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 with 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 from the second planting are collected and undergoing CLas quantification. Results from the 2nd generation plantings detailed below also show the included mThionin lines with significantly improved growth and CLas titer numbers compared to WT when grafted to commercial scions. 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 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 including increased ACP mortality (up to 95% from TBL, 96% from PKT, 60% from BLT and 70% from TPK) and decreased transmission of CLas into the leaf. Analysis of the ACP endosymbionts show a reduction in titer that may indicate a mode of action for ACP mortality (outlined in Objective 4). The best performing of these lines have been moved forward into greenhouse trials as described below. 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. Two follow on greenhouse studies are underway. The first is an improved ACP-inoculated trial with the best performing `74′ and `188′ lines. The second is a larger graft-inoculated study directly comparing the best performing 3rd generation chimeras (TPK and TBL) with the earlier 1st (Mthionin) and 2nd (`74′ and `188′) generation lines. For this, a total of 420 grafted plants (all on WT Carrizo rootstock for uniformity) were made and grafted with CLas+ RL for uniform transmission. In the earlier 6 and 9 month samplings, plants were beginning to test CLas positive and 12 month assessments are underway. An additional two rounds of rooted cuttings (totaling >1600 replicates) have been made from those same lines for paired ACP-inoculated greenhouse and field trials. ACP-inoculations will be done in free flying cages for a 2 month availability window, a protocol developed in a parallel project that has yielded up to a 100% infection rates. Both studies will begin as soon as the plants are large enough. The best performing PKT and LBT lines have also been replicated (>200 plants) for following the same greenhouse to field study pipeline, with graft inoculations as soon as the plants are of sufficient size. 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. At the 1 year assessment, Hamlin plants with transgenic Carrizo rootstocks showed noticeably reduced CLas titer (in scion midribs) when compared to those on conventional rootstocks. WT Hamlin on `74′ Carrizo had a 10x reduction in CLas titer, WT Hamlin on MThionin a 15x reduction and WT Hamlin on `188′ a 144x reduction. Plants grafted on MThionin-Carrizo also showed better trunk diameter, tree height and canopy volume compared to WT/WT grafts. Because of the high ACP mortality seen in detached leaf assays, field plantings of all chimera are also being surveyed to determine the rates of successful ACP colonization on new flush. An additional planting of transgenic scions (Hamlin) on WT rootstocks is also being prepared to complement these plantings. 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) and down-regulation of DMR6 genes (to enhance disease resistance). 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, 27 transgenic Carrizo and Hamlin lines highly expressing Onyx (a code name to protect IP), a peptide with both antimicrobial and insecticide activity, were evaluate by DLA. The 5 Onyx-Carrizo lines showing high ability to kill ACP (to 83% mortality) were selected for further evaluation. Strongly performing lines were replicated as rooted cuttings (250 Onyx and 189 Topaz plants) that will soon enter greenhouse trials. The available Onyx transgenic material is being further expanded through production of additional constitutive (13 Valencia and 6 Ray Ruby) and phloem specific lines (25 Carrizo, 5 Hamlin, 8 Valencia, and 13 Ray Ruby). Down regulated DMR6 Carrizo, either by stable expression of specific hairpin RNA (for RNA interference) or by Cas9-sgRNA genome editing were generated, cloned, and are being assessed. Since DMR6 is a broad immune suppressor, down-regulated plants could be expected to have heightened immune response. To test this, they were first evaluated for potential Canker resistance which is both a quicker assay and a desirable trait in its own right. After Xanthomonas challenge on detached leaves, both hairpin and gene edited DMR6 lines showed reduced bacterial titers, statistically significant reductions in Canker symptoms and higher expression of some down-stream defense genes compared to WT controls. Several transgenic lines developed no disease symptoms whatsoever. A planting of 190 trees (including WT controls) from the best performing genome edited line (40 trees) and hairpin knockdown lines (150 trees) is being prepared; the plants have been replicated as rooted cuttings and will go into the field once regulatory requirements are met. An amendment to the current BRS permit has been submitted for their addition and is currently under review. As an effort to accelerate development of non-transgenic HLB resistant plants using gene editing, we transformed early flower transgenic plants (carrying FT-Mcherry or FT-scFv gene) with the DMR6 targeting CRISPR construct. Cotyledons from FT-Mcherry line #3 seeds were transformed and 51 putative positive plants are now growing in soil. These plants are already showing strong early flowering phenotypes, but must still be confirmed for presence of the CRISPR transgene. Seeds from 4 more FT-Mcherry lines are now being germinated for additional transformations. 