The goal of this project is to transform the Arabidopsis and citrus NPR1 genes (AtNPR1 and CtNPR1), and the rice XIN31 gene into citrus, and to evaluate their resistance to both citrus canker (caused by Xanthomonas axonopodis pv. citri (Xac)) and greening diseases. The first year objectives include: (1) Molecular characterization of the transgenic plants; (2) Inoculation of the transgenic plants with Xac. (3) Inoculation of the transgenic plants with the HLB pathogen and monitoring of the bacterium in planta with quantitative PCR; (4) Transformation of SUC2::NPR1 into citrus. (5) Plant maintenance. Overall the project is doing very well. To date, we have transformed AtNPR1, CtNPR1, XIN31, and XIN31K into the susceptible citrus cultivar ‘Duncan’ grapefruit. A total of seven lines for AtNPR1, 15 lines for CtNPR1, 12 lines for XIN31, and eight lines for XIN31K have been produced. All of these lines are maintained in green houses located at the Citrus Research and Education Center in Lake Alfred, Florida. More transgenic lines for each gene are to be available. A PCR system for identifying the presence of the transgenes has been established. A preliminary screening of part of the transgenic population has led to the identification of three, three, eight positive lines for the AtNPR1, CtNPR1, and XIN31 genes, respectively. Among them, the six PCR-positive lines for the NPR1 genes have been multiplied by grafting. Additionally, we have obtained the promoter construct of the Arabidopsis SUC2 gene from Dr. Jude Grosser’s lab. Construction of SUC2::NPR1 is in progress.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce. A new 8 acre site has been bedded, supplied with irrigation, and a ground cover established. Several acres in the far NE corner have been prepared for Dr. Dawson’s proposed field test of modified CTV expression vectors designed to produce anti-microbial peptides in citrus host plants. APHIS specified that Dr. Dawson’s site be as far from existing commercial citrus groves as possible, and recommended the NE corner of the Picos Farm. Answers have been provided to numerous questions from regulators to facilitate field testing approval. Cooperators have been made aware that the site is ready for planting.
The diseases Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of genes conferring resistance to these diseases or the HLB insect vector is a promising way to solve these problems. Transformation vectors, suitable for incorporating genes into citrus trees, have been prepared for five antimicrobial peptides (AMPs) with several promoters and are being used to generate transformants of rootstock and scion genotypes. Many thousands of putatively transformed shoots have been developed to produce citrus resistant to HLB and CBC or citrus psyllid. Many have been micrografted to grow shoots for propagation and further evaluation. Large numbers of D4E1 transformed rootstocks are being challenged with HLB and citrus canker. Initial trials indicate significantly reduced susceptibility to citrus canker from about 20% of the D4E1 transformed citrus. The first steps have been taken to use information from Liberibacter sequence data to develop a transgenic solution for HLB-resistance. In addition, collaboration has been initiated with a molecular biology team at the Western Regional Research Center in Albany, CA to develop constructs which minimize commercial IP conflicts, regulatory problems, and consumer concerns in transgenic citrus resistant to HLB and CBC. The strategy is called ÒcisgenicsÓ and uses genes from citrus. The genes are identified from citrus genomic data. A total of 39 antimicrobial peptides (AMPs) have been assessed in-vitro for activity in suppressing growth of the bacteria causing CBC and two surrogates for Liberibacter that are closely related alpha-proteobacters. In the initial studies, the synthetic AMPs D4E1 and D2A21 were among the most active, with minimum inhibitory concentrations at 1 µM or less across all test bacteria. Following these studies, an agreement was developed with AgroMed (the producers of these peptides) and a citrus industry partner, Southern Gardens Citrus. An additional 20 synthetic AMPs have been assessed as part of this agreement, revealing several AMPs that were highly active against all test species; transformation constructs will soon be prepared to produce transgenic citrus. Hemolysis assays have been conducted with 36 of the AMPs to gauge potential for human health risks: most of the AMPs showed slight or negligible hemolytic activity. The tomato cultivar ÔM82Õ was transformed with the AMP D4E1 and Garlic Lectin as a model system for more quickly assessing resistance than is possible using citrus. D4E1-transformed tomatoes were challenged by inoculations with Agrobacterium tumefaciens: no immune plants were identified, but some produced only very small galls and overall gall mass was 30% lower in D4E1-transformed vs. control ÔM82Õ. Transformed plants have been propagated and D4E1- transformed vs. control plants will also be challenged with Xanthomonas campestris pv. vesicatoria to assess resistance. Garlic-lectin-transformed tomatoes vs. control ÔM-82Õ will be tested for effects on populations of the phloem-feeding whitefly pest (Bemisia species). High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ÒnaturalÓ psyllid inoculation in the field are being compared. A material transfer agreement has been established with Texas A&M University to permit lab and greenhouse comparisons with the Spinach Defensin expressing grapefruit and ‘Hamlin’. Since this material is well down the regulatory pathway, it makes no sense to move forward with any transformed citrus which is not markedly superior to this benchmark material.
