The size of the cobalt chloride paper will be the same for each of the co-ordinates, 2cm long by 1cm wide. This will make a fair test for observing a colour change from blue to pink.
The co-ordinates for sampling will be chosen at random using the random button on the calculator. To further make this a fair test, the same co-ordinates used on the upper epidermis will be used on the lower epidermis.
Proposed Method:
The rate of transpiration will be measured first using a strip of anhydrous cobalt chloride paper. This needs to be dried and so will be placed in the microwave for two minutes before being applied to five different positions on the upper and lower sides of the leaf using sticky tape. The co-ordinates of the positions on the leaf will be obtained using the random button on the calculator.
Anhydrous cobalt chloride paper is blue and turns pink on hydration. The paper turning from blue to pink will show the emission of water from the leaf. When the paper has been attached to the positions on the leaf, the stop clock will be started. The stop clock will be stopped as soon as the colour change has taken place at all the positions. The time taken for the colour to change from blue to pink will be recorded. The upper and under sides of the leaf will be done individually. This will be repeated once more to give two sets of results. By doing this, a better average of results will be obtained.
Stomatal distribution will be measured by making a replica of the leaf using clear nail varnish. A thin layer of nail varnish will be applied onto both sides of the leaf, left to dry, and then peeled off carefully using forceps. The film of nail varnish will be placed onto a slide, covered with a cover slip and viewed under a microscope. The number of stomata will be counted in a given field of view. The number of stomata will be counted at five different views of each co-ordinate.
To calculate the area of the field of view under the microscope, the diameter will be measured first using a transparent ruler. Using the formula πr², the area can be calculated. This enables the number of stomata per mm² to be calculated. The stomatal densities of the lower and upper epidermis can be compared.
Apparatus:
Xeromorphic plant
Anhydrous cobalt chloride paper Cover slips
Stop clock
Nail varnish
Graticule
Ruler
Sticky tape
Graph paper
Calculator
Slides
Microscope
Forceps
Petri dishes
Method:
Random numbers were selected using the random button on a calculator. The first number selected was used as the y co-ordinate in centimetres and the second was used as the x co-ordinate in millimetres. Only four points were selected on the calculator. The fifth point was the centre of the leaf. Two of the points were plotted above the centre and the other two below. These points were plotted onto the leaf using a ballpoint pen. These co-ordinates were the points where the experiment would be carried out.
The rate of transpiration was measured using anhydrous cobalt chloride. The strips were measured using a ruler and cut 2cm long by 1cm wide. The paper was handled with fine forceps at all times in order to avoid getting any moisture onto it, resulting in inaccurate results.
The first part of the experiment was to create a control. A piece of cobalt chloride was picked up with forceps and placed into a petri dish. This was placed into the microwave with the lid half open. The microwave was turned on at high power for 2 minutes. After this, the petri dish was taken out of the microwave and placed in air until the cobalt chloride paper turned fully pink, at which point it was fully hydrated.
Each strip of cobalt chloride was dried in the microwave for 2 minutes before it was applied to the co-ordinates using sticky tape. It was made sure that the space between the leaf and the sticky tape was airtight in order to avoid any moisture from the air getting in, as this would affect the results. As the strips of cobalt chloride were applied the timer was started. When the colour change took place and the colour of the strips matched the control, the timer was stopped the time taken for the colour change was noted. This enabled the rate of transpiration to be calculated. This was carried out for each of the co-ordinates on the upper and lower epidermis of the leaf twice and the results were tabulated.
The stomatal density was measured by making a replica of the leaf using clear nail varnish. A thin layer of nail varnish was painted onto the leaf using the brush found in the bottle. This was left to dry for ten minutes. The nail varnish was peeled off carefully, at the co-ordinates, with fine forceps and placed onto a slide with a cover slip on top. The slide was placed on the stage of a microscope and the number of stomata was counted. At
each co-ordinate the number of stomata were counted at five different fields of view. This procedure was carried out at each co-ordinate on the upper and lower epidermis of the leaf.
The same leaf used in the experiment was drawn around onto graph paper carefully without taking the leaf off the plant, as this would be biologically unethical. This was done to enable the area of the leaf and the total number of stomata to be calculated.
