During the last quarter of 2015 Core Citrus Transformation Facility (CCTF) continued to operate as planned and produced transgenic Citrus plants. The number of orders received for the last three months jumped to nine. Two of those orders were for transgenic Duncan grapefruit and the rest are the set of seven orders for production of genetically modified Pineapple orange. CCTF still has not received two groups of orders announced by prospective clients about 5-6 months ago. Also, one set of orders that included the use of three different vectors was withdrawn and the client is working on re-cloning of all vectors. CCTF produced 51 plants within last quarter. These plant belong to multitude of orders, altogether nine of them, some of which are old and some newer. Six of the produced plants were Valencia oranges, 11 were Duncan grapefruit, and the rest were Carrizo citrange. As I have previously reported to Dr. H. Browning, the analysis of transgenic plants carrying NPR1 gene was successfully completed by Yosvanis Acanda from Mature tissue transformation facility. Out of 67 tested transgenic rootstock plants produced by the CCTF, 23 were shown to be high expressers of NPR1. These plants are being kept in the CCTF s greenhouse until they reach the size at which they can be propagated so that they can serve as rootstocks for both transgenic and WT scion plants.
The mature citrus transformation facility continues to produce transgenic events for its clients. Productivity has significantly improved using vectors with reporter genes. Transgenics produced via Agrobacterium can now be supplemented with transgenics produced using biolistics in immature and mature citrus. Scientists have been encouraged to submit vectors with all plant sequences and no pest sequences, which might lessen regulatory hurdles. Genes are now being stacked in an effort to prevent the pathogen from easily overcoming tolerance or resistance. A manuscript is being prepared describing biolistics in immature citrus. Currently, all transgenic events are being transferred to scientists directly without secondary grafting or propagation, unless otherwise requested. To improve efficiencies and lessen potential micrografting incompatibilities, sweet orange is being micrografted onto decapitated sweet orange rootstock. Micrografting losses in mature rootstock have significantly decreased when young shoots are micrografted onto decapitated rootstock grown in high sucrose solution. We are in the process of introducing new breeder lines in which to produce transgenics. Three sweet orange varieties and one grapefruit are being introduced via shoot-tip grafting. Once plants are established, they will be used in budding mature citrus onto rootstock to obtain budstick for transformations. Our website is being updated to reflect current prices and technologies employed.
The mature citrus transformation facility continues to produce transgenic events for its clients. Productivity has significantly improved using vectors with reporter genes. Transgenics produced via Agrobacterium can now be supplemented with transgenics produced using biolistics in immature and mature citrus. Scientists have been encouraged to submit vectors with all plant sequences and no pest sequences, which might lessen regulatory hurdles. Genes are now being stacked in an effort to prevent the pathogen from easily overcoming tolerance or resistance. A manuscript is being prepared describing biolistics in immature citrus. Currently, all transgenic events are being transferred to scientists directly without secondary grafting or propagation, unless otherwise requested. To improve efficiencies and lessen potential micrografting incompatibilities, sweet orange is being micrografted onto decapitated sweet orange rootstock. Micrografting losses in mature rootstock have significantly decreased when young shoots are micrografted onto decapitated rootstock grown in high sucrose solution. We are in the process of introducing new breeder lines in which to produce transgenics. Three sweet orange varieties and one grapefruit are being introduced via shoot-tip grafting. Once plants are established, they will be used in budding mature citrus onto rootstock to obtain budstick for transformations. Our website is being updated to reflect current prices and technologies employed.
