Source: CLEMSON UNIVERSITY submitted to NRP
MICRORNA-MEDIATED TRANSGENE CONTAINMENT IN IMPORTANT PERENNIAL GRASSES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1027003
Grant No.
2021-33522-35342
Cumulative Award Amt.
$500,000.00
Proposal No.
2021-04334
Multistate No.
(N/A)
Project Start Date
Sep 1, 2021
Project End Date
Mar 31, 2026
Grant Year
2021
Program Code
[HX]- Biotechnology Risk Assessment
Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
(N/A)
Non Technical Summary
Turfgrass and switchgrass are among the most important perennial grasses significantly impacting agriculture production, agriculture economy, sustainable energy and environment. Like in many row crops, genetic engineering of both switchgrass and turfgrasses using transgenic technologies offers the opportunity to incorporate many economic and agronomic benefits that are difficult or impossible to achieve through traditional breeding techniques. However, the risk of transgene escape and the unforeseen environmental consequence by the use of transgenic technology in perennial grasses require development of strategies for transgene containment. To this end, specific measures to induce male sterility or total sterility for transgene containment usually need to be implemented in parallel with the introduction of genes of interest for trait modifications in transgenics, which not only complicate the whole process of plant genetic engineering, but also cause accumulation of unwanted foreign DNAs in the host genome. The major objective of this project is to develop and evaluate a novel approach that explores the use of different miRNA genes taking advantage of their dual roles in plant reproductive development and beneficial agronomic trait improvement to produce self-contained superior transgenic switchgrass and turfgrass with significantly improved multiple agronomic traits. Specifically, a microRNA gene, miR396 that regulates both plant sterility and abiotic stress responses will be introduced into switchgrass and turfgrass together with three additional miRNA genes, miR319, miR528 and miR393 that are all positive regulators of plant abiotic stress responses. Transgenic lines with the stacked miRNA genes will be evaluated in the greenhouse in "pollen-cage" study and in the field trial gene flow study for the efficacy of miRNA396-mediated sterility induction and transgene containment as well as the overall impacts of all miRNA genes on beneficial agronomic traits. Results from this researchwill allow the establishment of a novel gene containment system in grasses and produce environmentally safe, self-contained superior transgenic perennial grasses with improvement of multiple beneficial agronomic traits. The system developed from this research is also transferable and could easily be adapted to other perennial grasses and food crop species to develop self-contained transgenic plants with significantly improved multiple traits. The project directly addresses USDA BRAG program area #1 "Management practices to minimize the environmental risk of genetically engineered (GE) organisms" and specifically, "Development or evaluation of effective strategies, including molecular and/or genetic, to limit gene transfer (gene flow) or outcrossing to sexually compatible organisms or transfer of genetic material between viruses, insects, or microorganisms" (1d).
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2061629104070%
2061629108030%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1629 - Perennial grasses, other;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;
Goals / Objectives
The major objective of this project is to explore the use of different miRNA genes to develop a cost-effective new system for transgene self-containment in switchgrass and turfgrass, producing environmentally safe transgenic (TG) products with significantly improved multiple agronomic traits. There are four major objectives proposed in this project:Objective #1: Design and synthesize two chimeric gene constructs, p35S/miR396-35S/miR319-Ubi/hyg and p35S/miR528-35S/miR393-Ubi/bar for switchgrass and turfgrass transformation (Year 1).The miR396- and miR319-containing construct, p35S/miR396-35S/miR319-Ubi/hyg, and the miR528- and miR393 construct, p35S/miR528-35S/miR393-Ubi/bar, will be prepared for use in switchgrass and turfgrass transformation. The two chimeric gene constructs will be prepared and validated using standard molecular biology techniques and DNA sequencing.Objective #2: Produce transgenic switchgrass and turfgrass harboring two (miR396 and miR319) or four miRNA genes (miR396, miR319, miR528 and