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 many promoters have been used to generate transformants of rootstock and scion genotypes. Thousands of putatively transformed shoots have been developed to produce citrus resistant to HLB and CBC or citrus psyllid. Many hundreds have been micrografted and some further propagated for replicated evaluation. D35S/D4E1 transformed rootstocks have been challenged with HLB and CBC. Initial trials on CBC resistance were inconclusive. HLB-inoculated transformed plants grew significantly better than controls but developed HLB symptoms. More active promoters are in the pipeline to achieve better results. Tests of garlic-lectin transformed citrus are underway to determine effect on psyllid feeding and development. Efforts are underway to use Liberibacter sequence data to develop a transgenic solution for HLB-resistance, targeting a transmembrane transporter. Collaboration is underway with a USDA team in Albany, CA to provide constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns. Genes are also being identified from citrus genomic data to permit transformation and resistance using citrus-only sequences. 39 antimicrobial peptides (AMPs) have been assessed in-vitro for activity in suppressing growth of the bacteria causing CBC and two bacteria related to Liberibacter. 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. An additional 20 synthetic AMPs were assessed, revealing several AMPs that were highly active against all test species, with negligible hemolytic activity. Transformation constructs will be prepared to produce citrus with these AMP transgenes, having completed an agreement with entities who posses the rights to these AMPs. 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 be challenged with Xanthomonas campestris pv. vesicatoria to assess resistance. Garlic-lectin-transformed tomatoes vs. control ÔM-82Õ will be tested for effects on the phloem-feeding whitefly. 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. High-throughput CBC screening methods are being compared, with the hope that CBC-resistance will be correlated with HLB resistance in transgenics driven by constitutive promoters. 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’. They also are providing the spinach defensin AMPs for in-vitro analysis. 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.
We completed the construction of two quarantine houses, one for Citrus saplings infected by psyllids collected from the field, and another for Citrus grafted to infected root stock, and ramped up rearing to high numbers of insects with Ct values indicative of infection (Stansly). Two infected colonies were initiated in April 2009: (1) ÔCarrizo’ citrange and ‘Pineapple’ sweet orange plants (total 33) were inoculated with Candidatus Liberibacter asiaticus through grafting bark tissue from a PCR positive HLB infected plant, and (2) 300 field collected psyllids that tested 10% HLB positive by PCR (N=52) were released in two cages, each containing 6 ‘Carrizo’ plants. All plants tested negative for HLB before they were inoculated and became infected. After about one month, dark green mature leaves were PCR tested and 85% (orange) and 95% (‘Carrizo’) grafted plants, respectively, were HLB positive, whereas, plants exposed to infected psyllids were negative. Then, grafted plants were pruned to induce new shoots that tested 31% and 35% HLB positive at 2wk and 90% and 100% positive at 4wk when 97% of mature leaves also tested positive. Two months after exposure, 83% psyllid-infested plants tested HLB positive based on qPCR using dark green mature leaves. We optimized qPCR by developing a standardized curve to determine threshold levels of detection in adult and nymphs (Roberts). Adult psyllids collected on 8/12/09 from both infected colonies and a control colony reared on Murraya paniculata were qPCR tested, and reported to be 61% and 67% HLB positive, grafted and psyllid-infected plants, respectively. Our efforts to maintain quarantined colonies with a high HLB titer have been tracked by lengthy and systematic qPCR. The latest report consisted of 148 reps from 3 HLB infected colonies and 1 uninfected colony, as well as 35 corroborative replicates tested in Riverside, CA. We performed exploratory qPCR analysis of psyllid organs (guts) for the presence of HLB. Detailed methodology is per Li et al. (2006) for plant material and Manjaunth et al. (2008) for psyllid samples (Roberts; Stansly). Isolation and purification of mRNA from 1000 non-infected adult D. citri gut extirpations (#1UNIFadult-gut), previously reported, yielded ~10 ug of total RNA (511 ng/ul in a total volume of 20 ul), from 1ml of Trizol sample #4INFwhole-adult (50-100 mg adult psyllids), at 572 ng/ul in 20 ul (or enough for cDNA synthesis) (Gang, Brown). Two cDNA libraries have been successfully constructed from samples #1 and #4. These libraries were sent for DNA sequencing using 454 technology ~2 weeks ago. The first DNA sequence data will soon be assembled and processed. We are developing a new protocol for total RNA extraction from psyllid larval samples #5INFlarv and #6UNINFlarv. Preparations yielded ~10 ug of total RNA (~500 ng/ul in a total volume of 20 ul) from 1ml of Trizol sample (50-100 mg psyllids). Sequencing of libraries under construction will commence once we are sure of the quality of the current data. To complement the 1000 uninfected guts extirpated during the last quarter, we have commenced extirpation of 1000 guts from adults reared from eggs on grafted Citrus. We have stockpiled 200 guts to date. Only adults raised from egg on graft-infected plants were used. This follows the finding of Inoue et al (2009) that Liberibacter multiplies to high titers in 5th instars but not in adults transferred from uninfected plants (Cicero, Brown).
