In cloning the three SA genes, EDS1, SID2, and WIN3, we currently confirmed the cloning of the full-length ctEDS1 and are in the process of moving the sequence to the binary vector for plant transformation. We showed in the last progress report that we obtained 3′ end RACE sequence for ctWIN3 and 5′ end RACE sequence for ctSID2. In order to amplify the other ends of the two genes, we tried to design different primers for RT-PCR. We also performed TAIL PCR, in which we used citrus genomic DNA as a template in a series of PCR in order to obtain the missing regions of the two genes. However, these attempts were unsuccessful. With Carrizo sequence database (http://citrus.pw.usda.gov/) recently available, we have been doing bioinformatics analysis and have identified additional SA genes that have citrus homologs with available sequence. We are currently design primers to further amplify these additional SA genes. For ctEDS5/pBINplusARS transformation, we obtained 5 Col-0 and 5 eds5-1 carrying the transgene. We are in the process of screening T0 seeds for additional independent transformants. In the meantime, we planted these 10 transgenic plants for disease resistance assay with Pseudomonas infection. We continue to characterize the transgenic plants overexpressing ctNDR1/pBINplusARS, ctNPR1/pBINplusARS, or ctPAD4/pBINplusARS. We obtained 4 homozygous ndr1 + ctNDR1/pBINplusARS and performed disease resistance assay. The recent data confirmed our earlier report that ctNDR1 complemented Arabidopsis ndr1 mutant. Additional analysis will be conducted to verify this result and to further characterize the defense phenotypes of the transgenic plants. For plants overexpressing ctNPR1/pBINplusARS or ctPAD4/pBINplusARS, we did not observe complementation of npr1 or pad4 mutant with transgenic plants currently obtained. We reason that overexpression of these two genes may be toxic or citrus cDNA clones may not be well expressed in Arabidopsis. We are currently trying to clone the genomic fragments of these two genes. We will repeat npr1 or pad4 complementation once we obtain the genomic clones.
During the 2nd quarter of funding, the Core Citrus Transformation Facility (CREC) continued it’s mission of producing transgenic Citrus plants according to the orders from multiple clients. The demand for genetically transformed citrus plants remains high. Most recently, CCTF received three new orders to produce transgenic grapefruit carrying genes harbored in following vectors: p19-5; p20-7; and p21-1. The bulk of the work presently revolves around orders placed in the previous quarter but work also goes on to complete older orders. Out of presently serviced orders, all except two are concerned with resistance of different citrus cultivars to diseases, primarily HLB and canker. The following transgenic citrus plants were delivered to various researchers: Resistance to bacterial diseases-canker and HLB: 1) N1* gene: two Duncan plants; 2) pCIT108P3 vector: two Flame plants; 3) NPR1: three Flame plants and superNPR1-four Hamlin plants; 4) AS7 gene: eight Duncan plants and A13* gene: four Duncan plants; 5) pMOG800 vector: two Duncan plants. Resistance to CTV: 1) Gene in p33 vector: 18 Mexican limes, 16 C. macrophylla, and seven Hamlin plants. Orders not associated with citrus disease resistance: 1) CL1 gene: one Duncan. 2) pHK vector: 12 Mexican limes. During this quarter, more than forty recovered new transgenic plants were soil-adapted, and are ready for PCR testing to confirm the presence of the trasngene of interest. Please be informed that the person directly managing the CCTF (and co-PI) is Dr. Vladimir Orbovic.
