Our progresses we have made for this project: 1) We have used the Kn1 gene to drastically improves shoot regeneration efficiently from transgenic cells of citrus. We have successfully used a maize knotted1 (KN1) gene to enhance genetic transformation efficiencies of juvenile tissues of six citrus varieties, Pineapple, Hamlin, Sucarri, Valencia, Carrizo and Eureka lemon via Agrobacterium-mediated infection. Our results demonstrate that expression of the KN1 gene improved transformation efficiencies from 3- to 15-fold compared to a control vector, 3- to 11-fold relative to the highest transformation efficiencies previously reported for the same citrus varieties. Stable incorporations of T-DNA into our transgenic plants have been confirmed with both histochemical staining of GUS activity and molecular analyses. The majority of KN1 over-expressing citrus plants grow and develop normally at young seedling stages, similar to those of the wild type plants. With all six genotypes of citrus tested including Eureka lemon, a cultivar difficult to transform, we have demonstrated that the kn1 gene can be an effective molecular tool for enhancing the genetic transformation of juvenile citrus tissues. Using mature shoot segments of Valencia and other cultivars as explants, we also found that the KN1 gene can improve transformation efficiencies compared to the control vector BUT an increase in efficiency is lower than what has been observed in juvenile citrus tissues. 2) We have demonstrated that manipulation of auxin transport can significantly enhances shoot regeneration of citrus. We have observed that the apical ends of epicotyl segments regenerated more shoots than the basal ends, and we therefore hypothesized that auxin transport and/or endogenous auxin concentration may play a key role in shoot regeneration of citrus explants. We tested some auxin transport modulators and identify one modulator that improved shoot regeneration. However, when the modulator was included in the transformation experiment, the transformation efficiency did not improve (i.e., number of transgenic shoots produced per explant). We hypothesize that the auxin modulator may inhibit Agrobacterium infection or T-DNA integration. 3) We have shown that an epigenetic modulator may be used to enhance shoot regeneration and transformation of mature citrus tissue. When we used an epigenetic modulator in transformation experiments with mature tissues, we observed increases in transformation efficiency of several citrus cultivars including Valencia and Washington Navel oranges. We have further demonstrated that the epigenetic modulator can lead to increases in shoot regeneration efficiency of mature citrus tissues when compared to the controls. 4) We have demonstrated that low Agrobacterium infection and T-DNA integration efficiencies are limiting factors for mature citrus transformation. As described above, we have developed some tools for enhancing shoot regeneration from mature citrus tissues. However, when these tools were used in mature citrus tissue transformation, the increase in transformation efficiency was lower than in juvenile tissues. We have further shown that the Agrobacterium infection and DNA integration are a major factor limiting transformation efficiency of mature citrus tissues, which provides a basis for our future experimentation to improve transformation efficiency of mature citrus tissues. We have published one manuscript reporting that Kn1 can drastically improve genetic transformation efficiencies of six citrus cultivars including a lemon cultivar: Hu et al (2016): Kn1 gene overexpression drastically improves genetic transformation efficiencies of citrus cultivars. Plant cell, Tissue and Organ Culture. 125: 81-91. The second manuscript reporting the effects of poplar transport of endogenous auxin and an auxin transport modulator on citrus regeneration and transformation will be submitted in 2-3 weeks. The third one is currently under preparation.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter,all the transgenic lines produced have been confirmed for gene integration by conventional PCR and analyzed for gene expression using qPCR. 35% of the lines tested have been determined to be high expressers while the rest were medium to low in expression. Cuttings from all the better performing lines have been made and are being rooted in the mist bed for future challenge with Diaprepes. A number of other potential root specific promoters are being evaluated. Several have been cloned and transformation vectors are being produced. Results from our studies have been presented in the World Congress on In vitro Biology.
