Journal of Applied Research on Medicinal and Aromatic Plants 10 (2018) 49–58

Contents lists available at ScienceDirect

Journal of Applied Research on Medicinal and Aromatic

Plants
journal homepage: www.elsevier.com/locate/jarmap

Role of chitosan on the growth, physiological parameters and enzymatic T

activity of milk thistle (Silybum marianum (L.) Gaertn.) in a pot experiment
⁎
Sara Safikhana, Korous Khoshbakhta, , Mohammad R. Chaichib, Abbas Aminic,
Babak Motesharezadehd
a
Department of Agroecology, Shahid Beheshti University, Environmental Sciences Research institute, Shahid Beheshti University, Evin, Tehran, Iran
b
Department of Crop Science, College of Agriculture, California State University, Pomona, USA
c
Centre for Infrastructure Engineering, Western Sydney University, Penrith 2751, NSW, Australia
d
Department of Soil Science and Engineering, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: Salt stress is the destructive factor in plant growth and physiological activities. Using biobased stimulants, such
Salinity as chitosan, is important to reduce the adverse effects of salinity. This study was carried out in a greenhouse
Ions concentration located at the University of Tehran, in 2016. The goal of this study was to evaluate the effect of chitosan
H2O2 application on modification of adverse effects of soil salinity on growth and physiological characteristics of milk
Proline
thistle (Silybum marianum (L.) Gaertn.). A pot experiment with a factorial arrangement of treatments was con-
Root growth
ducted based on a randomized complete block design (RCB) with three replications Four irrigation water salinity
Soluble sugars
levels were control (tap water 0.8), 4, 8 and 12 dS/m and four levels of chitosan were mixed with dry soil to yield
0, 0.01, 0.05 and 0.1% chitosan (DW/DW). The results showed that salinity reduced root dry weight; shoot dry
weight, total plant biomass, and increased soluble sugars, proline content, CAT spell out first use and POD spell
out first use enzyme activity and H2O2 concentration in leaves. The use of chitosan led to a reduction of salinity
adverse effects and increased plant growth and improved physiological traits. Chitosan application at 0.01%
increased chlorophyll a and total chlorophyll and at 0.05% level increased chlorophyll b compared to other
chitosan treatments. The highest concentration of soluble sugars and proline was achieved by chitosan appli-
cation across all salinity levels. Chitosan application under 0.01 and 0.05% enhanced the enzymatic activity and
decreased H2O2 concentration in leaves. The results illustrated that chitosan could protect plants from salt stress
damage by modulating intracellular ion concentration and by enhancing the capacity of antioxidant enzyme
activities. It seems that the average concentration of chitosan as a bio-stimulant (0.01 and 0.05%) plays a
positive role in reducing salinity and enhancing growth in milk thistle.

1. Introduction thistle is known as a low-input annual crop which is tolerant to various

environmental stresses (Karkanis et al., 2011; Afshar et al., 2015).
Milk thistle (Silybum marianum (L.) Gaertn.) is a recognized med- Salinity is one of the major abiotic stresses limiting crop production,
icinal plant belonging to the Asteraceae family and originated in the particularly in arid and semi-arid regions. Salt stress affects plant
Mediterranean Basin. Silymarin, a derivative of milk thistle, has been physiology at both whole plant and cellular levels through osmotic and
used as an herbal remedy to treat liver disorders for more than 2000 ionic stress (Murphy and Durako, 2003). Salinity also reduces the plant
years (Afshar et al., 2014). Milk thistle is commercially grown in Europe growth and development through specific ion effects, nutritional im-
where Poland is the biggest producer of milk thistle seeds and its de- balance, low osmotic potential of soil solution, and combinations of
rivatives in the world with cultivation area of more than 2000 ha these factors (Ashraf and Harris, 2004). All of these factors caused by
(Andrzejewska et al., 2011). In addition to silymarin, milk thistle seeds high salt contents can affect various major plant processes like photo-
contain relatively high amounts of oil which need to be removed from synthesis, protein synthesis and also energy and lipid metabolisms (Li
the seeds before extraction of silymarin (Hadolin et al., 2001). Milk et al., 2008). Ghavami and Ramin (2008) showed that growth

⁎
Corresponding author at: Department of Agroecology, Shahid Beheshti University, Environmental Sciences Research Institute, Shahid Beheshti University, Post
Box: 1983963113, Evin, Tehran, Iran.
E-mail addresses: ssafi[email protected] (S. Safikhan), [email protected] (K. Khoshbakht), [email protected] (M.R. Chaichi),
[email protected] (A. Amini), [email protected] (B. Motesharezadeh).

