Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. A journal manuscript was submitted on research showing that use of the antioxidant lipoic acid significantly improves transformation efficiency in Mexican lime; experiments to test this with commercial sweet oranges are underway. We continued with experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency, including: kanamycin, hygromycin, mannose and phosphinothricin. ‘New publications supported by this grant:1. Dutt, M., D.H. Lee and J.W. Grosser. 2010. Bifunctional selection-reporter systems for genetic transformation of citrus: mannose and kanamycin based systems. In Vitro Cellular & Developmental Biology-Plant 46:467-476; 2.Orbovic, V., M. Dutt and J.W. Grosser. 2010. Seasonal effects of seed age on regeneration potential and transformation success rate in three citrus cultivars. Scientia Horticulturae 127: 262-266 Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Working with Mr. Orie Lee and a commercial harvesting company (w/ Frank Rogers), a plan to collect meaningful yield and fruit quality from the St. Helena project was developed – with harvest expected later this month. Approximately 10 acres of trees planted 2.8 years ago include a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks. The majority of trees have a significant yield and the trial is showing significant rootstock affects on precocious bearing and early fruit quality – the best selections from this trial will be ideal candidates for testing with juvenile transgenics. Also of interest is the cultural program being used at the St. Helena project that mimics OHS principals but with reduced input. The trees have been grown with a UF research slow-release fertilizer mix (in cooperation with Harrell’s Fertilizer) and daily irrigation. Two trees were confirmed with HLB the first year; but even with bad neighbors, there has been no detected additional spread of HLB during the past year. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL sweet oranges (a group of clones with Rhode Red quality that show high solids in young trees) were regenerated and successfully micrografted for further study of early flowering and transgene expression. Approximately 25 transgenic sweet orange trees from OLL selections were produced containing four different gene constructs. Progress was also made transforming OLL clones with the alternative embryogenic culture transformation system, as numerous transformed somatic embryos have been recovered. Progress was also made in the regeneration and characterization of plants containing the FDT transgenes for early flowering.
Objective 1: A comparative study of two susceptible hosts, Duncan grapefruit (DG, C. paradisi), and Rough lemon (RL, C. jambhiri) and two resistant species of kumquat (Fortunella spp.), ‘Meiwa’ and ‘Nagami has been conducted to evaluate the basis for resistance to Xanthomonas citri subsp. citri (Xcc). The type of resistance occurring in kumquats is a hypersensitive response (HR) that develops within 48-72 h. This is based on the phenotype of the lesion, histological changes at the cellular level of infected tissue and the early expression of genes related to programmed cell death (PCD). In kumquats but not in DG and RL, several HR-related genes are expressed at 4 h post-inoculation (pi) with Xcc at 108 cfu/ml. The genes that are over-expressed are lipoxygenase, glutathione transferase, metacapcase, acid chitinase and peroxidases that have been linked with PCD. Later at 24 h, additional genes related to plant defense like PR-2 (betaglucanase) are highly expressed in kumquats but less so in DG and RL and their activity continues to increase up to 48 h pi. Different sets of genes are expressed in susceptible DG an RL related to the host pathogen interaction. The gene expression analyses have been extended to include comparisons of susceptible RL 8166 with the RL cybrid (RL + Valencia orange as the cytoplasm donor). Validation of the inheritance of resistance for the RL 8166 cybrid and newly developed cybrids of susceptible Ruby red grapefruit (RG) with Valencia orange (VO) is underway. The resistance inherited from VO is not HR (qualitative) but instead a degree of quantitative resistance compared to highly susceptible RG. Evidence for this is based on an intermediate lesion phenotype for cybrids in vitro and in-planta. In contrast to development of callus in susceptible RL and RG, the inoculated area develops more necrosis by 10 days pi. Xcc populations in the cybrids plateau at a level below populations in RL or RG. The lesion type is a mixture of necrotic and callus tissue that indicates that some cell death occurs and arrests the proliferation of Xcc. Expression of HR- and host pathogen interaction related genes in the RL cybrid is intermediate between kumquats and RL. The different pattern of gene expression suggest an interaction between the parent nuclear genes with the heterologous mitochondria and chloroplast from the cytoplasmic donor (VO). Field trials with several Ruby red grapefruit cybrids planted in canker-affected locations on the east coast continue to show less foliar disease incidence than the adjacent Red grapefruit trees. The production of cybrids lines using a new callus line of Meiwa kumquat as the cytoplasmic donor is underway in the Grosser lab. A new device for precise inoculation of Xcc bacteria into citrus leaves has been published and the prototype of the newly designed instrument is currently being used in several projects related to citrus canker.
