Appl Biochem Biotechnol (2014) 173:968979

DOI 10.1007/s12010-014-0890-6

Ascorbic Acid and Salicylic Acid Mitigate NaCl Stress

in Caralluma tuberculata Calli
Riaz Ur Rehman & Muhammad Zia &
Bilal Haider Abbasi & Gang Lu &
Muhammad Fayyaz Chaudhary

Received: 2 February 2014 / Accepted: 24 March 2014 /

Published online: 18 April 2014
# Springer Science+Business Media New York 2014

Abstract Plants exposed to salt stress undergo biochemical and morphological changes even
at cellular level. Such changes also include activation of antioxidant enzymes to scavenge
reactive oxygen species, while morphological changes are determined as deformation of
membranes and organelles. Present investigation substantiates this phenomenon for
Caralluma tuberculata calli when exposed to NaCl stress at different concentrations.
Elevated levels of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate
peroxidase (APX), and glutathione reductase (GR) in NaCl-stressed calli dwindled upon
application of non-enzymatic antioxidants; ascorbic acid (AA) and salicylic acid (SA).
Many fold increased enzymes concentrations trimmed down even below as present in the
control calli. Electron microscopic images accentuated several cellular changes upon NaCl
stress such as plasmolysed plasma membrane, disruption of nuclear membrane, increased
numbers of nucleoli, alteration in shape and lamellar membrane system in plastid, and
increased number of plastoglobuli. The cells retrieved their normal structure upon exposure
to non-enzymatic antioxidants. The results of the present experiments conclude that NaCl
aggravate oxidative molecules that eventually alleviate antioxidant enzymatic system.
Furthermore, the salt stress knocked down by applying ascorbic acid and salicylic acid
manifested by normal enzyme level and restoration of cellular structure.
Keywords Antioxidant enzymes . Caralluma tuberculata calli . NaCl stress . ROS .
Ultra-structure

R. U. Rehman
Horticulture and Floriculture Institute, Government of Punjab, Rawalpindi, Pakistan
M. Zia (*) : B. H. Abbasi
Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan 45320
e-mail: ziachaudhary@gmail.com
G. Lu
College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China
M. F. Chaudhary
Preston Institute of Nanoscience and Technology, Preston University, Islamabad, Pakistan

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Abbreviations
AA
Ascorbic acid
APX
Ascorbate peroxidase
CAT
Catalase
FWGR Fresh weight growth rate
GR
Glutathione reductase
ROS
Reactive oxygen species
SA
Salicylic acid
SOD
Superoxide dismutase
POD
Peroxidase
Introduction
Salinity is among the sternest factors influencing crop efficiency, even in well-watered soils.
Considerable changes in water balance and ionic form result damage at molecular level and
severely affect the growth in stressed plants. Consequently, the plant tissues die and death of
plant may occur in severe saline conditions [1]. Such stresses result in interference of growth
and metabolism by triggering secondary responses like the production of highly reactive
oxygen species (ROS).
The production of ROS such as the hydrogen peroxide (H2O2), the superoxide radical
(O2), and the hydroxyl radical (OH1) are critical; however, enzymatic or non-enzymatic
ROS-scavenging systems in plants efficiently wipe out these hazardous components. ROS,
mainly hydrogen peroxide (H2O2), also act as important signal in both biotic and abiotic stress
responses [2]. The major antioxidant enzymes are superoxide dismutase (SOD) catalyzing the
dismutation of O2 to H2O2; catalase (CAT) that dismutase H2O2 to oxygen and water; and
ascorbate peroxidase (APX) that reduces H2O2 to water by utilizing ascorbate as particular
electron donor. Moreover, other enzymes involved are glutathione reductase (GR),
monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX), and glutathione-D-transferase, which are significant in protecting cell
against oxidative stress [3].
In salt-affected cell/plants, biochemical as well as physiological changes occur i.e., dehydration at cellular level, swelling and structural collapse of membranes, disorder of the outer
chloroplast envelope, thinning of partitions, adhesion within the grana, decrease in chloroplast
volume [46], swelling of thylakoids at earlier stage [7, 8], and deformation of other organelles. Such physiological changes have been observed both in salt-sensitive and salt-adaptive
cell lines. Osmoregulation mechanism is a complex process; however, the adaptive capacity to
maintain membrane integrity during a long period of water deficit may be an essential
biological trait for drought tolerance.
Salicylic acid (SA) and ascorbic acid (AA) are small antioxidant molecules, which are
water soluble and act as a principal substrate in non-enzymatic detoxification of hydrogen peroxide in the cyclic pathway. Consistent findings have reported the valuable effect
of ascorbic acid application used exogenously in improving the adverse effects on
growth due to salt stress [9]. Salicylic acid also intervenes the oxidative rupture that
causes death of the cells in the oversensitive reaction and proceeds as signal to develop
complete internal resistance [10]. It also plays an important role in many abiotic stresses
to survive the plants against these pressures [11]. However, unexpectedly, little is known
about the role of these antioxidative compounds in callus stress adaptation. The aims of
the present study were to investigate the antioxidant enzyme status in the callus of
Caralluma tuberculata, under NaCl stress, alleviation of NaCl-stress by ascorbic acid

