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) A total of 85 plants including 65 transgenic plants and 20 non-transgenic control plants were planted into the field on May 20, 2021. These transgenic plants include replicates of three transgenic lines that have been shown to have robust tolerance to HLB in the greenhouse. Ten replicates of another transgenic line that expresses a different disease resistance gene were also included. This transgenic line has shown HLB tolerance in the greenhouse for more than eight years. In addition, we have eight transgenic Carrizo lines that express three different disease resistance genes. These lines have been replicated and grafted with Valencia. The three constructs was added onto the field trial permit. These plants will be transplanted into the field in the Fall of 2021. The transgenic plants that were transplanted in 2019 were examed. The plants grow well in the field and none of the plants has shown HLB symptoms. We plan to collect samples for CLas titer assay in this fall.
(2) The citrus gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was cloned and sequenced. The two conserved amino acids T177 and P181, which are responsible for glyphosate tolerance, were changed to isoleucine (I) and serine (S), respectively. The resulting citrus TIPS EPSPS gene was cloned into a T-DNA binary vector, which was introduced into Agrobacteria. To test if the citrus TIPS EPSPS gene can provide tolerance to glyphosate, we transformed it into Arabidopsis, since it will take shorter time to know the result in Arabidopsis. We will have plants for glyphosate tolerance test in the next quarter.
(3) Transgenic citrus plants expressing the Arabidopsis nicotinamide adenine dinucleotide-binding receptor were inoculated with CLas using psyllids. We are waiting for the HLB symptom development. Meanwhile, we repeated nicotinamide adenine dinucleotide-binding experiment for the citrus lectin receptor kinase proteins using Monolith NT.115. We plan to use radiolabeled nicotinamide adenine dinucleotide to confirm the binding results in the next quarter.