Herbaceous Crops Research

Development of In Vitro Culture Systems for Switchgrass (Panicum virgatum)

Research Funded by: U.S. Department of Energy (DOE), Office of Fuels Development through the Oak Ridge National Laboratory

Project Manager: S. B. McLaughlin, 865-574-7358, mclaughlinsb@ornl.gov

Performing Organization: University of Tennessee, Department of Plant and Soil Sciences, Knoxville, TN 37901-1071

Principal Investigator(s): B.V. Conger, 865-974-8833, congerbv@utk.edu

Contract Number: 11X-SY161C

Contract Period: 07/97-12/02

Contract Funding: FY 2000: $33,360

Objective: Develop efficient and repeatable regeneration systems from cell and tissue cultures of switchgrass (Panicum virgatum) and utilize these systems for genetic transformation of this species.

Approach/Background: Research efforts to improve biomass production of switchgrass should also include the potential applications of biotechnology. Within six months after initiation of the project in 1992, we had developed protocols for plant regeneration through both organogenesis and somatic embryogenesis from tissues and cells of various explants cultured in vitro. Although whole plants were regenerated from several genotypes, we found that, in general, lowland cultivars, such as Alamo and Kanlow, were much more amenable to tissue culture manipulations than upland cultivars such as Cave-in-Rock and Blackwell.

During the next 5-6 years, we developed several systems for regenerating plants. These included the production of inflorescences directly from cultured split top nodes of tillers in the two to four node stage and axillary shoot production from split half nodes below the top node. The latter represents an excellent potential system for micropropagation because the shoots proliferate directly from the primary; explant without an intervening callus. This reduces the possibility of inducing genetic and chromosomal changes that frequently result from cells going through cycles of dedifferentiation and redifferentiation (so-called somaclonal variation). Other regeneration systems include multiple shoot formation from both young seedlings and directly from mature caryopses and regenerable suspension cultures by somatic embryogenesis. The production of thousands of embryos in liquid offers the potential of mass propagation on a much larger scale than axillary shoot formation (mentioned above) and also provides targets for genetic transformation. Development of the above systems has allowed us to move to the next step of initiating gene transfer experiments. These experiments were first conducted using microprojectile bombardment with two different constructs and then in early 2000, with Agrobacterium.

Developed protocols with regard to medium formulations incubation conditions etc., have been utilized in attempts to obtain haploid and/or doubled haploid plants from anther cultures. Several thousand anthers from genotypes of both upland and lowland types have been plated. However, limited success has been obtained in regenerating plants. A more recent approach involves isolated microspore culture utilizing protocols developed for wheat, triticale, rye, and timothy.

Status/Accomplishments: Gene transfer experiments were conducted in the latter part of 1999 and 2000 with microprojectile bombardment. Genes utilized were the reporter uidA (gus) in which tissues stain blue when incubated with a substrate, X-gluc, the reporter gene gfp (green fluorescent protein) and the selectable marker bar which confers tolerance to phophinothricin based selective agents. Transformation was obtained with the constructs, pAHC25 (gus and bar genes) and a new plasmid (gfp and bar genes) constructed in our laboratory. GUS was expressed in callus tissue and floral parts including pollen grains, ovaries and lodicules. GFP was expressed in callus and in leaf tissue and pollen of T₁ plants. Plants tolerant to the herbicide Basta (bar gene) were obtained from both constructs

Sexual transmission of both the gfp and bar genes and their expression in T₁ progeny was demonstrated and their presence was confirmed by Southern blot hybridization. A Southern analysis, utilizing the restriction enzyme KpnI which cuts the GFP-BAR plasmid only once, indicated copy numbers of gfp ranging from as few as 3-4 to numerous. Plants with the lowest copy numbers of transgenes had an approximate 1:1 ratio of GFP fluorescing: nonfluorescing pollen grains and a higher rate of sexual transmission of transgenes than plants with high copy numbers.

Genetic transformation experiments beginning in early 2000 were also focused on use of Agrobacterium tumefaciens. The strain AGL1 containing the 18.15 kb transformation vector pDM805 was used to infect various explants. Success was obtained in most experiments for both gus and bar genes. Transient GUS expression was observed in tissues (calluses, caryopses, leaf and seedling segments, etc.) incubated with the bacterium. GUS expression was also observed in pollen and ovaries of T₀ plants. Calluses selected on medium containing 10 mg^-l bialaphos regenerated plants that were tolerant to Basta. Transformation frequencies ranged from 14.5 to 25% indicating high efficiency. Presence of both the bar and gus genes in T₀ plants was confirmed by Southern blot hybridization.

