USDA Ft. Pierce (Neidz) Agrobacterium-mediated transformation of mature tissue explants: Transformation of mature internode explants from greenhouse trees has been demonstrated in four citrus types including Valencia sweet orange (1 plant), Ruby Red grapefruit (1 plant), US-942 (8 plants), and Etrog citron (8 plants). Current efforts are directed toward characterizing this system for routine transgenic plant production. 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 entitled, ‘Bud emergence and shoot growth from mature citrus nodal stem segments’ was accepted for publication by the journal Plant Cell, Tissue and Organ Culture. The paper 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. 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. Mineral effect on shoots regeneration: Preliminary results suggest that mineral nutrition significantly affects in vitro culture response. The effects on transformation are currently being studied. University of Florida (Moore, Grosser, Gmitter) Efforts continue with greenhouse grown tissue (CREC) Rootstock effect on mature tissue transformation: the experiment conducted to determine if vigorous allotetraploid rootstocks could increase transformation efficiency was compromised by endogenous fungal contamination. We are now testing coconut fiber, sterile liquid nutrition, and low humidity in a clean environment for growing mature tissue explants in efforts to minimize problems with fungal contamination. Characterization of mature-tissue transgenic ‘Hamlin’ plants: recovered mature-tissue derived transgenic ‘Hamlin’ plants from previous experiments were propagated via micro-grafting for further characterization. Research continues on using cell penetrating peptides (CPPs) to deliver cargo (proteins, chemicals, plasmid) to existing citrus cells (Gainesville). Using the easily visualized GUS enzyme, we have found that we can efficiently get protein imported into a number of citrus tissues, using several different CPPs. Currently we are testing import of plasmid DNA, which should let us test clones and constructs before we do stable transformation. Based on a recent report on woody plants, we are also investigating whether we can produce cultures of rapidly proliferating cambial cells from citrus (Lee et al. 2010. Nature Biotechnology 28:1213).
We reported last quarter the cloning of the ctEDS1 gene in the binary vector pBINplusARS. We have already transformed the Arabidopsis eds1-2 mutant with this construct. The T0 seeds were harvested and will be selected for the transgenic plants in the next few weeks. Additional newly cloned genes include ctSID2, encoding the major biosynthetic enzyme for salicylic acid biosynthesis, and ctNHL1, which is a homolog of NDR1. These two genes were obtained from RACE followed by RT-PCR. The cDNA fragments of these genes are now in the pGEM T-easy vector and were confirmed with sequencing. The next step will be to clone these cDNA fragments to the binary vector pBINplusARS for plant transformation. Since the recent release of the Citrus sinensis (sweet orange) and clementine genome sequence, we have conducted extensive bioinformatics analysis on defense related genes in citrus based on published literature. Such analysis confirmed citrus defense genes that have already been cloned in my laboratory with this support. In addition, we found that most published defense genes are present in citrus with full-length sequences available. Therefore, we anticipate that our further cloning and functional characterization of citrus defense genes should be greatly expedited. We have so far selected additional 10 candidate citrus defense genes for the next round of cloning and complementation analysis.
Huanglongbing (HLB) is a serious and devastating disease of citrus caused by Candidatus Liberibacter spp. and vectored by the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The disease has the potential to greatly limit the production of citrus in Florida and other citrus growing regions worldwide. Current control of ACP and HLB is inadequate, but identifying and incorporating traits from uncultivated Citrus spp. and Citrus relatives that confer resistance to ACP is a potential strategy to manage the disease. In a study by USDA-ARS, 87 genotypes primarily in the Rutaceae orange subfamily Aurantioideae, were assessed in the field in South Florida for resistance to natural populations of ACP. The majority of genotypes hosted all three life stages of ACP, however there were differences among genotypes in the mean ranks for eggs (F = 3.13, df = 86, P < 0.001), nymphs (F = 9.01, df = 86, P < 0.001), and adults (F = 4.21, df = 86, P < 0.001). Very low levels of ACP were found on two genotypes of Poncirus trifoliata, 'Simmon's trifoliate' and 'little-leaf'. Poncirus trifoliata, the trifoliate orange, readily forms hybrids with Citrus spp. and is