ORIGINAL PAPER

Biochemical, physiological and growth changes in responseto salinity in callus cultures of Sesuvium portulacastrum L.

Vinayak Haribhau Lokhande •

Tukaram Dayaram Nikam • Suprasanna Penna

Received: 2 June 2009 / Accepted: 20 January 2010 / Published online: 6 February 2010

� Springer Science+Business Media B.V. 2010

Abstract In vitro-grown cells of Sesuvium portulacastrum

L., an important ‘salt accumulator’ mangrove associate,

were incubated on a medium containing different levels of

salt, including 0, 100, 200, or 400 mM NaCl, in order to

evaluate biochemical, physiological, and growth responses.

A significant decrease in callus growth, water status, and cell

membrane damage was observed under salt stress. Osmotic

adjustment was revealed by the accumulation of inorganic

ions, such as sodium (Na?), and organic osmolytes (proline,

glycine betaine, and total soluble sugars) in NaCl-treated

calli compared to control. However, accretion of osmolytes

and inorganic ions did not support growth of calli under NaCl

stress conditions. The observed reduced growth rate in calli

subjected to stress, up to 200 mM NaCl, was coupled with

lower catalase and ascorbate peroxidase activities and with a

significantly higher superoxide dismutase activity. These

findings suggested that S. portulacastrum cell cultures

exhibited higher osmotic adjustment to salt stress.

Keywords Antioxidative enzymes � In vitro � Osmolytes �Salt tolerance � Sesuvium portulacastrum � Water status

Introduction

Sesuvium portulacastrum L., commonly called seapurslane,

belongs to the family Aizoaceae, and is a herbaceous

facultative halophyte (Lokhande et al. 2009a, b). It grows

naturally in the subtropical, Mediterranean, coastal and

warmer areas around the world, and in India, it grows along

the coastal sides of both eastern and western regions

(Lokhande et al. 2009b). The plant plays a major role in the

environmental protection such as sand dune fixation, saline

soil stabilization, desalination, desert greenification,and

landscaping in an ornamental capacity (Menzel and Leith

1999), and acts as an alternative culture to problematic soil

in the arid and semiarid regions of the world. Of the var-

ious categories of mangroves, Sesuvium comes under ‘salt

accumulators’ as the plants accumulate high concentrations

of salts in their cells and tissues and overcome salt toxicity

by developing succulence (Ashraf 2009).

Salinity is one of the most important environmental

constraints affecting more than 800 million ha of arable

land (Munns and Tester 2008) and, therefore, the agricul-

tural productivity (Sairam et al. 2002). Thus, studies aimed

at understanding physiological and biochemical mecha-

nisms under salt stress has generated considerable interest.

Generally, plants under saline conditions are stressed by an

increase in the osmotic potential in the rooting medium and

by the toxic effect of high concentration of saline ions

(Munns 2002). Osmotic adjustment under salt stress is an

adaptation mechanism operated by both halophytes and

glycophytes in order to maintain their water balance

(Flowers and Colmer 2008). The best characterized bio-

chemical response of plant cells to osmotic stress is accu-

mulation of some inorganic ions such as sodium (Na?) and

compatible organic solutes like proline, glycine betaine

(GB), and soluble sugars (Flowers and Colmer 2008).

These compatible solutes can accumulate to high levels

without disturbing intracellular biochemistry (Bohnert and

Jensen 1996), protecting sub-cellular structures, mitigating

oxidative damage caused by free radicals (Attipali et al.

V. H. Lokhande � T. D. Nikam

Department of Botany, University of Pune, Pune 411 007, India

V. H. Lokhande � S. Penna (&)

Functional Plant Biology Section, Nuclear Agriculture &

Biotechnology Division, Bhabha Atomic Research Centre,

Trombay, Mumbai 400 085, India

e-mail: prasanna@barc.gov.in; penna888@yahoo.com

123

Plant Cell Tiss Organ Cult (2010) 102:17–25

DOI 10.1007/s11240-010-9699-3

2004), and maintaining the enzyme activities under salt

stress (Yokoi et al. 2002).

