Progress 12/15/17 to 09/14/22
Outputs
Target Audience: Other scientists including those performing research on crop improvement, domestication, plant seed oil and meal production, Brassicas and oilseed crops; Start-up and established companies that develop crops; Agribusinesses including Oilseed Processors; Farmers; Educators; Students; Federal and State Legislators; Policy Makers; Reporters; the Public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the project extension period, a total of 16 undergraduate students, 3 graduate students, and 3 postdoctoral fellows gained experience on this project. This has benefited their professional development, providing them with valuable scientific training, data for publications, and networking connections for their next career steps. How have the results been disseminated to communities of interest?Two Field Days: April 22, 2022. Illinois State University Field Day. ISU Lexington, IL farm. May 26, 2022. Western Illinois University Field Day. WIU farm, Macomb, ILhttp://www.wiu.edu/pennycress/field-days/2022-field-day.php Oral Presentations: February 2, 2022. Sedbrook, J. "Developing pennycress (Thlaspi arvense) into an oilseed cash cover crop named Covercress," Daybreak Rotary Club, Bloomington/Normal, IL. Zoom. March 2, 2022. Sedbrook, J. "Developing pennycress into an oilseed cash cover crop named Covercress." Donald Danforth Center seminar series, St. Louis, MO. April 13, 2022. Sedbrook, J. Illinois State University Community Partners Breakfast hosted by ISU President Kinzy. "Developing pennycress into an oilseed cash cover crop named CoverCress." Bone Student Center, Illinois State University. April 19, 2022. Sedbrook, J. "Using agricultural biotechnology to rapidly domesticate pennycress and the opportunity that this cover crop and oilseed crop has to mitigate climate change" Asia-Pacific Economic Cooperation High Level Policy Dialogue on Agricultural Biotechnology. Agriculture & Food Systems Institute. Malaysia. Zoom. July 26, 2022. Sedbrook, J. "Domesticating pennycress into the new oilseed cash cover crop named CoverCress,"Westminster Community Meeting, Bloomington, IL. Poster Presentations: January 8-12, 2022. Gautam, B., Hafner, A., Sloman, K., Bayliss, R., and Sedbrook, J. Generating and characterizing larger seed size mutants in the oilseed plant pennycress (Thlaspi arvense). Plant and Animal Genome Conference, Virtual. February 23, 2022. Hafner, A., Sloman, K., McWilliams, A., Bayliss, R., Gautam, L., Jarvis, B., Williams, D., and Sedbrook, J. "Decreasing glucosinolate content in the oilseed crop pennycress (Thlaspi arvense L.).," Phi Sigma Research Symposium, ISU, Normal, IL. July 9-13, 2022. Gautam, B., Hafner, A., Sloman, K., Bayliss, R., Phippen, W., and Sedbrook, J. Generating and characterizing larger seed size mutants in the oilseed plant pennycress (Thlaspi arvense). American Society of Plant Biologists (ASPB) International Meeting, Portland, OR What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We requested and were approved a no-cost extension for this project, with the final project end date being 09/14/2022. This extension allowed us to work on five manuscripts, with two of those manuscripts being published in 2022. This extension also allowed us to collect and analyze data from pennycress lines planted in four locations (near St. Paul, MN, Macomb, IL, Normal, IL, and Arenzville, IL) for the 2021-2022 field season. These data validated our hypotheses that specific domestication traits mutations to reduce seed coat fiber content, reduce seed oil erucic acid content, increase seed oil oleic acid content,increase total seed oil content, reduce seed glucosinolate content, improve seed germination and seedling establishment, and reduce seed pod shatter would be phenotypically stable at multiple geographic locations and not negatively impact plant health or seed yields. Our hypotheses were confirmed. Over the course of this project, we have obtained patent protection on these genetic changes. CoverCress, Inc. has licensed much of this intellectual property and incorporated the genetic changes discovered by this project into the commercial varieties of CoverCress™. Moreover, CoverCress Inc. was acquired by Bayer, Chevron, and Bunge (publicly announced August 1st, 2022: https://www.bayer.com/media/en-us/bayer-expands-existing-investment-to-acquire-majority-share-in-sustainable-lower-carbon-oilseed-producer-covercress-inc/ ). In summary, the discoveries we were able to make with this USDA NIFA funding have been instrumental in developing and commercializing domesticated pennycress (aka CoverCress™).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Nunn, A., Rodr�guez-Ar�valo, I., Tandukar, Z., Frels, K., Contreras-Garrido, A., Carbonell-Bejerano, P., Zhang, P., Ramos-Cruz, D., Jandrasits, K., Lanz, C., Brusa, A., Mirouze, M., Dorn, K., Jarvis, B., Sedbrook, J., Wyse, D., Otto, C., Langenberger, D., Stadler, P., Weigel, D., Marks, M. David, Anderson, J., Becker, C., and Chopra, R. (2022). Chromosome-level Thlaspi arvense genome provides new tools for translational research and for a newly domesticated cash cover crop of the cooler climates. Plant Biotechnol. J. 20: 944-963. https://doi.org/10.1111/pbi.13775
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Phippen, W. B., Rhykerd, R., Sedbrook, J. C., Handel, C., and Csonka, S. (2022). From farm to flight: CoverCress as a low carbon intensity cash cover crop for sustainable aviation fuel production. A review of progress towards commercialization. Frontiers Energy Res. 939. https://doi.org/10.3389/fenrg.2022.793776
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Progress 12/15/20 to 12/14/21
Outputs
Target Audience: Other scientists including those performing research on crop improvement, domestication, plant seed oil and meal production, Brassicas and oilseed crops; Start-up and established companies that develop crops; Agribusinesses including Oilseed Processors; Farmers; Educators; Students; Federal and State Legislators; Policy Makers; Reporters; the Public. Changes/Problems:We requested and were approved for a no-cost extension for this project, with the end date now being 09/14/2022. This extension will allow us to collect and analyze data from pennycress lines planted in the 2021-2022 field season as well as complete writing and submit related manuscripts. What opportunities for training and professional development has the project provided?ISU Three PhD students, 2 Master's student, and 6 undergraduate students participated on this project this past year, including performing wet bench experiments in the laboratory and field phenotypic analyses. Presentations at meetings external to ISU were limited to Zoom due to COVID. Two of the PhD students graduated in 2021, with one then beginning a postdoc at the Carnegie Institution at Stanford University and the other becoming director of a medical diagnostics laboratory. One Master's student graduated in 2021 and entered a PhD program in molecular biology. UMN One graduate student and one postdoc at the UMN worked to complete the objectives of this project. The postdoc received opportunities to develop project management and mentoring skills and further develop their research and writing skills. The postdoc landed a job at CoverCress Inc., leading their molecular breeding group. We have employed undergraduates interested in agricultural sciences and plant breeding and genetics. WIU Three undergraduate students performed independent research on pennycress in 2021. In addition, 12 students enrolled in Principles of Plant Breeding course used the EMS mutant lines to conduct independent projects looking at breeding traits and selection techniques. Students even conducted controlled crosses to develop their own pennycress lines. An additional 4 students helped support the pennycress program by maintain plants in the greenhouse and field, cleaning seed, and taking laboratory measurements. UW 1 postdoc conducted experiments on the project this past year. How have the results been disseminated to communities of interest?Presentations January 12th, 2021. Phippen, W. Invited speaker, BioAg Winter meeting, Macomb, IL. "Pennycress as a viable cover crop. 25+ producers from West central Illinois." March 16, 2021. Phippen, W. Invited speaker, Galesburg, IL Sunrise Rotary (producers and businesses). "Advances in New crops - Pennycress development". May 15th, 2021. Sedbrook, J. presentation. NSF Convergent Food Systems Conference. "Developing pennycress into an oilseed cash cover crop named Covercress for the U.S. Midwest". June 16, 2021. Phippen, W., Webinar, Commercial Aviation Alternative Fuel Initiative (CAAFI), "Pennycress crop readiness for commercial aviation". July 19th, 2021. Sedbrook, J. presentation. Agriculture & Food Systems Institute workshop entitled, Gene Edited Plants: Context