1a. Objectives (from AD-416):
To reduce potentially negative effects of transgene insertion and genomic presence.
1b. Approach (from AD-416):
We will 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. Single copy transgenic Carrizo citrange founder lines will be generated containing the selection gene cassette flanked by fused recognition sites. An exchange venctor will be biolistcally transformed into the various Carrizo citrange founder lines. RMCE will be examined by negative slection 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.
3. Progress Report:
This project is related to Objective 1: Create new genetic combinations of citrus, Objective 2: Screen germplasm for important traits and select superior individuals, Sub objective 1 D: Create new scions and rootstocks with potential resistance to Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) by genetic transformation. The targeting construct containing the recombinase platform has been completed and consists of the recombinase recognition sites for integration upstream of the positive/negative selection genes, Kan::codA. Kan is used for positive selection of transgenic citrus production while the codA gene will be used to detect successful recombination events. Downstream of the selection system are recognition sites for recombinase-mediated excision. Also included between the recombinase recognition sites is a minimal promoter beta-glucuronidase-Plus gene. This gene will allow detection of the targeting construct inserting into hyperactive chromosomal expression loci. This type of locus is desirable for high expression of transgenes. Due to the placement of the minimal promoter beta-glucuronidase-Plus between the recombinase recognition sites it will be removed during recombinase-mediated targeting. More recently a second version of the targeting construct has been completed with an improved selectable expression cassette. Efforts are now underway to complete the agro-bacterium based pEXCH vector, which will be used to deliver both recombinases (for Recombinase-Mediated Cassette Exchange) and genes of interest (for genomic insertion). Carrizo has been transformed with the preliminary construct, to provide material for initial testing. To date more than 2,500 explants have been treated and 157 potential lines from Carrizo and 149 from Hamlin have been micrografted exvitro with more potentially transformed shoots continually being developed. Two independent transformation events with pCTAGV-KCN3 vector were realized and DSRed expression could be detected transiently during co-cultivation (Figure 4). Well-developed shoots were induced after 35 days in selection medium, 57 explants from Carrizo and 48 explants from Hamlin.