Update for 9/30/10: This project will assess a range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. The largest seedlings of these accessions have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination. They are now being grown in a climate controlled, DPI-certified greenhouse and monitored for symptom development; to date, no symptoms have been observed. Additional seedlings now large enough for inoculation have been moved into our certified HLB-testing greenhouse, and inoculations are about to take place. Further, we have been granted DPI permission to plant out the Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test, at the Picos Road Farm near Ft. Pierce, with USDA-ARS. This population is of significant interest as the trifoliate orange and some of its hybrids are very HLB-tolerant, and this experiment is an opportunity to map genetic components responsible for the tolerance. Testing the material for HLB and CTV has been completed and only pathogen free selections safe for field planting have been identified and budwood samples sent to the USDA-ARS for propagation and subsequent field planting. Approximately 100 selections were chosen and trees have been budded in late August. We continue to seek additional germplasm resources, to expand the breadth and depth of the material categories we described in our proposal; a source for new C. latipes hybrids has been identified. We are still exploring other options within Florida for a field trial, but no secure, long-term commitments have been forthcoming, despite multiple discussions with growers throughout the state. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have expanded, and are continuing to expand, the number of types we wish to challenge. We will be developing new information about potentially tolerant/resistant germplasm that can lead to expanded efforts to capture and exploit the genetic basis for this phenomenon.
Transgenic studies have proceeded, with several hundred plants containing various combinations of natural or synthetic genes and promoters produced; many of these are currently in greenhouse testing and field trial locations. Additional transgenic plants have been propagated for new hot psyllid greenhouse tests, and for field planting. New candidate genes have been identified from citrus and other plants for HLB and canker resistance; vectors have been constructed for a next round of transgenic plant production, already underway. Citrus-specific promoters, transcription factors, and other genetic elements have been identified and incorporated into some of the new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. The sweet orange citrus genome sequence was mined to identify genes controlling anthocyanin expression, in an effort to develop visual and citrus-derived markers for genetic transformation; several candidates have been identified for further experiments. More than 875 transgenic plants have now been planted with a collaborator in Martin county, and these are being monitored regularly, along with a second site in Indian River county. The plant materials growing out include sweet oranges, grapefruit and mandarin hybrids. Several new rootstock trials with more than 15,000 trees were planted throughout Florida using advanced selections, to assess their adaptation to evolving advanced citrus production systems; these trials are monitored regularly, and data has been collected on their early performance. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests; rootstock hybrids are showing diverse responses when grafted with HLB-infected Valencia, ranging from extreme sensitivity to high levels of tolerance; four complex tetraploid rootstocks have shown some repression of HLB in greenhouse tests (one was symptom-free up to 22 months ). We initiated a program to rotate new germplasm (rootstock and transgenic) through a ‘hot psyllid’ house (in collaboration with Dr. Stelinski) to ensure HLB inoculation prior to approved field planting; two groups of 50 trees have been rotated through so far, scheduled for planting at a collaborators field site, under permit from DPI. Rootstock candidates that produce nucellar seedlings have been identified using SSR markers; these rootstocks were preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora. Hybrid plants for rootstock improvement from the previous season were planted, and new crosses made 2010 were planted in the field. Previous work to develop rootstocks against other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) continues, as we collected data from replicated trials and plantings. Final data have been collected from a field trial of various Valencia somaclones and seedless Midsweet selections, and following final analysis the most consistently high yielding clones from each will be moved forward for release; most candidates have already moved through the DPI-Parent Tree Program. New pummelo-grapefruit seedless hybrids have been selected, some showing field tolerance to canker; their fruit have been assayed for furanocoumarin content and several with good fruit quality have been found FC-free, potentially producing grapefruit cultivars that alleviate drug interaction concerns. Patents have been issued by the US-PTO for Valquarius (SF14W-62) and Valenfresh (N7-3), very early- and late- maturing Valencia selections respectively, and licensing is in process. Patent applications and documents for release were developed for 7 new cultivars, and these were approved by the UF-IFAS Cultivar Release Committee for release and commercialization according to UF-IFAS policy.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. ‘ We have greatly improved our efficiency of screening . ‘ We are modifying the vector to express more than one anti-HLB gene. ‘ We are modifying the vector to allow addition of a second vector. ‘ We are preparing to put trees into the field for testing as soon as potential freezes are over. ‘ We continue to supply infected and healthy psyllids to the research community.
