1a.Objectives (from AD-416):
The immediate objectives of this cooperative research project are to test and deploy novel promoter and site-specific recombination systems into crop plants. The ultimate goal is to make genetic engineering more precise, more useful in improving crop traits, and more likely to find applications in plant production. The first phase of the research will focus on establishing a recombination platform in soybean that will allow precise site-specific insertion of gene sequences into the genome and deletion of unneeded sequences such as marker genes after genetic transformation.
1b.Approach (from AD-416):
The proposed research will develop the platforms needed to use two novel pairs of unidirectional recombinase enzymes, namely Bxb 1 and ParA, for performing precise genetic engineering of crops. These recombinases can perform controlled DNA integration into or excision from plant chromosomes based on the presence and orientation of specific recognition target sites. Agrobacterium-mediated transformation will be used to generate multiple candidate founder transgenic lines in which the recombination platforms can be tested and shown to function for generating transgenic plants with valuable new traits and lacking superfluous foreign DNA.
The plasmid constructs required for initial transformation were made and introduced into soybean cotyledons using Agrobacterium. Two transgenic lines have been identified so far, but only one of them has a single copy of the target transgene. That line is being grown for seed increase. More transformation experiments have been initiated to identify more single-copy target lines. In preliminary experiments to establish parameters for the conditional negative selection using marker gene codA, wild type soybean cotyledon-derived callus was cultured on a range of concentrations of the selection chemicals. The minimal concentration for toxicity was determined and this will be the level used to select codA transgenics. This research relates to Objective 1 of the parent project, the development of recombination systems for plants.