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

Abstract

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:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States); Energy Frontier Research Centers (EFRC) (United States). Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1618907
Alternate Identifier(s):
OSTI ID: 1416724
Report Number(s):
NREL/JA-2700-70582
Journal ID: ISSN 1746-4811; 113; PII: 263
Grant/Contract Number:  
SC0000997; AC36-08GO28308
Resource Type:
Published Article
Journal Name:
Plant Methods
Additional Journal Information:
Journal Name: Plant Methods Journal Volume: 13 Journal Issue: 1; Journal ID: ISSN 1746-4811
Publisher:
Springer Science + Business Media
Country of Publication:
United Kingdom
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; switchgrass; bioenergy crop; red fluorescent protein; agrobacterium-mediated transformation; β-Glucuronidase (GUS)

Citation Formats

Lin, Chien-Yuan, Donohoe, Bryon S., Ahuja, Neha, Garrity, Deborah M., Qu, Rongda, Tucker, Melvin P., Himmel, Michael E., and Wei, Hui. Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum). United Kingdom: N. p., 2017. Web. doi:10.1186/s13007-017-0263-6.
Lin, Chien-Yuan, Donohoe, Bryon S., Ahuja, Neha, Garrity, Deborah M., Qu, Rongda, Tucker, Melvin P., Himmel, Michael E., & Wei, Hui. Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum). United Kingdom. doi:10.1186/s13007-017-0263-6.
Lin, Chien-Yuan, Donohoe, Bryon S., Ahuja, Neha, Garrity, Deborah M., Qu, Rongda, Tucker, Melvin P., Himmel, Michael E., and Wei, Hui. Tue . "Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum)". United Kingdom. doi:10.1186/s13007-017-0263-6.
@article{osti_1618907,
title = {Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum)},
author = {Lin, Chien-Yuan and Donohoe, Bryon S. and Ahuja, Neha and Garrity, Deborah M. and Qu, Rongda and Tucker, Melvin P. and Himmel, Michael E. and Wei, Hui},
abstractNote = {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.},
doi = {10.1186/s13007-017-0263-6},
journal = {Plant Methods},
number = 1,
volume = 13,
place = {United Kingdom},
year = {2017},
month = {12}
}

Journal Article:
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DOI: 10.1186/s13007-017-0263-6

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