Plant Improvement


Establish early-stage field trials for new Huanglongbing-tolerant canker-resistant transgenic scions

Report Date: 05/13/2021   Project: 18-017   Year: 2021

Establish early-stage field trials for new Huanglongbing-tolerant canker-resistant transgenic scions

Report Date: 05/13/2021
Project: 18-017   Year: 2021
Percentage Completion: 0.41
Category: Plant Improvement
Author: Zhonglin Mou
Sponsor: Citrus Research and Development Foundation

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, the following activities have been conducted: (1) Transgenic citrus plants for field trials were maintained in the greenhouse. These plants will be transplanted into the field on May 20, 2021. (2) Cloning the citrus gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). We have constructed a vector based on citrus DNA sequences for generating cisgenic or intragenic citrus plants. However, the transformation efficiency of the vector is extremely low. We plan to develop a transformation selection method based on citrus DNA sequences to facilitate this process. It has been shown in other plant species that an EPSPS variant is able to provide tolerance to glyphosate. We thus cloned the citrus EPSPS gene full-length coding sequence from sweet orange total cDNA. This gene will be mutated to create a similiar citrus EPSPS variant. (3) Optimizing conditions for analyzing nicotinamide adenine dinucleotide-binding activities of a group (10) of citrus lectin receptor kinase proteins using Monolith NT.115. Four different buffers with a pH value in the range of 5-8 have been tested. Although binding activity was detected for some of the proteins using the binding test model, a reliable Kd value has not been achieved. Our goal is to find the functional citrus nicotinamide adenine dinucleotide-binding recptor for generation of intragenic or cisgenic citrus plants. We are testing the transgenic citrus plants expressing the Arabidopsis nicotinamide adenine dinucleotide-binding receptor for HLB resistance/tolerance.



Optimization of the CRISPR technology for citrus genome editing

Report Date: 05/05/2021   Project: 18-025   Year: 2021

Optimization of the CRISPR technology for citrus genome editing

Report Date: 05/05/2021
Project: 18-025   Year: 2021
Percentage Completion: 0.87
Category: Plant Improvement
Author: Nian Wang
Sponsor: Citrus Research and Development Foundation

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. We have further optimized LbCas12a. 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. Genome modified lines in canker are being regenerated. We are editing 6 putative HLB susceptibility genes for sweet orange.  



Part B - The UF/CREC-Citrus Improvement Program's Field Trial Evaluations (Complementary to Part A- the UF/CREC Core Citrus Improvement Program)

Report Date: 05/04/2021   Project: 18-039-C   Year: 2021

Part B - The UF/CREC-Citrus Improvement Program's Field Trial Evaluations (Complementary to Part A- the UF/CREC Core Citrus Improvement Program)

Report Date: 05/04/2021
Project: 18-039-C   Year: 2021
Percentage Completion: 80
Category: Plant Improvement
Author: Jude Grosser
Sponsor: Citrus Research and Development Foundation

