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.
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.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter,most of the transgenic lines produced have been confirmed for gene integration by conventional PCR and analyzed for gene expression using qPCR. 40% of the lines tested have been determined to be high expressers while the rest were medium to low in expression. Cuttings from the larger lines have been made and are being rooted in the mist bed for future challange with Diaprepes. A number of other potential root specific promoters have been identified from the phytozome database. qPCR gene expression analyses on non-transgenic leaves, flowers, fruit, phloem, seeds and roots have identified some that can potentially be used in the future for root specific gene expression. Results from some of these studies will be presented in the World Congress on In vitro Biology in the summer.
Our project is focused on the following objectives: 1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions. 2. Breeding of HLB tolerant/resistant processing sweet orange-like hybrids. 3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. 4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. The project began on 1 November 2015. In the late autumn, and through the winter and spring, we collected data from several ongoing field trials of rootstock cultivars throughout the state. Trees were assessed for HLB incidence and severity through all plantings, and yields and fruit quality was determined in selected replicated trials. Hybrid families planted in the field were evaluated to identify selections producing fruit that resembled sweet orange in appearance, and exhibiting few or no HLB symptoms. Fruit from the best of these were tested for juice quality and a few were also analyzed for volatile components and compared with standard sweet orange. Fruit samples were provided to a major juice company for processing and assessment of the juice for flavor. The entire collection of raw material produced by the breeding program and growing in groves at several different locations throughout the growing areas of Florida has been assessed for HLB symptoms, to identify individuals displaying few or no symptoms of HLB, regardless of parentage; ~4.3% of all trees were characterized as tolerant based on tree health and overall appearance. Specific highlights in the reporting period are below. The GFC/Indiantown project has been terminated and removed. A narrative with figures will be prepared on posted on the CREC website under Varieties and Rootstocks. Yield data and HLB ratings were obtained from the Wheeler multi-scion/rootstock cooperative trial. Difference among rootstocks and scion-rootstock interactions were apparent even among these 3 year old trees. Yield data were obtained from 3 year old trees on various rootstocks planted 8 x 15 in a CPI cooperative project. The CREC plant breeding team s collective field evaluation records have been reviewed, consolidated and organized for more efficient record keeping. A 3.5 year old planting of 220 OLL seedlings was assessed for HLB; two HLB-free seedling trees were identified and propagated for further study. 2016 crosses included several crosses of HLB-tolerant mandarins with sweet orange-like juice profiles with OLL oranges in efforts to develop seedless HLB-tolerant sweet orange-like hybrids for processing and fresh market. 2016 rootstock crosses (diploid and tetraploid) featured HLB tolerant parents, and included crosses of LB8-9 with Phytophthora resistant parents. Two more sets of gauntlet rootstocks were rotated into a hot psyllid house, approximately 100 trees were prepared for field planting (this quarter). Gauntlet trees at Picos Farm were assessed; 10 selections 3-4 years old are promising and being further monitored. Yield and fruit quality data were collected again from 6 and 7.5 year old trees in the St. Helena Project. A public Field Day was held to update interested growers on progress with the trial and selected UFRs.
Objective 1. (Greenhouse experiment): seedlings of the required rootstocks: x639, Swingle, WGFT+50-7, UFR-3 and UFR-15 were moved up to round citripots and moved into the HLB screening greenhouse. Liners are approaching stick-grafting size, and it should be possible to begin grafting in 2-3 months. Objective III: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). All the pretreatment data have been collected on trees, leaf and soil nutrient analysis were done to obtain a complete pretreatment tree assessment. First fertilizer applications were made at both the field locations and data was collected. All the data and photographs have been recorded for mid-year evaluation. Second round of applications are being made currently. All the micro-nutrient and foliar application products have been collected. Macro-nutrient applications will be made in August. Currently, all the data from pre-treatment and mid-year are being analyzed to confirm any differences among the treatments. Ojective 5. (funded by Orie Lee, 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. The second treatment was applied May 30th, 2016. There was no further activity during this period. 1. Control no extra nutrition 2. Harrells St. Helena mix (2lbs per tree) 3. Harrells St. Helena mix (2lbs.)+ 2x TigerSul manganese (90 gm) 4. Harrells St. Helena mix (2lbs.) + 2x Florikan polycoated sodium borate (32 gm) 5. Harrells St. Helena mix (2lbs.) + 2x TigerSul manganese (90 gm) + 2x FL sodium borate (32 gm) 6. 4x TigerSul manganese (180 gm) 7. 4x Florikan polycoated sodium borate (64 gm) 8. 4xTigerSul manganese (180 gm) + 4x Florikan polycoated sodium borate (64 gm) Baseline yield data was taken from each plot in January, 2016; pfd is severe and will impact the 2017 harvest. Donated micronutrient treatments were also applied at the Hughes Post Office block – where there were yield increases this past season, enhanced by specific treatments, especially those containing both boron and manganese. Trees look exceptional at present, and we expect to see a yield increase for the 2nd year in a row since we began the study.