and Communication for Plant Breeding Innovation. "Employing CRISPR genome editing to rapidly domesticate pennycress into an oilseed cash cover crop called Covercress". August 25th, 2021. Sedbrook, J. presentation. American Chemical Society AGRO Division, 262nd ACS National Meeting & Exposition. Zoom. John Sedbrook invited presentation entitled, "Employing CRISPR genome editing to rapidly domesticate pennycress into an oilseed cash cover crop called Covercress". September 16th, 2021. Sedbrook, J. Participant, virtual strategy session focused on the development of a government wide Roadmap to support the development and deployment of the Sustainable Aviation Fuels (SAF) Roadmap. October 11th, 2021. Sedbrook, J. presentation. Washington State University Department of Crop and Soil Sciences seminar series. "Developing pennycress into an oilseed cash cover crop named Covercress". December 2, 2021. Sedbrook, J. presentation. CSHL Plant Genomes, Systems Biology & Engineering, Cold Spring Harbor, New York. "Developing pennycress (Thlaspi arvense) into an oilseed cash cover crop named Covercress." December 2, 2021. Sedbrook, J. presentation. Canola Week, Canola Industry Meeting, Canola Council, Saskatoon, Alberta, Canada. "Developing pennycress into an oilseed cash cover crop named Covercress." Stakeholder Engagement April 16th, 2021. Sedbrook, J. Participant, Cover Crops Field Day with a focus on pennycress. Illinois State University Lexington Farm. May 2021: Dr. Phippen conducted the 12th annual In-Person Pennycress Field Day at the WIU farm. May 7th, 2021, CoverCress Field Showing. CoverCress breeding and seed increase plots, Burrus Seed Farm near Arenzville, IL, organized by J. Sedbrook. Attendees included Congressmen Rodney Davis and Darren LaHood, farmers, agribusiness representatives, and local and international press. May 13th, 2021. Sedbrook, J. Participant, Cooperative Governance of Gene Editing (with a focus on pennycress). Hosted by Dr. Nicholas Jordan, University of Minnesota. August 11th, 2021. Sedbrook, J. Participant, CoverCress, Inc. Advisory Board Meeting. St. Louis, MO. Dr. Phippen created 2 short videos highlighting pennycress production and research program at WIU. https://www.iprefercap.org/videos/ Popular press articles: April 20, 2021, Kay Shipman, FarmWeek, "ISU offers cover crop practicalities in central Illinois". https://www.farmweeknow.com/environmental/isu-offers-cover-crop-practicalities-in-central-illinois/article_182f4fe6-a153-11eb-8780-a73e9b82fd6c.html April 30, 2021, Phyllis Coulter, Illinois Farmer Today, "Agronomist: Creating new crop like flying plane while building it". https://www.agupdate.com/illinoisfarmertoday/news/crop/agronomist-creating-new-crop-like-flying-plane-while-building-it/article_9389b3e8-a860-11eb-bd1e-c735fd4156a8.html May 7, 2021, Samantha McDaniel-Ogletree, Jacksonville Journal-Courier, "A new crop soon could grow an additional source of revenue for Illinois farmers". https://www.agupdate.com/illinoisfarmertoday/news/crop/new-crop-establishes-new-harvest-season/article_89d463ca-d920-11eb-90d4-c309e8bdddde.html May 26, 2021, Rod Nickel and Karl Plume, Reuters, "Stinkweed to false flax: oilseeds race to reap biofuel bonanza". https://www.reuters.com/business/energy/stinkweed-false-flax-oilseeds-race-reap-biofuel-bonanza-2021-05-26/ June 3, 2021. Science Christian Monitor cover story, "Climate versus jobs? Not in this heartland state". https://www.csmonitor.com/Environment/2021/0603/Climate-versus-jobs-Not-in-this-heartland-state July 1, 2021, Phyllis Coulter, Illinois Farmer Today, "New crop establishes new harvest season". https://www.agupdate.com/illinoisfarmertoday/news/crop/new-crop-establishes-new-harvest-season/article_89d463ca-d920-11eb-90d4-c309e8bdddde.html September 23rd, 2021. International Seed Federation Stories from the Ground short film series, "The Grass is Greener" episode highlights the development of Covercress, interviewing John Sedbrook. https://www.youtube.com/watch?v=x8ILAacfACE What do you plan to do during the next reporting period to accomplish the goals?Work remains in phenotypically assessing lines currently growing in the field, the analyzing seed compositions and compiling all of the data for the 2021-2022 field season. Four related manuscripts are being written for submission to high quality peer reviewed journals in 2022.
Impacts
What was accomplished under these goals?
We tested dozens of pennycress CRISPR-edited lines and EMS-mutagenized lines chosen for improvements in core agronomic traits. These lines contained either single mutations or multiple mutations combined through genetic crossing and/or CRISPR genome editing. We assessed a variety of plant growth (field and growth chamber-grown at UMN, WIU, and ISU) and seed compositional traits including germination and stand establishment, sizes of rosette leaves entering into winter, overwintering resilience, first flower, seed yields, total seed oil content, improved fatty acid compositions, reduced seed fiber content, increased seed meal protein content, increased seed size, reduced seed and vegetative glucosinolate content, and reduced pod shatter. These studies have confirmed genetics that improve each of these traits without compromising plant growth and resilience. Vetted mutations have been introduced into the crop CoverCress (domesticated pennycress), which was planted commercially in Illinois in the fall of 2021 by CoverCress, Inc. The trait improvements incorporated into CoverCress, originating from this groundbreaking research and in collaboration with CoverCress, Inc., include reduced seed dormancy, reduced seedpod shatter, low erucic acid seed oil, reduced seed fiber content, reduced seed glucosinolate content, and increased seed oil content. Additional details: Glucosinolates: CRISPR genome edited single and double mutant lines have been identified and confirmed as having seed glucosinolate levels reduced by 30 to 40 percent each without impacting plant growth and seed yields. These mutations have been combined by genetic crossing and CRISPR genome editing in both laboratory lines and CoverCress commercial varieties. Isolation of triple homozygous mutant lines is underway, after which determinations will be made as to whether the combined mutations have additive effects on reducing glucosinolate and if the plants remain healthy. Other mutation combinations are also being generated with the same end goal of reducing seed glucosinolate level to near or below 30 umol/gm. These mutations are also being stacked with the core pennycress domestication traits (low seed fiber, low erucic acid, reduced pod shatter, reduced seed dormancy) arising from this USDA NIFA-funded research which are in commercial CoverCress (domesticated pennycress) varieties that were planted in Illinois on about 1,000 acres in the fall of 2021 by CoverCress, Inc. We have screened thousands EMS treated lines for reduced glucosinolates using NIRS. Early on we found a line with very low levels of glucosinolates with a mutation in the AOP gene that encodes an enzyme responsible for completing the last biochemical step leading to the biosynthesis of sinigrin, the predominant glucosinolate in pennycress. However, the EMS allele was associated with a linked detrimental mutation that reduced seed size, plant stature, and delayed flowering for plants grown in the field. We have recently identified lines containing recombinant breaks between aop and the detrimental mutation that behave better under field conditions. We are now in the process introgressing this cleaning aop allele into lines containing multiple domestication traits. During the next few months lines containing improved germination, reduced fiber, reduced seedpod shatter, high oleic oil, reduced glucosinolates, and early flowering/maturing will be ability for testing. Such domesticated pennycress lines should be suitable for commercial production and will serve as key starting material for longer term conventional breeding efforts. Seed germination: We have taken a range of approaches to identify parameters influencing germination, including seed treatments, light treatment and the effect of transparent testa mutations. For seed treatment, we performed priming of seeds with water, GA4 and hydrogen peroxide overnight on WT MN106 seeds from three years of harvest. Primed seeds were placed on soil; we found that seeds primed with GA4 or hydrogen peroxide had 50% germination 24hrs earlier than the control. In addition, we found that seed source had confounding effects on germination. To understand seed aging or deterioration effects on germination, we performed seed longevity test. Results were inconclusive due to the storage conditions of available seed sources. For light treatment, we used red light exposure for different time intervals on water and GA imbibed seeds. We found that imbibed seeds