Tobacco shoot regeneration from calli in temporary ... · Tobacco shoot regeneration from calli in...

Tobacco shoot regeneration from calli in temporary

immersion culture for biosynthesis of heterologous

biopharmaceuticals

Sherwin Savio Barretto

Thesis submitted for the Degree of Doctor of Philosophy PhD

Imperial College London

Department of Life Sciences

Faculty of Natural Sciences

Imperial College London

2014

1

Declaration of Originality

I hereby declare that this thesis, submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy of Imperial College London, represents my own work and has not been

previously submitted to this or any other institute for any degree, diploma or other

qualification.

Sherwin Savio Barretto

2

Copyright Declaration

The copyright of this thesis rests with the author and is made available under a Creative

Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy,

distribute or transmit the thesis on the condition that they attribute it, that they do not use it

for commercial purposes and that they do not alter, transform or build upon it. For any reuse

or redistribution, researchers must make clear to others the licence terms of this work.

3

Abstract

‘Molecular farming’, the use of transgenic plants to produce biopharmaceutical proteins is

emerging as a new biotechnological paradigm. Transgenic plants offer several advantages

over conventional microbial and mammalian cell host technologies. In particular,

transplastomic plants, with transformed plastid genomes, are capable of massive expression

of foreign proteins and represent a promising platform for biopharmaceutical synthesis.

The main theme of this PhD thesis is the investigation of in vitro regeneration of tobacco

(Nicotiana tabacum) shoots from callus tissue in temporary immersion (TI) culture for

heterologous biopharmaceutical synthesis. There is special emphasis on subunit vaccine

expression in transplastomic tobacco, in which foreign protein accumulation is correlated

with chloroplast number and development during the organogenesis process.

Studies using transplastomic N. tabacum expressing TetC (tetanus toxin fragment C)

investigated the influence of several culture parameters on biomass regeneration and

recombinant protein expression. The parameters investigated include medium nitrogen source

ratio, sucrose concentration and hydrodynamics. These studies highlight the sensitivity of

transplastomic protein yields to the culture microenvironment, and provide a starting point

for further optimisation. Further studies demonstrated the feasibility of TI culture for

biosynthesis of proteolytically-unstable transplastomic subunit vaccines, p24 (HIV antigen)

and VP6 (rotavirus antigen). TI culture is also demonstrated as a means for nuclear

expression of functional Guy’s 13 monoclonal antibody. Finally, the use of TI culture as the

basis of novel technological innovations is investigated. This includes the demonstration of

transplastomic protein expression in a prototype large-scale mechanical temporary immersion

bioreactor. Encapsulation of callus aggregates in an alginate matrix for long-term germplasm

preservation was trialled, prior to temporary immersion regeneration.

Overall, this work presents a novel in vitro propagation method for the contained large-scale

biosynthesis of biopharmaceutical proteins, as a potential alternative to conventional plant

propagation platforms based on agricultural cultivation or cell suspension culture.

4

Acknowledgements

I gratefully acknowledge all the individuals who have provided the assistance and support I

needed to complete this complex assignment.

First of all, I would like to express my sincere gratitude and deep appreciation to my

supervisor, Prof. Peter Nixon, for providing me with the opportunity to pursue this exciting

and challenging PhD project, and for all his guidance and support. I am equally grateful to

my co-supervisor Prof. Klaus Hellgardt whose guidance and invaluable technical ‘know-

how’ has helped me tremendously. I would like to give my deepest thanks to Dr Franck

Michoux, the post-doc who started this project and who in many ways has acted as my

‘unofficial supervisor’. I am extremely grateful for his assistance, support, time and patience.

I would like to thank all members of the 7th floor of the Ernst Chain Building, past and

present, for their cooperation, encouragement, support and friendship. I would especially like

to thank Hussain Haji Taha, Dr Jianfeng Yu, Shengxi Shao, Dr Niaz Ahmad, Dr Steven

Burgess, Dr Marko Boehm, Dr Charlotte Ward, Dr Agripina Banda, Sana Asghar, Xu Zhao,

Alexandros Papagiannakis, Jiyao Gan, Chi Zeng, Zheng-Yi Wei, Jayasudha Nagarajan,

Marin Sawa, Charlie Cotton, Jeffery Douglass, Sven Dc, Dr Tanai Cardona Londono, Dr

Karim Maghlaoui, Dr Alison Telfer, Katharina Brinkert, Dr Wojciech Bialek, Dr Andreas

Fantuzzi, Dr Gillian Young, Ruiqiong An, Dr Masooma Rasheed, Ewelina Krysztofinska,

Mostafa Jamshidiha, Dr Lisa Hale, Dr Justin Yeoman, Dr James MacDonald, Bhavish Patel,

Dr Christian Richard, Amanda Koslovaite and all other lab-fellows, past and present, whom I

have had the greatest pleasure crossing paths with. In particular, I will always remember Prof.

Bill Rutherford for his carefree attitude, advice and chats.

I would like to thank all my collaborators, for their help, guidance, materials and equipment. I

would like to thank Prof. John Gray, University of Cambridge for providing me with the

transplastomic seeds for the expression of p24 and VP6 and the accompanying antibodies. I

would like to thank Prof. Julian Ma and Pascal Drake for providing me with the transformant

seeds for the expression of Guy’s 13 monoclonal antibody. These collaborative efforts have

helped in the advancement of the scientific endeavour.

5

I would like to thank the Biotechnology and Biological Sciences Research Council (BBSRC)

Targeted Priority Studentships initiative for funding this work.

I am deeply indebted to my family for their love, support, encouragement and prayers for my

success.

Finally, I would like to give thanks to Almighty God, for the innumerable graces and

blessings bestowed upon me to enable me to undertake this pursuit.

6

Table of Contents

Table of Contents .................................................................................................................................... 6

List of Figures ........................................................................................................................................ 14

List of Tables ......................................................................................................................................... 17

List of Abbreviations ............................................................................................................................. 18

Chapter 1. Introduction .................................................................................................................. 23

1.1 The Biotechnology revolution and recombinant biopharmaceuticals ................................. 23

1.2 Transgenic plants .................................................................................................................. 25

1.2.1 The scope of transgenic plants as an alternative host technology ............................... 25

1.2.2 The benefits of transgenic plant host systems relative to conventional platforms ..... 27

1.2.3 Bioprocessing of plant-derived biopharmaceuticals .................................................... 29

1.2.3.1 Choice of localisation target in transgenic plant host systems ................................. 29

1.2.3.2 The choice of in vitro or soil-based cultivation for growth of transgenic plants ...... 30

1.2.3.3 The use of bioreactors in cell and tissue cultures ..................................................... 32

1.2.3.4 The temporary immersion culture format ................................................................ 33

1.2.3.5 Downstream processing ........................................................................................... 37

1.2.4 Chloroplast transformation ........................................................................................... 38

1.2.4.1 An overview of plant genetic transformation strategies .......................................... 38

1.2.4.2 The plastid genome as a target for genetic transformation ..................................... 39

1.2.4.2.1 Plastids organelles in higher plants .................................................................... 39

1.2.4.2.2 Features of the plastid genome .......................................................................... 40

1.2.4.2.3 The plastid genome as a novel target for genetic engineering .......................... 42

1.2.4.2.4 Methods for transformation of the chloroplast ................................................ 42

1.2.4.2.5 Benefits of plastid transformation ...................................................................... 44

1.2.4.2.5.1 Hyperexpression of transgenic proteins in plastids ..................................... 44

7

1.2.4.2.5.2 Post-transcriptional regulation of plastid protein synthesis and implications

for transformation vectors ................................................................................................ 45

1.2.4.2.5.3 Polycistronic transcription of plastid genes and opportunities for metabolic

pathway engineering ........................................................................................................ 46

1.2.4.2.5.4 Maternal inheritance of the chloroplast genome and implications for

biosafety 47

1.2.4.2.6 Applications of plastid genetic engineering ........................................................ 48

1.2.4.2.6.1 Plastid engineering for improvement of agronomic traits and Rubisco

activity 48

1.2.4.2.6.2 Molecular farming of protein biopharmaceuticals ...................................... 49

1.2.4.2.6.3 Expression of fusion proteins with affinity tags........................................... 50

1.3 Background to PhD Project ................................................................................................... 51

1.3.1 The PhD project in the context of previous studies ...................................................... 51

1.3.2 Aims of Study ................................................................................................................ 52

Chapter 2. Materials and Methods ................................................................................................. 54

2.1 Stock Solutions ...................................................................................................................... 54

2.1.1 Standard solutions and buffers ..................................................................................... 54

2.1.2 Antibodies ..................................................................................................................... 54

2.2 Cultivation of transgenic Nicotiana tabacum ....................................................................... 56

2.2.1 Nicotiana tabacum growth conditions ......................................................................... 56

2.2.2 In vitro micropropagation of transgenic N. tabacum ................................................... 57

2.2.2.1 In vitro germination of sterile seedlings .................................................................. 57

2.2.2.2 Callus induction and proliferation, and suspension cultures ................................... 57

2.2.2.3 Temporary immersion regeneration of shoots from callus ..................................... 58

2.2.2.4 Temporary immersion organogenesis from encapsulated calli (modified procedure)

58

2.2.2.5 Regeneration of shoots in a large-scale hydraulic bioreactor ................................. 59

2.2.2.5.1 Construction of large-scale bioreactor ............................................................... 59

2.2.2.5.2 Operation of large-scale bioreactor .................................................................... 59

8

2.2.2.6 Harvest of regenerated shoots, and fresh and dry weight determination .............. 60

2.3 Protein Analysis ..................................................................................................................... 61

2.3.1 Total Soluble Protein Extraction ................................................................................... 61

2.3.2 Determinination of protein concentration ................................................................... 61

2.3.3 SDS-PAGE ...................................................................................................................... 62

2.3.4 Staining of polyacrylamide gels .................................................................................... 62

2.3.5 Immunoblotting and Enhanced Chemiluminescence (ECL) Detection ......................... 62

2.3.6 Indirect ELISA to assess functional activity of plant-expressed monoclonal antibodies

63

2.4 Analysis of in vitro regenerated plant biomass ..................................................................... 64

2.4.1 Viability assay of in vitro regenerated shoots ............................................................... 64

2.4.2 Chlorophyll Fluorometry ............................................................................................... 65

Chapter 3. Parameters affecting the dynamics of biomass growth and transplastomic protein

accumulation in temporary immersion culture .................................................................................... 66

3.1 Introduction .......................................................................................................................... 66

3.1.1 In vitro differentiated plant tissues for molecular farming .......................................... 66

3.1.2 Nt-pJST12 as a rational model host system for investigating the impact of the culture

environment on recombinant protein expression ........................................................................ 67

3.2 In vitro shoot regeneration via organogenesis of N. tabacum callus and the influence on

transplastomic protein expression ................................................................................................... 68

3.2.1 Callus organogenesis as a developmental pathway for in vitro plantlet regeneration 68

3.2.2 In vitro morphogenesis dynamics during temporary immersion culture ..................... 69

3.2.2.1 Design of experiment .................................................................................................... 69

3.2.2.2 Results and Discussion .................................................................................................. 69

3.2.2.2.1 Dynamics of biomass growth and morphogenesis ............................................. 69

3.2.2.2.2 Differential expression of TetC during in vitro organogenesis ........................... 72

3.3 Impact of Hyperhydricity on Expression of TetC .................................................................. 75

3.3.1 The hyperhydricity phenomenon in in vitro micropropagation ................................... 75

3.3.2 TetC accumulation in vitrified and non-vitrified leaves ................................................ 76

9

3.3.3 Discussion on the influence of hyperhydricity on transplastomic protein expression . 78

3.4 The Impact of Tissue Culture Media on Morphogenesis and TetC yield .............................. 79

3.4.1 The impact of ammonium : nitrate ratio on TetC yield ................................................ 79

3.4.1.1 The nitrogen requirements of in vitro plant growth ............................................... 79

3.4.1.2 Design of experiment ................................................................................................ 80

3.4.1.3 Results ...................................................................................................................... 80

3.4.1.3.1 Influence of nitrogen source ratio on growth and TetC expression ................... 80

3.4.1.3.1.1 Influence of nitrogen source ratio on biomass accumulation and

morphogenesis .................................................................................................................. 80

3.4.1.3.1.2 Influence of nitrogen source ratio on yield of TetC ..................................... 83

3.4.1.3.2 pH shift of media during temporary immersion culture..................................... 85

3.4.1.4 Discussion on the influence of nitrogen pool on in vitro regeneration and

transplastomic protein expression ........................................................................................... 86

3.4.2 Effect of initial media pH on biomass growth and TetC expression ............................. 88

3.4.2.1 Design of experiment ............................................................................................... 88

3.4.2.2 Results and Discussion ............................................................................................. 89

3.4.2.2.1 The influence of initial medium pH on biomass growth and TetC expression ... 89

3.4.3 The Impact of Varying Sucrose and Irradiance for Photomixotrophic Propagation

Regimes 91

3.4.3.1 The importance of exogenous saccharides and irradiance in in vitro plant tissue

culture 91

3.4.3.2 Design of experiment ............................................................................................ 92

3.4.3.3 Results and Discussion .......................................................................................... 92

3.4.3.3.1 Effect of sucrose and irradiance on biomass accumulation and TetC expression

92

3.4.3.3.2 Effect of sucrose and irradiance on TetC expression .......................................... 94

3.4.3.3.3 Pulse amplitude modulation (PAM) fluorometry to assess photosynthetic

activity of in vitro regenerated shoots .................................................................................. 97

3.4.3.4 Implications of these findings on transplastomic molecular farming ................... 98

10

3.4.4 The influence of altered MS media strength on biomass accumulation and yield ...... 99

3.4.4.1 Design of experiment ............................................................................................... 99

3.4.4.2 Results and Discussion ............................................................................................. 99

3.4.4.2.1 Influence of altered MS media strength on biomass accumulation and TetC

expression 99

3.5 Effect of Temporary Immersion Culture Hydrodynamics on Viability, Growth and

Transplastomic Protein Expression in early-stage Callus Morphogenesis ...................................... 103

3.5.1 The importance of hydrodynamics in plant cell and tissue cultures ................................. 103

3.5.2 Investigation of the effects of fluid hydrodynamics on callus morphogenesis, viability and

heterologous protein turnover during pneumatic immersion ................................................... 104

3.5.2.1 Aims of experiment ................................................................................................. 104

3.5.3 Characterisation of key parameters............................................................................ 105

3.5.3.1 Characterisation of the rheological and hydrodynamic properties of the pneumatic

submersion of plant biomass .................................................................................................. 105

3.5.3.2 Characterisation of the rheological properties of tissue culture media ................ 105

3.5.3.3 Parameters for characterising the hydrodynamic flow field ................................. 106

3.5.3.4 Estimation of average shear rate ........................................................................... 106

3.5.3.5 Estimation of specific power input ........................................................................ 107

3.5.3.6 Cumulative Energy Dissipation .............................................................................. 107

3.5.4 Design of experiment ................................................................................................. 107

3.5.5 Results ......................................................................................................................... 108

3.5.5.1 Effect of shear rate and power dissipation on biomass accumulation .................. 108

3.5.5.2 Effect of shear rate and gassed power input on mitochondrial activity ................ 109

3.5.5.3 Effect of shear rate and gassed power input on TetC expression ......................... 110

3.6 Discussion on in vitro morphogenesis of callus in TIBs and transplastomic protein

expression, and the various parameters affecting these ............................................................... 114

Chapter 4. Expression and assembly of Guy’s 13 monoclonal antibody via temporary immersion

shoot regeneration ............................................................................................................................. 117

4.1 Introduction : Monoclonal antibody production in transgenic plants................................ 117

11

4.1.1 Monoclonal antibodies as biopharmaceuticals .......................................................... 117

4.1.2 Plant systems for antibody production ....................................................................... 119

4.1.3 Guy’s 13 monoclonal antibody as a topical immunotherapy agent for prevention of

dental caries ................................................................................................................................ 120

4.2 Expression and assembly of Guy’s 13 mAb by temporary immersion regeneration of N.

tabacum cv. Xanthii ........................................................................................................................ 121

4.2.1 Design of experiment .................................................................................................. 121

4.2.2 Results ......................................................................................................................... 121

4.2.2.1 Non-reducing Western Immunoblotting to confirm expression and assembly of

Guy’s 13 IgG1 in transgenic tobacco ....................................................................................... 121

4.2.2.2 Antigen binding assay for functional studies of expressed Guy’s 13 monoclonal

antibody 123

4.2.3 Discussion ................................................................................................................... 126

4.2.3.1 Plants possess the relevant machinery for expression of functional antibodies... 126

4.2.3.2 The impact of hyperhydricity on functional Guy’s 13 mAb titre in temporary

immersion regeneration ......................................................................................................... 127

4.2.3.3 Demonstration of mAb production in in vitro shoot regeneration via temporary

immersion culture ................................................................................................................... 128

Chapter 5 Expression of transplastomic proteolytically unstable proteins via temporary

immersion shoot regeneration ........................................................................................................... 130

5.1 Introduction ........................................................................................................................ 130

5.1.1 In planta proteolysis of recombinant proteins ........................................................... 130

5.1.2 Transplastomic expression of vaccine subunits susceptible to proteolytic degradation

131

5.1.2.1 VP6 as a potential subunit vaccine against rotavirus infection ............................. 132

5.1.2.2 p24 as a subunit vaccines against HIV.................................................................... 132

5.2 Expression of transplastomic proteins susceptible to degradation via temporary immersion

shoot regeneration ......................................................................................................................... 133

5.2.1 Accumulation of plastid-expressed rotavirus VP6 via temporary immersion shoot

regeneration and comparison to soil-grown seedlings .............................................................. 133

12

5.2.1.1 Design of experiment ............................................................................................. 133

5.2.1.2 Results .................................................................................................................... 134

5.2.1.2.1 VP6 stability in soil-grown tobacco leaves ........................................................ 134

5.2.1.2.2 Expression of VP6 in in vitro temporary immersion-regenerated biomass ...... 135

5.2.2 Accumulation of HIV-1 p24 antigen via temporary immersion shoot regeneration and

comparison to soil-grown seedlings ........................................................................................... 136

5.2.2.1 Design of experiment ............................................................................................. 136

5.2.2.2 Results .................................................................................................................... 137

5.2.2.2.1 p24 stability in soil-grown tobacco leaves ........................................................ 137

5.2.2.2.2 Expression of p24 in temporary immersion-regenerated shoots ..................... 138

5.2.3 Discussion on the expression of proteolytically unstable transplastomic proteins via TI

regeneration ............................................................................................................................... 140

Chapter 6. Developing new tools for in vitro molecular farming ................................................. 143

6.1 Development of large-scale mechanical bioreactor ........................................................... 143

6.1.1 The need for scale-up of in vitro organogenesis for molecular farming purposes ..... 143


6.1.3 Results ......................................................................................................................... 145

6.1.3.1 Biomass Accumulation and Organogenesis ........................................................... 145

6.1.3.2 Comparison of biomass accumulation between the mechanical bioreactor and RITA

147

5.1.3.3 Comparative analysis of TetC expression in the mechanical bioreactor ............... 148

6.1.3.4 Discussion on the development of a large-scale bioreactor ............................... 150

6.2 The influence of pre-culture preservation of encapsulated callus on temporary immersion

morphogenic potential and TetC expression .................................................................................. 152

6.2.1 Synthetic seed technology .......................................................................................... 152


6.2.3 Results ......................................................................................................................... 153

6.2.3.1 Influence of duration and temperature of encapsulated callus preservation on

growth and morphogenesis in temporary immersion culture ............................................... 153

13

6.2.3.2 Influence of Influence of duration and temperature of encapsulated callus

preservation on TetC expression in regenerated shoots ........................................................ 155

6.2.3.3 Discussion on the influence of alginate encapsulation on temporary immersion

regeneration and TetC expression .......................................................................................... 155

6.3 Discussion on described studies and how they relate to new developments in in vitro

molecular farming ........................................................................................................................... 156

Chapter 7 Summary and future directions ...................................................................................... 158

7.1 In vitro plant tissue culture as an alternative platform for biosynthesis of

biopharmaceuticals ......................................................................................................................... 158

7.2 The influence of temporary immersion shoot regeneration on biosynthesis of

transplastomic proteins .................................................................................................................. 162

7.3 Scale-up of callus-to-shoot regeneration for biopharmaceutical expression .................... 164

7.4 Biosynthesis and assembly of functional monoclonal antibodies in temporary immersion

shoot regeneration ......................................................................................................................... 165

7.5 Implementation of robust bioprocesses for biopharmaceutical synthesis ........................ 167

Bibliography ........................................................................................................................................ 169

14

List of Figures

Figure 1.1 Technological design and operational principle of Twin‐Flask system .............................. 35

Figure 1.2 Technological design and operational principle of RITA® system ..................................... 36

Figure 1.3 Technological design and operational principle of bioreactor of immersion by bubbles

system ................................................................................................................................................. 36

Figure 1.4 Downstream processing routes for seed and leaf crops ................................................... 38

Figure 1.5 Gene map of plastid genome from tobacco (N. tabacum). ............................................... 41

Figure 1.6 Schematic representation of the chloroplast expression cassette .................................... 46

Figure 2.1 Operation of large-scale mechanical bioreactor ............................................................... 60

Figure 3.1 Callus-meristemoid transition and shoot bud formation. ................................................. 71

Figure 3.2 Logistic increase of fresh and dry biomass accumulation during in vitro organogenesis in

RITA® TIBs .......................................................................................................................................... 72

Figure 3.3 SDS-PAGE and immunoblot showing differential expression of TetC. .............................. 74

Figure 3.4 Increase in TetC volumetric yield and fresh biomass. ....................................................... 74

Figure 3.5 Visual comparison between non-vitrified and vitrified shoots .......................................... 76

Figure 3.6 Investigation of hyperhydricity on TetC accumulation at different time intervals of

temporary immersion culture, by SDS-PAGE and immunoblot. ......................................................... 77

Figure 3.7 Effect of hyperhydricity on TetC accumulation at various sucrose concentrations and

irradiances. .......................................................................................................................................... 78

Figure 3.8 Effect of NO3-: NH4

+ ratio on developmental status of N. tabacum regenerated shoots

after 40-day temporary immersion culture. ....................................................................................... 82

Figure 3.9 Influence of NO3-: NH4

+ ratio on fresh and dry biomass accumulation. ............................ 83

Figure 3.10 SDS-PAGE and immunoblot analysis of lysates to assess TetC expression under various

NO3-: NH4

+ ratios. ................................................................................................................................ 84

Figure 3.11 Densitometric quantification of TetC intrinsic yields and volumetric yields under various

NO3-: NH4

+ ratios, from immunoblot data in Figure 3.10. ................................................................... 84

Figure 3.12 Shift in medium pH over temporary immersion culture period.. .................................... 85

Figure 3.13 Influence of media pH on fresh and dry biomass accumulation ..................................... 90

Figure 3.14 SDS PAGE and immunoblot analysis of of media pH effects on TetC expression. ........... 90

Figure 3.15 Visual demonstration of the effect of sucrose concentration on shoot morphogenesis

after 40-day temporary immersion culture ....................................................................................... 93

Figure 3.16 Effect of sucrose concentration and irradiance on fresh and dry biomass accumulation.

… ......................................................................................................................................................... 94

15

Figure 3.17 SDS-PAGE and immunoblot showing the effect of sucrose and light on Tetc expression.

…. ......................................................................................................................................................... 95

Figure 3.18 Influence of sucrose and light levels on TetC intrinsic yield (ng TetC per µg total soluble

protein (TSP)), determined densitometrically. ................................................................................... 96

Figure 3.19 Influence of sucrose and light levels on estimated absolute TetC yield (µg TetC per

volume of bioreactor) ......................................................................................................................... 96

Figure 3.20 Effect of MS media strength on fresh and dry biomass accumulation. ......................... 101

Figure 3.21 TIB cultures at 40 days (prior to harvest) at 100%, 50%, 25% MS medium strength .... 101

Figure 3.22 Visual comparison of 40-day temporary immersion cultures at 100% and 200% MS

medium strength .............................................................................................................................. 102

Figure 3.23 SDS-PAGE and immunoblot showing the effect of MS media strength on TetC

expression. ........................................................................................................................................ 102

Figure 3.24 Plots showing the influence of average shear rate and energy dissipation rate on fresh

and dry biomass accumulation, after 3, 20 and 40-day cultures ...................................................... 109

Figure 3.25 Plots showing the influence of average shear rate, energy dissipation rate and total

energy dissipation (after 20 days culture only) on mitochondrial respiratory activity after 0, 3, 20

and 40-day cultures .......................................................................................................................... 110

Figure 3.26 SDS-PAGE and immunoblots showing the effect of air flow rate on TetC expression. .......

. ....................................................................................................................................................... 111

Figure 3.27 Impact of hydrodynamics on TetC intrinsic yield (ng/µg) after 40-day culture………………..

. ....................................................................................................................................................... 112

Figure 3.28 Absolute recombinant protein yield depends on both intrinsic yield and biomass growth

... ....................................................................................................................................................... 116

Figure 4.1 Structure of immunoglobulin G (IgG)............................................................................... 119

Figure 4.2 SDS-PAGE and Western Immunoblot of lysates of in vitro and soil-cultivated biomass

under non-reducing conditions. ....................................................................................................... 123

Figure 4.3 Lysate titration curve showing the binding of Guy’s 13 mAb to the purified SWCF

fragment of SA I/II. ............................................................................................................................ 124

Figure 5.1 SDS-PAGE and immunoblot showing VP6 accumulation in the leaves of an 8-week old

soil-grown plant. ............................................................................................................................... 135

Figure 5.2 SDS-PAGE and Western immunoblot for demonstration of the expression of VP6 in TIB-

grown biomass ................................................................................................................................. .136

Figure 5.3 SDS-PAGE and immunoblot showing p24 accumulation in the leaves of an 8-week old

soil-grown plant. ............................................................................................................................... 138

16

Figure 5.4 SDS-PAGE and Western immunoblot for demonstration of the expression of p24 in TIB-

grown biomass. ................................................................................................................................. 139

Figure 6.1 Large 60 l mechanical bioreactor in operation ................................................................ 144

Figure 6.2 Schematic showing operation of large bioreactor. .......................................................... 145

Figure 6.3 Visual demonstration of shoot morphogenesis in large- and small-scale temporary

immersion bioreactors. ..................................................................................................................... 146

Figure 6.4 Increase in fresh and dry biomass accumulation in the mechanical temporary immersion

bioreactor after 50 and 80 days culture ........................................................................................... 147

Figure 6.5 Comparison of fresh and dry biomass accumulation in large bioreactor and RITA® culture

vessels. .............................................................................................................................................. 148

Figure 6.6 SDS-PAGE and immunoblot demonstrating TetC expression in large mechanical TIB ..........

. ....................................................................................................................................................... 149

Figure 6.7 Comparison of TetC yield in large bioreactor and RITA® culture vessels. ....................... 150

Figure 6.8 Callus aggregates encapsulated in a sodium alginate matrix. ......................................... 153

Figure 6.9 Effect of callus encapsulation duration and temperature on fresh and dry biomass

accumulation in temporary immersion shoot regeneration cultures. ............................................. 154

Figure 6.10 SDS-PAGE and immunoblot demonstrating TetC expression in shoot biomass

regenerated from encapsulated callus stored for various durations and temperatures. ................ 155

17

List of Tables

Table 2.1 List of antibodies used. ....................................................................................................... 55

Table 3.1 Variable fluorescence / maximal fluorescence (Fv/Fm) measurements for leaves

regenerated by temporary immersion culture of Nt-pJST12 under different photomixotrophic

treatments .......................................................................................................................................... 98

Table 4.1 EC50 titres and EC50 dilutions of plant lysates with standard errors, derived from 4-

parameter logistic curve fitting to titration curves ........................................................................... 125

18

List of Abbreviations

% (v/v) % volume / volume

% (w/v) % weight / volume

3’-UTR 3’ untranslated region

5’-UTR 5’ untranslated region

µmol.m-2.s-1 Micromoles of photons per square metre, per second (units of

photosynthetic photon flux)

aadA gene encoding aminoglycoside 3’’-adenylyl transferase for

spectinomycin resistance (in plastid transformation cassettes)

AMV Alfalfa Mosaic Virus

APS Ammonium persulfate

ATP Adenosine triphosphate

atpB Plastidial gene encoding beta subunit of ATP synthase

BIB® Bioreactor of immersion by bubbles

BiP Binding protein (molecular chaperone)

BIT® Twin flasks bioreactor system

BSA Bovine serum albumin

CCD Charge-coupled device (digital imaging system)

CPMV Cowpea Mosaic Virus

dH2O Distilled water

DNA Deoxyribonucleic acid

DTP diphtheria–tetanus–pertussis (vaccine)

DW Dry weight

cGMP ‘current good manufacturing practice’

CHO Chinese hamster ovary cells

CIM Callus induction medium

CTB cholera toxin B subunit

E. coli Escherichia coli

DSP Downstream processing

DTT Dithiothreitol

EBA Expanded bed adsorption

EC50 Half maximal effective concentration

19

ECL Enhanced Chemiluminescence

EDTA Ethylenediaminetetraacetic acid

ELISA Enzyme-linked immunosorbent assay

ER Endoplasmic reticulum

FDA United States Food and Drug Administration

FFD fractional factorial design

Fc crystallizable fragment

Fd ferredoxin

Fv Variable fragment (of immunoglobulin)

Fv/Fm Maximum PSII quantum yield

FW Fresh weight

GFP+ Variant of green fluorescent protein

GM Genetically modified

GMO Genetically modified organism

GMP ‘Good manufacturing practice’

GOGAT glutamine 2-oxoglutarate amino transferase (or glutamate synthase)

GS glutamine synthetase

GST glutathione-S-transferase

H Heavy chain (of antibody)

H2L2 fully assembled antibody composed of two heavy and two light chains

Hc Heavy chain (of TetC)

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HIC Hydrophobic interaction chromatography

His6-MBP His-tagged derivative of the maltose binding protein

HRP Horseradish peroxidase

HSA Human serum albumin

hST Human somatotropin

Ig Immunoglobulin

IGF Insulin-like growth factor

IgG Immunoglobulin G

IMAC Immobilized metal ion affinity chromatography

IR Inverted repeat (region of plastome)

IVIG intravenous immunoglobulin

kb Kilobase

20

kDa Kilodalton

L Light chain

Lc Light chain (of TetC)

LSC Large single-copy (region of plastome)

M Molar / moles per litre; unit of concentration

MBP Maltose binding protein

MES 2-(N-morpholino)ethanesulfonic acid

mRNA Messenger RNA

MS medium Murashige & Skoog medium

N. tabacum Nicotiana tabacum

NAA 1-napthaleneacetic acid

NME new molecular entity

NMR Nuclear magnetic resonance

ORF Open reading frame

OspA Outer surface protein A

p24 HIV surface antigen

PAM Pulse amplitude modulated (fluorometry)

PBS Phosphate buffered saline

PE Polyethylene

PEB-A Protein extraction buffer

PEG Polyethylene glycol

PGI Plastid-genome incompatibility

PHB Polyhydroxybutyric acid

PPF Photosynthetic photon flux [units: µmol photons m-2s-1]

PPM ™ Plant Preservative Mixture, a broad-spectrum biocide supplied by Plant

Cell Technology, Inc.

Prrn Promoter of plastid-encoded ribosomal RNA gene

PsbA Plastid gene encoding D1 subunit of photosystem II

ptDNA Plastid DNA

PSII Photosystem II

PTOX plastid terminal oxidase

PVDF polyvinylidene fluoride

PVX Potato Virus X

ORF Open reading frame

21

rbcL Plastidial gene encoding Rubisco large sububit

RBS Ribosomal binding site

RER rough endoplasmic reticulum

RITA® Recipient for Automated Temporary Immersion (translated from

French), lab-scale TIB supplied by CIRAD.

RNA Ribonucleic acid

RO Reverse osmosis

ROS Reactive oxygen species

Rpm Revolutions per minute; frequency of rotation

rrn Plastidial gene encoding ribosomal RNA

RSM Response surface methodology

Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase

SA I/II Streptococcal antigen

SBB Slug Bubble Bioreactor

scFv Single-chain Fv antibody fragment

SD Standard deviation

SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis

SE Somatic embryogenesis

spp. species

SSC Small single-copy (region of plastome)

T7G10 Gene 10 from bacteriophage T7

TBS Tris-buffered saline

TBSV Tomato Bushy Stunt Virus

TDZ Thidiazuron

TEMED N,N,N,N-tetramethylenediamine

TeNT Tetanospasmin (also known as TeTx)

TetC Tetanus toxin fragment C

TF triphenylformazan

TI Temporary immersion

TIB Temporary immersion bioreactor

TIS Temporary immersion system

TMB 3,3′,5,5′-Tetramethylbenzidine

TMV Tobacco Mosaic Virus

TRI bioreactor Temporary root zone immersion bioreactor

22

TSP Total soluble protein

TTC 2,3,5-triphenyltetrazolium chloride

UTR Untranslated region

VP6 Rotavirus surface antigen

WUB Wave and Undertow Bioreactor

WT Wild-type

23

Chapter 1. Introduction

1.1 The Biotechnology revolution and recombinant biopharmaceuticals

The etymology of ‘biotechnology’ is a fusion of ‘biology’ and ‘technology’, and true to its

name, biotechnology is concerned with the exploitation and industrialisation of biological

agents for useful ends. The term ‘biotechnology’ was coined in 1919 by a Hungarian

engineer, Karl Ereky, who envisioned a great ‘biochemical age’, analogous to the stone and

iron ages (Kennedy, 1991; Webb and Atkinson, 1992). Biotechnology encompasses a broad

and divergent array of technologies with various levels of sophistication, which makes a

precise definition of the field rather difficult (Gavrilescu and Chisti, 2005). Biotechnology

may be defined as the “controlled and deliberate application of simple biological agents –

living or dead cells or cell components – in technically useful operations, either of productive

manufacture or as service operations” (Campbell, 1992). According to the European

Federation of Biotechnology, biotechnology can be considered “the integrated use of

biochemistry, microbiology and engineering sciences in order to achieve technological

application of the capabilities of microorganisms, cultured tissues / cells and parts thereof.”

(Klingenberg, 1984). Whichever definition is invoked, the common theme is the utilisation of

biological systems for some useful end, such as producing valuable products (such as

biopharmaceuticals) or performing a useful service (such as bioremediation).

Biotechnology can be divided into four major market segments: biopharmaceutical,

agricultural, environmental and industrial (Colwell, 2002). Although thought of as one of the

hallmarks of the modern era, biotechnology can be considered to be one of the oldest of

human activities. Primitive microbial and processing technologies have been used for

millennia, in activities such as grain milling, brewing, cheese-making, baking, food

preservation and traditional folk medicine, which over time became highly sophisticated

semi-empirical industries (Hulse, 2004). These so-called ‘old biotechnology’ applications are

the precursors to the modern biotechnology revolution, which has emerged and proliferated

over the last 40 years, through a convergence of four distinct fields of activity, namely

genetic engineering, protein engineering, metabolic pathway engineering and biochemical

(bioprocess) engineering (Gavrilescu and Chisti, 2005). The modern biotechnology field was

24

arguably born in 1973, when the first recombinant DNA was produced by cloning a gene into

a bacterial plasmid (Cohen et al., 1973). What distinguishes the ‘old’ from the ‘new’

biotechnology paradigms is that the former focuses on the production of low- to medium-

value products in bulk quantities, whereas the latter is usually geared towards extremely high-

value products that are required in minute quantities for pharmaceutical or diagnostic

purposes (Webb and Atkinson, 1992). Many countries are realising the socio-economic

benefits of biotechnology, and implementing integrated programmes for developing their

own native biotechnology industries, for the purpose of industrial regeneration, job creation,

social progress, sustainability and enhanced quality of life for their citizens through novel

medical and commercial products (Gavrilescu and Chisti, 2005).

Pharmaceuticals are defined as substances that are used for diagnosis, treatment, cure or

prevention of diseases or substances used to enhance physical or mental well-being (Nagels

et al., 2012), and may be broadly categorised as small molecule chemical therapeutics and

protein biopharmaceuticals. Protein biopharmaceuticals may be categorised into four major

groups (Leader et al., 2008; Strohl and Knight, 2009): (1) protein therapeutics with enzymatic

or regulatory activity (e.g. insulin, growth hormone, factor IX replacement therapies); (2)

proteins with specific targeted activity (e.g. monoclonal antibodies and immunoadhesins); (3)

vaccines (such as fragment C of tetanus toxin); and (4) protein diagnostics for biomarkers

such as glucagon, and imaging agents such as technetium-labeled antibodies. Of all the

market segments for recombinant proteins, the biomedical segment is the one which is

growing most rapidly (Colwell, 2002). Recombinant biopharmaceuticals represent a

modernisation of an old technology. Prior to the advent of recombinant DNA technology,

biotherapeutics were derived in very small amounts from largely unsafe human or animal

sources. The use of variolation with infectious smallpox scab material for centuries (Colwell,

2002), and anti-dipthera animal sera since at least 1895 (Strohl and Knight, 2009) were

primitive attempts at vaccination. In the early 20th century therapeutics were derived from

human or animal sources, such as blood clotting factors, human serum albumin from plasma,

insulin from porcine or bovine pancreas, and glucocerebrosidase from placenta (Sethuraman

and Stadheim, 2006). United States Food and Drug Administration (FDA) approval of the

world’s first commercial biopharmaceutical products, a monoclonal antibody-based

diagnostic kit in 1981, soon followed by Genentech’s bacterially produced insulin in 1983

(Johnson, 1983), set the pace for an exponential increase in biopharmaceuticals to enter the

market in the coming decades.

25

The three major industrial host platforms for producing recombinant proteins are bacteria

(mainly Escherichia coli), yeast and mammalian cells (Sabalza et al., 2014). Mammalian

cells are commonly used for producing biopharmaceuticals for which the glycosylation

pattern is of some relevance to the molecule’s intrinsic activity (Sodoyer, 2004). The large-

scale production of recombinant pharmaceutical proteins is often hampered by the poor

expression of their mature, active forms in prokaryotic hosts such as E. coli and by the high

costs and the limited scalability of traditional fermenter-based platforms using mammalian

cells (Merlin et al., 2014). An emerging alternative to these incumbent expression systems are

transgenic plants, which may address these technical issues (Sharma and Sharma, 2009).

1.2 Transgenic plants

1.2.1 The scope of transgenic plants as an alternative host technology

The genetic transformation of plants was first demonstrated in the early 1980s (Fraley et al.,

1983), and since then, an intense programme of research and development has been

undertaken, involving several industry and academic parties, to exploit transgenic plant host

systems. The original focus of transgenic plant biotechnology was improvement of crop

plants, either agronomic traits or improving their nutritional quality (Clarke and Daniell,

2011; Uncu et al., 2013). Although these are still active fields of research, the same molecular

technologies are being applied to the biosynthesis of high-value biopharmaceuticals. Until

recently, mammalian cells have commanded a near monopoly on production of complex

biopharmaceutical proteins, especially those requiring post-translational modifications

(Kuystermans et al., 2007) and antibodies (Birch and Racher, 2006; Zhang and Shen, 2012).

However, in recent decades, transgenic plants have emerged as a promising platform for the

efficient production of biopharmaceutical proteins (Rybicki, 2010). Plants have been used to

express a diverse range of mammalian proteins, including monoclonal antibodies, therapeutic

enzymes, blood proteins, cytokines, growth factors and growth hormones (Davies, 2010; Ko

et al., 2009; Xu et al., 2012b). Plants, being eukaryotic, are capable of post-translational

processing required for bioactivity, such as protein folding, disulfide bond formation, subunit

assembly, proteolytic cleavage, and glycosylation (Xu et al., 2012b). The use of plants to

26

produce recombinant pharmaceutical proteins has been termed ‘molecular farming’ (Spök et

al., 2008a).

Molecular farming can be rightly considered a ‘disruptive technology’ paradigm as it seeks to

take market share away from conventional biotechnologies, not through incremental

improvements, but by changing the game plan (Fischer et al., 2013). The field emerged and

gained momentum in the early 1990s, when Prodigene started producing avidin commercially

in maize seeds (Hood et al., 1997), followed by a host of other analytical proteins, including

β-glucuronidase, laccase and trypsin (Twyman et al., 2013; Witcher et al., 1998). However,

several roadblocks, including early technological inefficiencies, initially low yields,

regulatory barriers, hyped expectations, public recalcitrance to genetically-modified crops

and high-profile environmental transgressions caused the original molecular farming bubble

to burst (Fischer et al., 2013; Sabalza et al., 2014). The demand for recombinant biologics has

instead been met by incremental innovations in mammalian cell technology (Stoger et al.,

2014; Zhang and Shen, 2012). However, this bubble did not define the industry, but rather

represented early ‘teething’ problems intrinsic in every novel technological platform and

presented an opportunity to re-evaluate and reposition molecular farming in the context of the

biopharmaceutical landscape. The molecular farming paradigm has since re-emerged in the

2000s, with the convergence of transplastomics, a plethora of various in vitro and agricultural

cultivation platforms, bioprocess design, improved molecular biology strategies and yield

improvements providing solutions to the aforementioned problems.

The prospect of plants as competitive and commercially viable biopharmaceutical platform

has recently been realised, after decades of research and incremental improvements. In 2006,

the US Department of Agriculture approved a poultry vaccine against Newcastle disease

made by Dow Agrosciences (IN, USA), manufactured in tobacco cell suspensions, which was

the first license ever issued to a veterinary vaccine produced in plant cells (Katsnelson et al.,

2006). Although the vaccine was never commercialised because the company withdrew from

animal vaccine research, this represents a regulatory milestone in the acceptance of plants as

a manufacturing platform (Ritala et al., 2014). In 2012, the first recombinant plant-based

biopharmaceutical was approved for human use, by the FDA (Anon., 2012). Taliglucerase

alfa (ELELYSO™) is a carrot cell-expressed human β-glucocerebrosidase used as an enzyme

replacement therapy for Gaucher disease, produced by the Israeli company Protalix

BioTherapeutics (Zimran et al., 2011). The utility of plant-made vaccines in pandemic

27

situations was recently demonstrated when the experimental passive vaccine ZMapp, was

rolled out as treatment during the 2014 Ebola virus outbreak in West Africa, despite not

having gone through clinical trials. ZMapp (Kentucky BioProcessing), a combination of three

monocolonal antibodies produced in Nicotiana benthamiana, was found to be effective in

reversion of advanced Ebola virus disease in nonhuman primates (Qiu et al., 2014). A

number of clinical trials of plant-derived biopharmaceuticals are currently underway. A Phase

I clinical trial was recently completed in January 2014, for a personalised plant-derived

vaccine for the treatment of non-Hodgkin’s lymphoma, a product jointly developed by Icon

Genetics GmbH (Halle, Germany) and Bayer Innovation GmbH (Düsseldorf, Germany)

(Ritala et al., 2014).

As well as high-value pharmaceutical synthesis, transgenic plants are seen as a cost-

competitive source of low-value products required in moderate or bulk quantities, such as

industrial enzymes (Hood et al., 2007; Howard et al., 2011), analytical proteins (Hood and

Woodard, 2005), nutraceuticals (Ajjawi and Shintani, 2004; Zhao, 2007), chemical

feedstocks (Lynd et al., 1999) and biodegradable polymers (Mooney, 2009) with the potential

to displace petroleum-derived sources. The sustainability credentials of plant host systems are

often cited (Mooney, 2009). Plants are presented as carbon-neutral solar-powered systems

that, as photosynthetic organisms require little in the way of energy or material inputs

(Mooney, 2009; Sharma and Sharma, 2009). This is only partially true, as plant growth

approaches (both micropropagation and agricultural growth) are still very intensive

operations, in terms of fertiliser / media supply, mechanisation, downstream processing,

storage and supply chaining and the many ancillary operations surrounding cultivation.

However, the establishment of plant-derived chemicals would represent a step towards a

sustainable bio-economy and an improvement to the current petroleum-based system, which

is characterised by increasing feedstock prices and greenhouse gas emissions (Ragauskas et

al., 2006).

1.2.2 The benefits of transgenic plant host systems relative to conventional platforms

Transgenic plants have a number of benefits that set them apart from conventional bacterial,

yeast and mammalian cell-based host systems. A major advantage of molecular farming is

that plant cultivation does not require a high degree of technological input, and basic

28

agricultural techniques can be used (Ma et al., 2013). Some species can grow in almost any

soil or climate (Koprowski, 2005). Moreover, if field cultivation is not pursued,

micropropagative techniques are well-established and straightforward compared to equivalent

microbial or mammalian cell fermentation plants. The microeconomics of transgenic plant

cultivation enables them to be cost-effective host systems, offering order-of-magnitude cost

savings compared to conventional microbial systems (Twyman et al., 2003). It has been

suggested that agricultural production of recombinant protein could be 10-50 times cheaper

than E. coli fermentation (Kusnadi et al., 1997). The elimination of fermenters and other

sophisticated intensive equipment associated with standard ‘stainless steel’ biotechnology

facilities reduces capital expenditure and running costs (Twyman et al., 2003). This is even

true for micropropagation, as plant tissue culture vessels have fewer energy and resource

demands than fermenters. Although set-up costs for cultivation are high, further maintenance

costs are minimal, as only standard agricultural or micropropagation practices are involved

(Stoger et al., 2002). Scalability is an important consideration for any host system. In

principle, plant cultivation is infinitely scalable (Rybicki, 2009), limited only by light, space

and nutrients. In vitro micropropagation allows a ‘scale-out’ approach, through simple

multiplication of culture vessels. In comparison, fermentation systems and transgenic animals

have limits to capacity increase. Moreover, the speed and flexibility of scale-up is important.

It may take several years to achieve ten-fold scale-up of a herd of transgenic sheep through

natural breeding cycles, but transgenic plants can be scaled up 100-1000 fold in a single

generation through soil-based or in vitro regeneration from seeds, callus or explants

(Schillberg et al., 2002; Twyman et al., 2003). If scale-down is required, surplus transgenic

animals must be sacrificed or maintained at a loss, whereas the capacity devoted to

agriculture or micropropagation can be adjusted as required (Twyman et al., 2003). In terms

of regulatory fulfilment, biopharmaceutical production in planta for administration to humans

and animals provides an additional margin of safety as compared to biopharmaceuticals

produced in animal tissues (Koprowski, 2005). Importantly, plant cells do not harbour any

known human pathogens (Xu et al., 2011). This is a significant limitation of mammalian cell

culture and was brought to light in 2009, when Genzyme halted their CHO cell production of

their Gaucher disease therapeutic Cerezyme® after infection with calicivirus (Rybicki, 2010).

29

1.2.3 Bioprocessing of plant-derived biopharmaceuticals

1.2.3.1 Choice of localisation target in transgenic plant host systems

An advantage of transgenic plants as multicellular differentiated host systems is the

possibility of localising foreign protein to almost any organ or cellular compartment,

including leaves, stems, seeds, hairy roots, cell suspension cultures, the apoplast, transformed

chloroplasts and seed protein storage vacuoles (Jiang and Sun, 2002; Rybicki, 2009). Two

plant organs that are emerging as the target host organs of choice are seeds and leaves (Lau

and Sun, 2009; Xu et al., 2012b). Leaves as host organs are amenable to transplastomic and

transient expression (Xu et al., 2012b). However, leaves suffer one important disadvantage

that affects their storage and distribution; leaf proteins are generally unstable without further

processing (Jamal et al., 2009). Seeds are suitable tissues for expressing and storing foreign

proteins, as they have intrinsically high levels of protein accumulation during seed

development (Bewley, 1997; Lau and Sun, 2009; Wilken and Nikolov, 2012). The plant’s

intrinsic capability to partition proteins into specific tissue sinks (Hood and Woodard, 2005)

can be exploited to effectively concentrate heterologous proteins. For example in transgenic

maize kernels, trypsin expression in the embryos is over 100-fold greater than in the

endosperm, and the embryo makes up less than 10% of the kernel dry weight, so separation

of the embryo from the endosperm greatly concentrates the protein (Hood and Woodard,

2005). Compared to leafy biomass, protein localisation in seed crops will result in lower

yields, though increased protein stability in dessicant storage organelles devoid of proteases

allows for long-term storage. Recombinant hirudin expressed as an oleosin fusion in canola

was stable and unmodified for at least three years (Parmenter et al., 1996). Single-chain Fv

(scFv) antibodies expressed in tobacco seeds were found to retain their functionality after

storage at room temperature for 18 months (Ramírez et al., 2001). The degree of protein

stability in seeds presents a decoupling point between cultivation and downstream processing

as biomass does not need to be processed immediately after harvesting (Boothe et al., 2010;

Nikolov and Hammes, 2002; Wilken and Nikolov, 2012). For vaccines, the degree of stability

provided by seeds can circumvent the need for cold-chain refrigeration (Boothe et al., 2010).

30

1.2.3.2 The choice of in vitro or soil-based cultivation for growth of transgenic plants

In contrast to microbial or mammalian cell expression systems, there is a large plethora of

plant expression systems that can be exploited for foreign protein production. There are two

main options for commercial transgenic plant biomass production: soil-based cultivation of

whole plants or in vitro culture, which include micropropagation (plant tissue culture), hairy

root and cell suspension cultures (Xu et al., 2012b).

Soil-based cultivation is the most straightforward and scalable method of cultivation as

standard agricultural techniques can be used. Agricultural soil-based cultivation of transgenic

plants is an extremely low-cost platform, and on the basis of process economics alone, can

compete with established bacterial and mammalian cell fermentation systems (Doran, 2000).

With soil-based cultivation, there are two options: field or greenhouse cultivation. Field

cultivation provides the best process economy, though this is balanced against risks posed by

pests, parasites, anthropogenic pollution and environmental variation which can reduce batch-

to-batch consistency and product quality (Fischer et al., 2012). Greenhouse cultivation is

considerably more expensive than field growth but conditions can be more easily controlled

for greater reproducibility (Doran, 2000; Fischer et al., 2012). It is estimated that a

monoclonal antibody expressed in greenhouse-grown alfalfa will cost US$500–600 g-1 based

on operations in a 250 m2 greenhouse including heating, labour and consumables for protein

extraction and purification (Khoudi et al., 1999). Tobacco and Arabidopsis are being firmly

established model organisms for molecular farming (Budzianowski, 2010; Demeyer, 2011).

However, they are annual crops, completing their life cycle within one year (Craufurd and

Wheeler, 2009; Håkansson, 2003; Wheeler et al., 2000). This may limit the potential for

large-scale agricultural cultivation, as only one harvest a year is allowed. The establishment

of transformation schemes for perennial plant species has been advocated, as these remain

viable for long periods can be cultivated perpetually and the reduced number of breeding

cycles can mitigate genetic instability in transformant lines (D'Aoust et al., 2004; Doran,

2000; Xu et al., 2012b). The perennial legume forage crop, Alfalfa (Medicago Sativa) has

been successfully transformed (Busse et al., 2002; Khoudi et al., 1999; Vlahova et al., 2005).

One potential limitation of soil-based cultivation is gene segregation associated with sexual

reproduction, which can cause variation in protein yields and product quality. This is

especially true with the production of multimeric proteins such as monoclonal antibodies, in

31

which large variations of the fully assembled product have been reported over successive

generations (De Neve et al., 1998; Khoudi et al., 1999).

