r - 1 - GENE TRANSFER BY ELECTROPORATION IN PLANT PROTOPLASTS AND TISSUES BY MICHIEL THEODOOR JAN DE BOTH 1990 A thesis submitted for the degree of Doctor of Philosophy of the University of London and for the Diploma of Membership of the Imperial College Department of Pure and Applied Biology Imperial College of Science and Technology London, SW7 - 2 - ABSTRACT GENE TRANSFER BY ELECTROPORATION IN PLANT PROTOPLASTS AND TISSUES Direct gene transfer to protoplasts by electroporation offers an alternative to Agrobacterium-mediated transformation for the intro­ duction of foreign DNA into plant cells. In the present study an effi­ cient system for the electroporation of tobacco protoplasts has been established by testing various pulse parameters and different pulse types. The isolation and regeneration of tobacco mesophyll protoplasts were optimized. The most important parameters to obtain high plating efficiencies are the composition of the culture medium, the culture density, culture in agarose 'beads', and the dilution of the culture after 8 days. Fertile plants have been regenerated from these proto­ plasts. Subsequently, electroporation experiments are described using short (microsecond) rectangular and exponentially decaying pulses on wheat and tobacco protoplasts. No transient expression of chloram­ phenicol acetyltransferase (CAT) was detected in either protoplast system. Transient expression of CAT in wheat protoplasts was obtained after PEG-mediated transformation. Stably transformed tobacco plants were regenerated after electroporation with neo as a selectable marker. Molecular evidence for transformation was obtained by the polymerase chain reaction (PCR) on the R1 offspring. Exponentially decaying pulses of low initial field strength and millisecond duration resulted in good transient expression of CAT in tobacco protoplasts. A systematic study of the pulse conditions re­ sulted in a standard electroporation protocol, using three pulses of 225 V.cnT1 and 46 ms duration, on 2 x l 0 6 protoplasts at a density of 106 mL-1 in 20 rrM KC1. The optimal field strength is discussed in terms of the critical membrane breakdown potential. Using these pulse conditions, stably transformed colonies at a relative transformation frequency of 10“h were obtained. Some electroporation experiments with whole tissues are also described. However, these did not lead to positive results. The importance of the pulse length for the transfer of DNA into protoplasts by an electric field-driven flow is discussed. - 3 - ACKNOWLEDGEMENTS Many people have been of great assistance or have given invaluable advice, both during the period of practical work for and the writing-up phase of this thesis. First of all, I would like to thank my supervisors, Dr Mike G.K. Jones of Rothamsted Experimental Station and Professor K.W. Buck of Imperial College, who have followed the progress of the work with great interest, and advised and encouraged me frequently. Valuable technical advice was also received from many people in the Biochemistry Department of Rothamsted, especially from Dr Mike Burrell, Dr Brian Forde, Dr Janice Forde, Dr Martin Kreis, Dr Gert Ooms, Dr Stan Pierpoint, and Dr Dave Twell, from Dr Steven Gleddie, PBI, Cambridge, UK, and from Dr Michiel Tempelaar, State University of Groningen, the Netherlands. I would like to thank all those who worked in the Emrys Thomas Lab during my stay there, in particular Dr Melody Clarke, Dr Stephany Cooper-Bland, Dr Luc de Bry, Rachel Dunckley, Dr Colin Eady, Dr Neil Fish, Dr Dave Foulger, Dr Patrick Gallois, Dr Heddwyn Jones, Dr Bruce Lee, Dr Keith Lindsey, Dr Sheila Maddock, Dr Renee Malone, Clair Marris, Kate Murdoch, Dr Angela Karp, Dr Rob Potter, Sue Steele, Corine Symonds, and Dr Jennifer Topping, for the pleasant discussions and collaborations. The valuable technical assistance of Sue Steele, Caroline Sparks, Sarah Waller, and Kate Murdoch is gratefully acknowl­ edged. All these people did a lot to make my stay in Rothamsted a pleasant and memorable one. A special word of thanks is due to Dr Qinsheng Wu, with whom, during two years, I collaborated very amicably on the transformation of wheat protoplasts. Thanks is also due to the Rothamsted Photographic Department for their excellent work, and to Julian Franklin for taking care of the greenhouse plants. A special word of thanks goes to Reinette, my wife, for encour­ agements, enormous help during the writing-up phase, and the tremen­ dous patience during all those years. The financial support of BIOSEM, Groupe Limagrain, and the encouragements of Joel Perret are also acknowledged with gratitude. - 4 - TABLE OF CONTENTS A page Abstract 2 Acknowledgements 3 Table of Contents 4 List of Tables 8 List of Figures