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Title: Agrobacterium-Mediated Transformation of Prairie Cord Grass for Guar Production

 [1];  [2];  [1];  [3];  [1]
  1. Los Alamos National Laboratory
  2. NMC
  3. El Dorado Biofuels
Publication Date:
Research Org.:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
OSTI Identifier:
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DOE Contract Number:
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Resource Relation:
Conference: LANL Annual Student Symposium ; 2017-08-09 - 2017-08-09 ; Los Alamos, Arizona, United States
Country of Publication:
United States
Biological Science; Agrobacterium, Guar Gum, Spartina pectinata, Prairie Cordgrass

Citation Formats

Mettler, Jacquelyn Gloria, Massan, Joseph, Daligault, Hajnalka, Laur, Paul, and Starkenburg, Shawn. Agrobacterium-Mediated Transformation of Prairie Cord Grass for Guar Production. United States: N. p., 2017. Web.
Mettler, Jacquelyn Gloria, Massan, Joseph, Daligault, Hajnalka, Laur, Paul, & Starkenburg, Shawn. Agrobacterium-Mediated Transformation of Prairie Cord Grass for Guar Production. United States.
Mettler, Jacquelyn Gloria, Massan, Joseph, Daligault, Hajnalka, Laur, Paul, and Starkenburg, Shawn. 2017. "Agrobacterium-Mediated Transformation of Prairie Cord Grass for Guar Production". United States. doi:.
title = {Agrobacterium-Mediated Transformation of Prairie Cord Grass for Guar Production},
author = {Mettler, Jacquelyn Gloria and Massan, Joseph and Daligault, Hajnalka and Laur, Paul and Starkenburg, Shawn},
abstractNote = {},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = 2017,
month = 7

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  • Background: Because of interest in the production of renewable bio-hydrocarbon fuels, various living organisms have been explored for their potential use in producing fuels and chemicals. The oil-producing (oleaginous) yeast Lipomyces starkeyi is the subject of active research regarding the production of lipids using a wide variety of carbon and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements using the tools of synthetic biology and metabolic engineering. However, using these tools for strain improvement requires the establishment of effective and reliable transformation methods with suitable selectable markers (antibiotic resistance ormore » auxotrophic marker genes) and the necessary genetic elements (promoters and terminators) for expression of introduced genes. Chemical-based methods have been published, but suffer from low efficiency or the requirement for targeting to rRNA loci. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. Results: In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species and that the introduced DNA can be reliably integrated into the chromosomes of these species. The gene deletion of Ku70 and Pex10 was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial -glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1 promoter was also stably expressed in seven different Lipomyces species. Conclusion: The results from this study clearly demonstrate that Agrobacterium-mediated transformation is a reliable genetic tool for gene deletion and integration and expression of heterologous genes in L. starkeyi and other Lipomyces species.« less
  • 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 evaluatingmore » 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. 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