A study to investigate the effect of increasing sodium chloride (NaCl) concentration

(50mM, 100mM,150mM and 200mM) on the morphological and biochemical properties

of rice seedling (Oryza sativa).

RESEARCH QUESTION: To what extant does increasing concentration of NaCl (0mM,

50mM, 100mM, 150mM, and 200 mM) affect the shoot length and the chlorophyll content of

Gosaba rice seedlings (Oryza sativa) under lab conditions for seven days?

SUBJECT AREA: BIOLOGY

Word Count: 3958

1
 ABSTRACT

Rice is cultivated in 114 nations all around the world having an incredible financial

importance. Rice is vulnerable to salinity, at the early vegetative stages. With the aim of

creating salt-tolerant rice cultivars, a few morphological examinations have been

practiced. This paper seeks to focuses on how increasing concentration of sodium

chloride (0mM, 50mM, 100mM, 150mM, and 200 mM) affects the shoot length and the

chlorophyll content of Gosaba rice seedlings (Oryza sativa) under laboratory conditions

for a period of seven days?

Rice (Oryza sativa) seedlings were grown and treated with different concentration of sodium

chloride solution . 100g of the rice seedling was separated into five groups and treated with

different concentration of sodium chloride. After a week the shoot length and chlorophyll

content was measured by using Arnon’s equation.

The results of the investigation showed; with the increase of NaCl concentration,
the shoot length and the chlorophyll content decreased. Even at NaCl concentration of

200mM, the rice seedling showed growth as well as sufficient amount of chlorophyll content.

Sundarbans is a region with affected by high levels of salinity. There is a relation because as

the salt stress increases more and more plants develop tolerance against it.

(Word count :200 words)

2
 Table of Contents:

Chapter 1…………………………………………………………………………………...1-5
Introduction

Chapter 2 …………………………………………………………………………………6-14
Materials and Method

Chapter 3 ……………………………………………………………………………......15-26
Results

Chapter 4………………………………………………………………………………...27-29
Evaluation and Conclusion

Bibliography……………………………………………………………………………..30-33

Appendix…………………………………………………………………………………34-38

3
 CHAPTER 1

INTRODUCTION:

1
Rice is one of the most important cereal crops, a staple food that is consumed by more than

half of the world’s population. 2There are essentially two species of rice that is cultivated for

human consumption namely: Oryza sativa which is grown worldwide and Oryza glaberrima

which is grown in some parts of West Africa. It is an annual grass of the ‘Gramineae’ family.

3
The main rice-producing countries are; India, China, Indonesia, Bangladesh and Philippines.

Rice can be produced in a wide range of locations from the wettest areas to the driest of

deserts. 4It is grown along the Arakan Coast that receives an annual rainfall of more than

5,100mm. 5In contrast to this, it is also grown in Saudi Arabia that receives an annual rainfall

of less than 100mm. It is popularly grown in the Upper Sind in Pakistan where the mean

temperature is 33 ℃, it is grown in Otaku in Japan which has an average temperature of 17℃.

It is also grown under a broad range of solar radiation ranging from 25% to 95%. 6Rice is

classified into different varieties depending on the altitude and water source used to cultivate

it.
7
About 79 million hectares of land is covered by irrigated lowland rice, which is responsible

for 75% of the rice production worldwide. This method of growing rice has been one of the

1
Shakeela, B. S., Chachar, Q.I, S. D., Solangi,, A.B, Solangi, and J.A. Effect of Salinity (NaCl) Stress on
Physiological Characteristics of Rice (Oryza Sativa L.) at Early Seedling Stage. Effect of Salinity (NaCl) Stress
on Physiological Characteristics of Rice (Oryza Sativa L.) at Early Seedling Stage. International Journal of
Agricultural Technology, n.d. Web. 1 Nov. 2018.
2
. "Rice as a Plant." Ricepedia. Research Program on Rice, n.d. Web. 18 Jan. 2019.
3
Cago, Lanessa. "10 Largest Rice Producing Countries." World Atlas. Worldatlas, 27 Jan. 2016. Web. 2 Jan.
2019.
4
"CCAFS: CGIAR Research Program on Climate Change, Agriculture and Food Security." Impact of Water
Scarcity in Australia on Global Food Security in an Era of Climate Change. Climate Change Agriculture and
Food Security, 13 Dec. 2018. Web. Nov.-Dec. 2018.
5
Maclean, J.L, B. Hardy, and G.P Hettel. "Importance of Rice." Rice Almanac Source Book for Most Important
Economic Activity on Earth. By D.C Dawe. 3rd ed. N.p.: CABI, 2002. 4-5. Print.
6
Guang-Jiong, Hou. "Classifying Rice Soils on the Basis of Their Physiological Characteristics." Proceedings
of Symposium on Paddy Soils (1981): 387-91. Print.
7
Bouman, B. A. M., R. M. Lampayan, and T. P. Tuong. Preface. Water Management in Irrigated Rice: Coping
with Water Scarcity. Los Baños, Philippines: International Rice Research Institute, 2007. Iv. Print.

