How Do Altering Salicylic Acid Concentrations (0.0%, 0.5%, 1.0%, 1.5%, 2.0%) Affect The Germination

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Title: Investigating the effect of salicylic acid concentrations (0.0%, 0.5%, 1.0%, 1.5%, 2.0%) on the
germination of Capsicum annuum longum seeds by measuring the radicle length (mm) of the sprouts over
7 days.
Research Question: How do altering salicylic acid concentrations (0.0%, 0.5%, 1.0%, 1.5%, 2.0%) affect
the germination of Capsicum annuum longum seeds measuring the radicle length (mm) of the sprouts of
over 7 days?
1. Introduction
I’m strongly engaged with the Turkish culture, and a big part of our culture is
food. One of the most important ingredients used in our food culture is a Turkish
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cultivar of Capsicum annuum longum (Capsicum annuum l.) “sweet pepper”,
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known as “pointed” pepper in Turkey. “Pointed” peppers are important as they
show the most phenolic content, high amounts of Vitamin C, and antioxidant
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capacity (Frary et al. 631). In many areas of Turkey drought is being faced and as
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a result, the agricultural sector is regressing. Plants are facing innumerable
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Figure 1: Capsicum
challenges: such as underwatering and lack of humidity. Therefore, there are
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annuum l. seeds
fewer agricultural products produced and exported. Turkey’s biggest economic (“Capsicum Annuum”)
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income source is usually from the agricultural sector. However, the agricultural
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sector is continuously decreasing. This decrease has extremely worried me. Increasing the germination rate
of seeds is often discussed to improve the agricultural sector. On the other hand, several experiments reveal
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that salicylic acid (SA) affects plants' growth (Raskin 440). Several sources also show that SA may have
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important roles in plants such as DNA repairing, fruit yield, germination, abiotic stress tolerance, and
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potential germination rate (Koo et al. 2). However, there aren’t any findings which investigate the
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relationship between SA and seed germination. This is crucial since seed germination is one of the most
important stages of plant growth and is extremely important regarding crop yield and crop quality.
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Therefore, understanding different factors that affect germination rate held great importance to improve
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the agricultural sector. This study aims to determine the relationship between various concentrations of
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salicylic acid and the germination rate of C. annuum longum seeds. The findings may possibly contribute
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to the agricultural sector if optimum concentrations of the acid are found to increase germination rate, and
hence hold importance. Therefore, the aim of this investigation is determining the relationship between
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salicylic acid concentrations and find the optimum concentration(s) that will result in optimum radicle
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length and hence germination.

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2. Investigation
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2.1 Background Information:

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Enzymes are biological catalysts that speed up the rate of biochemical reactions by lowering the activation
energy (the minimum energy required to initiate a reaction) and producing an alternative pathway (Allot
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and Mindorff 96). For instance, germination is also a process that is controlled by enzymes as the enzymes
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control the imbibition of water for the development of the radicle. Without enzymes germination can’t
continue. Germination is defined as the emergence of the radicle via seed coat (Joshi 1481). Therefore,
radicle elongation is a factor which determines germination. On the contrary, inhibitors slow down or stop
enzymatic rate activity by binding to enzymes. There are 2 types of reversible inhibitors: competitive and
non-competitive. Competitive inhibitors bind to the enzyme's active site since they resemble the shapes of
substrates, which prevent the substrates from binding to the active sites, so enzymatic activity is slowed
down (Ahern and Rajagopal 1). Non-competitive inhibitors, on the other hand, function differently. They
don’t resemble the shape of substrates, instead they bind to the allosteric site of the enzyme hence altering
the shape of the enzyme, so substrates don’t recognize the active site (Ahern and Rajagopal 2). Therefore,
increasing substrate concentration doesn’t affect enzyme activity with non-competitive inhibition, unlike
competitive inhibitors.
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Salicylic acid is a hormone that plays a role in plant defense, resistance, and
immunity that is an example of a competitive inhibitor. Due to these
effects of this hormone on plants, it’s also called “phytohormones” (Koo et
al. 1). When looking at its metabolic pathway, the acid inhibits enzymes
such as peroxidase and polyphenol oxidase, which are involved in the
debilitation of plant defense (War et al. 1789). It also inhibits catalases and
pathogen growth to provide plant immunity. Due to this pathway, plants can
successfully survive under biotic and abiotic conditions. Moreover, SA
affects plant growth, photosynthesis, glycolysis, and flowering (Klessig and Figure 2: Structural formula of
Jocelyn 1439). On the other hand, SA has a molecular formula of C7H6O3 salicylic acid (“Salicylic Acid”)
and is a carboxylic acid belonging to the “plant phenolics” group, with an
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aromatic ring and a hydroxyl group. The hydroxyl group increases the
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compound’s solubility properties.
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2.2 Hypothesis:
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H1: As salicylic acid concentration increases; the radicle length will rapidly increase and there will be a
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positive relationship. At high concentration, the radicle elongation will start to decrease. This hypothesis
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can be supported by a past experiment where optimal salicylic acid concentrations resulted in an increased
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growth rate and high acid levels negatively affected plant growth (Koo et al. 6).
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H0: Increasing concentrations of salicylic acid have no effect on the germination rate of Capsicum. annuum
l.
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3. Methodology
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3.1 Experimental Variables:

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3.1.a Independent Variable: Concentration of salicylic acid (0.0%, 0.5%, 1.0%, 1.5%, 2.0%)
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Concentration Mass of Volume of

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of salicylic acid salicylic acid water (ml)

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(%) (g) (± 0.1 g) (±1 ml)

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0.00 0.00 100

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0.50 0.50 100

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1.00 1.00 100

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1.50 1.50 100

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2.00 2.00 100

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Table 1: Mass of salicylic acid required for the needed amount of concentration in the
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solutions [Given in 2 decimal places]
Same volume of water was used with different masses of salicylic acid to compare the different
concentrations. The required mass was calculated prior to starting the experiment. Concentration was used
as a unit to make the calculations simpler. To observe precise values, concentration intervals were
determined evenly. Using a concentration of 0.0% provides a control group, which can be used to distinctly
identify the independent variable’s impact on the dependent variable, the germination rate. Concentrations
were also chosen considering past experiments. In a past experiment, the salicylic acid's inhibitory effect
peroxidase activity started at 0.2% concentration (Raskin 449). Also, high levels of concentration (>
%1.50) started to affect plant growth negatively (War et al. 1789). A concentration of 2.00% as the
maximum was still chosen, to observe the germination when the optimal concentration was exceeded.
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3.1.b Dependent Variable: The germination over a period of 5 days, determined by measuring the radicle
length (mm) of the Capsicum annuum l. sprouts. Mean germination time could also be chosen as a
dependent variable to determine germination. However, it wasn’t chosen as to read in an article, mean
germination time is not an appropriate method in calculating germination. It leads to mistakes in post
statistical tests since it doesn’t show the time to a specific germination percentage (Soltani et al. 4).
3.1.c Controlled Variables: The controlled variables are demonstrated below in Table 2:
Controlled Reason of Control Method of Control

Variable
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The acid’s inhibitory activity is dependent upon pH. The pH was measured for
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pH of the Different solutions of the pH result in different inhibition each trial of each
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solution rates and the data would significantly vary. Neutral form of concentration every day using
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SA shows the greatest germination activity (Raskin 443). a pH probe.
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Because different seeds have different genetic The same packages of
Genus and characteristics, they have different optimal conditions that Capsicum annuum l. were
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species of the affect germination rates differently. This means that their bought from the same
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seed (Seed type) radicle elongation and germination rates would vary
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significantly.
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The number of Using a set number of seeds ensures that the data collected is
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seeds reliable. Inaccurate conclusions can be drawn about the Number of seeds was counted
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effect of salicylic acid concentration on plant growth since by hand before each trial.
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more plant numbers indicate more sprouting.

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Studies show that seed size has a significant effect on seed Seeds that had similar sizes
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Size of seeds germination, where large seeds generally show the most were chosen and seeds were
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(mm) rapid germination rate and develop into large seedlings more measured before as
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quickly (Saeed and Shaukat 292). approximately 5.0 mm (±0.5

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mm).
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Prior to preparing the

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solutions, 100 ml of water

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Amount of water Using different amounts of water gives solutions that are was measured using a 200 ml
(ml) more/less dilute than each other. The only factor that graduated cylinder (± 2 ml)
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determines the concentration of the solution must be amount for each trial and
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of salicylic acid for the results to be valid for comparison. concentration. Same amount
of water (5 ml) was
additionally added to the
spray tubes every day.
Source of water impacts growth rate. In a past experiment, Distilled water was used
there were different germination rates using distilled and tap throughout the whole
Source of water water (Emerson and Lewis 809). If water from different experiment.
sources is used, there could be false conclusions made for
the effect of salicylic acid on germination rate since the
particles in tap water can interfere with inhibitory activity.
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The time should be controlled since germination will

continue if the time is extended. Therefore, the impact of the The experiment was ended
Time (days) calculations on the germination rate will be less accurate. after 7 days at the exact same
It’s also important to ensure that the reason why radicle time as it started (9:00 a.m.)
elongation & germination rate are different is because
salicylic acid concentrations, not because the time periods
are different.
Moisture absorption will differ with using different seed Cotton pads were used for
Seed germination germination mediums. Using the same germination medium each trial and each
medium is important to reduce other factors that affect the concentration.
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germination rate of seeds.
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Table 2: Table containing the controlled variables of the experiment with their reasoning and
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method of control
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3.1.d. Uncontrolled Variables: The uncontrolled variables of the experiment are demonstrated
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below in Table 3:
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Uncontrolled Significance & Explanation
Variable
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Past experiments suggest that fluctuating temperatures affect germination rate (Koger
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et all. 989). Optimum temperatures of Capsicum annum l. seed germination are
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between 21°C and 27°C (Whitlock 1). If temperature fluctuation is too significant,
germination rate will differ, leading to incorrect data collected. Temperature is also
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important to control because it affects enzymatic activity. Each plant has optimum
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Temperature (°C) temperature for its growth rate. Though the seeds were placed next to a windowsill,
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room temperature couldn’t be kept constant since the setup had to be left unattended
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during the weekends and the windows were often closed and opened. The room
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temperature was measured using a thermometer and was generally between 20°C and
25°C.
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An experiment in an article has shown faster germination rate in higher light

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Light availability intensities (Emerson and Lewis 812). In the experiment, the seeds relied on sunlight
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as a source of energy as they were placed next to windowsills. and there is no way to
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control sunlight intensity, as it varies every day.

