The stomatal density of a dicotyledonous plant growing in both Sunny and Shady conditions
CONTENTS
Section Page
Abstract 2
Plan Proposal 4
Introduction 7
Apparatus 11
Method 12
Results 18
Analysis of Results 20
Discussion of Results 25
Conclusion 28
ABSTRACT
This project investigated the stomatal density of a dicotyledonous plant growing in both Sunny and Shady conditions. The stomatal density of a batch of leaves taken from the main branches of the plant was estimated using a light microscope and an eyepiece graticule.
Transparent nail varnish was used to obtain replicas of the different adaxial (lower) leaf surfaces by carefully applying it to the same area of the different sample and carefully removing it with the aid of a pair of forceps after drying. The field of view to be used for the stomatal counts was calibrated by placing the stage micrometer on the microscope and counting the calibrations that occupied the graticule scale at magnification X40. The dried nail varnish was viewed under a microscope at magnification X40 and the number of stomata seen in the field of view of the microscope was counted using a hand held tally counter. The counts in the same field of view was repeated twice again and averaged to increase the accuracy in the results.
The stomatal density of the adaxial (lower) leaf surfaces of plants found in the sun was found to be higher than the stomatal density on the adaxial (lower) leaf surfaces of plants found in the shade. From the results obtained the average number of stomata seen in the field of view was 39 stomata in the sun and 27 stomata in the shade this thereby supports the above statement. Further evidence of this was given from calculations which indicates that the stomatal density of the adaxial (lower) leaf surfaces of the plant exposed to the sun was approximately 5x105 stomata per square centimeter and the stomatal densities on the adaxial (lower) leaf surfaces of the plants in the shade were approximately 3.4x105 stomata per cm2.
A Student t-test was used to test the reliability of the Null and Alternate Hypothesis `Reject the Null Hypothesis and accept the Alternative Hypothesis if the positive test value of t is greater than the critical value at P%. Therefore, the Null Hypothesis which states that there will be less stomata on leaves exposed to the sun and more stomata on leaves exposed to the shade;' was rejected and the Alternate Hypothesis which states that `there will be more stomata in the lower epidermis of leaves exposed to the sun and less stomata on leaves exposed to the shade;' was accepted
Therefore the conclusion drawn for this experiment was that the stomatal distribution depends on the position of the leaves on the plant. Hence there will be more stomata in leaves that are found in the sun than in leaves that are found in the shade.
Plan Proposal
TITLE: An investigation to compare stomatal distribution in shady and sun plants of the same species
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.
ALTERNATE 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.
INITIAL PLAN OF METHOD
The leaves chosen will be cut from a specific plant/bush.
A plant exposed to both sunny and shady conditions will be chosen. Since leaves in the shade will be larger than the leaves in the sun, the surface area of the leaves will be measured to limit errors.
The surface area of the leaves will be traced on a piece of graph paper and the number of squares counted which makes up the leaf surface area.
When the leaves are excised from the plant, they will be placed in a rubber bag and labelled. Back in the laboratory, the underside of the same part of the leaves will be painted with clear nail varnish and allowed to dry for 30 minutes. This will be done to make the nail varnish dry properly and prevent it from being sticky and hard to remove.
Using a pair of forceps, the nail varnish will carefully be removed from the leaf and placed onto a microscope slide. The slide will be labelled according to what specimen it will be taken from.Before viewing the slides under the microscope, the field of view will be calculated using an eyepiece micrometer and a graticule.The area will be calculated using the formula r2.
PRECAUTIONS
Leaves used must be of the same size and nail varnish should be applied to the same place of each leaf.
Nail Varnish has a strong vapour and it can easily ...
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Using a pair of forceps, the nail varnish will carefully be removed from the leaf and placed onto a microscope slide. The slide will be labelled according to what specimen it will be taken from.Before viewing the slides under the microscope, the field of view will be calculated using an eyepiece micrometer and a graticule.The area will be calculated using the formula r2.
PRECAUTIONS
Leaves used must be of the same size and nail varnish should be applied to the same place of each leaf.
Nail Varnish has a strong vapour and it can easily catch fire so it must be used away from fire.
Nail varnish must be completely dry to prevent it from tearing when it is being removed.
RESULTS AND ANALYSIS
A table showing the number of samples and stomatal counts will be drawn and graphs to show the relevance of the results will also be drawn. Statistical tests such as the Student t-test will be used to prove or disprove the hypothesis.
Number of stomata in leaves exposed to the sun
SAMPLE
Count 1
Count 2
Count 3
Average number of stomata in the sun
Total
Average
DISCUSSION AND CONCLUSION
The trends and patterns in the data will be recognized and commented upon.
Limitations and their effects on the influence on the accuracy of the experiment will be discussed.
Statistical tests will be used to prove or disprove the hypothesis and any anomalies accounted for with reasonable arguments. And hence the statistical tests will therefore lead to the drawing up of the Conclusion.
REFERENCES
This section will contain authors name, title of book and dates of publishing where information for introduction, discussion and conclusion taken from will be
found.
