Grantee Research Project Results

Final Report: Development of Herbicide-Tolerant Energy and Biomass Crops

EPA Grant Number: R829479C013
Subproject: this is subproject number 013 , established and managed by the Center Director under grant R829479
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Water Innovation Network for Sustainable Small Systems
Center Director: Reckhow, David A.
Title: Development of Herbicide-Tolerant Energy and Biomass Crops
Investigators: Weeks, Donald P.
Institution: University of Nebraska at Lincoln
EPA Project Officer: Aja, Hayley
Project Period: July 1, 2002 through June 30, 2004 (Extended to December 31, 2005)
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Targeted Research , Hazardous Waste/Remediation

Objective:

The objectives of the project were to clone, sequence, and express in plants the three genes involved in the first step in the degradation of dicamba by Pseudomonas maltophilia, stain DI-6.

Summary/Accomplishments (Outputs/Outcomes):

Previously, we had isolated the dicamba O-demethylase from P. maltophilia, strain DI-6, and performed initial enzyme characterization. This led to the discovery that the enzyme was composed of three separate components, an oxygenase (now named dicamba monooxygenase, DMO), a ferredoxin, and a reductase. We demonstrated requirements for dicamba, oxygen, nicotinamide adenine dinucleotide (NADH), and Mg²⁺ for the reaction in which dicamba is converted into the herbicidally inactive compound, 3,6-dichlorosalicylic acid. Each of the three components was purified to near-homogeneity and subjected to amino acid sequence analysis by Edman degradation procedures. The peptide sequences obtained allowed for preparation of degenerate oligonucleotide probes, which were used to select bacterial clones carrying each of the genes encoding the three dicamba O-demethylase components.

Each of the genes encoding the three components of dicamba O-demethylase were genetically engineered for expression in higher plants. Agrobacterium tumefaciens-based transformation protocols were utilized to place the genes in regenerated, transgenic tobacco plants. Spraying the transgenic plants with dicamba revealed that only the presence of the genetically modified DMO gene was needed to provide plants with protection against treatment with dicamba. Further analyses demonstrated that high levels of tolerance to dicamba could be obtained. That is, while control, nontransgenic tobacco plants were killed quickly after treatment with dicamba at 0.5 lb/acre. Several transgenic tobacco plants expressing the genetically engineered DMO gene were tolerant to treatments with at least 5 lbs/acre (i.e., 10 times the rate used by farmers to kill broadleaf weeds in their field). These experiments were repeated using tomatoes. Results were nearly identical to those obtained with tobacco.

The next step was to determine if the modified DMO gene could be inserted into soybean plants and provide this valuable crop with resistance to dicamba. In brief, these experiments were successful. Several lines of transgenic soybean plants fully tolerant to levels of dicamba used by farmers were obtained. Plants were tested under field conditions where they also showed high levels of tolerance to treatment with dicamba.

We also have moved forward with analysis of the biochemical properties of DMO and its associated ferredoxin and reductase. This research has demonstrated that DMO is a member of the Rieske nonheme-iron monooxygenase family of enzymes. The kinetic and physical properties of DMO, ferredoxin, and reductase components have been determined.

Conclusions:

The dicamba-resistance gene has been successfully genetically engineered for expression in several plants including tomato, tobacco, Arabidopsis, and soybeans. Initial field tests with dicamba-tolerant soybean plants have shown excellent tolerance to treatments with concentrations of dicamba well above those used by farmers for weed control. Most of the characterization of dicamba O-demethylase and its three constituent enzyme components has been accomplished and submitted for publication. Likewise, the cloning and characterization of the genes encoding the three components has been accomplished. A future manuscript will deal with the genetic engineering of the dicamba monooxygenase gene for expression in model plants and crop plants.

Journal Articles:

No journal articles submitted with this report: View all 1 publications for this subproject

Supplemental Keywords:

sustainable industry, waste, agricultural engineering, bioremediation, environmental engineering, new technology, innovative technology, bioaccumulation, biodegradation, bioenergy, bioengineering, biotechnology, phytoremediation, plant biotechnology,, RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Waste, TREATMENT/CONTROL, Remediation, Environmental Chemistry, Technology, Hazardous Waste, Biochemistry, Hazardous, hazardous waste treatment, chemical plants, microbial degradation, transgenic plants, intrinsic security, reaction system, computational fluid dynamics, biotechnology, phytoremediation

Relevant Websites:

http://www.cpbr.org Exit

Progress and Final Reports:

Original Abstract

2003 Progress Report

2004 Progress Report

2005

Main Center Abstract and Reports:

R829479    Water Innovation Network for Sustainable Small Systems

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829479C001 Plant Genes and Agrobacterium T-DNA Integration
R829479C002 Designing Promoters for Precision Targeting of Gene Expression
R829479C003 aka R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
R829479C005 Development of Novel Plastics From Agricultural Oils
R829479C006 Conversion of Paper Sludge to Ethanol
R829479C007 Enhanced Production of Biodegradable Plastics in Plants
R829479C008 Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
R829479C009 Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
R829479C010 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals
R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C014 Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
R829479C015 Coordinated Expression of Multiple Anti-Pest Proteins
R829479C016 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C017 Molecular Improvement of an Environmentally Friendly Turfgrass
R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
R829479C019 Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
R829479C021 Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C024 Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
R829479C025 Chemical Induction of Disease Resistance in Trees
R829479C026 Development of Herbicide-Tolerant Hardwoods
R829479C027 Environmentally Superior Soybean Genome Development
R829479C028 Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
R829479C029 Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
R829479C030 Engineered Plant Virus Proteins for Biotechnology