nitrogen until use.

The effect of the above extraction protocol on viral particle yield was examined to determine if either grinding virus in a mortar and pestle or adsorption of virus to ground

144 hairy root biomass resulted in significant reductions in recoverable virus. Virus was added to the viral extraction buffer and one mL of the virus-containing extraction buffer was ground in the presence of acid-purified sand (0.05 g) and 10% (w/v) fresh weight

N. benthamiana hairy roots (frozen) in a cold mortar and pestle for 30 seconds. Ground samples were centrifuged in a refrigerated centrifuge (4ºC) at 10 000 × g for 20 minutes. The concentrations of virus in sample supernatants were compared to the concentration of virus in the unprocessed extraction buffer. Results are shown in

Appendix 2.

2.17.3.2 Sample extraction for the detection of GFP in plant biomass and for virus detection using Western blot

Hairy roots and whole plant material was frozen in liquid nitrogen prior to extraction.

Frozen material was ground in a cold mortar and pestle in the presence of a small amount of 50–150-mesh acid-purified sand and PBS containing a protease inhibitor cocktail for plant cell and tissue extracts (Sigma-Aldrich). The protease inhibitor cocktail contained 4-(2-aminoethyl)benzenesulfonyl fluoride, bestatin, pepstatin A, trans-epoxysuccinyl-L-leucylamido(4-guaridino)butane, leupeptin and

1,10-phenanthroline. For each 0.1 g of plant biomass, 967 µL PBS and 33 µL protease inhibitor cocktail was used for extraction. The extract was centrifuged in a refrigerated centrifuge (4ºC) at 10 000 × g for 20 minutes. The supernatant was collected and frozen in liquid nitrogen until analysis.

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2.17.3.3 Sample extraction for assessment of total soluble protein

Suspension cells, hairy roots or whole plant material was frozen in liquid nitrogen prior to processing. Frozen material was ground in a cold mortar and pestle in the presence of a small amount of 50–150-mesh acid-purified sand and PBS, using 1.0 mL PBS to extract 0.1 g plant biomass. The extract was centrifuged in a refrigerated centrifuge

(4ºC) at 10 000 × g for 20 minutes. The supernatant was collected and frozen in liquid nitrogen until use.

2.17.4 Quantification and analysis of virus

2.17.4.1 Quantification of TMV using ELISA

TMV accumulation in plant biomass and culture medium was quantified using a commercially available direct-sandwich ELISA designed to detect TMV vulgare

(TMV-c) (Agdia, USA). The amount of viral antigen in extracts from TMV-infected plant biomass, suspension cells and protoplasts detected using ELISA has been reported to correspond strongly to virus detected using bioassay (Sander and Mertes, 1984). The manufacturer’s methods were followed with minor modifications. The method used is outlined below.

High-binding 96-well microtitre plates (Nunc-immunoTM plate MaxisorpTMsurface:

Nunc, Denmark) were coated with a polyclonal chicken anti-TMV-c antibody diluted according to the manufacturer’s instructions in carbonate buffer, pH 9.6, which consisted of 1.59 g L-1 sodium carbonate (Ajax Finechem) and 1.59 g L-1 sodium hydrogen carbonate (Ajax Finechem). One hundred µL of diluted coat antibody was applied per well, and the plates were incubated overnight at 4ºC. The coat antibody was removed from the plates by flicking and the wells washed three times with 150 µL

146

PBS containing 0.05% (w/v) Tween 20. Plant extracts (Section 2.17.3.1) were applied to the microtitre plates and serially diluted using viral extraction buffer (Section

2.17.3.1). Purified TMV (Section 2.5.2) with a known concentration determined using

UV absorption spectrophotometry (Section 2.5.3) was used as a standard, and was also serially diluted using viral extraction buffer. TMV standards were applied at initial concentrations of between 3.0 × 10-3 and 3. 0 × 10-4 µg mL-1 depending on the concentration of TMV expected in the extracts examined. Microtitre plates with samples applied were incubated overnight at 4ºC. The samples were removed from the plates by flicking and the wells washed three times with 150 µL PBS containing

0.05% (w/v) Tween 20. A horseradish-peroxidase-labelled polyclonal chicken anti-TMV-c antibody diluted according to the manufacturer’s instructions was applied to the plates and incubated for 2 hours at 37ºC. The labelled antibody was removed from the plates and the wells washed three times with 150 µL PBS containing

0.05% (w/v) Tween 20. The ELISA was developed using o-phenylenediamine dihydrochloride and the reaction was stopped using 3 M sulphuric acid (BDH). The developed ELISA was read at 490 nm using an Emax precision microplate reader

(Molecular Devices, USA). Non-specific background reactions attributable to non-viral components in the plant tissue extracts were low.

When the viral concentration in plant culture medium was assayed, samples were serially diluted in PBS containing 0.05% (w/v) Tween 20 instead of viral extraction buffer.

Both the capture antibody and the enzyme-labelled antibody employed in this ELISA were polyclonal and raised using whole TMV particles. Although the antibodies were

147 raised using full-length virus particles, because TMV particles are composed of multiple copies of the coat protein arranged in a helical manner around the viral genomic RNA (Section 1.6.1), the assay would not have been specific for full viral particles. The polyclonal capture antibody would have been able to pull down full

TMV particles, truncated particles of less-than-full-length and free coat protein molecules. Similarly, the enzyme-labelled antibody would have bound captured full- length particles and truncated particles. The polyclonal enzyme-labelled antibody would probably also have been able to bind captured free coat protein molecules or aggregated coat protein, although steric hindrance could have interfered with some interactions.

When investigating the effect that the truncation of viral particles would have on virus titres determined using an ELISA with polyclonal capture and enzyme-labelled antibodies, Thomas and Warren (1994) observed that truncated particles formed by mechanically damaging particles gave an “appreciably” higher titre than an equivalent mass of full length TMV particles and that free coat protein molecules formed by the disruption of virus particles with alkali gave a titre twice that of an equivalent mass of whole virus.

The potential ability of the ELISA used in this investigation to detect truncated particles and free coat protein has some implications for the accuracy of the titres obtained, as the presenceof these elements could result in artificially high viral titres. Although it was expected that the presence of truncated particles and free coat protein molecules in cell extracts could result in a small artificial increase in the calculated titre, due to the inherent inaccuracy and limitations of other detection methods, it was difficult to

148 determine the degree of error. It was however expected that the yield determined using

ELISA would reflect well the actual yield for a number of reasons;

1. The percentage of less-than-full-length viral particles in the sap from hairy roots

was found to be relatively low (less than 20%) (Section 3.9.4.2). The processing

of biomass (Section 2.17.3.1) either did not result in particle truncation or

truncated particles did not give a higher titre than an equivalent mass of whole

virus in the ELISA (Section 2.17.3.1 and Appendix 2).

2. The purified virus preparation used as the standard in the ELISA contained

particles of less-than-full length and therefore the effect of truncated virus in the

samples on the final titre would be reduced. The percentage of particles with a

length less than that of full TMV particles (approximately 25%) was higher than

that that observed in the sap of hairy roots. The higher proportion of particle

truncation than in sap may have been attributable to particle disruption during

virus extraction and purification. The concentration of the standard virus

preparation was determined using UV absorption spectrophotometry (Section

2.5.3) and the contribution of truncated viral particles to the total viral yield would

have been proportional to their RNA content

3. The concentration of free coat protein should have been relatively low in root

extracts because within the cells most coat protein is assembled into viral particles

(Shalla and Amici, 1964; Hills et al., 1987). High particle stability would have

resulted in only limited disintegration of viral particles after extraction. Non-

particle associated coat protein would probably have been in the form of small

aggregates (Butler, 1984, 1999).

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2.17.4.2 Western blot detection of viral coat protein

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and Western blotting were utilised to detect viral coat proteins from

TMV and tobacco mild green mosaic virus (from TMV-GFPC3) (Section 1.7.1).

Extracts from plant biomass were prepared for electrophoresis by mixing samples

(Section 2.17.3.2) with Laemmli’s sample buffer (Laemmli, 1970) containing 5% (v/v)

β-mercaptoethanol (Sigma-Aldrich) using a sample:buffer volume ratio of 1:1.

Samples were heated at 100ºC for 3 minutes. Depending on the virus being analysed, either diluted purified TMV (Section 2.5.2) or TMV-GFPC3 (Section 2.6.4) was used as positive controls. Diluted purified virus was prepared for electrophoresis in the same manner as biomass samples. SeeBlue®Plus2 pre-stained standard (Invitrogen, USA) and MagicMarkTM Western Protein Standard (Invitrogen) were used as molecular weight markers.

Twenty µL (or a volume suitable to the well volume) of prepared sample–Laemmli’s buffer mixture were loaded into the wells of 10% Tris-glycine minigels (Invitrogen).

TMV (27.4 ng) and/or 9.15 ×107 viral particles of TMV-GFPC3 was used as a positive control. Molecular weight markers were applied to the gels according to the manufacturer’s instructions. Gels were run at a constant voltage of 125 volts for

100 minutes at room temperature using a Novex xCell IITM Mini Cell (Novex, USA).

After electrophoresis, the proteins were transferred to 0.45-µm Nitrocellulose Extra

Blotting Membranes (Sartorius) using a custom-made semi-dry blotting apparatus with

Tris-glycine transfer buffer. The Tris-glycine transfer buffer consisted of 5.8 g L-1

Tris-base (Sigma-Aldrich), 2.9 g L-1 glycine (Ajax Finechem) and 0.37 g L-1 lauryl

150 sulfate sodium salt (SDS) (Sigma-Aldrich ) in 200 mL methanol (Ajax Finechem) made to 1 L with Milli-Q water. Transfer was carried out for 3 hours at a constant current of

2 milliamperes per cm2 of gel.

After protein transfer, the membranes were blocked at room temperature overnight by shaking in blocking buffer, which consisted of 50 mM Tris-base, pH 7.5 (Sigma-

Aldrich) containing 150 mM sodium chloride and 2% (w/v) BSA. Membranes were washed three times for 10 minutes each wash in washing buffer, which consisted of

50 mM Tris-base, pH 7.5, containing 150 mM sodium chloride and 0.05% (w/v)

Tween 20. The membranes were then incubated with unlabelled polyclonal rabbit anti-

TMV antibody (Agdia) at a concentration of approximately 0.4 µg mL-1 in washing buffer for 2 hours at room temperature with gentle shaking. The anti-TMV antibody was raised using whole viral particles from a number of TMV strains and was reactive against a variety of tobamoviruses. Membranes were rinsed twice and washed once for

15 minutes and three times for 5 minutes each wash in a large volume of washing buffer. The membranes were then incubated for 2 hours at room temperature with gentle shaking in horseradish-peroxidase-labelled goat anti-rabbit antibody

(DakoCytomation, Denmark) diluted in washing buffer according to the manufacturer’s recommendation. Membranes were rinsed twice and washed once for 15 minutes and three times for 5 minutes each wash in a large volume of washing buffer.

Blots were developed using ECL development solution (Amersham Biosciences,

England) according to the manufacturer’s instructions. X-ray film was exposed to the membrane for 1–2 minutes, developed using GBX developer and replenisher

151

(Eastman Kodak, USA) and fixed with GBX fixer and replenisher (Eastman Kodak).

The film was rinsed with Milli-Q water prior to drying.

TMV-GFPC3 was quantified from Western blots, using SynGene GeneTools Analysis

Software (Version 3.03.03: SynGene Laboratories, USA). Four serially diluted standards containing known numbers of TMV-GFPC3 particles were used to form the standard curve for quantification.

2.17.4.3 Particle size analysis

Transmission electron microscopy was performed with the assistance of Mrs. Margaret

Budanovic, Electron Microscopy Unit, University of New South Wales.

The size and conformation of TMV particles extracted from hairy roots was examined using transmission electron microscopy. The medium was removed from hairy roots infected with TMV using suction filtration through Whatman No. 1 filter paper. A small sample of roots was crushed between two clean glass slides and the sap collected in a microfuge tube and centrifuged in a refrigerated centrifuge (4ºC) at 10 000 × g for

20 minutes to remove cell debris. A drop of the clarified sap was placed on parafilm and a formvar-(ProSciTech, Australia) coated 200-mesh copper grid (ProSciTech) was placed on the drop. After 60 seconds, the grid was removed and excess sap was drawn off using filter paper. The grid was negatively stained using 4% uranyl acetate (BDH) in water by floating the grid on a drop of stain for 30 seconds. Excess stain was drawn off using filter paper. Prepared grids were examined using a Hitachi 7000 transmission electron microscope (Hitachi, Japan) and micrographs taken of all observed viral particles.

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The size of viral particles in purified viral preparations was also examined using the same sample staining methodology.

The size of virus particles extracted from N. benthamiana hairy roots was determined by measuring the length of particles in micrographs. Measurements were made using a ruler with 0.5 mm gradations and compared with the micrograph scale to determine actual particle length. Particle length data were presented in the form of a relative frequency distribution with class size of 40 nm. Forty nm class sizes were selected to reflect the inaccuracy of particle length determination using the above measurement method. Relative frequency distributions were prepared using 90–150 particle measurements obtained from grids prepared from triplicate hairy root cultures.

2.17.4.4 Viral infectivity using local lesion assays

Infectivity assays were used to determine if viral protein levels detected using ELISA corresponded to the levels of infectious virus in biomass samples and culture media.

The methods used were based on those reported by Noordam (1973) and Dijkstra and de Jager (1998). Half-leaf assays, where the samples to be analysed and a standard

TMV preparation were applied to different halves of the same leaf, were used to determine infectivity because different leaves can exhibit different susceptibilities to local lesion formation (Noordam, 1973).

Similarly-sized leaves were detached from N. glutinosa plants (Section 2.4.2), cut in half along the midrib and dusted with sterile carborundum (500 mesh). Biomass extracts (Section 2.17.3.1), medium samples (10 µL) or purified TMV-GFPC3

(Sections 2.6.4) were applied to the base of half leaves and spread over the leaf surface

153 with a gloved finger. An appropriately diluted preparation of purified TMV (Section

2.5.2) that resulted in the formation of readily countable numbers of lesions was applied as described above to the remaining half leaves. The sample and standard preparations were rinsed from the leaves with sterile water and both halves of the leaves placed on damp filter paper in Petri dishes. The dishes were sealed with parafilm and incubated under continuous fluorescent (Cool White) illumination (650 lux) at 23ºC until lesions appeared and could be counted (4–5 days). Each sample was applied to six half leaves.

Results for viral infectivity were reported as infectivity or relative infectivity.

Infectivity was reported when the ability of virus in a sample to initiate viral infection was of interest. Varying concentrations of virus in the samples were not taken into consideration as the concentration of infectious virus in a preparation and not the infectivity per unit virus was of interest. Results were expressed as total infectivity or concentration of infectious virus. Infectivity was determined using the following formula:

Sample count Infectivity = (2.6) Standard count

where sample count and standard count refer to the number of local lesions on corresponding half leaves inoculated, respectively, with sample (biomass, medium or purified TMV-GFPC3) and standard TMV preparation.

Relative infectivity refers to the intrinsic viral infectivity of a sample relative to that of

TMV from plants. The relative infectivity is intrinsic because it is not dependant on the

154 viral concentration or volume applied in the assay, but reflects only the intrinsic properties of the virus. Relative infectivity does not vary with virus concentration.

Relative infectivity was determined using the following formulae:

amount of virus applied in sample Normalised standard count = standard count × (2.7) amount of virus applied in standard

sample count Relative infectivity = (2.8) normalised standard count

where normalised standard count refers to the lesion count that would have been obtained for the standard if the amount of standard virus (as measured by ELISA

Section 2.17.4.1 or electron microscopy Section 2.17.6) applied to the leaf had been the same as the amount of virus in the sample (as measured by ELISA or electron microscopy); amount of virus applied in the sample/standard refers to the total amount

(particles or mg) of virus applied to the leaf, and sample count and standard count refer to the number of local lesions on individual half leaves inoculated, respectively, with sample and standard TMV preparations.

2.17.5 Detection of GFP

2.17.5.1 Detection of GFP using ELISA

The concentration of GFP in biomass and medium samples was quantified using an indirect sandwich ELISA. The procedure used was based on the method described by

Su et al. (2004) with some modifications.

Ninety-six-well microtitre plates (Nunc-immunoTM plate MaxisorpTMsurface) were coated with 0.2 µg mL-1 mouse anti-GFP monoclonal antibody (Molecular Probes,

155

USA) diluted with PBS containing 0.05% (w/v) Tween 20, 5% (w/v) sucrose and

1% (w/v) BSA. Diluted antibody (100 µL per well) was applied to the microtitre plates and incubated overnight at 4ºC. The coat antibody was removed from the plates by flicking and the wells were washed three times with 150 µL PBS containing 0.05%

(w/v) Tween 20, with the plates being flicked between washes. The wells were blocked using 100 µL blocking solution which contained PBS with 0.05% (w/v) Tween 20,

5% (w/v) sucrose and 1% (w/v) BSA, and incubated for 1 hour at room temperature.

The blocking buffer was removed and the wells washed three times with 150 µL PBS containing 0.05% (w/v) Tween 20. One hundred µL samples were applied to the plates and diluted with PBS containing 0.05% (w/v) Tween 20. As a standard, recombinant

GFP (BD Bioscience, USA) was applied to the plates at a concentration of 62.5 ng mL-1 and serially diluted in PBS containing 0.05% (w/v) Tween 20. Samples were incubated for 2 hours at room temperature. The samples were removed and the wells washed three times with 150 µL PBS containing 0.05% (w/v) Tween 20. One hundred µL of

0.4 µg mL-1 rabbit anti-GFP polyclonal antibody (Molecular Probes) diluted in PBS containing 0.05% (w/v) Tween 20, 5% (w/v) sucrose and 1% (w/v) BSA was added to the wells and incubated for 2 hours at room temperature. The wells were washed three times with 150 µL PBS containing 0.05% (w/v) Tween 20. One hundred µL of goat anti-rabbit immunoglobulin with a horseradish-peroxidase-label (DakoCytomation) was diluted with PBS containing 0.05% (w/v) Tween 20 and 1% (w/v) BSA, applied at a concentration of 1.5 × 10-4 mg mL-1 and incubated for 2 hours at room temperature.

The wells were washed three times with 150 µL PBS containing 0.05% (w/v) Tween 20 and the ELISA was developed with o-phenylenediamine dihydrochloride. The ELISA development reaction was stopped with 3 M sulphuric acid and read at 490 nm. The concentrations of GFP in extracted biomass and medium samples were determined by

156 comparing sample titration curves with the titration curve obtained for the GFP standard.

2.17.5.2 Detection of GFP using Western blotting

SDS-PAGE under reducing conditions and Western blotting was utilised to determine if

GFP was produced in hairy root cultures infected with the viral vector TMV-GFPC3.

Hairy root or whole plant biomass extracted in PBS containing a protease inhibitor cocktail (Section 2.17.3.2) and medium samples were prepared for electrophoresis by mixing with Laemmli’s sample buffer (Laemmli, 1970) containing 5% (v/v)

β-mercaptoethanol using a sample:buffer volume ratio of 1:1. Samples were heated at

100ºC for 3 minutes. Recombinant GFP was used as a positive control and standard and was prepared for electrophoresis in the same manner as the biomass samples. The molecular weight markers used were SeeBlue®Plus2 pre-stained standard and

MagicMarkTM Western Protein Standard.

Twenty µL of the prepared sample–laemmli’s buffer mixture were loaded into the wells of 10% Tris-glycine minigels. Ten ng of GFP was applied as a positive control.

Molecular weight markers were applied to the gels according to the manufacturer’s directions. Gels were run at a constant voltage of 125 volts for 100 minutes at room temperature. After electrophoresis, the proteins were transferred from the gel to nitrocellulose membrane (Section 2.17.4.2).

After protein transfer, the membranes were blocked at 4ºC overnight in a blocking buffer containing 50 mM Tris-base, pH 7.5, 150 mM sodium chloride, 0.1% (w/v)

157

Tween 20 and 2% BSA. The membranes were washed three times for 10 minutes each wash in washing buffer (Section 2.17.4.2). The membranes were incubated for 2 hours with mouse anti-GFP monoclonal antibody (Molecular Probes, USA) at a concentration of 0.2 µg mL-1 in blocking buffer. The membranes were rinsed twice and washed once for 15 minutes and then three times for 5 minutes each wash in a large volume of washing buffer. The membrane was then incubated with rabbit anti-mouse antibody labelled with horseradish peroxidase (DakoCytomation) at a concentration of

6.5 × 10-4 mg mL-1 in blocking buffer for 2 hours at room temperature. Membranes were rinsed twice and washed once for 15 minutes and three times for 5 minutes each wash in a large volume of washing buffer.

Blots were developed using ECL development solution according to the manufacturer’s instructions. X-ray film was exposed to the membrane for 1–2 minutes, developed using GBX Developer and Replenisher and fixed with GBX Fixer and Replenisher. The film was rinsed with Milli-Q water prior to drying.

2.17.5.3 Detection of GFP using fluorescence microscopy

Wet-mounted plant material (root and leaf) was examined using an Olympus BH2-RFL

(reflected light fluorescence) microscope with ultraviolet illumination, using a UV

[DM-400 (L-420) – DM-455 (y-455)] diachroic mirror unit with a built-in barrier filter attachment and excitation filter U(UG-1)V. Photographs were taken using Fujicolour

Superia X-TRA 400 ASA film (Fuji, Japan) and Olympus OM-1 camera (Olympus,

Japan). For some samples, root material was stained with propidium iodide (Sigma-

Aldrich) to reduce background auto-fluorescence.

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2.17.6 Quantification of virus using scanning electron microscopy

The concentration of TMV utilised as inocula in the culture experiments was determined spectrophotometrically (Section 2.5.3). Spectrophotometric determination of virus concentration provides a measure of the mass concentration of virus in units of mg mL-1, not the number concentration of virus. Virus concentrations determined by comparative methods of quantification such as ELISA and Western-blots are dependent on the units of the standard preparation. TMV-GFPC3 has a longer genome than TMV and therefore the assembled TMV-GFPC3 particle is longer than the TMV particle. In order to ensure that similar number concentrations of viral particles were used to inoculate cultures when using TMV and the modified TMV-GFPC3 scanning electron microscopy was used to determine the number concentrations of the purified suspensions of TMV (Section 2.5.2) and TMV-GFPC3 (Section 2.6.4) used for culture inoculation. The method used was based on that of Luria et al. (1951) with modification.

Polystyrene microspheres, 0.11 µm in diameter (Spi Supplies, USA) and at a concentration of 6.0 × 1012 particles mL-1, were diluted 1:1000 in Milli-Q water filtered using a 0.2-µm syringe filter (Sartorius). The suspended microspheres were mixed with an equal volume of purified virus diluted in filtered (Sartorius Midistart, 0.2 µm) 0.02%

(w/v) BSA prepared in Milli-Q water. The viral preparations (TMV and TMV-GFPC3) used were purified using the PEG precipitation method outlined in Section 2.5.2. The diluted microsphere and virus suspensions were sprayed onto formvar-(ProSciTech) coated brass stubs using a glass chromatographic solvent sprayer (Quickfit B24/29) as shown in Figure 2.2. The stubs were air-dried and sputter-coated with chromium using an Emitech K575X Peltier coating unit.

159

Figure 2.2 Glass chromatographic solvent sprayer used to spray suspensions containing virus and microspheres onto brass stubs for virus quantification.

The coated stubs were examined using an S900 Hitachi scanning electron microscope.

Micrographs of whole droplets were taken. Microspheres and viral particles were counted from droplets on a number of stubs. For TMV preparations particles of approximately 300 nm were counted, but significantly truncated virus particles were not counted. For TMV-GFPC3 preparations particles 300 nm or longer were counted, but significantly truncated particles were not included in the count. Approximately 640 viral particles and 470 microspheres were counted to determine the particle number concentration for TMV. Approximately 1700 viral particles and 1850 microspheres were counted to determine the particle number concentration for TMV-GFPC3. A micrograph of a droplet containing microspheres and TMV-GFPC3 particles is shown in Figure 2.3.

The concentration of virus was determined using the following relationship:

160

-1 n V Viral particles (mL ) = ⋅[]B ⋅ D V (2.9) n B

where nV is the total number of viral particles in the droplets, nB is the total number of microspheres in the droplets, [B] is the number concentration of diluted microspheres in

-1 the suspension (mL ) and Dv is the dilution factor of the virus.

Figure 2.3 Micrograph of a droplet containing microspheres (spherical) and TMV-GFPC3 (rod shaped) particles.

2.17.7 Total soluble protein

The total soluble protein concentration in selected hairy root and whole plant extracts

(Section 2.17.3.3) and culture medium samples was determined using Coomassie Plus

(Pierce, USA), Bradford assay reagent. The assay was performed according to the manufacturer’s procedure with minor modifications. The method used is outlined below.

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Ten µL of extracted hairy root or plant sample or BSA standard was added to each well of 96-well microtitre plates (Sarstedt, Australia). Coomassie Plus Reagent (250 µL) equilibrated to room temperature was added to each well and the samples mixed for

30 seconds. Plates were incubated for 10 minutes at room temperature prior to the measurement of sample adsorbance at 595 nm using a microplate reader. BSA

(Albumin Standard Ampoules, Pierce) at an initial concentration of 1.0 mg mL-1 was used as a standard. Sample protein concentration was determined by comparing sample absorbance to a titration curve of BSA standards.

When culture medium was assayed, due to the low protein content of medium, an increased sample volume was used. One hundred and forty µL medium or diluted BSA standard was mixed with 140 µL of Coomassie Plus Reagent. The remainder of the assay was performed as outlined above.

2.18 Statistical Analysis

Data are presented as mean ± error

Standard and maximum errors were used to reflect the scatter of datum points when replicate measurements were taken. When data were obtained from three or more samples standard error was calculated. When data were obtained from only two samples, maximum error was used to reflect the scatter of datum points. Maximum error is the difference between each datum point and the mean of the two duplicate points.

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Differences in sample means were assessed for significance using the Student’s t-test and Analysis of Variance (ANOVA). For the purpose of statistical analysis, the underlying distribution was assumed to be normal.

Comparisons between the means of two groups of data were performed using the

Student’s t-test (Wardlaw, 1985). Significance was determined using a 95% confidence interval. Prior to the use of the Student’s t-test, the variances of the samples in the two groups of data were examined using the F-test to ensure that the variances did not differ significantly.

Comparisons between more than two groups of data were performed using ANOVA

(Wardlaw, 1983). Significance was determined using a 95% confidence interval.

When ANOVA indicated that significant differences existed between groups of data,

Duncan’s multiple-range test (Miller and Miller, 2004) was used to determine where the significant differences were located.

The relative variabilities of data sets were compared by expressing the standard deviation as a percentage of the sample mean (coefficient of variation) (Keller and

Warrack, 1991).

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CHAPTER 3 – RESULTS

3.1 Tobacco Mosaic Virus Accumulation in Nicotiana tabacum Plants

Results for the concentration of accumulated viral coat protein and the concentration of total soluble protein in Nicotiana tabacum plants systemically infected with tobacco mosaic virus (TMV) are shown in Figure 3.1. The accumulated viral coat protein is also expressed as a percentage of total soluble protein. TMV particles are composed of

95% coat protein and 5.0% genomic RNA.

The concentrations of accumulated viral coat protein and total soluble protein in the biomass and the accumulated viral protein as a percentage of total soluble protein were

180

160

140 dry weight) dry

-1 120

100

20 percentage of total soluble protein Accumulated viral coat protein as a total soluble protein (mg g protein soluble total Concentrations of viral coat protein and and protein coat of viral Concentrations 0 Leaf Stem Root

Figure 3.1 Concentration of accumulated viral coat protein (), concentration of total soluble protein (), and accumulated viral coat protein as a percentage of total soluble protein () in N. tabacum plants systemically infected with TMV. The error bars indicate standard errors for triplicate samples of stem and root, and eleven leaf samples.

164 not significantly different (p < 0.05) for N. tabacum leaves and roots. However these results for stems were significantly lower (p < 0.05) than for the leaves and roots.

3.2 TMV Accumulation in N. tabacum Suspension and Hairy Root Cultures

3.2.1 Accumulation of TMV in N. tabacum suspension cultures

3.2.1.1 Growth of N. tabacum suspension cultures

Results for the growth of N. tabacum suspension cultures in shake flasks are shown in

Figure 3.2. Data are plotted using semi-logarithmic coordinates to show exponential growth kinetics. After inoculation into fresh medium, cultures exhibited a slight lag in growth (Day 0 to Day 2). Growth was exponential from Day 2 to Day 12 with a maximum specific growth rate of 0.24 day-1, which corresponds to a doubling time of

2.9 days. Biomass decreased significantly (p < 0.05) between Day 14 and Day 22.

0.8 1

0.7

0.6

0.5

0.4 0.1

0.3

0.2 Biomass (g dry weight)

0.1

0.0 0.01 0 3 6 9 12 15 18 21 24 Time (days)

Figure 3.2 Growth of N. tabacum suspension cultures presented using linear coordinates () and semi-logarithmic coordinates (□). The error bars indicate standard errors from triplicate cultures.

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3.2.1.2 Infection of N. tabacum suspension cultures with TMV

N. tabacum suspension cultures were inoculated with virus by vortexing the cells in the presence of 1.5 µg mL-1 TMV. Viral inoculum was either retained in the suspension culture for the duration of the experimental period or removed after 2 hours by exchanging the medium. Culture growth and virus accumulation were examined

8 hours after culture infection and when the cultures were in the early exponential growth phase (Day 5), late exponential growth phase (Day 10), and the stationary phase of growth (Day 14 and Day 19).

Results for the growth of TMV-infected and control cultures are shown in Figure 3.3.

There were no significant differences (p < 0.05) between the growth of non-infected control cultures and cultures infected with TMV, irrespective of whether the TMV inoculum was retained in the culture medium or removed by exchanging the medium.

Results for the amount and concentration of virus accumulated in the biomass of

N. tabacum suspension cultures infected with TMV are shown in Figure 3.4. Unless otherwise indicated, virus accumulation levels refer to biomass-associated virus only.

When the viral inoculum was retained in the culture medium, the amount of TMV in the cell biomass (Figure 3.4A) increased 35-fold relative to that observed 8 hours after virus addition (Day 0), to reach a maximum of (5.5 ± 1.2) × 10-3 mg TMV after

19 days. When the TMV inoculum was removed 2 hours after inoculation, the amount of TMV associated with the biomass 8 hours after inoculation was 3-fold lower than at a similar time post-infection in cultures in which the TMV inoculum was retained in the medium. Subsequent viral accumulation within the biomass was also lower than when

166

0.8

0.7

0.6

0.5

0.4

0.3

0.2 Biomass (g dry weight) (g dry Biomass

0.1

0.0 Day 0 Day 5 Day 10 Day 14 Day 19 Time

Figure 3.3 Growth of TMV-infected N. tabacum suspension cultures and non- infected control cultures. () TMV-infected cultures with viral inoculum retained in the medium; () TMV-infected cultures with viral inoculum removed 2 hours after inoculation using medium exchange; () non-infected control cultures. The error bars indicate standard errors from triplicate cultures.

the virus was retained. Over the culture period when the viral inoculum was removed from the medium, the amount of TMV in the biomass increased 26-fold relative to that observed 8 hours after virus addition, to reach a maximum of (1.3 ± 0.23) × 10-3 mg

TMV on Day 14.

Results for the concentration of virus accumulated in the biomass of N. tabacum suspended cells are shown in Figure 3.4B. In both TMV-inoculated cultures, the maximum concentration of virus in the biomass was observed 5 days after culture infection. The concentration of virus accumulated in the biomass when the inoculum virus was removed was substantially lower than when the inoculum was retained.

167

0.008

0.007

0.006

0.005

0.004

0.003

Amount of virus (mg) Amount of virus 0.002

0.001

0.000 Day 0 Day 5 Day 10 Day 14 Day 19 Time

0.030

0.025

0.020

0.015 dry weight) dry -1 0.010 (mg g Concentration of virus of virus Concentration 0.005

0.000 Day 0 Day 5 Day 10 Day 14 Day 19 Time

Figure 3.4 Amount of virus in the biomass (A) and concentration of virus in the biomass (B) of N. tabacum suspension cultures infected with TMV. () TMV-infected suspension cultures with the viral inoculum retained in the medium; () TMV-infected cultures with the viral inoculum removed 2 hours after inoculation using medium exchange. The error bars indicate standard errors from triplicate cultures.

168

In Figure 3.5, the total amount of virus and the amounts of virus detected in the biomass and medium of N. tabacum suspension cultures are shown for cultures in which the TMV inoculum was retained in the medium. After the addition of TMV, a rapid decrease in the amount of virus in the medium was observed, with only 32% of the inoculum virus detectable in the medium 8 hours after addition. After the initial decrease in medium virus, total virus levels remained relatively constant for the next

9 days, after which time the amount of virus present in the cultures decreased. The total amount of virus after 19 days was equivalent to only 13% of the initial viral inoculum.

These data indicate that, rather than net propagation or replication of the virus, there was a substantial loss of virus from the cultures during the culture period. Most of this loss occurred immediately after inoculation.

0.08

0.07

0.06

0.05

0.04

0.03

Amount of virus (mg) Amount of virus 0.02

0.01

0.00 0 5 10 15 20 Time (days)

Figure 3.5 Amount of virus in N. tabacum suspension cultures infected with TMV and with inoculum virus retained in the medium. (¡) Total virus (biomass and medium); (z) virus in the biomass; () virus in the medium; (▬▬) inoculum virus. The error bars indicate standard errors from triplicate cultures.

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3.2.2 Accumulation of TMV in N. tabacum hairy roots

3.2.2.1 Growth of N. tabacum hairy roots

Results for the growth of N. tabacum hairy roots in shake flasks are shown in

Figure 3.6. Data are plotted using semi-logarithmic coordinates to show exponential growth kinetics. Hairy root growth was exponential from Day 0 to Day 18, with a maximum specific growth rate of 0.16 day-1, which corresponds to a doubling time of

4.3 days. Hairy root growth continued with a decreased growth rate from Day 18.

Within the period examined (36 days), growth did not enter stationary phase.

0.50 1 0.45 0.40 0.35 0.30 0.25 0.1 0.20 0.15

Biomass (g dry weight) (g dry Biomass 0.10 0.05 0.00 0.01 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.6 Growth of N. tabacum hairy roots presented using linear coordinates () and semi-logarithmic coordinates (□). The error bars indicate standard errors from triplicate cultures.

3.2.2.2 Infection of N. tabacum hairy root cultures with TMV

N. tabacum hairy roots were inoculated with virus by vortexing the roots in the presence of 1.5 µg mL-1 TMV. The viral inoculum was either retained in the hairy root

170 cultures for the duration of the experimental period or removed after 4 hours by exchanging the medium.

Results for the growth of TMV-infected N. tabacum hairy roots and non-infected control cultures at selected times during the growth period are shown in Figure 3.7.

The observed differences in the biomass of cultures 7 hours after inoculation (Day 0) and at 5, 14, 22 and 32 day post-inoculation were generally not significant (p < 0.05).

0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15

Biomass (g dry weight) (g dry Biomass 0.10 0.05 0.00 Day 0 Day 5 Day 14 Day 22 Day 32 Time

Figure 3.7 Growth of TMV-infected N. tabacum hairy root cultures and non- infected control cultures. () TMV-infected cultures with viral inoculum retained in the medium; () TMV-infected cultures with viral inoculum removed after 4 hours using medium exchange; () non-infected control cultures. The error bars indicate standard errors from triplicate cultures (Days 0, 5, 14 and 22) and quadruplicate cultures (Day 32).

171

Results for the amount and concentration of virus accumulated in the biomass of

N. tabacum hairy root cultures are shown in Figure 3.8. Viral accumulation is shown for cultures in which the TMV inoculum was either retained in the medium or removed after 4 hours using medium exchange. Viral accumulation in replicate cultures was highly variable, as indicated by the large standard errors.

The amount of accumulated TMV in N. tabacum hairy roots is shown in Figure 3.8A.

In cultures where the viral inoculum was retained in the medium, the amount of TMV accumulated in the cells increased 213-fold relative to the that observed 7 hours after virus addition (Day 0) [(8.0 ± 0.53) × 10-5 mg TMV], accumulating to a maximum of

(1.7 ± 1.0) × 10-2 mg TMV 14 days post-infection. When the TMV inoculum was removed from cultures 4 hours after inoculation, the amount of TMV associated with the biomass 7 hours after inoculation (Day 0) [(3.8 ± 1.1) × 10-5 mg TMV] was significantly lower (p < 0.05) than in cultures where the TMV inoculum was retained in the medium. During the culture period the amount of TMV accumulated in the biomass increased 713-fold relative to the amount of virus associated with the roots 7 hours after culture infection with TMV, accumulating to (2.7 ± 1.7) × 10-2 mg TMV 22 days post- infection. The amount of virus accumulated in N. tabacum hairy root cultures at each time point was not significantly affected (p < 0.05) by inoculum removal.

Results for the concentration of virus accumulated in the biomass of N. tabacum hairy root cultures are shown in Figure 3.8B. The patterns of virus accumulation were similar when viral inoculum was retained in the medium and when inoculum was removed. During the culture period the concentration of virus accumulated in the biomass increased above that observed 7 hours after infection with TMV irrespective of

172

0.050 0.045 0.040 0.035 0.030 0.025 0.020 0.015 Amount of virus (mg) Amount of virus 0.010 0.005 0.000 Day 0 Day 5 Day 14 Day 22 Day 32 Time

0.22 0.20 0.18 0.16 0.14 0.12

dry weight) dry 0.10 -1 0.08

(mg g 0.06 Concentration of virus of virus Concentration 0.04 0.02 0.00 Day 0 Day 5 Day 14 Day 22 Day 32 Time

Figure 3.8 Amount of virus in the biomass (A) and the concentration of accumulated virus in the biomass (B) of N. tabacum hairy root cultures infected with TMV. () TMV-infected hairy root cultures with the viral inoculum retained in the medium; () TMV-infected hairy root cultures with the viral inoculum removed after 4 hours using medium exchange. The error bars indicate standard errors from triplicate cultures (Days 0, 5, 14 and 22) and quadruplicate cultures (Day 32).

173 whether the viral inoculum was retained in the medium or removed by medium exchange. However the high variability of viral accumulation between replicate cultures largely prevented identification of significant accumulation patterns and trends.

In Figure 3.9, the total amount of virus and the amounts of virus detected in the biomass and medium of N. tabacum hairy root cultures are shown for cultures in which the TMV inoculum was retained in the medium. After the addition of TMV, a rapid decrease in the amount of virus in the medium was observed, with only 21% of the inoculum virus detectable 7 hours after addition. The total amount of virus (biomass and medium) was relatively constant for the remainder of the examined period. The total amount of virus after 32 days was equivalent to only 35% of the initial viral

0.08

0.07

0.06

0.05

0.04

0.03

Amount of virus (mg) Amount of virus 0.02

0.01

0 0 5 10 15 20 25 30 35 Time (days)

Figure 3.9 Amount of virus in N. tabacum hairy root cultures infected with TMV and with inoculum virus retained in the medium. (¡) Total virus (biomass and medium); (z) virus in the biomass; () virus in the medium; (▬▬) inoculum virus. The error bars indicate standard errors from triplicate cultures (Days 0, 5, 14 and 22) and quadruplicate cultures (Day 32).

174 inoculum. These data indicate that, rather than net propagation or replication of the virus, there was a substantial loss of virus from the cultures during the culture period.

Most of this loss occurred immediately after inoculation.

3.3 TMV Accumulation in Nicotiana benthamiana Suspension Cultures

Nicotiana benthamiana suspension cultures were examined for their ability to become infected with, and subsequently accumulate, TMV.

3.3.1 Growth of TMV-infected N. benthamiana suspension cultures

Results for the growth of N. benthamiana suspension cultures infected with TMV and non-infected control cultures are shown in Figure 3.10. Data are plotted in Figure

3.10B using semi-logarithmic coordinates to show exponential growth kinetics. Both

TMV-infected and non-infected control cultures exhibited an exponential growth phase

(Day 2 to Day 14) followed by a short period of decelerated growth. The cultures had similar maximum specific growth rates, 0.17 day-1 and 0.16 day-1, for TMV-infected and non-infected suspensions, respectively. The maximum specific growth rates corresponded to biomass doubling times of 4.1 days and 4.3 days, for TMV-infected and non-infected suspensions, respectively. Virus-infected and control cultures also exhibited a stationary phase when the biomass remained relatively constant, prior to a gradual decrease in biomass dry weight. The observed differences in biomass between infected and non-infected cultures were generally not significant (p < 0.05).

175

A. 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15

Biomass (g dry weight) 0.10 0.05 0.00 0 5 10 15 20 25 30 35 40 Time (days)

1.00

0.10 Biomass (g dry weight)

0.01 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.10 Growth of TMV-infected () and non-infected control (□) N. benthamiana suspension cultures presented using linear coordinates (A) and semi-logarithmic coordinates (B). The error bars indicate standard errors from quadruplicate TMV-infected suspension cultures and triplicate non-infected control suspension cultures.

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3.3.2 Virus accumulation in TMV-infected N. benthamiana suspension cultures

Results for the amount and concentration of virus accumulated in the biomass of

N. benthamiana suspension cultures infected with TMV are shown in Figure 3.11.

Data are plotted using semi-logarithmic coordinates in Figure 3.11B to show the range of values measured and exponential viral growth kinetics. During the first 4 days of the culture period, the amount of virus accumulated in the biomass did not increase significantly (p < 0.05). Virus accumulated exponentially from Day 4 until Day 18 with a maximum specific accumulation rate of 0.28 day-1, corresponding to a constant doubling time of 2.5 days. The specific accumulation rate was modelled using exponential kinetics and reflects the maximum slope of linear regions, when the data are plotted using semi-logarithmic coordinates (Figure 3.11B). Increases in the amount of virus accumulated in the biomass from Day 18 were not significant (p < 0.05). Over the culture period, the amount of accumulated virus increased 120-fold relative to that observed 3.5 hours after virus addition (Day 0) [(8.2 ± 3.3) × 10-5 mg TMV], to reach a maximum of (9.9 ± 3.8) × 10-3 mg TMV on Day 24.

The concentration of accumulated virus in the biomass did not change significantly

(p < 0.05) from Day 0 to Day 4 (Figure 3.11). The concentration of virus accumulated in the biomass increased exponentially in the biomass from Day 4 to Day 18 (Figure

3.11B). An initial peak in the concentration of virus accumulated in the biomass occurred between Day 20 and Day 24. Changes in the concentration of accumulated virus in the biomass after Day 18 were not significant (p < 0.05).

177

0.035

0.030

0.025

0.020 dry weight) weight) dry

-1 0.015

(mg g 0.010 Concentration of virus of virus Concentration Amount of virus (mg) and (mg) and Amount of virus 0.005

0.000 0 5 10 15 20 25 30 35 40 Time (days)

0.1

0.01

0.001 dry weight) weight) dry -1

(mg g 0.0001 Concentration of virus Amount of virus (mg) and (mg) and Amount of virus

0.00001 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.11 Amount of accumulated virus in the biomass () and the concentration of accumulated virus in the biomass (¡) of N. benthamiana suspension cultures infected with TMV. The data are presented using linear coordinates (A) and semi- logarithmic coordinates (B). The error bars indicate standard errors from quadruplicate cultures.

178

The relationship between the amount of virus contained in individual (replicate) TMV- infected N. benthamiana suspension cultures and the biomass of the cultures is shown in Figure 3.12. Virus accumulation was not directly proportional to suspension culture growth (Figure 3.12A). When plotted using semi-logarithmic coordinates (Figure

3.12B), these data show a first-order relationship during the first 24 days of the culture period, during which the amount of virus in the suspension biomass doubled for every

0.069 g dry weight of biomass formed. Data from Days 26–36 although included in

Figure 3.12B were not utilised to determine the relationship between the accumulation of virus in the biomass and culture growth because age-related cell lysis was occurring during this period.

179

0.016

0.014

0.012

0.010

0.008

0.006

0.004

0.002

Virus accumulated in the biomass (mg) 0.000 0.0 0.1 0.2 0.3 0.4 0.5 Biomass (g dry weight)

0.1

0.01

0.001

0.0001

Virus accumulated in the biomass (mg) 0.00001 0.0 0.1 0.2 0.3 0.4 0.5 Biomass (g dry weight)

Figure 3.12 Relationship between the amount of virus in the biomass and the amount of biomass in TMV-infected N. benthamiana suspension cultures. Data for individual replicate cultures Days 0–24 () and Days 26–36 (□), and averaged values from replicate cultures at a given time point for Days 0–24 (¡) and Days 26–36 (). The trend line shown is for individual samples from Days 0–24. The data are plotted using linear coordinates (A) and semi-logarithmic coordinates (B). The error bars indicate standard errors from quadruplicate cultures.

180

3.4 Initiation and Selection of N. benthamiana Hairy Root Cultures

3.4.1 Initiation of N. benthamiana hairy roots

Twenty hairy root clones were generated from N. benthamiana stem explants, leaf explants and rooted stem sections (Section 2.3.3.1). Nine of the clones were initiated using Agrobacterium rhizogenes strain A4 and eleven were initiated using

A. rhizogenes strain 15834. Only six of the clones exhibited rapid growth characteristics and remained free of A. rhizogenes contamination during successive subcultures and were therefore suitable for further investigation. Five of these clones were initiated using A. rhizogenes strain A4, whereas the remaining clone was initiated using A. rhizogenes strain 15834. Figure 3.13 shows hairy roots emerging from a wound site on a rooted stem section of N. benthamiana after infection with

A. rhizogenes strain 15834.

Figure 3.13 Hairy roots emerging from the site of infection with A. rhizogenes strain 15834 on a rooted N. benthamiana stem section.

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3.4.2 Infection of N. benthamiana hairy root clones with TMV

Growth and TMV accumulation in six N. benthamiana hairy root clones initiated using

A. rhizogenes strains 15834 and A4 (Section 3.4.1) were examined and compared with the results for N. tabacum hairy root cultures (Section 2.9.1). For ease of reference, the

N. benthamiana clones are labelled with a reference number (1 to 6), and with the

A. rhizogenes strain used to initiate the cultures, e.g. N. benthamiana (1-15834).

3.4.2.1 Growth of TMV-infected N. benthamiana hairy roots

In Figure 3.14, results for the growth of TMV-infected N. benthamiana hairy root cultures, a TMV-infected N. tabacum hairy root culture and a non-infected N. tabacum hairy root culture are shown. After 14 days, the seven different TMV-infected hairy root cultures could be divided into four significantly different groups (p < 0.05) according to biomass accumulation. After 14 days, the growth of TMV-infected and non-infected control N. tabacum hairy roots was significantly lower (p < 0.05) than the growth of the TMV-infected N. benthamiana hairy roots.

N. benthamiana hairy roots could be divided into three groups, the first containing

N. benthamiana clones (1-15834), (3-A4) and (4-A4), the second group containing

N. benthamiana (6-A4) and the third group containing N. benthamiana clones (2-A4) and (5-A4). The growth of hairy roots within each group did not differ significantly

(p < 0.05), but growth between the groups was significantly different (p < 0.05).

N. benthamiana hairy roots 3-A4 and 4-A4 exhibited root fragmentation when grown in liquid medium in shake flasks.

182

0.6

0.5

0.4

0.3

0.2

Biomass (g dry weight) 0.1

0.0 l ro m 4) ) ) ) ) ) nt cu 83 -A4 -A4 -A4 -A4 -A4 co ba 15 (2 (3 (4 (5 (6 m ta 1- na na na na na cu N. a ( ia ia ia ia ia ba ian am am am am am ta m th th th th th N. ha en en en en en nt . b . b . b . b . b be N N N N N N.

Figure 3.14 Growth of TMV-infected N. tabacum and N. benthamiana hairy root cultures and a non-infected control N. tabacum hairy root culture. () Day 0; () Day 5; () Day 14. The error bars indicate standard errors from triplicate (Days 0 and 5) and quadruplicate cultures (Day 14).

3.4.2.2 TMV accumulation in N. benthamiana hairy roots

Results for the concentration of accumulated virus in N. benthamiana and N. tabacum hairy root cultures are shown in Figure 3.15. Shortly after TMV infection (4 hours) and 5 days after infection, the differences in virus concentration in the different hairy root cultures were not significant (p < 0.05). After 14 days, in four of the

N. benthamiana hairy root clones (1-15834, 2-A4, 3-A4 and 5-A4) the TMV concentrations did not differ significantly (p < 0.05) from those in the N. tabacum hairy roots. After 14 days, virus concentration in N. benthamiana hairy root clone 6-A4 was significantly higher (p < 0.05) than in most of the other N. benthamiana and N. tabacum hairy root cultures. The concentration of virus accumulated in the N. benthamiana hairy root clone 4-A4 was significantly higher (p < 0.05) than in the N. tabacum hairy

183 root culture and was not significantly different (p < 0.05) from the concentration of accumulated virus in any of the other N. benthamiana hairy root clones, but the culture exhibited root fragmentation.

N. benthamiana hairy root clone 6-A4 was selected for further examinations of viral accumulation in tissue culture due to its relatively high growth rate, suitable growth characteristics in shake flasks and high levels of TMV accumulation.

