4142 in Biosciences 8(16), Print : ISSN 0974-8431,
Trends Trends in Biosciences
4142-4147, 2015 8 (16), 2015
Effect of Salicylic Acid on Germination of Phaseolus vulgaris and Cicer
arietinum Under Salt Stress
ONKAR GROVER, INDU GAUR, JOYEETA MITRA AND P. K. PAUL
Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector 125,
Gautam Buddha Nagar, NOIDA, India
email: onkar_grover@yahoo.co.in; indugaur03@gmail.com; joyeetamimi@gmail.com;
prabir_kp@rediffmail.com
ABSTRACT
Plants respond to salt stress through changes in
physiological and metabolic characteristics. Salicylic
acid acts as growth promoter in plants during various
developmental stages. The objective of present study
was to demonstrate the effect of three salt
concentrations (0%, 0.85% and 3.0% NaCl) on
germination percentage, germination rate, radicle
and hypocotyl length of kidney bean (Phaseolus
vulgaris) and chickpea (Cicer arietinum). Role of
salicylic acid (0, 1.5, 3.0 and 4.5 mg/l) on above
parameters for both seed varieties, with and without
exposure to salt stress was also investigated. Results
demonstrated a significant delay in the germination
process and elevation in total soluble cytoplasmic
protein concentration with increase in salinity, which
was significantly improved by application of salicylic
acid up to a specific concentration (3.0 mg/l).
Therefore, salicylic acid has high potency of balancing
the damage caused by salt stress during germination.
Key words Salt stress; Germination; salicylic acid;
Cytoplasmic proteins; Kidney bean;
Chickpea.
Salinity is one of the most serious factors
limiting the productivity of agricultural crops, with
adverse effects on germination, plant vigour and
crop yield. Worldwide, more than 45 million
hectares of irrigated land have been damaged by
salt, and 1.5 million hectares are taken out of
production each year as result of salinity (Munns,
and Tester, 2008). Exposure to salt stress alters
various metabolic processes in plant, leading to
reduction of cell division and expansion (Hasegawa,
et al., 2000; Munns, 2002; Zhu, 2007). These
effects reduce overall plant growth, development
and survival probability.
Germination is critical part of plant life. The
ability of seeds to germinate at high salt
concentration in soil is of crucial importance for
the survival and growth of plant. Seed germination
and seedling growth are known to be more sensitive
to salt stress compared with later developmental
stages (Ashraf, 1994). During germination the
stored protein from cotyledons is hydrolysed to
provide nutrition to the emerging plantlet. Therefore
it is important to understand the impact of salt stress
on this physiological process since any alteration
would drastically impact the development of
embryo to a seedling. Also, a treatment with
capability of improving the seed germination and
its subsequent establishment would enhance the
crop yield.
Salicylic acid (SA), a plant based phenolic
compound is considered as a potent endogenous
growth regulator due to its role in plant defense
mechanism(s) against several biotic and abiotic
stress. Intracellular SA concentration and SA
induced signalling pathway(s) are associated with
controlling cell growth, cell death and defense
(Chen, et al., 2001). Application of SA in various
crops is associated with enhanced resistance of
plant towards drought and salt stress through
activation of antioxidative mechanism(s). Studies
have shown that inclusion of small concentration
of SA in growth medium improves the germination
process in plants under salt stress. Hence, the role
of SA in improving the germination of two
important seed varieties in present study was
investigated by analysing various parameters
associated with germination process.
MATERIALS AND METHODS
Plant material
Seeds of kidney bean and chickpea were
procured and surface- sterilised using 0.6% (w/v)
sodium hypochlorite solution for 30 seconds
followed by rinsing twice with sterile distilled water.
 GROVER et al., Effect of Salicylic Acid on Germination of Phaseolus vulgaris and Cicer arietinum Under Salt Stress 4143

