The site has been prepared and the contract has been signed to initiate construction.
The Core Citrus Transformation Facility (CCTF) continues to serve the community of researchers exploring ways to improve Citrus plants and make them tolerant/resistant to diseases. CCTF does its service by producing transgenic material. Within the last quarter, the CCTF facility worked on producing transgenic plants of the following combinations: produced the following transgenic citrus plants (transgene in parenthesis): Mexican lime (pHK vector); Duncan grapefruit (ELP3 gene); Duncan (MKK7 gene); Duncan plants (p7 gene); Duncan (p10 gene); Mexican lime and Hamlin (p33 gene); Duncan (SUC-CitNPR1 gene); Duncan (pWG19-5 vector); Duncan plants (pWG20-7 vector); Duncan (pWG21-1 vector); Duncan (pWG22-1 vector); Duncan (pWG24-13 vector); and Duncan (pWG25-13 vector). All of these plants are for researchers funded by CRDF in the battle against HLB and canker.
McTeer trial – (3.5-year old SugarBelle trees on 15 rootstocks, nearly 100% HLB infected as of September (2011)- remediation program initiated in January by application of southern pine biochar and Harrell’s UF mix slow release fertilizer): Slow release fertilizer was applied to all trees in January. Unlike sweet oranges, HLB-impacted SugarBelle fruit does not drop from the tree, and the impacted crop is still hanging on the trees, months after the typical harvest date. Rootstock effects on longer-term tree health are being monitored, trees on Orange #19 still look the best at present. St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 5.0 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). CREC scouts assessed HLB again in March. HLB is spreading much more quickly in the zone with the traditional tree spacing (15×25); whereas it was very low in the highest density spacing (9×20). Big differences continued in HLB infection rates per rootstock, with the control commercial rootstocks continuing to show the highest infection rates, with most >80% infected. Sour orange-like hybrids (now 3.5 years old) are growing off very well with lower than expected HLB infection frequencies (mostly Pummelo x Shekwasha mandarin hybrids). Harrell’s UF mix was applied to all trees in January, and TigerSul was purchased for application in the next quarter (to avoid minor zinc deficiency observed last summer). Yield and fruit quality data collection was completed in March; statistical analysis underway. Greenhouse Experiments – Rootstock liners (test rootstocks and controls) were moved into the HLB-greenhouse and grafting with HLB-infected Valencia budsticks was completed for the rootstock comparison experiment (trees growing on slow release fertilizer). Liners of rootstock Orange #15 for the individual nutrient experiment were moved to the HLB-greenhouse – grafting will begin this quarter. Protection of seed source trees: The release of new and improved rootstocks to the Florida Industry will require a large and stable source of viable nucellar seeds for our nurseries. Since seed source trees will be growing in the HLB environment, such trees should be protected from HLB. 1. Agrobacterium mediated transformation to produce transgenic tetraploid Orange 16 plants containing the Snowdrop Lectin insecticidal gene (GNA) have been conducted. In addition, we have produced several Orange 16 plants containing the GNA gene (snowdrop lectin) fused with a Tobacco PR1b signal peptide for improved extracellular secretion of the GNA protein by plant cells and the GNA gene fused with a HDEL C-terminal extension for retention of the GNA protein in the endoplasmic reticulum. 2. Transgenic Carrizo containing a construct containing the Snowdrop Lectin insecticidal gene stacked with the antimicrobial gene CEMA (AMP) have been produced and are being propagated for analysis.
