Removal of Pollutants with Transgenic Trees

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Removal of Pollutants with Transgenic Trees
Catey Dominguez Sabrina Koga November 6, 2008
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Benzene

• Important solvent
• Used in production of drugs, plastic, synthetic
  rubber, and dyes
• Comes from combustion or evaporation of fuels
• Carcinogen

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Vinyl chloride

• Used chiefly in the production of polyvinyl
  chloride (PVC)
• Evaporates easily and can dissolve in water in
  small amounts
• Is a carcinogen

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Trichloroethylene

• Cleaning solvent
• Adhesives, paint remover, correction fluids, spot
  removers
• Easily evaporates
• Remains in ground for an extended time
• Possible carcinogen, adverse effects on heart,
  lungs, liver, kidneys, nerves, and can cause death

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Chloroform

• Used to make chemicals
• Evaporates easily
• Slow break down process
• Dissolves in water
• Possible carcinogen
• Harmful to liver, kidneys, skin

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Carbon tetrachloride

• Used in industrial applications
• Easily evaporates doesnt dissolve well
• Stable in air, quickly breaks down in soil/water
• Harmful to atmosphere
• Kidneys, liver, CNS, possible coma and/or death
• Carcinogen

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Phytoremediation
Phytoremediation the treatment of environmental
problems through the use of plants

• Metabolize
• Sequester
• Volatize

Genus, Populus

• Natural removal of trichloroethylene and CCl4 in
  a
• process similar to mammals
• Rapid growth
• High water uptake

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Enhanced phytoremediation of volatile
environmental pollutants with transgenic trees
Sharon L. Doty, C. Andrew James, Allison L.
Moore, Azra Vajzovic, Glenda L. Singleton,
Caiping Ma, Zareen Khan, Gang Xin, Jun Won Kang,
Jin Young Park, Richard Meilan, Steven H.
Strauss, Jasmine Wilkerson, Federico Farin, and
Stuart E. Strand
PNAS, Vol.104 no.43
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Goal of study
Increase effectiveness of phytoremediation in
poplars

• Transform with genes involved in
  pollution- metabolism from other organisms
• Mammalian Cytochrome P450 2E1

Broad specificity (trichloroethylene, chloroform,
carbon tetrachloride, benzene, vinyl chloride,
and others)
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Creation of transgenic poplars
Experimental Poplars

• rabbit CYP2E1 cDNA inserted into pSLD50-6, under
• cauliflower mosaic virus 35S promoter
• pSLD50-6 inserted into A. tumefaciens
• trees transformed with Agrobacterium

Null Poplars

• pKH200 replaced pSLD50-6

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Assess activity of phytoremediation (metabolism)
Trichloroethylene (TCE) Metabolism

• Cuttings of null and experimental trees sealed in
  VOA vials
• 50 mg/ml TCE added to vials
• Poplar cuttings were allowed one week of exposure
• Flash-frozen and ground
• Extraction of trichloroethanol from plant tissue

trichloroethanol metabolite of tricloroethylene,
in early stages of metabolism
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TCE metabolism
Average TCE metabolism of CYP2E1 lines was nearly
45-fold greater than in the control cuttings
Lines 20 and 78 had metabolism of over 100 fold
greater than control
Functionality of phytoremediation greatest in
poplar lines 20 and 78, which are transgenic for
mammalian cytochrome P450 2E1
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Measurement of CYP2E1 expression
RNA extraction from 100 mg leaf samples from the
following poplars

• wild type
• null vector
• CYP2E1-4
• CYP2E1-8
• CYP2E1-20
• CYP2E1-78

RT-PCR performed and quantified

• housekeeping gene 18S
• probe CYP2E1

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RT-PCR of the transgene CYP2E1

• Gene expressed in all transgenic lines
• Highest expression in line 78
• Not expressed in WT or null

Expression levels correlate with the productivity
of the transgenic enzyme
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With what efficiency can pollutants be collected
from the ground?
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TCE uptake

• 8 cm tall cuttings of null and rCYP2E1 lines
• Received same dosage of TCE as metabolism
  experiment
• Samples from medium taken at two time points

4 hours 7 days

• TCE extracted from samples

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TCE uptake from plant medium
CYP2E1 transgenic plants removed TCE at a much
quicker rate from the medium than null
Lines 4, 20 and 78 had the greatest percent and
rate of removal from medium
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Removal of chloroform

• Cuttings with 4-6 leaves, 8 cm tall
• Null and CYP2E1 20 78 lines
• Chloroform added to hydroponic solution (3mg/ml)
• Chloroform content of solution measured daily

After 1 week, the transgenic plants removed 99
of the chloroform from medium 1820 removed by
controls
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Removal of CCl4

• Cuttings with 4-6 leaves, 8 cm tall
• CCl4 added to hydroponic solution
• (1.6mg/ml)
• CCl4 content of solution monitored
• daily

After 1 week of exposure, the transgenic plants
removed 9294 of the carbon tetrachloride from
medium 1022 removed by the controls
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With what efficiency can pollutants be collected
from the air?
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Removal of TCE from air

• Small rooted poplar plants in
• hydroponic solution placed in
• dessicators
• CYP2E1 line 78, null, and unplanted
• 82ml of TCE-saturated water in test
• tubes placed in dessicators
• 500ml gas from dessicator injected
• into SRI 8610C GC flame ionization
• detector

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Volatile TCE removed
The CYP2E1 plants removed 79 of TCE from the
air Null vector control plants did not show any
statistically significant uptake of TCE compared
with the unplanted soil control
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Removal of benzene from air

• Plant cuttings placed in flasks containing
• 225ml hydroponic solution in dessicator
• 200ml benzene-saturated water in test
• tubes
• 500ml gas from dessicator injected into
• SRI 8610C GC-flame ionization
• detector

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GC analysis of benzene removal
36-46 of volatile benzene removed by CYP2E1 line
78 Control plants removed an average of 13
benzene
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Vinyl chloride

• Cuttings of null and line 78 transgenic poplars
• 350mg vinyl chloride in test tube inserted in
  dessicator
• The pollutant in air measured by GC analysis

• However, the transgenic poplars
• underwent blackening of their tissue
• Toxic byproducts created due to the
• metabolism of vinyl chloride
• High dose will not be found in nature
• Unlikely to be a problem in nature

After 5 days 49 of the vinyl chloride was
removed by the CYP2E1 lines Average of 29
removed by vector control cuttings.
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Conclusions

• Inserting mammalian cytochrome P450 2E1
  successfully increased the productivity and rate
  of phytoremediation in poplars
• Transgenic lines had increased uptake of
  pollutants from both soil and air
• Intermediates created as byproducts during
  metabolism of pollutants are potentially toxic

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Acknowledgements
Becker, H. (2004). Phytoremediation Using plants
to clean up soils. USDA Agricultural
Research Service. 13 August.
Cupp, M. J. Tracy, T. S. (1998). Cytochrome
P450 New nomenclature and clinical
implications. American Family Physician. January.
Doty, S., James, C., Moore, A., Vajzovic, A.,
Singleton, G., Ma, C., Khan, Z., Xin, G.,
Kang, J., Park, J., Meilan, R., Strauss, S.,
Wilkerson, J., Farin, F. Strand, S.
(2007). Enhanced phytoremediation of volatile
environmental pollutants with transgenic
trees. PNAS. 104(43). 1681616821.
Reigate Banstead Borough Council (2007).
Pollutants hydrocarbons. Reigate
Banstead Borough Council. 24 July.