ABSTRACT
The arrangement of this project was designed to investigate the effect of salinity on germination rate of kang kong. Different concentrations of sodium chloride (NaCl) of 1.0%, 2.5%, 5.0%, 7.5% and 10.0% are prepared. The seeds of kang kong is soaked in the salt solutions for 12 hours, dried and then sown Petri dishes. The seeds were left to germinate for 72 hours and 15ml of their respective salt solutions were added at 24 hours interval. The results were recorded and the germination rate was determined. Using Pearson product-moment correlation coefficient (PMCC), a statistical data can be produced. It shows a significant negative linear relationship between concentration of salts and the germination rate of kang kong. The r value calculated was greater than critical value at 5% significant level.
Key words- Salinity, Seed germination, Germination rate, Ipomoea aquatica, Sodium chloride
RESEARCH AND RATIONALE
The objective of this work is to conduct a bioassay to investigate the relationship between salinity and the germination rate of kang kong seed hence determining the best salinity for maximum seed germination.
Bioassay is a test method employed in measuring response of living animal or plant tissue to the toxicity of chemical contaminants. In a bioassay, certain number of individuals of a sensitive species is exposed to specific concentration of the contaminant for a specific period to examine the toxic effects.
Definition taken from: http://www.businessdictionary.com/definition/bioassay.html
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Soil salinity is the amount of salt in the soil. The source of salt in the soil are usually the build up of minerals such as Na+, K+, Ca2+, Mg2+, Cl-, HCO3- and SO24-. High soil salinity means high concentrations of these minerals in the soil. This may happen because low water content in the soil due to inadequate rainfall in the area and mass usage of inorganic fertilisers. Researcher claim that increases in salinity will decreases the rate of germination of seeds15. For this investigation, concentration of sodium chloride will act as the manipulative variable.
Sodium chloride, also known as salt, common salt, table salt, or halite, is an ionic compound with the formula NaCl. Sodium chloride is the salt most responsible for the salinity of the ocean and of the extracellular fluid of many multicellular organisms. As the major ingredient in edible salt, it is commonly used as a condiment and food preservative.
Definition taken from: http://en.wikipedia.org/wiki/Sodium_chloride
Figure 1: Ionic structure of NaCl.
Image taken from: http://www.school-for-champions.com/chemistry/bonding_types.htm
The principal effect of salts on germination is osmotic. However, toxic effects are associated with excess salinity.2
Salinity stress affects germination by preventing the uptake of water owing to the osmotic potential, as well as by allowing entry of toxic ions into the developing embryo or seedling. The seeds of plants adapted to higher salts stresses are found to germinate only when the salt content of their habitat reaches a low level; for example, after heavy rainfall.
Quoted from: Babasaheb B. Desai, Seeds Handbook: Biology, Production, Processing and Storage, Second Edition, New York: Marcel Dekker Inc. Page 77
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Figure 2: Concept of osmosisOsmosis is the movement of water through semi-permeable membrane from low solute concentration (hypotonic) to high solute concentration (hypertonic). If the seeds were immersed in high salinity (hypertonic), water will move out from the seed making the seed dry and making the condition unfavourable for germination.
Image taken from: http://www.islandnet.com/~tiger/Tiger/RO/how_ro_works.htm
In the next decade, World may faces many challenges like the shortage of supply of fresh water and quality lands to plant crops, so more attention will be given to use low quality of both water and soil. Soil salinity also considered as one of the important obstacle to overcome in order to increases crops productivity. Investigation of salinity on seed germination will give detail about the effects and this knowledge can be useful in the near future.
In this experiment, I choose to use Ipomoea aquatica seeds because it has fast rate of germination and they are easy to handle. Hence, the result can be obtained quickly and accurately.
The dependent variable for this experiment is germination rate and it is expressed in percentage. It shows the number of seeds that germinate in the experiments. Germination may be defined as an emergence of embryo from seed by starting a variety of anabolic and catabolic activities, including respiration, protein synthesis, and mobilisation of food reserves after it has absorbed water1.
Hence this investigation is an attempt to find the optimum salt concentration in the soil for maximum crops germination.
