Progress 09/15/01 to 09/15/06
Outputs
There are strategic as well as economic incentives to develop a renewable, United States-based supply of natural rubber. Currently, nearly all commercial natural rubber comes from a single species, the Brazilian rubber tree (Hevea brasiliensis) and the USA is almost completely dependent on imports from distant countries. Rubber-producing crop plants ideally would be fast-growing, produce high biomass, and be annual crops. Annual crops can be more readily included in on-farm crop rotations and farming systems and planted and plowed-out in response to market needs and farmer production considerations. Perennial cropping systems, such as those for trees, that must remain in place for many years are not well suited to changing market needs and price fluctuations. Global natural rubber production will be best served in the future by multiple natural rubber crops. Scientists working under this project have optimized transformation and regeneration protocols in order to
genetically manipulate guayule (Parthenium argentatum) and sunflower (Helianthus annuus). Three guayule breeding lines were transformed to increase the availability of initiator molecules that are part of the isopreniod pathway and that are used in rubber synthesis. The tissue-culture-derived transgenic plants and their seed-generated progeny were grown in separate field experiments. Transformation with the genes for the initiators of rubber synthesis did not increase rubber concentration or yield. Sunflower is not amenable to genetic transformation and is complicated by the difficulty in combining regeneration and transformation within the same cells. However, comprehensive studies elucidated important variables needed to improve the success rate for sunflower transformation. Stable transformants incorporating marker and rubber biosynthesis genes have been produced in sunflower. In bioreactors, it has been shown that by deleting the Mg transporters from Saccharomyces cerevisiae, it
is possible to have different levels of total intracellular Mg. It has also been shown that the total intracellular level of the strains with deletion of Mg transporter depends on the amount of Mg supplemented to the medium. In addition to that, the total intracellular Mg can also be regulated by expressing Mg transporters from Arabidopsis thaliana. The results indicate that if the Mg levels in the plant can be regulated, the molecular weight of natural rubber could also be subject to manipulation and regulation. A rubber extraction and quantification protocol has been developed using acetone, methanol, and hexane, each with unique extraction temperatures, times, and number of extractions. Extractions using a Dionex ASE 200 were sequential using first acetone, followed by methanol, and last with hexane. The optimized protocol is being used to evaluate transgenic sunflowers. This research project has provided a deeper insight into the rubber biosynthetic pathway. Additionally, we have
developed protocols, methods, and new, novel genetic material to further the objective of developing a United States-based production and supply of natural rubber.
Impacts
New crops developed for natural rubber will require a more thorough understanding of the biosynthetic pathway of natural rubber. Most rubber-producing plants make low quality, low molecular weight polymers and only a few species make the high molecular weights required for high quality product manufacturing. The research conducted under this project provided the building blocks that may lead to transgenic guayule and sunflower lines with improved rubber production. A sunflower transformation system has been employed using explants from mature seeds to produce transformants through T3 seed. These transformants carry genes that are part of the isoprenoid pathway. Sunflower transformation efficiency remains low, which will continue to impede the development of sunflower as a rubber-producing crop. This impact of this project is summarized by the training of five graduate students who successfully completed their graduate degrees, publication of numerous scientific papers,
presentation of numerous scientific papers at meetings, raising public and scientific awareness of the importance of natural rubber to society, development of new and improved methods for rubber determination, safe standard operating protocols for complying with regulations for transgenic plants that produce industrial products such as rubber, and most importantly the development of new transgenic guayule and sunflower lines. Lastly, this research project has allowed us to leverage additional funding from other government and non-goverment sources to continue our research on this importanct topic.
Publications
- da Costa, B.M.T., Keasling, J. D., and Cornish, K. 2005. Magnesium Regulation of in vitro Rubber Synthesis by Parthenium argentatum, In: M.J. Pascual-Villalobos, F.S. Nakayama, C.A. Bailey, E. Correal, and W.W. Schloman, Jr. (eds.) Industrial Crops and Rural Development. Proceedings of the 2005 Annual Meeting for the Association for the Advancement of Industrial Crops 17-21 September 2005. Murcia, Spain. pp 629-638.
- Dong, N., and Cornish, K. 2003. Using leaf disk for guayule transformation. Congress on In Vitro Biology. Abstract No. P-2039. p. 46-A.
