2007 Annual Report 1a.Objectives (from AD-416) Rapid and efficient methods will be developed to maximize production of significant quantities of purified-phytochemicals from plants, processed agricultural products and co-products, and plant tissue culture enhanced for phytochemical production. These phytochemicals will be used to evaluate their mode of action, determine dose requirements, and subsequently in the phytochemical evaluation of crude and processed agricultural products. Characterized phytochemicals, such as the saponins from soy and glucosinolates from crucifers, will be used to develop new agricultural pest control products, chemical feed stocks for industrial processes, and designer functional foods for humans and animals. The isolation and evaluation methods developed here will be used with minimal modification for the evaluation of other phytochemicals in biologically-active plant extracts. A key feature of this research is the development of collaborations with our analytical team, industrial; processors, bioassay researchers for both pest control and in mammalian cell systems, and mammalian nutritional research. This research will result in defined roles for phytochemicals and new/enhanced functional products and foods. 1b.Approach (from AD-416) Develop new or enhance existing protocols for the identification, quantitation, and elucidation of metabolic forms of phytochemicals from plant tissues, products, and by-products. Characterize phytochemicals from plants that demonstrate effective biological activity against agricultural pests; demonstrate biological activity in cell culture or nutritional studies; or have chemical functionality in industrial processes. Develop or optimize protocols for the induction of secondary metabolites and important economically-valuable phytochemicals and improve growth and morphogenesis in cultured plants, plant cells, plant tissues and organs. 3.Progress Report Significant progress has been made on the development of methods to purify gram quantities of specific soy isoflavones and soy saponins. This material is being provided to collaborators at Iowa State University and the University of Illinois for evaluation of their biological effects. A number of papers and funded grants have resulted from this interaction. The analytical methodology has been applied to measuring levels of saponins and isoflavones in a variety of soy products. Significant progress has been made on the development of methods to purify gram quantities of a variety of glucosinolates from crucifer species as well. This material is being used as analytical standards and as a source to prepare degradation products for evaluation in pest control, industrial antioxidant production and for evaluation in human disease prevention. This material is being provided to collaborators at Bradley University and the University of Illinois Medical School Research Facility in Peoria, IL. We are expanding our research efforts in the evaluation of the chemical and biological activity of compounds found in dry corn distillers grains. These materials have been shown to contain potent antioxidant activity, and preliminary laboratory and limited field work has shown that this material contains potent pesticidal activity against weedy species as well as providing nutrient enrichment to the soil. Biologically active compounds (antioxidants) are being isolated and characterized from corn distillers solubles/solids in collaboration with researchers at the University of Illinois. The analytical methodology available in this research project has been used to identify and quantitate phytochemicals in several other collaborative projects. It is generally believed that occurrence of high biomass (primary metabolism) coupled with high phytochemical production are mutually exclusive. Plants require carbon obtained from atmospheric photosynthesis for both primary and secondary metabolism. Within the tissue culture environment, exogenous sugar provides the carbon for these processes. We have found a strong correlation between secondary metabolism, primary metabolism and sugar content in the media. We are continuing our studies to exploit these observations to increase other secondary metabolite levels, coupled to high biomass production in vitro and in vivo. In order to obtain high yields of secondary products from plant foliage in vitro, we studied the influence of the physical environment with and without supplemental carbon dioxide additions. The in vitro environment being sterile necessitates the occurrence of high chamber relative humidity (about 100%). These high humidity levels significantly retard normal growth and foliage development, which correspondingly reduces secondary metabolism. By intermediate air applications, normal foliage growth and development as well as higher secondary metabolism occurs. We are currently exploiting these findings to improve secondary metabolism to develop model plant systems to study. 