4.5 4.0 3.5 3.0 2.5

dry weight) dry 2.0 -1 1.5

(mg g 1.0 Concentration of virus 0.5 0.0 m 4) ) ) ) ) ) cu 83 -A4 -A4 -A4 -A4 -A4 ba 15 (2 (3 (4 (5 (6 ta 1- na na na na na N. a ( ia ia ia ia ia an am am am am am mi th th th th th ha en en en en en nt . b . b . b . b . b be N N N N N N.

Figure 3.15 Concentration of accumulated virus in the biomass of TMV-infected N. tabacum and N. benthamiana hairy root cultures. () Day 0; () Day 5; () Day 14. The error bars indicate standard errors for triplicate (Days 0 and 5) and quadruplicate cultures (Day 14).

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3.5 Summary of Preliminary Studies with N. tabacum and N. benthamiana

Suspension and Hairy Root Cultures

The maximum viral concentrations observed in N. tabacum and N. benthamiana suspension and hairy roots cultures are presented in Table 3.1. The concentrations of virus accumulated in hairy root cultures of N. tabacum and N. benthamiana were higher than the concentrations of virus accumulated in the corresponding suspension cultures.

The concentration of virus accumulated in TMV-infected N. benthamiana hairy root cultures was considerably higher than in the other cultures examined. Subsequent investigations of viral accumulation in cultured cells were performed using

N. benthamiana hairy roots as preliminary investigations indicated that viral accumulation in this culture was superior.

Table 3.1 Maximum virus concentrations observed in N. tabacum and N. benthamiana suspension and hairy root cultures.

Culture type Maximum virus concentration (mg g-1 dry weight)

N. tabacum suspensions (1.7 ± 0.79) × 10-2 (Day 5)

N. tabacum hairy roots (1.4 ± 0.57) × 10-1 (Day 14)

N. benthamiana suspensions (2.6 ± 0.21) × 10-2 (Day 33)

N. benthamiana hairy roots 3.4 ± 0.69 (Day 14)

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3.6 Incubation of N. benthamiana Hairy Roots with Non-Infectious TMV

To assess the contribution of passive viral adsorption onto the surfaces of plant roots to the measured accumulation of virus in hairy root biomass, experiments were performed using deactivated TMV. TMV was deactivated by exposure to UV radiation

(Section 2.13.1). At low doses of UV radiation, damage to viral RNA alone can result in loss of infectivity. However as the dose of UV radiation is increased, protein damage can occur that prevents the virus from uncoating to release RNA. Exposure to high UV radiation can also result in the failure of viral particles to combine with antibodies due to coat protein modification and virus particle disintegration (Gibbs and Harrison,

1976). It was therefore of concern that the UV treatment applied was sufficient to prevent TMV propagation and replication without compromising the ELISA methods used for viral quantification.

3.6.1 Deactivation of TMV

The properties of TMV as a function of time of exposure to UV radiation are shown in

Figure 3.16. TMV was characterised in terms of its infectivity as measured using local lesion assays (Section 2.17.4.4), spectrophotometric properties (A260/280: Section 2.5.3), and the ratio of the virus concentration measured using ELISA (Section 2.17.4.1) to the virus concentration measured spectrophotometrically (Section 2.5.3).

The relative infectivity of the virus preparation decreased rapidly after exposure to UV radiation. After only 1 hour, the relative infectivity had decreased from 0.87 (relative infectivity of the original preparation) to zero. No infection events were subsequently identified by local lesion formation when UV-exposed virus was rubbed onto the surface of indicator leaves.

186

1.6 1.6

1.4 1.4

1.2 1.2

1.0 1.0

0.8 0.8 260/280 A 0.6 0.6

Relative Infectivity 0.4 0.4 measured by ELISA:virus Ratio of virus concentration

0.2 0.2 spectrophotometry absorption concentration determined by UV UV by determined concentration

0.0 0.0 024681012 Time (hours)

Figure 3.16 Properties of TMV during exposure to UV radiation. (¡) Relative infectivity; (▲) A260/280; () ratio of the virus concentration measured using ELISA to the virus concentration measured spectrophotometrically.

The ratio of the absorbance of virus preparations at 260 and 280 nm (A260/280) generally reflects the purity of a virus preparation (Dijkstra and de Jager, 1998). The RNA/DNA component of a virus has a maximum absorbance at 260 nm and the protein component has a maximum absorbance at 280 nm. Consequently, the RNA:protein ratio and therefore the A260/280 value in purified virus preparations should be constant. When the virus preparations were exposed to UV radiation, a small gradual increase in the A260/280 value was observed with increasing exposure time, indicating that some alteration in the composition of the virus preparation occurred in response to UV exposure. Over the 12 hour irradiation period, the A260/280 value increased by approximately 19%.

If the ability of viral coat protein to bind with specific antibodies is unaltered and the

RNA content of the preparation remains the same after UV irradiation, the

187 concentration of a TMV preparation measured using ELISA should be similar to that measured using UV absorbance spectrophotometry. Discrepancies between the concentrations determined using these two methods could indicate that the virus coat protein and/or RNA content were altered.

During the period over which the virus preparations were irradiated, the volumes were reduced by evaporation. Therefore, the ratio of the virus concentration measured using

ELISA and the virus concentration measured using UV adsorption spectrophotometery was used to determine if the virus coat protein and/or RNA content of the preparation were altered. As can be seen in Figure 3.16, this ratio remained-relatively constant for the first 6–8 hours of UV irradiation; however exposure for longer times resulted in a rapid decrease in the ratio, indicating that damage to the virus may have occurred.

Based on these results, TMV was deactivated using exposure to UV radiation for

6–8 hours. Six to 8 hours exposure provided a high degree of confidence that the virus was no longer infectious, while alterations to the viral coat protein that could affect virus association with plant cells would be minimised.

3.6.2 Incubation of N. benthamiana hairy root cultures with non-infectious

TMV

TMV particles deactivated by exposure to UV radiation for 6–8 hours were added to

N. benthamiana hairy roots in Gamborg’s B5 medium (Section 2.13.2). The concentrations of deactivated virus in the biomass and medium were examined to determine if (or during which period of the time course) inoculum virus contributed significantly to the results observed when hairy roots are inoculated with infectious

188

TMV. The use of non-infectious virus allowed the association of virus with the hairy roots and virus longevity in the medium to be examined without new virus being accumulated in the biomass or released into the medium.

The nature of the interactions between TMV and hairy roots are not known but may involve adsorption or active uptake, although, to date, no active uptake mechanisms have been identified for plant viruses in whole plants, suspension cells or protoplasts

(Shaw, 1999). Deactivated virus that is detected with the biomass fraction of a culture is referred to as being associated with the biomass and no conclusion about the nature of the biomass–virus interaction is inferred.

3.6.2.1 Over 12 hours

Results for the amounts of TMV in the biomass and medium over the first 12 hours after the addition of deactivated virus to N. benthamiana hairy root cultures are shown in Figure 3.17.

The amount of virus in the culture medium decreased rapidly after inoculum virus addition to the cultures: within 30 seconds of virus addition, only 33% of the added virus was detected in the medium. The amount of virus in the medium varied little over the subsequent 12 hours.

The loss of virus from the medium was not accounted for by a corresponding increase in the amount of TMV associated with the hairy roots. When the amount of virus associated with the biomass was at its maximum level (12 hours post-inoculation) the total virus associated with the hairy roots accounted for only 0.052% of the total

189 inoculum virus added to the cultures. Increases in the amount of virus associated with the biomass after 2 hours were not significant (p < 0.05).

0.00007 0.08

0.00006 0.07

0.06 0.00005 0.05 0.00004 0.04 0.00003 (mg) 0.03

the biomass (mg) 0.00002 0.02

0.00001 0.01 medium in the Amount of virus Amount of virus associated with

0.00000 0.00 024681012 Time (hours)

Figure 3.17 Amount of virus during the incubation of N. benthamiana hairy roots with deactivated TMV for 12 hours. (¡) Amount of virus associated with the biomass; () amount of virus in the medium. The error bars indicate standard errors from quadruplicate cultures.

In Figure 3.18, results for the concentration of TMV associated with the hairy root biomass are shown per gram biomass dry weight. The concentration of virus associated with the hairy roots increased rapidly within the first 2 hours after virus addition but did not increase significantly (p < 0.05) thereafter.

190

0.016

0.014

0.012

0.010 dry weight) dry -1 0.008

0.006

0.004

0.002 the biomass (mg g

Concentration of virus associated with 0.000 02468101214 Time (hours)

Figure 3.18 Concentration of virus associated with the biomass during the incubation of N. benthamiana hairy roots with deactivated virus for 12 hours. The error bars indicate standard errors from quadruplicate cultures.

3.6.2.2 Over 36 days

Figure 3.19 shows results for hairy root growth, the amount of virus associated with the biomass and in the medium, and the concentration of virus associated with the biomass in N. benthamiana hairy root cultures over a 36-day period after the addition of deactivated virus.

The amount of virus in the medium decreased rapidly immediately after deactivated virus was added to the medium (Figure 3.19B). After Day 12, the amount of virus in the medium did not change significantly (p < 0.05) and was close to zero.

191

0.7 0.6 0.5 0.4 0.3 0.2 0.1

Biomass (g dry weight) (g dry Biomass 0.0 0 5 10 15 20 25 30 35 40 Time (days)

0.0030 0.08 0.0025 0.07 0.06 0.0020 0.05 0.0015 0.04 0.0010 0.03 0.02 medium (mg) medium 0.0005 0.01 Amount of biomass- associated virus (mg) 0.0000 0.00 in the virus of Amount 0 5 10 15 20 25 30 35 40 Time (days)

0.030 0.025 0.020 0.015 dry weight) dry -1 0.010 0.005 (mg g

Concentration of virus 0.000 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.19 Growth (A), amounts of virus associated with the biomass and in the medium (B) and concentration of virus associated with the biomass (C) for N. benthamiana hairy root cultures inoculated with deactivated TMV. (▲) Hairy root biomass; (¡) amount of virus associated with the biomass; () amount of virus in the medium; (z) concentration of virus associated with the biomass. The error bars indicate standard errors from triplicate cultures (Days 6, 12, 18, 21 and 24) and quadruplicate cultures (Days 0, 3, 8, 15, 27, 30, 33 and 36).

192

The amount of biomass-associated virus (Figure 3.19B) increased for 6 days after virus addition. The amount of virus associated with the biomass after 6 days was significantly higher (p < 0.05) than the amount of virus associated with the biomass after 12 hours. For the remainder of the experimental period, the amount of virus associated with the biomass did not differ significantly (p < 0.05) from the value observed on Day 6. The relatively constant amounts of biomass-associated virus observed from Day 6 to Day 36 coincided with a period of significant (p < 0.05) biomass accumulation (Figure 3.19A).

Results for the concentration of virus associated with the biomass are shown in

Figure 3.19C. As observed for the amount of virus associated with the biomass in

Figure 3.19B, the concentration of biomass-associated virus increased for 6 days after deactivated virus was added to the cultures. The concentration of virus associated with the biomass 6 days after virus addition was not significantly higher (p < 0.05) than the concentration 2 hours after inoculation (Figure 3.18). This indicates that the initial

(Day 0.5 to Day 6) increase in the amount of virus associated with the biomass observed in Figure 3.19B reflected an increase in the amount of hairy root biomass that was associated with an approximately constant concentration of virus per unit mass.

After Day 6, the concentration of biomass-associated virus decreased rapidly suggesting that biomass-associated virus was diluted by growth. The concentration of biomass- associated virus was low for the remainder of the culture period (Day 9 to Day 36).

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3.7 Effect of Root Age on Viral Accumulation

Hairy root inocula used in investigations examining the effect of inoculum root age on viral accumulation were obtained from cultures 6, 10, 14, and 21 days post-hairy-root-subculture (pre-cultures). Pre-cultures were initiated using inoculum roots from 14-day-old cultures. Six, 10, 14 and 21 days post-initiation, the hairy root cultures were in mid-exponential, late exponential, early decelerating and late decelerating phases of growth, respectively (Appendix 3).

3.7.1 Hairy roots in fresh medium

3.7.1.1 Effect of root age on hairy root culture growth

Results for the growth of TMV-infected N. benthamiana hairy root cultures and non-infected control cultures initiated using fresh medium and root inocula from cultures 6, 10, 14 and 21 days after subculture are shown in Figure 3.20. After 7 days, the biomass in virus-infected cultures initiated using root inocula from 6-day and

10-day cultures was significantly higher (p < 0.05) than the biomass in cultures initiated using 14-day and 21-day roots (Figure 3.20A). The biomass in virus-infected cultures initiated using root inocula from 14-day cultures was significantly (p < 0.05) higher than the biomass in cultures initiated using 21-day roots. This pattern was also observed on Day 11. Fifteen days post-culture-initiation, only cultures initiated using

21-day hairy roots exhibited a biomass significantly (p < 0.05) lower than the biomass observed in the other hairy root cultures. From Day 19, cultures initiated using variously-aged hairy root inocula did not differ significantly (p < 0.05) with regard to biomass accumulation.

194

0.7

0.6

0.5

0.4

0.3

0.2 Biomass (g dry weight) (g dry Biomass 0.1

0.0 0 5 10 15 20 25 Time (days)

0.7

0.6

0.5

0.4

0.3

0.2 Biomass (g dry weight) 0.1

0.0 0 5 10 15 20 25 Time (days)

Figure 3.20 Growth of TMV-infected N. benthamiana hairy root cultures (A) and non-infected control hairy root cultures (B) initiated using variously-aged root inocula. (¡) 6-day-old inocula; () 10-day-old inocula; (▲) 14-day-old inocula; (z) 21-day-old inocula. Error bars indicate standard errors from quadruplicate cultures when cultures were infected with TMV and triplicate cultures for the non-infected controls.

195

For non-infected control hairy root cultures after Day 7, the biomass of cultures initiated using root inocula from 6-day and 10-day roots was significantly higher

(p < 0.05) than the biomass in cultures initiated using 14-day and 21-day roots (Figure

3.20B). The biomass of non-infected control cultures initiated using root inocula from

14-day cultures was significantly (p < 0.05) higher than the biomass in cultures initiated using 21-day roots. From Day 11 the biomass of non-infected control cultures initiated using root inocula from 10-day, 14-day and 21-day cultures generally did not differ significantly (p < 0.05). The final biomass of the non-infected culture initiated using inoculum from 21-day cultures was significantly lower than the biomass of the other non-infected control cultures.

Overall, the growth of N. benthamiana hairy roots cultures initiated using variously- aged inocula was not significantly affected (p < 0.05) by TMV infection (Figure 3.20).

3.7.1.2 Viral accumulation in hairy root cultures initiated using variously-aged root inocula

Results for the amount and concentration of virus accumulated in the biomass when

N. benthamiana hairy root cultures were initiated using variously-aged root inocula are shown in Figure 3.21. Considerable variability was observed in replicate cultures from the same treatment group. As a consequence, the differences in the amounts and concentrations of accumulated virus observed within a treatment group and between different treatment groups were generally not statistically significant (p < 0.05).

196

0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 Amount of virus (mg) Amount of virus 0.10 0.05 0.00 0 5 10 15 20 25 Time (days)

1.0 0.9 0.8 0.7 0.6 0.5 dry weight) dry -1 0.4 0.3 (mg g

Concentration of virus 0.2 0.1 0.0 0 5 10 15 20 25 Time (days)

Figure 3.21 Amount of virus accumulated in the biomass (A) and concentration of virus accumulated in the biomass (B) when variously-aged N. benthamiana hairy roots were used as inocula for cultures subsequently infected with TMV. (¡) 6-day-old inocula; () 10-day-old inocula; (▲) 14-day-old inocula; (z) 21-day-old inocula. The error bars indicate standard errors from quadruplicate cultures.

197

When cultures were initiated using 10-day-old and 14-day-old hairy root inocula, the maximum amounts and concentrations of accumulated virus were low and did not differ significantly (p < 0.05) from the amounts and concentrations of biomass-associated virus observed 5 hours after culture inoculation (Day 0). In cultures initiated using

6-day-old and 21-day-old hairy root inocula, increases in both the amount and concentration of accumulated virus in the biomass appeared larger than when cultures were initiated using 10-day-old and 14-day-old hairy root inocula, but these were still generally not statistically significant (p < 0.05) relative to the results at Day 0.

Subsequent investigations were performed using inocula from 21-day-old hairy root cultures. Although virus accumulation levels were similar when 6- and 21-day-old hairy roots were used as inocula, 21-day-old roots were selected because the longer culture period required for inoculum preparation provided larger amounts of biomass.

3.7.2 Effect of infection of variously-aged hairy roots with TMV in conditioned medium on subsequent viral accumulation

In the previous experiment (Section 3.7.1), TMV-infected N. benthamiana hairy root cultures were initiated by adding roots to fresh medium just prior to the addition of

TMV. However, viral infection of established hairy root cultures in conditioned medium may be more conducive to culture infection with TMV and subsequent virus accumulation. The infection of variously-aged hairy roots by co-incubation of virus with roots in conditioned medium was examined to determine if virus accumulation was affected by the use of conditioned medium.

198

3.7.2.1 Stability of TMV in conditioned media from variously-aged hairy root cultures

The stability and infectivity of TMV were examined in fresh Gamborg’s B5 medium and in conditioned Gamborg’s B5 media removed from hairy root cultures 6, 10, 14, and 21 days after inoculation of 0.2 g fresh weight of N. benthamiana hairy roots into fresh medium.

Protein concentration in conditioned medium

Results for the concentration of total soluble protein in fresh and conditioned

Gamborg’s B5 media are shown in Figure 3.22. No protein was detected in fresh medium. Protein was detected in the conditioned media, with the concentration increasing with increasing culture age. The total soluble protein concentrations in each examined medium were significantly (p < 0.05) different from those in all the other media.

0.016

) 0.014 -1

0.012

0.010

0.008

0.006

0.004

Protein concentraion (mg mL (mg concentraion Protein 0.002

0.000 Day 0 Day 6 Day 10 Day 14 Day 21 Time

Figure 3.22 Concentration of total soluble protein in fresh medium (Day 0) and conditioned Gamborg’s B5 media from hairy root cultures 6, 10, 14 and 21 days after culture initiation. Error bars indicate standard errors from triplicate cultures.

199

Stability of virus in conditioned plant media

Results for the stability of TMV in fresh and conditioned Gamborg’s B5 media over a

12-hour period are shown in Figure 3.23. The concentration of TMV in the different media decreased rapidly after virus addition. Within 5 minutes of virus addition, the virus concentration was reduced to between 9.3 and 20% of that added initially.

Differences in the concentration of virus in the variously-aged media were not significant (p < 0.05).

0.0016 )

-1 0.0014

0.0012

0.0010

0.0008

0.0006

0.0004

0.0002 Concentration of virus (mg mL of virus Concentration

0.0000 02468101214 Time (hours)

Figure 3.23 Concentration of TMV in fresh and conditioned Gamborg’s B5 media. (¡) Fresh medium (Day 0); (¡) medium from 6-day-old N. benthamiana hairy root cultures; () medium from 10-day-old N. benthamiana hairy root cultures; (▲) medium from 14-day-old N. benthamiana hairy root cultures; (z) medium from 21-day-old N. benthamiana hairy root cultures. The error bars represent standard errors from triplicate flasks.

200

Virus infectivity in conditioned media

Results for the infectivity (Section 2.17.4.4: Equation 2.6) of TMV in fresh and conditioned Gamborg’s B5 media are shown in Figure 3.24. The infectivity of virus in each medium did not change significantly (p < 0.05) over the examined period. The infectivity of TMV in the four conditioned media did not differ significantly (p < 0.05) but the infectivity of TMV in fresh Gamborg’s B5 medium 10 and 29 hours after addition of the virus was significantly (p < 0.05) higher than the infectivity of TMV in the conditioned media 10 and 27 hours after addition.

3 Infectivity

0 0 5 10 15 20 25 30 Time (hours)

Figure 3.24 Infectivity of TMV in fresh and conditioned Gamborg’s B5 media. (¡) Fresh medium (Day 0); (¡) medium from 6-day-old N. benthamiana hairy root cultures; () medium from 10-day-old N. benthamiana hairy root cultures; (▲) medium from 14-day-old N. benthamiana hairy root cultures; (z) medium from 21-day-old N. benthamiana hairy root cultures. The error bars represent standard errors from 6 replicates from each of triplicate cultures (n = 18).

201

3.7.2.2 Growth of variously-aged hairy roots in conditioned media

Results for the growth of TMV-infected and non-infected control N. benthamiana hairy root cultures initiated using root inocula from 6-, 10-, 14- and 21-day cultures in conditioned medium from cultures of the same age are shown in Figure 3.25.

At culture initiation, there were no significant (p < 0.05) differences in the biomass of the inoculum roots for the different TMV-infected cultures. However, by Day 7 and for the remainder of examined period, the biomass of cultures initiated using 6-day-old hairy roots and conditioned medium was significantly (p < 0.05) higher than the biomass of the other cultures. The biomass of cultures initiated using inocula from

10-, 14-, and 21-day-old hairy root cultures and conditioned media were low and did not differ significantly (p < 0.05).

There were no significant (p < 0.05) differences between the growth of virus-infected hairy root cultures and the corresponding non-infected control cultures.

The biomass of TMV-infected hairy root cultures initiated using inocula from 10-, 14-, and 21-day-old hairy roots in conditioned media were significantly (p < 0.05) lower than the biomass produced by hairy roots of the same age infected with TMV and cultured in fresh Gamborg’s B5 medium (Figure 3.20A). In contrast, the final biomass of TMV-infected hairy root cultures initiated using 6-day-old hairy roots in conditioned medium was not significantly (p < 0.05) lower than that of TMV-infected cultures initiated using 6-day-old inoculum roots in fresh medium.

202

0.7

0.6

0.5

0.4

0.3

0.2 Biomass (g dry weight) 0.1

0.0 0 5 10 15 20 25 Time (days)

0.7

0.6

0.5

0.4

0.3

0.2 Biomass (g dry weight) 0.1

0.0 0 5 10 15 20 25 Time (days)

203

Figure 3.25 Growth of N. benthamiana hairy root cultures initiated using variously- aged root inocula in conditioned media from hairy root cultures of the same age (A) infected with TMV or (B) non-infected control cultures. (¡) 6-day-old hairy roots inoculated into conditioned medium from 6-day-old root cultures; () 10-day-old hairy roots inoculated into conditioned medium from 10-day-old root cultures; (▲) 14-day-old hairy roots inoculated into conditioned medium from 14-day-old root cultures; (z) 21-day-old hairy roots inoculated into conditioned medium from 21-day-old root cultures. The error bars indicate standard errors from quadruplicate cultures when cultures were infected with virus and triplicate cultures of non-infected controls.

204

3.7.2.3 Viral accumulation when variously-aged hairy roots in conditioned media were infected with TMV

Results for the amount of virus accumulated in the biomass when variously-aged

N. benthamiana hairy roots in conditioned media from root cultures of the same age were infected with TMV are shown in Figure 3.26.

0.10 0.09 0.08 0.07 0.06 0.05 0.04 0.03 Amount of virus (mg) Amount of virus 0.02 0.01 0.00 0 5 10 15 20 25 Time (days)

Figure 3.26 Amount of virus accumulated in N. benthamiana hairy root cultures initiated using variously-aged root inocula in conditioned media from cultures of the same age. (¡) 6-day-old hairy roots inoculated into conditioned medium from 6-day-old root cultures; () 10-day-old hairy roots inoculated into conditioned medium from 10-day-old root cultures; (▲) 14-day-old hairy roots inoculated into conditioned medium from 14-day-old root cultures; (z) 21-day-old hairy roots inoculated into conditioned medium from 21-day-old root cultures. The error bars represent standard errors from quadruplicate cultures.

205

The accumulation of virus in replicate hairy root cultures from the same treatment group was highly variable. The amount of virus accumulated in the biomass of cultures initiated using 10-, 14-, and 21-day-old inoculum roots in conditioned media was very low. The amount of virus accumulated in the biomass of cultures initiated using

6-day-old hairy roots in conditioned medium was higher than in the other treatments; however the large variability in replicate samples resulted in statistically significant differences (p < 0.05) being observed on Days 7 and 25 only. At these times, the amount of virus in cultures initiated using 6-day-old roots in conditioned medium was greater than in the cultures initiated using 14- and 21-day-old roots in conditioned media.

The amount of virus accumulated in the biomass of cultures initiated in conditioned media (Figure 3.26) appeared to be lower than the amount of virus accumulated in the biomass of cultures initiated using roots of the same age in fresh medium (Figure

3.21A). This result can be attributed at least in part to the reduced biomass of cultures grown in conditioned media (Figure 3.25A) compared with cultures grown in fresh medium (Figure 3.20A). However, because of the variability between replicate samples, the differences in the total amount of virus accumulated in the biomass when hairy roots were infected and cultured in conditioned medium and when roots were infected and grown in fresh medium were not significantly (p < 0.05).

The concentration of virus accumulated in the biomass when variously-aged

N. benthamiana hairy roots in conditioned media from cultures of the same age and fresh Gamborg’s B5 medium were infected with TMV are shown in Figure 3.27.

206

0.80

0.70

0.60

0.50

0.40 dry weight) dry -1 0.30

(mg g 0.20 Concentration of virus of virus Concentration

0.10

0.00 0 5 10 15 20 25 Time (days)

0.22 0.20 0.18 0.16 0.14 0.12

dry weight) dry 0.10 -1 0.08

(mg g 0.06 Concentration of virus of virus Concentration 0.04 0.02 0.00 0 5 10 15 20 25 Time (days)

207

0.18

0.16

0.14

0.12

0.10

dry weight) dry 0.08 -1 0.06 (mg g

Concentration of virus 0.04

0.02

0.00 0 5 10 15 20 25 Time (days)

1.00 0.90 0.80 0.70 0.60 0.50 dry weight) dry -1 0.40 0.30 (mg g

Concentration of virus of virus Concentration 0.20 0.10 0.00 0 5 10 15 20 25 Time (days)

Figure 3.27 Concentration of virus accumulated in the biomass of TMV-infected N. benthamiana hairy root cultures initiated using (A) 6-day-old hairy root inocula, (B) 10-day-old hairy root inocula, (C) 14-day-old hairy root inocula, (D) 21-day-old hairy root inocula, in conditioned medium from corresponding root cultures (¡) or fresh Gamborg’s B5 medium (). The error bars represent standard errors from quadruplicate cultures.

208

The concentration of virus accumulated in the biomass of hairy root cultures initiated using variously-aged root inocula in conditioned media from cultures of the same age did not differ significantly (p < 0.05). TMV appeared to accumulate to higher concentrations in the biomass of cultures initiated in fresh medium than in the biomass of cultures initiated using roots of the same age grown in conditioned media. However due to the variability seen in viral accumulation within treatment groups, these differences were generally not significant (p < 0.05).

The infection and growth of roots in conditioned media did not improve levels of viral accumulation in hairy root cultures compared with the use of fresh Gamborg’s B5 medium.

3.7.3 TMV accumulation in hairy root cultures initiated using 21-day

N. benthamiana hairy roots and infected by co-incubation with TMV

3.7.3.1 Growth of N. benthamiana hairy roots – examined over a 52-day period

The accumulation of TMV in N. benthamiana hairy roots was examined over an extended period, to allow the accumulation or loss of virus to be examined during culture growth and non-growth periods.

Results for the growth of N. benthamiana hairy root cultures initiated using 21-day-old roots and infected with TMV (1.5 µg mL-1) by co-incubation of the inoculum roots with the virus in fresh Gamborg’s B5 medium are shown in Figure 3.28. Data are plotted in

Figure 3.28B using semi-logarithmic coordinates to show exponential growth kinetics.

Virus-infected hairy root cultures exhibited exponential growth from Day 3 until

Day 10 with a maximum specific growth rate of 0.35 day-1, which corresponds to a

209

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0.1

0.01 Biomass (g dry weight) (g dry Biomass

0.001 0 5 10 15 20 25 30 35 40 45 50 55 Time (days)

Figure 3.28 Growth of N. benthamiana hairy root cultures inoculated with TMV by co-incubation of roots with virus () and non-infected control cultures (□). The data are presented using linear coordinates (A) and semi-logarithmic coordinates (B). The error bars indicate standard errors from quadruplicate cultures when cultures were infected with virus and triplicate cultures for the non-infected controls.

210 biomass doubling time of 2.0 days. Hairy root growth continued with a decreasing specific rate of growth until Day 22. The hairy root biomass did not change significantly (p < 0.05) from Day 22. The growth of TMV-infected cultures was not significantly different (p < 0.05) from that of non-infected control cultures.

3.7.3.2 Virus accumulation in N. benthamiana hairy roots – examined over a 52-day period

Results for the amount and concentration of virus accumulated in the biomass when

N. benthamiana hairy root cultures initiated using 21-day-old roots were infected with

TMV are shown in Figure 3.29. Data are plotted using semi-logarithmic coordinates

(Figure 3.29B) to show the range of values measured and exponential viral growth kinetics.

The amount of virus accumulated in the biomass increased exponentially in

N. benthamiana hairy roots for 18 days after culture infection with TMV. The maximum specific rate of virus accumulation was 0.50 day-1, which corresponds to a virus doubling time of 1.4 days. From Day 18 to Day 28, the amount of virus accumulated in the biomass fluctuated considerably within the hairy root cultures, but the amounts of virus did not differ significantly (p < 0.05). The maximum amount of accumulated virus (1.1 ± 0.66 mg) was observed 28 days after culture initiation.

Changes in the amount of virus in the biomass observed from Day 28 were not significant (p < 0.05).

The concentration of virus accumulated in the biomass was relatively low and constant for the first 10 days of the culture period (corresponding to the period of exponential

211

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-1 1.5

(mg g 1.0 Amount of virus (mg) Amount of virus Concentration of virus of virus Concentration 0.5

0.0 0 5 10 15 20 25 30 35 40 45 50 55 Time (days)

0.1

0.01 dry weight) weight) dry -1 0.001 (mg g Amount of virus (mg) Amount of virus Concentration of virus of virus Concentration 0.0001

0.00001 0 5 10 15 20 25 30 35 40 45 50 55 Time (days)

Figure 3.29 Amount of virus accumulated in the biomass (¡) and the concentration of virus accumulated in the biomass () of N. benthamiana hairy root cultures. The data are presented using linear coordinates (A) and semi-logarithmic coordinates (B). The error bars indicate standard errors from quadruplicate cultures.

212 hairy root growth: Figure 3.28B) then increased rapidly from Day 10 until Day 20.

The maximum observed virus concentration was 1.9 ± 0.28 mg g-1 dry weight (Day 22).

From Day 22, the pattern of virus accumulation per gram dry weight (concentration of virus) was similar to the pattern observed for the amount of virus accumulated in the biomass, as the biomass weight changed only slightly from this time (Figure 3.28A).

3.8 Effect of Hairy Root Injury on Virus Accumulation

Infection of plants with TMV requires that the plant cell walls be breached to allow virus particles to enter the cells and initiate viral infection. When hairy roots were prepared for use as inocula for new cultures, the roots were removed from Erlenmeyer flasks, filtered through a Buchner funnel and separated into appropriately sized portions. This process resulted in unavoidable root injury (subculture trauma). These injuries could provide sites for viral entry into the cells. The effect of intentionally increasing the level of root injury on subsequent virus accumulation in hairy root cultures was examined by vortexing hairy roots in the presence of an abrasive and by drawing the root mass over sandpaper prior to viral infection.

3.8.1 Effect of intentional root injury on subsequent root growth

Results for the growth of N. benthamiana hairy root cultures after the inoculum roots had been subjected to normal subculture trauma only, vortexed in the presence of an abrasive or injured by drawing over sandpaper (Section 2.9.3.2) are shown for non- infected control cultures and TMV-infected cultures in Figure 3.30 and Figure 3.31, respectively. Data are plotted using semi-logarithmic coordinates to show exponential growth kinetics.

213

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0.2 Biomass (g dry weight) (g dry Biomass 0.1

0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.30 Growth of N. benthamiana hairy root cultures initiated using hairy root inocula that had been subjected to general subculture trauma (¡), vortexed in the presence of an abrasive () or injured by drawing over sandpaper (▲). The error bars indicate standard errors from triplicate cultures.

Additional injury of the hairy root inoculum did not significantly (p < 0.05) affect culture growth, with all non-infected control cultures exhibiting similar patterns of growth and final biomass (Figure 3.30).

In hairy root cultures initiated using root inoculum with general subculture trauma and roots injured by vortexing in the presence of an abrasive, subsequent infection with

TMV did not significantly (p < 0.05) affect hairy root growth (Figure 3.31). TMV- infected cultures initiated using hairy roots treated with sandpaper had significantly lower (p < 0.05) hairy root biomass between Day 9 and Day 15. However the final biomass of the TMV-infected cultures initiated using hairy roots treated with sandpaper

214

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0.0 0 5 10 15 20 25 30 35 40 Time (days)

0.1

0.01 Biomass (g dry weight) (g dry Biomass

0.001 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.31 Growth of TMV-infected N. benthamiana hairy root cultures initiated using hairy root inocula that had been subjected to general subculture trauma (¡), vortexed in the presence of an abrasive () or injured by drawing over sandpaper (▲). The data are presented using a linear coordinates (A) and semi-logarithmic coordinates (B). The error bars indicate standard errors from triplicate and quadruplicate cultures.

215 was not significantly (p < 0.05) different from the final biomass of the other TMV- infected cultures or the corresponding non-infected control culture.

3.8.2 Virus accumulation in hairy root cultures when root inocula were intentionally injured

Results for the amount and concentration of virus accumulated in the biomass are shown in Figure 3.32 for hairy root cultures with inocula subjected to normal subculture trauma, vortexed in the presence of an abrasive or injured by drawing over sandpaper prior to infection with TMV. The differences in both the amount and the concentration of virus accumulated in the biomass were not significant (p < 0.05) for the different treatments.

As these data showed that additional injury of inoculum roots prior to TMV infection had no significant (p < 0.05) effect on viral accumulation in N. benthamiana hairy roots, subsequent experiments were performed using roots subjected to subculture trauma only and no further intentional injury.

216

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1.0 Amount of virus (mg) Amount of virus 0.5

0.0 0 5 10 15 20 25 30 35 40 Time (days)

5.0 4.5 4.0 3.5 3.0

dry weight) dry 2.5 -1 2.0

(mg g 1.5 Concentraion of virus 1.0 0.5 0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.32 Amount of accumulated virus in the biomass (A) and the concentration of accumulated virus in the biomass (B) when TMV-infected N. benthamiana hairy root cultures were initiated using hairy root inocula that had been subjected to general subculture trauma (¡), vortexed in the presence of an abrasive () or injured by drawing over sandpaper (▲). The error bars indicate standard errors from triplicate and quadruplicate cultures.

217

3.9 Characteristics of Hairy Root Growth and Viral Accumulation in

N. benthamiana Hairy Roots over 36 Days

Results from preliminary experiments allowed general parameters that were conducive to viral accumulation in hairy root cultures to be identified. Examination of viral accumulation in cultures initiated using variously-aged root inocula indicated that roots from 21-day cultures inoculated into fresh medium facilitated the multiplication and accumulation of TMV. Intentional injury of inoculum hairy roots prior to TMV infection was observed not to significantly increase (p < 0.05) viral accumulation compared to roots subjected to subculture trauma only.

General inoculation procedures were identified, by which 21-day-old roots were infected with virus by co-incubation with TMV in fresh medium. Inoculum roots were not intentionally injured prior to infection with virus. When the above inoculation conditions were utilised, maximum concentrations of accumulated virus in the biomass were generally observed between Day 18 and Day 27, although concentrations of accumulated virus observed during this period fluctuated considerably (Figure 3.29).

When the culture period was extended beyond Day 27, the concentration of accumulated virus in the biomass was not significantly lower (p < 0.05) than the maximum concentrations observed between Day 18 and Day 27, but was less variable.

To allow both the maximum levels of viral accumulation (Day 18 to Day 30) and the less variable region (from Day 30) to be examined, a 36-day culture period was selected. Full culture parameters were examined using the above conditions over a

36-day period.

218

3.9.1 Growth of N. benthamiana hairy roots

N. benthamiana hairy root cultures infected with TMV and non-infected control cultures are shown at various times after culture initiation in Figure 3.33.

N. benthamiana hairy roots infected with TMV were not visually different from the control cultures in either extent of growth, morphology or colour.

Results for the growth of TMV-infected N. benthamiana hairy root cultures and non-infected controls are shown in Figure 3.34. Data are plotted in Figure 3.34B using semi-logarithmic coordinates to show exponential growth kinetics. TMV-infected hairy roots exhibited exponential growth from Day 3 to Day 9 with a maximum specific growth rate of 0.41 day-1, which corresponds to a biomass doubling time of 1.7 days-1.

Hairy root growth continued at a decreasing specific rate until Day 24. The growth of

TMV-infected N. benthamiana hairy roots did not differ significantly (p < 0.05) from the growth of non-infected control hairy roots.

219

A1 B1

A2 B2

A3 B3

220

A4 B4

A5 B5

A6 B6

Figure 3.33 N. benthamiana hairy root cultures (A) infected with TMV and (B) non-infected control cultures. (1) One day after culture initiation; (2) 9 days after culture initiation; (3) 15 days after culture initiation; (4) 20 days after culture initiation; (5) 28 days after culture initiation; (6) 37 days after culture initiation. TMV was added to the cultures at the time of root inoculation.

221

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0.01 Biomass (g dry weight) (g dry Biomass

0.001 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.34 Growth of TMV-infected N. benthamiana hairy root cultures () and non-infected control cultures (□). The data are presented using linear coordinates (A) and semi-logarithmic coordinates (B). The error bars indicate standard errors from quadruplicate TMV-infected cultures and triplicate non-infected control cultures.

222

3.9.2 Medium characteristics

3.9.2.1 Sugar utilisation

Results for the amount of total sugar in the medium and the utilisation of component sugars in TMV-infected N. benthamiana hairy root cultures and non-infected control cultures are shown in Figure 3.35. The results are presented as amount of sugars in the medium rather than the concentration of sugars because a significant (p < 0.05) reduction in medium volume occurred (28%) over the 36-day experimental period. The total amount of sugar in the growth medium did not decrease significantly (p < 0.05) for the first 6 days of the culture period, as would be expected when the increase in hairy root biomass was small. From Day 6, the total amount of sugar in the medium of both

TMV-infected and non-infected cultures decreased rapidly. Utilisation of sugar by

TMV-infected N. benthamiana hairy root cultures was not significantly (p < 0.05) different from that by non-infected control cultures.

Sucrose was almost completely hydrolysed to fructose and glucose by Day 12 in

TMV-infected and non-infected cultures. Glucose was preferentially utilised by the hairy roots and was completely consumed by Day 27 in both cultures. By Day 36, fructose had also been completely utilised by both cultures. Differences in the hydrolysis of sucrose between TMV-infected hairy roots and non-infected controls were not significant (p < 0.05). Similarly, differences in the utilisation of glucose and fructose by TMV-infected and non-infected control hairy roots were generally not significant (p < 0.05).

223

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0.4 glucose and fructose (g) and fructose glucose 0.2 Amount of total sugars, sucrose, sucrose, Amount of total sugars,

0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.35 Sugar utilisation by TMV-infected hairy root cultures (A) and non- infected control hairy root cultures (B). (¡) Amount of total sugar in the medium; () amount of sucrose in the medium; (z) amount of glucose in the medium; (▲) amount of fructose in the medium. The error bars indicate standard errors from quadruplicate TMV-infected cultures and triplicate non-infected control cultures.

224

3.9.2.2 Medium pH and conductivity

Results for medium pH and medium conductivity for TMV-infected N. benthamiana hairy root cultures and non-infected control cultures are shown in Figure 3.36. In virus-infected cultures, medium pH increased from 5.5 ± 0.037 at culture initiation to

6.8 ± 0.12 15 days after culture initiation. Medium pH then decreased until Day 27 to pH 6.00 ± 0.10, before increasing again. Similar changes in pH were observed in the medium of non-infected control cultures. There was no significant difference (p < 0.05) in medium pH between the TMV-infected and non-infected control hairy root cultures.

8 4 ) -1 7 3

6 2 5 Medium pH Medium

1 4 Medium conductivity (mS cm (mS conductivity Medium

3 0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.36 Medium pH and conductivity in TMV-infected hairy root cultures and non-infected control cultures. () Medium pH in TMV-infected cultures; ( ) medium pH in non-infected control cultures, (z) medium conductivity in TMV-infected cultures; ({) medium conductivity in non-infected control cultures. The error bars indicate standard errors from quadruplicate TMV-infected cultures and triplicate non-infected control cultures.

225

Medium conductivity in TMV-infected cultures decreased slowly from an initial level of 3.4 ± 0.037 mS cm-1 for the first 6 days of the culture period. From Day 6, medium conductivity decreased rapidly until Day 15, after which time only small changes in medium conductivity were observed. The rapid decrease in medium conductivity

(Day 6 to Day 15) coincided with a rapid increase in hairy root biomass (Figure 3.34).

A similar decrease in medium conductivity was observed in non-infected control cultures. There was no significant (p < 0.05) difference in medium conductivity between the TMV-infected and non-infected control hairy root cultures.

3.9.3 Virus accumulation in N. benthamiana hairy roots

3.9.3.1 Characteristics of virus accumulation in hairy root biomass

Results for the amount of virus accumulated in the biomass (average of at least three replicate cultures) and the amount of virus from individual replicate hairy root cultures are shown in Figure 3.37. Data from the individual replicate cultures are presented to show the variability in the amount of virus accumulated in individual cultures. Data for the amount of inoculum virus and the average amount of accumulated virus in cultures from Day 21 to Day 36 are also shown in Figure 3.37.

When the amount of virus accumulated in a culture was greater than the amount of virus added as an inoculum, viral multiplication and accumulation within the culture were indicated. When N. benthamiana hairy roots were infected by co-incubation with virus, for the first 9 days of the culture period the amount of virus accumulated in the biomass and the amount of virus accumulated in the individual replicate cultures were lower than the amount of inoculum virus (0.075 mg). From Day 12, the amount

(average) of virus accumulated in the biomass was higher than the amount of inoculum

226

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2.0

1.5

1.0 Amount of virus (mg) of virus Amount 0.5

0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.37 Amount of virus accumulated in N. benthamiana hairy root biomass, and virus accumulated in individual replicate samples. () Average amount of virus accumulated in the biomass at each time point calculated from individual replicate cultures; (z) amount of virus accumulated in the biomass in individual replicate cultures; (- - - -) average amount of virus accumulated in the biomass from Day 21 to Day 36; (— —) amount of inoculum virus added to the cultures at time zero. The error bars indicate standard errors from quadruplicate cultures for the amount of virus accumulated and from 24 cultures for the average amount of accumulated virus between Days 21 and 36.

virus, and in all but three individual cultures (from Day 12 and Day 36) the amounts of virus accumulated in the biomass were higher than the amount of virus added to the cultures as an inoculum. This indicates that virus was replicating within the biomass of

TMV-infected N. benthamiana hairy roots.

Considerable variability in the virus content of individual cultures sampled at any particular time was observed. This was particularly evident after Day 21 when a small

227 number of replicate cultures accumulated relatively large amounts of virus (greater than

1.5 mg), whereas most cultures accumulated only moderate amounts of virus

(approximately 0.5 mg). A small number of replicate cultures also accumulated low levels of virus (less than 0.1 mg). As high variability in the amount of accumulated virus in the biomass was observed routinely, variability was considered to be a feature of virus accumulation in this system and individual cultures accumulating unusually high or low amounts of virus were not excluded as outliers when determining the average virus accumulation at particular time points. As a result, large standard errors are often associated with values for average amounts of accumulated virus in the biomass.

When accumulation of virus was to be examined using different culture types and treatments, the large fluctuations in the virus accumulation levels at different time points and the large standard errors made comparisons difficult. To assist in analysis, in some investigations, average amounts of virus accumulated over a designated time period are reported. Approximate plateauing of viral accumulation during the later period of the time course allowed an average approximation of viral accumulation to be determined. Average maximum amounts of accumulated virus in the biomass were calculated from the first peak in accumulation, which was generally observed between

Day 18 and Day 24, to the end of the investigation period (Day 36). When no distinct early peak in accumulation was observed, data were averaged from the time when accumulated virus first approached levels that were similar to the maximum accumulation levels. Outliers were not removed. This is illustrated in Figure 3.37, where data are averaged between Days 21 and 36 to give an average maximum viral

228 content of 0.86 ± 0.14 mg. Data were similarly averaged to obtain average maximum concentrations of biomass-associated virus.

The relationship between the amount of virus contained in individual TMV-infected

N. benthamiana hairy root cultures and the biomass of the cultures is shown in

Figure 3.38. Virus accumulation was not directly proportional to hairy root growth.

When plotted using semi-logarithmic coordinates (Figure 3.38B), after an initial period corresponding to the short period of delayed root growth after culture initiation

(Day 1 to Day 3) (Figure 3.34), these data show a first-order relationship throughout the remainder of the culture period. When cultures were newly initiated and biomass was below 0.10 g dry weight, the amount of virus in the biomass doubled with every

0.015 g dry weight increase in biomass, but when the culture biomass increased above

0.10 g dry weight the amount of virus in the roots doubled with every 0.063 g dry weight increase in biomass.

3.9.3.2 Virus in the biomass

Results for the amount and concentration of virus accumulated in the biomass of

N. benthamiana hairy root cultures infected with TMV are shown Figure 3.39.

The amount of virus accumulated in the biomass increased exponentially from Day 3 to

Day 12 after culture infection with TMV with a specific accumulation rate of

0.68 day-1, which corresponds to a virus doubling time of 1.0 day. The period of exponential viral accumulation coincided with the period of exponential hairy root growth (Day 3 to Day 9) and continued into the period of decelerated root growth.

Although viral accumulation was exponential from Day 3 to Day 12, the amount of

229

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1.0 Amount of virus (mg) Amount of virus 0.5

0.0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Biomass (g dry weight)

0.1

0.01 Amount of virus (mg) Amount of virus 0.001

0.0001 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Biomass (g dry weight)

Figure 3.38 Relationship between the amount of virus in the biomass and the biomass of TMV-infected N. benthamiana hairy root cultures. Data from individual cultures (); averaged values from replicate cultures for a given time point (¡). The data are presented using linear coordinates (A) and semi-logarithmic coordinates (B). The error bars indicate standard errors from quadruplicate cultures.

230

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10 10

1 0.1 dry weight) dry -1 0.01 0.1 (mg g Amount of virus (mg) Amount of virus Concentration of virus 0.001 Biomass (g dry weight)

0.0001 0.01 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.39 Amount of virus accumulated in the biomass (¡), concentration of virus accumulated in the biomass () and the hairy root biomass (▲) of N. benthamiana hairy root cultures infected with TMV. The data are presented using linear coordinates (A) and semi-logarithmic coordinates (B). The error bars indicate standard errors from quadruplicate cultures.

231 virus accumulated in the hairy roots was very low during this period. Virus continued to accumulate in the biomass at a reduced rate until Day 21, by which time

0.92 ± 0.36 mg of virus had accumulated. For the remainder of the growth period, the amount of accumulated virus in the biomass fluctuated but remained relatively high.

The concentration of virus accumulated in the biomass increased exponentially from

Day 3 to Day 21. Accumulation was exponential when the amount of virus in the biomass was increasing exponentially (Day 3 to Day 12) and continued to increase exponentially when the rate of viral accumulation was decreasing (Day 12 to Day 21) because the root growth rate was also decreasing during this period. The maximum virus concentration in the biomass, 2.2 ± 0.77 mg g-1 dry weight, was observed 27 days post-infection. The large standard errors associated with the amount of accumulated virus were also observed for the concentration of accumulated virus.

3.9.3.3 Virus in the medium

When hairy roots were infected with virus by co-incubating the inoculum roots with virus in the medium, for the remainder of the culture period detectable virus in the culture medium would consist of residual inoculum virus and hairy-root-produced virus that had been released into the medium. In Figure 3.40, results for the amount of medium virus are shown. The results are presented as amount of virus in the medium rather than concentration of virus because a significant (p < 0.05) reduction in medium volume occurred (28%) over the 36-day experimental period.

232

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0.05

0.04

0.03

Amount of virus (mg) Amount of virus 0.02

0.01

0.00 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.40 The amount of virus in the medium when N. benthamiana hairy roots were infected with TMV by co-incubating inoculum roots with virus in the medium. The error bars indicate standard errors from quadruplicate cultures.

Twenty-three hours after TMV addition to the cultures, the amount of virus in the medium had decreased from the inoculated level of 7.5 × 10-2 mg to

(1.2 ± 0.12) × 10-2 mg. The amount of virus in the medium remained relatively constant over the remainder of the experimental period.

The amount of virus in the medium of N. benthamiana hairy root cultures inoculated with infectious TMV was generally significantly higher (p < 0.05) than the amount of virus in cultures inoculated with UV-deactivated virus (Section 3.6.2.2 and

Figure 3.19B). Rapid initial decreases in medium virus levels were observed when both infectious and deactivated TMV were added to cultures. However, while the amount of deactivated virus in the medium continued to decrease until virus was close to zero (Day 12), when cultures were inoculated with infectious virus after the initial

233 decrease in the medium virus levels, the amount of virus in the medium remained relatively constant. The significantly higher (p < 0.05) amount of virus in the medium of cultures inoculated with infectious TMV may have been due to the release of root produced virus into the medium.

