Source: UNIVERSITY OF NEVADA submitted to
IMPROVING TEFF GRASS:A FORAGE FODDER & HIGHLY NUTRITIOUS, LOW-GLUTEN GRAIN CROP
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1006591
Grant No.
(N/A)
Project No.
NEV00386
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2015
Project End Date
Jun 30, 2020
Grant Year
(N/A)
Project Director
Cushman, J.
Recipient Organization
UNIVERSITY OF NEVADA
(N/A)
RENO,NV 89557
Performing Department
Biochemistry
Non Technical Summary
As the driest state in the U.S., all crop production in Nevada relies upon supplemental irrigation, so improving the drought tolerance of food and forage crops is an important goal for regional agriculture to maximize productivity while protecting limited water resources. Tef is a warm season, C4-photosynthesis grass that is gaining popularity in the U.S. as a high-quality summer forage, fodder, and grain. The long-term goals of the proposed integrated research and extension project are to improve the relative drought tolerance and lodging resistance of tef (Eragrostis ssp., Poaceae). The specific objectives and approaches are to determine the relative drought tolerance of three Eragrostis species with increased capacity to withstand prolonged water-deficit stress and evaluate their relative biomass productivity and seed yield under three different field irrigation regimes in Nevada. Field studies will also analyze the potential of each species for use as forage evaluated under different grazing frequencies and for productivity and nutritive value when grown as a hay crop. Lodging is a common problem in tef; therefore, mutant collections will be created using an elite variety of tef and screened for plants with reduced stature and thus, reduced susceptibility to lodging and greater harvest indices. In addition, varieties with reduced lodging and greater seed yields (harvest indices) will be developed by introducing an induced mutation that results in a semi-dwarf "Green Revolution" phenotype. The expected outcome of this project is the generation of tef germplasm with improved drought tolerance and lodging resistance to improve forage, fodder, and grain yield stability in Nevada and the arid western U.S. This research proposal addresses three out of six of the NAES research priority areas including: Agricultural Production and Environmental Sciences in a Semi-arid Environment, Economic Development with Emphasis in Rural Areas, and Natural Resources Management in the Great Basin and Sierran Ecosystems. This proposal specifically addresses both the need to develop low water-use alternative crops that also provide a value-added component for farming and ranching activities to expand Nevada's narrow economic base and the need for agricultural development and economic diversification strategies by investing in advanced basic research.
Animal Health Component
0%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20215991040100%
Knowledge Area
202 - Plant Genetic Resources;

Subject Of Investigation
1599 - Grain crops, general/other;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
Tef (or Teff) is a warm season, C4-photosynthesis grass that is gaining popularity in the U.S. as a high-quality summer forage, fodder, and gluten-free grain. The long-term goals of the proposed integrated research and extension project are to improve the relative drought tolerance and lodging resistance of tef (Eragrostis ssp., Poaceae). The primary goals of the proposed research are to: 1) Determine the relative drought and dehydration tolerance of three Eragrostis species that differ in their ability to withstand water-deficit stress. 2) Evaluate the relative biomass productivity and seed yield of three Eragrostis species that differ in drought-stress tolerance under three different field irrigation regimes in Nevada. Field studies will also analyze the potential of each species for use as forage evaluated under different grazing frequencies and for productivity and nutritive value when grown as a hay crop. The secondary goal is develop more lodging-resistant tef. Lodging is a common problem in tef; therefore, two additional aims will be to: 3) Generate and evaluate mutant collections for plants with reduced stature and thus, reduced susceptibility to lodging, and greater harvest indices. 4) Develop elite E. tef varieties with reduced lodging and greater seed yields (harvest indices) by introducing an induced mutation to create a dominant-negative gibberellin response modulator. Once developed, these improved cultivars of tef will be evaluated under field conditions in Nevada. The expected outcome of this project is to develop tef germplasm with improved drought tolerance and lodging resistance to improve forage, fodder, and grain yield stability in Nevada, the arid western U.S, and other semi-arid growing areas around the world.
Project Methods
Aim 1) Determine the relative drought and dehydration tolerance of three Eragrostis species that differ in their ability to withstand prolonged periods of water-deficit stress. The hypothesis to be tested is that more drought-tolerant varieties of tef can be identified and will provide new drought-tolerant germplasm for cultivar improvement efforts. The approach will involve determination of the longest interval without irrigation that Eragrostis tef var. 'Tiffany' (Williams love grass, moderately drought tolerant), Eragrostis curvula (weeping love grass, drought tolerant), and Eragrostis nindensis (strong drought- and poikilochlorophyllous desiccation-tolerant) can survive. Each species will be evaluated for growth rates, seed yield, and overall height and biomass parameters under greenhouse conditions. The three species will also be evaluated under field production conditions (see Aim 2). To assess drought and dehydration tolerance, plants will be evaluated for relative and absolute water content (RWC, AWC), electrolyte leakage, and photosynthetic activity of leaves at regular intervals during drying and upon rewatering and recovery to assess rates and limits of water loss, leaf damage, and photosynthetic capacity (maintenance of primary production).Task 1: Measure the inter-irrigation survival interval for each species after withholding water for progressively longer periods prior to rewatering the plants and assessing their ability to attain active regrowth. Task 2: Measure the relative and absolute water content (RWC, AWC), electrolyte leakage, and photosynthetic activity of leaves at regular intervals during drying and upon rewatering and recovery.Aim 2) Conduct field trials to assess the relative biomass productivity and seed yield of three Eragrostis species that differ in their drought-stress tolerance under three different irrigated growing conditions in northern Nevada.Task 1: Determine the relative biomass productivity and seed yield for three Eragrostis species in response to three different irrigation regimes.Treatments and Experimental Design. Eragrostis tef var. 'Tiffany', Eragrostis curvula, and Eragrostis nindensis will be evaluated under three irrigation regimes [100, 75, and 50% of reference evapotranspiration (ET)] after the plants have fully emerged (100% ground cover) in a 3 × 3 factorial run as a pseudo-randomized complete block design experiment with four replications of each treatment combination.Plot Establishment and Management. Plot sizes will be ~6 × 1.5 m, with each plot separated by 1-m alleyways and 2-m alleyways between each block.Data Collection. Data that will be collected include: plant density/plant emergence, plant height, lodging severity, days to flowering, days to maturity, branches plant-1, seed heads per plant-1, panicle length, seed yield, seed weight, crop biomass, weed population, weed dry biomass, and light interception.Task 2: Determine the grazing frequency effects on productivity for three Eragrostis species in northern NevadaTreatments and Experimental Design. Eragrostis tef var. 