Investigation into the effects of auxin (IAA) growth regulator on plant tissue.

In week 8 of our Botany Practicals we looked at the effects of auxin (IAA) growth regulator on plant tissue. The experiment was carried out over 2 weeks. The first week involved the initial set up and the second week, observing and recording the results.

What are Auxins?

Indole acetic acid

Auxins are a class of plant hormones involved in plant-cell elongation, apical dominance, and rooting.

One of the most common auxins is Indole Acetic Acid, or IAA, which is produced in the apical meristem of the shoot. Developing seeds produce IAA, which stimulates the development of a fleshy fruit. IAA is produced in actively growing shoot tips and developing fruit, and it is involved in elongation. Before a cell can elongate, the cell wall must become less rigid so that it can expand. IAA triggers an increase in the elasticity of cell walls, allowing elongation to occur. Auxin is able to do this by promoting the intake of water, by the stimulation of hydrogen secretion from the cells into the cell walls, lowering the pH of the cell wall. This increases the elasticity of the cell wall.

IAA has different effects on different plant organs:

Low concentrations of IAA - stimulate root growth and have no effect on the shoot.

Higher concentrations of IAA - stimulate shoot growth but inhibit root growth.

Below is the experiment as it was carried out in the lab:

The Experiment

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Week 1: Setting up the experiment

Materials & Equipment:

• Radish (Raphanus sativus) seeds
• IAA solution (0.01% w/v) 

[Note: IAA is insoluble in water. To make a 0.01% soln., weigh out 0.1g of IAA powder and place it in a glass beaker. Then add 2ml of ethanol and stir to dissolve. Add 800ml of distilled water and heat to 80 degrees C for 5 mins, to evaporate the ethanol. After cooling at room temp., plave in a volumetric flask and make up to 1L with distilled water.]

• Distilled water
• 8 small graduated bottles
• Thermometer
• Beaker
• 8 Petri dishes
• 8 circular acetate grids
• 8 filter papers
• Absorbent cotton wool
• Adhesive tape
• Incubator (25 degrees C)

Procedure:

1. Firstly, the Petri dishes and tubes are labelled as follows:

10^2 ppm, 10 ppm, 1 ppm, 10^-1 ppm, 10^-2 ppm, 10^-3 ppm, 10^-4 ppm, distilled water control.

Then using serial dilutions we can examine the effects of different concentrations of auxin on radish seed growth and development.

Serial dilutions is the method by which the concentration of a solution is decreased along a line of test tubes. This allows us to examine the effects of different concentrations of IAA on the seeds.

Fig. 1: Serial dilution method

                                    

The serial dilution is carried out as follows:

• Add 10 ml of 0.01% w/v IAA solution to the 1st tube.
• Add 9 ml of distilled water to the next 7 tubes.
• Using a Pasteur pipette, remove 1 ml of the IAA soln. from the 1st tube and add it to the 2nd tube. Place the cap on the tube and mix.
• Then using a clean Pasteur pipette, remove 1ml of soln. from the 2nd tube and add it to the 3rd tube. Place cap on and mix.
• Using a clean Pasteur pipette each time, repeat this serial dilution for the 4th, 5th, 6th and 7th tubes. Discarding 1 ml of soln. from the 7th bottle. Each tube now contains 9 ml of solution.

2. The circular acetate grids are fit inside each lid of each Petri dish and 5 radish seeds are placed along a grid line in each dish as shown in Fig. 2.

Fig. 2: Radish seeds on grid line

3. Then using clean Pasteur pipettes, 2 ml of each soln. is added to the dish with the corresponding IAA soln. label.

4. A piece of cotton wool is then spread, about 0.5 cm thick and to fit each dish, on top of the filter paper in each dish. The remaining 7 ml soln. is then added to the cotton wool in the appropriate dishes.

5. The dishes are left for a few minutes to ensure that the cotton wool absorbs all the soln.

6. Then the base of each dsh is put in place and secured with adhesive tape.

7. The dishes are left standing vertically on their edge to ensure that the roots grow down. 

8. The dishes are them placed in an incubator at 25 degrees C for 2-3 days.

Week 2: Observing and recording the results

When we returned to the lab to observe the results of the experiment, we started by measuring the lengths of the shoots and roots of the seedlings in each dish by using the acetate grids.
We recorded our findings and then we calculated the total length and the average length of the roots and shoots in each dish and recorded it.
We then estimated the percentage stimulation or inhibition of the roots and shoots growth in each dish using the following formula:

% stimulation =   (average length - average length of control) x 100

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                                                  (average length of control)

We recorded the results in Table 1 and 2 below:

SHOOT
Concentration of IAA	% stimulation/inhibition
10^-4	0.12
10^-3	5.73
10^-2	-19.21
10^-1	-87.01
1	-91.17
10	-77.00
10^2	-100

Table 1.

ROOT
Concentration of IAA	% stimulation/inhibition
10^-4	-34.11
10^-3	-68
10^-2	-90.2
10^-1	-87.11
1	-99.8
10	-87
10^2	-100

Table 2.




We also graphed the percentage stimulation/inhibition of shoot and root growth against IAA concentration:

Graph 1: % stimulation/inhibition of shoot

Graph 2: % stimulation/inhibition of root