All transgenic rootstocks (Swingle and Carrizo) expressing the AtNPR1 gene, developed using mature tissue transformation and juvenile transformation have been evaluated using qPCR. Several of the promising lines have also been evaluated for the trans-protein production using western blotting techniques. Cuttings of several lines have been propagated and budded with non-transgenic ‘Valencia’ sweet orange. Many others are currently being propagated in the mist bed. To produce seed source trees, trees are being produced by budding onto US802 rootstock. These will be planted in a USDA-APHIS approved field site when ready. Transgenic rootstocks expressing other potent plant derived transgene(s) are also being produced using the mature tissue transformation method.
Citrus Transformation Facility (CTF) continued its operation without interruptions. In the first three months of 2019, CTF received nine orders. Five of these orders were for production of transgenic Duncan grapefruit plants and four for production of transgenic Valencia plants. Within this period, CTF produced 77 transgenic citrus plants. Out of this number, 42 were Duncan grapefruit plants, 19 Mex lime plants, 11 Pomelo plants, four Kumquat plants, and one Pineapple sweet orange plant. These plants are result of work on 15 different orders.In February, CTF purchased one bin of Duncan grapefruits and stored them in the cold room for the supply of seeds. For experiments requiring Valencia seedlings, we are picking Valencia fruit from the trees on the CREC property to get seeds. The seeds of other cultivars are obtained through purchase from Lyn Citrus nursery in California or by picking fruit from DPI Arboretum in Winter Haven.One of the employees with the lowest FTE has additionally decreased her attendance and will leave the lab at the end of June.
We continue to produce Agrobacterium-mediated mature transgenics for customers, test different approaches for increasing efficiency, & develop biolistics as a tool in citrus. During this quarter, 11 mature transgenics were produced with Agrobacterium & have been micrografted, & a remaining 42 positive shoots were produced using Agrobacterium & must still be micrografted once/if they get larger. These two genetic constructs had GFP & were transformed into Hamlin (one of our better cultivars for transformation). An inordinate number of transgenics were produced, which is very unusal in mature citrus & must have been the result of cultivar x genetic construct interaction. The GFP reporter makes identification of transgenics easy. A new initiative from the CREC Director is to produce mature citrus transgenics with stacked disease resistance genes using biolistics for faster deregulation. We have been able to produce transgenics for Dr. Mou using a promising, new disease resistance gene & the genetic construct did not rearrange in mature citrus. (Unfortunately it rearranged for Dr. Orbovic in immature citrus). We used a RecA- Agro strain because we suspected there might be a problem. In a couple of transgenics, the gene of interest (GOI) was lost, but the majority of transgenics have the transgene. This is very similar to the deletion of the GOI from Dr. McNellis’ genetic construct using the mature citrus transformation protocol. During that instance, Dr. Orbovic was able to produce immature transgenics that had the GOI, while it rearranged in mature transformation, even using a RecA- Agro strain. One advantage of biolistics over Agro transformation is that the GOI does not rearrange as easily as in Agro. A manuscript was accepted & revised for In Vitro Cellular & Developmental Biology-Plant. This manuscript showed that improved selection with phosphomannose isomerase after biolistics in immature citrus drastically increased transformation efficiency after bombardment (a mean of ~2 transgenics per paired shot). We are now testing phosphomannose isomerase (PMI) as a new selectable marker in mature citrus. Once the optimal mannose/sucrose concentrations have been determined in mature citrus, we will test PMI selection after Agrobacterium-mediated transformation. We are testing a new protocol for precipitating DNA onto gold particles for biolistics that increased efficiency in other species. This protocol will also be useful for gene editing using biolistic transformation. We must buy a new soil sterilizer & a new ultra low -80C freezer. Both are essential to this program. An external review of the two transformation labs was conducted March 28 & 29 & presumably there will be a report issued at some time in the future.
