A large-scale trial of greening-tolerant citrus cultivars is addressing the need of Indian River growers to know what are the best rootstocks and scions for growing fresh fruit. The project has two objectives: (i) Assess the performance of new grapefruit cultivars with certain rootstocks in the IR district; and (ii) Evaluate the influence of UFR and other recent rootstocks on grapefruit, navel, and mandarin in the IR in comparison to legacy/standard rootstocks. There are four trials: Trial 1) 18 grapefruit cultivars on three rootstocks; Trial 2) 32 rootstocks with Ray Ruby grapefruit as the scion; Trial 3) 31 rootstocks with Glenn F-56-11 navel orange as the scion; and Trial 4) 31 rootstocks with UF-950 mandarin as the scion. In June 2021, 400 tress were planted to add to the 4,500 trees planted the previous two years. The final 90 grapefruit trees on UFR-8 rootstock will be planted in September 2021. In August 2021, leaf and soil samples were collected from each experimental plot to properly manage fertilizer requirements. Controlled-release polycoated fertilizer was applied appropriately in July 2021. All trees were treated biweekly with appropriate agrochemicals to manage canker, Asian citrus psyllid, mites, and citrus leafminers. Tree height, tree width in cardinal directions (E-W/N-S), and trunk diameter were measured on three middle trees in each experimental plot in June 2021 to quantify canopy volume and tree size. Results to date were presented by MSc student Martin Zapien at the 2021 American Society for Horticultural Sciences in Denver, CO, in August 2021. Martin placed 3rd in the Scholar’s Ignite Competition. Tree size data from June 2021 show some scion/rootstock combinations are exhibiting significant differences in canopy volume. In Trial 1, Pummelette UF-5-1-99-2 grapefruit on US-942 is 9.1X larger (7.3 m3) than US 1-83-179 grapefruit hybrid on sour orange (0.8 m3). In Trial 2, grapefruit on UFR-15 is 2.5X larger (5.5 m3) than on UFR-17 (2.2 m3). In Trial 3, navel orange on US-802 is 2.2X larger (3.7 m3) than on Willits (1.7 m3). In Trial 4, mandarin on C-22 is 2.2X larger (4.3 m3) than on 46×20-04-6 (2.0 m3). Longer-term evaluation is needed to identify the most promising scions and rootstocks to determine their profitability and capability of meeting grower and market needs. Greening disease is spreading in the field as visual greening symptoms are on average 25% (up to 60%) of the total tree canopy volume. Leaf samples for quantifying CLas titer were collected in March 2021 and sent to Southern Gardens for analysis. Trees that are CLas-free (ct values >32) and CLas-infected (ct values of 26-32) can be found in the same plots, but many symptomatic trees are developing vigorous canopies. Many trees are bearing their first fruits. The incidence of Asian citrus psyllids, Diaprepes root weevils, whiteflies, leafminers, and citrus canker was quite apparent during the summer due to abundant rainfall and the emergence of a large amount of flush. Leafminer damage has been substantial. Nonetheless, tree growth has not been significantly adversely affected by these pests due the biweekly application of agrochemicals. The second field day to exhibit this project will take place on October 14, 2021.
Evaluation of Newly Developed and/or Released Citrus VarietiesSept 2021 Investigator: Nabil KillinyIn this project we are profiling the new scions and rootstocks for their tolerance to HLB by studying the metabolite content by GC-MS, and challenging new varieties with psyllids and HLB.Progress on Objectives: Objective 1To understand the mechanism behind the tolerance of different varieties toward HLB. The comparison between the varietal responses will allow us to determine the mechanism of tolerance to CLas. This quarter we continued our focus in four areas: 1) new mandarin hybrid Lucky (SugarBelle x Nava x Osceola); 2) new Valencia varieties, 3) CUPS new grapefruit varieties, 4) rootstock evaluations. In addition, we tried again to graft Marathon Mandarin onto Swingle and Carrizo unsuccessfully (0/10). We have not been able to get even a small number of Marathon from the citrus nurseries, so we assume they are also having problems with propagation.Findings: 1) For the mandarin hybrid we call Lucky and its parents, Sugar Belle and Nava × Osceola, we completed the volatile organic compound (VOC) analysis to determine if Lucky will have any of the chemical properties of Sugar Belle, which is considered tolerant to HLB. We found it has slightly more total VOCs than either of the parents, and about the same as `Valencia’ sweet orange, which we used for a comparison. We detected 50 VOCs and the dominant leaf volatiles were sabinine, linalool, trans-ß-ocimene, and ß-caryophyllene in the mandarin hybrid. Like other mandarins, they are low in d-limonene and almost completely lack neral and geranial, the aldehydes that dominate `Valencia’. In addition, we repeated the ACP choice experiment beginning in June using CLas-infected ACPs. The experiment just concluded with the final samples being collected on 9/3/21. We are looking for any chemical clues that indicate an induction of defense compounds after infestation with ACP. To achieve this, leaf samples from the 15 plants were collected 5 days, two weeks, and one month after adding psyllids to the cages. Thereafter, monthly leaf samples were taken in parallel with ACP population counts. The leaf samples need to be processed now for volatiles and HLB status. Currently there are no HLB symptoms, but the trees will be followed closely to determine how quickly they develop