Plant Improvement

Comparison of field performance of citrus trees on rootstocks propagated by seedlings, cuttings, and tissue culture

Final Report (Due 5/31/2024)- Project # 23-037: Fruit, Yield, and Quality Assessment for Advanced Selections and Drone Re-flight for Hurricane-Affected Sites.

Quarterly Project # 21-025: Transgenic capable field site to assess HLB-resistant and other improved citrus

1. Please state project objectives and what work was done this quarter to address them: Project # 21-025 focuses on developing HLB-resistant and other improved citrus cultivars to mitigate the devastating effects of HLB on the Florida citrus industry. The project utilizes transgenic and non-transgenic approaches, and all experiments are conducted at the secure field site of the USDA/ARS Picos Research Farm. The goal is to provide sustainable solutions to combat HLB, safeguard the citrus industry, and support economic citrus production.This quarter, The test site received routine horticultural care, and trees were exposed to HLB and psyllid vectors to assess their resistance in a real-world environment. All BRS permits were upheld, and the site underwent inspections to ensure compliance with regulatory requirements. In addition Dr. Zhonglin Mou planted a total of 23 transgenic rootstock lines were planted in the farm on July 24, 2024. These lines include 14 NPR3 RNAi rootstock lines, three NPR3 RNAi rootstock lines top-grafted with Hamlin, and six NPR1 rootstock lines. These transgenic lines were generated through mature tissue transformation. There were planted in the field for fruit/seed production. Dr. Mou’s existing transgenics were also assessed. The NPR1 scion lines: 69 trees were planted in May 2019, 98 in May 2021, and 27 in October 2021. Among those planted in 2019, 15 were produced through mature tissue transformation, and 11 survived. These trees have flowered and are producing fruits. Two lines were chosen for propagation. Budsticks from these two lines have been submitted to DPI on July 15, 2024. Leaf and fruit samples were taken from the mature trees on July 26, 2024 for CLas titer assays. One tree from juvenile transformation has flowered this spring and currently has three fruits on the tree. Other juvenile trees have not flowered. 2. Please state what work is anticipated for next quarter:Next quarter, the team will analyze the data collected from the current field trials to evaluate the efficacy of different HLB-resistant candidates. Researchers will continue to have access to the test site for data collection and analysis purposes. Discussions with Soilcea are still continuing as the USDA continues to review contract documents for the planting of CRSPR trees for trial at this site in a true Florida citrus growing region. Upcoming  meetings with Dr. Manjul Dut will be held during the end of August to discuss additional plantings at the site and plans for evlautating existing plantings. 3. Please state budget status (underspend or overspend, and why):This quarter, the project is ontrack for spending. We expect that we will be on track for spending for the remainder of the contract period.   4. Please show all potential commercialization products resulting from this research, and the status of each: The trangenic test site is consistently monitiored and researchers are contacted when their population(s) of trees show promising results for commericalization. This research has the potential to yield HLB-resistant transgenic and non-transgenic citrus cultivars suitable for commercial production. Some materials may be immediately suitable for commercial citrus production. The University of Florida has shown increased interest in the commricalization of some transgenic scions planted in the field that show promising single tree data. The data gathered from the field trials will guide recommendations for advancing materials to larger replicated trials, ensuring the development of commercially viable HLB-resistant citrus varieties.     

Generation of non-transgenic HLB-resistant sweet orange varieties using CRISPR-Cas technology

