Recent evaluation of root production on HLB-affected trees compared to presumed healthy trees confirms that root loss is due to reduced root longevity. This root loss is exacerbated by biotic and abiotic stresses in the rhizosphere. Increased susceptibility of Las’infected roots to Phytophthora spp. is evidenced by statewide populations that have fluctuated from unprecedented highs in the 2011 season to an unprecedented low in 2013 compared to 25 years of pre-HLB soil populations. Phytophthora propagules per soil volume and per root resurged in 2014 in response to a more than doubling of root density based on intensive (i.e., local repeated measures) and extensive (i.e., Syngenta statewide survey) sampling compared to 2013. Substantially higher root density in Florida groves in spring and summer 2014 has so far this harvest resulted in less fruit drop than in the previous two seasons. Both field surveys and greenhouse experiments show that HLB increases the susceptibility of the citrus root system to the damaging root pathogen P. nicotianae. Increased P. nicotianae infection suggests that root system damage occurs more quickly in the presence of both pathogens. Chemical control of P. nicotianae slows infection of the root system and may slow yield decline in HLB-affected trees in Phytophthora-infested groves, however, HLB reduces the effectiveness of fungicides for control of Phytophthora root rot as a consequence of increased root susceptibility to P. nicotianae infection.
For 2015 season, FireWall 50WP (50% streptomycin; Agrosource, Inc.) has been granted an EPA section 18 registration for control of citrus canker in Florida grapefruit. The label for FireWall restricts use to no more than two applications per season. As a condition for FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance in treated groves. The objective of this survey is to apply our published protocol (Behlau et al., 2012) for sampling canker-infected grapefruit leaves for isolation and detection of streptomycin resistant Xcc. The survey for 2015 season will be conducted in November 2015 and the report of results submitted to FDACS. Greenhouse trials to measure the residual systemic activity of streptomycin against Xcc in leaves after foliar spray will be repeated as in 2014 to confirm trans-cuticular and upward movement of streptomycin into new foliage via the xylem.
Objective 1. As reported previously, methyl accepting chemotaxis proteins (MCPs) were identified for wide (Xcc62, Xcc306) and narrow host range strains of Xanthomonas citri subsp. citri (Xcc) compared to X. fuscans subsp. aurantifolii B and C strains, X. alfalfae sbsp. citrumelonis (Xac) or X. campestris pv. campestris (Xc). All Xcc strains showed similar MCP content except Xcc306 which lacked some of the MCPs. MCP analysis was expanded to include Xcc strains from different geographical areas. 37 strains from Argentina (27%), Uruguay (32.5%), Brazil (27%) and 5 isolates from citrus of uncertain origin (13.5%) were analyzed for 15 MCPs. Only one of the MCPs was conserved among all Xanthomonas strains (XCV1951), and two of them were specific of Xc (XCC0324 and XCC1954). Objective 2. The role of extracellular DNA (eDNA) was evaluated in the formation and stabilization of the biofilm matrix. The presence of eDNA in biofilm was confirmed for several Xcc strains and Xac. DNAse treatments of Xcc strains and Xac reduced biofilm formation at the initial stage of development as well as disrupted preformed biofilm. The DNAse effect on formation or disruption varied among Xcc strains and Xanthomonas species and indicates differing roles for eDNA. Differences in fiber structures containing eDNA in biofilms, bacterial cultures, and in twitching motility assays were detected by SYTO-9 staining and fluorescence microscopy. The proposed roles for eDNA in the early stages of biofilm formation are as an adhesin and in mature biofilms as a structural component. .