New hybrids between citrus relatives and citrus cultivars were studied for tolerance to HLB disease in greenhouse testing. Some new hybrids that were studied remain healthy and vigorous despite HLB infection and have been selected for further field and greenhouse testing. These hybrids will be used as parental material for breeding high quality rootstock and scion cultivars with tolerance. In this quarter, yield, fruit quality, tree size, and other performance information were collected from existing USDA/cooperative rootstock and scion field trials. Trees were propagated to establish new field trials for new rootstock and scion cultivars. Studies continue to assess rootstock tolerance of Huanglongbing (HLB) under field conditions using existing trials that have become infected with HLB through natural spread. Some differences among rootstocks for HLB response were noted and are being examined more carefully. A field experiment continued to identify rootstocks with resistance to the Phytophthora-Diaprepes Complex. Crosses were completed for development of improved citrus rootstocks focused on developing an improved sour orange (Supersour) rootstock and introgressing HLB resistance from exotic citrus relatives. In a coordinated effort between this grant and the FCATP transgenic citrus grant to USDA, selected anti-microbial and insect resistance genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with resistance to HLB and Citrus Bacterial Canker (CBC). An early flowering gene, FT, was transformed into outstanding citrus breeding material to facilitate rapid introgression of favorable traits, such as disease resistance, into new cultivars and increase early productivity of scion cultivars. Research is continuing to follow leads generated by the HLB gene expression study completed last year, including cloning of selected genes and promoters strongly expressed in response to HLB infection. These sequences are being used as targets for novel exotic genes, to help identify endogenous resistance genes, and to selectively express transgenes when and where the tree is infected by HLB. Work was continued to study the inheritance of fruit quality factors in sweet orange-type material using more than 1000 trees from populations of hybrids between high quality pummelo and mandarin parents. Work was initiated to study gene expression in seedless cultivars and identify genes that may be specifically associated with seedlessness. One promising new seedless scion cultivar, ‘Early Pride’, was released for commercial use. One highly productive dwarfing rootstock, US-942, is in the final stages of official release.
One of the major problems in genetically transforming mature citrus tissue in the hot and humid conditions of Florida and Brazil is obtaining clean explant material to initiate uncontaminated tissue cultures. Therefore initial efforts on this project are focused on methods to produce mature citrus tissue that is uncontaminated by bacteria, fungi, or insects. USDA scientists are developing in vitro systems for the continuous and semi-continuous production of mature tissue. Mature tissue derived from in vitro shoots is aseptic and will require little or no disinfestation. In addition, such tissue may respond well to tissue culture manipulations and be highly suitable for genetic transformation. UF and Brazilian scientists are growing trees under very clean conditions that prevent infestation by pests. Tissue derived from these trees should be better suited for transformation since the duration and intensity of disinfestation required should be minimal. In both the in vitro and in vivo systems, the 1) addition of compounds such as disinfectants, biocides, and antibiotics for control of pests without harm to the citrus tissue and, 2) the pretreatments, medium constituents, and environmental conditions required to effectively grow and manipulate this material is being investigated.
