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. The following work has been conducted in this quarter: (1) New replicates of the transgenic lines that have been inoculated by CLas-infected psyllids were maintained in the greenhouse for symptom development. Background: We have generated transgenic �Hamlin� sweet orange and �Duncan� grapefruit and screened the transgenic lines for HLB resistance or tolerance. We did not find any resistant line but identified three independent lines (two �Hamlin� lines and one �Duncan� line) that exhibit robust tolerance to HLB. This result indicates that the Arabidopsis NPR1 gene is able to create HLB tolerance in citrus. Since we only have HLB-tolerant �Hamlin� and �Duncan�, we decided to transform the NPR1 gene into other cultivars including sweet orange �Pineapple� and �Valencia� as well as grapefruit �Ray Ruby� (These plants were generated by the mature tissue transformation lab). The following table shows the new lines that have been inoculated by CLas-infected psyllids and are maintained in the greenhouse for symptom development: Replicate Genotype Transgene Parental line A1 Ray Ruby NPR1 21 81 Pineapple NPR1 15 75 Pineapple NPR1 16 66 Pineapple NPR1 17 78 Pineapple NPR1 18 84 Valencia NPR1 20 76 Hamlin NPR1 8 73 Hamlin NPR1 10 67 Hamlin NPR1 11 70 Hamlin NPR1 12 87 Hamlin NPR1 13 79 Hamlin NPR1 14 (2) Propagated plants from those that have been treated with CTV-FT3 and have produced flowers for later heat treatment to remove CTV and CLas. Background: The three independent lines (�Duncan� 57-28, �Hamlin� 13-3, and �Hamlin� 13-29) with robust tolerance to HLB have been treated with CTV that carrying the FT3 gene, which promotes conversion from juvenile to mature tissues. The three lines have all developed blooms. The flowering-promoting CTV and HLB bacterial pathogen (CLas) in the transgenic plants need to be removed before producing healthy progenies. The following table shows the new lines received from the transformation lab and will be replicated and screened for HLB responses: Transgenic line Genotype Transgene Maturation Replicates made 57-28 Duncan NPR1 Yes 4 13-3 Hamlin NPR1 Yes 6 13-29 Hamlin NPR1 Yes 3 (3) More transgenic lines received from the transformation lab were transplanted into bigger pots and will be analyzed for the transgenic protein accumulation. Background: We have not obtained multiple independent lines for �Valencia� and �Ray Ruby� and have requested more from the mature tissue transformation lab. The following table shows the new lines received from the transformation lab and will be replicated and screened for HLB responses: Transgenic line Genotype Transgene A99 Valencia NPR1 A100 Valencia SuperNPR1(a) A102 Valencia SuperNPR1 A101 Valencia SuperNPR1 A72 Valencia ELP3 (b) A73 Valencia ELP3 A97 Hamlin SupperNPR1 A98 Hamlin SupperNPR1 (a) SupperNPR1 is a more active version of the NPR1 gene. (b) ELP3 encodes a disease resistance regulator, which appears to also provide tolerance to HLB. A manuscript titled �Overexpression of the Arabidopsis NPR1 protein in citrus confers tolerance to Huanglongbing� has been written and submitted to the Journal of Citrus Pathology in this quarter.