Seed Round-Up:
The Generatoin of a
Superweed through
Herbicide Misuse
Plant growth and genetics
Can plants develop resistance
to Glyphosate through low-level
exposure across multiple generations?
Roy Johnson
8/6/20
Seed Round-Up: The Geration of a Superweed through Herbicide Misuse
Abstract:
The use of glyphosate as an Herbicide in the United States has more than doubled in
the past 30 years. In the course of this experiment Arabadopsis thaliana was used an
investigational tool to track the development of herbicide resistant genes through
exposure to small amounts of glyphosate at two key points in the growth cycle of A.
thaliana; shortly after germination and close to the point of flower development and
tracking the growth and development of the treated plants. A. thaliana seeds were
placed into one of four test groups or a control group. Each group received a designated
concentration of gylphosate between 1-51% or no glyphosate for the control group.
Plant growth showed an inverse relatoinship to the concentration of glyphosate applied
with more glyphosate leading to lower growth rates. Plants treated with low
concentrations (less than 25%) of glyphosate were able to produce viable seeds which
may be capable of confering resistance to glyphosate in future generational studies.
Responsible use of herbicides is important to ensure that the efficacy of the chemicals
does not decline with increasing genetic resistance.
Introduction:
Herbicides have come to have a common place in the garages and barns of both
families and farmers alike. Although the use of herbicide can be a useful tool in
combating weeds, the misuse or overuse of herbicide may have dramatic
consequences. Through the course of this project, the ability of a plant to develop
resistance to the herbicide glyphosate is tested. Using Arabadopsis thaliana as a model
organism, tiered amounts of glyphosate are applied to plants during germination and
flowering to examine the effects of the glyphosate on plant growth and development.
This project will be continued as a longitudinal study to track the development of
glyphosate resistance through minimal exposure over multiple generations. The
implications of glyphosate resistance developing in the course of a few generations
could render this herbicide useless in a matter of decades.
Research Paper:
RoundUp is a commercially available herbicide manufactured by Monsanto Corporation.
The use of herbicides, in particular glyphosate has increased several fold over the past
three decades (Berqkvist). Herbicides have several practical applications for
commercial and residential purposes. Many farmers use herbicides to remove weeds
and reduce the amount of tilling and effort in growing crops. Roundup is the brand name
for the active chemical compound contained in the herbicide known as glyphosate. The
chemical formula for Glyphosate is C3H8NO5P and is known by the IUPAC name N(phosphonomethyl)-glycine.
When applied to plants, glyphosate is absorbed through the leaves and cuticles of the
plant and is readily transported throughout the plant by phloem tissues. The result of an
application of glyphosate include yellowing and wilting of the plant leaves followed by
eventual plant death. The disruption of the protein synthesis pathway inhibits the
production of vital aromatic amino acids necessary for plant growth (National Pesticide
Information Center).
Glyphosate kills plants in 4-20 days by inhibiting an enzyme vital to the plant’s protein
synthesis pathway. The targeted enzyme is a part of the shikimic acid pathway known
as 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase. EPSP synthase is essential
in the production of the three vital aromatic amino acids L-tryptophan, L-phenylalanine,
and L-tyrosine. Without these essential amino acids a plant will die soon after exposure
to glyphosate.
Common uses for glyphosate include ornamental weed control and widespread use in
crop production. Many households use glyphosate to control weed growth along
driveways or in gardens. For large scale applications glyphosate is used commercially
to control plant growth along roadways and railways.
Many plants today have been genetically engineered to be “RoundUp Ready.” These
plants have a genetic mutation which conveys resistance to the active ingredient
glyphosate. With “RoundUp Ready” plants, you can “spray Roundup agricultural
herbicides in-crop from emergence through flowering”(Roundup Ready System).
Glyphosate can persist in soil for several days or weeks. Various studies have found
half-lives ranging from 3- 130 days in soil, with an average half-life persistence of 44-60
days (Schuette). Although the toxicity of glyphosate in plants is well understood, the
effect of glyphosate on animal life is still a hotly debated topic. Some studies have
shown an increased risk of certain types of cancer after animals are exposed to
glyphosate over long periods of time. In areas where repeated applications are
common, the impact of glyphosate on animal life needs more study.