In comparison with soil-grown plants, in vitro approaches, such as cell suspension and

micropropagation, allow propagation in sterile culture vessels under defined conditions,

giving greater process consistency and reduced batch-to-batch variability (Xu et al., 2011). In

vitro plant and tissue cultures have been used for over 50 years for the synthesis of a wide

range of secondary metabolites, with important roles in pharmaceuticals, cosmetics,

perfumeries, dyeing, and flavour industries (Eibl and Eibl, 2008; Steingroewer et al., 2013).

Commercially successful products include ginseng saponins, shikonin, berberine and

paclitaxel (‘Taxol’) (Fujita, 1988; Kim et al., 1991; Zhang and Zhong, 2004; Zhong, 2002).

In contrast, the use of in vitro cultures for recombinant protein expression is still in its

infancy (Eibl and Eibl, 2008), although a wide diversity of foreign proteins have been

produced, including monoclonal antibodies (Boivin et al., 2010; Holland et al., 2010; Sharp

and Doran, 2001a; Vasilev et al., 2013) and subunit vaccines (Lai and Chen, 2012; Michoux

et al., 2013; Michoux et al., 2011). Carrot cell suspension cultures are used by Protalix

(Israel) for the production of their key product, Gaucher disease therapeutic ELELYSO™

(Zimran et al., 2011). Both suspension and micropropagative cultures circumvent the ‘genetic

instability’ problem, as they are based on asexual (vegetative) reproduction. Plant cell

suspension culture involves agitation of friable callus tissue in bioreactors or shake flasks to

form small aggregates and single cells (Hellwig et al., 2004; Xu et al., 2011).

Micropropagation is defined as the culture of somatic cells, tissues or organs under controlled

in vitro conditions for the generation of clonal progeny plants, in a relatively short time

(Dubranszki and da Silva, 2010). ‘Micropropagation’ and ‘plant tissue culture’ are often used

interchangeably, though in the literature, ‘micropropagation’ is usually used in reference to

generating elite progeny plantlets in vitro for ex vitro transfer or because the plants

themselves are a commercial product (Abbasin et al., 2010; da Silva et al., 2007; Dubranszki

and da Silva, 2010; Santana-Buzzy et al., 2007; Zych et al., 2005). Micropropagative tissue

culture hinges on two intrinsic properties of plant tissues, totipotency, the ability of somatic

cells to divide and regenerate whole plants, and plasticity to generate one type of tissue from

another (Georgiev et al., 2009). Regeneration can follow two morphogenic pathways:

organogenesis or somatic embryogenesis (Ziv, 2000). There are five main types of cultured

tissues: seedlings, isolated embryos, organs, explants (tissue or callus cultures), and isolated

cells or small aggregates in liquid suspension (suspension culture) (Sajc et al., 2000).

32

1.2.3.3 The use of bioreactors in cell and tissue cultures

The use of in vitro suspension culture and micropropagation for biomass production

facilitates the utilisation of bioreactor technologies, allowing the tightly controlled growth

conditions for enhanced target protein expression. Compared to traditional protocols,

bioreactor technologies are time and labour-saving, scalable, allow improved nutrient and

oxygen transfer, and facilitate enhanced growth and multiplication (Akin-Idowu et al., 2009).

Originally, stainless steel stirred tank, bubble column and airlift reactors used for microbial

fermentation were employed, after minor modifications, for plant cell suspension cultures

(Eibl and Eibl, 2008). At high biomass concentrations exceeding 30 g dry weight l−1, poor

oxygen transfer and heterogeneous biomass distribution is observed in airlift bioreactors and

bubble column reactors (Eibl and Eibl, 2008; Tanaka, 1981). Therefore stirred tanks are

preferable for cell suspension cultures at high cell densities. Variations on these conventional

reactors as well as non-traditional designs have become more common in recent years,

especially for micropropagation and root culture. The issues associated with impeded oxygen

transfer and high shear has led to the development of liquid-dispersed and gas-phase

bioreactors, also known as ‘spray’ and ‘mist’ bioreactors, which are used mainly in hairy root

culture (Towler et al., 2006; Weathers et al., 2008; Weathers et al., 2010). In particular, the

‘balloon’ type bubble bioreactor has been adopted by several Korean companies for ginseng

root culture at the 10,000–20,000 l scale, and can also be operated in ‘ebb-and-flow’ mode

for micropropagation (Choi et al., 2006; Weathers et al., 2010). Rotating drum bioreactors

have been conventionally used for fermentations, though they have been adapted to plant

suspension and tissue culture (Weathers et al., 2010).

One of the most momentous changes in plant cell and tissue bioprocessing is the shift

towards disposable single-use reactors, often based on plastic bags (Eibl and Eibl, 2008; Eibl

et al., 2010; Weathers et al., 2010). Capital costs of disposable culture systems are far less

than for the usual stainless steel tanks. The stringent good manufacturing practice (GMP)

requirements for therapeutic compounds necessitate dedicated vessels or costly cleaning

operations between runs (Weathers et al., 2010). Although disposable reactors may have

limited scalability, their lower costs allow multiple units to be used (Weathers et al., 2010).

33

Mechanically-driven bag bioreactors, characterised by wave-induced motion, include the

BioWave, AppliFlex, Tsunami-Bioreactor, Optima and OrbiCell (Eibl and Eibl, 2006). The

BioWave was found to be effective for cultivating tobacco, grape, apple and yew cells up to

10 l culture volume, achieving a 40g fresh weight l-1 day-1 maximum productivity (Eibl and

Eibl, 2008). The Wave and Undertow Bioreactor (WUB) operates by a wave-and-undertow

mechanism, whereas agitation and aeration in a Slug Bubble Bioreactor (SBB) occurs by

movement of large-diameter ‘Taylor-like’ or slug bubbles through a column; both were

successfully used to express isoflavones in tobacco and soya suspension cells (Terrier et al.,

2007).

Bioreactors have allowed enormous scale-up of cell suspension cultures, which often

mitigates against low yields. The 200 l Orbshake device was used for the 100 l culture of

tobacco BY-2 suspensions, giving cell growth and recombinant protein yields comparable to

shake flasks, hence allowing over 100-fold scaling without loss of productivity (Ritala et al.,

2014; Schillberg et al., 2013). Culture volumes of up to 70 m3 were achievable when

microbial fermenters were adapted for plant cells (Eibl and Eibl, 2008). Phyton Biotech in

Germany produces taxanes and paclitaxel in stainless steel stirred tank reactors at the world’s

largest GMP plant cell culture facility (Huang and McDonald, 2012). In comparison to cell

suspensions, the complexity of differentiated tissue has constrained their scale-up potential,

and as a result, bioreactors for tissue culture tend to remain bench-scale (Steingroewer et al.,

2013), although there are exceptions. The mass propagation of Stevia rebaudiana shoots in a

500 l bioreactor and Lilium bulblets in 5-20 l non-stirred reactors has been described (Akita et

al., 1994; Lim et al., 1997). Commercial micropropagation processes have typically involved

scaled-out multiplication of plantlets in multiple small or medium-scale vessels (da Silva et

al., 2007; Escalona et al., 1999; Firoozabady and Gutterson, 2003).

1.2.3.4 The temporary immersion culture format

Of all the various plant bioreactor designs, those based on temporary immersion have several

features making them most amenable to semi-automated micropropagation (Aitken-Christie,

1991; Watt, 2012). In temporary immersion (TI) culture, plant biomass is not permanently

submerged in medium, which may adversely affect growth and morphogenesis and induce

hyperhydricity (vitrification), but is periodically immersed (Watt, 2012; Weathers et al.,

34

2010). In TI systems (TIS), nutrient uptake occurs from a thin film of medium retained on the

surface of the plant tissues by capillarity, whose chemical composition is renewed with each

immersion (Debergh, 1983; Teisson and Alvard, 1995). Temporary immersion systems

facilitate adequate oxygen supply (owing to the fact that biomass is not permanently

submerged), reduced shear damage and reduced risk of contamination (Etienne and

Berthouly, 2002; Teisson and Alvard, 1999). Temporary immersion culture has been

acclaimed as “the most natural tissue culture approach”, as tissue morphogenesis under

reduced exposure to liquid medium more closely resembles soil-based plant cultivation

(Arencibia et al., 2008; Watt, 2012; Ziv, 2000; Ziv, 2005). Temporary immersion culture has

been demonstrated to produce high-quality plantlets at high multiplication rates for several

species, including Siraitia grosvenorii (Yan et al., 2010), sugarcane (Lorenzo et al., 1998;

Mordocco et al., 2009), coffee (Coffea Arabica) (Albarran et al., 2005), pineapple (at 300%

and 400% greater multiplication rates than liquid and solid cultures) (Escalona et al., 1999)

and Musa spp. (Alvard et al., 1993; Escalant et al., 1994). Temporary immersion is

efficacious for stimulating shoot proliferation such as for Pinus radiata (Aitken-Christie and

Jones, 1987) and banana (Alvard et al., 1993).

One of the earliest liquid culture systems which was a precursor to modern TI systems

involved growth of Pinus radiata on solid medium with periodic liquid medium

replenishment and allowed monthly harvesting of shoots (Aitken-Christie and Jones, 1987).

The earliest TI bioreactors were modified, adapted Nalgene two-compartment filtration units,

used for Musa propagation (Alvard et al., 1993) and Hevea brasiliensis somatic

embryogenesis (Etienne et al., 1997), or more recently for Curcuma zedoaria and Zingiber

zerumbet plantlet propagation (Stanly et al., 2010). A number of different bioreactor systems

have been adopted for the micropropagation of several species of horticultural, conservation

or pharmaceutical significance (Watt, 2012). Three popular configurations, based on

pneumatic medium transfer, are the Twin Flasks System (BIT®), Recipient for Automated

Temporary Immersion (RITA®) and bioreactor of immersion by bubbles (BIB®) (Escalona

et al., 1999; Mordocco et al., 2009; Soccol et al., 2008; Zhu et al., 2005). The Twin-Flask

system (BIT®) (Escalona et al., 1999) consists of two containers, one for plant biomass and

the other as a reservoir for liquid medium. When a solenoid valve is opened and compressed

air is turned on, the medium is forced into the first flask, immersing the plants. The process is

reversed when another solenoid valve is opened and air pressure forces the medium back into

the original reservoir (Watt, 2012). The RITA® (VITROPIC, France) bioreactor is made of

35

two compartments, an upper one containing biomass and a lower one containing liquid

medium, linked in such a manner that overpressure applied to the lower compartment pushes

the medium into the upper compartment. Immersion of plantlets is achieved through

application of sterile air overpressure. During the immersion period, air flow replaces the

atmosphere inside the vessel as overpressure escapes through an outlet at the top of the

apparatus. When air flow is stopped, the medium returns to the lower compartment under

gravity (Teisson and Alvard, 1999). A more recent TI system, similar to the RITA®, is the

Plantima® (A-Tech Bioscientific Co. Ltd., Taipei, Taiwan) (Yan et al., 2010).The BIB® is

an entirely new TI system based on immersion of biomass in foam, as opposed to liquid

medium (Georgiev et al., 2014). The BIB® has two chambers divided transversely by a

porous plate (Soccol et al., 2008). The system uses a system interlinked by hoses of flexible

rubber for air flow and medium is delivered to the biomass through bubbling (Scheidt et al.,

2011; Scheidt et al., 2009). As well as these three popular configurations, there is a number of

variations of these as well as more ‘unorthodox’ designs (Georgiev et al., 2014; Watt, 2012).

These include the thermo-photo-bioreactor, based on the two-chamber but including a water

bath for temperature control and integrated UV light source, hybrid Ebb-and-Flow with

saturated tubular convective flow, rocker systems, low-cost disposable systems as well as

custom-made temporary immersion systems for individual labs (Georgiev et al., 2014;

Navarro et al., 2011).

Figure 1.1 Technological design and operational

principle of Twin‐Flask system (Georgiev et al., 2014)

(A) period of exposure. The whole volume of liquid medium

is located into the medium storage tank. Air lines of both

containers are closed and the solenoid valves are opened to

atmosphere; (B) dislocation of liquid medium from medium

storage tank to culture chamber. The air line of cultivation

chamber is closed, and the air line of medium storage tank is

opened. The overpressure moves the medium into the

cultivation chamber; (C) period of immersion. The propagules

are immersed into the liquid medium. The medium storage

tank is empty. Air lines for both containers are closed and the

solenoid valves are opened to atmosphere; (D) draining out

the nutrient medium back to the culture medium tank. The air

line of cultivation chamber is opened, whereas the air line of

medium storage tank is closed. The overpressure moves

back the medium into the medium storage tank.

36

Figure 1.2 Technological design

and operational principle of RITA®

system (Georgiev et al., 2014)

(A) period of exposure; (B) Dislocation of

liquid medium. Air pressure is applied to

the bottom compartment through the

central pipe. The liquid medium is moving

to the upper compartment; (C) period of

immersion; (D) draining out the nutrient

medium. The air flow is stopped and the

medium flows back to the bottom

compartment due to gravity.

Figure 1.3 Technological design and

operational principle of bioreactor of

immersion by bubbles system (Georgiev et

al., 2014)

(A) period of exposure; (B) period of

immersion. Air is supplied and foam is formed.

The explants are immersed by culture

medium in a form of bubbles. When aeration

stops, the foam density decreases with time

due to liquid drainage and the explants are

exposed to gaseous environment.

37

1.2.3.5 Downstream processing

Downstream processing (DSP), involving the extraction and purification of recombinant

proteins, is an integral part of any bioprocess, though it was largely ignored by the early

pioneers of the molecular farming boom (Fischer et al., 2012). The exception was Prodigene

Inc., purveyors of plant-derived technical reagents (Menkhaus et al., 2004; Nikolov and

Woodard, 2004). Huge economic savings can be made through optimisation of downstream

processing, since it accounts for approximately 80% of the final product costs (Evangelista et

al., 1998; Kusnadi et al., 1997). Plant cultivation-to-purification processing procedures

should be standardized to ensure the same final product for therapeutic or diagnostic purposes

(Ko and Koprowski, 2005). The downstream processing schemes of most plant-based

bioprocesses can be divided into three general stages: plant material pre-processing, protein

extraction and protein purification (Nikolov and Woodard, 2004). Pre-processing normally

involves grinding or fractionation of plant tissue. The choice of primary recovery operation

differs between leaf and seed-based bioprocesses. Leafy tissue is typically macerated

followed by centrifugation clarification (Nikolov and Woodard, 2004). For seeds, extraction

can be undertaken concurrently with wet grinding or subsequent to dry grinding and

fractionation for volume reduction (Menkhaus et al., 2004). Two important purification

methods are immunoprecipitation and affinity chromatography (Ko and Koprowski, 2005).

These may be supplemented by additional steps for further purification of captured proteins,

such as hydrophobic interaction chromatography (HIC), immobilized metal ion affinity

chromatography (IMAC), ion-exchange and ceramic hydroxyapatite purification (Wilken and

Nikolov, 2012). The integration of clarification and adsorption is possible by using expanded

bed adsorption (EBA) (Bai and Glatz, 2003; Menkhaus and Glatz, 2005; Valdés et al., 2003).

In contrast to conventional ‘fixed bed’ chromatographic modes, EBA involves fluidisation of

chromatography resins, facilitating capture of target proteins from viscous or high-particulate

mixtures (such as macerated biomass) and reducing fouling of resins (Bai and Glatz, 2003;

Menkhaus and Glatz, 2005; Valdés et al., 2003). Protein A or G affinity chromatography

column operations have been ubiquitously used for capture of plant-made antibodies (IgGs),

often followed by an anion exchange polish step (Chen, 2008; Nikolov et al., 2008; Wilken

and Nikolov, 2012).

38

Figure 1.4 Downstream processing routes for seed and leaf crops (Nikolov and Woodard,

2004)

1.2.4 Chloroplast transformation

1.2.4.1 An overview of plant genetic transformation strategies

Stable nuclear transformation, involving integration of a foreign gene construct within the

nuclear genome, thereby conferring stably inheritable traits, has been the most common

method of plant transformation (Obembe et al., 2011). However, there are significant

limitations with nuclear transformation. T-DNA, the section of Agrobacterium Ti plasmid

that is transferred to the host plant genome, can be integrated as multiple copies, as direct or

inverted repeats or other complex patterns, which can lead to transgene silencing (Husaini et

al., 2011). Likewise, transgene silencing is often observed with nuclear transformation by

microprojectile bombardment, caused by integration of multiple copies of the transgene

(Husaini et al., 2011). Nuclear transgene expression is unpredictable and characterised by low

and highly variable expression levels. Therefore nuclear transformation programmes often

have long development times for generation of stable transformants, due to the complex

genetics associated with identifying and stabilising transgenic lines (Hiatt and Pauly, 2006).

In order to circumvent the issues related to nuclear transformation, transient expression

(epichromosomal transformation) may be used (Fischer et al., 2012; Lico et al., 2005).

Epichromosomal transformation does not involve integration of heterologous genes into the

39

plant nuclear genome and therefore does not allow transgene inheritance (Lico et al., 2005).

Epichromosomal transformation may be mediated by a number of vectors: viruses which

infect a number of plant species such as Bromovirus, Hordivirus (BSMV: Barley Stripe

Mosaic Virus), Tobamovirus (TMV: Tobacco Mosaic Virus), Potyvirus, Potexvirus (PVX:

Potato Virus X), Comovirus (CPMV: Cowpea Mosaic Virus), Tombusvirus (TBSV: Tomato

Bushy Stunt Virus) and Alfamovirus (AMV: Alfalfa Mosaic Virus) (Lico et al., 2005; Pogue

et al., 2002) or Agrobacterium tumefacians (“Agroinfiltration”) (Lee and Yang, 2006).

Transient expression is rapid, and process development is not delayed by regeneration times

and the need to establish stable lines by breeding and seed banking (Fischer et al., 2012). As

there is a short time interval between transformation and expression, transient expression is

suited to the rapid, large-scale and cost-effective production of strain-specific vaccines,

especially in the developing world (Merlin et al., 2014). However, there are important

limitations of transient systems, related to the process complexity introduced by the

infiltration process. The large-scale use of GM bacteria or viruses imposes a huge regulatory

and technical burden, related to their containment, fermentation and proper disposal (Fischer

et al., 2012).

Another approach which circumvents the problematic protein expression associated with

stable nuclear transformation, and non-inheritability and technical problems associated with

epichromosomal (transient) expression, is transformation of plastids.

1.2.4.2 The plastid genome as a target for genetic transformation

1.2.4.2.1 Plastids organelles in higher plants

In plants and algae, plastids are semi-autonomous double-membrane bound organelles, which

evolved from free-living cyanobacteria that were acquired by the eukaryotic host through

endosymbiosis (Gould et al., 2008). In plants, plastids play important roles in photosynthesis,

amino acid and lipid synthesis, starch and oil storage, fruit and flower coloration, gravity

sensing, stomatal functioning, and environmental perception (Wise, 2006).

There is a wide diversity of plastid types. All plastids are derived from small undifferentiated

plastids termed proplastids, found in dividing cells in plant meristems. During cell division,

40

proplastids differentiate into the specialised plastid type depending on the type of cell they

reside in (Osteryoung and Pyke, 2014). The different plastid types can be distinguished by

their pigment composition, biochemical contents and structural features. For instance, green

chloroplasts in leaves contain chlorophyll, etioplasts which are formed under low light

conditions lack pigments, white amyloplasts store starch and possess a minimal internal

membrane system, oil-storing elaoiplasts are small and round, and brightly coloured

chromoplasts found in flowers and fruit contain high concentrations of various carotenoids

(Wise, 2006).

Chloroplasts, the plastids found in green leaves and algae, are the best understood plastid type

in terms of biochemistry and morphology. Chloroplasts are important as the site of oxygenic

photosynthesis. Chloroplasts are observable as plano-convex discs (Wise, 2006). Chloroplasts

are delineated by a galactolipid double membrane envelope (Dörmann, 2001), which contain

an internal membranes system known as thylakoids, (Menke, 1962) surrounded by the

stroma, an aqueous matrix. The thylakoids consist of a continuous 3-D membrane

architecture surrounding a single thylakoid lumen. Thylakoid membranes contain cylindrical

stacked membranes known as grana which are connected by unstacked stroma lamellae.

Mature chloroplasts may contain 40 to 60 grana stacks with diameters of 0.3 – 0.6 µm.

Within the thylakoids large protein-chlorophyll complexes are embedded, containing light-

harvesting complexes, photosystem II, photosystem I, cytochrome b6/f and ATPase which

carry out the light reactions of photosynthesis (Dekker and Boekema, 2005). The stroma is

the location of the Calvin-Benson carbon-fixation reactions (Jablonsky et al., 2011).

1.2.4.2.2 Features of the plastid genome

Plastids possess their own genome and ribosomal machinery for protein synthesis, which is

homologous to that of prokaryotes (Harris et al., 1994). The plastid genome (plastome,

plastid DNA or ptDNA) is a double stranded circular DNA molecule of 130 – 160 kb (Tissot

et al., 2008). The plastid genome contains 120 – 130 genes, which mainly orchestrate the

process of photosynthesis and genetic system ‘housekeeping’ functions (Shimada and

Sugiura, 1991). Around 100 genes encode proteins in land plants, and between 30 – 50 genes

encode RNAs (Rivas et al., 2002; Sugiura, 1992). Genes are arranged in operons and are

transcribed polycistronically (Barkan, 1988). An almost universal feature of the plastid

41

genome is a large inverted repeat (IR) sequence, 10 – 25 kilobase pairs (kb) in size, which

separates the remainder of the genome into single copy regions of approximately 80 kb and

20 kb, the large single-copy (LSC) and small single-copy (SSC) regions (Palmer, 1983;

Palmer, 1985). The plastome has significantly reduced in size with evolution, because many

cyanobacterial genes were incorporated into the nuclear genome (Martin et al., 2002). The

ptDNA is localised to membranes in clusters of approximately 10 genomes, known as

nucleoids (Roh and Choi, 2004).

Figure 1.5 Gene map of plastid genome from tobacco (N. tabacum).

Arrows depict direction of transcription. The map was drawn using OGDRAW v1.1

(http://ogdraw.mpimp-golm.mpg.de/index.shtml) using Genbank accession number of N. tabacum

plastome Z00044. Abbreviations: LSC = large single copy; SSC = small single copy; IR = inverted

repeat region.

http://ogdraw.mpimp-golm.mpg.de/index.shtml

42

1.2.4.2.3 The plastid genome as a novel target for genetic engineering

The science of plastid genetic engineering is known as ‘transplastomics’ (Chamberlain and

Stewart, 1999). Plastid genetic engineering was first attempted in the microalga

Chlamydomonas reinhardtii (Boynton et al., 1988). After the tobacco plastid genome was

fully sequenced (Shinozaki et al., 1986), chloroplast transformation was successfully

undertaken in tobacco (Nicotiana tabacum) (Svab et al., 1990). Since then, tobacco has been

firmly established as the de facto standard model system for transplastomics (Bock, 2014),

though this has brought both benefits and constraints. Tobacco is a leafy plant with high

biomass yields and high soluble protein, with well-established protocols for chloroplast

modification (Tremblay et al., 2010). However, tobacco is an unpalatable crop which

produces toxic compounds, and is therefore not amenable to the production of ‘edible

vaccines’ (Mishra et al., 2008). Moreover, the scope of the transplastomics field depends on

the diversity of crop species that can be transformed. Chloroplast transformation has been

extended to relatively few other species, including Arabidopsis thaliana (Lutz et al., 2011;

Sikdar et al., 1998), potato (Nguyen et al., 2005; Valkov et al., 2011), oilseed rape (Cheng et

al., 2010), carrot (Kumar et al., 2004), cabbage (Liu et al., 2007), lettuce (Kanamoto et al.,

2006; Ruhlman et al., 2007) and tomato (Ruf et al., 2001) and cauliflower (Nugent et al.,

2006) and rice (Oryza sativa) (Lee et al., 2006). Unfortunately, straightforward, reliable and

reproducible transformation protocols for monocot plants, which include agriculturally

important cereals have been lacking (Bock, 2014; Clarke and Daniell, 2011).

1.2.4.2.4 Methods for transformation of the chloroplast

Stable delivery of transgenes into plastids can be undertaken by tissue bombardment with a

particle gun (biolistics) (Svab et al., 1990), treatment of protoplasts with polyethylene glycol

(PEG) (Golds et al., 1993; Kofer et al., 1998; Spörlein et al., 1991), microinjection or

grafting. Biolistics is the classical and most widely adopted method, as it is less time-

consuming and easier in terms of tissue culture procedures. Plasmids are coated on the

surface of gold or tungsten microparticles (0.4 – 1.0 μm) and shot into leaves using a gene

gun. PEG-mediated transformation is occasionally used, being technically demanding and

laborious (Bock, 2014). A rarely used technique, transformation of plastids by microinjection

has resulted in transient gene expression but stable plastome transformation has not been

43

reported (Knoblauch et al., 1999; Maliga, 2003). A new method of plastid transformation by

horizontal genome transfer has been developed, based on the recent observation that

chloroplast DNA (and presumably chloroplasts) can migrate between cells in grafted plants

(Bock, 2014; Stegemann and Bock, 2009). Plastid transformation by grafting may be

instrumental in extending the technology to new species. However, the applicability of

grafting is restricted by potential incompatibilities between the plastid and nuclear genomes

of distantly related species, given the tight functional and regulatory interactions between the

plastids and nucleus (Greiner and Bock, 2013). The causes for plastid-genome

incompatibility (PGI) may be due to poor complementarity between nuclear and plastidial

proteins that are involved in photosynthetic regulation or plastid protein synthesis (Greiner

and Bock, 2013).

Transgenes are incorporated into the plastome by homologous recombination. Flanking

sequences ensure site-specific insertion of the construct into the inverted repeat region of the

plastid genome (Daniell et al., 1998), eliminating ‘position effects’ observed in nuclear

transformed plants. Moreover, ‘gene silencing’ has not been observed in recombinant plastids

(Daniell et al., 2002). Homologous recombination requires prior knowledge of the sequence

of the inverted repeat region of the plastome. One impediment is the lack of plastome

sequence information for several crop plants (Sabir et al., 2014). In the past 25 years, plastid

genome sequences of just 50 crop plastid genomes have been published, compared to 250

non-crop genomes (Jansen and Ruhlman, 2012; Sabir et al., 2014; Saski et al., 2005). As well

as limited successful transformation of such crop species, this also restricts the availability of

plastome sequences for the design of transformation constructs, further reducing the scope for

a ‘universal vector’ for plastid transformation (Sabir et al., 2014). In the quest for a universal

vector, transformation efficiency may be adversely affected if flanking sequences of a

different species are used. For example, when inverted regions from the petunia plastome

were used as flanking regions for tobacco chloroplast, the transformation efficiency was only

7% (DeGray et al., 2001).

To obtain genetically stable transformed plants, the desired genetic modification must be

present in each ptDNA copy in every cell, a situation known as homoplasmy (also known as

homoplastomy) (Ahmad and Mukhtar, 2013). Failure to achieve homoplasmy results in rapid

somatic segregation and genetic instability. Usually, 2 – 3 rounds of selection and

regeneration allow elimination of residual wild-type genomes still present in primary

44

transformants (Bock and Khan, 2004). Selective enrichment of transformed ptDNA is

undertaken via tissue culture, using an antibiotic selection pressure. The transformation

cassette contains an antibiotic-detoxifying gene which confers a selective advantage to

plastids containing transgenic plastomes. Antibiotic selection pressures such as

spectinomycin, streptomycin or kanamycin are used as these inhibit plastid protein synthesis

(Maliga, 2002). The aadA gene encoding a aminoglycoside 3’’-adenylyl transferase which

confers spectinomycin resistance to transformed chloroplasts is the most commonly used

selectable marker gene included in vector constructs (Svab and Maliga, 1993). Transformants

exhibit green shoots indicating photosynthetically functional chloroplasts and can easily be

distinguished from untransformed shoots which are bleached and display depressed growth

(Maliga, 2004). The tissue culture medium triggers cell division, yielding meristemic cells

with 10 to 14 proplastids, each of which carries only one or two nucleoids. Reduction in

plastid number accelerates plastid sorting during cell division. In the presence of the

antibiotic selection pressure, transgenic plastids divide at a faster rate than plastids containing

only wild-type ptDNA. Non-transformed plastids are lost by dilution during cell division

(Maliga and Bock, 2011). The absence of segregation of antibiotic-resistance is considered a

confirmation of homoplasmy (McCabe et al., 2008). Once homoplasmy is established,

rooting of cultured transformant shoots, followed by ex vitro transfer of regenerated whole

plants is undertaken to produce seeds which can then be banked for future propagation.

1.2.4.2.5 Benefits of plastid transformation

1.2.4.2.5.1 Hyperexpression of transgenic proteins in plastids

One of the most alluring features of plastids for biopharmaceutical production is their

enormous capacity to hyperexpress and accumulate foreign proteins. This is due to

polyploidy with approximately 100 chloroplasts per tobacco leaf cell containing a total of

10,000 copies of ptDNA (Shaver et al., 2006). Successful examples of protein overexpression

include accumulation of insecticidal protein Cry2Aa2 to 46% TSP (total soluble protein) (De

Cosa et al., 2001), tetanus toxin fragment C (TetC) to 25% TSP (total soluble protein) of leaf

tissue (Tregoning et al., 2003) and GFP to 38% TSP (Yabuta et al., 2008). Maximum

transgenic hyperexpression ever achieved was that of antibacterial lysin at over 70% TSP,

which even compromised production of endogenous proteins (Oey et al., 2009). Moreover,

45

chloroplasts can perform post-translational modifications (important for proteins of

eukaryotic origin), including correct folding and disulfide bonding, such as for human

somatotropin (hST) (Staub et al., 2000) and cholera toxin B subunit (CTB) (Daniell et al.,

2001a). Heterologous proteins expressed in plants via nuclear transformation or viral

transfection are susceptible to glycosylation with potentially allergenic non-mammalian

glycans (Sriraman et al., 2004). In contrast, plastid-encoded proteins do not undergo

glycosylation, though a disadvantage is that it precludes the synthesis of glycosylated

antibodies.

1.2.4.2.5.2 Post-transcriptional regulation of plastid protein synthesis and

implications for transformation vectors

Chloroplast protein synthesis is largely regulated at the post-transcriptional level (Stern et al.,

1997; Tillich et al., 2010). To optimise transgene expression, chloroplast transformation

schemes exploit a number of endogenous and heterologous regulatory elements, many of

which influence mRNA processing (Ruhlman et al., 2010), such as 3’- and 5’-untranslated

regions (UTRs). Unlike bacteria, the 3’-UTRs in plastids act as processing and stabilising

elements, but do not terminate transcription (Stern and Gruissem, 1987), so it is possible to

extend existing operons by one or more genes. 5’-untranslated regions (5’-UTRs) are

important for transcript stability and translation efficiency (Singh et al., 2001) and contain a

sequence that forms a stem-loop for binding to ribosomes, analogous to bacterial Shine-

Dalgarno sequences (Eibl et al., 1999; Hirata et al., 2004) . Many vectors contain an intact or

truncated 5’-UTR of a highly expressed plastid gene such as psbA, atpB, rbcL (Kuroda and

Maliga, 2001b; Staub and Maliga, 1994), or gene 10 from the bacteriophage T7 (T7G10)

(Kuroda and Maliga, 2001a). The strong plastid rRNA operon (rrn) promoter (Prrn) is the de

facto standard promoter included in most plastid transformation vectors (Maliga, 2003). The

plastid psbA promoter (PpsbA) and chimeric E. coli trc promoter (Ptrc) have also been used

(Staub and Maliga, 1994; Newall et al., 2003). Where polycistronic transcription is required,

a short intercistronic expression element sequence may be inserted to facilitate intercistronic

mRNA cleavage (Zhou et al., 2007).

46

Figure 1.6 Schematic representation of the chloroplast expression cassette. Map of the chloroplast

expression vector shows possible integration sites (flanking regions), promoters, selectable marker

genes, regulatory elements, and genes of interest (adapted from Verma and Daniell, 2007).

1.2.4.2.5.3 Polycistronic transcription of plastid genes and opportunities for

metabolic pathway engineering

Most plastid genes are organised in operons and are co-transcribed to produce polycistronic

mRNAs (Bogorad, 2000). This allows the possibility for stacking several transgenes in

operons and expressing entire biosynthetic or functional pathways, through a single

transformation event. The scope for metabolic engineering applications is huge (Bock, 2014).

In transformed tobacco chloroplasts expressing Cry2Aa2, the cry2Aa2 operon contains a

small open reading frame (ORF) immediately upstream of the cry2Aa2 gene. This ORF

encodes a putative chaperonin which facilitates the folding of Cry2Aa2 into proteolytically-

stable cuboidal crystals, protecting the foreign protein from protease damage, as well as

aiding downstream purification (De Cosa et al., 2001). The generation of transplastomic

tomatoes with elevated β-carotene (provitamin A) levels represents a milestone in plastid

metabolic pathway engineering for generation of nutritionally enhanced foods. Lycopene, a

caretonoid abundant in ripe red tomato fruits, can be enzymatically converted to β-carotene

by lycopene β-cyclases. A chromoplast transformation scheme which incorporated a β-

47

cyclase transgene from daffodil (Narcissus pseudonarcissus) yielding β-carotene levels

reaching 1 mg g-1 dry weight (Apel and Bock, 2009) was found to be most efficient after

testing cyclase transgenes from various other carotenoid-synthesising bacterial, fungal and

plant species (Wurbs et al., 2007). Proof-of-principle studies have assessed the potential for

transplastomic biosynthesis of novel carotenoids (Wilson and Roberts, 2012). Astaxanthin is

a high-value ketocarotenoid used as a food and feed additive. It is not naturally synthesised in

higher plants but does accumulate in some marine bacteria and algae, though it is chemically

synthesised for commercial use. Hasunuma et al. (2008) succeeded in producing over 0.5%

(dry weight) astaxanthin in tobacco leaves by co-expressing genes encoding β-carotene

ketolase and β-carotene hydroxylase, two enzymes involved in astaxanthin biosynthesis. One

of the most complex metabolic pathways transferred into the plastome, is that for the

synthesis of polyhydroxybutyric acid (PHB), a bacterial biopolyester, introduced into tobacco

chloroplasts (Atkin and Cummins, 1994; Lossl et al., 2005). An operon of three genes phbC-

phbB-phbA, encoding three enzymes derived from Ralstonia eutropha has been introduced

into tobacco chloroplasts. The cytoplasmic mevalonate pathway, consisting of six enzymes,

has been incorporated into tobacco chloroplasts, enabling synthesis of isoprenoids (Kumar et

al., 2012).

1.2.4.2.5.4 Maternal inheritance of the chloroplast genome and implications for

biosafety

Despite the self-evident economic and technical benefits of agricultural cultivation of

transgenic plants, concerns with genetically modified organism (GMO) containment are

hindering its widespread implementation. This issue has been especially poignant in the

recent history of molecular farming, with a number of high-profile ‘leaks’ into the biosphere

(Fox, 2003). Transplastomic plants do provide a greater level of biosafety than nuclear

transformed plants, since plastid genes are largely maternally inherited, limiting the risk of

dissemination of transgenes by pollination (Daniell et al., 2002; Hagemann, 2004). It used to

be assumed that chloroplast transformation offered total transgene containment (Daniell et

al., 2002), although studies have shown that in rare cases, low-level leakage of transgenes in

pollen may occur (Avni and Edelman, 1991; Ruf et al., 2007; Svab and Maliga, 2007).

Another possibility is the transfer of transgenes from the plastome to the nuclear genome

(Huang et al., 2003; Sheppard et al., 2008; Stegemann et al., 2003). Although transplastomic

48

plants do not offer ‘absolute’ biosafety, they may be used in conjunction with in vitro cell

suspension, tissue culture or greenhouse cultivation under fully-contained conditions, as part

of an integrated biosafety strategy (Martine et al., 2009).

1.2.4.2.6 Applications of plastid genetic engineering

Transplastomic plants have huge scope to revolutionise biotechnology, with many innovative

applications currently being envisaged and assessed. The potential applications of this

emerging paradigm hinge upon (but are not limited to) three main areas, engineering crop

plants for improved agronomic traits and nutritionally-enhanced foods, ‘molecular farming’

of biopharmaceuticals, metabolic engineering approaches, and expression of commercial

enzymes and other proteins (Bock, 2007; Bock, 2014).

1.2.4.2.6.1 Plastid engineering for improvement of agronomic traits and Rubisco

activity

Plastid engineering can potentially provide an environmentally benign way to improve the

agronomic traits of field-grown crop plants such as herbicide and insect resistance, since

maternal inheritance precludes dissemination of transgenes through pollination.

Resistance to herbicides is a promising application, yielding extremely herbicide-tolerant

strains of crop plants (Bock, 2007). Successful examples of plastid-encoded herbicide

tolerance include resistances to glyphosate (Ye et al., 2001), sulfonyl-urea herbicides

(Sharma and Shanker Dubey, 2005) and PPT-based herbicides (Lutz et al., 2001). A recent

innovation is the development of a plastid resistance gene against D-amino acids that could

be used as a herbicide (Bock, 2014; Gisby et al., 2012).

Strategies for insect resistance are usually based on the synthesis of Bacillus thuringiensis-

derived Cry insecticidal proteins, and have proved very effective (De Cosa et al., 2001;

Scragg et al., 1988; Steward et al., 1999). Transplastomic plants expressing Cry2Aa2 could

kill insects which were tolerant to insecticidal proteins at concentrations 40,000 times higher

than normal (Zhong et al., 1994).

49

Despite being otherwise nutritionally rich, grain legumes contain limited quantities of the

sulphur-containing amino acids, methionine and cysteine. As well as reducing sulphur

deficiency in humans, methionine and cysteine-enriched legumes as ruminant feed may

increase wool, milk and beef yields in sheep and cows (Higgins et al., 1989; Onodera, 1993;

Pickering and Reis, 1993; Rogers et al., 1979). Attempts to upregulate metabolism of

sulphur-containing amino acids and clone high-sulphur storage proteins have enhanced the

nutritional profile of some species, but improvements have been modest (Sabir et al., 2014).

For example, nuclear expression of sunflower seed albumin in T. subterraneum was 0.3%

TSP (Rafiqul et al., 1996), and 0.2% TSP in Festuca arundinacea Schreb. (Wang et al.,

2001). The demonstrated hyperaccumulation of foreign proteins in chloroplasts may mean

that transplastomic plants may be key in enhancing human and animal diets (Ruhlman and

Daniell, 2007).

Efforts to improve global crop productivity through enhancing photosynthetic efficiency are

underway, focusing on the naturally inefficient carbon-fixing enzyme, Rubisco. Although

little progress has been made, there is huge scope for engineering Rubisco (whose large

subunit is plastid-encoded) through transplastomic approaches (Whitney et al., 2011). A

recent development has been the replacement of tobacco Rubisco large subunit (plastid-

encoded) with the cyanobacterial version, which resulted in more efficient carbon fixation

(Lin et al., 2014).

1.2.4.2.6.2 Molecular farming of protein biopharmaceuticals

Transplastomic plants can provide an ideal platform for high-yield synthesis of

biopharmaceutical proteins such as antibodies, vaccines and anti-microbials (Bock, 2007). An

exciting prospect is the development of whole plant ‘edible vaccines’ that require little or no

downstream purification, no expensive refrigerated storage, and facilitate straightforward

needle-free delivery (Daniell et al., 2005; Mason et al., 2002). Edible vaccines do not require

a high degree of purity, so may be enriched or partially-purified, or simply delivered as a

tissue homogenate without any purification (Walmsley and Arntzen, 2003). Vaccination with

tissue homogenates confers bioencapsulation of the active antigen, which may provide some

protection against degradation or dilution in the digestive system (Gonzalez-Rabade et al.,

50

2011). Alternatively, enrichment strategies may be used, including acid precipitation and ion-

exchange chromatography followed by freeze-drying, which increases the concentration of

the recombinant antigen and removes >95% of potentially toxic polyphenols and alkaloids

(Gonzalez-Rabade et al., 2011). However, an enrichment scheme would remove the

bioencapsulation advantage. For orally administered whole plant vaccines, there is a need to

move away from transplastomic tobacco, which is unpalatable and contains potentially toxic

metabolites such as polyphenols and alkaloids, towards palatable food crops such as lettuce

or tomato (Gonzalez-Rabade et al., 2011). Subunit vaccines against HIV (Gonzalez-Rabade

et al., 2011), tetanus (Michoux et al., 2011; Tregoning et al., 2003), cholera (Daniell et al.,

2001a), anthrax (Kamarajugadda and Daniell, 2006; Koya et al., 2005), plague (Daniell et al.,

2005), Lyme disease (Michoux et al., 2013) and rotavirus (Birch-Machin et al., 2004) have

been successfully expressed in transplastomic plants.

1.2.4.2.6.3 Expression of fusion proteins with affinity tags

Affinity (biospecific) tags are widely used used in lab-scale protein purification for functional

proteomics and structural biology studies, and are becoming increasingly common in

bioprocessing (Waugh, 2005; Wood, 2014). Affinity tags allow selective capture of a fusion

protein, through binding of the tag polypeptide to a ligand, followed by elution and cleavage

of the tag from the target protein using enzymatic or chemical methods (Esposito and

Chatterjee, 2006; Frey and Gorlich, 2014; Waugh, 2005; Wood, 2014). Expression of fusion

tags is especially beneficial in transplastomic plant systems (Ahmad, 2012; Ahmad et al.,

2012a; Daniell et al., 2009; Wilken and Nikolov, 2012). Fusion tags can stabilise and protect

target proteins against proteolytic degradation in plastids and facilitate simplified affinity-

based purifications (Wilken and Nikolov, 2012). Insulin-like growth factor (IGF) fused to the

Z-domain of Staphylococcus aureus, expressed in tobacco chloroplasts, was purified in a

simple procedure using two ammonium sulphate precipitation steps and an affinity column

(Daniell et al., 2009), followed by chemically cleavage with hydroxylamine to release the Z-

domain. Transplatomic fusion protein, cholera toxin B–proinsulin localised as inclusion

bodies in tobacco leaves, and underwent in vitro solubilisation and refolding before

purification of the fusion protein by an IMAC-Ni packed-bed column (Boyhan and Daniell,

2011). Studies undertaken by the Nixon group involved transformation and expression of two

affinity tags glutathione-S-transferase (GST) and a His-tagged derivative of the maltose

51

binding protein (His6-MBP) in tobacco chloroplasts, followed by straightforward affinity-

based purification (Ahmad, 2012; Ahmad et al., 2012a). Elution of GST using a glutathione

resin resulted in 50% recovery when a high DTT concentration of 5 mM in the PEB-A

extraction buffer (Ahmad, 2012; Ahmad et al., 2012a). His6-MBP was purified in a two-step

procedure using immobilised nickel affinity chromatography to bind the His6-tag giving 95%

recovery, followed by an amylose column to bind MBP (Ahmad, 2012; Ahmad et al., 2012a).

1.3 Background to PhD Project

1.3.1 The PhD project in the context of previous studies

The Nixon Group, Imperial College London, has a special interest in biolistic chloroplast

transformation, for the overexpression of a wide variety of proteins. Previous studies have

included the transplastomic expression of the following proteins: GST and His6-MBP affinity

tags (Ahmad, 2012; Ahmad et al., 2012a); membrane proteins plastid terminal oxidase

(PTOX) and NADPH dehydrogenase from Chlamydomonas reinhardtii (Ahmad et al.,

2012b); G-protein coupled receptors (Ahmad, 2012); fragment C of tetanus toxin (TetC)

(Michoux et al., 2011; Tregoning et al., 2004; Tregoning et al., 2003); a variant of green

fluorescent protein (GFP+) (Michoux et al., 2011); a subunit vaccine antigen against tetanus,

and outer surface protein A (OspA) from Borrelia burgdorferi which can be used as a

vaccine antigen against Lyme disease (Michoux et al., 2013). The tobacco species Nicotiana

tabacum has been used as the model organism for all aforementioned studies.

The expression of TetC in chloroplasts is one of special significance as it represents the first

reported successful expression of a subunit vaccine antigen in plant chloroplasts (Tregoning

et al., 2003). This is an attempt to produce a potent vaccine against tetanus that could

potentially be administered mucosally (Tregoning et al., 2004). Initial proof-of-concept

studies demonstrated intrinsic yields of 10% and 25% total soluble protein (TSP) in soil-

grown plants, depending on whether plasmid constructs with AT-rich or GC-rich codons

were used, respectively (Tregoning et al., 2003). The regeneration of shoots from callus

tissue in temporary immersion bioreactors represented the next step in this programme, and

52

TetC yields of 8% total soluble protein were reported (Michoux et al., 2011). This was the

first report of in vitro culture of differentiated shoots used as a host platform for

transplastomic protein synthesis. Further studies, in collaboration with Prof. Heribert

Warzecha (Technische Universität Darmstadt, Germany), demonstrated the expression of

Lyme disease vaccine candidate, OspA (Michoux et al., 2013). Temporary immersion culture

has been demonstrated to result in high-yield synthesis of transplastomic proteins in a

contained and controlled manner. The studies outlined in this PhD thesis are in continuity

with these preliminary experiments. The studies outlined in this thesis continue in the

investigation of transplastomic overexpression of TetC, under a wider range of culture

treatments, as well as the expression of a number of other proteins of biotherapeutic

significance.

1.3.2 Aims of Study

The successful establishment of molecular farming as a route to inexpensive

biopharmaceuticals will require the effective integration of a plant genetic engineering with

plant propagation and conventional bioprocessing approaches (such as those associated with

established microbial and mammalian cell host platforms). In particular, this PhD dissertation

focusses on the convergence of transplastomics and in vitro micropropagation for the high-

yield biosynthesis of biopharmaceuticals. The studies described in this PhD dissertation are

based on the in vitro Nicotiana tabacum shoot regeneration from callus in temporary

immersion culture as a platform for the biosynthesis of biopharmaceutical proteins with

special emphasis on transplastomic (plastid-encoded) vaccine antigens. Chapter 3 investigates

various in vitro culture conditions and the influence on biomass growth and transplastomic

TetC protein expression, and relates the synthesis of transplastomic proteins to the maturation

of chloroplasts and increase in chloroplast number during the in vitro morphogenesis process.

Chapter 4 investigates the nuclear expression and assembly of fully-functional monoclonal

antibodies via temporary immersion shoot regeneration, and discusses the implications of in

vitro differentiated shoots for industrial production of complex proteins such as antibodies.

Chapter 5 focusses on a major bottleneck in transplastomic protein expression, the proteolytic

degradation of transplastomic proteins, within the context of in vitro temporary immersion

shoot regeneration, through investigation of two proteins particularly susceptible to

proteolysis, VP6 and p24. Chapter 6 will outline experiments entailing novel developments in

53

in vitro shoot production for molecular farming, scale-up of culture systems and germplasm

preservation, and the impact of biomass growth and TetC expression. The scale-up of

temporary immersion shoot regeneration from 0.5 l to 60 l in a mechanical bioreactor will be

described, which represents one of the first attempts of scaling-up in vitro organogenesis for

biopharmaceutical production. Finally, the feasibility of alginate encapsulation of callus

germplasm for medium-term preservation prior to temporary immersion regeneration was

investigated.

54

Chapter 2. Materials and Methods

2.1 Stock Solutions

2.1.1 Standard solutions and buffers

All buffers, solutions and media were prepared in reverse osmosis (RO) filtered water

(Neptune model L993162, Purite, UK). All chemicals were procured from Sigma-Aldrich

Chemicals (USA), Melford Laboratories (UK), Thermo Fisher Scientific (USA), or Merck

Chemicals (Germany), unless otherwise stated.

2.1.2 Antibodies

Primary and secondary antibodies used in immunoblotting and ELISA studies are listed in

Table 2.1. All antibodies are diluted in Tris-buffered saline (TBS) or phosphate-buffered

saline (PBS) to the required dilution. All secondary antibodies are horseradish-peroxidase

(HRP) conjugated.

55

Table 2.1 List of antibodies used

Primary

antibody

Epitope

Primary

antibody

working

dilution

Source

Secondary

antibody

Secondary

antibody

working

dilution

α-TetC

Fragment C of

tetanus antigen

(TetC)

1:3,000

Prof. N.

Fairweather,

Imperial College

London

anti-rabbit

IgG

1:10,000

α-p24

HIV-1 p24 (HIV

antigen)

1:500

D7320, Aalto

Bioreagents,

Dublin, Ireland

(donated by

Prof. J. Gray,

Cambridge

University)

sheep anti-

goat

monoclonal

antibody

(A9452,

Sigma)

1:1000

α-VP6 (rabbit

polyclonal anti-

rotavirus VP6

antiserum)

VP6 bovine

rotavirus antigen

1:3000

Prof. J. Gray,

Cambridge

University

anti-rabbit

IgG

1;10,000

Guy’s 13

monoclonal

antibody in

transgenic

tobacco lysates

(i.e. analyte is the

primary

antibody)

For indirect

ELISA,

Streptococcal

antigen SA I/II

(conformational

epitope)

N/A for

Western

blot; serial

10-fold

dilution

series from

120 µg/ml

or neat.

Tobacco lysates

from temporary

immersion

culture; lines

donated from

Prof. J. Ma,

SGUL.

Anti-mouse

IgG

1:1,000

56

2.2 Cultivation of transgenic Nicotiana tabacum

2.2.1 Nicotiana tabacum growth conditions

Nicotiana tabacum cv. Petit Havana was used in these studies, except in the monoclonal

antibody expression studies where N. tabacum cv. Xanthii was used. All in vitro

micropropagated plants including seedlings, calli, suspension cultures and regenerated

plantlets were grown in an indoor air-conditioned plant cultivation facility at 25ºC, under a 16

hour photoperiod, at an approximate light intensity of 45 – 120 µmol photons m-2 s-1. Soil-

grown seedlings used as positive or negative controls were grown in a research greenhouse

facility at 25 ºC / 20 ºC (day / night) under 16 hour photoperiod, irradiance of 120 µmol m-2 s-

1 and 40% humidity.

Media at all stages of in vitro micropropation were based on MS basal medium (Duchefa

Biochemie, Netherlands) supplemented with 3% (w/v) sucrose (30 g l-1) and set at pH 5.8

(although certain TI cultures deviated from this recipe). Solid cultures, such as seedling

germination and callus induction, were performed on MS medium supplemented with 8 g l-1

agar (Melford Laboratories Ltd, Suffolk, England) as a gelling agent. To exclude microbial

contamination, 500 mg l-1 spectinomycin and 1 ml l-1 Plant Preservative Mixture™ broad

spectrum biocide (PPM) (supplied by Plant Cell Technology, Washington DC, USA) were

added. PPM is a mixture of two isothiazolones, methylisothiazolone and

chloromethylisothiazolone, which have been found to be effective at eradicating surface and

endophytic bacteria and fungi in plant tissue cultures, while having minimal impact on

explant growth and morphogenesis of several species (Compton and Koch, 2001; George and

Tripepi, 2001; Miyazaki et al., 2010; Niedz, 1998; Niedz and Bausher, 2002). Sucrose and all

plant growth regulators were provided by Sigma (St. Louis, MO, USA). All media, culture

vessels and other materials were sterilised by autoclaving at 120 ºC (103 kPa) for 20 minutes.