True sweet oranges: New OLL sweet orange candidates repeated for early-mid season maturity (consecutive years), including OLL-FB-4-13 (mentioned in previous report) and OLL-FB-9-33.  OLL-FB-4-13, OLL-FB-9-33, and OLL-FB-1-22 were selected for inclusion in the CRDF scion trial, and were entered into the Parent Tree Program (using special slots provided by Ben Rosson and Kristen Aslan).  A new selection OLL-FB-4-08 was identified with January 1 maturity (first time fruiting). Potential HLB resistance from ‘gauntlet’ rootstock candidates:  qPCR was run on leaves sampled from more than 625 gauntlet rootstock trees at the USDA Picos Farm, and 54% tested questionable or negative for active infection.  Among these, qPCR was run on roots on 60 of the best performing trees.  Among these, 13 trees had root ct values of 40, meaning no bacteria was detected in the roots. Two of these also had leaves that tested negative and 4 others questionable. We also identified 19 gauntlet rootstocks in this group that were negative for active root infections, and among these 4 had no active infections in the leaves, and 9 had questionable leaf infections.  Considering the severe fruit quality problems, especially from young trees the past 2 seasons in Florida, we also focused on identifying the best of these producing high brix fruit right off the bat.  3 potentially resistant rootstocks were identified producing high brix fruit: N+HBPxOrange19-12-3 (a backcross of UFR-4 onto it’s mom), B11-R5-T25-11-3 (a Flying Dragon hybrid with potential for high density plantings), and A+HBPxCH+50-7-12-11 (also tree-size controlling, with UFR-6 pollen parent).   Several gauntlet rootstocks are also showing an apparent tolerance mechanism, as they show consistently good tree health, but had higher CLas titers in the roots than the trees above; these include a few hybrids made with SugarBelle.  Identification of probable zygotic rootstocks at St. Helena showing good HLB tolerance:  5 trees on apparent zygotic rootstocks were identified producing 3 – 3-5 boxes of Vernia/Valquarius fruit per tree with 12 – 12.5 brix.  One is a tetraploid from Orange 12 (Nova+HBP-derived), two from HBPummelo x Shekwasha, and two from HBPummelo (open pollination).  One rootstock genotype has been recovered, and scaffold roots of the other selections have been cut in efforts to retreive the rootstock germplasm.   Fruit quality data was collected from the Trailer Park trial – two combinations of 3.75 year-old trees made 6.0 and 5.9 lbs solids per box: Valencia B9-65/UFR-4 and OLL-4/UFR-4, respectively. Yield data was collected from the St. Helena trial.  There was no issue with fruit drop, and lbs. solids across more than 50 rootstocks averaged around 6.0 lbs. solids per box.  Overall yield increased slightly.  These positive results were attributed to the continued year-round use of CRF containing an enhanced micronutrient package. Yield data was collected from the Peace River Valencia/rootstock trial, and the Duda trials.  Data analysis and entry onto the Rootstock Data Website: annual updates included: Duda Valencia APS rootstock trial, Duda Vernia rootstock trial, Smoak Valencia rootstock trial, Bryan Paul Doe Hill multi-scion rootstock trial, and the IMG navel/grapefruit rootstock trial. Data analyses for subsequent trial updates was performed for (in progress): Banack multi-scion rootstock trial, Hidden Golf Trailer Park trial, Wheeler Bros. scion/rootstock trial, Orie Lee OLL clone/rootstock demonstration trial, Teaching Block scion/rootstock trial, Bryan Paul Doe Hill Grove multi-scion/rootstock trial, Citra (PSREU) scion/rootstock trial, Hammond IR Minneola rootstock trial, IR Marsh grapefruit/rootstock trial, the St. Helena rootstock survey trial, and trial and the Peace River Valencia/rootstock trial.    



Fort Pierce Field Test Site for Validating HLB and/or ACP Resistance

Report Date: 04/15/2021   Project: 18-085C   Year: 2021

Fort Pierce Field Test Site for Validating HLB and/or ACP Resistance

Report Date: 04/15/2021
Project: 18-085C   Year: 2021
Percentage Completion: 0.75
Category: Plant Improvement
Author: Ed Stover
Sponsor: Citrus Research and Development Foundation

Update for this quarter:A substantial infrastructure renewal project is underway at the Picos location on USDA base funding. A full renovation of the water management system has been completed and will provide improved storm protection. Drainage channels were re-dug and cleared of vegetation. Culverts were inspected and replaced if damaged. The road surfaces have also improved to provide better vehicle access. Tree maintenance and field trials have received priority support under current conditions. Full assessments have been conducted on USDA transgenic plantings by onsite personnel. UF collaborators have been permitted into the test site; samples and data have been collected. A manuscript detailing results from the canker resistance assessment of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside was accepted for publication in Hortscience as “Incidence of Asiatic Citrus Canker on Trifoliate Orange and its Hybrid Accessions in a Florida Field Planting.” This site is also participating in a trial program to use drone based aerial photography for mapping and HLB assessment. The primary BRS permit, which covers the transgenic materials planted by Z. Mou, J. Jones, T. McNellis as well as USDA scientists has been renewed (AUTH – 0000043619  effective 1/27/2021).  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. 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. 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. A manuscript has been prepared and submitted from this data. 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) Grafted trees of conventional sweet orange and grapefruit scions on transgenic rootstock expressing antimicrobial citrus-thionin and bacterial recognition domain fusion proteins (165 trees with controls) as a collaboration between USDA and Innate Immunity.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) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State14)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.   



