Progress 09/01/02 to 05/27/04
Outputs
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? Natural rubber is a strategic raw material used in enormous amounts in over 40,000 applications. The United States, with no natural rubber domestic production of its own, is completely dependent upon imports. The supply of natural rubber to the United States, which uses over 20% of the world supply, is threatened by many factors. Over 90% of global production is in South East Asia. Natural rubber shortages would seriously impact defense, transportation, medicine and consumer markets. No other high performance elastomers can be used successfully in place of natural rubber in many of these applications. Industrial growth in Asia, particularly China, continues to squeeze supplies and drive up prices. Our overall goal is to develop commercially-viable natural rubber- producing crops suitable for cultivation
in the temperate climate of the United States. To this end, our research supports the commercial development of guayule as a source of hypoallergenic latex, especially for the medical products market, and the development of annual rubber- producing crops. Our research also explores novel properties and new value-added uses for guayule natural rubber. The research goals require manipulation of the biochemical pathways involved in the regulation of rubber yield and quality, and, therefore, identification of the enzymes and genes that regulate these parameters. The research goals also require physical and chemical characterization of rubber and latex materials. During 2004, the addition of a second SY to the project broadened our capability in this area. Our characterization, processing, and product development activities will enhance crop development through feedback and focus on the most productive, highest quality rubber- producing species. Development of bioproducts and biofuels
from resin and fiber co-products will profitably consume the non-rubber plant components, minimizing agricultural waste and the associated disposal costs and environmental impact. Successful accomplishment of this goal will reduce US dependence upon natural rubber imports, minimize reliance on tenuous supplies of this essential and strategic raw material from developing countries, solve life-threatening Type I latex allergies to protein contaminants in rubber products, and address the need for higher-performing rubber products than currently available in a wide range of commercial and strategic applications. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004) Transform Parthenium argentatum with deregulated 3-hyroxy-methyl-glutary- CoA (HMGR). Deliver transgenic shoots to U of A for rooting and field trials. Clone genes to the three subunits of the P. argentatum rubber transferase complex. Complete analysis of pre-existing transgenics (allylic
pyrophosphate synthase overexpression), for gene expression, enzyme activity, biomass, rubber/latex yield and quality, and resin yield. Transform Helianthus annuus cv. HA 300 and other lines using GUS constructs. Test and refine transformant rescue and rooting methods. Deliver transformants to CSU for proliferation and rooting, including grafting. Provide genes of the three identified subunits of the rubber transferase complex to Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington (KTRDC) for transformation of Nicotiana tabacum. Make a P. argentatum genomic library from cold-induced bark tissue. Expand H. annuus transformation efforts to bombardment of HA300 and CMS (male sterile) cultivars. Test N. tabacum nuclear and plastidic transformation methods. Begin cloning the melavonate (MEV) and methylerythritol (MEP) pathway genes from H. annuus and P. argentatum (using available EST's). Identify plastid pseudo-gene(s) in H. annuus and P. argentatum.
Develop constructs using green fluorescent protein (GFP) as transgenic marker. Characterize latex and latex films from P. argentatum and H. brasiliensis, chemically and physically. Analyze and assess rubber and resin latex sub-components and resin composition. Initiate design of comprehensive applications matrix database for latex formulation performance. Perform broad assessment of chemical and physical properties for single polymer and blended lattices. Determine optimum stabilizer type and level for P. argentatum. Determine the temperature ranges over which raw and compounded P. argentatum and H. brasiliensis latex and films retain malleability. Determine whether the low temperature property can be transferred to other latices by mixing latex subcomponents. Test a range of unlabeled IPP analogs from University of Alberta in rubber transferase inhibition assays. Select promising analogs for labeling. Extract and characterize resin fraction from guayule bagasse. Incorporate bagasse
and fiber into pulped paper products, and novel films. Isolate and deliver guayule bagasse samples before and after resin extraction for fractionation and fermentation biofuels trials. Year 2 (FY 2005) Transform P. argentatum with native and/or synthetic genes (from UCB) to subunits of the rubber transferase complex. Deliver transgenic shoots to University of Arizona for rooting and field trials. Provide genes encoding subunits to *UCB for overexpression in microbial systems. Evaluate transgenic HMGR P. argentatum samples from field trials and select best performers. Transform H. annuus using the same constructs as for P. argentatum. Select, proliferate and deliver half to Colorado State University for joint efforts on rooting. Develop methods to improved transformation and rescue efficiencies. Test materials and extracts from transgenic N. tabacum produced at KTRDC. Isolate the genes encoding three subunits of rubber transferase complex. Insert yeast MEV genes into N. tabacum
plastids via plastid integration vectors. Deliver rooted transgenic H. annuus to Ohio State University and Carl Hayden Bee Research Center, Tucson (CHBRC). Determine if transgenes are expressed in H. annuus and P. argentatum pollen. Identify native genes for carotenoids and/or anthocyanins and clone. Grow and evaluate transgenic plants. Complete cloning of MEP and MEV pathway genes. Complete applications matrix testing for P. argentatum latex formulation performance: mechanical properties, fluid resistance, barrier properties, service temperature range, fatigue and failure, and degradation by thermal, chemical, or mechanical mechanisms. Develop specific model latex formulations related to target applications and compare to H. brasiliensis and synthetic polyisoprene (SP). Incorporate microfibril nanocomposites into P. argentatum latex formulations and compare to H. brasiliensis and SP. Determine the underlying causes of the low temperature malleability of P. argentatum rubber
particle cores. Begin product application research. Test labeled IPP analogs in incorporation assays. Select incorporated analogs for synthesis of larger amounts of 13C labeling. Design new analogs with University of Alberta. Test guayule resin fraction in adhesives and coatings applications. Evaluate resin-lignin stream from bagasse fractionation. Complete fractionation and fermentation biofuels trials from bagasse samples before and after resin extraction. Year 3 (FY 2006) Evaluate transgenic HMGR P. argentatum samples from field trials and select best performers. Make constructs with genes favorably affecting rubber biosynthesis identified by University of Nevada Reno (if discovered). Transform best P. argentatum lines and ship to University of Arizona for field trials. Evaluate rubber transferase subunits overexpressed in microbial systems by UCB in rubber transferase assays. Evaluate greenhouse-grown transgenic H. annuus. Make additional transgenics as indicated by results.
Develop methods to improve transformation and rescue efficiencies. Test materials and extracts from rubber-producing transgenic N. tabacum produced at KTRDC for rubber yield and molecular weight. Isolate the promoter region for the genes encoding three subunits of the rubber transferase complex. Use EST's and PCR to clone the genes to the P. argentatum FPP synthase and sesquiterpene cyclase. Insert rubber biosynthesis genes into plastids of N. tabacum, H. annuus and P. argentatum with and without a functioning MEP pathway. Make constructs with native genes for carotenoids and/ or anthocyanins and test with pollen-specific and constitutive promoters. Grow and evaluate transgenic plants for gene expression. Characterize latex and latex films from greenhouse grown H. annuus, chemically and physically, including the rubber and resin latex sub- components. Develop methodology for preparation of dry rubber crumb or sheet from P. argentatum for experimental designs. Develop thermoplastic
elastomers from P. argentatum rubber. Incorporate microfibril nanocomposites into P. argentatum latex formulations and compare to H. brasiliensis and SP. Determine the breadth of application over which guayule latex and rubber is a significantly better-performing elastomer than H. brasiliensis latex and rubber at cold temperatures. Incorporate novel/functionalized rubber polymer analogs into rubber for chemical and physical tests. Explore additional guayule bagasse high value applications. Explore biofuels results with commercial partner, possible high value applications, including activated carbon and charcoal. Year 4 (FY 2007) Evaluate transgenic HMGR P. argentatum samples from field trials and select best performers. Evaluate transgenic rubber transferase P. argentatum samples from field trials. Analyze microbes transformed with all three subunits by UCB for rubber. Generate transgenic H. annuus and evaluate for improvement in rubber yield and/or quality. Develop research plan to
develop commercially viable rubber-producing tobacco. Transform P. argentatum with genes to a cold-induced promoter and anti- sense FPP synthase and sesquiterpene cyclase. Deliver proliferated transgenic shoots to U of A for rooting and field trials. Grow and evaluate transgenic plants for rubber yield and quality. Design approaches to transfer effective abatement methods to improved lines of H. annuus and P. argentatum. Chemically and physically characterize rubber crumb from P. argentatum, including sub-components. Produce prototype parts and test for performance in new applications deriving benefits from P. argentatum latex. Evaluate P. argentatum rubber formulations vs. H. brasiliensis and SP, with a focus on processing/mixing, cure kinetics and cost. Benchmark low temperature physical property of cold temperature elastomer performance vs. silicone rubber. Identify potential application areas based on physical and chemical test results on novel/functionalized rubber polymers.
