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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC IMPROVEMENT OF CITRUS
2013 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 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.


4.Accomplishments
1. 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.

2. 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.

3. 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.


Review 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.

Last Modified: 12/22/2014
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