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EXPERIMENTAL HYPOTHESIS
The higher the concentration on Sodium chloride, the lower the rate of germination of kang kong.
NULL HYPOTHESIS
Germination rate of kang kong is not affected by the concentration of Sodium chloride.
PLANNING
Trial
A trial experiment had been conducted to determine the best range of concentrations of sodium chloride to be used and the suitability of the methods and procedures.
Methods involved
Preparing the various concentration of sodium chloride.
The concentrations of sodium chloride chosen for the trial were: 0% (distilled water), 5.0%, 10.0%, 15.0%, 20.0%, and 25.0%. The concentrations were manipulated by mixing different mass of sodium chloride with 100ml distilled water in 250ml beaker. Glass rod was used to stir the mixture until dissolved. The mass used is shown in the table below.
Salt concentration (%)
Mass of sodium chloride used (gram)
Volume of distilled water (millilitre)
0.0
0
100
5.0
5
100
10.0
10
100
15.0
15
100
20.0
20
100
25.0
25
100
Table 1: Mass of sodium chloride used to manipulate the concentrations.
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Soaking and sowing the seeds.
6 Petri dishes were prepared containing 30ml of salt solution of each concentration. The seeds were soaked in the Petri dishes for 12 hours. Then, 6 Petri dishes were prepared and labelled. Layers of cotton wools were placed in the dishes and moisten with 30ml of the different concentrations of sodium chloride for each dish according to their label. The soaking solutions were discarded and 20 seeds of about the same size were chosen, dried and sown in the labelled Petri dishes. 15ml of salt solution of each concentration were added at 24 hours interval to respective labelled dishes.
Readings
Result was collected after 72 hours. Germination was observed to occur when the radicle was visibly extended beyond the surface of the seeds. The numbers of seeds germinated were counted and the germination rate can be determined by calculation. The result was tabulated.
TRIAL RESULTS
Number of seed used in each concentrations = 20
After 72 hours
Salt concentration (%)
Number of seeds germinate
Germination rate (%)
0
19
95
5
14
70
10
2
10
15
0
0
20
0
0
25
0
0
Table 2: The germination rate for each concentration.
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From the trials, I found that more seeds are needed to be used for the result to be more statistically significant. 30 seeds were used per concentration for the real investigation instead of 20 seeds. I also found that concentrations above 10% gave the same result with 0% germination rate. A high concentration of salt has inhibition effect on germination.
Hence, subsequent experiments were done by using concentration of sodium chloride lower than 10%. Main experiment was done twice (replicate 1 and 2) using the same method for more reliable results. I decided to use Pearson product-moment correlation coefficient (PMCC) to demonstrate the data statistically.
MAIN EXPERIMENT
VARIABLES
Manipulated: The concentration of sodium chloride solution, %
Different mass of chloride were mixed with 100ml of distilled water to manipulate the concentration of salt solution. The control was distilled water (0% concentration) for comparison.
Measuring cylinder was used to measure 100ml of water and electronic balance was used to measure the mass of sodium chloride needed.
Responding: Germination rate, %
It was calculated from the equation:
A seed germinate was indicated by the emergence of its radicle after a fixed time (72 hours)
Controlled: Number of seeds used, volume of solution used for soaking (30ml per Petri dish), volume of solution used for sowing (30ml per Petri dish), environmental factors (temperature, light intensity, humidity)
The Petri dishes were left on a laboratory table at 25ËšC for 72 hours and the volumes used were kept constant.
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MATERIALS AND APPARATUS
Materials
Apparatus
Distilled water
Petri dish
Kang kong (Ipomoea aquatica) seeds
Measuring cylinder
Sodium chloride (NaCl)
Electronic balance
Glass rod
Cotton wool
Spatula
250ml beaker
Table 3: List of materials and apparatus used
SAFETY PRECAUTION
Spatula was used to transfer sodium chloride into the beaker to prevent it from being contaminated.
The electronic balance is extremely sensitive. The surface of the balance is clean before measurement to make sure no particle present that can affect the reading.
