Name:
Pd:
Clover Lab – Cyanogenesis Assay Pre-Lab
http://pages.wustl.edu/cloverproject
Essential Questions
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What adaptations have evolved in plants to provide protection from herbivory?
What is the genetic basis of the protective adaptations?
What factors might cause the genes for those adaptations to vary within a
species?
What is Clover Cyanogenesis?
White clover (Trifolium repens) is a forage plant, native to Eurasia, which has spread
throughout the world. It is very tasty to many herbivores including, insects, aphids,
mollusks, and large mammals (horses love it!).
1. Together with your lab partner, brainstorm, then list, different types of
protective defenses that plants have evolved to prevent herbivory (being
eaten by herbivores).
2. Based on the appearance of clover, which of the protective adaptations
from your list above do you think clover would use to avoid being eaten by
herbivores?
The Cellular Biology
White clover produces a poisonous compound called hydrogen cyanide. Cyanide is a
very fast acting poison that inhibits enzymes in the mitochondrial electron transport
chain, and hence blocks electron transport. Without the electron transport chain, most
organisms cannot produce ATP. The inability to produce ATP quickly leads to death.
Small organisms that eat cyanide producing clover either die from eating too much or
leave the plant alone in favor of other acyanogenic (non-cyanide producing) clover or
other herbaceous plants.
The ability to produce cyanide in clover is the direct result of two genes that control the
production of two compounds required for cyanogenesis, as well as the absence or
presence of herbivores in the area. One gene codes for a cyanogenic glucosides (sugar
compounds with a cyanide containing group attached), and the other gene codes for an
enzyme that is required to cleave the cyanide from the sugar so that the cyanide can be
released.
3. Do you think cyanide will kill the plant cells? Why or why not?
4. Why would having two genes to control the release of the poisonous
cyanide be advantageous to the plant?
5. Recall what you learned about plant cell structure. How might a clover
plant keep the two compounds separate within the cell?
6. How would grazing on clover cause the release of the cyanide?
The Genes
In order to produce cyanide, a plant needs a copy of the dominant allele for gene
controlling production of the cyanide containing sugar and a dominant allele for the
enzyme to release the cyanide from the sugar. In clover, the cyanide-sugar compound
is called linamarin/lotaustralin and the enzyme is linamarase. For simplicity purposes,
we will use the letter C for the cyanide-sugar compound and E for the enzyme.
7. Which of the following genetic combinations would result in the production
of cyanide?
Genotype
CCEE
CCee
CcEe
ccEE
ccEe
ccee
Ccee
CCEe
Cyanide-sugar
Production (y/n)
Enzyme Production
(y/n)
Cyanide Produced
(y/n)
The Environment
Cyanogenic clover plants are found more often in warmer climates. The frequency of
cyanogenesis decreases as one moves further north into cold climates as well as at
higher elevations.
8. Why would it be a disadvantage to a plant to produce cyanide in climates
where the temperature regularly falls below freezing?
9. Using the climate map below, make a prediction about the percentage of
your clover plants you expect to be cyanogenic (produce cyanide). Use the
data below as a guide for your prediction.
Location
Predicted % of
Cyanogenesic
Clover Plants
New Orleans, LA
St. Louis, MO
Nassau, WI
The Metabolism
Biosynthesis is the production of biological compounds by organisms. Clover plants, in
order to be cyanogenic, must synthesize both compounds – the cyanide-sugar and the
enzyme. Synthesis is an energy consuming process.
10. Based on the fact that synthesis requires energy, do you think it would be
an advantage to a clover plant to produce the linamarase enzyme if the
cyanide containing sugar is not present or to produce the sugar if the
enzyme is not present?
11. Based on your answer, make a prediction about the relative frequency of
the following genotypes.
Genotype
Predicted Relative Frequency
C_ee
ccE_
C_E_
ccee
12. Cyanogenic glucosides can be used as a way of storing energy and so can
serve a function besides cyanogenesis. Linamarase, on the other hand, is
only useful in cyanogenesis and is energetically especially expensive to
produce. Based on this information, would you expect to find more C_ee
plants or more ccE_ plants in nature? Explain why?