symptoms and when HLB can be detected by PCR. 2) For the evaluation of the new sweet oranges Valquarius and Vernia, the leaf samples for analysis of volatiles have been run on the GC-MS and integrated. The data analysis was completed. Qualitatively, the three sweet orange varieties are very similar, but the total VOC content was highest in Valencia, followed by Valquarius, then Vernia. We identified 47 VOCs in the hexane-extracted leaves of the three varieties. We did not detect any unique compounds among the three varieties. Those in the highest concentrations included sabinene, linalool, neral, geranial, ß-elemene, a-sinensal, and phytol. Many differences (37/47) were statistically significant because of the high content found in the traditional Valencia variety as compared to the two newer varieties. We have previously associated higher levels of VOCs to ACP attraction, so there may be a slight advantage to the newer varieties in having a lower VOC content. We plan to perform ACP choice tests on the new varieties by pairing them with Valencia in a new choice test apparatus we made for this purpose. 3) New grapefruit variety from CUPS – UF914 is being evaluated and compared to traditional Duncan, Ruby Red and Ray Ruby varieties for volatile and non-volatile metabolite content. UF-914 is a low furanocoumarin variety so special attention is being paid to these compounds. We have detected several coumarins and sterols in the grapefruit extracts and quantification is in progress. Objective 2 4) To understand the role of rootstocks in citrus tolerance to HLB. The comparison between rootstock metabolites will allow us to determine the best scion/rootstock combinations for tolerating CLas. a. The rootstock seeds from the USDA (US-802, 812, 897, 942, 1283, 1284, 1516) for metabolite profiling and HLB/nematode screening were moved outside to encourage growth, repotted into larger pots, and are now about 6 inches tall and 7 months old. We hope they will be ready for evaluations soon. We plan to challenge these rootstocks with ACP to determine their response to HLB.b. The grapefruit leaf samples collected from CUPS included Duncan on four rootstocks, and Ray Ruby on two rootstocks. We will make comparisons to see if there are any differences in the volatile and non-volatile metabolites due to the different rootstocks.
We generated raw sequence data for Valencia orange (S, sensitive), Ruby Red grapefruit (S), Clementine mandarin (S), LB8-9 Sugar Belle® mandarin hybrid (T, tolerant), and Lisbon lemon (T) and preliminary assemblies and analyses were carried out. Because of reduced sequencing costs, we were able to enter additional important genomes into the pipeline beyond those originally proposed, including Carrizo citrange, sour orange, and Shekwasha (an important breeding parent for HLB tolerance); these also have now been sequenced and preliminarily assembled. We now have the transcriptome data for two of our target genomes, using both Illumina and PacBio sequencing platforms, and genome annotation (i.e., identify all the genes within the genome) is continuing. Hi-C sequencing of the 7 remaining target genomes is underway; once completed these data will be integrated with the PacBio assembly, to produce improved chromosome scale assemblies. RNA samples of these 7 additional genomes have been prepared to generate the transcriptome data required for genome annotation, and further characterization of large-scale structural variations within and among the genomes upon which we are focused.In a related effort, our team recently compared 69 new east Asian genomes and other mainland Asian citrus to reveal a previously unrecognized wild sexual species native to Japan’s Ryukyu Islands: C. ryukyuensis, which hybridized with an ancient east Asian mandarin to produce Shekwasha (shiikuwasha) mandarin, a powerful source of HLB tolerance in rootstock breeding. Further, by studying the genomes of C. ryukyuensis-derived hybrids and other citrus. we traced the origin and spread of apomixis (nucellar embryony, a trait that is required for seed propagation of citrus rootstocks) from Mangshanyeju wild mandarins in China a few million years ago through most of the commonly known contemporary citrus types (orange, grapefruit, lemon, etc.). This work resulted in deeper understanding and new genome-based tools that can be exploited for two critically important traits in citrus genetic improvement, nucellar embryony and most importantly HLB tolerance; the research was published in Nature Communications in July 2021 (see https://doi.org/10.1038/s41467-021-24653-0). We used the PacBio Sequel platform to sequence full-length gene transcripts in the leaf tissues of sweet orange and trifoliate orange and reconstructed their leaf transcriptomes. We identified novel full-length transcripts that were not present in the published reference transcriptomes. We found that some NBS-encoding genes (nucleotide binding site genes, one typical class of disease resistance genes in plants) underwent alternative splicing. One alternatively spliced NBS transcript expressed in HLB symptomatic leaf and fruit of sweet orange, and another alternatively spliced NBS transcript was differentially expressed in CLas-infected trifoliate orange samples, suggesting that isoforms of some NBS-encoding genes may play an important role in HLB tolerance of trifoliate orange, or alternatively HLB susceptibility in sweet orange. The new transcriptomes will be useful to identify candidate genes for disease resistance that have been missed in the published citrus genomes and transcriptomes.