'Tiffany', Eragrostis curvula, and Eragrostis nindensis will be evaluated for productivity under different grazing frequencies based on herbage accumulation and nutritive value.Plot Establishment and Management. Plots will be ~5 × 3.5 m in size with each plot separated by 1-m alleyways and 3?m alleyways between each block.Data Collection. Data that will be collected include: herbage accumulation (HA), sward height, forage nutritive value, weed density (botanical composition), morphological characteristics, and light interception.Task 3: Determine the harvest frequency, stubble height, and nitrogen application rate effects on forage component responses of three tef species grown as hay crops in Nevada.Treatments and Experimental Design. Eragrostis tef var. 'Tiffany', Eragrostis curvula, and Eragrostis nindensis will be evaluated for productivity and nutritive value grown as hay crops under different harvest frequencies, stubble heights, and N application rates.Plot Establishment and Management. Plot size will be ~5 × 1.5 m with each plot separated by 1-m alleyways and 2-m alleyways between each block.Data Collection. The data that will be collected for this experiment include: forage harvested, samples for forage nutritive value, plant height, morphological characteristics, weed population (botanical composition), and light interception data.Statistical Analysis. The response variables for both experiments will be analyzed by fitting mixed models using PROC MIXED in SAS.Aim 3) Generate and evaluate mutant collections for plants with reduced stature and thus, reduced susceptibility to lodging and greater harvest indices. The approach will involve generating induced mutant (e.g., by EMS) collections in the Eragrostis tef var. 'Tiffany' genetic background and screening this population for short-statured varieties. The hypothesis to be tested is that natural variation for stature is relatively low in existing tef germplasm collections, and that induced mutations will be needed to create sufficient genetic variation to allow selection of altered gibberellin-synthesis or signaling genes and thereby allow identification and isolation of short-statured lines that are more resistant to lodging by phenotypic screening. To facilitate screening of this EMS-mutant population, pooled DNA samples will be collected and screened for candidate mutations in target dwarfing genes known to reduce plant stature.Task 1: Generation of EMS-mutant collection of Eragrostis tef var. 'Tiffany'. The chemical mutagen ethyl methanesulfonate (EMS) will be used to create a mutant population with greater sequence diversity from which to isolate mutant alleles of dwarfing genes. Task 2: Phenotypic screening of EMS-mutant collection of Eragrostis tef var. 'Tiffany'. Approximately 20,000 M1 plants will be grown out in one-liter pots under greenhouse conditions and under field conditions at the Valley Road field laboratory starting in early June.Task 3: Genotypic screening of EMS-mutant collection of Eragrostis tef var. 'Tiffany'. The draft genome and transcriptome of E. tef has been published (Cannarozzi et al., 2014). Therefore, the most efficient way to identify useful sequence polymorphisms within target dwarfing genes that could overcome lodging is to resequence target loci within the EMS-mutagenized population using high-throughput next-generation DNA sequencing.Aim 4) Develop elite E. tef varieties with reduced lodging and greater seed yields (harvest indices) by introducing a mutant, dominant-negative gibberellin response modulator. The approach will be to introduce a mutant dominant-negative version of the Reduced height-1 (Rht-1a and Rht-1b) genes from tef, which have deletions or stop codons in the N-terminal region of protein-encoding gene, into Eragrostis tef var. 'Tiffany' by Agrobacterium-mediated transformation. These Della domain-containing proteins function in gibberellin-mediated plant growth regulation (Peng et al., 1999) and are expected to result in plants with reduced stature and improved harvest index.Task 1: Design, construction, and introduction of dominant-negative dwarfing genes into tef.Dominant-negative versions of the Reduced height-1 (Rht-1a and Rht-1b) genes from tef (Smith et al., 2012) will be created to generate dwarf plants that exhibit reduced gibberellin response.Task 2: Screening for dominant-negative dwarfing transformed tef lines. At least 20 independent transgenic plants will be identified for each construct tested, transferred to soil and grown under greenhouse conditions.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The project results and outcomes are being targeted to the scientific community at local, regional, national and international scientific meetings, specifically to research scientists and undergraduate and graduate students and post-doctoral researchers conducting research on alternative crops for arid lands. However, we would also like to introduce Eragrostis tef (tef) to the lay public and local farmers, stakeholders and the general public to foster access to the details of the research project as they develop. Therefore, a project website called "Improving Tef: A forage, fodder, and highly nutritious, low-gluten grain crop" was created (http://naes.unr.edu/teff/) that targets scientists and the general public. This website features a general description of the project, the major project goals, key results, lead personnel, events, and contact information. Changes/Problems:Changes: Aim 3) Generate and evaluate mutant collections for plants with reduced stature and thus, reduced susceptibility to lodging and greater harvest indices. This aim will not be initiated. Instead, efforts will be focused on direct mutagenesis strategies using CRISPR/Cas9 genome editing strategies. Problems: Agrobacterium-mediated (and biolistics) transformation of embryogenic callus E. tef have been reported. Initial attempts to develop embryogenic callus from explants of the E. tef genotype from which the reference genome was derived, the Tsedey cultivar (DZ-Cr-37), was heavily contaminated with fungi and could not be used for transformation and regeneration trial. An alternative, high performing accession has now been selected instead for these experiments. Weed infestation in the field was a major problem during the growing season even after herbicide application. Weed abatement measures are ongoing to reduce the weed seed loading on these plots. Fall armyworm decimated the grain component of all the experimental plots. Therefore, pesticide application will occur at regular intervals prior to and during anthesis. Application will be performed at night when the army worms are active. What opportunities for training and professional development has the project provided?Dr. John Cushman (Department of Biochemistry and Molecular Biology, UNR) serves as PI on this project along with Co-PI Juan Solomon (Department of Agriculture, Nutrition, and Veterinary Sciences). During the current reporting period, three graduate students (Mitiku Mengistu, Dhurba Neupane, and Christina Igono) were trained. Also, four undergraduate students (Austin Zell, Keisha Swift, Thomas Iida, and Hailey Hermann) received research training under this project. How have the results been disseminated to communities of interest?Scientific poster presentations: Iida T, Mengistu MA, and Cushman JC. Phenotypic analysis of Eragrostis tef (Zucc.) to improve grain and forage production. Nevada Undergraduate Research Symposium. April 20, 2017. Reno, NV. Iida T, Mengistu MA, and Cushman JC. Genetic analysis and inter-irrigation interval determination of drought tolerant lines of Eragrostis tef (Zucc.). Summer Undergraduate Research Symposium. August 9, 2017. Reno, NV. Mengistu MA, Iida T, Hermann H, Thrift K, Cushman JC. Phenotypic diversity analysis and development of semi-dwarf Eragrostis tef mutant lines using CRISPR/Cas9 genome-editing technology. Molecular Biosciences Graduate Student Retreat, August 25, 2017. Reno, NV. Greenhouse tour featuring Tef Research Project: "VentureBeats Group", February 24, 2017, 20 participants. Greenhouse tour featuring Tef Research Project: "GEOL 407: Earth resources and Energy Class", April 13, 2017, 30 students. Greenhouse tour featuring Tef Research Project: "CABNR FIT", August 21-23, 2017, 90 students. Greenhouse tour featuring Tef Research Project: "UNR Foundation Planned Giving Council", October 25, 2017, 30 participants. Field tour featuring Tef Research Project: UNR undergraduates, September 15, 2017. 