Source: UNIV OF CONNECTICUT submitted to
UNDERSTANDING GENE FLOW IN GRASSES AS A COMPONENT OF ECOLOGICAL RISK ASSESSMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0190816
Grant No.
(N/A)
Project No.
CONS00737
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2006
Project End Date
Sep 30, 2009
Grant Year
(N/A)
Project Director
Auer, C. A.
Recipient Organization
UNIV OF CONNECTICUT
(N/A)
STORRS,CT 06269
Performing Department
PLANT SCIENCE
Non Technical Summary
The first genetically-modified turfgrass could be approved by the federal government by 2007. There is dispute about the potential benefits and risks associated with biotechnology-derived perennial grasses. This project will provide scientific information that will strengthen the ecological risk assessment process and provide new information about gene flow in turfgrasses.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2052130107050%
2052130108050%
Knowledge Area
205 - Plant Management Systems;

Subject Of Investigation
2130 - Turf;

Field Of Science
1070 - Ecology; 1080 - Genetics;
Goals / Objectives
The first genetically-modified turfgrass, a herbicide-tolerant creeping bentgrass (Agrostis stolonifera) for golf courses, could be approved by the federal government by 2007. The approaching commercialization of transgenic turfgrasses has triggered debate about the ecological risk assessment process, gene flow and future environmental impacts. This project is justified because Connecticut has about 180 public and private golf courses and these facilities are adjacent to many different native and managed plant communities throughout the state. The expected advantages of GM turfgrasses include decreased pesticide use and labor costs for the golf course industry. The potential ecological risks include gene flow that would transfer the herbicide-resistant trait to native grasses, introduced grasses and conventional creeping bentgrass. These transgenic bentgrass populations could alter weed management in sensitive natural areas such as wetlands. This project will generate evidence about the potential for gene flow and risks derived from gene flow. Based on preliminary research, gaps in knowledge exist for ecological risk assessment for herbicide-tolerant creeping bentgrass (HTCB) in Connecticut. For example, there is no information about the potential for gene flow from HTCB to Agrostis perennans, a common native perennial bentgrass. At least six native or introduced bentgrass species in Connecticut can hybridize with HTCB. Herbicide-tolerant bentgrass populations represent a potential environmental hazard in wetlands and other areas where invasive plants and weeds are managed by herbicides. This study expects to achieve the following research objectives: 1) characterizing bentgrass species biogeography and gene flow in Connecticut, 2) characterizing wetlands and other plant communities where herbicide-tolerant bentgrass could negatively effect land management and/or the environment, and 3) establishing field sites and methods for monitoring gene flow. This project will add to the science-based information for ecological risk assessment of genetically-modified turfgrasses.
Project Methods
The research objectives and proposed experiments are summarized below. Objective 1. Ecological Risk Assessment for Agrostis stolonifera (creeping bentgrass): Gene Flow, Hazard Identification and Monitoring Studies. A. Characterize the biogeography of native and introduced bentgrasses (Agrostis and Polypogon species) in Connecticut. B. Identify natural and managed plant communities containing bentgrasses, especially habitats that might be managed by herbicides. C. Study gene flow using traditional plant breeding methods between creeping bentgrass and Agrostis perenanns, a common and widely distributed native grass. D. Study gene flow between bentgrasses using molecular methods: Molecular marker methods do not rely on the presence of transgenes and have been used in many studies with natural plant populations. RAPD and AFLP methods will be employed. E. Development of field sites and methods to monitor gene flow. Objective 2. Ecological Risk Assessment for Kentucky Bluegrass: Biogeography and Gene Flow Studies. A. Characterize the biogeography of Kentucky Bluegrass (Poa pratensis) in Connecticut. B. Identify the habitats and plant communities in Connecticut containing Kentucky bluegrass and its sexually-compatible relatives. C. Gene flow Studies: Develop methods to study gene flow in Poa species.

