ObjectiveThis project has one major objective for the Juvenile Tissue Citrus Transformation Facility (JTCTF) to stay open and offer the service for production of transgenic citrus plants to scientific community. By serving the researchers involved in work to find solutions against huanglongbing (HLB) and citrus canker, JTCTF remains integral part of the overall effort to assist Citrus Industry to stay profitable in this period when it is challenged by the threat of emerging diseases. Major accomplishments per objectiveThe objective set for this project was only partially met in 2020. Due to the circumstances associated with the COVID19 pandemic, JTCTF was not open at full capacity for the big part of the year. During the first quarter of 2020, JTCTF worked according to normal schedule up to March 13th. The facility was mostly closed for two weeks after that. Throughout the month of April and the beginning of May, the lab was visited on a daily basis to make sure the equipment was in order, to manage plants in the greenhouse and in the lab, and to take care of cultures from previously performed experiments. Upon the approval of the low level (Phase1) re-opening in May, JTCTF resumed some activities. Because of the surface area of the lab where most of the activities of JTCTF take place, being in Phase1 meant that only one employee can be in the lab at the time. Under such work schedule, it was not possible to run new transformation experiments. We proceeded to process the data from experiments that were already done. In the month of July, JTCTF started operating under the rules of Phase3 and it remained in that mode of operation until the end of 2020. Throughout the period between July and December, facility was able to work at about 40% of the capacity. This level of work was higher than needed because of the lack of new orders. One CREC faculty member announced the possibility of multiple orders but retracted that statement in the fall. This group also cancelled two orders and the work on two other orders was halted in the early phase until the transition of the facility into EBA unit is complete and new price list goes into effect. Employees of the facility paid from the USDA grant, continued to do transformation experiments as the time allowed them.Number of orders that JTCTF worked on in 2020 is seven, and they were all associated with the USDA grants where V. Orbovic is a co-PI. The orders were for production of transgenic Duncan grapefruit, Hamlin sweet orange, and Indian curry leaf plant (Murraya koenigii).The JTCTF produced 108 transgenic plants in 2020. Most of these plants belonged to Duncan grapefruit cultivar, there were also 19 Valencia orange plants, two M. koeinigii plants and one Pomelo plant (Table 1). Considering the role of JTCTF in the common effort of fighting citrus tree diseases, all produced plants were potentially resistant/tolerant to HLB or had ability to kill Asian citrus psyllids. None of the plants were sent outside of the state of Florida and will be used to estimate the effect of transgenes on the ability of citrus plants to sustain HLB. Table 1. Plants produced by CTF in 2020Cultivar Number of plants produced Duncan grapefruit 86 Valencia sweet orange 19 Pomelo 1 M. koenigii 2 During the 2020, JTCTF has done 54 experiments and used 88 appropriate bacterial strains for co-incubation of explants. For those experiments we cut about 80,000 explants. There were three experiments where all explants were contaminated and for seven experiments we had partial of full loss of data because they were not processed on time due to COVID19-related closure of CREC. About 25,000 explant were inspected for the presence of GFP fluorescence and about 9,000 for the pale/white phenotype. We also preformed about 700 PCR reactions with different primer pairs in search for JJ7 transgenic Valencia shoots.In an effort to secure sufficient supply of Duncan grapefruit seeds, I purchased 25 half-bushel bags of Duncan fruit in the beginning of 2020. Because of the low demand, most of the fruit rotted and we had to throw it away during the summer. We also got some Duncan fruit from CUPS at CREC. As opposed to 2019, we asked A. Schuman’s crew to harvest and deliver them to us in small installments during the fall and we managed to utilize all of them. For experiments done with the Hamlin material, we got some seeds from the people who ordered those plants while the rest came from fruit we picked from Block 22, a grove that belongs to CREC. M. koeinigii seeds were extracted from fruit harvested from trees grown at CREC.Major shortcomings, unfinished businessThe flux of employees working in the CTF remained high. One full-time employee left JTCTF in the middle of 2020 and she was not replaced. Another employee for whom there was not sufficient funding left facility at the end of 2020. The labor force projected for the demand of transgenic citrus plants in 2021 is at the level of 1.6 FTE which translates into one full time employee and one part-time employee.The transition of JTCTF into an EBA (educational business activity) unit mandated by UF administration will be completed soon. This process has been going on for the last eight months and once complete will fully re-define the operation of the facility. The opportunities going forwardMy efforts to attract business and get more people to order transgenic plants for the last year and-a-half were not successful. Others involved in management of JTCTF on the higher level are also investing efforts for that to happen. Whether these efforts will yield more orders for JTCTF remains to be seen. Disappearance of COVID19 as a threat may increase activities on all fronts and as a consequence lead to more orders. Publications from this project1) Sinn, J., Held, J., Vosburg, C., Klee, S., Orbovic, V., Taylor, E., Gottwald, T., Stover, E., Moore, G., McNellis, T. (2020) Flowering Locus T chimeric protein induces floral precocity in edible citrus. Plant Biotechnology Journal.2) Zhang, F., Rossignol, P., Huang, T., Wang, Y., May, A., Dupont, C., Orbovic, V., Irish, V. (2020) Reprogramming of stem cell activity to convert thorns into branches. Current Biology, 30:2951-2961.
