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.
A large-scale trial of greening-tolerant citrus cultivars is addressing the need of Indian River growers to know what are the best rootstocks and scions for growing fresh fruit. The 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. In June 2021, 400 tress were planted to add to the 4,500 trees planted the previous two years. The final 90 grapefruit trees on UFR-8 rootstock will be planted in September 2021. 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 July 2021. 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 three middle trees in each experimental plot in June 2021 to quantify canopy volume and tree size. Results to date were presented by MSc student Martin Zapien at the 2021 American Society for Horticultural Sciences in Denver, CO, in August 2021. Martin placed 3rd in the Scholar’s Ignite Competition. Tree size data from June 2021 show some scion/rootstock combinations are exhibiting significant differences in canopy volume. In Trial 1, Pummelette UF-5-1-99-2 grapefruit on US-942 is 9.1X larger (7.3 m3) than US 1-83-179 grapefruit hybrid on sour orange (0.8 m3). In Trial 2, grapefruit on UFR-15 is 2.5X larger (5.5 m3) than on UFR-17 (2.2 m3). In Trial 3, navel orange on US-802 is 2.2X larger (3.7 m3) than on Willits (1.7 m3). In Trial 4, mandarin on C-22 is 2.2X larger (4.3 m3) than on 46×20-04-6 (2.0 m3). Longer-term evaluation is needed to identify the most promising scions and rootstocks to determine their profitability and capability of meeting grower and market needs. Greening disease is spreading in the field as visual greening symptoms are on average 25% (up to 60%) of the total tree canopy volume. Leaf samples for quantifying CLas titer were collected in March 2021 and sent to Southern Gardens for analysis. Trees that are CLas-free (ct values >32) and CLas-infected (ct values of 26-32) can be found in the same plots, but many symptomatic trees are developing vigorous canopies. Many trees are bearing their first fruits. The incidence of Asian citrus psyllids, Diaprepes root weevils, whiteflies, leafminers, and citrus canker was quite apparent during the summer due to abundant rainfall and the emergence of a large amount of flush. Leafminer damage has been substantial. Nonetheless, tree growth has not been significantly adversely affected by these pests due the biweekly application of agrochemicals. The second field day to exhibit this project will take place on October 14, 2021.
Evaluation of Newly Developed and/or Released Citrus VarietiesSept 2021 Investigator: Nabil KillinyIn this project we are profiling the new scions and rootstocks for their tolerance to HLB by studying the metabolite content by GC-MS, and challenging new varieties with psyllids and HLB.Progress on Objectives: Objective 1To understand the mechanism behind the tolerance of different varieties toward HLB. The comparison between the varietal responses will allow us to determine the mechanism of tolerance to CLas. This quarter we continued our focus in four areas: 1) new mandarin hybrid Lucky (SugarBelle x Nava x Osceola); 2) new Valencia varieties, 3) CUPS new grapefruit varieties, 4) rootstock evaluations. In addition, we tried again to graft Marathon Mandarin onto Swingle and Carrizo unsuccessfully (0/10). We have not been able to get even a small number of Marathon from the citrus nurseries, so we assume they are also having problems with propagation.Findings: 1) For the mandarin hybrid we call Lucky and its parents, Sugar Belle and Nava × Osceola, we completed the volatile organic compound (VOC) analysis to determine if Lucky will have any of the chemical properties of Sugar Belle, which is considered tolerant to HLB. We found it has slightly more total VOCs than either of the parents, and about the same as `Valencia’ sweet orange, which we used for a comparison. We detected 50 VOCs and the dominant leaf volatiles were sabinine, linalool, trans-ß-ocimene, and ß-caryophyllene in the mandarin hybrid. Like other mandarins, they are low in d-limonene and almost completely lack neral and geranial, the aldehydes that dominate `Valencia’. In addition, we repeated the ACP choice experiment beginning in June using CLas-infected ACPs. The experiment just concluded with the final samples being collected on 9/3/21. We are looking for any chemical clues that indicate an induction of defense compounds after infestation with ACP. To achieve this, leaf samples from the 15 plants were collected 5 days, two weeks, and one month after adding psyllids to the cages. Thereafter, monthly leaf samples were taken in parallel with ACP population counts. The leaf samples need to be processed now for volatiles and HLB status. Currently there are no HLB symptoms, but the trees will be followed closely to determine how quickly they develop symptoms and when HLB can be detected by PCR. 2) For the evaluation