My investigation was prompted by observing that Lemna minor L. grew in a thick blanket in a pond on my farm. I wanted to discover what was causing the plant to grow in such abundance, which led to my research into its ecological niche.
For my experiment, I will investigate the role of phosphorus in reproduction. Phosphorus enters the plant through root hairs and tips and also directly through the leaf cells. It is absorbed mainly as H2PO4-, the primary orthophosphate ion. To a lesser extent,
some is also absorbed as HPO42-. Once absorbed, phosphorus is utilized as a component of many organic molecules in the plant. In these various organic forms, phosphorus is used for many chemical processes. These include:
Energy Reactions: Phosphorus is a part of the ADP and ATP, which are alternate forms of the molecule that is responsible for all chemical reactions within the plant that require energy.
Photosynthesis: the process by which sunlight is captured in ATP by chlorophyll and is then used to convert carbon dioxide and water to glucose and oxygen. Photosynthesis provides the glucose needed for respiration in plant cells.
Genetic Transfer: Phosphorus is a key component of many of the organic molecules that form genes and chromosomes, which are effectively the instruction manual for how the plant is formed. In order for DNA to be transferred between cells, there must be a sufficient supply of phosphorus. [5]
When these processes are happening more quickly, the plant will grow and asexually reproduce more quickly. The rate at which these processes occur depends on the supply of phosphorus. Therefore as the concentration of phosphorus increases, the rate of growth and reproduction will be greater in Lemna minor L.
Aim: To investigate the effect of different concentrations of calcium phosphate on the reproduction rate of Lemna minor L.
Hypothesis: By increasing the amount of calcium phosphate, the rate of reproduction and therefore number of Lemna minor L. fronds, will increase because phosphorus (in calcium phosphate) has a vital role in the growth of plants.
Null Hypothesis: Increasing the amount of calcium phosphate will have no effect on the reproduction rate of Lemna minor L. and the number of fronds in the different concentrations of calcium phosphate will increase at a similar rate.
Method:
1. Assemble Sach's nutrient solution [6] without the Calcium phosphate (Ca3(PO4)2) by dissolving the following in 5L of distilled water using a magnetic stirrer to ensure that the nutrients are well dispersed throughout the solution.
-2.5g NaCl
-2.5g CaSO
-2.5g MgSO4
-2.5g KNO3
-5 drops FeCl3 (1% solution)
2. The next step is to make up the solutions with different amounts of calcium phosphate representing the five different trophic states according to the Environment Waikato Classification of Trophic States (Figure 1). This is done by serial dilution.
Trophic State
Secchi depth
(m)
Total Phosphorus
(mg/m3)
Total Nitrogen
(mg/m3)
Chlorophyll a
(mg/m3)
Oligotrophic
> 7.0
< 10
< 200
< 2
Mesotrophic
3.0 - 7.0
10 - 20
200 - 300
2 - 5
Eutrophic
1.0 - 3.0
20 - 50
300 - 500
5 - 15
Supertrophic
0.5 - 1.0
50 - 100
500 - 1500
15 - 30
Hypertrophic
< 0.5
> 100
> 1500
> 30
Figure 1.
http://www.ew.govt.nz/environmental-information/Rivers-lakes-and-wetlands/Learn-about-our-lakes/Water-quality-glossary/
Start by taring the scale and measuring 1x10-1gL-1 of calcium phosphate. Add this to 1L of the Sach's solution labeling clearly 0.1gL-1 calcium phosphate solution. Measure 1L of Sach's solution using the most accurate conical flask possible. From this 1L take 1mL using a 1mL pipette and discard. Rinse the pipette with 0.1g/L calcium phosphate solution to prevent dilution of this solution, then measure 1mL of 0.1g/L calcium phosphate solution using a 1mL pipette and add to the 999mL of Sach's solution, labeling clearly, 1x10-4gL-1 solution (C1). Mix using a magnetic stirrer for 30 minutes. This is the concentration that represents hypertrophic conditions.(figure 1)
3. Using a 500mL measuring cylinder and reading from the meniscus to prevent parallax error, measure 275mL of Sach's solution and add to a 1L conical flask. Also using a 500mL measuring cylinder, measure 225mL of C1 and add to the conical flask. Stir thoroughly using a magnetic stirrer. The concentration of this solution is 4.5x10-5 gL-1, which represents supertrophic conditions. (figure 1).
-Measure 425mL of Sach's solution using a 500mL measuring cylinder and add to a 1L conical flask. Measure 75mL of C1 using a 100mL measuring cylinder and add to the conical flask also. Stir thoroughly using a magnetic stirrer. The concentration of this solution is 1.5x10-5gL-1, which represents eutrophic conditions. (figure 1).