Potassium plays a major role in the biochemical and

physiological processes in plants. Under typical physio-

logical conditions, plant cells require high K? (100–

200 mM) and lower Na? (less than 1 mM) and, accord-

ingly, the high cytosolic K?/Na? ratio to maintain the

osmotic balance (Tester and Davenport 2003) for proper

functioning of the cell. To maintain a high K?/Na? ratio in

the cytosol, plant cells employ primary active transport,

mediated by channels and co-transporters for Na? extru-

sion and/or the intracellular compartmentalization of Na?

into the vacuole (Blumwald 2000). However, under salt

stress, ion ratios are altered by the uncontrolled influx of

Na? through K? pathways. Halophytes being continuously

exposed to high salinity conditions undergo osmotic

adjustment, preferably through higher accumulation of

Na? and its efficient sequestration into the vacuole at low

external water potential as well as by low potassium use

efficiency as compared to glycophytes.

In addition to osmotic stress, salt stress also causes oxi-

dative damage, thereby affecting the cellular membrane

integrity, enzyme activities, and functioning of plant pho-

tosynthetic apparatus (Jitesh et al. 2006). The antioxidant

enzymes like superoxide dismutase (SOD), catalases (CAT),

ascorbate peroxidase (APX) and other enzymes are effi-

ciently involved in scavenging of reactive oxygen species

(ROS) produced by salt stress, and act as one of the main

tolerance mechanisms against oxidative stress in plants.

The response to osmotic and oxidative stresses in plants

has involved a suite of adaptations at cellular, tissue, and

whole plant level (Hasegawa et al. 2000; Borsani et al.

2003). Many studies have revealed that the salt tolerance

mechanism operating at whole plant level also worked at

the cellular level in both halophytes and non-halophytes

(Tonon et al. 2004; Arzani 2008). However, there is mea-

gre information available on the cellular responses to salt

and involvement of antioxidant defence system in man-

groves. Recently, Zhao et al. (2009) reported the effect of

salt stress at cellular level on growth and osmotic regula-

tion in Thellungiella halophila. In Sesuvium, studies at

whole plant level have demonstrated the role of osmolytes

and inorganic ions in osmotic adjustment under salinity

stress (Messedi et al. 2004), but there is no information at

the cellular level on the involvement of these osmolytes

and especially antioxidant enzyme defence capacitance to

salinity tolerance. The assessment of intracellular changes

operating at cellular levels under salt stress can be a useful

means to segregate the specific effect of salt and to identify

specific physiological and biochemical traits associated

with salt tolerance at whole plant level. Thus, the present

investigation was carried out with the objective of using in

vitro cultures to evaluate the responses to salt-induced

stress in Sesuvium. Specifically, we studied the effect of

salt on growth, osmotic adjustment, and antioxidant

defence mechanism to look into the mechanism used by in

vitro-cultured cells of this halophyte, Sesuvium portula-

castrum.

Materials and methods

Plant material

The nodal sectors (*3.0 cm) of S. portulacastrum were

collected from the coastal region of Maharashtra state,

India, and maintained at the Botanic Garden, Department

of Botany, University of Pune, Pune-07 (Lokhande et al.

2009a). The nodal explants (*1.0 cm) from established

plantlets were harvested and surface sterilized with 0.1%

(w/v) mercuric chloride (HgCl2) solution for 5 min, and

rinsed five times with sterile distilled water. The explants

were cultured vertically onto the shoot multiplication

medium (MS; Murashige and Skoog 1962) supplemented

with 20 lM 6-benzyladenine (BA) and 30 g l-1 sucrose.

The pH of the medium was adjusted to 5.8 and solidified

with 0.2% gelrite (Sigma–Aldrich, USA) prior to auto-

claving at 121�C for 15 min. The cultures were incubated

at 25 ± 2�C under a 16-h photoperiod (30 lmol m-2 s-1

PFD) and 70% relative humidity.

Callus induction, proliferation and maintenance

After 4 weeks of culture, callus induction was observed on

the shoot multiplication medium at the lower cut end on

nodal explant and was hard, compact, greenish and non-

proliferated. The callus was incubated on Murashige and

Skoog (MS) (1962) solid medium consisting of 10 lM 2,4-

dichlorophenoxyacetic acid (2,4-D) and 5.0 lM BA and

30 g l-1 sucrose, for its proliferation. After 2 weeks of

culture, the callus underwent proliferation and was soft,

fragile and whitish in nature. Subsequently, the callus was

regularly sub-cultured at intervals of 21 days on the freshly

prepared callus proliferation medium (MS medium con-

taining 10 lM 2,4-D and 5.0 lM BA) and, after mainte-

nance for more than 8 months under control conditions,

was used for salt stress analyses.

