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, with CLas+ ACP feeding, as well as studying them in established greenhouse and field studies. Greenhouse studies (With 9 Carrizo lines and 4 Hamlin lines, 98 total plants with controls) include graft inoculation of Carrizo rooted cuttings with CLas+ rough lemon, no-choice caged ACP inoculation of Carrizo rooted cuttings, and no-choice caged ACP inoculation of Hamlin grafted on Carrizo with all combinations of WT and transgenic. Data collection continues from Mthionin field plantings. Results from the first round of field plantings (45 plants) of Mthionin transgenic Carrizo root-stock grafted with non-transgenic rough lemon show transgenics maintaining higher average CLas CT values (2.5 CT higher @ 18 months), but with a high degree of variability. A large second planting of Mthionin transgenics went into the ground in April 2019, including transgenic Carrizo with WT Hamlin scions (81 plants), transgenic Hamlin on non-transgenic Carrizo root-stock (108 plants) and WT/WT controls (16 plants). Scheduled assessments for both field plantings is being prioritized under current covid-19 pandemic conditions. The 24 month field assessment of the first planting and 12 month assessment for the second planting are completed. Leaf samples from both populations have been collected and are being processed for Clas quantification. Additional grafts of WT Hamlin and Ray Ruby scions to Mthionin root-stock have been made and are included in the imminent chimera planting discussed in Objective 2. The Mthionin construct has also been extensively transformed into Valencia, Ray Ruby and US-942 to provide transgenic material of these critical varieties. The first 51 putative lines are now in soil and are undergoing expression analysis. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, have been conducted and repeated for lines expressing chimera constructs TPK, PKT, CT-CII, TBL, BLT, LBP/’74’, `73′, and `188′ (as well as scFv-InvA, scFv-TolC) using adjusted protocols to improve sensitivity and transmission rates (See section 4). Further detached leaf assays are being run to compare the relative effectiveness between each generation of chimera constructs and to expand the number of lines tested from each. DLA testing has allowed us to identify lines from several constructs with significant effects on CLas transmission and even increased ACP mortality. Recent results include up to 95% mortality in ACP after 7 days feeding on detached leaves of the 3rd generation TBL transgenics and 70% for TPK. Lines from promising constructs have been moved forward into greenhouse studies based on DLA results, as noted below. Initial ACP inoculations conducted on 8 lines of citrus Thionin-lipid binding protein chimeras (`73′, and ’74’) showed a statistically significant reduction (13x) in CLas titer for `74′ transgenics vs WT in the CLas+ plants. However, many plants remained CLas negative at 6 months post infestation, indicating a low inoculation efficiency. All ACP inoculated greenhouse experiments are now using an improved protocol using a combination of smaller plants, more aggressive trimming and close observation to safely extend the caged ACP infestation time from 7 days to 21 without harming the plants. Additional greenhouse studies are also being prepared in parallel using bud inoculations. In June, 150 plants representing the best performing 7 lines of `188′ and 6 lines of `74′ were no-choice caged ACP inoculated using the new protocol. At 3 months, control plants tested positive at twice the rate of the earlier inoculation; 6 month tissue samples are now collected and processed, awaiting qPCR analysis. The large additional greenhouse study will directly compare the best performing 3rd generation chimera (TPK and TBL) with the earlier 1st (Mthionin) and 2nd (`74′ and `188′) lines. A total of 420 grafted plants (all on WT Carrizo rootstock for uniformity) have been made and will be bud inoculated as soon as the scions are sufficiently grown. We are also emphasizing parallel field trials for all phenotyping efforts. A field planting of ~400 `74′, `188′ and Mthionin transgenics is underway. The first 165 plants (WT Hamlin and Ray Ruby on transgenic Carrizo) went into the soil in August 2020 and will be undergoing their first assessment by February 2021. 200 more grafts of `74′ and `188′ transgenic Hamlin on WT root-stocks are being made to complete the planting. Fifteen new transformations, totaling over 5000 explants, have been completed to generate sufficient events of Valencia, Ray Ruby, US-942, and Hamlin (when not already complete) lines expressing `74′, `188′, TBL, TPK and other advanced chimera constructs. Over 200 new putative transgenic lines including 74-Valencia, 74-Ray Ruby, 74-US-942, 74-Hamlin, 188-Ray Ruby, 188-Valencia, 188-US-942, TBL-US-942, TBL-Hamlin, and TPK-Hamlin are now in soil and undergoing expression analysis. Objective 3, ScFv Constructs: Greenhouse studies on the 5 scFv lines in the 1st round of ACP-inoculation has been completed with the best performing lines showing significantly reduced CLas titer over the 12 month period (up to 250x reduction) and a much higher incidence of no CLas rDNA amplification in all tissue types. The best Carrizo lines have been grafted with WT Ray Ruby scions and are now in the ground at the Picos farm location undergoing field trials. An additional 129 rooted cuttings are propagated for follow up plantings with a Hamlin scion. A second round of greenhouse trials (150 plants from 12 lines)have been bud inoculated with HLB+ RL. A third set of 370 plants for greenhouse trials has been propagated with the first 54 plants to reach a suitable size already inoculated using the new ACP inoculation protocol. Tissue for testing CLas titer from both sets of plants has been collected and processed; now awaiting qPCR analysis. 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. Several peptides variants being screened through this assay have shown significant ability to reduce CLas titer by foliar application to grapefruit trees in initial testing- conducted by CRADA partners. Hamlin and Valencia trees have been selected and blocked for trunk application trials with these peptides. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; increasing from 10 to 20 ACP per leaf, decreasing the feeding period (7 days to 3) and adding a 4 day incubation period between feeding and tissue collection. In order to better investigate the effects of peptides producing ACP mortality, we have expanded the analysis of ACP bodies to include quantification of other major endosymbionts (Wolbachia, Profftella, and Carsonella) in addition to CLas. 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 and potential therapeutic effects. The best performing lines of Mthionin, chimeras `74′ and `188′ 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) and Carrizo/Mthionin (2 lines) have been returned certified clean. In addition to the use of the AMP Mthionin, its variants and chimeric proteins, new strategies have been implemented in our Laboratory to fight HLB, including the evaluation of insecticidal peptides to control the ACP (CLas vector), as well as the downregulation of the DMR6 genes to enhance defense responses against HLB disease. 54 independent transgenic lines of Carrizo, Hamlin and Ray Ruby expressing the insecticide peptide Topaz (a code name to protect IP), under constitutive and phloem specific promoter (SCAmpP-3) were evaluated for their ability to kill ACP and 12 lines (4 event of each genotype) were selected to move up in the screening pipeline for HLB/ACP tolerance, since they showed significant ACP mortality. Also 24 Carrizo transgenic events highly expressing Onyx (a code name to protect IP), a peptide with antimicrobial and insecticide activity, were evaluate by DLA, and 5 lines showing high ability to kill ACP (to 83% mortality) are being propagated for further evaluation. Onyx has been introduced also in Hamlin, Ray Ruby and Valencia , under SCAmpP-3 promoter. Down regulated DMR6 Carrizo transgenic citrus, either by expression of specific hairpin RNA or by specific Cas9-sgRNA were generated and are ready to be assessed for HLB resistance by grafting with infected scion. Objective 5, Transgenic product Characterization: Transgenic Carrizo lines expressing His6 tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), and PKT (20 lines) and His6/Flag tagged variants of scFv-InvA (22 lines) and scFv-TolC (18 lines) constructs have been generated and confirmed for transgene expression by RT-qPCR. Total protein samples have been extracted from His-tagged transgenic lines and sent to our CRADA partner for testing. Experiments are underway using these plants to track the movement and distribution of transgene products in parallel to direct antibody based approaches.
1. Please state project objectives and what work was done this quarter to address them:Objective 1: Determine how different cover crop mixtures impact soil and root health and weed cover in established commercial citrus groves.The cover crops planted in November 2019 were mowed in May 2020, and the summer/fall cover crops were planted on May 15 and 19, 2020. Legume cover crops were sunnhemp and cowpea, and non-legumes were brown top millet, Egyptian wheat (similar to sorghum sudangrass), and buckwheat. Germination counts and weed density measurements were made in July, and data is being analyzed. After 1 year of cover crop treatments, soil organic matter significantly increased in the row middles treated with cover crops for both locations. In addition, there were significant changes to the abundance of soil bacteria, particularly bacteria critical for nitrogen cycling, and soil nitrogen concentrations in the row middles planted with the legume cover crop mixes. Planting cover crop mixtures improved the biodiversity of vegetation in the row-middles and changed the composition of weeds. Sedges and grasses were the predominant weed species in row-middles without cover crop plantings, whereas more broad-leaved vegetation was found in cover cropped row-middles. Objective 2: Examine the impact of eco-mowing in conjunction with cover crops on soil and root health and weed cover in established commercial citrus groves.Eco-mowing occurred in early May 2020 when cover crops were mowed in anticipation of planting the next set of cover crops. After a year of treatment, soil organic matter slightly increased (in the range of 0.3-0.5%) under the tree canopy receiving eco-mowing compared to regular cover crop treatments; however, no significant differences were detected between treatments. Visual root growth assessments show continued root growth under cover cropping and eco-mowing, but analysis is ongoing. Soil moisture appears to be similar across all treatments, possibly due to the presence of a high water table at both sites. Objective 3: Quantify the effect of cover crops and eco-mowing on tree growth and production.After 1 year of study, we have not yet observed differences in fruit yield, fruit quality, canopy volume, and trunk size. This is not unexpected, as trees of this age could take at least two years to show responses to treatments. We will continue to assess canopy volume and trunk size, and harvest data will be collected again in Spring 2021. Objective 4: Identify the economic benefits of using cover cropsA student was trained on partial budgeting and valuing soil health. The student began work on developing a citrus budget that is appropriate for comparing management strategies with cover crops relative to business as usual. We found that differences in how the budgets are reported limits historical data collection to five years. These data will be used as a benchmark when doing partial budgeting, which is now in its early stages. In addition to budget ta.60sks, the relevant literature was reviewed and incorporated into a draft of the adoption survey. The survey is ready for review and IRB approval. Objective 5: Communicate results to growers using field days and extension materialsPreliminary results are being presented at the Citrus Expo in August. Initial results on weed suppression by cover crops was included in a presentation at this years Citrus Growers Institute. Information on cover crops and preliminary data will be included in two articles for the Citrus Industry magazine in September. Discussions are underway about how to