Fundamental and Applied Agriculture
Vol. 6(4), pp. 336–348: 2021
doi: 10.5455/faa.128175
P LANT S CIENCE|O RIGINAL A RTICLE

Chitosan mitigates salt stress in rice by enhancing antioxidant
defense system
Md Khaleduzzaman 1 , Mohammad Anowar Hossain 1* , Muhammad Javidul Haque
Bhuiyan 1 , Sakil Mahmud 1 , Md Tahjib-Ul-Arif 1 , Yoshiyuki Murata 2
1 Department of Biochemistry and Molecular Biology, Bangladesh Agricultural University, Mymensingh 2202,

Bangladesh
2 Graduate School of Environmental and Life Science, Okayama University, Kita-ku, Okayama 700-8530, JapanA RTICLE I NFORMATIONA BSTRACTArticle History
Submitted: 29 Sep 2021
Accepted: 29 Nov 2021
First online: 30 Dec 2021

Salinity is one of the major constraints for rice (Oryza sativa L.) cultivation
globally, as rice is highly sensitive to salinity than other cereal crops. In order
to elucidate the improvement mechanism of salinity tolerance by chitosan(CS), we analyzed the morphological, biochemical and antioxidant enzymatic
activities of salt-stressed rice at germination and seedling stages of four cultivars: BRRI dhan28 and BRRI dhan29 as salt-sensitive and Pokkali and Pengek
as salt-tolerant. Salinity inhibited seed germination as well as growth of riceseedlings. At the germination stage, CS increased the germination percentage
by enhancing amylase activity and significantly accelerated shoot growth
but not root growth under salt stress condition. In the seedling stage, 50 ppm
of CS mitigated the growth inhibition of seedling of the sensitive cultivars
and the reduction of chlorophyll (both chl ‘a’ and chl ‘b’) contents. Moreover,
CS increased the activities of catalase (CAT) and ascorbate peroxidase (APX)
but not the activities of peroxidase (POX). However, the accumulation of
proline and phenols in the seedlings was increased under saline condition
but not under saline condition supplemented with CS. Principal component
analysis revealed that a relatively stronger association between 50 ppm CS
treatment with the non-stress condition than that of 25 ppm CS treatment
both in sensitive and tolerant cultivars which suggested that 50 ppm CS
treatment was more effective. Our results suggest that exogenous CS can beused to mitigate salt-induced adverse effects in rice as well as in other crop
plants.

Academic Editor
Md Nesar Uddin
nesar.uddin@bau.edu.bd*Corresponding Author
Mohammad Anowar Hossain
anowar.biochem@bau.edu.bd

Keywords: Salinity, chitosan, antioxidant system, salinity tolerance, rice
Cite this article: Khaleduzzaman M, Hossain MA, Bhuiyan MJH, Mahmud S, Tahjib-Ul-Arif M,
Murata Y. 2021. Chitosan mitigates salt stress in rice by enhancing antioxidant defense system.
Fundamental and Applied Agriculture 6(4): 336–348. doi: 10.5455/faa.128175

1

Introduction

Salinity is a serious threat to the crop production in
the southern region of Bangladesh (Haque et al., 2014)
and it is one of the adverse environmental factors that
affect plants spanning from seed germination to productivity. More than 6% of the world’s total cultivablearea is affected by over accumulated salt (Munns andTester, 2008), and this problem prolongs to worsen
due to secondary salt, which accumulates in irrigatedsoils as water evaporates (Yang and Guo, 2017).

Seed germination plays important roles in the establishment of seedlings and their subsequent growth.
Salinity impairs plant growth and development via
water scarcity, cytotoxicity due to excessive uptake ofions such as sodium (Na+) and chloride (Cl–), and nutritional imbalance (Hasegawa et al., 2000). However,salinity hampers seed germination by affecting major
events of germination such as imbibition, metabolism
activation, the emergence of embryonic tissues andseedling establishment (Hasanuzzaman et al., 2013;

Khaleduzzaman et al.

Fundam Appl Agric 6(4): 336–348, 2021

Wahid et al., 2011). It has been revealed that salinity inhibits seed germination by reducing α-amylase
activity, which is the major reason for poor hydrolysis or imbibitions of stored substances (Hua-long
et al., 2014; Liu et al., 2018). Other studies reported
that salt stress reduced root length, root dry weight,
shoot length and shoot dry weight in both sensitive
and tolerant rice cultivars where the magnitude of
growth reduction is much higher in sensitive cultivar
compared to tolerant (Mahmud et al., 2016; Kumar
and Khare, 2016).
Salinity causes oxidative stress in plants throughthe production of reactive oxygen species (ROS) or
free radicals, such as superoxide (O2·− ), singlet oxygen (1 O2 ), and hydrogen peroxide (H2O2) (Noctor
and Foyer, 1998). Plants encounter salinity stressthrough enzymatic and non-enzymatic antioxidant
mechanisms (Gupta and Huang, 2014; Bose et al.,2013; Kibria et al., 2017; Dreyer and Dietz, 2018).
Non-enzymatic antioxidants catalyze redox reactions
and rely on electron donation via reduction of lowmolecular-weight antioxidants, such as glutathione,
flavonoids, phenols and free proline (Noctor andFoyer, 1998; Symes et al., 2018). Superoxide dismutase (SOD), catalase (CAT), peroxidase (POX) andascorbate peroxidase (APX) have been previously reported as the major antioxidant enzymes that serveas an antioxidant defense system in many plants under salt stress (Parida et al., 2004; Chawla et al., 2012;
Mahmud et al., 2017; Wang et al., 2017; Foyer, 2018).
Various exogenous compounds are applied to promote plant growth and induce abiotic resistance (Yasmeen et al., 2012). Chitosan similar to plant growth
regulators, is a partially deacetylated form of chitin,a natural biopolymer from the exoskeleton of crustaceans and fungal cell walls, which is biocompatible,
biodegradable and a sustainably renewable cheap resource that has many applications, including plant
defense (Katiyar et al., 2014; Malerba and Cerana,
2015; Ray et al., 2016b), confer resistance against abiotic stresses such as water deficit, salinity, heat stress,and heavy metal toxicity (Malerba and Cerana, 2015).
It has been revealed that exogenous chitosan could
mitigate salt stress by increasing antioxidant enzyme(SOD, POX, and CAT) and also enhancing photosynthetic pigments i.e. chlorophyll a and b (Ma et al.,2011). Despite all this knowledge, there are still many
points that need to be clarified about the stage, organ,and variety-specific alleviating capability of chitosan
under salt stress.
The present study aims to elucidate the chitosan’sdose-specific response to the antioxidative capacityof the salt-sensitive and salt-tolerant rice cultivars at
the seedling stage.

