Characterization of Transgenic Peanuts Expressing Oxalate Oxidase for Governmental Approval of Their Release for Control of Sclerotinia Blight

Abstract

<i>Sclerotinia minor</i> Jagger is a fungal pathogen of cultivated peanut (<i>Arachis hypogaea</i> 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 <i>Sclerotinia</i> spp. With little to no naturally occurring resistance to Sclerotinia blight in <i>Arachis</i> 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 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.