The transgenic plants to be developed for this project are now growing in two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped from the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL, to Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, in early October, 2014. An additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped to Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. The plants at both locations are growing well. At Penn State, all the transgenic lines are being propagated as vegetative cuttings. We have achieved over 50% success rate in rooting cuttings from the plants, and rooted cuttings are now growing for all of the independent lines. The rooting process for cuttings takes 3-4 months in our facility at Penn State. We have performed two protein immunoblots with an antibody that will detect the FLT-antiNodT fusion protein (anti-cmyc antibody). We detected protein of the correct size (~50 kD) as a single band without degradation products in two different lines, and not in control plant samples. These results are very promising and are consistent with successful expression of full-length FLT-antiNodT fusion protein in ‘Duncan’ grapefruit. During the next quarter, we will be repeating these experiments with all of the transgenic lines and using improved protein extraction techniques to develop better western blot images and documentation.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we continued optimizing the NAD+ treatment conditions. Both soil drench and foliar spraying of NAD+ were tested again. Foliar spraying did not provide significant protection against citrus canker, soil drench induced strong resistance against the pathogen. We previously observed strong systemic protection against canker by NAD+ one month after the treatment in upper new flushes and are repeating the experiment to confirm the systemic effects. We are also testing different recipes for stabilizing NAD+ on the surface of leaves or in soil, since this chemical is not stable under aqueous conditions. For objective 2, transgenic citrus plants expressing the Arabidopsis nonhost resistance genes are growing in greenhouse and will be tested for canker resistance. We are also propagating the transgenic plants to produce progenies for HLB resistance test. Citrus homologs of the Arabidopsis nonhost resistance genes have been cloned and sequenced. To test their functionality, we have cloned the citrus homologs into plant expression vector and have transformed into the corresponding Arabidopsis mutants. T1 plants are growing.
Analysis and writing continue in preparation for the end of this project in June. One student is completing her MS degree, and with her data completed and analyzed, manuscripts are being developed. These may not be completed by the termination date of the project, but we hope to have at least first drafts done. The FT3 gene is also being put into the CTV vector and is being put into CRISPR/Cas systems in collaborations with others.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Two (2) misting chambers to propagate candidate, rootstock materials as rooted-cuttings have been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Funds from this project were used to support the construction of a new greenhouse at the IRREC. This greenhouse is completed and operational. – The first cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation are currently being propagated. – The second cohort of advanced, tetrazygous citrus rootstock materials for en masse propagation have been identified and are being prepared to have cuttings taken from them. – In addition to the 1st & 2nd cohorts of tetrazygous rootstocks, promosing diploid rootstocks have also been identified and are being prepared to have cuttings taken from them. As of March 2015, plants are still growing in the green house and no significant changes to report.
A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP were obtained. Exposure to canker inoculum showed remarkable resistance in chimera compared to control. RNA was isolated from 16 transgenic Hamlin containing Chimera. RT-qPCR showed 50% of them have relative high gene expression. One of them showed over hundred times higher expression compare to plant expressing the lowest level of chimera. These promising transgenic lines were replicated by grafting for HLB challenge. About 30 Hamlin transformed with thionin also were obtained. Twenty transgenic lines were confirmed containing thionin gene by PCR. RNA was isolated from 16 transgenic Hamlin containing thionin. Six of them have relative high gene expression by RT-qPCR. These transgenic lines will be replicated for HLB challenge. Two new chimeral peptide have been developed and is used to transform citrus. Many transformed Carrizo shoots with new chimera construct were obtained. Some were transferred to the rooting medium. Replicated trangenic lines expressing chimera, thionin and D4E1were grafted with HLB infected rough lemon. Las tilter will be checked by qPCR periodically. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Many putative transformants were generated on the selective media. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slight canker resistance by infiltration test. Spray inoculation was tried and some of them show obvious canker resistance. To confirm that high ROS production was not due to variability in Hamlin, we examined 40 Hamlin seedlings and no or very low level ROS production was detected. In contrast, relatively higher ROS production was detected from wild-type Carrizo seedings compared to Hamlin seedlings. Two potential FLS2 orthologues were identified in Hamlin and their expression was shown much lower compare to nbFLS2. Replicated trangenic Carrizo lines expressing nbFLS2 were challenge with ACP. Las titer will be checked by qPCR periodically. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter is being used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only.
