The project has five objectives:(1) Remove the flowering-promoting CTV and the HLB bacterial pathogen in the transgenic plants(2) Graft CTV- and HLB-free buds onto rootstocks(3) Generate a large number of vigorous and healthy citrus trees(4) Plant the citrus trees in the site secured for testing transgenic citrus for HLB responses(5) Collect the field trial data In this quarter, the following activities have been conducted: (1) Transgenic citrus plants for field trials were maintained in the greenhouse. These plants will be transplanted into the field on May 20, 2021. (2) Cloning the citrus gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). We have constructed a vector based on citrus DNA sequences for generating cisgenic or intragenic citrus plants. However, the transformation efficiency of the vector is extremely low. We plan to develop a transformation selection method based on citrus DNA sequences to facilitate this process. It has been shown in other plant species that an EPSPS variant is able to provide tolerance to glyphosate. We thus cloned the citrus EPSPS gene full-length coding sequence from sweet orange total cDNA. This gene will be mutated to create a similiar citrus EPSPS variant. (3) Optimizing conditions for analyzing nicotinamide adenine dinucleotide-binding activities of a group (10) of citrus lectin receptor kinase proteins using Monolith NT.115. Four different buffers with a pH value in the range of 5-8 have been tested. Although binding activity was detected for some of the proteins using the binding test model, a reliable Kd value has not been achieved. Our goal is to find the functional citrus nicotinamide adenine dinucleotide-binding recptor for generation of intragenic or cisgenic citrus plants. We are testing the transgenic citrus plants expressing the Arabidopsis nicotinamide adenine dinucleotide-binding receptor for HLB resistance/tolerance.