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 primary focus was on conducting field trials for promising HLB-resistant candidates from various collaborating institutions, including Texas A&M University, University of Florida, and USDA/ARS. Researchers continued to maintain and collect data from ongoing field trials, including monitoring CLas titer and HLB progression in the test trees. 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. 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. Any required adjustments or changes resulting from the observations will be managed individually without compromising the overall project objectives. If any candidates show promising results, they may be included in larger replicated trials for further validation. 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 transition in personnel caused some disruptions in data collection and analysis for the ongoing field trials. However, the project team was able to adapt and allocate the remaining budget efficiently to maintain the test site and ensure the continued progress of the research. 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 immediately suitable for commercial citrus production, while others may serve as breeding parents for further improvement. 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.
1. Please state project objectives and what work was done this quarter to address them: Project # 21-025 continues its focus on developing HLB-resistant and other improved citrus cultivars to combat the devastating effects of HLB on the Florida citrus industry. The project employs transgenic and non-transgenic approaches and conducts experiments at the secure field site of the USDA/ARS Picos Research Farm. The goal remains to provide sustainable solutions to combat HLB, safeguard the citrus industry, and support economic citrus production.This quarter, the primary emphasis was on analyzing the data collected from the ongoing field trials for various HLB-resistant candidates. Researchers thoroughly evaluated the efficacy of different strategies to combat HLB and its psyllid vectors based on the data obtained. The test site continued to receive the necessary horticultural care and maintained exposure to HLB and psyllid vectors to ensure realistic evaluations of the candidates’ resistance. The team continued to adhere to BRS permits and underwent inspections to ensure compliance with regulatory requirements. 2. Please state what work is anticipated for next quarter: Next quarter, the team will finalize the analysis of data from the current field trials. Based on the results, the project will make recommendations for advancing promising HLB-resistant candidates to larger replicated trials for further validation. Researchers will have continued access to the test site for any additional data collection and analysis requirements. There will be an update to trangenic field markers sinage and labeling to insure ease of finding material and compliance with BRS permits. We will also be conducting a transgenic field day with all of the cooperators that have material at the site. The project team will also initiate the process of requesting a six-month no-cost extension to the project timeline. 3. Please state budget status (underspend or overspend, and why): This quarter, there was an underspend mainly due to underspend in the previous quarters. Given the temporary disruptions caused by changes in PI and leadership at USHRL, the project team is requesting a six-month no-cost extension to the project timeline. The extension will allow sufficient time to complete the ongoing field trials, finalize data analysis, and make informed recommendations for advancing potential HLB-resistant candidates. The no-cost extension will ensure the project’s successful completion without additional financial burden to the funding agency. 4. Please show all potential commercialization products resulting from this research, and the status of each: The research has shown promising results in identifying HLB-resistant transgenic and non-transgenic citrus cultivars. Some of the candidate materials display a high likelihood of being suitable for immediate commercial citrus production. These HLB-tolarant cultivars could become valuable assets to the citrus industry, providing a sustainable solution to combat the devastating impact of HLB. Other materials may serve as important breeding parents for further improvement in HLB resistance and other commercially significant traits.The data gathered from the field trials will drive recommendations for advancing the most promising materials to larger replicated trials, ensuring the development of commercially viable HLB-resistant citrus varieties.
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.Rhizotron pictures were taken in both commercial trials and image analyses is continuing. 2. Please state what work is anticipated for next quarter: Tree growth measurements will be conducted. Leaves will be collected for nutrient analyses. Roots will be collected for specific root length analysis. 3. Please state budget status (underspend or overspend, and why): Budget status is as expected.