2

Materials and Methods

2.1

Experimental layout

337

The experiment was conducted using two saltsensitive (BRRI dhan28 and BRRI dhan29) and twosalt-tolerant rice cultivars (Pokkali and Pengek). The
uniform-sized rice seeds were sorted out and thenseeds were sterilized using 5% sodium hypochlorite +2% Tween-20 for 25 min, followed by five times washing with distilled water. Subsequently, the seeds were
presoaked in distilled water for 24 h and kept for 3
days on a wet filter paper covered with aluminum foil
at 27±2 °C to induce germination. Three days after
germination, seeds were transferred to the mentioned
treatments and the number of seeds germinated wascounted until the next 5th day. For seedlings parameters, uniformly germinated seeds were placed in plastic pots (10.5 cm × 12.5 cm) containing 8 kg of soil
(silt loam, pH of 6.18, 0.15 dS m−1 EC, 8.4% CEC, exchangeable Na+ of 0.35 milliequivalents per 100 g soil,
exchangeable K+ of 0.14 cmol kg−1 ) in a net-house (average temperature 25 °C and relative humidity 60%).
The 21-day-old seedlings were separated into four
treatment groups as follows “C”, foliar spray with
distilled water (non-stress condition); “S”, 9 dS m−1
NaCl + foliar spray with distilled water; “S+25CS” 9
dS m−1 NaCl + foliar spray with 25 ppm chitosan;and “S+50CS” 9 dS m−1 NaCl + foliar spray with 50
ppm chitosan. In the case of salt treatment, 2 L ofdesired concentrations of NaCl was added to the top
surface of the soil. Leaf of the 21 days old seedlings
was sprayed with 25 mL of chitosan once with thedesired concentration. The seedlings were grown forseven days more before harvesting, and then the fresh
leaves were collected to assess the parameters mentioned. CS was dissolved in 1.0% acetic acid to get astock solution. The pH of the solution was adjusted
to 6.5 with NaOH. Both the germination and seedlingstage experiments were conducted in completly randomized design with three independent replications
for each treatment.

2.2

Germination percentage (GM%)

To test CS-induced salinity tolerance, 20 seeds of each
cultivar were placed in a 9 cm Petri dish with an
imbibed filter paper containing the four groups oftreatment as follows “C”, with distilled water (nonstress condition); “S”, 12 dS m−1 NaCl; “S+25CS” 12
dS m−1 NaCl + 25 ppm chitosan; and “S+50CS” 12
dS m−1 NaCl + 50 ppm chitosan. Germination percentage is calculated from the following formula:
G
× 100
(1)
T
where GM (%) = germination percentage, G = total
number of seeds germinated, and T = total numberof seeds placed for germination.
GM (%) =Khaleduzzaman et al.

2.3

Fundam Appl Agric 6(4): 336–348, 2021

Determination of α-amylase activity

Alpha-amylase was determined spectrophotometrically by using 3, 5 dinitrosalicylic acid (DNSA) following the modified procedure of Bernfeld (1955).
Endosperm tissues were used for amylase extraction.
Five hundred milligrams of endosperm tissue werecrushed with 10 mL of 0.02 M phosphate buffer at pH
6.0. One gram soluble starch as a substrate was dissolved in 100 mL of 0.02 M phosphate buffer, pH 6.9
containing 0.0067 M NaCl. One milliliter of enzyme
extract and 1.0 mL of substrate solution were taken
in a test tube. The reaction mixture was incubated at
30 °C for 15 minutes and the reaction was stopped
by adding 5 mL of DNSA reagent. The homogenate
was centrifuged at 3000×g for 20 minutes. The supernatant was used to determine the enzyme activity.
The optical density of the color developed was read at
540 nm and maltose liberated (mg) were determined.

2.4

Analysis of plant growth parameters

The length from the shoot base to the leaf tip was
measured for shoot length (SL) determination. Likewise, the root length (RL) was determined by measuring the length from the root base to the root tip.
From each experiment, 25 seedlings were collected
and weighed to determine the dry weight (DW). The
DW of shoot and root was determined after oven drying at 60 °C for four days. The DWs of shoots androots were expressed as mg seedling−1 .

2.5

Determination of antioxidant enzyme activity

Antioxidative defense mechanisms by supplemented
CS were assessed by examining major antioxidant
enzymes such as CAT (EC 1.11.1.6) (Aebi, 1984), POX(EC 1.11.1.7) and APX (EC 1.11.1.11) (Nakano andAsada, 1981). Fresh leaf sample (50 mg) was collected
and homogenized with 3 mL of 50 mM phosphatebuffer (pH 8.0) (PB) in an ice-chilled mortar and pestle. The homogenate was centrifuged at 11,000×g for
10 min. The clear supernatant was used for assaying
the enzyme activity. For the CAT activity assay, thereaction mixture was made of 0.7 mL of PB, 0.1 mL
of EDTA and 0.1 mL of H2O2. For POX, 0.6 mL of
50 mM PB, 0.1 mL of EDTA, 0.1 mL of H2O2 and 0.1
mL of guaiacol and for APX assay, 0.6 mL of PB, 0.1
mL of EDTA, 0.1 mL of H2O2, and 0.1 mL of ascorbate. All the mixture was made in an Eppendorf tube.
The reaction was started by adding 0.1 mL of enzyme
extract and changes in absorbance were recorded immediately at 240 nm, 470 nm, and 290 nm for CAT,
POX, and APX assay, respectively, at 30 s intervals fortwo min. The activity of CAT and APX was calculated
from the decrease whereas POX activity was from theincrease in absorbance.

2.6

338

Determination of proline

Proline content was determined following the modified method of Bates et al. (1973). Leaf sample (50mg) was homogenized in a chilled mortar with pestle using 10 mL of 3% sulfosalicylic acid. The homogenate was centrifuged (4500×g for 10 min) andthen filtered through filter paper. Two milliliters of
the filtered extract were pipette into the test tube followed by 2 mL acid ninhydrin and 2 mL glacial acetic
acid and then incubated for 1 hour at 100 °C. The
reaction was started by adding 4 mL of toluene. The
absorbance of the collected toluene was measured at
520 nm in a vis/uv spectrophotometer (Shimadzu,
UV-1201; Japan).