Preliminary work to explore optimal parameters for genetic transformation of Murraya has been carried out, including assessments of shoot sensitivity to the selection agent kanamycin using untransformed shoots, determinations of bacterial growth curves, and appropriate and effective antibiotic concentrations for bacterial selection. Using the recently optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments have been initiated using the plasmid pTLAB 21 harbored in Agrobacterium tumefaciens strain EHA 101. This vector contains the genes for kanamycin resistance as a selection agent and GFP (green fluorescent protein) as an easily observed marker. Various factors, including a range of OD values (cell density or concentration in liquid culture) of Agrobacterium cultures, the duration of explant incubation in bacterial cultures, duration of co-cultivation period, and the composition of co-cultivation and regeneration media were tested, to attempt establishment of an optimal and standardized transformation protocol. Optimal conditions for transformation using shoot tips and lateral buds, to develop an alternative method using a different tissue source should the organogenic approach prove too difficult or inefficient for transformation, were also explored. Regeneration of buds and some shoots has occurred from organogenic cultures of longitudinally cut seedling epicotyl segments, following these transformation experiments. Observations of the regenerating cultures have revealed several buds and shoots displaying green fluorescence, indicating successful genetic transformation. Their growth is being monitored, as well as the stability and uniformity of GFP expression over time, and further production of new transgenic events is underway.
Because of successful efforts to achieve acceptable seed germination rates and to produce more vigorous seedlings in vitro, higher quality seedling explant materials have been obtained. This accomplishment has enabled work to proceed to establish a suitable and efficient protocol for in vitro regeneration of Murraya, which can then be used for the various planned transformation experiments. Research was conducted to develop a suitable and robust organogenic shoot regeneration protocol, using various parts of germinated seedlings including hypocotyls (longitudinally cut vs. no cut), epicotyls (longitudinally cut vs. no cut), roots, leaves, and cotyledons. These explants were cultured on MS basal medium supplemented with various concentrations of BA, TDZ alone, or in combination with NAA. Two suitable and reasonably effective shoot regeneration protocols have been developed and optimized for transformation experiments (a manuscript is in preparation). Simultaneously, the capacity of shoot tips and lateral buds to undergo multiple shoot formation and multiplication in vitro has been examined. Shoot tips and lateral buds were excised directly from in vitro germinated seedlings and cultured on MS medium supplemented with BA at 0, 1, 3, and 6 mg/l. Newly formed shoots were subcultured on the same medium at 3 week intervals to determine optimal conditions for maximal shoot multiplication.
Quarterly report April 2011: Reserach to explore optimal various parameters for genetic transformation of Murraya was carried out, including assessments of shoot sensitivity to the selection agent kanamycin using untransformed shoots, determinations of bacterial growth curves, and appropriate and effective antibiotic concentrations for bacterial selection. Using the recently optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments have been initiated using the plasmid pTLAB 21 harbored in Agrobacterium tumefaciens strain EHA 101. This vector contains the genes for kanamycin resistance as a selection agent and GFP (green fluorescent protein) as an easily observed marker. Various factors, including a range of OD values (cell density or concentration in liquid culture) of Agrobacterium cultures, the duration of explant incubation in bacterial cultures, duration of co-cultivation period, and the composition of co-cultivation and regeneration media were tested, and we established an optimal, standardized transformation protocol. Optimal conditions for transformation using shoot tips and lateral buds, to develop an alternative method using a different tissue source should the organogenic approach prove too difficult or inefficient for transformation, were also explored. Regeneration of buds and some shoots occurred from organogenic cultures of longitudinally cut seedling epicotyl segments, following these transformation experiments. Observations of the regenerating cultures revealed several buds and shoots displaying green fluorescence, indicating successful genetic transformation. Their growth was monitored, as well as the stability and uniformity of GFP expression over time. Nearly all of these transformation events proved to be either chimeric or transient, so further production of new transgenic events is being pursued.