Salt stress treatment

For the availability of adequate nutrient and oxygen supply

to inner part of the callus, the calli were cut into small

pieces of approximately 4–5 mm size. Only the fresh,

viable and actively growing and proliferating calli pieces

were weighed (*1.0 g) and cultured on solid callus pro-

liferation medium (MS consisting of 10 lM 2,4-D and

18 Plant Cell Tiss Organ Cult (2010) 102:17–25

123

5.0 lM BA) supplemented in the absence or presence of

various levels of salt (0, 100, 200, or 400 mM NaCl) in

addition to 30 g l-1 sucrose as a source of carbon. The pH

of the medium was adjusted to 5.8 and solidified with 0.2%

gelrite prior to autoclaving at 121�C for 15 min. The

cultures were incubated under controlled conditions as

described earlier. Calli were evaluated after 15 days of salt

treatment for growth rate, accumulation of proline, glycine

betaine, and total soluble sugars. Damage to membrane

was measured in terms of relative electrolytic leakage, and

antioxidant enzyme activities were analyzed for CAT, APX

and SOD.

Relative growth rate measurements

Samples were weighed to calculate relative growth rate

(RGR). Calli were weighed initially at the time of their

transfer (Wi) and finally after 15 days of salt treatment

(Wf), and the mean callus RGR calculated by: [(Wf - Wi)/

Wi] 9 100%.

Measurement of membrane damage rate

Membrane damage was determined in terms of relative

electrolytic leakage (REL) according to the method of

Sullivan (1972). This technique was based on the increase of

cellular membrane permeability and concomitantly greater

electrolyte diffusion out of cells when tissue is injured by a

stress situation. The determination of REL is the measure of

change in the electrical impedance and electrolyte leakage

to detect stress injury of cell membrane. To measure the

REL, calli clumps of approximately equal size were incu-

bated for 24 h in a test tube (25 9 150 mm) containing

10 ml of distilled water at room temperature (25�C) and the

initial electrical conductivity (EC1) was measured after

incubation period. Then, the samples were killed by auto-

claving for 15 min at 121�C temperature to release all the

ions from the tissue and final electrical conductivity (EC2)

was measured when cooled to room temperature. The REL

was calculated as: (EC1/EC2) 9 100.

Determination of tissue water content

The fresh weight (FW) of the calli was determined

immediately after removal from the medium and blotted

with tissue paper to remove the excess water. Dry weight

(DW) was recorded after drying the calli at 60�C in the hot

air oven for 48 h. The percent tissue water content (TWC

%) of the calli was determined using the following

equation:

TWC %ð Þ ¼ FW� DWð Þ=FW½ � � 100

Measurement of osmotic regulation

Free proline content

Free proline content was measured spectrophotometrically

according to the method of Bates et al. (1973). About

500 mg of fresh calli were homogenized in 3% (w/v)

aqueous 50-sulfosalicylic acid, and the residue was

removed by centrifugation at 10,000g for 10 min at 4�C.

The supernatant (2 ml) was reacted with 2 ml each of acid

ninhydrin and glacial acetic acid by incubating at 100�C in

a hot water bath for 1 h. The reaction was terminated in an

ice bath and allowed to cool at room temperature. The

reaction mixture was extracted with 4 ml toluene and

mixed vigorously with a stirrer for 10–15 s. The chromo-

phore containing toluene was aspirated from the aqueous

phase and warmed to room temperature. The optical den-

sity was measured at 520 nm (UV-1700 PharmaSpec, UV–

Visible spectrophotometer; Shimadzu, Japan) using toluene

as a blank. The amount of proline was determined from a

standard curve using L-proline and expressed as lg of

proline g-1 FW.

Glycine betaine (GB) analysis

Accumulation of GB in response to salt stress was deter-

mined according to Grieve and Grattan (1983). The fresh

calli (500 mg) were ground in liquid nitrogen and the finely

ground powder was mechanically shaken with 20 ml of

deionised water for 16 h at 25�C. Samples were filtered,

and filtrates (500 ll) were diluted (1:1) with 2N H2SO4.