50 additional plants from FT-scFv/CRISPR stacking transformations are also in soil awaiting confirmation. The early flowering trait will greatly decrease the time needed to produce an edited but non-transgenic offspring. A set of 30 confirmed positive plants from this gene stacking effort will be evaluated. 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 one year assessment is now complete and several scFv lines are showing significantly improved growth characteristics, leaf tissue was collected and is 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 in both whole plant and detached leaf assays. 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. To further investigate the impact of transgenic or exogenous chimeral application, a DLA protocol to assess changes in ACP inoculability is also being developed. A manuscript detailing the protocol and findings is being prepared. 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, DMR6 knockdowns 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), Carrizo/’74’ (1 line), Carrizo/’188′ (1 line) and Hamlin/’74’ (1 line) have been returned certified clean. Objective 5, Transgenic Product Characterization: Experiments are also underway to 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 (34 lines) and scFv-TolC (30 lines) have been generated and confirmed by RT-qPCR. Total protein samples have been extracted from His-tagged transgenic lines and sent to our collaborator for testing.
This project has two objectives: (i) Assess the performance of new grapefruit cultivars with certain rootstocks 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. There are four trials: Trial 1) 18 grapefruit cultivars on three rootstocks; Trial 2) 32 rootstocks with `Ray Ruby’ grapefruit as the scion; Trial 3) 31 rootstocks with ‘Glenn F-56-11′ navel orange as the scion; and Trial 4) 31 rootstocks with `UF-950 mandarin as the scion.There are now 4,900 trees in the grove. The final 90 grapefruit trees on UFR-8 rootstock are growing slowly in the nursery and are expected to be planted in February or March 2022. In August 2021, leaf and soil samples were collected from each experimental plot to properly manage fertilizer requirements. Controlled-release, polycoated fertilizer was applied appropriately in early November 2021 based on lab results. All trees were treated biweekly with appropriate agrochemicals to manage canker, Asian citrus psyllid, mites, and citrus leafminers.Tree height, tree width in cardinal directions (E-W/N-S), and trunk diameter were measured on the three middle trees in each experimental plot in October 2021 to quantify canopy volume and tree size. Some scion/rootstock combinations are exhibiting significant differences in canopy volume. In Trial 1, `Pummelette UF-5-1-99-2′ grapefruit on US-942 is 5.6X larger (11.2 m3) than `US 1-83-179’ grapefruit hybrid on sour orange (2.0 m3). In Trial 2, grapefruit on UFR-15 is 2.6X larger (8.9 m3) than on 46×20-04-6 (3.4 m3). In Trial 3, navel orange on US-802 is 1.8X larger (5.3 m3) than on Willits, UFR-16, and UFR-1 (2.9 m3). In Trial 4, mandarin on US-942 is 2.6X larger (6.2 m3) than on 46×20-04-6 (2.4 m3). Many trees are developing vigorous canopies despite HLB symptoms. Results will be presented at the annual Florida Citrus Show in January 2022 in Fort Pierce, FL. Visual HLB symptoms are apparent on approximately 25% of the total tree canopy volume for most of the plots. However, trees look vigorous and maintain a bright green foliage. Leaf samples from Spring flush were collected in September 2021 and sent to Southern Gardens Labs for quantifying CLas titer. The ct values ranged 35-40 (CLas-free >32). Leafminer damage was substantial during the summer months but decreased recently with cooler temperatures. Nevertheless, tree growth has not been significantly adversely affected by these pests due the biweekly application of agrochemicals.Trees are bearing their first fruits, and we expect to collect this information in the first quarter of 2022, which will serve well to establish potential yields and marketability as well as HLB damage. Long-term evaluation is needed to identify the most promising scions and rootstocks to determine their profitability and capability of meeting grower and market needs.The Citrus Horticulture Lab organized the annual drive-through Millennium Grove Field Day on 14 October 2021 to showcase the results to growers and stakeholders. More than 50 attendees coming from local and neighboring counties (St. Lucie, Okeechobee, Polk) toured the grove, received an explanatory handout, and met with personnel involved in the project.
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 preliminarily assembled. The transcriptome data for two of our target genomes, produced previously by a commercial vendor on their PacBio platform was found to be inadequate. So, we are in the process of finding another vendor to redo the long-read sequencing on the most recent PacBio platform. Hi-C sequencing of the 7 remaining target genomes is underway; two were completed. These Hi-C results will be combined with the PacBio assemblies using Hi-Rise software, to produce improved chromosome scale assemblies. RNA samples of these 7 additional genomes have been prepared from young and old leaf tissue 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. However, we are attempting to increase the types and numbers of tissues sampled to have a fuller representation of the gene content of each genome.Finally, we have collaborated with the USDA Germplasm Repository for Citrus and prepared genomic DNA from more than 120 citrus species, varieties, and relatives for enrichment and sequencing of specifically targeted genes. Sequencing libraries have been prepared and are in que for processing. Results from this effort will provide important insights for the evolution and domestication of select genes that are important for citrus resistance or susceptibility to Huanglongbing and other diseases. The results will also provide template gene sequences for genome editing for Huanglongbing resistance.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Seed from a first group of rootstock crosses was harvested and planted in the calcareous/Phytophthora soil as the first step in the `gauntlet’ screen; parents included several previously selected but unreleased HLB-tolerant rootstocks, as well as some of the UFRs, HLB-tolerant pummelos, and US-897 and US-942. Seed were harvested from a second group of crosses, using LB8-9 Sugar Belle® as a seed parent with pollen from various hybrids of Poncirus trifoliata with citrus accessions, Citrus ichangensis (Ci), different Cleopatra mandarin x Ci hybrids, and a Palestine sweet lime x Ci hybrid; these will be planted next into the `gauntlet’ screen. In collaboration with researchers at IFAPA in Spain, new information has been generated regarding performance of selected UFRs and other unreleased rootstock hybrids from our program in response to drought and flooding, Phytophthora, and salinity. Three new rootstock candidates were entered into the Parent Tree Program, including the first LB8-9 Sugar Belle® x trifoliate orange hybrid selected through the `gauntlet’ pipeline.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. We removed previously planted and tested scions from our program, grown in the Trailer Park block, that do not warrant further scrutiny. We planted 60 new scion selections from the program that have gone through the DPI PTP cleanup and certification; these include true oranges and orange-like hybrids, grapefruit and hybrids, mandarins, lemons, pummelos, and acid fruit. All trees are on UFR-5 rootstock. Following extensive phenotyping of a replicated planting of hybrids between Monreal Clementine and an accession of Citrus latipes (perhaps the most HLB-tolerant citrus), we have found at least two hybrids that remain PCR-negative after 6 years under high pressure in the field. They fruited last season and produced large fruit, somewhat resembling sweet orange but with high acidity. Pollen was collected from these and used to make crosses this past spring with two low-acid selections in our breeding program. Several thousand seeds were produced and will be planted soon. We plan to screen the seedlings with markers previously developed for fruit quality, acid content, and potential HLB tolerance. We completed micrografting somaclone seedling-derived populations of early maturing (January), high soluble solids OLL sweet orange clones to UFR-4 rootstock liners, in efforts to generate an even earlier maturing OLL clone. More than 100 individual seedlings successfully grafted to UFR-4 were stepped up to 4×4 citripots for spring planting at St. Helena. Parent Tree Program scion entries included 2 HLB-tolerant Valencia mutants produced by irradiation and selected from a small, replicated trial, and 1 September-maturing mandarin hybrid. Finally, 125 triploid hybrids, including sweet orange-like, mandarins and grapefruit, were prepared for field planting at the CREC. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We have more than 70 5-year-old-trees of `Marathon’ mandarin on sour orange, that set huge crops of fruit this season. Although all trees have HLB, there are few to no obvious disease symptoms in fruit, leaves, or canopy, demonstrating a high degree of tolerance thus far. We have followed closely their performance, and individual trees yielded more than 300 pounds of fruit. A first harvest was made in late September, and another planned for October. We will determine fruit size distribution, and we will follow post-harvest behavior and fruit quality. 4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. Several new genetic constructs have been developed using newly identified citrus specific promoters (phloem and root tissue), and new putative disease resistance genes, or downstream genes. Transgenic plants have been produced with some of these constructs, and additional transformation experiments are underway with several sweet oranges, grapefruit, and rootstocks. Finally, we have completed metabolomic studies, in collaboration with Dr. Y. Wang, to gain insight into the underlying mechanisms of HLB-tolerance and sensitivity; please see doi: 10.3389/fpls.2021.710598 and doi: 10.1021/acs.jafc.1c02875.
