Journal of Medicinal Plants and By-products (2017) 2: 153-163
Original Article
In vitro Response of Asparagus breslerianus to NaCl
Seyyed Javad Mousavizadeh*, Mohammad Reza Hassandokht and Abdolkarim Kashi
Department of Horticultural Sciences, University College of Agriculture and Natural Resources, University of
Tehran, Karaj, Iran.
Article History: Received: 21 July 2016 /Accepted in revised form: 09 January 2016
© 2013 Iranian Society of Medicinal Plants. All rights reserve
Abstract
Asparagus breslerianus a wild species in Iran, exhibited tolerance to salt in dry gypsum hills and dry
lands. In order to check for salt tolerance threshold via in vitro conditions, the A. breslerianus callus was
subjected to NaCl (sodium chloride) treatments. Six weeks old calli derived from male spear bud, were exposed
to 0, 21.88, 43.76, 65.64, 76.58, 87.52, 109.40, 131.28, 153.16 and 175.04 mM NaCl in MS (Murashige and
Skoog) basal medium supplemented with 0.88 µM BA (6-Benzylaminopurine) and 1.07 µM NAA (1Naphthalene acetic acid). According to results, friable and compact type, green and white green calli were
obtained up to 87.52 mM NaCl. Soft type, yellow and cream calli were observed with increasing salinity further
109.4 mM NaCl. Calli growth and plantlets regeneration were high in media up to 109.4 mM NaCl. In term of
mineral accumulation, sodium content increased with an increase in NaCl levels. It indicates that A.
breslerianus calli could acclimatize to salt stress by high osmotic adjustment. These suggested that, under in
vitro salt-induced osmotic stress, A. breslerianus is reflected as salt resistant which tolerate NaCl up to 109.4
mM.
Key words: Asparagus, Salt tolerance, Callus type, Callus growth
Introduction
Asparagus L. species (Asparagaceae family) in
compare to other vegetable crops, has high capacity
to salt tolerance [1], and it is divided into halophyte
plants [2]. It was previously stated that A.
officinalis L. seeds capable to germinate in water
salinity up to 9.4 dSm-1 [3]. Even applications of
NaCl stimulated earliness, total yield, marketable
yield and number of spears in A. officinalis [4].
In vitro salinity-induced cultures are preferable
tool for explore stress mechanism [5-6], which are
unconnected to external environment. The
investigation on asparagus in vitro salt stress was
focused on A. officinalis [7-8], and A. maritimus
Pall. [9].
There is a special species in Asparagus family
named A. breslerianus it is infrequent that habits in
dry gypsum hills and dry lands [10]. It was
mentioned A. breslerianus as a rare and saline
habitat species, which need preservation [11].
Recently a wild population of A. breslerianus has
been investigated form Iran and their plants have
shown different morphological characters in
compare to other Asparagus species [12].
In our previous study, octoploid level (2n=8x=80)
was identified by flow cytometry in A. breslerianus
[13]. A distinct polyploid series of diploid (2x),
tetraploid (4x), hexaploid (6x) and even
dodecaploid (12x) species were previously reported
in the Asparagus genus [14-15-16-17]. A.
breslerianus was early reported as diploid [18].
Octoploid level in this species is not occurring very
often [13].
Because of both salt tolerance capacity and ploidic
level, A. breslerianus germplasm should be
conservation in gene banks or botanical gardens.
This can be more important if we know that they
Corresponding author: Department of Horticultural Sciences, University College of Agriculture and Natural Resources,
University of Tehran, Karaj, Iran
Email Address: mousavizadeh@ut.ac.ir
154
Journal of Medicinal Plants and By-products (2017) 2: 153-163
are in risk of disappear due to livestock grazing and
erosion [12].
The objective of the present study was in vitro
indices of NaCl using callus culture to broad
information about salt tolerance mechanism in
this halophytic vegetable.
Material and Methods
Plant Material
Young spears (15-20 cm) of male gender of wild
Asparagus breslerianus Schult. & Schult.f.,
emerging in the spring were gathered from natural
zone of Iran (36° 01′ N and 56° 02′) as declared in
the Flora of Iran [19-10].
