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, 15911597, 2023. https://doi.org/10.1038/s41477-023-01520-y). Among the 8 target genes, we have done transformation again on 5/18, 5/25, 6/25, 7/27, and 8/17. The transformants are under 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, and 8/17 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. 3. Please state budget status (underspend or overspend, and why):On schedule.
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-two USDA replicated rootstock trials are active. For the current quarter, canopy health was collected from all trials. Cropping and fruit quality data was collected from two early mandarin rootstock trials, and two lemon rootstock trials. Juice analysis of frozen samples continued from trials for the 2022-23 season. A new auto-titrator and spectrophotometer for use in fruit quality analysis were purchased from base USDA funds, and will greatly accelerate and improve the evaluation of fruit quality in rootstock trials.During this quarter, SSR assessment of trueness-to-type from seed was completed for the SuperSour rootstocks that are available now and will be released this year. This information on trueness-to-type from seed will be published shortly after the official release of the two new rootstocks. 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 new USDA rootstocks, US SuperSour 4 and US SuperSour 5 were released by USDA on September 5th, 2023, and are already clean and available in the DPI program to commercial nurseries. Four 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. Selected new crosses were completed in spring 2023, and seed extracted this quarter. About 230 hybrid seedlings from 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 33 advanced selection SuperSour rootstocks with Valencia scion continue growing in the nursery, with a plan for field planting in summer 2024.Establish replicated field trials. One new replicated field trial with a new high quality mandarin scion on 17 rootstocks was established this quarter at the Whitmore farm in Lake County.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 Hamlin scion, continuing propagation of trees for future trials, and care of existing trees in trials. Data analysis of tree performance in the 32 trials is underway, to evaluate SuperSour candidates for official release in 2025-26, and compare performance of those commercially available. Cooperative work continues to evaluate USDA rootstocks for tolerance/resistance to sting nematode, citrus nematode, salinity, cold hardiness, 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. One employee left the program, and a recruitment is underway to take on those duties. Work is progressing as planned.
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-two USDA replicated rootstock trials are active. For the current quarter, canopy health was collected from all trials. Cropping and fruit quality data was collected from two early mandarin rootstock trials, and two lemon rootstock trials. Juice analysis of frozen samples continued from trials for the 2022-23 season. A new auto-titrator and spectrophotometer for use in fruit quality analysis were purchased from base USDA funds, and will greatly accelerate and improve the evaluation of fruit quality in rootstock trials.During this quarter, SSR assessment of trueness-to-type from seed was completed for the SuperSour rootstocks that are available now and will be released this year. This information on trueness-to-type from seed will be published shortly after the official release of the two new rootstocks. 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 new USDA rootstocks, US SuperSour 4 and US SuperSour 5 were released by USDA on September 5th, 2023, and are already clean and available in the DPI program to commercial nurseries. Four 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. Selected new crosses were completed in spring 2023, and seed extracted this quarter. About 230 hybrid seedlings from 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 33 advanced selection SuperSour rootstocks with Valencia scion continue growing in the nursery, with a plan for field planting in summer 2024.Establish replicated field trials. One new replicated field trial with a new high quality mandarin scion on 17 rootstocks was established this quarter at the Whitmore farm in Lake County.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 Hamlin scion, continuing propagation of trees for future trials, and care of existing trees in trials. Data analysis of tree performance in the 32 trials is underway, to evaluate SuperSour candidates for official release in 2025-26, and compare performance of those commercially available. Cooperative work continues to evaluate USDA rootstocks for tolerance/resistance to sting nematode, citrus nematode, salinity, cold hardiness, 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. One employee left the program, and a recruitment is underway to take on those duties. Work is progressing as planned.