Create new candidate hybrids. During this quarter, hybrids from previous cycles were selected, and propagation planned for the next cycle of Stage 1 field trials. New seed source trees were propagated for advanced selections and planted in the field. Based on new information on performance of different parental combinations in rootstock field trials, additional crosses among the best parental material are planned for next spring, as resources are available. Propagate and plant new field trials. Budwood increase trees of selected scions were grown, in preparation for budding trees for new rootstock trials. Trees for Stage 2 rootstock trials with Valencia and Hamlin on selected released rootstocks and the best of the next generation hybrids are being grown in the greenhouse in preparation for field planting in 2021. One new Stage 2 trial with Valencia scion and one new Stage 2 trial with grapefruit scion were planted in the field. Nursery trees for two new Stage 1 trials with 60 new rootstocks and Valencia were propagated in the greenhouse in preparation for field planting in fall 2021. Some planned propagation for new trials was delayed because of USDA institutional Coronavirus shutdown. Collect data from field trials. Extensive information on tree performance is collected from established field trials, and includes measurement of tree size, fruit crop, fruit quality, and pathogen titer, HLB symptoms, and assessments of tree health. Cropping data is collected during the time of scion harvest, and during this quarter none of the active trials have scions for which crop would normally be harvested. Assessments of tree health were completed on 23 trials during this quarter. All the backlog of fruit quality analysis (caused by institutional Coronavirus restrictions) from frozen juice samples in the previous season was completed, with full data on brix, acid, and color from 15 replicated trials. Because the data collection load from field trials was lower this quarter, additional effort was put into tree maintenance, and updates to irrigation and drainage systems in selected trial blocks.Evaluate effectiveness for seed propagation of new rootstocks and develop seed sources. Some of the newest hybrid rootstocks can be uniformly propagated by seed, but others cannot. As the best rootstocks are identified through field trials, seed sources are established and used to determine trueness-to-type from seed. Studies were continued this quarter to evaluate seed propagation for 20 of the most promising SuperSour hybrid rootstocks. Progeny groups were scored, and tissue samples were prepared for SSR analysis of progeny to determine frequency of nucellar polyembryony and uniformity from seed. This work is progressing more slowly than planned because of institutional Coronavirus restrictions. Cooperative work continues to compare field performance of rootstocks propagated by seed, cuttings, and tissue culture. Evidence indicates that performance of rootstocks is primarily determined by rootstock cultivar, and not much influenced by propagation method.Posting field trial results for grower access. The USDA rootstock trials produce large amounts of information that is useful to identify the most promising of the new hybrids, as well as comparative information on the relative performance of many commercially available rootstocks. During this quarter, updated trial summaries were prepared for uploading to the website https://www.citrusrootstocks.org/, and a presentation on rootstock performance in field trials was given at the Florida Grower Citrus Show.Release of superior new rootstocks for commercial use. Release of new USDA rootstocks is based on robust data from multiple trees in replicated field trials over multiple years, including information on tree survival and health, canopy size, fruit yield and fruit quality, and observations on tolerance of disease and other biotic and abiotic threats. Outstanding performance in industry plantings from some of the previously released USDA rootstocks, especially US-942 and US-812, indicate the process is effective. Several of the 350 advanced Supersour rootstock hybrids in field trials are exhibiting outstanding performance in comparison with the commercial standard rootstocks. Information on some of the most promising of the next-generation USDA hybrid rootstocks was presented at the Florida Grower Citrus Show. Performance data continues to be collected, and it is anticipated that 2-3 of the most outstanding of these will be officially released in 2022-23.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Stepped up 160 candidate rootstock hybrids from the first step of the Gauntlet screen (high pH, calcareous soil inoculated with Phytophthora), from different crosses into citripots; crosses were made using LB8-9 (Sugar Belle®) as a seed parent with either trifoliate hybrids or salt tolerant sour orange (pummelo-mandarin hybrids) types. Three new Gauntlet rootstock candidates, all showing very good HLB tolerance and possibly resistance, were entered into the PTP. These hybrids arose from similar crosses as described above, combining genetics from pummelo, Shekwasha mandarin, LB8-9, and trifoliate orange. All 3 hybrids are showing excellent health and ability to suppress CLas replication in their root systems and in grafted Valencia scion. 2. Develop new, HLB-tolerant scion cultivars from sweet orange germplasm, as well as other important fruit types such as grapefruit, mandarins, and acid fruit. We are in the process of removing previously planted and tested scions from our program that do not warrant further scrutiny, grown in the Trailer Park block. Newly available spaces will be planted in late summer with 60 new scion selections from the program that have gone through the DPI PTP cleanup and certification; these include true oranges and orange-like hybrids, grapefruit and hybrids, mandarins, lemons, pummelos, and acid fruit. All trees have been grown on UFR-5 by a commercial nursery. Somaclone seedling derived populations of January-maturing OLL sweet orange clones are being propagated on UFR-4 rootstock, to attempt to produce an even earlier maturing clone; to date >100 individual seedlings have been propagated.3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We have explored some new approaches to quantifying tree responses to HLB, in addition to the previously used subjective approaches. Specifically, we have begun measuring photosynthetic parameters and leaf canopy indexes, to produce repeatable and reliable quantitative data in support of further genetic analyses of tolerant types. Analysis of collected data is underway, in consultations with plant physiologists familiar with the techniques and data interpretation. This work will improve the precision with which we can define HLB tolerance genes. 4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. Using the quantitative data described in 3. above, we are conducting additional GWAS to validate previously identified, or to identify new, genomic regions associated with HLB tolerance and/or sensitivity. Several new genetic constructs have been developed using newly identified citrus specific promoters (phloem and root tissue), and new putative disease resistance genes, or downstream genes. Transgenic plants have been produced with some of these constructs, and additional transformation experiments have been begun. Finally, the very early response of citrus to the CLas, vectored by infective ACP, was evaluated for the first time, thus allowing the changes in gene expression relating to the primary mechanisms of susceptibility and host -pathogen interactions to be studied, and without the secondary effects caused by the development of complex whole plant symptoms; please see doi: 10.3389/fpls.2021.635153.