The project has three objectives: (1) Confirm HLB resistance/tolerance in transgenic citrus lines. (2) Determine the chimerism of the HLB-resistant/tolerant transgenic lines. (3) Confirm HLB resistance in citrus putative mutants (nontransgenic lines). For objective 1, we have been working on five transgenic lines (HAM 13-3, HAM 13-29, and DUN 57-25, DUN 205-25c, and DUN 207-8) that exhibit tolerance to HLB. HAM 13-3: we had some difficulty with propagation of this particular line. We have repeated the propagation and are waiting for the progenies to become old enough for HLB test. HAM 13-29: we have generated 21 progenies. After HLB inoculation, 11 of these plants have no HLB symptoms and others exhibited mild symptoms. DUN 57-25: four progenies have been made and tested. Three plants have no HLB symptoms and one plant was crappy and discarded. We are continuing propagating this line. DUN 205-25c: this transgenic plant became dark green and the leaves are thick and extremely curly. We are propagating this plant. DUN 207-8: eight progenies have been generated. These plants have been inoculated with HLB. For objective 2, we confirmed the chimerism of the transgenic plants using real-time quantitative PCR. Results showed that HAM 13-3, HAM 13-29, DUN 57-25, DUN 205-25c, and DUN 207-8 are homogeneous (not chimeric). For objective 3, three Ray Ruby grapefruit putative mutants (#3, #6, and #93) showed tolerance to HLB. Seven, 10, and 10 progenies were generated for mutant line 3, line 16, and line 93, respectively. The progenies were tested for HLB resistance/tolerance. Unfortunately, none of these mutated plants exhibited resistance/tolerance to HLB.
Citrus Huanglongbing (HLB) poses the greatest threat to the survival of the Florida and US citrus industry. Research to incorporate HLB resistance/tolerance into citrus has been recommended by the National Research Council as one of the top priority topics for addressing the HLB threat. A number of Poncirus and Citrus cultivars have been found to be tolerant to HLB. Microarray-based profiling of the transcriptomes of two cultivars with HLB tolerance (Poncirus hybrid US-897and rough lemon) and two cultivars without HLB tolerance have identified ~1,200 genes that are differentially expressed in HLB-tolerant cultivars. These genes constitute a valuable pool of potential candidates from which true HLB tolerance genes can be identified. The original project proposal aimed to identify approximately 1,200 potential candidate genes ~differentially expressed in HLB-tolerant Poncirus and rough lemon, conduct massively parallel sequencing of the ~candidate genes, perform genetic association and linkage analysis to find most likely candidate gene(s) for HLB tolerance, and clone these candidates for function validation. We have identified up-regulated genes in rough lemon in response to CLas inoculation. By combining our new gene expression data with 14 other published data sets, using two gene expression meta-analysis tools, we identified 3,122 probe sets differentially expressed between HLB-tolerant and susceptible varieties or in response to CLas inoculation. These probe sets correspond to 2,147 genes in the Clementine genome. To capture the sequence polymorphisms in these candidate genes for association and linkage analysis and to obtain full-length sequences for gene cloning, we sequenced the genomes of more than 40 Poncirus and Citrus accessions from 25-50x coverage and obtained partial genome sequences of 30 Poncirus and Citrus accessions from our collaborators, resulting in >700 Gb of sequence data. We have assembled the sequence reads into contigs and mapped these to the candidate genes for 48 Poncirus and Citrus accessions. To perform association and linkage analysis, in collaboration with USDA colleagues we collected HLB severity and CLas Ct data from more than 70 Poncirus and Citrus hybrids and accessions planted at the Picos Road Farm (USDA, Ft. Pierce). These accessions were replicated in 8 randomized complete blocks. For approximately 60% of the accessions, data were collected for two growing seasons, in addition to recently published information from the same planting. For the remaining 40%, plants were poorly established initially, and we will continue to collect phenotypic data for one additional growing season on HLB severity and CLas Ct value. Gene cloning efforts have focused on genes expressed at significantly higher levels in HLB-tolerant accessions or after CLas inoculation and with known functions in plant defense or immunity. However, some of the selected genes seemed to encode proteins toxic to E. coli or Agrobacterium cells, resulting in failure in gene cloning. To overcome this, gene expression vectors were modified. Two gene constructs have been developed and are being introduced into citrus to produce transgenic plants. To speed up the cloning of additional genes at full length, we have begun to complete a high-quality reference genome sequence of Poncirus, which currently does not exist. Based on these results, we have published two refereed papers and several abstracts. HLB-tolerant citrus so engineered are expected to be deregulated at much quicker speeds and less expense and be better accepted by consumers. Combining tolerance genes from Poncirus and rough lemon in the same citrus cultivars may result in augmented tolerance, even resistance. The primers and new DNA markers derived from HLB tolerance candidate genes also can be used to screen existing rootstock and scion breeding populations to facilitate development of non-engineered HLB-tolerant/resistant citrus scion or rootstock cultivars by conventional breeding.
