Plant Growth Regul (2007) 53:53–63
DOI 10.1007/s10725-007-9204-0
ORIGINAL PAPER
Iso-osmotic effect of NaCl and PEG on growth, cations
and free proline accumulation in callus tissue of two indica
rice (Oryza sativa L.) genotypes
Muhammad Sajid Aqeel Ahmad Æ Farrukh Javed Æ
Muhammad Ashraf
Received: 11 April 2007 / Accepted: 4 July 2007 / Published online: 22 July 2007

Springer Science+Business Media B.V. 2007
Abstract One-month old calli of two indica rice
genotypes, i.e., Basmati-370 and Basmati-Kashmir
were subjected to two iso-osmotic concentrations
( 0.57 MPa and 0.74 MPa) created with 50 and
100 mol m 3 NaCl or 10 and 18% solutions of PEG-
8000. Both genotypes tolerated only low levels of
stress and showed severe growth suppression at
0.74 MPa. The degree of stress tolerance of both
genotypes was greater for PEG induced stress than
for NaCl induced stress. The relative growth rate of
callus was reduced under both stresses, however, the
reverse was true for callus dry weight. Sodium (Na+)
content of the callus tissue was increased only under
NaCl induced stress. Salt induced stress reduced K+
and Ca2+ contents, but the PEG induced stress
increased them. Higher levels of stress increased the
proline content many folds with more increase being
under PEG stress than that under NaCl. Water and
osmotic potentials of the callus tissue decreased,
whereas turgor potential increased under both abiotic
stresses. Overall, Basmati-370 was more tolerant to
both NaCl and PEG induced stresses than Basmati-
Kashmir, because of less reduction in growth and
more dry weight. Moreover, Basmati-370 accumu-
lated higher amounts of cations, free proline, and
maintained maximum turgor as compared to
M. Sajid Aqeel Ahmad (&)
F. Javed
M. Ashraf
Department of Botany, University of Agriculture,
Faisalabad 38040, Pakistan
e-mail: sajidakeel@yahoo.com
Basmati-Kashmir. In conclusion, at cellular level,
mechanism of NaCl induced osmotic stress tolerance
was found to be associated with more ionic accumu-
lation of inorganic solutes and that of PEG induced
osmotic stress tolerance with the accumulation of free
proline, as an important osmolyte in the cytosol.
Keywords Indica basmati rice
In vitro