miR393) (Year 1-2).The chimeric gene construct, p35S/miR396-35S/miR319-Ubi/hyg, assembled from objective #1 will be introduced into switchgrass and creeping bentgrass to produce multiple independent transgenic lines in both species. Simultaneously, we will also co-transform switchgrass and creeping bentgrass with the two Agrobacterium strains containing p35S/miR396-35S/miR319-Ubi/hyg and p35S/miR528-35S/miR393-Ubi/bar, respectively, generating multiple independent transgenic lines harboring both constructs.Objective #3: Evaluate the effectiveness of miRNA396-mediated sterility in transgenic plants generated in Objectives #2 and conduct "pollen cage" studies to assess how efficient the miR396-mediated sterility in transgene containment (Year 2-3).We will vernalize all transgenic creeping bentgrass lines together with wild type control plants and then observe and analyze, along with switchgrass plants, plant flowering and sterility. Since both switchgrass and creeping bentgrass are out-crossing species, transgenic plants alone or together with wild type plants will be placed in "pollen cages" to determine the effectiveness of miR396-mediated sterility and the efficacy of male sterility in mitigating transgene flow in switchgrass and creeping bentgrass.Objective #4: Conduct field trial gene flow study to evaluate the efficiencies of miR396-mediated male sterility in the prevention of transgene escape from genetically modified grass (Year 3-4). We will also conduct field trial gene flow study, using sterile transgenic plants harboring four miRNA genes together with non-transgenic plants, under isolated conditions. Effectiveness of miR396-mediated male sterility under field conditions and its usefulness in transgene containment will be assessed.Objective #5: Performance evaluation of the transgenic plants generated in Objectives #2 that express multiple miRNA genes under normal conditions and various environmental adversities compared to wild type (WT) controls (Year 3-4).To further assess the possible additive effect of the stacked miRNA genes on transgenic plants stress response, we will evaluate plant performance under drought, salt, heat stress and N starvation/over-fertilization.
Project Methods
Objective #1: Design and synthesize two chimeric gene constructs, p35S/miR396-35S/miR319-Ubi/hyg and p35S/miR528-35S/miR393-Ubi/bar for switchgrass and turfgrass transformation (Year 1).To demonstrate and evaluate sterility induction in switchgrass and creeping bentgrass by miR396 when stacking with other miRNA genes, we will first prepare two chimeric gene constructs containing either miR396 and miR319 or miR528 and miR393 for plant transformation.The two vectors prepared as described above will be introduced into Agrobacterium tumefaciens by electroporation for use in turfgrass and switchgrass transformation via infection of embryogenic callus initiated from mature seeds.Objective #2: Produce transgenic switchgrass and turfgrass harboring two (miR396 and miR319) or four miRNA genes (miR396, miR319, miR528 and miR393) (Year 1-2).Genetically engineering switchgrass and creeping bentgrass by transforming plants with the miR396- and miR319-containing constructs and co-transformation of plants with the two chimeric constructs assembled from objective #1 to introduce all four miRNA genes, miR396, miR319, miR528 and miR393, producing multiple independent transgenic lines in both cases following previously established Agrobacterium-mediated plant transformation protocols via infection of embryogenic callus initiated from mature seeds and plant tissue culture, selection and regeneration procedures. The regenerated T0 plants will be transferred into soil and grown in the greenhouse. We will conduct molecular characterization of these T0 transformants to demonstrate the insertion, copy number and expression of the introduced foreign genes by PCR, Southern, Northern and RT-PCR analyses using genomic DNA and RNA extracted from leaves. the gene hyg, bar, miR396, miR319, miR528 or miR393 will be used as a probe in Southern and Northern analyses.Objective #3: Objective #3: Evaluate the effectiveness of miRNA396-mediated sterility in transgenic plants generated in Objectives #2 and conduct "pollen cage" studies to assess how efficient the miR396-mediated sterility in transgene containment (Year 2-3).We will vernalize all transgenic creeping bentgrass lines together with wild type control plants in a cold room at 5°C in an 8-h-ligh/16-h-dark photoperiod for 15 weeks, and then move the plants back to room temperature under long-day conditions (16h/8h, light/dark) to observe and analyze, along