-Measure 462.5mL of Sach's solution using a 500mL measuring cylinder and add to a 1L conical flask. Measure 37.5mL using a 50mL measuring cylinder and add to the conical flask also. Stir thoroughly using a magnetic stirrer. The concentration of this solution is 7.5x10-6gL-1, which represents mesotrophic conditions. (figure 1).
-Measure 487.5mL of Sach's solution using a 500mL measuring cylinder and add to a 1L conical flask. Measure 12.5mL of C1 using a 20mL measuring cylinder and add this to the conical flask also. Stir thoroughly using a magnetic stirrer. The concentration of this solution is 2.5x10-6gL-1, which represents oligotrophic conditions. (figure 1).
4. Measure 100mL of Sach's solution using a 100mL measuring cylinder and add to a plastic cup. Repeat this three times, labeling N1, N2 and N3. This solution has no calcium phosphate and is the control solution.
Measure 100mL of each of the calcium phosphate solutions using a 100mL measuring cylinder, repeating twice for each concentration, so that there are 18 labeled cups of the six different concentrations of calcium phosphate solution.
5. Rinse the sample of Lemna minor L, (taken as recently as possible from the source), with distilled water. This ensures that no nutrients from the source affect the results of the experiment. Using tweezers, count out twenty fronds and add to the first cup. A frond will be defined as any elliptical leaf, including those growing from the budding pouches. Repeat this process for each of the other 17 cups. N.B. make sure there are similar amounts of smaller and larger fronds and roots in each cup, so the conditions in each cup (apart from (Ca3(PO4)2) concentration) are the same. Also ensure that you only add green fronds to the cups. Brown, black and clear fronds are dead. Measure and record the temperature and pH of each cup. Cover each cup with gladwrap to prevent evaporation.
6. The Lemna minor L. will be grown in the glasshouse. Placing is important- for the first row, place cups in the order - N1, O1, M1, E1, S1, H1. In the next row- O1, M1, E1, S1, H1, N1.In the next row- M1, E1, S1, H1, N1, O1. This random formation ensures that if light intensity varies, it won't affect the results overall, as the positioning is random.
7. Draw up a table and record the number of fronds in each beaker every Monday, Wednesday and Friday for twenty days.
Results:
Trophic State
Conc. (Ca3(PO4)2)
(gL-1)
Reproduction rate
(fronds per day)
N/A
0
4.10
Oligotrophic
0.0000025
3.95
Mesotrophic
0.0000075
3.75
Eutrophic
0.000015
3.65
Supertrophic
0.000045
3.60
Hypertrophic
0.0001
3.95
Table 1.
Graph 1.
Conclusion:
My results show that the concentration of phosphorus has had no effect on the rate of reproduction of Lemna minor L. in this experiment. The reproduction rate of Lemna minor L. in varying concentrations of phosphorus was very similar. The highest reproduction rate was in the solution with no calcium phosphate and the lowest reproduction rate was in the solution representing supertrophic conditions. The difference between the highest and lowest reproduction rate was less than one frond per day (0.5 fronds per day). This insignificant difference indicates that there is no relationship between calcium phosphate concentration and reproduction rate in Lemna minor L. Therefore, I will accept my null hypothesis.
Discussion:
My investigation was prompted by an observation that Lemna minor L. Grew in abundance in a pond on my farm. So I decided to look at the effect of the concentration of calcium phosphate on the reproduction rate, or increase in number of fronds of Lemna minor L. My results indicated that changing the calcium phosphate concentration has had no effect on the reproduction rate of Lemna minor L. (Graph 1). Below, I have outlined why I believe this is the case.