True sweet oranges: With the passing of Louise Lee, the trial block containing the OLL somaclone seedling population is in jeopardy of being sold for development. Efforts to rescue the most promising clones from this trial are underway, and permission was obtained from DPI (Ben Rosson) to propagate the rescued trees at the CREC. Clones being rescued include those showing earlier maturity, higher soluble solids, and better HLB tolerance. Potential HLB resistance from ‘gauntlet’ rootstock candidates: qPCR was completed on root samples from 45 additional promising gauntlet rootstock candidates. 18 rootstock candidates had ct values above 30, including 6 that had ct values of 40 (no bacterial detected). Among these, 5 rootstock candidates showed suppression of CLas in the Valencia scion; including 3 vigorous pummelo x latipes hybrids (13-76, 13-53 and 13-43), and two [Amblycarpa+HBPummelo] x [sour orange + rangpur] hybrids (13-15 and 13-12). Note that C. latipes and the sour orange+rangpur are highly HLB tolerant parents. Pathogen-free material of top gauntlet rootstocks sent to TC labs for micropropagation include potentially HLB resistant S10xS15-12-25, S11x50-7-16-12, S11x50-7-16-6, and A+HBPxCH+50-7-12-11; and potentially HLB tolerant LB8-9(SugarBelle)xS13-15-16, LB8-9(SugarBelle)xS10-15-9, S10xS15-12-34, and A+HBPxCH+50-7-12-39. Note that S10, S11, S13 & S15 are all salt tolerant pummelo/mandarin parents. Molecular marker analysis of the promising super-root mutant UFR-1 clone#28 (Fast 28, discovered by Beth Lamb at the Rucks TC lab) showed that it is a deletion mutant of UFR-1; this vigorous rootstock continues to look promising in the field with sweet orange and grapefruit scions. Additional liners are being propagated at the Rucks TC lab for advanced trials. Identification of probable zygotic rootstocks at St. Helena showing good HLB tolerance (mentioned in previous report): 6 trees on apparent zygotic rootstocks were identified producing 3 – 3-5 boxes of Vernia/Valquarius fruit per tree with 12 – 12.5 brix. One is a tetraploid from Orange 12 (Nova+HBP-derived), one from Purple 2 (Nova+HBPxCleo+SO), two from HBPummelo x Shekwasha, and two from HBPummelo (open pollination). Since the staked-up scaffold roots did not sprout, we cut the tops (scion) off the trees as necessary to recover the rootstock genotypes. Two of the trunks have begun to sprout. We plan to generate pathogen-free material of these selections, followed byTC micropropagation to generate material for advanced trials. We also identified a truly stellar Valquarius tree on rough lemon; we are conducting a molecular marker test on the roots to see if the rootstock is a unique zygotic. If so, it will be treated as the above rootstocks. St. Helena: The entire trial was assessed tree by tree, and all under-performing trees were pink-flagged for removal. This has created approximately 2000 rotational spaces, and new rootstock candidates along with new early-mid season sweet orange candidates will be planted. Tree removal and replanting are expected to get underway this quarter. Field Trial Data Collection: Tree size data was collected from the following trials: Bryan Paul, Smoak, Greene River Citrus(lemon), Tom Hammond, Post Office and Peace River. Tree health assessment data was collected from the Mislevy trial. Data analysis and entry onto the Rootstock Data Website: annual updates included: Heller Bros., Peace River, Bryan Paul Doe Hill, Smoak, Post Office, and Tom Hammond. Trial data being uploaded and analyzed included data from the follwoing trials: St. Helena, Premier Citrus, Greene River, Lee Family Groves, IMG, Banack, Cutrale and Wayne Simmons.
The project has five objectives:
(1) Remove the flowering-promoting CTV and the HLB bacterial pathogen in the transgenic plants
(2) Graft CTV- and HLB-free buds onto rootstocks
(3) Generate a large number of vigorous and healthy citrus trees
(4) Plant the citrus trees in the site secured for testing transgenic citrus for HLB responses
(5) Collect the field trial data
In this quarter, the following activities have been conducted:
(1) A total of 85 plants including 65 transgenic plants and 20 non-transgenic control plants were planted into the field on May 20, 2021. These transgenic plants include replicates of three transgenic lines that have been shown to have robust tolerance to HLB in the greenhouse. Ten replicates of another transgenic line that expresses a different disease resistance gene were also included. This transgenic line has shown HLB tolerance in the greenhouse for more than eight years. In addition, we have eight transgenic Carrizo lines that express three different disease resistance genes. These lines have been replicated and grafted with Valencia. The three constructs was added onto the field trial permit. These plants will be transplanted into the field in the Fall of 2021. The transgenic plants that were transplanted in 2019 were examed. The plants grow well in the field and none of the plants has shown HLB symptoms. We plan to collect samples for CLas titer assay in this fall.
