14 - Gibberellin on the production of amylase

What is the role of gibberellin during germination?

It is secreted by the embryo, then diffuses to the aleurone layer of the endosperm to stimulate the production of amylase, which hydrolyses starch to maltose

What equipment is needed for this practical?

• Seeds

• Sodium hypochlorite solution

• Beaker, scalpel & forceps

• Sterile containers (to soak seeds)

• Tile

• 1M gibberellic acid

• Distilled water

• Petri dishes

• Adhesive tape

• Potassium iodide solution

• Measuring cylinder 

What is the method for this practical?

1. Dilute the stock gibberellin solution to several set concentrations & place in labelled sample bottles

2. Cut seeds in half & use only the half with the endosperm (discard the half with the embryo)

3. Sterilise the seed halve by placing it in sodium hypochlorite solution & leave for 3 minutes

4. Wash the seed halve with distilled water 5 times until there is no smell of bleach

5. Place the seed halves in the gibberellin solution & leave for 12-48 hours. Leave lids of sample bottles loose to prevent conditions becoming anoxic

6. Use sterile forceps to move seed halves onto a sterile petri dish with starch agar. Partially tape the lids (prevents conditions becoming anoxic) & leave for 12-48 hours

7. Pour potassium iodide onto the plates & measure the clear zone around the seed half

What does the clear zone suggest?

Starch has been hydrolysed by amylase into maltose, hence not stained by iodine → the larger the clear zone, the higher the amylase concentration

Why are the seeds placed in sodium hypochlorite solution?

To sterilise the seeds

What is the effect of gibberellin concentration on amylase production?

Increasing the gibberellin concentration increases the area of the clear zone, suggesting an increased production of amylase & more starch hydrolysed into maltose

What are some sources of error in this practical?

The existing gibberellin & amylase content of the seeds may be different

What are some control variables of this practical?

• Time allowed to soak in gibberellin

• Time left on starch agar plate

• Source & age of seeds

What is the risk, safety precaution & in emergency for the hazard: biohazard

Risk:

• allergies, soil bacteria & contamination

Safety precaution:

• wash hands after practical

In emergency:

• seek assistance

What is the risk, safety precaution & in emergency for the hazard: scalpel

Risk:

• cuts from sharp object

Safety precaution:

• cut away from fingers

• use forceps to hold sample whilst cutting

In emergency:

• elevate cuts & apply pressure

• seek medical assistance

What is the risk, safety precaution & in emergency for the hazard: sodium hypochlorite/gibberellic acid

Risk:

• irritant

Safety precaution:

• wear eye protection & gloves

In emergency:

• wash eyes & skin with cold water

What type of graph should be plotted?

Gibberellin concentration against area of clear zone

What is the conclusion from this practical?

• As gibberellin concentration increases, the amount of amylase produced by the aleurone layer increases

• So, the area of starch from the agar around the seed half that is hydrolysed into amylose also increases

Why does a clear zone appear around the seed half when iodine is added to the agar plate?

Iodine stains starch blue/black, so the area where starch has been hydrolysed appeared as a clear area around the seed half