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INVESTIGATOR: Muthan, B.

PERFORMING INSTITUTION:
WEST VIRGINIA STATE UNIVERSITY
PO BOX 1000
INSTITUTE, WEST VIRGINIA 25112

GENETIC IMPROVEMENT OF SWITCHGRASS FOR ENERGY USES

NON-TECHNICAL SUMMARY: Plant oils represent one of the most energy-rich sources of renewable hydrocarbons; they are stored in the form of triacylglycerols (TAGs) which are used as alternative feedstocks for biofuel or bioproduct production. Switchgrass (Panicum virgatum L.) is a perennial, warm-season, dedicated bioenergy crop capable of producing a higher biomass suitable as a bioenergy feedstock. Studies have shown the promise of growing switchgrass on West Virginia's reclaimed surface mine lands--and even of producing biomass greater than that of a comparable target yield on agricultural lands. In switchgrass, trait improvement by traditional breeding is hampered due to genetic complexity and availability of resources. Biotechnology, however, offers creative ways for the genetic improvement of switchgrass focusing on the desired traits that are not present in nature. It is possible to alter a specific pathway in switchgrass by ectopic introduction of an Arabidopsis enzyeme/transcription factor. Our overall goal is to generate oil rich switchgrass plants capable of growing on large areas of Appalachian surface coal mine lands. Development of tools such as gene constructs and genome edited transgenic lines, will be of importance to scientists working in basic and applied energy research. Sharing of large-scale sequencing data in this project with the broader scientific community will support research projects beyond oils. In accordance with our mission as an 1890 land grant institution, our principal educational objective is to create STEM-centric learning and vocational opportunities for minority (and often first-generation) college students who come from a region with the lowest college attendance rate in the nation. Educational activities will include advanced training in experimental methods, field experience in academic laboratories, mentorships, and travel to make presentations at state, regional, and national conferences.

OBJECTIVES: The goals of this research are to explore a variety of genomics, biochemical, genetic, and molecular approaches to understanding fatty acids and triacylglycerols (TAGs) synthesis in vegetative tissues of plants. We will use this knowledge to reprogram leaves to accumulate TAGs through the alteration of key transcription factors/genes central to converting sugars to oils in high biomass-yielding crop switchgrass capable of growing on large areas of Appalachian surface coal mine lands. Our project will be designed to achieve the following objectives: 1) investigate lipid metabolism in switchgrass in response to coal mine soils; 2) engineer fatty acids and TAGs biosynthesis in switchgrass; and 3) develop a gene editing system for switchgrass.

APPROACH: The Muthan lab in collaboration with Co-PD lab, will study the morphological, physiological and metabolic indicators of switchgrass mine soils tolerance. Data on plant height, leaf length, leaf width, leaf sheath length, leaf relative water content (RWC), electrolyte leakage (EL), photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (Tr), intercellular CO2 concentration (Ci), water use efficiency (WUE), and biomass yield will purse to understand the mechanism underlying acid mine tolerance and identify genes that can be used for future molecular breeding. We will conduct RNA Seq., post-sequence analysis, data interpretation, and qRT-PCR analysis for candidate genes. Further functional analysis will be done in model plant Arabidopsis using T-DNA mutant screening and overexpression/ RNAi lines. We will use candidate gene approach to increase triacylglycerols (TAGs) accumulation in switchgrass by use of versatile binary vectors with monocot-specific promoters. Total genomic DNA and RNA, for Southern blotting and qRT-PCR analysis respectively, isolated from leaves of transgenic switchgrass plants and untransformed plants will be used to detect the integration and sufficient expression of transgenes in the transgenic lines. Changes in metabolites such as oils, starches, free sugars and glucose will be measured in the transgenic switchgrass lines expressing fatty acids, and TAG/oil biosynthesis genes or enzymes alone or in combination. Bomb calorimeter or elemental analysis will used to determine changes in the energy density of biomass. We will test gene-editing strategies in switchgrass.