Much was accomplished during the first quarter of 2019. Due to the urgency of needing data from these large-scale field trials, much effort was put into preparing for harvesting `Hamlin’ fruit in the months prior to start of funding. A statistician was consulted to advise in the selection of replicate trees for data collection. Existing field maps were modified and new worksheet were created containing unique tree identification numbers. Multiple field trips were conducted to survey trials and to label individual trees with plastic tags containing the unique identification numbers. In December 2018, we were therefore able to harvest fruit and collect yield data in a well-organized and statistically valid fashion for the `Hamlin’ Fort Basinger location (Highlands County). In January 2019, yield data were collected in the same fashion from the `Hamlin’ Lake Wales location (Polk County). Data were sent to CREC for growers’ access on the https://citrusresearch.ifas.ufl.edu/trial-overview/ website. Two field technicians and one student were brought on board and commenced work on this project in January 2019. All personnel was trained regarding trial design, statistical design, and procedures of horticultural data collection. We have begun our horticultural assessments according to the objectives outlined in the proposal. These include tree height, canopy volume, and canopy health ratings. Due to the large scale of the trials, these measurements are taking considerable time. Considerable time is also being dedicated to accurately document and organize data for statistical analyses. In March 2019, fruit were collected to conduct fruit quality analyses from `Valencia’ trees at both Fort Basinger and Lake Wales. Due to the high costs of fruit quality analysis and for statistical purposes, only trees on replicated rootstock cultivars were sampled and six replications per rootstock were included. Harvest of Valencia trees is scheduled for April 2019.
Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is continuing apace. Detached leaf assays, with CLas+ ACP feeding, have been conducted and lines with the most promising results have begun greenhouse studies. These 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 grafted Hamlin on Carrizo with all combinations of WT and transgenic.The first field plantings with Mthionin transgenic Carrizo (45 plants) have been made with leaves of non-transgenic rough lemon on transgenics showing higher average CLas CT, significantly decreased leaf mottle and significantly increased health values after 6 months. Plants for follow up field plantings of transgenic Hamlin on WT Carrizo (112 plants), WT Hamlin on transgenic Carrizo (84 plants), WT Ray Ruby on transgenic Carrizo (118 plants) and WT Valencia on transgenic Carrizo (118 plants) with WT controls are being propagated.Seeds for additional scion variety transformations have been collected and germinated. Shoots will be developed enough to yield epicotyl tissue for Mthionin construct transformations in 2 (Hamlin), 4 (Ray Ruby) and 6 (Valencia) weeks. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, were conducted on lines representing chimera constructs TPK, PKT, CT-CII, TBL, LBP/’74’, `73′, and `188′. Multiple lines from several constructs were moved forward into greenhouse studies based on these results as noted below. Definitive results for TPK, PKT, CII, and TBL were hindered by low inoculation rates. Assays for these constructs are being repeated to identify which lines of each are best suited for greenhouse studies. Detached leaf feeding assay protocols have also been adjusted to improve sensitivity (See section 4)No-choice caged ACP inoculation has been conducted on 8 lines of citrus Thionin-lipid binding protein chimeras (`73′, and ’74’). Three month data has been collected, while many plants are yet to show CLas DNA amplification, there is a statistically significant reduction (13x) in CLas titer for transgenics vs WT in the CLas+ plants. An additional 475 rooted cuttings have been propagated from chimera constructs (6 lines of `188′, 7 lines of `74′ and 12 lines of `73′) for the next round of ACP inoculation trials. 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 at the conclusion of the study. The best lines have been used as rootstock for WT Ray Ruby scions and will be moved to the field once the graft union is strong enough. An additional 129 rooted cuttings are propagated for additional grafts and field plantings. ACP inoculations were conducted on 150 more plants from 12 scFv lines. Data from 3 months post inoculation has been collected, but too few plants are testing positive at this time for a conclusive analysis. An additional 370 rooted cuttings have been propagated from the remaining scFv constructs/lines to be tested and will soon be mature enough for ACP inoculation. Objective 4, Screening Development and Validation: Details of the high throughput ACP homogenate assay, and its use for selecting lytic peptides for activity against CLas, has been submitted for publication and remains in use for early screening of therapeutics in the lab. 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. The use of a phloem specific endogene would allow for samples to be normalized to phloem cells instead of total citrus cells, more accurately evaluating bacterial titer and potential therapeutic effects with the phloem limited CLas. 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) have been generated and confirmed for transgene expression by RT-qPCR. These plants will be used for generating data on the movement and distribution of transgene products in parallel to antibody based approaches.
The research agreement contract was finalized in January, 2019, and funds became available in February, 2019. Dr. McNellis initiated a search for a graduate student to perform the work through the Department of Plant Pathology and Environmental Microbiology graduate program and the Plant Biology graduate program at Penn State. A suitable candidate was interviewed in February, 2019, and an offer of admission was made in March, 2019. A rotation student in the Intercollege Program in Plant Biology at Penn State also expressed interest in the project and began a rotation in Dr. McNellis’ lab on March 5, 2019, which will continue for at least the remainder of the spring semester. It is likely that one or both these students may become available to work on the project. The grapefruit trees expressing the FT-scFv anti-HLB antibody protein were continuously maintained at Penn State and at the USHRL in Ft. Pierce, FL, and continue to grow normally and are ready for analysis.
