1. Please state project objectives and what work was done this quarter to address them:Objective 1: Determine how different cover crop mixtures impact soil and root health and weed cover in established commercial citrus groves.Preparation has begun for the next planting of cover crops for fall/winter 2020. Herbicide has been applied to row middles, and seeds should be planted by the end of Nov 2020. The mix will include: sunnhemp, pea, daikon radish, oats, and winter rye. Results from our August 2020 sampling are still being analyzed, but preliminary assessment indicates similar patterns to Year 1: increased bacterial abundance in cover crop treatments, and increased abundance of nitrogen cycling genes with cover crops. In addition, we also found an increase in the abundance of bacteria under the canopy of trees in the legume+non-legume treatment. Weed density survey data collected from the latest sampling interval (July 2020) is currently being analyzed. This survey’s preliminary observations suggest the continued suppression of weed emergence and spread by cover crop plantings in citrus row-middles compared to non-cover cropped controls. Objective 2: Examine the impact of eco-mowing in conjunction with cover crops on soil and root health and weed cover in established commercial citrus groves.Eco-mowing will next occur in November 2020 with the planting of the next round of cover crops. Data from Year 2 (collected in Aug 2020) is still being analyzed. Weed data were collected in July 2020, and the impacts of the eco-mowing on weed emergence and coverage in citrus tree rows are being analyzed. Visual root growth assessments show continued root growth under cover cropping and eco-mowing, but analysis is ongoing. Soil moisture appears to be similar across all treatments, possibly due to the presence of a high water table at both sites. We will report the trends in the next quarter. Objective 2: Examine the impact of eco-mowing in conjunction with cover crops on soil and root health and weed cover in established commercial citrus groves.Eco-mowing occurred in early May 2020 when cover crops were mowed in anticipation of planting the next set of cover crops. After a year of treatment, soil organic matter slightly increased (in the range of 0.3-0.5%) under the tree canopy receiving eco-mowing compared to regular cover crop treatments; however, no significant differences were detected between treatments. Visual root growth assessments show continued root growth under cover cropping and eco-mowing, but analysis is ongoing. Soil moisture appears to be similar across all treatments, possibly due to the presence of a high water table at both sites. Objective 3: Quantify the effect of cover crops and eco-mowing on tree growth and production.After 1 year of study, we have not yet observed differences in fruit yield, fruit quality, canopy volume, and trunk size. This is not unexpected, as trees of this age could take at least two years to show responses to treatments. We will continue to assess canopy volume and trunk size, and harvest data will be collected again in Spring 2021. Objective 4: Identify the economic benefits of using cover cropsA student was trained on partial budgeting and valuing soil health. The student began work on developing a citrus budget that is appropriate for comparing management strategies with cover crops relative to business as usual. We found that differences in how the budgets are reported limits historical data collection to five years. These data will be used as a benchmark when doing partial budgeting, which is now in its early stages. In addition to budget ta.60sks, the relevant literature was reviewed and incorporated into a draft of the adoption survey. The survey is ready for review and IRB approval. Objective 5: Communicate results to growers using field days and extension materialsPreliminary results are being presented at the Citrus Expo in August. Initial results on weed suppression by cover crops was included in a presentation at this years Citrus Growers Institute. Information on cover crops and preliminary data will be included in two articles for the Citrus Industry magazine in September. Discussions are underway about how to host a field day, or a virtual field day, at some point in the next year. 2. Please state what work is anticipated for next quarter: The most recent collection of soils, leaves, and roots for microbial and nutrient analysis will be completed by the end of August 2020. Microbial DNA will be extracted from soils collected in August 2020 and analysis of soil microbes important in nitrogen cycling will begin. Soils collected in August 2020 will also be analyzed for soil organic matter and nutrients. Analysis of data from weed density measurements will be performed. Canopy and trunk size measurements and leaf nutrient status along with root image collections and soil moisture monitoring will continue in the next quarter. A graduate student and postdoctoral research associate on the project will be presenting results of the project at American Society of Agronomy virtual annual meeting in November 2020. The next set of cover crops are scheduled to be planted in October. The composition of the mixes is still being discussed. The economics team expects to execute the adoption survey and begin analysis. They will also construct the framework for partial budgeting and assessing the cost of cover crop use and continue to collect data. 3. Please state budget status (underspend or overspend, and why): We are on track with our planned budget spending.
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 In this quarter, we focused on the following greenhouse and laboratory work: (1) Took care of transgenic plants in the greenhouse. We now have two batches of transgenic plants. One batch have been prepared for the proposed field trial, but transplanting was delayed due to COVID-19. The other batch are newly produced, expressing a regulatory gene of systemic acquired resistance. These plants were regularly watered and fertilized. (2) Laboratory work was focused on cloning the extracellular domains of a group (10) of citrus homologs of an Arabidopsis disease resistance gene. This gene encodes a receptor-like kinase with the extracellular domain binding nicotinamide adenine dinucleotide. We found that overexpression of this receptor-like kinase increases resistance to bacterial pathogens and have thus generated transgenic citrus plants overexpressing the Arabidopsis receptor-like kinase gene. The citrus genome encodes more than ten homologs of this receptor-like kinase. To find out the functional homolog(s) of the receptor-like kinase gene, we cloned the extracelllar domains of the closest ten homologs in an E. coli expression vector. We are optimizing the protein expression conditions and will express and purify these fusion proteins from E. coli. The next step will be to test their nicotinamide adenine dinucleotide-binding activity. The protein(s) that binds nicotinamide adenine dinucleotide will be the citrus functional homolog and will be used to generate intragenic/cisgenic citrus plants.