1. Please state project objectives and what work was done this quarter to address them: The purpose of this project is to generate non-transgenic HLB resistant Valencia and Hamlin sweet orange plants using CRISPR-Cas technology. Objective 1. Generate non-transgenic HLB resistant/tolerant Valencia and Hamlin sweet orange plants by mutation of HLB susceptibility genes. In total, six putative S genes were proposed to be edited and we have lately added more targets. Constructs needed for CRISPR genome editing are being made. Multiple edited lines were generated for ACD2 gene. However, further confirmation demonstrated none of them was biallelic/homozygous mutant. We are genenerating more lines for ACD2 and other target genes. To speed up the process, we have further optimized the transgene-free CRISPR genome editing using Cas12a/crRNA ribonucleoprotein. We have developed an efficient co-editing strategy for generating transgene-free, gene-edited plants via Agrobacterium-mediated transient expression of cytosine base editor (CBE)/gRNA-Cas12a/crRNA-GFP in planta.  Specifically, CBE/gRNA was used to base edit the ALS gene to confer resistance to herbicide chlorsulfuron as a selection marker, which has no negative effects on plant phenotypes; Cas12a/crRNA was used for editing genes(s) of interest; GFP was used for selecting transgene-free transformants. Using this approach, transgene-free genome-edited plants can be relatively easily generated for citrus in the T0 generation.  Whole genome sequencing further confirmed transgene-free and absence of off-target mutations in the edited plants. We are also using this strategy for genome editing of our target genes. This has been published by Nat. Plants (9, 1591–1597, 2023. https://doi.org/10.1038/s41477-023-01520-y). Among the 8 target genes, we have done transformation again on 5/18/23, 5/25/23, 6/25/23, 7/27/23, 8/17/23, 11/16/23, 12/07/23, 1/18/24, 2/15/2024, 2/22/2024, 3/7/2024, 4/11/2024, 4/25, 5/9, 5/23, 6/6, 6/20, 7/5, 7/19. The transformants done before August 2023 have successufully generated shoots for target genes. Deep sequencing of the edited protoplasts showed a mutation rate of 16-78%. Two targets have near zero mutation rates. We have successfully generated non-transgenic lines for two of the S genes. The plants have been confirmed to be 100% biallelic mutants and have been grafted on US-42 in the greenhouse. A third target gene has generated more than 20 shoots and been micrografted. In addition, we have generated Ray Ruby embryogenic protoplasts which are being tested for efficacy of transformation, editing, and regeneration. Objective 2. Generate cisgenic genome modified Valencia and Hamlin sweet orange plants by knock-in the gene encoding MaSAMP from Microcitrus. We are optimizing the knock-in method using the CRISPR technology. We have made some progress in knock-in methodology using non-transgenic approach. We have conducted multiple transformations useing the developed method for knockin with MaSAMP. We have demonstrated the knock-in works using GFP as an insert. We have conducted knock-in for MaSAMP on 5/18/23, 8/17/23, 11/16/23, 12/07/23, 1/18/24, 2/15/2024, 2/22/2024, 3/7/2024, 4/11/2024, 5/23, 6/20, 7/19 which are under regeneration. We are also testing to improve the insertion using priming editing technology.  2. Please state what work is anticipated for next quarter:More and more regenerated shoots have been generated. We will conduct more micrografting which will be repoted in the greenhouse. We will propogate and conduct CLas inoculation after we have acqauire 6 biological replicates per line for the non-transgenic genome edited plants.  3. Please state budget status (underspend or overspend, and why):On schedule.     

Consolidation of citrus breeding plant material to vacate space for Stage I and Stage II field trials and to exploit tolerant germplasm for gene editing strategies

