2008 Annual Report 1a.Objectives (from AD-416) Development of tightly regulated gene switches for trait improvement and functional genomics applications in forage plants: (1) Development of a EcR gene switch for use in monocot system. (2) Testing of EcR gene switch in functional genomics application using zinc finger genes as a model system. 1b.Approach (from AD-416) Expression of transgenes is becoming a powerful tool in many biotechnological applications in agriculture. Constitutive promoters, those that are expressed all the time in all tissues, are presently the primary means used to express transgenes in plants. Metabolic energy waste, negative pleiotropic effects and potential gene escape are some of the disadvantages associated with the use of constitutive promoters. To counter these problems, gene switches that can regulate the expression of transgenes through hormone analogs and antibiotics have been developed. Ligands that are suitable for regulation of transgenes for large-scale production and field use are the commercially available non-steroidal ecdysone agonists. Ecdysone agonist and ecdysone receptor (EcR) have been shown to regulate gene expression in tobacco, Arabidopsis and corn. In a previous project, highly sensitive EcR gene switches that can support the induction of transgenes with nanomolar concentration of ecdysone agonist in both Arabidopsis and tobacco plants were developed. This work aims to test the use of these gene switches in monocots, ultimately to be used in forage based systems. The first approach will be used to use the gene switch in rice as a model monocot system with routine transformation procedures. A second approach will be to use the gene switches to evaluate the performance of the EcR gene switch system in functional genomics using a zinc finger transcription factors. This will be tested as a model to system to evaluate its use for transcription factors, and will be tested in Arabidopsis, and if successful, will be converted for use in rice and Lolium ssp. 3.Progress Report Performance of this agreement is monitored by USDA-ARS, FAPRU scientists being actively involved as collaborators in the agreement project, and meeting with the collaborator on an as needed basis to alter experimental design. Information exchange and planning is via face to face meetings, teleconferences or email on a regular basis. Most genes in plants are under temporal or tissue/organ/cell specificity, and only those genes essential for cellular function are expressed ubiquitously. Development of transgenic approaches will require the use of gene expression systems that can be manipulated, and one approach is through the application of exogenous compounds. The goal is the development of an inducible promoter system that can be used in forage based systems in the field. An ecdysone-based system has been initially developed at the University of Kentucky for use in tobacco and Arabidopsis and will now be tested for suitability in forage applications. The first approach toward this goal is to determine whether the ecdysone inducible promoter functions in monocots by using rice as a model system. Rice plants containing the inducible promoter have been recovered. Analysis is ongoing to determine the level of expression of the reference gene (GUS) in these plants. Tissue culture and transformation techniques for tall fescue have been problematic, but are in progress.