- Dong, N., Williams, N., McMahan, C., Rath, D., Pearson, C., and Cornish, K. 2005. Factors Affecting Agrobacterium-mediated Sunflower Transformation. Paper P-1017, Invitro. In Vitro Cell. Dev. Biol. Plant. 2005 In Vitro Biology Meeting Abstract Issue, 41, p. 30-A.
- Dong, N., Montanez, B., Creelman, R. A., and Cornish, K. 2006. Low light and low ammonia are key factors for guayule leaf tissue shoot organogenesis and transformation. Plant Cell Reports 25:26-34.
- Mau, C.J.D., Garneau, S., Scholte, A. A., van Fleet, J., Vederas, J. C., and Cornish, K. 2003. Protein farnesyl transferase inhibitors interfere with farnesyl diphosphate binding by rubber transferase. European Journal of Biochemistry. 270:3939-3945.
- Veatch-Blohm, M. E., and Ray, D. T. 2005. Water stress effects on rubber concentration and rubber distribution in guayule. In M. J. Pascual-Villalobos, F. S. Nakayama, C. A. Bailey, E. Correal, and W. W. Schloman, Jr. (Eds.) Industrial Crops and Rural Development, p. 607-617.
- McMahan, C. M., Cornish, K., McCoy, III., R. G., Brichta, J. L., Wilson, S., Coffelt, T. A., Nakayama, F. S., and Ray, D. T. 2005. Post-harvest storage conditions on guayule latex quality from agronomic trials. In M. J. Pascual-Villalobos, F. S. Nakayama, C. A. Bailey, E. Correal and W. W. Schloman, Jr. (Eds.) Industrial Crops and Rural Development, p. 575-596.
- Ray, D. T. 2004. Natural Rubber. Encyclopedia of Plant and Crop Science, Crops and Industrial Uses Section, Marcel Dekker, Inc., pages 778-780.
- Jorge, M. H. A., Veatch-Blohm, M. E. Ray, D. T., and Foster, M. A. 2006. Guayule seed germination under different conditioning treatments. Industrial Crops and Products 24:60-65.
- Veatch-Blohm, M. E., Ray, D. T., and McCloskey, W. B. 2006. Water-stress induced changes in resin and rubber concentration and distribution in greenhouse grown guayule. Agron. J. 98:766-773.
- Jorge, M. H. A, and Ray, D. T. 2005. Germination characterization of guayule seed by morphology, mass and X-ray analysis. Industrial Crops and Products 22:59-63.
- Veatch-Blohm, M. E. 2005. Genetic and Environmental Effects on Growth, Resin and Rubber Production in Guayule (Parthenium argentatum, Gray). Ph.D. Thesis. Department of Plant Sciences. The University of Arizona.
- Marcal Henrique Amici Jorge. 2005. Germination and Characterization of Guayule (Parthenium argentatum Gray) Seed. Ph.D. Thesis. Department of Plant Sciences. The University of Arizona.
- William Joseph Liesenbein. 2006. Development of a Methodology to Screen for Transgenic Guayule (Parthenium argentatum Gray) Seedlings, M.S. Thesis. Department of Plant Sciences. The University of Arizona.
- Cornish, K, McMahan, C. M., Pearson, C. H., Ray, D. T., and Shintani, D. K. 2005. Biotechnological development of domestic rubber producing crops. Rubber World 233:40-44.
- Ray, D. T., Coffelt, T. A., and Dierig, D. A. 2005. Breeding guayule for commercial production. Industrial Crops and Products 22:15-25.
- Coffelt, T. A., Ray, D. T., Nakayam, F. S., and Dierig, D. A. 2005. Genotypic and environmental effects on guayule (Parthenium argentatum) latex and growth. Industrial Crops and Products 22:95-99.
- Veatch, M. E., Ray, D. T., Ma, C.J.D., and Cornish, K. 2005. Growth, rubber and resin evaluation of two-year-old transgenic guayule. Industrial Crops and Products 22:65-74.
- da Costa, B.M.T., Keasling, J. D., McMahan, C. M., and Cornish, K. 2006. Magnesium ion regulation of in vitro rubber biosynthesis by Parthenium argentatum Gray. Phytochemistry 67:1621-1628.
- Scholte, A.A., Eubanks, L. M., Poulter, C. D., Vederas, J. C. 2004. Synthesis and Biological Activity of Isopentenyl Diphosphate Analogues. Bioorg. Med. Chem. 12:763-770.
