Expression in grasses of multiple transgenes for degradation of munitions compounds on live-fire training ranges

Abstract

The deposition of toxic munitions compounds, such as hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX), on soils around targets in live-fire training ranges is an important source of groundwater contamination. Plants take up RDX but do not significantly degrade it. Reported here is the transformation of two perennial grass species, switchgrass (Panicum virgatum) and creeping bentgrass (Agrostis stolonifera), with the genes for degradation of RDX. These species possess a number of agronomic traits making them well equipped for the uptake and removal of RDX from root zone leachates. Transformation vectors were constructed with xplA and xplB, which confer the ability to degrade RDX, and nfsI, which encodes a nitroreductase for the detoxification of the co-contaminating explosive 2, 4, 6-trinitrotoluene (TNT). The vectors were transformed into the grass species using Agrobacterium tumefaciens infection. All transformed grass lines showing high transgene expression levels removed significantly more RDX from hydroponic solutions and retained significantly less RDX in their leaf tissues than wild-type plants. Soil columns planted with the best-performing switchgrass line were able to prevent leaching of RDX through a 0.5-m root zone. These plants represent a promising plant biotechnology to sustainably remove RDX from training range soil, thus preventing contamination of groundwater.

Keywords: RDX; TNT; monocot promoters; phytoremediation; stacked genes; switchgrass.

Figure 1

Construction of vectors for transformation of the grasses. (a) T‐DNA region of the binary vector plasmid pRCS2‐ABNR‐HR. The RDX degradation gene xplA, flavodoxin reductase gene xplB and TNT‐detoxifying nitroreductase gene nfsI were constructed into versatile cloning vector pSATs (Chung et al., 2005). Arrows show the direction of transcription. (b) The Osact, Zmubi and Pvubi promoters were used to replace the promoters in the pSAT vectors resulting in pNSAT1a, pNSAT3a and pNSAT6a, respectively. (c) T‐DNA region of the binary vector plasmid pRCS2‐NABNR. The hpt, xplA, xplB and nfsI genes were constructed into pNSAT1a, pNSAT6a, pNSAT3a and pSAT4a, respectively. The expression cassettes of these genes were integrated into the binary vector pPZP‐RCS2 to produce pRCS2‐NABNR. Abbreviations: 35s, CaMV 35s; rbc, Rubisco small subunit; act, actin; ags, agropine synthase; Osact, Oryza sativa actin promoter; Zmubi, Zea mays ubiquitin promoter; Pvubi, Panicum virgatum (switchgrass) ubiquitin promoter; RB left border; RB right border.

Figure 2

Functional evaluation of the pNSATs vectors using transient expression reporter genes in the cytosol of epidermal onion cells. (a) Fluorescence microscopy showing GPF expression following particle bombardment with pNSAT1a‐GFP (OsAct‐GPF‐35S). (b) Histochemical staining of GUS expression following particle bombardment with pNSAT3a/6a‐GUS (ZmUbi‐GUS‐Mas; PvUbi‐GUS‐rbc).

Figure 3

Production of transgenic creeping bentgrass and switchgrass. (a) Appearance of embryogenic calli of creeping bentgrass infected with Agrobacterium harbouring pRCS2‐NABNR after 3 weeks of culture on callus induction medium with hygromycin. (b) Hygromycin‐resistant calli on regeneration medium with hygromycin and (c) Transgenic plants in soil. (d) Appearance of embryogenic calli of switchgrass infected with Agrobacterium harbouring pRCS2‐NABNR after 4 weeks of culture on callus induction medium with hygromycin. (e) Hygromycin‐resistant plantlets on regeneration medium with hygromycin and f) Genetically transformed plants in soil.

Figure 4

Molecular characterization of xplA ‐ xplB‐nfsI ‐transformed switchgrass. (a) Transcript abundance measured using quantitative RT‐PCR on plant lines transformed with xplA, xplB and nfsI. Values were normalized to the switchgrass reference gene eIF‐4a (Gimeno et al., 2014). Arabidopsis values were normalized to the reference gene ACT2. All values are relative to the expression levels of the xplA‐xplB‐nfsI expressing Arabidopsis line 7D (Rylott et al., ,; n = 4 ± SE). (b) Western blot analysis on leaf blades of switchgrass lines expressing XplA, XplB and nitroreductase (NR) protein. (c) Band intensities were quantified for XplA and XplB expression. Levels were normalized to the Coomassie‐stained Rubisco large subunit; results are from three replicate blots ± SE.

Figure 5

Uptake of RDX by xplA ‐ xplB‐nfsI ‐transformed creeping bentgrass grown in liquid culture. (a) Concentration of RDX in culture medium over the course of the experiment. After three days, the medium from line N19 contained significantly (P = 0.010) less RDX than medium from wild‐type plants, and after nine days, lines N5 and N18 had also removed significantly more RDX from the media than wild type (P = 0.03 for N5 and 0.05 for N18). (b) Concentration of RDX in creeping bentgrass tissue after 16 days. Letters indicate that RDX concentrations in tissue were significantly different (P < 0.05) from other lines (n = 3 ± SE, N/D = none detected).

Figure 6

Uptake of RDX by xplA ‐ xplB‐nfsI ‐transformed switchgrass grown in liquid culture. (a) Concentration of RDX in culture medium over the course of the experiment. All three transgenic lines removed RDX from the medium at significantly faster rates than the wild‐type plants (P = 0.051, 0.0014 and 0.0016 for lines N1, 2 and 3, respectively, at day 3; P = 0.0043 for line N1 at day 7). (b) Concentration of RDX in switchgrass tissue after 14 days (n = 3 ± SE, N/D = none detected).

Figure 7

Recovery of RDX applied to wild‐type and xplA ‐ xplB‐nfsI ‐transformed switchgrass in column experiments. (a) Mass of RDX applied as solutions containing 30 mg/L, and mass recovered in the leachate by flushing each column with 5 L water. (b) RDX level was significantly less (P = 0.0044) in the transgenic leaf tissue compared with the wild‐type leaf tissue in the column experiments after 14 days (n = 4 ± SE, N/D = none detected).