Objective 1: A comparative study of two susceptible hosts, Duncan grapefruit (DG, C. paradisi), and Rough lemon (RL, C. jambhiri) and two resistant cultivars of kumquat (Fortunella spp.), ‘Meiwa’ and ‘Nagami’, evaluated the mechanisms involved in the resistance of kumquat to the citrus canker. MK and Nagami NK developed a hypersensitive response (HR), with necrotic lesions with population of Xanthomonas citri subsp. citri (Xcc) < 5 log units after 168 h in detached leaf and attached leaf assays. Early expression of genes related to programmed cell death associated with HR were identified in MK and NK. The resistance in kumquats has several characteristics associated with HR: 1) Rapid necrosis of leaf tissue in 48-72 h post inoculation in vitro or 72-96h in planta; 2) Disruption of epidermal and mesophyll cells by 72 h; 3) Xcc bacterial ingress limited to few cell layers below the epidermis; 4) Xcc population growth arrested at 72 h coincident with the cellular disruption; 4) Light microscopy and TEM, show death of the cells adjacent to the inoculation site with very few bacteria proliferating; 6) 5) HR-related genes and other putative resistance-related genes expressed early in resistant KN but not in susceptible DG. Behavior of susceptible DG and RL was: 1) No symptoms are detect in susceptible until 72 hr after inoculation and water soaked developes at 168 hr; 2) pustular callus-like lesions erupted through the cuticle by 10-16 days post inoculation (dpi); 4) Xcc populations reach 6 log cfu of Xcc per inoculation site at 168 hr; 5) Xcc population increases up to 15 dpi. Objective 2: Validate the inheritance or resistance for cybrids with susceptible Red grapefruit (RG) and RL with Valencia orange (VO). The putative RL cybrid has been recently been fully analyzed and determined using Single stranded repeat (SSR) analysis to be a cybrid from a mislabeled callus line of Valencia orange and not Meiwa kumquat. Hence the inheritance of resistance is not the HR type but is one of moderate susceptibility compared to high susceptibility in RG. Evidently, there is a definite expression of resistance in the cybrid inherited as a result of presence of the heterologous mitochondrial or chloroplast genome from the VO callus line. Evidence for this is as follows: Intermediate lesion symptoms are observed for RL+VO cybrid in vitro and in-planta. In contrast to development of callus, the inoculated area develops necrosis by 10 dpi. Xcc population plateaus by 10 dpi below the bacteria populations susceptible RL or Red GF. Two types of lesion were observed: necrotic and also callus, suggesting that cell death occurs and arrests the proliferation of Xcc. Expression of HR-related genes is intermediate between MK and RL, further substantiating that some yet to be determined elements of resistance have been inherited in the cybrids. Finally, the current set of Ruby red grapefruit cybrids with VO planted in canker-affected locations on the east coast continue to be more resistant than Red grapefruit trees around them. To expedite and standardize the evaluation of resistance in citrus germplasm, a prototype needle-free device was designed and evaluated for delivery of Xcc into the leaves of cultivars susceptible and resistant to citrus canker. The device delivered a precisely controlled volume of bacterial suspension through infiltration of stomates by injection with pressurized gas. The device produced a uniform inoculation of bacteria into the leaves as measured by the volume of infiltration and diameter of the infiltrated area. No damage to the leaves was observed after inoculation with the automated device, even though a higher number of canker lesions developed compared to a hand-held needleless syringe injection method. The level of practice needed for operation of the automated device was minimal compared to considerable skill required to perform the hand-held injection. Results from inoculations with the automated device are in accord with the results for the hand-held syringe method.
This is a 3-year project with 2 specific aims: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, the pBI1.4T-AtMKK7 construct has been mobilized into the Agrobacterium strain EHA105. The culture of the Agrobacterium was used for co-incubation with ‘Duncan’ grapefruit explants. About 1700 explants were incubated and 17 shoots were tested with PCR. Nine of these shoots were positive in the initial screen and all of them were grafted onto Carrizo. The transgenic plants are growing. Further confirmation of the presence of the AtMKK7 gene in the transgenic plants by PCR and analysis of the expression levels of AtMKK7 in each transgenic line will be performed. Resistance of the transgenic lines to citrus canker and greening (HLB) will be characterized when the transgenic plants are available. For objective 2, we previously did irradiation for the first batch of ‘Duncan’ grapefruit hypocotyl cuttings with a irradiation dosage of 40G. The irradiated cuttings generated significantly fewer shoots than the control, suggesting that the irradiation caused severe damage to the hypocotyl cuttings. Calli were formed on both irradiated cuttings and the control. The shoots and calli generated on both the irradiated cuttings and the control have been transferred onto selective medium containing 0.2 mM of sodium iodoacetate. We prepared another batch of explants (90 tubes of ‘Duncan’ grapefruit seedlings) for irradiation. A total of 90 plates of hypocotyl cuttings (each plate with 40-50 stem pieces) was irradiated with a dosage of 30G on 06/28/10. The irradiated stem pieces were placed on non-selective shooting medium. Ten plates of hypocotyl cuttings were kept as non-irradiated controls, for future comparison. The plates are kept under 14 hour photoperiod. Shoots generated from the irradiated hypocotyls were transferred onto selective medium with 0.2 mM of sodium iodoacetate. We are also preparing the third batch of hypocotyls for irradiation.