commonly incorporated into rootstock varieties. The field experiment was followed by no-choice tests in which female ACP had the opportunity to lay eggs for six days on five genotypes of Poncirus trifoliata, three genotypes from the Citrus genera that were not represented in the field, and a control (Citrus macrophylla) to determine whether any genotypes were resistant to ACP. Numbers of eggs on the five genotypes of P. trifoliata (means between 7-60) were lower than on the control (mean = 281.3; .2= 59.5, P < 0.001), which indicates that genotypes of P. trifoliata show some resistance to ACP. Numbers of eggs laid on the three genotypes of Citrus (means 129-200) were not significantly lower than on the control (.2= 4.37, P = 0.23). An additional 107 genotypes, including 81 genotypes of P. trifoliata and trifoliate hybrids, were planted mid-January and will be screened for resistance to oviposition by ACP in the coming months. Studies have been initiated to compare plant volatiles associated with plant genotypes that are readily colonized by the psyllid to those less colonized by the psyllid. Collaborators with the Fujian Academy of Agricultural Sciences in Fuzhou, China, initiated two experiments on resistance to ACP within the Rutaceae. Forty genotypes were evaluated in a free-choice experiment conducted in a screen house. Citrus tankan Hort. (cultivar Fuyouxuan Jiagan) was completely avoided by adults, and no eggs or nymphs were ever observed on this cultivar. No eggs or nymphs were observed on the following: C. reticulata Blanco (cultivars Bayueju, Xiang Ponkan, and Mashuiju); C. mitis (cultivars Chengshi Calamondin and Variegated Calamondin); C. sinensis (cultivars Navelia Navel orange and Skaggs Bonanza Navel Orange); C. grandis (cultivar Chandler Pummelo), and Fortunella hindsii var Chintou. The most heavily colonized genotypes included: C. reticulata Blanco (cultivar Fina Sodea Clementine); C. sinsensis (cultivar Fengcai anliucheng); and C. grandis Osbeck (cultivar HB Pummelo). A free-choice field experiment was established comparing 31 genotypes. Due to prolonged cool weather, no results have yet been obtained from this experiment. A delegation from FAAS will be visiting USDA-ARS during April or May to coordinate research efforts.
Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: In addition to the SSI4 obtained from Arabidopsis thaliana var. Columbia genomic DNA, we created two new constructs (5-4) AtSUC2/SSI4 and AtSUC2/ssi4 mutant derived from Nossen genomic DNA (lines obtained from Dr. Klessig). Out of 49 ssi4 transgenics, 18 showed a reduced stature phenotype. However, none of the ssi4 expressing transgenics displayed a severe dwarf phenotype seen when expressed using their native promoter. We visited Dr. Orbovic’s laboratory at the UF CREC at Lake Alfred and resolved a technical problem regarding the PCR-based screening of citrus transformants. Dr. Orbovic has subsequently identified transgenic citrus for our two clones: AtSUC2/snc1 and AtSUC2/ssi4 mutants. Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. By further restricting expression of the R protein to the single cell that is pierced by the insect stylet, we anticipate that a defense can be mounted without a manifestation of a dwarf phenotype. The PAD4/ssi4 transgenics were difficult to obtain. We screened three times as many To seeds to obtain 18 plants. From these, 50% developed the “smaller stature” phenotype. Conversely, almost all transgenic plants containing SSI4 (wt) developed normally. Of the 33 transgenics with the PAD4/SNC1 construct, the majority showed no detrimental effect on growth. However, from the 37 PAD4/snc1 mutant transgenics, 10 were smaller than normal. We obtained 27 transgenics with the PAD4-reporter and analyzed GUS expression. Originally, the PAD4 promoter was selected based on literature reports of its phloem-specific expression and wound-inducibility upon the insect feeding. Our results indicated four basic groups of PAD4/GUS reporter expression in transgenic Arabidopsis leaves as follows: 1) phloem-specific expression with no wound induction; 2) universal expression with strong wound induction; 3) restricted expression with no wound induction; and 4) strong wound expression only. Conclusions: Our GUS analyses of two phloem-specific promoters (AtSUC2 and AtPAD4) revealed that transgenic plants do not maintain strict phloem-specific expression. Moreover, we found no PAD4 plants with the expected phenotype of wound-specific expression limited to phloem cells. However, substitution of the native promoters for ssi4 and snc1 genes with AtSUC2 and AtPAD4 did result in a significant reduction in the severity of the growth retardation phenotypes. Evaluation of more transformants will give us a better picture of the feasibility of utilizing these two promoters to obtain the desired expression patterns in citrus.