CCTF continued its expanded operation with the new organization of the work reported in the previous quarterly report. The facility continued to service orders mostly for FCPRAC funded researchers wanting transgenic plants with various disease resistance constructs. The list of transgenic plants that were produced during the quarter and confirmed by the presence of the reporter gene and appropriate validating PCR reaction: LIMA gene: 1 Flame grapefruit; LIMA gene: 9 Mexican lime; CIT108 gene: 1 Valencia sweet orange; CIT108p gene: 3 Flame; CIT108p3 gene: 5 Flame; CIT108p17 gene: 16 Flame; PITA gene: 3 Duncan grapefruit; F3* gene: 2 Duncan; AF1 gene: 1 Hamlin sweet orange, 1 Duncan; NPR1 gene: 1 Duncan; p6 gene; 3 Sour orange; pTLAB21 vector: 3 Duncan; pTLAB32 vector: 3 Duncan. CCTF also produced approximately 30 soil-adapted plants that were not tested by PCR for the presence of the transgene of interest. These fall into two groups: 1) plants that were selected on the basis of the presence of reporter gene, 2) plants obtained after co-incubation of explants with the Agrobacterium strain that carried binary vector with the reporter gene.
This is a 3-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (MKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, we have subcloned the Arabidopsis MKK7 gene into the CTV-based expression vector and transition expression of MKK7 in citrus leaves is underway. We have also subcloned the Arabidopsis MKK7 gene into the plant binary vector pBI1.4T (a pBI121 derivative) and transformed the MKK7 gene into citrus using the Agrobacterium-mediated approach. The MKK7 transgenic plants are growing and characterization of the MKK7 transgenic citrus plants is underway. For objective 2, we have decided to use the Hamlin suspension cells as starting materials for the selection. The Hamlin cell suspension culture has been scaled up in Murashige and Tucker (MT) liquid medium. Several flasks of the culture are maintained for subculture. To determine the concentrations that will be used in the selection, the Hamlin cells from the suspension culture were grown on MT medium plates supplemented with different concentrations of sodium iodoacetate ranged from 0 to 0.2 mM. Hamlin suspension cells were found to be highly sensitive to the inhibitor. A concentration of 0.1 mM of sodium iodoacetate could completely arrest their growth. Therefore, 0.1 mM of sodium iodoacetate has been used in the selection. We are also testing hypocotyls of citrus seedlings because plants can be easily regenerated from hypocotyl-derived callus cells. Hypocotyls were grown on MT medium plates supplemented with different concentrations of sodium iodoacetate ranged from 0 to 0.4 mM. Hypocotyls were also very sensitive to the inhibitor. A concentration of 0.2 mM could completely inhibit the growth of calli generated from hypocotyls. We will use 0.2 mM of sodium iodoacetate in selection of hypocotyl-derived calli. We are currently generate mutations in the hypocotyls using fast neutron-mediated mutagenesis. Calli will be generated from fast neutron-treated hypocotyls. The hypocotyl-derived callus cells will be selected on MT medium plates supplemented with 0.2 mM sodium iodoacetate.