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and salicylic acid, and to investigate the intracellular changes resulted by the stress in
callus tissues of C. tuberculata.

Materials and Methods

Plant Material and Explant Preparation
The plant material of C. tuberculata used for the study was obtained from the local market of
Quetta (Balochistan, Pakistan) and was identified by Prof. Dr. Mir Ajab Khan, Department of
Plant Sciences, Quaid-i-Azam University Islamabad, Pakistan. The plant material brought to
lab was multiplied in earthen pots in greenhouse for continuous supply of explants.
The methodology to produce callus was adopted as described by Rehman et al. [12]. In
detail, before starting the experiment, the plants collected from the earthen pots were washed
under running tap water for 30 min to remove all adhering contaminants following washing
with 0.2 % liquid detergent (Triton X-100) for about 15 min. Thereafter, the plants were rinsed
with distilled water and treated with bevistin (a fungicide) for 30 min followed by rinsing with
water. These plantlets were now treated with 0.1 % HgCl2 solution for 10 min followed by a
55 min rinsing with sterilized distilled water under aseptic conditions. Thereafter, the shoot
tip portion (10 mm long) of the plants was isolated aseptically and cultured on MS medium
containing different concentrations of plant growth regulators.
Culture Media and Culture Conditions
The MS medium [13] supplemented with 4.44 M 6-benzyl amino purine (BAP)+9.04 M
2,4-dichloro-phenoxy acetic acid (2,4-D) along with 9.08103 M thidiazuron (TDZ) was
used to induce callus from shoot tip explants of C. tuberculata. Sucrose (3 %) was added as a
carbon source, and pH was adjusted at 5.70.1 using 0.1 N KOH or HCl. The media was
solidified with 0.7 % noble agar (Merck) and autoclaved at 121 C under pressure of
103.42 kPA for 20 min. All the cultures were maintained in culture room at 252 C under
4 ft long 40 W tubes (Philips) and incandescent bulb (25 W) at 3,500 lx intensity of
illumination using 16 h light photoperiod.
After 28 days of initiation of calli, small pieces (approx. 1 g) were transferred on plant
growth regulators supplemented MS medium (as described above) along with different
concentrations of NaCl (100300 mM) for 15 days. To analyze the effect of stress alleviators,
calli were transferred on MS medium containing 300 mM NaCl with ascorbic acid (AA 100
and 200 M) and salicylic acid (SA 100 and 200 M) for 15 days. The weight of callus
measured before and after the application of NaCl alone and in combination of antioxidants
and the change in fresh weight were calculated in percentage.
Determination of Antioxidant Activities
For determination of antioxidant activities, callus was ground in chilled mortar and pestle with
homogenization buffer. The homogenized callus was centrifuged at 10,000g for 20 min at
4 C. Supernatant was used to determine the activity of SOD, POD, APX, CAT, and GR as
well as protein contents.
Superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed by using the photochemical
NBT method [14]. The samples (0.5 g) were homogenized in 5.0 ml extraction buffer
consisting of phosphate 50.0 mM, pH 7.8. The assay mixture (3.0 ml) contained 50.0 mM

