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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Insects and Horticulture Research » Research » Research Project #425052

Research Project: Genetic Improvement of Citrus for Enhanced Resistance to Biotic and Abiotic Stresses

Location: Subtropical Insects and Horticulture Research

2015 Annual Report

1. Create new genetic combinations of citrus germplasm via conventional breeding, mutation, and transformation, to include rootstock and scion development and evaluation for essential traits of disease resistance and horticultural qualities. 1.A. Use sexual hybridization to create new germplasm from diverse parental types with useful horticultural characteristics. 1.B. Create new scions with useful traits through mutation. 1.C. Create new scions and rootstocks with potential resistance to huanglongbing and citrus bacterial canker by genetic transformation. 2. Develop and evaluate methods to improve citrus transformation, including the use of proliferating in vitro shoot cultures, as a novel source for genetic transformation and germplasm preservation. 2.A. Develop methods to produce proliferating in vitro shoot cultures of rootstock and scion types. 2.B. Determine the transformation efficiency of in vitro shoot cultures. 3. Develop and evaluate new methods to efficiently screen germplasm for important traits, improve the process of citrus variety development, and apply appropriate methods to select superior individuals. 3.A. Refine and evaluate methods to assess huanglongbing tolerance/resistance, and apply appropriate methods to select superior individuals. 3.B. Develop and apply methods to test selections for abiotic stress, including high pH. 4. Evaluate field performance and other traits for rootstock and scion selections and release new cultivars as appropriate.

New citrus selections will be created by sexual hybridization, mutation, and genetic transformation from existing cultivars and species. Sources of tolerance or resistance to huanglongbing will be emphasized in choice of parents for hybrids. Genes with potential to induce tolerance or resistance to huanglongbing will be emphasized in transformation, including anti-microbial peptides, chimeral anti-microbial peptides, citrus genes that respond to infection by the pathogen, but with regulation altered to increase resistance, and genes that target specific metabolic components of the pathogen. Methods will be developed to improve citrus transformation, including the use of proliferating in vitro shoot cultivars. Hybrids and other new types will be assessed for important traits, including the use of molecular markers, and greenhouse, laboratory, and field assays. Methods to assess huanglongbing tolerance or resistance and tolerance of high pH will be refined and applied to new hybrids and transgenics. Promising selections will be entered into long-term field trials at multiple locations, and data will be 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 Report
This is the second year of a project that continues the long-term goals of previous projects to develop new citrus scion and rootstock cultivars with traits critical for successful commercial production and marketability. The current project also initiates new work to address current threats and problems in U.S. citrus production. Hybridizations for improved citrus scion production were conducted using 68 parental combinations and about 1400 flowers in 2015. About three thousand new hybrid seedlings were produced from 2014 crosses for scion and rootstock development. Greenhouse studies continued to assess rootstock and scion tolerance of Candidatus Liberibacter asiaticus infection and huanglongbing disease. Greenhouse and field studies continued to indicate significant differences among plant genotypes with respect to tree tolerance or resistance to huanglongbing, the disease caused by Candidatus Liberibacter asiaticus. Influence of scion genotype on tolerance to huanglongbing appeared of a larger magnitude, but rootstock influence on tolerance was also significant. Work also continued to assess rootstock tolerance to Citrus tristeza virus and calcareous soils. Data on fruit crop, tree size, and health were collected from more than ten established rootstock and scion field trials. Three new rootstock field trials were planted. Trees were prepared for planting of four additional field trials with new hybrid rootstocks later in 2015. About 14,000 new propagations of new hybrid rootstocks were completed to prepare trees for budding and planting in additional field trials in 2016. Cooperative work continued with three commercial nurseries to multiply promising hybrid rootstocks to produce trees for medium-scale commercial plantings. Cooperative work was initiated under external funding with a university partner to establish twelve new rootstock field trials in Florida with the most promising rootstocks for huanglongbing tolerance. Progress has been made to characterize apparent tolerance to huanglongbing in conventional citrus suitable for use as scions, including about 4,000 that are sweet orange-like. Collaborations continue to show that the aroma volatiles in many of the advanced sweet-orange-like selections are very similar to true sweet orange, making it likely they can be officially designated as “sweet orange” for commercial use. Several of these selections appear to be much more resistant to huanglongbing (HLB) than are true sweet oranges. Initiated replicated commercial plantings of our most advanced sweet orange-like hybrids and mandarins alongside standards. Grant funded studies continued to produce and test transgenic rootstocks and scions, targeting increased tolerance to huanglongbing and canker diseases. More than five hundred new transgenic rootstocks and scions were produced. Transformation of scions included work with single chain fragment variable antibodies that bind to specific regions of huanglongbing pathogen proteins. Genes expressing 18 different single chain fragment variable antibodies were used to create hundreds of transgenic citrus plants. We will test the hypothesis that these proteins can interfere with the activity of these proteins in transgenic citrus and thereby protect them from colonization by the bacteria that causes huanglongbing. Transformation of new rootstocks focused on manipulation of the citrus resistance genes CtNDR1, CtMPK4, CtTGA7, CtNHL125, CtFAD7, CtNHO1, CtMIR2, and CtOSM34. Testing of selections for Liberibacter tolerance was conducted by controlled inoculation in the greenhouse using the vector Asian citrus psyllid. Three separate inoculation tests were conducted, involving nearly one thousand plants and more than twenty thousand caged psyllids. Monitoring and data collection continued on previous groups of transgenic plants that have been inoculated with Liberibacter. Several transgenic rootstock selections showing increased resistance to Liberibacter have been identified from groups transformed with other resistance genes, and are also being prepared for further testing. Demonstrated reduced susceptibility to citrus canker in transgenic citrus expressing the FLS2 receptor from Nicotiana benthamiana. Transgenic citrus expressing a modified citrus protein and a chimera of that protein show substantially reduced susceptibility to citrus canker, and in preliminary studies, appear remarkably tolerant to huanglongbing. Focused work continued with twelve new promising rootstock hybrids that were identified as tolerant to huanglongbing disease by superior performance in replicated field trials. Five new huanglongbing (HLB) tolerant rootstocks were released for commercial use in Florida and plant material is being distributed through the Florida agency that regulates distribution of citrus within the state. Proliferating in-vitro shoot cultures (stage 2 cultures) were established from juvenile and mature explants of two rootstock selections, Carrizo and US-942, to be used for transformation, germplasm conservation, and micropropagation. We are currently multiplying these cultures to determine their use in genetic transformation of citrus.