The objective of this project is to determine if Carrizo rootstocks, either wild type or overexpressing the Arabidopsis NPR1 gene, and so having an enhanced, inducible defense response, have any effect on gene expression and/or the defense response of wild type (non transgenic) grapefruit scions to HLB. It has long been recognized in the traditional growing of citrus that rootstock selection has a large effect on scion phenotype. However, it is not understood at the molecular level how rootstocks provide their advantages or disadvantages. For this purpose we have propagated transgenic Carrizo rootstock plants (lines 776, 854 and 857) that express the AtNPR1 gene. This gene is central in plant defense and these lines also show enhanced expression of the marker PR1 gene. Additionally, one transgenic line that lacks AtNPR1 expression and PR1 overexpression (line 884) and one transformed but nontransgenic line (i.e. lacks the AtNPR1 transgene, line 859) and wild type Carrizo planst were also propagated as negative controls. These lines have been grafted with wild type ÔDuncanÕ grapefruit. Other controls consist of nongrafted clones of AtNPR1 Carrizo (all lines mentioned above), wild type Carrizo, and non grafted grapefruit. The plants are being maintained in a containment facility at UF in Gainesville. We have also multiplied our HLB inoculum in ÔDuncanÕ grapefruit. The next step is to challenge the different graft combinations and to examine them for phenotype and gene expression with and without HLB challenge.
Juvenile sweet oranges were grafted to several rootstocks with potential to induce scion precosity. Trees will subsequently be transferred to our early fruiting structure to encourage precocious flowering and fruit set. Seed of the same clones were planted to provide juvenile seedlings for planned experiments. Transformation experiments comparing transformation efficiency of various selected sweet oranges were initiated, including several clones that have previously shown precocious bearing in field trials. In Arabidopsis and other plants, the overexpression of a FLOWERING LOCUS T (FT) gene in transgenic plants leads to accelerated flowering. In the Moore lab we have cloned and sequenced the three citrus FT genes and placed them individually in transformation vector plasmids. We have initiated Agrobacterium-mediated transformation experiments with Carrizo citrange, sweet orange, and grapefruit.
This project has two main objectives: 1) Development of genetic transformation systems for mature tissues of the most important sweet orange varieties of Florida, namely Hamlin and Valencia, using Pineapple as a control for which a transformation system already exists, and of the most important rootstock genotype, Carrizo citrange. Procedures would be first developed in Spain and then transferred to Florida, where a new greenhouse would be constructed and implemented for this purpose. 2) Overexpression of meristem-identity genes in Pineapple sweet orange to generate more compact and productive varieties. RNA interference-mediated downregulation of a gibberellin biosynthesis gene in Carrizo citrange to produce semidwarf/semidwarfing rootstocks. For the first objective, preparation of plant material for transformation was initiated about one year ago in Spain and this permitted us starting transformation experiments of the three sweet orange varieties 3 months ago. Both Hamlin and Valencia mature tissues are being highly responsive to organogenic regeneration. As we started to work with Valencia, the first GUS-positive mature Valencia transformants have been already generated and are growing in vitro. Due to space limitations, preparation of starting material of mature Carrizo citrange is a bit delayed. In Florida, we have provided to The University of Florida IFAS facilities all the specifications for the greenhouse and are in the planning stages. Moreover, we have to hire a manager for the mature transformation facility and allow this scientist to begin training in the Spanish laboratory. A job description has been written, a search committee selected, and the position is awaiting approval by University of Florida bureaucracy. We expect that this position will be filled by late summer. For the second objective, we have initiated experiments to ectopically over-express CsAP1 (AP1 from sweet orange) and CsFT (FT from sweet orange) in transgenic Pineapple sweet orange. The formalization of the master agreement between the IVIA and the Department of Citrus has suffered an unanticipated delay of several months mainly due to IVIA bureaucracy.
