Source: UNIVERSITY OF TENNESSEE submitted to
SWITCHGRASS BIOCONFINEMENT: DELAYED FLOWERING, SELECTIVE MALE- AND SEED-STERILITY, AND CONDITIONAL TOTAL BIOCONFINEMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1000875
Grant No.
2013-33522-20997
Project No.
TEN2013-03566
Proposal No.
2013-03566
Multistate No.
(N/A)
Program Code
HX
Project Start Date
Sep 1, 2013
Project End Date
Aug 31, 2017
Grant Year
2013
Project Director
Stewart, C. N.
Recipient Organization
UNIVERSITY OF TENNESSEE
2621 MORGAN CIR
KNOXVILLE,TN 37996-4540
Performing Department
Plant Sciences
Non Technical Summary
Genes that are engineered into crops typically serve the purpose of adding value to the crop or product. There remains a concern among regulators of biotechnology, some scientists and citizens, about gene flow from biotech crops into other non-engineered plants, including free-living wild plants and weeds. In a crop such as switchgrass, where wild switchgrass exists in the areas of cultivation, the gene flow concern can be addressed by engineering plants so that they do not flower, or that pollen, seed, or both are non-viable. We will test three different systems to achieve these endpoints. The first uses a gene that delays flowering 'microRNA 156,' that also increases biomass production. The second system utilizes an enzyme to degrade DNA that will be targeted to be produced specifically in pollen or seed. This DNA chopper--the restriction enzyme 'EcoRI'--has proven to render male sterility in a test plant system and thus, it will be applied to switchgrass. The third system to limit gene flow is to produce a switchable system--a 'synthetic circuit' in engineered plants. This circuit will allow plants to flower and set seed when someone wants them to reproduce, say, when breeding to produce seed to sell to farmers, but be activated to limit gene flow in production fields. The systems could be combined to increase effectiveness of gene flow limitation. Taken together these systems will advance our technological toolkit and knowledge to limit gene flow in engineered crops.
Animal Health Component
0%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20116291040100%
Goals / Objectives
Goals / Objectives The overall goal of the project is to develop systems to bioconfine transgenes into the intended host of switchgrass. Specific objectives of the project include the following: 1. Delayed or no flowering. Switchgrass plants engineered for moderate overexpression of microRNA156 (miR156), which renders delayed or no flowering, will be field-tested. 2. Male- and seed-sterility. A restriction endonuclease, EcoRI, will be specifically expressed in pollen or seed tissues and the efficacy for ablating target cells will be assessed, first in rice as a grass model and then translated to switchgrass. 3. Conditional total transgene bioconfinement. A novel system in which EcoRI-based seed sterility is repressible by chemical treatment for breeding (in trans) will be developed and assessed in rice and then translated to switchgrass.
Project Methods
We will use a combination of methods from the computational-based genetic engineering and synthetic biology to field experiments. The three technologies focus on 1) delayed-to-no flowering, 2) male- and seed-specific sterility approaches, and 3) conditional total bioconfinement. The total bioconfinement system is a default-on system that can be toggled to the off position for breeding and on for deployment. Success in one or more of these approaches will translate to important steps for bioconfinement of transgenes for switchgrass and other crops. The proposed research is based on recent discoveries and preliminary biotechnological data demonstrating that certain key components for bioconfinement are effective and show promise for additional research. While the delayed-to-no flowering transgenic system in switchgrass is most advanced in its development, we need to better understand its limits of seed production and transcriptomic effects. Male sterility and seed sterility are based on new technologies that are designed for switchgrass. Conditional total bioconfinement based on toggling is the "grail" for bioconfinement, since it would enable breeding and seed production by companies, yet, if effective, provide complete transgene bioconfinement in production fields. All research will follow NIH Guidelines and USDA APHIS regulations.

