2005 Annual Report
1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter?
Worldwide reserves of oil are finite and the production of oil is projected to decline in coming decades as easily accessible deposits are depleted. In addition, there are significant environmental penalties associated with burning fossil fuels. Renewable fuels derived from plant matter have the potential to decrease our dependence on oil, but major engineering and agricultural obstacles must first be overcome by research.
Maintaining a sustainable fuel supply is critical for the U.S. economy and all the citizens of the U.S. Even a modest increase in domestic renewable fuel production would have major implications for the national economy, the environment, the maintenance of jobs throughout the nation, the vitality of rural America, and would contribute to enhanced national security by lessening a dependence on imported oil.
The primary aims of this project seek to improve the efficiency of biomass utilization through improved crop genetics. To accomplish this, the project is building on existing knowledge of cell wall biosynthesis to create genetically modified lines of a high-yielding energy crop Panicum virgatum (switchgrass) that are more easily converted to ethanol. To increase fundamental understanding of grass cell wall biosynthesis, a molecular genetic approach for identifying genes that alter cell wall composition has been adopted which utilizes the model temperate grass Brachypodium distachyon.
This project contributes strongly to National Program 307, Bioenergy and Energy Alternatives. This project will apply a variety of biotechnological approaches to the problem of producing renewable and affordable energy from plant matter. Traits that are being manipulated in potential energy crops are also important in forage grasses and legumes that are covered in National Program 205 (Rangeland, Pasture, and Forages). In addition, the scope and wide applicability of some of the molecular and genomics technologies that this project is developing will contribute directly and indirectly to all other programs that involve, or depend on, genetic or molecular data, functional genomics, gene discovery, and germplasm development; i.e., National Programs 301 (Plant, Microbial, and Insect Genetic Resources, Genomics and Genetic Improvement), 302 (Plant Biological and Molecular Processes), 304 (Crop Protection and Quarantine), 306 (Quality and Utilization of Agricultural Products), and 107 (Human Nutrition).
2.List the milestones (indicators of progress) from your Project Plan.
Milestones Objective 1: Alteration of the activity of key lignin biosynthetic enzymes
• Gene cloning
• Switchgrass cultivar Alamo transformation
• Analysis of transgenic switchgrass for degree of silencing
• Development of switchgrass transformation technology (including new accessions)
• Selection and propagation of lines for field trials
• Initiate field trials
Milestones Objective 2: Link transgenes to male sterility
• Gene cloning
• Swithgrass cultivar Alamo transformation
• Analyze male sterility constructs for pollen viability
• Analyze transgenics for lignin composition/degree of silencing
• Selection of lines for field trials
Milestones Objective 3: Identify novel genes affecting cell wall composition
• Optimize Brachypodium transformation and begin generating mutants
• Optimize FTIR screen and begin screening mutants
• Isolate Brachypodium promoters
• Characterize and prioritize Brachypodium mutants
• Clone Brachypodium genes/begin characterization
4a.What was the single most significant accomplishment this past year?
Breeding progress and biotechnological approaches to crop improvement in switchgrass are hampered by a lack of significant information about its genetic structure and makeup. To create a resource for genetic studies and biotechnological approaches, scientists at the Western Regional Research Center, in collaboration with ARS and non-ARS scientists in Lincoln and Kearney, Nebraska sequenced 11,990 expressed sequence tags (ESTs) that represented over 7000 unique genes. These will be used as a resource for creating gene based markers to facilitate breeding efforts as well as for isolating genes with critical roles in biomass production or utilization that can be altered via modern molecular techniques. These gene-based markers are the first such resources available to identify switchgrass germplasm correlated with valuable traits such as biomass yield, drought tolerance, and seed production. The same ESTs are a source of candidate genes for engineering traits such as lignin content, and identified sequences for genes in the lignin synthetic pathway are being used to reduce lignin content for more efficient switchgrass biomass conversion.
4b.List other significant accomplishments, if any.
To allow researchers to utilize Brachypodium as a modern model organism to study cell wall composition it is necessary to develop genomic resources. In collaboration with non-ARS scientists at the University of Nebraska, Kearney we constructed cDNA libraries from five different tissues and began determining expressed sequences. Approximately, 16,000 raw sequences have been determined to date with a goal of obtaining 20,000 sequences. These sequences will be used to generate molecular markers to facilitate mapping in Brachypodium, to identify genes that may play a role in cell wall composition and to provide anchors for the development of a physical map. In addition, the cDNA libraries can be used to identify genes of interest that have not been identified during the sequencing project.
4c.List any significant activities that support special target populations.
5.Describe the major accomplishments over the life of the project, including their predicted or actual impact.
The major accomplishments of the project to date are the transformation of switchgrass with constructs designed to decrease lignin content, the development of a transformation protocol for Brachypodium, and the development of genomic resources for switchgrass and Brachypodium.
To produce switchgrass with decreased lignin content, we cloned partial sequences for four lignin biosynthetic genes from switchgrass. These sequences were then used to construct transgenes designed to down-regulate the corresponding endogenous genes. These constructs were introduced into switchgrass and the resulting transgenic plants are in the process of being characterized for gene expression and lignin content. This work will lead to enhanced switchgrass lines that biomass producers, and forage breeders will use to more efficiently produce energy, or livestock.
To develop Brachypodium as an effective tool to study the genetic basis of cell wall composition, we developed a high-efficiency Agrobacterium-mediated transformation protocol. This method will be used to generate a large mutant population that will be screened to identify genes involved in determining cell wall composition. In addition, this method allows Brachypodium to be used as a model to quickly test ideas before applying them to temperate grasses and cereals with longer generation times and more difficult transformation protocols.
Underpinning genetic progress and understanding of genome structure in Brachypodium and switchgrass is the need for more sequence data in these relatively uncharacterized species. We have initiated gene sequencing in these species and these efforts have already provided a large gene inventory and have helped to identify sequences for development as molecular markers. Ultimately the markers will aid in genetic characterization and breeding efforts while the genes themselves can be manipulated in biotechnological approaches to crop improvement.
6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
All results from this project are made available to all researchers and the public through publication of results in the scientific literature. Information on all sequences generated by this project is deposited with GenBank (National Library of Medicine) for unlimited public access. Preliminary results and important results prior to publication are made available to other laboratories as appropriate through informal contacts, seminars, and meetings. No constraints to the adoption and durability of the results are known.
Switchgrass transformation expertise developed by Drs. John Vogel and Christian Tobias has resulted in inquiries from several corporations and we have effectively facilitated their efforts in this area. CRADA project planning is underway with two companies that in the long term will result, if successful, in commercialization of improved switchgrass varieties with value added traits.
7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
Vogel, J.P., Raab, T.K., Somerville, S.C. 2004. Mutations in pmr5 result in powdery mildew resistance and altered cell wall composition. Plant Journal. 40:968-978.
Tobias, C.M., Chow, E.K. 2004. Structure of the Cinnamyl-Alcohol Dehydrogenase Gene Family in Rice and Promoter Activity of a Member Associated with Lignification. Planta. 220:678-688.