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-two USDA replicated rootstock trials are active. For the current quarter, canopy health was collected from all trials. Cropping and fruit quality data was collected from two early mandarin rootstock trials, and two lemon rootstock trials. Juice analysis of frozen samples continued from trials for the 2022-23 season. A new auto-titrator and spectrophotometer for use in fruit quality analysis were purchased from base USDA funds, and will greatly accelerate and improve the evaluation of fruit quality in rootstock trials.During this quarter, SSR assessment of trueness-to-type from seed was completed for the SuperSour rootstocks that are available now and will be released this year. This information on trueness-to-type from seed will be published shortly after the official release of the two new rootstocks. 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 new USDA rootstocks, US SuperSour 4 and US SuperSour 5 were released by USDA on September 5th, 2023, and are already clean and available in the DPI program to commercial nurseries. Four 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. Selected new crosses were completed in spring 2023, and seed extracted this quarter. About 230 hybrid seedlings from 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 33 advanced selection SuperSour rootstocks with Valencia scion continue growing in the nursery, with a plan for field planting in summer 2024.Establish replicated field trials. One new replicated field trial with a new high quality mandarin scion on 17 rootstocks was established this quarter at the Whitmore farm in Lake County.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 Hamlin scion, continuing propagation of trees for future trials, and care of existing trees in trials. Data analysis of tree performance in the 32 trials is underway, to evaluate SuperSour candidates for official release in 2025-26, and compare performance of those commercially available. Cooperative work continues to evaluate USDA rootstocks for tolerance/resistance to sting nematode, citrus nematode, salinity, cold hardiness, 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. One employee left the program, and a recruitment is underway to take on those duties. Work is progressing as planned.
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.Leaves were collected for nutrient analysis. Roots were collected for root length analysis. Tree size measurements were conducted. Rhizotron analysis continued. CLas analyses are in progress. All trees in one of the commercial sites were injected with OTC by the grower collaborator this year. 2. Please state what work is anticipated for next quarter: Lab analyses and data analyses will continue. Tree health will be monitored. 3. Please state budget status (underspend or overspend, and why): Budget status is on track.
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 rto BRS permits and underwent inspections to uphold regulatory compliance. A significant milestone during this quarter was the hosting of a field day, where researchers and stakeholders were provided with insights into successful trial materials and the data collection processes. Moreover, signage and field markers were updated to enhance collaborator ease and streamline documentation processes. There were over 60 in attendance at this first field day event. 2. Please state what work is anticipated for next quarter:Efforts will also focus on updating transgenic field markers signage and labeling to ensure compliance. Additionally, the project aims to submit a permit to BRS for additional planting in late summer and early fall, enabling further research expansion (Material from Dr. Robert Shatters). 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 experienced a slight underspend primarily due to complications in hiring USDA personnel and historical underspend trends assoicated with this project. The technition on this project is currently budgeted and continuously working on permitting assoicated improvment of this block for the USDA and cooperators. 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.
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 rto BRS permits and underwent inspections to uphold regulatory compliance. A significant milestone during this quarter was the hosting of a field day, where researchers and stakeholders were provided with insights into successful trial materials and the data collection processes. Moreover, signage and field markers were updated to enhance collaborator ease and streamline documentation processes. There were over 60 in attendance at this first field day event. 2. Please state what work is anticipated for next quarter:Efforts will also focus on updating transgenic field markers signage and labeling to ensure compliance. Additionally, the project aims to submit a permit to BRS for additional planting in late summer and early fall, enabling further research expansion (Material from Dr. Robert Shatters). 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 experienced a slight underspend primarily due to complications in hiring USDA personnel and historical underspend trends assoicated with this project. The technition on this project is currently budgeted and continuously working on permitting assoicated improvment of this block for the USDA and cooperators. 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.
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 rto BRS permits and underwent inspections to uphold regulatory compliance. A significant milestone during this quarter was the hosting of a field day, where researchers and stakeholders were provided with insights into successful trial materials and the data collection processes. Moreover, signage and field markers were updated to enhance collaborator ease and streamline documentation processes. There were over 60 in attendance at this first field day event. 2. Please state what work is anticipated for next quarter:Efforts will also focus on updating transgenic field markers signage and labeling to ensure compliance. Additionally, the project aims to submit a permit to BRS for additional planting in late summer and early fall, enabling further research expansion (Material from Dr. Robert Shatters). 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 experienced a slight underspend primarily due to complications in hiring USDA personnel and historical underspend trends assoicated with this project. The technition on this project is currently budgeted and continuously working on permitting assoicated improvment of this block for the USDA and cooperators. 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.