https://doi.org/10.1016/j.jarmap.2018.06.002
Received 27 November 2017; Received in revised form 29 May 2018; Accepted 9 June 2018
Available online 30 August 2018
2214-7861/ Crown Copyright © 2018 Published by Elsevier GmbH. All rights reserved.
S. Safikhan et al. Journal of Applied Research on Medicinal and Aromatic Plants 10 (2018) 49–58

parameters such as plant height, the number of leaves per plant, the 2. Material and methods
number of capitula per plant, main shoot capitulum diameter, and seed
yield and yield components per plant were reduced with salinity greater 2.1. Experimental design
than 9 dS/m. In another study, they reported that percentage of ger-
mination and the number of normal seedlings at different salt treatment A pot experiment with a factorial arrangement of treatments was
at 15.8 °C were higher than at 25 °C or 35.8 °C. The mean time to 50% conducted based on a randomized complete block design (RCB) with
germination was lowest at 15.8 °C temperature. Their results suggested three replications. All pots were seeded on Oct. 10, 2016. The experi-
that best germination indices and seedling emergence (50%) were mental period continued to Dec. 20, 2016. During the experimental
achieved at salinity levels up to 9 dS/m at 15.8 °C (Ghavami and Ramin, period, pots were kept in a glass greenhouse under natural light. The
2007). Root length of milk thistle decreased as the level of water sali- minimum and maximum temperatures of the greenhouse ranged be-
nity increased compared to control. A negative correlation between tween 20 and 25 °C, respectively, and the relative humidity was kept at
germination and salinity level was reported by El-Garhy et al. (2016). ∼50%. Four irrigation water salinity levels were control (tap water
Application of biostimulants is one of the approaches to decrease 0.8), 4, 8 and 12 dS/m and four levels of chitosan were mixed with dry
the negative effect of abiotic stress and increase yield and quality of soil to yield 0, 0.01, 0.05 and 0.1% chitosan (DW/DW). Irrigation water
many crops. Chitosan can be obtained by partial deacetylation of chitin treatments were applied all through the growing period (planting of
(poly-N-acetyl-D-glucosamine) from crustacean shells and it is the seeds to plant physiological maturity). Water salinity levels were cre-
second most abundant polysaccharide after cellulose. There are an es- ated by NaCl application to tap water. To maintain a constant soil EC at
timated 10 gigatons of chitin recycled in nature each year (Ruiz-Herrera the four saline levels, sufficient irrigation was applied to cause leaching.
et al., 2002). Chitosan is a biodegradable, renewable polysaccharide Constant soil EC during the study that was produced by the four saline
that generally is considered to be biocompatible and non-toxic (Kean irrigation treatments was confirmed by measuring soil EC following
and Thanou, 2010). irrigation of additional pots treated same four saline irrigation levels.
Chitosan was first categorized as an elicitor in plants activating
genes that underlie the biosynthetic pathways of secondary metabolites 2.2. Chitosan characterization
(Yin et al., 2011). Chitosan can be used both in vivo and in vitro and can
be sprayed on plant aerial organs to induce the accumulation of Chitosan treatments were created by application of different
bioactive secondary metabolites (Yin et al., 2011). The report indicated amounts of the material based on soil dry weight in pots. To separate
that chitosan reduced plant transpiration in pepper plants, resulting in the rhizosphere soil from the bulk soil, a cylindrical rhizobag (13 cm
26%–43% reduction in water use without a reduction in dry matter diameter, 11 cm height, and 60 mesh (0.25 mm) pores), was utilized in
yield (Bittelli et al., 2001). These results suggested that chitosan might each pot (Fig. 2a, b). The rhizobag methodology was adopted from
be an effective antitranspirant for reducing the consumption of irriga- Wenzel et al. (2001). Each rhizobag was filled with 1000 g sieved soil
tion water in agriculture. and fixed vertically into the pots. Rhizobags did not prevent nutrient
Reactive Oxygen Species (ROS) contain superoxide anion radicals, absorption by the roots (Ohta et al., 2004). Four levels of chitosan (0%
hydroxyl radicals, and hydrogen peroxide that are generated as by- (control), 0.01%, 0.05% and 0.1%) based on the soil dry weight in the
products of metabolic processes inside cells or in environmental Rhizobag (1 kg/pot) were added and mixed with the soil.
sources. Antioxidant activity of chitosan has also been described (Park Chitosan with CAS Number 9012-76-4 and MDL number
et al., 2004). Chitosan modulates the plant response to several abiotic MFCD00161512 was purchased from Sigma-Aldrich, USA. The physi-
stresses including salt and water stress (Dzung et al., 2011; Ruan and cochemical properties of chitosan were beige to orange color, powder
Xue, 2002). Feng et al. (2007) indicated that water-soluble chitosan is a form, viscosity 800–2000 cps and high molecular weight. The scanning
natural antioxidant and that its antioxidant activity depends on its electron microscopy (SEM) examination (a) as well as the chemical
molecular weight. formula of chitosan (b) are presented in Fig. 1.
The effectiveness of chitosan application depends on the con-
centration of the compound, substrate, water content, temperature, 2.3. Soil characterization
stage of rooting and plant development during exposure to drought
stress. Chitosan application improved the chlorophyll content, number A combined soil sample was collected from Research Station of
of nodes and root establishment in grapevine plant under drought stress College of Agriculture, University of Tehran, Karaj, Iran, and sieved to
(Gornik et al., 2008). The research carried out by Borkowski et al. ≤2 mm. The sampled field was mainly allocated to cereal crops pro-
(2006) indicated that chitosan increased fruit yield of tomato and duction. Soil moisture percentage, field capacity (F.C.) and permanent
cabbage. Chitosan affects biochemical reactions which modify the ad- wilting point (P.W.P.) were determined based on the Klute (1986)
verse effects of salinity in plants (Amiri et al., 2016). Increased root and method. The physical/chemical properties of the experimental soil are
shoot dry weight, germination, leaf area index and chlorophyll content presented in Table 1.
in maize and bean crops have been affected by chitosan (Amiri et al.,
2016). Photosynthetic pigments and leaf relative water content are 2.4. Plantation
affected by salinity. Salt reduces the amount of chlorophyll in chamo-
mile (Kovacik et al., 2009; Sarani et al., 2013). The use of biological Milk thistle (Silybum marianum (L.) Gaertn.) seeds were purchased
stimulators, such as chitosan, could alleviate the adverse effects of from Pakatan Bazr Seed Company, Isfahan, Iran. Fifteen seeds of milk
biotic and abiotic stresses. thistle were sown 2 cm deep in each plastic pots (23 cm diameter ×
However, few positive results of chitosan application on plant 24 cm height) containing 8.0 kg of soil. Following seeding, tap (0.8 dS/
growth and development under stressed conditions have been reported. m) and prepared saline water (4, 8 and 12 dS/m) were added to each
This experiment was performed due to a lack of information on the pot according to the corresponding treatments to reach saturation and
application of chitosan on milk thistle, especially in salt stress condi- drain to F.C.
tions. The objective of this study was to evaluate the effect of chitosan During the growing period, to prevent excessive salt accumulation
application on growth and physiological characteristics (biochemical, in rhizosphere, pots were irrigated with distilled water in two occa-
physiological, and enzymatic activities) of milk thistle under salinity sions. To prevent any drought stress incidence in fresh and saline water
stress. treatments, the soil moisture in pots were kept at 80% of the F.C. all
through the experimental period.
During the experimental period, all the pots were kept inside a glass