Objective 1. Create hybrid rootstocks which combine germplasm from parental material with good rootstock traits and HLB tolerance, propagate the most promising of these hybrids, and establish replicated field trials with commercial scions. During Oct-Dec 2018, six new replicated rootstock trials were field planted, including three on the East coast region, two in the Central ridge region, and one in the Southwest region. Replicated randomized experimental designs were used and included several commercial standard rootstocks for comparison.Multiple trees of about 120 selected new rootstock hybrids were budded and grown in the nursery, in preparation for field planting new rootstock field trials. These nursery trees included many of the most promising SuperSour hybrids identified in ongoing trials established in previous years, as well as several commercial standard rootstocks. These nursery trees also include several new and different hybrids chosen because of newly available information about parentage and characteristics best associated with outstanding traits. Three new field trials with sweet orange scion will be planted from these trees in 2019, including one trial in the East coast region, one in the Central ridge region, and one in the Southwest region. A rootstock trial with grapefruit scion is also planned for planting during spring 2019 in the East coast region. Objective 2. Collect field performance data from early-stage replicated rootstock field trials and release new rootstock cultivars as justified by superior performance in multiyear field trials. Seventeen rootstock trials planted prior to summer 2018 (as described in the Proposal Appendix ii) were monitored and used for data collection on field performance, as appropriate during this quarter for the scion involved. Yield and fruit quality data was collected from four Hamlin and one pummelo field trial.Three new USDA rootstocks were released on 2 November 2018, identified as US SuperSour 1 (SS1), US SuperSour 2 (SS2), and US SuperSour 3 (SS3), and are freely available without restriction. An informational sheet with performance data on the three rootstocks was prepared and distributed widely to industry, and provided to CRDF. All three new rootstocks demonstrated good fruit quality with sweet orange and superior fruit yield in trials, compared with Sour orange, Swingle, and Cleopatra rootstocks. Seed source trees have been planted with all three new rootstocks, although initially propagation will be limited to cuttings and micropropagation from FDACS clean source material.
A number of successes have been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, and continues to replant with new advanced material (Grosser, personal comm.). Using trees planted at the test site, transgenic overexpression of an Arabidopsis defense gene was reported to enhance citrus HLB resistance (Dutt et al., 2015). The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material planted this spring that has shown great promise in the greenhouse (Hao, Stover and Gupta, 2016). A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 6 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Ramadugu et al., 2016), P. trifoliata displayed reduced colonization by ACP (Westbrook et al., 2011), and measures of HLB-tolerance were associated with percentage citron in accession pedigrees (Miles et al., 2017). A UF-Gmitter led association mapping study is underway using the same planting, to identify loci/genes associated with HLB- and ACP-resistance. A broad cross-section of other Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside. 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. A manuscript reporting identified HLB resistance associated QTLs has been published (Huang et al., 2018). David Hall assessed ACP colonization on a subset of plants and further documented host morphological traits associated with ACP-colonization in Poncirus (Hall et al., 2017a&b). Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruits that approach commercial quality, were planted within the citrange site. As of April 2014 at the Picos Test Site, several of these USDA hybrids had grown to a height of seven ft (one now released as US SunDragon), with dense canopies and good fruit set, while sweet oranges were stunted (3 ft) with very low vigor. These differences largely continue and the observations have encouraged aggressive use of this and other trifoliate hybrids as parents (Stover et al., unpublished). A 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 replicated planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. HLB phenotyping and growth data have been collected and genotyping will be conducted under a new NIFA grant. 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.). Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site. New transgenics from Jeffrey Jones and Zhonglin Mou of UF, Tim McNellis of PSU will be planted in the next month. Availability of this resource will continue to b
Objective 1. (Greenhouse experiment): qPCR analysis was completed on all trees to determine CLas titers, and results were received from the Southern Gardens Diagnostic Laboratory. Surprisingly, 44 trees tested negative for CLas, mostly from WGFT+50-7, UFR-3, X639 and Swingle, especially with treatments 5 &6. This suggests that over time, slow release of strong micro-nutrient packages can have a therapeutic effect. Trees have been trimmed and made ready for field planting in the spring, in a possible collaboration with AllTech via Ed Dickinson (and will require a DPI permit). Objective 3: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). In this time period, we did final the final round of fertilization (3rd application). We also collected the final tree health data for 2018 including leaf nutrient analysis. The results with some treatments/locations showing yield and fruit quality improvements, were presented to CRDF board meeting in October 2018. Results were also presented at Nutrition day in December, which was followed by the field day at the Fort Meade trial location. Field day was very well received by growers and grower feedback was very good. Objective 5. (funded by Lee Groves, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. Positive results showing a therapeutic affect from overdoses of manganese against HLB were presented at the annual ASHS meeting in Washington DC, and a manuscript has now been accepted for publication in HortScience pending acceptable revisions.