Despite the short time period from the initial funding of our FCATP grant, we have made progress on all areas of the project: 1. Isolate TAL effectors: We have obtained one of the primary Xc TAL effector genes, PthAw,and it has been subcloned into an expression vector. Additional Xc TAL effectors are in the process of being isolated by PCR and subcloned into expression vectors. 2. Identify TAL effector binding sites: A binding site determination has been made for PthAw. Additional binding sites will be determined as the TAL effectors become available. 3. Construction of modified Bs3 promoter constructs: The binding site for PthAw has been introduced into the Bs3 promoter, upstream of the native UPA box which mediates induction by AvrBs3. 4. Rapid testing for promoter function: The new construct was tested with the GUS reporter gene in the standard N. benthamiana assay and was found to be inducible by PthAw in planta. Importantly, the modified promoter was completely inactive in the absence of PthAw. Furthermore, much work has been done to develop a transient assay system in citrus leaves. Conditions have been established that allow the assessment of the function of individual Xc effectors upon delivery into citrus leaves by Agrobacterium. This method is now being used to test Bs3 constructs. One issue that has come to light is that the native pepper strain, Xanthomonas euvesicatoria 85-10, causes a non-specific hypersensitive response, and so delivery of TAL effectors to citrus must come from other bacterial strains, such as Agrobacterium. Additional transient assays are in progress in citrus cell cultures and protoplasts, and Bs3 constructs using the GFP reporter gene will be tested for both GFP and Bs3 activity. The determination of Bs3 activity in citrus is our main objective for the present time. 5. Stable citrus transformation: Initial work on this has been initiated with test constructs. Until constructs with Xc TAL effector binding sites are available, stable transformants are being tested using AvrBs3-inducible constructs. A first project meeting was convened in Gainesville on June 15th with PI’s Horvath, Jones, Stall and Moore. We discussed the initial data reported here and reviewed the goals and milestones for this period. The emphasis on determining the activity of BS3 in citrus was highlighted and experiments planned. An additional meeting will be convened in July in Quebec with PI’s Horvath, Lahaye, and Jones’ lab postdoc Figuieredo.
Objective II: Website creation and development. A Citrus Greening/HLB Genome Resources Website has been created and can be accessed at http://www.citrusgreening.org/. Significant aspects of this objective achieved during this quarter include the following: A. Development of a policy for data sharing. Concerns about publication priority, reluctance to release data that might be in error, and the slow pace at which sequence is generated have all contributed to a scarcity of publicly available genome data. In collaboration with the program manager, a policy was developed whereby the genome resources website would provide a venue where preliminary data and analyses generated from these data could be deposited, with access restricted to registered users. Prior to viewing the restricted data, users would be required to acknowledge the preliminary nature of the data and agree not to publish analyses based on these data without contacting the depositors. B. Website organization. The http://www.citrusgreening.org/ website has been designed to accommodate both information with unrestricted access and data limited to registered users. Web pages with unrestricted access include those containing general information and links on the disease, pages providing guidelines and links for commonly used analytical tools such as BLAST analysis and genome viewing with the Artemis genome viewer, and pages summarizing the range of Ca. Liberibacter sequences that have been deposited at NCBI. With the exception of a 1.2 Mb draft genome sequence for Ca. L. asiaticus, all of the Ca. Liberibacter sequences currently available at NCBI fall into one of three gene clusters, diagrams of which have been posted on the website together with hyperlinks to their component accession records at NCBI. In order to restrict access to pages containing preliminary data, a registration page has been set up requiring users, pending acceptance of the data sharing policy, to enter contact information and choose a user name and password. Once reviewed by the PI, the registrants are granted access to the restricted pages and their contact information added to the list of registered users. Information on data submission is included on the pages with unrestricted access. While it is expected that the site will grow significantly as more data and analyses become available, the basic website structure has been designed to accommodate future growth. Objective III: Bioinformatic analysis. Using the draft genome sequence for Ca. L. asiaticus deposited at NCBI, a single pseudomolecule has been generated from the deposited contigs to aid users in viewing the 34 contigs in their totality (draft sequences are deposited at NCBI as independently numbered and annotated contigs). Various feature files have been produced for the pseudomolecule, so that users can view annotations generated by the MG-RAST server as well as BLAST hits independent of gene calls. Functional roles predicted by the MG-RAST server have been mapped to KEGG metabolic maps and the results compared to maps generated for two fastidious plant pathogenic bacteria and four insect endosymbiotic bacteria. Comparison reveals that the deposited Ca. L. asiaticus sequence encodes only a small percentage of enzymes involved in amino acid, carbohydrate, energy, glycan, lipid, and nucleotide metabolism relative to these other organisms. This limited metabolic capability suggests that the deposited sequence may not represent a complete genome. Objective I: Assess community needs. Various members of the citrus greening community have provided input during website development. Further conversations between the PI and community members about data deposition and desired analyses are expected at the special session on citrus greening to be held at the meeting of the American Phytopathological Society (August 1-5).