and Plates 10 List of Abbreviations 13 CHAPTER 1. INTRODUCTION 14 1.1. GENERAL INTRODUCTION 15 1.2. GENETIC TRANSFORMATION OF PLANTS 15 1.2.1. Agrobacterium-mediated transformation 15 1.2.2. Direct gene transfer 20 1.2.3. Other transformation techniques 25 1.3. PHYSICS OF ELECTROPORATION 26 1.3.1. Membrane permeabilization 26 1.3.2. Critical membrane potential 27 1.3.3. Membrane resealing 29 1.3.4. Pulse types 30 1.4. OUTLINE OF THIS WORK 31 CHAPTER 2. MATERIALS AND METHODS 33 2.1. GENERAL TECHNIQUES 34 2.1.1. Chemicals and solutions 34 2.1.2. Aseptic techniques 36 2.2. MATERIALS 38 2.2.1. Bacterial strains 38 2.2.2. Plasmid vectors 39 2.2.3. Axenic shoot cultures of Nicotiana tabacum L. 48 2.2.4. Cell suspension cultures of Triticum aestivum L. 48 2.2.5. Immature embryos of Triticum aestivum L. 49 2.3. TISSUE CULTURE TECHNIQUES 50 2.3.1. Isolation and regeneration of tobacco mesophyll protoplasts 50 2.3.2. Isolation and culture of wheat suspension protoplasts 57 2.4. DIRECT GENE TRANSFER TECHNIQUES 58 2.4.1. Electroporation equipment for rectangular pulses 58 2.4.2. Electroporation equipment for exponentially de­ caying pulses 59 2.4.3. Electroporation procedures 60 - 5 - TABLE OF CONTENTS (c o n tin u e d ) £age 2.4.4. PEG transformation of wheat protoplasts 62 2.4.5. Uptake of phenosafranin 62 2.4.6. Selection 63 2.5. MICROBIOLOGICAL TECHNIQUES 63 2.5.1. Growth and maintenance of bacterial strains 63 2.5.2. Plasmid DNA isolation 65 2.5.3. Transformation of E.coli 68 2.6. BIOCHEMICAL TECHNIQUES 69 2.6.1. Restriction enzyme analysis and gel electro­ phoresis 69 2.6.2. Assay for chloramphenicol acetyltransferase 70 2.6.3. Assay for 3-glucuronidase 71 2.6.4. Molecular analysis of transformed plants by PCR 71 CHAPTER 3: THE ISOLATION AND REGENERATION OF TOBACCO PROTO­ PLASTS 74 3.1. INTRODUCTION 75 3.2. PROTOPLAST ISOLATION AND PURIFICATION 76 3.2.1. Source material 76 3.2.2. Isolation of the protoplasts 79 3.2.3. Protoplast purification 83 3.2.4. Morphology of the isolated protoplasts 90 3.3. PROTOPLAST CULTURE AND COLONY FORMATION 91 3.3.1. Protoplast density 91 3.3.2. Culture media 94 3.3.3. Development of the protoplasts 100 3.3.4. Culture of protoplasts in agarose beads 104 ♦ 3.3.5. Dilution of the protoplast culture 108 3.4. PLANT REGENERATION 110 3.4.1. Shoot induction 110 3.4.2. Rooting 116 3.4.3. Growth to maturity . 120 3.5. DISCUSSION 123 3.5.1. Protoplast yields 123 3.5.2. Plating efficiencies 124 3.5.3. Regeneration 126 CHAPTER 4: SELECTION LEVELS 128 4.1. DETERMINING SELECTION LEVELS FOR TOBACCO PROTOPLASTS 129 4.1.1. Effect of kanamycin sulphate on the plating efficiency 129 - 6 - TABLE OF CONTENTS (c o n tin u e d ) page 4.1.2. Effect of kanamycin sulphate on the regeneration and shoot development 129 4.1.3. Reconstruction experiment 131 4.2. DETERMINING SELECTION LEVELS FOR WHEAT PROTOPLASTS 132 CHAPTER 5: ELECTROPORATION WITH SHORT RECTANGULAR PULSES 135 5.1. OUTPUT TESTS OF THE PULSE GENERATORS 136 5.1.1. Introduction 136 5.1.2. The performance of the generators at different resistance values 136 5.1.3. Rise in temperature due to the electric pulse 142 5.2. TESTING THE EFFECT OF RECTANGULAR PULSES ON PROTO­ PLAST MEMBRANES 144 5.2.1. Protoplast fusions 144 5.2.2. Uptake of phenosafranine 145 5.3. ELECTROPORATION OF TOBACCO PROTOPLASTS WITH DNA 148 5.3.1. Transient expression of CAT 148 5.3.2. Stably transformed colonies 148 5.4. ELECTROPORATION OF WHEAT PROTOPLASTS WITH DNA 154 5.4.1. Transient expression of CAT 154 5.4.2. Stably transformed colonies 157 5.5. DISCUSSION 157 5.5.1. Membrane permeabilization induced by rectangu­ lar DC pulses 157 5.5.2. DNA uptake following rectangular DC pulses 159 5.5.3. Literature reports on electroporation of proto­ plasts with rectangular pulses 161 CHAPTER 6: ELECTROPORATION WITH EXPONENTIALLY DECAYING PULSES 163 6.1. PHYSICS OF EXPONENTIALLY DECAYING PULSES 164 6.1.1. Capacitance and pulse length 164 6.1.2. Heat effects 166 6.2. ELECTROPORATION WITH PULSES OF SHORT DURATION 166 6.2.1. Tobacco protoplasts 166 6.2.2. Wheat protoplasts 169 6.3. ELECTROPORATION WITH PULSES OF LONG DURATION 173 6.3.1. Transient expression of CAT in tobacco proto­ plasts 173 6.3.2. Optimization of the physical electroporation parameters 175 6.3.3. Optimization of the chemical and biological para­ meters 183 - 7 - TABLE OF CONTENTS (c o n tin u e d ) Page 6.3.4. Transient expression of GUS in tobacco proto­ plasts 188 6.3.5. Stable transformation of tobacco protoplasts 189 6.4. DISCUSSION 191 6.4.1. Standard electroporation conditions for transient expression 191 6.4.2. High voltage - short duration pulses versus low voltage- long duration pulses 192 6.4.3. Wheat protoplasts 193 CHAPTER 7: A NOTE ON THE ELECTROPORATION OF WHOLE TISSUES 194 7.1.