1
most sustainable agricultural systems in the world retaining the nutrient cycle. 8Irrigation

water contains different kinds of dissolved salts the most common ones being table salt

(NaCl) and gypsum (CaSO4). The most common cations are calcium (Ca2+), magnesium

(Mg2+) and sodium (Na+). 9,10

These ions are released into the soil through the weathering of

rocks or human activity, which is one of the main causes of soil salinity.
11
Salinity is the presence of salt in soil and water.12 It is one of the most crucial factors

limiting agriculture in the arid and semi-arid regions of the world. There are two kinds of

salinity; i) primary salinity that occurs naturally such as irrigation water. ii) secondary

salinity that is caused due to human activity such as the use of fertilizers. It is expected to

vigorously affect the plant productivity in the coming years.13 14At present about 800 million

hectares of total surface land in the word is affected by salinity which reduces the average

production potential of crops. Every year more and more land become susceptible to soil
15,16
salinity. In India out of 187.8 million hectares of land, 10.1 million hectares of land is

affected by soil salinity. Increasing soil salinity also affects water resources.17The major

8
Grattan, Stephen R. Irrigation Water Salinity and Crop Production. Rep. no. 8066. Davis: U of California
Agriculture and Natural Resources, 2002. Print.
9
"Salts and Trace Elements." Soil and Water Quality an Agenda for Agriculture. Washington, D.C.: National
Academy, 1993. 361. Print.
10
Thien, S.J J., and J. G. Graveel. "ENVIRONMENTAL INFLUENCES ON SOIL FORMATION." Little
Crow. Laboratory Manualfor Soil Scienc, n.d. Web. 9 Oct. 2018.
11
Queensland, The State of. "Types of Salinity | Salinity." Transport and Motoring | Queensland Government.
CorporateName=The State of Queensland; Jurisdiction=Queensland, 08 Oct. 2013. Web. 24 Oct. 2018.
12
Phap, Vu Anh., Hohen Landwirtschaftlichen Fakultät, Der Agrarwissenschaften, Dr, and Rheinischen
Friedrich-Wilhelms-Universität. Institut Für Molekulare Physiologie Und Biotechnologie Der Pflanzen - Der
Universität Bonn, Aug. 2006. Web. 29 Dec. 2018.
13
Ahmad, Parvaiz, and Mohd Rafiq. Wani. "Effect of Salinity on Plants and the Role of Arbuscular Mycorrhizal
Fungi and Plant Growth-Promoting Rhizobacteria in Alleviation of Salt Stress." Physiological Mechanisms and
Adaptation Strategies in Plants under Changing Environment. Vol. 1. New York, NY: Springer, 2014. 116.
Print.
14
Thi My Linh Hoang, Thach Ngoc Tran, Thuy Kieu Tien Nguyen, Brett Williams, Penelope Wurm 3, Sean
Bellairs, and Sagadevan Mundree 1,*. Improvement of Salinity Stress Tolerance in Rice: Challenges and
Opportunities. Agronomy, 2016. Web. 28 Nov. 2018. <https://www.mdpi.com/2073-4395/6/4/54/htm>.
15
Bhawan, Krishi. "Introduction." Introduction. Overnment of India Ministry of Agriculture & Farmers Welfare
Department of Agriculture, Cooperation& Farmers Welfare (Natural Resource Management Division). New
Delhi: n.p., 2016. 1-2. Print.
16
"Introdution." N.p., n.d. Web. 19 Jan. 2019.
<http://shodhganga.inflibnet.ac.in/bitstream/10603/23739/7/07_chapter_01.pdf>.
17
Székely, Árpád, Tímea Szalóki, János Pauk, and Mihály Janice. Effect of Salinity on Rice (Oryza Sativa L.) in
Seedling Stage. National Agricultural Research and Innovation Centre, Research Department of Irrigation and

2
inhibitory effect caused due to salinity is- osmotic effect, ion toxicity, and nutritional
18
imbalance. This results in low crop productivity creating famine-like conditions, resulting

in the loss of fertility in cultivable lands. This can be disastrous with a growing population.

Rice can tolerate some amount of salinity without affecting much in its growth,19 20,21
it is

affected mainly at its early vegetative state, as it grows it becomes more tolerant to salt.
22
Salinity is found to cause physiological changes in respiration, photosynthesis, nitrogen

fixation causing growth inhibition and yield loss. In the study conducted by; Y. Ali, Z.

Aslam, M. Y. Ashraf and G. R. Tahir they concluded that there is a reduction in chlorophyll

content due to the inhibitory effect caused by various salts.

23
The effect of salinity on rice growth include - decrease in the germination rate, reduced

plant height, poor root growth and increase in the sterility of the rice seedling.

