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. This method has been succefully used to generate non-transgenic biallelic mutants of sweet orange. 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 further optimized the CRISPR/Cas9 system. Now, the biallelic mutation rate reaches 89% for Carrizo citrange and 79% for Hamlin sweet orange. We have generated one homozygous line in the promoter region of canker susceptibility genes of Hamlin. We have successfully generated one biallelic mutant of grapefruit that is canker resistant. We also successfully generated multiple biallelic and homozygous mutant lines of sweet orange that are canker resistant. 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. Base editors have been successfully used to precisely edit specific residues of citrus genes. ErCas12a has been succes for non-transgenic gene editing of embryogenic Hamlin sweet orange protoplast cells. We are editing 6 putative HLB susceptibility genes for sweet orange. One biallelic mutant line has been generated for ACD2.
1. Please state project objectives and what work was done this quarter to address them: Objective 1. Investigate effects of rootstock propagation method and the interaction with rootstock on root structure, root growth, and tree performance during the first 3 years of growth in the field.Bimonthly root growth measurements with the minirhizotron imaging system continued in all trials. Trials 2 and 3 third-year tree size data collection was completed. Fruit quality analysis was completed for trial 3. We are working on completing the analysis of the year 3 data of trials 2 and 3 to be included in a publication of all three years of data. A Citrus Industry Magazine article sumarizing findings of this project was published (July issue). A virtual seminar was presented on June 21 that included information on this project. Objective 2. Investigate if trees on rootstocks propagated by tissue culture or cuttings differ in susceptibility to Phytophthora-induced decline or wind-induced blow-over compared with trees on rootstocks propagated by seed.Monthly root growth measurements with the rhizotron imaging system continued. We are still working on the PCR and ELISA assays. 2. Please state what work is anticipated for next quarter: We will continue with our minirhizotron root imaging analysis and. We will continue with the statistical analysis and interpretation of all data collected. 3. Please state budget status (underspend or overspend, and why): Approximately 67% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.
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.Fruit collection and fruit quality analysis for the Valencia trials was completed.Harvest was completed for both Valencia trials. Tree/canopy size and trunk diameter measurements were completed for this year.We are still working on the leaf CLas analysis in for trees in the Valencia trials.The statistical analysis of all data is nearly completed and is being prepared for publication. Objective 2. Develop outreach to transfer information to growers and other industry clientele.A one hour virtual seminar on rootstocks, including a summary of results from this project, was presented on June 21. 2. Please state what work is anticipated for next quarter: We will continue with the data analysis. Data are being prepared for a presentation at the Citrus Expo in August. 3. Please state budget status (underspend or overspend, and why): Approximately 70% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.
Update for this quarter:A significant USDA-funded infrastructure project at the Picos Farm location is now complete. Improvements included a full renovation of the water management system; with drainage channels re-dug and cleared of vegetation, culverts inspected and replaced as needed, and new irrigation controls with remote access capability added. This will significantly improve storm and flood protection. Site management and field trials are progressing well. Four new transgenic trials were planted this quarter; two antimicrobial peptide expressing designs from the Stover team and two enhanced disease resistance designs from UF researchers. With these trials, the transgenic site is now operating at full capacity (some UF and other non-transgenics have been managed under this grant in adjoining blocks, exceeding 10 acres total for three years). The UCRiverside-led trifoliate and trifoliate hybrid trial has concluded, a manuscript regarding identified HLB-tolerance is in preparation, and trees will be removed to make space available for future plantings. The primary BRS permit has been amended to include a new UF gene construct that has shown a reduction in CLas titer in greenhouse trials. All regulatory protocols are being observed and the annual site review from APHIS/BRS has been conducted. Recent quarters:An additional permit has been approved (AUTH – 0000043620 effective 12/17/2020) for material with “Confidential Business Information” for a project led by R. Shatters. The primary BRS permit AUTH – 0000043619 has been renewed. Stover analyzed data on canker incidence for as Incidence of Asiatic Citrus Canker on Trifoliate Orange and its Hybrid Accessions in a Florida Field Planting. 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. 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. A manuscript detailing these results has been published in HortScience DOI: 10.21273/HORTSCI15684-20. Previously established at the site:A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. 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 USDA permits.2) Under the Stover permit, a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. 4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project.5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and will continue to be conducted under a new NIFA grant.6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).7) In a project led by Fred Gmitter, there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo.10) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls.11) Multiple plantings with grafted trees of l Hamlin, Valencia and grapefruit scions on transgenic rootstock expressing antimicrobial citrus-thionin and bacterial recognition domain fusion proteins (219 trees with controls) as a collaboration between USDA and the New Mexico Consortium.12) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type.13) An additional planting from Zhonglin Mou of UF that includes transgenic grapefruit (31 plants) and sweet orange (33 plants) scions expressing two different resistance genes and grafted on WT swingle rootstocks; as well as non-transgenic controls. 14) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State15)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