This project has three objectives: 1) gap closure of Ca. Liberibacter asiaticus (Las) found in Florida; 2) complete genomic sequencing to closure of Ca. L. americanus (Lam) strain S’o Paulo from Brazil, and 3) comparative genome analysis of Las and Lam to attempt to determine common factors enabling pathogenicity to citrus. Objective 1 Progress: Within the recently published Las strain psy62 chromosomal genome (Duan et al. 2009), many unique genes of unknown function are found, and several phage related genes are found integrated into the chromosome, but no replicating phage DNA was found. We reported last quarter the finding of two complete circular lytic phage genomes, SC1 and SC2, with the copy number of SC1 replicating an average 10X higher in citrus and 20X higher in periwinkle, than when the phage is integrated into the chromosome in infected psyllids. [Gabriel & Zhang, 2009. Phytopathology 99 (6): S38]. Neither phage were previously reported. Phage SC1 is 40 kb in size. SC2 is 39 kb in size and appears to be a degenerate variant of SC1. Phage particles, 25 nm in size, and likely corresponding with infectious SC1 phage, have now been documented inside Las cells, inside swollen Las cells that appear to be lysing and outside of Las cells by electron microscopy in Las-infected periwinkle phloem cells. Phage particles were not seen in Las-infected citrus. In citrus, the phage are replicating to relatively high copy number but not lysing Las cells, as they are in periwinkle. Since some of the SC-1 and SC-2 phage genes were found by Duan et al (2009) to be integrated into the Las genome, both phage likely have a lysogenic cycle. Lysogenic phage are known to mobilize bacterial genes when they repackage into infectious particles and then horizontally transfer these genes between different bacteria that they infect. Any genes carried on a phage that replicates to a level 10X higher than the host chromosome also replicates the copy number of all genes it carries to a level 10X higher than the rest, dramatically increasing gene expression levels, possibly affecting virulence. Following that lead, several new Las genes were found on SC1 and SC2. Detailed genomic analysis has revealed a total of six outer membrane proteins, five of which are predicted to form beta barrel structures, several of which were not previously reported in the psy62 genome. One of these appears to be an enzyme that is predicted to modify the bacterial outer membrane structure; another is a predicted porin, which form structures through which molecules can passively diffuse. A third, and previously unreported protein is a predicted outer membrane anchored protein with a collagen triple helix repeat. This unusual protein may be capable of forming a fibrous protein matrix, perhaps causing Las aggregation and trapping Las within phloem cells. We observed Las cells within periwinkle phloem by EM to occupy well over 50% of the cell cytoplasm. Objective 2 Progress: In collaboration with Fundecitrus in Brazil, Lam strain ‘S’o Paulo’ DNA samples extracted from citrus and purified on pulsed-field gels by Dr. Nelson Wulff has resulted in 78 kb of partially confirmed Lam genomic sequence obtained by 454 sequencing assembled into contigs greater than 1 kb. Dr. Wulff recently brought new Lam DNA to our lab for additional sequencing and comparison with the Las genome and the yet to be published sequence from the Liberibacter causing “zebra chip” disease of potato (Objective 3). We are on track to meet target deadlines of the original proposal.
