Objective: A single objective of this project is to assure the presence of active site that will provide un-interrupted service for production of transgenic citrus plants to researchers involved in fight against huanglongbing (HLB) and citrus canker. Through its services, Citrus Transformation Facility (CTF) offers a place for groundwork for the scientific community. For the laboratories without transformation capabilities, CTF makes their projects possible by producing transgenic plants. CTF staff also participate in other research projects that had to do with production of transgenic citrus plants and when needed, offer advising and training services.
Major accomplishments per objective: The uninterrupted operation of CTF that resulted in production of transgenic citrus plants is the major accomplishment for the 2019. Throughout the whole year, the facility was open and ready to accept the orders and start working on them almost immediately. Altogether, the CTF received 25 orders during last year. Placed orders included requests for transgenic Duncan grapefruit, Valencia orange, Mexican lime, and Indian curry leaf plant (Murraya koeinigii). The number of produced transgenic plants is 246 (Table 1). We have produced additional 17 plants (10 Duncan and seven Valencia) that were designated as transgenic and included in one of our quarterly reports. However, upon additional testing we decided those plants are not carrying the genes they were supposed to and we deducted them from the final count. Those plants that were produced belong to following cultivars: Duncan grapefruit, Mexican lime, Valencia sweet orange, Pomelo plants, Kumquat plants, Pineapple sweet orange Carrizo citrange, and plants of Indian curry leaf plant. All of the plants produced by CTF were the result of research that has a goal of fighting the HLB disease. These plants have the potential to either be tolerant or resistant to HLB, or in the case of Indian curry leaf plants, they produce chemicals that can kill Asian Citrus psyllids. All plants stayed in the state of Florida where further tests will be conducted to test desired traits resulting from introduction of transgenes.
Table 1. Plants produced by CTF in 2019
Cultivar Number of plants produced
Duncan grapefruit 155
Mexican lime 32
Valencia sweet orange 20
Pomelo 21
Kumquat 4
Pineapple sweet orange 4
M. koenigii 10
Number of co-incubation experiments done with explants of different cultivars and appropriate bacterial strains was 150. About 150,000 explants were used in those experiments. In only one experiment all the explants were contaminated and in three others there was a partial loss of material. The data from 136 experiments were collected in 2019. One hundred and ten experiments included green fluorescent protein (GFP) as a reporter gene and because of that we inspected under the microscope about 100,000 shoots and buds that sprouted from treated explants for the presence of GFP fluorescence. Altogether there were 1914 transgenic shoots and buds but 1293 were chimeric and 621 were exhibiting GFP fluorescence in all tissues. We have also preformed about 950 PCR reactions with primers specific to LOB gene and to GUS gene during selection of Duncan grapefruit shoots positive for gene carried by JJ8 binary vector. Additional 925 PCRs were done with Valencia shoots using primers specific for sequences carried by the JJ7 vector. Furthermore, 570 GUS assays were also done with samples from Valencia shoots in search of those transformed with sequences from the JJ7 vector.
In February of 2019, CTF purchased one bin of Duncan grapefruits and stored them in the cold room for supply of seeds that lasted until November. In September, the crew working in A. Schumann’s CUPS harvested half of the yield from Duncan grapefruit trees we have there. Since CUPS-produced fruit do not tolerate well storage conditions of cold room in CREC’s packinghouse, we lost more than 50% of fruit in a short period of time. At the end of January of 2020 when the second half of Duncan grapefruit gets harvested from CUPS, we will concurrently use them for experiments and extract seeds that will be stored. In order to secure sufficient supply of Duncan seeds, we will purchase half of the box of fruit in February 2020. For experiments requiring Valencia seedlings, we are picking Valencia fruit from the trees on the CREC property to get seeds. The seeds of other cultivars are obtained through purchase from Lyn Citrus nursery in California or by picking fruit from DPI Arboretum in Winter Haven.
Major shortcomings, unfinished business: High majority of work done in CTF on production of transgenic citrus plants includes GFP as a reporter gene. Numbers reported in the above section best describe why. Almost all plants produced in the 2019 were selected based on the GFP fluorescence. All of the PCR reactions and GUS assays performed last year for orders that did not use GFP, lead to production of just a few plants. These tests also resulted in some false positives I described above. CTF is at the point where we can relatively easy satisfy the orders for some citrus cultivars and produce about 10 transgenic plants with desired gene within nine months if the GFP is a reporter gene. GFP is a powerful tool that researchers are holding on to, because it helps them get the results (transgenic plants) fast. CTF has no leverage to steer people away from using GFP as a selection tool in the process of production of transgenic citrus plants. Such an effort would also be counterproductive until equally efficient reporter gene is available.
