The Effect of pH and Temperature on the Germination of

Phaseolus vulgaris Seeds

Alessio Artusi and Kate Barber

Macomb Mathematics Science Technology Center

Biology

Section 9B

Mr. Acre/Mr. Estapa/Mrs. Gravel

19 May 2016
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Introduction

On average, farmers work 50.4 hours a week producing food (Moran). Majority of this

time is spent planting seeds and tending to the growth of these seeds. The objective of this

experiment was aimed to lessen hours farmers work by increasing the speed at which crops can

be produced. The first step in doing this is to find the conditions best fit to minimize the time

Phaseolus vulgaris seeds spend germinating to have them start growing quicker.

The goal was to germinate Phaseolus vulgaris seeds with varying temperature and pH to

find the the conditions that would yield the fastest germination. Three different waters with

different pH values (5, 6, 7) were mixed up. Paper towel was dampened and then 5 seeds were

placed in the middle of the paper towel, that was put into a bag and placed in an incubator, or left

at room temperature (22, 25, 28 degrees Celsius). Originally it was hypothesized that the seeds

would germinate fastest with high temperature and low pH.

The experiment uses different pH values (5, 6, and 7) of water used to germinate these

seeds. The reason the values are more acidic is because Phaseolus vulgaris seeds grow best in

soil of pH from 6 to 7 (Caines). This fact helps support the original hypothesis of a lower pH.

The reason pH matters when germinating seeds is because the pH slightly changes the

shape of enzymes used to germinate . These deformities mostly slow down the germination but

at extremes the seeds would die before germination ("Introduction to Enzymes”).

Temperature, on the other hand, affects germination speed because seeds go into

dormancy, or prevent germination because of outside conditions, and germination is stunted

(Roberts). Dormancy of a seed before it is germinated can potentially lasts days making

temperature a huge factor in germination.

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The temperatures used in this experiment were 22, 25, and 28 degrees celsius. The reason

these values were chosen is because Phaseolus vulgaris germinates best between 21 and 28

degrees Celsius (Caines). The reason a higher temperature was chosen for the hypothesis

opposed to a low temperature is because at a high temperature molecules and enzymes would

move faster during germination.

Since the objective of this experiment is to maximize the speed at which seeds germinate,

it was decided that the data should be based on how many seeds germinated on the first day of

germination (second day after incubation). The reason the data was taken like this, is because it

gives a more diverse range in data compared to using average days until germination which

would usually be between 2 and 3 days, which makes comparing data very difficult.
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Problem Statement

Problem:

What would the ideal level of pH in water and temperature be in regards to the

germination rate of Phaseolus vulgaris?

Hypothesis:

If the pH level of the water decreases and the temperature the seeds are exposed to

increases, then the Phaseolus vulgaris seeds will have a shorter germination period.

Data Measured:

The effect of the pH level of water and temperature on the germination of Phaseolus

vulgaris seeds is what is being measured. The pH will be measured by using pH testers, the

temperature will be measured using a thermometer, and the germination period is going to be

measured in how many days it took for the seeds to germinate. The high and low standards were

chosen because the average temperature they grow best in is 21-27 degrees Celsius, and pH level

of 6.0 to 7.0.
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Experimental Design
Materials:
(300) Phaseolus vulgaris Seeds
(60) Ziploc Plastic Bags
(2) Incubators
(1) Baking Soda 16 oz
(1) Citric Acid 4 oz
(2) Distilled Water 1 gallon
(3) Bounty Paper Towel
(20) pH Testers

Procedure:

1. Clean out three separate containers used to hold the different pH waters.
2. Fill up each container with distilled water.
3. Add citric acid to a container of distilled water to change the pH of one of the
containers

4. Test the pH and continue to add citric acid until the pH reaches 6, if the pH
goes under 6 add baking soda and test again.

