Decades of military activity on live-fire training ranges have resulted in the contamination of land and groundwater by recalcitrant high explosives, particularly TNT and RDX. TNT and its transformation products are highly toxic, but tend to bind strongly to clay and organic matter in soil, so are largely contained at the site of contamination. RDX, however, poses a major concern, because of its high mobility through the soil water table and consequent potential for contamination of groundwater. RDX contamination of training ranges is now proving to be a significant threat to drinking water sources. Currently, there are no cost-effective processes to contain RDX or remediate large areas of contaminated land on training ranges.

The objective of this project was to engineer transgenic grasses to remove and degrade RDX in the root zone of soil contaminated with explosives. The expression in plants of a novel, RDX-degrading cytochrome P450 gene, xplA, was investigated. This enzyme, along with its redox partner (XplB), can degrade RDX to harmless metabolites. Since TNT often occurs as a co-contaminant alongside RDX, it was also necessary to engineer resistance to TNT, because this explosive is highly toxic to plants.

Wild type and transgenic plants expressing XplA growing on soil with and without RDX. The transgenic plants show minimal signs of toxicity and removed significant amounts of RDX from the soil

Under a previous SERDP project, researchers demonstrated that expression of the rhodococcal gene, xplA, in the model plant Arabidopsis, conferred the ability to tolerate and degrade high concentrations of RDX. In addition, researchers showed that expression of the bacterial nitroreductase gene, nfsI, in tobacco and Arabidopsis conferred resistance to toxic levels of TNT.

This project used a variety of different approaches to improve and extend the capacity of plants to detoxify RDX and TNT, while addressing some of the practical and regulatory issues involved in the development of this technology. For example, attempts were made to improve the RDX-degrading efficiency of the XplA protein itself. In other work, researchers tried to improve the efficiency of the nitroreductase enzyme by targeting its expression specifically to root tissues. Microarray studies and mutant screens were used to identify additional genes involved in TNT detoxification.

The problem of biocontainment of transgenes was addressed, using a chloroplast transformation approach in tobacco. A bioassay system was used to investigate herbivory of transgenic and RDX-treated Arabidopsis plants. Model plant systems, such as Arabidopsis and tobacco, were used to develop the technologies because of their efficient transformation methods and well-developed genomics tools. However, the major objective of this project was to generate transgenic plants that can phytoremediate TNT and RDX pollution in field conditions, on training ranges. For this purpose, plants needed to be robust enough to withstand fire and tolerate the effects of disruption by heavy machinery. Hence, researchers aimed to introduce the xplA/B and nfsI genes into suitable grass species that could be grown on range land in order to decontaminate soil over a large area. The effectiveness of RDX-removal by the transgenic grasses was tested in demonstration-scale field experiments. Rational engineering based on the structure of XplA and random mutagenesis and directed evolution techniques were used to characterize XplA, with the aim of improving the ability of the enzyme to degrade RDX. A possible mode of action for denitration of RDX was suggested. 

Liquid culture and soil experiments confirmed that Arabidopsis plants containing the transgenes xplA/B and nfsI could remove and degrade RDX from soil and leachate in the presence of TNT.

In microarray experiments, members of several candidate gene families were up-regulated in response to TNT. These included GTases (UDP-glycosyltransferases), OPRs (oxophytodienoate reductases), GSTs (glutathione-S-transferases), and cytochromes P450. Arabidopsis plants engineered to over-express GTases, OPRs or GSTs were more tolerant to TNT than wild-type. Production of different TNT-glutathione conjugates was investigated, in the context of possible manipulation of GSTs to optimize TNT-detoxification capacity.

Mutant screens identified an additional gene (MDHAR6), whose loss-of-function conferred resistance to TNT, as confirmed by liquid culture and soil-based experiments. Accumulation in the mitochondria of superoxide produced by MDHAR6 with TNT as substrate was proposed as the main mechanism for TNT toxicity in plants.

Experiments were carried out with tissue-specific promoters, whereby nfsI was targeted to the roots of Arabidopsis, using a root-specific promoter (RolD). Liquid culture experiments and soil studies showed that the transformed plants had increased tolerance to TNT, suggesting that such promoters could be a useful candidate for targeted transgene expression in dicot species.

Attempts were made to address some of the regulatory and public acceptance issues surrounding the release of transgenic plants. For example, experiments were carried out to engineer biocontainment of transgenes by chloroplast transformation. The protocol and vectors were optimized for successful introduction of xplA/B and nfsI into tobacco chloroplasts. This conferred tolerance to TNT and improved TNT uptake from solution, although the capacity of the plants to take up and degrade RDX was no better than wild-type. Studies were undertaken to investigate whether a generalist insect herbivore (the locust Schistocerca gregaria) showed any feeding preference for or against Arabidopsis plants transformed with xplA/B and/or treated with RDX. The locusts showed no preference for any of the treatment combinations, compared with wild-type. In other experiments, attempts to assess the impact of the genetically modified plants on soil microflora showed that expression of nfsI by tobacco plants did not have a non-target effect on soil microbial communities.

Western wheatgrass and switchgrass were chosen as the most suitable grasses to engineer for phytoremediation purposes on range land. Creeping bentgrass was used as a model to develop the technology. Tissue culture methods, transformation protocols, and regeneration techniques were developed and optimized during the course of the project. The xplA/B and nfsI genes were successfully transferred into the above grasses, using a range of newly-designed vectors with monocot promoters. Transformed switchgrass was able to remove and degrade RDX from liquid culture medium. Preliminary experiments suggested that transgenic switchgrass could also remove RDX from soil leachate.

Methods for seeding grasses on ranges were developed, using seedballs to seed remote areas. Seedball construction and formulation were optimized to improve germination and establishment.

Field studies were initiated under an ESTCP project to validate the technology at demonstration scale. In this demonstration, the optimal spacing of the grass plants in the trials was determined and test cells were designed and constructed at Fort Drum (NY, USA) for outdoor trials. 

Transgenic grasses were developed with unique abilities to detoxify TNT and degrade RDX. Such plants have the potential to provide a self-sustaining, inexpensive and environmentally-friendly method of range restoration that can be used over large areas of land for preventing groundwater contamination. Engineering plants to remove explosives could provide an efficacious means to clean up land contaminated through military activities, and presents an effective potential solution to a serious environmental problem.