Once we have been able to establish at IVIA the procedures and conditions to transform mature Hamlin, Pineapple and Valencia sweet oranges, we are in conditions to transfer the basic protocols to Florida. We believe they can be reproduced with little or no modification at the new laboratory that is being set up at the CREC. As we have also developed a genetic transformation system for mature Carrizo citrange, we are now incorporating a construct of interest into this genotype. The second objective of our project was to develop genetic engineering strategies to improve citrus tree management. In this sense, we proposed to reduce endogenous gibberellin levels in transgenic rootstocks to make them dwarf or semidwarf. Such rootstocks could provide reduced size to non-transgenic scion varieties grafted into them. With this aim, we have incorporated a hairpin construct into Carrizo citrange to silence an endogenous GA20-oxidase gene and them reducing bioactive gibberellin levels in growing shoots. After Agrobacterium-mediated transformation, the explants regenerated abundant callus and showed prolific shoots formation. Around 70% of the explants regenerated shoots in the light step. At this moment there are several transgenic (PCR-positive) shoots micrografted in vitro. They are still pending of grafting on vigorous rootstocks in the greenhouse and Southern blot verification in coming weeks/months. New experiments will be run with this construct and fresh starting plant material within the next couple of months. For improving citrus tree management, we also proposed to over-express flowering-time genes in both the Carrizo citrange rootstock and the Pineapple sweet orange scion. This objective was initiated one year ago or so, and we have now at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange expressing either FT or AP1 flowering-time genes already established in the greenhouse. We continue characterizing them in detail. The PI and his greenhouse/growth room manager, Josep Peris, travelled to Florida last March 20-27th to visit the CREC and supervise the last steps of the growth room construction before been finalized (by the end of April, we guess). We suggested to revise minor details to make the facility more reliable and helpful for operators. We also short-trained the tissue culture technicians in horticultural practices. We checked substrate, seed stock and plant nutrition issues with the manager Dr. Zapata.
A comparative study of two susceptible hosts, Duncan grapefruit (DG, Citrus 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 early expression of genes related to programmed cell death (PCD). In kumquats but not in DG, several genes linked to PCD (lipoxygenase, glutathione transferase, metacapcase, acid chitinase and peroxidases) are expressed at 4 h post-inoculation (pi) with Xcc at 108 cfu/ml. Later at 24 h, additional genes related to plant defense (e.g. betaglucanase) are highly expressed in kumquats but less so in DG and RL and their activity continues to increase up to 48 h pi. Additional sets of genes related to PCD and the host pathogen interaction will be investigated this coming year. A cybrid is an asymmetric hybrid that contains the nucleus of one parent in combination with the mitochondrion and/or chloroplast of the cytoplasm donor parent. Twenty cybrids of highly susceptible Red grapefruit (RG) and the more tolerant Valencia orange (VO, C. sinensis) as the cytoplasm donor, were screened for their susceptibility to Xcc. Tolerance inherited from VO appeared to be quantitative based on an intermediate lesion phenotype in selected cybrids. In contrast to the callus-like lesions typical for susceptible RG, lesions were more necrotic for VO and the cybrids. This lesion phenotype indicated cell death arrested the proliferation of Xcc. Populations of Xcc at 14 days post inoculation in cybrids (7.2 Log cfu), were similar to VO (7.6 Log cfu) and one log unit lower than RG (8.4 Log cfu). Expression of genes related to host pathogen interaction in VO and cybrids differed from RG. The contrasting pattern suggested a differential interaction of genes from the nucleus with the mitochondria and chloroplast genes from the cytoplasm donor. Mitochondria and chloroplasts have a central role in stress and PCD signaling. The response of cybrids to Xcc confirms inheritance of resistance from VO may be expressed at different levels depending on whether mitochondrial and/or chloroplast genomes are transferred in the cybridization process. Field trials with several Ruby red grapefruit cybrids planted in canker-affected locations on the east coast were showing less foliar disease incidence than the adjacent Red grapefruit trees, but were severely damaged by freezes. Additional material has been planted out in canker prone locations. Production of cybrids lines using a new callus line of Meiwa kumquat as the cytoplasmic donor is underway in the Grosser lab. The resultant cybrids will be evaluated for inheritance of HR resistance using the same approaches