2.7

Determination of total phenol

Total phenol was determined according to the modified method developed by Sadsivam and Manickam,(1996). Freeze-dried leaf sample (50 mg) was mixed
with 10 times a volume of 80% ethanol and centrifuged at 10,000×g for 20 min. Dissolved pellets
were mixed with 0.5 mL of folin-ciocalteau reagent
followed by 2 ml of 20% sodium carbonate. The material was mixed thoroughly and tubes were placed inboiling water exactly for one minute. The tubes werethen cooled and the absorbance was measured at 650
nm spectrophotometer.

2.8

Determination of chlorophyll content

Chlorophyll content was determined according to themethod developed by Coombs et al., (1985). Freshleaf sample (50 mg) was taken into a test tube containing 10 mL of 80% acetone and was covered byaluminum foil, and preserved in the dark for 7 to 10
days. Spectrophotometric reading was taken at 645
nm and 663 nm wavelengths.

2.9

Statistical analysis

The data were subjected to a one-way or two-way
analysis of variance using Minitab 17 (Pennsylvania, USA), followed by Tukey’s test (P < 0.05). The
heatmap with hierarchical clustering and principal
component analysis (PCA) was performed by R 3.6.1
using ‘heatmap’, ‘ggplot2’ and ‘factoMineR’ packages.

3

Results

3.1

Alpha-amylase activity of rice seeds

The protective role of CS on the germination capacityof rice seeds under salinity stress was evaluated bydetermining the GM% and α-amylase activity (Fig. 1ab). The GM% was recorded higher under non-stressKhaleduzzaman et al.

Fundam Appl Agric 6(4): 336–348, 2021

condition compared to saline condition. The highest
GM% was observed in Pokkali (100%), whereas thelowest (75%) was in BRRI dhan28 under saline condition. However, there were significant increases of
GM% in all sensitive and tolerant cultivars with supplementation of 25 ppm and 50 ppm of CS compare tothat of saline condition. Salinity drastically reduced
the α-amylase activity in all cultivars compared tonon-stress conditions. The highest amylase activity
was observed in Pokkali under non-stress condition.
However, the lowest amylase activity was observedin the saline condition in BRRI dhan28. Supplementation of CS with saline condition remarkably increased
the amylase content compared to that of saline condition. There was a significant increase in amylase
content in all cultivars supplemented with CS while
no variations were found between two different concentrations.

3.4

Growth and biomass of rice plants

To determine whether CS could alleviate the toxic effect of salinity, we monitored the growth and biomassof rice seedlings in terms of shoot length (SL), shoot
dry weight (SDW), root length (RL) and root dry
weight (RDW) (Fig. 2a-d). The SL as well as SDW,
was recorded higher in the tolerant cultivars under
non-stress conditions. Salinity induced the reductionof these parameters both in the tolerant as well as sensitive cultivars. Supplementation of CS with salinecondition significantly increased SL and SDW in thetolerant cultivars. In BRRI dhan28, both 25 and 50
ppm CS treatments enhanced SL and SDW, whereas
only 50 ppm CS treatment improved the SL and SDW
in BRRI dhan29 significantly compared to saline condition. Salinity reduced RL and RDW significantly insensitive cultivars but these values remain unchangedin the tolerant cultivars under salt stress conditions.
Supplementation of CS at both concentrations could
not alleviate the salt-induced reduction of RL and
RDW. From these points, we suppose that CS might
not interact with salt stress in the root growth zone.

3.3

Phenotype of rice plants

Salinity caused a significant reduction in plant
growth, leaf expansion and variation in leaf color
in all the treatments compared with their respective
control plants during growth stages (Fig. 3). However, the tolerant cultivars showed lesser degree of
reduction in these traits that indicating their considerable adaptability in stressed conditions. Supplementation of CS on salt-stressed rice seedlings improved
the growth in all cultivars. Saline condition supplemented with 50 ppm of CS displayed improved
growth of rice seedlings than 25 ppm of CS. Amongthe cultivars, Pengek and Pokkali showed superiority
over the other two cultivars.

pigmentsinrice

We determined the Chl content of rice leaves to know
the role of CS in protecting photosynthetic machinery
under salt stress (Fig. 4a-b). The content of Chl ‘a’and Chl ‘b’ was found higher in the sensitive cultivars compared to tolerant cultivars. Salt stress caused
a drastic reduction in Chl ‘a’ and Chl ‘b’ contents
compared to non-stress condition in the sensitive cultivars, whereas tolerant cultivars showed a slight reduction. On the contrary, supplementation of CS
showed significant alleviation of Chl ‘a’ and Chl ‘b’content more likely in the sensitive cultivars. Salinecondition supplemented with 50 ppm of CS enhanced
the upregulation of chlorophyll content.

3.5
3.2

Photosynthetic
plants339

Proline and phenolic contents

Salt stress increased the proline content comparedwith that of the non-stress condition (Fig. 5a). Interestingly, salt-sensitive cultivars had higher proline
content compared to tolerant cultivars under nonstress condition. Supplementation of CS could significantly reduce the proline content in all the cultivars.
Saline conditions supplemented with both 25 and 50
ppm of CS had a similar trend of decrease in proline
content in BRRI dhan28. However, saline condition
supplemented with only 50 ppm of CS reduced theproline content in BRRI dhan29 compare to saline condition. Salinity increased total phenolic contents in all
the cultivars (Fig. 5b). Conversely, supplementationof both 25 and 50 ppm of CS with the saline condition
could significantly lower the phenolic contents to its
basal level in the tolerant cultivars, whereas supplementation of 50 ppm of CS with the saline condition
could significantly decrease the phenolic contents insensitive cultivars.

3.6

Antioxidant activities in rice plants

The rice seedlings exposed to salt stress had increased CAT and APX activity in salt-tolerant cultivars whereas sensitive cultivars unable to increase
CAT and APX activity (Fig. 6a, b). In contrast, CS
supplementation to saline condition showed enhancement of CAT and APX activity in sensitive cultivars
as compared with only salt-stressed seedlings. Supplementation of 50 ppm of CS showed more effect on
CAT and APX activity. Salt stress increased POX activity in salt-tolerant cultivars as compared with thenon-stress condition, whereas sensitive cultivars wereunable to increase POX activity under salt-stress conditions (Fig. 6c). Supplementation of CS with salinecondition did not affect POX activity in salt-tolerant
as well as salt-sensitive cultivars as compared withsaline condition.

Khaleduzzaman et al.