Progress 09/01/13 to 08/31/17

Outputs
Target Audience:The target audience for the project includes other plant biotechnology researchers as well as geneticists, ecologists, and those interested in crop improvement in agriculture. Extension agents and the general public are interested in genetic engineering and methods to assure its biosafety. Changes/Problems:We accomplished more results than expected from the miRNA156-control of plant flowering, which could lead to a followup study. We accomplished less than planned on the inducible promoter and synthetic circuit research owing to technical difficulties. The inducible promoter work is wrappling up, but fewer promoters seem to function in the crop (rice) than we expected. The synthetic circuit work was predicated on the inducible promoter success, which was slower than expected, and also complicated by the need to insert introns into some of the EcoRI constructs to prevent the constructs from killing the bacterial vectors (E. coli and Agrobacterium). Nonetheless, a subset of the promoters are functional and appear to be stable in transgenic plants. We are currently collecting data on the successful promoters to determine efficacy of the gamete-level bioconfinement systems in Objectives 2 and 3. What opportunities for training and professional development has the project provided?Post-doc: Yi Sang Graduate students: Jennifer Trumbo, Chelsea Johnson, and Tammy Stackhouse Undergraduate students: Morgan Peck, Ellie Terry-Emert, Kacie Reynolds, Francisco Palacios High school students: Walker Nowell One postdoc and three graduate students were provided with substantial training under this grant. In addition four undergraduate students and one high school students performed research. How have the results been disseminated to communities of interest?The results have been published in peer-reviewed papers. In addition, the PI went on a National Academies-sponsored speaking tour of Hawaii in 2017 that was associated with the 2016 "GE Crops" publication in which the PI served on the authoring committee. There, members of the public, concerned citizens, lawyers, and scientists were reached. What do you plan to do during the next reporting period to accomplish the goals?Overall Impact (there was not enough character space left in the "What was accomplished" field. The scientific problem addressed was how to contain genetically engineered switchgrass genes in the fields the crop is planted. The project addressed ways to control flowering to prevent transgene spread, as well as making pollen and seeds that do not carry transgenes. The transgene containment issue is important for regulators of biotechnology as well as for other stakeholders. The funding addressed the problem of transgene containment bu performing laboratory and field research to target multiple modalities of containment. Research was performed to field test existing genetically engineered (GE) switchgrass for delayed flowering and to perform growth of these plants in simulated topical-to-termperate zones of the GE switchgrass in growth chambers to see if controlled breeding activities could be performed in areas outside of switchgrass cultivation. New promoters that are active in seeds and pollen were cloned to determine if they could disrupt seed and pollen cells in rice, which was used as a proxy for switchgrass. The major activites ranged from extensive molecular biology and biotechnology laboratory work to identify promoters, make DNA constructs and genetically engineer plants to growth chamber, greenhouse and field experiments to assess plant performance. Further laboratory work was perfomed to characterize the plants, including analysis of all the genes expressed in field-grown switchgrass engineered for delayed flowering. The following people were involved in the project: PIs Neal Stewart and Zeng-Yu Wang, Collaborator Yuhong Tang Research associate Reginald Millwood Post-doc Yi Sang Graduate students Jennifer Trumbo, Chelsea Johnson, and Tammy Stackhouse Undergraduate students Morgan Peck, Ellie Terry-Emert, Kacie Reynolds, Francisco Palacios High school student Walker Nowell We used a number of innovative techniques. A complete transcriptomics analysis to understand the totality of gene expression during growth of the GE plants for delayed flowering in the field was among the largest studies of its kind, which was made possible from the allowence of USDA regulators for plants to flower. The allowance was made because of the remoteness and natural boundaries of the field site. The use of a restriction enzyme to chop pollen and seed DNA is novel. The scientific scientific knowledge built upon existing discoveries and techniques, which were translated to switchgrass. We learned that the regulation of gene expression level of the miR156 gene is crucial to decrease flowering (up to over 90 less) while not decreasing biomass of switchgrass. Those data and GE plants for decreased flowering suggest that using a constitutive promoter will not be enough to cease flowering completely and suggests new strategies to further control the miR156 transgene by using more precise promoters that are controlled by plant development and environmental cues. We learned that many seed-specific and pollen specific promoters actually have "leaky" expression that kills plants and the bacterium (Agrobacterium) used to engineer plants. Because of this finding, we had to add new DNA into the EcoR1 restriction enzyme that chops up DNA--an intron--to prevent it from killing the bacteria. We had to reject the promoters that led to plant death. These promoters resulted in plants that yielded progeny with apparent bioconfinement (no transgenes) that are being further analyzed. The outputs of our project were new GE plants that can be further analyzed and scientific papers and data that can be further analyzed. The research was impactful in a number of ways, but mostly to increase the knowledgebase around the problem of bioconfinement of transgenes. The findings were published in some of the best journals--most rigorous and highly cited journals--in the field of plant biotechnology and bioenergy, and so we thing the scientific knowledge creation is substantial and important. The papers were 'complete stories' that included substantial data sets. GE switchgrass with a trait was allowed to flower in the field for the first time which allowed the collection of delayed flowering data in these plants in a relevant field site in Tennessee, which also yielded a substantial gene expression dataset. Ultimately, we expect that assuring biosafety of GE crops will yield significant environmental and sustainability outcomes. Most of the impacts are at the national level, but the international level is relevant too as developers of plant biotechnology look for technologies to assure biosafety and regulatory compliance. The scientists who study biosafety of GE crops, regulators, developers of biotechnology and the concerned public will be impacted. The completion of the stated objectives should be useful to federal regulatory agencies in making decisions about GE organisms. Switchgrass is a relatively new bioenergy crop that has only recently (past 10 years) been genetically engineered and field tested. Regulators are eager to learn basic information about its biology, gene flow and preventing gene flow. To date, the studies performed under this BRAG grant provide, by far, the greatest body of work to engineer mechanisms in the plant to substantially decrease gene flow and increase confinement of GE elements in switchgrass (or any other species). We produced many outputs. Nine publications, ten students were trained (including the postdoc and the research associate who is also a PhD student), Around 20 presentations, including those without abstracts (i.e., presentations made by the PI to universities, companies, the public, and other venues) were made, and one patent was filed.

