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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC IMPROVEMENT OF CITRUS

Location: Subtropical Insects and Horticulture Research

2010 Annual Report


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
This report documents research conducted under the parent citrus breeding project of the USHRL 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. Over the past year, efforts have focused on finding resistance to Candidatus Liberibacter asiaticus, the organism believed to cause the disease huanglongbing, as well as methods to make breeding progress in the presence of the disease. Genetic transformation with genes active against huanglongbing disease or its vector was targeted in the transgenic program. To date, thousands of transformants with antimicrobial genes have been produced and are in the process of being tested. Studies of host plant changes in gene expression following infection with huanglongbing are being used to identify citrus gene targets that may be manipulated to increase host plant resistance to the disease. Transgenic citrus trees have already been produced targeting one such gene, citrus PP2, and are being tested. Liberibacter sequence data are being used to develop a transgene that will target a Liberibacter-specific transmembrane transporter. Peptides have been made specific to the extra-membrane sequence and are being tested in E. coli containing the Liberibacter gene. 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. The long term safety and integrity of the USDA citrus breeding material, which comprises perhaps the greatest diversity of citrus hybrids in the world, has been threatened by Huanglongbing disease. Efforts have focused on testing the breeding collection for the presence of Liberibacter, protection of germplasm, and research on methods to eliminate the pathogen from infected, but highly valuable material. A simple approach involving identifying Liberibacter negative branches on Liberibacter positive trees coupled with antibiotic treatment of buds has proven effective. Differences in susceptibility to huanglongbing among commercial varieties have been observed. Using surveys of field trees, Minneola and sweet orange tend to be most uniformly high in incidence of Liberibacter infection, while the cultivar Temple typically is lowest in Liberibacter infection. Poncirus trifoliata and some of its hybrids have been observed to tolerate and/or significantly suppress Liberibacter infection. Transcriptome analysis was conducted for early fruit development in three seedy citrus genotypes and their seedless mutants: several differences were consistent across all seedy vs. seedless comparisons. Eight seedling trees of each of 106 different diverse accessions from the citrus repository have been planted in a randomized complete block design at the Ft. Pierce farm, with data collected on HLB and psyllid infestation.


4.Accomplishments
1. Released New Cultivar US-942 citrus rootstock. Florida citrus production is threatened by the recently-introduced bacterial disease huanglongbing. High fruit productivity on young compact trees is one strategy that may allow profitable citrus production of existing susceptible varieties in the presence of the disease. One promising new hybrid citrus rootstock with outstanding fruit productivity on compact trees was developed by ARS researchers in Ft. Pierce, Florida and released in 2010 for commercial use. This new rootstock, US-942, has exhibited outstanding performance on several different sites and with several different scions. There is large commercial interest in this new citrus rootstock in the U.S.

2. Created Transgenic Rootstocks and Scions for resistance to Huanglongbing disease. More than one thousand transgenic citrus rootstocks and scions have been produced by ARS researchers in Ft. Pierce, Florida with engineered genes that have good potential to induce resistance to huanglongbing, citrus bacterial canker, or asian citrus psyllid. Antimicrobial peptide D4E1-transformed sweet orange has displayed resistance to citrus canker. Other antimicrobial peptides and more active promoters have been identified and used in recent transformations. Collaboration with an ARS team in Albany, CA is providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Citrus genomic data has been produced to identify the appropriate citrus genes for this approach. Transgenic citrus cultivars with resistance to hungalongbing and commercial acceptability will be of great benefit for the continued production of citrus in the U.S.

3. Identified Antimicrobial Peptides active against the pathogens causing citrus canker and Huanglongbing disease. ARS researchers in Ft. Pierce Florida have used in vitro assays to identify antimicrobial peptides with strong activity against the pathogen causing citrus bacterial canker and bacteria closely related to the bacteria causing huanglongbing. More than 20 synthetic antimicrobial peptides were assessed. Two synthetic antimicrobial peptides were among the most active, along with a crustacean antimicrobial peptide, Tachyplesin, with minimum inhibitory concentrations at 1 micromolar or less across all test bacteria. Antimicrobial peptides with strong activity against the pathogens causing citrus canker and huanglongbing are invaluable for the development of cultivars resistant to these diseases and the long-term success of the U.S. citrus industry.

4. Determined the effects of Candidatus Liberibacter asiaticus on citrus juice quality. When huanglongbing disease was confirmed to be present in Florida questions arose regarding impacts the disease may have on juice quality. There was speculation that the juice from diseased trees produced novel off-flavor compounds that would have negative effects on juice flavor. Studies were conducted by ARS researchers in Ft. Pierce, Florida to compare chemical and flavor components of juice from healthy and HLB-affected trees. Results indicate that the most consistent difference between juice from healthy or diseased trees was a reduction in total sugar content. No novel off-flavor compounds were produced. Although trained taste panelists could distinguish between juice produced from healthy or diseased fruit, differences were minimal and related to less sweetness and more acidity. These results indicate that production of good-tasting citrus and citrus juice may continue from existing cultivars even though they become infected with huanglongbing. This information is of critical importance to citrus grove managers, citrus juice processors and citrus consumers.

5. Developed improved methods for citrus transformation. Genetic improvement of citrus by transformation is critical to the rapid development of new cultivars resistant to citrus canker, huanglongbing, and other disease problems. Current methods for citrus transformation are relatively inefficient and can only be used with juvenile tissue. ARS Researchers at Ft. Pierce, Florida identified plant growth regulator types, proportions, and concentrations that resulted in a substantial increase in shoot regeneration in sweet orange and grapefruit epicotyl explants cultures used in genetic transformation experiments. For mature tissue transformation:.
1)Four populations of adult phase trees were established in the greenhouse;.
2)A decontamination protocol was developed that results in >90% clean explants, sufficient for tissue culture studies and practical applications;.
3)A system was developed for the production of in vitro adult phase shoots from cultured nodes of greenhouse trees; and.
4)A system was developed for the production of shoots from cultured internodes from greenhouse trees. The system results in shoot and bud formation in 70-90% of the explants. These improved methods for citrus transformation will be of tremendous benefit in the development of transgenic citrus cultivars with resistance to hungalongbing.


Review Publications
Aradhya, M.K., Stover, E.W., Velasco, D., Koehmstedt, A. 2010. Genetic structure and differentiation in cultivated fig (Ficus carica L.). Genetica. 138:681-694

Zhang, M., Duan, Y., Turechek, W., Stover, E.W., Powell, C.A. 2010. Screening molecules for control of citrus Huanglongbing (HLB) using an optimized regeneration system for 'Candidatus Liberibacter asiaticus' infected periwinkle (Catharunthus roseus) cuttings. Phytopathology. 100:239-245.

Niedz, R.P., Evens, T.J. 2010. The effects of benzyladenine and meta-topolin on in vitro shoot regeneration of a citrus citrandarin rootstock. Research Journal of Agricultural & Biological Sciences. 6:45-53.

Niedz, R.P., Evens, T.J. 2010. Effect of Agrobacterium culture and inoculation density on transformation efficiency of a citrange (Citrus reticulata x Poncirus trifoliata). Journal of Forestry and Horticulture. 2(3):30-37.

Stover, E.W., Dangl, G., Yang, J., Bautista, J.C., Aradhya, M.K. 2010. Investigations into the origin of 'Norton' grape using SSR markers. Proceedings of Florida State Horticultural Society. 122:19-24.

Last Modified: 4/17/2014
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