An investigation to compare stomatal distribution in shady and sunny sides in Bougainvillea Glabr. How does the stomatal density vary in the sunny and shady parts of paper flowers? (Bougainvillea Glabra)
Bougainvillea Glabra are evergreen flowering plants surrounded by colorful bracts including red, yellow, golden and white. They belong to Nyctaginaceae Family and are angiosperms. The average height is 10-12ft with spread of 8ft in hot and humid places (such as Bangkok where the experiment is conducted). There is a lot of variation in its leaf shape; leaves from these plants can be globular, elliptical, chordate or obviate and leaves can be up to 4 inches long. The plant was originated from South America. They are frost sensitive and foliage can be damaged below 32°F. [1]
http://www.plantoftheweek.org/image/bouga1.jpg
Fig: Bougainvillea Glabra
In shade, there is less sunlight reaching the leaves of the plant hence less photosynthesis. In order for photosynthesis to be efficient larger leaves are needed hence leaves in the shade have more stomata. In this experiment, the same sizes of leaves will be chosen from both sides of the plant hence limiting the error of surface area affecting the results due to differences in the leaf. The aim of this experiment is to find out if stomatal distribution in leaves is affected by the position of the sun.
Hypothesis
There will be more stomata per unit surface area in the lower epidermis of leaves exposed to the sun and less stomata on leaves exposed to the shade.
Explanation: This is because Photosynthesis occurs under the following conditions: light, chlorophyll in chloroplasts, carbon dioxide and water. A suitable temperature is also relevant in photosynthesis because it involves biochemical process. In sunlight, potassium ions build up inside the guard cells by active transport, the guard cells contain chloroplasts when these absorb sunlight photosynthesis takes place which leads to the production of sugars. In areas exposed to the sun, there will be a higher rate of photosynthesis. When there is more photosynthesis there is a greater need for gases to be exchanged at a faster rate and hence there will be more stomata.
Null Hypothesis: There will be less stomata per unit surface area in the lower epidermis of leaves exposed to the sun and more stomata on leaves exposed to the shade
Graph 1. Predictive graph of the effect of sunlight exposure on Bougainvillea glabra.
Independent Variables
The independent variable is either the leaf being from the Sunny (exposed to the sunlight) or Shady (not exposed to sunlight) part of the plant.
Dependent Variables
The dependent variable is the stomata count in 0.44 mm field of view of a sample taken from the center of the leaf.
Control Variables
Variable
Effect
How it is controlled
Plant species
Different species may have different structure or adaptability, changing the amount of stomata present in their leaf.
Sample of leaves are collected from the same species.
Leaf size
If one leaf is bigger than another, this may mean that the stomatal count was affected in growth.
Ensure that all sample leaves are of a similar size.
Part from which the sample is obtained
Different parts of the leaf may have different stomatal count because of environment correlates
To reduce the error of stomatal count in different part of the leaf, sample should only be taken from a certain part. (In this case, the center of the leaf)
The condition of the leaf
If the leaf is wilted or damaged, the stomata count may vary compared to a healthy leaf.
Collect and store leaves that are not wilted or damaged. Store in water to make sure they do not wilt.
The size of the leaves
A larger leaf may have more stomata than a smaller leaf.
Area of the leaf from both conditions should be measured in a graph paper and has to be similar to limit the error of surface area affecting the results due to differences in the leaf
Room temperature
The temperature in which the leaf is stored may affect (wilt) the leaf
The room temperature must correlate to the natural environment from which the leaf was obtained.
Microscope
The type of microscope and the magnification will affect the stomata count.
The microscope should be same for all the leaves examined and in the same power. (high power lens)
Humidity
Stoma open when the environment is more humid. Hence, all samples should be collected at the same level of humidity
All leaf samples are obtained in the same time of the day.
Plant
Plant from the same species may have different stomatal count.
The samples are collected from the same plant.
Age of the plant
If the plant it too old, the plant will be in a state of dying and hence will have stomata that are not operational
The plant’s age should be known.