The objectives of this project 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 Obj 1: Transgenic rootstocks (n=12) that were budded with non-transgenic scion were side grafted with HLB infected budwood and maintained in the greenhouse. 6 months following infection trees were tested for the presence of HLB and were also evaluated for PR1 gene expression. PR1 is a SAR marker. We observed that all scions (with transgenic rootstock or non-transgenic control) were infected within a year of inoculation. There was no statistical difference between the treatments and control for the first 18 months. In several transgenic rootstock lines, Ct values did not decline at the same rate as controls after 18 months of infection. In several transgenic rootstock – non transgenic scion combinations, there was also an enhanced expression of the PR1 gene, which indicated an active defense mechanism. At the termination of the project, all lines with enhanced PR1 gene expression were alive, albeit infected. 4 of the 12 controls died while 6 more exhibited enhanced HLB symptoms. Only one control could be considered an outlier with mild HLB symptoms at the termination of the project. Obj 2: This objective was in progress at the termination of the project with rootstock lines clonally propagated in the mist bed and ready to be budded with non-transgenic scion. Obj 3: 61 transgenic lines (Carrizo, US942) with different genes stacked with NPR1 were produced by the mature transformation lab. Most of these tested were determined to produce adequate transprotein. At the termination of the project, all lines were being sized up in the greenhouse for clonal propagation. Additionally, several select lines were budded onto standard trifoliate rootstocks for field planting. This was being planned for seed production. Obj 4: This objective – field based under USDA BRS permit could not be initiated due to termination of the project
True sweet oranges: With the passing of Louise Lee, the trial block containing the OLL somaclone seedling population is in jeopardy of being sold for development. Efforts to rescue the most promising clones from these trial blocks continued. Multiple selected OLL and Vernia clones have been propagated under permit from DPI, and will be planted at St. Helena. Potential HLB tolerance/resistance from ‘gauntlet’ rootstock candidates: Rootstock sprouts were recovered from 12 superior gauntlet trees that had the tops cut off to induce sprouting for rootstock recovery. All have been successfully grafted to recover the 12 diverse rootstock genotypes (seed trees of these selection were not available). Following clean-up, these rootstocks will be propagated by cuttings and TC micropropagation for avanced trials. 10 more promising rootstock selections were provided to Agromillora for TC micropropagation initiation. These include 3 more promising gauntlet SugarBelle hybrids (one SugarBelle x trifoliate orange 50-7 hybrid), and hybrids of [salt tolerant HBPummelo x Shekwasha] with trifoliate orange 50-7 and x639. Agromillora has already generated 3500 shoots from the promising S10xS15-12-25 gauntlet rootstock hybrid previously introduced. This collaboration with Agromillora will greatly accelarate the planting of important stage 2 rootstock trials. CREC Trailer Park Trial: 10 trees each of 65 promising new scions available in the Parent Tree Program (selected based on feedback from Fruit Displays and breeder intuition), all on UFR-5 rootstock, were wrapped, planted and mapped. Trees were grown at Southern Citrus Nursery. Overage trees will be planted at 3 other diverse locations. This will provide a valuable resource to our new hire Dr. John Chater regarding advanced trials and commercialization of the best selections.Rootstock candidate identified from Strang/Gapway trial. In November of 2019, we planted approximately 100 HLB+ Valencia trees on selected rootstocks (good trees left over from a CRDF-funded greenhouse nutrition/rootstock study) in the Gapway grove near the CREC, under permit from DPI. Most of the trees were successfully established. This summer we identified one superior tree, that even with a 24 ct value before planting, grew off twice as fast as any of the other trees, and already set a good crop of fruit, and showed no HLB symptoms. We cut this tree to induce rootstock sprouts, and the