18 participants. Nevada Field Day: September 30, 2017. Many participants visited our booth for explanation on our work pertaining to Tef evaluation. Leveraged Research Funds: Thomas Iida (Cushman Lab) - Nevada Undergraduate Research Award (NURA) proposal awarded to undergraduate student Thomas Iida for the 2016-2017 Academic Year: Determining the Drought Tolerance and Forage Quality of Desiccation-tolerant Tef Grass. Amount: $1500. Mitiku Mengistu (Cushman Lab) - Borlaug LEAP Fellowship awarded to graduate student Mitiku Mengistu for the 2016-2017 Academic Year: Improving drought tolerance and lodging in tef grass: A forage, fodder, and highly nutritious, gluten-free grain crop. Amount: $19,559. Thomas Iida (Cushman Lab) - Nevada Undergraduate Research Award (NURA) proposal and EPSCOR Undergraduate Research Opportunity (UROP) proposal awarded to undergraduate student Thomas Iida for the 2017-2018 Academic Year and 2017 Summer, respectively: Eragrostis Tef Chronic Water Stress Screening, Genotypic Analysis, and Root Formation. Amount: $4,750 Project website: The project website was created in 2017 using word press and can be accessed at: https://naes.unr.edu/teff/. The following impacts will be summarized there. Project impacts to date: Preliminary analysis (descriptive) indicates that there is a wide variation for plant height, panicle length and form, floret distribution across the panicle, fresh above-ground biomass, dry above-ground biomass, grain yield, and seed color varies widely among the 367 Eragrostis tef accessions when grown under greenhouse conditions. The existence of such variability among the accessions is critical for the improvement of Tef's productivity and adaptability in the drier areas. This information will be used to select for high-yielding accessions for replicated field trials for human food and animal forage. Grain yield was found to be related to panicle morphology. Five different panicle morphologies were recognized (e.g., very loose, loose, semi-loose/semi-compact, compact, and very compact). Grain yield was highest in very loose and loose panicle types. Most accessions had either very loose or loose panicle forms. Regardless of the panicle type, average spiklets/floret were always higher in the top of the panicle. Six (6) accessions with enhanced drought tolerance were identified relative to a control variety used for commercial production in Nevada. Four plastid gene markers (rbcL, matK, psaB, and trnK) were isolated, amplified, and sequenced from four drought-tolerant Eragrostis tef accessions, Eragrostis curvula (drought tolerant control line), and cultivar Dessie (drought susceptible control line). Biomass production differed among Eragrostis tef entries (accessions and cultivars) in 2017. The two commercial cultivars evaluated did not produce greater biomass than any of the nine accessions during this first year of data collection. This information will assist in the selection process of high-yielding accessions for breeding development for human food (grain) and animal feed (biomass) for local and regional producers. Some high-yielding accessions were extremely sensitive to water deficit based upon field observation. Such information will be essential for the identification of potential drought tolerant lines suitable for Nevada growing conditions. A total of 98 accessions did not grow or had extremely limited harvestable material during this evaluation leading us to speculate that moisture deficits in those areas of the field may have led to this trend. CRISPR/Cas9 sgRNAs were successfully designed and cloned for two target genes involved in regulating plant stature including the dominant-negative gibberellin response modulator Reduced height (Rht-1a and Rht-1b) genes and GA20 oxidase (Sd-1a, Sd-1b, and Sd-1c) genes. High quality protoplasts were isolated (for the rapid evaluation of CRISPR/Cas9 constructs before the actual transformation of Eragrostis tef calli) from Eragrostis tef mesophyll cells using an optimized protocol. What do you plan to do during the next reporting period to accomplish the goals?Aim 1) Determine the relative drought and dehydration tolerance of three Eragrostis species that differ in their ability to withstand prolonged periods of water-deficit stress. The relative drought or desiccation tolerance of Eragrostis tef var. 'Dessie' and 'Tiffany', and the six drought tolerant accessions will now be reevaluated for growth rates, seed yield, and overall height and biomass parameters under field conditions (see Aim 2). To assess drought/dehydration tolerance mechanisms, accessions will be reevaluated carefully under greenhouse conditions for relative and absolute water content (RWC, AWC), electrolyte leakage, and photosynthetic activity of leaves at regular intervals under acute and chronic water-deficit stress to assess rates and limits of water loss, leaf damage, and photosynthetic capacity. Aim 2) Conduct field trials to assess the relative biomass productivity and seed yield of three Eragrostis species that differ in their drought-stress tolerance under three different irrigated growing conditions in northern Nevada. The top 30 best-performing accessions of the USDA E. tef germplasm collection will be reevaluated under field conditions during the 2018 growing season. In addition, selected lines will be evaluated under three irrigation regimes [100, 75, and 50% of reference evapotranspiration (ET)] after the plants have fully emerged (100% ground cover) in a 3 × 3 factorial experiment conducted as a pseudo-randomized complete block design experiment with four replications of each treatment combination. Plot sizes will be ~6 × 1.5 m, with each plot separated by 1-m alleyways and 2-m alleyways between each block. Data to be collected include: plant density/plant emergence, plant height, lodging severity, days to flowering, days to maturity, branches plant-1, seed heads per plant-1, panicle length, seed yield, seed weight, crop biomass, weed population, weed dry biomass, and light interception. Selected Eragrostis tef accessions will also be evaluated for productivity under different grazing frequencies based on herbage accumulation and nutritive value, productivity and nutritive value grown as hay crops under different harvest frequencies, stubble heights, and N application rates. Aim 4) Develop elite E. tef varieties with reduced lodging and greater seed yields (harvest indices) by introducing a mutant, dominant-negative gibberellin response modulator. For CRISPR/Cas9 genome editing of the two target genes involved in regulating plant stature (dominant-negative gibberellin response modulator Reduced height (Rht-1a and Rht-1b) genes and GA20 oxidase (Sd-1a, Sd-1b, and Sd-1c) genes), mutagenesis frequencies at target sites will be tested in protoplasts prior to the creation of stable transformation lines. In order to reduce or avoid regulatory impediments associated with the deployment of transgenic plants here in the US and in Africa, Agrobacterium-mediated transformation approaches will be avoided. Instead, CRISPR/Cas9 constructs will be introduced using protoplast or Biolistics transformation approaches using embryogenic callus using explants from immature embryos (Bebre et al., (2013) South African Journal of Botany. 87: 9-17). Regeneration trials of E. tef from protoplasts and embryogenic callus cultures are also in progress.