24
One of the most important physiological activity affected by salinity is photosynthesis.

Water Management. Cereal Research Non-profit Ltd., 1 Nov. 2017. Web. 18 Jan. 2019.
<http://columella.mkk.szie.hu/archives/columella_7_suppl_2017/Szekely_etal.pdf>.
18
"Soil Salinity." NeuroImage. Academic Press, n.d. Web. 7 Dec. 2018.
<https://www.sciencedirect.com/topics/earth-and-planetary-sciences/soil-salinity>.
19
Thi My Linh Hoang, Thach Ngoc Tran, Thuy Kieu Tien Nguyen, Brett Williams, Penelope Wurm 3, Sean
Bellairs, and Sagadevan Mundree 1,*. Improvement of Salinity Stress Tolerance in Rice: Challenges and
Opportunities. Agronomy, 2016. Web. 28 Nov. 2018. <https://www.mdpi.com/2073-4395/6/4/54/htm>.
20
"Effect of Salinity on Morphological and Physiological Characteristics in Correlation to Selection of Salt
Tolerance in Rice (Oryza Sativa L.)." Enzymes Application in Diagnostic Prospects. Academic Journals Inc.,
USA, n.d. Web. 17 Sept. 2018. <https://scialert.net/fulltextmobile/?doi=ijar.2011.780.788#28412_ja>.
21
Linghe Zeng, and Michael C. Shannon. Salinity Effects on Seedling Growth and Yield Components of Rice.
Alliance of Crop, Soil and Environmental Science Societies. American Society of Agronomy, July 2000. Web.
Dec.-Jan. 2018-2019. <https://dl.sciencesocieties.org/publications/cs/abstracts/40/4/996>.
22
’Mechanism of Salinity Tolerance in Plants: Physiological, Biochemical and Molecular Characterization.’’
International Journal of Genomics n.d. Web. 1Jan 2019.
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3996477/>
23
"Salinity." By-products - IRRI Rice Knowledge Bank. Your Information Source for Farming. Rice Knowledge
Bank, n.d. Web. 29 Nov. 2018. <http://www.knowledgebank.irri.org/decision-tools/rice-doctor/rice-doctor-fact-
sheets/item/salinity>.
24
M. Djanaguiraman, and R. Ramadass. EFFECT OF SALINITY ON CHLOROPHYLL CONTENT OF RICE
GENOTYPES. Agric. Sci. Digest. Department of Crop Physiology, Tamil Nadu Agricultural University,
Coimbatore, n.d. Web. 23 Aug. 2018. <https://arccjournals.com/uploads/articles/asd243006.pdf>.

3
25
High salt concentration in the soil makes it hypertonic which decreases the water potential

within the cell, thus increasing the concentration of ions inside the cell.

26
Chlorophyll is a green color photosynthetic pigment found in cyanobacteria and green

plants which helps absorb sunlight. Chlorophyll is the key part for the plant’s survival and

growth. Their molecules are specifically arranged in and around photosystems that are

embedded in the thylakoid membranes of chloroplasts. It plays a very important role in

photosynthesis, which is one of the most important physiological processes. There are five

types of chlorophyll; chlorophyll a and chlorophyll b which is found in green plants whereas

chlorophyll c, chlorophyll d and chlorophyll e is found in green bacteria and algae.

27
1.Chlorophyll a (C55H77O5N4): it is the primary photosynthetic pigment, it directly absorbs

light during photosynthesis. .

2.Chlorophyll b (C55H70O6N4Mg): it is an accessory pigment that acts indirectly in

photosynthesis by transferring the light to chlorophyll a.

25
M.A Hakim, Abdul Shukor Juraimi, M.M Hanafi, Mohz Razi Ismail, M.Y Rafil, Fasad Aslani, and A.
Selamat. The Effect of Salinity on Chlorophyll, Proline and Mineral Nutrients in Common Weeds of Coastal
Rice Fields in Malaysia. Journal of Environmental Biology. N.p., n.d. Web. 24 Oct. 2018.
<http://www.jeb.co.in/journal_issues/201409_sep14/paper_13.pdf>.
26
"Chlorophyll." ScienceDaily. ScienceDaily, n.d. Web. 30 Nov. 2018.
<https://www.sciencedaily.com/terms/chlorophyll.htm>.
27
"The Difference between Chlorophyll A & B and Photosynthesis Overview." Dyna-Gro. N.p., 07 Apr. 2018.
Web. 19 Jan. 2019. <https://dyna-gro.com/the-difference-between-chlorophyll-a-b-and-photosynthesis-
overview/>.

4
 28

Molecular structure of chlorophyll 28

This research paper focuses on how does increasing concentration of NaCl (0mM, 50mM,

100mM, 150mM, and 200 mM) affect the shoot length and the chlorophyll content of

Gosaba5 rice seedlings (Oryza sativa) under lab conditions for seven days?

28
"Lab Report on Isolation of Chlorophyll and Beta Carotene." Artxy. N.p., n.d. Web. 30 Dec. 2018.
<http://www.art-xy.com/2013/03/isolation-of-chlorophyll-and-beta.html>.

5
 CHAPTER 2:

Materials and method

Objective of the study:

Objective 1: To study the change in the shoot length of the rice seedling (Oryza sativa) with

increasing salt stress.

Null Hypothesis: The shoot length of all the rice seedling (Oryza sativa)remains the same.

Alternate Hypothesis: The shoot length of rice seedling will decrease with the increase of salt

stress (50mM, 100mM, 150mM and 200mM).

Objective 2: To observe the effect in total chlorophyll content in the rice seedling (Oryza

sativa) with increasing soil salinity.

Null Hypothesis: The total chlorophyll content of the rice seedling remains same with the

increase in salt stress (50mM, 100mM, 150mM and 200mM).

Alternate Hypothesis: The total chlorophyll content in the rice seedling would decrease as the

salt stress increases.

Objective 3: A comparative study between the concentration of chlorophyll a and chlorophyll

b with an increase in the salt stress.

Null Hypothesis: The concentration of both chlorophyll a and chlorophyll b remains the same

with an increase in the salt stress.

Alternate Hypothesis: The concentration of chlorophyll b decreases more than that of

chlorophyll a with an increase in the salt stress.

