TRANSFORMATION OF BARLEY (HORDEUM VULGARE) USING THE WHEAT PUROINDOLINE GENE by Yusuke Odake A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Plant Pathology MONTANA STATE UNIVERSITY Bozeman, Montana May 2004 ii APPROVAL of a thesis submitted by Yusuke Odake This dissertation has been read by each member of the dissertation committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the College of Graduate Studies. Dr. John E. Sherwood Approved for the Department of Plant Sciences and Plant Pathology Dr. John E. Sherwood Approved for the College of Graduate Studies Dr. Bruce R. McLeod iii STATEMENT OF PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a master’s degree at Montana State University, I agree that the Library shall make it available to borrowers under rules of the Library. If I have indicated my intention to copyright this thesis by including a copyright notice page, copying is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for permission for extended quotation from or reproduction of this thesis in whole or in parts may be granted only by the copyright holder. Yusuke Odake May 17, 2004 iv ACKNOWLEDGEMENTS I would like to thank my committee chair, Dr. John E. Sherwood for providing me with the opportunity to work on the project and awakening my interest in molecular biology and plant pathology. Furthermore, I want to thank him for his assistance in the molecular technique and scientific technical writing. I wish to express my sincere gratitude to Dr. Michael J. Giroux and Dr. William E. Grey, the members of my thesis committee, for thesis guidance and suggestions. Thanks to Dr. David C. Sands for opening the door for me to study at Montana State University- Bozeman. To Shirley Gerhardt, Dr. Thamir Al-Niemi, Dr. Carlotta Balconi, Dr. Paula Kosted, and fellow graduate students Todd Osmundson and Dr. Rebecca Bargabus, I am greatly indebted for their generously sharing molecular techniques, the joy of learning science, and guidance in technical writing in English and countless hours of instruction. I also appreciate the other graduate students in Ag-Bio Science building and faculty and staff of the Plant Sciences and Plant Pathology Department for all their help and advice regarding my studies and research. I would like to express my appreciation to the Dr. William O. Dingwall foundation, and the Department of Plant Sciences and Plant Pathology for the financial support of my study in the United States. Finally, I would like to thank Dr. Paul V. Griesy, and Dr. Masateru Ono, my undergraduate university mentors, for their advice, and my family and friends, for their encouraging words and love. v TABLE OF CONTENTS LIST OF TABLES............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii ABSTRACT....................................................................................................................... ix 1. LITERATURE REVIEW ................................................................................................1 BARLEY (HORDEUM VULGARE L. EMEND. BOWDEN).....................................................1 Barley Diseases........................................................................................................2 FUSARIUM HEAD BLIGHT...............................................................................................2 GENETIC ENGINEERING OF PLANTS...............................................................................4 Agrobacterium-mediated Transfer of DNA.............................................................5 Biolistic (Particle) Bombardment Method...............................................................7 Electroporation and Polyethylene Glycol Treatment Method .................................8 Selection...................................................................................................................8 CONSTRUCT (PLASMID)................................................................................................10 Promoter.................................................................................................................11 Terminator..............................................................................................................14 RECOMBINANT BARLEY ...............................................................................................15 PHYTOPATHOLOGY.......................................................................................................17 Genetic Approach ..................................................................................................17 Candidate Anti-disease Genes for Barley Transformation ....................................19 LIPID-BINDING (TRANSFER) PROTEINS ........................................................................19 Puroindolines .........................................................................................................21 Hordoindolines.......................................................................................................24 2. MATERIALS AND METHODS...................................................................................27 BARLEY VARIETY AND GROWING CONDITIONS .........................................................27 CALLUS INDUCTION AND REGENERATION ..................................................................27 PLASMID AMPLIFICATION ............................................................................................29 BOMBARDMENT (PARTICLE PREPARATION)................................................................30 GUS Exression ......................................................................................................31 Transformation of Barley with the Puroindoline a Gene.......................................33 DNA EXTRACTION .......................................................................................................35 RNA EXTRACTION........................................................................................................36 SOUTHERN HYBRIDIZATIONS.......................................................................................37 NORTHERN HYBRIDIZATIONS ......................................................................................39 3. RESULTS ......................................................................................................................42 OPTIMIZATION ..............................................................................................................42 vi TABLE OF CONTENTS – CONTINUED GUS EXPERIMENTS ......................................................................................................46 BOMBARDMENT EXPERIMENT .....................................................................................48 SCREENING FOR TARGET GENES IN PUTATIVE TRANSGENIC PLANTS.......................52 4. DISCUSSION................................................................................................................57 LITERATURE CITED ......................................................................................................66 vii LIST OF TABLES Table Page 1. Concentration of 2,4-D and BAP in Callus Induction (CI) Medium......................28 2. Concentration of 2,4-D and BAP in Osmoticum Medium (OM) and Callus Regeneration (CR) Medium. .......................................................................29 3. Callus Induction Frequency (CIF%) Callus Growth Rate (CGR, mg/embryo/day) and Callus Quality Frequency (CQF%) on CI Media.....43 4. Green Callus Frequency (GCF%) Observed on Intermediate Media (IM), Callus Regeneration Frequencies (CRF%) Observed on CR Media .....................45 5. GUS Experiment with pScBVuidA and pRQ6 Plasmids .......................................47 6. GUS Experiment with Plasmid pScBVuidA and Three Rupture Disks (650, 900, 1100 psi) .............................................................47 7. Particle Bombardment with Plasmids pUbipinA and p35Shph..............................48 8. Particle Bombardment with Double and Single Shots with Plasmids pUbipinA and p35Shph ..................................................................49 9. List of Putative Transgenic Plants ..........................................................................51 viii LIST OF FIGURES Figure Page 1. Plasmid pRQ6.........................................................................................................31 2. Plasmid pScBVuidA...............................................................................................32 3. Plasmid pUbipinA ..................................................................................................33 4. Plasmid p35Shph ....................................................................................................34 5. Southern Blot Analysis of 35 Putative Transgenic Lines.......................................54 6. Northern Blot of pinA Expression in 35 Putative Transgenic Lines ......................55 7. Northern blot of hph Expression in 35 Putative Transgenic Lines.........................56 ix ABSTRACT Feed and malting barley are major crops in Montana. Harrington,