Determining and Augmenting Salt Tolerance in

Triticum aestivum using Calcium Carbonate

Objective

Abstract
Salt tolerance is defined as the relative growth of an organism in
the presence of salinity. The higher the tolerance the better an
organism can survive in the presence of NaCl. Rising sea levels
will be detrimental to economically important crops like wheat.
Previous research has shown that calcium is involved with
cellulose composition and regulation of salt impaired genes like
SOS1, TaNHX1, SOS3, TaSC, and CesA gene. This research
attempts to find an ideal NaCl: Ca ratio to allot for optimal wheat
growth in saline conditions. Ninety-nine Triticum aestivum seeds
were planted in 500 mL of organic soil each. Eleven groups of
nine seeds each were maintained at variedcombinations 0, 3.0
and 9.0 ppt of salinity and 70, 100, 130, and 160 ppm of calcium.
At the end of three months, wheat plants were taken and
measured for cellulose composition via acid spectrophotometry
and genes were monitored via real-time PCR. Increased calcium
levels proportional to NaCl levels demonstrated to allow wheat
plants to grow better significantly under salt stress. Overall,
results suggest that increased calcium levels proportional to NaCl
levels could be implemented to allow for wheat plants to grow
under salt stress.

Background
Salt tolerance is the relative growth of an organism in the presence of salinity
(Salt Tolerance of Plants). The higher the salt tolerance, the better an organism
can survive in the presence of salt. Excessive salt ions will cause ionic stresses in
living organisms as Na+ and Cl- ions inhibit metabolic processes and protein
synthesis. The salt ions hinder osmotic activities. This will cause the plant cells to
shrink and dehydrate due to a lack of water. As more cells dehydrate, they will
experience a shortage of chloroplast to produce glucose leading to death (Hannik).
Global climate change is the increase of Earth's average surface temperature
due to greenhouse gases (Global Warming). Over the last few decades, humans
have increased Earths temperature by 0.74 degrees Celsius through fossil fuel
combustion (Global Warming). This increases the rate at which ice caps meltincreasing sea level. The Ocean with salt levels of 35 parts per thousand cannot be
used by plants (Ocean). Due to global climate change, sea levels are intruding
into freshwater systems and increasing salt content of soils across the globe. This
will be harmful to plants that are near the coast and cannot tolerate high salinity.
The rising sea levels will be harmful to wheat (Grains). Wheat, 689 million tons
produced a year, constitute a large portion of the worlds food supply (Goldstein).
Wheat are glycophytes and cannot tolerate salinity levels over 6 dS/m. Much of the
wheat grown in the world are grown near coastal areas in countries like China,
India, Australia, USA, and France.

increase the salt tolerance of wheat and isolate the ideal sodium chloride: calcium ratio.

2. Determine cellulose composition, genes expressed by salt intrusion, and calcium levels in wheat samples during salt stress

Methods

Crop like wheat to need have a greater salt tolerance

Controversy behind GMO

Solution to increase salt tolerance of wheat by calcium

Experimental setup and explanation

Soil calcium analysis

Wheat seeds were planted 0.5cm deep in long plastic 500 mL pint
containers with 400mL of Viagro Organic Plant Compost. This study
utilized 99 containers; thus, 99 wheat crops. According to Ranks,
wheat crops requires moist soil and full sunlight. Thus, the 99 cups
were watered at 6:00 MT and left under
a 125-watt Hydrofarm grow lamp for 12
hours everyday (Ranks). Due to the
declining temperature
and various
other
environmental variables, crops
were grown indoors with a dehumidifier
to main humidity at 50%. There were

Calcium is used by plants to maintain a strong and

healthy cell wall. The stronger the cell wall, the higher the
salt exclusion. As salt exclusion increases salt tolerance
increases. Thus, calcium was monitored to determine if
there is an ideal relationship between calcium and salt
tolerance. Previous wheat growing experience has shown
that it takes wheat about 2 days to germinate. After
germination, the crops were treated with their designated
sodium chloride concentrations. Three days later, day 7
after planting, the crops were treated with their designated
calcium concentrations. The 3 days lapse will enable the
crops to adjust to the salt. At 12 day intervals after the
initial calcium treatment, cross sections were cut from
each wheat crop and analyzed for calcium. mRNA was
extracted and examined using a RNA extraction kit from
Quiagen and real time PCR.

Cellulose is a long polymer of sugars. Commonly found in the cell

walls of plants, cellulose is the structural material for plants. When a
plant undergoes salt stress, the first line of defense is the cell wall
(Senese). A strong cell wall means better defense compared to a
weak cell wall. Thus, it is hypothesized that the greater the cellulose
content the better the support of the plant in saline soils. A Semimicro
method by Dr. Updegraff was utilized to determine cellulose
composition. Readings were plotted on a standard curve to determine
cellulose composition. See Semimicro determination of cellulose in
biological materials for complete procedure. This analysis was
conducted with wheat crops from each group.

Cellulose composition analysis

Discuss results obtained and data analysis

Discuss real world applications and future research

11 seeds per group and 9 groups in

total. Wheat setup is depicted in
figures
and. The
Figure 6: wheat setup with a 125experimental setup is
watt Hydrofarm as the light source
divide into sets with different salinity
levels that are further divided into
different
calcium levels. The wheat
were
watered with their specific
calcium and sodium chloride solutions once every 3 days after
germination. pH, soil salinity (dS/m), soil and air temperature, soil
moisture, nitrates, sulfates and phosphates were monitored once
every 3 days to limit variability. Plant growth were measured once
every 5 day after germinations. Growth was measured from the height
of the plant to the soil (Grains).