Researchers at the USDA Ft. Pierce: 1. Source of mature tissue. Four populations of adult phase trees were maintained in the greenhouse including Valencia sweet orange/Sun Chu Sha (73 trees), Ruby Red grapefruit/US812 (62 trees), US-942 citrange rootstock/Cleo (32 trees), Calamondin (31 trees), and Etrog Arizona 861-S1 citron (67 trees). In vitro bud emergence and growth manuscript accepted for publication. A manuscript was submitted to the journal Plant Cell, Tissue and Organ Culture that documents the system developed for producing in vitro adult phase shoots from cultured nodes of greenhouse trees. Shoot regeneration from mature tissue explants. A system was developed for the production of shoots from cultured internodes from greenhouse trees. The system results in shoot and bud formation in 70-90% of the explants. A manuscript is in preparation that documents this research. Agrobacterium-mediated transformation of mature tissue explants. Transformation of mature internode explants from greenhouse trees has been demonstrated in grapefruit (1 plant), US-942 (4 plants), and Etrog citron (4 plants) using the beta-glucuronidase reporter gene. Current efforts are now directed toward identifying the factors important for a system of sufficient efficiency for routine transgenic plant production. New tissue culture method of Agrobacterium-mediated transformation of tissue explants. Preliminary results using alternative culture methods suggest improved transformation efficiencies. These approaches will be further explored. At the CREC in the Gmitter lab, work is continuing on the use of Thin Cell Layers (TCLs) as explants for mature tissue transformation. Experiments have been done to induce regeneration in the TCLs by manipulating the amount of growth regulators, carbon source and also by pre-treating the TCLs with BA but regeneration is still problematic from these explants. In the Grosser lab, research confirmed the work of others showing that the flush used to generate transformation explants was critical. In the Machado laboratory in Brazil, research on the regeneration ability of various sweet oranges continues. These differences are present in both mature and juvenile tissues. In the Moore laboratory in Gainesville, experiments still are focused on using small peptides as vehicles to deliver cargos to plant tissues. If these techniques could be worked out they would have a number of applications for citrus transformation, perhaps even eventually allowing the transfer of genes or gene products to existing trees. A transient transformation expression system has also been developed using citrus leaves.
Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. This objective is proposed as an alternative strategy in case constitutive expression of the mutant R proteins proves to be detrimental to the growth or vigor of the plant. By restricting expression of the R protein to the single cell that is pierced by the insect stylet, a defense can be mounted without endangering the overall health of the plant. In the long run, this may be the most effective strategy in fighting Liberibacter infection since the response can engineered to be quite robust. Results: To further insure more specific expression of the R proteins in the phloem, we identified a wound-inducible, phloem-specific promoter that triggers expression upon wounding resulting from aphid feeding. We substituted the AtSUC2-940 promoter (phloem-specific) with the AtPAD4-1002 promoter (PAD4) in SNC1, snc1, SSI4, and ssi4 R gene constructs in the pCAMBIA1305.1 vector. Additionally, we created a PAD4/GUSplus construct to verify phloem- and wound-specific activation. The PAD4 constructs were introduced to Arabidopsis plants, grown to seeds and harvested this week (Jan 2011). The transgenic seeds will be subjected to selective screening for R gene transformants. Due to the cloning limitations (restriction site configuration), all PAD4/R constructs had to be first assembled in the 1305.1 pCAMBIA vector prior to transfer into the 2301 pCAMBIA for transformation into citrus. We selected two constitutive R protein mutants and PAD4/GUSplus constructs, and transferred them to the Citrus Research and Education Facility at Lake Alfred (Dr. Vladimir Orbovic). Preliminary reports indicated that transformants are proving difficult to obtain, potentially due to the lethal nature of at least one R protein mutant. Conclusions: Our hypothesis is that phloem-restricted expression of the R protein constitutive mutants would limit the potential negative impacts on plant growth. Use of the PAD4 promoter to express the R protein constructs should further increase the stringency of transcriptional control over expression of the potentially harmful R proteins. Our goal is have only the single phloem cell that is penetrated by the stylet express the constitutively active R protein, hence limiting potentially detrimental effects and focusing a robust defense at the source of Liberibacter introduction. Additionally, these constructs with the PAD4 promoter may reduce spurious expression in callus cells during the transformation protocol into citrus.