Fundam Appl Agric 6(4): 336–348, 2021

340

Figure 1. Effect of CS supplementation on (a) germination percentage (GM%) and (b) α-amylase activity of riceseed grown under normal or salt stress condition. Data represented as the means of three
independent replicates for each treatment (n = 3). The vertical bars indicate standard errors of means.
Different letters on the top of each bar denote statistically significant differences at P < 0.05, based on
Tukey’s test. “C”, with distilled water (non-stress condition); “S”, 12 dS m−1 NaCl; “S+25CS” 12 dS
m−1 NaCl + 25 ppm chitosan; and “S+50CS” 12 dS m−1 NaCl + 50 ppm chitosan

Figure 2. Effect of CS supplementation on (a) root length, (b) root dry wt., and (c) shoot length and d) shoot dry
wt. of rice seedlings grown under normal or salt stress condition. Data represented as the means of
three independent replicates for each treatment (n = 3). The vertical bars indicate standard errors ofmeans. Different letters on the top of each bar denote statistically significant differences at P < 0.05,
based on Tukey’s test. “C”, with distilled water (non-stress condition); “S”, 12 dS m−1 NaCl;“S+25CS” 12 dS m−1 NaCl + 25 ppm chitosan; and “S+50CS” 12 dS m−1 NaCl + 50 ppm chitosan

Khaleduzzaman et al.

Fundam Appl Agric 6(4): 336–348, 2021

341

Figure 3. Effect of CS supplementation on the phenotypes of (a) Pengek, (b) Pokkali, (c) BRRI dhan28, and (d)BRRI dhan29 rice seedlings grown under normal or salt stress condition. “C”, foliar spray with
distilled water (non-stress condition); “S”, 9 dS m−1 NaCl + foliar spray with distilled water;“S+25CS” 9 dS m−1 NaCl + foliar spray with 25 ppm chitosan; and “S+50CS” 9 dS m−1 NaCl + foliar
spray with 50 ppm chitosan

3.7

Heatmap with hierarchical clusteringand PCA

Mean values of all morpho-physiological and biochemical parameters were used to perform heatmap
with hierarchical clustering and PCA. Hierarchicalclustering categorized different treatment × cultivars
into two clusters (Cluster-A and Cluster-B) (Fig. 7a).
Cluster-A contains all treatments combination withsalt-sensitive BRRI dhan28 and BRRI dhan29 except‘S+50CS’×BRRI dhan29. On the other hand, cluster-B
is composed of all treatments combined with salttolerant Pokkali and Pengek. It is visible that the hierarchical clustering separated tolerant and sensitive
genotypes in separate clusters irrespective of treatments.
The first two components of PCA combinedly explained 74.2% of the data variability (Fig. 7b). The
PCA biplot revealed the association of the different morpho-physiological and biochemical parameters with the treatment groups of different cultivars.
The growth-related morpho-physiological parameters were strongly associated with ‘C’, ‘S+25CS’ and‘S+50CS’ treatments of Pokkali and Pengek comparedto that of all treatments of BRRI dhan28 and BRRI
dhan29. The ‘S’ treatment of Pokkali and Pengek
strongly interlinked with CAT, APX and POX activity,and phenol content.

4

Discussion

Improvement of crop cultivation technologies is focused on increasing crop productivity and improving
yield quality, and at the same time on minimizing
risks posed to the natural environment. The use ofbiostimulants contributes to the increase in the pro-ductivity of plants, especially under their exposure
to stress induced by negative environmental stimuli such as salinity, drought, high and low temperature etc. It is well reported that CS supplementation
upregulate many physiological processes, and here
we investigated the effect of CS supplementation on
salt stress mitigation in rice. Salinity hampers seedgermination by affecting major events of germination such as imbibition, metabolism activation, theemergence of embryonic tissues and seedling establishment. However, the negative effect of salinity
on seed germination may vary due to various factors, such as varietal variation, level of salinity andother external factors. Poor hydrolysis also limits thetranslocation of food reserve from storage tissue todevelop embryo that also negatively affects germination and seedling establishment. Salinity inhibits
amylase activity, which is the major reason for poor
hydrolysis or imbibition of stored substances. In thisstudy, salt-stress reduced germination percentage aswell as α-amylase activity whereas both GM% and αamylase activity were increased with the supplementation of CS in salt-stressed rice seedlings. (Fig. 1a-b).
Increased α-amylase activity by supplemented CS isprobably due to the increment of GM% as well as thesurvival of rice seedlings under salt stress.
Determination of morphological parameters suchas growth and biomass of plants are used to assess theadverse effects of abiotic stress (Akram et al., 2019).
In the present investigation, the RL, RDW, SL andSDW were reduced in salt-stressed rice seedlings compared with that of non-stress plants (Fig. 2a-d) whichmight happen due to the reduced chlorophyll contents (Fig. 4a-b) (Roy et al., 2019; Tahjib-Ul-Arif et al.,2019).

Khaleduzzaman et al.

Fundam Appl Agric 6(4): 336–348, 2021

342

Figure 4. Effect of CS supplementation on photosynthetic pigments (a) Chl a and (b) Chl b contents of theleaves of rice seedlings grown under normal or salt stress condition. Data represented as the meansof three independent replicates for each treatment (n = 3). The vertical bars indicate standard errorsof means. Different letters on the top of each bar denote statistically significant differences at P < 0.05,
based on Tukey’s test. “C”, foliar spray with distilled water (non-stress condition); “S”, 9 dS m−1
NaCl + foliar spray with distilled water; “S+25CS” 9 dS m−1 NaCl + foliar spray with 25 ppm
chitosan; and “S+50CS” 9 dS m−1 NaCl + foliar spray with 50 ppm chitosan

Figure 5. Effect of CS supplementation on (a) proline and (b) phenol contents of the leaves of rice seedlings
grown under normal or salt stress condition. Data represented as the means of three independent
replicates for each treatment (n = 3). The vertical bars indicate standard errors of means. Different
letters on the top of each bar denote statistically significant differences at P < 0.05, based on Tukey’s
test. “C”, foliar spray with distilled water (non-stress condition); “S”, 9 dS m−1 NaCl + foliar spraywith distilled water; “S+25CS” 9 dS m−1 NaCl + foliar spray with 25 ppm chitosan; and “S+50CS” 9
dS m−1 NaCl + foliar spray with 50 ppm chitosan

Khaleduzzaman et al.