The overall aim of this project is to develop and evaluate oil-in-water (o/w) microemulsion (ME) formulations to deliver antimicrobial essential oils (EOs) to the phloem of HLB-infected trees. In the previous quarter we improved the oil loading and temperature stability of our formulations, performed method development for estimating essential oil uptake and transport in citrus plants and also developed dye doped formulations to investigate penetration of essential oils into citrus leaves by fluorescence microscopy. Previously we had reported that the formulations had minimum inhibitory concentrations (MIC) in the range of 50-100 ppm of oil concentration against L.crescens. Since the minimum bactericidal concentrations (MBC) are about 4 times greater than MIC, we tested the formulations at 500 ppm oil concentration in order to estimate the MBC. All the oil formulations as well as controls (formulation with the oil containing just surfactant) were effective indicating that MBC of the formulations is less than 500 ppm of oil concentration. Tests to assess efficacy of the formulations in HLB infected citruses via a bud graft technique were undergoing at Indian River Citrus and Education Center, Ft. Pierce. The results showed the formulations as well controls (formulation with the oil containing just surfactant) killed the bacteria in the bud stick. EO-A and thyme oil were also phytotoxic to the buds at the concentration evaluated (5000 and 500 ppm), since none of them grew. However, the scions treated with just control did grow indicating that the surfactants used are not phytotoxic to the plants. In field tests using trunk application EO-A and EO-B were ineffective. We tested the potential of combining the currently used thermal therapy (Dr. Ehsani, UF) with our EO formulations which could lead to enhanced essential oil penetration into leaves and kill the bacteria in plant parts like roots where thermal therapy is ineffective. Thyme oil formulations were diluted to 2000 ppm of thyme oil in field and sprayed using a hand sprayer till an even coverage was achieved which was followed by thermal treatment. QPCR tests are undergoing to assess the efficacy of such a combined technique. A leaf assay test is also being conducted with EO-A formulation at Dr. Gonzalez’s lab as a quick study to evaluate the performance of the formulation at different EO-A concentrations.
Image acquisition and PCR analysis: Individual leaves (213 total) from seedlings from the first run (Optical Sensing Project-1; OSP-1) were photographed using three emission filters (500nm, 690nm and 730nm) using three excitation wavelengths (UV 364nm, Blue 470nm, Green 527nm) in addition to polarization images at 594nm Approximately 1,500 images were examined for correlations between HLB infection and fluorescence emissions and optical rotation of 594 nm light (polarization images). The leaves were processed by isolation of the midveins and the tissue pulverized in liquid nitrogen and stored at -80 C for PCR analysis at a later date. A second trial (OSP-2) was started Dec 15th by placing the seedlings in William Dawson’s psyllid room at the CREC at Lake Alfred, FL.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over five years. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015 (Grosser, personal comm.). The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material is being planted this spring that has shown great promise in the greenhouse (unpublished). A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 5 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Lee et al., in preparation), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). A new UF-Gmitter led association mapping study has just been initiated using the same planting, to identify genes associated with HLB- and ACP-resistance. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished) and David Hall is now assessing ACP colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. As of April 2014 at the Picos Test Site, several of these USDA hybrids had grown to a height of seven ft, with dense canopies and good fruit set, while sweet oranges are stunted (3 ft) with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population will be planted at the Picos Test Site this spring in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting will also include a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