with switchgrass plants, plant flowering and sterility. Since both switchgrass and creeping bentgrass are out-crossing species, transgenic plants alone or together with wild type plants will be placed in "pollen cages" to determine the effectiveness of miR396-mediated sterility and the efficacy of male sterility in mitigating transgene flow in switchgrass and creeping bentgrass.Objective #4: Conduct field trial gene flow study to evaluate the efficiencies of miR396-mediated male sterility in the prevention of transgene escape from genetically modified grass (Year 3-4).To evaluate effectiveness of miR396-mediated male sterility and its usefulness in transgene containment under field conditions, we will conduct field trial gene flow study, using sterile transgenic plants harboring four miRNA genes together with non-transgenic plants, under isolated conditions. Approximately 350 non-transgenic plants will be planted in transects around approximately 300 TG plants. Once the plants have finished flowering, inflorescences of the non-TG plants will be enclosed in hybridization bags. Any remaining, un-bagged, inflorescences will be cut. The inflorescences from each non-TG plant will then be harvested, dried and planted and screened in the greenhouse for herbicide resistance. By this method we will recover, if any existing, seedlings produced through transgene flow for subsequent molecular verification to confirm the presence of the bar gene using PCR and Southern blot analysis. The percentage of resistant seedling progeny will be calculated as the number of survivors divided by the total number of seedlings germinated.Objective #5: Performance evaluation of the transgenic plants generated in Objectives #2 that express multiple miRNA genes under normal condition and various environmental adversities compared to WT controls (Year 3-4).To further assess the possible additive effect of the stacked miRNA genes on transgenic plant stress response, we will evaluate plant performance under drought, salt, heat and N starvation/over-fertilization. To this end, three independent lines from TG switchgrass and creeping bentgrass harboring either two (miR396 and miR319) or four miRNA genes (miR396, miR319, miR528 and miR393) will first be clonally propagated and split into multiple replicates in pots, and 5 replicates will be used for evaluation under each treatment of stress conditions. Wild type plants will also be clonally propagated and used as controls for comparison in all the evaluations.

Progress 09/01/24 to 08/31/25

Outputs
Target Audience:The primary target audiences for this project are plant biotechnologists in academia, industry and regulatory agencies, bioenergy crop and turfgrass breeders, bioenergy and turfgrass industries concerned about the risks associated with genetically engineered perennial grass species and interested in commercializing transgenic switchgrass, turfgrass and other perennial grass species. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The Principal Investigator of the project has been responsible for organizing and supervising all the research activities for this project. A Research Associate and a PhD student have continued to work on the project and further developed their professional research skills under direction and mentorship by the Principal Investigator of the project. Six undergraduate students have been trained for their directed research while working together with the research associate and the graduate student since the beginning of this project. An undergraduate summer research intern has also been trained working together with the graduate student on this project. How have the results been disseminated to communities of interest?The preliminary results and related research data obtained so far have been presented as posters and oral presentations in international conferences, academic institutions, and peer-reviewed papers in academic journals. What do you plan to do during the next reporting period to accomplish the goals?So far, we have completed all tasks outlined in Objective #1, Objective #2, Objective #3, Objective #5 and Objective #6. Our research progress is in line with the time frame proposed in our proposal. We have now propagated enough transgenic plants expressing all four miRNA genes (miR319, miR528, miR393 and miR396) in the greenhouse, and they are currently being vernalized under cold conditions for five months in preparation for field trial study evaluating efficiencies of miR396-mediated male sterility in the prevention of transgene escape from genetically modified grass outlined in the Objective #4.

