Evaluation of parameters affecting switchgrass tissue culture [electronic resource] : toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum).

Published:: Washington, D.C. : United States. Dept. of Energy. Office of Basic Energy Sciences, 2017.
Oak Ridge, Tenn. : Distributed by the Office of Scientific and Technical Information, U.S. Dept. of Energy
Physical Description:: Article numbers 113 : digital, PDF file
Additional Creators:: National Renewable Energy Laboratory (U.S.), United States. Department of Energy. Office of Basic Energy Sciences, and United States. Department of Energy. Office of Scientific and Technical Information

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Summary:

Switchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection. After evaluating the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N₆ macronutrient/B₅ micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50-100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical β-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines. The process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.

Report Numbers:

E 1.99:nrel/ja--2700-70582
nrel/ja--2700-70582

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Published through SciTech Connect.
12/19/2017.
"nrel/ja--2700-70582"
Plant Methods 13 1 ISSN 1746-4811 AM
Chien-Yuan Lin; Bryon S. Donohoe; Neha Ahuja; Deborah M. Garrity; Rongda Qu; Melvin P. Tucker; Michael E. Himmel; Hui Wei.

Funding Information:

AC36-08GO28308
SC0000997

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