We have propagated by cuttings the following ÔCarrizoÕ citrange AtNPR1 transgenic lines: 854, 857, 859 and 884. Lines 854 and 857 show high overexpression of the endogenous marker gene PR1 (considered a marker of SAR). Lines 859 and 884 do not express the AtNPR1 transgenic gene and do not show overexpression of the endogenous PR1 gene, hence are considered as negative controls. Subsequently we have grafted a number of these plants with wild type (WT) ÔDuncanÕ grapefruit. We also grafted WT ÔCarrizoÕ plants with ÔDuncanÕ grapefruit as controls. We have also treated the plants with either salicylic acid (SA) or water (as negative control) and are in the process of comparing their response using TaqMan Real Time PCR. In preparation for the real time experiments we have also sequenced a number of genes of interests (NPR1, NPR3 and PR1) from both ÔCarrizoÕ and ÔDuncanÕ to guaranteed that the target probe/primer sequences within the genes are identical and that any observed differences in expression are not due to differential efficiency in annealing of the probes and/or amplification. We have also standardized the real time reaction for the marker PR1 gene and continue to do so for the rest of the genes. This will allow us to analyze the response of the plants as proposed in objective one. The same group of plants will subsequently be analyzed as proposed in objectives 2 and 3. For this purpose we have also been propagating HLB-infected material and have standardized the real time PCR detection of the pathogen so we are confident we can conduct the proposed experiments.
Assessment of Bs3 activity in citrus remains our primary initial objective. We have three approaches under development: 1) Transient transformation by Agrobacterium infiltration. We have been working to establish a reproducible Agrobacterium-based citrus transformation system. Our first efforts have focused on leaf infiltrations with fluorescent (GFP) reporter gene constructs and examination by confocal microscopy. The method has not performed adequately thus far, with the appearance of fluorescent materials in the GFP channel which may be phenolic compounds. Although we can subtract out autofluorescence, these compounds are still visible and complicate the interpretation of the experiments. Consequently we resynthesized gene constructs with the GUS reporter gene. We continue to test the GFP constructs with appropriate controls, but we have now also begun the use of the GUS constructs. We have also tested transient transformation by dip inoculation of various citrus plant parts, as well as infiltration of intact citrus fruits. Most of the testing has been with 35S:GUS marker gene constructs, and we have observed GUS staining of albedo in sweet orange and other citrus fruits. We will continue to examine the Bs3 GFP and GUS constructs in the fruit assay to assess the function of both marker genes and Bs3 by these approaches. 2) Transient transformation using particle bombardment. We have initiated this additional transient approach to test a standard assay for resistance gene function – co-bombardment of reporter genes with or without a resistance gene that cause cell death. In these assays, a GUS reporter gene produces individual transformed cells that turn blue, whereas in the presence of the R gene, the blue signal is lost due to a highly localized hypersensitive response. These experiments require a different set of gene constructs, which have now been made and will be testing with a particle gun at UF. 3) Stable transformation using Agrobacterium. Our stable transformation protocol requires citrus seedlings, which must be germinated from seasonally-available seeds. Seeds are now available, and the first experiments have been set up. These experiments included the following constructs: (i) 35S:Bs3-GFP (constitutively expressed Bs3 gene) (ii) 35S:Bs3m-GFP (constitutively expressed inactive Bs3 gene) and (iii) Bs3:Bs3-GFP (the Bs3 gene under control of its own promoter). We will assess GFP expression, cell death, and growth of X. citri. First results will be available later in October. In addition to the transformation work, we have completed assembly of a multi X. citri strain-inducible Bs3 gene construct. The original Bs3 promoter, which has an activation site for AvrBs3, was modified to contain fourteen additional activation sites for TAL effectors from all four X. citri A strains, as well as A*, Aw, C and B strains (Table 2 of our Project Narrative). This promoter is currently used to drive the GUS reporter gene, will be used in planta to test which X. citri strains activate the complex promoter. The design is facilitated by the recent discovery of a simple code between amino acid residues in TAL effectors and specific DNA bases defining protein-DNA interaction, in the manner of zinc finger proteins (in press, Science). The results will inform commercial constructs with Bs3 conferring disease resistance. 2Blades has gained the capability to do whole genome sequencing in our new laboratory, and we will begin sequencing of several X. citri strains to examine TAL effector sequence diversity. Coordination of the project is facilitated by participation of all the parties in regular conference calls to discuss results and next steps.
