2011 Annual Report
1a.Objectives (from AD-416)
The overall objective of the proposed research is to develop improved plant germplasm and advanced process technology enabling domestic production of two strategic industrial raw materials – domestic natural rubber and castor oil, along with their valuable co-products. The crops for industrial production of rubber are Parthenium argentatum (guayule), Taraxacum kok-saghyz (Russian dandelion) and Hevea brasiliensis (Hevea), and for castor oil are Ricinus communis (castor) and Lesquerella fendleri (Lesquerella).
Domestic production of both castor and rubber has taken place in the past, yet critical issues denied sustainability of these important industrial crops. We propose to address these via the following objectives:
Objective 1: Develop germplasm that enables domestic commercial production of natural rubber and ricinoleate by metabolic engineering and conventional breeding.
Objective 2: Develop technologies to enable the domestic production of natural rubber and ricinoleate with properties as good as or better than existing sources for industrial applications. Identify chemical processes and/or modifications to develop superior performance properties of natural rubber from domestic sources. Identify processing methods to enhance the value of castor oil byproducts.
1b.Approach (from AD-416)
Improvements in natural rubber yield will be sought through genetic manipulation of guayule and Russian dandelion plants. The biosynthesis of ricinoleate in Lesquerella will be metabolically engineered. Transformation methods will be developed and optimized; efficacious high expression and tissue-specific promoters will be identified. Genetically altered lines will be phenotypically characterized for alterations in enzymatic activity and production of target metabolites. Promising lines will be tested in controlled environment greenhouse and/or field trials on site or at collaborator locations. Improved germplasm will also be developed/identified through characterization of NPGS castor and Russian dandelion lines. Mutagenesis will be used to develop high yield, low toxin and allergen castor lines.
More specific knowledge of the biosynthetic pathways for production of natural rubber and hydroxy fatty acids will be achieved through physiological, cell biology, and biochemical studies.
Knowledge of the detailed physical and chemical characteristics of natural rubber from alternative crops will be developed to elucidate the mechanisms for differences from incumbent materials and to provide strategies for technological equivalence.
The processing of castor seed meal by bioconversion to reduce toxin and allergen content of byproducts will be researched.
Replacing 5325-41000-048-00D and 5325-21000-016-00D (June, 2010).
FY2011 research was conducted under the new project 5325-21410-020-00D toward sustainable technologies and processes for two key biobased products:.
1)natural rubber and.
Under Objective 1: Germplasm development, seed development in Lesquerella was documented, including seed oil type and content, and the expression of genes controlling the types of oil made by the plant. A procedure for stable genetic transformation of the plant was advanced, and a strategy created that may lead to production of castor oil free of toxin and allergens in this alternative species. Key to this strategy is a newly-discovered seed-specific promoter (Pnapin) that was successfully incorporated into Lesquerella. Meanwhile, investigations of existing castor germplasm characterized oil content of over 150 publically-available lines and over 1000 mutant seeds that could lead to identification of high oil content, low toxin, low allergen plants.
In germplasm development for natural rubber crops, testing of rubber and inulin (5C sugar) content was initiated for the USDA Russian dandelion collection, under curation at Pullman, WA. Plans are to make all lines available to researchers and breeders along with information relating their genetics to natural rubber and sugar production. Improvement of guayule, a US-native rubber-producing crop, focused on genetic modification. ARS researchers successfully incorporated five different genes for increasing biosynthesis of rubber precursors, and two genes for decreasing competing pathways, in hopes of improving overall rubber yield. Evaluations of these plants so far confirm the importance of coupling specific genes to effective control elements (promoters).
Objective 2: Technology/process development for utilization, sought to accelerate the domestic utilization of natural rubber from guayule. While the polymer is identical to that from imported Hevea, naturally-occurring non-rubber constituents in Hevea have a profound effect on industrial use. In FY2011 collaborative research revealed that blends of low-protein guayule latex and high-protein Hevea latex exhibit intermediate physical properties (tensile strength, gel, and thermo-oxidative stability) related to the beneficial effects of proteins. This suggests that proteins, or their peptide and amino acid degradation products, in Hevea latex are capable of physical and chemical interaction with guayule rubber in the blend.
New analytical methods identified previously-unknown components of castor seed oil featuring similar chemical functions (hydroxy fatty acids) as conventional castor oil. These oils may have value in industrial applications. Specifically, diacylglycerol and triacylglycerol containing 11,12,13-trihydroxy-9,14-octadecadienoic acid were found in castor oil. Triacylglycerols in sesame oil were identified for a visiting scientist from Korea. The fatty acid methyl esters of algal oil were also analyzed.
Finally, detoxification of castor oil byproducts could impact suitability for domestic production. In FY2011 conditions were identified for solubilizing 70% of castor stem biomass for subsequent processing by bioconversion.
Stable genetic transformation of Lesquerella fendleri. Seed oil of Lesquerella fendleri (Lf ) contains hydroxyl fatty acids which have valuable industrial uses, for example in manufacture of lubricants, plastics, and cosmetics. Genetic transformation, a key technology for crop improvement, is only successful if all plant cells are modified in a permanent manner. ARS researchers in Albany, CA, developed protocols to overcome the hurdles of formation of mixtures of transformed/untransformed cells in (Lf) transformation, and were the first to generate stably transformed Lf lines. The transformation system will further commercialization efforts by allowing the engineering of varieties with herbicide and disease resistance, and production of oils that have not be obtained through traditional breeding.
Chen, G.Q., Lin, J.T. 2010. Use of Quantitative PCR for Determining Copy Numbers of Transgenes in Lesquerella fendleri. American Journal of Agricultural and Biological Science. 5(3):415-421.
Lin, J.T., Chen, G.Q. 2011. Identification of diacylglycerol and triacylglycerol containing 11,12,13-trihydroxy-9-14-octadecadienoic acids in castor oil. New Biotechnology. 28(2):203-208. DOI: 10.1016/j.nbt2010.10.004.
Chen, G.Q., Lu, C., Lin, J.T. 2011. Hydroxy fatty acid synthesis and lipid gene expression during seed development in Lesquerella fendleri (L.). Industrial Crops and Products. doi:101.1016/j.indcrop.2010.08.03.
Chan, A.P., Crabtree, J., Zhao, Q., Lorenzi, H., Orvis, J., Puiu, D., Melake-Berhan, A., Jones, K.M., Redman, J., Chen, G.Q., Cahoon, E.B., Gedil, M., Stanke, M., Wortman, J.R., Fraser-Liggett, C.M., Ravel, J., Rabinowicz, P.D. 2010. Draft genome sequence of the oilseed species Ricinus communis. Nature Biotechnology. 28(9):951-956.
Chen, G.Q. 2011. Effective Elimination of Chimeric Tissue in Transgenics for the Stable Genetic Transformation of Lesquerella fendleri. HortScience. 46(1):86-90.
Galiani, P.D., Martins, M.A., De Souza Goncalves, P., Mcmahan, C.M., Mattoso, L.C. 2011. Seasonal and clonal variations in technological and thermal properties of raw Hevea natural rubber. Journal of Applied Polymer Science. 122(4): 2749-2755.
Ray, D.T., Foster, M.A., Coffelt, T.A., Mcmahan, C.M. Guayule: Culture, Breeding and Rubber Production, Industrial Crops and Uses (2010), pp. 384-410.