Impacts
What was accomplished under these goals? Objective 1. Delayed or no flowering. Switchgrass plants engineered for moderate overexpression of microRNA156 (miR156), which renders delayed or no flowering, will be field-tested. (100% complete) Planned for years 1-3, completed in year 4 during the NCE. A field experiment was completed after two growing seasons. The experiment was designed to examine flowering and overall plant growth for genetically engineered delayed flowering switchgrass. In this unique field study of transgenic switchgrass that was permitted to flower, two low (T14 and T35) and two medium (T27 and T37) miR156 overexpressing 'Alamo' lines, along with nontransgenic control plants, were grown in eastern Tennessee. miR156 expression was positively associated with decreased and delayed flowering in switchgrass. Line T27 did not flower during the two-year study. Line T37 did flower, but not all plants produced panicles. Flowering was delayed in T37, resulting in 70.6% fewer flowers than controls during the second field year with commensurate decreased seed yield: 1,205 seeds per plant vs. 18,539 produced by each control. These results are notable given that line T37 produced equivalent vegetative aboveground biomass to the controls. An in-depth examination showed the overexpression of miR156 affected other genes associated with flowering [five miR156 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL)] which had suppressed expression in one or more of the transgenic lines. Line T27, which had the highest miR156 overexpression, showed a significant reduction for all five SPL flowering genes. On the contrary, line T35 had the lowest miR156 overexpression and had no significant change in any of the flowering genes. Because of the research field's geographical features, this study was the first instance of any genetically engineered trait in switchgrass, in which experimental plants were allowed to flower in the field in the eastern U.S.; USDA-APHIS-BRS regulators allowed open-flowering (USDA-APHIS-BRS release permits (13-046-104r-a1 and 16-056-103r). We found medium overexpression of miR156, e.g., line T37, resulted in delayed and reduced flowering accompanied by high biomass production. Although this phenotype is high desirable from a regulatory standpoint, this could be challenging for plant breeders that likely need some sexual reproductive capacity for crop improvement as well as seed production for commercial field establishment. In our previous experiments, we have observed accelerated flowering in switchgrass when exposed to warm temperatures. Therefore, it may be possible to rescue the flowering phenotype through light and temperature treatments. To investigate light and temperature variations on miR156 switchgrass, we simulated a latitudinal cline in controlled growth chamber experiments for various individual sites from the tropics to cool-temperate conditions which included weekly average high and low temperatures and day lengths over the switchgrass growing season for each simulated site: Guayaquil, Ecuador; Laredo, Texas, USA; and Brattleboro, Vermont, USA. Flowering and reproduction among transgenic line with low (T-14 and T-35)-to-moderate (T-27 and T-37) overexpression of miR156 were assessed. Lower simulated latitudes (higher temperatures with low-variant day length) and long growing seasons promoted flowering of the miR156 transgenic switchgrass lines. Tropical conditions rescued the flowering phenotype in all transgenic lines except T-27. Higher numbers of plants in lines T-35 and T-37 and the controls produced panicles, which also occurred earlier in the study as temperatures increased and day length decreased. Line T-14 was the exception as more plants flowered in the cool-temperate (Vermont) conditions. Increased biomass was found in transgenic lines T-35 and T-37 in tropical conditions. No difference in biomass was found in subtropical (Texas) chambers, and two lines (T-14 and T-35) produced less biomass than the control in cool-temperate conditions. Objective 2. Male- and seed-sterility. A restriction endonuclease, EcoRI, will be specifically expressed in pollen or seed tissues and the efficacy for ablating target cells will be assessed, first in rice as a grass model and then translated to switchgrass. (90% complete) Planned for years 1-3, nearly completed in year 4 during the NCE, will be in early 2018. Male- and seed-sterility experiments were conducted using rice (Oryza sativa L.) as a model for switchgrass. Agrobacterium-mediated transformation has been successful in rice using vectors containing the EcoRI gene controlled by one of several pollen-specific (e.g., tomato LAT52, rice OsRTS and Osg6b, wheat TaPSG719) or seed-specific promoters (e.g. barley HvLtp1, and switchgrass PvGlb1). Although BRAG funding has ended, transgenic plant analysis will continue as a student is finishing up her masters degree on other funding. Hand-crossing is underway between transgenic T0 plants and emasculated nontransgenic rice plants. Seeds collected from crossing will be screened for transgene presence, and the rate of transgene escape calculated for each promoter listed above. Once crossing is complete, each plant with be self-pollinated. To estimate transgene copy number segregation analysis will be performed with selfed seeds. Once progeny analysis is complete, tissue-specific expression of EcoRI will be evaluated through qRT-PCR analysis, and phenotypic measures will be taken and compared to nontransgenic controls. Objective 3. Conditional total transgene bioconfinement. A novel system in which EcoRI-based seed sterility is repressible by chemical treatment for breeding (in trans) will be developed and assessed in rice and then translated to switchgrass. (90% complete) Planned for years 1-3, nearly completed in year 4 during the NCE, will be in early 2018. Two independent Agrobacterium transformation vectors have been constructed to evaluate conditional total transgene bioconfinement. The first vector contains the maize ZmUbi1 constitutive promoter controlling the expression of an RNAi-EcoRI repressive fragment. The second vector is identical except the ZmUbi1 promoter has been replaced with the glyphosate-inducible pM10 promoter. Rice has been transformed with each vector and several transgenic plants have been recovered for each. Once seed-specific EcoRI transgenics are down-selected to the best performing lines, they will be crossed with each of the RNAi-EcoRI transgenic types to evaluate conditional bioconfinement. Overall Impact This section is included in the field " What do you plan to do the next reporting period" since including it here was unacceptable to the system (went over the 8000 character limit).