5. Repeat steps 4 - 5 in a different container until the pH reaches 5 instead of 6.

6. Leave the third beaker with distilled water (test to be sure the pH is 7).
7. Get 24 pieces of paper towel and dampen them in the water of 7 pH.
8. Evenly distribute 5 seeds on each dampened paper towel.
9. Place each piece of paper towel inside a Ziploc bag with the seeds folded inside of
. the towel, tightly close the Ziploc bag afterwards.

10. Repeat steps 8 - 10, this time dampening each piece of paper towel in the water of
. 5 pH.

11. Repeat steps 8 - 10, this time dampening only 10 pieces of paper towel in the water
. of 6 pH.

12. Put 12 of the 5 pH bags into an area of room temperature and the other 12 bags
. into an incubator set to 28°C.

13. Put 12 of the 7 pH bags into an area of room temperature and the other 12 bags
. into an incubator set to 28°C.

14. Put all 10 of the 6 pH bags into an incubator set to 25°C.

15. Check each towel daily.
16. Record the number the number of seeds that have germinated each day for each
. trial.
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Diagrams:

Water (5 pH and
6 pH)

Paper
Towel
Distilled
Water

pH Ziploc
Testers Bags

Seed Baking
Packets
Figure 1. Materials Soda

Figure 1 shows the materials used in the experiment, not included was the citric acid used

to lower the pH of the distilled water. The other materials are used to change the pH of water, set

up the seeds in the paper towel, and finally put the seeds in a Ziploc bag to germinate.
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Incubat
or
Bags of
Seeds

Figure 2.Extra materials

Figure 2 shows how the incubators were used to maintain a constant heat, the figure also

shows how the seeds were folded into dampened paper towel and then put into the incubator.

Data and Observations

Data:
Table 1
Design of Experiment Values
Temperature (Celsius) pH
- Standard + - Standard +
22 26 28 5 6 7
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Table 1 shows the value for each treatment used in the experiment. They were chosen

after pre-trials because the previous values had very slow germination because they were too hot.

The values were experimented slightly and resulted in a higher temperature and slight decrease

in pH. These values were also supported by the article "In What Conditions Will Pinto Beans

Grow Best?” (Caines) in which she explained how Phaseolus vulgaris grew best in higher

temperatures between 22 and 28 degrees Celsius. It also stated that the seeds grew more acidic,

which affected the pH levels used. The pH the water that was used to germinate the seeds was

changed by either adding baking soda to raise the pH or citric acid to lower it. In addition the

temperature was changed by using an incubator and keeping one trial in room temperature.

Table 2
DOE Data
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Table 2 shows that there were 12 DOEs done for the experiment, each DOE consisted of

5 different trials, ( +,+ ), ( +,- ), ( -,+ ), ( -,- ), and the standard. Each trial was done 12 times, and

the average was found for each. The data shows how many seeds germinated based on the pH of

the water used, and temperature the seeds were exposed to.

Observations:
Table 3
Observations
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Date Observation
The seeds exposed to high temperature had formed condensation while seeds
2/25/2016
in room temperature had not
Seeds in high temperature, once germinated, grew fast, the embryo for those
3/2/2016 seeds on the day they germinated were three times as long as seeds in
temperature held low.
3/3/2016 Brown spots were spotted under the seeds after germination.
Some seeds have had the “skin” of the shell peel off, some other ones had
3/9/0216 cracked where the embryo attempted to penetrate and therefore failed
germination.

Table 3 shows the observations made during trials. The bags that were involved in trials

with high temperature had condensation around the bag, while the bags in room temperature did

not have a notable amount of condensation. There were also brown spots on the paper towel after

germination had occurred.

Figure 3. Before Germination

Figure 3 shows the seeds before germination. The seeds that are shown are (+,+) right

before they were put into the incubator. 5 seeds were put onto each towel and 12 towels were

used in the experiment (except for the Standard which only had 10).
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Figure 4. After Germination

Figure 4 shows the seeds after they germinated. The seeds shown are the (+ ,+) group

after two days. The other trials were not pictured but it was evident that seeds in which

temperature was held high had more germination than any other.The embryo for these trials were

also much longer than any other. The trials in which temperature was held low had much less

germinated seeds (usually around half of the seeds) and had much smaller embryos. Finally the

standard trials were in between, having most seeds germinate with not as long of an embryo. The

difference in pH for the trials did not have any differences notable by eye.
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Data, Analysis, and Interpretation

A two-factor Design of Experiment (DOE) was used to analyze the data that was

collected. A two-factor DOE tests how two variables and their interaction effect a response

variable. The experiment tested how many Phaseolus vulgaris seeds would germinate while

being affected by different levels of pH and temperature (Celsius).

