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) Preparing and maintaining transgenic plants for field trials in the greenhouse. Transgenic plants prepared earlier for the proposed field trial were maintained in the greenhouse, waiting for transplanting. Newly produced transgenic plants expressing a regulatory gene of systemic acquired resistance were regularly watered and fertilized. A group of transgenic rootstocks were produced. We plan to graft scions onto these rootstocks in the coming year. (2) Expressing and purifying the extracellular domains of a group (10) of citrus homologs of an Arabidopsis disease resistance gene that encodes a receptor-like kinase with the extracellular domain binding nicotinamide adenine dinucleotide. It has been shown that overexpression of this receptor-like kinase increases resistance to bacterial pathogens. The citrus genome carries more than ten homologs of this receptor-like kinase. We have cloned in the last quarter the extracelllar domains of the closest ten homologs in an E. coli expression vector. In this quarter, we optimized the protein expression conditions, expressed and purified these fusion proteins from E. coli. These proeins are fused with GFP. We are currently testing the nicotinamide adenine dinucleotide-binding activities of these proteins using Monolith NT.115. The homolog(s) with nicotinamide adenine dinucleotide-binding activity will be the citrus functional homolog and will be used to generate intragenic/cisgenic citrus plants. We have generated transgenic citrus plants expressing the Arabidopsis nicotinamide adenine dinucleotide-binding receptor.s
The fall cover crop mix planted in early November at both locations has had excellent germination and growth. This mix included daikon radish, coker, Wrens grain rye, and dove millet. Sunnhemp, alyce clover, sesbania, dixie crimson clover, and yellow sweet blossom clover were included in the mixes for the nitrogen-fixation treatments (1/2 of the rows). Germination counts and biomass samples have been collected, and are currently being analyzed.
Measurements of the abundance of specific N-cycling genes has begun for soil samples collected at the last annual collection. After one year of cover crops, the abundance of nitrification genes (ammonia-oxidizing archaea and bacteria) significantly increased in treatments with cover crops when compared to the grower standard control, suggesting an increase in soil nitrogen availability. Measurements are in progress to determine the abundance of nitrogen-fixing genes (important with legumes since they are related to changes in the content of ammonium in soils) and denitrification genes (often linked to changes in soil organic carbon, organic matter, and nitrogen losses from the soils). Multivariate statistical analyses are also in progress to evaluate the influence of abiotic (soil properties) on biotic (microbial gene abundance) variables in treatments with and without cover crops.
Dataloggers and soil moisture probes continue to record soil moisture every hour. Root growth measurements using the mini-rhizotron tubes installed in both groves were performed in March and July 2019 and Feb 2020. Data on these measurements are currently being analyzed and will continue in Fall 2020. Initial analysis of yield, trunk area and canopy volume found no significant differences after 1 year of cover crops for one location. However, preliminary data indicates a greater canopy volume and trunk area at the second field site in treatments with the cover crop + legume mixture compared to other treatments. There were no significant differences in soil phosphorous, potassium, or magnesium concentrations between treatments at either location after 1 year of cover crops. Additional nutrient measurements are still in progress.
Weed density measurements from both study locations were collected in Aug 2019 and Jan 2020. Preliminary analysis of weed density data suggests that cover crops significantly reduced (up to 84%) weed pressure in treated row-middles when compared to non-treated controls. Moreover, cover crops improved the biodiversity in the treated row middles. Annual biomass data collection was also performed in both study locations in Jan 2020. Cover crop and weed biomass was harvested from the plots and bagged. In the lab they were sorted and weighed out to gather information on total weed/cover crop biomass from the treatment/control plots.
The first harvest since cover crops have been planted will occur in Spring 2020 (likely end of March). A project meeting with the grove managers will be held after harvest to discuss the next planting of cover crops. Some root rizhotrons and data loggers were damaged at one of the sites by hogs and some heavy equipment. These problems will be rectified in the next quarter to ensure continuity of of measurements in some treatments.