A graticule slide was used to measure the diameter of the field of view. This allowed the area of the field of view to be calculated using the formula:
Area of a circle = π r ²
Where π is 3.142 (3dp) and r is the radius of the circle
The number of stomata per mm² was then calculated. Finally the stomatal densities on the upper and lower epidermis were compared.
Table 1:
The time taken for the cobalt chloride paper to become fully hydrolysed on the upper epidermis. Rate of transpiration on the upper epidermis calculated using the formula:
Rate of transpiration = 1/Time
Table 2:
The time taken for the cobalt chloride paper to become fully hydrolysed on the lower epidermis. Rate of transpiration on the lower epidermis calculated using the formula:
Rate of transpiration = 1/Time
Table 3:
The number of stomata counted at five different fields of view at each of the five co-ordinates on the upper epidermis of the leaf
Table 4:
The number of stomata counted at five different fields of view at each of the five co-ordinates on the lower epidermis of the leaf
Table 5:
Shows the area of the leaf being tested
Table 6:
Shows the number of stomata per mm² and the total number on the upper epidermis
Table 7:
Shows the number of stomata per mm² and the total number on the upper epidermis
Discussion:
The results show that the rate of transpiration was higher on the upper epidermis compared to the lower epidermis. There were more stomata present on the lower epidermis than on the upper epidermis.
Graph 1 shows that the rate of transpiration was greater on the lower epidermis. The rate of transpiration at co-ordinate 1 was 2.5 x 10ˉ on the upper epidermis, whereas on the lower epidermis, rate of transpiration was 3.29 x 10ˉ at the same co-ordinate. This is a difference of 0.72 x10ˉ secˉ . The differences in rate of transpiration were similar for each of the 5 co-ordinates. The peak rate of transpiration on the lower epidermis was 3.62 x10ˉ secˉ at co-ordinate 2, whereas the rate of transpiration on the upper epidermis peaked at 2.74 x10ˉ at co-ordinate 3. The lowest rate of transpiration was 2.57x10ˉ on the upper epidermis and 3.29x10ˉ on the lower epidermis. There were no anomalous results for the rate of transpiration.
Graph 2 shows the overall mean rate of transpiration on the upper and lower epidermis of the leaf. The mean rate of transpiration on the upper epidermis was 2.63x10ˉ , whereas the lower epidermis had a mean transpiration rate of 3.48x10ˉ . The difference between the average rate of transpiration on the upper and lower epidermis is 0.85x10ˉ . This is a percentage difference of 13.9%. This shows that the lower epidermis has a greater rate of transpiration than the upper epidermis, more water is lost by evaporation from the lower epidermis. This statement correlates with the number of stomata on the lower and upper epidermis of the leaf.
Graph 3 shows the mean number of stomata, at each of the five co-ordinates, on the upper and lower epidermis of the leaf. At co-ordinate 1, there were 11 stomata/mm² on the upper epidermis and 41 stomata/mm² on the lower epidermis. This is a difference of 30 stomata. The peak number of stomata was 14 on the upper epidermis. This was at
co-ordinate 3. This was also the co-ordinate at which the rate of transpiration on the upper epidermis peaked. The lowest number of stomata on the upper epidermis was 11. There was a difference between the peak number of stomata and the lowest number of stomata on the upper epidermis of only 3 stomata. The peak number of stomata on the lower epidermis was 46 at co-ordinate 4. The lowest number of stomata on the lower epidermis was 33. The difference between the peak number of stomata at co-ordinate 4 and the lowest number of stomata at co-ordinate 3 on the lower epidermis was 13.
Graph 4 shows the overall mean number of stomata on the upper and lower epidermis of the leaf. The upper epidermis has an average of 63,630 stomata and the lower epidermis has an average of 199,980 stomata. The difference in stomata number between the upper and lower epidermis is 136,350. This means that the lower epidermis has about 3.143 times more stomata than the upper epidermis. This graph is based on results tables 6 and 7.
Graph 5 clearly shows that there is a close relationship between the number of stomata and the rate of transpiration. The graph also shows that the greater the number of stomata, the faster the rate of transpiration.