The amount of virus in the medium only contributed significantly (p < 0.05) to the total amount of virus (medium and biomass) in N. benthamiana hairy root cultures from

Day 0 to Day 6 when relatively large amounts of the inoculum virus were retained in the medium and when the amount of virus in the biomass was low. From Day 9 the total amount of virus (medium and biomass) was not significantly different (p < 0.05) from the amount of virus in the biomass, with medium virus contributing only

1.5–4.3% of the total amount of virus (medium and biomass). Accordingly, the amount of virus in the medium was not routinely determined.

3.9.3.4 Biomass-associated viral protein expressed as a percentage of total soluble protein

In Figure 3.41, results for the concentration of viral coat protein associated with the biomass and the concentration of total soluble protein in the biomass are shown. Viral coat protein is also expressed as a percentage of total soluble protein.

By mass, TMV contains approximately 95% coat protein and 5% genomic RNA.

During the culture period, the concentration of viral coat protein in the biomass increased from (1.2 ± 0.24) ¯ 10-2 mg g-1 dry weight 4.5 hours (Day 0) after the inoculation of cultures with TMV, to 1.9 ± 0.91 mg g-1 dry weight on Day 27. The concentration of total soluble protein in hairy roots was initially low (Days 0 and 3).

234

30 g dry weight) weight) g dry

-1 25

5 percentage of total soluble protein of total soluble percentage Accumulated viral coat protein as a Concentration of viral coat protein and and protein coat of viral Concentration total soluble protein (mg protein soluble total 0 Day 0 Day 3 Day 9 Day 12 Day 15 Day 21 Day 27 Day 33 Time

Figure 3.41 Concentration of accumulated viral coat protein (), concentration of total soluble protein (), and accumulated viral coat protein as a percentage of total soluble protein () in N. benthamiana hairy roots infected with TMV. The error bars indicate standard errors for quadruplicate cultures.

Relatively low concentrations of total soluble protein were also observed in older largely senescent, hairy root cultures (Days 21, 27 and 33). Total soluble protein levels in rapidly growing hairy roots (Days 9, 12 and 15) were at least double the levels observed at other culture times.

Accumulated viral coat protein as a percentage of total soluble protein increased throughout the culture period. Accumulated viral coat protein as a percentage of soluble protein on Days 27 and 33 were high, (26 ± 10)% and (19 ± 3.8)%, respectively, due to the high concentration of accumulated viral coat protein and the decreased total soluble protein concentrations in the older hairy roots.

235

The concentration of total soluble protein and the concentration of viral coat protein within the hairy root biomass on Day 27 were approximately 6- and 10- fold lower, respectively, than in the roots of TMV-infected N. tabacum plants (Figure 3.1). Viral coat protein as a percentage of total soluble protein in the roots of whole plants

[(38 ± 5.5)%] did not differ significantly (p < 0.05) from the result for Day 27 hairy roots; however, it was significantly higher (p < 0.05) than the results for Day 33 hairy roots. Virus as a percentage of total soluble protein in the leaves of TMV-infected

N. tabacum plants was not significantly different from the results for Day 27 and

Day 33 TMV-infected hairy roots.

3.9.4 Viral infectivity and particle integrity in TMV-infected N. benthamiana hairy roots

Virus infectivity and particle length were examined in extracts and sap from

TMV-infected N. benthamiana hairy roots to assess the integrity and efficacy of the viral particles.

3.9.4.1 Infectivity of virus in hairy root extracts

Results for the relative infectivity (Section 2.17.4.4: Equation 2.8) of virus in hairy root extracts are shown in Figure 3.42. Relative infectivity did not change significantly

(p < 0.05) during the first 15 days of the culture period but decreased thereafter. By

Day 36, a 10-fold reduction in the relative infectivity of the virus compared with the average relative infectivity of virus from Day 1 to Day 15 was observed. The reduction in relative infectivity over the experimental period was significant (p < 0.05).

236

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0.8

0.6 Relative infectivity 0.4

0.2

0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.42 Relative infectivity of accumulated virus in hairy root extracts. The error bars indicate standard errors for 6 replicates from each of quadruplicate cultures (n = 24).

TMV produced in plants (N. tabacum) was used as the standard in local lesion assays when the relative infectivity of hairy-root-produced TMV was determined.

A relative infectivity of 1 for hairy-root-produced TMV indicates that the infectivity was the same as plant-produced TMV. Initially, the infectivity of hairy-root-produced virus was similar to the infectivity of plant-produced TMV. However as the hairy root cultures aged, TMV in the biomass was less infectious than plant-produced TMV.

The concentration of infectious virus in TMV-infected N. benthamiana hairy roots is shown in Figure 3.43. Infectivity (Section 2.17.4.4: Equation 2.6) of virus is expressed as a function of biomass dry weight. The concentration of infectious virus within the cultures increased significantly (p < 0.05) between Day 1 and Day 21.

237

16000

14000

12000

10000 dry weight) dry

-1 8000

6000

4000 (infectivity g 2000 Concentration of infectious virus virus of infectious Concentration

0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.43 Concentration of infectious virus in the hairy root biomass. The error bars indicate standard errors for 6 replicates from each of quadruplicate cultures (n = 24).

3.9.4.2 Particle length

The length of virus particles in TMV-infected N. benthamiana hairy roots was examined using transmission electron microscopy. Sample preparation methods were selected to reduce particle fragmentation and minimise particle size alteration (Section

2.17.4.3). Frequency distributions of virus particle length in 15-, 20- and 28-day cultures are shown in Figure 3.44. Insufficient particle numbers were observed in sap from cultures 10 days after infection to allow the analysis to be performed at earlier culture times. Samples were not concentrated for the purpose of analysis as this could have resulted in particle fragmentation and an alteration in particle length distribution.

238

100

40 Frequency 20

0 0 0 0 0 0 0 0 0 0 0 0 0 4 80 6 0 2 6 2 8 2 4 8 120 1 2 240 280 3 3 400 440 48 5 560 600 64 6 7 760 800 8 8 920 Virus particle length (nm)

100

40 Frequency 20

0 0 0 0 0 0 0 0 0 0 0 0 0 4 80 6 0 2 6 2 8 2 4 8 120 1 2 240 280 3 3 400 440 48 5 560 600 64 6 7 760 800 8 8 920 Virus particle length (nm)

100

40 Frequency 20

0 0 0 0 0 0 0 0 0 0 0 0 0 4 80 6 0 2 6 2 8 2 4 8 120 1 2 240 280 3 3 400 440 48 5 560 600 64 6 7 760 800 8 8 920 Virus particle length (nm)

Figure 3.44 Frequency distributions of virus particle length 15 days (A), 20 days (B), and 28 days (C) after N. benthamiana hairy root cultures were infected with TMV.

239

Full-length infectious TMV particles have a particle length of 300 nm (Stubbs, 1999).

As can be seen in Figure 3.44, the majority of virus particles, 76%, 74% and 67%, respectively, from Day 15, 20 and 28 hairy roots were located in the 280–320 nm class.

Most particles not in the 280–320 nm class were shorter than full-length TMV.

Respectively, 14%, 17% and 17% of particles, in the Day 15, 20 and 28 cultures were shorter than full-length TMV particles. The proportion of sub-viral particles in the hairy roots was relatively small and does not account for the low relative infectivity of virus (Figure 3.42) observed in cultures.

Thomas and Warren (1999) attributed increases in TMV antigen in plant suspension culture as determined using ELISA to a decrease in the number of full-length virus particles, and an increase in sub-viral particles. Although an increase in the proportion of sub-viral particles was observed from Day 15 to Day 28 in this study, the increase was small and therefore would not have accounted for the large increase in viral antigen concentration determined using ELISA over this period (Figure 3.39).

3.9.5 Distribution of TMV in hairy roots

The concentration of virus accumulated in different regions of Erlenmeyer-flask-grown hairy root biomass was examined to determine if virus accumulated uniformly in the biomass.

240

3.9.5.1 Distribution of virus in different concentric regions

Results for the concentration of virus accumulated in different concentric regions of the circular root mat of flask-grown TMV-infected N. benthamiana hairy roots

(Section 2.15.1) are shown in Figure 3.45. The inner core region generally contained older hairy roots including the inoculum hairy roots; the middle concentric and outer concentric regions generally contained younger hairy root material. The concentration of virus in the biomass of different root mats was variable. However, in a majority of the root mats the concentration of virus accumulated in the biomass decreased with increasing distance from the centre of the root mat. In root mats in which the concentration of virus decreased with increasing distance from the centre, the concentrations of virus in the outer concentric region were 1.7–2.5-fold lower than in roots from the inner core region.

3.9.5.2 Distribution of virus in radial segments

Results showing the concentration of virus in eight different root segments from the root mats of three Erlenmeyer-flask-grown TMV-infected N. benthamiana hairy roots

(Section 2.15.2) are shown in Figure 3.46. The average concentration of virus in the root mat for each culture is also shown. The concentration of virus in root segments from individual root mats was variable, indicating that virus was not distributed evenly within the root biomass.

To minimise the effect of uneven viral distribution on virus quantification, at least 6% of the fresh culture biomass in advanced cultures (Day 18 onwards) and up to 50% of the fresh culture biomass in less mature cultures were analysed. Virus accumulation was also generally examined in quadruplicate cultures.

241

A. B.

0.35 0.70

0.30 0.60

0.25 0.50

0.20 0.40 dry weight) dry 0.15 weight) dry 0.30 -1 -1

0.10 0.20 (mg g (mg g Concentration of virus of virus Concentration 0.05 of virus Concentration 0.10

0.00 0.00 Inner Middle Outer Inner Middle Outer

C. D.

0.35 1.00

0.30 0.80 0.25

0.20 0.60

dry weight) dry 0.15 weight) dry -1 -1 0.40 0.10

(mg g (mg g 0.20 Concentration of virus of virus Concentration 0.05 of virus Concentration

0.00 0.00 Inner Middle Outer Inner Middle Outer

0.6

0.5

0.4

0.3 dry weight) -1 0.2 (mg g

Concentration of virus 0.1

0.0 Inner Middle Outer

Figure 3.45 Concentration of virus in different concentric regions of Erlenmeyer- flask-grown TMV-infected N. benthamiana hairy roots. Results are shown for the inner core, middle region and outer region from 5 replicate hairy root cultures.

242

0.6

0.5

0.4

0.3 dry weight) dry -1 0.2

(mg g 0.1 Concentraion of virus

0.0 12345678Average

0.6

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0.3 dry weight) dry -1 0.2

(mg g 0.1 Concentraion of virus

0.0 12345678Average

0.6

0.5

0.4

0.3 dry weight) dry -1 0.2

(mg g (mg 0.1 Concentraion of virus of virus Concentraion

0.0 12345678Average

Figure 3.46 Concentration of virus in adjacent radial segments of TMV-infected N. benthamiana hairy root mats from three cultures (A, B and C) and the average virus concentration in the root mat.

243

3.10 Medium Conditions at the Time of Viral Infection and Subsequent

TMV Accumulation

3.10.1 Removal of TMV inoculum after an “inoculation phase”

Medium conditions conducive to viral infection of hairy roots may not be the same as those conducive to hairy root growth. The use of separate media during the infection and growth phases requires an exchange of medium and removal of the viral inoculum contained within the infection-phase medium. The effect of inoculum virus removal shortly after infection on subsequent viral accumulation in the biomass was examined.

Viral inoculum was removed from hairy root cultures using medium exchange 16 hours after inoculation of cultures with TMV (Section 2.9.5.1).

3.10.1.1 Hairy root growth

Results for the growth of TMV-infected N. benthamiana hairy root cultures and non-infected control cultures when the medium into which the inoculum was added was exchanged for fresh medium are shown in Figure 3.47.

Observed differences in hairy root growth between the cultures in which the medium was exchanged and cultures in which the medium was not exchanged were not significant (p < 0.05) for TMV-infected or non-infected control cultures.

3.10.1.2 Virus accumulation

Results for the amount and concentration of virus accumulated in the biomass of

N. benthamiana hairy roots with and without inoculum virus removal using medium exchange are shown in Figure 3.48.

244

0.7

0.6

0.5

0.4

0.3

0.2 Biomass (g dry weight) (g dry Biomass 0.1

0.0 0 5 10 15 20 25 30 35 40 Time (days)

0.7

0.6

0.5

0.4

0.3

0.2 Biomass (g dry weight) 0.1

0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.47 Growth of TMV-infected N. benthamiana hairy roots (A) and control non-infected hairy roots (B) when medium was exchanged for fresh Gamborg’s B5 medium 16 hours after the addition of inoculum virus or buffer. () Hairy root growth when medium was exchanged; (¡) hairy root growth without medium exchange. The error bars indicate standard errors for quadruplicate cultures when cultures were infected with virus and triplicate cultures for non-infected control cultures.

245

2.5

2.0

1.5

1.0 Amount of virus (mg) Amount of virus 0.5

0.0 0 5 10 15 20 25 30 35 40 Time (days)

4.0

3.5

3.0

2.5

2.0 dry weight) dry -1 1.5

(mg g 1.0 Concentration of virus

0.5

0.0 0 5 10 15 20 25 30 35 40 Time (days)

246

Figure 3.48 Amount of virus accumulated in the biomass (A) and the concentration of virus accumulated in the biomass (B) when inoculum virus was removed from the medium 16 hours after addition using medium exchange () or retained in the culture medium (¡). Open symbols and dashed lines in (B) represent the average maximum concentration of accumulated virus in the biomass when viral inoculum was removed using medium exchange (Days 18–36) ( ) and when the inoculum was retained in the medium (Days 21–36) (). The error bars indicate standard errors from quadruplicate or triplicate cultures for amount and concentration of accumulated virus. When average concentrations of accumulated virus are presented, the error bars indicate the standard errors determined for 24 and 23 samples, respectively, when the virus was removed from the medium and retained in the medium.

247

The removal of the viral inoculum from cultures using medium exchange did not significantly affect (p < 0.05) the amount or the concentration of virus accumulated in the biomass. The average maximum concentrations of virus in the biomass when the inoculum virus was removed or retained in the medium were, respectively, 0.91 ± 0.24 mg g-1 dry weight (Day 18 to Day 36) and 1.58 ± 0.25 mg g-1 dry weight (Day 21 to Day

36). The average maximum concentration of virus accumulated in the biomass when the inoculum virus was removed from the medium was not significantly lower

(p < 0.05) than the average maximum concentration of virus accumulated in the biomass when the inoculum virus was retained in the medium.

Results for the amount of virus in the medium of hairy root cultures with and without

TMV removal are shown in Figure 3.49. Data are plotted using semi-logarithmic coordinates to show the range of values measured and exponential viral growth kinetics.

The results are presented as amount of virus in the medium rather than concentration because a significant (p < 0.05) reduction in medium volume occurred over the 36-day experimental period.

When inoculum virus was removed from the cultures using medium exchange, TMV was not detectable in the medium (using ELISA) for 6 days. The amount of virus in the medium then increased exponentially from Day 9 until Day 21. The increase in medium virus coincided with TMV accumulation in the hairy roots (Figure 3.48), indicating that virus was released from the infected roots. When the viral inoculum was removed from the culture medium, the proportion of the plant-produced virus that was released into the medium was very low (0.0–5.3)%. The total amount of virus

248

(medium and biomass) was not significantly different (p < 0.05) from the amount of virus in the biomass alone.

Throughout the examined period the amount of virus in the medium was generally significantly lower (p < 0.05) in cultures in which the inoculum virus had been removed using medium exchange than in cultures where the inoculum virus was retained in the medium.

0.025 0.1

0.020

0.01 0.015

0.010 0.001 Amount of virus (mg) Amount of virus (mg) Amount of virus 0.005

0.000 0.0001 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.49 Amount of accumulated virus in the medium of hairy root cultures when inoculum virus was removed from the medium 16 hours after addition using medium exchange (, □) or when inoculum virus was retained in the medium (¡, ). The data are presented using linear and semi-logarithmic coordinates, with closed symbols and solid lines used to represent data presented using linear coordinates and open symbols and dashed lines representing data shown using the semi-logarithmic coordinates The error bars represent standard errors from quadruplicate cultures.

249

3.10.2 Infection of N. benthamiana hairy roots with TMV in different

“media”

When TMV was added to Gamborg’s B5 medium containing inoculum N. benthamiana hairy roots, the amount of virus in the medium was observed to decrease rapidly

(Figure 3.17 and Figure 3.40) so that only a small proportion of the inoculum virus remained free in the medium to initiate new infections. The stability of TMV was examined in sterile Gamborg’s B5 medium, Murashige and Skoog (MS) medium and

0.01 M phosphate buffer, pH 7.4, the storage buffer for TMV, to examine if similar decreases in medium virus concentrations were observed in different “media” and in the absence of hairy roots. To determine if the medium conditions at the time of infection affected subsequent viral accumulation in hairy root cultures, N. benthamiana hairy roots were infected with virus in Gamborg’s B5 medium and in 0.01 M phosphate buffer, pH 7.4.

3.10.2.1 Stability of TMV in different media

Results for the concentration of TMV in sterile Gamborg’s B5 medium, MS medium and 0.01 M phosphate buffer, pH 7.4, over a 29-hour period after the addition of

1.5 × 10-3 mg mL-1 TMV are shown in Figure 3.50. In all treatments, the concentration of virus decreased rapidly after virus addition. During the first 9 hours of the incubation period the concentration of virus did not differ significantly (p < 0.05) between the three media examined. However, the amount of virus detected in phosphate buffer was significantly higher (p < 0.05) than in Gamborg’s B5 medium and

MS medium 10.5, 12 and 29 hours after virus addition to the medium.

250

0.0016 )

-1 0.0014

0.0012

0.0010

0.0008

0.0006

0.0004

0.0002 Concentration of virus (mg mL of virus Concentration

0.0000 0 2 4 6 8 1012141618202224262830 Time (hours)

Figure 3.50 Concentration of TMV in different media. Gamborg’s B5 medium (¡); MS medium (); 0.01 M phosphate buffer, pH 7.4 (▲). The error bars represent standard errors from triplicate flasks.

Average residual concentrations of virus in the different media were determined after virus was no longer decreasing rapidly. The average residual concentrations of TMV

(2–29 hours) retained in Gamborg’s B5 medium, MS medium and 0.01 M phosphate buffer, respectively, corresponded to 23%, 23% and 32% of added inoculum virus.

Medium and buffer used in this investigation did not contain hairy roots and were heat- sterilised to ensure that proteases were denatured.

3.10.2.2 Infectivity of TMV in different media

Results for the infectivity of TMV (Section 2.17.4.4: Equation 2.6) in Gamborg’s B5 medium, MS medium and 0.01 M phosphate buffer, pH 7.4, 0.06, 10.5 and 29 hours after virus addition to the media are shown in Figure 3.51. The infectivity of TMV in the individual media did not change significantly (p < 0.05) over the examined period.

251

3 Infectivity

0 0 5 10 15 20 25 30 Time (hours)

Figure 3.51 Infectivity of TMV in different media. (¡) Gamborg’s B5 medium; () MS medium; (▲) and 0.01 M phosphate buffer, pH 7.4. The error bars represent standard errors for 6 replicates from each of triplicate cultures (n = 18).

The infectivity of TMV in Gamborg’s B5 medium appeared to be higher than in MS medium and phosphate buffer; however, because of the large variability between replicate samples for Gamborg’s B5 medium, the difference was generally not significant (p < 0.05).

3.10.2.3 Infection of hairy roots in phosphate buffer and subsequent medium exchange

Hairy root growth and viral accumulation were examined when N. benthamiana hairy roots were infected with TMV during a separate “inoculation phase” performed in phosphate buffer. The viral inoculum was added directly to 0.01 M phosphate buffer, pH 7.4, containing the root inoculum. Phosphate buffer was replaced with Gamborg’s

B5 medium using medium exchange 23 hours after TMV addition. Results for the

252 growth of N. benthamiana hairy root cultures that were infected with TMV while the hairy root inoculum was in 0.01 M phosphate buffer are shown in Figure 3.52. Data are plotted using semi-logarithmic coordinates in Figure 3.52B to show exponential growth kinetics. The growth of N. benthamiana hairy root cultures infected with TMV in

Gamborg’s B5 medium when the viral inoculum was retained in the medium, or removed using medium exchange, are also shown in Figure 3.52 for comparison.

The biomass of cultures infected with TMV in phosphate buffer was generally not significantly lower (p < 0.05) than the biomass of cultures infected with TMV in

Gamborg’s B5 medium when the viral inoculum was retained in the medium, or removed using medium exchange. The growth of non-infected control hairy roots cultures (data not shown) was generally not significantly different (p < 0.05) from that of the corresponding TMV-infected hairy root cultures.

Results for the amount and concentration of virus accumulated in the biomass when

N. benthamiana hairy roots were infected with TMV in 0.01 M phosphate buffer are shown in Figure 3.53. The phosphate buffer and inoculum virus were removed from the cultures 23 hours after infection and replaced with fresh Gamborg’s B5 medium.

The accumulation of virus in N. benthamiana hairy roots infected with TMV in

Gamborg’s B5 medium when the inoculum virus was retained in the medium throughout the experimental period, or removed using medium exchange after 23 hours, are also shown in Figure 3.53 for comparision.

253

0.8

0.7

0.6

0.5

0.4

0.3

0.2 Biomass (g dry weight) 0.1

0.0 0 5 10 15 20 25 30 35 40 Time (days)

0.1 Biomass (g dry weight)

0.01 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.52 Growth of N. benthamiana hairy roots when: (▲) hairy roots were infected with TMV in 0.01 M phosphate buffer, with TMV inoculum and phosphate buffer removed and replacement with Gamborg’s B5 medium 23 hours after infection; (¡) hairy roots were infected with TMV in Gamborg’s B5 medium with the TMV inoculum retained in the medium; () hairy roots were infected with TMV in Gamborg’s B5 medium with the TMV inoculum removed 23 hours after infection. The data are presented using linear coordinates (A) and semi-logarithmic coordinates (B). The error bars represent standard errors from triplicate and quadruplicate cultures.

254

2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 Amount of virus (mg) Amount of virus 0.4 0.2 0.0 0 5 10 15 20 25 30 35 40 Time (days)

3.5

3.0

2.5

2.0 dry weight) dry

-1 1.5

(mg g 1.0 Concentration of virus 0.5

0.0 0 5 10 15 20 25 30 35 40 Time (days)

255

Figure 3.53 Amount of virus accumulated in the biomass (A) and the concentration of virus accumulated in the biomass (B) in N. benthamiana hairy root cultures when: (▲) hairy roots were infected with TMV in 0.01 M phosphate buffer, with the TMV inoculum and phosphate buffer removed and replaced with Gamborg’s B5 medium 23 hours after infection; (¡) hairy roots were infected with TMV in Gamborg’s B5 medium with the TMV inoculum retained in the medium; () hairy roots were infected with TMV in Gamborg’s B5 medium with the TMV inoculum removed after 23 hours using medium exchange. Open symbols (B) represent the average maximum concentration of accumulated virus in the biomass from Day 25 to Day 36 in cultures with the corresponding symbol type. The error bars indicate standard errors from triplicate or quadruplicate cultures for the amount and concentration of accumulated virus in the biomass, and between 15 and 20 cultures for the average maximum concentration of accumulated virus.

256

The removal of inoculum virus when cultures were infected in Gamborg’s B5 medium, as observed in Section 3.10.1.2, was not associated with a significant (p < 0.05) reduction in the amount or concentration of virus accumulated in the hairy root biomass. The infection of hairy roots with TMV in 0.01 M phosphate buffer, pH 7.4, prior to inoculum virus removal and buffer replacement with Gamborg’s B5 medium was associated with an apparent reduction in viral accumulation compared with the results obtained using medium exchange alone. However, due to the considerable variability between replicate samples, the reductions in both the amount and concentration of accumulated virus in the biomass observed when hairy roots were inoculated with TMV in phosphate buffer were generally not significant (p < 0.05)

(Figure 3.53A).

The average maximum concentration of virus accumulated in the biomass (Day 25 to

Day 36) of cultures infected with TMV in Gamborg’s B5 medium with and without subsequent inoculum removal using medium exchange did not differ significantly

(p < 0.05) (Figure 3.53B). When hairy roots in phosphate buffer were infected with

TMV, and the viral inoculum and buffer were replaced with Gamborg’s B5 medium

23 hours after infection, the average maximum concentration of virus accumulated in the biomass (Day 25 to Day 36) was significantly lower (p < 0.05) than the average maximum concentration of virus accumulated in the biomass (Day 25 to Day 36) of cultures infected with TMV in Gamborg’s B5 medium with and without inoculum removal (Figure 3.53B).

In Table 3.2, the proportions of individual replicate cultures exhibiting active viral infections are presented when hairy roots in 0.01 M phosphate buffer were infected

257 with TMV and when hairy roots in Gamborg’s B5 medium were infected with TMV, with and without inoculum virus removal using medium exchange.

Table 3.2 Percentage of individual replicate cultures (from Day 9) with active viral infections when different media and conditions were used for hairy root infection with TMV.

Inoculation method Percentage of replicate cultures in which the concentration of accumulated virus (mg g-1 dry weight) exceeded the average concentration of virus associated with the biomass 3 days after culture infection

Hairy roots infected with TMV in Gamborg’s B5 100% medium with TMV inoculum retained for the duration of the culture period

Hairy roots infected with TMV in Gamborg’s B5 medium with the TMV inoculum removed after 92% 23 hours using medium exchange

Hairy roots infected with TMV in 0.01 M phosphate buffer, pH 7.4, with the TMV 33% inoculum and buffer removed after 23 hours using medium exchange

When hairy root cultures were inoculated with virus, a proportion of the cultures exhibited very low levels of viral accumulation. This was assumed to indicate that some cultures failed to develop a viral infection that was able to spread throughout the

258 developing root mass and result in high levels of viral accumulation, although primary infection events may have occurred. In infected cultures virus associated with the biomass can be either produced in the roots or taken up from the inoculum virus. When cultures were inoculated with deactivated virus (Section 3.6.2.2), maximum concentrations of root-associated inoculum virus were observed between Day 3 and

Day 6 post-infection (Figure 3.19C). Although differences in the association of active and deactivated virus may have existed, for the purpose of defining active infection, the average concentration of virus in a particular culture 3 days post-culture-initiation plus the standard error was assumed to represent the maximum concentration of adsorbed virus in a culture, as at this time point the concentration of inoculum virus associated with the biomass would have been high and the concentration of root-produced virus would still be low. From Day 9, if the concentration of virus accumulated in an individual culture was lower than the average concentration of accumulated virus and the standard error 3 days post-infection for the same treatment group, it was assumed that a culture did not have an active viral infection. When hairy roots in Gamborg’s B5 medium were infected with TMV, a majority of the replicate cultures were classified as having active infections. In contrast, when an inoculation phase in phosphate buffer was utilised, only 33% of individual cultures were classified as having active infections

(Table 3.2).

The low level of virus accumulated in the biomass and the small proportion of cultures exhibiting active infections when an inoculation phase in phosphate buffer was utilised indicates that the use of a phosphate buffer was not conducive for the initiation of infection or the subsequent accumulation of virus in N. benthamiana hairy roots.

259

3.11 Alteration of Inoculum Virus Concentration

3.11.1 Association of virus with hairy root biomass over 12 hours when the concentration of the viral inoculum was altered

Varying concentrations of TMV were added to 0.2 g N. benthamiana hairy roots in

50 mL Gamborg’s B5 medium and the concentration of virus associated with the biomass examined over a 12-hour period to determine the effect of virus inoculum concentration on virus association with the hairy root biomass. Figure 3.54 shows results for the concentration of TMV associated with the hairy root biomass when TMV was added to the medium at concentrations of 0.75 µg mL-1, 1.5 µg mL-1, 3.0 µg mL-1,

6.0 µg mL-1 and 9.0 µg mL-1. The mechanisms by which inoculum virus interacts with the biomass fraction of a culture during the first 12 hours of the culture period are unknown. Virus that is detected with the biomass is referred to as being associated with the biomass and no conclusion about the nature of the biomass–virus interaction is inferred.

The concentration of virus associated with the hairy root biomass generally increased for the first 7–10.5 hours after virus addition then remained relatively constant for the remainder of the examined period. The relatively constant concentrations were considered to represent approximate plateau regions. Increasing the concentration of inoculum virus generally resulted in an increase in the concentration of virus associated with the biomass, although there was some evidence of saturation in the

6.0–9.0 µg mL-1 TMV treatments. The observed increases in the concentration of biomass-associated virus when the inoculum concentration was increased from 0.75 to

3.0 µg mL-1 were not significant (p < 0.05). Similarly, the observed increases in the concentration of biomass-associated virus when the inoculum concentration was

260 increased from 6.0 to 9.0 µg mL-1 were not significant (p < 0.05). The concentration of virus associated with the biomass was significantly different (p < 0.05) between the two groups (0.75–3.0 µg mL-1 and 6.0–9.0 µg mL-1).

0.09

0.08

0.07

0.06

0.05

dry weight) dry 0.04 -1

0.03 (mg g

Concentration of virus 0.02

0.01

0.00 02468101214 Time (hours)

Figure 3.54 Concentration of TMV associated with the biomass when the concentration of the virus inoculum was altered. Inoculum concentrations were (¡) 0.75 µg mL-1 TMV; () 1.5 µg mL-1 TMV; (▲) 3.0 µg mL-1 TMV; (z) 6.0 µg mL-1 TMV; (¡) 9.0 µg mL-1 TMV. The error bars represent standard errors from quadruplicate cultures.

Results for the average maximum concentrations of biomass-associated virus within the plateau regions (from 7 or 10.5 hours after inoculation) when viral inoculum was added to hairy root cultures at different concentrations are presented in Figure 3.55. When the inoculum concentration was increased from 0.75 µg mL-1 TMV to 6.0 µg mL-1, the concentration of biomass-associated virus increased approximately linearly. When the viral inoculum concentration was increased above 6.0 µg mL-1 to 9.0 µg mL-1, the average maximum concentration of biomass-associated virus in the plateau region

261 increased only slightly, indicating that saturation of the root surfaces available for virus association may have occurred.

0.07

0.06

0.05

0.04 dry weight) dry -1 0.03

0.02 virus (mg g 0.01

Concentration of biomass-associated 0.00 0246810 Inoculum virus concentration (μg mL-1)

Figure 3.55 Plateau concentrations of biomass-associated virus when the inoculum virus concentration ranged from 0.75 µg mL-1 TMV to 9.0 µg mL-1 TMV. The error bars represent standard errors from 16, 16, 8, 20 and 20 cultures when cultures were inoculated with 0.75, 1.5, 3.0, 6.0 and 9.0 µg mL-1 TMV, respectively.

3.11.2 Effect of altering the concentration of inoculum virus on hairy root growth and virus accumulation

3.11.2.1 Effect of inoculum virus concentration on hairy root growth

Results for the growth of hairy root cultures infected using various concentrations of virus (0.75 µg mL-1 TMV to 9.0 µg mL-1 TMV) and a non-infected control culture are shown in Figure 3.56. The pattern and rates of growth of the infected hairy root cultures and the non-infected control were similar. The final culture biomass

(Day 33 and 36) did not differ significantly (p < 0.05) between the cultures.

262

0.7

0.6

0.5

0.4

0.3

0.2 Biomass (g dry weight) (g dry Biomass 0.1

0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.56 Growth of TMV-infected N. benthamiana hairy root cultures infected using different concentrations of viral inoculum. (¡) 0.75 µg mL-1 TMV; () 1.5 µg mL-1 TMV; (▲) 3.0 µg mL-1 TMV; (z) 6.0 µg mL-1 TMV; (¡) 9.0 µg mL-1 TMV; (z) non-infected control hairy root culture. The error bars represent standard errors from quadruplicate cultures when cultures were infected with virus and triplicate cultures for the non-infected controls.

3.11.2.2 Accumulation of TMV in hairy root cultures when the inoculum virus concentration was altered

Results for the concentration of accumulated virus in the biomass when the concentration of the inoculum virus used to infect N. benthamiana hairy root cultures was altered (0.75 µg mL-1 TMV to 9.0 µg mL-1 TMV) are shown in Figure 3.57. The average maximum concentration of accumulated virus and the concentration of virus in individual replicate cultures are also shown in Figure 3.57.

263

2.5

2.0

1.5 dry weight) dry -1 1.0 (mg g Concentraion of virus of virus Concentraion 0.5

0.0 0 5 10 15 20 25 30 35 40 Time (days)

6.0

5.0

4.0

3.0 dry weight) dry -1 2.0 (mg g Concentration of virus of virus Concentration 1.0

0.0 0 5 10 15 20 25 30 35 40 Time (days)

264

C. 8.0

7.0

6.0

5.0

4.0 dry weight) dry -1 3.0

(mg g 2.0 Concentration of virus

1.0

0.0 0 5 10 15 20 25 30 35 40 Time (days)

15 dry weight) dry -1 10 (mg g

Concentration of virus of virus Concentration 5

0 0 5 10 15 20 25 30 35 40 Time (days)

265

E. 16

8 dry weight) dry -1 6 (mg g

Concentration of virus 4

0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.57 Concentration of virus accumulated in the biomass (¡), average maximum concentration of virus in the biomass (- - - -) and concentration of virus in individual replicate cultures (z) when N. benthamiana hairy roots were inoculated using (A) 0.75 µg mL-1 TMV; (B) 1.5 µg mL-1 TMV; (C) 3.0 µg mL-1 TMV; (D) 6.0 µg mL-1 TMV; (E) 9.0 µg mL-1 TMV. The error bars represent standard errors from quadruplicate cultures for concentration of accumulated virus. Error bars for the average maximum concentration of accumulated virus represent standard errors determined using between 28 and 32 cultures.

As the viral inoculum concentration was increased, a general increase in the concentration of biomass-accumulated virus was observed. Maximum specific rates of virus accumulation of 0.42 (Day 0–15), 0.58 (Day 0–12), 0.52 (Day 0–15), 0.62

(Day 0–12) and 0.61 day-1 (Day 0–12) were observed, respectively, when cultures were inoculated using 0.75, 1.5, 3.0, 6.0 and 9.0 µg mL-1 TMV.

The proportion of individual replicate cultures accumulating moderate concentrations of virus (above 1.0 mg g-1 dry weight) increased as the concentration of the viral inoculum

266 was increased. The proportion of individual replicate cultures that exhibited active viral infections (Section 3.10.2.3) also increased as the inoculum concentration was increased. As in Section 3.10.2.3, individual replicate cultures from Day 9 that exhibited concentrations of biomass-associated virus above the average Day 3 concentration of biomass-associated virus plus the standard error from the same treatment group were assumed to exhibit an active viral infection. The Day 3 virus concentrations used to determine active infection were 3.9–7.2-fold higher than the average maximum concentrations of biomass-associated virus observed in the short- term (12 hour) investigation of virus association with hairy roots (Figure 3.54 and

3.55).

When cultures were infected using 0.75 µg mL-1 TMV (Figure 3.57A) only 53% of replicate cultures exhibited active viral infections. Increasing the inoculum concentration to 1.5 µg mL-1 increased the percentage of cultures with active viral infections to 84%. Increasing the inoculum concentration further to 3.0, 6.0 and

9.0 µg mL-1 TMV resulted in only relatively small increases in the percentage of cultures displaying active TMV infections to 85, 90 and 94%, respectively.

As the inoculum virus concentration was increased, the average maximum concentration of biomass-associated virus also increased. As previously outlined

(Section 3.9.3.1), cultures exhibiting low levels of virus accumulation were retained when determining average virus concentrations as their presence appears to be indicative of the efficiency of virus infection and subsequent viral accumulation under the designated growth and inoculation conditions.

267

In Figure 3.58, results for the average maximum concentrations of virus accumulated in the biomass of hairy root cultures are presented as a function of virus inoculum concentration. In Figure 3.59, results for the average maximum concentration of virus accumulated in the biomass are presented as a function of the average maximum concentration of inoculum virus associated with the biomass in short-term (12 hour) experiments (Figure 3.55).

3 dry weight) -1 2 (mg g

1 Concentration of accumulated virus 0 0246810 Inoculum virus concentration (μg mL-1)

Figure 3.58 Average maximum concentration of virus accumulated in the biomass when N. benthamiana hairy root cultures were infected using different concentrations of inoculum virus. The error bars represent standard errors for between 28 and 32 cultures.

As the concentration of the inoculum virus was increased, the average maximum concentration of biomass-associated virus in the hairy root cultures also increased. A similar trend was observed when the average maximum concentration of virus accumulated in the biomass was plotted against the average maximum concentration of inoculum virus associated with the biomass after 7–10.5 hours (Figure 3.55). These

268 results indicate that a relationship exists between the concentration of inoculum virus associated with the biomass soon after inoculation and the maximum concentration of virus accumulated in the biomass.

3 dry weight) dry -1 2 (mg g 1

Concentration of accumulated virus 0 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07

Concentration of biomass-associated inoculum virus (mg g-1 dry weight)

Figure 3.59 Average maximum concentration of virus accumulated in the biomass of N. benthamiana hairy root cultures as a function of average maximum concentration of inoculum virus associated with the biomass when hairy root cultures were infected using different inoculum virus concentrations. Y error bars represent standard errors for between 28 and 32 cultures for average maximum concentration of virus accumulated in the biomass. X error bars represent standard errors from between 8 and 20 cultures for the average maximum concentration of biomass-associated inoculum virus.

From Figure 3.58, the average maximum concentration of virus accumulated in the hairy root biomass when cultures were infected using 0.75 µg mL-1 TMV was significantly lower (p < 0.05) than the average maximum concentration of virus accumulated in the biomass when cultures were infected using 1.5 µg mL-1 TMV.

When cultures were infected using 3.0 µg mL-1 TMV, the result was not significantly

269

(p < 0.05) different from that when an inoculum of 1.5 µg mL-1 TMV was used, but was significantly (p < 0.05) lower than for an inoculum of 6.0 µg mL-1 TMV. The average maximum concentrations of virus accumulated in the biomass observed when hairy root cultures were inoculated with 6.0 µg mL-1 and 9.0 µg mL-1 TMV were not significantly

(p < 0.05) different.

In Figure 3.60, results for the average maximum ratio of accumulated virus in the biomass to inoculum virus per microgram of inoculum virus are shown. Although the average maximum concentrations of accumulated virus increased as the inoculum concentration was increased (Figure 3.58), the efficiency of viral accumulation per microgram of inoculum TMV did not change significantly (p < 0.05) (Figure 3.60).

0.022 0.020 0.018 0.016 0.014 0.012 viral inoculum) viral -1

g 0.010 μ to inoculum virus to inoculum virus 0.008 (mg 0.006 0.004 Maximum ratio of accumulated virus 0246810 Inoculum virus concentration (μg mL-1)

Figure 3.60 Efficiency of viral accumulation per microgram of inoculum virus when N. benthamiana hairy roots cultures were infected using various inoculum virus concentrations (0.75–9.0 μg mL-1 TMV). The error bars represent standard errors for between 28 and 32 cultures.

270

3.12 Proportional Scale-Up in Shake Flasks

The scale-up of viral infection of hairy roots was examined in shake flasks by keeping constant the ratio of root weight:amount of virus:medium volume (Section 2.9.7). The medium volumes were selected so that the gas–liquid oxygen mass transfer coefficient when medium was placed in differently-sized flasks would be similar to that when

50 mL medium was used in 250-mL Erlenmeyer flasks. The flask sizes, medium volumes, root inoculum weights and viral inocula used are summarised in Table 2.1.

For simplicity, treatments in the scale-up series are referred to by the medium volume.

3.12.1 Association of inoculum virus with hairy roots using scale-up conditions

The association of inoculum virus with the hairy root inoculum when cultures were scaled-up proportionally was examined over a 12 hour-period (Section 2.12.2). Results for the amount and concentration of biomass-associated virus when cultures were scaled-up are presented in Figure 3.61. Reflecting the range of root weights used as inocula (0.11 g to 0.54 g fresh weight), the amount of virus associated with the biomass increased significantly (p < 0.05) as the medium volume was increased (Figure 3.61A).

To determine if virus was associated with the inoculum hairy roots proportionally in the scaled-up cultures, the concentration of virus associated with the biomass was determined (Figure 3.61B). The observed differences in the concentration of biomass- associated virus were not significant (p < 0.05) 4 and 7.5 hours after virus addition; however, significant (p < 0.05) differences were observed between several of the treatment groups from 9 hours.

271

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Figure 3.61 Amount of biomass-associated virus (A) and concentration of biomass- associated virus (B) when shake-flask inoculations were scaled-up proportionally: (¡) 27 mL medium; () 50 mL medium; (▲) 82 mL medium; (z) 134 mL medium. The error bars represent standard errors from quadruplicate cultures.

272

In Figure 3.62, results for the average maximum concentration of virus in the plateau regions of Figure 3.61B are presented as a function of the medium volume. Average maximum concentrations of biomass-associated virus were calculated using the data from 7.5 hours after virus addition when culture medium volumes of 27 mL and 50 mL were used, and 9.0 hours after virus addition when culture medium volumes of 82 mL and 134 mL were used. Average maximum concentrations of virus associated with the biomass increased significantly (p < 0.05) with each increase in medium volume. The concentration of virus associated with the biomass increased linearly as cultures were scaled-up over the range examined.

Proportional scale-up did not result in the same degree of interaction between inoculum virus and roots during the first 12 hours of the incubation period. As the virus

0.016

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0.008 dry weight) dry -1 0.006

(mg g 0.004 Concentration of virus of virus Concentration

0.002

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Figure 3.62 Average maximum concentration of biomass-associated virus during scale-up. The error bars represent standard errors from 16, 17, 12 and 12 cultures, respectively, when medium volumes 27 mL, 50 mL, 82 mL and 134 mL were used.

273 concentration in the medium was maintained at 1.5 µg mL-1, the increased association of virus with the roots as the medium volume was increased indicates that more virus was available in the medium and able to associate with the hairy root biomass or, when the root inoculum size was increased, that the root surface area available for virus association may not have increased proportionally with the inoculum fresh weight.

3.12.2 Hairy root growth and viral accumulation during proportional scale-up in shake flasks

When proportional scale-up of viral infection in shake flasks was examined, time- course curves were produced for growth and viral accumulation using medium volumes of 27 mL, 50 mL and 82 mL. Only a partial time-course curve was generated using

134 mL of medium due to limited materials. For 134-mL control cultures, growth was monitored from Day 0 to Day 18 to allow the effect of scale-up on growth rate to be determined. For 134-mL TMV-infected cultures, growth and viral accumulation were examined from Day 21 to Day 36 to allow average maximum viral accumulation levels to be determined.

3.12.2.1 Hairy root growth

Results for the growth of scaled-up TMV-infected N. benthamiana hairy root cultures and non-infected control cultures are shown in Figure 3.63. Scaled but non-infected hairy root cultures (Figure 3.63B), showed similar patterns of growth. Roots exhibited exponential growth from Day 3 to Day 9, after which time the rate of root growth decelerated. Maximum specific growth rates were similar for all cultures but showed a decreasing trend as the medium volume increased. Maximum specific growth rates

274

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Figure 3.63 Growth of N. benthamiana hairy root cultures infected with 1.5 µg mL-1 TMV (A) and non-infected control cultures (B) when shake-flask cultures were scaled- up proportionally. (¡) 27 mL medium; () 50 mL medium; (▲) 82 mL medium; (z) 134 mL medium. The error bars indicate standard errors from quadruplicate cultures when cultures were infected with virus and triplicate cultures for non-infected control cultures.

275 were 0.39 day-1, 0.38 day-1, 0.38 day-1 and 0.33 day-1, respectively, using medium volumes of 27 mL, 50 mL, 82 mL and 134 mL.

The pattern of growth of TMV-infected hairy root cultures (Figure 3.61A) was similar to that observed in non-infected roots (Figure 3.61B) and differences in growth were not significant (p < 0.05). However, an increase in maximum specific growth rates with increasing medium volumes was observed. The maximum specific growth rates were

0.26 day-1, 0.31 day-1 and 0.39 day-1, respectively, using volumes of 27 mL, 50 mL and

82 mL.

Scale-up was generally proportional as determined by the biomass yield (Yxs) for

TMV-infected cultures and non-infected control cultures when medium volumes were

27mL, 50 mL, 82 mL and 134 mL. The biomass yields from total sugars for

TMV-infected cultures were 0.39, 0.38, 0.34 and 0.37 g dry weight g-1, respectively, using medium volumes of 27 mL, 50mL, 82 mL and 134 mL. The biomass yields for non-infected cultures were 0.39, 0.38 and 0.35 g dry weight g-1, respectively, using medium volumes of 27 mL, 50mL and 82 mL. The biomass yields were calculated assuming that the initial sucrose concentrations were 0.03 g mL-1 and that sucrose utilisation was complete.

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3.12.2.2 Virus accumulation

Results for the amount and concentration of virus accumulated in the biomass when the infection of N. benthamiana hairy roots in shake-flask cultures was scaled-up proportionally are shown in Figure 3.64.

The amount of virus accumulated in the biomass increased when cultures were scaled-up from 27 mL to 134 mL (Figure 3.64A). This is not unexpected because, if efficient infection of hairy roots with virus occurred in all cultures, the amount of virus accumulated in the biomass would be expected to increase with scale-up, due to increased biomass.

The concentration of virus accumulated in hairy root cultures appeared to increase as the cultures were scaled-up (Figure 3.64B). However, the differences between the concentrations of accumulated virus in the scaled cultures at discrete times post- infection were not significant (p < 0.05). In Figure 3.65, the average maximum concentrations of accumulated virus in the biomass obtained from Figure 3.64B are plotted against the medium volume. Overall, the average maximum concentrations of virus accumulated in the biomass after culture increased linearly with volume when the cultures were scaled-up proportionally. However, because of the large errors observed, the average maximum concentration of accumulated virus did not differ significantly

(p < 0.05) when medium volumes of 50 mL (Day 15 to Day 36), 82 mL (Day 15 to Day

36) and 134 mL (Day 21 to Day 36) were used. The average maximum concentration of virus accumulated in the biomass when the medium volume was 27 mL (Day 18 to

Day 36) was significantly (p < 0.05) lower than in the other medium volumes.

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Amount of virus (mg) Amount of virus 1.5 1.0 0.5 0.0 0 5 10 15 20 25 30 35 40 Time (days)

5.0 4.5 4.0 3.5 3.0 2.5 dry weight) dry -1 2.0 1.5 (mg g

Concentration of virus 1.0 0.5 0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.64 Amount of virus accumulated in the biomass (A) and the concentration of virus accumulated in the biomass (B) when infection of N. benthamiana hairy roots with TMV was scaled-up proportionally in shake flasks. (¡) 27 mL medium; () 50 mL medium; (▲) 82 mL medium; (z) 134 mL medium. The error bars represent standard errors from quadruplicate cultures.

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Figure 3.65 Average maximum concentration of accumulated virus in the biomass after culture during scale-up. The error bars represent standard errors from 28, 32, 31 and 22 cultures, respectively, when medium volumes 27 mL, 50 mL, 82 mL and 134 mL were used.

In Figure 3.66, the average maximum concentration of virus accumulated in the biomass after culture (Figure 3.64B) and the corresponding average maximum concentration of inoculum virus associated with the biomass in the plateau region

(Figure 3.61B) are shown as infection was scaled-up. As observed also in Figure 3.59, a linear relationship between the average maximum concentration of inoculum virus associated with the biomass and the average maximum concentration of virus accumulated in the biomass was observed.

279

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Concentration of biomass-associated inoculum virus (mg g-1 dry weight)

Figure 3.66 Average maximum concentration of accumulated virus in the biomass after culture and the corresponding average maximum concentration of inoculum virus associated with the biomass as inoculation was scaled-up. Y error bars represent standard errors from 28, 32, 31 and 22 cultures, respectively, when medium volumes were 27 mL, 50 mL, 82 mL and 134 mL. X error bars represent standard errors from 16, 17, 12 and 12 cultures, respectively, when medium volumes were 27 mL, 50 mL, 82 mL and 134 mL.

280

3.13 Accumulation of TMV in N. benthamiana Hairy Root Cultures with

Established Infections

Growth and viral accumulation in N. benthamiana hairy roots were examined when non-infected hairy roots were infected with TMV at the point of culture initiation

(primary viral infection) and in subsequent generations of roots that had previously been infected with TMV (established viral infection) (Section 2.10).

3.13.1 Accumulation of TMV in two generations of hairy root cultures with established TMV infections and in a hairy root culture with a primary TMV infection

3.13.1.1 Growth of hairy root cultures with established TMV infections

Results for the growth of N. benthamiana hairy root cultures with a developing primary viral infection and two successive generations of cultures with established viral infections are shown in Figure 3.67. The growth of non-infected control cultures is also shown in Figure 3.67. Growth of cultures with primary and established infections, although overlapping, was not coincident.

The growth of cultures with a primary TMV infection and the first generation cultures with an established virus infection did not differ significantly (p < 0.05) during the examined period. However, the final biomass (Days 33 and 36) and the biomass during the active growth phase (Day 6 to Day 15) of the second generation hairy root culture with an established viral infection was significantly (p < 0.05) higher than in the other two cultures. As growth of the virus-infected hairy root cultures did not differ significantly (p < 0.05) from growth of the corresponding non-infected control cultures, the observed differences in growth of the second generation cultures with established

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Figure 3.67 Growth of N. benthamiana hairy root cultures with a primary TMV infection and established TMV infections (A) and non-infected control cultures (B). (¡) Hairy root culture with a primary TMV infection in (A) or its corresponding control in (B); () first generation hairy root culture with an established TMV infection in (A) or its corresponding control in (B); (▲) second generation hairy root culture with an established TMV infection in (A) or its corresponding control in (B). The error bars represent standard errors from quadruplicate cultures when cultures were infected with virus and triplicate cultures for non-infected controls.

