Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 204 - Plant Product Quality and Utility (Preharvest) | 920 - Orange | 1080 - Genetics (excludes breeding) | 40% |
| 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants | 910 - Grapefruit | 1000 - Biochemistry and biophysics | 30% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1160 - Pathology | 30% |
Agrobacterium-mediated genetic transformation will be used on nucellar citrus shoot tissue to reduce survival, proliferation, and/or virulence of the causal pathogens for HLB and citrus canker. Efforts will include antimicrobial peptides and proteins (AMPs); transgenes associated with the pathogens and possibly those that trigger host plant disease responses. A parallel strategy is incorporating transgenes that deter or kill the psyllid vector. Both constitutive and phloem-specific promoters will be used. Transgenic plants will be tested for resistance in the laboratory, greenhouse, and field. Huanglongbing (HLB) is now well-established in FL and it is predicted that essentially all FL citrus will be infected in 7-10 years. It is unlikely that the disease or its vector will be eradicated and, there are no known management strategies to control HLB or maintain acceptable yields with HLB present. No HLB-resistance has been identified in cultivated citrus, making transgenic solutions the priority for plant material to permit sustained economic citrus production where HLB is endemic. Canker is now endemic and an important factor in economic value and productivity, especially for fresh-market citrus.
This project is related to inhouse project objective 1: Create new genetic combinations of citrus, objective 2: Screen germplasm for important traits and select superior individuals, Sub objective 1 D: Create new scions and rootstocks with potential resistance to huanglongbing (HLB) and citrus bacterial cancker (CBC) by genetic transformation. Numerous transgenic plants have been created using numerous constructs. Many verified transgenics are micrografted and replicated. Plants from the initial round of scion transformations are now replicated and are being exposed to Huanglongbing, using graft inoculations and Liberibacter infected psyllids in greenhouse and field environments. Challenge with HLB through exposure to infected Acian citrus psyllid (ACP) is being conducted on a replicated set of 33 independent Hamlin transformants, 5 Valencia transformants, 4 midseason transformants, and 3 non-transformed controls. Several events continue to grow better than all controls at 12 months after initiating the challenge, with 35 percent greater trunk- cross-sectional area increase than the overall experimental average and 64 percent greater growth than the mean of the controls, but do not show immunity to Liberibacter development. A series of promoters were tested with the beta-glucuronidase (GUS) gene. The three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives unusually consistent and high GUS activity. D35S produces the highest level of expression but with great variability between events. The manuscript will be submitted before the end of the fiscal year. Liberibacter sequence data target a transmembrane transporter as a possible transgenic solution for HLB resistance. In E. coli expressing the Liberibacter (CLas) translocase, two exterior epitope-specific peptides suppressed adenosine triphosphate (ATP) uptake by 60+percent and significantly suppressed CLas growth in culture. After verification these will be used to create transgenes. Anthocyanin regulatory genes, give bright red shoots and were tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Unfortunately, when antibiotics were left out of regeneration media, almost no red shoots were recovered. However, high anthocyanin apples are reported to have field resistance to bacterial fire-blight, presumably due to high levels of phenolic compounds. Red citrus transgenics will be tested for Huanglongbing, Asian citrus psyllid and canker resistance. High throughput evaluation of Huanglongbing (HLB) resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and natural psyllid inoculation in the field are being compared. The first trial has been in the field for 34 months and a repeated trial has been in the field for 22 months. Leaf samples have been collected monthly and Polymerase chain reaction (PCR) analysis of CLas conducted. Comparison of field-grown and greenhouse-grown Valencia following graft-inoculation show much more rapid CLas development in greenhouse-grown trees. Several new collaborations are being explored to feed new Huanglongbing- suppressing transgenes and novel strategies into the citrus transformation pipeline. Sequence data are being mined for citrus antimicrobial peptides and defensins to test in-vitro and ultimately in- planta.