Media and Culture Condition
Vessels used for the culture were jam jar with a
capacity of 30 ml MS basal medium (Murashige
and Skoog). Ten NaCl treatments based on local
soil EC (7.2 dSm-1) carry out for salt stress
responsive. For this aim, media supplemented with
0, 21.88, 43.76, 65.64, 76.58, 87.52, 109.40,
131.28, 153.16 and 175.04 mM NaCl. Then,
uniform size calli (diameter ranking from 8 to 10
mm, derived from spears bud of A. breslerianus)
were individually weighed and were transferred to
media along with 0.88 µM BA and 1.07 µM NAA
as used for callus growth and development. The
cultured media were incubated in white fluorescent
light (illumination at 3000 Lux) in a growth room
at 25± 2 oc. The developed calli were inoculated on
the same MS medium to evaluate their response on
vegetative bud multiplication potential. Induced
shoots were subcultured on the same medium
fortified with 3.9 µM Ancymidol and 1.07 µM
NAA for rooting [9]. The multiple shoots with root
were subcultured for rapid clonal multiplication.
The derived plants were acclimated in pots
containing cocopeat: perlite: vermiculate (1:1:1)
and maintained in growth chamber under
controlled conditions with fluorescent light and a
photon flux of approximately 3000 Lux, at 25±2
°C, under a 16‑ hour photoperiod for eight weeks.
Callus Growth, Plantlet Regeneration and Number
of Shoot Initiated
The frequency of calli formation per explants was
recorded through average samples after four weeks
of culture. The plantlet regeneration capacity was
determined after six weeks, as the frequency of
calli that showed shoot on their surface. Shoots per
callus were counted and recorded as number of
shoot initiated.
Chlorophyll Content
Chlorophyll a, b and ab content were extracted by
acetone 80%, from 1 g of calli. The amount of
these pigments were measured by Arnon (1956)
method spectrophotometerily (UV/Vis 2100) at 645
and 663 nm and expressed as mgg-1 fresh weight
[20].
Dry Matter (DM) and Ash Percentage
Fresh weight of calli were determined. The calli
were dried in an oven at 70oC for 48 h and dry
matter measured. Dry matter and ash content were
expressed in percentage of fresh weight and dry
matter basis, respectively, according to the
following formula:
DM = Dry matter/Fresh weight×100
Ash = Ash weight/Dry matter×100
Callus Relative Growth Rate (RGR)
Callus relative growth rate (RGR) was determined
on fresh weight (FW). The RGR of the callus was
calculated as:
RGR = [In (final weight) – In (initial weight)]/day
[21].
The calli were harvested from saline medium after
54 days of treatments and fresh weight of the callus
tissue calculated. The RGR was expressed as mg
per fresh weight of callus.
Index of Tolerance (IT)
To compare callus responses to different levels of
stress condition, an index of tolerance (IT), based
on RGR was calculated according to the following
formula:
IT = RGRtreatment/RGRcontrol [21].
Relative water content (RWC)
Callus relative water content (RWC) was calculated
as a percentage of fresh weight. The callus water
content was calculated as:
RWC = (Fresh weight – dry matter) /dry matter ×
100 [18].
K+ and Na+ analysis
K+ and Na+ concentrations were determined by ash
at 550 °C for 24 h, dissolving the ash in 10 ml HCl
2 M, and assaying the solution obtained using a
flame photometer [22].
Experimental Design and Statistical Analysis
155
Data were analyzed statistically using analysis of
variance in a completely randomized design with
10 treatments (10 levels of NaCl) and three
replications (Three jars from each treatment with
three explants in each jar). The data of number of
shoot initiated were normalized by root square
( X ). The regression model was fitted to the
data using the Proc Reg of SAS. The changes of
traits over long-term simulation versus salt
condition were describable using polynomial
regression model (y = a + bx + cx2). In one case for
the K+ and Na+ content a simple, linear regression
model (y = a + bx) was used. Differences among
the means were determined for significance at
P<0.05 using least significant difference (LSD 5%)
test and the system program SAS 9.1 software
(SAS Institute Inc., Cary, NC, USA).
Results and Discussions
Callus Type
Different callus textures (compact, hard, soft and
friable) were evaluated after four weeks. Colors of
callus were also noted. Calli without any obvious
organ regeneration are typically called friable or
compact depending on their tissue characteristics
[23]. In this experiment, callus texture was
generally observed either compact or friable (Fig.