Impacts
What was accomplished under these goals? Objective #1.Design and synthesize two vectors combining different miRNA genes for switchgrass andturfgrass transformation. This objective had been planned for year 1 of the project and we successfully completed the task of producing two chimeric gene constructs. The first construct p35S/miR396-35S/miR319-Ubi/hyg, consists of a CaMV35S-driven miR396 gene linked to a CaMV35S-driven miR319 gene and a corn ubiquitin promoter (Ubi)-driven hygromycin resistance marker gene, hyg. The second construct p35S/miR528-35S/miR393-Ubi/bar, consists of a CaMV35S-driven miR528 gene linked to a CaMV35S-driven miR393 gene and a corn Ubi promoter-driven herbicide resistance marker gene, bar. These two constructs were then respectively introduced into Agrobacterium and used to infect embryogenic callus initiated from mature seeds of switchgrass and turfgrass as planned in Objectve #2, generating transgenic plant lines harboring and expressing either two miRNA genes (miR396 and miR319 or miR528 and miR393) or all four miRNA genes (miR396, miR319, miR528 and miR393). Three representative transgenic lines from each group have been selected, clonally propagated in greenhouse and compared for their development, responses to abiotic stresses including drought and salinity assessing the impact of stacked miRNA genes on various agronomic traits as outlined in Objective #5. The clonally propagated plants were also placed under 4°C, 16h/8h light/dark conditions for 6-month vernalization and then moved back to the greenhouse. The vernalized plants flowered in the greenhouse but are all male-sterile shedding only few non-viable pollens, indicating stacking of different miRNA genes does not impact how miR396 modulates plant sterility. Simultaneously, we also initiated work for the task outlined in Objective #6 by clonally propagating previously produced transgenic creeping bentgrass lines containing miR319, miR528 and miR393, respectively. These transgenic lines were maintained in the cold room for 6-month vernalization and then moved back to the greenhouse. Transgenic lines overexpressing miR319, miR528 and miR393, respectively, all flowered. Examination of flowering revealed no difference from wild type controls, indicating that overexpression of miR319, miR528 or miR393 does not impact plant flowering. The vernalized transgenic plants expressing all four miRNA genes (miR396, miR319, miR528 and miR393) were used for "pollen cage" study as outlined in Objective #3. None of the tested transgenic plants in the pollen cage produced any seeds indicating the complete male sterility in transgenic plants expressing all four miRNA genes (miR396, miR319, miR528 and miR393). We also have propagated enough transgenic plants expressing all four miRNA genes (miR319, miR528, miR393 and miR396) in the greenhouse, and they are currently being vernalized under cold conditions for five months in preparation for field trial study evaluating efficiencies of miR396-mediated male sterility in the prevention of transgene escape from genetically modified grass outlined in the Objective #4.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Chen, X., Chen, Z., Watts, R., Luo, H. (2025) Non-coding RNAs in plant stress responses: molecular insights and agricultural applications. Plant Biotechnology Journal, https://doi.org/10.1111/pbi.70134.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Zhao, J., Xiong, Y., Xu, M., Gou, W., Xiong, Y., Dong, Z., Pan, L., Sha, L., Luo, H., Ma, X. (2025) Organelle genome characteristics and phylogenetic analysis of a warm-season turfgrass Eremochloa ophiuroides (Poaceae). Biology 14:975, https://doi.org/10.3390/biology14080975.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Chen, X., Chen, Z., Fiorentino, A., Kuess. M., Tharayil, N., Kumar, R., Leonard, E., Noorai, R., Hu, Q., Luo, H. (2024) MicroRNA169 integrates multiple factors to modulate plant growth and abiotic stress responses. Plant Biotechnology Journal 22:2541-2557.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Chen, Z., Chen, X., Hu, Q., Luo, H. (2025) Functional characterization of plant specific nuclear factor Y subunit alpha (NFYA) transcription factor in creeping bentgrass (Agrostis stolonifera). Poster presentation in 2025 Society for In Vitro Biology (SIVB) Meeting, June 7-10, 2025, Norfolk, VA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Chen, X., Pickrel, A., Hu, Q., Kuess, M, Luo, H. (2025) Off-target effects of the site-specific recombinases in perennial grasses. Poster presentation in 2025 Society for In Vitro Biology (SIVB) Meeting, June 7-10, 2025, Norfolk, VA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Watts, R., Chen, Z., Chen, X., Hu, Q., Luo, H. (2025) RNA demethylase AsALKBH1A regulates plant development and environmental stress adaptation in Agrostis stolonifera. Poster presentation in 2025 Society for In Vitro Biology (SIVB) Meeting, June 7-10, 2025, Norfolk, VA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2025 Citation: Luo, H. (2025) Biotechnology for stress mitigation in grasses. Invited speaker in the International Pastureland and Forage Industry Congress 2025 (IPFIC 2025), July 10-13, 2025, Hulunbuir, China.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Luo, H. (2025) Molecular strategies for transgene containment and removal in plants. Invited speaker in the session Biotechnology approaches for animal and crop improvement and environmental risk assessment of genetically engineered organisms of the 2025 Society For In Vitro Biology (SIVB) Meeting, June 7-10, 2025, Norfolk, VA.