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. First, LbCas12a was used to modify the CsPDS gene successfully in Duncan grapefruit via Xcc-facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBEP thA4 -CsLOBP) of CsLOB1. A single crRNA was selected to target a conserved region of both Type I and Type II CsLOBPs, since the protospacer adjacent motif of LbCas12a (TTTV) allows crRNA to act on the conserved region of these two types of CsLOBP. CsLOB1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 in Xanthomonas citri by binding to EBEP thA4 -CsLOBP. A total of seven 35S-LbCas12a-transformed Duncan plants were generated, and they were designated as #D35 s1 to #D35 s7, and ten Yao-LbCas12a-transformed Duncan plants were created and designated as #Dyao 1 to #Dyao 10. LbCas12a-directed EBEP thA4 -CsLOBP modifications were observed in three 35S-LbCas12a-transformed Duncan plants (#D35 s1, #D35 s4 and #D35 s7). However, no LbCas12a-mediated indels were observed in the Yao-LbCas12a-transformed plants. Notably, transgenic line #D35 s4, which contains the highest mutation rate, alleviates Xcc.pthA4:dCsLOB1.4 infection. Finally, no potential off-targets were observed. 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.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 are further characterizing the citrus U6 promoters and testing their efficacy in driving sgRNA and crRNA in genome editing of citrus. We have significantly increased the transient expression efficiency. 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. One manuscript is in preparation for publication.
1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Twelve new rootstock crosses were made in spring 2019 and seeds were harvested and planted into the calcareous soil/ Phytophthora screen, the first stage of the gauntlet screening protocol. Seeds were harvested from many of the UFRs for distribution to nurseries. Seeds from various unreleased rootstock candidates, showing good performance (HLB tolerance, good yields and fruit quality, some tree size controlling with more efficient canopies) in several field trials throughout the industry were harvested to be available for future trials with interested industry partners in Florida, as well as for new trials in other citrus production areas. New hybrids from 2018 crosses for rootstock improvement were field planted. 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. Spring crosses for this objective were numerous. We harvested fruit from more than 2 dozen interploid crosses, including several designed to produce sweet orange-like fruit, using HLB tolerant parents selected by our program previously. In addition, selected HLB and canker tolerant pummelo breeding parents were crossed with diploid and tetraploid grapefruit, to produce new grapefruit hybrids with enhanced tolerance of HLB. Embryo rescue has been completed and plant production is underway. Over 100 new somaclones have been regenerated in vitro from EV1 and EV2, for further selection. Four new grapefruit hybrid selections were made in this autumn 2019 that produce fruit very similar to grapefruit in appearance, color and flavor, but with improved fruit quality attributes and substantially more tolerant trees, and these have been entered into the DPI Parent Tree Program for cleanup. Finally, the OLL-20 sweet orange was approved for release to the Florida citrus industry. 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. This work is ongoing, and it will improve the precision with which we can define HLB tolerance genes. We have evaluated fruit quality of the more tolerant types of sweet orange-like hybrids, as well as mandarins and grapefruit hybrids, and identified some worthy of further evaluation as potential new cultivars. 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 preparing to conduct 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.
Update for this quarter:
Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State were planted.
Fruit were harvested for transgenic gene flow experiment and seed extracted.
Five seedlings resulted from FF-5-51-2 x early-flowering transgenic Carrizo (cross in test site) and one resulting seedling has already flowered.
Gmitter group has begun to collect data on Clementine x C. latipes.
BRS visited site to orient new staff.
Previous two quarters
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. Renewal and approval for BRS permit effective 9/1/19 through 8/31/20. 4) 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.
11) 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 Stover permit.
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. Likely 2019 will be the last year for data collection.
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) 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.
12) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State
13)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.
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% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations.
Most recent quarter:
Stover lab:
· 5300 for Stover lab – for inoculations to screen antimicrobial transgenes
Stover lab:5300 ACP for inoculating 100 detahced leaves, 90 no-choice small trees and six homogenate assays of peptides.
Bowman lab:
First test group of sweet orange on rootstocks was scored in November 2019 (at 4 mai) and PCR being processed now. Grafted trees for groups 2, 3, and 4 are growing in greenhouse now. Will begin ACP inoculation of group 2 in April.Rootstock liners for groups 5, 6, and 7 are being grown in greenhouse now.
