Enhanced Transformation of TNT by Tobacco Plants Expressing a Bacterial Nitroreductase

Abstract

The manufacture, disposal, and detonation of explosives have resulted in the pollution of large tracts of land and groundwater. Historically, 2,4,6-trinitrotoluene (TNT) is the most widely used military explosive and is toxic to biological systems and recalcitrant to degradation. To examine the feasibility of enhancing the ability of plants to detoxify the explosive TNT, we created transgenic tobacco (Nicotiana tabacum) constitutively expressing the nsfI nitroreductase gene from Enterobacter cloacae. The product of TNT reduction by the nitroreductase was found to be 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT). Characterization of the transgenic lines in sterile, aqueous conditions amended with TNT demonstrated that these plants were able to remove all of the TNT from the medium at an initial concentration of 0.5 mM (113 mg L⁻¹) TNT. In contrast, growth was suppressed in wild-type plants at 0.1 mM (23 mg L⁻¹). Following uptake, transgenic seedlings transformed TNT predominantly to 4-HADNT and its high levels appeared to correlate with enhanced tolerance and transformation of TNT. Transformation products of TNT were subsequently conjugated to plant macromolecules to a greater degree in transgenic tobacco, indicating enhanced detoxification compared to the wild type.

KEY WORDS:

• phytodetoxification
• phytoremediation
• 2,4,6-Trinitrotoluene (TNT)
• explosives

ACKNOWLEDGEMENTS

The authors thank Richard Williams and Stephen Nicklin for their useful comments about the transformation and analysis of TNT.

This work was supported by the Biotechnology and Biological Sciences Research Council, U.K. Ministry of Defense, and by the U.S. Department of Defense, through the Strategic Environmental Research and Development Program (SERDP).

Notes

^aAs TNT was not added to these flasks, weight measurements were taken at the same time as the addition of TNT to the other flasks.

^bAnalysis of variance showed that the weight index was significantly different at 0.25 mM (P = 0.05).