2006 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? 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 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. The research goals require state of the science physical and chemical characterization of rubber and latex materials. Our research also explores novel properties and new value-added uses for guayule natural rubber. 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. The research addresses ARS National Program 306, Quality and Utilization of Agricultural Products, Research Component, New Processes, New Uses and Value-added Foods and Biobased Products. The research also contributes to the goals of National Program 302, Plant Biological and Molecular Processes. Development of new and improved rubber-producing crops, including annual crops, using metabolic engineering will increase latex and rubber yield and quality. 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. In addition, the project incorporates biotechnological risk assessment and abatement to reduce or eliminate the environmental impact of transgenes. 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 by year the currently approved milestones (indicators of research progress) FY2004 Milestone 1.1 Development of improved guayule lines Transform Parthenium argentatum with deregulated rubber biosynthesis gene. Deliver transgenic shoots to U of Arizona 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. Milestone 1.2 Development of rubber producing sunflower Transform Helianthus annuus cv. HA 300 and other lines using GUS constructs. Test and refine transformant rescue and rooting methods. Deliver transformants to Colarado State University for proliferation and rooting, including grafting. Milestone 1.3 Creation of rubber producing tobacco Provide genes of the three identified subunits of the rubber transferase complex to KTRDC for transformation of Nicotiana tabacum. Milestone 1.4 Production of supra-high molecular weight rubber Make a P. argentatum genomic library from cold-induced bark tissue. Milestone 2.0 Biotechnological risk assessment and abatement 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. Milestone 3.1 Characterization of latex and rubber and compounded formulations from guayule and sunflower Characterize latex and latex films from P. argentatum, 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. Milestone 3.2 New rubber products, compounds, blends and composites Perform broad assessment of chemical and physical properties for single polymer and blended lattices. Determine optimum stabilizer type and level for P. argentatum. Milestone 3.3 Cold temperature elastomers 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. Milestone 3.4 Novel and/or functionalized rubber polymers Test a range of unlabeled IPP analogs from U of Al in rubber transferase inhibition assays. Select promising analogs for labeling. Milestone 4.1 Co-product development Extract and characterize resin fraction from guayule bagasse. Incorporate bagasse and fiber into pulped paper products, and novel films Milestone 4.2 Biofuels Isolate and deliver guayule bagasse samples before and after resin extraction for fractionation and fermentation trials. FY2005 Milestone 1.1 Development of improved guayule lines 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. Milestone 1.2 Development of rubber producing sunflower 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. Milestone 1.3 Creation of rubber producing tobacco Test materials and extracts from transgenic N. tabacum produced at KTRDC. Milestone 1.4 Production of supra-high molecular weight rubber Isolate the genes encoding three subunits of rubber transferase complex. . Use EST’s and PCR to clone the genes to the P. argentatum FPP synthase and sesquiterpene cyclase. Milestone 2.1 Biotechnological risk assessment and abatement 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. Milestone 3.1 Characterization of latex and rubber and compounded formulations from guayule and sunflower 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. Milestone 3.2 New rubber products, compounds, blends and composites 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. Milestone 3.3 Cold temperature elastomers Determine the underlying causes of the low temperature malleability of P. argentatum rubber particle cores. Begin product application research. Milestone 3.4 Novel and/or functionalized rubber polymers 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. Milestone 4.1 Co-product development Test guayule resin fraction in adhesives and coatings applications. Evaluate resin-lignin stream from bagasse fractionation. Milestone 4.2 Biofuels Complete fractionation and fermentation trials from bagasse samples before and after resin extraction. FY2006 Milestone 1.1 Development of improved guayule lines 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. Milestone 1.2 Development of rubber producing sunflower Develop methods to improve transformation and rescue