2019-12_18-066C – Citrus Transformation Facility remains integral part of the efforts to combat HLB disease through production of transgenic Citrus plants

Citrus Transformation Facility remains integral part of the efforts to combat HLB disease through production of transgenic Citrus plants

Report Date: 01/15/2020
Project: 18-066C   Year: 2019
Category: Plant Improvement
Author: Vladimir Orbovic
Sponsor: Citrus Research and Development Foundation, Inc.

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.

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