Investigation into the effect of varying concentrations of potassium nitrate solution, on the germination and subsequent root radicle growth of cress seeds

The following investigation is based on seed germination and growth. In this particular study seeds will be subjected to a variety of different solution concentrations, the aim being to find out how this affects their growth and germination.

Introduction

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The germination of seeds is dependent on the presence of three things; a sufficient water supply, a suitable temperature and an appropriate partial pressure of oxygen. Germination also relies on the maturity of the embryo as well as the presence of the three factors listed above.

The picture above shows a seed in trans-section. The embryo consists of a shoot (plumule,) a root (radicle) and 1 or 2 seed leaves. The seed’s food store is either contained in the endosperm (tissues surrounding embryo) or within the seed leaves. The role of the testa is to enclose and protect the seed’s contents.

Water, which is absorbed through the microphyle and testa, is essential for activating the enzymes that catalyse the biochemical reactions of germination. When water is absorbed it stimulates the production of gibberelin (a plant growth regulator, synthesised in most parts of the plant.) The gibberelin, in turn, stimulates the synthesis of amylase by the cells in the aleurone layer. The amylase hydrolyses the starch molecules in the endosperm, converting them to soluble maltose molecules. Finally, the maltose is converted into glucose, providing a source of carbohydrate for respiration. Gibberellin causes this effect by regulating the genes that are involved in the synthesis of amylase.

The role of temperature in germination is to provide the optimum conditions for the enzymes involved in food mobilisation. Enzymes are biological catalysts, meaning they speed up a chemical reaction whilst remaining unchanged themselves at the end of the reaction. They possess a special feature called an active site (usually a cleft or depression.) Substrate molecules can bind, temporarily, to this active site and, whilst binded, the enzyme changes the structure of the substrate. Enzymes work by providing an alternative, lower energy route along which a reaction can take place. This lowering of the activation energy increases the number of molecules which have enough energy to react. Therefore, more molecules react and more products can be formed.

The diagram above shows the effect of temperature on an enzyme-controlled reaction. It demonstrates the importance of temperature in seed germination. At very low temperatures the enzyme reactions inside the seed can take place only very slowly. This is because molecules have very little energy and so move very slowly. Collisions between substrates and enzymes are infrequent and, even when they do collide they may not possess enough energy to react. At these low temperatures the seed is unlikely to be able to germinate as the rate of its biochemical reactions, such as food mobilisation, will be too slow.

As the temperature rises the enzymes and substrates move faster, collisions become increasingly frequent and, as a result, the rate of germination steadily rises as the temperature rises.

However, above a certain temperature the enzymes vibrate so energetically that some of the bonds holding the enzyme in its precise shape begin to break. The substrate can no longer fit into the enzymes active site so no reaction can occur. The enzyme is said to be denatured. If a seed were subjected to such temperatures it would be unable to survive as none of the necessary biochemical reactions could occur.

Like all living organisms, seeds need to respire to survive. Respiration makes energy for growth and metabolism available. The process involves the breaking down of organic molecules in a series of energy-releasing stages. The final stage requires oxygen which diffuses through the testa. However, the rate of this diffusion may be too slow and the seed may have to rely on some anaerobic respiration until the testa ruptures. Glucose, produced via the actions of gibberelin as explained earlier, is used as the fuel in respiration.

Preliminary work

A pilot test was carried out in order to ensure the efficiency and success of the actual experiment. In the pilot experiment it was decided to investigate a number of different factors in order to determine the best combination of these factors for use in the actual experiment. First, two different seed types (cress and carrot) were selected. The two seed types were subjected to the same conditions and then analysed after 5 days. The seed type with most apparent germination and growth was selected for the final experiment.

Next, two different solutions were chosen (potassium nitrate and copper nitrate.) The solution in which the seeds germinated most successfully was used in the final experiment.

In the next stage, two solution concentrations were chosen, one high (ratio 6:2) and one low (ratio 2:6.) It was hoped that this would give some indicator of a suitable range to use in the actual practical.