The extract was cooled in an ice water for 1 h and then

mixed with 200 ll of I2-KI reagent (mixed with 20%

potassium iodide and 15.7% iodine). The tubes were gently

mixed and stored at 4�C for 16 h followed by centrifuga-

tion at 10,000g for 15 min at 0�C. Periodide crystals were

dissolved in 9.0 ml of 1, 2-dichloroethane, and after 2 h,

absorbance was measured spectrophotometrically at

365 nm (UV-1700 PharmaSpec, UV–Visible spectropho-

tometer; Shimadzu). The GB content (lg g-1 FW) was

determined from a standard curve prepared using glycine

betaine (Sigma–Aldrich) as standard.

Total soluble sugars (TSS) content

TSS was estimated as per the anthrone method (Watanabe

et al. 2000) with some modifications. About 200 mg of

calli were homogenized with ice-chilled 80% ethanol in a

mortar and pestle. The extract was prepared by centrifu-

gation at 5,000g for 10 min at 4�C, and final volume was

adjusted to 10 ml with 80% ethanol. Then 1 ml of super-

Plant Cell Tiss Organ Cult (2010) 102:17–25 19

123

natant was reacted with 3 ml of freshly prepared anthrone

reagent by incubating the reaction mixture for 10 min at

100�C in a hot water bath. The reaction was terminated by

quick cooling in an ice bath and allowed to cool at room

temperature. The optical density was measured spectro-

photometrically at 620 nm (UV-1700 PharmaSpec, UV–

Visible spectrophotometer; Shimadzu). A standard curve

was prepared using D-glucose; the TSS was calculated and

expressed as mg g-1 FW.

Measurement of inorganic ions concentration

For the quantification of major inorganic ions (Na?, K?

and Ca?), calli harvested from four individual petriplates

per treatment were oven-dried at 80�C for 48 h.

Samples (100 mg) from each treatment were acid diges-

ted in 35% (v/v) HNO3 at 100�C for 30 min; and the

ion content was analyzed by flame atomic absorption

spectrophotometry.

Measurement of antioxidant enzyme activities

Extraction

The treated and control calli (500 mg) were homogenized

in 5 ml of ice cold 50 mM sodium phosphate buffer (pH

7.0) containing 0.1 mM EDTA and 1% (w/v) Polyvinyl-

pyrrolidone (PVP) with chilled mortar and pestle. The

homogenate was filtrated with single layered cheese cloth

and centrifuged at 15,000g for 20 min at 4�C. An appro-

priate aliquot/dilution of the supernatant was used as a

crude enzyme(s) for determination of antioxidant enzyme

activities. Soluble protein content in the enzyme extract

was determined according to Bradford (1976) using Bovine

Serum Albumin (BSA) as a standard.

Catalase (CAT) assay

CAT (EC 1.11.1.6) activity was measured by following

the decomposition of hydrogen peroxide (H2O2) as

described by Cakmak and Marschner (1992) with minor

modifications. The activity was measured in a reaction

mixture (1 ml) containing 50 mM phosphate buffer (pH

7.0) and 15 mM H2O2. The reaction was initiated by

adding 50 ll enzyme extract and the activity was deter-

mined as a result of H2O2 decomposition by monitor-

ing the decrease in absorbance at 240 nm (e = 36

mM-1 cm-1) for 2 min at an interval of 15 s. The slope

of readings between the time interval considered as DA

and the enzyme activity was expressed as lKat of CAT

activity mg-1 protein.

Ascorbate peroxidase (APX) assay

APX (EC 1.11.1.11) activity was determined according to

Nakano and Asada (1981). The reaction mixture (1 ml)

contained 50 mM phosphate buffer (pH 7.0), 0.5 mM

ascorbate, 0.1 mM H2O2, and 0.1 mM EDTA. The reaction

was started by adding 100 ll of crude enzyme. Ascorbate

oxidation was monitored for 1 min by measuring the

decrease in absorbance at 290 nm at every 15 s

(e = 2.8 mM-1 cm-1). The enzyme activity was expressed

as lKat of APX activity mg-1 protein.

Superoxide dismutase (SOD) assay

The total SOD (EC 1.15.1.1) was assayed according to

Becana et al. (1986) by inhibition of the photochemical

reduction of nitroblue tetrazolium (NBT). The reaction

mixture (3 ml) containing 50 mM phosphate buffer (pH

7.8) and 0.1 mM EDTA to which an oxygen-generating

system containing 14.3 mM methionine, 82.5 lM NBT,

and 2.2 lM riboflavin, prepared freshly in situ, was added.