ACKNOWLEDGMENTS
This section will contain names of people who helped in project and will be thanked and acknowledged
INTRODUCTION
This project was set out to investigate the stomatal distribution in a Plant whose leaves are found in different positions to the sun; that is leaves found in the sun and leaves found in the shade.
Stomata are important for a plants gaseous exchange by regulating what enters and what exits the leaf be it oxygen, carbon dioxide, water vapour or any other gas from the leaf. Each stomatal pore is surrounded by two curve shaped cells called guard cells.
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 pottassium 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.
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 were 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.
TITLE: Comparing stomatal distribution in shady and sun plants of the same species
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.
ALTERNATE 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.
APPARATUS:
Quantity
Item
5
Leaves on the sunny side of plant
5
Leaves on the shady side of the plant
Nail varnish
30
Microscope slides
30
Cover slips
Forceps
Light Microscope
Eye Piece Graticule
Stage Micrometer
Knife
Tally Counter
METHOD
SELECTING THE DIFFERENT BATCHES OF
LEAVES FOR COUNTING
The leaves were all chosen from the same plant, which was found near the School's Administration block. It had all the conditions that my investigation required. The same plant had some of its leaves receiving sunlight and the other part not receiving sunlight but shade from the building.
Since leaves in the shade are 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.
The leaves chosen for this investigation were cut from the branches of the plant and placed in a bucket of water.
In the laboratory, the surface area of the leaves selected, was determined by placing each leaf on a graph sheet and carefully tracing out its outline. The number of squares on the graph sheet were counted and used to select the leaves suitable for the investigation. Selected leaves used in the experiment ranged from 112 to 116 squares, which is about 15- 20 cm2.
SETTING UP THE MICROSCOPE
A microscope is an instrument used to view objects that are too small to be seen with the naked eye but visible through a microscope. The microscope enlarges these small objects to sizes that can be visible to the naked eye.
The microscope was taken out of the cupboard and placed on the desk. The microscope slides were also taken out and cleaned with a tissue. The slides were then labelled accordingly. The samples taken from the sun were labelled Sunny no 1,2,3,4... and samples from the shade were labelled no 1,2,3,4... The microscope was then plugged into an electrical socket and the slides mounted on it. This therefore led to an orderly arrangement of apparatuses and easier examining of samples.
CALIBRATING THE EYE PIECE GRATICULE
The eyepiece graticule was 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 was mounted with the eyepiece graticule in position, and the stage micrometer was brought into focus, and altered such that its initial graduation coincided with the initial mark on the graticule.
Counting was 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.
Since each division on the stage micrometer is 100mm, it followed that: one division on the graticules, scaled (in mm)
= Number of divisions on stage micrometer X100
Number of divisons on the eyepiece graticule scale
DETERMINING THE STOMATAL DENSITY
A leaf was removed from the plant and each leaf was painted on the dorsal surface with a transparent nail varnish and allowed to dry up.
After a few minutes, the dry nail varnish was carefully peeled off from both surfaces of the respective leaf using a fine pair of forceps. The thin films were viewed under the microscope. The number of stomata seen in the field of the microscope was counted using a tally counter and the results were recorded.
CALCULATIONS
m x100 = 100m
m=10-6m
00mm = 10-6m x
= 1 x 10-4m
Area of microscope field of view =
=
= 7.854 x 10-6cm2
STOMATA DENSITY OF LEAVES EXPOSED TO THE SUN
LOWER SURFACE
Average No. Of Stomata =
(40+44+50+21+51+41+36+50+45+30+32+31+38+41+42)
= 39.4 Stomata
? 39 Stomata
7.854 x 10-5 cm2 = 39 Stomata
?1cm2 = 39 Stomata X 1cm2
7.894 x 10-5cm2
=4.965 x 105
?5 x105 Stomata
?Stomata density = 5 x 105 stomata per 1cm2
STOMATA DENSITY HAVE LEAVES IN SHADE CONDITION
LOWER SURFACE
Average number of Stomata
(32+21+20+25+25+32+28+21+32+16+31+30+27+34)
= 26.6 Stomata
?27 Stomata
7.854 x 10-5 cm2 = 27 Stomata
?1cm2 = 27 Stomata x 1cm2
7.854 x 10-5 cm2
= 3.4 x 10-5 Stomata
?Stomata density = 3.4 x 105 Stomata 1cm2
Sources of eror and precautions
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.