Fundam Appl Agric 6(4): 336–348, 2021

Figure 6. Effect of CS supplementation on (a)catalase (CAT) activity, (b) ascorbate
peroxidase (APX) activity, and (c)peroxidase (POX) activity of the leaves ofrice plants grown under normal or salt
stress condition. Data represented as themeans of three independent replicates foreach treatment (n = 3). The vertical bars
indicate standard errors of means.
Different letters on the top of each bar
denote statistically significant differences
at P < 0.05, based on Tukey’s test. “C”,
foliar spray with distilled water (non-stresscondition); “S”, 9 dS m−1 NaCl + foliar
spray with distilled water; “S+25CS” 9 dS
m−1 NaCl + foliar spray with 25 ppm
chitosan; and “S+50CS” 9 dS m−1 NaCl +foliar spray with 50 ppm chitosan

343

On the contrary, supplementation of CS with salinecondition improved the growth performance of saltstressed rice seedlings as evident by their improved
phenotypic appearance (Fig. 3a-d) and growthrelated features, including RL, RDW, SL and SDW(Fig. 2a-d), perhaps by increasing chlorophyll contents of rice plants (Fig. 4a-b). Moreover, our results were verified by PCA, which indicated that saltstressed rice plants supplemented with CS exhibiteda stronger correlation with chlorophyll content compared with salt-stressed plants in all four rice cultivars (Fig. 7b). Consistent with our results, it has also
been reported that exogenous CS increased root andshoot length as well as root and shoot dry weights
in wheat and maize plants (jing Guan et al., 2009;
Lizárraga-Paulín et al., 2011; Ma et al., 2011; Peykani
and Sepehr, 2018), rice (Ruan and Xue, 2002; Boonlertnirun et al., 2008), beans (Phaseolus vulgaris L) (Zayed
et al., 2017) and improves the tolerance of seedlings
to stress conditions.
To withstand salt stress, plants accumulate compatible solutes such as proline, which decreases the cytoplasmic osmotic potential, facilitating water absorption, and scavenges reactive oxygen species (ROS)(Qureshi et al., 2013; Pottosin et al., 2014). As an osmoprotectant with potential antioxidant activity, proline
plays a vital role in abiotic stress tolerance in plants(Hasanuzzaman et al., 2014; Nahar et al., 2016; Rayet al., 2016b). Proline is accumulated preferentially inleaves to maintain chlorophyll level and cell turgor toprotect photosynthetic activity under salt stress (SilvaOrtega et al., 2008). In this investigation, proline content was increased in salt-stressed rice seedlings compared to that of non-stressed plants which is more
pronounced in salt-sensitive cultivars than in salttolerant cultivars (Fig. 5a). This was also supported
by PCA where proline content was more closely associated with saline-stressed sensitive species than inother treatment groups (Fig. 7b). The accumulationof proline in plants under salt stress is caused either
by the induction of expression of proline biosynthesis genes (P5CS, P5CR) or by the repression of thegenes of its degradation pathway (PDH silencing)(Marco et al., 2015). The elevated proline content due
to salt stress may also be an adaptation to compensate
the energy for growth and survival and thereby help
plant tolerate stress, as reported earlier
in Crotalaira striata Chandrashekar and Sandhyarani(1996) and spinach leaves (Ozturk and Demir, 2003).
However, in salt-stressed rice seedlings, foliar supplementation of CS decreased the proline content
which suggested that CS somehow prevent water
imbalance in plants thus reducing the need for excess proline accumulation. Similar results were
found in salt-affected rice seedlings (Hasanuzzaman
et al., 2014; Rahman et al., 2016; Shams Peykani andFarzami Sepehr, 2018; Sen et al., 2020).
dummy text dummy text dummy text dummyKhaleduzzaman et al.

Fundam Appl Agric 6(4): 336–348, 2021

344

Figure 7. (a) Heatmap with hierarchical clustering and (b) principal component analysis (PCA) show thetreatment-variable interaction of different cultivars. In hierarchical clustering two distinct clusters(cluster-A and -B) were identified at the treatment × cultivars level. The colour scale of heatmap
displays the intensity of normalized mean values of different parameters. The entire dataset was
analyzed using PCA. The variables included germination percentage (GM), α-amaylase content(Amyl), shoot length (SL), root length (RL), shoot dry weight (SDW), root dry weight (RDW),
chlorophyll a (Chl a), chlorophyll b (Chl b), proline (Pro), total phenol content (Phenol), catalase(CAT), ascorbate peroxidase (APX), and peroxidase (POX). “C”, foliar spray with distilled water(non-stress condition); “S”, NaCl + foliar spray with distilled water; “S+25CS” NaCl + foliar spraywith 25 ppm chitosan; and “S+50CS” NaCl + foliar spray with 50 ppm chitosan. ‘28’, BRRI dhan28;‘29’, BRRI dhan29, ‘P’, Pengek; and ‘Po’, Pokkali
The non-enzymatic antioxidants such as phenolic
compounds play a significant role in the mitigation ofexcessive cellular ROS activities caused by salt stress.
Plants need to adapt different abiotic stresses andpolyphenols accumulate in response to these stresses
helping plants to acclimatize to unfavorable environments (Lattanzio, 2013; Pereira, 2016). Plants understress can biosynthesize more phenolic compounds
in comparison to plants growing under normal conditions (Selmar, 2008). These compounds have antioxidative properties and are capable of scavenging
free radicals and reducing cell membrane peroxidation (Schroeter et al., 2002), hence protecting plant
cells from adverse effects of oxidative stress. In thisstudy, salt stress increased total phenolics contents inboth rice genotypes compared with that of non-stresscondition (Fig. 5b). The increased phenolics contents
in salt stress conditions might be regulated by thealtered activities of key enzymes of phenolic biosynthetic pathways like PAL (phenylalanine ammonia
lyase) (Mrázová et al., 2017). On the contrary, supplementation of CS reduced the phenolic contents compared with that of salt-stressed rice seedlings (Fig. 5b).

The increase of antioxidant activity is related tobetter oxidant management under stressed conditions(Ahmad et al., 2010; Engwa, 2018). In this investigation, CAT and APX activities were increased in salttolerant cultivars but not in salt-sensitive cultivars
under salt stress conditions. The PCA also revealed
that tolerant cultivars had a stronger positive association with POX, APX, and CAT activity than sensitive cultivars (Fig. 7b). However, supplementation of
CS increased CAT and APX activities in salt-stressed
rice seedlings of salt-sensitive cultivars (Fig. 6a-b).
Turk (2019) recently reported that CS increased CAT
and APX activities in salt-stressed wheat seedlingsin comparison to non-stress seedlings and increase
tolerance to salt stress. It has been revealed that low
concentration CS treated seeds of safflower and sunflower can alleviate the oxidative stress caused bysalt stress by reducing enzyme (CAT and POX) activities in both crops (Jabeen and Ahmad, 2012). Several
studies have also reported that pretreatment withCS during salinity stress results in increased antioxidant enzyme activities which ultimately reduces thenegative effect caused by salt stress in Oryza sativa

Khaleduzzaman et al.