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Millwood RJ, Moon HS, Poovaiah CR, Muthukumar B, Rice JH. Abercrombie JM, Abercrombie LL, Green WD, Stewart CN, Jr. 2016. Engineered selective plant male sterility through pollen-specific expression of the EcoRI restriction endonuclease. Plant Biotechnology Journal 14: 1281-1290. http://onlinelibrary.wiley.com/doi/10.1111/pbi.12493/epdf


Progress 09/01/15 to 08/31/16

Outputs
Target Audience:The target audiences for our project are farmers, biofuel industry developers and others in the private sector interested in sustainable large-scale farming of transgenic switchgrass and other biofuel feedstocks as well as other crops. We will pursue patenting and licensing opportunities to enable commercial development to help. Additionally, regulators interested in the reduction or possible elimination of the risk of outcrossing (via pollen flow and successful pollination and fertilization) of future plantings of transgenic switchgrass with compatible wild relatives. Lastly, conservation biologists from federal and non-federal entities (including NGOs) interested in the prevention of invasiveness traits in agronomic x wild hybrids, as well as the relatedness of agronomic cultivars and wild populations. Efforts undertaken in this reporting period include the PD's participation in the annual PD meeting, and a poster presentation at the Plant Biology 2016 Conference in Austin, Texas hosted by The American Society of Plant Biologists. Changes/Problems:While we are making progress on the grant, there have been delays caused by both personnel and technical issues. Postdoc Yi Sang had difficulties launching Objective 1 because he was troubleshooting unexpected significant issues in Objectives 2 and 3. Then Dr. Sang left for a faculty position at Lanzhou University, China in 2015. We replaced the postdoc with a research scientist and a graduate student in place to finish the project by August 2017. We list the status of each research objective along with the unexpected issues. Objective 1. Delayed or no flowering. Switchgrass plants engineered for moderate overexpression of microRNA156 (miR156), which renders delayed or no flowering, will be field-tested. ISSUES: see above. STATUS: We will complete the second year of the field study Dec 2016, with a manuscript submitted in 2017. Objective 2. Male- and seed-sterility. A restriction endonuclease, EcoRI, will be specifically expressed in pollen or seed tissues and the efficacy for ablating target cells will be assessed, first in rice as a grass model and then translated to switchgrass. ISSUES: EcoRI apparently affected the growth of Agrobacterium when under the control of several of our experimental promoters, which, unexpectedly, led to the death of Agrobacterium. This occurrence was surprising since we did not experience it using our initial promoter. We have mitigated this problem by inserting an intron into the EcoRI gene so that it is not expressed in Agrobacterium. This caused a lengthy delay and cost issues. STATUS: Several rebuilt promoter/EcoRI constructs are in transgenic rice and the rice is being grown for characterization for bioconfinement. We are still hopeful to produce transgenic switchgrass with the most effective bioconfinement constructs. Objective 3. Conditional total transgene bioconfinement. A novel system in which EcoRI-based seed sterility is repressible by chemical treatment for breeding (in trans) will be developed and assessed in rice and then translated to switchgrass. ISSUES: The key promoters responsible for chemical induction were found to not be working reliably in rice, so alternate versions have been cloned and are being characterized. STATUS: It appears that a longer version of a chemical inducible promoter is working better, so we have begun cloning the synthetic circuit vector for switchable bioconfinement. Under the circumstances, it is impossible to complete any the objectives by August 2016. We will be fortunate to complete two-to-three objectives by August 2017, but that plan will be far more productive than abandoning the project in September 2016. What opportunities for training and professional development has the project provided?The graduate student attended the Switchgrass III conference in Knoxville, Tennessee in 2015 and ASPB in 2016. This allowed our student to present current findings and network with switchgrass researchers.? How have the results been disseminated to communities of interest?Through conferences such as the annual ASPB Plant Biology meetingand the annualBRAG meeting. What do you plan to do during the next reporting period to accomplish the goals?The overall goal of the project is to develop systems to bioconfine transgenes into the intended host of switchgrass. Specific objectives of the project include the following: Delayed or no flowering. Switchgrass plants engineered for moderate overexpression of microRNA 156 (miR156), which renders delayed or no flowering, will be field-tested--80% complete At the end of the 2016 growing season we will collect the following data, which will be analyzed using a one-way ANOVA to test for significant differences among germplasm within each site. The characteristics are time to first flower each season, panicles per plant, end-of-season above-ground biomass, maximum plant height (with and without flowers), lignin content/composition, saccharification efficiency, forage quality, persistence and subsampling of plants for miR156 expression level by qRT-PCR analysis using the appropriate statistical treatment. We will also examine, if the flowering phenotype seems to be variable among replicate plants, an association between miR156 level and flowering phenotypes. These are all routine analyses we currently perform in our switchgrass research. The overexpression of miR156 may affect many other genes and metabolic processes. To identify downstream genes and to assess the potential for off-target, non-target or unintended effects of miR156 overexpression, we will analyze and compare global gene expression pro?ling and metabolic profiling of plants grown under field conditions (2016 season). Global gene expression pro?ling will be carried out by using the new Affymetrix switchgrass chips at the Noble Foundation. For a comprehensive analysis of large numbers of metabolites, leaf tissues from transgenic and control plants will be harvested, immediately frozen in liquid nitrogen, lyophilized, and stored at -80°C. Dry tissues will be extracted using various solvents and subjected to GC-MS and LC-MS analyses. Male- and seed-sterility. A restriction endonuclease, EcoRI, will be specifically expressed in pollen or seed tissues and the efficacy for ablating target cells will be assessed, first in rice as a grass model and then translated to switchgrass 60% complete To simplify downstream analysis, those phenotypically normal T0 transgenic rice plants will be subjected to Southern blot analysis to select single copy insertion lines. For each construct, a transgene escape rate value will be obtained from individual transgenic events, and the efficiency of each construct/promoter will be compared by using Chi-squared tests. The purpose of the rice experiments is to down-select to one or two candidate promoter-EcoRI constructs each for selective pollen and seed ablation that would be effective in switchgrass. Conditional total transgene bioconfinement. A novel system in which EcoRI-based seed sterility is repressible by chemical treatment for breeding (in trans) will be developed and assessed in rice and the translated to switchgrass. 30% complete The two constructs described previously will be evaluated in rice to determine whether RNAi-EcoRI constructs are sufficient to rescue viable seed. To this end, we will choose the two best pollen and seed promoters identified from Objective 2 to perform crosses and downstream germination analyses discussed earlier.