6
Variables:

Independent Variable:

Variable Unit/ Method of Measurement

Uncertainty

The seeds were exposed to different concentration of

Sodium Chloride NaCl solutions- (0mM , 50mM, 100mM, 150mM,

(NaCl) ±0.04mM 200mM)

concentration

Dependent Variable:

Variable Unit/ Uncertainty Method of Measurement

Shoot Length ± 0.01cm Using a 30cm ruler.

Total Chlorophyll ± 0.03 mg/l The chlorophyll was extracted and its absorbance

Content was measured using a visible spectrophotometer

The chlorophyll content was estimated using

Arnon’s equation.

Controlled Variable:

Variable Unit/ Uncertainty Method of control It’s Impact if not controlled

Rice Variety -- Only one variety of Different rice variety have

rice (Gosaba) was difference tolerance to salt

used. giving different results

7
Temperature ± 0.1°C The rice seedlings The increase or decrease in

were kept under a temperature effects the

constant growth. At high or low

temperature in the temperatures the

lab at 25°C photosynthetic enzymes do

not work efficiently as they

lose their shape and

function.

Light Intensity --- The rice seedlings Decrease in light intensity

were placed under reduces the plant growth,

the same light development and yield as

source decrease in light intensity

reduces the rate of

photosynthesis.

Volume of NaCl ± 0.05ml 20ml of sodium Adding excess or less of

solution added in chloride (NaCl) NaCl would affect the

the paper cups. solution was added absorption affecting the

to each of the set. growth.

Size of the paper -- All the paper cups Using different paper cups

cups used were of the of different sizes would

same length, width, have different volume

and height. would, change the space for

growth thus giving irregular

growth patterns.

Nutrients. -- Only sodium Using different salts would

8
 chloride (NaCl) allow the uptake of other

solution was added. nutrients affecting the

morphological and

biochemical and

physiological properties of

the plant.

Number of rice -- Only five rice If different number of rice

seedling used to seedling were seedling were taken to test

take the shoot chosen at random the shoot length and the

length. length and to test total chlorophyll then the

the total results obtained could not

chlorophyll be compared.

content.

Mass of the ±0.05 100g of the rice If different mass of the rice

seedling taken for seedling was seedling was taken then

growth in NaCl divided into five there would be irregular

solution. group each pattern of germination and

containing 20g the results obtained could

using a digital be compared.

weighing balance.

Time period for the The rice seedling if the time period is not

growth of rice were allowed to maintained for all the plants

seedling. grow for a period then the shoot length

of seven days. obtained cannot be

9
 compared to each other,

Materials Required:

Experimental Sample

Sample Quantity Unit

Oryza sativa seeds
(Gosaba) collected from
29 150 G
Gosaba Rice Research
Center.

CHEMICALS USED:

Name of the chemical Quantity Unit

Sodium Chloride (NaCl) 500 mM
Hogland Solution 1.63 G
Distilled Water 2000 Ml
80 % Acetone 80 Ml
sodium hypochlorite (NaOCl) 50 Ml

INSTRUMENTS USED:

Instrument Quantity Unit Uncertainty

Visible Spectrophotometer 800nm 1 nm ±0.02
Centrifuge machine (REMI R8C)
1 rpm ±0.001
5000rpm
Digital Weighing Balance 1 g ±0.05
APPARATUS USED:

Apparatus Quantity Unit Uncertainty

Measuring Cylinder 500 ml ±0.05
Tips 50 ml ±0.001
Paper Cups 50 - -
Eppendorf 2 ml -
Motor and pestle - - -

29 Refer to appendix A

10
MISSELENIOUS ITEMS:

Item Quantity Unit Uncertainty

Ruler 1 cm ±0.01
blotting paper - - -
Paper Cups 50 - -

Procedure:

Collection of seeds (sample for experiment):

 150g of Gosaba rice seeds were collected from the 30

Gosaba Research Rice Center,

located in Sunderbans, in West Bengal.

Preparation of different concentration of NaCl solution:

 200mM of sodium chloride (NaCl) was prepared by dissolving 11.69g of NaCl in

1000ml of distilled water.

 To prepare 50mM/100mM/150mM of NaCl, the volumes were adjusted by using the

formula V1S1=V2S2 where;

 S1= the concentration of stalk solution 200mM

 V2= the required volume 100mL

 S2= concentration of the desired salt (50mM/100mM/150mM)

 100ml of sodium chloride solution was measured using a measuring cylinder from

each of the concentration. (50mM, 100mM, 150mM and 200mM)

Germination of seeds and growth of seedling:

 100g of the rice seeds were weighed using a digital weighing balance and placed in a

petri dish.

30 Refer to Appendix A for details

11
  The seeds were washed in normal tap water, the surface was sterilized with 5%

sodium hypochlorite (NaOCl) for 15 minutes.

 They were then washed with sterile distilled water thrice and placed in a petri dish.

 10% of Hoagland media solution was prepared by dissolving 1.63 g of Hoagland salt

in 1000 ml of water.

 The Hoagland solution was added to the germinating seeds and allowed to grow for

seven days under laboratory conditions.

 After the seeds germinated they were separated and 20g of the germinated seeds were

put into five different cups and labeled based on the sodium chloride (NaCl)

concentration that was used. The seeds were grown only in the presence of sodium

chloride solution.

 After a week the readings were taken for morphological and biochemical properties.

Measurement of shoot length:

 The rice seedlings were taken out of the cup and washed with normal tap water

 The rice seedling was then gently tapped with a blotting paper until the surface of

the roots dried.

