This research project is directed towards controlling psyllids using biologically-based control strategies that employ the use of RNAi technology against key biological control pathways, peptide hormones and protein inhibitors that, if expressed in transgenic citrus, would enhance plant resistance to psyllid feeding. Both protein-based and RNAi strategies were tested by feeding psyllids artificial diets. To support the artificial diet assays, we optimized the diet composition by adding an antimicrobial agent to eliminate fungal growth that is introduced by the psyllids during the assay period. Using this approach we identified suitable buffers and optimal diet pH during the feeding period. In separate experiments, Tryspin Modulating Oostatic factor (TMOF), a mosquito decapeptide hormone, and cysteine protease inhibitor (CPI) from the Asian Citrus psayllids that was identified in our laboratory were added to artificial diets on which psyllids were allowed to feed. After 10 days of feeding, 100% mortality was observed in psyllids feeding on diets containing TMOF or CPI, whereas, 40% mortality was found in psyllids feeding on the control diets. CPI fed psyllids caused a significant higher mortality than the controls after 7 days of feeding. Based on these observations a collaborative project with Dr. Bill Dawson’s laboratory (Univ. of Florida, IFAS, CREC, Lake Alfred, FL) was initiated to use a Citrus tristeza virus (CTV) expression vector to produce TMOF and CPI in the in the citrus phloem. Clones containing the sequences of mosquito TMOF and CPI (cathepsin protease inhibitor) (both shown to be effective against psyllids) were provided to Dr. Dawson’s laboratory. Once CTV vectors are constructed and used to infect citrus plants, Dr. Dawson’s laboratory will provide us with the plants to evaluate the effect on psyllids while feeding on these plants. In parallel to these studies, we synthesized dsRNA molecules targeting 11 different psyllids essential genes encoding three different classes of proteins (alpha-tubuliln, V-ATPase, and Cathepsins). Initial feeding studies with alpha-tubulin dsRNA and V-ATPase dsRNA caused ~60% psyllids mortality as compared to only ~30% mortality for psyllids fed a control diet containing an equal amount of dsRNA not specific to the psyllid. Using this information, we have initiated experiments to produce citrus plants that express these dsRNAs in citrus phloem cells. A collaborative project with Dr. Bill Dawson’s laboratory (Univ. of Florida, IFAS, CREC, Lake Alfred, FL) was initiated to use a Citrus Tristeza Virus (CTV) expression vector to produce the psyllid dsRNAs in the citrus phloem. Clones containing the sequences of a psyllid Vacuolar ATPase and Cathepsin protease (both shown to be effective dsRNA targets) were provided to Dr. Dawson’s laboratory. Once CTV vectors are constructed and used to infect citrus plants, Dr. Dawson’s laboratory will provide us the plants to evaluate the effect on psyllids while feeding on these plants. We have also designed expression vectors for the production of transgenic plants expressing the psyllid dsRNAs through Agrobacterium-mediated plant transformation. These will be used to initiate the production of transgenic citrus plants that constitutively produce these dsRNAs in the phloem.
New lemon selections suitable for the California desert climate are needed to diversify production. Desert lemons occupy an important early-season market niche, which could be lost to international competitors. Lemons are also an important source of fruit for packinghouses located in other areas of the state. This ongoing project, which addresses the CRB ‘Production’ priority for New Variety Development, was designed to evaluate twelve lemon selections under desert conditions. The immediate objectives of the project are to provide the industry with information on the tree growth, yield, packout, and fruit quality characteristics for the lemon selections in the California desert. These include: ‘Allen Eureka’, ‘Variegated Pink-Fleshed Eureka’, ‘Corona Foothills’ (a bud sport of ‘Villafranca’), ‘Limoneira 8A Lisbon’, ‘Walker Lisbon’, ‘Femminello Santa Teresa’, ‘Interdonato’, ‘Limonero Fino 49′, Limonero Fino 95’, ‘Messina’, ‘Seedless’ lemon and ‘Yen Ben’. There are 20 trees of each variety, grouped into five replications of four trees each. All are budded to Citrus macrophylla rootstock. Leaves were collected for nutrient analysis for all 12 varieties in September, 2010; these are currently being processed and analyzed. The first harvest for 2010 took place on November 2, 2010, using professional harvesters from a local packinghouse. We collected yield from each group of four trees by counting the numbers of whole and fractional picking sacks harvested from each group. Also, we collected fruit packout and exterior fruit quality from each variety using a portable fruit line. For the first harvest, ‘Corona Foothills’, Limonero Fino 49′ and ‘Walker Lisbon’ had the greatest yields ranging from 200 to 250 lbs. per tree, while ‘Seedless’ lemon, ‘Variegated Pink-Fleshed Eureka’ and ‘Yen Ben’ had the least yield of less than 75 lbs. per tree With regards to fruit packout, ‘Messina’ had the largest size, peaking on sizes 75 and 95, while ‘Corona Foothills’, Interdonato’, ‘Limonero Fino 49’ and ‘Limonero Fino 95’ peaked on sizes 95 and 115. ‘Variegated Pink-Fleshed Eureka’ and ‘Yen Ben’ had the smallest sized fruit, peaking on size 140. There was little effect of selection upon fruit exterior quality. Samples of 20 fruit per group of four trees were collected for determination of interior fruit quality, including peel thickness, peel smoothness, percentage juice, juice pH and TSS/TA ratio. These data have not yet been analyzed. Several growers attended a Field day and saw the trees first-hand at the experimental block on September 28, 2010.
