Biotechnology to Generate Disease Resistant Mature Citrus as a Service

Biotechnology to Generate Disease Resistant Mature Citrus as a Service

Report Date: 10/03/2019
Project: 18-067C   Year: 2019
Category: Horticultural & Management
Author: Janice Zale
Sponsor: Citrus Research and Development Foundation

To decrease our dependence on CRDF funding, two research proposals were submitted to two federal agencies: NSF and NIFA. It should be ~Christmas before we hear whether the NSF was successful, but NIFA will likely be delayed because of its move to Kansas City. I am working on a third proposal that will be submitted to the USDA SCRI Citrus whenever the RFP is announced. This RFP will also be delayed because of USDA’s move to Kansas City. CRDF funding obligations would be decreased if these proposals were funded. I have contacted scientists at UF, the USDA in Fort Pierce, Texas A&M, University of Arizona, Standford, UC Davis & UC Riverside to increase our clientele. In the near future, our prices will increase as suggested by the CREC Director, the CRDF, and the External Review committee. The actual dollar amount will be determined in consultation with the CREC Director.

It was a productive quarter for the transgenic event production. Forty-four transgenic shoots were identified, 18 surived micrografting, 6 died, and 10 are still healing. Use of the GFP reporter partially accounts for this increased productivity. In spring & summer, our productivity also naturally inceases because citrus is more vigorous. In addition, a new staff member was screening for transgenics & this might have contributed to increased productivity. We received two new vectors from Dr. Mou. Dr. Bonning contacted me about potential work in the future. Dr. Wang’s group is interested in transforming mature grapefruit for commercialization, but we had to introduce grapefruit cultivars first (see below) & then determine which cultivars are amenable to transformation. Dr. Dutt has provided three vectors for our collaborative CRDF project together.

We discovered that Dr. Grosser’s OLL4 and Valquarius cultivars have relatively high Agrobacterium transformation efficiencies. UF15 rootstock is recalcitrant to Agrobacterium transformation with no shoots forming in tissue culture. We introduced new budwood cultivars from FDACS. The budwood introductions were Marsh, Flame, Ray Ruby, and Duncan grapefruits, Temple sweet orange (zygotic embryos), and US-942. We will introduce OLL20 in the future because it is important to the juice industry.

We continue to conduct biolistic transformation of mature scions with citrus reporter genes & selectable markers with the ultimate goal being cis/intragenic production. Next week, we will test whether Dr. Dutt’s reporter can be used without a selectable marker. A new plant – derived selectable marker has been tested with excellent results. Replication of these experiments is currently underway. A similar vector with a citrus-derived selectable marker will be tested in the future. This system is an alternative to E. coli genes for antibiotic resistance & selection. Using citrus-derived sequences should also be more consumer friendly. A manuscript should be produced from this research. PEG-mediated DNA precipitation is being tested to increase biolistic transformation efficiency rather than protamine sulfate or spermidine.

Dr. Wu performed Southern blots for another scientist to show copy number of the transgenes for a scientific publication.

RC Webview, the electronic system that runs the growth facility, has been offline for ~1 week & the reasons are unclear. UF Gainesville is supposedly in the process of connecting a new server. With no other choice available, we have to water with city water which might contain slime mold spores, does not contain enough chlorine, and hasn’t passed through 2 UV filters.

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