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phosphate buffer (pH 7.8), 1.0 M EDTA, 26.0 mM L-methionine, 750.0 M NBT, and
20.0 M riboflavin. The photoreduction (formation of purple formazan) of NBT was measured
at 560 nm through spectrophotometer, and an inhibition curve was made against different
volumes of extract. One unit of SOD is defined as the volume of extract present in reaction
mixture that causes inhibition of the photoreduction of NBT by 50 %.
Volume of 3.0 ml guaiacol was used as a substrate to measure the peroxidase (POD; EC
1.11.1.7) activity. A reaction mixture was constituted by mixing of 1 % guaiacol, 0.4 % H2O2,
50.0 mM potassium phosphate buffer (pH 6.1), and enzyme extract. Guaiacol oxidized and
increase in absorbance was measured at 470 nm through spectrophotometer. Activity of the
enzyme was found at 252 C in micromolar of guaiacol oxidized per minute per gram fresh
weight [15].
The assay for ascorbate peroxidase (APX; EC 1.11.1.11) activity was carried out according
to the method of Nakano and Asada [15]. In a reaction mixture (3.0 ml) containing 100.0 L
enzyme extract, 100.0 mM phosphate (pH 7), 0.3 mM ascorbic acid, 0.1 mM EDTA-Na2, and
0.06 mM H2O2. In this reaction mixture, H2O2 was added, and after 30 s of this addition, the
change in absorption was recorded through spectrophotometer at 290 nm.
Assay to find catalase (CAT; EC 1.11.1.6) activity was done by the method of Cakmak and
Marschner [16]. In this assay, 25.0 mM buffer of potassium phosphate containing 0.1 mM
EDTA (pH 7.0) was mixed with 10.0 mM H2O2 and the enzyme extract. Within 1 min of
mixing the enzyme extract, the reduction in absorbance of H2O2 (E=39.4 mM1 cm1) was
recorded at 240 nm on spectrophotometer.
Assay of glutathione reductase (GR; EC 1.6.4.2) was followed by the method of Foyer and
Halliwell [17]. Reduction in absorbance was monitored at 340 nm through spectrophotometer.
This reduction in absorbance was recorded due to oxidation of NADPH (E=6.2 mM1 cm1).
The reaction was carried out by mixing 25.0 mM buffer of potassium phosphate. This buffer
was formulated at pH 7.8 by the addition of 0.2 mM EDTA. Enzyme aliquot was added and
absorbance was recorded.
The measurement of concentration of soluble protein was done by following the method of
Bradford [18]. In this assay, bovine serum albumin was used as standard. Stable dyealbumin
complex is the base of this assay. The stable dyealbumin could be measured at 590 nm
spectrophotometrically. A dye which is known as Coomassie brilliant blue G-250 was weighed
0.01 % (w/v) and was mixed together with ethanol 4.7 % (w/v) and 8.5 % (w/v) phosphoric
acid to make protein-dye reagent.
Transmission Electron microscopy of Treated Calli
The callus treated with NaCl and alleviated by ascorbic acid (AA) and salicylic acid (SA) for
15 day were selected for fixation. Callus (23 mm2) was fixed in 2.5 % glutaraldehyde (v/v) at
room temperature in 0.1 M sodium phosphate buffer (pH 7.4) and then rinsed three times with
same sodium phosphate buffer. The washed callus samples were post fixed in 1 %
osmium(VIII) oxide (OsO4) for 1 h. After 1 h, the samples were again washed three times
with 0.1 M sodium phosphate buffer. The three rinses were given in a way that there should be
10 min difference in each rinse. After washing, the samples were dried for 1520 min interval
in a graded ethanol series (50, 60, 70, 80, 90, 95, and 100 %) and in the end step 20 min in
absolute acetone. The samples were then penetrated and implanted in Spurrs resin for whole
night. The specimen was heated at 70 C for 9 h to prepare very slim cuttings
(80 nm) of the specimens. Copper grids were used to mount these ultra-thin specimens for screening in the transmission electron microscope (JEOL TEM-1230EX) at
an accelerating voltage of 60.0 kV.

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Data Analysis
Percent variation for growth protein content and antioxidant enzymes was calculated as
follows:
% variationfor NaCl stress value for treated calliuntreated calli=untreated calli  100
% variationfor mitigants value of treated callicalli at 300 mM NaCl=calli at 300 mM NaCl  100

All the experiments were performed in triplicate, and the results are presented as mean
standard error. The values were analyzed by LSD test with P<0.05.

