1. Please state project objectives and what work was done this quarter to address them: Objective 1. Investigate effects of rootstock propagation method and the interaction with rootstock on root structure, root growth, and tree performance during the first 3 years of growth in the field.Bimonthly root growth measurements with the minirhizotron imaging system continued in all trials. Leaves were collected in trials 2 and 3 for CLas detection; samples are being analyzed. Trials 2 and 3 third-year horticultural data collection (tree size, health ratings, etc.) was completed. We also counted and collected fruits for fruit quality analysis and yield determination in trials 2 and 3. There were very few fruit in trial 2, but fruit quality analysis was completed for trial 3. We are working on completing the analysis of the year 3 data of trials 2 and 3 to be included in a publication of all three years of data. Objective 2. Investigate if trees on rootstocks propagated by tissue culture or cuttings differ in susceptibility to Phytophthora-induced decline or wind-induced blow-over compared with trees on rootstocks propagated by seed.Monthly root growth measurements with the rhizotron imaging system continued. We are still working on the PCR and ELISA assays. 2. Please state what work is anticipated for next quarter: We will continue with our minirhizotron root imaging and other data collection. We will continue with the statistical analysis and interpretation of all data collected. A Citrus Industry Magazine article will be prepared sumarizing findings of this project. 3. Please state budget status (underspend or overspend, and why): Approximately 65% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.
1. Please state project objectives and what work was done this quarter to address them: Objective 1. Investigate rootstock effects on horticultural performance of Valencia and Hamlin trees commercially grown under HLB-endemic conditions using standardized field data collection procedures.We conducted additional HLB foliar disease symptom and canopy color ratings at both Valencia trials for seasonal comparison. We also collected leaves for additional nutrient analysis and CLas detection.Valencia fruit samples were collected at the Basinger location for fruit quality analysis in advance of the harvesting. Fruit quality analysis was completed for these samples at the CREC pilot plant.We are working on the leaf CLas analysis.We are continuing with the statistical analysis of all new data and are working on a manuscript for publication of three years of Valencia data. Objective 2. Develop outreach to transfer information to growers and other industry clientele.Nothing to report in this quarter. 2. Please state what work is anticipated for next quarter: Fruits will be collected from the Valencia trees at the Lake Wales (Camp Mack) location for fruit quality analysis, and yield will be assessed. We will continue with the data analysis. A rootstock seminar including trial updates will be given in June at SWFREC (or virtual, depending on the Covid-19 situation). 3. Please state budget status (underspend or overspend, and why): Approximately 64% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.
We generated raw sequence data for Valencia orange (S, sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, tolerant), and Lisbon lemon (T) and preliminary assemblies and analyses were carried out. Because of reduced sequencing costs, we were able to enter additional important genomes into the pipeline beyond those originally proposed, including Carrizo citrange, sour orange, and Shekwasha (an important breeding parent for HLB tolerance); these also have now been sequenced and assembled. We performed Hi-C sequencing with two genomes and incorporated these data with PacBio sequence of one of our target genomes resulting in an improved chromosome scale assembly. The two parental chromosomes of the target genome were phased/separated using Illumina short reads from citrons, pummelos and mandarins. By genome alignment and comparison to the Poncirus assembly (see below), minor assembly errors in repetitive regions have been fixed, resulting in a polished assembly; transcript sequencing for annotation (i.e., identify all the genes within the genome) currently is in progress. The availability of high-quality assemblies for the 3 basic species (C. medica, reticulata, and maxima) will allow a more thorough and complete characterization of large-scale structural variation (SVs: deletions, insertions, etc.) in genomes of commercial interest. These SVs are the driving force for phenotypic diversity especially among somatic mutants (e.g., different oranges, grapefruits), and this information will become more important as we test different sweet orange mutants exhibiting enhanced tolerance of HLB. A manuscript is in preparation on this work. As an example of the utility of these quality-improved new citrus genome assemblies, we have examined the polyembryonic allele associated with a MITE transposon insertion in the promoter of the CitRKD1 gene in the mandarin lineage, for sequence completeness. Previous reference assemblies either lacked this allele (the MITE insertion obviously is absent in the monoembryonic Clementine reference) or were missing important sequence in the promoter region of the gene (in the sweet orange reference assembly from China). Full knowledge of the polyembryony gene in citrus is important because it is the basis for rootstock propagation by nucellar seedlings, and it is an impediment to breeding by preventing the ability to make hybrids using polyembryonic seed parents. More importantly, this example demonstrates the quality of our assemblies; lacking promoter sequence for important HLB-resistance candidate genes could lead to CRISPR editing failures, which is exactly what we proposed to prevent through successful achievement of the objectives of this project. We completed and published on the first ever high-quality reference genome of Poncirus trifoliata using the same pipeline used for this project, and a manuscript was published in The Plant Journal (https://doi.org/10.1111/tpj.14993), and the sequence has been released to the global citrus research community through Phytozome and the Citrus Genome Database. By mining this new genome, we identified candidate genes within previously identified chromosomal regions for HLB tolerance, including a transcription factor gene and one disease resistance-like gene that are up-regulated by CLas and positively selected in trifoliate orange. These genes are promising candidate genes for further research and were highlighted in our published manuscript, so other researchers may also begin to explore their potential.