- Scholte, A.A., and Vederas, J. C. 2006. Incorporation of Deuterium-labelled Analogs of Isopentenyl Diphosphate for the Elucidation of the Stereochemistry of Rubber Biosynthesis. Org. Biomol. Chem.4:730-742.
- Bushman, B.S., Scholte, A. A., Cornish, K., Scott, D. J., Brichta, J. L., Vederas, J. C., Ochoa, O., Michelmore, R. W., Shintani, D., and Knapp, S. J. 2006. Identification and Comparison of Natural Rubber from Two Lactuca Species. Phytochemistry 67:2590-2596.
- Scholte, A.A. 2006. Chemical Biology Studies on Prenyltransferase Enzymes. Ph.D. Thesis. University of Alberta. 255 pages.
- Scott, D.J., da Costa, B.M.T., Espy, S. C., Keasling, J. D., and Cornish, K. 2003. Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates. Phytochemistry 64:123-134.
- da Costa, B.M.T., Keasling, J. D., and Cornish, K. 2005. Regulation of rubber bbiosynthetic rate and molecular weight in Hevea brasiliensis by metal cofactor. Biomacromolecules 6:279-289.
- Espy, S.C., Keasling, J. D., Castillon, J., and Cornish, K. 2006. Initiator-independent and initiator-dependent rubber biosynthesis in Ficus elastica. Arch Biochem Biophys. 448:13-22.
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Progress 01/01/05 to 12/31/05
Outputs
Sunflower (H. annuus) produces a small amount of rubber naturally but is quite recalcitrant to genetic modification. Enhancement of the quality and quantity of rubber produced could provide a route to domestic natural rubber production in the USA. Genes targeted to increase latex quantity and quality such as HMGR co-A reductase (HMGR) and FPP synthase (FPPS) were used to transform sunflower and guayule in 2005. Kanamycin (antibiotic) selection was used for sunflower and guayule transformation in place of the of herbicide selection used in 2004 (glufosinate). A series of vectors containing the marker gene and target genes have been made. In 2005, HMGR and HMGR/FPP double construct plasmids were used; positive PCR results confirm transformation of guayule with the HMGR constructs. This accomplishment will speed the development and release of improved guayule lines for the American farmer by enhancing rubber yields in guayule. High expression level promoters for
sunflower have been identified. Vectors with these promoters have been constructed and successfully validated in tobacco. Promoter 409 was highly expressed in meristem tissue. Promoter 427 expressed lower in normal conditions but expressed higher when wounding. The shoot tip transformed by P409-NPTII was more resistant to kanamycin than P35S-NPTII. Plants transformed by P427-tAN-2A-FPP can express HMGR and FPPS in lower level in normal conditions but express even higher level when the bark is peeled (wounding). The Agrobacterium EHA101 harboring vector 2A-tAN-FPP-NPTII has been successfully used to transform sunflower HA300. More than sixty putative herbicide and antibiotic-resistant transformants from 6 independent events were successfully rooted. PCR confirmation of transformation was positive for the F1 and F2 generation plants for the GUS construct, and at the end of 2005, for a double construct of HMGR co-A reductase/FPP synthase. Sunflower leaves were tested and found to be PCR
positive for both the NPTII gene and HMGR gene. This is the first time HMGR-transformed sunflower has been obtained. Greenhouse trials comparing rubber production for transformed, control, and wild type plants are planned for 2006. Analytical capabilities developed in 2004-2005 for quantifying sunflower rubber production will provide key indicator data from those trials. In February 2005, Dr. Colleen McMahan, USDA-ARS, Albany, CA and Dr. Calvin Pearson, Colorado State University, planned and executed the third Albany Rubber Workshop, a meeting of scientists and collaborators active in domestic rubber research. Participants included: University of California-Berkeley, University of Nevada-Reno, Yulex Corporation, University of Edmonton-Alberta, Colorado State University, Arizona State University, and USDA-ARS Western Regional Research Lab personnel.
Impacts
A more complete understanding of rubber biosynthesis in gauyule has been achieved based on research conducted in this project. During 2005, sunflower was successfully transformed using two genes important in the isoprenoid pathway (HMGR and FPP). The research efforts of this project over the past year have led to patent applications, development of new protocols for rubber determination in sunflower, consistent tissue culture techniques for sunflower, and a better understanding of rubber biosynthesis and rate limiting production factors for rubber production in plants. The 2005 research efforts are providing additional building blocks to allow us to conduct additional research leading to rubber-producing guayule and sunflower lines. Thus, even more powerful research accomnplishments are expected in the next year.