The purpose of this project is to optimize the CRISPR technology for citrus genome editing. This study is related to the CRDF RMC-18 Research Priorities 4AB. Objective 1. Expanding the toolbox of citrus genome editing. In this study, we will adapt StCas9, NmCas9, AsCpf1 (from Acidaminococcus), FnCpf1 (from Francisella novicida) and LbCpf1 (from Lachnospiraceae) on genome modification of citrus. Lately, we have shown CRISPR-Cpf1 can be readily used as a powerful tool for citrus genome editing. In our recent study, we employed CRISPR-LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. Our study showed that CRISPR-LbCas12a can readily be used as a powerful tool for citrus genome editing. One manuscript entitled CRISPR-LbCas12a-mediated modification of citrus has been published on Plant Biotechnol J. We are currently further optimizing LbCas12a-crRNA-mediated genome editing to make homologous biallelic mutations. We are also testing AsCpf1 and FnCpf1 for their application in citrus genome editing and generating homologous biallelic mutations. We have successfully generated both homozygous and biallelic mutations in the EBE region of LOB1 gene in pumlo. This work has been submitted for publication. We are in the process of generating homozygous and biallelic lines of other citrus varieties.Recently, we have developed multiplex genome editing toolkits for citrus including a PEG mediated protoplast transformation, a GFP reporter system that allows rapid assessment of the CRISPR constructs, citrus U6 promoters with improved efficacy, tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we have successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, indicating that the CRISPR-mediated citrus genome editing technology is mature and could be implemented in citrus genetic improvement as a viable approach. In addition, our study lay the foundation for non-transgenic genome editing of citrus. One manuscript entitled Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations has been published on Plant Molecular Biology.We have successfully developed base editing tools for citrus genome editing. Objective 2. Optimization of the CRISPR-Cas mediated genome editing of citrus. In this study, we are testing different promoters including INCURVATA2 promoter, the cell division-specific YAO promoter, and the germ-line-specific SPOROCYTELESS promoter, and ubiquitin promoter in driving the expression of Cas9 and Cpf1 orthologs. To optimize the expression of sgRNA and crRNA, we have identified multiple citrus U6 promoters and two of the citrus U6 promoters showed higher efficacy in driving gene expression in citrus than 35S promoter and Arabidopsis U6 promoter. We have further increased the mutation efficacy to 50%. We have generated one homozygous line in the promoter region of canker susceptibility genes of Hamlin. Objective 3. Optimization of the CRISPR technology to generate foreign DNA free genome editing in citrus. We have conducted transient expression of Cas9/sgRNA plasmid and Cas9 protein/sgRNA ribonucleoprotein complex in citrus protoplast. We are also conducting citrus genome editing using Cpf1/crRNA plasmids and ribonucleoprotein complex in citrus protoplast. The plasmid-transformed protoplast has 1.7% editing efficiency, and the RNP-transformed samples have approximately 3.4% efficiency. The genome modified protoplast cells are undergoing regeneration. We aim to increase the efficacy to over 20% and eventually generate non-transgenic genome modified citrus. One patent has been filed on the CRISPR-Cas mediated genome editing of citrus. We have lately optimized the citrus protoplast isolation and manipulation, our data showed that more than 98% of the isolated protoplasts were alive. We regularly obtained a transfection efficiency of approximately 66% or above. Genome modified lines in canker and HLB S genes are being regenerated.
Within the third quarter of 2020, the Juvenile Tissue Citrus Transformation Facility (JTCTF) has operated at about 40% of its capacity. In accordance with regulations established by the University of Florida (UF) as a response to COVID19 epidemic, JTCTF transitioned to Phase3 during this period. We have started doing new transformation experiments to the extent allowed by the available labor force and starting material. No new orders for production of transgenic citrus were received.Transition of the JTCTF to the Phase3 meant that there could two employees in the lab at the time. According to UF regulations, there can be one person per 150 ft2 of working space. Surface area of the JTCTF is such that allows only two employees to be there at the same time. Labor force of JTCTF includes two employees who work on USDA grants and they also had to be included into the work schedule. As a result, number of experiments that can be done per week is lower than the usual output of JTCTF. Another factor that affected productivity was available starting material. The fruit we had stored in the packing house has gone bad much faster than usual because the cold room broke down at one point during summer. Seeds extracted form fruit that is going bad were also bad and germinated seedlings carried endopathogens. This led to contamination of material used in transformation experiments and loss of data. Sixteen transgenic plants were produced this quarter. These plants included 14 Duncan grapefruit and two Valencia oranges. These plants were the results of work on eight different orders/vectors: BB3, BB4, ZM15-2, ZM16, HGJ87, HGJ88, JJ8, and NADR2.I continued to work on the development of the EBA form. After multiple meetings with the CREC leadership and other participants in the process of transition of service labs to EBA units, the EBA form was completed. It was submitted to IFAS personnel responsible for the first step evaluation. There were no changes in the JTCTF staff.