To determine the capability of a plant to develop genetic resistance to glyphosate
applications, Arabidopsis thaliana will be used as a model organism to track
susceptibility across multiple generations. A. thaliana is widely used as a model for plant
genetics due to a short generational time and the large amount of available research
into the A. thaliana genome (Meinke 1998). Some plants are known to be naturally
glyphosate resistance as alternate pathways for the production of the essential amino
acids blocked by glyphosate have been established. The genome of A. thaliana does
not naturally contain genes resistant to glyphosate. Because A. thaliana can grow from
seed to flowering plant in less than six weeks it is possible to expose multiple
generations to low levels of glyphosate to see if resistance can develop through multiple
generations by the creation of alternate pathways for amino acid synthesis.
The aromatic amino acids L-tryptophan, L-phenylalanine, and L-tyrosine are created
through unique pathways in plants and are not naturally synthesized in mammals.
Because the pathway to create the essential amino acids is not present in mammals it
was an ideal target for herbicide development without causing harm to mammalian
development (Maeda 2012).
Experimental Plan:
Purpose

Can plants develop resistance to low level exposure to glyphosate across
multiple generations?
Hypothesis

If Arabadopsis thaliana is exposed to low concentrations of glyphosate over
multiple generations, then mutations which provide resistance to glyphosate will
develop.
Variables
Independent Variable:

Glyphosate exposure titrated to different levels
Dependent Variable:

Glyphosate Resistance +/-
Controls:
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Raphanus sativus plants not exposed to glyphosate
Soil, Water, Light Temperature Conditions
Materials:
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Seed starter tray x 100
1 Liter plant pots
Organic soil
Arabadopsis thaliana seeds
Raphanus sativus seeds
Grow light
Light Timer
Glyphosate
Gloves
Goggles
5 misting sprayers
Water
fan
Procedure:
1. Fill seed starter tray with 50g of organic soil per cup (fill 40 cups)
2. Place 1 A. thaliana seed in a cup of soil.
3. Repeat step “2” twenty (50) times.
4. Add 25mL of water to each cup containing a seed.
5. Separate seeds into groups of 10.
6. Separate each seed group with plastic walls to prevent cross pollination
7. Label Seed Groups A, B, C, D, E
8. Place seeds under grow light.
9. Turn on fan to keep plants cool and provide wind for pollination.
10. Water seeds with 25mL of water every 24 hours.
11. Allow seeds to germinate and grow for 4 days.
12. Measure and record height and condition of each plant.
13. Mix glyphosate with water to create strengths A (1%), B (10%), C (25%), D(51%)
**Wear googles and gloves when handling glyphosate**
14. Apply 1 metered spray (1 ml) of solution A to each of the 10 plants in group A.
15. Repeat step 14 three times for Groups B, C, and D
**Plant group E is not exposed to glyphosate.**
16. Continue to water plants every 24 hours.
17. Measure and record height and condition of each plant 24 hours after glyphosate
exposure
18. Measure and record height and condition of each plant 4 days after glyphosate
exposure
19. Measure and record height and condition of each plant 8 days after glyphosate
exposure
20. Measure and record height and condition of each plant 12 days after glyphosate
exposure
21. Measure and record height and condition of each plant 16 days after glyphosate
exposure
22. Measure and record height and condition of each plant 20 days after glyphosate
exposure
23. Measure and record height and condition of each plant 24 days after glyphosate
exposure
24. Measure and record height and condition of each plant 28 days after glyphosate
exposure
25. Carefully transfer each seed starter into a 1 liter pot.
26. Label each pot with Group A, B, C, D, or E, and type of plant.
27. Measure and record height and condition of each plant 32 days after glyphosate
exposure
28. Measure and record height and condition of each plant 36 days after glyphosate
exposure
29. Measure and record height and condition of each plant 40 days after glyphosate
exposure
30. Apply 1 metered spray (1 mL) per plant of each strength to each group A-D
31. Measure and record height and condition of each plant 44 days after glyphosate
exposure
32. Measure and record height and condition of each plant 48 days after glyphosate
exposure
33. Measure and record height and condition of each plant 52 days after glyphosate
exposure
34. Measure and record height and condition of each plant 56 days after glyphosate
exposure
35. Measure and record height and condition of each plant 60 days after glyphosate
exposure
36. Collect any seeds from plants to create F1 generation.
37. Repeat steps 1-38 for F1 generation.
38. Repeat steps 1-37 for F2 generation.
39. Compile all data.
Discussion:
The application of glyphosate at the standard concentration of 51% contained in
commercially available herbicide RoundUp is highly effective in killing plants. One plant
in the group receiving the 51% glyphosate application grew at a normal rate. It is
possible that the surviving plant in the 51% group did not receive an adequate
application of glyphosate or that the sprayer malfunctioned to allow this plant to survive.