Impacts
What was accomplished under these goals? Aim 1) Determine the relative drought and dehydration tolerance of three Eragrostis species that differ in their ability to withstand prolonged periods of water-deficit stress. USDA Eragrostis tef germ plasm collection containing 367 accessions (mainly from Ethiopia) obtained from Vicki Bradley (USDA-ARS Western Region Plant Introduction Station, Washington State University, Pullman, WA) was screened in triplicate under greenhouse conditions in pots. The following data were collected: plant height, panicle (inflorescence) length, panicle form, distribution of florets across the panicle, fresh above-ground biomass, dry above-ground biomass, grain yield, seed color. A fourth replication of this study was completed and the results are being evaluated in the greenhouse, harvesting and data collection to be completed soon. Acute drought stress: A total of six (6) accessions with notable drought tolerance were subjected to acute water-deficit stress analysis using replicates (n = 48). E. tef variety "Dessie" (William's love grass) was included as a drought-sensitive control accession. One hundred-day-old plants were subjected to multiple weeks of acute water-deficit stress and then rewatered and grown to maturity. The relative drought tolerance of the accessions was then ranked to discover which lines had the greatest ability to withstand acute water-deficit stress as determined by dry weight biomass accumulation and seed production. Chronic drought stress: A total of six (6) accessions with notable drought tolerance were subjected to chronic water-deficit stress analysis using replicates (n = 48). E. tef variety "Dessie" (William's love grass) was included as a drought-sensitive control accession. Sixty-day-old plants were subjected to chronic water-deficit stress by watering the plants at 100%, 75%, 50%, 25%, and 0% soil holding capacity and grown to maturity. The relative drought tolerance to this chronic water-deficit stress of the accessions was then ranked to discover which line had the greatest ability to withstand chronic water-deficit stress as determined by dry weight biomass accumulation and seed production. In order to evaluate the phylogenetic relationships of those drought tolerant tef accessions with susceptible genotype (Eragrostis tef variety Dessie) and a highly drought-tolerant species (Eragrostis curvula), four plastid marker genes were identified for use including rbcL, psaB, matK, and trnK. The marker genes were amplified from the drought tolerant tef accessions and sent for sequencing. Aim 2) Conduct field trials to assess the relative biomass productivity and seed yield of three Eragrostis species that differ in their drought-stress tolerance under three different irrigated growing conditions in northern Nevada. Grain and biomass production of Tef accessions and cultivars screened under two nitrogen application rates: This study was carried out at the UNR Main Station Field Lab in 2017 using nine of the highest yielding tef [Eragrostis tef (Zuccagni)] accessions, based on grain and biomass yield under greenhouse evaluation, and two commercially available cultivars (Dessie and Tiffany). Accessions were seeded at a rate of 5 kg ha-1 on June 14, 2017. This experiment was a split-plot arrangement of a randomized complete block design with four replications. Treatments were the nine accessions and two cultivars as whole plot and N rates (80 and 120) as subplots. Three weeks after germination, N fertilizer at the two rates (80 and 120 kg N ha-1) was applied using urea (46-0-0) by hand. Data collected were mid-season leaf area index, SPAD chlorophyll index, samples for nutritive value, and end of season plant height, grain, and biomass yield (harvest area = 2.8 m2). Collection of grain yield was not possible because fall armyworm (Spodoptera frugiperda) decimated the grain component in the entire study. Data were analyzed using PROC MIXED of SAS. In 2017, Tef biomass production was different (P = 0.0229) among entries, but N application rate or interaction with Entry did not affect (P = 0.2581) biomass production. Forage dry matter yield was similar for entries PI 494286, PI 494289, PI 494349, PI 494369, and PI 494456 (average yield = 9214 kg DM ha-1). Entry PI 494456 (9955 kg DM ha-1) produced greater biomass than PI 195936, PI 494348, PI 494351, PI 494448, Dessie and Tiffany (average = 7875 kg DM ha-1). Entry PI 195936 (7417 kg DM ha-1) produced less biomass than entries PI 494286, PI 494289, PI 494349, and PI 494369 (average = 9028 kg DM ha-1). The two commercial cultivars did not produce greater biomass than any of the accessions during this first year of data collection. Nitrogen application rate did not influence biomass production in this study. Aim 3) Generate and evaluate mutant collections for plants with reduced stature and thus, reduced susceptibility to lodging and greater harvest indices. This aim has not yet been initiated and is being replaced by CRISPR/Cas9 approaches instead. Nothing to report. Aim 4) Develop elite E. tef varieties with reduced lodging and greater seed yields (harvest indices) by introducing a mutant, dominant-negative gibberellin response modulator. In order to reduce plant stature to lessen yield losses due to lodging, CRISPR/Cas9 constructs were designed that target genes involved in regulating plant stature including dominant-negative gibberellin response modulator (Reduced height (Rht-1a and Rht-1b) genes and GA20 oxidase (Sd-1a, Sd-1b, and Sd-1c) genes, which impair GA biosynthesis. Three guide RNAs were designed for each gene by careful alignment and evaluation of multiple gene copies for each gene due to the allotetraploid nature of the E. tef genome. sgRNAs were synthesized for each gene and were successfully cloned into sgRNA vectors for expression under the control of the wheat U6 promoter. This CRISPR/Cas9 genome editing approach is being used to avoid regulatory impediments associated with the deployment of transgenic plants here in the US and in Africa created by Agrobacterium-mediated transformation approaches. Growth conditions and protoplast preparation for E. tef were optimized for PEG-mediated transformation of protoplast in order to introduce and rapidly test the efficacy of the CRISPR/Cas9 constructs. In addition, regeneration trials of E. tef from transfected protoplasts and Agrobacterium-mediated or Biolistics®-mediated transformation of various explant tissues will be initiated shortly.