Our presentations at the Second International Research Conference on HLB in Orlando documented recent progress in characterizing HLB resistance/tolerance as found in Poncirus trifoliata and hybrids of that species with citrus. Some trifoliate hybrid citrus rootstocks, including US-802 and US-897, were demonstrated to become infected by HLB more slowly, to develop smaller amounts of bacterial even after infection, and to exhibit dramatically less symptoms in response to HLB infection than other citrus cultivars. A greenhouse experiment was initiated to compare the utility of the trifoliata-type resistance in the rootstock and scion position in grafted trees. Evidence was presented that documents significantly higher levels of some antimicrobial metabolites in the HLB-resistant trifoliate hybrid germplasm. Gene expression and metabolomic studies are underway to further characterize the genes and metabolites responsible for the resistance that can be used in creation and selection of conventional and transgenic varieties with improved tolerance or resistance to HLB. In this quarter, fruit quality, yield, and/or tree size data were collected from fourteen rootstock and scion field trials. Data was collected from one large cooperative rootstock trial with Ray Ruby grapefruit scion to evaluate rootstock effect on grapefruit quality and sheepnosing. Scion growth was measured on a greenhouse experiment to develop a more rapid way to evaluate CTV resistance and to evaluate supersour rootstocks for tolerance to four different decline strains of CTV. Rooted cuttings of supersour rootstock hybrids were budded with scions to propagate trees for field trials. One hundred supersour-type hybrids were selected from among new progeny for propagation and additional testing. A field trial was planted to compare HLB reaction of standard rootstock varieties to that of new transgenic rootstocks. Budded greenhouse trees for field trials were grown to planting size. Experiments continued to assess the utility of different methods for testing germplasm for resistance or tolerance to Asian Citrus Psyllid (ACP) and HLB disease. Data continued to be collected from four field experiments to assess the HLB tolerance of sweet orange trees on 15 different rootstocks. A field experiment continued to identify rootstocks with resistance to the Phytophthora-Diaprepes Complex. In coordinated research between this grant and the FCATP transgenic citrus grant to USDA, selected anti-microbial genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with resistance to HLB and Citrus Bacterial Canker (CBC). Rootstocks were transformed with an early flowering gene to produce selections that will allow more rapid advancement through generations and thus, more rapid genetic improvement. Selected transgenic rootstocks were challenged with HLB to assess potential resistance, including constructs with two new bacterial resistance genes. Research is continuing to use HLB responsive genes and promoters identified in a previously published gene expression study for engineering resistance in citrus. A microarray analysis was completed on gene expression in HLB-susceptible and HLB-tolerant selections to identify differences that can help guide selection from conventional breeding and transgenic efforts. This data is being analyzed now for publication and to guide future breeding and transformation research. The new hybrid rootstock US-942 was officially released for commercial use, based on outstanding performance in several different field trials. Seed of US-942 was provided for distribution to Florida citrus nurseries.