The flux of employees working in the CTF remained high. Two employees who worked at CTF in the beginning of 2019 have left. One of these employees was funded from the USDA grant where there was money left over upon his departure. We are presently in the final stages of hiring a replacement. In the spring of 2019, one OPS employee was hired on a temporary basis for six months. This person left the facility on November 1st although some funds remained available. New employee was already hired as a replacement.
The opportunities going forward: Future opportunities for the CTF reflect the needs of Florida Citrus Industry. The most important thing that CTF can do is to participate in fight against HLB and citrus canker by producing trees with increased tolerance and/or resistance to these diseases regardless of methodology used.
Researchers using CRISPR for editing of citrus genome are still trying to produce homozygous plants that do not have in their cells any “leftovers” from the process of genetic modification. Even when this gets accomplished, that should be just the beginning of the use of this technology in the improvement of citrus. This was, and still is, the great opportunity for the CTF to play its role by producing citrus plants with edited genes for the benefit of all stakeholders in the citrus industry.
The CTF was the first site where cisgenic citrus plants were produced. These plants contain only the DNA from citrus even after genetic modification. Since it is not known whether CRISPR can be used successfully in all efforts for improvement of elite citrus cultivars, introduction or modification of genes from same or related species remains as valid approach. CTF is ready for such efforts at any given time.
Publications from this project
1) Jia, H., Orbović, V., Wang, N. (2019) CRISPR-LbCas12a-mediated modification of citrus. Plant Biotechnology Journal, doi: 10.1111/pbi.13109
2) Song, G., Prieto, H., Orbović, V. (2019) Agrobacterium-mediated transformation of tree fruit crops: Methods, progress, and challenges. Frontiers in Plant Science, 10:226.
The objectives of this project are to produce mature citrus transgenics that will flower & fruit naturally using Agrobacterium as a service for customers, increase transformation efficiency, & conduct research to further biolistic transformation, so that it will also become an efficient service. Plants produced using biolistic transformation can be deregulated faster at less expense. Approximately 36 Agrobacterium-mediated transgenics were produced & micrografted this quarter, 11 survived, unfortunately 10 died, & it is too soon to tell if the remaining 15 will survive. In the near future, a new staff member will devote much time to increasing micrografting efficiency since it is critical because mature shoots will not root. Many of the transgenics produced were not for customers this quarter, but were to determine the optimal concentration of a new selection agent for biolistic transformation of mature citrus in tissue culture. Before Christmas, a number of Agrobacterium-mediated transgenic plants were delivered to Dr. Mou. His latest vector(s) have a prospensity to rearrange in Agrobacterium, therefore we must do PCR for detection of the transgene. Typically 2/3 of the plants regenerated plants retain his transgene whereas the rest lose it. Dr. Wang’s lab would like grapefruit transgenics, so we introduced more grapefruit cultivars (Flame, Duncan, Marsh, Ray Ruby). However, before we produce transgenics for Dr. Wang’s lab, we must first test the different cultivars to see which ones are amenable to Agrobacterium transformation since there is cultivar dependency using it. We have already tested Ruby Red grapefruit, Ray Ruby grapefruit & Dr. Grosser’s new Red Grapefruit with discouraging results. Thus the CRDF can see that not all of our efforts in the mature lab bring in money from customers as there usually is preliminary work to do first.
Mature Valencia sweet orange has a low transformation efficiency. In an attempt to increase it, we tested zeatin riboside hormone rather than BAP. Unfortunately this hormone did not significantly improve efficiency & zeatin riboside is prohibitively expensive anyway. Fortunately Dr. Grosser’s new cultivars derived from Valencia (EV1, EV2, Valquarius) all have relatively high Agrobacterium transformation efficiencies & are alternatives to Valencia.