Publications


    Progress 10/01/15 to 09/30/16

    Outputs
    Target Audience:The project results and outcomes are being targeted to the scientific community at national and international scientific meetings, specifically to research scientists and undergraduate and graduate students and post-doctoral researchers conducting research on alternative crops for arid lands. However, we would also like to introduce Eragrostis tef (tef) to the lay public and local farmers, stakeholders and the general public to foster access to the details of the research project as they develop. Therefore, a project website was created (http://naes.unr.edu/teff/) that targets scientists and the general public. This website features a general description of the project, the major project goals, key results, lead personnel, events, and contact information. Changes/Problems:Changes: Aim 3) Generate and evaluate mutant collections for plants with reduced stature and thus, reduced susceptibility to lodging and greater harvest indices. This aim will not be initiated. Instead, efforts will be focused on direct mutagenesis strategies using CRISPR/Cas9 genome editing strategies. Problems: Agrobacterium-mediated (and biolistics) transformation of embryogenic callus E. tef have been reported. If transformation cannot be performed with high efficiency, then the progress in generating transgenic lines might be delayed. Protoplast transformation and regeneration approaches using E. tef have not been reported. If these steps cannot be performed with high efficiency, then progress in the generation of CRISPR/Cas9 knockout lines might be delayed. Weed infestation in the field was a major problem during the growing season even after herbicide application. Because of the number of accessions evaluated, more manual inputs are needed for best plot management practices. Seed threshing equipment (purchase in progress) and repairs to Wiley mills are vital for sample preparation for nutritive value analysis. What opportunities for training and professional development has the project provided?Dr. John Cushman (Department of Biochemistry and Molecular Biology, UNR) serves as PI on this project along with Co-PI Juan Solomon (Department of Agriculture, Nutrition, and Veterinary Sciences). During the current reporting period, three graduate students (Mitiku Mengistu, Dhurba Neupane, and Christina Igono) were trained. Also, four undergraduate students (Austin Zell, Keisha Swift, Thomas Iida, and Hailey Hermann) received research training under this project. Nevada Undergraduate Research Award (NURA) proposal awarded to undergraduate student Thomas Iida for the 2016-2017 Academic Year: Determining the Drought Tolerance and Forage Quality of Desiccation-tolerant Tef Grass. Amount: $1500. Borlaug LEAP Fellowship awarded to graduate student Mitiku Mengistu for the 2016-2017 Academic Year: Improving drought tolerance and lodging in tef grass: A forage, fodder, and highly nutritious, gluten-free grain crop. Amount: $19,559. How have the results been disseminated to communities of interest?Scientific poster presentation: Mengistu, MA, Solomon JKQ, and Cushman JC. Improving Tef Grass: A forage, fodder, and highly nutritious, gluten-free grain crop. Molecular Biosciences Graduate Student Retreat, August 26, 2016. Reno, NV. Lay poster presentation: Mengistu M, Neupani D, Igolo C, Solomon J, Davison J, Oliver MJ, Cushman JC. Tef grass: A forage, fodder, and highly nutritious, low-gluten grain crop. Nevada Agricultural Experiment Station, Ag Field Day, September 17, 2016. Reno, NV. Greenhouse tour featuring Tef Research Project: "New Comers Club", May 17, 2016, 40 participants. Greenhouse tour featuring Tef Research Project: "New Graduate Student Orientation Lab Tour (Graduate Association of the Biosciences (GAB))", August 24, 2016, 15 participants. Greenhouse tour featuring Tef Research Project: "DOE Officials Lab Tour- Food-Energy-Water Nexus", October 12, 2016, 12 participants. What do you plan to do during the next reporting period to accomplish the goals?Aim 1) Determine the relative drought and dehydration tolerance of three Eragrostis species that differ in their ability to withstand prolonged periods of water-deficit stress. The relative drought or desiccation tolerance of Eragrostis tef var. 'Dessie' and 'Tiffany', six drought tolerance accessions, and the desiccation tolerant species will be reevaluated for growth rates, seed yield, and overall height and biomass parameters under greenhouse conditions. These species and accessions will also be reevaluated under field production conditions (see Aim 2). To assess drought/dehydration tolerance and inter-irrigation survival interval, plants will be evaluated for relative and absolute water content (RWC, AWC), electrolyte leakage, and photosynthetic activity of leaves at regular intervals during acute drying and upon rewatering and recovery and following chronic water-deficit stress at fixed irrigation levels to assess rates and limits of water loss, leaf damage, and photosynthetic capacity. Aim 2) Conduct field trials to assess the relative biomass productivity and seed yield of three Eragrostis species that differ in their drought-stress tolerance under three different irrigated growing conditions in northern Nevada. The top 30 best-performing accessions of the USDA E. tef germplasm collection will be reevaluated under field conditions during the 2017 growing season. In addition, selected lines will be evaluated under three irrigation regimes [100, 75, and 50% of reference evapotranspiration (ET)] after the plants have fully emerged (100% ground cover) in a 3 × 3 factorial run as a pseudo-randomized complete block design experiment with four replications of each treatment combination. Plot sizes will be ~6 × 1.5 m, with each plot separated by 1-m alleyways and 2-m alleyways between each block. Data to be collected include: plant density/plant emergence, plant height, lodging severity, days to flowering, days to maturity, branches plant-1, seed heads per plant-1, panicle length, seed yield, seed weight, crop biomass, weed population, weed dry biomass, and light interception. Selected Eragrostis tef accessions will also be evaluated for productivity under different grazing frequencies based on herbage accumulation and nutritive value, productivity and nutritive value grown as hay crops under different harvest frequencies, stubble heights, and N application rates. Aim 4) Develop elite E. tef varieties with reduced lodging and greater seed yields (harvest indices) by introducing a mutant, dominant-negative gibberellin response modulator. Following the successful cloning of CRISPR/Cas9 sgRNAs for two target genes involved in regulating plant stature (dominant-negative gibberellin response modulator Reduced height (Rht-1a and Rht-1b) genes and GA20 oxidase (Sd-1a, Sd-1b, and Sd-1c) genes), the next step will be to test the mutagenesis frequencies at target sites in protoplasts prior to the creation of stable transformation lines. In order to reduce or avoid regulatory impediments associated with the deployment of transgenic plants here in the US and in Africa, Agrobacterium-mediated transformation approaches will be avoided. Instead, CRISPR/Cas9 constructs will be introduced using protoplast or Biolistics transformation approaches using embryogenic callus using explants from immature embryos (Bebre et al., (2013) South African Journal of Botany. 87: 9-17). Regeneration trials of E. tef from protoplasts and embryogenic callus cultures are in progress.

