Progress 04/04/08 to 04/03/13
Outputs
Progress Report 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. 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. This project is terminating during this FY13 annual report cycle. The most significant accomplishments over the five years of the project were: 1) Released one new disease-resistant hybrid rootstock, US-942, in 2010. The rootstock filled a much-needed niche for a semi-dwarfing rootstock with good productivity and field tolerance to the Phytophthora-Diaprepes complex; 2) Released a new very low-seeded mandarin. �US Early Pride� is a new, very low-seeded mandarin that was developed and evaluated by the USDA, ARS and will provide US citrus growers with an improved option for an early-season mandarin resulting in greater consumer appeal and a competitive advantage in the market place; and 3) Identified symptoms and gene expression changes associated with huanglongbing disease and tolerance. Five papers were published in refereed journals documenting tolerance to huanglongbing, field performance of different rootstocks when challenged by the pathogen, and gene expression changes associated with disease development and the tolerance reaction. A new project is in place for the next 5 years. During FY 2013, more than 1500 seedlings were produced from 2012 crosses to develop improved citrus scions or rootstocks, and are being grown out for evaluation. In 2013, 1400 crosses were made emphasizing genetic seedlessness for scions, and huanglongbing tolerance for scions and rootstocks. Data on fruit crop, tree size, and health was collected from more than twenty rootstock and scion field trials. Several new promising selections were identified among scion and rootstock hybrids in field testing. Progress has been made to characterize apparent tolerance to huanglongbing in some conventional citrus germplasm. Poncirus trifoliata continues to test as the most huanglongbing-resistant citrus. Numerous replicated field trials have been established with hybrids of Poncirus trifoliata as either the rootstock or scion, and are being evaluated for tolerance to huanglongbing. In field trials under severe disease pressure, several new Poncirus trifoliata hybrid rootstocks have provided the best yield with sweet orange scion, while one new Poncirus trifoliata hybrid scion with commercial quality fruit displays the greatest growth in a scion trial. More than 900 new selections have been produced by genetic transformation of good commercial citrus scion and rootstock cultivars using genes expected to improve resistance to bacterial diseases such as huanglongbing. Testing of transgenic plants was initially conducted in greenhouses using graft or psyllid-inoculation with huanglongbing. Clones that appear resistant were retested and placed into field trials. Several transgenic sweet oranges grow significantly better than controls under severe huanglongbing pressure. Factors potentially important for efficient genetic transformation of juvenile and mature citrus tissue were investigated and included types, proportions and concentrations of plant growth regulators, mineral nutrients, carbon sources, explant sources, preconditioning treatments, culture matrix, and genotype. Accomplishments 01 Rootstocks that significantly improved fruit production under severe huanglongbing disease pressure were identified in field trials. Huanglongbing disease is widespread in Florida and severely affects health in infected citrus trees. Numerous studies were completed by ARS researchers in Ft. Pierce, Florida comparing tree health and fruit cropping of sweet orange and other cultivars grafted on numerous rootstocks in field trials growing in areas severely affected by huanglongbing. The studies demonstrated significant rootstock differences in fruit production under conditions of severe huanglongbing. The results indicate that some rootstocks enable citrus trees to better tolerate huanglongbing disease, and suggest that use of a tolerant rootstock will be one component of successful citrus production management in the presence of that disease. 02 Identified the stage of the transformation process where there is a significant difference between recalcitrant and amenable citrus types. Citrus types vary widely in their transformability. For example, Poncirus and Poncirus hybrids (e.g., Carrizo citrange) are amenable to genetic transformation and routinely have a transformation efficiency > 50%. Conversely, scion types such as sweet orange grapefruit have efficiencies of 1% or less. Improving transformation of the scion types by an order of magnitude would greatly reduce the resources required to widely utilize transgenic technology in citrus. Numerous factors and stages were examined and compared between sweet orange, grapefruit, and Carrizo. Though Carrizo has a higher shoot regeneration capacity, this property is not sufficient to explain the difference in transformability. These results suggest that these citrus types respond differently at the early stages of transformation. Thus, improving the transformation efficiency of the scion types probably requires modified treatment at these early stages. 03 Four new sweet orange-like hybrids with high quality fruit were selected. ARS citrus breeders at Ft. Pierce, Florida have identified four hybrids with high-quality fruit similar to sweet orange in appearance and flavor. All established sweet oranges are selected mutations of the original progenitor orange, and are almost identical genetically. Sweet oranges are among the most highly susceptible citrus to the devastating disease huanglongbing, and the new sweet-orange-like hybrids have entered tests to determine whether they have greater tolerance/resistance. Working with other ARS Researchers, these hybrids have been shown to have aroma volatiles very similar to existing sweet oranges, making it likely they can be officially classified as sweet orange and marketed commercially as sweet oranges. One hybrid is easily peeled by hand. Clean budwood was developed and trees are being produced for testing in growers� orchards.