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Table 3: Table containing the controlled variables of the experiment with their reasoning and
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method of control
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3.2 Materials & Apparatus

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1x Safety goggles 1x VELP® Heating magnetic stirrer

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10x Gloves 1x Magnetic needle

1x Laboratory coat 1x Stopwatch (± 0.1 s)
1x Thermometer (±0.3°C) 1x Pen
550 mL of Distilled water 5x Spray bottles
2x 250 mL Graduated cylinder (±2 mL) 250x Capsicum annuum l. seeds
1x 150 mL Beaker (±5 mL) 1x Ruler (± 0.5 mm)
5g of Salicylic Acid 25x Petri dish
1x Electric mass balance (± 0.1 g) 25x Cotton wool pad
1x Watch glass 1x Tweezer
1x Spatula
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3.3 Formation of the Experiment

A preliminary experiment was conducted to achieve feasibility of the main experiment and pre-identity
potential problems. It was concluded that the seeds are successful of germination. Through the preliminary
experiment and existing research, the concentration of salicylic acid to be used was determined. The
experiment was mainly adapted from an article in “Landes Bioscience” by War et al. The study presented
the effect of salicylic acid to antioxidant enzymes (POD and PPO) in Cicer arietinum (chickpea) plants,
which are a type of legume. There was no existence on the effect of salicylic acid concentration on a plant
from the Nightshade family, hence Capsicum annuum l. was chosen as the seed. Salicylic acid has been
chosen as an independent variable to investigate since it has been suggested to affect growth under stress
through nutrient uptake, flowering, and stomatal regulation in plants (War et al. 1787). 0.0% concentration
solutions were used for treating plants with water as a control group to make more accurate comparisons
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and conclusions conduct statistical tests on growth. As read in an article, concentration differences were
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chosen to be small since high concentrations (>1.5%) negatively affected plant growth (Koo et al. 6). Since
the chosen concentrations of salicylic acid are inexpensive and available for commercial purchase, the
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findings can be applied to the economical & agricultural sector. Another finding in the preliminary
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experiment was that salicylic acid can’t easily dissolve in water. Therefore, a heating magnetic stirrer was
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decided to be used and putting a magnetic needle inside it further speeded up the dissociation rate.
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3.4 Procedure
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1. Preparation of the SA Solutions
1.1: Gather materials on a spacious, clean environment, and ensure that the risk assessments are
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complete.
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1.2: Measure the needed amount of distilled water (mL) using the graduated cylinder, referring to
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Table 1 [see page 3].

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1.3: Pour the distilled water inside the beaker.

1.4: Measure the required amount of salicylic acid using the scale, watch
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glass and spatula, referring to Table 1 [see page 3].

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1.5: Pour the salicylic acid inside the beaker with distilled water.
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1.6: Plug in the power cord of the heating magnetic stirrer and turn it on.
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1.7: Place the beaker on the countertop.

1.8: Put the magnetic needle inside the beaker.
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1.9: Adjust the heating to 250°C, and the stirrer to 9.5. Simultaneously, start
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the stopwatch and continue for 10 minutes.

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1.10: Turn the heating magnetic stirrer off and simultaneously stop the
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stopwatch.
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1.11: Using the pen, label the spray bottles according to the varying
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concentrations (0.0%, 0.5%, 1.0%, 1.5%, 2.0%).

1.12: Take the beaker from the heating magnetic stirrer using the tweezer. Figure 3: Picture of the beaker
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in the heating magnetic stirrer

1.13: Pour the solution from the beaker inside the corresponding spray bottle.
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[Note: For the 0.0% salicylic acid concentration, only steps 1.1-1-4, 1.11 and 1.13 should be
followed.]
[Note: Prior to using the graduated cylinder & beaker for each new concentration, clean it with
distilled water and later rinse it with a napkin.]
2. Preparation of the Petri Dishes
2.1: Measure 10 Capsicum annuum l. seeds that are 5 mm using the ruler.
2.2: Put the Capsicum annuum l. seeds inside the beaker and leave them overnight with distilled
water.
2.3: Put a cotton wool pad inside the petri dish.
2.4: Take the Capsicum annuum l. seeds from the beaker and place them on the surface of the cotton
wool pad.
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2.5: Label the petri dish to its corresponding salicylic acid concentration (0.0%, 0.5%, 1.0%, 1.5%,
2.0%) using the pen.
2.6: Repeat steps 2.1-2.5 for 5 trials.
2.7: Repeat steps 2-1-2.6 for all salicylic acid concentrations (0.0%, 0.5%, 1.0%, 1.5%, 2.0%)
2.8: Place all dishes close to each other in a windowsill, where the sun is available.
3. Collection of data
3.1: For 7 days, spray each cotton pad with its corresponding salicylic acid concentration (0.0%,
0.5%, 1.0%, 1.5%, 2.0%) at 9:00 am, using the spraying bottles.
[Add 5 mL of distilled water to the spraying bottles everyday.]
[Ensure that the solutions are sprayed evenly.]
[Shake the spray bottles prior to spraying.]
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3.2: After 7 days, count how many seeds have successfully
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germinated.
3.3: Extract the germinated seeds that have formed sprouts using
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the tweezer and place them straight and upright onto a flat
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surface.
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3.4: For each sprout, measure the radicle length using the ruler. Figure 4: Self-made diagram demonstrating the
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part of the sprout that should be measured
[Refer to Figure 5.]
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[Ensure that the only the radicle is measured, not the shoot.]
3.5 Safety, Environmental & Ethical Considerations:

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Safety Considerations: Don’t allow smoking, eating, and drinking inside the laboratory. The hazard
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statements of salicylic acid show that skin contact should be avoided (“Safety Data Sheet”). Therefore,
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gloves should be worn. If the skin gets contact skin rinse with water. If the solution gets on clothes,
immediately take off contaminated clothing. To be protected from this, wear a laboratory coat. Salicylic
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acid can also cause serious eye damage so eye contact should be avoided (“Safety Data Sheet”). In case
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eye contacts salicylic acid, flush eyes with water for 10-15 minutes. The salicylic acid is harmful if
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swallowed as it can cause abdominal pain, nausea, and vomiting (“Safety Data Sheet”). In case of ingestion,
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rinse mouth with water and seek medical advice. If any of the glass material is broken and hence and
injuries occur, seek the first aid to attend the wounds. To prevent burning the skin, use tweezers to pick up
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the beakers from the heating magnetic stirrers. If boiled water is spilled on the skin, put in under lukewarm
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water.
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Environmental Considerations: Avoid the solutions from contacting drains, surface, and ground water.
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Place the materials & apparatus used in appropriate containers for disposal. Allow the Capsicum annum l.
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sprouts to be disposed to the nature so that they can decompose and continue to be a part of the ecosystem.
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Ethical Considerations: A significant amount of Capsicum annum l. seeds (250) are used for experimental
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purposes rather than for consumption. This may be considered unethical since there are shortages for seeds
in many areas of the world, which is why the agriculture sector is regressing. Also, wastage of water should
be minimized since water scarcity is a pressing problem in today’s world. To overcome these unethical
factors, calculations were done to ensure that sufficient amounts were used for the experiment.
4. Data
4.1 Qualitative Data
• 2.0% concentration of salicylic acid solution turned cloudy, and a part of the salicylic acid was floating in
the bottom of the bottle.
• Seeds that were treated with solutions with higher salicylic acid concentrations started to germinate quickly
(day 2).
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• Seeds that were treated with 0.0% salicylic acid took the longest time to germinate (4 days).
• After 10 days, all seeds successfully germinated and formed sprouts.
• Part of the radicles partially broke after picking them up with the tweezer, which resulted in random uncertainties.
[See 6. Evaluation]
4.2 Quantitative Raw Data
0.0% Radicle length of sprout (mm) (± 0.5 mm)

Concentration of Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
Salicylic Acid
Sprout 1 7 8 8 6 7
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Sprout 2 8 8 8 9 6
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Sprout 3 8 8 8 8 6
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Sprout 4 7 6 6 7 8
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Sprout 5 8 8 8 7 6
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Sprout 6 9 8 8 7 7
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Sprout 7 9 8 8 8 6
Sprout 8 6 6 8 8 8
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Sprout 9 7 7 7 8 8
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Sprout 10 7 6 da 8 8 7
Table 3: Table demonstrating the radicle length of the sprouts which were treated with 0.0%
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concentration of salicylic acid

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[Check the Appendix to see Tables 4,5,6,7 for the experimental results of all concentrations
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of salicylic acid]
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4.3 Processed Data Calculations [0.5% concentration was used as the sample]
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To calculate the mean radicle length for each concentration, the following steps were used:
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Calculating the mean radicle length for each trial for 1. Add up the radicle length (mm) of each sprout in the
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a particular concentration particular trial, referring to Table 4.

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# For example, for Trial 1:

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x̄ = ' x! (17 + 16 + 19 + 17 + 19 + 18 + 15 + 16 + 17 + 15) = 169

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!$%
• x! = The average radicle length (mm) of the sprout 2. Divide the sum found in step 1 to the number of sprouts
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for the particular trial (10)

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• N = Number of sprouts for the particular trial

169
= +,. . mm
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10
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Calculating the mean radicle length of the particular 1. Add up the mean radicle length (mm) of each trial in
concentration the particular concentration, referring to the previous
# calculation.
1
x̄ = ' x! (16.9 + 16.2 + 17.0 + 16.6 + 16.3) = 83
N
!$%
2. Divide the sum found in step 1 to the number of trials
• x! = The average radicle length (mm) of the sprout (5).
for the particular concentration 83
= +,. , mm
• N = Number of trials for the particular concentration 5
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Calculating the standard deviation: 1. For each data point of radicle length (mm), calculate
the square of its distance to the mean (x̄ ).
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σ = 8# ∑#
!$%(x! − x̄ )
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(16.9 − 16.6)& = 0.09
(16.2 − 16.6)& = 0.16
• x̄ = The average radicle length (mm) for the (17.0 − 16.6)& = 0.16
particular concentration (16.6 − 16.6)& = 0
• N = Number of trials (16.3 − 16.6)& = 0.09
2. Add the results from step 1.
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(0.09 + 0.16 + 0.16 + 0 + 0.09) = 1.31
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3. Divide the number from step 2 by the number of trials
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(5).
1.31
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≈ 0.26
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4. Take the square root of the number found in step 2
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√0.26 ≈ =. >++.
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[When square rooting a number with x significant

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figures, the answer produced has x+1 significant figures]

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[Calculations were rounded to 3 significant figures.]