Two new replicated field trials with Valencia scion were planted with SuperSour-type rootstocks at two locations, one in the east coast area and one in the west coast area. The west coast trial is on high pH soil and should give good information about the tolerance of the SuperSour rootstocks to high pH, in addition to HLB tolerance. Field performance information is being collected on more than 400 new rootstocks in 17 different replicated field trials. Performance attributes being assessed include tree growth, tree health, fruit yield, fruit quality, and tolerance or resistance to HLB and other diseases. New rootstocks are only appropriate for large-scale grower use when outstanding performance has been documented by statistically replicated trials over multiple years. It is anticipated that at least one of the best new SuperSour rootstocks will be released for commercial use within 3 years. In the meantime, outstanding performance has been documented for US-802 and US-942 rootstocks over multiple years in trials affected by HLB, and these rootstocks are available in large numbers through commercial nurseries. During this quarter, 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. A new paper providing a comprehensive comparison of field performance for the new USDA rootstocks with other standard rootstocks, was submitted and accepted for publication in a refereed journal. This publication will be a very valuable reference for use by growers and nurseries making decisions about the best rootstocks to use for new plantings. A new replicated field trial was established to collect detailed information about Valencia tree performance on the most HLB-tolerant rootstocks under optimum management conditions. Focused study in this trial will help to more clearly measure the ways in which tree performance is affected by HLB and estimate the economic viability of commercial production on the most tolerant rootstocks. 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 2017. New trials in propagation continue to focus on sweet orange scion, but include some plantings to assess performance with new scions that have better tolerance to HLB. Nursery experiments were conducted with promising new rootstocks to determine nursery-related traits important for commercial use. Cooperative work continued with commercial nurseries involved with micropropagation, to facilitate more rapid deployment of the best new rootstocks. A cooperative project is underway with Dr. Ute Albrecht (UF, Immolakee) to compare trees on rootstocks propagated by seed, cuttings, and micropropagation, so that growers can have confidence that rootstocks propagated by the different methods will have equivalent performance. Cooperative planning continued with UF researchers, to submit grant proposals to USDA NIFA to help fund expanded rootstock research and development efforts. Cooperative grant-funded work continued with UF researchers and a commercial nursery to propagate trees for use in multiple rootstock field trials sponsored by the HLB MAC program.
Good progress was made in the validation of the effectiveness of metabolite profiles for selection of HLB tolerant rootstocks. Focused studies were continued to identify key metabolic compounds in 12 specific rootstocks of known HLB-tolerance, as well as scions grafted on them, and collect the detailed information to be used in the validation process. In this quarter, the second set of leaf and root samples from greenhouse and field tests with different rootstocks were processed and shipped to the West Coast Metabolomic Center for metabolomic analysis by gas chromatography-time of flight (GC-TOF) mass spectrometry (MS). The GC-TOF-MS analysis for the first cycle of samples will be completed in the next quarter and results available for detailed analysis by our research team in Ft. Pierce and Immokalee. The experimental design of the first and second set of samples collected in the first year of the project will allow comparison of metabolite profiles of the same genotypes in different seasons, in grafted and ungrafted trees, and in different ages and sizes of plants, providing critical information about how metabolite profiles change under different conditions. In preparation for the second year of the project, clean seedlings and budded trees were prepared for greenhouse studies, and budded trees were prepared for field planting. Selected trees within the experiments were inoculated with Las, and tissue was collected to analyze for Las infection by qPCR. The material in this study will provide valuable insight into metabolite changes during the early stages of HLB disease development. Following approval by CRDF, the project was revised to include a new researcher at University of Florida in Immokalee, Dr. Ute Albrecht, who has experience and special expertise in this research field. Grant funds will be provided through a Non-Assistance Cooperative Agreement to support this work in Immokalee. A post doc has been hired in Immokalee to work on the project. The project changes actually reduce the overall cost, but will significantly increase productivity and the opportunity for success. In this quarter, a study by our team of metabolic differences among rootstocks was published, entitled “Metabolic variations in different citrus rootstock cultivars associated with different responses to Huanglongbing”. That study used GC-TOF-MS analysis to compare metabolite profiles of six rootstock cultivars infected and not infected with Las. The study identified numerous chemical compounds that appeared strongly associated with Las tolerance, and offers good opportunities for further study and incorporating the tolerance behavior into other rootstocks. The study demonstrated large metabolic differences between an HLB-sensitive rootstock and the HLB-tolerant rootstocks US-897 and US-942, both with and without Las infection, and will serve as a foundation for continuing work under this grant.
