Highest priority is being placed on advanced stage work to develop hybrid rootstocks that can be made available for release to growers in the next two years, especially selections from the SuperSour project. Based on outstanding performance in field trials, three Supersour rootstocks will be released in 2018, tentatively identified as US SuperSour 1, 2, and 3. I am working with FDACS-DPI to get clean plant material of these rootstocks to nurseries now, to ensure that trees will be available to growers soon after release. During the quarter, performance data was collected from established field trials, including crop size, fruit quality, tree size, health, and infection status by qPCR. New information on rootstocks and field performance was presented to growers at the Florida Citrus Show in January and the Peace River Packers Field Day on March 7. A summary of performance from one of the trials is given below. Trees in the USHRL nursery are being prepared for planting three additional field trials with the most promising SuperSour rootstocks later in 2018. Additional information is available on the USDA rootstock breeding project, on request.
1) Trees have been in the ground for 4 years in a trial of 50 selections and cultivars on US-802 following no-choice ACP inoculation and several months in an ACP house. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a semi-annual basis. HLB is now widespread and the trees looking healthiest include a full sib of our best mandarin selection (unfortunately not in this trial), and several of our best grapefruit-like hybrids. The one true grapefruit is the least healthy selection in the trial. The best performers include hybrids containing Poncirus, and conventional hybrids which are predominately mandarin or pummelo. It may take 2-3 more years to clearly distinguish tolerant material. These trees are cropping this year, though Irma dropped a lot of fruit, and fruit will be used in a complementary project exploring synthesis of orange-like juice from HLB-tolerant types. The 5 selections with the highest percent of full canopy volume ratings had 46% to 57% increase in canopy volume over the last year while the 7 with the lowest percent of full canopy volume ranged from 0 to 23% increase. The r2 for percent of full canopy volume vs. last year s % growth was 0.37 across the entire planting. Several selections that had looked good last year suddenly dropped to a very low canopy increase, and it appears this may be a relatively early indicator of HLB tolerance. 2) In June 2015 a field planting was established of: seedling trees of 133 Fortune x Fairchild hybrids from an earlier mapping study, seedlings of 27 Ponkan-like accessions, budded trees of 10 advanced ARS selections that are predominately mandarin, and budded trees of Fortune, Fairchild and Valencia. Data collection is underway. A NIFA grant proposal was submitted and funded to mobilize tolerance for industry use, and this planting will contribute. It is a collaboration between ARS, UF and UC Riverside. 3) Replicated trials in multiple locations are established of our best sweet-orange-like cultivars and mandarin-types. Volatiles from sweet-orange-like hybrids are so similar to sweet orange that likely can be legally named sweet orange. 4) RNA-seq compared transcriptome responses in HLB moderately tolerant Sun Chu Sha mandarin and susceptible Duncan grapefruit, to Xcc-flg22 and CLas-flg22 (most active epitope from the pathogen flagella; project initiated with Gloria Moore at University of Florida). Differential expression of a number of genes occurred between tolerant and susceptible citrus infected with CLas, suggesting their involvement in HLB tolerance. In addition, several genes were similarly regulated by CLas-flg22 and CLas treatments. Genes identified were recently published and are valuable for studying HLB tolerance mechanisms and potential for screening for HLB-tolerant citrus using CLas-flg22 as a pathogen proxy. A population of mandarin hybrids from a 2015 cross was used to test for CLas-flg22 responsiveness. Leaf injections of CLas-flg22 and Xcc-flg22 were conduct on about 60 seedlings, and 8 randomly selected plants were subject to expression analysis of marker genes identified by transcriptomics previously. CLas-flg22 injection induced marker genes in several plants, although the number of markers induced was inconsistent. The analysis of the remaining 50 plus plants is underway. 5) A group of CLas effector (28) was predicted using bioinformatics tools. The study of transcriptional level of these effectors in infected citrus samples was initiated. Many of the effectors were successfully detected by RT-qPCR in citrus RNA, suggesting their roles in pathogen virulence and host response modulation. The initial detection was achieved from 2 weeks to 10 weeks after inoculation in multiple citrus genotypes. A comparison between root and leaf CLas expression was carried out. Results indicated different deployment of effectors between above- and underground CLas infections. Using a detached leaf assay for ACP-mediated inoculation, we also detected the expression the effector as early as 6h after ACP exposure, and observed continuous expression of effectors by CLas from 6h to 7 days. In addition, expression patterns of effectors were different between HLB susceptible and tolerant/resistant citrus hosts, which was validated by testing, although only a few genotypes were tested. Correlation between effector expressione and host tolerance/susceptibility is underway analyzing citrus types with various genetic backgrounds. The number of effectors detected has a positive linear relationship with bacterial titer, but less association with infection stage within the first week of CLas inoculation. Consistently detected effectors may have important roles in pathogen virulence. Citrus transformation for expression of CLas effectors will permit investigation of host modulation and discovery of binding targets in citrus. 