Download Experimental Objective: Life in Bloom Module Experiment Duration

Life in Bloom Module
Experiment Duration: 9 days
Difficulty: Moderate
Seeds require the right conditions to germinate. Temperature, light, and water are the external
forces that influence germination. The plant hormone Gibberellic Acid (GA) is a critical
component of the internal control of the developmental processes, acting to promote
germination, plant growth and development. This experiment will explore the effects of several
mutations in genes responsible for GA synthesis and signaling on germination. You will be able
to see the difference between mutations that affect the early (ga1) and late (ga5) stages of GA
synthesis and compare these with a mutation involved in GA sensing and response (gai-1).
These differences will be demonstrated by observing germination in water versus GA solution of
seeds that are grown either in the dark or under continuous light.
Experimental Objective:
This experiment will give you an understanding of plant hormone biosynthetic pathways,
hormone sensing and mutant analysis, using a model plant Arabidopsis thaliana (Arabidopsis).
Materials:
Below is a list of materials required for this experiment. Make sure that you have all materials
ready before you start.
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Seeds (Table 1)
Petri Dishes
Filter Paper
Water
GA Solution
Toothpicks
Wax Paper
• Micropore Tape or
Parafilm
• Aluminum Foil
• Microscope or
Magnifying Glass
• Masking Tape
• Pen
Table 1: Seeds
Genotype
Stock
Number
Ler (Wild type) CS20
ga1-2
CS3103
ga1-4
CS3105
ga5-1
CS62
gai-1
CS63
Experimental Protocol:
Treatments and Experimental Design
Order seeds from ABRC (abrcoutreach.org/educational-kits) well in advance to when you plan to
start the experiment. You should receive around 200 seeds for each of 5 Arabidopsis genotypes
in small tubes labeled with the ABRC stock number (Table 1 shows the corresponding genotypes
for each of the ABRC stock numbers). Students will be testing how well each of the 5
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Arabidopsis genotypes germinate in petri dishes that contain either water or 200 µM (micro
molar) GA solution and then are either kept in complete darkness or are placed under direct light.
Separate students into 4 groups. Each group will perform one of four experimental treatments on
3 replicate petri dishes (Table 2). It is important for each group to replicate their experimental
treatment at least 3 times so there are enough results to statistically analyze the data.
Table 2: Experimental Treatments
Light
Dark
Water
Group 1, 3 replicates Group 3, 3 replicates
GA 200 µM Group 2, 3 replicates Group 4, 3 replicates
Experimental Timetable
This experiment is designed to take nine days (Figure 1). It is best to have students start the
experiment on a Thursday and then remove the petri dishes from cold treatment on Sunday.
Students can then count the results of the Petri dishes in the Light treatments on Monday-Friday.
Since groups 3 and 4 will only count the results of their Petri dishes on Friday, they can recount
the Petri dishes of groups 1 and 2 on Monday-Thursday, to double check their results.
Figure 1: Timetable
Preparation of Petri Dishes
1. Print out a copy of the Petri dish template, provided as a link on the Life in Bloom kit
detail page (abrcoutreach.org/educational-kits).
2. Cut the template as indicated and give each group the template that corresponds to their
treatment (Table 2).
3. Give each group 3 Petri dishes and 12 pieces of filter paper.
4. Have each group label 3 separate pieces of filter paper exactly as is indicated by the
template they receive, making sure to fill in the current date and the appropriate replicate
number (Figure 2 gives an overview of how a final Petri-Dish will look).
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Figure 2: Petri dish layout
5. Have students stack 2 blank pieces of filter paper together and place the stacked pieces
into a Petri dish.
6. Students then need to use their finger to smooth the edges of the filter paper in the Petri
dish (Figure 3A).
7. Take one of the labeled pieces of filter paper and stack this on top of another blank piece
of filter paper and smooth these pieces into the Petri-dish on top of the 2 blank pieces that
were already smoothed into the dish (Figure 3B, C).
8. Repeat this process for all three of their Petri dishes (all finished dishes should resemble
Figure 3D). Each dish should contain 4 pieces of filter paper, 3 blank and 1 labeled.
Figure 3: Putting filter paper in Petri Dishes
9. Let groups 1 and 3 prepare 30 ml of water.
10. Let groups 2 and 4 prepare 30 ml of 200 µM GA solution.
11. Have students soak the filter paper in each of their Petri dishes with 10 ml of the liquid
they were given.