Results and Discussion

Growth Characteristics of Calli, Protein Content, and Antioxidant Enzymes
The calli of C. tuberculata responded quickly to salt stress, and a linear reduction in the weight
of calli and total soluble protein was recorded. The NaCl stress considerably enhanced the
levels of antioxidant enzymes. However, application of antioxidant molecules (salicylic acid or
ascorbic acid) mitigated the callous salt effects; increase in fresh weight and reduction in
antioxidant enzymes was observed.
Application of 100 mM NaCl in callus culture media reduced fresh weight up to 18.3 %,
and this reduction rose up to 67.4 % at 300 mM NaCl as compared with control (Table 1).
Total soluble protein contents also reduced up to 35.7 % at 200 mM NaCl; however, this
reduction was less (14.2 %) in 300 mM NaCl-treated calli. Several studies on different plant
species have reported similar growth inhibition kinetics upon exposure of cultured cells to high
levels of NaCl i.e., Suaeda nudiflora [19], Nitraria tangutorum, and Oryza sativa callus [20].
Visually, it was also observed that the C. tuberculata calli generated in the presence of NaCl
was smaller, harder, and desiccated. Upon salt stress due to cellular dehydration, the packed
cell volume decreases following inflammation and structural collapse [21]. Proteins involve in
osmotic balance and signaling pathway specifically express on salt stress, reducing the soluble
protein contents [20, 22]. Such variations, reduction in weight, and protein content have been
reported in both salt stressed and salt adaptive calli [23].
Table 1 Effects of NaCl and antioxidant treatments on fresh weight (g) and total soluble protein (mg/g FW) of
Caralluma tubarculata calli. The results are presented as averagestandard deviation of triplicate values
Treatment

Fresh weight (g)

Total soluble protein (mg/g FW)

Control

19.51.1 bc

1.40.3 d

NaCl 100 mM

15.90.8 cd

1.10.2 e

NaCl 200 mM

10.51.0 d

0.90.18 f

NaCl 300 mM
NaCl 300 mM AA 100 M

6.40.7 e
17.21.2 c

1.20.3 e
1.50.2 d

NaCl 300 mM+AA 200 M

20.71.6 b

2.30.5 b

NaCl 300 mM+SA 100 M

19.10.9 bc

2.10.3 c

NaCl 300 mM+SA 200 M

23.10.7 a

3.70.5 a

Means followed by same small letters are not significantly different by the LSD test at P0.05
AA ascorbic acid, SA salicylic acid

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Addition of salicylic acid (SA) and ascorbic acid (AA) antagonized the negative effects of NaCl
stress on callus growth. AA and SA at higher concentration (200 M) also showed a growth
promoting effect as it gave the callus growth rate even higher than that of unstressed calli (control).
The FW increased up to 223 and 260 % when calli were cultured in presence of 200 M AA and
SA, respectively, in addition with 300 mM NaCl (Table 1). These non-enzymatic antioxidants (SA
and AA) are important for plant growth and development along with antioxidant capacity [24, 25].
Salicylic acid also works as signal due to which plants develop internal resistance against biotic and
abiotic stresses [10]. A research on chick pea indicated that the additional ascorbic acid (4.0 mM)
gave strength to the stem and roots and improved fresh and dry biomass of salt-stressed plants [26].
Exogenous application of AA also modulates salt-stressed undesired effects on growth, cell
division, and cell enlargement [9]. NaCl stress had negative effect on total soluble protein contents,
which showed significant reduction in stressed callus. However, compared to control as well as
stressed calli, a significant increase in protein contents was observed with the addition of antioxidants. SA proved comparatively better for protein contents of C. tuberculata callus showing the
highest value of protein contents (3.7 mg/g FW; 208 % increase as compared with 300 mM NaClstressed calli) at 200 M. The non-enzymatic antioxidants provide shield against oxidative burst
and also stimulate biomass accumulation, increasing fresh and dry weight [27, 28]. Therefore,
appropriate concentration is important for optimum results.
The calli grown in the presence of NaCl varied antioxidant enzymes response. As the
concentration of NaCl in the culture media increased, a boost in peroxidase, ascorbate
peroxidase, and catalase activities were observed in the calli. A maximum increase of 134.4,
123.5, and 153.5 % was observed in peroxidase, ascorbate peroxidase, and catalase, respectively, in the calli grown at 300 mM NaCl concentration (Fig. 1). Concentrations of these
enzymes decreased when AA and SA were also applied in combination with 300 mM NaCl.
The reduction was more pronounced by applying 200 M as compared with 100 M. It was
also observed that ascorbate peroxidase reduced at high rate (7582 %) as compared with
peroxidase (2684 %) and catalase (4361 %). The figure also shows that the reduction in
ascorbate peroxidase was consistent irrespective to type and concentration of mitigant.
Enhanced concentration of salt increased the POD activity as compared to control.
Application of 300 mM NaCl in the culture media increased POD activity up to 134 %. To
reduce injurious effect of NaCl, SA functioned better as compared with AA and higher
concentration was optimum. It was observed that application of 200 M SA or AA decreased
the POD activity below the level present in control calli (Fig. 1). In salinized cells of
S. nudiflora and cotton, NaCl-induced enhancement of POD activity to decompose H2O2
produced (Cherian and Reddy 2003; Lin and Kao 1999). Increase in POD activity confers salt
tolerance ability in plant species and protection against oxidative stress [29, 30].
Ascorbate peroxidase (APX) reflected a gradual rise in its activity in response to enhanced NaCl
concentrations, and at 300 mM NaCl, a fourfold increase in APX activity was observed as compared
with control. However, application of SA and AA decreased APX activity five to six times as
compared with control (300 mM NaCl). It was observed that decrease in APX activity was not
dependent on the type of antioxidant and concentration. Stimulation of APX indicates that the
enzyme has a critical role in plant cells dissimulating H2O2 produced during O2 scavenging [31].
Such variations have already been observed in pea [32], cotton [33], and rice [34]. However, level of
APX is determined by salt concentration, time of stress, type of tissue, and age of plant [35]. Ascorbic
Fig. 1 Effects of salinity (NaCl, 0300 mM) and antioxidants (salicylic acid and ascorbic acid; 100 and 200 M) b
on SOD, POD, APX, CAT, and GR activities in Caralluma tubarculata calli. Data are the meanSD of three
replicates. Small letters marked on each bar are not significantly different by the LSD test at P0.05. Control calli
(CC),. Ascorbic acid (AA), salicylic acid (SA)

