New harvest data was collected from field trials for the 2013-14 season and confirmed outstanding performance of several new USDA rootstocks in comparison with standard rootstocks under heavy HLB inoculation pressure. The studies included measures of fruit yield, fruit quality, tree survival, and tree size. The rootstocks US-896, US-1279, US-1281, US-1282, US-1283, US-1284, US-1318, US-1319, and US-1321 were outstanding, as compared to the common commercial rootstocks. The new USDA rootstocks will be submitted for commercial release in 2014. These promising selections have been provided to Florida DPI to establish clean budwood sources and have been propagated by USDA to multiply material for further testing. These selections are also being made available for the CRDF Product Development Project to establish large scale commercial field trials. Cooperative arrangements are being made with commercial nurseries for large scale vegetative propagation of the most promising rootstocks, as needed to meet commercial demand. Three new rootstock field trials were planted in spring 2014. About 800 Valencia trees on Supersour rootstocks were prepared for planting field trials in St. Lucie County in summer 2014. About 120 new Supersour rootstock hybrids were created in 2013-14 and established in the greenhouse nursery at Ft. Pierce. Ten thousand propagations of Supersour rootstock selections were completed to prepare trees for controlled testing and additional field trials in 2015. Cooperative work continued with a commercial nursery to multiply promising Supersour rootstocks to produce trees for medium-scale commercial plantings. Greenhouse studies continue to assess Supersour tolerance of CTV, salinity, and calcareous soils. Data was collected from a greenhouse test of Supersour rootstocks to measure quick decline reaction in response to CTV infection. Studies were conducted on defense-related citrus genes associate with HLB infection. Data was collected on localized expression in shoots and roots of trees with different scion/rootstock combinations. Genes studied include those identified by our expression studies as being associated with HLB response, such as RDR1, RAP4, CSD1, and CtCDR1, and also genes identified in collaborative work with a University of Maryland team. Genes that appear particularly promising are used for transformation of citrus to manipulate expression of susceptibility and tolerance. A study of the interaction between rootstock tolerance and scion tolerance/susceptibility has been completed and will be published in 2014. A study on the effect of grafting height in HLB tolerance was initiated in the greenhouse with a similar study being prepared for field planting in 2014. New transgenic citrus rootstock selections are produced at about 50 new transgenics per month in our program. The citrus resistance genes CtSID2, CtSFD1, CtPAD3, and CtCDR1 were primary focus for transformation in this quarter. Following promising preliminary testing, propagations of fifteen new transgenic rootstocks with overexpression of citrus gene CtNDR1 were propagated and entered into a replicated greenhouse test with HLB. Monitoring and data collection continued on previous groups of transgenic plants that have been inoculated with HLB. Several transgenic rootstock selections with AMP gene expression have been identified that result in reduced infection, reduced disease population, more vigorous growth, or reduced HLB symptoms in the transgenic plants or grafted sweet orange scions.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports 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. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. As of April 9, 2014, a total of 5,314 transgenic plants have passed through inoculation process. A total of 106,250 bacteriliferous 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. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. As an offshoot of the research, damage by western flower thrips was so severe that research was conducted to validate damage by this pest to developing flush and facultative predation on ACP, which led to the following publication: Hall, D. G. 2014. Interference by western flower thrips in rearing Asian citrus psyllid: damage to host plants and facultative predation. Crop Protection. 60: 66-69. A thrips predator, Orius insidiosus, proved to feed aggressively on immature ACP, thus would be incompatible for thrips control in an ACP rearing operation.
