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.
Significant progress has been made establishing a website, presenting data from our field trials. Data from ten trials listed below is now available; and data from 6 more trees was analyzed with presentations prepared for entry to the website in the next quarter (11-16 below). Website access: https://citrusresearch.ifas.ufl.edu/rootstock-trials/
Trial # Online Title Location – County Date planted
1 Basinger ‘Vernia’ Rootstock Trial Highlands 2006-2007
2 Central Ridge Multi-Sweet Orange Scion Rootstock Trial Polk 2013
3 Indian River ‘Marsh’ Grapefruit Rootstock Trial Indian River 2007
4 Indian River ‘Vernia’ Rootstock Trial St. Lucie 2011
5 Indian River Lemon Rootstock Trial Indian River 2017
6 Large-scale ‘Hamlin’ and ‘Valencia’ Rootstock Trials Polk 2015
7 Multi-Sweet Orange Scion SW Flatwoods Roostock Trial Hendry 2014
8 Peace River ‘Valencia’ Roostock Trial Polk 2017
9 SW Flatwoods Sweet Orange Roostock Trial Hendry 2014
10 SW Florida Sweet Orange Roostock Planting Lee 2014
Data analyzed and Presentations prepared for entry:
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
Data from the St. Helena trial supports the commercial release of additional UF rootstock candidates Blue 1, Cleo+Carrizo somatic hybrid, Orange 14, White 1, WGFT+50-7 and Amblycarpa + HBJL-2B. Collection of botanical data on these selections was initiated as necessary for future release. Data from St. Helena also shows superior HLB tolerance from two of the Foguet rootstocks from Argentina (Orange 1804 and Aqua 1803). The possibility of getting more seed of these two selections from Argentina is being pursued. Traditional Kuharske rootstock showed good HLB recovery potential at St. Helena under the utlizized enhanced CRF program. UFR-14 continued to perform exceptionally well at St. Helena, and since there are no seed trees, it was introduced into the tissue culture propagation program at Agromillora (and expected to be introduced at the Rucks Nursery TC lab next quarter). Data from St. Helena suggests that with good a root nutrition program, growers could expect annual yields exceeding 500 boxes/acre from multiple rootstocks, with good fruit quality. UFR-5 is emerging as a good candidate rootstock for both the ridge and flatwoods soils (supported by multiple trials.).
The present reporting period runs from June 16 – September 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, September 12 – 15, 2019. During this trip, he set up plant propagations sufficient for 1-2 runs of an HLB resistance test for all the FT-scFv grapefruit lines. Existing propagated plants were cut back to induce a new flush of growth, which is essential for a successful HLB infection test. Personnel at the USHRL will initiate the HLB screenhouse test when the plants have reached the optimal stage of re-growth. Chad also worked with personnel at the USHRL to set up a field test of two of the FT-scFv lines. Chad is now working on mastering PCR skills for CLas detection that will be part of the HLB infection tests in the lab at Penn State. We anticipate that the first run of an HLB resistance test will beinitiated 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 studies of the stabilty and expression of the FT-scFv protein in the grapefruit lines. He optimized western blotting and antibody selection and plans to test for movement of FT-scFv across the graft union using immunoprecipitation during the next reporting period. Finally, we initiated tests to check whether FT-scFv plants are fertile. Initial fruits obtained from the FT-scFv plants were seedless, possibly because they are being grown in greenhouses or growth chambers. Jeremy performed some cross-pollinations of FT-scFv flowers in the spring of 2019, and one of these fruits was cut open during the reporting period and found to have normal seed set. This is good news if the FT-scFv construct were ever to be used as a citrus breeding acceleration tool, since precocity is one of the phenotypes of FT-scFv plants. Jeremy also tested pollen viability from FT-scFv plants and found the pollen to be viable and able to germinate in the laboratory in pollen germination medium.
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 as outlined ion the grant.
We already planted approximately 3,600 trees and are awaiting for the remaining trees to become available from the nursery. Our Accomplishments were recently highlighted at CBS12.com (https://cbs12.com/news/local/largest-citrus-greening-grapefruit-experiment-starts-in-fort-pierce) and at the Citrus Industry (http://citrusindustry.net/2019/10/01/new-grapefruit-rootstock-scion-experiment/?fbclid=IwAR0LTWTZDfLcXoxx6jFn7RiRanJLIfXKdfV6IMQn1GJdi_EfR4XZN72mLMo).