Iso-osmotic stresses
NaCl
PEG
Introduction
Different plants use different strategies to counteract
osmotic stresses (salinity and drought). For example,
mechanism of stress tolerance has been reported to be
associated with hyper-accumulation of compatible
solutes such as glycine betaine and proline, exclusion
of toxic ions, increased accumulation of total soluble
carbohydrates and total soluble proteins (Binzel and
Rauveni 1994; Gangopadhyay and Basu 2000, Javed
2002). It is now well evident that these solutes
contribute to prevent dehydration and cellular dam-
age by osmotic adjustment (Santos-Diaz and Ochoa-
Alejo 1994a, b; Ashraf and Foolad 2007).
Osmotic stress affects numerous plant physiological
and biochemical processes both at cellular and tissue
level (Lutts et al. 1996; Reddy and Vajranabhajah
1996), however, the study of these processes at whole
plant level is rather difficult. Whole plant contains
mostly non-growing cells, which make it difficult to
123
54
characterize the biochemical processes that are oper-
ating in growing cells in response to abiotic stresses
(Turner and Jones 1980; Neumann 1997). In vitro
culture technique provides a controlled and uniform
environment for studying physiological and biological
processes in plants, particularly at cellular level under
salt (Stavarek and Rains 1984) and drought (Hasegawa
et al. 1984) induced osmotic stresses. Furthermore, it
provides means of careful growth measurement of
actively growing callus cells in response to abiotic
stresses (Stephen et al. 2002; Shibli and Al-Juboory
2002).
Osmotic stress effects in plant cell and tissue
culture studies can be investigated by using either
ionic and penetrating (e.g., NaCl, KCl), non-ionic and
penetrating (e.g., mannitol and sorbitol) and non-
ionic and non-penetrating [e.g., polyethylene glycol
(PEG)] stress agents (Gangopadhyay et al. 1997a, b).
Among these osmotic agents, PEG is the most widely
used osmoticum to study the water status of plants
(Handa et al. 1982; Bhaskaran et al. 1985; Newton
et al. 1986, 1989) PEG is an inert, non-ionic and non-
toxic chemical of high molecular weight. It is highly
soluble in water, and is available in a wide range of
molecular weights (e.g., PEG-4000, PEG-4500, PEG-
6000, and PEG-8000) (Lawlor 1970). It simulates
water deficit conditions in cultured cells in a similar
manner to that observed in the cells of intact plants
subjected to true drought conditions (Hohl and
Schopfer 1991; Attree et al. 1991). PEG of high
molecular weight (more than 4,000) induces water
stress in plants by decreasing the water potential of
the nutrient solution without being taken up and with
evidence of toxicity (Lawlor 1970). On the other
hand, NaCl is an ionic and penetrating stress agent
and is well known to produce specific ionic toxicities
in plant culture studies. In addition, it can be easily
taken up by the cultured cells and can cause ionic as
well as osmotic effects (Turner and Jones 1980;
Neumann 1997).
Rice represents a group of plants in which the
correlation of stress tolerance compared at cellular
and whole plant level is opposite, i.e., isolated cells
are much more tolerant to stress than the whole plants
(Neumann 1997). Cells with increased tolerance to
different abiotic stresses can be selected from different
genotypes by in vitro culture and can be regenerated
to whole plants (Binzel and Rauveni 1994; Do-Quang
2000) with enhanced stress tolerance.
123
Plant Growth Regul (2007) 53:53–63
Although, considerable work has already been
done to elucidate the mechanism of osmotic stress
tolerance in cereals, these studies are mostly at whole
plant level (Singh and Singh 1995; Xu et al. 1995;
Folkard et al. 1999) and still more experimentation is
required to understand these mechanisms at cellular
level. In the present study, we aimed to compare the
effects of iso-osmotic stresses generated by an ionic
and penetrating (NaCl) and non-ionic and non-
penetrating (PEG) osmotic stress agents on growth
and physiological responses of two indica Basmati-
rice genotypes at cellular level.
Materials and methods
The cellular behavior of the two indica rice genotypes
calli toward the iso-osmotic stress induced by either
NaCl or polyethylene glycol (PEG) was studied in the
Tissue Culture Laboratory, Department of Botany,
University of Agriculture Faisalabad. The seeds
(mature embryos) of two indica-rice (Oryza sativa
L.) genotypes, Basmati-Kashmir and Basmiti-370
were obtained from the Mutation Breeding Division,
Nuclear Institute for Agriculture and Biology
(NIAB), Faisalabad, Pakistan. A preliminary exper-
iment was conducted in petriplates for the selection
of genotypes and osmotic stress levels. Four Basmati
rice genotypes (i.e., Basmati-370, Basmati-Kashmir,
Basmati-Karnal, and Basmati-385) were exposed to
iso-osmotic solutions of 0, 0.44 [50 mol m 3 NaCl
and 10% PEG], 0.57 [100 mol m 3 NaCl and 18%
PEG], 0.71 [150 mol m 3 NaCl and 23% PEG] and
0.86 MPa [200 mol m 3 NaCl and 29% PEG] of
both NaCl and PEG. On the basis of germination
percentage, days to 50% germination, plumule and
radicle length, and fresh and dry weights of plumules
and radicles (data not reported), genotype Basmati-
370 was categorized as salt tolerant and Basmati-
Kashmir as salt sensitive. They were chosen for
further experimentation. Similarly, 0.57 and 0.74
MPa levels of both osmotic agents were found to be
the most suitable for further studies.
The surface sterilized dehusked seeds of the two
selected genotypes were dried on autoclaved filter
paper and placed in culture tubes for callus initiation
and maintenance. The basal salt medium used for the
initiation and maintenance of callus was LS (Linsmaier
and Skoog 1965) basal medium, supplemented with
Plant Growth Regul (2007) 53:53–63
2.5 mg l 1 2,4-D (2,4-dichlorophenoxyacetic acid)
and 0.5 mg l 1 6-furfurylaminopurine (kinetin) (Nigel
et al. 1999). The culture tubes were sealed with cotton
plugs and incubated under continous white fluorescent
light (PAR 300 lmol m 2 s 1) in a growth room at
25 ± 2C for 30 days. One-month old calli were
aseptically cultured in Erlenmeyer flasks (100 ml,
Pyrex) containing 40 ml of liquid LS-medium (sup-
plemented with the same concentration of 2,4-D and
kinetin as used for callus initiation) and iso-osmotic
treatments developed by NaCl and polyethylene glycol
(PEG-8000) along with a control. These osmotic
agents were used in two iso-osmotic concentrations
( 0.57 MPa or 0.74 MPa) estimated by a vapor
pressure osmometer (VAPRO 5520, Wescor Inc.,
Logan, Utah, USA) and corresponded to the final
osmotic potentials of 0.57 MPa [50 mol m 3 NaCl
and 10% PEG-8000] and 0.74 MPa [100 mol m 3
NaCl and 18% (w/v) PEG-8000]. The osmotic
potential of the control solution was 0.17 MPa. The
flasks were placed on gyratory shaker for 15 days
under the iso-osmotic stress treatments and incubated
under continuous white fluorescent light (PAR
300 lmol m 2 s 1) in a growth room at 25C ± 2C.
The experiment was laid down in a completely
randomized manner with five replications.
Calli after harvesting from the liquid saline
medium were washed with distilled deionized water
for 2 min with continuous agitating and then dried
with tissue papers. Fresh weights of calli were
determined and relative growth rate was calculated
as the difference of natural logs of final fresh weight
and the initial weight of the callus. Calli were dried in
an oven at 65C for 72 h and their dry weights
deterimined. For the deterimination of osmotic, water
and turgor potentials, fresh calli, and their respective
media were put in centrifuge tubes and were frozen
for 7 days at 70C in an ultralow freezer. The calli
and the media were then defrosted and 10 ll of the
extracted sap from calli and of the medium were used
to deterimine the osmotic potential using a vapor
pressure osmometer (VAPRO-5520, Wescor Inc.,
Logan, Utah, USA). Water potential of the callus was
assumed to be equal to the solute potential of the
culture medium (as callus tissues are in unconfined
system and osmotic potential of the medium corre-
sponds to the water potential of the tissue). Callus
turgor potential was calculated as the difference
between water potential (ww) and osmotic potential
55
(ws) of the callus. Extraction and estimation of
proline was conducted according to the procedure
described by Bates et al. (1973). Na+, K+, and Ca2+
were deterimined in acid digest (HNO3) using an
atomic absorption spectrophotometer (AAnalyst 300,
Perkin-Elmer, Germany).
Statistical analysis
Analysis of variance (ANOVA) of the data was
computed using a STATISTICA package for Win-
dows, Kernel Release 5.5A (StatSoft, Inc., Tulsa, OK
74104, USA). The Visual General Linear Model
(GLM) was fitted using the same statistical package
for assessing variation within treatments and interac-
tion terms by splitting their degree of freedom into
contrast comparisons.
Results
Relative growth rate of the calli of both indica rice
genotypes was significantly reduced by increasing
osmotic stress in the culture medium. Both levels of
the NaCl and PEG differed significantly as well as
from control (Table 1). The reduction in relative
growth rate was greater at 0.74 MPa as compared
to that at 0.57 MPa of both solutes. Genotype
Basmati-Kashmir exhibited more reduction as com-
pared to Basmati-370 at both levels. Overall, the
reduction in relative growth rate was greater in
osmotic stress induced by NaCl than that by PEG
(Fig. 1). In contrast, dry weight of the calli of both
genotypes increased with increasing level of osmotic
stress induced by both solutes. This increase in dry
weight was also significantly different within the
levels as well as within osmotic agents (Table 1).
Genotype Basmati-370 performed better than Bas-
mati-Kashmir under both levels of osmotic stress and
exhibited more increase in dry weight as compared to
Basmati-Kashmir. However, this increase was greater
in osmotic stress induced by PEG than that by NaCl.
Increase in dry weight was more in 0.74 MPa than
in 0.57 MPa of both the osmotic agents (Fig. 1)
compared to their respective controls.
Osmotic potential decreased (more negative) sig-
nificantly with increasing concentration of both
osmotic agents in the culture medium. The decrease
was also significantly different within the levels of
123
 56

