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: 01/04/2022
Project: 18-066C   Year: 2021
Category: Plant Improvement
Author: Vladimir Orbovic
Sponsor: Citrus Research and Development Foundation

  ObjectiveThis project had only one objective which was to support the operation of the facility that would provide service for production of transgenic citrus plants to researchers working towards solution against huanglongbing (HLB) and citrus canker. The Citrus Transformation Facility (CTF) has been a part of the multipronged approach to fight citrus diseases by producing genetically transformed citrus plants that were used to gain important knowledge about possible introduction of tolerance/resistance traits. This knowledge represented a foundation for understanding of effects that introduced gene(s) could have on the growth of transgenic plants and on their ability to sustain and survive diseases. Within the scientific community working in this field there are laboratories without transformation capabilities, and for those research groups CTF was the place where the ideas from projects came to life because of the production of transgenic plants. Major accomplishments per objectiveThe major accomplishment of this project is that it has facilitated the existence of the CTF throughout the last three years. The CTF has undergone the transition into the Educational Business Activity (EBA) unit within the University of Florida that overhauled the way it provides service and operates as business. This transition together with the wide-ranging effects of COVID-19 pandemic have negatively affected operation of the CTF and funding that came from this project together with the funds provided by the University of Florida have kept the CTF open. Within the last three years, the CTF has produced 388 transgenic plants (Table 1). Number of plants produced in 2019 was 246, in 2020 it was 108, and in 2021 it was 36. Without exception,    all the plants we created were associated with the research directed towards finding a solution to HLB and citrus canker diseases. Some of our transgenic plants including some Duncan grapefruits and all Pomelos exhibited increased resistance to citrus canker. The other plants we produced have the potential to either be tolerant or resistant to HLB, or in the case of Indian curry leaf plants (Murraya koeinigii), they produce chemicals that can kill Asian Citrus psyllids. Besides Duncan grapefruits and Pomelos, we also produced transgenic Mexican lime, Valencia sweet orange, Kumquats, Pineapple sweet oranges, and Indian curry leaf plants. All of these plants stayed in the state of Florida and were used in tests to determine the efficiency of introduced transgenes on desired traits.Table 1. The plants produced by CTF in the three-year period (2019-2021).Cultivar         Number of plants produced         Duncan grapefruit             257         Mexican lime                           32         Valencia sweet orange      51         Pomelo                           22         Kumquat                             4         Pineapple sweet orange     10         M. koenigii                            12         Total                                      388          The number of orders placed at the CTF was 57. In 2019, there were 25 orders, in 2020 there were seven, and in 2021 there were 25 orders. The work done on these orders was performed within 266 experiments that included 358 co-incubations of seedling explants with appropriate bacterial strains. Altogether, 314,000 explants were processed in these experiments. Approximately, 170,000 explants were inspected under the microscope equipped with the blue light source for the presence of GFP-fluorescing shoots and buds. Close to 3,000 PCR reactions were done in search of shoots either carrying or expressing a transgene of interest. Also, 1,750 GUS histochemical assays were performed to confirm the transgenic nature of shoot and plants. In average, the transformation rate was about 1% and that number included both chimeric and fully transformed shoots. The percentage of shoots that were successfully grafted was low in the range of 50%.With the help of staff from CREC’s Business office, during the year 2021 I was able to organize the system of payments for the work performed by the CTF as an EBA and get first payments processed. 


Your browser does not support pdfs, click here to download the file.