The soaked seeds were very fragile and thus need to be handle very carefully.
Apparatus that made up of glasses are very fragile. They can break easily and might cause injury. Extra care is needed when using them.
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DATA COLLECTION IN MAIN STUDY
Number of seeds used per concentration = 30
Soaking for 12 hours
Result collected after 72 hours
Concentration (%)
Number of seeds germinated
Mean number of seeds germinate
Germination rate (%)
Replicate 1
Replicate 2
0.0
26
24
25
83.33
1.0
27
27
27
90.00
2.5
22
23
23
76.67
5.0
24
25
25
83.33
7.5
13
11
12
40.00
10.0
4
5
5
16.67
Table 4: Germination rate for each concentration
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SAMPLE CALCULATION
Germination rate, % = mean number of seeds germinate/30 X 100
= 25/30 X 100
= 83.33%
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STATISTICAL ANALYSIS
The product moment correlation coefficient (PMCC) can be used to tell us how strong the correlation between two variables is.
A positive value indicates a positive correlation and the higher the value, the stronger the correlation. Similarly, a negative value indicates a negative correlation and the lower the value the stronger the correlation.
If there is a perfect positive correlation (in other words the points all lie on a straight line that goes up from left to right), then r = 1.
If there is a perfect negative correlation, then r = -1.
If there is no correlation, then r = 0. r would also be equal to zero if the variables were related in a non-linear way (they might lie on a quadratic curve rather than a straight line, for example).
The strength between the variables in my experiment (concentration and germination rate) can be calculated using Pearson product-moment correlation coefficient (PMCC).
Definition taken from: http://www.mathsrevision.net/alevel/pages.php?page=67
*SS = sum of squares
x
0.0
1.0
2.5
5.0
7.5
10.0
∑x=26.00
y
83.33
90.00
76.67
83.33
40.00
16.67
∑y=390.00
x2
0
1.00
6.25
25.0
56.25
100.0
∑x2=188.5
y2
6943.89
8100
5878.29
6943.89
1600
277.89
∑y2=29743.96
xy
0
90
191.675
416.65
300
166.7
∑xy=1165.025
SS (x) =
= 188.5 - (26)2/6
= 75.8333
SS (y) = ∑ y2 -
= 29743.96 - (390)2/6
= 4393.96
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SS (xy) = ∑ xy -
= 1165.025 - (26)(390)/6
= -524.975
Using formula to find the correlation coefficient, r
= -524.975/(75.83331/2 X 4393.961/2)
= -0.8577 (negative sign indicates negative correlation between variables)
r = 0.8577 > 0.811 (critical value) for 5% significant level (from Table 5)
Degree of freedom = n - 2
n = # pairs of data
= 6 - 2 = 4
Level of significance for two-tailed test
.10
.05
.02
.01
1
.988
.997
.9995
.9999
2
.900
.950
.980
.990
3
.805
.878
.934
.959
4
.729
.811
.882
.917
5
.669
.754
.833
.874
6
.622
.707
.789
.834
Table 5: Critical value for PMCC
The r value calculated shows a significant negative linear relationship between concentration of sodium chloride and germination rate since r value calculated was greater than critical value at 5% significant level.
Therefore, null hypothesis can be rejected.
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DATA ANALYSIS
From Table 4, it can be seen that germination rate is high only in the low concentration of sodium chloride. The kang kong seeds germination rate falls below 50% at concentration greater than 5%. The statistical analysis using PMCC further verified the negative correlation between the two variables, the higher the concentration of sodium chloride the lower the germination rate of the kang kong seeds.
Graph 1 also illustrates the same trend of relationship between the two variables. The highest germination rate (90%) was recorded when the lowest concentration of sodium chloride (1%) was used.
However, it also can be seen that the line is fluctuating downward but the germination rate at 5.0% is the same as the controlled with 83.33% where it supposed to be lower than that. This inconsistency may arise due to many errors such as:
Some of the seeds may be initially damaged before the experiment. Hence, germination is not possible.
An error occurred during weighting the mass of sodium chloride for preparation of the solution and this may cause the concentration to be altered.