Table 1 Analyses of variance (ANOVA) and contrast comparisons for various growth, physiological and biochemical attributes of two rice genotypes calli subjected to

123
iso-osmotic stress generated by NaCl or PEG
General effect d.f. RGR Dry weight Osmotic potential Water potential Turgor potential Na+ content K+ content Ca2+ content Proline

Intercept 1 7.128*** 218586.5*** 28.035*** 14.25*** 2.310*** 3808220*** 6054524*** 353531*** 634288.3***
Genotypes (G) 1 0.036*** 466.34*** 0.252*** 0.104*** 0.0323*** 43595*** 34417*** 6300.25*** 8306.4***
Treatment (T) 4 0.124*** 519.79*** 0.678*** 0.531*** 0.0105*** 602536*** 39588*** 20399.77** 37146.8***
C versus All 1 0.268*** 1085.58*** 1.90*** 1.564** 0.0164*** 586415*** 231 ns 5497* 73564.2***
C versus NaCl 1 0.319*** 759.19*** 1.195*** 1.046** 0.0053** 1512224*** 24869*** 37307*** 21243.6***
C versus PEG 1 0.144*** 1062.86*** 2.02** 1.593** 0.0259*** 28354* 16892* 892 ns 122107.7**
NaCl versus PEG 1 0.516*** 38.22** 0.163*** 0.881*** 0.0115*** 1689659*** 124132*** 57110*** 62232.8***
V1 C versus V1 NaCl 1 0.15*** 991.21*** 0.780** 0.674** 0.004* 983465*** 6637* 5958* 13709.6**
V1 C versus V1 PEG 1 0.325** 1341.79*** 1.252** 0.969** 0.0181*** 13648* 12707** 1128 ns 83392.9***
V2 C versus V2 NaCl 1 0.212*** 74.21*** 0.439** 0.386** 0.0017 ns 558599*** 20037** 18065*** 7927.4**
V2 C versus V2 PEG 1 0.127*** 89.78*** 0.799** 0.642** 0.0085*** 17715*** 5052* 75 ns 42190.5***
V1 NaCl1 versus V1 NaCl2 1 0.307*** 196.89*** 0.187*** 0.146*** 0.0026* 91790*** 10985* 8121* 5641.3**
V1 PEG1 versus V1 PEG2 1 0.345*** 421.18*** 0.240*** 0.154*** 0.0096*** 98.82ns 9340** 247 ns 2240.0***
V2 NaCl1 versus V2 NaCl2 1 0.324*** 13.82*** 0.1014*** 0.078*** 0.0016 ns 45890** 8993** 10437* 3743.0*
V2 PEG1 versus V2 PEG2 1 0.845** 179.08** 0.135*** 0.104*** 0.0019 ns 425 ns 5070* 256 ns 1322.9*
G · T interaction 4 0.005** 101.55*** 0.011*** 0.007*** 0.0006 ns 14750.63** 544.86 ns 172.83 ns 1578.7*
V1 NaCl versus V2 NaCl 1 0.152** 266.59*** 0.136*** 0.068*** 0.0116*** 98455*** 19903*** 4070* 1399.5*
V1 PEG versus V2 PEG 1 0.361*** 527.35*** 0.140*** 0.058*** 0.018*** 82 ns 14043*** 2632 ns 11060.0**
V1 NaCl versus V2 PEG 1 0.0001 ns 223.29** 0.007*** 0.001 ns 0.0020 ns 1167635*** 14241*** 12416** 11048.1**
V1 PEG versus V2 NaCl 1 0.101*** 576.85*** 0.433** 0.212*** 0.0389*** 574140*** 143583*** 51319*** 61348.8***
Error 40 0.001 2.84 0.0005 0.0004 0.0005 2507.34 925.51 737.07 941.80
Significant at *0.05, **0.01, and ***0.001 levels. Ns = non-significant
V1 = Basmati-370, V2 = Basmati-Kashmir, C = Control, PEG = Poly-ethylene glycol-8000, NaCl1 = 50 mol m 3, NaCl2 = 100 mol m 3, PEG1 = 10%, PEG2 = 18%
The 4 degree of freedom have been split into contrasts to check the variation within treatments (T) or variety · treatment (V · T) interaction
Plant Growth Regul (2007) 53:53–63
Plant Growth Regul (2007) 53:53–63 57

Fig. 1 Effect of NaCl or Basmati-370 Basmati-Kashmir

PEG induced iso-osmotic 1

Relat ive increase in f . w t . ( g g -1)

stress on the growth LSD V x T (5%) = 0.054
parameters of two rice 0.8 a a -7.57%
genotype callus tissues. -25.11% b -30.54%
Bars represent SE values 0.6 cd -46.48% ab de
ab
(n = 5). Percent increase or f
0.4 c
decrease is shown. Means e -19.54%
sharing same letters do not
0.2 -36.89% fg
differ significantly at 5% g
-54.23%
-58.38%
0