About 500 supersour-type (SS) rootstock hybrids have been selected for propagation and further testing. Selected SS rootstocks are being evaluated for tolerance to CTV quick decline and propagated for placement into field trials. Commercial cooperators are being identified who will host early stage trials of some SS rootstocks. Rootstock liners were budded with scions to prepare trees for trials. Budded greenhouse trees for field trials were grown to planting size. A new field trial was planted to assess the interaction of rootstocks and scion cultivars on tree performance under an open hydroponic management system. Data on tree size and HLB titer were collected from several rootstocks trials to assess rootstock effect on tree growth under HLB disease pressure. Data were collected from a trial planted on trellis to examine the effect of tree manipulations on the length of time for transition from juvenility to maturity. This information will be valuable to accelerate the pace of development for new rootstocks and scions. Studies continue to assess citrus germplasm tolerance to Huanglongbing (HLB) and Phytophthora/Diaprepes in the greenhouse and under field conditions. In a new greenhouse study, Poncirus trifoliata, Cleopatra mandarin, and several hybrid selections were inoculated with HLB to further evaluate the apparent HLB tolerance in some trifoliata-type selections revealed in a previously completed greenhouse study. Greenhouse trees inoculated with Citrus tristeza virus (CTV) were tested for virus titer in preparation for CTV-induced decline evaluation of supersour rootstocks. More than fifty citrus genotypes and citrus relatives, as well as thousands of progeny from crosses, have been challenged by natural inoculation with Liberibacter in the field, and data are being collected on HLB symptoms and Liberibacter titer by PCR. Detailed information is being collected on HLB tolerance and tree performance in four rootstock field trials. All citrus germplasm and cultivars become infected with Liberibacter when inoculated, but different germplasm responds to HLB infection at different rates and with different symptom severity. Greenhouse and field studies are continuing to determine the most efficient methods to evaluate new citrus germplasm from crosses and transformation for resistance or tolerance to HLB. Preliminary evaluations were completed on gene expression in HLB-susceptible and HLB-tolerant selections to identify differences that can help guide selection from conventional breeding and transgenic efforts. 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. Efforts continue to transform trees with seven different promoters and three new anti-bacterial genes targeted at producing HLB-resistant cultivars. Testing of transgenic plants for HLB resistance continues under the resources provided by this grant. Genetic transformation was used to introduce the citrus FT gene for induction of early flowering into citrus scion and rootstock germplasm. Commercial field plantings of the new seedless mandarin cultivar ‘Early Pride’ were planned with cooperators. The final revision of the release notice for US-942 rootstock was prepared and submitted for official approval.
Although Year 2 of this project officially began on July 1, 2010, funds did not reach any of the researchers until 9/9/10, and at this time, only the UF PI (Moore) and the UF subcontractors (Grosser, Gmitter) received funding. The USDA subcontractors did not get their funds until late in September due to procedures at UF and USDA. Therefore little new research was started in this quarter. Other funds were found to maintain materials and personnel.