This project began with funding from the FCPRAC to support participation by the PI in the International Citrus Genome Consortium (ICGC), to sequence a citrus genome for the benefit of the global citrus research community, primarily to enable broad research targeting HLB and other critical citrus disease threats. CRDF continued to support the project until recently. The ICGC selected a haploid derived from Clementine mandarin, to simplify sequence assembly, gene model prediction, and annotation. The first genome was sequenced using Sanger technology, the platinum standard for quality data, in collaboration with the US DOE Joint Genome Institute (JGI) and its affiliate HudsonAlpha (USA), Genoscope (France), and Istituto Genomica Applicata (IGA; Italy). Support from the citrus community came from researchers in Florida, Brazil, France, Spain, and Italy. As sequencing technology rapidly evolved, we were able to leverage the project to use the first pyrosequencing technology from Roche/454, and a second genome was added to the project, that of Ridge Pineapple sweet orange. Roche/454 contributed substantially to this project, as did UF-ICBR and JGI. Both genomes were made freely available to the public in January of 2011, at JGI’s genomic web portal, Phytozome, and also in the Citrus Genome Database of Tree Fruit GDR (tfGDR), an SCRI-supported project. Since then, scores of papers have been published globally on various aspects of citrus biology and genetics, including many manuscripts focused on HLB research, which would not have been possible without the valuable genomic resources from this project. To illustrate the significant impact of these genomes, since January 1, 2013 alone there have been over 31,000 unique pageviews, and nearly 1600 downloads of the datasets at Phytozome. And in the 3 years since posting, the Citrus Genome Database of tfGDR was visited over 15,000 times by nearly 9000 unique users from 135 countries. Since then, as technology costs decreased even further, the ICGC added 6 more genomes; comparative analysis revealed the underlying genomic structure of sweet orange, pointing the way to new strategies to reconstruct orange with genetic contributions from HLB resistant citrus or relatives by sexual hybridization without the use of GMO strategies, or to potentially modify existing cultivars by engineering with constructs made entirely from citrus DNA. Though CRDF support for the work has terminated, the ICGC now has sequenced more than 30 citrus genomes, and work is underway to analyze these sequences and to provide a very rich resource for disease resistance gene identification and manipulation into the future. The Florida citrus growers who were the very first group in the world to fund citrus genome sequencing are to be commended for their bold foresight and financial contributions; they enabled development of universally valuable research tools for the global citrus community. In addition to developing a high quality reference citrus genome, the project proposed to use the genomic resources for research aimed at understanding further the interaction of various host citrus plants with the presumed causal agent of HLB, Candidatus Liberibacter asiaticus, to identify potential genetic targets for modification, and to identify early signals of infection that might be used to develop early detection methods. No fewer than 12 manuscripts have been published by the PI in collaboration with other labs, all of which have relied on the reference genome sequences. These works have explored and compared metabolic, transcriptomic, and proteomic responses of HLB-tolerant and sensitive plants, providing new insights to the mechanisms of disease and tolerance. New CRDF-funded projects underway to map and potentially identify specific genetic targets for intercession, to develop consumer-friendly HLB resistant cultivars, are feasible because of the genome resources developed. The culmination of this project is a manuscript accepted for publication this year in Nature Biotechnology, which compares the genomes of the fundamental citrus species, and elaborates their evolutionary and taxonomic relationships. Such understanding will support and enable more rapid and targeted improvement of commercial citrus cultivars as new threats to the industry appear.
The productivity of the Core Citrus Transformation Facility (CCTF) in the time between January and April of 2014 was higher than it was in the previous quarter. We have continued to produce transgenic plants for different research groups from the state of Florida and beyond. The work done within this quarter concentrated mostly on old orders. In communication with one research group, which is the biggest client at this time, the decision was made to prioritize the work on some of the orders they previously placed so CCTF had to shift the efforts towards specific orders and disregard time when the orders were placed. Also, through the communication with the CREC Director, CCTF received direct order from CRDF to produce some rootstock plants transformed with the NPR1 gene. These efforts are being coordinated with the Mature Tissue Transformation Lab (MTTL). Considering the importance of this project, CCTF started working on it immediately. Initial co-incubation experiments were done just a few days upon receipt of this order with the plant material obtained from MTTL. However, the quality of seedlings obtained from the MTTL was not good and results from that series of experiments had to be discarded. Rootstock cultivars of Carrizo, Swingle, and Citrus macrophylla are being used in this project. C. macrophylla seeds used for production of seedlings, that are the source of explants for co-incubation experiments, seem to be carrying some endophyte as most of the material obtained from these seeds ended up being contaminated. Because of this observation, the decision was made to put the emphasis on the Carrizo and Swingle cultivars. The work on this order is continuing at high pace. Plants produced by the CCTF within this three months period belong to the following orders: pNah-10 plants, pN9-seven plants, pN18-six plants, pX11- eight plants, pW14- three plants, pX20- one plant, pX28- one plant, pN7- one plant, pHGJ4- one plant, pMed16- one plant, pMed14- two plants, pELP3-G-nine plants, pELP4-G- three plants, pTMN1-five plants, and pMG105- two plants. Altogether 60 plants were produced and they were all Duncan grapefruit.