During the final quarter of the year, the Core Citrus Transformation Facility (CCTF) maintained its level of performance and produced transgenic citrus plants for many satisfied customers. During the 4th quarter, the CCTF continued to utilize the provided funding to process the orders for transgenic plants, mostly servicing CRDF funded researchers studying transgenes with potential to generate resistance to HLB. Orders processed during this quarter include: pY46-Carrizo; pY102-Carrizo; pY141-Carrizo; pY150-Carrizo; pCitIntra-Duncan; pAZI-Duncan; pAtBI-Duncan; pBCR2-Duncan; pDPR1-Duncan; pLP1-Hamlin; pLP1-C-mac; pLP2-Hamlin; pLP2-C-mac. During the last quarter of this funding year, work was mostly concentrated on recent orders. Fourteen Duncan plants were produced carrying a gene of interest from the p35S-TRX vector and 23 more Duncan plants were produced carrying a gene from the pSucTRX vector. Multiple Duncan plants were produced toward satisfaction of ‘WG’ group of orders: eight-pWG22-1 plants, three-pWG21-1, and four pWG25-13 plant. Also, following Carrizo plants were produced for the ‘Yale’ order: nine plants with the gene from the pY46 vector and 11 plants with the gene from the pY102 vector. Eighteen Duncan plants were produced after treatment with bacteria harboring pBCR2 vector. Three more Duncan plants were produced with the EDS5 gene and six Mexican limes with the P35 gene. Four additional Duncan plants carrying a gene from pSUC-CitNPR1 were produced. The impact of citrus diseases on Florida citriculture is rapidly growing, and our participation in this battle is growing with some positive results already being published. Continued funding for CCTF which is an integral part of this community and contributes greatly towards common goal will allow for the progress to go on by keeping production of transgenic material un-interrupted and at high levels.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. A number of plant derived insecticidal transgenes, each driven by a root specific promoter were incorporated into Carrizo citrange. As part of this project we cloned four insecticidal genes and two plant derived promoters. Constructs were initially tested on N. benthamiana for confirmation of transgene activity. Carrizo citrange was subsequently transformed utilizing the conventional Agrobacterium mediated transformation process. A total of 123 putative transgenic lines were generated. PCR screening identified 74 of these lines to be transgenic. 54 of these transgenic lines had a high level of transgene expression as determined by qPCR. We have also identified a number of putative root specific genes from Citrus clementina by data mining the phytozome database. Of them several putative sequences were selected for characterization using qPCR. RNA was extracted from mature and juvenile roots from non-transgenic Swingle citrange. Each of the identified sequences were characterized in these different tissues. In addition, transcript levels in the leaves were also measured. The Cic1867m gene was determined to be very root specific and a 1.2 kb fragment of its promoter was cloned from Clementine genomic DNA using PCR. Deletion analyses identified a 0.8 kb fragment from that promoter fragment to be sufficient for root specific activity and transgenic plants were produced using this promoter. Cuttings from all the better performing lines have been made and are being rooted in the mist bed. These clones will be sized up for Diaprepes feeding experiments. Clonally propagated plants will be force fed with Diaprepes neonates – when available and root damage / insect mortality evaluated.