1. Please state project objectives and what work was done this quarter to address them: Objective 1: Destroy declining plant material with no direct use to the citrus industry to make space for Stage I and Stage II trials (Years 1 and 2).Since project planning and the beginning of the project period, several thousand citrus trees have been pushed and destroyed at CREC, a portion of which is for making space for Stage I and Stage II trials.  Of these several thousand trees, approximately thousands are slated to be pushed and destroyed since the beginning of the last reporting period.  Hundreds of trees to up to 1,000 have been identified as being suitable for conservation, movement with the tree spade (or other means) and sourced for budwood.  Trees were identified by both drone and by visual inspection.  The nursery is aware of the need to collect and propagate uncertified budwood and they have given the go-ahead to transfer budwood immediately.  UFarm charges have conserved and saved all materials needed for data mining for tolerant genes and other variants. Objective 2: Conserve, move, consolidate, and propagate tolerant and resistant citrus trees to a centralized location. (Years 1 and 2).The block where consolidation will take place has been reserved for this purpose.  The nursery will receive the first budwood on Tuesday 8.6.24.  When the buds take, we will move forward with tree movement with tree spade and other means.  We do not have possession of the tree spade.  The tree spade has been repaired and we are waiting for the UF/IFAS payment to process before bringing it to the CREC to begin practicing moving trees of the appropriate size.  2. Please state what work is anticipated for next quarter: We anticipate using the USDA-ARS tree spade to move the first trees and transferring budwood to the nursery for propagation, which will start on this coming Tuesday.  1,000 budeyes from 84 accessions are planning to be transferred to the nursery.  We also anticipate removing thousands of additional citrus trees with no immediate value to the citrus industry to continue to make space for Stage I and II trials as well as breeding populations.    3. Please state budget status (underspend or overspend, and why): We are no longer underspent (29% time spent versus 40% funded expended) according to the UF/IFAS grants tracking system. The budget is being expended at a rate at which all funding will be expended by the end of the project period.   The payment has been made to the contractor, and we are on track for spending.    

Preliminary Field Trial to Evaluate the Ability of HLB Tolerant Interstocks to Protect Commercial Scions Against HLB

Understanding the HLB tolerance and reduced fruit drop in Parson Brown and evaluation of other early season sweet oranges

Evaluation of Potential HLB Tolerant Grapefruit Rootstock/Scion Combinations in Florida

1. Please state project objectives and what work was done this quarter to address them: The overall goal is to discover which of the seven (7) grapefruit scion and six (6) rootstock combinations planted in 41 blocks perform well under the endemic HLB disease pressure in Florida. The objectives are to 1) measure tree growth (using drone technology) and ratings of canopy thickness, yellowing, and HLB disease symptom severity, and 2) estimate fruit drop and tree yields as they come into production through preharvest fruit counts and measurement of fruit weight, size distribution and fruit quality from representative fruit samples.  Work done:   In February 2024 we continued with the fruit sampling for sizing and external and internal fruit quality analysis, to calculate fruit yield, plus the estimation of fruit drop by counting fruits on the tree and on the ground. In March and April 2024, we processed and analyzed the data collected in the previous months, and completed collection of disease index ratings, and intiated documentation of tree growth. A program is been developed to extract the data from the drone spreadsheets. Yield and disease index data were presented to Florida Citrus Show attendees on April 3, 2024.Preliminary results were presented by Dr. Zambon at the Polytechnic Univeristy of Valencia on May 22, 2024, and to the Valencian Grower’s Association on the May 23, 2024.    Drone data was collected on May 29, 2024.   2. Please state what work is anticipated for next quarter: Next quarter will comprise of the drone data analysis, continuation of the ground documentation of tree growth and initiating the HLB based on a quadrant-evaluation rating (disease index).    3. Please state budget status (underspend or overspend, and why): Spending delayed a little to allow for the second round of drone flights and maintain personnel until year two funds are available. A no-cost extension was approved. Expenses for the other half of the drone measurements will be paid upon receipt of the invoice, expected early June.