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Following the above-mentioned calculations, the following results were reached for each concentration
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(%) of salicylic acid on the effect of radicle length (mm):

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Concentration of salicylic Mean radicle length (mm) Standard deviation

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acid (%) (± 0.5 mm)

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0.0 7.34 0.5142

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0.5 16.6 0.5119

1.0 24.0 1.694
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1.5 31.6 0.8660

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2.0 21.3 1.241

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Table 8: Table demonstrating the mean radicle length (mm) and standard deviation for each
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salicylic acid concentration (%) of the experiment

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[Note: Mean radicle lengths (mm) were rounded to 3 significant figures, whereas the standard
deviations were rounded to 4 significant figures.]
4.4 Uncertainty Calculations [0.5% concentration was used as the sample]

To calculate percentage uncertainty (%) for each mean radicle length (mm) the following steps were used:
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Absolute Uncertainty 1. Divide the absolute uncertainty to the mean radicle

Relative Unceterainty =
Value length (mm), referring to Table 8.
Percentage Uncertainty = Relative Uncertainty × 100 0.5

≈ 0.0301
16.6
2. Multiply the number found in step 1 by 100.

0.0301 × 100 ≈ %A. =+
Using these calculations for each concentration, (0.0%, 0.5%, 1.0%, 1.5%, 2.0%) the following
results were reached:
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Concentration of salicylic acid (%) Percentage Uncertainty (%)
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0.0 6.81
0.5 3.01
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1.0 2.08
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1.5 1.58
2.0 2.35
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Table 9: Table demonstrating the concentration of salicylic acid and its percentage
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uncertainty in the experiment
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4.5 Graph
A scatter plot was plotted for the data to visualize the general relationship between salicylic acid
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concentration (0.0%, 0.5%, 1.0%, 1.5%, 2.0%). Later, the best-fit line was found using Excel to also
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evaluate the R2 value.

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Mean Radicle Length (mm) (± 0.5 mm)
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25
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10 y = -10,7x3 + 21,0x2 + 7,83x + 7,623

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R² = 0.982
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0
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0 0,5 1 1,5 2 2,5

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Concentration of salicylic acid solution (%)
Graph 1: Graph demonstrating the relationship between varying concentrations of salicylic

acid (%) and mean radicle length (mm) (± 0.5 mm) [The pink points are the data points and
the dashed lines are the best fit polynomial line that excel suggested.] [The equation & R²
was rounded to 3 significant figures.]
4.6 Graph & Data Analysis

From Graph 1 and Table 8, it can be concluded that there’s a strongly positive polynomial correlation
between the concentration of salicylic acid (%) with the radicle length (mm). However, this is only valid
up to 1.5% concentration. Starting from 0.0% concentration the radicle length (mm) rapidly increases until
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1.5% concentration, where it starts to decrease. Therefore, the peak/optimum salicylic concentration for
germination is 1.5%.
Considering the rapid increase until 1.5% concentration, the strongly positive relationship can be supported
by the best-fit model Excel suggested. The R2 value (coefficient of determination) can be used to measure
the model or line’s accuracy since it’s essentially a measure of how well a model “fits” a dataset (Zach).
The closer the R2 value is to 1, the better fit the data are for the model/line. Therefore, the R2 value of 0.982
shows that the data points are extremely close to the plotted line of best fit. This also further proves the
string positive polynomial correlation between salicylic acid concentration (%) and radicle length (mm),
hence the germination.
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Some of the error bars are long and some are shorter. Between 0.5% - 1.0% concentrations and between
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1.0% - 1.5% as the error bars don’t overlap, there’s a statistical significance between the concentrations.
Though the concentrations of 1.0% and 2.0% overlap, generally the error bars don’t overlap so generally
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the results are statistically significant and the data can be said to be reliable.
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4.7 Statistical Analysis
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It has been shown that there’s a strongly positive relationship between salicylic acid concentrations (0.0%,
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0.5%, 1.0%, 1.5%, 2.0%) and mean radicle length (mm) hence the germination rate. To test the statistical
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significance of this relationship, the single factor ANOVA test was performed. To conduct the test, the
assumption that the trials were independent of each other had to be made. Moreover, the following
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hypotheses were followed:
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• H1: Salicylic acid concentrations have a statistically significant effect on the radicle length (mm) of
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Capsicum annuum l strouts.