Reinvigorated scions have thorns (L. Pena, personal commun) & one objective was to bud for thorniness to increase efficiency. However, there was too much variability after budding within and between cultivars to achieve this objective. As an example, you might find no thorns at the stem base & thorns at the top of the stem in one plant vs thorns the entire length of the scion in another plant. This objective could not be accomplished because of this issue.
We have tested different DNA precipitation methods (spermidine, PEG) to precipitate DNA onto gold particles prior to bombardment. Spermidine is the standard precipitation protocol & protamine sulfate is not significantly better, according to Dr. Wu who previously tested it. PEG warrants further investigation in the near future.
Prices were increased before Christmas & will probably be increased again ~ March, 2020 after consulting with Dr. Rogers. Although we have increased transformation efficiency significantly, it must become even more productive to cover our costs. Mature transformation is notoriously high input & relatively low productivity. But we do not want to raise prices so high that we lose customers. Biolistic transformation should generate additional customers, so we are hoping to make improvements to this protocol as quickly as possible.
I applied for an FDACS funding opportunity, which would decrease costs somewhat to CRDF. We will not know the results of this competition until ~ Sept 2020 & if funded, the funding would start in January 2021.
The present reporting period runs from September 15 – December 15, 2019. Mr. Chad Vosburg is the M.S. degree student in the Penn State Department of Plant Pathology graduate program who is working on the project. Chad took a trip to Fort Pierce, FL, November 15 – 25, 2019. During this trip, he set up additional plant propagations for 1-2 runs of an HLB resistance test for all the FT-scFv grapefruit lines. Existing propagated plants that had been cut back to induce a new flush of growth were not yet growing out in order to allow for the setup of an HLB infection test. Personnel at the USHRL are monitoring the plants to determine when they have reached the optimal stage of re-growth to start an HLB resistance test using psyllid-mediated transmission. Chad also learned how to propagate citrus by grafting. In particular, he grafted the highest expressing line of FT-scFv to rough lemon rootstocks, since this line propagates very poorly by rooted cuttings. This will provide sufficient plants to test for HLB resistance of this highest expressing line, which would be the most likely to have a strong resistance phenotype. In addition, Chad grafted the next two highest expressing lines to rough lemon rootstocks known to be HLB infected. Rough lemon supports a relatively low CLas titer and this will form an additional test for HLB resistance, as we wait for psyllid-mediated tests to commence. A field test of two of the FT-scFv lines is now set up at the Pecos Farm at the USHRL, with 10 plants per transgenic line, plus non-transgenic controls, having been planted in the field. Chad has sampled these plants for time zero measurements of CLas titers. We anticipate that the first run of an HLB resistance test will be initiated during the next reporting period. Mr. Jeremy Held, a Ph.D. student in the Intercollege Graduate Program in Plant Biology at Penn State, continued his analysis of graft-transmissibility of the FT-scFv protein in the grapefruit lines. Initial tests for FT-scFv transmissibilty to non-transgenic scions did not yield any FT-scFv protein detection signal. However, we are now optimizing the protein isolation procedure to enrich the protein samples for for phloem tissue content. The FT-scFv protein is expected to be limited to the phloem in the non-transgenic scion, making it more difficult to detect than in the transgenic FT-scFV rootstock plants.
Tree evaluations at the three trial locations (1) SWFREC, (2) Hendry County – Duda & Sons, and (3) Polk County – Peace River Packing Co. were continued during the fourth quarter of 2019. Tree evaluations included monthly (SWFREC) or bimonthly (Hendry County and Polk County) root growth measurements using rhizotrons and leaf flush ratings as well as biannual tree/canopy size and trunk diameter measurements.
The SFWREC field trial was terminated after 2 years of growth following a final horticultural assessment. A subset of 72 trees was excavated over a period of 3 weeks using compressed air (Airspade), which allows the fibrous roots to remain intact. A detailed analysis of the root architectures, including the number and length of lateral roots and size distribution of roots, was conducted to investigate potential differences among propagation methods and rootstocks, but also between bed side and swale side. Differences among different propagation methods were statistically significant only for the length per lateral root which was highest for rootstocks propagated from seed. Significant differences were also found among rootstock cultivars for the primary root diameters which was largest for US-802 and US-942 and smallest for US-897, US-1516, and Swingle. A trend for a larger number of primary roots and primary root length on cuttings propagated roots was observed. For many of the measured root variables significant differences were also observed between swale side and bed side. Roots growing into the bed side were more numerous, longer, and thinner compared with roots growing into the swale side. Information from these excavations will be presented during the 2019 Citrus Show in Fort Pierce.
