This is an image of the Broad Beans we used after they were split
Title:
The use of starch agar and milk agar to show digestive activity during seed germination.
Abstract:
During this laboratory session, we set up an experiment to investigate and show the activity of digestive enzymes during germination. For this experiment four soaked broad beans were used; 2 of which remained raw and the other 2 were boiled and four plates were used; 2 sterile starch agar plates (1 Raw: 1 Boiled) and 2 skimmed milk agar plates (1 Raw: 1 Boiled).The sterile seeds were placed in the appropriate plates facing downward in the agar (using aseptic techniques) and the 4 plates were incubated at 18-20˚C for 48 hours. After 48 hours the plates were retrieved and the activity of the digestive enzymes was shown using 2 reagents;
a) Iodine solution showing the starch digestion in the starch plates
b) Biuret reagent showing protein digestion in the skimmed milk plates
Materials and Methods used:
4 soaked broad bean seeds | Boiling water bath |
2 sterile starch agar plates | Thermometer |
2 sterile skimmed milk plates | Incubator (18-20˚C) |
Iodine solution | Forceps |
Biuret reagent | Backed blade |
Disinfectant | Marker |
Sterilized water | Paper towels |
Mild disinfectant solution | Timer |
2 beakers | Burner |
· The bench was swabbed with disinfectant.
· Label one of the sterile starch plates “Raw” and also label the plate with your name and date.
· Label the other starch plate “Boiled” and also label the plate with your name and date.
· Two of the seeds were killed by boiling them for 5 minutes; these will act as the controls.
· Each seed was spilt in half, to separate the cotyledons.
· The seeds were all sterilized by soaking them in a disinfectant solution for ten minutes and then they were rinsed twice using distilled water.
· The forceps which were to be used were sterilized by flaming them and were then allowed to cool.
· With minimal opening of the Petri dish lid, to minimize contamination by microorganisms, the forceps were used to place all the seed halves facing down on the agar in the appropriate places. The forceps were re-flamed and the bench was re-swabbed.
· The plates were incubated at 18-20˚C for 48 hours.
· This procedure was repeated with the milk agar plates.
· After 48 hours, the plates were retrieved and flooded the starch plates with iodine solution.
· These plates were left for 2 minutes and then the iodine solution was poured off.
· The pattern of the starch digestion was observed by holding the plate up to the light.
· The skimmed milk plates were flooded with biuret reagent and left for 2 minutes.
· The reagent was poured off and the pattern of protein digestion was observed by holding the plate up to the light.
Results:
Starch Agar Plates:
The plate with the raw seeds had good growth with radicles growing to a maximum size of 3cm, a root growing to 2cm and I also got growth of a 1cm plumule. The plate with the boiled seed had no growth. When the starch agar plates were retrieved a pale, aqueous iodine solution was poured over the plates to show where the starch has been hydrolyzed. Only 4 small seed shapes remained clear indicated by a colorless halo around the seed halves. The starch agar plate that had the boiled seeds in it turned blue/black when the iodine was added. The starch plate agar with the unboiled seeds remains yellow/brown underneath where the beans had been.
Skimmed Milk Plates:
On the plate with the boiled seeds, there was no germination as when the Biuret reagent was poured onto the plate the plate stayed brownish in color. The plate with the raw seeds had only slight germination. There was evidence of small radicles which were pale white in color. When the Biuret reagent was poured onto the plates most of the agar turned green due to a sulphur compound but there were 4 small clear areas proving that the bean seeds had absorbed some of the protein to help growth. Trypsin from the embryo will digest protein in the milk.
A Bean seed contains a lot of starch. Starch is insoluble. When the seeds begin to germinate, the enzymes amylase is secreted. This breaks the starch into maltose, which is soluble. The maltose can then be absorbed by the growing embryo, which can break it down to glucose. The seedling can then use it to supply energy for growth, and also to build up cellulose to make up cells walls for the new cells that are made as the seeding grows.
The results of the treatments of the starch agar plates show that when the embryo begins to germinate amylase is produced, whereas this is not the case in boiled seeds because the boiled seeds are dead and unable to synthesize proteins. The amylases in the seeds had diffused out and converted the starch around them into maltose. The rest of the dish went blue/black, but not the maltose. In relation to the milk skimmed plates, the raw seeds can synthesize proteolytic enzymes which can digest the milk protein in the agar.