Other users:
· 500 for Dean Gabriel – UF
· 4200 for Yongping Duan – inoculations of resistant Duncan Grapefruit
· 1440 for Randy Niedz – inoculations of Ridge Pineapple and Duncan Grapefruit
Previous quarter:
Over 11,560 ACP infected ACP were used in the last quarter, to screen trees in no-choice inoculation of transgenic citrus and prescreen transgenic events using detached leaf assays
As of December 21, 2018, a total of 14,111 plants had passed through the inoculation process. A total of 361,255 psyllids from colonies of CLas-infected ACP had been used in inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations were many used to refine inoculation procedures, which provided insight into the success of our inoculation methods and strategies for increasing success. After inoculations, plants were returned to the breeders and subsequently subjected to further inoculations when they are transplanted to the field.
In addition to inoculating germplasm, infected psyllids were supplied to other researchers for other purposes. This side of the project grew over time, and detailed records were not maintained on how many were given out until 2018. More than 10,000 infected psyllids were supplied to the research community for an array of experiments during 2018. Recipients included researchers with USDA in Fort Pierce, Ithaca and Beltsville, UF in Gainesville, Cornell in Ithaca, University of California, and University of Nevada.
Create new candidate hybrids. Sexual hybridization is completed between selected elite parents during spring flowering and seed collected in the fall. During this quarter, seed were collected from crosses of elite rootstock hybrids US-942 and US-897 with pummelos. Selected hybrids will be grown-out for propagation, testing, and establishment of seed trees. Emphasis of hybridization in the USDA rootstock program is among parents with superior tolerance to HLB, CTV, and Phytophthora, along with showing favorable effects on grafted tree yield, fruit quality, and tree size. Propagate and plant new field trials. Replicated multi-year field trials with commercial scions are essential to evaluate performance of rootstocks, both to determine whether each new rootstock should be released for commercial use, and to develop comparative performance information among new and existing rootstocks for a diversity of scions, soils, and management conditions. Most of the rootstock field trials are planted with a single scion representing a common commercial type on each of 40-60 different rootstocks. Adequate replication is considered a critical factor in the USDA rootstock trials, with 6-7 replications the minimum and 12 replications the optimum to provide an acceptable level of reliability for results. Three new rootstock trials with sweet orange scion on promising rootstocks were planted in this quarter. Nursery trees for three additional rootstock trials are being prepared in the greenhouse for planting in spring 2020.Collect data from field trials. 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. Measurements related to cropping are on an annual cycle based on the scion, while measurements of health and tree size are on a schedule determined by the specific conditions and goals of the trial. Cropping data was collected from 6 trials with early-maturing scions during Nov 2019-Jan 2020. Assessments of tree health and measurements of tree size were completed on nine trials during this quarter.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 testing, seed sources are established and used to determine trueness-to-type from seed. Studies were initiated this quarter to evaluate seed propagation for 25 of the most promising SuperSour hybrid rootstocks.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. I recognize that this information is of great value to citrus growers and nurseries, and am working to create summaries from 38 trials that will be posted onto a website for easy access by Florida growers. It is anticipated that the first stage of that information will be posted to https://citrusrootstocks.org/ soon. Release of superior new rootstocks for commercial use. Several of the 300 advanced Supersour rootstock hybrids in field trials are exhibiting outstanding performance in comparison with the commercial standard rootstocks. Perforance data continues to be collected, but it is anticipated that 2-3 of the most outstanding of these will be officially released in 2021-22.
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 performed:
(1) To ensure the quality of the citrus plants for the field trial, protein levels in the propagated plants were analyzed. Leaf tissues were collected for total protein extraction. After extraction, total protein was separated in SDS-PAGE gel. The proteins were transferred onto a nitrocellulose memebrane. The blot was probed with an antibody against the overexpressed protein. It was found that all progeny plants express good amount of the transgenic protein. The plants were carefully managed by regular watering and fertilization. This batch of plants will be transplanted into the field at the end of April or early May of 2020.
(2) Purify antibody against a major citrus defense protein. One boost was conducted to the rabbits. Serum was extracted form both rabbits and purified with protein A beads. The purifed antibody was tested using the citrus protein transiently expressed in Nicotiana benthamiana. It was also tested using total protein extracted from the sweet orange ‘Hamlin’. The titer of the antibody has been improved by the boost. The antibody can now be used to analyze other citrus materials.