120 38.64% 48.72%

22.36% b 24.91% a
LSD V x T (5%) = 2.870
100 c c
d d
80
Dry w eight ( mg )

c c
d 15.24% d 15.94%
60 e e
0.71% 1.62%
40

20

0
-0.17 MPa -0.57 MPa -0.74 MPa - 0.17 MPa - 0.57 MPa - 0.74 MPa
0 50 100 0 10 18
NaCl (mol m -3) PEG (%)

solutes, and also within the solutes (Table 1). Both Na+ content of the callus increased with increase
genotypes showed a consistent decrease in osmotic in the intensity of stressful environment (NaCl and
potential with increasing intensity of the stresses. PEG) of the culture medium. Genotypes and treat-
Basmati-370 showed more decrease in osmotic ment means showed highly significant differences
potential compared to Basmati-Kashmir at both (Table 1). Both genotypes exhibited a consistent
levels of both stresses. However, reduction in osmotic increase in Na+ content with increasing stress level.
potential was more under PEG induced stress com- Rice calli showed greater accumulation of Na+ under
pared with salt induced osmotic stress (Fig. 2). salt induced osmotic stress than that in PEG induced
Highly significant differences were observed stress. Basmati-370 accumulated more Na+ compared
between the genotypes and treatments with respect to Basmati-Kashmir at both osmotic levels of NaCl or
to water potential. Decrease in water potential was PEG induced stresses (Fig. 3).
statistically significant for both levels as well as for Although K+ content of the callus decreased with
both osmotic agents (Table 1). Basmati-370 exhib- increasing intensity of NaCl induced osmotic stress in
ited more and consistent reduction in water potential the growth medium, the reverse was true in PEG
than Basmati-Kashmir under both stresses. Water induced osmotic stress. The sources, levels, osmotic
potential was more negative under PEG induced agents and interaction were all statistically highly
osmotic stress compared to NaCl induced osmotic significant (Table 1) for both NaCl and PEG induced
stress (Fig. 2). stresses. Under both levels of salt induced osmotic
In contrast, turgor potential of the callus tissue stress, Basmati-370 exhibited relatively less reduction
increased with increasing intensity of both stresses, in K+ content. In contrast, the same genotype showed
but the extent was greater under PEG than under more increase than Basmati-Kashmir at both levels of
NaCl induced osmotic stress. Genotypes and treat- drought (PEG) induced osmotic stress. Higher levels
ments differed significantly under both stresses. This of osmotic stress seemed to be more effective in
increase in turgor was variable for levels as well as reducing K+ content of the callus tissue (Fig. 3).
for osmotic agents (Table 1). Basmati-370 main- Similar results were obtained for Ca2+ content of
tained greater turgor as compared to Basmati-Kash- the callus for both genotypes. Ca2+ contents
mir at all levels of both stresses (Fig. 2). decreased under salt induced osmotic stress, but

123
58 Plant Growth Regul (2007) 53:53–63

Fig. 2 Effect of NaCl or Basmati-370 Basmati-Kashmir

201.49%
PEG induced iso-osmotic
162.87% a
stress on water relations of 1.6
two rice genotypes callus b LSD V x T (5%) = 0.038 120.18%

O smot ic p ot ent ial ( - MPa)

1.4
d
tissues. Bars represent SE 1.2
91.05%
values (n = 5). Percent f
1 c
increase or decrease is
0.8 179.98%
shown. Means sharing same e
h f
letters do not differ 0.6 h g 137.73% 110.88%
significantly at 5% 0.4 77.91%
0.2 i i
0
1.2 310.39% 360.67% a
LSD V x T (5%) = 0.034
b
225.84%
Water pot ent ial ( - MPa)

1
179.07% d
0.8 e c

0.6 d 306.43%
242.54% e
0.4 f 190.64%
g g
142.69%
0.2
g g
0
0.45 LSD V x T (5%) = ns 53.14%
0.4
25.39% 21.72%
Tu rg or p ot ent ial ( MPa)

0.35
9.03%
0.3
0.25
40.48%
0.2 22.10% 22.90%
6.45%
0.15
0.1
0.05
0
-0.17 MPa -0.57 MPa -0.74 MPa - 0.17 MPa - 0.57 MPa - 0.74 MPa
0 50 100 0 10 18
NaCl (m ol m -3 ) PEG (%)

increased under drought induced stress. Genotypes Discussion

and treatments showed highly significant differences
for Ca2+ content in response to osmotic stress induced From the results of the present study, it is evident that
by both agents (Table 1). Genotype Basmati-Kashmir iso-osmotic stress developed by both agents (NaCl
exhibited more reduction in Ca2+ compared to and PEG) significantly reduced the callus fresh
Basmati-370 at both NaCl level. However, the same weight of both indica Basmati-rice genotypes. How-
genotype showed an increase in Ca2+ accumulation ever, the reverse was true for callus dry weight. The
under drought induced osmotic stress (Fig. 3). increase in callus dry weight due to PEG and NaCl
Proline content of both rice genotypes calli induced stresses has already been found in a number
increased with increasing concentration of both of studies (Pushpalatha and Padmanabhan 1998;
solutes (NaCl and PEG) in the culture medium. Chauhan and Prathapasenan 2000; Pushpam and
Highly significant differences were observed for the Rangasamy 2000b; Shankhdhar et al. 2000). In this
treatment and genotype means (Table 1). Basmati- study, increase in dry weight under both conditions
370 showed more increase in proline content com- was almost equal. Thus, it can be postulated that
pared to Basmati-Kashmir under both stresses. The enhanced ionic uptake mainly Na+ and Cl- (Almansouri
accumulation of proline was slightly greater under et al. 2000; Javed 2002) and increased production of
PEG induced osmotic stress than that under NaCl proline (Al-Khayri 2002; Al-Khayri and Al-Bahrany.
induced stress (Fig. 3). 2002) might have played a vital role in increasing