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. Five putative transgenics have survived and passed a PCR screen, and these will soon be grafted onto rootstocks. 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. EFR, XA21, and XA21 + EFR constructs have been re-created with the inclusion of a GFP marker for confirmation of transformants; selection is underway. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to coexpress Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs have been provided to the Lake Alfred transformation facility, and selection of transformants in Duncan grapefruit is underway. 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. Four CRISPR constructs are being created in the Staskawicz lab: C1) A construct targeting two sites that will produce a 100 bp deletion in Bs5 in both Carrizo and Duncan (the bs5 transgene will be added); C2) A construct targeting a site overlapping the two conserved leucines; C3) C2 with the addition of a bs5 repair template for Carrizo that will not be cut; and C4) C2 with a similar repair template for Duncan grapefruit. The constructs have been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties, and verified to function. To aid in the selection of positive transgenics, a GFP reporter has been added into each CRISPR construct. The constructs with GFP will be re-verified in N. benthamiana and sent for transformation.
The citrus relatives planting (85 seed source genotypes from the gene bank) has been assessed for growth and apparent HLB tolerance. Within the genus Citrus, measures of tolerance msuch as canopy density, health, and tree size, correlate positively with % citron in pedigree, with r2 of 0.3-0.6. A manuscript describing apparent tolerance to HLB in citrus and citrus-related germplasm is ready for submission. Chemical, morphological and transcriptome characteristics are being assessed to determine what factors are associated with observed tolerance (three distinct projects), so they can be used in early screening and possibly directed transgenesis. A paper describing HLB resistance in this population has just been published in Plant Disease. 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. HLB is now widespread and trees of more vigorous scion types are generally the healthiest at this point in time. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is underway. Generation of new chimeras has been difficult. An existing periclinal chimera (Satsuma and Poncirus) has been imported and is now being grown at USHRL 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 detectable in most plants at 3 weeks By nine weeks after exposure, susceptible genotypes can be clearly distinguished from reported resistant material by higher CLas levels in roots. Averaged across genotypes and tissues, total CLas per tree was 5 billion in week 3 after ACP exposure and ddoubled every 3 weeks through week 12. 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). 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. 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. A mapping population of Fortune x Fairchild has been planted (collaborating Roose and Gmitter) along with related material, in an effort to identify genes associated with tolerance in the mandarin phenotypic group. Seedlings with a range of pedigree contributions from Microcitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, and are being grown for field testing of HLB resistance.
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 for HLB challenge. Propagated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Twelve months after graft inoculation, Las titer was examined and compared in old leaves (most with HLB symptom), young expanded leaves (with or without HLB symptom) and fibrous roots of transgenic and control plants. Our results showed again that transgenic citrus expressing Mthionin has lower Las titer compared to control and transgenic plant expressing chimera. These data suggest transgenic plants expressing thionin are promising for HLB resistance (The manuscript for this research will be published in Frontiers in Plant Biology). Antibody against thionin will be produced for investigating the correlation of thionin expression and HLB resistance. 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 obtained. DNA was isolated from 46 plants and 40 of them are PCR positive. Furthermore, the third generation chimeral peptides were designed based on citrus thionins, the vector construction were finished and citrus transformation are underway. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Trees expressing NbFLS2 showed significant canker resistance to spray inoculation. Replicated Carrizo and Hamlin were challenged with ACP feeding. Leaves were taken six months after ACP feeding inoculation. DNA was isolated and Las titer was tested. Our preliminary results showed that transgenic trees expressing NbFLS2 can reduced Las titer. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing AHLs 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 protein p235 with a nuclear-localization sequence has been identified and studied. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR , correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product targets to citrus chloroplasts. Transcription data were obtained by RNA-Seq. Data analysis and comparison are underway. 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.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. The data has been published by Plant Biotechnology Journal Transgenic Valencia transformed by Cas9/sgRNA has been established in our lab. Three transformants have been verified by PCR. The PthA4-binding site in CsLOB1 promoter was modified as expected, only one transgenic line seems to be bi-allelic mutant. The EBE modifed transgenic line is being evaluated for resistance against Xac. One Cas9/sgRNA binary vector, which is designed to target CsLOB1 open reading frame, designated as GFP-Cas9/sgRNA:cslob1, was used to transform Duncan grapefruit epicotyls by Agrobacterium-mediated method. Several transgenic citrus lines were created, verified by PCR analysis and GFP detection. Cas9/sgRNA:cslob1-directed modification was verified on the targeted site, based on the direct sequencing of PCR products and the chromatograms of individual colony. Upon Xcc infection, some transgenic lines showed delayed canker symptom development. We are currently analyzing the genome modified plants using transgenic approaches including off-targets. To generate non-transgenic DNA free canker resistant citrus, Cas9 containing nucleus localization signal was overexpressed and purified. The purified Cas9 showed activity in cutting target sequence and are being used to generate canker resistant plants. We have conducted multiple tries of genome editing using protoplast. Currently, we are optimizing the condition to conduct genome editing using protoplast. We also tested different sgRNAs to generate deletion in the coding region of CsLOB1.