Good progress was made in continuing the development of new hybrid rootstocks that support early and high fruit yield and fruit quality in the scion, have good soil adaptability, and are resistant to important diseases, especially HLB. This work was begun previously under CRDF project 508. As requested by CRDF, this new project 15-002 will place highest priority on hybrid rootstocks already created in the USDA program and being considered for release to growers over the next six years. This will include about 400 Supersour-type rootstocks that have been previously created and selected to include in field trials and other specialized testing. During this quarter, a new support scientist was hired under the grant to replace a support scientist, previously on staff, that was leaving to accept a permanent job at a University. Other personnel working on the project remained in place to continue tree care, experiment establishment, and data collection. During this quarter, tree care continued in established rootstock field trials, and tree size measurements were taken from selected trials. Trees in field trials were scored for health, HLB symptoms, and samples were collected from some groups for PCR detection of Las infection. Analysis was completed on data from several established trials to assess relative rootstock performance, rootstock effects on yield, fruit quality, tree size, and HLB symptom development. Nursery experiments were conducted with promising new rootstocks to determine nursery-related traits important for commercial use. Greenhouse experiments continued to assess rootstock tolerance to CTV and high pH. Analysis was completed on data from one cooperative field trial with 17 rootstocks that became 100% infected with Las and was determined to be a good comparison of rootstock performance with severe HLB challenge. The two best rootstocks in the trial were US-942 and US-1516. Based on this and other data, US-1516 rootstock was submitted by Bowman and McCollum for unrestricted commercial release by USDA. US-1516 is available as clean budwood from DPI, and some seed is available from Bowman. Outstanding performance by US-942 in that cooperative trial reinforced previous observations that US-942 provides better tree health and productivity than most other rootstocks when trees become infected with Las. A manuscript was prepared for publication to provide thorough documentation of the results from the cooperative trial. A manuscript was prepared for publication to provide documentation of US-1516 rootstock. During this quarter, cooperative work continued with UF researchers to propagate trees in a commercial nursery to use in multiple rootstock field trials sponsored by the HLB MAC program. Trees from the commercial nursery are scheduled to be planted into six cooperative field trials in 2016. Trees in the USDA nursery on a large number of advanced rootstock selections, especially Supersour-type, were continued in propagation for field trials to be planted in 2015 and 2016.
The project will focus on work to identify metabolite profiles associated with tolerance to HLB and other stresses in advanced rootstock selections, and validate the effectiveness of these metabolite profiles for selection by comparison of existing rootstock selections within the USDA program. As requested by CRDF, the project will place highest priority on work with new hybrid rootstocks already created, selected, and included in field trials. As requested by CRDF, other parts of the original proposal that involved integration with the breeding program, recurring selection, and discovery of biomarkers and antimicrobial compounds were eliminated, with a consequent simpler focus and smaller budget. In this quarter, data from a preliminary metabolomic study was organized and prepared for publication. This study was conducted under a previous CRDF funded grant, and demonstrated striking metabolic differences between HLB sensitive and HLB tolerant genotypes, both with and without Las infection. This data will serve as a foundation for continuing work under this grant. The work under this grant will focus on collecting an extended set of metabolomic data that will be adequate to allow validation of the approach by the end of project year 3. Environment may have strong effects on metabolomic data, so field testing, greenhouse testing, and information on seasonal variation will be critical for success in validating the approach. To clarify environmental effects, greenhouse studies will be needed, and a greenhouse suitable for the work is being constructed. In this quarter, plans were drawn up, and contract issued. The new greenhouse is expected to be completed in February 2016. In this quarter, plans for were made for specific studies to identify key metabolic compounds and collect the first stage of information to be used in the validation process. For the first year of the project, three greenhouse studies with potted trees, and five field studies (with established trees of different ages) will be conducted, to collect information about expression in leaf and root tissue, as well as seasonal effects on expression. The work will focus on 12 rootstocks that are of commercial importance and where previous studies have identified relative differences in tolerance to Las infection. Established field plantings are available with these 12 rootstocks suitable for the testing needed. In this quarter, trees in the selected field trials were scored for HLB symptoms and sampled for PCR testing, so that disease and infection status can be included in the choice of trees for metabolic testing. In preparation for the greenhouse studies, trees were propagated in another greenhouse, so that they can be moved into the new house for immediate experimentation, as soon as the new house is completed.