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2.2.2 In vitro micropropagation of transgenic N. tabacum

2.2.2.1 In vitro germination of sterile seedlings

Seeds from stable transformant lines were obtained from the Nixon group or from

collaborators. Seeds were germinated in vitro in sterile Magenta vessels on semi-solid MS

medium containing 8 g l-1 agar (Melford Laboratories Ltd., Suffolk, England) gelling agent.

Prior to sowing, seeds were surface sterilised in mild bleach (50% v/v) with 0.1% (v/v)

Tween-20 for 15 minutes, and washed in 3 times for 5 minutes in autoclaved dH2O.

Seedlings were grown for 3 – 4 weeks to provide donor material for generation of callus

germplasm.

2.2.2.2 Callus induction and proliferation, and suspension cultures

For callus initiation, proliferation and shaken suspension, ‘callus induction’ medium (CIM)

was used (4.4 g l-1 MS basal medium, 3% (w/v) sucrose, 1 mg l-1 1-napthaleneacetic acid

(NAA), 0.1 mg l-1 kinetin, pH 5.8). Sections of vascularised leaves from sown plantlets were

cut with a sterile scalpel and plated on culture plates, containing semi-solid CIM (with 8 g l-1

agar), abaxial side down. After 2 – 4 weeks, undifferentiated primary callus, induced mainly

at the cut edges of leaf sections, was plated to new solid CIM for further growth for 2 – 3

weeks. In fresh medium, isolated from the original functional leaf tissue, callus tissue rapidly

proliferates. This may be repeated 2 – 3 times if necessary.

Further liquid callus suspension cultures were undertaken to generate fine uniform callus

aggregates as suitable inocula for temporary immersion culture. Erlenmeyer flasks were

loaded with 200 ml of CIM and small pieces of friable callus from the second or third callus

subculture. Erlenmeyer flasks were orbital shaken at 140 rpm for a period of 2 – 3 weeks to

generate inocula for regeneration. Suspension cultures may be subcultured to fresh CIM 2 – 3

times. Replenishment of media and dilution of waste products extend the duration of the

‘exponential phase’ and allow generation of large quantities of fine aggregates.

58

2.2.2.3 Temporary immersion regeneration of shoots from callus

Plantlet regeneration from undifferentiated callus was undertaken in temporary immersion

(TI) cultures in RITA® two-compartment bioreactors (Vitropic, France), with a biomass

chamber volume of 0.5 l. Air for immersion of cultures is delivered using a vacuum pump

and manifold via silicone tubing. Periodic pneumatic immersion is performed with

application of air over-pressure via an air-pump and solenoid valve medium delivery from a

lower compartment containing medium to an upper biomass chamber. Air bubbled into the

plant chamber during immersion also provides gentle agitation and renews the headspace

atmosphere (Etienne and Berthouly, 2002), providing high oxygen concentrations (Roels et

al., 2006). An immersion duration and frequency of 4 mins every 8 hours was used.

Using a sterile scalpel, 0.5g of fine callus aggregates from suspension culture and 300 ml of

‘plantlet regeneration’ medium were loaded into each RITA® bioreactor. The composition of

‘plantlet regeneration’ medium is 4.4 gl-1 MS basal medium, 3% sucrose and 0.1 µM

thidiazuron (TDZ) at pH 5.8. The duration of temporary immersion culture was 40 days,

during which the callus undergoes organogenesis and vegetative biomass accumulation.

2.2.2.4 Temporary immersion organogenesis from encapsulated calli (modified

procedure)

A variation of the above temporary immersion regeneration procedure was employed using

callus inoculum encapsulated in an alginate matrix. For generation of the calcium alginate

matrix, 3% (w/v) low-viscosity sodium alginate (Sigma, St. Louis, MO) was used as the

gelling matrix and 100 mM CaCl2 (Sigma, St. Louis, MO) as the complexing agent (both

autoclaved solutions) (Hung and Trueman, 2012; Naik and Chand, 2006; Rai et al., 2008;

Singh et al., 2010; Singh et al., 2009). Callus suspensions were filtered using the Corning®

filter system. Using a sterile scalpel, 0.5g of calli were incubated for at least 10 minutes in

sterile falcon tubes containing 25 ml of plantlet regeneration medium supplemented with 3%

sodium alginate (per bioreactor). The solution was carefully decanted. The calli were then

transferred to sterile falcon tubes containing 25 ml 100 mM CaCl2, and incubated for 30

minutes to allow the complexation reaction to occur. The alginate matrix is hardened through

ion exchange between Ca2+ and Na+. The CaCl2 solution was then discarded and the

59

encapsulated calli were washed twice in autoclaved water. The calli encapsulated in the

alginate matrix was then dried for 3 hours by air flow in a laminar flow hood. 0.5 g of

encapsulated callus inoculum was loaded into each RITA bioreactor, and allowed to undergo

organogenesis for 40 days, as per the standard procedure.

2.2.2.5 Regeneration of shoots in a large-scale hydraulic bioreactor

2.2.2.5.1 Construction of large-scale bioreactor

We invented, constructed and operated a prototype large-scale ‘box-in-a-bag’ mechanical

temporary immersion bioreactor for the regeneration of N. tabacum shoots. This consisted of

a biomass-containing chamber within a sealed, sterile plastic bag containing liquid plantlet

regeneration medium. Nutrient delivery to the biomass worked according to the principle of

periodic contacting of biomass with liquid medium through deformation of the bag, which

was achieved through vertical displacement of the box-bag assembly by a hydraulic car jack.

The biomass chamber consisted of a 60 l polypropylene box. Large holes were drilled in the

base and a stainless steel 0.5 mm wire mesh placed on the inner surface of the base to allow

flooding and drainage of medium. The chamber was placed in a 200 l sterile BIOEAZE®

Polyethylene (PE) Bag (Sigma, Aldrich, St. Louis, MO, USA), which was heat-sealed to

close any openings and maintain sterility. Sterile air filters were placed at the inoculation

ports of the BIOEAZE® bag. The box-bag assembly was sterilised by gamma irradiation

(Synergy Health, Swindon, UK). The displacement mechanism was provided by an

Automatic Jack ™ mechanical car jack (product no. 86025, Universal Power Group, Texas,

USA), connected to a mains operated universal digital timer (Tempatron, Maldon, UK). The

mechanical jack was modified to lift to a maximum of 12 cm. The box-bag assembly was

placed upon the car jack piston to facilitate the up / down vertical displacement, and housed

in a larger 145 l box for support and stability.

2.2.2.5.2 Operation of large-scale bioreactor

16 l of plantlet regeneration medium was loaded into the BIOEAZE® Polyethylene (PE) Bag,

followed by inoculation of 60 g callus suspension. Medium and inoculum addition was

60

undertaken via an autoclaved funnel and inoculation port of the bag placed in a laminar flow

hood (this was a compromise as it is physically impossible to place the entire box-bag

assembly in the hood). Shoot regeneration culture was undertaken for 50 – 80 days. The

biomass media immersion mechanism occurred as follows. In between immersions, the

mechanical jack and biomass chamber is raised in the default ‘up’ position. The liquid

medium settles under gravity in the ‘bulge’ beneath the biomass chamber. During

immersions, the mechanical jack piston descends to the ‘down’ position, so that biomass

chamber and bag are level. The deformation of the bag and the disappearance of the ‘budge’

cause the medium to be displaced so that it contacts the biomass and nutrient delivery can

take place. When the immersion cycle has ended, the jack piston and biomass chamber are

once more raised to their default position and the liquid medium drains away from the

chamber, settling at the bottom of the bag. Similar to the RITA temporary immersion system,

in this system, the callus inoculum undergoes organogenesis and vegetative biomass

accumulation.

Figure 2.1 Operation of large-scale mechanical bioreactor

(A) When the mechanical jack piston is in the default ‘up’ position, the biomass chamber and bag

assembly are raised and the medium settles in the bulges beneath the biomass chamber.

(B) The piston is lowered for nutrient delivery. The displacement of liquid medium and the space

constraint causes the biomass to be immersed.

2.2.2.6 Harvest of regenerated shoots, and fresh and dry weight determination

At the end of the allotted culture period, plantlet biomass was harvested and weighed to

determine fresh weight. Small representative samples of shoot tissue were excised and frozen

at -80°C for further analysis. Additionally, large healthy leaves were excised for chlorophyll

fluorescence analysis. After fresh weight determination, plantlets were dried in an oven at

A B

61

80ºC for 24 hours, for total moisture removal from tissues. Dried biomass was then allowed

to cool to room temperature and weighed. The ratio of dry-to-wet weight was taken, and the

dry weight is calculated.

2.3 Protein Analysis

2.3.1 Total Soluble Protein Extraction

Total soluble protein (TSP) extractions were undertaken from frozen regenerated plant

biomass (healthy leaves, vitrified leaves, undifferentiated shoot primordia and representative

samples of total biomass) harvested from temporary immersion culture. Frozen plant extracts

were ground with a pestle and mortar in liquid nitrogen. The resulting fine powder was mixed

with protein extraction buffer (PEB-A) (50 mM HEPES-KOH (pH 7.5), 2 mM DTT, 1 mM

EDTA, 10 mM potassium acetate, 5 mM magnesium acetate, and 1 tablet of cOmplete Mini

protease inhibitors EDTA-free cocktail (Roche Applied Sciences, Germany) per 5 ml buffer)

in a ratio of approximately 100 mg biomass to 100 µl. Samples were vortex mixed for 1

minute. Samples were centrifuged at 18,000 g for 30 minutes. The supernatants were

collected and the pellets were discarded. The supernatants were centrifuged again to remove

any residual pellets. Lysate extracts containing total soluble protein (TSP) were subjected to

SDS-PAGE as described in 2.3.3.

2.3.2 Determinination of protein concentration

The concentration of proteins in the lysates was determined using the Bradford Assay (Sigma

Aldrich, St. Louis, MO, USA), according to the manufacturer’s instructions (Bradford, 1976).

A dilution series of known concentrations of bovine serum albumin (BSA) were used as a

standard. The absorbance at 595 nm was plotted against the concentration to compute the

extinction coefficient against which the protein concentrations of the samples were

calculated.

62

2.3.3 SDS-PAGE

Soluble proteins were separated by sodium dodecyl sulphate polyacrylamide gel

electrophoresis (SDS-PAGE) using the Bio-Rad mini-gel electrophoresis system (Bio Rad

Laboratories, USA). 15% (w/v) gels were made. Resolving gel recipe (6 mini-gels): 9.38 ml

40% acrylamide; 5 ml 3 M Tris-HCl buffer (pH8.9); 300 µl 10% SDS; 6 M urea; dH2O added

to 25 ml; 0.01% (w/v) N,N,N,N-tetramethylenediamine (TEMED); 0.1% (w/v) ammonium

persulphate (APS). 5% stacking gel recipe (6 mini-gels): 1.25ml 40% acrylamide; 1.2 ml 1 M

Tris-HCl (pH 6.8), 100 µl 10% SDS; H2O added to 10 ml; 0.01% (w/v) TEMED; 0.1% (w/v)

APS. Prior to loading onto gels, TSP samples were solubilised in 4 × solubilisation buffer

(250 mM Tris-HCl (pH 6.9), 8% (w/v) SDS, 40% (w/v) glycerol, 0.1% (w/v) bromophenol

blue and 10% (w/v) β-mercaptoethanol (added freshly for each use)) for 5 mins at 100 °C. 8–

12 µg of protein were loaded into each lane. Gels were run at room temperature at 130 V for

2–3 hours in SDS-PAGE running buffer (25 mM Tris-HCl (pH 8.3), 190 mM glycine, 0.1%

(w/v) SDS). Precision Plus Protein™ Dual Color Standards (Bio Rad Laboratories, USA)

were used to determine molecular weights of migrated protein bands. Mini-gels were run as

duplicates, with one mini-gel stained and the other for immunoblot transfer.

2.3.4 Staining of polyacrylamide gels

Gels were stained for 3 – 5 hours with Coomassie blue (0.2% (w/v) Coomassie Brilliant Blue

‘R-250’, 40% (v/v) ethanol and 10% (v/v) acetic acid) or SYPRO® Orange Protein Gel Stain

(Life Technologies) (5,000 x dilution of concentrate in 10% (w/v) acetic acid). Prior to

visualisation, Coomassie-stained gels were de-stained overnight or SYPRO® Orange-stained

were de-stained for 5 minutes, in both cases with 10% (v/v) acetic acid. Visualisation of total

protein staining was undertaken using a standard gel imager for Coomassie or LAS-3000

CCD digital imaging system (FujiFilm, USA) for SYPRO® Orange, according to the

manufacturer’s instructions.

2.3.5 Immunoblotting and Enhanced Chemiluminescence (ECL) Detection

At all stages after protein electrotransfer, all buffers used, including blocking, incubation and

washing are based on TBS-Tween (2.42 g l-1 Tris-base, 80 g l-1 NaCl, pH 7.6, 0.1% (v/v)

63

Tween-20) unless otherwise stated. Following electrophoresis, ‘wet’ transfer of proteins to a

0.2 µm nitrocellulose membrane was conducted using the Bio-Rad Mini Trans-Blot®

Electrophoretic Transfer Cell (Bio-Rad Laboratories, USA) at 60 V for 1 hour, according to

the manufacturers’ instructions, using transfer buffer (0.84 g l-1 NaHCO3, 0.30 g l-1 Na2CO3,

20% (v/v) methanol) (Towbin et al., 1979). The membranes were blocked in 5% (w/v) dried

skimmed milk (Marvel brand) for 1 hour. Alternatively, proteins were transferred to a

polyvinylidene fluoride (PVDF) membrane using the iBlot® Dry Blotting System

(Invitrogen, USA). The membranes were then incubated in primary antibody overnight at 4

ºC or for 1 hour at room temperature, washed 3 × 20 minutes, and subsequently incubated

with secondary antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room

temperature. The membranes were washed 3 × 10 minutes in TBS-Tween, followed by a 10

minute final wash in TBS without Tween.

Enhanced Chemiluminescence (ECL) was undertaken using the Enhanced

Chemiluminescence (ECL) Detection kit (Amersham Pharmacia, UK), according to the

manufacturer’s instructions. Chemiluminescent detection was conducted using a LAS-3000

CCD digital imaging system (FujiFilm, USA), according to manufacturer’s instructions.

Alternatively, chemiluminescent detection may be undertaken on X-ray film (Kodak, USA)

developed in a Curix60 table top processor (AGFA Healthcare, Belgium), according to the

manufacturers’ instructions.

For semi-quantitative analysis of target proteins, densitometry was conducted on exposures

(digital or film) that were not overexposures, using ImageJ software (National Institutes of

Health, USA). Normalisation to stained total protein was used as a loading control (Aldridge

et al., 2008).

2.3.6 Indirect ELISA to assess functional activity of plant-expressed monoclonal

antibodies

Costar® 3603 96-well polystyrene plates (Corning) were used as the solid phase in all

instances. Phosphate buffer saline (PBS) (7.5 mM Na2HPO4, 2.5 mM NaH2PO4, 150 mM

NaCl, pH 7.4) was used as a universal buffer at all stages, including coating, blocking,

64

antibody incubations and washing, unless otherwise stated. TSP lysates containing expressed

antibodies were prepared according to the procedure described in 2.3.1 and total protein

concentrations were determined, as described in 2.3.2. Serial dilutions of lysates were made

in PBS. The antigen, diluted 1/1000 to approximately 5 µg ml-1, was immobilised (‘coated’)

to the bottom of the wells by passive adsorption, for 2 hours at room temperature, or 4ºC

overnight. After washing twice with distilled water, the wells were blocked with 5% (w/v)

dried skimmed milk (Marvel brand) for 3 hours at room temperature or overnight at 4ºC,

followed by 2 × washes with distilled water with 0.1% (v/v) Tween-20. Wells were then

incubated in plant TSP lysates as serial 10-fold or 2-fold dilutions (‘log10’ or ‘log2’ dilutions

respectively) for 2 hours at room temperature, to facilitate binding of the immobilised antigen

to antibodies expressed in the lysates. After washing 5 times with distilled water and 0.1%

Tween-20, incubation of horseradish peroxidase (HRP) conjugated secondary antibody

(‘conjugate’) with 5% milk was undertaken for 2 hours to allow binding to the primary

antibody. Detection was undertaken by adding equal volumes of 3,3′,5,5′-

Tetramethylbenzidine (TMB) solution (Merck-Millipore, USA) and stop solution (1 N

H2SO4), and reading absorbances at 450 nm, according to the manufacturers’ instructions.

2.4 Analysis of in vitro regenerated plant biomass

2.4.1 Viability assay of in vitro regenerated shoots

This viability assay is based on the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to

insoluble red triphenylformazan (TF) by mitochondrial dehydrogenase activity, as an

indicator of plant cell viability (Clemensson-Lindell and Persson, 1995). 400 mg of fresh

biomass were incubated in 7.5 ml of 0.1 M sodium phosphate buffer (pH 7.0) with 0.6%

(w/v) TTC and 0.05% Tween-20, for 20 hours at 30°C in darkness (Ruf and Brunner, 2003).

After incubation, the formazan was extracted by decanting the buffer, grinding biomass in

liquid nitrogen, transferring the powder to eppendorf tubes, addition of 1.7ml 96% ethanol,

and centrifugation at 10,000g for 2 minutes. The supernatants containing TF were collected

and the absorbance at 520 nm was determined.

65

2.4.2 Chlorophyll Fluorometry

Chlorophyll fluorescence measurements were undertaken using a pulse-modulated

fluorometer (DUAL PAM 2000, Walz Germany) with DUAL-PAM-100 measuring system

and DUAL-PAM v1.11 software. Recordings of photosystem II (PSII) parameters such as Fm

(maximum fluorescence), Y(II) (effective PSII quantum yield), Fv/Fm (maximum PSII

quantum yield) were taken (Maxwell and Johnson, 2000).

Medium or large healthy TIB-regenerated leaves were used for measurements. Leaves were

dark-adapted for at least 15 minutes beforehand. Fluorometry was undertaken at room

temperature. The minimal fluorescence (F0) and maximum fluorescence (Fm) were taken

using a saturation pulse of 6000 µmol photons m-2 s-1 for 0.6 seconds in dark-adapted leaves.

66

Chapter 3. Parameters affecting the dynamics of

biomass growth and transplastomic protein accumulation

in temporary immersion culture

3.1 Introduction

3.1.1 In vitro differentiated plant tissues for molecular farming

With the advent of plant-made biopharmaceuticals, there is a need to establish robust

bioprocessing and regulatory strategies (Spök et al., 2008b). Within the bioproduction

process, the ‘upstream’ steps involving cultivation and harvesting require the most innovation

and development, in order to exploit the distinctive features of the plant host system (Colgan

et al., 2010). In contrast, for the later downstream processing (DSP) steps involving crude

extraction and further purification, much of the established approaches from existing

microbial and mammalian cell-based platforms can be used as they will not differ greatly

from plant-based bioprocesses (Fischer et al., 2012). There is increasing interest in the in

vitro regeneration and differentiation of shoots as the heart of this suite of upstream

bioprocessing technologies for large-scale biosynthesis of biopharmaceutical proteins (Doran,

2000; Steingroewer et al., 2013). Fortunately, decades of expertise from the commercial

micropropagation industries can be applied to new molecular farming applications. There are

several challenges that the in vitro plant regeneration paradigm must face, if it is to compete

with established recombinant host systems. Existing bacterial, fungal and mammalian cell

biotechnologies are built on decades of bioprocess innovation and infrastructure, which have

resulted in fairly in-depth biological and process characterisation of fermentation culture

systems (Shuler and Kargi, 2002). Moreover, conventional bacterial and yeast hosts are

unicellular and undergo little significant developmental change during bioprocessing,

whereas plant morphogenic growth involves a greater degree of complexity which

profoundly impacts product yield. This is especially true with cell and tissue culture in which

culture parameters can be evaluated in a systematic way and ‘tuned’ in order to optimise

heterologous protein yield (Doran, 2000). The studies outlined in this chapter describe the

67

influence of various temporary immersion culture parameters on the growth and

morphogenesis of Nicotiana tabacum callus tissue and TetC biosynthesis.

3.1.2 Nt-pJST12 as a rational model host system for investigating the impact of the

culture environment on recombinant protein expression

Clostridium tetani, a bacterium widespread in nature, produces tetanus toxin (also known as

tetanospasmin, TeNT or TeTx), an extremely potent neurotoxin, which is responsible for the

clinical syndrome of tetanus. C. tetani spores germinate and synthesise TeNT under

conditions of low oxygen tension, slight acidity and nutrient availability such as wounds and

skin ruptures (Popoff, 1995). Tetanus usually occurs after an acute injury, and is

characterised by prolonged contraction of skeletal muscle fibres and over-activity of the

autonomic nervous system, which causes spasms and vasoconstriction, leading to a rise in

blood pressure (Tregoning et al., 2004). Tetanus toxin may constitute more than 5% of the

total mass of the organism. It is a single polypeptide of approximately 150 kDa and 1315

amino acid residues, which forms a two-chain activated molecule composed of a heavy chain

(Hc) and light chain (Lc) linked by a disulphide bond (Calvo et al., 2012). Parenteral

immunisation has been very effective at preventing tetanus, by causing production of anti-

tetanus toxin antibodies, thus blocking its action. Conventionally, vaccine preparations

against tetanus are produced by formaldehyde detoxification of tetanus toxoid, although this

is a time-consuming and intensive process (Anderson et al., 1996) and causes a reduction in

antigenicity (Metz et al., 2013). Tetanus vaccine antigens have been routinely administered as

a component of the trivalent diphtheria–tetanus–pertussis (DTP) vaccine since the 1940s

(Bernstein et al., 1995; Brodzik et al., 2009; Orenstein et al., 1990). Fragment C, also known

as TetC, is a non-toxic 47 kDa cleavage product of tetanus toxin that can be used as a subunit

vaccine against tetanus (Makoff et al., 1989; Tregoning et al., 2003). TetC has been

successfully expressed in chloroplasts, in the stable N. tabacum cv. Petit Havana line Nt-

pJST12, generated by the Nixon group, Imperial College London (Tregoning et al., 2003).

This transplastomic line was used in all the studies described in this chapter.

68

3.1.3 Aims of studies

Preliminary studies undertaken by the Nixon group into the feasibility of in vitro shoot

morphogenesis in RITA® temporary immersion bioreactors (TIBs) for high-level and

contained expression of TetC have proved effective (Michoux et al., 2013). Moreover the

yield of TetC can be modulated by changing various culture conditions. The aim of these

studies, using TetC expression in Nt-pJST12 as a model system, is to:

gain understanding of the in vitro morphogenesis process in TIBs, and the

implications on transplastomic protein yields;

investigate the influence of various culture conditions on biomass growth and

transplastomic protein yields.

Nt-pJST12 shoot biomass was regenerated from callus tissue under a range of different

conditions in RITA® temporary immersion bioreactors, through changing the composition of

the standard culture media or the culture protocol.

3.2 In vitro shoot regeneration via organogenesis of N. tabacum callus

and the influence on transplastomic protein expression

3.2.1 Callus organogenesis as a developmental pathway for in vitro plantlet

regeneration

As a biotechnological host, plants are unique, in terms of growth and development.

Organogenesis is a defining feature of plants, owing to their multicellularity, while being

absent in alternative bacterial and yeast expression systems. Although organogenesis is a

feature of animals, the developmental strategies of plants are entirely different. Most land

plants (embryophytes) undergo indeterminate growth as a mode of irreversible volume

increase, involving the continuous formation of new tissues and organs as a result of

perpetual meristemic activity throughout their lifetime (Kutschera and Niklas, 2013). In

contrast, animals undergo determinate growth, in which body size and body size and organ

number are predetermined, and organogenesis only occurs during embryogenesis (Woodward

69

et al., 2006). In plant tissue culture, the indeterminate mode of growth is closely related to

totipotency, the capacity to regenerate organs and even whole plants from differentiated cells

(George et al., 2007). From a biotechnological perspective, indeterminate growth of plants in

organogenic tissue culture from callus tissue is an advantageous feature, as it facilitates

continual formation and proliferation of shoot tissue for recombinant protein expression.

3.2.2 In vitro morphogenesis dynamics during temporary immersion culture

3.2.2.1 Design of experiment

In order to determine growth kinetic parameters during temporary immersion organogenesis,

several temporary immersion cultures in RITA® bioreactors were undertaken in parallel,

using the same callus suspension inoculum in order to minimise batch-to-batch variation.

Temporary immersion cultures were allowed to grow for an allotted time interval, and then

the biomass was harvested for fresh and dry weight determination and total soluble protein

(TSP) extraction.

3.2.2.2 Results and Discussion

3.2.2.2.1 Dynamics of biomass growth and morphogenesis

Callus tissue is a disorganised, apparently undifferentiated cell mass, which in nature is

generated as a stress response to wounding (Stobbe et al., 2002). The totipotency of callus

tissue is widely exploited in tissue culture, and callus formation is induced through

exogenous application of auxin and cytokinin (Ikeuchi et al., 2013). In vitro morphogenesis

involves the transition of callus to fully-differentiated shoot tissue. This is a complex

phenomenon, involving morphological, physiological and metabolic changes as a

consequence of reprogramming of gene expression and induction of the totipotent state of

somatic cells (Ovečka et al., 1997).

70

In vitro morphogenesis involves localisation of rapidly dividing cells, and aggregation of

friable calli, accompanied with an increase in metabolism (Fournier et al., 1991; George et

al., 2007). This localisation into clusters was observed to result in formation of meristemoids.

These are calluses in which there is a layer of proliferating shoot meristems overlaying an

inner core of vacuolated cells acting as a mechanical and nutritional support (George et al.,

2007). This observation is consistent with previous reports of a meristemoid stage in organ-

forming cultures of N. tabacum (Ovečka et al., 1997; Ross et al., 1973; Yoshikawa and

Furuya, 1983). The formation of meristemoids in temporary immersion culture involves the

transition of callus aggregates to compact clusters, and subsequently the formation of

primordia leading to shoot buds (Ovečka et al., 1997; Yoshikawa and Furuya, 1983).

Primordia represent an intermediate stage between undifferentiated calli and fully

differentiated shoots, having the appearance of nodular outgrowths that will develop into

shoot buds. In temporary immersion culture, the meristemoid developmental sequence, with

zones of preferential cell division and subsequent shoot primordia formation is not entirely

age dependent, but occurs continuously over several days, starting early in some calluses but

later in others. Localisation of cell division on the surface of callus clusters leading to

meristem formation was observed between days 6 and 17, with subsequent formation and

proliferation of shoot primordia occurring between days 12 and 25. This apparently continual

developmental phenomenon without clear-cut milestones is consistent with other reports

(Ross et al., 1973). In vitro morphogenesis is instigated by 0.1 µM thidiazuron (TDZ), a

cytokinin. TDZ is known to be effective at promoting shoot regeneration (Ivanova and van

Staden, 2008; Thomas and Katterman, 1986) and somatic embryogenesis (Gill and Saxena,

1993).

The kinetics of increase in biomass accumulation during a temporary immersion culture can

be modelled using a logistic growth model, similar to bacterial growth models (Figure 3.2).

There is a ‘lag’ phase between days 0 – 20, followed by an exponential increase in biomass

from approximately day 25 to 35. The increase in biomass growth slows down from

approximately day 40 to 100. In practice, TIB cultures are normally harvested at day 40. The

biomass growth curves are reflective of the morphological and physiological changes that

accompany the transition of callus aggregates to differentiated shoots that were observed

during morphogenesis.

71

Figure 3.1 Callus-meristemoid transition and shoot bud formation during morphogenesis. (A)

Callus aggregates on day 0 (inoculum from callus suspension culture) (B) Callus aggregates on day 6.

(C) Primordia formation at meristems in callus clusters on day 17. (D) Shoot bud formation in

meristemoids on day 25. (E) Leaf and shoot formation on day 30.

(F) Fully-differentiated shoot clusters on day 40.

72

Figure 3.2 Logistic increase of fresh and dry biomass accumulation during in vitro

organogenesis in RITA® TIBs.

3.2.2.2.2 Differential expression of TetC during in vitro organogenesis

SDS-PAGE and immunoblot analysis was undertaken to demonstrate the differential

expression of TetC at different time intervals during the morphogenesis process in TIB

culture (Figure 3.3). A comparative analysis of TetC ‘absolute’ volumetric yield (milligrams

per litre of bioreactor) (estimated using densitometry of immunoblots) and fresh biomass

increase is shown in Figure 3.4. There is a clear increase in expression between days 0 and

25, closely following the transition of calli to shoots. The intrinsic yield of TetC (as a

proportion of total soluble protein) is 15-fold higher in shoot clusters than in undifferentiated

calli used as inoculum. A small decline in TetC expression is observed in older tissues, from

73

90 to 100 days. This is possibly due to senescence-related proteolysis in the chloroplasts.

TetC is exhibited as a doublet of two bands of size 43 kDa and 47kDa, consistent with

previous work undertaken by the Nixon group (Michoux et al., 2011; Tregoning et al., 2003).

The doublet may be attributed to partial degradation of TetC in the chloroplast or during total

soluble protein (TSP) extraction (Michoux et al., 2011; Tregoning et al., 2003).

The increase in intrinsic TetC yield during morphogenesis reflects the maturation of plastids,

involving the transition of proplastids to mature chloroplasts, accompanied by development

of photosynthetic apparatus and increased plastidial protein synthesis (Ladygin et al., 2008).

The number of plastids and nucleoids increase during in vitro cell and tissue culture. There is

a striking correlation between transplastomic protein expression and the increase in

chloroplast number and developmental status. This may be inferred by the increasing

abundance of chloroplast-encoded Rubisco large subunit, which as one of the most abundant

proteins in plants, is represented by a distinct band at approximately 50 kDa on a gel (Ma et

al., 2009). It has been previously observed that plastid copy number increases rapidly in

exponential phase N. tabacum cell cultures (Takeda et al., 1999), with the highest frequency

of chloroplast division occurring in the early exponential phase. Moreover, the intrinsic

features of the transplastomic transformation vector can greatly influence expression. Prrn,

the plastid 16S rRNA promoter included in most plastid transformation vectors is similar to

the 16S rRNA promoter in rice (Michoux et al., 2011) which has seven-fold lower activity in

rice embryogenic calli compared to leaves (Silhavy and Maliga, 1998). It was found that

plastid-encoded mRNA levels of embryogenic rice calli were much lower than in leaves, for

rbcL (153-fold lower), atpB (37-fold lower), 16SrDNA (7-fold lower) (Silhavy and Maliga,

1998).

74

Figure 3.3 SDS-PAGE and immunoblot showing differential expression of TetC. TetC

expression during (A) callus-to-shoot morphogenesis and (B) stationary phase. 12% acrylamide;

LWM marker (A); Precision Plus marker (B); 8µg protein loading per well; Coomassie staining.

A B

Figure 3.4 Increase in TetC volumetric yield and fresh biomass. Error bars

denote standard errors.

75

3.3 Impact of Hyperhydricity on Expression of TetC

3.3.1 The hyperhydricity phenomenon in in vitro micropropagation

Hyperhydricity (sometimes known as ‘vitrification’) is a common and problematic

phenomenon in micropropagative tissue culture, much to the chagrin of commercial

micropropagators. Ever since Debergh et al. (1981) first reported and defined the

phenomenon of ‘vitrification’, many comprehensive studies have been undertaken to attempt

to understand and reduce this phenomenon. Although a number of factors have been

implicated in the induction of hyperhydricity, it is not entirely predictable (Olmos et al.,

1997). Hyperhydricity encompasses a range of morphological disorders, characterised by a

thick, translucent, brittle stems and leaves (Kevers et al., 2004; Olmos and Hellın, 1998; van

den Dries et al., 2013). In temporary immersion culture, vitrified shoots are easily

distinguished by translucency of shoots and leaves, thick waterlogged leaves with little

deposition of epicuticular waxes (Figure 3.5). It is recognised that the hyperhydricity

phenomenon in in vitro culture is due, in part, to high humidity, the presence of liquid media,

low air exchange and sealed culture vessels (Kevers et al., 2004; Olmos and Hellın, 1998; van

den Dries et al., 2013). In the temporary immersion culture system, regular infiltration of

biomass with liquid medium contributes to hyperhydricity.

76

Figure 3.5 Visual comparison between non-vitrified and vitrified shoots

A, B, C: Non-vitrified biomass

D, E, F: Vitrified biomass

3.3.2 TetC accumulation in vitrified and non-vitrified leaves

Regenerated biomass harvested from a number of TIB cultures was separated into vitrified

and non-vitrified (healthy) samples and TSPs were extracted for SDS-PAGE and immunoblot

analysis. Hyperhydricity was found to have little notable influence on the expression of TetC,

at different stages of morphogenesis (Figure 3.6) or at different sucrose and light irradiances

(Figure 3.7). However, the immunoblots indicate that the intensity of the lower 44 kDa band

of the TetC doublet is lower in vitrified shoots compared to healthy shoots. As the expected

A B C

D E F

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size of TetC is 47 kDa, this suggests that hyperhydricity may inadvertently lead to increased

TetC stability.

Figure 3.6 Investigation of hyperhydricity on TetC accumulation at different time intervals

of temporary immersion culture, by SDS-PAGE and immunoblot. (A) 28 days; (B) 33 days; (C)

40 days. 8-10 µg protein analysed by SDS-PAGE on 12% (w/v) polyacrylamide gel followed by

Coomassie or Sypro Orange staining.

A B C

78

Figure 3.7 Effect of hyperhydricity on TetC accumulation at various sucrose

concentrations and irradiances. 11 µg protein loading; 12% acrylamide; Sypro Orange staining;

Precision Plus marker.

3.3.3 Discussion on the influence of hyperhydricity on transplastomic protein

expression

These studies indicate that hyperhydricity conditions have little significant impact on

transplastomic protein expression (Figures 3.6 and 3.7). The reasons for this are unknown,

although this may be associated with the expression and localisation of the heterologous

proteins in the plastids. Hyperhydric waterlogging of plant tissues is a largely apoplastic

phenomenon (van den Dries et al., 2013) and although there is increased expression of stress-

associated genes, such as hypoxia-responsive genes (van den Dries et al., 2013) and

antioxidant enzymes (Balen et al., 2009; Dewir et al., 2006), these are mainly nuclear-

79

encoded. The transcriptional regulatory circuits which cause upregulated expression of

certain proteins under stress conditions mainly act on nuclear genes (Aarts and Fiers, 2003;

Ciarmiello et al., 2011), and not the plastid genome. Therefore incorporation of the transgenic

construct into the plastid genome may offer protection against the stress-related changes in

protein synthesis. The observation that hyperhydricity may contribute to increased stability of

transplastomically expressed proteins is unexpected and intriguing. The reasons behind this

observation are currently unknown, though worthy of future investigation.

3.4 The Impact of Tissue Culture Media on Morphogenesis and TetC

yield

3.4.1 The impact of ammonium : nitrate ratio on TetC yield

3.4.1.1 The nitrogen requirements of in vitro plant growth

Nitrogen (N) is an essential element for plant life. After carbon, N is required in largest

quantity by plants, being a constituent of proteins, DNA, RNA, chlorophylls, co-enzymes,

phytohormones and secondary metabolites (Hawkesford et al., 2012). Fertilisers and culture

media usually include inorganic nitrogen, in the form of nitrate (NO3-) or ammonium (NH4

+),

though organic sources such as glutamine or urea may be used. Standard Murashige & Skoog

(1 ×) media contains 60 mM inorganic nitrogen, a higher amount than other in vitro culture

media and more than 2/3 of the total mineral content (Murashige and Skoog, 1962), in a ratio

of 40 mM NO3- : 20 mM NH4

+. Despite the ubiquity of MS media in plant tissue culture, very

few studies have attempted to establish a physiological basis for this particular nitrogen pool

(George et al., 2007). However, the absolute and relative amounts of nitrogen have been

found to significantly influence growth and morphogenesis (George et al., 2007), in in vitro

tissue culture (Chaleff, 1982; Evans, 1993; Ivanova and Van Staden, 2009), cell suspensions

(Gorret et al., 2004; Holland et al., 2010; Vasilev et al., 2013) and seedling cultivation (Bar-

Yosef et al., 2009; Tan et al., 2000).

80

The aim of this set of studies is to investigate the influence of the inorganic nitrogen pool

composition on biomass growth, organogenesis and TetC expression in temporary immersion

culture. The first study involved assessing the change in media pH over the course of a 40-

day TI culture duration, and inferring the nitrogen assimilation kinetics from this. The second

study entailed investigation of biomass accumulation and transplastomic protein expression at

various NO3-: NH4

+ ratios, while keeping the total inorganic nitrogen content at 60 mM.


The ratio of NO3-: NH4

+ was modulated, while keeping the total concentration of inorganic

nitrogen constant at 60 mM, as in standard MS media (Murashige and Skoog, 1962). MS

basal media including vitamins was made de novo, to the same composition as the

commercial version (M0222, Duchefa Biochemie) (http://www.duchefa-

biochemie.com/product/details/number/M0222), though without inorganic nitrogen. Nitrogen

was added later according to the required ratio of NO3-: NH4

+. TIB shoot regeneration

cultures were undertaken at seven ratios of NO3-: NH4

+ – 0:60, 10:50, 20:40, 30:30, 40:20,

50:10, and 60:0 (mM : mM). The ionic balance of the media was maintained using K+ and Cl-

as balancing ions when individual minerals were removed. Shoot regeneration cultures were

undertaken as duplicates for each ratio tested, in RITA® TIBs. After a 40-day culture period,

biomass was harvested and TSPs were extracted for SDS-PAGE and immunoblot analysis.

3.4.1.3 Results

3.4.1.3.1 Influence of nitrogen source ratio on growth and TetC expression

3.4.1.3.1.1 Influence of nitrogen source ratio on biomass accumulation and

morphogenesis

The biomass growth of N. tabacum was highly affected by the NO3-: NH4

+ ratio at an initial

total nitrogen content of 60 mM (Figure 3.9). There is a positive relationship between NO3-

content and fresh and dry biomass accumulation, and extent of morphogenesis (Figure 3.8).

No morphogenesis occurs at 0:60 and 10:50; no differentiated leaf tissue was observed for

http://www.duchefa-biochemie.com/product/details/number/M0222

http://www.duchefa-biochemie.com/product/details/number/M0222

81

these treatments. At all treatments between 20:40 and 60:0, fully-differentiated leaf and shoot

tissue is observed, with no visually notable differences between them. When NH4+ was used

as the sole nitrogen source, there was virtually no biomass accumulation, 16.8 g l-1 fresh

weight and 0.42 g l-1 dry weight. At 10:50, the biomass accumulation was 212 g l-1 fresh

weight and 8 g l-1 dry weight, but biomass consisted of undifferentiated callus clusters.

Interestingly, the callus biomass observed at 0:60 and 10:50 exhibited a deep orange colour,

indicating a lack of chlorophyll and presumably little photosynthetic capacity. The curve

plateaus at 20:40, with maximum fresh biomass accumulation of 291 g l-1 observed at this

ratio (Figure 3.9). Between 10:50 and 60:0, there was a marginal increase in dry weight, from

9 g l-1 to 11.6 g l-1.

It is obvious that in vitro regenerated tobacco has a preference for nitrate, compared to

ammonium, as a N source. Quantitatively, this preference may be determined on the basis of

total mean dry biomass generated in ammonium alone, as compared to nitrate alone. For the

sake of symmetry around ‘zero’ and for simple comparison between species or propagation

regimes, the preference ratio is log2-transformed as:

𝑙𝑜𝑔2𝑃𝑅 = 𝑙𝑜𝑔2 (𝐷𝑊𝑎𝑚𝑚𝑜𝑛𝑖𝑢𝑚

𝐷𝑊𝑛𝑖𝑡𝑟𝑎𝑡𝑒)

with log2PR denoting the log2 preference ratio (Bartelheimer and Poschlod, 2014). In this

study, the log2PR for in vitro regenerated tobacco was found to be -4.77, indicating a strong

preference for nitrate.

82

Figure 3.8 Effect of NO3-: NH4

+ ratio on developmental status of N. tabacum regenerated

shoots after 40-day temporary immersion culture. (A) 0:60; (B) 10:50; (C) 20:40; (D) 30:30; (E)

40:20; (F) 50:10; (G) 60:0; (H) proliferation of callus aggregates lacking chlorophyll under 10:60

treatment; (I) fully-differentiated leaves observed under 60:0 treatment.

83

Figure 3.9 Influence of NO3-: NH4

+ ratio on fresh and dry biomass accumulation. Error bars

represent standard errors.

3.4.1.3.1.2 Influence of nitrogen source ratio on yield of TetC

Immunoblot analysis was undertaken to assess the impact of NO3-: NH4

+ ratio on TetC

expression after a 40-day culture period (Figure 3.10). Hyperexpression of TetC is observed

in shoots having undergone morphogenesis, at NO3-: NH4

+ ratios between 20:40 and 60:0. In

comparison, very low expression is observed in undifferentiated callus at NO3-: NH4

+ ratios

0:60 and 10:50 (high ammonium treatments). Densitometric analysis was undertaken to

quantify TetC expression and estimate volumetric yields (Figure 3.11). The nitrate-related

shoot regeneration resulted in TetC intrinsic yields of 7–9 ng µg-1, compared to 2 ng µg-1 in

undifferentiated biomass, an approximate 4-fold increase. In absolute terms, the estimated

volumetric TetC yield is 2–2.5 mg l-1 in regenerated tissues, compared to approximately 100

µg l-1 in undifferentiated callus when NH4+ is the sole nitrogen source, a 20 – 25 fold

increase.

84

Figure 3.10 SDS-PAGE and immunoblot analysis of lysates to assess TetC expression

under various NO3-: NH4

+ ratios. 12% acrylamide gel; 7 µg protein loading; Sypro Orange staining;

Precision Plus ladder.

Figure 3.11 Densitometric quantification of TetC intrinsic yields and volumetric yields

under various NO3-: NH4

+ ratios, from immunoblot data in Figure 3.10. Error bars represent

standard errors.

85

3.4.1.3.2 pH shift of media during temporary immersion culture

The preference of N. tabacum biomass for nitrate is further validated by the observed shift in

pH during temporary immersion regeneration culture, shown in Figure 3.12. 1 ml samples of

media were withdrawn from duplicate TIB cultures every 2-3 days and the mean pH was

determined. It was observed that during temporary immersion regeneration, the pH of the

media steadily rises from approximately pH 5.5 (post-autoclave pH) to 7.0. Gradual media

alkalinisation during liquid culture is a well-documented phenomenon in plant suspension

culture (George et al., 2007; McDonald and Jackman, 1989; Srinivasan et al., 1995). The

nitrogen assimilation dynamics can be inferred, as an increase in pH is related to uptake of

nitrates. Uptake of nitrate takes place effectively in an acid pH, but is accompanied by

extrusion of anions, leading to the medium gradually becoming more alkaline. In contrast,

uptake of ammonium results in the cells excreting protons into the medium, making it more

acidic (George et al., 2007). This study indicates that in vitro culture for N. tabacum callus

organogenesis has a preference for nitrate as a nitrogen source, highlighted by the gradual pH

rise of the media. It is widely reported that for suspension cultures there is an initial sharp

uptake of NH4+ characterised by a sharp fall in pH to approx. 4.2-4.6 (George et al., 2007),

followed by a gradual alkalinisation during which NO3- absorption is stimulated. Although

there is a small decrease in pH to 5.4, this is not as pronounced as in other studies (Gorret et

al., 2004; McDonald and Jackman, 1989; Srinivasan et al., 1995), indicating a very weak

preference for NH4+.

Figure 3.12 Shift in medium pH over temporary immersion culture period. Error bars denote

standard errors.

4.00

4.50

5.00

5.50

6.00

6.50

7.00

7.50

0 2 4 7 9 11 14 16 18 21 23 25 28 30 32 35 37

pH

Time (days)

86

3.4.1.4 Discussion on the influence of nitrogen pool on in vitro regeneration and

transplastomic protein expression

These studies have demonstrated the preference for NO3- over NH4

+ for in vitro N. tabacum

shoot differentiation, organogenesis and transplastomic protein expression. It was found that

NH4+ as a sole nitrogen source is ineffective for growth, whereas NO3

- alone was sufficient to

induce morphogenesis. Although a NO3-:NH4

+ ratio of 10:50 resulted in high biomass

accumulation, no differentiation and functionalization of leaves occurred. High biomass

growth and shoot regeneration were observed for every NO3-:NH4

+ ratio tested between 20:40

and 60:0.

The superiority of nitrate over ammonium for promoting growth and protein expression is

consistent with previous several previous studies in plant tissue culture. Successful shoot

formation and proliferation has been reported when NO3- was the only nitrogen source

(Cousson and Van, 1993; Ivanova and Van Staden, 2009; Nagakubo et al., 1993; Ramage and

Williams, 2002; Tsai and Saunders, 1999; Woodward et al., 2006). Our study demonstrates

high shoot regeneration when a combination of NO3- and NH4

+ are used, indicating a

synergistic effect between the two ions (Gamborg, 1970; Ivanova and Van Staden, 2009;

Ramage and Williams, 2002; Shirdel et al., 2011). The inclusion of NH4+ mitigates the

alkalinisation of the media, and thus may contribute towards enhanced NO3- assimilation

(Shirdel et al., 2011). The highest regeneration of Aloe polyphylla shoots occurred with

NH4+:NO3

- (mM) of 20:40, 30:30 and 40:20 (Ivanova and Van Staden, 2009) and greatest

shoot formation of tobacco leaf discs was reported when media containing 40mM NO3-

:20mM NH4+ was used (Ramage and Williams, 2002). Nitrogen assimilation in plants occurs

through progressive reduction of NO3- to NO2

- (nitrite) by nitrate reductase and then to NH4+

by nitrite reductase, followed by ammonium assimiliation into amino acids (Masclaux-

Daubresse et al., 2010; Xu et al., 2012a). Despite this, ammonium as the sole nitrogen source

has been found to inhibit growth and shoot morphogenesis in a number of species (Castro-

Concha et al., 2006; Chaleff, 1982; Cousson and Van, 1993; Murthy et al., 1998; Ramage and

Williams, 2002; Richter et al., 2007; Walch-Liu et al., 2000). This is the result of reduced cell

division and elongation (Walch-Liu et al., 2000). Reduced growth potential caused by high

NH4+ levels is attributed to a number of possible factors including acidification of media and

87

corresponding growth inhibition, the toxic effects of intracellular free ammonia (NH3) which

is related to disturbances in pH homeostasis, uncoupling of photophosphorylation and a

reduction of photosynthesis (Ivanova and Van Staden, 2009; Walch-Liu et al., 2000). It has

also been suggested that at low pH, NH4+ inhibits uptake of essential inorganic elements or

causes leakage of nitrogenous metabolites (Murthy et al., 1998). However, assimilation of

NH4+ as the sole nitrogen source is possible in buffered media or at a pH close to neutrality

(to mitigate the influence of acidification), or in the presence of an organic acid citrate,

malate or pyruvate (George et al., 2007).

These studies demonstrate the associations between inorganic nitrogen source, chloroplast

maturation, developmental status of biomass and expression of transplastomic proteins. The

complex interactions between these factors may be explained when considering that nitrogen

assimilation in plants is developmentally regulated, and the chloroplasts play a major role in

this (Xu et al., 2012a). Reduction of nitrite to ammonium is catalysed by nitrite reductase

localised in chloroplasts (Meyer and Stitt, 2001). Assimilation of ammonium to amino acids

by the so-called GS/GOGAT cycle also occurs in the chloroplast. Ammonium is fixed to a

glutamate molecule by glutamine synthetase (GS) to form glutamine (Masclaux-Daubresse et

al., 2010). This glutamine reacts subsequently with 2-oxoglutarate to form two glutamate

molecules, catalysed by glutamine 2-oxoglutarate amino transferase (GOGAT) (Masclaux-

Daubresse et al., 2010). In chloroplasts, GS and GOGAT are both reduced by the electron

carrier protein ferrodoxin (Fd) or NADPH, which must first be reduced by photosystem I

(Hanke et al., 2004). Moreover, GS is dependent on hydrolysis of ATP (Eisenberg et al.,

2000). Organic assimilation of nitrogen into amino acids requires the availability of carbon

skeletons and especially keto-acids (Masclaux-Daubresse et al., 2010). Hence, the link

between photosynthesis and plastidial nitrogen assimilation, in the context of transplastomic

protein expression, becomes apparent. The supply of ATP, Fd, NADPH and carbon skeletons

for nitrogen assimilation requires actively photosynthesising chloroplasts. It is known that

nitrate reductase (a cytosolic enzyme) and nitrite reductase are induced in the presence of

nitrate (Faye et al., 1986; George et al., 2007; Grimes and Hodges, 1990); this is corroborated

by these studies. The reduced capability of the chloroplasts to undertake nitrogen reduction

and amino acid synthesis explains the decreased TetC expression when NH4+ was the sole

nitrogen source and when 50 mM NH4+ : 10 mM NO3

-.

88

The demonstrated requirement of nitrate for the expression of recombinant protein expression

is consistent with previous reports. Nitrate enrichment of the culture medium resulted in

increased accumulation of accumulation of 2G12 monoclonal antibody in N. tabacum BY-2

cell suspension cultures (Holland et al., 2010). Through a combination of fractional factorial

designs (FFDs) and response surface methodology (RSM), it was found that KNO3 and

NH4NO3 were among the nutrients that has the most significant influence on human antibody

M12 accumulation in N. tabacum BY-2 cell suspension cultures (Vasilev et al., 2013). The

estimated volumetric yield of TetC observed when NO3- and NH4

+ are used in combination is

similar to that when NO3- alone is used, with the optimum yield observed at 20:40 NO3

-

:NH4+, suggesting a synergistic effect of using both ions on protein metabolism. From the

perspective of plants as protein factories, NH4+ is the more ideal nitrogen source. NO3

-

uptake requires expenditure of considerable ATP (Atkin and Cummins, 1994). In

comparison, NH4+ uptake can be driven by the electrochemical gradient generated during

normal metabolism, although energy is required for re-establishment of the original gradient

(Raven et al., 1992). NH4+ directly goes towards amino acid synthesis (and therefore protein

synthesis) via glutamate, without first being reduced (George et al., 2007). Unfortunately, in

this study low assimilation of NH4+ was observed. This may be partially attributed to initial

low pH of 5.8 and the further possible acidification during culture, limiting uptake of NH4+.

3.4.2 Effect of initial media pH on biomass growth and TetC expression


As a standard practice, plant tissue culture media is titrated to slightly acid conditions, pH

5.4–5.8, prior to autoclaving (George et al., 2007). In our protocol, the medium pH is set at

5.8. Many in vitro plant cells and tissues will tolerate pH in the range of 4.0-7.2 (Butenko et

al., 1984). The medium pH is an important parameter which determines a range of metabolic,

biotransport and morphogenic activities in cultured tissues (George et al., 2007). As

demonstrated in 3.4.1.3.2, the pH is expected to drift over the course of the culture period as

well as after autoclaving, especially in unbuffered medium. The impact of initial medium pH

on biomass accumulation and TetC expression was investigated, by undertaking 40-day

temporary immersion regeneration cultures in media titrated to a range of pH levels, 3.8, 4.8,

89

5.8 and 6.8 (pre-autoclave pH). Cultures were undertaken as triplicates, and TSPs from

regenerated biomass were used to undertake SDS-PAGE and immunoblot analysis.