In the second quarter of 2016 Core Citrus Transformation Facility (CCTF) continued to operate without interruption although prospective moving date for the lab was June 17th. Eventually the date was pushed back to July 21st so facility is still in its old location. Due to the very high number of orders placed in the last quarter and increased work load, I have hired one more employee who was trained in the lab during the month of April. This new employee is working full time. However, another employee was taken back from 1.0 FTE to 0.4 FTE at her own request. The number of orders placed at the CCTF remained high. We have received 12 orders within the last 3 months. Seven of those orders were paid in advance although no material associated with transformation was received. Customer just wanted to secure the place in our work schedule for time when they are ready to send us plasmid constructs. The plants produced within the last quarter are almost all from the experiments associated with orders placed within last 9-12 months. We produced 67 plants: nine Carrizo citranges, six Swingle citrumelos, and 52 Duncan grapefruits. Transgenic rootstock plants carrying NPR1 produced in our facility are still in our greenhouse. They are at the stage when they could easily be propagated by cuttings. I am awaiting further instructions on what to do with these plants.
This project (Hall-15-016) is an extension of a project that came to a close last summer (Hall-502). The driving force for this project is the need to evaluate citrus transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. Briefly, individual plants to be inoculated are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer, and finally the plants are transplanted to the field where evaluations of resistance continue. 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: Two technicians funded by the grant have been fully trained in establishing and maintaining colonies of infected psyllids, conducting qPCR assays on plant and psyllid samples, and running the inoculations. As of June 7, 2016, a total of 8,694 plants have passed through inoculation process. A total of 170,895 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. As reported in the last progress report and reiterated here, research recently showed that seedling citrus with flush is significantly more prone to contracting the HLB pathogen than seedling citrus without flush: Hall, D. G., U. Albrecht, and K. D. Bowman. 2016. Transmission rates of Ca. Liberibacter asiaticus by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. J. Econ. Entomol. 109: 558-563. doi: 10.1093/jee/tow009. Therefore, the program has been changed to ensure that plants to be inoculated have flush. Current research indicates that the no-choice inoculation step used in our program is successful an average of 79% of the time when approximately 70% of ACP placed on a plant test positive for CLas (Ct <36) and have CLas titers of around CT=26 to 29 (success contingent on flush being present on a plant).
This project (Hall-15-016) is an extension of a project that came to a close last summer (Hall-502). The driving force for this project is the need to evaluate citrus transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. Briefly, individual plants to be inoculated are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer, and finally the plants are transplanted to the field where evaluations of resistance continue. 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: Two technicians funded by the grant have been fully trained in establishing and maintaining colonies of infected psyllids, conducting qPCR assays on plant and psyllid samples, and running the inoculations. As of June 7, 2016, a total of 8,694 plants have passed through inoculation process. A total of 170,895 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. As reported in the last progress report and reiterated here, research recently showed that seedling citrus with flush is significantly more prone to contracting the HLB pathogen than seedling citrus without flush: Hall, D. G., U. Albrecht, and K. D. Bowman. 2016. Transmission rates of Ca. Liberibacter asiaticus by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. J. Econ. Entomol. 109: 558-563. doi: 10.1093/jee/tow009. Therefore, the program has been changed to ensure that plants to be inoculated have flush. Current research indicates that the no-choice inoculation step used in our program is successful an average of 79% of the time when approximately 70% of ACP placed on a plant test positive for CLas (Ct <36) and have CLas titers of around CT=26 to 29 (success contingent on flush being present on a plant).
