Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): Objective 1. Breed new citrus rootstock and scion cultivars with improved traits for successful commercial fruit production, including resistance or tolerance to diseases, including huanglongbing (HLB). 1.A. Select clones from among progeny groups and enter into replicated field trials. 1.B. Collect performance information from replicated field trials and identify outstanding clones for commercial release. Objective 2. Introduce useful traits into citrus populations by hybridization, transformation, and/or gene editing, and recombine traits to obtain combinations of traits representing superior new types. 2.A. Complete sexual crosses for new scion and rootstock types and establish into field for first evaluation. 2.B. Evaluation of transgenic citrus in field test site at USDA Picos farm. 2.C. Develop embryogenic cell lines for citrus CRISPR applications. Objective 3. Design novel breeding strategies such as genomic selection and create molecular genetic markers for fruit quality trait evaluation of citrus populations and efficient selection of elite materials. 3.A. Collect phenotypic data suitable for mapping and developing molecular genetic markers for traits associated with useful scion and rootstock characteristics 3.B. Map or associate genomic regions with useful traits and identify molecular markers Objective 4. Improve methodology to manage and evaluate new rootstock and scion germplasm for valued traits with a focus on tolerance/ resistance to HLB and to employ these methods to develop and commercialize new cultivars. 4.A. Develop an in vitro HLB system. 4.B. Develop a UAV aerial imaging system for field trial data collection. 4.C. Develop a high throughput phenotyping method and data storage for commercial citrus scion and rootstock traits. Approach (from AD-416): This project will focus on development of new citrus rootstock and scion cultivars with traits urgently needed for commercial production, especially improved marketability, and resistance to huanglongbing (HLB). These new rootstock and scion cultivars will enhance the competitiveness of the U.S. citrus industry by improving production efficiency, plant health, and market value of U.S. citrus crops. Breeding citrus for resistance to HLB is particularly difficult because of the long-life cycle of citrus, predominant apomixis among most cultivars, and lack of significant HLB resistance in the important cultivated citrus. The primary objectives of this project are to generate new citrus genotypes through conventional breeding, mutation, and genetic modification technologies; evaluate them as candidate cultivars for commercial use; and release new superior cultivars. In addition, improved methods will be developed for genetic modification of citrus, early selection of the most promising new hybrids using molecular markers, and more efficient field evaluation. Improved methods to pre-select and evaluate candidate hybrids will increase the success and pace of developing cultivars with the most important traits. Among global efforts to develop new citrus cultivars with HLB resistance, this program has the unique advantage of 1) numerous replicated field trials with hundreds of hybrids from which performance data is being collected for development of molecular markers and selection of superior new cultivars, 2) advanced breeding lines (produced by more than a century of introgression) which contain alleles for resistance to HLB and other critical traits from related species, and 3) excellent infrastructure including research field sites in two different production regions where the promising new scion and rootstock cultivars are evaluated. The planned research project will capitalize on these advantages to release superior new scion and rootstock cultivars, and to develop new germplasm, tools, and techniques that will accelerate the next generation of citrus breeding. Objective 1.A. ARS researchers at Fort Pierce, Florida selected clones from among progeny groups and enter into replicated field trials. New sweet orange and rootstock clones were planted in 3 new replicated field trials. These trials included the scions Donaldson, Robel, and Parson Brown oranges selected from other trials. Additionally, 32 new selections from breeding populations were made this season. These selections included 20 orange-like varieties, 5 seedless and low-seeded mandarins, 2 low-seeded irradiated selections of grapefruit-like hybrids with dark red internal color, and 5 new rootstocks. The new trials are critical for assessing the performance and potential commercial viability of these new clones in various conditions. Objective 1.B. Collect performance information from replicated field trials and identify outstanding clones for commercial release. Rigorous phenotypic data was collected from 300 scion cultivars in replicated plantings at four locations, and from 350 rootstocks in replicated plantings at five locations. This extensive data collection is crucial for identifying the best performing clones. This has resulted in an elite list of high brix orange-like selections suitable for commercial orange juice production, and new hybrid rootstocks that can increase tree health and production of high-quality citrus fruit. This work includes collaboration with industry stakeholders and underscores the importance of industry partnerships in advancing agricultural research and development. Objective 2.A. Complete sexual crosses for new scion and rootstock types and establish into field for first evaluation. Twenty-four parental combinations were crossed to create useful HLB tolerant variation, with 1120 flowers pollinated from these combinations in 2023. Seed harvested from the crosses was planted into the greenhouse. Hybrid seedlings from previous year�s crosses were planted into the field for fruiting out and first evaluation. Objective 2.B. Evaluate transgenic citrus in field test site at USDA Picos farm. Trees were evaluated quarterly for huanglongbing tolerance and metrics related to tree size and health in this test area. These trials in this test area included assessments of rootstock and scion trials targeted at huanglongbing tolerance, focusing on canopy density and tree health. The continuous evaluation of transgenic citrus at the USDA Picos farm provides valuable data on the effectiveness of genetic modifications in enhancing huanglongbing tolerance, which is vital for developing resilient citrus varieties. Objective 2.C. Develop embryogenic cell lines for citrus clustered regularly interspaced short palindromic repeats (CRISPR) applications. Embryogenic cell lines from varieties important to the Florida and California citrus industries were started, including Hamlin (early season) , Valencia (late season), Pineapple (midseason), Donaldson and Tobias. Donaldson is being widely propagated for testing as a huanglongbing- tolerant sweet orange. Tobias flowers within 2 years making it a potential breeding parent to reduce juvenility. Embryogenic potential