Laboratory 1
(Part B)
Allelopathy
Exercise Goals
You should be able to do the following after completing this exercise:
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Explain the concept of a bioassay.
Prepare simple extracts and serial dilutions of plant compounds.
Explain basic experimental design, control groups, and treatment groups.
Describe allelopathy and relate it to interference competition.
Assess bioassay data for evidence of allelopathy.
Background reading:
Text:
Krebs, pp. 81-85
Allelopathy refers to the direct inhibition of the development or growth of organisms by toxic
substances produced and released by other organisms. The term describes any situation in which one
organism interferes with another through the use of chemicals. Allelopathy is most frequently used in
the context of plant interaction in which on plant releases noxious compounds that inhibit the
germination or growth of other plants. For example, trees of black walnut (Juglans nigra) contain the
compound hydroxyjuglone, which, when released into the environment, is oxidized into juglone.
Juglone inhibits the germination and growth of certain species of plants, so that many plant species are
able to live under or near black walnuts. The release of natural products into the environment and the
interaction of these substances with living organisms fall under the heading “chemical ecology”.
Allelopathic compounds directly inhibit other organisms and provide an example of interference
competition, in which one organism gains access to a greater range or quantity of resources by
physically or chemically preventing another organism from using those resources. Allelopathy can
thus be defined as interference competition brought about through the chemical modification of local
environments. Allelopathic effects are typically selective, hindering certain species but not others. In
some instances, researchers have identified the active allelopathic compounds, isolated them, and
studied their biosynthesis. In other instances, circumstantial evidence suggests that allelopathy is
occurring, although detailed experimental and chemical data are lacking.
In this exercise, we will perform a simple experiment to determine whether compounds present in
crude extracts of specific plants have allelopathic effects on cucumber germination. This
Reference
Rice, E. L. 1984. Allelopathy. Academic Press, New York. (On 2-hour reserve in Library.)
approach takes advantage of a bioassay, in which organisms maintained under standardized laboratory
conditions are used to test for the effect of a particular substance or treatment. We will use cucumber
seeds as a bioassay to test for the effects of various plant extracts on cucumber development and
growth.
Seeds to which an extract is applied make up a treatment group. We will compare germination and
growth of each treatment group with germination and growth of a control group, consisting of
cucumber seeds handled and maintained in the same way as the treatment groups, except that no plant
compounds will be applied to the control seeds. A comparison of results for treatment and control
groups makes it possible to determine if there is an effect of a plant compound on germination and
growth, over and above the effects of other aspects of the experimental procedure common to both the
control and treatment seeds.
PROCEDURE
You will prepare aqueous extracts from parts of three different species of plants available in your
laboratory. Your instructor will provide the common and scientific names of the plants and will
specify the plant parts to be used in preparing the extracts.
1.
Weigh out a 20g sample of the plant.
2.
Place the sample in a blender and add 200ml of distilled water.
3.
Blend the sample fully and allow the resulting mixture to sit in the blender for 60 minutes.
4.
Vacuum-filter the mixture through cheesecloth and rapid filter-paper placed in a Buchner
funnel, as demonstrated by your instructor. Transfer the filtered aqueous extract to a
bottle labeled with the species name and the phrase “undiluted extracts”.
Technical note: The cheesecloth removes coarse particles that would clog the filter
paper placed in the Buchner funnel.
5.
Use our extract to prepare a serial dilution, which is a series of increasingly dilute
extracts.
a.
b.
c.
6.
50% dilution
i.
Place 50ml of undiluted extract in a graduated cylinder and pour
the 50ml of extract into a 100ml volumetric flask.
ii.
Add distilled water until the solution level reaches the white line.
iii.
Cover the volumetric flask tightly with parafilm and invert the
flask several times until the solution is thoroughly mixed.
iv.
Transfer the diluted extract from the volumetric flask to a glass
bottle labeled with the species name and the percent dilution.
10% dilution
i.
Pipet 10ml of undiluted extract into a 100ml volumetric flask.
ii.
Perform steps ii through iv listed above under “50% dilution”.
2% dilution
i.
Pipet 2ml of undiluted extract into a 100ml volumetric flask.
ii.
Perform steps ii through iv listed above under “50% dilution”.
Obtain 13 petri dishes.
7.
Place a piece of filter paper into each petri dish.
8.
Scatter 10 cucumber seeds into each petri dish.
9.
Add 10ml of distilled water to one of the petri dishes, cover it, and label it with the word
“control” and your name, the date, and your laboratory section.
10. For each plant species, repeat step 9 for the undiluted extract, the 50% dilution, the 10%
dilution and the 2% dilution. Label each of the resulting 12 petri dishes with the word
“treatment”, the plant species and dilution, your name, the date and your lab section.
Technical note: The top part of the petri dish has the larger diameter. Make sure the
top of each petri dish is seated firmly against the bottom piece, so that evaporation
from the dish is minimized.
11. Store the petri dishes at room temperature in the lab drawers indicated by your instructor.
12. After one week:
a.
Count the number of cucumber seeds that germinated in each petri dish. In
Table 1, record the number of seeds that germinated, whether the petri dish is a
control or treatment, and if it is a treatment dish, the species and dilution.
b.
Using a ruler, measure the length of the developing root (radicle) of each
germinated seed. In Table 2, record your measurements, whether the petri dish
is a control or treatment, and if it is a treatment dish, the species and dilution.
For a given species and dilution, place all of the radicle lengths in the single box
in the right-hand column of the table separating each length by a comma.
TABLE 1 – NUMBERS OF GERMINATED SEEDS IN CONTROL AND TREATMENT GROUPS
Control or Treatment
Species
Dilution
Number of Germinated
Seeds
TABLE 2 – RADICLE LENGTHS OF GERMINATED SEEDS IN CONTROL AND TREATMENT GROUPS
Control or Treatment
Species
Dilution
Radicle Lengths of
Germinated Seeds
(cm)