The reaction was initiated by adding 100 ll of crude

enzyme. The entire system was kept 30 cm below the light

source (six 15-W fluorescent tubes) for 30 min. The reac-

tion was stopped by switching off the light. For light blank,

all the reactant without enzyme extract was incubated in

light as for the samples, whereas all the reactants along

with 100 ll enzyme extract were incubated in dark for dark

blank. The reduction in NBT was measured by monitoring

the change in absorbance at 560 nm. The readings of light

blank were used in calculation of enzyme units. 1 U SOD

enzyme was defined as amount that produces 50% inhibi-

tion of NBT reduction under the assay condition and

expressed as U SOD activity mg-1 protein.

Statistical analyses

The experiment was laid out in a completely randomized

design (CRD). The analyses were repeated with three inde-

pendent biological samples. The data were analyzed by one-

way analysis of variance (ANOVA) using the statistical

software SPSS 10.0, and the treatment means were compared

by using Duncan’s multiple range test (DMRT) at P B 0.05.

Data were expressed as mean ± standard error (SE).

Results

Effect of salt stress on calli growth, membrane damage

rate (MDR) and water status

Fresh, actively proliferating calli cut into small pieces of

approximately 4–5 mm size were used for the analyses

20 Plant Cell Tiss Organ Cult (2010) 102:17–25

123

ensuring availability of adequate nutrient and oxygen

supply to inner parts of the callus. Observations after

15 days of salt treatment showed a significant decrease in

RGR of stressed calli with increase in the salt levels, and

were the least at 400 mM NaCl, almost 21.55-fold less as

compared to control (Fig. 1a). Total protein content was

significantly the highest in calli stressed with 100 mM

NaCl compared to control and further decreased with

increased salt concentrations thereafter (data not shown).

The MDR measured in terms of REL was appreciably

higher at different levels of salt as compared to control, and

was almost two-fold in the calli treated with 400 mM salt

compared with the control (Fig. 1b). The percent TWC of

salt-treated calli decreased significantly with increase in

salt levels in comparison to control calli (Fig. 1c). Simi-

larly, the FW/DW ratio was notably affected with elevated

levels of salt (Fig. 1d).

Osmotic adjustment through accumulation of osmolytes

(proline, GB and TSS)

Significantly higher levels of proline and GB content were

observed in the calli grown under salt stress (Fig. 2). The

calli grown on MS medium supplemented with 200 and

400 mM salt showed significantly the highest proline

content (more than 12- and seven-fold, respectively) in

comparison to control calli (Fig. 2). A similar pattern

was also observed for GB content. Salt-stressed calli

accumulated appreciably the highest GB content in the calli

treated with 200 mM (14.38 fold) and 400 mM (11.30

fold) salt compared to control calli (Fig. 2). The accumu-

lation of TSS was stress response dependent, where salt-

stressed calli accumulated significantly more TSS in

comparison to control calli. At higher levels of salt

(400 mM), the accumulation of TSS was significantly the

highest by almost 2.75-fold (Fig. 3).

84

85

86

87

88

89

90

91

92

Wat

er c

onte

nt (

%) (C) a

bb

c

0

2

4

6

8

10

12

FW

/DW

rat

io

NaCl concentrations (mM)

(D)a

b b,cc

0

50

100

150

200

250

Rel

ativ

e gr

owth

rat

e (%

)

(A)a

b

c c

0102030405060708090

100

0 100 200 4000 100 200 400

0 100 200 400

Mem

bran

e da

mag

e ra

te (

%)

NaCl concentrations (mM)

(B) ab

c

d

Fig. 1 The relative growth rate (a), membrane damage rate (b),

percent water content (c), and FW/DW ratio (d) of Sesuvium calli

under different salt concentrations. Callus (*1.0 g) was transferred to

media containing 0–400 mM NaCl, and relative growth rate,

membrane damage rate, percent water content, and FW/DW ratio

were measured after 15 days of culture. Error bars SE (n = 3).