Six Alamo clones were used for anther culture experiments. From 15,720 anthers plated, hundreds of green plants were regenerated that could be established in soil. However, these were produced from only a very few anthers. Also, the period between the initial plating of anthers and receiving the first response was very long, 4-6 months. Furthermore, growth of the plantlets was slow and the mortality rate was high. As mentioned, a new approach utilizing isolated microspore culture is being investigated.

Yield trials that included Alamo and our 2-_, 4-_, and 20-clone synthetics, which originated from the original establishment of regenerated plants in the field, plus three experimental breeding lines from Charles Taliaferro’s program at Oklahoma State University were successfully established at Knoxville and at the Highland Rim Experiment Stations at Springfield, Tennessee. Dry matter yields obtained in 2000 (the establishment year) indicated that the best of our synthetics are superior to Alamo and at least equal to those from Oklahoma State.

Project Location(s): Most of the work is being conducted on the University of Tennessee campus at Knoxville where the P.I. is located. A field experiment (mentioned above) was established at Springfield and another is planned for the West Tennessee Experiment Station at Jackson, Tennessee.

Publications and Presentations:

1. Conger, B. V. 2000. Genetic transformation experiments in orchardgrass and switchgrass. Agronomy Abstracts, p. 176.
2. Somleva, M. N., and B. V. Conger. 2000. Agrobacterium mediated transformation of orchardgrass and switchgrass. Agronomy Abstracts, p. 181.
3. Conger, B. V., H. A. Richards V. Rudas, and J. K. McDaniel. 2000. Transformation of switchgrass with GFP-BAR. 3^rd Intern. Crop Sci., Congress Book of Abstracts, p.240.
4. McDaniel, J. K., H. A. Richards, H. Sun, and B. V. Conger. 2000. A GFP-BAR construct for switchgrass transformation. Abstract. InVitro Cell. Dev. Biol. 36(3) Part II p.62A.

Summary Date: June 2001

Development of Optimal Establishment and Cultural Practices for Switchgrass and Other Energy Crops

Research Funded by: U.S. Department of Energy (DOE), Office of Fuels Development through Oak Ridge National Laboratory.

Project Manager: S. B. McLaughlin, 865-574-7358, mclaughlinsb@ornl.gov

Performing Organization: Auburn University, Auburn, AL 36849-5145, http://www.auburn.edu

Principal Investigator(s): David Bransby 334-844-3935, dbransby@acesag.auburn.edu

Contract Number: 19X-XY164C

Contract Period: 06/97 - 05/02

Contract Funding: FY 2000: $139,800

Objective: The goals of this project are (a) to improve establishment procedures and management practices for switchgrass, (b) to evaluate different varieties of switchgrass for optimal biomass production, (c) to establish the relationship between research plot yields of switchgrass and yields from commercial fields, and (d) to evaluate alternative species to switchgrass for energy.

Approach/Background: Establishment procedures under evaluation include use of different planters, and the effect of insecticide application at planting. Variety tests are being conducted with Alamo, Kanlow and Cave-in-Rock switchgrass at five locations, and with eight varieties at one location.

Management practices under evaluation include frequency, height and date of harvesting, effects of row spacing, and effects of nitrogen (N) fertilization. Three commercial scale tests are being conducted to compare yields with small research plots.

New perennial species to be evaluated as alternatives to switchgrass include giant reed (Arundo donax), Miscanthus x giganteus and sericea lespedeza (Lespedeza cuneata). Annual species to be evaluated include ryegrass (Lolium multiflorum), forage soybeans, sunn hemp and kenaf.

Status/Accomplishments: Extremely severe drought was experienced throughout Alabama in the summer of 2000. No distinct advantage has been identified for any particular seeding equipment, or for the application of insecticides at the time of seeding. Alamo yields continue to be equal or better than that of all other varieties at all locations.

Wide row spacing provided equal or better yields than solid stands. Yields from one and two cuts per year were generally similar, but basal cover was lower in one-cut plots. Yield increased with N fertilization up to 112 kg of N/ha, but there was little response to higher rates, although this was dependent on row spacing.

Yields from commercial scale fields were generally 10% to 30% lower than those observed in small research plots. Yield of cotton, corn and soybeans were equal or higher when these crops followed switchgrass in a rotation, compared to being planted on land that had previously been fallow.

Sericea lespedeza and Arundo donax provided the best yields from the alternative perennial crops tested, and ryegrass and kenaf provided the best yields among the annuals.

Project Location(s): Decatur, AL (Tennessee Valley); Crossville, AL (Sand Mountain); Camp Hill, AL (Piedmont); Tallassee, AL (E. V. Smith Research Center); Marion Junction, AL (Black Belt); Headland, AL (Wiregrass); Fairhope, AL (Gulf Coast).