High-Throughput Inoculation of Citrus Germplasm for HLB Resistance Screening

Report Date: 04/15/2021   Project: 18-065C   Year: 2021

High-Throughput Inoculation of Citrus Germplasm for HLB Resistance Screening

Report Date: 04/15/2021
Project: 18-065C   Year: 2021
Percentage Completion: 0.78
Category: Plant Improvement
Author: Ed Stover
Sponsor: Citrus Research and Development Foundation

This quarter:The Bowman lab has established three new experiments with grafted Valencia trees on groups of rootstocks which were inoculated with ACP during the quarter (Janyuary to March 2021).  Each experiment compares replications on nine different rootstocks. For that, 189 trees  were inoculated with 3,780 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.). Six detached leaf assays experiments, envolving individual 290 leaves, were inoculated  using 2,900 Clas infected ACPs in this quarter. Transgenic material tested in DLAs were of three constructs (ONYX and two Chimerical Thionin), and a total of 46 independent events were tested alongside WT controls.The balance of Covid-19 restricted lab hours was spent processing the leaf and ACP samples in preparation for CLas qPCR.  We continue to see substantial ACP mortality from feeding on CLas-killing transgenic leavesIn addition 600 CLas+ ACP were provided to Dr. YongPing Duan of USDA.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.  Previous quarter:Dean Gabriel of UF, and USDA scientists Kim Bowman, Ed Stover and G avin Poole have all run experiments totalling ~7,300 ACP. Samples have all been collected on-time from ongoing experiments. All samples collected, that have not been analyzed, have been processed for qPCR.   



Fort Pierce Field Test Site for Validating HLB and/or ACP Resistance

Report Date: 04/15/2021   Project: 18-085C   Year: 2021

Fort Pierce Field Test Site for Validating HLB and/or ACP Resistance

Report Date: 04/15/2021
Project: 18-085C   Year: 2021
Percentage Completion: 0.75
Category: Plant Improvement
Author: Ed Stover
Sponsor: Citrus Research and Development Foundation

Update for this quarter:A substantial infrastructure renewal project is underway at the Picos location on USDA base funding. A full renovation of the water management system has been completed and will provide improved storm protection. Drainage channels were re-dug and cleared of vegetation. Culverts were inspected and replaced if damaged. The road surfaces have also improved to provide better vehicle access. Tree maintenance and field trials have received priority support under current conditions. Full assessments have been conducted on USDA transgenic plantings by onsite personnel. UF collaborators have been permitted into the test site; samples and data have been collected. A manuscript detailing results from the canker resistance assessment of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside was accepted for publication in Hortscience as “Incidence of Asiatic Citrus Canker on Trifoliate Orange and its Hybrid Accessions in a Florida Field Planting.” This site is also participating in a trial program to use drone based aerial photography for mapping and HLB assessment. The primary BRS permit, which covers the transgenic materials planted by Z. Mou, J. Jones, T. McNellis as well as USDA scientists has been renewed (AUTH – 0000043619  effective 1/27/2021).  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. 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. 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. A manuscript has been prepared and submitted from this data. 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) Grafted trees of conventional sweet orange and grapefruit scions on transgenic rootstock expressing antimicrobial citrus-thionin and bacterial recognition domain fusion proteins (165 trees with controls) as a collaboration between USDA and Innate Immunity.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) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State14)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.   



High-Throughput Inoculation of Citrus Germplasm for HLB Resistance Screening

Report Date: 04/15/2021   Project: 18-065C   Year: 2021

High-Throughput Inoculation of Citrus Germplasm for HLB Resistance Screening

Report Date: 04/15/2021
Project: 18-065C   Year: 2021
Percentage Completion: 0.78
Category: Plant Improvement
Author: Ed Stover
Sponsor: Citrus Research and Development Foundation

This quarter:The Bowman lab has established three new experiments with grafted Valencia trees on groups of rootstocks which were inoculated with ACP during the quarter (Janyuary to March 2021).  Each experiment compares replications on nine different rootstocks. For that, 189 trees  were inoculated with 3,780 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.). Six detached leaf assays experiments, envolving individual 290 leaves, were inoculated  using 2,900 Clas infected ACPs in this quarter. Transgenic material tested in DLAs were of three constructs (ONYX and two Chimerical Thionin), and a total of 46 independent events were tested alongside WT controls.The balance of Covid-19 restricted lab hours was spent processing the leaf and ACP samples in preparation for CLas qPCR.  We continue to see substantial ACP mortality from feeding on CLas-killing transgenic leavesIn addition 600 CLas+ ACP were provided to Dr. YongPing Duan of USDA.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.  Previous quarter:Dean Gabriel of UF, and USDA scientists Kim Bowman, Ed Stover and G avin Poole have all run experiments totalling ~7,300 ACP. Samples have all been collected on-time from ongoing experiments. All samples collected, that have not been analyzed, have been processed for qPCR.   