As I stated in my introduction, phosphorus is not the only macronutrient in plants. Nitrogen and potassium are also needed in large amounts for growth and reproduction. Because of this, they are both major components of plant fertilisers. Although they have different roles, all of these nutrients contribute to growth and reproduction in the plant. As well as these primary macronutrients, there are also secondary macronutrients, which are usually provided in sufficient amounts by the soil without being added to fertilisers. These are calcium, magnesium and sulfur. Then there are the micronutrients, which are needed in much smaller amounts, which are copper, iron, chlorine, manganese, boron, zinc and molybdenum. [7] . Lemna minor L. is structurally adapted so that these nutrients are absorbed directly by the frond as well as the root, for a high rate of nutrient absorption. Lemna minor L. is predominantly composed of metabolically active cells so the "tissue contains twice the protein, fat, nitrogen and phosphorus of other plants" [8] , which is why having a large supply of nutrients is important. I used the Environment Waikato Tropic State Index (figure 1) classifications for my concentrations of calcium phosphate. I used a standard nutrient solution (Sach's solution) to dilute the calcium phosphate from a well-known biology textbook (Evelyn, Morholt and Brandwein, 1966). However; Sach's nutrient solution contains nitrogen, which is also an indicator of trophic state and a primary macronutrient, in a concentration that exceeds the concentration of nitrogen found in hypertrophic conditions. Hypertrophic is the highest level of nutrient enrichment. (figure 1) So in every solution, the amount of nitrogen was extremely high compared to much smaller amounts of phosphorus, even in the solution with the greatest concentration of calcium phosphate. Therefore, even though I changed the concentration of calcium phosphate, the effect of the nitrogen from the potassium nitrate would have caused a high rate of growth and reproduction in all of the cups making the effect of the phosphorus in the calcium phosphate negligible. So my results have biological significance, as they show that nitrogen also has a very important role in the growth and reproduction of Lemna minor L, which is why Lemna minor spreads rapidly in water with a high level of nitrogen as well as phosphorus. [9] The high level of nitrogen relative to the phosphorus concentration is the probable reason I got the results I did. These results accept my null hypothesis, rejecting my hypothesis that increasing the concentration of calcium phosphate would increase the reproduction rate in Lemna minor L.
To increase the amount of phosphorus, I also had to increase the amount of calcium. Had it turned out that my results accepted my hypothesis, this could have been a problem, as if changing the calcium phosphate concentration had increased the rate of reproduction, there
would have been no way of distinguishing whether it was the calcium or phosphorus that have caused the result. If this had been a problem, it could be overcome by further investigation. This could be done by performing several more experiments using different phosphorus compounds. If the results were very similar, this would prove that it is the phosphorus that is affecting the results, not the other chemical in the compound.
Validity of Conclusion:
Conc. (gL-1)
O
E
O-E
(O-E)2
(O-E)2/E
0
4.10
3.83
0.27
0.0729
0.0190
0.0000025
3.95
3.83
0.12
0.0144
0.00376
0.0000075
3.75
3.83
-0.08
0.00640
0.00167
0.000015
3.65
3.83
-0.18
0.0324
0.00846
0.000045
3.60
3.83
-0.23
0.0529
0.0138
0.0001
3.95
3.83
0.12
0.0144
0.00376
Table 2.
The Chi-squared Test:
To ensure the validity of my conclusion, I used the Chi-Squared Test to do a statistical anaylsis of my data. (Table 2). The figure I calculated for the sum of (O-E)2/E with five degrees of freedom was 0.0505. This corresponds to a probability of 0.95, which is well above the critical probability for determining whether to accept or reject my null hypothesis. Therefore I can say that my conclusion is valid and I must accept my null hypothesis.
Linear Regression:
To further prove the validity of my conclusion, I also used a linear regression test. The R-squared value indicates how well the line fits the data, a value of 1 being a perfect fit, which would show a perfect trend. My R-squared value is 0.002. This value is very low, which indicates that only 0.25% (barely any) of the variation in my results can be explained by calcium phosphate concentration. This means there is virtually no pattern to my data and I must accept my null hypothesis.
Graph 2.
Evaluation of Method:
In my initial method, there were several problems. I hadn't considered the variables, other than the concentration of calcium phosphate that could affect my results, such as pH, temperature and light intensity. In my final method, I took the temperature and pH of the solutions before I started my experiment to ensure they were all the same. I also arranged my cups so that the varying light intensity in the greenhouse didn't affect my results overall. For each concentration I had three cups, so in each row, rather than placing three of each concentration in a row, I had the cups randomly positioned so that light intensity didn't alter my results.
In my initial method, the number of fronds I started with in each cup was thirty. I had to change this in my final method to fifteen, as if I had started with thirty, I would have spent an unnecessarily long amount of time counting fronds.
In my initial method, I stirred my solutions using a stirring rod. Because the concentrations of calcium phosphate were so small, this would not have ensured that the calcium phosphate was evenly distributed throughout the solution, which could have altered the accuracy of my results. I overcame this problem by using a magnetic stirrer for thirty minutes for each of the calcium phosphate solutions, so they were thoroughly mixed.
Another problem I hadn't considered in my initial method was evaporation. If I simply left the cups in the greenhouse, some of the water would have evaporated, changing the concentrations of my solutions and giving inaccurate results. In my final method, I covered the cups with gladwrap to prevent evaporation, ensuring that each of the different concentrations stayed the same for the duration of the experiment.