Publications and Presentations:

1. Ma, Z., C. W. Wood and D. I. Bransby. 2000. Impacts of soil management on root characteristics of switchgrass. Biomass and Bioenergy 18: 105-112

Summary Date: July 2001

Evaluation of Switchgrass Cultivars and Cultural Methods for Biomass Production in the South Central United States

Research Funded by: U.S. Department of Energy (DOE), Office of Fuels Development through Oak Ridge National Laboratory

Project Manager: S. B. McLaughlin, 865-574-7358, mclaughlinsb@ornl.gov

Performing Organization: Texas A&M University Agricultural Experiment Station, 3507 HWY 59 E., Beeville, TX 78102

Principal Investigator(s): William R. Ocumpaugh, 361-358-6390, ocumpaug@fnbnet.net

Contract Number: Subcontract 19XSY091C

Contract Period: May 1992 - July 2002

Contract Funding: FY 2000: $155,000

Objective: Evaluate both upland and lowland switchgrass ecotypes from the breeding program at OSU (Dr. Charles Taliaferro) compared to the best upland and best lowland cultivar at Stephenville, Dallas, and College Station, TX; Hope, AR; and Clinton, LA. Determine Best Management Practices necessary to improve the reliability of establishing a stand of switchgrass. Conduct controlled environment studies to develop Science-Based knowledge regarding switchgrass stand establishment. Select for specific plant traits that are thought to control seedling success. Determine the effect of five N rates and five dates of fall harvest (one harvest per season) on switchgrass biomass yield and stand persistence, and determine the effect of N rates and time of fall harvest on switchgrass biomass composition (biofuel quality). Evaluate the potential response to targeted irrigation on land that is considered prime switchgrass growing land. Determine the effects of switchgrass production on soil C sequestration, soil microbial biomass C and N, and soil N dynamics compared to other forage grasses and cropping/vegetation systems. This objective will be carried out on both long-term and seasonal bases. Utilize the above measurements plus other selected soil properties to estimate effects of switchgrass production on soil quality as compared to other forage grasses and vegetation systems. Since Switchgrass stands cannot be maintained at the southern locations in Texas, we will evaluate Bundleflower, Desmanthus virgatus and D. subulatus, and shrubby legume that is native to Northern Mexico and Southern Texas, and Giant Reed, Arundo donax, a C-3 perennial that is found along the roadsides all over the eastern half of Texas. Document stover yields from corn and sorghum crops grown at four locations in Texas and compare them to the grain yields on the same plots.

Approach/Background: The efficient production of biofuel crops require the complete management package from how to consistently establish the crop, the proper fertility requirements, as well as when and how to harvest it (including the consequences/benefits of not harvesting it when yield is maximized).

Alamo switchgrass is native to Texas, and has been documented to be a very productive plant throughout the South once it is established. A major constraint to more rapid adoption of switchgrass as a biofuel crop has been the difficulty with which both researchers and producers have had with obtaining a good stand. Rainfall in much of the western part of the South is NOT reliable, so obtaining a good stand is sometimes limited by poor moisture conditions following seeding, and this unreliable rainfall also limits yield potential of already established stands. In research plots, even with irrigation, stand failures are still common, indicating that the plant has other inherent problems. In addition, we do not have a single herbicide that will control the weeds and not be phytotoxic to our lowland ecotypes of switchgrass. These issues must be resolved before switchgrass will have any value as a biofuel crop. The best management practices to most effectively grow and harvest switchgrass were also lacking when this project was initiated. The indirect benefit of growing switchgrass as crop needed to be evaluated to document the long-term effects on soil quality and sequestration of organic carbon. We have approached this entire project at multiple locations in Texas as well as one location each in Arkansas, and Louisiana.

Status/Accomplishments: We have obtained enough location-years of data to indicate there may be a critical period when rainfall, if deficient, has a devastating effect on production of established switchgrass. As total rainfall can be low, and we can still have good yields, if there is adequate rain in the late spring.

Alamo switchgrass is one of our best variety in the long-term studies. In the newer variety trials, most of Taliaferro's Lowland types continue to show promise. The Upland types are ALL inferior to the Lowland types, with the yield difference in the 2X to 3X range between these two types. Preliminary yields of some alternative species appear promising including Arundo donax with yields in the range or 20,000 kg/ha and up. We have the first years yield estimates on sorghum and corn stubble from four location in Texas, and they look like they are high enough to justify harvesting if this year is any indication of normal stubble yields.