Table 1. Effect of three NaCl concentrations (%w/v) on germination of kidney bean at four
time intervals (hours post treatment). The values represent mean of data collected
from three replicates.
GP GR RL HL
Sampling hours Sampling hours Sampling hours Sampling hours
NaCl (%) 4 24 48 72 4 24 48 72 4 24 48 72 4 24 48 72
0 0 56 66 80 0 32 36 39.5 0 2 8 14 0 0 1 4
0.85 0 30 40 54 0 20 23.3 26.3 0 1 5 6 0 0 0 1
3.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
 GP- Germination percentage; GR- Germination rate; RL- Radicle length; HL- Hypocotyl length

Treatments and germination rate (GR) were calculated for both

varieties as mentioned below:
Dried seeds of both varieties were treated
separately under following conditions- GP = (Number of seeds germinated/total
(a) NaCl concentration- 0.85 and 3.0% (w/v) number of seeds) x 100
(b) SA concentration- 1.5, 3.0 and 4.5 mg/l GR = X1/Y1+(X2-X1)/Y2+…+ (Xn-Xn-1)/
(c) Combination of NaCl and SA- Yn
0.85% (w/v) NaCl+1.5 mg/l SA Where Xn = GP on Nth day and Yn = number
0.85% (w/v) NaCl+3.0 mg/l SA of day from first day of treatment (Mguire, 1962).
3.0% (w/v) NaCl+1.5 mg/l SA Also, radicle and hypocotyl length of both
3.0% (w/v) NaCl+3.0 mg/l SA seed varieties were measured. Three replicates were
taken for each variety and its respective treatment.
(d) Control- Sterile distilled water
Isolation of Cytoplasmic Proteins
Seeds were soaked in plastic trays with 50
seeds each in all sets. The trays were kept under Seeds were crushed in sterilized pre-chilled
controlled conditions at 25±1ºC, RH 70% and 12h mortar. 50mg of the crushed seed was mixed with
(L/D) photoperiod. The seeds were treated with 400μL of extraction buffer containing 0.5M Tris
respective solutions every alternate day. chloride (pH 6.8), 2.5% Sodium Dodecyl Sulphate
Experimental set up was kept in triplicate. The (SDS), 10% glycerol and 5% â-mercaptoethanol
complete germination process was observed for (freshly added). The contents were vortexed for
two weeks from soaking in all sets. 5min followed by incubation at 4ºC for 30min. The
mixture was then centrifuged at 12,000xg for 5min
Sampling
at 4ºC. The supernatant was collected and protein
Germinating seeds of both varieties were concentration was estimated by Bradford method
collected randomly from all sets at interval of 4, (Bradford, 1976) using BSA as standard.
24, 48 and 72h. The germination percentage (GP)

Table 2. Effect of three NaCl concentrations (%w/v) on germination of chickpea at four time
intervals (hours post treatment). The values represent mean of data collected from
  three replicates.
GP GR RL HL
Sampling hours Sampling hours Sampling hours Sampling hours
NaCl
(%) 4 24 48 72 4 24 48 72 4 24 48 72 4 24 48 72
0 0 64 76 90 0 28 31.3 34.8 0 14 30 45 0 0 10 22
0.85 0 40 54 66 0 15 18.3 21.8 0 10 22 27 0 0 2 10
3.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

GP- Germination percentage; GR- Germination rate; RL- Radicle length; HL- Hypocotyl length
4144 Trends in Biosciences 8 (16), 2015

Table 3. Effect of four SA concentrations (w/v) on germination of kidney bean at four time
intervals (hours post treatment). The values represent mean of data collected from
three replicates.

GP GR RL HL
Sampling hours Sampling hours Sampling hours Sampling hours
SA(mg/l) 4 24 48 72 4 24 48 72 4 24 48 72 4 24 48 72
0 0 56 66 80 0 32 36 39.5 0 2 8 14 0 0 1 4
1.5 0 72 81 89 0 36 39 41 0 5 12 16 0 2 7 9
3.0 0 31 39 60 0 15.5 18.2 23.4 0 0 3 7 0 0 0 1
4.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
  Germination percentage; GR- Germination rate; RL- Radicle length; HL- Hypocotyl length
GP-