 Five seedling were chosen at random from each of the set and separated.

 The selected rice seedling was placed on the blotting paper with a ruler beside it

and the shoot length of the rice seedling was marked.

Chlorophyll estimation:

 50mg of the sample from each of the Sodium chloride (NaCl) concentration was

weighed.

12
  80% acetone was prepared by adding 80ml of acetone and 20ml water in a measuring

cylinder.

 The rice seedling from each of the set (from different concentrations of NaCl) was

crushed in the motor pestle with 2ml of 80 % acetone.

 2ml of the crushed sample were poured into an eppendorf and placed in the centrifuge

machine and centrifuged at 3000rpm for 6 minutes, using the centrifuge machine.

 Once the chlorophyll pigment comes in the supernatant, and the tubes were taken out.

 The spectrophotometer absorbance specification was set at 645 nm.

 80% acetone was poured into a cuvette and placed in the visible spectrophotometer as

blank.

 The centrifuged solution was poured in the cuvette and placed in the visible

spectrophotometer at 645nm.

 The absorbance was measured at 654nm and the data was collected.

 The wavelength was set to 663 nm and the absorbance value of the samples were

noted.

 For the shoot length; the mean of the different concentrations was taken out and a for

shoot length v/s sodium chloride concentration was plotted on a bar graph.

 For chlorophyll estimation Arnon’s formula was used. For Total Chlorophyll; 31Total

Chl (mg/l) = 0.0202 Absorbance 663 + 0.00802 Absorbance 645 was used and mean

was taken out and plotted on a bar graph .

 For chlorophyll a; Chl a (mg/l) = 0.0127 Absorbance 663 – 0.00269 Absorbance 645

was used and then mean was taken out and plotted on a bar graph.

31 Dr. Stephen G. Saupe. "Plant Physiology BIOLOGY (372)." Structure & Reactivity .auto-style6 { Font-
family: "Times New Roman", Times, Serif; } .auto-style3 { Margin-left: 80px; } .auto-style4 { Font-family:
"Times New Roman", Times, Serif; Font-weight: Bold; } .auto-style7 { Text-decoration: Underline; } .auto-
style8 { Margin-left: 40px; }. College of St. Benedict/ St. John's University; Biology Department, n.d. Web. 06
Feb. 2019. <https://employees.csbsju.edu/ssaupe/biol327/Lab/photosyn/chlorophyll_quant.htm>.

13
  For chlorophyll b; Chl b (mg/l) = 0.0029 Absorbance 663 – 0.00468 Absorbance

645.32

 The mean values were taken out and a bar graph was plotted for all.

 Two correlation studies were performed to test i)the relationship between shoot length

and increasing NaCl concentration and ii) the relationship between total chlorophyll

content and increasing NaCl concentration.

 A t-test was performed to compare the concentration of chlorophyll a with respect to

chlorophyll b.

Safety procedure.

 gloves and lab coats were worn, while working with the seeds and preparing the

chemicals

 Caution was taken while washing the seedling as roots and the leaves may detach

from the seedling.

32 Refer to appendix B

14
 CHAPTER 3

Results

Qualitative Data:

The result expressed shows that the shoot length decreases with the increase in sodium

chloride concentration. The increase in salinity severely affected the shoot length.

Oryza sativa in control

setup (0mM)

Oryza sativa treated

with 50mM NaCl

Oryza sativa treated

with 100mM NaCl

Oryza sativa treated

with 150mM NaCl

Oryza sativa treated

with 200mM NaCl

Figure 1: Oryza sativa treated with different concentrations of sodium

chloride (NaCl)solution

15
Quantitative Data:

Table 1: A table to show the shoot length of the rice seedling after being treated with

different concentrations of sodium chloride (NaCl).

Concentration of Shoot Length Mean of the shoot Standard deviation

NaCl (Mm) ±0.1 cm length ±0.1 cm

±0.04mM

Control 19.00

16.80

17.20 18.08 1.03

18.40

19.00

50 16.40

20.00

16.20 16.34 2.39

15.90

13.30

100 13.60

14.30

12.10 14.12 1.45

16.10

14.50

150 10.8

12.9

12.00 12.20 1.33

16
 12.10

11.20

200 9.00

10.60

9.70 9.80 0.73

10.50

9.20

Figure 2 showing the decrease of the shoot length with the increase in salt salinity.

The effect of increasing salt stress on the shoot length of rice seedlings.
Control
50
100
Shoot Length (±0.1 cm )

150
200

Concentration of NaCl(±0.04mM)

Figure 2: A graph to show the effect of increasing salt stress on the shoot length of
rice seedlings. (the error bars signify the standard deviation)

Figure 2 provides support to the experimental hypothesis in objective 1. The error bars

signify the standard deviation. The first evidence is the decreasing trend line showing a

negative correlation; it indicates that the shoot length is inversely proportional to salt

concentration. The raw data bolsters this, as the group of rice seedling, which was not treated

17
with sodium chloride (NaCl), had the highest shoot length - 18.08 cm. Whereas the rice

seedling treated with the highest sodium chloride (NaCl) concentration had the lowest shoot

length 9.80cm. The percentage decrease in shoot length was 12.5%, 21.9%, 32.5% and 45.8%

at 50mM, 100mM, 150mM and 200mM respectively.

Table 2: A table showing chlorophyll content of rice seedling in different sodium chloride

(NaCl) concentrations at the light intensity of 645 nm and 663nm.

Concentration of NaCl Absorbance of the Absorbance of the chlorophyll

±0.04mM chlorophyll content of the content of the rice seedling at

rice seedling at 645nm 663 nm ±0.02𝑛𝑚

±0.02𝑛𝑚

Control 0.31 0.61

0.29 0.58

0.28 0.65

0.27 0.57

0.28 0.57

50 0.20 0.47

0.21 0.45

0.25 0.47

0.24 0.46

0.25 0.49

100 0.21 0.41

0.19 0.42

0.20 0.43

0.22 0.42

18
 0.22 0.42

150 0.20 0.37

0.20 0.41

0.20 0.39

0.19 0.38

0.21 0.37

200 0.12 0.29

0.21 0.30

0.20 0.26

0.19 0.28

0.18 0.29

Table 3: a table showing the decrease total chlorophyll content of rice seedlings at different

sodium chloride (NaCl) concentrations

Concentration of Total chlorophyll Mean of the Total Standard deviation

NaCl ±0.04mM content ± 0.03 mg/l Chlorophyll

content ± 0.03 mg/l

Control 11.29 10.73 0.40

10.69

10.92

10.26

10.47

50mM 7.86 8.36 0.52

7.80

19
 8.72

8.45

8.96

100mM 7.51 7.54 0.30

7.07

7.53

7.86

7.75

150mM 7.09 7.14 0.13

7.33

7.12

6.97

7.17

200mM 4.76 5.94 0.69

6.59

6.22

6.04

6.07

20
Figure 3 showing the decrease of total chlorophyll content of rice seedling at with increasing

sodium chloride (NaCl) concentrations.

The effect of increasing sodium chloride (NaCl) concentrations on the

Total Chlorophyll Content of rice seedlings control
Total Chlorophyll content (± 0.03 mg/l )

50
100
150
200

Concentration of NaCl (±0.04Mm)

Figure 3 A graph to show the effect of increasing sodium chloride (NaCl)

concentrations on the total chlorophyll content of rice seedlings. (the error bars signify the
standard deviation)

Figure 3 provides support to the experimental hypothesis in objective 2 . The error bars

signify the standard deviation The first evidence being the decreasing trend line showing a

negative correlation; it shows that the total chlorophyll content and the salt stress are

inversely proportional. The group of rice seedling that was not treated with sodium chloride

(NaCl) had the highest chlorophyll content of 10.73 mg/l. Whereas the group of rice seedling

that was treated with the highest concentrations sodium chloride (NaCl) concentration had

the lowest chlorophyll content of 5.94 mg/l.

21
Table 4: A table comparing chlorophyll a and chlorophyll b values of the rice seedling at

different sodium chloride (NaCl) concentrations.

Concentration of NaCl Chlorophyll a ± 0.03 mg/l Chlorophyll b ± 0.03 mg/l

±0.04mM

Control 6.99 4.30

6.67 4.03

7.55 3.37

6.58 3.69

6.56 3.91

50 5.48 2.34

5.21 2.59

5.28 3.44

5.19 3.28

5.53 3.44

100 4.58 2.94

4.79 2.29

4.89 2.65

4.78 3.08

4.68 3.07

150 4.16 2.94

4.64 2.69

4.38 2.75

4.32 2.66

22
 4.20 3.07

200 3.34 1.42

3.27 3.33

2.78 3.58

3.01 3.03

3.28 2.69

Figure 4 showing the comparison between the two chlorophyll a and chlorophyll b values

with increasing sodium chloride (NaCl) concentrations. 33

Comparison between chlorophyll a and chlorophyll b

with the increase in sodim chloride (NaCl).
Concentration of chlorophyll a and

9
chlorophyll b (± 0.03 mg/l )

8
7
6
5
4 chlorophyll a
3
chlorophyll b
2
1
0
control 50 100 150 200
Concentration of NaCl(±0.04mM )

Figure 4 : A comparative study between chlorophyll a and chlorophyll b in with

the increase in sodium chloride (NaCl) concentrations. (the error bars signify the
standard deviation)

Figure 4 supports the experimental hypothesis in objective 3 The error bars signify the

standard deviation. The group of rice seedling not treated with sodium chloride (NaCl) had

the highest chlorophyll a and chlorophyll b value; chlorophyll a value is 6.87 mg/l whereas

33 Raw data table for chlorophyll a and chlorophyll b in appendix b.

23
chlorophyll b content equal to 3.86 mg/l. The group of rice seedling that was treated with the

highest sodium chloride (NaCl) concentrations (200mM) had the lowest chlorophyll a and

chlorophyll b value; chlorophyll a value is 3.14 mg/l and chlorophyll b value being 2.79 mg/l.

Overall chlorophyll a content was greater than the chlorophyll b content.

Statistical Analysis:

Figure 5: Correlation between shoot length with increasing NaCl concentrations

Correlation between shoot length and different NaCl concentrations .

y = 21.738e-0.152x
Shoot Length (±0.1 cm )

R² = 0.9819

Shoot Length(cm)

Expon. (Shoot
Length(cm))

Concentration of NaCl(±0.04mM )

Figure 5:Correlation between shoot length in increasing sodium chloride (NaCl)

concentrations

24
Figure 6: correlation between Total Chlorophyll Content (mg/l) in increasing sodium chloride

(NaCl) concentrations

Correlation between Total Chlorophyll Content in different NaCl concentrations

Total Chlorophyll Content (± 0.03 mg/l )

y = -1.08x + 11.182
R² = 0.9141

Total Chlorophyll Content (nm)

Linear (Total Chlorophyll

Content (nm))

Concentration of NaCl(±0.04mM )

Figure 6: correlation between Total Chlorophyll Content (mg/l) in increasing sodium

chloride (NaCl) concentrations
Analysis and Evaluation:

Objective 1: To study the change in the shoot length of the rice seedling (Oryza sativa) with

increasing salt stress.

In this experiment, the correlation was used to see the effect of increasing salt stress on the

shoot length. The equation of the line y = 21.738e-0.152x where ‘y’ represents the total

chlorophyll content and the ‘x’ axis shows the different sodium chloride concentration. As

seen in figure 5, there is a decrease in the trend line showing a negative correlation; the shoot

length and salt stress are inversely proportional to each other, as the salt stress increases from

0mM to 200mM the shoot length decreases. The shoot length decreases from 18.08 cm to

9.80 cm as the NaCl concentration increases. The R2(0.9819)obtained was more than the

critical R-value (0.99080775128). Thus, the experimental hypothesis will be accepted which

25
states that the shoot length would decrease with the increase in NaCl concentration satisfying

objective1.

Objective 2: To observe the effect in total chlorophyll content in the rice seedling (Oryza

sativa) with increasing soil salinity.

As visible in figure 6, the Total Chlorophyll content decreases with the increase in salt stress.

The equation of line y = -1.08x + 11.182 where ‘y’ represents the total chlorophyll content

and the ‘x’ axis shows the different sodium chloride concentration. In figure 6 the trend line

obtained shows a decrease with increase in salt stress. The R2 (0.9141) value was more than

the R-Critical value (0.95607008111). They are inversely proportional to each other. The

highest chlorophyll content was marked in control 0mM that is 10.73 mg/l while the lowest

was for 200mM that is 5.94 mg/l. Thus, the experimental hypothesis will be accepted which

states that the total chlorophyll content will decrease with the increase in NaCl concentration

from 0mM to 250mM .

Objective 3: A comparative study between the concentration of chlorophyll a and chlorophyll

b with an increase in the salt stress.

As seen in figure 4 the concentration of chlorophyll a is more than that of chlorophyll b. The

highest concentration of chlorophyll a is in control 0mM that is 6.86 mg/l and the lowest is

for 200mM 3.14mg/l. As compared to chlorophyll a, in chlorophyll b the highest

concentration was in control 0mM that is 3.86 mg/l and the lowest was present in 200mM

that is 2.79. On performing the t-test the value received was 2.79mg/l which is more than the

critical value (2.31).Thus, the experimental hypothesis will be accepted that is; chlorophyll a

value is more than chlorophyll b value.

26
 CHAPTER 4

Evaluation and Conclusion

Evaluation:

Error Effect on Improvement

Data

Number of trials High Only five trials could have been conducted as the time

period was quite limited. At least 10 to 15 trials could

have been conducted to make the data more valid.

Accuracy and Low Since the samples were crushed manually, and stirred

precision of the manually, it could have been improved upon by using

method automated apparatus like magnetic stirrers and

hydraulic press could have been used.

Conditions for Low The laboratory in which the samples were germinated

germination only had a set of lights and an apparatus to measure

humidity. Since there would be more environmental

conditions such as pressure acting as extraneous

variables on the seedlings a more controlled

environment in a specialized laboratory should be used.

CONCLUSION:

The aim of this experiment was to investigate the effect of increasing salt stress on the shoot

length and the total chlorophyll content. A part of the investigation also included a

comparative study between chlorophyll a and chlorophyll b. Salinity is one of the major

27
factors that affect plant growth and metabolism, leading to severe damage and a loss in

productivity.

The study showed that salinity had a significant effect on growth and on the photosynthetic

pigments. As the salt concentration in the soil increased, the shoot length and the chlorophyll
34
content decreased. The main reason for this is osmosis; it is the movement of water

molecules across the semi-permeable membrane from a more dilute solution to a less dilute

solution. The osmotic effect due to salinity was the main inhibitory factor that reduced the

germination of seeds.

Increase in NaCl concentration also affects the rate of photosynthesis. The area of leaf

affected under the salt- stress increases the oxidative stress (increase in the reactive oxygen

species) which causes the depletion of chloroplast and loss of chlorophyll.

There is an overall decrease in the concentration of both chlorophyll a and chlorophyll b but

the concentration of chlorophyll a is more than that of chlorophyll b. In the t-test the value

obtained was 2.79, which was more than the critical value (2.31) thus the experimental
35
hypothesis in objective 3 could be accepted. The reason why the concentration of

chlorophyll a is more than that of chlorophyll b is because chlorophyll a is the primary

pigment that absorbs the light for photosynthesis, chlorophyll b is an accessory pigment that

absorbs the light and pass it onto chlorophyll a.

34
"Water Uptake and Transport in Vascular Plants." Nature News. Nature Publishing Group, n.d. Web. 19 Nov.
2018. <https://www.nature.com/scitable/knowledge/library/water-uptake-and-transport-in-vascular-plants-
103016037>
35
"The Difference between Chlorophyll A & B and Photosynthesis Overview." Dyna-Gro. N.p., 07 Apr. 2018.
Web. 19 Jan. 2019. <https://dyna-gro.com/the-difference-between-chlorophyll-a-b-and-photosynthesis-
overview/>

28
36
Salinity is one of the biggest environmental problem at present, projects have been

undertaken to fight this problems like:

1. Changing the environment where the crop is grown:

2. Genetically modifying the crop so that it can be made tolerable to such conditions:

this involves breeding the crop varieties with those that have a tolerant to salinity.

This makes the crop tolerant to salinity to some extent thus allowing some production

to take place.

3. By growing varieties of rice like Pobbeli, Indonesia; IR2151, India; Bicol that is

tolerant to salinity.

37
It can be concluded that one of the main reasons why the productivity of the rice crops

decreases with and increase in salinity which is because of the osmotic pressure. It decreases

the chlorophyll content and its ability to trap sunlight also decreases, thus decreasing the

metabolic activities of the plants thus reducing its activity and ability.

36
"Stress and Disease Tolerance." By-products - IRRI Rice Knowledge Bank. N.p., n.d. Web. 1 Jan. 2019.
<http://www.knowledgebank.irri.org/ricebreedingcourse/Breeding_for_salt_tolerance.htm>.
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Biljana Bojović, Gorica Đelić, Marina Topuzović, and Milan Stanković. EFFECTS OF NaCl ON SEED
GERMINATION IN SOME SPECIES FROM FAMILIES Brassicaceae and Solanaceae. Nstitute of Biology and
Ecology, Faculty of Science, University of Kragujevac,. N.p., n.d. Web. 30 Nov. 2018.

29
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33
 APPENDIX

APPENDIX A

A: Gosaba Research Rice Center

38

39
“The Sundarbans is the largest mangrove forest in the world and is shared between India

and Bangladesh. The Indian Sundarbans is inhabited by nearly 5 million people distributed

over 50 odd islands. The major occupations of the local community are agriculture and

capture fisheries. The Sundarbans is a World Heritage site of UNESCO, a Biosphere Reserve

declared by Government of India and also an important Tiger Reserve.

The main goal of this project was to develop sustainable agricultural methods among rice

farmers in the Sundarbans, primarily through the development of indigenous varieties (and

salt tolerant varieties of rice in particular) which could be grown without the use of chemical

pesticides and chemical fertilizers.’’

38
‘’Map of Gosaba Block’’
<https://www.researchgate.net/publication/319527711_Social_Capital_and_Disasters_Analysis_of_th
e_Villages_in_Satjelia_Gram_Panchayat_in_Gosaba_Block_of_the_Indian_Sundarbans>
39
"ENDEV. Ociety for Environment & Development 329, Jodhpur Park, Kolkata 700 068 E-Mail:
Cedkolkata@yahoo.com Cedendev@gmail.com URL: Www.cendev.org."
Http://cendev.org/endev/newsletter/15.pdf. N.p., n.d. Web. 19 Jan. 2019.

34
40
“This rice research station was established in the year 1965 under the Directorate of

Agriculture, Government of West Bengal. The objective of this station is to identify varieties

suitable for irrigated and rainfed ecosystems.”

40
"Rice Research Station, Directorate of Agriculture West Bengal." CHINSURAH.
N.p., n.d. Web. 19 Jan. 2019. <http://www.icar-
iirr.org/AICRIP/Centers/45%20Chinsurah.pdf>.

35
APPENDIX B:

Raw data table for chlorophyll a and chlorophyll b and sample calculation for rice seedling

treated with 200mM NaCl.

B: Calculations:

Chlorophyll a (mg/l): 0.0127 x 0.29- 0.00269 x 0.12

Chlorophyll b (mg/l): 0.0029 x 0.29 – 0.00468 x 0.12

Total Chlorophyll (mg/l)- 0.0202 x 0.29+ 0.00802 x 0.12.

Table 7 : A table depicting the mean chlorophyll a value of the rice seedling at different

sodium chloride (NaCl) concentrations.

Concentration of NaCl Chlorophyll a Standard deviation

±0.04mM ± 0.03 mg/l

Control 6.87 0.42

50 5.34 0.16

100 4.74 0.12

150 4.34 0.19

200 3.14 0.24

Graph 3 depicting the decrease of chlorophyll a concentration with an increase in sodium

chloride (NaCl) concentration.

36
 Mean chlorophyll a(mg/l)

control
50
chlorophyll a (0.03 mg/l )

100
150
200

Concentration of NaCl (±0.04mM )

Graph 3 A graph showing the decrease of chlorophyll a concentration with an

increase in NaCl concentrations (the error bars signify the standard deviation)

Table 8: the mean chlorophyll b value of the rice seedling at different sodium chloride (NaCl)

concentration.

Concentration of NaCl Chlorophyll b ± 0.03 mg/l Standard deviation

±0.04mM

Control 3.86 0.35

50 3.03 0.52

100 2.81 0.34

150 2.82 0.18

200 2.79 0.85

Graph 4 showing the decrease in chlorophyll b with the increase in NaCl concentrations.

37
 Mean chlorophyll b (mg/l)
control
Chlorophyll b (0.03 mg/l mg/l)

50
100
150
200

Concentration of NaCl(±0.04mM )

Graph 4 A graph showing the decrease of chlorophyll b concentration with an increase

in NaCl concentrations (the error bars signify the standard deviation)

38