The Mature Citrus Facility has made significant progress producing transgenics for clients as a service although we are attempting to make even greater progress. Since July 1, 2015, ~100 transgenics were produced with Agrobacterium, which exceeds previous production. The increase in productivity is primarily due to superior vectors with reporter genes, stronger promoters driving expression of the nptII selectable marker, and an increase in our micrografting efficiencies to 75 -77%. Our clients include Drs. Grosser, Dutt, Louzada, McNellis, Wang, and Mou. After optimizations for biolistic transformation of mature citrus have concluded, these transgenics will augment those generated with Agrobacterium. Our project objectives of increasing micrografting efficiencies, propagating transgenic events into replicates, applying for external funding, and service work have been met. Service work will continue for the same clients in the next funding cycle. A manuscript describing the biolistic transformation of immature citrus has been published, and another manuscript on the selection of transgenics without reporter genes in temporary immersion bioreactors is being submitted. An additional manuscript is in preparation describing the development of a quantitative in situ 4-MUG assay for transgenic, mature citrus shoots. The Mature Citrus Facility protocols have changed in an effort to speed the growth of mature scions. There is a tremendous growth advantage if rootstocks are not removed. After budding mature buds, rootstocks are left attached for the two flushes of stem growth. Mature buds will break and stems can be used in transformations within 6-8 weeks rather than 12-16 weeks specified in the earlier protocol. We continue to optimize for the PMI selectable marker using biolistics and Agrobacterium transformations. The number of nontransformed, escaped shoots appears to be significantly lower than with nptII as a selectable marker. Various treatments (cold treatments and hormone applications) were tried to in an effort to increase regeneration rates and transformation efficiencies in recalcitrant mature citrus scions, but none were satisfactory. However, a citrus DNA sequence drastically increases the number of transgenics in recalcitrant scions. An expression vector is being prepared to test in co-transformations. New breeder lines (3 sweet orange and 1 grapefruit) were introduced through shoot-tip grafting and are being budded for transformations. Protocols will initially follow those used for Hamlin and Valencia, but might still have to be optimized for these new cultivars. Some clients have asked for each transgenic event to be budded onto immature rootstock into replicates, and then flowering seems to be delayed. Every time mature citrus is budded onto immature rootstock, it is reinvigorated and this may potentially delay flowering. An experiment is being conducted to determine how many months flowering is delayed by grafting flowering tissue onto immature rootstock. This result will influence our recommendations to clients. Our lab will be moving to the packinghouse in July, 2016 in order to fix the AC in our current lab. This move will cause disturbances to plant production, but we will do everything within our power to minimize disturbances to the mature citrus transformation pipeline.
The project has three objectives: (1) Confirm HLB resistance/tolerance in transgenic citrus lines. (2) Determine the chimerism of the HLB-resistant/tolerant transgenic lines. (3) Confirm HLB resistance in citrus putative mutants (nontransgenic lines). For objective 1, we continued propagating the transgenic lines that overexpress Arabidopsis defense genes and inoculated the previously generated progenies. The new progeny plants are growing in the greenhouse. The progenies obtained in the last quarter have been inoculated with Las-infected psyllids for two months and moved back to the greenhouse for symptom development. HLB symptoms on the plants have been carefully monitored and recorded. For objective 2, we performed the second round of real-time quantitative PCR (qPCR) to determine the chimerism of the HLB-resitant/tolerant transgenic lines. The results indicated that several lines of the HLB-resitant/tolerant transgenic lines are not chimeric. If these lines are confirmed to be HLB-resitant/tolerant in objective 1, they will be able to be propagated by grafting for industry use. For objective 3, we continued propagating the gamma ray-mutagenized mutant lines that are likely resistant/tolerant to HLB and inoculated previously generated progenies. The new progeny plants are growing in the greenhouse. As for the transgenic progenies, those obtained earlier were inoculated with Las-infected psyllids and are currently in the greenhouse for symptom development.
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