of the new sweet oranges Valquarius and Vernia, the leaf samples for analysis of volatiles have been run on the GC-MS and integrated. The data analysis was completed. Qualitatively, the three sweet orange varieties are very similar, but the total VOC content was highest in Valencia, followed by Valquarius, then Vernia. We identified 47 VOCs in the hexane-extracted leaves of the three varieties. We did not detect any unique compounds among the three varieties. Those in the highest concentrations included sabinene, linalool, neral, geranial, ß-elemene, a-sinensal, and phytol. Many differences (37/47) were statistically significant because of the high content found in the traditional Valencia variety as compared to the two newer varieties. We have previously associated higher levels of VOCs to ACP attraction, so there may be a slight advantage to the newer varieties in having a lower VOC content. We plan to perform ACP choice tests on the new varieties by pairing them with Valencia in a new choice test apparatus we made for this purpose. 3) New grapefruit variety from CUPS – UF914 is being evaluated and compared to traditional Duncan, Ruby Red and Ray Ruby varieties for volatile and non-volatile metabolite content. UF-914 is a low furanocoumarin variety so special attention is being paid to these compounds. We have detected several coumarins and sterols in the grapefruit extracts and quantification is in progress. Objective 2 4) To understand the role of rootstocks in citrus tolerance to HLB. The comparison between rootstock metabolites will allow us to determine the best scion/rootstock combinations for tolerating CLas. a. The rootstock seeds from the USDA (US-802, 812, 897, 942, 1283, 1284, 1516) for metabolite profiling and HLB/nematode screening were moved outside to encourage growth, repotted into larger pots, and are now about 6 inches tall and 7 months old. We hope they will be ready for evaluations soon. We plan to challenge these rootstocks with ACP to determine their response to HLB.b. The grapefruit leaf samples collected from CUPS included Duncan on four rootstocks, and Ray Ruby on two rootstocks. We will make comparisons to see if there are any differences in the volatile and non-volatile metabolites due to the different rootstocks.
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. We now have the transcriptome data for two of our target genomes, using both Illumina and PacBio sequencing platforms, and genome annotation (i.e., identify all the genes within the genome) is continuing. Hi-C sequencing of the 7 remaining target genomes is underway; once completed these data will be integrated with the PacBio assembly, to produce improved chromosome scale assemblies. RNA samples of these 7 additional genomes have been prepared 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.In a related effort, our team recently compared 69 new east Asian genomes and other mainland Asian citrus to reveal a previously unrecognized wild sexual species native to Japan’s Ryukyu Islands: C. ryukyuensis, which hybridized with an ancient east Asian mandarin to produce Shekwasha (shiikuwasha) mandarin, a powerful source of HLB tolerance in rootstock breeding. Further, by studying the genomes of C. ryukyuensis-derived hybrids and other citrus. we traced the origin and spread of apomixis (nucellar embryony, a trait that is required for seed propagation of citrus rootstocks) from Mangshanyeju wild mandarins in China a few million years ago through most of the commonly known contemporary citrus types (orange, grapefruit, lemon, etc.). This work resulted in deeper understanding and new genome-based tools that can be exploited for two critically important traits in citrus genetic improvement, nucellar embryony and most importantly HLB tolerance; the research was published in Nature Communications in July 2021 (see https://doi.org/10.1038/s41467-021-24653-0). We used the PacBio Sequel platform to sequence full-length gene transcripts in the leaf tissues of sweet orange and trifoliate orange and reconstructed their leaf transcriptomes. We identified novel full-length transcripts that were not present in the published reference transcriptomes. We found that some NBS-encoding genes (nucleotide binding site genes, one typical class of disease resistance genes in plants) underwent alternative splicing. One alternatively spliced NBS transcript expressed in HLB symptomatic leaf and fruit of sweet orange, and another alternatively spliced NBS transcript was differentially expressed in CLas-infected trifoliate orange samples, suggesting that isoforms of some NBS-encoding genes may play an important role in HLB tolerance of trifoliate orange, or alternatively HLB susceptibility in sweet orange. The new transcriptomes will be useful to identify candidate genes for disease resistance that have been missed in the published citrus genomes and transcriptomes.