50
S. Safikhan et al. Journal of Applied Research on Medicinal and Aromatic Plants 10 (2018) 49–58

Fig. 1. (a) SEM image of HM (high molecular) Chitosan and (b) Chemical formula of chitosan.

greenhouse under natural light. The minimum and maximum tem- using a UV-160 A UV–vis recording spectrometer (Shimadzu UV 180).
peratures of the greenhouse were maintained at 20 and 25 °C in day and The total chlorophyll and its components were calculated using the
night, respectively; the RH was maintained at ∼50%. After germina- following equations.
tion, when plants reached 4-leaf stage, the seedlings were thinned to
Chlorophyll a: 12.7 (A663) – 2.69 (A645)
five plants per pot and in couple of weeks they were thinned to three
plants per pot Chlorophyll b: 22.9 (A645) – 4.68 (A663)

Total Chlorophyll: 20.2 (A645) + 8.02 (A663)

2.5. Studied traits

2.5.1. Root and shoot growth 2.5.3. Determination of soluble sugar

After 72 days of planting, plants with a uniform growth were se- After 72 days of saline water (NaCl) application, soluble sugar was
lected to continue the experiment. The plant root and shoot were cut at measured by the following procedure: 0.5 g of leaf samples were cut up
the base and weighed to determine the dry root and shoot weight. and heated at 100 °C for 30 min in 5 mL distilled water. The extract was
Samples were dried at 60 °C for 48 h, and their mean root and shoot dry diluted 5-fold for determination. A mixture of 500 μL diluents, 1 mL 5%
weight were recorded for each treatment and replicate. phenol and 5 mL sulfuric acid was made and after standing for 3 min,
the absorbance was read at 485 nm. Soluble sugar concentration was
2.5.2. Leaf chlorophyll content quantified by comparison with a standard curve using the criterion of
Leaf chlorophyll was measured 72 days after planting. Total chlor- glucose.
ophyll, as well as chlorophyll a and b concentrations, were measured
according to Arnon (1986). One gram of fresh leaves was taken in the 2.5.4. Proline content
middle of the flowering period, ground with 10 mL of 80% acetone and Proline was extracted according to the procedure of Irigoyen et al.
then centrifuged at 5000 rpm for 5 min. The absorbance of the solution (1992) using 0.3 g of leaf sample and 6 mL of extraction medium.
was read at 645 nm and 663 nm against the solvent (acetone) blank Proline was quantified by spectrophotometry at 515 nm by a

Fig. 2. (a) Rhizobag structure, (b) Vertically set into pot.