Impacts
What was accomplished under these goals? Delayed or no flowering miR156 switchgrass 80% complete Progress has been made with the field experiment of delayed or nonflowering miR156 switchgrass under a USDA APHIS BRS release permit started June, 2015. Four overexpression miR156 transgenic lines [two low expressing lines (T14 and T35) and two medium expressing lines (T27 and T37)] were planted in the field along with nontransgenic 'Alamo' control plants in a randomized design with a total of 20 plots. A single plot contained 10 plants of the same type, and each plant type/plot was replicated 4 times. Also included in the center of each plot were two nontransgenic ST2 clones (derived from the 'Alamo' cultivar), which serve as pollen recipients to assess proximal pollination from experimental plants. Seeds from ST2 plants were collected at the end of the season and screened for transgenicity. After one growing season, we completed data collection to assess plant growth performance, flowering phenotype and gene flow/bioconfinement efficacy. In our findings, we observed line T14 produced 50% less above-ground biomass than the control whereas no other transgenic line had different biomass than the control plants. When assessing flowering time, we found the medium overexpression lines exhibited the greatest delay in flowering. The control plants flowered two weeks post-transplanting, whereas T37 required 13 weeks to flower and T27 did not produce any flowers. In addition, all transgenic lines produced significantly fewer panicles than the control. Notably, line T27 produced no panicles and line T37 produced 10 fewer panicles on average than control plants. Lastly, we have screened seeds collected from ST2 pollen-recipient plants for half of the total plots. We observed T37 plots had a similar percentage of transgenic seed compared to 'Alamo' control plots (19% and 14%; respectively). All seed collected from the remaining transgenic plots contained a higher percentage (T14=38%; T27=55%; T35=32%) of transgenic individuals. In summary, we conclude that miR156 line T37 holds great potential from a bioconfinement standpoint because of its high biomass output, delayed flowering time, low panicle production, and low transgene flow percentage. Although line T27 seemed promising because no panicles were produced, the plants were very small and biomass output was very low. From a bioenergy feedstock view this transgenic line does not make the cut. We assessed the flowering phenology of miR156 switchgrass lines under simulated tropical, sub-tropical, and northern conditions in growth chamber experiments. Transgenic lines (T14, T27, T35, T37) and nontransgenic 'Alamo" control plants discussed above were grown in environmentally controlled growth chambers. Growth conditions were programmed to mimic field conditions in cool-temperate, subtropical, and tropical climates (e.g., southern Vermont, southern Texas, and coastal Ecuador, respectively). These experiments were started in July 2015 for cool-temperate and subtropical, and the tropical climate experiment was started in August 2015. The growing season length was based the temperature range specific to switchgrass growth and development in the respective region. Typically, switchgrass growth begins when weekly average temperatures are above 15/10?C for day/night and ends when weekly minimum temperatures average below 15?C. Temperature and day length were adjusted weekly to mimic seasonal change during the growth seasons corresponding to each representative climate area. Flowering times, total panicle production, and above-ground biomass were recorded and compared for each plant type. The cool-temperate and subtropical experiments were completed for one growing season and the tropical climate experiment will conclude in August 2016. Transgenic lines behaved similarly in both the cool-temperate and subtropical experiments in regards to flowering time and panicle production. In both experiments lines T27 and T37 did not flower. Under both climate conditions, line T14 flowered slightly earlier than 'Alamo' control plants, and line T35 flowered two weeks after controls. In addition, no significant differences were observed in the total number of panicle produced for lines T14 and T35. However, significant differences were observed in biomass production under cool-temperate conditions. Lines T14 and T35 produced significantly (59 g per plant on average) less biomass compared to control plants while no differences were detected in the remaining transgenic lines. When biomass production was evaluated under subtropical conditions, no significant differences were observed among all plant types. Male- and seed-sterility 60% complete Switchgrass was transformed using microprojectile bombardment with the EcoRI gene driven by the Zm13 maize pollen-specific promoter. Twenty transgenic events were recovered and evaluated for biomass production and bioconfinement efficacy in the greenhouse and growth chambers. Plants were clonally propagated and ten plants were included for each plant type. A completely randomized design was used, and the experiment concluded after 29 weeks of growth. At the end of the growth period, we observed two lines (E8 and E9) consistently performed as well the control plants, whereas the other lines were less productive. Additionally, to evaluate bioconfinement potential hand-crossing experiments were performed with all 20 transgenic lines under greenhouse conditions. A reciprocal cross was performed between transgenic Zm13-EcoRI plants paired to nontransgenic ST2 clones. Each cross was replicated three times. We found a high transgenic percentage of seeds collected from both parents. Our negative control crosses performed as expected as we observed no transgenic seeds. We concluded the Zm13 promoter did not drive EcoRI expression to sufficient levels to prevent transgenic pollen production/pollination. Therefore, we eliminated this promoter from further analysis. Several additional pollen-specific (e.g. tomato LAT52, rice OsRTS and Osg6b, wheat TaPSG719, and switchgrass PvPS1) and seed-specific