2006 Progress Report: Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants

EPA Grant Number: R829479C028
Subproject: this is subproject number 028 , 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: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Center Director: Schumacher, Dorin
Title: Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
Investigators: Doty, Sharon
Institution: University of Washington
EPA Project Officer: Lasat, Mitch
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:

There are no published reports of willow transformation, so the primary aim of this project is to develop effective transformation protocols. The specific aims are to: propagate sterile explants of the willow clones in tissue culture; begin embryonic callus culture; optimize transformation and regeneration methods; verify transgene presence and expression in transformed willow; and evaluate transformation frequencies from the different protocols. The genes we will introduce to enhance biofuel production and phytoremediation are an antisense 4CL-1 gene cloned from willow and a cytochrome P450 2E1 gene, respectively.

Progress Summary:

Since the funding did not become available until mid-April and this annual report is due in mid-October, the following progress report covers only the first 6 months. The five willow clones and cuttings of wild-grown willow grew well and readily rooted in standard plant growth medium (Figure 1). However, only one of the willow lines responded well to the auxin treatment to induce callus culture (Figure 2). Although callus formed from all of the willow lines, most were brown or grayish in color and failed to grow (Figure 3). We therefore turned to cytokinin treatment to induce shoot growth. Salix clones SV-1 and Sx61 responded especially well to this treatment, producing shoots from leaf explants.

Salix  Clone, SV-1, Growing Well in Tissue Culture.

Figure 1. Salix Clone, SV-1, Growing Well in Tissue Culture

Callus From Salix Clone, Sx61, on  Medium Containing Low Levels of 2,4-D

Figure 2. Callus From Salix Clone, Sx61, on Medium Containing Low Levels of 2,4-D

Callus From Salix Clone, 94006,  Grew Poorly

Figure 3. Callus From Salix Clone, 94006, Grew Poorly

Agrobacterium-mediated transformation of willow seems to be ineffective because all of the willow lines responded with severe necrosis. This was also noted by other groups that attempted willow transformation. Therefore, we will turn to the use of the biolistic transformation method.

In order to increase the efficiency of biofuel production, we are attempting to clone one of the lignin biosynthesis genes in order to downregulate its expression in transgenic willow. We designed “universal primers” for the 4CL-1 gene, prepared genomic DNA of all the willow clones, and performed PCR. Several of the clones did yield PCR product of the appropriate size. We are in the process of cloning these gene fragments, and will verify them by sequencing.

Our work with enhancing phytoremediation in aspen trees by overexpressing CYP2E1 has been very effective. This project, sponsored by the U.S. Department of Energy and the National Institute of Environmental Health Sciences, is nearly ready for publication. We will introduce this gene into willow when the transformation method has been developed.

Journal Articles:

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

Supplemental Keywords:

Scientific Discipline, Waste, TREATMENT/CONTROL, POLLUTANTS/TOXICS, Treatment Technologies, Technology, Chemicals, Biochemistry, Bioremediation, Molecular Biology/Genetics, Biology, plant-based remediation, transgenic plants, plant uptake studies, biotechnology, plant biotechnology, phytoremediation

Progress and Final Reports:

Original Abstract
  • 2005
  • 2007
  • Final

  • Main Center Abstract and Reports:

    R829479    The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program

    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