Progress with the rapid flowering system (pvc pipe scaffolding system) in the greenhouse: Several of the transgenic plants have reached the top of the scaffold, and the apical stems have been trained to grow down (expected to encourage early flowering). The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Another rootstock with strong potential to influence juvenility was identified (Nova+HBP x sour orange + Flying Dragon). Seeds have been planted. Experiments to efficiently stack promising transgenes are underway. Experiments to efficiently stack promising transgenes are underway. Transgenic sweet orange plants containing a construct with CEME gene (AMP) stacked with the NPR1 (SAR inducer) gene have been evaluated. We have recovered 10 transgenic lines that contain both genes incorporated into the genome. We have also transformed our newly released sweet orange somaclone OLL#8 with this construct. Also, constructs containing the AttacinE gene stacked with the NPR1 gene and the CEMA gene stacked with the NPR1 gene have been produced, and transformation of OLL#8 and Valencia Sweet Orange is currently underway. Correlation of transgene expression with disease resistance response: Western blot analysis for plants containing LIMA and GNA are nearly completed, data is showing a strong correlation between transgene expression and desired phenotype. This supports the dogma that fairly large populations of transgenic plants are necessary (for each transgene/cultivar) to obtain adequate transgene expression while maintaining cultivar integrity. Improved transformation methodology (for seedless or recalcitrant cultivars, and eventually marker-free or ‘all plant’ consumer-friendly transformation): 1. In efforts to reduce transgene mediated metabolic load on the plant, we have transformed Hamlin suspension cultures with constructs containing our reporter gene (grape anthocyanin gene) driven by either an embryo-specific Carrot DC3 promoter or an embryo-specific Arabidopsis At2S2 promoter. It is expected that plants obtained from these constructs will not produce the reporter protein once a transgenic plant has been selected. Currently putatively transgenic embryos have germinated and are being grown to size for analysis. 2. The binary vector for an inducible cre-lox based marker free selection is under construction. We anticipate transformation experiments with this vector in the following quarter. Targeted transgene expression: ‘ additional transgenic plants of Duncan, Carrizo, Pineapple, Hamlin, and Valencia (produced with Agrobacterium-mediated transformation) containing the LIMA gene (AMP) controlled by AtSUS2 promoter (phloem specific) have been propagated by micrografting. Plant characterization and molecular analysis on these plants will begin the next quarter. In greenhouse evaluation (Southern Gardens w/ Mike Irey) of transgenic plants exposed to HLB positive psyllids, we observed several transgenic LIMA and NPR1 lines driven by a phloem specific AtSUC2 phloem-limited promoter to be HLB tolerant. Most of these lines were negative to qPCR after 2 years of evaluation and did not demonstrate visible disease symptoms.
Several anti-NodT scFv single-chain antibodies are now in hand. The anti-NodT antibody with the highest affinity for the target 30 amino-acid peptide has been selected and has been expressed successfully in E. coli. The plant gene expression construct consisting of the 35S Cauliflower Mosaic Virus promoter driving expression of a fusion of the flowering locus T protein with best-performing anti-NodT scFv protein has been produced. We are now in the process of transferring this 35S::FLT-scFv expression cassette into the pTLAB citrus transformation construct. We have been working on this construct for several months during January – April 2013, but still have not completed this step, which has proven to be unexpectedly difficult. We anticipate that we will be able to complete this step during the next quarter, using some alternate techniques. Once the construct is completed, production of transgenic plants will begin.
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 75 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We are attempting to develop methods to be able to screen genes faster. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids. Preliminary results suggest that the RNAi approach against psyllids will work. We also continue to screen transgenic plants for other labs.
This is a joint project between CREC and USDA, Fort Pierce. The objective of this project is to find poncirus hybrids that exist now that are sufficiently tolerant and of sufficient horticultural and juice quality to be used now for new planting in the presence of high levels of Huanglongbing (HLB) inoculum. We believe there is a good chance that there mature budwood exists with these properties that could be available immediately for new plantings. Although these trees are not likely to be equal in juice and horticultural qualities of the susceptible varieties of sweet oranges grown in Florida, with their tolerance to HLB they could be an acceptable crutch until better trees are developed. We surveyed the trees at the Whitney field station and found 5 lines that we thought could be acceptable for juice. Those have been propagated and are being screened for tolerance and horticultural properties. The hybrid plants are being incubated in the psyllid containment room to allow multiple psyllids to inoculate the plants with HLB. So far the chosen hybrids appear to be tolerant.
