1a. Objectives (from AD-416)
1. Create new genetic combinations of citrus germplasm via conventional breeding, mutation, and transformation. a. Improve and enhance methods to evaluate and screen new priority commercial citrus scions and rootstock cultivars for resistance and/or tolerance to Huanglongbing and the Asian citrus psyllid. 2. Screen germplasm for important traits and select superior individuals. 3. Evaluate selections for field performance and other traits. 4. Release new scion and rootstock varieties for commercial use. 5. Develop new, more effective testing methods (especially RE-PCR) for screening and identifying host-plant resistance to Huanglongbing and citrus bacterial canker, and apply these testing methods to discover novel resistance genes from elite citrus germplasm, unstudied citrus wild species, traditional dooryard varieties, etc.
1b. Approach (from AD-416)
New citrus selections will be created by sexual hybridization, mutation, and genetic transformation from existing cultivars and species. Hybrids and other new types will be tested for important traits using molecular markers, greenhouse and laboratory assays. Promising selections from these assays will be entered into long-term field trials at multiple locations and data collected on tree health, size, fruit yield and quality. Selections that appear to have desirable combinations of traits will be released for commercial or dooryard use.
3. Progress Report
Progress on the project included making crosses for new hybrids, growing hybrid seedlings, planting field trials for evaluation of new hybrids, collecting performance information from trials for new selections, and conducting research to develop foundational information that will improve the development of new citrus cultivars. A high priority for new cultivars is to include resistance or tolerance to the devastating disease huanglongbing (HLB), caused by the bacterium Candidatus Liberibacter asiaticus (CLas). Work has been conducted to compare incidence and severity of HLB among citrus scion and rootstock genotypes in replicated field and greenhouse trials. Poncirus trifoliata, a related species sexually compatible with citrus, has been shown to have tolerance to CLas infection and has been incorporated broadly into breeding materials and advanced rootstock and scion selections. Genetic transformation is being employed to introduce several types of bacterial resistance genes into citrus cultivars with the goal of producing cultivars resistant to CLas. Factors potentially important for efficient genetic transformation of juvenile and mature tissue were investigated, and included types, proportions and concentrations of plant growth regulators, mineral nutrients, carbon sources, explant source, preconditioning treatments, and Agrobacterium growth phase and inoculation density. A series of transformants were produced that linked several different promoters to the beta glucuronidase (GUS) gene, with the goal of identifying which promoter will be most useful in controlling expression of bacterial resistance genes introduced in citrus. Selected hybrids with Poncirus trifoliata and selected transformants with antibacterial genes are being challenged with CLas to indentify relative levels of susceptibility. Challenge with CLas through exposure to infected Asian Citrus Psyllid is being conducted on replicated sets of antimicrobial peptide transformants. Studies were completed to identify differences between HLB-susceptible and tolerant genotypes in gene expression and metabolites. Several genes were identified that appear key in determining tolerance, and will be used to design targets for transformation and selection. Considerable effort was devoted to rescue and preservation of elite citrus germplasm that was present only in the field and in the presence of HLB, citrus canker, citrus tristeza virus as well as insect pests and the possibility of devastating freezes or hurricanes.
1. Characterized pollination requirements of ‘US Early Pride’ seedless mandarin. ‘US Early Pride’ is a new seedless mandarin released by the USDA in 2009. Although ‘US Early Pride’ consistently produces good yields of seedless fruit in the presence of alternate pollenizers, it was not known if ‘US Early Pride’ requires a pollenizer for fruit production nor if ‘US Early Pride’ can serve as a source of pollen for other mandarins that require cross pollination. We determined that ‘US Early Pride’ pollen is limited in abundance and of low viability compared to other mandarin types. In addition, we found that when bees were prevented from visiting ‘US Early Pride’ flowers, no fruit were produced. Taken together these results indicate that it is likely that ‘US Early Pride’ will require an alternate source of pollen to provide acceptable cropping. This information is of critical importance to citrus growers who are interested in producing ‘US Early Pride’.
2. Determined susceptibility to Huanglongbing pathogen Candidatus Liberibacter asiaticus (CLas). CLas infection among sixteen citrus scion genotypes. To screen citrus germplasm for resistance to CLas, an effective method to quantify differences in pathogen abundance among genotypes is essential. In a greenhouse study, we exposed sixteen citrus genotypes to Asian citrus psyllids that were known to be infected with CLas and followed the incidence of infection over one year. We found that detectable levels of the pathogen did not develop until about five months of exposure to the citrus psyllids. Among the genotypes, we were able to detect consistent differences in abundance of the pathogen. Results of this work will be important in the design of experiments to compare resistance to CLas infection among new citrus germplasm that is being created.
3. Identified breeding sources of resistance and tolerance to Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas). We characterized resistance to CLas and Asian citrus psyllid in diverse genetic material in multiple experiments in the greenhouse and the field. Among extensive material tested, Poncirus trifoliata and some of its hybrids display tolerance to CLas infection, resistance to proliferation of CLas, and/or resistance to colonization by Asian citrus psyllid. Poncirus trifoliata has been utilized extensively in USDA breeding efforts, and it is expected that some advanced hybrid selections approaching commercial fruit quality may already have useful resistance or tolerance to CLas and its insect vector. We have identified genes associated with this resistance, through comparison of gene expression in tolerant hybrid and susceptible selections. These genes are being incorporated into new desirable cultivars through hybridization and transformation to help sustain the U.S. citrus industry in the face of HLB.
Maul, D.P., Mccollum, T.G., Guy, C.L., Porat, R. 2011. Temperature conditioning alters transcript abundance of genes related to chilling stress in 'Marsh' grapefruit flavedo. Postharvest Biology and Technology. 60:177-185.