Production of amylase in germinating cereal grains


Independent variable: Concentration of gibberellins solution
Dependent variable: Area of clear zone created


Background information


Cereal grains contain a store of starch which is insoluble. This needs to be transported to the

embryo so it has a supply of energy for growth. In order to be transported across the grain, the

starch needs to be made soluble which occurs when the developing embryo releases a hormones

called gibberellins which stimulate other cells causing the release of amylase - the enzyme that

digests starch.

In this experiment, the cereal grains will be soaked in different concentrations of gibberellins, thus

causing different amounts of amylase to be released and so a different amount of starch

digested. The starch digestion can be observed when the agar plates containing the soaked seeds

are washed with potassium iodine in iodide solution which turns blue black in the presence of

starch. Therefore, the more starch that is digested, the larger the clear zone around the

seeds will be created.


Equipment list


  • Gibberellic acid solution (1g/dm3)
  • Distilled water
  • Pipettes
  • Cereal grains
  • Small bottles
  • Scalpel
  • White tile
  • Sodium hypochlorite solution
  • Stopwatch
  • Sterile water
  • Muslin or gauze
  • Sterile forceps or tweezers
  • Petri dishes containing starch agar jelly
  • Tape

  • Marker pen
  • Iodine in potassium iodide solution
  • Ruler


Method


Before the experiment the different concentrations of gibberellins must be made up using varying

volumes of distilled water and gibberellin acid.




Day 1

1. Use a pipette to add the prepared solutions to each small sample bottle and label the bottle

with its concentration, using a marker pen.

2. Collect the number of seeds you need for the experiment and use a scalpel to cut them in half

as shown in the diagram. Discard the embryo halves.

3. Place the endosperm halves in a solution of sodium hydrochlorite for 5 minutes to sterilise

them.




4. Now rinse the seeds 5 separate times through with sterilised water, draining them 

carefully using a muslin after each wash.

5. Use tweezers to place 3 seeds in each gibberellin solution and leave them to soak for 24-48

hours, place a lid on the solution bottles but leave it slightly unscrewed to allow oxygen

to enter.


Day 2


6. After the seeds have been soaked take 6 Petri dishes and label each one with a different

gibberellin concentration and use sterile tweezers or forceps to place the 3 seeds soaked in

each bottle to the Petri dish labelled with the same concentration.

7. When placing the seeds in the Petri dish be sure to place them with the cut face down.

Use adhesive tape to secure the lids of the dishes in place at 2 points.

8. Incubate the Petri dishes for 24-48 hours.


Day 3


9. Take the incubated plates and one at a time slightly open the lid and use a syringe to pour

potassium iodine in potassium iodide solution on the surface of the agar plate.

10.Where starch is present the solution will turn blue black, where the starch has been

digested it will leave a clear zone.

11. Measure the diameter of the clear zone created for each seed and record it in a suitable

table.





To calculate the area of the clear zone use the formula:

Area = 几r2