This project produces transgenics, cis/intragenics & subgenics, in agronomically acceptable cultivars, for field testing & potential commercialization.The original proposal was for a 3 year funding period, but the project was only funded for 1 year because the CRDF wanted transgenics made by a company in Brazil. In 2016, after the CRDF realized that logististically, transgenics could not be easily made in Brazil, the mature citrus facility (MCF) was funded 2 more years. In total, 3 proposals were written for this project. Because of instability in funding, similar to what is presently occurring with short-term contracts, it has been difficult to keep good employees & maintain productivity. The significant objectives for the 1st & 2nd funding periods were: Mature plant production as a service using with Agrobacterium harboring vectors with disease resistance genes & molecular analyses to show copy number of the transgenes & gene expression; Plant propagation to form replicates for field testing; Increase micrografting efficiencies, bypass it altogether, or root mature scion; Test different selectable markers & reporters; Develop a biolistics protocol for immature/mature citrus; Introduce new, high yielding cultivars for tests in transformation; Apply for external funding. At the beginning of this funding cycle, new customers were charged a nominal fee for transgenics because previously our services were free. All of the abovementioned objectives have been addressed. Plant production for customers, for technology development, & for increasing efficiency produced ~437 transgenics for this 3 year period (~558 in total since 2014). An additional 400 transgenics were propagated for customers, either through budding or rooting. Mature scion cannot be rooted, micrografting cannot be bypassed, but micrografting efficiencies are stable at 77% by 1 operator. Molecular analyses were conducted for customers (~120 qPCR assays for 1 customer) & for publications, & thousands of endpoint PCRs for the gene of interest conducted. Several grants proposals were submitted & 2 small proposals were funded. Biolistic transformation was developed for immature & mature citrus, & an new selectable marker significantly increased efficiency. This was the first report of biolistic transformation of citrus with plant regeneration. This objective will become increasingly important considering new 2018 USDA APHIS guidelines in which APHIS will not oversee field tests for cis/intragenics/subgenics that do not carry plant pest or vector sequences, & these trees can be fast-tracked to growers at reduced expense, essentially similar to cultivars produced with traditional breeding. High yielding cultivars from the Plant Improvement Team were introduced & Agrobacterium & biolistics transformation efficiencies determined. There is one new scion cultivar that has an extermely high Agrobacterium transformation efficiency & another scion cultivar has reproduciby high efficiency. Most cultivars can be transformed using biolistics, although none have exceedingly high transformation efficiency yet. A cisgenic selectable marker, constructed by Drs. Zale & Dutt, is being tested in mature citrus to increase transformation efficiency. An intragenic citrus reporter from Dr. Dutt works well to replace GUS. A UF CREC Initiative during this timeframe was to stack two genes in transgenics to prevent the bacteria from overcoming resistance of one gene. An unfunded UF scientist, complied with the request to make the stacked gene constructs, & the MCF produced ~150 transgenics to meet this UF CREC Initiative. Biolistic-mediated gene editing of the PDS gene was achieved in immature Carrizo & Valencia. One customer’s Agrobacterium vector had a tendency to rearrange, in Agrobacterium prior to transformation. A total of 33 trees were produced for this scientist, but the transgenes rearranged in all but 2 events. This mutation was documented by restriction digests of vector DNA grown in E. coli vs Agrobacterium. A consecutive double budding method was devised so that mature citrus is reinvigorated well prior to experimentation. New services were added that offer biolistic transformation of minimal cis/intragenic expression cassettes. This service, if utilized by scientists, will provide significantly more monetary revenue for the mature citrus facility. However the CRDF should encourage scientists to use bioistics for cis/intragenics for faster & cheaper deregulation. Growth room maintenance is expensive & it is expensive to staff the MCF. A number of publications were generated by this project.