The plants receiving lower concentrations of glyphosate were able to grow to greater
heights at an inverse relationship to the amount of glyphosate applied. Flower formation
results are also similar to the results for height. The more glyphosate the plants
received, the lower the percentage of plants that were able to propagate flowers. After
60 days of growth 90% of the plants in the control group receiving no glyphosate
produced flowers. The plant groups receiving 1%, 10%, 25%, and 51% grew flowers at
rates of 80%, 60%, 20% and 10% respectively. Seed production followed a similar trend
as well. Plants receiving 1%, 10%, 25%, and 51% glyphosate produced seeds at rates
of 50%, 20%, 1%, and 0%. 80% of the control group receiving no glyphosate were able
to produce flowers. The collected seeds will be used to create the F1 generation to
follow for development of genetic resistance to glyphosate.
1 plant in the 25% glyphosate group did not sprout and affected the average recorded
height. The un-sprouted seed was likely not affected by the application of glyphosate
and was simply a bad seed.
The effect of glyphosate of the plants was also seen in the appearance of the leaves
following application. Glyphosate was applied to the plants 4 days after planting and 40
days after planting. The first application of glyphosate stunted the growth of the plants in
proportion to the concentration of glyphosate used. In plants that were surviving but
growing weakly following the first application, the second application of glyphosate on
day 40 was effective in causing death in the majority of the weakened plants. Plant
death was evident as the leaves of the plant changed colors from green to yellow then
brown, and turgidity of the leaves changed from firm to weak to wilted.
The development of glyphosate resistance is a concern for commercial and residential
applications of herbicide. In the United States there are >40 species of weeds that are
known to be glyphosate resistant. As the use of glyphosate continues to increase, more
plant species will be inadvertently exposed to low levels through misuse or overuse.
Although the lower concentrations may not be fatal to the plant, the possible genetically
resistant weeds that may develop have far graver consequences.
Future studies will follow the seeds obtained through a similar course to track the
development of glyphosate resistance.
Conclusion:
At high concentrations glyphosate is highly toxic to plants. Lower concentrations of
glyphosate below 25% allow for flower production in a majority of plants. The creation of
seeds in plants treated with glyphosate, even at low levels, occurs at 50% of normal
levels. The seeds obtained from plants exposed to glyphosate may or may not contain
mutations which confer resistance to glyphosate. Further study is needed to track A.
thaliana’s ability to survive higher concentrations of glyphosate. In plants that display
resistance, DNA sequencing can be performed to confirm if mutations have occurred in
the ESRB gene to confer resistance to glyphosate.
Acknowledgements:
I would like to give a special thanks to Amanda Lane-Cline for inspiring this project.
Recognition is also due to my wife Kate for her support and help in collecting data.
Works Cited
National Pesticide Information Center. Oregon State University, USEPA. September
2010. 4 August 2014. <http://npic.orst.edu/factsheets/glyphotech.html>.
Roundup Ready System. Monsanto. 2014. 4 August 2014.
<http://www.monsanto.com/weedmanagement/pages/roundup-ready-system.aspx>.
Schuette, Jeff. “ENVIRONMENTAL FATE OF GLYPHOSATE”. California Department of
Pesticide Regulation, Environmental Monitoring and Pest Management. November
1998.
Meinke, David; et. Al. “Arabidopsis thaliana: A Model Plant for Genome Analysis.”
SCIENCE. VOL 282 23 OCTOBER 1998. 681.
Maeda H, Dudareva N. “The shikimate pathway and aromatic amino Acid biosynthesis
in plants.” Annual Review of Plant Biology. 2012.
Nandula, VK; et. Al. “Glyphosate-resistant and –susceptible soybean (Glycine max) and
canola (Brassica napus) dose response and metabolism relationships with glyphosate”.
Journal of Agricultural Food Chemistry. May 2 2007. 3540-5.
Peter Bergkvist. KEMI Swedish Chemicals Agency 12/15/2011.
<http://www.kemi.se/en/Content/Statistics/Statistics-in-brief/Statistics-in-brief--Substances-and-substance-groups/Glyphosate/>. August 5, 2014