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 rto BRS permits and underwent inspections to uphold regulatory compliance. A significant milestone during this quarter was the hosting of a field day, where researchers and stakeholders were provided with insights into successful trial materials and the data collection processes. Moreover, signage and field markers were updated to enhance collaborator ease and streamline documentation processes. There were over 60 in attendance at this first field day event. 2. Please state what work is anticipated for next quarter:Efforts will also focus on updating transgenic field markers signage and labeling to ensure compliance. Additionally, the project aims to submit a permit to BRS for additional planting in late summer and early fall, enabling further research expansion (Material from Dr. Robert Shatters). 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 experienced a slight underspend primarily due to complications in hiring USDA personnel and historical underspend trends assoicated with this project. The technition on this project is currently budgeted and continuously working on permitting assoicated improvment of this block for the USDA and cooperators. 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.
1. Please state project objectives and what work was done this quarter to address them: Objective 1: Production of interstock trees for evaluation in grower plots Interstock grafts have been produced on most of the rootstocks. Some of the rootstock liners growing in the greenhouse were damaged due to a hard to control thrip infestation in the greenhouse which led to malformed leaves and shoots. We have controlled the greenhouse infestation but the grafting work has been slightly delayed due to this issue. It is anticipated that all interstock grafting will be completed in the next quarter. 2. Please state what work is anticipated for next quarter: All rootstock liners should be stick grafted with the HLB tolerant interstocks in the third quarter of this project. 3. Please state budget status (underspend or overspend, and why): We are on track with the budget spending as funds have been committed for plant propagation.
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: Per the project timeline, we have been preparing for September and October grove visits and tree and fruit measurements. We have also developed a contract with Aerobotics to collect drone measurements on all experimental blocks in September and October of this year. 2. Please state what work is anticipated for next quarter: Next quarter will be extremely busy conducting tree measurements (via drone), taking tree measuremnts and, for trees with fruit production, record fruit numbers on sample trees (not including buffer trees) and raking underneath the trees to later measure fruit drop. 3. Please state budget status (underspend or overspend, and why): Currently underspending because no field work yet underway this quarter. Heavy use of personelle (and expenses) begin in September (next quarter) and wil continue throughout the winter. In addition, expense for drone measurements have not yet been incurred.
1. Please state project objectives and what work was done this quarter to address them: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-one USDA replicated rootstock trials are active. For the current quarter, canopy health and tree size data was collected from all trials. Juice analysis continued from trials for the 2022-23 season. Some problems developed with the autotitrator and spectrophotometer used for the fruit quality analysis which slowed down the juice analysis. New equipment is being purchased with USDA funding to provide higher throughput analysis of juice samples beginning in the 2023-24 season.During this quarter, SSR assessment of trueness-to-type from seed was completed for the SuperSour rootstocks that are available now and will be released this year. This information is being prepared for publication that will coincide with the official release of the new rootstocks. Additional work on trueness-to-type assessment and salinity tolerance of new rootstocks was continued.Preparation for release. The most promising USDA rootstock selections are provided to the FDACS-DPI program for shoot tip grafting and disease testing. The two new USDA rootstocks planned for release this year are already clean and available in the DPI program. Four other promising new USDA rootstocks were provided to DPI for STG and testing this quarter, in preparation for possible release in future years.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 rootstocks US SuperSour 4 and US SuperSour 5 have moved to the final stage of approval for release by USDA.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. Selected new crosses were completed in spring 2023, and good fruit set was achieved. Fruit will be monitored and harvested for seed extraction later in 2023.Propagate hybrids. Budded trees continued to be grown in the nursery for a rootstock trial with a new high quality scion, with field planting scheduled for August. Trees for two new stage 2 rootstock trials with thirty SuperSour rootstocks were budded with Valencia in this quarte, with a plan for field planting in 2024.Establish replicated field trials. One new replicated field trial with a new high quality scion on 14 rootstocks was established with a commercial cooperator this quarter.Tree care in trials. Periodic care was applied in the 31 field trials to maintain tree health and productivity, and manage weeds.2. Please state what work is anticipated for next quarter:For the coming quarter, work will focus on harvesting seed from crosses, beginning collection of 2023-24 season yield and fruit quality data on trials with early scions, continuing propagation of trees for future trials, and care of existing trees in trials. Official release of US SuperSour 4 and US SuperSour 5 is planned for the next quarter.3. Please state budget status (underspend or overspend, and why):This is the first quarter of the second year, and budget is on schedule for spending. One employee left the program, and a new employee was hired to take on those duties. The project is fully staffed, and work is progressing as planned.