For EDS1 cloning, we currently confirmed by sequencing the cloning of the full-length ctEDS1 and already moved the sequence from the pGEM T-easy vector to the binary vector pBINplusARS for plant transformation. With Carrizo sequence database (http://citrus.pw.usda.gov/) recently available, we designed primes to clone the 3′ end sequence of ctSID2, which we were previously unable to obtain with several methods. We performed the RACE reaction and have now obtained this missing sequence. We will subsequently design primers to amplify the full-length sequence of ctSID2. In addition, we did bioinformatics analysis and identified additional 9 citrus homologs of Arabidopsis defense genes that have available sequences in the database. We designed primers for these citrus genes and have been conducting RACE in order to amplify the genes. So far we have obtained the 3′ end sequences for ctNHL1, ctSFD1, and ctFAD7. Further cloning of 5′ end and/or 3′ end sequences of these citrus defense genes are currently underway. We continue to characterize the transgenic plants expressing ctNDR1/pBINplusARS. We obtained 5 homozygous ndr1 + ctNDR1/pBINplusARS. Through HR test and disease resistance assay with infection of the avirulent strain P. syringae avrRpt2, we further confirmed that over-expression of ctNDR1 could complement Arabidopsis ndr1 mutant. We are going to further characterize the defense phenotypes of these transgenic plants.
Over the past quarter, we have continued to develop all aspects of our project. In particular we have progressed in the following areas: 1. Building and testing additional TAL effector and promoter constructs. We have synthetically assembled a number of TAL effector genes matching X. citri TAL effectors and showed that they transcriptionally activate our broad recognition or “super” promoter in a Nicotiana benthamiana system. We have also assembled promoters with individual TAL effector binding sites to test activity and specificity. 2. Testing activation of gene constructs against a diverse world wide collection of X. citri isolates. Using the transient transformation method that we have developed, we have tested the reaction of thirty X. citri isolates on grapefruit leaves. We see a very high correlation between isolates which are capable of inducing disease in standard susceptible germplasm and recognition by our promoter constructs, indicating that the resistance constructs we have created will be able to confer broad resistance to diverse strains of citrus canker. Additionally, we are preparing and testing X. citri strains with single or multiple disruptions in their TAL effector complement to test the role of specific TAL effector proteins in the disease and resistance process. 3. Stable transformations. The transformed lines generated last Fall and Winter have progressed through selection, shoot formation and rooting, and are now growing in soil. These lines are tested by PCR as they reach adequate size, and positively scored lines have been subjected to pathogen testing by pin-prick assay with X. citri. We have identified several canker resistant transgenic lines. We are currently setting up additional transformations to generate more transgenic material for line testing and with new promoter constructs. 4. Manuscript preparation We are in the process of drafting a manuscript of our results.
In the last few months, we have continued working on genetic transformation of mature material from the three sweet orange genotypes (Valencia, Hamlin and Pineapple) with the aim of improving transgenic regeneration efficiency and having a reliable mature transformation procedure for each type that could be reproduced in Florida. After some last refining, Valencia sweet orange is routinely transformed at IVIA now. The transgenic nature of the first plants acclimated to the greenhouse has been confirmed through Southern blot analysis. Hamlin is more difficult to transform but with appropriate modifications of the tissue culture media and the source material used we have been able to produce already many transgenic plants, as confirmed also by Southern blot. Pineapple is routinely transformed at IVIA since the 90’s and is being used as control. Transformation of mature Carrizo citrange was initiated later, simply because we had not enough space and personnel to work with all the genotypes at the same time. During the last quarter, we have been more focused on developing a reliable transformation system for this genotype. More than 100 mature transformants (PCR-positive shoots) have been produced so far. The key in this case is using proper source material. We have preparing new source material to attempt transformation of mature citrumelo and grapefruit in the coming months. Regarding our second objective, at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange expressing either FT or AP1 flowering-time genes are established in the greenhouse and we are now characterizing them in detail (genetic and phenotypically). Additionally, a hairpin construct aimed to induce RNA interference to silence and endogenous GA20-oxidase gene and them reducing gibberellin biosynthesis has been synthesized and incorporated into Agrobacterium tumefaciens. It will be used to transform Carrizo citrange. In Florida, construction of the growth room has been finally initialed and according to the schedule it will be finalized before the end of February. The PI and his greenhouse manager are planning to travel to Florida next March to supervise and setting up plant growth conditions, to set up the healthy citrus mother materials, and to establish substrate, fertirrigation and phytosanitary treatments.
