Biology Individual Investigation

Effect of increasing concentrations of L-ascorbic acid helping

alleviate the salt stress on germination of Pennisetum glaucum.

BACKGROUND INFORMATION:
India is a country that is geographically bound by oceans on three sides and hence as a result
is being exceedingly prone to costal inundation. Over the years the phenomena of heating
being trapped in the earth due to carbon emissions, burning of fossil fuels and deforestation
also known as global warming is resulting in an increasing rise in temperatures worldwide.
This rise in temperatures world wide is causing glaciers and ice caped mountains to melt. This
melting is causing a rise in the sea level leading to about 20% of the worlds cultivated land
being affected by the salinity and saline soil 1, this number will only keep on increasing. If this
continues, a country like India, which is bounded by water on three sides, the estimate
2
damage done in the year 1994 was 21.7Mha (Million Hectare Area) as the years have
progressed these figures would have only increased. With a population of 1,266,883,598 3 and
growing a significant part of the population is dependent on farming as an occupation it is
essential to sustain this profession. The number of farmers committing suicide due to being
unable to pay back loans as a result of an successful, poor quality and or damaged crop is also
escalating. Salt stress is usually caused due to various reasons; the two main reasons for this
are both natural and man-made. Man-made salt stress occurs due to excess use of fertilizers
while natural salt stress on the other hand occurs due to seawater reaching the soil from the
ocean.

RESEARCH QUESTION:
To what extent does increasing concentrations of antioxidants (Vitamin C/L-ascorbic acid)
help alleviate the salt stress on germination of Pennisetum glaucum (monocot)?

HYPOTHESIS:
If the seeds are exposed to high concentration of antioxidants L-ascorbic acid more will seep

1 ttp://www.eolss.net/sample-chapters/c10/e5-01a-04-05.pdf
2Sehgal and Abrol, (1994) -
http://www.ipublishing.co.in/jesvol1no12010/EIJES2071.pdf
3 http://www.indexmundi.com/india/demographics_profile.html

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in and enter the soil and seeds helping alleviate the effect that the salt stress will have on the
germination of Pennisetum glaucum allowing the crop to grow better and faster despite the
exposure to salty conditions.

VARIABLES:
Independent variable:
Salt Stress:
 NaCl solution with a salinity of 3.5%
Vitamin C
 Vitamin C concentrations of 10-8M, 10-6M, 10-4M, 10-2M
Dependent variables:
Number of seed germinated

 The Dependent Variable, being the number of seeds does not require measurement and
is counted in numbers.

Length of seed germinated:

 The length of the seeds germinated is measured in mm using a 10 cm long ruler.

Controlled Variables:
Water
 Reason to control: The amount of water a seed receives affects the rate of
germination and it is also the source of minerals to help a seed germinate.
 Method to control: All the water was obtain from the same source and water them
at the plants were watered at same time of the day, i.e. 12:00.

Seeds
 Reason to control: The Pennisetum glaucum seeds serve as the basis of the
investigation and it is most important to ensure their quality and source
 Method to control: Seeds from the same batch, same variety was used and it was
ensured that they are stored at the same temperature.

Sunlight

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 Reason to control: the amount of sunlight a plant receives affects its germination
rate of a seedling that is being observed during the course of this investigation.
 Method to control: The four IV settings and the control are kept in a straight line
close to each other, which ensures they get the same amount of sunlight.

Time:
 Reason to control: If some seeds were left to germinate for longer the data
obtained at the end of the investigation would be in accurate. The data was
collected everyday at the dame time hence giving them the same to grow and
leading to accurate data being collected.
 Method to control: The investigation was conducted over duration of 72 hours, and
observations were made after a span of 12 hours.

Pretreatment of Pennisetum glaucum Seeds:

 Reason to control: Different incubation time periods will lead to different levels of salt
being absorbed which will result in
 Method to control: Use the same stop watch for all the replicates carried out to ensure
the pretreatment time is the same.

Uncontrolled Variables:
 Pressure
 Temperature
However, the 4 IV settings and the controlled variable were kept at a small distance from one
another in the same area so the pressure and temperature will be the same.