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Seeds were extracted from 27 candidate rootstock seed source trees, including several UFRs and other experimental rootstocks, that were fund to be free of seed transmissible pathogens by DPI testing. We are authorized to distribute seeds to licensed nurseries for the UFRs, and to collaborative nurseries and other organizations within Florida, in the US, and globally, to establish advanced trials to compare our best rootstock selections with industry standards. We updated and added new data to existing rootstock trial files and added new files to our website (https://crec.ifas.ufl.edu/citrus-research/rootstock-trials/), currently there is information from 24 locations. We planted seed from 2020 diploid and tetraploid rootstock crosses into calcareous, high pH soil inoculated with two species of Phytophthora, the first step of the `gauntlet’ screening. We stepped up rooted cuttings from more than 50 gauntlet rootstock candidates to citripots in preparation for grafting of replicate trees as necessary to meet the new CRDF guidelines for Stage 1 rootstock evaluations (approximately 500 liners). 2. Develop new, HLB-tolerant scion cultivars from sweet orange germplasm, as well as other important fruit types such as grapefruit, mandarins, and acid fruit. Embryo rescue from 37 interploid crosses made using selected HLB tolerant plants in 2020 was completed, including 10 crosses for red grapefruit improvement, 10 for sweet orange improvement, and 10 for mandarin improvement. Shoots have been generated from many of the excised embryos, and flow cytometry was used to verify triploidy among some of the hybrid populations. Micrografting of the earliest to germinate normal shoots has begun and will continue through the next quarter. Seeds harvested from 2 diploid crosses made for sweet orange improvement were planted and have germinated. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. Evaluations of existing breeding populations in the field were somewhat restricted by UF COVID travel regulations, but we nonetheless went through our materials and made selections for further evaluations and testing. Four new HLB tolerant red grapefruit hybrids, one sweet-orange like hybrid, and three seedless easy to peel mandarins were selected and submitted to the DPI Parent Tree Program for cleanup and production of certified budwood for future trials. We have continued monitoring a unique hybrid family of more than 400 individuals (with many of these planted as 3 tree replicates) from the cross of Clementine mandarin with a wild species reported by numerous sources to be nearly resistant to CaLas attacks, by collecting detailed HLB phenotypic data, including Ct values and other tree health measures as described previously. The frequency of CaLas-negative trees remains unexpectedly high.4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. We selected ~ 450 mandarin hybrids for GWAS studies, using the data referred to in Obj 3 above. DNA samples were prepared, each individual was genotyped using the citrus Axiom SNP array, and GWAS analysis is proceeding. This work will validate previously identified, or identify new genomic regions, associated with HLB tolerance or sensitivity. 5. Other related activities. We have continued testing the alternating temperature chamber regime to produce CaLas- and CTV-free lines of promising new scion and rootstock candidates for propagation and to hasten their use in advanced field trials. Five Vernia seedling selections, one grapefruit hybrid, and ten rootstocks were treated, and all were PCR negative, and will now be tested for CTV.
1. Please state project objectives and what work was done this quarter to address them: This report covers the period of December 1, 2020 – February 28, 2021. The objective of this project is to test transgenic ‘Ducan’ grapefruit trees expressing an anti-HLB antibody fused to the FT (Flowering Locus T) protein (FT-scFv protein). Several accomplishments were made during this reporting period. Graduate student Mr. Chad Vosburg was able to take a trip to Florida to assess trees, take samples, and send samples for qPCR analysis at Southern Gardens. Chad visited Fort Pierce for more than one month from late December, 2020, to late January, 2021. He took samples from a field trial natural inoculation, an Asian citrus psyllid (ACP) infection in the greenhouse, and a graft challenge with FT-scFv scions grafted to HLB-infected rough lemon rootstocks. These samples were prepared and sent to Southern Gardens for qPCR detection and quantification of ‘Candidatus Liberibacter asiaticus’ (CLas) within the tissues. Data are anticipated to be received from these samples in March, 2021. In addition, growth data were taken from FT-scFv scions on infected rough lemon. The transgenic scions appeared to be consistently growing faster and more robustly than the control non-transgenic scions. However, these data are still being analyzed and we anticipate full analysis in the next reporting period. A second run of this experiment was initiated by additional grafting of transgenic and control scions to infected and uninfected rough lemon during Chad’s most recent visit in order to determine whether this effect is replicable. Finally, our publication of the accelerated blooming phenotype of FT-scFv transgenics was published in the February issue of Plant Biotechnology Journal and was honored with being selected as the cover feature of that issue. A copy of the finalized publication and journal cover is included in this report. 2. Please state what work is anticipated for next quarter:qPCR data from the field, graft, and psyllid-transmission HLB challenge tests will be received from Southern Gardens in March, 2021. These data will be analyzed during the next reporting period. Additional sampling of plants from the psyllid-inoculated and field grown trees will be performed with help from collaborators at University of Florida and the USDA USHRL. These will be sent to Southern Gardens for qPCR CLas quantification for an additional infection time point. In March, 2021, we plan to submit a request for a second, six-month, no-cost extension in order to complete the time courses for CLas quanitification and characterization of any potential HLB tolerance of the FT-scFv transgenic lines. This no cost extension request will be especially critical to completing the field test of the transgenics, since so far we do not detect CLas infections in those trees. In addition, it will be important to re-test the graft-transmission response of the FT-scFv scions to CLas infection, since it appears that they may have some tolerance to CLas infection. A no-cost extension would also enable a summer visit by a student or the PI to Fort Pierce to perform additional sampling and tree assessments. 3. Please state budget status (underspend or overspend, and why):Budget spending is on track, considering delays due to COVID-19. An amended budget by was developed and submitted in February, 2021.