Finally, two different solution depths were chosen (12cm� and 4cm�) to see whether the seed grew best in deep or shallow volumes.

Equipment used in pilot 1

* 12 petri dishes

* 24cm� potassium nitrate.

* 16cm� copper nitrate

* 56cm� water (distilled)

* 105 cress seeds

* 75 carrot seeds

* Blotting paper

* Marker pen

* 2 measuring cylinders

* 2 measuring syringes

Method used in pilot 1

The 12 petri dishes were laid out on a table with their lids removed, and 12 circles were cut from the blotting paper each with a diameter slightly smaller than that of the petri dish. One circle of blotting paper was then placed in each dish. The blotting paper was chosen because of its absorbent properties. 15 seeds were placed in each dish and 8cm� of a solution was added to each. The lids were then replaced and each dish labeled using the marker pen.

The 12 dishes were set up as shown in the table:

Dishes 11 and 12 were set up as controls. They did not contain any solution, just water.

The dishes were labeled as follows;

1. Cress – high concentration of potassium nitrate

2. Cress – low concentration of potassium nitrate

3. Cress – high concentration of copper nitrate

4. Cress – low concentration of copper nitrate

5. Carrot – high concentration of potassium nitrate

6. Carrot – low concentration of potassium nitrate

7. Carrot – high concentration of copper nitrate

8. Carrot – high concentration of copper nitrate

9. Cress – large volume of solution

10. Cress – small volume of solution

11. Cress – control

12. Carrot – control

The lids of the dishes were then replaced and the dishes were placed in a tray lined and covered with wet newspaper. The wet newspaper makes the environment damp so minimising evaporation by decreasing the water potential gradient between inside and outside the dish.

Results

The seeds were checked after one day and the newspaper had completely dried out. None of the seeds showed any signs of germination. The newspaper was re-dampened and the seeds left for a further 3 days.

After day 4 the control groups had fully germinated with the cress seeds having an average root length of 2.75cm and the carrot seeds an average root length of 1.96cm. It was noted that some seeds had slid under the blotting paper and showed no signs of germination.

None of the other seeds showed any signs of germination. It was therefore difficult to decide which solution and at what volume to use in the actual experiment. However, due to the success of the controls, it seemed that a volume of 8cm� was adequate. Potassium nitrate was chosen, at random, after concluding that the type of solution did not particularly matter as long as it was consistent throughout all experiments.

Pilot 1 showed that seeds are more likely to germinate in low concentrations. The concentrations used in this experiment were far too high as none of the seeds germinated. It was decided that another pilot be carried out, varying only the solution concentrations but using much lower concentrations, with a larger range.

Pilot test 2

In pilot 2 five dishes were used. The dishes were set up as in pilot 1 but only cress seeds were used and the solution concentrations were as follows.

Dish 1 – 0.1 mols

Dish 2 – 0.01 mols

Dish 3 – 0.001 mols

Dish 4 – 0.0001 mols

Dish 5 – 0.00001 mols

This time the tray was placed in a sealed plastic bag, to help prevent the newspaper drying out.

Results of pilot 2

The newspaper did not dry out throughout the entire 5 days. All the seeds germinated and the average root radicle length in each dish was calculated. It was noted that some seeds had overlapped as they grew, and these tended to be shorter than the majority. These seeds were not included in calculations of the mean.

There was a general increase in root radicle length as the solution concentrations decreased, the exception being dishes 3 and 4, between which there was a slight decrease. The reason for this exception is unknown and may have been a result of the decreased accuracy brought about by the small sample size. On the other-hand it may not have been an inaccuracy at all.

Prediction

Based on the research and preliminary studies, detailed above, the following prediction was made.

‘Subjecting the seeds to an increase in the concentration of potassium nitrate solution will result in a decrease of seed germination and subsequent length of root radicle grown. Over the concentration range 0.1 – 0.00001 mols, the average root radicle length will increase by approximately 2cm.’