(2) The citrus gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was cloned and sequenced. The two conserved amino acids T177 and P181, which are responsible for glyphosate tolerance, were changed to isoleucine (I) and serine (S), respectively. The resulting citrus TIPS EPSPS gene was cloned into a T-DNA binary vector, which was introduced into Agrobacteria. To test if the citrus TIPS EPSPS gene can provide tolerance to glyphosate, we transformed it into Arabidopsis, since it will take shorter time to know the result in Arabidopsis. We will have plants for glyphosate tolerance test in the next quarter.
(3) Transgenic citrus plants expressing the Arabidopsis nicotinamide adenine dinucleotide-binding receptor were inoculated with CLas using psyllids. We are waiting for the HLB symptom development. Meanwhile, we repeated nicotinamide adenine dinucleotide-binding experiment for the citrus lectin receptor kinase proteins using Monolith NT.115. We plan to use radiolabeled nicotinamide adenine dinucleotide to confirm the binding results in the next quarter.
The purpose of this project is to optimize the CRISPR technology for citrus genome editing. This study is related to the CRDF RMC-18 Research Priorities 4AB. Objective 1. Expanding the toolbox of citrus genome editing. In this study, we will adapt StCas9, NmCas9, AsCpf1 (from Acidaminococcus), FnCpf1 (from Francisella novicida) and LbCpf1 (from Lachnospiraceae) on genome modification of citrus. Lately, we have shown CRISPR-Cpf1 can be readily used as a powerful tool for citrus genome editing. In our recent study, we employed CRISPR-LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. Our study showed that CRISPR-LbCas12a can readily be used as a powerful tool for citrus genome editing. One manuscript entitled CRISPR-LbCas12a-mediated modification of citrus has been published on Plant Biotechnol J. We are currently further optimizing LbCas12a-crRNA-mediated genome editing to make homologous biallelic mutations. We are also testing AsCpf1 and FnCpf1 for their application in citrus genome editing and generating homologous biallelic mutations. We have successfully generated both homozygous and biallelic mutations in the EBE region of LOB1 gene in pumlo. This work has been submitted for publication. We are in the process of generating homozygous and biallelic lines of other citrus varieties.Recently, we have developed multiplex genome editing toolkits for citrus including a PEG mediated protoplast transformation, a GFP reporter system that allows rapid assessment of the CRISPR constructs, citrus U6 promoters with improved efficacy, tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we have successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, indicating that the CRISPR-mediated citrus genome editing technology is mature and could be implemented in citrus genetic improvement as a viable approach. In addition, our study lay the foundation for non-transgenic genome editing of citrus. One manuscript entitled Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations has been published on Plant Molecular Biology.We have successfully developed base editing tools for citrus genome editing. This method has been succefully used to generate non-transgenic biallelic mutants of sweet orange. Objective 2. Optimization of the CRISPR-Cas mediated genome editing of citrus. In this study, we are testing different promoters including INCURVATA2 promoter, the cell division-specific YAO promoter, and the germ-line-specific SPOROCYTELESS promoter, and ubiquitin promoter in driving the expression of Cas9 and Cpf1 orthologs. To optimize the expression of sgRNA and crRNA, we have identified multiple citrus U6 promoters and two of the citrus U6 promoters showed higher efficacy in driving gene expression in citrus than 35S promoter and Arabidopsis U6 promoter. We have further increased the mutation efficacy to 50%. We have further optimized the CRISPR/Cas9 system. Now, the biallelic mutation rate reaches 89% for Carrizo citrange and 79% for Hamlin sweet orange. We have generated one homozygous line in the promoter region of canker susceptibility genes of Hamlin. We have successfully generated one biallelic mutant of grapefruit that is canker resistant. We also successfully generated multiple biallelic and homozygous mutant lines of sweet orange that are canker resistant. Objective 3. Optimization of the CRISPR technology to generate foreign DNA free genome editing in citrus. We have conducted transient expression of Cas9/sgRNA plasmid and Cas9 protein/sgRNA ribonucleoprotein complex in citrus protoplast. We are also conducting citrus genome editing using Cpf1/crRNA plasmids and ribonucleoprotein complex in citrus protoplast. The plasmid-transformed protoplast has 1.7% editing efficiency, and the RNP-transformed samples have approximately 3.4% efficiency. The genome modified protoplast cells are undergoing regeneration. We aim to increase the efficacy to over 20% and eventually generate non-transgenic genome modified citrus. One patent has been filed on the CRISPR-Cas mediated genome editing of citrus. We have lately optimized the citrus protoplast isolation and manipulation, our data showed that more than 98% of the isolated protoplasts were alive. We regularly obtained a transfection efficiency of approximately 66% or above. Base editors have been successfully used to precisely edit specific residues of citrus genes. ErCas12a has been succes for non-transgenic gene editing of embryogenic Hamlin sweet orange protoplast cells. We are editing 6 putative HLB susceptibility genes for sweet orange. One biallelic mutant line has been generated for ACD2.