1. Please state project objectives and what work was done this quarter to address them:The project objectives are: 1.Evaluate existing transgenic Carrizo and Swingle AtNPR1 overexpressing rootstocks in the laboratory and greenhouse. 2. Conduct a replicated field trial with the best transgenic rootstocks budded with non-transgenic `Valencia’ and test for GMO gene products in the fruit or juice. 3. Produce additional transgenic rootstock lines and stack other gene(s) responsible for SAR using mature transformation. 4. Evaluate transgene segregation analyses of the rootstock progeny and large-scale propagation of select lines.We have continued on the evaluation of transgenic trees in the greenhouse and field. Trees in the greenhouse have been tested against HLB and Ct values have not significantly changed from the earlier sampling. We have detected lower Ct in some lines but the majority have remained similar to last sampling. Western blotting of several newer lines has been conducted to identify high expressing lines. Most transgenic lines with a stacked construct (NPR1 with other transgene) have been produced. AtNPR1 overexpressing seed source trees will be planted in field for production of seed as some have had to be rebudded due to failure of the bud union or tree mortality from Phytophthora infection. We have produced several trees to serve as interstocks and they will be evaluated in the next cycle 2. Please state what work is anticipated for next quarter: We will continue on our evaluation of the transgenic lines, propagate additional lines for evaluation and test the interstock trees against HLB. 3. Please state budget status (underspend or overspend, and why): We are on track with the budget and have not overspent.
1. Please state project objectives and what work was done this quarter to address them: The objectives of this project are to produce disease resistant, commercially & agronomically acceptable, mature citrus transgenics & intragenics that will flower & fruit naturally using Agrobacterium & biolistics for research & commercialization.The research focus of this project is to improve Agrobacterium & biolistic transformation efficiency of mature citrus, so that the mature citrus protocols become more productive, decrease prices for scientists, & contribute more to financial self-sufficiency of our lab. We made great strides in increasing transformation efficiency of some cultivars in Year 2. Another objective was to find a citrus selectable marker that functions well in citrus transformation as an alternative to the nptII gene & this was also accomplished in Year 2. During this last quarter, we have been short-staffed since Jan 21 because our growth room assistant was in a motorcycle crash & he was on a ventilator for 3 weeks & he had to undergo rehab. Thankfully he will be ok. We have been busy in the growth room with much physical labor because mature transformation process requires it. Thankfully Dr. Dutt had a visiting student who assisted in the growth room during this time. One genetic construct submitted by a faculty member did not work at all, so no transgenic tissues or plants were produced. Another 3 genetic constructs submitted by a different faculty member produced shoots, but he did not want these transgenic shoots micrografted. He only wanted photos of the shoots. Because of these unusal orders, we have not been producing plants for significant monetary gain. However, we did produce ~41 transgenic scion shoots for ourselves, testing our new selection method. We have also been testing different rootstocks developed by Dr. Grosser for micrografting to see if there are better alternatives to Carrizo to lessen losses in micrografting transgenic shoots. We tested different basal tissue culture media for grapefruit & found that one works best for regenerating shoots & that it is superior to the standard sweet orange protocol. It still remains to be seen whether Agrobacterium-mediated transgenics can be produced using this medium or whether mature grapefruit cultivars are recalcitrant to Agrobacterium. We have conducted numerous biolistic transformations & tested different variables to enhance transformation efficiency. Bombardments were conducted with different vectors, cultivars, & treatments, but results are still pending. A spontaneous mutant of Early Valencia 1 (EV1) was discovered in the growth room. The leaves are enormous compared to the non-mutant (wild-type) EV1, so perhaps it has become a tetraploid. This tree had only undergone the process of shoot-tip grafting for plant introduction in previous years, but there was no 2,4-D in the shoot-tip grafting medium to induce mutations. Apparently Valencia derivatives undergo spontaneous mutations frequently. We will bud this mutant & the OLL20 mutant previously discovered from FDACS, challenge them with HLB, to determine whether they have HLB disease resistance. Mutations are the most frequent source of new cultivars in citrus. 2. Please state what work is anticipated for next quarter: Work will continue for two scientists who have submitted a number of vectors. Another scientist might be interested in testing Arobacterium transformation of grapefruit now that we have optimized the medium for shoot production. We are also finishing two manuscripts on the plant selectable marker & enhanced transformation of mature citrus. 3. Please state budget status (underspend or overspend, and why): CRDF funding is sufficient, however we will probably overspend the Director’s account since we have not been producing plants for much monetary gain, unless we can make up for this in later quarters.