The fourth field trial was established in November at the Florida Center for Sustainable Agriculture in Vero Beach (collaborator: Mr. Bob Adair). This trial includes the 3 rootstock varieties US-942, US-812, and US-1516, which was modified from the originally proposed cultivars (US-802, US-897, Swingle) which were not available as tissue culture propagated rootstocks. All rootstocks were propagated by seed, cuttings, and tissue culture. Therefore, the design was a 3 x 3 design, including 3 propagation methods and 3 rootstocks. For each combination 12 grafted trees were available, which were arranged in 6 replicates of 2. The scion cultivar is Valencia. Baseline tree measurements were conducted immediately after planting. Two weeks after planting, rhizotrons were inserted next to one of each of the trees in each replicate.
Tree evaluations at the two locations (Fort Basinger and Lake Wales) were continued during the fourth quarter of 2019. Hamlin and Valencia trees in both locations were rated for canopy density, canopy color, and foliar HLB disease symptoms. Ratings were conducted on a scale of 1 to 5 on each side of a tree as outlined in the appendix of the RFP. The average ratings for HLB foliar disease symptoms ranged from 1.2 to 1.6, indicating an overall low percentage of disease symptoms. Although significant differences were found among rootstocks, they varied depending on the location and the scion variety. The average ratings for canopy density and canopy color ranged from 4.0 to 4.9, indicating a healthy canopy. No significant differences were found among rootstocks for the canopy color ratings. Canopy density ratings varied significantly among rootstocks, but results varied depending on the location and the scion variety.
In addition to the tree health ratings, leaves were collected from trees in all four trials and sent to a service lab (Waters lab) for leaf nutrient analysis. For many of the macro- and micro-nutrients significant differences were found among rootstocks, but there was no clear trend. For all nutrients, significant differences were also found between the two locations and between the two scion varieties.
For the Hamlins, a subset of fruit from each experimental unit was collected in December at both the Lake Wales and Fort Basinger location. Fruit were analyzed for the following quality parameters: fruit weight, % juice, % solids, % acid, brix/acid ratio, and juice color. Fruit quality analyses were conducted at the CREC Processing Pilot Plant. Overall, extensive fruit drop was observed, which will be assessed in January immediately before the harvest. Weight per fruit was 156 g (5.5 oz) on average. The average percentage of juice was 54.5%. Average acid was 0.47-0.50% and average TSS was 8.7-9.1. The resulting brix/acid ratio was 17.4-19.5. For all measured parameters significant differences were found among rootstocks but results also varied between the two locations.
Significant progress has been made with additions to our website, presenting data from our field trials. Data from the 6 trials listed blow has been entered onto the website during this quarter. Website access: https://citrusresearch.ifas.ufl.edu/rootstock-trials/
Trial # Online Title Location – County Date planted
11 Vero Beach Navel Orange and Grapefruit Roostock Trials Indian River 2010
12 Charlotte County Multi-Scion Rootstock Trial Charlotte 2014
13 LaBelle Valencia APS Roostock Trial Hendry 2009-2010
14 LaBelle ‘Vernia’ Roostock Trial Hendry 2010
15 South Ridge Valencia Rootstock Trial Highlands 2014
16 St. Helena Rootstock Survey Trial Polk 2008 & 2010
Seed fruit was harvested from UFR rootstocks and a few other promising rootstock selections including the gauntlet selection A+VolkxOrange19-11-8, and the seed was extracted and treated by Southern Citrus Nurseries. UFR seed will be distributed to participating nurseries. Seed from Cleo+Carrizo was also extracted, and will be used in experiments in efforts to solve the granulation problem in Seedless Snack and the granulation/low brix problem in Florida navels.