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. The following work has been conducted in this quarter: (1) We have started to treat the three independent transgenic lines ( Duncan 57-28, Hamlin 13-3, and Hamlin 13-29), which have gone through the long-term HLB test and exhibited robust tolerance to HLB disease. The first batch of plants, including two replicates of the transgenic line 57-28, three replicates of the line 13-3, and one replicate of the line 13-29, have been treated under the alternating temperature conditions (25 C for 4 hours and 42 C for 4 hours) for two months. These plants have generated some new shoots during the treatment. We have tested if heat treatment is able to remove CTV and the CLas bacteria. So far the new shoots are negative for both CTV and CLas, indicating that the treatment is effective. The new shoots will be used for generating citrus trees for field trials. (2) We have screened 28 new transgenic lines against HLB-infected psyllids. These lines were generated by the mature transformation laboratory. The following lines still look great and haven’t shown any HLB symptoms: #82-6 Hamlin, #70-4 Hamlin, #26 Hamlin, #65 Hamlin, #82 Hamlin, #73-5 Hamlin, #11 Pineapple, #33 Pineapple, #73-5 Pineapple, #78 Pineapple. Based on the nymph production phenotype, these plants should have been infected by HLB. We have tested bacterial titers in these plants by qPCR, and indeed, the majority of these plants are CLas positive. The bacterium-free plants (with low CLas titers) will be inoculated again. (3) The eight new transgenic lines (A99, A100, A102, A101, A72, A73, A97, and A98) were irrigated and fertilized regularly. After they reach appropriate size, they will be screened against HLB-infected psyllids. (4) The manuscript titled Overexpression of the Arabidopsis NPR1 protein in citrus confers tolerance to Huanglongbing has been revised and published in the Journal of Citrus Pathology in this quarter.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. Major accomplishments per objective (1) Obtain mature tissues of the best transgenic lines: successfully achieved. The citrus flower-promoting gene FT3 was previously cloned into the CTV vector by the Dawson lab. The CTV-FT3 construct was introduced into Agrobacterium. Tobacco leaves were infiltrated with the resulting Agrobacterium. CTV-FT3 recombinant virions were purified from systemically infected tobacco leaves and bark flap inoculated into C. macrophylla seedlings, which began blooming in about five months. Buds from the matured C. macrophylla were grafted onto the original plants of all transgenic lines (EDS5-Dun-205-9, ELP3-Dun-207-8, ZMSN-Ham-73-1, ZMSN-Dun-137-2, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25). All plants began blooming in 6-18 months. We have successfully achieved this objective and also demonstrated that CTV-FT3 is efficient for converting juvenile tissues to mature tissues. The CTV-FT3-infected C. macrophylla plants that have bloomed can be used as bud source for promoting maturation. The three independent transgenic lines (NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25) that have shown robust tolerance to HLB have been treated under the alternating temperature conditions (25 C for 4 hours and 42 C for 4 hours) to remove CTV and the CLas bacteria. The resulting clean germplasms will be used to generate trees for field trials. (2) Determine whether transgenics prevent psyllids from being infected: accomplished with negative results. CLas-infected psyllids can transfer the CLas bacteria to the next generation of psyllids by inoculating the flush area in which the nymphs develop, which allows the next generation of psyllids to continue spreading HLB without the need for another source plant. In our experiments, we noticed that several of the transgenic lines exhibit delayed or reduced levels of CLas after infection. We started to test if the delayed or reduced production of CLas is sufficient to prevent or reduce the infection of the progeny psyllids. CLas bacteria-carrying transgenic plants were placed in cages, and clean psyllids (not infected by CLas) were moved into the cages. The progenies of the psyllids were collected and tested for CLas titers. A total of six rounds of cage experiments with vegetatively propagated plants from the transgenic lines ELP3-Dun-207-8, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25 were conducted. Results showed that none of the transgenes was able to pre-vent psyllids from being infected by CLas. (3) Continue testing generations of vegetative propagation from the best transgenic lines: successfully achieved. Three independent transgenic lines, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25, have gone through at least six rounds of HLB inoculation. Three generations of progenies (18 replicates for NPR1-Ham-13-3, 31 replicates for NPR1-Ham-13-29, and 25 replicates for NPR1-Dun-57-25) were inoculated with CLas-infected psyllids. The inoculation was repeated until all plants were CLas positive based on qPCR. All progeny plants have shown no or minor HLB symptoms. The three transgenic lines are thus highly tolerant to HLB and will be put into the field for field trials.