51
S. Safikhan et al. Journal of Applied Research on Medicinal and Aromatic Plants 10 (2018) 49–58

Table 1
Physical and chemical properties of soil filled in both pots and rhizomes.
PWP Field capacity Total nitrogen Available phosphorous Available potassium pH Electrical conductivity Organic matter Soil Texture (Loam)
(%) (%) (%) (mg kg−1) (mg kg−1) (EC) (%)
ds/m Clay (%) Silt Sand (%)
(%)

10.42 22.40 0.09 14.1 151 8 1.70 0.84 22.5 43.34 34.16

colorimetric reaction with ninhydrin (Irigoyen et al., 1992). The reac- levels; control (tap water), 4, 8, and 12 dS m−1. The combination of all
tion mixture contained 1.5 mL of 25% (w: v) ninhydrin, 1.5 mL acetic levels of all factors resulted in 16 treatments.
acid and 0.5 mL of the extract. Samples were incubated for 1 h in a All the data were statistically analyzed using SAS 9.1 software. The
boiling water bath, and thereafter they were cooled on ice. Then 2 mL means of each trait were compared according to the Duncan multiple
toluene were added to the reaction mixture, vigorously agitated and ranges at p ≤ 0.05. All graphs were created using Excel.
finally the upper organic phase was extracted to measure the absor-
bance. For the calculation of proline concentration, a standard curve 3. Results and discussion
was prepared with L-proline.
3.1. Plant height
2.5.5. Enzyme activity
The antioxidant enzyme activities were measured using leave sam- Plant height was significantly inhibited under salinity treatments
ples. Approximately 250 mg of fresh leaf tissue was randomly taken compared to control (P < 0.05) (Table 2, Fig. 3). However, plant
from plants in each treatment, frozen in liquid nitrogen, and stored at height was increased when treated with chitosan under salt stress. At
80 °C for further analyses. The extraction of CAT, POD (Spell out for control level (no salinity stress), no significant effect of different chit-
first use of enzymes) was acquired according to the method proposed by osan treatments was observed. The highest plant height at 4 dS/m
He et al. (2001) with minor modifications. Briefly, frozen leaves were salinity treatment was obtained by 0.05% of chitosan application. As
homogenized with 4 mL of 150 mMice-cold phosphate buffer (pH 7.0) the severity salinity treatments increased to 8 dS/m, the highest plant
with its mortar and pestle and centrifuged at 15,000×g, for 20 min at height was observed at 0.05 and 0.1% chitosan application. Ad-
4 °C. The supernatant was collected and used for the characterization of ditionally, the highest plant height at 12 dS/m salinity level was ob-
enzyme activity. served at 0.01% chitosan application. Nguyen Van et al. (2013) re-
ported a positive effect of chitosan application on plant height and leaf
2.5.5.1. Catalase activity assay. CAT activity was measured by area in coffee plant. Also, chitosan spraying with 20–40 microgram/mL
monitoring the H2O2 decomposition at 240 nm in 3 mL reaction had a positive effect on total fresh weight, leaf area, plant height, root
mixture containing 50 mmol L−1 phosphate buffer (pH 7.0), 15 mmol length, soluble protein and soluble sucrose in leaves, while the content
L−1 H2O2, 100 mL enzyme extract and 0.1% (V/V) Triton X-100 (Aebi, of crude fiber decreased in Chinese cabbage (Ouyang and Langlai,
1984). The activity was expressed in terms of mmol H2O2 reduced min 2003). Results revealed that plant height and leaf number per plant,
mg −1 protein −1. both under pot and field conditions, increased with chitosan application
in okra plants (Mondal et al., 2012). Abdel-Mawgoud et al. (2010)
2.5.5.2. Peroxidase activity assay. For characterizing the peroxidase (EC showed that chitosan application improved plant height, the leaf
1.11.1.7) activity, a mixture of 25 mmol L−1 phosphate buffer (pH 7.0), number, leaf fresh and dry weights, and yield in strawberry.
0.05% Guaiacol, 10 mmol L−1 H2O2 and enzyme were prepared. This
activity was determined by measuring the increase in the absorbance at 3.2. Root dry weight
470 nm due to Guaiacol oxidation (E = 26.6 mM–1 cm–1) (Hemeda and
Klein, 1990). Saline water (12 dS/m) decreased root dry weight by 55.3% com-
pared to control (Table 3). When exposed to salt, plants showed serious
2.5.5.3. Quantification of H2O2. According to the method proposed by cellular damage, a great decrease in biomass, root growth, water status,
Loreto and Velikova (2001), one g fresh leaf tissue was thoroughly and photosynthesis. However, chitosan application across all the con-
homogenized in liquid nitrogen and extracted with 3 mL of 1% (w/v) centrations increased root dry weight compared to control. Chitosan
trichloroacetic acid (TCA). Then, it was centrifuged at 10,000 x g for application enhanced the salinity tolerance of the plant and alleviated
15 min at 4 °C; the supernatant was collected at the end. To the assay stress symptoms. Previous studies reported that the root system can
mixture, which contained 10 mM potassium phosphate buffer (pH 7) interact with salt stress (Chatzistathis et al., 2013; Contreras-Cornejo
and 1 M potassium iodide (KI), an appropriate volume of the et al., 2014; Wu et al., 2015). Our study showed that salt stress de-
supernatant was added to maintain an equal amount of protein; this creased root dry weight, which suggested that the root system was in-
was followed by incubation in dark for 15 min. The absorbance was jured to some extent in the process of interacting with salinity. To adapt
measured at 390 nm, and the content of H2O2 was determined using the to the environment, the root system adjusted its morphology to
standard curve; the H2O2 concentration was expressed as mg g−1 of the strengthen its absorption and survival through increasing the root
fresh weight (FW). length and root surface area.
Probably other places for the consistent use of unit abbreviations
(min; d; diam; normally P for probability; F.C.; etc.). Also, use of Latin 3.3. Shoot dry weight
name when first reference of a plant type.
The maximum shoot dry weight was achieved in 4 dS/m with 0.01%
2.6. Statistical analysis chitosan concentration treatment. However, in other salinity levels,
chitosan application increased shoot dry weight of milk thistle plants
The data were statistically evaluated by a two way ANOVA with two (Fig. 4). At control level (no salinity stress) no significant effect of
factors and their levels. Factor A: chitosan with four levels (0.1, 0.05, different chitosan treatments was observed. The highest shoot dry
0.01 and 0% (control). Factor B: Conductivity (salinity) with four weight at 4 dS/m salinity treatment was obtained by 0.01% of chitosan