We conducted a comprehensive soil testing for the fertilization assessment, and hired an agricultural field assistant. A local, domestic student was not identified. The PI opened the search for any student available at the HOS Department.
I am going to postpone the HLB leaf diagnostic in a few months due to the limited amount of biomass available from the young trees.
During the third quarter of 2019 multiple field trips were conducted to continue tree evaluations and complete measurements of trunk cross sectional areas of rootstocks and scion. The statistical analysis of data was continued. A subset of trees with selected scion/rootstock combinations was used for additional root analyses (root mass and specific root length). Several field trips were also conducted to meet with the grower and plant breeders to decide which rootstocks are of no commercial value and may be removed.
Updates on this project including information on tree size, yield, and fruit quality were presented at three grower/industry events: 1) a presentation at the Citrus Advisory Committee meeting at SWFREC (12th July) titled “Evaluation of citrus rootstock performance in large-scale field trials, 2) a presentation at the Citrus Expo in Fort Myers (14th Aug) titled “Bigger is not always better – large-scale rootstock evaluations on a Ridge and flatwoods site”, and 3) a one-hour seminar including printed educational information at SWFREC (15th Sep) titled “Which rootstock to plant in an HLB-endemic environment”. A new EDIS publication “Citrus nursery production guide, chapter 4: Rootstock selection” was completed and is available at https://edis.ifas.ufl.edu/hs1340.
Between the July and October, Citrus transformation Facility continued its operation without any interruptions. Within this period, we accepted three new orders. All orders required production of transgenic Duncan grapefruit plants.
In the last three months, CTF produced 74 plants. Among the produced plants there were: six Duncan plants (BB3), 18 Duncan plants (BB4), seven Duncan plants (HGJ34), three Duncan plants (HGJ74), two Duncan plants (HGJ87), three Duncan plants (HGJ88), ten Duncan plants ZM14, four Mexican lime plants (M2SF), one Mexican lime plant (contSF), one Murraya (BB1), one Pomelo plants (HGJ68), five Valencia plants (BB3), five Valencia plants (BB4), eight Valencia plants (JJ7). Codes in parentheses represent names of different binary vectors (genes) used in transformation experiments.
One of the CTF employees working on the USDA grant left the lab in the first week of September. After securing additional funding from other participants of that USDA grant, I have submitted position description to HR department and expect to hire new employee before the end of the year. Currently, there are five employees in the CTF.
Most of the fruit of Duncan grapefruit and Valencia orange that was stored in the cold room at CREC rotted and started growing mold. Only six bins of Valencia and ten bins of grapefruit were kept as a source of seeds. The crew working in A. Schumann’s CUPS harvested half of the yield from Duncan grapefruit trees we have there. These fruits will be used for seed extraction in a matter of weeks. We will ask A. Schumann’s crew to harvest the rest of Duncan grapefruit from CUPS for us in January.
To decrease our dependence on CRDF funding, two research proposals were submitted to two federal agencies: NSF and NIFA. It should be ~Christmas before we hear whether the NSF was successful, but NIFA will likely be delayed because of its move to Kansas City. I am working on a third proposal that will be submitted to the USDA SCRI Citrus whenever the RFP is announced. This RFP will also be delayed because of USDA’s move to Kansas City. CRDF funding obligations would be decreased if these proposals were funded. I have contacted scientists at UF, the USDA in Fort Pierce, Texas A&M, University of Arizona, Standford, UC Davis & UC Riverside to increase our clientele. In the near future, our prices will increase as suggested by the CREC Director, the CRDF, and the External Review committee. The actual dollar amount will be determined in consultation with the CREC Director.