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 All rootstock trees as needed for this project have been sourced and are being sized up for interstock budding. The US942 trees were larger in size and most of those have been stick grafted for interstock production. It is anticipated that all interstock rootstock trees will be stick grafted in second quarter of 2023 2. Please state what work is anticipated for next quarter: All rootstock liners will be stick grafted with the HLB tolerant interstocks in the second 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: Objective 1: Study the changes in the reactive oxygen species (ROS) and investigate the correlation between ROS accumulation, sucrose levels and callose deposition in early season sweet oranges Budded trees from Hamlin, Parson Brown and Roble sweet oranges have been produced and infected with CaLas through stick grafting. Control samples before infection have been collected and the next set will be collected once trees are HLB+. These trees will also be used for Objective 3 analysis. Objective 2: Quantify fruit drop and canopy yield efficiency in Parson Brown as compared to Hamlin. Understand the underlying mechanism of better fruit retention and yield efficiency in Parson Brown as compared to Hamlin. This experiment will be initiated from Mid June. Objective 3: Determine the mechanism underpinning the tolerance of Parson Brown to HLB by leaf volatiles and non-volatile metabolite analysis using GC-MS.Trees have been produced for both objective 1 and 3 and analysis is underway. Objective 4: Evaluate other early season sweet orangesLeaf samples have been collected from a Roble sweet orange grove in Zephyrhills. Additionally, samples from several survivor trees from a grove in Lake County have been collected. We have analysed the HLB status of these trees and the biochemical profile of the leaf samples. 2. Please state what work is anticipated for next quarter: Fruit drop studies will be intiated on field trees. Also, molecular and biochemical analysis of the HLB infected trees will be initiated. 3. Please state budget status (underspend or overspend, and why): We are on track with the budget. A postdoc has been hired to work on several of the parameters in this project from the second quarter.
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 data was collected from all trials, and for the ten bearing trials with Valencia scion, fruit yield and fruit quality data was also collected from about 4500 experimental units (replications). Juice quality evaluations continued on samples from other trials with early maturing scions.During this quarter, seedlings of a select group SuperSour rootstocks with outstanding performance were grown for assessment of trueness-to-type from seed in preparation for cultivar release for commercial use.Preparation for release. The most promising USDA rootstock selections that may be released in the next 2 years were previously provided to the FDACS-DPI program for shoot tip grafting and disease testing. Those have been completed in the DPI program and are currently available for commercial nursery propagation, following the decision for official release by USDA.Release of new rootstocks. New rootstocks will be officially released by USDA for commercial use when justified by superior peformance in multi-year replicated field trials. The rootstocks US-1688 and US-1709 have been proposed for release, and are in the second stage of approval by USDA. It is anticipated that these rootstocks will be named US SuperSour 4 and US SuperSour 5, and will be released and commercially available sometime in 2023.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 this quarter with twenty high-priority parental combinations based on the most recent information about heritability of important rootstock traits. Fruit set was scored and will be followed until harvest of the hybrid seed.Propagate hybrids. Budded trees in the nursery for a rootstock trial with a new high-quality scion continued to be grown and developed for field planting in 2023. New rootstocks with the most superior performance were selected from stage 1 trials and liners propagated, in preparation for budding of trees with Valencia in summer of 2023. It is anticipated that these trees will be ready for field planting of two new rootstock field trials in spring 2024.Establish replicated field trials. No new field trials were planted 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 completing the juice quality analysis from the Valencia field trials, collection of tree size and canopy health data from all the trials, continuing propagation of trees for future trials, and care of existing trees in trials. Greenhouse and laboratory work will also continue with focus on assessment of trueness-to-type from seed for the most promising rootstocks using SSR methodology.3. Please state budget status (underspend or overspend, and why):Budget is on schedule to be spent out by the end of the first year. The project was fully staffed, and work is progressing as planned.
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. 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. 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. Using the developed method for knockin with MaSAMP. 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: 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.In both commercial trials, fruit were harvested to determine yield and fruit quality was analyzed. Trials had few fruit, either due to the impacts of hurricane Ian and/or because of decline from HLB. Trees at SWFREC have continued to decline from HLB and did not bear any fruits. 2. Please state what work is anticipated for next quarter: Tree health and growth will continue to be monitored. 3. Please state budget status (underspend or overspend, and why): Budget status is as expected.
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, and during this quarter, canopy health data was collected from all trees in the trials. For the trials with Hamlin and lemon scion, fruit yield and fruit quality data was collection during this quarter. Preparations began for the collection of yield and fruit quality data from trials with Valencia scions, which will be completed during the next quarter.
During this quarter, seedlings of the most promising advanced rootstock selections were grown for assessment of trueness-to- type or uniformity from seed, and will be evaluated by morphology and molecular markers.
Preparation for release. The most promising USDA rootstock selections are provided to the FDACS-DPI program for shoot tip grafting and disease testing. Those that have completed the process are held as trees at FDACS and USDA, Ft. Pierce, for use as seed trees and sources of clean budwood. Two new USDA rootstocks tentatively planned for release in 2023 have already gone through the FDACS program, and are available as clean budwood.