host a field day, or a virtual field day, at some point in the next year. 2. Please state what work is anticipated for next quarter: The most recent collection of soils, leaves, and roots for microbial and nutrient analysis will be completed by the end of August 2020. Microbial DNA will be extracted from soils collected in August 2020 and analysis of soil microbes important in nitrogen cycling will begin. Soils collected in August 2020 will also be analyzed for soil organic matter and nutrients. Analysis of data from weed density measurements will be performed. Canopy and trunk size measurements and leaf nutrient status along with root image collections and soil moisture monitoring will continue in the next quarter. A graduate student and postdoctoral research associate on the project will be presenting results of the project at American Society of Agronomy virtual annual meeting in November 2020. The next set of cover crops are scheduled to be planted in October. The composition of the mixes is still being discussed. The economics team expects to execute the adoption survey and begin analysis. They will also construct the framework for partial budgeting and assessing the cost of cover crop use and continue to collect data. 3. Please state budget status (underspend or overspend, and why): We are on track with our planned budget spending.
The 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. 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%. 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.
Create new candidate hybrids. During this quarter, seed from last year’s crosses were grown-out in the greenhouse in preparation for propagation, testing, and establishment of seed trees. Emphasis of hybridization in the USDA rootstock program is among parents with superior tolerance to HLB, CTV, and Phytophthora. Propagate and plant new field trials. Budwood increase trees of selected scions were propagated in our nursery, in preparation for budding trees for new rootstock trials in the fall and spring. Plantings of new field trials this quarter were delayed because of institutional Coronavirus shutdown. Nursery trees for rootstock trials with Valencia, Star Ruby grapefruit, Eureka lemon, and Navel orange are being prepared in the greenhouse for field planting sometime later in 2020.Collect data from field trials. 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 this quarter is not usually used for harvest of any citrus crops in Florida. Assessments of tree health and measurements of tree size were completed on 2 trials during this quarter, which was reduced from the normal because of the institutional Coronavirus shutdown.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 testing, seed sources are established and used to determine trueness-to-type from seed. Studies were continued this quarter to evaluate seed propagation for 25 of the most promising SuperSour hybrid rootstocks. SSR analysis of progeny was delayed because of institutional Coronavirus shutdown.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, the website https://www.citrusrootstocks.org/ was updated with new summaries of performance information from the USDA rootstock trials. Release of superior new rootstocks for commercial use. Several of the 300 advanced Supersour rootstock hybrids in field trials are exhibiting good performance in comparison with the commercial standard rootstocks. Performance data continues to be collected, but it is anticipated that 2-3 of the most outstanding of these will be officially released in 2021-22.
Update for this quarter: Test site grant- USDA has mandated that all employees be on maximum telework since March 2020 due to the Covid-19 pandemic. Therefore, existing plots have been maintained, but the planned additional planting has been postponed. UF collaborators have been permitted into the test site and samples and data have been collected. Samples we previously collected have been processed by technicians at home (with APHIS-BRS permission) and are ready for qPCR including many of them 2400 seeds collected for the transgenic pollen-flow experiment. We held a virtual site-visit from APHIS-BRS, showing photographs of the site and were found to be in compliance. A new “efile” APHIS-BRS permit request has been submitted for all test-site plantings (except CREC-Dutt, Grosser and Gmitter who maintain a separate permit), it is under review and should be approved by Sept. 2020. Previous quarter Stover analyzed data on canker incidence in a block of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside, from data collected 8/17 and 9/19. Most notably: Almost all accessions with lower ACC lesion incidence were hybrids vs. pure trifoliate, though a few pure Poncirus had lower ACC than most. Based on chloroplast genome data from 57K Affymetrix SNP chip, provided by M. Roose, 11 of 33 reported seed parentage for hybrids was inaccurate, convention of female first was not followed. Of 34 hybrids validated, similar numbers had Poncirus, grapefruit, and sweet orange chloroplasts. Chloroplast type did not affect ACC incidence, but in each year accessions with grapefruit chloroplasts had small but statistically higher ACC severity than those with Poncirus chloroplasts. Hybrids of Citrus with Poncirus have markedly reduced ACC sensitivity compared to Poncirus, indicating that this trait is readily overcome in breeding. Seed from fruit harvested for transgenic gene flow experiment coninue to be processed for PCR. Previously established at the site: A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. Renewal and approval for BRS permit effective 9/1/19 through 8/31/20. 4) Continuation of an experiment on pollen flow from transgenic trees. FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. Early-flowering transgenic Carrizo (flowered ex-vitro within five months of seed sowing, and used at 12 months) was used to pollinate some of the same FF-5-51-2 What should be the final samples from the C. Ramadugu-led Poncirus trial (#3 below) completed preparation and were shipped in ethanol to UC Riverside. Availability of the test site for planting continues to be announced to researchers. Plantings: 1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the Stover permit. 2) Under the Stover permit a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. Likely 2019 will be the last year for data collection. 4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project. 5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and will continue to be conducted under a new NIFA grant. 6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.). 7) In a project led by Fred Gmitter there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo. 10) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls. 11) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type. 12) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State13)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