Develop detailed plan to pursue and expand promising guaylae bagasse co- product applications. Develop detailed plan to pursue and expand promising biofuels applications. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. Year 1 (FY 2004) Parthenium argentatum was successfully transformed with deregulated 3- hyroxy-methyl-glutary-CoA (HMGR). Transgenic shoots delivered to U of A for rooting and field trials. The three subunits of the P. argentatum rubber transferase complex were not cloned. Protein sequence analysis of the three subunits of the P. argentatum trasferase complex has proven more difficult than anticipated. Early sequence data on small subunits has been accomplished and yielded unusual sequence. The subsequent highly degenerative probes were used to screen
for genes by PCR unsuccessfully. Efforts to find the genes will be improved by the use of an improved primary library of cold-induced bark. Twenty putative herbicide-resistant Helianthus annuus transformants, have been successfully rooted in the greenhouse at CSU. The milestone of provide genes of the three identified subunits of the rubber transferase complex to Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington (KTRDC) for transformation of Nicotiana tabacum has not been met because the genes have not been cloned. As genes become available during the next year, these will be provided to KTRDC. cDNA libraries were successfully constructed from the cold-induced bark of Parthenium argentatum (USDA & University of Nevada Reno) and will be used for screening the rubber transferase genes. A cDNA library was constructed for Ficus elastica and work is planned for Hevea brasiliensis. Nuclear transformation and chloroplast transformation of H. annuus were
successfully obtained using biolistic transformation methods. N. tabacum was similarly tested. Cloning of the MEP pathway dxr gene from P. argentatum has been completed, as the first progress in the series of genes needed for chloroplast rubber biosynthesis. Constructs that will be used to individually capture the genes in MEV and MEP from H. annuus and P. argentatum are almost completed. From collaboration with UNR, we have several genes from P. argentatum associated with rubber biosynthesis. We also have constructs of these genes with GFP. These genes are farnesyl pyrophosphate synthase (fps), short rubber particle protein (srp), and allene oxide synthase (aos). Srp and aos are marker genes for the rubber particle and will be helpful in the development of plastid for making rubber in tobacco. Characterization of latex and latex films from P. argentatum, chemically and physically ,and analysis rubber and resin latex sub- components and resin composition are essentially complete.
A comprehensive applications matrix database for latex formulation performance has been designed. Chemical and physical properties for single polymer and blended lattices have been assessed. Determination of optimum stabilizer type and level for P. argentatum has been delayed unitl 2005. Determination of the temperature ranges over which raw and compounded P. argentatum and H. brasiliensis latex and films retain malleability and determination whether the low temperature property can be transferred to other latices by mixing latex subcomponents were not completed in 2004. We did successfully determine that the low-temperature malleability of P. argentatum particles is not due to differences in the rubber glass transition behavior. Both P. argentatum and H. brasiliensis produce cis 1, 4-polyisoprene with glass transitions at 70 C. Tests of a range of unlabeled IPP analogs from University of Alberta in rubber transferase inhibition assays were completed. Promising analogs for labeling
were selected. The milestones concerning extraction and characterization resin fraction from guayule bagasse and incorporation of bagasse and fiber into pulped paper products and novel films were not met in 2004 due to other priorities. Training for Accelerated Solvent Extraction (ASE) techniques was completed, and we plan to isolate and characterize guayule reins in 2005. Isolated and delivered guayule bagasse samples before and after resin extraction for fractionation and fermentation biofuels trials. Latex- extracted bagasse samples have been provided to Dr. A. A. Boateng, Crop Conversion Science & Engineering Unit at the USDA/ARS Eastern Regional Research Center, in a newly-established collaboration. Dr. Boateng will measure syngas/hydrogen from bagasse by pyroprobe measurements. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Year 2 (FY
2005) Transform Parthenium argentatum with native and/or synthetic genes (from University of California Berkeley (UCB)) to subunits of the rubber transferase complex. Deliver transgenic shoots to University of Arizona for rooting and field trials. Provide genes encoding subunits to UCB for overexpression in microbial systems. Evaluate transgenic HMGR P. argentatum samples from field trials and select best performers. Transform Helianthus annuus using the same constructs as for P. argentatum. Select, proliferate and deliver half to Colorado State University for joint efforts on rooting. Develop methods to improved transformation and rescue efficiencies. Provide genes of the three identified subunits of the rubber transferase complex to Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington (KTRDC) for transformation of Nicotiana tabacum. Insert yeast MEV genes into N. tabacum plastids via plastid integration vectors. Deliver rooted transgenic H. annuus
to Ohio State University and Carl Hayden Bee Research Center, Tucson (CHBRC). Determine if transgenes are expressed in H. annuus and P. argentatum pollen. Identify native genes for carotenoids and/or anthocyanins and clone. Grow and evaluate transgenic plants. Complete cloning of MEP and MEV pathway genes. Complete applications matrix testing for P. argentatum latex formulation performance: mechanical properties, fluid resistance, barrier properties, service temperature range, fatigue and failure, and degradation by thermal, chemical, or mechanical mechanisms. Determine optimum stabilizer type and level for P. argentatum. Develop specific model latex formulations related to target applications and compare to H. brasiliensis latex and synthetic polyisoprene (SP). Determine the underlying causes of the low temperature malleability of P. argentatum rubber particle cores. Begin product application research. Test labeled IPP analogs in incorporation assays for novel and/or