Objective 1. (Greenhouse experiment): seed was extracted, seed coats removed using a chemical-removal procedure and planted of the needed rootstocks: x639, Swingle, WGFT+50-7 and UFR-3. Seeds germinated well and healthy seedlings are now being transferred to larger pots as necessary to begin the treatments/grafting. Objective III: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). All the pretreatment data have been collected on trees, leaf and soil nutrient analysis are done to obtain a complete pretreatment tree assessment. First fertilizer applications have been made at both the locations and data is being collected. Second round of applications will be made in June 2016. Ojective 5. (funded by Orie Lee, 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. The second treatment was recently applied (May 30th, 2016). 1. Control no extra nutrition 2. Harrells St. Helena mix (2lbs per tree) 3. Harrells St. Helena mix (2lbs.)+ 2x TigerSul manganese (90 gm) 4. Harrells St. Helena mix (2lbs.) + 2x Florikan polycoated sodium borate (32 gm) 5. Harrells St. Helena mix (2lbs.) + 2x TigerSul manganese (90 gm) + 2x FL sodium borate (32 gm) 6. 4x TigerSul manganese (180 gm) 7. 4x Florikan polycoated sodium borate (64 gm) 8. 4xTigerSul manganese (180 gm) + 4x Florikan polycoated sodium borate (64 gm) Baseline yield data was taken from each plot in January, 2016; pfd is severe and will impact the 2017 harvest. Donated micronutrient treatments were also applied at the Hughes Post Office block – where there were yield increases this past season, enhanced by specific treatments, especially those containing both boron and manganese.
During this reporting period (January, February, and March, 2016), control plants that have been through the transformation process, but not containing the transgene, were generated and sent to Penn State, and they are growing well at the Penn State location. These plants are the best comparison to the FLT-antiNodT plants in terms of plant behavior and disease resistance. We call these the “transformation control” trees. The transgenic plants being produced 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. Dr. McNellis has applied for and been granted an APHIS BRS permit to send propagated FLT-antiNodT plants to Florida for replicated testing for HLB resistance in Dr. Gottwald’s lab. However, before sending the plants, we must obtain the needed Florida state permit (FDACS 08084), and this is in progress. Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) transformed ‘Hamlin’ sweet orange and the ‘Carrizo’ rootstock with the FLT-antiNodT expression construct, and we received these plants at Penn State in early April, 2016. During the next reporting period, we will test these plants for expression of the FLT-antiNodT anti-HLB protein. Dr. McNellis will also produce rooted cuttings of all these lines for later testing for HLB resistance in Florida.
During this reporting period (January, February, and March, 2016), control plants that have been through the transformation process, but not containing the transgene, were generated and sent to Penn State, and they are growing well at the Penn State location. These plants are the best comparison to the FLT-antiNodT plants in terms of plant behavior and disease resistance. We call these the “transformation control” trees. The transgenic plants being produced 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. Dr. McNellis has applied for and been granted an APHIS BRS permit to send propagated FLT-antiNodT plants to Florida for replicated testing for HLB resistance in Dr. Gottwald’s lab. However, before sending the plants, we must obtain the needed Florida state permit (FDACS 08084), and this is in progress. Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) transformed ‘Hamlin’ sweet orange and the ‘Carrizo’ rootstock with the FLT-antiNodT expression construct, and we received these plants at Penn State in early April, 2016. During the next reporting period, we will test these plants for expression of the FLT-antiNodT anti-HLB protein. Dr. McNellis will also produce rooted cuttings of all these lines for later testing for HLB resistance in Florida.