1. The goal of this project is to protect citrus from Candidatus Liberibacter asiaticus by inducing the phloem to produce anchored, single-chain antibodies that will bind and immobilize the bacteria, allowing the plant to destroy them by natural defense mechanisms. Anchoring is accomplished by expressing the antibodies as extensions of native, phloem-specific sieve element occlusion (SEO) proteins. By the end of the first quarter of the grant period we produced over 30 transgenic Carrizo lines. We selected 8 lines for further analysis: 3 untransformed controls transformed without the binding proteins and 5 transformed lines with varying degrees of construct expression. On June 13, 2023, the plants were sent (with a USDA interstate transport permit) to the Amit Levy lab at the Citrus Research and Education Center at the University of Florida. 2. The Levy lab is now continuing to grow the plants and in the next month will expose them to Diaphorina citri carrying CLas.We also began the second phase of the program, to test additional antibody constructs (although this phase cannot be completed in the 1-year timeline). One objective is to use different phloem-specific promoters to increase the number of anchored antibodies in the phloem. A second strategy is to create dual antibodies, a strategy that has been shown to dramatically suppress bacterial growth. 3. The budget status is as anticipated with funds neither underspent nor overspent. 4. Commercialization products: None were anticipated for this grant, although it may be possible to commercialize the transformed Carrizo citrus as rootstocks if they are sufficiently protected by our strategy.
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 will be edited. 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 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. Among the 8 target genes, we have done transformation again on 5/18, 5/25, or 6/25. The transformants are under 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, which is 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. 3. Please state budget status (underspend or overspend, and why):On schedule.
1. Please state project objectives and what work was done this quarter to address them: The primary objective of Project # 21-025 is to address the devastating impact of huanglongbing (HLB) on the Florida citrus industry. The disease has caused significant economic losses and threatens the sustainability of citrus production in the region. The project aims to test and validate HLB-resistant transgenic and non-transgenic citrus materials in a secure field site at the USDA/ARS Picos Research Farm. This will enable researchers to evaluate the efficacy of various strategies to combat HLB and its psyllid vector, with the ultimate goal of sustaining economic citrus production in HLB-endemic regions.The research team continued to maintain and collect data from ongoing field trials. The 10-acre test site at the USDA/ARS Picos Research Farm was secured and equipped for the upcoming experiments. The team provided pre-planting planning assistance to researchers to ensure compliance with transgenic regulations and offered annual transgenic-control training sessions. 2. Please state what work is anticipated for next quarter: Routine sample and data collection will continue to track the CLas titer and HLB progression in the test trees.Researchers will be granted access to the site for data collection and data analysis purposes.The team will conduct inspections and maintain compliance with the APHIS Biotechnology Regulatory Services (BRS) permits. Farm staff and the employed technician will receive further training on handling transgenic field trials. 3. Please state budget status (underspend or overspend, and why): This quarter, there was an underspend of the allocated budget due to changes in PI and leadership at USHRL. The change in personnel led to some delays in the implementation of field trials and preparation of the test site. However, the project team managed to reallocate the budget effectively and continued to make progress in planning and preparing for the upcoming experiments. 4. Please show all potential commercialization products resulting from this research, and the status of each: The research has the potential to yield HLB-resistant transgenic and non-transgenic citrus cultivars suitable for commercial production. Some materials may be ready for immediate commercial use, while others will serve as breeding parents for further improvement. Intellectual property rights of researchers will be preserved, and no expectations of inclusion in related patents will be imposed by USDA/ARS.
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