Goals of this project: 1. Evaluate existing transgenic Carrizo and Swingle AtNPR1 overexpressing rootstocks in the laboratory and greenhouse. 2. Conduct a replicated field trial with the best transgenic rootstocks budded with non-transgenic `Valencia’ and test for GMO gene products in the fruit or juice. 3. Produce additional transgenic rootstock lines and stack other gene(s) responsible for SAR using mature transformation. 4. Evaluate transgene segregation analyses of the rootstock progeny and large-scale propagation of select lines. Cumulative (December 1, 2018 to September 30, 2020) progress report: Objective 1: Evaluate existing transgenic Carrizo and Swingle AtNPR1 overexpressing rootstocks in the laboratory and greenhouse. Results: Production of budded plants for the study: We generated a population of AtNPR1 overexpressing transgenic lines prior to the start of this project. Transgenic lines were generated using the Carrizo citrange and Swingle citrumelo cultivars. Molecular analyses: Two populations of transgenic AtNPR1 expressing citrus have been used in this study. The first is a population of Juvenile tissue derived lines (2300-NPR1-x) and the other a population of mature tissue derived (Ax and ZMx) lines. Transgenic lines have been evaluated during this period for AtNPR1 expression using qPCR (Table 1). Table 1: Mean AtNPR1 Ct values from the different mother trees. PCR was performed for 40 cycles. Experiment was repeated thrice. All the lines were tested for the PR1 and PR2 expression. These PR (Pathogenesis related) genes are markers for the Systemic acquired resistance (SAR) pathway and their overexpression indicates that our NPR1 gene is active. Selected lines with high PR1 and PR2 expression are being used for the greenhouse assays to understand if the transgenic rootstock can confer resistance to the non-transgenic scion. Ct values of the HLB infected budsticks range from 22.5 to 25.1. The first set of data was collected in May 2020. The second set of data was collected in September 2020 (Table 2) Subsequently we analyzed protein expression in selected A and ZM series mature tissue derived transgenic lines using western blot with AtNPR1 specific antibody. All transgenic lines tested positive when leaf samples were analyzed (Figure 2). When roots were evaluated, A22, A47, A48, ZM26 and ZM30 had the best AtNPR1 protein expression. (Figure 3). Figure 3: Western blotting of root samples with AtNPR1 specific antibody. In the first round of clonal rootstock propagation, we focused on the juvenile tissue derived transgenic lines, which had been produced before the start of the current project and were thus bigger in size, compared to the A and ZM series of mature tissue derived transgenic lines. Cuttings from selected lines with low Ct values were propagated under mist. It has been observed that some transgenic lines do not perform well after propagation. They either do not root or the rate of growth is very slow. This is often related to higher trans-protein production in these lines. These lines were discarded and removed from the study. In the second round of propagation, we have focused on propagating cuttings from the A and ZM series of mature transgenic lines. Discussion: A greenhouse study is in progress with selected transgenic lines budded with HLB infected sweet orange budsticks. The objective of this study is to understand whether the transgenic AtNPR1 rootstock can protect the aboveground scion against HLB. Three metrics are being evaluated: 1) HLB (Clas) titer, 2) PR-1 gene expression and 3) PR-2 gene expression. The CLas measurements were conducted twice. The first sampling was done in late May 2020 and the second sampling was done in late September 2020. Leaf sampling should have been preferably done in March – April, but due to COVID related and other restrictions had to be delayed. According to our results, all transgenic lines were HLB positive at the time of the initial sampling in late May 2020 (Summer Leaf Ct values – Table 2). When leaves were again sampled in September 2020 (Fall Leaf Ct values – Table 2), Ct values were either statistically similar or had increased in most transgenic rootstock lines. Ct values in the control lines significantly decreased during this period. Thus, this preliminary data provides an indication that transgenic rootstocks could potentially protect the non-transgenic scion from HLB. Although the trees still have HLB, the Ct values have not decreased in the same manner as we see in the controls. This can only be because of the effect of the transgenic rootstock on the scion. Line 2300-NPR1-25 had very high Ct values in the September evaluation, changing from 26.8 to 31.2 within 3 months. We will pay special attention to this line following our next sampling in December – January to confirm the validity of the fall results. PR-1 and PR-2 gene expression were stable in the budded scions, when compared to the HLB infected non-transgenic control. Samples were collected in March, June and September 2020. We saw decreased gene expression at the 6 month sampling in June and it may be due to a seasonal fluctuation. Even with the fluctuations, there was consistent upregulation of both genes in our trees. At this point, we cannot correlate enhanced gene expression to the HLB Ct results. Future directions: Leaves will be sampled at a 6 monthly interval till the end of the grant cycle and pertinent results will be provided in the quarterly report. An in-depth RNA expression study (RNAseq) on selected lines and control using an illumina HiSeq platform will be conducted to understand differential gene expression in the non-transgenic scion. This data will be essential in getting a detailed understanding of the genetic regulation of the SAR pathways and can provide conclusive evidence on the SAR activity. Additionally, we plan to study the impact of CLas infection on non-transgenic Valencia leaf metabolites using gas chromatography mass spectrometry by a process developed by Nabil Killiny. Expression analysis for genes involved in jasmonic acid (JA), salicylic acid (SA), and proline-glutamine pathways will also be conducted to correlate results observed in the RNAseq and GCMS data. Objective 2: Conduct a replicated field trial with the best transgenic rootstocks budded with non-transgenic `Valencia’ and test for