Image acquisition and PCR analysis: Conducted polarized light analysis of OSP-3 seedlings (8 weeks psyllid exposure). In consultation with Alireza Poureza (postdoc assigned to the project from the Lee lab), the exposure conditions were standardized to facilitate image analysis. In addition, we switched to using an 80 mm fixed lens to increase image resolution. Preliminary inspection of the fluorescence images from OSP-1 failed to show strong correlations with HLB positive plants. All of our emphasis was shifted to the use of polarized light at 594 nm using methods recently perfected in the Lee laboratory (Poureza doctoral dissertation). Multiple image sets were acquired for the OSP-3 seedlings in the process of standardizing the exposure and lens settings. Three leaves from each seedling were processed for quantitative PCR analysis which will be reported in the next reporting period. OSP-4 seedling were started in the psyllid growth room and moved closer to the lights and the canopies of the infected feeder plants to enhance infection rates. Image analysis for OSP-3 was initiated, first looking at simple properties of image texture such as the distribution of pixel intensities and associated standard deviations. The results of image texture analysis will be reported in the final quarter (April 2015). A preliminary look at the data suggests that the polarization approach is valid as a means for detecting HLB infection in seedlings.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional to the above parameters, 3. Evaluate effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. The experiment was planted 3-4 July on a 10-acre block at 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. Experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: (1) insecticide alone, (2) foliar nutrition alone, (3) insecticide + nutrition, and (4) untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3.5 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on replicated single-row plots to repel Asian citrus psyllid and, together with a drip irrigation/fertigation system, to increase citrus growth rate over the unmulched control. The block was planted 3-4 July 2012 and monitoring of ACP with flush inspection and sticky cards commenced 13 Aug. Normal grove care operations included Kocide sprayed monthly for control of canker, and one application of Intrepid for leafminer control. Baythroid, Portal, and Admire were applied to insecticides plots for ACP control. Sticky cards (3 per subplot) are monitored foACP and other common citrus pests and replaced every other week. 4,394 psyllids have been found on sticky cards so far of which greater than 65% are in no-mulch plots while only 16% have been found in plots that receive insecticide and 4% in plots with both mulch and insecticide All of 103,412 shoots have been examined so far of which 12,638 were infested with ACP and 1813 were infested with Aphids, both predominately in no-mulch plots with more than 65% infestation. In contrast, 1473 ACP and 593 aphid-infested flush were found on insecticide-treated trees and 486, and196 on trees on mulch and receiving insecticide drenches. Tap samples were initiated 15 July 2014 shows that the mulch is still deterring psyllids although less in the top canopy than the sides. Thus far, 2752 adult ACP have been counted by tap sample. Of 288 leaf samples for HLB testing collected February 2015 (9 per subplot), 99 were positive: 34 from mulched treatments and 29 from insecticide plots. Only 14 of an expected 72 trees were positive in plots with both insecticide and mulch. Trunk cross sectional area is now 7% larger for trees receiving extra foliar nutrition than those without (P = 0.089) and larger for trees with mulch than without (P = 0.081). Similarly, trees receiving insecticide applications have significantly larger trunks than without (P = 0.005). Lack of a bigger effect of mulch on growth may be due to an irrigation system failure during 13 days in Feb and 15 in May which caused more drought stress to trees on mulch while the unmulched trees benefited more from rainfall (1.53 inches at FAWN SWFREC). Visual rating of trees with mulch were 27% less symptomatic of HLB than trees without mulch. Similarly trees receiving insecticides were rated 27% less symptomatic than trees without insecticide while foliar nutrition had no effect. A manuscript entitled “Role of Metalized Reflective Mulch with Nutritional and Insecticidal Treatments in Mitigation of HLB in New Citrus Plantings is in preparation. Talks were presented at the state and national entomological society meetings and a poster at the HLB meeting.