52
S. Safikhan et al. Journal of Applied Research on Medicinal and Aromatic Plants 10 (2018) 49–58

application. At the salinity of 8 dS/m, the highest shoot dry weight was

F value

261.2
3.17

3.98
0.60
observed at 0.05 and 0.1% chitosan application levels. This is while the

–
–
–
lowest shoot dry weight was observed at control (no chitosan applica-

3.7377**
0.0569**
tion) compared to all different levels of chitosan application at 12 dS/
0.0453

0.0085
0.0143

0.1409
Prolin

m.

6.01
MS

Positive and additive effect of chitosan application were reported on

maize (Guan et al., 2009), okra (Mondal et al., 2012), safflower (Amiri
F value

570.7

et al., 2016) plants. The mechanism of action of chitosan on growth is

Soluble carbohydrate

0.06

5.34
0.44

not clear. It was also found that chitosan may induce a signal to syn-
–
–
– thesize plant hormones such as gibberellins and enhance growth and
1848.8029**

development by some signaling pathway related to auxin biosynthesis

17.2884**
0.1930

1.4104
3.2390

2.1217

(Uthairatanakij et al., 2007). Moreover, plants treated with chitosan

2.52

may be less prone to stress evoked by unfavorable conditions, such as

MS

drought, salinity, low or high temperature (Jabeen and Ahmad, 2013;

F value

Pongprayoon et al., 2013). Chitosan leads to physiological and bio-

469.3
14.31
0.03

0.67

chemical changes through the stimulation of biological processes,

–
–
–

which ultimately leads to changes in the molecular level and the ex-
0.064910**
0.001970**

pression of the gene (Limpanavech et al., 2008; Nguyen Van et al.,

0.000004

0.000092
0.000138
Total ch

0.0138

2013; Salachna and Zawadzinska, 2014).

1.06
MS

3.4. Total biomass

F value

93.17
0.47

4.82
0.93

As we hypothesized, application of chitosan alleviated the negative

–
–
–

impact of salt stress on total dry weight of milk thistle. Application of

0.00604**
0.00031*
0.00003

0.00006
0.00006
2.41359

chitosan with 0.01% concentration resulted in 37.41% higher biomass

0.0091
Ch b

on plants grown under 12 dS/m salt stress treatment, compared to

MS

control (Fig. 5). Furthermore, plants treated with chitosan with 0.01%
F value

concentration exhibited higher tolerance to salt stress in 4 dS/m level.