Fundam Appl Agric 6(4): 336–348, 2021

(Martínez González et al., 2015), (Al-Tawaha et al.,2018), Vigna radiata (Ray et al., 2016a), Trachyspermum
ammi (Mahdavi and Rahimi, 2013) and Plantago ovata(Mahdavi, 2013). Further, a hydroponic experiment
done on wheat showed that CS treated seed resulted
in positive effects by significantly increasing antioxidant enzymzes (POX and CAT) during salt-induced
stress and was able to alleviate the oxidative stress(Ma et al., 2011).

5

Conclusion

Increasing the tolerance of rice against salinity is an
important task for rice researchers to reduce the effectof climate change on rice production. In summary,
our study provides the evidence that supplementation of CS comparatively ameliorated salt stressinduced growth inhibition and biomass loss by regulating several physiological and biochemical mechanisms, including (i) improvement of germination byincreasing α-amylase activity, (ii) protection of photosynthetic pigments, (iii) maintaining osmoprotection
and stimulation of phenolics productions, and (iv)enhancement of the activities of ROS-scavenging enzymatic antioxidants (CAT, APX and POX). Taken together, these results suggest that CS supplementation
could be a viable cost-effective technology that canbe employed for the mitigation of salt stress-caused
adverse effects on crop performance to ensure sustainable agriculture in saline-prone areas. However,further studies at the field level using different modesof CS supplementation to a range of crop species under various regimes of salinity should be needed toascertain the beneficial roles of CS in the managementof salinity. Additionally, it would be fascinating to
identify whether CS application positively alters seedbiochemical and nutrient contents of rice that may
help us deal with the undernutrition problems of thepeople living in many developing countries.

Acknowledgments
The authors are thankful to Dr. Hari Pada Seal (Professor, Department of Agricultural Chemistry, BAU,
Mymensingh) for providing the chitosan. The authors
would like to extend their sincere appreciation to the
Bangladesh Agricultural University Research System
for funding this research (Project No. 2016/13/AUGC).

Conflict of Interest
The authors declare that there is no conflict of interests regarding the publication of this paper.

345

References
Ahmad P, Jaleel CA, Salem MA, Nabi G, Sharma S.
2010. Roles of enzymatic and nonenzymatic antioxidants in plants during abiotic stress. Critical Reviews in Biotechnology 30:161–175. doi:
10.3109/07388550903524243.
Akram R, Fahad S, Masood N, Rasool A, Ijaz M, Ihsan MZ, Maqbool MM, Ahmad S, Hussain S,
Ahmed M, Kaleem S, Sultana SR, Mubeen M,
Saud S, Kamran M, Nasim W. 2019. Plant growthand morphological changes in rice under abiotic stress. In: Advances in Rice Research for
Abiotic Stress Tolerance. Elsevier. p. 69–85. doi:
10.1016/b978-0-12-814332-2.00004-6.
Al-Tawaha AR, Turk MA, Al-Tawaha ARM, Alu’datt
MH, Wedyan M, Al-Ramamneh E, Hoang AT.
2018. Using chitosan to improve growth ofmaize cultivars under salinity conditions. Bulgarian Journal of Agricultural Science 24:437–
442.
Bates LS, Waldren RP, Teare ID. 1973. Rapid determination of free proline for water-stressstudies.
Plant and Soil 39:205–207.
doi:
10.1007/bf00018060.
Bernfeld P. 1955. Amylases, α and β. In: Methods in Enzymology. Elsevier. p. 149–158. doi:
10.1016/0076-6879(55)01021-5.
Boonlertnirun S, Boonraung C, Suvanasara R. 2008.
Application of chitosan in rice production. Journal of metals, materials and minerals 18:47–52.
Bose J, Rodrigo-Moreno A, Shabala S. 2013. ROS
homeostasis in halophytes in the context of salinity stress tolerance. Journal of Experimental
Botany 65:1241–1257. doi: 10.1093/jxb/ert430.
Chandrashekar KR, Sandhyarani S. 1996. Salinity
induced chemical changes in Crotalaria striata
DC plants. Indian Journal of Plant Physiology
1:44–48.
Chawla S, Jain S, Jain V. 2012. Salinity induced oxidative stress and antioxidant system in salttolerant and salt-sensitive cultivars of rice (Oryza
sativa L.). Journal of Plant Biochemistry andBiotechnology 22:27–34. doi: 10.1007/s13562012-0107-4.
Dreyer A, Dietz KJ. 2018. Reactive oxygen species andthe redox-regulatory network in cold stress acclimation. Antioxidants 7:169. doi: 10.3390/antiox7110169.
Engwa GA. 2018. Free radicals and the role of plant
phytochemicals as antioxidants against oxidative stress-related diseases. In: Phytochemicals -Khaleduzzaman et al.

Fundam Appl Agric 6(4): 336–348, 2021

Source of Antioxidants and Role in Disease Prevention. InTech. doi: 10.5772/intechopen.76719.
Foyer CH. 2018.
Reactive oxygen species,
oxidative signaling and the regulationof photosynthesis.
Environmental andExperimental Botany 154:134–142.
doi:
10.1016/j.envexpbot.2018.05.003.
Gupta B, Huang B. 2014. Mechanism of salinity
tolerance in plants: Physiological, biochemical, and molecular characterization. International Journal of Genomics 2014:1–18. doi:
10.1155/2014/701596.
Haque MA, Jahiruddin M, Hoque MA, Rahman MZ,
Clarke D. 2014. Temporal variability of soil
and water salinity and its effect on crop at kalapara upazila. Journal of Environmental Science and Natural Resources 7:111–114. doi:
10.3329/jesnr.v7i2.22218.
Hasanuzzaman M, Alam MM, Rahman A, Hasanuzzaman M, Nahar K, Fujita M. 2014. Exogenous
proline and glycine betaine mediated upregulation of antioxidant defense and glyoxalasesystems provides better protection against saltinduced oxidative stress in two rice (Oryza sativa
L) varieties. Research International 2014:1–17.
doi: 10.1155/2014/757219.
Hasanuzzaman M, Nahar K, Fujita M, Ahmad P,
Chandna R, Prasad MNV, Ozturk M. 2013. Enhancing plant productivity under salt stress:
Relevance of poly-omics. In: Salt Stress in
Plants. Springer New York. p. 113–156. doi:
10.1007/978-1-4614-6108-1_6.
Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ. 2000.
Plant cellular and molecular responses to highsalinity. Annual Review of Plant Physiology
and Plant Molecular Biology 51:463–499. doi:
10.1146/annurev.arplant.51.1.463.
Hua-long L, Han-jing S, Jing-guo W, Yang L, De-tang
Z, Hong-wei Z. 2014. Effect of seed soaking
with exogenous proline on seed germination ofrice under salt stress. Journal of Northeast Agricultural University 21:1–6. doi: 10.1016/s10068104(14)60062-3.
Jabeen N, Ahmad R. 2012. The activity of antioxidant enzymes in response to salt stress in safflower (Carthamus tinctorius L.) and sunflower(Helianthus annuus L.) seedlings raised from seedtreated with chitosan. Journal of the Scienceof Food and Agriculture 93:1699–1705. doi:
10.1002/jsfa.5953.
jing Guan Y, Hu J, ju Wang X, xia Shao C. 2009.
Seed priming with chitosan improves maize346