(3) Analyze the transgenic plants already planted in the field. The size of the plants in the field at Ft Pierce USDA ARS were measured. Leaf samples were collected from each plant. Total protein and RNA have been extracted. Assays of the transgenic protein levels and CLas titers are ongoing.
The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require regulatory approval.
Based on two year’s graft-inoculation assays in greenhouse with two HLB bacterial isolates and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected seedlings showed no significant difference from their maternal trees.
The first group of the propagates on three different rootstock from the selections of Scott Grove’s seedling variants were grown at our research farm, Picos Farm, where the plants are under extreme high HLB disease pressure with very aggressive HLB pathogens. These new plantings (July, 2017; Nov, 2017; and May, 2018) showed different disease index, the longer the planting was, the higher the disease index, which was also highly correlated with the titers of Ca. Liberibacter asiaticus (Las) in infected plants. It is worth noting that the new HLB isolate from Picos Farm caused severe HLB disease on most of grapefruit selections of seedlings and bud sports in our latest, graft-based greenhouse evaluation. Those selections were either resistant or tolerant to the previous HLB isolates we maintained in greenhouse. Prelimilnary data showed some of the selections are better than the others with either lower disease index or better canopy growth. Some of the selections showed much lower infection rate (less than 20%) than the control (40%) and poor performers (40-50%) after planting in Picos research farm for 26 months. Among the Scott’s seedling selections, one of the 4 selections displayed its outperformance than the others with the lowest disease rate of ca. 13.0% and better growth canopy. All the plants were verified by Las-specific qPCR assays, and there were no significant difference among infected genotypes in term of Las bacterial titers. It is worth noting that some of the plants propagated via cutting (without a rootstock) showed some promising outcome with less disease index. However, there was no significant difference observed among the three rootstocks in the trials. Some of the genotypes planted in Nov. 2017 (about 10%) bears fruits, and the fruits from these genotype selections were collected, and they are going to be evaluated for juice quality via taste panels and instruments. In summary, we have yet oberved at least two selections (one from bud sport) that are significantly better than the others (including control grapefruit plants).
The second group of the propagates on the three different root stocks mentioned above (Ca. 750 plants) were planted in Scott Groves in two lots, with this experiment fully planted by mid-September 2019. All of the propagates have been tested for the presence of Las via qPCR. All the 700 plus plants in Scott grove grow as expectedand and will be further evaluated.
Objective: A single objective of this project is to assure the presence of active site that will provide un-interrupted service for production of transgenic citrus plants to researchers involved in fight against huanglongbing (HLB) and citrus canker. Through its services, Citrus Transformation Facility (CTF) offers a place for groundwork for the scientific community. For the laboratories without transformation capabilities, CTF makes their projects possible by producing transgenic plants. CTF staff also participate in other research projects that had to do with production of transgenic citrus plants and when needed, offer advising and training services.
Major accomplishments per objective: The uninterrupted operation of CTF that resulted in production of transgenic citrus plants is the major accomplishment for the 2019. Throughout the whole year, the facility was open and ready to accept the orders and start working on them almost immediately. Altogether, the CTF received 25 orders during last year. Placed orders included requests for transgenic Duncan grapefruit, Valencia orange, Mexican lime, and Indian curry leaf plant (Murraya koeinigii). The number of produced transgenic plants is 246 (Table 1). We have produced additional 17 plants (10 Duncan and seven Valencia) that were designated as transgenic and included in one of our quarterly reports. However, upon additional testing we decided those plants are not carrying the genes they were supposed to and we deducted them from the final count. Those plants that were produced belong to following cultivars: Duncan grapefruit, Mexican lime, Valencia sweet orange, Pomelo plants, Kumquat plants, Pineapple sweet orange Carrizo citrange, and plants of Indian curry leaf plant. All of the plants produced by CTF were the result of research that has a goal of fighting the HLB disease. These plants have the potential to either be tolerant or resistant to HLB, or in the case of Indian curry leaf plants, they produce chemicals that can kill Asian Citrus psyllids. All plants stayed in the state of Florida where further tests will be conducted to test desired traits resulting from introduction of transgenes.