3.4.2.2.1 The influence of initial medium pH on biomass growth and TetC expression

High shoot morphogenesis was observed in all treatments tested (Figure 3.13). Initial media

pH was found to have no significant effect on either fresh or dry biomass accumulation.

There was also a small impact on TetC intrinsic yield, with more acidic conditions favouring

TetC expression (Figure 3.14). A small increase in TetC expression is observed with pH 3.8

and 4.8, in comparison to 5.8 and 6.8.

Media pH is an important parameter as it influences the uptake of nutrients and

phytohormones and regulation a wide range of biochemical reactions in plant tissues,

especially those catalysed by enzymes (Owen et al., 1991). However, in this study, pH was

found to have no discernable influence on growth or recombinant protein expression. This

study is in concordance with others in which initial pH has little influence on biomass growth

in suspension and regeneration cultures (Bhatia and Ashwath, 2005; Butenko et al., 1984;

Kaul and Staba, 1968; Martin and Rose, 1976). These results suggest that In vitro cultured N.

tabacum callus and shoots are tolerant to a wide range of acidic pH.

90

Figure 3.13 Influence

of media pH on fresh

and dry biomass

accumulation. Error bars

represent standard errors.

Figure 3.14 SDS PAGE

and immunoblot analysis

of of media pH effects on

TetC expression. 12%

acrylamide gel; 14µg protein

loading; Sypro Orange

staining.

91

3.4.3 The Impact of Varying Sucrose and Irradiance for Photomixotrophic

Propagation Regimes

3.4.3.1 The importance of exogenous saccharides and irradiance in in vitro plant

tissue culture

Light and sugars both play an important role in plant grown and development. As well as

being the main source of energy via photosynthesis, light plays a signalling and regulatory

role in several developmental processes (Franklin et al., 2005). High light may be a source of

stress and damage the photosynthetic apparatus through photoinhibition (Vass et al., 2007).

Sugars also play a major role in metabolism, as respiratory substrates and precursors for

synthesis of metabolically important compounds such as amino acids, fatty acids and

structural compounds such as cellulose. In vitro tissue culture usually involves addition of the

dissacharide sucrose to the medium and growth in sterile airtight vessels (Pospóšilová et al.,

1999), in order to promote an increase in multiplication rate. However, features of this culture

environment include low air exchange rates, high humidity (>95%), low photosynthetic

photon flux (PPF) (50-100 µmol photons m-2 s-1), low photoperiod CO2 concentrations,

ethylene accumulation and stagnant air movements (Zobayed et al., 2000). These conditions

can cause several physiological and metabolic abnormalities. Photoautotrophic culture

methods have been advocated to overcome these problems and enable plantlets to develop

full photosynthetic capabilities to produce endogenous carbohydrates for growth.

Photoautotrophic growth conditions are regarded to promote growth and physiological

development, a higher multiplication rate than conventional heterotrophic culture regimes

and successful ex vitro acclimatization (Kozai et al., 1991). Photoautotrophy is induced

through using sugar-free medium and can be promoted through forced ventilation or

enhanced diffusive ventilation (Kozai and Kubota, 2001), CO2-enriched air (Kozai, 1991;

Solárová and Pospíšilová, 1997) and high light intensities. Photoautotrophy is difficult to

achieve in practice, so a directed photomixotrophic culture strategy may be used, without

eliminating sugar from the medium completely (Gonzalez-Olmedo et al., 2005). Most

micropropagative protocols involve photomixotrophic growth of plant tissues, which

assimilate carbon via both exogenous sugars and photosynthesis.

92


In typical in vitro cell, tissue and organ cultures, 3% (w/v) or 30 g l-1 sucrose is typically

used, the de facto standard level since Murashige and Skoog first formulated their MS media

(Murashige and Skoog, 1962). This is the case with our TI culture method for heterologous

protein expression in N. tabacum (Michoux et al., 2013; Michoux et al., 2011). It appears that

this 3% sucrose level is based mainly on tradition, without reference to the intrinsic

photosynthetic capabilities of in vitro regenerated plantlets. In typical micropropagation

facilities including our own, irradiance levels are typically low, approximately 45 µmol m-2 s-

1. Hence, for the purposes of this study, 3% sucrose and irradiance of 45 µmol m-2 s-1 will be

considered ‘standard’ conditions, and other sets of sucrose and light intensities will be

considered ‘deviations’ from standard conditions.

The aim of this study is to investigate the impact of sucrose concentration and light level on

vegetative biomass growth in temporary immersion culture and yield of TetC. Temporary

immersion organogenesis cultures were undertaken under varying sucrose concentrations, 0

(0%), 7.5 (0.75%), 15 (1.5%), 30 (3%) or 60 g/l (6% w/v), denoted S0, S7.5, S15, S30 and S60

treatments and three irradiance levels under three difference irradiance levels, 45, 75 and 120

µmol m-2 s-1. PAM fluorometry was undertaken on regenerated leaves by temporary

immersion culture to confirm their photosynthetic activity.


3.4.3.3.1 Effect of sucrose and irradiance on biomass accumulation and TetC

expression

When cultured in sucrose-free media, tobacco callus displayed virtually no growth and

underwent necrosis, demonstrating that exogenous carbohydrates are essential for shoot

multiplication (Figure 3.15). This may be due to insufficient light intensity to establish truly

photoautotrophic cultures. These results in are agreement with those of Schnapp and Preece

(1986) who showed that reducing the sucrose concentration to 0 produced fewer and shorter

axillary shoots in in vitro grown carnation plants (Schnapp and Preece, 1986). The supply of

exogenous sucrose positively influenced in vitro growth, both fresh and dry biomass

93

accumulation (Figure 3.16). The data show that there is an apparently linear relationship

between sucrose concentration and dry biomass accumulation, for all three irradiances

investigated. The positive association between exogenous sucrose and vegetative growth

traits is consistent with findings by previous authors (Galzy and Compan, 1992; Klughammer

and Schreiber, 1994; Kovtun and Daie, 1995; Kubota et al., 2002; Schnapp and Preece, 1986;

Tichá et al., 1998). Notably, the fresh biomass accumulation for S15 treatments was almost

identical to S30, despite having half the sugar supply. The regenerated shoots for both S15 and

S30 treatments showed visually healthy phenotypes, with a high degree of differentiation and

expansion. A reduction in fresh weight accumulation is observed for S60 under all light levels

tested, indicating that high sucrose levels exert an inhibitory effect on growth. Several

authors have reported that excessive sugar-feeding can be detrimental and impede in vitro

biomass accumulation, although the optimum level of sucrose varies between species and

culture protocol (Kovtun and Daie, 1995; Park et al., 2004b; Zhang et al., 1996; Zych et al.,

2005).

Figure 3.15 Visual demonstration of the effect of sucrose concentration on shoot

morphogenesis after 40-day temporary immersion culture at 45 µmol m-2 s-1. (A) 0 g/l (0%); (B)

7.5 (0.75%); (C) 15 g/l (1.5%); (D) 30 g/l (3%) (E) 60 gl-1 (6% w/v).

94

Figure 3.16 Effect of sucrose concentration and irradiance on fresh and dry biomass

accumulation. Error bars denote standard errors.

3.4.3.3.2 Effect of sucrose and irradiance on TetC expression

Densitometric analysis of immunoblots (Figures 3.17, 3.18 and 3.19) show that under

conditions of sucrose deprivation (0.75% and 1.5% sucrose), transplastomic protein

expression is greatly increased. From a standard 3% sucrose concentration, a 2-fold reduction

to 1.5% results in approximately 50% increase in TetC yield, whereas a 4-fold reduction to

0.75% gave an approximate 4-fold increase in TetC expression. The impact of sucrose

reduction on transplastomic protein yield increase was so great that at 45 µmol m-2 s-1

irradiance, a 3-fold increase in absolute TetC yield from 2 mg l-1 to 6 mg l-1 is possible by

reducing sucrose concentration from 3 to 0.75% w/v, even after accounting for a 32%

reduction in fresh biomass. Conversely, high sucrose concentrations seem to inhibit

heterologous protein expression, though this may be associated with reduction in growth

95

potential. The doubling of sucrose concentration from 3% to 6% resulted in approximately

86% and 89% reduction in intrinsic and absolute TetC yields. Irradiance levels were not

found to profoundly influence TetC expression levels. The light levels investigated in this

study were very low (characteristic of typical micropropagative facilities), and may not

greatly influence the development of photosynthetic capabilities.

Figure 3.17 SDS-PAGE and immunoblot showing the effect of sucrose and light on Tetc

expression. 10% acrylamide gel; 7 µg protein loading; Coomassie staining.

96

Figure 3.18 Influence of sucrose and light levels on TetC intrinsic yield (ng TetC per µg total

soluble protein (TSP)), determined densitometrically. (A) sucrose concentration as independent

variable; (B) irradiance as independent variable. Error bars denote standard errors.

Figure 3.19 Influence of sucrose and light levels on estimated absolute TetC yield (µg TetC

per litre of bioreactor)

0

1000

2000

3000

4000

5000

6000

0 1 2 3 4 5 6

Estimated absolute

TetC yield (µg/l)

Sucrose concentration (% w/v)

A B

97

The suppression of transplastomic protein expression with increasing sucrose supply may be

due to a number of factors. Firstly, increased sucrose supply is associated with a reduction in

photosynthetic capacity, through a number of mechanisms. Importantly, there is

downregulation of plastidial gene transcription associated with photosynthesis (Sheen, 1990).

The repression of transplastomic gene expression by exogenously supplied sugars is clearly

observed, though the exact mechanism is not fully known. In plants, the end-products of

photosynthesis, sucrose and monosaccharide cleavage products glucose and fructose are the

main molecules in carbohydrate metabolism, translocation and polysaccharide formation

(Roitsch and González, 2004). Triose phosphates, products of the photosynthetic Calvin-

Benson cycle are substrates for sucrose biosynthesis in the cytosol. Sucrose is then

translocated in phloem to sinks or stored in the vacuole (Wind et al., 2010). The Calvin-

Benson cycle intermediate fructose 6-phosphate is a precursor for plastidial starch

biosynthesis (Zeeman et al., 2010). Given their important metabolic roles, sucrose and its

derivatives are important in sensing and signalling, especially with regulation of

photosynthesis (Häusler et al., 2014). In soil-grown plants, accumulated saccharides are

implicated in repressing photosynthesis (Neales and Incoll, 1968), which is generally

accepted to be a feedback mechanism to ensure balanced carbon flow between the source and

sink (Ainsworth and Bush, 2011; Jang and Sheen, 1994; Paul and Pellny, 2003). In tissue

culture, this effect is even more pronounced with the artificially high supply of exogenous

sucrose. High exogenous sucrose levels have been shown to reduce the photosynthetic rate of

in vitro plantlets (Arigita et al., 2002; de la Viña et al., 1999).

3.4.3.3.3 Pulse amplitude modulation (PAM) fluorometry to assess photosynthetic

activity of in vitro regenerated shoots

‘Pulse amplitude modulation’ (PAM) fluorometry for determination of chlorophyll a (Chl a)

fluorescence is a powerful tool to elucidate information on photosystem II activity and the

potential for photosynthetic electron flow (Schreiber et al., 1986). Chlorophyll fluorescence

measurements confirmed that in vitro regenerated plantlets are photosynthetically active,

despite the presence of exogenous sucrose. Chlorophyll fluorescence studies demonstrated

the photochemical functionality of photosystem II. Fv/Fm is a quantitative measure of the

intrinsic (maximum) photochemical efficiency of photosystem II (Maxwell and Johnson,

2000). Maximum photochemical efficiency was measured for S30 and S15 treatments at 45

98

and 120 µmol m-2 s-1 (Table 3.1). The Fv/Fm values for all treatments measured varied greatly

between 0.4-0.7, though there is no clear relationship between sucrose concentration and

irradiance levels. This apparent independence of PSII photochemical efficiency and sugar and

irradiance in in vitro micropropagated plantlets is consistent with previous findings

(Kadleček et al., 2003). The great variation in Fv/Fm is thought to be due to differences in the

in vitro culture environment of the temporary immersion bioreactors (despite our best efforts

to maintain uniformity). The estimated value of Fv/Fm for higher C3 plants of several species,

subject to optimal growing conditions, is remarkably constant at 0.832±0.004 (Guerra et al.,

2001). The reduced PSII photochemical efficiency suggests physiological stress under

temporary immersion culture conditions.

Average Fv/Fm Standard

Deviation

30g/l sucrose; 120 µmol m-2 s-1 0.575 0.091 (n=9)




Table 3.1 Variable fluorescence / maximal fluorescence (Fv/Fm) measurements for leaves

regenerated by temporary immersion culture of Nt-pJST12 under different photomixotrophic

treatments.

3.4.3.4 Implications of these findings on transplastomic molecular farming

Sugars are considered to be a major modulator of plant cell division and growth (Riou-

Khamlichi et al., 2000), because their availability to proliferating cells in meristems is

indicative of overall photosynthetic activity and hence prevailing growth conditions (Koch,

1996). It is not surprising that sucrose wields such great influence on the vegetative growth

and transplastomic protein expression in in vitro shoot regeneration. However, the finding

that sucrose causes repression of transplastomic protein expression was unexpected.

Expression in the plastids provides an explanation for this phenomenon. Plastidial protein

expression is dependent on the developmental status of chloroplasts (Barkan, 2011; Klein and

Mullet, 1987; Møller et al., 2014; Silhavy and Maliga, 1998). The maturation of chloroplasts

99

is influenced by conditions favouring photosynthesis, and conditions of high sucrose supply

and low irradiances common in plant tissue culture, can impede plastid development and

photosynthetic capacity. These studies suggest, perhaps counter-intuitively, that for high

transplastomic protein biosynthesis, exogenous sucrose levels at a quarter of the standard

level (0.75% as opposed to 3%) are required. Light level was found to have little influence on

TetC expression, although far higher irradiances are probably required to stimulate

photosynthetic activity.

3.4.4 The influence of altered MS media strength on biomass accumulation and yield


Murashige & Skoog (MS) media is the de facto standard basal media for micropropagation

(Murashige and Skoog, 1962). Although initially developed for the optimal growth of

tobacco callus, this formulation has successfully been used to satisfy the micro- and macro-

nutrient requirements for a range of cell and tissue culture methods and several species

(George et al., 2007). 4.4 g l-1 MS media (i.e. 1 × or 100% MS strength) corresponds to the

standard concentration used in most protocols. Deviations from this standard, 25%, 50%,

100% and 200% MS media strength were investigated for the impact on temporary

immersion biomass regeneration and TetC expression. For each treatment, cultures were

undertaken as duplicates for 40-day durations, and TSPs from harvested biomass were used

for SDS-PAGE and immunoblot analysis.


3.4.4.2.1 Influence of altered MS media strength on biomass accumulation and

TetC expression

Biomass accumulation is positively correlated with %MS media strength, and the fresh

biomass accumulation curve (Figure 3.20) demonstrates a classic rectangular hyperbolic

relationship, with marginal yield increases at higher strengths. Shoots grown on ½ and ¼ MS

basal medium strength were green-yellow in colour with high differentiation but little

100

expansion (Figures 3.21 and 3.22). The biomass fresh weights for both ½ and ¼ strength

media were much reduced compared to the full-strength medium, at 59% and 26% of that of

the full-strength medium, indicating a rapid and fairly linear decline in biomass generation

with medium dilution. Visually, the health of plantlets grown on half and quarter- strength

medium can be considered as poor, relative to the control. The discolouration indicates

chlorosis, insufficiency to produce chlorophyll, possibly due to nitrogen, iron or magnesium

deficiency (Kumar Tewari et al., 2006; Petrović, 2013; Tagliavini et al., 2000; Will, 1966).

However, there is no significant difference in dry weight accumulation, between full- and

half-strength MS concentrations, despite nearly double fresh weight difference. In contrast to

these findings, some authors have reported that half-strength MS medium has resulted in

reduced necrotic or discoloured tissue formation (Abbasin et al., 2010; North et al., 2011),

possibly due to reduced osmotic damage from lowered salt concentrations. There is only a

marginal increase in biomass accumulation of 5% upon doubling the strength from 100% to

200%, though this is probably due to the space limitations of the TIB.

Immunoblot analysis reveals that the relationship between TetC intrinsic yield and media

strength is fairly linear (Figure 3.23). Using densitometry, it has been determined that dilution

of media from 100% (standard) to 50% and 25% results in % reductions in TetC expression

of 63% and 84% respectively. Conversely, doubling the MS strength from 100% to 200%

results in a 61% increase in TetC yield.

This investigation shows that a simple way of enhancing transplastomic protein yields is to

double the MS concentration from the standard 4.4 g/l. There is also the need to establish an

‘optimum’ MS strength which results in maximum TetC yield. From this study, it is clear that

the ‘optimum’ medium concentration is greater than 100% MS. Further studies would need to

be undertaken to establish this optimum.

101

Figure 3.20 Effect of MS media strength on fresh and dry biomass accumulation. Error bars

denote population standard errors.

Figure 3.21 TIB cultures at 40 days (prior to harvest) at 100%, 50%, 25% MS medium strength,

from left to right.

102

Figure 3.22 Visual comparison of 40-day temporary immersion cultures at 100% and 200% MS

medium strength, from left to right.

Figure 3.23 SDS-PAGE and immunoblot showing the effect of MS media strength on Tetc

expression. 10% acrylamide gel; 14 µg protein loading; Coomassie staining. WT culture used as

negative control.

103

3.5 Effect of Temporary Immersion Culture Hydrodynamics on

Viability, Growth and Transplastomic Protein Expression in early-stage

Callus Morphogenesis

3.5.1 The importance of hydrodynamics in plant cell and tissue cultures

Mechanical forces are known to have profound impacts on cell structure, physiology and

metabolism (Gloe et al., 2002), so from a biotechnological perspective, it is important to gain

insight into these phenomena. Culture of microbes, plant suspensions or mammalian cells

requires effective nutrient and oxygen transfer, and homogeneous cell suspensions. This is

achieved through mixing, via impeller agitation and sparging of air in stirred tank bioreactors

and sparging alone in bubble column and airlift reactors (Dufourmantel et al., 2007).

Hydrodynamic shear stresses are required for adequate mixing, associated with velocity

gradients within the agitated or sparged liquid (Merchuk, 1991). However, numerous studies

regarding microbial, mammalian and plant cell cultures have demonstrated that

hydrodynamic shear can adversely affect growth, metabolism and product formation. For

plant aggregate suspensions, hydrodynamic shear forces can cause changes in morphology,

release of intracellular compounds, alterations of morphology and productivity, and loss of

cellular viability (Meijer et al., 1993).

Consideration of rheology is important for understanding the fluid mechanical and biological

aspects of hydrodynamic shear stress. Shear stress τ is a force per unit area acting on a

surface in the tangential direction (dimensions, N m-2 or Pa), that arises from velocity

fluctuations in the fluid. The spatial velocity gradient is known as the shear rate and is

expressed as

𝛾 =𝑑𝑢𝑥

𝑑𝑦

Where 𝑢𝑥 is the velocity in the x direction and y is perpendicular to x (dimensions, s-1)

(Dufourmantel et al., 2007). The shear stress is directly proportional to the shear rate; this

relationship, Newton’s law of viscosity, may be expressed as

𝜏 = −𝜇𝑑𝑢𝑥

𝑑𝑦 or 𝜏 = −𝜇𝛾

104

in which the proportionality constant, µ is the viscosity of the fluid (dimensions, N m-2 s or Pa

s). This only applies to Newtonian fluids, in which µ is considered constant. An extension of

this, the two-parameter Ostwald-de Waele model (power law) (Coulson et al., 1999) explains

the rheological behaviour of non-Newtonian fluids

𝜏 = 𝑘𝛾𝑛

In which k is the consistency coefficient and n is the power-law index. For Newtonian fluids,

𝑛 = 1 and k is equivalent to the viscosity µ, and this correlation simplifies to Newton’s law

of viscosity.

A major limitation in the large-scale culture of plant cells, either as suspension cultures or in

the initial phases of plantlet regeneration is their fragile nature coupled with the shear stresses

of the hydrodynamic flow field of the culture vessel. The shear sensitivity of plant cells has

been extensively investigated, and the concept of ‘viability’ is often invoked in such studies.

‘Viability’ is generally considered the propensity of cells to grow and divide (Dunlop et al.,

1994), though it is possible for cells to remain viable without discernable biomass increase

(Liu et al., 2008). Alternative viability assays based on membrane integrity, metabolic

activity or ATP content (Crouch et al., 1993) are useful in determining the health of cells

after imposition of stress conditions, though these parameters do not determine whether cells

can undergo growth and morphogenesis. Arguably, characterisations of cellular viability

should include the potential for growth, division and (where appropriate in micropropagation

applications) morphogenesis, as these are more important, from a biotechnological viewpoint

(Kieran et al., 2000). Many studies have attempted to quantify the viability of plant

suspensions (Kota et al., 1999; Liu et al., 2008). Although insightful, flow regimes are far too

complex to draw a simple relationship between the fluid dynamics and cell viability (Sowana

et al., 2001).

3.5.2 Investigation of the effects of fluid hydrodynamics on callus morphogenesis,

viability and heterologous protein turnover during pneumatic immersion

3.5.2.1 Aims of experiment

In temporary immersion culture, periodic suspension of liquid media is the mode of nutrient

transfer to plant biomass. This occurs through pneumatic power input provided by the

105

isothermal expansion of sparged air (Sánchez Pérez et al., 2006). Observations of previous

cultures undertaken by the Nixon group suggest that during early temporary immersion

culture, excessive air flow damages cell aggregates and inhibits growth and morphogenesis

(data not shown). Previous to the study described below, there has not been any quantitative,

systematic investigation into these complex hydrodynamic phenomena and the biological

responses in terms of morphogenesis and product formation.

This study aims to correlate N. tabacum biomass growth, morphogenesis, viability and

heterologous protein turnover with pneumatic immersion hydrodynamics. Mechanisms

accounting for shear damage of callus aggregates and organogenic clusters are postulated.

Furthermore, the results of this study will provide the context of potential strategies for the

scale-up of pneumatic immersion culture for biopharmaceutical synthesis.

3.5.3 Characterisation of key parameters

3.5.3.1 Characterisation of the rheological and hydrodynamic properties of the

pneumatic submersion of plant biomass

As gas inflow is the only source of fluid motion during periodic media suspension in

temporary immersion bioreactors (Sánchez Pérez et al., 2006), gas flow rate is indicative of

the prevailing shear conditions. In order to gain insight into the hydrodynamic flow field of

the biomass chamber during pneumatically driven suspension of media and the biological

responses of biomass, it is first necessary to determine a number of rheological and

hydrodynamic parameters.

3.5.3.2 Characterisation of the rheological properties of tissue culture media

The hydrodynamics of multiphase reactors such as temporary immersion bioreactors depend

on the rheological properties of the media and its density (Sánchez Pérez et al., 2006). The

tissue culture media, composed of 1 × MS media (4.4 g l-1) and 3% (w/v) (30 g l-1) sucrose, is

a Newtonian fluid; hence Newton’s law of viscosity applies (Kato et al., 1978; Rodrıguez-

Monroy and Galindo, 1999; Tanaka, 1982; Trejo-Tapia et al., 2001). For aqueous solutions

106

and suspensions, viscosity is a function of solute concentration (Jones and Talley, 1933). The

viscosity (µ) of the media was estimated using Mooney’s commonly used semi-empirical

equation relating viscosity to solute concentration (Mooney, 1951):

μ = μsexp (2.5φ

1 −φφ∗

)

In this, µs is the viscosity of the solvent (water), and φ is the volume concentration of

spherical solute particles. This theory describes the effect of infinite viscosity increase when

approaching a critical volume concentration of spherical particles, φ∗ = 0.74, corresponding

to the close packing of uniform spheres (de Bruijn, 1942). In this case, µs (viscosity of water)

is 0.000891 Pa s at 25ºC. The pre-dissolution volume of the non-aqueous components of the

media, 1 × MS basal salt mixture (4.4 g/l) and 30 g l-1 sucrose was determined to be 35 ml,

giving a volume concentration φ of 0.035 (35 ml l-1). Inputting these values into the Mooney

expression, the viscosity of media is estimated to be 0.000976 Pa s. The density (ρ) of the

tissue culture media is 1.034 g l-1.

3.5.3.3 Parameters for characterising the hydrodynamic flow field

In bubble column type bioreactors, the pneumatic air supply is the only source of power,

through isothermal expansion of gas. Two important parameters, the average shear rate and

volumetric power input can be determined from the superficial gas velocity (Ug) (m s-1)

(Sánchez Pérez et al., 2006). The superficial gas velocity is simply the volumetric flow rate

(m3 s-1) divided by the cross-sectional area (m2).

3.5.3.4 Estimation of average shear rate

In bubble-column reactors, the average shear rate of a Newtonian fluid depends on the

superficial gas velocity Ug, as follows this mechanistically-derived relationship (Sánchez

Pérez et al., 2006):

γ = [gρUg

μ]

1 2⁄

107

3.5.3.5 Estimation of specific power input

The specific pneumatic power input, also known as the rate of energy dissipation (Pg

V) (power

per unit volume) (W m-3) which applies when the isothermal expansion of gas is the

predominant source of power (Chisti and Moo-Young, 1989; Sánchez Pérez et al., 2006), is

given by:

Pg

V= ρgUg

This can also be expressed in terms of as energy dissipation per unit mass (ϵ) (W kg-1) by

dividing the above equation by the fluid density.

3.5.3.6 Cumulative Energy Dissipation

It may be more appropriate to assess cumulative biological responses against cumulative

energy dissipation (instead of specific power dissipation) (J kg-1) (Dunlop et al., 1994;

Sowana et al., 2001). This is simply a product of the specific power dissipation (ϵ) (W kg-1)

and the exposure time (s). In this study the total energy dissipation over the initial 20 days of

TI culture is easily calculated, given the immersion duration / frequency of 4 mins / 8 hours.

The total exposure time over the initial 20 days is 14400 s (4 h).

3.5.4 Design of experiment

Temporary immersion cultures were undertaken at various air flow rates during pneumatic

immersion. Differential air flow rates of 38, 45, 165, 376 and 440 ml / min, correspond to

average shear rates of 28.5, 31.0, 59.2, 89.3 and 96.7 s-1 respectively, isothermal gas

expansion power rates of 0.77, 0.90, 3.31, 7.53 and 8.82 mW kg-1 respectively, and total

energy dissipation over an initial 20-day duration of 11.1, 13.0, 47.6, 108.5 and 127.0 J

respectively. Cultures were undertaken as duplicates and biomass was harvested after 3, 20 or

40-day durations. The estimated shear rates and power dissipation rates are the initial rates at

the start of the culture period, because callus aggregates are most susceptible to shear damage

just after inoculation, and before formation of functional organs. Since each TIB is inoculated

with only 0.5 g callus, the impact of inocula on system properties is assumed to be negligible

and the system is considered a two-phase system. After the allotted culture durations,

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biomass was harvested and weighed. Samples were taken for analysis of mitochondrial

activity (as a measure of biomass viability) and TetC expression. Mitochondrial activity

assays were based on mitochondrial activity based on the reduction of 2,3,5-

triphenyltetrazolium chloride (TTC) to insoluble red triphenylformazan (TF) (Towill and

Mazur, 1975). SDS-PAGE and immunoblot analysis was undertaken to assess TetC

expression.

3.5.5 Results

3.5.5.1 Effect of shear rate and power dissipation on biomass accumulation

Accumulation of fresh and dry biomass declined with increasing flow rate for both 20 and

40-day old cultures, corresponding to increasing shear rate and energy dissipation rates

(Figures 3.24). An increase of air flow rate from 38 and 376 ml min-1 (corresponding to a

28.5 to 89.3 s-1 change in shear rate, or 0.77 to 7.53 mW kg-1 change in energy dissipation),

resulted in 50% and 14% reductions in fresh weight at 20 and 40 days respectively. However,

the most significant decrease in growth occurred at 440 ml min-1 (equivalent to a shear rate of

96.7 s-1 or energy dissipation rate of 8.82 mW kg-1), giving fresh weights of only 4.2 g and

25.9 g per bioreactor at 20 and 40 days, respectively, corresponding to 82% and 80%

decreases relative to that at 38 ml/min. No significant reductions in biomass growth (either

fresh or dry weights) were observed in 3-day cultures.

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Figure 3.24 Plots showing the influence of average shear rate and energy dissipation rate

on fresh and dry biomass accumulation, after 3, 20 and 40-day cultures. Error bars denote

standard errors.

3.5.5.2 Effect of shear rate and gassed power input on mitochondrial activity

Mitochondrial dehydrogenase activity, an indicator of overall cellular viability and metabolic

activity was expressed in terms of absorbance (520 nm) of reduced triphenylformazan

(Castro-Concha et al., 2006; Ruf and Brunner, 2003; Towill and Mazur, 1975). Plots showing

the effect of hydrodynamic parameters on mitochondrial dehydrogenase activity after 3, 20

and 40-day cultures are in Figure 3.25. Mitochondrial activity was largely unaffected by air

flow rate (and derived parameters, shear rate and energy dissipation rate) at 3 and 20 days

between 38 and 376 ml min-1, though a shallow increase in mitochondrial activity with

increasing hydrodynamics was observed for the 40 days culture. Significant impairment of

mitochondrial function is observed at a 440 ml min-1, corresponding to an average shear rate

of 96.7 s-1 and energy dissipation rate of 8.82 mW kg-1. At 20 and 40 days, the mitochondrial

activity is 60% and 67% of that at 376 ml/min. This is reflective of the reduction in biomass

growth at this flow rate. It is better to express cell viability as a function of total cumulative

energy dissipation (Sowana et al., 2001), which is a product of the energy dissipation rate and

exposure time. A plot of mitochondrial activity against total energy dissipation over the first

20 days of temporary immersion culture is presented in Figure 3.24. The drop in

mitochondrial function is observed between 108.5 and 127 J kg-1.

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3.5.5.3 Effect of shear rate and gassed power input on TetC expression

SDS-PAGE and immunoblot analysis was undertaken on TSPs extracted from biomass

grown at various air flow rates at 3, 20 and 40-day intervals (Figure 3.26). For 3 and 20 days,

air flow rate was found to have no discernable effect on TetC expression. However, for the

40-day cultures, TetC yields decrease with increasing air flow rate. Densitometric analysis

was undertaken on this immunoblot to visualise the trend in TetC yield reduction with

parameters derived from air flow rate, shear rate and energy dissipation rate after 40-day

culture. Apparent exponential decay relationships are observed for both intrinsic and

volumetric TetC yields with respect to both parameters (Figure 3.27).

Figure 3.25 Plots showing the

influence of average shear rate,

energy dissipation rate and total

energy dissipation (after 20 days

culture only) on mitochondrial

respiratory activity after 0, 3, 20 and

40-day cultures. Error bars denote

standard errors.

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A B

Figure 3.26 SDS-PAGE and

immunoblots showing the effect

of air flow rate on TetC

expression. (A) 3-day TI culture;

(B). 20-day TI culture; (C) 40-day TI

culture. 10% acrylamide gel; 7 µg

protein loading; Coomassie staining.

A B

C

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Figure 3.27 Decline in intrinsic and volumetric TetC yields with hydrodynamics after 40-day

culture. Intrinsic yield values (ng/µg) were estimated densitometrically, from immunoblot data.

Volumetric yields (g l-1) were determined as the product of intrinsic TetC yield (ng µg-1), total soluble

protein extracted (µg/g fresh biomass) and fresh biomass per unit volume (g/l). Intrinsic and

volumetric yields were expressed as proportions of the maximum yield (obtained at 38 ml/min air flow

rate), on a relative scale between 0 and 1 (A) Average shear rate as independent variable; (B) Energy

dissipation rate as independent variable.

3.5.6 Discussion of the influence of hydrodynamic shear on temporary immersion

regeneration of N. tabacum shoots and TetC expression

Most previous studies investigating shear effects in in vitro plant culture have focussed on

suspension cultures. This is the first known attempt to systematically quantify the impact of

hydrodynamics on in vitro organogenesis, especially in the context of transplastomic protein

synthesis. Most protocols involving pneumatically-driven temporary immersion culture for

differentiated plant growth recommend a low gas flow rate for pneumatic immersion

(Steingroewer et al., 2013), although this is usually anecdotal and lacks any quantitative

basis. This study represents an attempt to empirically correlate the hydrodynamic flow field

associated with periodic pneumatic infiltration with cumulative biological responses in terms

of differentiated biomass growth, metabolic activity and transplastomic protein expression,

revealing important implications for industrial bioprocessing and scale-up.

In pneumatic two-phase systems such as TIBs, the hydrodynamics are determined by the air

flow rate (Sánchez Pérez et al., 2006). Although the fluid dynamics are too complex to fully

characterise, two parameters mechanistically-derived from the aeration rate, the average shear

rate and isothermal energy dissipation may be correlated against biological responses.

Importantly, the shear environment had a major impact in terms of reduction in biomass

growth. This may be attributed to decreased cell division under stress conditions or cell lysis.

It is speculated that differentiation of plant tissues may protect cells against lysis; therefore

for the 40-day old culture, reduced cell division may be the primary mode of damage. An

alternative explanation is that lysis of undifferentiated callus clusters during the early phases

of culture would reduce the amount of viable inocula for further biomass growth. Between 38

and 376 ml min-1 mitochondrial activity was largely unaffected by increasing shear for 3-day

and 20-day cultures, although a steady increase was noted for 40-day cultures. This increased

respiratory activity may be due to increased protein synthesis as a plant stress response (Aarts

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and Fiers, 2003). At a critical flow rate of 440 ml min-1, corresponding to an average shear

rate of 96.7 s-1 and energy dissipation rate of 8.82 mW kg-1, significant cell damage is

observed for both 20 and 40-day cultures, indicated by reduction in biomass accumulation

and mitochondrial activity. Several authors have suggested the need to establish critical

values for energy dissipation to avoid, and thus reduce excessive shear damage of cells

(Dunlop et al., 1994; Kieran et al., 2000; MacLoughlin et al., 1998; Sowana et al., 2001).

These results also suggest a critical total energy dissipation of 127 J kg-1. Total energy

dissipation is a more reliable critical parameter than energy dissipation rate, as biological

responses against shear damage tend to be cumulative over time (Sowana et al., 2001). Total

energy dissipation over 20 days was chosen because the initial 20 days of culture

approximate to the ‘lag’ phase of organogenesis, when there is very little biomass increase

and virtually no morphogenesis, hence the culture can be approximated to a two-phase

system. During this phase, the callus inoculum slowly proliferates just prior to the rapid

increase in meristemic shoot formation. Indeed, this early phase is important in determining

the later physiological and metabolic status of the biomass. TetC expression was found to be

especially sensitive to shear damage, and exhibits an apparent exponential decrease in yield

with increasing shear conditions. Unlike growth and mitochondrial activity, which exhibited

a significant decline with a high air flow rate, the reduction in TetC intrinsic yield was across

the entire range of conditions tested.

Although no known comprehensive studies of hydrodynamic effects in callus morphogenesis

have been undertaken, the results of this study indicate a number of fundamental approaches

that can be employed for the design and scale-up of pneumatic immersion cultures. These

results indicate that low aeration rates promote growth and transplastomic protein expression.

These results suggest that to avoid critical cell damage, it is advisable not to exceed an

average shear rate of 89.3 s-1, or total energy dissipation of 108.5 J kg-1, with bioprocess

scale-up. However, a steady decline in transgenic protein expression was observed, even at

moderate aeration rates, which demonstrates that low air flow conditions are necessary to

maintain high yields of transplastomic protein expression.

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3.6 Discussion on in vitro morphogenesis of callus in TIBs and

transplastomic protein expression, and the various parameters

affecting these

Conventional transgenic plant host systems, based on nuclear or transient expression, have

unfortunately been hampered by low yields which have rendered these systems uneconomical

compared to existing bacterial, fungal and mammalian cell host systems. In order to compete

with established bioproduction technologies, it is important that transgenic plant expression

systems provide heterologous protein yields comparable to these incumbents. Transplastomic

plants have been proven to be technically feasible protein ‘factories’, able to provide

recombinant protein hyperexpression in chloroplasts, several orders of magnitude greater

than nuclear transformants (Bock, 2014). These studies have shown that for in vitro

transplastomic plant systems, recombinant protein expression can be modulated, by tuning

plastid development and morphogenic status of biomass through culture conditions.

These studies have presented in vitro morphogenesis of shoots from calli (shoot regeneration)

in RITA® temporary immersion bioreactors (TIBs) as a viable method for the biosynthesis of

transplastomic proteins. The intrinsic yield of TetC in TIB regenerated shoots is

approximately 15-fold greater than in callus suspensions. This is of significance, as plant

suspension cultures are an alternative promising host technology. In addition, TIB

regeneration provides 500-fold biomass increase after 40-day culture. The increase in

transplastomic protein yields is associated with the increase in chloroplast number and

maturation of chloroplasts’ photosynthetic capabilities during morphogenesis. As a future

work, this association may be quantitatively determined through correlating transplastomic

protein expression against a highly expressed plastid-encoded protein such as Rubisco large

subunit (encoded by rbcL), D1 subunit of photosystem II (encoded by PsbA), ATP synthase

beta subunit (encoded by atpB), P700 reaction centre of photosystem I (encoded by psaA) or

chloroplast RNA polymerase alpha subunit (encoded by rpoA). These proteins would be ideal

candidates as their expression is directly related to the chloroplast’s ability to undertake

photosynthesis, or in the case of RNA polymerase, related to the chloroplast’s functional

development (Erickson, 1998; Kim et al., 1993; Tiller and Bock, 2014; Weihe et al., 2012).

115

In both in vitro and soil-based plant cultivation, the intuitive approach has been to optimise

growth conditions for visually-observable plant phenotypic health and vigour. The traditional

agricultural and horticultural industries have focussed their efforts on the production of high

quality plant biomass, because the biomass itself is a useful product. This approach has

extended to micropropagative tissue culture (which inherently imposes stresses on plants), in

which plantlet vigour is often measured in terms of ex vitro survival rates. However, as these

studies have shown, there is little association between visual plantlet health and heterologous

protein turnover. This is an important theme in the emerging molecular farming field,

consistent with findings of other authors (Colgan et al., 2010). This principle is especially

evident in the observations that hyperhydricity has little effect on transplastomic protein

expression, and that increased shoot development at high sucrose concentrations did not lead

to improved transplastomic protein expression. Although plant health per se is not wholly

important for recombinant protein expression, there is a strong correlation between

chloroplast development, photosynthetic capability, and plantlet developmental status and

transplastomic protein synthesis. This is demonstrated with the sucrose inhibition of TetC

expression, and the increased TetC expression observed in regenerated shoots when both

NO3- and NH4

+ are used as a nitrogen source. Moreover, these studies suggest that the

imposition of excessive abiotic stresses, such as high shear during pneumatic immersion of

biomass will impinge upon product synthesis.

When investigating the role of certain bioprocess parameters on transplastomic protein

expression, it is clear that overall yield is dependent on two factors, the intrinsic yield of

heterologous protein and overall biomass yield (Sabalza et al., 2014; Twyman et al., 2013).

The intrinsic yield is the heterologous protein yield as a proportion to total soluble protein

(TSP) (often expressed as % TSP), or as a proportion to fresh or dry biomass. For the

purposes of these studies, intrinsic yield is understood to mean yield as a proportion of total

soluble protein, for simple comparison between treatments. The relationship between

‘absolute yield’, intrinsic yield and fresh biomass yield can be demonstrated with this simple

equation.

Absolute heterologous protein yield (g

l) = Intrinsic yield (

ng

ng) × TSP from fresh biomass (

g

l)

Any approach to increase heterologous protein must focus on maximising either or both of

these variables (Figure 3.28). Rational approaches to increasing transgenic protein outputs

may be based on recombinant DNA technology (such as codon optimisation, the utilisation of

116

strong promoters and strategic use of plastid gene regulatory elements such as 5’-UTRs) or

optimisation of growth conditions. These studies have focussed on the latter approach.

Figure 3.28 Absolute recombinant protein yield depends on both intrinsic yield and biomass

growth. (A) This equation underpinning protein yield is presented graphically. Intrinsic yield is often

expressed as %TSP. (B) The absolute yield can be thought of as a function of culture conditions.

A B

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Chapter 4. Expression and assembly of Guy’s 13

monoclonal antibody via temporary immersion shoot

regeneration

4.1 Introduction : Monoclonal antibody production in transgenic plants

4.1.1 Monoclonal antibodies as biopharmaceuticals

The discovery of monoclonal antibodies (mAbs) in hybridoma cell lines (Kohler and

Milstein, 1975) and their subsequent exploitation have revolutionised medicine and

biochemistry (Reichert et al., 2005). Monoclonal antibodies and antibody fragments are

amongst the most important biotherapeutic agents used in the treatment of a number of

diseases. Immunized sera and intravenous immunoglobulin (IVIG) have been conventionally

used to provide passive immunity to patients with immunoglobulin deficiency and infectious

diseases (Casadevall and Scharff, 1995; Goswami et al., 2013), though these have been

largely replaced by mAb therapeutics. There is resurgence in the development of antibody-

vaccines against infectious diseases, which has been driven by a number of worrying

paradigms: the increasing resistance of pathogens to multiple antibiotics, the emergence of

new pathogens, and the growing population of immunocompromised individuals (Berry,

2005; Berry and Gaudet, 2011; Casadevall, 1998). For example, patients at high-risk from

severe respiratory syncytial virus (RSV) infection, one of the leading causes of hospital

admissions for paediatric respiratory illness, are given a mAb, palivizumab, which binds to

the RSV F protein and thereby directs the immune-mediated clearance of the virus from the

body (Leader et al., 2008). A major application of mAb therapeutics is the treatment of

inflammatory diseases, by targeting tumour necrosis factor (TNF), a cytokine that stimulates

increased activity of the immune system. These include Infliximab (Remicade;

Centocor/Merck) and adalimumab (Humira; Trudexa/Abbott), which are routinely used to

treat rheumatoid arthritis, as well as Crohn's disease and plaque psoriasis. MAbs have

become one of the largest classes of new therapies in oncology applications (Pillay et al.,

2011). Over 40 mAbs and mAb fragments have been approved by the Food and Drug

Administration (FDA) as therapeutics and diagnostics over the past 25 years (Brorson and

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Jia, 2014). One of the major strengths of antibody therapeutics is their relatively high

approval success rates, currently 23% for canonical antibodies, and 35% for antibody drug

conjugates (ADCs) (Reichert, 2014), compared to 7% for small molecule “new molecular

entities” (NMEs) (Hay et al., 2014). In principle, mAbs represent ‘ideal’ pharmaceutical

agents. They bind to specific targets, have slow clearance rates and elicit reduced side effects

than many small molecule drugs (Brorson and Jia, 2014). However, mAbs generally exhibit

low potencies, and are therefore required in high doses for chronic diseases (Jain and Kumar,

2008). This means that mAbs are among the most expensive therapeutics. For example, two

licenced antibodies, palivizumab and infliximab, are used at doses of 10 – 15 mg/kg body

weight (Ma et al., 2005a). For some mAb therapeutics, intensive large-scale

biomanufacturing processes producing 100 – 1000 kg/year would be required to cope with

market demands (Ma et al., 2005a).

Antibodies, also known as immunoglobulins (Ig), comprise of five main classes, IgG, IgA,

IgM, IgD and IgE, of which IgG is the most abundant in humans (Schroeder and Cavacini,

2010). The general structure of an immunoglobulin G (IgG) molecule is shown in Figure 4.1

(Nelson et al., 2008). An IgG molecule consists of the constant Fc (crystallizable fragment)

and an antigen binding domain comprising the Fv (variable fragment) and the Fab fragment

(antibody fragment) (Figure 4.1) (Nelson et al., 2008). Antibodies are heteromultimeric

proteins comprised of two heavy chains and two light chains. Therefore production of

antibodies requires not only gene expression but also oligomerization of subunits for

functional antibodies. To avoid challenges associated with antibody assembly and reduce the

size and complexity of the molecule, antibodies have been engineered by connecting two of

the variable, antigen-binding domains of the light and heavy chain with a small linker peptide

to generate a continuous polypeptide chain (Bird and Walker, 1991). However, this single-

chain Fv will often have a lower affinity for the antigen than the parent antibody, which may

be a significant bottleneck in applications requiring high affinity binding (Bird and Walker,

1991; Tavladoraki et al., 1993). Although the mechanisms of N-linked glycosylation differ in

plants and mammals (Schoberer and Strasser, 2011), high mannose type N-glycans in plants

have structures identical to those in other eukaryotes.

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Figure 4.1 Structure of immunoglobulin G (IgG) (Nelson et al., 2008)

4.1.2 Plant systems for antibody production

Currently, mAbs have been expressed mainly in mammalian cells culture, particularly

myeloma cell lines and Chinese hamster ovary cells (Dinnis and James, 2005). However,

mammalian cell culture is slow, expensive and productivities are low (Dietmair et al., 2012).

Alternative expression systems, such as yeast and bacteria, do not have the specific

machinery for post-translational modifications of active (or partially-active) mAbs. In 1989,

pioneering work undertaken by Hiatt and colleagues revealed the prospect of expressing

functional antibodies in plants (Hiatt et al., 1989). Since then, sustained research efforts on

this theme have demonstrated the feasibility of using transgenic plant host systems for

producing therapeutic antibodies. Plant systems offer several advantages including low

upstream cost inputs, an absence of human or animal pathogen contaminants, and the ability

to employ post-translational modifications such as glycosylation and disulphide bond

formation (Brodzik et al., 2006; Ko et al., 2003; Obembe et al., 2011; Richter et al., 2000). To

express complete antibodies in plants, the original approach was to clone light and heavy

chains into separate plants and subsequent crossing. This approach is time-consuming. Co-

transformation of heavy and light chains is a faster alternative, but may suffer from a large

proportion of low antibody expressors (Engelen et al., 1994; Neve et al., 1993; Nicholson et

al., 2005). Nicholson et al. (2005) transformed rice to express the components of a secretory

antibody, with the transgenes on different plasmids delivered simultaneously. Approximately

20% of transformants carried the four transgenes encoding all the components required for

expression of a complete secretory antibody, namely the light chain (LC), heavy chain (HC),

joining chain (JC) and secretory component (SC).

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4.1.3 Guy’s 13 monoclonal antibody as a topical immunotherapy agent for prevention

of dental caries

Dental caries are caused by colonisation of the tooth surface by cariogenic bacteria of the

mutans streptococci group, such as Streptococcus mutans and S. sobrinus (Featherstone,

2000; Loesche, 1986). Adhesion of Streptococcus mutans to the tooth surface is mediated by

a cell surface glycoprotein, SA I/II (Munro et al., 1993). Guy’s 13 is a mouse monoclonal

antibody (IgG1) which recognises streptococcal antigen, SA I/II. Guy’s 13 is a promising

immunotherapy agent for prevention of dental caries, which could have the potential to

revolutionise dental health. Guy’s 13, when used as a passive mucosal vaccine, could affect

the adhesion function of SA I/II, inhibiting S. mutans colonisation of the buccal cavity. It is

suggested that Guy’s 13 may block adhesion epitopes by exerting their effect, either locally

(through steric hindrance) or at a distance (through induced conformational changes in the

SA I/II molecule) (van Dolleweerd et al., 2004). In 1994, pioneering research by Prof. Julian

Ma and colleagues led to the expression and assembly of Guy’s 13 monoclonal in Nicotiana

tabacum plants (Ma et al., 1994). A major disadvantage of biosynthesis of Guy’s 13 in soil-

grown plants is secretion of the antibody into the soil, which is a regulatory and

environmental bottleneck, calling for the development of in vitro or contained growth

systems. Continuous innovation by Prof. Ma’s lab (St. George’s University of London) have

led to expression of Guy’s 13 in hydroponic rhizosecretion, hairy root cultures, teratomas,

cell suspensions and hydroponic seedlings (Drake et al., 2003; Drake et al., 2009; Sharp and

Doran, 2001b). An IgA/G chimeric secretory variant of Guy’s 13 is in commercial

development by Planet Biotechnology Inc. (USA) under the trade name CaroRx™ (Ma et al.,

2005b). The studies outlined below demonstrate the expression of Guy’s 13 in in vitro shoots

regenerated from callus tissue, in RITA® temporary immersion cultures.

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4.2 Expression and assembly of Guy’s 13 mAb by temporary immersion

regeneration of N. tabacum cv. Xanthii


Temporary immersion shoot regeneration cultures of Nicotiana tabacum cv. Xanthii callus

suspensions expressing Guy’s 13 mAb were undertaken. Seeds from N. tabacum lines which

were demonstrated to stably express the monomeric Guy’s 13 antibody (Ma et al., 1994) were

kindly donated by Prof. Julian Ma and Dr. Pascal Drake of St. George’s University of

London for this purpose. The gene constructs and transformation procedures used for

generating the antibody-producing plant have been described in Ma et al. (1994). Briefly,

tobacco leaf disks were infected with Agrobacterium tumefaciens containing Guy’s 13 heavy-

or light-chain cDNA including the native (mouse) immunoglobulin leader sequence, then

regenerated plants expressing heavy chains were crossed with those expressing light chains to

generate progeny plants which produce the full antibody (Ma et al., 1994).

After a 40-day culture period in RITA® temporary immersion bioreactors, harvest of

biomass, and total soluble protein extractions were undertaken, followed by SDS-PAGE and

Western immunoblotting under non-reducing conditions to confirm expression and assembly

of Guy’s 13, followed by functional ELISA to investigate antigen binding activity of the

expressed antibody. For both immunoblotting and ELISA protocols, immunodetection of the

expressed antibody was undertaken using a HRP-conjugated anti-mouse IgG (no ‘primary’

antibody is necessary as the expressed Guy’s 13 is a murine antibody).

4.2.2 Results

4.2.2.1 Non-reducing Western Immunoblotting to confirm expression and

assembly of Guy’s 13 IgG1 in transgenic tobacco

Non-reducing SDS-PAGE and immunoblot analysis (Figure 4.2) demonstrates the expression

and oligomerisation of the heavy chain (H) (~57 kDa), light chain (L) (~25 kDa), and the

122

full-length IgG1, in both tissue culture-grown (temporary immersion culture) and soil-grown

biomass. Guy’s 13 mAb expression and assembly is observed in both healthy and vitrified

shoots. An additional band observed at 80 kDa, might correspond to the HL assembly

intermediate. The accumulation of the full-length antibody is significantly greater than

accumulation of unassembled heavy and light chains for all samples, confirming that

transgenic plants possess the apparatus for folding, assembly, disulphide bond formation and

stabilisation of complex multimers such as antibodies. This is in agreement with several

previous reports of full antibody production in plant systems. However, this is the first report,

to our knowledge, of immunoglobulin production in leaves generated by in vitro

organogenesis from callus tissue. The presence of bands corresponding to H, L and HL

fragments may be attributed to assembly intermediates (Khoudi et al., 1999; Neve et al.,

1993; Wongsamuth and Doran, 1997) or proteolysis of the expressed antibody possibly

during sample homogenisation (Ma et al., 1994; Sharp and Doran, 2001a). For tobacco-

expressed Guy’s 13 mAb, additional bands have been observed in previous studies, notably at

40, 45, 120 and 135 kDa (Sharp and Doran, 2001a; Wongsamuth and Doran, 1997). The

absence of these bands in this study suggests that the fragments observed are not attributable

to in planta proteolysis. In mice, the order of immunoglobulin multimer assembly varies

according to the heavy chain isotype. IgM and IgG2b assemble first as HL and then H2L2,

whereas for IgG1 and IgG2a, H2 dimers are formed first, then H2L and finally H2L2 (Baumal

et al., 1971; Percy et al., 1976). The subunit assembly pathway of Guy’s 13 IgG1 is unknown

in plants (Wongsamuth and Doran, 1997), but these results suggest assembly of HL (putative

80 kDa fragment), analogous to murine IgM and IgG2b, though this is open to conjecture.