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters have been used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and are now replicated and are being exposed to HLB, using graft inoculations and CLas infected psyllids in greenhouse and field environments. Challenge with HLB through exposure to infected ACP (in collaboration with D. Hall) is being conducted on a replicated set (8 plants of each) of 33 independent Hamlin transformants, 5 Valencia transformants, 4 midseason transformants, and 3 non-transformed controls. It is anticipated that statistical analysis of CLas levels and symptoms will permit identification of material with significant resistance, for further testing. A series of promoters has been tested with the GUS gene to see how effective they are. As expected, the three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives unusually consistent and high GUS activity. Use of this promoter may reduce the number of independent transformants needed. CLas sequence data were used to target a transmembrane transporter (Duan collaboration),as a possible transgenic solution for HLB-resistance. Radiolabelled ATP is being used assess effect of identified peptides in E. coli expressing the CLas translocase. Collaboration with a USDA team in Albany, CA is: providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns; providing genes from citrus genomic data, from Carrizo citrange sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences; citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic Carrizo and tobacco and are being tested in citrus scions. Anthocyanin production genes,give bright red shoots (Gray collaboration) and are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 27 months and a repeated trial has been in the field for 15 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, where HLB and ACP are widespread. The first trees have been in place for more than fourteen months. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser has just planted an additional 89 tress including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been propagated for a replicated trial in collaboration with Fred Gmitter of UF and are growing well in the greenhouse. These will be planted in the spring of 2011, and monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. An experimental attract/kill product, to disrupt citrus leaf miner (CLM) without disrupting ACP, was not effective last year. Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids. CLM damage also compromises ability to view HLB symptoms. Several applications of Admire are being used to encourage an undamaged flush on transgenic trees. We are still learning how to grow trees for best assessment of HLB-resistance.
Previously we standardized quantitative real time PCR assays for 18 genes (AZI1, BLI, CHI, COI1, EDR1, EDS1, EDS5, JAR1, NDR1, NPR1, NPR3, PBS1, PR1, R13032, R20540, RAR1, RdRp, and SGT1) associated with SAR and plant defense. Using this technique and based on our previous results on defense gene expression during pathogen and PAMP inoculations we selected a few genes (AZI1, CHI, EDS1, NPR1, PBS1, R13032 and RdRp) that are differentially expressed during PTI/ETI/SAR and best characterize this response in citrus. We treated 24 ‘Carrizo’ citrange AtNPR1 transgenic lines with Candidatus Liberibacter asiaticus flagellin 22 peptide (L-flg22, as a proxy for the pathogen) and analyzed the expression of these 7 genes. The idea was to determine how the transgenic lines responded L-flg22, an inducer of PTI/SAR, and which transgenic lines showed an enhanced response compared to wild type plants. Several transgenic lines showed expression levels that were much higher for most of the genes compared to the wild type plants, further indicating that AtNPR1 seems to modify the defense response in citrus. We also studied the response grapefruit plants grafted on transgenic ‘Carrizo’ AtNPR1 plants infiltrated with L-flg22. The results are being analyzed.
During the past year we developed and standardized 8 more gene expression assays for the study of defense response in citrus using real time PCR. The 18 genes we have selected are either important in the early induction and regulation of SAR (AZI1, EDR1, EDS1, EDS5, NDR1, NPR1, NPR3, PBS1, R13032, R20540, RAR1, and SGT1), are targets of the regulatory SAR pathway (BLI, CHI, PR1 and RdRp) or are components of the jasmonic acid (JA) pathway (COI1 and JAR1) that works antagonistically to SAR. Several of these genes were previously undescribed for citrus, however our microarray studies indicated that these sequences were differentially regulated by chemical and pathogen treatment. Additionally, we continued to propagate more AtNPR1 ‘Carrizo’ citrange transgenic plants. To our previous lines (854, 857, 859 and 884) we have added 757, 775, 854, 890, 896 and also more of 857, the most promising line. We also studied the response of lines 854, 857, 859, 884 and wild type (WT) to Actigard (Syngenta Corporation), a commercial version of the SAR inducer salicylic acid (SA). We studied the effect of Actigard on gene expression levels either alone or followed by treatment with a Candidatus Liberibacter asiaticus Flagellin-like peptide (L-Fgl, as a proxy for the pathogen). In general, Actigard significantly induced some SAR genes (For example: CHI, EDS1, PBS1, RAR1 and RdRp) compared to water treated plants, these same genes were induced significantly higher in transgenic lines, specially 857 and these effects were observed as early as 6 hours after treatment . We are still analyzing the effect of the L-Fgl, although it seems to repress the expression of some SAR target genes, at least in the WT plants. We have not started the HLB inoculation experiments yet as the number of plants is limited and we wanted to characterize their response before the plants were permanently confined to a containment facility. However, we will initiate this part of the research as soon as we receive the last year of funding for this project.