It was a productive quarter for the transgenic event production. Forty-four transgenic shoots were identified, 18 surived micrografting, 6 died, and 10 are still healing. Use of the GFP reporter partially accounts for this increased productivity. In spring & summer, our productivity also naturally inceases because citrus is more vigorous. In addition, a new staff member was screening for transgenics & this might have contributed to increased productivity. We received two new vectors from Dr. Mou. Dr. Bonning contacted me about potential work in the future. Dr. Wang’s group is interested in transforming mature grapefruit for commercialization, but we had to introduce grapefruit cultivars first (see below) & then determine which cultivars are amenable to transformation. Dr. Dutt has provided three vectors for our collaborative CRDF project together.
We discovered that Dr. Grosser’s OLL4 and Valquarius cultivars have relatively high Agrobacterium transformation efficiencies. UF15 rootstock is recalcitrant to Agrobacterium transformation with no shoots forming in tissue culture. We introduced new budwood cultivars from FDACS. The budwood introductions were Marsh, Flame, Ray Ruby, and Duncan grapefruits, Temple sweet orange (zygotic embryos), and US-942. We will introduce OLL20 in the future because it is important to the juice industry.
We continue to conduct biolistic transformation of mature scions with citrus reporter genes & selectable markers with the ultimate goal being cis/intragenic production. Next week, we will test whether Dr. Dutt’s reporter can be used without a selectable marker. A new plant – derived selectable marker has been tested with excellent results. Replication of these experiments is currently underway. A similar vector with a citrus-derived selectable marker will be tested in the future. This system is an alternative to E. coli genes for antibiotic resistance & selection. Using citrus-derived sequences should also be more consumer friendly. A manuscript should be produced from this research. PEG-mediated DNA precipitation is being tested to increase biolistic transformation efficiency rather than protamine sulfate or spermidine.
Dr. Wu performed Southern blots for another scientist to show copy number of the transgenes for a scientific publication.
RC Webview, the electronic system that runs the growth facility, has been offline for ~1 week & the reasons are unclear. UF Gainesville is supposedly in the process of connecting a new server. With no other choice available, we have to water with city water which might contain slime mold spores, does not contain enough chlorine, and hasn’t passed through 2 UV filters.
The overall goal of this project is to understand if a non-transgenic scion can be protected against HLB following budding to a transgenic SAR inducing rootstock. Progress has been made on all proposed objectives according to the time frame. This third quarter report of year 1 funding details progress made so far. All available transgenic lines expressing the AtNPR1 transgene have been evaluated in the laboratory for gene expression using qPCR. We have identified three distinct expression levels – low, medium and high. Selected lines that have a high level of NPR1 expression are also being evaluated by Western Blot using NPR1 specific antibody for confirmation. In some lines there is good corellation between NPR1 production in leaves vs roots while in others root NPR1 production is lower than leaves. Transgenic Kuharske and Swingle rootstock lines that highly express the AtNPR1 transgene have been clonally propagated and budded with non-transgenic Valencia scions. We detected enhanced levels of the PR1 gene following qPCR on the non-transgenic valencia scions. Additional lines are being clonally propagated for budding with non-transgenic Valencia in 2020. A subset of the existing trees is being side grafted with HLB infected budwood (Ct between 22.5 to 25.1) in the greenhouse while another set is being planted out in the field (Obj 1). A USDA-APHIS permit has been obtained for evaluation of these transgenic lines and the first set of trees will be planted in late 2019 (Obj 2). Three additional stacked gene constructs with NPR1 and the other listed genes in the project proposal have been produced and provided to the mature tissue laboratory for production of transgenic rootstocks expressing these transgenes (Obj 3). Trees are being produced with these genetic constructs. AtNPR1 overexpressing seed source trees budded onto US802 rootstock have been produced for field evaluation and will also be planted in late 2019 (Obj. 4). Additionally, transgenic NPR1 lines that have been observed to be tolerant to HLB under field conditions are also being propagated for use as interstocks in this study. In summary, several years of research has resulted in the develoment of this study and our progress thus far has been satisfactory.