In this proposal our objective is to find citrus versions for the two proteins that make up the functional components of a chimeric antimicrobial protein (CAP) previously described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We have successfully identified a suitable replacement for the first component, the human neutrophil elastase (HNE). We obtained a close match with the tomato PR14a protein that is highly conserved in both Citrus sinensis (Cs) and Citrus clementina (Cc) genomes. We have completed the construction of two synthetic genes that encode this protein, one that just contains the coding region of CsP14a and the other is a chimeric version that contains the CsP14a coding region linked to the CecropinB (CecB) protein. We have also completed the construction constructing two CaMV35S expression cassettes to express both these synthetic genes so that the expressed proteins can be secreted. We have used CLASP to identify a citrus replacement component for CecB. Since CB has no enzymatic activity, we could not use a well-constrained motif like an active site. We chose instead the structural motif Lys10, Lys11, Lys16, and Lys29 a unique feature of CecB. We have used an approach similar to that described earlier to identify a replacement for HNE to identify a replacement component for CB. However, instead of comparing the reactive atoms as was done for the HNE matching algorithm, in this case we match for the C-alpha atoms of specific residues based on the overall shape of CB. Thus, we adopted a slightly different computational flow in this case. First, we did a keyword search, ‘plants’ in http://www.pdb.org/ that yielded about 1000 proteins. Each pdb was expanded on basis of each chain. For example, PDB X.pdb with chains A and B resulted in PDB files XA.pdb and XB.pdb. This list was filtered based on a 80% similarity redundancy. Then proteins larger than 60 aa in length were ignored. APBS (Adaptive Poisson-Boltzmann Solver) software developed to examine electrostatic properties of proteins at a nanoscale) was run on each pdb (the ones that failed electrostatic analyses, a few, were excluded). We were able to extract 200 proteins which we then analyzed further using CB motif. The CB structure is characterized by two helices. We chose specific residues from these two helices such that the residues were polar, and had stereochemical matches. For example, Lys can be replaced by either Arg or His, and thus constitutes a good candidate. We chose, Lys10, Lys11, Lys16 and Lys29 as the input motif from CB, allowing Lys to be matched by Lys, Arg or His. Since the 52 aa H+ATPase (HAT) is large so we cannot have it cheaply synthesized as a protein. We are developing additional computational tools to better evaluate individual alpha helical domains so we can identify smaller proteins that can be more cheaply synthesized for testing. We have also begun making a CAP construct where CB is replaced by HAT. This will be expressed in plants to evaluate antimicrobial properties.
Our accomplishments are: 1) We have transformed juvenile explants of succari sweet orange and carrizo citrange using transformation enhancing genes (K and I genes) we have constructed. The use of the K gene leads to 7-15 fold increases in transformation efficiency while the use of the I gene had 4-9 fold increases in transformation efficiency when compared to a conventional Ti-plasmid vector containing no K or I gene. However, many adventitious shoots produced from the I gene containing Ti-plasmid vector are abnormal. 2) We conducted one transformation experiment using explants from adult trees but the results were not satisfactory due to some unexpected problems. We are preparing more tissues from adult trees grown in greenhouse for transformation using these constructs. 3) We are constructing the other transformation enhancing gene constructs and they should be done soon for testing.