promoters (e.g. maize ZmOle16, barley HvLtp1, and switchgrass PvOle16 and PvGlb1) are currently under evaluation for tissue-specific expression of EcoRI. Each of these promoters along with the EcoRI gene have been cloned into Agrobacterium transformation vectors and rice transformation is currently underway. Once tightly controlled tissue-specific expression has been confirmed by RT-PCR and qRT-PCR analyses, hand-crossing experiments will be performed to evaluate bioconfinement potential. Conditional total transgene bioconfinement 30% complete Two independent Agrobacterium transformation vectors have been constructed to evaluate conditional total transgene bioconfinement. The first vector contains the maize ZmUbi1 constitutive promoter controlling the expression of an RNAi-EcoRI repressive fragment. The second vector is identical except the ZmUbi1 promoter has been replaced with the glyphosate-inducible pM10 promoter. Both vectors have been used in rice transformation. Once pollen- and seed-specific EcoRI transgenic lines are produced in rice they will be crossed with each of the RNAi-EcoRI transgenic types to evaluate conditional bioconfinement.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Johnson, C.R., R.J. Millwood, Y. Sang, Z-Y. Wang, C.N. Stewart, Jr. 2016. Reproduction and bioconfinement of miR156 transgenic switchgrass. Plant Biology 2016, Austin, Texas, USA.


Progress 09/01/14 to 08/31/15

Outputs
Target Audience:The target audiences for our project are primarily scientists, regulators and developers of plant biotechnology in the private sector. Research is comunicated tothe target audiences by presentations at conferences, such as the annual BRAG PD meeting, theSwitchgrass III meeting in Knoxville, and publications. Changes/Problems:Postdoc Yi Sang left the project for a tenure-track faculty position earlier than expected. While this is good news from a trainee development perspective, it left a gap in the more advanced science aspects of the project that is currently being addressed. There will likely be a delay in completing the goals of the project. What opportunities for training and professional development has the project provided?One postdoc was trained.Dr. Sang is now an assistant professor in the Institute of Plant Biology and Plant Physiology at Lanzhou University, China. Two graduate students, Jennifer Trumbo (now a graduate student in another program at UTK) and Chelsea Johnson, were trained. How have the results been disseminated to communities of interest?Publications and presentations at scientific meetings, including the BRAG PI meeting in 2015 in Riverdale, and theCereal Engineering Consortium Workshop, Cambridge, Mass., June 8 & 9 2015. What do you plan to do during the next reporting period to accomplish the goals?The overall goal of the project is to develop systems to bioconfine transgenes into the intended host of switchgrass. Specific objectives of the project include the following: Delayed or no flowering. Switchgrass plants engineered for moderate overexpression of microRNA 156 (miR156), which renders delayed or no flowering, will be field-tested. At the end of the 2015 growing season and for the remaining seasons for both field and growth chambers experiments we will collect the following data, which will be analyzed using a one-way ANOVA to test for significant differences among germplasm within each site. The characteristics are time to first flower each season, panicles per plant, end-of-season above-ground biomass, maximum plant height (with and without flowers), lignin content/composition, saccharification efficiency, forage quality, persistence and subsampling of plants for miR156 expression level by qRT-PCR analysis using the appropriate statistical treatment. We will also examine, if the flowering phenotype seems to be variable among replicate plants, an association between miR156 level and flowering phenotypes. These are all routine analyses we currently perform in our switchgrass research. The overexpression of miR156 may affect many other genes and metabolic processes. To identify downstream genes and to assess the potential for off-target, non-target or unintended effects of miR156 overexpression, we will analyze and compare global gene expression profiling and metabolic profiling of plants grown under field conditions (second year, 2016 season). Global gene expression profiling will be carried out by using the new Affymetrix switchgrass chips at the Noble Foundation. For a comprehensive analysis of large numbers of metabolites, leaf tissues from transgenic and control plants will be harvested, immediately frozen in liquid nitrogen, lyophilized, and stored at -80°C. Dry tissues will be extracted using various solvents and subjected to GC-MS and LC-MS analyses. Male- and seed-sterility. A restriction endonuclease, EcoRI, will be specifically expressed in pollen or seed tissues and the efficacy for ablating target cells will be assessed, first in rice as a grass model and then translated to switchgrass. To simplify downstream analysis, those phenotypically normal T0 transgenic rice plants will be subjected to Southern blot analysis to select single copy insertion lines. For each construct, a transgene escape rate value will be obtained from individual transgenic events, and the efficiency of each construct/promoter will be compared by using Chi-squared tests. The purpose of the rice experiments is to down-select to one or two candidate promoter-EcoRI constructs each for selective pollen and seed ablation that would be effective in switchgrass. Conditional total transgene bioconfinement. A novel system in which EcoRI-based seed sterility is repressible by chemical treatment for breeding (in trans) will be developed and assessed in rice and the translated to switchgrass. The two constructs described previously will be evaluated in rice to determine whether RNAi-EcoRI constructs are sufficient to rescue viable seed. To this end, we will choose the two best pollen and seed promoters identified from Objective 2 to perform crosses and downstream germination analyses discussed earlier.