True sweet oranges: The most significant results from the grant period were the identification of new sweet orange candidates that repeated for early maturity (consecutive years), and are candidates to replace Hamlin: Nine December 1 – maturity Vernia clones identified (seedlings from selected Vernia somaclones, growing in the Mathew Block at the Lee Family Alligator Grove with no psyllid control): R25-T2,R25-T5, R25-T7, R25-T9, R25-T12, R26-T2, R26-T10, R26-T12 & R26-T14. A January-maturity OLL clone also repeated for early maturity: FB-4-13 (seedling selection from selected OLL somaclones, growing in Frank’s Block 13-E at Lee Family Groves in St. Cloud with no psyllid control). Four of the early Vernia clones and the early OLL clone were entered into the Parent Tree Program (PTP), using out-of-cycle slots kindly provided by Ben Rosson and Kristen Aslan at DPI. Orange-like hybrids: A seedless triploid orange-like hybrid C4-8-25 [SugarBelle x (Succari sweet orange + Murcott)] was identified with October maturity, showing 11.85 brix, 5.99 soluble solids and 35+ color score. This selection was entered into the PTP. Another seedless triploid orange-like hybrid C4-7-29 [Nules Clementine x (Page + Ortanique)] was identified with December/January maturity, showing 15.19 brix, 7.98 lbs. solids and 43+ color score. This hybrid is showing good HLB tolerance and is already through the PTP, available for expanded trials. Both of these selections have good juice blending and fresh market potential. Another orange-like hybrid 1859 with good HLB tolerance continued to be a favorite with both processors and fresh fruit packers in our fruit displays. This selection is just through PTP, and budwood is being increased for advanced trials. Cybrid Flame Red Grapefruit clone C4-3-32 fruit repeated for making 12 brix, and was highly rated in our January fruit display. The original tree continues to show better HLB tolerance than standard grapefruit, and this selection should be through the PTP sometime this summer. Tree health index data was collected from the following trials: Duda, Wheeler Bros., Peace River Growers, IMG, Tom Hammond Minneola, Banack, Smoak, Steve Brewer, Bryan Paul and Citra trials. Juice quality data was collected from the following trials: Wheeler Bros., Citra, Heller Bros., and the Castle Teaching Block Trial. Yield data was collected from Wheeler Bros., IMG, Tom Hammond Minneola, and Bryan Paul trials. Data analysis and entry onto the Rootstock Data Website: Lykes, Premier, Heller Bros., Greene, Wayne Simmons, Peace River, Jackson Citrus, English Bros., Citrus, IMG, Duda, Lee Family Groves, & Citra trials were reposted to correct UFR codes and/or to add new data; new trials posted included: Banack Fellsmere Scion Rootstock Trial #25; and Raley Vernia Rootstock Trial #26 (Dundee). Data analyses for subsequent trial updates was performed for (in progress): Wheeler Bros., Heller Bros., Lykes Camp Mack and Basinger, Wayne Simmons, Bryan Paul Doe Hill, Duda, Smoak, Teaching Block, & Tom Hammond Trials. Data analyses also conducted on Steve Brewer, Hidden Golf Trailer Park, Orange Co. Somaclone, Cody Estes, Serenoa and Post Office trials.
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 is addressing the need of establishing field plantings to generate regional, updated information for the Indian River IIR) 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) 36 rootstocks with Ray Ruby grapefruit as the scion, T3) 30 rootstocks with Glenn 56-11 navel orange, and T4) 30 rootstocks with UF-950 mandarin.
We planted 3,400 trees in Sep/2019 and 1,100 trees in Aug/2020 and are waiting for the remaining trees on UFR rootstocks from 7 through 14 to be delivered by the nursery (Spring/2021). Masters student started on Jan/2020. Controlled-release poly coated fertilizer was applied in Sep/2019, Jan, May and Sep/2020, Jan/2021. Irrigation controller, sand media filtration system and water flow meter were installed. 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. Hoop boom was modified to spray young trees with higher accuracy, reducing the waste of agrochemical products. We created a location map and began production and distribution of QR tags to be used with scanner codes during data collection in the field.
Tree height, tree width in two positions (E-W/N-S), and trunk diameter were measured in three central trees from each experimental plot in Feb, July and Nov/2020, and Feb/2021, and canopy volume calculated. have been presented at the 2020 ASHS annual conference (https://ashs.confex.com/ashs/2020/poster/eposter.cgi?eposterid=367). The following summary data reflect the best and worst treatments in each trial during the first 12 months of growth.
Preliminary results from Oct/2020 show some scion-rootstock combinations are exhibiting significant differences in canopy volume. In Trial 1, Pummelette UF-5-1-99-2 grapefruit on US-942 was 4x larger (0.2 m3) than Star Ruby Gft DPI-60 on X-639 (0.05 m3) (P<0.0001). In Trial 2, Ray Ruby grapefruit 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). In Trial 3, Glenn F-56-11 navel orange on 2247x6070-02-2 was ~5x larger (0.25 m3) than on Willits (0.04 m3) (P<0.0001). In Trial 4, UF-950 mandarin on US-897 was ~5x larger (0.25 m3) than on WGFT+50-7 (0.04 m3) (P<0.0001). Cultivars grafted on different rootstocks have no differences in tree phenological stage. Leaf samples were taken for HLB diagnostic and bacterial titer quantification, and on average treatments resulted negative (Cycle threshold > 32) by Sep/2020, despite the infection is spreading and individual plants are getting infected.
Asian citrus psyllids, Diaprepes root weevils, whiteflies, and other insects are less abundant in the field, except for leafminers, which caused severe damage due to the excessive rainfall and wind gusts that made pesticide application challenging during this quarter. Nonetheless, tree growth has not been significantly affected by these pests.
