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.
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.Leaves were collected for nutrient analysis, tree size measurements and HLB disease/health ratings were conducted. Fibrous roots were collected to assess root growth. Rhizotron pictures were taken. Data analysis was continued. 2. Please state what work is anticipated for next quarter: Rhizotron measurements will continue and data analysis will continue. Post-hurricane fruit drop evaluations will be conducted. 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. Three of the 31 established replicated trials listed (Appendix ix. Table 1) on the original proposal were determined to be complete, and were removed (WFF 2011, WFF 2012, WFF 2014 HB). During this quarter, canopy health data was collected from all trees in the other 28 active rootstock trials. Collection of brix and acid fruit quality data was completed during this quarter for the 2022 season on the active Valencia trials by using frozen juice samples. 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 is highly desired. This will help inform appropriate sites and management for commercial use. During this quarter, rootstock plants were propagated in preparation for a study to evaluate salinity tolerance among selected promising new rootstocks. During this quarter, rootstock plants were also propagated in preparation for a study to evaluate sting nematode tolerance among selected promising new rootstocks (primarily funded by a different CRDF grant). Preparation for release. Uniform seed propagation is not a requirement for new rootstocks, but is a highly desired trait for convenience of propagation. During this quarter, work continued to evaluate uniformity from seed for the most promising SuperSour hybrids, some of that in collaboration with Dr. Albrecht and a graduate student. In the past, the FDACS budwood program has cleaned up and maintained a large number of USDA rootstock selections during the field testing process and in preparation for commercial release. That FDACS program has announced changes that would mostly remove USDA rootstock holdings that are not already commercially used, which will require some significant adjustments to the USDA preparations for rootstock release. How that will be managed is still being sorted out.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 three new USDA rootstocks. New USDA clones are assigned official names at release, and these new names 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 on testing of hybrids previously created, and with funding uncertainty, no new hybrids were created in spring 2022. 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. Plant material was prepared in the nursery for three new Stage 2 trials, including commercially available rootstocks and the most promising of the new SuperSour hybrids. It is anticipated that these trees will be ready for field planting in spring 2023. Establish replicated field trials. Two new replicated field trials were planted this quarter, one is a Stage 2 trial with Hamlin scion at the Whitmore Farm in Lake County, and the second is a Stage 1 trial with Valencia scion at a collaborator site in Desoto County. These will be added to the list for field performance data collection.
1. Please state project objectives and what work was done this quarter to address them: The main objective is to achieve better fruit retention by applying different formulations of advanced Zn-based products that will result in better yields.Two nutritional Zn product formulations, technical-grade FertiZink and NuZinc, were produced by our industry collaborator and delivered to support the proposed field trial on grapefruit and orange. Commercial trial sites have been identified and mapped by the research team in three different FL locations with the help of CRDF staff. Foliar application at two rates (400ppm and 800 ppm) has begun as proposed. Routine quality control and characterization of materials as represented in tank-mix conditions has also begun in lab settings. 2. Please state what work is anticipated for next quarter: In coming months, we anticipate to:(i) assess HLB and canker severity, determine canker lesion incidence of fruit, quantify fruit drop on a weekly basis. (ii) determine grapefruit yield, quality, and shelf-life at the end of growing season.(iii) continue to perform quality control and characterization of materials as represented in tank-mix conditions 3. Please state budget status (underspend or overspend, and why): Underspend as the UF-subcontracting process just completed.