Function of individual X. citri transcription activator like effectors (TALEs): The activity and specificity of specific X. citri TALE proteins PthA1-4 have been tested using two different approaches. In one approach, activity was tested transiently using a reporter assay in Duncan grapefruit leaves. In these assays, plants were co-inoculated with a reporter construct consisting of a 14 TALE binding element (EBE) version of the Bs3 promoter driving the GUSi reporter gene together with Agrobacterium containing individual pthA genes or combinations of genes. Our results from these studies show that on its own Xc PthA4 is the most effective activator of gene expression, however co-inoculation with other individual proteins increases expression. We also used a second approach in which stable transgenic Nicotiana benthiamiana plants containing a 4 EBE promoter:GUS construct were inoculated with individual citrus TALEs introduced via X. campestris pv. campestris. We then quantified activity using GUS leaf disc staining and fluorescence MUG assays. In both assays, we could observe that individual TALEs did trigger expression of the GUS gene, so long as the corresponding EBE was present. This system also permitted us to examine the activity of pthA genes from a range of strains, including the sequenced Brazilian A 306 strain, A44 from Argentina, a typical A strain from Miami, an unusual strain isolated from Etrog in Florida,and a C strain designated #93 Brazil. TALEs from these strains triggered expression of the construct when matching EBEs were present. These data show that the promoter constructs are functioning as designed, with specificity for individual TALEs from a wide range of strains. We also now have a number of assays to evaluate TALE-promoter interactions to evaluate the roles of TALEs individually and in combination. Transformation and production of stable citrus lines: We have experienced difficulty in recovering functional stable transgenic citrus with from our transformation efforts. We have obtained many transformants, but to date we have not identified a line with a functional, intact transgene construct. In response, we are pursuing several alternative approaches to obtain stable transgenics, including further examination and testing of original constructs, preparation of new constructs in alternative vectors, additional controls, optimization of the transformation protocol, and new methods of transformation. We continue to regularly set up transformation experiments and analyze transformants by PCR, sequencing and pathogen testing. Given the successful function of constructs in transient reporter and disease resistance assays and the success of stable transformation with other constructs, we expect to recover functioning stable transformants through continued optimization of the transformation process.
The goals of our integrated core program are to develop and evaluate new citrus scion and rootstock cultivars suitable for California conditions. Research on citrus scion breeding continued on schedule. An important development is addition of Dr. Soon Park to the breeding team as the lead scion breeder, replacing Tim Williams who has retired but remains active as a volunteer. To obtain low-seeded cultivars we irradiated budwood of Robinson, Lee, and Sidi Aissa mandarins and Cocktail grapefruit, and then propagated trees at Lindcove and UCR. New pollinations during spring 2013 are still in progress and more than 900 flowers have been pollinated. Evaluation of existing hybrids identified 22 promising selections; of these, three grapefruit hybrids were submitted to CCPP for cleanup prior to multi-location evaluation. Selections evaluated in multi-location trials included low-seeded selections of Lisbon lemon, Encore, Nova, Clementine Oroval , Kinnow, Fremont and several hybrids. No new varieties were released. For the low-seeded lemon selection, among eight locations mean seed counts ranged from 0.4 to 2.9 seeds per fruit. Although most fruit had 0-2 seeds, some fruit had 8-10 at several locations. Additional data is needed to determine whether to release this selection. Trees at Lindcove will be screened to evaluate productivity and seed content when not cross-pollinated. Rootstock breeding activities included propagation of over 800 new hybrids to be field-planted in 2013. Additional crosses made in 2012 produced about 1000 seeds. Seedlings from 32 varieties were grown for a Phytophthora tolerance trial. New rootstock trials for Clementine and DaisySL were planted at Lindcove and for DaisySL at CVARS (Thermal). We completed evaluation of field trials of Fukumoto navel at Lindcove and Tango mandarin at Porterville. Packline data was collected on the Porterville Tango and Lindcove Fukumoto trials but has not yet been analyzed. Seeds of rootstock selections were collected to propagate trees for new rootstock trials for lemons and possibly one for navels. We completed two harvests (Oct. 16-17 and Dec. 16- 17, 2012) of the replicated trial initiated in 2006 to evaluate 12 lemon selections for the California desert. Yield, fruit packout and exterior fruit quality data was collected at each harvest and fruit of each selection from the first sample date were also evaluated for interior fruit quality measurements. Evaluations of fruit from 48 cultivars introduced from outside California and corresponding commercial standards were completed including 23 Satsuma mandarins, 8 Clementine and other early season mandarins, 2 grapefruit hybrids, 9 navel oranges, 4 mid- and late season mandarins and 2 commercial type lemons. Results of our evaluations of 19 new Satsuma introductions in comparison with two commercial standards will be published in the March/April 2013 issue of Citrograph. Fruit of scion cultivars under development and new introductions were available for growers to see and taste at the Lindcove Research and Extension Center field day (Dec. 13, 2012), World Ag Expo (Feb. 11-14, 2013), UCR Citrus Day (Jan. 31 2013) and the California Citrus Mutual Citrus Showcase (March 7, 2013).