  • Type: Other Status: Other Year Published: 2024 Citation: Luo, H. (2024) Towards robust crops under environmental adversities. Invited speaker in the University of Maryland Department of Plant Science and Landscape Architectures Plant Science Lecture Series. November 18, 2024.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Luo, H. (2024) Epigenetic modification of grasses for new germplasm development. Invited speaker in the 2024 International Symposium on Grassland Ecological Management and Grassland Biodiversity, November 15-18, 2024, Lanzhou, China.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Luo, H. (2024) Epigenetic control of plant development and stress response in grasses. Invited speaker in the 2024 International Conference on the Cooperation and Integration of Industry, Education, Research and Application: Green Low-Carbon and Sustainable Development Sub-venue, October 22-23, 2024, Chengdu, China.


Progress 09/01/23 to 08/31/24

Outputs
Target Audience:The primary target audiences for this project are plant biotechnologists in academia, industry and regulatory agencies, bioenergy crop and turfgrass breeders, bioenergy and turfgrass industries concerned about the risks associated with genetically engineered perennial grass species and interested in commercializing transgenic switchgrass, turfgrass and other perennial grass species. Changes/Problems:No changes for the proposed research in this period of time. What opportunities for training and professional development has the project provided?The Principle Investigator of the project has been responsible for organizing and supervising all the research activities for this project. A Research Associate and a PhD student have continued to work on the project and further developed their professional research skills under the direction and mentorship by the Principle Investigator of the project. Six undergraduate students have been trained for directed research while working together with the research associate and the graduate student since the beginning of this project. An undergraduate summer research intern has also been trained working together with the graduate student on this project. How have the results been disseminated to communities of interest?The preliminary results and related research data obtained so far have been presented as posters and oral presentations atinternational conferences, academic institutions, and peer-reviewed papers in academic journals. What do you plan to do during the next reporting period to accomplish the goals?So far, we have completed all tasks outlined in the Objective #1, Objective #2, Objective #3, Objective #5 and Objective #6. Our research progress is in line with the time frame proposed in our proposal. We are now propagate transgenic plants expressing all four miRNA genes (miR319, miR528, miR393 and miR396) in preparation for field trial outlined in the Objective #4.

Impacts
What was accomplished under these goals? Objective 1.Design and synthesize two vectors combining different miRNA genes for switchgrass andturfgrass transformation. This objective had been planned for year 1 of the project and we successfully completed the task producing two chimeric gene constructs. The first construct, p35S/miR396-35S/miR319-Ubi/hyg, consists of a CaMV35S-driven miR396 gene linked to a CaMV35S-driven miR319 gene and a corn ubiquitin promoter (Ubi)-driven hygromycin resistance marker gene, hyg. The second construct, p35S/miR528-35S/miR393-Ubi/bar, consists of a CaMV35S-driven miR528 gene linked to a CaMV35S-driven miR393 gene and a corn Ubi promoter-driven herbicide resistance marker gene, bar. These two constructs were then respectively introduced into Agrobacterium and used to infect embryogenic callus initiated from mature seeds of switchgrass and turfgrass as planned in Objectve #2, generating transgenic plant lines harboring and expressing either two miRNA genes (miR396 and miR319 or miR528 and miR393) or all four miRNA genes (miR396, miR319, miR528 and miR393). Three representative transgenic lines from each group have been selected, colonally propagated in greenhouse and compared for their development, reponses to abiotic stresses including drought and salinity assessing the impact of stacked miRNA genes on various agronomic traits as outlined in Objective #5. The colonally propagated plants were also placed under 4°C, 16h/8h light/dark conditions for 6-month vernalization, and then moved back to the greenhouse. The vernalized plants flowered in the greenhouse but are all male-sterile shedding only few non-vialble pollen, indicating stacking of different miRNA genes does not impact how miR396 modulates plant sterility. Simultaneously, we also initiated work for the task outlined in Objective #6 by colonally propagating previously produced transgenic creeping bentgrass lines containing miR319, miR528 and miR393, respectively. These transgenic lines were maintained in the cold room for 6-month vernalization, and then moved back to the greenhouse. Transgenic lines overexpressing miR319, miR528 and miR393, respectively, all flowered. Examination of flowering revealed no difference from wild type controls, indicating that overexpression of miR319, miR528 or miR393 does not impact plant flowering. The vernalized transgenic plants expressing all four miRNA genes (miR396, miR319, miR528 and miR393) were used for "pollen cage" study as outlined in Objective #3. None of the tested transgenic plants in the pollen cage produced any seeds indicating the complete male sterility in transgenic plants expressing all four miRNA genes (miR396, miR319, miR528 and miR393).

