Skip to main content
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

2017 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 fourth 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 involves new work, especially focused on developing new rootstocks and scions resistant or tolerant to the disease huanglongbing. Data on tree size, health, and cropping was collected from ten established rootstock and scion field trials. Three new replicated field trials were planted. Trees were prepared for planting of ten additional rootstock field trials in 2017-2018. Work expanded under material transfer agreements to cooperatively test new scions and rootstocks in field trials with university and private partners. Cross hybridizations for improved scions and rootstocks were conducted and new hybrid seedlings were planted in the greenhouse. Material was selected from existing promising greenhouse and field trees for further study. Greenhouse and field studies continued to assess rootstock and scion tolerance of Candidatus Liberibacter asiaticus infection and huanglongbing disease. Greenhouse and field work indicate highly significant effects of rootstock and scion genotype on tree tolerance or resistance to huanglongbing, the disease caused by Candidatus Liberibacter asiaticus. Over 11,000 new scion program hybrids have been planted in the last seven years, with crosses from 2011 and 2012 starting to fruit. These are the first hybrids created specifically to target resistance/tolerance to huanglongbing. As our understanding of huanglongbing-tolerance advances we make better crosses each year. For example, we are now actively using breeding parents with citron in their pedigree, reflecting new data indicating citron confers tolerance. Outstanding fruit quality and early evidence of huanglongbing-tolerance is evident in many populations. A large replicated trial of 50 advanced selections and cultivars is in the fourth year of severe huanglongbing challenge and a number of our selections are showing excellent growth despite infection. Large replicated plantings of our most-advanced selections were planted at five grower sites, and trees are being prepared for very large scale replicated trials using funds from a grant. Large replicated plantings continue to be phenotyped, in an effort to ultimately identify genes associated with huanglongbing tolerance and resistance. Advances have been made in developing methods to expedite screening for genotype response to huanglongbing and rapid screening of potential transgenic products and other therapies. In FY 17, transient intermittent water- or nutrient-stress was shown to accelerate huanglongbing development in graft-inoculated trees by two months compared to non-stressed trees. Interestingly, the greatest stress treatment eliminated the pathogen over time. Infected detached leaves and disks punched from infected leaf petioles are showing promise as methods to screen peptides for elimination of the pathogen that may be expressed in transgenic plants, or as a therapy for eliminating the pathogen in existing trees. Progress continues in developing transgenic citrus with huanglongbing-resistance. In the previous year, transgenic Carrizo rootstock plants expressing a modified plant thionin (Mthionin) were grafted with scions infected with the huanglongbing-pathogen and after one year the control Carrizo roots had 1800 X higher pathogen titer than the transgenic Carrizo. A patent application has been completed on Mthionin. Hamlin, Valencia, and Ray Ruby transgenics expressing this peptide have been created. Replicated trees have been prepared for field planting using the Mthionin Carrizo and non-transgenic scions. Transgenics expressing chimeral peptides (containing separate lytic and gram negative membrane recognition sequences) have been created combining Mthionin or citrus thionin with a citrus lipid binding protein. Small trees have been exposed to no-choice feeding by Candidatus Liberibacter asiaticus-infected Asian citrus psyllid, and effects on pathogen suppression will be determined. A promoter from the most highly expressed gene in citrus phloem has been characterized and is being used in creating transgenic plants targeting Candidatus Liberibacter asiaticus. Using a reporter gene, this promoter (396ss) had 500x greater messenger Ribonucleic acid (RNA) in the midrib compared to the laminar area, vs. identical laminar and petiole expression from a more common promoter. The 396ss promoter is being actively used to create transgenic citrus and a patent application for 396ss is in development. New transgenic plants have also been created expressing peptides identified by ARS researchers in Ft. Pierce, Florida, as preventing Candidatus Liberibacter asiaticus acquisition in Asian citrus psyllid. Citrus stage 1 and stage 2 in vitro shoot cultures have been developed for some important citrus rootstock types – Carrizo, X-639, and US-942. Plant in vitro culture recognizes 5 stages as follows: 1) Stage 0 – Preparative stage. Mother plants are grown and treated to minimize microbial contamination and maximize the in vitro response of the explants. 2) Stage 1 – Initiation of axenic culture stage. Axenic cultures are established followed by some growth. 3) Stage 2 – Multiplication stage. The cultures produce sufficient shoots for subsequent propagation (or source material for transformation) as well as shoots to maintain the stock. Note: There are few reports that clearly describe this stage in citrus. 4) Stage 3 – Preparation for ex vitro growth stage. Shoots are prepared, generally elongated, for in/ex vitro rooting or grafting (commonly done in citrus). 