Within each set of experiments, means with different letters are

significantly different at P \ 0.05

d

c

a

b

C C

B

A

0.0

0.5

1.0

1.5

2.0

2.5

0 100 200 400

Pro

line,

GB

(mg

g-1 F

W)

NaCl concentrations (mM)

Proline

Glycine Betaine

Fig. 2 Proline and glycine betaine contents of Sesuvium calli under

different salt concentrations. Callus (*1.0 g) was transferred to

media containing 0–400 mM NaCl, and proline and glycine betaine

content was measured after 15 days of culture. Error bars SE

(n = 3). Within each set of experiments, bars with different letters on

same color column are significantly different at P \ 0.05

Plant Cell Tiss Organ Cult (2010) 102:17–25 21

123

Mineral ions (Na?, K?, Ca?) concentration

Considerable differences were observed in the accumula-

tion of sodium, potassium, and calcium ions in the salt-

treated and untreated calli. The sodium as well as calcium

ion concentrations were significantly increased (4.3- and

3.0-fold, respectively) with increase in the levels of salt

compared to control calli (Fig. 4). However, potassium ion

concentration reduced appreciably in the salt-stressed calli

in comparison to control (Fig. 4).

Salinity induced changes in antioxidant enzymes

activities

Salt stress to Sesuvium calli significantly affected antioxi-

dant enzyme activities after 15 days of treatment (Fig. 5).

CAT activity was significantly the highest in control calli

as compared to salt-stressed calli (Fig. 5a). The activity of

APX reduced progressively and significantly at 100 and

200 mM salt; however, the activity increased significantly

in calli stressed at 400 mM (Fig. 5b). SOD activity was

also increased considerably under salt stress. Significantly,

the highest SOD activity was observed in the 200 mM salt-

stressed calli, whereas the activity at 400 mM NaCl was at

par with control (Fig. 5c).

Discussion

Studies with halophytes and non-halophytes have shown

that the extent of salt stress adaptation observed in the

c

bb

a

0

5

10

15

20

25

30

35

40

0 100 200 400

Tot

al so

lubl

e su

gars

(mg

g-1 F

W)

NaCl concentrations (mM)

Fig. 3 Total soluble sugar content of Sesuvium calli under different

salt concentrations. Callus (*1.0 g) was transferred to media

containing 0–400 mM NaCl, and TSS content was measured after

15 days of culture. Error bars SE (n = 3). Means with differentletters are significantly different at P \ 0.05

0

20

40

60

80

100

120

140

0 100 200 400

Ion

conc

. (m

mol

g-1

DW

)

NaCl concentrations (mM)

Sodium Potassium Calcium

Fig. 4 Mineral ions (sodium, potassium, and calcium) concentration

of Sesuvium calli under different salt concentrations. Callus (*1.0 g)

was transferred to media containing 0–400 mM NaCl, and sodium,

potassium, and calcium content was measured after 15 days of culture

b

c c

a

0

1

2

3

4

5

6

7

AP

X a

ctiv

ity (

µK

at m

g-1

prot

ein) APX activity

a

b

cc

0

0.05

0.1

0.15

0.2

0.25

CA

T a

ctiv

ity (m

Kat

mg

-1pr

otei

n)

CAT activity

cd

a

b

0

5

10

15

20

25

30

0 100 200 400

SOD

act

ivity

(U

mg-1

prot

ein)

NaCl concentrations (mM)

SOD activity

(A)

(B)

(C)

Fig. 5 Antioxidant enzyme (CAT, APX, and SOD) activities of

Sesuvium calli under different salt concentrations. Callus (*1.0 g)

was transferred to media containing 0–400 mM NaCl, and CAT (a),

APX (b), and SOD (c) activities were measured after 15 days of

culture. Error bars SE (n = 3). Within each experiment, means with

different letters are significantly different at P \ 0.05

22 Plant Cell Tiss Organ Cult (2010) 102:17–25

123

whole plant is also exhibited in callus tissue (Tonon et al.

2004; Arzani 2008); however, few in vitro culture systems

have been established from mangrove plants for studying

their salt tolerance at the cellular level (Cherian and Reddy

2003; Amor et al. 2005; Zhao et al. 2009). The study of salt

tolerance mechanism at the field level may not be preferred

as the salinity levels in field vary depending on season and

soil depth. Therefore, salt stress studies are preferably

conducted in soil-less cultures (like callus or axillary

shoots) with nutrient solutions of known salt concentrations

(Vijayan et al. 2003). In vitro culture provides a controlled

and uniform environment for studying physiological and

biological processes in plants particularly at the cellular

level under salt-induced osmotic stress (Ahmad et al.