4.Accomplishments 1) Evaluation of biodiesels from several oilseed sources as environmentally-friendly contact herbicides. Postemergence contact herbicidal activities of biodiesels from several oilseed sources were examined for potential use to control broadleaf weeds in turfgrasses. Cuphea, Lesquerella, meadowfoam (Limnanthes alba), milkweed (Asclepias syriaca and Ascelepias speciosa), and soybean oils were applied as 1 and 2% volume per volume aqueous emulsions plus a nonionic surfactant to two-week-old seedlings of perennial ryegrass, velvetleaf (Abutilon theophrasti) and sicklepod (Senna obtusifolia) in the greenhouse. All five biodiesels were more phytotoxic to sicklepod and velvetleaf than to perennial ryegrass. These results indicate that biodiesels may be useful as environmentally-friendly contact herbicides in turfgrasses, as there are few contact herbicide options available for the homeowner market which will not cause turf injury. This effort directly supports National Program 306, Quality and Utilization of Agricultural Products, Component 2-New Processes, New Uses, and Value-Added Foods and Biobased Products, Problem Area 2b-New Uses for Agricultural By-products. 2) Alkaloid production via plant tissue culture in a laboratory facility In many cases, plants are the only source of many important pharmaceuticals, such as the alkaloids which are derived from poppy. In order to secure morphine alkaloids within a laboratory setting, novel plant tissue culture technology was developed. A unique plant tissue culture technology was developed whereby alkaloids were harvested from leafy and shoot material while in a green, actively-growing, non-senescent stage. This invention demonstrates the employment of in vitro leafy plant material to produce commercial pharmaceuticals. Implementation of this technology will result in guaranteed product yield within secured laboratory commercial facilities. This effort directly supports National Program 306, Quality and Utilization of Agricultural Products, Component 2-New Processes, New Uses, and Value-Added Foods and Biobased Products, Problem Area 2b-New Uses for Agricultural By-products. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of new CRADAs and MTAs 4 Number of active CRADAs and MTAs 2 Number of invention disclosures submitted 2 Number of U.S. patents granted 1 Number of non-peer reviewed presentations and proceedings 6 Number of newspaper articles and other presentations for non-science audiences 6 Review Publications Jadhav, U., Ezhilarasan, R., Vaughn, S.F., Berhow, M.A., Mohanam, S. 2007. Iberin induces cell cycle arrest and apoptosis in human neuroblastoma cells. International Journal of Molecular Medicine 19(3):353-361. Jadhav, U., Ezhilarasan, R., Vaughn, S.F., Berhow, M.A., Mohanam, S. 2007. Dietary isothiocyanate iberin inhibits growth and induces apoptosis in human glioblastoma cells. Journal of Pharmacological Sciences 103(2):247-251. Kim, S., Berhow, M.A., Kim, J., Chi, H., Chung, I. 2006. Evaluation of soyasaponin, isoflavone, protein, lipid, and free sugar accumulation in developing soybean seeds. Journal of Agricultural and Food Chemistry 54:10003-10010. Vaughn, S.F., Holser, R.A. 2007. Evaluation of biodiesels from several oilseed sources as environmentally-friendly contact herbicides. Industrial Crops and Products 26(1):63-68. Prasongsuk, S., Berhow, M.A., Dunlap, C.A., Weisleder, D., Leathers, T.D., Eveleigh, D.E., Punnapayak, H. 2007. Pullulan production by tropical isolates of Aureobasidium pullulans. Journal of Industrial Microbiology and Biotechnology. 34(1):55-61. Johnson, E.T., Berhow, M.A., Dowd, P.F. 2007. Expression of a maize Myb transcription factor driven by a putative silk-specific promoter significantly enhances resistance to Helicoverpa zea in transgenic maize. Journal of Agricultural and Food Chemistry. 55(8):2998-3003. Sun, Y., Cheng, J.J., Himmel, M.E., Skory, C.D., Adney, W.S., Thomas, S.R., Tisserat, B., Nishimura, Y., Yamamoto, Y.T. 2006. Expression and characterization of Acidothermus cellulolyticus E1 endoglucanase in transgenic duckweed Lemna minor 8627. Bioresource Technology. Available: http://www.sciencedirect.com, doi:10.1016/j.biortech.2006.09.055