282 infection were probably related to differences in culture medium or other non-biotic conditions during the experimental period. Maximum specific growth rates (Day 3 to

Day 12) for cultures with primary and first and second generation established viral infections were 0.30, 0.35 and 0.35 day-1, respectively. These correspond to biomass doubling times of 2.3, 1.9 and 1.9 days, respectively, for cultures with primary and first and second generation established viral infections.

The growth of the non-infected control cultures (Figure 3.67B) did not differ significantly (p < 0.05) during the examined period.

3.13.1.2 Viral accumulation in hairy root cultures with primary and established viral infections

Amount of virus in the biomass

Results for the amount of virus accumulated in hairy root cultures with a primary virus infection and in two subsequent, consecutive generations of hairy root cultures with established viral infections are shown in Figure 3.68. Data are presented using linear and semi-logarithmic coordinates to allow differences in the initial and maximum amounts of virus in the biomass and the different patterns of viral accumulation to be observed.

Viral accumulation in hairy roots with a primary viral infection followed the general pattern observed in previous experiments. The amount of virus accumulated in the biomass increased exponentially from Day 0 until Day 21, with a maximum specific accumulation rate of 0.53 day-1. This corresponds to a virus doubling time of 1.3 days.

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Figure 3.68 Amount of accumulated virus in hairy root biomass of cultures with a primary TMV infection and in two subsequent consecutive generations of hairy root cultures with established viral infection. The data are plotted using linear coordinates (A) and semi-logarithmic coordinates (B). (¡) Hairy root culture with a primary TMV infection; () first generation hairy root culture with an established TMV infection; (▲) second generation hairy root culture with an established TMV infection. The error bars represent standard errors from quadruplicate cultures.

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The patterns of virus accumulation in the first and second generation cultures with established TMV infections were similar. The initial amount of virus (Day 0) was higher than in hairy roots with a primary viral infection. The amount of virus accumulated in the biomass increased exponentially from Day 3 to Day 12 for first generation cultures with an established infection, and from Day 0 to Day 12 for second generation cultures. The maximum specific rate of viral accumulation of 0.31 day-1, which was observed for both cultures with an established infection, was lower and occurred over a shorter time period than the maximum rate of viral accumulation in cultures with a primary TMV infection. The maximum specific virus accumulation rate corresponds to a virus doubling time of 2.2 days.

The amount of virus accumulated in cultures with established TMV infections at individual time points was not significantly different (p < 0.05) from the amount of virus accumulated in the cultures with a primary TMV infection at corresponding times post-culture-initiation. The average maximum amount of virus accumulated in the biomass of cultures with first generation established infections (0.99 ± 0.16 mg: Day 27 to Day 36) and cultures with second generation established infections (1.4 ± 0.22 mg:

Day 27 to Day 36) were not significantly different (p < 0.05) from the average maximum amount of virus accumulated in the biomass of cultures with a primary TMV infection (1.8 ± 0.33 mg: Day 21 to Day 36).

Concentration of virus in the biomass

Results for the concentration of accumulated virus in the biomass of hairy root cultures with a primary virus infection and in two subsequent, consecutive generations of hairy root cultures with established viral infection are shown in Figure 3.69. Data are

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286

Figure 3.69 Concentration of virus accumulated in the hairy root biomass of cultures with a primary TMV infection, and in two subsequent, consecutive generations of hairy root cultures with established viral infections. The data are plotted using linear coordinates (A) and semi-logarithmic coordinates (B). (¡) Hairy root culture with a primary TMV infection; () first generation hairy root culture with an established TMV infection; (▲) second generation hairy root culture with an established TMV infection. Open symbols (A) represent the average maximum or average concentration of virus accumulated in the biomass of cultures with the corresponding symbol type. The error bars for concentration of accumulated virus indicate standard errors from quadruplicate cultures. Error bars for the average concentrations of accumulated virus indicate standard errors determined using 20 or 51 cultures.

287 presented using linear and semi-logarithmic coordinates to allow differences in the initial and the maximum concentration of virus in the biomass and the different patterns of viral accumulation to be observed.

Virus accumulation per gram dry weight in hairy root cultures with primary TMV infections followed the general pattern observed previously in similarly-infected cultures (Figure 3.29). The concentration of accumulated virus increased initially

(Day 0 to Day 3), but then remained relatively constant until Day 9 as both the amount of virus and the biomass increased exponentially. The concentration of virus in the biomass increased exponentially from Day 9 until Day 21 as hairy root growth slowed.

The average maximum concentration of accumulated virus in the biomass of hairy root cultures with primary TMV infections (Day 21 to Day 36) was 3.4 ± 0.63 mg g-1 dry weight.

First and second generation hairy root cultures with established viral infections exhibited high concentrations of accumulated virus at culture initiation, reflecting the concentration of virus in Day 21 roots from the previous generation culture.

Accumulated virus concentrations in both the first and second generation cultures did not vary significantly (p < 0.05) throughout the experimental period and were not significantly (p < 0.05) different from each other. Average concentrations of accumulated virus of 2.0 ± 0.27 mg g-1 dry weight (Day 0 to Day 36) and 1.6 ± 0.18 mg g-1 dry weight (Day 0 to Day 36), respectively, were observed for first and second generation cultures with established infections.

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The average maximum concentration of virus accumulated in the cultures with the primary virus infection (Day 21 to Day 36) was significantly (p < 0.05) higher than the average concentration of virus accumulated in the first and second generation cultures

(Day 0 to Day 36) with established viral infection. However the average maximum concentration of virus accumulated in the biomass of cultures with primary viral infections, calculated after the initial peak in accumulation (Day 24 to Day 36), was not significantly different (p < 0.05) from the average maximum concentrations of virus in cultures with first and second generation established viral infections calculated over a similar period (Day 24 to Day 36).

Cultures with established TMV infections (first and second generation) were initiated using root inocula from each of four pre-cultures from the previous generation of TMV- infected hairy roots (primary or first generation established). The pre-cultures from which the inoculum roots were obtained varied with regard to the concentration of virus accumulated in the hairy root biomass, reflecting the general variation in viral yields between replicate cultures observed in this work (Section 3.9.3.1). At each sample time, cultures initiated using inoculum roots from each of the four inoculum pre- cultures were harvested and the concentration of accumulated virus determined. In

Figure 3.70 the average concentration of virus accumulated in progeny first and second generation cultures with established infections are shown as a function of the concentration of virus in the inoculum (pre-culture) roots. A loose relationship between the concentration of virus in the inoculum roots and the concentration of virus accumulated in the progeny cultures was observed.

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Figure 3.70 Average concentration of virus in inoculum (pre-culture) roots and the corresponding average concentration of virus (Day 0 to Day 36) in progeny cultures. In (A) root inoculum was obtained from cultures with primary viral infections 21 days post-infection and progeny cultures were first generation cultures with established infections. In (B) root inoculum was obtained from cultures with first generation established infections 21 days post-initiation and progeny cultures were second generation cultures with established infections. X error bars represent standard errors from quadruplicate samples in (A) and maximum errors for duplicate samples in (B). Y error bars represent standard errors from 12 or 13 cultures.

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In Figure 3.71, the concentration of virus in individual replicate cultures from first generation cultures with established TMV infections initiated using root inocula from separate pre-cultures are shown. For clarity, results from cultures initiated using root inocula from only three of the four replicate cultures are presented. The concentration of virus accumulated in the biomass of cultures initiated using inocula obtained from individual pre-cultures fluctuated over the examined period but, generally, virus accumulation in cultures initiated using low virus-accumulating inocula (reflected by viral accumulation in cultures at Day 0) was generally low, and virus accumulation in cultures initiated using high virus-accumulating inocula was comparably high.

Significant (p < 0.05) differences in the concentrations of accumulated virus were observed between replicate cultures initiated using different inoculum cultures. The concentration of virus in the inoculum culture is reflected by the concentration of virus at Day 0.

3.13.2 Effect of increasing the viral inoculum concentration used to initiate a primary viral infection in hairy roots on viral accumulation in a subsequent-generation hairy root culture with an established viral infection

The average maximum concentration of virus accumulated in a culture with a primary infection can be increased by increasing the concentration of the inoculum virus

(Figure 3.58). The effect of increasing the concentration of the viral inoculum used to initiate a primary infection in N. benthamiana hairy roots on the accumulation of virus in subsequent cultures with established viral infections was examined, to determine if a similar relationship also applied between generations of root cultures.

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Figure 3.71 Concentration of virus in the biomass of replicate second generation cultures with established TMV infections initiated using root inocula from three different first generation cultures with established TMV infections. The origin of the inoculum roots used to initiate the second generation cultures is indicated by (▲, ¡, ), each representing one inoculum root source.

3.13.2.1 Hairy root growth

In Figure 3.72A, results for the growth of N. benthamiana hairy roots with a primary viral infection initiated using a viral inoculum of 9.0 µg mL-1 TMV, and growth of a subsequent hairy root culture with an established viral infection, are shown. The growth of non-infected control hairy root cultures is shown in Figure 3.72B. Growth of the culture with a primary viral infection did not differ significantly (p < 0.05) from growth of the cultures with the established viral infection. Maximum specific growth rates (Day 3 to Day 12) of cultures with primary and established viral infections were

0.36 and 0.31 day-1, respectively. These correspond to biomass doubling times of

1.9 and 2.3 days, respectively for cultures with primary and established viral infections.

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Figure 3.72 Growth of N. benthamiana hairy root cultures with a primary TMV infection initiated using 9.0 μg mL-1 TMV and an established TMV infection (A) and non-infected control cultures (B). (¡) Hairy root cultures with a primary TMV infection in (A) or its corresponding control in (B); () first generation hairy root culture with an established TMV infection in (A) or its corresponding control in (B). The error bars represent standard errors from quadruplicate cultures when cultures were infected with virus and triplicate cultures for non-infected control cultures.

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TMV infection of hairy roots, whether primary or established, did not significantly

(p < 0.05) affect the growth of hairy roots compared with the non-infected controls.

3.13.2.2 Virus accumulation

Amount of accumulated virus

The amount of virus accumulated in hairy root cultures with a primary viral infection initiated using a viral inoculum of 9.0 µg mL-1 TMV and a subsequent (first) generation hairy root culture with an established viral infection are shown in Figure 3.73. Data are presented using linear and semi-logarithmic coordinates to allow differences in the initial and maximum amounts of virus in the biomass and the different patterns of viral accumulation to be observed.

The patterns of virus accumulation (amount) in cultures with primary and established

TMV infections were similar to those observed in Figure 3.68. In the culture with the primary TMV infection, virus accumulated exponentially for 15 days after infection, with a maximum specific accumulation rate of 0.66 day-1. This corresponds to a virus doubling time of 1.1 days. In the hairy root culture with the established viral infection, the initial amount of virus in the cultures was significantly (p < 0.05) higher than in cultures with a primary infection. The amount of accumulated virus in the cultures with established infections increased exponentially from Day 3 to Day 12; however the specific rate of accumulation was lower (0.37 day-1) than in cultures with the primary viral infection. The virus doubling time was 1.9 days.

294

2 Amount of virus (mg) Amount of virus 1

0 0 5 10 15 20 25 30 35 40 Time (days)

0.1

0.01

0.001 Amount of virus (mg) Amount of virus 0.0001

0.00001 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.73 Amount of virus accumulated in hairy root cultures with a primary viral infection initiated using a viral inoculum of 9.0 µg mL-1 TMV (¡) and in a subsequent hairy root culture with an established viral infection (). The data are plotted using linear coordinates (A) and semi-logarithmic coordinates (B). The error bars represent standard errors from quadruplicate cultures.

295

The amount of virus accumulated in the hairy roots with the developing primary viral infection was generally not significantly different (p < 0.05) from the amount of virus accumulated in the hairy root culture with the established TMV infection at corresponding time points. The average maximum amount of virus accumulated in cultures with a developing primary infection, 1.9 ± 0.31 mg (Day 15 to Day 36), was not significantly different (p < 0.05) from that achieved in cultures with an established viral infection, 1.5 ± 0.25 mg (Day 21 to Day 36).

Concentration of accumulated virus

The concentration of virus accumulated the in hairy root culture with a primary viral infection initiated using a viral inoculum of 9.0 µg mL-1 TMV and a subsequent generation hairy root culture with an established viral infection are shown in

Figure 3.74. Data are presented using linear and semi-logarithmic coordinates to allow differences in the initial and maximum concentrations of virus in the biomass and the different patterns of viral accumulation to be observed.

The patterns of viral accumulation in the cultures with primary and established viral infections were similar to those observed in Figure 3.69. The concentration of virus in the hairy root cultures with the established viral infection did not change significantly

(p < 0.05) over the experimental period. The average maximum concentration of virus in the biomass (Day 15 to Day 36) of hairy roots with the primary viral infection

(3.5 ± 0.62 mg g-1 dry weight) was not significantly (p < 0.05) different from the average concentration of virus in the biomass (Day 0 to Day 36) of the hairy roots culture with the established viral infections (2.5 ± 0.28 mg g-1 dry weight).

296

6 dry weight) dry -1 4 (mg g Concentration of virus 2

0 0 5 10 15 20 25 30 35 40 Time (days)

100

1 dry weight) dry -1 0.1 (mg g

Concentration of virus 0.01

0.001 0 5 10 15 20 25 30 35 40 Time (days)

297

Figure 3.74 Concentration of virus in a hairy root culture with a primary viral infection initiated using a viral inoculum of 9.0 µg mL -1 TMV (¡), and a subsequent generation hairy root culture with an established viral infection (). The data are plotted using linear coordinates (A) and semi-logarithmic coordinates (B). Open symbols (A) represent the average maximum or average concentration of accumulated virus in the biomass of cultures with the corresponding symbol type. The error bars indicate standard errors from triplicate cultures for hairy roots with a primary viral infection and quadruplicate cultures for hairy roots with established viral infections. The error bars for the average virus concentrations indicate standard errors determined using 23 and 52 cultures for cultures with primary infections and established infections, respectively.

298

In this investigation, increasing the concentration of the inoculum virus used to initiate the primary viral infection from 1.5 µg mL-1 TMV (Figure 3.69) to 9.0 µg mL-1 TMV

(Figure 3.74) did not result in a significant (p < 0.05) increase in the average maximum concentration of virus accumulated in the biomass. Average maximum concentrations of accumulated virus were 3.4 ± 0.63 mg g-1 dry weight (Day 21 to Day 36) and

3.5 ± 0.62 mg g-1 dry weight (Day 15 to Day 36), respectively, when the inoculum concentrations were 1.5 µg mL-1 TMV and 9.0 µg mL-1 TMV. Similarly, the average concentration of virus accumulated in the biomass of cultures with first generation established viral infections initiated using root inocula from primary cultures infected using 1.5 µg mL-1 TMV (2.0 ± 0.27 mg g-1 dry weight) (Figure 3.69) was not significantly different from the average concentration of virus accumulated in the biomass of cultures with first generation established viral infections initiated using root inocula from primary cultures infected using 9.0 µg mL-1 TMV (2.5 ± 0.28 mg g-1 dry weight) (Figure 3.74).

299

3.14 Infection of N. benthamiana Hairy Roots with TMV-GFPC3 in Shake

Flasks

3.14.1 Production, concentration and infectivity of TMV-GFPC3 preparations

3.14.1.1 Production and concentration of TMV-GFPC3 preparations

TMV-GFPC3 particles were produced in N. clevelandii plantlets inoculated with RNA transcripts from 30B-GFPC3 plasmids (Section 2.6.3). Cycle 3 GFP was expressed in inoculated leaves and in upper un-inoculated leaves. Figure 3.75 shows a Western blot of a crude extract of leaf material in which GFP was detected, confirming

TMV-GFPC3 production in N. clevelandii leaves.

1 2 3 4

40 kDa 30 kDa 20 kDa

Figure 3.75 Western blot probed with anti-GFP antibody, showing GFP in a crude extract of N. clevelandii leaf from a plant inoculated with RNA transcript from 30B-GFPC3 plasmids. Lane 1 Molecular weight marker; Lane 2 10 ng recombinant GFP; Lane 3 Crude extract from a non-infected leaf; Lane 4 Crude extract from a leaf of a plant inoculated with RNA transcript from the 30B-GFPC3 plasmid.

300

In experiments in which cultures were infected with TMV, the concentration of the inoculum virus was reported as a mass concentration determined using spectrophotometric methods (Section 2.5.3). As TMV-GFPC3 particles are longer than

TMV particles, use of the same inoculum mass concentration as for TMV

(1.5 μg mL-1 TMV) would result in the addition of fewer TMV-GFPC3 particles to the cultures. Therefore, the particle number concentration of the TMV inoculum was determined so that an equivalent number concentration of TMV-GFPC3 could be used as viral inoculum. The particle number concentrations (particles mL-1) of TMV and

TMV-GFPC3 were determined using scanning electron microscopy (Section 2.17.6).

As the weight concentration (mg mL-1) of TMV preparations could be measured spectrophotometrically (Section 2.5.3) the number of particles of TMV per milligram could be determined. A TMV mass of 1.0 mg was equivalent to 2.65 × 1012 full-length

TMV particles. The number concentration of TMV particles equivalent to a mass concentration of 1.5 µg mL-1 TMV was 3.97 × 10 9 particles mL-1.

When the mass concentration of TMV-GFPC3 was determined, all particles with lengths greater than or the same as that of native TMV (300 nm) were counted as full particles, although the true length of TMV-GFPC3 particles determined proportionally from RNA genome length would be closer to 360 nm (Dawson et al., 1989). Shorter particles were included because the findings of Rabindran and Dawson (2001) indicated that particles of length 300–360 nm are probably hybrid vectors which, while unable to express Cycle 3 GFP, would retain infectivity. Of the particles included in the count, fewer than 31% had lengths approaching the 360 nm length expected for full-size

TMV-GFPC3 particles.

301

3.14.1.2 Infectivity of TMV-GFPC3 purified from plants

The infectivity (Section 2.17.4.4) of purified TMV-GFPC3 (Section 2.6.4) was compared to the infectivity of purified TMV (Section 2.5.2) used as a viral inoculum.

The relative infectivity of TMV-GFPC3 compared to inoculum TMV was

(3.8 ± 2.1) × 10-3. Therefore, purified plant-produced TMV was approximately

(260 ± 140)-fold more infectious than an equivalent number of TMV-GFPC3 particles.

The proportion of infectious TMV-GFPC3 particles that expressed Cycle 3 GFP was not determined.

3.14.1.3 Expression of GFP in plants infected with purified TMV-GFPC3 particles

Leaves of N. benthamiana plantlets were inoculated with either RNA transcript from the 30B-GFPC3 plasmid, purified TMV-GFPC3 particles, or a crude N. clevelandii leaf extract containing TMV-GFPC3 particles. After 22 days, the leaves were examined using a fluorescence microscope (Section 2.17.5.3). Figure 3.76 shows micrographs of leaf sections from N. benthamiana plantlets infected with RNA transcript or purified

TMV-GFPC3. In the low-magnification micrographs, the red chlorophyll auto-fluorescence appears dull due to rapid quenching of the chlorophyll signal and the use of short exposure times that were optimal for imaging GFP.

When N. benthamiana plants were infected using RNA transcript from the 30B-GFPC3 plasmid, GFP was expressed uniformly throughout the inoculated and upper plant leaves (Figure 3.76E). Only small regions (if any) in the upper leaves showed the red background auto-fluorescence of chlorophyll rather than the green fluorescence associated with GFP expression. When inoculated and non-inoculated upper leaves from N. benthamiana plantlets infected with purified TMV-GFPC3 particles were

302

A. B.

0.5 mm 0.5 mm

C. D.

0.5 mm 0.2 mm

E. F.

0.5 mm 0.5 mm

303

Figure 3.76 Micrographs of wet-mounted N. benthamiana leaf sections from plants infected using either RNA transcript from the 30B-GFPC3 plasmid or purified TMV-GFPC3 particles. The photographs were taken under UV illumination using a reflected-light fluorescence microscope. (A) Area of bright green fluorescence against a red fluorescent background in a leaf inoculated with purified TMV-GFPC3 particles (1 second exposure); (B) area of dull green fluorescence against a red fluorescent background in a leaf inoculated with purified TMV-GFPC3 particles (1 second exposure); (C) areas of bright and dull green fluorescence against a red fluorescent background in a leaf inoculated with purified TMV-GFPC3 particles (1 second exposure); (D) individual fluorescent cells and small clusters of fluorescent cells in an upper leaf of a plant inoculated with purified TMV-GFPC3 particles (0.5 second exposure); (E) upper leaf of a plant inoculated with RNA transcript showing bright green fluorescence throughout the leaf (0.5 second exposure); (F) control leaf from a non-infected plant showing red chlorophyll auto-fluorescence (1 second exposure).

304 examined, only small regions of bright green fluorescence (Figures 3.76A and 3.76C) or dull green fluorescence (Figures 3.76B and 3.76C) were observed in the inoculated leaves. The upper leaves showed very little fluorescence, with only trace amounts of fluorescence observed in individual cells (Figure 3.76D). The limited expression of

GFP in leaves infected with purified TMV-GFPC3 and the lack of GFP expression in upper plant leaves compared with the extensive expression of GFP in plants infected with RNA transcript, suggest that only a proportion of the purified TMV-GFPC3 particles were able to facilitate the expression of GFP and that the genetic stability of the infectious particles was also limited.

3.14.2 Infection of N. benthamiana hairy roots with TMV-GFPC3 particles

N. benthamiana hairy roots were inoculated with TMV-GFPC3 so that the number of particles added to the medium was either the same as the number of TMV particles in

75 µg TMV (1.98 × 1011 particles) (equivalent to 1.5 µg mL-1 TMV in 50 mL medium), or twice that number (3.97 × 1011 particles). Hairy roots were added to the medium just prior to virus addition, and the virus was retained in the medium for the duration of the culture period.

3.14.2.1 Growth of N. benthamiana hairy roots inoculated with TMV-GFPC3 particles

Results for the growth of N. benthamiana hairy root cultures inoculated with two concentrations of TMV-GFPC3 and the growth of non-infected control cultures are shown in Figure 3.77. The differences in growth between the cultures were not significant (p < 0.05).

305

0.7

0.6

0.5

0.4

0.3

0.2 Biomass (g dry weight) (g dry Biomass 0.1

0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.77 Growth of N. benthamiana hairy root cultures infected using viral inocula of 1.98 × 1011 TMV-GFPC3 particles (¡) and 3.97 × 1011 TMV-GFPC3 particles (), and a non-infected control culture (▲). The error bars represent standard errors from quadruplicate cultures when hairy roots were infected with TMV-GFPC3 and triplicate cultures for non-infected controls.

3.14.2.2 Sugar utilisation by N. benthamiana hairy root cultures inoculated with

TMV-GFPC3

The utilisation of sugar by N. benthamiana hairy root cultures inoculated with two concentrations of TMV-GFPC3 and non-infected control cultures are shown in

Figure 3.78. The differences in sugar utilisation between the cultures were generally not significant (p < 0.05).

306

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4 Amount of total sugars (g) Amount sugars of total 0.2

0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.78 Amount of total sugar in the medium of N. benthamiana hairy root cultures infected using viral inocula of 1.98 × 1011 TMV-GFPC3 particles (¡) and 3.97 × 1011 TMV-GFPC3 particles (), and a non-infected control culture (▲). The error bars represent standard errors from quadruplicate TMV-GFPC3 infected cultures and triplicate cultures for non-infected control cultures.

The hydrolysis and utilisation of component sugars (sucrose, glucose and fructose) by hairy root cultures inoculated with TMV-GFPC3 followed similar patterns to those observed for TMV-infected cultures (Figure 3.35A) and generally did not differ significantly (p < 0.05) from the component sugar utilisation by non-infected control hairy root cultures (data not shown).

307

3.14.2.3 TMV-GFPC3 accumulation in N. benthamiana hairy root cultures

TMV-GFPC3 levels in root extracts and medium were determined using Western blots.

Purified inoculum TMV-GFPC3 with particle number concentration determined using scanning electron microscopy (SEM) (Section 2.17.6) was used as a standard for quantification. Because the standard contained virus particles shorter than 300 nm that were not included in SEM quantification, and because particles that were included in

SEM quantification were of variable length, the concentration of virus determined using

Western blots was approximate only and may have represented a lower viral protein concentration than was actually contained in samples. In addition, protein quantification from Western blots can be relatively inaccurate due to varying efficiencies of protein transfer from Tris-glycine gels. Despite these limitations, relative concentrations of viral protein after different treatments were obtained using the western blot method.

The amount of virus accumulated in the biomass and the concentration of virus accumulated in the biomass of N. benthamiana hairy roots inoculated using two concentrations of TMV-GFPC3 are shown in Figure 3.79 and Figure 3.80, respectively. Data are plotted using semi-logarithmic coordinates in Figure 3.79B and

Figure 3.80B to show the range of values measured and exponential virus growth kinetics.

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2.5 ) 11 2.0

1.5

1.0

0.5 Amount of virus (particles × 10 (particles Amount of virus

0.0 0 5 10 15 20 25 30 35 40 Time (days)

10 ) 11 1

0.1

0.01

0.001 Amount of virus (particles × 10 (particles Amount of virus

0.0001 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.79 Amount of virus accumulated in the biomass when N. benthamiana hairy root cultures were inoculated using 1.98 × 1011 TMV-GFPC3 particles (¡) and 3.97 × 1011 TMV-GFPC3 particles (). Data are plotted using linear coordinates (A) and semi-logarithmic coordinates (B). The error bars represent standard errors from quadruplicate cultures.

309

4.0

) 3.5 11

3.0

2.5

2.0 dry weight × 10 weight dry -1 1.5

1.0 Concentration of virus of virus Concentration

(particles g 0.5

0.0 0 5 10 15 20 25 30 35 40 Time (days)

10 ) 11

0.1 dry weight × 10 weight dry -1

0.01 Concentration of virus of virus Concentration (particles g

0.001 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.80 Concentration of virus accumulated in the biomass when N. benthamiana hairy roots were inoculated using 1.98 × 1011 TMV-GFPC3 particles (¡) and 3.97 × 1011 TMV-GFPC3 particles (). Data are plotted using linear coordinates (A) and semi-logarithmic coordinates (B). The error bars represent standard errors from quadruplicate cultures.

310

When N. benthamiana hairy root cultures were inoculated using 1.98 × 1011

TMV-GFPC3 particles, the amount of biomass-associated virus increased rapidly between Days 0 and Day 12, with a maximum specific accumulation rate of 0.53 day-1.

This corresponds to a virus doubling time of 1.3 days. Virus accumulation continued at a reduced specific rate until Day 30, although increases in the amount of virus accumulated in the biomass beyond Day 18 were not significant (p < 0.05). The concentration of accumulated virus in the biomass (Figure 3.80) increased until Day

12. The concentration of accumulated virus in the hairy root biomass did not increase significantly (p < 0.05) above the concentration observed 9 days after infection.

When N. benthamiana hairy root cultures were inoculated using 3.97 × 1011

TMV-GFPC3 particles, the initial (until Day 21) patterns for the amount of virus accumulated in the biomass and the concentration of virus accumulated in the biomass were similar to those observed when cultures were inoculated using 1.98 × 1011 TMV-

GFPC3 particles. The amount of biomass-associated virus increased rapidly between

Day 0 and Day 12 with a maximum specific accumulation rate of 0.61 day-1, corresponding to a virus doubling time of 1.1 days. Virus accumulation continued at a reduced rate until Day 21. The amount of biomass-associated virus increased rapidly between Day 21 and Day 30, and remained relatively high for the remainder of the culture period.

When the amount and concentration of accumulated virus in the biomass were compared for the cultures inoculated using two concentrations of TMV-GFPC3, the differences in accumulated virus between Day 0 and Day 18 and on Day 24 were not significant (p < 0.05). However on Days 30 and 36, hairy roots inoculated using

311

3.97 × 1011 TMV-GFPC3 particles contained significantly (p < 0.05) higher amounts and concentrations of virus than did cultures inoculated using 1.98 × 1011

TMV-GFPC3 particles. The average maximum concentration of virus accumulated in the biomass of cultures inoculated using 3.97 × 1011 TMV-GFPC3 particles,

(1.8 ± 0.44) × 1011 particles (Day 24 to Day 36), was approximately 3-fold higher than the average maximum concentration of virus accumulated in the biomass of cultures inoculated using 1.98 × 1011 particles, (6.4 ± 0.36) × 1010 particles (Day 21 to Day 36).

3.14.2.4 Medium virus

In Figure 3.81, results for the amount of virus in the medium are shown for

N. benthamiana hairy root cultures inoculated using two concentrations of

TMV-GFPC3. When cultures were inoculated with 3.97 × 1011 TMV-GFPC3 particles, the amount of virus in the medium decreased rapidly and only 40% of the added virus was detected in the medium 2.5 hours after virus addition. When cultures were inoculated with 1.98 × 1011 particles, the amount of virus in the medium decreased but,

2.5 hours after virus addition, 73% of the inoculum virus was retained in the medium.

The initial decrease in the amount of virus retained in the medium was not as pronounced with TMV-GFPC3 as when TMV was added to the cultures (Figure 3.40).

Medium TMV-GFPC3 levels showed a generally decreasing trend over the culture period for both TMV-GFPC3 concentrations tested. Medium virus levels were higher when 3.97 × 1011 TMV-GFPC3 particles were added to the cultures; the differences were significant (p < 0.05) 12 and 36 days after virus addition. After 36 days, the amount of TMV-GFPC3 in the medium of both cultures was equivalent to 32% of the added inoculum virus.

312

4.5

) 4.0 11

3.5

3.0

2.5

2.0

1.5

1.0

0.5 Amount of virus (particles × 10 (particles Amount of virus

0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.81 Amount of virus in the medium when N. benthamiana hairy root cultures were inoculated using 1.98 × 1011 TMV-GFPC3 particles (¡) and 3.97 × 1011 TMV-GFPC3 particles (). The error bars represent standard error from quadruplicate cultures.

3.14.2.5 Total amount of virus

The total amounts of virus in N. benthamiana hairy root cultures inoculated with two concentrations of TMV-GFPC3 are shown in Figure 3.82. More virus was detected in the medium than in the biomass in both cultures. These data indicate that there was a net loss of virus from cultures inoculated with TMV-GFPC3.

3.14.2.6 GFP expression in hairy root cultures inoculated with TMV-GFPC3

Hairy roots inoculated with 1.98 × 1011 and 3.97 × 1011 TMV-GFPC3 particles were examined for GFP expression using fluorescent microscopy, ELISA and Western blotting (Section 2.17.5). The culture medium was also analysed for GFP. GFP was not detected in the hairy root biomass or culture medium.

313

2.5 ) 11 2.0

1.5

1.0

0.5 Amount of virus (particles × 10 (particles Amount of virus

0.0 0 5 10 15 20 25 30 35 40 TIme (days)

4.5

) 4.0 11

3.5

3.0

2.5

2.0

1.5

1.0

0.5 Amount of virus (particles × 10 (particles Amount of virus

0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.82 Total amount of virus in N. benthamiana hairy root cultures inoculated using (A) 1.98 × 1011 TMV-GFPC3 particles and (B) 3.97 × 1011 TMV-GFPC3 particles. (¡) Total virus (biomass and medium); (z) virus in the biomass; () virus in the medium; (▬▬) inoculum virus. The error bars represent standard errors from quadruplicate cultures.

314

3.14.3 Viral accumulation in N. benthamiana hairy root cultures inoculated with equal numbers of TMV-GFPC3, TMV and deactivated TMV particles

To allow comparisons between TMV and TMV-GFPC3 levels, virus accumulation in

N. benthamiana hairy root cultures infected with an equal number of TMV-GFPC3,

TMV and deactivated TMV particles was expressed in terms of particle number. The conversion factor of 2.65 × 1012 TMV particles per milligram of TMV was determined using scanning electron microscopy (Section 2.17.6). Only full-length (and therefore potentially infectious) viral particles were counted when determining this conversion factor, even though a small number of truncated particles was present in the purified

TMV preparation. The majority of TMV particles from hairy root cultures were full-length particles (Figure 3.44); however some fragmented particles were also present. Due to the presence of fragmented particles in the standard, the conversion of

TMV virus weight (as determined using ELISA with a spectrophotometrically- determined standard concentration) to virus particle number may give a slightly lower particle concentration than that which would represent the total viral protein in the sample.

Results for the amount of virus associated with the biomass when N. benthamiana hairy root cultures were inoculated with TMV-GFPC3, TMV and deactivated TMV at a concentration of 3.96 × 109 particles mL-1 are shown in Figure 3.83. The accumulation patterns, but not the amounts of accumulated virus, were similar for

TMV- and TMV-GFPC3-infected hairy root cultures. Virus accumulation in both these cultures occurred exponentially until Day 12 (Figures 3.39B and 3.79B), but

TMV-infected hairy roots had a higher maximum specific viral accumulation rate of

0.68 day-1 compared with 0.53 day-1 for TMV-GFPC3-infected cultures. The average

315

4.5

) 4.0 12

3.5

3.0

2.5

2.0

1.5

1.0

0.5 Amount of virus (particles × 10 (particles Amount of virus

0.0 0 5 10 15 20 25 30 35 40 Time (days)

6.0 ) 10 5.0

4.0

3.0

2.0

1.0 Amount of virus (particles × 10 (particles Amount of virus

0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.83 Amount of virus in the biomass in N. benthamiana hairy root cultures inoculated with (A) 3.96 × 109 particles mL-1 TMV-GFPC3, TMV, and deactivated TMV and (B) 3.96 × 109 particles mL-1 TMV-GFPC3 and deactivated TMV. (¡) TMV; () TMV-GFPC3; (▲) deactivated TMV. The error bars represent standard errors from quadruplicate cultures.

316 maximum amount of biomass-accumulated virus (Day 21 to Day 36) in the

TMV-infected hairy root cultures was approximately 65-fold higher than that for

TMV-GFPC3 over the same period.

The pattern of accumulation of virus in the biomass when hairy root cultures were inoculated with deactivated virus differed significantly to that observed when cultures were inoculated with infectious TMV and TMV-GFPC3. When cultures were inoculated with deactivated TMV, the amount of virus associated with the biomass increased for 6 days after infection but did not change significantly (p < 0.05) thereafter. The average maximum amount of biomass-associated virus (Day 21 to Day

36) in TMV-GFPC3-infected hairy roots was approximately 6.7-fold greater than that achieved (Day 21 to Day 36) in cultures inoculated with deactivated TMV

(Figure 3.83B).

The concentration of virus in the biomass of hairy root cultures inoculated with

TMV-GFPC3 and deactivated TMV are shown in Figure 3.84. Viral accumulation patterns per gram dry weight in TMV-GFPC3- and deactivated TMV-inoculated hairy root cultures were different. When hairy root cultures were infected with

TMV-GFPC3, the concentration of TMV-GFPC3 increased for the first 12 days of the culture period and then remained relatively constant for the remainder of the experimental period. However, when hairy root cultures were inoculated with deactivated TMV, the concentration of deactivated virus per gram dry weight increased for 3 to 6 days after virus addition before decreasing and remaining low for the remainder of the experimental period. From Day 12, the average maximum

317 concentration of biomass-associated virus in cultures infected with TMV-GFPC3 was approximately 5.8-fold higher than in roots inoculated with deactivated TMV.

1.2 )

11 1.0

0.8

0.6 dry weight × 10 weight dry -1 0.4 Concentration of virus of virus Concentration 0.2 (particles g

0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.84 Concentration of biomass-associated virus in N. benthamiana hairy root cultures inoculated with 3.96 × 109 particles mL-1 TMV-GFPC3 () and deactivated TMV (▲). The error bars represent standard errors from quadruplicate cultures.

Results for the amount of virus in the medium when hairy root cultures were inoculated with equal numbers of TMV-GFPC3, TMV and deactivated TMV particles are shown in Figure 3.85. The data for TMV-GFPC3 in the culture medium were dissimilar to those for TMV and deactivated TMV. The initial decrease in the amount of virus in the medium was more pronounced when TMV and deactivated TMV were added to cultures than when TMV-GFPC3 was added. Throughout the examined period, the amount of TMV-GFPC3 in the medium was significantly (p < 0.05) higher than the amount of deactivated TMV. The amount of TMV-GFPC3 in the medium was also significantly (p < 0.05) higher than the amount of TMV in the medium soon after

318 addition (Days 0 and 1, respectively, for TMV-GFPC3 and TMV) and on Day 12 and

Day 24. By the end of the culture period (Day 36), the amount of TMV-GFPC3 in the medium was only 1.3-fold and not significantly (p < 0.05) higher than the amount of

TMV in the medium, but was 56-fold and significantly (p < 0.05) higher than the amount of deactivated TMV in the medium.

2.5 ) 11 2.0

1.5

1.0

0.5 Amount of virus (particles × 10 (particles Amount of virus 0.0 0 5 10 15 20 25 30 35 40 Time (days)

Figure 3.85 Amount of virus in the culture medium when N. benthamiana hairy root cultures were inoculated with 1.98 × 1011 TMV-GFPC3 (), TMV (¡) and deactivated TMV (▲) particles. The error bars represent standard errors from quadruplicate cultures.

319

3.15 Infection of N. benthamiana Hairy Roots with TMV and TMV-GFPC3 in

Bioreactors

Accumulation of virus (TMV and TMV-GFPC3) in hairy roots was examined in

2-L stirred bioreactors (Section 2.16). Hairy roots (8.0 g) were inoculated with virus by co-incubation in the bioreactor vessel. The inoculum virus concentration,

1.5 μg mL-1 TMV or 3.96 × 109 particles mL-1 TMV-GFPC3, was the same as that used in the standard shake-flask investigations. As biomass samples could not be taken from the bioreactor during operation, viral levels in the biomass were analysed only when the cultures were terminated. Culture progression was assessed by monitoring sugar utilisation, medium conductivity and medium pH.

3.15.1 Hairy root growth and sugar utilisation

The growth of TMV-infected hairy roots in a 2-L bioreactor over a 31-day period is shown in Figure 3.86. The hairy roots were initially contained within a cylindrical mesh basket (Figure 3.86A). The stainless steel mesh used for construction of the basket allowed root penetration, and hairy roots were able to grow towards the bioreactor walls and also towards the base of the vessel (Figure 3.86B). The magnetic stirrer bar initially limited hairy root growth towards the base of the vessel by shearing the tips off the growing roots; however, as the root density in the bioreactor increased, the cleared region near the base of the bioreactor decreased in size until movement of the stirrer bar was limited. Growth of TMV-GFPC3-infected and non-infected control hairy roots did not appear to differ from that for TMV-infected hairy roots shown in

Figure 3.86.

320

A. B.

C. D.

Figure 3.86 Growth of TMV-infected N. benthamiana hairy roots in a stirred 2-L bioreactor at various times after culture initiation. (A) Day 2; (B) Day 12; (C) Day 15; (D) Day 27.

321

Results for the final biomass of N. benthamiana hairy root cultures infected with TMV and TMV-GFPC3 and also non-infected control cultures are shown in Figure 3.87.

Infection of hairy root cultures with virus did not result in significant (p < 0.05) alterations in final biomass when compared to non-infected root cultures.

10 Biomass (g dry weight) (g dry Biomass 5

0 TMV TMV-GFPC3 Control

Figure 3.87 Final hairy root biomass in TMV- and TMV-GFPC3-infected N. benthamiana hairy root cultures and non-infected control cultures in 2-L stirred bioreactors after 31 days. The error bars represent maximum errors from duplicate cultures. A single bioreactor culture was carried out using TMV-GFPC3.

Results for the concentration of sugar in the medium from TMV- and

TMV-GFPC3-infected and non-infected control N. benthamiana hairy root cultures grown in bioreactors are shown in Figure 3.88. Although the medium volume decreased throughout the experimental period due to absorption of water by the growing roots, the results are expressed as concentrations as the medium volumes during the cultures period are unknown. For the first 6 days of the cultures, the concentration of sugar (sucrose, fructose and glucose) in the medium remained

322 relatively high and did not differ significantly (p < 0.05) between the different cultures.

For the remainder of the experimental period (Day 6 to Day 31) the concentration of sugar in the medium of TMV-infected cultures was generally significantly (p < 0.05) higher than in the control cultures. When cultures were infected with TMV-GFPC3, the concentration of sugar in the medium was initially (Day 9 to Day 15) significantly

(p < 0.05) higher than the concentration of sugar in the medium from control cultures; however, from Day 21 differences in the concentrations of total sugar in the medium were not significant (p < 0.05).

40 ) -1 35

5 Concentration of total sugars (g L sugars of total Concentration 0 0 5 10 15 20 25 30 Time (days)

Figure 3.88 Total sugar utilisation by TMV- () and TMV-GFPC3- (▲) infected N. benthamiana hairy roots and non-infected control hairy roots (¡) in a 2-L stirred bioreactor. For TMV-inoculated and non-infected control bioreactors, the error bars indicate standard errors for duplicate samples from duplicate bioreactors. For the TMV-GFPC3-inoculated bioreactor, the error bars indicate maximum errors from duplicate samples from one bioreactor.

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3.15.2 Medium pH and conductivity

Results for the pH of medium from TMV- and TMV-GFPC3-infected and non-infected control N. benthamiana hairy root cultures grown in 2-L stirred bioreactors are shown in Figure 3.89. All cultures exhibit similar medium pH profiles; the observed differences in medium pH between the different cultures were not significant (p < 0.05).

Medium pH Medium 6

3 0 5 10 15 20 25 30 Time (days)

Figure 3.89 Medium pH when TMV- () and TMV-GFPC3- (▲) infected N. benthamiana hairy roots and non-infected control (¡) hairy roots were grown in 2-L stirred bioreactors. The error bars represent maximum errors from duplicate bioreactors for TMV-infected and non-infected control cultures.

324

Results for the conductivity of medium from TMV- and TMV-GFPC3-infected and non-infected control N. benthamiana hairy root cultures grown in bioreactors are shown in Figure 3.90. The conductivity of the medium from TMV-GFPC3-infected cultures and non-infected control cultures did not differ significantly (p < 0.05) during the examined period. The conductivity of the medium from TMV-infected cultures was significantly (p < 0.05) higher than in the other cultures from Day 12 to Day 21, but from Day 24 the differences were not significant (p < 0.05).

4.5

) 4.0 -1 3.5

3.0

2.5

2.0

1.5

1.0

Medium conductivity (mS cm (mS conductivity Medium 0.5

0.0 0 5 10 15 20 25 30 Time (days)

Figure 3.90 Medium conductivity when TMV- () and TMV-GFPC3- (▲) infected N. benthamiana hairy roots and non-infected control (¡) hairy roots were grown in 2-L stirred bioreactors. Error bars indicate maximum errors from duplicate bioreactors for TMV-infected cultures and non-infected control cultures.

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3.15.3 Accumulation of virus

3.15.3.1 Accumulation of virus in hairy root biomass

Virus accumulation in N. benthamiana hairy roots from cultures infected with TMV and TMV-GFPC3 was determined 31 days after inoculation. Results for the concentration of virus in root samples taken from different regions of the bioreactor

(Section 2.16.2) are shown in Figure 3.91. Viral concentration is expressed as milligrams per gram dry weight for cultures inoculated with TMV and as particles per gram dry weight for cultures inoculated with TMV-GFPC3.

As indicated in Figure 3.91A, TMV did not accumulate uniformly in the hairy root biomass. The highest concentrations of virus were found in hairy roots in the mesh basket and near the base of the bioreactor. These concentrations did not differ significantly (p < 0.05) from each other; however, the concentration of virus accumulated in hairy roots from the mesh basket and the base of the bioreactor were significantly (p < 0.05) higher than in roots from the other examined regions.

Results for the concentration of virus accumulated in biomass sampled from different regions of the TMV-GFPC3-infected N. benthamiana hairy root cultures are shown in

Figure 3.91B. The concentrations of virus accumulated in different regions of the bioreactor were not significantly different (p < 0.05).

326

0.6 0.5 0.4 0.3 dry weight) dry

-1 0.2 0.1 (mg g

Concentration of virus 0.0

p se et et et all To a sk sk sk w B a a ba or h b h b h ct s s es ea me me m r e e bio th th he to nd t nt a t to ce all en ja r w c ad to dja ht ac t a eig re gh -h io ei n b -h Mid ee Mid etw t b igh he d- Mi

) 10 9.0 8.0 7.0 6.0 5.0 4.0

dry weight × 10 weight dry 3.0 -1 2.0 1.0 0.0 Concentration of virus Concentration t t t ll p se ke ke ke a To Ba as as as r w (particles g b b b to h h h ac s es s re Me m me io he he e b t d t th t to n o n ll a t t ce a en ja r w c ad to dja ht ac t a eig re h -h io eig n b -h Mid ee tw Mid be ht eig -h Mid

Figure 3.91 Concentration of virus accumulated in hairy roots from different regions of the bioreactor containing (A) TMV-infected hairy roots (B) TMV-GFPC3-infected hairy roots. For roots infected with TMV, the error bars indicate standard errors for triplicate samples from duplicate bioreactors. For roots infected with TMV-GFPC3, the error bars indicate standard errors for triplicate samples from a single bioreactor.

327

The concentration of TMV in bioreactor-grown hairy roots from near the base of the bioreactor (0.39 ± 0.13 mg g-1 dry weight) was approximately 4-fold lower than the average maximum concentration of accumulated virus (Day 21 to Day 36) in shake- flask-grown hairy roots (1.6 ± 0.25 mg g-1 dry weight) inoculated using the same concentration of TMV (Figure 3.39). However the concentration of TMV-GFPC3 in bioreactor-grown hairy roots from near the base of the bioreactor [(7.4 ± 0.52) × 1010 particles g-1 dry weight] was similar to the average maximum concentration of

TMV-GFPC3 (Day 21 to Day 36) in shake-flask-grown hairy roots [(6.4 ± 0.36) × 1010 particles g-1 dry weight] inoculated using the same concentration of TMV- GFPC3

(Figure 3.80).

In Figure 3.92, a comparison of the concentrations of accumulated virus in the biomass of bioreactor-grown N. benthamiana hairy roots inoculated with TMV and

TMV-GFPC3 is shown. For the purpose of comparison, viral accumulation in hairy roots infected with TMV is expressed as particles per gram dry weight, using the relationship of 2.65 × 1012 particles per milligram of TMV (Section 3.14.1.1). The concentration of virus in the biomass was significantly (p < 0.05) higher in hairy roots from near the base of the bioreactor and from the mesh basket when hairy roots were infected using TMV compared with TMV-GFPC3. All of the mid-height samples inoculated with TMV and TMV-GFPC3 did not differ significantly (p < 0.05) in concentration of accumulated virus. TMV-GFPC3-infected hairy roots from the top of the bioreactor accumulated significantly (p < 0.05) higher concentrations of virus than similarly-positioned TMV-infected hairy roots.

328

1.6 ) 12 1.4 1.2 1.0 0.8 dry weight ×10 weight dry

-1 0.6 0.4

Concentration of virus Concentration 0.2

(particles g 0.0 t t t ll op se ke e ke a T Ba s sk s r w ba ba ba to h h ac es esh es re M m e m bio the th e o d th t t an to en ll nt ac a e dj r w jac t a cto ad gh ea t ei or igh -h bi he id n d- M ee Mi etw t b igh he d- Mi

Figure 3.92 Comparison of the concentration of virus accumulated in hairy roots from different regions of the bioreactors infected with TMV or TMV-GFPC3. () Hairy roots infected with TMV; () hairy roots infected with TMV-GFPC3. For roots infected with TMV, the error bars indicate standard errors for triplicate samples from duplicate bioreactors. For roots infected with TMV-GFPC3, the error bars indicate standard errors for triplicate samples from a single bioreactor.

3.15.3.2 Viral accumulation in the medium

Results for the concentration of virus in the culture medium when N. benthamiana hairy roots were infected with TMV or TMV-GFPC3 in bioreactors are shown in Figure

3.93. The concentration of TMV is expressed as both milligrams per milliliter and viral particles per milliliter using the relationship of 2.65 × 1012 particles per milligram of

TMV (Section 3.14.1.1).

329

5.0 1.8 4.5 1.6 4.0 )

9 1.4

3.5 ) -3

× 10 1.2

-1 3.0 × 10

1.0 -1 2.5 2.0 0.8

0.6 (mg mL

Concentration of Concentration 1.5 (particles mL TMV and TMV-GFPC3 and TMV-GFPC3 TMV 1.0 0.4 of TMV Concentration 0.5 0.2 0.0 0.0 0 5 10 15 20 25 30 Time (days)

Figure 3.93 Concentration of virus in the culture medium when hairy roots were infected with virus in bioreactors. () TMV; (▲) TMV-GFPC3. For cultures inoculated with TMV, the error bars indicate standard errors for duplicate samples from duplicate bioreactors. For cultures inoculated with TMV-GFPC3, the error bars indicate maximum errors for duplicate samples from one bioreactor.

As observed in shake flasks (Figure 3.40), when TMV was added to the bioreactors, a rapid decrease in the concentration of TMV in the medium was observed. After the initial decrease, the concentration of TMV remained low and relatively constant for the remainder of the culture period. The average amount of TMV in the medium 31 days after culture initiation was equivalent to approximately (10 ± 0.58)% of the virus added to the medium as inoculum.