The data was collected by putting five seeds surrounded by a damp paper towel (with

varying pH levels), into a Ziploc bag and putting it into an incubator (every trial had a different

amount of pH, and a different incubation temperature).

Each trial was completed twelve times, while the standard was only ran ten times, The

averages were then taken which were used to represent each trial and the standard, which makes

it easier to understand the data as a whole.

Table 4
Factors and values

Table 4 shows the low, high, and standard values for the factors (temperature and pH)

used in the experiment. For temperature, the low was held at 22° Celsius, the high was held at

28° Celsius, and standard was held at 25° Celsius. When the pH was low, it was held at 5, the

standard was held at 6, and high was held at 7. These values were chosen because during pre-

trials these differences resulted in an easily observable difference in trials.

Table 5
Averages
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Table 5 shows the different trials and their results as averages. When both variables were

held high, the average amount of seeds that germinated was 5, when they were both held low,

the average was 2.1666, when the pH was high and the temperature was low, the average was

2.6666, and when the pH was low and the temperature was high, the average was 5. The grand

average (average of all the other averages) was 3.7083, this value will be used to later predict the

result of a similar experiment that used different values for the variables.

Figure 5. Standard Data

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Figure 5 shows a graph of the standard data, The data seems to have straight trend

meaning that the experiment was likely carried out well and that there were likely no lurking

variable (outside effects that grow or lessen as trials proceed). The reason this is known is

because the variance, or extent that points differ, is low.

The range of the data can be found by subtracting the highest value (5) by the lowest

value (4) giving the range of data, which is 1. The range of standards will be used to find the

statistical significance of the independent variables in the experiment. When a factor is

significant it means the effect it had could not be by chance alone, in addition the factor could

have still had a practical significance.

Table 6
Effect of pH

Figure 6. Effect of pH
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Table 6 shows the average amount of seeds germinated when pH is held high and low. By

subtracting the average when held high by the average when held low (3.8333 - 3.5833) the

effect value of pH is found, which is .25. The effect value shows how much of an effect the

change in pH had in the experiment. If the effect is found in between twice the amount of the

positive and negative range of standards then the factor is not statistically significant, in addition

the closer to 0 the value is, the less of an effect it had. Since .25 is in between 2 and -2 pH is is

not a statistically significant value.

Figure 6 shows a visual representation of the effect of pH in a graph. It is apparent that

the slope of the line is small, and as the pH changes (Represented in -1 and 1 on the X axis),

almost the same amount of seeds are germinating on average. Looking at the effect in a practical

view and not statistically it still seems that the pH either had a small effect on the germination,

meaning the values chosen for pH were too similar, or the slight increase of germination was

mostly done by chance.

Table 7
Effect of temperature

Figure 7. Effect of Temperature

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Table 7 shows the average amount of seeds germinated when temperature is held high

and low. By subtracting the high average by the low average the effect value of temperature is

found, which is equal to 2.5833. This value means that on average 2.5833 more seeds germinated

when temperature is held high then when it is held low. The effect is also significant as it is not

found in between 2 and -2. What this significance represents is that this difference could not have

happened by chance alone.

Figure 7 shows a visual representation of the effect of temperature in a graph. This line

has a huge slope relative to the one in figure two meaning there is a bigger difference in ger-+

mination when temperature is held high compared to when it is held low. Looking at it

practically the temperature has a huge effect in this experiment. When held high temperature

wields more than double the amount of seeds then when it is held low.

