University of Alberta Environmental biosafety of genetically engineered crops: Flax (Linum usitatissimum L.) as a model system by Amitkumar Jayendrasinh Jhala A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Science Department of Agricultural, Food and Nutritional Science ©Amitkumar Jayendrasinh Jhala Spring 2010 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission. Examining Committee Dr. Linda Hall, Agricultural, Food and Nutritional Science Dr. Randall Weselake, Agricultural, Food and Nutritional Science Dr. Lloyd Dosdall, Agricultural, Food and Nutritional Science Dr. Jocelyn Hall, Biological Sciences Dr. Robert Blackshaw, Agriculture and Agri-Food Canada, Lethbridge Dedicated to my Madam for her continuous love, support and encourangement Abstract Flax (Linum usitatissimum L.) is considered as a model plant species for multipurpose uses with whole plant utilization for several purposes including industril, food, animal feed, fiber, nutraceutical, pharmaceutical, and bioproduct markets. Therefore, flax is in the process of genetic engineering to meet the market requirements. Prior to commercial release of genetically engineered (GE) flax, a risk assessment was conducted to determine intra- and inter-specific pollen-mediated gene flow and for quantifing and mitigating the adventitious presence (AP) of volunteer flax in canola (Brassica napus L.). The results of pollen-mediated gene flow study (crop-to-crop) suggest that about 1.85% outcrossing would occur in adjunct area, when two flax cultivars were grown in close proximity of 0.1 m apart. Some rare gene flow events were recorded maximum up to 35 m distance from the pollen source but at a very low frequency. The genus Linum has several wild and weedy species, distributed in many parts of the world. A meta-analysis was conducted to determine the potential for gene introgression from GE flax to wild relatives, the occurrence, the phylogeny of flax wild relatives and reported interspecific hybridization. The results demonstrated that cultivated flax has ability to hybridize and form viable F1 plants with at least nine species of Linum; however, none of these species have been reported to occur in Canada. Hybridization of flax with many other wild relatives has either not been studied or reported. However, based on the evidence of reported work, gene flow from GE flax to wild or weedy relatives may occur elsewhere depending on species distribution, sympatry, concurrent flowering, ploidy level and sexual compatibility. The results of the experiments to mitigate the adventitious presence of flax volunteers in canola suggest that combinations of pre-plant followed by post-emergence herbicides were most effective for reducing volunteer flax density and AP in glufosinate-resistant canola. Post-emergence application of imazamox+imazethapyr, however, was not effective for controlling volunteer flax in imidazolinone-resistant canola. Best management practices were developed to mitigate transgene movement from GE flax to ensure co-existance of GE, conventional and organic flax without market harm. Acknowledgement I express my sincere gratitude to my academic supervisor Dr. Linda M. Hall for her valuable guidance, constructive suggestions, motivation, encouragements and support throughout the period of my doctoral research. Her close observations, dynamic personality and tireless efforts as a mentor are truly appreciated. I feel highly privileged in expressing my profound sense of gratitude for my supervisory committee members Drs. Randall Weselake and Jocelyn Hall for their invaluable suggestions and insightful criticism to review my dissertation. I am also thankful to Dr. Robert E. Blackshaw and Dr. Lloyd Dosdall for his suggestions to improve this thesis. I am indebted to Mr. Keith Topinka for teaching me Canadian agriculture and his help and support. I am also thankful to Dr. Paul Dribnenki and Susan McEachern for providing us breeder seeds of flax for gene flow studies and their assistance with the thiobarbituric acid test. I gratefully acknowledge the technical help and kind cooperation provided by Lisa Raatz, Debby Topinka, Judy Irving, Jaime Crow, Jody Dexter, Marc McPherson, Ryan Nielson and Vanessa Kavanagh. Their invaluable suggestions, timely support and helpful attitude created a very friendly ambience during my research. I also acknowledge the timely help and constant encouragement