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.A manuscript with the detailed findings of trial 1 has been accepted for publication in the journal HortScience and can be accessed at: https://doi.org/10.21273/HORTSCI15507-20.Bimonthly root growth measurements with the minirhizotron imaging system continued in trial 2 (Hendry County) and trial 3 (Polk County). The root core data analysis to assess fibrous root traits was completed.DNA extraction and CLas detection by PCR was completed.Leaf nutrient results were obtained and data analysis was completed. Data are currently assembled into one manuscript to be published later this year. 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.Tee horticultural measurements were conducted. Leaves and roots were collected for pathogen analysis. Leaves were collected for nutrient analysis. 2. Please state what work is anticipated for next quarter: We will continue with our minirhizotron root imaging. We will continue with the statistical analysis and interpretation of all data collected. We will start processing tissue samples for PCR/ELISA analysis. 3. Please state budget status (underspend or overspend, and why): Approximately 56% of funds have been spent which is slightly 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.Canopy color, disease and thickness visual ratings were completed in all trials. Trials were prepared for upcoming fruit sampling and harvesting, and tree identification tags were replaced in all four trials. Fruit samples were collected in the Hamlin trial at Basinger for fruit quality analysis at the CREC pilot plant. Hamlin trees at the Fort Basinger location were harvested in collaboration with the Lykes commercial harvesting crew.Objective 2. Develop outreach to transfer information to growers and other industry clientele.The complete datasets from years 1-2 were provided to the UF/CREC citrus breeding team during the last quarter and should now be available on their citrus field trial website. I continued to be engaged with CRDF representatives and attended plant improvement committee meetings to provide input on rootstock selection. A presentation Effect of rootstock on the health of HLB-infected citrus trees and the interaction with biotic and abiotic soil factors was given during the Materials Innovation for Sustainable Agriculture (MISA) 2020 virtual Symposium (Nov 2020) which was attended by CRDF representatives.The manuscript Field performance of `Hamlin’ orange trees grown on various rootstocks in HLB-endemic conditions by Kunwar SG, Grosser J, Gmitter FG Jr., Castle W.S, and Albrecht U. containing the first two years of Hamlin data was accepted for open-access publication in HortScience and will be available online soon. 2. Please state what work is anticipated for next quarter: Fruits will be collected from the Hamlin trees at the Lake Wales (Camp Mack) location and trees will be harvested. We will continue with the data analysis. 3. Please state budget status (underspend or overspend, and why): Approximately 57% of funds have been spent, which is slightly 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: This report covers the period of September 1 – November 30, 2020. 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. Work on the project was slowed a bit by COVID-19 restrictions. However, tissue sampling and phenotypic analysis was continued on three HLB inoculation tests underway for the FT-scFv plants: 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. Samples taken from the field trial trees were all negative for ‘Candidatus Liberibacter asiaticus’ (CLas) infection by qPCR at the 6-month point after planting the trees at the test site in November of 2019. Samples were taken and frozen for later analysis in November, 2020. We applied for and received permission for travel by graduate student Mr. Chad Vosburg to Florida from Pennsylvania to perform research at the Univerersity of Florida Indian River Research and Extension Center (IRREC) in Fort Pierce, FL. This included permissions from Penn State and the University of FL. To make this work possible, permission was applied for and recieved to transfer most of the citrus trees for this project from the next door United Sttaes Horticultral Research Laboratory (USHRL). Trees were moved in part in November, 2020. Photographic documentation of control and FT-scFv scions on HLB-infected rough lemon was performed in October, 2020. We also applied for permits to move replacement trees from Florida to Pennsylvania for transgene protein expression and target interaction properties. A no-cost extension was approved to extend the project operation period to the end of May, 2021. 2. Please state what work is anticipated for next quarter: Graduate student Mr. Chad Vosburg will visit IRREC for the first half of January,2021, to perform a replicate graft transmission experiment, which yielded promising results in the first run of the experiment. He will also sample trees from the field site and the psyllid-mediated infection tests, test some of these for CLas levels by qPCR, and send a remainder of them to a centralized CLas quantification facility for analysis. Trees will be transferred to our Pennsylvania lab for characterization of transgenic protein expression. Quantitative CLas analysis in field-grown, graft-inoculated, and ACP-challenged FT-scFv plants and control plants will be obtained. Plans will be made for a trip to Florida by Chad in May, 2021. 3. Please state budget status (underspend or overspend, and why): Budget spending is on track, considering delays due to COVID-19. We will submit an amended budget by January, 2021.
The project was granted a no cost extension of Year 1 into July 2020; we are now into Year 2. A comprehensive overview of progress since project inception through October 2020 follows below. Relevant Objectives are summarized below.Year 1: Objective 1: Produce suitable plant materials, isolate high-quality genomic DNA samples for sequencing, generate PacBio genome sequence data, and assemble PacBio sequence reads as sequencing data are received. Objective 2: Prepare RNA libraries for transcriptome sequencing, generate full-length transcript sequencing data on Nanopore sequencers. Year 2: Objective 1: Produce HI-C sequence data, polish and finalize genome assemblies at the chromosomal scale. Objective 2: Assemble transcriptomes, analyze gene structures, and annotate. Objective 3: Complete haplotype phasing, conduct structural and relational genomic studies, generate a list of all genes in each of the five genomes. Write manuscripts. Results Plant materials were produced and HMW-DNA samples were prepared from Valencia orange (S, sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, tolerant), and Lisbon lemon (T). We generated raw sequence data for all 5 genomes and preliminary assemblies and analyses were carried out. For four of the five genomes, the results exceeded the quality of any other publicly available citrus reference genomes, even before Dovetail Hi-C proximity ligation sequencing to finalize assembly at the chromosome level. However, the quantity of grapefruit sequence was insufficient, so we prepared a new sample Ruby Red grapefruit HMW-DNA. Technical issues with the PacBio Sequel II platform at the UCB sequencing facility, and it turns out at other sequencing centers as well, were encountered and resolved; there is now sufficient grapefruit sequence to proceed with assembly and other downstream activities. 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). 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 have been 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; materials have been collected for transcript sequencing for annotation (i.e. identify all the genes within the genome). 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). A manuscript is in preparation on this work. A previously funded CRDF project supported the initiation of a project producing the first ever high-quality reference genome of Poncirus trifoliata using the same pieline, and under this current project the task was completed; a manuscript was accepted for publication in The Plant Journal, and the sequence will be released to the global citrus research community through Phytozome upon publication. 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. Conclusions1. We completed all genome sequencing work under Year 1, Objective 1. Additional important genomes have been entered into the pipeline because of reduced costs for sequencing, and sequence reads have been produced.2. We have not yet generated all the full-length transcript sequence data, as proposed for Year 1. This goal was compromised for several reasons beyond our control, but progress is now being made.3. Hi-C sequencing for proximity ligation was completed for two genomes, and along with Illumina short reads resulted in a phased chromosome scale assembly of one target genome. 4. We have produced a genome assembly of Poncirus, an important source of resistance to CLas and HLB that exceeds the quality of all previously produced citrus genomes, using our pipeline. This genome assembly was used to repair minor assembly errors in repetitive regions of the genome mentioned in 3 above, and by mining the sequence we have identified genes to be targeted for HLB resistance.
