Most commonly grown citrus cultivars are sensitive to Huanglongbing (HLB). However, some citrus species and related genera are substantially more tolerant, such as Citrus jambhiri (rough lemon), Poncirus trifoliata and Microcitrus australis. Genome analysis will improve our understanding of the HLB tolerance mechanisms. Nuclear DNA from Citrus jambhiri was used to generate more than 235 million paired-end reads (2 X 100 nt) of the rough lemon genome. A reference-guided method was used to assemble the rough lemon genome. Based on analysis of the SNPs identified, rough lemon was found to have originated from the interspecific hybridization of mandarin, citron and pummelo, and its chloroplast is probably derived from mandarin. RNA-sequencing data was used for gene annotation, and some differentially expressed (DE) genes were identified. Most of DE genes were up-regulated in HLB affected trees, compared with non-affected trees. These DE genes were mainly involved in response to stress, carbohydrate metabolic process, response to abiotic stimulus, cell wall organization or biogenesis, ion transport and signaling. Based on our meta-analysis and co-expression network analysis of previously published gene expression data, we have shortlisted 2,000 HLB-responsive candidate genes in citrus (Du et al., 2015; Rawat et al., 2015). To identify sequence polymorphisms and validate the expression patterns of candidate genes, we have isolated genomic DNA and RNA from 20 citrus accessions. The DNA and RNA preps are being evaluated for quality and are to be sent to a commercial DNA sequencing company for large-scale sequencing on the HiSeq 2500. The sequencing data of these samples will be analyzed with the focus on 2000 candidate genes. The genomic indel variations and SNPs will be identified within these sequenced accessions for the selected candidate genes.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, several transgenic lines expressing each of our test genes have been regenerated and many of them have rooted. The rooted material has been successfully acclimatized to the greenhouse for growth and further testing. We are in the process of confirming the gene expression levels in these transgenic lines to isolated lines that have excellent gene expression. These selected lines will be propagated for subsequent evaluation with Diaprepes neonates.
The main accomplishments during this quarter: We repeated and confirmed the effects of K and I genes on genetic transformation for cultivars Valencia and Washington oranges and observed drastic increases in transformation efficiencies if compared to a conventional Ti-plasmid vector containing no K or I gene. We have confirmed that the K and I genes can drastically enhance transformation efficiencies of juvenile explants of 5 different citrus cultivars. We started test the effects of the K gene on transformation efficiency of a lemon cultivar. Lemon is difficult for genetic transformation. Our major efforts have been in testing the effects of the K and I genes and other factors on mature tissues. We used K and I genes to do genetic transformation of mature Pineapple orange. The K gene resulted in about two fold increases in transformation efficiency while the I gene produced about three fold increases in efficiency compared to control vector. We have also started testing effects of other factors on transformation of mature tissues in combination with the K gene. We have made some significant progress but microbial contaminations of adult tissues harvested from greenhouse grown trees have sometimes caused problems for us. One example is that we have repeated and confirmed the effects of the transport of an endogenous plant hormone in explants on shoot regeneration efficiency. We observed that manipulating that process improves shoot regeneration and transformation efficiency of juvenile citrus explants. We are testing the effects of the same manipulation on transformation efficiency of adult tissues of citrus.
The overall objective of this project was to use the PAMP receptors EFR and XA21 to engineer citrus plants resistant to both HLB (causal agent Candidatus Liberibacter asiaticus, CLas) and citrus canker (Xanthomonas axonopodis pv citri, Xac). Since neither receptor recognizes a PAMP from CLas, the first objective was to engineer a variant of EFR (EFR+) to recognize the elf18 peptide from CLas. This novel receptor would then be combined with XA21 or an XA21-EFR chimera that recognizes a PAMP from Xac. A number of strategies to engineer an EFR+ variant that recognized elf18-Clas were tested, but none were successful. These included PCR mutagenesis, screening of natural variants in an extensive Arabidopsis accession collection, creating targeted mutations based on the modeled interactions among elf18, EFR, and BAK1, and testing high-throughput strategies such as phage display and FACS. However, we were able to successfully create a functional XA21-EFR chimera. Although we did not generate an EFR+ variant that recognized elf18-CLas, expression of EFR and XA21 may still provide significant protection against citrus canker. Therefore, we transformed three constructs into citrus: EFR alone, EFR with XA21, and EFR with the XA21-EFR chimera described above. The latter two constructs have the potential of providing stronger and more durable resistance than EFR alone. Some transgenic events have been obtained in Duncan grapefruit and sweet orange, and these have been transferred to Dr. Jeff Jones lab at the University of Florida for testing with citrus canker.