We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project will significantly expand the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in regulating citrus defense. We have three specific objectives in this proposal: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. Currently we have cloned 8 full-length genes in these categories in the entry vector pJET and two of the genes were further cloned to the binary vector pBIN19plusARS. Transformation of Arabidopsis and citrus plants will be simultaneously performed to obtained transgenic plants over-expressing the constructs for further analysis of plant disease resistance. In addition, we are continuing to generate and/or characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project, although the support of this previous project has already been terminated. A manuscript describing the cloning and characterization of the citrus NDR1 ortholog was recently under revision in the journal Frontiers in Plant Science.
Fruit quality, yield, tree health, and tree size data were collected from eight late-season rootstock field trials. All trials appear significantly affected by HLB. Some selections, including US-942, appear to perform better than other standard rootstocks in locations being affected by HLB. Rootstocks that appear to have more tolerance to HLB were identified, and selected rootstocks propagated for increase and inclusion into more and larger scale field trials. Fruit quality data was collected at multiple harvest dates for a large grapefruit rootstock trial in Indian River County. Significant differences were observed among the rootstocks for many fruit quality traits. For example, sour orange induced an intermediate level of total soluble solids early season and relatively high solids late season. US-897 induced the highest soluble solids early season, and US-812 induced the highest soluble solids late season, while X-639 and US-852 rootstocks induced the lowest soluble solids throughout the season. A field trial with new supersour rootstocks was planted at a site in Indian River County and will be used to evaluate trees for tolerance of Diaprepes, Phytophthora, and HLB. Rootstock liners were budded in preparation for planting in three new rootstock field trials later this year. Several thousand propagations of supersour rootstocks at USHRL were prepared for budding and use in field trials. Cooperative work continued with a commercial nursery to multiply promising supersour rootstocks to prepare trees for medium-scale commercial plantings. Work continues to assess supersour tolerance of CTV, salinity, and calcareous soils. The most promising new USDA rootstocks were identified for a cooperative effort with CRDF to place new rootstocks with HLB tolerance into larger scale commercial plantings. Detailed evaluation of specific defense-related citrus genes continued, including genes identified by expression studies as being associated with HLB response, such as CtCDR1 and CtPDF2. Constructs designed to alter expression of these citrus defense genes are being used to transform citrus for improvement of HLB tolerance and resistance, and derived transgenics will be tested using the pathogen. In a collaborative study with a University of California team and funded by CRB, we are comparing gene expression for trees infected with HLB to those infected with CTV. There are some common elements to the two different diseases that present good opportunities to understand citrus defense response. A study to understand the interaction of rootstock tolerance with scion tolerance/susceptibility is being completed and will be published later this year. Work continues to examine the effect of grafting height on tree response when the rootstock is an HLB-tolerant type. More than 200 new transgenic rootstock selections with potential resistance to HLB were produced this quarter, targeting increased expression of the citrus resistance genes CtNPR1, CtEDS1, CtMOD1, CtEDS5, CtPAD4, CtNDR1, and CtACD1. Eighteen new transgenic rootstocks with selected antimicrobial genes were propagated and entered into a replicated greenhouse test with ACP inoculation to assess tolerance to HLB. Evaluation and indexing of three previous groups of transgenic rootstocks under test with HLB continued, with selected transgenics showing some promise. A paper was published describing the overexpression of a citrus NDR1 ortholog and increased disease resistance. Presentations were made at Florida Citrus Show, Ft. Pierce and Florida Citrus Growers Institute, Avon Park, with exciting new information regarding rootstock effect on tree performance with HLB.