exemplified by my friends Jigar Patel, Sarthak Patel, Ketul Chaudhry, Japan Trivedi, Amit Singh, Mridul and Mrunal Jain. Financial support from the Alberta Ingenuity Scholarship Fund, Alberta Agriculture and Food, Alberta Advance Education and the University of Alberta is gratefully acknowledged. Finally, I thank my family for their support, motivation and encourangement throughout my study. Table of Contents Chapter 1 Environmental biosafety of genetically engineered crops: Flax (Linum usitatissimum L.) as a model system Introduction……………………………………………………………….1 Global status of genetically engineered (GE) crops………………………3 Environmental biosafety and risk assessment of GE crops……………….8 Major benefits of GE crops……………………………………………...10 Major consequencies of GE crops……………………………………….14 Flax- A model crop………………………………………………………18 Research objectives……………………………………………………...23 References……………………………………………………………….31 Chapter 2 Flax (Linum usitatissimum L.): Current uses and future applications.....................................................................................................41 Introduction……………………………………………………………...41 Area and production of flax ……………………………………………..42 Flax for human consumption ……………………………………………43 Flax for edible oil………………………………………………..............45 Flax for functional food …………………………………………………46 Flax fiber and its uses………………………………………………........49 Flax as animal feed ………………………………………………...........52 Flax for industrial uses ……………………………………………….....53 Conclusion………………………………………………....................….55 References………………………………………………....................….60 Chapter 3 Potential hybridization of flax (Linum usitatissimum L.) with weedy and wild relatives: An avenue for movement of engineered genes?..................66 Introduction……………………………………………………………...66 Taxonomy and phylogeny ………………………………………………69 Variability in chromosome numbers ……………………………………72 Center of origin and evolution of L. usitatissimum……………………...73 Interspecific hybridization in Linum…………………………………….75 Linum hybrids among taxa with n=15……………………………..76 Linum hybrids among taxa other than (n=15)……………………...78 Geographic distribution …………………………………………………79 Linum species in the New World…………………………………..81 Biology and ecology …………………………………………………….83 Linum usitatissimum L. (2n=30)…………………………………...84 Linum perenne L. (2n=18)…………………………………………86 Linum sulcatum Riddell (2n=30)…………………………………...87 Soil persistence of flaxseed……………………………………………...88 Conclusion……………………………………………….........................89 References……………………………………………….......................104 Chapter 4 Pollen-mediated gene flow in flax (Linum usitatissimum L.) and strategy to confine transgene movement: Can biotech and organic flax co-exist? …………………………………………………………………....115 Introduction…………………………………………………………….115 Materials and Methods…………………………………………………120 Plant Material………………………………………………….......120 Field experiments………………………………………….............120 Seed screeing………………………………………………………121 Statistical analysis…………………………………………………122 Results………………………………………………………………….124 Discussion………………………………………………………...........127 Stratagy to reduce transgene movement...……………………………...130 References……………………………………………………………...143 Chapter 5 Adventitious presence: Volunteer flax (Linum usitatissimum L.) in herbicide resistant canola (Brassica napus L.)…………………...............150 Introduction ……………………………………………………………135 Materials and methods ……………………………………...................155 Volunteer flax seed viability test..…………………………...........158 Statistical analysis….……………..……………............................158 Results and discussion ………………………………………................160 Volunteer flax control. ……………………………………...........160 Crop response……………………………………………………..161 Volunteer flax seed production, viability, yield and AP………….162 Sources of materials ………………………………………...................168 References………………………………................……………….......173 Chapter 6 Genetically engineered flax (Linum usitatissimum L.): Potential benefits, risks, regulations and mitigation of transgene movement………............178 Introduction………………………………..………………...................178 History and current status of genetic engineering of flax ……………..182 Potential benefits of GE flax ………………………………………......186 Increased agronomic performance ………………………………..186 Linseed oil for industrial applications…………………………….188 Novel oil products………………………………………………...188 Flax for the nutraceutical market……………………………….....189