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Title: Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum)

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 N 6 macronutrient/B 5 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,more » 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.« less
Authors:
 [1] ;  [1] ;  [2] ;  [2] ;  [3] ;  [4] ;  [1] ;  [1]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States). Biosciences Center
  2. Colorado State Univ., Fort Collins, CO (United States). Department of Biology
  3. North Carolina State Univ., Raleigh, NC (United States). Department of Crop and Soil Sciences
  4. National Renewable Energy Lab. (NREL), Golden, CO (United States). National Bioenergy Center
Publication Date:
Report Number(s):
NREL/JA-2700-70582
Journal ID: ISSN 1746-4811
Grant/Contract Number:
AC36-08GO28308; SC0000997
Type:
Accepted Manuscript
Journal Name:
Plant Methods
Additional Journal Information:
Journal Volume: 13; Journal Issue: 1; Journal ID: ISSN 1746-4811
Publisher:
BioMed Central
Research Org:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; switchgrass; bioenergy crop; red fluorescent protein; agrobacterium-mediated transformation; β-Glucuronidase (GUS)
OSTI Identifier:
1416724