rootstock genotype has been recovered. SSR marker analysis shows that the rootstock is from x639, but flow cytometry analysis suggests that there has been a deletion, as the tree repeatedly shows less than the typical diploid amount of DNA. Further investigation is underway. St. Helena: Following removal of all under-performing trees, the irrigation system was repaired and the grove prepared for the major resetting operation. Approximately 600 reset trees are ready to plant, including a new population of Vernia somaclone-derived seedlings, rescued promising selected OLL and Vernia somaclone seedling-derived clones from the Orie Lee trials in St. Cloud (mentioned above, permission granted from DPI to plant rescued sweet orange genotypes at this location), new sweet orange/rootstock combinations, and some new early and mid-season orange-like hybrid scion candidates. Planting is expected to begin this quarter. CREC Block 16: a new population of 175 protoclones of EV-1 and EV-2 were planted in efforts to identify a more robust clone of Early Valencia, and possibly a seedless clone. At present, the available EV clones are showing an HLB response similar to Hamlin regarding fruit drop. Field Trial Data Collection, etc.: Tree height data was collected from the Duda, Peace River and CREC Teaching Block trials. Seed fruit from UFR and a few other promising rootstock selections was harvested for seed extraction (seed to be extracted by Southern Citrus Nursery). Fruit was sampled from all scion trial blocks, and promising early-maturing selections were identified and included in the October Fruit Display – data forthcoming. Data analysis and entry onto the Rootstock Data Website: annual updates included: Premier Indian River grapefruit trial, Greene Citrus lemon trial, IMG navel & grapefruit/rootstock trial, Duda Vernia/rootstock trial, St. Helena rootstock Survey trial, and CREC scion/rootstock trial. Trial data from 20 additional trials is being prepared for website updates, including 8 new trials not yet posted. Initiated creation of a database using Microsoft Access with information from all field trials.
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. 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. We have successfully adapted the adenine base editors (ABE) to modify the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit and Hamlin sweet orange. Inoculation of the TATA-edited plants with the canker pathogen Xanthomonas citri subsp. Citri (Xcc) demonstrated that the TATA-edited plants were resistant to Xcc. In addition, cytosine base editors (CBE) was successfully used to edit the acetolactate synthase (ALS) gene of Carrizo citrange, a hybrid of Citrus sinensis `Washington’ sweet orange X Poncirus trifoliata. Editing the ALS genes conferred resistance of Carrizo to the herbicide chlorsulfuron. Two ALS-edited Carrizo plants did not show green florescence although the starting construct for transformation contains a GFP expression cassette. We performed PCR amplification for Cas9 gene in the mutant plants and found that Cas9 gene was undetectable in the herbicide resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression.
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 were performed:
(1) A total of 27 transgenic rootstock plants were planted into the field on October 8, 2021. These transgenic plants include eight transgenic Carrizo lines that express three different disease resistance genes. The transgenic rootstocks were replicated and grafted with Valencia. The transgenic plants that were transplanted in 2019 and earlier in 2021 were examed. The plants grow well in the field and one plant from the 2019 planting has shown HLB symptoms. Tissues will be collected in the fall for CLas titer assay.
(2) To test if the citrus 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mutant (called EPSPS TIPS) could provide tolerance to glyphosate, we have transformed a T-DNA construct carrying the citrus EPSPS TIPS mutant gene into Arabidopsis. Seeds from the first generation (T1) plants were screened on kanamycin plates to identify single T-DNA insertion lines. Six independent single T-DNA insertion lines were identified. We are currently screening for homozygous plants with the seeds collected from T2 plants and will test their glyphosate tolerance once homozyous lines are obtained.