Reviewing Mendelian Genetics and Natural Selection
First, define the following terms in your own words.
Heredity
Allele
Genotype
Phenotype
Natural
Selection
Evolution
Adaptation
Applying Mendel’s Concepts
13. There are 2 alleles of the gene that controls the ability to produce the
cyanide containing sugar. In order to express this trait, the clover must
have a dominant allele at the C gene. Using upper-case or lower-case
letters for dominant and recessive alleles, write the allele combinations that
could be found in a clover plant. Underline those that would produce the
cyanogenic glucoside.
14. There are 2 alleles of the E gene which controls the production of the
enzyme required to break down the cyanogenic glucosides and release
cyanide. In order to express this enzyme, the clover must have a dominant
allele. Using upper-case and lower-case letters, write the allele
combinations that could be found in a clover plant. Underline those that
would produce the cyanide-releasing enzyme.
15. Cross a clover plant that is homozygous dominant for both the cyanogenic
glucoside and linamarase enzyme trait with a clover plant that is recessive
for the cyanogenic glucosides and enzyme trait. What genotype and
phenotype ratios could we expect in the offspring?
Applying Darwin’s Theory of Natural Selection
Natural selection occurs in populations of living things as a result of genetic variation
and an overproduction of offspring within the population. This results in a struggle for
existence, which leads to differential survival and reproduction within the population.
Over time, this process can result in big changes in a population.
16. Based on your understanding of how natural selection and heredity works,
explain how cyanogenesis in clover (and other plants) might have evolved.
Name:
Pd:
Clover Lab – Cyanogenesis Assay Procedure
Essential Questions



What adaptations have evolved in plants to provide protection from herbivory?
What is the genetic basis of the protective adaptations?
What factors might cause the genes for those adaptations to vary within a
species?
Materials
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

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4 clover plants
48 well tissue culture plate with lid
2 x 20 µl pipette tips (or small disposable pipettor)
1 x 20 µl + pipettor (or small disposable pipettor)
24 toothpicks
550 µl (~11 drops) linamarin solution (keep refrigerated until use)
550 µl (~11 drops) linamarase solution (keep refrigerated until use)
1 sheet of Feigl-Anger test paper
6 large binder clips
Distilled water
Note: Be sure to keep test paper in a cool dry location, wrapped in foil at all times!
Procedure
1. Label the clover plants #1-4.
2. Inspect the 48 well culture plate. Find the letters and numbers that identify the
rows and columns. Label the 48 well culture plate and lid with your lab group
number. Label each Row as follows (see Individual Data Sheet Diagram):
A and B=
C and D=
E and F=
“TO” for tissue only
“E” for positive for Enzyme
C” for positive for Cyanide Compound
3. Label the well plate diagram across top with the identity of each of your 4 clover
plants.
1 leaf = 3 leaflets
4. For plant 1, place 3 fresh, young, green leaves into each of the wells in column
#1, a total of 6 wells. These will be wells A1-F1. Skip a Column of wells and
Place 3 leaves from plant #2 in wells A3-F3 and so on, for all four plants. Label
your diagram carefully, as to which plant is in each well.
5. Place the lid on top of the plate and place it in a -20 ºC freezer (normal freezer)
for 45 minutes. [Optional stopping point: Plates can be left in freezer until next
class, or even longer if necessary.]
6. Remove the plate from the freezer and thaw the samples by placing the plate in a
37 ºC incubator for 10 minutes. If an incubator is not available, plants can be
thawed in a warm, dry area of the room for about 20 minutes, or teacher can
remove them prior to the class period.
7. Remove plate from incubator and add the reagents as follows:
a. In wells A and B: Marked “TO” leaf tissue only + 20 l or 1 drop of distilled
water (no reagents added).
b. In wells C and D: Marked “E” leaf tissue + 20 µl or 1 drop of linamarin
solution.
c. In wells E and F: Marked “C” leaf tissue + 20 µl or 1 drop of linamarase
solution Repeat for all plants and all wells.