exposed to red light for 5 min increased germination in WT MN106. We have found several transparent testa mutants that have increased germination with this treatment. With these experiments, we have successfully determined two of the major factors that influence pennycress seed germination. These results were recently published in Industrial Crops and Products - lead author Ott. We wanted to evaluate the effects of seed treatments when pennycress seeds are buried at 0.75 cm. This is extremely important as wild type pennycress seeds germinate poorly if buried. This limits water availability to surface-planted seeds as the surface dries there faster than subsurface soil. This experiment was first planted in the field in the Fall of 2019 and we included wild-type and several mutant candidates. We found ~15% germination for the wild-type seeds without any seed priming whereas hydro-priming with or without hormones increased the germination rates by 2-3 folds respectively. Using this approach, we have found two lines that have 80% germination rate with hydropriming, and we are continuing to further understand genetics behind this trait. These lines were planted in the field in the fall of 2021 at UMN, WIU, and ISU locations and are under evaluation. This study has allowed us to answer several key questions about germination in pennycress. In addition, we have begun introgressing the domestication traits into these enhanced germination lines. Plant height: We have screened several hundred lines for reduced plant height mutants in pennycress. During the screens we found several candidates for this trait. Unfortunately, many of them did not breed true the following season and we suspect a possible influence of spring germination on this trait. In total, we have identified four candidate lines - A7-111, A7-76, A7-77 and E5-497 that have at least 25% reduction in height compared to the wild-type plants. We have established causal relationship between the plant height and the mutation in a known dormacy gene. We have performed nitrogen rate study on the wild type (MN106) for two years and two locations and results suggest the increase in nitrogen content will increase the total seed yields (Ott et al. - Unpublished). We are in the process of testing nitrogen effects on A7-77 dwarf mutant in the greenhouse setup. In addition, we have combined the A7-77 mutation with another mutation that greatly speeds maturation. Alone, this latter mutation results in taller plants that are prone to excessive lodging. Preliminary data indicates that combining the semi dwarf trait with the early maturing traits results in a line that matures early while exhibiting reduced lodging compared to the early maturing parental line. Current we are introgressing the domestication traits into this double mutant and it is currently undergoing additional field testing. We hope to finish these studies by the end of this summer and will be communicating these results in peer-reviewed journals. Early maturity: We have found several early flowering mutants from our previous screens. We have continued our work on two candidate lines A7-25 and A7-248. We have performed extensive characterization of these lines and found candidate genes responsible for this phenotype. In addition, we have performed three years of planting date effect on these lines across 2 locations (Ott et al. - unpublished). Results from this study will be communicated to journals by the end of this year.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Ott, M.A., Gardner, G., Rai, K.M., Wyse, D.L., Marks, M.D., and Chopra, R. (2021) TRANSPARENT TEST 2 allele confers major reduction in pennycress (Thlaspi arvense L.) seed dormancy. Industrial Crops and Products, 174 https://doi.org/10.1016/j.indcrop.2021.114216
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Jarvis, B.A., Romsdahl, T.B., McGinn, M., Nazarenus, T.J., Cahoon, E.B., Chapman, K.D., and Sedbrook, J.C. (2021) CRISPR/Cas9-induced fad2 and rod1 mutations stacked with fae1 confer high oleic acid seed oil in pennycress (Thlaspi arvense L.). Frontiers Plant Sci. 12:652319. https://doi.org/10.3389/fpls.2021.652319
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Marks, M.D., Chopra, R., and Sedbrook, J.C. (2021) Technologies enabling rapid crop improvements for sustainable agriculture: Example pennycress (Thlaspi arvense L.). Emerg. Top. Life Sci. 5: 325-335. https://doi.org/10.1042/ETLS20200330
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Esfahanian, M., Nazarenus, T.J., Freund, M.M., McIntosh, G., Phippen, W.B., Phippen, M.E., Durrett, T.P., Cahoon, E.B., and Sedbrook, J.C. (2021) Generating pennycress (Thlaspi arvense) seed triacylglycerols and acetyl-triacylglycerols containing medium-chain fatty acids. Front. Energy Res. 10:620118. https://doi.org/10.3389/fenrg.2021.620118
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Chopra, R., Folstad, N., and Marks, M.D., (2020) Combined genotype and fatty-acid analysis of single small field pennycress (Thlaspi arvense) seeds increases the throughput for functional genomics and mutant line selection. Industrial Crops and Products 156 https://doi.org/10.1016/j.indcrop.2020.112823
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Progress 12/15/19 to 12/14/20
Outputs
Target Audience:Other scientists including those performing research on crop improvement, domestication, plant seed oil and meal production, Brassicas and oilseed crops; Start-up and established companies that develop crops; Oilseed processors; Farmers; Educators; Students; Federal and State Legislators; Reporters; the Public. Changes/Problems:We requested and were approved for a no-cost extension for this project, with the end date now being 08/31/2022. This extension will allow us to collect and analyze data from pennycress mutant lines planted in the 2021-2022 field season. COVID-19 restrictions continue to hamper student involvement and productivity. What opportunities for training and professional development has the project provided?ISU: 2 PhD students, 2 Master's student, and 6 undergraduate students participated on this project this past year, including performing wet bench experiments in the laboratory and field phenotypic analyses. Presentations at meetings were limited due to COVID. One PhD student will be graduating in 2021 and going to the Carnegie Institution at Stanford University for a postdoc. One Master's student graduated in 2020 and is working at an agriculture diagnostics laboratory. UMN: One graduate student and one post-doc at the UMN are working to complete the objectives of this project. The post docs have received opportunities to develop project management and mentoring skills and further develop their research and writing skills. We have employed undergraduates interested in agricultural sciences and plant breeding and genetics. WIU: 1 undergraduate student conducted experiments on the project this past year. An additional 5 students helped support the pennycress program by maintain plants in the greenhouse and field, cleaning seed, and taking laboratory measurements. UW-Madison: 1 postdoc conducted experiments on the project this past year. How have the results been disseminated to communities of interest?News stories: AGRINEWS. Associated Press. Nov. 18, 2020. Illinois State, elected officials praise cover crop research. https://www.agrinews-pubs.com/2020/11/18/illinois-state-elected-officials-praise-cover-crop-research/a2b0bi6/ The Pantagraph. Lenore Sobota. Oct. 13, 2020. 'Wonder weed' research at Illinois State University called a 'game changer'. https://www.pantagraph.com/news/local/education/watch-now-wonder-weed-research-at-illinois-state-university-called-a-game-changer/article_0982bba5-b904-501b-918a-5dd12becd438.html WEEK News (Television and on-line). Alex Menke. Oct. 12, 2020. Cashing in on Pennycress. https://week.com/2020/10/12/cashing-in-on-pennycress/ Illinois Farmer Today. Phyllis Coulter. Oct. 2, 2020. More dollars go into developing pennycress. https://www.agupdate.com/illinoisfarmertoday/news/crop/more-dollars-go-into-developing-pennycress/article_e60236fe-028e-11eb-803e-d756825d3a06.html Brownfield Ag News. Rhiannon Branch. Aug. 14, 2020. Domesticated Pennycress: A new cash cover crop in development. https://brownfieldagnews.com/news/domesticated-pennycress-a-new-cash-cover-crop-in-development/ ISU: Sedbrook, J. "Pennycress domestication traits and related CRISPR resources," DOE Integrated Pennycress Resilience Project (IPReP) seminar series, International, Zoom (December 15, 2020). Sedbrook, J. (Organizer/Presenter) ISU Ag Innovation briefing to legislatures and ag stakeholders. Illinois State University Lexington Farm. Creating economic opportunities in Illinois. Presenters: Dietz, L. (President, Illinois State University); Csonka, S. (Commercial Aviation Alternative Fuel Initiative - CAAFI); Handel, C. (CoverCress, Inc.), Lurkins, L. (Illinois Farm Bureau), Whittaker, S. (Illinois Geological Survey), Jordan, B. (Great Plains Institute), B. (Presenter) " Illinois State University Ag Innovation Briefing: Creating economic opportunities in Illinois," Symposium, Workshop. (Oct. 12, 2020). Sedbrook, J. "Pennycress - Biofuel for a Greener Future," Illinois State University Club, Zoom, (October 9, 2020). Williams, D. (Presenter), Sedbrook, J. "Elucidating the roles of the HIGH ALOPHATIC GLUCOSINOLATE (HAG) genes in glucosinolate production in pennycress (Thlaspi arvense).," Illinois State University Cell and Molecular Biology Seminar Series, Zoom (March 25, 2020). Sedbrook, J. "Advancing Field Pennycress as a New Oilseed Biofuels Feedstock that Does Not Require New Land Commitments.," USDA NIFA/DOE Project Director/Principal Investigator Meeting, Washington, D.C. (February 24, 2020). Sedbrook, J. "Employing CRISPR Gene Editing to Rapidly Domesticate Pennycress into an Oilseed Cash Cover Crop," National Alliance of Independent Crop Consultants (NAICC) Conference, San Antonio, TX. (January 22, 2020). UMN: We have reported preliminary results of our phenotypic analysis to university researchers at several seminars, symposiums, conferences, and in pennycress virtual field day webinars in lieu of a 2020 in-person pennycress field day. We will continue to disseminate results as we complete additional field and greenhouse trials. A detailed list of seminars and publications are listed below. Professional conferences/meetings: Bayer Poster Presentation on 8 Jan 2020. Pennycress Yield Peaks At Low Nitrogen Rate. Matthew A. Ott*, Katherine Frels, RatanChopra, M. David Marks, M. Scott Wells ASA-CSSA-SSSA presentation 12 Nov 2020. Transparent Testa Alleles Confer Major Increases in Pennycress Germination Rates. Matthew A. Ott, Ratan Chopra, Gary Gardner, Donald Wyse, M. David Marks. ASA-CSSA-SSSA presentation 12 Nov 2020. Multi-locular Trait in Field Pennycress. Ratan Chopra and M. David Marks. WIU: May 2020: 11th annual Pennycress Field Day virtual presentations. Created 3 short videos highlighting pennycress production in lieu of in-person events. February 2020: W. B. Phippen. 2020. Pennycress as a new oilseed feedstock for the Midwest. Carinata 2020 and SPARC annual meeting, Tampa Bay, Florida. February 25-26, 2020 (oral). What do you plan to do during the next reporting period to accomplish the goals?ISU: Continue to characterize mutants we have in hand. Continue to stack mutations in top breeding lines using CRISPR and genetic crossing. Identify additional mutations to further improve agronomic traits. Disseminate findings through additional publications, presentations, and interactions with stakeholders. UMN: We are screening for additional mutant lines with new gene targets and these lines are being grown in the field. Continue to stack and characterize traits already identified. Continue screens for lines showing improved germination. Disseminate findings through additional publications, presentations, and interactions with stakeholders. Two manuscripts are under preparation and we will be working on these papers during the next period. WIU: Complete 2021 field trails and evaluations of the current advanced breeding lines for improved stand establishment and early flowering. Screen and evaluate the top 30 EMS mutant plants under field conditions for desirable agronomic traits. Continue evaluations of spring nitrogen application timing. Continue to increase seed volumes of promising yellow seeded GE lines. UW-Madison: Complete seed fiber analyses for two manuscripts.
Impacts
What was accomplished under these goals?
We have completed comprehensive analyses of CRISPR and EMS-induced mutations in 12 pennycress transparent testa genes, performing phenotypic analyses on seed fiber, oil, and protein content/composition, germination efficiencies, seed yields, and other traits under both growth chamber and field conditions. A large manuscript for this study is on track for submission in 2021. Many of these mutants perform remarkably well, improving multiple composition and performance traits. Mutations in one gene in particular will be in the first Covercress commercial varieties to be planted in the fall of 2021 and stacked with other mutations that this project has identified and confirmed as being agronomically relevant/viable including mutations conferring low erucic and high oleic seed oil. Along with the transparent testa manuscript, we are on track to submit a manuscript focused on our efforts to stack core domestication traits (low fiber, low erucic, reduced glucosinolate) into pennycress using CRISPR gene editing. During year three of this project (this report), we published five papers, including a paper in Nature Food highlighting our efforts in generating domestication traits in pennycress using ethyl methanesulfonate (EMS) mutagenesis (Chopra et al., 2020). We were also successful in 2020 at being issued a patent for the pennycress low fiber trait and methods of making. Other highlights: To understand germination in pennycress, we have taken a range of approaches to identify parameters that influence uniform germination. These approaches include seed treatments and light treatment. We have optimized protocols for seed germination under water stress and at a greater depth compared to the traditional planting depths. In this past year, we have found several mutants that have increased germination compared to the wild-type plants when grown under dark conditions and reduced water conditions. With these experiments, we have successfully determined three major candidate genes that influence germination of pennycress seeds. We also evaluated the fresh seed dormancy in pennycress and found that the transparent testa mutants have substantially reduced fresh seed dormancy compared to the co-grown wild type. In fact, some transparent testa mutants do not require GA treatment to break seed dormancy, which is a notable advancement in that commercial varieties (called Covercress) will not require GA treatment before plants thereby saving money and simplifying logistics.We also have identified several mutants that show variation in seed size and oil composition. Those mutant lines are being tested for their role in pennycress germination. We continue to make changes in seed composition, plant architecture, and flowering time in pennycress. We have planted a range of mutants in the field at UMN, ISU, and WIU during the growing season of 2020-2021 and are evaluating these lines throughout their growth, along with analyses of seed yields and compositions during the summer 2021. For example, at WIU, 54 pennycress breeding lines and selections from 2019 were field evaluated during the 2019-2020 growing season.Seed yields ranged from 55-1,038 lbs./acre. Though spring-type lines have more uniform germination and stand establishment in the fall and earlier flowering in the spring, their seed yield tends to be lower than the winter types. Six new mutant pennycress lines selected for early establishment, early flowering, above ground biomass, tillering, branching, plant height, stem thickness, and seed yield performed very well under field conditions in Macomb, Illinois. The top performing line 'ELIZA-891-13' had an average height of 59 cm, harvest index of 34% and a total seed yield of 1,038 lbs of seed/acre compared to the control of 'Elizabeth' which only had a yield of 479 lbs. acre. A nitrogen experiment investigating six fertilizer rates on a single winter variety resulted in the highest yield coming from a N application of 50 lbs./acre in the fall and additional application of 50lbs./acre of nitrogen in the spring. Additional nitrogen application timing experiments of the spring application are planned for the 2021 growing season. Results of applying a protectant fungicide (Piaxor) late season did not have a significant effect on seed yield in 2020.We experienced another quick dry down season and disease pressure was not seen in the trails this year. Also at WIU in the greenhouse, a very large screen of EMS mutants was conducted to identify possible new traits. A total of 681 individual mutant plants were evaluated for early flowering, tillering, floral and stem branching, plant height, stem thickness, disease susceptibility (black spot), seed coat color, seed yield, and total seed oil content. The top performing 30 lines were selected and are growing in outdoor field trials in the fall to spring 2020-2021 season. We continue to introgress and stack a variety of domestication traits into single lines for further analyses including yield performance andabiotic stress resilience interrogation within the USDA-NIFA CAP Integrated Pennycress Research Enabling Farm and Energy Resilience (IPREFER) project and DOE BER-funded Integrated Pennycress Resilience Project (IPReP).