This quarter:This quarter , the colony of CLas infected psyllids supplied a total of 5,045 ACPs used for (1) evaluation of rootstock breeding material, (2) transgenic events evaluation, (3) applied research for CLas control in citrus performed by USDA and University researchers; and (4) monitoring the colony quality.The Bowman lab continues the experiments with grafted Valencia trees on groups of rootstocks which were inoculated with ACP during the previous quarter (Janyuary to March 2021). Each experiment compares replications on nine different rootstocks. This quarter 63 trees were inoculated with 1,280 ACP from the positive ACP colonies. Periodic evaluation of tree health, growth, and CLas titer via PCR were conducted on trees from rootstock experiments that were inoculated during the previous months following a set schedule. Periodic colony checks were conducted by PCR to maintain CLas positive colonies. The Stover lab conducted weekly detached leaf assays (DLAs) challenging transgenic citrus with CLas inoculated by infected ACP in the lab, which is used to identify best performing transgenic events (transgenics varying by position of transgene insertion etc.) expressing antimicrobial peptides and defensive proteins targeting CLas, as well as natural insecticide peptides to control ACP. Six detached leaf assays experiments, involving individual 240 leaves, were inoculated using 2,400 CLas infected ACPs in this quarter. Transgenic material tested in DLAs were Carrizo and Hamlin plants expressing ONYX peptide under constitutive and phloem specific promoter. A total of 48 independent events were tested alongside WT controls. The leaves (midribs) and ACPs are being processed and submitted to qPCR for CLas titer after each DLA to better understand the effect of the transgenic peptide in bacteria control and transmission. These trials have being very useful in terms of providing information that allow to select the best transgenic events (ones causing high ACP mortality and/or low CLas transmission to plant) for propagation and further evaluation at greenhouse environment. We continue to see substantial ACP mortality from feeding on CLas-killing transgenic leaves. Research involving evaluation of the microbiome of ACPs fed on transgenic causing high insect mortality was conducted this quarter using 230 ACPs fed in a set of 22 transgenic leaves. In addition 775 CLas+ ACP were provided to researcher collaborators (600 ACPs for Dr. YongPing Duan of USDA and 175 for Florida International University, for Jessica Dominguez, a Ph.D. student, who is developing a thesis in alternative compounds to control CLas bacteria). Also, to monitor colony quality, 360 ACPs were used for Clas detection by qPCR this quarter. In this quarter, seven new colony cages (with 250 ACPs/cage) were set up to renew and support the demand of the hot ACPs for future use. 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% (I don’t think Anna Sara has 50% of her time to take care of the colonies) to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Previous quarter:United States Department of Agriculture scientists Kim Bowman, Ed Stover and YongPing Duan have all run experiments totalling 7,280 ACPs. Samples have all been collected on-time from ongoing experiments. All samples collected, that have not been analyzed, have been processed for qPCR.
This quarter, the colony of CLas infected psyllids supplied a total of 6,840 ACPs used for (1) transgenic events evaluation, (2) applied research for CLas control in citrus performed by USDA and University researchers; and (3) monitoring the colony quality by qPCR.The Stover lab conducted weekly detached leaf assays (DLAs) challenging transgenic citrus with CLas inoculated by infected ACP in the lab, which is used to identify best performing transgenic events (transgenics varying by position of transgene insertion etc.) expressing antimicrobial peptides and defensive proteins targeting CLas, as well as natural insecticide peptides to control ACP. Seven detached leaf assays experiments, involving individual 220 leaves, were inoculated using 2,200 CLas infected ACPs in this quarter. Transgenic material tested in DLAs were Carrizo plants expressing ONYX peptide and chimeric AMP TS, both under SCAmp-P3 phloem specific promoter. A total of 36 independent events were tested alongside WT controls. The leaves (midribs) and ACPs are being processed and submitted to qPCR for CLas titer after each DLA to better understand the effect of the transgenic peptide in bacteria control and transmission. These trials have being very useful in terms of providing information that allow to select the best transgenic events (ones causing high ACP mortality and/or low CLas transmission to plant) for propagation and further evaluation in the greenhouse environment. We continue to see substantial ACP mortality from feeding on CLas-killing transgenic leaves. Research involving evaluation of the microbiome of ACPs fed on transgenic causing high insect mortality was conducted this quarter using 450 ACPs fed in a set of 45 transgenic leaves. A research paper has been prepared (Rapid in vivo screening for huanglongbing resistance in genetically modified citrus by detached leaf assay- J.Krystel, M. Grando, Q. Shi, E. Cochrane, E. Stover) in order to report important modifications implemented into the DLAs using Clas+ ACPs to evaluate transgenic plants and investigate the mode of actions of peptide in controlling the psyllids. In addition, 780 CLas+ ACP were provided to researcher collaborators (100 ACPs for Dr. Michelle Heck USDA-Ithaca, NY; 480 for Florida International University, for Jessica Dominguez, a Ph.D. student, who is developing a thesis in alternative compounds to control CLas bacteria) and 200 ACP were furnished to Dr. Randy Niedz (USDA Fort Pierce) for activities in a HLB NIFA project. Also, ten new colony cages were set up to renew and support the demand of the hot ACPs. For that 37 HLB positive plants were infested with 2.400 ACPs. Periodic colony checks were conducted by PCR to maintain CLas positive colonies. This quarter 960 ACPs were used for Clas detection by qPCR to monitor colony quality. In this quarter, 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 ~30% of her time to the program in order to maintain colonies (including watering, setting up new cages, terminating old cages, cleaning growth chambers and cages). USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Previous quarter: United States Department of Agriculture scientists Kim Bowman, Ed Stover and Yong Ping Duan have all run experiments totaling 5,045 ACPs. Samples have all been collected on-time from ongoing experiments. All samples collected, that have not been analyzed, have been processed for qPCR.