1, a and b). Independent of the media NaCl, four
morphological different callus types could be
distinguished: 1) a friable, type with green, white
green colored (Fig. 1, a), 2) a compact, hard, friable
type, with green or white green colored, 3) a
compact, hard type with white green, cream
colored (Fig. 1, b), 4) a compact, soft type with
cream, yellow colored either surrounded with water
(Table 1). Data in Table 1 showed that up to 100%
of the calli were formed compact and in media
including media with 0 to 87.52 mM NaCl had
friable callus. Soft type, yellow and cream colored
calli were observed with increasing salinity in more
than 109.4 mM (Table 1). These results are in
agreement with Odyssea paucinervis callus growth
in salt condition [24]. Soft, friable and cream
colored calli were reported in Salvadora persica
under NaCl stress condition [25].
Dry Matter (DM), Relative Water Content (RWC)
and Ash Percentage
Considerable variations were observed in callus
DM, RWC and ash among salinity conditions
Mousavizadeh et al.
culture. Results (Table. 2) showed that there was a
significant difference among 10 applied NaCl
concentrations for DM, RWC (P<0.01), and ash
percentage (P<0.05).
As seen in Fig. 2, the ash was increased until 87.52
mM NaCl. Then, ash decreased with NaCl rising to
175.04 mM. Based on X2 and X slope, ash was
increased rapidly. On the other hand, Aparagus
callus ash at different media NaCl was rapidly
raised and then slowly reduced than 87.52 mM. As
shown in Fig. 3, the highest callus DM% was
obtained in 131.28 to 175.04 mM. Below the 87.52
mM by rising DM, ash increased, but up to 87.52
mM, ash decreased by DM decreasing. The
remarkable point is that DM has not decreased with
increasing salinity. May be Na+ accumulation at
high concentrations of salt, increases the callus
density, and increased DM at salt levels. Calli dry
matter of A. officinalis cv. UC.157 gradually
increased as salt concentration increased up to 6000
ppm salt mixture (3 NaCl:1 (3 MgCl2:1 CaCl2)) [8].
The increase in callus dry matter under salinity
stress has already been observed in rice [26],
tomato [27] and Salvadora persica [25].
As seen in Fig. 3, the RWC response towards salt
increase was high until 109.4 mM NaCl. Then,
RWC decreased with NaCl rising to 175.04 mM.
Aparagus callus RWC at different media NaCl was
slowly reduced than 109.4 mM. Results of
correlation coefficient showed that a significantly
negative correlation exists between callus RWC
and DM%, and positive correlation exists between
callus RWC and callus growth, callus volume,
plantlet regeneration, number of shoot initiated, K+
and RGR (Table 3). RWC at highest salt stress
(175.04 mM NaCl) five percent decreased
compared to control (Fig. 3). Asparagus with
decreasing water potential and osmotic adjustment
reduces injuries under drought stress [28]. In
another plant for example in sugarcane, callus
growth and water content decreased under NaCl
stress [29].
Callus Growth, Callus Volume and Plantlet
Regeneration
Callus formation was started after one week of
culture. Significant differences were recorded
(Table. 2) among NaCl concentrations for callus
growth percentage (P<0.05), callus volume
(P<0.01) and plantlet regeneration percentage as
regeneration capacity of callus (P<0.01).
Journal of Medicinal Plants and By-products (2017) 2: 153-163
156
Table 1 Callus form of Asparagus breslerianus Schult. & Schult.f. in NaCl treated media.
NaCl
concentration (mM)
0
21.88
43.76
65.64
76.58
87.52
109.4
131.28
153.16
175.04
Callus color
Callus type
White green
White green
Green, White green
Green, White green
White green
White green
White green
Yellow, Cream
Yellow, Cream
Cream
Friable, Compact
Friable, Compact
Friable, Compact
Compact, Hard, Friable
Compact, Hard, Friable
Compact, Hard, Friable
Compact, Hard
Compact, Hard
Compact, Soft
Compact, Soft
Fig. 1 Asparagus breslerianus callus subjected to NaCl concentration after six week of culture in MS medium
supplemented with 0.88 µM BA and 1.07 µM NAA.
(a) White green and friable callus derived from medium under 43.76 mM NaCl.
(b) Yellow, cream and compact, hard type callus derived from medium under 131.28 mM NaCl.
(c) Shoots initial and development after eight weeks of culture in medium under 65.64 mM NaCl.
(d) High shoot development after eight weeks of culture in medium under 87.52 mM NaCl.
(e) Shoots initial and development after eight weeks of culture in medium without NaCl (control).
(f) Root development after shoot initiated and subculture in medium containing 76.58 mM NaCl supplement with 3.9 µ M
Ancymidol and 1.07 µM NAA for eight weeks.
Table 2 Analysis of variance of callus culture of Asparagus breslerianus Schult. & Schult.f. in NaCl treated media.
