Submitted to: International Research Conference on Huanglongbing
Publication Type: Abstract Only
Publication Acceptance Date: 10/20/2014
Publication Date: N/A
Technical Abstract: We have designed an innovative system to to deploy a novel pair of recombinase enzymes, namely Bxb1 and CinH, for performing precise genetic engineering of citrus (Thomson et al. 2012). They control the integration and the excision of sequences based on the presence and orientation of specific recognition target DNA sites allowing the precise integration of transgenes, with the simultaneous removal of marker genes and/or any other unneeded sequences (Wang et al. 2011). Their unidirectional activity ensures that the integration and excision events produced are non-reversible and stable, generating citrus lines with reliably and uniformly expressed introduced traits. The binary pTAG vector contains the double enhanced 35S promoter constitutively expressing the fusion gene codA:nptII, and a DsRed visual reporter gene expressed by GmUbi3 promoter, which are flanked by fused recognition sites, with the integration recognition sites (attP - for Bxb1) on the 5’ side and the excision recognition sites (Res - for CinH) on the 3’ side. The translationally fused codA:nptII selectable marker gene, provides both positive selection (via the antibiotic kanamycin in the medium, for which nptII provides resistance) and negative selection (via 5-FC, which is made toxic by codA ). s Single copy insertion lines containing TAG have been generated, and will be tested for re-transformation with use of the negative selection to select against lines where the marker has not been properly excised. Epicotyl explants from Carrizo and Hamlin were transformed using Agrobacterium tumefaciens strain EHA105 carrying pTAG. A total of three hundred and thirty five candidate founder lines were characterized by Southern blot analysis to identify lines with a single copy of the recombinase platform in the genome. These founder lines will be utilized in developing transgenic plants with priority traits such as huanglongbing resistance.