APPARATUS:
Sample
1. Pennisetum glaucum seeds X300
Chemicals:
1. NaCl powder
2. L-ascorbic acid powder

TABLE 1: GLASSWARE
NAME QUANTITY CAPACITY
Beaker 5 100 cm3

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Measuring Cylinder 2 10 cm3

Petridish 5 -

TABLE 2: MISCELLANEOUS REQUIRMENTS

NAME QUANTITY CAPACITY
Digital weighting 1 -
balance
Cotton 1 roll -
Measuring scale 1 -
Syringe 5 10 cm3
Dropper 1 -
Stirrer 1 -

PROCEDURE AND METHODOLOGY:

Preparation of NaCl solution
1. Keeping in mind that salt water has a salinity of 3.5%4 the NaCl solution prepared
must have the same concentration.
2. Measure 3.5 grams of NaCl powder using a weighing balance
3. Measure 1000 cc of water using a measuring cylinder
4. Using a stirrer mix the NaCl into the water and stir until the salt is dissolved.
Preparation of Vitamin C solution
1. Measure 1.76 grams of Vitamin C powder in 10 ml of tap water to form the stock solution
of IM concentration
2. Using the process of serial dilution method (1:100) take 1ml of the stock solution to 99ml
of distilled water to form a 10-2 M solution.
3. Step 2 Vitamin C is repeated to obtain different Vitamin C solutions of different Vitamin C
concentrations i.e. 10-4 M 10-6 M 10-8 M solutions
Pretreatment of Pennisetum glaucum Seeds
1. Take 300 Pennisetum glaucum seeds and soak them in 3.5% NaCl solution
2. Let the sample soak in the solution of duration of 12 hours.
Vitamin C and pretreated seeds
1. Add 10 pretreated Pennisetum glaucum seeds to each beaker or container
10-2M, 10-4M, 10-6M and 10-8M solution.
4 https://www.sciencedaily.com/terms/seawater.htm

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1. Add 10 pretreated Pennisetum glaucum seeds to each breaker or container.

2. Incubate the solution at room temperature.
3. After 72 hours measure the length of the seedlings and calculate the mean of all the 10
Pennisetum glaucum seeds per concentration to be used for analysis.

SET UP:
 The 4 IV settings and the control variable were kept in petridishes on cotton.
 Each petridish was covered with a lid and left in the sunlight to germinate.

IMAGE 1: Set up for the investigation.

SAFETY AND ETHICAL CONSIDERATIONS:

The investigation conducted does not cause any allergic response to the human body neither
was any species endangered nor is a side effect of Vitamin C on health that need to be
addressed. However, whilst preparing the salt and Vitamin C it would be best if gloves and the
required apparatus were used.

RESULTS:
Qualitative Observations:

IMAGE 2: Samples of concentrations-10-2M and 10-8M

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Figure 2 shows the plant with the highest concentration of L-ascorbic acid grew the best, the
shoot looked way healthier than the others that received lower concentrations.
The sample with highest concentration of L-ascorbic acid has bright green leaves
and a long shoot while in the other sample not all the seeds germinated, the shoot
did not bear leaves and the shoot length was much shorter.

Quantitative Observations:
TABLE 3: RAW SEEDS: LENGTH
Length of seeds germinated (mm) ±0.05cm3
TRIAL Control Sample 1 Sample 2 Sample 3 Sample 4
-8 -6 -4
10 M 10 M 10 M 10-2M
1 12 12 14 13 18
2 13 12 15 16 18
3 13 11 15 14 15
4 14 13 16 16 17
5 13 12 15 16 18

TABLE 4: PRE-TREATED SEEDS: LENGTH

Length of seeds germinated (mm) ±0.05cm3
TRIAL Control Sample 1 Sample 2 Sample 3 Sample 4
-8 -6
10 M 10 M 10-4M 10-2M
1 1 2 3 5 8
2 2 2 4 6 8
3 3 3 4 6 9
4 2 1 4 5 8
5 2 2 5 6 7

TABLE 5: RAW SEEDS: NUMBER OF SEEDS GERMINATED

Total number of seeds germinated
TRIAL Control Sample 1 Sample 2 Sample 3 Sample 4
-8 -6
10 M 10 M 10-4M 10-2M
1 5 2 1 3 4
2 5 1 2 3 4
3 4 1 2 4 4
4 5 1 1 4 3
5 3 2 2 3 4

TABLE 6: PRE-TREATED SEEDS: NUMBER OF SEEDS

GERMINATED
Total number of seeds germinated

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TRIAL Control Sample 1 Sample 2 Sample 3 Sample 4

10-8M 10-6M 10-4M 10-2M
1 3 1 3 4 4
2 4 1 2 5 5
3 3 0 3 5 5
4 3 1 3 4 4
5 5 2 2 5 5

TABLE 7: MEAN LENGTH OF SEEDS GERMINATED (mm)

SAMPLE PRE-TREATED SEEDS RAW SEEDS
Control 2 13
Sample 1 2 12
10-8M
Sample 2 4 15
10-6M
Sample 3 6 16
10-4M
Sample 4 8 18
10-2M

TABLE 8: MEAN OF NUMBER OF SEEDS GERMINATED

SAMPLE PRE-TREATED SEEDS RAW SEEDS
Control 4 5
Sample 1 1 1
10-8M
Sample 2 2 3
10-6M
Sample 3 3 2
10-4M
Sample 4 5 5
10-2M