efficiencies. Evaluate greenhouse-grown transgenic H. annuus. Make additional transgenics as indicated by results. Develop methods to improve transformation and rescue efficiencies. Milestone 1.3 Creation of rubber producing tobacco Test materials and extracts from rubber-producing transgenic N. tabacum produced at KTRDC for rubber yield and molecular weight. Milestone 1.4 Production of supra-high molecular weight rubber 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. Milestone 2.1 Biotechnological risk assessment and abatement 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. Milestone 3.1 Characterization of latex and rubber and compounded formulations from guayule and sunflower 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. Milestone 3.2 New rubber products, compounds, blends and composites Develop thermoplastic elastomers from P. argentatum rubber. Incorporate microfibril nanocomposites into P. argentatum latex formulations and compare to H. brasiliensis and SP. Milestone 3.3 Cold temperature elastomers 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. Milestone 3.4 Novel and/or functionalized rubber polymers Incorporate analogs into rubber for chemical and physical tests. Milestone 4.1 Co-product development Explore additional high value applications. Milestone 4.2 Biofuels Explore with commercial partner, possible high value applications, including activated carbon and charcoal. FY2007 Milestone 1.1 Development of improved guayule lines Evaluate transgenic 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. Milestone 1.2 Development of rubber producing sunflower Generate transgenic H. annuus and evaluate for improvement in rubber yield and/or quality. Milestone 1.3 Creation of rubber producing tobacco Develop research plan to develop commercially viable rubber-producing tobacco. Milestone 1.4 Production of supra-high molecular weight rubber 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. Milestone 2.1 Biotechnological risk assessment and abatement 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. Milestone 3.1 Characterization of latex and rubber and compounded formulations from guayule and sunflower Chemically and physically characterize rubber crumb from P. argentatum, including sub-components. Milestone 3.2 New rubber products, compounds, blends and composites Produce prototype parts and test for performance in new applications deriving benefits from P. argentatum latex. Evaluate P. argentatum rubber formulations vs. Hevea brasiliensis and SP, with a focus on processing/mixing, cure kinetics and cost. Milestone 3.3 Cold temperature elastomers Benchmark low temperature physical property performance vs. silicone rubber. Milestone 3.4 Novel and/or functionalized rubber polymers Identify potential application areas based on physical and chemical test results. Milestone 4.1 Co-product development Develop detailed plan to pursue and expand promising applications. Milestone 4.2 Biofuels Develop detailed plan to pursue and expand promising applications. FY2008 Milestone 1.1 Development of improved guayule lines Evaluate transgenic rubber transferase P. argentatum samples from field trials and select best performers. Patent and release improved guayule lines Milestone 1.2 Development of rubber producing sunflower Continue to generate transgenic H. annuus and evaluate for improvement in rubber yield and/or quality. Milestone 1.3 Creation of rubber producing tobacco Determine next steps based on initial results. Milestone 1.4 Production of supra-high molecular weight rubber Evaluate transgenic P. argentatum (over-expressed rubber transferase) in field trials for rubber yield and molecular weight. Milestone 2.0 Biotechnological risk assessment and abatement Implement abatement methods and continue evaluation of transgenic plants. Milestone 3.1 Characterization of latex and rubber and compounded formulations from guayule and sunflower Complete applications matrix results for P. argentatum rubber compounds: mechanical properties, dynamic mechanical, electrical, optical, surface, fluid resistance, service temperature range, weatherability, fatigue and failure performance. Milestone 3.2 New rubber products, compounds, blends and composites Pursue and expand promising applications Milestone 3.3 Cold temperature elastomers Produce prototype parts and test in key applications deriving benefits from low temperature properties Milestone 3.4 Novel and/or functionalized rubber polymers Continuing research to pursue and expand promising applications leveraging performance properties. Milestone 4.1 Co-product development Pursue and expand promising applications. Milestone 4.2 Biofuels Pursue and expand promising applications. FY2009 Project ends in FY2008. 