Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum)
Abstract 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 gen...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2017-12-01
|
| Series: | Plant Methods |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13007-017-0263-6 |
| _version_ | 1818877587740426240 |
|---|---|
| author | Chien-Yuan Lin Bryon S. Donohoe Neha Ahuja Deborah M. Garrity Rongda Qu Melvin P. Tucker Michael E. Himmel Hui Wei |
| author_facet | Chien-Yuan Lin Bryon S. Donohoe Neha Ahuja Deborah M. Garrity Rongda Qu Melvin P. Tucker Michael E. Himmel Hui Wei |
| author_sort | Chien-Yuan Lin |
| collection | DOAJ |
| description | Abstract 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 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. 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. |
| first_indexed | 2024-12-19T14:00:40Z |
| format | Article |
| id | doaj.art-02fd39642873401c9c1c94323205a630 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-19T14:00:40Z |
| publishDate | 2017-12-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-02fd39642873401c9c1c94323205a6302022-12-21T20:18:28ZengBMCPlant Methods1746-48112017-12-0113111910.1186/s13007-017-0263-6Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum)Chien-Yuan Lin0Bryon S. Donohoe1Neha Ahuja2Deborah M. Garrity3Rongda Qu4Melvin P. Tucker5Michael E. Himmel6Hui Wei7Biosciences Center, National Renewable Energy LaboratoryBiosciences Center, National Renewable Energy LaboratoryDepartment of Biology, Colorado State UniversityDepartment of Biology, Colorado State UniversityDepartment of Crop and Soil Sciences, North Carolina State UniversityNational Bioenergy Center, National Renewable Energy LaboratoryBiosciences Center, National Renewable Energy LaboratoryBiosciences Center, National Renewable Energy LaboratoryAbstract 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 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. 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.http://link.springer.com/article/10.1186/s13007-017-0263-6SwitchgrassBioenergy cropRed fluorescent protein (RFP)β-Glucuronidase (GUS) stainingAgrobacterium-mediated transformation |
| spellingShingle | Chien-Yuan Lin Bryon S. Donohoe Neha Ahuja Deborah M. Garrity Rongda Qu Melvin P. Tucker Michael E. Himmel Hui Wei Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum) Plant Methods Switchgrass Bioenergy crop Red fluorescent protein (RFP) β-Glucuronidase (GUS) staining Agrobacterium-mediated transformation |
| 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 | Switchgrass Bioenergy crop Red fluorescent protein (RFP) β-Glucuronidase (GUS) staining Agrobacterium-mediated transformation |
| url | http://link.springer.com/article/10.1186/s13007-017-0263-6 |
| work_keys_str_mv | AT chienyuanlin evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT bryonsdonohoe evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT nehaahuja evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT deborahmgarrity evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT rongdaqu evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT melvinptucker evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT michaelehimmel evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum AT huiwei evaluationofparametersaffectingswitchgrasstissueculturetowardaconsolidatedprocedureforagrobacteriummediatedtransformationofswitchgrasspanicumvirgatum |