2007). Therefore, in the present investigation, cellular

growth responses of Sesuvium under salt stress were stud-

ied. The results of the present investigation revealed that

Sesuvium calli respond fairly differently to salt stress in

comparison to responses observed in whole plant under

salinity or drought stress (Messedi et al. 2004; Slama et al.

2008). The higher levels of tissue organization in the whole

plant might efficiently distribute the effect of toxic saline

ions into different parts of the plants through its seques-

tration into the vacuoles and/or extrusion and hence show

better salt tolerance capacity which was not found at the

cellular level. In addition, restriction to entry of excess

toxic ions inside the plant at the root surface reduces the

heritable damage of the whole plant, while in the case of

callus cultures, the unorganised mass of calli is directly

exposed to toxic concentrations of saline ions in the med-

ium and hence loses its compatibility for sequestration of

these ions into the vacuoles. Furthermore, there are possi-

bly several physiological factors operating at the whole

plant level conferring salt tolerance which are lacking in

the dedifferentiated and unorganised callus cultures (Zhang

et al. 2004). This could be the reason that callus cultures

and whole plant respond differentially to salt stress.

One of the strategies developed by plants under salt

stress is to slow down their growth rate, which has been

observed in a number of in vitro systems of halophytes and

non-halophytes (Yasumoto et al. 1999; Khayri 2002; Zhang

et al. 2004; Patade et al. 2008; Zhao et al. 2009). The

reduction in growth not only helps the plant to save the

energy for defence purposes but also limits the risk of

heritable damage (May et al. 1998). The present study has

shown reduction in growth kinetics of Sesuvium calli in

terms of RGR (Fig. 1a), as has been observed in different

plant species: halophytes such as Sonnertia alba (Yasumoto

et al. 1999), Populus euphratica (Zhang et al. 2004),

Thellungiella halophila (Zhao et al. 2009,) and glycophytes

like tobacco (Watad et al. 1991), Saccharum officinarum

(Patade et al. 2008), and Arabidopsis thaliana (Zhao et al.

2009). The hyper-osmotic stress reduces the water

availability to the cell in the medium (Chamandoosti 2007),

as a result of which the FW and water content (Fig. 1c) of

salt-stressed calli decreased in comparison to control calli.

Furthermore, the view about a significant decrease in FW/

DW ratio in the Sesuvium stressed calli (Fig. 1d) was sup-

ported by the reduced FW/DW ratio observed in the callus

cultures of Sonnertia alba (Yasumoto et al. 1999). The

reduction in callus growth (FW and DW) under salt stress

might be a result of nutritional imbalance due to an inter-

ference of excess salt ions, with essential nutrients involved

in both uptake and translocation (Patade et al. 2008). The

reduction in water content of the cell under salt stress was

correlated with the increased cell membrane permeability of

plants growing under salt stress (Tabaei-Aghdaei et al.

2000; Farooq and Azam 2006) and consequently the injury

to the cell membrane which is measured in terms of REL.

Similarly, we observed a progressive increase in REL and

reduction in water content of Sesuvium calli with increasing

salinity levels. This was consistent with the higher values

recorded for REL under salt stress in wheat (Sairam et al.

2002; Farooq and Azam 2006) as well as in callus cultures

of sugarcane (Patade et al. 2008) and tobacco (Watad et al.

1991). The results suggest that, under salt stress conditions,

the membrane permeability of Sesuvium calli increases

producing more leakage of electrolytes outside the cell and

reduction in the growth as well as the water content of the

cell.

The low water content of the cell was apparently

adjusted by accumulating the organic osmolytes such as

proline, GB, and soluble sugars for the survival of the cells

under saline conditions. Accumulation of osmolytes under

salt stress is an indication of oxidative damage which

provides protection to cytosol from dehydration through

the development of compatible cytoplasmic osmoticum. In

the present study, significantly higher accumulation of

proline and GB as observed in the salt-stressed calli

(Fig. 2) might be an adaptive feature for the survival and

maintenance of reduced growth rate and water balance of

Sesuvium calli under salt stress conditions. Proline and GB

act as osmotica in presence of low water content and play a

major role in maintenance of osmotic balance, which was

found to be higher in salt-tolerant species than in salt-

sensitive species (Zhang et al. 2004). Zhao et al. (2009)