The N-rate by fall harvest management study conducted at Dallas, Hope, and Yoakum document a small yield losses if harvest is delayed into the fall and winter, but not as large as we had indicated in earlier reports. The 1998 and 1999 data from Dallas suggests there are losses are larger than we observed in 2000 at Dallas or any other location in any year. We are fairly confident that these earlier losses were related to very high populations of rats and rabbits at the Dallas site. The fitted dry matter yield losses for all other locations and years are in the 10 to 11 kg/ha/day range or less. Suggesting that if you delayed harvest by 100 days you will expect to give up about 1000 to 1100 kg/ha in harvestable dry matter. The N response has been fairly flat and quadratic, with the fitted data indicating that yields maximize at about 165 kg of N per hectare, but since these responses are quadratic, economic maximums are likely closer to 120 kg/ha. In addition, lodging is quite severe at some sites when the N rates are pushed beyond the 120 kg/ha.

Intensive interest currently exists in the use of soil biological parameters as indicators of soil quality because these parameters respond more rapidly to changes in soil management than does soil organic C (SOC). Soil microbial biomass (SMB) is the most active fraction of soil organic matter and is responsible for nutrient cycling/turnover in soils, is a source/sink of N, and may be used to predict changes in soil quality long before differences are observable as changes in SOC. Initial results from soil samples collected at four Texas locations and one location each in Louisiana and Arkansas showed that total SOC under switchgrass may be lower compared to other cropping scenarios, especially other adapted forage grasses and forest. Switchgrass had greater SOC than cultivated treatments, however, SOC under switchgrass is expected to become similar to long-term grass and forested systems with time. Although SOC was generally not highest for switchgrass, SMB C/SOC was proportionally greater for switchgrass at most locations, implying potential improvement in soil quality and more active nutrient cycling with switchgrass. The portion of SOC that exists as SMB C has been used as an indicator of soil quality, with increasing proportions indicating enhanced quality.

Switchgrass establishment failures continue to plague our research. We feel our problem is associated with poor seedling traits, incomplete knowledge about proper planting time and other cultural practices (lack of science-based Best Management Practices for switchgrass establishment), and not having a good selective herbicide to reduce the tremendous weed load that we regularly encounter. We are addressing all of these issues in several sets of experiments. Although we do not have all the issues solved, we have found promising results. We have identified and cloned 24 lines of switchgrass with low seed dormancy; we plan to produce seed on these 24 clones at four sites in 2001. We have identified individual plants with improved seedling mass at two-weeks after emergence, and have cloned these for further development. We have identified some promising herbicides and will continue to evaluate these with different management approaches.

Project Location(s): Beeville, TX, Yoakum, TX, College Station, TX, Overton, TX, Dallas, TX, Stephenville, TX, Hope, AR, and Clinton, LA.

Publications and Presentations:

1. Cassida, K. A., W.R. Ocumpaugh, and J. Grichar. 2000. Using herbicides for improving establishment of switchgrass. Proc. American Forage and Grassland Council. 9:196-200. July 15-17, 2000. Madison, WI.
2. Lobo-Alonzo, P .J., F. M. Hons, W. R. Ocumpaugh, M. A. Hussey, J .P. Muir, J. C. Read, B. C. Venuto, K. A. Cassida, and W. J. Grichar. 2000. Changes in soil carbon and nitrogen under switchgrass. p. 247, Agronomy Abstracts. Madison WI.
3. Nerada, J. D., W. J. Grichar, W. R. Ocumpaugh, K. A. Cassida, G. W. Evers, J. N. Rahmes, and V. B. Langston. 2000. Tolerance of switchgrass to herbicides. Tex. Plant Prot. Conf. 12:31.
4. Ocumpaugh, W. R., M. A. Hussey, J. C. Read, J. P. Muir, F. M. Hons, G. W. Evers, K. A. Cassida, B. A. Venuto, and W. J. Grichar, Jr. 2000. Switchgrass for biofuel: A Texas perspective. p. 177 Agronomy Abstracts. Madison WI.
5. Ocumpaugh, W. R., M. Hussey, J. Read, J. Muir, F. Hons, G. Evers, K. Cassida, D. Kee, B. Venuto, and J. Grichar. 2000. Evaluation of switchgrass cultivars and cultural methods for biomass production in the south central U.S. (Annual report 1999.) Submitted to Oak Ridge national Laboratory, Oak Ridge, TN. February 2000. 90 pages.
6. Sanderson, M.A., and R.L. Reed. 2000. Switchgrass growth and development in response to water, nitrogen, and plant density. J. Range Management 53:221-227.

Summary Date: June 2001

Genetic Variation Among Switchgrasses for Agronomic Traits, Forage Quality, and Biomass Fuel Production

Research Funded by: U.S. Department of Energy (DOE), Office of Fuels Development through the Oak Ridge National Laboratory

Project Manager: S.B. McLaughlin, 865-576-8143, mclaughlinsb@ornl.gov

Performing Organization: Agricultural Research Service, U.S. Dept. of Agriculture, 344 Keim Hall, P.O. Box 830937, University of Nebraska, Lincoln, NE 68583-0937.