1. Please state project objectives and what work was done this quarter to address them: Objective 1. Investigate effects of rootstock propagation method and the interaction with rootstock on root structure, root growth, and tree performance during the first 3 years of growth in the field.Bimonthly root growth measurements with the minirhizotron imaging system continued in all trials. Trials 2 and 3 third-year tree size data collection was completed. Fruit quality analysis was completed for trial 3. We are working on completing the analysis of the year 3 data of trials 2 and 3 to be included in a publication of all three years of data. A Citrus Industry Magazine article sumarizing findings of this project was published (July issue). A virtual seminar was presented on June 21 that included information on this project. Objective 2. Investigate if trees on rootstocks propagated by tissue culture or cuttings differ in susceptibility to Phytophthora-induced decline or wind-induced blow-over compared with trees on rootstocks propagated by seed.Monthly root growth measurements with the rhizotron imaging system continued. We are still working on the PCR and ELISA assays. 2. Please state what work is anticipated for next quarter: We will continue with our minirhizotron root imaging analysis and. We will continue with the statistical analysis and interpretation of all data collected. 3. Please state budget status (underspend or overspend, and why): Approximately 67% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.
1. Please state project objectives and what work was done this quarter to address them: Objective 1. Investigate rootstock effects on horticultural performance of Valencia and Hamlin trees commercially grown under HLB-endemic conditions using standardized field data collection procedures.Fruit collection and fruit quality analysis for the Valencia trials was completed.Harvest was completed for both Valencia trials. Tree/canopy size and trunk diameter measurements were completed for this year.We are still working on the leaf CLas analysis in for trees in the Valencia trials.The statistical analysis of all data is nearly completed and is being prepared for publication. Objective 2. Develop outreach to transfer information to growers and other industry clientele.A one hour virtual seminar on rootstocks, including a summary of results from this project, was presented on June 21. 2. Please state what work is anticipated for next quarter: We will continue with the data analysis. Data are being prepared for a presentation at the Citrus Expo in August. 3. Please state budget status (underspend or overspend, and why): Approximately 70% of funds have been spent, which is somewhat underspent due to Covid-19 related complications that affected research, travel, and hiring of personnel.
Update for this quarter:A significant USDA-funded infrastructure project at the Picos Farm location is now complete. Improvements included a full renovation of the water management system; with drainage channels re-dug and cleared of vegetation, culverts inspected and replaced as needed, and new irrigation controls with remote access capability added. This will significantly improve storm and flood protection. Site management and field trials are progressing well. Four new transgenic trials were planted this quarter; two antimicrobial peptide expressing designs from the Stover team and two enhanced disease resistance designs from UF researchers. With these trials, the transgenic site is now operating at full capacity (some UF and other non-transgenics have been managed under this grant in adjoining blocks, exceeding 10 acres total for three years). The UCRiverside-led trifoliate and trifoliate hybrid trial has concluded, a manuscript regarding identified HLB-tolerance is in preparation, and trees will be removed to make space available for future plantings. The primary BRS permit has been amended to include a new UF gene construct that has shown a reduction in CLas titer in greenhouse trials. All regulatory protocols are being observed and the annual site review from APHIS/BRS has been conducted. Recent quarters:An additional permit has been approved (AUTH – 0000043620 effective 12/17/2020) for material with “Confidential Business Information” for a project led by R. Shatters. The primary BRS permit AUTH – 0000043619 has been renewed. Stover analyzed data on canker incidence for as Incidence of Asiatic Citrus Canker on Trifoliate Orange and its Hybrid Accessions in a Florida Field Planting. Most notably: Almost all accessions with lower ACC lesion incidence were hybrids vs. pure trifoliate, though a few pure Poncirus had lower ACC than most. Based on chloroplast genome data from 57K Affymetrix SNP chip, provided by M. Roose, 11 of 33 reported seed parentage for hybrids was inaccurate, convention of female first was not followed. Chloroplast type did not affect ACC incidence, but in each year accessions with grapefruit chloroplasts had small but statistically higher ACC severity than those with Poncirus chloroplasts. Hybrids of Citrus with Poncirus have markedly reduced ACC sensitivity compared to Poncirus, indicating that this trait is readily overcome in breeding. A manuscript detailing these results has been published in HortScience DOI: 