Microarray time course studies comparing tolerant rough lemon to susceptible sweet orange have been completed. More genes were differentially expressed early in HLB-affected rough lemon than sweet orange but the opposite was observed at later time points; many of these were associated with defense responses, carbohydrate metabolism, and disease resistance pathways. Microscopy showed callose deposition in phloem of infected plants of both species, but phloem transport was much greater in lemon leaves than orange. Proteomic analysis of infected orange revealed greater abundance of several stress related proteins, and microarray data indicated that their underlying genes were also upregulated; these results are leading to better understanding of the underlying causes of HLB disease. Transgenic studies have proceeded, with several hundred plants containing various combinations of natural or synthetic genes and promoters produced; many of these are currently in greenhouse testing and field trial locations. Additional transgenic plants have been propagated for new hot psyllid greenhouse tests, and for field planting. New candidate genes have been identified from citrus and other plants for HLB and canker resistance; vectors have been constructed for a next round of transgenic plant production. Citrus-specific promoters, transcription factors, and other genetic elements have been identified and incorporated into some of the new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. An international project to produce a high density genetic linkage map to aid genome sequence assembly and validation has been completed, with >900 sequence based markers. Several new rootstock trials with more than 15,000 trees were planted throughout Florida using advanced selections, to assess their adaptation to evolving advanced citrus production systems; these trials are monitored regularly. Rootstock candidates that produce nucellar seedlings have been identified using SSR markers; these rootstocks were preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora. Four complex tetraploid rootstocks have shown some repression of HLB in greenhouse tests (one 22 months symptom-free). Hybrid plants for rootstock improvement from the previous season were planted, and new crosses made 2010 are growing off. Previous work to develop rootstocks against other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) continues, as we collected data from replicated trials and plantings. Final data have been collected from a field trial of various Valencia somaclones and seedless Midsweet selections, and following final analysis the most consistently high yielding clones from each will be moved forward for release; most candidates have already moved through the DPI-Parent Tree Program. New pummelo-grapefruit seedless hybrids have been selected, some showing field tolerance to canker; their fruit have been assayed for furanocoumarin content and several with good fruit quality have been found FC-free, potentially producing grapefruit cultivars that alleviate drug interaction concerns. Patents have been issued by the US-PTO for Valqarius (SF14W-62) and Valenfresh (N7-3), very early- and late- maturing Valencia selections respectively, and licensing is in process. Patent applications and documents for release approval have been developed for 2 new sweet oranges.
