The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. Major accomplishments per objective (1) Obtain mature tissues of the best transgenic lines: successfully achieved. The citrus flower-promoting gene FT3 was previously cloned into the CTV vector by the Dawson lab. The CTV-FT3 construct was introduced into Agrobacterium. Tobacco leaves were infiltrated with the resulting Agrobacterium. CTV-FT3 recombinant virions were purified from systemically infected tobacco leaves and bark flap inoculated into C. macrophylla seedlings, which began blooming in about five months. Buds from the matured C. macrophylla were grafted onto the original plants of all transgenic lines (EDS5-Dun-205-9, ELP3-Dun-207-8, ZMSN-Ham-73-1, ZMSN-Dun-137-2, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25). All plants began blooming in 6-18 months. We have successfully achieved this objective and also demonstrated that CTV-FT3 is efficient for converting juvenile tissues to mature tissues. The CTV-FT3-infected C. macrophylla plants that have bloomed can be used as bud source for promoting maturation. The three independent transgenic lines (NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25) that have shown robust tolerance to HLB have been treated under the alternating temperature conditions (25�C for 4 hours and 42�C for 4 hours) to remove CTV and the CLas bacteria. The resulting clean germplasms will be used to generate trees for field trials. (2) Determine whether transgenics prevent psyllids from being infected: accomplished with negative results. CLas-infected psyllids can transfer the CLas bacteria to the next generation of psyllids by inoculating the flush area in which the nymphs develop, which allows the next generation of psyllids to continue spreading HLB without the need for another source plant. In our experiments, we noticed that several of the transgenic lines exhibit delayed or reduced levels of CLas after infection. We started to test if the delayed or reduced production of CLas is sufficient to prevent or reduce the infection of the progeny psyllids. CLas bacteria-carrying transgenic plants were placed in cages, and clean psyllids (not infected by CLas) were moved into the cages. The progenies of the psyllids were collected and tested for CLas titers. A total of six rounds of cage experiments with vegetatively propagated plants from the transgenic lines ELP3-Dun-207-8, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25 were conducted. Results showed that none of the transgenes was able to pre-vent psyllids from being infected by CLas. (3) Continue testing generations of vegetative propagation from the best transgenic lines: successfully achieved. Three independent transgenic lines, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25, have gone through at least six rounds of HLB inoculation. Three generations of progenies (18 replicates for NPR1-Ham-13-3, 31 replicates for NPR1-Ham-13-29, and 25 replicates for NPR1-Dun-57-25) were inoculated with CLas-infected psyllids. The inoculation was repeated until all plants were CLas positive based on qPCR. All progeny plants have shown no or minor HLB symptoms. The three transgenic lines are thus highly tolerant to HLB and will be put into the field for field trials.