5) Stage 4 – Transfer to ex vitro growth stage. Identify the conditions required to transition an in vitro plant to ex vitro growth (e.g, greenhouse). Identifying the appropriate conditions is important since significant losses can occur at this stage. Stages 1 and 2 have not been used in citrus breeding and genetics but would be quite useful in citrus for a number of applications including genetic transformation. Many of the advantages of using these sorts of cultures is that they are both clonal and vegetative; thus, genetic transformation could be applied to all citrus types, not just those that produce polyembryonic seed (the only citrus types now transformed). One observation from this research is that the physiological transition from a stage 1 culture to a stage 2 culture takes from 1 to 2 years. This is a very long time, and something not expected or reported in the literature. A Carrizo culture was developed that took 13 months to adapt. We have a citron culture that is now 13 months old and still has not adapted. Most plant species adapt in under 3 months. We now have three citrus rootstock varieties established as stage 2 cultures – Carrizo, X-639, and US-942. Citron is being developed as a model system because of its high susceptibility to huanglongbing (HLB), and thus it’s potential as a useful model in HLB research. Psyllid infestation was attempted a number of times but the resulting contamination could not be eliminated. Psyllids readily fed on in vitro shoots and laid eggs that resulted in larva. Further, electrofeeding experiments were conducted to determine how feeding on in vitro shoots compared to feeding on ex vitro feeding. A mineral nutrition software tool was developed. ARS-Media for Excel was developed to solve mineral nutrition linear programming equations. An Excel application solves the mineral nutrient linear programming calculations required for in vitro mineral nutrition research for improving the growth of in vitro shoot cultures. Use of this software is required for all mineral nutrient experiments that involve the culture of organisms (such as citrus and huanglongbing (HLB)).

1. Superior multi-year field performance for two USDA citrus rootstocks. Many existing rootstocks decline rapidly when affected by huanglongbing disease, and this has become a devastating problem for the Florida citrus industry. Results from long-term field trials conducted by ARS researchers and affected by huanglongbing, indicate that the rootstocks US-802 and US-942 provide improved yield, fruit quality, and tree health as compared with standard commercial rootstocks. Both rootstocks are commercially available in limited quantities and ARS researchers are working with nurseries and micropropagation companies to expand production of the rootstocks to meet the large commercial demand. 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. Identified sweet-orange-like hybrids as breeding parents. These hybrids are very similar to sweet-orange in flesh quality, produce only hybrid seed, have high gamete viability, produce sweet-orange-like progeny, and have little inbreeding depression in crosses with parents having a high proportion of sweet-orange in their pedigrees. Conventional sweet orange is a very poor breeding parent due to high levels of apomixis (which results in seedlings that are genetically identical to parents, and not hybrids) and also have a very narrow genetic base, making Florida citrus essentially a mono-culture. Three of these new selections have been released for use as sweet-orange breeding parents. These should contribute to enhanced breeding of sweet-orange types around the world and will contribute to developing citrus with sweet-orange traits but greater disease resistance.

3. Established stage 1 and stage 2 in vitro cultures. Three citrus rootstocks Carrizo, X-639, and US-942 were established as stage 1 and stage 2 cultures. Citron and Valencia sweet orange were established as stage 1 cultures. Studying huanglongbing in the greenhouse and field is difficult and expensive because of citrus biology (long juvenile period, inbreeding depression, very inefficient scion genetic transformation methods, and as a tree crop requires substantial greenhouse and field infrastructure for experimentation), and long times required to test and validate potential control and treatment strategies. Also, the pathogen that causes huanglongbing, Candidatus Liberibacter asiaticus, cannot be cultured. The use of in vitro shoot cultures for huanglongbing research will provide new methods that are less costly and more rapid than current greenhouse- and field-based approaches and will compliment these approaches.

Review Publications
Niedz, R.P. 2016. ARS-Media: A spreadsheet tool for calculating media recipes based on ion-specific constraints. PLoS One. 11(11):e0166025. doi:10.1371/journal.pone.0166025.
Bowman, K.D., Faulkner, M.L., Kesinger, M. 2016. New citrus rootstocks released by USDA 2001-2010: field performance and nursery characteristics. HortScience. 51(10):1208-1214.
Stover, E.W., Shatters, R.G., Gruber, B., Kumar, Moore, G.A. 2016. Influence of photoperiod duration and phloem disruption through scoring on growth, disease symptoms and bacterial titer in citrus graft-inoculated with Candidatus Liberibacter asiaticus. HortScience. 51:1215-1219.
Stover, E.W., Lin, Y., Yang, X., Vashisth, T. 2016. Hydrogen cyanamide on citrus: preliminary data on phytotoxicity and influence on flush in potted and field trees. HortTechnology. 26:839-845.
Miles, G., Stover, E., Keremane, M., Ramadugu, C., Lee, R.F. 2017. Apparent tolerance to huanglongbing in citrus and citrus-related germplasm. HortScience. 52:31-39.