Release of new rootstocks. New rootstocks will be officially released by USDA for commercial use when justified by multi-year performance in the early-stage replicated field trials. Based on outstanding performance in field trials, the proposed release documentation has been submitted for two new USDA rootstocks, coded as US-1688 and US-1709. It is anticipated that these two rootstocks will be released during 2023.
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 are planned for spring 2023, with parental combinations based on new information and breeding values of particular clones assembled over the past year.
Propagate hybrids. New hybrids to be used in trials will be propagated by uniform nucellar seed or stem cuttings. Trees were budded in the nursery with two new superior scion cultivars on 20 rootstocks, in preparation for field planting in 2023. Plant material was prepared in the nursery for one new Stage 2 trial with Valencia scion including the important commercially available rootstocks and the most promising of the new rootstocks. It is anticipated that these trees will be ready for field planting in spring 2024.
Establish replicated field trials. No new field trials were planted this quarter, but tree preparation continued for two trials to be planted in summer 2023.
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 making new crosses, cropping data collection from Valencia field trials, continuing propagation of trees for future trials, and care of existing trees in trials.
3. Please state budget status (underspend or overspend, and why):
Budget is on schedule to be spent out by the end of the first year. The project is fully staffed, and work is progressing as planned.
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. 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 will use the developed method for knockin wiht MaSAMP. 2. Please state what work is anticipated for next quarter:To generate more genome edited lines for ACD2 and other target genes. Using the developed method for knockin with MaSAMP. 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: Knocking out disease susceptibility genes (S genes) has resulted in broad disease resistance in multiple crops. The bottleneck to using CRISPR for engineering HLB resistance in citrus has been the lack of suitable and validated gene targets. Identifying suitable gene targets has been the most urgent task for achieving HLB resistance through gene editing. We are the first group in the world that have knocked out two S genes, DMR6 and SWEET1, in HLB-susceptible `Duncan’ grapefruit and produced dmr6 and sweet1 mutants. The objectives of this project are to evaluate the resistance of four `Duncan’ mutants to HLB after graft inoculation (Objective 1a) and exposure to infected Asian citrus psyllids (ACP) (Objective 1b,) and to assess potential side effects of these mutations on citrus plant growth and morphology (Objective 2). The overall goal of this project is to determine the effectiveness of editing DMR6 and SWEET1 for engineering HLB resistance in citrus. Experiment 1 is being conducted to achieve Objective 1a. Four `Duncan’ mutants and one control wildtype line were grafted onto HLB-free sour orange rootstock in two batches. The first batch of clonal plants were graft-inoculated with CLas-positive buds or CLas-free buds (mock inoculation) on June 17, 2023, resulting in 29 graft-inoculated plants and 10 mock-inoculated plants. These plants have been grown in a temperature-controlled greenhouse. Data on plant height, trunk diameter below and above graft union, HLB symptoms, and CLas titer were collected on June 6 (before inoculation), September 21, (3 months post inoculation), and December 21, 2023 (6 months post inoculation). The Ct value of these mutants and wildtype was approximately 40, indicating undetectable CLas in these inoculated plants. This result was unexpected, and it suggests that the graft inoculation did not perform as expected, even though the CLas inoculum source plants continued to test positive, with a Ct values of 27 to 28. Consequently, these plants were re-inoculated in December 2023; tender shoots were fed upon by 10 female and 10 male hot Asian citrus psyllids (ACP) per plant for 10 days, then the ACPs were collected and analyzed for CLas titer. On average, 63% of these ACPs tested CLas-positive. These re-inoculated plants will be tested for CLas titer in March 2024 (3 months post re-inoculation). The second batch of clonal plants (total 34, with 4 to 8 per mutant) were propagated onto sour orange on July 24, 2023. These plants have grown to a height of 16 to 49 inches; when new shoots become available in spring 2024, we will use hot ACP to inoculate them. Experiment 2 and Experiment 3 are being conducted for Objective 1b and 2, respectively. These experiments require 20 clonal plants of similar stem diameter for each mutant or