The main accomplishments during this quarter: 1) We were continuing to infect and transform mature tissues of of citrus using Agrobacterium with the shoot enhancing genes we constructed. The explants used were greenhouse grown Washington Navel, Pineapple and Valencia. More calli formed than with the regular vectors. However, because the numbers of calli produced were relatively small, rates of shoot regeneration between the control vector and transformation enhancing vectors had not been compared. We were preparing a large number of adult explants for future infection experiments. We also started to use a number of techniques to reduce the contamination problems and a large number of explants of adult tissues for infection. 2) We were characterizing the enhancement of transformation efficiency of juvenile tissues of citrus using our regenreation enhancing genes in detail and also verified some of the results obtained previously. 3) Verification experiment for the role of an endogenous plant hormone in citrus regeneration from juvenile tissues upon transformation was performed and some progress had been made. However, more time is needed to generate significant results. We hope this study could shed some lights on the role of that particular hormone in adult tissue generation after infection. If so, the experimental results may guide designs of additional gene constructs for enhancing adult tissue transformation.
The main accomplishments during this quarter: 1) We improved a sterilization technique used for greenhouse-grown mature/adult shoot tissues and the contamination problems have been significantly reduced. 2) We have infected mature/adult tissues of Valencia and Washington orange using our transformation enhancing genes (K and I genes). Our preliminary results show that the use of the K and I genes we developed lead to drastic increases in transformation efficiency of mature tissues when compared to the conventional Ti-plasmid vector containing no K or I gene. 3) We have observed that the transport of an endogenous plant hormone in explants plays an important role in shoot regeneration efficiency. We also observed that manipulating the transport of that hormone improves shoot regeneration and genetic transformation efficiency of juvenile citrus explants. We are currently testing the effects of the same manipulation on adult tissues of citrus. We hope that manipulation can further enhance transformation efficiency of adult citrus tissues. 4) We are writing one manuscript that reports the drastic enhancement of citrus transformation efficiency of juvenile tissues of citrus. We will work on the second one, the effects of the transport and its manipulation for an endogenous plant hormones in explant tissues, once the first one is submitted.
We have concluded our phase 1 search employing our recently developed bioinformatics tools PAGAL and SCALPEL that led to the identification of 3 potential citrus candidate proteins that could serve as replacements for the CecB lytic peptide domain of our previously described chimeric antimicrobial protein (CAP; Dandekar et al., 2012 PNAS 109(10): 3721-3725). Using the same tools we have further refined our search within these particular proteins to identify a smaller segment that was tested for antimicrobial activity after chemical synthesis of the protein candidates. The following citrus proteins were chemically synthesized a 22 aa version of HAT (CsHAT22; a 52 aa segment of this protein was previously identified) a 15 aa segment of ISS (CsISS15 ‘ a negative control) and 20 aa segment of PPC (CsPPC20). These proteins were used to test the efficacy of their antimicrobial activity using the following bacteria, Xanthomonas, Xylella, E.coli and Agrobacterium. Using the same search criteria we identified a 22 aa N-terminal segment of the 34 aa Cecropin B (CBNT22) protein and a 12 aa segment of cathaylecitin (CATH15), representing protein with known antimicrobial activity that could serve a positive control for our bioassays. CsHAT22 and CsPPC20 were able to inhibit bacterial growth at levels comparable to CBNT22 and CATH15, however, CsISS15 displayed no detectable antimicrobial activity (as expected). We have recently obtained Liberibacter crescens and will soon test the antimicrobial activity with this bacteria as a surrogate for CaLas the causative agent of HLB. We have designed 3 constructs 1) CsP14a with a secretion sequence and Flag tag, 2) CsP14a ‘ CecB (this is construct 1 expressed as a CAP with the original CecB and 3) CsP14a-CsHAT52 (the 52 aa version of the CsHAT protein from Citrus) for testing in CTV vectors system and in transgenic plants (tobacco and citrus rootstock). These three constructs have been successfully incorporated into CTV vectors and are being infected to develop plant materials that can be used for challenge with HLB. All three of the above constructs have been introduced into binary vectors and then incorporated into Agrobacterium strains and these are being used to transform tobacco and Carrizo rootstocks. The plant transformation process in underway and will culminate in the isolation of transgenic plants that can be tested for disease resistance efficacy.