Field variety trials are a simple but effective tool to test plant horticultural performance under different environmental conditions and enhance the commercial adoption of new cultivars. Large-scale, rapid implementation of HLB-tolerant cultivars depends on reliable data, and the Millennium Block project is addressing the need of establishing field plantings to generate regional, updated information for the Indian River Citrus District. The project has two objectives: (i) Assess performance of new grapefruit cultivars with certain rootstocks under HLB endemic conditions in the IR district and (ii) ) Evaluate the influence of UFR and other recent rootstocks on grapefruit, navel, and mandarin in the IR in comparison to legacy/standard rootstocks. Trials tested: T1) grapefruit cultivars on three rootstocks, T2) 38 rootstocks with `Ray Ruby’ grapefruit as the scion, T3) 36 rootstocks with ‘Glenn 56-11′ navel orange, and T4) 36 rootstocks with `UF-950 mandarin. We planted 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. 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 tree 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 and N-S), and trunk diameter were measured in three central trees from each experimental plot in Feb, July and Nov/2020, and canopy volume calculated. The following summary data reflect the best and worst treatments in each trial during the first months of growth. Statistical significance is omitted for the reporting purposes, and data have been presented at the 2020 ASHS annual conference (https://ashs.confex.com/ashs/2020/poster/eposter.cgi?eposterid=367).Although the trials are independent, we observed rootstock performance varied according to the scions used. In Trial 1 (grapefruit), `UF 5-1-99-2′ Pummelette (DPI-435-86) and `US 6-16-172′ Pummelo Hybrid (DPI-847-6172) on US-942 are producing the most vigorous trees. Canopy volume is 8-fold larger (4.28 m3) than `US 1-83-179’ Grapefruit Hybrid (DPI-847-US 1-83-179) on US-942 (0.5 m3). In Trial 2 (grapefruit), trees on A+Volk×Orange 19-11-8 have developed the most robust canopy, with elongated branches that can reach the ground. Overall, trees on A+Volk×Orange 19-11-8 are 3-fold larger (3.91 m3) than Orange 16 (1.24 m3). In Trial 3 (navel orange), C-22 is outperforming all rootstocks with the development of a dark green, thick canopy that is 2.5 times larger (2.67 m3) than the poorest performer, Willits (0.95 m3). In Trial 4 (mandarin), US-812 grew 3-fold larger (2.01 m3) than the lower performers Sun Chu Sha and Cleopatra mandarins, US-1283, C-57 Furr and Orange 16 (0.13 m3) in the mandarin trial. 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 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 in the project on 10/09/2020 with 49 growers attending (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 counties. 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. 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.
Tree health index data was collected from the following trials: IMG, Citra, Steve Brewer (Land-O-Lakes scion trial), Banack, Hidden Golf Course (multi-scion trial), Smoak, Wheeler Bros., Peace River, and Greene Citrus (lemon trial). All ‘gauntlet’ trees were scored for health index; large fruit size was noted on a few selections that also had exceptionally healthy trees. Tree size/canopy volume data was collected from the Orie Lee OLL clone/rootstock trial. Our field crew assisted with roof repairs to our certified greenhouses and to our repository; this was necessary to maintain their legal status as sources for pathogen-free trees to be planted across the state. Propagation of best ‘gauntlet’ rootstocks: Additional cuttings were produced from 3 promising SugarBelle hybrids and two S11 (salt tolerant Pummelo x Cleo) x trifoliate orange 50-7 hybrids. Two large grapefruit/rootstock experiments, including all of our PTP red grapefruit clones and new candidate rootstocks were planted with Bryan Paul citrus and English Bros. Citrus (many of the trees for the English Bros. trial were propagated by our team at the CREC, all others by Briteleaf Nursery). Significant emphasis was placed on data analysis and entry onto the website:Trials posted or updated in the website: August-September-October Trial Online Title Notes 1 Basinger ‘Vernia’ Rootstock Trial Format and presentation updated 3 Indian River ‘Marsh’ Grapefruit Rootstock Trial Format and presentation updated 4 Indian River ‘Vernia’ Rootstock Trial Added: 2019-20 Yield; HLB rating 5 Indian River Lemon Rootstock Trial Format and presentation updated 8 Peace River ‘Valencia’ Rootstock Trial Added 2019-20 Yield and HLB rating and RSTK table, 2020/21 HLB rating 16 St. Helena Rootstock Survey Trial – Addendum Added 2019-20 Valencia & Vernia PS/box, PS/acre, 7y cumTrials worked on during August-September-October – data analyses Trial Online Title Notes 2 Waverly Scion Rootstock Trial Added: 2020-21 HLB rating 5 Indian River Lemon Scion Trial with Two Rootstocks Format and presentation updated and new data 6 Large-scale ‘Hamlin’ and ‘Valencia’ Rootstock Trials Added 2019/20: evaluation of all parameters. 7 Multi-Sweet Orange SW Flatwoods Rootstock Trial Updated 12 Charlotte County Multi-Scion Rootstock Trial Added 2019-20 and 2020-21 Tree health rating 15 South Ridge Valencia Rootstock Trial Added 2019-20 Yield and fruit drop and 2020-21: HLB rating 23 Indian River [IR] Minneola Rootstock Trial Added 2020-21 HLB rating and % of Dead trees. 25 Fellsmere Scion-Rootstock Trial# 25_12-6-20 Added HLB Ratings, size Jan 2020 and Oct 2020 MAC City Block New data, HLB rating etc. Hidden Golf Trailer Park Trial 2018-19 Yield, HLB and size ratings, 2019-20 HLB rating Cutrale Amelia grove Started organizing data: canopy diameter, tree size, etc.
Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is ongoing. As the most proven of our transgenics, we continue to use them as a reference in detached leaf assays, with CLas+ ACP feeding, as well as studying them in established greenhouse and field studies. Greenhouse studies (With 9 Carrizo lines and 4 Hamlin lines, 98 total plants with controls) include graft inoculation of Carrizo rooted cuttings with CLas+ rough lemon, no-choice caged ACP inoculation of Carrizo rooted cuttings, and no-choice caged ACP inoculation of Hamlin grafted on Carrizo with all combinations of WT and transgenic. Data collection continues from Mthionin field plantings. Results from the first round of field plantings (45 plants) of Mthionin transgenic Carrizo root-stock grafted with non-transgenic rough lemon show transgenics maintaining higher average CLas CT values (2.5 CT higher @ 18 months), but with a high degree of variability. A large second planting of Mthionin transgenics went into the ground in April 2019, including transgenic Carrizo with WT Hamlin scions (81 plants), transgenic Hamlin on non-transgenic Carrizo root-stock (108 plants) and WT/WT controls (16 plants). Scheduled assessments for both field plantings is being prioritized under current covid-19 pandemic conditions; the 24 month field assessment of the first planting and 12 month assessment for the second planting are completed. Leaf samples from both populations have been collected and are being processed for Clas quantification. Additional grafts of WT Hamlin and Ray Ruby scions to Mthionin root-stock 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 51 putative lines are now in soil and are undergoing expression analysis. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, have been conducted and repeated for lines expressing chimera constructs TPK, PKT, CT-CII, TBL, BLT, LBP/’74’, `73′, and `188′ (as well as scFv-InvA, scFv-TolC) using adjusted protocols to improve sensitivity and transmission rates (See section 4). Further detached leaf assays are being run to compare the relative effectiveness between each generation of chimera constructs and to expand the number of lines tested from each. We have completed DLA testing of all 35s and many phloem specific (ScampP) promoter driven 3rd generation Carrizo lines. These assays have 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 are now in greenhouse studies based on DLA results, as noted below. Initial ACP inoculations conducted on 8 lines of citrus Thionin-lipid binding protein chimeras (`73′, and ’74’) showed a statistically significant reduction (13x) in CLas titer for `74′ transgenics vs WT in the CLas+ plants. However, many plants remained CLas negative at 6 months post infestation, indicating a low inoculation efficiency. All ACP inoculated greenhouse experiments are now using an improved protocol using a combination of smaller plants, more aggressive trimming and close observation to safely extend the caged ACP infestation time from 7 days to 21 without harming the plants. Additional greenhouse studies are also being prepared in parallel using bud inoculations. In June, 150 plants representing the best performing 7 lines of `188′ and 6 lines of `74′ were no-choice caged ACP inoculated using the new protocol. At 3 months, control plants tested positive at twice the rate of the earlier inoculation; 6 month tissue samples are now collected and processed, awaiting qPCR analysis. A large additional greenhouse study is now 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+ Rough Lemon. The same lines are now being replicated for additional greenhouse and field trials. This is part of our efforts to emphasize parallel field trials for all phenotyping efforts. Towards this goal, a field planting of ~400 `74′, `188′ and Mthionin transgenics is underway. The first 165 plants (WT Hamlin and Ray Ruby on transgenic Carrizo) went into the soil in August 2020 and will be undergoing their first assessment by February 2021. 200 more grafts of `74′ and `188′ transgenic Hamlin on WT root-stocks are being made to complete the planting. 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 275 new putative transgenic lines including 74-Valencia, 74-Ray Ruby, 74-US-942, 74-Hamlin, 188-Ray Ruby, 188-Valencia, 188-US-942, TBL-US-942, TBL-Hamlin, TBL-Ray Ruby, TPK-Ray Ruby, TPK-US-942 and TPK-Hamlin are now in soil and undergoing expression analysis. Objective 3, ScFv Constructs: Greenhouse studies on the 5 scFv lines in the 1st round of ACP-inoculation has been completed with the best performing lines showing significantly reduced CLas titer over the 12 month period (up to 250x reduction) and a much higher incidence of no CLas rDNA amplification in all tissue types. The best Carrizo lines have been grafted with WT Ray Ruby scions and are now in the ground at the Picos farm location undergoing field trials. They are scheduled to receive their first assessment in March 2021. An additional 129 rooted cuttings are propagated for follow up plantings with a Hamlin scion. A second round of greenhouse trials (150 plants from 12 lines) have been bud inoculated with HLB+ RL. A third set of 370 plants for greenhouse trials has been propagated with the first 54 plants to reach a suitable size already inoculated using the new ACP inoculation protocol. Tissue for testing CLas titer from both sets of plants has been collected and processed; now awaiting qPCR analysis. Objective 4, Screening Development and Validation: A protocol using a high throughput ACP homogenate assay for selecting lytic peptides for activity against CLas is now in use. A manuscript on the protocol has been published in Plant Methods (DOI: 10.1186/s13007-019-0465-1) to make it available to the HLB research community. Several peptides variants screened through this assay have now shown significant ability to reduce CLas titer by foliar application to grapefruit trees in initial testing- conducted by CRADA partners. Hamlin and Valencia trees have been selected and blocked for trunk application trials with these peptides. 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 trunk application trials. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; increasing from 10 to 20 ACP per leaf, decreasing the feeding period (7 days to 3) and adding a 4 day incubation period between feeding and tissue collection. In order to better investigate the effects of peptides producing ACP mortality, we have expanded the analysis of ACP bodies to include quantification of other major endosymbionts (Wolbachia, Profftella, and Carsonella) in addition to CLas. An array of phloem specific citrus genes has been selected for investigation as potential reference genes to improve detached tissue and plant sampling techniques. Multiple sets of sequence specific qPCR primers for each gene have been synthesized and tested for efficiency. Six varieties of citrus have been propagated for endogene stability testing. A phloem specific endogene would allow normalizing to phloem cells, more accurately evaluating CLas titer and potential therapeutic effects. The best performing lines of Mthionin, chimeras `74′ and `188′ and scFv transgenics have been submitted to Florida Department of Plant Industry for shoot-tip graft cleanup in preparation for future field studies. Hamlin/Mthionin transgenics (3 lines) and Carrizo/Mthionin (2 lines) have been returned certified clean. In addition to the use of the AMP Mthionin, its variants and chimeric proteins, new strategies have been implemented in our Laboratory to fight HLB, including the evaluation of insecticidal peptides to control the ACP (CLas vector), as well as the downregulation of the DMR6 genes to enhance defense responses against HLB disease. 54 independent transgenic lines of Carrizo, Hamlin and Ray Ruby expressing the insecticide peptide Topaz (a code name to protect IP), under constitutive and phloem specific promoter (SCAmpP-3) were evaluated for their ability to kill ACP and 12 lines (4 event of each genotype) were selected to move up in the screening pipeline for HLB/ACP tolerance, since they showed significant ACP mortality. Also 24 Carrizo transgenic events highly expressing Onyx (a code name to protect IP), a peptide with antimicrobial and insecticide activity, were evaluate by DLA, and 5 lines showing high ability to kill ACP (to 83% mortality) were selected for further evaluation. These high performing lines from Topaz and Onyx have now been replicated as rooted cuttings for greenhouse trials. Approximately 400 cuttings were made from these transgenic lines in September and October, and around 40% are already rooted and producing new shoots. Onyx has been introduced also in Hamlin, Ray Ruby and Valencia, under SCAmpP-3 promoter. A set of 30 new putative transgenic events are well acclimatized and are undergoing transgene expression analysis. Newly regenerated transformed plants have being transfer to soil.Down regulated DMR6 Carrizo transgenic citrus, either by expression of specific hairpin RNA or by specific Cas9-sgRNA were generated and are ready to be assessed for HLB resistance by grafting with infected scion. Objective 5, Transgenic product Characterization: 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 tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), and PKT (20 lines) and His6/Flag tagged variants of scFv-InvA (22 lines) and scFv-TolC (18 lines) constructs have been generated and confirmed for transgene expression by RT-qPCR. Total protein samples have been extracted from His-tagged transgenic lines and sent to our CRADA partner for testing. Experiments are also underway to use these plants to track the movement and distribution of transgene products as are parallel efforts with antibody based approaches.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. We have had 27 candidate rootstock seed trees tested by DPI. All have received a clean bill of health; no seed transmissible pathogens were found. We have harvested fruit from these selections, and seed extractions are in progress. Once completed, we are authorized to distribute seeds 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 continuously update and add new data to existing and new rootstock trial files to our website (https://crec.ifas.ufl.edu/citrus-research/rootstock-trials/), currently there is information from 24 locations. 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 2019 crosses yielded shoots from >1750 germinating embryos coming from 36 interploid crosses (including 11 targeting sweet orange-like hybrids, and 8 targeting grapefruit improvement), Micrografting to rootstocks has been completed, and nearly all have been transferred to the greenhouses to grow off. Embryo rescue from 37 interploid crosses made using selected HLB tolerant plants in 2020 is nearly completed, including 10 crosses for red grapefruit improvement, 10 for sweet orange improvement, and 10 for mandarin improvement. In addition, 2 diploid crosses were also made for sweet orange improvement. Populations of protoclones from EV1 and 2, were produced to seek more robust early maturing Valencia types; these trees have been prepared for field planting. Cybridization experiments were conducted to combine Meiwa kumquat cytoplasm with OLL and EV sweet oranges, and red grapefruit clone N11-7, to attempt improvements in citrus canker resistance; plant regeneration is well underway, and we expect micrografting and molecular characterization to proceed in early 2021. Three HLB tolerant red grapefruit hybrids and two mandarins were submitted to the DPI Parent Tree Program for cleanup and the production of certified budwood for future trials.3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We used new approaches to quantify tree responses to HLB, in addition to the previously used subjective approaches; we measured photosynthetic parameters and leaf canopy indexes, to produce repeatable and reliable quantitative data in support of further genetic analyses of tolerant types. Objective quantitative data of tree responses provides more reliable information that improves the precision with which we can associate genome regions with tolerance or sensitivity; see Obj 4. We also have been collecting detailed HLB phenotypic data, including Ct values and other tree health measures as described above, from 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.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 from these trees were prepared, and each individual was genotyped using the citrus Axiom SNP array. GWAS analysis is proceeding. This work will validate previously identified, or identify new genomic regions, associated with HLB tolerance or sensitivity. Further, we have contributed budwood of 54 UF-CREC selections to a top-working trial of approximately 200 selections in total, that is a collaboration with USDA-ARS Ft. Pierce and supported by industry. These have been topworked in a commercial grove and are now growing; this smaller but more diverse population will be used for GWAS in the future. We assisted with the collection of DNA samples to fingerprint the plantings to be assured that the trees are properly labeled and coincide with the original clones and budwood sources at DPI. Samples have also been prepared for genotyping all individuals using the GBS approach, to be fed into GWAS analysis in the future.
