Continued Funding for the Mature Citrus Facility to Produce Disease Tolerant, Transgenic Citrus

Continued Funding for the Mature Citrus Facility to Produce Disease Tolerant, Transgenic Citrus

Report Date: 03/28/2016
Project: 15-045C   Year: 2016
Category: Horticultural & Management
Author: Janice Zale
Sponsor: Citrus Research and Development Foundation

We continue to produce transgenic, mature citrus trees and transfer them to scientists (Drs. Dutt, Louzada, McNellis, Mou, Wang) as soon the primary or secondary grafts heal. Mature scion transformation efficiencies have increased to 7.6%, and micrografting efficiencies have improved to 77%. Approximately 154 transgenics (primary transgenics and vegetative progeny) have been transferred to Dr. Dawson’s lab for additional testing, and another ~50 will be transferred next month. For out-of-state transport of transgenics, USDA APHIS permits were obtained by scientists prior to shipping. Shipping certification was also obtained through UF. Transgenic, mature citrus has been shipped to Dr. McNellis at Penn State. A manuscript was submitted to a scientific journal describing biolistic transformation of immature citrus rootstock, never previously reported in the literature. Biolistic transformation to produce transgenics will augment those produced with Agrobacterium. A rapid, high throughput, nondestructive MUG assay is being developed to screen whole putative transgenic citrus shoots for GUS expression. It is quantifiable and more sensitive than using X-Gluc as substrate. Fluorescence can be quantified on a plate reader, or visualized on a gel doc with known controls. It is anticipated that GUS expression will correlate to copy number similar to NPTII expression. It remains to be determined whether the shoots will survive immersion in the MUG substrate and subsequent micrografting, but minimal exposure to the substrate, followed by rinsing, might not be harmful. Data are being collected describing the method for potential publication. We are still optimizing the PMI selectable marker using biolistics and Agrobacterium transformations in immature and mature citrus transformation. The results so far look promising and shoot growth doesn’t appear to be negatively impacted like shoot growth on kanamycin medium. Sour orange and Volkameriana seed have been purchased for the growth room because seed of our preferred rootstock varieties have been sold-out. It remains to be determined if these varieties perform well in the growth room.


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