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: 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.
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