As observed in shake flasks (Figure 3.81), when TMV-GFPC3 was added to the bioreactor, only a small initial decrease in the concentration of medium virus was

330 observed. The amount of TMV-GFPC3 in the medium 31 days after culture initiation was equivalent to approximately 48% of the virus added to the medium as inoculum.

3.15.4 GFP expression in bioreactor grown hairy roots inoculated with

TMV-GFPC3

GFP was not detected in the culture medium (Day 0 to Day 31) or the hairy root biomass (Day 31) of bioreactor cultures infected with TMV-GFPC3.

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CHAPTER 4 – DISCUSSION

4.1 Accumulation of Virus in Hairy Roots and Suspended Cells

Examinations of viral accumulation in cultured cells were performed using Nicotiana tabacum var. Hicks and N. benthamiana suspended cell and hairy root cultures. Both

N. tabacum var. Hicks and N. benthamiana are permissive hosts for tobacco mosaic virus (TMV) and systemic viral infections can be established in both plants. However

N. benthamiana is hyper-susceptible to infection with tobamoviruses (Yang et al.,

2004) and the heterologous protein concentrations obtained using some tobamovirus- based-vectors have been reported to be higher when N. benthamiana rather than

N. tabacum was utilised as a host plant (Fitzmaurice, 2002).

For both species, it was observed that viral accumulation was superior in hairy root cultures compared with cell suspensions (Table 3.1). Comparisons of viral accumulation between N. tabacum hairy root (Figure 3.8) and suspension cultures

(Figure 3.4) were not straightforward because of infrequent sampling and because the virus:biomass ratio at inoculation was not consistent between investigations.

N. benthamiana hairy root clones were identified in which significantly higher concentrations of virus were accumulated compared with N. tabacum hairy roots

(Figure 3.15). N. benthamiana hairy roots were selected for further investigation because TMV accumulation levels were at least 24-fold higher than in the other cultures tested (Table 3.1). N. benthamiana hairy roots were also the only culture type in which the total accumulated virus was observed to exceed the amount of inoculum virus added to the cultures (Figure 3.37).

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A caveat on these findings is that multiple N. benthamiana hairy root clones were screened for their ability to accumulate virus whereas the N. tabacum hairy root and suspension cultures and N. benthamiana suspension culture examined were chosen because they exhibited advantageous growth characteristics. It is possible, therefore, that with further screening, N. tabacum and N. benthamiana cultures could be identified with improved viral accumulation characteristics. This may be particularly so for suspension cultures, where culture morphology has been identified previously to influence virus accumulation (Beachy and Murakishi, 1973).

4.2 Viral Accumulation in N. benthamiana Hairy Roots

N. benthamiana hairy roots initiated from virus-free plant material were able to become infected with TMV and the virus was able to replicate and accumulate to moderate levels within the roots (Figure 3.37). Infection of hairy roots was achieved by co-incubating roots with virus in liquid growth medium. Intentional injury of hairy roots was not required (Figure 3.32), although the division of the root mat to obtain the root inoculum would have resulted in considerable root injury.

When N. benthamiana hairy roots were inoculated by co-incubation with TMV at a concentration of 1.5 μg mL-1, at culture times greater than 12 days post culture initiation both the average amount of virus in the biomass and the amount of virus accumulated in the biomass in a majority of the replicate cultures exceeded the amount of virus added in the inoculum (75 μg TMV) (Figure 3.37). An increase in the amount of virus in the hairy roots above the level added as inoculum is indicative of viral multiplication. The average maximum amount of TMV accumulated in the hairy root biomass,

0.86 ± 0.14 mg (Day 21 to Day 36), was approximately 3000-fold higher than the

333 amount of virus associated with the biomass 23 hours after virus addition to the cultures and 11-fold higher than the amount of inoculum virus added.

4.2.1 Kinetics of TMV accumulation in N. benthamiana hairy roots

The infection methodology by which 0.2 g fresh weight of N. benthamiana hairy roots from 21-day-old cultures were infected by co-incubation with 75 μg TMV in 50 mL

Gamborg’s B5 medium was used as the standard infection method throughout this investigation. When this method was applied, a relatively high proportion of inoculated cultures developed active infections (Sections 3.10.2.3 and 3.11.2.2) and virus accumulation per microgram of inoculum virus was efficient (Figure 3.60). Within this section, the kinetics and other aspects of viral accumulation within hairy roots will be discussed.

Comparisons of virus accumulation patterns in N. benthamiana hairy root cultures with those reported for virus-infected plant cell suspension and callus cultures are limited by the differing methodologies used to measure viral accumulation. When virus accumulation in suspension and callus cultures was monitored and quantified by the incorporation of [H3]-uridine into the viral genome and/or the incorporation of

[C14]-leucine into the viral coat protein (Beachy and Murakishi, 1973; Pelcher et al.,

1972; Wu and Murakishi, 1979), direct comparisons could be readily made with patterns of viral accumulation determined by Enzyme Linked Immunosorbent Assay

(ELISA) (Section 2.17.4.1) as all methods provide a quantitative measure of the amount of virus or viral constituents. However, in previously published studies, viral accumulation in callus and suspension cultures was more frequently monitored by quantifying infectious virus using local lesion assays, with the results generally

334 presented as the concentration of infectious virus in cell extracts and medium

(Murakishi et al., 1971; White et al., 1977; Wu et al., 1959, 1960; Wu and Murakishi,

1978). These results can be compared with infectivity but not relative infectivity data determined for virus accumulated in N. benthamiana hairy root biomass and medium

(Section 2.17.4.4). Comparisons between trends observed for infectious virus concentration and total virus concentrations determined using ELISA can be made; however, as only a proportion of total virus is infectious and that proportion changes throughout the culture period (Figure 3.42), divergence in trends would be expected.

Culture infection and the kinetics of viral accumulation will be discussed in four sections:

• Inoculum virus in the medium

• Association of inoculum virus with the biomass

• Virus accumulation in the hairy root biomass

• Virus accumulation in the culture medium

4.2.1.1 Inoculum virus in the medium

Hairy root inoculation by co-incubation was achieved by adding inoculum virus directly to medium in Pyrex Erlenmeyer flasks. Inoculum roots were added to the medium prior to virus addition. A rapid and substantial decrease in the amount of virus in the medium was observed when TMV and deactivated TMV were added to medium containing hairy roots (Figures 3.17 and 3.40). Decreases in detectable virus in the medium were not accounted for by an equivalent association of TMV or deactivated

TMV with the hairy root biomass. The maximum amounts of virus (infectious or deactivated) associated with the biomass within the first 12 hours after inoculation

335 accounted for less than 0.5% of the inoculum virus (Figures 3.17 and 3.61A) although only 20% of the inoculum virus was detected in the medium (Figures 3.17 and 3.40).

Comparable decreases in medium virus were observed when inoculum virus was added to sterile plant culture media and 0.01 M phosphate buffer in Pyrex Erlenmeyer flasks

(Figure 3.50), indicating that plant-produced proteases were not responsible for virus depletion from the medium.

Thomas and Warren (1994) reported that TMV exhibited long-term stability in sterile modified MS medium as measured by infectivity. In the current work, the infectivity of

TMV in various media was observed to be relatively stable (Figure 3.51); however, the rapid depletion of virus from the medium occurred prior to sampling and it is possible that the stability of virus reported by Thomas and Warren (1994) and observed in this work represented the stable retention of residual inoculum virus in the medium.

Poor retention of proteins in plant culture medium has been reported previously.

Protease activity (Kwon et al., 2003b; Lee et al., 2002; Sharp and Doran 1999, 2001a;

Shin et al., 2003; Terashima et al., 1999b), protein instability (James et al., 2000;

LaCount et al., 1997; Tsoi and Doran, 2002; Wahl et al., 1995), protein insolubility or aggregation (Sharp and Doran, 2001a) and surface adsorption of proteins (Doran,

2006a; Magnuson et al., 1996; Sharp and Doran 2001a) have been suggested and/or demonstrated to be responsible. The mechanism(s) contributing to inoculum virus depletion from plant culture media were not identified. However, the depletion of virus from sterile medium free of hairy root biomass indicates that the loss was not due to association of virus with the hairy root biomass, nor degradation of virus by root- released proteases. Plant proteases co-purified with the inoculum virus could have

336 contributed to virus degradation; however, as significant reductions in virus concentration were observed when low concentrations of purified virus were incubated in sterile storage buffer in Erlenmeyer flasks (Figure 3.50) but reductions in the concentration of viral stock preparations (Section 2.5.2) were not observed upon storage (data not shown), virus degradation by co-purified protease appears unlikely.

Rapid reductions in the amount of virus in the medium were most likely caused by the adsorption of inoculum virus to flask surfaces: to confirm this, desorption of virus from flask surfaces would need to be demonstrated. Significant levels of non-specific adsorption of baculovirus to the surfaces of tissue culture plates have been reported

(Dee and Shuler, 1997).

4.2.1.2 Association of inoculum virus with the biomass

In short-term studies (0–12 hours), when hairy roots were exposed to deactivated virus, the concentration of virus associated with the biomass initially increased rapidly

(0–2 hours), and then appeared to plateau (Figure 3.18). Examination over a longer time period (Figure 3.19C) indicated that the concentration of deactivated virus associated with the biomass did not increase significantly (p < 0.05) above the level observed 2 hours post-infection. The behaviour of infectious virus in culture medium and its interaction with hairy roots were assumed to be similar to those of deactivated virus, although possible alteration of the TMV coat protein during UV inactivation

(Gibbs and Harrison, 1976) could have resulted in differing behaviours. When hairy roots were inoculated with infectious TMV, because interactions between roots and virus were assumed to be similar to those observed for deactivated virus, the apparent plateaux in the concentrations of root-associated virus observed within 12 hours of inoculation (Figures 3.54 and 3.61B) were assumed to represent the saturation

337 concentration of inoculum virus associated with the biomass. The time interval over which the plateau concentration of infectious inoculum virus was determined was not extended beyond 12 hours post-infection to ensure that significant amounts of progeny virus would not be included in the plateau concentrations as virus multiplication has been observed within 24 hours of infection in protoplasts (Harrison and Mayo, 1983;

Takebe and Otsuki, 1969; Wu and Shaw, 1996) and suspended cells (Murakishi et al.,

1971; Pelcher et al., 1972).

The relationship observed between the average maximum concentration of inoculum virus associated with the roots within 12 hours and the average maximum concentration of virus accumulated after culture of the biomass (Figures 3.59 and 3.66) indicates that the concentration of inoculum virus associated with the biomass during the apparent adsorption “plateau period” may be proportional to the level of initial cell infection

(primary cell infection). Nevertheless, the concentration of biomass-associated virus observed during the plateau period is probably much higher than the concentration of virus entering the cells and initiating infection within the hairy roots. Halliwell and

Gazaway (1975) determined that, to achieve 100% infection of suspended plant cells, the entry of 620 viral particles per cell was required. It is probable that only a small proportion of the inoculum virus associated with the hairy root biomass was intracellular due to the difficulties of virus entry into walled cells, and that only a small proportion of intracellular virus particles initiated infection. The possibly small proportion of inoculum virus that entered cells and initiated infection may have been undetectable by ELISA (Section 2.17.4.1) due to rapid uncoating of viral particles after entry into cells (Section 1.6.3).

338

Virus infection of suspension cultures occurs rapidly after culture exposure to virus

(Murakishi et al., 1971), with virus thought to enter cells through the transient breaks in cell walls caused by the rupture of plasmodesmata. Infection of hairy roots also appears to occur relatively rapidly after culture exposure to inoculum virus. Inoculum virus removal 16 and 23 hours after addition did not result in significant reductions in the average maximum concentration of virus accumulated in the hairy roots (Figures 3.48B and 3.53B), indicating that a majority of infection events initiated by inoculum virus

(primary infection events) occurred prior to 16 hours post-inoculation. It was assumed that entry of virus into the roots occurred via injured root cells; however, the nature of the injuries through which infection occurred (i.e. injuries to root hairs through mixing or subculture trauma) and the availability of suitable injuries throughout the culture period are unknown.

4.2.1.3 Virus accumulation in the hairy root biomass

In suspension cultures of N. tabacum L. var. Havana 38 inoculated with TMV, viral replication and accumulation have been reported to occur within 24 hours of infection

(Murakishi et al., 1971; Pelcher et al., 1972). Small increases in virus concentration were detected in the biomass of these cultures within 24 hours of infection and, because excess inoculum virus was removed from cultures soon after infection (Murakishi et al.,

1970) and in some investigations virus quantification methods that differentiated between inoculum and cell-culture-synthesised virus were used (Pelcher et al., 1972), the increases could be attributed to viral multiplication. In the present work

N. benthamiana hairy roots were inoculated with TMV by co-incubating roots with inoculum virus (Section 2.9). When the inoculum virus was removed from culture medium 16 and 23 hours after addition, the amount of virus in the biomass 3 days after

339 infection was not significantly (p < 0.05) different from the amount of virus associated with the biomass shortly after inoculum virus removal (Day 1) (Figures 3.48A and

3.53A) and because the virus detection method used (ELISA against viral coat protein:

Section 2.17.4.1) does not differentiate between inoculum and root-synthesised progeny virus, the accumulation of root-synthesised virus in the biomass during the first three days of the culture period could not be confirmed. When inoculum virus was retained in the medium for the duration of the experimental period, significant

(p < 0.05) increases in the amount of virus in the biomass were observed during the first three days of the incubation period; however, because similar significant (p < 0.05) increases were also observed when cultures were inoculated with deactivated inoculum virus, the increase could not be attributed to the replication of virus in the roots.

From Day 3 to Day 12, the amount of virus accumulated in the hairy root biomass increased exponentially (Figure 3.39B). For most of this period (Day 3 to Day 9), hairy root growth was also exponential (Figure 3.34B). The concentration of accumulated virus in the biomass was frequently, although not always, observed to increase exponentially during this period (Figures 3.39B and 3.69B). Exponential increases in the concentration of accumulated virus were only observed if the specific rate of virus accumulation exceeded the specific rate of biomass growth. The concentration of infectious virus (Figure 3.43) also increased rapidly during this period, indicating that the increased concentration of virus observed using ELISA was due at least in part to the accumulation of full-length infectious viral particles.

From Day 12 to Day 21, the amount of virus accumulated in the hairy root biomass continued to increase and hairy root growth also continued; however both occurred with

340 reduced specific rates compared with those observed before Day 12 (Figure 3.39B).

The concentration of virus accumulated in the biomass continued to accumulate exponentially during this period (Figure 3.39B). The concentration of infectious virus also increased (Figure 3.43); however the relative infectivity of virus, which had previously remained relatively constant, began to decrease from Day 15 (Figure 3.42).

Factors contributing to the decreased specific accumulation rate of TMV after Day 12 have not been confirmed; however the reduction in the rate of hairy root growth as roots entered the decelerated growth phase (Day 9 to Day 21) (Figure 3.34B) was closely followed by a reduction in the accumulation rate of TMV (Day 12 to Day 15), indicating that the reduced rate of viral accumulation may have been associated with reduced rates of root growth. Decreased rates of protein and RNA synthesis in the hairy roots associated with nutrient and in particular oxygen limitation may have resulted in reduced rates of viral multiplication.

From Day 21, the amount and concentration of virus in the biomass generally did not increase significantly (p < 0.05) (Figure 3.39A) and the hairy root biomass remained relatively constant (Figure 3.34A). The relatively constant levels of virus observed are consistent with a cessation, or only very low levels, of viral replication in cells that, as a result of nutrient or oxygen limitation, were no longer growing rapidly. Although the amount of virus in the aging cultures remained high, the decrease in the concentration of infectious virus (Figure 3.43) and the low relative infectivity of virus (Figure 3.42) during this period also indicate that the ability of the virus accumulated in the hairy roots to initiate new infections was declining.

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An association between hairy root growth and viral accumulation was observed in this investigation, with maximum rates of viral accumulation coinciding with the period of exponential root growth and decreases in root growth rate followed by reductions in the rate of viral accumulation (Figure 3.39). As viral replication requires the use of host cell replication and protein synthesis machinery, the consistency of the relationship between hairy root growth and viral accumulation may reflect the availability of nucleotides and amino acids within the root cells. Although virus appears only to accumulate in actively growing hairy roots (Figures 3.39 and 3.68), the virus yields observed in hairy root cultures may not have been significantly limited by the cessation of hairy root growth. When TMV-infected hairy roots were transferred to fresh medium, the concentration of virus in the biomass did not continue to increase

(Figures 3.69 and 3.74). These results indicate that while virus accumulation appears to occur only in actively growing hairy roots the cessation of viral accumulation in newly infected hairy root cultures may have been independent from the cessation of root growth.

A relationship between virus doubling and increased biomass was also observed in hairy root cultures (Figure 3.38). Shortly after culture initiation, when cultures were in the lag and early exponential phases of growth, the amount of virus in the biomass doubled with every 0.015 g increase in biomass dry weight. For the remainder of the culture period, a first order relationship between virus accumulation and biomass was observed, with virus doubling with every 0.063 g increase in biomass. The significance of the constant virus doubling with biomass growth is unclear; however a correlation between viral accumulation and biomass growth is evident.

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There are similarities between the pattern of TMV accumulation observed in

N. benthamiana hairy root cultures and those reported for TMV in suspension and callus cultures of a permissive strain of tobacco, N. tabacum L. var. Havana 38

(Murakishi et al., 1971; Pelcher et al., 1972; White et al., 1977) and for southern bean mosaic virus (SBMV) in callus and suspension cultures of Glycine max var. Harosoy 63

(White et al., 1977; Wu and Murakishi, 1978) (Section 1.9.2.1). A single peak in virus accumulation was observed in N. benthamiana hairy roots that was similar to the single accumulation peak reported to occur in N. tabacum L. var. Havana 38 and Glycine max var. Harosoy 63 suspensions infected with TMV and SBMV, respectively.

In TMV-infected N. tabacum L. var. Havana 38 suspension cultures rapid increases in viral particle (Day 1 to Day 5) (Pelcher et al., 1972) and infectious virus (Day 0.5 to

Day 7) (Murakishi et al., 1971) concentrations were observed as two different phases of accumulation: an initial very rapid phase followed by a phase of slower accumulation.

In N. benthamiana hairy roots infected with TMV the rapid increase in viral particle concentration (Day 3 to Day 21: Figure 3.39) and infectious virus concentration (Day 6 to Day 21: Figure 3.43) was observed as one phase, although it is possible that an additional phase analogous to the initial very rapid phase of viral accumulation reported in N. tabacum L. var. Havana 38 suspensions occurred in N. benthamiana hairy roots between Day 0 and Day 3, but was undetected because of the high background concentrations of root-associated but not infecting inoculum virus and the low sensitivity if the virus detection methods used.

The total viral accumulation phase observed in N. benthamiana hairy roots was lengthened compared with that in N. tabacum L. var. Havana 38 suspensions. In

343

N. benthamiana hairy roots maximum yields were observed 21 days post-infection, compared with 6 to 8 days (Murakishi et al., 1971) and 5 days (Pelcher et al., 1972) post-infection in N. tabacum suspensions. The lengthening of the viral accumulation phase observed in root cultures may have been attributable to greater nutrient availability and the relatively long growth phase (Day 3 to Day 24) of the root cultures

(Figure 3.34A). Neither culture growth kinetics nor doubling times were reported for virus-infected N. tabacum L. var. Havana 38 suspension cultures; however, the availability of sugar in the hairy root cultures was higher than that reported for the

N. tabacum suspensions and the root inoculum was also relatively small (Table 4.1).

Extended growth (Figure 3.10A) and viral accumulation phases (Figure 3.11A) were also observed for N. benthamiana suspension cultures infected with TMV.

Table 4.1 Culture parameters for TMV-infected cell cultures

Culture Inoculum Available Total Reference biomass sugar (g) inoculum (g fresh weight) virus (μg) N. tabacum 0.3 0.09 36 Murakishi et al., suspension 1971 N. tabacum 4.0 0.6 450 Pelcher et al., suspension 1972 N. tabacum 1.0 0.6 45 White et al., suspension 1977 Daucus carota 0.25 0.3 195 Warren and Hill, suspension 1989 N. benthamiana 0.2 1.5 75 This work hairy roots N. benthamiana 1.1 1.29 225 This work suspension

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4.2.1.4 Virus accumulation in the culture medium

In the medium of cultures from which the inoculum virus was removed hairy-root- produced virus was first detected 9 days after culture initiation (Figure 3.49). The amount of virus in the medium increased exponentially from Day 9 to Day 21 and then remained relatively constant for the remainder of the experimental period. The period of TMV accumulation in the medium (Day 9 to Day 21) coincided with the period of viral accumulation in the biomass (Figure 3.48). The average maximum amount of virus in the medium (Day 21 to Day 36) was only 1.1% of the average maximum amount of virus accumulated in the biomass. In comparison, the average maximum amount of virus in the medium (Day 18 to Day 36) of cultures in which the viral inoculum was retained in the medium for the culture duration was 1.9% of the average maximum amount of virus accumulated in the biomass (Figures 3.49 and 3.48). It is possible that the amount of virus released from the hairy roots into the medium was larger than indicated in Figure 3.48, but that ongoing virus instability resulted in the depletion of root released virus from the medium (Section 4.2.1.1).

Virus accumulation in the medium during culture of virus-infected protoplasts was reported to roughly parallel the accumulation of virus in the protoplasts and was attributed to the release of virus as a result of protoplast disintegration (Takebe and

Otsuki, 1969). Murakishi et al. (1971) also reported the accumulation of small amounts of virus in suspension culture medium; however in suspension cultures, virus only began to accumulate in the medium as the maximum virus titre in the biomass was approached, and the titre of virus in the medium continued to increase as suspended cells lost their integrity and the viral titre in the aging biomass decreased. Even so, the

345 maximum virus titre in the medium represented only 0.15% of the maximum titre in the biomass (Murakishi et al., 1971).

The release of virus from hairy roots into the medium may have occurred as a result of cell lysis. However, the detection of root-produced virus in medium from rapidly growing cultures (Day 9) (Figure 3.47) indicates that, at least initially, the release of virus from hairy roots may have resulted from mechanical injury to the roots rather than age-related lysis. It is possible that a mechanism other than cell lysis contributed to virus entry into the medium. Protein can be secreted from hairy roots into the culture medium; however, assembled TMV particles are significantly larger than the largest recombinant proteins demonstrated to be actively secreted from hairy root biomass

(Sharp and Doran, 2001b). As TMV is able to cross the cell membrane and wall during cell-to-cell movement (Section 1.6.4.2), virus entry into the medium may be able to occur by a yet unidentified mechanism.

The low proportion of virus released from the roots would limit the feasibility of purifying viral particles directly from culture medium. However in viral vector systems where recombinant proteins are produced in free cytosolic form, the addition of appropriate signal sequences could facilitate protein secretion, allowing the purification of the recombinant protein directly from the medium.

4.2.2 Hairy root growth and viral accumulation

At the level of virus accumulation currently achieved, the accumulation of TMV in

N. benthamiana hairy roots had no discernable effect on hairy root growth

(Figure 3.34). This suggests that cell physiology was not detrimentally altered by the

346 cytoplasmic accumulation of virus, and that virus accumulation did not deplete the intracellular pool of nucleotides and amino acids sufficiently to reduce hairy root growth rates. However, as the concentration of virus accumulated in the biomass was relatively low compared to that observed in whole plants (Figure 3.1) and the proportion of root cells involved in the infection may also have been low, it cannot be assumed that growth effects would not be observed if virus yields were to increase. It also cannot be assumed that the effect of genetically transformed viral vectors on culture growth would be similar to those of the parental virus, as some vectors have been reported to initiate more severe symptoms in host plants than the parent virus

(Bendahmane et al., 1999; Hendy et al., 1999). Expression of foreign proteins using viral vectors could reduce growth as a result of the detrimental effects of heterologous protein accumulation on plant cells.

4.2.3 Variability in viral accumulation within cultures

Variability in the concentration of accumulated virus was observed within individual

N. benthamiana hairy root mats infected with TMV by co-incubation. Hairy root inocula used to initiate N. benthamiana hairy root cultures in shake flasks contained a large number of roots which, although obtained from 21-day-old hairy root cultures, would have been morphologically and developmentally heterogeneous. Within

Erlenmeyer flasks incubated with orbital shaking growth of hairy roots occurs predominantly from the generally clumped, centrally located inoculum roots either; from existing primary root tips or from newly formed lateral roots (Figure 3.33).

Newly formed roots rapidly fill the base region of the flasks. Plagiatropic growth also occurs, resulting in the formation of aerial roots. As cultures age, extensive lateral root formation and interweaving of roots occurs, resulting in the formation of dense root

347 mats. Long roots that wind around the inner circumference of the vessels are frequently observed. Roots of varying age are found throughout the root mat, although inoculum roots are primarily located near the radial centre.

When mature root mats were divided into three concentric regions, the concentration of accumulated virus within the different concentric regions in individual root mats was generally observed to decrease with increasing distance from the centre of the root mat

(Figure 3.45 and Section 2.15.1). The concentrations of virus in the outer concentric region were 1.7–2.5-fold lower than in the inner concentric region. The mechanisms by which virus became distributed throughout the hairy root mat were not determined, but virus movement probably occurred primarily via cell-to-cell movement from cells with a primary infection to adjacent non-infected cells in a cascading manner. However, assuming cell-to-cell movement in N. benthamiana hairy roots occurred at a rate no greater than that observed in N. benthamiana leaf cells (25 μm h-1; Cheng et al., 2000) and movement occurred at this rate during the entire 27 day culture period, the maximum lineal progression of the infection front would be approximately 1.6 cm from the site of primary infection. As the radius of the root mat was approximately 4.2 cm, cell-to-cell movement alone would probably be insufficient to account for TMV movement into the outer concentric regions of the root mat. Initiation of new sites of primary infections by inoculum virus retained in the culture medium or by released root-produced virus, combined with subsequent cell-to-cell movement, could facilitate dissemination of virus throughout the root mat. Additionally, the presence of vascular tissue within mature roots in liquid culture (Butcher and Street, 1964; White, 1936) could possibly allow a limited form of long-distance movement of virus to occur within the hairy roots, although the lack of transpirational driving force in liquid-grown roots

348 would limit the rate at which virus could move through the vascular tissue. The relatively low concentrations of virus accumulated in the outer concentric regions of the root mat indicate that on-going initiation of new infection sites as a result of new primary infection events or long-distance viral movement does not occur at a level that results in uniform concentrations of virus accumulating throughout the biomass.

Although the results for the distribution of virus in the different concentric regions of the root mat indicated that TMV was able to move within the hairy roots, albeit in a somewhat limited manner, when root mats were divided into radial segments the concentration of virus within different segments of the same root mat varied considerably (Figure 3.46). Inhibition of viral accumulation, poor viral movement characteristics and uneven distribution of primary viral infection events could possibly result in heterogeneous viral distribution within different roots and between different roots. However, it is unclear whether these factors could have contributed to the uneven viral distribution pattern observed in radial segments from the root mats, and it is possible that non-uniform accumulation may have been caused by an uncontrolled abiotic factor.

Uneven distribution of virus in the hairy root mat may be attributable to the inhibition of TMV replication in roots. Valentine et al. (2002) observed that when normal roots of N. benthamiana seedlings were infected with TMV-GFPC3 (30B-GFPC3), mature lateral roots were able to become infected with virus, but that virus accumulation in developing lateral primordia and lateral roots developed from these primordia was inhibited by a gene-silencing-type mechanism. MacFarlane and Popovich (2000) also reported that the distribution of TMV-GFP (30B-GFP) in plant roots was patchy; this

349 was attributed to poor virus movement and the failure of virus to invade meristematic regions in the roots. Hairy roots used in this investigation were clonal; however variation in the type and position of root injury or root breakage during the preparation of the inoculum and during culture could have resulted in considerable variability in the morphology of individual roots, particularly with regards to lateral root formation (Falk and Doran, 1996; Kino-Oka et al., 1999). As hairy roots exhibit a high degree of lateral branching, inhibition of viral replication in lateral roots could have resulted in variable distribution of virus within different roots in the root mat and also reduced the potential viral yield. If gene silencing in developing lateral roots was responsible for the uneven

TMV distribution observed between radial segments, a more uniform distribution may be achieved by co-infecting cultures with viruses that are able to suppress gene silencing (Valentine et al., 2002). Alternatively, tobraviruses such as tobacco rattle virus that are able to move efficiently within roots and infect root tips (MacFarlane and

Popovich, 2000) could be used to infect hairy root cultures and possibly result in more uniform accumulation.

Uneven distribution of virus could also be influenced by uneven initial (primary) virus infection levels and the ability of virus to replicate in the inoculum roots. Inoculum root heterogeneity with regard to morphology, injury levels and age may result in an unequal susceptibility to infection and therefore distribution of primary infection events in inoculum roots. Differing levels of primary infection and subsequent rates of viral replication, particularly if infection levels were generally low, may have significantly affected the final amount of virus within individual roots. Roots developing from inoculum roots which were not infected by virus or displayed only low levels of initial virus infection may have continued to show low levels of viral accumulation.

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Detailed examination of viral distribution in individual hairy roots and hairy root mats using labelled virus would be required to identify the causes of uneven distribution and develop techniques to reduce variability.

4.2.4 Variability in viral accumulation between cultures

Considerable variability in the yield of virus accumulated in the biomass of replicate hairy root cultures was observed (Section 3.9.3.1 and Figure 3.37). The variability in yield was most evident from Day 15, but occurred throughout the experimental period.

The viral yield between replicate N. benthamiana suspension cultures was also variable

(Figure 3.11A); however the average coefficient of variability (Day 0 to Day 36) for suspension cultures (31%) was lower than that for hairy roots (66%) indicating that the variability in viral accumulation between replicate suspension cultures was lower than between replicate hairy root cultures. The observed variability in virus yield between hairy root cultures may reflect the uneven distribution of virus in the root mat (Section

4.2.3) and the use of only a fraction of the root mat for analysis of viral levels.

However, as viral yields between replicate cultures varied considerably prior to Day 15 when the proportion of the root mat used for virus quantification was relatively large

(Section 2.9), and because moderate variability was also observed between replicate suspension cultures, it is unlikely that uneven virus distribution in the root mat alone was responsible for the observed results. Variability in virus yield has been reported previously in some transiently infected suspension cultures (Wu et al., 1960), and was attributed to culture heterogeneity caused by sub-optimal culture conditions (Warren and Hill, 1989). Variability in the virus yield in hairy root cultures could be influenced by variation in infection or accumulation efficiency due to uncontrolled abiotic or biotic factors.

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4.2.5 Particle length and infectivity

The concentration of TMV determined using ELISA (Figure 3.39) and the concentration of infectious virus (Figure 3.43) in TMV-infected hairy root cultures were observed to increase for the first 21 days of the culture period. Particle size analysis performed on sap from hairy root cultures 15, 20 and 28 days post-infection indicated that full-length TMV accounted for a majority (74–76%) of viral particles

(Figure 3.44). Only 15–17% of particles were shorter than full length and the length of these particles varied considerably. The high proportion of full-length particles indicated that the increase in virus concentration determined using ELISA represented a true increase in concentration rather than the accumulation of truncated particles as reported by Thomas and Warren (1994) (Section 1.9.2.1). The nature of the small population of truncated particles found in the current work was not examined, but they may have represented particles physically disrupted during extraction (Warren et al.,

1992), packaged sub-genomic RNA or host RNA (Beachy and Zaitlin, 1977; Siegel,

1971), or a mutant sub-population of virus with a length shorter than full length.

Although both the concentration of viral particles and the concentration of infectious virus increased in cultures until Day 21, the maximum fold increase in the concentration of virus (Day 1 to Day 21) determined using ELISA (100-fold) (Figure

3.39) was greater than the maximum fold increase in the concentration of infectious virus (32-fold) (Figure 3.43). Examination of the relative infectivity of accumulating virus (Figure 3.42) showed that, for the first 15 days of the culture period corresponding roughly to the period of exponential culture growth and viral accumulation (Figure 3.39), the infectivity of virus extracted from the hairy roots was similar to that of the standard virus preparation isolated from systemically infected

352 leaves of a permissive host (N. tabacum var. Hicks). However after Day 15, the relative infectivity of virus in the hairy roots decreased significantly (p < 0.05). The reduction in relative infectivity coincided with reductions in hairy root growth rate and the rate of viral accumulation in the biomass.

The reduction in the relative infectivity of virus accumulating in the hairy roots may have been caused by the preferential accumulation of a mutant sub-population of TMV that had a reduced ability to initiate primary infection that is required for local lesion formation, but which retained the ability to accumulate in cells once infection was initiated. Particle length analysis (Figure 3.44) indicates that any numerically significant mutant virus must have been near full length. Schneider and Roossinck

(2000) in an analysis of TMV population diversity reported that sequence diversity developed rapidly in plant-produced TMV, but that over successive subcultures in the same host species mutations did not become fixed, indicating that the introduced mutations did not confer selective advantages on the virus sub-populations. The inoculum virus used in this investigation (Section 2.5.2) would most likely have contained a variety of mutant viruses. It is possible that in N. benthamiana hairy roots, a mutant sub-population present in the inoculum produced in N. tabacum or a mutant virus that developed as TMV replicated in the hairy roots could have had a selective advantage that resulted in the mutant becoming numerically dominant in the hairy roots.

Analysis of viral genome sequences early and late in the root growth cycle would be required to determine if a numerically dominant mutant population had developed.

Additionally, if a numerically dominant mutant population was identified, it would be necessary to confirm that the mutant’s ability to initiate infection was reduced compared with that of the inoculum virus generated in N. tabacum.

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As the majority of virus particles extracted from the biomass were full length, and the reduction in relative infectivity occurred rapidly from Day 15, the reduction in virus infectivity is more likely to be due to the effect of cellular components on either virus infectivity or the susceptibility of test plants to viral infection than virus mutation. A reduction in the infectivity of cherry ringspot virus in cucumber cotyledons similar to that observed in the N. benthamiana hairy roots has been reported and attributed to the accumulation of inactivating compounds in the leaves (Fulton, 1967). Cellular components including oxidised polyphenols (o-quinones) and tannins can inactivate or precipitate virus particles, and specific cellular components can reduce the susceptibility of test plants to infection (Dijkstra and de Jagar, 1998; Fulton, 1967;

Gibbs and Harrison, 1976). Many phenolics and tannins are secondary plant metabolites (Loomis, 1974), and their concentration would be expected to increase in hairy roots as the cultures aged. Reductions in the relative infectivity of virus in extracts from aging roots without concurrent reductions in the concentration of virus is consistent with the accumulation of inhibitory or inactivating phenolic and tannin-like compounds within the roots.

Some interaction between virus particles and either tannins or o-quinones could have occurred during the culture period; however greater interaction probably occurred during sample extraction when intracellular compartments were disrupted. Buffers used for the extraction of virus from the hairy root biomass contained sodium sulphite to limit polyphenol oxidation and bovine albumen and polyvinylpyrrolidone to limit the association of tannins with the virus and the subsequent precipitation of virus.

Nevertheless, as the cultures aged, the potentially higher concentrations of inhibitory and interfering components and the greater time required for extraction of samples with

354 increasing mass (Section 3.9.5.2) may have resulted in increased interaction between virus particles and inhibitory cellular components, resulting in reduced intrinsic viral infectivity.

4.2.6 Virus yield

The concentrations of TMV accumulated in systemically infected leaves from

N. tabacum plants and reported in the literature (1 mg g-1 fresh weight: Gooding and

Hebert, 1967; 16.5–23.5 mg g-1 dry weight: Copeman et al., 1969) are similar to those observed in this work in systemically infected leaf (24 ± 6.6 mg g-1 dry weight) and root

(20 ± 2.9 mg g-1 dry weight) tissue from N. tabacum var. Hicks infected using the same

TMV isolate as used to infect hairy root cultures (Figure 3.1). These results provide confirmation that the TMV isolate used in this work was able to accumulate to high levels within whole plant biomass. TMV coat protein accounted for (27 ± 7.4)% and

(38 ± 5.5)% of total soluble protein in systemically infected leaf and root material, respectively, from N. tabacum var. Hicks. N. tabacum rather than N. benthamiana was used to determine TMV accumulation in whole plants because, although the latter is highly susceptible to viral infection, it is not generally used for the commercial field propagation of TMV or TMV-based vectors due to unsuitable field-growth characteristics (Gleba et al., 2004; Fitzmaurice, 2002).

When N. benthamiana hairy root cultures were infected with TMV by co-incubation using a virus inoculum concentration of 1.5 μg mL-1 (Section 2.9), the maximum concentration of virus accumulated in the biomass (Figure 3.39) was

2.2 ± 0.77 mg g-1 dry weight (0.11 ± 0.039 mg g-1 fresh weight) and the average maximum concentration of accumulated virus (Day 21 to Day 36) was

355

1.6 ± 0.25 mg g-1 dry weight (0.075 ± 0.012 mg g-1 fresh weight). Variation in the concentration of accumulated virus was observed when cultures were inoculated using standard conditions (Section 2.9) and a virus inoculum concentration of 1.5 μg mL-1, but the average maximum concentration of accumulated virus in the biomass averaged across all investigations (1.7 ± 0.34 mg g-1 dry weight) was similar to the concentration reported above. The highest average (Day 18 to Day 36) maximum concentration of virus accumulated in the biomass, 4.2 ± 0.60 mg g-1 dry weight (0.21 ± 0.029 mg g-1 fresh weight), was obtained when the viral inoculum concentration was increased to 9.0

μg mL-1 TMV (Figure 3.57E); however the efficiency of virus accumulation relative to the inoculum virus level was no greater than when an inoculum concentration of 1.5 μg mL-1 TMV was used (Figure 3.60).

In this work, the average maximum concentration of TMV accumulated in hairy root cultures was approximately 5.7 (9.0 μg mL-1 inoculum) –15 (1.5 μg mL-1 inoculum)- fold lower than the concentration of TMV accumulated in systemically infected leaves of N. tabacum var. Hicks infected with the same isolate of TMV. Although the level of virus accumulation in the biomass of hairy roots was lower than that observed in whole plants, viral coat protein accounted for a maximum of (26 ± 10)% of total soluble protein in the hairy roots inoculated using 1.5 μg mL-1 TMV (Figure 3.41), which was not significantly different (p < 0.05) from that observed in the roots and leaves of whole plants (Figure 3.1).

It is possible that the relatively low total soluble protein levels observed in the hairy roots (Figure 3.41) may have limited virus accumulation in the root cultures. The concentrations of total soluble protein in N. benthamiana hairy roots were between

356

2.1- and 8.2-fold lower than those in the roots of N. tabacum var. Hicks (Figure 3.1).

Low protein levels have also been observed in suspension cultures and have been implicated in low viral yield in these cultures (Kassanis, 1957). Gene silencing

(Valentine et al., 2002), which may have limited the dissemination and accumulation of

TMV throughout the hairy root biomass, could also have resulted in low viral yield in hairy root cultures; the effects of gene silencing on virus distribution may be eliminated by careful vector selection (MacFarlane and Popovich, 2000; Valentine et al., 2002).

Production of virus in hairy root cultures does not represent an alternative to whole- plant production of wild-type virus or viral vectors in situations where field production adequately addresses containment and product safety. This is because of the lower viral yields obtained in hairy root cultures and the significantly higher production costs associated with tissue culture production systems compared with field-based agricultural systems. However, if currently attained TMV yields in hairy roots could be replicated using transgenic viruses, foreign protein or vaccine production at levels competitive with those currently achieved using stably transformed plant cell suspension and hairy root cultures (Table 1.1) may be possible.

In Table 4.2, TMV yields observed in N. benthamiana hairy root and suspension cultures in this work and previously reported virus yields in plant suspension cultures are presented. Virus yields in liquid-grown suspension cultures only are shown, as these represent a potential alternative to large-scale hairy root cultures, although viral yields in agar-grown cultures can be higher than in liquid-grown cultures (Murakishi et al.,

1971). Yields are reported per gram fresh biomass to allow total yield comparisons and also per microgram of inoculum virus to indicate the efficiency of accumulation.

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Process viral productivity is also presented in Table 4.2. Inoculation methods can also

affect yield and are listed in Table 1.3. The concentration of TMV accumulated in N.

benthamiana hairy roots is comparable to that achieved previously in some suspension

cultures (Murakishi et al., 1971; Thomas and Warren, 1994; Warren and Hill, 1989).

The efficiency of accumulation per microgram of inoculum virus in N. benthamiana

hairy root cultures was similar to that observed previously in suspension cultures

infected by co-incubation with virus (Thomas and Warren, 1994; Warren and Hill,

1989) but considerably lower than those observed

Table 4.2 Accumulation of virus in plant tissue cultures Culture type Inoculum virus (TMV) Maximum Yield Process Reference Viral Productivity Total Concentration mg g-1 fresh weight mg μg-1 mg (g-1 fresh -1 (μg) mg mL-1 μg g-1 inoculum weight) day medium fresh virus weight biomass N. benthamiana 75 1.5 375 (7.5 ± 1.2) × 10-2* 1.0 × 10-3 * (3.6 ± 0.57) × This work hairy root (Days 21–36) 10-3 b 300 6 1500 0.17 ± 0.036 * 5.5 × 10-4 * (1.1 ± 0.24) × This work (Days 15–36) 10-2 b 450 9 2250 0.21 ± 0.029 * 4.7 × 10-4 * (1.2 ± 0.17) × This work (Days 18–36) 10-2 b N. benthamiana 225 4.5 200 (6.5 ± 0.51) × 10-4* 3.1 × 10-6 * (3.7 ± 0.29) × This work suspension (Days 18–36) 10-5 b N. tabacum L. 36 12 120 0.39 ± 0.09 1.1 × 10-2 (5.6 ± 1.3) × Murakishi var. Havana 38 (Day 7) 10-2 et al., 1971 suspension Daucus carota 195 19.5 780 0.047 (Day 21) 2.4 × 10-4 2.2 × 10-2 Warren and L. suspension Hill, 1989 N. tabacum L. 200 4 800 0.14 (Day 4) 7.2 × 10-4 3.5 × 10-2 Thomas and var. White 0.25 a (Day 14) 1.3 × 10-3 1.8 × 10-2 Warren, 1994 Burley suspension N. tabacum NR NR NR Approximately 2.0 - Approximately Warren suspension (Day 21) 0.10 et al., 1992

* Data represents average maximum concentrations a Reported yield may be falsely high due to the presence of fragmented virus b When virus yield was expressed as average maximum concentration, process viral productivity was calculated using the average maximum virus concentration and the earliest time from which the average concentration was calculated NR Data not reported

358 when cultures were inoculated using vibratory inoculation (Murakishi et al, 1971). The

TMV concentration in the most productive N. benthamiana hairy root cultures tested in the current work was still one order of magnitude lower than the concentration of TMV reported by Warren et al. (1992) in suspended N. tabacum cells. The viral yield in

N. benthamiana suspension cultures is discussed in Section 4.7.

This similarity of viral yields in N. benthamiana hairy root cultures and in N. tabacum and D. carota suspensions indicate that hairy roots could represent a competitive culture-based system for the production of virus, particularly as there is considerable potential for yield increases in the roots by optimisation of infection methods and altering medium composition to favour viral accumulation.

Whereas the average maximum concentrations of accumulated virus observed in hairy root cultures were 5.7–15-fold lower than in the leaves of systemically infected

N. tabacum plants, high concentrations of accumulated virus were found in some individual replicate cultures. In individual cultures inoculated using 1.5 μg mL-1 and

6.0 μg mL-1 TMV, concentrations of accumulated virus as high as 5.0 mg g-1 dry weight

(0.25 mg g-1 fresh weight) (Figure 3.57B) and 23 mg g-1 dry weight (1.1 mg g-1 fresh weight) (Figure 3.57D), respectively, were obtained. These results indicate that, despite the generally low protein concentrations in root cultures, it is possible to achieve high levels of virus in hairy roots. The factors contributing to the high yields of virus in these cultures have not been identified; however if they could be identified and duplicated, a culture system in which virus yields were equivalent to those in whole plants would be obtained. Additional improvements to virus yields in hairy roots may be achieved by applying methods that have resulted in improved yields in suspension

359 cultures. In particular, modifications to the culture medium to favour virus accumulation were not examined in this investigation but, in suspension cultures, were associated with significant increases in yield (Hill et al., 1990). Viral titres in green pigmented suspension and callus cultures have also been reported to be higher than in similar unpigmented cultures (Murakishi et al., 1971), and this approach may be able to be applied to hairy root cultures.

4.3 Modifications to Infection Procedures

A range of modifications were made to the standard inoculation procedures

(Section 2.9) to determine what, if any, effect they had on viral accumulation in hairy root cultures. Modifications were predominantly, although not exclusively, aimed at increasing the level of primary infection within cultures.

4.3.1 Intentional root injury

The mechanism by which TMV enters the cells of plants is not fully defined; however the entry of virus into plant cells is widely considered to occur through breaches in the cell wall and protoplast. Cell injury must be sufficient to allow virus entry into the cell protoplast but must not result in cell death. Accumulation of virus in hairy root cultures inoculated using standard inoculation procedures (Figure 3.39 and Section 2.9) indicates that the inoculation method used resulted in adequate root injury to allow virus infection to occur, although the nature of the injuries through which infection occurred are unknown. The effect of applying intentional root injury prior to infection with TMV was examined to determine if increased virus accumulation was observed

(Sections 2.9.3.2 and 3.8).

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The two methods of intentional injury applied to inoculum hairy roots did not result in accumulation of significantly increased (p < 0.05) concentrations of virus compared with the yields obtained when normal hairy roots were inoculated via co-incubation with virus (Figure 3.32B). These results indicate either that increasing the level of primary infection did not result in increased accumulation or, more likely, that increasing the injury level did not increase the level of primary infection because wounds formed using the injury methods applied were unsuitable or unavailable for infection. It is also possible that the availability of potential infection sites did not limit primary infection (Section 4.3.5).

Vortexing of inoculum roots in the presence of an abrasive, and drawing inoculum roots over sandpaper, may have increased the level of lethal cell injury in hairy root cultures, although not sufficiently to significantly affect (p < 0.05) subsequent culture growth

(Figure 3.30). The type of sub-lethal injury suitable for culture infection by virus may not have been increased by these treatments. Throughout this investigation, it was assumed that primary viral infection of hairy roots occurred through physical injuries to the hairy roots that occurred during the preparation of the root inoculum for subculture; however cell damage through which infection could occur, particularly to root hairs, may have occurred during subsequent root incubation in liquid medium. Alternatively, additional suitable sub-lethal cell damage caused by intentional root injury may have been unavailable as a site of primary viral infection because the cell injury and viral infection phases did not occur concurrently. Murakishi (1968) determined that the time between the dispersion of callus in liquid medium, which would have ruptured the plasmodesmata between some cells (Murakishi et al., 1971), and the exposure of cells to virus affected the virus titre, with high titre observed only when the time separating

361 cell dispersion and exposure was short (less than 10 minutes). Murakishi (1968) concluded that injuries made to callus cells during dispersion were only receptive to virus infection for a short period. Injuries to hairy root cells, although presumably not of the same type as those formed in disrupted suspension cultures, may have only been transiently available for infection. Investigations into viral inoculum concentration

(Section 4.3.5 and Section 3.11.2.2) also suggest that the inoculum concentration used in this investigation (1.5 μg mL-1) may not have resulted in the saturation of available infection sites. If inoculum virus was limiting, even under normal injury levels, further increases in the available infection sites would not have resulted in enhanced infection.

Although intentional injury of hairy roots did not result in increased viral accumulation in this investigation, if the nature of the cell injuries involved in infection could be identified, improved mechanisms of cell injury may be developed that increase the level of suitable injuries for virus infection. Enhanced cell injury, if adequate levels of inoculum virus were provided, may still result in increased yield.

4.3.2 Separation of the initial infection phase and culture growth

To achieve high levels of viral accumulation, both efficient infection and subsequent viral replication are required and these may be best achieved using different medium conditions.

4.3.2.1 Medium exchange

The exchange of culture medium after hairy root infection with virus results in the removal of inoculum virus that would otherwise be retained in the medium. When inoculum virus was removed from suspension cultures by a similar process of medium

362 exchange soon after infection, Murakishi et al. (1971) reported that the virus titre in cultures at a defined time post-infection was not affected, with similar titres observed when the inoculum was removed immediately after addition (0 minutes) and 12 hours after addition, indicating that the infection process in vortex-disrupted cell aggregates occurred rapidly. The removal of inoculum virus from hairy root cultures

16 and 23 hours after addition (Figures 3.48 and 3.53, respectively) did not significantly (p < 0.05) affect subsequent viral accumulation. This indicates that a majority of primary infection events initiated by inoculum virus occurred prior to inoculum virus removal and that the process of inoculum virus removal did not affect virus replication and accumulation. Further investigations would be required to determine the minimum period required for root and inoculum virus co-incubation to achieve optimal hairy root infection prior to inoculum virus removal.