Huanglongbing (HLB) is a serious and devastating disease of citrus caused by Candidatus Liberibacter spp. and vectored by the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The disease has the potential to greatly limit the production of citrus in Florida and other citrus growing regions worldwide. Current control of ACP and HLB is inadequate, but the identification and incorporation of ACP resistance traits from uncultivated Citrus spp. and Citrus relatives is seen as a potential disease management strategy. In a study by USDA-ARS, 87 genotypes primarily in the Rutaceae orange subfamily Aurantioideae, were assessed in the field for resistance to natural South Florida populations of ACP. The majority of genotypes surveyed hosted all three life stages of ACP, however there were significant differences among genotypes in the mean ranks for ACP eggs (F = 3.13, df = 86, P < 0.000), nymphs (F = 9.01, df = 86, P < 0.000), and adults (F = 4.21, df = 86, P < 0.000). The only sampled genotype that was completely avoided by all life stages of ACP was Casimiroa edulis, commonly known as white sapote, which was one of the few plants included in the study belonging to the Rutaceae subfamily Toddalioideae. Although not completely avoided, very low levels of ACP were found on two surveyed genotypes of Poncirus trifoliata, 'Simmon's trifoliate' and 'little-leaf'. Poncirus trifoliata, the trifoliate orange, readily forms hybrids with Citrus spp. and is commonly incorporated into rootstock varieties. The identification of partial resistance in this species to ACP may prove useful in future citrus breeding programs efforts aimed at controlling the damage caused by reducing the incidence and spread of HLB. In recent psyllid no-choice oviposition studies using six different genotypes of Poncirus trifoliata, with Citrus aurantium and C. macrophylla as susceptible controls, a greatly reduced number of ACP eggs were laid on the Poncirus trifoliata selections when compared with the controls. The post doc assigned to the project resigned in December to take a permanent job elsewhere. A new post doc has been hired and is set to start during late January. A major thrust of upcoming work will be on no-choice experiments with Poncirus trifoiata selections. Collaborators with the Fujian Academy of Agricultural Sciences in Fuzhou, China, initiated two experiments on resistance to ACP within the Rutaceae : one with 31 citrus varieties and one with 40 citrus varieties. Both experiments are free-choice studies under greenhouse conditions. A delegation from FAAS will be visiting USDA-ARS during April to coordinate research.
Production of transgenic Citrus plants in the Core Citrus Transformation Facility (CREC) continues to be at the rate of about 100 plants per three months. Orders are being serviced for clients based both in Gainesville and in Lake Alfred. The demand for transgenic material is holding steady. Additional four orders were taken to produce transgenic grapefruit carrying genes harbored in following vectors: pWG22-1; pWG24-13; and pWG25-13, and pWG27-3. However, most of the activities of the facility are directed towards completion of previously placed orders. New orders are being serviced according to the order they were placed. The list of transgenic plants that were delivered within the last quarter includes those concerned with resistance to both bacterial diseases and CTV. Canker and HLB: 1) N1* gene: one Duncan plants; 2) NPR1: three Flame plants and superNPR1-six Flame plants; 3) AS7 gene: two Duncan plants and A13* gene: four Duncan plants; 4) pMKK7 vector: 20 Duncan plants; 5) pMOD1 vector: seven Duncan plants; 6) pNAC1 vector: one Duncan plants; 7) pSuc-NPR1 vector: three Duncan plants. CTV: 1) Gene in p33 vector: 26 Mexican limes, 16 C. macrophylla, and five Hamlin plants. CCTF also produced and delivered eight more Mexican lime plants for the pHK vector order. Change in genotype of these plants is not involved in response to plant pathogens. There are about twenty soil-adapted plants that will be tested by the PCR to confirm the presence of gene of interest in their tissue. Publication supported by this grant: Orbovic, V., M. Dutt and J.W. Grosser. 2010. Seasonal effects of seed age on regeneration potential and transformation success rate in three citrus cultivars. Scientia Horticulturae 127: 262-266