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 at least one complete circular phage genome and a large contig that appeared to represent another phage. Neither were previously reported. We have now quantified the copy number per cell of the two phage DNAs relative to the main chromosome, and have confirmed that the SC-1 phage DNA appears to replicate in Las when infecting citrus to a level averaging 10X higher than the Las chromosome when Las is extracted from infected psyllids, and that SC-1 replicates to a level averaging 20X higher than the Las chromosome when Las is extracted from infected periwinkle. The SC-2 phage DNA appears to replicate in Las to a level 2-3X higher than the Las chromosome in both citrus and periwinkle. These data strongly indicate that we have discovered two lytic phage and was reported at the last APS meeting [Gabriel & Zhang, 2009. Phytopathology 99 (6): S38]. 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, encoding: 1) a hemagglutinin; 2) an ABC dipeptide secretion component of a Type I secretion system; 3) a porin type major outer membrane protein; 4) a lysozyme and 5) a fatty acid desaturase, any of which may contribute to pathogenicity. None were reported in the psy62 genome. The hemagglutinin was found on both SC-1 and SC-2. Hemagglutinins are secreted by Type I secretion systems and can be effectors of pathogenicity. Complete Type I systems are found in the Las genome. Since this new hemagglutinin is encoded by two different phage, and since the phage can replicate to ca. 10X-20X copy number in plants per Las cell, this hemagglutinin will be investigated experimentally for a potential role in HLB disease. 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. Additional sequencing is currently being performed, and will be analyzed together with data from our previously reported Lam fosmid libraries in 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.
In this quarter, three new field trials were planted at the Whitmore Farm in Lake County to study inheritance of fruit quality factors in sweet orange-type material from populations of hybrids between high quality pummelo and mandarin parents. One of these was planted on trellis, to also examine the effect of tree manipulations on the length of time for transition from juvenility to maturity. Tree performance information was collected from existing USDA rootstock field trials. Greenhouse trees were inoculated for assessment of new Supersour rootstock hybrids for tolerance to CTV. Cuttings were made from new Supersour rootstock hybrids to propagate trees for field trials. Seedling and cutting liners of rootstock selections in the greenhouse were budded with Valencia, Hamlin, Ray Ruby, and Minneola to prepare trees for field trials. Studies continue to assess rootstock and scion tolerance of Huanglongbing (HLB) under field conditions and in the greenhouse. Some rootstocks and scions appear more tolerant to HLB infection than others. Some new hybrids that were studied remain healthy and vigorous despite HLB infection and have been selected for further field and greenhouse testing. US-897 rootstock has exhibited strong tolerance to HLB infection and preliminary information suggests it confers increased HLB tolerance on trees with susceptible scions. Experiments were conducted to assess the effectiveness of different methods for testing new cultivars for resistance to Asian citrus psyllid and HLB disease. A field experiment continued to identify rootstocks with resistance to the Phytophthora-Diaprepes Complex. In coordinated research 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). Selected transgenic rootstocks from this effort were challenged with HLB and CBC to assess resistance potential. Some transgenics were found to have potential resistance or tolerance. Research is continuing to follow leads generated by the HLB gene expression study completed last year, including cloning of a selected promoter strongly expressed in response to HLB infection, and combining that promoter with selected resistance genes to produce transgenic citrus with a rapid resistance response. An early flowering gene, FT, was transformed into superior citrus breeding material to facilitate rapid introgression of favorable traits, such as disease resistance, into new cultivars and increase early productivity of scion cultivars. Four new hybrid rootstocks, US-1235, US-1239, US-1225, and US-1241, were identified as especially promising for expanded field trials in the coming year.