functionalized rubber polymers. Select incorporated analogs for synthesis of larger amounts of 13C labeling. Design new analogs with University of Alberta. Extract and characterize resin fraction from guayule bagasse. Incorporate bagasse and fiber into pulped paper products, and novel films. Complete fractionation and fermentation trials from bagasse samples before and after resin extraction for biofuels. Year 3 (FY 2006) Evaluate transgenic HMGR P. argentatum samples from field trials and select best performers. Make constructs with genes favorably affecting rubber biosynthesis identified by University of Nevada Reno (if discovered). Transform best P. argentatum lines and ship to University of Arizona for field trials. Evaluate rubber transferase subunits overexpressed in microbial systems by UCB in rubber transferase assays. Evaluate greenhouse-grown transgenic H. annuus. Make additional transgenics as indicated by results. Develop methods to improve transformation and rescue
efficiencies. Isolate the promoter region for the genes encoding three subunits of the rubber transferase complex. Use EST's and PCR to clone the genes to the P. argentatum FPP synthase and sesquiterpene cyclase. Insert rubber biosynthesis genes into plastids of N. tabacum, H. annuus and P. argentatum with and without a functioning MEP pathway. Make constructs with native genes for carotenoids and/ or anthocyanins and test with pollen-specific and constitutive promoters. Grow and evaluate transgenic plants for gene expression. Characterize latex and latex films from greenhouse grown H. annuus, chemically and physically, including the rubber and resin latex sub- components. Develop methodology for preparation of dry rubber crumb or sheet from P. argentatum for experimental designs. Incorporate clay nanocomposites into P. argentatum latex formulations and compare to H. brasiliensis and SP. Incorporate microfibril nanocomposites into P. argentatum latex formulations and compare to H.
brasiliensis and SP. Determine the breadth of application over which guayule latex and rubber is a significantly better-performing elastomer than H. brasiliensis latex and rubber at cold temperatures. Incorporate analogs of novel and/or functionalized rubber polymerinto rubber for chemical and physical tests. Test guayule resin fractions of guayule bagasse in adhesives and coatings applications. Evaluate resin-lignin stream from bagasse fractionation. Explore with commercial partner, possible high value applications of bagasse for biofuels, including activated carbon and charcoal. Year 4 (FY 2007) Evaluate transgenic HMGR P. argentatum samples from field trials and select best performers. Evaluate transgenic rubber transferase P. argentatum samples from field trials. Analyze microbes transformed with all three subunits by UCB for rubber. Generate transgenic H. annuus and evaluate for improvement in rubber yield and/or quality. Transform P. argentatum with genes to a cold-induced
promoter and anti- sense FPP synthase and sesquiterpene cyclase. Deliver proliferated transgenic shoots to U of A for rooting and field trials. Grow and evaluate transgenic tobacco plants for rubber yield and quality. Design approaches to transfer effective abatement methods to improved lines of H. annuus and P. argentatum. Chemically and physically characterize rubber crumb from P. argentatum, including sub-components. Produce prototype parts and test for performance in new applications of new rubber compounds deriving benefits from P. argentatum latex. Evaluate P. argentatum rubber formulations vs. H. brasiliensis and SP, with a focus on processing/mixing, cure kinetics and cost. Develop thermoplastic elastomers from P. argentatum latex or rubber. Benchmark low temperature physical property performance of elastomers vs. silicone rubber. Identify potential application areas of novel and/or functionalized rubber polymers based on physical and chemical test results. Explore additional
high value applications of guayule bagasse. Develop detailed plan to pursue and expand promising applications of bagasse in biofuels. 4. What were the most significant accomplishments this past year? A. Development of commercially-viable natural rubber-producing crops in the United States will reduce dependence upon natural rubber imports, minimize reliance on supplies of this essential and strategic raw material from developing countries, solve life-threatening Type I latex allergies in rubber products, and address the need for higher-performing rubber products than currently available in a wide range of applications. One key to development of rubber-producing crops such as guayule, sunflower, and perhaps tobacco, is a mechanistic understanding of rubber biosynthesis, the roadmap for metabolic engineering. In 2004, project researchers at the ARS in Albany, California, in collaboration with the University of California at Berkeley, made an important discovery of the role of
magnesium in rubber biosynthesis, i.e., a magnesium-dependant conformational change in rubber transferase enzyme impacts both the rate of biosythesis and the rubber molecular weight. Through just a doubling in magnesium concentration, a conformational change occurs in the physical structure of the active site such that the binding affinity increases 60 fold; inhibition of the enzyme at higher metal ion concentrations indicates an optimum desired magnesium level. Resesarch will have a considerable impact on rubber production in plants as well as the development of rubber-producing crops. B. Other significant accomplishment(s), if any. Sunflower (Helianthus annuus) is an agronomically important crop in the USA that produces rubber naturally. Enhancement of the quality and quantity of rubber produced could provide an additional route to domestic natural rubber. However, H. annuus is not amenable to genetic transformation because of difficulty combining regeneration and transformation
within the same cells; i.e. an efficient transformation system is currently not available. Agrobacteruml-mediated transformation of H. annuus was successfully achieved by ARS scientists in 2004. Twenty glufosinate-resistant sunflower plants were obtained via Agrobacterium- mediated transformation with a vector containing the GUS gene. One of these glufosinate resistant plants showed GUS activity, while most of the herbicide resistant plants showed GUS positive in PCR analysis. The biotechnological improvement of crops requires transformation of genes critical to the rubber biosynthetic pathway into the target plants. Improved transformation methods have been developed and published in 2004. Moreover, ARS scientists in Albany, California have successfully transformed a key enzyme in the rubber biosynthetic pathway into Parthenium argentatum using these methodologies. Overproduction of the enzyme may produce significantly higher quantities of a substrate for rubber biosynthesis,