In the past few months, the most significant progress made on the FT project was the completion of the new FMVcDNA27 construct, which contains an FT3 cDNA insert in the pCAMBIA2201 vector with a constitutive FMV promoter. This construct was created as a first step towards the development of a new FT3 construct with an inducible promoter. In order to ensure that the cDNA was as effective as the genomic, this FMVcDNA27 construct will be compared to the original p27 construct which contains a genomic FT3 insert in the pCAMBIA2201 vector with the FMV promoter. Transformation of Carrizo and tobacco tissue is already underway in order to compare the action of these two constructs. Additionally, we have arranged for the materials transfer of two inducible promoter systems from the Danforth Foundation. Both of these promoters are inducible by the chemical methoxyfenozide, a widely-available pesticide, approved for field use. One system is driven by the CsMV constitutive promoter, and the other by the RTBV vascular-specific promoter. Once we have verified that the smaller and more manageable cDNA is as effective as the original genomic version of the FT3 gene, we will begin development of the inducible promoter constructs.
USDA-ARS-USHRL, Fort Pierce Florida has thus-far produced over 3,000 scion or rootstock plants transformed to express peptides that might mitigate HLB, and many additional plants are being produced. 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 two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants 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. This report marks the end of the third quarter of the project, during which we have began large-scale production of CLas positive ACP. To date on this project, a technician dedicated to the project has been hired, a second career technician has been assigned part-time, two small air-conditioned greenhouses for rearing psyllids are completed and are functioning well, and 18 individually caged CLas-infected plants are being used to rear ACP for infestations. A total of 2,124 transgenic plants have passed through the screening program. A total of 43,680 psyllids have been used in no-choice inoculations. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Maintaining an open infestation of infected psyllids (phase 2 of the inoculation process) was challenging this past quarter because of surprise pest problems, notably western flower thrips which invaded the greenhouse initially attacking new flush and then first instar psyllids (facultative predation). Tamarixia radiata invaded the house during January, killing a majority of psyllid nymphs. Measures have been taken to limit these unwanted pests, but these measures are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects for the control of spider mites and thrips.
The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, has spread to citrus growing regions nearly worldwide and adults transmit phloem-limited bacteria (Candidatus Liberibacter spp.) that are putatively responsible for citrus greening disease (huanglongbing). Host plant resistance ultimately may provide the most effective, economical, environmentally safe, and sustainable method of control. In earlier experiments we identified genotypes of Poncirus trifoliata and xCitroncirus sp. (hybrids of P. trifoliata and another parent species) that were resistant to ACP. One mechanism we investigated to see whether it contributed to this resistance was plant hormones. We sprayed salicylic acid, methyl jasmonate, and abscisic acid, which are all common plant hormones, on susceptible citrus plants to test the influence on host choice, oviposition, development, and survival of ACP. Abscisic acid cut the life span of adult ACP in half compared to untreated control plants. The plant hormones had no strong effects on host choice, oviposition, or development of ACP. We are preparing the data for publication in a peer-reviewed journal. Another mechanism that may confer resistance to ACP is the structure of the scelerenchyma (a fibrous ring) surrounding the phloem. The scelerenchyma in non-citrus plant species was found to prevent feeding by herbivorous insects. We are collaborating with a Research Entomologist at our facility to test whether ACP choose different feeding positions on leaves of resistant P. trifoliate versus suceptible xCitroncirus sp. We completed two replications and found differences in feeding position. Our collaborator is currently evaluating the position of salivary sheaths from ACP and the scelerenchyma in leaves using scanning electron microscopy. Collaborators at the Fujian Academy of Agricultural Sciences conducted free-choice tests with all major groups of citrus and found differences among and within groups. Most groups of citrus were colonized by ACP, but lemons were the most preferred group and sour oranges and kumquats were the least preferred. The differences among citrus varieties within a group may be useful because volatile and phloem contents that differ between the least and most preferred species can be compared. FAAS continues their screening of germplasm including CRC accessions. To date, there is obvious resistance to ACP in Poncirus and among some distant relatives of Citrus. There is also at least a little ACP resistance within most of the Citrus groups but, in general, there is no evidence that any Citrus germplasm would offer as much genetic resistance to ACP as Poncirus. Among trifoliate hybrids, there is some material that appears as resistant as Poncirus itself, indicating that traits of interest (greatly reduced oviposition, reduced longevity) were genetically inherited.
Construction of primary structure is completed except for fabric. Fabric has been delivered.
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