Two trees have been found growing in HLB-ravaged orchards in Guangdong and one other in Guangxi province, that appeared to be free of HLB symptoms, while all other trees planted at the same time were either dead or declining, and replants likewise were afflicted. The trees from Guangdong were propagated at the Guangdong Institute of Fruit Tree Research facilities, and are being grown to conduct new tests of their reaction to HLB following deliberate inoculations. These trees have been tested twice after propagation using standard RT-PCR protocols, and they remain PCR negative for HLB. Two propagations of one of the selections have been replanted in an infected orchard location. The tree in Guangxi has been transplanted to a protected location in Guilin, at the Guangxi Citrus Research Institute. A valuable side benefit of this project has been the opportunity in our search for “survivors” to survey regions where HLB devastation is severe and quite widespread, and in doing so we have also visited orchards that appear to be nearly completely unaffected by HLB though surrounded by severely declining orchards. These surprising locations have been visited both in Guangxi and Guangdong. We have been investigating the nature of their management programs that has enabled them to survive to eight years of age or more in apparently good health. We interviewed growers, pathologists, horticulturists, and entomologists associated with these healthy orchards. We have reported on our experiences and the answers to our questions in recent editions of “Citrus Industry”. Although located in different provinces several hundred miles apart, the key elements outlined to us were the same. These include critically timed pesticide applications, use of pathogen-free planting materials, and maintenance of tree health through good nutrition. Our impressions have been presented likewise through talks given at various grower meetings in Florida.
At this point, we have collected and germinated seeds from several of the candidate categories listed in the proposal, including pummelos, intergeneric hybrids with Poncirus, and others; seedlings are being grown in DPI-certified greenhouses at the CREC to provide budwood for topworking and young trees to plant directly in the field. We have not been able to acquire seed of all the selections we intend to test, because the project approval came some time after the season for seed acquisition. We had an agreement with one grower in Florida to plant out the range of genetic diversity we hoped to test, but that agreement has been terminated, and we are currently looking for other options within Florida. We are working on the list of materials that can be sent to our collaborators in China, without compromising UF-IFAS intellectual property rights.
The seeds of Murraya paniculata were procured from the USDA-ARS National Clonal Germplasm Repository for Citrus and Dates, as well as from local sources in Florida. Seeds have been available only periodically for our experiments. These seeds have been germinated in vitro and used to establish cultures for experiments designed first to improve the efficiency of plantlet regeneration from epicotyl explants, which is a prerequisite for experiments to develop successful genetic transformation protocols for Murraya. Following this step, we conducted a first round of transformation experiments using the plant regeneration protocol that was developed. A tissue culture medium designated as M10 (Murashige and Skoog’s (MS) standard medium supplemented with predetermined levels of BA and NAA) was used as the regeneration medium for all the transformation experiments. In efforts to standardize the genetic transformation protocol, experiments were carried out with plasmids previously used successfully for citrus genetic transformation in our lab; specifically we used pCAMBIA2301 and pGreen0029 harbored in Agrobacterium tumefaciens strain AGL-1. Various factors were tested in efforts to develop a standard protocol for transformation, such as varying OD values of the Agrobacterium cultures, the duration of explant incubation time, duration of co-cultivation, and the amount of antibiotic used for selection of transgenic shoots and for Agrobacterium removal. Unfortunately, we were not successful in recovering transgenic plantlets from any of these experimental treatments. Currently, we have designed and implemented new experiments using a number of different Agrobacterium strains to which Murraya may be more susceptible, as well as other plasmid vectors that may be more compatible with this system. Our original intention was to test methods and materials that are routinely used successfully for citrus transformation, to avoid additional and possibly lengthy experiments to assess various other Agrobacterium strains and plasmid constructs. We did so, according to plans, but found that the assessment of other strains and plasmids is unavoidable.
