Plan/Hypothesis/Justification
Preliminary tests were carried out on cress seeds using fresh tomatoes and water and propanone to make the extracts. The greatest effect on germination and growth of cress was observed when the pulp surrounding the seeds was used in conjunction with propanone. The propanone helps to extract the inhibitor by breaking the cell walls of the tomato. ABA is lipid soluble therefore it dissolves perfectly in propanone.
The aim of this experiment is to treat cress seeds with variable concentrations of extracts and to measure its direct effect upon their germination and growth. The independent variable used will be the extract concentration; the dependent variables will be percentage germination and germination and growth rate. A Spearman rank correlation test will be used to determine whether the association between two variables is significant.
I predict the greater the concentration of extract, the greater the inhibitory effect it will have upon the germination and growth. The highest concentration will halt germination and growth. As the concentration becomes lower, the rate of germination and growth will become greater; tomato seeds do not germinate whilst under the highest concentration of extract within their fruit and also my preliminary results showed that 0 % germination occurred at this concentration. This will happen because amylase production or action is inhibited preventing glucose to be produced; When the extract is more dilute, less inhibitor is present to effect amylase production or action upon the starch reserves, hence more glucose can be produced and a faster rate of respiration can occur; this will increase the germination rate and growth rate.
Apparatus
42 petri dishes and lids
42 labels
1 marker pen
84 fresh tomatoes
Propanone
Distilled water
Food processor
Large funnel and Filter paper discs
Cotton wool
650 cress seeds
Transparent incubator with controlled oxygen pump/filter
Large chopping tile
Metal teaspoon
Scalpel and blade
Stirring rod
Sieve
Safety goggles
Plastic gloves
100cm³ measuring cylinder
Test Tube holder
6 test tubes and stoppers
2 10ml syringes
Top pan electronic balance (reads to 3 decimal places)
Weighing dish
Thermostatically controlled oven
Glass dish
Large conical flask
Large metal spoon
Dessiccator
Method
- Scoop pulp from half of tomatoes, sieve to discard seeds and place in clean processor. Discard remaining flesh.
- Measure 250 ml of propanone and add to pulp. Process for 3 minutes. Pour mixture into glass dish and cover to prevent evaporation.
- Repeat above procedure for remaining tomatoes and add mixture to glass dish. Mix thoroughly with large spoon.
- Filter the mixture using the funnel lined with filter paper and pour extracts into the conical flask.
- Label 1 syringe ‘water’ and 1 syringe ‘extract’ to avoid successive contamination of extracts and control.
- Label 6 tests tubes in holder :
- Control
- 20%
- 40%
- 60%
- 80%
- 100%
- Using the ‘water’ syringe, add 10 ml distilled water to tube 1. Using the ‘extract’ syringe add 2 ml of extract tube 2, 4 ml of extract to tube 3, 6 ml of extract to tube 4, 8 ml of extract to tube 5 and 10 ml of extract to tube 6. Seal the tubes.
- Line each petri dish with cotton wool of equal amounts and depth. Place 15 cress seeds in each dish and cover (ensure you weigh each set of seeds before adding to each dish; write this mass on corresponding dish).
- Divide dishes into 7 groups of 6. In each group label each of the 6 dishes as the test tubes were previously labelled.
- Also label each dish in the:
1st group ‘Day 0 to day3’
2nd group ‘day 4’
3rd group ‘day 5’
4th group ‘day 6’
5th group ‘day 7’
6th group ‘day 8’
7th group ‘day 9’
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Using the extracts in the test tubes, add them to their corresponding dish in the 1st group, ensuring all the seeds are covered by the extract equally for a fair test. Cover immediately to avoid evaporation of propanone.
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Repeat distribution of extracts for the 2nd to the 7th group.
- Add 10 ml of water to every dish in every group and cover.
- Place all dishes in incubator at 21°c.
- It is imperative that the seeds receive 10 ml of water every other day for germination to occur.
The incubator must be transparent like a greenhouse; hence the seeds can remain in the incubator whilst photosynthesising. The incubator should be placed on a large window sill free from obstruction of other objects allowing equal amounts of light to penetrate.
- After 2 days, count the number of germinated seeds in each dish. Replace dishes and repeat the following day. Record results in table. Discard any non-germinated seeds. Uncover all dishes.
- In the afternoon of day 3, take the seeds from group 1 and calculate its dry mass.
To calculate dry mass, place only germinated seedlings in an oven proof dish (including seed coat) into the oven at 70 °c for 30 minutes. Place seeds in the dessiccator to cool to avoid condensation build up. Weigh the seedlings. Repeat oven and weighing process until constant mass is observed. Discard group seedlings after baking.
Record constant mass in table. Calculate mass per seed (see table below)
- The dry mass stage must be completed for each successive group each following day until group
7 has been weighed.
It is imperative that all drying stages take place at the same time every afternoon to create a fair test.
Produce the following table for each % of extract including the control.
Please note, to compensate for the very small mass of a cress seed, multiply figures by 15 throughout (use previous table for mean mass per seed data).
Table to show % change in mass per day for 20% extract on cress seeds.
Plot a graph of % mass loss or gain (y-axis) against days for each extract.
By calculating the gradient of each graph you can discover the rate of change of mass (g day –¹).
Comparing each rate from each graph you will be able to see which concentration of inhibitor had the most inhibitory effect on growth. The smallest rate should correspond with the 100% concentration.
Using day 2 and 3 percentage germination data calculate mean % germination and plot a graph of extract (x-axis) against mean % germination (y-axis). From this you should be able to see a negative correlation between the variables. This will tell you there is a direct inhibitory effect on germination by the extracts. Graphs could also be drawn to display rate of germination.
Risk assessment and safety
Propanone is an irritant to eyes skin and lungs and is highly flammable.
- Extracts should be produced and used in the fume cupboard/ if unavailable wear a mouth mask.
- Extracts should be kept away from flames and dishes and bottles covered
- Wipe away spillages with paper towels
- Wear safety goggles and gloves
- Handle the scalpel and blade carefully
