Evaluation of existing cultivar/rootstock combinations for HLB resistance/tolerance has revealed potentially valuable tolerance and indicates that early HLB symptoms and earlier CLas titer are unrelated to growth and cropping. In August 2010, the plants were established at Pico s farm in Ft. Pierce FL. Despite the high incidence of mottle in SugarBelle /SourOrange, it had the greatest overall increase in diameter. ‘SugarBelle’ and ‘Tango’ (which were not on the same stock as ‘Hamlin’ and so results should be viewed as comparing cultivar/rootstock combinations) were the healthiest in overall appearance in 10/15 and had the most fruit (88 per tree). All cultivars except sweet oranges and grapefruit are progressing in production, but production was compromised in all varieties by the severe HLB pressure at this site, and commercial value of the observed tolerance remains uncertain. A mapping population of Fortune x Fairchild has been planted (collaborating Roose and Gmitter) along with related material, in an effort to identify genes associated with tolerance in the mandarin phenotypic group. The citrus relatives planting (85 seed source genotypes from the gene bank) has been assessed for growth and apparent HLB tolerance. Most trees containing citron in their pedigree have markedly greater canopy densities and greater tree size than other accessions in the Genus citrus. One alleged standard sour orange looks much healthier and is much larger than other sour oranges. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and have been transferred to the field at Ft. Pierce FL. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a quarterly basis. HLB is now widespread and trees of more vigorous scion types are generally the healthiest at this point in time. Progress continues on the antibiotic treatment of HLB infected bud-wood to compare growth at different levels of CLas infection. HLB-infected budwood was treated with various concentrations of antibiotics and grafted on sour orange rootstock using 3 fairly HLB-resistant ( Temple , GnarlyGlo , and Nova ) 3 tolerant ( Jackson , FF-5-51-2, and Ftp 6-17-48), and 3 susceptible ( Flame , Valencia , and Murcott ) genotypes. Standard growth measurements and disease severity are evaluated and leaves sampled for qPCR analysis on a quarterly basis. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is underway. One hundred and fifty etiolated seedlings of the trifoliate Rubidoux and the sweet orange Hamlin have been approach grafted together. Generation of new chimeras has been difficult. An existing periclinal chimera (Satsuma and Poncirus) has been imported,has been with DPI two years, but has not yet been released to us for testing. A method for the rapid identification of potential sources of HLB resistance is being developed. This project involves the screening of citrus seedlings at the 3 to 5 leaf stage, or very small micrografted trees, that are exposed to HLB infect ACP feeding. CLas titer levels, using real time PCR, are easily detectable in most plants at 3 weeks Seedlings of Hamlin and Dancy show marked CLas proliferation and systemic movement from 3-6 weeks after exposure to ACP. Trees of seemingly HLB resistant/tolerant sweet orange-like hybrids and mandarin -types were propagated on x639. Replicated trials with standards have been established, in cooperation with G. McCollum. Six locations each of all sweet orange-like together and 4 with all mandarins were established in replicated block plantings with 6-8 trees of each cultivar at each site (in Ridge, IR and Gulf coast). Seedlings with a range of pedigree contributions from Microcitrus and Eremocitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, and are germinating for field testing of HLB resistance.
A 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 six 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 has been planted that has shown great promise in the greenhouse and the permit has been updated to plant many new transgenics. 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 6 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, with the last samples collected this week), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). Marked tolerance to HLB is apparent in many accessions with citron in their pedigree. All replicates of one alleged “standard sour orange” looks remarkably healthy and may permit comparison of more susceptible and tolerant near-isogenic variants. 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 broader cross-section of Poncirus-derived genotypes are on the site in a project led by UC Riverside/USDA-ARS Riverside, in which half of the trees of each seed source were graft-inoculated prior to planting. 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). In July 2015 David Hall led assessment of ACP colonization across the entire planting, and the Gmitter lab will map markers associated with reduced colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. Several of these USDA hybrids have grown well, with dense canopies and good fruit set but copious mottle, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population was just planted at the Picos Test Site in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released related cultivars. Valencia on UF Grosser tetrazyg 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.).
