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 were performed:
(1) A total of 27 transgenic rootstock plants were planted into the field on October 8, 2021. These transgenic plants include eight transgenic Carrizo lines that express three different disease resistance genes. The transgenic rootstocks were replicated and grafted with Valencia. The transgenic plants that were transplanted in 2019 and earlier in 2021 were examed. The plants grow well in the field and one plant from the 2019 planting has shown HLB symptoms. Tissues will be collected in the fall for CLas titer assay.
(2) To test if the citrus 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mutant (called EPSPS TIPS) could provide tolerance to glyphosate, we have transformed a T-DNA construct carrying the citrus EPSPS TIPS mutant gene into Arabidopsis. Seeds from the first generation (T1) plants were screened on kanamycin plates to identify single T-DNA insertion lines. Six independent single T-DNA insertion lines were identified. We are currently screening for homozygous plants with the seeds collected from T2 plants and will test their glyphosate tolerance once homozyous lines are obtained.
(3) The citrus genome encodes several putative nicotinamide adenine dinucleotide-binding receptors. Nicotinamide adenine dinucleotide-binding activities of two of the putative receptors were tested once. These putative receptors appeared to have binding activity, though the result needs to be confirmed. CLas-inoculated transgenic citrus plants expressing the Arabidopsis nicotinamide adenine dinucleotide-binding receptor were maintained in the greenhouse and no HLB symptom has been developed. We plan to graft the transgenic scions onto sweet orange rootstocks for easier detection of HLB resistance or tolerance.