Densitometric analysis suggests that biosynthesis of the full antibody in TIB-regenerated

shoots is comparable to that in soil-grown plants. Despite the inclusion of TDZ in the media,

which promotes shoot formation and supresses rooting, a small degree of adventitious rooting

was observed (<1% of total fresh weight). Yield of the full antibody is approximately 2.72-

fold higher in adventitious roots than in healthy (non-vitrified) shoots, determined

densitometrically. Despite this, given the extremely low proportion of biomass comprised of

roots, the current system would not be feasible for the large-scale root production.

123

Figure 4.2 SDS-PAGE and Western Immunoblot of lysates of in vitro and soil-cultivated

biomass under non-reducing conditions. Assembly of full-length IgG is confirmed, as well as

fragment intermediates. 7.5 µg TSP loaded per well. Coomassie-stained 15% acrylamide gel with

LMW ladder. No β-mercaptoethanol or DTT was added, to maintain non-reducing conditions.

4.2.2.2 Antigen binding assay for functional studies of expressed Guy’s 13

monoclonal antibody

Functional ELISAs were undertaken to investigate the antigen-binding specificities of

expressed Guy’s 13 mAb, on lysate TSPs from healthy non-vitrified leaves, vitrified leaves

both regenerated via temporary immersion culture, and leaves from soil-cultivated plants. A

positive control, high purity Guy’s 13 mAb derived from the supernatant of hybridoma

124

culture (kindly donated by Dr. P. Drake, SGUL) was used and temporary immersion-grown

biomass which does not express Guy’s 13 was used as a negative control. Samples were run

as duplicates, and the data from four different microplate assays conducted in parallel were

combined. Figure 4.3 shows the binding titration curves of the lysates, based on average

response absorbance. The titration curves of the temporary immersion-grown tissue (non-

vitrified and vitrified) are similar to that of soil-grown plants, indicating comparable antigen-

binding activity of in vitro and soil-grown biomass.

Figure 4.3 Lysate titration curve showing the binding of Guy’s 13 mAb to the purified SWCF

fragment of SA I/II. (A) Antigen binding as a function of protein concentration, with 4-PL logistic

curves fitted. (B) Antigen binding as a function of a relative dilution from 120 µg/ml starting

concentration. The plotted data produced sigmoid curves for transformant lines grown as in vitro TIB

cultures and as soil grown plants. The positive control, Guy’s 13 derived from mouse hybridoma

culture supernatant gave a steep sigmoidal curve. Error bars denote the population standard

deviation.

A

B

125

4-parameter logistic (4-PL) (sigmoidal) models were fitted to the antibody titration curves

using a Levenberg Marquardt iteration algorithm and weighting, with OriginPro 9.1. The

equation describing the 4-PL model is as follows:

𝑦 = 𝐷 +𝐴 − 𝐷

(1 + (𝑥𝐶)

𝐵

)

in which y is the response (absorbance at 450 nm), x is the protein concentration, D is the

response at infinite antibody concentration, A is the response at zero antibody concentration,

C is the inflection point or the concentration at which the response is half-way between A and

D, and B is the Hill slope factor (Findlay and Dillard, 2007). C is the EC50 value, the serum

(or lysate) titre corresponding to 50% antigen binding to the antibody. The lysate EC50 values

are shown in Table 4.1. The lysate EC50 of vitrified shoots is 35.7% of that of non-vitrified

shoots, demonstrating a 2.8-fold enhanced avidity of vitrified shoot-expressed Guy’s 13. The

avidity of Guy’s 13 antibody expressed in soil-grown plants is comparable to that in vitrified

shoots.

Non-vitrified shoots

Vitrified shoots

Soil-grown plant

Positive control

EC50 titre of

lysate (µg/ml)

117.71 (±16.36)

42.00 (±1.09)

49.68 (±0.41)

1.29 (±0.05)

EC50 dilution

(from 120

µg/ml initial

concentration)

1.02

2.86

2.42

92.76

Table 4.1 EC50 titres and EC50 dilutions of plant lysates with standard errors, derived from

4-parameter logistic curve fitting to titration curves

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4.2.3 Discussion

4.2.3.1 Plants possess the relevant machinery for expression of functional

antibodies

A number of studies have observed that plants have the capability to synthesise and assemble

functional immunoglobulins, despite the absence of homologous proteins in plants,

suggesting that the mechanisms of protein assembly in plants must be similar to mammals

(Hiatt et al., 1989). This is the first known study of the expression and assembly of functional

immunoglobulins in in vitro regenerated leaves from callus tissue. This study demonstrates

that suitable post-translational machinery required for synthesis of complex proteins can be

developed by in vitro organogenesis from callus in TIBs. Oligomerisation of antibodies is

important for increasing the functional affinity to antigens (Batra et al., 2002), giving

improved pharmacokinetics. In mammals, immunoglobulins are synthesised in B-cells and

plasma cells. The heavy and light chains are synthesised independently. This is followed by

translocation to the lumen of the rough endoplasmic reticulum (RER), directed by N-terminal

signal sequences. In the RER, formation of disulphide bonds and folding and assembly of the

full immunoglobulin occurs, guided by a number of chaperones and foldases. The assembled

antibodies are transported to the Golgi and then post-Golgi vesicles, for export by exocytosis.

Throughout this process, carbohydrates are continuously added to H-chains, from the nascent

chain being attached to the ribosome to the point of secretion. It is thought that a similar

pathway occurs in plants, although this is only partially understood (Ma and Hein, 1995). In

plants, it is known that a significant amount of expressed antibodies accumulate in the

apoplastic space (Hein et al., 1991). Antibody engineering approaches may be employed for

enhanced apoplastic accumulation, through inclusion of a secretory component such as for

IgA (Ma et al., 1994). It has been demonstrated that targeting recombinant proteins to the

apoplast can reduce proteolytic degradation, due to the lower abundance of proteases

compared to that in the cytosol (Benchabane et al., 2008).

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4.2.3.2 The impact of hyperhydricity on functional Guy’s 13 mAb titre in

temporary immersion regeneration

The observation that hyperhydricity positively influences functional mAb titre is an

unexpected finding. The reasons for this are unknown, although a number of explanations can

be put forward, including an increase in chaperone activity or provision of a more favourable

environment for antibody accumulation in the apoplast.

It is known that upregulated synthesis of BiP (binding protein), a 78 kDa ER-resident

molecular chaperone and member of the Hsp70 family occurs under various stress conditions,

including water stress (Alvim et al., 2001). Importantly, it has been shown that

hyperhydricity significantly induced synthesis of BiP in a number of species, including

pepper (Fontes et al., 1999) and eggplant (Picoli et al., 2001). It is possible that the same

phenomenon is occurring in the RITA® temporary immersion culture system. Elevated

activity of BiP (and perhaps other chaperones) may be enhancing correct assembly of Guy’s

13 H2L2 multimers, and thus improving folding of the epitope-binding sites. This would

account for the increased avidity of expressed antibodies.

In temporary immersion culture, infiltration with liquid media and high humidity associated

with a sealed culture vessel exacerbate the hyperhydricity syndrome. Hyperhydric (vitrified)

plants are unable to maintain a correct water balance and accumulate water (Rojas-Martinez

et al., 2010). In particular, excess water is localised mainly in the apoplast (Fukao and Bailey-

Serres, 2004; Gribble et al., 1998). Previous studies have confirmed that Guy’s 13 antibody

accumulates in the apoplast and is even rhizosecreted from hairy roots (Drake et al., 2009),

despite the absence of secretory domains (Ma et al., 1994). It is possible that the unnaturally

large influx of water in the apoplast may provide a stabilised environment for accumulated

antibodies through dilution effects. This may be because of reduced proteolysis by dilution of

intercellular proteases. It was found that the majority of proteolytic degradation of

monoclonal antibodies occurs in the apoplastic space (Hehle et al., 2011). Alternatively,

reduced aggregation of assembled antibody through dilution may contribute to increased

functional activity.

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Although hyperhydric conditions are known to induce changes in protein synthesis, related to

photosynthesis, cellulose and lignin synthesis and stress responses (Rojas-Martinez et al.,

2010), non-reducing Western immunoblot analysis suggests similar abundances of the full

antibody in vitrified and non-vitrified tissue. The increased titre of the expressed antibody

(and its fragments) in hyperhydric shoots, as demonstrated by ELISA, is probably due to

greater avidity of the synthesised antibody.

4.2.3.3 Demonstration of mAb production in in vitro shoot regeneration via

temporary immersion culture

The majority of studies involving expression of mAbs in plants have involved soil cultivation

of whole plants (Artsaenko et al., 1998; Busse et al., 2002; Ko et al., 2009; Stoger et al.,

2000), although there have been some innovations in the development of root rhizosecretion

systems (Drake et al., 2003; Drake et al., 2009) and cell suspensions (Holland et al., 2010;

Vasilev et al., 2013). This is the first known report of mAb production by in vitro

regeneration of leaves from callus. These studies demonstrate that in vitro regeneration can

yield titres of functional antibody comparable to that from soil-grown plants. This suggests

that TIB biomass growth can be a feasible alternative to conventional agricultural growth

methods. The observation that hyperhydricity results in higher titres presents the possibility

of promoting hyperhydric conditions in tissue culture to modulate the functional antibody

titre. If Guy’s 13 mAb was to be commercialised, a production throughput of over 1,000 kg a

year would be needed, just for administration to the child population of Europe alone (Ma et

al., 2005a). Scaled-out production in TIBs may be the only reasonable way of achieving this

target. Vitrified biomass from just one RITA® TIB can produce enough antibody for an

estimated 60 patient doses at the EC50 dosage. This is based on the assumptions that

approximately 1 mg total soluble protein is extractable from 1 g fresh biomass (based on

previous observations), dilution of lysates to the EC50 dosage, and there are no downstream

losses of the Guy’s 13 mAb.

As the ‘plantibody’ paradigm progresses, plant suspension cultures have also been implicated

as being industrially feasible for mAb expression. Despite being more expensive and giving

lower mAb yields than production in soil grown plants, plant cell culture allows shorter

production cycles as well as higher batch-to-batch consistency (Boivin et al., 2010; Magy et

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al., 2014). These studies demonstrate the feasibility of in vitro shoot regeneration as a viable

alternative to plant suspension culture, offering the same process consistency under

controlled environmental conditions as in vitro suspension culture (bioreactors and

micropropagative multiplication), while yielding titres comparable to that of soil-grown

plants. Scaled-out temporary immersion culture systems may be a feasible way of large-scale

biosynthesis of Guy’s 13 necessary to provide enough doses for a global oral health

campaign.

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Chapter 5 Expression of transplastomic proteolytically

unstable proteins via temporary immersion shoot

regeneration

5.1 Introduction

5.1.1 In planta proteolysis of recombinant proteins

Proteolytic degradation of recombinant proteins is one of the most significant technical

challenges in expression of biopharmaceuticals in plant systems (Benchabane et al., 2008).

The accumulation of foreign proteins in planta can span several orders of magnitude, from

less than 0.02% total soluble protein (TSP) for human serum protein C, interferon β,

erythropoietin and epidermal growth factor (Daniell et al., 2001b) to 47% TSP for plastid-

encoded Bt Cry2Aa2 (De Cosa et al., 2001) and over 70% for a plastid-encoded phage lytic

protein (Oey et al., 2009). The great variability in recombinant protein accumulation is due,

in part, to proteolysis by native proteases. Hundreds of plant genes encode proteins involved

in proteolytic processes (Rawlings et al., 2008), with over 800 protease genes in Arabidopsis

(van der Hoorn, 2008). The number of susceptible cleavage sites accessible to endogenous

proteases for peptide bond cleavage is important in determining a foreign protein’s tendency

to undergo complete hydrolysis or ‘partial trimming’ (Benchabane et al., 2008). The specific

tissue or organ in which recombinant protein expression or accumulation occurs has a strong

influence on yield and integrity (Potenza et al., 2004). Although leaves are the target organ of

choice for recombinant proteins (especially transplastomic proteins), the lower metabolic

rates of seeds and tubers seem to confer greater foreign protein stability due to a lower

abundance of proteases (Artsaenko et al., 1998; Stoger et al., 2005). Additionally, protein

sequestration in specific organelles or cellular compartments determines the stability and

yield of foreign proteins in planta (Benchabane et al., 2008).

Transplastomic host systems offer several benefits over nuclear transformants. One major

advantage is the hyperexpression of foreign proteins via the plastid genome and apparently

greater stability of sequestered protein. It is observed that when nuclear-encoded proteins are

targeted to the chloroplast they accumulate to high levels. For example, targeting a fungal

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xylanase to the chloroplasts with the ribulose-1,5-bisphosphate carboxylase/oxygenase

(Rubisco) activase transit peptide resulted in high accumulation in Arabidopsis (Hyunjong et

al., 2006). In a similar manner, transferring human growth hormone to the plastids with a

Rubisco small subunit chloroplast transit peptide resulted in improved turnover in N.

benthamiana (Gils et al., 2005). However, there are a number of endogenous proteases in the

chloroplast (Adam et al., 2006), which can potentially degrade plastid-encoded foreign

proteins. A number of studies have reported the age-related proteolysis of transplatomic

proteins in mature or senescing leaves (Birch-Machin et al., 2004; De Cosa et al., 2001; Zhou

et al., 2008). However, in relative terms, degradation of foreign proteins may not be a

significant problem, since high net accumulation will usually still occur given the high rate of

plastidial protein synthesis.

5.1.2 Transplastomic expression of vaccine subunits susceptible to proteolytic

degradation

In the following studies, the impact of temporary immersion shoot regeneration on

transplastomic protein accumulation was investigated for two proteins known to be

susceptible to age-related proteolytic degradation in the chloroplast, VP6 bovine rotavirus

capsid protein and HIV-1 p24 antigen. Both proteins are of special significance as subunit

vaccines against rotavirus and HIV respectively. N. tabacum cv. Petit Havana lines

expressing VP6 and p24 were obtained from Professor John Gray, University of Cambridge.

In previous studies, accumulation of VP6 was found to be reduced in maturing leaves of N.

tabacum cv. Petit Havana despite constant mRNA transcript levels, possibly as a result of

proteolytic degradation in the chloroplasts. Similarly, it was observed that p24 accumulation

in N. tabacum cv. Petit Havana and N. tabacum cv. Maryland Mammoth were susceptible to

declining accumulation in mature leaves, probably due to degradation or translational

limitations (McCabe et al., 2008; Zhou et al., 2008). These previous investigations

confirming the age-related degradation of transplastomic proteins were undertaken by Prof.

Gray’s group. In the following described studies, biomass of lines expressing VP6 and p24

were grown via in vitro organogenesis in 40-day temporary immersion culture, and intrinsic

yields were compared to soil-grown plants.

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5.1.2.1 VP6 as a potential subunit vaccine against rotavirus infection

Rotavirus infection is the most common cause of severe diarrhoea in infants and young

children and is responsible for the deaths of about 600,000 children every year (Dennehy,

2008). Over 80% of all rotavirus deaths occur in developing countries in south Asia and sub-

Saharan Africa (Parashar et al., 2006). The development of a vaccine is therefore a high

priority (Shann and Steinhoff, 1999). Vaccines based on live, attenuated rotavirus strains

have been commercially available since 2005-2006, including Rotarix (GlaxoSmithKline)

and RotaTeq (Merck) (Ward and McNeal, 2010). In 1999, the Rotashield vaccine was

withdrawn from the market after one year of universal use because of associations with

intussusception, even though the probability of this is very low and several lives could have

been saved (Rennels, 2000). Though current vaccines are found to be fairly safe and

efficacious (Justino et al., 2012), the ‘Rotashield’ incident does demonstrate the existence of

safety concerns with live rotavirus vaccines. While there is no urgent requirement to

withdraw current live vaccines, given their life-saving potential and minimal safety risks,

there is a need to develop alternative candidates, with high immunogenicity and fewer safety

issues (Ward and McNeal, 2010), such as subunit vaccines. One such subunit candidate is the

rotavirus inner coat protein VP6. VP6 is highly abundant with 760 molecules in the inner

capsid layer, making up over half the total protein mass in the rotavirus particle and is a

highly conserved protein (Inka Borchers et al., 2012). VP6 has been found to be highly

immunogenic (Choi et al., 2000) and the majority of antibodies generated on rotavirus

infection are against VP6 (Svensson et al., 1987). Oral administration of VP6 is found to

induce serum IgG and mucosal IgA immunoglobulins in mice and calves thus protect against

rotavirus infection (Dong et al., 2005; Gonzalez et al., 2010; Zhou et al., 2010). VP6 is thus

an ideal subunit vaccine against rotavirus infection.

5.1.2.2 p24 as a subunit vaccines against HIV

The acquired immunodeficiency syndrome (AIDS) resulting from human immunodeficiency

virus (HIV) infection is arguably the greatest medical and scientific challenges to face

humankind over the past three decades (Meyers et al., 2008). Over 30 years since the

discovery of HIV and AIDS, no effective vaccine has been developed. HIV has a high

mutation rate and there are various subtypes prevalent in different geographical regions,

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complicating the potential for a universal vaccine (Kalish et al., 1995; Spira et al., 2003). A

multi-component vaccine comprising several proteins may be necessary to elicit immunity.

Current efforts in vaccine development have focussed on subunit vaccines which target

epitopes within conserved regions of the virus. The HIV-1 Gag precursor protein Pr55 Gag as

well as its derivatives, proteins p17 (matrix protein) and p24 (capsid protein) resulting from

cleavage of Pr55Gag by the viral protease, are potentially good vaccine candidates (Meyers et

al., 2008). Moreover Pr55Gag and possibly p17-p24 fusion proteins can form non-infectious

highly immunogenic virus-like particles (VLPs) morphologically similar to immature HIV

particles (Morikawa et al., 2000), which are potent stimulators of cellular and humoral

responses (Doan et al., 2005). The 24 kDa capsid protein p24 is an ideal candidate as there is

80% conservation of identical residues across HIV-1 clades because of structure / function

constraints (Hanke and McMichael, 2000). A number of serological studies have

demonstrated that the risk of AIDS increases with falling serum titres of anti-p24 antibodies

(Cheingsong-Popov et al., 1991; De Wolf et al., 1987; Dyer et al., 2002; Novitsky et al.,

2003), indicating that high anti-p24 antibody titres are required for a disease-free state

(Meyers et al., 2008).

5.2 Expression of transplastomic proteins susceptible to degradation via

temporary immersion shoot regeneration

5.2.1 Accumulation of plastid-expressed rotavirus VP6 via temporary immersion

shoot regeneration and comparison to soil-grown seedlings


A stable transplastomic transformant N. tabacum (cv. Petit Havana) line, Nt- Prrn-VP6 line

7A, which expresses VP6 was used in this study. This line was generated by Professor John

Gray at Cambridge University as described in Birch-Machin et al. (2004). Seeds donated by

Prof. John Gray were used as donor material for the induction of callus germplasm. Plantlet

regeneration from callus via 40-day temporary immersion culture in RITA® bioreactors was

undertaken to investigate the expression of VP6. In parallel, seedlings were grown on soil.

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Biomass was harvested and total soluble protein was extracted for SDS-PAGE and

immunoblot analysis.

5.2.1.2 Results

5.2.1.2.1 VP6 stability in soil-grown tobacco leaves

To assess the accumulation of VP6 in leaves of different ages, total soluble protein extracts

were prepared from a single 8 week-old plant grown on soil under greenhouse conditions,

and subjected to SDS-PAGE and Western immunoblotting (Figure 5.1). Leaves were

numbered from the bottom (leaf 1) upwards towards the youngest leaf (leaf 12) closest to the

shoot apex. Leaves 1-3 had presumably undergone senescence-related proteolysis, as TSP

levels were too low for comparative analysis, therefore TSPs of leaves 4-12 were analysed.

The chronological ‘distance’ between each leaf represents a plastochron, the period of time

between the initiation of two successive leaf primordia (Hill and Lord, 1990). Therefore, leaf

number is a good indication of morphologic development. On an immunoblot, VP6 was

detectable as a band at approximately 40 kDa. The amount of VP6 detected is fairly constant

in the youngest leaves, leaves 8-12. VP6 accumulation is significantly lower in the oldest

leaves (leaves 4-7), evidenced by reduction in immunoblot band intensity, suggesting

extensive proteolytic breakdown of synthesised protein in the chloroplast. Despite relative

stability in the youngest leaves (8-12), the observation of doublet bands at approximately 40

kDa suggests a partial cleavage. The age-related degradation of VP6 observed in this study is

in agreement with previous immunoblot studies undertaken by Birch-Machin et al. (2004).

Previous Northern blot analysis has revealed similar VP6 mRNA levels at each leaf

development stage, indicating that reduction in VP6 accumulation is probably because of

age-related protein degradation and not decreased transcription rates (Birch-Machin et al.,

2004).

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Figure 5.1 SDS-PAGE and immunoblot showing VP6 accumulation in the leaves of an 8-week

old soil-grown plant. 12% acrylamide gel; Precision Plus marker; Coomassie staining. Leaf number

corresponds to number of plastochrons, with leaf 1 (1st plastochron) being the oldest and leaf 12 (12th

plastochron) being the youngest. The immunoblot demonstrates a gradient in VP6 accumulation,

suggesting age-related proteolytic degradation of VP6 in the chloroplasts.

5.2.1.2.2 Expression of VP6 in in vitro temporary immersion-regenerated biomass

SDS-PAGE and Western immunoblot analysis of TSPs was undertaken to assess the impact

of temporary immersion callus-to-shoot regeneration on VP6 accumulation. Samples from

the youngest leaves of a soil-grown seedling (leaf 12) and sterile in vitro seedling were

included for comparison (Figure 5.2 (A)). Expression of VP6 was detectable in TIB-grown

shoots, albeit at a much lower level than that in both soil-grown and in vitro plants. This may

be because of proteolytic degradation by plastidial proteases. The low expression of VP6 in

TI-regenerated leaves suggests that, despite different developmental pathways, age-dependent

proteolysis of recombinant proteins still occurs. This may occur during the later stages of 40-

day temporary immersion plantlet regeneration.

SDS-PAGE and immunoblot analysis demonstrate the increased expression of VP6 in

regenerated leaves compared to callus suspension (inoculum) (Figure 5.2 (B)). VP6

expression in undifferentiated calli is negligible, and below the limit of detection for Western

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immunoblot. This dependence of transplastomic protein expression on plant physiological

development is expected, and reflects the upregulated plastidial gene expression associated

with chloroplast maturation.

Figure 5.2 SDS-PAGE and Western immunoblot for demonstration of the expression of VP6 in

TIB-grown biomass. (A) Comparison of VP6 accumulation in in vitro grown biomass and

seedlings. Accumulation of VP6 was compared between shoots grown in RITA® temporary

immersion bioreactors (TIBs), the youngest leaf of an in vitro seedling and the youngest leaf (leaf 12)

of an 8-week old soil-grown seedling. (B) Comparison of VP6 expression in callus and in vitro

regenerated shoots. Approx. 11 µg protein loading. 12% acrylamide; Coomassie staining; Precision

Plus marker.

5.2.2 Accumulation of HIV-1 p24 antigen via temporary immersion shoot

regeneration and comparison to soil-grown seedlings


A stable transplastomic transformant N. tabacum (cv. Petit Havana) line, Nt-pZSJH1p24 line

which expresses p24 was used in this study. This line was generated by Professor John Gray

at Cambridge University (Zhou et al., 2008). Seeds were kindly donated by Prof. John Gray

A

B

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were used as donor material for the induction of callus germplasm. Plantlet regeneration from

callus via 40-day temporary immersion culture in RITA® bioreactors was undertaken to

investigate the expression of p24. In parallel, seedlings were grown on soil. Biomass was

harvested and total soluble protein was extracted for SDS-PAGE and immunoblot analysis.

5.2.2.2 Results

5.2.2.2.1 p24 stability in soil-grown tobacco leaves

The impact of leaf development on p24 expression in soil-grown Nt-pZSJH1p24

transformants was investigated. SDS-PAGE and Western immunoblot analysis was

undertaken on total soluble protein extracts, prepared from a single 8 week-old seedling

grown under greenhouse conditions (Figure 5.3). Leaves were numbered from the bottom

(leaf 1) upwards towards the youngest leaf (leaf 12) closest to the shoot apex. TSP levels in

the oldest leaves, leaves 1 to 3, were too low to undertake any meaningful assessment, so

TSPs from leaves 4-12 were analysed. On an immunoblot, bands at 24 kDa represent the

expression of the p24 capsid protein. Accumulation of p24 was found to be dependent on leaf

age, similar to accumulation of VP6 in Nt- Prrn-VP6, as described previously. There is a

steady, proportional decrease in p24 accumulation with leaf age, with barely detectable levels

in leaves 4 and 5. These observations corroborate with previous studies demonstrating the

age-related degradation of p24 in both Maryland Mammoth and Petit Havana varieties of N.

tabacum (McCabe et al., 2008; Zhou et al., 2008).

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Figure 5.3 SDS-PAGE and immunoblot showing p24 accumulation in the leaves of an 8-

week old soil-grown plant. 15% acrylamide gel; Precision Plus marker; Coomassie staining. 11µg

protein loading. Leaf number corresponds to number of plastochrons, with leaf 1 (1st plastochron)

being the oldest and leaf 12 (12th plastochron) being the youngest. The immunoblot demonstrates a

gradient in VP6 accumulation, suggesting age-related proteolytic degradation of VP6 in the

chloroplasts.

5.2.2.2.2 Expression of p24 in temporary immersion-regenerated shoots

SDS-PAGE and Western immunoblot analysis was undertaken on TSPs to investigate the

impact of in vitro TI callus-to-shoot organogenesis on p24 expression (Figure 5.4 (A)). For

comparison, leaves 12 and 6 from a soil-grown plant were included, corresponding to the

most recent plastochron cycle (youngest leaf) and the plastochron mid-way through plant

development, respectively. Expression of p24 in TIB-regenerated shoots is comparable to that

in leaf 6, suggesting that vegetative biomass growth provides some mitigation against

proteolytic decay of the recombinant protein. Expression in the in vitro grown plantlet is

analogous to that in the youngest leaves.

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The effect of developmental status of TIB-grown biomass on p24 accumulation was also

investigated (Figure 5.4 (B)), comparing p24 intrinsic yields in callus suspension (TIB

inoculum), shoot primordia and regenerated shoots after a 40-day culture period.

Accumulation of p24 is detected in leaves and shoot primordia buds (i.e. intermediate

between callus and fully differentiated leaves). In comparison, no detectable expression was

observed in callus suspension. Accumulation in leaves was greater than that in primordia.

This analysis demonstrates the dependence of transplastomic protein expression on the

degree of morphogenesis of plant biomass. This reflects the upregulated plastidial gene

expression associated with chloroplast development.

Figure 5.4 SDS-PAGE and Western immunoblot for demonstration of the expression of p24 in

TIB-grown biomass. (A) Comparison of p24 accumulation in in vitro grown biomass and

seedlings. Accumulation of p24 was compared between shoots grown in RITA® temporary

immersion bioreactors (TIBs), the youngest leaf of an in vitro seedling and leaves 6 and 12 of an 8-

week old soil-grown seedling. (B) The influence of in vitro callus morphogenesis on p24

expression in TIB. Approx. 11 µg protein loading. 15% acrylamide; Coomassie staining; Precision

Plus marker.

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5.2.3 Discussion on the expression of proteolytically unstable transplastomic proteins

via TI regeneration

These studies involving VP6 and p24 synthesis in transplastomic tobacco confirm previous

reports of proteolytic instability of the foreign protein in soil-grown plants. In vitro

micropropagative shoot regeneration is an alternative cultivation technology, and so

experiments were performed to assess the impact of this different developmental pathway on

transplastomic protein expression. For p24 expression, in vitro organogenesis seems to

provide some protection against proteolytic degradation, though this effect is less apparent

for VP6. VP6 appears to be more susceptible to proteolytic degradation than p24, as

evidenced by lower expression relative to the soil-grown equivalent. Despite recombinant

protein expression in both transplastomic strains being dependent on the same plastid gene

expression regulatory mechanisms and similar plastid protease environments, VP6 and p24

exhibit different accumulation levels. It must be remembered that the intrinsic physico-

chemical properties of recombinant proteins also influence their susceptibility to degradation,

e.g. number of endopeptidase sites, steric access of proteases, tendency to aggregate. For p24

expression via in vitro biomass growth, protease degradation has little real significance, as

the elevated expression associated with morphogenesis and chloroplast development far

exceeds the rate of degradation.

The differences in proteolytic landscapes between in vitro regeneration and soil cultivation

may be explained by invoking the different morphological developmental pathways. Seedling

germination and growth involves bud initiation culminating in leaf formation at the shoot

apical meristem. This occurs in a sequential manner, giving a linear stem with nodes giving

rise to petioles and leaves, and the internodes between each leaf representing the plastochrons

between each successive bud initiation. In contrast, in vitro morphogenesis in TIB culture

involves bud initiations from multiple meristemic nodes in callus clusters. Moreover there is

absence of apical dominance, TDZ-induced suppression of root formation and lack of

tropisms (due to continual displacement of biomass). This gives rise to complex phyllotaxic

arrangements, since several leaves appear concurrently within a short space of time. The in

vitro rapid proliferation of shoots and leaves does not promote age-related protease activity.

In contrast, the oldest leaves in soil-cultivated plants are prone to senescence, involving high

protease activity. From a regulatory perspective, the gradient in transplastomic protein

expression with successive plastochrons in soil-grown plants is unfavourable, as this reduces

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batch-to-batch consistency and makes it difficult to accurately determine overall recombinant

protein yields. In comparison, rapid shoot proliferation in temporary immersion culture

should be more reproducible. Accumulation of both VP6 and p24 are greater in soil-grown

plants than in vitro regenerated shoots. The reduced protein yield in in vitro regenerated

shoots may be a result of the imposition of aberrant environmental conditions (compared to

greenhouse or field conditions) (Gaspar et al., 2002).

It must be remembered that differential intracellular protein accumulation represents the

difference between protein synthesis and protein degradation (Doran, 2006). There has been

much progress in the development of molecular biology strategies for modulating

heterologous protein expression. However, high transcription does not guarantee high levels

of heterologous protein accumulation for a number of transgenic plant systems (Doran,

2006). The literature contains several examples of transgenic plant systems in which there is

little correlation between mRNA transcript level and protein yield for both nuclear

transformants (Ohtani et al., 1991; Outchkourov et al., 2003; Richter et al., 2000) and

chloroplast transformants (Birch-Machin et al., 2004), suggesting that proteins are effectively

expressed but subsequently degraded.

Green leaves have become the de facto host organ of choice for molecular farming

applications, in spite of the high metabolic rate and the proteolytic activity associated with

endogenous protein and turnover. This is because of their rapid growth rate, the application of

conventional agricultural methods, and the availability of numerous regulatory sequences

adapted to transgene expression in the leaf cell environment (Benchabane et al., 2008). As

demonstrated in Chapter 3, leaves are especially suitable for transplastomic protein

expression, due to transgene hyperexpression associated with upregulation of plastid gene

expression during photosynthetic development. For transplastomic proteins, although

proteolysis is a major issue, its impact is largely mitigated by the inherently high protein

synthesis rates.

To summarise the results of the studies outlined in this chapter, expression of subunit vaccine

candidates VP6 and p24, against rotavirus and HIV respectively, is possible via temporary

immersion regeneration. Although the intrinsic yields for both proteins are less than that for

soil cultivation, it must be remembered that high biomass yields are possible with TIB

culture, 250 – 300 g/l, with near 100% volume occupancy of the culture vessel. Moreover,

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since in vitro shoot proliferation occurs in a short time, there is a reduced gradient of

expression with increasing age of biomass, giving reduced batch-to-batch variability. With

optimisation of culture conditions or cellular engineering strategies such as protease-

knockout, it may be possible to undertake large scale manufacture of subunit vaccines for

vaccination programmes in endemic regions. VP6 and p24 are of special importance, as

subunit vaccines against two highly dangerous viruses, especially prevalent in the developing

world. Viral capsid proteins, like VP6 and p24 are ideal candidates for subunit vaccines

because of their high immunogenicity and conservation as structural proteins between virus

subtypes (Novitsky et al., 2001). In a number of viral infections, immune responses directed

against viral coat proteins have been found to be protective, like hepatitis B and influenza

(Gottlieb and Ben-Yedidia; Iwarson et al., 1985; Russell and Liew, 1980).

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Chapter 6. Developing new tools for in vitro molecular

farming

6.1 Development of large-scale mechanical bioreactor

6.1.1 The need for scale-up of in vitro organogenesis for molecular farming purposes

In principle, field-cultivated plants as biopharmaceutical protein biofactories should have

almost infinite scalability, in stark contrast to conventional microbial and mammalian cell-

based systems (Fischer et al., 2013; Rybicki, 2009). However, the world’s productive

agricultural land is not unlimited, and is set to be under considerable pressure from a rising

population, intensification of food production as well as demand for biofuels (Lymbery,

2014). Although field production to provide cheap vaccines to developing countries is

morally laudable, it is possible that at a large-scale, this will divert agricultural capacity away

from much-needed food crops, and may cause food prices to rise, not to mention the

possibility of transgene release. In this context, the strength of in vitro plant growth platforms

is the decoupling of biomass growth from agricultural resources. Nevertheless, the challenge

of scalability still remains. For the supply of high-value vaccines and biotherapeutics from in

vitro micropropagative approaches to be feasible, high-throughput bioprocessing will be

needed. In Chapter 3, we saw that with in vitro shoot regeneration in RITA® temporary

immersion bioreactors, typical yields of 2 mg l-1 of TetC antigen were possible, which is

enough to provide enough vaccine doses, either intranasally or orally for 250 or 20

individuals, respectively. An estimated 3,350 RITA® cultures alone would be required to

supply intranasal tetanus vaccines against all the babies born in the UK in 2013 (not factoring

yield losses in downstream processing). There is a need for a high-yield biomass growth

platform capable of large-scale biotherapeutics production. This study describes the

demonstration of a custom-made 60 l mechanical temporary immersion bioreactor for large-

scale transplastomic protein synthesis.

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A prototype 60 l mechanical (hydraulic) temporary immersion bioreactor was custom-built

(Figure 6.1). Duplicate temporary immersion shoot regeneration cultures of Nt-pJST12

biomass were undertaken according to the methods described in section 2.2.2.5, for 50 and 80

days respectively. After the allotted time periods, the biomass was harvested, weighed and

total soluble proteins (TSPs) were extracted for SDS-PAGE and immunoblot analysis of TetC

expression. Biomass growth and TetC yields were compared to that of standard RITA®

TIBs. We invented, designed and constructed the large temporary immersion bioreactor for

this study, as described in section 2.2.2.5.1.

Figure 6.1 Large 60 l mechanical bioreactor in operation

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Figure 6.2 Schematic showing operation of large bioreactor.

(A) When the mechanical jack piston is in the default ‘up’ position, the biomass chamber and bag

assembly are raised and the medium settles in the bulges beneath the biomass chamber.

(B) The piston is lowered for nutrient delivery. The displacement of liquid medium and the space

constraint causes the biomass to be immersed.

6.1.3 Results

6.1.3.1 Biomass Accumulation and Organogenesis

After 50 or 80 days culture in the 60 l mechanical bioreactor, the biomass was harvested,

visually analysed and weighed. Regenerated biomass underwent complete morphogenesis,

similar to regenerated biomass grown in 0.5 l RITA® bioreactors (Figure 6.3). Shoots

displayed a higher degree of leaf formation and expansion than those grown in the RITA®.

This is probably related to reduced space limitations in the small-scale vessel.

A B

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Fresh and dry biomass accumulation were 1183.4 g and 56.1 g, and 2324.2 g and 98.1 g, after

50 and 80 days respectively. Between 50 and 80 days, biomass accumulated by 96% and 75%

for fresh and dry biomass, respectively. However, the proportion of fresh biomass composed

of hyperhydric (vitrified) shoots increased from 47.5% to 63% from day 50 to 80.

A B

Figure 6.3 Visual demonstration of shoot morphogenesis in large- and small-scale temporary

immersion bioreactors. (A) 60 l mechanical bioreactor; (B) 0.5 l RITA® TIB.

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Figure 6.4 Increase in fresh and dry biomass accumulation in the mechanical temporary

immersion bioreactor after 50 and 80 days culture

6.1.3.2 Comparison of biomass accumulation between the mechanical bioreactor

and RITA

Figure 6.5 shows a comparative analysis of fresh and dry biomass accumulation in the 60 l

mechanical and 0.5 l RITA bioreactors. Although comparatively, the mechanical bioreactor

produces 9.8- and 19.3-fold higher fresh biomass yields at days 50 and 80, respectively, than

that in the RITA® temporary immersion bioreactor, 120.1 g (Figure 6.5 (A)), this distinction

is superficial due to the size difference in culture vessel. For a more reliable analysis, the

biomass yields should be normalised. Figures 6.5 (B) and (C) show fresh and dry biomass

accumulation normalised against medium volume and the ‘floor space’ area of the vessels.

The volumes of medium used in the RITA® system and mechanical bioreactor system are 0.3

l and 16 l respectively. The estimated space footprints of the RITA® and mechanical

bioreactor are 0.015 m2 and 0.502 m2, respectively. Although the external diameter and actual

cross-sectional area of the RITA® is 0.124 m and 0.015 m2, since the cross-section is

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circular, the area was adjusted to that of a square to account for ‘lost’ space between stacked

vessels. The footprint of the mechanical bioreactor is based on the larger 60 l box. After

normalisation against medium volume or area, the adjusted biomass yield of the RITA® is

greater than that in the mechanical bioreactor. The fresh and dry biomass yields per litre of

medium are 2.8- and 2.6-fold greater in the RITA® bioreactor than the mechanical

bioreactor. The fresh and dry biomass yields per square metre of ‘floor space’ are 1.7- and

1.6-fold greater in the RITA® bioreactor than the mechanical bioreactor.

5.1.3.3 Comparative analysis of TetC expression in the mechanical bioreactor

SDS-PAGE and immunoblot analysis confirms the expression of TetC in shoot biomass

regenerated in the large mechanical bioreactor, in vitrified and non-vitrified shoots (Figure

6.6). Densitometric quantification of TetC yields was undertaken to estimate intrinsic yields

and ‘absolute’ yields in the large bioreactor (Figure 6.7 (A, B)). The absolute yield (per

bioreactor vessel) was normalised to volume of medium and ‘floor space’ area, just as for

biomass yields (Figure 6.7 (C, D)).

Figure 6.5 Comparison of fresh

and dry biomass accumulation in

large bioreactor and RITA® culture

vessels. (A) biomass accumulation per

vessel (non-normalised); (B) biomass

accumulation normalised to medium

volume; (C) biomass accumulation

normalised to space footprint (area).

A B

C

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Estimated intrinsic yields of TetC in the large TIB were 24.5 and 10.4 ng µg-1 TSP at days 50

and 80, compared to 7.1 ng µg-1 TSP in standard 40-day RITA culture. Hence in the large

TIB, at 50 and 80 days, there is a 3.4-fold and 46% increase, representively, compared to in

RITA culture. The estimated absolute yields of TetC per vessel at days 50 and 80 in the large

TIB are 28.0 and 23.3 mg, representing a 34- and 28- fold difference compared to in the

RITA, though this is largely due to the difference in size between these vessels. When the

absolute TetC yields are normalised to volume of medium or area, the yields in the large TIB

and RITA are comparable.

Figure 6.6 SDS-PAGE and immunoblot demonstrating TetC expression in large

mechanical TIB. Comparison of TetC yields between large mechanical TIB (50 days and 80 days

culture), 40-day RITA® TIB and young leaves of a soil-grown plant. 12% acrylamide; LWM marker

(A); Precision Plus marker (B); 14 µg protein loading per well; Coomassie staining.

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6.1.3.4 Discussion on the development of a large-scale bioreactor

Scaling of transgenic plant-based bioprocesses will be necessary to providing mass quantities

of inexpensive vaccines and other biotherapeutics, as well as deriving ‘economies of scale’

(Xu et al., 2012b). When scaling in vitro plant tissue culture processes for molecular farming

applications, there are two approaches that can be taken, scale-out and scale-up. Scaling-out

of field-grown plants is straightforward, by simply devoting more agricultural land to

cultivation, whereas for in vitro micropropagation it would involve multiplication of vessels

(and possibly growth facilities). Scaling-up is appropriate for in vitro cell suspension and

tissue cultures undertaken in bioreactors (Akita et al., 1994; Hellwig et al., 2004; Reuter et

Figure 6.7 Comparison of TetC yield in large bioreactor and RITA® culture vessels. (A) TetC

intrinsic yield (ng TSP / µg TSP) determined from densitometric analysis of immunoblots (B) Estimated

‘absolute’ TetC yield per bioreactor (mg TetC) (non-normalised) (C) Estimated TetC yield normalised to

medium volume (mg/l) (D) Estimated TetC yield normalised to space footprint (area). Error bars denote

standard errors.

A B

C D

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al., 2014), and approaches can be adapted from conventional microbial and mammalian cell

bioprocesses (Glacken et al.; Junker, 2004; Thiry and Cingolani, 2002). This study describes

the scale-up of shoot regeneration for TetC expression from 0.5 l (RITA® TIB) to 60 l

(mechanical bioreactor) and the implications on biomass growth and heterologous protein

yields.

In this study, the mechanism of medium immersion was altered upon scale-up. In the small-

scale RITA®, immersion is pneumatically-driven (Watt, 2012), whereas in the large

bioreactor, immersion was hydraulic, involving physical displacement of the medium. The

change in mode of operation resulted in no loss of TetC yield. In fact there was a 3.4-fold

increase in TetC intrinsic yield between the RITA® and large TIB at 50 days culture. This

may be associated with reduced imposition of abiotic stresses related to scaling of the culture

environment (Chaves et al., 2002; Ciarmiello et al., 2011). The large TIB presumably

experienced reduced humidity as a result of a larger headspace and the increased space

promoted organogenesis (Figure 5.3). When TetC yield is normalised to medium volume or

‘floor space’, the large TIB and RITA system give comparable yields. Relative to the

‘floorspace’ footprint, the actual TetC yield, after 50 days culture in the large TIB, was

roughly equivalent to that in the RITA. The 57% reduction in TetC intrinsic yield in the large

mechanical TIB between days 50 and 80 (Figure 5.7 (A)) may be related to age-related

proteolysis in the chloroplast. However, this is largely offset by exponential biomass growth,

so in terms of absolute yields, there is a 17% decrease in TetC yield between days 50 and 80.

Scale-up of RITA®-type bioreactors may be difficult due to the high energy requirements

required for gas-powered suspension of liquid media (Majumdar, 1996). This study

demonstrates the straightforward construction and operation of a large bioreactor using

simple, easily obtainable components. Although this is a prototype, similar inexpensive low-

tech micropropagation approaches may be adopted in developing countries for large-scale

production of plant-made vaccines (Savangikar, 2004). Alternatively, a scale-out approach

involving multiplication of small-scale RITA® or similar vessels may be used.

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6.2 The influence of pre-culture preservation of encapsulated callus on

temporary immersion morphogenic potential and TetC expression

6.2.1 Synthetic seed technology

The concept of a “synthetic seed” was first proposed in 1977 (Murashige, 1977), and has

since evolved into a field of intense research and experimentation within the field of

micropropagation. A synthetic (artificial) seed is defined as artificially encapsulated somatic

embryo, shoot bud or any other meristemic tissue that can be used as a functionally mimic

seed for sowing and possesses the ability for conversion to a plant under in vitro or ex vitro

conditions and that can retain this potential even after storage (Hicks, 1994; Munetaka, 1999).

Originally, synthetic seeds were used for encapsulation of somatic embryos, though has since

been extended to other non-embryonic vegetative propagules such as apical shoot buds, nodal

segments as well as calli (Babaoglu and Yorgancilar, 2000; Choffe et al., 2000; Park et al.,

2004a; Standardi and Piccioni, 1998). Despite great advances in the development of synseed

technology, a true analog to natural seeds is yet to be realised (Kumar et al., 2005). Synthetic

seed technology could provide an efficient, cost-effective means for mass clonal propagation

of plant material, if proliferation, rooting and conversion are well controlled (Piccioni, 1997;

Standardi and Piccioni, 1998). Encapsulation of in vitro-derived plant tissues can have

several applications in micropropagation, such as cultivation independent of natural and

seasonal conditions, conservation of germplasm, long-term storage of plant material through

cryopreservation and exchange of sterile material between laboratories (Teng, 1999).

There are two main types of synthetic seed, hydrated and dessicated. A number of coating

agents such as sodium alginate, potassium alginate, carrageenan, sodium alginate with

gelatin, sodium pectate, carboxymethyl cellulose are used for encapsulation, though sodium

alginate is most extensively used (Teng, 1999). An alginate hydrogel is frequently selected as

a matrix due to characteristics including moderate viscosity and low spinnability of the

solution, low toxicity and quick gelation (Onishi et al., 1994). Alginate beads can be made by

the ‘droplet hardening’ method, through dropping propagules with sodium alginate solution

(0.5-5.0% w/v) into CaCl2 solution (30-100 mM) (Onishi et al., 1994; Redenbaugh et al.,

1993). It has been observed that the gel capsule provides hindrance to the emergence of the

shoot and root.

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This study described below explains the application of ‘synthetic seed’ technology to the

long-term storage of Nicotiana tabacum callus germplasm of the Nt-pJST12 transplastomic

line and the subsequent in vitro regeneration of shoots in RITA® TIBs for biosynthesis of

transplastomic TetC protein.


A major application of synthetic seed technology in micropropagation is the long-term

storage of germplasm prior to tissue culture, such as hairy roots, somatic embryos

(Vdovitchenko and Kuzovkina, 2011).

0.5 g callus aggregates were encapsulated in sodium alginate according to the method

described in 2.2.2.4 and stored at 4°C or 25°C, for 0, 9, 18, 40 or 138 days. Subsequently,

encapsulated callus propagules were inoculated into RITA temporary immersion bioreactors

and shoot regeneration cultures were undertaken, as duplicates. After 40-day temporary

immersion cultures, biomass was weighed, and SDS-PAGE and immunoblots of TSPs were

undertaken to assess the influence of germplasm storage time and temperature.

6.2.3 Results

6.2.3.1 Influence of duration and temperature of encapsulated callus preservation on

growth and morphogenesis in temporary immersion culture

Encapsulation of calli within an alginate matrix without storage had no apparent effect on its

capacity for expansion and morphogenesis in temporary immersion culture. The

Figure 6.8 Callus aggregates

encapsulated in a sodium

alginate matrix.

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morphogenesis dynamics exhibited by the encapsulated callus inocula were similar to those

without having undergone encapsulation. After a lag phase of approximately 10 days in

which little expansion is observed, expansion of the callus mass and development of

meristemic nodules was observed between days 10-25. Between days 20 and 25, formation of

shoot buds and differentiation of leaves were observed.

Although alginate encapsulation had little effect on the morphogenesis dynamics, the extent

of growth was affected by storage duration and temperature. The fresh and dry biomass

accumulation of temporary immersion regenerated shoots is indicative of the shoot

regeneration potential of the encapsulated callus germplasm (Figure 6.9). There is a decline

in temporary immersion culture regeneration potential, with increasing germplasm storage

time. Moreover, the regeneration potential is greater after storage at 4°C than at 25°C. After

storage for 18 days at 4°C and 25°C respectively, the fresh biomass accumulation is 72% and

70% of that having undergone no storage (day 0). After storage for 138 days at 4°C and 25°C

respectively, the fresh biomass accumulation is 37% and 5% of that having undergone no

storage (day 0).

Figure 6.9 Effect of callus encapsulation duration and

temperature on fresh and dry biomass accumulation in

temporary immersion shoot regeneration cultures. Error bars

denote standard error.

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6.2.3.2 Influence of Influence of duration and temperature of encapsulated callus

preservation on TetC expression in regenerated shoots

SDS-PAGE and immunoblot analysis was undertaken to assess the influence, if any, of callus

encapsulation duration and temperature on TetC intrinsic yield (Figure 5.10). No significant

differences were observed between the various treatments. The TetC intrinsic yields under all

storage durations and temperatures are comparable to that in biomass grown in the control

culture (inoculum was not encapsulated).

6.2.3.3 Discussion on the influence of alginate encapsulation on temporary

immersion regeneration and TetC expression

With alginate encapsulation for callus preservation, there is a trade-off in terms of the gradual

loss of regeneration potential with increased germplasm storage time, which is heightened at

room temperature compared to 4°C, as these results have shown. This is due to reduced

Figure 6.10 SDS-PAGE and

immunoblot demonstrating

TetC expression in shoot

biomass regenerated from

encapsulated callus stored for

various durations and

temperatures. TetC control

culture was inoculated with non-

encapsulated callus. Negative

control is TIB-grown WT biomass.

12% acrylamide; LWM marker

(A); Precision Plus marker (B); 8

µg protein loading per well; Sypro

orange staining.

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viability of callus germplasm with prolonged storage. However, alginate encapsulation may

still be useful as a viable method for short-term germplasm preservation, in spite of reduced

growth potential. For example, 40 days alginate preservation of callus at 4°C will result in

fresh biomass accumulation of 178.6 g l-1, compared to 277.8 g l-1 if no storage was

undertaken. Although this represents a 36% loss in fresh weight, the 40-day decoupling of

inoculum generation and biopharmaceutical manufacture may be invaluable in satisfying

unpredicted increases in demand. This reduction in growth potential of encapsulated

germplasm with increased storage time is consistent with previous studies (Naik and Chand,

2006; Perveen and Anis, 2014; Rai et al., 2008; Zych et al., 2005).

6.3 Discussion on described studies and how they relate to new

developments in in vitro molecular farming

These studies have highlighted two areas of intense research and innovation in the

micropropagation field, the development of large-scale cell and tissue culture capabilities,

and the preservation of germplasm. (Dodds, 1988; George et al., 2007; Murashige, 1977;

Patel et al., 2000). Such developments in micropropagative technology can be easily adapted

to the high throughput biosynthesis of plant-produced biopharmaceuticals.