Publications
- Veatch, M.E., D.T. Ray, C.J.D. Mau, and K. Cornish. 2005. Growth, rubber, and resin of evaluation of two-year-old transgenic guayule. Industrial Crops and Products 22:65-74.
- Blohm, M.E.V. 2005. Genetic and Environmental Effects on Growth, Resin and Rubber Production in Guayule (Parthenium argentatum Gray). Ph.D. Dissertation. Univ. of Arizona, Tuson, AZ.
- Scholte, A. A., 2005. Chemical Biology Studies of Prenyl Transferase Enzymes. Ph.D. thesis, University of Alberta, Canada.
- Dong, N, and Cornish, K. 2005. Transformation Methods for Guayule Using Agrobacterium and Reduced Light to lower the Wounding Response and Enhance Recovery. U.S. Patent Application, USDA Docket No. 0109.03.
- Williams, N., McMahan, C., Rath D., Pearson, C., and Cornish, K. 2005. Factors Affecting Agrobacterium-mediated Sunflower Transformation. N. Dong, P-1017 presented at Society of In Vitro Biology Annual Meeting, June 4-7, 2005 , Invitro: 2005 In Vitro Biology Meeting Abstract Issue, 41, p. 30-A, Spring 2005.
- Rath, D.J., Pearson, C. H., Cornish, K., McMahan, C.M., and Scott, D.J. 2005. Quantifying natural rubber in Helianthus annuus L. using the Dionex ASE 200. Proceedings of the 167th Technical Meeting of the Rubber Division, American Chemical Society, San Antonio, Texas, May 16-18, 2005.
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Progress 01/01/04 to 12/31/04
Outputs
The heritability of yield components in 97 progeny plants from 22 transgenic parent guayule plants are being tested at the Maricopa Agricultural Center, Arizona. Growth in the progeny, as measured by height and width, is less than was measured in the parent plants at the equivalent age (approximately 15-20 cm less). Resin concentration appears to be the most heritable (least environmental influence), and rubber concentration was found to be less variable then height and width, but more variable than resin concentration. In greenhouse experiments, no differences were found in rubber and resin production between any of the transgenic lines in either cold night or warm night treatments. Water stress reduced growth, resin and rubber production, but no differences were found between the non-transformed control and transgenic lines. A wide range of isolation and purification techniques were tested to purify the three subunits of the rubber transferase complex from Ficus and
guayule. Four peptide sequences for one of the small rubber transferase proteins have been obtained (Ficus elastica) one of the small subunits. The University of Alberta very recently reported 3 partial sequences for the other small subunit. Early sequence data on the two small subunits from guayule yielded unusual sequence. The two small subunits are very tightly associated with each other (HPLC was not able to separate them. One peptide sequence has been obtained from the larger of the two subunits. Efforts to obtain internal sequence form the N-termainal blocked and heavily glycosylated laterest subunit are focused on deglycosylation (enzymatic and chemical) followed chemical and/or proteolytic digestion. Fragments are sequenced by mass spectrometry. cDNA libraries were constructed from cold-induced bark of Parthenium argentatum (USDA & University of Nevada Reno) and will be used for screening the rubber transferase genes. Efforts to find the genes will be improved by the use of an
improved primary library for guayule. P. argentatum HMGR transformations have been moderately successful; transformed explants are in boxes for three confirmed Aspergillus /HMGR (Niu). Four additional Saccharomyces /HMGR are growing on selectable media but not yet large enough to PCR. However, plant development has been slower than anticipated; root and shoot development has frequently been slower than hoped. An HMGR-FPP synthase-BAR gene construct for plant transformations has been made. Limited attempts so far to transform guayule with this vector. Assays for transient GUS expression indicated that nuclear transformation can be successfully obtained for Parthenium argentatum using biolistic transformation methods. This can serve as a backup if needed.