Principal Investigator(s): Kenneth P. Vogel, 402-472-1564, kpv@unlserve.unl.edu

Contract Number: DE-AI05-90OR2194

Contract Period: August 1, 1997 - September 30, 2001

Contract Funding: FY 2000: $358,285

Objective: Develop improved switchgrass cultivars and associated management practices for use in the agricultural production of biomass for energy in the northern Great Plains and Midwest States. Develop information on the economic costs associated with the production of switchgrass biomass in the central and northern Great Plains using information obtained from farmers in on-farm trials.

Approach/Background: The current research is the third phase of a research program that was initiated in 1990. The first phase of this research, 1990-1992, evaluated all available cultivars and elite strains and identified cultivars and strains that had the most potential for use as biofuel crops. The second phase of research was for the period August 1993 to September 30, 1997. Research focused on five specific areas to address specific breeding, genetics, and production concerns. The five areas were: (1) Molecular genetic studies on classifying switchgrass germplasm and developing molecular genetic markers; (2) determine the feasibility of producing F1 switchgrass hybrid cultivars, (3) determine the optimum rate of N fertilization and stage of maturity at harvest that will result in the largest biomass yields per acre; (4) determine the effectiveness of new herbicides in aiding switchgrass establishment by reducing weed competition; (5) determine if vesicular-arbuscular micorrhizae (VAM) improve the establishment of switchgrass and the efficiency of nutrient utilization. Research on objectives 1, 2, and 4 is continuing. Research on objectives 3 and 4 has been completed.

In this phase, the project has the following objectives: (1) continue to determine the feasibility of producing F1 switchgrass hybrid cultivars, (2) continue the development of switchgrass cultivars for use as biofuel crops USDA Hardiness Zones (HZ) 2, 3, 4, and 5; (3) continue research on developing molecular markers and genetic tools to be utilized in switchgrass breeding; (4) conduct cooperative switchgrass breeding work for the Lake States in cooperation with the University of Wisconsin. Switchgrass is photoperiod responsive so cultivars have to be developed for specific hardiness zones. Cooperative research at the University of Wisconsin is enhancing the capability to develop improved switchgrass cultivars for research for HZ 2 and 3. In addition, research was initiated to compare the carbon budget of switchgrass grown as a biomass fuel crop with corn.

Economic analysis conducted by the Oak Ridge National Laboratory, U.S. Department of Energy and the Economic Research Service, U.S. Department of Energy (Walsh et al., 1998) indicated that herbaceous biomass crops such as switchgrass could compete favorably with conventional crops in the upper Great Plains. On the basis of this analysis, in 1999, the project was expanded at the request of the Oak Ridge National Laboratory to include: (5) development of a white paper on the assessment of the potential for genetic modification of herbaceous plants for fuel feedstock; (6) establish switchgrass field trials in North Dakota in cooperation with the USDA-ARS Northern Plains Research Laboratory, Mandan, ND (ARS-Mandan) to assess the biomass yield of switchgrass cultivars in North Dakota; (7) establish switchgrass field trials in South Dakota in cooperation with South Dakota State University (SDSU) via a Specific cooperative Agreement to assess the biomass yield of switchgrass cultivars in South Dakota.

In additional, a proposal was requested to significantly expand research on developing switchgrass as a biomass fuel crop for the Northern Plains area in FY 2000. The expanded projects for FY 2000 included (8) determine the biomass yields of switchgrass grown on commercial scale production fields in potential production regions of Nebraska, South Dakota, and North Dakota and determine the economic costs associated with switchgrass biomass production using commercial scale farm equipment by participating farmers. Research managed and coordinated by ARS-NE and the Univ. of Nebraska. (9) Determine and quantify the environmental benefits associated with switchgrass biomass production in the upper plains including changes in soil carbon levels and other changes in soil quality. (10) Evaluate the yield potential and production economics of managing former CRP grasslands as a biomass cropping system. Determine the best agronomic practices for converting and managing former CRP for use in biomass production. Determine effects of management practices including harvesting schemes on wildlife habitat quality. Determine effect of management of former CRP as a biomass crop on soil quality including changes in soil carbon. Research managed and coordinated by South Dakota State University (SDSU). (11) Develop new technologies and plant germplasm to address specific production problems including research on seed quality, mineral requirements, and diseases of switchgrass.

Status/Accomplishments: The main accomplishment in 2000 was developing the research plans for the new research areas, establishing cooperative agreements with cooperating institutions and scientists, and implementing the research. The new research involves cooperative research with the South Dakota State University, the University of Wisconsin, University of Nebraska, USDA-ARS at Mandan, ND, and the USDA-NRCS. Research was initiated in all the new areas. Field scale trials were established on four farms in Nebraska and plans were developed for establishing additional on-farm trials in South Dakota and North Dakota in 2001. Research plans and arrangements were made for determining the feasibility of converting various types of Conservation Reserve Program (CRP) into biomass production fields and fieldwork was implemented.