428.7
16.77
0.29

0.55

At control level (no salinity stress) no significant effect of different

–
–
–

chitosan treatments was observed. The highest total biomass at 4 dS/m

0.03142**
0.00122**

salinity treatment was obtained by 0.01% of chitosan application. As

0.00002

0.00004
0.00007

0.0098
Analysis of variance of growth and biochemical characteristics of milk thistle affected by salt stress and chitosan.

the salinity treatments increased to 8 dS/m, the highest total biomass

Ch a

1.11
MS

was observed at 0.05 and 0.1% chitosan application treatments. At

12 dS/m salinity level, the lowest total biomass was observed at control
F value

672.6
19.28

(no chitosan application) compared to all different levels of chitosan

0.57

4.52

application.
–
–
–
Total biomass

This polymer has been reported to enhance and regulate plant

3.44377**
0.09869**
0.02326**
0.00289

0.00512

growth, development, and yield (Gornik et al., 2008; Cabrera et al.,

0.0843
5.56

2013; Wang et al., 2015). Chitosan has been used as a bio promoter to
MS

stimulate plant growth, an abiotic water stress modifier, and a pathogen

F value

resistant agent. However, these responses are complex and depend on

302.5
12.21
0.61

3.63
Shoot dry weight

chitosan-based structures and concentrations as well as the plant spe-

–
–
–

cies and developmental stage (Qavami et al., 2017). Application of

2.10137**
0.08479**
0.02522**

chitosan to some extent modified yield reduction in plants grown under

0.00425

0.00694

0.0982

stress conditions (Emami Bistgani et al., 2017). The mechanism in-

8.84
MS

volved in plant growth stimulation under chitosan elicitation is not

known. Chitosan application at various stages of plant development was
F value

499.3

demonstrated to stimulate plant growth and development: Seed

1.91

5.63
0.25
Root dry weight

–
–
–

priming with chitosan enhance seedling root and shoot growth

(Manjunatha et al., 2008; Ma et al., 2014).
0.22976**
0.00259**

*and **: Significant at 5% and 1% probability levels.

0.00087

0.00012
0.00046

0.0252
6.07
MS

3.5. Effect of salt stress and chitosan on pigments

F value

Chlorophyll content is widely used as an index of abiotic tolerance

123.2
14.69
0.58

3.30

indicator in plants. Plants exposed to stressed environments such as

–
–
–
Plant height

salinity result in decreased chlorophyll concentration, thereby leading

123.28**
14.69**

to overall growth retardation. As shown in Table 3, increasing salinity

1.5143
3.30**
0.58

1.65
7.88

stress up to 4 dS/m did not significantly reduce chlorophyll a, b and

MS

total. With increasing of salinity stress up to 8 and 12 dS/m, the

D.F.

chlorophyll content in plants followed a decreasing trend. The

30
2
3
3
9

–
–

minimum chlorophyll a, b and total chlorophyll (0.709, 0.306 and

1.015 mgg−1 FW, respectively) were observed in 12 dS/m salinity level
Salt × Chitosan

(Table 3).
Salt stress
Chitosan

Chitosan application at 0.01% concentration increased chlorophyll

Table 2

S.O.V.

Block

Error

a (0.782 mgg−1 FW) as well as total chlorophyll (1.115 mgg−1 FW).

C.V.
CD

Also, application of 0.05% of chitosan warrantied maximum

53
S. Safikhan et al. Journal of Applied Research on Medicinal and Aromatic Plants 10 (2018) 49–58

Fig. 3. Effect of salt stress and exogenous chitosan on plant height of milk thistle.

chlorophyll b in treated plants (0.341 mgg−1 FW). These results were 3.7. Effect of chitosan on proline
associated with the significant increase of magnesium and total ni-
trogen in the leaves (Dzung et al., 2011; Nguyen Van et al., 2013) as Salt stress increased proline accumulation in milk thistle leaves. The
these are important elements in the composition of chlorophyll. The maximum proline content was observed in 12 dS/m (Table 3). Chitosan
same results were also shown on other plants. Chitosan oligomer in- application stimulated proline accumulation. Proline levels in milk
creased the chlorophyll content of soybean and peanut by 17.9% and thistle leaves increased by 8.0% when treated with 0.1% chitosan
23.0%, respectively (Dzung and Thang, 2004; Dzung, 2005). Also, compared to control. We found a synergy between imposed salt stress
chitosan application increased chlorophyll content, photosynthetic and and chitosan application with respect to proline levels. The highest
chloroplast enlargement in the leaves of Dendrobium orchid plants level of proline was accumulated in plants grown under 12 dS/m salt
(Limpanavech et al., 2008) and coffee (Nguyen Van et al., 2013). stress and sprayed with 0.1% chitosan. Thus, the effect of chitosan on
Chitosan appears to improve the tolerance of plants, such as saf- modification of salt stress was due to its least stimulatory influence on
flower and sunflower (Jabeen and Ahmad, 2013) to salt stress. In this proline accumulation. Application of 400 μL L−1 chitosan increased
experiment, plants treated with salt, exhibited significant decreases in proline content in thyme (Thymus defenses) plants grown under drought
chlorophyll a, b and total compared with control, whereas chitosan stress (Emami Bistgani et al., 2017).
with different concentrations led to a significant increase under NaCl The accumulation of ions requires the accumulation of solutes in the
stress. These results correspondence with Zou et al. (2015) observation cytosol playing a role in both osmoprotective and osmotic adjustment
on wheat. under abiotic stress (Flowers and Colmer, 2008; Munns and Tester,
2008). This accumulation of osmolytes especially that of proline, is a
common phenomenon in plants. Besides its role as an osmolyte, proline
3.6. Effect of salt stress and chitosan on soluble carbohydrates contributes to scavenging ROS, stabilizing subcellular structures,
modulating cell redox homeostasis, supplying energy and functioning
Under salinity stress, soluble sugar content in leaves of milk thistle as a signal (2008; Szabados and Savoure, 2010; Sharma et al., 2011).
increased by 43.6% compared with control (P < 0.05) (Table 3). When Proline is accumulated preferentially in leaves in order to maintain
the samples were treated with chitosan at different concentrations, chlorophyll level and cell turgor to protect photosynthetic activity
soluble sugar content significantly increased (P < 0.05). Soluble sugar under salt stress (Silva-Ortega et al., 2008). Proline plays also a po-
content in plants treated with 0.05% concentration of chitosan, in- tential role in scavenging ROS products (Soshinkova et al., 2013).
creased by 4.0% compared to control. Zou et al. (2015) reported in-
creasing of soluble carbohydrates by application of chitosan on wheat
plants. Chitosan application induced a decline in malondialdehyde 3.8. Effect of chitosan application on enzymatic activities
content, altered the relative permeability of the plasma membrane and
increased the concentrations of soluble sugars, proline, peroxidase, and The activities of several representative antioxidant enzymes, in-
catalase activities on Maize plants (Guan et al., 2009). cluding CAT, POD concentration were measured in milk thistle to de-
termine the physiological effect of exogenous chitosan on these anti-
oxidant enzymes within the context of salt stress. The results showed