Juvenile Tissue Citrus Transformation Facility (JTCTF) completed second quarter of operation as an EBA unit. The lab is open for customers who need transgenic citrus material and are ready to pay for services based on the EBA price list. At the same time, JTCTF still does work based on the research grants that were received in previous years. JTCTF resumed pre-COVID operation in regard to presence of work force in the lab well ahead of the date set by UF/CREC administration. However, one of the employees still works the night shift. There are still four employees on the roster although one of them has not worked since December because of unresolved immigration status.Within the first half of the 2021, JTCTF has charged clients for the work it performed as the EBA unit and multiple payment were successfully processed. The Business office staff at CREC helped in this process and made it work. As we receive more orders, the support from the business office in processing of payments will be very important.Between April and July, JTCTF accepted five new orders. Client are still mostly interested in transgenic Duncan grapefruit and Valencia sweet orange. Three of five placed orders were associated with the existing USDA grant, while two orders came from clients through EBA. The facility is still expecting the influx of orders that were going to be placed already based on the fact that JTCTF was designated as subcontractor on the research grants. Those orders are delayed because the work on those projects was also delayed due to COVID-19.The output of the JTCTF in the last 90 days was eight transgenic plants. Six plants were Duncan grapefruit, one was Hamlin orange, and the last one was Valencia orange. The number of transgenic shoots JTCTF produced within the last six months was big. However, those experiments were done for the proof of concept and transgenic shoots were not grafted and grown into plants. The output of the facility would have much been better if these plants were developed.
1. Please state project objectives and what work was done this quarter to address them: This report covers the period of March 1, 2021 – May 31, 2021. 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 (FT-scFv protein). Several accomplishments were made during this reporting period. Graduate student Mr. Chad Vosburg was able to take a trip to Florida to assess trees again, take samples, and send samples for qPCR analysis at Southern Gardens. Chad visited Fort Pierce for several days in May, 2021. The samples taken and/or processed, and then sent to Southern Garderns included those from a field trial natural inoculation, an Asian citrus psyllid (ACP) infection in the greenhouse, and a repeate experiment of graft challenge of FT-scFv scions grafted to HLB-infected rough lemon rootstocks. These samples were prepared and sent to Southern Gardens for qPCR detection and quantification of ‘Candidatus Liberibacter asiaticus’ (CLas) within the tissues. Data are anticipated to be received from these samples in July-August 2021. In addition, growth data were taken from FT-scFv scions on infected rough lemon. The transgenic scions appeared to be consistently growing faster and more robustly than the control non-transgenic scions. However, this difference did not meet a standard cutoff for statistical significance. The FT-scFv protein appears to provide a mild benefit to the grapefruit trees when inoculated by grafting, even if not statistically significant. The replicate grafting inoculation experiment begun in January, 2021, has additional replicates and may provide more statistically robust results. In March, 2021, we applied for a no-cost extension through November of 2021, and it was granted in May, 2021. 2. Please state what work is anticipated for next quarter:Additional qPCR data from the field, graft, and psyllid-transmission HLB challenge tests will be received from Southern Gardens in the summer of 2021. These data will be analyzed during the next reporting period. Additional sampling of field grown trees will be performed with help from collaborators at the USDA USHRL. These will be sent to Southern Gardens for qPCR CLas quantification for an additional infection time point. 3. Please state budget status (underspend or overspend, and why):This project is running under budget thanks for wage support from Penn State for Chad Vosburg’s work on the project, which resulted in some salary funds not being expended.
