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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

2014 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 first year of a new project. The new project continues the long-term goals of a previous project (6618-21000-013-00D) to develop new citrus scion and rootstock cultivars with traits critical for successful commercial production and marketability. The new project also initiates new work to address current threats and problems in U.S. citrus production. Data on fruit crop, tree size, and health was collected from more than twenty established rootstock and scion field trials. Five new rootstock field trials were planted. Over three thousand new hybrid seedlings were field planted for evaluation of scion traits. Trees were prepared for planting of three additional field trials with new hybrid rootstocks later in 2014. About twelve thousand new propagations of new hybrid rootstocks were completed to prepare trees for budding and planting in additional field trials in 2015. Work began to construct an additional greenhouse to propagate hybrid rootstocks for field trials. Cooperative work continued with three commercial nurseries to multiply promising hybrid rootstocks to produce trees for medium-scale commercial plantings, and an agreement is being set up with a fourth commercial nursery to begin similar work. Greenhouse studies continued to assess hybrid rootstock tolerance of Candidatus Liberibacter asiaticus infection and huanglongbing disease. Work also continued to assess rootstock tolerance to Citrus tristeza virus, calcareous soils, and salinity. Progress has been made to characterize apparent tolerance to Liberibacter-huanglongbing in conventional citrus. A citrus hybrid with commercial-quality fruit has grown extremely well compared to standards in a replicated trial under heavy Liberibacter-huanglongbing pressure. Trees of this new hybrid are large and are visibly fully healthy and cropping heavily despite Liberibacter infection. In contrast, sweet orange trees are much smaller, have substantial dieback, and no fruit. Grant funded studies continued on defense-related genes and small RNAs associated with Liberibacter-huanglongbing, in collaboration with University of Maryland and University of California research groups. A study of localized defense gene expression in shoots and roots provided evidence of striking differences that are a major advance in understanding, and yield strong insights into ways to overcome the disease. A study of the interaction between rootstock tolerance and scion tolerance/susceptibility provides additional insights into disease progression and the potential for improved management. During the year, more than 2000 seedlings were produced from 2013 crosses to develop improved citrus scions or rootstocks, and are being grown out for evaluation. In 2014, crosses were made with 48 parental combinations, emphasizing genetic seedlessness for scions, and Liberibacter-huanglongbing tolerance for scions and rootstocks. Three hundred to five hundred buds for each of eight advanced scion selections have been irradiated to generate low-seeded or better-colored variants. Seven new quality scion selections were identified and provided to a collaborator to be shoot-tip grafted for disease clean-up. Ten new promising rootstock hybrids were identified by superior performance in replicated field trials, and clean material was prepared for release and commercial distribution. Cooperative projects were established with commercial nurseries to begin propagation of the most promising new rootstocks. 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. Experiments to develop proliferating in vitro shoot cultures have been initiated for juvenile Carrizo citrange and Citrus macrophylla. Nutritional and plant growth regulator effects are being determined in these experiments. As conditions are identified that result in vigorous stage 2 cultures, mature tissue and a broader range of types will be examined.

1. New sweet orange-like hybrid with tolerance to huanglongbing (HLB) disease. ARS researchers at Ft. Pierce, Florida have developed a new hybrid with high-quality sweet orange-like fruit that displays excellent tolerance to HLB disease. The selection is a hybrid between citrus and trifoliate orange, but unlike other similar hybrids, has commercial quality fruit which is comparable with sweet orange in flavor. The selection has grown extremely well compared to standards in a replicated trial under heavy HLB pressure. Trees have been propagated at a commercial nursery and will be placed in replicated plantings on six grower sites with other advanced sweet-orange-like selections and industry standards in 2015.

2. Nine new citrus rootstocks with tolerance to huanglongbing (HLB) disease. ARS researchers at Ft. Pierce, Florida have identified nine new citrus rootstocks that display much higher sweet orange fruit productivity and tree health in field trials growing in areas severely affected by HLB, as compared with commercial standard rootstocks. These rootstocks, US-1279, US-1281, US-1282, US-1283, US-1284, US-1293, US-1317, US-1319, and US-1321 have been entered into 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, and suggest that use of a tolerant rootstock will be one component of successful citrus production management in the presence of that disease.

3. Developed a liquid culture method suitable for genetic transformation. Citrus types vary widely in their transformability on agar media, with scion types such as sweet orange and grapefruit having low transformation efficiencies, often much less than one percent. Improving transformation of the scion types would greatly reduce the resources required to widely utilize transgenic technology in citrus and may be facilitated by using a liquid culture system. ARS researchers at Ft. Pierce, Florida examined numerous factors and determined their effects on shoot regeneration and/or genetic transformation efficiency. A liquid culture system was developed and found to be suitable for shoot regeneration and genetic transformation. The liquid system resulted in in vitro responses that were comparable to agar, and in some cases some improvement was observed. For example, genetic transformation efficiency was generally higher in the liquid system, possibly due to fewer escapes.

Review Publications
Albrecht, U., Hall, D.G., Bowman, K.D. 2014. Transmission efficiency of Candidatus Liberibacter asiaticus and progression of Huanglongbing disease in graft and psyllid-inoculated citrus. HortScience. 49(3):367-377.
Stover, E.W., Richardson, M.L., Driggers, R., Hall, D.G., Duan, Y.P., Lee, R.F. 2014. Incidence and severity of Asiatic citrus canker on citrus and citrus–related germplasm in a Florida field planting. HortScience. 49:4-9.
Lu, H., Zhang, C., Albrecht, U., Shimizu, R., Wang, G., Bowman, K.D. 2013. Overexpression of a citrus NDR1 ortholog increases disease resistance in Arabidopsis. Frontiers in Plant Science. 4(157):1-10.