Progress 10/01/06 to 09/30/09

Outputs
OUTPUTS: Research results have been disseminated to scientists, federal agencies, and individuals involved in land management. Information has been provided to the public through both presentations at local, regional, and national conferences. Presentations on these research topics have been made to two federal agencies (USDA, EPA). PARTICIPANTS: Graduate student: Collin Ahrens, Plant Science, University of Connecticut. The graduate student has received training through laboratory experiments, field studies, and relevant coursework. TARGET AUDIENCES: Target audiences: scientists, land managers, crop breeders, biotechnology companies, and federal agencies. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
A number of research projects have been initiated and completed during the grant period. The major outcomes of these projects are summarized here. Genetically-engineered perennial grasses are being developed for future commercialization as turfgrasses and biofuels crops. Thus, there is a critical need for scientific information that can be used to characterize potential ecological risks from exposure pathways (e.g. pollen mediated gene flow, seed dispersal, plant dispersal) and hazards (e.g. increased weediness, invasion). Research sponsored by this grant has focused on one type of genetically-engineered grass, herbicide resistant creeping bentgrass (Agrostis stolonifera). Major accomplishments have included: 1) the characterization of Agrostis distribution relevant to pollen-mediated gene flow and hybridization in the northeastern U.S., 2) the characterization of habitat favorable to Agrostis species in both natural and managed landscapes, 3) production of the first Agrostis species identification manual, and 4) training in molecular methods that will support future research on Agrostis population genetics and gene flow.

Publications

  • No publications reported this period


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Activities accomplished: Research to build a landscape-scale Habitat Suitability Model (HSM) was conducted incorporating geospatial information, ecological data, and mathematical modeling (multivariate logistical regression). Researchers visited 290 random plots (100 m2) within the 8.5 square km HSM study site in Bloomfield, Connecticut and recorded the presence/absence of bentgrass species and other ecological variables. The data is being used to create a map predicting the likelihood that bentgrasses occur in any location. As expected, survey data showed that the non-native bentgrasses preferred open, sunny habitats. Just over half of the plots with bentgrasses also contained at least one invasive plant species. Nearly half of the plots with bentgrasses occurred in public parks and utility right-of-ways. The study also showed that bentgrass populations co-occurred with nine state-listed species in the CT Natural Diversity Database in the HSM study site. Products: One oral presentation to the Connecticut Invasive Species Council. Two oral presentations to federal agencies with responsibilities for regulation of genetically-modified crops. One poster presentation at the annual meeting of the Botanical Society of America. One peer-reviewed scientific paper. PARTICIPANTS: One graduate student was supported during the academic year. The graduate student received training through coursework, field work and participation in professional meetings. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Bentgrasses (Agrostis genus) are common grasses in golf courses, agricultural fields, disturbed sites, roadsides, and grasslands. In the near future, the federal government may approve an application to commercialize genetically-modified, herbicide-resistant (HR) creeping bentgrass. This project aims to predict ecological risk by characterizing bentgrass distribution in Connecticut and the probability that pollen-mediated gene flow and seed dispersal will move the HR trait into feral and cultivated bentgrass populations. A landscape-scale Habitat Suitability Model (HSM) is being developed using geospatial information, ecological data, and mathematical modeling (multivariate logistical regression). The project will continue to collect and analyze data to characterize the potential future impact of HR creeping bentgrass on weed management, the protection of endangered species, and the removal of invasive plants. If validated, the HSM method could become an important tool to support predictive ecological risk assessments, stewardship plans, and decision-making for other types of genetically-engineered plants.

Publications

  • Auer, C.A. 2008. Ecological risk assessment and regulation for genetically-modified ornamental plants. Critical Reviews in Plant Sciences 27:255-271


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Genetically-engineered perennial grasses are being developed for future commercialization as turfgrasses, forage crops and biofuel feedstocks. This project is focusing on ecological risk assessment for an herbicide resistant creeping bentgrass that could be commercialized for use on golf courses. The potential long-term ecological risk from transgenic creeping bentgrass needs to be carefully considered because bentgrasses are not highly domesticated, are already naturalized and/or weedy in many ecosystems, and have sexually-compatible relatives in diverse plant communities. The challenge for risk assessors is predicting if the herbicide resistance trait will escape from cultivation, spread to native and naturalized bentgrass species, negatively impact the environment and/or alter future land management practices. The first accomplishment from this project was the documentation of Agrostis (bentgrass) demography and the production of a tool to identify the ten native and naturalized species in Connecticut. Information was gathered from herbaria and other sources. Plants were collected and photographed to produce a visual tool for species identification. Seeds were collected from a variety of habitats for future research projects. The second accomplishment was completion of preliminary work for experiments that will test the hypothesis that two non-native bentgrass species (A. stolonifera and A. gigantea) will alter their patterns of gap colonization and plant fitness (e.g. seed production) in sites managed with herbicides. This research will help predict how Agrostis populations carrying an herbicide resistance trait will behave in two ecological settings (agricultural and wet meadow). Current work includes greenhouse experiments to characterize the adaptability of Agrostis species to stress gradients. PARTICIPANTS: Dr. Carol Auer, Associate Professor, Department of Plant Science, University of Connecticut Mr. Collin Ahrens, PhD student, Department of Plant Science, University of Connecticut TARGET AUDIENCES: Risk assessors focusing on the potential ecological risks from genetically-modified plants.