Development of Next-Generation SuperSour rootstocks with tolerance to HLB

Objective 1. Collect field performance data from replicated rootstock field trials and release new rootstock cultivars as justified by superior performance in multiyear field trials.Collect field performance data.  Thirty USDA replicated rootstock trials are active.  For February-April 2024, tree survival, cropping and juice data was collected from eight Valencia rootstock trials.  Juice analysis of frozen samples was conducted for early trials from the 2023-24 season.  Renovation of the fruit processing lab at USHRL was initiated, and will involve installation of a new autotitrator, refractometer, spectrophotometer, and data management system that will greatly improve workflow and increase the capacity to process fruit samples.  During this quarter, planting of stored seed began on candidate SuperSour rootstocks that will be used for morphology and SSR assessment of trueness-to-type from seed for selections tentatively considered for release over the next three years.  Additional work on trueness-to-type assessment and salinity tolerance of new rootstocks was also continued.  Cooperative work was continued to assess US rootstocks for resistance/tolerance to citrus nematode, sting nematode, and other traits.Release of new rootstocks. New rootstocks will be officially released by USDA for commercial use when justified by superior performance in multi-year replicated field trials.  The most promising USDA rootstock selections are provided to the FDACS-DPI program for shoot tip grafting and disease testing in advance of potential release.  The two newest USDA rootstocks, US SuperSour 4 and US SuperSour 5 released in September 2023, were propagated for planting in upcoming demonstration plantings and field trials.  Plans are also being made to include these newest rootstocks in the next phase of CRAFT.  Eight other promising new USDA rootstocks are planned to provide to DPI for STG and testing in 2024, in preparation for possible release in future years.Objective 2. Create hybrid rootstocks that combine germplasm from parental material with good rootstock traits and HLB tolerance, propagate the most promising of these hybrids, and establish replicated field trials with commercial scions.Create hybrids. About 230 hybrid seedlings from 2023 high priority crosses are being grown in the greenhouse this quarter for preliminary evaluation and further testing.Propagate hybrids.  About 1500 trees for two new stage 2 rootstock trials with 35 advanced selection SuperSour rootstocks and Valencia scion continue growing in the nursery, with a plan for field planting in June 2024.Establish replicated field trials. No new trials were field planted this quarter, but two are planned for planting in the next quarter.Tree care in trials. Periodic care was applied in the 30 field trials to maintain tree health and productivity, and manage weeds.  Two young rootstock trials are being grown with IPCs.  One rootstock trial is being used for a comparison of tree performance of different rootstocks with and without OTC injection.2. Please state what work is anticipated for next quarter:For the coming quarter, work will focus on processing brix, acid, and color for about 5000 juice samples from rootstock trials in the 2023-24 season, continuing propagation of trees for future trials, collection of data on tree size and health in trials, and care of existing trees in trials.  Two new Valencia rootstock trials with the advanced SuperSour selections are planned for field planting in the next quarter.  Data analysis of tree performance in the 30 trials is underway, to evaluate SuperSour candidates for official release in 2025-26, and to compare performance among commercially available rootstocks. Cooperative work continues to evaluate USDA rootstocks for tolerance/resistance to sting nematode, citrus nematode, salinity, cold hardiness, nursery performance, and other traits.  Work continues to coordinate with other laboratories in mapping of important rootstock traits that will facilitate more effective citrus breeding in the future.3. Please state budget status (underspend or overspend, and why):The budget is on schedule and spent out all second-year funds at the end of the second year of the project.  Work is progressing as planned. 

Transgenic capable field site to assess HLB-resistant and other improved citrus

1. Please state project objectives and what work was done this quarter to address them:This quarter, Project #21-025 continued its dedicated efforts towards developing HLB-resistant and improved citrus cultivars to mitigate the detrimental impact of HLB on the Florida citrus industry. The project employed both transgenic and non-transgenic approaches and conducted experiments at the secure field site of the USDA/ARS Picos Research Farm. The primary focus remained on collecting data from ongoing field trials for various HLB-resistant candidates.  To ensure realistic evaluations of candidates’ resistance, the test site received necessary horticultural care and maintained exposure to HLB and psyllids. Additionally, the team adhered to BRS permits and underwent inspections to uphold regulatory compliance. A significant milestone during this quarter was contact with the company Soilcea for the planting of transgenic CRISPR rootstocks and scions at the Picos Transgenic site. We are currently in the paperwork process surrounding this relationship and look forward to having these materials at the Picos transgenic site to sever as an additional location and a valuable evaluation resource.  2. Please state what work is anticipated for next quarter:Efforts will also focus on removing materials that are abandon or no longer being utilized to make way for Soilcea trees. We are hoping to be able to initiate a planting of Soilcea trees by the end of the summer.  Data collection on active trials will continue, with allocated labor for monitoring and permitting to ensure smooth progress.  3. Please state budget status (underspend or overspend, and why):The project is currently on tract to be spent down by the end of September 2024. The delays in the initiation of this project have been remedied and the project is moving forward.   4. Please show all potential commercialization products resulting from this research, and the status of each:The research conducted under Project #21-025 has yielded promising results in identifying HLB-resistant transgenic and non-transgenic citrus cultivars. Some of these materials show potential for commercial citrus production, while others  may serve as breeding parents for further improvement in HLB resistance and other commercially significant traits. Data gathered from the field trials will continue to guide the project in advancing the most promising materials to larger trials, ultimately contributing to the development of commercially viable HLB-resistant citrus varieties and safeguarding the Florida citrus industry’s future.   

