Plant Improvement

Support role of the Citrus Core Transformation Facility remains crucial for research leading to production of Citrus plants that may be tolerant or resistant to diseases

This project had a single objective to assure un-interrupted production of transgenic citrus plants through the services of Citrus Transformation Facility (CTF). Continued operation of the facility was going to guarantee a foundation regarding this aspect of research for the scientific community involved in fight against huanglongbing (HLB) and citrus canker. By producing transgenic plants and bringing into life the ideas of scientists working in different laboratories, CTF would facilitate the process of search for candidate plants that would be tolerant/resistant to diseases and provide major relief for Florida Citrus Industry. The objective of this project was successfully accomplished. Throughout the funding period of three and-a-half years, CTF operated without major interruptions and provided service in the form of production of transgenic plants. The level of production stayed at expected, satisfactory level and resulted in creation of 781 citrus plants that represent independent transgenic events. These plants belong to eight different cultivars: Duncan grapefruit, Carrizo citrange, Pineapple sweet orange, Mexican lime, Valencia sweet orange, Swingle citrumelo, Kumquat, and Pomelo.During this project, CTF worked on 103 orders that were placed by 10 clients and this group included eight faculty members based at University of Florida, one faculty from University of California and one Foundation. The number of projects for which the plants were produced was higher than the number of clients. For example, Dr. Nian Wang was one of the clients but orders from his lab were the part of at least five different projects done by his post-doctoral associates.All produced transgenic plants were associated only with research that has to do with disease resistance. This is a clear indication of the role CTF plays in the efforts to overcome effects of HLB and citrus canker.CTF supported the projects from Dr. Nian Wang’s lab that resulted in successful application of CRISPR/Cas9-mediated genome editing technology in Citrus. Plants produced this way will be the most market-friendly as they only have minor modifications of their own gene(s) and in the near future will be free of any other inserted DNA. The work done in CTF lead to production of Duncan grapefruit plants that exhibit high degree of resistance to citrus canker. Other commercially important cultivars could be made with improved resistance to canker based on results of this project.As a part of another research project aimed at control of Asian Citrus Psyllid (ACP) in citrus groves, transgenic Indian curry leaf plants were also produced in CTF. There are six such plants that are being tested for the ability to kill ACP after they were allowed to feed on their leaves. All these successes were achieved even though CTF experienced high flux of employees, had to move to temporary location for six months, and was exposed to adverse effects of hurricane Irma.Future opportunities for the CTF are determined by the needs of Florida Citrus Industry. As those needs change from the search for the solution to HLB and canker to a development of specialty fruit with consumer-oriented traits in post-HLB era, CTF will be there to support all these efforts.

Fort Pierce Field Test Site for Validating HLB and/or ACP Resistance

Update for this quarter: The Stover team has submitted paperwork to renew their primary BRS permit, which covers the transgenic materials planted by Z. Mou, J. Jones, T. McNellis as well as USDA scientists. A new permit has been approved (AUTH – 0000043619  effective 12/17/2020) for material with “Confidential Business Information” for a project led by R. Shatters. The new BRS permitting system called e-file has used us a first trial and it has been very arduous and time-consuming. Information was provided for the BRS review of the transgenics at Picos Farm for CREC (Dutt, Grosser and Gmitter) who maintain a separate permit. UF collaborators have been permitted into the test site and samples and data have been collected. Data were collected on McNellis trees by USDA. Samples we previously collected have been processed by technicians at home (with APHIS-BRS permission) and are ready for qPCR. A manuscript has been prepared and submitted for canker data collected in a block of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside. Previously Stover analyzed data on canker incidence in a block of replicated trifoliate and trifoliate hybrids planted in collaboration with NCGR-Citrus/Dates and UCRiverside, from data collected 8/17, 9/19, and 9/20,  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. Of 34 hybrids validated, similar numbers had Poncirus, grapefruit, and sweet orange chloroplasts.   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. Seed from fruit harvested for transgenic gene flow experiment coninue to be processed for PCR. 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.   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 State13)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.     

Genes, Software, and a Playbook: Combating HLB in Florida Orchards