Vegetative material of promising gauntlet rootstocks S10xS15-12-25, 8-1-99-2B X C-22-12-11 and LB8-9xS13-15-16 (the first gauntlet rootstock made with SugarBelle as a parent) was provided to Agromillora and the Phillip Rucks Nursery Tissue Culture laboratory for tissue culture initiation. Another promising selection recovered from Balm (non-gauntlet) 2247 x 2075-02-7 was also provided. Seed trees are not available for any of these rootstocks. The ‘S’ designations in the above parentages indicate salt tolerant parents, as rootstock hybrids stacked with abiotic stress genes continue to perform exceptionally well in the HLB screen. Four other candidates available from the DPI Parent Tree Program were also made available to the TC companies; these include A+HBJL-2B (tetraploid sour orange-type), Blue 1, White 1 and Cleo+Carrizo. Seed was also extracted from several Flying Dragon hybrids (and planted for advanced trials, these have potential for tree size control).
The first trees were planted in our new PTS (Parent Tree Structure), where we will keep superior breeding parents (both diploid and tetraploids) free of HLB and other diseases; guaranteeing their availability for future crosses.
Field Planting at the USDA Picos Farm: 150 new gauntlet rootstock selections were planted (grafted with HLB+Valencia); these include 40 new hybrids made with SugarBelle LB8-9, and many of them also include one of the salt tolerant parents (S10: HB Pummelo x Shekwasha; and S13: HB Pummelo x Cleopatra).
The project has been proceeding well and mostly is on track to achieve the first year’s objectives, to generate PacBio genome sequence data, to begin assembly of the genomes as data are generated, and to prepare plant materials for the next phases of the project (which will include Omni-C library construction and sequencing for chromosome scale assemblies, and for transcriptome sequencing to inform annotation. Using optimized protocols for DNA purification, we have produced the quantity of HMW DNA, and quality of the preps required for PacBio long read sequencing. Samples were sent to the UC Berkeley Genome Sequencing Laboratory, and sequencing libraries were constructed there. Contractual issues between UF and UCB/LBNL, based on language in the UF-CRDF contract, delayed the start of the sequencing runs. Sequencing runs were finally authorized in mid-September, despite the lack of resolution of the contractual issues. The first genomes were finished running in late October. Preliminary output from the first genome run showed that >250 Gbp of sequence were produced, yielding potentially > 83X coverage of the genome, very near our target of 85X coverage; the remaining genomes are in queue and should be completed in November-December. Plant materials are being prepared for samples to be used for Omni-C sequencing, and for RNA isolations and sequencing to enable annotation of assembled sequences.
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 mainly 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 or standard rootstocks. We are in the process of planting four independent studies at the UF/IFAS IRREC. This report is mainly related to the grove planting operations and initial tree care since the study was just implemented. We planted approximately 3,600 trees and are awaiting for the remaining trees to become available from the nursery. Slow release poly coated fertilizer applied and tree wraps added. We installed the irrigation controller and activated field valves making irrigation system automation fully operable. Sand media filtration and water meter were purchased and delivered. Installation will be performed soon. We applied imidacloprid to prevent leaf minor and psillids. The grove has been continuously scouted for pests such as orange dogs and ants. Hoop boom was modified to spray young trees with higher accuracy, reducing the waste of agrochemical products. We created a tree location map and began production and distribution of QR tags to be used with scanner codes during data collection in the field. The group met with the certified crop advisor to develop a spray program schedule based on time of year and conditions to be applied as determined by IPM scouting. Masters student was selected, interviewed and hired; start date Jan/2020.
Objective 1, Mthionin Constructs:
Assessment of the Mthionin transgenic lines is ongoing. Detached leaf assays, with CLas+ ACP feeding, have been conducted and lines with the most promising results have begun 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 the first round of field plantings (45 plants) of Mthionin transgenic Carrizo rootstock grafted with non-transgenic rough lemon. Initial results show transgenics maintaining higher average CLas CT, significantly decreased leaf mottle and significantly increased health values after 6 months. A large second planting of Mthionin transgenics went into the ground in April, including transgenic Carrizo with WT Hamlin scions (81 plants), transgenic Hamlin on non-transgenic Carrizo rootstock (108 plants) and WT/WT controls (16 plants). The next significant data collection will be at one year in field, April 2020. Additional grafts of WT Ray Ruby (118 plants) and WT Valencia (118 plants) on transgenic rootstock are growing in the greenhouse
Additional Mthionin construct transformations have also been completed on 450 Valencia, 300 Ray Ruby, and 415 US-942 explants to provide additional transgenic material of these critical varieties.