Objective: Determine if Carrizo rootstocks, either wild type or over-expressing the Arabidopsis NPR1 gene (with an enhanced, inducible defense response) have any effect on gene expression and/or the defense response of wild type (non transgenic) grapefruit scions to HLB. We recently started to propagate new lines from cuttings of 9 individually transformed plants: lines 757, 761, 763, 775, 854, 857, 890, 896 and 897, all transformed with the AtNPR1 (We had to wait until the plants were large enough to withstand the taking of multiple cuttings). However, we have found that propagation by cuttings is difficult with certain lines and, even when it is possible, it may take several months for new growth on the cuttings. This process is still underway. Concurrently, we have standardized more probes and primers for the detection of SAR-associated citrus genes. Making these primers and probes requires knowledge of nucleotide sequence of the genes. Then the primers and probes must be tested and conditions optimized before experiments can be done. The list of genes we can test now includes: AZI1, BLI, CHI, EDR1, EDS1, EDS5, NDR1, NPR1, NPR3, PBS1, PR1, R13032, R20540, RAR1 and SGT1, in addition to our controls 18S and COX. We chose these 15 genes because they are either important in the early induction and regulation of SAR (AZI1, EDR1, EDS1, EDS5, NDR1, NPR1, NPR3, PBS1, R13032, R20540, RAR1 and SGT1) or are targets of the regulatory SAR pathway (BLI, CHI and PR1). In Objective 1 of this project, we propose to compare the response of AtNPR1 transgenic plants vs. wild type plants to the treatment of the SAR inducer salicylic acid (SA), by testing for expression of the above listed genes. This has been accomplished for the first set of lines. We intend to repeat this experiment with the increased number of transgenic lines once they are ready, with the increased number of genes we have identified, and using the commercial version of SA (Actigard, Syngenta Corporation).
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 have completed several large screenings of antibacterial peptides against Las in sweet orange trees. About 50 different antibacterial constructs have been tested in trees. We have found two peptides that appear to effectively protect sweet orange trees from HLB. However, we and other labs continue screening for better genes that more effectively control HLB and can be approved for use in a food crop. In the California lab, we developed methods to rapidly screen anti-bacterial peptides against Ca. L. psyllaurous in tobacco plants. Tobacco plants were either inoculated with Ca. L. psyllaurous by using the tomato psyllid (Bactericerca cockerelli) and challenged one week later with recombinant Tobacco mosaic virus (TMV) expressing the specific peptides, or the plants first were inoculated with recombinant TMV, followed one week later by using B. cockerelli to inoculate Ca. L. psyllaurous. These assays are being analyzed presently. We also are improving the CTV-based vector to be able to produce multiple genes at the same time. This could allow expression of genes against HLB and canker or multiple of genes against HLB. Another major goal is to do a field test of the CTV vector with antibacterial peptides, which is an initial step in obtaining EPA and FDA approval for use in the field. After some delays, we have received permission for USDA APHIS and are now establishing the field test.
This project has three objectives: 1) gap closure of Ca. Liberibacter asiaticus (Las) found in Florida; 2) complete genomic sequencing to closure of Ca. L. americanus (Lam) strain S’o Paulo from Brazil, and 3) comparative genome analysis of Las and Lam to attempt to determine common factors enabling pathogenicity to citrus. Objective 1 Progress: The recently published Ca. Liberibacter asiaticus (Las) strain psy62 genome, derived from a psyllid, revealed a prophage-like region of DNA in the genome, but phage have not been associated with Las to date. In the present study, shotgun sequencing and a fosmid DNA library of curated Las strain UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in Las and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. The SC-2 phage DNA appeared to stably replicate as an excision plasmid at a level 2-3X higher in planta than in psyllids. Objective 2 Progress: Similar phage DNA sequences (corresponding to both SC1 and SC2 and including putative lysogenic conversion genes) were found in Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected citrus in Brazil in collaboration with Dr. Nelson Wulff at Fundecitrus. We now have approximately 86% of the predicted Lam genome confirmed. Interestingly, both the SC1 and SC2 Las phage were found in Lam, and the gene order of the phage was also highly conserved. The 5 new and potentially pathogenicity related genes found in SC2 were also found on the equivalent Lam phage. This may be further evidence of the importance of these phage in lysogenic conversion of Liberibacter to become more virulent. This Lam genome is about one year from completion.
Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. The rationale is that by constitutive expression of an R protein, the plant innate immunity response will be at a high state of alert and will be able to mount a robust defense against infection by phloem pathogens. Overexpression of R proteins often results in lethality or in severe stunting of growth. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: We sequenced all of the constructs introduced into Arabidopsis which consisted of the AtSUC2 promoter (940 upstream from ATG) driving expression of the mutated (constitutive) and wild type forms of SSI4 and SNC1 (R proteins). For SSI4, which is derived from the Nossen cultivar of arabidopsis, two closely related genes (MUF8.3 and 8.2) are present in the Columbia cultivar. We cloned both, and the original from the Nossen cultivar, and cloned them behind the AtSUC2 promoter. These wild type versions of SSI4 will be used as controls for non-active forms of the R protein (pathogen activated). The four AtSUC2/R protein constructs (mutant and wild type of each of the two R proteins) were transferred from the pCAMBIA1305.1 vector, which confers hygromycin resistance, into pCAMBIA2301 with kanamycin resistance since the former is detrimental to transformation into citrus. The four constructs were submitted to the UF Citrus Research Facility at Lake Alfred for transformation into citrus. Transformation of these constructs into the Duncan variety of grapefruit is currently in progress at the Lake Alfred Citrus Research and Education Center (Dr. Vladimir Orbovic). Conclusions: Our hypothesis was that phloem-restricted expression of the R protein constitutive mutants would limit potential negative impacts on growth. The Arabidopsis transgenic plants expressing R protein mutants did not seem to be significantly affected in the majority of cases. Approximately 8% of the snc1 transgenics exhibited a stunted phenotype, very similar to the snc1 mutant expressed from its native promoter (not phloem specific). The first series of ssi4 transgenics (construct 5-2) had a point mutation (C>T) in the coding region that generated a premature stop codon and shortened the protein by 78 aa in the C-terminal region, past the leucine rich repeat (LLR). Five percent of these truncated ssi4 transgenics showed phenotypic differences mostly in the rosette appearance and lighter green, splotchy coloring. However, this effect will be investigated again in the new full-length ssi4 transgenic plants.
1- The physical construction/renovation of the growth room did not start yet. A draft of the final layout of the lamps and benches was presented. Several scenarios about air filtration and distribution were discussed as well as safety, security and the irrigation system. Regarding irrigation, it seems like there are concerns about the amount of run-off water leaving the growth room. A holding tank was proposed as a way to contain the water that is coming out of the facility, however the existence of a retention pond south of the building might be the best solution. No decision has been reached at this point regarding the disposal of the water. We make clear to everyone that we need hose bibs in the growth room and there will approximately 2000 gallons/week of run off water coming from the growth room, this is inclusive of watering the plants and maintaining cleanliness. 2 – The first material to establish mother plants from Hamlin 1-4-1 was released from Dr. Peggy Sieburth lab. The clean shoot tips are approximately 4 weeks old and they will need to be grafted in approximately 4 more weeks on rootstocks that are currently growing inside the lab. These rootstocks are being maintained inside the lab for 6 months under laboratory conditions and they needed to be transferred to bigger pots 2 months ago. Under these conditions the grafting will be delay until the growth room is available. Since these rootstocks are already suffering, a new batch was started as a backup. We hope that the shoot tips can hold a few months more until the grafting can be performed. We will need to transfer them several times which in normal conditions is not required. Maintaining shoot tips for a long time on in vitro conditions might also induce juvenility, which we will want to avoid. 3 – Construction of the growth room will start on October 25th according to information provided by the CREC maintenance supervisor. 4 – A grower was selected and he will be at CREC in a few weeks.