A number of genetic constructs have been received from various scientists, with and without supporting documentation (i.e. plasmid maps, sequence, publications). The genetic constructs for which we have supporting documentation have been put in the queue. The constructs without supporting documentation have been appropriately stored until information is received. As a new step in quality control, we are sequencing some plasmids, particularly those lacking the pVS1 replicon for stability in Agrobacterium. A genetic construct obtained from Dr. Mou has been transformed into mature scions of Hamlin, Valencia, Ray Ruby and mature rootstocks of Carrizo and Swingle. As advised, we have discontinued work with Pineapple sweet orange. Shoots will be pre-screened once they are bigger. Transgenic shoots of Valencia or Hamlin be micro-propagated and budded in different combinations with transgenic or wild-type Carrizo or Swingle immature rootstock. These trees will be submitted for disease screening to determine which have superior disease tolerance and whether transgenic rootstocks confer graft transmissable tolerance. Different cytokinins will be tested in scion and rootstock micro-propagation. Progress in being made towards increasing the productivity of the lab and the growth room. In the lab, mature scion and rootstock stem explants are being cut to 0.6 cm rather than the standard 1.0 cm for tissue culture. These smaller explants survive the Agrobacterium transformation protocol well and regenerate plantlets. This approach increases the explants available for each weekly transformation from ~600 to 1000. In the growth room, rootstocks are being budded with mature scion at an earlier age and the results look promising. Using this approach, we only need to transplant the rootstock on which the mature scion bud has opened. In an additional effort to increase our productivity, small experiments are being conducted to determine whether we can regenerate shoots from calli derived from mature leaf tissue after Agrobacterium treatment. Plantlets have been regenerated from the calli in one variety and are being elongated. These plants should still flower and fruit early because they have undergone the phase transition from immature to mature. We have also been able to root some varieties. NPTII immunostrip tests were conducted in sweet orange and grapefruit trees transformed with marker genes. Out of 47 transgenics tested, 34 tested positive for expression of the NPTII gene. Genomic DNA extraction and Southern blots to show transgene integration are underway.
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 three years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/8th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. In a project led by Richard Lee, an array of seedlings from the Germplasm Repository are in place, with half preinoculated with Liberibacter. Additional plantings are welcome from the research community.
Several new USDA rootstocks had outstanding performance in field trial comparisons with standard rootstocks using Hamlin or Valencia scion. Field performance was examined in trials under heavy HLB inoculation pressure and incorporated measures of fruit yield, fruit quality, tree survival, and tree size over a period of several years. The rootstocks US-896, US-1279, US-1281, US-1282, US-1283, US-1284, US-1318, US-1319, and US-1321 have exhibited outstanding yield and fruit quality under disease pressure from HLB, as compared to the most common commercial rootstocks. The new USDA rootstocks will be submitted for commercial release in 2014. These promising selections have been provided to Florida DPI to establish clean budwood sources and have been propagated by USDA to establish multiple seed source trees and multiply material for further testing. Seed trees of these new rootstock selections have been or will be established at the USDA farms at Ft. Pierce and Leesburg. Cooperative arrangements are being made with commercial nurseries for large scale vegetative propagation of the most promising rootstocks, as needed to meet commercial demand. New supersour rootstock hybrids were created and established in the greenhouse nursery at Ft. Pierce. Budded nursery trees of advanced rootstock selections were grown off in preparation for planting in three new rootstock field trials in spring 2014. Two thousand propagations of supersour rootstocks were budded with Valencia for use in field trials to be planted in summer 2014. Cooperative work continued with a commercial nursery to multiply promising supersour rootstocks to produce trees for medium-scale commercial plantings. Work continues to assess supersour tolerance of CTV, salinity, and calcareous soils. Data was collected from a greenhouse test of supersour rootstocks to measure quick decline reaction in response to CTV infection. Investigations continued on specific defense-related citrus genes, including localized expression in shoots and roots. Genes studied include those identified by our expression studies as being associated with HLB response, such as RDR1, RAP4, CSD1, and CtCDR1, and also genes identified in collaborative work with a University of Maryland team. Genes that appear particularly promising are used for transformation of citrus to manipulate (increase or decrease) expression of particular genes involved in citrus response to HLB. In a collaborative study with a University of California team and funded by CRB, we continued work to compare gene expression for trees infected with HLB to those infected with CTV. A study of the interaction between rootstock tolerance and scion tolerance/susceptibility has been completed and will be published in 2014. A preliminary study to examine the effect of HLB tolerant rootstock grafting height on tree response to HLB was completed in the greenhouse. A second, more thorough study of grafting height was initiated with a similar study being prepared for field planting in 2014. New transgenic citrus rootstock selections are produced at about 50 new transgenics per month in our program. The citrus resistance genes CtNHL1, CtJAR1, CtMOD1, CtACD1, and CtEDS1 were primary focus in this quarter. Following promising preliminary testing, propagations of 25 transgenic rootstocks with overexpression of citrus gene NDR1 were prepared for further testing with HLB. Twelve new transgenic rootstocks with selected antimicrobial genes were propagated and entered into a replicated greenhouse test with HLB. Monitoring and data collection continued on previous groups of transgenic plants that have been inoculated with HLB. Several transgenic rootstock selections have been identified that support vigorous growth and no HLB symptoms in grafted sweet orange scions.
