Grantee Research Project Results
2006 Progress Report: Development of Herbicide-Tolerant Hardwoods
EPA Grant Number: R829479C026Subproject: this is subproject number 026 , 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: Duke University Center for Environmental Implications of NanoTechnology
Center Director: Wiesner, Mark R.
Title: Development of Herbicide-Tolerant Hardwoods
Investigators: Meilan, Richard
Institution: Purdue University
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2004 through September 30, 2007 (Extended to December 31, 2007)
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research
Objective:
The objectives are to:
- Engineer improved walnut genotypes with constructs containing genes that impart tolerance to Arsenal®.
- Screen transgenic plantlets in the field for commercially useful levels of herbicide tolerance.
- Determine if the introduced trait is expressed stably over time and in different environments.
Progress Summary:
Tissue Culture
In order to genetically engineer a plant, one must be able to insert a gene into a chromosome of an individual plant cell and then cause that cell to differentiate into a whole plant. The former process is referred to as transformation; the latter, regeneration. Before inducing cells in an explant to differentiate into whole plants, it is necessary to identify those cells that have been transformed. This is most often accomplished by inserting an antibiotic resistance gene (usually NPTII, which imparts resistance to kanamycin) along with the gene of interest. Introducing antibiotic resistance genes into plants is not only controversial, but assembly of the necessary genetic constructs can be cumbersome. To avoid these difficulties, we decided to use the acetohydroxyacid synthase (AHAS) gene, which imparts resistance to the active ingredient in the herbicide Arsenal® (Imazapyr), both as an in vitro selectable marker and to confer a commercially useful trait.
To use this approach, though, we needed to conduct a dose-response study in which we determined the concentration that would effectively kill non transformed cells without having any detrimental effects on our transgenic lines. This was accomplished after obtaining purified Imazapyr from our industrial partner, BASF. Untransformed somatic embryos from two lines of walnut (genotypes 21 and 91) were used to repeatedly test five concentrations of Imazapyr, and 1 μM (0.26 mgL-1) was found to be most effective.
In our original proposal, we stated that we expected to obtain 25 independent transgenic lines (events) in each of two elite black walnut (Juglans nigra L.) genotypes. This has proven to be more problematic than we had anticipated. Our black walnut transformation and regeneration protocol was developed using genotypes other than those needed for the completion of this project. It is widely known that protocols optimized for one genotype generally cannot be used effectively with others; with each new genotype, the protocol must be re-optimized. This has certainly proven to be the case for walnut.
One of our problem areas has been maturation and germination of somatic embryos that were induced to form in vitro. Using four black walnut genotypes (designated 21, 83, 89, and 91), we have conducted experiments that mimic the conditions required for zygotic embryo germination in nature, including treatments such as desiccation, cold temperatures, darkness, and various types of media. These studies have yielded encouraging results. For example, shoot and root production from embryos increased from 2.5 percent to 25 percent as the length of cold treatment increased from 0 to 20 weeks. In addition, a full-strength medium was found to be more effective for both germination and regeneration.
Once embryos germinate in vitro, it is imperative that they develop sufficient secondary roots (i.e., fine roots), so the resulting plantlets have adequate water absorption capacity to survive ex vitro. To boost secondary root production, we have tested various auxin treatments, light regimes, and rooting media. These studies have also yielded positive results and are now complete.
Once rooted plantlets have been acclimated in soil, it has been exceedingly difficult to induce additional shoot development. To overcome this difficulty, we have experimented with a product known as Promalin, which is a mixture of biological active gibberellins (a class of plant hormones that regulate shoot elongation). This work is ongoing.
We are now in the process of converting a large number of globular-stage embryos to cotyledonary-stage embryos. This step takes approximately 2 months to complete and is a necessary prerequisite to transformation. Once this is done, we will utilize the AHAS-containing construct (see below) to transform these explants and then regenerate whole, transgenic plants using the information derived from the experiments described above.
Construct Assembly
Before we can transform walnut explants, we need to assemble a binary (plasmid) vector that will be inserted in Agrobacterium tumefaciens, a vehicle for moving genes into plant cells. We plan to include not only our gene of interest (AHAS) in the transferred DNA (T-DNA) region of the binary vector but also a reporter gene b-glucuronidase (GUS), so we can visualize transformed cells and thus avoid the production of chimeric plants. We encountered some unexpected restriction endonuclease sites in the backbone we were using for the assembly of our binary vector, which precluded its further use. We then discovered that our secondary backbone did n ot posses an intron-containing version of the GUS gene. This is necessary to avoid the selection of false-positive plants resulting from residual A. tumefaciens that may be present in plant tissues (prokaryotes cannot excise introns, which prevent them from producing a functional gene product). We have identified, ordered, and obtained a vector backbone that contains an abundance of unique restriction sites and an intron-containing version of GUS. We are now well on our way to assembling the binary vector that we will need to transform black walnut. It will be ready when the cultures described above are receptive to A. tumefaciens infection.
Future Activities:
During the next 12-month period, we will: (1) finish the assembly of our binary vector; (2) transform the binary vector into A. tumefaciens; (3) produce transgenic plants; and (4) perform molecular characterizations of these transgenic plants.
We plan to conduct field tests of the transgenic plants under a no-cost extension agreement.
Supplemental Keywords:
Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Genetics, Geochemistry, Technology, New/Innovative technologies, Agricultural Engineering, bioengineering, transgenic plants, herbicide tolerance, plant genes, bahiagrass, biotechnology, plant biotechnology, cloning, environmentally friendly turfgrassProgress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R829479 Duke University Center for Environmental Implications of NanoTechnology 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
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.