Leaf samples for determining HLB incidence were taken from a pool of trees from each experimental plot in May and Sept/2020, and Feb/2021 and sent to the Southern Gardens lab for analysis; on average, all samples tested negative (no trees with Ct values <32) but there are several positive trees with visible symptoms. Fruit phenology, pests and diseases have been monitored monthly. Canopy thickness, canopy color and HLB incidence have been measured quarterly in all experimental plots. The Ferrarezi Lab organized a very successful drive-thru field day to showcase the results to growers on 10/09/2020 with 49 attendees (limited by covid-19 regulations). An estimated 24,000 acres of citrus were represented at the event (70% of the current grapefruit industry acreage, highlighting the importance of the event and my program engagement with the industry). Attendees came from local and neighboring counties including St. Lucie, Charlotte, and Okeechobee. Another field day took place on 12/10/2020 with 4 large growers and industry leaders. Overall, trees are building up vigorous canopies, and morphological differences among scions/rootstocks are beginning to show. The study is providing paramount information on young scions and rootstocks with remarkable horticultural attributes. However, longer-term evaluation is required to identify the most promising scions and rootstocks to determine their profitability and capability of meeting grower and market needs.
Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is ongoing. As the most proven of our transgenics, we continue to use them as a reference in detached leaf assays, as well as studying them in established greenhouse and field trials. 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 has continued for the two Mthionin field plantings. The first plantings of transgenic or control Carrizo grafted with non-transgenic rough lemon scions (45 total plants) have shown transgenics maintaining higher average CLas CT values (2.5 CT higher @ 18 months), but with a high degree of variability. The larger second planting of transgenic Carrizo with WT Hamlin scions, transgenic Hamlin on non-transgenic Carrizo and WT/WT controls (205 total plants) have shown very encouraging results; with the transgenic Hamlin on WT Carrizo having statistically better trunk diameter, tree height and canopy volume compared to controls. Scheduled assessments for both plantings have been prioritized under pandemic conditions, allowing the 36 month field assessment of the first planting and 24 month assessment for the second planting to be completed. Leaf samples associated with these assessments have also been collected and are being processed for CLas quantification. Additional grafts of WT Hamlin and Ray Ruby scions to Mthionin root-stock were made and are included in the ongoing 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 56 putative lines from these transformations are in soil and undergoing expression analysis. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, have been conducted for lines expressing chimera constructs TPK, PKT, CT-CII, TBL, BLT, LBP/’74’, ’73’, and ‘188’ (as well as scFv-InvA, scFv-TolC, Topaz and Onyx). esting of all 35s driven 3rd generation Carrizo lines is complete and the analysis of phloem specific and scion-type lines is well underway. This work has already identified numerous lines with significant effects on CLas transmission and increased ACP mortality (up to 95% from TBL and >70% from TPK). The best performing lines have been moved forward to greenhouse trials. Initial ACP-inoculated greenhouse trials on 8 lines of citrus Thionin-LBP chimeras (’73’, and ’74’) showed a statistically significant reduction (13x) in CLas titer for ’74’ transgenics vs WT in the CLas+ plants. However, many plants remained CLas negative due to low inoculation efficiency. In June, 150 plants representing the best performing 7 lines of `188′ and 6 lines of `74′ were no-choice caged ACP inoculated using a new protocol to improve inoculation rates. At 3 months, control plants tested positive at twice the rate of the earlier inoculation; 6-12 month tissue samples are now collected and processed, awaiting qPCR analysis. A large greenhouse study is underway to 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) were made and bud inoculated with CLas+ RL to ensure high transmission. The first leaf collection will be collected in March. An additional ~1200 rooted cuttings have been made from those same lines for matching ACP-inoculated greenhouse and field trials. An earlier field planting of 1st and 2nd generation lines (~400 plants of Mthionin, `74′, and `188′) is also underway. The first 165 plants (WT Hamlin and Ray Ruby on transgenic Carrizo) went into the soil in August 2020, a second set of 70 WT Valencia on transgenic Carrizo are ready to be moved to the field this season and the remaining 200 transgenic Hamlin on WT root-stocks are being grafted. Eighteen new transformations, totaling over 6200 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 325 new putative transgenic lines from 74-Valencia, 74-Ray Ruby, 74-US-942, 74-Hamlin, 188-Ray Ruby, 188-Valencia, 188-US-942, TBL-US-942, TBL-Hamlin, TBL-Ray Ruby, TPK-Ray Ruby, TPK-US-942 and TPK-Hamlin are now in soil and undergoing expression analysis. Objective 3, ScFv Constructs: ACP inoculated greenhouse studies on 5 scFv lines has been completed with transgenics showing significantly reduced CLas titer (up to 250x reduction) and a significantly higher incidence of no CLas rDNA amplification in roots and leaves compared to WT. These lines have been grafted with WT Ray Ruby scions and are undergoing field trials at Picos farm. Their first assessment will be in March 2021. An additional 129 rooted cuttings are propagated for follow up plantings with a Hamlin scion. A second greenhouse trial (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 ACP-inoculated using the improved protocol. Tissue from both trials for testing CLas titer 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 screened through this assay have since been confirmed to reduce CLas titer by foliar application to grapefruit trees in tests performed by CRADA partners. Citron, Hamlin and Valencia trees have been selected and blocked for trunk application trials using these peptides. Transgenic Nicotiana benthamiana plants expressing His-6 tagged variants of the chimeras TBL, TPK, PKT and LBP have also been generated to produce sufficient protein extracts for use in exogenous applications. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; adjusting feeding period and ACP numbers. We have also expanded the analysis of ACP bodies to include quantification of other major endosymbionts (Wolbachia, Profftella, and Carsonella) to better investigate the activity of peptides causing CLas mortality. 