We have proposed construction of several fusion genes and test them individually or in combination for their effects on improvement of transient expression or stable incorporation of T-DNA genes in citrus tissues. We have shown that the use of some genes we constructed can lead to 2- to 4-fold increases in transient expression activity of T-DNA genes or stable transformation efficiency using juvenile and mature citrus tissues as explants. However, our results are not always consistent in mature shoot tissues of Washington Navel and Valencia oranges. We believe that physiological conditions of explants may play a key role for the observed variations and inconsistence. We have also combined some of these genes and tested their effects. We did not see significant synergistic effects on transient expression or stable transformation efficiencies in mature citrus tissues of Washington Navel and Valencia oranges. We also used a Kn1 gene along with other genes in mature tissues of Washington Navel and Valencia oranges. Little synergistic effects in promoting transformation efficiencies have been observed. Some of the genes we identified may be of useful in improving transformation efficiencies of mature citrus tissues.
We have used root specific auxin biosynthetic gene, root specific-CKX fusion gene (cytokinin oxidase gene), a combination of both the root-specific iaaM and root-specific CKX genes, and other genes produce transgenic Carrizo citrange and HLB-tolerant rootstock US-897. We have produced a large number of independent transgenic citrus plant lines, respectively. Some of these plants have significant increases in root number and root length compared to the WT plants, consistent with our tobacco plant results. We have observed that adventitious root initiation of some of these transgenic citrus plants is faster and enhanced. Our results have also showed that the auxin overproducing plants have improved success rates of grafting. Enhanced root growth and biomass of rootstock may enhance citrus trees’ tolerance to abiotic and biotic stresses. Improved grafting success is desirable for commercial citrus planting because most citrus trees used for fruit production require grafting. We are currently using transgene free genome editing technologies to create the same traits in citrus rootstock.
We have tested a large number of chemical regulators on transient gene expression in mature citrus tissues. Among these chemicals and their combinations, we have identified that some can enhance Agrobacterium-mediated transient gene expression levels. Enhanced transient gene expression activity is important for producing non-transgenic gene-edited mutant citrus plants from mature explants. We have developed a method to produce non-transgenic genome edited mutants without going through sexual reproduction (Chen et al., 2018). We have also observed that that some chemical manipulations can promote shoot regeneration and stable transformation efficiencies of mature citrus tissues.
We have published two articles and two more are under preparation.
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 from the first round of field plantings with Mthionin transgenic Carrizo rootstock (45 plants) with non-transgenic rough lemon continues. 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) with data collection scheduled in October. Additional grafts of WT Ray Ruby (118 plants) and WT Valencia (118 plants) on transgenic rootstock are being made for follow up plantings. Mthionin construct transformations have also been completed on 450 Valencia and 300 Ray Ruby 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 on lines representing chimera constructs TPK, PKT, CT-CII, scFv-InvA, scFv-TolC, TBL, BLT, LBP/’74’, `73′, and `188′. Detached leaf feeding assay protocols were adjusted to improve sensitivity and increase 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 plants remained CLas negative at 6 months post infestation, indicating a low inoculation efficiency. All ACP inoculations for greenhouse experiments are now using an improved protocol. 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. We expect this longer infestation period to greatly improve inoculation rates. In June, 150 plants representing the best performing 7 lines of `188′ and 6 lines of `74′ were no-choice caged ACP inoculated using this improved protocol with the first samplings and data collection scheduled for 9.26.19. We are also emphasizing parallel field trials for all phenotyping efforts. Preparations are now being made for a field planting of ~400 `74′ and `188′ transgenics is scheduled for spring 2020. Completed so far are 196 grafts of WT scions (Hamlin, Valencia, and Ray Ruby) onto transgenic Carrizo root stocks. 200 more grafts of `74′ and `188′ transgenic Hamlin on WT rootstocks are underway. All plants will be ready for the post Hurricane planting season.Seven new transformations, totaling over 3000 explants, have been completed to generate 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 qPCR sampling is scheduled for November 2019. The 370 scFv rooted cuttings already propagated for a 3rd round of ACP-inoculations will use the higher pressure 21 day infestation protocol described above. Objective 4, Screening Development and Validation: A protocol using a high throughput ACP homogenate assay for selecting lytic peptides for activity against CLas is now in use. A manuscript on the protocol has been published in Plant Methods (DOI: 10.1186/s13007-019-0465-1) to make it available to the HLB research community. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; increasing from 10 to 20 ACP per leaf, decreasing the feeding period (7 days to 3) and adding a 4 day incubation period between feeding and tissue collection.An array of phloem specific citrus genes has been selected for investigation as potential reference genes to improve detached tissue and plant sampling techniques. The use of a phloem specific endogene would allow for samples to be normalized to phloem cells instead of total citrus cells, more accurately evaluating bacterial titer and potential therapeutic effects with the phloem limited CLas.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. 3 lines of Hamlin Mthionin transgenics and 2 lines of Carrizo/Mthionin 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 both His6 and Flag tagged variants of scFv 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.