Table 3
Growth and biochemical characteristics of milk thistle affected by salt stress and chitosan.
Treatments Root dry weight Ch a Ch b Total Ch Soluble carbohydrates Prolin
−1 −1
(g) (mgg FW) (mgg FW) (mgg −1 FW) (mgg −1 FW) (mgg −1 FW)

Salt stress
0 ds/m 0.539 a 0.811 a 0.353 a 1.165 a 61.403 c 1.512 c
4 ds/m 0.377 b 0.810 a 0.350 a 1.160 a 62.550 c 1.585 c
8 ds/m 0.255 c 0.742 b 0.324 b 1.066 b 72.966 b 2.157 b
12 ds/m 0.241 c 0.709 c 0.306 c 1.015 c 88.180 a 2.707 a
Chitosan
0 Control 0.333 b 0.757 c 0.329 b 1.087 b 69.867 c 1.890 b
0.01 % 0.369 a 0.782 a 0.333 b 1.115 a 70.831 bc 2.029 a
0.05 % 0.355 a 0.768 b 0.341 a 1.109 a 72.661 a 2.001 a
0. 1 % 0.354 a 0.764 bc 0.331 b 1.095 b 71.745 ab 2.041 a

Different letters in each column for each factor indicate significant difference at P ≤ 0.05. by Duncan test.

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Fig. 4. Effect of salt stress and exogenous chitosan on shoot dry weight of milk thistle.