Based on the results and looking at the graphs, it can be concluded that the hypothesis, stating that a greater stomatal density will have a greater rate of transpiration, has been shown. The null hypothesis, stating that there will be no correlation between the number of stomata and the rate of transpiration, has shown to be incorrect.
Limitations:
The cobalt chloride paper was dried in the microwave for two minutes at first. However, as the microwave became hot, the petri dishes begun to melt after only one minute. This is when the cobalt chloride paper was placed in the microwave for only one minute. The reduction in time might not have allowed the cobalt chloride paper to dry as much as it had dried when placed in the microwave for two minutes. This could have affected the time calculated for the rate of transpiration.
Before placing the cobalt chloride paper onto the leaf, it had to be taken out of the microwave, sticky tape had to be placed onto it and then it was taken and placed onto the leaf. This took about 20 seconds. During this time, some hydration of the cobalt chloride paper could have taken place. This would have given some inaccuracy in the calculation of the rate of transpiration.
There were three people carrying out experiments around the same plant. This would have increased the concentration of carbon dioxide around the plant causing stomata to close and therefore reducing the rate of transpiration.
When looking at the nail varnish imprints under the microscope, counting the number of stomata with the naked eye could have led to human error. This would have definitely occurred whilst counting the number of stomata on the lower epidermis, since there are so many. This error could have affected the skewness of the results whilst comparing the number of stomata to the rate of transpiration.
Also, whilst counting the number of stomata under the microscope, the number of stomata had to be counted at five different fields of view. The same field of view may have been used to count the number of stomata when the slide was moved around. This would have given an inaccurate approximation as to the number of stomata on the epidermis of the leaf.
The rate of transpiration was measured at five different co-ordinates on the upper epidermis and five co-ordinates on the lower epidermis. These results were used to calculate the overall mean rate of transpiration on the surface of the leaf. However, if more co-ordinates were used, the mean rate of transpiration would have been more accurate.
Only one leaf of the plant was experimented on. If more leaves were used, there would be more of an idea as to the rate of transpiration and the number of stomata on different leaves of the same plant. Rate of transpiration and number of stomata could also have been compared to the size and surface area of the leaf.
The investigation could also have been improved if different varieties of xerophytes were used. The number of stomata, rate of transpiration and the adaptations of the plants could have been investigated further.
Transpiration is necessary for transporting nutrients, cooling the plant, moving sugar and chemicals, and keeping upward water pressure. Water is pulled into the roots because of evaporation and hydrogen bonding. Evaporation then pulls on this chain of molecules.
The rate of transpiration is dependent on the size of the stomatal aperture and the diffusion gradient between the leaf and the atmosphere. The internal factors of the plant, which affect the rate of transpiration, include surface area of the leaf, thickness of the cuticle and stomatal density. The larger surface area, the higher the rate of transpiration. A thick cuticle reduces the rate of cuticular transpiration. The greater the number of stomata per unit area of leaf, the greater the rate of transpiration.
Stomata control the rate at which transpiration occurs. Guard cells gain and lose water, which close and open the stomata respectively. When the water pressure in the guard cells becomes greater than in the surrounding cells the stomata open allowing transpiration. Almost 90% of water contained in a plant is lost through transpiration.
Xerophytes are plants adapted to survive in dry conditions of unfavourable water balance. They have the effect of reducing the rate of transpiration to conserve water. Xeromorphic adaptations include thicker cuticles on leaves and stems, reduction in the size of leaves, curling or rolling of the leaves into a cylindrical shape, presence of epidermal hairs, the number and distribution of the stomata in pits or grooves.
Xerophytes such as evergreens have a thick cuticle to reduce cuticular transpiration and are often shiny so they reflect sunlight causing leaf temperature and therefore transpiration to fall.
Xerophytes like Ammophilia and Calluna can roll their leaves, which lengthens the path for diffusion and traps moist air. Ammophilia also have protective hairs on the surface of the leaf, which traps moist air.
These results show that the plant is adapted to its environment because the upper epidermis has less stomata, which minimises water loss from the plant. The upper epidermis receives more sunlight, than the lower epidermis, during the day. This means that the upper epidermis is exposed to more light and higher temperatures than the lower epidermis. With the upper epidermis having less stomata, water loss is minimised. This shows that this xerophyte is well adapted to its environment.