Practical 3

AMYLASE ACTIVITY IN GERMINATING BARLEY

Question 1:

Weigh 10 germinating barley seeds and record the mass. Using a mortar and pestle, crush the
seeds. Add 10mL of phosphate buffer slowly and crushing the seeds simultaneously. Filter
the mixture through a funnel with filter paper into a measuring cylinder. Record the filtrate
collected. This will be amylase extract. Perform a five-fold dilution by adding 20mL of
phosphate buffer and 5mL of the amylase extract into a measuring cylinder and mix well.
This will be the diluted amylase extract.

Prepare the control by pipetting 5mL of the diluted amylase extract into a test tube and place
it in a boiling water bath for 10 mins. The reason for using the boiled control is to compare
and see the changes to iodine if inactive enzymes were used together with 0.5% starch and
buffer.

Place one drop of iodine into each of the labelled wells in the ceramic test plates. Preparing
the reaction mixture in a test tube by mixing 1mL of 0.5% starch solution and 5mL of
phosphate buffer solution. Add a drop of the amylase reaction mixture to the iodine on the
ceramic plate to test if starch is present.

To prepare amylase reaction mixture, add 1mL of diluted amylase reaction mixture and mix.
Start with the well labelled 0, at 1-minute intervals, using a Pasteur pipette, add a drop of the
amylase reaction mixture into each well until the achromic point has been achieved.
Achromic point is achieved when iodine no longer changes its colour. If the achromic point is
below 5 minutes, reduce the volume of amylase extract added. If the achromic point is above
20 minutes, the amylase extract should be increased, keeping in mind that the volume is to be
constant.

Repeat the experiment using the boiled enzymes, followed by equal amounts of buffer and
starch solution.

To calculate amylase activity, divide 5mg of starch by the product of time taken to reach the
achromic point (mins) and the weigh of barley seeds used (grams), determined by the volume
of amylase extracted and considering the five-fold dilution performed (multiply by 1/5).

(347 words)
Question 2:

Since the concentration of the starch solution is 0.5% (500mg/ 100mL), amount of starch
added to the reaction tube is 1mL, therefore, weigh of starch added to reaction tube is 5mg.

5 mgof starch
=0.402 mg of starch hydrolysed per min
12.43 minutes

1 mL of starch solution 5 mLof amylase extract

× ×1.083 g
8.3 mL of amylase extract 25 mL of diluted amylase extract

¿ 0.026 g of barley tissue

0.402mg of starch hydrolysed per min

=15.41 mg of starch hydrolysed per min per gram of barley tissue
0.026 g of barley tissue

Question 3:

A. The aim of this experiment was to investigate and analyse the amylase activity in
dormant, germinating and growing barley seedlings. The results obtained show that
germinating seeds have the highest amylase activity, followed by whole barley seedlings,
finally, dormant seeds. The rate of amylase activity was determined by the time taken to
reach the achromic point. The shorter the time taken to reach the achromic point, the
higher the amylase activity. Hydrolysis of starch into glucose with the presence of
enzyme amylase provides energy for germinating seeds to grow. Growing barley seeds
have leaves, which help them to photosynthesise (Kura-Hotta et al 1987). During
germination, energy is required, thus, cellular respiration occurs. Breakdown of amylase
produces starch and glucose which then be used to undergo cellular respiration. Dormant
seeds are not germinating, therefore, no amylase activity occurs as starch is not required
(Dehghanpour et al 2001). Abscisic acid being the factor that dormant seeds are not
germinating as it stops the seed from transitioning from embryonic to germinating stage
(Kermode 2005).

The aim of this experiment was well-fulfilled as iodine was chosen to test for the
presence of starch which shows an obvious colour change from brown to dark blue.
Achromic point has reached when iodine in the wells shows a gradient from dark blue to
brown, indicating that as time passes by, starch is hydrolysed. Another additional strength
 was that the experimental data was collected from other groups as well, as different
people perceives colour change differently, the judging of achromic point was
dependable.

One of the weaknesses of this experiment was that the amount of iodine added to the
wells were unequal. One drop from Pasteur pipette does not give the same volume each
time, thus, this might affect the experimental results.

(295 words)

B. One of the ways that could possibly improve this experiment is to drop the reaction
mixture into the wells at 30 seconds intervals instead of 1 minute as this would give a
more accurate result for the achromic point. Besides, instead of using 10 barley seeds, use
20 barley seeds as this could reduce the errors in the case that few of the seeds might not
be suitable to test for its amylase activity as some dormant seeds may still have enzymatic
activity. In addition, instead of using a Pasteur pipette, a 1mL pipette could be used to
ensure all the wells contain the same volume of iodine. The concentration of starch could
be increased eg: more than 2%, as this would give more apparent results in terms of the
colour change in iodine when the starch has been hydrolysed. With this, determining the
colour change of iodine would be easier. The experiment should be repeated for at least
one time to improve the accuracy of the achromic point and to exclude the errors.
(175 words)

References
Kermode AR (2005) Role of abscisic acid in seed dormancy. Role of ABA in The Promotion
of Developmental Processes, Prevention Of Precocious Germination and Induction of Seed
Dormancy 24, 321-322

Dehghanpour FH, Tavakkol AR, Sarifzadeh F, Chavoshinasab S (2011) Germination

improvement in dormant and non-dormant seeds of oregano. Australian Journal of Crop
Science 5(4), 421-422. doi: https://doi.org/10.2136/sssaj2004.0215

Kura-Hotta M, Satoh K, Katoh S (1987) Relationship between photosynthesis and

chlorophyll content during leaf senescence of rice seedlings. Plant and Cell Physiology
28,1321-1329. doi: https://doi.org/10.1093/oxfordjournals.pcp.a077421