The objective of this project is to screen citrus germplasm for resistance to the Asian citrus psyllid (ACP). Although citrus huanglongbing (HLB) is a century-old disease and the control of ACP is the key factor for HLB management, there is little information regarding citrus host resistance to ACP. Our preliminary results indicate there is ACP resistance in citrus germplasm. Historically, most (if not all) citrus resistance to HLB has been evaluated using graft inoculations, which sometimes resulted in plants becoming infected that appeared to have resistance in the field. What is needed is research to find varieties with resistance to the psyllid. Greenhouse and field evaluations of the USDA germplasm collection will be conducted. There already exists in China individual citrus plants that are thought to be HLB-resistant,but likely some of these are ACP resistant. The identification of a psyllid-resistant variety (or individual mutant) could revolutionize HLB management strategies. If we can identify ACP resistant germplasm, we can identify resistance genes for use in traditional and molecular breeding. Our intentions are to screen hundreds of sources of USDA and Chinese germplasm for resistance to ACP and to field test these to see if resistance to the psyllid negates HLB or greatly facilitates control. We expect to identify psyllid resistant citrus genotypes (or individual mutants from field-resistant collections) and/or citrus relatives within the Rutaceae that have psyllid resistance. We expect to determine traits that confer resistance and to identify traits that might be transferred to citrus varieties currently grown in order to make them resistant to the psyllid and thus less prone to contracting HLB. The ultimate return from this project would be an effective management strategy to control ACP and HLB that is less costly and friendlier to the environment and non-target organisms than the repetitive use of broad spectrum insecticides. A post doc was found and hired to conduct this research. A trip was made to China to firm up research plans with the Dr. Liu Bo and the Fujian Academy of Agricultural Sciences. Seeds representing the entire citrus/citrus relative collection at USDA-ARS-NCGR were obtained and have been planted in Ft Pierce. At the time this report was prepared, the Chinese government had not yet approved importation of germplasm from USA. A field planting of 87 citrus genotypes and relatives, primarily of the orange subfamily Aurantioideae, in Ft Pierce was screened for psyllid infestations during June, July, and August. There were significant differences in susceptibility of the genotypes as measured by the categorical rating of adults (F = 3.97, df = 86, P = 0.0001), nymphs (F = 7.56, df = 86, P = 0.0001) and eggs (F = 2.17, df = 86, P = 0.0001). Many of the genotypes were highly susceptible to infestations by the psyllid, but Glycosmis pentaphylla, Clausena harmandiana, and two genotypes of Poncirus trifoliata were completely avoided by the psyllid. The genera Glycosmis and Clausena are not members of the “true citrus fruit trees” and are not sexually compatible with Citrus however these groups could still serve as a source of psyllid resistance genes. However, Poncirus trifoliata, the trifoliata orange, readily forms hybrids with Citrus spp., is the dominant root stock in China and since 1892 has been used in Florida either alone or in hybrid form. This cold hardy genotype is highly resistant to citrus tristeza virus, Phytophthora-induced diseases, and citrus nematode. In recent psyllid no-choice oviposition studies using six different genotypes of Poncirus trifoliata, with Citrus aurantium and C. macrophylla as susceptible controls, a greatly reduced number of eggs were laid on the Poncirus trifoliata selections when compared with the controls.
1- The first objective of the second year was to build a plant growth room at the Citrus Research and Education Center in Florida (CREC). The physical construction/renovation of the growth room has not been started so far. The construction is expected to start at the end of September. The rootstocks that are currently growing in the lab are big and we will need to start growing a new batch until the growth room is ready. They cannot be transferred to another greenhouse because it will defeat the purpose of growing under controlled conditions. 2- Training of the manager Dr. Zapata has been completed at the IVIA under the supervision of Dr. Pena. 3- Initial material to establish the mother plants are being produced at the Department of Agriculture with Dr. Peggy Sieburth. She will start releasing the in vitro plants in September. Ideally these plants should be used immediately for grafting on the rootstocks; plants will be kept in vitro until the growth room is ready. The plants cannot be kept for more than 4-5 months on in vitro conditions. 4- A search for a full growth room technician started. The final hiring process will be completed once the growth room construction starts. We expect this technician to go for 2 weeks of training in Spain. At this moment, construction/renovation of the growth room is a major bottleneck for the progress of the project. Clean materials for the most important scion varieties of Florida have been obtained with the help of Dr. Peggy Siebuth through shoot-tip grafting. Clean rootstocks (stored at the lab at this moment-the only clean area we count with) are already 6-month-old and will be ready to be grafted with the clean scions for next October. However, we do not know yet when the growth room will be finalized. Only after this facility is fully operative, we will be able to perform the grafts with the clean materials. If the construction of the growth room is delayed further (more than 3 months), we have the risk of losing both the scions and the rootstocks, and consequently lose months of work (that could not be repeated until next year due to Dr. Sieburth’s agenda) and a lot of money. These delays (no growth room after 1.5 years of project) are making impossible to fulfil our objective for the end of this 3-year project. This situation is out of my understanding- Leandro Pe’a