The main accomplishments during this quarter: 1) We did three Agrobacterium infections using adult tissues of Washington Navel, Pineapple and Valencia from greenhouse-grown plants. With the K and I genes, we observed more calli formed than with the regular vectors, which is a positive sign. However, the numbers of calli produced are relatively small comparing to those of the juvenile explants. We started the step to regenerate shoots from calli we have already produced but no significant results can be reported at this time. 2) We have observed endogenous concentrations of a hormone may play a role in citrus regeneration efficiency from juvenile tissues. We have started additional experiments to verify that observation. If that is true, we will modify the gene cassettes we originally designed accordingly.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. We now are examining the possibility of treating infected plants with antimicrobial peptides to allow them to recover from an HLB infection. We are beginning to work with a couple of teams of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. We are screening a large number of transgenic plants for other labs. We have promising transgenic plants that are being rescreened to ensure efficacy against HLB. We have had a collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We have found that three different lines appear to be giving strong tolerance against HLB. We are propagating the plants for more extensive analysis.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, October 2013. The lab has been in operation for more than 7 years, and as of October 2015, we have processed more than 2,300 grower samples. Additionally, more than 34,000 samples have been received for research for the entire period of diagnostic service supported by grant funding of individual researchers for more than 71,475 samples processed. Grower samples are typically processed and reports returned within a two to four week time period. Numbers specific to this report are 1605 samples received from growers. This number represents an increase from the previous two years. The increased number is likely due to the increased efforts to mitigate the HLB-associated tree stresses. Grower in this area, and most other regions, currently have one or more HLB mitigation program that they are evaluating. These growers are using the HLB lab to evaluate the effectiveness of their efforts. The HLB Diagnostic Lab webpage was updated to announce the service of detection of CLas in psyllids as funded in this grant.
The proposal funded the establishment of an 8″ well, pump, diesel engine, and associate irrigation work to provide freeze protection for IFAS rootstock trials. The project was completed in June 2014. The well easily will protect the 20-acre plot in its entirety. It is 700′ deep and will deliver 800 gal/minute.
Transgene Stacking for Long-Term Stable Resistance: Transgenic plants containing the NPR1 gene (best gene in our program for HLB resistance) stacked with the CEME transgene (best gene in our program for canker resistance) have been clonally propagated for further study (7 lines). Improving Consumer Acceptance: 1. The inducible cre-lox based marker free selection system: Molecular analysis of the putative marker free plants developed containing the cre gene driven by a Soybean heat shock gene promoter have demonstrated clean integration of the transgene in a majority of the regenerated plants. Leaky gene expression using this heat shock promoter system has however been observed in a few cases. This has not hampered our ability to regenerate marker free plants. This vector is being modified to incorporate the NPR1 gene from Arabidopsis, already proven to make plants resistant to HLB. 2. Transformation of Hamlin and W Murcott with a binary vector containing Dual T-DNA borders for gene segregation and marker free transformation of citrus suspension cells: We observed one of four scenarios when plants were analyzed using PCR 1) Most plants contains only the T-DNA of interest 2) Several plants contains both T-DNAs integrated into the genome 3) plants containing only the selectable marker T-DNA without any transgene of interest and 4) A few escapes that did not contain any transgene. Plants were obtained in a ratio of 6:2:1:1. Our results demonstrated the ability to produce marker free plants using this system, although we generated a number of escapes. Improvement of this protocol is currently underway to reduce the number of escapes and speed up the plant regeneration process. Induction of early flowering (Carrizo transformed with the FT gene): A majority of the plants flower prematurely in the tissue culture medium. These plants with apical flower development do not further develop in vitro. We are currently modifying the tissue culture medium to prevent in vitro flowering and successfully regenerate transgenic plants containing the FT gene for greenhouse evaluation. Transformation experiments are also underway with modified constructs containing weaker promoters driving the FT gene. Efforts to establish a new transgenic field site at the Southwest Research and Education Center: Working with Dr. Phil Stansly, we successfully renewed our transgenic field permit with APHIS to add this additional field site (the 4th site approved). Approximately 400 transgenic citrus plants were wrapped and tagged for planting during the next quarter. Field site preparation is underway.