S.O.V
df
Dry matter
Salt
9
10.9**
Error
20
3.01
CV%
26.36
**
(P<0.01), * (P<0.05).
Continue Table 2
S.O.V
df
Chlorophyll
b
Salt
9
1.56**
Error
20 0.165
CV%
27
**
(P<0.01), * (P<0.05).
Ash
RWC
Callus
induction
Callus
volume
Bud
induction
46.88*
16.95
24.61
10.9**
3.01
1.85
242.3*
92.33
10.17
18.34**
2.82
23.5
5828.3**
192.6
21.96
Chlorophyll
ab
Na+
K+
1.21**
0.128
27.02
327.17**
7.17
8.72
145.3**
10.39
23.18
Callus
relative
growth rate
90.56**
5.06
13.69
Number
of shoot
initiated
22.91**
1.13
29.76
Index
tolerance
0.25**
0.01
13.69
Chlorophyll
a
1.14**
0.118
30.02
of
Number of
rooted
plants
288.6**
10.70
28.36
Mousavizadeh et al.
157
Fig. 2 Ash and dry matter (DM) content of Asparagus breslerianus callus subjected to NaCl treated media. Bars represent
SD values.
Fig. 3 Relative water content (RWC) of Asparagus breslerianus callus subjected to NaCl treated media. Bars represent SD
values.
Callus growth and plant regeneration percentage
were in maximum level in media with 0 to 109.4
mM NaCl. In opposite media from 131.28 to
175.04 mM NaCl had the lowest callus growth
without any plant regeneration percentage (Fig. 4).
A very high shoot multiplication rate was attained
on media exposed with 0 to 109.4 mM. Shoots and
leaves were regenerated into young plantlets (Fig.
1, c). Based on correlation coefficient results,
significant correlation observed between callus
growth and plantlet regeneration with DM, RWC,
callus volume, number of shoot initiated, K+ and
RGR (Table 3). Callus volume in medium without
NaCl (0 mM) was high. Callus volume in the
media that subjected to NaCl from 21.88 up 175.04
mM, were significantly decreased (Fig. 5).
Negative correlation observed between callus
volume with DM and Na+. Also, significant
correlation observed between callus volume with
RWC, callus growth, plantlet regeneration, number
of shoot initiated, K+ and RGR (Table 3). It was
reported that in vitro shoot production and growth
of A. officinalis were inhibited at 2% NaCl [7].
Mahon-Demias, a genotype of bread wheat
(Triticum aestivum), reacts moderately decrease in
the capacity of callus proliferation which reached
25% at 15 g·L −1 NaCl treatment [30]. Plantlet
regeneration of BRRI dhan genotype of Rice was
80, 20 and 0 % at 0, 100 and 150 mM NaCl,
respectively [31].
Number of Shoot Initiated
Number of shoot initiated was affected
significantly (P<0.01) with the increasing level of
salt in the cultural medium (Table 2). Maximum
158
Journal of Medicinal Plants and By-products (2017) 2: 153-163
shoot initiated was recorded in medium that
supplemented with 87.52 mM NaCl (Fig. 1, d).
There was not any shoot formation in NaCl from
131.28 to 175.04 mM (Fig. 6). Significant
correlation observed between number of shoot
initiated with DM, RWC, callus growth, plantlet
regeneration, callus volume, K+ and RGR.
Correlation
between
plantlet
regeneration
(regeneration capacity of callus) and number of
shoot initiated clearly confirms that these
characteristics are dependent together (Table 3).
The results stated that low content of neutral salt
stress was useful for growth of A. breslerianus
which also was reported previously in asparagus
[1]. The highest number shoots in A. officinalis
proliferated at 2000 ppm salt mixture (3 NaCl:1 (3
MgCl2:1 CaCl2)) in comparison to control cultures
[32]. Also, a positive association between
photosynthesis and yield under saline conditions
has been found in A. officinalis [18].
K+ and Na+ Content
Considerable variations (P<0.01) were observed in
K+ and Na+ among salinity condition culture (Table
2). As seen in Fig. 7, the Na+ content was increased
and the K+ content was decreased by NaCL
application. The lowest K+ content was obtained at
175.04 mM NaCl. The maximum accumulation of
Na+ was observed at 175.04 mM NaCl. Finding
results agree with some species were able to
substitute K+ by Na+ to guarantee the osmotic
adjustment which also was reported previously in
Medicago sativa L., [21] and Saccharum sp. [29].
Salinity stress increased the rice callus sodium
content (Na+) while potassium (K+) content
decreased [26]. Both Na+ and Cl- increased with
salinity treatment in tissues of friable and compact
callus cells of asparagus [7]. Potassium has an
important role in the plants biochemical and
physiological mechanisms [33]. Below common
conditions, plant cells require 100 - 200 mM K+
and less than 1 mM Na+ to maintain the osmotic
balance [34]. Under salt stress, halophytes
subjected to high salinity conditions accumulate
high content of Na+ undergo osmotic adjustment
[33]. Nutritional imbalance such as Na+ and Clmay cause a reduction in callus growth under salt
condition [29].
Chlorophyll Content
Significant differences (P<0.01) were recorded for
chlorophyll a, b and ab content in response to NaCl
treatments (Table 2). Previous researchers reported
that 100 mM NaCl did not affect on chlorophyll
content of in vitro propagated shoots of Eucalyptus
camaldulensis [39]. According to Fig.8,
Chlorophyll a, b and ab content were obtained a
decreasing trend in first levels of NaCl (21.88 and
43.76 mM) and then increased up to 109.4 mM.
The chlorophyll was declined with NaCl rising
from 131.28 to 175.04 mM. On the other hands, the
results demonstrated that chlorophyll was declined
slowly by lowest (21.88 and 43.76 mM) and
exactly by highest (131.28 to 175.04 mM) levels of
NaCl. Significant correlation observed between
chlorophyll content with DM, RWC, callus growth,
plantlet regeneration, callus volume, number of
shoot initiated, K+ and RGR (Table 3). Correlation
between chlorophyll content and K+ clearly
confirms that chlorophyll may have vital character
in osmotic adjustment in A. breslerianus under salt
stress. Close correlation between salt tolerance and
chloroplasts has been found. So that chlorophyll
content increased in the resistant cultivar [35]. At
maximum level of NaCl, breakdown of chlorophyll
occurs mainly due to Cl accumulation in tissue [3637]. Chlorophyll content could be applied to as a
sensitive index of cellular that exposed to salt
condition; therefore, chlorophyll decrease means
toxicity in tissues due to accumulation of Na+ and
Cl- [38], so, A. breslerianus is tolerant to salinity
up to 109.4 mM NaCl (Fig. 8).
Relative Growth Rate (RGR)
Based on results (Table 2) and Fig. 9, RGR
significantly (P<0.01) decreased with NaCl rising
to 109.4 mM. Calli RGR at different media NaCl
were reduced over 109.4 mM. A significantly
negative correlation was observed between callus
RGR and DM%, and positive correlation was
obtained between callus RGR and RWC, callus
growth, callus volume, plantlet regeneration,
number of shoot initiated and K+ (Table 3). The
NaCl influenced the RGR value that is mostly
might be due to the selective accumulation of Na+
ion. With agreement to these results, callus relative
growth rate (RGR; fresh) of rice indicated a
progressive decrease in salt condition [26]. A
significant decrease reported in RGR of Sesuvium
portulacastrum L. calli in salt levels [33]. Any
increase in salinity levels in tomato media was led
to decrease of calli RGR [27].
Mousavizadeh et al.
159
LSD 5% = 16.36
LSD 5% = 23.64
Fig. 4 Callus growth and plant regeneration percentage of Asparagus breslerianus subjected to NaCl treated media.
LSD 5% = 2.86
Fig. 5 Callus volume of Asparagus breslerianus subjected to NaCl treated media.
LSD 5% = 24.09
Fig. 6 Number of shoot initiated of Asparagus breslerianus callus subjected to NaCl treated media.
Journal of Medicinal Plants and By-products (2017) 2: 153-163
160
Fig. 7 K+ and Na+ content of Asparagus breslerianus callus subjected to NaCl treated media. Bars represent SD values.
Fig. 8 Chlorophyll a, b and ab content of Asparagus breslerianus callus subjected to NaCl treated media. Bars represent SD
values.
Fig. 9 Relative growth rate (RGR) of Asparagus breslerianus callus subjected to NaCl treated media. Bars represent SD
values.
Mousavizadeh et al.
161
Fig. 10 Index of tolerance (IT) of Asparagus breslerianus callus subjected to NaCl treated media. Bars represent SD values.
Fig. 11 Number of rooted plants of Asparagus breslerianus in vitro shoot initiated subjected to NaCl treated media.
Table 3 Pearson correlation coefficients among traits of Asparagus breslerianus callus culture in salinity condition.