Impacts
What was accomplished under these goals? The overall goal of the project is to develop systems to bioconfine transgenes into the intended host of switchgrass. Specific objectives of the project include the following: Delayed or no flowering. Switchgrass plants engineered for moderate overexpression of microRNA 156 (miR156), which renders delayed or no flowering, will be field-tested. Male- and seed-sterility. A restriction endonuclease, EcoRI, will be specifically expressed in pollen or seed tissues and the efficacy for ablating target cells will be assessed, first in rice as a grass model and then translated to switchgrass. Conditional total transgene bioconfinement. A novel system in which EcoRI-based seed sterility is repressible by chemical treatment for breeding (in trans) will be developed and assessed in rice and the translated to switchgrass. Accomplishments Delayed or no flowering miR156 switchgrass Progress has been made on the field-test of delayed or nonflowering miRNA156 switchgrass. A USDA APHIS BRS release permit was obtained, and the field experiment was planted in June, 2015. Four overexpression miR156 transgenic lines will be evaluated and compared to the nontransgenic cultivar 'Alamo' control plants. All transgenic plants were produced in the cultivar Alamo. Two low expressing lines (T14 and T35) and two medium expressing lines (T27 and T37) were planted in the field along with control plants. Data were collected time to first flower and mid-season data were collected for plant height and plant circumference. For flowering time, the low miR156 line T14 was the first to flower at 2 weeks post-transplanting. The control plants produced panicles 4 weeks post-transplanting and the low miR156 line T35 produced panicles at 8 weeks. All plants for these plant types were flowering at 12 weeks post-transplanting. Medium miR156 expressing lines exhibited different flowering pattern. At 14 weeks post-transplanting two of ten plants per plot had produced panicle for line T37 and line T27 produced no panicles. Additionally, differences were observed for both plant height and plant circumference. Lines T14, T27, and T37 were all shorter than the control type although lines T14 and T37 were only slightly shorter with differences ranging from 10-20 cm. Line T27 was much shorter standing at half the size of the control plant. Differences in plant circumference were much less severe. Lines T14 and T27 were the only ones to significantly differ from the control type. A smaller circumference was observed for both lines with less than a 10% decrease in circumference for line T14 and nearly a 20% decrease for line T27. The results of the mid-season analyses indicate that line T37 offers the best combination of bioconfinement potential and biomass production. T37 plants are only slightly similar in size compared to the control type yet flowers 10 weeks later. An additional set of experiments were started to evaluate the flowering patterns of miR156 switchgrass grown under conditions that mimic climates other than the field site in Tennessee. Transgenic lines (T14, T27, T35, T37) and control plants discussed above will be grown under growth chamber conditions that mimic field conditions in cool-temperate, sub-tropical, and tropical climates. These experiments were started in July 2015 and will run for two growing seasons in growth chambers programmed for appropriate season and day length. One concept of operation is that miR156 plants would not flower in mid-temperate North America, i.e., in the "switchgrass belt,"but could conceivably flower and be bred in the tropics or subtropics for seed production. Therefore, flowering times and total panicle production will be recorded and compared for each plant type as well as biomass quantities. Male- and seed-sterility Switchgrass has been transformed through microprojectile bombardment with the EcoRI gene driven by the Zm13 maize pollen specific promoter. Twenty transgenic events were recovered and these events will be evaluated for tissue-specific expression of the EcoRI gene first through reverse transcription polymerase chain reaction (RT-PCR). Once lines have been selectively eliminated by RT-PCR, the remaining lines will be evaluated by quantitative RT-PCR analysis (qRT-PCR). Hand-crossing experiments are also underway under greenhouse conditions to evaluate bioconfinement potential. Several pollen-specific (e.g. tomato LAT52, rice OsRTS and Osg6b, wheat TaPSG719, and switchgrass PvPS1) and seed-specific promoters (e.g. maize ZmOle16, barley HvLtp1, and switchgrass PvOle16 and PvGlb1) will be evaluated for tissue-specific expression of EcoRI. Each of these promoters along with the EcoRI gene have been cloned into Agrobacterium transformation vectors and rice transformation has begun. Once tightly controlled tissue-specific expression has been confirmed by RT-PCR and qRT-PCR analyses, hand-crossing experiments will be performed to evaluate bioconfinement potential. Conditional total transgene bioconfinement Two independent Agrobacterium transformation vectors have been constructed to evaluate conditional total transgene bioconfinement. The first vector contains the maize ZmUbi1 constitutive promoter controlling the expression of an RNAi-EcoRI repressive fragment. The second vector is identical except the ZmUbi1 promoter has been replaced with the glyphosate-inducible pM10 promoter. Both vectors have been used in rice transformation. Once pollen- and seed-specific EcoRI transgenic lines are produced in rice they will be crossed with each of the RNAi-EcoRI transgenic types to evaluate conditional bioconfinement.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Millwood, R.J., H.S. Moon, C.R. Poovaiah, B. Muthukumar, J.H. Rice. J.M. Abercrombie, L.L Abercrombie, W.D. Green, C.N. Stewart, Jr. Engineered selective plant male sterility through pollen-specific expression of the EcoRI restriction endonuclease. Plant Biotechnology Journal.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: 212. Trumbo, J.L., B. Zhang, and C.N. Stewart, Jr. 2015. Manipulating microRNAs for improved biomass and biofuels from plant feedstocks. Plant Biotechnology Journal 13: 337-354.