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. Results showed that the use of the antioxidant lipoic acid significantly improves transformation efficiency in Mexican lime, and a manuscript reporting this was accepted with revision for publication in PCTOC. Recovered transgenic Mexican lime trees 1.5 years after removal from tissue culture were girdled, which induced flowering about 4 months later; with complete flowering and fruit set in less than two years. 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. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Working with Mr. Orie Lee and Faryna Harvesting, yield and fruit quality data was collected from the St. Helena project. Approximately 10 acres of trees planted 3.0 years ago include a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks. The best rootstocks identified to show positive affects on rapid tree growth with precocious bearing and good early fruit quality (higher lb. solids) were somatic hybrids Changsha mandarin+trifoliate orange 50-7, white grapefruit+trifoliate orange 50-7, and sour orange+Carrizo, and tetrazygs White#4 (Nova+HBPummelo x Succari sweet orange+Argentine trifoliate orange), Orange#13, Orange#14,Orange#18 and Orange#19 (Orange series all Nova+HBPummelo x Cleo+Argentine trifoliate orange). Trees on these rootstocks averaged more than a half-box of fruit per tree with juice brix values great than 11. These rootstocks will now be tested to determine if they can shorten juvenility in transgenic plants produced from juvenile explant. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL and Vernia sweet oranges were successfully micrografted to Carrizo citrange or experimental Tetrazyg rootstocks and are growing well in the greenhouse. These will now be clonally propagated onto the rootstocks mentioned above for further study of early flowering and transgene expression. Horticultural manipulations on these plants will include the RES (Rapid Evaluation System) growth method plus girdling. Transformation with early-flowering genes: ‘Citrus has at least 3 FT genes. Cloning and characterization of all 3 (genomic clones) has been completed. We have put them into transformation vectors with a constitutive promoter and performed transformation experiments with Carrizo and Duncan. Although only a few transgenic plants were recovered, we know the constructs work because we had previously tested them in tobacco, where we recovered early flowering phenotypes, as well as some other phenotypic alterations. Our current theory is that expression with the 35S promoter is too strong in citrus (in some cases we got flowering directly on the initial explant!). We are currently testing a poplar FT with a weaker inducible promoter (a heat shock promoter shown to be inducible in Oregon); which will hopefully solve the promoter problem.
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.
When dsRNA targeting either a psyllid cathepsin or a psyllid vacuolar ATPase gene are fed in artificial diets to the Asian citrus psyllid, an increase in psyllid mortality is realized. The oral uptake of ~300 bp dsRNA fragments matching the coding region to either psyllid Vacuolar ATPase or cathepsin can induce mortality in the Asian citrus psyllid. Comparisons were made to determine the optimal dsRNA size. Psyllids were fed either the ~300 bp dsRNAs directly or after processing to siRNAs with the Dicer enzyme. Results showed that the 300 bp dsRNAs induced greater mortality and than that observed with processed siRNAs. Furthermore, non-linear dose dependent toxicity of the ~300 bp dsRNAs suggesting complex interactions that have not yet been characterized with respect to dsRNA induced toxicity in insects.