The main accomplishments during this quarter: We continued testing the effect of the K gene on transformation efficiency of a lemon cultivar. In general, lemon cutilvars are difficult to be genetically transformed. We have observed the K gene can drastically improve the transformation efficiency of the lemon cultivar used, similar to the effects of the K gene on the other orange cultivars tested. We continued to repeat the effects of K and I genes on transformation efficiency of mature citrus explants of mature Pineapple orange. The effects of the K and I genes have been confirmed. We have also tested effects of a non-conventional regulator of gene expression on regeneration efficiency of Washington navel orange and Valencia orange. We observed several fold increases in shoot regeneration efficiencies of both cultivars. Our goal is to use this regulator and also its combination with the K gene to improve transformation efficiencies of of both juvenile and mature citrus tissues One manuscript reporting the drastically improvement of several citrus cultivars has been submitted in the end of August. We have started to write the second manuscript from the project.
During this reporting period (July, August, and September, 2015), the transgenic plants to be developed for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. These plants are continuing to be propagated at both Ft. Pierce and Penn State. Our collaboration with Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) to transform varieties important to the Florida citrus industry, including the ‘Valencia’ and ‘Hamlin’ sweet orange varieties and the ‘Citrumello’ and ‘Carrizo’ rootstocks with the FLT-antiNodT expression construct, has had initial success. Hamlin and Carrizo transformants are now growing at Lake Alfred. Dr. Zale will maintain the original transformants, and will send propagated cuttings to Penn State for molecular analysis over the next 3-6 months. We will also send some of the propagated sweet orange and rootstock plants to Ft. Pierce for HLB resistance testing in collaboration with Dr. Tim Gottwald and possibly Ed Stover. During this reporting period, we also initiated development of an FLB-antiNodT expression cassette in the transformation construct pBI121, which has a history of successful approval for transgenic plant development. We anticipate that this construct could be completed during the next reporting period, and we would forward it to Dr. Zale immediately upon completion for further citrus transformations. In August, Dr. McNellis presented a poster at the annual meeting of the American Phytopathological Society in Pasadena, CA, describing the results so far, including successful expression of the FT-scFv protein in grapefruit with minimal or no negative effects on plant phenotype.
During this reporting period (July, August, and September, 2015), the transgenic plants to be developed for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. These plants are continuing to be propagated at both Ft. Pierce and Penn State. Our collaboration with Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) to transform varieties important to the Florida citrus industry, including the ‘Valencia’ and ‘Hamlin’ sweet orange varieties and the ‘Citrumello’ and ‘Carrizo’ rootstocks with the FLT-antiNodT expression construct, has had initial success. Hamlin and Carrizo transformants are now growing at Lake Alfred. Dr. Zale will maintain the original transformants, and will send propagated cuttings to Penn State for molecular analysis over the next 3-6 months. We will also send some of the propagated sweet orange and rootstock plants to Ft. Pierce for HLB resistance testing in collaboration with Dr. Tim Gottwald and possibly Ed Stover. During this reporting period, we also initiated development of an FLB-antiNodT expression cassette in the transformation construct pBI121, which has a history of successful approval for transgenic plant development. We anticipate that this construct could be completed during the next reporting period, and we would forward it to Dr. Zale immediately upon completion for further citrus transformations. In August, Dr. McNellis presented a poster at the annual meeting of the American Phytopathological Society in Pasadena, CA, describing the results so far, including successful expression of the FT-scFv protein in grapefruit with minimal or no negative effects on plant phenotype.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. All of these constructs had the peptide gene inserted between the coat protein genes, which is positioned sixth from the 3′ terminus. However, we have found that much more foreign protein can be made from genes positioned nearer the 3′ terminus. Based on that we built constructs with the peptide gene next to the 3′ terminus. These constructs produced much greater amounts of peptide and provided more tolerance to Las. Unfortunately, they are less stable. So now we are rebuilding constructs with the peptide gene inserted at an intermediate site hoping for a better compromise of amounts of production and stability. We have produced a large amount of inoculum for a large field test via Southern Gardens Citrus. We are screening a large number of transgenic plants in collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We are propagating a progeny set of plants of the promising candidates for a final greenhouse test.