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• H0: Salicylic acid concentrations don’t have an effect on the radicle length (mm) of Capsicum annuum l
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strouts.
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The ANOVA test was done with significant value 0.05 and the following results were reached:
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Figure 5: Results of the ANOVA test [The complete results are in the Appendix]
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Analyzing the results, it’s evident that the P-value is less than the significant value of 0.03. Moreover, the
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F value is greater than the F-critical value. These indicate that the null hypothesis (H0) is rejected, and the
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alternative hypothesis (H1) is supported so there is a statistically significant effect of salicylic acid
concentrations on the radicle length (mm) of Capsicum annuum l strouts.
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5. Conclusion
H1 stated that as salicylic acid concentration increases; the radicle length will rapidly increase and there
will be a positive relationship. At high concentration, the radicle elongation will start to decrease. H0 stated
that increasing concentrations of salicylic acid have no effect on the germination rate of Capsicum. annuum
l. As shown in in the processed data calculations and Graph 1, H1 is supported and H0 is rejected. In
conclusion, the alternative hypothesis (that as salicylic acid concentration increases the radicle length will
rapidly increase and there will be a positive relationship, and at high concentration the radicle elongation
will start to decrease) is supported by the experimental & processed data. The data is sufficient to answer
the research question: there’s a strongly positive polynomial correlation between salicylic acid
concentrations (%) and radicle elongation (mm)/germination. The effect of salicylic acid concentrations
(%) on the radicle length (mm) of Capsicum annuum l is statistically significant. The optimum salicylic
10
acid concentration for germination is 1.5%. However, at high concentration (2.0%) the germination is
inhibited. As both were concluded, the aim was reached.
6. Evaluation
The high R2 value (0.982) with the polynomial equation suggests that the data is representative
by the line of best fit. Consequently, the strongly positive and polynomial correlation between the variables
is justified. The conclusion that as salicylic acid concentration increases the radicle length (and
germination) increases, and that at high concentration the germination starts to be inhibited is supported by
a past study. The obtained results are in accordance with other published data. A past experiment suggests
that optimal salicylic acid concentrations of salicylic acid resulted in an increased growth rate and high
concentrations started to negatively affect plant growth (Koo et al. 6). The effect of salicylic acid
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concentrations on germination and plant growth seems to be similar.
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The reason why there’s a strongly positive relationship between salicylic acid concentration and
germination can be explained by the fact that salicylic acid is a competitive inhibitor, as mentioned in 2.1
y.
Background Information [see page 2]. Salicylic acid is a competitive inhibitor that inhibits the enzymatic
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activity of debilitation of defense (War et al. 1789). It also inhibits pathogen growth. Consequently, the
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seed gets more optimum germination conditions, which is why the mean radicle length increases as
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salicylic acid concentration increases. In other words, salicylic acid increases the survival rate of Capsicum
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annuum l. seeds.
6.1 Strengths of the Experiment

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• The standard deviations in the experiment are moderately low (between 0.5142 and 1.94). This is
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extremely important as it ensures that the collected data has high precision which leads to an accurate &
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reliable conclusion.
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• 10 seeds with 5 trials were investigated for each salicylic acid concentration (0.0%, 0.5%, 1.0%, 1.5%,
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2.0%). Due to the repetition of the experiment, 250 datapoints were collected in total. This decreases the
standard deviation and hence increases the precision of the data. Also, the possibility of random errors is
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lowered which lead to accuracy of the results.

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• Apparatus & materials used throughout the experiment have low absolute uncertainties, leading to low
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percentage uncertainties. Demonstrated in Table 9, [see page 10] the percentage uncertainties in the
experiment are between %1.58 and %6.81. This means that the random errors in the experiment are low.
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This is significant since it ensures that the conclusion was drawn with accurate data.
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• Doing the preliminary experiment led to pre-identifying the potential problems in the main experiment.
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It was verified that the seeds were able to germinate and the concentration intervals to be used in the main
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experiment were determined.

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6.2 Weaknesses, sources of errors & improvements of the experiment

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Source of Weakness Reasoning & Justification Modifications

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Though at last there were 250 datapoints

Number of trials collected, doing more trials reduce the influence Increasing the number of
(systematic error) of random errors on the results. Also, the trials.
standard deviation can be reduced which will
lead to more precise data.
A spray bottle wide a more
Diameter of the spray The spray bottle had a wide diameter. Therefore, narrow diameter can be
bottles (systemic error) not all concentrations received equal amounts of utilized. Alternatively, a
the solution as some of it sank to the bottom. syringe can be used to inject
the solution to the petri
dishes.
11
Cotton wool pads were placed in petri dishes. If

soil were used, many of the growing media and Soil can be planted in
Germination medium needed nutrients in seed germination would be flowerpots. Consequently, the
(systematic error) provided. Even different types of soils could soil will be the germination
affect the germination since different types of medium for the seeds.
soils have different moistures.
Continuing to observe how much the radicle
continues to grow would allow the conclusion to The experiment can be
Length of the experiment be more accurate. The results found in 7 days repeated with the length
(systematic error) may change over time, therefore the relationship being 2-3 weeks.
between the salicylic acid concentration and
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radicle length can’t be assumed to strongly
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positive forever.
Certain assumptions had to be made due to
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uncontrollable reasons which limited the
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accuracy of the investigation. Temperature The experiment can be done
couldn’t be monitored since the setup had to be in a room with air
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left unattended. This is crucial since fluctuating conditioning, where the
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temperatures affect germination rate and the temperature can be altered.
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Uncontrolled variables radicle length (Koger et al. 989). Enzymatic The petri dishes can be put
(systematic error) activity also increases by temperature, and this is inside an incubator where
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important since the only factor that affects temperature is controlled.
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enzymatic activity must be the inhibitor Instead of using the sun as the
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(salicylic acid) in the experiment to make energy source, artificial light

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accurate conclusions. The light source in the such as lamps could be used.
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experiment couldn’t also be controlled since the

sunlight intensity is uncontrollable. In higher
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light intensities, there’s faster germination rate

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(Emerson and Lewis 812).