A white paper on the feasibility of genetically modifying plants for use as feedstocks for energy production was completed and submitted to DOE (see below publication by Vogel and Jung). In summary, a review of research on breeding improved forages and the genetic systems controlling cell wall composition indicated that it should be highly feasible to genetically modify the feedstock quality of switchgrass and other herbaceous plants using both conventional and molecular breeding techniques. Effectiveness of breeding to modify herbage of switchgrass and other perennial and annual herbaceous species has already been demonstrated. The use of molecular markers and transformation technology will greatly enhance the capability of breeders to modify the plant structure and cell walls of herbaceous plants. It will be necessary to monitor gene flow to remnant wild populations of plants and have strategies available to curtail gene flow if it becomes a potential problem. It also will be necessary to monitor plant survival and long term productivity as affected by genetic changes that improve forage quality. Breeding for improved feedstock quality will likely affect the rate of improvement of biomass production per acre. If the same level of resources are used, multi-trait breeding simply reduces the selection pressure and hence the breeding progress that can be made for a single trait. An expanded version is available as: Vogel, K.P. and H.G. Jung. 2001. Genetic modification of herbaceous plants for feed and fuel. Critical Reviews in Plant Sciences 20:15-49.

Project Location(s): Lincoln, Mead, Concord, and Clay Center, NE, Madison, Arlington, and Marshfield, WI, Brookings, Highmore, Bristol, Pierre, Cottonwood, SD, Mandan, Streeter, ND

Publications and Presentations:

1. Vogel, Kenneth P. 2000. Improving warm-season grasses using selection, breeding, and biotechnology. p. 83-106. In: K.J. Moore and B. Anderson (eds.) Native warm-season grasses: Research trends and issues. Crop Science Special Publication Number 30. Crop Science Society of America and American Society of Agronomy, Madison, WI.
2. Vogel, K.P. and H.G. Jung. 2000. Genetic improvement of switchgrass and other herbaceous plants for use as a biomass fuel feedstock. Oak Ridge National Laboratory, U.S. Dept. of Energy Publication ORNL/Sub/90-90OR21954/1. 39 p.
3. Brejda, John J. and Kenneth P. Vogel. 2000. Switchgrass fertilization and harvest management as a biofuel crop in the Midwest. Speaker and Poster Abstracts. Carbon: Exploring the Benefits to Farmers and Society. August 29-31, Des Moines, IA.
4. Vogel, K.P. 2000. Variation for winter survival in a high IVDMD switchgrass population Annual Meetings Abstracts ASA/CSS/SSSA. p. 176.
5. Smart, A.J., L.E. Moser, and K.P. Vogel. 2000. The effects of divergent selection for tiller number in big bluestem and switchgrass seedlings. Annual Meetings Abstracts ASA/CSS/SSSA. p. 94.
6. Brejda, J.J., K.P. Vogel, and D.T. Walters. 2000. Yield and N removal by switchgrass managed for biomass production. Annual Meetings Abstracts ASA/CSS/SSSA. p. 128.

Summary Date: July 2001

Selection and Breeding of New Switchgrass (Panicum virgatum) Varieties for Increased Biomass Production

Research Funded by: U.S. Department of Energy (DOE), Office of Fuels Development through the Oak Ridge National Laboratory

Project Manager: Samuel B. McLaughlin, 865-574-7358, mclaughlinsb@ornl.gov

Performing Organization: Oklahoma State University, Department of Plant & Soil Sciences, Stillwater, OK 74078-6028, http://clay.agr.okstate.edu/plantsoilsci/plasosci.html

Principal Investigator(s): Charles Taliaferro, 405-744-9627, cmt@mail.pss.okstate.edu

Contract Number: 19X-SY162C

Contract Period: 08/97-7/02

Contract Funding: FY 2000: $37,250

Objective: To develop new switchgrass varieties with enhanced biomass yield potential for the central and southern United States. Supporting objectives include: 1) the development of a comprehensive switchgrass germplasm collection that is fully characterized for major performance traits, 2) elucidation of the breeding behavior of switchgrass, and 3) identification of best breeding procedures for switchgrass.

Approach/Background: Switchgrass is a polymorphic, perennial, cross-pollinated species comprised of many ecotypic forms adapted to a range of climatic and edaphic conditions. Biomass production in switchgrass is a quantitative trait governed by many genes. Genotypic recurrent selection (GRS) is used to increase biomass yield in breeding populations by increasing the frequency of genes in those populations governing high yield capability. Selection of plants with high breeding value is based on the performance of their progeny. Recurring breeding cycles (selection and inter-mating of superior plants) effect incremental improvement for biomass yield. We are conducting GRS in upland and lowland ecotypic switchgrass populations to provide improved varieties for different climatic and edaphic conditions. Switchgrass breeding is facilitated by an ongoing effort to improve the quantity and quality of switchgrass germplasm available for research and by investigations of basic genetic and reproductive characteristics of the species.