Table 1. Plants produced by CTF in 2019
Cultivar Number of plants produced
Duncan grapefruit 155
Mexican lime 32
Valencia sweet orange 20
Pomelo 21
Kumquat 4
Pineapple sweet orange 4
M. koenigii 10
Number of co-incubation experiments done with explants of different cultivars and appropriate bacterial strains was 150. About 150,000 explants were used in those experiments. In only one experiment all the explants were contaminated and in three others there was a partial loss of material. The data from 136 experiments were collected in 2019. One hundred and ten experiments included green fluorescent protein (GFP) as a reporter gene and because of that we inspected under the microscope about 100,000 shoots and buds that sprouted from treated explants for the presence of GFP fluorescence. Altogether there were 1914 transgenic shoots and buds but 1293 were chimeric and 621 were exhibiting GFP fluorescence in all tissues. We have also preformed about 950 PCR reactions with primers specific to LOB gene and to GUS gene during selection of Duncan grapefruit shoots positive for gene carried by JJ8 binary vector. Additional 925 PCRs were done with Valencia shoots using primers specific for sequences carried by the JJ7 vector. Furthermore, 570 GUS assays were also done with samples from Valencia shoots in search of those transformed with sequences from the JJ7 vector.
In February of 2019, CTF purchased one bin of Duncan grapefruits and stored them in the cold room for supply of seeds that lasted until November. In September, the crew working in A. Schumann’s CUPS harvested half of the yield from Duncan grapefruit trees we have there. Since CUPS-produced fruit do not tolerate well storage conditions of cold room in CREC’s packinghouse, we lost more than 50% of fruit in a short period of time. At the end of January of 2020 when the second half of Duncan grapefruit gets harvested from CUPS, we will concurrently use them for experiments and extract seeds that will be stored. In order to secure sufficient supply of Duncan seeds, we will purchase half of the box of fruit in February 2020. For experiments requiring Valencia seedlings, we are picking Valencia fruit from the trees on the CREC property to get seeds. The seeds of other cultivars are obtained through purchase from Lyn Citrus nursery in California or by picking fruit from DPI Arboretum in Winter Haven.
Major shortcomings, unfinished business: High majority of work done in CTF on production of transgenic citrus plants includes GFP as a reporter gene. Numbers reported in the above section best describe why. Almost all plants produced in the 2019 were selected based on the GFP fluorescence. All of the PCR reactions and GUS assays performed last year for orders that did not use GFP, lead to production of just a few plants. These tests also resulted in some false positives I described above. CTF is at the point where we can relatively easy satisfy the orders for some citrus cultivars and produce about 10 transgenic plants with desired gene within nine months if the GFP is a reporter gene. GFP is a powerful tool that researchers are holding on to, because it helps them get the results (transgenic plants) fast. CTF has no leverage to steer people away from using GFP as a selection tool in the process of production of transgenic citrus plants. Such an effort would also be counterproductive until equally efficient reporter gene is available.
The flux of employees working in the CTF remained high. Two employees who worked at CTF in the beginning of 2019 have left. One of these employees was funded from the USDA grant where there was money left over upon his departure. We are presently in the final stages of hiring a replacement. In the spring of 2019, one OPS employee was hired on a temporary basis for six months. This person left the facility on November 1st although some funds remained available. New employee was already hired as a replacement.
The opportunities going forward: Future opportunities for the CTF reflect the needs of Florida Citrus Industry. The most important thing that CTF can do is to participate in fight against HLB and citrus canker by producing trees with increased tolerance and/or resistance to these diseases regardless of methodology used.
Researchers using CRISPR for editing of citrus genome are still trying to produce homozygous plants that do not have in their cells any “leftovers” from the process of genetic modification. Even when this gets accomplished, that should be just the beginning of the use of this technology in the improvement of citrus. This was, and still is, the great opportunity for the CTF to play its role by producing citrus plants with edited genes for the benefit of all stakeholders in the citrus industry.