Publications

  • Type: Other Status: Other Year Published: 2023 Citation: Luo, H. (2023) Gene pyramiding for boosted plant growth and broad abiotic stress tolerance. Invited speaker in the 2023 International Conference on Frontier in Grassland Science, October 15-17, 2023, Beijing, China.
  • Type: Other Status: Other Year Published: 2023 Citation: Luo, H. (2023) Towards robust grasses under environmental adversities. Invited speaker in the International Conference on Grassland and Forage Science, November 14-17, 2023, Lanzhou, China.
  • Type: Other Status: Other Year Published: 2024 Citation: Luo, H. (2024): Mechanisms of turfgrass stress tolerance and advances in its genetic improvement. Invited speaker in the First World Grassland Conference, July 13, 2024, Hohhot, China.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Watts, R., Chen, Z., Chen, X., Hu, Q., Miao, S., Luo, H. (2024) Epitranscriptomic regulation of gene expression in perennial grass development and stress adaptation. Poster presentation in 2024 World Congress on In Vitro Biology (SIVB) Meeting, June 8-12, 2024, St. Louis, MS.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Chen, X., Chen, Z., Watts, R., Hu, Q., Kuess, M, Luo, H. (2024) The off-target phenotypic and genetic effects of the site-specific DNA recombinases in plants. Poster presentation in 2024 World Congress on In Vitro Biology (SIVB) Meeting, June 8-12, 2024, St. Louis, MS.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Chen, Z., Chen, X., Miao, S., Hu, Q., Luo, H. (2024) Pyramiding microRNAs Enhances Plant Growth and Broad Abiotic Stress Resistance. Invited oral presentation in 2024 World Congress on In Vitro Biology (SIVB) Meeting, June 8-12, 2024, St. Louis, MS.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Liu, C., Dong, K, Du, H., Wang, X., Li, X., Huang, N., Hu, Q., Luo, H., Sun, X. (2024) AsHSP26.2, a chloroplast small heat shock protein positively regulates growth and development in creeping bentgrass. Plant Cell Reports 43:32.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Zhao, G., Liu, Y., Li, L., Che, R., Douglass, M., Benza, K., Angove, M., Luo, K., Hu, Q., Chen, X., Henry, C., Hu, Q., Li, Z., Ning, G., Luo, H. (2024) Gene pyramiding for boosted plant growth and broad abiotic stress tolerance. Plant Biotechnology Journal 24:678-697.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Chen, X., Chen, Z., Fiorentino, A., Kuess. M., Tharayil, N., Kumar, R., Leonard, E., Noorai, R., Hu, Q., Luo, H. (2024) MicroRNA169 integrates multiple factors to modulate plant growth and abiotic stress responses. Plant Biotechnology Journal https://doi.org/10.1111/pbi.14367.


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:The primary target audiences for this project are plant biotechnologists in academia, industry and regulatory agencies, bioenergy crop and turfgrass breeders, bioenergy and turfgrass industries concerned about the risks associated with genetically engineered perennial grass species and interested in commercializing transgenic switchgrass, turfgrass and other perennial grass species. Changes/Problems:No changes for the proposed research in this period of time. What opportunities for training and professional development has the project provided?The Principle Investigator of the project has been responsible for organizing and supervising all the research activities for this project. A Research Associate and a PhD student have continued to work on the project and further developed their professional research skills under the direction and mentorship by the Principle Investigator of the project. Six undergraduate students have also been trained for directed research while working together with the research associate and the graduate student since the beginning of this project. A high school summer research intern has also been trained working together with the graduate student on this project. How have the results been disseminated to communities of interest?The preliminary results and related research data obtained so far have been presented as posters and oral presentations at international conferences, academic institutions, and a peer-reviewed paper in an academic journal. What do you plan to do during the next reporting period to accomplish the goals?So far, we have completed all tasks outlined in the Objective #1 and Objective #2 as well as part of the tasks outlined in the Objective #6. Our research progress is completely in line with the time frame proposed in our proposal. Over the next year, we will analyze all the transgenic lines generated to evaluate the feasibility of using different miRNA genes to develop a cost-effective new system for transgene self-containment in switchgrass and turfgrass, producing environmentally safe transgenic products with significantly improved multiple agronomic traits as planned in the Objectives # 3 and #4 and #5. We will also continue research outlined in the Objective #6 to analyze vernalized transgenic creeping bentgrass lines containing miR319, miR528 and miR393, respectively for flowering characterization.