1. Please state project objectives and what work was done this quarter to address them: This report covers the period of March 1, 2021 – May 31, 2021. 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 (FT-scFv protein). Several accomplishments were made during this reporting period. Graduate student Mr. Chad Vosburg was able to take a trip to Florida to assess trees again, take samples, and send samples for qPCR analysis at Southern Gardens. Chad visited Fort Pierce for several days in May, 2021. The samples taken and/or processed, and then sent to Southern Garderns included those from a field trial natural inoculation, an Asian citrus psyllid (ACP) infection in the greenhouse, and a repeate experiment of graft challenge of FT-scFv scions grafted to HLB-infected rough lemon rootstocks. These samples were prepared and sent to Southern Gardens for qPCR detection and quantification of ‘Candidatus Liberibacter asiaticus’ (CLas) within the tissues. Data are anticipated to be received from these samples in July-August 2021. In addition, growth data were taken from FT-scFv scions on infected rough lemon. The transgenic scions appeared to be consistently growing faster and more robustly than the control non-transgenic scions. However, this difference did not meet a standard cutoff for statistical significance. The FT-scFv protein appears to provide a mild benefit to the grapefruit trees when inoculated by grafting, even if not statistically significant. The replicate grafting inoculation experiment begun in January, 2021, has additional replicates and may provide more statistically robust results. In March, 2021, we applied for a no-cost extension through November of 2021, and it was granted in May, 2021. 2. Please state what work is anticipated for next quarter:Additional qPCR data from the field, graft, and psyllid-transmission HLB challenge tests will be received from Southern Gardens in the summer of 2021. These data will be analyzed during the next reporting period. Additional sampling of field grown trees will be performed with help from collaborators at the USDA USHRL. These will be sent to Southern Gardens for qPCR CLas quantification for an additional infection time point. 3. Please state budget status (underspend or overspend, and why):This project is running under budget thanks for wage support from Penn State for Chad Vosburg’s work on the project, which resulted in some salary funds not being expended.
1. Please state project objectives and what work was done this quarter to address them:The project objectives are: 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.We have continued on the evaluation of transgenic trees in the greenhouse and field. Trees in the greenhouse and field have not been been tested against HLB in this quarter as sampling is done bi-annually. We have produced several trees with each gene as outlined in the proposal and objective 3 is almost complete. RNA has been isolated from all the new lines and we are preparing to propagate selected lines that have enhanced gene expression. Western blot analysis of some prior propagated lines have been done. All previously identified lines with high gene expression have been clonally propagated by budding onto rootstock for field planting to provide more seed source trees. Additionally seedlings from several existing seed source trees have been produced and budded with Valencia sweet orange. We are also exploring the ability of some of the high expressing lines as interstocks to protect the non-transgenic scion against HLB. White grapefruit is being used in this study due to its extreme susceptibility to HLB. 2. Please state what work is anticipated for next quarter: We will continue on our evaluation of the transgenic lines, propagate additional lines for evaluation and test the interstock trees against HLB. 3. Please state budget status (underspend or overspend, and why): We are on track with the budget and have not overspent.
1. Please state project objectives and what work was done this quarter to address them: Since this CRDF proposal was written in 2018 & the external review occurred, the status of our lab has changed at UF, & in the future, we will conduct less research & focus more on service. In any event, the objectives of this project were to produce disease resistant, commercially & agronomically desirable, mature citrus transgenics, cisgenics, & intragenics that will flower & fruit naturally using Agrobacterium & biolistics as a service for research & commercialization. The research focus of this project is to improve Agrobacterium & biolistic transformation efficiency of mature citrus, so that the mature citrus protocols become more productive, decrease prices for scientists, & contribute more to financial self-sufficiency of our lab. We made great strides in increasing Agro-mediated transformation efficiency of some cultivars in Year 2 (after testing virB5 & plant hormones) & found a completely unrelated method that improves transformation efficiency. A manuscript was submitted for review to my supervisor. An all-citrus selectable marker that functions well in transformation, as an alternative to nptII, was also elucidated in Year 2. A manuscript is being written describing the new selection system. The objective of research into DNA-free gene editing will be continued only in collaboration with a customer(s). From one customer, we received four vectors, performed five transformations, none of his vectors worked & no transgenics were produced. This scientist is using a new vector backbone that doesn’t function in mature citrus scions or rootstocks. We are in the process of assisting him troubleshoot this issue. In addition, the Agro strain he is using, EHA105, tends to rearrange the vectors & the genes of interest can be lost. We transformed some of his vectors into the AGL1 strain, which is RecA- & doesn’t rearrange as often. From now on, we will always include a control transformation with a reliable vector that consistently yields results. Another scientist gave us three vectors to transform into mature citrus & we produced 50 transgenic shoots. He did not want these shoots grafted onto rootstock, but we took high quality photographs for him. The scientist wrote a manuscript with the photographs of the transgenics & included us as co-authors. For another order, we produced ~82 transgenic shoots in one rootstock transformation, an excess of 40 were micrografted, & it is too soon to tell how many will survive. 2. Please state what work is anticipated for next quarter: A number of vectors have been transformed into mature citrus & we are awaiting results. In addition, we have more orders for Agro-mediated transgenics, so we are budding mature citrus for these transformations. In addition, two potential orders for biolistics are pending, however the efficiency of biolistics is still being increased by testing different osmotic treatments & cultivars. Unfortunately the lab member responsible for biolistics is away on sick leave, so we must conduct the optimizations for him. Budding of Kuharske, Carrizo, US942, Hamlin, Valencia, & EV1 was performed for biolistic transformation in order to collect stem tissue explants more appropriate for bombardments. In addition, Dr. Dutt is currently making genetic constructs that will be tested for one biolistic order. We might get additional orders once the outcomes of grant competitions are announced. 3. Please state budget status (underspend or overspend, and why): CRDF funding is sufficient, however we will probably overspend the Director’s account since we lost time & money doing five transformations that didn’t work, unless we can make up for it in later quarters.
