Cargando…
Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum)
BACKGROUND: 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 mod...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2017
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5735814/ https://www.ncbi.nlm.nih.gov/pubmed/29270209 http://dx.doi.org/10.1186/s13007-017-0263-6 |
_version_ | 1783287274974216192 |
---|---|
author | Lin, Chien-Yuan Donohoe, Bryon S. Ahuja, Neha Garrity, Deborah M. Qu, Rongda Tucker, Melvin P. Himmel, Michael E. Wei, Hui |
author_facet | Lin, Chien-Yuan Donohoe, Bryon S. Ahuja, Neha Garrity, Deborah M. Qu, Rongda Tucker, Melvin P. Himmel, Michael E. Wei, Hui |
author_sort | Lin, Chien-Yuan |
collection | PubMed |
description | BACKGROUND: 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. RESULTS: 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, 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. CONCLUSION: 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13007-017-0263-6) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5735814 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-57358142017-12-21 Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum) Lin, Chien-Yuan Donohoe, Bryon S. Ahuja, Neha Garrity, Deborah M. Qu, Rongda Tucker, Melvin P. Himmel, Michael E. Wei, Hui Plant Methods Methodology BACKGROUND: 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. RESULTS: 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, 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. CONCLUSION: 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13007-017-0263-6) contains supplementary material, which is available to authorized users. BioMed Central 2017-12-19 /pmc/articles/PMC5735814/ /pubmed/29270209 http://dx.doi.org/10.1186/s13007-017-0263-6 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology 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) |
title | Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum) |
title_full | Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum) |
title_fullStr | Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum) |
title_full_unstemmed | Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum) |
title_short | Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum) |
title_sort | evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for agrobacterium-mediated transformation of switchgrass (panicum virgatum) |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5735814/ https://www.ncbi.nlm.nih.gov/pubmed/29270209 http://dx.doi.org/10.1186/s13007-017-0263-6 |
work_keys_str_mv | AT linchienyuan evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT donohoebryons evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT ahujaneha evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT garritydeborahm evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT qurongda evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT tuckermelvinp evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT himmelmichaele evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT weihui evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum |