A Correlation Study of the Number of Stomates and the Rate of Transpiration in

Calamansi (Citrus microcarpa) and Pomelo (Citrus maxima) Plants1

Rochelle Mae M. Piñon

Group 2 22L

December 4, 2019

______________________
1
A Scientific paper submitted in partial fulfillment of the requirements in BIO 11.1 (Investigative
Biology Laboratory) laboratory under Prof. Aimee Cagalawan, 1st semester, 2019-2020.
 Abstract

This study determined the relationship between the number of

stomates in plants of the Citrus family and their rates of transpiration. Using
the transpiration set-up, transpiration rates of Calamansi (Citrus microcarpa)
and Pomelo (Citrus maxima) plants were determined. Leafy shoots from the
plants were obtained and let to transpire for 20 minutes, measuring their
volume displacement in 4-minute intervals. Using the imprinting method, the
number of stomates in the Calamansi and Pomelo leaves were counted. Since
internal factors affect the rate of transpiration in plants, then the number of
stomates would have a significant impact on the rate of transpiration in plants,
particularly in the Citrus species. Furthermore, an increase in the number of
stomates also increases its transpiration rate. Results show that Pomelo plants
have a higher transpiration rate with 0.038cm3/min, than Calamansi plants
with 0.009 cm3/min. With regards with number of stomates on their abaxial
sides, Calamansi leaves have a higher average stomatal count with 45.5 than
Pomelo leaves which only have an average stomatal count of 26. The results
satisfy the hypothesis. A significant relationship between the number of
stomates present and their transpiration rates exists. However, an increase in
the number of stomates did not prove that there is also an increase in their
transpiration rate. The results presented were inversely proportional to that of
the hypothesis. It presents that for every increase in the number of stomates,
there is a decrease of 0.936 cm3/min on the plant’s transpiration rate.
Therefore, the hypotheses were partially accepted.

INTRODUCTION

Plants are one of the most vital components of the Earth. Their importance could never be

neglected because they are the ones responsible for the survival of other Earth inhabitants. One of

their tasks is to provide food for other living organisms. This is made possible through

photosynthesis. In photosynthesis, light energy is being captured from the sun and is converted

into chemical compounds, such as carbohydrates, which other organisms use to power themselves

(Choi & Douglas 2018). In addition, plants also provide raw materials such as, firewood, timber,

fibers, medicines, dyes, pesticides, oils, and rubber that people process to produce different

products useful in everyday life (Wilkin & Brainard 2019). Moreover, plants also provide habitat

for both animals and humans. For instance, forests all over the world serve as a home to different

1
A Scientific paper submitted in partial fulfillment of the requirements in BIO 11.1 (Investigative
Biology Laboratory) laboratory under Prof. Aimee Cagalawan, 1st semester, 2019-2020.
species of wildlife (Greentumble 2019). Plants also play a huge role in the recycle of matter in

biogeochemical cycles, such as transpiration. In transpiration, water is loss through the stomatal

openings of the leaves by evaporation (Taylor et al. 2018). These stomates, found on both sides

of a leaf, are guarded by a pair of guard cells. Guard cells are kidney beans shaped structures that

aid in the process of transpiration. They have a “bacon effect” mechanism where, its composition,

a thick and thin cell wall forms a bent shaped due to their width differences. K+ accumulation

marks the beginning of the opening of the stomates. Afterwards, osmosis inflates the guard cells

and the uneven cell wall enables the opening of the stomates. These stomates close when, K+ ions

already exit the cell and when, there is a decrease in the water content inside its vacuoles (Shipunov

2018).

Approximately, 98% of a plant’s energy is used in transpiration. It functions for cooling

the plant and the distribution of water and minerals to the leaves for photosynthesis. Too much

heat disrupts the metabolic processes that occur in plants, which in turn results to its stunted growth

rate (Brawner 2011). Extreme heat conditions could even lead to the death of the plant. In these

situations, transpiration comes in handy. Water evaporation through transpiration greatly helps in

cooling the plant, but it could also lead to water loss if not properly regulated. This could also

affect the amount of water distributed among various parts in a plant that could be utilized for

photosynthesis.

There are several factors that affect the transpiration rate in plants. It can be classified into

two: Internal and External factors. Internal factors include: the number, size, position and

movement of stomates, thickness of the boundary layer of still air hugging the surface of the leaf,

and the thickness of its cuticle layer. External factors include: air temperature, relative humidity,

wind speed and radiation intensity (Duka et al.2018). External factors are conditions to which the

1
A Scientific paper submitted in partial fulfillment of the requirements in BIO 11.1 (Investigative
Biology Laboratory) laboratory under Prof. Aimee Cagalawan, 1st semester, 2019-2020.
plants are subjected to. Temperature may affect the transpiration rate in two ways. Firstly, warmer

temperatures speed up the rate of evaporation, thus increasing the transpiration rate. Secondly,

when warm air and cold air have the same amount of water, cold air tend to have a shallow water

concentration gradient while the warm air may be able to hold more water vapor, thus having a

steeper water concentration gradient. Another external factor, relative humidity affects the

transpiration rate inversely. When the relative humidity is high, transpiration rate is slower. On the

other hand, if the relative humidity is low, thus, a dry atmosphere, a presence of a steep water

concentration gradient between the humid inside of a stomata and the outside air will create a faster

transpiration rate. Wind speed affects the transpiration rate proportionally. When water is lost from

a leaf, this forms a thin layer at the surface of the leaf. Faster wind speeds remove this layer much

faster, thus producing a faster transpiration rate. Radiation intensity increases the rate of

photosynthesis, which in turn, increases the amount of glucose stored in guard cells. This leads to

a lower water potential in a leaf. As its water potential decreases, more water enters the guard cells

resulting to its turgidity. The turgor pressure in these guard cells lead to the opening of the

stomates, thus a faster transpiration rate. On the other hand, the internal factors which may affect

transpiration depend on the structure of a plant and its species. Since internal factors affect the rate

of transpiration in plants, then the number of stomates would have a significant impact on the rate

of transpiration in plants, particularly in the Citrus species. Furthermore, an increase in the number

of stomates would also increase their transpiration rate.

This study aimed to determine the number of stomates and determine the transpiration

rate in the Citrus species. Specifically, the relationship between the number of stomates present in

their leaves and their transpiration rates. It was conducted at The Institute of Biological Sciences,

Wing C, room 127 in the University of the Philippine Los Baños last November 21, 2019.

1
A Scientific paper submitted in partial fulfillment of the requirements in BIO 11.1 (Investigative
Biology Laboratory) laboratory under Prof. Aimee Cagalawan, 1st semester, 2019-2020.
 MATERIALS AND METHODS

Figure 1.1 Schematic diagram of the research methodology

In determining the transpiration rates of the Citrus species, two set-ups were made from

each plant sample, Calamansi (Citrus microcarpa) and Pomelo (Citrus maxima). This corresponds

to the replicates used in the experiment. To measure their transpiration rates, leafy shoots from

each mature plant was obtained. The size of the leaves was taken into account since, Pomelo plants

have larger leaves, a 1:3 ratio of Pomelo: Calamansi leaves was employed. Theses shoots were

cut diagonally in their stem to allow for a longer surface area for the uptake of water. Thereafter,

the shoots were immediately placed inside the flasks with a 2-hole rubber stopper filled with

distilled water. The shoot was inserted in one hole while the glass tube was inserted into the other

hole. The upper portion of the flasks were sealed with paraffin wax to accurately measure volume

displacement. To measure their volume displacements each set-up was made to transpire for 20

minutes, measuring their volume displacement in 4-minute intervals.

1
A Scientific paper submitted in partial fulfillment of the requirements in BIO 11.1 (Investigative
Biology Laboratory) laboratory under Prof. Aimee Cagalawan, 1st semester, 2019-2020.
To determine the volume of water lost during transpiration the following formula was used:

𝑉 = 𝜋𝑟 2 ℎ

Where:

r= the radius of the glass tube

h= the height of the water lost in the glass tube per time interval

In determining their transpiration rates the following formula was used:

𝐹𝑖𝑛𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒 − 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒

𝑅𝑎𝑡𝑒 =
𝑇𝑖𝑚𝑒

Figure 2.1 Transpiration set-ups of Calamansi (Citrus microcarpa) and Pomelo

(Citrus maxima) plants respectively

To determine the number of stomates, the imprinting method was employed. To do this, a

fresh leaf sample from each plant was obtained. A 1 cm2 area of the leaves’ abaxial and adaxial

side were painted with a clear nail polish and were made to dry. Afterwards, the nail polish was

removed using a clear plastic tape and was placed on a clean glass slide. The slides were observed

under HPO.

1
A Scientific paper submitted in partial fulfillment of the requirements in BIO 11.1 (Investigative
Biology Laboratory) laboratory under Prof. Aimee Cagalawan, 1st semester, 2019-2020.
 RESULTS AND DISCUSSION

A B

C D
Figure 3.1 Stomates found on the abaxial side of: (a&b) C. microcarpa (c&d) C. maxima

The stomates of Citrus species were found to be abundant in the abaxial side of the leaves.