10.21273/HORTSCI15684-20. Previously established at the site:A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. Continuation of an experiment on pollen flow from transgenic trees. FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. Early-flowering transgenic Carrizo (flowered ex-vitro within five months of seed sowing, and used at 12 months) was used to pollinate some of the same FF-5-51-2 What should be the final samples from the C. Ramadugu-led Poncirus trial (#3 below) completed preparation and were shipped in ethanol to UC Riverside. Availability of the test site for planting continues to be announced to researchers. Plantings:1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the USDA permits.2) Under the Stover permit, a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. 4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project.5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and will continue to be conducted under a new NIFA grant.6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).7) In a project led by Fred Gmitter, there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo.10) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls.11) Multiple plantings with grafted trees of l Hamlin, Valencia and grapefruit scions on transgenic rootstock expressing antimicrobial citrus-thionin and bacterial recognition domain fusion proteins (219 trees with controls) as a collaboration between USDA and the New Mexico Consortium.12) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type.13) An additional planting from Zhonglin Mou of UF that includes transgenic grapefruit (31 plants) and sweet orange (33 plants) scions expressing two different resistance genes and grafted on WT swingle rootstocks; as well as non-transgenic controls. 14) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State15)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
This quarter:This quarter , the colony of CLas infected psyllids supplied a total of 5,045 ACPs used for (1) evaluation of rootstock breeding material, (2) transgenic events evaluation, (3) applied research for CLas control in citrus performed by USDA and University researchers; and (4) monitoring the colony quality.The Bowman lab continues the experiments with grafted Valencia trees on groups of rootstocks which were inoculated with ACP during the previous quarter (Janyuary to March 2021). Each experiment compares replications on nine different rootstocks. This quarter 63 trees were inoculated with 1,280 ACP from the positive ACP colonies. Periodic evaluation of tree health, growth, and CLas titer via PCR were conducted on trees from rootstock experiments that were inoculated during the previous months following a set schedule. Periodic colony checks were conducted by PCR to maintain CLas positive colonies. The Stover lab conducted weekly detached leaf assays (DLAs) challenging transgenic citrus with CLas inoculated by infected ACP in the lab, which is used to identify best performing transgenic events (transgenics varying by position of transgene insertion etc.) expressing antimicrobial peptides and defensive proteins targeting CLas, as well as natural insecticide peptides to control ACP. Six detached leaf assays experiments, involving individual 240 leaves, were inoculated using 2,400 CLas infected ACPs in this quarter. Transgenic material tested in DLAs were Carrizo and Hamlin plants expressing ONYX peptide under constitutive and phloem specific promoter. A total of 48 independent events were tested alongside WT controls. The leaves (midribs) and ACPs are being processed and submitted to qPCR for CLas titer after each DLA to better understand the effect of the transgenic peptide in bacteria control and transmission. These trials have being very useful in terms of providing information that allow to select the best transgenic events (ones causing high ACP mortality and/or low CLas transmission to plant) for propagation and further evaluation at greenhouse environment. We continue to see substantial ACP mortality from feeding on CLas-killing transgenic leaves. Research involving evaluation of the microbiome of ACPs fed on transgenic causing high insect mortality was conducted this quarter using 230 ACPs fed in a set of 22 transgenic leaves. In addition 775 CLas+ ACP were provided to researcher collaborators (600 ACPs for Dr. YongPing Duan of USDA and 175 for Florida International University, for Jessica Dominguez, a Ph.D. student, who is developing a thesis in alternative compounds to control CLas bacteria). Also, to monitor colony quality, 360 ACPs were used for Clas detection by qPCR this quarter. In this quarter, seven new colony cages (with 250 ACPs/cage) were set up to renew and support the demand of the hot ACPs for future use. Project rationale and focus: The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~50% (I don’t think Anna Sara has 50% of her time to take care of the colonies) to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Previous quarter:United States Department of Agriculture scientists Kim Bowman, Ed Stover and YongPing Duan have all run experiments totalling 7,280 ACPs. Samples have all been collected on-time from ongoing experiments. All samples collected, that have not been analyzed, have been processed for qPCR.