1. Released five new citrus rootstocks with tolerance to huanglongbing disease. ARS researchers at Ft. Pierce, Florida identified and released five new citrus rootstocks that display much higher sweet orange fruit productivity and tree health in multiyear field trials growing in the Florida east coast area severely affected by huanglongbing, as compared with commercial standard rootstocks. The new rootstocks, US-1279, US-1281, US-1282, US-1283, US-1284, are available from the Florida clean budwood program, and are being propagated for entry into large scale grower trials in 2015. The results from field testing indicate that these rootstocks enable sweet orange trees to better tolerate huanglongbing disease. Trees on these rootstocks become infected with the disease, but after infection these trees yield more fruit that have higher sugar content in comparison to orange trees on other standard rootstocks. The results suggest that use of these or other tolerant rootstocks will be a key component of successful citrus production management in the presence of huanglongbing disease.

2. Developed ARS-MEDIA for calculating media recipes based on ion-specific constraints. ARS researchers at Ft. Pierce, Florida developed ARS-MEDIA, an ion solution calculator that uses Microsoft Excel to generate recipes of salts for complex ion mixtures specified by the user. The spreadsheet is based on an algorithm published, by the author, in Nature Methods. The spreadsheet was made available to the public via the ARS software website ( The software is required for any experiment designed to determine ion specific effects. One major use of the software is in developing improved fertilizer compositions and mineral nutrient formulations for use in various agricultural applications – plant tissue culture, hydroponic, greenhouse & field fertilizer studies, and algal growth. Fields where ion-specific effects are important include agriculture, biology, ecology & environmental science, medicine, animal science, chemistry, and physics.

3. Developed transgenic citrus with reduced susceptibility citrus canker disease. Transgenic citrus plants were produced expressing the FLS2 receptor from Nicotiana benthamiana. These transgenic plants show substantially reduced susceptibility to citrus canker, and increased tolerance to huanglongbing disease. These disease resistant or tolerant cultivars are of great interest because of the large commercial importance of these two diseases in the U.S. citrus industry.

Review Publications
Furr, J., Reece, P., Kahn, T., Siebert, T., Barry, G., McCollum, G., Castle, W., Stover, E. 2014. ‘US Furr’ and ‘US Furr-ST’ mandarin. Journal of American Pomological Society. 68:198-203
Bai, J., Baldwin, E.A., Hearn, J., Driggers, R.E., Stover, E.W. 2014. Volatile Profile Comparison of USDA Sweet Orange-like Hybrids versus ‘Hamlin’ and ‘Ambersweet’. HortScience. 49(10):1262-1267.
Stover, E.W., Mccollum, T.G., Ramos, J.E., Shatters, R.G. 2015. Growth, health and Liberibacter asiaticus titer in diverse citrus scions on mandarin vs. trifoliate hybrid rootstocks in a field planting with severe huanglongbing. Proceedings of Florida State Horticultural Society. 127:53-59.
Stover, E.W., McCollum, T.G., Driggers, R.E., Lee, R.F., Shatters, R.G., Duan, Y.P, Ritenour, M.A., Chaparro, J., Hall, D.G. 2015. Resistance and tolerance to huanglongbing in citrus. Acta Horticulturae. 1065:899-903.