Design Aspect 2: Method
Equipment:
20 Leaves on the sunny side of plants (15- 20 cm2)
20 Leaves on the shady side of the plant (15- 20 cm2)
Clear Nail varnish (9ml)
Cover slips
Forceps
Clear Tape
Light Microscope (1) and Microscope slides (40)
Eye Piece Graticule
Stage Micrometer
Tally Counter
Temperature Probe
Light meter
Graph paper
Ruler (20cm, +/-0.5 cm error)
pH paper
2
Design Aspect 3:
The data sample is ample, the independent variable is changed 5 times in both Sunny and Shady conditions and the experiment is repeated 20 times. No plants were harmed during the experiment because the sample was taken from a plant that is not about to be extinct. No chemical was used to get the sample of leaves.
Method:
Collect leaves from the same plant, the same plant needs to have some of its leaves receiving sunlight and the other part not receiving sunlight but shade (in this condition: shade from the building).
Since leaves in the shade will be larger than the leaves in the sun, the surface area of the leaves is measured to limit errors. This is so because larger leaves have different stomatal distribution than smaller leaves and this investigation is set out to compare the stomatal density of leaves of the same size in different conditions.
Cut the leaves and place it in water.
Measure the light intensity surrounding the Plant in the Sunny and Shady condition
Measure the pH of the soil in which the plant grows.
Calculate the surface area of the leaf by counting the area covered on a graph paper. The number of squares on the graph sheet is counted to select the leaves suitable for the investigation.
Leaves used in the experiment ranged from 112 to 116 squares, which is about 15- 20 cm2.
Label the samples taken from the sun and the shade, # 1,2,3,4…
Plug microscope into an electrical socket and mount the slides on it. This therefore leads to an orderly arrangement of apparatuses and easier examining of samples
The eyepiece graticule is calibrated by placing a micrometer on the stage of the light microscope slide and securing it in place with the pair of stage clips.
The eyepiece is mounted with the eyepiece graticule in position, and the stage micrometer is brought into focus, and altered such that its initial graduation coincided with the initial mark on the graticule.
Counting is done along both scales until a point was reached where there was another coincidence between a graduation mark on the graticule scale and another on the stage micrometer
A leaf is removed from the plant and each leaf is painted on the center of the lower epidermis (ventral surface) with a transparent nail varnish and allowed to dry up.
After a few minutes, the dry nail varnish is carefully peeled off from both surfaces of the respective leaf using a tape. Take the tape off with a forcep. The thin film is viewed under the microscope under 0.45mm diameter, at 400x magnification. The number of stomata seen in the field of the microscope is counted and using a tally counter, the results should be recorded.
When counting the number of stomata in the field of view it must be counted with precision and it has to be counted more than once to improve the accuracy.
The nail varnish must be carefully removed to prevent it from tearing and carefully placed on the slide to prevent it from creasing and damaging the trace.
Position the nail polish impression on a microscope slide, and count the stomata within a field of view of 0.45mm diameter, at 400x magnification. Record this number into a data table.
Conduct steps 9-16 , 20 times for both condition of leaves from Sunny and Shady condition.
Data Processing
Observational data
Field of view: 0.44mm diameter
Magnification: 400x
Temperature: 25 degrees Celsius
Light Intensity at sunlight: 983LUX
Light Intensity at 1cm: 300.4 LUX
pH 7
Table 1. Raw data to compare stomatal distribution in shady and sun plants of the same species
Number of Stomata (+/-1)
Sample
Stomata count in samples from Sunny side
Stomata count in samples from Shady side
1
40
20
2
53
20
3
42
21
4
43
27
5
50
32
6
21
25
7
3
17
8
51
32
9
46
21
10
31
28
11
30
32
12
38
16
13
43
31
14
40
30
15
36
34
16
54
27
17
43
27
18
43
32
19
21
15
20
51
25
Analysis of Data
Table 2. Processed data of Stomata count in both Sunny and Shady condition with average and standard deviation calculated.