The project has five objectives:(1) Remove the flowering-promoting CTV and the HLB bacterial pathogen in the transgenic plants(2) Graft CTV- and HLB-free buds onto rootstocks(3) Generate a large number of vigorous and healthy citrus trees(4) Plant the citrus trees in the site secured for testing transgenic citrus for HLB responses(5) Collect the field trial data During the project period, we conducted the following activities. Objectives (1) to (4) were accomplished. Objective (5) was delayed due to the Covid-19 pandemic. We will continue collecting filed trial data, although the project has been terminated. (1) We previously generated three HLB-tolerent transgenic lines, ‘Hamlin’ 13-3, 13-29, and Duncan ’57-28′. The transgenic plants carried both the HLB-causing bacterium CLas and the flowering-promoting CTV vector. To remove CTV and CLas, the plants were treated in a growth chamber with alternating temperatures of 25 and 42 degree C every 4 hours for a total of 60 to 90 days. New shoots formed on the treated plants were tested for the presence of CLas and CTV by quantitative PCR (qPCR) with specific primers. The alternating temperature treatment is known to be highly efficient for removing CTV. We found that it is also effective for eliminating CLas. The new shoots obtained were free of both CTV and CLas. (2) & (3) Budwoods from the new shoots were grafted onto ‘Swingle’ rootstocks. A total of 83 transgenic plants were produced using CTV- and CLas-free budwoods of the above mentioned transgenic lines (28 for 13-3, 31 for 13-29, and 28 for 57-28). Moreover, 13 plants propagated from a new NPR1 transgenic line generated through mature tissue transformation, 17 plants from an EDS5 transgenic line, and 7 plants from an ELP3 transgenic line were produced. All these transgenic lines showed HLB tolerance in the greenhouse. In addition, a total of 27 transgenic rootstock plants were produced. These transgenic plants include eight transgenic ‘Carrizo’ lines that express three different disease resistance genes. The transgenic rootstocks were replicated and grafted with ‘Valencia’. All plants were grown and maintained in the greenhouse for two to three years. (4) The transgenic plants were planted in The Picos Farm in 2019 and 2021 (no plants were planted in 2020 due to Covid-19). A total of 69, 98, and 27 plants were planted on May 9, 2019, May 20, 2021, and October 8, 2021, respectively. (5) This objective is still ongoing with funding from other resources. The transgenic plants transplanted on May 9, 2019 and May 20, 2021 were examined on April 24, 2022. The plants grow well in the field and one plant from the 2019 planting has shown HLB symptoms. Leaf tissues were collected on April 24, 2022 and analyzed for CLas titers. We will continue monitor the transgenic plants in the field and periodically analyze leaf samples in the coming years. Besides the proposed objectives, we continue working on development of techniques that are able to produce consumer friendly citrus products. These techniques include CTV-delivered gene silencing, transgene-free CRISPR, and cisgenesis or intragenesis (cis/intragenesis). Our CTV and CRISPR projects are supported by USDA. The cis/intragenesis project is partially supported with the CRDF funds. We have previously created an intragenic vector. Unfortunately, the efficiency of the vector is extremely low. Our goal is to develop a strategy to significantly improve the efficiency. We are using the citrus 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mutant (called EPSPS TIPS) that provides tolerance to glyphosate as a selective marker to increase the intragenic vector efficiency. A strong citrus promoter is needed to drive the EPSPS TIPS gene, we are building a system to identify such citrus promoters. Once the efficiency of the intragenic vector is improved, it can be used to either silence or overexpress a target gene. We are using CTV-delivered gene silencing to identify targets for silencing, which is supported by USDA. We are also screen for genes for overexpression through cis/intragenesis. We recently discovered two nicotinamide adenine dinucleotide (NAD)-binding receptors in the model plant Arabidopsis, which when overexpressed, increase resistance to bacterial pathogens. With the support of the CRDF funds, we generated transgenic citrus expressing the Arabidopsis NAD receptors. The transgenic citrus plants were inoculated with CLas-infected psyllids and are maintained in the greenhouse for HLB symptom development. We are grafting the transgenic scions onto sweet orange rootstocks for easier detection of HLB resistance or tolerance. Furthermore, the citrus genome encodes several putative NAD-binding receptors. NAD-binding activities of two of the putative receptors were tested. These citrus receptors will be overexpressed using the intragenic vector to create HLB tolerance. We also developed highly efficient citrus microRNA (miRNA) vectors. These vectors will be combined with the intragenic vector to specifically silence negative immune regulators to created HLB tolerance in citrus. In summary, we have accomplished four of the five proposed objectives and will finish field data collection (Objective 5) in the coming years using funding from other sources.