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
Jarvis, B.A., Romsdahl, T.B., McGinn, M., Nazarenus, T.J., Cahoon, E.B., Chapman, K.D., and Sedbrook, J.C. (In Press) CRISPR/Cas9-induced fad2 and rod1 mutations stacked with fae1 confer high oleic acid seed oil in pennycress (Thlaspi arvense L.). Frontiers Plant Sci.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Marks, M.D., Chopra, R., and Sedbrook, J.C. (2021) Technologies enabling rapid crop improvements for sustainable agriculture: Example pennycress (Thlaspi arvense L.). Emerg. Topics Life Sci. ETLS20200330. doi: https://doi.org/10.1042/ETLS20200330
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Esfahanian, M., Nazarenus, T.J., Freund, M.M., McIntosh, G., Phippen, W.B., Phippen, M.E., Durrett, T.P., Cahoon, E.B., and Sedbrook, J.C. (2021) Generating pennycress (Thlaspi arvense) seed triacylglycerols and acetyl-triacylglycerols containing medium-chain fatty acids. Front. Energy Res. 10:620118. https://doi.org/10.3389/fenrg.2021.620118
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Sedbrook, J.C. and Durrett, T.P. (2020) Pennycress, carbon wise: Labeling experiments reveal how pennycress seeds efficiently incorporate carbon into biomass. J. Exp. Botany. 71: 28422846. https://doi.org/10.1093/jxb/eraa136
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Chopra, R., Johnson, E.B., Emenecker, R., Cahoon, E.B., Lyons, J., Kliebenstein, D.J., Daniels, E., Dorn, K.M., Esfahanian, M., Folstad, N., Frels, K., McGinn, M., Ott, M., Gallaher, C., Altendorf, K., Berroyer, A., Ismail, B., Anderson, J.A., Wyse, D.L., Ulmasov, T., Sedbrook, J.C., and Marks, M.D. (2020) Identification and stacking of crucial traits required for the domestication of pennycress. Nature Food. 1: 84-91. https://doi.org/10.1038/s43016-019-0007-z This paper was the subject of a News & Views article: https://doi.org/10.1038/s43016-019-0016-y
- Type:
Theses/Dissertations
Status:
Awaiting Publication
Year Published:
2020
Citation:
ELUCIDATING THE ROLE OF THE HIGH ALIPHATIC GLUCOSINOLATE (HAG) GENES IN PENNYCRESS (THLASPI ARVENSE L.) GLUCOSINOLATE PRODUCTION
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
B.C. Phippen and W.B. Phippen. 2020. Impact of Nitrogen and Sulfur on the Biofuel Cover Crop Field Pennycress (Thlaspi arvense L.). Calvin University Undergraduate Research Day, November 2020 (Poster).
|
Progress 12/15/18 to 12/14/19
Outputs
Target Audience:Other scientists including those performing research on crop improvement, domestication, plant seed oil and meal production, Brassicas and oilseed crops; Start-up and established companies that develop crops; Oilseed processors; Farmers; Educators; Students; the Public. Changes/Problems:ISU: We expect reductions in our abilities to perform lab work and field work due to the COVID-19 pandemic. UMN : The late spring in MN greatly delayed flowering time and maturity date of field grown plants. This limited the value of the field data collected for flowering and plant height during the summer of 2019. We expect reductions in our abilities to perform lab work and field work due to the COVID-19 pandemic. WIU: Significant rainfall in fall delayed pennycress planting and had an impact on plot establishment. Significance of impact will be assessed in the early spring. UW-Madison: COVID-19 restrictions are hampering experiments. What opportunities for training and professional development has the project provided?ISU: 2 PhD students, 2 Master's students, and 9 undergraduate students participated on this project this past year, including performing wet bench experiments in the laboratory and field phenotypic analyses, and presenting their findings at local, regional, and international meetings. One high-profile publication (Chopra et al., 2020) also arose from their work. UMN: 1 PhD student, 2 post docs, 2 technicians and five undergraduates on the project this past year. Of note, two of the undergraduates have continued onto graduate school obtain advanced degrees in plant science (UWISC and NCSU). WIU: 2 undergraduate students conducted experiments on the project this past year. An additional 4 students helped support the pennycress program by maintain plants in the greenhouse and field, cleaning seed, and taking laboratory measurements. UW-Madison: 1 postdoc conducted experiments on the project this past year. How have the results been disseminated to communities of interest?Sedbrook (ISU) - Oral Presentations: Sedbrook, J. (Presenter) "Employing CRISPR Gene Editing and EMS Mutagenesis to Rapidly Domesticate Pennycress As an Oilseed­ Producing Cash Cover Crop.," ASA, CSSA and SSSA International Annual Meeting, San Antonio, TX. (November 12, 2019). Sedbrook, J. (Organizer/presenter) "Covercress (domesticated pennycress) Economic, environmental, and societal opportunities throughout Illinois.," Briefing, Illinois Farm Bureau, Bloomington, IL. (October 29, 2019). Sedbrook, J. (Interview) "From Weed to Cash: Researchers Genetically Engineer Pennycress.," WGLT News story on USDA funding of $10 million IPREFER project. (October 3, 2019). Sedbrook, J., Esfahanian, M., Jarvis, B., Williams, D. (Interview) "ISU researchers see potential to make dollars with pennycress.," Pantagraph News story on USDA funding of $10 million IPREFER project. (October 2, 2019). Sedbrook, J., Esfahanian, M., Jarvis, B., Williams, D. (Interview) "https://isu10news.com/tv-10-news-wednesday-september-18-2019/," TV-10 News story on USDA funding of $10 million IPREFER project. (September 18, 2019). Sedbrook, J. (Organizer/presenter) "Covercress (domesticated pennycress) Economic, environmental, and social opportunities throughout Illinois.," Lab/facilities tour and briefing to U.S. Congressional Representative Rodney Davis, State of Illinois Senate Minority Leader Bill Brady, and State of Illinois Representative Dan Brady, School of Biological Sciences, Illinois State University. (August 20, 2019). Esfahanian, M. (Presenter), Sedbrook, J. "Identifying mutants having reduced seed coat fiber and increased protein content to improve seed meal nutritional value of the oilseed crop pennycress (Thlaspi arvense)." American Society of Plant Biologists Annual Meeting, San Jose, CA. (August 5, 2019). Sedbrook, J. (Presenter) "Innovation in Agriculture: What Does Gene Editing Mean for the Future?," Congressional Briefing, House Agriculture Research Caucus, Washington D.C. Capital Hill. (July 16, 2019). Sedbrook, J. (Presenter) "Pennycress as a Cash Cover Crop: Value Proposition," Cooperative Governance Workshop on gene editing, University of Minnesota. (June 3, 2019). Sedbrook, J. (Presenter) "Progress in developing pennycress as an oilseed cash cover crop.," Western Illinois University Pennycress Field Day, Macomb, IL. (May 23, 2019). Sedbrook, J. (Presenter) "Pennycress as a new oilseed cash cover crop," ISU Farm Field Day, Lexington, IL. (April 20, 2019). Jarvis, B. (Presenter), Sedbrook, J. "Improving Pennycress Seed Oil Composition Using CRISPR Gene Editing, EMS Mutagenesis, and Transgenic Approaches," Gordon Research Conference on Plant Lipids, Galveston, TX. (January 29, 2019). Sedbrook lab (ISU) - Poster Presentations: Sedbrook, J. (Presenter), Chopra, R., Esfahanian, M., Jarvis, B., Johnson, E., Daniels, E., McGinn, M., Lyons, J., Suo, T., Messmer, M., Ulmasov, T., Phippen, W., marks, D. "Rapidly domesticating pennycress as an oilseed-producing winter cash cover crop that does not require new land commitments." American Society of Plant Biologists Annual Meeting, San Jose, CA. (August 5, 2019). Esfahanian, M. (Presenter), McGinn, M., Jarvis, B., Suo, T., Sedbrook, J. "Utilizing CRISPR genome editing to rapidly domesticate the winter annual oilseed crop pennycress (Thlaspi arvense)." Phi Sigma Research Symposium, Illinois State University. (April 12, 2019). Janowiak, K. (Presenter), Suo, T., Esfahanian, M., Chopra, R., Woodworth, J., Haag, K., King, A., Marks, D., Sedbrook, J. "Reducing seed coat fiber content to improve seed meal nutritional value of the oilseed crop pennycress (Thlaspi arvense)." Phi Sigma Research Symposium, Illinois State University. (April 12, 2019). Jarvis, B. (Presenter), Marchiafava, D., Williams, D., Sedbrook, J. "Improving Pennycress Seed Oil Composition Using CRISPR Gene Editing, EMS Mutagenesis, and Transgenic Approaches." Phi Sigma Research Symposium, Illinois State University. (April 12, 2019). Williams, D. (Presenter), Jarvis, B., McGinn, M., Chopra, R., Joe Lyons, J., Ulmasov, T., Marks, D., and Sedbrook, J. Altering the Glucosinolate Biosynthetic Pathway to Improve Palatability and Nutritional Value of Pennycress (Thlaspi arvense) Seed Meal. Integrative Plant Biology and Bioenergy Symposium, Illinois State University. (April 26, 2019). Sedbrook, J. (Presenter) "Advancing