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acid plays a vital role in mediating the undesired affects of salt on plant metabolism and growth.
These mitigating effects are attributed by stimulation of reaction by enzymes [36] along with the
stabilization and protection of organs responsible for photosynthesis from damage due to oxidation
[37]. The results also show that AA and SA reduced the POD level many times and diminished toxic
effects of NaCl. However, type and concentration of mitigant did not affect at higher extinct.
Catalase also takes away H2O2 into water and oxygen produced inside the cell [38], and this
reaction takes place at higher extent in biotic and abiotic stress conditions [39]. An overall
increase in CAT activity was observed when C. tubarcaulata calli was subcultured in the presence
of NaCl. A threefold increase (153 %) in CAT activity was calculated on 300 mM NaCl
concentration as compared with control. Submission of AA and SA reduced the CAT activity
and trimmed down CAT level approximately equal to control calli (Fig. 1). Statistically, not much
difference was observed between both non-enzymatic antioxidants and concentrations.
In case of SOD and GR, an increase in activities was observed at 100 and 200 mM NaCl.
Further increase in NaCl concentration (300 mM) decreased the enzyme values (Fig. 1). An
increase (54 %) in GR activity was observed in stressed calli (200 mM) as compared to control,
while at 300 mM NaCl, the activity was equal to control. In comparison, much increase in SOD
activity was not observed at 200 mM NaCl stress (16 % increase); however, 26 % decrease in
SOD activity was observed in calli regenerated at 300 mM NaCl. Application of AA and SA (100

Fig. 2 Electron micrographs of Caralluma tuberculata calli describing modifications in cell wall, cell membrane, and vauoles: a control, b exposed to 300 mM NaCl alone, c exposed to 300 mM NaCl+ascorbic acid
(200 M), d exposed to 300 mM NaCl+salicylic acid (200 M). Cell wall (CW), cell membrane (CM),
mitochondria (Mt), plastids (P), vacuole (Vac), plastid (P)