    Impacts
    What was accomplished under these goals? Aim 1) Determine the relative drought and dehydration tolerance of three Eragrostis species that differ in their ability to withstand prolonged periods of water-deficit stress. USDA Eragrostis tef germ plasm collection containing 367 accessions (mainly from Ethiopia) obtained from Vicki Bradley (USDA-ARS Western Region Plant Introduction Station, Washington State University, Pullman, WA) was screened in triplicate under greenhouse conditions in pots. The following data were collected: plant height, panicle (inflorescence) length, panicle form, distribution of florets across the panicle, fresh above-ground biomass, dry above-ground biomass, grain yield, and seed color. Four short statured accessions of Eragrostis tef and variety "Dessie" (a red-seeded variety commonly known as William's love grass, moderately drought tolerant) were screened by flow cytometry for genome size, which ranged from 690-743 Mb. No diploid accessions were identified within the USDA-ARS collection. The USDA E. tef germ plasm collection was screened for drought or desiccation tolerance under greenhouse growing conditions. E. tef variety "Dessie" (Williams love grass, moderately drought tolerant) was included as a drought-sensitive control accession. Two-month-old plants after planting were subjected to chronic water-deficit stress by withholding water for 90 days. Plants were then rewatering for 14 days and then evaluated for survival and regrowth. Water potential was tracked during the dry-down period until plants reached air dry conditions. A total of six (6) accessions with notable drought tolerance were identified. Seeds for Eragrostis curvula (weeping love grass, drought tolerant) were purchased from two different commercial sources for future evaluation. Eragrostis nindensis (strong drought- and poikilochlorophyllous desiccation-tolerant) was requested from Jill Farrant. A total of six (6) accessions with notable drought tolerance were subjected to repeated analysis using replicates (n = 10). E. tef variety "Dessie" (Williams love grass, moderately drought tolerant) was included as a drought-sensitive control accession. Sixty day-old plants were subjected to 70 days of chronic water-stress. Water potential and chlorophyll retention were tracked during the dry-down period under plants reached air dry conditions. The relative drought tolerance of these selected accessions was then ranked. Aim 2) Conduct field trials to assess the relative biomass productivity and seed yield of three Eragrostis species that differ in their drought-stress tolerance under three different irrigated growing conditions in northern Nevada. USDA Eragrostis tef germ plasm collection containing 367 accessions was grown under field conditions in individual small plots (0.9 m long by 0.6 m wide) seeded on June 10, 2016. Because of the quantity of seed available, one replication of each accession was arranged in a completely randomized design experiment. Fertilizer N was applied at a rate of 80 kg N ha-1. Plots were irrigated using a water-reel three times per week during the first 60 days and thereafter, twice per week for the next 60 days after which no supplemental irrigation was done. Plots were harvested on October 24, 2016 (137 days from seeding to harvest). Data collected were seed quantity per plot, biomass, and samples for nutritive value evaluation. Only biomass production data is reported thus far because manual seed cleaning will take a considerable amount of time for all the tef accessions and grinding mill is needed for sample preparation for nutritive value analysis. Aim 3) Generate and evaluate mutant collections for plants with reduced stature and thus, reduced susceptibility to lodging and greater harvest indices. This aim has not yet been initiated and is being replaced by CRISPR/Cas9 approaches instead. Nothing to report. Aim 4) Develop elite E. tef varieties with reduced lodging and greater seed yields (harvest indices) by introducing a mutant, dominant-negative gibberellin response modulator. In order to reduce plant stature to lessen yield losses due to lodging, CRISPR/Cas9 constructs were designed that target genes involved in regulating plant stature including dominant-negative gibberellin response modulator (Reduced height (Rht-1a and Rht-1b) genes and GA20 oxidase (Sd-1a, Sd-1b, and Sd-1c) genes, which impair GA biosynthesis. Three guide RNAs were designed for each gene by careful alignment and evaluation of multiple gene copies for each gene due to the allotetraploid nature of the E. tef genome. sgRNAs were synthesized for each gene and were successfully cloned into sgRNA vectors for expression under the control of the wheat U6 promoter. This CRISPR/Cas9 genome editing approach is being used to avoid regulatory impediments associated with the deployment of transgenic plants here in the US and in Africa created by Agrobacterium-mediated transformation approaches. Growth conditions and protoplast preparation for E. tef are being optimized in anticipation of PEG-mediated transformation of protoplast in order to introduce and rapidly test the efficacy of the CRISPR/Cas9 constructs. In addition, regeneration trials of E. tef from transfected protoplasts and Agrobacterium-mediated or Biolistics®-mediated transformation of various explant tissues will be initiated shortly. Project impacts to date: Preliminary analysis (descriptive) indicates that there is a wide variation for plant height, panicle length and form, floret distribution across the panicle, fresh above-ground biomass, dry above-ground biomass, grain yield, and seed color varies widely among the 367 Eragrostis tef accessions when grown under greenhouse conditions. The existence of such variability among the accessions is critical for the improvement of Tef's productivity and adaptability in the drier areas. This information will be used to select for high-yielding accessions for advanced field trials for human food and animal forage. Data will be subjected to various statistical analyses including analysis of variance, correlation, clustering, and principal component analysis. All the tested accessions were found to be allotetraploids. No diploid accessions of Eragrostis tef have been identified to date. Grain yield was found to be related to panicle morphology. Five different panicle morphologies were recognized (e.g., very loose, loose, semi-loose/semi-compact, compact, and very compact). Grain yield was highest in very loose and loose panicle types. Most accessions have either very loose or loose panicle forms. Regardless of the panicle type, average spiklets/floret were always higher in the top of the panicle. Six (6) accessions with enhanced drought tolerance were identified relative to a control variety used for commercial production in Nevada. Biomass production among Eragrostis tef accessions was observed to vary widely under field conditions and this information will assist in the selection process of high-yielding accessions for advanced field for human food and animal forage. The highest yielding 32 accessions averaged 1.2 ± 0.30 kg DM m-2. This value is from replication 1 only, and may change when the values of replication 2 and 3 are combined. Some accessions were extremely sensitive to moisture deficit based upon field observation. Such information will be essential for the identification of potential drought tolerant lines suitable for Nevada growing conditions. A total of 98 accessions did not grow or had extremely limited harvestable material during this evaluation leading us to speculate that moisture deficits in those areas of the field may have led to this trend. CRISPR/Cas9 sgRNAs were successfully designed and cloned for two target genes involved in regulating plant stature including the dominant-negative gibberellin response modulator Reduced height (Rht-1a and Rht-1b) genes and GA20 oxidase (Sd-1a, Sd-1b, and Sd-1c) genes.

