Transient Gene Expression Following DNA Transfer to Plant Cells: The Phenomenon; Its Causes and Some Applications. A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Cellular and Molecular Biology in the University of Canterbury. RICHARD JOHN WELD 2000 1 ..... ACKNOWLEDGEMENTS ..... I would like to thank Dr Jack Heinemann, Dr Colin Eady and Dr Sandra Jackson for their supervision of this project, for their criticism and their encouragement. I would also like to thank Dr Ross Bicknell for his advice and support. I acknowledge the generosity of Dr Jim Haseloff for the gift of plasmid pBINmgfp5-ER, Dr Ed Morgan for the gift of Nicotiana plumbaginifolia suspension cells and advice on their culture, Dr Steve Scofield for the gift ofpSLJll 01, Dr Nicole Houba-Herin for the gift ofpNT103 andpNT804, Dr Jerzy Paszkowski for the gift ofpMDSBAR, Dr Andrew Gleave for the gift ofpART8 and pART7, Dr n Yoder for the gift ofpAL144 and Dr Bernie Carroll for information on the construction of pSLJ3 621. This project was made possible by funds provided by a FfRST Doctoral Fellowship provided through the New' Zealand Institute for Crop and Food Research and a University of Canterbury Doctoral Scholarship. I would like to offer my grateful thanks to all those staff and students of the Plant and Microbial Sciences Department and those staff and students at the New Zealand Institute for Crop and Food Research at Lincoln who have assisted me in various ways during the course of my research and to those who have offered their friendship, support and encouragement. In particular I would like to thank: Dougal Holmes for help with computer graphics; Ruth Butler for assistance with statistical analysis; Andrew Catenach for information and advice regarding Hieracium aurantiacum; Jackie Healy, Tonya Frew and Meeghan Pither-Joyce for technical advice. Especial gratitude to my partner Bridget who held the fort during those times when I locked myself away. - 1 FEB 200'1 2 ..... TABLE OF CONTENTS..... Acknowledgements.................................................................................... ............... 1 Table of contents..................................................................................................................... 2 List of figures........................................................................................................................... 4 List of tables............................................................................................................................. 8 List of abbreviations............................................................................................................... 9 Abstract...................................................................................................................................... 10 1. General Introduction......................................................................................................... 11 1.1. Project Overview.................................................................................................... 11 1. 2. Transient T -DNA Expression in Plant Cells ...................................................... .. 16 1.3. Plant Transformation ............................................................................................ 26 1.4. T-DNA Transfer from Agrohacterium tumefaciells ........................................... .. 28 1.5. Integration ofT-DNA into the Plant Genome ................................................. 29 1.6. Epigenetic Modification of Transgene Expression.......... ,.................................. .. 31 1. 7. Improved Integration Strategies.......................................................................... .. 33 1. 8. ActivatorlDissociatioll Transposons....................................................................... 35 1. 9. Dissociatioll as a Vector for Allium cepa Transformation................................. .. 36 1.10. Dissociatioll Elements and Gene Tagging ............................................................. 37 1.11. Transformation and Tissue Culture of Allium cepa ........................................... .. 39 1.12. Transformation and Tissue Culture of Hieracium auralltiacum ....................... .. 40 1. 13. Transformation and Tissue Culture of Nicotiana plumhaginifolia .................... 41 1. 14. References ............................................................................................................... .. 41 2. Dissociation (Ds) Elements as Vectors for Transformation of Onion (A.llium cepa)...... ...... ......... ......... ............ ...... .................... ....... ..... 63 2. 1. Introduction............................................................................................................... 63 2. 2. Materials and Methods............................................................................................. 64 2. 3. Results........................................................................................................................ 67 2. 4. Discussion............................................ ...................................................................... 71 2. 5. References.................................................................................................................. 74 3 3. Ds transposition Mediated by Transient Transposase Expression in Hieracium aurantiacum................................................................................................... 77 3.1. Introduction......................................................................................................... .. 77 3.2. Materials and Methods........................................................................................ 79 3.3. Results ................................................................................................................... 83 3.4. Discussion ............................................................................................................. .. 111 3.5. References............................................................................................................. 118 4. Transient GFP Expression in Nicotiana plumbaginifolia Suspension Cells Following Co-cultivation with Agrobacterium tumefaciens: the Role of Gene Silencing, Cell Death and T -DNA Loss....................................................... 126 4. 1. Introduction........................................................................................................... 126 4. 2. Materials and Methods......................................................................................... 127 4.3. Results................................................................................................................... 131 4.4. Discussion ............................................................................................................. 155 4.5. References............................................................................................................. 159 5. General Discussion........................................................................................................... 166 5.1. General Discussion ............................................................................................... 166 5.2. References.............................. .'.~.~ ......................................................................... .. 172 4 ...... LIST OF FIGURES ..... Figure 1. 1. 1. Transposition of Ds elements as a strategy to transform A. cepa..................................................................... ... 12 Figure 1. 1. 2. Transposition of Ds elements as a strategy to recover H. aurantiacum cells that transiently expressed the Ac transposase gene........................................................ ... 13 Figure 1.1. 3. TransientAc transposase expression as a method to mobilise Ds elements for gene-tagging in H. aurantiacum ................. .. 14 Figure 1.1. 4. Tracking N. plumbaginijolia cells that transiently expressed the m-gf'p5-ER gene....................................................... 15 Figure 3. 3. 1. Map ofT-DNA of Plasmid pSLJ3621............................................. 95 Figure 3. 3. 2. Autoradiograph of HpaIlBglJI digested H. aurantiacum DNA hybridized to a labelled probe homologous to the aadA spectinomycin resistance gene......................................................... 96 Figure 3. 3. 3. H. aurantiacum R4 721 co-cultivated with pNE5 and stained for /3-glucuronidase activity................................................. 97 Figure 3. 3. 4. H. aurantiacum A3 3621 leaf explant cultured on HR medium supplemented with 600mgll spectinomycin for 4 weeks................. 98 Figure 3. 3. 5. H. aurantiacum A3 3621 shoots regenerating on HR medium supplemented with 600mg/l spectinomycin...................... 99 Figure 3.3.6. H. aurantiacum A3 3621 shoots regenerated after co­ cultivation with A. tumefaciens (pSLJ1111) and selection for spectinomycin resistance............................................. 100 5 Figure 3. 3.7. H. aurantiacum A3 3621 shoots regenerated after co­ cultivation with A. tumefaciens (pSLJllll) not forming roots on HO medium supplemented with 600mgll spectinomycin. it.................. .......................................... .. 101 Figure 3. 3. 8. DNA sequences of Ds excision sites in three spectinomycin resistant H. aurantiacum plants....... ......... ..... ..... ................... 102 Figure 3. 3. 9. H. aurantiacum A3 3621 shoots regenerated