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, all transgenic lines as described in the project proposal have been regenerated and most plants have been acclimatized and transferred into the greenhouse for further growth. A few putative transgenic lines have been transferred to fresh in vitro rooting medium in efforts to stimulate root production. Root samples from 21 lines have been analyzed for gene expression using qPCR. Of them, 12 were determined to be high expressers while the rest were medium to low in expression. We are in the process of evaluating the remaining greenhouse acclimated lines for gene expression. The high expresser lines will be propagated for subsequent evaluation with Diaprepes neonates.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics have survived and passed a PCR screen, and these have been grafted onto rootstocks. Grafted plants are currently growing, and will be tested for responsiveness to the elf18 ligand for EFR and for canker resistance. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and the protocol is being optimized. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to co-express Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs were provided to the Lake Alfred transformation facility, but the transformation attempts have so far been unsuccessful, possibly due to negative effects of the constructs in Agrobacterium or in citrus. Troubleshooting of these transformations with a negative control construct is underway. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we are targeting mutating the native citrus Bs5 alleles while simultaneously replacing the gene with the effective resistance allele. Two editing constructs have been created, one targeting a site overlapping the two conserved leucines, and one targeting two flanking sites to create a deletion in Bs5. Both constructs have been verified to function by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties. These constructs have been prioritized for transformation into Carrizo citrange, and transformations are underway at UC Davis. Transformants with mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
The project has three objectives: (1) Confirm HLB resistance/tolerance in transgenic citrus lines. (2) Determine the chimerism of the HLB-resistant/tolerant transgenic lines. (3) Confirm HLB resistance in citrus putative mutants (nontransgenic lines). For objective 1, we are propagating a number of citrus transgenic lines overexpressing Arabidopsis defense genes. Our previous results indicated that these transgenic lines are likely resistant or highly tolerant to HLB. The progeny plants are growing in the greenhouse. For objective 2, we performed on round of real-time quantitative PCR (qPCR) to determine the chimerism of the HLB-resitant/tolerant transgenic lines. The results showed some of the lines may be chimeric. We are repeating the qPCR experiment. For objective 3, we are propagating the previously generated gamma ray-mutagenized mutant lines that are likely resistant/tolerant to HLB. The progeny plants are growing in the greenhouse.
During the last quarter of 2015 Core Citrus Transformation Facility (CCTF) continued to operate as planned and produced transgenic Citrus plants. The number of orders received for the last three months jumped to nine. Two of those orders were for transgenic Duncan grapefruit and the rest are the set of seven orders for production of genetically modified Pineapple orange. CCTF still has not received two groups of orders announced by prospective clients about 5-6 months ago. Also, one set of orders that included the use of three different vectors was withdrawn and the client is working on re-cloning of all vectors. CCTF produced 51 plants within last quarter. These plant belong to multitude of orders, altogether nine of them, some of which are old and some newer. Six of the produced plants were Valencia oranges, 11 were Duncan grapefruit, and the rest were Carrizo citrange. As I have previously reported to Dr. H. Browning, the analysis of transgenic plants carrying NPR1 gene was successfully completed by Yosvanis Acanda from Mature tissue transformation facility. Out of 67 tested transgenic rootstock plants produced by the CCTF, 23 were shown to be high expressers of NPR1. These plants are being kept in the CCTF s greenhouse until they reach the size at which they can be propagated so that they can serve as rootstocks for both transgenic and WT scion plants.
The mature citrus transformation facility continues to produce transgenic events for its clients. Productivity has significantly improved using vectors with reporter genes. Transgenics produced via Agrobacterium can now be supplemented with transgenics produced using biolistics in immature and mature citrus. Scientists have been encouraged to submit vectors with all plant sequences and no pest sequences, which might lessen regulatory hurdles. Genes are now being stacked in an effort to prevent the pathogen from easily overcoming tolerance or resistance. A manuscript is being prepared describing biolistics in immature citrus. Currently, all transgenic events are being transferred to scientists directly without secondary grafting or propagation, unless otherwise requested. To improve efficiencies and lessen potential micrografting incompatibilities, sweet orange is being micrografted onto decapitated sweet orange rootstock. Micrografting losses in mature rootstock have significantly decreased when young shoots are micrografted onto decapitated rootstock grown in high sucrose solution. We are in the process of introducing new breeder lines in which to produce transgenics. Three sweet orange varieties and one grapefruit are being introduced via shoot-tip grafting. Once plants are established, they will be used in budding mature citrus onto rootstock to obtain budstick for transformations. Our website is being updated to reflect current prices and technologies employed.
The mature citrus transformation facility continues to produce transgenic events for its clients. Productivity has significantly improved using vectors with reporter genes. Transgenics produced via Agrobacterium can now be supplemented with transgenics produced using biolistics in immature and mature citrus. Scientists have been encouraged to submit vectors with all plant sequences and no pest sequences, which might lessen regulatory hurdles. Genes are now being stacked in an effort to prevent the pathogen from easily overcoming tolerance or resistance. A manuscript is being prepared describing biolistics in immature citrus. Currently, all transgenic events are being transferred to scientists directly without secondary grafting or propagation, unless otherwise requested. To improve efficiencies and lessen potential micrografting incompatibilities, sweet orange is being micrografted onto decapitated sweet orange rootstock. Micrografting losses in mature rootstock have significantly decreased when young shoots are micrografted onto decapitated rootstock grown in high sucrose solution. We are in the process of introducing new breeder lines in which to produce transgenics. Three sweet orange varieties and one grapefruit are being introduced via shoot-tip grafting. Once plants are established, they will be used in budding mature citrus onto rootstock to obtain budstick for transformations. Our website is being updated to reflect current prices and technologies employed.