6) Seedlings with a range of pedigree contributions from Microcitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, are being grown, and will be planted soon for field testing of HLB resistance. 7) Our putative chimeras have not proven to be successful. We identified a chimera (Satsuma and Poncirus) from the Citrus genebank, arranged its importation, and we finally got permission to accept this material and maintain it in a quarantine death house. Cuttings of the chimera and each separate component (Owari and Poncirus) have been rooted and will be challenged by hot ACP feeding in the next quarters.
1) Assessed use of isolated leaf inoculation and small plant destructive sampling: Isolated leaf inoculations with ACP do not readily distinguish between resistant and susceptible citrus selections, but prove useful in assessing CLas-killing transgenics. Within a week, such assays have shown marked reductions in CLas in leaves and in ACP. Small plant destructive inoculation assays now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. 2) Data collection continues on transgenics. Transgenic plants expressing a modified thionin are promising for HLB resistance and they have been extensively propagated for testing in the greenhouse and the field. 67 plants representing 13 independent events and wild types (4-5 replicates each) were transferred into larger pots and are getting ready for field planting and resistance evaluation. The remaining plants were used for bud-inoculation tests as transgenic root stock and/or scion. About 100 small rooted cuttings were grafted with CLas+ rough lemon for identification of the most resistant lines. Another 805 rooted cuttings were created at the end of 2017 from 18 events for future experimentations. Tissue specific constructs of the very promising Mthionin gene have been developed. The phloem-specific variant has been transformed into Carrizo and is currently being transformed into other citrus types including Hamlin and Ray Ruby. The root specific variant is being transformed only into Carrizo. 3) Transgenics expressing LuxI from Agrobacterium, and an array of ScFv transgenics (more in 4 below) have been propagated and are in replicated testing. New chimeral peptides (citrus only genes) have been used to produce many Carrizo plants and shoots of Hamlin, Valencia and Ray Ruby. A total of 35 lines of Carrizo with citrus thionin V2-LBP construct, and 20 lines with citrus thionin V1-LBP construct have been generated. A total of 18 independent Carrizo lines, each expressing citrus thionin-EDS and citrus thionin D2A21 chimeras respectively, were produced with confirmation of high level transgene expressions. Using the detached leaf ACP-inoculation assay, it was shown than transgenic Carrizo expressing citrus thionin V1-LBP chimera have significantly less CLas titer after 1 week of ACP feeding than the wild type controls. His-6 affinity tagged variants of citrus thionin-BPI/LBP expressing constructs have been created with C-terminal and N-terminal thionin orientations. These constructs have been transformed into benthamiana for efficient in plantae production and purification of protein for use in detached tissue assays with multiple lines for each construct confirmed as transgenic and currently undergoing analysis for expression levels. 4) Antibodies (ScFv) to the CLas invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. Two representative constructs, one targeting each gene, have been challenged by CLas + ACP. At six and nine months post inoculation, transgenic plants are showing consistent and significant decreases in bacterial titer (300x) as measured by qPCR and a much higher incidence of plants with no measurable bacterial DNA amplification. Additional plants representing 21 independent events from all 7 constructs have been replicated as rooted cuttings for ACP challenge of whole plants. A second round of ACP inoculations has been conducted on 150 plants replicated from twelve independent transformation events representing three different ScFv constructs. Additional lines will be inoculated once sufficient mature transgenic material becomes available. 5) Arabidopsis DMR6 (downy mildew resistance 6)-like genes were previously shown to be downregulated in more tolerant Jackson compared to susceptible Marsh grapefruit. DMR6 acts as a suppressor of plant immunity and it is upregulated during pathogen infection. In a gene expression survey of DMR6 orthologs in Hamlin , Clementine , Carrizo , rough lemon, sour orange and citron, expression levels were significantly higher in all CLas-infected trees compared with healthy trees in each citrus genotype. We developed 2 RNA silencing (hairpinRNA) constructs targeting citrus DMR6 and DLO1 respectively. Citrus DMR6 is silenced in hairpin transgenic plants and with an average silencing efficiency of 41.4%. DMR6 silenced Carrizo plants (28 independent so far) exhibit moderate to strong activation of plant defense response genes. Determination of silencing efficiency of DLO1 in transgenic plants (20 plants so far) are ongoing. Carrizo plants carrying these constructs with multiple events each were transferred into larger pots to stimulate growth in early 2018 and subsequent propagations 6) Budwood from our best performing Mthionin transgenics and citrus gene chimeras have been sent to DPI for cleanup and then broad field testing.