Number of Stomata (+/-1)
Sample
Stomata count in samples from Sunny side
Stomata count in samples from Shady side
1
40
20
2
53
20
3
42
21
4
43
27
5
50
32
6
21
25
7
3
17
8
51
32
9
46
21
10
31
28
11
30
32
12
38
16
13
43
31
14
40
30
15
36
34
16
54
27
17
43
27
18
43
32
19
21
15
20
51
25
Average
38.95
25.6
SD
12.35505969
5.825804665
The formula used to calculate average:
Where,
N= Number of data
Excel was used to calculate Standard Deviation:
Standard Deviation Formula
The equation used to determine the stoma per millimeter
Example: S (Sunny) = 38.95/0.242
= 160.95
= 161 (correct to 1 decimal place)
S (Shady)= 25.6/0.242
=105.7
=106 (correct to 1 decimal place)
The equation for the field of view:
= (0.44/2)^2 * 5
= 0.242
Where,
http://www.esa.org/tiee/vol/v1/experiments/stomata/stomata_big1.jpg
Figure: Showing the Stomata view in the microscope.
Sunny Side
Shady side
Stomatal density
161
106
Table: Showing the calculated Stomatal density on 0.44mm view for the Sunny side and Shady side of leaves from a sample of 20 leaves per variable.
Graph 2: represents the stomatal count of the 0.44mm² field of view on the lower epidermis of Bougainvillea glabra leaves. The errors are show the spread of the data which is calculated from the Standard Deviation.
High Point
Low point
Sunny Condition
190
138
Shady Condition
138
81
Table: Key for the error bars in tabular format
Conclusion
The findings of this investigation show that stomatal density in leaves exposed to the sun were higher than that of leaves in the shade which supports my hypothesis.
This is because; in general, the greater the number of stomata/unit area, the greater is the rate of stomatal transpiration.
On a plant such as Bougainvillea glabra , we know that the stomatal density is the same on each leaf if the environment was either only Shady or only Sunny. However, due to the condition of Sunny or Shady the stomatal density differs. This is due to the fact that the plants are sited at different locations: those plants facing the sun have a higher stomatal distribution than those that are in the shade.
Those in direct sunlight will transpire more so need to be able to facilitate the loss of water vapour. Therefore, there is more stoma on the sunny part of the leaf.
For the plant to photosynthesize, carbon dioxide needs to be taken up from the help of stomata, since sunlight is needed for photosynthesis, more stomata will be open in the parts where sunlight is directly focused.
From the small range of SD for the Shady condition we can see that the central tendency of the data (5.825804665) is greater and the data is concentrated around the mean. The data is not spread out and very close. However, for the Sunny condition, we can see that the data is spread out because of the high range of SD (5.825804665). This is represented in the error bars.
Evaluation
Weakness
Effect
Suggestions for improvement
Appropriate age of the plant
This may have reduced the stomata count.
Samples can be taken from a plant with appropriate age known
Light intensity
Since light intensity surround each leaf was not measured, this may vary the stomata count in different leaves
To get perfect result, light intensity near each leaf can be taken even though this would be time consuming.
Humidity
Since the experiment was conducted on 2 different days, the humidity could have changed triggering the guard cells to open/close stomata.
All samples should be taken in the same day the experiment is conducted.
Statistical test
Tells how precise the result/conclusion is.
Even though the standard deviation tells the central tendency of data, the variance can be seen by completing a t-test.
Difficulties finding enough leaves from a single light intensity.
The same light intensity for the individual conditions is vital, because the light intensity is the independent variable, and therefore must remain the same throughout.
Choosing from a larger Bougainvillea glabra may have increased the chances of finding enough leaves within the particular light intensity.
Exact Stomata count
Since the stomata count is done through the microscope, the stomata count may not be exact due to human error.
The Stomata count should be repeated at least twice to avoid this error in data.
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