The project has five objectives:(1) Remove the flowering-promoting CTV and the HLB bacterial pathogen in the transgenic plants(2) Graft CTV- and HLB-free buds onto rootstocks(3) Generate a large number of vigorous and healthy citrus trees(4) Plant the citrus trees in the site secured for testing transgenic citrus for HLB responses(5) Collect the field trial data During the project period, we conducted the following activities. Objectives (1) to (4) were accomplished. Objective (5) was delayed due to the Covid-19 pandemic. We will continue collecting filed trial data, although the project has been terminated. (1) We previously generated three HLB-tolerent transgenic lines, ‘Hamlin’ 13-3, 13-29, and Duncan ’57-28′. The transgenic plants carried both the HLB-causing bacterium CLas and the flowering-promoting CTV vector. To remove CTV and CLas, the plants were treated in a growth chamber with alternating temperatures of 25 and 42 degree C every 4 hours for a total of 60 to 90 days. New shoots formed on the treated plants were tested for the presence of CLas and CTV by quantitative PCR (qPCR) with specific primers. The alternating temperature treatment is known to be highly efficient for removing CTV. We found that it is also effective for eliminating CLas. The new shoots obtained were free of both CTV and CLas. (2) & (3) Budwoods from the new shoots were grafted onto ‘Swingle’ rootstocks. A total of 83 transgenic plants were produced using CTV- and CLas-free budwoods of the above mentioned transgenic lines (28 for 13-3, 31 for 13-29, and 28 for 57-28). Moreover, 13 plants propagated from a new NPR1 transgenic line generated through mature tissue transformation, 17 plants from an EDS5 transgenic line, and 7 plants from an ELP3 transgenic line were produced. All these transgenic lines showed HLB tolerance in the greenhouse. In addition, a total of 27 transgenic rootstock plants were produced. These transgenic plants include eight transgenic ‘Carrizo’ lines that express three different disease resistance genes. The transgenic rootstocks were replicated and grafted with ‘Valencia’. All plants were grown and maintained in the greenhouse for two to three years. (4) The transgenic plants were planted in The Picos Farm in 2019 and 2021 (no plants were planted in 2020 due to Covid-19). A total of 69, 98, and 27 plants were planted on May 9, 2019, May 20, 2021, and October 8, 2021, respectively. (5) This objective is still ongoing with funding from other resources. The transgenic plants transplanted on May 9, 2019 and May 20, 2021 were examined on April 24, 2022. The plants grow well in the field and one plant from the 2019 planting has shown HLB symptoms. Leaf tissues were collected on April 24, 2022 and analyzed for CLas titers. We will continue monitor the transgenic plants in the field and periodically analyze leaf samples in the coming years. Besides the proposed objectives, we continue working on development of techniques that are able to produce consumer friendly citrus products. These techniques include CTV-delivered gene silencing, transgene-free CRISPR, and cisgenesis or intragenesis (cis/intragenesis). Our CTV and CRISPR projects are supported by USDA. The cis/intragenesis project is partially supported with the CRDF funds. We have previously created an intragenic vector. Unfortunately, the efficiency of the vector is extremely low. Our goal is to develop a strategy to significantly improve the efficiency. We are using the citrus 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mutant (called EPSPS TIPS) that provides tolerance to glyphosate as a selective marker to increase the intragenic vector efficiency. A strong citrus promoter is needed to drive the EPSPS TIPS gene, we are building a system to identify such citrus promoters. Once the efficiency of the intragenic vector is improved, it can be used to either silence or overexpress a target gene. We are using CTV-delivered gene silencing to identify targets for silencing, which is supported by USDA. We are also screen for genes for overexpression through cis/intragenesis. We recently discovered two nicotinamide adenine dinucleotide (NAD)-binding receptors in the model plant Arabidopsis, which when overexpressed, increase resistance to bacterial pathogens. With the support of the CRDF funds, we generated transgenic citrus expressing the Arabidopsis NAD receptors. The transgenic citrus plants were inoculated with CLas-infected psyllids and are maintained in the greenhouse for HLB symptom development. We are grafting the transgenic scions onto sweet orange rootstocks for easier detection of HLB resistance or tolerance. Furthermore, the citrus genome encodes several putative NAD-binding receptors. NAD-binding activities of two of the putative receptors were tested. These citrus receptors will be overexpressed using the intragenic vector to create HLB tolerance. We also developed highly efficient citrus microRNA (miRNA) vectors. These vectors will be combined with the intragenic vector to specifically silence negative immune regulators to created HLB tolerance in citrus. In summary, we have accomplished four of the five proposed objectives and will finish field data collection (Objective 5) in the coming years using funding from other sources.