wildtype line. To produce the required clonal plants, 50 to 75 cuttings were taken from each mutant stock plant and stuck into potting mix in early July 2023. The cuttings were rooted under an intermittent misting system in a secure greenhouse for 3 months. For each mutant, six to 37 cuttings have rooted and produced new shoots. To produce additional clonal plants for Objective 1b and 2, another batch of cuttings were taken on November 3, 2023. These cuttings were rooted in Oasis Rootcubes. Now, enough numbers of rooted cuttings have been produced for all four mutants and wildtype `Duncan’. These cuttings have been potted and are being promoted to grow and produce tender shoots for ACP-based inoculation and plant morphological comparison. 2. Please state what work is anticipated for next quarter: Objective 1a (Experiment 1) -1st batch of inoculated plants: This batch of plants was re-inoculated in December 2023 by exposing them to infected ACP. All plants are growing well in a secure air-conditioned greenhouse. Data on plant height, trunk diameter, HLB symptom will be collected in March 2024. Leaves from each plant will be sampled for DNA isolation and qPCR analysis to quantify CLas titer 3 months post re-inoculation. Objective 1a (Experiment 1) – 2nd batch of clonal plants: This batch of plants is growing well and will be inoculated with CLas by 10 days’ feeding of hot ACP. Our initial plan was to inoculate this group of plants in January 2024, but the infected ACP colonies were ruined by invading ants. Our collaborating entomology group is raising new hot ACPs, which are expected to be ready for us in March 2024. After inoculation, the plants will be grown in a temperature-controlled greenhouse. Data on plant growth, trunk diameter, HLB symptom, and CLas titer will be collected 3 months post inoculation. Objective 1b (Experiment 2): Rooted cuttings are being pushed to grow rapidly for ACP inoculation in April 2024, as described above. After inoculation, the plants will be grown in a temperature-controlled greenhouse. Data on plant growth, trunk diameter, HLB symptom, and CLas titer will be collected 3 months post inoculation. Objective 2 (Experiment 3): Rooted cuttings will be potted up in containers in 4 weeks and grown in a secure greenhouse at CREC. Plant growth and leaf and shoot morphology of mutants will be monitored closely and compared with the wildtype to determine potential side effects from the edited S genes. 3. Please state budget status (underspend or overspend, and why): The total spendings by far add to $15,301.04, about 15.4% of the total budget. This is below the expected spending for the second quarter of the project. The primary reason was the difficulty we experienced in propagating the mutants and the wildtype to produce clonal plants for Objective 1b and 2 during the hot summer and fall months. Without enough numbers of clonal plants, the planned CLas inoculation, DNA extraction, qRT-PCR, and horticultural experiments had to be postponed to the third quarter of the project.
1. Please state project objectives and what work was done this quarter to address them: Knocking out disease susceptibility genes (S genes) has resulted in broad disease resistance in multiple crops. The bottleneck to using CRISPR for engineering HLB resistance in citrus has been the lack of suitable and validated gene targets. Identifying suitable gene targets has been the most urgent task for achieving HLB resistance through gene editing. We are the first group in the world that have knocked out two S genes, DMR6 and SWEET1, in HLB-susceptible `Duncan’ grapefruit and produced dmr6 and sweet1 mutants. The objectives of this project are to evaluate the resistance of four `Duncan’ mutants to HLB after graft inoculation (Objective 1a) and exposure to infected Asian citrus psyllids (ACP) (Objective 1b,) and to assess potential side effects of these mutations on citrus plant growth and morphology (Objective 2). The overall goal of this project is to determine the effectiveness of editing DMR6 and SWEET1 for engineering HLB resistance in citrus. Experiment 1 is being conducted to achieve Objective 1a. Four `Duncan’ mutants and one control wildtype line were grafted onto HLB-free sour orange rootstock in two batches. The first batch of clonal plants were graft-inoculated with CLas-positive buds or CLas-free buds (mock inoculation) on June 17, 2023, resulting in 29 graft-inoculated plants and 10 mock-inoculated plants. These plants have been grown in a temperature-controlled greenhouse. Data on plant height, trunk diameter below and above graft union, HLB symptoms, and CLas titer were collected on June 6 (before inoculation), September 21, (3 months post inoculation), and December 21, 2023 (6 months post inoculation). The Ct value of these mutants and wildtype was approximately 40, indicating undetectable CLas in these inoculated plants. This