This quarter: USDA has mandated that all employees be on maximum telework since March 2020 due to the Covid-19 pandemic. Therefore, existing experiments and ACP colonies have been maintained, but all planned new experiments have been postponed. Samples have all been collected on-time from ongoing experiments. All samples collected, that have not been analyzed, have been processed for qPCR. Project rationale and focus: The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~50% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Most recent quarter:A partial shut-down of USHRL was initiated 3/20/2020, as a response to the Covid-19 pandemic. ACP colonies are Stover lab:5460 ACP used for inoculating 390 detached leaves, 78 no-choice small trees ,and seven homogenate assays of peptides. Bowman lab: Prepared a group of grafted plants and planned to ACP-inoculate in March, but this experiment was disrupted by the Covid-19 slowdown. These ill be inoculated when personnel are allowed more extensive time at USHRL Other users:· 180 for Robert Shatters · 500 for Yongping Duan
The objectives of this project are to produce disease resistant, commercially & agronomically acceptable, mature citrus transgenics & intragenics that will flower & fruit naturally using Agrobacterium & biolistics as a service for customers. The research components of this project are to increase transformation efficiency & to develop biolistic transformation protocols, so that the biotech products produced in our lab for research & commercialization require less federal deregulation. During this quarter, we identified two mature cultivars that have high transformation efficiency with Agrobacterium. In addition, we identified two citrus selectable markers that function well for intragenics. One hundred fifty transgenic shoots were produced & micrografted this quarter, of which 66 survived, 26 it is too soon to tell, & 58 died. We are losing transgenic shoots in micrografting & have hired someone new who will be trained in micrografting once the pandemic subsides. Thirty-five transgenics have already been secondary grafted. Currently UF labs have limitied return to work schedules as the Covid-19 pandemic in FL is peaking with increased cases & deaths. Our lab is still working in shifts. Once the pandemic subsides, we will hopefully return to a more normal work schedule with people able to work together in one lab. However wearing masks, social distancing & disinfecting surfaces are directives that we must follow in the workplace now. We are on track monetarily. Actually by year’s end, we will be slightly overspent in salaries, possibly due to raises paid to deserving staff members last year. Next quarter, the Mature Lab will test variables affecting biolistic transformation efficiency using Dr. Dutt’s all-citrus promoters & terminators to produce intragenic citrus. USDA APHIS BRS does not examine these trees in expensive, replicated field trials, because new federal regulations state that intragenic trees, without vector sequences, will be considered similar to trees produced through traditional plant breeding. In addition, we introduced OLL-20, which is a favorite of the juice industry, & we will determine if it does well in biolistic & Agrobacterium transformation experiments. OLL-8 is recalitrant in Agrobacterium transformation experiments, but it can be transformed with the gene gun. In contrast, OLL-4 does very well in Agrobacterium transformation & we have yet to test it with the gene gun.
In the three month period between April and July of 2020, the activity in the Juvenile Tissue Citrus Transformation Facility (JTCTF) was low due to special regime of work established by the University of Florida (UF) as a response to COVID19 epidemic. No new transformation experiments were done and no orders were received.Until May, the work done in the JTCTF included only the activities performed by the essential personnel. The essential personnel were in the lab five times a week for the period of few hours. Under such conditions I organized the employees to take care of plants in the greenhouse and in the lab. In May, UF approved low level (Phase1) re-opening of JTCTF. Because of the surface area of the lab where most of the activities of JTCTF take place, being in Phase1 meant that one employee can be in the lab at the time. Such time table with insufficient presence of labor force does not allow for new experiments to be done. I organized the staff to process the material and the data from experiments that have already been completed. For some experiments where material was abandoned in early stages of experiments, we lost both the material and the data. There were experiments where we were able to salvage both. Altogether, we produced 26 transgenic plants this quarter. These plants included 22 Duncan grapefruit and four Valencia oranges. These plants were the results of work on six different orders/vectors: BB3, BB4, ZM15-2, ZM16, ZM17, and NADR2.My efforts to transition JTCTF to EBA unit were mostly hampered by the low level of assistance from UF administrators in Gainesville and unclear situation with funding. I hope that within the next quarter I will have final version of EBA form or the transition will be delayed until January of next year. In May, JTCTF lost a long-term full-time employee. I have asked an employee who is already employed as an OPS in the facility to take over some of the responsibilities until more permanent solution is reached.