Impacts
The primary product is the production of a Agrostis (bentgrass) identification manual that could be used by ecologists, turfgrass specialists, land managers and others. The bentgrasses are extremely difficult to identify based on vegetative and/or reproductive structures. This new manual provides the first detailed photographs of flowers, inflorescences and vegetative features that aid in identification. Taxonomic keys and distribution maps focus on the ten species found in Connecticut, but the book could be used across the U.S. because some introduced, non-native species are widely distributed.

Publications

  • Ahrens, C., Auer, C. 2007. Gene flow and the future of bentgrasses in Connecticut. Newsletter of the Connecticut Botanical Society 34:1-6
  • Ahrens, C., Auer, C. 2007. The Bentgrasses of Connecticut. A manual for identification of Agrostis and closely related species. Storrs Agricultural Experiment Station, Storrs, Connecticut


Progress 09/30/06 to 12/31/06

Outputs
In the first three months of the project, efforts have focused on characterizing the distribution and habitats of the bentgrass species reported in Connecticut. The ten closely-related species in Connecticut include three native species and seven introduced species that are currently classified in three genera: Agrostis (8 species), Apera (1) and Polypogon (1). Seeds are being obtained and plants established in the greenhouse, along with other preliminary work essential for future field work and other projects.

Impacts
Ecological risk assessment is based upon scientific knowledge regarding potential hazards and exposure routes. Information about bentgrass plant distribution and the probability of hybridization between species will provide a basis for assessing gene flow and environmental risk.

Publications

  • No publications reported this period


Progress 01/01/05 to 12/31/05

Outputs
This project is investigating the physiological, developmental and genetic basis for de novo shoot organogenesis, a process through which plant cells divide and organize into entire shoots. Experiments have been conducted to examine the effect of cyclin-dependent kinase inhibitors with structural similarity to the plant hormone cytokinin on plant development. These cytokinin analogs have been shown to alter cell cycle and cytokinin metabolism in plants. A mutant screen identified four roscovitine-resistant mutants from a T-DNA tagged population of Arabidopsis thaliana. Previous work characterized the mutant phenotypes, cytokinin oxidase activity and endogenous cytokinin levels. Current research is focusing on the mutant line ror1. Using a plasmid rescue strategy and BLAST search, a tagged gene with unknown function was identified on chromosome three in the ror1 line. A domain in this gene suggests that the protein may be a glucosyltransferase. Further analysis determined that the ror1 mutant contains only one tagged gene and that the ror1 gene is expressed in both mutant and wild type plants. Real-time PCR is being conducted to quantify expression of the ror1 gene in the mutant line. Production of the recombinant protein encoded by ror1 has been slowed by technical problems. A straightforward approach using E. coli has provided only slow-growing cell colonies. A new approach is being tried to produce large amounts of the ror1 protein for cytokinin metabolism assays. The long-term goal is to understand the function of the ror1 gene in cytokinin regulation, plant growth and organogenesis.

Impacts
The relationship between cyclin-dependent kinase inhibitors, cytokinins and shoot organogenesis has received little attention. These experiments may provide new strategies and insights to improve shoot organogenesis in plant species important to horticulture, forestry and agriculture.