Impacts
(N/A)
Publications
- Zhao, H., Sun, R., Albrecht, U., Padmanabhan, C., Wang, A., Coffey, M.D., Girke, T., Wang, Z., Close, T.J., Roose, M., Yokomi, R.K., Folimonova, S., Vidalakis, G., Rouse, R., Bowman, K.D., Jin, H. 2013. Small RNA profiling reveals phosphorus deficiency as a contributing factor in symptom expression for Citrus Huanglongbing Disease. Molecular Plant. 6(2):301-310.
- Petracek, P.D., Sun, D., Dou, H., Stover, E.W. 2012. Gibberellic acid (GA3) effects on late season grapefruit peel oil composition. In: Proceedings of the 38th Annual Meeting of the Plant Growth Regulation Society of America. 38:108-116.
- Albrecht, U., Bowman, K.D. 2011. Transcriptional response of susceptible and tolerant citrus to infection with Candidatus Liberibacter asiaticus. Plant Science. 185-186:118-130.
- Benyon, L.S., Stover, E., Bowman, K.D., Niedz, R.P., Shatters, R.G., Zale, J.,M., Belknap, W.R. 2013. GUS expression driven by constitutive and vascular specific promoters in citrus hybrid US-802. In Vitro Cellular and Developmental Biology Plants. 49:255-265.
- Stover, E., Stange, R.R., McCollum, T.G., Jaynes, J., Irey, M., Mirkov, E. 2013. Screening antimicrobial peptides in-vitro for use in developing transgenic citrus resistant to huanglongbing and citrus canker. Journal of the American Society for Horticultural Science. 138:142-148.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. About 1200 seedlings were produced from crosses made in 2011. In 2012, made 49 types of crosses directed at major market types emphasizing genetic seedlessness and huanglongbing-tolerance. Top-quality selections were identified: a fine-flavored large mandarin; a late ripening mandarin; and four sweet-orange- and grapefruit-like trifoliate hybrids. To free them of virus, 21 priority selections were shoot-tip grafted by a collaborator including a peelable orange-type. Progress has been made to characterize apparent tolerance to huanglongbing in some conventional citrus scion cultivars. Several rootstock selections were identified with significant tolerance or resistance to huanglongbing disease. Hybrids with trifoliate orange continue to test as the most huanglongbing- resistant citrus, and a replicated trial of 114 hybrids and standards has been established and genotyped with cooperators. Tree health, size, and cropping information were collected from numerous established field trials. More than one thousand transgenic citrus selections were produced, including numerous gene constructs with potential to induce resistance to huanglongbing disease and other useful traits. Factors potentially important for efficient genetic transformation of juvenile and mature citrus tissue were investigated and included types, proportions and concentrations of plant growth regulators, mineral nutrients, carbon sources, explant source, preconditioning treatments, culture matrix, and genotype. Many transgenic selections have been propagated for further testing, including a select group that was planted into a field trial. Several transgenic sweet oranges grow significantly better than controls under severe huanglongbing pressure. Metabolomic analysis of juice from fruit affected by huanglongbing disease revealed significant differences in the concentrations of sugars, amino and organic acids, limonin glucoside, and limonin. With the exception of phenylalanine, limonin glucoside, and limonin, oranges from huanglongbing infected trees had lower concentrations of metabolites compared to fruit from uninfected trees. Experiments were conducted to determine pollination requirements of the recently released cultivar, US Early Pride mandarin. US Early Pride was hand pollinated using eleven different pollen sources. Results indicate that each pollen parent induced fruit set and could serve as a pollenizer for US Early Pride. In a second experiment US Early Pride trees growing in the field were enclosed in screen cages to exclude alternate sources of pollen; bees were placed in the cages to ensure movement of US Early Pride pollen; results suggest that US Early Pride is self-fertile, but cropping may be improved with the use of pollenizers. Accomplishments 01 Some rootstocks provide improved sweet orange tree tolerance to huanglongbing. Huanglongbing disease is widespread in Florida and severely affects health in infected citrus trees. A study was completed by ARS researchers in Ft. Pierce, Florida comparing tree health and frui cropping on sweet orange trees grafted on numerous rootstocks in four field trials growing in an area severely affected by huanglongbing. The study demonstrated significant rootstock differences in ability to tolerate huanglongbing disease. The results indicate that some rootstoc enable citrus trees to better tolerate huanglongbing disease, and sugges that use of a tolerant rootstock will be one component of successful citrus production management in the presence of that disease. 