Funding is now in place among all the partners of the International Citrus Genome Consortium (US, Brazil, Spain, France, and Italy) to move forward with the project to sequence a haploid citrus genome. DNA samples for sequencing have been prepared, and the strict quality control standards required by the sequencing centers (JGI in the US, Genoscope in France, and IGA in Italy) have been met. DNA samples have been shipped only to Genoscope and IGA at this point. The University of Florida and Brazilian interests remain in negotiations with JGI over contract language, before JGI can proceed with their portion of the sequencing project. Meanwhile, work has proceeded at the UF-CREC to produce sample materials needed for the microarray experiments planned, using Affymetrix GeneChips, a new array platform developed by the co-PIs at UF using Agilent technology, and for the cDNA platform available through our co-PI in Spain. To this end, two sets of plants of sweet orange, rough lemon, and Volkamer lemon, representing the more susceptible and more tolerant types respectively, have been inoculated with budwood from HLB-infected Carrizo citrange (Carrizo is resistant to CTV, so viral interaction complications will be avoided) in an environmentally controlled greenhouse. Samples of RNA have been prepared from all of the plants at regular intervals, to be used in microarray experiments. Plants have been observed for symptoms, and qPCR has identified some that were successfully infected. The HarvEST Citrus EST database is in process of being updated, to provide an improved database for gene expressions studies. The EST sequences from our colleagues in Brazil and Japan have all been downloaded and reassembled, increasing the number of publicly available citrus ESTs to more than 465,000. The plans to exploit genome sequence information for a better understanding of the interactions of citrus plants with the pathogen causing HLB are ultimately most dependent on having the genome assembled and annotated; for this reason, our main focus will be on accomplishing that goal, while continuing to establish the experiments and collecting the samples that will be used for subsequent microarray analyses and deep transcriptome sequencing.
We received an APHIS permit to plant genetically modified citrus trees in the field. More than 900 transgenic citrus trees, containing 15 different constructs with potential to provide resistance to HLB and/or canker, have been propagated for field planting at 3 locations where HLB/canker pressure is severe. Planting will begin in a few weeks. Several new genetic constructs have been prepared for new transformation experiments, including lima b, a more ‘consumer-friendly’ sister to the lima a that has shown some promise already against HLB. With tests completed proving function two new phloem limited promoters, there are 4 currently available that could be combined with AMPs to target HLB in phloem tissue. Propagation and testing of previously produced transgenic plants continues, with several showing either delayed, reduced or no symptoms of HLB following inoculation. Transgenic plants have also been produced containing insecticidal genes and these are being prepared for tests against aphids and psyllids. Transgenic and citrus hybrid plants have been tested by canker inoculations, and those showing few or no symptoms are being retested. A series of experiments have been established to provide materials needed for research aimed at further characterization of gene expression differences resulting when susceptible citrus are challenged with HLB. RNA and DNA samples have been prepared at regular intervals to follow gene expression changes over time. These experiments may provide genetic targets to induce resistance to HLB, or at least to develop markers for early detection of disease. Studies into the metabolism of sugars and starch in HLB-infected plants have been initiated. Previous research microarray experiments were analyzed and have revealed more information on the defense machinery within kumquat cells. Several of the target genes have been fully cloned for further experiments including genetic transformation, following a more careful study of their gene expression patterns. New DNA samples from a genetic population have been received for genotyping to contribute to the International Citrus Genome Consortium sequencing project in collaboration with colleagues in Spain and France. Hybrid plants have been produced for rootstock improvement from the previous season, and new crosses made this spring 2009 have been harvested and planted. Previous work to develop rootstocks tolerant of or resistant to other maladies (such as CTV, blight, Phytophthora/Diaprepes, calcareous soils, etc.) continues, as we collected data from replicated trials and other plantings. New rootstock trials were planned, propagated, and planted, to test the suitability of experimental rootstocks for advanced production technologies; tree size control and broad-spectrum disease resistance and tolerance of adverse environmental conditions are critical to these efforts. Some trials include the recently released UF cultivar Sugar Belle (Tm), but most are with sweet orange. A new greenhouse screen for tolerance to Phytophthora/soil adaptation