Create new candidate hybrids. During this quarter, hybrids from previous cycles were selected, and propagation planned for the next cycle of Stage 1 field trials. New seed source trees were propagated for advanced selections and planted in the field. Based on new information on performance of different parental combinations in rootstock field trials, additional crosses among the best parental material are planned for next spring, as resources are available. Propagate and plant new field trials. Budwood increase trees of selected scions were grown, in preparation for budding trees for new rootstock trials. Trees for Stage 2 rootstock trials with Valencia and Hamlin on selected released rootstocks and the best of the next generation hybrids are being grown in the greenhouse in preparation for field planting in 2021. One new Stage 2 trial with Valencia scion and one new Stage 2 trial with grapefruit scion were planted in the field. Nursery trees for two new Stage 1 trials with 60 new rootstocks and Valencia were propagated in the greenhouse in preparation for field planting in fall 2021. Some planned propagation for new trials was delayed because of USDA institutional Coronavirus shutdown. Collect data from field trials. Extensive information on tree performance is collected from established field trials, and includes measurement of tree size, fruit crop, fruit quality, and pathogen titer, HLB symptoms, and assessments of tree health. Cropping data is collected during the time of scion harvest, and during this quarter none of the active trials have scions for which crop would normally be harvested. Assessments of tree health were completed on 23 trials during this quarter. All the backlog of fruit quality analysis (caused by institutional Coronavirus restrictions) from frozen juice samples in the previous season was completed, with full data on brix, acid, and color from 15 replicated trials. Because the data collection load from field trials was lower this quarter, additional effort was put into tree maintenance, and updates to irrigation and drainage systems in selected trial blocks.Evaluate effectiveness for seed propagation of new rootstocks and develop seed sources. Some of the newest hybrid rootstocks can be uniformly propagated by seed, but others cannot. As the best rootstocks are identified through field trials, seed sources are established and used to determine trueness-to-type from seed. Studies were continued this quarter to evaluate seed propagation for 20 of the most promising SuperSour hybrid rootstocks. Progeny groups were scored, and tissue samples were prepared for SSR analysis of progeny to determine frequency of nucellar polyembryony and uniformity from seed. This work is progressing more slowly than planned because of institutional Coronavirus restrictions. Cooperative work continues to compare field performance of rootstocks propagated by seed, cuttings, and tissue culture. Evidence indicates that performance of rootstocks is primarily determined by rootstock cultivar, and not much influenced by propagation method.Posting field trial results for grower access. The USDA rootstock trials produce large amounts of information that is useful to identify the most promising of the new hybrids, as well as comparative information on the relative performance of many commercially available rootstocks. During this quarter, updated trial summaries were prepared for uploading to the website https://www.citrusrootstocks.org/, and a presentation on rootstock performance in field trials was given at the Florida Grower Citrus Show.Release of superior new rootstocks for commercial use. Release of new USDA rootstocks is based on robust data from multiple trees in replicated field trials over multiple years, including information on tree survival and health, canopy size, fruit yield and fruit quality, and observations on tolerance of disease and other biotic and abiotic threats. Outstanding performance in industry plantings from some of the previously released USDA rootstocks, especially US-942 and US-812, indicate the process is effective. Several of the 350 advanced Supersour rootstock hybrids in field trials are exhibiting outstanding performance in comparison with the commercial standard rootstocks. Information on some of the most promising of the next-generation USDA hybrid rootstocks was presented at the Florida Grower Citrus Show. Performance data continues to be collected, and it is anticipated that 2-3 of the most outstanding of these will be officially released in 2022-23.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Stepped up 160 candidate rootstock hybrids from the first step of the Gauntlet screen (high pH, calcareous soil inoculated with Phytophthora), from different crosses into citripots; crosses were made using LB8-9 (Sugar Belle®) as a seed parent with either trifoliate hybrids or salt tolerant sour orange (pummelo-mandarin hybrids) types. Three new Gauntlet rootstock candidates, all showing very good HLB tolerance and possibly resistance, were entered into the PTP. These hybrids arose from similar crosses as described above, combining genetics from pummelo, Shekwasha mandarin, LB8-9, and trifoliate orange. All 3 hybrids are showing excellent health and ability to suppress CLas replication in their root systems and in grafted Valencia scion. 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 are in the process of removing previously planted and tested scions from our program that do not warrant further scrutiny, grown in the Trailer Park block. Newly available spaces will be planted in late summer with 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 have been grown on UFR-5 by a commercial nursery. Somaclone seedling derived populations of January-maturing OLL sweet orange clones are being propagated on UFR-4 rootstock, to attempt to produce an even earlier maturing clone; to date >100 individual seedlings have been propagated.3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We have explored some new approaches to quantifying tree responses to HLB, in addition to the previously used subjective approaches. Specifically, we have begun measuring photosynthetic parameters and leaf canopy indexes, to produce repeatable and reliable quantitative data in support of further genetic analyses of tolerant types. Analysis of collected data is underway, in consultations with plant physiologists familiar with the techniques and data interpretation. This work will improve the precision with which we can define HLB tolerance genes. 4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. Using the quantitative data described in 3. above, we are conducting additional GWAS to validate previously identified, or to identify new, genomic regions associated with HLB tolerance and/or sensitivity. 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 have been begun. Finally, the very early response of citrus to the CLas, vectored by infective ACP, was evaluated for the first time, thus allowing the changes in gene expression relating to the primary mechanisms of susceptibility and host -pathogen interactions to be studied, and without the secondary effects caused by the development of complex whole plant symptoms; please see doi: 10.3389/fpls.2021.635153.
