1. Please state project objectives and what work was done this quarter to address them: Since this CRDF proposal was written in 2018 & the external review occurred, the status of our lab has changed at UF, & in the future, we will conduct less research & focus more on service. In any event, the objectives of this project were to produce disease resistant, commercially & agronomically desirable, mature citrus transgenics, cisgenics, & intragenics that will flower & fruit naturally using Agrobacterium & biolistics as a service 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 Agro-mediated transformation efficiency of some cultivars in Year 2 (after testing virB5 & plant hormones) & found a completely unrelated method that improves transformation efficiency. A manuscript was submitted for review to my supervisor. An all-citrus selectable marker that functions well in transformation, as an alternative to nptII, was also elucidated in Year 2. A manuscript is being written describing the new selection system. The objective of research into DNA-free gene editing will be continued only in collaboration with a customer(s). From one customer, we received four vectors, performed five transformations, none of his vectors worked & no transgenics were produced. This scientist is using a new vector backbone that doesn’t function in mature citrus scions or rootstocks. We are in the process of assisting him troubleshoot this issue. In addition, the Agro strain he is using, EHA105, tends to rearrange the vectors & the genes of interest can be lost. We transformed some of his vectors into the AGL1 strain, which is RecA- & doesn’t rearrange as often. From now on, we will always include a control transformation with a reliable vector that consistently yields results. Another scientist gave us three vectors to transform into mature citrus & we produced 50 transgenic shoots. He did not want these shoots grafted onto rootstock, but we took high quality photographs for him. The scientist wrote a manuscript with the photographs of the transgenics & included us as co-authors. For another order, we produced ~82 transgenic shoots in one rootstock transformation, an excess of 40 were micrografted, & it is too soon to tell how many will survive. 2. Please state what work is anticipated for next quarter: A number of vectors have been transformed into mature citrus & we are awaiting results. In addition, we have more orders for Agro-mediated transgenics, so we are budding mature citrus for these transformations. In addition, two potential orders for biolistics are pending, however the efficiency of biolistics is still being increased by testing different osmotic treatments & cultivars. Unfortunately the lab member responsible for biolistics is away on sick leave, so we must conduct the optimizations for him. Budding of Kuharske, Carrizo, US942, Hamlin, Valencia, & EV1 was performed for biolistic transformation in order to collect stem tissue explants more appropriate for bombardments. In addition, Dr. Dutt is currently making genetic constructs that will be tested for one biolistic order. We might get additional orders once the outcomes of grant competitions are announced. 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 lost time & money doing five transformations that didn’t work, unless we can make up for it in later quarters.