The first study focussed on the development of a large-scale hydraulically-driven temporary

immersion bioreactor for the biosynthesis of transplastomic vaccines. Although there has

been notable progress in the development and scale-up of plant cell suspension cultures for

recombinant protein and small molecule synthesis over the last 30 years (Boivin et al., 2010;

Eibl and Eibl, 2008; Kieran et al., 1997; Kwok et al., 1992; Magy et al., 2014; Moon et al.,

1999; Scragg et al., 1988; Srinivasan et al., 1995; Terrier et al., 2007), there has been little

equivalent innovation in large-scale culture of differentiated plant tissues (Huang and

McDonald, 2012; Steingroewer et al., 2013). Most projects involving recombinant protein

expression in whole tissues have focussed on field or greenhouse seedling propagation

(Artsaenko et al., 1998; Busse et al., 2002; Ko and Koprowski, 2005), although as an

exception, there has been moderate progress in the development of systems for culturing

hairy roots / rhizosecretion (Drake et al., 2003; Drake et al., 2009; Wongsamuth and Doran,

1997) and mosses (Decker and Reski, 2004; Decker and Reski, 2007; Hohe and Reski, 2002).

It is likely that in the near future, tissue culture scale-up will become an intense field of

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research, as the need for high-throughput in vitro differentiated biomass growth systems for

biopharmaceutical synthesis will increase. This is especially relevant for transplastomic

protein expression, which is highly dependent on the development of mature chloroplasts in

differentiated leafy tissue. This study demonstrates a very straightforward strategy for the

scale-up of temporary immersion culture, which provides transplastomic protein yields

comparable to that in 0.5 l RITA cultures. Such a system does not require sophisticated

equipment, instrumentation or ‘stainless steel’ infrastructure associated with microbial

fermentation plants and is amenable to a disposable single-use bioprocessing approach (Eibl

et al., 2010; Huang and McDonald, 2012; Kwon et al., 2013). Single-use approaches confer

technical benefits relating to simplified bioprocess set-up such as ease of validation, less

capital investment for stainless steel vessels, reduced turnover time between each run, and the

potential use of integrated processes for more robust processes with shorter development time

and increased throughput (Eibl et al., 2010; Huang and McDonald, 2012).

The short-term to mid-term preservation of germplasm is an important part of any in vitro

micropropagation programme. Cryopreservation and alginate encapsulation are two

important methods for preservation of elite plant lines in commercial micropropagation and

plant conservation, though the latter is the simpler and cost-effective option (Perveen and

Anis, 2014). Hence alginate encapsulation protocols for vegetative propagules have been

established for a number of woody and non-woody species (Gardi et al., 1999; Hung and

Trueman, 2012; Kim and Park, 2002; Nagamori et al., 1999; Perveen and Anis, 2014). In

terms of in vitro tissue culture for biopharmaceutical production, preservation of germplasm

may confer several technical benefits. Alginate encapsulation may be employed in the

banking of stable transformant lines, after genetic stability has been established. Alginate

encapsulation can be used in the maintenance of a stable inventory of germplasm. Hence,

when demand for a biotherapeutic peaks, a source of tissue culture inoculum is readily

available to scale-up biosynthesis to satisfy demand. In practical terms, encapsulated

propagules may be easily transported between labs and culture facilities with minimal loss of

viability.

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Chapter 7 Summary and future directions

7.1 In vitro plant tissue culture as an alternative platform for

biosynthesis of biopharmaceuticals

The ‘molecular farming’ paradigm started in the early 1980s, when the first successful higher

plant transformation was reported, (Fraley et al., 1983), soon followed by the earliest reports

of expression of antibodies in plants (During, 1988; Hiatt et al., 1989). Since then, an intense

global programme of research and development into plants as suitable hosts for foreign

protein expression has been underway (Faye and Gomord, 2010; Ritala et al., 2014;

Schillberg et al., 2013; Spök et al., 2008a; Xu et al., 2012b). However, despite the

demonstrated advantages over conventional host technologies (Xu et al., 2012b), 30 years

later, only a small number of plant-produced therapeutics have been licensed, notably a

poultry vaccine against Newcastle disease (though this was never marketed) (Dow

Agrosciences, USA) (Katsnelson et al., 2006; Ritala et al., 2014), ELELYSO™ enzyme

replacement therapy for Gaucher disease (Protalix BioTherapeutics, Israel, in collaboration

with Pfizer) (Zimran et al., 2011) and ZMapp monoclonal antibody treatment against Ebola

(Qiu et al., 2014). A number of reasons behind the apparent decoupling of research and

commercialisation of plant-made biopharmaceuticals have been cited, including low initial

yields of foreign proteins, recalcitrance of industry leaders to replace firmly-established

microbial or mammalian bioprocesses with plant-based platforms, biosafety concerns related

to open field cultivation, and regulatory frameworks tailored to non-plant host systems

(Fischer et al., 2013; Martine et al., 2009; Soria-Guerra et al., 2011; Spök et al., 2008a; Spök

et al., 2008b; Xu et al., 2012b; Xu et al., 2011). It must be remembered that many of these

apparent bottlenecks hampering the adoption of molecular farming approaches are directly

related to the host tissue and cultivation method (Doran, 2013).

Historically, conventional soil-based cultivation of whole plants has been implicated as being

pertinent for the high-yield production of plant-made pharmaceuticals, as standard

agricultural procedures represent a very straightforward, low-tech approach and field

cultivation is highly scalable (Doran, 2000; Fischer et al., 2012; Rybicki, 2009; Stoger et al.,

2002; Xu et al., 2012b). Soil cultivation is the standard and most intuitive approach to both

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leafy and seed-based biomass growth (Xu et al., 2012b). The preference for soil-grown

seedlings appears to be reflected in much of the ‘molecular farming’ research zeitgeist over

the last 30 years (Chargelegue et al., 2005; Ma et al., 1994; Villani et al., 2009). In principle,

the case for agriculturally cultivated transgenic plants is compelling, though in reality, a

number of practical issues persist. Long development times, dependence of biomass yields on

prevailing environmental (biotic and abiotic) conditions and season, and genetic variation as

a result of sexual reproduction can affect product quality and consistency in accordance with

GMP principles (Fischer et al., 2012). Moreover transformational biosafety strategies, based

on transplastomic maternal inheritance (Daniell et al., 2002), or male sterility through RNA

silencing or barnase-induced (Commandeur et al., 2003; Gleba et al., 2004), are not absolute

guarantees against transgene pollution. Indeed, recalcitrance from both the public and

regulatory bodies have hindered large-scale field cultivation of transgenic biopharmaceutical

crops, especially in the European Union (Sang et al., 2013; Spök et al., 2008a). It is hoped

that the proliferative global adoption of genetically modified food crops (also known as

‘biotech’ crops) since the 1990s, especially USA, Brazil, Argentina, India, Canada, and

China, (James, 2013) will set the precedence for a similar adoption of biopharmaceutical

crops.

The strength of in vitro plant growth compared to agricultural propagation is in the ability to

control environmental conditions. Suspension culture has been pursued as a viable alternative

to agricultural plant propagation by researchers and industrialists (Hellwig et al., 2004;

Schillberg et al., 2013; Weathers et al., 2010; Xu et al., 2011). Suspension culture combines

the benefits of whole plant systems with those of microbial or mammalian cells (Hellwig et

al., 2004; Xu et al., 2011). Dedifferentiated callus aggregates can be cultured under scalable

tightly controlled conditions in a similar manner to industrial microbial fermentations, and

the same approaches to bioprocess optimisation can be applied (Xu et al., 2011). Moreover,

suspension cultures have fewer regulatory and environmental compliance hurdles for

ensuring product quality and safety than soil-grown plants (Xu et al., 2011). This must be

balanced against considerably higher capital costs than for soil-grown plants (Weathers et al.,

2010). Cell suspension culture has a relatively long history of over 50 years, initially as a

means for commercial high-value metabolite synthesis (Georgiev et al., 2009), and in recent

years as a platform for recombinant protein expression (Hellwig et al., 2004; Holland et al.,

2010; Schillberg et al., 2013; Vasilev et al., 2013; Weathers et al., 2010). Despite decades of

160

bioprocess optimisation, low recombinant protein yields are still a major limitation of cell

suspensions (Twyman et al., 2013).

While agriculturally produced whole plants have been the host system of choice for

preclinical and clinical development of plant-made biopharmaceuticals (Karg and Kallio,

2009), and cell suspension technologies are emerging as a technically superior alternative

(Xu et al., 2011), there has been relatively little development in the development and scale-up

of plant tissue and organ cultures for biopharmaceutical production (Steingroewer et al.,

2013). While micropropagation techniques for multiplication and ex vitro transfer of plantlets

have been developed and optimised for decades, these have been applied mainly to

commercial horticulture and conservation of rare plants (Akin-Idowu et al., 2009; Dubranszki

and da Silva, 2010; Escalona et al., 1999; Kumar et al., 2006; Zych et al., 2005). There has

been some precedence of in vitro differentiated plant tissue culture for synthesis of bioactive

metabolites, though this has been largely limited to hairy roots and adventitious roots

(Steingroewer et al., 2013; Weathers et al., 2010). There has been considerable investigation

of hairy root culture for synthesis of bioactive metabolites, though scaled-up and

commercially feasible technologies are currently lacking (Bourgaud et al., 2001; Choi et al.,

2006; Georgiev et al., 2008; Steingroewer et al., 2013). Adventitious roots have been

particularly successful for commercial production of several metabolites, with over 45 tonnes

fresh weight per year reported by CBN Biotech Company (South Korea) for ginseng

production (Baque et al., 2012; Steingroewer et al., 2013). Although the in vitro culture of

differentiated explants such as shoots, plantlets, bulbs, microtubers and embryos have been

undertaken in commercial micropropagation for decades (George et al., 2007), and more

recently for the synthesis of bioactive metabolites (Steingroewer et al., 2013), there are few

reports of these used for the expression of recombinant proteins. Recent studies undertaken

by the Nixon group demonstrated that in vitro shoots regenerated from callus in temporary

immersion bioreactors could result in the overexpression of transplastomic proteins,

including TetC tetanus antigen, GFP+ (Michoux et al., 2011) and Lyme disease vaccine

antigen (Michoux et al., 2013). These were the first reports of in vitro shoots regenerated in

bioreactors being used for the expression of foreign proteins and the studies presented in this

PhD dissertation follow on from these pioneering studies.

Perhaps an explanation for the limited exploitation of in vitro tissue and organ cultures for

molecular farming lies in the intrinsic features of in vitro differentiated plant tissues (Huang

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and McDonald, 2012; Steingroewer et al., 2013). Suspension cultures, being composed of

undifferentiated callus aggregates have been relatively simple to grow in accordance with

GMP principles, borrowing heavily from bioprocessing strategies of established microbial

fermentations and mammalian cell cultures (Hellwig et al., 2004; Schillberg et al., 2013;

Weathers et al., 2010; Xu et al., 2011). In contrast, tissue cultures are morphologically and

biochemically complex. Traditional bioreactor configurations used for cell suspensions such

as stirred-tank reactors are not suited to differentiated tissues, as mechanical agitation,

hydrodynamics and reduced mass transfer and oxygen supply limitations in liquid culture can

be damaging to plantlet morphology at high biomass concentrations (Huang and McDonald,

2012; Steingroewer et al., 2013; Weathers et al., 2010). However, with modifications to

design and operation of stirred tank reactors, stirred tank bioreactors may be adapted to

differentiated tissues (Steingroewer et al., 2013). For example, low impeller speeds of 30-100

min-1 are recommended and tolerable tip speeds vary between 1 and 2 m s-1 (Steingroewer et

al., 2013). It is recommended to keep power input per unit volume under 1000 W m-3 for

reduced shear damage (Steingroewer et al., 2013). Pneumatic bioreactors, such as bubble

column reactors, have simple designs and operations, and may be used to alleviate the issues

associated with mechanical agitation, though there may be mass transfer limitations at high

tissue densities, caused by gas channelling among dense organs (Choi et al., 2006;

Sivakumar, 2006; Steingroewer et al., 2013; Vlaev and Fialova, 2003; Wang and Zhong,

2007). For metabolite production in hairy root culture, ‘unorthodox’ designs such as liquid-

dispersed and gas-phase bioreactor configurations have been used to alleviate such issues

(Weathers et al., 2010). Avoidance of stress conditions related to permanent submersion,

through the use of temporary immersion micropropagation has also been applied for a variety

of explants (Watt, 2012), though research undertaken in the Nixon group represents the first

application of this for biopharmaceutical production (Michoux et al., 2013; Michoux et al.,

2011).

The studies presented in this dissertation demonstrate the feasibility of in vitro tissue culture

via temporary immersion culture as an alternative to both agricultural propagation and cell

suspension, providing the advantages of differentiated tissue cultivation with the ease of

manipulating production conditions associated with suspension culture in bioreactors. In this

system, biomass and recombinant protein yields can easily be modulated through

manipulation of various culture parameters. The temporary immersion organogenesis system

can be a viable platform for large-scale GMP production of biopharmaceuticals, in a

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technically competent and economical manner. This system is especially suitable for the

biosynthesis of transplastomic proteins, as intrinsic protein expression is approximately 15-

fold higher in differentiated shoots than callus suspension in the case of TetC, reflecting

chloroplast maturation during morphogenesis. Moreover, 500-fold fresh weight biomass

accumulation is observed in this system.

7.2 The influence of temporary immersion shoot regeneration on

biosynthesis of transplastomic proteins

Chapters 3, 5 and 6 of this PhD dissertation have focussed on the biosynthesis of

transplastomic proteins in temporary immersion culture of N. tabacum shoots. In particular,

Chapter 3 focussed on the effects of various culture parameters on yield of TetC.

In the early development of the molecular farming paradigm, low intrinsic yields of foreign

proteins rendered transgenic plants uneconomical host systems compared to established

mammalian and microbial systems, and hindered the adoption of the new technology by

industry (Sabalza et al., 2014). Currently, these low yields have been addressed a number of

genetic strategies, transplastomics for overexpression in plastids (Bock, 2007; Bock, 2014;

Clarke and Daniell, 2011; Maliga and Bock, 2011), transient expression (Boivin et al., 2010;

Davies, 2010; Joh et al., 2005), inclusion of genetic regulatory elements in transplastomic and

nuclear transformation constructs for more efficient transcription and translation (Parra et al.,

2011; Ruhlman et al., 2010; Sharma et al., 2008). Plant growth conditions are also important

in determining yields of foreign protein (Sabalza et al., 2014).

As was emphasised in Chapter 3, ‘absolute’ foreign protein yield is the multiplication of

intrinsic yield (target protein as percentage of total soluble protein or per unit biomass) by

total biomass (expressed as fresh weight or soluble protein equivalent). Both depend on the

complex interplay of several genetic, biochemical, physiological and environmental factors.

The intrinsic yield is a measure of accumulation of protein, which depends on the rate of

protein synthesis balanced against protein degradation (Sabalza et al., 2014). In vitro biomass

accumulation depends on several metabolic and physiological factors including nutrient

163

assimilation, photosynthetic capacity and cytokinin-induced morphogenesis (George et al.,

2007).

Various culture treatments and factors were found to affect transplastomic protein

accumulation (both intrinsic and absolute), to various degrees. Transplastomic protein yields

were especially sensitive to nitrogen source ratio, sucrose concentration, irradiance, MS basal

medium concentration, hydrodynamics and protease activity. Other treatments were found to

have little or no influence on transplastomic protein accumulation, including water stress-

induced hyperhydricity, medium pH, mechanical immersion (as opposed to pneumatic

immersion) and scale-up, and encapsulation of callus inocula in alginate.

It is possible that protein localisation in the chloroplast stroma may isolate the foreign protein

from certain cellular phenomena which would otherwise adversely affect protein

accumulation. For example, hyperhydricity, a predominantly apoplastic phenomenon, was

demonstrated to have no effect on TetC expression in the plastids. Encapsulation of callus in

alginate presumably had no influence on chloroplast development, and therefore did not

affect transplastomic protein expression (though it did influence the extent of shoot

regeneration). Transplastomic protein expression was enhanced at reduced sucrose

concentrations, which is directly related to elevated photosynthetic capacity and development

of chloroplast thylakoids (Arigita et al., 2002). The requirement of both nitrate and

ammonium for biomass morphogenesis and transplastomic protein expression is related to the

assimilation of nitrogen for synthesis of amino acid and proteins and nitrogen metabolism in

the chloroplasts.

The experiments investigating the influence of hydrodynamics on biomass accumulation and

transplastomic protein were particularly intriguing, with implications for scale-up of

pneumatic temporary immersion culture. This was the first reported attempt to quantify

biological responses against hydrodynamics for in vitro shoot regeneration, although similar

studies have been undertaken for plant suspension cultures (Dunlop et al., 1994;

MacLoughlin et al., 1998; Scragg et al., 1988; Sowana et al., 2001). For 40-day temporary

immersion cultures, a critical air flow rate of 440 ml min-1 was identified, corresponding to

an average shear rate of 96.7 s-1 and energy dissipation rate of 8.82 mW kg-1 and total energy

dissipation of 127 J kg-1 (over first 20 days), which resulted in significantly reduced biomass

accumulation, mitochondrial activity and transplastomic protein yields. If scale-up of

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pneumatic temporary immersion regeneration is to be undertaken, it is recommended not to

reach these values. The steady decline in transplastomic protein intrinsic and volumetric

yields even at moderate air flow rates suggests that operating at low air flow rates is

advisable.

These studies investigating the influence of various parameters on transplastomic protein

yields can serve as “prior art” for further optimisation of culture conditions for maximisation

of protein yields. These studies involved deviations from standard culture conditions,

involving the classical approach of alteration of one parameter at a time. The limitation of

this approach is that it does not account for interactions between factors tested (Vaidya et al.,

2009). However, these studies can be the starting point for medium optimisation by

‘statistical experimental design’ techniques, such as fractional factorial design or response

surface methodology, which can give insight into synergistic effects between medium

components (Dinarvand et al., 2013; Jeon et al., 2014; Vasilev et al., 2013). A combination of

fractional factorial design and response surface methodology was applied to the optimisation

of culture medium for tobacco BY-2 cells producing a secretory antibody (Vasilev et al.,

2013).

7.3 Scale-up of callus-to-shoot regeneration for biopharmaceutical

expression

Chapter 6 of this PhD dissertation described the scale-up of temporary immersion shoot

morphogenesis for transplastomic biopharmaceutical expression in a hydraulically-driven

mechanical bioreactor, from 0.5 l (standard bench-scale RITA® culture) to 60 l (volume of

the biomass chamber, though the volume of the entire set-up was 145 l). This is the first

reported attempt of the scale-up of in vitro callus-to-shoot morphogenesis, and could be the

basis of large-scale biopharmaceutical synthesis in differentiated tissues. Importantly, it was

observed that biomass accumulation and transplastomic protein yields were comparable to

that in small-scale RITA® bioreactors.

While suspension cultures have been successfully scaled up from shake flasks to working

volumes of 50 – 100 l or greater, with little impact on growth kinetics (Terrier et al., 2007),

tissue and organ cultures are known to be difficult to scale-up (Steingroewer et al., 2013;

165

Weathers et al., 2010). This has not been a pertinent issue in commercial horticultural

micropropagation, in which multiplication of small-scale culture vessels has been common

(George et al., 2007). To date, little progress has been made in the scale-up of shoot cultures.

Shoot propagules are sensitive to shear damage and form dense clumps in which mass

transfer may be limited. Mass propagation of Stevia rebaudiana shoots to 65 kg fresh weight

were reported in a 500 l bioreactor without mechanical agitation (Akita et al., 1994;

Takayama and Akita, 1994; Takayama and Akita, 2006). More modest attempts to scale-up

micropropagation, such as producing high quality orchid plantlets in medium-scale rocker

boxes, have been reported (Adelberg, 2006; Weathers et al., 2010).

The choice of explant is also important in scale-up of tissue culture for biopharmaceutical

expression and can affect product quality and consistency. The 500 l culture of Stevia used

shoots as inocula, though this may be inadvisable for biopharmaceutical expression. Direct

organogenesis using shoots as inocula may be difficult to scale-up for a number of reasons.

Shoots have only a limited number of meristems, thereby limiting regenerative potential.

Shoots have complex morphologies, which complicates scale-up approaches as well as

handling and inoculation (Akita et al., 1994; Takayama and Akita, 1994; Takayama and

Akita, 2006). Moreover, the competence to form shoot buds can be highly variable and

depend on length of leaves (Sreedhar et al., 2008), and regenerative potential decreases with

age of leaves (Ibrahim and Debergh, 2001; Sreedhar et al., 2008). As a compromise,

primordia can be used as inocula, being more convenient for inoculation and able to form

many shoots (Akita et al., 1994; Takayama and Akita, 1994). In the studies outlined in this

PhD thesis, dedifferentiated callus was used as inocula. Callus tissue is an advisable choice of

explant for molecular farming purposes. Callus is a relatively homogeneous, unorganised

tissue with high regenerative potential, containing multiple meristemic nodes (George et al.,

2007; Ikeuchi et al., 2013), and therefore better suited to GMP production of

biopharmaceuticals.

7.4 Biosynthesis and assembly of functional monoclonal antibodies in

temporary immersion shoot regeneration

30 years of previous studies have confirmed that plants possess the relevant cellular

‘machinery’ to express, assemble and post-translationally modify antibodies with antibody-

166

binding function (Artsaenko et al., 1998; Brodzik et al., 2006; Drake et al., 2003; Drake et al.,

2009; Hiatt et al., 1989; Hiatt and Pauly, 2006; Holland et al., 2010; Khoudi et al., 1999; Ko

and Koprowski, 2005; Ko et al., 2003; Ma et al., 1998; Ma et al., 1994; Magy et al., 2014;

Qiu et al., 2014; Tavladoraki et al., 1993; Vasilev et al., 2013). However, the studies

described in Chapter 4 describe the first reported attempt to express functional monoclonal

antibodies in in vitro regenerated shoots, using expression of nuclear-encoded Guy’s 13

antibody in N. tabacum as a model system. These studies demonstrate the potential

application of temporary immersion regeneration of shoots from callus for monoclonal

antibody expression, giving antibody titres comparable to soil-grown plants. Until now,

studies involving expression of mAbs in plants have involved soil cultivation of whole plants

(Artsaenko et al., 1998; Busse et al., 2002; Ko et al., 2009; Stoger et al., 2000), although there

have been some innovations in the development of root rhizosecretion systems (Drake et al.,

2003; Drake et al., 2009) and cell suspensions (Holland et al., 2010; Vasilev et al., 2013).

Unexpectedly, hyperhydricity (vitrification) of shoots resulted in increased antibody titres,

which presents an opportunity to enhance titres through imposition of water stress.

The expression of antibodies in transgenic plants (“plantibodies”) is one of the most exciting

technological developments of the molecular farming field (Ko et al., 2009; Ko and

Koprowski, 2005). This study presents an alternative in vitro biomanufacturing platform

which is amenable to GMP standards of quality and consistency. Glycosylation, the covalent

linkage of sugar moieties to proteins, is an important post-translational modification of

immunoglobulins in mammals (Wright and Morrison, 1997). In plant cells, glycosylation

occurs in the secretory pathway in the ER and Golgi. However, the mechanisms of N-linked

glycosylation differ in plants and mammals (Schoberer and Strasser, 2011). Plants add α(1,3)

fucose and β(1,2) xylose residues to the N-glycan of their glycoproteins, whereas mammals

add α(1,6) fucose moieties, glucose and sialic acid residues to the N-glycan (Obembe et al.,

2011). Plant glycans are immunogenic in several mammals, although their role in human

allergies has not been clarified (Bardor et al., 2003; Sabalza et al., 2014; van Ree et al.,

2000). Nonetheless, the potential impact of plant glycans on immunogenicity and adverse

allergic reactions, there is a need to engineer plants to emulate human N-glycosylation

(Gomord et al., 2010; Sabalza et al., 2014). One strategy is to include the fusion of the ER-

retention signal KDEL, to ensure glycosylation is only of the universal ‘high mannose type’

(Petruccelli et al., 2006; Saint-Jore-Dupas et al., 2007; Triguero et al., 2005). Another

approach is ‘glycoengineering’, in which host plants have been engineered to prevent

167

addition of plant-type glycans or add human-type glycans (Fischer et al., 2012; Ko et al.,

2008). However, plant glycosylation can even be beneficial for enhanced antibody avidity, as

is the case of Protalix’s Gaucher’s disease therapeutic, ELELYSO™ (taliglucerase alfa), a

recombinant glucocerebrosidase produced in carrot cells (Protalix BioTherapeutics, Israel)

(Fischer et al., 2012; Sabalza et al., 2014). The glucocerebrosidase is targeted to localise in

vacuolar compartments for terminal addition of plant-specific mannose for improved uptake

(Sabalza et al., 2014; Shaaltiel et al., 2007). The challenges associated with glycosylation of

plant-produced antibodies are considered one of the major regulatory bottlenecks of the

“plantibody” paradigm (Obembe et al., 2011). In our study, the impact of temporary

immersion shoot regeneration on glycosylation of Guy’s 13 antibody was not characterised.

As a future work, the glycosylation pattern and glycan quantitative ratio can be determined

by exoglycosidases digestion followed by HPLC analysis or GC-MS (Shaaltiel et al., 2007).

7.5 Implementation of robust bioprocesses for biopharmaceutical

synthesis

The studies outlined in this dissertation have investigated the feasibility of temporary

immersion shoot regeneration from callus as the basis for a bioprocess platform for high-

yield synthesis of biotherapeutics. Temporary immersion culture of N. tabacum biomass in

small-scale pneumatic systems (RITA® bioreactors) and a large-scale mechanical system

was undertaken. Such systems can be implemented with little capital equipment,

instrumentation or expertise, as opposed to conventional ‘stainless steel’ microbial

fermentation facilities. Standard temperature-controlled micropropagation or greenhouse

facilities can be used, such as those used in commercial horticulture, though laminar flow

hoods and extensive light and space would be required. Power requirements would be

considerably less than standard fermentation facilities. A simple inexpensive low-power air

pump can provide pneumatic suspension of liquid media for approximately 25 RITA®.

Although lighting would be a major energy sink, low power light-emitting diodes (LED)

could be used; these have been shown to effectively stimulate growth in a number of

micropropagative processes (Nhut et al., 2003; Okamoto et al., 1996; Tan Nhut et al., 2001).

Low-tech micropropagative facilities can be set up easily in developing countries, for

example, to provide cheap vaccines for the populace.

168

Much of the focus of molecular farming research has been optimisation of recombinant

protein yields, through molecular or growth strategies (Twyman et al., 2013). Unfortunately,

relatively little development of downstream processes for product extraction and purification

has been undertaken (Fischer et al., 2012). Downstream processing routes must conform to

GMP standards and result in a sufficiently pure and homogeneous pharmaceutical product,

according to regulatory requirements. Robust downstream processing technologies are well-

established for endotoxin removal from bacteria and viruses from mammalian cells (Fischer

et al., 2012). Since plants do not contain endotoxins or mammalian viruses, it is expected that

downstream processes for plant-based systems can be simplified from established

technologies. Downstream processing can account for 80% of the cost of plant-based

bioprocesses, so there are significant savings to be gained from optimisation (Evangelista et

al., 1998; Kusnadi et al., 1997).

169

Bibliography

Aarts, M.G.M., and M.W.E.J. Fiers. 2003. What drives plant stress genes? Trends Plant Sci. 8:99-102. Abbasin, Z., S. Zamani, S. Movahedi, G. Khaksar, and B.E.S. Tabatabaei. 2010. In vitro

micropropagation of yew (Taxus baccata) and production of plantlets. Biotechnology. 9:48-54.

Adam, Z., A. Rudella, and K.J. van Wijk. 2006. Recent advances in the study of Clp, FtsH and other proteases located in chloroplasts. Current Opinion in Plant Biology. 9:234-240.

Adelberg, J. 2006. Agitated, Thin-Films Of Liquid Media For Efficient Micropropagation. In Plan Tissue Culture Engineering. Vol. 6. S.D. Gupta and Y. Ibaraki, editors. Springer Netherlands. 101-117.

Ahmad, N. 2012. Developing new tools for the expression of foreign proteins in tobacco chloroplasts. Imperial College London.

Ahmad, N., F. Michoux, J. McCarthy, and P. Nixon. 2012a. Expression of the affinity tags, glutathione-S-transferase and maltose-binding protein, in tobacco chloroplasts. Planta. 235:863-871.

Ahmad, N., F. Michoux, and P.J. Nixon. 2012b. Investigating the Production of Foreign Membrane Proteins in Tobacco Chloroplasts: Expression of an Algal Plastid Terminal Oxidase. PLOS ONE. 7:e41722.

Ahmad, N., and Z. Mukhtar. 2013. Green factories: plastids for the production of foreign proteins at high levels. Gene Therapy and Molecular Biology. 15:14-29.

Ainsworth, E.A., and D.R. Bush. 2011. Carbohydrate Export from the Leaf: A Highly Regulated Process and Target to Enhance Photosynthesis and Productivity. Plant Physiol. 155:64-69.

Aitken-Christie, J. 1991. Automation. 363-388 pp. Aitken-Christie, J., and C. Jones. 1987. Towards automation - radiata pine shoot hedges in vitro. Plant

Cell Tissue Organ Cult. 8:185-196. Ajjawi, I., and D. Shintani. 2004. Engineered plants with elevated vitamin E: a nutraceutical success

story. Trends Biotechnol. 22:104-107. Akin-Idowu, P.E., D.O. Ibitoye, and O.T. Ademoyegun. 2009. Tissue culture as a plant production

technique for horticultural crops. Afr. J. Biotechnol. 8:3782-3788. Akita, M., T. Shigeoka, Y. Koizumi, and M. Kawamura. 1994. Mass propagation of shoots of Stevia

rebaudiana using a large scale bioreactor. Plant Cell Reports. 13:180-183. Albarran, J., B. Bertrand, M. Lartaud, and H. Etienne. 2005. Cycle characteristics in a temporary

immersion bioreactor affect regeneration, morphology, water and mineral status of coffee (Coffea arabica) somatic embryos. Plant Cell Tissue Organ Cult. 81:27-36.

Aldridge, G.M., D.M. Podrebarac, W.T. Greenough, and I.J. Weiler. 2008. The use of total protein stains as loading controls: An alternative to high-abundance single-protein controls in semi-quantitative immunoblotting. Journal of Neuroscience Methods. 172:250-254.

Alvard, D., F. Cote, and C. Teisson. 1993. Comparison of methods of liquid-medium culture for banana micropropagation - effects of temporary immersion of explants. Plant Cell Tissue Organ Cult. 32:55-60.

Alvim, F.C., S.M.B. Carolino, J.C.M. Cascardo, C.C. Nunes, C.A. Martinez, W.C. Otoni, and E.P.B. Fontes. 2001. Enhanced Accumulation of BiP in Transgenic Plants Confers Tolerance to Water Stress. Plant Physiol. 126:1042-1054.

Anderson, R., X.M. Gao, A. Papakonstantinopoulou, M. Roberts, and G. Dougan. 1996. Immune response in mice following immunization with DNA encoding fragment C of tetanus toxin. Infection and Immunity. 64:3168-3173.

170

Anon. 2012. Taliglucerase (Elelyso) for Gaucher Disease. Medical Letter on Drugs and Therapeutics. 54:56-56.

Apel, W., and R. Bock. 2009. Enhancement of Carotenoid Biosynthesis in Transplastomic Tomatoes by Induced Lycopene-to-Provitamin A Conversion. Plant Physiol. 151:59-66.

Arencibia, A.D., A. Bernal, L. Yang, L. Cortegaza, E.R. Carmona, A. Pérez, C.-J. Hu, Y.-R. Li, C.M. Zayas, and I. Santana. 2008. New role of phenylpropanoid compounds during sugarcane micropropagation in Temporary Immersion Bioreactors (TIBs). Plant Science. 175:487-496.

Arigita, L., A. González, and R.S. Tamés. 2002. Influence of CO2 and sucrose on photosynthesis and transpiration of Actinidia deliciosa explants cultured in vitro. Physiologia Plantarum. 115:166-173.

Artsaenko, O., B. Kettig, U. Fiedler, U. Conrad, and K. Düring. 1998. Potato tubers as a biofactory for recombinant antibodies. Molecular Breeding. 4:313-319.

Atkin, O.K., and W.R. Cummins. 1994. The Effect of Nitrogen Source on Growth, Nitrogen Economy and Respiration of Two High Arctic Plant Species Differing in Relative Growth Rate. Functional Ecology. 8:389-399.

Avni, A., and M. Edelman. 1991. Direct selection for paternal inheritance of chloroplasts in sexual progeny of Nicotiana. Mol. Gen. Genet. 225:273-277.

Babaoglu, M., and M. Yorgancilar. 2000. TDZ-specific plant regeneration in salad burnet. Plant Cell, Tissue and Organ Culture. 63:31-34.

Bai, Y., and C.E. Glatz. 2003. Capture of a recombinant protein from unclarified canola extract using streamline expanded bed anion exchange. Biotechnol. Bioeng. 81:855-864.

Balen, B., M. Tkalec, D. Pavoković, B. Pevalek-Kozlina, and M. Krsnik-Rasol. 2009. Growth Conditions in In Vitro Culture Can Induce Oxidative Stress in Mammillaria gracilis Tissues. J Plant Growth Regul. 28:36-45.

Baque, M.A., S.H. Moh, E.J. Lee, J.J. Zhong, and K.Y. Paek. 2012. Production of biomass and useful compounds from adventitious roots of high-value added medicinal plants using bioreactor. Biotechnol Adv. 30:1255-1267.

Bar-Yosef, B., N.S. Mattson, and H.J. Lieth. 2009. Effects of NH4 : NO3 : urea ratio on cut roses yield, leaf nutrients content and proton efflux by roots in closed hydroponic system. Scientia Horticulturae. 122:610-619.

Bardor, M., C. Faveeuw, A.C. Fitchette, D. Gilbert, L. Galas, F. Trottein, L. Faye, and P. Lerouge. 2003. Immunoreactivity in mammals of two typical plant glyco-epitopes, core alpha(1,3)-fucose and core xylose. Glycobiology. 13:427-434.

Barkan, A. 1988. Proteins encoded by a complex chloroplast transcription unit are each translated from both monocistronic and polycistronic mRNAs. The EMBO journal. 7:2637.

Barkan, A. 2011. Expression of plastid genes: organelle-specific elaborations on a prokaryotic scaffold. Plant Physiol. 155:1520-1532.

Bartelheimer, M., and P. Poschlod. 2014. The response of grassland species to nitrate versus ammonium coincides with their pH optima. Journal of Vegetation Science. 25:760-770.

Batra, S.K., M. Jain, U.A. Wittel, S.C. Chauhan, and D. Colcher. 2002. Pharmacokinetics and biodistribution of genetically engineered antibodies. Current Opinion in Biotechnology. 13:603-608.

Baumal, R., M. Potter, and M.D. Scharff. 1971. Synthesis, assembly, and secretion of gamma globulin by mouse myeloma cells. 3. Assembly of the three subclasses of IgG. The Journal of Experimental Medicine. 134:1316-1334.

Benchabane, M., C. Goulet, D. Rivard, L. Faye, V. Gomord, and D. Michaud. 2008. Preventing unintended proteolysis in plant protein biofactories. Plant Biotechnol. J. 6:633-648.

Bernstein, H.H., E.P. Rothstein, P.P. Associates, M.E. Pichichero, J.L. Green, E. Pediatrics, K.S. Reisinger, M.M. Blatter, J. Halpern, A.M. Arbeter, D.I. Bernstein, V. Smith, S.S. Long, H. Rathfon, and D.S. Krause. 1995. Reactogenicity and immunogenicity of a three-component

171

acellular pertussis vaccine administered as the primary series to 2, 4 and 6 month old infants in the United States. Vaccine. 13:1631-1635.

Berry, J.D. 2005. Rational monoclonal antibody development to emerging pathogens, biothreat agents and agents of foreign animal disease: The antigen scale. The Veterinary Journal. 170:193-211.

Berry, J.D., and R.G. Gaudet. 2011. Antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology. New Biotechnology. 28:489-501.

Bewley, J.D. 1997. Seed germination and dormancy. The plant cell. 9:1055. Bhatia, P., and N. Ashwath. 2005. Effect of medium pH on shoot regeneration from the cotyledonary

explants of tomato. Biotechnology. 4:7-10. Birch-Machin, I., C.A. Newell, J.M. Hibberd, and J.C. Gray. 2004. Accumulation of rotavirus VP6

protein in chloroplasts of transplastomic tobacco is limited by protein stability. Plant Biotechnol. J. 2:261-270.

Birch, J.R., and A.J. Racher. 2006. Antibody production. Advanced Drug Delivery Reviews. 58:671-685. Bird, R.E., and B.W. Walker. 1991. Single chain antibody variable regions. Trends Biotechnol. 9:132-

137. Bock, R. 2007. Plastid biotechnology: prospects for herbicide and insect resistance, metabolic

engineering and molecular farming. Current Opinion in Biotechnology. 18:100-106. Bock, R. 2014. Genetic engineering of the chloroplast: novel tools and new applications. Current

Opinion in Biotechnology. 26:7-13. Bock, R., and M.S. Khan. 2004. Taming plastids for a green future. Trends Biotechnol. 22:311-318. Bogorad, L. 2000. Engineering chloroplasts: an alternative site for foreign genes, proteins, reactions

and products. Trends Biotechnol. 18:257-263. Boivin, E.B., E. Lepage, D.P. Matton, G. De Crescenzo, and M. Jolicoeur. 2010. Transient expression of

antibodies in suspension plant cell suspension cultures is enhanced when co-transformed with the tomato bushy stunt virus p19 viral suppressor of gene silencing. Biotechnol Prog. 26:1534-1543.

Boothe, J., C. Nykiforuk, Y. Shen, S. Zaplachinski, S. Szarka, P. Kuhlman, E. Murray, D. Morck, and M.M. Moloney. 2010. Seed-based expression systems for plant molecular farming. Plant Biotechnol. J. 8:588-606.

Bourgaud, F., A. Gravot, S. Milesi, and E. Gontier. 2001. Production of plant secondary metabolites: a historical perspective. Plant Science. 161:839-851.

Boyhan, D., and H. Daniell. 2011. Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide. Plant Biotechnol. J. 9:585-598.

Boynton, J.E., N.W. Gillham, E.H. Harris, J.P. Hosler, A.M. Johnson, A.R. Jones, B.L. Randolphanderson, D. Robertson, T.M. Klein, K.B. Shark, and J.C. Sanford. 1988. Chloroplast transformation in chlamydomonas with high-velocity microprojectiles. Science. 240:1534-1538.

Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72:248-254.

Brodzik, R., M. Glogowska, K. Bandurska, M. Okulicz, D. Deka, K. Ko, J. van der Linden, J.H.W. Leusen, N. Pogrebnyak, M. Golovkin, Z. Steplewski, and H. Koprowski. 2006. Plant-derived anti-Lewis Y mAb exhibits biological activities for efficient immunotherapy against human cancer cells. Proceedings of the National Academy of Sciences. 103:8804-8809.

Brodzik, R., S. Spitsin, N. Pogrebnyak, K. Bandurska, C. Portocarrero, K. Andryszak, H. Koprowski, and M. Golovkin. 2009. Generation of plant-derived recombinant DTP subunit vaccine. Vaccine. 27:3730-3734.

Brorson, K., and A.Y. Jia. 2014. Therapeutic monoclonal antibodies and consistent ends: terminal heterogeneity, detection, and impact on quality. Current Opinion in Biotechnology. 30:140-146.

172

Budzianowski, J. 2010. Tobacco -a highly efficient producer of vaccines. Przeglad lekarski. 67:1071-1076.

Busse, U., V. Levee, S. Trepanier, and L. Vezina. 2002. Production of Antibodies in Alfalfa (Medicago Sativa). In Molecular Farming of Plants and Animals for Human and Veterinary Medicine. L. Erickson, W.J. Yu, J. Brandle, and R. Rymerson, editors. Springer Netherlands. 237-257.

Butenko, R.G., A.K. Lipsky, N.D. Chernyak, and H.C. Arya. 1984. Changes in culture medium pH by cell suspension cultures of Dioscorea deltoidea. Plant Science Letters. 35:207-212.

Calvo, A.C., S. Oliván, R. Manzano, P. Zaragoza, J. Aguilera, and R. Osta. 2012. Fragment C of Tetanus Toxin: New Insights into Its Neuronal Signaling Pathway. International Journal of Molecular Sciences. 13:6883-6901.

Campbell, P.N. 1992. Biochemistry and molecular biology. Biochemical Education. 20:158-165. Casadevall, A. 1998. Antibody-based therapies as anti-infective agents. Expert opinion on

investigational drugs. 7:307-321. Casadevall, A., and M.D. Scharff. 1995. Return to the Past: The Case for Antibody-Based Therapies in

Infectious Diseases. Clinical Infectious Diseases. 21:150-161. Castro-Concha, L., R. Escobedo, and M. Miranda-Ham. 2006. Measurement of Cell Viability in In Vitro

Cultures. In Plant Cell Culture Protocols. Vol. 318. V. Loyola-Vargas and F. Vázquez-Flota, editors. Humana Press. 71-76.

Chaleff, R.S. 1982. Induction, maintenance, and differentiation of rice callus cultures on ammonium as sole nitrogen source. Plant Cell, Tissue and Organ Culture. 2:29-37.

Chamberlain, D., and C.N. Stewart. 1999. Transgene escape and transplastomics. Nat Biotech. 17:330-331.

Chargelegue, D., P.M.W. Drake, P. Obregon, A. Prada, N. Fairweather, and J.K.-C. Ma. 2005. Highly Immunogenic and Protective Recombinant Vaccine Candidate Expressed in Transgenic Plants. Infection and Immunity. 73:5915-5922.

Chaves, M.M., J.S. Pereira, J. Maroco, M.L. Rodrigues, C.P.P. Ricardo, M.L. Osório, I. Carvalho, T. Faria, and C. Pinheiro. 2002. How Plants Cope with Water Stress in the Field? Photosynthesis and Growth. Annals of Botany. 89:907-916.

Cheingsong-Popov, R., C. Panagiotidi, S. Bowcock, A. Aronstam, J. Wadsworth, and J. Weber. 1991. Relation between humoral responses to HIV gag and env proteins at seroconversion and clinical outcome of HIV infection. 23-26 pp.

Chen, Q. 2008. Expression and purification of pharmaceutical proteins in plants. Biol Eng. 1:291-321. Cheng, L., H.P. Li, B. Qu, T. Huang, J.X. Tu, T.D. Fu, and Y.C. Liao. 2010. Chloroplast transformation of

rapeseed (Brassica napus) by particle bombardment of cotyledons. Plant Cell Rep. 29:371-381.

Chisti, Y., and M. Moo-Young. 1989. On the calculation of shear rate and apparent viscosity in airlift and bubble column bioreactors. Biotechnol. Bioeng. 34:1391-1392.

Choffe, K., J. Victor, S. Murch, and P. Saxena. 2000. In vitro regeneration of Echinacea purpurea L.: Direct somatic embryogenesis and indirect shoot organogenesis in petiole culture. In Vitro Cellular & Developmental Biology - Plant. 36:30-36.

Choi, A.H.C., M. Basu, M.M. McNeal, J. Flint, J.L. VanCott, J.D. Clements, and R.L. Ward. 2000. Functional Mapping of Protective Domains and Epitopes in the Rotavirus VP6 Protein. Journal of Virology. 74:11574-11580.

Choi, Y.-E., Y.-S. Kim, and K.-Y. Paek. 2006. Types and designs of bioreactors for hairy root culture. In Plan Tissue Culture Engineering. Springer. 161-172.

Ciarmiello, L.F., P. Woodrow, A. Fuggi, G. Pontecorvo, and P. Carillo. 2011. Plant Genes for Abiotic Stress.

Clarke, J.L., and H. Daniell. 2011. Plastid biotechnology for crop production: present status and future perspectives. Plant Mol Biol. 76:211-220.

Clemensson-Lindell, A., and H. Persson. 1995. Fine-root vitality in a Norway spruce stand subjected to various nutrient supplies. Plant Soil. 168-169:167-172.

173

Cohen, S.N., A.C.Y. Chang, H.W. Boyer, and R.B. Helling. 1973. Construction of Biologically Functional Bacterial Plasmids In Vitro. Proceedings of the National Academy of Sciences. 70:3240-3244.

Colgan, R., C. Atkinson, M. Paul, S. Hassan, P.W. Drake, A. Sexton, S. Santa-Cruz, D. James, K. Hamp, C. Gutteridge, and J.-C. Ma. 2010. Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals. Transgenic Res. 19:241-256.

Colwell, R.R. 2002. Fulfilling the promise of biotechnology. Biotechnol. Adv. 20:215-228. Commandeur, U., R.M. Twyman, and R. Fischer. 2003. The biosafety of molecular farming in plants.

AgBiotechNet. 5:1-9. Compton, M., and J. Koch. 2001. Influence of plant preservative mixture (PPM)TM on adventitious

organogenesis in melon, petunia, and tobacco. In Vitro Cellular & Developmental Biology - Plant. 37:259-261.

Coulson, J.M., J.F. Richardson, J.R. Backhurst, and J.H. Harker. 1999. Coulson and Richardson's Chemical Engineering Volume 1 - Fluid Flow, Heat Transfer and Mass Transfer (6th Edition). Elsevier. 108.

Cousson, A., and K.T.T. Van. 1993. Influence of ionic composition of the culture medium on de novo flower formation in tobacco thin cell layers. Canadian Journal of Botany. 71:506-511.

Craufurd, P., and T. Wheeler. 2009. Climate change and the flowering time of annual crops. Journal of Experimental Botany. 60:2529-2539.

Crouch, S.P.M., R. Kozlowski, K.J. Slater, and J. Fletcher. 1993. The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. Journal of Immunological Methods. 160:81-88.

D'Aoust, M.-A., U. Busse, M. Martel, P. Lerouge, D. Levesque, and L.-P. Vézina. 2004. Perennial Plants as a Production System for Pharmaceuticals. In Handbook of Plant Biotechnology. John Wiley & Sons, Ltd.

da Silva, A.B., M. Pasqual, J.B. Teixeira, and A.G. De Araujo. 2007. Micropropagation methods of pineapple. Pesqui. Agropecu. Bras. 42:1257-1260.

Daniell, H., S. Chebolu, S. Kumar, M. Singleton, and R. Falconer. 2005. Chloroplast-derived vaccine antigens and other therapeutic proteins. Vaccine. 23:1779-1783.

Daniell, H., R. Datta, S. Varma, S. Gray, and S.B. Lee. 1998. Containment of herbicide resistance through genetic engineering of the chloroplast genome. Nat. Biotechnol. 16:345-348.

Daniell, H., M.S. Khan, and L. Allison. 2002. Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci. 7:84-91.

Daniell, H., S.B. Lee, T. Panchal, and P.O. Wiebe. 2001a. Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. Journal of Molecular Biology. 311:1001-1009.

Daniell, H., G. Ruiz, B. Denes, L. Sandberg, and W. Langridge. 2009. Optimization of codon composition and regulatory elements for expression of human insulin like growth factor-1 in transgenic chloroplasts and evaluation of structural identity and function. BMC Biotechnol. 9:33.

Daniell, H., S.J. Streatfield, and K. Wycoff. 2001b. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 6:219-226.

Davies, H.M. 2010. Commercialization of whole-plant systems for biomanufacturing of protein products: evolution and prospects. Plant Biotechnol. J. 8:845-861.

de Bruijn, H. 1942. The viscosity of suspensions of spherical particles. (The fundamental η-c and φ relations). Recueil des Travaux Chimiques des Pays-Bas. 61:863-874.

De Cosa, B., W. Moar, S.B. Lee, M. Miller, and H. Daniell. 2001. Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat. Biotechnol. 19:71-74.

de la Viña, G., F. Pliego-Alfaro, S.P. Driscoll, V.J. Mitchell, M.A. Parry, and D.W. Lawlor. 1999. Effects of CO2 and sugars on photosynthesis and composition of avocado leaves grown in vitro. Plant Physiology and Biochemistry. 37:587-595.

De Neve, M., H. Van Houdt, A.-M. Bruyns, M. Van Montagu, and A. Depicker. 1998. Screening for Transgenic Lines with Stable and Suitable Accumulation Levels of a Heterologous Protein. In

174

Recombinant Proteins from Plants. Vol. 3. C. Cunningham and A.R. Porter, editors. Humana Press. 203-227.

De Wolf, F., J. Goudsmit, D.A. Paul, J. Lange, C. Hooijkaas, P. Schellekens, R.A. Coutinho, and J. van der Noordaa. 1987. Risk of AIDS related complex and AIDS in homosexual men with persistent HIV antigenaemia. British medical journal (Clinical research ed.). 295:569.

Debergh, P.C. 1983. Effects of agar brand and concentration on the tissue-culture medium. Physiologia Plantarum. 59:270-276.

Decker, E.L., and R. Reski. 2004. The moss bioreactor. Current Opinion in Plant Biology. 7:166-170. Decker, E.L., and R. Reski. 2007. Moss bioreactors producing improved biopharmaceuticals. Current

Opinion in Biotechnology. 18:393-398. DeGray, G., K. Rajasekaran, F. Smith, J. Sanford, and H. Daniell. 2001. Expression of an antimicrobial

peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol. 127:852-862.

Dekker, J.P., and E.J. Boekema. 2005. Supramolecular organization of thylakoid membrane proteins in green plants. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1706:12-39.

Demeyer, R. 2011. Evaluation of Arabidopsis spp. as a production platform for molecular farming. Ghent University.

Dennehy, P.H. 2008. Rotavirus Vaccines: an Overview. Clinical Microbiology Reviews. 21:198-208. Dewir, Y.H., D. Chakrabarty, M.B. Ali, E.J. Hahn, and K.Y. Paek. 2006. Lipid peroxidation and

antioxidant enzyme activities of Euphorbia millii hyperhydric shoots. Environmental and Experimental Botany. 58:93-99.

Dietmair, S., L.K. Nielsen, and N.E. Timmins. 2012. Mammalian cells as biopharmaceutical production hosts in the age of omics. Biotechnology Journal. 7:75-89.

Dinarvand, M., M. Rezaee, M. Masomian, S.D. Jazayeri, M. Zareian, S. Abbasi, and A.B. Ariff. 2013. Effect of C/N Ratio and Media Optimization through Response Surface Methodology on Simultaneous Productions of Intra- and Extracellular Inulinase and Invertase from Aspergillus niger ATCC 20611. BioMed Research International. 2013:13.

Dinnis, D.M., and D.C. James. 2005. Engineering mammalian cell factories for improved recombinant monoclonal antibody production: lessons from nature? Biotechnol. Bioeng. 91:180-189.

Doan, L.X., M. Li, C. Chen, and Q. Yao. 2005. Virus-like particles as HIV-1 vaccines. Reviews in Medical Virology. 15:75-88.

Dodds, J.H. 1988. Tissue culture technology: Practical application of sophisticated methods. American Potato Journal. 65:167-180.

Dong, J.-L., B. Zhou, G. Sheng, and T. Wang. 2005. Transgenic Tobacco Expressing a Modified VP6 Gene Protects Mice Against Rotavirus Infection. Journal of Integrative Plant Biology. 47:978-987.

Doran, P.M. 2000. Foreign protein production in plant tissue cultures. Current Opinion in Biotechnology. 11:199-204.

Doran, P.M. 2006. Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol. 24:426-432.