GMO gene products in the fruit or juice. Transgenic lines were planted in the field under USDA permit in March 2020. USDA permit allows us to harvest and evaluate seed and fruits from the transgenic lines as necessary for the success of this project. We obtained 250 control trees (non-transgenic Valencia budded onto non-transgenic Kuharske) from Brite leaf nursery to plant as border row trees as stipulated in the transgenic permit in addition to the transgenic rootstocks inside the trial. Another 20% control non transgenic trees were planted within the trial. Future directions: COVID related travel restrictions had resulted in a unique management situation for the transgenic field trees soon after trees were planted in March 2020. We are however getting back on track for management of this site. More trees will be planted in Spring 2021, including most of the rootstock seed source lines that have good transgenic expression. These will be planted for fulfilling objective 4. Field trees are managed according to set USDA protocols and leaves will be sampled in March 2021 for year 1 data collection. Subsequently leaves will be sampled at a 6-monthly interval (every spring and fall) and samples will be sent to Southern Gardens diagnostic lab for HLB analysis. RNA from fruit samples will be isolated and tested for presence of AtNPR1 using qPCR. Objective 3: Produce additional transgenic rootstock lines and stack other gene(s) responsible for SAR using mature transformation. The mature citrus facility (MCF) is a dedicated facility for mature citrus transformation and we have utilized their services for this project. The MCF received four vectors, each containing two stacked, disease resistance genes conferring Systemic Acquired Resistance (SAR). The four vectors are GNS (gus-NPR1,SABP2), GNO (gus-NPR1,OBF5), GNA (gus-NPR1,AZL1) and GND (gus, NPR1, DIR1). Table 3 lists the transgenic lines produced by the mature transformation facility and available for this project. Trees will be evaluated by qPCR for gene expression and the best lines will be clonally propagated for evaluation in 2021. Objective 4: Evaluate transgene segregation analyses of the rootstock progeny and large-scale propagation of select lines. Most of the transgenic rootstocks that show enhanced PR-1 and PR-2 expression have been propagated by budding on US802 rootstock. Trees are to be planted in the field in spring 2021. Several mature tissue derived transgenic lines have already flowered in the greenhouse and fruit harvested from 3 lines. Seedlings were planted from each line. Gus staining and AtNPR1 gene expression of the lines have indicated genetic stability in the progeny. A uniform population has now been generated for budding with non-transgenic scions. Since preliminary results indicate normal seed germination and growth, we are hopeful that overexpression of the AtNPR1 transgene in rootstocks will not have any detrimental effect on the subsequent generations. Selected transgenic lines are also being mass propagated using in vitro tissue culture to generate a large population of transgenic lines for field testing in two additional USDA approved field sites. Conclusions: According to the time frame proposed in our project proposal, we are on track with this project. Year one was spent establishing the framework for this study, getting the plants ready, doing the background molecular analyses and infecting plants with HLB. We were also able to obtain an USDA permit for field trials and have planted a population of trees in the field. In year 2, we demonstrate that CLas titers do not significantly fluctuate following infection of the non-transgenic scions budded onto the transgenic rootstocks. Results obtained are only for 12 months after budding and further evaluation will conclusively demonstrate the validity of this hypothesis. Transgenic rootstock lines with the stacked SAR inducing genes have been produced by the mature citrus facility. Also all existing transgenic lines that have been observed to have enhanced PR-1 and PR-2 gene expression have been propagated. Seedlings from the first batch of transgenic lines have been phenotypically normal and genotypically stable. Thus, from preliminary indications, it seems the AtNPR1 transgene does not have any deleterious effect on the seedling progeny.
1. Please state project objectives and what work was done this quarter to address them: Objective 1. Investigate rootstock effects on horticultural performance of Valencia and Hamlin trees commercially grown under HLB-endemic conditions using standardized field data collection procedures.Leaf samples were collected from all four trials for nutrient analysis. Data analysis and interpretation is in progress. Manuscripts with all data are being prepared for open-access publication. Objective 2. Develop outreach to transfer information to growers and other industry clientele.A Citrus Industry magazine article was published in the July 2020 issue for an update on the trials: https://citrusindustry.net/2020/07/08/rootstock-effects-on-valencia-and-hamlin-in-large-scale-commercial-plantings/ Another update was presented during the virtual Citrus Expo (> 380 views): Is Bigger Ultimately Better? An update on large-scale rootstock evaluations on a ridge and flatwoods site (U Albrecht); https://citrusexpo.net/citrus-session-1/; https://citrusindustry.net/2020/10/02/long-term-rootstock-evaluation-is-best/ A presentation was made during the American Society for Horticultural Sciences (ASHS) annual conference, which was held virtually during 10-13 Aug: “Assessing Rootstock Effects on the Horticultural Performance of `Valencia’ Orange Trees Grown Commercially in HLB Endemic Conditions” (Presenter/authors: Kunwar S, Grosser W, Albrecht U) The complete datasets from years 1-2 were provided to the UF/CREC citrus breeding team for inclusion on their citrus field trial website. The same data were also provided to CRDF. 2. Please state what work is anticipated for next quarter: We will continue with manuscript preparation (for open-access publication of trial data). We will be preparing for fruit quality analysis and harvest of the Hamlin trials. 3. Please state budget status (underspend or overspend, and why): Approximately 50% of funds have been spent, which is slightly underspent because of COVID-related complications.