    Publications

    • Type: Theses/Dissertations Status: Accepted Year Published: 2016 Citation: Keisha Thrift. Biochemistry and Molecular Biology Senior Thesis. Drought Tolerance Testing and Biomass Analysis of Eragrostis tef (Zucc.) Trotter Accession to Determine Potential Grain and Forage Production. May 2016.
    • Type: Websites Status: Published Year Published: 2016 Citation: Improving Tef: A forage, fodder, and highly nutritious, low-gluten grain crop


    Progress 07/01/15 to 09/30/15

    Outputs
    Target Audience:The project results and outcomes are being targeted to the scientific community at national and international scientific meetings, specifically to research scientists and undergraduate and graduate students and post-doctoral researchers conducting research on alternative crops for arid lands. However, we would also like to introduce Eragrostis tef (tef) to the lay public and local farmers, stakeholders and the general public to foster access to the details of the research project as they develop. Therefore, a project website is currently under development (http://naes.unr.edu/tef/) and targets scientists and the general public. This website will feature a general description of the project, the major project goals, key results, lead personnel, events, and contact information. Changes/Problems:Changes: Aim 3) Generate and evaluate mutant collections for plants with reduced stature and thus, reduced susceptibility to lodging and greater harvest indices. This aim will not be initiated. Instead, efforts will be focused on direct mutagenesis strategies using CRISPR/Cas9 genome editing strategies. Problems: Agrobacterium-mediated and biolistics transformation of embryogenic callus E. tef have been reported. If transformation cannot be performed with high efficiency, then the progress in generating transgenic lines might be delayed. Protoplast transformation and regeneration approaches using E. tef have not been reported. If these steps cannot be performed with high efficiency, then progress in the generation of CRISPR/Cas9 knockout lines might be delayed. What opportunities for training and professional development has the project provided?Dr. John Cushman (Department of Biochemistry and Molecular Biology, UNR) serves as PI on this project along with Co-PI Juan Solomon (Department of Agriculture, Nutrition, and Veterinary Sciences). During the current reporting period, three graduate students (Mitiku Mengistu, Dhurba Neupane, and Christina Igono) were trained. Also, two undergraduate students (Austin Zell and Keisha Swift) received research training under this project. How have the results been disseminated to communities of interest?As this project was initiated only recently, no results have yet be disseminated to the community; however, a lay poster presentation is under development. What do you plan to do during the next reporting period to accomplish the goals?Aim 1) Determine the relative drought and dehydration tolerance of three Eragrostis species that differ in their ability to withstand prolonged periods of water-deficit stress. The relative drought or desiccation tolerance of Eragrostis tef var. 'Dessie' and 'Tiffany' and the desiccation tolerance accession will be evaluated for growth rates, seed yield, and overall height and biomass parameters under greenhouse conditions. The three species will also be evaluated under field production conditions (see Aim 2). To assess drought and dehydration tolerance, inter-irrigation survival interval, plants will be evaluated for relative and absolute water content (RWC, AWC), electrolyte leakage, and photosynthetic activity of leaves at regular intervals during drying and upon rewatering and recovery to assess rates and limits of water loss, leaf damage, and photosynthetic capacity. Aim 2) Conduct field trials to assess the relative biomass productivity and seed yield of three Eragrostis species that differ in their drought-stress tolerance under three different irrigated growing conditions in northern Nevada. All 367 accessions of the USDA E. tef germplasm collection will be evaluated again under both greenhouse and field conditions during the 2016 growing season. In addition, selected lines will be evaluated under three irrigation regimes [100, 75, and 50% of reference evapotranspiration (ET)] after the plants have fully emerged (100% ground cover) in a 3 × 3 factorial run as a pseudo-randomized complete block design experiment with four replications of each treatment combination. Plot sizes will be ~6 × 1.5 m, with each plot separated by 1-m alleyways and 2-m alleyways between each block. Data that will be collected include: plant density/plant emergence, plant height, lodging severity, days to flowering, days to maturity, branches plant-1, seed heads plant-1, panicle length, seed yield, seed weight, crop biomass, weed population, weed dry biomass, and light interception. Selected Eragrostis tef accessions will also be evaluated for productivity under different grazing frequencies based on herbage accumulation and nutritive value, productivity and nutritive value grown as hay crops under different harvest frequencies, stubble heights, and N application rates. Aim 4) Develop elite E. tef varieties with reduced lodging and greater seed yields (harvest indices) by introducing a mutant, dominant-negative gibberellin response modulator. In order to accomplish this aim, three different sgRNAs will be designed for each target gene manually and validated using CRISPR-PLANT and CasOT web-based tools. Target sites with restriction sites will be targeted to facilitate detection of mutated sites and to estimate mutagenesis frequencies in protoplasts prior to the creation of stable transformation lines. pJIT163-2NLSCas9 and pAct1-HPT vectors along with pOsU3-SgRNA and pTaU6-sgRNA vectors have been obtained from AddGene. In order to reduce or avoid regulatory impediments associated with the deployment of transgenic plants here in the U.S. and in Africa, Agrobacterium-mediated transformation approaches will be avoided. Instead, CRISPR/Cas9 constructs will be introduced using protoplast or Biolistics® transformation approaches using embryogenic callus using explants from immature embyros (Bebre et al., (2013) South African Journal of Botany. 87: 9-17). Thus, regeneration trials of E. tef from protoplasts and embryogenic callus cultures will be initiated shortly.