Impacts
What was accomplished under these goals? Objective 1.Design and synthesize two vectors combining different miRNA genes for switchgrass andturfgrass transformation. This objective was planned for year 1 of the project and we have successfully completed the task producing two chimeric gene constructs. The first construct, p35S/miR396-35S/miR319-Ubi/hyg, consists of a CaMV35S-driven miR396 gene linked to a CaMV35S-driven miR319 gene and a corn ubiquitin promoter (Ubi)-driven hygromycin resistance marker gene, hyg. The second construct, p35S/miR528-35S/miR393-Ubi/bar, consists of a CaMV35S-driven miR528 gene linked to a CaMV35S-driven miR393 gene and a corn Ubi promoter-driven herbicide resistance marker gene, bar. Simultaneously with the construction of the two transformation vectors, we also initiated plant tissue culture experiments in switchgrass and turfgrass to produce embryogenic callus for use in plant genetic transformation planned in Objectve #2. Agrobacterium-mediated plant transformation with the construct, p35S/miR396-35S/miR319-Ubi/hyg, and co-transformation with the two different constructs, p35S/miR396-35S/miR319-Ubi/hyg and p35S/miR528-35S/miR393-Ubi/bar using embryogenic callus prepared from both switchgrass and turfgrtass was then conducted. Potentially transformed cells were regenerated into plants. Molecular analysis of the regenerated plants identified transgenic switchgrass and creeping bentgrass lines harboring and expressing either two miRNA genes (miR396 and miR319) or all four miRNA genes (miR396, miR319, miR528 and miR393). Three representative transgenic lines from each group have been selected and are currently being colonally propagated in greenhouse for further analysis to reveal whether or not stacking of different miRNA genes would impact how miR396 modulates plant sterility, and assess the impact of stacked miRNA genes on various agronomic traits. Simultaneously, we also initiated work for the task outlined in Objective 6 by colonally propagating previously produced transgenic creeping bentgrass lines containing miR319, miR528 and miR393, respectively. These transgenic lines were then maintained in the cold room for 6-month vernalization. Currently, they have been moved back to the greenhouse, and we expect they would flower under normal growth conditions allowing for flowering characterization.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Liu, C., Dong, K, Du, H., Wang, X., Li, X., Huang, N., Hu, Q., Luo, H.*, Sun, X.* (2023) AsHSP26.2, a chloroplast small heat shock protein positively regulates growth and development in creeping bentgrass. Plant Cell Reports (in press) doi: 10.22541/au.168311178.81983493/v1.
  • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Zhao, G., Liu, Y., Li, L., Che, R., Douglass, M., Benza, K., Angove, M., Chen, X., Henry, C., Hu, Q., Li, Z., Luo, H. (2023) Gene pyramiding for boosted plant growth and broad abiotic stress tolerance. Plant Biotechnology Journal (in press) doi.org/10.1111/pbi.14216.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Liu, H., Todd, J. L., Luo, H. (2023) Turfgrass salinity stress and tolerance - A review. Plants 12:925.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Luo, H. (2023) Towards robust crops under environmental adversities. Invited oral presentation in 2023 Society For In Vitro Biology (SIVB) Meeting, June 10-14, 2023, Norfolk, VA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Chen, X., Yeung, J., Fiorentino, A., Hu, Q., Kuess, M, Luo, H. (2023) MiR169-NF-Y module associates with creeping bentgrass biomass production. Invited oral presentation in 2023 Society For In Vitro Biology (SIVB) Meeting, June 10-14, 2023, Norfolk, VA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Chen, Z., Chen, X., Hu, Q., Kuess, M., Luo, H. (2023) Pyramiding microRNAs for transgene containment and broad plant abiotic stress resistance. Poster presentation in 2023 Society For In Vitro Biology (SIVB) Meeting, June 10-14, 2023, Norfolk, VA.
  • Type: Other Status: Published Year Published: 2022 Citation: Luo, H., (2022) Grass genetic engineering to tackle environmental adversities. Invited seminar (virtual) in Qingdao Agricultural University, Qingdao, P. R. of China, December 2, 2022.