1- The growth room construction started on October 22nd, 2010, projected finish date is February 11th, 2011. The construction is already one week behind according to the schedule. They are approximately half way done with the wall insulation, the ceiling insulation has not yet begun. We set up a meeting to discuss disposal of the waste stream for the grow room. The director of UF/IFAS Pesticide Information Office, the coordinator of the UF facilities planning and operations and a representative of the EPA were involved in the discussions. A final list of pesticides and chemicals to be used in the grow room was finalized in order to comply with all environmental regulations. 2 – All in vitro clean shoot tips (Hamlin 1-4-1, Valencia 1-14-19 and Pineapple F-60-3) to establish the mother plants were released from Dr. Peggy Sieburth lab, from the Department of Agriculture, Winter Haven. They are still in test tube conditions. The shoot tips are already 3-month old and they are ready for grafting onto rootstocks grown in pots. Since the growth room is not ready, we have transferred them to fresh medium to keep them alive until our growth room is completed. A second transfer of the shoot tips to fresh media is scheduled for January. As mentioned in earlier reports, this material is needed to be grafted on rootstocks in pots at approximately 2 months of growth. Another factor we are worried about is the current rootstock growth conditions. In the lab where they are developing, the lack of appropriate light, temperature, and space to grow them is jeopardizing 6 months worth of work. They are growing but with extreme difficulty. The initial planting material was discarded since it was getting too old and new batches are already growing but until we don’t have a better place where growing them on clean conditions we will continue to struggle. Even if we can occupy the new growth room the last week of January, we are still going to be concerned about achieving our desirable results. 3 – The growth room technician was finally hired and will start working the first week of January. He will go to Spain for training next Spring. 4 – The lab is 80% set up and, after removal of all plants to the growth room, we will clean and finish setting up the lab for full in vitro culture purposes. Supplies and equipment for the growth room will be purchased once it is completed.
Two full genome sequences have been assembled and annotated. The first is the haploid Clementine selected by the ICGC partners (US, Brazil, Spain, France, and Italy) for sequencing by JGI (US), Genoscope (FR), and IGA (IT). Sanger technology was used to produce the highest quality assembly to serve as THE reference genome for all subsequent citrus genomics efforts. This genome will soon be released through the Phytozome portal at JGI, as well as Tree Fruit GDR, and will be presented at the coming International Plant and Animal Genome (PAG) Conference in January 2011. The current version, Citrus clementina 0.90, is based on ~6.4x coverage, and is a very preliminary product released to enable citrus research community access. Additional work in 2011 will vastly improve the assembly through inclusion of BAC end sequences and integration with a high-density genetic linkage map, to yield a chromosome-based assembly with improved annotations. Fifteen BAC clones are being sequenced and assembled to compare with the assembled genome for validation of the assembly. The second citrus genome is from sweet orange, through collaboration between UF, Roche/454, JGI, and the Georgia Institute of Technology using the 454 platform. This genome sequence is based on ~30x depth of sequence coverage and was assembled using Newbler software; it covers 319 Mb spread over 12,574 scaffolds. Half of the genome is accounted for by 236 scaffolds 251 kb or longer. The current gene set (orange1.1) integrates 3.8 million new ESTs (produced this year) with homology and ab initio-based gene predictions; 25,376 protein-coding loci have been predicted generating a total of 46,147 transcripts. The sweet orange genome also will be presented at PAG in January 2011, and can be accessed through the portals indicated above. The 3.8 million sweet orange ESTs came from 17 different libraries that were produced and sequenced using the 454 platform. They represent various biotic/abiotic challenges including psyllid feeding on young seedlings, canker inoculation, and treatment with salicylic acid, among others. Substantial progress has also been made on the other objectives of this project. Studies comparing the time courses of gene expression in two sets of HLB-inoculated sweet orange and rough lemon plants, representing more susceptible and more tolerant types respectively, have been completed using Affymetrix and Agilent citrus chips (the latter developed by us at UF); some differentially expressed genes have been confirmed by RT-PCR. In addition, comparisons of carbohydrate metabolism and anatomical changes associated with gene expression differences in these same plants have been completed; manuscripts are in submission. Our collaborators at UCR have updated the HarvEST-Citrus database, including sequences from Brazil and Spain, to provide an improved database for gene expression studies containing more than 465,000 publicly available ESTs.