The transgenic plants to be developed for this project are now growing in 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. The plants at both locations are growing well. At Penn State, all the transgenic lines have been successfully propagated as vegetative cuttings. All of the lines growing at Penn State have been found to express the FLT-antiNodT fusion protein, with five of the seven lines expressing very high levels of the protein. We must continue to let these plants grow a bit more before starting the HLB resistance tests. We have initiated a collaboration with Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) to transform varieties important to the Florida citrus industry, including the ‘Valencia’ and ‘Hamlin’ sweet orange varieties and the ‘Citrumello’ rootstock with the FLT-antiNodT expression construct. We have immediately started transformations with the available transformation construct used to transform ‘Duncan’ grapefruit, in plasmid pTLab21. In addition, we are developing an FLB-antiNodT expression cassette in the transformation construct pBI121, which has a history of successful approval for transgenic plant development. In June, Dr. McNellis submitted a Stakeholder Relevance Statement to the USDA Specialty Crop Citrus Disease Research and Extension program to further develop this project. However, a full proposal was not invited. Dr. McNellis will present a poster describing the results of this project to date at the American Phytopathological Society conference in Pasadena, California, August 1-5, 2015.
The project has two objectives: (1) Increase citrus disease resistance by activating the natural SAR inducer-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we treated citrus plants with the natural SAR inducer using three different approaches: leaf infiltration, foliar spray, and soil drench. Three concentrations were tested: 1, 5, and 10 mM. For leaf infiltration, the infiltrated leaves were inoculated 1 day later with the canker bacterial pathogen; for foliar spray, treated leaves were inoculated 3 days later; and for soil drench, leaves on treated plants were inoculated 7 days later. For each treatment, 5 plants were used. Three leaves on each plant were inoculated and 6 inoculations on each leaf were conducted. A total of 90 inoculations were used for each treatment. Fourteen days after inoculation, numbers of lesions formed on the inoculated leaves were counted. Results showed that all concentrations of the SAR inducer induced strong resistance to citrus canker. The inoculated plants have been cut back. Systemic residual resistance will be tested on the new flushes. We are repeating this comprehensive testing experiment and will identify the most efficient treatment method. For objective 2, transgenic citrus plants expressing the Arabidopsis nonhost resistance genes have been propagated. The progenies are growing in greenhouse and will be tested for disease resistance.