  • Type: Other Status: Published Year Published: 2023 Citation: 8. Luo, H., (2022) Towards robust grasses under environmental adversities. Invited seminar (virtual) in Nanjing Agricultural University, Nanjing, P. R. of China, September 2, 2022.


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:The primary target audiences for this project are plant biotechnologists in academia, industry and regulatory agencies, bioenergy crop and turfgrass breeders, bioenergy and turfgrass industries concerned about the risks associated with genetically engineered perennial grass species and interested in commercializing transgenic switchgrass, turfgrass and other perennial grass species. Changes/Problems:No changes for the proposed research in this period of time. What opportunities for training and professional development has the project provided?The Principle Investigator of the project has been responsible for organizing and supervising all the research activities for this project. A Research Associate and a PhD student have continued to work on the project and further developed their professional research skills under the direction and mentorship by the Principle Investigator of the project. Three undergraduate students have also been trained for directed research while working together with the research associate and the graduate student since the beginning of this project. A high school summer research intern has also been trained working together with the graduate student on this project. How have the results been disseminated to communities of interest?The preliminary results and related research data obtained so far have been presented as posters and oral presentations in international conferences. What do you plan to do during the next reporting period to accomplish the goals?So far, we have completed tasks outlined in the Objective #1 and started research as planned in the Objective #2. We have also started research outlined in Objective #6. Over the next year, we will continue switchgrass and turfgrass genetic transformation with the chimeric gene construct, p35S/miR396-35S/miR319-Ubi/hyg, and co-transformation with the two different constructs, p35S/miR396-35S/miR319-Ubi/hyg and p35S/miR528-35S/miR393-Ubi/bar, generating multiple independent transgenic lines expressing two or four different miRNA genes (Objective #2). These transgenic lines will then be used to evaluate the feasibility of using different miRNA genes to develop a cost-effective new system for transgene self-containment in switchgrass and turfgrass, producing environmentally safe transgenic products with significantly improved multiple agronomic traits as planned in the Objectives # 3 and #4 and #5.

Impacts
What was accomplished under these goals? Objective 1.Design and synthesize two vectors combining different miRNA genes for switchgrass andturfgrass transformation. This objective was planned for year 1 of the project and we have successfully completed the task producing two chimeric gene constructs. The first construct, p35S/miR396-35S/miR319-Ubi/hyg, consists of a CaMV35S-driven miR396 gene linked to a CaMV35S-driven miR319 gene and a corn ubiquitin promoter (Ubi)-driven hygromycin resistance marker gene, hyg. The second construct, p35S/miR528-35S/miR393-Ubi/bar, consists of a CaMV35S-driven miR528 gene linked to a CaMV35S-driven miR393 gene and a corn Ubi promoter-driven herbicide resistance marker gene, bar. Simultaneously with the construction of the two transformation vectors, we also initiated plant tissue culture experiments in switchgrass and turfgrass to produce embryogenic callus for use in plant genetic transformation planned in Objectve #2. Agrobacterium-mediated plant transformation with the construct, p35S/miR396-35S/miR319-Ubi/hyg, and co-transformation with the two different constructs, p35S/miR396-35S/miR319-Ubi/hyg and p35S/miR528-35S/miR393-Ubi/bar using embryogenic callus prepared from both switchgrass and turfgrass have been conducted. Currently, Agrobacterium-infected plant calli are being cultured and selected for potentially transformed cells, which would be subjected for regeneration into plants.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Xiaotong Chen, Jason Yeung, Andrew Fiorentino, Qian Hu, Morgan Kuess, Hong Luo (2022) Constitutive expression of a miR169 gene alters plant development and enhances drought and salt tolerance in transgenic creeping bentgrass. Poster presemtation in the 2022 Meeting of the Society for In Vitro Biology (SIVB), June 4-7, San Diego, CA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Xiaotong Chen, Charles Henry, Annalise Enger, Qian Hu, Zhengyuan Pan, Xiaoyuan Gao, Lynda McMaster-Schuyler, Peiyu Zeng, Hong Luo (2022) A dual recombination system for transgene containment and elimination in perennial grasses. Poster presentation in the 2022 Meeting of the Society for In Vitro Biology (SIVB), June 4-7, San Diego, CA.