|Thomson, James - Jim|
Submitted to: Citrograph
Publication Type: Trade Journal
Publication Acceptance Date: 9/4/2015
Publication Date: 1/1/2016
Citation: Oliveira, M.L., Stover, E.W., Thomson, J.G. 2016. Utilization of founder lines for improved Citrus biotechnology via RMCE. Citrograph. 7:56-60.
Technical Abstract: On October 1st 2011 the CRB chose to fund a unique research project, the development of citrus cultivars specifically for genetic engineering (GE). The objective of this research was to develop GE citrus ‘Founder Lines’ containing DNA sequences that will allow the precise insertion of genes for desired traits using biotechnology. Founder Lines insure predictable gene expression because a single copy of the transgene(s) is inserted in a region of the citrus genome that provides high and consistent transgene activity without disrupting the activity of nearby native genes. This research was previously discussed in the 2013 4(1) Citrograph article entitled ‘Founder lines for improved citrus biotechnology’. Since the initial inception of this project a number of goals have been accomplished. Most importantly, 335 transgenic plants from Carrizo and 15 transgenic sweet orange plants have been obtained. Of these, 123 transgenic lines from Carrizo and all 15 candidate Founder Lines in sweet orange were confirmed to each contain a single copy of the transgene. The article details the strategy for assembling the DNA construction used to create the Founder Lines, including the importance of target site orientation and how all the parts function to assist the recombinase mediated cassette exchange (RMCE) targeting process. Figures are provided that demonstrate how successful transformation can be visualized by fluorescence encoded by the DsRed gene and how the codA gene is used to select for successful RMCE swapping events. Also discussed is the genetic transformation process itself, why it’s necessary, its current limitations, and potential expansions of the method to other cultivars and types of explants.