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′ using adjusted protocols to improve sensitivity and transmission rates (See section 4). Multiple lines from several constructs have been moved forward into greenhouse studies based on these 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 control plants remained CLas negative at 6 months post infestation, indicating a low inoculation efficiency. All greenhouse experiments are now using an improved protocol to enhance inoculation. Through a combination of selecting smaller plants, more aggressively trimming larger plants and close observation, we have been able to extend the caged ACP infestation time from 7 days to 21 without severe mold or cage damage to the plants. In June, 150 plants representing the best performing 7 lines of `188′ and 6 lines of `74′ were no-choice caged ACP inoculated using the new protocol. At 3 months, control plants are now testing positive at twice the rate of the earlier inoculation and 6 month samples will be collected December 2019.
Plants are grafted and in the greenhouse for a field planting of ~200 `74′ and `188′ transgenics which is scheduled for spring 2020, with WT scions (Hamlin, Valencia, and Ray Ruby) on transgenic Carrizo root stocks. 200 more grafts of `74′ and `188′ transgenic Hamlin on WT rootstocks are underway. These plants will be ready for planting fall 2020.
Seven new transformations, totaling over 3000 explants, have been completed to expand lines of Valencia, Ray Ruby, and Hamlin (when not already complete) lines expressing `74′, `188′, TBL, TPK and other advanced chimera constructs.
Objective 3, ScFv Constructs:
Greenhouse studies on the 5 scFv lines in the 1st round of ACP-inoculation has been completed with the best performing lines showing significantly reduced CLas titer over the 12 month period (up to 250x reduction) and a much higher incidence of no CLas rDNA amplification in all tissue types. The best Carrizo lines have been grafted with WT Ray Ruby scions and, with all appropriate permitting now completed, will be moved to the field after hurricane season. An additional 129 rooted cuttings are propagated for follow up plantings.
The 3 month data from the 150 plants from the 2nd group of scFv lines (12 lines) that were initially no-choice ACP inoculated showed an insufficient infection rate. These plants have now been graft inoculated with HLB+ RL and are undergoing the first post-inoculation analysis. An additional 370 rooted cuttings were propagated for the third round of ACP-inoculations. From which, the first group of 54 plants large enough to use have been inoculated with the higher pressure 21 day protocol.
Objective 4, Screening Development and Validation:
A protocol using a high throughput ACP homogenate assay for selecting lytic peptides for activity against CLas is now in use. A manuscript on the protocol has been published in Plant Methods (DOI: 10.1186/s13007-019-0465-1) to make it available to the HLB research community. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; increasing from 10 to 20 ACP per leaf, decreasing the feeding period (7 days to 3) and adding a 4 day incubation period between feeding and tissue collection.
An array of phloem specific citrus genes has been selected for investigation as potential reference genes to make comparisons focusing on phloem tissue only . Multiple sets of sequence specific qPCR primers for each gene have been synthesized and tested for efficiency. Six varieties of citrus have been propagated for endogene stability testing. A phloem specific endogene would allow normalizing to phloem cells, more accurately evaluating CLas titer and potential therapeutic effects.
The best performing lines of Mthionin, chimeras `74′ and `188′ and scFv transgenics have been submitted to Florida Department of Plant Industry for shoot-tip graft cleanup in preparation for future field studies. Hamlin/Mthionin transgenics (3 lines) and Carrizo/Mthionin (2 lines) have been returned certified clean.
Objective 5, Transgene Characterization:
Transgenic Carrizo lines expressing His6 tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), and PKT (20 lines) and His6/Flag tagged variants of scFv-InvA (22 lines) and scFv-TolC (18 lines) constructs have been generated and confirmed for transgene expression by RT-qPCR. Experiments are underway using these plants to track the movement and distribution of transgene products in parallel to direct antibody based approaches.