This quarter, the colony of CLas infected psyllids supplied a total of 6,840 ACPs used for (1) transgenic events evaluation, (2) applied research for CLas control in citrus performed by USDA and University researchers; and (3) monitoring the colony quality by qPCR.The Stover lab conducted weekly detached leaf assays (DLAs) challenging transgenic citrus with CLas inoculated by infected ACP in the lab, which is used to identify best performing transgenic events (transgenics varying by position of transgene insertion etc.) expressing antimicrobial peptides and defensive proteins targeting CLas, as well as natural insecticide peptides to control ACP. Seven detached leaf assays experiments, involving individual 220 leaves, were inoculated using 2,200 CLas infected ACPs in this quarter. Transgenic material tested in DLAs were Carrizo plants expressing ONYX peptide and chimeric AMP TS, both under SCAmp-P3 phloem specific promoter. A total of 36 independent events were tested alongside WT controls. The leaves (midribs) and ACPs are being processed and submitted to qPCR for CLas titer after each DLA to better understand the effect of the transgenic peptide in bacteria control and transmission. These trials have being very useful in terms of providing information that allow to select the best transgenic events (ones causing high ACP mortality and/or low CLas transmission to plant) for propagation and further evaluation in the greenhouse environment. We continue to see substantial ACP mortality from feeding on CLas-killing transgenic leaves. Research involving evaluation of the microbiome of ACPs fed on transgenic causing high insect mortality was conducted this quarter using 450 ACPs fed in a set of 45 transgenic leaves. A research paper has been prepared (Rapid in vivo screening for huanglongbing resistance in genetically modified citrus by detached leaf assay- J.Krystel, M. Grando, Q. Shi, E. Cochrane, E. Stover) in order to report important modifications implemented into the DLAs using Clas+ ACPs to evaluate transgenic plants and investigate the mode of actions of peptide in controlling the psyllids. In addition, 780 CLas+ ACP were provided to researcher collaborators (100 ACPs for Dr. Michelle Heck USDA-Ithaca, NY; 480 for Florida International University, for Jessica Dominguez, a Ph.D. student, who is developing a thesis in alternative compounds to control CLas bacteria) and 200 ACP were furnished to Dr. Randy Niedz (USDA Fort Pierce) for activities in a HLB NIFA project. Also, ten new colony cages were set up to renew and support the demand of the hot ACPs. For that 37 HLB positive plants were infested with 2.400 ACPs. Periodic colony checks were conducted by PCR to maintain CLas positive colonies. This quarter 960 ACPs were used for Clas detection by qPCR to monitor colony quality. In this quarter, Project rationale and focus: The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses.CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~30% of her time to the program in order to maintain colonies (including watering, setting up new cages, terminating old cages, cleaning growth chambers and cages). USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Previous quarter: United States Department of Agriculture scientists Kim Bowman, Ed Stover and Yong Ping Duan have all run experiments totaling 5,045 ACPs. Samples have all been collected on-time from ongoing experiments. All samples collected, that have not been analyzed, have been processed for qPCR.
Juvenile Tissue Citrus Transformation Facility (JTCTF) completed second quarter of operation as an EBA unit. The lab is open for customers who need transgenic citrus material and are ready to pay for services based on the EBA price list. At the same time, JTCTF still does work based on the research grants that were received in previous years. JTCTF resumed pre-COVID operation in regard to presence of work force in the lab well ahead of the date set by UF/CREC administration. However, one of the employees still works the night shift. There are still four employees on the roster although one of them has not worked since December because of unresolved immigration status.Within the first half of the 2021, JTCTF has charged clients for the work it performed as the EBA unit and multiple payment were successfully processed. The Business office staff at CREC helped in this process and made it work. As we receive more orders, the support from the business office in processing of payments will be very important.Between April and July, JTCTF accepted five new orders. Client are still mostly interested in transgenic Duncan grapefruit and Valencia sweet orange. Three of five placed orders were associated with the existing USDA grant, while two orders came from clients through EBA. The facility is still expecting the influx of orders that were going to be placed already based on the fact that JTCTF was designated as subcontractor on the research grants. Those orders are delayed because the work on those projects was also delayed due to COVID-19.The output of the JTCTF in the last 90 days was eight transgenic plants. Six plants were Duncan grapefruit, one was Hamlin orange, and the last one was Valencia orange. The number of transgenic shoots JTCTF produced within the last six months was big. However, those experiments were done for the proof of concept and transgenic shoots were not grafted and grown into plants. The output of the facility would have much been better if these plants were developed.
1. Please state project objectives and what work was done this quarter to address them: This report covers the period of March 1, 2021 – May 31, 2021. The objective of this project is to test transgenic ‘Ducan’ grapefruit trees expressing an anti-HLB antibody fused to the FT (Flowering Locus T) protein (FT-scFv protein). Several accomplishments were made during this reporting period. Graduate student Mr. Chad Vosburg was able to take a trip to Florida to assess trees again, take samples, and send samples for qPCR analysis at Southern Gardens. Chad visited Fort Pierce for several days in May, 2021. The samples taken and/or processed, and then sent to Southern Garderns included those from a field trial natural inoculation, an Asian citrus psyllid (ACP) infection in the greenhouse, and a repeate experiment of graft challenge of FT-scFv scions grafted to HLB-infected rough lemon rootstocks. These samples were prepared and sent to Southern Gardens for qPCR detection and quantification of ‘Candidatus Liberibacter asiaticus’ (CLas) within the tissues. Data are anticipated to be received from these samples in July-August 2021. In addition, growth data were taken from FT-scFv scions on infected rough lemon. The transgenic scions appeared to be consistently growing faster and more robustly than the control non-transgenic scions. However, this difference did not meet a standard cutoff for statistical significance. The FT-scFv protein appears to provide a mild benefit to the grapefruit trees when inoculated by grafting, even if not statistically significant. The replicate grafting inoculation experiment begun in January, 2021, has additional replicates and may provide more statistically robust results. In March, 2021, we applied for a no-cost extension through November of 2021, and it was granted in May, 2021. 2. Please state what work is anticipated for next quarter:Additional qPCR data from the field, graft, and psyllid-transmission HLB challenge tests will be received from Southern Gardens in the summer of 2021. These data will be analyzed during the next reporting period. Additional sampling of field grown trees will be performed with help from collaborators at the USDA USHRL. These will be sent to Southern Gardens for qPCR CLas quantification for an additional infection time point. 3. Please state budget status (underspend or overspend, and why):This project is running under budget thanks for wage support from Penn State for Chad Vosburg’s work on the project, which resulted in some salary funds not being expended.
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