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Transgenic Duncan grapefruit and sweet orange lines carrying either EFR alone or EFR plus an XA21-EFR chimera were tested for canker resistance in the greenhouse. The two most promising Duncan grapefruit lines carrying EFR were selected for further testing in the field in collaboration with Dr. Ed Stover at the USDA ARS. Some new Duncan grapefruit transformants carrying EFR, XA21, or both genes have been produced at the Core Citrus Transformation Facility at UF Lake Alfred, and any that survive will be analyzed for canker resistance. No transformants were obtained for CSPR, and this construct has been abandoned. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Work on these constructs has been discontinued due to negative effects of the constructs in citrus. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement Our gene editing target is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we chose to mutate the native citrus Bs5 alleles while simultaneously introducing the effective resistance allele as a transgene, rather than to attempt precise gene editing. Two editing constructs were created, one targeting the two conserved leucines, and one targeting two sites in the second exon to create a deletion in Bs5. The constructs were transformed into Carrizo citrange, and transformants are currently being analyzed at UC Berkeley. To date, we have identified one plant with mutations knocking out both alleles of the native Bs5 gene and several other candidate plants that may also have a loss of function of both alleles. More plants remain to be analyzed. Any plants confirmed to have biallelic loss of function mutations in Bs5 plus the introduced bs5 resistance allele will be propagated and shipped to Dr. Jeff Jones’ lab at UF Gainesville for canker testing.
The driving force for this project (Hall-15-016) is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus breeders at USDA-ARS-USHRL, Fort Pierce Florida continue producing germplasm that needs to be evaluated. The more rapidly germplasm can be evaluated, the sooner breeders can identify HLB-tolerant germplasm for the Florida citrus industry. The purpose of this project is to support a high-throughput facility to evaluate citrus germplasm for HLB resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. The original inoculation program called for individual plants to be caged with 20 infected psyllids for a two-week infestation, and then housed for six months in a greenhouse with an open infestation of infected psyllids. As indicated below, the open infestation step was abandoned. After the caged inoculation step, plants are moved into a psyllid-free greenhouse and evaluated for growth, HLB symptoms and CLas titer, and finally the plants are transplanted to the field where evaluations of resistance continue and additional inoculations by field psyllids occurs. CRDF funds for the inoculation program cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career technician is assigned ~50% to the program. USDA provides for the program two small air-conditioned greenhouses, two walk-in chambers, and a large conventional greenhouse. Currently 18 individual colonies of infected psyllids are maintained. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected citron plants. Update: As of March 15, 2018, a total of 11,888 plants have passed through inoculation process. A total of 326,295 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations are many plants inoculated over the past year to assess transmission rates, which has provided insight into the success of our inoculation methods and strategies for increasing success. We have abandoned the greenhouse open-infestation step because of continual problems mainly with invasive pests such as thrips, scales and parasitoids. Research indicated that the no-choice inoculation step should usually average around 74% effective and gets plants back to the breeders faster. The plants are subjected to further inoculations in the field. The no-choice inoculation procedure was evaluated monthly for 12 months, and success in getting seedlings infected was evaluated six months after each monthly infestation. The results indicated a 74% average success rate in getting seedlings infected when flush (immature leaves) was present, with success ranging from 40 to 100%. Success was significantly related to how many ACP on a seedling tested positive for CLas, thus greater success rates would be expected using more than 20 ACP per seedling. Based on the results of the research, an infestation shorter than two weeks would be as effective, which would be advantageous for guarding against excessive ACP damage to seedlings if more than 20 per seedling were used. It remains possible that modifications to the no-choice inoculation procedure would increase success rates and reduce variability, for example 25 to 30 ACP per seedling for a 1 week period, perhaps with larger seedlings in larger cages.