result was unexpected, and it suggests that the graft inoculation did not perform as expected, even though the CLas inoculum source plants continued to test positive, with a Ct values of 27 to 28. Consequently, these plants were re-inoculated in December 2023; tender shoots were fed upon by 10 female and 10 male hot Asian citrus psyllids (ACP) per plant for 10 days, then the ACPs were collected and analyzed for CLas titer. On average, 63% of these ACPs tested CLas-positive. These re-inoculated plants will be tested for CLas titer in March 2024 (3 months post re-inoculation). The second batch of clonal plants (total 34, with 4 to 8 per mutant) were propagated onto sour orange on July 24, 2023. These plants have grown to a height of 16 to 49 inches; when new shoots become available in spring 2024, we will use hot ACP to inoculate them. Experiment 2 and Experiment 3 are being conducted for Objective 1b and 2, respectively. These experiments require 20 clonal plants of similar stem diameter for each mutant or wildtype line. To produce the required clonal plants, 50 to 75 cuttings were taken from each mutant stock plant and stuck into potting mix in early July 2023. The cuttings were rooted under an intermittent misting system in a secure greenhouse for 3 months. For each mutant, six to 37 cuttings have rooted and produced new shoots. To produce additional clonal plants for Objective 1b and 2, another batch of cuttings were taken on November 3, 2023. These cuttings were rooted in Oasis Rootcubes. Now, enough numbers of rooted cuttings have been produced for all four mutants and wildtype `Duncan’. These cuttings have been potted and are being promoted to grow and produce tender shoots for ACP-based inoculation and plant morphological comparison. 2. Please state what work is anticipated for next quarter: Objective 1a (Experiment 1) -1st batch of inoculated plants: This batch of plants was re-inoculated in December 2023 by exposing them to infected ACP. All plants are growing well in a secure air-conditioned greenhouse. Data on plant height, trunk diameter, HLB symptom will be collected in March 2024. Leaves from each plant will be sampled for DNA isolation and qPCR analysis to quantify CLas titer 3 months post re-inoculation. Objective 1a (Experiment 1) – 2nd batch of clonal plants: This batch of plants is growing well and will be inoculated with CLas by 10 days’ feeding of hot ACP. Our initial plan was to inoculate this group of plants in January 2024, but the infected ACP colonies were ruined by invading ants. Our collaborating entomology group is raising new hot ACPs, which are expected to be ready for us in March 2024. After inoculation, the plants will be grown in a temperature-controlled greenhouse. Data on plant growth, trunk diameter, HLB symptom, and CLas titer will be collected 3 months post inoculation. Objective 1b (Experiment 2): Rooted cuttings are being pushed to grow rapidly for ACP inoculation in April 2024, as described above. After inoculation, the plants will be grown in a temperature-controlled greenhouse. Data on plant growth, trunk diameter, HLB symptom, and CLas titer will be collected 3 months post inoculation. Objective 2 (Experiment 3): Rooted cuttings will be potted up in containers in 4 weeks and grown in a secure greenhouse at CREC. Plant growth and leaf and shoot morphology of mutants will be monitored closely and compared with the wildtype to determine potential side effects from the edited S genes. 3. Please state budget status (underspend or overspend, and why): The total spendings by far add to $15,301.04, about 15.4% of the total budget. This is below the expected spending for the second quarter of the project. The primary reason was the difficulty we experienced in propagating the mutants and the wildtype to produce clonal plants for Objective 1b and 2 during the hot summer and fall months. Without enough numbers of clonal plants, the planned CLas inoculation, DNA extraction, qRT-PCR, and horticultural experiments had to be postponed to the third quarter of the project.
1. Please state project objectives and what work was done this quarter to address them: This is a continuation of project 18-028C. 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 our regular observations on root growth using minirhizotrons. Tree health ratings and tree measurements were conducted. 2. Please state what work is anticipated for next quarter: Rhizotron measurements and data analysis will continue and tree ratings will be conducted. Depending on grower collaborator’s schedule, we will harvest the fruit and analyse fruit quality. 3. Please state budget status (underspend or overspend, and why): Budget status is as expected.
Objective 1. Collect field performance data from Stage 1 and 2 replicated rootstock field trials and release new rootstock cultivars as justified by superior performance in multiyear field trials.