1. Please state project objectives and what work was done this quarter to address them: Objective 1. Investigate effects of rootstock propagation method and the interaction with rootstock on root structure, root growth, and tree performance during the first 3 years of growth in the field. Bimonthly root growth measurements with the rhizotron imaging system continued in trial 2 (Hendry County) and trial 3 (Polk County). All 2-year horticultural evaluations for trials 2 and 3 were completed and data analysis is in progress. Preliminary data suggest that the propagation method does not have a significant influence on tree growth and health during the first 2 years of growth. In contrast, significant rootstock effects were observed. For some of the rootstocks, effects differed by location. For example, US-942 and X-639 appear to perform better at the Hendry County location than the Polk County location. A Citrus Industry magazine article was submitted, which is to appear in the August 2020 issue. The article contains information on root architectures of field-ready plants and after 2 years of field growth (trial 1). These data are currently being prepared for publication in a peer-reviewed open-access journal. 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. Trial 4 (planted in Nov 2019) is in progress and trees are growing well. Root growth measurements with the rhizotron imaging system continued. 2. Please state what work is anticipated for next quarter: We will be collecting root core samples for root mass determination and analysis of fibrous root structure in all trials. Rhizotron image analyses will continue. Leaf samples will be collected for nutrient analysis. 3. Please state budget status (underspend or overspend, and why): Approximately 40% of funds have been spent. One personnel left and we were not able to rehire yet due to covid-19 related complications.
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 growth measurements were completed in all four trials using standard procedures. Valencia harvest data and fruit quality data were received, and most of the statistical analyses were completed. Objective 2. Develop outreach to transfer information to growers and other industry clientele.A Citrus Industry magazine article was submitted for the July 2020 issue. The article summarized the most recent data from the Hamlin trials and the previous season’s data from the Valencia trials (this season’s data were not available at the time of article submission). 2. Please state what work is anticipated for next quarter: We will collect leaf samples in all trials for leaf nutrient analysis. This year’s Valencia data will be summarized for a virtual/recorded presentation at the Citrus Expo. In addition, a student presentation will be given at the ASHS conference in August (to be held virtually). 3. Please state budget status (underspend or overspend, and why): Approximately 45% of funds have been spent, which is mostly in accord with the timeline. Some funds could not be spent as fruit harvest and quality analysis had to be outsourced due to the covid-19 imposed research halt.
This report covers the period of March 1, 2020 – May 31, 2020. During this period, M.S. student Chad Vosburg made a visit to the USDA United States Horticultural Research Laboratory (USHRL) in Fort Pierce, FL, to help initiate a Asian citrus psyllid (ACP) transmission experiment for HLB resistance testing of the FT-scFv transgenic ‘Duncan’ grapefruit lines produced for this project. In addition, he continued work on an HLB graft transmission test and analysis of field trees at the USHRL Picos farm in Fort Pierce. Continuing work collaborating with personnel at USHRL (Greg McCollum, Ed Stover, Earl Taylor) has produced some initial preliminary patterns in the HLB tests. Two lines were tested by ACP-mediated transmission, along with controls. As of this writing, all the trees except for three trees of one FT-scFv line were exhibiting some level of HLB symptoms at 3 months into the experiment. This indicates that the HLB transmission was highly successful. In addition, ACP nymphs were observed on all the trees, indicating that the HLB-carrying psyllids had actively colonized and reproduced on the test plants, which is conducive to HLB transmission. Symptom severity differed between the transgenic lines and the ‘Duncan’ control trees, but has not yet been quantified. We hope to obtain quantitative symptom measurements in the next reporting cycle. The trees planted outdoors at the Picos farm were tested by PCR for presence of ‘Candidatus Liberibacter asiaticus’ (CLas) and all found to be negative at about 6 months after plating, including control trees. The same two lines used in the field test were also grafted to rough lemon heavily infected with CLas, along with control grafts. Buds of the two transgenic lines grew vigorously and showed no or limited HLB symtpoms so far, while control buds showed severe stunting or failed to grow out. We are cautiously optimistic about that result, but the experiment will need to be repeated. The experiment is ongoing and bud graft growth, symptoms, and CLas titers will be measured. The PI also applied for and received the necessary permits to bring additional FT-scFv transgenic lines to Pennsylvania for analysis. We hope to be able to do a transfer in the next reporting period. It should be noted that the Covid-19 shutdowns of Penn State and USHRL in late March, 2020, has affected work progress. In mid June, work has cautiously resumed at Penn State. However, this situation has caused some delay of experimentation and limits access to plants at USHRL. We anticipate that it will very likely be necessary to request a no-cost extension to be able to complete the project objectives.