1. Please state project objectives and what work was done this quarter to address them: The objectives of this project are to produce disease resistant, commercially & agronomically acceptable, mature citrus transgenics & intragenics that will flower & fruit naturally using Agrobacterium & biolistics as a service for customers. The research focus of this project is to increase Agrobacterium & biolistic transformation efficiency of mature citrus, so that biotech products produced in our facility for commercialization can reach the Florida Citrus Growers sooner, at less expense. During this quarter, we tested two new, citrus selectable markers in Agrobacterium transformation & produced ~55 transgenics that survived micrografting. The number of transgenics produced with these citrus genes appeared to be higher than the standard nptII selectable marker although we have to finish data analyses to conclusively make this statement. These selectable markers are important for intragenics using all citrus sequences. The next step will be to characterize these transgenics at the molecular level, & to determine whether extra doses of these genes have any effect on phenotype, particularly the fruit. In addition, ~70 transgenics were produced for another CRDF project, however our portion of that funding is set to expire by the year’s end. We introduced OLL-20 from FDACS, which is a favorite of the juice industry, but the mother plants produced from shoot-tip grafts & all of the budded plants were chimeric. We are currently trying to resolve the chimeras by cutting off the chimeric tissue. FDACS has been alerted to this issue in the event that their trees were chimeric. The Educational Business Account (EBA) has been completed & it is now up to UF officials to approve it. Once approved, our new price list will be implemented, which should allow for a more reasonable fee collection that contributes more to salaries & consumables. Currently UF labs have limited return to work schedules as the Covid-19 pandemic in FL is still causing a significant number of cases & deaths. Our lab continues to work in shifts. Once the pandemic subsides, we will hopefully return to a more normal work schedule with people able to work together in one lab. Wearing masks, social distancing & disinfecting surfaces are directives that we must follow in the workplace now. 2. Please state what work is anticipated for next quarter: We will continue to produce transgenics for clients. For one client, this has involved testing different, commercially important mature grapefruit cultivars (Marsh, Flame, and Ray Ruby). Thus far, one mature grapefruit cultivar regenerated the most shoots in tissue culture & might have the highest transformation efficiency. We are also performing experiments to increase the efficiency of biolistic transformation of mature citrus by altering the osmotic pretreatment & finding cultivars amenable to the process. The two citrus selectable markers are being tested in biolistic transformation. The USDA APHIS BRS does not examine cis/intragenic trees, because the new federal SECURE RULE states that intragenic trees, without vector sequences, will be considered similar to trees produced through traditional plant breeding. 3. Please state budget status (underspend or overspend, and why): We are on track monetarily.
1. Please state project objectives and what work was done this quarter to address them: The objectives of this project are to produce disease resistant, commercially & agronomically acceptable, mature citrus transgenics & intragenics that will flower & fruit naturally using Agrobacterium & biolistics as a service for customers. The research focus of this project is to increase Agrobacterium & biolistic transformation efficiency of mature citrus, so that biotech products produced in our facility for commercialization can reach the Florida Citrus Growers sooner, at less expense. During this quarter, we tested two new, citrus selectable markers in Agrobacterium transformation & produced ~55 transgenics that survived micrografting. The number of transgenics produced with these citrus genes appeared to be higher than the standard nptII selectable marker although we have to finish data analyses to conclusively make this statement. These selectable markers are important for intragenics using all citrus sequences. The next step will be to characterize these transgenics at the molecular level, & to determine whether extra doses of these genes have any effect on phenotype, particularly the fruit. In addition, ~70 transgenics were produced for another CRDF project, however our portion of that funding is set to expire by the year’s end. We introduced OLL-20 from FDACS, which is a favorite of the juice industry, but the mother plants produced from shoot-tip grafts & all of the budded plants were chimeric. We are currently trying to resolve the chimeras by cutting off the chimeric tissue. FDACS has been alerted to this issue in the event that their trees were chimeric. The Educational Business Account (EBA) has been completed & it is now up to UF officials to approve it. Once approved, our new price list will be implemented, which should allow for a more reasonable fee collection that contributes more to salaries & consumables. Currently UF labs have limited return to work schedules as the Covid-19 pandemic in FL is still causing a significant number of cases & deaths. Our lab continues to work in shifts. Once the pandemic subsides, we will hopefully return to a more normal work schedule with people able to work together in one lab. Wearing masks, social distancing & disinfecting surfaces are directives that we must follow in the workplace now. 2. Please state what work is anticipated for next quarter: We will continue to produce transgenics for clients. For one client, this has involved testing different, commercially important mature grapefruit cultivars (Marsh, Flame, and Ray Ruby). Thus far, one mature grapefruit cultivar regenerated the most shoots in tissue culture & might have the highest transformation efficiency. We are also performing experiments to increase the efficiency of biolistic transformation of mature citrus by altering the osmotic pretreatment & finding cultivars amenable to the process. The two citrus selectable markers are being tested in biolistic transformation. The USDA APHIS BRS does not examine cis/intragenic trees, because the new federal SECURE RULE states that intragenic trees, without vector sequences, will be considered similar to trees produced through traditional plant breeding. 3. Please state budget status (underspend or overspend, and why): We are on track monetarily.
