Development of Citrus Cultivars Resistant to Huanglongbing Disease by Transformation with Amp Genes

New citrus cultivars with resistance to the devastating disease Huanglongbing (HLB) are urgently needed in Florida and everywhere HLB is found.  Surveys of citrus germplasm for resistance to HLB that may be used in conventional breeding have not yet identified any reliable sources of strong resistance to this bacterial disease.  Even if sources of resistance are identified, developing new citrus cultivars through conventional breeding may take too long to wait for the desperately needed new cultivars. The most promising approach appears to be transformation of good existing cultivars with trangenes that confer resistance to HLB or its vector.  Antimicrobial peptide (AMP) genes have been selected for our initial work in transformation because of their broad activity against bacterial pathogens and preliminary work indicating suppression of bacteria closely related to the Liberibacter pathogen that causes HLB.  Methods for the Agrobacterium-mediated genetic transformation of citrus using seedlings of the rootstocks US-802 and US-812 were refined to obtain about 4 putative transformed shoots in vitro per explant seedling of US-802 and 5 shoots per explant seedling of US-812.  Candidate transformed shoots of the rootstocks were rescued, after antibiotic selection, by an improved micrografting technique.  Approximately 30% and 86% of the shoots from US-802 and US-812, respectively, survived the micrografting and transition to the greenhouse. Testing of established micrografted plants in the greenhouse for transgene insertion, indicated that 37% and 76% of the US-802 and US-812 greenhouse plants, respectively, were transformed with the target AMP transgene.  Selected transgenic citrus plants with the AMP transgene were challenged with HLB disease using psyllid and graft inoculation under greenhouse conditions.  During preliminary testing, some AMP transgenics appeared fully susceptible to HLB infection, while others appeared to have some resistance.   Testing of the AMP transgenic citrus for resistance to HLB is continuing.