There was no funding received until the end this quarter, so little progress could be made.
This project has been difficult from the beginning and when we were coming up on the third year, all of my Co-PIs decided they did not to continue on this project for various reasons. I did want to continue because my part of the project was going well. It took some time to sort this out but by May 9, 2012 I had e-mailed Dr. Browning for guidance. He indicated that I could submit by myself. I did this on June 19. I know it was received because Ms. Nowicki had me change something in the budget. And the executed contract was sent. Then I heard nothing. On September 28, Dr. Turpen called me about why I had not submitted paperwork. I told him I had and resent the third year paperwork to him. Again I heard nothing until an NOA was sent in the middle of November for $39,000. When someone in DSR asked Ms. Nowicki about a no-cost extension, she said there would not be one, I had until December 31 to spend the money because I had been so late in getting in the paperwork. I was not extremely late in getting in the paperwork! And of course I could not spend all of the funding in six weeks (during the holiday season) without doing things I am not comfortable doing because I have no desire to fail an audit or get in trouble with either CRDF or UF. A 6 month no-cost extension was finally executed. This allowed us to continue preliminary work on cell penetrating peptides, which led to the funding of Project 752. How to write the reports was problematic because of the gaps in funding and although I asked for guidance, I did not receive any.
There was no funding received during this quarter, so progress could not be made.
Oral uptake of dsRNA targeting specific Asian citrus psyllid genes can induce psyllid mortality and reduce Liberibacter titer in infected psyllids. Significant progress has been made with research on the use of RNAi as a means of Asian citrus psyllid control. We have made use of a Citrus tristeza virus (CTV) dsRNA expression system which when inoculated into C.Marcrophylla results in leaf phloem containing dsRNAs which target essential ACP genes. When ACP feed on leaves from these “paratrangenic” (CTV transfected) citrus plants, mortality was double that observed when the ACP fed on artificial diets containing this synthetic dsRNA, greater than 80% mortality. Currently, new versions of paratransgenic citrus are being produced to determine the best sequence to use targeting this essential gene. The use of the Ion Torrent (by Life Technologies) next generation sequencing has provided rich insight into the transcription profile of “paratransgenic” fed “sick” ACP using comparative RNA Seq analysis. The data support the specific down-regulation of the dsRNA target gene as the cause of mortality as seen by the significant perturbation of genes in the molecular pathway in which this gene functions. RNA Seq data continues to be collected from ACP fed on a variety of gene specific dsRNA containing diets. Experimentation has also begun using artificial diets containing dsRNAs synthesized by a novel “mass-production” technique that would make it practical for the use of RNAi technology as a field application. The use of topical application in conjunction with “paratransgenic” plant varieties presents a strategy for effective delivery and a multiple gene targeting employment of the RNAi pest control technology.