Activities are reported by project objectives below. 1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions. Seedlings are being grown of over one dozen unreleased rootstocks already shown to control tree size support good fruit loads and to have minimal HLB symptom expression. These will be propagated with sweet orange scions for field planting at the St. Helena site next season. As part of the gauntlet screening, we stick-grafted approximately 75 new candidate rootstock hybrids produced from HLB-tolerant parents in 2017 with HLB+ Valencia sweet orange for HLB screening. We produced rooted cuttings of approximately 100 gauntlet candidate rootstock hybrids including 47 hybrids combining HLB-tolerant LB8-9 Sugar Belle with complementary rootstock germplasm (including salt tolerant pummelo/mandarin hybrids and trifoliate orange 50-7). Replicated cuttings will be used for further HLB-tolerance assessment. Super Root Mutants of 10 selections of UFR and other rootstocks discovered by Beth Lamb at the Phillip Rucks Nursery Tissue Culture Lab were potted up; this group includes 3 mutants of UFR-1, 3 of UFR-3, one of UFR-4, one of UFR-17 and one of SO+50-7. These lines are producing feeder roots at a much higher density than the standard clones, and we will screen these selections for potentially enhanced HLB tolerance. 2. Breeding of HLB tolerant/resistant processing sweet oranges and orange-like hybrids. New hybrids produced have been potted up to grow until field planting next season. 3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. The data from this continued effort are being analyzed cumulatively across multiple seasons to more accurately identify and characterize tolerant individuals. 4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. We continued focused effort on field trial data management, analysis and interpretation. Files from more than 80 sites have been opened, conditions of the trials have been noted, and plans for 2018-19 field data collection have been developed and prioritized, based on this information. Efforts to review and summarize data have continued, and information is being organized for inclusion in our soon-to-be available website. We completed data analysis from evaluation of young resets of new sweet orange selections on multiple rootstocks in LaBelle/Immokalee trials, for tree size, health and yield: UFR-17, UFR-15 and 46×20-04-42 (pummelo x Cleo) were outstanding performers here. UFR-4 also did well. The best combinations for tree health and early fruit production were Valencia B9-65/UFR-17 and OLL-20/UFR-17. Our field team visited 10 field trial sites this summer, assessed trees for condition, and contacted grove management personnel, to make them aware of our continued interest in the trials. We planted one new rootstock trial, an additional 400+ trees in a fresh fruit scion trial, and 250 grapefruit hybrids at the IRREC.We collected detailed botanical and morphological information to support IP protection for release of 2 new rootstocks, one sweet orange and a deep red grapefruit.Finally, a very substantial effort was undertaken this year to rescue promising individual trees of diverse scion and rootstock germplasm from our 50-acre research block at the GCREC in Balm. These blocks have not been irrigated since early fall of 2017, and we were forced to leave the site. All trees in this block were subjectively assessed for potential HLB tolerance, as well as general overall health and appearance, using a 0-4 scale (0=dead; 4=completely heathy appearing tree). We harvested budwood from ~2300 individuals with scores = 3 and propagations for field planting in another location. Between June and end of September 2018, we had to revisit the block and collect additional budwood from trees that were not successfully propagated. We are growing off these trees for planting in 2019 at a new location.
Objective 1. (Greenhouse experiment): qPCR analysis was conducted on all trees to determine CLas titers. The combination of Valencia on WGFT+50-7 rootstock showed significantly reduced Clas populations, especially in Treatment #5 (Harrell s 12-3-9- St. Helena mix). Many plants of this combination in Treatment #5, and in a few other treatments showed no active infections (trees previously showing active infections). Lower numbers of trees of other combinations also showed reduced CLas titers; but all the data was just received, so the complete analysis is still underway. Evidence accumulating that nutrition can indeed suppress CLas titers. Objective 3: Field trials of CRF/TigerSul blends: Use of CRF with tiger micronutrient seems promising – data recently presented to CRDF board at Arcadia. 20-50% higher rates of micronutrients seem to improve the fruit yields Soil applied fertilizer program seems to be beneficial for HLB-affected trees Nutrition does have an effect on fruit quality! Focus should be on constant supply of nutrients Either frequent application of conventional fertilizer or adding some amount of CRF is beneficial Uninterrupted fertigation is supported by data Objective 5. (funded by Lee Groves, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. Positive results showing a therapeutic affect from overdoses of manganese against HLB is being presented at the annual ASHS meeting in Washington DC, and a manuscript has now been submitted to HortScience. Another round of treatments was applied to the trees, although this round we substituted Florikan polycoated Mn for TigerSul Mn to get longer distribution.