123
Plant Growth Regul (2007) 53:53–63 59

Fig. 3 Effect of NaCl or Basmati-370 Basmati-Kashmir

PEG induced iso-osmotic 1200 1020.06%

Na+ co nt en t ( µ mo l g -1 d. w t .)
stress on cations and free 1000
a LSD V x T (5%) = 85.28
proline of two rice 714.17%
genotypes callus tissues. 800 b
Bars represent SE values
(n = 5). Percent increase or 600
b
decrease is shown. Means 400 850.05%
sharing same letters do not c 97.13% 107.16%

differ significantly at 5% 200 608.65% de d

ef ef
f f 109.55% de127.21%
def
0

700
K+ co nt ent (µ mol g -1 d. w t .)

LSD V x T (5%) = ns 25.12%

600 8.49%
-3.13%
500
-21.15%
18.22%
400 4.87%
300 -14.09%
200 -31.88%

100

450
4.72% 8.21%
Ca2+ cont ent (µ mol g -1 d . w t .)

LSD V x T (5%) = ns
400
-9.09%
350
-29.12% 3.56%
300 0.11%
250 -15.09%
200
150 -38.47%
100
50
0

350 473.06%
Proline cont ent (µ mo l g -1 d. w t .)

LSD V x T (5%) = 10.12

300 391.27% a
250 c
240.12%
200 e
110.33% b
150
g d 351.95%
f 286.66%
100
i 193.32% i
50 h
i 83.50% i
0
-0.17 MPa -0.57 MPa -0.74 MPa - 0.17 MPa - 0.57 MPa - 0.74 MPa
0 50 100 0 10 18
NaCl (mol m -3) PEG (%)

dry weight under NaCl induced osmotic stress Osmotic and water potentials of the calli became
(Figs. 2 and 3). On the other hand a significant more negative with increasing level of osmotic stress.
increase in free proline content (Al-Khayri 2002; Al- However, the turgor potential of the callus tissue was
Khayri and Al-Bahrany 2002) along with other free maintained and/or increased under both osmotic
amino acids (Willadino et al. 1996a; Willadino et al. stresses. Callus tissue of Basmati-370 showed greater
1996b; Juhasz et al. 1997), soluble proteins (Garcia maintenance of turgor potential than that of Basmati-
et al. 1999) and soluble carbohydrates (Liu and Kashmir at both stress levels induced by both agents.
Staden 2001; Javed 2002) may have played an This decrease in osmotic and water potentials of the
important role in increasing dry weight under PEG callus tissue was greater in PEG induced osmotic
induced osmotic stress. stress than in that of NaCl. Osmotic potential was