Update for this quarter: Test site grant- USDA has mandated that all employees be on maximum telework since March 2020 due to the Covid-19 pandemic. None-the-less, several new plantings of USDA transgenics were made this quarter. UF collaborators have been permitted into the test site and samples and data have been collected. Data were collected on McNellis trees by USDA. Samples we previously collected have been processed by technicians at home (with APHIS-BRS permission) and are ready for qPCR including many of them 2400 seeds collected for the transgenic pollen-flow experiment. A new “efile” APHIS-BRS permit request has been submitted for all test-site plantings (except CREC-Dutt, Grosser and Gmitter who maintain a separate permit), it is under review and should be approved soon. We were the first to use this new permitting system and it was very arduous. Additional canker data were collected in a block of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside. Previously Stover analyzed data on canker incidence in a block of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside, from data collected 8/17 and 9/19. Most notably: Almost all accessions with lower ACC lesion incidence were hybrids vs. pure trifoliate, though a few pure Poncirus had lower ACC than most. Based on chloroplast genome data from 57K Affymetrix SNP chip, provided by M. Roose, 11 of 33 reported seed parentage for hybrids was inaccurate, convention of female first was not followed. Of 34 hybrids validated, similar numbers had Poncirus, grapefruit, and sweet orange chloroplasts. Chloroplast type did not affect ACC incidence, but in each year accessions with grapefruit chloroplasts had small but statistically higher ACC severity than those with Poncirus chloroplasts. Hybrids of Citrus with Poncirus have markedly reduced ACC sensitivity compared to Poncirus, indicating that this trait is readily overcome in breeding. Seed from fruit harvested for transgenic gene flow experiment coninue to be processed for PCR. Previously established at the site: A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. Renewal and approval for BRS permit effective 9/1/19 through 8/31/20. 4) Continuation of an experiment on pollen flow from transgenic trees. FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. Early-flowering transgenic Carrizo (flowered ex-vitro within five months of seed sowing, and used at 12 months) was used to pollinate some of the same FF-5-51-2 What should be the final samples from the C. Ramadugu-led Poncirus trial (#3 below) completed preparation and were shipped in ethanol to UC Riverside. Availability of the test site for planting continues to be announced to researchers. 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 Stover permit. 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. Likely 2019 will be the last year for data collection. 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 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) 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. 12) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State13)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
This quarter: USDA has mandated that all employees be on maximum telework since March 2020 due to the Covid-19 pandemic. Therefore, existing experiments and ACP colonies have been maintained, but new experiments have been relatively few. Dean Gabriel of UF, and USDA scientists Kim Bowman, Ed Stover and G avin Poole have all run experiments totalling ~2000 ACP. Samples have all been collected on-time from ongoing experiments. All samples collected, that have not been analyzed, have been processed for qPCR. Project rationale and focus: The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~50% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Most recent quarter:A partial shut-down of USHRL was initiated 3/20/2020, as a response to the Covid-19 pandemic. ACP colonies are Stover lab:5460 ACP used for inoculating 390 detached leaves, 78 no-choice small trees ,and seven homogenate assays of peptides. Bowman lab: Prepared a group of grafted plants and planned to ACP-inoculate in March, but this experiment was disrupted by the Covid-19 slowdown. These ill be inoculated when personnel are allowed more extensive time at USHRL Other users:· 180 for Robert Shatters · 500 for Yongping Duan
1. Please state project objectives and what work was done this quarter to address them: .This report covers the period of June 1 – August 31, 2020. The objective of this project is to test transgenic ‘Ducan’ grapefruit trees expressing an anti-HLB antibody fused to the FT (Flowering Locus T) protein. Work on the project was slowed a bit by COVID-19 restrictions. However, tissue sampling and phenotypic analysis was continued on three HLB inoculation tests underway for the FT-scFv plants: a field trial natural inoculation; an Asian citrus psyllid (ACP) infection in the greenhouse; and a graft challenge with FT-scFv scions grafted to HLB-infected rough lemon rootstocks. Samples taken from the field trial trees were all negative for ‘Candidatus Liberibacter asiaticus’ (CLas) infection by qPCR at the 6-month point after planting the trees at the test site in November of 2019. The graft transmission test has yielded promising initial data. While 93% of the FT-scFv scions grew out and were healthy and vigorous with only slight to no HLB symptoms, only 67% of the control buds grew out, and those that did grow were relatively short and slow growing. The non-growing buds remained green, indicating a successful grafting, but failed to grow. This raises the possibility that the FT-scFv protein will counteract growth of CLas when the plant is mounting a defense reaction to Clas, as may be the case because rough lemon is tolerant to CLas. Also during this reporting period, a manuscript was written, revised, and then accepted for publication in Plant Biotechnology Journal describing our finding that the FT-scFv transgenic plants have a strong precocious blooming phenotype (https://doi.org/10.1111/pbi.13463). This is a very useful output of the project that will enable rapid-cycle breeding in citrus. Citrus breeding programs are already using the technique developed through this project to accelerate breeding for HLB resistance. Although this outcome was not an original objective of the project, it is a fortunate finding that solves a long-standing challenge of producing precocious edible citrus varieties. 2. Please state what work is anticipated for next quarter: Next quarter we anticipate that the graduate student will be able to visit ACP vector infected trees at Fort Pierce in November, 2020. These trees will be moved from the United States Horticultural Research Laboratory to the Indian River Research and Extension Center next door the week of October 12-16, 2020. This will allow us to have direct access to these trees. We will also continue sampling of tissues an qPCR monitoring of CLas titers in trees from all tests, and measure phenotypes of FT-scFv scions grafted onto the HLB-positive rough lemon rootstocks, including shoot length and HLB symptom severity. We also plan to set up a repeat of the rough lemon graft transmission experiment, given the promising results of the first run of the experiment. 3. Please state budget status (underspend or overspend, and why): The budget is 40% spent. This is partially due to an approximately 8 month delay to the start of the project while a graduate student was recruited to be the full-time researcher on the project. Travel has been limited since March, 2020 due to COVID-19, resulting in reduced travel costs. In addition, the project director elected to redirect all the budget for his salary to research support. This project will need a one-year no-cost extension to complete thorough testing of the HLB resistance tess already initiated and to complete a repeat of the graft-inoculation test. The existing budget will be sufficient to cover conclusion of all experiments and support the graduate student to the end of his M.S. degree.