Update for 6/30/10: Two microarray platforms (our Agilent chip containing disease resistant genes and the Affymetrix Citrus GeneChip) are being used to compare gene expression over time in response to canker and HLB. Comparisons are being made between resistant kumquat and susceptible grapefruit for canker, and HLB tolerant rough lemon and susceptible sweet oranges are being compared. Comparisons of carbohydrate metabolism in HLB-infected and healthy plants revealed that starch, sucrose, and glucose accumulated in infected leaves, maltose decreased and fructose levels were unchanged. Comparisons of cell-wall bound invertase, a critical enzyme involved in sucrose metabolism and plant defenses, have shown its activity to be induced 4-fold in both symptomatic and asymptomatic leaves of infected sweet orange. Further, the expression profiles of 2 starch breakdown genes are downregulated. Transgenic studies are proceeding, with several hundred plants containing various new combinations of natural or synthetic genes and promoters produced; these are currently being propagated for field trials or are already in greenhouse testing. New candidate genes have been identified from citrus and other plants for HLB and canker resistance, from gene expression studies and data mining of public EST databases; vectors are being produced for a next round of transgenic plant production. Phloem-specific promoters are being identified for incorporation in constructs to produce more consumer friendly transgenic plants, by limiting transgene expression to specific tissues. Previously, microarray experiments highlighted canker defensive genes in kumquat; real-time PCR confirmed their roles, and these will be used in new transformation experiments to produce canker resistant citrus. Cybrid grapefruit plants showing tolerance to canker in greenhouse tests have been planted in the Indian River area to assess field tolerance to canker. New DNA samples from a genetic population are being genotyped with previously developed SSR markers to develop a high density genetic linkage map to aid genome sequence assembly and validation; this is part of the international genome sequencing collaboration. Several new rootstock trials with more than 15,000 trees have been or will be planted throughout Florida using advanced selections, to assess their adaptation to evolving advanced citrus production systems; these trials are monitored regularly. Rootstock candidates that produce nucellar seedlings are being identified routinely using SSR markers; these rootstocks were preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora. Four complex tetraploid rootstocks are showing some suppression of HLB symptoms in greenhouse tests that are still underway. New crosses for rootstock improvement were made in 2010, using previously proven parental lines. New pummelo-grapefruit seedless hybrids selected the past 2-3 years, as well as breeding parents, are being assayed for fruit furanocoumarin content; several have been found FC-free. Genetic studies are underway using in silico expression approaches to EST data analysis to define the genetic controlling elements of this trait, which seems to be simply inherited based on preliminary results.
Update for 9/30/10: Data from microarray time course studies comparing tolerant rough lemon to susceptible sweet orange are being analyzed. Microscopic studies of anatomical features in HLB-infected rough lemon and sweet orange plants are underway, along with experiments to study carbohydrate loading into phloem tissues using fluorescence labeled sugars; the results from these experiments are being compared with the results of microarray time course experiments to elucidate the mechanisms underlying HLB-disease symptom development. Transgenic studies are proceeding, with several hundred plants containing various new combinations of natural or synthetic genes and promoters produced; these are currently being propagated for field trials or are already in greenhouse testing. New candidate genes have been identified from citrus and other plants for HLB and canker resistance, using results from gene expression studies and bioinformatic data mining of public EST databases; vectors are being produced for a next round of transgenic plant production. Citrus-specific promoters, transcription factors, and other genetic elements are being identified and some of these will be incorporated into new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. Previously, microarray experiments highlighted canker defensive genes in kumquat; real-time PCR confirmed roles of one kumquat R-gene, two kinases, and one transcription factor, and these will be prepared as new constructs for transformation experiments to produce canker resistant citrus. Cybrid grapefruit plants were produced with kumquat, and preliminary results show that some behave like kumquat in challenges. Previously tested cybrids showing some tolerance to canker in greenhouse tests have been planted in the Indian River area to assess their field tolerance to canker. New DNA samples from a genetic population are being genotyped with previously developed SSR markers to develop a high density genetic linkage map to aid genome sequence assembly and validation; this is part of the international genome sequencing collaboration. Several new rootstock trials with more than 15,000 trees have been or will be planted throughout Florida using advanced selections, to assess their adaptation to evolving advanced citrus production systems; these trials are monitored regularly. Rootstock candidates that produce nucellar seedlings are being identified using SSR markers; these rootstocks were preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora. Four complex tetraploid rootstocks are showing some suppression of HLB symptoms in greenhouse tests that are still underway. Hybrid plants for rootstock improvement from the previous season were planted, and new crosses made in 2010 have been harvested. Seed from other candidate rootstock plants have been planted, and additional ones collected, to study their nursery performance attributes. Data collected from a field trial of various Valencia somaclones and seedless Midsweet selections across the past several years have pointed to candidate oranges likely to be released in 2011 have entered the DPI-Parent Tree Program.