Continued efforts to improve transformation efficiency: ‘ The effect of antioxidants lipoic acid, glycine betaine and glutathione are being evaluated for increased transformation efficiency. Some treatments are showing a significant increase in transformation efficiency across a range of citrus genotypes.’ We have successfully developed an efficient transformation system for embryogenic citrus callus, (publication in Plant Cell Reports in press). This system works well for polyembryonic mandarin types (i.e. W. Murcott, Ponkan) that are seedless or more recalcitrant using the common Agrobacterium-mediated citrus method. The method should also work well for lemons. Horticultural manipulations to reduce juvenility in commercial citrus: The RES (Rapid Evaluation System). ‘ Commercial sweet orange, grapefruit, and specialty mandarin cultivars were propagated and planted in the RES. Several juvenile hybrids from our breeding program flowered and set fruit after only one year – our goal is to force flowering and fruit set in juvenile sweet oranges and grapefruits in 1-2 years. If successful, the same approach could be applied to transgenics. A two-year old field trial using a juvenile Valencia budline on more than 70 rootstocks is showing significant rootstock affects on precocious bearing. Transformation of precocious but commercially important sweet orange clones: transgenic plants of precocious ‘Vernia’ sweet orange somaclones were regenerated and micrografted for further study of early flowering. Transgenic approaches to reduce juvenility: ‘ Whole plants generated from ciFT and empty vector control transformation experiments of Carrizo are being evaluated by both PCR amplification assays and a repeat of the screening histochemical GUS assay. No obvious phenotypes have yet been observed among the whole plants, however, flowers have occasionally been observed to occur on in vitro shoots. ‘ Putative transformed Duncan grapefruit whole plants in soil and shoots being rooted in vitro have been generated. We will be doing additional transformation experiments as soon as fresh seed becomes available. We will shortly have T1 seed from additional plants and will soon be able to proceed with assays to phenotype this generation and compare the effects that each of the ciFT genes has on expression and morphology. ‘Through a project being conducted by an HHMI-sponsored undergraduate student, Melanie Pajon, we will also be cloning the tomato FT ortholog and using it to obtain citrus transformed by a heterologous FT gene.Transformation of Samsun tobacco with the ciFT genes has resulted in a number of T0 plants of each of the 3 ciFT constructs, some of which have produced T1 seeds. We will shortly have T1 seed from additional plants and will soon be able to proceed with assays to phenotype this generation and compare the effects that each of the ciFT genes has on expression and morphology. Phenotypes of the T0 plants have ranged from early flowering, multi-branching, dwarfs to ones very similar in architectures in the wild type parent.
During the first quarter of funding, Core Citrus Transformation Facility (CCTF) continued to process the orders for transgenic Citrus material. Demand for genetically transformed citrus plants stayed high resulting in influx of new orders listed here by names of genes of interest or plasmids: p6; p33; p7; p10; pMOG8000; pAS7; pAS13*; pNAC1; pMKK7; pMOD1; and pSucNPR1. The work also continued on the old orders that were previously partially completed. Considering that transgenic Duncan plants carrying NPR1 gene exhibited significant resistance to Citrus canker, CCTF received order for production of commercially important Flame grapefruit cultivar transformed with the same NPR1 gene (order completed-NPR1 gene: 10 plants). A gene thought to be superior to NPR1 (so-called superNPR1) was introduced into Duncan grapefruit (superNPR1 gene: 12 plants). Introduction of NPR1 into Hamlin orange cultivar and superNPR1 into Flame is half completed. Work on the order pAS7 that is associated with HLB tolerance/resistance is half done (5 Duncan plants). Order that included use of pLC plasmid is also completed (8 Hamlin orange plants). Ten Mexican limes transformed with gene in pHK plasmid were produced, but satisfaction of this order will include production of more plants per client’s request. Five plants of Mexican lime transformed with p33 gene were also produced. CCTF produced more plants for the old orders: N1* gene: 3 Duncan; C5*: 3 Duncan; CL1 gene: 2 Valencia, 3 Duncan; CL2 gene: 1 Duncan; PiTA gene: 1 Valencia; CIT108p: 1 Flame. About thirty more soil-adapted plants will be submitted to the PCR testing as a secondary proof of their transgenicity before delivery to clients. Some of the funds from the grant were used to hire additional help through the summer resulting in seasonal increase of CCTF capabilities. Please be informed that the person directly managing the CCTF (and co-PI) is Dr. Vladimir Orbovic.
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