Collect field performance data. Since the beginning of the project, three of the 31 replicated trials listed on the original proposal were removed, and three new replicated trials have been established. So the current list of active replicated rootstock trials for the project remains at 31. During this quarter, canopy health data was collected from all trees in the trials. For two trials with an early mandarin scion, fruit yield and fruit quality data was collection during this quarter. Preparations began for the collection of yield and fruit quality data from trials with Hamlin and lemon scions, which will be completed during the next quarter.
Supplemental testing of promising selections. When rootstock selections appear promising in field trials and are being considered for commercial release, additional information about stress tolerance and other traits is highly desired. This will help inform appropriate sites and management for commercial use. During this quarter, rootstock plants were prepared for a study to evaluate salinity tolerance among selected promising new rootstocks. During this quarter, seedlings of the most promising advanced rootstock selections were grown for assessment of trueness-to- type or uniformity from seed, and will be evaluated by morphology and molecular markers.
Preparation for release. The most promising USDA rootstock selections are provided to the FDACS-DPI program for shoot tip grafting and disease testing. Those that have completed the process are held as trees at FDACS and USDA, Ft. Pierce, for use as seed trees and sources of clean budwood. The introduction of new plant material to this FDACS program was temporarily suspended in 2022, but is expected to begin again soon. The two new USDA rootstocks tentatively planned for release in 2023 have already gone through the FDACS program, and are available as clean budwood.
Release of new rootstocks. New rootstocks will be officially released by USDA for commercial use when justified by multi-year performance in the early-stage replicated field trials. Based on outstanding performance in field trials, the proposed release documentation is being prepared for two new USDA rootstocks, coded as US-1688 and US-1709. It is anticipated that these two rootstocks will be released during 2023. New USDA clones are assigned official names at release, and the new names for these have not yet been approved.
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. Current focus of USDA rootstock work is primarily on testing of hybrids previously created. Selected new crosses are planned for spring 2023, with parental combinations based on new information and breeding values of particular clones assembled over the past year.
Propagate hybrids. New hybrids to be used in trials will be propagated by uniform nucellar seed or stem cuttings. Trees were budded in the nursery with two new superior scion cultivars on 20 rootstocks, in preparation for field planting in 2023. Plant material was prepared in the nursery for one new Stage 2 trial with Valencia scion including the important commercially available rootstocks and the most promising of the new rootstocks. It is anticipated that these trees will be ready for field planting in spring 2024.
Establish replicated field trials. No new field trials were planted for this reporting quarter.
Tree care in trials. Periodic care was applied in the 31 field trials to maintain tree health and productivity, and manage weeds.
This is the final quarterly report for the project. We were lacking sufficient PacBio long read coverage for Ruby Red grapefruit and Shiikuwasha, to complete the series of seven commercially important citrus for which we aimed to provide the highest possible quality genome assemblies to a chromosome scale and with the greatest accuracy and contiguity technically possible. New HMW DNA preparations were made from the last two selections, and PacBio sequencing has begun. To provide the best possible annotation of the genomes, we required broader collections of tissue types to maximize the number of expressed genes we can find for annotation of the assemblies. We collected samples of tender flush, mature leaves, flowers, young and nearly mature fruit, bark, and for some accessions leaf tissue with and without symptoms of CLas infection and citrus canker. RNA samples were prepared and for two of the seven genomes, we have completed pooled sample RNAseq using the PacBio Seq IIe platform to have full-length transcript reads, and tissue-specific barcoded samples Illumina short reads, to enable tissue specific gene expression studies as well. Five of the seven genomes have been assembled using both the PacBio and Hi-C sequencing and assembly using Hi-Rise; preliminary assemblies of the last two listed above will be completed once the new PacBio reads are available. We have focused on finalizing two of the seven genomes that are most near to full completion, while continuing to generate genome and transcriptome data for the remaining five These two have had their chromosomes properly phased, multiple previous unanchored sequence contigs have now been integrated, centromeric regions have been defined, telomeric regions have been polished, and we have resolved nearly all highly repetitive sequences in these regions on a scale unmatched by any other citrus genome assembly. We are confident that these two are the most perfect and accurate citrus genome assemblies possible with current sequencing technologies and assembly tools. The RNAseq data for these two, mentioned above, are being used currently to complete the annotation of the assembly, a manuscript is being written, and we anticipate making these two assemblies public in the near future. The same steps applied to these two as described above, will be implemented with the remaining five genomes, and we will take these all to the same level of completeness and perfection.
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