������� This project produces transgenics, cis/intragenics & subgenics, in agronomically acceptable cultivars, for field testing & potential commercialization.The original proposal was for a 3 year funding period, but the project was only funded for 1 year because the CRDF wanted transgenics made by a company in Brazil. In 2016, after the CRDF realized that logististically, transgenics could not be easily made in Brazil, the mature citrus facility (MCF) was funded 2 more years. In total, 3 proposals were written for this project. Because of instability in funding, similar to what is presently occurring with short-term contracts, it has been difficult to keep good employees & maintain productivity.������� The� significant objectives for the 1st & 2nd funding periods were: Mature plant production as a service using with Agrobacterium harboring vectors with disease resistance genes & molecular analyses to show copy number of the transgenes & gene expression;� Plant propagation to form replicates for field testing; Increase micrografting efficiencies, bypass it altogether, or root mature scion; Test different selectable markers & reporters; Develop a biolistics protocol for immature/mature citrus; Introduce new, high yielding cultivars for tests in transformation; Apply for external funding. At the beginning of this funding cycle, new customers were charged a nominal fee for transgenics because previously our services were free.��������� All of the abovementioned objectives have been addressed.� Plant production for customers, for technology development, & for increasing efficiency produced ~437 transgenics for this 3 year period (~558 in total since 2014).� An additional 400 transgenics were propagated for customers, either through budding or rooting.� Mature scion cannot be rooted, micrografting cannot� be bypassed, but micrografting efficiencies are stable at 77% by 1 operator. Molecular analyses were conducted for customers (~120 qPCR assays for 1 customer) & for publications, & thousands of endpoint PCRs for the gene of interest conducted. Several grants proposals were submitted & 2 small proposals were funded. Biolistic transformation was developed for immature & mature citrus, & an new selectable marker significantly increased efficiency. This was the first report of biolistic transformation of citrus with plant regeneration. This objective will become increasingly important considering new 2018 USDA APHIS guidelines in which APHIS will not oversee field tests for cis/intragenics/subgenics that do not carry plant pest or vector sequences, & these trees can be fast-tracked to growers at reduced expense, essentially similar to cultivars produced with traditional breeding.�������� High yielding cultivars from the Plant Improvement Team were introduced & Agrobacterium & biolistics transformation efficiencies determined. There is one new scion cultivar that has an extermely high Agrobacterium transformation efficiency & another scion cultivar has reproduciby high efficiency. Most cultivars can be transformed using biolistics, although none have exceedingly high transformation efficiency yet.� A cisgenic selectable marker, constructed by Drs. Zale & Dutt, is being tested in mature citrus to increase transformation efficiency.� An intragenic citrus reporter from Dr. Dutt works well to replace GUS.� A UF CREC Initiative during this timeframe was to stack two genes in transgenics to prevent the bacteria from overcoming resistance of one gene. An unfunded UF scientist, complied with the request to make the stacked gene constructs, & the MCF produced ~150 transgenics to meet this UF CREC Initiative. Biolistic-mediated gene editing of the PDS gene was achieved in immature Carrizo & Valencia.� One customer’s Agrobacterium vector had a tendency to rearrange, in Agrobacterium prior to transformation.� A total of 33 trees were produced for this scientist, but the transgenes rearranged in all but 2 events. This mutation was documented by restriction digests of vector DNA grown in E. coli vs Agrobacterium.� A consecutive double budding method was devised so that mature citrus is reinvigorated well prior to experimentation.������� New services were added that offer biolistic transformation of minimal cis/intragenic expression cassettes. This service, if utilized by scientists, will provide significantly more monetary revenue for the mature citrus facility. However the CRDF should encourage scientists to use bioistics for cis/intragenics for faster & cheaper deregulation. Growth room maintenance is expensive & it is expensive to staff the MCF.� A number of publications were generated by this project.��