The sodium chloride is not fully dissolved in the distilled water making the concentration inaccurate.
In this experiment, distilled water (0% concentration) acted as control so that comparison can be made with other concentrations. The result shows that highest germination rate is at the range of 0% to 1%. This suggested that the seeds have optimum germination rate at low salinity soils. Soils which have salinity more than 1% may give difficulties for seeds to germinate.
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EVALUATION
Seed germination depends on both internal and external conditions. The most important external factors include temperature, water, oxygen and sometimes light or darkness21.
Sufficient amount of water is needed by a seed before cellular metabolism and growth can resume. Water activates enzymes in the seed so that they are rapidly released to digest the stored food into smaller molecule that can be transported and converted into energy needed for growth3.
The main books that I refer to during my investigation are Babasaheb B. Desai, Seeds Handbook: Biology, Production, Processing and Storage, Second Edition, New York: Marcel Dekker Inc. (page 7-89) and Michael Fenner, Seeds: The Ecology of Regeneration in Plant Communitites, Southampton: C.A.B International. (page 327-348). These sources were indeed reliable because they have been written by experts in botany world and also being revised, reorganised, and undergone careful editing before being published.
The uptake of water by seeds is called imbition. Altering the concentration of sodium chloride may affect the water and osmotic potential. If the seeds are hyponic (lower concentration of solute in the membrane) to its surrounding medium, imbition of water will be prevented. Instead, water will move out of the seeds into their surroundings. Salinity stress affects germination by preventing the uptake of water owing to the osmotic potential, as well as by allowing entry of toxic ions into the developing embryo or seedling1.
In severe case of salinity, the embryo of the seed can be damaged by the excess ions. The common ions responsible for the toxic effect of salinity are Na+ and K+. Physiologically absolute ratio of K+/Na+ in the tissue is important18. The increase in salinity shortens this ratio and probably caused injury to embryo18. Maintaining the ratio of these ions in the embryo is essentials in order for the seed to germinate.
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In this investigation, I found some limitations that include the uncontrolled temperature of surroundings. The temperature in the laboratory may fluctuate over time. As a result, it may affect the germination time of the seeds and the numbers of seeds germinate.
The other limitation is that variability of the health of kang kong seeds used in the experiment. The embryo of some seeds may be initially damaged before the experiments being conducted. Hence, it cannot be determined whether the seeds did not germinate due to the inhibitory effect of sodium chloride or not.
However, the large sample gave enough replicates to support the conclusion with anomalies were ignored. Thus, the result is quite reliable as it was repeated and controlled. In this investigation, the effect of sodium chloride on seed germination was tested in a proper condition without exposing the seeds to any other stresses such as exposure to UV light.
Other species of plant seeds can also be used to test the hypothesis. This is because different plat species may respond differently to salinity stress. Therefore, using seeds from only one species of plants may gives wrong indication about salinity stress on seed germination.
CONCLUSION
Based on the result obtained and statistical analysis, it can be concluded that the higher the concentration of sodium chloride, the lower the germination rate of kang kong seeds, Ipomoea aquatica sp as concentration sodium chloride will affect osmotic potential of the seeds and the imbition of water. The best salinity is below 1% level.
A statistically significant negative linear relationship between concentration of sodium chloride and germination rate was proven using PMCC since the r value calculated was greater than crirical value at 5% significant level.
Experimental hypothesis is accepted and the null hypothesis is rejected.
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APPENDICES
C:\Users\acer\Pictures\DSC01902.JPG
Image of the kang kong seed used
EVALUATION OF SOURCES
My campus library provides access to online and written journal for my investigation such as Science Direct and Springerlink (source 23, 24 and 25). Science Direct and Springerlink are the leading full-text scientific database offering journal articles and book chapters from more than 2,500 peer-reviewed journals and more than 11,000 books. There are currently more than 9.5 million articles/chapters. Hence, it highly regarded as reliable source.
Wikipedia is a reference site that been visited by millions of people (source 11, 12, 19, and 21). The information in the website regarded as reliable as it is cited and linked to many other literatures and academic research around the globe.
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