Objective 1. Identify key metabolites that are associated with rootstock traits. Accomplishments: Metabolite profiles of rootstock seedlings of ten rootstock cultivars in two separate experiments were assessed for primary metabolites in collaboration with West Coast Metabolomics Center (WCMC), UC-Davis. Results from the original study were published (Albrecht, Fiehn, Bowman. Plant Physiology and Biochemistry 107:33-44). The data obtained are large and complex and are being used for additional detailed analysis. An additional manuscript was submitted to Plant Physiology and Biochemistry in this quarter. An excerpt from this analysis is shown below. Objective 2. Investigate the effect of grafting on metabolite profiles. Accomplishments: Eleven rootstocks were analyzed as greenhouse and field-grown grafted trees in combination with Valencia and other scions. Samples were submitted to WCMC for analysis, and metabolic profiles of leaves and roots of the grafted trees are being compared. These studies will aid in identifying rootstock-scion interactions. The data sets are large and complex, but a publication is expected to be submitted in the next few months. Objective 3. Establish metabolite profiles of trees on different rootstocks in response to HLB. Accomplishments: An experiment consisting of 300 trees of Valencia grafted on ten rootstocks and grown in greenhouses was completed. Trees were inoculated with Las. Leaf and root tissue of Las-infected and uninfected plants were collected at 30 weeks after inoculation. Samples from these experiments were sent to WCMS for GC-TOF-MS analysis of primary metabolites and secondary metabolites in 2017. Partial results have been received, with the remaining results expected soon. Results from these analyses are being used for detailed analysis of metabolite profiles associated with HLB. A publication of the results is being prepared. Additional information is available on request.
Objective 1. (Greenhouse experiment): Another round of PCR samples were taken from every tree and sent to the Southern Gardens diagnostic laboratory to determine HLB status – results are just back and are now being analyzed. Leaf nutritional samples were sent to Waters Lab for analysis, results are back and are now being analyzed and interpreted. Most trees, even though infected, are growing exceptionally well, except for treatment #3, where trees are not showing much growth and are beginning to yellow. Objective 3: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). The nutrition treatments have been applied for 2 year now. The third year first quarter applications of all the treatments have been completed and third year tree health parameters have been collected. The fruits at both the locations have been harvested and evaluated. Currently, we are in process of data analysis and interpretation. The results from year 2 nutritional trial will be presented at OJ Break on May 15th, 2018 Ojective 5. (funded by Lee Groves, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. Positive results will be presented at ASHS meeting this summer, and we initiated preparation of a journal manuscript to present the results to the scientific community.