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and 200 M) increased SOD activity in the stressed calli as compared with control (300 mM
NaCl); however, in case of GR increase was observed at 100 M AA and 200 M SA. It was
observed that both non-oxidants did not much favored SOD to mitigate injurious effects of NaCl.
An increase in SOD activity due to salt stress has been documented in Buddleja parviflora and
Bruguiera gymnorrhiza [40], Avicennia marina [41], and Rhizophora stylosa [42], and such
increase has also been reported in cotton, tomatoes, and pea genotypes which are salinity tolerant
[6, 35, 43]. The roles of GR and glutathione in the H2O2 scavenging in plant cells have been well
established in HalliwellAsada pathway [44]. GR catalyzes the rate limiting the last step of
ascorbate-glutathione pathway. Reduced glutathione is a very efficient scavenger of ROS as it is a
powerful reductant. Non-enzymatic antioxidants like reduced ascorbate and glutathione scavenged superoxide radicals generated in plants. However, the APX and glutathione reductase
exhibit enhanced activities [42]. The results also show that in C. tuberculata calli, GR plays a
major role to fight against oxidative molecules as compared with SOD.
Ultra-Structural Modifications of Calli Upon Salt and Antioxidant Treatments
Ultra-structural observations of Caralluma cell revealed modifications in NaCl-treated cells,
and these modifications were more obvious on the cell wall, nucleus, and plastids. In the cells

Fig. 3 Electron micrographs of Caralluma tuberculata calli describing variations in nucleus a control, b exposed
to 300 mM NaCl alone, c exposed to 300 mM NaCl+ascorbic acid (200 M), d exposed to 300 mM NaCl+
salicylic acid (200 M). Cell wall (CW), nucleus (N), nucleolus (Nu)

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of the untreated callus (control), the cytoplasm looked granular and thick with several
subcellular organelles. These cells possessed a thick cell wall, continuous and smooth cell
membranes, a central vacuole, a well-shaped nucleus, and few numbers of mitochondria and
plastids (Figs. 2, 3, and 4).
A considerable reduction in the cell wall thickness with increased vacuolization was evident
under NaCl stress (Fig. 2). Plasma membrane was plasmolysed and was either absent or
insignificant; however, disruption of nuclear membrane and increased numbers of nucleoli
were some of the other obvious changes observed in the nucleus of NaCl-treated cells (Fig. 3).
The plastids of NaCl-treated cells showed alteration in shape and in lamellar membrane system
with increased number of plastoglobuli (Fig. 4). In case of AA treatment along with NaCl, the
cell wall was relatively better in shape, with slight shrinkage of cytoplasmic and increased
number of mitochondria. Addition of AA improved the shape of plastids and nucleus. The
lamellar membrane was more compact although high amount of plastoglobuli was present.
However, SA proved better, where the cell wall was almost fully recovered and numbers of
vacuoles were reduced. Both AA and SA recovered damaged to nucleus and plastid. The

Fig. 4 Electron micrographs of Caralluma tuberculata calli describing variations in plastids: a control, b
exposed to 300 mM NaCl alone, c exposed to 300 mM NaCl+ascorbic acid (200 M), d exposed to 300 mM
NaCl+salicylic acid (200 M). Cell wall (CW), cell membrane (CM), plastids (P), plastoglobuli (PG), lamellar
membrane (LM)

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shape of plastid restored, and it turned into elongated with reduced plastoglobuli, while
lamellar membrane became compacted again. The number of plastids remained constant,
and no change was observed in any treatment. Electron microscopy had been used to assess
damages at the ultra-structural and tissue levels to make the foundation of examination
macroscopically on which the damage rating is based [45]. Many reports describe variations
in cellular structure due to salt stress e.g., alteration in the cell wall [46] reduced thickness in
the cell wall [47], increased number of micro bodies and mitochondria [48], swelling of
thylakoid [49], etc. It has been postulated that increase in salt concentration induces
enhanced F-ATPase activity by increase in mitochondria number to provide excessive
energy supply for osmotic adjustment [50]. While plastids are considered to be at high
risk by oxidative stress due to electron flux, elevated levels of oxygen might be the
reasons for swelling of plastids [51], break down of thylakoid membrane, and higher
number of plastoglobuli.

Conclusions
In conclusion, the higher activities of SOD, CAT, GR, POD, and APX in response to salinity
stress play an important role in salt tolerance in the calli of C. tuberculata. The physiological
effects at cellular level include cell membrane damage, disruption of nuclear membrane,
variation in nucleoli number, and deformation of plastids. The antioxidant molecules (SA
and AA) successfully mitigated salt toxicity and improved the growth of C. tuberculata calli
revealed by normal distribution of antioxidant enzymes and revival of cellular structure.

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