Doran, P.M. 2013. Therapeutically important proteins from in vitro plant tissue culture systems. Current medicinal chemistry. 20:1047-1055.

Dörmann, P. 2001. Galactolipids in Plant Membranes. In eLS. John Wiley & Sons, Ltd. Drake, P.M., D.M. Chargelegue, N.D. Vine, C.J. van Dolleweerd, P. Obregon, and J.K. Ma. 2003.

Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco roots. Plant Mol Biol. 52:233-241.

Drake, P.M.W., T. Barbi, A. Sexton, E. McGowan, J. Stadlmann, C. Navarre, M.J. Paul, and J.K.-C. Ma. 2009. Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco. The FASEB Journal. 23:3581-3589.

Dubranszki, J., and J.A.T. da Silva. 2010. Micropropagation of apple - A review. Biotechnol. Adv. 28:462-488.

175

Dufourmantel, N., M. Dubald, M. Matringe, H. Canard, F. Garcon, C. Job, E. Kay, J.-P. Wisniewski, J.-M. Ferullo, B. Pelissier, A. Sailland, and G. Tissot. 2007. Generation and characterization of soybean and marker-free tobacco plastid transformants over-expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance. Plant Biotechnol. J. 5:118-133.

Dunlop, E.H., P.K. Namdev, and M.Z. Rosenberg. 1994. Effect of fluid shear forces on plant cell suspensions. Chemical Engineering Science. 49:2263-2276.

During, K. 1988. Wound-inducible expression and secretion of T4 lysozyme and monoclonal antibodies in Nicotiana tabacum. Inaugral Dissertation to Obtain a Doctorate at the Mathematisch-Naturwissenchaftlichen Fakultät der Universität zu Köln. Köln ed:1-90.

Dyer, W.B., H. Kuipers, M.W. Coolen, A.F. Geczy, J. Forrester, C. Workman, and J.S. Sullivan. 2002. Correlates of antiviral immune restoration in acute and chronic HIV type 1 infection: sustained viral suppression and normalization of T cell subsets. AIDS research and human retroviruses. 18:999-1010.

Eibl, C., Z.R. Zou, A. Beck, M. Kim, J. Mullet, and H.U. Koop. 1999. In vivo analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency. Plant J. 19:333-345.

Eibl, R., and D. Eibl. 2008. Design of bioreactors suitable for plant cell and tissue cultures. Phytochemistry Reviews. 7:593-598.

Eibl, R., S. Kaiser, R. Lombriser, and D. Eibl. 2010. Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology. Applied Microbiology and Biotechnology. 86:41-49.

Eisenberg, D., H.S. Gill, G.M.U. Pfluegl, and S.H. Rotstein. 2000. Structure–function relationships of glutamine synthetases. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1477:122-145.

Engelen, F., A. Schouten, J. Molthoff, J. Roosien, J. Salinas, W. Dirkse, A. Schots, J. Bakker, F. Gommers, M. Jongsma, D. Bosch, and W. Stiekema. 1994. Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco. Plant Mol.Biol. 26:1701-1710.

Erickson, J. 1998. Assembly of Photosystem II. In The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Vol. 7. J.D. Rochaix, M. Goldschmidt-Clermont, and S. Merchant, editors. Springer Netherlands. 255-285.

Escalant, J.V., C. Teisson, and F. Cote. 1994. Amplified somatic embryogenesis from male flowers of triploid banana and plantain cultivars (Musa spp.). In Vitro Cell. Dev. Biol.-Plant. 30P:181-186.

Escalona, M., J.C. Lorenzo, B. Gonzalez, M. Daquinta, J.L. Gonzalez, Y. Desjardins, and C.G. Borroto. 1999. Pineapple (Ananas comosus L-Merr) micropropagation in temporary immersion systems. Plant Cell Reports. 18:743-748.

Esposito, D., and D.K. Chatterjee. 2006. Enhancement of soluble protein expression through the use of fusion tags. Current Opinion in Biotechnology. 17:353-358.

Etienne, H., and M. Berthouly. 2002. Temporary immersion systems in plant micropropagation. Plant Cell Tissue Organ Cult. 69:215-231.

Etienne, H., M. Lartaud, N. MichauxFerriere, M.P. Carron, M. Berthouly, and C. Teisson. 1997. Improvement of somatic embryogenesis in Hevea brasiliensis (Mull Arg) using the temporary immersion technique. In Vitro Cell. Dev. Biol.-Plant. 33:81-87.

Evangelista, R.L., A.R. Kusnadi, J.A. Howard, and Z.L. Nikolov. 1998. Process and Economic Evaluation of the Extraction and Purification of Recombinant β-Glucuronidase from Transgenic Corn. Biotechnology Progress. 14:607-614.

Evans, N.E. 1993. A Preliminary Study on the Effects of Nitrogen Supply on the Growth In Vitro of Nine Potato Genotypes (Solanum spp). Journal of Experimental Botany. 44:837-841.

176

Faye, L., and V. Gomord. 2010. Success stories in molecular farming—a brief overview. Plant Biotechnol. J. 8:525-528.

Faye, M., A. David, and A. Lamant. 1986. Nitrate reductase activity and nitrate accumulation in in vitro produced axillary shoots, plantlets and seedlings of Pinus pinaster. Plant Cell Rep. 5:368-371.

Featherstone, J.D.B. 2000. The science and practice of caries prevention. The Journal of the American Dental Association. 131:887-899.

Findlay, J.W.A., and R.F. Dillard. 2007. Appropriate calibration curve fitting in ligand binding assays. AAPS J. 9:E260-E267.

Firoozabady, E., and N. Gutterson. 2003. Cost-effective in vitro propagation methods for pineapple. Plant Cell Reports. 21:844-850.

Fischer, R., S. Schillberg, J.F. Buyel, and R.M. Twyman. 2013. Commercial aspects of pharmaceutical protein production in plants. Curr Pharm Des. 19:5471-5477.

Fischer, R., S. Schillberg, S. Hellwig, R.M. Twyman, and J. Drossard. 2012. GMP issues for recombinant plant-derived pharmaceutical proteins. Biotechnol. Adv. 30:434-439.

Fontes, M.A., W.C. Otoni, S.M.B. Carolino, S.H. Brommonschenkel, E.P.B. Fontes, M. Fári, and R.P. Louro. 1999. Hyperhydricity in pepper plants regenerated in vitro: involvement of BiP (Binding Protein) and ultrastructural aspects. Plant Cell Reports. 19:81-87.

Fournier, D., C. Bonnelle, and Y. Tourte. 1991. Ultrastructural features of soybean somatic cells at the beginning of an organogenic process: toward a new concept. Biology of the Cell. 73:99-105.

Fox, J.L. 2003. Puzzling industry response to ProdiGene fiasco. Nat Biotech. 21:3-4. Fraley, R.T., S.G. Rogers, R.B. Horsch, P.R. Sanders, J.S. Flick, S.P. Adams, M.L. Bittner, L.A. Brand, C.L.

Fink, J.S. Fry, G.R. Galluppi, S.B. Goldberg, N.L. Hoffmann, and S.C. Woo. 1983. Expression of bacterial genes in plant cells. Proceedings of the National Academy of Sciences. 80:4803-4807.

Franklin, K.A., V.S. Larner, and G.C. Whitelam. 2005. The signal transducing photoreceptors of plants. The International journal of developmental biology. 49:653-664.

Frey, S., and D. Gorlich. 2014. Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases. Journal of chromatography. A. 1337:106-115.

Fujita, Y. 1988. Shikonin: Production by Plant (Lithospermum erythrorhizon) Cell Cultures. In Medicinal and Aromatic Plants I. Vol. 4. Y.P.S. Bajaj, editor. Springer Berlin Heidelberg. 225-236.

Fukao, T., and J. Bailey-Serres. 2004. Plant responses to hypoxia – is survival a balancing act? Trends Plant Sci. 9:449-456.

Galzy, R., and D. Compan. 1992. Remarks on mixotrophic and autotrophic carbon nutrition of Vitis plantlets cultured in vitro. Plant Cell, Tissue and Organ Culture. 31:239-244.

Gamborg, O.L. 1970. The Effects of Amino Acids and Ammonium on the Growth of Plant Cells in Suspension Culture. Plant Physiol. 45:372-375.

Gardi, T., E. Piccioni, and A. Standardi. 1999. Effect of bead nutrient composition on regrowth of stored in vitro-derived encapsulated microcuttings of different woody species. Journal of Microencapsulation. 16:13-25.

Gaspar, T., T. Franck, B. Bisbis, C. Kevers, L. Jouve, J.F. Hausman, and J. Dommes. 2002. Concepts in plant stress physiology. Application to plant tissue cultures. Plant Growth Regul. 37:263-285.

Gavrilescu, M., and Y. Chisti. 2005. Biotechnology—a sustainable alternative for chemical industry. Biotechnol. Adv. 23:471-499.

George, E.F., A. Hall, and G.J. de Klerk. 2007. Plant Propagation by Tissue Culture: Volume 1. The Background. Springer.

177

George, M.W., and R.R. Tripepi. 2001. Plant Preservative Mixture™ can affect shoot regeneration from leaf explants of Chrysanthemum, European birch, and Rhododendron. Hortscience. 36:768-769.

Georgiev, M.I., J. Weber, and A. Maciuk. 2009. Bioprocessing of plant cell cultures for mass production of targeted compounds. Applied Microbiology and Biotechnology. 83:809-823.

Georgiev, V., M. Ilieva, T. Bley, and A. Pavlov. 2008. Betalain production in plant in vitro systems. Acta Physiol. Plant. 30:581-593.

Georgiev, V., A. Schumann, A. Pavlov, and T. Bley. 2014. Temporary immersion systems in plant biotechnology. Engineering in Life Sciences:n/a-n/a.

Gill, R., and P.K. Saxena. 1993. Somatic embryogenesis in Nicotiana tabacum - induction by thidiazuron of direct embryo differentiation from cultured leaf-disks. Plant Cell Reports. 12:154-159.

Gils, M., R. Kandzia, S. Marillonnet, V. Klimyuk, and Y. Gleba. 2005. High-yield production of authentic human growth hormone using a plant virus-based expression system. Plant Biotechnol. J. 3:613-620.

Gisby, M.F., E.A. Mudd, and A. Day. 2012. Growth of transplastomic cells expressing D-amino acid oxidase in chloroplasts is tolerant to D-alanine and inhibited by D-valine. Plant Physiol. 160:2219-2226.

Glacken, M.W., R.J. Fleischaker, and A.J. Sinskey. Mammalian cell culture: engineering principles and scale-up. Trends Biotechnol. 1:102-108.

Gleba, Y., S. Marillonnet, and V. Klimyuk. 2004. Design of safe and biologically contained transgenic plants: tools and technologies for controlled transgene flow and expression. Biotechnology & genetic engineering reviews. 21:325-367.

Gloe, T., H.Y. Sohn, G.A. Meininger, and U. Pohl. 2002. Shear Stress-induced Release of Basic Fibroblast Growth Factor from Endothelial Cells Is Mediated by Matrix Interaction via Integrin αVβ3. Journal of Biological Chemistry. 277:23453-23458.

Golds, T., P. Maliga, and H.U. Koop. 1993. Stable plastid transformation in peg-treated protoplasts of Nicotiana tabacum. Bio-Technology. 11:95-97.

Gomord, V., A.-C. Fitchette, L. Menu-Bouaouiche, C. Saint-Jore-Dupas, C. Plasson, D. Michaud, and L. Faye. 2010. Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnol. J. 8:564-587.

Gonzalez-Olmedo, J.L., Z. Fundora, L.A. Molina, J. Abdulnour, Y. Desjardins, and M. Escalona. 2005. New contributions to propagation of pineapple (Ananas comosus L. Merr) in temporary immersion bioreactors. In Vitro Cell. Dev. Biol.-Plant. 41:87-90.

Gonzalez-Rabade, N., E.G. McGowan, F. Zhou, M.S. McCabe, R. Bock, P.J. Dix, J.C. Gray, and J.K.C. Ma. 2011. Immunogenicity of chloroplast-derived HIV-1 p24 and a p24-Nef fusion protein following subcutaneous and oral administration in mice. Plant Biotechnol. J. 9:629-638.

Gonzalez, D.D., M.V. Mozgovoj, D. Bellido, D.V. Rodriguez, F.M. Fernandez, A. Wigdorovitz, V.G. Parreño, and M.J. Dus Santos. 2010. Evaluation of a bovine rotavirus VP6 vaccine efficacy in the calf model of infection and disease. Veterinary Immunology and Immunopathology. 137:155-160.

Gorret, N., A.K. bin Rosli, S.F. Oppenheim, L.B. Willis, P.A. Lessard, C.K. Rha, and A.J. Sinskey. 2004. Bioreactor culture of oil palm (Elaeis guineensis) and effects of nitrogen source, inoculum size, and conditioned medium on biomass production. Journal of Biotechnology. 108:253-263.

Goswami, S., W. Wang, T. Arakawa, and S. Ohtake. 2013. Developments and challenges for mAb-based therapeutics. Antibodies. 2:452-500.

Gottlieb, T., and T. Ben-Yedidia. Epitope-based approaches to a universal influenza vaccine. Journal of Autoimmunity.

Gould, S.B., R.F. Waller, and G.I. McFadden. 2008. Plastid Evolution. Annual Review of Plant Biology. 59:491-517.

178

Greiner, S., and R. Bock. 2013. Tuning a menage a trois: co-evolution and co-adaptation of nuclear and organellar genomes in plants. BioEssays : news and reviews in molecular, cellular and developmental biology. 35:354-365.

Gribble, K., J. Tingle, V. Sarafis, A. Heaton, and P. Holford. 1998. Position of water in vitrified plants visualised by NMR imaging. Protoplasma. 201:110-114.

Grimes, H.D., and T.K. Hodges. 1990. The Inorganic NO3-: NH4

+ ratio Influences Plant Regeneration and Auxin Sensitivity in Primary Callus Derived from Immature Embryos of Indica Rice (Oryza sativa L.). J. Plant Physiol. 136:362-367.

Guerra, M.P., L.L. Dal Vesco, J.P.H.J. Ducroquet, R.O. Nodari, and M.S.D. Reis. 2001. Somatic embryogenesis in goiabeira serrana: genotype response, auxinic shock and synthetic seeds. Revista Brasileira de Fisiologia Vegetal. 13:117-128.

Hagemann, R. 2004. The Sexual Inheritance of Plant Organelles. In Molecular Biology and Biotechnology of Plant Organelles. H. Daniell and C. Chase, editors. Springer Netherlands. 93-113.

Håkansson, S. 2003. Annual and Perennial crops. Weeds and weed management on arable land: an ecological approach:14-15.

Hanke, G.T., Y. Kimata-Ariga, I. Taniguchi, and T. Hase. 2004. A Post Genomic Characterization of Arabidopsis Ferredoxins. Plant Physiol. 134:255-264.

Hanke, T., and A.J. McMichael. 2000. Design and construction of an experimental HIV-1 vaccine for a year-2000 clinical trial in Kenya. Nat Med. 6:951-955.

Harris, E.H., J.E. Boynton, and N.W. Gillham. 1994. Chloroplast ribosomes and protein synthesis. Microbiological reviews. 58:700.

Häusler, R.E., L. Heinrichs, J. Schmitz, and U.-I. Flügge. 2014. How Sugars Might Coordinate Chloroplast and Nuclear Gene Expression during Acclimation to High Light Intensities. Molecular Plant. 7:1121-1137.

Hawkesford, M., W. Horst, T. Kichey, H. Lambers, J. Schjoerring, I.S. Møller, and P. White. 2012. Chapter 6 - Functions of Macronutrients. In Marschner's Mineral Nutrition of Higher Plants (Third Edition). P. Marschner, editor. Academic Press, San Diego. 135-189.

Hay, M., D.W. Thomas, J.L. Craighead, C. Economides, and J. Rosenthal. 2014. Clinical development success rates for investigational drugs. Nat Biotech. 32:40-51.

Hehle, V.K., M.J. Paul, P.M. Drake, J.K. Ma, and C.J. van Dolleweerd. 2011. Antibody degradation in tobacco plants: a predominantly apoplastic process. BMC Biotechnol. 11:128.

Hein, M.B., Y. Tang, D.A. McLeod, K.D. Janda, and A. Hiatt. 1991. Evaluation of immunoglobulins from plant cells. Biotechnology Progress. 7:455-461.

Hellwig, S., J. Drossard, R.M. Twyman, and R. Fischer. 2004. Plant cell cultures for the production of recombinant proteins. Nat Biotech. 22:1415-1422.

Hiatt, A., R. Caffferkey, and K. Bowdish. 1989. Production of antibodies in transgenic plants. Nature. 342:76-78.

Hiatt, A., and M. Pauly. 2006. Monoclonal antibodies from plants: A new speed record. Proceedings of the National Academy of Sciences. 103:14645-14646.

Hicks, G. 1994. Shoot induction and organogenesis in vitro: A developmental perspective. In Vitro Cellular & Developmental Biology - Plant. 30:10-15.

Higgins, T.J., P.A. O’Brien, D. Spencer, H.E. Schroeder, H. Dove, and M. Freer. 1989. Potential of Transgenic Plants for Improved Amino Acid Supply for Wool Growth. In The Biology of Wool and Hair. G.E. Rogers, P.J. Reis, K.A. Ward, and R.C. Marshall, editors. Springer Netherlands. 441-445.

Hill, J.P., and E.M. Lord. 1990. A Method for Determining Plastochron Indices During Heteroblastic Shoot Growth. American Journal of Botany. 77:1491-1497.

Hirata, N., D. Yonekura, S. Yanagisawa, and K. Iba. 2004. Possible Involvement of the 5′-Flanking Region and the 5′UTR of Plastid accD Gene in NEP-Dependent Transcription. Plant and Cell Physiology. 45:176-186.

179

Hohe, A., and R. Reski. 2002. Optimisation of a bioreactor culture of the moss Physcomitrella patens for mass production of protoplasts. Plant Science. 163:69-74.

Holland, T., M. Sack, T. Rademacher, K. Schmale, F. Altmann, J. Stadlmann, R. Fischer, and S. Hellwig. 2010. Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full-size antibody in BY-2 suspension culture. Biotechnol. Bioeng. 107:278-289.

Hood, E., D. Witcher, S. Maddock, T. Meyer, C. Baszczynski, M. Bailey, P. Flynn, J. Register, L. Marshall, D. Bond, E. Kulisek, A. Kusnadi, R. Evangelista, Z. Nikolov, C. Wooge, R. Mehigh, R. Hernan, W. Kappel, D. Ritland, C. Ping Li, and J. Howard. 1997. Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification. Molecular Breeding. 3:291-306.

Hood, E.E., R. Love, J. Lane, J. Bray, R. Clough, K. Pappu, C. Drees, K.R. Hood, S. Yoon, A. Ahmad, and J.A. Howard. 2007. Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed. Plant Biotechnol. J. 5:709-719.

Hood, E.E., and S. Woodard. 2005. Commercialization of a protein product from transgenic maize. Natl Agric Biotechnol Council. 17:147-157.

Howard, J.A., Z. Nikolov, and E.E. Hood. 2011. Enzyme Production Systems for Biomass Conversion. In Plant Biomass Conversion. John Wiley & Sons, Inc. 227-253.

Huang, C.Y., M.A. Ayliffe, and J.N. Timmis. 2003. Direct measurement of the transfer rate of chloroplast DNA into the nucleus. Nature. 422:72-76.

Huang, T.-K., and K.A. McDonald. 2012. Bioreactor systems for in vitro production of foreign proteins using plant cell cultures. Biotechnol. Adv. 30:398-409.

Hulse, J.H. 2004. Biotechnologies: past history, present state and future prospects. Trends in Food Science & Technology. 15:3-18.

Hung, C.D., and S.J. Trueman. 2012. Alginate encapsulation of shoot tips and nodal segments for short-term storage and distribution of the eucalypt Corymbia torelliana x C. citriodora. Acta Physiol. Plant. 34:117-128.

Husaini, A.M., Z. Rashid, R.u.R. Mir, and B. Aquil. 2011. Approaches for gene targeting and targeted gene expression in plants. GM Crops. 2:150-162.

Hyunjong, B., D.-S. Lee, and I. Hwang. 2006. Dual targeting of xylanase to chloroplasts and peroxisomes as a means to increase protein accumulation in plant cells. Journal of Experimental Botany. 57:161-169.

Ibrahim, R., and P.C. Debergh. 2001. Factors controlling high efficiency adventitious bud formation and plant regeneration from in vitro leaf explants of roses (Rosa hybrida L.). Scientia Horticulturae. 88:41-57.

Ikeuchi, M., K. Sugimoto, and A. Iwase. 2013. Plant Callus: Mechanisms of Induction and Repression. The Plant Cell Online. 25:3159-3173.

Inka Borchers, A.M., N. Gonzalez-Rabade, and J.C. Gray. 2012. Increased accumulation and stability of rotavirus VP6 protein in tobacco chloroplasts following changes to the 5′ untranslated region and the 5′ end of the coding region. Plant Biotechnol. J. 10:422-434.

Ivanova, M., and J. van Staden. 2008. Effect of ammonium ions and cytokinins on hyperhydricity and multiplication rate of in vitro regenerated shoots of Aloe polyphylla. Plant Cell, Tissue and Organ Culture. 92:227-231.

Ivanova, M., and J. Van Staden. 2009. Nitrogen source, concentration, and NH4+ : NO3

− ratio influence shoot regeneration and hyperhydricity in tissue cultured Aloe polyphylla. Plant Cell, Tissue and Organ Culture. 99:167-174.

Iwarson, S., E. Tabor, H.C. Thomas, P. Snoy, and R.J. Gerety. 1985. Protection against hepatitis B virus infection by immunization with hepatitis B core antigen. Gastroenterology. 88:763-767.

Jablonsky, J., H. Bauwe, and O. Wolkenhauer. 2011. Modeling the Calvin-Benson cycle. BMC Syst Biol. 5:1-13.

Jain, E., and A. Kumar. 2008. Upstream processes in antibody production: Evaluation of critical parameters. Biotechnol. Adv. 26:46-72.

180

Jamal, A., K. Ko, H.-S. Kim, Y.-K. Choo, H. Joung, and K. Ko. 2009. Role of genetic factors and environmental conditions in recombinant protein production for molecular farming. Biotechnol. Adv. 27:914-923.

James, C. 2013. Global Status of Commercialized Biotech/GM Crops. ISAAA Brief 46. Jang, J.C., and J. Sheen. 1994. Sugar sensing in higher plants. The Plant Cell Online. 6:1665-1679. Jansen, R.K., and T.A. Ruhlman. 2012. Plastid Genomes of Seed Plants. In Genomics of Chloroplasts

and Mitochondria. Vol. 35. R. Bock and V. Knoop, editors. Springer Netherlands. 103-126. Jeon, J.Y., J.-S. Kwon, S.T. Kang, B.-R. Kim, Y. Jung, J.G. Han, J.H. Park, and J.K. Hwang. 2014.

Optimization of culture media for large-scale lutein production by heterotrophic Chlorella vulgaris. Biotechnology Progress. 30:736-743.

Jiang, L., and S.S.M. Sun. 2002. Membrane anchors for vacuolar targeting: application in plant bioreactors. Trends Biotechnol. 20:99-102.

Joh, L.D., T. Wroblewski, N.N. Ewing, and J.S. VanderGheynst. 2005. High-level transient expression of recombinant protein in lettuce. Biotechnol. Bioeng. 91:861-871.

Johnson, I.S. 1983. Human Insulin from Recombinant DNA Technology. Science. 219:632-637. Jones, G., and S.K. Talley. 1933. The Viscosity of Aqueous Solutions as a Function of the

Concentration. Journal of the American Chemical Society. 55:624-642. Junker, B.H. 2004. Scale-up methodologies for Escherichia coli and yeast fermentation processes.

Journal of Bioscience and Bioengineering. 97:347-364. Justino, M.C.A., E.C. Araújo, L.-J. van Doorn, C.S. Oliveira, Y.B. Gabbay, J.D.A.P. Mascarenhas, Y.S.

Miranda, S.d.F.S. Guerra, V.B.d. Silva, and A.C. Linhares. 2012. Oral live attenuated human rotavirus vaccine (RotarixTM) offers sustained high protection against severe G9P8 rotavirus gastroenteritis during the first two years of life in Brazilian children. Memórias do Instituto Oswaldo Cruz. 107:846-853.

Kadleček, P., B. Rank, and I. Tichá. 2003. Photosynthesis and photoprotection in Nicotiana tabacum L. in vitro-grown plantlets. J. Plant Physiol. 160:1017-1024.

Kalish, M.L., A. Baldwin, S. Raktham, C. Wasi, C.C. Luo, G. Schochetman, T.D. Mastro, N. Young, S. Vanichseni, H. Rubsamen-Waigmann, and et al. 1995. The evolving molecular epidemiology of HIV-1 envelope subtypes in injecting drug users in Bangkok, Thailand: implications for HIV vaccine trials. AIDS (London, England). 9:851-857.

Kamarajugadda, S., and H. Daniell. 2006. Chloroplast-derived anthrax and other vaccine antigens: their immunogenic and immunoprotective properties.

Kanamoto, H., A. Yamashita, H. Asao, S. Okumura, H. Takase, M. Hattori, A. Yokota, and K.-I. Tomizawa. 2006. Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids. Transgenic Res. 15:205-217.

Karg, S.R., and P.T. Kallio. 2009. The production of biopharmaceuticals in plant systems. Biotechnol. Adv. 27:879-894.

Kato, A., S. Kawazoe, and Y. Soh. 1978. Viscosity of the Broth of Tobacco Cells in Suspension Culture: Biomass Production of Tobacco Cells (Part IV). Journal of fermentation technology. 56:224-228.

Katsnelson, A., J. Ransom, P. Vermij, and E. Waltz. 2006. News In Brief. Nat Biotech. 24:233-234. Kaul, B., and E.J. Staba. 1968. Dioscorea tissue cultures. 1. Biosynthesis and isolation of diesgenin

from Dioscorea deltoidea callus and suspension cells. Lloydia. 31:171-179. Kennedy, M.J. 1991. The evolution of the word ‘biotechnology’. Trends Biotechnol. 9:218-220. Kevers, C., T. Franck, R.J. Strasser, J. Dommes, and T. Gaspar. 2004. Hyperhydricity of

Micropropagated Shoots: A Typically Stress-induced Change of Physiological State. Plant Cell, Tissue and Organ Culture. 77:181-191.

Khoudi, H., S. Laberge, J.-M. Ferullo, R. Bazin, A. Darveau, Y. Castonguay, G. Allard, R. Lemieux, and L.-P. Vézina. 1999. Production of a diagnostic monoclonal antibody in perennial alfalfa plants. Biotechnol. Bioeng. 64:135-143.

181

Kieran, P., D. Malone, and P. MacLoughlin. 2000. Effects of Hydrodynamic and Interfacial Forces on Plant Cell Suspension Systems. Adv. Biochem. Eng. Biotechnol. 67:139-177.

Kieran, P.M., P.F. MacLoughlin, and D.M. Malone. 1997. Plant cell suspension cultures: some engineering considerations. Journal of Biotechnology. 59:39-52.

Kim, D.-I., H. Pedersen, and C.-K. Chin. 1991. Development of process strategies for berberine production in plant cell suspension cultures. Journal of Biotechnology. 21:201-207.

Kim, M., D. Christopher, and J. Mullet. 1993. Direct evidence for selective modulation of psbA, rpoA, rbcL and 16S RNA stability during barley chloroplast development. Plant Mol.Biol. 22:447-463.

Kim, M., and J. Park. 2002. High Frequency Plant Regeneration of Garlic (Allium sativum L.) Calli Immobilized in Calcium Alginate Gel. Biotechnology and Bioprocess Engineering. 7:206-211.

Klein, R., and J. Mullet. 1987. Control of gene expression during higher plant chloroplast biogenesis. Protein synthesis and transcript levels of psbA, psaA-psaB, and rbcL in dark-grown and illuminated barley seedlings. Journal of Biological Chemistry. 262:4341-4348.

Klingenberg, P. 1984. A. T. Bull, G. Holt und M. D. Lilly: Biotechnology — international trends and perspectives. 84 Seiten. OECD Publ., Paris 1982. Preis: 28,— DM. Food / Nahrung. 28:900-900.

Klughammer, C., and U. Schreiber. 1994. An improved method, using saturating light pulses, for the determination of photosystem I quantum yield via P700+-absorbance changes at 830 nm. Planta. 192:261-268.

Knoblauch, M., J.M. Hibberd, J.C. Gray, and A.J.E. van Bel. 1999. A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes. Nat Biotech. 17:906-909.

Ko, K., M.H. Ahn, M. Song, Y.K. Choo, H.S. Kim, K. Ko, and H. Joung. 2008. Glyco-engineering of biotherapeutic proteins in plants. Molecules and cells. 25:494-503.

Ko, K., R. Brodzik, and Z. Steplewski. 2009. Production of Antibodies in Plants: Approaches and Perspectives. In Plant-produced Microbial Vaccines. Vol. 332. A. Karasev, editor. Springer Berlin Heidelberg. 55-78.

Ko, K., and H. Koprowski. 2005. Plant biopharming of monoclonal antibodies. Virus Research. 111:93-100.

Ko, K., Y. Tekoah, P.M. Rudd, D.J. Harvey, R.A. Dwek, S. Spitsin, C.A. Hanlon, C. Rupprecht, B. Dietzschold, M. Golovkin, and H. Koprowski. 2003. Function and glycosylation of plant-derived antiviral monoclonal antibody. Proceedings of the National Academy of Sciences. 100:8013-8018.

Koch, K.E. 1996. Carbohydrate-modulated gene expression in plants. Annu. Rev. Plant Physiol. Plant Molec. Biol. 47:509-540.

Kofer, W., C. Eibl, K. Steinmüller, and H.-U. Koop. 1998. PEG-mediated plastid transformation in higher plants. In Vitro Cellular & Developmental Biology - Plant. 34:303-309.

Kohler, G., and C. Milstein. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 256:495-497.

Koprowski, H. 2005. Vaccines and sera through plant biotechnology. Vaccine. 23:1757-1763. Kota, M., H. Daniell, S. Varma, S.F. Garczynski, F. Gould, and W.J. Moar. 1999. Overexpression of the

Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proceedings of the National Academy of Sciences. 96:1840-1845.

Kovtun, Y., and J. Daie. 1995. End-Product Control of Carbon Metabolism in Culture-grown Sugar Beet Plants (Molecular and Physiological Evidence on Accelerated Leaf Development and Enhanced Gene Expression). Plant Physiol. 108:1647-1656.

Koya, V., M. Moayeri, S.H. Leppla, and H. Daniell. 2005. Plant-Based Vaccine: Mice Immunized with Chloroplast-Derived Anthrax Protective Antigen Survive Anthrax Lethal Toxin Challenge. Infection and Immunity. 73:8266-8274.

182

Kozai, T. 1991. Photoautotrophic micropropagation. In Vitro Cellular & Developmental Biology - Plant. 27:47-51.

Kozai, T., K. Iwabuchi, K. Watanabe, and I. Watanabe. 1991. Photoautotrophic and photomixotrophic growth of strawberry plantlets in vitro and changes in nutrient composition of the medium. Plant Cell Tissue Organ Cult. 25:107-115.

Kozai, T., and C. Kubota. 2001. Developing a photoautotrophic micropropagation system for woody plants. J. Plant Res. 114:525-537.

Kubota, C., M. Ezawa, T. Kozai, and S.B. Wilson. 2002. In Situ Estimation of Carbon Balance of In Vitro Sweet Potato and Tomato Plantlets Cultured with Varying Initial Sucrose Concentrations in the Medium. Journal of the American Society for Horticultural Science. 127:963-970.

Kumar, M.B.A., V. Vakeswaran, and V. Krishnasamy. 2005. Enhancement of synthetic seed conversion to seedlings in hybrid rice. Plant Cell, Tissue and Organ Culture. 81:97-100.

Kumar, S., A. Dhingra, and H. Daniell. 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., F.M. Hahn, E. Baidoo, T.S. Kahlon, D.F. Wood, C.M. McMahan, K. Cornish, J.D. Keasling, H. Daniell, and M.C. Whalen. 2012. Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts. Metabolic engineering. 14:19-28.

Kumar Tewari, R., P. Kumar, and P. Nand Sharma. 2006. Magnesium deficiency induced oxidative stress and antioxidant responses in mulberry plants. Scientia horticulturae. 108:7-14.

Kumar, V., M.M. Naidu, and G.A. Ravishankar. 2006. Developments in coffee biotechnology - in vitro plant propagation and crop improvement. Plant Cell Tissue Organ Cult. 87:49-65.

Kuroda, H., and P. Maliga. 2001a. Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs. Nucleic Acids Res. 29:970-975.

Kuroda, H., and P. Maliga. 2001b. Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts. Plant Physiol. 125:430-436.

Kusnadi, A.R., Z.L. Nikolov, and J.A. Howard. 1997. Production of recombinant proteins in transgenic plants: Practical considerations. Biotechnol. Bioeng. 56:473-484.

Kutschera, U., and K.J. Niklas. 2013. Cell division and turgor-driven stem elongation in juvenile plants: A synthesis. Plant Science. 207:45-56.

Kuystermans, D., B. Krampe, H. Swiderek, and M. Al-Rubeai. 2007. Using cell engineering and omic tools for the improvement of cell culture processes. Cytotechnology. 53:3-22.

Kwok, K.H., P. Tsoulpha, and P.M. Doran. 1992. Limitations associated with conductivity measurement for monitoring growth in plant tissue culture. Plant Cell, Tissue and Organ Culture. 29:93-99.

Kwon, J.Y., Y.S. Yang, S.H. Cheon, H.J. Nam, G.H. Jin, and D.I. Kim. 2013. Bioreactor engineering using disposable technology for enhanced production of hCTLA4Ig in transgenic rice cell cultures. Biotechnol Bioeng. 110:2412-2424.

Ladygin, V., N. Bondarev, G. Semenova, A. Smolov, O. Reshetnyak, and A. Nosov. 2008. Chloroplast ultrastructure, photosynthetic apparatus activities and production of steviol glycosides in Stevia rebaudiana in vivo and in vitro. Biol. Plant. 52:9-16.

Lai, H., and Q. Chen. 2012. Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations. Plant Cell Reports. 31:573-584.

Lau, O.S., and S.S.M. Sun. 2009. Plant seeds as bioreactors for recombinant protein production. Biotechnol. Adv. 27:1015-1022.

Leader, B., Q.J. Baca, and D.E. Golan. 2008. Protein therapeutics: a summary and pharmacological classification. Nat Rev Drug Discov. 7:21-39.

183

Lee, M., and Y. Yang. 2006. Transient Expression Assay by Agroinfiltration of Leaves. In Arabidopsis Protocols. Vol. 323. J. Salinas and J. Sanchez-Serrano, editors. Humana Press. 225-229.

Lee, S.M., K. Kang, H. Chung, S.H. Yoo, X.M. Xu, S.B. Lee, J.J. Cheong, H. Daniell, and M. Kim. 2006. Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny. Molecules and cells. 21:401-410.

Lico, C., A. Desiderio, S. Banchieri, and E. Benvenuto. 2005. Plants as biofactories: Production of pharmaceutical recombinant proteins. In Proceedings of the International Congress “In the Wake of the Double Helix: From the Green Revolution to the Gene Revolution. 577-593.

Lim, S., J. Seon, K. Paek, B. Han, and S. Son. 1997. Development of pilot scale process for mass production of Lilium bulblets in vitro. In International Symposium on Biotechnology of Tropical and Subtropical Species Part 2 461. 237-242.

Lin, M.T., A. Occhialini, P.J. Andralojc, M.A. Parry, and M.R. Hanson. 2014. A faster Rubisco with potential to increase photosynthesis in crops. Nature. 513:547-550.

Liu, C.W., C.C. Lin, J.J.W. Chen, and M.J. Tseng. 2007. Stable chloroplast transformation in cabbage (Brassica oleracea L. var. capitata L.) by particle bombardment. Plant Cell Reports. 26:1733-1744.

Liu, C.W., C.C. Lin, J.C. Yiu, J.J.W. Chen, and M.J. Tseng. 2008. Expression of a Bacillus thuringiensis toxin (cry1Ab) gene in cabbage (Brassica oleracea L. var. capitata L.) chloroplasts confers high insecticidal efficacy against Plutella xylostella. Theor. Appl. Genet. 117:75-88.

Loesche, W.J. 1986. Role of Streptococcus mutans in human dental decay. Microbiological Reviews. 50:353-380.

Lorenzo, J.C., B.L. Gonzalez, M. Escalona, C. Teisson, P. Espinosa, and C. Borroto. 1998. Sugarcane shoot formation in an improved temporary immersion system. Plant Cell Tissue Organ Cult. 54:197-200.

Lossl, A., K. Bohmert, H. Harloff, C. Eibl, S. Muhlbauer, and H.U. Koop. 2005. Inducible trans-activation of plastid transgenes: Expression of the R. eutropha phb operon in transplastomic tobacco. Plant and Cell Physiology. 46:1462-1471.

Lutz, K.A., A. Azhagiri, and P. Maliga. 2011. Transplastomics in Arabidopsis: Progress Toward Developing an Efficient Method. In Chloroplast Research in Arabidopsis. Vol. 774. R.P. Jarvis, editor. Humana Press. 133-147.

Lutz, K.A., J.E. Knapp, and P. Maliga. 2001. Expression of bar in the plastid genome confers herbicide resistance. Plant Physiol. 125:1585-1590.

Lymbery, P. 2014. Farmageddon: The True Cost of Cheap Meat. Bloomsbury Publishing. Lynd, L.R., C.E. Wyman, and T.U. Gerngross. 1999. Biocommodity Engineering. Biotechnology

Progress. 15:777-793. Ma, J.K.C., E. Barros, R. Bock, P. Christou, P.J. Dale, P.J. Dix, R. Fischer, J. Irwin, R. Mahoney, M.

Pezzotti, S. Schillberg, P. Sparrow, E. Stoger, and R.M. Twyman. 2005a. Molecular farming for new drugs and vaccines. EMBO reports. 6:593-599.

Ma, J.K.C., P. Christou, R. Chikwamba, H. Haydon, M. Paul, M.P. Ferrer, S. Ramalingam, E. Rech, E. Rybicki, A. Wigdorowitz, D.-C. Yang, and H. Thangaraj. 2013. Realising the value of plant molecular pharming to benefit the poor in developing countries and emerging economies. Plant Biotechnol. J. 11:1029-1033.

Ma, J.K.C., P.M.W. Drake, D. Chargelegue, P. Obregon, and A. Prada. 2005b. Antibody processing and engineering in plants, and new strategies for vaccine production. Vaccine. 23:1814-1818.

Ma, J.K.C., and M.B. Hein. 1995. Immunotherapeutic potential of antibodies produced in plants. Trends Biotechnol. 13:522-527.

Ma, J.K.C., B.Y. Hikmat, K. Wycoff, N.D. Vine, D. Chargelegue, L. Yu, M.B. Hein, and T. Lehner. 1998. Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nat Med. 4:601-606.

184

Ma, J.K.C., T. Lehner, P. Stabila, C.I. Fux, and A. Hiatt. 1994. Assembly of monoclonal antibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants. European Journal of Immunology. 24:131-138.

Ma, Z., C. Cooper, H.-J. Kim, and D. Janick-Buckner. 2009. A Study of Rubisco through Western Blotting and Tissue Printing Techniques. CBE Life Sciences Education. 8:140-146.

MacLoughlin, P.F., D.M. Malone, J.T. Murtagh, and P.M. Kieran. 1998. The effects of turbulent jet flows on plant cell suspension cultures. Biotechnol. Bioeng. 58:595-604.

Magy, B., J. Tollet, R. Laterre, M. Boutry, and C. Navarre. 2014. Accumulation of secreted antibodies in plant cell cultures varies according to the isotype, host species and culture conditions. Plant Biotechnol. J. 12:457-467.

Majumdar, S.R. 1996. Pneumatic Systems: Principles and Maintenance. McGraw-Hill. Makoff, A.J., S.P. Ballantine, A.E. Smallwood, and N.F. Fairweather. 1989. Expression of Tetanus

Toxin Fragment C in E. coli: Its Purification and Potential Use as a Vaccine. Nat Biotech. 7:1043-1046.

Maliga, P. 2002. Engineering the plastid genome of higher plants. Curr Opin Plant Biol. 5:164-172. Maliga, P. 2003. Progress towards commercialization of plastid transformation technology. Trends

Biotechnol. 21:20-28. Maliga, P. 2004. Plastid transformation in higher plants. Annual Review of Plant Biology. 55:289-313. Maliga, P., and R. Bock. 2011. Plastid Biotechnology: Food, Fuel, and Medicine for the 21st Century.

Plant Physiol. 155:1501-1510. Martin, S.M., and D. Rose. 1976. Growth of plant cell (Ipomoea) suspension cultures at controlled pH

levels. Canadian Journal of Botany. 54:1264-1270. Martin, W., T. Rujan, E. Richly, A. Hansen, S. Cornelsen, T. Lins, D. Leister, B. Stoebe, M. Hasegawa,

and D. Penny. 2002. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc. Natl. Acad. Sci. U. S. A. 99:12246-12251.

Martine, G., H. Philippe, and S. Myriam. 2009. Biosafety considerations associated with molecular farming in genetically modified plants. Journal of Medicinal Plants Research. 3:825-838.

Masclaux-Daubresse, C., F. Daniel-Vedele, J. Dechorgnat, F. Chardon, L. Gaufichon, and A. Suzuki. 2010. Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Annals of Botany. 105:1141-1157.

Mason, H.S., H. Warzecha, T. Mor, and C.J. Arntzen. 2002. Edible plant vaccines: applications for prophylactic and therapeutic molecular medicine. Trends in Molecular Medicine. 8:324-329.

Maxwell, K., and G.N. Johnson. 2000. Chlorophyll fluorescence—a practical guide. Journal of Experimental Botany. 51:659-668.

McCabe, M.S., M. Klaas, N. Gonzalez-Rabade, M. Poage, J.A. Badillo-Corona, F. Zhou, D. Karcher, R. Bock, J.C. Gray, and P.J. Dix. 2008. Plastid transformation of high-biomass tobacco variety Maryland Mammoth for production of human immunodeficiency virus type 1 (HIV-1) p24 antigen. Plant Biotechnol. J. 6:914-929.

McDonald, K.A., and A.P. Jackman. 1989. Bioreactor studies of growth and nutrient utilization in alfalfa suspension cultures. Plant Cell Reports. 8:455-458.

Meijer, J.J., H.J.G. ten Hoopen, K.C.A.M. Luyben, and K.R. Libbenga. 1993. Effects of hydrodynamic stress on cultured plant cells: A literature survey. Enzyme and Microbial Technology. 15:234-238.

Menke, W. 1962. Structure and Chemistry of Plastids. Annual Review of Plant Physiology. 13:27-44. Menkhaus, T.J., Y. Bai, C. Zhang, Z.L. Nikolov, and C.E. Glatz. 2004. Considerations for the Recovery of

Recombinant Proteins from Plants. Biotechnology Progress. 20:1001-1014. Menkhaus, T.J., and C.E. Glatz. 2005. Antibody Capture from Corn Endosperm Extracts by Packed

Bed and Expanded Bed Adsorption. Biotechnology Progress. 21:473-485. Merchuk, J. 1991. Shear effects on suspended cells. In Bioreactor Systems and Effects. Vol. 44.

Springer Berlin Heidelberg. 65-95.

185

Merlin, M., E. Gecchele, S. Capaldi, M. Pezzotti, and L. Avesani. 2014. Comparative Evaluation of Recombinant Protein Production in Different Biofactories: The Green Perspective. BioMed research international. 2014.

Metz, B., W. Tilstra, R. van der Put, N. Spruit, J. van den Ijssel, J. Robert, C. Hendriksen, and G. Kersten. 2013. Physicochemical and immunochemical assays for monitoring consistent production of tetanus toxoid. Biologicals. 41:231-237.

Meyer, C., and M. Stitt. 2001. Nitrate Reduction and signalling. In Plant Nitrogen. P. Lea and J.-F. Morot-Gaudry, editors. Springer Berlin Heidelberg. 37-59.

Meyers, A., E. Chakauya, E. Shephard, F.L. Tanzer, J. Maclean, A. Lynch, A.-L. Williamson, and E.P. Rybicki. 2008. Expression of HIV-1 antigens in plants as potential subunit vaccines. BMC Biotechnol.

Michoux, F., N. Ahmad, A. Hennig, P. Nixon, and H. Warzecha. 2013. Production of leafy biomass using temporary immersion bioreactors: an alternative platform to express proteins in transplastomic plants with drastic phenotypes. Planta. 237:903-908.

Michoux, F., N. Ahmad, J. McCarthy, and P.J. Nixon. 2011. Contained and high-level production of recombinant protein in plant chloroplasts using a temporary immersion bioreactor. Plant Biotechnol. J. 9:575-584.

Mishra, N., P.N. Gupta, K. Khatri, A.K. Goyal, and S.P. Vyas. 2008. Edible vaccines: A new approach to oral immunization. Indian J Biotechnol. 7:283-294.

Miyazaki, J., B. Tan, and S. Errington. 2010. Eradication of endophytic bacteria via treatment for axillary buds of Petunia hybrida using Plant Preservative Mixture (PPMTM). Plant Cell Tiss Organ Cult. 102:365-372.

Møller, S.G., J. Maple, and D. Gargano. 2014. Biogenesis of Chloroplasts. In The Structural Basis of Biological Energy Generation. Springer. 435-449.

Moon, K.H., H. Honda, and T. Kobayashi. 1999. Development of a bioreactor suitable for embryogenic rice callus culture. Journal of Bioscience and Bioengineering. 87:661-665.

Mooney, B.P. 2009. The second green revolution? Production of plant-based biodegradable plastics. The Biochemical journal. 418:219-232.

Mooney, M. 1951. The viscosity of a concentrated suspension of spherical particles. Journal of Colloid Science. 6:162-170.

Mordocco, A.M., J.A. Brumbley, and P. Lakshmanan. 2009. Development of a temporary immersion system (RITA) for mass production of sugarcane (Saccharum spp. interspecific hybrids). In Vitro Cell. Dev. Biol.-Plant. 45:450-457.

Morikawa, Y., D.J. Hockley, M.V. Nermut, and I.M. Jones. 2000. Roles of matrix, p2, and N-terminal myristoylation in human immunodeficiency virus type 1 Gag assembly. J Virol. 74:16-23.

Munetaka, S. 1999. Organogenesis in vitro. Current Opinion in Plant Biology. 2:61-64. Munro, G.H., P. Evans, S. Todryk, P. Buckett, C.G. Kelly, and T. Lehner. 1993. A protein fragment of

streptococcal cell surface antigen I/II which prevents adhesion of Streptococcus mutans. Infection and Immunity. 61:4590-4598.

Murashige, T. 1977. Plant cell and organ cultures as horticultural practices. In Symposium on Tissue Culture for Horticultural Purposes 78. 17-30.

Murashige, T., and F. Skoog. 1962. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum. 15:473-497.

Murthy, B.N.S., S.J. Murch, and P. Saxena. 1998. Thidiazuron: A potent regulator of in vitro plant morphogenesis. In Vitro Cellular & Developmental Biology - Plant. 34:267-275.

Nagakubo, T., A. Nagasawa, and H. Ohkawa. 1993. Micropropagation of garlic through in vitro bulblet formation. Plant Cell, Tissue and Organ Culture. 32:175-183.

Nagamori, E., H. Honda, and T. Kobayashi. 1999. Release of embryogenic carrot cells with high regeneration potency from immobilized alginate beads. Journal of Bioscience and Bioengineering. 88:226-228.

186

Nagels, B., K. Weterings, N. Callewaert, and E.J.M. Van Damme. 2012. Production of Plant Made Pharmaceuticals: From Plant Host to Functional Protein. Crit. Rev. Plant Sci. 31:148-180.

Naik, S.K., and P.K. Chand. 2006. Nutrient-alginate encapsulation of in vitro nodal segments of pomegranate (Punica granatum L.) for germplasm distribution and exchange. Scientia Horticulturae. 108:247-252.

Najafpour, G.D. 2006. Biochemical Engineering and Biotechnology. Elsevier. 294. Navarro, G.E.Z., A.E.S. Honorato, A.G.C. Oyarzun, E.A.T. Rodriguez, H.G.P. Encalada, and P.A.Z.

Cantillana. 2011. Thermo-photo-bioreactor and method for the culture and mass micropropagation of Deschampsia antarctica in vitro. Google Patents.

Neales, T.F., and L.D. Incoll. 1968. The control of leaf photosynthesis rate by the level of assimilate concentration in the leaf: A review of the hypothesis. Bot. Rev. 34:107-125.

Nelson, D.L., A.L. Lehninger, and M.M. Cox. 2008. Lehninger principles of biochemistry. Macmillan. Neve, M., M. Loose, A. Jacobs, H. Houdt, B. Kaluza, U. Weidle, M. Montagu, and A. Depicker. 1993.

Assembly of an antibody and its derived antibody fragment in Nicotiana and Arabidopsis. Transgenic Res. 2:227-237.

Nguyen, T.T., G. Nugent, T. Cardi, and P.J. Dix. 2005. Generation of homoplasmic plastid transformants of a commercial cultivar of potato (Solanum tuberosum L.). Plant Science. 168:1495-1500.

Nhut, D.T., T. Takamura, H. Watanabe, K. Okamoto, and M. Tanaka. 2003. Responses of strawberry plantlets cultured in vitro under superbright red and blue light-emitting diodes (LEDs). Plant Cell, Tissue and Organ Culture. 73:43-52.

Nicholson, L., P. Gonzalez-Melendi, C. Van Dolleweerd, H. Tuck, Y. Perrin, J.K.C. Ma, R. Fischer, P. Christou, and E. Stoger. 2005. A recombinant multimeric immunoglobulin expressed in rice shows assembly-dependent subcellular localization in endosperm cells. Plant Biotechnol. J. 3:115-127.

Niedz, R.P. 1998. Using Isothiazolone Biocides to Control Microbial and Fungal Contaminants in Plant Tissue Cultures. HortTechnology. 8:598-601.

Niedz, R.P., and M.G. Bausher. 2002. Control of in vitro contamination of explants from greenhouse- and field-grown trees. In Vitro Cellular & Developmental Biology - Plant. 38:468-471.

Nikolov, Z., and D. Hammes. 2002. Production of recombinant proteins from transgenic crops. In Plants as Factories for Protein Production. Springer. 159-174.

Nikolov, Z.L., J.T. Regan, L.F. Dickey, and S.L. Woodard. 2008. Purification of Antibodies From Transgenic Plants. In Process Scale Purification of Antibodies. John Wiley & Sons, Inc. 387-406.

Nikolov, Z.L., and S.L. Woodard. 2004. Downstream processing of recombinant proteins from transgenic feedstock. Current Opinion in Biotechnology. 15:479-486.

North, J.J., P.A. Ndakidemi, and C.P. Laubscher. 2011. Effects of various media compositions on the in vitro germination and discoloration of immature embryos of bird of paradise (Strelitzia reginae). Plant Omics. 4:100-113.