resulting in higher yields and potentially higher molecular weight rubber. This accomplishment will speed the development and release of improved guayule lines for the American farmer by enhancing rubber yields in guayule and facilitating the creation of annual rubber-producing crops for the United States. Introduction of transgenic rubber-producing plants requires biological risk assessment, and identification of approaches to minimize risks, including the potential for spread of transgenes via pollen. Chloroplast transformation, genetic engineering outside of the cell nucleus, is a key element in our strategy to contain the spread of transgenes. Experiments probing the feasibility of biolistically-mediated nuclear and chloroplast transformation for P. argentatum (guayule) were performed. Initial results indicate the successful transformation of both the guayule nuclear genome and plastome (chloroplast DNA). Further experiments will be done to verify whether this will be the first
demonstration of reproducible biolistic-based transformation methods. The ability to utilize an alternative to Agrobacterium-mediated nuclear transformation will significantly reduce the potential for bacterial contamination and its impact on the development of transgenic rubber-producing plants. In partnership with the University of Nevada-Reno, researchers successfully developed a method to prepare of active guayule washed rubber particles (WRPs). The protocol was further modified for Taraxacum kok-saghyz (Russian dandelion), and is now used routinely with good success, providing a constant supply of material for WRP purification and proteomic analyses. Russian dandelion is of interest as an excellent model system for US industrial crop development. Rubber biosynthetic gene expression during Russian dandelion root development was studied in vitro over a six-month period. Patterns of rubber latex accumulation, in vitro rubber transferase activity, and the expression of
rubber/isoprenoid biosynthetic genes revealed that young roots produce very little rubber. However in the period between 2 and 2.5 months, rubber accumulates rapidly; the rate of accumulation levels after 2.5 months. These results provide the building blocks for the next phase of the project: biochemical characterization of Russian dandelion rubber transferase, and analyses of transgenic plants. C. Significant activities that support special target populations. Introduction of guayule as a commercial American agricultural crop provides a potential source of income for rural Native Americans. Guayule is indigenous to US southwest desert land now part of Native American reservations. Field trial plots have been planted on reservation land in Arizona. The first pilot bioprocessing plant came on line at Maricopa, Arizona in July of 2003, with increases in production capacity planned in 2005 and 2006. These production facilities will be located in the guayule growing region enhancing
the positive impact on rural development. The research under the subordinate project "Cost-effective industrial crops for small-scale farmers" Project Number 5325-41000-019-03 R, is specifically targeted to guayule farming by black small farmers in South Africa. D. Progress Report See input for subordinate projects under separate report. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Field trials have been completed for the first guayule lines genetically altered to confer higher rubber yield. Those trials continue with the most promising second-generation plants. Genetic manipulation is expected to increase rubber yields beyond the 10% maximum achieved by conventional plant breeding. Guayule has been successfully transformed with a key rubber biosynthesis gene. Parameters for maximizing latex yield through optimization of agronomic practices, post-harvest handling and bioprocessing have been established. The latex
extraction process has been scaled-up leading to the first commercial pilot processing plant. That plant is currently producing significant volumes of (pre-commercial) latex. Biochemical mechanisms underlying the regulation of yield (rate) and quality (molecular weight) have been elucidated. This research will improve the profitability of commercial production of nonallergenic guayule latex, enhance rural development, and increase the supplies of latex products safe for use by the estimated 20 million Americans with Type I latex allergy. Also, the research should lead to improved metabolic engineering strategies enabling the regulation of rubber yield and molecular weight in new domestic rubber-producing crops plants. The research may lead to the production of sunflowers with commercially viable yield of high quality rubber, and to the production of rubber-producing tobacco, to provide a large-scale alternative crop use for tobacco farmers. 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? ARS scientists continue to provide technical information to the exclusive licensee of the latex production and product patents, in support of guayule latex commercialization. A CRADA with the licensee specialty chemicals company has provided the mechanism for a larger role specific to latex processing and production. Test methods critical to quality assurance of the pre-commercial latex have been transferred. In 2004 two key process improvements have been tested in the ARS laboratory and scaled up to the licensee's pilot plant. That same partner continues to benefit from agronomic developments and publications by ARS and collaborators, for example, subordinate IFAFS project "Development of Nonallergic Latex Products from Guayule"
(58-5325-1-0001), and our International Latex Conference Paper, "Characterization of guayule latex: variation across lines and plant tissues". A second CRADA has been signed with a biotechnology company to generate improved guayule lines using proprietary transcription factors. The new guayule lines are expected to lead to patents and licensing to the developing guayule latex industry. This technology is still in the laboratory; a minimum of 2 years of field trials will be required to fully demonstrate the potential. Our extractions and purification technologies are already in use at Yulex Corporation, the University of Arizona, Tucson, the University of Nevada, Reno, the Colorado University Experimental Station, Fruita, Oregon State University, and the University of Alberta, Edmonton. Techniques and methodologies for improved transformation efficiency of P. argentatum have been shared with the University of California, Berkeley, Colorado State University, and our CRADA partner. 7.