12. Pour off excess liquid from their Petri dishes. Excess liquid may cause seeds to move
around and ruin the experiment.
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13. Give each group at least 30 seeds of each genotype (Table 1). Be careful when
transporting seeds as they are very small and can easily be blown away. It is best not to
transport the seeds in an open container and they should never be carried around
when poured on weighing paper.
14. Pour out the seeds from one genotype onto a piece of weighing paper.
15. Take a toothpick and wet its tip using the wet filter paper of a Petri dish prepared in Steps
11 and 12 .
16. Using the wet tip of the toothpick pick up one seed at a time from the weighing paper and
put it in its corresponding place on the Petri dish.
17. Place 10 seeds in their appropriate sections for each genotype on each of the 3 Petri
dishes for their group, as shown in Figure 2.
18. Place the lid on the Petri dishes and wrap the edges of each Petri dish with Parafilm or
Micropore tape completely sealing them shut.
19. Each group should stack their 3 Petri dishes on top of one another and wrap their groups
stack completely with aluminum foil so that no light reaches the seeds.
20. Each stack of wrapped Petri dishes should be labeled, using masking tape, with the
corresponding treatment, group number and date.
21. Put each stack of wrapped Petri dishes in a cold room or refrigerator at 4°C-6°C. This
process is called stratification. Stratification is important for breaking seed dormancy
and synchronizing the germination of the seeds. Be extremely careful when preparing,
transferring or working on Petri dishes with seeds. Rough handling can cause seeds to
shift from their intended position and ruin the experiment.
22. All Petri dishes should be kept in cold treatment for a minimum of 2 days and a
maximum of 6 days.
Data Collection:
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After cold treatment is complete remove Petri dishes from refrigerator or cold room.
Remove the aluminum foil for Group 1 and 2 Petri dishes (LIGHT treatments) and place
these dishes unstacked under bright light (For best results use a light rack).
Place the Group 3 and 4 wrapped Petri dishes (DARK treatments) in a drawer or cabinet
away from any light source.
The day Petri dishes are removed from cold treatment is considered Day 0.
On Days 1-4 Groups 1 and 2 should count the number of seeds germinated for each of the
genotypes on each of their Petri dishes (LIGHT treatments) and record the results each day.
Groups 3 and 4 can recount Group 1 and 2 Petri dishes double checking the results.
Counting germinated seeds can be challenging. Arabidopsis seeds are extremely small and
the early stages of germination cannot be observed by the naked eye. Use a large magnifying
glass or a dissecting microscope to examine the seeds.
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Condensation will often form inside the lid of the Petri dish. If you need to, remove the lid to
count germinating seeds. Carefully close and reseal edges of opened Petri dishes with
Parafilm or Micropore tape after you are done counting.
On Day 5 remove the Group 3 and 4 Petri dishes (DARK treatments) from the dark storage
location and remove the aluminum foil.
Have each group count the germinated seeds for their own groups Petri dishes and record the
results.
Seed Germination:
Figure 4: Arabidopsis seed germination stages
A
B
C
Germination is the process by which plants emerge from their seeds. Figure 4 represents what
you should see when observing Arabidopsis seeds under a microscope during different stages of
germination.
To count germinating seeds look for the emergence of root radicles (root tips) from the seeds.
You can see the difference between an intact seed that has not germinated yet (Figure 4A), and a
germinating seed (Figure 4B). As the experiment progresses you will observe later stages of
germination as the seedling elongates, begins to green and forms root hairs (Figure 4C).
Only count seeds where you clearly observe the emergence of the root radicle. If you are not
sure about a particular seed, do not count it and wait for the following day when its germination
status should be clearer. Discuss your counts with the rest of your group and be consistent!
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Germination Data Analysis:
Figure 5: Germination data examples
Light, GA 200 µM
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
B
CS20 Ler
CS3103 ga1-2
CS3105 ga1-4
CS62 ga5-1
Germination %
Germination %
A
CS63 gai-1
Day 1 Day 2 Day 3 Day 4 Day 5
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
GA (200 µM)
5 Day Light
5 Day Dark
CS20 CS3103 CS3105 CS62
Ler ga1-2 ga1-4 ga5-1
CS63
gai-1
Create X-Y Scatter plots and bar charts to compare the germination data you have collected.
