1. Phytoremediation: Remediation of contaminated areas using plants
(Doty 2008).
Backed up by the California Biome Grass experiment as well as
proof of inoculation, the 5 types of perennial grasses can be in-
oculated with PTA1 to test for TNT toxicity resistance.
2,4,6 –trinitrotoluene
(TNT)
Explosive compound used as munitions.
Classified possible human carcinogen and proven
toxic to all organisms. (reviewed in Rylott 2011)
US Dept. of Def. estimates 24.6 million acres of active military rang-
es contaminated with unexploded ordnance (UXOs). (Rylott 2008)
Estimated cost using traditional methods: $16 ～ $165 billion.
There could be more than 15 million acres of closed sites with
UXOs, also vast areas in sea.
Fig 2. Structural formula
of trinitrotoluene
molecule.
Advantages to
phytoremediation
Methods of Tradi-
tional Environmen-
tal Cleanup
Solar drivenIncineration of soil
Costs 10-20% of
mechanical meth-
ods
Removing soil to
landfill
Soil usable after
treatment
Composting
Generates less sec-
ondary wastes
Capping soil
Table 1. Advantages of phytoremediation (Source: Chappell 1998, Rylott
2008)
Figure 1. Main methods of phytoremediation. (Source: Nature
Education. Model of different phytoremediation. Digital
image. Phytoremediation. Scitable, 2011. Web. )
Introduction
Review of Literature
Fig 4. The three phases of the ‘green liver’ model: hypo-
thetical pathway representing the metabolism of trini-
trotoluene (TNT) in plant tissues: phase I: activation of
TNT by reduction to 2-amino-4,6-dinitrotoluene; phase
II: conjugation with a plant molecule, for example, glu-
cose; phase; phase III: sequestration of the conjugate in-
to the vacuole or cell wall. (Reviewed in Aken 2009)
Primary site of entry is roots.
Compared to rhizosphere bacteria,
have a closer interaction with host
(Doty 2008, Weyens 2009).
Plant provides nutrients + endophyte
benefits plant growth, resistance.
Perennial grass: revegetation. Used in military training lands due
to durability, large spread, high weed control, ease of growth,
and low maintenance.
Agropyron sibericum (siberian wheatgrass), Pascopyrum smithii
(western wheatgrass) and Elymus trachycaulus (slender wheat-
grass) are all used at military training sites (Waldron 2005, Palazzo 2009).
Panicum virgatum L. (switchgrass) and Agrostis stolonifera L.
(creeping bentgrass) are also widely used grasses for experi-
ments.
Endophyte PTA1
Isolated from Populus tremula x alba 717-1B4 culture.
Identified as a Pseudomonas strain with a 99% match. (Doty, unpublished)
Experiments with “California Biome” grass and PTA1: proof of
inoculation and inoculated grew better than uninoculated.
Fig 5. PTA1 colonies in plant tissue (Photograph
by Zareen Khan)
Fig 6. California Biome Grass exposed to TNT
(Photograph by Zareen Khan)
Uninoculated Inoculated
Endophytic bacteria:
Mutualistic/communalistic
bacteria living within plant
tissues.

2. Methodology
1) PTA1 inoculated grasses will show higher resistance to TNT
compared to uninoculated grasses.
1) Determine if the introduction of endophyte PTA1 enhances
resistance of 5 types of grasses to the phytotoxic TNT.
Grass varieties
Fig 19. Inoculated samples of seeds. (1 Mar. 2012. Personal Photo)
Fig 24
Fig 25
Figure 26 displays the average final
weight of each grass type compared
between inoculated vs uninoculat-
ed. Standard error bars are shown.
Creeping bent grass, Siberian
wheatgrass, and western wheat-
grass shows a difference between
inoculated and uninoculated.
Purpose Hypothesis
Results
Fig 20. Setup of agar plates
for germination of seeds
Pascopy-
rum smithii
(western
wheatgrass)
Ely-
mus trachycaul
us (slender
wheatgrass)
Panicum
virgatum L.
(switchgrass)
Ag-
rostis stolonifera
L. (creeping
bentgrass)
Agropyron siber-
icum (siberian
wheatgrass)
Procedure
>0.1 OD600
Fig 18. Diode Array Spectro-
photometer (12 Mar. 2012. Personal Photo)
400uMTNT
Uninoculated
TNT
Inoculated
TNT
Uninoculated
Control
(NO TNT)
Inoculated
Control
(NO TNT)
Luria-Bertani broth
with mannitol-
glutamic acid: MG/L
broth (for growth of
bacteria)
Trial: 7 days.