In a study by Inyama et al. (2015), the stomates found on the Citrus family were hypostomatic and

anomocytic. This indicates that it is best to measure the number of stomates found on the abaxial

side of a leaf. Since, transpiration occurs through the help of stomates, counting the number of the

stomates found on the abaxial side of a leaf, would be beneficial in measuring their transpiration

rates and accounting for the factors that would affect their rates.

1
A Scientific paper submitted in partial fulfillment of the requirements in BIO 11.1 (Investigative
Biology Laboratory) laboratory under Prof. Aimee Cagalawan, 1st semester, 2019-2020.
Table 1.1 Volume of water lost in the transpiration set-up and the number of stomates

present in the Calamansi (Citrus microcarpa) and Pomelo (Citrus maxima) species
Plant Calamansi A Calamansi B Pomelo A Pomelo B

species

Water level Volume Water level Volume Water Volume Water Volume
height (cm) of water height (cm) of water level of water level of water
lost lost height lost (cm3) height lost
(cm3) (cm3) (cm) (cm) (cm3)
0 min 0.0 0.000 0.0 0.000 0.0 0.000 0.0 0.000

4 mins 0.3 0.059 0.3 0.059 1.2 0.236 1.1 0.216

8 mins 0.6 0.118 0.6 0.118 2.1 0.412 2.1 0.412

12 mins 0.8 0.157 0.8 0.157 2.9 0.569 2.8 0.550

16 mins 1.0 0.196 0.9 0.177 3.8 0.746 3.7 0.726

20 mins 1.2 0.236 1.0 0.196 4.7 0.923 4.6 0.903

Number of 50 41 26 26
Stomates
present
Average 45.5 26
number of
stomates

Pomelo and Calamansi plants have a significant difference in the number of stomates

present in their abaxial sides. Table 1.1 presents that Calamansi leaves have an average of 45.5

stomates per unit area, while Pomelo leaves have 26 stomates per unit area. In a study by Inyama

et al. (2015), the number of stomates present per unit area in the Citrus species such as C. sinensis

is 64-70; 60-70 in C. limon; 55-66 in C. aurantifolia; 14-26 in C. reticulata; and 40-50 in C.

paradisi. However, it was stated that Citrus species such as C. limon and C. sinensis share the same

affinity with the highest number of stomates per unit area. This proves that even though, C.

1
A Scientific paper submitted in partial fulfillment of the requirements in BIO 11.1 (Investigative
Biology Laboratory) laboratory under Prof. Aimee Cagalawan, 1st semester, 2019-2020.
microcarpa and C. maxima share the same family, they do not necessarily share the same affinity

for the number of stomates present.

Figure 3.2 Transpiration rates of Calamansi and Pomelo plants within 20 minutes

0.06
0.057

0.05
0.047
0.044 0.044
VOLUME (CM3/MIN)

0.04
0.037

0.03

0.02
0.015 0.015
0.01 0.01
0.008 0.007

0 0
0 4 8 12 16 20
TIME (MIN)

Calamansi Pomelo

There is a difference between the transpiration rates of Calamansi and Pomelo plants.

Figure 3.2 shows that Pomelo yielded a faster transpiration rate as compared to Calamansi plants.

The values obtained for the transpiration rate of Pomelo was higher than that of the Calamansi.

Pomelo plants have an average rate of 0.038 cm3/min, while Calamansi plants only have a 0.009

cm3/min transpiration rate. In a study by Aina & Malik (2013), Citrus species such as C. sinensis

and C. limon were tested against Myrtaceae species such as E.torelliana and E.camadulensis to

determine their transpiration rates. It was stated that the Myrtaceae species have a complex

stomatal type with five or more epidermal cells having direct contact with guard cells, produced

faster transpiration rates as compared to Citrus species with no such stomates. This indicates that

1
A Scientific paper submitted in partial fulfillment of the requirements in BIO 11.1 (Investigative
Biology Laboratory) laboratory under Prof. Aimee Cagalawan, 1st semester, 2019-2020.
transpiration rates may also be related to the number of epidermal cells in direct contact with the

guard cells.

Table 1.2 Analysis of Variance of the Recorded Data

ANOVA

Source of SS df MS F P-value F crit

Variation
Between Groups 0.00119025 1 0.00119025 952.2 0.001 18.5128

Within Groups 0.00000250 2 0.00000125

Total 0.00119275 3

Table 1.2 presented the results of the One-way Analysis of Variance (ANOVA) used in

determining the significant difference of the Citrus species’ transpiration rate. The computed p-

value is 0.001 which is less than the level of significance of 0.05. Thus, the null hypothesis is

rejected. Instead, it shows that there is a significant difference on the transpiration rates of the

Citrus species.