RESULTS
A table showing the average number of stomata counted in the sun
Number of stomata in leaves exposed to the sun
SAMPLE
Count 1
Count 2
Count 3
Average number of stomata in the sun
40
39
40
40
2
42
45
46
44
3
53
49
48
50
4
21
21
21
21
5
50
52
51
51
6
43
39
41
41
7
36
36
37
36
8
51
50
49
50
9
46
42
47
45
0
29
30
30
30
1
31
33
32
32
2
30
31
32
31
3
38
38
38
38
4
40
41
41
41
5
42
42
42
42
Total
592
588
595
592
Average
39
39
39
39
A table showing the average number of stomata counted in the shade
Number of stomata in leaves exposed to the shade
Sample
Count 1
Count 2
Count 3
Average no of stomata in the shade
31
31
33
32
2
20
22
21
21
3
21
20
20
20
4
27
25
24
25
5
27
24
25
25
6
25
24
28
25
7
32
32
32
32
8
28
27
28
28
9
21
21
21
21
0
32
32
32
32
1
5
7
7
6
2
31
30
31
31
3
30
30
30
30
4
27
26
27
27
5
34
34
35
34
Total
401
395
404
399
Average
27
26
27
27
Analysis of Results
Graphical Representation
Graph 1
This graph shows the average number of stomata in the sun that was counted
Graph 2
This graph shows the average number of stomata in the shade that was counted
Graph 3
This graph shows a relationship between the average number of stomata counted in the sun and the average number of stomata counted in the shade.
Statistical Representation
Student t-test
t
s=
in the sun =7
in the shade =-6
s1=variance in sun
s2=variance in shade
n1=15
n2=15
In the sun
s= = = s=
s1=8.6
In the shade
s= = = s=
s2=8.8
s1=8.6 s2=8.8
t
t
t
tcal= 3.82
tcrit=1.701
Null hypothesis was rejected since tcal= 3.82 is greater than tcrit=1.701 thus the
alternate hypothesis accepted if the positive test value of t is greater than the critical value. The Null Hypothesis which states that there will be less stomata on leaves exposed to the sun and more stomata on leaves exposed to the shade;' was rejected and the alternate hypothesis which states that `there will be more stomata in the lower epidermis of leaves exposed to the sun and less stomata on leaves exposed to the shade;' was accepted
DISCUSSION OF RESULTS
The findings of this investigation show that stomatal density in leaves exposed to the sun were higher than that of leaves in the shade.
It was observed from Graph 1 that the highest number of stomata counted happened to be 52 whereas in Graph 2 the highest number of stomata was 34. Also from Graph 1 the lowest number of stomata recorded was 21 and from Graph 2 the lowest number of stomata recorded was 16. In addition to this, Graph 3 shows that the stomatal density on leaves exposed to the sun was more than the stomatal density of leaves found in the shade. This is so because plants normally have more stomata in leaves exposed to the sun due to a higher rate of photosynthesis and transpiration. Thus leaves exposed to the sun need more stomata for gaseous exchange during photosynthesis unlike leaves found in the shade, which do not receive as much sunlight as leaves in the sun hence has a smaller amount of stomata as compared to the leaves in the sun.
In general, the greater the number of stomata/unit area, the greater is the rate of stomatal transpiration, however their stomatal density is also important. For example, the lower surfaces of dicotyledonous leaves usually have more stomata than their upper sufaces. (Biological Science)
In order to ascertain the null hypothesis that the stomatal density would not vary from species to species, the Student t-test, which is one tailed, was used at the interval data. For this to be adopted as the hypothesis for the experiment, the value of tcrit had to be less than the value of tcal.
On a tree, the stomatal density is the same on each leaf. But for different species, 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. Hence an increase in the number of stomata on those plants. This was verified by Simkins and Williams, in 1989.
Accordingly, some trees have developed adaptations, such as fleshy stems, and thickened barks, in order to lower their transpiration rate. These plants in their efforts to retain water have very few stomata on their leaves, if they have any leaves at all. Other trees located in wetlands and marshes have high transpiration rates so will need a high stomatal density to get rid of the excess water vapour. Also, the surface area of the leaves of each different species varies. As the surface area to volume ratio of the leaves increases so does the number of stomata on the leaf. This is because a larger number of stomata will be needed to aid in transpiration from the larger leaves.
Most stomata occur in the leaves. In the dorso-ventrally flattened leaves typical at most dicotyledons, stomata are present mainly in the lower epidermis (Taylor et al, 1997). In the experiment performed, it was found out that the stomatal density was highest on the lower epidermis, of leaves exposed to the sun than to leaves found in the shade.
The Biological Significance in this experiment is that the knowledge of stomatal distribution can help farmers practice photoperiodism. If they do practice this, their crops will grow better because the leaves in the shade were larger hence will receive more sunlight and will therefore photosynthesise better to produce more glucose which is stored as starch.
Experiments to be carried out in the future which may expand on the theories presented should include investigations in which samples of the stomatal traces should be taken from the adaxial(lower) and adaxial(upper) surfaces of the leaves. Also samples should be taken from different types of plants not one type of plant to makes the results more accurate.
Conclusion
The statistical test carried out on the result obtained rejected the null hypothesis. The alternate hypothesis which was consistent with the observations made was thus accepted. Hence it can be concluded that leaves that are exposed to the sun have a higher stomatal distribution than leaves found in the shade have a higher surface area than leaves exposed to the sun.
References
A.C. Dutta, Botany for Degree Students, Fifth Edition 1979, Publishers:Oxford University Press