We completed computational comparative analysis of the three genome categories: Liberibacter, Citrus and Psyllid. The results are available at the website
The HLB-tolerant rootstocks US-1279, US-1281, US-1282, US-1283, and US-1284 were released by USDA in 2014, and are available from FDACS-DPI as clean sources for vegetative propagation and establishment of seed trees. Plant material was also provided to commercial nurseries for micropropagation. Fruit yield of Hamlin trees infected by HLB on these rootstocks was 2-4 times the yield of trees on Swingle, and the trees on these rootstocks also had fruit that is larger in size and higher in sugar content. The previously released USDA rootstocks, US-942, US-802, and US-897 were demonstrated in greenhouse testing and field trials to exhibit better tolerance to HLB than many of rootstocks commonly used in Florida. The promising new USDA rootstocks, along with other new HLB-tolerant rootstocks from USDA and Univ. of Florida will be used in a series of grower-cooperator field trials with funding provided by the HLB-MAC program in 2016-17. An updated rootstock selection guide including information about these and other new rootstocks was developed cooperatively with University of Florida and released in 2015 as an extension publication, “Florida Citrus Rootstock Selection Guide, 3rd Edition”. Yield, fruit quality, tree health, and other performance information was collected from 10-20 established rootstock field trials each year, and used to assess new rootstock performance at different sites. Thousands of budded nursery trees were prepared with Supersour and other new rootstocks, and numerous new field trials were planted in 2013-2015. About fifteen thousand new propagations of Supersour and other rootstocks were prepared for greenhouse testing for disease tolerance and budding for additional field trials in 2016. Greenhouse and field studies were used to evaluate the phytophthora tolerance of 70 new Supersour and other rootstocks. Greenhouse tests were used to assess Supersour tolerance of CTV. Trees were planted into the field to establish seed sources for the most promising Supersour selections. Studies of defense gene and metabolomic profiles of hybrid rootstocks that are highly tolerant to HLB were studied, so as to improve our ability to create and select conventional hybrid and transgenic rootstocks that possess a high level of tolerance. Additional studies were conducted on defense-related gene expression and small RNAs associated with HLB infection, in collaboration with University of Maryland and University of California research groups. Several research studies were published to document the new important information. New grant proposals were submitted to NIFA-SCRI and other funding agencies to follow up on new research opportunities in these areas. Thousands of new transgenic rootstocks were produced, including the novel genes for antimicrobial peptides and constructs that would optimize expression of natural citrus defense genes. Optimizing of citrus defense genes was directed in significant part by results from other research under this grant to identify defense gene expression and metabolic profiles associated with tolerance to HLB. Fifteen transgenic rootstock selections showing increased resistance to HLB have been identified from preliminary test groups with transgenes, and trees are being propagated for additional testing. Several hundred additional transgenic citrus have been produced and are awaiting screening with HLB, including transgenics with optimized expression of the citrus defense genes CtSID2, CtSFD1, CtPAD3, CtCDR1, CtMPK4, CtTGA7, CtDIR1, CtERF1, CtFAD7, CtFMO1, CtAZL1, CtRDR1, CtRAP4, CtCSD1, CtNHL3, CtNHO1, and CtNHL25. Work is continuing under the new CRDF-funded screening project to test these transgenic citrus for tolerance to HLB.
Progress has been made in identifying conventional citrus that is quite tolerant to HLB. Evaluation of existing standard and non-standard cultivars ( Hamlin , Temple , Fallglo , Sugar Belle , Tango , and Ruby Red ) for HLB resistance/tolerance is complete. In August 2010, the plants were established at Pico s farm in Ft. Pierce Fl. Data on the growth rate, disease severity, and Candidatus Liberibacter asiaticus (CLas) titer levels have been collected since April 2012. During the 4-year period, there were significant differences in disease severity, stem diameter, and CLas levels among the varieties. All trees exhibited symptoms of HLB and tested positive for CLas, with similar titers measured at most recent sample dates. Fallglo had the lowest incidence of HLB symptoms, whereas Ruby Red had the highest incidence. Ruby Red also appears to be in significant decline. Despite the high initial titer levels found in SugarBelle , it had the greatest overall increase in diameter and was the healthiest in overall appearance. In Nov. 2014 Temple trees had significantly greater fruitload, with 26 fruit/tree, followed by Tango with 10 fruit /tree, Hamlin/Kinkoji with 5 fruit/tree and all others with 0-1.4 fruit/tree. All cultivars except sweet oranges and grapefruit are progressing in production, but production was compromised in all varieties by the severe HLB pressure at this site, and commercial value of the observed tolerance remains uncertain. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and have been transferred to the field at Ft. Pierce Fl. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a monthly basis. As of December 2014, the first HLB symptoms are apparent. Progress has been made on the antibiotic treatment of HLB infected bud-wood to compare growth at different levels of CLas infection. HLB-infected budwood was treated with various concentrations of antibiotics and grafted on sour orange rootstock using 3 fairly HLB-resistant ( Temple , GnarlyGlo , and Nova ) 3 tolerant ( Jackson , FF-5-51-2, and Ftp 6-17-48), and 3 susceptible ( Flame , Valencia , and Murcott ) genotypes. Standard growth measurements (stem diameter and height), disease severity were evaluated and leaves were sampled for qPCR analysis. Evaluations and sampling will continue on quarterly basis. Trees will be field planted July 2015. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is underway. One hundred and fifty etiolated seedlings of the trifoliate Rubidoux and the sweet orange Hamlin have been approach grafted together. Generation of new chimeras has been difficult. Several adventitious buds have emerged from the treated graft region, with several appearing to be chimeral. The newly emerged plants will be tested using LC/MS to determine the origin of the three layers. To increase the success rate, additional plants will be grafted over the next twelve months. An existing periclinal chimera has been imported and will be released from DPI. 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 evaluated at 3, 6, and 9 weeks Seedlings of Hamlin and Dancy show early CLas proliferation and systemic movement. Only low levels of CLas have been observed in Carrizo. Trees of seemingly HLB resistant/tolerant sweet orange-like hybrids and mandarin -types have been 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 will be established with 6-8 trees of each cultivar at each site. The first trials have been established with cooperators (in Ridge, IR and Gulf coast) for replicated block plantings at each site.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over five years. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015 (Grosser, personal comm.). The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material is being planted this spring that has shown great promise in the greenhouse (unpublished). A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 5 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Lee et al., in preparation), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). A new UF-Gmitter led association mapping study has just been initiated using the same planting, to identify genes associated with HLB- and ACP-resistance. A broader cross-section of Poncirus-derived genotypes are on the sire in a project led by UC Riverside/USDA-ARS Riverside, in which half of the trees of each seed source were graft-inoculated prior to planting. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished). In July 2015 David Hall will be leading assessment of ACP colonization across the entire planting, and the Gmitter lab will map markers associated with reduced colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. As of April 2014 at the Picos Test Site, several of these USDA hybrids had grown to a height of seven ft, with dense canopies and good fruit set, while sweet oranges are stunted (3 ft) with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population will be planted at the Picos Test Site in July in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting will also include a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
Chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) are being tested. Carrizo transformed with a chimera AMP showed remarkable resistance in citrus canker compared to control. RT-qPCR showed 50% of 16 chimera transgenic Hamlin have relatively high gene expression, with 100x difference between high and low expressers. These promising transgenic lines were replicated by grafting for HLB challenge. Twenty transgenic Hamlin lines were confirmed to contain thionin gene by PCR, and six have high gene expression by RT-qPCR. Transgenic Hamlin lines expressing thionin were grafted onto Carrizo for HLB challenge. Replicated transgenic Transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon. Promising resistance to HLB was observed based on plant growth and phenotype. Las titer is being checked from root and new flush rough lemon leaves. Two new chimeral peptide from citrus genes only were developed and used to produce many Carrizo plants and Hamlin shoots. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Many putative transformants were generated on the selective media: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. There is only slight canker resistance by infiltration, but considerably resistance to spray inoculation. To confirm that high ROS production was not due to variability in Hamlin, we examined l 40 Hamlin seedlings and no or very low level ROS production was detected. Two potential FLS2 orthologues were identified in Hamlin and their expression was shown much lower compare to nbFLS2. Primers were designed for two citrus FLS2 orthologues. They showed low gene expression in transgenic and nontransgenic citrus. Results on FLS2 transgenics against canker disease were summarized and a manuscript was written and submitted to MPMI. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing. 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 is being used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only.