TABLE 9: GERMINATION PERCENTAGE

SAMPLE PRE-TREATED SEEDS RAW SEEDS
Control 72% 88%
Sample 1 20% 28%
10-8M
Sample 2 48% 32%
10-6M
Sample 3 52% 52%
10-4M

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Sample 4 92% 76%

10-2M

Microsoft Excel was used to calculate the standard deviation for the values in the tables
above.
IMAGE 3: Screenshot of Excel

GRAPHICAL REPRESENTATION:

Mean length of seeds germinated (mm)

30
Length of seeds germinated (mm)

25

20

15

10

0
Control Sample 1 10-8M Sample 2 10-6M Sample 3 10-4M Sample 4 10-2M

Concentration of L-ascorbic acid

PRE-TREATED SEEDS RAW SEEDS

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Mean number of seeds germinated

12
Number of seeds germinated

10

0
Control Sample 1 10-8M Sample 2 10-6M Sample 3 10-4M Sample 4 10-2M
Concentration of L-ascorbic acid

PRE-TREATED SEEDS RAW SEEDS

Germination Percentage
180%
160%
Germination Percentage

140%
120%
100%
80%
60%
40%
20%
0%
Control Sample 1 10-8M Sample 2 10-6M Sample 3 10-4M Sample 4 10-2M

Concentration of L-ascrobic acid

GERMINATION PERCENTAGE PRE-TREATED SEEDS

GERMINATION PERCENTAGE RAW SEEDS

EVALUATION:
Strengths:
 The experiment was repeated multiple times with the same variables and controls to
make the data as accurate as possible.
 The investigations set up was simple and data was easy to collect.
 Majority of the variables have been controlled to minimize errors and increase
reliability
 The research papers referred to suggest the investigation would be successful
Limitations:

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 There was scope for an error to be made while taking readings of the shoot length
using a meter rule.
 A mistake could be made while counting the seeds and calculating the average and
germination percentage due to the large number of seeds.
 Variables like temperature, pressure, sunlight, and water can differ for each sample.
 The exact amount of L-ascorbic acid per sample could not be determined.
Errors:
Random Errors:
These errors are the ones that occur while using lab equipment’s for instance when using a
rule, beaker and measuring cylinder. These errors in apparatus could lead to inaccurate
readings being taken resulting in experimental inaccuracy.
TABLE 10: STANDARD DEVIATION IN APPARATUS USED
APPARATUS STANDARD DEVIATION
Beaker ±1 cm 3
3
Measuring cylinder ± 0.1cm
Digital weighing balance ± 0.01cm3
3
Measuring ruler ± 0.05 cm
Systematic Errors:
 The possibility of parallax errors is very high due to the use of apparatus in every
stage of the investigation despite taking the readings slowly and immense care
 The amount of water, Ascorbic acid and salt poured must not be exactly the same.
Improvements:
 The intermediate values should be considered.
 Repeated replicates should be done to reduce the scope of errors to the maximum
possibility to obtain data as accurate as possible
 Parallax errors should be avoided by placing the ruler flat on a sturdy surface and
readings from measuring cylinders should be taken at eye level.

FURTHER SCOPE:
Costal inundation is a problem that is leading to many farmers loosing their source of income
and multiple suicides across a large developing nation like India. If this research is done on a
larger scale and orange peels’ ascorbic acid content is resourcefully used and investigation is
conducted the crop yield in India can increase by a large scale and crops will be available in
abundance. This experiment on a much larger level can also extend itself to the effect of
multiple determinants on germination and plant growth, however, correlation does not imply
causation. On a smaller scale this experiment design with moderations could be tested to

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further complement this data one could study the effect of such salt stress and Vitamin C on
various other seeds and plants that are similar in nature to Pennisetum glaucum .

CONCLUSION:
The research conducted through this investigation shows that high concentration of Vitamin C
helps seed germination by helping overcome the inhibitory effect that salt stress has on the
Pennisetum glaucum seeds kept under observation for a period of 72 hours.

BIBLIOGRAPHY:
http://www.kew.org/science-conservation/plants-fungi/pennisetum-glaucum-pearl-
Pennisetum glaucum
 "WHat Is the Uncertainty in a Metre Rule?" Physics Forums. N.p., n.d. Web. 21 Sept. 2016.
 http://www.knockhardy.org.uk/sci_htm_files/08error.pdf
 http://scholarworks.umass.edu/cgi/viewcontent.cgi?article=1035&context=jmap
 http://www.arpnjournals.com/jabs/research_papers/rp_2015/jabs_0415_720.pdf
 http://www.halophyte.org/pdfs/drkhan_pdfs/178.pdf
 https://www.ukessays.com/essays/biology/effect-of-different-concentrations-of-vitamin-c-
solution-biology-essay.php

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