List your most important publications in the popular press and presentations to organizations and articles written about your work. "Rubber", MODERN MARVELSRG, The History Channel, June 9, 2004. "The Desert's Potential: Arizona Desert Shrub A Source For Rubber", Jane Larsen, The Arizona Republic, June 9, 2004, p. D1. "Development of Domestic Rubber Moves Forward", YUMA SUN, Staff Report, May 30, 2004. "Researchers Aim to Turn Rabbit Brush to Rubber", Zack Hall, Reno Gazette-Journal, February 13, 2004, p. 1D. "Grant Funds Guayule Project", Miles Moore, Rubber & Plastics News, November 17, 2003. "Mendel Pumps Up Rubber Plant", Janis Mara, The Oakland Tribune, October 9, 2003. "U.S. Rubber Source Could Bloom: CSU Scientist Seeks to Boost Sunflowers' Production of a Much-needed Commodity", Lori Cumpston, The Denver Post, October 6, 2003.
Impacts
(N/A)
Publications
- Imam, S.H., Glenn, G.M., Shey, J., Klamczynski, A., Nguyen, T.T., Cornish, K., Orts, W.J. 2004. Characterization and performance of starch-poly- (vinyl alcohol) (pva) blends with agricultural waste fiber. Society of Plastics Engineers Proceedings. 2437-2441.
- Cornish, K., Mcmahan, C.M., Mccoy, R.G., Van Fleet, J.E., Fowler, J.L. Characterization of guayule latex: variation across lines and plant tissues. Latex International Conference Proceedings. p. 239-254. 2004
- Scott, D.J., Da Costa, B.M., Espy, S.C., Keasling, J.D., Cornish, K. 2003. Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates. Phytochemistry. 64:123-124.
- Cornish, K., Myers, M.D., Kelley, S.S. 2004. Latex quantification in homogenate and purified latex samples from various plant species using near infrared reflectance spectroscopy. Industrial Crops and Products. 19(3):283-296.
- Mau, C.J., Garneau, S., Scholte, A., Van Fleet, J.E., Vederas, J., Cornish, K. Protein farnesyltransferase inhibitors interfere with farnesyl diphosphate binding by rubber transferase. 2003. European Journal of Biochemistry. 270(19):3939-3945.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? The United States is completely dependent upon imported natural rubber, a strategic, irreplaceable raw material used in enormous quantities. The US consumes over 20% of the total world supply. To reduce our dependence upon imported rubber, domestic rubber-producing crops must be developed. To this end, our research supports the commercial development of guayule as a source of hypoallergenic latex, especially for the medical products market, and the development of annual rubber-producing crops. Our research also explores novel properties and new value-added uses for guayule natural rubber. The research goals require manipulation of the biochemical pathways involved in the regulation of rubber yield and quality, and, therefore, identification of the enzymes and genes that regulate these parameters. The research goals also require quality testing and characterization of rubber and latex
materials. 2. How serious is the problem? Why does it matter? Natural rubber is a strategic raw material used in enormous amounts in over 40,000 applications. The United States, with no natural rubber domestic production of its own, is completely dependent upon imports. The supply of natural rubber to the United States, which uses over 20% of the world supply, is threatened by many factors. Over 90% of global production is in South East Asia. Natural rubber shortages would seriously impact defense, transportation, medicine and consumer market requirements. No other high performance elastomers are available that could be used instead of natural rubber. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? The research seeks to develop new domestic sources of natural rubber, including novel uses and products, so contributing to the Bio-based Products National Program 306 (70%) and uses biotechnological metabolic engineering
approaches to generate new plant varieties with improved rubber yield and quality, so contributing to National Program 302 Plant Biological and Molecular Processes, Component II - Biological processes that determine plant productivity and quality (30%). 4. What were the most significant accomplishments this past year? A. The creation or improvement of domestic natural rubber-producing crops requires the identification, sequencing and cloning of the genes that encode rubber transferase, the enzyme responsible for making rubber in plants. ARS scientists in Albany, California have purified three unusual proteins involved in the synthesis of natural rubber from three different rubber producing species, and distributed the proteins amino acid sequencing to other ARS scientists in Albany, and to collaborators at the University of Berkeley, California, the University of Nevada, Reno, and the University of Alberta, Edmonton, Canada. Partial amino acid sequence of the purified proteins has
