1. Please state project objectives and what work was done this quarter to address them: The objectives of this project are to produce disease resistant, commercially & agronomically acceptable, mature citrus transgenics & intragenics that will flower & fruit naturally using Agrobacterium & biolistics for research & commercialization. It should also be understood that it is through transgenics (gene over/under-expression) that gene function is determined & this will become an important service in the future. The research focus of this project is to improve Agrobacterium & biolistic transformation efficiency of mature citrus, so that the mature citrus protocols become more productive & decrease prices for scientists. This was accomplished for some rootstocks (Kuharski and US942) and scions (Hamlin and EV1) by selection in liquid rather than solid medium in Year 2. Another objective was to find a citrus selectable marker that functions well in citrus transformation as an alternative to the nptII gene & this was also accomplished in Year 2. Two manuscripts are in preparation: the new plant selectable marker; genetic transformation of mature citrus & selection in liquid culture. ~Thirty scion transgenics were produced, micrografted & secondary grafted this quarter. PCR of ~150 Agro-mediated transgenics was conducted to confirm the presence/absence of the transgenes in plants produced in liquid culture. Southern blots are being performed to show gene integration into the genome & copy number of the T-DNA into the genome for a manuscript. We have been working on biolistic transformation to increase transformation efficiency in anticipation of a future order from California. Numerous bombardments have been conducted, testing different mature cultivars (Hamlin, Valencia, Kuharski & US942), testing liquid culture, & testing the new citrus selectable marker. These experiments are currently in progress, so results cannot be provided yet. We might have to alter the standard protocol that we developed for immature citrus to achieve more efficient biolistic transformation of mature citrus. There are no publications of transformation of mature grapefruit. However, we attempted to produce grapefruit transgenics for Dr. Nian Wang’s group. We tested mature Duncan, Marsh, Ray Ruby & Flame using Dr. Leandro Pena’s tissue culture protocol for sweet orange, which utilizes MS or MT medium, but it did not yield results. In fact, none of the grapefruit cultivars (Ruby grapefruit, Grosser’s Red grapefruit) have ever performed well. The next step would be to test other tissue culture medium (WPM, DPW, DBA3), which differ in macro/micro-nutrients. Since Dr. Nian Wang’s group is trying to produce mature transgenics, perhaps it will test the different media. Because mature grapefruit is recalcitrant to Agrobacterium transformation, I will no longer accept orders for grapefruit until the system can be optimized. 2. Please state what work is anticipated for next quarter: I am waiting to hear from one scientist in California to see if she is interested in using our facility for a biolistic transformation project. Another scientist in California was potentially interested in Agrobacterium transformations for research (I contacted her last year) but she said Dr. Nian Wang, who is Co-PI on their grant, will probably produce their transgenics. Last week, we received a fairly large order from Dr. Zhonglin Mou, and we are working on an order for Dr. Manjul Dutt. 3. Please state budget status (underspend or overspend, and why): The CRDF budget has been received, but I am still waiting for the chart field numbers for the UF Educational Business Account (EBA). As I understand it, my EBA application is still in the CFO’s office pending signatures.