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 this trial are underway, and permission was obtained from DPI (Ben Rosson) to propagate the rescued trees at the CREC. Clones being rescued include those showing earlier maturity, higher soluble solids, and better HLB tolerance. Potential HLB resistance from ‘gauntlet’ rootstock candidates: qPCR was completed on root samples from 45 additional promising gauntlet rootstock candidates. 18 rootstock candidates had ct values above 30, including 6 that had ct values of 40 (no bacterial detected). Among these, 5 rootstock candidates showed suppression of CLas in the Valencia scion; including 3 vigorous pummelo x latipes hybrids (13-76, 13-53 and 13-43), and two [Amblycarpa+HBPummelo] x [sour orange + rangpur] hybrids (13-15 and 13-12). Note that C. latipes and the sour orange+rangpur are highly HLB tolerant parents. Pathogen-free material of top gauntlet rootstocks sent to TC labs for micropropagation include potentially HLB resistant S10xS15-12-25, S11x50-7-16-12, S11x50-7-16-6, and A+HBPxCH+50-7-12-11; and potentially HLB tolerant LB8-9(SugarBelle)xS13-15-16, LB8-9(SugarBelle)xS10-15-9, S10xS15-12-34, and A+HBPxCH+50-7-12-39. Note that S10, S11, S13 & S15 are all salt tolerant pummelo/mandarin parents. Molecular marker analysis of the promising super-root mutant UFR-1 clone#28 (Fast 28, discovered by Beth Lamb at the Rucks TC lab) showed that it is a deletion mutant of UFR-1; this vigorous rootstock continues to look promising in the field with sweet orange and grapefruit scions. Additional liners are being propagated at the Rucks TC lab for advanced trials. Identification of probable zygotic rootstocks at St. Helena showing good HLB tolerance (mentioned in previous report): 6 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), one from Purple 2 (Nova+HBPxCleo+SO), two from HBPummelo x Shekwasha, and two from HBPummelo (open pollination). Since the staked-up scaffold roots did not sprout, we cut the tops (scion) off the trees as necessary to recover the rootstock genotypes. Two of the trunks have begun to sprout. We plan to generate pathogen-free material of these selections, followed byTC micropropagation to generate material for advanced trials. We also identified a truly stellar Valquarius tree on rough lemon; we are conducting a molecular marker test on the roots to see if the rootstock is a unique zygotic. If so, it will be treated as the above rootstocks. St. Helena: The entire trial was assessed tree by tree, and all under-performing trees were pink-flagged for removal. This has created approximately 2000 rotational spaces, and new rootstock candidates along with new early-mid season sweet orange candidates will be planted. Tree removal and replanting are expected to get underway this quarter. Field Trial Data Collection: Tree size data was collected from the following trials: Bryan Paul, Smoak, Greene River Citrus(lemon), Tom Hammond, Post Office and Peace River. Tree health assessment data was collected from the Mislevy trial. Data analysis and entry onto the Rootstock Data Website: annual updates included: Heller Bros., Peace River, Bryan Paul Doe Hill, Smoak, Post Office, and Tom Hammond. Trial data being uploaded and analyzed included data from the follwoing trials: St. Helena, Premier Citrus, Greene River, Lee Family Groves, IMG, Banack, Cutrale and Wayne Simmons.
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) A total of 85 plants including 65 transgenic plants and 20 non-transgenic control plants were planted into the field on May 20, 2021. These transgenic plants include replicates of three transgenic lines that have been shown to have robust tolerance to HLB in the greenhouse. Ten replicates of another transgenic line that expresses a different disease resistance gene were also included. This transgenic line has shown HLB tolerance in the greenhouse for more than eight years. In addition, we have eight transgenic Carrizo lines that express three different disease resistance genes. These lines have been replicated and grafted with Valencia. The three constructs was added onto the field trial permit. These plants will be transplanted into the field in the Fall of 2021. The transgenic plants that were transplanted in 2019 were examed. The plants grow well in the field and none of the plants has shown HLB symptoms. We plan to collect samples for CLas titer assay in this fall.
(2) The citrus gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was cloned and sequenced. The two conserved amino acids T177 and P181, which are responsible for glyphosate tolerance, were changed to isoleucine (I) and serine (S), respectively. The resulting citrus TIPS EPSPS gene was cloned into a T-DNA binary vector, which was introduced into Agrobacteria. To test if the citrus TIPS EPSPS gene can provide tolerance to glyphosate, we transformed it into Arabidopsis, since it will take shorter time to know the result in Arabidopsis. We will have plants for glyphosate tolerance test in the next quarter.
(3) Transgenic citrus plants expressing the Arabidopsis nicotinamide adenine dinucleotide-binding receptor were inoculated with CLas using psyllids. We are waiting for the HLB symptom development. Meanwhile, we repeated nicotinamide adenine dinucleotide-binding experiment for the citrus lectin receptor kinase proteins using Monolith NT.115. We plan to use radiolabeled nicotinamide adenine dinucleotide to confirm the binding results in the next quarter.
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