Impacts
A more complete understanding of rubber biosynthesis in Parthenium argentatum has been achieved based on research conducted in this project. A reliable protocol for solvent rubber extraction and determination has been developed for sunflower using the Dionex ASE 200 and this will speed our research efforts. After 2 years of work and much effort, IBC approval has been obtained for producing transgenic sunflower at Colorado State University. This approval opens the way for producing transgenic sunflowers in the lab and greenhouse in quantities that will allow us to evaluate rubber production in transgenics. The research efforts over the past year in this project have led to patent applications, development of new protocols for rubber determination in sunflower, better tissue culture techniques for sunflower, a latex extraction method in sunflower, and a better understanding of rubber biosynthesis and rate limiting production factors for rubber production in plants. The
research conducted in this project over the past year is providing the building blocks to allow us to conduct additional research leading to rubber-producing guayule and sunflower lines. Thus, even more powerful research achievements are expected in the next year or two.
Publications
- da Costa, B.M.T., J.D. Keasling, and K. Cornish, 2005. Regulation of rubber biosynthetic rate and molecular weight in Hevea brasiliensis by metal cofactor. Biomacramolecules 6(1):279-289.
- Scott, D.J., Da Costa, B.M.T., 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.
- M. E. Veatch, D.T. Ray, C.J.D. Mau, and K. Cornish, 2004. Growth, rubber, and resin evaluation of two-year-old transgenic guayule. Industrial Crops and Products, In Press, Corrected Proof, Available online 28 October 2004.
- Mau, C.; Garneau, S.; Scholte; A., van Fleet J; Vederas, J.C., and Cornish, K., Protein Farnesyl Transferase Inhibitors Interfer with Farnesyl Diphosphate Binding by Rubber Transerase, Eur. J. Biochem., 270, 3939-3945 (2003).
- Scholte, A.A.; Eubanks, L.M.; Poulter, C.D.; and Vederas, J.C., Synthesis and Biological Activity of Isopentenyl Diphosphate Analogues, Bioorg. Med. Chem., 12, 763-770 (2004).
- Rath, D.J., C.H. Pearson, and K.F. Cornish. 2004. Extracting Rubber from Sunflower using the Dionex ASE 200. Paper no. 4543. Annual Meeting Abstracts. Poster presentation was made at the ASA-CSSA-SSSA Annual Meetings in Seattle, Washington. November 1, 2004.
- Rath, Donna, and Calvin Pearson. 2004. Inducing Shoot Production in Sunflower using TDZ in Tissue Culture. p. 13-20. In: Western Colorado Research Center 2003 Research Report. Colorado State University, Agricultural Experiment Station and Cooperative Extension, Technical Report TR04-05. Fort Collins, Colorado.
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Progress 01/01/03 to 12/31/03
Outputs
A new method using leaf tissue for guayule transformations has been developed and a patent application and paper are in preparation. A BAR selective marker and an intron containing the GUS reporter gene have been introduced into the guayule lines G7-11 (aka AZ2 and AZ101CL). Transgenic guayule plants from this method were resistant to the herbicide glufosinate, stained positively with GUS, and gave positive PCR results. Efforts to efficiently transform sunflower moved on from the cotyledon method previously reported, to testing other methods because the transgenic shoots obtained failed before rooting. We have successfully regenerated sunflower plants from tissue cultures of shoot apices but no transgenic plants were obtained. Continued work with cotyledons totaling 4,000 attempts have led to only a single transformed plantlet. We have shown that affinity of the rubber transferase of H. brasiliensis for rubber substrate depends on the concentration of Mg present. At
low levels of Mg concentration, rubber transferase has a low affinity for isopentenyl pyrophosphate (IPP). This suggests that Mg leads to a conformational change in the enzyme, which increases the affinity for IPP. At high Mg concentrations (30 mM), the rubber transferase affinity for IPP is decreased, thus a decrease in the activity of rubber transferase is observed. This observation could be explained by Mg ion binding to the catalytic site and preventing the Mg/IPP complex from interacting with the catalytic site. We have exploited F. elastica to investigate the kinetics of initiator-dependent and initiator-independent rubber biosynthesis, and the influence of cation cofactor on these processes. As observed for H. brasiliensis, cofactor concentration affects the binding affinity of the enzyme for both substrates, although the differences are less dramatic in this species. Also, the binding affinity for the IPP monomer is considerably higher in the absence of initiator than in its
presence, indicating that the initiator probably competes with IPP at the IPP binding site. A series of IPP analogs have been synthesized and tested, including a series of IPP analogs in which the terminal methyl group has been replaced by various subtituents such as ethyl and vinyl. The ethyl analog has also been synthesized in 13C-labelled form and has been used in incorporation experiments with rubber transferase from P. argentatum. Solid state 13C-NMR experiments showed that although excellent spectra of rubber were obtained, insufficient quantities of analog were incorporated to be detectable by this approach. Use of radioactive 3H or 14C analogs are being examined. Non-radioactive deuterium-labeled IPP analogs have also been synthesized to examine the 3D mechanism of rubber biosynthesis. Product analysis will employ both solid state 13C NMR spectroscopy as well as ozonoltyic degradation and solution phase investigation using NMR and masss spectrometry. Using mass spectrometry,
segments of rubber transferase proteins have been sequenced. Promising results have been obtained on a Parthenium 266 kDa protein and on Fiscus 3.5 kDa and 13 kDa proteins.