(3) The citrus genome encodes several putative nicotinamide adenine dinucleotide-binding receptors. Nicotinamide adenine dinucleotide-binding activities of two of the putative receptors were tested once. These putative receptors appeared to have binding activity, though the result needs to be confirmed. CLas-inoculated transgenic citrus plants expressing the Arabidopsis nicotinamide adenine dinucleotide-binding receptor were maintained in the greenhouse and no HLB symptom has been developed. We plan to graft the transgenic scions onto sweet orange rootstocks for easier detection of HLB resistance or tolerance.
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. We are working on completing the analysis of data from trials 2 and 3 to be included for publication of three years of field data. The most time-consuming task is the analysis of root images captured with the minirhizotron system, which is still ongoing. 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.We continued our monthly root growth measurements with the rhizotron imaging system and their analysis. We are still working on the PCR and ELISA assays.Much of the time in this quarter was spent on devising a methodology to simulate wind-induced uprooting for our upcoming evaluations. We practiced on similar-sized trees at our research center to test different strategies and finalized a method that uses winching to pull trees while measuring and recording the exact force over time; this method will be combined with videotaping each excavation event. 2. Please state what work is anticipated for next quarter: We will conduct the pull-over (to simulate wind-induced uprooting) of trees in our 2-year-old field trials. Prior to pulling-over the trees, biometric data will be collected (tree height, canopy volume, trunk diameters) and leaves will be collected for nutrient analysis and CLas detection. After pulling-over the trees, we will severe the root crowns for detailed analysis in the lab (to examine the relationship of root structure and anchorage strength). 3. Please state budget status (underspend or overspend, and why): Approximately 75% of funds have been spent, which is 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.Tree measurements for the Hamlin trials were completed.The statistical analysis of some of the data is still in progress. Data are being prepared for open access publication. Objective 2. Develop outreach to transfer information to growers and other industry clientele.Three-year data of the Valencia trials were presented in August at the Citrus Expo in Fort Myers. 2. Please state what work is anticipated for next quarter: We will continue with the data analysis and preparation of 3-year Valencia data for open access publication. We will prepare for the Hamlin harvest. 3. Please state budget status (underspend or overspend, and why): Approximately 80% of funds have been spent, which is underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.
Update for this quarter:Site management and field trials are progressing well. Fall assessments of the USDA transgenic and conventional plantings are underway (trial # described below; #8, #9, #10, #11, and #15). Stover lab transgenic plantings are also undergoing a survey to assess ACP colonization rates. Detached leaf assays showed high ACP mortality (>90%) after 7 days of feeding on these and similar plants, which may translate to significant changes to the field ACP populations. Two additional plantings of sweet oranges expressing disease resistance genes designed by UF researchers were made this quarter as replicates of those made earlier in the season (#13). All regulatory protocols for the care and disposal of transgenic material are being properly observed. Recent quarters:A significant USDA-funded infrastructure project has been completed, fully renovating the water management systems and significantly improving storm and flood protection. An additional permit has been approved (AUTH – 0000043620, effective 12/17/2020) for material with confidential business information(CBI) for a project led by R. Shatters. The primary BRS permit (AUTH – 0000043619) has been renewed and amended to include a new construct from UF. The annual site review from APHIS/BRS has been conducted. With the recent planting of four new transgenic trials, the transgenic site is operating at full capacity. The UCRiverside-led trifoliate and trifoliate hybrid trial has concluded, a manuscript regarding identified HLB-tolerance is in preparation; and these trees can be removed as needed to make space available for future plantings. Stover analyzed data on canker incidence for this trial. Notable results: 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. We continue investigation of potential pollen flow from transgenic trees. A selection of FF-5-51-2 trees slightly more than 1000 ft from the US-802 planting, are self-incompatible, mono-embryonic and flower concurrently. If no pollination from transgenic trees is seen in 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. Trial plantings:1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the USDA permits.2) Under the Stover permit, a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. 4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project.5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and analysis will continue to be conducted under a new NIFA grant.6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).7) In a project led by Fred Gmitter, there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo.10) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls.11) Multiple plantings with grafted trees of l Hamlin, Valencia and grapefruit scions on transgenic rootstock expressing antimicrobial citrus-thionin and bacterial recognition domain fusion proteins (219 trees with controls) as a collaboration between USDA and the New Mexico Consortium.12) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type.13) Planting from Zhonglin Mou of UF that includes transgenic grapefruit (31 plants) and sweet orange (60 plants) scions expressing two different resistance genes and grafted on WT swingle rootstocks; as well as non-transgenic controls. 14) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State15)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
1. Please state project objectives and what work was done this quarter to address them: This report covers the period of June 1, 2021 – August 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). We have inoculation tests completed using graft transmission and Asian citrus psyllid (ACP) transmission of hte pathogen (Candidatus Liberibacter asiaticus, or CLas), and a natural infection trial in a grove at USHRL. The major accomplishment in this quarter was that we received data returned form Southern Gardens on all the infected trees from our greenhouse tests and those from a field trial natural inoculation. These are PCR data showing CLas titers in the infected plants. 2. Please state what work is anticipated for next quarter:We will use the next quarter to analyze the data on CLas titers in all our test trees and compare transgenics with controls. 3. Please state budget status (underspend or overspend, and why):The project is running under budget due to support received from Penn State for graduate student Chad Vosburg. We plan to request an additional no-cost extension to cover publication costs only into 2022.