Track every 3 days except
last day.
Grass lined horizontally
Plate vertical to allow growth
PTA1
Incubate Until
Germination
Control
Inoc + TNT
Inoc control Uninoc control
Uninoc +
TNT
Figures 22-25 are observational data showing comparisons between plant groups and growth in
length. (Mar 2012. Personal Photo).
Trial1Trial2
Fig 7 to Fig 16. All retrieved from USDA The PLANTS Database and bugwood.org
Fig 7 Fig 9
Fig 11
Fig 13
Fig 15
Fig 16
Fig 17. Broth of PTA1 (12
Mar. 2012. Personal Photo)
MG/L
Broth
Fig 22
Fig 23
Inoc + TNT
Inoc control
Uninoc control
Uninoc +
TNT
1 wk
Developing 'Super Plants' with Symbiosis:
Endophyte-Assisted Phytoremediation of TNT
Fig 8
Fig 10
Fig 12 Fig 14
Fig 26
Fig 21. Left to right: slender, Siberian, creeping, switch, and western.
(3 Mar. 2012. Personal Photo)

3. Limitations:
-Germination time: Grass varieties germinated on different days.
-Short testing period: The plate experiment only ran for a week,
longer trial could give data comparing TNT dose and plant survival.
-Undetermined colonization: While there is proof that colonization
occurs with PTA1, there is no evidence that colonization occurred.
-Lack of data: Initial grass weights were not calculated. Future ex-
periments will include this data.
-Vertical Setup: The water from the agar solution seems to have
evaporated under incubation lights then condensed afterwards.
May have caused discrepancies.
Why is this research important?
The use of natural endophytes is desirable due to non-
invasiveness. Transgenic plants and endophytes are being re-
searched, but natural endophytes are easier for in vivo research
and have less stringent policies.
Phytoremediation as a remediation method is cost effective, more
aesthetically pleasing, and is known to cause less damage to the
environment compared to traditional methods.
1)PTA1 inoculated grasses showed higher resistance
to TNT compared to uninoculated grasses.
2) Inoculation with PTA1 can lead to
greater biomass and plant length even in
the absence of a toxic material.
PTA1 Experiment:
-Visual length data: looks great!
-Trial 1: difference between inoc. dosed vs. uninoc. dosed.
400uM TNT did not reach LD50.
-Trial 2: difference between inoc. vs. uninoc.; possibly due to
phytohormones (auxins)
-Weight data: Inoc better than uninoc, but high variation.
-TNT has huge effect on plant growth (as expected).
-Slender wheatgrass did not germinate at all. One reason: old seeds.
Discussion
I would like to thank my mentor, Dr. Sharon Doty, in the School of Environmental and Forest Sciences at the
University of Washington for all her work and cooperation with me.
Special thanks to Dr. Khan, Mr. Jun Won Kang, and Mr. Ronald Cuie for their past and still going support.
Thank you all very much to the rest of the Doty lab as well.
Thank you to Ms. Valerie Holmes, Ms. Martha Strachan and Ms. Mary Glodowski for their ongoing help.
Thank you to my parents for their ongoing encouragement.
Questions:
Is there a toxicity dose limit for TNT (and RDX) for these grasses?
What about RDX?
Future research:
RDX is a co-contaminator along with TNT. Both are
often found together on military sites, but they are
both phytotoxic, causing a huge problem in their
phytoremediation (Aken 2009, Rylott 2008). Most effective
method for degradation: a plant tolerant to both
compounds and degrading both compounds.
-One idea: co-inoculation with PTA1 and Rhodococcus, a bacteria
that can grow in RDX by using it as a nitrogen source.
Fig 27. Chemical
Structure of RDX.
PTA1PTA1PTA1
Fig 28. Rhodococcus on
plate. (Doty 2008)
RhodococcusRhodococcusRhodococcus
(Nitroreductase(Nitroreductase(Nitroreductase
gene)gene)gene)
TNT
DegradeDegradeDegrade
RDXRDXRDX
AndAndAnd
TNTTNTTNT
RDX
Fig 5.
Conclusion
Acknowledgements