2019-12_18-068C – Biotechnology to Generate Disease Resistant Mature Citrus as a Service

Biotechnology to Generate Disease Resistant Mature Citrus as a Service

Report Date: 01/15/2020
Project: 18-067C   Year: 2019
Category: Plant Improvement
Author: Janice Zale
Sponsor: Citrus Research and Development Foundation, Inc.

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.

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