Table 8
Interaction Effect

Figure 8. Interaction effect

Table 8 compares the data when pH is held low and high to when temperature is held low

and high. These values will be used to form lines which show the interaction between the factors

held low and high. When subtracting the slope of the line labeled solid by the slope of the line
 Artusi - Barber 16

labeled dotted (1.1666 - 1.4166) the interaction effect is found, which is -.25. Because -.25 is in

between 2 and -2 the interaction is not significant.

Figure 8 shows a visual representation of how pH and temperature interact with each

other and how that effects the experiment. Since the slopes of the two lines are similar it means

that there does not appear to be an interaction effect happening. In addition the interaction effect

(-.25) is close to zero meaning even when viewing it practically there likely is not an interaction

effect.

Figure 9. Test of Significance

Figure 9 shows which of the effects of the variables were significant and which were not.

All of the effects that are located in between the two fences are insignificant and the effects

outside of the fences are significant. The fences were found by doubling the range of standards

(1) and setting up the fences at the positive and negative of that value (2 and -2). The effect of

pH (.25) and interaction effect (-.25) are not significant because they are in between 2 and -2

while the effect of temperature (2.5833) is significant because it is greater than 2.

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Y = 3.7081 + 2.5833/2(x) + “noise”

Y = 3.7081 + 2.5833/2(.5) + “noise”

Y = 4.031 + “noise”

Figure 10. Interpolation Parsimonious Equation

Figure 10 shows an interpolated parsimonious prediction equation. The equation find

what the experiment would yield when using halfway points of significant independent variables.

In this experiment only the temperature the seeds were exposed to was significant (because its

effect value was outside of the fences created by twice the range of standards). The equation is

represented by the grand average plus half of all significant values times x (or y with multiple

variables) plus “noise” which is an unquantifiable variable which represents inaccuracies in the

experiment. X is replaced by either .5 (when finding factor in between high and standard) and -.5

(when finding factor in between low and standard). Here .5 five was plugged in and the answer

become 4.031, this means that if the trial was done again with temperature at 26.5° Celsius it

would likely yield 4.031 seeds.

Observing all the data it is apparent that the pH barely affected the experiment, even

when looking at it practically not by significance, the values of the lows and highs for pH were

almost the same, meaning that the difference in pH did not matter. The same thing can be said for

the interaction of pH and temperature. The temperature, however, did have a huge effect on the

germination of seeds, when the temperature was low not many seeds would germinate quickly

but when the temperature was high all seeds germinated quickly.
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Conclusion

Originally it was hypothesized that the Phaseolus vulgaris would germinate fastest when

exposed to a high temperature and a low pH level. This hypothesis has proven to not be true

because the high, low ( + , - ) average was tied for highest with the high, high ( + , + ) average

with 5 seeds germinated because it was a tie, the original hypothesis has been rejected.

This experiment was the first, small step in ultimately lessening the hours worked by

farmers by speeding up the germination of a seed. Another researcher could add onto this 12

research by using the same factors to find the temperature and pH that Phaseolus vulgaris plants

grow fastest in, the reason for this is so that farmers spend less time tending to the plants and

work less. Researchers could also replicate this experiment but have a bigger difference in pH

levels in order to get a better understanding in what pH Phaseolus vulgaris seeds germinate

fastest in.

This experiment was the first, small step in ultimately lessening the hours worked by

farmers by speeding up the germination of a seed. Another researcher could add onto this

research by using the same factors to find the temperature and pH that Phaseolus vulgaris plants

grow fastest in, the reason for this is so that farmers spend less time tending to the plants and

work less. Researchers could also replicate this experiment but have a bigger difference in pH

levels in order to get a better understanding in what pH Phaseolus vulgaris seeds germinated

fastest in.