Objective 1. (Greenhouse experiment): PCR samples were taken from every tree and sent to the Southern Gardens diagnostic laboratory to determine HLB status. Nutritional profile as well as infection status of all grafted plants were analyzed in this quarter. qPCR values indicated that there was no significant differences in the infection rate amongst the different treatments at this time. The Harrell s nursery mix supplemented with Boron has lower overall infection rates as estimated by higher Ct values in all the rootstocks except UFR15, where the nursery mix supplemented with Manganese performed better. It is still too early to see much treatment and rootstock differences. Objective 3: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). The total 2nd year fertilizer applications were completed. Year 2 yield and fruit quality data collection will begin this quarter. Ojective 5. (funded by Lee Groves, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. Potential breakthrough – qPCR was run on each tree – significant bacterial titer differences were found among the treatments. The best treatment was a 4x overdose of manganese, producing an average ct value of 32.75; whereas the control tree average ct value was 23.19 (indicating severe infections). Seven of twelve trees in the high manganese treatment exhibited ct values of 32 or above, and were considered not to have an active infection; whereas no control trees reached 32. Other treatments containing high manganese also did much better than the controls, except for the treatment with both 4x manganese and 4x boron. This level of boron seemed to be antagonistic to the uptake of manganese, as demonstrated by the leaf mn content data. Yield and fruit quality data from these plots are being collected this quarter – results should be quite interesting.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. The following work has been conducted in this quarter: (1) Analyzed another batch of rootstock transgenic lines (54 Carrizo lines, 7 Swingle lines, and 3 C-MAC lines). Two more Swingle lines and three C-MAC lines accumulated high levels of transgene products. More than half of the Carrizo lines accumulated high levels of transgene products. These rootstock transgenic lines will be analyzed based on the protein levels. (2) Conducted another cage experiment for replicates of the transgenic lines with low numbers of psyllid progenies. (3) Made more replicates of the transgenic lines tolerant to HLB.
The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require the regulatory approval. Based on two year’s graft-inoculation assays in greenhouse and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). All these propagates (ca. 250 trees) are growing well in greenhouse, and expected to go to field next month. These propagates will be tested by qPCR before going to fields. The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected seedlings showed no significant difference from their maternal trees. New propagations of the selections from the the Scott grove seedlings, including 245 Red Ruby and 51 white Marsh plants on three different rootstock and 38 Red Ruby cuttings were planted in our research farm on Nov.15, 2017. Before going to field, samples were taken from these plants, and processed for qPCR detection. We will monitor the disease incidence and Las titers if there is an infection. Meanwhile we will go second round of selection and propagations on Scott seedlings, and then plant them in Scott grove for field evaluation.
Activities are reported by project objectives below. 1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions. Planted ~2000 seeds from 20 2017 rootstock crosses in calcareous/Phytophthora infested soil (“Gauntlet”), and completed stick grafting of approximately 100 new gauntlet rootstock candidates selected from 2015 crosses. Propagated 5 “super-root” mutants from tissue cultured UFR1, UFR-3, and UFR-6, and begun testes for HLB tolerance. 2. Breeding of HLB tolerant/resistant processing sweet oranges and orange-like hybrids. Recovered ~ 400 plants from interploid crosses targeting HLB tolerant sweet orange/mandarin type hybrids. Planted 203 transgenic plants (73 independent lines, mostly oranges but some Duncan and W. Murcott) containing Valencia-derived BG or LTP at out APHIS approved transgenic site at the SWFREC. 3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. We continue to monitor our germplasm collection and breeding families for performance against HLB. We have initiated a genomic selection effort based on phenotypic assessments and using a high density SNP chip for genotyping. Interesting genomic regions have been highlighted that contain putative disease resistance and defense mechanism genes. Worked with Dr. Johnny Ferrarezi to plant the entire population of cybrid grapefruit clones (kumquat cytoplasm) at the IRREC. These include cybrids of Flame, Ruby somaclone N11-11 and White Marsh showing significantly improved canker tolerance in greenhouse assays. The original N11-11 somaclone, derived from Ruby red, has shown a strong recovery response from HLB; thus the cybrids made from this clone are potentially improved for both canker and HLB tolerance. In addition to the original population, propagations of selected clones were also planted, and many were on the rootstocks propagated via the previous CRDF grant with Barrett Gruber. Approximately 340 trees were planted. 4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. Field planted promising new OLL sweet oranges/new rootstock candidates in 2 locations. Planted 150 E.V. (Early Valencia) trees on promising new rootstocks in a high Diaprpres pressure area, in an effort to identify a rootstock that doesn t drop fruit after HLB infection. Harvested seed from all available UFR seed trees; seed kindly extracted by John Gose @ Lykes, now being distributed to participating licensed nurseries. Other related activities: Scored trees in a high-peel oil lemon selection trial in the IR region, after 7 days of Irma flooding. This will serve as a baseline for future evaluations of flood damage over time. Speaking of Irma, we spent much of September going through all CREC plots and field trials, standing up and staking trees blown over by the storm.