Field Pennycress as a New Oilseed Biofuels Feedstock that Does Not Require New Land Commitments.," USDA NIFA/DOE Project Director/Principal Investigator Meeting, Washington, D.C. (February 26, 2019). Sedbrook, J. (Presenter), Ulmasov, T., Phippen, W., Marks, D. "CoverCress® (domesticated pennycress) as an oilseed cash cover crop for the U.S. Midwest.," Gordon Research Conference on Plant Lipids, Galveston, TX. (January 29, 2019). Marks (UMN) - Oral Presentations: November 2019: Progress report to UMN Forever Green Group Marks lab (UMN) - Presentations: Ratan Chopra - Agronomy and Plant Genetics Departmental Seminar. (March 2019) Ratan Chopra - oral presentation, ASA-CSSA-SSSA International Annual Meeting, Nov. 10-13, San Antonio, Texas. Title - "Progress Toward the Identification and Stacking of Crucial Domestication Traits in Pennycress". Matthew Ott - oral presentation, ASA-CSSA-SSSA International Annual Meeting, Nov. 10-13, San Antonio, Texas. Title - "Acceleration of Flowering Time and Maturity in a Pennycress (Thlaspi arvense) Line and Identification of the Causative Gene" Matthew Ott - poster presentation, ASA-CSSA-SSSA International Annual Meeting, Nov. 10-13, San Antonio, Texas. Title - "Pennycress (Thlaspi arvense) Yield Peaks at Low Nitrogen Rate". Phippen (WIU) - Oral Presentations: May 2019, 10th annual Pennycress Field Day and Tour, Macomb, IL September 2019, Advancement of Industrial Crops Annual Meeting, Tucson, Arizona December 2019, CAAFI webinar, Pennycress a new cash cover crop What do you plan to do during the next reporting period to accomplish the goals?ISU: Continue to characterize mutants we have in hand. Continue to stack mutations in top breeding lines using CRISPR and genetic crossing. Identify additional mutations to further improve agronomic traits. Disseminate findings through additional publications, presentations, and interactions with stakeholders. UMN: Continue to stack and characterize traits already identified. Continue screens for lines showing improved germination. Disseminate findings through additional publications, presentations, and interactions with stakeholders. WIU: Complete 2020 field trails and evaluations of the current advanced breeding lines for improved stand establishment and early flowering. Screen and evaluated 1,080 EMS mutant plants in greenhouse for desirable agronomic traits. Continue evaluations of nitrogen applications and fungicide treatments in the field. Advance ALS resistance to final greenhouse trials. Continue to increase seed volumes of promising lines. UW-Madison: Further elucidate the composition of NDF and ADF in wild type pennycress seed coats and how that composition varies in different mutants.
Impacts
What was accomplished under these goals?
ISU We have generated many different CRISPR protospacer combinations to target in pennycress 1) multiple genes at once, 2) single genes in multiple locations, and 3) promoter regions ("promoter bashing" to produce partial loss of function). We found that two protospacers targeting chromosomal locations near each other often times produced deletions between the two protospacers. These deletions can easily be scored by PCR product size differences. Using the above CRISPR strategies, we generated and initiated characterization of homozygous mutations in genes hypothesized to be involved in synthesizing seed coat fiber (4 genes targeted), seed glucosinolate (7 genes targeted), pod shatter (5 genes targeted), high oleic acid (2 genes targeted - promoter bashing of 1 because total knockout was found to produce slow plant growth). Detailed phenotypic analyses have shown that, while mutations in all three genes may be commercially-viable, mutations in one gene in particular confer multiple agronomic benefits including better germination and stand establishment in the field, higher seed oil content, substantially reduced seed coat fiber content, and growth and seed yields comparable to or better than wild type (manuscript in preparation). Reducing glucosinolate has been complicated by the fact that two genetic solutions required mutations in two and three redundant genes, respectively. We have generated these combinations and are in the process of performing detailed phenotypic analyses. Field studies of the aop2 EMS mutant uncovered a developmental delay during flowering; we hypothesize this unexpected phenotype may be due to a second site mutation. We currently have CRISPR-generated aop2 mutants growing in the field, which are growing like wild type. For reducing pod shatter, we have used CRISPR to knock out five genes in total - one of the five produced no phenotype, whereas knockout mutations in the other four genes completely abolished pod shatter -this extreme phenotype is not commercially viable because the seeds cannot be extracted using a combine. Fortunately, a partial lof EMS mutation appears commercially viable (Chopra et al., 2020). We continue to search for a CRISPR solution by attempting to generate partial lof alleles with promoter regions deletions and CRISPR base substitution editing; this will allow rapid introduction of the reduced pod shatter trait into top commercial varieties thereby forgoing time consuming genetic crosses. UMN Seed germination: We have taken a range of approaches to identify parameters influencing germination, including seed treatments and light treatment. For seed treatment, we performed priming of seeds with water, GA4 and hydrogen peroxide overnight on WT MN106 seeds from three years of harvest. Primed seeds were placed on soil; we found that seeds primed with GA4 or hydrogen peroxide had 50% germination 24hrs earlier than the control. In addition, we found that seed source had confounding effects on germination. To understand seed aging or deterioration effects on germination, we performed seed longevity test. Results were inconclusive due to the storage conditions of available seed sources. For light treatment, we used red light exposure for different time intervals on water and GA imbibed seeds. We found that imbibed seeds exposed to red light for 5 min increased germination in WT MN106. We have found several mutants that have increased germination with this treatment. With these experiments, we have successfully determined two of the major factors that influence pennycress seed germination. We wanted to evaluate the effects of seed treatments when pennycress seeds are buried at 0.75 cm. This is extremely important as wild type pennycress seeds germinate poorly if buried. This limits water availability to surface-planted seeds as the surface dries there faster than subsurface soil. This experiment was planted in the field in the Fall of 2019 and we included wild-type and several mutant candidates. We found ~15% germination for the wild-type seeds without any seed priming whereas hydro-priming with or without hormones increased the germination rates by 2-3 folds respectively. Using this approach, we have found two lines that have 80% germination rate with hydropriming, and we are continuing to further understand genetics behind this trait. This study has allowed us to answer several key questions about germination in pennycress and we will be repeating these studies before publications. Plant height: We have screened several hundred lines for reduced plant height mutants in pennycress. During the screens we found several candidates for this trait. Unfortunately, many of them did not breed true the following season and we suspect a possible influence of spring germination on this trait. In total, we have identified four candidate lines - A7-111, A7-76, A7-77 and E5-497 that have at least 25% reduction in height compared to the wild-type plants. We have established causal relationship between the plant height and the mutation in RGA1 gene. We have performed nitrogen rate study on the wild type (MN106) for two years and two locations and results suggest the increase in nitrogen content will increase the total seed yields (Ott et al. - Unpublished). We are in the process of testing nitrogen effects on A7-77 dwarf mutant in the greenhouse setup. We hope to finish these studies by the end of this summer and will be communicating these results in peer-reviewed journals. Early maturity: We have found several early flowering mutants from our previous screens. We have continued our work on two candidate lines A7-25 and A7-248. We have performed extensive characterization of these lines and found candidate genes responsible for this phenotype. In addition, we have performed three years of planting date effect on these lines across 2 locations (Ott et al. - unpublished). Results from this study will be communicated to journals by the end of this year. Domestication Traits: We identified lines with reduced seed pod shatter, reduced erucic acid, reduced glucosinolates, reduced fiber, early flowering and reduced PUFAs (Chopra et al. 2020). We combined many of these traits in single background and are currently field testing. Some combinations include tt8/aop, aop/fae1/rod1/ind1, aop1/elf6/fae1/ind1, and aop1/fae1/rod1. WIU 54 pennycress breeding lines and selections were field evaluated in replicated plots in 2019. 