This project addressed CPDC priority 3.C. Evaluate rootstock performance derived from tissue culture compared with conventional propagation materials. The purpose of the project was to determine whether the method of rootstock propagation impacts growth and performance of grafted field-grown citrus trees. Four field trials were established in different production regions. One field trial was established at SWFREC (Collier County) in 2017 and included different rootstock cultivars in combination with Valencia scion. The rootstocks had been propagated from seed and vegetatively by cuttings and tissue culture. Two more field trials were established with grower collaborators on a central ridge site in Polk County and on a flatwoods-type site in Hendry County in April 2018. These trials also consisted of Valencia scion in combination with different rootstocks generated by seed, cuttings, or tissue culture. An additional field trial was established in November 2019 in Indian River County. We conducted detailed measurements on above-ground tree traits using standard horticultural methods of evaluation. We also examined the roots structures in detail by excavating trees at the SWFREC and Indian River County location. The overall objective of the project was to investigate effects of rootstock propagation method and the interaction with rootstock on root structure and tree performance during the early years of growth in the field. The project was designed to help growers and citrus nurseries resolve concerns about the quality of citrus trees propagated by methods other than by seed, specifically tissue culture. After three years of field growth, we did not measure any differences in tree growth, health, and productivity due to the rootstock propagation method that may raise concerns against using cuttings or tissue culture propagated trees in commercial citrus production. In contrast to the propagation method, the rootstock cultivar had a considerable influence on tree growth and productivity, reiterating the importance of choosing the proper rootstock for each production site. Regardless of how the rootstocks are propagated, it is strongly recommended to purchase only high-quality trees from registered citrus nurseries, inspect root structures prior to transplanting, use proper planting practices, and good tree care when establishing a new grove. Results from this project were disseminated in numerous extension presentations, trade journal articles, and peer-reviewed journal publications.
This project addressed CPDC priority 3.C. Evaluate rootstock performance derived from tissue culture compared with conventional propagation materials. The purpose of the project was to determine whether the method of rootstock propagation impacts growth and performance of grafted field-grown citrus trees. Four field trials were established in different production regions. One field trial was established at SWFREC (Collier County) in 2017 and included different rootstock cultivars in combination with Valencia scion. The rootstocks had been propagated from seed and vegetatively by cuttings and tissue culture. Two more field trials were established with grower collaborators on a central ridge site in Polk County and on a flatwoods-type site in Hendry County in April 2018. These trials also consisted of Valencia scion in combination with different rootstocks generated by seed, cuttings, or tissue culture. An additional field trial was established in November 2019 in Indian River County. We conducted detailed measurements on above-ground tree traits using standard horticultural methods of evaluation. We also examined the roots structures in detail by excavating trees at the SWFREC and Indian River County location. The overall objective of the project was to investigate effects of rootstock propagation method and the interaction with rootstock on root structure and tree performance during the early years of growth in the field. The project was designed to help growers and citrus nurseries resolve concerns about the quality of citrus trees propagated by methods other than by seed, specifically tissue culture. After three years of field growth, we did not measure any differences in tree growth, health, and productivity due to the rootstock propagation method that may raise concerns against using cuttings or tissue culture propagated trees in commercial citrus production. In contrast to the propagation method, the rootstock cultivar had a considerable influence on tree growth and productivity, reiterating the importance of choosing the proper rootstock for each production site. Regardless of how the rootstocks are propagated, it is strongly recommended to purchase only high-quality trees from registered citrus nurseries, inspect root structures prior to transplanting, use proper planting practices, and good tree care when establishing a new grove. Results from this project were disseminated in numerous extension presentations, trade journal articles, and peer-reviewed journal publications.