Citrus Transformation Facility remains integral part of the efforts to combat HLB disease through production of transgenic Citrus plants

Report Date: 04/14/2021   Project: 18-066C   Year: 2021

Citrus Transformation Facility remains integral part of the efforts to combat HLB disease through production of transgenic Citrus plants

Report Date: 04/14/2021
Project: 18-066C   Year: 2021
Percentage Completion: 0.75
Category: Plant Improvement
Author: Vladimir Orbovic
Sponsor: Citrus Research and Development Foundation

Starting with the 2021, Juvenile Tissue Citrus Transformation Facility (JTCTF) started operating as an EBA unit. As a result of this transition, relationship between the clients and JTCTF is redefined in a sense that clients will be paying for the work JTCTF has done and not for the produced plants. Towards this goal, JTCTF has created a price list for the services it offers. In accordance with changed status of JTCTF, administration at CREC has established an account that will be used for financial transaction associated with facility’s operation. In the first three months of 2021, Juvenile Tissue Citrus Transformation Facility (JTCTF) accepted five new orders. Four of those orders were for transgenic sweet orange plants and one for Duncan grapefruit plants. One of the orders is associated with the existing USDA grant, while four other orders will be paid for. Certain number of orders that were expected because JTCTF was designated subcontractor have not materialized because those clients have not started working on projects for which they received funding as they had difficulties in hiring personnel.At this time, JTCTF has four employees but one has not worked since December of 2020 because of unresolved immigration status. Another employee is working during the night which has allowed us to return to the level of attendance of pre-COVID period. As a result, the facility operated at about 75% capacity. Between January and April, JTCTF produced 10 plants. Those include five Duncan grapefruit plants transformed with HGJ87 and HGJ88 plasmids. The rest are sweet orange plants transformed with pXH-H1 and pXH-H2 plasmids.  



Citrus Transformation Facility remains integral part of the efforts to combat HLB disease through production of transgenic Citrus plants

Report Date: 04/14/2021   Project: 18-066C   Year: 2021

Citrus Transformation Facility remains integral part of the efforts to combat HLB disease through production of transgenic Citrus plants

Report Date: 04/14/2021
Project: 18-066C   Year: 2021
Percentage Completion: 0.75
Category: Plant Improvement
Author: Vladimir Orbovic
Sponsor: Citrus Research and Development Foundation

Starting with the 2021, Juvenile Tissue Citrus Transformation Facility (JTCTF) started operating as an EBA unit. As a result of this transition, relationship between the clients and JTCTF is redefined in a sense that clients will be paying for the work JTCTF has done and not for the produced plants. Towards this goal, JTCTF has created a price list for the services it offers. In accordance with changed status of JTCTF, administration at CREC has established an account that will be used for financial transaction associated with facility’s operation. In the first three months of 2021, Juvenile Tissue Citrus Transformation Facility (JTCTF) accepted five new orders. Four of those orders were for transgenic sweet orange plants and one for Duncan grapefruit plants. One of the orders is associated with the existing USDA grant, while four other orders will be paid for. Certain number of orders that were expected because JTCTF was designated subcontractor have not materialized because those clients have not started working on projects for which they received funding as they had difficulties in hiring personnel.At this time, JTCTF has four employees but one has not worked since December of 2020 because of unresolved immigration status. Another employee is working during the night which has allowed us to return to the level of attendance of pre-COVID period. As a result, the facility operated at about 75% capacity. Between January and April, JTCTF produced 10 plants. Those include five Duncan grapefruit plants transformed with HGJ87 and HGJ88 plasmids. The rest are sweet orange plants transformed with pXH-H1 and pXH-H2 plasmids.  



Comparison of field performance of citrus trees on rootstocks propagated by seedlings, cuttings, and tissue culture

Report Date: 04/12/2021   Project: 18-028C   Year: 2021

Comparison of field performance of citrus trees on rootstocks propagated by seedlings, cuttings, and tissue culture

Report Date: 04/12/2021
Project: 18-028C   Year: 2021
Percentage Completion: 0.65
Category: Plant Improvement
Author: Ute Albrecht
Sponsor: Citrus Research and Development Foundation

 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.  Leaves were collected in trials 2 and 3 for CLas detection; samples are being analyzed. Trials 2 and 3 third-year horticultural data collection (tree size, health ratings, etc.) was completed. We  also counted and collected fruits for fruit quality analysis and yield determination in trials 2 and 3. There were very few fruit in trial 2, but 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.   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 and other data collection. We will continue with the statistical analysis and interpretation of all data collected. A Citrus Industry Magazine article will be prepared sumarizing findings of this project.  3. Please state budget status (underspend or overspend, and why): Approximately 65% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.  