Create new candidate hybrids. Seedlings from 2019 crosses continued to be grown-out in the greenhouse in preparation for propagation, testing, and establishment of seed trees. Selections were made from 2018 cross seedlings and prepared for making cuttings to enter into Stage 1 trials. Emphasis of hybridization in the USDA rootstock program is among parents with superior tolerance to HLB, CTV, and Phytophthora. Some of the best performing of the newest hybrids in Stage 1 field trials are hybrids of US-942.Propagate and plant new field trials. Budwood increase trees of selected scions were grown, in preparation for budding trees for new rootstock trials. Four new replicated field trials were planted in St. Lucie County this quarter, at Stage 1 trial with Valencia orange, and Stage 2 trials with Bearss lemon, Eureka lemon, and Washington Navel orange. Other planned plantings were delayed because of institutional Coronavirus shutdown. Nursery trees for Stage 2 rootstock trials with Valencia orange, Hamlin orange, and Star Ruby grapefruit are being prepared in the greenhouse for field planting in 2021.Collect data from field trials. Information on tree performance is collected from established field trials, and includes measurement of tree size, fruit crop, fruit quality, and pathogen titer, HLB symptoms, and assessments of tree health. Cropping data is collected during the time of scion harvest, and this quarter was not the time for cropping data with any of the trials. Assessments of tree health and measurements of tree size were completed on 3 trials during this quarter, which was reduced from the normal because of the institutional Coronavirus shutdown. Assessments of brix, acid, and color for the fruit quality analysis of last season data from Stage 1 and Stage 2 trials resumed this quarter after being on hold during the institutional Coronavirus shutdown the past 6 months. The pace of working through his backlog of fruit quality assessments will be slow because of continuing institutional Coronavirus restrictions, and the influx of new fruit quality assessments on the new season crop that begins in the coming weeks.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.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 summaries for five trials were prepared for uploading to the website https://www.citrusrootstocks.org/. Release of superior new rootstocks for commercial use. Several of the 350 advanced Supersour rootstock hybrids in field trials are exhibiting good performance in comparison with the commercial standard rootstocks. Performance data continues to be collected, but it is anticipated that 2-3 of the most outstanding of these will be officially released in 2021-22. Release of new rootstocks is based on the summation of performance in 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.
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.Preparation has begun for the next planting of cover crops for fall/winter 2020. Herbicide has been applied to row middles, and seeds should be planted by the end of Nov 2020. The mix will include: sunnhemp, pea, daikon radish, oats, and winter rye. Results from our August 2020 sampling are still being analyzed, but preliminary assessment 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, we also found an increase in the abundance of bacteria under the canopy of trees in the legume+non-legume treatment. Weed density survey data collected from the latest sampling interval (July 2020) is currently being analyzed. This survey’s preliminary observations suggest the continued suppression of weed emergence and spread by cover crop plantings in citrus row-middles compared to non-cover cropped controls. 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 will next occur in November 2020 with the planting of the next round of cover crops. Data from Year 2 (collected in Aug 2020) is still being analyzed. Weed data were collected in July 2020, and the impacts of the eco-mowing on weed emergence and coverage in citrus tree rows are 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. We will report the trends in the next quarter. Objective 2: Examine the impact of eco-mowing in conjunction with cover crops on soil and root health and weed cover in established commercial citrus groves.Eco-mowing occurred in early May 2020 when cover crops were mowed in anticipation of planting the next set of cover crops. After a year of treatment, soil organic matter slightly increased (in the range of 0.3-0.5%) under the tree canopy receiving eco-mowing compared to regular cover crop treatments; however, no significant differences were detected between treatments. Visual root growth assessments show continued root growth under cover cropping and eco-mowing, but analysis is ongoing. Soil moisture appears to be similar across all treatments, possibly due to the presence of a high water table at both sites. Objective 3: Quantify the effect of cover crops and eco-mowing on tree growth and production.After 1 year of study, we have not yet observed differences in fruit yield, fruit quality, canopy volume, and trunk size. This is not unexpected, as trees of this age could take at least two years to show responses to treatments. We will continue to assess canopy volume and trunk size, and harvest data will be collected again in Spring 2021. Objective 4: Identify the economic benefits of using cover cropsA student was trained on partial budgeting and valuing soil health. The student began work on developing a citrus budget that is appropriate for comparing management strategies with cover crops relative to business as usual. We found that differences in how the budgets are reported limits historical data collection to five years. These data will be used as a benchmark when doing partial budgeting, which is now in its early stages. In addition to budget ta.60sks, the relevant literature was reviewed and incorporated into a draft of the adoption survey. The survey is ready for review and IRB approval. Objective 5: Communicate results to growers using field days and extension materialsPreliminary results are being presented at the Citrus Expo in August. Initial results on weed suppression by cover crops was included in a presentation at this years Citrus Growers Institute. Information on cover crops and preliminary data will be included in two articles for the Citrus Industry magazine in September. Discussions are underway about how to host a field day, or a virtual field day, at some point in the next year. 2. Please state what work is anticipated for next quarter: The most recent collection of soils, leaves, and roots for microbial and nutrient analysis will be completed by the end of August 2020. Microbial DNA will be extracted from soils collected in August 2020 and analysis of soil microbes important in nitrogen cycling will begin. Soils collected in August 2020 will also be analyzed for soil organic matter and nutrients. Analysis of data from weed density measurements will be performed. Canopy and trunk size measurements and leaf nutrient status along with root image collections and soil moisture monitoring will continue in the next quarter. A graduate student and postdoctoral research associate on the project will be presenting results of the project at American Society of Agronomy virtual annual meeting in November 2020. The next set of cover crops are scheduled to be planted in October. The composition of the mixes is still being discussed. The economics team expects to execute the adoption survey and begin analysis. They will also construct the framework for partial budgeting and assessing the cost of cover crop use and continue to collect data. 3. Please state budget status (underspend or overspend, and why): We are on track with our planned budget spending.