6 promising new EMS mutant pennycress lines selected for early establishment, early flowering, above ground biomass, tillering, branching, plant height, stem thickness, and seed yield were evaluated for the first time under field conditions. Seed yields were from 756-1192 lbs/acre. The top performing line 590-20 had an average height of 79 cm, harvest index of 31% and a total seed yield of 1,192 lbs of seed /acre compared to the control of 'Elizabeth' which only had a yield of 561 lbs/acre. An additional 48 pennycress lines were planted in replicated trials for field evaluation in 2019 with seed yields ranging from 18-1052 lbs./acre. In the greenhouse, seed increases were conducted on yellow seed coat mutants and other early flowering selections or other experiments. Selections and evaluations were continued for herbicide resistance to ALS in pennycress. Several winter type progeny show excellent resistance to ALS in the initial screenings. UW-Madison In analyzing the seed coat fiber content of the various pennycress tt mutants and WT, we were obtaining inconsistent results with respect to Acid Detergent Fiber (ADF) content and Neutral Detergent Fiber (NDF) content. We hypothesized this was due to residual protein content that was not being totally extracted using the standard methods. Therefore, we developed a novel method that assured complete ADF extraction, obtaining consistent results showing the tt mutants have considerable reductions in ADF, making the meal comparable to canola.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Chopra, R, Folstad, N, Lyons, J, Ulmasov, T, Gallaher, C, Sullivan, L, McGovern, A, Mitacek, R, Frels, K, Altendorf, K, Killiam, A, Ismail, B, Anderson, JV, Wyse, DL, and Marks, MD (2019) The adaptable use of Brassica NIRS calibration equations to identify pennycress variants to facilitate the rapid domestication of a new winter oilseed crop. Industrial Crops and Products 128: 5561. https://doi.org/10.1016/j.indcrop.2018.10.079
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Chopra, R., Johnson, E.B., Emenecker, R., Cahoon, E.B., Lyons, J., Kliebenstein, D.J., Daniels, E., Dorn, K.M., Esfahanian, M., Folstad, N., Frels, K., McGinn, M., Ott, M., Gallaher, C., Altendorf, K., Berroyer, A., Ismail, B., Anderson, J.A., Wyse, D.L., Ulmasov, T., Sedbrook, J.C., and Marks, M.D. (2020) Identification and stacking of crucial traits required for the domestication of pennycress. Nature Food. 1: 84-91. https://www.nature.com/articles/s43016-019-0007-z This paper was the subject of a News & Views article: https://www.nature.com/articles/s43016-019-0016-y
|
Progress 12/15/17 to 12/14/18
Outputs
Target Audience:Other scientists including those performing research on crop improvement, domestication, plant seed oil and meal production, Brassicas and oilseed crops; Start-up and established companies that develop crops; Oilseed processors; Farmers; Educators; Students; the Public. Changes/Problems:ISU Nothing major to speak of. There is some concern our aop2 knockout mutants may have delayed flowering. The UMN aop2 partial loss of function EMS mutant appears to grow fine plus has glucosinolate levels below the 30 µmol/gram regulatory limit. Fortunately, preliminary data suggest our other genes' loss of function mutants grow like wild type; further testing including field testing is necessary to confirm. UMN The late spring caused by April snows in MN greatly delayed flowering time and maturity date of field grown plants. This limited the value of the field data collected during the summer of 2018 WIU Dramatic freeze thaw cycles during the winter of 2018-2019 may have impact on surviving plants in spring 2019. UW-Madison Nothing to report. What opportunities for training and professional development has the project provided?UMN: 2 PhD students, 1 post doc, 1 technician, and 4 undergraduates participated on the project this past year. ISU: 2 PhD students, 2 Master's students, and 17 undergraduate students participated on this project this past year. One of the PhD students graduated and landed a job at a start-up company (CoverCress, Inc.). 5 of the undergraduate students either moved to another lab (1), graduated (4) and entered professional schools (2) or teaching (1), or stayed in the lab as a technician (1). All four graduate students benefited from internships with the start-up company CoverCress, Inc. WIU: 2 undergraduate students conducted experiments on the project this past year. An additional 4 students helped support the pennycress program by maintain plants in the greenhouse and field, cleaning seed, and taking laboratory measurements. UW-Madison: 1 PhD student conducted experiments on the project this past year. How have the results been disseminated to communities of interest?Sedbrook (ISU) - Oral Presentations: May 9, 2018: Sedbrook, J. "Employing synthetic biology approaches to facilitate value-added oil production in the oilseed cover crop pennycress.," AOCS (American Oil Chemists' Society) Annual Meeting, Minneapolis, MN. May 8, 2018: Sedbrook, J. "Improving agronomic traits of the oilseed-producing winter cover crop pennycress using CRISPR-Cas9 genome editing and EMS mutagenesis.," University of Minnesota Plant and Microbial Biology Colloquium Series, University of Minnesota, St. Paul, MN. March 2018: Esfahanian, M. (PhD student in Sedbrook lab) "Improving agronomic traits of the oilseed-producing winter cover crop pennycress (Thlaspi arvense) using CRISPR-Cas9 genome editing and EMS mutagenesis.," American Society of Plant Biologists (ASPB) Midwest Meeting, Ames, IA. ? February 27, 2018: Sedbrook, J. "Advancing Field Pennycress as a New Oilseed Biofuels Feedstock that does not Require New Land Commitments.," USDA NIFA/DOE Project Director/Principal Investigator Meeting, Washington D.C. Sedbrook lab (ISU) - Poster Presentations: March 3-4, 2018: Suo, T. (Presenter), Esfahanian, M., Chopra, R., Woodworth, J., Haag, K., King, A., Janowiak, K., Marks, D., Sedbrook, J. "Reducing seed coat fiber content to improve seed meal nutritional value of the oilseed crop pennycress (Thlaspi arvense).," American Society of Plant Biologists (ASPB) Midwest Meeting, Ames, IA.? January 13-17, 2018: Esfahanian, M. (Presenter), McGinn, M., Jarvis, B., Suo, T., Sedbrook, J. "Utilizing CRISPR genome editing to rapidly domesticate the winter annual oilseed crop pennycress (Thlaspi arvense).," Plant and Animal Genome (PAG) Conference, San Diego, CA. Marks (UMN) - Oral Presentations: March 2018: Progress report to UMN Forever Green Group July 2018: Danforth Research Center, St. Louis, MO September 2018: Department of Biochemistry, University of Nebraska-Lincoln Phippen (WIU) : May 17th, 2018: Field Day showcasing pennycress at the Western Illinois University farm. Phippen (WIU) - Oral Presentations: May 2018: 9th annual Pennycress Field Day and Tour, Macomb, IL November 2018: Crop Science Society of America Annual Meeting, Invited speaker, Biofuels symposium Baltimore, MD. What do you plan to do during the next reporting period to accomplish the goals?ISU: Continue to characterize mutants we have in hand. Continue to stack mutations in top breeding lines using CRISPR and genetic crossing. Identify additional mutations to further improve agronomic traits. Disseminate findings through additional publications, presentations, and interactions with stakeholders including a Field Day at the ISU farm showcasing pennycress on April 20, 2019. UMN: We have one additional publication under review. Continue to stack and characterize traits already identified. Continue screens for lines showing improved germination. Disseminate findings through additional publications, presentations, and interactions with stakeholders. WIU: Complete 2019 field trails and evaluations of the current advanced breeding lines for improved stand establishment and early flowering. Continue evaluations of spring type pennycress lines for improved traits. Advance ALS resistance to final greenhouse trials. Continue to increase seed volumes of promising lines. Disseminate findings through additional publications, presentations, and interactions with stakeholders including a Field Day at the WIU farm showcasing pennycress in May, 2019. UW-Madison: Further elucidate the composition of ADF in wild type pennycress seed coats and how that composition varies in different mutants.