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Though the ruler had a low uncertainty, (± 0.5

mm) the measurements still were rounded to the The radicals should be
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Measurement of the nearest millimeter. This has led the data into carefully straightened. The
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radicals (random error) being less precise and less accurate. Also, part of tweezers should be used
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the radicles broke when picking them up with carefully to extract the
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the tweezer and therefore couldn’t be completely strouts.

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straightened. So, the accuracy of measuring the

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radicles were limited.

Table 10: Table demonstrating the weaknesses, sources of errors & improvements of the
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experiment
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7. Extensions
In the investigation, it has been shown that radicle length (mm) and hence germination increases with
salicylic acid concentration. The optimum concentration (1.5%) can be used in Turkey or other countries
where agriculture issues are on the rise, to acquire faster germination and plant growth. However, salicylic
acid concentration’s possible side effects also need to be investigated as concentrations exceeding 1.5%
seem to decrease radicle length. The inhibition activity that salicylic acid causes under varying
temperatures on seed germination could also be investigated. Also, the effect of other types of acid or
treatments on the radicle elongation could be investigated.
12
Works Cited
Ahern, Kevin and Rajagopal, Indira. Chem 4320/5320: Biochemistry 1. Open Education Resource, 2023. LibreTexts.
“Capsicum Annuum | Dried Botanical ID.” Idtools.org, 2020,

idtools.org/dried_botanical/index.cfm?packageID=1096&entityID=2358. Accessed 10 Mar. 2023.
Emerson, Robert, and Charlton M. Lewis. “Factors Influencing the Efficiency of Photosynthesis.” American Journal
of Botany, vol. 26, no. 10, Dec. 1939, pp. 808, 822.
Frary, Anne, et al. “Water-Soluble Antioxidant Potential of Turkish Pepper Cultivars.” HortScience, vol. 43, no. 3,
2008, pp. 631, 636. ResearchGate.
e
.k
Joshi, Renu. “Role of Enzymes in Seed Germination.” IJCRT, vol. 6, no. 2, 2018, pp. 1481-1485.
ac
https://doi.org/https://www.ijcrt.org/papers/IJCRT1812620.pdf. Accessed 7 Apr. 2023.
y.
m
Klessig, Daniel F., and Jocelyn Malamy. “The Salicylic Acid Signal in Plants.” Plant Molecular Biology, vol. 26, no.
de
26, 1994, pp. 1439, 1458.
ca
na
Koger, Clifford H., et al. “Factors Affecting Seed Germination, Seedling Emergence, and Survival of Texasweed
(Caperonia Palustris).” Weed Science, vol. 52, no. 6, Dec. 2004, pp. 989–95, https://doi.org/10.1614/ws-03-139r2.
tio
da
Koo, Young Mo, et al. “Salicylic Acid as a Safe Plant Protector and Growth Regulator.” The Plant Pathology Journal,
un
vol. 36, no. 1, Feb. 2020, pp. 1–10, https://doi.org/10.5423/PPJ.RW.12.2019.0295.

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PubChem. “Salicylic Acid.” @Pubchem, PubChem, 2023, pubchem.ncbi.nlm.nih.gov/compound/Salicylic-acid.

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Accessed 15 Jan. 2023.

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m
Raskin, I. “ROLE of SALICYLIC ACID in PLANTS.” Plant Molecular Biology, vol. 43, no. 43, 1992, pp. 439, 463.
o@
“Safety data sheet” Safe Work Australia, 2022, https://www.carlroth.com/medias/SDB-9268-AU-

ng
EN.pdf?context=bWFzdGVyfHNlY3VyaXR5RGF0YXNoZWV0c3wyNDY0NTB8YXBwbGljYXRpb24vcGRm
ra
fHNlY3VyaXR5RGF0YXNoZWV0cy9oODUvaDE4LzkwNjk5NTc1NDYwMTQucGRmfGZhMjY0MzdlZjExZ
.o
GE4M2M5MGE5ZTBlNWQ0MDFhZTFlMzQ3Y2I0N2I4MjZmOTZkNWQwZjk2NTQzZGUzYTA5MGQ.
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Accessed 20 Mar. 2023.

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Soltani, Elias, et al. “Problems with Using Mean Germination Time to Calculate Rate of Seed Germination.”
ResearchGate, CSIRO Publishing, 2015,
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www.researchgate.net/publication/285202523_Problems_with_using_mean_germination_time_to_calculate_rate_
st
of_seed_germination. Accessed 12 Mar. 2023.

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War, Abdul Rashid, et al. “Role of Salicylic Acid in Induction of Plant Defense System in Chickpea (Cicer
ArietinumL.).” Plant Signaling & Behavior, vol. 6, no. 11, Nov. 2011, pp. 1787–92,
https://doi.org/10.4161/psb.6.11.17685. Accessed 25 Mar. 2023.
Whitlock, Linda. “How to Grow Peppers - Part 2.” MSU Extension, 2016,
www.canr.msu.edu/resources/how_to_grow_peppers_part_2#:~:text=Peppers%20thrive%20when%20temperatur
es%20are,70%C2%B0%20F%20at%20night. Accessed 19 Mar. 2023.
Zach. “What Is a Good R-Squared Value?” Statology, 24 Feb. 2019, www.statology.org/good-r-squared-value/.