Status/Accomplishments: Accessions within a comprehensive switchgrass germplasm collection were grouped into core subsets based on cluster analysis of descriptor data taken over the past several years. Crossing blocks of each core set were field established to provide foundation seed. The core subsets provide a means for long-term maintenance of the genetic diversity within the collection and will be made available to support breeding and other research.

Ongoing breeding and evaluation studies indicate that recurrent selection within upland and lowland ecotypic populations has increased biomass yield potential. Multi-environment field-testing of several experimental cultivars synthesized using elite parents chosen from breeding populations strengthened the database on their performance. Seed scale-up was continued for two synthetics, and initiated for a third synthetic, in preparation for potential release as new commercial cultivars. Isolated field crossing blocks were established to produce seed of 25 new synthetic cultivars.

New cycles of GRS were initiated for three lowland and two upland breeding populations. Heterosis for biomass yield was further documented for some F₁ hybrids, indicating potential for developing hybrid cultivars. A program of inbreeding in conjunction with selection was continued as a potential means of facilitating breeding improvement.

The number of tillers and width and length of leaves were identified as the principal components contributing to switchgrass biomass yield. Evaluation of clonal switchgrass plants (half-sib plants) under high and low yield environments indicated the effects of environment on relative yield performance to be small.

Project Location(s): Stillwater, OK; Perkins, OK; Chickasha, OK; Haskell, OK; Tipton, OK; Collaborator sites include: Mandan, ND; Madison, WI; Lincoln, NE; Manhattan, KS; Booneville, AR; College Station, TX; Dallas, TX; Stephenville, TX; Blacksburg, VA; Harpenden Hertfordshire United Kingdom

Publications and Presentations:

1. Taliaferro, C. M., K. P. Vogel, J. H. Bouton, S. B. McLaughlin, and G. A. Tuskan. 1999. Reproductive characteristics and breeding improvement potential of switchgrass. Pp. 147-153, In
2. Overend, R. P. and E. Chornet (Eds.), Proc. 4^th Biomass Conf. Americas. Aug. 29 - Sept. 2, 1999.
3. Das, M. K. and C. M. Taliaferro. 2000. Heritability of biomass yield in switchgrass. P. 101, Agronomy Abstracts, Amer. Soc. Agron., Madison, WI.
4. Taliaferro, C. M. and A. Parco. 2000. Inheritance of Allozyme markers in switchgrass. P. 104. Agronomy Abstracts, Amer. Soc. Agron., Madison, WI.
5. Taliaferro, C. M. 2000. Breeding seed- and Vegetatively-propagated bermudagrasses and switchgrass. P. 176, Agronomy Abstracts, Amer. Soc. Agron., Madison, WI.
6. Parco, Arnold. Current. 2000. Inheritance of allozyme markers in switchgrass (Panicum virgatum). M.S. Thesis, Oklahoma State University, Stillwater, OK.

Summary Date: July 2001

Switchgrass as a Biofuels Crop for the Upper Southeast: Variety Trials and Cultural Improvements

Research Funded by: U.S. Department of Energy (DOE), Office of Fuels Development through Oak Ridge National Laboratory

Project Manager: S.B. McLaughlin, 865-574-7358, mclaughlinsb@ornl.gov

Performing Organization: Virginia Polytechnic Institute and State University, Blacksburg, VA 24061

Principal Investigator(s): D.J. Parrish, (540) 231-9778, dparrish@vt.edu; D.D. Wolf, (540) 231-9741, dwolf@vt.edu; J.H. Fike, (540) 231-8654, jfike@vt.edu; and W.L. Daniels, (540) 231-7175, wdaniels@vt.edu

Contract Number: 19X-SY163C

Contract Period: 5/97 to 7/2002

Contract Funding: FY 2000: $34,000

Objective: This project is evaluating the production potential of switchgrass grown in the upper southeastern US by measuring yields of common varieties and new breeder's lines under various management regimes. Establishment practices are also being refined, and long-term, sustainable management strategies are also being investigated.

Approach/Background: Switchgrass has excellent potential as a biofuels feedstock, but information is needed on its establishment and management to optimize productivity regionally. The species has a reputation for being difficult to establish. Our experience suggests that proper attention to key factors can greatly improve the odds for successful establishment and early productivity. Those factors include planting date, seed dormancy, and pest management.