Our last report mentioned the work evaluating three Pummelo x Citrus latipes hybrids for use as double duty grove windbreak trees that would also attract ACP. Our manuscript entitled Double Duty: Production of psyllid-attracting windbreak citrus trees for the control of Diaphorina citri, the vector of huanglongbing was submitted for publication. Objective(s) pursued: 1. To evaluate the tolerance of newly released/developed citrus cultivars to CLas pathogen.2. To evaluate the tolerance of newly released/developed citrus cultivars to D. citri.3. To determine the mechanism underpinning the tolerance of the newly developed cultivar to HLB. To achieve these objectives, we are challenging these varieties with ACP and graft inoculating them with CLas-infected material to study their responses. We also are extracting and analyzing the leaf polar metabolites, leaf stored volatiles and the released volatiles of new flush via GC-MS. The information received will allow us to predict the success of the variety, similar to our studies on Sugar Belle and Bingo hybrids. In a recent study we investigated the effect of rootstocks on citrus tolerance to citrus greening pathogen by studying the metabolite profile of `Sugar Belle’ mandarin hybrid using gas-chromatography mass spectrometry (GC-MS). The principle component analysis showed that the metabolite profiles of the `Sugar Belle’ mandarin hybrid on the three selected rootstocks were different from each other. These results indicated that rootstocks could affect the primary and secondary metabolites of citrus scions, and consequently could affect scion tolerance to pathogens. The data was published in Plant Signaling and Behavior.Progress on Objectives: Rootstock evaluationsWe received 7 rootstocks for evaluation in the form of seeds, which have been germinated and are growing well: UFR-1, -2, -4, -5, -6, -15 and -17; 46 x 20- 04-6; 46 x 20-04-29Scion evaluations C2-2-1; OLL8; N40-6-3; RBB7-34; Grapefruit 914 – we grafted the budwood from these scions onto UF-2 in November 2019. Lucky – the parents of this cross are Sugar Belle mandarin hybrid and Nava x Osceola (pollen). We have 15 plants of each of the three (the two parents and the hybrid cross) ready for evaluation. We planned to make the first VOC analyses during spring flush but were unable due to the COVID closure. We will continue with evaluations of the mature leaves. Newly released varietiesWe recently received `Marathon’ Mandarin kindly donated from Southern Citrus nursery for propagation and evaluation. Marathon is susceptible to HLB but continues to have good yield after infection. The seed parent was the mandarin variety `Daisy’, which was produced by crossing the mandarin varieties `Fortune’ and `Fremont’. The pollen parent was the small-fruited mandarin cultivar `Seedless Kishu’, which is also known as `Mukakukishu’ in Japan. The leaf and phloem chemical composition will be evaluated on this new variety as soon as we have propagated enough plants for good analytical replication. We expect to have enough by fall to begin analyes.
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, with CLas+ ACP feeding, as well as studying them in established greenhouse and field studies. Greenhouse studies (With 9 Carrizo lines and 4 Hamlin lines, 98 total plants with controls) include graft inoculation of Carrizo rooted cuttings with CLas+ rough lemon, no-choice caged ACP inoculation of Carrizo rooted cuttings, and no-choice caged ACP inoculation of Hamlin grafted on Carrizo with all combinations of WT and transgenic. Data collection continues from Mthionin field plantings. Results from the first round of field plantings (45 plants) of Mthionin transgenic Carrizo root-stock grafted with non-transgenic rough lemon show transgenics maintaining higher average CLas CT values (2.5 CT higher @ 18 months), but with a high degree of variability. A large second planting of Mthionin transgenics went into the ground in April 2019, including transgenic Carrizo with WT Hamlin scions (81 plants), transgenic Hamlin on non-transgenic Carrizo root-stock (108 plants) and WT/WT controls (16 plants). Scheduled assessments for both field plantings is being prioritized under current covid-19 pandemic conditions. The 24 month assessment of the first planting has been completed and 12 month assessment for the second planting is underway. Additional grafts of WT Hamlin and Ray Ruby scions to Mthionin root-stock have been made and are included in the imminent chimera planting discussed in Objective 2. The Mthionin construct has also been extensively transformed into Valencia, Ray Ruby and US-942 to provide transgenic material of these critical varieties. The first 51 putative lines are now in soil and are undergoing expression analysis. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, have been conducted and repeated for lines expressing chimera constructs TPK, PKT, CT-CII, scFv-InvA, scFv-TolC, TBL, BLT, LBP/’74’, `73′, and `188′ using adjusted protocols to improve sensitivity and transmission rates (See section 4). Further detached leaf assays are being run to compare the relative effectiveness between each generation of chimera constructs and to expand the number of lines tested from each. DLA testing has allowed us to identify lines from several constructs with significant effects on CLas transmission and even increased ACP mortality. Recent results include up to 95% mortality in ACP after 7 days feeding on detached leaves of the 3rd generation TBL transgenics and 70% for TPK. Lines from promising constructs have been moved forward into greenhouse studies based on DLA results, as