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). Boxes and lbs. solids per canopy volume (2013/2014 season yield) were calculated for each rootstock for both Valquarius and Vernia scions. Highest yield efficiency was on rootstocks SO+50-7 and FG-1731 (UFR-13), both tree-size controlling rootstocks that are good candidates for ACPS. Since we have no seed tree for FG-1731, field tree roots were sprouted, and the rootstock genotype has been recovered by micrografting (also successful for FG-1733, both original seed trees destroyed by the canker eradication program). The progressively modified nutrition program continues to produce good results, as there is an excellent fruit set across the trial; we expect a yield increase this year if fruit drop is contained. The addition of the Schumann blend of TigerSul micros (iron, zinc and manganese) seems to be making a significant difference in overall tree health, as even control trees on Swingle and Volk are improving. The new well funded by CRDF is now completed and on-line. Greenhouse Experiments – Nutritional study: preliminary results continue to be quite interesting on the growth of the HLB infected Valencia trees on Orange #15 rootstock. The 3x overdose of TigerSul manganese treated trees are showing the best growth; trees treated with polymer-coated sodium borate (Florikan) are producing large dark green leaves. The 2nd 6-month application of various overdose treatments were applied to the trees. The trial will be broken down in November. Infected symptomatic trees on other rootstocks were treated with 3x TigerSul manganese and polymer-coated sodium borate. Protecting Seed Source Trees: Transgenic rootstock Orange #4 plants containing the GNA insecticidal transgene have been clonally multiplied as rooted cuttings and are being sized up for evaluation. Transgenic Orange #16 and Orange #19 tetrazyg plants transformed with GNA are being clonally multiplied in a mistbed to provide replicated plants for evaluation.
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 have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. We now are examining the possibility of treating infected plants with antimicrobial peptides to allow them to recover from an HLB infection. We are screening a large number of transgenic plants for other labs. We have promising transgenic plants that are being rescreened to ensure efficacy against HLB. We are beginning to work with a couple of teams of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las.
This is a joint project between CREC and USDA, Fort Pierce. The objective of this project was 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 7 lines that we thought could be acceptable for fruit or juice. Those have been propagated and are being screened for tolerance and horticultural properties. Each of the hybrids has been propagated onto four rootstocks (US 812, US 942, Swingle, and Sour Orange) with multiple replicates of each rootstock scion combination. These trees are ready for secondary evaluation. A second set of the hybrids was propagated from the first set onto Kuharski rootstock, there are 16 replicates of each scion hybrid and the trees will soon be ready for filed evaluation.T he hybrid plants are being incubated in the psyllid containment room to allow multiple psyllids to inoculate the plants with HLB. All of the plants are tolerant to CLas. We resurveyed the trees at the Whitney field station. Most of the hybrids are HLB infected but have only minor symptoms. The fruit on the chosen trees were again considered to have acceptable fruit taste. Most of the chosen trees which lacked HLB symptoms were found to contain the HLB bacterium. These plants, which contain 1/8 poncirus, are tolerant to HLB and have acceptable taste. At the very least, these plants should be acceptable for back-yard trees, which would allow home owners to have citrus fruit trees in the presence of HLB.