Publications

  • Dwivedi, S., Auer, C. 2005. Use of ror1 mutant to understand the N-glucosylation pathway in Arabidopsis. Cytokinins and Auxins in Plant Development. Second International Symposium. Prague, Czech Republic


Progress 01/01/04 to 12/31/04

Outputs
This project is investigating the physiological, developmental and genetic basis for de novo shoot organogenesis, a process through which plant cells divide and organize into entire shoots. Experiments have been conducted to examine the effect of cyclin-dependent kinase inhibitors (olomoucine and roscovitine) with structural similarity to cytokinins on Arabidopsis plants. These cytokinin analogs have been shown to alter cell cycle and cytokinin metabolism in a variety of species. A mutant screen identified four roscovitine-resistant mutants from a T-DNA tagged population of arabidopsis. In the past year, emphasis has been placed on characterizing the phenotype and cytokinin metabolism in these mutants. For example, the roscovitine-resistant mutants show a lower concentration of the degradation enzyme cytokinin oxidase compared to wild-type arabidopsis. Identification of the T-DNA tagged gene in one mutant line has been accomplished through a plasmid rescue strategy. Using the rescued DNA sequence and a BLAST search, a gene with unknown function was identified on chromosome three. A domain in this gene suggests that the protein may be a glucosyltransferase. The long-term goal is to understand the function of the tagged gene in each of the roscovitine-resistant mutants on cytokinin regulation and shoot organogenesis.

Impacts
The relationship between cyclin-dependent kinase inhibitors, cytokinins and shoot organogenesis has received little attention. These experiments may provide new strategies to improve regeneration of plants important to horticulture, forestry and agriculture.

Publications

  • Dwivedi, S., Auer, C.A. 2004. Understanding cytokinin N-glucosyltransferase metabolism using Arabidopsis mutants. American Society of Plant Biology, Orlando, Florida


Progress 01/01/03 to 12/31/03

Outputs
This project is investigating the physiological, developmental and genetic basis for de novo shoot organogenesis, a process through which plant cells divide and organize into entire shoots. In the first three months of this project, experiments have been conducted on the physiological and developmental aspects of shoot organogenesis using Arabidopsis genetic mutants generated from previous work and obtained from outside sources. Experiments are examining the effect of cyclin-dependent kinases (CDKs) with structural similarity to cytokinins on Arabidopsis plants in vitro. Dose-response experiments for the CDKs olomoucine and roscovitine have shown that roscovitine is active at much lower concentrations than olomoucine. In some experiments, olomoucine and roscovitine give opposite responses with roscovitine inhibiting developmental events and olomoucine promoting growth. A mutant screening protocol has been developed to obtain CDK-resistant Arabidopsis mutants. Another part of this project is detailed developmental studies of shoot organogenesis in Arabidopsis. Preliminary histological work has revealed some aspects of Arabidopsis shoot organogenesis, but key features such as the initial location and pattern of cell division and pattern of vascular tissue development are still under investigation.

Impacts
The relationship between cyclin-dependent kinases, cytokinins and shoot organogenesis has received little attention. These experiments may provide new strategies to improve shoot organogenesis in plant species important to horticulture, forestry and agriculture.

Publications

  • No publications reported this period


Progress 01/01/02 to 12/31/02

Outputs
This project continues to utilize unique mutant lines of arabidopsis to understand the genetic and developmental basis for de novo shoot organogenesis. One of the critical barriers to applying biotechnology and gene transfer to woody plants and other species is the inability to reliably regenerate whole shoots from single transformed cells. Our long-term goal is to identify genes essential to de novo shoot organogenesis in all plants. Currently, we are working with five unique, non-allelic, shoot-organogenesis-deficient (sod) arabidopsis mutants which are blocked in the very early steps of de novo shoot organogenesis. In two mutants, we know that single recessive mutations block development during the stage of cellular competence or induction. Experiments with STM-GUS constructs showed the mutations blocked stm gene expression during the differentiation phase. We are using a histological approach to determine the temporal and spacial pattern of cell division leading to shoot organogenesis in arabidopsis cotyledon explants. Wild-type and sod mutants are being compared to understand how the sod mutations affect cell division patterns. Other work includes the production of backcrossed populations and studies of 20 arabidopsis mutants that have aberrant patterns of shoot organogenesis.

Impacts
A complete understanding of developmental events requires a combination of experimental approaches. We have established an exciting genetic approach to understanding de novo shoot organogenesis and are currently using a histological approach to look at the first stages of cell division leading to shoot formation.

Publications

  • Auer, C.A. 2002. Discoveries and dilemmas concerning cytokinin metabolism. Journal of Plant Growth Regulation 21:24-31
  • Zhao, Q., R. Fisher and C. Auer. 2002. Developmental phases and STM expression during Arabidopsis shoot organogenesis. Plant Growth Regulation 37:223-231