noted below. Initial ACP inoculations conducted on 8 lines of citrus Thionin-lipid binding protein chimeras (`73′, and ’74’) showed a statistically significant reduction (13x) in CLas titer for `74′ transgenics vs WT in the CLas+ plants. However, many plants remained CLas negative at 6 months post infestation, indicating a low inoculation efficiency. All greenhouse experiments are now using an improved protocol to enhance inoculation. Through a combination of selecting smaller plants, more aggressively trimming larger plants and close observation, we have been able to extend the caged ACP infestation time from 7 days to 21 without severe mold or cage damage to the plants. In June, 150 plants representing the best performing 7 lines of `188′ and 6 lines of `74′ were no-choice caged ACP inoculated using the new protocol. At 3 months, control plants tested positive at twice the rate of the earlier inoculation; 6 month tissue samples are now collected and processed, awaiting qPCR analysis. We are beginning a large greenhouse study to directly compare the best performing 3rd generation chimera (TPK and TBL) with the earlier 1st (Mthionin) and 2nd (`74′ and `188′) lines. All lines are being grafted onto WT Carrizo root-stock for uniformity. A total of 420 grafts (150 completed, 270 under way) will be bud inoculated with CLas+ RL and analyzed for resistant phenotypes. We are also emphasizing parallel field trials for all phenotyping efforts. A field planting of ~400 `74′, `188′ and Mthionin transgenics is underway. 165 grafted plants (WT Hamlin and Ray Ruby on transgenic Carrizo) are made and ready for the field. The ground is being prepared and plantings will begin as soon as conditions allow. 185 grafts of WT scions (Hamlin, Valencia, and Ray Ruby) onto transgenic Carrizo root stocks. 200 more additional grafts of `74′ and `188′ transgenic Hamlin on WT root-stocks are being made to complete the planting. Fifteen new transformations, totaling over 5000 explants, have been completed to generate Valencia, Ray Ruby, US-942, and Hamlin (when not already complete) lines expressing `74′, `188′, TBL, TPK and other advanced chimera constructs. Over 200 new putative transgenic lines including 74-Valencia, 74-Ray Ruby, 74-US-942, 74-Hamlin, 188-Ray Ruby, 188-Valencia, 188-US-942, TBL-US-942, TBL-Hamlin, and TPK-Hamlin are now in soil and undergoing expression analysis. Objective 3, ScFv Constructs: Greenhouse studies on the 5 scFv lines in the 1st round of ACP-inoculation has been completed with the best performing lines showing significantly reduced CLas titer over the 12 month period (up to 250x reduction) and a much higher incidence of no CLas rDNA amplification in all tissue types. The best Carrizo lines have been grafted with WT Ray Ruby scions and, with all appropriate permitting now completed and the plants sized up, will be moved to the field after hurricane season. An additional 129 rooted cuttings are propagated for follow up plantings with a Hamlin scion. The 3 month data from the 150 plants from the 2nd group of scFv lines (12 lines) that were initially no-choice ACP inoculated showed an insufficient infection rate. These plants have now been bud inoculated with HLB+ RL. An additional 370 rooted cuttings were propagated for the third round of ACP-inoculations. From which, the first group of 54 plants large enough to use have been inoculated with the higher pressure 21 day protocol. Tissue for testing CLas titer from both sets of plants has been collected and processed; now awaiting qPCR analysis. 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. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; increasing from 10 to 20 ACP per leaf, decreasing the feeding period (7 days to 3) and adding a 4 day incubation period between feeding and tissue collection. 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 and potential therapeutic effects. The best performing lines of Mthionin, chimeras `74′ and `188′ 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) and Carrizo/Mthionin (2 lines) have been returned certified clean. Objective 5, Transgene Characterization: Transgenic Carrizo lines expressing His6 tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), and PKT (20 lines) and His6/Flag tagged variants of scFv-InvA (22 lines) and scFv-TolC (18 lines) constructs have been generated and confirmed for transgene expression by RT-qPCR. Total protein samples have been extracted from His-tagged transgenic lines and sent to our CRADA partner for testing. Experiments are underway using these plants to track the movement and distribution of transgene products in parallel to direct antibody based approaches.
Field variety trials are a simple but effective tool to test plant horticultural performance under different environmental conditions and enhance the commercial adoption of new cultivars. Large-scale, rapid implementation of HLB-tolerant cultivars depends on reliable data, and the Millennium Block project is addressing the need of establishing field plantings to generate regional, updated information for the Indian River Citrus District. The project has two objectives: (i) Assess performance of new grapefruit cultivars with certain rootstocks under HLB endemic conditions in the IR district and (ii) ) Evaluate the influence of UFR and other recent rootstocks on grapefruit, navel, and mandarin in the IR in comparison to legacy/standard rootstocks. Trials tested: T1) grapefruit cultivars on three rootstocks, T2) 38 rootstocks with Ray Ruby grapefruit as the scion, T3) 36 rootstocks with Glenn 56-11 navel orange, and T4) 36 rootstocks with UF-950 mandarin.