The results of pilot 2 suggest that as the concentration of solution decreases there will be a steep increase in seed length at first, but that the increase will become less apparent as the concentration continues to decrease. This information, however, will not be used to make a quantitative prediction as the accuracy of the pilot tests is uncertain, due to the lack of repeats.

The prediction above is based on the results of the pilot experiments and the following background knowledge:

Water absorption – Water is an essential mineral to a germinating seed. As described in the introduction, it is needed to initiate the work of the enzymes, which catalyse all the biochemical reactions of a plant. Increasing the volume of water, therefore, increases the rate at which biochemical reactions occur thus causing an increase in the rate of germination, which relies on these reactions.

Water is also a reagent in the hydrolysis of stored food substances and is required for the translocation of hydrolysed food reserves to the sites of growth in the embryo. An increase in water volume means more food is hydrolised and transported to the embryo. The food is used in respiration, producing energy for use in growth. Providing additional food increases the rate of respiration and so increases the rate of plant growth.

Referring back to the prediction, it is possible to relate the lowered plant growth in high solution concentrations to the availability of water.

A seed relies on a procedure, known as osmosis, to gain the water it requires to grow. Osmosis is a process by which water moves from an area where it is in high concentration to an area where it is in a lower concentration, in attempt to ‘even out’ the concentrations on both sides of a permeable barrier. The water molecules move by diffusion down a concentration gradient. The existence of a salt, such as potassium nitrate, lowers the water potential in the area where it is present. This, in turn, decreases the water potential gradient between the area where the salt is present and the adjacent, ‘low water potential’ area. As a result, less water moves by osmosis.

Inside a seed the concentration of water is low. Therefore, when put in a solution of water only, large volumes of water move into the seed, down a water potential gradient. When potassium nitrate is introduced to the solution the concentration of the water outside the seed is lowered and so is the water potential gradient between the seed and its environment. The higher the concentration of potassium nitrate outside the seed the lower the water potential gradient and the lower the volume of water that diffuses into the seed.

The need for fertilisers – Potassium nitrate is a fertiliser, often put on plants to aid growth. However, part 1 of the prediction, concluded that the presence of potassium nitrate, in fact slowed down the growth of the developing seeds. This is because the nitrogen, in fertilisers, is not needed by the plant until the embryo has developed and has begun to photosynthesise. Once the plant is developed, the nitrogen constitutes to essential biological molecules, such as proteins and nucleic acids.

Variables

Independent Variable –

* Concentration of Potassium Nitrate solution; 0.1mols, 0.01mols, 0.001mols, 0.0001mols and 0.00001mols.

Dependent Variables –

* Germination of seeds

* Length of root radicle grown, once germinated

* Appearance of a plumule (shoot.)

Control Variables –

* Temperature – room temperature

* Light – Dark

* Total volume of solution – 8cm�

* Solution used – potassium nitrate

* Water used – distilled

* Humidity – Quite humid due to wet newspaper

* Equipment used – Consistent throughout

Equipment

Method

1. Lay 7 of the 14 petri dishes out on the table and remove the lids. The remaining 7 will be used in the repeats.

2. Take the blotting paper and scissors and cut 14 circles, of equal diameter, out of the blotting paper. The circles should have a diameter slightly smaller than that of a petri dish, enabling them to fit neatly inside the base of a dish.

3. Using the marker pen, label the dishes with the following: Dish 1 – ‘0.1 mol, Potassium Nitrate’

Dish 2 – ‘0.01mols Potassium Nitrate’

Dish 3 – ‘0.001 mol, Potassium Nitrate’

Dish 4 – ‘0.0001 mol, Potassium Nitrate’

Dish 5 – ‘0.00001 mol, Potassium Nitrate’

Dish 6 – ‘8 cm� water’

Dish 7 – ‘8cm� Potassium Nitrate’