CHARACTERIZATION OF TRANSGENIC PEANUTS EXPRESSING OXALATE OXIDASE FOR GOVERNMENTAL APPROVAL OF THEIR RELEASE FOR CONTROL OF SCLEROTINIA BLIGHT Shanna Marie Chriscoe Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Master of Science In Life Science In Plant Pathology, Physiology and Weed Science Elizabeth A. Grabau, Co-Chair Patrick M. Phipps, Co-Chair Mary Ann Hansen John M. McDowell M. A. Saghai Maroof 8 July 2008 Blacksburg, VA Keywords: Arachis hypogaea, peanut, Sclerotinia minor, Sclerotinia blight, oxalate oxidase, transgenic plants, governmental regulation 0 Characterization of Transgenic Peanuts Expressing Oxalate Oxidase for Governmental Approval of Their Release for Control of Sclerotinia Blight Shanna Marie Chriscoe ABSTRACT Sclerotinia minor Jagger is a fungal pathogen of cultivated peanut (Arachis hypogaea L.) that can cause crop losses in excess of 50%. Fungicides are not completely effective at controlling the disease and can cost up to $311 per hectare for three applications. The ability to produce oxalic acid is necessary for the pathogenicity of some Sclerotinia spp. With little to no naturally occurring resistance to Sclerotinia blight in Arachis spp., a biotechnological approach was used to confer resistance to the disease. Peanut plants were transformed with a gene from barley encoding oxalate oxidase, an enzyme that degrades oxalic acid. Transformed peanuts showed resistance to S. minor and increased yields under disease pressure compared to the parental lines. Before the resistant varieties can be marketed, they must be reviewed and approved by the governmental regulatory system. Responsibility for regulation of transgenic plants in the U.S. is shared among the U.S. Department of Agriculture (USDA) through the Animal and Plant Health Inspection Service (APHIS), the Food and Drug Administration (FDA), and the Environmental Protection Agency (EPA). These agencies require several different data sets including molecular characterization and field studies before each transformation event can be commercialized. This project was designed to characterize three different transformation events, N70, P39 and W171. Molecular characterization included determination of insertion number, copy number, intactness of the expression cassette and stable inheritance of the transgene. N70 was found to have two insertions and two copies while W171 had one insertion with one copy. The P39 event has two ii insertions and two or more copies. Each of the three events was stable over multiple generations. Phenotypic comparisons of each transgenic line to the parent cultivar were carried out in field studies. Characteristics such as oxalate oxidase expression, yield and quality, hay quality, disease occurrence, aflatoxin content and plant height were assessed. Transgenic peanuts showed few differences from the parent cultivar other than resistance to Sclerotinia blight and yield under disease pressure. Outcrossing studies were completed to determine the rate and distance of cross pollination. Outcrossing rates in N70, P39 and W171 were less than 2.5% and occurred up to 19 rows or 17.4 m from the nearest transgenic row. The molecular characterization and field performance of N70, P39 and W171 have been assembled into a document to petition APHIS for determination of non-regulated status. iii TABLE OF CONTENTS ABSTRACT ....................................................................................................................................... ii TABLE OF CONTENTS ...................................................................................................................... iv LIST OF TABLES ............................................................................................................................. vii LIST OF FIGURES .............................................................................................................................. x CHAPTER 1 INTRODUCTION ............................................................................................................. 1 ARACHIS HYPOGAEA ........................................................................................................................ 2 BACKGROUND .............................................................................................................................. 2 PEANUT PRODUCTION AND DISEASE ............................................................................................ 4 OXALATE OXIDASE .......................................................................................................................... 6 PROTEIN CHARACTERIZATION ..................................................................................................... 6 EXPRESSION OF OXALATE OXIDASE ............................................................................................ 9 OXALATE OXIDASE AS A RESISTANCE GENE ............................................................................. 11 DEVELOPMENT OF TRANSGENIC PEANUT LINES ............................................................................ 13 TISSUE CULTURE AND TRANSFORMATION ................................................................................. 13 SELECTING LINES FOR MARKETING ........................................................................................... 14 GOVERNMENTAL REGULATION OF TRANSGENIC PLANTS .............................................................. 15 U.S. DEPARTMENT OF AGRICULTURE ........................................................................................ 16 FOOD AND DRUG ADMINISTRATION ........................................................................................... 16 ENVIRONMENTAL PROTECTION AGENCY ................................................................................... 17 SUBMITTING DATA FOR GOVERNMENTAL REVIEW .................................................................... 18 OBJECTIVES ................................................................................................................................... 21 REFERENCES .................................................................................................................................. 22 CHAPTER 2 PETITION FOR THE DETERMINATION OF NON-REGULATED STATUS ............................ 26 SUMMARY ...................................................................................................................................... 28 DIFFERENCE FROM A TYPICAL PETITION .................................................................................... 31 RATIONALE FOR THE DEVELOPMENT OF TRANSGENIC PEANUTS WITH SCLEROTINIA BLIGHT RESISTANCE ................................................................................................................................... 32 THE BASIS FOR THE REQUESTING NONREGULATED STATUS ...................................................... 32 RATIONALE FOR THE DEVELOPMENT OF N70, P39 AND W171 .................................................. 32 Effects of the 2002 Farm Bill on VA-Type Peanut Producers and the Benefits of using Transgenic Plants .................................................................................................................. 32 Rationale for Using Oxalate Oxidase to Control Sclerotinia minor ..................................... 34 ADOPTION OF N70, P39 AND W171 ........................................................................................... 35 SUBMISSIONS TO OTHER REGULATORY AGENCIES .................................................................... 35 Submission to the FDA ......................................................................................................... 35 Submission to the EPA ......................................................................................................... 36 iv Submissions to Foreign Governments .................................................................................. 36 THE PEANUT FAMILY ..................................................................................................................... 37 TAXONOMY................................................................................................................................ 37 PEANUT AS A CROP .................................................................................................................... 38 ORIGIN AND HISTORY OF PEANUT ............................................................................................. 39 GENETICS OF PEANUT ................................................................................................................ 40 POLLINATION OF PEANUT .......................................................................................................... 41 WEEDINESS OF PEANUT ............................................................................................................. 41 CHARACTERISTICS OF RECIPIENT PEANUT MATERIAL ............................................................... 41 NC 7 ...................................................................................................................................... 42 Wilson ................................................................................................................................... 42 Perry ...................................................................................................................................... 42 DEVELOPMENT OF TRANSGENIC PEANUT