Objective 1. Create hybrid rootstocks which combine germplasm from parental material with good rootstock traits and HLB tolerance, propagate the most promising of these hybrids, and establish replicated field trials with commercial scions.
Hybrid seed from 2018 crosses were grown, including SuperSour type parentage of unique HLB tolerant types, and 51 hybrids were selected for continuing work. Seed source trees were propagated for several new hybrids being prepared for release, and additional trees propagated as a source for cuttings. A study was completed evaluating seed propagation of some of the newest US rootstocks and presented at the FSHS meeting. Information was prepared and distributed to nurseries about accurately identifying many of the new citrus rootstocks.
Multiple budded trees of about 130 selected new rootstock hybrids were grown in the nursery, in preparation for field planting other new rootstock field trials later in 2019. These nursery trees included many of the most promising SuperSour hybrids identified in ongoing trials established in previous years, and several commercial standard rootstocks. These nursery trees also include other new and different hybrids chosen because of newly available information about parentage and characteristics best associated with outstanding traits. Three new field trials with sweet orange scion will be planted from these trees later in 2019, including one trial in the East coast region, one in the Central ridge region, and one in the Southwest region.
Objective 2. Collect field performance data from early-stage replicated rootstock field trials and release new rootstock cultivars as justified by superior performance in multiyear field trials.
Seventeen rootstock trials planted prior to summer 2018 (as described in the Proposal Appendix ii) were monitored and used for data collection on field performance, as appropriate during this quarter for the scion involved. During this quarter, data collection in established trials was focused on tree size and tree health. Plans were made to begin posting results from USDA rootstock field trials to the site www.citrusgreening.org, a webpage sponsored by USDA-NIFA, and that information will be updated at least once per year for each active trial. The first trial summary data for posting to this site has been assembled from a cooperative trial in the Ft. Meade area (Planted 2014, Valencia scion, 24 rootstocks, 7 replicates), and has been modified and provided in suitable format to the host Boyce Thompson Institute.
The three new USDA rootstocks released in November 2018, identified as SS1, SS2, and SS3, are being propagated in nurseries for numerous planned commercial plantings. Additional information is being collected from established trials and other experiments on these newest rootstocks. Made significant contributions to completion of 4th edition of the Florida Citrus Rootstock Selection Guide, the newest revision of the IFAS extension publication.
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 focused on the following three activities: (1) Fourty-nine transgenic plants (30 ‘Hamlin’ sweet orange plants expressing transgene 1, 6 ‘Duncan’ grapefruit plants expressing transgene 1, 6 ‘Duncan’ grapefruit plants expressing transgene 2, 6 ‘Duncan’ grapefruit plants expressing transgene 3, and 1 ‘Duncan’ grapefruit expressing transgene 4) were planted on May 9th, 2019 in the field in Ft Pierce USDA ARS with the help from Dr. Ed Stover. Twenty health plants (10 ‘Hamlin’ plants, 10 grapefruit plants) were purchsed from Briteleaf Nursery and were randomly planted together with the trangenic plants. All plants currently grow vey well and will be analyzed. (2) Continue propagating CTV and CLas free transgenic plants. More budwoods that are negative for CTV and CLas have been grafed onto rootstocks. We will keep propagating plants whenever budwoods are available. The propageted progeny plants will be analyzed for transgene expression by Western blotting. (3) A major citrus defense gene was cloned. This gene is expected to provide resistance or tolerance to HLB when overexpressed in citrus. We plan to generate cisgenic citrus plants using this gene. The full-length cDNA was sequenced and cloned into a bacterial expression vector. We are expressing protein for antibody development. The antibody would be very useful for identifying cisgenic citrus plants that accumulate high levels of the defense gene product, which would help shroten the time-consuming screening process.
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