123
60
more negative as compared to water potential of the
callus tissue. The reduction in osmotic and water
potential can be correlated with the accumulation of
solutes such as cations (Almansouri et al. 2000;
Javed 2002), anions (Cl and SO42 ) (Almansouri
et al. 2000; Javed 2002), proline (Hou et al. 1992;
Lutts et al. 1996), other free amino acids (Paek et al.
1988; Juhasz et al. 1997), soluble proteins (Paek
et al. 1988; Hou et al. 1992), carbohydrates (Alman-
souri et al. 2000; Javed 2002) and phenols (Gonzalez
and Garcia 1988). The accumulation of these solutes
leads to reduction in osmotic potential of the callus
tissue at cellular level and hence helps plant to
maintain growth under stressful environment. It is
known that maintenance of turgor is important under
stressful conditions in tolerant cultivars for enhanced
growth (Ikeda et al. 2002) which is brought about by
greater decrease in osmotic potential than water
potential of the tissue (Paek et al. 1988; Gupta et al.
1995). Those cultivars which cannot maintain turgor
are unable to grow and maintain growth at high
intensity of stress (Newton et al. 1987). In the present
study, the enhanced stress tolerance of the callus
tissue of the tolerant cultivar could have been due to
high solute accumulation and maintenance of turgor.
In the present study Na+ content of the callus
increased with increasing concentration of NaCl
induced osmotic stress in the culture medium. This
is similar to what has been earlier observed in a
number of studies (He and Yu 1995; Javed 2002;
Sathish et al. 1997). However, K+ and Ca2+ contents
were decreased as found in some other studies (Shah
et al. 1990; Almansouri et al. 2000). On the other
hand, the reverse was true for K+ and Ca2+ as well as
for Na+ under PEG induced osmotic stress. Such ion
imbalance due to drought has already been reported
in some earlier reports (Santos et al. 1999; Alman-
souri et al. 2000). It is now well established that one
of the most pronounced effects of salinity (NaCl) is a
consistent increase in Na+ and Cl contents along
with a concomitant decrease in K+ and Ca2+ content.
The higher levels of callus Na+ content at higher
salinity levels are thought to regulate growth and help
plant to tolerate high levels of salinity through ionic
compartmentation (He and Yu 1995; Lutts et al.
1996) and thus they enable the salt tolerant plants to
survive and grow better under high salinity stress
(Orton 1980; Thomas et al. 1992). A possible expla-
nation to this effect can be that the cell maintains the
123
Plant Growth Regul (2007) 53:53–63
balance between anions and cations. An increase in
the Cl content as a result of high salt levels is
accompanied by increased Na+ accumulation. As the
total charges are to be balanced in the cell, therefore,
increase in Na+ content would lead to decrease both
K+ and Ca2+ contents (Cramer et al. 1985). These
authors also found that at high salt concentration, Na+
can displace Ca2+ from plasma membrane, resulting
in a change in plasma membrane permeability that
can be detected as leakage of K+ from the cells. In
contrast, an increase in the osmotica under water
stress is accompanied by the accumulation of cations
which facilitate the plant to withstand the harmful
effects of water stress through osmotic adjustment. In
contrast, osmotic adjustment is solely developed in
response to tissue dehydration and is brought about
by decrease in the osmotic potential of the cell which
helps it to absorb water even at low water potential of
the external environment (Taiz and Zeiger 2002).
Published data reveal that there is a positive corre-
lation between abiotic stress tolerance and concen-
trations of vacuolar ionic (Muralitharan et al. 1993),
proline (YanJun et al. 2000; Al-Khayri and Al-
Bahrany 2002), other free amino acids (Juhasz and
Sarkadi 1994; Juhasz et al. 1995), soluble proteins
(Garcia et al. 1999), soluble carbohydrates (Smith
et al. 1984), or phenols (Gonzalez and Garcia 1988).
Proline is known to function as an osmoregulatory
molecule that prevents cellular dehydration through
osmotic adjustment. In addition, it may interact with
crucial macromolecules of the cell to maintain their
biological activity under stressful conditions. There-
fore, proline is known to maintain a hydration sphere
around the bio-polymers, and maintain their native
state thereby regulating growth under drought (PEG)
and salinity (NaCl) stresses (Rudulier et al. 1984;
Gangopadhyay and Basu 2000). Our studies revealed
that under both the osmotic stresses, proline content
increased in calli of both indica rice cultivars. Cv.
Basmati-370 accumulated more proline than cv.
Basmati-Kashmir under both NaCl and PEG induced
stresses. The extent of proline accumulation in the
two calli was more under drought (PEG) stress than
that in NaCl. Similar results have already been
reported by different researchers during their in vitro
experimentation to evaluate the effect of NaCl
induced osmotic stress (Pushpam and Rangasamy
2000a, 2000b; Shankhdhar et al. 2000; Basu et al.
2002) and drought stress (Lutts et al. 1999; Yoshida
Plant Growth Regul (2007) 53:53–63
et al. 1999; Garcia et al. 1999; Gangopadhyay and
Basu 2000) on rice calli. Proline is known to be
the most important osmoticstress-marker in in vitro
system (Gangopadhyay and Basu 2000). Its accumu-
lation increases many folds upon exposure to salt
(Al-Khayri 2002; Camara et al. 2000; Pushpam and
Rangasamy 2000a), and drought (Barakat and Abdel-
Latif 1995; He and Yu 1995). It is possible that
proline accumulation in excessive amount under
stressful condition could be due to its stimulated
synthesis from glutamate, lower rate of its oxidation
and slowed incorporation of proline into proteins
(Hanson and Hitz 1982). The higher levels of proline
at higher levels of salinity (NaCl) and drought
(PEG) are thought to regulate growth through
osmotic adjustment and reduce the acidity of the
cytoplasm (Vanekamp et al. 1989). On the basis of
our findings it could be concluded that proline
accumulation is not just a sign of cellular injury
resulted in response to drought or salt stress, but it is a
marker of stress tolerance having a definite osmo-
regulatory role thereby regulating growth under
stressful environments.