St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 5.5 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). Field day was held on December 12th, attendance was approximately 130. New HLB frequency of infection data was presented. Infection frequency of commercial rootstocks has now reached 89%. Overall, diploid rootstocks are now 67% infected, and tetraploid rootstocks are 57% infected. Four trifoliate orange x papeda hybrids from Sicily are still showing lower infections frequencies (31-59%). Additional seed of these selections has been acquired from Dr. Reforgiato-Recupero for additional trials. Most of the tetraploid rootstocks selected for release are still around 50% infected. New HLB severity assessment of all infected trees in the trial is underway. McTeer trial (ACPS trial of SugarBelle on 15 tree-size controlling rootstocks, nearly 100% HLB): every tree was scored for HLB severity. Rootstock White #4 (UFR-5) showed the overall best and most consistent tree health. Fruit quality was still poor in general, but there was a significant increase in harvestable fruit as compared to last year. Greenhouse Experiments – unsuccessful HLB-infected budstick grafts in the nutritional and rootstock studies were all regrafted (about 15% of the total trees). Most of the successfully grafted trees have flushed out, with minimal HLB symptoms in both experiments. Protecting Seed Source Trees: 1. The insecticidal Snowdrop Lectin gene is being employed in efforts to protect rootstock seed trees from psyllids (and thus Liberibacter inoculation), and for Diaprepes resistance. More than 100 ransgenic trees of Green #7, Orange #4, Orange #16 and Orange #19 containing the Snowdrop lectin gene were produced. Progress was also made stacking this gene with the CEME antimicrobial gene.
In collaboration with Mike Irey, we completed evaluation of a population of transgenic lines for their resistance to Clas. Transgenic trees were kept in a greenhouse containing CLas+ Asian Citrus psyllid (ACP) adults and trees were evaluated every 6 months for HLB for 2.5 years. In addition, psyllids were tested periodically for the presence of CLas. Although we had transgenic trees from four transgenes (LIMA, AttacinE, CEAD and NPR1) showing resistance to HLB, the results from NPR1 were the most promising. 27% of the trees containing the 35S-NPR1 construct and 57% of trees with the phloem specific AtSUC2-NPR1 construct survived and were PCR- after 30 months of inoculation. The HLB-free trees were moved to the SG field site under MTA and DPI petition (many in poor condition due to severe psyllid damage). Additional clones of the promising transgenic lines are currently being propagated for additional field testing in the Dunwoody Grove of Southern Gardens. In addition, Several newer NPR1 lines are already in the field test at the Picos Farms (USDA) and results there are promising as well. Our current goal is to combine transgenes that function by completely different mechanisms as to have a back-up to prevent the pathogen from overcoming single gene resistance in the field. We have produced 40 new transgenic plants of Hamlin cominging NPR1 with the successful AMP genes CEME or CEMA. We have successfully developed a heat inducible Cre/loxP site specific based recombination system for efficient excision of antibiotic resistance genes in citrus. In our construct, the nptII gene under the control of the NOS promoter and the Cre recombinase gene driven by either a soybean heat shock protein (hsp17.5E) promoter or a Arabidopsis thaliana small heat-shock protein (HSP20) gene promoter were flanked by two loxP recognition sites in direct orientation. An anthocyanin biosynthesis gene from Vitis vinifera (VvMYBA1) was placed outside the loxP sequence. Transformation efficiencies were similar using either soybean or the Arabidopsis promoter. Anthocyanin activity analysis on transformed Carrizo citrange (Citrus sinensis x Poncirus trifoliata) demonstrated that approximately 30-40% of transformation efficiency could be obtained following Agrobacterium mediated transformation and heat shock treatment. Molecular analyses have demonstrated that 100% selectable marker gene deletion occurred in all regenerated plants expressing anthocyanin. We have completed building a RES type structure in our greenhouse#7. Transgenic trees have reached the top of the structure and have been bent downwards. We did not observe any flowering this past year; however, downward growing branches on many of the trees have completely lost their thorns, indicating a rapid reduction in juvenility. Since this greenhouse is heated during the winter, flowering induction may be inhibited.
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