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional to the above parameters, 3. Evaluate effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. The experiment was planted 3-4 July on a 10-acre block at 23 x 9 ft spacing at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. Experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: (1) insecticide alone, (2) foliar nutrition alone, (3) insecticide + nutrition, and (4) untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch and no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3.5 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on replicated single-row plots to repel Asian citrus psyllid and, together with a drip irrigation/fertigation system, to increase citrus growth rate over the unmulched control. The block was planted 3-4 July 2012 and monitoring of ACP with flush inspection and sticky cards commenced 13 Aug. Normal grove care operations included Kocide sprayed monthly for control of canker, and one application of Intrepid for leafminer control. Baythroid, Portal, and Admire were applied to insecticides plots for ACP control. Sticky cards (3 per subplot) are monitored foACP and other common citrus pests and replaced every other week. 4,394 psyllids have been found on sticky cards so far of which greater than 65% are in no-mulch plots while only 16% have been found in plots that receive insecticide and 4% in plots with both mulch and insecticide All of 103,412 shoots have been examined so far of which 12,638 were infested with ACP and 1813 were infested with Aphids, both predominately in no-mulch plots with more than 65% infestation. In contrast, 1473 ACP and 593 aphid-infested flush were found on insecticide-treated trees and 486, and196 on trees on mulch and receiving insecticide drenches. Tap samples were initiated 15 July 2014 shows that the mulch is still deterring psyllids although less in the top canopy than the sides. Thus far, 2752 adult ACP have been counted by tap sample. Of 288 leaf samples for HLB testing collected February 2015 (9 per subplot), 99 were positive: 34 from mulched treatments and 29 from insecticide plots. Only 14 of an expected 72 trees were positive in plots with both insecticide and mulch. Trunk cross sectional area is now 7% larger for trees receiving extra foliar nutrition than those without (P = 0.089) and larger for trees with mulch than without (P = 0.081). Similarly, trees receiving insecticide applications have significantly larger trunks than without (P = 0.005). Lack of a bigger effect of mulch on growth may be due to an irrigation system failure during 13 days in Feb and 15 in May which caused more drought stress to trees on mulch while the unmulched trees benefited more from rainfall (1.53 inches at FAWN SWFREC). Visual rating of trees with mulch were 27% less symptomatic of HLB than trees without mulch. Similarly trees receiving insecticides were rated 27% less symptomatic than trees without insecticide while foliar nutrition had no effect. A manuscript entitled “Role of Metalized Reflective Mulch with Nutritional and Insecticidal Treatments in Mitigation of HLB in New Citrus Plantings is in preparation. Talks were presented at the state and national entomological society meetings and a poster at the HLB meeting.
We continue to conduct the weekly Agrobacterium-mediated transformations and screen putatively transformed mature scion and rootstock shoots for clients. The total number of transgenics produced and detected thus far (excluding the 157 reported earlier) is ~100, and ~ half of these survived primary and secondary grafting (excluding the 66 reported earlier). The transformation efficiency for a plasmid with no reporter gene and a weak promoter driving the npt11 selectable marker was 2.8% for mature scion and rootstock. As expected, mature rootstock gave a higher transformation efficiency than mature scion. As previously mentioned, the weaknesses of this protocol are the high number of escapes particularly with constructs having weak promoters driving the npt11 selectable marker, difficulties with micro-grafting, and for certain constructs with no reporter genes, the difficulties in screening by PCR. PCR is expensive, time-consuming, labor intensive and prone to error. After the standard tissue culture protocol, growth in selective liquid media has assisted in identifying 4 transgenic events, which indicates that better selection is important. GFP expressing transgenic shoots were regenerated from mature citrus after biolistics, but subsequently died after micro-grafting. We will consider purchasing mature scion from nurseries solely for biolistics because we cannot produce enough in the growth room to facilitate the weekly Agrobacterium transformations and optimize biolistics. This mature scion will not enter the growth facility unless it has been cleaned through shoot-tip grafting. Parameters still to be optimized include use of the hepta adapter, the number of bombardments per treatment, growth stages (days after culture initiation) of target explants, and osmotic treatment on medium containing sorbitol and mannitol (hours and concentrations). We have had to find an alternate California seed source because certain varieties that we regularly use in tissue culture are contaminated by a fungus. Spraying fungicide does not eliminate this seed borne fungus. A manuscript entitled, “Genetic Transformation of Commercially Important Mature Citrus Scions” authored by Hao Wu, Yosvanis Acanda, Alka Shankar, Michael Peeples, Calvin Hubbard, Vladimir Orbovi. and Janice Zale has been accepted for publication in Crop Science.
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