The data was statistically analysed for ANOVA
using general linear model procedure and least
square means test of statistical software SAS
(version 9.2). The data was represented as mean
values ± Standard error (SE) of the three replicates
for each treatment.
RESULTS AND DISCUSSION
Germination process
Variable NaCl concentration
The germination process of both varieties was
negatively impacted by salt stress. Seeds of kidney
beans soaked in distilled water showed maximum
germination with emergence of radicle and
hypocotyl within 72 hours of soaking. Seeds soaked
in 0.85% saline had a delayed germination compared
to those treated with distilled water but better than
those treated with 3.0% saline. Seeds soaked in
3.0% saline did not show any germination after 2
weeks of soaking (Table 1). The radicle and
hypocotyl length was decreased with increase in
salinity. Seeds soaked in 3.0% salt concentration
did not show any radicle/hypocotyl emergence.
Similar results were obtained for chickpea seeds
(Table 2). However the germination percentage was
found to be higher as compared to that of kidney
bean seeds under all 3 soaked conditions at all
sampling intervals.
Salinity is reported to influence seed
germination by decreasing the water uptake due to
lowered osmotic potentials of the medium further
causing changes in metabolic activity, perturbing
plant hormone balance, and reducing the utilization
of seed reserves (Kaya, et al., 2006). In the present
study, high germination was observed in distilled
water treatment which gradually decreased with
increase in salt concentration (0.85% and 3.0%
respectively). There was no germination at 3.0%
salinity again highlighting that salt stress effect the
normal germination process by preventing the water
uptake. Similar observations were reported by
Farhoudi, et al. (2015) in soy bean seeds. Results
demonstrated a higher germination rate in chickpea
than kidney bean because of its thinner seed coat.
Variable SA concentration
SA was found to promote the germination

Table 4. Effect of four SA concentrations (w/v) on germination of chickpea at four time intervals
(hours post treatment). The values represent mean of data collected from three
replicates.
GP GR RL HL
Sampling hours Sampling hours Sampling hours Sampling hours
SA(mg/l) 4 24 48 72 4 24 48 72 4 24 48 72 4 24 48 72
0 0 64 76 90 0 28 31.3 34.8 0 14 30 45 0 0 10 22
1.5 0 72 84 93 0 36 40 42.3 0 14 33 52 0 3 16 28
3.0 0 29 45 61 0 15 19.8 23.8 0 3 7 14 0 0 5 17
4.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
 
GP- Germination percentage; GR- Germination rate; RL- Radicle length; HL- Hypocotyl length
GROVER et al., Effect of Salicylic Acid on Germination of Phaseolus vulgaris and Cicer arietinum Under Salt Stress 4145

Table 5. Effect of SA on germination of Kidney bean exposed to two salt concentrations at

four time intervals (hours post treatment). The values represent mean of data
collected from three replicates.
GP GR RL HL
SA
(mg/l)+NaCl( Sampling hours Sampling hours Sampling hours Sampling hours
%)
4 24 48 72 4 24 48 72 4 24 48 72 4 24 48 72
1.5+0.85 0 38 47 63 0 19 22 26 0 1 6 10 0 0 1 2
1.5 + 3.0 0 10 17 21 0 5 7.3 8.3 0 0 2 5 0 0 0 1
3.0 +0.85 0 29 40 52 0 14.5 18.2 21.2 0 1 4 8 0 0 0 1
3.0+3.0 0 5 9 13 0 2.5 3.8 4.8 0 0 1 3 0 0 0 0
 GP- Germination percentage; GR- Germination rate; RL- Radicle length; HL- Hypocotyl length

