University of Alberta Environmental biosafety of field scale plant molecular farming with safflower {Carthamus tinctorius L.) by Marc Alexander McPherson © 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 Edmonton, Alberta Spring, 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-45565-4 Our file Notre reference ISBN: 978-0-494-45565-4 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada University of Alberta Library Release Form Name of Author: Marc Alexander McPherson Title of Thesis: Environmental biosafety of field scale plant molecular farming with safflower {Carthamus tinctorius L.) Degree: Doctor of Philosophy Year this Degree Granted: 2008 Permission is hereby granted to the University of Alberta Library to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. 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. Signature Abstract The oilseed crop safflower {Carthamus tinctorius L.) has been transformed to produce high-value proteins for plant molecular farming (PMF) and resistance to a broad- spectrum herbicide. There are many potential benefits of PMF to society and industry. However, prior to commercialization of crops intended for PMF safety of the food/feed system and the environment must be assured. As part of a preliminary biosafety assessment I conducted a literature review and experiments to quantify potential exposure, gene flow, via pollen and seed from transgenic safflower to the environment and the food/feed system. I evaluated the potential for pollen mediated gene flow (PMGF) from transgenic safflower to one or more of its wild/weedy relatives and to commodity safflower. Safflower is cross-compatible with several wild relatives in the Mediterranean, its center of origin. However, only two cross-compatible relatives occur in North America, C. oxyacanthus and C. creticus. PMGF from transgenic to commodity safflower was found to decline rapidly with distance from the pollen source. PMGF closest to the transgenic pollen source (0 to 3 m) ranged from 0.48 to 1.67% and rapidly declined to between 0.0024 to 0.03 % at distances of 50 to 100 m. To quantify potential seed mediated gene flow (SMGF) from transgenic safflower, I examined seed losses at harvest, seed persistence in soil, efficacy of herbicides to control safflower volunteer survival and fecundity in follow crops. Total seed loss at harvest in commercial fields was large equivalent or well above the recommended seeding rate for safflower (150 viable seeds m"2). Safflower has a relatively short longevity in the seed bank and viable seeds were not found in soil after two years. Safflower volunteer plants in commercial fields and small plot experiments did not survive chemical fallow, but in some cereal and broad leaved crops a small number of plants did survive and generate small amounts of viable seed. Modified cultural practices (best management practices) could be adopted to mitigate PMGF and SMGF from transgenic safflower in the agroecosystem, but thresholds of zero are not practically or biologically realistic. For Dad & Jaimie, and our loving memories of Mom. AKNOWLEDGEMENTS I would like to acknowledge the contribution of the thesis committee and my external examiner. I would like to thank Jocelyn Montgomery for moral support and understanding. I would like to extend my gratitude to Brian Otto, Jerry Kubik Jr., Dan Stryker and their families for use of their land and the time they provided during interviews, planning of experiments and helping to maintain plot areas. I would like to give special thanks to Keith and Debby Topinka for their hard work and dedication which often went above and beyond the call of duty. I really appreciate your support and belief in myself and the project. I would like to acknowledge the technical support from Lisa Raatz, Boris Henriquez, Greg Iwaasa and technical, agronomic, and scientific inputs from several people with Alberta Agriculture and Food. I would like to thank Hardeep Rai, Jeff Saarela, Jason Cathcart and Lisa Raatz for their critical review of a manuscript (Chapter 2) early in my program. I would like to thank a fellow student Ryan Nielson for his input throughout my PhD work. I would like to extend my gratitude to Bruce Alexander for tending my plants and advice on greenhouse experiments. I could not have completed this thesis without statistical advice from Rong-Cai Yang. I would like to extend my gratitude for his great patients and time spent providing me with guidance. I would like to thank Brad Hanson and Carol Mallory-Smith for statistical advice and SAS code used for the regression analysis of the outcrossing data set and Curtis Rainbolt, Donald Thill, Robert Zemetra and Dale Shaner for advice on analysis of the dose-response data. I would also like to thank Peter Blenis for several meetings regarding analysis and presentation of results. I would like to thank Allen Good, Ross McKenzie, Jason Cathcart and Jed Christianson for their scientific input throughout the project. This study was made possible by funds from the University of Alberta, Natural Sciences Engineering Research Council of Canada (NSERC), New Initiatives Fund Alberta Agriculture and Food (AAF), and SemBioSys Genetics Inc. I have learned an enormous amount from Linda and would like to thank her for guidance and support over these last four years. I appreciate the experimental work, scientific discussions and writing we have done together. We have had some great successes during the time I have worked and studied together. Table of Contents Chapter 1: Biosafety assessment of field scale plant molecular farming with safflower (Carthamus tinctorius L.) as a model crop 1 INTRODUCTION 1 DESCRIPTION OF SAFFLOWER 2 HERBICIDE RESISTANCE 4 PLANT MOLECULAR FARMING 8 Potential value of PMF 8 Potential products 11 Alternative production systems 14 Plant production platforms 15 ADVANTAGES, DISADVANTAGES AND FUTURE CHALLENGES FOR PMF.... 16 Optimization of heterologous protein yields in plants 16 Outlook for PMF 29 BENEFITS OF TRANSGENIC PLANTS 32 Biosafety of plants with novel traits (PNT) in Canada 34 Transgene movement in the environment and food/feed system 39 RISK ASSESSMENT 44 EXPERIMENTAL QUESTIONS 45 Pollen mediated gene flow 46 Seed mediated gene flow 48 CONCLUSIONS 49 REFERENCES 53 Chapter 2: Theoretical hybridization potential of transgenic safflower {Carthamus tinctorius L.) with weedy relatives in the New World 66 INTRODUCTION 68 RESULTS FROM THE LITERATURE 71 Taxonomy and phylogeny 71 Interspecific hybridization within Carthamus 74 GEOGRAPHIC DISTRIBUTION 82 BIOLOGY AND ECOLOGY 83 Carthamus tinctorius 83 Carthamus lanatus 84 SEED BIOLOGY OF SOME CARTHAMUS SPECIES 85 OUTCROSSING RATES 86 ECOLOGICAL NICHE 87 CONCLUSION 88 REFERENCES 100 Chapter 3: Issues of ferality and domestication for safflower intended for plant made pharmaceuticals 105 SAFFLOWER: FERALITY IN A PLANT MADE PHARMACEUTICAL PLATFORM 105 PLATFORM FOR PLANT MADE PHARMACEUTICALS 107 Safflower, systematics, biology, biogeography 107 Systematics and hybridization potential between cultivated safflower and its wild relatives 107 Carthamus: Biology and Biogeography 109 DOMESTICATION TRAITS IN CARTHAMUS Ill FERALITY OF SAFFLOWER 114 INFORMATION REQUIRED TO IMPROVE OUR PREDICTION FOR PMP SAFFLOWER FERALITY