of existing lines is tested annually using glycerol induction of somatic embryogenesis; globular embryos appear 4-6 weeks after culturing on media with glycerol as the carbon source if the cells are embryogenic. Embryogenic cells were provided to University of Maryland collaborators and are being used to develop CRISPR technology for citrus. Objective 3.A. Collect phenotypic data suitable for mapping and developing molecular genetic markers for traits associated with useful scion and rootstock characteristics. Detailed phenotypic and performance data was collected from 5,000 replications in rootstock field trials with sweet orange scion during the 2023-24 harvest season. That information was used for preliminary mapping-associated mapping for the citrus genome. Objective 3.B. Map or associate genomic regions with useful traits and identify molecular markers. A marker linked to sweet orange flavor in oranges and orange-like cultivars was identified and published in a manuscript on the chemical and genetic basis of orange flavor. A random forest model predicted untrained samples with 78% accuracy and identified 26 compounds crucial for orange flavor. Differential gene expression analysis identified a locus responsible for ester production in orange, and a DNA marker in the coding region accurately predicted phenotypes. This research significantly enhances our understanding of the genetic and chemical basis of orange flavor, providing new tools for breeding programs focused on improving citrus flavor. Objective 4.A. Develop an in vitro HLB system. The first experiment was to determine if stage I shoot cultures can be established from HLB tolerant and susceptible citrus types that are infected with the bacteria causing huanglongbing disease, Candidatus Liberibacter asiaticus. Stage 1 shoot cultures of infected and uninfected tissue were established from bud cultures of greenhouse citron trees. Shoots containing 3-4 buds were removed, sterilized, and the buds cultured in vitro. Shoots that emerged from the buds were excised and are being re-cultured every 30 days. The results indicated that shoot cultures from huanglongbing-infected trees can be established from citron. Citron was selected as it displays prominent leaf blotchy mottle symptoms, maintains a high titer of the bacteria, but the growth of the tree is not greatly affected. The next experiment is to determine if Stage 2 proliferating shoot cultures can be established from these Stage 1 shoot cultures. Establishing Stage 2 shoot cultures typically requires 1-2 years for citrus, which is unusually long compared to other plant species. Objective 4.B. Develop an unmanned aerial vehicle (UAV) aerial imaging system for field trial data collection. An aerial imaging system was developed using a commercially available drone. The UAV camera had a one inch complementary metal-oxide semiconductor sensor with an effective pixel number of 20 megapixels. Images for data analysis were acquired at an altitude of 46 meters and a speed of 12 kilometers/hour. Standardized 1-meter PVC pipes served as a size standard to allow for conversion of pixel number to any scale. Aerial images were taken automatically by vertical photography where the optical axis is vertical or near vertical to the tree. To minimize relief displacement a side-overlap of 70% and forward-overlap of 80% of the flight lines will be used. Images are standard red-green-blue (sRGB) and with a size 3:2 format of 5472 x 3648 pixels. Images were process using the open-source ImageJ software and 28 measures of canopy shape and size were captured. The next set of experiments will utilize this method to collect data from rootstock trials. Objective 4.C. Develop a high throughput phenotyping method and data storage for commercial citrus scion and rootstock traits. A high throughput phenotyping methodology was created to assess over 300 scion cultivars for traits linked to commercial success. As a result, 27 new varieties with commercial promise were selected, including sweet oranges, sweet orange-like hybrids, grapefruits, grapefruit-like hybrids, and mandarins. These selections underwent extensive evaluations and show great potential for commercial significance. The development of high throughput phenotyping methods is essential for efficiently screening large numbers of cultivars and identifying those with the most commercial potential. Artificial Intelligence (AI)/Machine Learning (ML) ARS researchers at Fort Pierce, Florida used Machine learning to process images taken from a UAV (Unmanned Aerial Vehicle). ACCOMPLISHMENTS 01 Released two new disease-tolerant hybrid citrus rootstocks with superior field performance. Sweet orange production in Florida has declined by more than 80 percent since the disease huanglongbing became spread throughout the state. Pre-existing citrus cultivars and rootstocks were mostly highly susceptible to the disease. Two new hybrid rootstocks, SuperSour 4 and SuperSour 5, demonstrated superior tree health and fruit productivity in replicated field trials affected by the disease, and were released for commercial use by ARS researchers at Fort Pierce, Florida. Sweet orange trees planted on these new rootstocks will have improved tolerance to huanglongbing disease, and improve fruit production and profitability of citrus growing in Florida and other areas affected by the disease. 02 Developed an aerial imaging system for field trial data collection. A system was developed by ARS researchers at Fort Pierce, Florida to capture aerial images and analyze those images using machine learning to extract multiple size and shape measures of the tree canopy. This system is being used to collect data from citrus scion and rootstock trials to supplement ground measures of tree height, width, and health. Over time, the evaluation of tree size and health in field trials will transition to using the aerial imaging system in place of ground measures, as a more accurate and less expensive and labor-intensive method of collecting field performance information. 03 Development of High Throughput Phenotyping Methods and Genetic Insights. A high throughput phenotyping methodology was developed by ARS researchers at Fort Pierce, Florida to assess over 300 new scion selections for traits linked to commercial success. This led to the selection of 27 new clones with commercial promise, including sweet oranges, sweet orange-like hybrids, grapefruits, grapefruit-like hybrids, and mandarins. Additionally, the research identified a molecular marker linked to sweet orange flavor in oranges and orange- like cultivars. The work involved a random forest model that predicted untrained samples with 78% accuracy and identified 26 compounds crucial for orange flavor. The combination of the new phenotyping methodology and a selectable marker for sweet orange flavor will greatly accelerate the development of valuable new hybrid cultivars.
Impacts
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Publications
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