Seed was collected from new crosses made the previous year to develop improved rootstocks and scions with tolerance to HLB and other improved traits. Selected hybrid seed was planted in the greenhouse. Promising SuperSour rootstock hybrids were identified and propagated for further testing. Cooperative work began with a commercial nursery to propagate the most promising SuperSour hybrids for large-scale commercial field trials. Fruit quality, yield, and tree size information were collected from 8 existing replicated rootstock and scion field trials. Performance data from field trials was summarized in a presentation at the Indian River Citrus Seminar. A field trial was planted in Indian River County to evaluate SuperSour selections in heavy flatwoods soil for tolerance of Phytophthora and Diaprepes weevil. A greenhouse study was completed to evaluate the tolerance of SuperSour rootstock hybrids to citrus tristeza virus. Data was collected from a replicated field trial in St. Lucie County to evaluate HLB tolerance of ten rootstocks exposed to natural infection in the field. Propagations from SuperSour rootstock hybrids were budded with ‘Hamlin’ scion to produce trees for disease testing and new rootstock field trials in the coming year. Field studies continued to assess the tolerance of different rootstocks to HLB with sweet orange scion under natural conditions, and work is beginning on a summary of those findings for publication. In coordinated research between this grant and the FCATP transgenic citrus grant to USDA, selected anti-microbial, insect resistance, and other genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with resistance to HLB and Citrus Bacterial Canker. Seed from the new USDA rootstocks US-812, US-897, US-802, and US-942 produced at the USDA Whitmore Farm was collected and provided to the Florida Citrus Nurserymen Association for commercial distribution. A study describing the tolerance of US-897 to HLB was published in the journal ‘HortScience’. In the study, both US-897 field trees naturally infected by HLB and greenhouse-grown trees that had been inoculated with Liberibacter through graft inoculation were observed to develop few or no symptoms of HLB, even though PCR clearly indicated the presence of Liberibacter infection. Typical symptoms of HLB found in other citrus, such as leaf blotchy mottle, shoot stunting and yellowing, and seed abortion were rare and not easily visible on US-897. It was noted that the HLB tolerance of US-897 probably is derived from its trifoliate orange parent. Experiments are planned to determine whether the tolerance to HLB found in US-897 results in increased tolerance for grafted trees with susceptible scions on US-897 rootstock. A greenhouse study is underway to compare the apparent HLB resistance of several different trifoliate hybrid rootstocks. Greenhouse studies continued to compare germplasm tolerance to HLB under carefully controlled conditions, and understand the impact of different factors on that tolerance. These studies will provide additional insights about how to engineer HLB resistant cultivars. Additional greenhouse and field studies are also underway to determine the most efficient methods to evaluate new citrus germplasm from crosses and transformation for resistance or tolerance to HLB. Greenhouse studies are underway to compare infection and symptom development in plants inoculated with Liberibacter by graft and Asian citrus psyllid.
1-The first objective of the second year was to build and start the operation of a plant growth room at the Citrus Research and Education Center in Florida (CREC). The growth room construction started on October 22nd 2010 and the projected finish date was February 11th 2011. There was a delay of a few weeks and the main contractor will finalize on March 25th, but the computer system contractor is still finishing the programming that will control the environmental conditions. We checked the growth room and it is working as expected however disposal of the waste stream will be a concern when the growth room is in full operation since the water that we will dispose needs to be collected in an external tank and test by the county to guarantee that we are not disposing contaminants that can affect the environment. We already started furnishing and placing equipment inside the building. Because of the delay in the construction, the growth room is not in fully operation yet. We believe this will take at least one additional month. The cost of the construction was higher than the original budget plan; the extra funding was provided by CREC and IFAS facilities as agreed initially. 2- A full time technician with nursery experience was hired after several months of searching. The process of hiring was slow. A first candidate was hired and the offer of employment was rejected due to a low salary offer. A second candidate was found but he quit two months after hiring. We hired a third technician with limited experience and he will start the first week of April. It seems like salary will be an important issue in the future to recruit and retain personal. 3- Training of the manager Dr. Zapata was completed at the IVIA under the supervision of Dr. Pena. It was emphasized during the training the improvement of transformation methods for more recalcitrant types, molecular analysis of the regenerants and plant material preparation at the greenhouse/growth room, including micrografting, phytosanitary treatments, fertilization and pruning. An annual schedule for completion of planting, transplanting, grafting and obtaining budsticks to transform was developed. 4- The Mature Transformation Laboratory was established, using an existing laboratory located at CREC. Two technicians with limited experience were hired and are currently being trained in the first tissue culture techniques, including culture media preparation, grafting, micrografting, explant preparation, culture and regeneration, etc. 5-Our selected varieties Hamlin 1-4-1, Pineapple S-F-60-3 and Valencia S-SPB-1-14-19 were subjected to cleaning through shoot tip-grafting at the Florida Department of Agriculture and Consumer Services (DOACS) with the help of Dr. Peggy Sieburth. They are kept at out lab and ready to be grafted on rootstocks when the growth room is fully operative. 6-Dr. Leandro Pena and his greenhouse and growth room manager Josep Peris made a visit of one week in March 2011 to supervise the last steps of the growth room construction before been finalized and suggested minor details to make the facility more reliable and helpful for operators. They also short-trained the tissue culture technicians in horticultural practices. They checked substrate, seed stock and nutrition issues with the manager Dr. Zapata.
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