Novitsky, V., P. Gilbert, T. Peter, M.F. McLane, S. Gaolekwe, N. Rybak, I. Thior, T. Ndung'u, R. Marlink, T.H. Lee, and M. Essex. 2003. Association between Virus-Specific T-Cell Responses and Plasma Viral Load in Human Immunodeficiency Virus Type 1 Subtype C Infection. Journal of Virology. 77:882-890.

Novitsky, V., N. Rybak, M.F. McLane, P. Gilbert, P. Chigwedere, I. Klein, S. Gaolekwe, S.Y. Chang, T. Peter, I. Thior, T. Ndung'u, F. Vannberg, B.T. Foley, R. Marlink, T.H. Lee, and M. Essex. 2001. Identification of Human Immunodeficiency Virus Type 1 Subtype C Gag-, Tat-, Rev-, and Nef-Specific Elispot-Based Cytotoxic T-Lymphocyte Responses for AIDS Vaccine Design. Journal of Virology. 75:9210-9228.

Nugent, G.D., S. Coyne, T.T. Nguyen, T.A. Kavanagh, and P.J. Dix. 2006. Nuclear and plastid transformation of Brassica oleracea var. botrytis (cauliflower) using PEG-mediated uptake of DNA into protoplasts. Plant Science. 170:135-142.

187

Obembe, O.O., J.O. Popoola, S. Leelavathi, and S.V. Reddy. 2011. Advances in plant molecular farming. Biotechnol. Adv. 29:210-222.

Oey, M., M. Lohse, B. Kreikemeyer, and R. Bock. 2009. Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. Plant J. 57:436-445.

Ohtani, T., G. Galili, J. Wallace, G. Thompson, and B. Larkins. 1991. Normal and lysine-containing zeins are unstable in transgenic tobacco seeds. Plant Mol.Biol. 16:117-128.

Okamoto, K., T. Yanagi, S. Takita, M. Tanaka, T. Higuchi, Y. Ushida, and H. Watanabe. 1996. Development of plant growth apparatus using blue and red LED as artificial light source. In International Symposium on Plant Production in Closed Ecosystems 440. 111-116.

Olmos, E., and E. Hellın. 1998. Ultrastructural differences of hyperhydric and normal leaves from regenerated carnation plants. Scientia Horticulturae. 75:91-101.

Olmos, E., A. Piqueras, J. Ramón Martınez-Solano, and E. Hellın. 1997. The subcellular localization of peroxidase and the implication of oxidative stress in hyperhydrated leaves of regenerated carnation plants. Plant Science. 130:97-105.

Onishi, N., Y. Sakamoto, and T. Hirosawa. 1994. Synthetic seeds as an application of mass production of somatic embryos. Plant Cell Tissue Organ Cult. 39:137-145.

Onodera, R. 1993. Methionine and lysine metabolism in the rumen and the possible effects of their metabolites on the nutrition and physiology of ruminants. Amino Acids. 5:217-232.

Orenstein, W.A., W. Atkinson, D. Mason, and R.H. Bernier. 1990. Barriers to vaccinating preschool children. Journal of health care for the poor and underserved. 1:315-330.

Osteryoung, K.W., and K.A. Pyke. 2014. Division and Dynamic Morphology of Plastids. Annual Review of Plant Biology. 65:443-472.

Outchkourov, N.S., B. Rogelj, B. Strukelj, and M.A. Jongsma. 2003. Expression of Sea Anemone Equistatin in Potato. Effects of Plant Proteases on Heterologous Protein Production. Plant Physiol. 133:379-390.

Ovečka, M., M. Bobák, and J. Šamaj. 1997. Development of shoot primordia in tissue culture of Papaver somniferum L. Biol. Plant. 39:499-506.

Owen, H.R., D. Wengerd, and A.R. Miller. 1991. Culture medium pH is influenced by basal medium, carbohydrate source, gelling agent, activated-charcoal, and medium storage method. Plant Cell Reports. 10:583-586.

Palmer, J.D. 1983. Chloroplast DNA exists in two orientations. Nature. 301:92-93. Palmer, J.D. 1985. Comparative organization of chloroplast genomes. Annu. Rev. Genet. 19:325-354. Parashar, U.D., C.J. Gibson, J.S. Bresee, and R.I. Glass. 2006. Rotavirus and severe childhood diarrhea.

Emerging infectious diseases. 12:304-306. Park, Y.-G., S.-J. Kim, Y.-M. Kang, H.-Y. Jung, D. Prasad, S.-W. Kim, Y.-G. Chung, and M.-S. Choi. 2004a.

Production of ginkgolides and bilobalide from optimized the <i>Ginkgo biloba</i> cell culture. Biotechnology and Bioprocess Engineering. 9:41-46.

Park, Y.-G., S.-J. Kim, Y.-M. Kang, H.-Y. Jung, D. Prasad, S.-W. Kim, Y.-G. Chung, and M.-S. Choi. 2004b. Production of ginkgolides and bilobalide from optimized the Ginkgo biloba cell culture. Biotechnology and Bioprocess Engineering. 9:41-46.

Parmenter, D.L., J.G. Boothe, and M.M. Moloney. 1996. Production and Purification of Recombinant Hirudin from Plant Seeds. Transgenic plants: a production system for industrial and pharmaceutical proteins:261.

Parra, G., K. Bradnam, A.B. Rose, and I. Korf. 2011. Comparative and functional analysis of intron-mediated enhancement signals reveals conserved features among plants. Nucleic Acids Res. 39:5328-5337.

Patel, A.V., I. Pusch, G. Mix-Wagner, and K.D. Vorlop. 2000. A novel encapsulation technique for the production of artificial seeds. Plant Cell Reports. 19:868-874.

Paul, M.J., and T.K. Pellny. 2003. Carbon metabolite feedback regulation of leaf photosynthesis and development. Journal of Experimental Botany. 54:539-547.

188

Percy, J.R., M.E. Percy, and R. Baumal. 1976. Assembly of three major subclasses of mouse immunoglobulin G: a theoretical model for covalent assembly in vivo. Can J Biochem. 54:688-698.

Perveen, S., and M. Anis. 2014. Encapsulation of internode regenerated adventitious shoot buds of Indian Siris in alginate beads for temporary storage and twofold clonal plant production. Acta Physiol. Plant. 36:2067-2077.

Petrović, A. 2013. Effect of nutrient starvation on some aspects of nitrogen metabolism in substrate-grown strawberry plantings cv. Nyoho. Ratarstvo i povrtarstvo. 50:24-30.

Petruccelli, S., M.S. Otegui, F. Lareu, O. Tran Dinh, A.C. Fitchette, A. Circosta, M. Rumbo, M. Bardor, R. Carcamo, and V. Gomord. 2006. A KDEL‐tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds. Plant Biotechnol. J. 4:511-527.

Piccioni, E. 1997. Plantlets from encapsulated micropropagated buds of M.26 apple rootstock. Plant Cell, Tissue and Organ Culture. 47:255-260.

Pickering, F., and P. Reis. 1993. Effects of abomasal supplements of methionine on wool growth of grazing sheep. Australian Journal of Experimental Agriculture. 33:7-12.

Picoli, E.A.T., W.C. Otoni, M.r.L. Figueira, S.M.B. Carolino, R.S. Almeida, E.A.M. Silva, C.R. Carvalho, and E.P.B. Fontes. 2001. Hyperhydricity in in vitro eggplant regenerated plants: structural characteristics and involvement of BiP (Binding Protein). Plant Science. 160:857-868.

Pillay, V., H.K. Gan, and A.M. Scott. 2011. Antibodies in oncology. New Biotechnology. 28:518-529. Pogue, G.P., J.A. Lindbo, S.J. Garger, and W.P. Fitzmaurice. 2002. Making an ally from an enemy:

Plant Virology and the New Agriculture. Annual Review of Phytopathology. 40:45-74. Popoff, M.R. 1995. Ecology of Neurotoxigenic Strains of Clostridia. In Clostridial Neurotoxins. Vol.

195. C. Montecucco, editor. Springer Berlin Heidelberg. 1-29. Pospóšilová, J., I. Tichá, P. Kadleček, D. Haisel, and Š. Plzáková. 1999. Acclimatization of

Micropropagated Plants to Ex Vitro Conditions. Biol. Plant. 42:481-497. Potenza, C., L. Aleman, and C. Sengupta-Gopalan. 2004. Targeting transgene expression in research,

agricultural, and environmental applications: Promoters used in plant transformation. In Vitro Cellular & Developmental Biology - Plant. 40:1-22.

Qiu, X., G. Wong, J. Audet, A. Bello, L. Fernando, J.B. Alimonti, H. Fausther-Bovendo, H. Wei, J. Aviles, E. Hiatt, A. Johnson, J. Morton, K. Swope, O. Bohorov, N. Bohorova, C. Goodman, D. Kim, M.H. Pauly, J. Velasco, J. Pettitt, G.G. Olinger, K. Whaley, B. Xu, J.E. Strong, L. Zeitlin, and G.P. Kobinger. 2014. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature. advance online publication.

Rafiqul, M., I. Khan, A. Ceriotti, L. Tabe, A. Aryan, W. McNabb, A. Moore, S. Craig, D. Spencer, and T.V. Higgins. 1996. Accumulation of a sulphur-rich seed albumin from sunflower in the leaves of transgenic subterranean clover (Trifolium subterraneum L.). Transgenic Res. 5:179-185.

Ragauskas, A.J., C.K. Williams, B.H. Davison, G. Britovsek, J. Cairney, C.A. Eckert, W.J. Frederick, J.P. Hallett, D.J. Leak, C.L. Liotta, J.R. Mielenz, R. Murphy, R. Templer, and T. Tschaplinski. 2006. The Path Forward for Biofuels and Biomaterials. Science. 311:484-489.

Rai, M.K., V.S. Jaiswal, and U. Jaiswal. 2008. Encapsulation of shoot tips of guava (Psidium guajava L.) for short-term storage and germplasm exchange. Scientia Horticulturae. 118:33-38.

Ramage, C.M., and R.R. Williams. 2002. Inorganic nitrogen requirements during shoot organogenesis in tobacco leaf discs. Journal of Experimental Botany. 53:1437-1443.

Ramírez, N., P. Oramas, M. Ayala, M. Rodríguez, M. Pérez, and J. Gavilondo. 2001. Expression and long-term stability of a recombinant single-chain Fv antibody fragment in transgenic Nicotiana tabacum seeds. Biotechnol. Lett. 23:47-49.

Raven, J.A., B. Wollenweber, and L.L. Handley. 1992. A Comparison of Ammonium and Nitrate as Nitrogen Sources for Photolithotrophs. New Phytologist. 121:19-32.

Rawlings, N.D., F.R. Morton, C.Y. Kok, J. Kong, and A.J. Barrett. 2008. MEROPS: the peptidase database. Nucleic Acids Res. 36:D320-D325.

189

Redenbaugh, K., J.A.A. Fujii, and D. Slade. 1993. Hydrated coatings for synthetic seeds. 35-46 pp. Reichert, J.M. 2014. Antibodies to watch in 2014: Mid-year update. mAbs. 6:799-802. Reichert, J.M., C.J. Rosensweig, L.B. Faden, and M.C. Dewitz. 2005. Monoclonal antibody successes in

the clinic. Nat Biotech. 23:1073-1078. Rennels, M.B. 2000. The Rotavirus Vaccine Story: A Clinical Investigator's View. Pediatrics. 106:123-

125. Reuter, L.J., M.J. Bailey, J.J. Joensuu, and A. Ritala. 2014. Scale-up of hydrophobin-assisted

recombinant protein production in tobacco BY-2 suspension cells. Plant Biotechnol. J. 12:402-410.

Richter, A.K., E. Frossard, and I. Brunner. 2007. Polyphenols in the woody roots of Norway spruce and European beech reduce TTC. Tree Physiol. 27:155-160.

Richter, L.J., Y. Thanavala, C.J. Arntzen, and H.S. Mason. 2000. Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat Biotech. 18:1167-1171.

Riou-Khamlichi, C., M. Menges, J.M.S. Healy, and J.A.H. Murray. 2000. Sugar Control of the Plant Cell Cycle: Differential Regulation of Arabidopsis D-Type Cyclin Gene Expression. Molecular and Cellular Biology. 20:4513-4521.

Ritala, A., S.T. Häkkinen, and S. Schillberg. 2014. Molecular pharming in plants and plant cell cultures: a great future ahead? Pharmaceutical Bioprocessing. 2:223-226.

Rivas, J.D.L., J.J. Lozano, and A.R. Ortiz. 2002. Comparative Analysis of Chloroplast Genomes: Functional Annotation, Genome-Based Phylogeny, and Deduced Evolutionary Patterns. Genome Research. 12:567-583.

Rodrıguez-Monroy, M., and E. Galindo. 1999. Broth rheology, growth and metabolite production of Beta vulgaris suspension culture: a comparative study between cultures grown in shake flasks and in a stirred tank. Enzyme and Microbial Technology. 24:687-693.

Roels, S., C. Noceda, M. Escalona, J. Sandoval, M.J. Canal, R. Rodriguez, and P. Debergh. 2006. The effect of headspace renewal in a Temporary Immersion Bioreactor on plantain (Musa AAB) shoot proliferation and quality. Plant Cell Tissue Organ Cult. 84:155-163.

Rogers, G.L., A.M. Bryant, and L.M. McLeay. 1979. Silage and dairy cow production. New Zealand Journal of Agricultural Research. 22:533-541.

Roh, K.S., and B.Y. Choi. 2004. Sucrose regulates growth and activation of rubisco in tobacco leaves in vitro. Biotechnology and Bioprocess Engineering. 9:229-235.

Roitsch, T., and M.-C. González. 2004. Function and regulation of plant invertases: sweet sensations. Trends Plant Sci. 9:606-613.

Rojas-Martinez, L., R.G. Visser, and G.-J. de Klerk. 2010. The hyperhydricity syndrome: waterlogging of plant tissues as a major cause. Propag. Ornam. Plants. 10:169-175.

Ross, M.K., T.A. Thorpe, and J.W. Costerton. 1973. Ultrastructural Aspects of Shoot Initiation in Tobacco Callus Cultures. American Journal of Botany. 60:788-795.

Ruf, M., and I. Brunner. 2003. Vitality of tree fine roots: reevaluation of the tetrazolium test. Tree Physiol. 23:257-263.

Ruf, S., M. Hermann, I.J. Berger, H. Carrer, and R. Bock. 2001. Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotech. 19:870-875.

Ruf, S., D. Karcher, and R. Bock. 2007. Determining the transgene containment level provided by chloroplast transformation. Proceedings of the National Academy of Sciences. 104:6998-7002.

Ruhlman, T., R. Ahangari, A. Devine, M. Samsam, and H. Daniell. 2007. Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts - oral administration protects against development of insulitis in non-obese diabetic mice. Plant Biotechnol J. 5:495-510.

Ruhlman, T., and H. Daniell. 2007. Plastid Pathways. In Applications of Plant Metabolic Engineering. R. Verpoorte, A.W. Alfermann, and T.S. Johnson, editors. Springer Netherlands. 79-108.

Ruhlman, T., D. Verma, N. Samson, and H. Daniell. 2010. The Role of Heterologous Chloroplast Sequence Elements in Transgene Integration and Expression. Plant Physiol. 152:2088-2104.

190

Russell, S.M., and F.Y. Liew. 1980. Cell cooperation in antibody responses to influenza virus. I. Priming of helper T cells by internal components of the virion. European Journal of Immunology. 10:791-796.

Rybicki, E.P. 2009. Plant-produced vaccines: promise and reality. Drug Discovery Today. 14:16-24. Rybicki, E.P. 2010. Plant-made vaccines for humans and animals. Plant Biotechnol. J. 8:620-637. Sabalza, M., P. Christou, and T. Capell. 2014. Recombinant plant-derived pharmaceutical proteins:

current technical and economic bottlenecks. Biotechnol. Lett.:1-13. Sabir, J., E. Schwarz, N. Ellison, J. Zhang, N.A. Baeshen, M. Mutwakil, R. Jansen, and T. Ruhlman.

2014. Evolutionary and biotechnology implications of plastid genome variation in the inverted-repeat-lacking clade of legumes. Plant Biotechnol. J. 12:743-754.

Saint-Jore-Dupas, C., L. Faye, and V. Gomord. 2007. From planta to pharma with glycosylation in the toolbox. Trends Biotechnol. 25:317-323.

Sajc, L., D. Grubisic, and G. Vunjak-Novakovic. 2000. Bioreactors for plant engineering: an outlook for further research. Biochemical Engineering Journal. 4:89-99.

Sánchez Pérez, J.A., E.M. Rodríguez Porcel, J.L. Casas López, J.M. Fernández Sevilla, and Y. Chisti. 2006. Shear rate in stirred tank and bubble column bioreactors. Chemical Engineering Journal. 124:1-5.

Sang, Y., R.J. Millwood, and C. Neal Stewart Jr. 2013. Gene use restriction technologies for transgenic plant bioconfinement. Plant Biotechnol. J. 11:649-658.

Santana-Buzzy, N., R. Rojas-Herrera, R.M. Galaz-Avalos, J.R. Ku-Cauich, J. Mijangos-Cortes, L.C. Gutierrez-Pacheco, A. Canto, F. Quiroz-Figueroa, and V.M. Loyola-Vargas. 2007. Advances in coffee tissue culture and its practical applications. In Vitro Cell. Dev. Biol.-Plant. 43:507-520.

Saski, C., S.-B. Lee, H. Daniell, T. Wood, J. Tomkins, H.-G. Kim, and R. Jansen. 2005. Complete Chloroplast Genome Sequence of Glycine max and Comparative Analyses with other Legume Genomes. Plant Mol.Biol. 59:309-322.

Savangikar, V. 2004. Role of low cost options in tissue culture. Low Costs Options for Tissue Culture Technology in Developing Countries:11-15.

Scheidt, G., A. Silva, Y. Oliveira, J. Costa, L.A. Biasi, and C.R. Soccol. 2011. In vitro growth of Melaleuca alternifolia Cheel in bioreactor of immersion by bubbles. Pak. J. Bot. 43:2937-2939.

Scheidt, G.N., A.H. Arakaki, J.S. Chimilovski, A.C.F. Portella, M.R. Spier, A.L. Woiciechowski, L.A. Biasi, and C.R. Soccol. 2009. Utilization of the bioreactor of immersion by bubbles at the micropropagation of Ananas comosus L. Merril. Brazilian Archives of Biology and Technology. 52:37-43.

Schillberg, S., N. Emans, and R. Fischer. 2002. Antibody molecular farming in plants and plant cells. Phytochemistry Reviews. 1:45-54.

Schillberg, S., N. Raven, R. Fischer, R.M. Twyman, and A. Schiermeyer. 2013. Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des. 19:5531-5542.

Schnapp, S.R., and J.E. Preece. 1986. In vitro growth reduction of tomato and carnation microplants. Plant Cell Tissue Organ Cult. 6:3-8.

Schoberer, J., and R. Strasser. 2011. Sub-Compartmental Organization of Golgi-Resident N-Glycan Processing Enzymes in Plants. Molecular Plant. 4:220-228.

Schreiber, U., U. Schliwa, and W. Bilger. 1986. Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynth Res. 10:51-62.

Schroeder, H.W., Jr., and L. Cavacini. 2010. Structure and function of immunoglobulins. The Journal of allergy and clinical immunology. 125:S41-52.

Scragg, A.H., E.J. Allan, and F. Leckie. 1988. Effect of shear on the viability of plant cell suspensions. Enzyme and Microbial Technology. 10:361-367.

191

Sethuraman, N., and T.A. Stadheim. 2006. Challenges in therapeutic glycoprotein production. Current Opinion in Biotechnology. 17:341-346.

Shaaltiel, Y., D. Bartfeld, S. Hashmueli, G. Baum, E. Brill-Almon, G. Galili, O. Dym, S.A. Boldin-Adamsky, I. Silman, J.L. Sussman, A.H. Futerman, and D. Aviezer. 2007. Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system. Plant Biotechnol J. 5:579-590.

Shann, F., and M.C. Steinhoff. 1999. Vaccines for children in rich and poor countries. Lancet. 354 Suppl 2:Sii7-11.

Sharma, A.K., and M.K. Sharma. 2009. Plants as bioreactors: Recent developments and emerging opportunities. Biotechnol. Adv. 27:811-832.

Sharma, M.K., N.K. Singh, D. Jani, R. Sisodia, M. Thungapathra, J.K. Gautam, L.S. Meena, Y. Singh, A. Ghosh, A. Tyagi, and A. Sharma. 2008. Expression of toxin co-regulated pilus subunit A (TCPA) of Vibrio cholerae and its immunogenic epitopes fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum). Plant Cell Reports. 27:307-318.

Sharma, P., and R. Shanker Dubey. 2005. Modulation of nitrate reductase activity in rice seedlings under aluminium toxicity and water stress: role of osmolytes as enzyme protectant. J. Plant Physiol. 162:854-864.

Sharp, J.M., and P.M. Doran. 2001a. Characterization of monoclonal antibody fragments produced by plant cells. Biotechnol. Bioeng. 73:338-346.

Sharp, J.M., and P.M. Doran. 2001b. Strategies for Enhancing Monoclonal Antibody Accumulation in Plant Cell and Organ Cultures. Biotechnology Progress. 17:979-992.

Shaver, J.M., D.J. Oldenburg, and A.J. Bendich. 2006. Changes in chloroplast DNA during development in tobacco, Medicago truncatula, pea, and maize. Planta. 224:72-82.

Sheen, J. 1990. Metabolic repression of transcription in higher plants. The Plant Cell Online. 2:1027-1038.

Sheppard, A.E., M.A. Ayliffe, L. Blatch, A. Day, S.K. Delaney, N. Khairul-Fahmy, Y. Li, P. Madesis, A.J. Pryor, and J.N. Timmis. 2008. Transfer of plastid DNA to the nucleus is elevated during male gametogenesis in tobacco. Plant Physiol. 148:328-336.

Shimada, H., and M. Sugiura. 1991. Fine structural features of the chloroplast genome: comparison of the sequenced chloroplast genomes. Nucleic Acids Res. 19:983-995.

Shinozaki, K., M. Ohme, M. Tanaka, T. Wakasugi, N. Hayashida, T. Matsubayashi, N. Zaita, J. Chunwongse, J. Obokata, K. Yamaguchi-Shinozaki, C. Ohto, K. Torazawa, B.Y. Meng, M. Sugita, H. Deno, T. Kamogashira, K. Yamada, J. Kusuda, F. Takaiwa, A. Kato, N. Tohdoh, H. Shimada, and M. Sugiura. 1986. The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. The EMBO Journal. 5:2043-2049.

Shirdel, M., A. Motallebi-Azar, S. Masiha, N. Mortazavi, M. Matloobi, and Y. Sharafi. 2011. Effects of inorganic nitrogen source and NH4

+ : NO3− ratio on proliferation of dog rose (Rosa canina). J

Med Plant Res. 5:4605-4609. Shuler, M., and F. Kargi. 2002. Bioprocess engineering: basic concepts. Prentice Hall international

series in the physical and chemical engineering sciences. Sikdar, S.R., G. Serino, S. Chaudhuri, and P. Maliga. 1998. Plastid transformation in Arabidopsis

thaliana. Plant Cell Reports. 18:20-24. Silhavy, D., and P. Maliga. 1998. Plastid promoter utilization in a rice embryogenic cell culture.

Current Genetics. 34:67-70. Singh, M., A. Boutanaev, P. Zucchi, and L. Bogorad. 2001. Gene elements that affect the longevity of

rbcL sequence-containing transcripts in Chlamydomonas reinhardtii chloroplasts. Proceedings of the National Academy of Sciences. 98:2289-2294.

Singh, S., M. Rai, P. Asthana, and L. Sahoo. 2010. Alginate-encapsulation of nodal segments for propagation, short-term conservation and germplasm exchange and distribution of Eclipta alba (L.). Acta Physiol. Plant. 32:607-610.

192

Singh, S.K., M.K. Rai, P. Asthana, S. Pandey, V.S. Jaiswal, and U. Jaiswal. 2009. Plant regeneration from alginate-encapsulated shoot tips of Spilanthes acmella (L.) Murr., a medicinally important and herbal pesticidal plant species. Acta Physiol. Plant. 31:649-653.

Sivakumar, G. 2006. Bioreactor technology: a novel industrial tool for high-tech production of bioactive molecules and biopharmaceuticals from plant roots. Biotechnol J. 1:1419-1427.

Soccol, C., G. Scheidt, and R. Mohan. 2008. Biorreator do tipo imersão por bolhas para as técnicas de micropropagação vegetal. Universidade Federal do Paraná. Patente,(DEPR. 01508000078).(in portuguese).

Sodoyer, R. 2004. Expression Systems for the Production of Recombinant Pharmaceuticals. BioDrugs. 18:51-62.

Solárová, J., and J. Pospíšilová. 1997. Effect of carbon dioxide enrichment during in vitro cultivation and acclimation to ex vitro conditions. Biol. Plant. 39:23-30.

Soria-Guerra, R.E., L. Moreno-Fierros, and S. Rosales-Mendoza. 2011. Two decades of plant-based candidate vaccines: a review of the chimeric protein approaches. Plant Cell Reports. 30:1367-1382.

Sowana, D.D., D.R.G. Williams, E.H. Dunlop, B.B. Dally, B.K. O’Neill, and D.F. Fletcher. 2001. Turbulent Shear Stress Effects on Plant Cell Suspension Cultures. Chemical Engineering Research and Design. 79:867-875.

Spira, S., M.A. Wainberg, H. Loemba, D. Turner, and B.G. Brenner. 2003. Impact of clade diversity on HIV-1 virulence, antiretroviral drug sensitivity and drug resistance. Journal of Antimicrobial Chemotherapy. 51:229-240.

Spök, A., S. Karner, A.J. Stein, and E. Rodríguez-Cerezo. 2008a. Plant molecular farming. Opportunities and challenges. JRC Scientific and Technical Reports.

Spök, A., R.M. Twyman, R. Fischer, J.K.C. Ma, and P.A.C. Sparrow. 2008b. Evolution of a regulatory framework for pharmaceuticals derived from genetically modified plants. Trends Biotechnol. 26:506-517.

Spörlein, B., M. Streubel, G. Dahlfeld, P. Westhoff, and H.U. Koop. 1991. PEG-mediated plastid transformation: a new system for transient gene expression assays in chloroplasts. Theor. Appl. Genet. 82:717-722.

Sreedhar, R., L. Venkatachalam, R. Thimmaraju, N. Bhagyalakshmi, M. Narayan, and G. Ravishankar. 2008. Direct organogenesis from leaf explants of Stevia rebaudiana and cultivation in bioreactor. Biol. Plant. 52:355-360.

Srinivasan, V., L. Pestchanker, S. Moser, T.J. Hirasuna, R.A. Taticek, and M.L. Shuler. 1995. Taxol production in bioreactors: Kinetics of biomass accumulation, nutrient uptake, and taxol production by cell suspensions of Taxus baccata. Biotechnol. Bioeng. 47:666-676.

Sriraman, R., M. Bardor, M. Sack, C. Vaquero, L. Faye, R. Fischer, R. Finnern, and P. Lerouge. 2004. Recombinant anti-hCG antibodies retained in the endoplasmic reticulum of transformed plants lack core-xylose and core-alpha(1,3)-fucose residues. Plant Biotechnol. J. 2:279-287.

Standardi, A., and E. Piccioni. 1998. Recent Perspectives on Synthetic Seed Technology Using Nonembryogenic In Vitro–Derived Explants. International Journal of Plant Sciences. 159:968-978.

Stanly, C., A. Bhatt, and C.L. Keng. 2010. A comparative study of Curcuma zedoaria and Zingiber zerumbet plantlet production using different micropropagation systems. Afr. J. Biotechnol. 9:4326-4333.

Staub, J.M., B. Garcia, J. Graves, P.T.J. Hajdukiewicz, P. Hunter, N. Nehra, V. Paradkar, M. Schlittler, J.A. Carroll, L. Spatola, D. Ward, G.N. Ye, and D.A. Russell. 2000. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat. Biotechnol. 18:333-338.

Staub, J.M., and P. Maliga. 1994. Translation of psbA mRNA is regulated by light via the 5′-untranslated region in tobacco plastids. Plant J. 6:547-553.

Stegemann, S., and R. Bock. 2009. Exchange of Genetic Material Between Cells in Plant Tissue Grafts. Science. 324:649-651.

193

Stegemann, S., S. Hartmann, S. Ruf, and R. Bock. 2003. High-frequency gene transfer from the chloroplast genome to the nucleus. Proceedings of the National Academy of Sciences. 100:8828-8833.

Steingroewer, J., T. Bley, V. Georgiev, I. Ivanov, F. Lenk, A. Marchev, and A. Pavlov. 2013. Bioprocessing of differentiated plant in vitro systems. Engineering in Life Sciences. 13:26-38.

Stern, D.B., and W. Gruissem. 1987. Control of plastid gene-expression - 3' inverted repeats act as messenger-RNA processing and stabilizing elements, but do not terminate transcription. Cell. 51:1145-1157.

Stern, D.B., D.C. Higgs, and J.J. Yang. 1997. Transcription and translation in chloroplasts. Trends Plant Sci. 2:308-315.

Steward, N., R. Martin, J.M. Engasser, and J.L. Goergen. 1999. Determination of growth and lysis kinetics in plant cell suspension cultures from the measurement of esterase release. Biotechnol. Bioeng. 66:114-121.

Stobbe, H., U. Schmitt, D. Eckstein, and D. Dujesiefken. 2002. Developmental stages and fine structure of surface callus formed after debarking of living lime trees (Tilia sp.). Ann Bot. 89:773-782.

Stoger, E., R. Fischer, M. Moloney, and J.K.-C. Ma. 2014. Plant Molecular Pharming for the Treatment of Chronic and Infectious Diseases. Annual Review of Plant Biology. 65:743-768.

Stoger, E., J.K.C. Ma, R. Fischer, and P. Christou. 2005. Sowing the seeds of success: pharmaceutical proteins from plants. Current Opinion in Biotechnology. 16:167-173.

Stoger, E., M. Sack, Y. Perrin, C. Vaquero, E. Torres, R. Twyman, P. Christou, and R. Fischer. 2002. Practical considerations for pharmaceutical antibody production in different crop systems. Molecular Breeding. 9:149-158.

Stoger, E., C. Vaquero, E. Torres, M. Sack, L. Nicholson, J. Drossard, S. Williams, D. Keen, Y. Perrin, P. Christou, and R. Fischer. 2000. Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Mol Biol. 42:583-590.

Strohl, W.R., and D.M. Knight. 2009. Discovery and development of biopharmaceuticals: current issues. Current Opinion in Biotechnology. 20:668-672.

Sugiura, M. 1992. The Chloroplast Genome. Plant Mol.Biol. 19:149-168. Svab, Z., P. Hajdukiewicz, and P. Maliga. 1990. Stable transformation of plastids in higher-plants.

Proc. Natl. Acad. Sci. U. S. A. 87:8526-8530. Svab, Z., and P. Maliga. 1993. High-frequency plastid transformation in tobacco by selection for a

chimeric aadA gene. Proceedings of the National Academy of Sciences. 90:913-917. Svab, Z., and P. Maliga. 2007. Exceptional transmission of plastids and mitochondria from the

transplastomic pollen parent and its impact on transgene containment. Proceedings of the National Academy of Sciences. 104:7003-7008.

Svensson, L., H. Sheshberadaran, S. Vene, E. Norrby, M. Grandien, and G. Wadell. 1987. Serum Antibody Responses to Individual Viral Polypeptides in Human Rotavirus Infections. Journal of General Virology. 68:643-651.

Tagliavini, M., J. Abadía, A. Rombolà, A. Abadía, C. Tsipouridis, and B. Marangoni. 2000. Agronomic means for the control of iron deficiency chlorosis in deciduous fruit trees. Journal of Plant Nutrition. 23:2007-2022.

Takayama, S., and M. Akita. 1994. The types of bioreactors used for shoots and embryos. Plant Cell, Tissue and Organ Culture. 39:147-156.

Takayama, S., and M. Akita. 2006. Bioengineering aspects of bioreactor application in plant propagation. In Plan Tissue Culture Engineering. Vol. 6. S.D. Gupta and Y. Ibaraki, editors. Springer Netherlands. 83-100.

Takeda, S., Y. Kaneko, H. Matsushima, Y. Yamada, and F. Sato. 1999. Cultured green cells of tobacco as a useful material for the study of chloroplast replication. Methods Cell Sci. 21:149-154.

194

Tan Nhut, D., T. Takamura, H. Watanabe, and M. Tanaka. 2001. Artificial light source using light-emitting diodes (LEDs) in the efficient micropropagation of Spathiphyllum plantlets. In II International Symposium on Biotechnology of Tropical and Subtropical Species 692. 137-142.

Tan, X.W., H. Ikeda, and M. Oda. 2000. The absorption, translocation, and assimilation of urea, nitrate or ammonium in tomato plants at different plant growth stages in hydroponic culture. Scientia Horticulturae. 84:275-283.

Tanaka, H. 1981. Technological problems in cultivation of plant cells at high density. Biotechnol. Bioeng. 23:1203-1218.

Tanaka, H. 1982. Oxygen transfer in broths of plant cells at high density. Biotechnol. Bioeng. 24:425-442.

Tavladoraki, P., E. Benvenuto, S. Trinca, D. De Martinis, A. Cattaneo, and P. Galeffi. 1993. Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature. 366:469-472.

Teisson, C., and D. Alvard. 1995. A new concept of plant in vitro cultivation liquid medium: Temporary immersion. In Current Issues in Plant Molecular and Cellular Biology. Vol. 22. M. Terzi, R. Cela, and A. Falavigna, editors. Kluwer Academic Publ, Dordrecht. 105-110.

Teisson, C., and D. Alvard. 1999. In vitro production of potato microtubers in liquid medium using temporary immersion. Potato Research. 42:499-504.

Teng, W.L. 1999. Source, etiolation and orientation of explants affect in vitro regeneration of Venus fly-trap (Dionaea muscipula). Plant Cell Reports. 18:363-368.

Terrier, B., D. Courtois, N. Henault, A. Cuvier, M. Bastin, A. Aknin, J. Dubreuil, and V. Petiard. 2007. Two new disposable bioreactors for plant cell culture: The wave and undertow bioreactor and the slug bubble bioreactor. Biotechnol. Bioeng. 96:914-923.

Thiry, M., and D. Cingolani. 2002. Optimizing scale-up fermentation processes. Trends Biotechnol. 20:103-105.

Thomas, J.C., and F.R. Katterman. 1986. Cytokinin Activity Induced by Thidiazuron. Plant Physiol. 81:681-683.

Tichá, I., F. Čáp, D. Pacovská, P. Hofman, D. Haisel, V. Čapková, and C. Schäfer. 1998. Culture on sugar medium enhances photosynthetic capacity and high light resistance of plantlets grown in vitro. Physiologia Plantarum. 102:155-162.

Tiller, N., and R. Bock. 2014. The Translational Apparatus of Plastids and Its Role in Plant Development. Molecular Plant. 7:1105-1120.

Tillich, M., S. Beick, and C. Schmitz-Linneweber. 2010. Chloroplast RNA-binding proteins Repair and regulation of chloroplast transcripts. RNA Biol. 7:172-178.

Tissot, G., H. Canard, M. Nadai, A. Martone, J. Botterman, and M. Dubald. 2008. Translocation of aprotinin, a therapeutic protease inhibitor, into the thylakoid lumen of genetically engineered tobacco chloroplasts. Plant Biotechnol. J. 6:309-320.

Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences. 76:4350-4354.

Towill, L.E., and P. Mazur. 1975. Studies on the reduction of 2,3,5-triphenyltetrazolium chloride as a viability assay for plant tissue cultures. Canadian Journal of Botany. 53:1097-1102.

Towler, M., Y. Kim, B. Wyslouzil, M. Correll, and P. Weathers. 2006. Design, Development, And Applications Of Mist Bioreactors For Micropropagation And Hairy Root Culture. In Plan Tissue Culture Engineering. Vol. 6. S.D. Gupta and Y. Ibaraki, editors. Springer Netherlands. 119-134.

Tregoning, J., P. Maliga, G. Dougan, and P.J. Nixon. 2004. New advances in the production of edible plant vaccines: chloroplast expression of a tetanus vaccine antigen, TetC. Phytochemistry. 65:989-994.

195

Tregoning, J.S., P. Nixon, H. Kuroda, Z. Svab, S. Clare, F. Bowe, N. Fairweather, J. Ytterberg, K.J. van Wijk, G. Dougan, and P. Maliga. 2003. Expression of tetanus toxin Fragment C in tobacco chloroplasts. Nucleic Acids Res. 31:1174-1179.

Trejo-Tapia, G., A. Jiménez-Aparicio, L. Villarreal, and M. Rodríguez-Monroy. 2001. Broth rheology and morphological analysis of Solanum chrysotrichum cultivated in a stirred tank. Biotechnol. Lett. 23:1943-1946.

Tremblay, R., D. Wang, A.M. Jevnikar, and S. Ma. 2010. Tobacco, a highly efficient green bioreactor for production of therapeutic proteins. Biotechnol. Adv. 28:214-221.

Triguero, A., G. Cabrera, J.A. Cremata, C.T. Yuen, J. Wheeler, and N.I. Ramirez. 2005. Plant-derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum-retention signal is N-glycosylated homogeneously throughout the plant with mostly high-mannose-type N-glycans. Plant Biotechnol J. 3:449-457.

Tsai, C.-J., and J. Saunders. 1999. Evaluation of sole nitrogen sources for shoot and leaf disc cultures of sugarbeet. Plant Cell, Tissue and Organ Culture. 59:47-56.

Twyman, R.M., S. Schillberg, and R. Fischer. 2013. Optimizing the Yield of Recombinant Pharmaceutical Proteins in Plants. Current Pharmaceutical Design. 19:5486-5494.

Twyman, R.M., E. Stoger, S. Schillberg, P. Christou, and R. Fischer. 2003. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 21:570-578.

Uncu, A.O., S. Doganlar, and A. Frary. 2013. Biotechnology for Enhanced Nutritional Quality in Plants. Crit. Rev. Plant Sci. 32:321-343.

Vaidya, B., S. Mutalik, R. Joshi, S. Nene, and B. Kulkarni. 2009. Enhanced production of amidase from Rhodococcus erythropolis MTCC 1526 by medium optimisation using a statistical experimental design. J Ind Microbiol Biotechnol. 36:671-678.

Valdés, R., B. Reyes, T. Alvarez, J. Garcıa, J.A. Montero, A. Figueroa, L. Gómez, S. Padilla, D. Geada, M.C. Abrahantes, L. Dorta, D. Fernández, O. Mendoza, N. Ramirez, M. Rodriguez, M. Pujol, C. Borroto, and J. Brito. 2003. Hepatitis B surface antigen immunopurification using a plant-derived specific antibody produced in large scale. Biochemical and Biophysical Research Communications. 310:742-747.

Valkov, V.T., D. Gargano, C. Manna, G. Formisano, P.J. Dix, J.C. Gray, N. Scotti, and T. Cardi. 2011. High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5' and 3' regulatory sequences. Transgenic Res. 20:137-151.

van den Dries, N., S. Giannì, A. Czerednik, F.A. Krens, and G.-J.M. de Klerk. 2013. Flooding of the apoplast is a key factor in the development of hyperhydricity. Journal of Experimental Botany.

van der Hoorn, R.A.L. 2008. Plant Proteases: From Phenotypes to Molecular Mechanisms. Annual Review of Plant Biology. 59:191-223.

van Dolleweerd, C.J., C.G. Kelly, D. Chargelegue, and J.K.-C. Ma. 2004. Peptide mapping of a novel discontinuous epitope of the major surface adhesin from Streptococcus mutans. Journal of Biological Chemistry. 279:22198-22203.

van Ree, R., M. Cabanes-Macheteau, J. Akkerdaas, J.P. Milazzo, C. Loutelier-Bourhis, C. Rayon, M. Villalba, S. Koppelman, R. Aalberse, R. Rodriguez, L. Faye, and P. Lerouge. 2000. Beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. The Journal of biological chemistry. 275:11451-11458.

Vasilev, N., U. Grömping, A. Lipperts, N. Raven, R. Fischer, and S. Schillberg. 2013. Optimization of BY-2 cell suspension culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method. Plant Biotechnol. J. 11:867-874.

Vass, I., K. Cser, and O. Cheregi. 2007. Molecular Mechanisms of Light Stress of Photosynthesis. Annals of the New York Academy of Sciences. 1113:114-122.

196

Vdovitchenko, M.Y., and I.N. Kuzovkina. 2011. Artificial seed preparation as the efficient method for storage and production of healthy cultured roots of medicinal plants. Russ J Plant Physiol. 58:524-530.

Villani, M.E., B. Morgun, P. Brunetti, C. Marusic, R. Lombardi, I. Pisoni, C. Bacci, A. Desiderio, E. Benvenuto, and M. Donini. 2009. Plant pharming of a full-sized, tumour-targeting antibody using different expression strategies. Plant Biotechnol. J. 7:59-72.

Vlaev, S.D., and M. Fialova. 2003. Bubble Column Bioreactors: Comparison with Stirred Fermenters Based on Local Gas Hold-up Distribution. The Canadian Journal of Chemical Engineering. 81:535-542.

Vlahova, M., G. Stefanova, P. Petkov, A. Barbulova, D. Petkova, P. Kalushkov, and A. Atanassov. 2005. Genetic modification of Alfalfa (Medicago sativa L.) for quality improvement and production of novel compounds. Biotechnology & Biotechnological Equipment. 19:56-62.

Walch-Liu, P., G. Neumann, F. Bangerth, and C. Engels. 2000. Rapid effects of nitrogen form on leaf morphogenesis in tobacco. Journal of Experimental Botany. 51:227-237.

Walmsley, A.M., and C.J. Arntzen. 2003. Plant cell factories and mucosal vaccines. Current Opinion in Biotechnology. 14:145-150.

Wang, S.-J., and J.-J. Zhong. 2007. Chapter 6 - Bioreactor Engineering. In Bioprocessing for Value-Added Products from Renewable Resources. S.-T. Yang, editor. Elsevier, Amsterdam. 131-161.

Wang, Z.Y., X.D. Ye, J. Nagel, I. Potrykus, and G. Spangenberg. 2001. Expression of a sulphur-rich sunflower albumin gene in transgenic tall fescue (Festuca arundinacea Schreb.) plants. Plant Cell Reports. 20:213-219.

Ward, R.L., and M.M. McNeal. 2010. VP6: A Candidate Rotavirus Vaccine. Journal of Infectious Diseases. 202:S101-S107.

Watt, M.P. 2012. The status of temporary immersion system (TIS) technology for plant micropropagation. Afr J Biotechnol. 11:14025-14035.

Waugh, D.S. 2005. Making the most of affinity tags. Trends Biotechnol. 23:316-320. Weathers, P., C. Liu, M. Towler, and B. Wyslouzil. 2008. Mist reactors: principles, comparison of

various systems, and case studies. Electronic Journal of Integrative Biosciences. 3:29-37. Weathers, P.J., M.J. Towler, and J.F. Xu. 2010. Bench to batch: advances in plant cell culture for

producing useful products. Applied Microbiology and Biotechnology. 85:1339-1351. Webb, C., and B. Atkinson. 1992. The role of chemical engineering in biotechnology. The Chemical

Engineering Journal. 50:B9-B16. Weihe, A., K. Liere, and T. Börner. 2012. Transcription and Transcription Regulation in Chloroplasts

and Mitochondria of Higher Plants. In Organelle Genetics. C.E. Bullerwell, editor. Springer Berlin Heidelberg. 297-325.

Wheeler, T.R., P.Q. Craufurd, R.H. Ellis, J.R. Porter, and P.V. Vara Prasad. 2000. Temperature variability and the yield of annual crops. Agriculture, Ecosystems & Environment. 82:159-167.

Whitney, S.M., R.L. Houtz, and H. Alonso. 2011. Advancing Our Understanding and Capacity to Engineer Nature's CO2-Sequestering Enzyme, Rubisco. Plant Physiol. 155:27-35.

Wilken, L.R., and Z.L. Nikolov. 2012. Recovery and purification of plant-made recombinant proteins. Biotechnol. Adv. 30:419-433.

Will, G.M. 1966. Magnesium deficiency: the cause of spring needle-tip chlorosis in young pines on pumice soils. New Zealand Forest Service.

Wilson, S.A., and S.C. Roberts. 2012. Recent advances towards development and commercialization of plant cell culture processes for the synthesis of biomolecules. Plant Biotechnol. J. 10:249-268.

Wind, J., S. Smeekens, and J. Hanson. 2010. Sucrose: Metabolite and signaling molecule. Phytochemistry. 71:1610-1614.

Wise, R.R. 2006. The Diversity of Plastid Form and Function. In The Structure and Function of Plastids. Vol. 23. R. Wise and J.K. Hoober, editors. Springer Netherlands. 3-26.

197

Witcher, D., E. Hood, D. Peterson, M. Bailey, D. Bond, A. Kusnadi, R. Evangelista, Z. Nikolov, C. Wooge, R. Mehigh, W. Kappel, J. Register, and J. Howard. 1998. Commercial production of β-glucuronidase (GUS): a model system for the production of proteins in plants. Molecular Breeding. 4:301-312.

Wongsamuth, R., and P.M. Doran. 1997. Production of monoclonal antibodies by tobacco hairy roots. Biotechnol. Bioeng. 54:401-415.

Wood, D.W. 2014. New trends and affinity tag designs for recombinant protein purification. Curr Opin Struct Biol. 26:54-61.

Woodward, A.J., I.J. Bennett, and S. Pusswonge. 2006. The effect of nitrogen source and concentration, medium pH and buffering on in vitro shoot growth and rooting in Eucalyptus marginata. Scientia Horticulturae. 110:208-213.

Wright, A., and S.L. Morrison. 1997. Effect of glycosylation on antibody function: implications for genetic engineering. Trends Biotechnol. 15:26-32.

Wurbs, D., S. Ruf, and R. Bock. 2007. Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome. Plant J. 49:276-288.

Xu, G., X. Fan, and A.J. Miller. 2012a. Plant Nitrogen Assimilation and Use Efficiency. Annual Review of Plant Biology. 63:153-182.

Xu, J., M.C. Dolan, G. Medrano, C.L. Cramer, and P.J. Weathers. 2012b. Green factory: Plants as bioproduction platforms for recombinant proteins. Biotechnol. Adv. 30:1171-1184.

Xu, J., X. Ge, and M.C. Dolan. 2011. Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures. Biotechnol Adv. 29:278-299.

Yabuta, Y., M. Tamoi, K. Yamamoto, K. Tomizawa, A. Yokota, and S. Shigeoka. 2008. Molecular design of photosynthesis-elevated chloroplasts for mass accumulation of a foreign protein. Plant and Cell Physiology. 49:375-385.

Yan, H.B., C.X. Liang, and Y.R. Li. 2010. Improved growth and quality of Siraitia grosvenorii plantlets using a temporary immersion system. Plant Cell Tissue Organ Cult. 103:131-135.

Ye, G.N., P.T.J. Hajdukiewicz, D. Broyles, D. Rodriguez, C.W. Xu, N. Nehra, and J.M. Staub. 2001. Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco. Plant J. 25:261-270.

Yoshikawa, T., and T. Furuya. 1983. Regeneration and in vitro flowering of plants derived from callus cultures of opium poppy (Papaver somniferum). Experientia. 39:1031-1033.

Zeeman, S.C., J. Kossmann, and A.M. Smith. 2010. Starch: Its Metabolism, Evolution, and Biotechnological Modification in Plants. Annual Review of Plant Biology. 61:209-234.

Zhang, R.Y., and W.D. Shen. 2012. Monoclonal Antibody Expression in Mammalian Cells. In Antibody Engineering. Vol. 907. P. Chames, editor. Humana Press. 341-358.

Zhang, Y.-h., J.-j. Zhong, and J.-t. Yu. 1996. Enhancement of ginseng saponin production in suspension cultures of Panax notoginseng: manipulation of medium sucrose. Journal of Biotechnology. 51:49-56.

Zhang, Z.-Y., and J.-J. Zhong. 2004. Scale-up of centrifugal impeller bioreactor for hyperproduction of ginseng saponin and polysaccharide by high-density cultivation of Panax notoginseng cells. Biotechnology Progress. 20:1076-1081.

Zhao, J. 2007. Nutraceuticals, nutritional therapy, phytonutrients, and phytotherapy for improvement of human health: a perspective on plant biotechnology application. Recent patents on biotechnology. 1:75-97.

Zhong, J.-J. 2002. Plant cell culture for production of paclitaxel and other taxanes. Journal of Bioscience and Bioengineering. 94:591-599.

Zhong, J.-J., K. Fujiyama, T. Seki, and T. Yoshida. 1994. A quantitative analysis of shear effects on cell suspension and cell culture of perilla frutescens in bioreactors. Biotechnol. Bioeng. 44:649-654.

198

Zhou, B., Y. Zhang, X. Wang, J. Dong, B. Wang, C. Han, J. Yu, and D. Li. 2010. Oral administration of plant-based rotavirus VP6 induces antigen-specific IgAs, IgGs and passive protection in mice. Vaccine. 28:6021-6027.

Zhou, F., J.A. Badillo-Corona, D. Karcher, N. Gonzalez-Rabade, K. Piepenburg, A.M.I. Borchers, A.P. Maloney, T.A. Kavanagh, J.C. Gray, and R. Bock. 2008. High-level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes. Plant Biotechnol. J. 6:897-913.

Zhou, F., D. Karcher, and R. Bock. 2007. Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons. Plant J. 52:961-972.

Zhu, L.H., X.Y. Li, and M. Welander. 2005. Optimisation of growing conditions for the apple rootstock M26 grown in RITA containers using temporary immersion principle. Plant Cell Tissue Organ Cult. 81:313-318.

Zimran, A., E. Brill-Almon, R. Chertkoff, M. Petakov, F. Blanco-Favela, E.T. Muñoz, S.E. Solorio-Meza, D. Amato, G. Duran, F. Giona, R. Heitner, H. Rosenbaum, P. Giraldo, A. Mehta, G. Park, M. Phillips, D. Elstein, G. Altarescu, M. Szleifer, S. Hashmueli, and D. Aviezer. 2011. Pivotal trial with plant cell–expressed recombinant glucocerebrosidase, taliglucerase alfa, a novel enzyme replacement therapy for Gaucher disease. 5767-5773 pp.

Ziv, M. 2000. Bioreactor technology for plant micropropagation. Horticultural Reviews. 24:1-30. Ziv, M. 2005. Simple bioreactors for mass propagation of plants. Plant Cell, Tissue and Organ Culture.

81:277-285. Zobayed, S.M.A., F. Afreen, and T. Kozai. 2000. Quality biomass program via photoautotrophic

micropropagation. Proceedings of the International Symposium on Methods and Markers for Quality Assurance in Micropropagation:377-386.

Zych, M., M. Furmanowa, A. Krajewska-Patan, A. Lowicka, M. Dreger, and S. Mendlewska. 2005. Micropropagation of Rhodiola Kirilowii plants using encapsulated axillary buds and callus. Acta Biologica Cracoviensia Series Botanica. 47:83-87.

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