1
Plant
regenera
tion
-
Number
of shoot
initiated
-
0.69**
0.68**
1
-
0.43*
0.71**
0.65**
Traits
DM
Ash
RWC
Callus
induction
Callus
volume
DM
Ash
RWC
Callus induction
Callus Volume
plant
regeneration
number of shoot
initiated
Na+
K+
RGR
Chl ab
1
-0.09ns
-1**
-0.38*
-0.70**
1
-0.09ns
-0.01ns
0.02ns
1
0.38*
0.70**
1
0.64**
-0.72**
0.07ns
0.72**
-0.60**
0.29ns
0.60**
ns
*
ns
0.31
0.39
-0.31
-0.57**
0.03ns
0.57**
**
ns
-0.69
0.31
0.69**
**
ns
-0.53
-0.02
0.53**
**
*
ns
(P<0.01), (P<0.05), (P>0.05).
ns
-0.29
0.68**
0.64**
0.74**
*
- 0.45
0.55**
0.68**
0.56**
ns
-0.34
0.82**
0.80**
0.82**
1
ns
-0.14
0.46**
0.56**
0.48**
Na+
K+
RGR
Chl
ab
-
-
-
-
-
-
-
-
-
-
-
-
1
- 0.42*
-0.28ns
-0.34ns
1
0.7**
0.64**
1
0.76**
1
162
Journal of Medicinal Plants and By-products (2017) 2: 153-163
Index of Tolerance (IT)
The index of tolerance (IT) revealed significant
main effect (P<0.01) for NaCl (Table 2). As seen in
Fig. 10, IT decreased with NaCl rising to 109.4
mM. Aparagus callus IT at different media NaCl
was reduced over 109.4 mM. Based on results,
RGR and IT correlated with K+ content (Table 3).
This clears that salt tolerance in A. breslerianus
enhanced by K+ uptake efficiency. IT index is used
in order to measure the sensitivity of plants to salt.
The less or negative the index indicates the
susceptibility of plant to salinity. Index of tolerance
(IT) is suitable for comparing responses to stress
[21].
Rooted Plants and Acclimation
Root induction was undertaken using 3.9 µM
Ancymidol and 1.07 µM NAA. Using of
Ancymidol was proposed for rooting of Asparagus
maritimus [9]. Also, Ancymidol (2 mg 1-1), KIN
0.1 (mg 1-1) and NAA (0.3 mg 1-1) were used for
induction of root in explants of asparagus wild
species [16]. Initiated shoots from calli were
transferred to MS medium containing the same
NaCl concentrations suppliment with 3.9 µM
Ancymidol and 1.07 µM NAA for rooting (Fig. 1,
f). Rooted plants were formed until 109.4 mM
NaCl after eight weeks growth periods on media.
The acclimatization asparagus rooted plants was
better when the micropropagated plantlets were
acclimatized after eight weeks incubation [32]. The
highest number of rooted plants was recorded at
control, 43.76 and 65.64 mM NaCl (Fig. 11). In the
media with 131.28, 153.16 and 175.04 mM NaCl
were not observed any rooted plants. During the
acclimation phase, a high survival rate was
achieved after six weeks in the growth chamber. It
is deserving to quote that the transfer of rooted
plantlets from aseptic growth chamber to an
external environment had always to be done
carefully. At the end a high multiplication rate can
be achieved by this technique.
Conclusion
NaCl induced elevation in callus dry matter (DM),
which clarify a cellular tolerance to salinity in A.
breslerianus. Relative water content (RWC) and
relative growth rate (RGR) of callus tissue were
found to be inversely correlated with DM and
directly correlated with K+ uptake in salt condition.
The highest RGR and RWC decreasing were
noticed less than 109.4 mM NaCl. These
consequences suggested that RGR restriction may
be due to the water availability being made fewer
and loss of turgor in cells. Loss of water, but
increased dry matter showed high metabolic
activity. It is notable that A. breslerianus callus at
high concentrations of salt is also able to survive
via the increase in DM (up to 10.26%), chlorophyll
content (up to 2.01 mgg-1 FW for chlorophyll ab),
preservation of the RWC (up to 89.73%) and K+
accumulation (41.64 mgg-1 DM). Our results
recommended that under in vitro salt stress
condition, the accumulation of Na+ and keeping of
a satisfactory water level can have vital characters
in osmotic adjustment in A. breslerianus as a
halophytic species.
Acknowledgements
This work has been supported by the University of
Tehran.
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