Impacts
A more complete understanding of rubber biosynthesis in Parthenium argentatum has been achieved based on research conducted in this project. A reliable protocol for solvent rubber extraction and determination has been developed for sunflower using the Dionex ASE 200 and this will speed our research efforts. After 2 years of work and much effort, IBC approval has been obtained for producing transgenic sunflower at Colorado State University. This approval opens the way for producing transgenic sunflowers in the lab and greenhouse in quantities that will allow us to evaluate rubber production in transgenics. The research efforts over the past year in this project have led to patent applications, development of new protocols for rubber determination in sunflower, better tissue culture techniques for sunflower, a latex extraction method in sunflower, and a better understanding of rubber biosynthesis and rate limiting production factors for rubber production in plants. The
research conducted in this project over the past year is providing the building blocks to allow us to conduct additional research leading to rubber-producing guayule and sunflower lines. Thus, even more powerful research achievements are expected in the next year or two.
Publications
- Cornish, K. Biotechnological production of domestic natural rubber-producing industrial crops. 2003. 6th Annual Internation Latex Conference, AKron, OH, July 29-30, Abstract No. 13.
- Dong, N., and Cornish, K. 2003. Transformation of guayule leaf sections. Association for the Advancement of Industrial Crops Annual Meeting. Portland, OR, October 12-15, 2003.
- Cornish, K., da Costa, B.M.T., and Scott, D.J. 2003. Biotechnological production of domestic natural rubber-producing industrial crops: regulation rate and molecular weight. Proceedings of the 6th International Latex Conference, July 29-30, 2003, Akron, Ohio.
- Dong, N. and Cornish, K. 2003. Using leaf disk for guayule transformation. In Vitro Cellular & Development al Biology, vol. 39:46A.
- Imam, S., Wood, D.F.,Shey, J., Greg Glenn, G.,Ingelsby, M., Orts, W., Nguyen, A., Ngoun, S., and Cornish, K. 2003. Slow-degrading water permeable biopolymer matrices containing renewable agricultural fibers. BioEnvironmental Polymer Society Annual Meeting, August 10-13, Denver, CO. p. 40.
- Mau, C.J.D., Garneau, S., Scholte, A.A., Vederas, J.C., and Cornish, K. 2003. Protein farnesyltransferase inhibitors interfere with farnesyl diphosphate binding by rubber transferase. European Journal of Biochemistry 270:3939-3945.
- Scott, D.J., da Costa, B.M.T., Espy, S.C., Keasling, J.D., and Cornish, K. 2003. Activation and inhibition of rubber transferases by metal cofactor and pyrophosphate substrate. Phytochemistry 64:121-132 (Harborne Memorial Issue).
