Core Practical 4: Investigate the effect of sucrose concentration on pollen tube growth
Objectives
Objectives
- Observe the growth of pollen grains
- Develop the skills of planning investigations and carrying out dilutions
- Carrying out research and cite sources of information to support planning and conclusions
- Ensure to use the correct concentrations of solutions as higher concentrations may be hazardous
- Work carefully to make sure you do not smash any glass slides as they may be sharp when broken
- Sucrose solution 2moldm-3
- Mineral salt culture medium
- Microscope
- Plants in flower
- Five Petri Dishes
- Filter paper
- Distilled water
- Measuring cylinders
- Stop clock
- Clean cavity slides
- Mounting needles
Procedure
- Carry out research into what concentrations of sucrose might be a suitable range to test in this experiment. Having found two websites[1] [2] [3]that discuss different sucrose concentrations we will use 0.0 , 0.2, 0.4, 0.8, 1.6 Sucrose solution mol dm-3 (%)
- Make up the solutions of sucrose and add mineral salt culture medium in a 1:1 ratio
- Label 5 petri dishes 0, 0.2, 0.4, 0.8 and 1.6 and add a filter paper which has been damped with their respective concentrations. This is to ensure the slides don’t dry out between observations, the petri dish acting as a humid chamber
- This is necessary as we do not use a cover slip for the pollen as we want to ensure aerobic conditions but this leads to the possible drying out of the slide
- Label one slide per sucrose concentration and pipette a few drops of the respective concentration solutions into the cavity of the slides
- Ensure that you do not touch the cavity in the centre of the slide as they must be clean as they may obscure the pollen
- Collect a flower that has mature anthers and is shedding pollen and rub the mounted needle over the anther allowing pollen to fall onto the cavity of the microscope slide. Use the same species of plant for each slide
- Every hour, check back on the pollen and record the growth of the pollen tubes under the x100 magnification, return the slide to the petri dish after every observation
Analysis of results
We can see from the graph that the most effective concentration for pollen tube growth was 0.4 mol dm-3 as the percentage of pollen cells with a tube formed was 36% compared to 30% of 0.2 mol dm-3. The experiment was done from 09:30 to 15:30, a period of 6 hours. After the period of 6 hours in the solution with no sucrose had shown no growth of the pollen tubes. 0.8 and 1.6 mol dm-3 showed relatively little growth with 8% and 2% respectively. The only difference between all the different experiments is the sucrose concentration that the pollen was kept in for the duration of the experiment. Therefore differences in observations would be due to this difference in sucrose concentration. It is clear that the for the higher sucrose concentrations of 1.6 and 0.8 the pollen tubes have severely stunted growth due to the fact that water travels from pollen to the surrounding through osmosis. This is due to the area surrounding the pollen being a low concentration of water. This stops formation of the tube for a majority of time however some pollen may be more resistant a large concentration difference of the water. For pollen in a solution of no sucrose the effect is the opposite. Relative to its surroundings the pollen is a low water concentration so, by osmosis, the water travels from the surroundings into the cytoplasm of the cell, this may end up lysing the cell if the concentration difference is significant enough. It appears, from our experiment that the concentration of the inside of the cell and the surroundings are similar enough, in the cases of 0.2 and 0.4 mol dm-3, that the pollen tubes can grow and form.
Evaluation
One issue that we found throughout our experiment was that the pictures we took at each concentration at each time are of different locations of the slide so it some parts of the slide the tubes may be developing at a quicker rate than others. Experimental problems such as this would be alleviated by making sure to observe the same location through telling other members of the group the specific area you focused. Also, if we did more experiments we could average the results minimising the effect of this problem. The second group doing the practical with us used pollen from a different flower that had been prepared differently. It is important, if we are to compare to use similar pollen, both in preparation and species. This is due to the fact that, as noted in a source linked previously, different species of flower have optimum sucrose concentration and would grow at different rates
[1] http://www.nuffieldfoundation.org/practical-biology/observing-growth-pollen-tubes
[2] http://www.saps.org.uk/attachments/article/83/Pollen%20germination%20across%20the%20seasons.pdf
[3] Brewbaker, James L., and Beyoung H. Kwack. “The Essential Role of Calcium Ion in Pollen Germination and Pollen Tube Growth.” American Journal of Botany, vol. 50, no. 9, 1963, pp. 859–865. www.jstor.org/stable/2439772.
We can see from the graph that the most effective concentration for pollen tube growth was 0.4 mol dm-3 as the percentage of pollen cells with a tube formed was 36% compared to 30% of 0.2 mol dm-3. The experiment was done from 09:30 to 15:30, a period of 6 hours. After the period of 6 hours in the solution with no sucrose had shown no growth of the pollen tubes. 0.8 and 1.6 mol dm-3 showed relatively little growth with 8% and 2% respectively. The only difference between all the different experiments is the sucrose concentration that the pollen was kept in for the duration of the experiment. Therefore differences in observations would be due to this difference in sucrose concentration. It is clear that the for the higher sucrose concentrations of 1.6 and 0.8 the pollen tubes have severely stunted growth due to the fact that water travels from pollen to the surrounding through osmosis. This is due to the area surrounding the pollen being a low concentration of water. This stops formation of the tube for a majority of time however some pollen may be more resistant a large concentration difference of the water. For pollen in a solution of no sucrose the effect is the opposite. Relative to its surroundings the pollen is a low water concentration so, by osmosis, the water travels from the surroundings into the cytoplasm of the cell, this may end up lysing the cell if the concentration difference is significant enough. It appears, from our experiment that the concentration of the inside of the cell and the surroundings are similar enough, in the cases of 0.2 and 0.4 mol dm-3, that the pollen tubes can grow and form.
Evaluation
One issue that we found throughout our experiment was that the pictures we took at each concentration at each time are of different locations of the slide so it some parts of the slide the tubes may be developing at a quicker rate than others. Experimental problems such as this would be alleviated by making sure to observe the same location through telling other members of the group the specific area you focused. Also, if we did more experiments we could average the results minimising the effect of this problem. The second group doing the practical with us used pollen from a different flower that had been prepared differently. It is important, if we are to compare to use similar pollen, both in preparation and species. This is due to the fact that, as noted in a source linked previously, different species of flower have optimum sucrose concentration and would grow at different rates
[1] http://www.nuffieldfoundation.org/practical-biology/observing-growth-pollen-tubes
[2] http://www.saps.org.uk/attachments/article/83/Pollen%20germination%20across%20the%20seasons.pdf
[3] Brewbaker, James L., and Beyoung H. Kwack. “The Essential Role of Calcium Ion in Pollen Germination and Pollen Tube Growth.” American Journal of Botany, vol. 50, no. 9, 1963, pp. 859–865. www.jstor.org/stable/2439772.