Two trees have been found growing in HLB-ravaged orchards in Guangdong and one other in Guangxi province, that appeared to be free of HLB symptoms, while all other trees planted at the same time were either dead or declining, and replants likewise were afflicted. The trees from Guangdong were propagated at the Guangdong Institute of Fruit Tree Research facilities, and are now planted in their research field to assess their reaction to natural inoculation with HLB. We visited these trees in October 2010 and observed no HLB symptoms, though surrounding trees showed obvious signs of disease. The tree in Guangxi that was transplanted to a protected location at the Guangxi Citrus Research Institute, was used to make several propagations, and these were deliberately inoculated by grafting infected material. These propagated trees have expressed symptoms of HLB now. They apparently are not resistant, but the question remains as to why the original source tree was not infected, and still appears symptom free. We visited with a citrus extension specialist from the Fujian Provincial Academy of Agricultural Sciences, Mr. Li, Jian, also in October 2010, to expand further our search for survivors, and to continue to learn about Chinese citrus industry adjustments in response to HLB. We visited the Pinghe County Guanximiyou (Chinese honey pummelo) production area, where we saw HLB being managed through good psyllid control, good nutrition and subsequent tree health, and the natural tolerance of this pummelo variety. We returned to specific orchards we visited previously in Guangdong and Guangxi. These were in very healthy condition in 2008, despite widespread infection of neighboring plots. The orchard in Guangdong was devastated with HLB, and we met the grower again; we were informed that fruit prices for his variety were very low and he simply stopped taking care of his trees. Orchards revisited in Guangxi were a different matter. These orchards have not only maintained their healthy condition, but they were substantially more productive and healthier in appearance than two years earlier. This confirms the utility and effectiveness of the management strategy being employed under direction of entomologist Deng of the Guangxi Citrus Research Institute. We interviewed growers, pathologists, horticulturists, and entomologists associated with these healthy orchards. The key elements outlined to us were critically timed pesticide applications, use of pathogen-free planting materials, and maintenance of tree health through good nutrition.
The objectives of this project are to develop, test and evaluate citrus rootstocks for disease and pest tolerance, and to select stocks that impart to the scion high yield, superior fruit quality, acceptable fruit size, and other essential traits. Activities involve production on new potential rootstocks by hybridization, screening these for valuable traits, and analysis of performance in replicated field trials. This report summarizes accomplishments from November 2010 to February 2011. We completed data collection for a rootstock trial with Moro blood orange scion. Yield, packline, and fruit quality analysis were completed for the Fukumoto rootstock trial at Lindcove. A trial of satsumas on 4 rootstocks was also harvested as planned. We continue to propagate trees of Washington navel on 28 rootstocks for a new trial to evaluate rootstock effects on early bearing and responses to high intensity management. This trial should be planted during summer of 2011. Seedlings for planned trials of Clementine and DaisySL mandarins are being grown at Lindcove, but these did not grow well after transplanting and we may have to start this trial again. Seeds were collected from standard rootstocks and new hybrids to be used in future rootstock trials and for future iron chlorosis and salinity tolerance screening trials. A Phytophthora citrophthora root rot resistance trial was planted in January.
TThe objectives of this project are to develop new mandarin, orange, lemon and grapefruit-type cultivars suitable for California conditions. The major approaches being used are hybridization-selection for mandarins and grapefruit, and mutation induction by gamma irradiation to obtain seedless forms of existing mandarins, oranges, and lemons. Promising selections are evaluated in field trials for tree size, yield, fruit quality, and disease susceptibility. This progress report summarizes accomplishments from November 2010 to February 2011. Hybridization during the 2010 season was quite successful and we have been harvesting and initiating culture of large numbers of new hybrid seed. No new irradiation has been conducted yet this year. Most work this year has involved evaluation of hybrids and selections from mutation breeding. Evaluation of new hybrid populations and trees grown from mutagen-treated buds is on schedule, with hundreds of trees evaluated to date. These include a population to be used to map genes influencing fruit quality and other traits. In the 6 replicated trials, detailed fruit quality studies are being conducted on DaisySL, FairchildLS, Nova IR10, KinnowLS, Encore IR6, 3 hybrid mandarins, and one grapefruit type, with Clementine and Tango as early and late season comparators. KinnowLS was released for propagation in California in late January 2010.
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.
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