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.
Work has been continued on the experiments initiated last quarter. Fresh seed has been obtained to generate new citrus seedlings and, as expected, germination rates are much improved. Therefore transient expression assays are being reported with larger numbers of seedlings and our new peptides. This quarter, I have been dividing work among between all of the proposal objectives. Plasmid constructs for a silencing experiment using the citrus analog of NPR1 are being verified and it should be possible to test silencing soon.
Transformations of citrus plants with the FLT-antiNodT fusion protein expression construct are now underway. The transformations are being performed at the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL. The FLT-antiNodT expression cassette is being introduced into ‘Duncan’ grapefruit by Agrobacterium tumefaciens – mediated transformation. Transformations were begun on November 5, 2013. The transformation construct includes a green fluorescent protein (GFP) marker. Within a few weeks of initiating the transformation experiment, clusters of green-fluorescing citrus cells were observed, indicating that the FLT-antiNodT fusion protein transformation vector was working. This was important, because we had encountered significant difficulties in the development of the FLT-antiNodT fusion protein transformation vector. All indications are that the transformation vector is working as expected, which is good news. Transgenic shoots were successfully regenerated that are green-fluorescing and presumably carry the FLT-antiNodT fusion protein expression cassette in their genome. As of December 13, 2013, micro-grafting of transformed shoots onto recipient plants was begun. Successful regeneration of shoots containing the FLT-antiNodT fusion protein expression cassette is critically important. Sometimes certain proteins and transgenes might interfere with the normal plant biology in such a way that regeneration of normal plant tissues cannot occur. We are pleased to report that this has not been a problem so far for this project. All indications are that we should be able to obtain transgenic plants suitable for testing for HLB resistance. This project has gone a bit more slowly than initially anticipated. All steps of the project took more time than initially predicted, from antibody development and cloning to the development of the transformation construct. It is likely that we will take at least until August, 2014, to obtain transformed plants that are suitable for HLB resistance testing. We will also need time to do laboratory experiments characterizing the expression of the FLT-antiNodT fusion protein in the transgenic plants. Therefore, we fully anticipate needing to request a minimum of one year no-cost extension to this project. A no-cost extension of the project will be necessary to achieve the goals of the project fully.
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. We have modified the CTV vector to produce higher levels of gene products to be screened. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We 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 are screening a large number of transgenic plants for other labs. We are beginning to work with a team of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. Since we are testing about 80 genes for induction of resistance or tolerance to HLB in citrus, we changing our focus of building new constructs to controlling psyllids until we have more conclusion from the peptides under screen. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. A recent advance is that has greatly speeded up our screen is that we now can estimate when plants become infected with HLB and can tell whether a peptide is working more quickly.
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. At this time, all 4 of 5 hybrids still have no symptoms. The inoculated plants are growing in the greenhouse as we wait to determine the degree of disease symptoms in each line.
Transformations of citrus plants with the FLT-antiNodT fusion protein expression construct are continuing at the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL. The FLT-antiNodT expression cassette has been introduced into ‘Duncan’ grapefruit by Agrobacterium tumefaciens – mediated transformation. Plants resistant to the kanamycin selection marker and expressing the green fluorescent protein have been regenerated successfully into plantlets. Plants are developing and appear to be healthy and normal, without any deleterious effects of the transgene noticed in the plant regeneration process so far. We anticipate that 1-foot tall plants will be available to begin testing as early as August of 2014. We are requesting a no-cost extension to the end of 2014 to have enough time to test the HLB resistance status of the trangenic plants.
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