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.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, & contribute more to financial self-sufficiency of our lab. We made great strides in increasing transformation efficiency of some cultivars 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. During this last quarter, we have been short-staffed since Jan 21 because our growth room assistant was in a motorcycle crash & he was on a ventilator for 3 weeks & he had to undergo rehab. Thankfully he will be ok. We have been busy in the growth room with much physical labor because mature transformation process requires it. Thankfully Dr. Dutt had a visiting student who assisted in the growth room during this time. One genetic construct submitted by a faculty member did not work at all, so no transgenic tissues or plants were produced. Another 3 genetic constructs submitted by a different faculty member produced shoots, but he did not want these transgenic shoots micrografted. He only wanted photos of the shoots. Because of these unusal orders, we have not been producing plants for significant monetary gain. However, we did produce ~41 transgenic scion shoots for ourselves, testing our new selection method. We have also been testing different rootstocks developed by Dr. Grosser for micrografting to see if there are better alternatives to Carrizo to lessen losses in micrografting transgenic shoots. We tested different basal tissue culture media for grapefruit & found that one works best for regenerating shoots & that it is superior to the standard sweet orange protocol. It still remains to be seen whether Agrobacterium-mediated transgenics can be produced using this medium or whether mature grapefruit cultivars are recalcitrant to Agrobacterium. We have conducted numerous biolistic transformations & tested different variables to enhance transformation efficiency. Bombardments were conducted with different vectors, cultivars, & treatments, but results are still pending. A spontaneous mutant of Early Valencia 1 (EV1) was discovered in the growth room. The leaves are enormous compared to the non-mutant (wild-type) EV1, so perhaps it has become a tetraploid. This tree had only undergone the process of shoot-tip grafting for plant introduction in previous years, but there was no 2,4-D in the shoot-tip grafting medium to induce mutations. Apparently Valencia derivatives undergo spontaneous mutations frequently. We will bud this mutant & the OLL20 mutant previously discovered from FDACS, challenge them with HLB, to determine whether they have HLB disease resistance. Mutations are the most frequent source of new cultivars in citrus. 2. Please state what work is anticipated for next quarter: Work will continue for two scientists who have submitted a number of vectors. Another scientist might be interested in testing Arobacterium transformation of grapefruit now that we have optimized the medium for shoot production. We are also finishing two manuscripts on the plant selectable marker & enhanced transformation of mature citrus. 3. Please state budget status (underspend or overspend, and why): CRDF funding is sufficient, however we will probably overspend the Director’s account since we have not been producing plants for much monetary gain, unless we can make up for this in later quarters.