In conclusion, callus tissues of Basmati-rice
genotypes tolerated only low level of osmotic stress
( 0.57 MPa) and sever growth reduction was
observed at high level of stress ( 0.74 MPa). The
response against salt induced osmotic stress was
comparatively better than that against PEG induced
osmotic stress. Basmati-370 performed better under
osmotic stress induced by both agents by showing
less reduction in growth, more dry weight, accumu-
lation of more cations and free proline, and mainte-
nance of high turgor. In addition, tolerance of rice
calli to NaCl induced osmotic stress seemed to be
associated with more accumulation and storage of
ions in the vacuole. On the other hand, PEG induce
osmotic stress tolerance seemed to be associated with
greater proline accumulation and a slight increase in
cations (Na+, K+, and Ca2+) in the callus tissues.
References
Agarwal RK, Gupta SC (1995) Plant growth substances as
osmoregulants under salt stress in callus cultures of
cowpea. Indian J Plant Physiol 4:325–327
Al-Khayri JM, Al-Bahrany AM (2002) Callus growth and
proline accumulation in response to sorbitol and sucrose-
induced osmotic stress in rice. Biol Plant 45:609–611
61
Al-Khayri JM (2002) Growth, proline accumulation, and ion
content in sodium chloride-stressed callus of date palm.
In Vitro Cell Develop. Biol Plant 38:79–82
Almansouri M, Kinet JM, Lutts S (2000) Physiological anal-
ysis of salinity resistance in Triticum turgidum var. durum
Desf.: Callus versus whole plant responses. Options
Mediterraneennes Ser A Seminaires Mediterraneens
40:263–265
Ashraf M, Foolad MR (2007) Roles of glycine betaine and
proline in improving plant abiotic stress resistance. Env
Exp Bot 59:206–216
Attree SM, Moore D, Sawhney VK, Fowke LC (1991) En-
hanced maturation and desiccation tolerance of white
spruce (Picea glauca (Moench) Voss) somatic embryos:
effect of a non-plasmolysing water stress and abscisic
acid. Ann Bot 68:519–522
Barakat MN, Abdel-Latif TH (1995) In vitro selection for
drought tolerant lines in wheat. I. Effect of polyethylene
glycol on the embryogenic cultures. Alexandria J Agric
Res 40:97–112
Basu S, Gangopadhyay G, Mukherjee BB (2002) Salt tolerance
in rice in vitro: implication of accumulation of Na+, K+
and proline. Plant Cell Tiss Organ Cult 69:55–64
Bates LS, Waldren RP, Teare ID (1973) Rapid determinations
of free proline for water stress studies. Plant Soil 39:
205–207
Bhaskaran S, Smith RH, Newton RJ (1985) Physiological
changes in cultured sorghum cells in response to induced
water stress I. Free proline. Plant Physiol 79:239–248
Binzel ML, Reuveni M (1994) Cellular mechanisms of salt
tolerance in plant cells. Hort Rev 16:33–69
Camara TR, Willadino L, Torne JM, Manick A, Santos MA
(2000) Effect of salt stress and exogenous proline in
maize callus. Revista Brasileira de Fisiologia Vegetal
12:146–155
Chauhan VA, Prathapasenan G (2000) Growth characteristic
and ion contents of rice callus under the influence of NaCl
and hydroxyproline. Acta Physiol Plant 22:39–44
Cramer GR, Lauchli A, Polito VS (1985) Displacement of Ca2+
by Na+ from plasmalemma of root cells: a primary
response to stress? Plant Physiol 79:207–211
Do-Quang B, Laszlo EH, Ibolya SK (2000) In vitro studies on
salt tolerance in rice (Oryza sativa L.). Cahiers Options
Mediterraneennes 8:25–27
Folkard A, Dingkuhn M, Wittstock C, Doerffling K (1999)
Sodium and potassium uptake of rice panicles as affected
by salinity and season in relation to yield and yield
component. Plant Soil 207:133–145
Gangopadhyay G, Basu S (2000) Proline as osmotic stress-
marker in in vitro system. In: Hemantaranjan A (ed)
Plant physiology, biochemistry and plant molecular biol-
ogy in 2000, vol. 3. Scientific Publishers, Jodhpur, India,
pp 283–304
Gangopadhyay G, Basu S, Gupta S (1997a) In vitro selection
and physiological characterization of NaCl- and mannitol-
adapted callus lines in Brassica juncea. Plant Cell Tiss
Organ Cult 50(3):161–169
Gangopadhyay G, Basu S, Mukherjee BB, Gupta S (1997b)
Effect of salt and osmotic shocks on unadapted and
adapted callus lines of tobacco. Plant Cell Tiss Organ Cult
49:45–52
123
62
Garcia A, Florido M, Perez I, Lara RM, Gonzalez MC, Marrero
MT (1999) Membrane stability and total protein content
in rice callus subjected to water stress. Cultivos Tropicales
20:17–20
Gonzalez S, Garcia E (1988) Effect of water stress on callus
and plantlets of sugarcane. Revista del Jardin Botanico
Nacional 9:57–66
Gupta SD, Auge RM, Denchev PD, Conger BV (1995) Growth,
proline accumulation and water relations of NaCl-selected
and non-selected callus lines of Dactylis glomerata L. Env
Exp Bot 35:83–92
Handa AK, Bressan RA, Handa S, Hasegawa PM (1982)
Characteristics of cultured tomato cells after prolonged
exposure to medium containing polyethylene glycol. Plant
Physiol 69:514–521
Hanson AD, Hitz WD (1982) Metabolic responses of meso-
phytes to plant water deficits. Annu Rev Plant Physiol
33:163–203
Hasegawa PM, Bressan RA, Handa S, Handa AK (1984)
Cellular mechanisms of tolerance to water stress. Hort-
Science 19:371–377
He DY, Yu SW (1995) In vitro selection of a high-proline-
producing variant from rice callus and studies on its salt
tolerance. Acta Phytophysiol Sinica 21(1):65–72
Hohl M, Schopfer P (1991) Water relations of growing maize
coleoptiles. Plant Physiol 95:716–722
Hou JH, Geng QH, Hu RH (1992) The effect of water stress on
callus of winter wheat. Acta Agric. Boreali-Sinica 7:52–56
Ikeda T, Fujime Y, Terabayashi S, Date S (2002) Water status
of garlic callus under various salt and osmotic stress
conditions. Hort Sci 37:404–405
Javed F (2002) In vitro salt tolerance in wheat I: growth and
ion accumulation in Triticum aestivum. Int J Agric Biol
4:459–461
Juhasz GA, Sarkadi L (1994) Changes in free amino acid
contents of bean callus cultures as affected by non-ionic
osmotic stress. Zoldsegtermesztesi Kutato Intezet Bullet-
inje 26:55–61
Juhasz GA, Sarkadi SL, Velich I, Varro P (1995) The effect of
non-ionic osmotic stress on bean callus cultures. Hort Sci
27:7–14
Juhasz GA, Simon-Sarkadi L, Velich I, Varro P (1997) Studies
of non-ionic osmotic stress on bean (Phaseolus vulgaris
L.) callus and seedlings cultures. Acta Hort 44:455–456
Lawlor DW (1970) Absorption of polyethylene glycols by
