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.
This year, several components needed to perform Recombinase-Mediated Cassette Exchange (RMCE) in soybean were developed. Transgenic soybeans were made using four different vectors and three different cultivars. The fluorescence protein DsRed has proven to be useful in identifying transformants during the shoot regeneration phase after the Agrobacterium co-cultivation. Two of transgenic lines exhibited inheritance consistent with a single locus containing the target transgene. One of the single-locus lines exhibits high transgene expression levels, indicating that its insertion site is located in a genomic environment that is “open” for expression and potentially, targeted integration. The transformed line is being grown for seed increase. In experiments to establish parameters for use of the negative selection marker gene codA to identify plants that have undergone RMCE, the minimal concentration for toxicity of the selection agent to wild-type soybean roots was determined. Transformants containing codA were generated and shown to be stunted by growth on levels of the selection agent that do not affect wild type soybeans. These experiments establish that the negative selection strategy can be used to identify transformants after transgene cassette exchange. Transient assays were used to show that the site-specific recombinases Bxb1, CinH, ParA and PhiC31 are functional in the soybean tissue. This research supports Objective 1 of the parent project: Develop and deploy in crop plants site-specific recombinase-based systems.