The CTF was the first site where cisgenic citrus plants were produced. These plants contain only the DNA from citrus even after genetic modification. Since it is not known whether CRISPR can be used successfully in all efforts for improvement of elite citrus cultivars, introduction or modification of genes from same or related species remains as valid approach. CTF is ready for such efforts at any given time.
Publications from this project
1) Jia, H., Orbović, V., Wang, N. (2019) CRISPR-LbCas12a-mediated modification of citrus. Plant Biotechnology Journal, doi: 10.1111/pbi.13109
2) Song, G., Prieto, H., Orbović, V. (2019) Agrobacterium-mediated transformation of tree fruit crops: Methods, progress, and challenges. Frontiers in Plant Science, 10:226.
The objectives of this project are to produce mature citrus transgenics that will flower & fruit naturally using Agrobacterium as a service for customers, increase transformation efficiency, & conduct research to further biolistic transformation, so that it will also become an efficient service. Plants produced using biolistic transformation can be deregulated faster at less expense. Approximately 36 Agrobacterium-mediated transgenics were produced & micrografted this quarter, 11 survived, unfortunately 10 died, & it is too soon to tell if the remaining 15 will survive. In the near future, a new staff member will devote much time to increasing micrografting efficiency since it is critical because mature shoots will not root. Many of the transgenics produced were not for customers this quarter, but were to determine the optimal concentration of a new selection agent for biolistic transformation of mature citrus in tissue culture. Before Christmas, a number of Agrobacterium-mediated transgenic plants were delivered to Dr. Mou. His latest vector(s) have a prospensity to rearrange in Agrobacterium, therefore we must do PCR for detection of the transgene. Typically 2/3 of the plants regenerated plants retain his transgene whereas the rest lose it. Dr. Wang’s lab would like grapefruit transgenics, so we introduced more grapefruit cultivars (Flame, Duncan, Marsh, Ray Ruby). However, before we produce transgenics for Dr. Wang’s lab, we must first test the different cultivars to see which ones are amenable to Agrobacterium transformation since there is cultivar dependency using it. We have already tested Ruby Red grapefruit, Ray Ruby grapefruit & Dr. Grosser’s new Red Grapefruit with discouraging results. Thus the CRDF can see that not all of our efforts in the mature lab bring in money from customers as there usually is preliminary work to do first.
Mature Valencia sweet orange has a low transformation efficiency. In an attempt to increase it, we tested zeatin riboside hormone rather than BAP. Unfortunately this hormone did not significantly improve efficiency & zeatin riboside is prohibitively expensive anyway. Fortunately Dr. Grosser’s new cultivars derived from Valencia (EV1, EV2, Valquarius) all have relatively high Agrobacterium transformation efficiencies & are alternatives to Valencia.
Reinvigorated scions have thorns (L. Pena, personal commun) & one objective was to bud for thorniness to increase efficiency. However, there was too much variability after budding within and between cultivars to achieve this objective. As an example, you might find no thorns at the stem base & thorns at the top of the stem in one plant vs thorns the entire length of the scion in another plant. This objective could not be accomplished because of this issue.
We have tested different DNA precipitation methods (spermidine, PEG) to precipitate DNA onto gold particles prior to bombardment. Spermidine is the standard precipitation protocol & protamine sulfate is not significantly better, according to Dr. Wu who previously tested it. PEG warrants further investigation in the near future.
Prices were increased before Christmas & will probably be increased again ~ March, 2020 after consulting with Dr. Rogers. Although we have increased transformation efficiency significantly, it must become even more productive to cover our costs. Mature transformation is notoriously high input & relatively low productivity. But we do not want to raise prices so high that we lose customers. Biolistic transformation should generate additional customers, so we are hoping to make improvements to this protocol as quickly as possible.
I applied for an FDACS funding opportunity, which would decrease costs somewhat to CRDF. We will not know the results of this competition until ~ Sept 2020 & if funded, the funding would start in January 2021.