4a.List the single most significant research accomplishment during FY 2006. Commercialization of Yulex Guayule Natural Rubber Latex Domestic natural rubber is a strategic priority in the United States. Domestic rubber production is now a commercial reality, supported by decades of USDA-funded research. In 2006, Yulex Corporation, the exclusive licensee of two USDA patents related to guayule rubber processing and products, supplied commercial quantities of guayule latex, YulexTM, to strategic development customers. Yulex has an ongoing collaboration with ARS scientists at WRRC funded by a CRADA that has further demonstrated that YulexTM Natural Rubber Latex is consistently of excellent quality, which has allowed pilot plant process development to move forward in scale-up operations. This research is part of NP306 Quality and Utilization of Agricultural Products, Component II, New Processes, New Uses, and Value-Added Foods and Biobased, Problem Statement A, Products, New products. 4b.List other significant research accomplishment(s), if any. Preferential substrate for rubber transferase enzyme confirmed Development of a domestic source of natural rubber is important because natural rubber is a strategic material in the US that is completely imported. This research is part of NP306 Quality and Utilization of Agricultural Products, Component II, New Processes, New Uses, and Value-Added Foods and Biobased, Problem Statement A, Products, New products. ARS scientists in Albany, CA, in collaboration with the University of Nevada, Reno and the University of Minnesota, are working together to improve rubber production in domestic crops. It was found that the enzyme rubber transferase that catalyzes rubber production in plants, works best with a particular isoprenoid molecule called farnesyl pyrophosphate. This finding will allow scientists to fine tune strategies for engineering domestic rubber-producing crop plants. Diversity of rubber productivity levels and quality in rubber trees Development of a domestic source of natural rubber is important because natural rubber is a strategic material in the US that is completely imported. This research is part of NP306 Quality and Utilization of Agricultural Products, Component II, New Processes, New Uses, and Value-Added Foods and Biobased, Problem Statement A, Products, New products. ARS scientists in Albany, CA in collaboration with EMBRAPA, University of California at Berkeley and University of Nevada, Reno performed research to understand the underlying physiological causes for the high variability in rubber production in plants. Scientists examined a series of related Brazilian Hevea brasiliensis trees and correlated rubber levels with differences in rubber biosynthetic activity, rubber molecular weight, and cellular magnesium concentrations. The optimal physiological conditions correlated with optimal rubber quantities and quality will allow scientists to fine tune strategies for engineering domestic rubber-producing crop plants. Optimal concentration of magnesium for rubber biosynthesis Development of a domestic source of natural rubber is important because natural rubber is a strategic material in the US that is completely imported. This research is part of NP306 Quality and Utilization of Agricultural Products, Component II, New Processes, New Uses, and Value-Added Foods and Biobased, Problem Statement A, Products, New products. To understand how rubber is synthesized in rubber-producing plants, ARS scientists in collaboration with the University of Nevada, Reno, further elucidated the role of magnesium as an important factor for rubber production. Scientists found that adding magnesium to isolated rubber synthesizing particles can restore rubber transferase activity. Scientists pinpointed the concentration of magnesium at which maximum rubber transferase activity occurs in rubber particles from three rubber-producing species (Parthenium, Hevea and Ficus). These results will allow scientists to fine tune strategies for engineering domestic rubber-producing crop plants. 4c.List 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. The Phase II Pilot Plant started production in 2005. In 2006, the first commercial shipments were made. Full-scale production is now in the manufacturing design stage. These production facilities will be located in the guayule growing region enhancing the positive impact on rural development. 4d.Progress report. See input for subordinate projects under separate report. 5.Describe the major accomplishments to date and their predicted or actual impact. From decades of USDA-funded research, domestic natural rubber is now a commercial reality in the United States. In 2006, Yulex Corporation, the excusive licensee of two USDA patents related to guayule rubber processing and products, supplied commercial quantities of Yulex™, a guayule latex product, to strategic development customers. Ongoing collaborative research at WRRC funded by a CRADA has further demonstrated that Yulex™ Natural Rubber Latex is consistently of excellent quality, which has allowed Yulex’s pilot plant to move forward to scale-up operations. Parameters for maximizing latex yield through optimization of agronomic practices, post-harvest handling and bioprocessing