germination and seedling growth in relation tophysiological changes under low temperaturestress. Journal of Zhejiang University SCIENCE
B 10:427–433. doi: 10.1631/jzus.b0820373.
Katiyar D, Hemantaranjan A, Singh B, Bhanu A.
2014. A future perspective in crop protection:
Chitosan and its oligosaccharides. Advancesin Plants & Agricultural Research 1:6. doi:
10.15406/apar.2014.01.00006.
Kibria MG, Hossain M, Murata Y, Hoque MA. 2017.
Antioxidant defense mechanisms of salinity tolerance in rice genotypes. Rice Science 24:155–162.
doi: 10.1016/j.rsci.2017.05.001.
Kumar V, Khare T. 2016. Differential growth andyield responses of salt-tolerant and susceptible
rice cultivars to individual (Na+ and Cl–) and additive stress effects of NaCl. Acta Physiologiae
Plantarum 38. doi: 10.1007/s11738-016-2191-x.
Lattanzio V. 2013. Phenolic Compounds: Introduction. In: Ramawat KG, Merillon J-M (Eds),
Natural Products: Phytochemistry, Botany andMetabolism of Alkaloids, Phenolics and Terpenes. Springer, Berlin/Heidelberg, Germany.
Liu L, Xia W, Li H, Zeng H, Wei B, Han S, Yin C. 2018.
Salinity inhibits rice seed germination by reducing α-amylase activity via decreased bioactive
gibberellin content. Frontiers in Plant Science
9:275. doi: 10.3389/fpls.2018.00275.
Lizárraga-Paulín EG, Torres-Pacheco I, MorenoMartínez E, Miranda-Castro SP. 2011. Chitosan
application in maize (Zea mays) to counteract theeffects of abiotic stress at seedling level. African
Journal of Biotechnology 10:6439–6446.
Ma L, Li Y, Yu C, Wang Y, Li X, Li N, Chen Q, Bu N.
2011. Alleviation of exogenous oligochitosan on
wheat seedlings growth under salt stress. Protoplasma 249:393–399. doi: 10.1007/s00709-0110290-5.
Mahdavi B. 2013. Seed germination and growth responses of Isabgol (Plantago ovata Forsk) to chitosan and salinity. International Journal of Agriculture and Crop Sciences 5:1084–1088.
Mahdavi B, Rahimi A. 2013. Seed priming with chitosan improves the germination and growth performance of ajowan. EurAsian Journal of Biosciences 7:69–76. doi: 10.5053/ejobios.2013.7.0.9.
Mahmud S, Sharmin S, Ch BLD, Hos MA. 2017. Effect of salinity and alleviating role of methyl
jasmonate in some rice varieties.
Asian
Journal of Plant Sciences 16:87–93.
doi:
10.3923/ajps.2017.87.93.

Khaleduzzaman et al.

Fundam Appl Agric 6(4): 336–348, 2021

347

Mahmud S, Sharmin S, Chowdhury BLD, Hossain
MA, Bhuiyan MJH. 2016. Mitigation of salt
stress in rice plant at germination stage byusing methyl jasmonate. Asian Journal of
Medical and Biological Research 2:74–81. doi:
10.3329/ajmbr.v2i1.27572.

Pereira A. 2016. Plant abiotic stress challenges fromthe changing environment. Frontiers in Plant
Science 7:1123. doi: 10.3389/fpls.2016.01123.

Malerba M, Cerana R. 2015. Reactive Oxygen andNitrogen Species in Defense/Stress Responses
Activated by Chitosan in Sycamore Cultured
Cells. International Journal of Molecular Sciences 16:3019–3034. doi: 10.3390/ijms16023019.

Peykani LS, Sepehr MF. 2018. Effect of chitosan on
antioxidant enzyme activity, proline, and malondialdehyde content in Triticum aestivum L. and
Zea maize L. under salt stress condition. Iranian
Journal of Plant Physiology 9:2661–2670.

Marco F, Bitrián M, Carrasco P, Rajam MV, Alcázar R,
Tiburcio AF. 2015. Genetic engineering strategies
for abiotic stress tolerance in plants. In: Plant
Biology and Biotechnology. Springer India. p.
579–609. doi: 10.1007/978-81-322-2283-5_29.

Pottosin I, Velarde-Buendia AM, Bose J, ZepedaJazo I, Shabala S, Dobrovinskaya O. 2014.
Cross-talk between reactive oxygen species andpolyamines in regulation of ion transport acrossthe plasma membrane: implications for plant
adaptive responses. Journal of Experimental
Botany 65:1271–1283. doi: 10.1093/jxb/ert423.

Martínez González L, Reyes Guerrero Y, Falcón Rodríguez A, Núñez Vázquez M. 2015. Effect ofseed treatment with chitosan on the growth ofrice (Oryza sativa L.) seedlings cv. INCA LP-5 insaline medium. Cultivos Tropicales 36:143–150.
Mrázová A, Belay SA, Eliášová A, Perez-Delgado C,
Kaducová M, Betti M, Vega JM, Pal’ove-Balang
P. 2017. Expression, activity of phenylalanineammonia-lyase and accumulation of phenolic
compounds in lotus japonicus under salt stress.
Biologia 72:36–42. doi: 10.1515/biolog-20170001.
Munns R, Tester M. 2008. Mechanisms of salinity tolerance.
Annual Review of Plant
Biology 59:651–681.
doi:
10.1146/annurev.arplant.59.032607.092911.
Nahar K, Hasanuzzaman M, Fujita M. 2016. Rolesof osmolytes in plant adaptation to drought
and salinity. In: Osmolytes and Plants Acclimation to Changing Environment: Emerging
Omics Technologies. Springer India. p. 37–68.
doi: 10.1007/978-81-322-2616-1_4.
Nakano Y, Asada K. 1981. Hydrogen peroxide isscavenged by ascorbate-specific peroxidase inspinach chloroplasts. Plant and cell physiology
22:867–880.
Noctor G, Foyer CH. 1998. Ascorbate and glutathione: Keeping active oxygen under control.
Annual Review of Plant Physiology and Plant
Molecular Biology 49:249–279. doi: 10.1146/annurev.arplant.49.1.249.
Ozturk L, Demir Y. 2003. Plant Growth Regulation
40:89–95. doi: 10.1023/a:1023078819935.
Parida AK, Das AB, Mohanty P. 2004. Defense potentials to NaCl in a mangrove, bruguiera parviflora: Differential changes of isoforms of someantioxidative enzymes. Journal of Plant Physiology 161:531–542. doi: 10.1078/0176-1617-01084.