02 Effects of nutritional treatments on huanglongbing (HLB). Nutritional treatments are being widely used in Florida to sustain production in citrus groves affected by huanglongbing disease. In one study, ARS researchers at Fort Pierce, Florida found that enhanced nutritional treatments of citrus trees affected by huanglongbing disease did not sustain tree health, yield, or fruit quality of Las-infected HLB- symptomatic trees. Moreover, since the nutritional supplements had no effect on Las titer, a major concern is that existing enhanced nutrition strategies have promoted area-wide buildup of inoculum and increased disease spread within and between citrus orchards. The findings of this research will have significant impact on management decisions for citrus growers dealing with huanglongbing affected orchards. 03 Two grapefruit-like cultivars perform better than standard grapefruit under disease pressure. Two diseases, huanglongbing and citrus canker, severely affect standard grapefruit varieties and threaten fruit production in Florida. ARS researchers at Ft. Pierce, Florida have identified grapefruit-like cultivars that perform far better than true grapefruit under severe pressure from huanglongbing and citrus canker. Tree health and productivity of Triumph and Jackson were greater than standard cultivars Marsh and Flame in a replicated field trial assessed for 3 years. In fruit quality assessments Triumph and Jackson fruit always met commercial maturity standards whereas Marsh and Flame fruit usually did not due to low total soluble solids and low Brix/acid ratios These results suggest that Triumph and Jackson or other grapefruit-like cultivars may be viable alternatives to standard grapefruit cultivars in the presence of severe huanglongbing and citrus canker.
Impacts
(N/A)
Publications
- Niedz, R.P., Evens, T.J., Hyndman, S., Adkins, S., Chellemi, D.O. 2011. In vitro shoot growth of Brugmansia x candida Pers. Physiology and Molecular Biology of Plants. DOl: 10.1007/s12298-011-0100-8.
- Albrecht, U., Mccollum, T.G., Bowman, K.D. 2012. Influence of rootstock variety on huanglongbing disease development in field-grown sweet orange (Citrus sinensis L.) osbeck trees. Scientia Horticulturae. 138:210-220.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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 yiel 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 fro visiting �US Early Pride� flowers, no fruit were produced. Taken togeth 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�. 02 Determined susceptibility to Huanglongbing pathogen Candidatus Liberibacter asiaticus (CLas). CLas infection among sixteen citrus scio 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 citru genotypes to Asian citrus psyllids that were known to be infected with CLas and followed the incidence of infection over one year. We found th detectable levels of the pathogen did not develop until about five month of exposure to the citrus psyllids. Among the genotypes, we were able t 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. 03 Identified breeding sources of resistance and tolerance to Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas). We characteriz resistance to CLas and Asian citrus psyllid in diverse genetic material 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 expect that some advanced hybrid selections approaching commercial fruit qualit may already have useful resistance or tolerance to CLas and its insect vector. We have identified genes associated with this resistance, throu 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 citrus industry in the face of HLB.
Impacts
(N/A)
Publications
- Stover, E.W., Portier, K. 2011. Design of field experiments: influence of treatment response relative to standard deviation and covariate characteristics on efficient blocking strategy. Journal of American Pomological Society. 65:2-16.
- 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.
- Castle, W.S., Bowman, K.D., Baldwin, J.C., Grosser, J.W., Gmitter, Jr., F. G. 2011. Rootstocks affect tree growth, yield, and juice quality of 'Marsh' grapefruit. HortScience. 46(6):841-848.
- Hert, M.M., Evens, T.J., McCollum, T.G., Niedz, R.P. 2011. Bud emergence and shoot growth from mature citrus nodal segments. Plant Cell Tissue And Organ Culture. 106(1):81-91.
- Stover, E.W., Mccollum, T.G. 2011. Levels of Candidatus Liberibacter asiaticus and Xanthomonas citri in diverse Citrus genotypes and relevance to potential transmission from pollinations. HortScience. 46(6):854-857.
- Westbrook, C.J., Hall, D.G., Stover, E.W., Duan, Y., Lee, R.F. 2011. Colonization of citrus and citrus-related germplasm by Diaphorina citri (Hemiptera: Psyllidae). HortScience. 46(7):997-1005.