salt stress and chitosan application had significant effect on there was not a significant difference between control and 4 dS/m
CAT(P < 0.01). While interaction effect of salt stress and chitosan salinity level at different concentrations of chitosan application. In
application had significant effect on POD (P < 0.05) (Table 4). With 12 dS/m treatment, minimum H2O2 concentration (1.127 mgg−1 FW)
increasing salinity levels CAT activity was increased (Fig. 6). Maximum was achieved in 0.01% chitosan application (Fig. 9). At medium (8 dS/
CAT activity (3.0315 IU mg −1 protein) was achieved in 12 dS/m. Si- m) and sever (12 dS/m) salinity stress treatments the lowest H2O2 ac-
milar to salt stress, chitosan application increased CAT activities in all tivities were observed at 0.05% and 0.1% chitosan applications, re-
levels of application (Fig. 7). spectively. Salt stress leads to the generation of reactive oxygen species,
Chitosan application in milk thistle increased the activity of per- such as H2O2, which cause lipid peroxidation and disturb normal cel-
oxidase in the leaves especially under salinity stress (4, 8, 12 dS/m) lular metabolism.
(Fig. 8). At control level (No salinity stress) no significant effect of Exposure of plants to abiotic and biotic stresses is often accom-
different chitosan treatments was observed. The lowest peroxidase ac- panied by an increase in ROS, and consequently oxidative stress. Plants
tivity was observed at control (no chitosan application) compared to all have evolved specific protective mechanisms, involving enzymatic and
different levels of chitosan application at 4 dS/m, As the salinity non-enzymatic antioxidants (Mittler, 2002). A key process in the anti-
treatments increased to 8 dS/m again the lowest peroxidase activity was oxidant defense system is an enzymatic conversion of O2 free radicals
observed at control (no chitosan application) compared to all different into H2O2, and it further detoxification by catalase and peroxidase to
levels of chitosan application. At 12 dS/m salinity treatment the highest H2O and O2 (Mittler, 2002). Our research showed that CAT and POD
peroxidase activity was observed at 0.01 and 0.05% chitosan applica- activities significantly increased in salt-stressed plants when treated
tion treatments. There was not a significant difference between chitosan with different concentrations of exogenous chitosan. By Enhancing the
levels under normal condition (none-saline). However, with increasing activities of CAT and POD showed better salt stress adaptation ability
salinity levels, the peroxidase activity increased with chitosan appli- with exogenous chitosan application, which promoted the conversion of
cation. Maximum activity of peroxidase (0.07133 IU mg −1 protein) ROS species and reduced H2O2 concentration in milk thistle plants.
was observed in 12 dS/m and 0.01% chitosan application. Sathiyabama et al., (2016) demonstrated chitosan sprayed plants had a
higher peroxidase activity in leaves of turmeric (Curcuma longa L.).
3.9. Effect of chitosan application on H2O2 concentration Anusuya and Sathiyabama (2016) reported that foliar application of
chitosan induced the activity levels of defense enzymes such as protease
Compared with the controls, salinity stress led to greater increases inhibitors (PI), β-1,3 glucanases, peroxidases (PO) and polyphenol
in H2O2 levels in milk thistle leaves. Treatment with exogenous chit- oxidase (PPO) in the leaves and rhizomes of turmeric (Curcuma longa L.)
osan reduced H2O2 salinity levels especially in 8 and 12 dS/m. plants. Numerous studies reported that chitosan has a potential for
However, this decrease was much smaller in control and 4 dS/m. So, scavenging system such as peroxidase; polyphenol oxidase (PPO)

Fig. 5. Effect of salt stress and exogenous chitosan on total biomass of milk thistle.

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S. Safikhan et al. Journal of Applied Research on Medicinal and Aromatic Plants 10 (2018) 49–58

Table 4
Enzymatic activities of milk thistle affected by salt stress and chitosan.
S.O.V. D.F. Catalase Peroxidase H2O2

MS F value MS F value MS F value

Block 2 0.00465 0.54 0.0000045 0.99 0.0000714 0.11

Salt stress 3 4.11316** 480.03 0.001603** 349.57 0.5276862** 803.51
Chitosan 3 0.08522** 9.95 0.000111** 24.40 0.005995** 9.13
Salt stress × Chitosan 9 0.008593 1.00 0.0000107* 2.34 0.001795* 2.73
Error 30 0.008568 – 0.00000459 – 0.000656 –
C.V. – 4.08 – 4.06 – 2.79 –

*and **: Significant at 5% and 1% probability levels.

superoxide dismutase and catalase (Agrawal et al., 2002; Ma et al.,

2014).
Chitosan suppresses antioxidant enzyme activities for mitigating
salt stress in mung bean varieties (Rani Ray et al., 2016). Results show
that free radical scavenging activity of chitosan was increased via the
amination process (Tamer et al., 2017). The promotion of antioxidant
activity was attributed to replacing hydroxyl groups with free amine
groups (Tamer et al., 2016). The destructive impact of reactive oxygen
species (ROS) against living cells brings about damage and ultimately
leads to cell death.

4. Conclusions

Fig. 6. Effect of salt stress on catalase activity of milk thistle. It is concluded that salt stress, especially under 8 and 12 dS/m,
decreased growth characteristics, chlorophyll content and increased
proline content, soluble carbohydrates enzymatic activity and H2O2
concentration in milk thistle leaves. However, chitosan application
especially under 0.01 and 0.05% enhanced the plant growth and de-
velopment, increased proline, soluble carbohydrates, enhanced enzy-
matic activity and decreased H2O2 concentration in leaves. The results
illustrated that chitosan could protect plants from salt stress damage by
enhancing the capacity of antioxidant enzymes activities. Hence, the
present study demonstrated that chitosan can be used as an eco-friendly
compound to protect milk thistle plants as well as to enhance growth
and biochemical parameters under salinity condition.

Acknowledgments

Fig. 7. Effect of exogenous application of chitosan on catalase activity of milk

The authors would like to express their appreciation to Shahid
thistle. Beheshti University for the financial support for this project.

Fig. 8. Effect of salt stress and chitosan application on peroxidase activity of milk thistle.

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Fig. 9. Effect of salt stress and chitosan application on H2O2 concentration of milk thistle.

Appendix A. Supplementary data oligomer on biophysical characteristics, growth, development and drought resistance
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online version, at doi:https://doi.org/10.1016/j.jarmap.2018.06.002. in Silybum marianum L. plants under abiotic elicitation. Journal of Plant Physiology
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