Progress 09/01/13 to 08/31/14

Outputs
Target Audience: The primary audience is government regulators and other scientists; ultimately the audience will consist of the concerned populace. The research will primarily initially be disseminated mainly through professional presentations and internationally recognized peer-reviewed journals, such as Plant Biotechnology Journal, BMC Biotechnology and Plant Cell Reports. The readers of these journals are scientists and regulators. The research will also be incorporated in various courses and outreach activities. Interviews will be granted with journalists that also serve as a conduit to disseminate research findings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Postdoc Yanhui Peng has received training and he also helping to mentor and undergraduate researcher and a new graduate student who is supported from other sources. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We will establish new plants in the field for the miR156 study and analyze the gene expression and flowering phenotypes. For Objectives 2 and 3, we will continue to build constructs and test them in rice, with the best ones going into switchgrass in year 2 of the grant.

Impacts
What was accomplished under these goals? 1. Delayed or no flowering. Switchgrass plants engineered for moderate overexpression of microRNA156 (miR156), which renders delayed or no flowering, will be field-tested. We have established one field experiment in a regulated site and have obtained a USDA APHIS BRS release-into-the-environment permit to establish a second site. The first site requres that panicles be removed as a bioconfinement mechanism. However, one transgenic event did not flower at all and another produced very few panicles. Based on early data, both of these transgenic events appeared to produce biomass comparable to the nontransgneic control line. 2. Male- and seed-sterility. A restriction endonuclease, EcoRI, will be specifically expressed in pollen or seed tissues and the efficacy for ablating target cells will be assessed, first in rice as a grass model and then translated to switchgrass. Greenhouse and field experiments confirm that EcoRI is able to completely ablate transgenic tobacco in pollen cells when under the control of the tomato LAT52 promoter. To translate this result to rice and switchgrass, a total of 7 pollen-specific and 6 embryo-specific promoters have been identified and 5 of these, controlling the expression of the EcoRI gene, have been transformed into rice. We found that the remainder of these were impossible to maintain in various strains of Agrobacterium tumefaciens strains. We hypothesize that 'leaky' expression is allowing EcoRI to be expressed and killing Agrobacterium. Therefore, a we constructed the EcoRI gene with an intron and have 9 new constructs that are stable in Agrobacterium. 3. Conditional total transgene bioconfinement. A novel system in which EcoRI-based seed sterility is repressible by chemical treatment for breeding (intrans) will be developed and assessed in rice and then translated to switchgrass. We have identified three inducible promoters and are characterizing these to couple with the best cassetted above to make a syntheticciruit-based GURT system. We have 5 glyphosate-inducible promoters under testing.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Sang, Y., R.J. Millwood, C.N. Stewart Jr. 2013 Gene use restriction technologies for transgenic plant bioconfinement. Plant Biotechnology Journal 11:649-658.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Ellstrand, N.C., P. Meirmans, J. Rong, D. Bartsch, A. Ghosh, T.J. de Jong, P. Haccou, B.-R. Lu, A.A. Snow, C.N. Stewart, Jr., J.L. Strasburg, P.H. van Tienderen, K. Vrieling, D. Hooftman. 2013. Introgression of crop alleles into wild or weedy populations. Annual Review of Ecology, Evolution, and Systematics 44: 325-345.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Gressel, J., C.N Stewart, Jr., L.V. Giddings, A.J. Fischer, J.C. Streibig, N.R. Burgos, A. Trewavas, A. Merotto, Jr., C.J. Leaver, K. Ammann, V. Moses, A. Lawton-Rauh. 2014. Overexpression of epsps transgene in weedy rice: insufficient evidence to support multiple conclusions about biosafety. New Phytologist 202:360-362.