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Abstract Detail



Applications of CRISPR technology across the plant tree of life

Shan, Shengchen [1], Hauser, Bernard [2], Barbazuk, W. Brad [2], Soltis, Pamela S. [3], Yang, Bing [4], Soltis, Douglas E. [5].

Developing Efficient Tissue Culture and CRISPR Systems in Tragopogon (Asteraceae), an Evolutionary Model for the Study of Polyploidy.

Tragopogon (Asteraceae) is an ideal system to examine the immediate consequences of polyploidy, or whole-genome duplication (WGD), which is a major evolutionary force in all eukaryotes. Naturally formed allotetraploid T. miscellus and T. mirus are only 90-100 years old. The development of an efficient genome editing system in Tragopogon will provide novel insights into the genetic consequences of WGD and facilitate studies of gene function in Asteraceae, the largest angiosperm family with many crop and ornamental species. In a previous study, using CRISPR/Cas9, we knocked out the phytoene desaturase (PDS) gene in both diploid and polyploid species of Tragopogon and regenerated shoots exhibiting the expected albino phenotype. However, the tissue culture process we initially used is cumbersome, and the root regeneration rate is extremely low. Here, we shortened the plant regeneration process by inducing shoots directly from explants (i.e., bypassing the callus stage) and successfully regenerated roots from shoots with a high efficiency. Pulse treatment of plant growth regulators (Indole-3-butyric acid [IBA] and synthetic cytokinin 6-benzylaminopurine [BAP]) was used for plant regeneration. To induce shooting, leaf explants were placed on COCO-1 medium (2.41 g/L woody plant salt and vitamins, 30 g/L sucrose, 5% coconut water, 2.5 mg/L BAP and 8.5 g/L agargellan; pH 5.7) for ~14 days. The explants were then transferred to MS medium (4.44 g/L MS salt and vitamins, 30 g/L sucrose and 5 g/L gelrite; pH 5.8) for ~16 days. Shoots were developed from explants with an average shoot regeneration rate of 33.4%. To induce rooting, shoots were excised from the explants and placed on COCO-R medium (2.41 g/L woody plant salt and vitamins, 30 g/L sucrose, 1.5 mg/L IBA and 8.5 g/L agargellan; pH 5.7) for 5 days. The shoots were then transferred to WPM medium (2.41 g/L woody plant salt and vitamins, 30 g/L sucrose and 8.5 g/L agargellan; pH 5.7) for 10 days. The shoots started rooting on WPM medium with an average root regeneration rate of 53.7%. With a high-throughput tissue culture system now in place, we are integrating plant regeneration with plant transformation and testing the genome editing efficiency of CRISPR by knocking out PDS and dihydroflavonol 4-reductase (DFR) genes. Developing the CRISPR system in Tragopogon holds enormous potential for examining the biological underpinnings of various gene retention patterns following WGD.


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1 - Florida Museum Of Natural History, 1659 Museum Rd, Gainesville, FL, 32611, United States
2 - Department of Biology, University of Florida, 220 Bartram Hall, Gainesville, FL, 32611
3 - University Of Florida, Florida Museum Of Natural History, PO Box 117800, Gainesville, FL, 32611, United States
4 - Division of Plant Sciences, University of Missouri, Columbia, MO, 65211
5 - Florida Museum of Natural History, 1659 Museum Rd, Gainesville, FL, 32611, USA

Keywords:
CRISPR
polyploidy
Tissue Culture
Tragopogon.

Presentation Type: Symposium Presentation
Session: SY6, Applications of CRISPR technology across the plant tree of life
Location: /
Date: Friday, July 23rd, 2021
Time: 1:00 PM(EDT)
Number: SY6008
Abstract ID:816
Candidate for Awards:A. J. Sharp Award,Katherine Esau Award,Margaret Menzel Award,Isabel Cookson Award,Edgar T. Wherry award,George R. Cooley Award,Emanuel D. Rudolph Award,Physiological Section Physiological Section Li-COR Prize,Maynard F. Moseley Award,Economic Botany Section best student paper,Physiological Section Best Paper Presentation


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