During the life of this project, 121 mature citrus transgenics were produced using genetic transformation with Agrobacterium. Sixty-six were transgenic for reporter genes and provided proof of concept that this protocol works in our hands. In the last 21 months, 55 transgenics with disease tolerance genes, most without reporters, were produced and micrografted onto rootstock. Agrobacterium transformation efficiencies were relatively low (3.47% positive shoots for constructs with reporters in scion, and 3.0% positive shoots for constructs with no reporters in scion and rootstock). Only 0.78% shoots/total explants plated were transgenic. Many more transgenics were probably produced, but were lost in the micrografting process. Attempts are being made to increase micrografting efficiencies. Although 9 vectors carrying disease tolerance genes were used in sizable transformation experiments, only 4 vectors yielded transgenics. The 55 mature citrus transgenics were produced with these 4 vectors to confer disease tolerance to HLB, canker, or both. These primary transgenics are being propagated into vegetative progeny to facilitate replicated field trials this fall. Numbers were low for Ray Ruby grapefruit (3 transgenics) and efforts are being made to optimize the tissue culture and transformation protocol for grapefruit. Experiments are underway to root mature scion because a larger scion could be easily micrografted onto rootstock with greater efficiencies. To increase our chance of success, we have been utilizing nurse cultures to supply additional nutrients to developing mature shoots. Additional vectors are being acquired from scientists around the country and worldwide. Budding is now done entirely in-house. A gene gun was purchased in July, 2014 to develop a high-throughput biolistics transformation system for mature citrus. Transient expression levels are relatively high, and a few stable transgenics have been produced. Optimizations for mature citrus have been hindered by the limited supply of mature scion in the growth room, which was primarily used for Agrobacterium transformations. In the future, we will purchase mature citrus from nurseries to continue optimizations. We have developed a high throughput screening system in which thousands of putative transgenics can be rapidly screened. A number of equipment expenditures were necessary to achieve this high level of efficiency. Equipment expenditures included a refrigerated centrifuge, a plate reader, a tissuelyser, a laminar flow bench and an incubator. A hybridization oven and dry baths were purchased for molecular analyses. Maintenance expenses for lighting, AC repair, sensors and expansion cards for RCWebview, and the water softener in the growth room are ongoing. This project depends upon a continual supply of healthy, viable rootstock seed and this was problematic last year. Rootstock of Swingle and Volkameriana had poor germination, and Macrophylla and Carrizo had disease/endophyte issues that negatively impacted budding of mature citrus and the tissue culture process.
The driving force for this project was the need to evaluate citrus transformed to express proteins that might mitigate HLB, which required citrus be inoculated with CLas. Although citrus can be manually inoculated by grafting with infected budwood, a program using CLas-infected ACP was preferred primarily because this is what occurs in nature. Nine colonies of infected ACP for inoculations were initially established in a walk-in chamber. Additional colonies of infected ACP were later established in two small greenhouses and in a second walk-in chamber. For each individual colony, a potted lemon or citron tree graft-inoculated with CLas (testing PCR-positive and showing HLB symptoms) was placed into a cage and trimmed to stimulate flush; ACP were introduced and allowed to reproduce; and the colonies were maintained over time by regularly trimming plants and introducing additional ACP as needed. The source of CLas for these lemon and citron trees was the original HLB-infected tree found during 2005 in south Florida. Germplasm to be challenged for HLB resistance was supplied by USDA-ARS citrus breeders. To inoculate plants, individual plants were caged for 2 wk with 20 psyllids from one of the infected colonies, after which the plants were held together for six months in a greenhouse with an open infestation of infected psyllids. After this two-step inoculation procedure, the plants were returned to the breeders. Challenges associated with the program have included variability in percentages of ACP testing CLas-positive in a single colony over time and among different colonies at any given time. Consequently for the first inoculation step we always used ACP from colonies with the highest percentages of infected ACP, and we increased the number of colonies to increase numbers having high percentages of infected ACP. Lots of qPCR assays were required to monitor infected ACP colonies and infected source plants. Growth chamber colonies generally have had greater percentages of infected ACP than greenhouse colonies, probably due to more stable environmental conditions in chambers. Another challenge has been that, among ACP that test PCR-positive for CLas, relatively low transmission rates have been reported (e.g., 12% or less). Therefore, caging an individual plant with 20 psyllids from an infected colony may not always result in inoculation. Success of the first inoculation step would likely be increased by increasing the number of ACP per plant. Subsequently holding the plants for six months in the greenhouse with free-roaming infected psyllids increases the probability of inoculation. With respect to the second inoculation step, population levels of ACP in the greenhouse were sometimes severely reduced as a consequence of damage to oviposition sites by western flower thrips or spider mites. Also, sometimes population levels of ACP eggs and nymphs were severely reduced by western flower thrips, and further reductions in populations of nymphs were sometimes caused by Tamarixia parasitoids that invaded the open infestation of ACP. Effectiveness of the inoculation program has been slow to gauge. A series of experiments was initiated during 2014 specifically to evaluate inoculation success. Meanwhile, recent feedback from inoculations of rootstock material gave some insight. Eleven groups of rootstock material (3,105 plants total) were passed through the inoculation program during 2011-2014. The average percentage of infected ACP used in step one was 52% (range 24 to 80%). qPCR 12 to 19 months after the two-step inoculation process indicated an average of 62% success in inoculating citrus. Plants that escaped inoculation had another opportunity for inoculation after being transplanted to the field.