Juvenile Tissue Citrus Transformation Facility (JTCTF) stayed open during the third quarter of 2021. However, the facility was idled for two weeks in August due to loss of employees and throughout that period did not perform any experiments. Within the last three months, JTCTF received five orders. None of these orders are associated with research grants and will be paid for. The work on these orders is starting. Requests for transgenic Valencia plants represented high majority and came at the time when JTCTF had in stock some Valencia fruits harvested earlier this year. These fruits started rotting in storage and extracted seeds had internal pathogens invisible to naked eye. This lead to contamination of starting material for our experiments, waste of lab resources, and slowdown in satisfaction of orders. For one of the orders, we were provided contaminated Agrobacterium culture which also resulted in contamination and disposal of plant material in multiple experiments. One of the orders was placed for transgenic Citrus macrophylla plants that are extremely difficult to produce and we are in the process of optimizing this process. For two orders placed earlier this year, we were not able to produce transgenic shoots and in consultation with the client the work with Valencia oranges was suspended and will begin with Duncan grapefruit. At the end of July, the work on multiple orders associated with the USDA grant where I am still an active participant stopped because of departure of employee who worked on them. Newly appointed person is expected to continue this work in October. For the initial phase of its existence as an EBA, JTCTF is envisioned to operate having two employees and a manager. One employee is full-time employee and the other is part time employee working three days a week. This labor force should be able to process 15-20 orders per year. In the month of September, JTCTF operated in this mode but was slowed down because of above-described circumstances. Because of the period of idling as well as time lost because of the contamination of experimental material, JTCTF has had very low productivity in the third quarter. Only six transgenic plants were produced including four Duncan grapefruit plants and two Valencia plants. What also contributed to this outcome is that for multiple earlier orders associated with the USDA grant, JTCTF produced transgenic shoots that were neither grafted nor rooted into plants. Results from these experiments served as a basis for design of new experiments. The Business office staff at CREC continued to provide assistance in processing of payments for the services JTCTF offers.
1. Please state project objectives and what work was done this quarter to address them: The objectives of this project are to produce disease resistant, commercially & agronomically desirable, mature citrus transgenics, intragenics, & non-GMO gene edited trees using Agrobacterium & biolistics as a service for research & commercialization. The research focus of this project is to improve transformation efficiencies, so that the mature citrus protocols become more productive, decrease prices for scientists, & contribute more to financial self-sufficiency of our lab. Our facility received an order from California to overexpress a citrus peptide in Hamlin, Kuharske & US942 using biolistic transformation to produce intragenics (all citrus sequences). The plants produced using this technology are not regulated & can be provided to the growers rapidly after field testing. We have started co-bombardments in these cultivars using other vectors until we receive the peptide. If this technology is successful, I will contact other scientists who might be interested in plant improvement using this method. For another scientist, 19 intragenics were produced using Agrobacterium transformation of mature EV1 scions. Twenty-four flowering shoots were produced & micrografted onto rootstock for another scientist. This quarter, a total of 52 transgenic or intragenic shoots were produced in EV1 or Kuharske and micrografted for different scientists. RNAi was also used to silence genes in another transformation experiment & a manuscript was published. A manuscript describing a protocol to increase transformation efficiency in mature citrus was submitted for review. Another manuscript will be submitted soon about a new intragenic selectable marker. 2. Please state what work is anticipated for next quarter: Co-bombardments to overexpress the citrus peptide will continue. We must finish some orders & new orders for Valencia, Early Valencia I, & Kuharske are pending. We just completed an experiment with Agrobacterium mediated gene editing & the results are pending. 3. Please state budget status (underspend or overspend, and why): CRDF funding is sufficient, but my EBA has been revised to better fund salaries. I am not sure how long it will take to move through the UF system. In the interim, Dr. Rogers funds the rest of our salaries.