been obtained. This accomplishment will allow the cloning of the rubber transferase genes which will in turn, enhance rubber yields in guayule and allow the creation of annual rubber- producing crops for the United States. B. The biotechnological improvement of crops requires efficient transformation systems to insert genes into the target plants. ARS scientists in Albany, California have developed a reproducible leaf disc transformation method which greatly improves the ease and efficiency of transformation in guayule. The transformation method has been used to transform guayule with two different genes. This accomplishment will speed the development and release of improved guayule lines for the American farmer. C. The research under the subordinate project "Cost-effective industrial crops for small-scale farmers" Project Number 5325-41000-019- 03 R, is specifically targeted to guayule farming by black small farmers in South Africa. D. Progress report ARS scientists in Albany,
California have continued research on guayule latex including analysis of transgenic guayule plants after their second winter rubber-production season, and assisting in equipment selection and optimization for the first commercial pilot bioprocessing plant. In collaboration with the US Water Conservation Lab they have continued latex quality analysis for guayule from different agronomic and post- harvest treatments. In collaboration with DOE-NREL, they demonstrated that rubber and resin yield can be quantified in guayule bark parenchyma in vivo using near infra-red spectroscopy. In biochemistry studies they found that the true rubber substrates are substrate-metal complexes but that the initiator substrate can bind and initiate rubber without metal. Magnesium concentration greatly impacts the rubber substrate binding constants and affects the rubber molecular weight. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The
first guayule lines genetically altered to confer higher rubber yield are in field trials. Genetic manipulation is expected to increase rubber yields beyond the 10% maximum achieved by conventional plant breeding. Parameters for maximizing latex yield through optimization of agronomic practices, post-harvest handling and bioprocessing have been established. The latex extraction process has been scaled-up leading to the first commercial pilot processing plant. Biochemical mechanisms underlying the regulation of yield (rate) and quality (molecular weight) have been elucidated. This research will improve the profitability of commercial production of nonallergenic guayule latex, enhance rural development, and increase the supplies of latex products safe for use by the estimated 20 million Americans with Type I latex allergy. Also, the research should lead to improved metabolic engineering strategies enabling the regulation of rubber yield and molecular weight in new domestic
rubber-producing crops plants. The research may lead to the production of sunflowers with commercially viable yield of high quality rubber, and to the production of rubber-producing tobacco, to provide a large-scale alternative crop use for tobacco farmers. 6. What do you expect to accomplish, year by year, over the next 3 years? FY 2004: Three subunits of the rubber transferase complex should be sequenced and cloned. The first set of guayule transgenics, previously metabolically-engineered with genes for initiator synthesis will be evaluated for rubber yield and quality as two year-old field grown plants after their second winter rubber induction season. Guayule lines will be transformed with a deregulated gene encoding the HMGCoA reductase which in turn regulates the level of rubber monomer in the cytosol. Biochemical analysis of rubber transferase enzymology and of the regulation of molecular weight will continue. Development of post-harvest crop models for guayule latex production
will continue. The underlying causes of the cold temperature malleability of the guayule latex rubber will be evaluated. Biotechnological risk assessment research will begin and strategies to control gene flow and use of foreign genes will be evaluated with collaborators at the Carl Hayden Bee research Center in Tucson, at Colorado and Ohio State Universities. FY 2005: Guayule HMGCoA reductase transgenic plants will move into field trials. Guayule will be transformed with the genes encoding the rubber transferase subunits. Synthetic genes for the two smaller subunits will have been synthesized and the authentic and synthetic genes prepared for overexpression in microbial systems with collaborators at UC Berkeley. An improved sunflower transformation system should be operating and sunflower will be metabolically engineered with genes for the rubber transferase subunits. Tobacco will be engineered with genes for the rubber transferase subunits and for substrate synthesis with