During the period of July, August, and September, 2017, Dr. McNellis submitted a full proposal to the USDA Citrus Specialty Crop program to expand and continue the existing project funded by CRDF, and this full grant proposal was submitted to the USDA on August 18, 2017. This project would be a collaboration between Dr. McNellis and Drs. Ozgur Batuman, Liliana Cano, and Rhuanito Ferrarezi. They have the expertise to do CLas infections and PCR-based quantification of CLas in plant tissues. In addition, Dr. McNellis has been working with Dr. Catherine Hatcher of the CRDF on a pre-proposal for the field testing and greenhouse testing for HLB resistance of the trees produced through this project, in anticipation that the current project will be concluded on December 31, 2017. Dr. McNellis’ lab also continued quantitative analysis of anti-HLB antibody protein expression in the transgenic lines (the FT-scFv fusion protein) by western blotting. It will be the objective and intention of the renewal project to test HLB resistance or tolerance status of ungrafted and grafted trees with the transgenic genotype.
Objective 1. (Greenhouse experiment): We reached the 6 month period since the stick grafting and first CRF application, so all CRF nutrition treatments were re-applied using all donated products (all trees), PCR samples were taken from every tree and sent to the Southern Gardens diagnostic laboratory to determine HLB status – hopefully we can report results in the next quarterly report. Composite leaf samples from each nutrition/rootstock combination were sent to Waters Lab for nutritional analysis. Overall, most trees are showing reasonably good health, with only minor symptoms appearing on some trees. The greenhouse and the experiment were not impacted by Irma. Objective 3: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). The third application of fertilizer treatments for the 2nd year was completed at both sites. There were no tree losses due to Irma, but there was some fruit blown off the trees, apparently fairly consistent across the replications within the trial. Ojective 5. (funded by Lee Groves, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. Trees still look relatively good, no significant impact by Irma. Photosynthesis rates were measured on all trees, and composite leaf samples were taken and sent to Water’s Lab for nutritional analysis.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. The following work has been conducted in this quarter: (1) Several propagations were made for the Swingle and Carrizo lines accumulating high levels of the transgenic protein. We noticed the Carrizo line that accumulates the highest level of the transgenic protein does not grow well, indicating that two much of the transgenic protein affects citrus growth and development. This provides a threshold for the transgenic protein. (2) The HLB-tolerant transgenic lines were further propagated. (3) Four TAIL-PCR experiments have been conducted with both right and left T-DNA border sequences to clone the gene suppressing psyllid reproduction on citrus plants. Unfortunately, not dominant PCR bands were obtained. The presence of the T-DNA insertion was confirmed using the kanamycin gene primers. We are determining the truncation sites of the T-DNA insertion and will clone the insertion site. (4) Cage experiment for replicates of the transgenic lines with low numbers of psyllid progenies is ongoing. More replicates will be generated.
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. Seven transgenics survived and passed a PCR screen, and these are currently being tested for canker resistance. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and therefore the putative transformants are being screened by RT-PCR. Eleven PCR-positive shoots have been grafted so far. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Work on these constructs has been discontinued due to negative effects of the constructs in citrus. 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 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we are targeting mutating the native citrus Bs5 alleles while simultaneously replacing the gene with the effective resistance allele. Two editing constructs have been created, one targeting the two conserved leucines, and one targeting two sites in the second exon to create a deletion in Bs5. Both constructs have been verified to function by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties. These constructs have been prioritized for transformation into Carrizo citrange, and transformations are underway at UC Davis, with several rooted plants obtained so far. Molecular characterization of the putative transformants will be carried out at UC Berkeley. Transformants with mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