Abstract Updates for this quarter:The support period for the dedicated testing site at the Picos farm has concluded. Over the course of this and previous projects, Picos has served well as a transgenic capable field testing site available to HLB and Citrus researchers. Full support for all associated projects was provided. The entire 10 acres was utilized, allowing government, university and private sector researchers to make numerous plantings of both transgenic and conventional citrus varieties with a high probability of HLB tolerance/resistance. Several of the earlier established trials are wrapping up. However, due to the long timeframe of citrus field trials, there remains work to be done on many of the plantings. Several of these trials will be continued under two new NIFA funded projects; 2020-70029-33176 Therapeutic Molecule Evaluation and Field Delivery Pipeline for Solutions to HLB and 2020-70029-3319 Providing Practical Solutions for HLB Treatment and Prevention. For the projects not supported under dedicated grants or appropriate for USDA-ARS base funding, new sources of funding will need to be secured in order to allow for their continuation. Recent quarters:A significant USDA-funded infrastructure project has been completed, fully renovating the water management systems and significantly improving storm and flood protection. USDA has also acquired a topper hedger to facilitate canopy management and reflect the best practices of commercial farms. Site management and field trials are progressing well. The site remains available for access to all researchers and all regulatory protocols for the care and disposal of transgenic material are being observed. The trees have been hedged and topped to promote growth, open canopies and access to nutritional sprays. The foliar spray program was applied every two weeks and including standard treatments for commercial groves. Crosses have been made with transgenic pollen to help elucidate if sexual embryos can be rescued from polyembryonic females, making use of the transgenic markers to determine if sexual hybridization is successful. A third year of crossings has also been made with the early flowering (FT) transgenics, continuing the work described below. An additional BRS transgenic release permit was approved (AUTH – 0000043620) for material with confidential business information (CBI) for a project led by R. Shatters. The primary BRS permit has also been renewed and amended to include a new construct from UF (Now AUTH – 0000206702). The annual site review from APHIS/BRS has been conducted successfully. Discussions have begun with APHIS-BRS to set conditions for new or expanded transgenic release permits in support of NIFA project 2020-70029-33176, Therapeutic Molecule Evaluation and Field Delivery Pipeline for Solutions to HLB, with field trials expected to begin later this year once all regulatory requirements are met. Four new plantings from UF expressing resistance genes and two new plantings from USDA-CRADA partners expressing antimicrobial peptides and anti-CLas plantibodies have been made. With recent plantings, the transgenic site is operating at full capacity. Fall 2021 assessments were completed for USDA plantings as described below for trials #8, #9, #10, #11 and #15. Fruit have been harvested from the second year of controlled crosses using pollen from early flowering (FT) transgenics on traditional varieties maintained in the testing site. Seeds from these fruit and those of future crossings will be used to assess inheritability of the phenotype and for CRISPR gene stacking to combine genome editing with accelerated breeding traits. The UCRiverside-led trifoliate and trifoliate hybrid trial has concluded, a manuscript regarding identified HLB-tolerance is in preparation; and these trees can be removed as needed to make space available for future plantings. Dr. Stover analyzed data on canker incidence for this trial and a manuscript detailing these results has been published in HortScience DOI: 10.21273/HORTSCI15684-20. Previously established at the site:A number of trials are underway at the CRDF funded Picos Test Site. A detailed current status is outlined below this paragraph. We continue investigation of potential pollen flow from transgenic trees to assess the possibility of reducing the isolation distances. Availability of the test site for planting continues to be announced to researchers. Supplemental: Full details on trial plantings.1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the USDA permits.2) Under the Stover permit, a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. 4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project.5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and analysis will continue to be conducted under a new NIFA grant.6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).7) In a project led by Fred Gmitter, there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo.10) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls.11) Multiple plantings with grafted trees of l Hamlin, Valencia and grapefruit scions on transgenic rootstock expressing antimicrobial citrus-thionin and bacterial recognition domain fusion proteins (219 trees with controls) as a collaboration between USDA and the New Mexico Consortium.12) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type.13) Planting from Zhonglin Mou of UF that includes transgenic grapefruit (31 plants) and sweet orange (60 plants) scions expressing two different resistance genes and grafted on WT swingle rootstocks; as well as non-transgenic controls. 14) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State15)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
