Grantee Research Project Results

2004 Progress Report: Molecular Improvement of an Environmentally Friendly Turfgrass

EPA Grant Number: R829479C017
Subproject: this is subproject number 017 , 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: Molecular Improvement of an Environmentally Friendly Turfgrass
Investigators: Altpeter, Fredy
Institution: University of Florida
EPA Project Officer: Aja, Hayley
Project Period: January 1, 2004 through December 31, 2004 (Extended to December 31, 2007)
Project Period Covered by this Report: January 1, 2004 through 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 this research project are to:

1. develop a genetic transformation protocol for turf-type bahiagrass;
2. and modulate the bioactive gibberellic acid content in transgenic turf-type bahiagrass for improved turf characteristics and reduced mowing requirements.

Progress Summary:

Bahiagrass is extensively used in the southern United States for utility turf along highways and for residential lawns. The popularity of bahiagrass is attributed to its excellent persistence supported by drought tolerance, heat tolerance, insect and disease resistance, and nematode suppression. Genetic engineering of turf-type bahiagrass is desirable to improve turf quality and reduce the mowing frequency. The cultivar Argentine was selected for genetic transformation because it is a commercially important turf-type bahiagrass cultivar and its tetraploid, apomictic nature should result in uniformity of the transgenic progeny and transgene containment resulting from sexual incompatibility with diploid wildtype bahiagrasses. We developed an efficient tissue culture system for regeneration of turf-type bahiagrass cultivar Argentine from mature seed-derived callus. Mature seed explants are preferred over other explants because they are readily available year-round, and the explant preparation prior to culture initiation is less labor intensive.

An efficient biolistic gene transfer protocol for turf-type bahiagrass (cultivar Argentine) also was developed. Transfer of a constitutive nptII, bar, or EPSPS expression cassette into seed-derived callus cultures was followed by selection with paromomycin sulfate, bialaphos, or glyphosate, respectively, during callus subculture and/or regeneration. Transgenic plants were confirmed, expressing bar, nptII, or EPSPS. Consequently, all three selectable markers supported the generation of transgenic plants. The comparison of three different selectable marker genes revealed significant differences in transformation efficiency. Immunodetection of transgene expression revealed that 1 to 10 transgenic plants were generated from the three different selectable marker genes per 20 bombarded Petri dishes with 20 callus pieces each. Transgenic plants were transferred to soil in approximately 17 to 20 weeks from the initiation of mature seed cultures. The transgenic nature of regenerated plants was confirmed with PCR of genomic DNA, using primers annealing to the transgene expression cassette and by immunoassays (ELISA or Quicksticks) of leaf protein extracts.

The highest transformation efficiency was obtained using nptII, and the lowest transformation efficiency was obtained using EPSPS as a selectable marker. The transgenic plants were transferred to soil and one EPSPS transgenic plant was confirmed with resistance to glyphosate using a 1 lb/acre application rate (no necrosis or growth reduction observed), whereas wildtype bahiagrass was eliminated with 0.1 lb/acre. Roundup resistance in transgenic bahiagrass will support the conversion of a conventional bahiagrass into improved dwarf bahiagrass turf.

The established transformation protocol currently is used to introduce a gibberellic acid catabolizing enzyme (GA-2-oxidase) under control of different regulatory promoter elements to achieve a reduction of bioactive gibberellic acid, which is expected to result in dwarfing, reduced mowing frequency, darker green color, enhanced drought, and shade tolerance.

The following vectors have been completed:

35S promoter — hsp70intron — GA2ox — NOS

Ubiquitin promoter — ubi1intron — GA2ox — NOS

These vectors currently are cointroduced together with a selectable marker gene into turf-type bahiagrass (cultivar Argentine).

Future Activities:

We will generate seed and vegetative progenies of transgenic lines. Transgene integration and expression of the progenies will be evaluated. Additional lines will be generated using alternative promoter—GA2 oxidase constructs to identify the lines, which combine the most desirable phenotype with persistence under mowing and good seed production.

Journal Articles:

No journal articles submitted with this report: View all 5 publications for this subproject

Supplemental Keywords:

molecular growth regulation, transgenic turfgrass, bahiagrass, dwarf, turf quality, mowing, sustainable industry, sustainable business, waste, agricultural engineering, bioremediation, environmental engineering, geochemistry, new technology, innovative technology, bioaccumulation, biodegradation, bioenergy, bioengineering, biotechnology, phytoremediation, plant biotechnology, sustainable industry/business, environmental chemistry,, Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Genetics, Technology, New/Innovative technologies, Ecology and Ecosystems, Agricultural Engineering, bioengineering, transgenic plants, plant genes, bahiagrass, biotechnology, plant biotechnology, cloning, environmentally friendly turfgrass

Relevant Websites:

http://www.cpbr.org Exit

Progress and Final Reports:

Original Abstract

2005 Progress Report

2006

Final Report

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