Figure 5 shows examples of a germination rate scatterplot for the five genotypes in the Light,
GA treatment (Figure 5A) and of a bar chart for the five genotypes in the GA treatment (Figure
5B). A number of graphs will automatically be created for you if you enter your results into the
data analysis spreadsheet available at abrcoutreach.osu.edu/educational-kits (choose the Life in
Bloom education kit to get access to a variety of downloadable resources). Compare the rates of
germination for the genotypes in the light treatments, and also compare the final germination
values in both the light and dark treatments. Observe the differences in germination for the
genotypes across the various treatment variables (Light, Dark, Water, and GA).
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GA & Germination:
Figure 6: GA Biosynthesis & Signaling
While analyzing the data in this experiment a few things should become clear. You will
first notice that without the addition of GA the ga1-4 and ga1-2 genotypes will not germinate.
You may notice that ga5-1 germinates more slowly than wild-type on water. You should see
that without the addition of GA nothing germinated in the dark (a few plants will occasionally
germinate in the “dark, water” treatment depending on how long you take to prepare and cover
your petri-dishes during plate preparation). You can also observe that adding GA in the dark
treatment did not rescue the germination of gai-1 (gai stands for GA insensitive), while it did
rescue the germination of all the other genotypes in the same conditions (Figure 5B). You can
compare your results with a set of example results available at abrcoutreach.osu.edu/educationalkits.
Figure 6 describes the GA synthesis and signaling pathway and shows which part is
disrupted for the various genotypes in the experiment. The ga1-2 and ga1-4 mutations block the
function of the same gene and demonstrate that both regions of the gene are necessary for the
gene function. Notice that the ga5-1 mutation affects a later stage of GA synthesis reducing the
production of several GA products. Discuss with your classmates the results obtained for the
ga5-1 mutant. Based on the position of the GA20 oxidase enzyme encoded by the GA5 gene, did
you expect to observe reduced germination rate for the ga5-1 mutant? Discuss different reasons
for a mild reduction of the germination rate of this genotype (e.g. A) a role of some of the GA
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precursors in stimulating germination or B) a nature of the ga5-1 mutation that might cause just a
knock-down of the GA20 oxidase function).
Figure 7: GA induces the degradation of DELLAs
Figure from Davière and Achard: Gibberellin signaling in plants. Development, 2013.
The DELLA proteins are a group of proteins involved in GA signaling with a role to
repress GA-dependent responses, including germination. There are 5 different DELLA proteins
in Arabidopsis: GAI, RGA, RGL1, RGL2, and RGL3. The binding of GA to its receptor
initiates a process by which DELLA proteins are degraded (Figure 7). Once DELLA proteins
are degraded, the genes previously blocked by DELLA become activated and germination can
occur. Therefore GA works in combination with the DELLA proteins to signal the activation of
genes responsible for germination. The gai-1 mutation acts to stabilize the GAI DELLA protein
and either prevent or reduce the ability of GA to induce its degradation (Figure 6). From the
experiment you should see that the gai-1 mutation prevents GA-induced germination in the dark.
Adding GA will not rescue this phenotype because the mechanism that responds to the presence
of GA is disrupted. What is the germination rate of gai-1 under light conditions? Is there any
difference between water and GA under light? Discuss why you think gai-1 seedlings germinate
in the light but not in the dark. Keep in mind what you observed for the other GA mutants. Is it
possible that light stimulates the production of larger amounts of GA than what was provided in
the experiment under dark conditions? Would adding more GA to the Petri dishes in the dark
stimulate germination of the gai-1 mutant? You may test this hypothesis by performing
additional experiments.
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Helpful Resources:
Web Resources available at abrcoutreach.osu.edu
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18 educational kits exploring a variety of topics and experiments
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Information about the ABRC education and outreach program
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Arabidopsis news and events
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Links to other useful resources
Specific Resources Available for this Experiment (Educational Tools  Life in Bloom)
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Protocol (PDF)
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Petri Dish Template (PDF)
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Data Collection Sheet (PDF)
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Data Analysis Spreadsheet (XLS)
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Example Results (XLS)
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Life in Bloom Protocol Video
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Button to order seeds for this experiment
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