The original goals this project addressed were to identify, propagate and test rootstock and/or scion HLB survivors, identified in the filed in Florida. The one year enhancement funding was to facilitate the project and to expand the information base of surviving trees. This was specifically focused on DNA marker fingerprinting using SSRs (simple sequence repeats) to assess trueness to type, particularly of the rootstocks. As a consequence, additional work in the labs and the greenhouses was conducted, and appropriate personnel worked on the project to provide the additional information promised in the enhancement request for funding. Methods were developed and refined for routine extraction of DNA and fingerprint analysis from feeder roots collected from beneath the survivor trees; this critical aspect was not a component of the original project proposal, but is providing helpful new information, to help determine whether the performance of these trees is based on rootstock variations. We have continued to monitor previously identified candidate survivor trees at the CREC, the GCREC, and some Polk County commercial groves where we have planted out materials from the CREC breeding program. Most of the trees now have begun to display symptoms after more than 24 months of observation, though there remain a few still unaffected as of June 2014. Trees at an abandoned location in Palm Beach County, that retain reasonably good condition and freedom from obvious symptoms, were sampled for both budwood and root tissues. Budwood samples were propagated onto healthy rootstock seedlings at the CREC. In addition, root samples were provided. We were able to collect root sprouts from some of the trees that were decapitated; we have propagated from these root sprouts, by budding and by rooted cuttings. These small trees are growing of for further propagations to test their responses to HLB. We extracted DNA from these sprouts and compared their DNA fingerprints with what we produced from feeder roots collected previously, as expected, the fingerprints were identical. New rootstock samples collected from trees were used to confirm nucellar embryony. Thus far, all except one of the rootstocks collected has been shown to be a nucellar seedling of the presumed rootstock. Routine fingerprinting of all scion varieties sampled has shown them all to be true to type; this does not discount the possibility of mutations for HLB tolerance/resistance. We have likewise collected feeder root samples from various survivor trees in other locations for fingerprinting. We have gathered information on many other possible survivors identified and in most cases it was decided to allow more time to determine whether the these trees warranted more careful analysis because they generally represented quite a few trees in each location. In other instances, the reported survivors actually exhibited more symptoms than would qualify as a ‘healthy’ survivor. New reports of survivors have come in from Highlands, Lee, Collier, Indian River, Lake, and Marion Counties; several have been documented, and some have been visited by the PI or extension personnel. In some cases, samples have been collected for propagation and further testing. In other cases, materials have not yet been collected, pending further assessment of disease expression in the autumn and winter seasons.
Transformations of citrus plants with the FLT-antiNodT fusion protein expression construct have been completed 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. Fifteen (15) independent transformant lines resistant to the kanamycin selection marker and expressing the green fluorescent protein have been regenerated successfully into plantlets. Of these 15 lines, 10 are strong expresses of the green fluorescent protein (GFP) transgenic marker, indicating successful transformation and expression of the transgenic marker genes. The other 5 show spotty expression of the GFP in cells of all tissues examined. This could indicate gene silencing might be affecting GFP expression in these plants. The effect that this might have on FLT-antiNodT fusion protein expression are not known, but will be tested later. The plantlets range from 5 – 15 cm in height, with the smaller plants being younger than the larger plants. All of the 15 lines appear to be growing and developing normally, which is a good sign that the FLT-antiNodT fusion protein is not having any unexpected deleterious effects on the plants. Permits to move the plants from Florida to Pennsylvania have been obtained, in order to study the FLT-antiNodT fusion protein expression levels and phloem-localization of the FLT-antiNodT fusion protein. Plans are being made to test the HLB resistance of these transgenic lines in collaboration with Dr. Tim Gottwald at the USDA Horticultural Research Lab at Ft. Pierce, FL.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we have been preparing citrus plants for root treatment with NAD+. We are testing NAD+ analogs to identify potential chemicals for citrus disease control. For objective 2, about 15 more transgenic lines have been generated. We are currently characterize the transgenic seedlings. For the 20 transgenic lines generated previously, we have confirmed 15 of them containing the transgene. Expression of the transgenes have also been tested. These plants are growing in the greenhouse and will be tested for canker resistance. We are cloning the citrus homologs and will confirm the sequences of the genes before transformation.
The topworking project was completed in mid-July. Trees with all failed grafts were re-grafted in early July, and the trees were cut to promote growth from new buds in late July. Approximately 275 trees were successfully topworked with 19 high quality scions showing potentially enhanced HLB tolerance, including 15 sweet orange clones, one triploid sweet orange-like hybrid and 3 red grapefruit clones. Successfully grafted trees are growing well and showing good health (no HLB symptoms at present, even though trees were topworked onto HLB-infected Hamlin scion and Carrizo rootstock). Controlled release fertilizer (Harrell’s UF mix combined with TigurSul micros iron, zinc and manganese) was purchased and applied to the trees in mid-July. This will carry the trees through to January. Trees will be monitored for HLB symptoms, and later by PCR analysis. Any scions showing enhanced HLB tolerance will of course be made available to the Florida citrus industry as quickly as possible.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