Consolidation of citrus breeding plant material to vacate space for Stage I and Stage II field trials and to exploit tolerant germplasm for gene editing strategies

1. Please state project objectives and what work was done this quarter to address them: Objective 1: Destroy declining plant material with no direct use to the citrus industry to make space for Stage I and Stage II trials (Years 1 and 2).Since project planning and the beginning of the project period, several thousand citrus trees have been pushed and destroyed at CREC, a portion of which is for making space for Stage I and Stage II trials.  Of these several thousand trees, approximately 1,000 trees were pushed and destroyed since the beginning of the project period.  Hundreds of trees have been identified as being suitable for conservation, movement with the tree spade (or other means) and sourced for budwood.  The nursery is aware of the need to collect and propagate uncertified budwood and we are waiting on the go-ahead from the nurserymen and -women to transfer budwood of the trees identified for conservation and consolidation to the nursery.  The drone project has identified trees that should be targeted for consolidation and ground truthing these trees has been completed at two large blocks of Plant Improvement land.  UFarm charges have conserved and saved all materials needed for data mining for tolerant genes and other variants. Objective 2: Conserve, move, consolidate, and propagate tolerant and resistant citrus trees to a centralized location. (Years 1 and 2).The block where consolidation will take place has been reserved for this purpose.  We are awaiting the nursery’s go-ahead to propagate the material.  Rootstocks have been selected.  When the buds take, we will move forward with tree movement with tree spade and other means.  We do not have possession of the tree spade.  According to the contractor who is repairing it, the tree spade will be ready in two to three weeks. We are developing contingency plans if the nursery delays the budwood transfer.  2. Please state what work is anticipated for next quarter: We anticipate taking possession of the USDA-ARS tree spade from the contractor and transferring budwood to the nursery for propagation.  We also anticipate removing thousands of citrus trees with no immediate value to the citrus industry to continue to make space for Stage I and II trials.    3. Please state budget status (underspend or overspend, and why): We are 1% underspent (15% time spent versus 14% funded expended) according to the UF/IFAS grants tracking system. The budget is underspent as we wait for the repairs of the tree spade.  Once that payment is made to the contractor, we should be on track for spending.  It was not anticipated for the tree spade repair to take more than three months.    

Evaluation of Potential HLB Tolerant Grapefruit Rootstock/Scion Combinations in Florida