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the HLB gauntlet : Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. Cuttings from the following ‘gauntlet’ rootstocks were treated with rooting hormone and place on the mistbed for rooting: 1. A+HBPxCH+50-7-12-14 2. 46×31-00-S10x46x31-00-S11-S5 (salt tolerant sour orange-type) 3. Orange 10 x Green 7-11-1 4. A+VolkxOrange19-11-5 5. A+HBJL2BxOrange14-09-7 6. A+HBJL2BxOrange19-09-31 7. A+HBJL1-09-14 8. A+FDxOrange19-11-11 Seedlings were grown from four promising sour orange-like rootstock candidates with promise for adaptation to IR soils, with a goal of providing a quick source of material for cuttings as follows: 1. 46×20-04-S22 2. 46×20-04-42 3. 46×20-04-48 4. 46×20-04-S13
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the HLB gauntlet : Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The original PI of this project Dr. Gruber has resigned, so Co-PI Dr. Jude Grosser is assuming responsibility for completing the project. Ms. Amy Dubois is the OPS assistant taking care of the trees at the IRREC, she will continue in this roll. All of the recovered cuttings and seedlings were evaluated and the inventories of liners that could make a good field tree are provided below. These will be grown to grafting size, grafted with selected scions (including a dark red grapefruit somaclone N11-15 showing possible tolerance/resistance to HLB) and planted in IR field trials. Viable cutting inventory: Rootstock # of liners recovered 1. A+HBPxCH+50-7-12-14 44 2. 46×31-00-S10x46x31-00-S11-S5 78 3. Orange 10 x Green 7-11-1 52 4. A+VolkxOrange19-11-5 90 5. A+HBJL2BxOrange14-09-7 71 6. A+HBJL2BxOrange19-09-31 14 7. A+HBJL1-09-14 25 8. A+FDxOrange19-11-11 50 Viable seedling inventory: 1. 46×20-04-S22 86 2. 46×20-04-42 94 3. 46×20-04-48 78 4. 46×20-04-S13 86
Citrus trees transformed with a chimera AMP and a thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated by grafting for HLB challenge. Replicated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Las titer was checked from new flush rough lemon leaves at six month after grafting. Las titer from 18.6-36.5 was detected in 90% of transgenics expressing the chimera. Some transgenic lines expressing thonin had the lower Las tilter(most in 33.3-36.4 ranges). Transgenic root sample were further tested and most were detected with las titer from 30 to 35. All root samples will be checked at 9 month for Las titer. Two new chimeral peptides (second generation) were developed and used to produce many Carrizo plants and Hamlin shoots. Transgenic carrizo plants carrying second generation AMPs were transferred to soil cones. DNA was isolated from 46 plants and 40 of them are PCR positive. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. Trees showed significant canker resistance to spray inoculation. Replicated Carrizo and Hamlin were challenged with ACP feeding. Las titer will be tested periodically. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Replicated transgenic Carrizo plants were challenged by ACP feeding. Las tilter will be tested soon. Transgenic Hamlin were propagated by grafting for HLB challenge. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. A Las expressed gene with a nuclear-localization sequence has been identified and studied, including creating transgenic citrus that express this p235 gene. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR amplification, correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product binds to citrus chloroplasts. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Putative transgenics are currently being verified by PCR in the Jones lab, and three PCR positive plants have been identified so far. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we will initiate more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Constructs are being created in the Staskawicz lab to express Bs2 under the 35S promoter and under a resistance gene promoter from tomato. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. Three CRISPR constructs are being created in the Staskawicz lab: 1) A construct targeting two sites that will produce a deletion in Bs5 in both Carrizo and Duncan (the bs5 transgene will be added); 2) A construct targeting a site overlapping the two conserved leucines, containing a bs5 repair template for Carrizo that will not be cut; and 3) a construct targeting the same site, with a repair template for Duncan grapefruit.
Chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) are being tested and are among the most promising transgenics we have created, along with thionin transgenics. Trees transformed with a chimera AMP showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated by grafting for HLB challenge. Transgenic Hamlin lines expressing thionin were grafted onto Carrizo for HLB challenge. Replicated transgenic Transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon. Promising resistance to HLB was observed based on plant growth and phenotype. Las titer is being checked from root and new flush rough lemon leaves. Two new chimeral peptides from citrus genes only were developed and used to produce many Carrizo plants and Hamlin shoots which will be tested soon as part of the next generation of this project. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. Trees showed significant canker resistance to spray inoculation. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. Transgenic plants of PP-2 hairpins (for suppression of PP-2 through RNAi to test possible reduction in vascular blockage even when CLas is present) and of PP-2 directly are grafted in the greenhouse. 40 putative transgenic plants transformed with citGRP1 were tested by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 had increased resistance to canker by detached leaf assay but do not appear as potent as some other AMPs. Transgenic Carrizo and Hamlin with peach dormancy genes show no evidence of enhanced or accelerated dormancy A Las expressed gene with a nuclear-localization sequence has been identified and studied, including creating transgenic citrus that express this p235 gene. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR amplification, correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product binds to citrus chloroplasts. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
Evaluation of existing cultivar/rootstock combinations for HLB resistance/tolerance has revealed potentially valuable tolerance and indicates that early HLB symptoms and earlier CLas titer are unrelated to growth and cropping. In August 2010, the plants were established at Pico s farm in Ft. Pierce FL. Data on the growth rate, disease severity, and Candidatus Liberibacter asiaticus (CLas) titer levels have been collected since April 2012. During the 5-year period, there were significant differences in disease severity, stem diameter, and at times CLas levels among the varieties. Despite the high incidence of mottle in SugarBelle /SourOrange, it had the greatest overall increase in diameter. ‘SugarBelle’ and ‘Tango’ (which were not on the same stock as ‘Hamlin’ and so results should be viewed as comparing cultivar/rootstock combinations) were the healthiest in overall appearance in 10/15 and had the most fruit (88 per tree). All cultivars except sweet oranges and grapefruit are progressing in production, but production was compromised in all varieties by the severe HLB pressure at this site, and commercial value of the observed tolerance remains uncertain. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and have been transferred to the field at Ft. Pierce FL. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a quarterly basis. As of December 2014, the first HLB symptoms were apparent. At 2 years since exposure, only a small proportion of trees of any genotype (including sweet orange), have marked HLB symptoms. Progress continues on the antibiotic treatment of HLB infected bud-wood to compare growth at different levels of CLas infection. HLB-infected budwood was treated with various concentrations of antibiotics and grafted on sour orange rootstock using 3 fairly HLB-resistant ( Temple , GnarlyGlo , and Nova ) 3 tolerant ( Jackson , FF-5-51-2, and Ftp 6-17-48), and 3 susceptible ( Flame , Valencia , and Murcott ) genotypes. Standard growth measurements and disease severity are evaluated and leaves sampled for qPCR analysis on a quarterly basis. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is underway. One hundred and fifty etiolated seedlings of the trifoliate Rubidoux and the sweet orange Hamlin have been approach grafted together. Generation of new chimeras has been difficult. Several adventitious buds have emerged from the treated graft region, with several appearing to be chimeral. o increase the success rate, additional plants will be grafted over the next twelve months. An existing periclinal chimera (Satsuma and Poncirus) has been imported,has been with DPI two years, but has not yet been released to us for testing. A method for the rapid identification of potential sources of HLB resistance is being developed. This project involves the screening of citrus seedlings at the 3 to 5 leaf stage, or very small micrografted trees, that are exposed to HLB infect ACP feeding. CLas titer levels, using real time PCR, are easily detetcable in most plants at 3 weeks Seedlings of Hamlin and Dancy show marked CLas proliferation and systemic movement from 3-6 weeks after exposure to ACP.. Trees of seemingly HLB resistant/tolerant sweet orange-like hybrids and mandarin -types were propagated on x639. Replicated trials with standards have been established, in cooperation with G. McCollum. Six locations each of all sweet orange-like together and 4 with all mandarins were established in replicated block plantings with 6-8 trees of each cultivar at each site (in Ridge, IR and Gulf coast). Seedlings with a range of pedigree contributions from Microcitrus and Eremocitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, and are germinating for field testing of HLB resistance.
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