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 from the second planting are collected and undergoing 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 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, 96% from PKT, 60% from BLT and 70% from TPK) and decreased transmission of CLas into the leaf. The ACP endosymbionts show a reduction in titer that may indicate a mode of action for ACP mortality when feeding on CLas-killing transgenics (outlined in Objective 4) as these are also gram-negative bacteria and ACP are dependent on their nutritional contributions. The best performing of these lines have been moved forward into greenhouse trials as described below. 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 have been collected and are undergoing qPCR analysis. A larger greenhouse study is also underway to directly compare the best performing 3rd generation chimeras (TPK and TBL) with the earlier 1st (Mthionin) and 2nd (`74′ and `188′) generation lines. A total of 420 grafted plants (all on WT Carrizo rootstock for uniformity) were made and graft- inoculated with CLas+ RL to ensure high transmission. The first 9 months of growth assessment and leaf collections are complete, with samples undergoing qPCR analysis. An additional two rounds of rooted cuttings (totaling >1600 replicates) have been made from those same lines for paired ACP-inoculated greenhouse and field trials. The best performing PKT and LBT lines have also been replicated (>200 grafted plants) for greenhouse studies using these same protocols, which will begin as soon as the plants are of sufficient size. 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. Because of the high ACP mortality seen in detached leaf assays, field plantings of all chimera are now undergoing a survey to determine the rates of successful ACP colonization. An additional 200 plants (transgenic Hamlin on WT rootstocks) are being produced to 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) and down-regulation of DMR6 genes (to enhance disease resistance). 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, 27 transgenic Carrizo and Hamlin lines highly expressing Onyx (a code name to protect IP), a peptide with both antimicrobial and insecticide activity, were evaluate by DLA. The 5 Onyx-Carrizo lines showing high ability to kill ACP (to 83% mortality) were selected for further evaluation. Strongly performing lines were replicated as rooted cuttings (250 Onyx and 189 Topaz plants) that will soon enter greenhouse trials. The available Onyx transgenic material is being further expanded through production of additional constitutive (13 Valencia and 6 Ray Ruby) and phloem specific lines (25 Carrizo, 5 Hamlin, 8 Valencia, and 13 Ray Ruby). Down regulated DMR6 Carrizo, either by stable expression of specific hairpin RNA (for RNA interference) or by Cas9-sgRNA genome editing were generated, cloned, and are being assessed. Since DMR6 is a broad immune suppressor, down-regulated plants could be expected to have heightened immune response. To test this, they were first evaluated for potential Canker resistance which is both a quicker assay and a desirable trait in its own right. After Xanthomonas challenge on detached leaves, both hairpin and gene edited DMR6 lines showed reduced bacterial titers, statistically significant reductions in Canker symptoms and higher expression of down-stream defense genes compared to WT controls. Several transgenic lines developed no disease symptoms whatsoever. A planting of 20 trees (including WT controls) from the best performing genome edited line is being prepared; the plants have been replicated as rooted cuttings and will go into the field once BRS permit amendments are completed. As an effort to accelerate development of non-transgenic HLB resistant plants using 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 in both whole plant and detached leaf assays. 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. To further investigate the impact of transgenic or exogenous chimeral application, a DLA protocol to assess changes in ACP inoculability is also being developed. 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, DMR6 knockdowns 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.
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