1. Please state project objectives and what work was done this quarter to address them: The overall goal is to discover which of the seven (7) grapefruit scion and six (6) rootstock combinations planted in 41 blocks perform well under the endemic HLB disease pressure in Florida. The objectives are to 1) measure tree growth (using drone technology) and ratings of canopy thickness, yellowing, and HLB disease symptom severity, and 2) estimate fruit drop and tree yields as they come into production through preharvest fruit counts and measurement of fruit weight, size distribution and fruit quality from representative fruit samples.  Work done:   In November and December 2023, and January 2024 we continued with the fruit sampling for sizing and external and internal fruit quality analysis, to calculate fruit yield, plus the estimation of fruit drop by counting fruits on the tree and on the ground. Drone data collected in October was accessible in late November, which is still to be analyzed due to the volume of data to be exported. We have initial data for fruit quality and yield for the eight blocks that were confirmed as ready to harvest. Due to raining conditions in December, the Holidays and vacation time of team members, data collection has been extended into January and February 2024. We completed data collection of 75% of the blocks, and expect to finish it by early March.Preliminary data was presented to the Indian River Citrus League Board of Directors meeting on February 10th, 2024.  2. Please state what work is anticipated for next quarter: Next quarter we will have another round of drone data collection, plus field measurements of tree growth and HLB symptoms based on a quadrant-evaluation rating (disease index).    3. Please state budget status (underspend or overspend, and why): Spending generally on track – payroll was started in October with the start of field evaluations and fruit harvests and is running a little behind schedule. Expenses for half the drone measurements have been paid.  

Generation of non-transgenic HLB-resistant sweet orange varieties using CRISPR-Cas technology

1. Please state project objectives and what work was done this quarter to address them: The purpose of this project is to generate non-transgenic HLB resistant Valencia and Hamlin sweet orange plants using CRISPR-Cas technology. Objective 1. Generate non-transgenic HLB resistant/tolerant Valencia and Hamlin sweet orange plants by mutation of HLB susceptibility genes. In total, six putative S genes were proposed to be edited and we have lately added more targets. Constructs needed for CRISPR genome editing are being made. Multiple edited lines were generated for ACD2 gene. However, further confirmation demonstrated none of them was biallelic/homozygous mutant. We are genenerating more lines for ACD2 and other target genes. To speed up the process, we have further optimized the transgene-free CRISPR genome editing using Cas12a/crRNA ribonucleoprotein. We have developed an efficient co-editing strategy for generating transgene-free, gene-edited plants via Agrobacterium-mediated transient expression of cytosine base editor (CBE)/gRNA-Cas12a/crRNA-GFP in planta.  Specifically, CBE/gRNA was used to base edit the ALS gene to confer resistance to herbicide chlorsulfuron as a selection marker, which has no negative effects on plant phenotypes; Cas12a/crRNA was used for editing genes(s) of interest; GFP was used for selecting transgene-free transformants. Using this approach, transgene-free genome-edited plants can be relatively easily generated for citrus in the T0 generation.  Whole genome sequencing further confirmed transgene-free and absence of off-target mutations in the edited plants. We are also using this strategy for genome editing of our target genes. This has been published by Nat. Plants (9, 1591–1597, 2023. https://doi.org/10.1038/s41477-023-01520-y). Among the 8 target genes, we have done transformation again on 5/18/23, 5/25/23, 6/25/23, 7/27/23, 8/17/23, 11/16/23, 12/07/23, 1/18/24. The transformants are under regeneration. Deep sequencing of the edited protoplasts showed a mutation rate of 16-78%. Two targets have near zero mutation rates. We are optimizing the guide RNA design. Some lines done in May 2023 are getting close to be micro-grafted.In addition, we have generated Ray Ruby embryogenic protoplasts which are being tested for efficacy of transformation, editing, and regeneration. Objective 2. Generate cisgenic genome modified Valencia and Hamlin sweet orange plants by knock-in the gene encoding MaSAMP from Microcitrus. We are optimizing the knock-in method using the CRISPR technology. We have made some progress in knock-in methodology using non-transgenic approach. We have conducted multiple transformations useing the developed method for knockin with MaSAMP. The protoplasts were being regenerated.We have demonstrated the knock-in works using GFP as an insert. We have conducted knock-in for MaSAMP on 5/18/23, 8/17/23, 11/16/23, 12/07/23, 1/18/24 which are under regeneration. 2. Please state what work is anticipated for next quarter:To generate more genome edited lines for ACD2 and other target genes using both RNP method and the co-editing method. Conductu regeneration for all the transformants. Conduct micro-grafting for shoots when ready.3. Please state budget status (underspend or overspend, and why):On schedule.     