Previously, we generated raw sequence data for Valencia orange (S, sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, tolerant), and Lisbon lemon (T) and preliminary assemblies and analyses were carried out. Because of reduced sequencing costs, we were able to enter additional important genomes into the pipeline beyond those originally proposed, including Carrizo citrange, sour orange, and Shekwasha (an important breeding parent for HLB tolerance); these also have now been sequenced and assembled. Now, we have completed Hi-C sequencing of two genomes, and by incorporating these data with PacBio sequence assembly from two of our target genomes we have produced improved chromosome scale assemblies and have phased the two parental chromosomes of the target genomes using Illumina short reads from citrons, pummelos and mandarins. Minor assembly errors in repetitive DNA regions have been repaired, by genome alignment and comparison to the Poncirus assembly we recently published, resulting in polished assemblies of these two accessions. Initial preliminary characterizations of the presence/absence variations among the four phased haploid genomes have been made. Notable variations among these four involve transposable elements including MITES with characteristic sizes. A genome graph-based approach is being pursued, with the goal of building a genome graph containing the different types of structural variations that are now being identified. The availability of high-quality assemblies for the 3 basic species (C. medica, reticulata, and maxima) will allow thorough and complete characterization of large-scale structural variations (SVs: deletions, insertions, etc.) in our target genomes of commercial interest. SVs are a driving force for phenotypic diversity especially among somatic mutants (e.g., different oranges, grapefruits, etc.), and this type of information will become more important as we test different sweet orange mutants exhibiting enhanced tolerance of HLB and attempt to determine the underlying causes of such tolerance. Currently, we are developing this approach using 27 previously published whole genome shotgun sequences of a particular citrus species and aligning them to our high-quality assembly of the same species. We have found several distinct basic genome types, with varying admixture patterns; a detailed characterization is in progress. We now have the transcriptome data for two of our target genomes, using both Illumina and PacBio sequencing platforms, and genome annotation (i.e., identify all the genes within the genome) is in progress. Samples have been prepared and collected, and plans are in place to proceed with Hi-C sequencing of the target genomes not yet completed and polished, as well as to generate the transcriptome data required for genome annotation, and further characterization of large-scale structural variations within and among the genomes upon which we are focused.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Seeds were extracted from 27 candidate rootstock seed source trees, including several UFRs and other experimental rootstocks, that were fund to be free of seed transmissible pathogens by DPI testing. We are authorized to distribute seeds to licensed nurseries for the UFRs, and to collaborative nurseries and other organizations within Florida, in the US, and globally. New trials will be established with these rootstocks in Australia (New South Wales and Queensland) and South Africa in the next year. A rootstock trial with previously exported rootstocks was propagated in Sicily this spring. These trials will provide information relating to performance under diverse soil and environmental conditions and can help development of recommendations for Florida conditions. More than 3000 flowers were pollinated in twenty-one new rootstock cross combinations of 13 different parents, to recombine multiple useful traits for rootstock improvement. We updated and added new data to existing rootstock trial files and added new files to our website (https://crec.ifas.ufl.edu/citrus-research/rootstock-trials/); currently there is information from 26 locations. Seedlings are being selected from 2020 diploid and tetraploid rootstock crosses in calcareous, high pH soil inoculated with two species of Phytophthora, the first step of the `gauntlet’ screening. More than 50 gauntlet rootstock candidates in citripots are growing off for grafting of replicate trees as necessary to meet the new CRDF guidelines for Stage 1 rootstock evaluations (approximately 500 liners). 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. Shoots generated from 37 interploid crosses made using selected HLB tolerant plants in 2020, including 10 crosses for red grapefruit improvement, 10 for sweet orange improvement, and 10 for mandarin improvement are in the process of micrografting on to 3 different rootstocks, and some have been already moved to the greenhouse. Seedlings from somaclone seedling-derived populations of early maturing (January), high soluble solids OLL selections were planted, to screen for even earlier maturing OLL types; seedling populations of a nearly seedless HLB-tolerant mandarin were also planted to get a completely seedless selection, and to extend the maturity season. A September-maturing pink grapefruit hybrid C2-53, with canker and HLB tolerance exceeding standard grapefruit, was approved for release by the IFAS cultivar release committee. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We have continued monitoring a unique hybrid family of more than 400 individuals (with many of these planted as 3 tree replicates) from the cross of Clementine mandarin with a wild species reported to be nearly resistant to CaLas attacks, by collecting detailed HLB phenotypic data, including Ct values and other tree health measures as described previously. The frequency of CaLas-negative trees remains unexpectedly high. We have revisited a replicated planting of sweet orange-Poncirus hybrids that was mapped for HLB tolerance QTLs (Huang et al. 2018) and are collecting new phenotypic data to study long term performance of these trees.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 were prepared, each individual was genotyped using the citrus Axiom SNP array, and GWAS analysis is proceeding. This work will validate previously identified, or identify new genomic regions, associated with HLB tolerance or sensitivity. We continued seedling and callus line transformation experiments using the BG gene, and have plants coming from 3 orange, 1 grapefruit, and 2 rootstock lines.
Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is ongoing. As the most proven of our transgenics, we continue to use them as a reference in detached leaf assays, as well as studying them in established greenhouse and field trials. The first MThionin field trial (45 plants, WT or transgenic Carrizo on rough lemon scions) has shown transgenics maintaining higher average CLas CT values (2.5 CT higher @ 18 months), but with a high degree of variability. The larger second MThionin planting (205 total grafted plants of transgenic Hamlin scions, transgenic Carrizo rootstock, or WT/WT controls) is producing encouraging results; with the transgenic Hamlin on WT Carrizo having statistically better trunk diameter, tree height and canopy volume compared to controls. Leaf samples have also been collected and are being processed for CLas quantification. The Mthionin construct has also been extensively transformed into additional varieties; with 10 confirmed transgenic lines of US-942 and 44 putative lines of Valencia and Ray Ruby now undergoing expression analysis. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, have been conducted to screen citrus lines expressing chimera constructs TPK, PKT, CT-CII, TBL, BLT, LBP/’74’, ’73’, and ‘188’ (as well as scFv-InvA, scFv-TolC, Topaz and Onyx). Testing of all 35s driven Carrizo lines is complete and the analysis of phloem specific and scion-types is well underway. This work has already identified numerous lines with significant effects on CLas transmission and increased ACP mortality (up to 95% from TBL and >70% from TPK). Endosymbiont analysis of high mortality causing transgenics (outlined in Objective 4) has also shown an accompanying reduction in Profftella titer that may indicate a mode of action. The best performing of these lines have been moved forward to greenhouse trials. Initial ACP-inoculated greenhouse trials on 8 lines of citrus Thionin-LBP chimeras (’73’, and ’74’) showed a statistically significant reduction (13x) in CLas titer for ’74’ transgenics vs WT. However, many plants of both treatments remained CLas negative due to low inoculation efficiency. In June, 150 plants representing the best performing 6 lines of `74′ and 7 lines of `188′ were no-choice caged ACP inoculated using a new protocol to improve transmission rates. At 3 months, control plants tested positive at twice the rate of the earlier inoculation; 6-12 month tissue samples are now collected, processed and ready for qPCR. A larger greenhouse study is also underway to directly compare the best performing 3rd generation chimera (TPK and TBL) with the earlier 1st (Mthionin) and 2nd (`74′ and `188′) lines. A total of 420 grafted plants (all on WT Carrizo rootstock for uniformity) were made and bud inoculated with CLas+ RL to ensure high transmission. The first (3 month) growth assessment and leaf collection is complete, with samples awaiting qPCR analysis. An additional ~1200 rooted cuttings have been made from those same lines for paired ACP-inoculated greenhouse and field trials. Field trials of 2nd generation chimeras (`74′, and `188′) with included MThionin plants are ongoing; with 165 plants (WT Hamlin and Ray Ruby on transgenic Carrizo) and 70 plants (WT Valencia on transgenic Carrizo) moved to the field in August 2020 and May 2021 respectively. An additional 200 plants (transgenic Hamlin on WT rootstocks) are being produced complete the planting. Eighteen new transformations, totaling over 6200 explants, have been completed to generate sufficient events of Valencia, Ray Ruby, US-942, and Hamlin lines expressing `74′, `188′, TBL, TPK and other advanced chimera constructs. From this effort, over 325 new lines from 74-Valencia, 74-Ray Ruby, 74-US-942, 74-Hamlin, 188-Ray Ruby, 188-Valencia, 188-US-942, TBL-US-942, TBL-Hamlin, TBL-Ray Ruby, TPK-Ray Ruby, TPK-US-942 and TPK-Hamlin are now in soil. Transgene expression analysis has confirmed the first 29 of these lines as positive with the remainder still being tested. In addition to the use of the Mthionin and its chimeric variants, new strategies have been implemented in our Laboratory to develop HLB resistant citrus. These efforts include the expression of insecticidal peptides to control ACP (CLas vector) and the downregulation of the DMR6 genes to enhance defense responses against HLB disease. 