Create new candidate hybrids. Sexual hybridization is completed between selected elite parents during spring flowering and seed collected in the fall. Selected hybrids are then grown-out for propagation, testing, and establishment of seed trees. Emphasis of current hybridization in the USDA rootstock program is among parents with superior tolerance to HLB, CTV, and Phytophthora, along with showing favorable effects on grafted tree yield, fruit quality, and tree size. US-942 and US-897 are two of the parents used in the most recent set of crosses with seed harvested in Oct 2019.Propagate and plant new field trials. Replicated multi-year field trials with commercial scions are essential to evaluate performance of rootstocks, both to determine whether each new rootstock should be released for commercial use, and to develop comparative performance information among new and existing rootstocks for a diversity of scions, soils, and management conditions. Because of the complexity of the new rootstock field trials, most USDA rootstock trials are propagated in the USDA citrus nursery in Ft. Pierce. Most of the rootstock field trials are planted with a single scion representing a common commercial type on each of 40-60 different rootstocks. Adequate replication is considered a critical factor in the USDA rootstock trials, with 6-7 replications the minimum and 12 replications the optimum to provide an acceptable level of reliability for results. Three new rootstock trials with promising rootstocks are being planted this fall. Nursery trees for four additional rootstock trials are being prepared in the greenhouse for planting in spring 2020.Collect data from field trials. Information collected from established field trials is collected by tree or by replication, and includes measurement of tree size, fruit crop, fruit quality, and pathogen titer, and assessments of tree health. Measurements related to cropping are on an annual cycle based on the scion, while measurements of health and tree size are on a schedule determined by the specific conditions and goals of the trial. There are currently 30 active USDA rootstock trials and containing about 300 new hybrid rootstocks being evaluated for potential release. Dr. Bowman is also a collaborator in many additional field trials for which data collection is managed by University of Florida researchers, and primarily funded by HLB-MAC Grants. Cropping data is being collected from 6 trials with early-maturing scions during Oct-Dec 2019.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. I recognize that this information is of great value to citrus growers and nurseries, and am working through several issues to post results from USDA rootstock field trials onto a website for easy access by Florida growers. It is anticipated that the first stage of that information will be posted to http://citrusrootstocks.org soon.
Collection of samples for the annual assessment of leaf and soil nutrient concentrations, trunk diameter, canopy size, and soil microbial community composition was finished in late August.
A project meeting with the grove managers was held in early September, and optimization for the planting of the next cover crop mix were discussed. The fall cover crop mix was planted in early November at both locations. This mix included daikon radish, coker, Wrens grain rye, and dove millet. Sunnhemp, alyce clover, sesbania, dixie crimson clover, and yellow sweet blossom clover were included in the mixes for the nitrogen-fixation treatments (1/2 of the rows). Immediately after planting, the summer cover crops were mowed, either with standard mowing or with the “eco-mowing” (aka reverse mowing) for ½ of the rows.
DNA was extracted from soil samples and sent for sequencing analysis. Measurements using qPCR of specific N-cycling genes have begun and will continue into next quarter.
Dataloggers and soil moisture probes continue to record soil moisture every hour. Root growth measurements using the mini-rhizotron tubes installed in both groves were performed in March and July 2019. Data on these measurements are currently being analyzed and will continue in Fall 2019.
Weed density and identification measurements were made in March and Aug 2019. The next data collection including weed density survey and biomass collection is scheduled towards the end of this year.
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 have conducted the following activities:
(1) Nurture small grafted transgenic plants in the greenhouse. The plants are regularly watered and fertilized to ensure vigorous growth. We still keep propagating the transgenic lines using budwoods free of CTV and CLas. We have generated enough progeny plants for this project. These plants will be transplanted in the Spring of 2020.
(2) Analyze rabbit serum against a major citrus defanse protein. The protein was purified and submitted for antibody development. The first batch of rabbit serum has been tested. Briefly, the citrus protein was transiently expressed in Nicotiana benthamiana. Total protein was extracted and separated in SDS-PAGE gel. After transferring the proteins onto a nitrocellulose membrane, the blot was probed with the rabbit serum. A strong specific band was detected in the sample from plants expressing the citrus defense protein, but not from plants transformed with an empty vector, indictaing that the antobody development is successful. Total protein was also extracted from the sweet orange ‘Hamlin’ and analyzed with the serum. The signal is rather weak. A boost has been requested to enhance the titer of the serum.
(3) Observe the transgenic plants already planted in the field. The 69 plants (49 transgenic plants and 20 controls) in the field at Ft Pierce USDA ARS were taken care of by the crew under the supervion of Dr. Ed Stover. The PI regularly communicate with Dr. Stover on the growth of the plants. We will measure the plants in the fourth quarter of this year.
