Genetic Improvement of Switchgrass Feedstock
for Biofuel Production
PI: Bingyu Zhao
Supported by American Electric Power(AEP)
Research team: Zhiyong Yang (Research Associate), Kerri Mill (Technician)
Primary research question:
To meet the feedstock requirement for sustainable biofuel production, we need to breed new switchgrass cultivars with high biomass yields, improved tolerance to disease, drought and salt stresses, and which can grow in marginal lands.
We employed both biotechnological and traditional plant breeding tools to develop new switchgrass germplasm with improved biofuel traits.
Research objectives & technical approach
(1) Optimize a switchgrass transformation protocol to improve the transformation efficiency of upland switchgrass cultivars;
(2) Characterize the transgenic switchgrass plants with improved drought tolerance;
(3) Develop and characterize transgenic switchgrass plants expressing fugal cellulase enzyme that can improve the efficiency of biofuel production;
(4) Generate new lowland switchgrass lines with different chromosome numbers through chromosome engineering
Background & significance
• Switchgrass (Panicum virgatum L.) is a native perennial grass of North America that is recognized as one of major biofuel crops. Switchgrass is expected to be planted in the marginal lands that can not be used for food production. One of the major challenges of growing plants in marginal lands is the limitation of water supply. Therefore, improve the water use efficiency and drought tolerance of switchgrass will ensure the sustainable biomass production grown in marginal lands.
• The bioconversion of switchgrass lignocellulosic feedstock into biofuels involves the enzymatic hydrolysis process which requires a large amount of enzymes such as cellulase
and cellobiohydrolase to convert cell wall cellulose and hemicellulose to fermentable sugar, such as glucose, which can be used for fermentation to produce ethanol or other biofuels. It is possible to co‐produce cellulase enzyme or other value added proteins in switchgrass plants which might reduce the cost of biofuel production and increase the profitability of the bioenergy industry. Optimize Switchgrass Transformation Protocol
for Improved Transformation Efficiency
Rationale: Transforming upland switchgrass cultivars is still challenging because genotypes competent for tissue culture are lacking. Combing of tissue culture selection and recurrent breeding will allow us to breed new switchgrass genotypes that are suitable for tissue culture and genetic transformation.
Selection of new upland switchgrass lines with high tissue culture capacity • About 10,000 switchgrass seeds of cv. Dacotah were selected for their tissue culture capacity. • 18 lines with the ability to produce embryogenic callus, that can be regenerated to green seedlings were selected.
• These lines will be crossed to each other, and hyrbid seeds will be further selected to further improve their tissue culture capacity
AtLov1 Transgenic Plants are More Tolerant to Drought Stress
Rationale: Improve the drought tolerance of switchgrass will allow switchgrass plants to grow and produce more biomass in marginal lands with minimum water use. We generate transgenic switchgrass plants expressing a Arabidopsis transcription factor AtLOV1 that can enhance the drought tolerance.
LOV1 transgenic
Wild type
AtLov1 Transgenic Plants Have Smaller Stomata Aperture with higher water use efficiency
WT
LOV1
AtLov1 Transgenic Plants Have Smaller Stomata Aperture with higher water use efficiency
Switchgrass leaf relative water content (RWC), PSII photochemical efficiency (PE), malondialdehyde (MDA) and abscisic acid (ABA) content in the transgenic plants over‐
expressing AtLov1 gene and wild type plants.
RWC
(%)
PE
(Fv/Fm)
MDA
(nmol g‐1 FW)
ABA
(ng g‐1 FW)
AtLov1 Transgenic 87.9a
0.572a
59.4b
39.6a
Wild type
79.3b
0.397b
85.2a
33.4b
Means followed by same letters within each column are not significantly different at P≤0.05.
Engineering Switchgrass Plants for Producing a Fungal‐derived Beta‐Endo‐glucanase
Rationale: Cellulose is a linear polysaccharide polymer with many glucose monosaccharide units. For biofuel production, we need break down cellulose into simple sugars by using the cellulase enzyme. We are trying to engineer switchgrass to produce cellulase enzyme that can facilitate the breakdown of cellulose.