54 independent transgenic lines of Carrizo, Hamlin and Ray Ruby expressing the insecticide peptide Topaz (a code name to protect IP), under constitutive and phloem specific (SCAmpP-3) promoters were evaluated by detached leaf assay. From these, 12 lines (4 event of each genotype) showed significant ACP mortality and were selected to move up in the screening pipeline for HLB/ACP tolerance. Also, 24 Carrizo transgenic events highly expressing Onyx (a code name to protect IP), a peptide with antimicrobial and insecticide activity, were evaluate by DLA. The 5 Onyx lines showing high ability to kill ACP (to 83% mortality) were selected for further evaluation. These strongly performing lines were replicated as rooted cuttings (103 Onyx and 189 Topaz plants) that will soon enter greenhouse trials. The available Onyx transgenic material is being expanded through production of additional constitutive (13 Valencia and 6 Ray Ruby) and phloem specific lines (25 Carrizo, 5 Hamlin, 5 Valencia, and 13 Ray Ruby). Down regulated DMR6 Carrizo, either by stable expression of specific hairpin RNA or by specific Cas9-sgRNA were generated, cloned, and are being assessed. Since DMR6 is a broad immune suppressor, downregulated plants were first evaluated for Canker resistance as a quicker assay. After Xanthomonas challenge, both transgenic and gene edited DMR6 lines showed reduced bacterial titers and statistically significant reductions in Canker symptoms compared to controls; including some lines that developed no symptoms whatsoever. As an effort to accelerate development of non-transgenic HLB resistant plants through gene editing, we transformed early flower transgenic plants (carrying FT-SCFV gene) with the DMR6 targeting CRISPR construct. The early flowering trait will greatly decrease the time needed to produce an edited but non-transgenic offspring. A set of 30 plants resulted from this gene stacking effort will be evaluated for the presence of both genes. Objective 3, ScFv Constructs: ACP inoculated greenhouse studies on 5 scFv lines have been completed with transgenics showing significantly reduced CLas titer (up to 250x reduction) and a significantly higher incidence of no CLas rDNA amplification in roots and leaves compared to WT. These lines have been grafted with WT Ray Ruby scions and are undergoing field trials at Picos farm. The first assessment was completed in March with leaf tissue collected and awaiting CLas quantification. An additional 129 rooted cuttings are propagated for follow up plantings with grafted Hamlin scions. A second greenhouse trial testing new lines (150 plants from 12 lines) have been bud inoculated with HLB+ RL. A group of 370 plants for a third greenhouse trial has been propagated with the first 54 plants to reach a suitable size ACP-inoculated using the improved protocol. Plant tissue from both second and third (partial) greenhouse trials has been collected and processed; now awaiting qPCR analysis for CLas quantification. Objective 4, Screening Development and Validation: A protocol using a high throughput ACP homogenate assay for selecting lytic peptides for activity against CLas is now in use. A manuscript on the protocol has been published in Plant Methods (DOI: 10.1186/s13007-019-0465-1) to make it available to the HLB research community. Transgenic Nicotiana benthamiana plants expressing His-6 tagged variants of the chimeras TBL, TPK, PKT and LBP have also been generated to produce sufficient protein extracts for use in exogenous applications. The detached leaf ACP-feeding assay (DLA) has undergone several small revisions to improve sensitivity and maintain consistent inoculation; adjusting feeding period and ACP numbers. We have also expanded the analysis of ACP bodies to include quantification of other major endosymbionts (Wolbachia, Carsonella and Profftella) to better investigate the activity of peptides causing CLas mortality. An array of phloem specific citrus genes has been selected for investigation as potential reference genes to improve detached tissue and plant sampling techniques. Multiple sets of sequence specific qPCR primers for each gene have been synthesized and tested for efficiency. Six varieties of citrus have been propagated for endogene stability testing. A phloem specific endogene would allow normalizing to phloem cells, more accurately evaluating CLas titer relative to Citrus DNA and potential therapeutic effects. The best performing lines of Mthionin, chimeras `74′,`188′, TPK, TBL and scFv transgenics have been submitted to Florida Department of Plant Industry for shoot-tip graft cleanup in preparation for future field studies. Hamlin/Mthionin transgenics (3 lines), Carrizo/Mthionin (2 lines) and Carrizo/’74’ (1 line) have been returned certified clean. Objective 5, Transgenic Product Characterization: Experiments are also underway track the movement and distribution of transgene products using antibodies and affinity tagged protein variants. CLas+ RL have been grafted as scions onto MThionin expressing Carrizo as a platform to test peptide movement and effects across the graft union. Transgenic Carrizo lines expressing His6 and/or Flag tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), PKT (20 lines), scFv-InvA (22 lines) and scFv-TolC (18 lines) have been generated and expression confirmed by RT-qPCR. Total protein samples have been extracted from His-tagged transgenic lines and sent to our CRADA partner for testing.
Field variety trials are a simple but effective tool to test plant horticultural performance under different environmental conditions and enhance the commercial adoption of new cultivars. Large-scale, rapid implementation of HLB-tolerant cultivars depends on reliable data, and the Millennium Block is addressing the need of establishing field plantings to generate regional, updated information for the Indian River IIR) Citrus District. The project has two objectives: (i) Assess performance of new grapefruit cultivars with certain rootstocks under HLB endemic conditions in the IR district and (ii) Evaluate the influence of UFR and other recent rootstocks on grapefruit, navel, and mandarin in the IR in comparison to legacy/standard rootstocks.
Trials tested: T1) grapefruit cultivars on three rootstocks, T2) 36 rootstocks with Ray Ruby grapefruit as the scion, T3) 30 rootstocks with Glenn 56-11 navel orange, and T4) 30 rootstocks with UF-950 mandarin.
We planted 3,400 trees in Sep/2019, 1,100 trees in Aug/2020, and 400 trees on Jun/2021. Only grapefruit trees on UFR-8 are going to be planted in Fall since not ready yet. Controlled-release poly coated fertilizer was applied in Sep/2019, Jan, May and Sep/2020, Jan and May/2021. Irrigation controller, sand media filtration system and water flow meter were installed. The group met with the certified crop advisor to develop a spray program schedule based on time of year and conditions to be applied as determined by IPM scouting. Hoop boom was modified to spray young trees with higher accuracy, reducing the waste of agrochemical products. We created a location map and began production and distribution of QR tags to be used with scanner codes during data collection in the field.
Tree height, tree width in two positions (E-W/N-S), and trunk diameter were measured in three central trees from each experimental plot in Feb, July and Nov/2020, and Feb/2021, and canopy volume calculated. have been presented at the 2020 and 2021 ASHS annual conferences. The study is on schedule, and the following summary data reflect the best and worst treatments in each trial during the first 18 months of growth.
Tree size data from Feb/2021 show some scion-rootstock combinations are exhibiting significant differences in canopy volume. In Trial 1, Pummelette UF-5-1-99-2 grapefruit on US-942 was 6x larger (4.7 m3) than US 1-83-179 grapefruit hybrid on Sour Orange (0.8 m3) (P<0.0001). In Trial 2, Ray Ruby grapefruit on US-812 was ~3x larger (4.1 m3) than on Orange 16 (1.3 m3) (P<0.0001). In Trial 3, Glenn F-56-11 navel orange on C-22 was ~2x larger (2.9 m3) than on Willits (1.2 m3) (P<0.0001). In Trial 4, UF-950 mandarin on US-942 was ~2x larger (2.5 m3) than on WGFT+50-7 (1.2 m3) (P<0.0001). Cultivars grafted on different rootstocks have no differences in tree phenological stage. HLB is spreading in the field and individual trees are getting infected as visual HLB symptoms are seen on some treatments in each trial. Leaf samples were taken in Mar/21 for HLB diagnostic and bacterial titer quantification. Asian citrus psyllids, Diaprepes root weevils, whiteflies, and other insects are less abundant in the field, except for leaf miners, which caused severe damage due to the excessive rainfall and wind gusts that made pesticide application challenging during this quarter. Nonetheless, tree growth has not been significantly affected by these pests. Leaf samples for determining HLB incidence were taken from a pool of trees from each experimental plot in May and Sept/2020, and Feb/2021 and sent to the Southern Gardens lab for analysis; on average, all samples tested negative (no trees with Ct values <32) but there are several positive trees with visible symptoms. Fruit phenology, pests and diseases have been monitored monthly. Canopy thickness, canopy color and HLB incidence have been measured quarterly in all experimental plots. The Ferrarezi Lab organized a very successful drive-thru field day to showcase the results to growers on 10/09/2020 with 49 attendees (limited by covid-19 regulations). An estimated 24,000 acres of citrus were represented at the event (70% of the current grapefruit industry acreage, highlighting the importance of the event and my program engagement with the industry). Attendees came from local and neighboring counties including St. Lucie, Charlotte, and Okeechobee. Another field day took place on 12/10/2020 with 4 large growers and industry leaders. Overall, trees are showing morphological differences among scions/rootstocks. Although HLB is spreading in the field, symptomatic trees are building up vigorous canopies. Longer-term evaluation is required to identify the most promising scions and rootstocks to determine their profitability and capability of meeting grower and market needs.
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