Impacts
What was accomplished under these goals?
ISU: We have adapted a CRISPR binary vector that is modular in nature, allowing for the inclusion of up to 8 protospacers thereby targeting edits at 8 chromosomal locations at the same time in a given plant. Initial use of this vector found it to be highly efficient, producing mutations at 3 of 4 protospacer targets in individual T1-generation plants. We believe the protospacer that did not produce mutations likely had a poorly-functioning promoter driving guide RNA expression. Therefore, we have replaced that promoter in subsequent constructs and are in the process of testing these new constructs in planta. Taken together, this is an exciting advancement in that we are now efficiently introducing multiple mutations at the same time into a single plant (e.g. fae1 target for low erucate seed oil, aop2 target for low glucosinolate, and tt8 target for reduced seed coat fiber into our top breeding lines), allowing us to stack core traits rapidly, avoiding the need for genetic crosses and generations of selection. We have made great progress in identifying and validating what appear to be commercial-grade mutations for the low fiber and low glucosinolate traits. For low fiber, we now have EMS and CRISPR-induced mutations in 9 different genes. Mutations in three of those gene targets have risen to the top of our list for potential commercialization. Each target has advantages. For example one seems to be the safest route since a naturally-occurring mutation was identified in the wild; another increases total oil content by 10% and protein by 35%; the third produces significantly larger seeds. All mutations dramatically reduce seed ADF content (by up to 50%) thereby making the pennycress seed meal nutritionally-equivalent to canola for use in animal feed. Being that the meal can represent one-third the value of the pennycress seed crop, this is an important advancement necessary for making pennycress profitable. With respect to the low glucosinolate trait, which is essential along with the low erucate trait in making the seed oil and meal equivalent in value to that of canola (we validated creation of the low erucate trait already), we have validated one EMS mutant as having substantially reduced glucosinolate as well as CRISPR-induced mutations in four different genes that reduce glucosinolate below the 30 µmol/gram regulatory limit. We are in the process of assessing plant growth as well as using the above-mentioned multi-hit binary vector to rapidly stack each of these mutations with the low erucate and low fiber traits in top pennycress breeding lines. We used CRISPR to mutate three genes known from other Brassicas to be involved in pod shatter. Knockouts of one of the genes surprisingly produced no changes in pod shatter compared to wild type. Mutations in the other two genes reduced pod shatter "too much" in that it may be too difficult to break the pods open for commercial harvest. We have introduced a rescue construct into plants carrying mutations in one of these genes in anticipation that there will be a range of rescue phenotypes with some being in the "sweat spot" of reduced pod shatter. Given that the partial rescue plants will be transgenic requiring costly regulatory approval, we continue screening for non-GMO partial loss-of-function pod shatter mutants in backgrounds having commercial utility. We have successfully gone through the USDA APHIS "Am I regulated" process in gaining clearance to field-plant some of our lines mutated by CRISPR that no longer carry any portions of the DNA construct. Those plants are currently in replicated field plots at Western Illinois University and Illinois State University and appear indistinguishable from wild type. UMN: Screens are underway to identify lines producing seeds that show more uniform germination. Forward screens have identified lines that germinate in the presence of inhibitors of GA. Reverse screens are assessing the influence of mutations in genes known to control germination in Arabidopsis. Selected lines are being tested for germination in the presence of PEG to mimic drought conditions. In addition, the lines are being tested for rate of germination at different depth of seeding. Selected lines will be assessed under field conditions in the fall of 2019. Two semi dwarf lines are being retested in the field for response of nitrogen. Meaningful results were not obtained in the spring of 2018 due to the late spring in MN. Bulked segregant analyses are underway to identify the causative mutations. Mutants showing reduced pod shatter showed a 90% reduction in loss over time during the summer of 2018. One key reduced pod shatter mutation has been introgressed into lines harboring mutations that result in improved oil fatty acid content, reduced glucosinolate, reduced seed fiber, and early maturity. These will be field tested in the fall of 2019. A low glucosinolate mutant with levels below 10 µmol/g in intact seeds has been identified. This trait has already been combined with the "zero erucic acid" trait previously identified to create a "double zero" pennycress. Numerous mutants with reduced condensed tannins (reduced fiber) have been characterized in the field for multiple generations. One of these is being stacked with the before mentioned traits. Beside elf6, two additional early flower mutants have been characterized for multiple seasons in the field. These two additional lines are currently being characterized by bulked segregant analyses to identify the causative mutations. WIU: Six promising pennycress plants selected for early establishment, early flowering, above ground biomass, tillering, branching, plant height, stem thickness, and seed yield were planted to the field in the fall 2018. Plants were selected from a large M2 population in summer of 2018. An additional 48 pennycress lines were also planted in replicated trails for field evaluation in 2019. A large nutrient experiment was initiated in the fall 2018 under field conditions comparing six fertilizer rates, two varieties, at three different planting densities. A fungicide trial was planted with 2 pennycress varieties, along with large strip trails of the top 10 performing pennycress lines. In the greenhouse, seed increases were conducted on yellow seed coat mutants and other early flowering selections. Selections and evaluations were continued for herbicide resistance to ALS in pennycress. Several winter type progeny show excellent resistance to ALS in the initial screenings. UW-Madison: 2D-NMR analyses were performed on extracted seed coat material of 6 pennycress reduced seed coat fiber mutants. The data showed clear fingerprints for lignin and other phenolics, but at levels much lower than others have published for Brassica seeds. Those published results were based on Acid Detergent Fiber (ADF) analysis, which cannot distinguish the composition of the fiber. Based on additional analyses we have done including interpretation of our 2D-NMR results, we now have a working hypothesis as to what is the major component of pennycress seed coat fiber, and will be working to obtain conclusive data in year 2 of this grant, as well as analyzing additional reduced seed coat fiber mutants to look for variations in composition.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
McGinn, M., Phippen, W., Chopra, R., Bansal, S., Jarvis, B., Phippen, M., Dorn, K., Esfahanian, M., Nazarenus, T., Cahoon, E., Durrett, T., Marks, M. David, Sedbrook, J. (2018). Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop. Plant Biotechnology Journal. https://doi.org/10.1111/pbi.13014
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Chopra, R., E.B. Johnson, E. Daniels, M. McGinn, K.M. Dorn, Maliheh Esfahanian, Nicole Folstad, Kirk Amundson, Kayla Altendorf, Kevin Betts, Katherine Frels, James A. Anderson, Donald L. Wyse, John C. Sedbrook, M. David Marks (2018) Translational genomics using Arabidopsis as a model enables the characterization of pennycress genes through forward and reverse genetics. The Plant Journal 96: 1091-1092. https://doi.org/10.1111/tpj.14172 (selected for the December cover and research highlights: https://onlinelibrary.wiley.com/doi/10.1111/tpj.14172)
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Chopra, R, Folstad, N, Lyons, J, Ulmasov, T, Gallaher, C, Sullivan, L, McGovern, A, Mitacek, R, Frels, K, Altendorf, K, Killiam, A, Ismail, B, Anderson, JV, Wyse, DL, and Marks, MD (2019) The adaptable use of Brassica NIRS calibration equations to identify pennycress variants to facilitate the rapid domestication of a new winter oilseed crop. Industrial Crops and Products 128: 5561. https://doi.org/10.1016/j.indcrop.2018.10.079
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