2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This CRIS project is part of National Program 302, Plant Biological and Molecular Processes. It has the goal of minimizing unintended and/or potentially negative impacts of plant transformation processes and of the introduction of new genes into the genome. Specifically we seek to understand and reduce or eliminate the effects of transgene insertion and expression on non-targeted plant processes, on the environment, and on the human food supply. Tools to be developed include those that. 1)confine expression of the newly introduced genes to the tissues and developmental stages or to the environmental situations in which they are needed;. 2)minimize the disruption of the plant genome by insertion of new sequences;. 3)eliminate extraneous foreign gene sequences not needed to make the desired change. The approach is a combination of molecular biology and genetic transformation. The characteristics of the resultant plants will be evaluated at the molecular and functional levels. The purpose of the research is to address the perceived lack of safety and predictability that currently limits the usefulness and public acceptance of biotechnology for the improvement of crops. One concern about the commercial release of transgenic crops is the possibility that they will have adverse impacts on the environment and on the food supply. A second concern is that introduction of new genes could have effects on plant metabolism other than those intended. This research will create tools that refine transformation technology and minimize these possibilities. New technologies will be developed to improve the predictability and control of the expression and location of transgenes in the host genome. An expected outcome is the ability to block or decrease transgene expression in plant tissues destined for human consumption, thereby increasing public acceptance of transgenic crops for commercial release. 2.List by year the currently approved milestones (indicators of research progress) Year 0 (FY 06): None, first four months of project. Year 1 (FY07): Milestone 1 - Characterize and document reporter gene expression in transgenic rice containing candidate native organ-specific promoters. Milestone 2 - Initiate research to identify several candidate crop promoters that are responsive to biotic and/or abiotic stress. Characterize and validate the stress responsive expression of selected candidates using northern blots or quantitative RT-PCR. Milestone 3 - Complete recombinase test constructs for excision in planta (pTC1), recombinase test constructs for integration in planta (pTC2, pTC3), recombinase test constructs for chromosomal integration in planta (pTC4) and the recombinase expression vectors (pRE-X) for the 6 recently discovered recombinase genes plus Cre and phiC31 as controls. Year 2 (FY08): Milestone 4 - Retrieve and sequence 5' flanking regions of validated stress responsive genes to use as putative promoters. Fuse candidate promoter regions to GFP and GUS reporter genes in Agrobacterium vectors and initiate plant transformation. Milestone 5 – Complete the transient excision assay using pTC1 in dicot and monocot protoplasts. Repeat with new pRE-X constructs if needed. Complete the transient integration assay using pTC2 and pTC3 in dicot and monocot protoplasts. Repeat with new pRE-X constructs if needed. Milestone 6 - Production of the transgenic plants for the genomic excision assay using pTC1, and the genomic integration assays using pTC2 and pTC3 as the chromosomal targets. Year 3 (FY09): Milestone 7 - Create transgenic wheat and/or barley plants containing candidate organ-specific promoters. Milesone 8 – Complete the the genomic excision assay using pTC1, and the genomic integration assays using pTC2 and pTC3 as the chromosomal targets. Repeat with new pRE-X constructs if needed. Milestone 9 - Complete the crosses for the genomic excision assay. Year 4 (FY10): Milestone 10 - Create transgenic wheat, barley and/or Brachypodium distachion plants containing pathogen-induced or abiotic stress-responsive promoters. Milesone 11 – Complete progeny analyses for genomic excision assay. Repeat with new pRE-X constructs if needed. Milestone 12 – Conduct recombinase mediated genomic integration with best pRE-X constructs. Year 5 (FY11): Milesone 13 - Characterize and document reporter gene expression in transgenic plants containing heterologous candidate organ-specific and stress responsive promoters. Milestone 14 - Analysis of pRE-X recombinase mediated genomic integration events. Additional recombinase optimization experiments if needed. 4a.List the single most significant research accomplishment during FY 2006. None, first four months of the new project. Please see report for in-house project 5325-21000-008-00D. 4b.List other significant research accomplishment(s), if any. None. 4c.List significant activities that support special target populations. None. 4d.Progress report. The OSQR project plan for the new 5-year plan was written and approved. The research for this project plan began in February, 2006. 5.Describe the major accomplishments to date and their predicted or actual impact. None. This report covers the first few months of this project. For previous project, see report for CRIS # 5325-21000-008-00D. Research in this project will contribute to Component 3 of NP 302 – Plant Biotechnology Risk Assessment, specifically Problem Statement 3A, Improving and Assessing Genetic Engineering Technology. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? We expect to patent all new discoveries from this project for use in the public domain by academic and biotechnology company scientists. Constraints: Even with these improvements, some members of the food industry and some consumers may continue to oppose genetically engineered crops. As the development time for GM crop plants takes a decade, new technology developed today will take some time before it will be incorporated into commercial products. For example, the marker removal technology was published by ARS in 1991, but it took 15 years for Monsanto to put out a first product using Cre-lox to remove the kanamycin resistance marker from a high lysine corn line scheduled for the commercial market in 2006. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). None yet for this project; see annual report for Project 5325-21000-008-00D.