Development of Next-Generation SuperSour rootstocks with tolerance to HLB

Objective 1. Collect field performance data from replicated rootstock field trials and release new rootstock cultivars as justified by superior performance in multiyear field trials.Collect field performance data.  Thirty USDA replicated rootstock trials are active.  For the current quarter, tree survival, cropping and juice data was collected from one mandarin rootstock trial and six Hamlin rootstock trials.  Juice analysis of frozen samples was completed for trials from the 2022-23 season, and begun on trials from the 2023-24 season.  During this quarter, seed was collected from more than 200 candidate SuperSour rootstocks.  This will be used for morphology and SSR assessment of trueness-to-type from seed.  Additional work on trueness-to-type assessment and salinity tolerance of new rootstocks was continued.Release of new rootstocks. New rootstocks will be officially released by USDA for commercial use when justified by superior performance in multi-year replicated field trials.  The most promising USDA rootstock selections are provided to the FDACS-DPI program for shoot tip grafting and disease testing in advance of potential release.  The two newest USDA rootstocks, US SuperSour 4 and US SuperSour 5 released in September 2023, were propagated for planting in upcoming demonstration plantings.  Two other promising new USDA rootstocks were provided to DPI for STG and testing this quarter, in preparation for possible release in future years.Objective 2. Create hybrid rootstocks that combine germplasm from parental material with good rootstock traits and HLB tolerance, propagate the most promising of these hybrids, and establish replicated field trials with commercial scions.Create hybrids. About 230 hybrid seedlings from 2023 high priority crosses will be grown in the spring for preliminary evaluation and further testing.Propagate hybrids.  Trees for two new stage 2 rootstock trials with 35 advanced selection SuperSour rootstocks with Valencia scion continue growing in the nursery, with a plan for field planting in spring-summer 2024.Establish replicated field trials. No new trials were field planted this quarter, but two are planned for planting in the next quarter.Tree care in trials. Periodic care was applied in the 32 field trials to maintain tree health and productivity, and manage weeds.  Two young rootstock trials are being grown with IPCs.  One rootstock trial is being used for a comparison of tree performance of different rootstocks with and without OTC injection.2. Please state what work is anticipated for next quarter:For the coming quarter, work will focus on collection of 2023-24 season yield and fruit quality data from trials with Valencia scion, continuing propagation of trees for future trials, and care of existing trees in trials.  Two new Valencia rootstock trials with the advanced SuperSour selections are planned for field planting in the next two quarters.  Data analysis of tree performance in the 30 trials is underway, to evaluate SuperSour candidates for official release in 2025-26, and compare performance of commercially available rootstocks. Cooperative work continues to evaluate USDA rootstocks for tolerance/resistance to sting nematode, citrus nematode, salinity, cold hardiness, nursery performance, and other traits.3. Please state budget status (underspend or overspend, and why):The budget is on schedule for spending all funds by the end of the currently-funded year.  Work is progressing as planned. 

Comparison of field performance of citrus trees on rootstocks propagated by seedlings, cuttings, and tissue culture

1. Please state project objectives and what work was done this quarter to address them: Objective 1: Assess rootstock propagation method and rootstock cultivar effects on growth, health, and productivity of grafted Valencia trees during the early production years (years 4-6 after planting) in two commercial citrus production environments. Objective 2: Assess rootstock propagation method effect on tree performance, root architectures and uprooting resistance of Valencia trees after 5 years of growth.We continued to monitor the trees. Rhizotron analysis continued. CLas analyses are in progress.  2. Please state what work is anticipated for next quarter:We will collect leaves for CLas analysis, conduct disease ratings, take rhizotron pictures, and continue with all data and lab analyses. We will also prepare for the harvest. 3. Please state budget status (underspend or overspend, and why):  Budget status is on track.