also demonstrated an increased accumulation of proline

and GB content in salt-stressed calli as compared to control

calli of mangrove, Thellungiella halophila. Accumulation

of TSS also imparts to the osmotic adjustment of the cell

under saline conditions (Fig. 3). Salinity-induced soluble

sugar accumulation has also been observed in P. euphra-

tica (Watanabe et al. 2000; Zhang et al. 2004). In addition

to accumulation of organic osmolytes, excess Na? accu-

mulation (Fig. 4) in the medium compensates for the loss

of K? ions, which have an added role of an osmoregulatory

Plant Cell Tiss Organ Cult (2010) 102:17–25 23

123

monovalent cation in the salt-stressed calli in comparison

to control calli. The K? outward rectifying channels

(KORCs), a class of low affinity K? channels which opens

during the depolarization of plasma membrane under salt

stress conditions, might mediate the efflux of K? and the

influx of Na? (Maathuis and Sanders 1997; Blumwald

2000) and hence alteration in the normal ratio of K?/Na?.

At the whole plant level in Sesuvium, sodium competes

with the potassium to enter inside the cell by affecting the

potassium absorption efficiency (Slama et al. 2008). The

reduced levels of K? as observed in the Sesuvium calli

under salt stress conditions may reduces osmotic pressure

brought about by K? accumulation within cells thus

affecting the cellular expansion, translocation of fixed

carbon, and turgor pressure of the cell (Szczerba et al.

2009). This might be one of the reasons for reduced growth

of the Sesuvium calli under salt stress condition.

Several studies have demonstrated that salt-tolerant spe-

cies increased their antioxidant enzyme activities and anti-

oxidant content in response to salt stress whereas salt-

sensitive species failed to do so (Yazici et al. 2007). In the

present investigation, CAT activity was significantly the

highest in control as compared to stressed calli (Fig. 5a)

suggesting the declining capacity of calli tissue to scavenge

H2O2 with increasing salt stress. The decrease in CAT

activity under increasing salinity has also been reported in

callus cultures of Suaeda nudiflora (Cherian and Reddy

2003) and Crithmum maritimum (Amor et al. 2005). APX use

ascorbate as a reductant to remove the excess H2O2 gener-

ated under salt stress. Several studies have reported a large

increase in APX activity after salinity stress in glycophytes;

however, studies on the role of APX in tolerance to salt stress

at cellular level in mangroves and other halophytes are

limited. In the present study, APX activity was found to be

low in the control calli as well as low levels of salt (100 and

200 mM NaCl) in the medium, whereas the activity was

significantly the highest in the calli treated with 400 mM salt

(Fig. 5b). The superoxide radicals formed at higher levels of

salt could be scavenged by increased SOD activity, and the

end product of this reaction, H2O2, is partially dismutated by

the increased activities of APX (Parida et al. 2004). Fur-

thermore, the increased SOD activity in stressed calli sug-

gested the view that the superoxide radicals were detoxified

efficiently. An increase in the APX activity and concomitant

increase in SOD activity is an adaptation to remove the

excess H2O2 generated due to increase in SOD activity under

excess salt stress (Jitesh et al. 2006).

Conclusions

The present results indicate that the cellular responses of

Sesuvium to salt tolerance were considerably different from

those reported at whole plant level by Messedi et al.

(2004). The RGR of calli reduced significantly as com-

pared to control calli under salt stress as revealed by

decreased water status, FW/DW ratio, damage to mem-

brane, disturbed osmotic balance, and altered antioxidant

enzyme activities. Accumulation of sodium (Na?) ions and

organic osmolytes (proline, GB, and TSS) in stressed calli

appears to be involved in osmotic adjustment under salt

stress at the cost of reduced growth rate and water content.

Moreover, the growth rate reduction observed in calli

stressed up to 200 mM NaCl was associated with lower

CAT and APX activities and significantly higher SOD

activity. Thus, the cultured cells of Sesuvium appeared to

possess better osmotic adjustment under salt stress. Further,

the results of this study illustrate the potential of using cell

and/or tissue culture for evaluation of salt tolerance at

cellular level, since responses are relatively fast, the gen-

eration times are short, and the environment is controlled.

Acknowledgment The senior author is grateful to the Department

of Atomic Energy (DAE), Board for Research in Nuclear Science

(BRNS) for financial support under the BARC-UOP collaborative

Ph.D. research programme.

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