We planted approximately 3,600 trees (Sep/19) and are waiting for the remaining trees to be delivered by the nursery (Summer/20). Masters student started on Jan/2020. Slow release poly coated fertilizer applied in Sep/19, Jan and May/20. Irrigation controller, sand media filtration system and water flow meter were installed. We applied imidacloprid to prevent leaf minor and psyllids, and followed with a spraying schedule as suggested by the certified crop advisor. The grove has been continuously scouted for pests such as psyllids, orange dogs and ants. Hoop boom was modified to spray young trees with higher accuracy, reducing the waste of agrochemical products. We created a tree location map and began production and distribution of QR tags to be used with scanner codes during data collection in the field. The group met with the certified crop advisor to develop a spray program schedule based on time of year and conditions to be applied as determined by IPM scouting.
Tree height, tree width in two positions (E-W and N-S), and trunk diameter were measured in three central trees from each experimental plot in Feb/20, and canopy volume calculated. Data only reflects the first 5-6 months of growth. On T1, ‘Pummelette UF-5-1-99-2’ on US-942 was 4x larger (0.2 m3) than ‘Start Ruby Gft DPI-60’ on X639 (0.05 m3) (P<0.0001). On T2, 'Ray Ruby Gft CGIP-103' on A+VolkxOrange 19-11-8 was ~3x larger (0.22 m3) than on UFR-17 (0.08 m3) (P<0.0001). On T3, 'Glenn Navel F-56-11' on 2247x6070-02-2 was ~5x larger (0.25 m3) than on Willets (0.04 m3) (P<0.0001). On T4, 'Mandarin UF-950' on US-897 was ~5x larger (0.25 m3) than on WGFT+50-7 (0.04 m3) (P<0.0001). Leaf samples for HLB diagnostic were taken from a pool of trees from each experimental plot and sent to the Southern Gardens lab, and all trees tested negative by May/20. Fruit phenology, pests and diseases have been monitored monthly. Canopy thickeness, canopy color and HLB incidence have been measured quarterly in all experimental plots.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Five rootstock 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. Similar crosses made in previous years have yielded several good candidates through the Gauntlet screen, and these new crosses were intended to explore new families or to expand on previously fruitful combinations. Two refereed manuscripts were published describing the positive impact of HLB-tolerant rootstocks from our program on juice quality and the metabolome, and the changes in the proteome of Valencia on tolerant vs. sensitive rootstocks. 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. Spring 2020 crosses for this objective were numerous. Forty-two interploid crosses were made for improving sweet orange-like, mandarin, grapefruit and acid fruit hybrids. Twelve crosses were made at the diploid level targeting sweet orange-like hybrid development. Embryo rescue from 2019 crosses resulted in shoots from >1750 germinating embryos from 36 interploid crosses (including 11 targeting sweet orange-like hybrids, and 8 targeting grapefruit improvement), all to be micrografted to rootstocks. Cybridization experiments were conducted to combine Meiwa kumquat cytoplasm with OLL and EV sweet oranges, to attempt improvements in citrus canker resistance; embryos have been recovered for next steps in plant regeneration. Somatic hybridization of Tango and W. Murcott suspension lines with leaf protoplasts of several CREC and other public cultivars, and advanced selections was attempted; several combinations already have produced embryos and a few shoots. Other materials, including grapefruit cybrids with Meiwa, and vigorous Vernia seedling selections, have been propagated for future field plating. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We have explored new approaches to quantifying tree responses to HLB, in addition to the previously used subjective approaches. Specifically, we measured photosynthetic parameters and leaf canopy indexes, to produce repeatable and reliable quantitative data in support of further genetic analyses of tolerant types. Objective quantitative data of tree responses provides more reliable information that improves the precision with which we can associate genome regions with tolerance or sensitivity; see Obj 4. To finish the current fruit season, we have evaluated fruit quality of the more tolerant types of sweet orange-like hybrids, as well as mandarins and grapefruit hybrids, and selected candidates in all categories worthy of further evaluation as potential new cultivars. 4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. We identified a set of ~ 500 individuals for GWAS studies, using the data referred to in Obj 3 above. Despite UF-CREC closure, we were able to collect leaf samples from these trees and DNA samples have been prepared to send to a commercial outfit to perform the SNP chip analysis. This work will validate previously identified, or to identify new, genomic regions associated with HLB tolerance 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 carried out. Finally, a renewal application has been submitted to the USDA for our multi-location transgenic field permit, to enable the program to continue to explore the impact of certain genetic modifications on HLB incidence, disease development, and potential tolerance or resistance, under real world field conditions.
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