Abstract Updates for this quarter:The support period for the dedicated testing site at the Picos farm has concluded. Over the course of this and previous projects, Picos has served well as a transgenic capable field testing site available to HLB and Citrus researchers. Full support for all associated projects was provided. The entire 10 acres was utilized, allowing government, university and private sector researchers to make numerous plantings of both transgenic and conventional citrus varieties with a high probability of HLB tolerance/resistance. Several of the earlier established trials are wrapping up. However, due to the long timeframe of citrus field trials, there remains work to be done on many of the plantings. Several of these trials will be continued under two new NIFA funded projects; 2020-70029-33176 Therapeutic Molecule Evaluation and Field Delivery Pipeline for Solutions to HLB and 2020-70029-3319 Providing Practical Solutions for HLB Treatment and Prevention. For the projects not supported under dedicated grants or appropriate for USDA-ARS base funding, new sources of funding will need to be secured in order to allow for their continuation. Recent quarters:A significant USDA-funded infrastructure project has been completed, fully renovating the water management systems and significantly improving storm and flood protection. USDA has also acquired a topper hedger to facilitate canopy management and reflect the best practices of commercial farms. Site management and field trials are progressing well. The site remains available for access to all researchers and all regulatory protocols for the care and disposal of transgenic material are being observed. The trees have been hedged and topped to promote growth, open canopies and access to nutritional sprays. The foliar spray program was applied every two weeks and including standard treatments for commercial groves. Crosses have been made with transgenic pollen to help elucidate if sexual embryos can be rescued from polyembryonic females, making use of the transgenic markers to determine if sexual hybridization is successful. A third year of crossings has also been made with the early flowering (FT) transgenics, continuing the work described below. An additional BRS transgenic release permit was approved (AUTH – 0000043620) for material with confidential business information (CBI) for a project led by R. Shatters. The primary BRS permit has also been renewed and amended to include a new construct from UF (Now AUTH – 0000206702). The annual site review from APHIS/BRS has been conducted successfully. Discussions have begun with APHIS-BRS to set conditions for new or expanded transgenic release permits in support of NIFA project 2020-70029-33176, Therapeutic Molecule Evaluation and Field Delivery Pipeline for Solutions to HLB, with field trials expected to begin later this year once all regulatory requirements are met. Four new plantings from UF expressing resistance genes and two new plantings from USDA-CRADA partners expressing antimicrobial peptides and anti-CLas plantibodies have been made. With recent plantings, the transgenic site is operating at full capacity. Fall 2021 assessments were completed for USDA plantings as described below for trials #8, #9, #10, #11 and #15. Fruit have been harvested from the second year of controlled crosses using pollen from early flowering (FT) transgenics on traditional varieties maintained in the testing site. Seeds from these fruit and those of future crossings will be used to assess inheritability of the phenotype and for CRISPR gene stacking to combine genome editing with accelerated breeding traits. The UCRiverside-led trifoliate and trifoliate hybrid trial has concluded, a manuscript regarding identified HLB-tolerance is in preparation; and these trees can be removed as needed to make space available for future plantings. Dr. Stover analyzed data on canker incidence for this trial and a manuscript detailing these results has been published in HortScience DOI: 10.21273/HORTSCI15684-20. Previously established at the site:A number of trials are underway at the CRDF funded Picos Test Site. A detailed current status is outlined below this paragraph. We continue investigation of potential pollen flow from transgenic trees to assess the possibility of reducing the isolation distances. Availability of the test site for planting continues to be announced to researchers. Supplemental: Full details on trial plantings.1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the USDA permits.2) Under the Stover permit, a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. 4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project.5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and analysis will continue to be conducted under a new NIFA grant.6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).7) In a project led by Fred Gmitter, there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo.10) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls.11) Multiple plantings with grafted trees of l Hamlin, Valencia and grapefruit scions on transgenic rootstock expressing antimicrobial citrus-thionin and bacterial recognition domain fusion proteins (219 trees with controls) as a collaboration between USDA and the New Mexico Consortium.12) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type.13) Planting from Zhonglin Mou of UF that includes transgenic grapefruit (31 plants) and sweet orange (60 plants) scions expressing two different resistance genes and grafted on WT swingle rootstocks; as well as non-transgenic controls. 14) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State15)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