using tetraploid rootstock candidates has been established. Seeds have been harvested and planted from several dozen newly available rootstock candidates with tree size control and productivity potential, for future rootstock trials particularly aimed at new production technologies and orchard design. New pummelo-grapefruit hybrids, some of which were previously found to be significantly more tolerant of canker than grapefruit, are being propagated for canker challenges. Several of these hybrids also have been found to contain little or no furanocoumarin compounds, suggesting that they might not result in harmful grapefruit-drug interactions; further, some of them produce seedless fruit. New grapefruit-type candidates have been found this season and preliminary selections have been made based on fruit quality and attributes, and several hundred more such hybrids have been produced through crosses made in the spring and subsequent seed collection or embryo rescue, using newly available breeding parents. We are well on our way to meeting project objectives and goals.
Several new sweet oranges have been approved for release by UF-IFAS; these include several from Brazil believed to have canker tolerance, as well as new Valencia types with earlier and later maturity. The former will be generally available, but the latter will be patented and available through license agreements; together, they give industry new options to produce distinctive, high-quality juice. Canker tolerance of the Brazilian selections is currently being assessed under controlled greenhouse conditions, and preliminary results indicate that some of them are, in fact, significantly more canker-tolerant than ordinary sweet oranges. Additional data was collected from other advanced sweet orange selections in replicated field trials, and the best performing of these are being cleaned of pathogens and indexed, to provide certified budwood sources following approval for their release. These include a range of Valencia-types with improved color, yield, and juice quality attributes, as well as some very productive, seedless Midsweet clones; fruit and juice of these selections have been included in several commercial taste panels and assessments, and have been shown to be superior in their quality attributes. Hybrid plants have been produced for rootstock improvement from the previous season, and new crosses were made this spring 2009, that utilized germplasm with potential canker and HLB resistance or tolerance. Previous work to develop rootstocks tolerant of or resistant to other maladies (such as CTV, blight, Phytophthora/Diaprepes, calcareous soils, etc.) continues, as we collected data from replicated trials and other plantings. New rootstock trials were planned, with plants being propagated or already planted, to test the suitability of experimental rootstocks for advanced production technologies; tree size control and broad-spectrum disease resistance and tolerance of adverse environmental conditions are critical to these efforts. Some trials include the recently released UF cultivar Sugar Belle (Tm), but most are with sweet orange. New transgenes have been developed or acquired, and experiments have been established to introduce them into HLB and canker susceptible citrus. Previously developed transgenic plants have been inoculated with HLB, and those thus far showing no symptoms and negative qPCR tests (after more than 18 months) are being propagated for further testing. Several newly developed transgenic lines are being propagated for canker inoculations and evaluation, as well. New pummelo-grapefruit hybrids, some of which were previously found to be significantly more tolerant of canker than grapefruit, are being propagated for canker challenges. Several of these hybrids also have been found to contain little or no furanocoumarin compounds, suggesting that they might not result in harmful grapefruit-drug interactions; further, some of them produce seedless fruit. A series of experiments have been established to provide materials needed for research aimed at further characterization of gene expression differences resulting when susceptible citrus are challenged with HLB. RNA and DNA samples have been prepared at regular intervals to follow gene expression changes over time. These experiments may provide genetic targets to induce resistance to HLB, or at least to develop markers for early detection of disease. Previous research revealed several target genes in kumquat associated with its canker resistance (hypersensitivity). Microarray experiments were analyzed and have revealed more information on the defense machinery within kumquat cells. Several of the target genes have been fully cloned for further experiments including genetic transformation, following a more careful study of their gene expression patterns. Publicly available citrus genome resources are being mined for genes that may be used to hasten development of citrus plants resistant to HLB, canker, and other production-limiting diseases. Additional molecular markers have been developed and mapped in collaboration with colleagues in Spain and France, in support of the International Citrus Genome Consortium sequencing project. We are well on our way to meeting project objectives and goals.
