1a. Objectives (from AD-416)
1. Create new genetic combinations of citrus germplasm via conventional breeding, mutation, and transformation. 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
This report documents research conducted under the parent citrus breeding project of the U.S. HORTICULTURAL RESEARCH LABORATORY in Ft. Pierce, Florida. Progress on the project included making crosses for new hybrids, growing hybrid seedlings, planting field trials for evaluation of new hybrids, and collecting performance information from trials for new selections. One new seedless mandarin cultivar, ‘US Early Pride’, was released for commercial use. Studies are underway on citrus tristeza virus resistance of new rootstock hybrids using reverse grafts. Greenhouse tests were completed to evaluate exotic germplasm resistance to Huanglongbing (HLB) disease. Data was collected on HLB infection and symptoms in selected field trials to evaluate potential rootstock influence on HLB-related decline. The outstanding new rootstock cultivars US-812 and US-942 and the standard scion cultivars Hamlin and Valencia were transformed with the resistance genes D4E1, garlic leaf lectin, and pyrrhocorcin. Multiple transgenic plants of each gene-cultivar combination were established in the greenhouse and are in the process of being tested for resistance to HLB and Citrus Bacterial Canker (CBC). A total of 39 antimicrobial peptides (AMPs) have been assessed in-vitro for activity in suppressing growth of the bacteria causing citrus canker and two surrogates for Liberibacter that are closely related alpha-proteobacters (Sinorhizobium melliotii and Agrobacterium tumefaciens):the synthetic AMPs D4E1 and D2A21 were among the most active, with minimum inhibitory concentrations (MIC) at 1µM or less across all test bacteria. Research is continuing to follow leads generated by the HLB gene expression study completed last year, including cloning of selected genes and promoters strongly expressed in response to HLB infection. These sequences are being used as targets for novel exotic genes, to help identify endogenous resistance genes, and to selectively express transgenes when and where the tree is infected by HLB. Work was continued to study the inheritance of fruit quality factors in sweet orange-type material using more than 1000 trees from populations of hybrids between high quality pummelo and mandarin parents. Work was initiated to study gene expression in seedless cultivars and identify genes that may be specifically associated with seedlessness. Juvenility in citrus seedlings is a major impediment to rapid selection and utilization of improved citrus cultivars, and a large field experiment has been established to test components of a reported juvenility reduction system (training upright, thorn removal weekly, and fall girdling). Two scion selections were sent to the United States Department of Agriculture, Agricultural Research Service Citrus Repository, Riverside, California. Five selections are ready for release from quarantine for field testing. A representative from the Yuma Mesa Agricultural Center, Arizona, visited the US Horticultural Research Laboratory research field sites to inspect potential selections for testing in Arizona. Two rootstock selections are under consideration for shipment to the Texas A&M University-Kingsville Citrus Center.
1. Release of New Cultivar ‘US Early Pride’ seedless mandarin. The objective of this work was to develop a seedless mutant of ‘Fallglo’ an important, but seedy mandarin variety. By irradiating budwood of ‘Fallglo’ a seedless mutant was produced. The seedless mutant has been evaluated for horticultural characteristics as well as consumer acceptance and found to be equivalent to ‘Fallglo’ in all aspects except for the absence of seeds. Seedless varieties of citrus fruit are preferred in the market and this seedless variety will expand marketing potential for commercial citrus growers and provide consumers with a novel, more convenient variety of citrus fruit.
2. Creation of Transgenic Rootstocks and Scions. The diseases Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the United States. Insertion of genes conferring resistance to these diseases or their insect vectors is a promising way to solve these problems. Transgenic citrus rootstock and scion cultivars containing the genes D4E1, garlic leaf lectin, or pyrrhocorcin have been created and established in the greenhouse. These transgenic plants may possess resistance to these diseases or insect vectors and are being tested with HLB, CBC, and Asian Citrus Psyllid.
3. Discovery of Exotic Germplasm with Tolerance to Huanglongbing Disease. The disease Huanglongbing (HLB) presents a serious threat to the future success of citrus production in the United States. New hybrids between the Papeda subgroup and citrus cultivars were observed to exhibit strong tolerance to Huanglongbing disease in greenhouse testing. These hybrids will be used as parental material for breeding high quality rootstock and scion cultivars with tolerance to Huanglongbing.
4. Development of antimicrobial peptide transformation vectors. The diseases Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the United States. Insertion of genes conferring resistance to these diseases or their insect vectors is a promising way to solve these problems. Transformation vectors for five antimicrobial peptides (AMPs) were constructed. These AMPs are D4E1, pyrrhocorcin, defensin, msrA1, and 6m1. These vectors are suitable for incorporating genes for each of these AMPs into citrus trees, and thus potentially creating new cultivars resistant to Huanglongbing and citrus bacterial disease.
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