Figure 29: SOS3 gene gel. Note the band position

Figure 30: SOS1 gene gel. Note group g band

between the yellow squares.

Conclusions

Figure 9: Stratagene PCR used for gene

length amplification and analysis

Figure 10: Gel electrophoresis of gene

for comparison

Research has shown that the genes SOS1, TaNHX1,

SOS3, TaSC, and CesA are expressed in wheat during
salt stress (Jian et al. 2012). Thus, PCR analysis was
conducted to determine the level of expression for those 7
genes compared to the standard levels of expression in
wheat not experiencing salt stress. This allows for
determination of whether Ca+2 had any effects on the
SOS1, TaNHX1, SOS3, TaSC, and CesA genes.

Figure 8: Same salinity setup as figure but

broken down according to calcium treatment

Figure 11: Wheat samples

Figure 12: Remains of a
Figure 13: SPECTRA
spectrometry containing only cellulose
sample void of cellulose
used to measure
absorbance
of cellulose

Statistical Analysis:
The data analysis package SPSS was used to run Post Hoc test after
ANOVA was used to determine statistical significance. Duncan t-test
and Tukey test were used to rank and sort the data groups according
to statistical significance and similarity.

Results

Wheat with calcium level of 100 ppm had the same cellulose and growth
compared to the control. T-test, Tukey test, and Duncan t-test confirmed the
statistical significance of the cellulose data and the wheat growth data. Explanation
to why salt tolerance was increased due to 100 ppm of calcium is that there was just
enough calcium to trigger the SOS1 gene to export salt, hence increased salt
tolerance. Results supported that calcium could be used to increase salt tolerance;
the second hypothesis of the proposed ideal ratio of 160 ppm calcium to 9.0dS/m of
sodium chloride was refuted. A calcium ratio of 100 ppm to a sodium chloride ratio of
9.0dS/m improved the salt tolerance of winter wheat by six fold.

Applications and Future Research

Due to the increasing sea level and salt intrusion, numerous crops like
wheat may die and leave the world with a shortage of food. Besides salt
intrusion, being able to increase the production of wheat in saline soils will
drastically increase the world food supply to eliminate hunger in many
countries. Thus, study sought to increase the salt tolerance of wheat through
non-genetic means. Genetic alterations have a lot of controversy as it
changes the genetic makeup of the organisms and that may be harmful for
consumption. However, this study increases the salt tolerance of wheat
through soil manipulation and does not involve any genetic changes. This
study demonstrated that calcium can be used as a mean to increase the salt
tolerance of wheat.
This method of increasing the soil calcium to increase salt tolerance may
be used to increase the salt tolerance of other cash crops. A future research
could attempt to find the ideal calcium ratio for other crops like rice and corn.
Further research could also elucidate the nutritional effects of manipulating
soil calcium levels. Currently, wheat is being grown at the ideal ratio until
harvest to determine the nutritional effects of altering calcium levels in the
soil. Furthermore, future research could also look into a smaller salt interval
that may confer a greater salt tolerance.

Hypothesis

Figure 15: Cellulose composition versus treatment type

It is hypothesized that calcium carbonate can act as a supplementary

nutrient to increase the salt tolerance of wheat when applied in the
correct sodium chloride: calcium ratio.

Table 1: Post Hoc Tukey Analysis

Variables and Control

Figure 16: Growth of Wheat (mm) during treatment

Table 2: Post Hoc Duncan T-test

Acknowledgements
Acknowledgements go to the following people and organizations for
their contributions to this study University of Sciences in Philadelphia,
HACH, Northeast Organic Farmers Association, Dr. Bologna, Dr.
Peethambaran, my high school, my research mentor, and my family.

References

-Wheat of Groups 12 and 13 are the experimental control without added NaCl or
CaCO3.
-Wheat of Groups A12 to D12 experience salt levels of 3.0 dS/m and varying
calcium levels
-Wheat of Groups E12 to H12 experience salt levels of 9.0 dS/m and varying
calcium levels

Figure 5:Phosphate testing via a colorimeter

-Soil temperature, air temperature soil moisture, nitrate, sulfate, phosphate, light
intensity, and pH were kept constant to alleviate variables and possible error

Cellulose Composition Analysis

Figure 2: Groundwater Depletion across the

contiguous United States

Figure 4:Sulfate testing via a colorimeter

Global warming to induce salt intrusion is a global issue

Soil Calcium Analysis

Rising sea levels will intrude onto freshwater systems and eradicate wheat crops.
This would cause a shortage of a vital food crop (Chinnusamy et al. 437). The
ability to grow wheat crops in areas with higher amounts of sodium chloride will
increase the production of wheat. This will support the large human population and
alleviate hunger in many poverty stricken areas like Africa, Indian, and Thailand.

Figure 3:Nitrate testing via a colorimeter

Wheat Set-Up and Growth Measurements

Figure 7: Wheat setup according to salinity (dS/m)

Figure 1: Amount of wheat grown in the world by

country

1. Determine whether calcium can be used to

Discussions

Figure 14: Progression of wheat through the treatment period

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