8. Use well F8 as a positive control by placing 20 µl or 1 drop linamarin solution +
20 µl or 1 drop linamarase, no plant tissue. Be sure to mix the contents of the
control well.
9. Using a new toothpick for each well, mash the tissue and reagents in each well to
mix compounds. Mash until a small amount of liquid is visible. Note: Be sure to
use a new toothpick for each well.
10. Wipe off the top of the plate to remove any moisture. Label your Feigl-Anger
paper with your initials, and Place the test paper on top of the well plate. Place
the lid over the paper on the plate and then clamp together using 6 large binder
clips (prevents bleed-over of HCN between wells).
11. Incubate the plate in a 37 ºC incubator for 30 minutes to 2 hours (or longer, if
needed, but not longer than 6 hours) then photograph or record results. If an
incubator is not available, cover plate with aluminum foil or place in a thick
envelope, and place in a warm dry area for approximately 90 minutes. Check
plate after 30 minutes and remove any tissue from already positive wells.
Note: For reliable scoring of positive results in weakly cyanogenic plants, a minimum of
90 minutes in an incubator and 3 hours at room temperature. Scoring plants after
shorter durations may yield false negative results for plants that produce low
levels of cyanogenic compounds.
Well-Plate Diagram for Individual Data Sheet
[TO]
Name:
Row A
Col 1
Col 2
Col 3
Col 4
Col 5
Col 6
Col 7
Col 8
Plant
1
X
Plant
2
X
Plant
3
X
Plant
4
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Control
C + E
Row B
[TO]
Row C
[E]
[E]
Period:
Row D
Row E
[C]
[C]
Date:
Row F
Note: The linamarin and linamarase to test for cyanide sugar control goes in F8.
Results
1. If cyanide is produced, a blue color change will appear on the paper. Carefully
inspect your paper. Record on your well-plate diagram which wells turned blue
and which did not. Inspect the paper from the underside, if results are
questionable.
a. If the clover has the ability to produce cyanide, which wells will be blue?
b. If the clover produces the sugar-cyanide compound, but not the enzyme,
which wells will be blue?
c. If the clover plant produces the enzyme, but not the sugar-cyanide
compound, which wells will be blue?
d. If the clover plant produces neither sugar-cyanide compound nor the
enzyme, which wells will be blue?
e. What is the purpose of well F8?
2. Record the phenotype and the genotype of your plants:
Plant
1
2
3
4
Cyanide
producer
(y/n)
Produces
Linamarase
only (y/n)
Produces
Linamarin
only (y/n)
Genotype
3. Collect class data:
Plant
Class Data
(Total number of
plants)
Cyanide producer
(y/n)
Produces
Linamarase only
(y/n)
Produces
Linamarin only
(y/n)
Does NOT
produce
Linamarin or
Linamarase
4. Calculate the percentage of plants that are cyanogenic.
5. Compare the percentage of plants that are cyanogenic to your hypothesis and to
the percentages from other climates.
6. Based on climate, do the results for your local area make sense? Explain.
7. Calculate the percentage of the plants that contain the gene for the linamarase
ONLY:
8. Calculate the percentage of the plants that contain the gene for cyanogenic
glucosides ONLY.
9. Calculate the percentage of the plants that contain NEITHER gene for
cyanogenic glucosides or linamarase.
10. Explain why natural selection would favor plants that produce cyanide in warm
climates, but not in cold climates.
11. Explain why natural selection would favor plants that produce neither compound
in cold climates versus a plant that produces only one of the compounds.
12. Assume more plants express the gene for the production of the sugar-cyanide
compound than express the gene for the production of the enzyme. What can
you infer about the relative metabolic “cost” of producing the enzyme?
13. Some plants in the class may have appeared stronger in the levels of cyanogenic
compounds produced in the class plants as a whole. What are some possibilities
as to why there would be varying levels of cyanogenic compounds in clover
plants growing in the same general area?