Hao, G., Stover, E.W., Gupta, G. 2016. Overexpression of a modified plant thionin enhances disease resistance to citrus canker and Huanglongbing (HLB). Frontiers in Plant Science. doi:10.3389/fpls.2016.01078.
Keremane, M.L., Ramadugu, C., Halbert, S., Duan, Y., Roose, M., Stover, E.W., Lee, R. 2016. Long term field evaluation reveals HLB resistance in Citrus relatives. Plant Disease.
Bai, J., Baldwin, E.A., Driggers, R.E., Hearn, J., Stover, E.W. 2016. Volatile and nonvolatile flavor chemical evaluation of USDA orange-mandarin hybrids for comparison to sweet orange and mandarin fruit. Journal of the American Society for Horticultural Science. 141(4):339-350.
Pisani, C., Ploetz, R., Stover, E., Ritenour, M., Scully, B. 2015. Laurel wilt in avocado: Review of an emerging disease. International Journal of Plant Biology and Research. 3(3):1043-1049.
Inch, S.A., Stover, E.W., Driggers, R.E., Lee, R.F. 2014. Freeze response of citrus and citrus-related genotypes in a Florida field planting. HortScience. 49:1010-1016.
Hao, G., Pitino, M., Ding, F., Lin, H., Stover, E.W., Duan, Y. 2014. Induction of innate immune responses by flagellin from the intracellular bacterium, ‘Candidatus Liberibacter solanacearum’. Biomed Central (BMC) Plant Biology. 14:211.
Niedz, R.P., Hyndman, S.E., Evens, T.J., Weathersbee, A.A. 2014. Mineral nutrition and in vitro growth of Gerbera hybrida (Asteraceae). In Vitro Cellular and Developmental Biology Plants. 50:458-470.
Niedz, R.P., Albano, J.P., Marutani-Hert, M. 2015. Effect of various factors on shoot regeneration from citrus epicotyl explants. Journal of Applied Horticulture. 17(2)121-128.
Rezzazadeh, R., Niedz, R.P. 2015. Protoplast isolation and plant regeneration of guava (Psidium guajava L.) using experiments in mixture-amount design. Plant Cell, Tissue And Organ Culture. 122(3):585-604
Wada, S., Maki, S., Niedz, R.P., Reed, B.M. 2015. Screening genetically diverse pear species for in vitro CaCl2, MgSO4 and KH2PO4 requirements. Acta Physiologiae Plantarum. 37, 63 DOI 10.1007/s11738-014-1754-y.
Wada, S., Niedz, R.P., Reed, B.M. 2015. Determining nitrate and ammonium requirements for optimal in vitro response of diverse pear species. In Vitro Cellular and Developmental Biology - Plants. 51(1):19-27.
Niedz, R.P., Evens T. 2016. Design of experiments (DOE) - history, concepts, and relevance to in vitro culture. In Vitro Cellular and Developmental Biology Plants. 52:547-562.
Mahmoud, S., Ramos, J.E., Shatters, R.G., Hall, D.G., Lapointe, S.L., Niedz, R.P., Rouge, P., Borovsky, D. 2016. Expression of Bacillus thuringiensis cytolytic toxin (Cyt2Ca1) in citrus roots to control Diaprepes abbreviatus larvae. Pesticide Biochemistry and Physiology. 136:1-11.
Stover, E.W., Hall, D.G., Shatters, R.G., Moore, G.A. 2016. Influence of citrus source and test genotypes on inoculations with Candidatus Liberibacter asiaticus. HortScience. 51:805-809.
Hall, D.G., Ammar, D., Bowman, K.D., Stover, E.W. 2017. Epifluorescence and stereomicroscopy of trichomes associated with resistant and susceptible host plant genotypes of the Asian citrus psyllid (Hemiptera: Liviidae). Journal of Microscopy and Ultrastructure. Available:
Hummer, K.E., Dempewolf, H., Bramel, P., Markham, R., Stover, E.W. 2015. Status of global strategies for horticultural fruit crops. Acta Horticulturae. 1101:147-152. doi: 10.17660/ActaHortic.2015.1101.22.
Belknap, W.R., Mc Cue, K.F., Harden, L.A., Vensel, W.H., Bausher, M.G., Stover, E.W. 2015. A family of small cyclic amphipathic peptides (SCAmpPs) genes in citrus. Genome. 16:303-313.
Pascacio, C., Lapointe, S.L., Williams, T., Sivinski, J.M., Niedz, R.P., Aluja, M. 2014. Mixture-amount design and response surface modeling to assess the effects of flavonoids and phenolic acids on developmental performance of Anastrepha ludens. Journal of Chemical Ecology. 40:297-306.