Qureshi MI, Abdin MZ, Ahmad J, Iqbal M.
2013. Effect of long-term salinity on cellular antioxidants, compatible solute and fatty
acid profile of Sweet Annie (Artemisia annua L.). Phytochemistry 95:215–223. doi:
10.1016/j.phytochem.2013.06.026.
Rahman A, Nahar K, Hasanuzzaman M, Fujita M. 2016. Calcium supplementation improves Na+/K+ ratio, antioxidant defense
and glyoxalase systems in salt-stressed riceseedlings. Frontiers in Plant Science 7:609. doi:
10.3389/fpls.2016.00609.
Ray SR, Bhuiyan MJH, Hossain MA, Hasan AK,
Sharmin S. 2016a. Chitosan ameliorates growthand biochemical attributes in mungbean varieties under saline condition. Research in Agriculture Livestock and Fisheries 3:45–51. doi:
10.3329/ralf.v3i1.27857.
Ray SR, Bhuiyan MJH, Hossain MA, Mahmud S,
Tahjib-Ul-Arif. 2016b. Chitosan suppresses antioxidant enzyme activities for mitigating salt
stress in mungbean varieties. IOSR Journal of
Agriculture and Veterinary Science 09:36–41. doi:
10.9790/2380-0909023641.
Roy PR, Tahjib-Ul-Arif M, Polash MAS, Hossen MZ,
Hossain MA. 2019. Physiological mechanismsof exogenous calcium on alleviating salinityinduced stress in rice (Oryza sativa L). Physiology and Molecular Biology of Plants 25:611–624.
doi: 10.1007/s12298-019-00654-8.
Ruan SL, Xue QZ. 2002. Effects of chitosan coating on
seed germination and salt tolerance of seedlingsin hybrid rice (Oryza sativa L). Acta Agron Sinica
28:803–808.

Khaleduzzaman et al.

Fundam Appl Agric 6(4): 336–348, 2021

Schroeter H, Boyd C, Spencer JP, Williams RJ, Cadenas E, Rice-Evans C. 2002. MAPK signaling inneurodegeneration: influences of flavonoids andof nitric oxide. Neurobiology of Aging 23:861–
880. doi: 10.1016/s0197-4580(02)00075-1.
Selmar D. 2008. Potential of salt and drought
stress to increase pharmaceutical significant
secondary compounds in plants. Landbauforschung Volkenrode 58:139–144.
Sen SK, Chouhan D, Das D, Ghosh R, Mandal P.
2020. Improvisation of salinity stress response inmung bean through solid matrix priming withnormal and nano-sized chitosan. International
Journal of Biological Macromolecules 145:108–123. doi: 10.1016/j.ijbiomac.2019.12.170.
Shams Peykani L, Farzami Sepehr M. 2018. Effectof chitosan on antioxidant enzyme activity, proline, and malondialdehyde content in Triticum
aestivum L. and Zea mays L. under salt stresscondition. Iranian Journal of Plant Physiology
9:2661–2670.
Silva-Ortega CO, Ochoa-Alfaro AE, Reyes-Aguero
JA, Aguado-Santacruz GA, Jiménez-Bremont JF.
2008. Salt stress increases the expression of p5cs
gene and induces proline accumulation in cactus pear. Plant Physiology and Biochemistry
46:82–92. doi: 10.1016/j.plaphy.2007.10.011.
Symes A, Shavandi A, Zhang H, Ahmed IM, AlJuhaimi F, Bekhit A. 2018. Antioxidant activities and caffeic acid content in New Zealand
Asparagus (Asparagus officinalis) roots extracts.
Antioxidants 7:52. doi: 10.3390/antiox7040052.
Tahjib-Ul-Arif M, Afrin S, Polash MAS, Akter T, Ray
SR, Hossain MT, Hossain MA. 2019. Role of exogenous signaling molecules in alleviating saltinduced oxidative stress in rice (Oryza sativa L.):c 2021 by the author(s). This work islicensed under a Creative Commons.
Attribution-NonCommercial 4.0
International (CC BY-NC 4.0) License348

a comparative study. Acta Physiologiae Plantarum 41. doi: 10.1007/s11738-019-2861-6.
Turk H. 2019. Chitosan-induced enhanced expression
and activation of alternative oxidase confer tolerance to salt stress in maize seedlings. Plant
Physiology and Biochemistry 141:415–422. doi:
10.1016/j.plaphy.2019.06.025.
Wahid A, Farooq M, Basra SMA, Rasul E, SiddiqueKHM. 2011. Germination of seeds and propagules under salt stress. In: Pessarakli M (Ed),
Handbook of plant and crop stress. CRC Press,
Boca Raton.
Wang N, Qiao W, Liu X, Shi J, Xu Q, Zhou H, Yan G,
Huang Q. 2017. Relative contribution of Na+/K+
homeostasis, photochemical efficiency and antioxidant defense system to differential salt tolerance in cotton (Gossypium hirsutum L.) cultivars.
Plant Physiology and Biochemistry 119:121–131.
doi: 10.1016/j.plaphy.2017.08.024.
Yang Y, Guo Y. 2017. Elucidating the molecular mechanisms mediating plant salt-stress responses. New Phytologist 217:523–539. doi:
10.1111/nph.14920.
Yasmeen A, Basra SMA, Farooq M, ur Rehman H,
Hussain N, ur Rehman Athar H. 2012. Exogenous application of moringa leaf extract modulates the antioxidant enzyme system to improve wheat performance under saline conditions. Plant Growth Regulation 69:225–233. doi:
10.1007/s10725-012-9764-5.
Zayed M, Elkafafi S, Zedan A, Dawoud S. 2017. Effectof nano chitosan on growth, physiological andbiochemical parameters of phaseolus vulgaris
under salt stress. Journal of Plant Production
8:577–585. doi: 10.21608/jpp.2017.40468.

The Official Journal of theFarm to Fork Foundation
ISSN: 2518–2021 (print)
ISSN: 2415–4474 (electronic)
http://www.f2ffoundation.org/faa