have been established. In addition, guayule has been successfully transformed with several key rubber biosynthesis genes. First and second-generation field trials on lines genetically altered to have enhanced initiator levels have shown that additional alterations are necessary to attain the goal of higher yield (>10%) of high quality rubber. We expect the research to lead to improved metabolic engineering strategies enabling the regulation of rubber yield and molecular weight in new domestic rubber-producing crops plant including sunflower. Transgenic sunflower lines are in greenhouse testing for validation of transformation, gene expression, and metabolic pathway effects. Overall, this research will improve the profitability of commercial production of non-allergenic 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. This research is part of NP306 Quality and Utilization of Agricultural Products, Component II, New Processes, New Uses, and Value-Added Foods and Biobased, Problem Statement A, Products. 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 commercial latex have been transferred. In 2006 agronomic and process improvements that were tested in ARS laboratories were scaled up to the licensee’s pilot plant. A second CRADA with a biotechnology company has generated guayule lines using four different proprietary transcription factors that will be tested in the field. These transgenic lines require a minimum of 2 years of field growth to perform field trials and fully demonstrate the potential. 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. 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. (NOTE: List your peer reviewed publications below). Popular Press Articles Cornish, K., McMahan, C.M., Pearson, C. H., Ray, D. T., Shintani, D. K. Biotechnological Development of Domestic Rubber-Producing Crops. Rubber World, November 2005, 40-44. "High Hopes of a Would-Be Rubber Baron," Alex Halperin, Business Week Online, December 27, 2005. "Natural-Rubber Latex Safe for Allergy Sufferers," Leslie Gordon, Associate Editor, Medical Design Magazine, October 2005. "Rubber Latex Is Safe for People Suffering from Allergies," Susan Shepard, Medical Product Manufacturing News, April 2006. Yulex Corporation was awarded the distinction of the "2005 Most Innovative New Product: Life Sciences" award by Connect, a San Diego business development organization founded by the University of California San Diego. The recognition was covered by the San Diego UnionTribune, (http://www.signonsandiego.com/uniontrib/20051210/news_1b10connect.html), the San Diego Metropolitan, and the Daily Transcript – as well as local and national television news. CONNECT is the globally recognized public benefits organization fostering entrepreneurship in the San Diego region by catalyzing, accelerating, and supporting the growth of the most promising technology and life sciences businesses. "Gloves Buyer's Guide: Global Raw Materials Prices Stretch Glove Makers'," Jeannie Akridge and Rick Dana Barlow, HEALTHCARE PURCHASING NEWS, April 2006 – Article notes an increase in natural rubber latex prices of 19% since January 2006 and over 79% from the beginning of 2005. It further points out an "option is emerging in the choice between natural rubber latex and synthetic models" in a "domestically grown natural rubber latex called guayule, manufactured and marketed under the brand name Yulex by Yulex Corp. (Carlsbad, CA)." "Tough Standard," Miles Moore, Rubber & Plastics News, June 26, 2006. A subcommittee of ASTM International has approved a new safety standard for natural rubber latex that, so far, only one company can pass. The standard defines a new Category 4 natural rubber latex that contains less than 200 micrograms total protein per gram dry weight of latex and no detectable protein under the ASTM method for detecting allergy-triggering proteins in latex. Carlsbad, Calif.-based Yulex holds the license from the U.S. Department of Agriculture to commercialize the USDA-patented method of manufacturing hypoallergenic latex from guayule. The agency's field tests have shown guayule latex to be completely safe when worn or used by latex-sensitive persons. United States Patent Application (Nov. 23, 2005) were successfully filed for Improved Guayule Plants, Products, and Derivatives. No. 700251456A. Inventors are Katrina Cornish and Niu Dong. Review Publications Dong, N., Montanez, B., Creelman, R., Cornish, K. 2005. Low light and low ammonium are key factors for guayule leaf tissue shoot organogenesis and transformation. Plant Cell Reports. 25(1):26-24 Cornish, K., McMahan, C.M., Pearson, C.H., Ray, D.T., Shintani, D.K. 2005. Domestic rubber producing crops. Rubber World. 233(2):40-44. Mcmahan, C.M., Cornish, K., Mccoy, R.G., Brichta, J.L., Wilson, S., Coffelt, T.A., Nakayama, F.S., Ray, D.T. 2005. Post-harvest storage effects on guayule latex quality from agronomic trials. International Conference on Industrial Crops and Rural Development Proceedings. p. 575-596.