Accessed 22 Mar. 2023.
13
Appendix

Concentration of
Salicylic Acid Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
Sprout 1 17 15 16 17 16
Sprout 2 16 16 18 18 19
Sprout 3 19 16 16 16 17
Sprout 4 17 17 18 17 16
Sprout 5 19 17 17 16 16
Sprout 6 18 17 18 18 16
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Sprout 7 15 18 17 17 16
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Sprout 8 16 16 18 15 16
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Sprout 9 17 15 16 15 14
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Sprout 10 15 15 16 17 17
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Concentration of
Salicylic Acid Trial 1 Trial 2
da Trial 3 Trial 4 Trial 5
Sprout 1 25 20 22 23 20
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Sprout 2 26 24 23 23 21
Sprout 3 25 24 20 23 23
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Sprout 4 24 24 26 27 24
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Sprout 5 23 22 23 25 24
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Sprout 6 24 24 25 26 25
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Sprout 7 26 24 27 24 20
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Sprout 8 26 25 25 24 23
Sprout 9 25 24 27 24 23
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Sprout 10 27 26 25 23 25
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Concentration of
Salicylic Acid Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
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Sprout 1 31 32 31 30 34
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Sprout 2 36 30 33 36 36
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Sprout 3 29 30 29 32 33
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Sprout 4 33 29 29 34 31
Sprout 5 31 31 33 30 30
Sprout 6 30 33 33 32 30
Sprout 7 33 33 32 31 29
Sprout 8 29 29 34 33 31
Sprout 9 31 31 33 33 32
Sprout 10 30 33 34 29 30
14

Concentration of
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
Salicylic Acid
Sprout 1 20 20 22 23 24
Sprout 2 19 20 21 23 20
Sprout 3 22 25 22 21 20
Sprout 4 19 22 22 23 22
Sprout 5 21 21 21 20 19
Sprout 6 21 21 23 23 22
Sprout 7 22 23 19 20 21
Sprout 8 21 20 23 24 19
Sprout 9 18 20 19 22 23
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Sprout 10 22 21 21 23 20
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Figure 5 Results of the ANOVA test [The full data is in the Appendix]
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15

How Do Altering Salicylic Acid Concentrations (0.0%, 0.5%, 1.0%, 1.5%, 2.0%) Affect The Germination

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length and hence germination.

2.1 Background Information:

3.1 Experimental Variables:

Concentration Mass of Volume of

of salicylic acid salicylic acid water (ml)

(%) (g) (± 0.1 g) (±1 ml)

0.00 0.00 100

0.50 0.50 100

1.00 1.00 100

1.50 1.50 100

2.00 2.00 100

solutions [Given in 2 decimal places]

Controlled Reason of Control Method of Control

more plant numbers indicate more sprouting.

quickly (Saeed and Shaukat 292). approximately 5.0 mm (±0.5

Prior to preparing the

solutions, 100 ml of water

The time should be controlled since germination will

An experiment in an article has shown faster germination rate in higher light

control sunlight intensity, as it varies every day.

3.2 Materials & Apparatus

1x Safety goggles 1x VELP® Heating magnetic stirrer

10x Gloves 1x Magnetic needle

3.3 Formation of the Experiment

Table 1 [see page 3].

1.3: Pour the distilled water inside the beaker.

glass and spatula, referring to Table 1 [see page 3].

1.7: Place the beaker on the countertop.

the stopwatch and continue for 10 minutes.

concentrations (0.0%, 0.5%, 1.0%, 1.5%, 2.0%).

in the heating magnetic stirrer

3.5 Safety, Environmental & Ethical Considerations:

4.2 Quantitative Raw Data

0.0% Radicle length of sprout (mm) (± 0.5 mm)

concentration of salicylic acid

a particular concentration particular trial, referring to Table 4.

# For example, for Trial 1:

x̄ = ' x! (17 + 16 + 19 + 17 + 19 + 18 + 15 + 16 + 17 + 15) = 169

for the particular trial (10)

• N = Number of sprouts for the particular trial

2. Add the results from step 1.

[When square rooting a number with x significant

figures, the answer produced has x+1 significant figures]

[Calculations were rounded to 3 significant figures.]

(%) of salicylic acid on the effect of radicle length (mm):

Concentration of salicylic Mean radicle length (mm) Standard deviation

acid (%) (± 0.5 mm)

0.0 7.34 0.5142

0.5 16.6 0.5119

1.5 31.6 0.8660

2.0 21.3 1.241

salicylic acid concentration (%) of the experiment

4.4 Uncertainty Calculations [0.5% concentration was used as the sample]

Absolute Uncertainty 1. Divide the absolute uncertainty to the mean radicle

Percentage Uncertainty = Relative Uncertainty × 100 0.5

2. Multiply the number found in step 1 by 100.

evaluate the R2 value.

10 y = -10,7x3 + 21,0x2 + 7,83x + 7,623

0 0,5 1 1,5 2 2,5

Concentration of salicylic acid solution (%)

Graph 1: Graph demonstrating the relationship between varying concentrations of salicylic

4.6 Graph & Data Analysis