4.3.2.2 Alternative infection media

The use of a specific medium for culture infection could result in increased viral accumulation if the level of primary infection within a culture was increased, for example by increasing the concentration of virus retained in the medium, increasing the infectivity of the virus, or increasing the susceptibility of cells within the roots to viral infection. The accumulation of TMV in N. benthamiana hairy roots was examined when infection was performed in 0.01 M phosphate buffer, pH 7.4. Phosphate buffer was selected as the “medium” for the infection phase because, under general storage conditions, TMV is stable in phosphate buffer and the presence of phosphate in buffers used for the infection of plant leaves with some viruses, including TMV, has been associated with increased lesion formation (Kado, 1972).

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The average maximum concentration of virus accumulated in hairy root cultures infected with TMV during a 23-hour inoculation phase in 0.01 M phosphate buffer was significantly lower (p < 0.05) than that measured in cultures infected with TMV while the roots were incubated in B5 medium, both when the inoculum virus was retained in the medium and when it was removed using medium exchange (Figure 3.53). In addition, only a small percentage (33%) of cultures infected using the phosphate buffer inoculation phase exhibited active infections (Table 3.2). Cultures classified as not having active viral infections may have contained replicating virus, but either a very low level of initial infection and/or subsequent low levels of virus multiplication may have been responsible for low levels of virus accumulation in the biomass. A similar reduction in the concentration of accumulated virus in the biomass and in the percentage of cultures with active infections was observed when hairy roots were infected with virus by co-incubation in Gamborg’s B5 medium, but when the inoculum virus concentration was reduced below 1.5 μg mL-1 TMV (Figure 3.57A), suggesting that a low level of primary infection may have contributed to the observed results.

TMV stability in sterile plant culture media (Gamborg’s B5 medium and MS medium) and 0.01 M phosphate buffer, pH 7.4, was examined using conditions similar to those used for viral inoculation of hairy root cultures (25°C in 250-mL Pyrex Erlenmeyer flasks). The retention of virus was poor in all the media examined (Figure 3.50).

Slightly higher levels of virus retention were observed using 0.01 M phosphate buffer compared with the examined plant culture media, but the differences were only significant (p < 0.05) from 10.5 hours after virus addition and, even in phosphate buffer, only approximately 32% (2–29 hours) of added virus was retained in the medium. The slightly improved retention may be attributable to improved virus

364 stability in the heavy-metal-free environment of phosphate buffer (Tsoi and Doran,

2002); however the use of phosphate buffer may not have reduced protein adsorption to the Pyrex surface of the Erlenmeyer flask, which is possibly a more significant contributing factor to protein instability in plant culture medium (Section 4.2.1.1). The infectivity of TMV in 0.01 M phosphate buffer was generally not significantly different from that in Gamborg’s B5 medium (Figure 3.51). Examination of TMV properties in phosphate buffer and Gamborg’s B5 medium indicated that differences in medium virus retention and infectivity were not large enough to explain the decreased virus accumulation observed in hairy roots infected in phosphate buffer (Figure 3.53). A caveat to these findings is that the infectivity of TMV in the different media prior to the rapid depletion of virus could not be determined.

The low level of virus accumulation and the low percentage of cultures displaying active infections when a phosphate buffer inoculation phase was used may have been contributed to by the nutritional status of the roots at or soon after infection. Hairy root inocula used in this investigation were obtained from 21-day-old cultures that were exhibiting near-stationary growth. The inoculum roots in Gamborg’s B5 medium were susceptible to virus infection and TMV was able to multiply and accumulate in the roots (Figure 3.53). The infection of inoculum roots in Gamborg’s B5 medium would have ensured that the nutrients that were required to enable the repair of cell damage, and to facilitate the rapid synthesis of viral RNA and protein needed for virus replication were available. When infection was performed in phosphate buffer, nutrient limitation would have been maintained, and rates of RNA and protein synthesis in the cells would probably have been low. It is possible that, due to continued nutrient limitation, injuries suitable for viral infection that may have been sub-lethal in non-

365 nutrient-limited cultures were lethal. Lower rates of protein and RNA synthesis may have also resulted in slower rates of virus replication and movement.

Although the use of phosphate buffer as an infection-phase medium did not result in increased viral accumulation, increases in accumulation may be achieved by the use of alternative media which increase culture susceptibility to virus infection or facilitate the retention of infectious virus in the medium.

4.3.3 Effect of hairy root pre-culture age on virus accumulation

Hairy root biomass from batch cultures contains roots of varying age and level of differentiation. The developmental profile of roots within batch-grown cultures changes as the culture matures. Although root inocula were obtained from cultures at defined times post-culture-initiation, the roots within the inoculum were highly heterogeneous with regard to both actual age and developmental status. Because viral accumulation was examined in cultures that were initiated using what could be regarded as a mixed root inoculum, it was not possible to determine which “age” of roots resulted in the highest level of virus accumulation. Nevertheless, cultures that were suitable for use as inocula could be identified and as the growth profile of cultures from which inocula were obtained was known, inferences regarding the susceptibility of inoculum roots to infection at varying times and subsequent viral accumulation could be made.

Viral accumulation in hairy roots was examined in cultures initiated by placing root inocula from 6-day-old cultures (mid-exponential growth phase), 10-day-old cultures

(late exponential growth phase), 14-day-old cultures (early decelerated growth phase),

366 and 21-day-old cultures (late decelerated growth phase) into fresh medium. In hairy root cultures, exponential growth is associated with the formation of a large number of elongating root tips (Payne, 1991) due to lateral root formation. The presence of large numbers of lateral root tips in root inocula from 6- and 10-day-old cultures was indicated by rapid biomass accumulation after transfer to fresh medium (Figure 3.20).

When cultures were initiated using inoculum roots from 14- and 21-day-old cultures, significantly lower (p < 0.05) culture biomass was initially observed (7 days post- inoculation) compared with cultures initiated using hairy root inocula from 6- and 10- day-old cultures (Figure 3.20), suggesting that the older inocula contained lower levels of root prymordia so that new lateral prymordia had to develop in these cultures before the cultures were able to exhibit rapid growth.

Warren and Hill (1989) reported that when carrot suspension cultures initiated using inocula from variously-aged cultures were infected with TMV, the concentration of accumulated virus and the pattern of accumulation were affected by the age of the cell inoculum. The concentration of accumulated virus decreased as the age of the inoculum increased. Clear trends regarding inoculum culture age and viral accumulation were not observed in hairy root cultures. Virus accumulated to low levels in cultures initiated using 10- and 14-day-old inocula and the concentration of virus accumulated in the biomass of these cultures did not increase significantly (p < 0.05) beyond the concentration observed 5 hours after culture infection (Figure 3.21B), indicating that, in general, these roots showed low susceptibility to infection, or that root development subsequent to infection was not conducive to high levels of viral accumulation. When TMV-infected cultures were initiated using inocula from 6- and

21-day-old cultures, the concentration of virus accumulated in the biomass increased

367 significantly (p < 0.05) above the concentration of virus associated with the biomass

5 hours after virus addition to the cultures (Figure 3.21B). Although the maximum concentration of virus in the biomass of cultures initiated using 6- and 21-day-old root inoculum was at least 3-fold higher than the concentration of virus accumulated in cultures initiated using 10- and 14-day-old inoculum roots, due to the variability in viral accumulation between replicate samples within each treatment group, the differences in accumulation were not significant (p < 0.05). During this experiment, sampling frequencies were lower than for the full time-course investigations and, because of the high variability between cultures (Section 4.2.4) the confidence in observed trends is low.

4.3.4 Culture initiation in conditioned medium

Inoculation of hairy roots with virus at the same time as hairy root subculture into fresh

Gamborg’s B5 medium resulted in the infection of roots with virus and the subsequent accumulation of virus in the biomass (Figure 3.39); however irrespective the age of the inoculum roots at the time of culture inoculation, shortly after transfer to fresh medium roots will contain a large number of actively growing cells. Wu et al. (1959, 1960) examined TMV infection and accumulation in N. tabacum × N. glutinosa suspension cultures infected with TMV at various times after culture initiation. The highest virus titres were found when cultures containing predominantly senescent cells were infected with virus; the lowest titres were obtained when cultures containing predominantly meristematic cells were infected with virus. The authors suggested that competition between the synthesis of plant protein and RNA and virus nucleoprotein synthesis in rapidly dividing meristematic cultures resulted in low levels of virus accumulation.

368

The absence of proteins in fresh plant culture medium is considered to be a contributing factor in the loss of secreted recombinant proteins (Doran, 2006b; Tsoi and Doran,

2002) due either to protease degradation (Kwon et al., 2003b; Lee et al., 2002; Sharp and Doran, 1999, 2001a; Shin et al., 2003; Terishima et al., 1999) or adsorption of recombinant proteins to the surfaces of growth vessels (Doran, 2006a). The addition of protein to culture medium has been reported to increase the stability of recombinant proteins in culture medium (James et al., 2000). Protein stability in conditioned plant medium has also been observed to be higher than in fresh plant medium, despite the presence of plant-produced proteases (Sharp and Doran, 2001a). The concentration of total soluble protein in conditioned media from N. benthamiana hairy root cultures increased over the 21-day period examined (Figure 3.22). The composition of the conditioned medium was not examined but it could be expected to contain root- produced proteins and other cellular components such as polysaccharides and tannins that were either actively secreted from the hairy roots or released as a result of cell lysis. Despite the presence of root-produced proteins in the conditioned medium

(Figure 3.22), the stability of TMV was not significantly greater in conditioned media than in fresh protein-free Gamborg’s B5 medium (Figure 3.23). James et al. (2000) did not report the concentration of added protein that was required to increase recombinant protein stability; however, even in conditioned medium obtained from hairy roots 21 days after infection, protein levels were relatively low [(1.3 ± 0.085) × 10-2 mg mL-1 medium] and may have been insufficient to significantly enhance virus stability.

Possible increases in viral stability conferred by increased medium protein concentrations may also have been negated by the effects of other components of the conditioned media.

369

Although TMV stability was not affected by incubation in conditioned media, TMV infectivity was reduced (Figure 3.24). The average infectivity of TMV in conditioned medium irrespective of the age of the culture from which the medium was obtained was

3–4-fold lower than the infectivity of TMV in fresh Gamborg’s B5 medium. Even though concentrations of virus in conditioned and fresh media were not significantly different (p < 0.05), the lower virus infectivity could have resulted in reductions in the number of primary infection events occurring when roots in conditioned medium were co-incubated with TMV. Factors affecting viral infectivity in conditioned media were not examined; however nucleases, tannins and polyphenols would be expected in conditioned media and could have contributed to reduced viral infectivity (Dijkstra and de Jager, 1999; Fulton, 1967; Gibbs and Harrison, 1976).

The concentration of virus accumulated in the biomass of the variously-aged roots inoculated with virus in conditioned medium appeared to be low compared with the concentration of virus observed when similarly aged cultures were inoculated with virus in fresh Gamborg’s B5 medium (Figure 3.27). However, when viral accumulation was compared at discrete times post-infection, viral accumulation in hairy roots that were infected in conditioned medium was not found to be significantly lower (p < 0.05) than in cultures infected in fresh medium. Because viral accumulation between replicate hairy root cultures within treatment groups was frequently observed to be highly variable (Section 3.9.3.1), differences in viral accumulation between treatment groups at discrete times were rarely found to be statistically significant, but significant differences were sometimes observed between the average maximum concentrations of accumulated virus. In this experiment, the average maximum concentrations of accumulated virus could not be determined because samples were taken relatively

370 infrequently. Because the variability between replicate samples was high, even though the culture biomass were significantly lower (p < 0.05) when cultures were initiated using inoculum roots from 10-, 14- and 21-day-old cultures and conditioned media of the same age compared with similarly aged roots in fresh medium (Figures 3.20 and

3.25), the total amount of virus in the biomass was not significantly different than when cultures were initiated using fresh Gamborg’s B5 medium (Figures 3.21A and 3.26).

The susceptibility of roots to infection in conditioned media, independent of subsequent viral replication and accumulation, was not determined in this investigation. However, as virus infectivity was reduced in conditioned medium (Figure 3.24) and hairy root growth was also generally reduced (Figures 3.25 and 3.20), although viral accumulation was not significantly lower, infection of hairy roots in conditioned medium cannot generally be considered advantageous for viral accumulation.

4.3.5 Virus Inoculum concentration

Increasing the concentration of the viral inoculum used to infect hairy root cultures over the concentration range examined (0.75–9.0 μg mL-1 TMV) was associated with significant (p < 0.05) increases in the average maximum concentration of virus accumulated in the biomass (Figures 3.57 and 3.58). Increasing the concentration of inoculum virus was also associated with increases in the percentage of inoculated cultures exhibiting active viral infections (Section 3.11.2.2).

When hairy root cultures are infected with virus via co-incubation, it is probable that primary infections develop in only a sub-set of cells within the root inoculum that are both injured and exposed to inoculum virus. The proportion of cells within the hairy

371 roots susceptible to primary viral infection is unknown. Cells from the root epidermis are exposed to inoculum virus and may be more subject to minor injury due to their position in the roots and therefore more susceptible to primary infection, although other cell types may also be exposed to virus and develop primary infections as a result of significant root injury.

In other virus-infected cell culture systems, including mammalian cells (Jung et al.,

2004), insect cells (Radford et al., 1997; Vallazza and Petri, 1999) and plant protoplasts

(Lesney and Murakishi 1981; Sander and Mertes, 1984), increasing the inoculum virus concentration within a certain range was associated with an increasing proportion of cells developing primary viral infections. In animal cell systems, each cell is assumed to be able to accumulate a finite amount of virus that is defined by the environmental conditions, so that virus yields are proportional to the number of infected cells in the culture (Jung et al., 2004; Radford et al., 1997). Each virus-infected plant-cell protoplast has also been reported to accumulate virus at a similar concentration, although the culture conditions can affect the concentration to which the virus accumulates (Huber et al., 1981). Hairy roots are a differentiated tissue and contain a variety of different cell types. It is unlikely that the amount of virus accumulated in different cell types is uniform, but if it is assumed that virus accumulates in each infected cell type at a relatively uniform maximum concentration, the increasing concentration of accumulated virus in hairy root cultures when the concentration of inoculum virus was increased (Figures 3.57 and 3.58) could be consistent with increasing numbers of virus-infected cells when virus accumulation ceases within the culture (Figure 3.39 and Section 4.2.1.3).

372

Examination of virus distribution in hairy root cultures (Figures 3.45 and 3.46) and viral accumulation in hairy root cultures after inoculum virus removal (Figure 3.48) indicate that TMV was able to move from cells with primary infections to other cells within the root mat; however, the uneven distribution of virus (Section 4.2.3 and

Figure 3.46) when the inoculum concentration was 1.5 μg mL-1 indicates that, when primary infection levels were relatively low, the mechanisms that facilitated viral movement within the root may have been insufficient to result in full viral dissemination throughout the root mat during the growth period. The increasing concentration of accumulated virus observed when the inoculum concentration was increased could result from an increased percentage of cells within a culture becoming infected as a result of viral dissemination from an increased number of primary infection sites. Increasing primary infection levels may have resulted in an increase in the total number of virus-infected cells within a culture and therefore increased virus yield.

As the inoculum concentration was increased from 0.75 μg mL-1 to 9.0 μg mL-1 TMV, the percentage of replicate cultures displaying active infections increased from 53% to

94% (Section 3.11.2.2). This may also be attributable to an increase in the level of primary infection in cultures as the inoculum concentration was increased. The threshold viral concentrations used to define active infection (Section 3.10.2.3) were arbitrary and were selected because investigations with deactivated virus (Figure

3.19C) indicated that, from Day 3, significant (p < 0.05) increases in the concentration of inoculum virus associated with the biomass would not be observed, and the concentration of replicating virus in the biomass would be low. Cultures from Day 9 that failed to accumulate this low level of virus were classified as not having an active

373 infection; however it is probable that virus was replicating within these cultures, but either because of a very low level of primary infection or some undefined form of inhibition of viral multiplication, accumulation remained very low.

In some animal cell cultures (Bleckwenn et al., 2003) and in plant protoplasts inoculated with virus in the absence of poly-L-ornithine (Sander and Mertes, 1984), increasing the virus inoculum concentration above a threshold level that resulted in the infection of all susceptible target cells did not result in further increases in virus or virus encoded protein accumulation. Within the viral inoculum concentration range examined for hairy root infection (0.75–9.0 μg mL-1 TMV), the average maximum concentration of virus accumulated in the biomass generally increased with increasing viral inoculum concentration (Figure 3.58). The failure to observe a clear plateau in accumulated virus concentrations indicates that further increasing the inoculum virus concentration could result in increases in accumulated virus.

Although a clear plateau in accumulation was not observed over the concentration range examined it is possible that the maximum yield achievable by increasing the inoculum concentration alone had been approached. When the inoculum virus concentrations were 0.75–6.0 μg mL-1, increasing the inoculum concentration was associated with linear increases in the average concentration of inoculum virus associated with the biomass during the first 12 hours of the culture period (Figure 3.55) and the average maximum concentration of virus accumulated in the biomass after culture

(Figure 3.57). The mechanisms by which inoculum virus enters susceptible cells in hairy roots are unknown; however, the linear relationship between the concentration of inoculum virus associated with the biomass and virus accumulation in the cultures

374 when the inoculum concentration was 0.75–6.0 μg mL-1 TMV (Figure 3.59) indicates that the number of cells that were infected with inoculum virus (primary infections) may have been proportional to the concentration of inoculum virus associated with the biomass. Inoculum virus associated with the biomass would include virus associated with the surface of the roots, virus which had entered cells but failed to initiate infections (Halliwell and Gazaway, 1975) and virus which entered cells and initiated primary infections. When the concentration of the inoculum virus was increased from about 6.0 μg mL-1 a plateau in the concentration of virus associated with the biomass within the first 12 hours of the culture period was observed (Figure 3.55), indicating that saturation of the root surface with virus may have occurred. The average maximum concentration of virus accumulated in the biomass after culture did not increase significantly (p < 0.05) as the inoculum virus concentration was increased from

6.0 to 9.0 μg mL-1, however a clear plateau in accumulation was not observed.

Investigations using higher inoculum virus concentrations will be required to determine if a plateau in accumulation occurs when the inoculum virus concentration is increased above 6.0 μg mL-1.

Although the average maximum concentration of virus accumulated in the biomass increased as the concentration of the viral inoculum was increased, the efficiency of viral accumulation per microgram of viral inoculum did not increase significantly

(p < 0.05) (Figure 3.60). Therefore, selection of an appropriate inoculum concentration for culture infection would be dependent on inoculum availability, the required confidence of infection and process costs. If virus inoculum was plentiful, the use of a high concentration of virus would result in high virus yields (Figure 3.58) and also a high assurance of successful infection (Section 3.11.2.2). Equivalent viral or

375 co-expressed protein yields could also be obtained after a shorter culture time

(Figure 3.57). Alternatively, if viral inoculum was less plentiful, for example if in vitro transcribed and assembled viral vectors were used, low viral inoculum concentration could be used for infection, with only small reductions in the confidence of active infection initiation (Section 3.11.2.2) but with the same proportional increase in progeny virus as obtained using a higher inoculum concentration (Figure 3.60).

However although efficiency of accumulation with regards to inoculum virus was similar over a range of inoculum concentrations, because production costs associated with culture growth would be similar irrespective of yield and the purification costs would be lower as the concentration of product increased, the unit cost of virus or co-produced proteins would be higher when lower inoculum concentrations were utilised.

Increased yields associated with the use of increased viral inoculum concentration may also be achievable by reducing the medium volume during infection. In insect cell cultures infected with baculovirus, an adsorption step in which the medium volume is reduced to increase the cell and viral density during an inoculation phase has been widely used. This technique has been associated with increased adsorption of inoculum virus to host cells and increased accumulation of virally expressed heterologous proteins (Petricevich et al., 2001). The use of a reduced volume inoculum phase was not examined for the hairy root cultures.

376

4.4 Accumulation of TMV in N. benthamiana Hairy Root Cultures with

Established Infections

Initiation of suspension and callus cultures using plant material systemically infected with virus has been widely reported (Hansen and Hildebrandt, 1966; Kassanis, 1957;

Murakishi and Carson, 1982; Raychaudhuri and Mishra, 1965; Reinert, 1966; Toyoda et al., 1989; Zelcer et al., 1981). Within systemically infected cultures, the concentration of accumulated virus in the biomass may be maintained at high levels

(Mühlbach and Sänger, 1981; Reinert 1966; Toyoda et al., 1989; Zelcer et al., 1981) or may gradually decline (Ball, 1967; Reinert, 1966) with repeated subculture.

Systemically infected suspension cultures in which virus is stably maintained have also been initiated using virus-infected protoplasts (Mühlebach and Sänger, 1981) and by subculturing newly infected callus cultures (Wu et al., 1960; Wu and Murakishi, 1978).

The effective infection of N. benthamiana hairy roots with TMV via co-incubation requires the use of relatively large amounts of inoculum virus and the resultant yield of accumulated virus can be somewhat unpredictable (Figures 3.37 and 3.39). Hairy root cultures infected with TMV were sub-cultured and accumulation of virus in subsequent generations of hairy roots was examined to determine how viral yield and the kinetics of virus accumulation were affected. Hairy roots were not initiated from systemically infected plant material as hairy root initiation is time consuming and initiated roots might not have been infected with virus, because hairy roots generally develop from individual transformed cells (Section 1.2.2.1) and, even in systemically infected plant material, cells can remain free of virus.

377

The kinetics of virus accumulation in cultures with established viral infections differed considerably from that in cultures with recently initiated primary infections.

In newly infected cultures in which viral infections were initiated using 1.5 μg mL-1

TMV, the amount of virus increased exponentially until Day 21 (Figure 3.68) with a maximum specific accumulation rate of 0.53 day-1. The maximum specific virus accumulation rate was higher than the maximum specific growth rate (0.30 day-1), and was maintained over a longer time period.

In cultures with established virus infections, the initial (Day 0) concentration of virus within the biomass reflected the concentration of virus accumulated in mature roots

(Day 21) from the previous-generation culture. The initial concentrations of virus in the first and second generation cultures, respectively, were 1300- and 550-fold higher than the concentration of virus associated with the biomass of roots with newly initiated primary infections approximately 3 hours after culture infection (Figure 3.69). The amount of virus in the established cultures increased exponentially from Day 3 to Day

12 and Day 0 to Day 12 for first and second generation cultures with established infections, respectively (Figure 3.68). The maximum specific rate of virus accumulation, 0.31 day-1 which was observed for both first and second generation cultures with established infections, was similar to the maximum specific growth rate of the established cultures (0.35 day-1). The concentration of virus in the biomass of cultures with established infections did not change significantly (p < 0.05) over the culture period; the increasing amount of virus within the cultures (Figure 3.69) primarily reflected the increase in culture biomass. In hairy root cultures with established infections, growth provides new cells which can become infected and in

378 which virus can accumulate to a similar concentration as in the inoculum hairy roots.

The fluctuations in the average amount and concentration of virus accumulated in the biomass that were frequently observed in cultures with newly initiated primary infections between Day 21 and Day 27 (Figure 3.29 and Section 3.7.3.2) were not observed in cultures with established infections. The pattern and kinetics of viral accumulation in cultures with an established viral infection initiated using inoculum from newly initiated cultures infected using 9.0 μg mL-1 TMV, were similar to those described above.

The variability between the concentrations of virus in replicate cultures observed when cultures were initiated by primary infection (Figure 3.37) was also observed but to a somewhat lesser extent in cultures with established infections. The average coefficients of variability (standard deviation /mean) for the concentration of virus in the biomass for cultures with recently initiated primary, first generation established and second generation established infections were 73, 61 and 68%, respectively, when determined from Day 0 to Day 36, and 89, 61 and 69%, respectively, when determined from

Day 21 to Day 36. The higher co-efficient of variability in cultures with recently initiated infections suggests that the initial steps of primary infection may contribute to subsequent variability in virus yields; although the variability between replicate cultures with established infections indicate that other significant sources of variability remain.

In this investigation cultures with established infections were initiated using root inocula from each of four virus-infected pre-cultures and at each sample time viral accumulation was examined in cultures initiated using roots from each of the pre- cultures. The concentrations of virus accumulated in the biomass of cultures with established virus infections varied between cultures initiated using root inocula from

379 different pre-cultures and also between cultures initiated using inoculum roots from a single pre-culture (Figure 3.70 and Figure 3.71). The continued variability in viral accumulation between different hairy root cultures with established infections would not be unexpected if the concentration of virus in pre-culture roots provided an indication of the potential ability of subsequently initiated cultures to accumulate virus.

Variability in viral accumulation within individual pre-cultures (Section 4.2.3 and

Figure 3.46) and between pre-cultures (Section 4.2.4) could be reflected in variable accumulation in the subsequent generation culture with an established infection. The distribution of virus in the root mats of cultures with established infections was not examined. The variability in the concentration of virus accumulated in cultures with established infections may be able to be reduced if the root inoculum used to initiate all the cultures was obtained from one pre-culture.

Using the subculture régime applied, cultures with established viral infections could be obtained in which the average virus concentration (Day 0 to Day 36) was not significantly different (p < 0.05) from the average maximum concentration of virus accumulated in newly initiated primary cultures (Figure 3.74). When cultures with established infections were derived from cultures with primary infections initiated using

1.5 μg mL-1 TMV , the average concentration of virus accumulated in the biomass

(Day 0 to Day 36) of first and second generation cultures with established infections, respectively, were 1.7- and 2.1-fold and significantly (p < 0.05) lower than the average maximum concentration of virus accumulated in the culture with the newly initiated primary infection (Figure 3.69); however, the average maximum concentration of virus accumulated in the recently initiated culture, 3.4 ± 0.63 mg g-1 dry weight (Day 21 to

Day 36), was unusually high (Section 4.2.6). The concentration of accumulated virus

380 in the established cultures could be maintained for at least two generations

(Figure 3.69).

Wu and Murakishi (1978) demonstrated in vortex-inoculated virus-infected suspension and callus cultures, in which virus accumulated at different rates because of different post-infection culture conditions, that if medium was renewed periodically to prevent nutrient limitation, the concentration of virus accumulated in the different cultures reached the same high level and then remained relatively constant. The similarity in the maximum concentration of virus accumulated in the biomass of hairy root cultures with primary and established infections indicates that the yield observed in the recently initiated primary cultures may have represented the maximum, or near maximum, yield achievable for the particular culture (Figures 3.69 and 3.74). These results also indicate that in culture with primary infections, nutrient limitation and the cessation of culture growth did not necessarily limit viral accumulation. A caveat to this finding is that the average maximum concentration of virus accumulated in the primary culture infected using 1.5 μg mL-1 TMV in this investigation (3.4 ± 0.63 mg g-1 dry weight) was unusually high (Section 4.2.6) and had the average maximum concentration been lower, an increase in the average concentration of accumulated virus may have been observed in the subsequently initiated cultures.

The observation that the concentration of virus accumulated in cultures with established infections was roughly related to the concentration of the virus in the roots used to inoculate the cultures (Figure 3.70) indicated that the concentration of virus accumulated in cultures with established infections could be increased by initiating cultures using root inocula obtained from high virus accumulating cultures. To

381 investigate whether this was the case, or if the viral yield in cultures with established viral infections was independent of the virus concentration in the inoculum roots, primary cultures were initiated using a high concentration of viral inoculum because this can result in increased viral accumulation (Figure 3.58). However, primary cultures initiated using 1.5 and 9.0 μg mL-1 TMV for these experiments were not initiated at the same time and, possibly as a result of uncontrolled abiotic factors or differences in inoculum roots or virus, increasing the inoculum concentration from

1.5 μg mL-1 to 9.0 μg mL-1 TMV did not result in a significant (p < 0.05) increase in the average maximum concentration of virus accumulated in the biomass (Figures 3.69 and

3.74). The average maximum concentration of virus in the first generation cultures with established infections initiated using inocula from cultures infected using 1.5 and

9.0 μg mL-1 TMV did not differ significantly (p < 0.05). Accordingly it was not possible to determine if the similarity in the concentration of accumulated virus in the cultures with established infections was related to the inoculum roots or represented the maximum achievable concentration of accumulated virus in cultures with established infections. Screening of root cultures with recently initiated primary infections to ensure high virus accumulation prior to their use as inocula for the initiation of cultures with established infections may be required to resolve this question.

Although virus concentrations similar to those observed in recently initiated cultures could be obtained in cultures with established infections and maintained over at least two successive generations (Figure 3.69 and 3.74), the potential application of cultures with established viral infections for vector or heterologous protein expression in hairy roots is currently limited using existing transgenic viral vectors. Viral sub-population selection has been reported when wild-type viruses were continuously propagated in

382 suspension cultures (Wu et al., 1960) and, therefore, long-term maintenance of virus in roots may not be desirable. Because the genomes of plant viral vectors generally exhibit relatively low levels of genetic stability (Section 1.5.1), the time required to initiate established infections in hairy root cultures could result in significant vector mutation prior to culture establishment. However, if viral vectors with enhanced stability were developed, although somewhat negating one of the advantages of using transgenic viral vectors as expression systems, which is the speed of foreign protein production, the use of cultures with established infections could be of particular benefit for foreign protein production using transgenic viral vectors. The use of cultures with established infections could reduce the amounts of high quality viral inoculum required, because inoculum virus would only be required to infect the initial primary cultures.

Additionally, as the concentration of virus in the biomass of even first generation cultures with established infections remains relatively constant, the length of the production cycle may be reduced below the 21 to 36 days required to achieve maximum viral accumulation in cultures with primary infections. Pre-culture selection prior to initiation of cultures with established infection may also result in the development of cultures that could reliably accumulate increased amounts of virus.

383

4.5 Infection of N. benthamiana Hairy Roots with a Transgenic Plant Viral

Vector

Replication of a plant-virus-based vector and foreign protein expression in cultured plant cells was examined in N. benthamiana hairy roots. Hairy root cultures were infected with the transgenic TMV-based vector TMV-GFPC3 by co-incubating the inoculum roots in Gamborg’s B5 medium with 1.98 × 1011 or 3.97 × 1011 viral particles.

The same infection protocol using equivalent number concentrations of TMV had resulted previously in efficient infection and moderate levels of viral accumulation in

N. benthamiana hairy root cultures (Section 3.11.2.2 and Figure 3.60).

Transgenic vector replication and subsequent accumulation to levels greater than those provided to the cultures could not be confirmed in hairy roots. The amount

(Figure 3.79) and concentration (Figure 3.80) of virus in the hairy root biomass increased over the culture period when roots were inoculated with both concentrations of TMV-GFPC3: the average maximum concentrations of virus associated with the biomass were 30- and 180-fold greater than the concentrations of virus associated with the biomass 2.5 hours after virus addition when cultures were inoculated using

1.98 × 1011 particles and 3.97 × 1011 particles, respectively. However, a net loss of virus was observed with the total amount of virus (biomass and medium) lower than the amount of virus added to the cultures as inoculum (Figure 3.82).

The detection of GFP in the hairy root biomass would have confirmed vector replication in cultures despite the observed net loss of virus observed. However, because of the instability of the TMV-GFPC3 vector (Rabindran and Dawson, 2001;

Shivprasad et al., 1999) (Section 1.7.3.1), the failure to detect GFP in the culture

384 biomass and medium (Section 3.14.2.6) does not necessarily mean that viral replication did not occur. The failure to detect GFP in the hairy roots was not entirely unexpected as the stability of the inserted foreign gene within the vector is known to be poor

(Rabindran and Dawson, 2001) and vector replication within the hairy root biomass was low, if present. Examination of the TMV-GFPC3 preparation used as inocula in the hairy root experiments indicated that considerable loss of the foreign gene insert had occurred. Rabindran and Dawson (2001) reported when investigating the stability of the 30B-GFP and 30B-GFPC3 vectors that GFP expression was lost after 2–3 serial passages in N. benthamiana when the virus was passaged every 2 weeks. Inoculum

TMV-GFPC3 particles used in the current study were purified from inoculated leaves, upper leaves and stems of Nicotiana clevelandii plants 23 days after infection with

RNA transcript (Sections 2.6.3 and 2.6.4). When N. benthamiana plantlets were inoculated with the purified vector preparation, inoculated leaves showed only patchy expression of GFP (Figures 3.76A, B and C), suggesting that the inoculum virus preparation contained particles with both the full TMV-GFPC3 genome and hybrid vectors in which the GFP sequence had been partially or fully deleted (Rabindran and

Dawson, 2001). Failure to detect extensive GFP expression in the upper non-inoculated leaves of N. benthamiana plantlets (Figure 3.76D) in which the full vector was reported to be able to move efficiently (Shivprasad et al., 1999) indicated that deletion of the

GFP gene sequence was ongoing. Although particle length analysis (Section 2.17.4.3) was not performed on the TMV-GFPC3 preparation used as inoculum for the hairy root cultures, it was observed during particle quantification (Section 2.17.6) that fewer than

31% of the counted particles (particle length of at least 300 nm) were approximately

360 nm in length, and therefore a similar length as full-length TMV-GFPC3 particles.

TMV-GFPC3 particles with lengths of 360 nm may be unable to express GFP due to

385 small deletions within the GFP gene; however, vectors shorter than 360 nm would be unlikely to express GFP as Rabindran and Dawson (2001) observed that recombination within the vector usually resulted in loss of the GFP gene. In addition to the poor initial quality of the inoculum TMV-GFPC3, the long culture period required to achieve maximum viral titres in N. benthamiana hairy roots (approximately 21 days) would have allowed mutations to continue to accumulate within the vector population and the final proportion of the vector particles able to express GFP, although not examined directly, would probably have been low.

Despite the low concentration of TMV-GFPC3 in the hairy root biomass and the failure to detect GFP in cultures, similarities between the pattern of virus accumulation when cultures were inoculated with 3.96 × 109 particles mL-1 TMV-GFPC3 (Figure 3.83B) and 1.5 μg mL-1 TMV (Figure 3.83A) and the differences in observed patterns when cultures were inoculated with TMV-GFPC3 and deactivated TMV that was unable to replicate in cultures (Figures 3.83B and 3.84) suggest that TMV-GFPC3 or its derivative hybrid vectors were replicating in the hairy root biomass, albeit at a low rate.

Radiolabeling of the replicating viral genome would be required to confirm this low level replication.

TMV and deactivated TMV both exhibited low retention in root-containing medium

(Figure 3.85), possibly as a result of adsorption to flask surfaces and minor adsorption to the biomass (Section 4.2.1.1). However, significantly higher (p < 0.05) amounts of

TMV-GFPC3 were retained in the medium for most of the culture period (Figure 3.85).

Factors contributing to the superior retention of TMV-GFPC3 in Gamborg’s B5 medium were not investigated; however, use of the coat protein of tobacco mild green

386 mosaic virus (TMGMV) to package the vector genome instead of the TMV coat protein

(Section 1.7.1) may have increased particle stability, or reduced the tendency of the virus to adsorb to the culture vessel and/or hairy roots. When cultures were inoculated with TMV and deactivated TMV, the rapid loss of virus from the medium would have limited the amount of virus available to associate with the hairy root biomass. In TMV-

GFPC3 inoculated cultures, the superior retention of virus in the medium (Figure 3.85) could have resulted in continued adsorption of virus to the biomass throughout the culture period, resulting in an increased amount of virus associated with the biomass.

Successful infection of plant cultures with virus requires that virus be able to enter plant cells, multiply efficiently within the infected cell and move from cells with a primary infection to previously uninfected cells by a combination of cell-to-cell and possibly long-distance movement. The lower accumulation of TMV-GFPC3 in N. benthamiana hairy root cultures compared with TMV (Figure 3.83A) indicates that one or a combination of the steps required to achieve efficient viral accumulation was less effective with TMV-GFPC3 than TMV.

Rabindran and Dawson (2001), when examining the stability of GFP and Cycle 3 GFP genes in the 30B vector, determined that the genes encoding GFP could be lost from the vectors as a result of recombination. The resultant mutant hybrid vectors that were able to accumulate to high levels within the biomass generally contained genes encoding for

TMV replicase, TMGMV coat protein and the 3´NTR (non-translated region), and often had a hybrid movement protein. The GFP gene and extra pseudoknots were frequently deleted. The hybrid particles were generally similar in length to parental TMV particles

387

(Figure 1.3) but displayed milder and delayed symptoms and were generally less competitive than parental TMV.

When the concentration of TMV-GFPC3 in the purified vector preparation used in this work was determined using electron microscopy (Section 2.17.6), particles with length

300 nm or greater were included in the count, although the actual particle length of

TMV-GFPC3 would have been closer to 360 nm (Section 3.14.1.1). The shorter particles were included in the concentration determinations because findings by

Rabindran and Dawson (2001) indicated that, while unable to express GFP, the hybrid vectors with length close to the parental virus were probably still infectious.

The relative infectivity of the TMV-GFPC3 inoculum preparation compared to that of the TMV inoculum (Equation 2.8) was estimated to be approximately (3.8 ± 2.1) × 10-3 and therefore the TMV-GFPC3 preparation was (260 ± 140)-fold less infectious than a similar number concentration of inoculum TMV (Section 3.14.1.2). The relative infectivity of TMV-GFPC3 may have been underestimated, as Rabindran and Dawson

(2001) reported that hybrid vectors generated from TMV-GFP only produced pin-point lesion in the test plant, N. glutinosa, whereas the parental TMV produced 1 mm lesions.

However, even if the relative infectivity of the vector inoculum was underestimated, as investigations with wild-type TMV indicated that halving the inoculum virus concentration from 1.5 μg mL-1 TMV to 0.75 μg mL-1 TMV resulted in significant reductions (p < 0.05) in the average maximum concentration of virus accumulated in the biomass (Figure 3.58) and the percentage of cultures exhibiting active viral infections (Section 3.11.2.2), even small reductions in the concentration of infectious virus in the TMV-GFPC3 inoculum could have resulted in considerable reductions in

388 the accumulation of the vector in the biomass and the percentage of cultures exhibiting active infections. The low infectivity of the TMV-GFPC3 inoculum may have resulted in a very low level of primary infection which would have contributed to the low level of viral vector accumulation in the hairy root biomass. Hairy root infection was not repeated using inoculum TMV-GFPC3 concentrations above 7.94 × 109 particles mL-1 due to limited supplies of the viral vector.

Reduced rates of replication of both the full TMV-GFPC3 vector (Toth et al., 2002) and the hybrid vectors generated from it by recombination (Rabindran and Dawson, 2001) compared with TMV, which have been reported previously to result in delayed symptom development in whole plants, could have further contributed to the low level of vector accumulation in infected cultures.

TMV-GFPC3 and TMV-GFP accumulation in the roots of whole plants has been studied by Valentine et al. (2002) and MacFarlane and Popovich (2000), respectively.

Both reported that expression of GFP in roots was patchy. This was attributed by

Valentine et al. (2002) to a gene-silencing-like mechanism that prevented the TMV- derived vector from becoming established in newly formed lateral roots. The existence of such a mechanism could limit vector accumulation in hairy roots, which are characterised by a high degree of lateral branching. Patchy GFP expression was reported by Macfarlane and Popovich (2002) when TMV-GFP was used as a vector, but was not observed when vectors based on tobraviruses, which are adapted for efficient movement in plant root systems, were utilised. These findings indicate that, as in the roots of whole plants, careful vector selection may allow efficient expression of heterologous proteins in hairy root cultures.

389

To achieve useful levels of heterologous protein expression in plant cell cultures, both low levels of vector accumulation and poor vector stability must be addressed.

Improved inoculation methods which allow the efficient infection of cell cultures using small viral inocula would also be advantageous. The problem of low levels of vector accumulation in hairy roots could be overcome by careful development and selection of vectors for use in plant roots. The vector used in this investigation, 30B-GFPC3, and the majority of other transgenic vectors have been developed to specifically facilitate high levels of vector or heterologous protein expression in plant leaves or fruiting bodies. However, vectors have been developed that allow efficient invasion of the root systems of certain plants (MacFarlane and Popovich, 2000) and the use of these or similar vectors may help improve viral vector accumulation.

The stability of vectors must also be addressed if high levels of heterologous protein expression are to be achieved in cultured cells. Using the TMV-GFPC3 vector, modification of the inoculum preparation methods such as reducing the time between plant infection with RNA transcript and harvest of the biomass (Section 2.6.3) may increase the proportion of full-length viral particles generated. Infection of cultures with inoculum obtained by packaging the RNA transcript in vitro (Toth et al., 2002) could result in a high proportion of infected cells containing vectors able to express

Cycle 3 GFP. However, even with these modifications, it is probable that over the course of the experimental period significant vector modification would occur, resulting in the loss of the GFP gene and associated reduction in foreign protein expression.

Because virus accumulation is lower in hairy roots than in whole plants (Section 4.2.6), vectors used to facilitate heterologous protein expression in hairy roots will have to

390 exhibit greater stability that vectors used to obtain similar levels of heterologous proterin expression in whole plants.

4.6 Infection Scale-up

4.6.1 Infection scale-up in shake flasks

Infection of hairy root cultures by co-incubation with virus in medium at a concentration of 1.5 μg mL-1 TMV was associated with relatively high confidence of infection (Section 3.11.2.2) and moderate viral yield (Figures 3.57 and 3.58) and inoculum virus requirements (Figure 3.60). Scale-up in shake flasks was performed with the concentration of virus in the medium maintained at 1.5 μg mL-1 TMV and the ratio of hairy root fresh weight to the amount of virus also kept constant (Section

2.9.7). Culture volumes were selected using the relationship for gas–liquid mass transfer in shake flasks determined by Henzler and Schedel (1991) so that, despite

altering the medium and flask volumes, the gas–liquid oxygen transfer coefficient (kLa) would be roughly the same as that observed using 50 mL of medium in a 250-mL

Erlenmeyer flask. Altering the dissolved oxygen concentration can significantly affect hairy root growth rates (Kanokwaree and Doran, 1997; Yu and Doran, 1994) and, because of the possible relationship between culture growth and viral accumulation

(Section 4.2.1.3), significant alterations to growth were undesirable. The scaled but non-infected control hairy root cultures exhibited similar growth kinetics to hairy roots in 50 mL medium in a 250-mL Erlenmeyer flask (Figure 3.63B and Section 3.12.2.1), indicating that either similar gas–liquid oxygen transfer coefficients were obtained upon scale-up, or the cultures were not highly sensitive to oxygen transfer conditions.

391

Scale-up of infection was performed so that the concentration of virus added to the medium was constant and the ratio of the amount of virus added to the biomass fresh weight was fixed. Had all conditions within the flasks remained similar during scale- up, the average maximum concentrations of virus in the adsorption plateau region observed in short-term experiments within 12 hours of culture infection would have been expected to remain constant for all medium volumes examined. However, the concentration of virus associated with the biomass increased significantly (p < 0.05) as the culture volume increased (Figures 3.61B and 3.62).

The significant differences observed in plateau virus concentration in short-term investigations may have been contributed to by non-uniform retention of virus in the medium or a non-proportional increase in available virus binding sites in the root biomass. When 1.5 μg mL-1 TMV was added to 50 mL of Gamborg’s B5 medium in a

250-mL Erlenmeyer flask, within minutes of virus addition only a small percentage of the added virus remained in the medium (Figure 3.50). Adsorption of inoculum virus to the flask surface is a possible factor contributing to this loss of virus (Section

4.2.1.1). During scale-up, medium volumes and flask sizes were selected so

that kL a was kept approximately constant; however, the ratio of medium volume to flask surface area contacting the medium when the cultures were at rest increased as the medium volume was increased. The ratio of the amount of virus to flask surface area therefore also increased as the medium volume increased. This may have resulted in an increase in the concentration of virus remaining in the medium and greater interaction between the hairy roots and virus as the medium volume was increased, consistent with the increasing concentrations of biomass-associated virus. Alternatively, the available sites or surface area for virus association in the hairy root biomass may have increased

392 disproportionately as the biomass fresh weight was increased. However, as the virus concentration added to the medium (1.5 μg mL-1 TMV) may have been below the level required to achieve saturation of binding and infection sites on the roots (Section 4.3.5), increasing the number of available binding sites may not have resulted in increases in the concentration of virus associated with the hairy root biomass.

The proportional scale-up of hairy root infection did not result in the accumulation of a constant concentration of virus in the hairy roots. However, the affect of scale-up on virus accumulation in hairy roots after cultures is equivocal. When the medium volume was 27 mL the average maximum concentration of virus accumulated in the biomass was significantly (p < 0.05) lower than when the medium volumes were 50–134 mL

(Figures 3.64B and 3.65). When medium volumes were between 50 and 134 mL, although the average maximum concentrations of virus accumulated in the biomass appeared to increase linearly as the medium volumes were increased (Figure 3.65), the differences in the concentrations were not significant (p < 0.05). The possible effect of further increases in medium volume on viral accumulation in the biomass after culture are unknown but, if the linear relationship between medium volume and concentration of virus accumulated in the biomass is maintained, further significant increases in the average maximum concentration of accumulated virus after culture may be observed.

The failure to observed constant concentrations of virus in hairy roots after culture when hairy root infection was scaled-up, may have been related to the non-uniform association of inoculum virus with inoculum roots (Figure 3.62). The relationship between the plateau concentration of inoculum virus associated with the biomass within

12 hours of root exposure to virus and the concentration of virus accumulated in the

393 biomass after culture, increased linearly as the medium volume was increased (Figure

3.66). The relationship between biomass-associated virus and accumulated virus upon scale-up (Figure 3.66) was similar to that observed when the viral inoculum concentration was increased in 250-mL flasks (Figure 3.59), suggesting that increases in accumulated virus may have resulted from increased levels of primary infection with scale-up. However, other contributing factors could also exist.

4.6.2 Accumulation of virus in bioreactor-infected and -grown hairy roots

Inoculation of hairy root cultures in bioreactors was performed via co-incubation of roots and virus using the same ratio of hairy root fresh weight:amount of virus:medium volume used for scale-up in shake flasks (Section 2.16.2).

4.6.2.1 Hairy root growth

The growth of hairy roots in the bioreactor system could not be monitored by measuring actual increases in biomass throughout the culture period; however, by examining sugar utilisation and medium pH and conductivity, hairy root growth could be monitored indirectly. When the cultures were terminated 31 days post-culture- initiation, the biomass dry weights of TMV- and TMV-GFPC3-infected and non- infected control hairy root cultures were not significantly (p < 0.05) different (Figure

3.87). However significant (p < 0.05) differences in the utilisation of medium sugars

(Figure 3.88) and decreases in medium conductivity (Figure 3.90) compared with the

TMV-GFPC3 infected culture and/or non-infected control cultures, indicate that the growth of TMV-infected hairy roots was initially delayed. This was unlikely to be due to the effect of accumulating virus on cell physiology because the accumulation of higher concentrations of virus in TMV-infected hairy roots in flask cultures (Figure

394

3.39) did not affect culture growth (Figure 3.34). Competition between virus and host cells for limited amino acids and nucleotides in nutrient limited root cells may have resulted in delayed growth of TMV-infected hairy roots in the bioreactor. Nutrient limitation in bioreactors used for hairy root culture is common and results from inefficient nutrient and in particular oxygen transfer to the cells, particularly when root density is high (Giri and Narasu, 2000; Wysokinska and Chmiel, 1997). Although the root inoculum used was relatively small (8.0 g fresh weight) it was contained within the small volume of the mesh basket, and fluid flow through the roots may have been limited by high root density and the presence of the mesh basket. Despite the possible initial delay in the growth of TMV-infected hairy roots, the final biomass achieved was not affected (Figure 3.87).

4.6.2.2 TMV accumulation

Virus accumulation in bioreactor-infected and -grown hairy roots (Figure 3.91A) was generally poor compared with that observed in flask cultures (Figure 3.39). TMV accumulated to concentrations of less than 0.1 mg g-1 dry weight in at least half of the hairy root biomass, and to only 0.39 ± 0.13 mg g-1 dry weight in roots growing towards the base of the bioreactor. The concentration of accumulated virus in the roots growing at the base of the bioreactor was approximately 4-fold lower than the average maximum concentration of virus (1.6 ± 0.25 mg g-1 dry weight) obtained in flask cultures. The concentration of virus accumulated in roots from within the inoculum tube was not significantly different (p < 0.05) from the concentration of virus accumulated in roots growing towards the base of the bioreactor.

395

The generally low levels of viral accumulation in bioreactors may be a reflection of poor primary viral infection of roots in the bioreactor, subsequent poor dissemination of virus throughout the developing biomass, low viral accumulation in infected cells, or a combination of these factors. Root inocula used in shake-flask and bioreactor experiments were obtained from 21-day-old shake-flask hairy root cultures and were prepared similarly prior to addition to fresh medium. In bioreactor and shake-flask investigations, virus was added to the medium after root addition. As the methods of inoculum preparation (root and virus) were similar for bioreactor and shake-flask cultures similar levels of primary infection would be expected, provided that the exposure of roots to inoculum virus was similar and the injuries that facilitated infection occurred during inoculum root preparation and were not caused by shake flask movement. The concentration of circulating virus in the bioreactor medium was not examined immediately after virus addition; however, after 24 hours, the concentration of virus in the medium from the bioreactor (Figure 3.93) was similar to the concentration of virus in medium from shake flasks, with 15% and 17% (data not shown) of the inoculum virus retained, respectively. The similarities in the proportions of inoculum virus retained in the medium in bioreactors and shake flasks suggest that primary infection in bioreactors was probably not inhibited by low medium virus concentrations. However, it is possible that contact between the inoculum virus and hairy roots was lower in the bioreactor than in shake flasks because of the moderately high root density in the mesh basket. Short-term interaction of inoculum virus with hairy roots within the bioreactor was not investigated. As virus association with the root biomass would be affected by both the concentration of virus retained in the medium and the exposure of the roots to the retained virus, comparison of adsorbed

396 virus in shake-flask-inoculated and bioreactor-inoculated cultures could indicate whether root exposure to inoculum virus in the bioreactor was lower.