Core Citrus Transformation Facility (CCTF) continued to operate at the high level it did in the previous period and produced transgenic material in a timely and dependable manner. CCTF maintained its standing as reputable partner that delivers transgenic material in reasonable amount of time and continued to be sought for services. This quarter marks the end of three year period that saw significant but expected increase in number of orders. With six new orders received most recently, CCTF has just surpassed its 200th order and 96 of those were placed within the last three years. Clients requested transgenic Duncan grapefruit and Valencia orange in the newest orders. The number of plants produced in this quarter is 85 bringing a total to 730 plants for the duration of the funding period. Most of the produced plants were of Duncan grapefruit cultivar, followed by Carrizo rootstock, Valencia orange, some Citrus macrophylla, Swingle citrumelo, and Mexican lime. This strong bias towards Duncan grapefruit points towards need of researchers to get fast answers regarding candidate gene that could potentially render Citrus plants tolerant/resistant to diseases. Since Duncan grapefruit is highly susceptible to both Huanglongbing (HLB) and citrus canker, challenging transgenic plants expressing gene of interest with bacteria causing these two diseases will quickly reveal if that gene holds any promise or not. Throughout the duration of this project, CCTF processed about 400000 explants. Approximately 5% of explants were lost to contamination which brings the total number of explants producing data to 380000. Overall transformation efficiency expressed as the percentage of positive shoots out of all of those tested was about 3% which is low. One of the reasons for this is the nature of orders received. Amongst 96 received orders, there were 34 for which total of just a few transgenic plants were produced. This was a result of withdrawal of 10 orders, presence of sequence detrimental for shoot development in binary vector used for transformation in six orders, and for 18 orders transformation was either not possible or achieved at the very low rate. The second reason for low transformation rate is the quality of seeds/seedlings resulting from the effect HLB infection has on fruit growing on trees. In the immediate future, CCTF will dedicate increasing efforts to acquire fruit/seeds of higher quality than what it was used recently. The labor force and the income of CCTF remained relatively stable during the last three years and changes in facility s staff never compromised the level of operation. Number of orders presently serviced by the CCTF and those that were either announced or anticipated in the near future is clear indication of sustaining interest of researchers in transgenic citrus plants. They also assure CCTF will stay busy for the years to come.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. All of these constructs had the peptide gene inserted between the coat protein genes, which is positioned sixth from the 3′ terminus. However, we have found that much more foreign protein can be made from genes positioned nearer the 3′ terminus. Based on that we built constructs with the peptide gene next to the 3′ terminus. These constructs produced much greater amounts of peptide and provided more tolerance to Las. Unfortunately, they are less stable. So now we are rebuilding constructs with the peptide gene inserted at an intermediate site hoping for a better compromise of amounts of production and stability. We have produced a large amount of inoculum for a large field test via Southern Gardens Citrus. We are screening a large number of transgenic plants in collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We are propagating a progeny set of plants of the promising candidates for a final greenhouse test.
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