Use of starch agar and milk agar to show digestive activity during seed germination.

Fig 1 : Diagram of enzyme activity in seed.

Back round information.

As a seed growths there are both physiological and metabolical changes that occur. These changes all occur in the seeds cells and tissue.The growth of seeds requires raw materials and energy and this comes form the expense of energy rich molecules eg. starch and lipids stored in the seeds cotyledons and endosperm.

 The first sign of growth seen in seedlings is when it intakes water in the right enviornment. If there is no water close to the seed the seed has the capability to create a small magnetic field in which it attracts water. What is usually observed is cell expansion of course this can probably only be seen under a microscope. After the initial growth we see other changes in the plant such as the growth of new cells and tissues and the granule and the pumule. Enzymes in the embryo are the catalyst in the seed to allow seed growth. Without them the food reserves need for growth would not be able to be transported. 

The enzymes break down starch and lipids and allow the seed to grow. The enzymes needed to breakdown the lipids and the starch are created by the release of Gibberelic acid. Gibberelic acid is released after inhibition activates specific genes thus releasing it. The absence of Gibberelic acid acts as a gene represser on a dormant form of growth. The production of hydrolytic enzymes results in the synthesis of new mRNA from DNA template. For example, transcription of the gene encoding for a-amylase, which digests starch in the endosperm, increases the response to gibberellin. thus, it is hypothesised that gibberellins, in this instance at least, are acting as depressors, turning on the genes for a-amylase production. Amylase plays a vital role in the growth of plants. In order for the hydrolysis of starch to become maltose amylase is needed to catalyse it. The effects of this can be seen in the experiment involving the starch agar plate and the raw seeds. The starch that is in the agar plates is digested by the amylase that is produced in the seed. When iodine is poured over the plates they will stain blue/black as a result of the presence of starch found in the agar plate. But under the seed where starch digestion should occur it will be clear as there is no starch left.

Release of amylase in barley.

Role of GA in seed germination.

Materials/equipment and preparation. 

• 4 soaked broad been seeds. Soak broad bean (Vicia faba) seeds in distilled water for two days, and then transfer seeds to a container with damp filter paper for one day allow germination to begin.
• 2 sterile starch agar plates. To prepare 4-5 plates boil 50 ml distilled water in a beaker, stirring continuously. Then mix 1 g agar powder with a little cold distilled water and add to the boiling starch solution, stirring continuously. Pour the starch agar into a conical flask, plugging the top with non-absorbent cotton wool. Sterilise the agar at 121 degreesC for 15 minutes and after cooling a little remove the cotton wool from the top of the flask and flame the top of the flask, then pour 20 ml of starch agar into each of the sterile petri dishes and allow to set.
• 2 sterile skimmed milk plates. ( To prepare 4-5 plates mix 1 g of skimmed milk powder with 50 m of cold distilled water in a beaker. Heat until dissolved but do not boil. Mix 1 g of agar powder with a little cold distilled water. Add the agar suspension to the hot skimmed milk solution, pour into a conical flask, plug the top with non-absorbent cotton wool and sterilise at 121 degreesC for 15 minutes. After cooling pour - 20ml into each of 5 plates using aseptic technique.
• Iodine solution. Dissolve 2 g of potassium iodine crystals in 25 ml water and add 1 g of iodine crystals. Make the total volume of the solution up to 100ml and store in a dark glass bottle.
• Biuret reagent.
• Disinfectant.
• Sterilised water.
• Mild disinfectant solution.
• 2 beakers.
• Boiling water bath.
• Thermometer.
• Incubator (18-20 degreesC )
• Forceps.
• Backed blade.
• Marker.
• Paper towels.
• Timer.
• Burner.

Procedure: 

Fig 2. broad beans.

4 petri dishes were used in this experiment 2 contained agar with milk 2 contained agar with starch. Milk has a lot of protein, casein been the most dominant. Proteases break down the polypeptide molecules to form smaller peptide bonds and aminos that the organism can use.

2 of the plates are used as a control and 2 to examine enzymatic activity.

Fig 3 

2 plates containing milk were labelled raw, and 2 plates containing starch were labelled boiled.

The petri dishes labelled boiled are used as controls. These plates need to be kept sterile and are sealed with parafilm before placing the seeds inside. Vicia faba (broad beans) sees were boiled 5 mins to inactivate/ kill them. An alcohol burner was used to sterilise the microenviornment before opening the sealed plates. The seeds are then cut in half with a sterilised scalpel to separate the cotyledons.

Fig 4.

2 more seeds were cut in half without boiling them. All 4 seeds need to be disinfected for 10 min. After 10 min the seeds are rinsed with distilled water. The sterilised seeds are then ready to be inserted into the media. Sterilise a forceps and allow it to cool before picking up the seeds.

With minimal opening of the petri dishes to avoid microbial contamination from the air, place the seeds face down into the agar. Place 2 halves of the boiled seeds into the milk containing agar and 2 halves into the starch agar. The same applies for the raw seeds 2 halves in the milk agar and 2 halves in the starch agar. Incubate all 4 plates at 18-20 degreesC for 48 hrs. This temperature should induce GA release and hence enzyme activity.

Fig 5.

Helpful hints.

• Take care in moving the agar plates with the seeds, to prevent the seeds from sliding.
• Have extra seeds boiled beforehand to allow for some being damaged during the procedure.
• Label the tops and bases of the plates and keep the labelling small.
• A blunt forceps is preferable to a sharp one as it is less likely to damage the seeds.
• A forceps can be used to hold the seed in place while splitting it with the backed blade.
• Have labelled petri dishes (`Raw' and `Boiled') to hand when splitting the seeds.

Sample experiment

Results:

After the incubation the seeds need to be removed and pour iodine into the starch plates, leave this for 2 minutes and pour of the excess iodine. If iodine is present it will turn the starch present to black colour, observe any areas where the seed was placed for starch digestion. If starch hydrolysed then there is a colourless halo around the seed areas. In this experiment it was observed that in the agar plates containing boiled seeds there was no clearing indicating amylase was not released. Because the raw seeds had germinated it released amylase but the dead boiled seeds were unable to produce enzymes. The skimmed milk plates were flooded with Bieuret agent. The biuret test is a chemical test, a copper iron is reduced to copper(1), which forms a complex with the nitrogens and carbons of the peptide bonds in alkaline solution. A violet colour indicates the presence of proteins. The flooded plates were left for 2 mins and the reagent was then poured off. In the boiled seeds the agar was violet with no clearings indicating no proteolytic enzymes were released. However in the raw seed agar plates there was a clearing around the seed area indicating proteins were degraded by protease.