The present reporting period runs from September 15 – December 15, 2019. Mr. Chad Vosburg is the M.S. degree student in the Penn State Department of Plant Pathology graduate program who is working on the project. Chad took a trip to Fort Pierce, FL, November 15 – 25, 2019. During this trip, he set up additional plant propagations for 1-2 runs of an HLB resistance test for all the FT-scFv grapefruit lines. Existing propagated plants that had been cut back to induce a new flush of growth were not yet growing out in order to allow for the setup of an HLB infection test. Personnel at the USHRL are monitoring the plants to determine when they have reached the optimal stage of re-growth to start an HLB resistance test using psyllid-mediated transmission. Chad also learned how to propagate citrus by grafting. In particular, he grafted the highest expressing line of FT-scFv to rough lemon rootstocks, since this line propagates very poorly by rooted cuttings. This will provide sufficient plants to test for HLB resistance of this highest expressing line, which would be the most likely to have a strong resistance phenotype. In addition, Chad grafted the next two highest expressing lines to rough lemon rootstocks known to be HLB infected. Rough lemon supports a relatively low CLas titer and this will form an additional test for HLB resistance, as we wait for psyllid-mediated tests to commence. A field test of two of the FT-scFv lines is now set up at the Pecos Farm at the USHRL, with 10 plants per transgenic line, plus non-transgenic controls, having been planted in the field. Chad has sampled these plants for time zero measurements of CLas titers. We anticipate that the first run of an HLB resistance test will be initiated during the next reporting period. Mr. Jeremy Held, a Ph.D. student in the Intercollege Graduate Program in Plant Biology at Penn State, continued his analysis of graft-transmissibility of the FT-scFv protein in the grapefruit lines. Initial tests for FT-scFv transmissibilty to non-transgenic scions did not yield any FT-scFv protein detection signal. However, we are now optimizing the protein isolation procedure to enrich the protein samples for for phloem tissue content. The FT-scFv protein is expected to be limited to the phloem in the non-transgenic scion, making it more difficult to detect than in the transgenic FT-scFV rootstock plants.
Tree evaluations at the three trial locations (1) SWFREC, (2) Hendry County – Duda & Sons, and (3) Polk County – Peace River Packing Co. were continued during the fourth quarter of 2019. Tree evaluations included monthly (SWFREC) or bimonthly (Hendry County and Polk County) root growth measurements using rhizotrons and leaf flush ratings as well as biannual tree/canopy size and trunk diameter measurements.
The SFWREC field trial was terminated after 2 years of growth following a final horticultural assessment. A subset of 72 trees was excavated over a period of 3 weeks using compressed air (Airspade), which allows the fibrous roots to remain intact. A detailed analysis of the root architectures, including the number and length of lateral roots and size distribution of roots, was conducted to investigate potential differences among propagation methods and rootstocks, but also between bed side and swale side. Differences among different propagation methods were statistically significant only for the length per lateral root which was highest for rootstocks propagated from seed. Significant differences were also found among rootstock cultivars for the primary root diameters which was largest for US-802 and US-942 and smallest for US-897, US-1516, and Swingle. A trend for a larger number of primary roots and primary root length on cuttings propagated roots was observed. For many of the measured root variables significant differences were also observed between swale side and bed side. Roots growing into the bed side were more numerous, longer, and thinner compared with roots growing into the swale side. Information from these excavations will be presented during the 2019 Citrus Show in Fort Pierce.
The fourth field trial was established in November at the Florida Center for Sustainable Agriculture in Vero Beach (collaborator: Mr. Bob Adair). This trial includes the 3 rootstock varieties US-942, US-812, and US-1516, which was modified from the originally proposed cultivars (US-802, US-897, Swingle) which were not available as tissue culture propagated rootstocks. All rootstocks were propagated by seed, cuttings, and tissue culture. Therefore, the design was a 3 x 3 design, including 3 propagation methods and 3 rootstocks. For each combination 12 grafted trees were available, which were arranged in 6 replicates of 2. The scion cultivar is Valencia. Baseline tree measurements were conducted immediately after planting. Two weeks after planting, rhizotrons were inserted next to one of each of the trees in each replicate.