Evaluation of citrus rootstock response to HLB in large-scale existing field trials

Report Date: 04/12/2021   Project: 18-029C   Year: 2021

Evaluation of citrus rootstock response to HLB in large-scale existing field trials

Report Date: 04/12/2021
Project: 18-029C   Year: 2021
Percentage Completion: 0.65
Category: Plant Improvement
Author: Ute Albrecht
Sponsor: Citrus Research and Development Foundation

 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.We conducted additional HLB foliar disease symptom and canopy color ratings at both Valencia trials for seasonal comparison. We also collected leaves for additional nutrient analysis and CLas detection.Valencia fruit samples were collected at the Basinger location for fruit quality analysis in advance of the harvesting. Fruit quality analysis was completed for these samples at the CREC pilot plant.We are working on the leaf CLas analysis.We are continuing with the statistical analysis of all new data and are working on a manuscript for publication of three years of Valencia data. Objective 2. Develop outreach to transfer information to growers and other industry clientele.Nothing to report in this quarter.  2. Please state what work is anticipated for next quarter: Fruits will be collected from the Valencia trees at the Lake Wales (Camp Mack) location for fruit quality analysis, and yield will be assessed.  We will continue with the data analysis. A rootstock seminar including trial updates will be given in June at SWFREC (or virtual, depending on the Covid-19 situation).  3. Please state budget status (underspend or overspend, and why): Approximately 64% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.  



Comparison of field performance of citrus trees on rootstocks propagated by seedlings, cuttings, and tissue culture

Report Date: 04/12/2021   Project: 18-028C   Year: 2021

Comparison of field performance of citrus trees on rootstocks propagated by seedlings, cuttings, and tissue culture

Report Date: 04/12/2021
Project: 18-028C   Year: 2021
Percentage Completion: 0.65
Category: Plant Improvement
Author: Ute Albrecht
Sponsor: Citrus Research and Development Foundation

 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.  Leaves were collected in trials 2 and 3 for CLas detection; samples are being analyzed. Trials 2 and 3 third-year horticultural data collection (tree size, health ratings, etc.) was completed. We  also counted and collected fruits for fruit quality analysis and yield determination in trials 2 and 3. There were very few fruit in trial 2, but 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.   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 and other data collection. We will continue with the statistical analysis and interpretation of all data collected. A Citrus Industry Magazine article will be prepared sumarizing findings of this project.  3. Please state budget status (underspend or overspend, and why): Approximately 65% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.  



Evaluation of citrus rootstock response to HLB in large-scale existing field trials

Report Date: 04/12/2021   Project: 18-029C   Year: 2021

Evaluation of citrus rootstock response to HLB in large-scale existing field trials

Report Date: 04/12/2021
Project: 18-029C   Year: 2021
Percentage Completion: 0.65
Category: Plant Improvement
Author: Ute Albrecht
Sponsor: Citrus Research and Development Foundation

 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.We conducted additional HLB foliar disease symptom and canopy color ratings at both Valencia trials for seasonal comparison. We also collected leaves for additional nutrient analysis and CLas detection.Valencia fruit samples were collected at the Basinger location for fruit quality analysis in advance of the harvesting. Fruit quality analysis was completed for these samples at the CREC pilot plant.We are working on the leaf CLas analysis.We are continuing with the statistical analysis of all new data and are working on a manuscript for publication of three years of Valencia data. Objective 2. Develop outreach to transfer information to growers and other industry clientele.Nothing to report in this quarter.  2. Please state what work is anticipated for next quarter: Fruits will be collected from the Valencia trees at the Lake Wales (Camp Mack) location for fruit quality analysis, and yield will be assessed.  We will continue with the data analysis. A rootstock seminar including trial updates will be given in June at SWFREC (or virtual, depending on the Covid-19 situation).  3. Please state budget status (underspend or overspend, and why): Approximately 64% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.  