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′,`188′, TPK, TBL 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 and its chimeric variants, new strategies have been implemented in our Laboratory to fight HLB. These efforts include the expression of insecticidal peptides to control ACP (CLas vector) and 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 (SCAmpP-3) promoters were evaluated for their ability to kill ACP. From these, 12 lines (4 event of each genotype) showed significant ACP mortality and were selected to move up in the screening pipeline for HLB/ACP tolerance. Also, 24 Carrizo transgenic events highly expressing Onyx (a code name to protect IP), a peptide with antimicrobial and insecticide activity, were evaluate by DLA. The 5 Onyx lines showing high ability to kill ACP (to 83% mortality) were selected for further evaluation. These strongly performing lines were replicated as rooted cuttings (55 Onyx and 131 Topaz plants) that will enter greenhouse trials as soon as the plants are of appropriate size. The available Onyx transgenic material is being expanded with 40 Hamlin and Carrizo plants transformed with the phloem specific version and 33 Ray Ruby and Valencia plants with the constitutive version undergoing expression analysis. Down regulated DMR6 Carrizo transgenic citrus, either by expression of specific hairpin RNA or by specific Cas9-sgRNA were generated, cloned, and are ready to be assessed. Since DMR6 is a broad immune suppressor, downregulated transgenic plants will be first evaluated for Canker resistance as a quicker assay. For that, clones from 5 selected lines are being inoculated with Xanthomonas citri and data is to be collected soon. As an effort to accelerate the development of non-transgenic HLB resistant plants through gene editing, we transformed early flower transgenic plants (carrying FT-SCFV gene) with the DMR6 targeting CRISPR construct. A set of 30 plants resulted from this gene stacking effort will be evaluated for the presence of both genes. Objective 5, Transgenic product Characterization: Experiments are also underway track the movement and distribution of transgene products using antibodies and affinity tagged protein variants. CLas+ RL have been grafted as scions onto MThionin expressing Carrizo as a platform to test peptide movement and effects across the graft union. Transgenic Carrizo lines expressing His6 and/or Flag tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), PKT (20 lines), scFv-InvA (22 lines) and scFv-TolC (18 lines) have been generated and expression confirmed by RT-qPCR. Total protein samples have been extracted from His-tagged transgenic lines and sent to our CRADA partner for testing
The purpose of this project is to optimize the CRISPR technology for citrus genome editing. This study is related to the CRDF RMC-18 Research Priorities 4AB. Objective 1. Expanding the toolbox of citrus genome editing. In this study, we will adapt StCas9, NmCas9, AsCpf1 (from Acidaminococcus), FnCpf1 (from Francisella novicida) and LbCpf1 (from Lachnospiraceae) on genome modification of citrus. Lately, we have shown CRISPR-Cpf1 can be readily used as a powerful tool for citrus genome editing. In our recent study, we employed CRISPR-LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. Our study showed that CRISPR-LbCas12a can readily be used as a powerful tool for citrus genome editing. One manuscript entitled CRISPR-LbCas12a-mediated modification of citrus has been published on Plant Biotechnol J. We are currently further optimizing LbCas12a-crRNA-mediated genome editing to make homologous biallelic mutations. We are also testing AsCpf1 and FnCpf1 for their application in citrus genome editing and generating homologous biallelic mutations. We have successfully generated both homozygous and biallelic mutations in the EBE region of LOB1 gene in pumlo. This work has been submitted for publication. We are in the process of generating homozygous and biallelic lines of other citrus varieties.Recently, we have developed multiplex genome editing toolkits for citrus including a PEG mediated protoplast transformation, a GFP reporter system that allows rapid assessment of the CRISPR constructs, citrus U6 promoters with improved efficacy, tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we have successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, indicating that the CRISPR-mediated citrus genome editing technology is mature and could be implemented in citrus genetic improvement as a viable approach. In addition, our study lay the foundation for non-transgenic genome editing of citrus. One manuscript entitled Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations has been published on Plant Molecular Biology.We have successfully developed base editing tools for citrus genome editing. We have further optimized LbCas12a. Objective 2. Optimization of the CRISPR-Cas mediated genome editing of citrus. In this study, we are testing different promoters including INCURVATA2 promoter, the cell division-specific YAO promoter, and the germ-line-specific SPOROCYTELESS promoter, and ubiquitin promoter in driving the expression of Cas9 and Cpf1 orthologs. To optimize the expression of sgRNA and crRNA, we have identified multiple citrus U6 promoters and two of the citrus U6 promoters showed higher efficacy in driving gene expression in citrus than 35S promoter and Arabidopsis U6 promoter. We have further increased the mutation efficacy to 50%. We have generated one homozygous line in the promoter region of canker susceptibility genes of Hamlin. We have successfully generated one biallelic mutant of grapefruit that is canker resistant. We also successfully generated multiple biallelic and homozygous mutant lines of sweet orange that are canker resistant. Objective 3. Optimization of the CRISPR technology to generate foreign DNA free genome editing in citrus. We have conducted transient expression of Cas9/sgRNA plasmid and Cas9 protein/sgRNA ribonucleoprotein complex in citrus protoplast. We are also conducting citrus genome editing using Cpf1/crRNA plasmids and ribonucleoprotein complex in citrus protoplast. The plasmid-transformed protoplast has 1.7% editing efficiency, and the RNP-transformed samples have approximately 3.4% efficiency. The genome modified protoplast cells are undergoing regeneration. We aim to increase the efficacy to over 20% and eventually generate non-transgenic genome modified citrus. One patent has been filed on the CRISPR-Cas mediated genome editing of citrus. We have lately optimized the citrus protoplast isolation and manipulation, our data showed that more than 98% of the isolated protoplasts were alive. We regularly obtained a transfection efficiency of approximately 66% or above. Genome modified lines in canker are being regenerated. We are editing several 6 putative HLB susceptibility genes for sweet orange.