The project has five objectives:(1) Remove the flowering-promoting CTV and the HLB bacterial pathogen in the transgenic plants(2) Graft CTV- and HLB-free buds onto rootstocks(3) Generate a large number of vigorous and healthy citrus trees(4) Plant the citrus trees in the site secured for testing transgenic citrus for HLB responses(5) Collect the field trial data In this quarter, we focused on the following greenhouse and laboratory work: (1) Took care of transgenic plants in the greenhouse. We now have two batches of transgenic plants. One batch have been prepared for the proposed field trial, but transplanting was delayed due to COVID-19. The other batch are newly produced, expressing a regulatory gene of systemic acquired resistance. These plants were regularly watered and fertilized. (2) Laboratory work was focused on cloning the extracellular domains of a group (10) of citrus homologs of an Arabidopsis disease resistance gene. This gene encodes a receptor-like kinase with the extracellular domain binding nicotinamide adenine dinucleotide. We found that overexpression of this receptor-like kinase increases resistance to bacterial pathogens and have thus generated transgenic citrus plants overexpressing the Arabidopsis receptor-like kinase gene. The citrus genome encodes more than ten homologs of this receptor-like kinase. To find out the functional homolog(s) of the receptor-like kinase gene, we cloned the extracelllar domains of the closest ten homologs in an E. coli expression vector. We are optimizing the protein expression conditions and will express and purify these fusion proteins from E. coli. The next step will be to test their nicotinamide adenine dinucleotide-binding activity. The protein(s) that binds nicotinamide adenine dinucleotide will be the citrus functional homolog and will be used to generate intragenic/cisgenic citrus plants.
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. 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. 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 and HLB S genes are being regenerated.
Within the third quarter of 2020, the Juvenile Tissue Citrus Transformation Facility (JTCTF) has operated at about 40% of its capacity. In accordance with regulations established by the University of Florida (UF) as a response to COVID19 epidemic, JTCTF transitioned to Phase3 during this period. We have started doing new transformation experiments to the extent allowed by the available labor force and starting material. No new orders for production of transgenic citrus were received.Transition of the JTCTF to the Phase3 meant that there could two employees in the lab at the time. According to UF regulations, there can be one person per 150 ft2 of working space. Surface area of the JTCTF is such that allows only two employees to be there at the same time. Labor force of JTCTF includes two employees who work on USDA grants and they also had to be included into the work schedule. As a result, number of experiments that can be done per week is lower than the usual output of JTCTF. Another factor that affected productivity was available starting material. The fruit we had stored in the packing house has gone bad much faster than usual because the cold room broke down at one point during summer. Seeds extracted form fruit that is going bad were also bad and germinated seedlings carried endopathogens. This led to contamination of material used in transformation experiments and loss of data. Sixteen transgenic plants were produced this quarter. These plants included 14 Duncan grapefruit and two Valencia oranges. These plants were the results of work on eight different orders/vectors: BB3, BB4, ZM15-2, ZM16, HGJ87, HGJ88, JJ8, and NADR2.I continued to work on the development of the EBA form. After multiple meetings with the CREC leadership and other participants in the process of transition of service labs to EBA units, the EBA form was completed. It was submitted to IFAS personnel responsible for the first step evaluation. There were no changes in the JTCTF staff.
Goals of this project: 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. Cumulative (December 1, 2018 to September 30, 2020) progress report: Objective 1: Evaluate existing transgenic Carrizo and Swingle AtNPR1 overexpressing rootstocks in the laboratory and greenhouse. Results: Production of budded plants for the study: We generated a population of AtNPR1 overexpressing transgenic lines prior to the start of this project. Transgenic lines were generated using the Carrizo citrange and Swingle citrumelo cultivars. Molecular analyses: Two populations of transgenic AtNPR1 expressing citrus have been used in this study. The first is a population of Juvenile tissue derived lines (2300-NPR1-x) and the other a population of mature tissue derived (Ax and ZMx) lines. Transgenic lines have been evaluated during this period for AtNPR1 expression using qPCR (Table 1). Table 1: Mean AtNPR1 Ct values from the different mother trees. PCR was performed for 40 cycles. Experiment was repeated thrice. All the lines were tested for the PR1 and PR2 expression. These PR (Pathogenesis related) genes are markers for the Systemic acquired resistance (SAR) pathway and their overexpression indicates that our NPR1 gene is active. Selected lines with high PR1 and PR2 expression are being used for the greenhouse assays to understand if the transgenic rootstock can confer resistance to the non-transgenic scion. Ct values of the HLB infected budsticks range from 22.5 to 25.1. The first set of data was collected in May 2020. The second set of data was collected in September 2020 (Table 2) Subsequently we analyzed protein expression in selected A and ZM series mature tissue derived transgenic lines using western blot with AtNPR1 specific antibody. All transgenic lines tested positive when leaf samples were analyzed (Figure 2). When roots were evaluated, A22, A47, A48, ZM26 and ZM30 had the best AtNPR1 protein expression. (Figure 3). Figure 3: Western blotting of root samples with AtNPR1 specific antibody. In the first round of clonal rootstock propagation, we focused on the juvenile tissue derived transgenic lines, which had been produced before the start of the current project and were thus bigger in size, compared to the A and ZM series of mature tissue derived transgenic lines. Cuttings from selected lines with low Ct values were propagated under mist. It has been observed that some transgenic lines do not perform well after propagation. They either do not root or the rate of growth is very slow. This is often related to higher trans-protein production in these lines. These lines were discarded and removed from the study. In the second round of propagation, we have focused on propagating cuttings from the A and ZM series of mature transgenic lines. Discussion: A greenhouse study is in progress with selected transgenic lines budded with HLB infected sweet orange budsticks. The objective of this study is to understand whether the transgenic AtNPR1 rootstock can protect the aboveground scion against HLB. Three metrics are being evaluated: 