plants and their effects on plant growth. New Phytol
69:501–514
Linsmaier EM, Skoog F (1965) Organic growth factor
requirement of tobacco tissue cultures. Physiol Plant
8:100–127
Liu T, Staden J van (2001) Partitioning of carbohydrates in
salt-sensitive and salt-tolerant soybean callus cultures
under salinity stress and its subsequent relief. Plant
Growth Regul 33:13–17
Lutts S, Kinet JM, Bouharmont J (1996) Effects of various salts
and of mannitol on ion and proline accumulation in
relation to osmotic adjustment in rice (Oryza sativa L.)
callus cultures. J Plant Physiol 149:186–195
Lutts S, Kinet JM, Bouharmont J (1999) Improvement of rice
callus regeneration in the presence of NaCl. Plant Cell
Tiss Organ Cult 57:3–11
123
Plant Growth Regul (2007) 53:53–63
Muralitharan MS, Chandler SF, Steveninck RFM (1993)
Physiological adaptation to high ion concentrations or
water deficit by callus cultures of high bush blueberry,
Vaccinium corymbosum. Aust J Plant Physiol 20:159–172
Neumann P (1997) Salinity resistance and plant growth
revisited. Plant Cell Environ 20:1193–1198
Newton RJ, Sen S, Puryear JD (1987) Free proline in water-
stressed pine callus. Tappi J 70:141–144
Newton RJ, Sen S, Puryear JD (1989) Solute contribution to
osmotic potential loblolly pine (Pinus taeda L.) callus. J
Plant Physiol 34:746–750
Newton RJ, Bhasakaran S, Puryear JD, Smith RH (1986)
Physiological changes in culture sorghum cells in re-
sponse to induced water stress: II. Soluble carbohydrates
and organic acids. Plant Physiol 81:626–629
Nigel WB, Jotham JP, Azhakanandam K, Power JB, Lowe KC,
Cocking EC, Davey MR (1999) Callus initiation, main-
tenance, and shoot induction in rice. In: Plant cell culture
protocols. ISBN 1-59259-583-9
Orton TJ (1980) Comparison of salt tolerance between Hord-
eum vulgare and H. jubatum in whole plants and callus
cultures. Zeitschrift fur Pflanzenphysiologie 98:105–118
Paek KY, Chandler SF, Thorpe TA (1988) Physiological
effects of Na2SO4 and NaCl on callus cultures of
Brassica campestris (Chinese cabbage). Physiol Plant
72:160–166
Pushpalatha T, Padmanabhan C (1998) Studies on in vitro
testing for salt tolerance in callus cultures of rice (Oryza
sativa L.). Current Agric 22:109–113
Pushpam R, Rangasamy SRS (2000a) Effect of salinity on
protein and proline content of callus and seedlings of rice.
Crop Res 20:192–196
Pushpam R, Rangasamy SRS 2000b In vitro response of rice
genotypes to salt stress. Madras Agric J 87:694–697
Reddy PC, Vajranabhaiah SN (1996) The role of polyamines,
ATP, kinetin and selection in stress tolerance of rice
(Oryza sativa L.) calli. Adv Plant Sci 9:143–147
Rudulier DL, Strom AR, Dandekar AM, Smith LT, Valentine
RC (1984) Molecular biology of osmoregulation. Science
224:1064–1068
Santos CL, Dos V, Caldeira G (1999) Comparative responses
of Helianthus annuus plants and calli exposed to NaCl. I:
growth rate and osmotic regulation in intact plants and
calli. J Plant Physiol 155:769–777
Santos-Diaz MS, Ochoa-Alejo N (1994a) Effect of water stress
on growth, osmotic potential and solute accumulation in
cell cultures from chili pepper (a mesophyte) and creosote
bush (a xerophyte). Plant Sci 96:21–29
Santos-Diaz MS, Ochoa-Alejo N (1994b) PEG tolerant cell
clones of chili pepper: growth, osmotic potential and
solute accumulation. Plant Cell Tiss Organ Cult 37:1–8
Sathish P, Gamborg OL, Nabors MW (1997) Establishment of
stable NaCl-resistant rice plant lines from anther culture:
distribution pattern of K+/Na+ in callus and plant cells.
Theo Appl Gene 95:1203–1209
Shah SH, Wainwright SJ, Merrett MJ (1990) The interaction of
sodium and calcium chlorides and light on growth,
potassium nutrition, and proline accumulation in callus
cultures of Medicago sativa L. New Phytol 116:37–45
Shankhdhar D, Shankhdhar SC, Mani RC (2000) In vitro
selection for salt tolerance in rice. Biol Plant 43:477–480
Plant Growth Regul (2007) 53:53–63
Shibli RA, Al-Juboory K (2002) Comparative responses of
‘‘Nabali’’ olive microshoot, callus and suspension cell
cultures to salinity and water deficit. J Plant Nut 25:61–74
Singh BR, Singh DP (1995) Agronomic and physiological re-
sponses of sorghum, maize and pearl millet to irrigation.
Field Crop Res 42:57–67
Smith RH, Bhaskaran S, Newton R, Miller F (1984) Sorghum
varieties screened in vitro for osmotic tolerance and
physiological studies. Plant Physiol 75:174
Stavarek SJ, Rains DW (1984) The development of tolerance
to mineral stress. Hort Sci 19:377–382
Stephen RG, Zeng L, Shannon MC, Roberts SR (2002) Rice is
more sensitive to salinity than previously thought. Annual
Report of U.S. Department of Agriculture’s Research
Service, p 481
Taiz L, Zeiger E (2002) Plant physiology, 2nd edn. Sinauer
Associates, Inc., Sunderland, MA
Thomas JC, Armand RL, Bohnert HJ (1992) Influence of NaCl
on growth proline and phosphoenol pyruvate carboxylase
level in Mesembryanthemum crystallinum suspension
cultures. Plant Physiol 98:626–631
Turner NC, Jones MM (1980) Turgor maintenance by osmotic
adjustment: a review and evaluation. In: Turner NC,
Kramer BJ (eds) Adaptation of plants to water and high
63
temperature stress. Willey Interscience, New York, pp
89–103
Vanekamp JH, Lampe JEM, Koot JHM (1989) Organic acids
as source of drought-induced proline synthesis in field
bean (Vicia faba L.) plants. J Plant Physiol 133:654–659
Willadino L, Camara T, Boget N, Claparols I, Santos M, Torne
JM (1996a) Polyamine and free amino acid variations in
NaCl-treated embryogenic maize callus from sensitive
and resistant cultivars. J Plant Physiol 149:179–185
Willadino L, Camara T, Boget N, Claparols I, Santos M, Torne
JM (1996b) Polyamine variations in sensitive embryo-
genic callus of maize as a response to NaCl stress. Revista
Brasileira de Fisiologia Vegetal 8:161–164
Xu SC, Dai JV, Shen XY, Wang LZ, Cui Q, Zhu YL (1995)
The effect of water stress on the photosynthetic charac-
teristics and yield of maize. Acta Agronomica Sinica
21:356–363
YanJun S, GuangMin L, ChunYan X (2000) Effect of water
stress on the callus of winter wheat with different drought
resistance. Acta Agriculturae Boreali-Sinica 15:47–52
Yoshida S, Kasai Y, Watanabe K, Fujino M (1999) Proline
stimulates albino regeneration from anther- and seed-derived
rice callus under high osmosis. J Plant Physiol 155:107–109
123