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 next set of cover crops were planted at the end of Nov 2020. These included sunnhemp, Austrian winter pea, daikon radish, oats, and winter rye. Analysis of the soil nutrient and microbial samples collected in August 2020 indicates similar patterns to Year 1: increased bacterial abundance in cover crop treatments, and increased abundance of nitrogen cycling genes with cover crops. In addition, preliminary analyses found that the types of microbes performing denitrification (part of the nitrogen cycle) are completely different under the legume+non-legume cover crops compared to the other treatments. Soil organic matter has also significantly increased under cover crop treatments. There has been an increase in the abundance of bacteria under the canopy of trees in the legume+non-legume treatment. Analysis of weed data from 2020 shows that cover crop planting has resulted in suppressing weeds up to 98% in one location and 69% in the second location. The observed trend in weed suppression is in line with the previous year. The germination and establishment of cover crops in the second location appear to be improving over time, as noted from the comparisons of cover crop density in planted row-middles between 2019 and 2020. 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 at the end of November 2020 with the planting of the next round of cover crops. Data from Year 2 (collected in Aug 2020) is still being analyzed. 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. Quantitative data on root growth (volume, length and area), root dieback, and recent soil moisture dynamics by treatments will be presented in the next quarter. Preliminary evaluation shows that conducting eco-mowing in the row-middles has unremarkable effects on weed emergence and coverage in the tree-rows. 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 cropsThe cover crops survey was drafted and has IRB approval. The survey was also reviewed but faculty, the project team, extension agents, and is waiting one growers comments. Qualtrics was engaged to administer the survey and next steps are outlined. Our initial estimates are that cover crop establishment is cost prohibitive. Therefore, the survey includes a question on willingness to participate in cost-share programs to help with establishment costs. The partial budgeting process is ongoing. Objective 5: Communicate results to growers using field days and extension materialsPreliminary results are being presented at the Soil Science Society of America/American Society of Agronomy meeting in November. Information on cover crops and preliminary data was 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, in the spring of 2021. 2. Please state what work is anticipated for next quarter: Analysis of soil inorganic N fractions (ammonium and nitrate) for samples collected in August 2020 continues and updates on other soil/leaf nutrient variables will be provided in the next quarter. DNA from soil samples collected in August 2020 has been extracted and microbial analysis has been completed (see above). Weed data analysis will be continued and weed density data collection for the 2021 spring planting will occur in early Summer 2021. Canopy and trunk size measurements and leaf nutrient status along with root image collections and soil moisture monitoring will continue, and quantitative differences reported in the next quarter. The next set of cover crops are scheduled to be planted in May 2021. 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. The cover crop survey will be administered, and data analysis will begin. Partial budgeting analysis will continue. 3. Please state budget status (underspend or overspend, and why): We are on track with our planned budget spending.
Create new candidate hybrids. Emphasis of hybridization in the USDA rootstock program is among parents with superior tolerance to HLB, CTV, and Phytophthora, and outstanding influence on fruit yield and quality. Some of the best performing of the newest hybrids in the Stage 1 field trials are hybrids of US-942 and US-802, so additional hybrids with this parentage are being selected for further testing. Seedlings from these crosses are being grown-out in the greenhouse in preparation for propagation, testing, and establishment of seed trees. Propagate and plant new field trials. Budwood increase trees of selected scions were grown, in preparation for budding trees for new rootstock trials. Nursery trees for Stage 1 with Valencia orange, and Stage 2 rootstock trials with Valencia, Hamlin, and Star Ruby grapefruit are being prepared in the greenhouse for field planting in 2021. Some planned propagation for new trials was delayed because of institutional Coronavirus shutdown. 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 during this quarter data on yield and fruit quality from six replicated trials with Hamlin scion. Early fruit drop data was also collected from Valencia scion trials. Assessments of tree health and measurements of tree size were completed on 10 trials during this quarter, which was reduced from the normal because of the institutional Coronavirus shutdown. Progress continued in working through the backlog of brix, acid, and color for the fruit quality analysis of last season fruit quality assessments caused by institutional Coronavirus restrictions.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 is progressing more slowly than planned because of institutional Coronavirus shutdown and restrictions. Cooperative work continues to compare field performance of rootstocks propagated by seed, cuttings, and tissue culture.Posting field trial results for grower access. The USDA rootstock trials produce large amounts of information that is useful to identify the most promising of the new hybrids, as well as comparative information on the relative performance of many commercially available rootstocks. During this quarter, updated trial summaries were prepared for uploading to the website https://www.citrusrootstocks.org/.Release of superior new rootstocks for commercial use. Release of new USDA rootstocks is based on robust data from multiple trees in replicated field trials over multiple years, including information on tree survival and health, canopy size, fruit yield and fruit quality, and observations on tolerance of disease and other biotic and abiotic threats. Several of the 350 advanced Supersour rootstock hybrids in field trials are exhibiting outstanding 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.