Virus accumulated at very low concentrations in hairy roots located at the mid-height and top of the bioreactor indicating that either virus was accumulating in only a small proportion of the root cells or that virus accumulated to only low levels in infected cells. Accumulation of virus in roots not contained within the mesh basket would be dependant on the movement of virus into the elongating roots via cell-to-cell or long- distance viral movement, or the initiation of new primary infections occurring as a result of root injury and exposure to either residual inoculum virus retained in the medium or released root-synthesised virus. Dissemination of virus throughout the hairy root biomass as a result of viral movement alone would probably require both cell-to- cell and some form of long-distance movement. As discussed previously (Section

4.2.3), the movement of TMV via cell-to-cell movement would result in only the slow dissemination of virus from sites of primary infection. If the rate of cell-to-cell movement in hairy root cultures was the same as or lower than that observed in leaves of N. benthamiana (Cheng et al., 2000), the maximum linear progression of the infection front would be less than 2 cm after 31 days. Therefore, in order for cells near the bioreactor wall (5.6 cm from the mesh basket wall) to become infected, either long- distance movement or the initiation of new primary infection events would be required.

In shake flask grown hairy roots the generally reducing concentration of virus accumulated in the biomass as radial distance from the centre of the root mat increased, indicated that long-distance movement and/or the initiation of new primary infections did not occur at a level that resulted in the uniform distribution of virus throughout the biomas (Figure 3.45 and Sections 4.2.3). The low concentration of virus in roots from

397 near the mid-height and top of the bioreactor indicate that as in shake-flask grown roots, long-distance movement and/or the initiation of new primary infections did not occur at a sufficient level to result in uniform viral accumulation throughout the biomass.

Environmental conditions unfavourable to viral accumulation in infected cells could also have existed within the bioreactor.

Virus accumulated to similar concentrations in hairy roots located at the base of the bioreactor and roots in the mesh basket. Stirrer bar movement was observed to result in the injury of roots growing towards the base of the bioreactor (Section 3.15.1 and

Figure 3.86). Both minor and major root injury may have facilitated the initiation of new primary infection events in the hairy root biomass near the base of the bioreactor.

Valentine et al. (2002) suggested that a gene-silencing-like mechanism originating in lateral root meristems inhibited the accumulation of virus in the roots of

N. benthamiana plants. The operation of a similar gene-silencing-type mechanism in hairy roots has not been confirmed; however, if the silencing mechanism was active in hairy roots and contributing to low or patchy viral accumulation the injury to, or excision of, lateral root meristems by the stir-bar may have lessened inhibition of viral replication and resulted in increased viral accumulation.

4.6.2.3 TMV-GFPC3 accumulation

Replication of TMV-GFPC3 in bioreactor-infected and -grown hairy roots could not be confirmed. The concentration of virus across the hairy root biomass was low (Figures

3.91B and 3.92) – approximately 57–130-fold lower than the average maximum concentration of TMV accumulated in flask-grown roots (Figure 3.39) – and GFP was not detected in the biomass or media (Section 3.15.4). The concentration of the vector

398 in the bioreactor-infected and -grown hairy roots was similar to the average maximum concentration of the vector in the biomass of flask grown roots inoculated using the same ratio of biomass to inoculum virus (Figure 3.80B).The uniform but low concentration of virus in the biomass of the hairy roots may have been attributable to the ongoing adsorption of inoculum virus with the hairy root biomass. Approximately

48% of the inoculum virus was retained in the medium (Section 3.15.3.2).

The failure of TMV-GFPC3 to accumulate to high levels in the bioreactor was not unexpected because TMV-GFPC3 accumulation in shake flasks was poor relative to

TMV accumulation, and results for accumulation of TMV in bioreactor and shake-flask infected hairy roots indicated that virus accumulation in shake-flask-grown roots was superior to that in bioreactor-grown roots.

4.7 Infection of N. benthamiana Suspended Cells with TMV

The pattern of TMV accumulation in N. benthamiana suspension cultures was similar to the pattern of viral accumulation reported in suspended cell cultures derived from permissive host plants (Section 1.9.2.1) (Murakishi et al., 1971; Pelcher et al., 1972;

White et al., 1977; Wu and Murakishi, 1978). The viral accumulation phase in the

N. benthamiana suspension culture was lengthened compared with those reported for

N. tabacum L. var. Havana 38 (Murakishi et al., 1971; Pelcher et al., 1972) and

Glycine max var. Harosoy 38 (White et al., 1977; Wu and Murakishi, 1978) suspension cultures. A similar lengthening of the accumulation phase was also observed in TMV- infected N. benthamiana hairy root cultures (Figure 3.39) and was attributed to a relatively long culture growth phase (Section 4.2.1.3). The lengthening of the accumulation phase in N. benthaminana suspensions may also be attributable to the long culture growth phase (Day 2 to Day 18) (Figure 3.10), although culture growth

399 kinetics and the relationship between viral accumulation and culture growth were not reported for the other permissive suspension cultures.

When N. benthamiana suspension cultures were infected with TMV by co-incubation the amount and concentration of virus in the biomass did not increase significantly

(p < 0.05) for the first 4 days of the culture period (Figure 3.11A). In other virus- infected suspension cultures in which the excess inoculum virus was removed from the medium and sensitive viral detection methods were used, replication and accumulation of TMV has been detected within 24 hours of infection (Murakishi et al., 1971; Pelcher et al., 1972); Therefore, it is probable that virus was replicating in N. benthamiana suspension cells with primary virus infections and possibly in neighbouring cells within cells aggregates during the first four days of the culture period. However, as the inoculum virus was retained in the medium, any small increases in the amount of virus in the biomass that may have resulted from the replication of infecting inoculum virus may not have been initially observed against a possible background of non-infecting inoculum virus that was associated with the biomass fraction of the culture.

From Day 4 to Day 18, the amount and concentration of virus in the suspension biomass increased exponentially (Figure 3.11B). Suspension culture growth was exponential between Day 2 and Day 14 and growth continued at a decelerated rate until the maximum biomass was observed 18 days post-infection (Figure 3.10). The amount and concentration of virus in the suspension biomass did not increase significantly

(p < 0.05) beyond Day 18. The close association between culture growth and virus accumulation in N. benthamiana suspension cultures (Figures 3.10 and 3.11) was also observed in hairy root cultures (Section 4.2.1.3 and Figure 3.39). Viral accumulation

400 was not examined when virus infected suspension cells were transferred to fresh medium and therefore it is not known whether the average maximum concentration of virus accumulated in the suspension cells [Day 18 to Day 36: (2.0 ± 0.11) × 10-2 mg g-1 dry weight] represented the maximum potential viral yield for this suspension culture or if viral accumulation within the culture had been limited by nutrient limitation and the cessation of suspension culture growth.

In N. benthamiana suspension cultures, despite the extended viral accumulation phase

(Day 4 to Day 18) the concentration of virus in the biomass increased only 11-fold and the amount of virus in the biomass increased only 90-fold over the experimental period.

The average maximum amount of virus in the biomass (Day 18 to Day 36) of

N. benthamiana suspension cultures [(7.4 ± 0.40) × 10-3 mg] was equivalent to only

3.3% of the amount of virus added to inoculate the cultures (0.225 mg). Virus multiplication could not therefore be confirmed.

Comparisons between the virus yields obtained in the N. benthamiana suspension cultures and previously reported yields in N. tabacum and D. carota suspensions

(Table 4.2) are not straightforward because the amount and concentration of virus per unit inoculum biomass differed between investigations and the concentration of virus used as inoculum can affect the viral yield (Murakishi et al., 1971). The concentration of virus in the biomass of N. benthamiana suspension cultures was considerably lower than the yields reported in other plant cell suspensions (Table 4.2). The concentration of virus in N. benthamiana suspension cultures was 600-fold lower than the concentration reported by Murakishi et al. (1971) in N. tabacum suspension cultures infected using vibratory inoculation and a lower amount of virus and concentration of

401 virus per gram fresh biomass. The maximum concentration of virus in N. benthamiana suspensions was also 72-fold lower that the concentration of TMV accumulated in

D. carota suspension cultures, which were infected by co-incubation of cells with inoculum TMV (Warren and Hill, 1989). D. carota suspension cultures were inoculated using a similar amount of virus, but a considerably higher concentration of virus per milliliter of medium and gram fresh biomass were utilised compared with

N. benthamiana suspension cultures (Table 4.2). The culture conditions used by

Warren and Hill (1989) would have also facilitated considerable culture growth (Table

4.1).

The relatively low TMV accumulation obtained using N. benthamiana suspension cultures in this study indicates that the initial infection of cultures and/or subsequent viral replication and accumulation were poor. Moderate levels of TMV accumulation were achieved in N. benthamiana hairy root cultures, indicating that virus could accumulate in cultured N. benthamiana cells; therefore, unless differences in the medium composition used in hairy root and suspension cultures significantly affected viral accumulation, virus would have been expected to be able to replicate and accumulate in infected suspended cells. Low yields observed in N. benthamiana suspension cultures could have been caused by low levels of primary infection. Cell inoculum used in this investigation was obtained from established suspension cultures which generally did not form large aggregates, rather than callus which was used by

Murakishi et al., (1971) and suspended cells were inoculated by co-incubating the suspension cells with the inoculum virus. The combination of these factors may have resulted in the rupture of only a small number of plasmodesmata and subsequent low levels of primary infection compared with cultures that were initiated using callus

402 and/or vortex inoculated. Thomas and Warren (1994) and Warren and Hill (1989) achieved higher levels of viral accumulation in cultures that were initiated using cell inoculum from established suspension cultures that were infected by co-incubation with virus than observed in N. benthamiana suspensions cultures (Table 4.2). Low virus yields in N. benthamiana suspension cultures may also be attributed to the accumulation of virus in only a small percentage of cells within the culture. The suspension culture used in this investigation was friable and did not tend to form large cell aggregates. This culture morphology would not have facilitated extensive virus dissemination as, in suspension cultures, virus movement is thought to occur predominantly via cell-to-cell movement which is possible only between cells within an aggregate. Increased viral accumulation in N. benthamiana suspension cultures may be obtained by the use of improved infection procedures that increase the proportion of cells in which primary infection occurs, and by the use of cultures that have a tendency to clump or culture conditions that favour clumping, thus facilitating virus movement.

Exposure of existing cultures to light, so that they develop green pigmentation

(Murakishi et al., 1971) and modification of medium composition (Hill et al., 1990) have also been associated with increased viral accumulation in suspension cultures and may facilitate increased viral accumulation in the existing N. benthamiana suspension.

4.8 Virus infection of and accumulation in N. benthamiana suspension and hairy root cultures

The average maximum concentration of virus accumulated in N. benthamiana hairy root cultures (Figure 3.39) was approximately 80 times higher than the average maximum concentration of virus accumulated in N. benthamiana suspension cultures

403

[Day 18 to Day 36: (2.0 ± 0.11) × 10-2 mg g-1 dry weight) (Figure 3.11). The higher accumulation of TMV in the hairy roots indicates that N. benthamiana hairy roots are a superior culture system for the accumulation of virus, although a caveat to this result is that only one N. benthamiana suspension culture was examined. An additional caveat to this result in that the hairy root inoculum was smaller than the suspended cell inoculum (Table 4.1) and therefore although the biomass dry weight:inoculum virus ratio was kept constant between investigations (Section 2.8) the amount and concentration of inoculum virus in the hairy root culture medium were lower than in suspension culture medium. Hairy root biomass was also able to undergo an increased number of doublings compared with the suspension biomass. Medium conditioning effects required the use of a higher inoculum biomass in suspension cultures and the amount of inoculum virus was increased so that the biomass:virus inoculum ratio was the same as that used in standard hairy root cultures. The concentration of inoculum virus added to the suspensions (4.5 μg mL-1 TMV) was three times that added to hairy root cultures (1.5 μg mL-1 TMV). The higher concentration of virus in the biomass of the hairy roots than suspensions after culture, despite the use of a lower inoculum virus medium concentration, further demonstrates the superiority of hairy roots as a culture system for the accumulation of virus.

When N. benthamiana suspension cultures were infected with TMV, it was expected that, because of the increased medium virus concentration (4.5 μg mL-1 TMV) and the high cell surface area exposed to the medium, contact between the virus and the biomass would be high resulting in a high concentration of inoculum virus associated with the biomass. However the concentration of virus associated with the suspension biomass shortly after virus addition (3.5 hours) (Figure 3.11) was 5.8-fold lower than

404 the concentration of virus associated with hairy roots inoculated using a lower concentration of virus (1.5 μg mL-1 TMV) at a comparable time after infection (Figure

3.54). The amount of inoculum virus associated with the hairy roots was also 1.4-fold higher than the amount of virus associated with the suspension cells, although the suspension biomass dry weight was 2.4-fold higher than the hairy root dry weight and the amount of virus added was 3-fold higher.

Despite the different concentrations of inoculum virus in the medium of suspension and general hairy root cultures, the low amount of virus associated with the suspension biomass shortly after infection suggests that N. benthamiana suspension cells have lower affinity for TMV than hairy roots. The mechanism by which virus interact with cultured plant cells is unknown, and the same mechanism may not be responsible for virus association with suspension cells and hairy roots. The apparently enhanced affinity of hairy roots for TMV may be related to the physiology of the differentiated root as an uptake organ, or root exudates may result in enhanced association of virus with the hairy root surface. Alternatively, although it had been assumed that a friable suspension culture would have a greater cell surface area exposed to the medium than hairy roots, the presence of root hairs on the roots may have resulted in the presentation of a relatively large surface area with which virus could associate. The higher virus accumulation in hairy roots could reflect an increased susceptibility to primary viral infection compared with suspended cells.

The specific virus accumulation rates in N. benthamiana suspension cultures of

0.28 day-1 (Day 4 to Day 18) and 0.079 day-1 (Day 18 to Day 24) were considerably lower then the specific virus accumulation rates in N. benthamiana hairy root cultures

405 of 0.68 day -1 (Day 3 to Day 12) and 0.22 day -1 (Day 12 to Day 21). In N. tabacum suspension cultures virus has been reported to move from infected cells into adjacent previously uninfected cells via cell-to-cell movement relatively rapidly (Plecher et al.,

1972; Russell and Halliwell, 1974). Russell and Halliwell (1974) reported that in chains of isolated suspension cells in which one cell was microinjected with TMV viral inclusions were formed in adjacent and non-adjacent cells as a result of cell-to-cell movement within 40–60 hours of initial infection. If virus dissemination from cells with primary infection to adjacent previously uninfected cells occurred at a similar rate in N. benthamiana cultures, the rates of viral accumulation reported above for

N. benthamiana suspension and hairy root cultures could represent viral accumulation in cells predominantly infected as a result of cell-to-cell viral movement. Some of the observed increase in virus in suspension cells could also have resulted from the accumulation of virus in dividing virus-infected cells (Pelcher et al., 1972). The higher virus accumulation rates in hairy root cultures may indicate that movement of virus from infected cells into previously uninfected cells occurs more efficiently in roots, possibly because of more extensive cell-to-cell contact than in suspension cultures. In suspension cells the progression of the viral infection via cell-to-cell movement would be limited by the small size and discrete nature of many cell aggregates.

Despite the differences in initial inoculum virus association with the hairy root and suspended cell biomass, and the different rates of viral accumulation within the two types of culture, in both suspension and hairy root cultures virus was observed to double with similar increases in biomass. In suspension cultures, the amount of virus in the biomass was observed to double with every 0.069 g increase in biomass dry weight

(Figure 3.12). In hairy root cultures, when the culture biomass increased above 0.1 g

406 dry weight, virus was observed to double with every 0.063 g increase in biomass

(Figure 3.38). The significance of virus doubling with defined increases in biomass and the similarity in the biomass increases required for viral doubling between suspension and hairy roots have not been determined.

407

CHAPTER 5 – CONCLUSIONS AND RECOMMENDATIONS

5.1 Conclusions

• TMV replicated and accumulated in Nicotiana benthamiana hairy roots. The ability

of a culture to become infected with and accumulate virus was affected by the plant

species and the form of culture. Characteristics of individual root lines (clones) also

influenced viral accumulation.

• Infection of hairy roots with TMV could be achieved by co-incubating roots and

virus in plant tissue culture medium. Primary infection events originating from the

inoculum virus occurred relatively soon after inoculation so that removal of

inoculum virus after 16 hours did not significantly affect subsequent virus

accumulation. The average maximum concentration of virus accumulated in hairy

roots was proportional to the plateau concentration of virus associated with the

hairy root biomass within 12 hours of infection. Uneven distribution of virus in root

mats harvested from shake flasks indicated that the initiation of primary infections

in newly formed roots distant from the original inoculum roots occurred with low

efficiency.

• Virus accumulated in actively growing hairy root cultures. Maximum viral

concentration and maximum culture biomass were often observed to coincide;

however investigations in which further culture growth in the absence of inoculum

virus was achieved by transferring infected roots to fresh medium indicated that

virus accumulation was not limited by the cessation of root growth. The average

concentration of virus accumulated in cultures with established virus infections was

408

generally although not necessarily significantly lower (p < 0.05) than the average

maximum concentration of virus accumulated in cultures in which infection was

newly initiated by primary infection.

• Considerable variability in the concentration of accumulated virus was observed

between replicate cultures and in different adjacent radial segments of the same root

mat, indicating the effect of uncontrolled biotic or abiotic factors on root infection

and subsequent viral movement and accumulation. The concentration of virus in

the hairy root biomass was generally higher towards the radial centre of the disc-

shaped root mat, reflecting the slow dissemination of TMV via cell-to-cell

movement and indicating that ongoing initiation of primary infection events and any

long-distance viral movement did not occur at a level sufficient to achieve uniform

accumulation of virus throughout the root mat.

• The concentration of virus accumulated in hairy roots and the percentage of cultures

exhibiting active viral infections were affected by the inoculum virus concentration

and the culture conditions at and shortly after infection. Manipulation of these

conditions may result in an increase in the percentage of cells within the inoculum

roots successfully infected with virus. Increasing the proportion of cells initially

infected with virus, could increase the number of sites within the roots from which

virus could disseminate, possibly resulting in increases in the percentage of cells

after culture in which virus was accumulating and the concentration of virus in the

biomass.

409

• Proportional scale-up of hairy root infection in shake flasks under constant oxygen

mass transfer conditions was associated with moderate levels of virus accumulation

in most scaled cultures; however, average maximum concentrations of virus after

culture were not constant at different scales. The plateau concentrations of

inoculum virus associated with the biomass within 12 hours of infection increased

as the cultures were scaled-up. The concentrations of virus accumulated in the

biomass of scaled cultures were generally proportional to the plateau concentrations

of inoculum virus associated with the biomass.

• Accumulation of TMV in bioreactor-infected and -grown hairy roots was poor

compared with accumulation in roots infected and grown in Erlenmeyer flasks.

Virus did not accumulate uniformly throughout the biomass in bioreactors.

• Viral vector (TMV-GFPC3) propagation and heterologous protein expression were

not confirmed in either flask or bioreactor hairy root cultures. Primary infection

levels may have been low because of low vector infectivity. Vector instability is

likely to have resulted in only a small proportion of infecting virus facilitating the

transcription and translation of Cycle 3 green fluorescent protein.

• Moderate viral yields were observed in N. benthamiana hairy roots. Average

maximum virus concentrations in hairy roots were 5.7–15-fold lower than in

systemically infected Nicotiana tabacum leaves. However, yields equivalent to

those observed in the leaves of whole plants were observed in some individual

cultures, indicating that high virus yields could be obtained in hairy roots. Virus

accumulated in N. benthamiana hairy roots at concentrations of the same order of

410

magnitude as those reported in the literature for N. tabacum suspension cultures.

Hairy roots represent a possible culture-based system for virus replication in

situations in which field-based agricultural systems do not adequately address issues

of containment or product safety.

5.2 Recommendations

• Further optimisation of hairy root infection protocols, with the objective of

increasing the level of primary infection in cultures and reducing the quantity of

inoculum virus required to achieve infection, would be desirable. Application of

optimised protocols may result in more efficient hairy root infection and subsequent

vector and heterologous protein accumulation. Co-expression of vector-encoded

marker proteins in hairy roots would allow further visual examination of the

characteristics of vector accumulation in hairy root cultures.

• The effect of plant growth medium composition, particularly the effect of medium

nitrogen levels, on viral accumulation in hairy roots should be examined to

determine if virus accumulation within infected cells can be increased. The

infection of green pigmented hairy roots with virus should be examined to

determine if viral accumulation is superior in pigmented cultures (Murakishi et al.,

1971).

• Efforts should be made to identify the factors contributing to the variability in virus

accumulation both within individual cultures and between replicate cultures.

Culture techniques and the preparation of suitable root inocula should be examined

in particular to determine if they contribute to variability. By understanding factors

411

involved, it may be possible to reduce variability and also increase yields. Other

hairy roots and clones could also be examined to determine if variability is

characteristic of TMV accumulation in hairy roots or specific to the clone used.

• The accumulation of other plant viruses in hairy roots could be examined to identify

viruses that accumulate to high levels in plant roots but with less variability than

TMV. Investigations could focus on viruses such as tobacco rattle virus that have

previously been demonstrated to replicate, accumulate to high concentrations, and

move efficiently in plant roots, and which are not subject to post-transcriptional

gene silencing.

• Transient expression vectors in which foreign protein encoding gene inserts are

stabily maintained could be developed specifically for use in hairy roots. Vectors

should also display high relative infectivity and good cell-to-cell movement

characteristics. The contribution of long-distance movement to viral dissemination

in hairy root cultures has not been determined, but provided that heterologous

proteins are not expressed as coat protein fusions, it may be possible to remove coat

protein genes from vectors without significantly effecting vector or heterologous

protein accumulation in cultured cell.

412

APPENDICES

Appendix 1 Filter Sterilisation of Purified TMV Preparations

The concentrations of virus in TMV preparations are shown before and after filter sterilisation in Figure A1.

0.0030 ) -1 0.0025

0.0020

0.0015

0.0010

0.0005 TMV concentration (mg mL concentration TMV

0.0000 Unfiltered TMV Filtered TMV

Figure A1 Concentration of virus in purified TMV preparations before and after filter sterilisation.

TMV preparations (2.44 ¯ 10-3 mg mL-1) were filtered through 0.2-µm Minisart filters

(Sartorius) prior to addition to plant cell cultures. Filtration of virus did not significantly effect (p < 0.05) the concentration of TMV in virus preparations.

413

Appendix 2 Extraction of TMV from Plant Biomass

The effect of the virus extraction method used to extract TMV from cell biomass on virus titres determined using ELISA was examined by extracting TMV-containing extraction buffer according to the extraction method provided in Section 2.17.3.1. The results are shown in Figure A2. When TMV-containing extraction buffer was processed in a cold mortar and pestle with 0.05 g acid-purified sand for 30 seconds and then centrifuged in a refrigerated centrifuge (4ºC) for 20 minutes at 10000 × g, the concentration of virus as determined using ELISA was not significantly different

(p < 0.05) from that in the unprocessed buffer:virus mix. When TMV-containing extraction buffer was processed in a cold mortar and pestle with 0.05 g acid-purified sand and 0.1 g fresh weight N. benthamiana hairy root biomass for 30 seconds and then centrifuged in a refrigerated centrifuge (4ºC) for 20 minutes at 10000 × g, the concentration of virus as determined using ELISA was not significantly different

(p < 0.05) from the concentration of virus in the unprocessed buffer:virus mix and the processed buffer:virus mix.

) 0.0025 -1

0.0020

0.0015

0.0010

0.0005

Virus concentration (mg mL 0.0000 Purified TMV in extraction Purified TMV in extraction Purified TMV in extraction buffer buffer, ground with sand for 30 buffer, ground with sand and seconds in a cold mortar and fresh hairy root biomass (10% pestle w/v) for 30 seconds in a cold mortar and pestle

Figure A2 Effect of extraction process on the concentration of TMV in extraction buffer.

414

Appendix 3 Growth of N. benthamiana hairy root cultures initiated using

14-day-old root inoculum

The growth of N. benthamiana hairy root cultures initiated using inoculum from

14-day-old roots is shown in Figure A3. Roots used in the examination of root age on viral accumulation were obtained from hairy root cultures initiated using inoculum from

14-day-old cultures, 6,10,14, and 21 days after culture initiation when the cultures were in the mid-exponential, late exponential, early decelerating and late decelerating phases of culture growth, respectively.

415

0.6

0.5

0.4

0.3

0.2 Biomass (g dry weight) (g dry Biomass 0.1

0.0 0369121518212427303336 Time (days)

0.1

0.01 Biomass (g dry weight) (g dry Biomass

0.001 0 3 6 9 121518212427303336 Time (days)

Figure A3 Results for growth of N. benthamiana hairy roots initiated using 14-day-old roots. Data are presented using linear (A) and semi-logarithmic (B) coordinates. Dashed lines indicate growth at 6, 10, 14 and 21 days after culture initiation. Error bars indicate standard errors from triplicate cultures.

416

REFERENCES

Agranovsky, A. and Morozov, S. (1999) Gene expression in positive strand RNA viruses: conventional and aberrant strategies. In: Mandahar, C. L (ed), Molecular Biology of Plant Viruses, Kluwer Academic Publishers, Massachusetts, pp 99–119. Anandalakshmi, R., Pruss, G. J., Ge, X., Marathe, R., Mallory, A. C., Smith, T. H. and Vance, V. B. (1998) A viral suppressor of gene silencing in plants. Proc. Natl. Acad. Sci. USA 95, 13079–13084. Angell, S. M. and Baulcombe, D. C. (1997) Consistent gene silencing in transgenic plants expressing a replicating potato virus X RNA. EMBO J. 16, 3675–3684. Arazi, T., Slutsky, S. G., Shiboleth, Y, M, Wang, Y., Rubinstein, M., Barak, S., Yang, J., Gal-On, A. (2001) Engineering zucchini yellow mosaic potyvirus as a non- pathogenic vector for expression of heterologous proteins in cucurbits. J. Biotechnol. 87, 67–82. Arazi, Y., Huang, P. L., Huang, P. L., Zhang, L., Shiboleth, Y. M., Gal-On, A., Lee- Huang, S. (2002) Production of antiviral and antitumor proteins MAP30 and GAP31 in cucurbits using the plant virus vector ZYMV-AGII. Biochem. Biophys. Res. Comm. 292, 441–448. Ball, E. (1967) Recovery of the tobacco callus cell and its derivatives from tobacco mosaic virus. Amer. J. Bot. 54, 639. Banerjee, S., Shang, T. Q., Wilson, A. M., Moore, A. L., Strand, S. E., Gordon, P. M. and Doty, S. L. (2002) Expression of functional mammalian P450 2E1 in hairy root cultures. Biotechnol. Bioeng. 77, 462–466. Batard, Y., Hehn, A., Nedelkina, S., Schalk, M., Pallett, K., Schaller, H., Werck- Reichart, D. (2000) Increasing expression of P450 and P450-reductase proteins from monocots in heterologous systems. Arch. Biochem. Biophys. 379, 161–169. Beachy, R. N. and Murakishi, H. H. (1970) Local lesion and viral crystal formation in tissue culture cells of different species and cultivars of Nicotiana. Phytopathology 60, 1283. Beachy, R. N. and Murakishi, H. H. (1971) Local lesion formation in tobacco tissue culture. Phytopathology 61, 877. Beachy, R. N. and Murakishi, H. H. (1973) Effect of cycloheximide on tobacco mosaic virus synthesis in callus from hypersensitive tobacco. Virology 55, 320–328.

417

Beachy, R. N. and Murakishi, H. H. (1976) Changes in soluble proteins in callus cells of hypersensitive tobacco inoculated with tobacco mosaic virus. In Vitro 12, 517–520. Beachy, R. N., Fitchen, J. H. and Hein, M. B. (1996) Use of plant viruses for delivery of vaccine epitopes. Ann. N. Y. Acad. Sci. 792, 43–49. Beachy, R. N. and Zaitlin, M. (1977) Characterization and in vitro translation of the RNAs from less-than-full-length, virus related, nucleoprotein rods present in tobacco mosaic virus preparations. Virology 81, 160–169. Belanger, H., Fleysh, N., Cox, S., Bartman, G., Deka, D., Trudel, M., Koprowski, H. and Yusibov, V. (2000) Human respiratory syncytial virus vaccine antigen produced in plants. FASEB J. 14, 2323–2328. Bendahmane, M., Koo, M., Karrer, E. and Beachy, R. N. (1999) Display of epitopes on the surface of tobacco mosaic virus: impact of charge and isoelectric point of the epitope of virus-host interactions. J. Mol. Biol. 290, 9–20. Berlin, J., Wray, V., Forche, E., Reng, H.-G., Schüler, H., Luckinger, R. and Mühlbach, H.-P. (1985) Production of potato spindle tuber viroid (PSTV) by large scale fermentation of PSTV-infected potato cell suspension cultures. J. Exp. Bot. 36, 1985–1995. Birnboim, H. C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513–1523. Bleckwenn, N. A., Bentley, W. E. and Shiloach, J. (2003) Exploring vaccina virus as a tool for large-scale recombinant protein expression. Biotechnol. Prog. 19, 130–136. Bodeutsch, T., James, E. A. and Lee, J. M. (2001) The effect of immobilization on recombinant protein production in plant cell culture. Plant Cell Rep. 20, 562–566. Bogorad, L. (2000) Engineering chloroplasts: an alternative site for foreign genes, proteins, reactions and products. TIBTECH 18, 257–263. Boothe, J. G., Saponja, J. A. and Parmenter, D. L. (1997) Molecular farming in plants: oilseeds as vehicles for the production of pharmaeutical proteins. Drug Development Res. 42, 172–181. Borisjuk, N. V., Borisjuk, L. G., Logendra, S., Petersen, F., Gleba, Y. and Ruskin, I. (1999) Production of recombinant proteins in plant root exudates. Nat. Biotechnol. 17, 466–469.

418

Boyko, V., Ferralli, J. and Heinlein, M. (2000) Cell-to-cell movement of TMV RNA is temperature-dependent and corresponds to the association of movement protein with microtubules. Plant J. 22, 315–325. Bruening, G., Beachy, R. N., Scalla, R. and Zaitlin, M. (1976) In vitro and in vivo translation of the ribonucleic acids of a cowpea strain of tobacco mosaic virus. Virology 71, 498–517. Buck, K. W. (1996) Comparison of the replication of positive-stranded RNA viruses of plants and animals. Adv. Virus Res. 47, 159–251. Buck, K. W. (1999) Replication of tobacco mosaic virus. Phil. Trans. R. Soc. Lond. B 354, 613–627. Butler, P. J. G. (1984) The current picture of the structure and assembly of tobacco mosaic virus. J. Gen. Virol. 65, 253–279. Butler, P. J. G. (1999) Self-assembly of tobacco mosaic virus: the role of an intermediate aggregate in generating both specificity and speed. Phil. Trans. R. Soc. Lond. B 354, 537–550. Butcher, D. N. and Street, H. E. (1964) Excised root culture. Bot. Rev. 30, 513–586. Cabanes-Macheteau, M., Fitchette-Lainé, A.-C., Loutelier-Bourhis, C., Lange, C., Vine, N. D., Ma, J. K. C., Lerouge, P. and Faye, L. (1999) N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants. Glycobiology 9, 365–372. Cardarelli, M., Spanò, L., Mariotti, D., Mauro, M. L., Van Sluys, M. A., Costantino, P. (1987) The role of auxin in hairy root induction. Mol. Gen. Genet. 208, 457–463. Carrington, J. C., Kasschau, K. D., Mahajan, S. K. and Schaad, M. C. (1996) Cell-to- cell and long-distance transport of viruses in plants. Plant Cell 8, 1669–1681. Carvalho, E. B., Holihan, S., Pearsall, B. and Curtis, W. R. (1997) Effect of root morphology on reactor design and operation for production of chemicals. In: Doran, P. M. (ed), Hairy Roots: Culture and Applications, Harwood Academic Publishers, Amsterdam, Netherlands, pp151–167. Casper, S. J. and Holt, C. A. (1996) Expression of the green fluorescent protein- encoding gene from a tobacco mosaic virus-based vector. Gene 173, 69–73. Chapman, S. N. (1998) Tobamovirus isolation and RNA extraction. In: Foster, G. D and Taylor, S. C. (eds), Plant virus protocols: from virus isolation to transgenic resistance, Humana Press, New Jersey, pp123–129.

419

Cheng, N.-H., Su, C.-L., Carter, S. A. and Nelson, R. S. (2000) Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana. Plant J. 23, 349–362. Chilton, M.-D., Tepfer, D. A., Petit, A., David, C., Casse-Delbart, F. and Tempé, J. (1982) Agrobacterium rhizogenes insets T-DNA into the genomes of the host plant root cells. Nature 295, 432–434. Choi, I.-R., Stenger, D. C., Morris, T. J. and French, R. (2000) A plant virus vector for systemic expression of foreign genes in cereals. Plant J. 23, 547–555. Christensen, A. H. and Quail, P. H. (1996) Ubiquitin promoter-based vectors for high- level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgen. Res. 5, 213–218. Christey, M. C. (2001) Use of RI-Mediated transformation for production of transgenic plants. Plant 37, 687–700. Citovsky, V. (1999) Tobacco mosaic virus: a pioneer of cell-to-cell movement. Phil. Trans. R. Soc. Lond. B 354, 637–642. Citovsky, V., Wong, M. L., Shaw, A. L., Prasad, B. V. V. and Zambryski, P. (1992) Visualisation and characterization of tobacco mosaic virus movement protein to single-stranded nucleic acids. Plant Cell 4, 397–411. Citovsky, V., Knorr, D., Schuster, G. and Zambryski, P. (1990) The P30 movement protein of tobacco mosaic virus is a single-stranded nucleic acid binding protein. Cell 60, 637–647. Commonwealth Myrological Institute Association of Plant Biologists: Description of plant Viruses Number 156. www.dpvweb.net/dpv/showadpv.php?dpvno=156 Copeman, R. J., Hartman, J. R. and Watterson, J. C. (1969) Tobacco mosaic virus concentration in inoculated and systemically infected tobacco leaves. Phytopathology 59, 1012–1013. Cramer, C. L., Boothe, J. G. and Oishi, K. K. (1999). Transgenic plants for therapeutic proteins: linking upstream and downstream strategies. Curr. Top. Microbiol. Immunol. 240, 95–118. Crameri, A., Whitehorn, E. A., Tate, E. and Stemmer, W. P. C. (1996) Improved green fluorescent protein by moleculer evolution using DNA shuffling. Nat. Biotechnol. 14, 315–319.

420

Culver, J. N., Lehto, K., Close, S. M., Hilf, M. E. and Dawson, W. O. (1993) Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes. Proc. Natl. Acad. Sci. USA 90, 2055–2059. Dalsgaard, K., Uttenthal, Ǻ, Jones, T. D., Xu, F., Merryweather, A., Hamilton, W. D. O., Langeved, J. P. M., Boshuizen, R. S., Kamstrup, S., Lomonossoff, C. P., Vela, C., Casal, J. I., Meloen, R. H. and Rodgers, P. B. (1997) Plant-derived vaccine protects target animals against a viral disease. Nat. Biotechnol. 15, 248–252. Daniell, H., Vivekananda, J., Nielsen, B. L., Ye, G. N., Tewari, K. K. and Sanford, J. C. (1990) Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors. Proc. Natl. Acad. Sci. USA 87, 88–92. Daniell, H. and Dhingra, A. (2002) Multigene engineering: dawn of an exciting new era in biotechnology. Curr. Opin. Biotech. 13, 136–141. Daniell, H., Carmona-Sanchez, O. and Burns, B. E. (2004) Chloroplast derived antibodies, biopharmaceuticals and edible vaccines. In: Fischer, R. and Schillberg, S. (eds), Molecular Farming: Plant-made Pharmaceuicals and Technical Proteins, Wiley-VCH Verlag GmbH and Co., Weinheim, pp 113–134. David, K. M. and Perrot-Rechemann, C. (2001) Characterisation of a tobacco Bright Yellow 2 cell line expression the tetracycline repressor at a high level for strict regulation of transgene expression. Plant Physiol. 125, 1548–1553. Dawson, W. O., Bubrick, P. and Grantham, G. L. (1988) Modification of the tobacco mosaic virus coat protein affecting replication, movement, and symptomology. Phytopathology 78, 783–789. Dawson, W. O., Lewandowski, D. J., Hilf, M. E., Bubrick, P., Raffo, A. J., Shaw, J. J., Grantham, G. L. and Desjardins, P. R. (1989) A tobacco mosaic virus-hybrid expresses and loses an added gene. Virology 172, 285–292. Dawson, W. O. (1992) Tobamovirus–plant interactions. Virology 186, 359–367. De Cosa, B., Moar, W., Lee, S.-B., Miller, M. and Daniell, H. (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat. Biotechnol. 19, 71–74. Dee, K. U. and Shuler, M. L. (1997) Optimisation of an assay for baculovirus titer and design of regimens for the synchronous infection of insect cells. Biotechnol. Prog. 13, 14–24.

421

Della-Cioppa, G. and Grill, L. K. (1996) Production of novel compounds in higher plants by transfection with RNA viral vectors. Ann. N. Y. Acad. Sci. 792, 57–61. Deom, C. M., Oliver, M. J. and Beachy, R. N. (1987) The 30-kilodalton gene product of tobacco mosaic virus potentiates virus movement. Science 237, 389–394. De Paolis, A., Mauro, M. L., Pomponi, M., Cardarelli, M., Spanò, L. and Costantino, P.

(1985) Localization of agropine-synthesizing functions in the TR region of the root- inducing plasmid of Agrobacterium rhizogenes 1855. Plasmid 13, 1–7. Dijkstra, J. and de Jager C. P (1998) Practical Plant Virology: Protocols and exercises. Springer-Verag, Heidelberg. Ding, S.-W., Rathjen, J. P., Li, W.-X., Swanson, R., Healy, H. and Symons, R. H. (1995) Efficient infection from cDNA clones of cucumber mosaic cucumovirus RNAs in a new Plasmid Vector. J. Gen. Virol. 76, 459–464. Dirnberger, D., Steinkeller, H., Abdennebi, L., Remy, J.-J. and van de Wiel, D. (2001) Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants. Eur. J. Biochem. 268, 4570–4579. Divéki, Z., Salánki, K. and Balázs, E. (2002) Limited utility of blue fluorescent protein (BFP) in monitoring plant virus movement. Biochimie 84, 997–1002. Donson, J., Kearney, C. M., Hilf, M. E. and Dawson, W. O. (1991) Systemic expression of a bacterial gene by tobacco mosaic virus-based vector. Proc. Natl. Acad. Sci. USA 88, 7204–7208. Doran, P. M. (2006a) Loss of secreted antibody from transgenic plant tissue cultures due to surface adsorption. J. Biotechnol. 122, 39–54. Doran, P. M. (2006b) Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol. 24, 426–432. Ehlers, U., Commandeur, U., Frank, R., Landsmass, J., Koenig, R. and Burgermesiter, W. (1991) Cloning of the coat protein gene from beet necrotic yellow vein virus and its expression in sugar beet hairy root. Theor. Appl. Genet. 81, 777–782. Evans, N. A. and McLaren, A. D. (1969) Quantum Yeilds for Inactivation of Tobacco Mosaic Virus Nucleic Acid by Ultraviolet Radiation (254 nm.) J. Gen. Virol. 4, 55–63. Falk, L. R. and Doran, P. M. (1996) Influence of inoculum morphology on growth of Atropa belladonna hairy roots and production of tropane alkaloids. Biotechnol. Lett. 18, 1099–1104.

422

Farran, I., Sánchez-Serrano, J. J., Medina, J. F., Prieto, J. and Mingo-Castel, A. M. (2002) Targeted expression of human serum albumin to potato tubers. Transgen. Res. 11, 337–346. Fecker, L. F., Kaufmann, A., Commandeur, U., Commandeur, J., Koenig, R. and Burgermeister, W. (1996) Expression of single-chain antibody fragment (scFv) specific for beet necrotic yellow vein virus coat protein or 25 kDa protein in Escherichia coli and Nicotiana benthamiana. Plant Mol. Biol. 32, 979–986. Fernández- Fernández, M. R., Martinez-Torrecuadrada, J. L., Casal, J. I. and Garcia, J. A. (1998) Development of an antigen presentation system based on plum pox potyvirus. FEBS Lett. 427, 229–235. Firek, S., Draper, J., Owen, M. R. L., Gandecha, A., Cockburn, B. and Whitelam, G. C. (1993) Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures. Plant Mol. Biol. 23, 861–870. Fischer, R., Emans, N., Schuster, F., Hellwig, S. and Drossard, J. (1999a) Towards molecular farming in the future: using plant-cell-suspension cultures as bioreactors. Biotechnol. Appl. Biochem. 30, 109–112. Fischer, R., Liao, Y.-C. and Drossard, J. (1999b) Affinity-purification of a TMV- specific recombinant full-size antibody from a transgenic tobacco suspension culture. J. Immunol. Meth. 226, 1–10. Fitzmaurice, W. P. (2002) Interspecific Nicotiana hybrids and their progeny. US Patent 6,344,597 B1. Francisco, J. A., Gawlak, S. L., Miller, M., Bathe, J., Russell, D., Chace, D., Mixan, B., Zhao, L., Fell, H. P. and Siegall, C. B. (1997) Expression and characterization of Bryodin 1 and a Bryodin 1-Based single-chain immunotoxin from tobacco cell culture. Bioconjugate Chem. 8, 708–713. Fulton, R. W. (1967) Transmision of plant viruses by grafting, dodder, seed and mechanical inoculation. In: Corbett, M. K. and Sisler, H. D. (eds), Plant Virology, University of Florida Press, Gainesville, pp 54–60. Gallie, D. R. (1996) Translational control of cellular and viral mRNAs. Plant Mol. Biol. 32, 145–158. Galun, E. (2005) RNA silencing in plants. Plant 41, 113–123 Galun, E. and Galun, E. (2001) The manufacture of medical and health products by transgenic plants, Imperial college press, London.

423

Gibbs, A. and Harrison, B. (1976) Plant Virology: the Principles. Edward Arnold, London. Gilleland Jr, H. E., Gilleland, L. B., Staczek, J., Harty, R. N., García-Sastre, A., Palese, P., Brennan, F. R., Hamilton, W. D. O., Bendahmane, M. and Beachy, R. N. (2000) Chimeric animal and plant virus expressing epitopes of outer membrane protein F as a combined vaccine against Pseudomonas aeruginosa lung function. FEMS Immunol. Med. Microbiol. 27, 291–297. Girard, L. S., Bastin, M. and Courtois, D. (2004) Expression of the human milk protein sCD14 in tobacco plant cell culture. Plant Cell Tiss. Organ Cult. 4621, 1–8. Giri, A. and Narasu, M. L. (2000) Transgenic hairy roots: recent trends and applications. Biotechnol. Adv. 18, 1–22. Gleave, A. P., Mitra, D. S., Markwick, N. P., Morris, B. A. M. and Beuning, L. L. (1998) Enhanced expression of the Bacillus thuringiensis cry9Aa2 gene in transgenic plants by nucleotide sequence modification confers resistance to potato tuber moth. Mol. Breed. 4, 459–472. Gleba, Y., Marillonnet, S. and Klimyuk, V. (2004) Engineering viral expression vectors for plants: the ‘full virus” and the “deconstructed virus” strategies. Curr. Opin. Plant Bio. 7, 182–188. Goelet, P., Lomonossoff, G. P., Butler, P. J. G., Akam, M. E., Gait, M. J. and Karn, J. (1982) Nucleotide sequence of tobacco mosaic virus RNA. Proc. Natl. Acad. Sci. USA 79, 5818–5822. Gooding Jr, G. V. and Hebert, T. T. (1967) A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology 57, 1285. Gomord, V., Fitchette, A.-C., Lerouge, P. and Faye, L. (2004) Glycosylation of plant- made pharmaceuticals. In: Fischer, R. and Schillberg, S. (eds), Molecular Farming: Plant-made Pharmaceuicals and Technical Proteins, Wiley-VCH Verlag GmbH and Co., Weinheim, pp 233–250. Gopinath, K., Wellink, J., Porta, C., Taylor, K. M., Lomonossoff, G. P. and van Kammen, A. (2000) Engineering cowpea mosaic virus RNA-2 into a vector to express heterologous proteins in plants. Virology 267, 159–173. Guo, H. S., López-Moya, J. J. and Garcia, A. (1998) Susceptibility to recombination rearrangement of a chimeric plum pox potyvirus genome after insertion of a foreign gene. Virus Res. 57, 183–195.

424

Halliwell, R. S. and Gazaway, W. S. (1975) Quantity of microinjected tobacco virus required for infection of single cultured tobacco cells. Virology 65, 583–587. Hamamoto, H., Sugiyama, Y., Nakagawa, N., Hashida, E., Matsunaga, Y., Takemoto, S., Watanabe, Y. and Okada, Y. (1993) A new tobacco mosaic virus vector and its use for the systemic production of angiotensin-I-converting enzyme inhibitor in transgenic tobacco and tomato. Bio/Technology 11, 930–932. Hansen, J. A. and Hildebrandt, A. C. (1966) The distribution of tobacco mosaic virus in plant callus cultures. Virology 28, 15–21. Harrison, B. D. and Mayo, M. A. (1983) The use of protoplasts in plant virus research. In: Helgeson, J. P. and Deverall, B. J. (eds), Use of tissue culture and protoplasts in plant pathology, Academic Press, Australia, pp 69–137. Harrison, B. D. and Wilson, T. M. A. (1999) Milestone in the research on tobacco mosaic virus. Phil. Trans. R. Soc. Lond. B 354, 521–529 Haynes, J. R., Cunningham, J., von Seefried, A., Lennick, M., Garvin, R. T. and Shen, S.-H. (1986) Development of a genetically-engineered, candidate polio vaccine employing the self-assembling properties of the tobacco mosaic virus coat protein. Bio/Technology 4, 637–641. Heinlein, M., Epel, B. L., Padgett, H. S and Beachy, R. N. (1995) Interaction of tobamovirus movement proteins with the plant cytoskeleton. Science 270, 1983–1985. Heinlein, M., Padget, H. S., Gens, J. S., Pickard, B. G., Casper, S. J., Epel, B. L and Beachy, R. N. (1998) Changing patterns of localisation of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection. Plant Cell 10, 1107–1120. Hellwig, S., Drossard, J., Twyman, R. M. and Fischer, R. (2004) Plant cell cultures for the production of recombinant proteins. Nat. Biotechnol. 22, 1415–1422. Hendy, S. Chun Chen, Z., Barker, H., Santa Cruz, S., Chapman, S., Torrance, L., Cockburn, W. and Whitelam, G. C. (1999) Rapid production of single-chain Fv fragments in plants using a potato virus X episomal vector. J. Immunol. Meth. 231, 137–146. Henzler, H.-J. and Schedel, M. (1991) Suitability of the shaking flask for oxygen supply to microbiological cultures. Bioprocess Eng. 7, 123–131.

425

Hiatt, A., Cafferkey, R. and Bowdish, K. (1989) Production of antibodies in transgenic plants. Nature 342, 76–78. Hibberd, J. M., Linley, P. J., Khan, M. S. and Gray, J. C. (1998) Transient expression of green fluorescent protein in various plastid types following microprojectile bombardment. Plant J. 16, 627–632. Hilf, M. E. and Dawson, W. O. (1993) The tobamovirus capsid protein functions as a host-specific determinant of long-distance movement. Virology 193, 106–114 Hill, S. J. L., Warren, M. K. and Phillips, M. K. (1990) The effect of culture parapeters on viral antigen accumulation in plant cell cultures inoculated with TMV. J. Exp. Bot. 41(supplement), 6. Hills, G. J., Plaskitt, K. A., Young, N. D., Dunigan, D. D., Watts, J. W., Wilson, T. M. A. and Zaitlin, M. (1987) Immunogold localization of the intracellular sites of structural and nonstructural tobacco mosaic virus proteins. Virology 161, 488–496. Hong, S.-Y., Kwon, T.-H., Lee, J.-H., Jang, Y.-S. and Yang, M.-S. (2002) Production of biologically active hG-CSF by transgenic plant cell suspension culture. Enzyme Microb. Technol. 30, 763–767. Huang, J., Nandi, S., Wu, L., Yalda, D., Bartley, G., Rodriguez, R., Lonnerdal, B. and Huang, N. (2002a) Expression of natural human lysozyme in rice grains. Mol. Breed. 10, 83–94. Huang, J., Sutliff, T. D., Wu, L., Nandi, S., Benge, K., Terashima, M, Ralston, A. H., Drohan, W., Huang, N. and Rodriguez, R. L. (2001) Expression and purification of functional human α-1-antitrypsin from cultured plant cells. Biotechnol. Prog. 17, 126–133. Huang, J., Wu, L., Yalda, D., Adkins, Y., Kelleher, S. L., Crane, M., Lonnerdal, B., Rodriguez, R. L. and Huang, N. (2002b) Expression of functional recombinant human lysozyme in transgenic rice cell culture. Transgen. Res. 11, 229–239. Huber, R., Hontelez, J., and van Kammen, A. (1981) Infection of cowpea protoplasts with both the common strain and the cowpea strain of TMV. J. Gen. Virol. 55, 241–245. Hughes, E. H., Hong, S.-B., Shanks, J. V., San, K.-Y. and Gibson, S. I. (2002) Characterization of an inducible promoter system in Catharanthus roseua hairy roots. Biotechnol. Prog. 19, 1183–1186.

