2008 Annual Report 1a.Objectives (from AD-416) Produce multiple independent transgenic cell lines and plants of peanut by microprojectile bombardment of embryogenic tissues cultured in vitro. Test for tissue specific expression of genes driven by tissue specific gene promoters in transformed peanut. Test peanut tissues transformed with fungal resistance genes for resistance to Aspergillus flavus/parasiticus. 1b.Approach (from AD-416) Embryogenic plant cell lines will be bombarded with DNA (containing genes for kanamycin or hygromycin resistance as selective markers) and stably transformed peanut tissues selected for antibiotic resistance. The tissue/organ specific expression of a beta-glucuronidase reporter gene driven by a soybean vegetative storage protein gene promoter will be tested in transgenic Arachis hypogaea progeny of the T1 and T2 generations. Other promoter/reporter or promoter/antifungal gene combinations will be similarly tested. As fungal resistance genes become available from cooperators, these genes will be tested for their effectiveness in inhibiting growth of Aspergillus flavus/parasiticus in peanut tissues transformed with these genes. 3.Progress Report Multiple genetic approaches to reduce aflatoxin contamination of peanut are being explored. Putative antifungal genes being tested include a human anti-apoptotic gene Bcl-xl, and a chloroperoxidase gene (CPO-P) from a bacterium, Pseudomonas pyrrocinia. The CPO-P gene under the control of the gene expression trigger called the CaMV35S promoter, which was introduced into peanut by a microprojectile bombardment technique (called genetic transformation), and multiple lines containing the gene, were tested for expression of the gene that was transferred (transgene). Expression was detected for both the direct gene expression product, ribonucleic acid (RNA), and protein. One line has been advanced for several generations, indicating that the gene insertion is stable and will be inherited in subsequent generations. This line also has shown a consistent 50 percent reduction in growth of the aflatoxin producing fungus, Aspergillus flavus, when seeds are inoculated in laboratory assays. The CPO-P line was planted in two aflatoxin tests in 2006. Each test contained 10 plots of the CPO-P line that were subjected to drought stress and will be assayed for aflatoxin production. The second protein introduced into peanut through genetic transformation was Bcl-xL, a human anti-apoptotic gene. Bcl-xL transgenic plants of other species show less negative responses to a wide range biotic and abiotic stresses. Our Bcl-xL transgenic peanut also demonstrated resistance to paraquat, a chloroplast-targeted herbicide. Research progress was monitored through teleconferencing, visits to cooperators labs, and scientific discussions at professional society meetings and the Aflatoxin Elimination Workshop sponsored by industry stakeholders.