PROGRESS: 2020/03 TO 2021/03
Target Audience:Undergraduate and graduate students; plant researchers in academia, scientists in biotechnology industries with a focus on renewable energy and post-coal mining reclamation companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided a training opportunity for undergraduate and graduate students during the reporting period. Classroom instruction of undergraduate class, graduate plant biotechnology classes incorporated active learning activities of plant transformation and bioenergy science using results and concepts from the research under this project. How have the results been disseminated to communities of interest?Publication: S. Sanjaya, B. Muthan. (2021). Creating Power Foods with Gene Technology, Scientia, Issue 148, March. 3, pp 15-18 (In Press). M.S. Thesis: Deo B. (2020). Genetic study of abiotic stress tolerance and phytoremediation mechanism in the bioenergy crops camelina and switchgrass in respond to coal mine soils. A Thesis Submitted to the Biotechnology Faculty of West Virginia State University in Partial Fulfillment of the Requirements for the Degree of Master of Science. Poster presentation: B. Muthan, Khathiwada, T. Heath, C. Challa, and S. Sanjaya. Increasing Energy Density in Switchgrass. Poster presentation in a symposium on Towards sustainable bioenergy production. Virginia Tech. February 2021. What do you plan to do during the next reporting period to accomplish the goals?Continue working on genetic, biochemical, and yield evaluation of switchgrass in response to the acid mine soils. Expression analysis for the transgenic switchgrass plants expressing fatty acid biosynthesis genes alone or in combination.

IMPACT: 2020/03 TO 2021/03
What was accomplished under these goals? With the harvest of fresh seed from the second year, three lowlands and three upland ecotypes were selected for a new experiment. This selection was based on the availability of seeds and the performance of ecotypes in the greenhouse. The germination percentage among lowland genotype on both coal mine and agriculture soil was calculated. Variety #7 recorded the highest germination percentage, followed by #11and #4 on both coal mine soil and agriculture soil. #40 has the highest germination rate among the upland varieties, followed by #29, while #44 had the lowest germination percentage. The height of the lowland and upland varieties was measured. Among lowland varieties, #4 and #7 had similar height on coal mine soil and #11 had slightly less height, while in agriculture soil, # 11 had the highest, and #4 had the lowest height. Among upland varieties, in coal mine soil, #29 and #40 were of similar heights, # 44 was lesser in height, while in agriculture soil #40 was the tallest, followed by #29 #44 was the shortest. When comparing upland and lowland varieties in coal mine soil, all were quite similar in height, with #44 being the shortest. When compared to agricultural soil, the upland varieties were longer than the lowland ones. The oven-dried sample was weighed for biomass calculation. Among the lowland genotypes, in coal mine soil, #11 has the highest biomass, followed by #4, and #7 has the least. The results were similar in agriculture soil, with #11 having the highest biomass followed by #4, and with #7 having the least. Among the upland varieties in coal mine soil, variety #40 had the highest biomass, followed by #29, while #44 has the least. The plants in agricultural soil followed the same trend. So, lowland #11 performed better than #4 and #7, while in the upland varieties, #40 showed the best performance. Among all lowland and upland varieties, # 40 was shown to outperform all other varieties. Our result aligned with previous studies that the upland genotype's biomass is higher than the lowland genotype in coal mine soil. In our study, # 11from lowland had high biomass and low NPQ, which suggested that it is less stressed in coal mine soil than the other two genotypes and had a high proline and higher photosynthesis rate. We have generated Agrobacterium-mediated transformation in switchgrass using callus developed by seeds and immature inflorescence callus and generated first-generation transgenic plants expressing transcription factor WRI1, DGAT, and lipid droplet protein alone and in combination. We confirmed the transgene's presence by genotyping using gene-specific primer sets, besides confirmed the presence of integration of transgene by Southern blot analysis using gene-specific probe prepared using DIG labeling. Further gene expression analysis is in progress. Young leaves from the selected transgenic switchgrass plants expressing transcription factor WRI1, DGAT, and lipid droplet protein alone and in combination and leaves from the wild-type plants used for lipid extraction. The TAG bands were considerably high in the transgenic plants as compared to wild-type plants. Subsequent experiments on the next generation progeny collection, lipid droplet analysis using confocal microscopy, TAG quantification using GC-FID, and calorific value analysis are in progress.

PUBLICATIONS (not previously reported): 2020/03 TO 2021/03
1. Type: Journal Articles Status: Awaiting Publication Year Published: 2021 Citation: S. Sanjaya, B. Muthan. (2021). Creating Power Foods with Gene Technology, Scientia, Issue 148, March. 3, pp 15-18 (In Press).
2. Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: Deo B. (2020). Genetic study of abiotic stress tolerance and phytoremediation mechanism in the bioenergy crops camelina and switchgrass in respond to coal mine soils. A Thesis Submitted to the Biotechnology Faculty of West Virginia State University in Partial Fulfillment of the Requirements for the Degree of Master of Science.