426

Hunter, T. R., Hunt, T., Knowland, J. and Zimmern, D. (1976) Messenger RNA for the coat protein of tobacco mosaic virus. Nature 260, 759–769. International Maize and Wheat Improvement Centre (www.cimmyt.cgiar.org/abc/protocols/plasmidminipreps.pdf) Jagadish, M. N., Hamilton, R. O., Fernandez, C. S., Schoofs, P., Davern, K. M., Kalnins, H., Ward, C. W. and Nisbet, I. T. (1993) High level production of hybrid potyvirus-like particles carrying repetitive copies of foreign antigens in Escherichia coli. Bio/Technology 11, 1166–1170. James, E. A., Wang, C., Wang, Z., Reeves, R., Shin, J. S., Magnuson, N. S. and Lee, J. M. (2000) Production and characterisation of biologically active human GM-CSF secreted by genetically modified plant cells. Protein Expr. Purif. 19, 131–138. James, E., Mills, D. R. and James E. L. (2002) Increased production and recovery of secreted foreign proteins from plant cell cultures using an affinity chromatography bioreactor. Biochem. Eng. J. 12, 205–213. Joelson, T., Ǻkerblom, L., Oxelfelt, P., Standberg, B. Tomenius, K. and Morris, T. J. (1997) Presentation of a foreign peptide on the surface of tomato bushy stunt virus. J. Gen. Virol. 78, 1213–1217. Johnson, J., Lin, T. and Lomonossoff, G. (1997) Presentation of heterologous peptides on plant viruses: genetics, structure and function. Annu. Rev. Phytopathol 35, 67–86. Jonard, G., Richards, K. E., Guilley, H and Hirth, L. (1977) Sequence from the assembly nucleation region of TMV RNA. Cell 11, 483–493. Jones, R. A., Rupert, E. A. and Barnett, O. W. (1981) Virus infection of Trifolium species in cell suspension cultures. Phytopathology 71, 116–119. Joshi, S., Pleij, C. W. A., Haenni, A. L., Chapeville, F and Bosch, L. (1983) Properties of the tobacco mosaic virus intermediate length RNA-2 and its translation. Virology 127, 100–111. Jouanin, L., Guerche, P., Pamboukdjian, N., Tourneur, C., Casse Delbart, F. and Tourneur, J. (1987) Structure of T-DNA in plants regenerated from roots transformed by Agrobacterium rhizogenes strain A4. Mol. Gen. Genet. 206, 387–392.

427

Jung, S., Behie, L. A., Lee, P. W. K. and Farrell, P. J. (2004) Optimisation of reovirus production from mouse L-929 cells in suspension culture. Biotechnol. Bioeng. 85, 750–760. Kado, C. I. (1972) Mechanical and Biological Inoculation principles. In: Kado, C. I. and Agrawal, H. O. (eds), Principles and Techniques in Plant Virology. Van Nostrand Reinhold, New York, pp 3–31. Kanokwaree, K. and Doran, P. M. (1997) The extent to which external oxygen transfer limits growth in shake flask culture of hairy roots. Biotechnol. Bioeng. 55, 520–526. Karpova, O. V., Ivanov, K. I., Rodionova, N. P., Dorokhov, YU., L. and Atabekov, J. G. (1997) Nontranslatability and dissimilar behaviour in plants and protoplasts of virus RNA and movement protein complexes formed in vitro. Virology 230, 11–21. Kassanis, B. (1957) The multiplication of tobacco mosaic virus in cultures of tumorous tobacco tissues. Virology 4, 5–13. Kassanis, B., Tinsley, T. W. and Quak, F. (1958) The inoculation of tobacco callus tissue with tobacco mosaic virus. Ann. Appl. Biol. 46, 11–19. Kassanis, B. and Macfarlane, I. (1964) Transmission of tobacco necrosis virus to tobacco callus tissues by zoospores of Olpidium brassicae. Nature 201, 218–219. Kassanis, B. (1967) Plant Tissue Culture. In: Maromorosch, K. and Koprowski, H. (eds), Methods in Virology Volume 1, Academic Press, New York, pp 537–564. Kay, R., Chan, A., Daly, M. and McPherson, J. (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236, 1299–1302. Kearney, C. M., Donson, J., Jones, G. E. and Dawson, W. O. (1993) Low level of genetic drift in foreign sequences replicating in an RNA virus in plants. Virology 192, 11–17. Keller, G. and Warrack, B. (1991) Essentials of business statistics: a systematic approach, Wadsworth Publishing Company, California. Khoudi, H., Laberge, S., Ferullo, J.-M., Bazin, R., Darveau, A., Castonguay, Y., Allard, G., Lemieux, R. and Vézina, L.-P. (1999) Production of a diagnostic monoclonal antibody in perennial alfalfa plants. Biotechnol. Bioeng. 64, 135–143.

428

Kieran, P. M., MacLoughlin, P. F. and Malone, D. M. (1997) Plant cell suspension cultures: some engineering considerations. J. Biotechnol. 59, 39–52. Kim, K.-Y., Kwon, S.-Y., Lee, H.-S., Hur, Y., Bang, J.-W. and Kwak, S.-S. (2003) A novel oxidative stress-inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco plants and cultured cells. Plant Mol. Biol. 51, 831–838. Kim, Y. K., Wyslouzil, B. E. and Weathers, P. J. (2002) Secondary metabolism of hairy root cultures in bioreactors. Plant 38, 1–10. Kino-Oko, M., Hitaka, Y., Ninomiya, K., Taya, M., Tone, S. (1999) Segmentation of plant hairy roots promotes lateral root emergence and subsequent growth. J. Biosci. Bioeng. 88, 690–692. Koivu, K. (2004) Novel sprouting technology for recombinant protein production. In: Fischer, R. and Schillberg, S. (eds), Molecular Farming: Plant-made Pharmaceuicals and Technical Proteins, Wiley-VCH Verlag GmbH and Co., Weinheim, pp 37–53. Koprowski, H. and Yusibov, V. (2001) The green revolution; plants as heterologous expression vectors. Vaccine 19, 2735–2741. Krebitz, M., Wiedermann, U., Essl, D., Steinkellner, H., Wagner, B., Turpen, T. H., Ebner, C., Scheiner, O. and Breiteneder, H. (2000) Rapid production of the major birch pollen allergen BET v 1 in Nicotiana benthamiana plants and its immunological in vitro and in vivo characterization. FASEB J. 14,1279–1288. Krebitz, M., Wagner, B., Ferreira, F., Peterbauer, C., Campillo, N., Witty, M., Kolarich, D., Steinkellner, H., Scheiner, O., Breiteneder, H., (2003) Plant-based heterologous expression of MAL d 2, a thaumatin-like protein and allergen of apple (Malus domestica), and its characterization as an antifungal protein. J. Mol. Biol. 329, 721–730. Kumagai, M. H., Turpen, T. H., Weinzettl, N., della-Cioppa, G., Turpen, A. M., Donson, J., Hilf, M. E., Grantham, G. L., Dawson, W. O., Chow, T. P., Piatak Jr., M. and Grill, L. K. (1993) Rapid, high-level expression of biologically active α- trichosanthin in transfected plants by an RNA viral vector. Proc. Natl. Acad. Sci. USA 90, 427–430.

429

Kumagai, M. H., Donson, J., Della-Cioppa, G., Harvey, D., Hanley, K. and Grill, L. K. (1995) Cytoplasmic inhibition of carotenoid biosyntheses with virus-derived RNA. Proc. Natl. Acad. Sci. USA 92, 1679–1683. Kumagai, M. H., Donson, J., della-Cioppa, G. and Grill, L. K. (2000) Rapid, high-level expression of glycosylated rice α-amylase in transfected plants by an RNA viral vector. Gene 245, 169–174. Kumar, S., Dhingra, A. and Daniell, H. (2004) Plastid-expressed Betaine Aldehyde Dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance. Plant Physiol. 136, 2843–2854. Kumar, S. G. B., Ganapathi, T. R., Revathi, C. J., Prasad, K. S. N. and Bapat, V. A. (2003) Expression of hepatitis B surface antigen in tobacco cell suspension cultures. Protein Expr. Purif. 32, 10–17. Kurata, H., Takemura, T., Furusaki, S. and Kado, C. (1998) Light-controlled expression of a foreign gene using the chalcone synthase promoter in Tobacco BY-2 cells. J. Ferment. Bioeng. 86, 317–323. Kusnadi, A. R., Nikolova, Z. L. and Howard, J. A. (1997) Production of recombinant proteins in transgenic plants: practical considerations. Biotechnol. Bioeng. 56, 473– 483. Kwon, T.-H., Shin, Y.-M., Kim, Y.-S., Jang, Y.-S. and Yang, M.-S. (2003a) Secretory production of hGM-CSF with a high specific biological activity by transgenic plant cell suspension culture. Biotechnol. Bioprocess Eng. 8, 135–141. Kwon, T. H., Seo, J. E., Lee, J. H., Jang, Y. S. and Yang, M. S. (2003b) Expression and secretion of the heterologous protein Interleukin-12 in plant cell suspension culture. Biotechnol. Bioeng. 81, 870–875. Kwon, T.-H., Kim, Y.-S., Lee, J.-H. and Yang, M.-S. (2003c) Production and secretion of biologically active human granulocyte-macrophage colony stimulating factor in transgenic tomato suspension cultures. Biotechnol. Lett. 25, 1571–1574. LaCount, W., An, G. and Lee, J., M. (1997) The effect of polyvinylpyrrolidone (PVP) on the heavy chain monoclonal antibody production from plant suspension cultures. Biotechnol. Lett. 19, 93–96. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

430

Larkin, P. J. and Scowcroft, W. R., (1981) Somaclonal variation – A novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60, 197–214. Lawrence, S. D and Novak, N. G. (2001) A rapid method for the production and characterization of recombinant insecticidal proteins in plants. Mol. Breed. 8, 139– 146. Lee, J.-H., Kim, N.-S., Kwon, T.-H., Jang, Y.-S. and Yang, M.-S. (2002) Increased production of human granulocyte-macrophage colony stimulating factor (hGM- CSF) by the addition of stabilizing polymer in plant suspension cultures. J. Biotechnol. 96, 205–211. Lehto, K., Bubrick, P. and Dawson, W. O. (1990a) Time course of TMV 30K protein accumulation in intact leaves. Virology 174, 290–293. Lehto, K., Grantham, G. L. and Dawson, W. O. (1990b) Insertion of sequences containing the coat protein subgenomic RNA promoter and leader in front of the tobacco mosaic virus 30K ORF delays its expression and causes defective cell-to- cell movement. Virology 174, 145–157. Leisner, S. M. (1999) Molecular basis of virus transport in plants. In: Mandahar, C. L (eds), Molecular Biology of Plant Viruses, Kluwer Academic Publishers, Boston, pp 161–181. Lesney, M. S. and Murakishi, H. H. (1981) Infection of soybean protoplasts from cell suspension culture with pod mottle virus. J. Gen. Virol. 57, 387–395. Lim, A. L., Tachibana, S., Watanabe, Y. and Wong, S. M. (2002) Expression and purification of a neuropeptide nocistatin using two related plant viral vectors. Gene 289, 69–79. Liu, F. and Lee, J. M. (1999) Effect of culture conditions on monoclonal antibody production from genetically modified tobacco suspension cultures. Biotechnol. Bioprocess Eng. 4, 259–263 Liu, L. and Lomonossoff, G. P. (2002) Agroinfection as a rapid method for propagating Cowpea mosaic virus-based constructs. J. Virol. Methods 105, 343–348. Locke, D. M. (1974) Viruses: the smallest enemy. Crown Publishers, New York. Loomis, W. D. (1974) Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. In: Fleischer, S. and Parker, L. (eds) Methods in Enzymology XXXI. Academic Press, New York, 528-555.

431

López-Moya, J. J., Fernández- Fernández, M. R., Cambra, M. and García, J. A. (2000) Biotechnological aspects of plum pox virus. J. Biotechnol. 76, 121-136. Luria, S. E., Williams, R. C. and Backus, R. C. (1951) Electron micrographic counts of bacteriophage particles. J. Bacteriol. 61,179–188. McCormick, A. A., Kumagai, M. H., Hanley, K., Turpen, T. H., Hakim, I., Grill, L. K., Tusé, D., Levy, S. and Levy, R. (1999) Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitope in tobacco plants. Proc. Natl. Acad. Sci. USA 96, 703–708. McCormick, A. A., Reinl, S. J., Cameron, T. I., Vojdani, F., Fronefield, M., Levy, R. and Tusé, D. (2003) Individualised human scFv vaccines produced in plants: humoral anti-idiotype responses in vaccinated mice confirm relevance to the tumor Ig. J. Immunol. Meth. 278, 95–104. McInnes, E., Davey, M. R., Mulligan, B. J., Davies, K., Sargent, A. W and Morgan, A. J. (1989) Use of a disarmed Ri plasmid vector in analysis of transformed root induction. J. Exp. Bot. 40, 1135–1144. McLain, L., Porta, C., Lomonossoff, G. P., Durrani, Z. and Dimmock, N. J. (1995) Human immunodeficiency virus type 1-neutralizing antibodies raised to a glycoprotein 41 peptide exposed on the surface of a plant virus. AIDS Res. Hum. Retroviruses 11, 327–334. McLean, B. G., Zupan, J. and Zambryski, P. C. (1995) Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells. Plant Cells 7, 2101–2114. MacFarlane, S. A. and Popovich, A. H. (2000) Efficient expression of foreign proteins in roots from tobravirus vectors. Virology 267, 29–35. Magnuson, N. S., Linzmaier, P. M., Gao, J.-W., Reeves, R., An, G. and Lee, J. M. (1996) Enhanced recovery of a secreted mammalian protein from suspension culture of genetically modified tobacco cells. Protein Expr. Purif. 7, 220–228. Magnuson, N. S., Linzmaier, P. M., Reeves, R., An, G., HayGlass, K. and Lee, J. M. (1998) Secretion of biologically active human Interleukin-2 and Interleukin-4 from genetically modified tobacco cells in suspension culture. Protein Expr. Purif. 13, 45–52.

432

Mallory, A. C., Parks, G., Endres, M. W., Baulcombe, D., Bowman, L. H., Pruss, G. J. and Vance, V. B. (2002) The amplicon-plus system for high-level expression of transgenes in plants. Nat. Biotechnol. 20, 622–625. Marathe, R., Anandalakshmi, R., Smith, T. H., Pruss, G. J. and Vance, V. B. (2000) RNA viruses as inducers, suppressors and targets or post-transcriptional gene silencing. Plant Mol. Biol. 43, 295–306. Masuta, C., Yamana, T., Tacahashi, Y., Uyeda, I., Sato, M., Ueda, S. and Matsumura, T. (2000) Development of clover yellow vein virus as an efficient stable gene- expression system for legume species. Plant J. 23, 539–546. Matsumoto, S., Ikura, K., Ueda, M. and Sasaki, R. (1995) Characterisation of a human glycoprotein (erythropoietin) produced in cultured tobacco cells. Plant Mol. Biol. 27, 1163–1172. Meyer, P., Heidmann, I., and Niedenhof, I. (1992) The use of African cassava mosaic virus as a vector system for plants. Gene 110, 213–217. Miller, I. and Miller, M. L. (2004) John E. Freund’s mathematical statistics with applications 7th edition. Pearson Prentice Hall, New Jersey, pp 505–507. Mitsuhashi, J. and Maramorosch, K. (1964) Inoculation of plant tissue cultures with aster yellow virus. Virology 23, 277–279. Modelska, A., Dietzschold, B., Sleysh, N., Fu, Z. F., Steplewski, K., Hooper, D. C., Koprowski, H. and Yusibov, V. (1998) Immunization against rabies with plant- derived antigen. Proc. Natl. Acad. Sci. USA 95, 2481–2485. Molina, A., Hervás-Stubbs, S., Daniell, H., Mingo-Castel, A. M. and Veramendi, J. (2004) High-yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts. Plant Biotechnol. J. 2, 141–153. Mor, T. S., Sum-Moon, Y., Palmer, K. E. and Mason, H. S. (2003) Geminivirus vectors for high level expression of foreign proteins in plant cells. Biotechnol. Bioeng. 81, 430–437. Mori, M., Fujihara, N., Mise, K. and Furusawa, I. (2001) Inducible high-level mRNA amplification system by viral replicase in transgenic plants. Plant J. 27, 79–86. Mühlbach, H.-P. (1982) Plant cell cultures and protoplasts in plant virus research. Curr. Top. Microbiol. Immunol. 99, 81–129.

433

Mühlbach, H.-P. and Sänger, H. L. (1981) Continuous replication of potato spindle tuber viroid (PSTV) in permanent cell cultures of potato and tomato. Biosci. Rep. 1, 79–87. Murakishi, H. H. (1968) Infection of tomato callus cells in suspension with TMV-RNA. Phytopathology 58, 993–996. Murakishi, H. H. and Carlson, P. S. (1982) In vitro selection of Nicotiana sylvestris variants with limited resistance to TMV. Plant Cell Rep. 1, 94–97. Murakishi, H. H., Hartmann, J. X., Beachy, R. N. and Pelcher, L. E. (1971) Growth curve and yield of tobacco mosaic virus in tobacco callus cells. Virology 43, 62–68. Murakishi, H. H., Hartmann, J. X., Pelcher, L. E and Beachy, R. N. (1970) Improved inoculation of cultured plant cells resulting in high virus titre and crystal formation. Virology 41, 365–367. Murray, C., Sutherland, P. W., Phung, M. M., Lester, M. T., Marshall, R. K. and Christeller, J. T. (2002) Expression of biotin-binding proteins, avidin and streptavidin, in plant tissues using plant vacuolar targeting sequences. Transgen. Res. 11, 199–214. Namba, K., Pattanayek, R. and Stubbs, G. (1989) Visulaization of protein-nucleic acid interactions in a virus. J. Mol. Biol. 208, 307–325. Needham, P. D., Atkinson, R. G., Morris, B. A. M., Gardner, R. C. and Gleave, A. P. (1998) GUS expression patterns from a tobacco yellow dwarf virus-based episomal vector. Plant Cell Rep. 17, 631–639. Nelson, R. S. and van Bel, A. J. E. (1998) The mystery of virus trafficking into, through and out of vascular tissue. Prog. Bot. 59, 476–533 Nemchinov, L. G., Liang, T. J., Rifaat, M. M., Mazyad, H. M., Hadidi, A. and Keith, J. M. (2000) Development of a plant-derived subunit vaccine candidate against hepatitis C virus. Arch. Virol. 145, 2557–2573. Ni, M., Cui, D., Einstein, J., Narasimhulu, S., Vergara, C. E. and Gelvin, S. B. (1995) Strength and tissue specificity of chimeric promotors derived from the octopine and mannopine synthase gene. Plant J. 7, 661–676.

434

Nicholas, B. L., Brennan, F. R., Martinez-Torrecuadrada, J. L., Casal, J. I., Hamilton, W. D. and Wakelin, D. (2002) Characterization of the immune response to canine parvovirus induced by vaccination with chimaeric plant viruses. Vaccine 20, 2727–2734. Nims, R. C., Halliwell, R. S. and Rosberg, D. W. (1967) Disease development in cultured cells of Nicotiana tabacum L. var. Samsun NN injected with tobacco mosaic virus. Cytologia 32, 224–235. Nims, C., Halliwell, R. S. and Rosberg, D. W. (1966) Disease development in cultured cells of Nicotiana tabacum ‘Samsun NN’ injected with tobacco mosaic virus. Phytopathology 56, 892–893. Nishiguchi, M., Langridge, W. H. R., Szalay, A. A. and Zaitlin, M. (1986) Electroporation-mediated infection of tobacco leaf protoplasts with tobacco mosaic virus RNA and cucumber mosaic virus RNA. Plant Cell Rep. 5, 57–60. Noordam, D. (1973) Identification of Plant Viruses. Methods and Experiments. Centre for Agricultural Publishing and Documentation, Wageningen, The Netherlands. Nurkiyanova, K. M., Ryabov, E. V., Commandeur, U., Duncan, G. H., Canto, T., Gray, S. M., Mayo, M. A. and Taliansky, M. E. (2000) Tagging Potato leafroll virus with the jellyfish green fluorescent protein gene. J. Gen. Virol. 81, 617–626. Odell, J. T., Nagy, F. and Chua, N.-H. (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313, 810–812. Okada, Y. (1999) Historical overview of research on the tobacco mosaic virus genome: genome organisation, infectivity and gene manipulation. Phil. Trans. R. Soc. Lond. B 354, 569–582. Oparka, K. J., Prior, D. A. M., Santa Cruz, S., Padgett, H. S. and Beachy, R. N. (1997) Gating of epidermal plasmosesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV). Plant J. 12, 781–789. Oparka, K. J. and Roberts, A. G. (2001) Plasmodesmata. A not so open-and-shut case. Plant Physiol. 125, 123–126. Osman, T. A. M. and Buck, K. W. (1997) The tobacco mosaic virus RNA polymerase complex contains a plant protein related to the RNA-binding subunit of yeast eIF-3. J. Virol. 71, 6075–6082.

435

Otsuki, Y., Shimomura, T. and Takebe, I. (1972) Tobacco mosaic virus multiplication and expression of the N gene in necrotic responding tobacco varieties. Virology 50, 45–50. Padidam, M. (2003) Chemically regulated gene expression in plants. Curr. Opin. Plant Bio. 6, 169–177. Palmer, K. E., Thompson, J.A. and Rybicki, E. P. (1999) Generation of maize cell lines containing autonomously replicating maize streak virus-based vectors. Arch. Virol. 144, 1345–1360. Payne, G. (1991) Plant Cell and Tissue Culture in Liquid Systems. Carl Hanser Verlag, Munich. Pelcher, L. E., Murakishi, H. H. and Hartmann, J. X. (1972) Kinetics of TMV-RNA synthesis and its correlation with virus accumulation and crystalline viral inclusion formation in tobacco tissue culture. Virology 47, 787–796. Pelham, H. R. B. (1978) Leaky UAG termination codon in tobacco mosaic virus RNA. Nature 272, 469–471. Pérez Filgueira, D. M., Zamorano, P. I. Domínguez, M. G., Taboga, O., Del Médico Zajac, M. P., Puntel, M., Romera, S. A., Morris, T. J., Borca, M. V. and Sadir, A. M. (2003) Bovine herpes virus gD protein produced in plants using a recombinant tobacco mosaic virus (TMV) vector possesses authentic antigenicity. Vaccine 21, 4201–4209. Perlak, F. J., Fuchs, R. L., Dean, D. A., McPherson, S. L. and Fischhoff, D. A. (1991) Modification of the coding sequence enhances plant expression of insect control protein genes. Proc. Natl. Acad. Sci. USA 88, 3324–3328. Phillips, R. L., Kaeppler, S. M. and Olhoft, P. (1994) Genetic instability of plant tissue cultures: breakdown of normal controls. Proc. Natl. Acad. Sci. USA 91, 5222– 5226. Pogue, G. P., Lindbo, J. A., Garger, S. J. and Fitzmaurice, W. P. (2002) Making an ally from and enemy: plant virology and the new agriculture. Annu. Rev. Phytopathol. 40, 45–74. Porta, C., Spall, V. E., Findlay, K. C., Gergerich, R. C., Farrance, C. E. and Lomonossoff, G. P. (2003) Cowpea mosaic virus-based chimeras: Effects of inserted peptides on the phenotype, host range, and transmissibility of the modified viruses. Virology 310, 50–63.

436

Porta, C., Spall, V. E., Lin, T., Johnson, J. E. and Lomonossoff, G. P. (1996) The development of cowpea mosaic virus as a potential source of novel vaccines. Intervirology 39, 79–84. Porta, C., Spall, V. E., Loveland, J., Johnson, J. E., Barker, P. J. and Lomonossoff, G. P. (1994) Development of cowpea mosaic virus as a high-yielding system for the presentation of foreign peptides. Virology 202, 949–955. Pruss, G., Ge, X., Shi, X. M., Carrington, J. C. and Vance, V. B. (1997) Plant viral synergism: The potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. Plant Cell 9, 859–868. Rabindran, S. and Dawson, W. O. (2001) Assessment of recombinants that arise from the use of a TMV-based transient expression vector. Virology 284, 182–189. Radford, K. M., Cavegn, C., Bertrand, M., Bernard, A. R., Reid, S and Greenfield, P. F. (1997) The indirect effect of multiplicity of infection on baculovirus expressed proteins in insect cells: secreted and non-secreted products. Cytotechnology 24, 73–81 Ramírez, N., Lorenzo, D., Palenzuela, D., Herrera, L., Ayala, M., Fuentes, A., Pérez, M., Gavilondo, J. and Oramas, P. (2000) Single-chain antibody fragments specific to the hepatitis B surface antigen, produced in recombinant tobacco cell cultures. Biotechnol. Lett. 22, 1233–1236. Raychaudhuri, S. P. and Mishra, M. D. (1965) Effect of some metabilites and their analogs on the infectivity of tobacco tissue culture containing chilli mosaic virus. Virology 25, 483–484. Reinert, R. A. (1966) Virus activity and growth of infected and healthy callus tissues of Nicotiana tabacum grown in vitro. Phytopathology 56, 731–733. Richards, K., Guilley, H., Jonard, G. and Hirth, L. (1978) Nucleotide sequence at the 5´ extremity of tobacco-mosaic-virus RNA. Eur. J. Biochem. 84, 513–519. Roggero, P., Ciuffo, M., Benvenuto, E. and Franconi, R. (2001) The Expression of a single-chain Fv antibody fragment in different plant hosts and tissues by using potato virus X as a vector. Protein Expr. Purif. 22, 70–74. Ruf, S., Hermann, M., Berger, I. J., Carrer, H. and Bock, R. (2001) Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat. Biotechnol. 19, 870–875.

437

Ruiz-Medrano, R., Xoconostle-Cazares, B. and Kragler, F. (2004) The plasmodesmatal transport pathway for homeotic proteins, silencing signals and viruses. Curr. Opin. Plant Bio. 7, 641–650. Russell, D. A. and Fromm, M. E. (1997) Tissue-specific expression in transgenic maize of four endosperm promoters from maize and rice. Transgen. Res. 6, 157–168. Russell, T. E. and Halliwell, R. S. (1972) Response of cultured cells of Nicotiana tabacum ‘Samsun’ and ‘Samsun NN’ to microinjection with tobacco mosaic virus. Phytopathology 62, 786. Russell, T. E. and Halliwell, R. S. (1974) Response of cultures cells of systemic and local lesion tobacco hosts to microinjection with TMV. Phytopathology 64, 1520– 1526. Saito, T., Hosokawa, D., Meshi, T. and Okada, Y. (1987) Immunocytochemical localisation of the 130K and 180K proteins (putative replicase components) of tobacco mosaic virus. Virology 160, 477–481. Saito, T., Yamanaka, K. and Okada, Y. (1990) Long-distance movement and viral assembly of tobacco mosaic virus mutants. Virology 176, 329–336 Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989a) Molecular Cloning: a Laboratory Manual Volume I Second edition. Cold Sprigs Harbour Laboratory Press, New York. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989b) Molecular Cloning: a Laboratory Manual Volume III Second edition. Cold Sprigs Harbour Laboratory Press, New York. Sanchez-Navarro, J., Miglino, R., Ragozzino, A. and Bol, J. F. (2001) Engineering of Alfalfa mosaic virus RNA 3 into an expression vector. Arch. Virol. 146, 923–939. Sander, E. and Mertes, G. (1984) Use of protoplasts and separate cells in plant virus research. Adv. Virus Res. 29, 215–262 Santa Cruz, S., Chapman, S., Roberts, A. G., Roberts, I. M., Prior, D. A. M. and Oparka, K. J. (1996) Assembly and movement of a plant virus carrying a green fluorescent protein overcoat. Proc. Natl. Acad. Sci. USA 93, 6286–6290. Schneider, W. L. and Roossinck, M. J. (2000) Evolution related sindbis-like plant viruses maintain different levels of population diversity in a common host. J. Virol. 74, 3130–3134.

438

Schouten, A., Roosien, J., van Engelen, F. A., de Jong, G. A. M. (Ineke), Borst- Vrenssen, A. W. M. (Tanja), Zilverentant, J. F., Bosch, D., Stiekema, W. J., Gommers, F. J., Schots, A. and Bakker, J. (1996) The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco. Plant Mol. Biol. 30, 781–793. Scragg, A. H. (1992) Bioreactors for the mass cultivation of plant cells. In: Fowler, M. W., Warren, G. S. and Moo-Young, M., (eds), Plant Biotechnology: Comprehensive Biotechnology Second Supplement, Pergamon Press, Oxford, pp 45–62. Sehnke, P. C. and Ferl, R. J. (1999) Processing of preproricin in trasngenic tobacco. Protein Expr. Purif. 15, 188–195. Sevón, N., Hiltunen, R. and Oksman-Caldentey, K.-M (1998) Somaclonal variation in transformed roots and protoplast-derived hairy root clones of Hyoscyamus muticus. Planta Med. 64, 37–41. Sevón, N. and Oksman-Caldentey, K.-M (2002) Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids. Planta Med. 68, 859–868. Shalla, T. A. and Amici, A. (1964) The distribution of viral antigen in cells infected with tobacco mosaic virus as revealed by electron microscopy. Virology 31, 78– 91. Sharp, J. M. and Doran, M. (1990) Characterisation of growth and Tropane Alkaloid Synthesis in Atropa belladonna Roots Transformed by Agrobacterium rhizogenes. J. Biotechnol. 16, 171–186. Sharp. J. M. and Doran, P. M. (1999) Effect of bacitracin on growth and monoclonal antibody production by tobacco hairy roots and cell suspensions. Biotechnol. Bioprocess Eng. 4, 253–258. Sharp, J. M. and Doran, P. M. (2001a) Strategies for enhancing monoclonal antibody accumulation in plant cell and organ cultures. Biotechnol. Prog. 17, 979–992. Sharp, J. M. and Doran, P. M. (2001b) Characterisation of monoclonal antibody fragments produced by plant cells. Biotechnol. Bioeng. 73, 338–346. Shaw, J. G. (1999) Tobacco mosaic virus and the study of early events in virus infections. Phil. Trans. R. Soc. Lond. B 354, 603–611.

439

Shin, Y.-J., Hong, S.-Y., Kwon, T.-H., Jang, Y.-S. and Yang, M.-S. (2003) High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in tansgenic rice suspension culture. Biotechnol. Bioeng. 82, 778–783. Shivprasad, S., Pogue, G. P., Lewandowski, D. J., Hidalgo, J., Donson, J., Grill, L. K. and Dawson, W. O. (1999) Heterologous sequence greatly affects foreign gene expression in tobacco mosaic virus-based vectors. Virology 255, 312–323. Siegel, A. (1971) Pseudovirions of tobacco mosaic virus. Virology 46, 50–59. Siegel, A., Hari, V. and Kolacz, K. (1978) The effect of tobacco mosaic virus infection on host and virus-specific protein synthesis in protoplasts. Virology 85, 494–503. Smith, M. L., Mason, H. S. and Shuler, M. L. (2002) Hepatitis B surface antigen (HBsAg) expression in plant cell cultures: kinetics of antigen accumulation in batch culture and its intracellular form. Biotechnol. Bioeng. 80, 812–822. Smolenska, L., Roberts, I. M., Learmonth, D., Porter, A. J., Harris, W. J., Wilson, T. M. A. and Santa Cruz, S. (1998) Production of a functional single chain antibody attached to the surface of a plant virus. FEBS Lett. 441, 379–382. Sommer, S., Köhle, A., Yazaki, K., Shimomura, K., Bechthold, A. and Heide, L. (1999) Genetic engineering of shikonin biosynthesis hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene. Plant Mol. Biol. 39, 683– 693. Sommer, S., Siebert, M., Bechthold, A and Heide, L. (1998) Specific induction of secondary product formation in transgenic plant cell cultures using an inducible promoter. Plant Cell Rep. 17, 891–896. Spencer, D. F. and Kimmins, W. C. (1969) Presence of plasmodesmata in callus cultures of tobacco and carrot. Can. J. Bot. 47, 2049–2051. Staczek, J., Bendahmane, M., Gilleland, L. B., Beachy, R. N. and Gilleland Jr, H. E. (2000) Immunization with a chimeric tobacco mosaic virus containing an epitope of outer membrane protein F of Pseudomonas aeruginosa provides protection against challenge with P. aeruginosa. Vaccine 18, 2266–2274. Staub, J. M. and Maliga, P. (1992) Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation. Plant Cell 4, 39–45. Staub, J. M. and Maliga, P. (1995) Expression of a chimeric uidA gene indicates that polycistronic mRNAs are efficiently translated in tobacco plastids. Plant J. 7, 845– 848.

440

Stoger, E., Sack, M., Perrin, Y., Vaquero, C., Torres, E., Twyman, R. M., Christou, P. and Fischer, R. (2002) Practical considerations for pharmaceutical antibody production in different crop systems. Mol. Breed. 9, 149–158. Stubbs, G. (1999) Tobacco mosaic virus particle structure and the initiation of disassembly. Phil. Trans. R. Soc. Lond. B 354, 551–557. Su, W. W., Guan, P. and Bugos, R. C. (2004) High-level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: characterization and sensing. Biotechnol. Bioeng. 85, 610–619. Subroto, M. A., Hamill, J. D. and Doran, P. M. (1996) Development of shooty teratomas from several solanaceous plants: growth kinetics, stoichiometry and alkaloid production. J. Biotechnol. 45, 45–57. Suehara, K-I., Takao, S., Nakamura, K., Uozumi, N. and Kobayashi, T. (1996) Optimal expression of Gus gene from methyl jasmonate-inducible promoter in high density culture of transformed tobacco cell line BY-2. J. Ferment. Bioeng. 82, 51–55. Sugiyama, Y., Hamamoto, H., Takemoto, S. Watanabe, Y. and Okada, Y. (1995) Systemic production of foreign peptides on the particle surface of tobacco mosaic virus. FEBS Lett. 359, 247–250. Sulzinski, M. A., Gabard, K. A., Palukaitis, P., Zaitlin, M. (1985) Replication of tobacco mosaic virus: VIII. Characterisation of a third subgenomic TMV RNA. Virology 145, 132–140. Sulzinski, M. A. and Zaitlin, M. (1982) Tobacco mosaic virus replication in resistant and susceptible plants: In some resistant species virus is confined to a small number of initially infected cells. Virology 121, 12–19. Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) Stable transformation of plastids in higher plants. Proc. Natl. Acad. Sci. USA 87, 8526–8530. Szécsi, J., Ding, X. S., Lim, C. O., Bendahmane, M., Cho, M. J., Nelson, R. S. and Beachy, R. N (1999) Development of tobacco mosaic virus infection sites in Nicotiana benthamiana. Mol. Plant-Microbe Interact. 12, 143 –152. Takamatsu, N., Ishikawa, M., Meshi, T. and Okada, Y. (1987) Expression of bacterial chloramphenical acetyltransferase gene in tobacco plants mediated by TMV-RNA. EMBO J. 6, 307–311.

441

Takamatsu, N., Watanabe, Y., Yanagi, H., Meshi, T., Shiba, T. and Okada, Y. (1990) Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector. FEBS Lett. 269, 73–76. Takebe, I. (1975) The use of protoplasts in plant virology. Annu. Rev. Phytopathol. 13, 105–125. Takebe, I. and Otsuki, Y. (1969) Infection of tobacco mesophyll protoplasts by tobacco mosaic virus. Proceedings of the National Academy of Sciences (Botany) 64, 843– 848. Terashima, M., Ejiri, Y., Hashikawa, N. and Yoshida, H. (1999a) Effect of osmotic

pressure on human α1-antitrypsin production by plant cell culture. Biochem. Eng. J. 4, 31–36. Terashima, M., Murai, Y., Kawamura, M., Nakanishi, S., Stoltz, T., Chen, L., Drohan,

W., Rodriguez, R.L. and Katoh, S (1999b) Production of functional human α1- antitrypsin by plant cell culture. Appl. Microbiol. Biotechnol. 52, 516–523. Thomas, P. and Warren, G. S. (1990) Growth profile and pattern of antigen accumulation in suspension cultures of tobacco inoculation with TMV. J. Exp. Bot. 41 (supplement), 6. Thomas, P. and Warren, G. S. (1994) Relationship between cell growth and viral antigen content of cultures of Nicotiana tabacum inoculated with tobacco mosaic virus. J. Exp. Bot. 45, 987–994. Torres, E., Vaquero, C., Nicholson, L., Sack, M., Stöger, E., Drossard, J., Christou, P., Fischer, R. and Perrin, Y. (1999) Rice cell culture as alternative production system for functional diagnostic and therapeutic antibodies. Transgen. Res. 8, 441–449. Toth, R. L., Chapman, S., Carr, F., Santa Cruz, S. (2001) A novel strategy for the expression of foreign genes from plant virus vectors. FEBS Lett. 489, 215–219. Toth, R. L., Pogue, G. P. and Chapman, S. (2002) Improvement of the movement and host range properties of a plant virus vector through DNA shuffling. Plant J. 30, 593–600. Toyoda, H., Chatani, K., Matsuda, Y. and Ouchi, S. (1989) Multiplication of tobacco mosaic virus in tobacco callus tissues and in vitro selection for viral disease resistance. Plant Cell Rep. 8, 433–436.

442

Trexler, M. M., McDonald, K. K. and Jackman, A. P. (2002) Bioreactor production of

human α1-Antitrypsin using metabolically regulated plant cell cultures. Biotechnol. Prog. 18, 501–508. Tsoi, B. M.-Y. and Doran, P. M. (2002) Effect of medium properties and additives on antibody stability and accumulation in suspended plant cell cultures. Biotechnol. Appl. Biochem. 35, 171–180. Turpen, T. H. (1999) Tobacco mosaic virus and the virescence of biotechnology. Phil. Trans. R. Soc. Lond. B 354, 663–673. Turpen, T. H., Reinl, S. J., Charoenvit, Y., Hoffman, S. L., Fallarme, V. and Grill, L. K. (1995) Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. Bio/Technology 13, 53–57. Twyman, R. M. (2004) Host plants, systems and expression strategies for molecular farming. In: Fischer, R. and Schillberg, S. (eds), Molecular Farming: Plant-made Pharmaceuicals and Technical Proteins, Wiley-VCH Verlag GmbH and Co., Weinheim, pp 191–216. Usha, R., Rohill, J. B., Spall, V. E., Shanks, M., Maule, A. J., Johnson, J. E. and Lomonossoff, G. P. (1993) Expression of an animal virus antigenic site on the surface of a plant virus particle. Virology 197, 366–374. Vain, P., Finer, K. R., Engler, D. E., Pratt, R. C. and Finer, J. J. (1996) Intron-mediated enhancement of gene expression in maize (Zea mays L.) and bluegrass (Poa pratensis L.). Plant Cell Rep. 15, 489–494. Valentine, T. A., Robert, I. M. and Oparka, K. J. (2002) Inhibition of tobacco mosaic virus replication in lateral roots is dependant on an activated meristem-derived signal. Protoplasma 219, 184–196. Vallazza, M. and Petri, T. (1999) Optimization of the production of triabin, a novel thrombin inhibitor, in High FiveTM insect cells infected with a recombinant baculovirus. Cytotechnology 29, 85–92. van Rooijen, G. J. H. and Moloney, M. M. (1995a) Structural requirements of oleosin domains for subcellular targeting to the oil body. Plant Physiol. 109, 1353. van Rooijen, G. J. H. and Moloney, M. M. (1995b) Plant seed oil-bodies as carriers for foreign proteins. Bio/Technology 13, 72–77.

443

Varma, A., Bhat, A. and Jain, R (2001) Plant viruses: unique genetic resource. International society of plant morphologists, Phytomorphology (Goldern Jubilee Issue 2001), 435–456. Verch, T., Yusibov, V. and Koprowski, H. (1998) Expression and assembly of a full- length monoclonal antibody in plants using a plant virus vector. J. Immunol. Meth. 220, 69–75. Verdelhan des Molles, D., Gomord, V., Bastin, M., Faye, L. and Courtois, D. (1999) Expression of a carrot invertase gene in tobacco suspension cells cultivated in batch and continuous culture conditions. J. Biosci. Bioeng. 87, 302–306. Verver, J., Wellink, J., Lent, J. V., Gopinath, K. and Van Kammen, A. (1998) Studies on the movement of cowpea mosaic virus using the jellyfish green fluorescent protein. Virology 242, 22–27. Vilaine, F. and Casse-Delbart, F. (1987) Independent induction of transformed roots by the TL and TR regions of the Ri plasmid of agropine type Agrobacterium rhizogenes. Mol. Gen. Genet. 206, 17–23. Wahl, M. F., An, G. and Lee, J. M. (1995) Effects of dimethyl sulfoxide on heavy chain monoclonal antibody production from plant cell cultures. Biotechnol. Lett. 17, 465–468. Wardlaw, A. C. (1985) Practical Statistics for Experimental Biologists. Wiley, New York. Warren, G. S. and Hill, S. J. L. (1989) Infection of suspension-cultured cells of carrot with tobacco mosaic virus. Physiol. Mol. Plant Pathol. 35, 287–292. Warren, G. S., Thomas, P., Herrera, M.-T., Hill, S. J. L. and Terry, R. F. (1992) The use of plant cell cultures for studying virus resistance, and enhancing the production of virus-resistant and virus-free plants. J. Biotechnol. 22, 171–200. Watanabe, Y., Emori, Y., Ooshika, I., Meshi, T., Ohno, T and Okada, Y. (1984) Synthesis of TMV-specific RNAs and proteins at the early stage of infection in tobacco protoplasts: transient expression of the 30K Protein and its mRNA. Virology 133, 18–24. Waterhouse, P. M., Graham, M. W. and Wang, M.-B. (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl. Acad. Sci. USA 95, 13959–13964.

444

Watson, J. D. (1954) The structure of tobacco mosaic virus. I. X-ray evidence of a helical arrangement of sub-units around the longitudinal axis. Biochem. Biophys. Acta 13, 10–18 White, F. F. and Sinkar, V. P. (1987) Molecular analysis of root induction by Agrobacterium rhizogenes. In: Schell, J. (eds), Plant DNA infectious agents, Springer-Verlag, New York, pp149–177. White, F. F., Taylor, B. H., Huffman, G. A., Gordon, M. P. and Nester, E. W. (1985) Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J. Bacteriol. 164, 33–44. White, J. L., Wu, F.-S. and Murakishi, H. H. (1977) The effect of low-temperature pre- incubation treatment of tobacco and soybean callus cultures on rates of tobacco- and southern bean mosaic virus synthesis. Phtopathology 67, 60–63. White, P. R. (1934a) Potentially unlimited growth of excised tomato root tips in a liquid medium. Plant Physiol. 9, 585–600. White, P. R. (1934b) Multiplication of the viruses of tobacco and aucuba mosaics in growing excised tomato root tips. Phytopathology 24, 1003–1011. White, P. R. (1936) Plant tissue cultures. Bot. Rev. 2, 419–437. Wigdorovitz, A., Pérez Filgueira, D. M., Robertson, N., Carrillo, C., Sadir, A. M., Morris, T. J. and Borca, M. V. (1999) Protection of mice against challenge with foot and mouth disease virus (FMDV) by immunization with foliar extracts from plants infected with recombinant tobacco mosaic virus expressing the FMDV structural protein VP1. Virology 264, 85–91. Willmitzer, L., Sanchez-Serrano, J., Bushfield, E. and Schell, J. (1982) DNA from Agrobacterium rhizogenes is transferred to and expressed in axenic hairy root plant tissue. Mol. Gen. Genet. 186, 16–22. Wolf, S., Deom, C. M., Beachy, R. N. and Lucas, W. J. (1989) Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit. Science 246, 377–379. Wongsamuth, R. and Doran, P. M. (1997) Production of monoclonal antibodies by tobacco hairy roots. Biotechnol. Bioeng. 54, 401–415.

445

Wright, K. E., Proir, F., Sardana, R., Altosaar, I., Dudani, A. K., Ganz, P. R. and Tackaberry, E. S. (2001) Sorting of glycoprotein B from human cytomegalovirus to protein storage vesicles in seeds of transgenic tobacco. Transgen. Res. 10, 177– 181. Wu, F. S. and Murakishi, H. H. (1978) Infection and synthesis rate of southern bean mosaic virus in soybean callus cells under selected cultural conditions. Phytopathology 68, 1389–1392. Wu, F. S. and Murakishi, H. H. (1979) Synthesis of virus and virus-induced RNA in southern bean mosaic virus-infected soybean cell cultures. J. Gen. Virol. 45, 149– 160. Wu, J. H., Hildebrandt, A. C. and Riker, A. J. (1959) Selection by tissue culture of a super mild strain of tobacco mosaic virus. Phytopathology 49, 555. Wu, J.-H., Hildebrandt, A. C. and Riker, A. J. (1960) Virus-host relationships in plant tissue culture. Phytopathology 50, 587–594. Wu, L., Jiang, L., Zhou, Z., Fan, J., Zhang, Q., Zhu, H., Han, Q. and Xu, Z. (2003) Expression of foot-and-mouth disease virus epitopes in tobacco by tobacco mosaic virus-based vector. Vaccine 21, 4390–4398. Wu, X. and Shaw, J. (1996) Bidirectional uncoating of the genomic RNA of a helical virus. Proc. Natl. Acad. Sci. USA 93, 2981–2984. Wu, X. and Shaw, J. G. (1997) Evidence that a viral replicase protein is involved in the disassembly of tobacco mosaic virus particles in vivo. Virology 239, 426–434. Wysokinska, H. and Chmiel, A. (1997) Transformed root cultures for biotechnology. Acta Biotechnol. 17, 131–159. Xiang, C., Miao, Z.-H. and Lam, E. (1996) Coordinated activation of as-1-type elements and tobacco glutathione S-transferase gene by auxins, salicylic acid, methyl-jasmonate and hydrogen peroxide. Plant Mol. Biol. 32, 415–426. Yang, S.-J., Carter, S. A., Cole, A. B., Cheng, N.-H., Nelson, R. S. (2004) A natural variant of a host RNA-dependant RNA polymerase is associated with increased susceptibility to viruses by Nicotiana benthamiana. Proc. Natl. Acad. Sci. USA 101, 6297–6302. Yano, A., Maeda, F. and Takekoshi, M. (2004) Transgenic tobacco cells producing the human monoclonal antibody to hepatitis B virus surface antigen. J. Med. Virol. 73, 208–215.

446

Yoshida, K., Kasai, T., Garcia, M. R. C., Sawada, S., Shoji, T., Shimizu, S., Yamazaki, K., Komeda, Y. and Shinmyo, A. (1995) Heat-inducible expression system for a foreign gene in cultured tobacco cells using the HSP18.2 promoter of Arabidopsis thaliana. Appl. Microbiol. Biotechnol. 44, 466–472. Yu, S. and Doran, P. M., (1994) Oxygen requirements and mass transfer in hairy-root culture. Biotechnol. Bioeng. 44, 880–887. Yusibov, V., Hooper, D. C., Spitsin, S. V., Fleysh, N., Kean, R. B., Mikheeva, T., Deka, D., Karasev, A., Cox, S., Randall, J., Koprowski, H. (2002) Expression in plants and immunologenicity of plant virus-based experimental rabies vaccine. Vaccine 20, 3155–3164. Yusibov, V., Modelska, A., Steplewiski, K., Agadjanyan, M., Weiner, D., Hooper, D. C. and Koprowski, H. (1997) Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1. Proc. Natl. Acad. Sci. USA 94, 5784–5788. Yusibov, V., Shivprasad, S., Turpen, T. H., Dawson, W. and Koprowski, H. (1999) Plant viral vectors based on tobamoviruses. In: Hammond, J., McGarvey, P. and Yusibov, V. (eds), Plant Biotechnology: New Products and Applications, Springer- Verlag, Berlin, pp 81–94. Zaitlin, M. (2000) Descriptions of Plant Viruses DPV 370 (www.dpvweb.net/dpv/showdpv.php?dpvbo-370) Zambryski, P. (1988) Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annual. Rev. Genet. 22, 1–30. Zelcer, A., van Adelsberg, J., Leonard, D. A. and Zaitlin, M. (1981) Plant cell suspension cultures sustain long-term replication of potato spindle tuber viroid. Virology 109, 314-322. Zhang, G., Leung, C., Murdin, L., Rovinski, B. and White, K. A. (2000) In planta expression of HIV-1 p24 protein using an RNA plant virus-based expression vector. Mol. Biotechnol. 14, 99–107. Zimmermann, S., Schillberg, S., Liao, Y.-C. and Fisher, R. (1998) Intracellular expression of TMV-specific single-chain Fv fragment leads to improved virus resistance in Nicotiana tabacum. Mol. Breed. 4, 369–379. Zimmern, D. (1975) The 5´ end group of tobacco mosaic virus RNA is m7G5´ppp5´Gp. Nucl. Acids Res. 2, 1189–1201.

447

Zimmern, D. (1977) The nucleotide sequence at the origin for assembly on tobacco mosaic virus RNA. Cell 11, 463–485.