1a.Objectives (from AD-416):
The research objectives are to reduce potentially negative effects of transgene insertion and genomic presence. The aim of this proposed research is to investigate the use of novel unidirectional recombinases Bxb1, CinH, ParA and phiC31 to implement a Recombinase-Mediated Cassette Exchange (RMCE) technique for precise integration with simultaneous marker removal.
1) To generate transgenic founder citrus Carrizo citrange lines containing the RMCE genetic platform for precise targeting.
2) To demonstrate proof of concept that the dual unidirectional RMCE is functional in Carrizo citrange.
1b.Approach (from AD-416):
Single copy transgenic Carrizo citrange founder lines will be generated containing the selection gene cassette flanked by fused recognition sites. An exchange vector will be biolistcally transformed into the various Carrizo citrange founder lines. Recombinase mediated cassette exchange will be examined by negative selection and used to score the most effective pairs. The most effective Carrizo citrange founder lines and recombinase pairs will be published and made publicly available.
The pCTAG plasmids that will be the targets for Recombinase Mediated Cassette Exchange (RMCE) have been constructed and transformed into citrus using Agrobacterium. Approximately 200 transgenic lines have been identified with selection so far. Molecular analysis of the pCTAG target citrus lines is underway. Identification of single copy insertion lines is a top priority and more transformation experiments have been initiated to increase the chances of finding them. In preliminary experiments to establish parameters for mature citrus tissue transformation, a range of concentrations for several selection agents are being tested for their ability to inhibit growth of citrus in culture. Transient assays demonstrated that the DsRed fluorescent protein can be used as a reporter gene in citrus. Construction of the Agrobacterium-based pEXCH vector is underway. This plasmid will be used to deliver genes of interest via RMCE for genomic insertion. This research supports Objective 1 of the parent project: Develop and deploy in crop plants site-specific recombinase-based systems.