We received an APHIS permit to plant genetically modified citrus trees in the field. More than 900 transgenic citrus trees, containing 15 different constructs with potential to provide resistance to HLB and/or canker, have been propagated for field planting at 3 locations where HLB/canker pressure is severe. Planting will begin in a few weeks. Several new genetic constructs have been prepared for new transformation experiments, including lima b, a more Òconsumer-friendlyÓ sister to the lima a that has shown some promise already against HLB. With tests completed proving function two new phloem limited promoters, there are 4 currently available that could be combined with AMPs to target HLB in phloem tissue. Propagation and testing of previously produced transgenic plants continues, with several showing either delayed, reduced or no symptoms of HLB following inoculation. Transgenic plants have also been produced containing insecticidal genes and these are being prepared for tests against aphids and psyllids. Transgenic and citrus hybrid plants have been tested by canker inoculations, and those showing few or no symptoms are being retested. A series of experiments have been established to provide materials needed for research aimed at further characterization of gene expression differences resulting when susceptible citrus are challenged with HLB. RNA and DNA samples have been prepared at regular intervals to follow gene expression changes over time. These experiments may provide genetic targets to induce resistance to HLB, or at least to develop markers for early detection of disease. Studies into the metabolism of sugars and starch in HLB-infected plants have been initiated. Previous research microarray experiments were analyzed and have revealed more information on the defense machinery within kumquat cells. Several of the target genes have been fully cloned for further experiments including genetic transformation, following a more careful study of their gene expression patterns. New DNA samples from a genetic population have been received for genotyping to contribute to the International Citrus Genome Consortium sequencing project in collaboration with colleagues in Spain and France. Hybrid plants have been produced for rootstock improvement from the previous season, and new crosses made this spring 2009 have been harvested and planted. Previous work to develop rootstocks tolerant of or resistant to other maladies (such as CTV, blight, Phytophthora/Diaprepes, calcareous soils, etc.) continues, as we collected data from replicated trials and other plantings. New rootstock trials were planned, propagated, and planted, to test the suitability of experimental rootstocks for advanced production technologies; tree size control and broad-spectrum disease resistance and tolerance of adverse environmental conditions are critical to these efforts. Some trials include the recently released UF cultivar Sugar Belle (Tm), but most are with sweet orange. A new greenhouse screen for tolerance to Phytophthora/soil adaptation using tetraploid rootstock candidates has been established. Seeds have been harvested and planted from several dozen newly available rootstock candidates with tree size control and productivity potential, for future rootstock trials particularly aimed at new production technologies and orchard design. New pummelo-grapefruit hybrids, some of which were previously found to be significantly more tolerant of canker than grapefruit, are being propagated for canker challenges. Several of these hybrids also have been found to contain little or no furanocoumarin compounds, suggesting that they might not result in harmful grapefruit-drug interactions; further, some of them produce seedless fruit. New grapefruit-type candidates have been found this season and preliminary selections have been made based on fruit quality and attributes, and several hundred more such hybrids have been produced through crosses made in the spring and subsequent seed collection or embryo rescue, using newly available breeding parents. We are well on our way to meeting project objectives and goals.
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