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. We have produced a list of candidate rootstock seed trees to be tested by DPI for seed transmissible pathogens. Receiving a clean bill of health for these trees will allow us to harvest and distribute seeds to collaborative nurseries and other organizations within Florida, in the US, and globally, to establish advanced trials to compare our best rootstock selections with industry standards. We continue to update and add new rootstock trial files to our website (https://crec.ifas.ufl.edu/citrus-research/rootstock-trials/), currently there is information from 24 locations. Two rootstock candidates selected from the Gauntlet screen for good HLB tolerance and tree performance have been entered into the DPI Parent Tree Program (DPI-PTP) for shoot-tip grafting.2. Develop new, HLB-tolerant scion cultivars from sweet orange germplasm, as well as other important fruit types such as grapefruit, mandarins, and acid fruit. Embryo rescue from 2019 crosses resulted in shoots from >1750 germinating embryos from 36 interploid crosses (including 11 targeting sweet orange-like hybrids, and 8 targeting grapefruit improvement), and nearly all have now been micrografted to rootstocks, and transferred to the greenhouse to grow off. Cybridization experiments were conducted to combine Meiwa kumquat cytoplasm with OLL and EV sweet oranges, to attempt improvements in citrus canker resistance; plant regeneration is underway. Somatic hybridization of Tango and W. Murcott suspension lines with leaf protoplasts of nine CREC and other public cultivars, and advanced selections, was attempted; most combinations already have produced embryos and shoots for propagation and establishment as new breeding parents for scion improvements. Transformation experiments using possible HLB resistance genes have taken place using seven different scion and rootstock selections, and some are nearing size for micrografting and establishment. Five new seedless and easy to peel mandarins have been entered into the DPI-PTP. Additionally, a nearly seedless Valencia orange mutant selected from an irradiation experiment in the 1990s, exhibiting unusual HLB tolerance compared with other oranges in a replicated planting, has also been entered for cleanup at DPI-PTP. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We used new approaches to quantify tree responses to HLB, in addition to the previously used subjective approaches; we measured photosynthetic parameters and leaf canopy indexes, to produce repeatable and reliable quantitative data in support of further genetic analyses of tolerant types. Objective quantitative data of tree responses provides more reliable information that improves the precision with which we can associate genome regions with tolerance or sensitivity; see Obj 4. 4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. We selected ~ 450 mandarin hybrids for GWAS studies, using the data referred to in Obj 3 above. DNA samples from these trees were prepared, and each individual was genotyped using the citrus Axiom SNP array. GWAS analysis is in progress now. This work will validate previously identified or identify new genomic regions associated with HLB tolerance or sensitivity. Further, we have contributed budwood of 54 UF-CREC selections to a top-working trial of approximately 200 selections in total, that is a collaboration with USDA-ARS Ft. Pierce and supported by industry. These have been topworked in a commercial grove and are now growing; this smaller but more diverse population will be used for GWAS in the future. Finally, our multi-location transgenic field permit has been renewed by the USDA; this will enable us to continue testing the effects of certain genetic modifications on HLB incidence, disease development, and potential tolerance or resistance, under real world field conditions.
The project has been granted a no cost extension into July 2020; year 1 activities, and some of year 2 objectives, have been nearly completed. A comprehensive overview of progress since project inception through July 2020 follows below. Timeline Year 1: Objective 1: Produce suitable plant materials for PacBio sequencing technical requirements, isolate high-quality genomic DNA samples for sequencing, generate PacBio genome sequence data, and assemble PacBio sequence reads as data are received. Objective 2: Prepare RNA libraries for transcriptome sequencing, generate full-length transcript sequence data on Nanopore sequencers. Results We have produced the plant materials and isolated the HMW-DNA from Valencia orange (S, sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, tolerant), and Lisbon lemon (T). We generated raw sequence data for all 5 genomes on the PacBio Sequel II. Preliminary assemblies and analyses were carried out. For four of the five genomes, the results exceeded the quality of any other publicly available citrus reference genomes, even before Dovetail Hi-C proximity ligation sequencing to finalize assembly at the chromosome level. However, the quantity of grapefruit sequence is insufficient, so we have prepared the Ruby Red grapefruit HMW-DNA and additional sequencing is in progress. We prepared materials from two genomes for the Dovetail Hi-C sequencing. Incorporating these data with PacBio sequence of one of our target genomes has resulted in an improved chromosome scale assembly. The two parental chromosomes of the target genome have been phased/separated using Illumina short reads from citrons, pummelos and mandarins. By genome alignment and comparison to the Poncirus assembly (see below), minor assembly errors in repetitive regions have been fixed, resulting in a polished assembly. The availability of high-quality assemblies for the 3 basic species (C. medica, reticulata, and maxima) will allow a more thorough and complete characterization of large-scale structural variation (SVs: deletions, insertions, etc.) in genomes of commercial interest. These SVs are the driving force for phenotypic diversity especially among somatic mutants (e.g. different oranges, grapefruits).A previously funded CRDF project supported the initiation of a project producing the first ever high-quality reference genome of Poncirus trifoliata, and under this current project we have completed the task; a manuscript is under review in The Plant Journal, and the sequence will be released to the global citrus research community upon publication. By mining this new genome, we identified candidate genes within previously identified chromosomal regions for HLB tolerance, including a transcription factor gene and one disease resistance-like gene that are up-regulated by CLas and positively selected in trifoliate orange. These genes are promising candidate genes for further research.Conclusions1. We completed all genome sequencing work under Year 1, Objective 1, except for generating more sequence data for the Ruby Red grapefruit, currently underway.2. We have not yet generated the full-length transcript sequence data, as proposed for Year 1, Objective 2. This goal was compromised for several reasons beyond our control but should progress in year 2.3. Hi-C sequencing for proximity ligation was completed for two genomes, and along with Illumina short reads resulted in a phased chromosome scale assembly of one target genome. 4. We have produced a very high-quality genome assembly of Poncirus, an important source of resistance to CLas and HLB. This genome assembly was used to repair minor assembly errors in repetitive regions, and by mining the sequence we have identified genes to be targeted for HLB resistance.