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. First, LbCas12a was used to modify the CsPDS gene successfully in Duncan grapefruit via Xcc-facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBEP thA4 -CsLOBP) of CsLOB1. A single crRNA was selected to target a conserved region of both Type I and Type II CsLOBPs, since the protospacer adjacent motif of LbCas12a (TTTV) allows crRNA to act on the conserved region of these two types of CsLOBP. CsLOB1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 in Xanthomonas citri by binding to EBEP thA4 -CsLOBP. A total of seven 35S-LbCas12a-transformed Duncan plants were generated, and they were designated as #D35 s1 to #D35 s7, and ten Yao-LbCas12a-transformed Duncan plants were created and designated as #Dyao 1 to #Dyao 10. LbCas12a-directed EBEP thA4 -CsLOBP modifications were observed in three 35S-LbCas12a-transformed Duncan plants (#D35 s1, #D35 s4 and #D35 s7). However, no LbCas12a-mediated indels were observed in the Yao-LbCas12a-transformed plants. Notably, transgenic line #D35 s4, which contains the highest mutation rate, alleviates XccΔpthA4:dCsLOB1.4 infection. Finally, no potential off-targets were observed. 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.
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 one of the citrus U6 promoters showed higher efficacy in driving gene expression in citrus than 35S promoter and Arabidopsis U6 promoter. We are further characterizing the promoter and testing its efficacy in driving sgRNA and crRNA in genome editing of citrus.
We are also developing a method to increase the transient expression efficiency. Transient expression of CRISPR constructs is useful to test whether some of the CRISPR constructs are working. Currently, the transient expression efficiency in citrus is very low, almost negelectable.
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. One manuscript is in preparation for publication.
The first steps in this project have taken place. As we are using the very latest DNA sequencing technologies, and we are attempting to produce nearly full-length genome sequences, it is important to begin with the highest quality DNA preparation as starting material for library construction for sequencing. We collected young leaves and young flush from the five cultivars selected for sequencing. This required multiple samplings because we were not always able to get the ideal tissue types; just slightly more mature vegetative tissue than the optimum did not give us the purity and the quantity of high-molecular weight (HMW) DNA required. We also optimized previous protocols we have used to be able to produce quantity and quality required. Finally, we were successful and samples have been sent to UC Berkeley Genome Sequencing Laboratory, and sequencing libraries have been constructed. Contractual issues between UF and UCB, based on language in the UF-CRDF contract, delayed the start of actual sequencing, while relevant officals at UF and UCB have worked toward resolution of the legalities. Sequencing runs are scheduled to begin in mid-September, once contractual issues are resolved. Plant materials are being prepared for RNA isolations, to enable annotation of assembed sequences.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Trees that were selected from the Gauntlet screen from 2018 crosses were stick grafted with CLas-infected Valencia budwood for further selection of tolerant types; those exhibiting severe HLB symptoms were discarded, others that are mildly symptomatic remain. We completed DNA fingerprinting to verify the origins of the “Super-Root Mutants” that have been selected from in vitro propagations in a commercial nursery and found some of these to be of zygotic origin, not resulting from mutations. Field performance of some has been encouraging, so we will continue this work.
2. Develop new, HLB-tolerant scion cultivars from sweet orange germplasm, as well as other important fruit types such as grapefruit, mandarins, and acid fruit. Spring crosses for this objective were numerous, and we are monitoring fruit and seed development. A handful of fruit from interploid crosses are being harvested to begin embryo rescue to recover the triploid hybrids; diploid crosses are being held on the tree until seeds are close to maturity, to maximize the recovery of new hybrids.
3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We have once again gone through the collection and evaluated tree health, to remove those trees that are failing from our list of tolerant types; the numbers of trees dropped from that list has been declining in the past evaluations, so our confidence in the performance of those remaining increases. There have been no collection-wide fruit quality evaluations conducted after early May, though we have noted a few selections with very late maturity.
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 have completed anatomical studies of HLB-tolerant LB8-9 and Bearss lemon and demonstrated that phloem regeneration is an obvious physical mechanism of their apparent tolerance. We have looked at the metabolomic profiles of this same set of plants, and these data have been analyzed. We are drawing conclusions in comparison with similar metabolic studies using different chemical techniques. Once completed, we will submit a manuscript detailing the findings, including information on potential biomarkers for HLB-tolerance in scion cultivars.
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