This project had a single objective to assure un-interrupted production of transgenic citrus plants through the services of Citrus Transformation Facility (CTF). Continued operation of the facility was going to guarantee a foundation regarding this aspect of research for the scientific community involved in fight against huanglongbing (HLB) and citrus canker. By producing transgenic plants and bringing into life the ideas of scientists working in different laboratories, CTF would facilitate the process of search for candidate plants that would be tolerant/resistant to diseases and provide major relief for Florida Citrus Industry. The objective of this project was successfully accomplished. Throughout the funding period of three and-a-half years, CTF operated without major interruptions and provided service in the form of production of transgenic plants. The level of production stayed at expected, satisfactory level and resulted in creation of 781 citrus plants that represent independent transgenic events. These plants belong to eight different cultivars: Duncan grapefruit, Carrizo citrange, Pineapple sweet orange, Mexican lime, Valencia sweet orange, Swingle citrumelo, Kumquat, and Pomelo.During this project, CTF worked on 103 orders that were placed by 10 clients and this group included eight faculty members based at University of Florida, one faculty from University of California and one Foundation. The number of projects for which the plants were produced was higher than the number of clients. For example, Dr. Nian Wang was one of the clients but orders from his lab were the part of at least five different projects done by his post-doctoral associates.All produced transgenic plants were associated only with research that has to do with disease resistance. This is a clear indication of the role CTF plays in the efforts to overcome effects of HLB and citrus canker.CTF supported the projects from Dr. Nian Wang’s lab that resulted in successful application of CRISPR/Cas9-mediated genome editing technology in Citrus. Plants produced this way will be the most market-friendly as they only have minor modifications of their own gene(s) and in the near future will be free of any other inserted DNA. The work done in CTF lead to production of Duncan grapefruit plants that exhibit high degree of resistance to citrus canker. Other commercially important cultivars could be made with improved resistance to canker based on results of this project.As a part of another research project aimed at control of Asian Citrus Psyllid (ACP) in citrus groves, transgenic Indian curry leaf plants were also produced in CTF. There are six such plants that are being tested for the ability to kill ACP after they were allowed to feed on their leaves. All these successes were achieved even though CTF experienced high flux of employees, had to move to temporary location for six months, and was exposed to adverse effects of hurricane Irma.Future opportunities for the CTF are determined by the needs of Florida Citrus Industry. As those needs change from the search for the solution to HLB and canker to a development of specialty fruit with consumer-oriented traits in post-HLB era, CTF will be there to support all these efforts.
Update for this quarter: The Stover team has submitted paperwork to renew their primary BRS permit, which covers the transgenic materials planted by Z. Mou, J. Jones, T. McNellis as well as USDA scientists. A new permit has been approved (AUTH – 0000043619 effective 12/17/2020) for material with “Confidential Business Information” for a project led by R. Shatters. The new BRS permitting system called e-file has used us a first trial and it has been very arduous and time-consuming. Information was provided for the BRS review of the transgenics at Picos Farm for CREC (Dutt, Grosser and Gmitter) who maintain a separate permit. UF collaborators have been permitted into the test site and samples and data have been collected. Data were collected on McNellis trees by USDA. Samples we previously collected have been processed by technicians at home (with APHIS-BRS permission) and are ready for qPCR. A manuscript has been prepared and submitted for canker data collected in a block of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside. Previously Stover analyzed data on canker incidence in a block of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside, from data collected 8/17, 9/19, and 9/20, Most notably: Almost all accessions with lower ACC lesion incidence were hybrids vs. pure trifoliate, though a few pure Poncirus had lower ACC than most. Based on chloroplast genome data from 57K Affymetrix SNP chip, provided by M. Roose, 11 of 33 reported seed parentage for hybrids was inaccurate, convention of female first was not followed. Of 34 hybrids validated, similar numbers had Poncirus, grapefruit, and sweet orange chloroplasts. Chloroplast type did not affect ACC incidence, but in each year accessions with grapefruit chloroplasts had small but statistically higher ACC severity than those with Poncirus chloroplasts. Hybrids of Citrus with Poncirus have markedly reduced ACC sensitivity compared to Poncirus, indicating that this trait is readily overcome in breeding. Seed from fruit harvested for transgenic gene flow experiment coninue to be processed for PCR. Previously established at the site: A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. Renewal and approval for BRS permit effective 9/1/19 through 8/31/20. 4) Continuation of an experiment on pollen flow from transgenic trees. FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. Early-flowering transgenic Carrizo (flowered ex-vitro within five months of seed sowing, and used at 12 months) was used to pollinate some of the same FF-5-51-2 What should be the final samples from the C. Ramadugu-led Poncirus trial (#3 below) completed preparation and were shipped in ethanol to UC Riverside. Availability of the test site for planting continues to be announced to researchers. Plantings: 1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the Stover permit. 2) Under the Stover permit a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. Likely 2019 will be the last year for data collection. 4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project. 5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and will continue to be conducted under a new NIFA grant. 6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.). 7) In a project led by Fred Gmitter there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo. 10) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls. 11) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type. 12) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State13)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