The idea of this experiment was to find the temperature and pH that Phaseolus vulgaris

would germinate fastest in. This was done by lowering or heightening the pH, then using those

solutions to dampen paper towel that would contain five seeds inside. Afterwards, the towels

were placed in Ziploc bags, which were then incubated in varying temperatures. Temperature and
 Artusi - Barber 19

pH were chosen because finding a preferable temperature could help farmers know when to plant

their seeds. In the article, "In What Conditions Will Pinto Beans Grow Best?" In the article, "In

What Conditions Will Pinto Beans Grow Best?" Caines says that optimum germination takes

place in 18.33-23.88 degrees Celsius. In addition, pH was chosen to help farmers know how

much they need to tend the acidity of the soil used. In the end, a high temperature and pH

resulted with the most seeds germinated with an average of 5 seeds, while a low temperature and

pH had the lowest result with an average of 2.167 seeds germinated. A high temperature was

expected to yield the best results, because the seed has a less likely chance of being dormant, and

the molecules and enzymes can work faster with a hotter temperature. Surprisingly, a high pH

yielded better results than a lower pH, because Phaseolus vulgaris, according to the article “In

What Conditions Will Pinto Beans Grow Best?”, prefers acidic conditions (Caines), because a

pH too high could negatively affect the shape of enzymes used during germination. These results

could be purely by chance though as the pH levels were very similar and results were not too

different.

Not many other experiments used both temperature and pH as factors for germinating

seeds but a handful have used one of the two for either germinating or growing seeds. In the

article “Does the pH of Water Affect the Growth of Bean Plants?” by Susan Shelley, the author

creates an experiment on the growth of plants with varying pH values. This article had very

similar design (relative to how pH was used) and had a similar way of collecting data. Another

article, by Tzvetanka titled, “Effects of High Temperature and Water Stress on Seed Germination

of the Invasive Species Mexican Sunflower” also used temperature on the growth and

germination of seeds. This article used intense temperatures to kill the seeds but the low end of

their temperature spectrum was similar to this experiment.

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It is important to also note that this experiment was in no way perfect. When it comes to

the design of the experiment there was not a big enough difference in the pH values used,

because of this the results for pH could have been by chance because the results were very

similar. There were also flaws in how the experiment was executed, an example of this is that in

a couple of the first trials the seeds were placed on top of the paper towel, not inside of it, this

made the water come into only one direction of the seed and negatively affect germination.

Another flaw was that the distilled water was kept in previously water bottles that could have

contained chemicals that would affect germination.

In the end, the experiment was a plethora of knowledge, not only because the results

show what conditions seeds germinate best, but also in how to design, conduct, and collect data

from an experiment. After every flaw, trial, or table created, more efficient ways to execute the

experiment were thought about. Essentially, if this experiment were to be replicated, or if another

one were to be made, it would run more smooth because of all the knowledge gained from this

one.

Works Cited
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Caines, Kimberly. "In What Conditions Will Pinto Beans Grow Best?”Homeguides. Web.

Jan.-Feb. 2016. <http://homeguides.sfgate.com/conditions-pinto-beans-grow-best-

74216.html>

Roberts, EH. "Temperature and Seed Germination." NCBI. 4 June 1988. Web. 10 May 2016.

<http://www.ncbi.nlm.nih.gov/pubmed/3077854>.

Shelly, Susan. "Does the PH of Water Affect the Growth of Bean Plants?" Infoplease. 5

Sept. 2003. Web. Jan.-Feb. 2016. <http://www.infoplease.com/cig/science-fair-

projects/does-ph-water-affect-growth-bean-plants.html>.

Tzvetanka, D. Dinkova. "Effects of High Temperature and Water Stress on Seed

Germination of the Invasive Species Mexican Sunflower." PLOS ONE. 28 Oct. 2015. Web.

Feb.-Mar. 2016. <http://journals.plos.org/plosone/article?

id=10.1371/journal.pone.0141567>.

"Introduction to Enzymes." Worthington Corporation, 6 Jan. 1972. Web. 15 Mar. 2016.

<http://www.worthington-biochem.com/introbiochem/introEnzymes.html>

Moran, Ciaran. "How Many Hours Do Farmers Actually Work per Week?" Agriland. 26

June 2015. Web. 19 Apr. 2016.