collaborators at the Tobacco Research and Development Center, University of Kentucky. Guayule latex, rubber, resin and bagasse will be further characterized and additional markets identified. Successful risk assessment strategies will be identified and developed. FY 2006: Rubber transferase proteins will be overexpressed in E. coli and yeast, then purified and tested for activity and antibody production. Guayule HMGCoA reductase transgenic plants will be assessed as one year- old plants in field trials. Sunflower and tobacco transgenics will have been evaluated as greenhouse and field plants. Sunflower will be metabolically engineered with genes for the substrate synthesis. The roles of the different subunits of the rubber transferase subunits will be established. Bioproducts will be developed from guayule latex, rubber, resin and bagasse. 7. 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? ARS scientists in Albany, California continued to provide information, to the exclusive licensee of the latex production and product patents, in support of guayule latex commercialization and have recently assisted with equipment selection and optimization for the first commercial pilot processing plant (construction completed June 3, 2003). Two new CRADA's have been negotiated with the licensee (a specialty chemicals company) in FY 2003, one in support of latex processing and production and the second to develop metabolically-engineered improved guayule lines. In addition, another CRADA has been signed with a biotechnology company to generate improved guayule lines using proprietary transcription factors. The new guayule lines are expected to lead to patents and licensing to the developing guayule latex industry. Our extractions and
purification technologies are already in use at Yulex Corporation, the University of Arizona, Tucson, the University of Nevada, Reno, the Colorado University Experimental Station, Fruita, Oregon State University, and the University of Alberta, Edmonton. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). "Domestic Natural Rubber Production and an Imminent Bagasse Mountain" by Katrina Cornish to DOE-NREL. Golden, CO. February 13, 2003. "Making Guayule Commercially Feasible." In Rubber Asia by Frank C. Saldo, November-December 2002. p. 119-123. "Sunflowers May Bounce Into The Future." In The Furrow (John Deere's popular publication) by Andy Markwart, Editor, Spring 2003. Vol. 108(4). p. 33. "Sunflower Rubber?" by Marcia Wood. Agricultural Research. 2002. "Sunflower Rubber." Voice of America, Special English AGRICULTURE REPORT by
George Grow. June 18, 2002. The Successful Farming Radio Program interview about the research progress and the prospects of rubber-producing sunflowers, interview by Darryl Anderson. The program aired on 132 affiliated stations. 2002. Chemistry published an article entitled "Rubber" including research on guayule natural rubber by Katrina Cornish. 2002. The issue also may be viewed at www.chemistry.org/Chemistry. Chemical Engineering Magazine interview by Gerald Dawkinson about the research progress and the prospects of rubber-producing sunflowers and hypoallergenic latex from guayule. 2002. Capital Press Agriculture Weekly interview by Vickie Horner, about the importance of natural rubber and research progress and prospects of rubber-producing sunflowers. 2002. A2C2 magazine (Advancing Applications in Contamination Control) interview by Paul Nesdore on latex allergies and the development of hypoallergenic guayule latex and latex products, for his editorial accompanying an article on
latex gloves from Motorola. 2002.
Impacts
(N/A)
Publications
- Imam, S.H., Wood, D.E., Shey, J., Glenn, G.M., Inglesby, M.K., Orts, W.J., Klamczynski, A., Nguyen, T.T., Ngoun, S.C., Cornish, K. Slow-Degrading Water Permeable Biopolymer Matrices Containing Renewable Agricultural Fibars, Bio Environmental Polymer Society Annual Meeting, Denver, CO. August 10-13, 2003. p. 40.
- Cornish K., Brichta, J.L., Chapman, M.H., Scott, D.J., Van Fleet, J.E., Xie, W.,Wood, D.F. Guayule latex: a clinically-proven, natural solution to Type I latex allergy. Paper 9. Proceedings of Latex. 2002. p. 119-132.
- Cornish, K., Backhaus, R.A. Induction of rubber transferase activity in guayule (Parthenium argentatum Gray) by low temperatures. Industrial Crops and Products 2003. v. 17. p. 83-92.
- Scott, D.J., da Costa, B.M.T., Espy, S.C., Keasling, J.D., Cornish, K. Activation and Inhibition of Rubber Transferases by Metal Cofactor and Pyrophosphate Substrate. Phytochemistry. 2003. v. 64. p. 121-132.
- Dong, N., Cornish, K. Using leaf disk for guayule transformation. Congress on In Vitro Biology. 2003. Abstract No. P-2039. p. 46-A.
- Cornish, K. Natural rubber biosynthesis and development of guayule as a commercially-viable crop. Terpnet 2003. Abstract No. 41.
- Shintani, D.K., Cornish, K. A reverse genetic approach to functionally identify plant rubber biosynthetic genes. Terpnet 2003. Lexington, KY. May 14-17, 2003. Abstract No. 43.
- Cornish K. Biotechnological production of domestic natural rubber- producing industrial crops. 6th Annual International Latex Conference. Akron, OH. July 29-30, 2003. Abstract No. 13.
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