Our research project is directed towards controlling psyllids using biologically-based control strategies that employ the use of RNAi technology against key biological control pathways, peptide hormones and protein inhibitors that, if expressed in transgenic citrus, would enhance plant resistance to psyllid feeding. During the first year of the grant’s period peptides, proteins and RNAi moieties were tested by feeding them to psyllids using artificial diets. The diet was optimized by adding an antimicrobial agent to eliminate fungal growth that is introduced by the psyllids during the assay period and we identified suitable buffers and optimal pH. Tryspin Modulating Oostatic factor (TMOF), a mosquito decapeptide hormone, and cysteine protease inhibitor (CPI) from Diaprepes abbreviatus, the citrus root weevil, were found to be excellent candidates; causing high mortalities when fed to psyllids by artifical diet. Ten psyllids genes representing three gene families of cathepsins (five genes), vacuolar ATPases (four genes), and tubulin (one gene) were targeted and their dsRNA (16 ng/’L) fed to psyllids using artificial diets. Three vacuolar ATPases and three cathepsins (B, L and F) showed significantly higher mortality than the controls. In the first quarter of the second year period our studies continued to characterize the cause of increased psyllid mortality induced by feeding of Double-stranded RNA (dsRNA) molecules targeting specific psyllid genes. Large scale experiments were conducted to harvest sufficient RNA for Northern blot characterization of the integrity and abundance of specific psyllid mRNAs that were targeted and showed enhanced insect morality. The Northern blot analyses although cumbersome and time consuming, are essential for complementing Q-RT-PCR based analyses of targeted transcript abundance. To further support and enhance our RNAi research observations using artificial feeding chambers, we developed a detached leaf assay that supports adult and nymph psyllid survival and allows dsRNA uptake into intact citrus leaves on which the psyllid are naturally feeding. Initial results suggest that transcript specific mortality induced by feeding dsRNA to psyllids in artificial diets can be reproduced using the detached citrus leaf assay. The assay was developed to show that low doses of dsRNA circulating within the phloem can shut down key biological genes in psyllids when ingested, and thus support the possibility that RNAi strategies can be developed to control psyllid feeding on citrus and, therefore, control the spread of HLB. As part of this research a dsRNA virus was also discovered in psyllids and was characterized. This virus is present in natural psyllid populations within Florida, but accumulates to higher levels when the psyllids are maintained in greenhouse colonies. Because it is possible that dsRNA viruses can suppress the RNAi machinery of an insect, we are currently developing dsRNA of virus free psyllid colonies to support future RNAi research in psyllids.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. These include Polyamines such as putrecine, spermine and spermidine; and Antioxidants such as lipoic acid, glycine betaine and glutathion. Lipoic acid continues to yield the best results. A carrot suspension culture overlay procedure is also being evaluated. Experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency are also underway, including: kanamycin, hygromycin, mannose and phosphinothricin. ‘New publication from work on alternative transformation systems: Dutt, M. and J.W. Grosser. 2010. An embryogenic suspension cell culture system for Agrobacterium mediated transformation of citrus. Plant Cell Reports. 29(11): 1251-1260. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ A field trial was established in collaboration with Mr. Orie Lee to evaluate sweet orange seedlings from six selected somaclones of precocious ‘Vernia’ sweet orange under commercial conditions. Juvenile Vernia trees are less thorny than other commercial sweet oranges, and our plan is to girdle the trees to induce early flowering and fruiting once the trees reach adequate size. The goal is to quickly establish a producing grove from juvenile budwood – as necessary to have a system for comparable transgenics. Significant progress was also made to identify rootstocks to enhance early production from juvenile scions, including subsequent transgenics. The 2.5 year old field trial using a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks is showing significant rootstock affects on precocious bearing – the best selections from this trial will be tested with juvenile transgenics, based on yield and fruit quality data to be taken in February. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious ‘Vernia’ sweet orange (including somaclones) were regenerated and successfully micrografted for further study of early flowering and transgene expression. 31 transgenic ‘Vernia’ trees were produced containing four different gene constructs. Progress was also made in the regeneration and characterization of plants containing the FDT transgenes for early flowering.
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