The largest component of this project is an eight-site, five-state, six-variety, cutting-management, and maximum-yield trial. Related fieldwork has involved studies on harvesting and fertilization practices that will foster long-term productivity and sustainability of switchgrass as a dedicated fuel crop. Laboratory efforts have been aimed at practical ways to overcome the seed dormancy that often makes establishment difficult. We have also investigated the fate of biomass left standing in the field after fall harvest.

Status/Accomplishments: A regional variety-screening study was established in 1992 at eight sites in Kentucky, North Carolina, Tennessee, Virginia, and West Virginia. Findings indicate that productivity varies greatly with location and to a lesser degree with ecotype and cutting management. The four lowland varieties show little or no yield increase with two cuttings. The two upland types in this study will yield 20 to 30% more if they are cut twice rather than once. With that boost in yield, they approach the lowland types in productivity (about 18 Mg/ha across locations in 1999). Cutting twice appears to deplete the plants' N reserves. A response to N fertilization is evident primarily in plots that are harvested twice per year, i.e., there is no yield benefit of adding more than 50 kg N/ha to plots that are harvested only after they senesce. In fact, we see evidence that 100 kg N/ha may be harmful to stands that are harvested only once per year. Nutrient flux studies suggest that switchgrass stands may be able to extract sufficient native N from the soil to produce good yields if they are allowed to conserve that N internally (by harvesting biomass only after the N has been translocated to below-ground parts).

Studies with delayed harvests have revealed that harvestable biomass declines about 10% between early September and the end of the season. This appears to be related to translocation of dry matter (and especially nitrogenous materials) to belowground parts. We have observed no significant decline in standing biomass from November through February in two different years at two locations in Virginia.

Recently harvested switchgrass seeds exhibit high levels of dormancy; germinability is often 10% or less. Dormancy can be broken by several treatments to include exposing wet seeds to 10^OC for 14 to 35 days (stratification) or holding dry seeds for extended storage times (after-ripening). An accelerated after-ripening (by exposing dry seeds to temperatures of 50^O to 60^OC for 5 to 30 days) is a feasible way to relieve much of the dormancy, but the exposure to higher temperatures poses risk of loss of vigor (aging), especially if seed moisture is elevated (>7%). Placing seeds in anoxic environments for the high-temperature exposure seems to reduce the aging, while allowing after-ripening to occur rapidly; but care must be taken to keep the seeds from becoming too dry during the high-temperature exposure. We have seen evidence for a remarkable plasticity of dormancy and germinability in switchgrass seeds; seeds can be moved reversibly from a dormant state to a more deeply dormant state and can, in fact, be moved from a germinable state to one that is deeply dormant. These findings have interesting scientific and practical implications.

Project Location(s): Blacksburg, VA; Orange, VA; Princeton, KY; Raleigh, NC; Jackson, TN; Knoxville, TN; Morgantown, WV

Publications and Presentations:

1. Gary, L.B. 2000. A gallon of gas at the pump? (UT plant scientist researches renewable fuels). U.T. Agriculture (Fall 2000) 15/2:18-19.
2. Parrish, D.J., D.D. Wolf, J.A. Balasko, J.T. Green, M. Rasnake, and J.H. Reynolds. 1999. Maximizing switchgrass biomass production. Agronomy Abstracts 2000: 114.
3. Parrish, D.J., D.D. Wolf, P.R. Peterson, and W.L. Daniels. 1999. Successful establishment of switchgrass and managing switchgrass as a biofuels feedstock. The Second Eastern Native Grass Symposium, Baltimore, MD. Abstract pp. 25-26.
4. Parrish, D.J., D.D. Wolf, and C. Garten. 2000. Root mass, C, N, and organic matter dynamics under switchgrass and tall fescue. Agronomy Abstracts 2000:313.
5. Parrish, D.J., D.D. Wolf, P.R. Peterson, and W.L. Daniels. Switchgrass as a biofuels crop for the upper Southeast: variety trials and cultural improvements. Annual Report for 1999. Oak Ridge National Laboratory. 36 pages
6. Reynolds, J.H., C.L. Walker, and M.J. Kirchner. 2000. Nitrogen removal in switchgrass biomass under two harvest systems. Biomass and Bioenergy 19:281-286.
7. Shen, Z.-X., D.J. Parrish, D.D. Wolf, and G.E. Welbaum. 2001. Stratification in switchgrass seeds is reversed and hastened by drying. Crop Science 41: (in press)
8. Wolf, D.D., D.J. Parrish, and C.T. Garten. 2000. Root mass, soil-C, soil-N, and soil organic matter dynamics under switchgrass and tall fescue. International Conference on Interactions in the Root Environment. IARC-Rothamsted, Harpenden, Herts, UK. 12 April 2000. Abstract p. B3.

Summary Date: July 2000