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 plasmid constructs required for initial citrus transformation were completed. Production of transgenic citrus containing the ‘TAG’ site required for recombinase mediated targeting was initiated. Currently over 1000 transformed citrus callus cultures are growing. They will be transferred to media to trigger plant regeneration. The pEXCH plasmid, required for Recombinase-Mediated Cassette Exchange with the genomic ‘TAG’ sites, is being constructed. The design will be modified to allow both biolistic and Agrobacterium-mediated delivery of the DNA. The compounds required for the conditional negative selection (using marker gene codA) were tested on wild type citrus cultures and the range of concentrations that are not toxic was established. This research relates to Objective 1 of the parent project, the development of recombination systems for plants.