    Impacts
    What was accomplished under these goals? Aim 1) Determine the relative drought and dehydration tolerance of three Eragrostis species that differ in their ability to withstand prolonged periods of water-deficit stress. Our initial approach was to compare the relative drought tolerance of Eragrostis tef var. 'Tiffany' (Williams love grass, moderately drought tolerant), Eragrostis curvula (weeping love grass, drought tolerant), and Eragrostis nindensis (strong drought- and poikilochlorophyllous desiccation-tolerant). The more drought or desiccation tolerant germplasm was represented by different species of Eragrostis, which would have required high-risk interspecific crosses and embryo rescue approaches. Ideally, the identification of a desiccation tolerant species of Eragrostis tef would streamline our future breeding efforts. Therefore, our first order of business was to screen the USDA Eragrostis tef germplasm collection containing 367 accessions (mainly from Ethiopia) for desiccation tolerance. This collection was obtained from Vicki Bradley (USDA-ARS Western Region Plant Introduction Station, Washington State University, Pullman, WA). This collection had not been screened for relative drought tolerance or ploidy level to our knowledge. Prior to screening, we evaluated E. tef variety "Dessie" (a red-seeded commercial variety with mild drought tolerance) for relative drought tolerance to determine when lethal water-deficit stress occurs. Plants were planted in pots on August 5, 2015 in the greenhouse, exposed to water-deficit stress on October 5, 2015 (2 months after planting), and evaluated for water-deficit stress tolerance. Plants were rewatered after 10, 14, 18, and 22 days of withholding water, rewatered for 7 days, and then photographed and evaluated for survival. All plants were killed after about 18 days of water-deficit stress. Next, all 367 accessions of the USDA E. tef germplasm collection were planted in pots on September 5, 2015 in the greenhouse. The plants were subjected to water-deficit stressed on November 5, 2015 (2 months after planting) and were evaluated for water-deficit stress tolerance by withholding water for 90 days. The relative water content of the plants was evaluated during the dry-down process using a Decagon Devices WP4C dewpoint hygrometer. Plants were drought stressed until the water potential of the plants attained an air-equilibrated state, which took 90 days and all plants appeared completely dry. Upon rewatering for 7 days, the collection was evaluated for survival and regrowth. Six candidate drought-tolerant lines were identified and one line displayed drought- and homeochlorophyllous desiccation-tolerance. These lines are being regrown as described above and will be subjected to further detailed analysis in the greenhouse and field. Small plants from the USDA E. tef germplasm collection were evaluated for ploidy in an effort to identify a diploid reference variety; however, all five accessions evaluated were found to be tetraploid. Aim 2) Conduct field trials to assess the relative biomass productivity and seed yield of three Eragrostis species that differ in their drought-stress tolerance under three different irrigated growing conditions in northern Nevada. All 367 accessions of the USDA E. tef germplasm collection were planted in pots on August 5, 2015 in the greenhouse under controlled conditions. The plants will be harvested on December 5, 2015 (4 months after planting), dried in drying ovens, and evaluated for biomass and seed production. Greenhouse-grown biomass ranged from 1 g to 98 g per plants dry weight. Plants were also evaluated for plant height, panicle morphology, floret number, and dry weight of vegetative biomass and seed number and weight per plant. Plant height ranged from 16 cm to 106 cm. Panicle length ranged from 14 cm to 93 cm. Number of florets per spiklet ranged from 2 to 11. In addition, all 367 accessions of the USDA E. tef germplasm collection were planted in pots on June 10, 2015 in the greenhouse and transplanted to the Valley Road field site on July 10, 2015 in a duplicated block design where each accession appeared twice in the field. The field accessions were harvested on October 30, 2015 (4 months, 10 days after planting) and are in the process of being evaluated for biomass and seed production. Thus far, field-grown biomass ranged from 68 g to 439 g per plant air-dry weight (preliminary results from those accessions weighed to date). Plant height ranged from 28 cm to 117 cm. Aim 3) Generate and evaluate mutant collections for plants with reduced stature and thus, reduced susceptibility to lodging and greater harvest indices. This aim has not yet been initiated. Nothing to report. Aim 4) Develop elite E. tef varieties with reduced lodging and greater seed yields (harvest indices) by introducing a mutant, dominant-negative gibberellin response modulator. Our initial approach was to introduce a mutant dominant-negative version of the Reduced height-1 (Rht-1a and Rht-1b) and GA20 oxidase genes. However, given the challenges associated with working with a tetraploid species such as tef and given the recent advances in CRISPR/Cas9 technologies, we have decided that the more direct approach of genome editing will be used to target these genes. In order to accomplish this aim, we have obtained single-guide (sgRNA) scaffold vectors that easily replace the ~20-nt target sequences of the sgRNA to target selected genomic sites (pOsU3-SgRNA and pTaU6-sgRNA vectors for rice and wheat, respectively) (Shan et al., 2014. Nature Protocols. 9: 2395-2410).

    Publications