Good progress was made in continuing the development of new hybrid rootstocks that support early and high fruit yield and fruit quality in the scion, have good soil adaptability, and are resistant to important diseases, especially HLB. This work was begun previously under CRDF project 508. As requested by CRDF, this new project 15-002 will place highest priority on hybrid rootstocks already created in the USDA program and being considered for release to growers over the next six years. This will include about 400 Supersour-type rootstocks that have been previously created and selected to include in field trials and other specialized testing. During this quarter, a new support scientist was hired under the grant to replace a support scientist, previously on staff, that was leaving to accept a permanent job at a University. Other personnel working on the project remained in place to continue tree care, experiment establishment, and data collection. During this quarter, tree care continued in established rootstock field trials, and tree size measurements were taken from selected trials. Trees in field trials were scored for health, HLB symptoms, and samples were collected from some groups for PCR detection of Las infection. Analysis was completed on data from several established trials to assess relative rootstock performance, rootstock effects on yield, fruit quality, tree size, and HLB symptom development. Nursery experiments were conducted with promising new rootstocks to determine nursery-related traits important for commercial use. Greenhouse experiments continued to assess rootstock tolerance to CTV and high pH. Analysis was completed on data from one cooperative field trial with 17 rootstocks that became 100% infected with Las and was determined to be a good comparison of rootstock performance with severe HLB challenge. The two best rootstocks in the trial were US-942 and US-1516. Based on this and other data, US-1516 rootstock was submitted by Bowman and McCollum for unrestricted commercial release by USDA. US-1516 is available as clean budwood from DPI, and some seed is available from Bowman. Outstanding performance by US-942 in that cooperative trial reinforced previous observations that US-942 provides better tree health and productivity than most other rootstocks when trees become infected with Las. A manuscript was prepared for publication to provide thorough documentation of the results from the cooperative trial. A manuscript was prepared for publication to provide documentation of US-1516 rootstock. During this quarter, cooperative work continued with UF researchers to propagate trees in a commercial nursery to use in multiple rootstock field trials sponsored by the HLB MAC program. Trees from the commercial nursery are scheduled to be planted into six cooperative field trials in 2016. Trees in the USDA nursery on a large number of advanced rootstock selections, especially Supersour-type, were continued in propagation for field trials to be planted in 2015 and 2016.
The project will focus on work to identify metabolite profiles associated with tolerance to HLB and other stresses in advanced rootstock selections, and validate the effectiveness of these metabolite profiles for selection by comparison of existing rootstock selections within the USDA program. As requested by CRDF, the project will place highest priority on work with new hybrid rootstocks already created, selected, and included in field trials. As requested by CRDF, other parts of the original proposal that involved integration with the breeding program, recurring selection, and discovery of biomarkers and antimicrobial compounds were eliminated, with a consequent simpler focus and smaller budget. In this quarter, data from a preliminary metabolomic study was organized and prepared for publication. This study was conducted under a previous CRDF funded grant, and demonstrated striking metabolic differences between HLB sensitive and HLB tolerant genotypes, both with and without Las infection. This data will serve as a foundation for continuing work under this grant. The work under this grant will focus on collecting an extended set of metabolomic data that will be adequate to allow validation of the approach by the end of project year 3. Environment may have strong effects on metabolomic data, so field testing, greenhouse testing, and information on seasonal variation will be critical for success in validating the approach. To clarify environmental effects, greenhouse studies will be needed, and a greenhouse suitable for the work is being constructed. In this quarter, plans were drawn up, and contract issued. The new greenhouse is expected to be completed in February 2016. In this quarter, plans for were made for specific studies to identify key metabolic compounds and collect the first stage of information to be used in the validation process. For the first year of the project, three greenhouse studies with potted trees, and five field studies (with established trees of different ages) will be conducted, to collect information about expression in leaf and root tissue, as well as seasonal effects on expression. The work will focus on 12 rootstocks that are of commercial importance and where previous studies have identified relative differences in tolerance to Las infection. Established field plantings are available with these 12 rootstocks suitable for the testing needed. In this quarter, trees in the selected field trials were scored for HLB symptoms and sampled for PCR testing, so that disease and infection status can be included in the choice of trees for metabolic testing. In preparation for the greenhouse studies, trees were propagated in another greenhouse, so that they can be moved into the new house for immediate experimentation, as soon as the new house is completed.