Update for 6/30/10: This project will assess a wide range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. The largest seedlings of these accessions have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination. They are now being grown in a climate controlled, DPI-certified greenhouse. Additional seedlings not yet large enough for inoculation are retained in a separate, DPI certified propagation greenhouse, for future inoculations in the greenhouse and for field planting once an acceptable site has been secured. Further, we have explored planting out the Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test, but we are waiting on DPI to grant permission for planting at the Picos Road Farm with USDA-ARS. This population is of significant interest as the trifoliate orange and some of its hybrids are very HLB-tolerant, and this experiment is an opportunity to map genetic components responsible for the tolerance. In advance of DPI-approval, we have begun testing for HLB and CTV, to ensure DPI that the plant materials are safe for field planting. Numerous somatic hybrids of citrus with related genera are also being prepared for inclusion in the plantings. We are in the process of acquiring additional germplasm resources, to expand the breadth and depth of the material categories we described in our proposal. Finally, we made a number of crosses in spring 2010 to produce segregating families of several purported tolerant and susceptible types to begin searching for evidence of genetic control of HLB tolerance/resistance within the citrus gene pool. Our collaborators in China have sought domestic funding support but have not been successful, so no materials will be sent there. One company in Florida that initially offered land for the project has since withdrawn its offer. We are currently exploring other options within Florida, to be followed up with agreements to move ahead; these represent locations where growers have decided not to remove HLB-infected trees, so we expect there to be opportunities to challenge our replicated materials. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have expanded, and are continuing to expand, the number of types we wish to challenge. We will be developing new information about potentially tolerant/resistant germplasm that can lead to expanded efforts to capture and exploit the genetic basis for this phenomenon.
This project is assessing a range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. Subsets of these families have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination, are being grown in a climate controlled, DPI-certified greenhouse and monitored for symptom development. Currently symptoms are being noted among some of the accessions, and data on disease progression will be collected. Additional seedlings that have reached sufficient size have now also been inoculated with the same HLB source. The individuals of the Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test at the Picos Road Farm near Ft. Pierce have been propagated. This population is of significant interest as the trifoliate orange and some of its hybrids are very HLB-tolerant, and this experiment is an opportunity to map genetic components responsible for the tolerance. We continue to seek additional germplasm resources, to expand the breadth and depth of the material categories we described in our proposal; a source for new C. latipes hybrids has been identified. We are still exploring other options within Florida for a field trial, but no secure, long-term commitments have been forthcoming, despite multiple discussions with growers throughout the state. Eleven crosses between 10 different susceptible and reputedly tolerant parents were made in spring 2010, and several thousand seeds were harvested. Populations of at least 150 from each cross have been planted and now are growing in a DPI-certified propagation house. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have expanded, and are continuing to expand, the number of types we wish to challenge. We will be developing new information about potentially tolerant/resistant germplasm that can lead to expanded efforts to capture and exploit the genetic basis for this phenomenon.
Update for 9/30/10: This project will assess a range of citrus germplasm and relatives for tolerance or resistance to HLB, through greenhouse assays and field tests; these germplasm resources were selected on the basis of research and observations in Asia and Florida. We have produced seedlings from 7 pummelo accessions (10-15 each), Citrus latipes (13 seedlings) and some hybrids of this species with trifoliate orange, 4 natural pummelo-mandarin introgression hybrids (9-16 each), 6 other miscellaneous wild citrus types (4-12 each), and various sweet orange lines for which there is anecdotal evidence of differential sensitivity to HLB. The largest seedlings of these accessions have been inoculated with HLB-infected, PCR positive budwood of Carrizo citrange to ensure freedom from CTV cross-contamination. They are now being grown in a climate controlled, DPI-certified greenhouse and monitored for symptom development; to date, no symptoms have been observed. Additional seedlings now large enough for inoculation have been moved into our certified HLB-testing greenhouse, and inoculations are about to take place. Further, we have been granted DPI permission to plant out the Core Citrus Mapping Population, a genetically well-characterized collection of more than 250 citranges that we proposed to test, at the Picos Road Farm near Ft. Pierce, with USDA-ARS. This population is of significant interest as the trifoliate orange and some of its hybrids are very HLB-tolerant, and this experiment is an opportunity to map genetic components responsible for the tolerance. Testing the material for HLB and CTV has been completed and only pathogen free selections safe for field planting have been identified and budwood samples sent to the USDA-ARS for propagation and subsequent field planting. Approximately 100 selections were chosen and trees have been budded in late August. We continue to seek additional germplasm resources, to expand the breadth and depth of the material categories we described in our proposal; a source for new C. latipes hybrids has been identified. We are still exploring other options within Florida for a field trial, but no secure, long-term commitments have been forthcoming, despite multiple discussions with growers throughout the state. To conclude, a wide range of genetic materials have been produced and prepared for greenhouse and field testing for their tolerance or susceptibility to HLB. We have expanded, and are continuing to expand, the number of types we wish to challenge. We will be developing new information about potentially tolerant/resistant germplasm that can lead to expanded efforts to capture and exploit the genetic basis for this phenomenon.