- Niedz, R.P., Evens, T.J. 2011. The effects of benzyladenine and meta- topolin on in vitro shoot regeneration of sweet orange. ARPN Journal of Agricultural and Biological Science. 6:64-73.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report 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. 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. 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. Accomplishments 01 Released New Cultivar US-942 citrus rootstock. Florida citrus productio 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 AR researchers in Ft. Pierce, Florida and released in 2010 for commercial u This new rootstock, US-942, has exhibited outstanding performance on several different sites and with several different scions. There is larg commercial interest in this new citrus rootstock in the U.S. 02 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 acti promoters have been identified and used in recent transformations. Collaboration with an ARS team in Albany, CA is providing constructs wit enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Citrus genomic data has been produced to identif 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 03 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 wit strong activity against the pathogen causing citrus bacterial canker and bacteria closely related to the bacteria causing huanglongbing. More th 20 synthetic antimicrobial peptides were assessed. Two synthetic antimicrobial peptides were among the most active, along with a crustace antimicrobial peptide, Tachyplesin, with minimum inhibitory concentratio at 1 micromolar or less across all test bacteria. Antimicrobial peptide with strong activity against the pathogens causing citrus canker and huanglongbing are invaluable for the development of cultivars resistant these diseases and the long-term success of the U.S. citrus industry. 04 Determined the effects of Candidatus Liberibacter asiaticus on citrus juice quality. When huanglongbing disease was confirmed to be present i 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 total sugar content. No novel off-flavor compounds were produced. Although trained taste panelists could distinguish between juice produce 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. 05 Developed improved methods for citrus transformation. Genetic improveme 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 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 establishe in the greenhouse; 2) A decontamination protocol was developed that results in >90% clean explants, sufficient for tissue culture studies an practical applications; 3) A system was developed for the production of vitro adult phase shoots from cultured nodes of greenhouse trees; and 4) system was developed for the production of shoots from cultured internod 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.
Impacts
(N/A)
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.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations 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 1�M 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. Technology Transfer Number of New Germplasm Releases: 1
Impacts
(N/A)
Publications
- Stover, E.W., Riaz, S., Walker, M. 2008. Pcr screening for xylella fastidiosa in grape genebank accessions collected in the Southeast US. American Journal of Enology and Viticulture.59:437-439.
- Niedz, R.P., Evens, T.J. 2008. The effects of nitrogen and potassium nutrition on the growth of nonembryogenic and embryogenic tissue of sweet orange (Citrus sinensis (L.) Osbeck). Biomed Central (BMC) Plant Biology. 8:126. doi:10.1186/1471-2229-8-126
- Gasic, K., Yuepeng, H., Kertbundit, S., Shulaev, V., Lezzoni, A., Stover, E.W., Bell, R.L., Wisniewski, M.E., Korban, S. 2009. Characteristics and transferability of new apple EST-derived SSRs to other Rosaceae species. Molecular Breeding. 23: 397-411.
- Hall, D.G., Gottwald, T.R., Nguyen, N., Ichinose, K., Le, Q.D., Beattie, G. A., Stover, E. W. 2008. Greenhouse investigations on the effect of guava on infestations of Asian citrus psyllid in grapefruit. Proceedings of Florida State Horticultural Society. 121:104-109
- Muganu, M., Dangl, G., Aradhya, M.K., Frediani, M., Scossa, A., Stover, E. W. 2009. Ampelographic and DNA Characterization of Local Grapevine Accessions of the Tuscia Area (Latium, Centre of Italy). American Journal of Enology and Viticulture. 60:110-115.
- Bautista, J., Dangl, G., Yang, J., Reisch, B., Stover, E.W. 2008. Use of Genetic Markers to Assess Pedigrees of Grape Cultivars and Breeding Program Selections. American Journal of Enology and Viticulture. 59:248- 254.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Develop new citrus scion and rootstock cultivars with needed horticultural traits, including enhanced tolerance of environmental stress, resistance to diseases and pests, and improved fruit quality and yield. Approach (from AD-416) Create new genetic combinations of citrus germplasm via conventional breeding, mutation, and transformation. Screen germplasm for important traits, such as fruit quality, yield, tree size, tolerance of cold and drought, and resistance to Citrus Tristeza Virus, Citrus Bacterial Canker, and Huanglongbing. Select and propagate superior individuals. Evaluate selections for field performance and other traits. Release promising new scion and rootstock varieties for commercial use. Significant Activities that Support Special Target Populations This project replaces Project 6618-21000-012-00D, which terminated in April, 2008. Progress on this new project since April 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. A study was completed on seed transmission of Huanglongbing and submitted for publication. Studies were initiated on citrus tristeza virus resistance of new hybrids using reverse grafts. Greenhouse tests were completed to evaluate germplasm resistance to Huanglongbing disease. Data was collected on Huanglongbing infection in selected field trials to evaluate potential cultivar influence on Huanglongbing-related decline. The rootstock cultivars US- 812 and US-802 were transformed with the anti-microbial peptide D4E1 and transgenic plants established in the greenhouse. All these areas of progress directly relate to the National Program 301 Plant Genetic Resources, Genomics, and Genetic Improvement, Component 3 Genetic Improvement of Crops, Problem Area 3C Germplasm Enhancement/Release of Improved Genetic Resources and Varieties.
Impacts
(N/A)
Publications
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