process as evident from the parameters under study varieties. However, the increase was more
Maximum germination rate was observed when prominent with 1.5mg/l treatment. Also, the
both seed varieties were treated with 1.5mg/l SA, response towards SA treatment against salinity was
followed by control and 3.0mg/l SA (Table 3, 4). more remarkable in kidney bean seeds compared
4.5mg/l SA showed total inhibition of seed to chickpea.
germination. Very high concentrations of SA
The present findings are in concordance with
(4.5mg/l) suppressed the germination rate of the
the previous studies. Borsani, et al., 2001 worked
seeds, with no development of radicle and
on Arabidopsis and reported that SA could increase
hypocotyl even after 2 weeks of germination.
the oxidative damage generated by NaCl. Similarly,
Previous work by Guan, and Scandalios, 1995
when low doses were applied exogenously, SA
(maize), McCue, et al., 2000 (garden pea),
could significantly improve Arabidopsis seed
Shakirova, et al., 2003 (wheat) and Hanieh, et al.,
germination from 50% to 80% under salt stress
2013 (sweet pepper) also supported the present
conditions (Rajjou, et al., 2006). Misra, and Saxena,
findings.
2009 observed that SA application could ameliorate
Combination of NaCl and SA the toxic effects generated from 100mM NaCl in
lentils and hence could be used as a potential growth
Since 4.5mg/l SA could not exhibit any
regulator to improve plant salinity tolerance.
growth to both the seed varieties, this treatment
was not taken into consideration for further studies. Protein concentration
Seed treatment with SA had positive effect on
Total soluble protein concentration of both the
germination under all saline conditions (Table 5,
seed varieties increased with increase in salinity (Fig.
6). SA at both concentrations (1.5mg/l and 3.0mg/
1) irrespective of the sampling time. Seeds soaked
l) enhanced the germination process in both seed

Table 6. Effect of SA on germination of chickpea exposed to two salt concentrations at four

time intervals (hours post treatment). The values represent mean of data collected
from three replicates.

GP GR RL HL
SA
(mg/l)+NaCl Sampling hours Sampling hours Sampling hours Sampling hours
(%)
4 24 48 72 4 24 48 72 4 24 48 72 4 24 48 72
1.5+0.85 0 38 57 69 0 19 25.3 28.3 0 9 24 37 0 0 2 11
1.5 + 3.0 0 27 39 47 0 13.5 17.5 19.5 0 6 14 20 0 0 1 7
3.0 +0.85 0 22 38 57 0 11 16.3 21.1 0 5 13 21 0 0 0 6
3.0+3.0 0 11 18 29 0 5.5 7.8 10.6 0 0 5 14 0 0 0 1
 
GP- Germination percentage; GR- Germination rate; RL- Radicle length; HL- Hypocotyl length
4146 Trends in Biosciences 8 (16), 2015
Fig. 1. Interactive effects of salinity and salicylic acid on germination percentage in kidney bean and
chickpea
SA concentration 1.5mg/l; SA concentration 3.0mg/l;
in 3.0% NaCl showed higher protein content
compared to those in distilled water (Pd”0.01) and
0.85% NaCl (Pd”0.05). In 1.5mg/l SA treated seeds,
there was initially dip in protein concentration as
compared to control. However, significant rise
(Pd”0.05) in soluble protein concentration was
observed in kidney bean and chickpea, when SA
concentration was increased to 3.0mg/l. Increased
SA levels against 0.85% and 3.0% NaCl stress
showed reduction in protein levels in both the
variety of seeds, although the protein levels were
higher as compared to control.
Total soluble protein content is reduced due
to increased activity of proteolytic enzymes,
resulting in degradation of abnormal or misfolded
proteins induced in response to stress. Under
hypersaline conditions, inefficient water uptake by
the plants severely impacts the functioning of these
enzymes resulting in the accumulation of misfolded
proteins (Hieng, et al., 2004). Together these
factors contribute to the increased protein
concentration in both seed varieties in 3.0% saline
condition.
Kidney bean possesses antioxidant,
antimutagenic, anticarcinogenic effects due to its
rich variety of phytochemicals (Heimler, et al.,
2005). Chickpea is a good source of essential dietary
components including vitamins and minerals
(Chibbar et al., 2010). It is evident from the current
SA concentration 4.5mg/
investigation that seed germination process in both
seed varieties, with potential beneficial effects on
human health, is influenced under salt stress which
results in delayed sprouting and significant change
in protein concentration. Also, SA up to a particular
concentration supports the two seed varieties to
overcome the salt induced stress thus improving
the overall germination process.
ACKNOWLEDGMENTS
The authors express their sincere thanks to
Amity Institute of Biotechnology, Amity University
Uttar Pradesh, NOIDA, India for providing the
necessary infrastructural facilities to carry out this
work.
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Received on 02-08-2015 Accepted on 08-08-2015