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Progress 01/01/02 to 12/31/02
Outputs
Research is being conducted at several labs that contribute to the objectives of the project. In Dr. Cornish's lab at Albany, CA, research has progressed in guayule and sunflower transformations including construction of a binary vector pND4 for Agrobacterium-mediated transformation and the development of a time-saving leaf disc transformation system in guayule. Transformant selection conditions have been established for the hygromycin selectable marker gene in both guayule and sunflower. Sunflower transformation has been achieved but with low efficiency. Also, experiments have clarified the role of cation cofactors in the regulation of rubber yield and molecular weight and provide the basis for optimizing these parameters in transgenic organisms. In Dr. Keasling's lab at the Univ. of Calif., Berkeley, the focus has been on developing three hosts for microbial rubber production: Escherichia coli, Saccharomyces cerevisiae, and Aspergillus nidulans. In E.coli, genes
have been cloned for a heterologous mevalonate pathway from S. cerevisiae and the genes have been expressed. Several different terpenes have been produced using the mevalonate pathway, indicating an increased amount of IPP and FPP produced by this organism. In A.nidulans, genes have been cloned for HMG-CoA reductase, IPP isomerase, and GGPP synthase. In S. cerevisiae, a reporter system has been developed to allow us to easily assay increased flux through the isoprenoid biosynthetic pathway. Also, HMG-CoA reductase has been cloned. The gene will be truncated (to remove the regulatory region of the enzyme) and then it will be expressed in S. cerevisiae. In Dr. Vederas's lab at the Univ. of Alberta-Canada, ten IPP analogs having carbon-linked dicarboxylate moieties in place of the pyrophosphate group have been chemically synthesized and they have been examined for inhibition of the enzyme. A labeled natural substrate of [4-13C]-isopentenyl pyrophosphate has been prepared and incorporated
into rubber. Two additional unnatural analogs have been chemically synthesized including methyl and vinyl-extended IPPs. A solid state 13C NMR spectra (300MHz and 500MHz) of 1-10mg samples has been obtained of unlabeled natural Hevea rubber prepared enzymatically by Dr. Cornish. This technique is expected to be crucial to our analysis of modified rubber molecules. In Dr. Ray's lab at the Univ. of Arizona a study is being conducted to determine if insertion of genes for precursors of rubber biosynthesis will increase overall rubber production in guayule. AZ101, G7-11, and N6-5 were transformed by USDA and to date the data support an effect of transformation on growth and resin production in guayule, which is most evident in AZ101 transformants. There could be a possible effect on latex production in N6-5 lines, but no definitive conclusions can be drawn prior to harvest of the entire plant in March 2003. In Dr. Pearson's lab at Colorado State Univ., protocols are being developed for
organogensis of sunflower and for rubber extraction using the Dionex ASE 200. A greenhouse has been modified to BL2-P to grow transgenic sunflowers and only minor compliance issues remain to be addressed to satisfy the IBC.
Impacts
In March 2002, a press release about this research project was prepared and sent out to a large number of media. The interest and response to the press release was worldwide. Newspaper and mazagine articles have appeared in many places across the country. Numerous inquiries about this effort have been received from countries such as Russia, Japan, Colombia, countries in Africa, Korea, European Union, and others. Scientists associated with this project have been interviewed for radio programs, newspapers and mazagine articles, and scientific newsletters. Clearly, a large and broad audience is keenly interested in this research and its progress. To assist and educate the public about this research and its progress, a website has been constructed:www.naturalrubber.info. The website is an efficient method to direct people for information about this research project. Six students are working towards graduate degrees as a part of this project. An open house was held for the
new laboratory at the CSU Western Colorado Research Center at Fruita and various dignitaries were invited to attend. As a research team, we have identified other scientists and partners that are interested in participating in this project in various ways. The success of this project will protect the U.S. from the attempts of the OPEC-like, recent tripartite efforts of Thailand, Malaysia, and Indonesia to manipulate the world's natural rubber market.
Publications
- Cornish, K., and Brichta, J.L. 2002. Rheological properties of latex from Parthenium argentatum Gray compared with latex from other Rubber-Producing Species. Journal of Polymers and the Environment 10:13-18.
- Cornish, K., and Wood, D.F. 2002. Visualization of the Malleability of the Rubber Core of Rubber Particles from Parthenium argentatum Gray and Other Rubber-Producing Species under extremely cold temperatures. Journal of Polymers and the Environment 10:155-162.
- Cornish K., Brichta, J.L., Chapman, M.H., Scott, D.J., Van Fleet, J.E., Wood, D.F., and Xie, W. 2002. Biological and physical characteristics of Parthenium argentatum (guayule) latex in comparison with latex from Hevea brasiliensis and Ficus elastica. Proceedings of the 5th International Latex Conference, July 30-31, Akron, Ohio, p 1-15.
- Cornish K., Brichta, J.L., Chapman, M.H., Scott, D.J., Van Fleet, J.E., Xie, W., and Wood, D.F. 2002. Guayule latex: a clinically-proven, natural solution to Type I latex allergy. Proceedings of Latex 2002, December 4-5. Berlin, Germany.
- Pearson C.H., Brichta, J.L., VanFleet, J.E., and Cornish, K. 2002. The potential of sunflower as a rubber-producing crop for the United States. Agronomy Abstracts. American Society of Agron. Madison, WI.
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