Transgenic studies have proceeded, with several hundred plants containing various combinations of natural or synthetic genes and promoters produced; many of these are currently in greenhouse testing and field trial locations. Additional transgenic plants have been propagated for new hot psyllid greenhouse tests, and for field planting. New candidate genes have been identified from citrus and other plants for HLB and canker resistance; vectors have been constructed for a next round of transgenic plant production, already underway. Citrus-specific promoters, transcription factors, and other genetic elements have been identified and incorporated into some of the new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. The sweet orange citrus genome sequence was mined to identify genes controlling anthocyanin expression, in an effort to develop visual and citrus-derived markers for genetic transformation; several candidates have been identified for further experiments. More than 875 transgenic plants have now been planted with a collaborator in Martin county, and these are being monitored regularly, along with a second site in Indian River county. The plant materials growing out include sweet oranges, grapefruit and mandarin hybrids. Several new rootstock trials with more than 15,000 trees were planted throughout Florida using advanced selections, to assess their adaptation to evolving advanced citrus production systems; these trials are monitored regularly, and data has been collected on their early performance. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests; rootstock hybrids are showing diverse responses when grafted with HLB-infected Valencia, ranging from extreme sensitivity to high levels of tolerance; four complex tetraploid rootstocks have shown some repression of HLB in greenhouse tests (one was symptom-free up to 22 months ). We initiated a program to rotate new germplasm (rootstock and transgenic) through a ‘hot psyllid’ house (in collaboration with Dr. Stelinski) to ensure HLB inoculation prior to approved field planting; two groups of 50 trees have been rotated through so far, scheduled for planting at a collaborators field site, under permit from DPI. Rootstock candidates that produce nucellar seedlings have been identified using SSR markers; these rootstocks were preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora. Hybrid plants for rootstock improvement from the previous season were planted, and new crosses made 2010 were planted in the field. Previous work to develop rootstocks against other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) continues, as we collected data from replicated trials and plantings. Final data have been collected from a field trial of various Valencia somaclones and seedless Midsweet selections, and following final analysis the most consistently high yielding clones from each will be moved forward for release; most candidates have already moved through the DPI-Parent Tree Program. New pummelo-grapefruit seedless hybrids have been selected, some showing field tolerance to canker; their fruit have been assayed for furanocoumarin content and several with good fruit quality have been found FC-free, potentially producing grapefruit cultivars that alleviate drug interaction concerns. Patents have been issued by the US-PTO for Valquarius (SF14W-62) and Valenfresh (N7-3), very early- and late- maturing Valencia selections respectively, and licensing is in process. Patent applications and documents for release were developed for 7 new cultivars, and these were approved by the UF-IFAS Cultivar Release Committee for release and commercialization according to UF-IFAS policy.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. ‘ We have greatly improved our efficiency of screening . ‘ We are modifying the vector to express more than one anti-HLB gene. ‘ We are modifying the vector to allow addition of a second vector. ‘ We are preparing to put trees into the field for testing as soon as potential freezes are over. ‘ We continue to supply infected and healthy psyllids to the research community.