Our transgenic efforts have evolved greatly in the three years of this project. As data accumulated and new ideas came to the forefront, efforts were focused on those aspects of the research proving most valuable, while several of the initial objectives were deemphasized. A new project was approved by CRDF (18-022) that is the next series of steps following up on the successes of project 15-026. To accelerate screening for CLas-killing transgenics, a detached leaf inoculation method was developed via CLas+ ACP no-choice infestation. This high throughput lab based method can test plants at small seedling stage, is non-destructive, and provides guiding information on assessment 6-12 month earlier than greenhouse based tests. To evaluate AMPs for potential use in CLas-killing transgenes, we have developed an in vitro procedure for directly measuring their ability to disrupt CLas cells. A homogeneous CLas suspension is recovered by macerating CLas+ ACP in a specially developed extraction buffer and removing cellular debris through spin filter centrifugation. Homogenates are exposed to AMPs vs controls for 4 hours. CLas cell integrity is determined by use of a photo-reactive DNA binding dye. Small plant destructive inoculation assays, where all plant tissues are weighed and sampled after no-choice CLas+ ACP feeding, now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. This method is being used in our transgenic efforts to validate the detached leaf assay results. A modified plant Thionin (Mthionin), was designed by G. Gupta using biophysical modeling. Transgenic expression of this peptide in Carrizo citrange showed a marked and highly statistically significant decrease in symptoms when challenged with Xanthomonas citri, the causal agent of citrus canker. When transgenic plants were challenged through graft inoculation with HLB infected material, both transgenic Carrizo tissues and non-transgenic scion (Rough Lemon) tissue showed reduced CLas titer up to 12 month (latest time-point) post graft when compared to control plants, with root CLas titer 1800 times greater in wild-type Carrizo. We are testing Mthionin transgenics in the field. Several of our best lines are at DPI for cleanup and broader field trialing, ideally head-to-head with transgenics from other programs Newer generations of AMPs, categorized as 2nd and 3rd generation, were designed (also by G. Gupta) using citrus native thionin as the foundation combined with other citrus genes with high affinity for CLas membranes. Numerous lines and events of 2nd and 3rd gen AMP transgenic citrus have been subject to the detached leaf assessments. Some showed promising CLas clearance; indicated by transgenic Carrizo leaves showing significantly lower CLas titer compared to wild type controls after a 7-day ACP infestation. Interestingly, bacterial quantity in the ACP bodies was also lower after feeding on the transgenic leaves, suggesting an uptake of AMPs into the insect body and disruption of gut bacterial cells. Several of our best lines are also at DPI for cleanup and broader field trialing. ScFv sequences targeting CLas outer membrane proteins were developed by J. Hartung and used for the creation of transgenes disrupting the CLas infection process. Transgenic plants are showing a consistent and statistically significant decrease in Clas titer twelve months after no choice CLas+ ACP inoculation (up to 250x reduction, measured by qPCR) and have a much higher incidence of plants with no measurable bacterial DNA amplification. Approximately 120 additional rooted cuttings were propagated for field trials, with the primary focus being rootstocks to protect conventional scions. We sought (with W. Belknap and J. Thomson) to identify highly expressed genes in the citrus phloem, reasoning that their promoters might be useful for transgenics combatting HLB. Through this work, a citrus gene family was identified and characterized encoding a group of Small Cyclic Amphipathic Peptides (SCAmpPs) with highly conserved gene structure, but considerable variation in the ultimate gene products. Variants of a tissue-specific SCAmpP promoter were tested using GUS as a reporter gene and resulted in excellent phloem-specific expression: 500x greater expression in leaf midribs/petioles compared to laminar area and visibly greater GUS gene product activity in midribs and vascular tissue compared to GUS driven by D35S. These citrus promoters are being used in all new transgenics from our program, usually with a parallel set of transgenics driven by D35S, to combat gram-negative pathogens in other citrus tissues.
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