Upgrading Citrus Genome Sequence Resources: Providing the Most Complete Tools Necessary for Genome Editing Strategies to Create HLB Resistant Cultivars

Report Date: 04/02/2021   Project: 18-010   Year: 2021

Upgrading Citrus Genome Sequence Resources: Providing the Most Complete Tools Necessary for Genome Editing Strategies to Create HLB Resistant Cultivars

Report Date: 04/02/2021
Project: 18-010   Year: 2021
Percentage Completion: 0.35
Category: Plant Improvement
Author: Fred Gmitter
Sponsor: Citrus Research and Development Foundation

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. We performed Hi-C sequencing with two genomes and incorporated these data with PacBio sequence of one of our target genomes resulting in an improved chromosome scale assembly. The two parental chromosomes of the target genome were 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; transcript sequencing for annotation (i.e., identify all the genes within the genome) currently is in progress. 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), and this information will become more important as we test different sweet orange mutants exhibiting enhanced tolerance of HLB. A manuscript is in preparation on this work. As an example of the utility of these quality-improved new citrus genome assemblies, we have examined the polyembryonic allele associated with a MITE transposon insertion in the promoter of the CitRKD1 gene in the mandarin lineage, for sequence completeness. Previous reference assemblies either lacked this allele (the MITE insertion obviously is absent in the monoembryonic Clementine reference) or were missing important sequence in the promoter region of the gene (in the sweet orange reference assembly from China). Full knowledge of the polyembryony gene in citrus is important because it is the basis for rootstock propagation by nucellar seedlings, and it is an impediment to breeding by preventing the ability to make hybrids using polyembryonic seed parents. More importantly, this example demonstrates the quality of our assemblies; lacking promoter sequence for important HLB-resistance candidate genes could lead to CRISPR editing failures, which is exactly what we proposed to prevent through successful achievement of the objectives of this project. We completed and published on the first ever high-quality reference genome of Poncirus trifoliata using the same pipeline used for this project, and a manuscript was published in The Plant Journal (https://doi.org/10.1111/tpj.14993), and the sequence has been released to the global citrus research community through Phytozome and the Citrus Genome Database. 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 and were highlighted in our published manuscript, so other researchers may also begin to explore their potential.  



Part A - The UF/CREC Core Citrus Improvement Program (Complementary to Part B - The UF/CREC Citrus Improvement Program's Field Trial Evaluations)

Report Date: 03/31/2021   Project: 18-011   Year: 2021

Part A - The UF/CREC Core Citrus Improvement Program (Complementary to Part B - The UF/CREC Citrus Improvement Program's Field Trial Evaluations)

Report Date: 03/31/2021
Project: 18-011   Year: 2021
Percentage Completion: 0.7
Category: Plant Improvement
Author: Fred Gmitter
Sponsor: Citrus Research and Development Foundation

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, to establish advanced trials to compare our best rootstock selections with industry standards. 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 24 locations. We planted seed from 2020 diploid and tetraploid rootstock crosses into calcareous, high pH soil inoculated with two species of Phytophthora, the first step of the `gauntlet’ screening. We stepped up rooted cuttings from more than 50 gauntlet rootstock candidates to citripots in preparation 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. Embryo rescue from 37 interploid crosses made using selected HLB tolerant plants in 2020 was completed, including 10 crosses for red grapefruit improvement, 10 for sweet orange improvement, and 10 for mandarin improvement. Shoots have been generated from many of the excised embryos, and flow cytometry was used to verify triploidy among some of the hybrid populations. Micrografting of the earliest to germinate normal shoots has begun and will continue through the next quarter. Seeds harvested from 2 diploid crosses made for sweet orange improvement were planted and have germinated. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. Evaluations of existing breeding populations in the field were somewhat restricted by UF COVID travel regulations, but we nonetheless went through our materials and made selections for further evaluations and testing. Four new HLB tolerant red grapefruit hybrids, one sweet-orange like hybrid, and three seedless easy to peel mandarins were selected and submitted to the DPI Parent Tree Program for cleanup and production of certified budwood for future trials. 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 by numerous sources 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.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. 5. Other related activities. We have continued testing the alternating temperature chamber regime to produce CaLas- and CTV-free lines of promising new scion and rootstock candidates for propagation and to hasten their use in advanced field trials. Five Vernia seedling selections, one grapefruit hybrid, and ten rootstocks were treated, and all were PCR negative, and will now be tested for CTV.