• endo‐beta‐1,4‐glucanase from fungus Trichoderma reesei was engineered into transgenic switchgrass plants. The endo‐
glucanase protein targets chloroplast. • Transgenic plants grow normal though with reduced biomass yield in greenhouse condition
Engineering Switchgrass Plants for Producing a Fungal‐derived Beta‐Endo‐glucanase
A diagram of the transgene construct
CK Endo1 Endo2
The transgenic plants were confirmed by RT‐PCR analysis 1 2 3 4 5 6
Endoglucanase gene
EF‐1a (ck)
Lane 1, Wild type CK
Lane 2‐6. are five different transgenic plants
Chromosome Engineering of New Lowland Switchgrass Lines
with Different Chromosome Numbers
Mature Switchgrass seeds
Seeds derived from tissue culture callus
Select embryogenic callus
Treat with chemical colchicine to induce chromosome doubling New switchgrass cultivar
Regeneration to green seedlings
Chromosome Engineering of New Lowland Switchgrass Lines
with Different Chromosome Numbers
Switchgrass callus treated with different colchicine concentrations Technical Highlights
We developed new 18 new lowland switchgrass lines with 72‐76 chromosomes. Most of these plants have delayed flowering time, and can accumulate significantly more biomass in greenhouse conditions.
Technical Highlights
Key take‐home points
1. We selected new upland switchgrass genotypes with improve tissue culture capacity.
2. Generated new transgenic switchgrass with improved drought tolerance.
3. Developed transgenic switchgrass plants to produce cellulase enzyme which may improve the efficiency of biofuel production. This is a proof‐of‐concept to use switchgrass for producing high value byproducts other than biofuel.
4. Generated novel autopolyploid switchgrass germplasm through chromosome engineering
Future research directions
We will further select the new upland switchgrass cultivar that will be used for genetic transformation research. The studies of drought tolerance switchgrass is currently supported by a new DOE‐USDA project for further research. We will select the endoglucase transgenic plants with improve agronomic traits.
Potential links with federal research priorities, sponsored research opportunities
The DOE‐USDA feedstock genomics program is specifically interested to support projects related to genetic improvement of switchgrass biomass yield, drought and salt tolerance. Two DOE projects was awarded to VT in 2011 and 2012, respectively.
Results
Highlight
•
•
•
•
We selected new upland switchgrass genotypes with improved tissue culture capacity.
Generated new transgenic switchgrass with improved drought tolerance.
Developed transgenic switchgrass plants to produce cellulase
enzyme which may improve the efficiency of biofuel production. Generated novel autopolyploid lowland switchgrass germplasm by chromosome engineering
Funded Projects
“Identifying Differences in Abiotic Stress Gene Networks between Lowland and Upland Ecotypes of Switchgrass”, DOE‐
USDA, $1.3M, 08/2012‐08/2015, Kevin Child (MSU), Robin Buell (MSU), Bingyu Zhao (VT) and Xunzhong Zhang (VT).
“An Integrated Approach to Improving Plant Biomass Production” DOE‐USDA, $1.33M, 08/2011‐08/2014, Jan Leach, John McKay, Daniel Bush, Hei Leung, Bingyu Zhao (VT), Andrew Kern
Publications
(1). Yang, Z., Z. Shen, H. Tetreault, L. Johnson, B. Friebe, T. Frazier, L.‐k. Huang, C. Burklew, X.‐Q. Zhang and B. Zhao (2013). "Production of Autopolyploid Lowland Switchgrass Lines Through In Vitro Chromosome Doubling." BioEnergy Research: 1‐11.
(2). Sathitsuksanoh, N., B. Xu, B. Zhao and Y. H. Zhang (2013). "Overcoming biomass recalcitrance by combining genetically modified switchgrass and cellulose solvent‐based lignocellulose pretreatment." PLoS One 8(9): e73523. (3). Xu, B., N. Sathitsuksanoh, Y. Tang, M. K. Udvardi, J. Y. Zhang, Z. Shen, M. Balota, K. Harich, P. Y. Zhang and B. Zhao (2012). "Overexpression of AtLOV1 in Switchgrass alters plant architecture, lignin content, and flowering time." PLoS ONE 7(12): e47399.
(4). Madhavi Dere, Ambika Mosale Venkatesh Murthy, Taylor Frazier, Zhiyong Yang, Catlin Burklew, Changhe Zhou, Zhengxing Shen, Foster Agblevor, Mike Zhang, Bingyu Zhao, Expression of Trichoderma ressi endoglucanase in transgenic switchgrass plants (In preparation).
Other Key Successes
We established a protocol for evaluation of drought and salt tolerance of switchgrass germplasm
Key Collaborations
• Kevin Child (MSU)
• Robin Buell (MSU)
• Jan Leach (CSU)