Table 1.3 Correlation Analysis of the Variables

Number of Stomates Transpiration Rate

Number of Stomates 1

Transpiration Rate -0.936952899 1

On the contrary, Table 1.3 showed the results of the correlation analysis to determine the

relationship between the number of stomates and the transpiration rates. It presents that for every

1
A Scientific paper submitted in partial fulfillment of the requirements in BIO 11.1 (Investigative
Biology Laboratory) laboratory under Prof. Aimee Cagalawan, 1st semester, 2019-2020.
increase in the number of stomates, there is a decrease of 0.936 cm3/min on the plant’s transpiration

rate. This contradicts a study by Mirbabaei (2016), wherein it was stated that the more stomates

present on a plant leaf, the higher its transpiration rate. This may be due to external factors such

as, air temperature, relative humidity, wind speed and radiation intensity which were not regulated

throughout the experiment.

SUMMARY AND CONCLUSION

Using the transpiration set-ups, transpiration rates of Calamansi (Citrus microcarpa)

and Pomelo (Citrus maxima) plants were obtained. Results presented that Pomelo plants have

an average transpiration rate of 0.038 cm3/min, while Calamansi plants have an average rate

of 0.009 cm3/min. Pomelo plants have a higher rate of transpiration as compared to Calamansi

plants. Regarding the number of stomates present on their abaxial sides, Calamansi leaves

contain a higher average number of stomates with 45.5, while Pomelo leaves only have an

average of 26 stomates. Results indicate that there is a significant relationship existing between

the number of stomates and their transpiration rates. However, the hypotheses were only

partially accepted, because an increase in the number of stomates did not necessarily mean that

there is also an increase in their transpiration rates. The results otherwise proved that, for every

increase in the number of stomates, there is a decrease of 0.936 cm3/min on the plant’s

transpiration rate. Other factors might have affected the results of the experiment. External

factors such as, air temperature, relative humidity, wind speed and radiation intensity which

were not maintained throughout the experiment. Factors such as diameter of the stomates,

movement of the stomates and direct contact of the epidermal cells with the guard cells were

1
A Scientific paper submitted in partial fulfillment of the requirements in BIO 11.1 (Investigative
Biology Laboratory) laboratory under Prof. Aimee Cagalawan, 1st semester, 2019-2020.
 also not taken into account. Future researchers, may wish to include these factors in their

future study. Moreover, an addition of the positive and negative control may also increase the

accuracy of the study.

Literature Cited

AINA DO & MALIK M. 2013. Stomatal Complex and transpiration rates in some members of Rutaceae
and Myrtaceae. https://www.ajbls.com/sites/default/files/AsianJBiolLifeSci_2_2_170.pdf
[accessed 02. December 2019].

BRAWNER M. 2011. Transpiration and Why It Matters. https://harlequinsgardens.com/transpiration-

and-why-it-matters/ [accessed 30. November 2019].

CLARK MA, CHOI J, DOUGLAS M. 2018. Biology 2e. OpenStax. 227-228pp.

DUKA IM, VILLA N, DELA VIÑA C, MANUEL MA, REAMILLO MC, RODRIGUEZ JJR, LADO J,
ADAJAR JC, GONZALES DR, MANALANG A, ALCABEDOS R, and PASCUAL E. 2018.
BIO 11.1 Laboratory Manual: Investigations in College Biology. 1st ed., University of the
Philippines Los Baños. 88p.

GREENTUMBLE. 2019. How Do Plants Help the Environment. https://greentumble.com/how-do-plants-

help-the-environment/ [accessed 29. November 2019].

INYAMA CN, OSUOHA VUN, MBAGWU FN, and DURU CM. 2015. Comparative Morphology of the
Leaf Epidermis in Six Citrus Species and its Biosystematic Importance. Med Aromat Plants 4:
194. doi:10.4172/2167-0412.1000194

MIRBABAEI D. 2016. The Effect of the Number of Stomata on the Transpiration Rate of Plants.
http://cssf.usc.edu/History/2016/Projects/S1811.pdf [accessed 02. December 2019].

SHIPUNOV A. 2018. Introduction to Botany. Minot State University. 88-90pp.

TAYLOR MR, DICKEY JL, SIMON EJ, HOGAN K, and REECE JB. 2018. Campbell Biology: Concepts
& Connections. 9th ed., Pearson Education, Inc. 650pp.

WILKIN D, and JEAN B. 2019. Importance of Plants. https://www.ck12.org/biology/importance-of-

plants/lesson/Importance-of-Plants-BIO/ [accessed 29. November 2019].

1
A Scientific paper submitted in partial fulfillment of the requirements in BIO 11.1 (Investigative
Biology Laboratory) laboratory under Prof. Aimee Cagalawan, 1st semester, 2019-2020.