Our significant progresses during this reporting time period are: 1) Using mature shoot segments of Valencia and Washington navel, we have demonstrated that the Kn1 gene can improve transformation efficiencies by approximately 2-fold compared to the control vector, which is much lower than those observed in juvenile citrus transformation. 2) We used an epigenetic modulator in our transformation experiments and observed about a 2- to 3-fold increase in overall transformation efficiency in mature tissues of Valencia and Washington navel oranges. We further demonstrated that the epigenetic modulator produced a 10-fold increase in shoot regeneration efficiency of mature citrus with no transformation when compared to the controls. 3) With expression of a 35S::GUS gene containing an intron as an indicator, we examined Agrobacterium infection and T-DNA integration activities in mature citrus using tobacco leaf discs and juvenile citrus tissues as references. Consistent with the fact that tobacco leaf discs can be efficiently transformed with Agrobacterium, we observed very high levels of transient and stable expression of GUS in the cut edges of tobacco discs. When juvenile citrus tissues were used for Agrobacterium infection, we observed reasonable levels of both transient and stable GUS gene expression. Using mature explants of Valencia, however, we observed extremely low levels of transient and stable expression of the GUS gene. As we have shown that although both the Kn1 and Ipt gene dramatically enhanced transformation efficiencies of juvenile citrus via increased shoot regeneration, they were far less effective at improving transformation on mature citrus tissue. Also, in mature Valencia and Washington navel oranges, we found that using an epigenetic modulator led to about 10-fold increase in shoot regeneration, but only a 2- to 3-fold increase in transformation efficiency (i.e., transgenic shoot production). We hypothesized that after improved shoot regeneration, Agrobacterium-mediated T-DNA integration remained the major challenges to improving mature citrus transformation. We are now working to enhance efficiencies of Agrobacterium-mediated stable T-DNA integration. Combining the various molecular tools we have, we would like to develop a ‘vector’ that is highly efficient and genotype-independent for mature citrus transformation. One manuscript reporting the drastically improvement of six citrus cultivars including a lemon cultivar has been published: Hu et al (2016): Kn1 gene overexpression drastically improves genetic transformation efficiencies of citrus cultivars. Plant cell, Tissue and Organ Culture. 125: 81-91. Two manuscripts are under preparation, reporting some of the results summarized above.
New field performance information is being collected on about 400 new SuperSour-type rootstocks in field trials. Performance attributes being assessed include tree growth, tree health, fruit yield, fruit quality, tolerance of high pH soil, 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, US-897, 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, trees in field trials were measured for tree size and scored for health, HLB symptoms, and samples were collected from some groups for PCR detection of Las infection. During this quarter, yield and fruit quality data were collected from 3 rootstock field trials with late maturing scion varieties. The most outstanding rootstock in a long-term Valencia trial on a flatwoods site in Collier County was US-802 rootstock, which yielded significantly more fruit than every other rootstock in the trial, including the second highest yielding rootstock, US-942. Every tree in this trial is infected with Las, but trees on US-802 still yield much more fruit than trees on any other rootstock. 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 comprehensive presentation on standard and new rootstocks was made at the Florida Citrus Growers Institute. A new paper was prepared, providing a comprehensive comparison of field performance for the new USDA rootstocks with other standard rootstocks, and was submitted for publication. 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 2016. 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 has been initiated 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. Greenhouse experiments continued to assess rootstock tolerance to HLB, CTV, and high pH. Research was initiated to more fully study the HLB-tolerance of trees formed by grafting HLB-susceptible scions on HLB-tolerant rootstocks. A better understanding of this behavior will help to more quickly identify new rootstocks with higher and more reliable levels of HLB tolerance in the field. Cooperative planning continued with UF researchers at several locations, to submit grant proposals to USDA NIFA to help fund expanded rootstock research and development efforts. Cooperative grant-funded work continued with UF researchers to establish acid fruit rootstock trials in South Florida. 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. Trees from the commercial nursery are scheduled to be planted into six cooperative field trials in 2016, and six more field trials in 2017.
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