1) HLB (Clas) titer, 2) PR-1 gene expression and 3) PR-2 gene expression. The CLas measurements were conducted twice. The first sampling was done in late May 2020 and the second sampling was done in late September 2020. Leaf sampling should have been preferably done in March – April, but due to COVID related and other restrictions had to be delayed. According to our results, all transgenic lines were HLB positive at the time of the initial sampling in late May 2020 (Summer Leaf Ct values – Table 2). When leaves were again sampled in September 2020 (Fall Leaf Ct values – Table 2), Ct values were either statistically similar or had increased in most transgenic rootstock lines. Ct values in the control lines significantly decreased during this period. Thus, this preliminary data provides an indication that transgenic rootstocks could potentially protect the non-transgenic scion from HLB. Although the trees still have HLB, the Ct values have not decreased in the same manner as we see in the controls. This can only be because of the effect of the transgenic rootstock on the scion. Line 2300-NPR1-25 had very high Ct values in the September evaluation, changing from 26.8 to 31.2 within 3 months. We will pay special attention to this line following our next sampling in December – January to confirm the validity of the fall results. PR-1 and PR-2 gene expression were stable in the budded scions, when compared to the HLB infected non-transgenic control. Samples were collected in March, June and September 2020. We saw decreased gene expression at the 6 month sampling in June and it may be due to a seasonal fluctuation. Even with the fluctuations, there was consistent upregulation of both genes in our trees. At this point, we cannot correlate enhanced gene expression to the HLB Ct results. Future directions: Leaves will be sampled at a 6 monthly interval till the end of the grant cycle and pertinent results will be provided in the quarterly report. An in-depth RNA expression study (RNAseq) on selected lines and control using an illumina HiSeq platform will be conducted to understand differential gene expression in the non-transgenic scion. This data will be essential in getting a detailed understanding of the genetic regulation of the SAR pathways and can provide conclusive evidence on the SAR activity. Additionally, we plan to study the impact of CLas infection on non-transgenic Valencia leaf metabolites using gas chromatography mass spectrometry by a process developed by Nabil Killiny. Expression analysis for genes involved in jasmonic acid (JA), salicylic acid (SA), and proline-glutamine pathways will also be conducted to correlate results observed in the RNAseq and GCMS data. Objective 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. Transgenic lines were planted in the field under USDA permit in March 2020. USDA permit allows us to harvest and evaluate seed and fruits from the transgenic lines as necessary for the success of this project. We obtained 250 control trees (non-transgenic Valencia budded onto non-transgenic Kuharske) from Brite leaf nursery to plant as border row trees as stipulated in the transgenic permit in addition to the transgenic rootstocks inside the trial. Another 20% control non transgenic trees were planted within the trial. Future directions: COVID related travel restrictions had resulted in a unique management situation for the transgenic field trees soon after trees were planted in March 2020. We are however getting back on track for management of this site. More trees will be planted in Spring 2021, including most of the rootstock seed source lines that have good transgenic expression. These will be planted for fulfilling objective 4. Field trees are managed according to set USDA protocols and leaves will be sampled in March 2021 for year 1 data collection. Subsequently leaves will be sampled at a 6-monthly interval (every spring and fall) and samples will be sent to Southern Gardens diagnostic lab for HLB analysis. RNA from fruit samples will be isolated and tested for presence of AtNPR1 using qPCR. Objective 3: Produce additional transgenic rootstock lines and stack other gene(s) responsible for SAR using mature transformation. The mature citrus facility (MCF) is a dedicated facility for mature citrus transformation and we have utilized their services for this project. The MCF received four vectors, each containing two stacked, disease resistance genes conferring Systemic Acquired Resistance (SAR). The four vectors are GNS (gus-NPR1,SABP2), GNO (gus-NPR1,OBF5), GNA (gus-NPR1,AZL1) and GND (gus, NPR1, DIR1). Table 3 lists the transgenic lines produced by the mature transformation facility and available for this project. Trees will be evaluated by qPCR for gene expression and the best lines will be clonally propagated for evaluation in 2021. Objective 4: Evaluate transgene segregation analyses of the rootstock progeny and large-scale propagation of select lines. Most of the transgenic rootstocks that show enhanced PR-1 and PR-2 expression have been propagated by budding on US802 rootstock. Trees are to be planted in the field in spring 2021. Several mature tissue derived transgenic lines have already flowered in the greenhouse and fruit harvested from 3 lines. Seedlings were planted from each line. Gus staining and AtNPR1 gene expression of the lines have indicated genetic stability in the progeny. A uniform population has now been generated for budding with non-transgenic scions. Since preliminary results indicate normal seed germination and growth, we are hopeful that overexpression of the AtNPR1 transgene in rootstocks will not have any detrimental effect on the subsequent generations. Selected transgenic lines are also being mass propagated using in vitro tissue culture to generate a large population of transgenic lines for field testing in two additional USDA approved field sites. Conclusions: According to the time frame proposed in our project proposal, we are on track with this project. Year one was spent establishing the framework for this study, getting the plants ready, doing the background molecular analyses and infecting plants with HLB. We were also able to obtain an USDA permit for field trials and have planted a population of trees in the field. In year 2, we demonstrate that CLas titers do not significantly fluctuate following infection of the non-transgenic scions budded onto the transgenic rootstocks. Results obtained are only for 12 months after budding and further evaluation will conclusively demonstrate the validity of this hypothesis. Transgenic rootstock lines with the stacked SAR inducing genes have been produced by the mature citrus facility. Also all existing transgenic lines that have been observed to have enhanced PR-1 and PR-2 gene expression have been propagated. Seedlings from the first batch of transgenic lines have been phenotypically normal and genotypically stable. Thus, from preliminary indications, it seems the AtNPR1 transgene does not have any deleterious effect on the seedling progeny.
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