Researchers at the USDA Ft. Pierce: 1. Source of mature tissue. Four populations of adult phase trees were maintained in the greenhouse including Valencia sweet orange/Sun Chu Sha (73 trees), Ruby Red grapefruit/US812 (62 trees), US-942 citrange rootstock/Cleo (32 trees), Calamondin (31 trees), and Etrog Arizona 861-S1 citron (67 trees). In vitro bud emergence and growth manuscript accepted for publication. A manuscript was submitted to the journal Plant Cell, Tissue and Organ Culture that documents the system developed for producing in vitro adult phase shoots from cultured nodes of greenhouse trees. Shoot regeneration from mature tissue explants. A system was developed for the production of shoots from cultured internodes from greenhouse trees. The system results in shoot and bud formation in 70-90% of the explants. A manuscript is in preparation that documents this research. Agrobacterium-mediated transformation of mature tissue explants. Transformation of mature internode explants from greenhouse trees has been demonstrated in grapefruit (1 plant), US-942 (4 plants), and Etrog citron (4 plants) using the beta-glucuronidase reporter gene. Current efforts are now directed toward identifying the factors important for a system of sufficient efficiency for routine transgenic plant production. New tissue culture method of Agrobacterium-mediated transformation of tissue explants. Preliminary results using alternative culture methods suggest improved transformation efficiencies. These approaches will be further explored. At the CREC in the Gmitter lab, work is continuing on the use of Thin Cell Layers (TCLs) as explants for mature tissue transformation. Experiments have been done to induce regeneration in the TCLs by manipulating the amount of growth regulators, carbon source and also by pre-treating the TCLs with BA but regeneration is still problematic from these explants. In the Grosser lab, research confirmed the work of others showing that the flush used to generate transformation explants was critical. In the Machado laboratory in Brazil, research on the regeneration ability of various sweet oranges continues. These differences are present in both mature and juvenile tissues. In the Moore laboratory in Gainesville, experiments still are focused on using small peptides as vehicles to deliver cargos to plant tissues. If these techniques could be worked out they would have a number of applications for citrus transformation, perhaps even eventually allowing the transfer of genes or gene products to existing trees. A transient transformation expression system has also been developed using citrus leaves.
Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. This objective is proposed as an alternative strategy in case constitutive expression of the mutant R proteins proves to be detrimental to the growth or vigor of the plant. By restricting expression of the R protein to the single cell that is pierced by the insect stylet, a defense can be mounted without endangering the overall health of the plant. In the long run, this may be the most effective strategy in fighting Liberibacter infection since the response can engineered to be quite robust. Results: To further insure more specific expression of the R proteins in the phloem, we identified a wound-inducible, phloem-specific promoter that triggers expression upon wounding resulting from aphid feeding. We substituted the AtSUC2-940 promoter (phloem-specific) with the AtPAD4-1002 promoter (PAD4) in SNC1, snc1, SSI4, and ssi4 R gene constructs in the pCAMBIA1305.1 vector. Additionally, we created a PAD4/GUSplus construct to verify phloem- and wound-specific activation. The PAD4 constructs were introduced to Arabidopsis plants, grown to seeds and harvested this week (Jan 2011). The transgenic seeds will be subjected to selective screening for R gene transformants. Due to the cloning limitations (restriction site configuration), all PAD4/R constructs had to be first assembled in the 1305.1 pCAMBIA vector prior to transfer into the 2301 pCAMBIA for transformation into citrus. We selected two constitutive R protein mutants and PAD4/GUSplus constructs, and transferred them to the Citrus Research and Education Facility at Lake Alfred (Dr. Vladimir Orbovic). Preliminary reports indicated that transformants are proving difficult to obtain, potentially due to the lethal nature of at least one R protein mutant. Conclusions: Our hypothesis is that phloem-restricted expression of the R protein constitutive mutants would limit the potential negative impacts on plant growth. Use of the PAD4 promoter to express the R protein constructs should further increase the stringency of transcriptional control over expression of the potentially harmful R proteins. Our goal is have only the single phloem cell that is penetrated by the stylet express the constitutively active R protein, hence limiting potentially detrimental effects and focusing a robust defense at the source of Liberibacter introduction. Additionally, these constructs with the PAD4 promoter may reduce spurious expression in callus cells during the transformation protocol into citrus.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