1. Please state project objectives and what work was done this quarter to address them: The objectives of this project are to produce disease resistant, commercially & agronomically acceptable, mature citrus transgenics & intragenics that will flower & fruit naturally using Agrobacterium & biolistics for research & commercialization. It should also be understood that it is through transgenics (gene over/under-expression) that gene function is determined & this will become an important service in the future. The research focus of this project is to improve Agrobacterium & biolistic transformation efficiency of mature citrus, so that the mature citrus protocols become more productive & decrease prices for scientists. This was accomplished for some rootstocks (Kuharski and US942) and scions (Hamlin and EV1) by selection in liquid rather than solid medium in Year 2. Another objective was to find a citrus selectable marker that functions well in citrus transformation as an alternative to the nptII gene & this was also accomplished in Year 2. Two manuscripts are in preparation: the new plant selectable marker; genetic transformation of mature citrus & selection in liquid culture. ~Thirty scion transgenics were produced, micrografted & secondary grafted this quarter. PCR of ~150 Agro-mediated transgenics was conducted to confirm the presence/absence of the transgenes in plants produced in liquid culture. Southern blots are being performed to show gene integration into the genome & copy number of the T-DNA into the genome for a manuscript. We have been working on biolistic transformation to increase transformation efficiency in anticipation of a future order from California. Numerous bombardments have been conducted, testing different mature cultivars (Hamlin, Valencia, Kuharski & US942), testing liquid culture, & testing the new citrus selectable marker. These experiments are currently in progress, so results cannot be provided yet. We might have to alter the standard protocol that we developed for immature citrus to achieve more efficient biolistic transformation of mature citrus. There are no publications of transformation of mature grapefruit. However, we attempted to produce grapefruit transgenics for Dr. Nian Wang’s group. We tested mature Duncan, Marsh, Ray Ruby & Flame using Dr. Leandro Pena’s tissue culture protocol for sweet orange, which utilizes MS or MT medium, but it did not yield results. In fact, none of the grapefruit cultivars (Ruby grapefruit, Grosser’s Red grapefruit) have ever performed well. The next step would be to test other tissue culture medium (WPM, DPW, DBA3), which differ in macro/micro-nutrients. Since Dr. Nian Wang’s group is trying to produce mature transgenics, perhaps it will test the different media. Because mature grapefruit is recalcitrant to Agrobacterium transformation, I will no longer accept orders for grapefruit until the system can be optimized. 2. Please state what work is anticipated for next quarter: I am waiting to hear from one scientist in California to see if she is interested in using our facility for a biolistic transformation project. Another scientist in California was potentially interested in Agrobacterium transformations for research (I contacted her last year) but she said Dr. Nian Wang, who is Co-PI on their grant, will probably produce their transgenics. Last week, we received a fairly large order from Dr. Zhonglin Mou, and we are working on an order for Dr. Manjul Dutt. 3. Please state budget status (underspend or overspend, and why): The CRDF budget has been received, but I am still waiting for the chart field numbers for the UF Educational Business Account (EBA). As I understand it, my EBA application is still in the CFO’s office pending signatures.
This quarter: The Bowman lab has established three new experiments with grafted Valencia trees on groups of rootstocks which were inoculated with ACP during the quarter. Each experiment compares replications on nine different rootstocks and made use of 2,520 ACP from the positive colonies. Periodic evaluation of tree health, growth, and CLas titer via PCR were conducted on trees from rootstock experiments that were inoculated during the previous months following a set schedule. Periodic colony checks were conducted by PCR to maintain CLas positive colonies. The Stover lab conducted weekly detached leaf assays (DLAs) challenging transgenic citrus with CLas inoculated by infected ACP in the lab, which is used to identify best performing transgenic events (transgenics varying by position of transgene insertion etc.). Eight detached leaf assays were run using 500 ACP each with a total of 4,000 ACP used in this quarter. Transgenic material tested in DLAs were of three constructs ( ONYX, 35s-BT and Scamp-ST), with seven events per construct equaling 56 individual plants tested alongside WT controls. The balance of Covid-19 restricted lab hours was spent processing the leaf and ACP samples in preparation for CLas qPCR. We continue to see substantial ACP mortality from feeding on CLas-killing transgenic leaves In addition 200 CLas+ ACP were provided to Dean Gabriel of UF and 600 were provided to YongPing Duan of USDA. Project rationale and focus: The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~50% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Previous quarter: Dean Gabriel of UF, and USDA scientists Kim Bowman, Ed Stover and G avin Poole have all run experiments totalling ~2000 ACP. Samples have all been collected on-time from ongoing experiments. All samples collected, that have not been analyzed, have been processed for qPCR.
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