The main accomplishments during this quarter: We continued testing the effect of the K gene on transformation efficiency of a lemon cultivar. In general, lemon cutilvars are difficult to be genetically transformed. We have observed the K gene can drastically improve the transformation efficiency of the lemon cultivar used, similar to the effects of the K gene on the other orange cultivars tested. We continued to repeat the effects of K and I genes on transformation efficiency of mature citrus explants of mature Pineapple orange. The effects of the K and I genes have been confirmed. We have also tested effects of a non-conventional regulator of gene expression on regeneration efficiency of Washington navel orange and Valencia orange. We observed several fold increases in shoot regeneration efficiencies of both cultivars. Our goal is to use this regulator and also its combination with the K gene to improve transformation efficiencies of of both juvenile and mature citrus tissues One manuscript reporting the drastically improvement of several citrus cultivars has been submitted in the end of August. We have started to write the second manuscript from the project.
During this reporting period (July, August, and September, 2015), the transgenic plants to be developed for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. These plants are continuing to be propagated at both Ft. Pierce and Penn State. Our collaboration with Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) to transform varieties important to the Florida citrus industry, including the ‘Valencia’ and ‘Hamlin’ sweet orange varieties and the ‘Citrumello’ and ‘Carrizo’ rootstocks with the FLT-antiNodT expression construct, has had initial success. Hamlin and Carrizo transformants are now growing at Lake Alfred. Dr. Zale will maintain the original transformants, and will send propagated cuttings to Penn State for molecular analysis over the next 3-6 months. We will also send some of the propagated sweet orange and rootstock plants to Ft. Pierce for HLB resistance testing in collaboration with Dr. Tim Gottwald and possibly Ed Stover. During this reporting period, we also initiated development of an FLB-antiNodT expression cassette in the transformation construct pBI121, which has a history of successful approval for transgenic plant development. We anticipate that this construct could be completed during the next reporting period, and we would forward it to Dr. Zale immediately upon completion for further citrus transformations. In August, Dr. McNellis presented a poster at the annual meeting of the American Phytopathological Society in Pasadena, CA, describing the results so far, including successful expression of the FT-scFv protein in grapefruit with minimal or no negative effects on plant phenotype.
During this reporting period (July, August, and September, 2015), the transgenic plants to be developed for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. These plants are continuing to be propagated at both Ft. Pierce and Penn State. Our collaboration with Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) to transform varieties important to the Florida citrus industry, including the ‘Valencia’ and ‘Hamlin’ sweet orange varieties and the ‘Citrumello’ and ‘Carrizo’ rootstocks with the FLT-antiNodT expression construct, has had initial success. Hamlin and Carrizo transformants are now growing at Lake Alfred. Dr. Zale will maintain the original transformants, and will send propagated cuttings to Penn State for molecular analysis over the next 3-6 months. We will also send some of the propagated sweet orange and rootstock plants to Ft. Pierce for HLB resistance testing in collaboration with Dr. Tim Gottwald and possibly Ed Stover. During this reporting period, we also initiated development of an FLB-antiNodT expression cassette in the transformation construct pBI121, which has a history of successful approval for transgenic plant development. We anticipate that this construct could be completed during the next reporting period, and we would forward it to Dr. Zale immediately upon completion for further citrus transformations. In August, Dr. McNellis presented a poster at the annual meeting of the American Phytopathological Society in Pasadena, CA, describing the results so far, including successful expression of the FT-scFv protein in grapefruit with minimal or no negative effects on plant phenotype.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. All of these constructs had the peptide gene inserted between the coat protein genes, which is positioned sixth from the 3′ terminus. However, we have found that much more foreign protein can be made from genes positioned nearer the 3′ terminus. Based on that we built constructs with the peptide gene next to the 3′ terminus. These constructs produced much greater amounts of peptide and provided more tolerance to Las. Unfortunately, they are less stable. So now we are rebuilding constructs with the peptide gene inserted at an intermediate site hoping for a better compromise of amounts of production and stability. We have produced a large amount of inoculum for a large field test via Southern Gardens Citrus. We are screening a large number of transgenic plants in collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We are propagating a progeny set of plants of the promising candidates for a final greenhouse test.
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. Cuttings from the following ‘gauntlet’ rootstocks were treated with rooting hormone and place on the mistbed for rooting: 1. A+HBPxCH+50-7-12-14 2. 46×31-00-S10x46x31-00-S11-S5 (salt tolerant sour orange-type) 3. Orange 10 x Green 7-11-1 4. A+VolkxOrange19-11-5 5. A+HBJL2BxOrange14-09-7 6. A+HBJL2BxOrange19-09-31 7. A+HBJL1-09-14 8. A+FDxOrange19-11-11 Seedlings were grown from four promising sour orange-like rootstock candidates with promise for adaptation to IR soils, with a goal of providing a quick source of material for cuttings as follows: 1. 46×20-04-S22 2. 46×20-04-42 3. 46×20-04-48 4. 46×20-04-S13
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