Significant genetic variation in OLL sweet orange seedling population identified (research block 13-W, Orie Lee Family Groves, St. Cloud): many of the trees again tested PCR negative for CLas (after 6 years in the field with no psyllid control). Clones showing high soluble solids and/or early maturity were propagated for further study. Significant genetic variation in Vernia sweet orange seedling population and various cybrids (Mathew Block, Lee Alligator Grove and CREC): multiple clones showing early maturity (December) and two clones with higher soluble solids were propagated for further study. Propagation of best ‘gauntlet’ rootstocks: The first gauntlet rootstock hybrid containing SugarBelle as a parent (LB8-9 x S13-15-16) planted in the field with a CLas-infected Valencia scion that has grown vigorously and shows no HLB symptoms after 2 years was successfully propagated by cuttings and also introduced for TC propagation with Agromillora and Phillip Rucks TC lab. The best performing ‘gauntlet’ selection S10xS15-12-25 (produced from a cross of salt tolerant HBPummelo x Shekwasha with salt tolerant HBPummelo x Cleo) was successfully propagated by cuttings, and also introduced for TC propagation with Agromillora. Both of these promising selections were also entered into the PTP. A few other promising ‘gauntlet’ rootstock genotypes were recovered by cutting off the top (scion portion) of field trees, as necessary for further propagation. Propagation of recent PTP selections from the UF/CREC breeding program (past 3 years): approximately 50 selections showing promise at fruit displays, etc. are now being propagated onto UFR-5 rootstock at Southern Citrus Nursery, 10 trees per selection (this includes all categories of fruit). Tree Planting: approximately 1000 trees of new hybrids were planted at the CREC and Riley Block (Haines City). New grapefruit and pummelo clones in the PTP were planted at the Trailer Park block. Approximately 200 additional rescued trees from the Balm/GREC were planted at the Eagle Lake Block; 500 trees of 6 scions on promising rootstock candidates were also planted at the Eagle Lake Block (part of the Tri-State project, not funded by CRDF). The following trials were posted/updated (with new seasonal data) on our rootstock data website: Basinger ‘Vernia’ Rootstock Trial; Waverly Scion/Rootstock Trial; Indian River ‘Marsh’ Grapefruit Rootstock Trial; Indian River ‘Vernia’ Rootstock Trial; Water Conserve II Valencia & Midsweet Scion Trial; Post Office Rootstock Trial; Indian River Minneola Rootstock Trial; and St. Cloud OLL Clone/Rootstock Demonstration Planting. The following trials have been updated and are awaiting final review for addition to the rootstock data website this quarter: Indian River Lemon Rootstock Trial; Peace River ‘Valencia’ rootstock Trial; SW Flatwoods Sweet Orange Rootstock Trial; Ver Beach Navel and Grapefruit Rootstock Trials; Charlotte County Multi-Scion rootstock Trial; LaBelle Valencia Advanced Production System (APS) Rootstock Trial; LaBelle Vernia rootstock Trial; South Ridge Valencia Rootstock Trial; St. Helena Rootsotck Survery Trial; and Teaching Block Scion and Rootstock Trial.
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 In this quarter, due to the COVID-19 travel restriction, we were unable to travel between lab and the field trial site. This limited our work in the field. We thus focused on laboratory and greenhouse work: (1) We have prepared a new batch of transgenic plants (65 in total) that were supposed to be transplanted into the field at the end of April or early May of 2020. However, due to the COVID-19 restriction, the transplanting was postponed. These plants were maintained in the greenhouse, and were regularly watered and fertilized during this quarter. (2) Tested the antibody against a major citrus defense protein with total protein extracts from several rootstocks including Citrus macrophylla. We are particulary interested in C. macrophylla, as we can easily silence genes in this species using the established CTV-RNAi method. It was found that the antibody is able to detect the protein in the rootstocks including C. macrophylla. This would be a good tool for identifying gene targets in citrus for both gene silencing and CRISPR/Cas9-mediated gene editing. (3) Created more transgenic citrus plants expressing a new key gene in systemic acquired resistance. These transgenic plants are growing in the greenhouse and characterization is unerway.
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