Tree evaluations at the two locations (Fort Basinger and Lake Wales) were continued during the fourth quarter of 2019. Hamlin and Valencia trees in both locations were rated for canopy density, canopy color, and foliar HLB disease symptoms. Ratings were conducted on a scale of 1 to 5 on each side of a tree as outlined in the appendix of the RFP. The average ratings for HLB foliar disease symptoms ranged from 1.2 to 1.6, indicating an overall low percentage of disease symptoms. Although significant differences were found among rootstocks, they varied depending on the location and the scion variety. The average ratings for canopy density and canopy color ranged from 4.0 to 4.9, indicating a healthy canopy. No significant differences were found among rootstocks for the canopy color ratings. Canopy density ratings varied significantly among rootstocks, but results varied depending on the location and the scion variety.
In addition to the tree health ratings, leaves were collected from trees in all four trials and sent to a service lab (Waters lab) for leaf nutrient analysis. For many of the macro- and micro-nutrients significant differences were found among rootstocks, but there was no clear trend. For all nutrients, significant differences were also found between the two locations and between the two scion varieties.
For the Hamlins, a subset of fruit from each experimental unit was collected in December at both the Lake Wales and Fort Basinger location. Fruit were analyzed for the following quality parameters: fruit weight, % juice, % solids, % acid, brix/acid ratio, and juice color. Fruit quality analyses were conducted at the CREC Processing Pilot Plant. Overall, extensive fruit drop was observed, which will be assessed in January immediately before the harvest. Weight per fruit was 156 g (5.5 oz) on average. The average percentage of juice was 54.5%. Average acid was 0.47-0.50% and average TSS was 8.7-9.1. The resulting brix/acid ratio was 17.4-19.5. For all measured parameters significant differences were found among rootstocks but results also varied between the two locations.
Significant progress has been made with additions to our website, presenting data from our field trials. Data from the 6 trials listed blow has been entered onto the website during this quarter. Website access: https://citrusresearch.ifas.ufl.edu/rootstock-trials/
Trial # Online Title Location – County Date planted
11 Vero Beach Navel Orange and Grapefruit Roostock Trials Indian River 2010
12 Charlotte County Multi-Scion Rootstock Trial Charlotte 2014
13 LaBelle Valencia APS Roostock Trial Hendry 2009-2010
14 LaBelle ‘Vernia’ Roostock Trial Hendry 2010
15 South Ridge Valencia Rootstock Trial Highlands 2014
16 St. Helena Rootstock Survey Trial Polk 2008 & 2010
Seed fruit was harvested from UFR rootstocks and a few other promising rootstock selections including the gauntlet selection A+VolkxOrange19-11-8, and the seed was extracted and treated by Southern Citrus Nurseries. UFR seed will be distributed to participating nurseries. Seed from Cleo+Carrizo was also extracted, and will be used in experiments in efforts to solve the granulation problem in Seedless Snack and the granulation/low brix problem in Florida navels.
Vegetative material of promising gauntlet rootstocks S10xS15-12-25, 8-1-99-2B X C-22-12-11 and LB8-9xS13-15-16 (the first gauntlet rootstock made with SugarBelle as a parent) was provided to Agromillora and the Phillip Rucks Nursery Tissue Culture laboratory for tissue culture initiation. Another promising selection recovered from Balm (non-gauntlet) 2247 x 2075-02-7 was also provided. Seed trees are not available for any of these rootstocks. The ‘S’ designations in the above parentages indicate salt tolerant parents, as rootstock hybrids stacked with abiotic stress genes continue to perform exceptionally well in the HLB screen. Four other candidates available from the DPI Parent Tree Program were also made available to the TC companies; these include A+HBJL-2B (tetraploid sour orange-type), Blue 1, White 1 and Cleo+Carrizo. Seed was also extracted from several Flying Dragon hybrids (and planted for advanced trials, these have potential for tree size control).
The first trees were planted in our new PTS (Parent Tree Structure), where we will keep superior breeding parents (both diploid and tetraploids) free of HLB and other diseases; guaranteeing their availability for future crosses.
Field Planting at the USDA Picos Farm: 150 new gauntlet rootstock selections were planted (grafted with HLB+Valencia); these include 40 new hybrids made with SugarBelle LB8-9, and many of them also include one of the salt tolerant parents (S10: HB Pummelo x Shekwasha; and S13: HB Pummelo x Cleopatra).
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