Journal of Medicinal Plants Research Vol. 5(16), pp.

3619-3628, 18 August, 2011

Available online at http://www.academicjournals.org/JMPR
ISSN 1996-0875 ©2011 Academic Journals

Full Length Research Paper

Isolation and characterization of NaCl-tolerant

mutations in two important legumes, Clitoria ternatea
L. and Lathyrus sativus L.: Induced mutagenesis and
selection by salt stress
Dibyendu Talukdar
Department of Botany, R. P. M. College (University of Calcutta), Uttarpara, Hooghly, 712258, West Bengal, India.
E-mail: dibyendutalukdar9@gmail.com.
Accepted 30 May, 2011

Six best performing mutants exhibiting NaCl-tolerance were identified in gamma ray induced M2
progeny of three different varieties of Clitoria ternatea L. and Lathyrus sativus L. each by preliminary
assessment of plant growth under salt stress. Salt tolerance of these six mutants, designated as CR1
and CR2 in C. ternatea and as LR1, LR2, LR3 and LR4 in L. sativus was evaluated by their responses to
22 different morpho-physiological and biochemical parameters under 130 mM NaCl treatment on 15-
day-old seedlings. A positive control set (mutant plants-watering only with distilled water) and a
negative control (unmutagenised mother variety- treated with 130 mM NaCl) was also maintained.
Among the six mutants, LR4 out performed all others showing significantly higher plant biomass
production, leaf K+/Na+ ratio and lower level of lipid perodixation and electrolyte leakage as compared to
positive control. It was closely followed by CR2, LR3 and LR2 seedlings. Enhanced salt tolerance of
these four mutants was attributed to increased activities of reactive oxygen species (ROS)-scavenging
enzymes-superoxide dismutase and ascorbate peroxidase along with non-enzymatic ascorbate and
carotenoids in leaves. Both CR1 and LR1 manifested moderate level of salt tolerance, but significantly
higher than negative control (NC) plants.

Key words: Induced mutation, NaCl-tolerance, growth, lipid peroxidation, antioxidant enzymes, Clitoria ternatea
L., Lathyrus sativus L.

INTRODUCTION

Clitoria ternatea L. or butterfly-pea belonging to family
Leguminosae is an important medicinal plant
commonly grown as a garden ornamental on fencerow.
It is an evergreen, herbaceous perennial legume
having slender, twinning stems with leaves of 5 to 7
leaflets elliptical to oblong in shape. Flowers are
usually solitary, axillary, bright blue or sometimes white
with a pale-yellow base. This plant has been used in
traditional as well as ‘Ayurvedic’ system of Indian
medicine for the treatment of chronic bronchitis,
dropsy, goiter, leprosy, mucous disorders, sight
weakness, skin diseases, sore throat and tumors (Jain
and De Filipps, 1991; Kulkarni et al., 1988). In Eastern
Himalayan states of India, its root power mixed with
water was taken orally in treatment of ascariasis and
fever, while root mixture with a milk product (‘ghee’) or
turmeric is generally used immediately after snake bite
(Dolui et al., 2004).
Lathyrus sativus L. or grass pea is one of the oldest
legume crops with cultivation period of more than 8000
years (Smartt, 1984). It has been widely used as ‘poor
man’s crop’ in Indian subcontinent, Australia, South
America, North Africa and The Mediterranean
countries for human consumption and animal feed
stock (Biswas, 2007; Campbell, 1997; Jackson and
Yunus, 1984; Smartt, 1984; Talukdar, 2009a). This
crop has astonishing combinations of many desirable
properties like requirement of very low agronomic
3620 J. Med. Plant. Res.
input, high N2 fixing capacity, high seed protein value
with number of important amino acids, presence of
antioxidant polyphenols, tolerance to various insects
and pests and capacity to grow at extreme
environmental conditions. A renewed interest in grass
pea cultivation and research in Europe and its
reintroduction in China have been justified by urgent
need to recover marginal and degraded lands over-
exploited by cereal cultivation (Granati et al., 2003;
Yang and Zhang, 2005). Its potentiality as a possible
phytoremediation for lead accumulation has recently
been explored (Brunet et al., 2008). Significant
accumulation of arsenic in plant parts, particularly in
roots, was also reported in Indian genotypes of grass
pea (Talukdar, 2011a).
Soil salinity is one of the most severe abiotic
stresses affecting production of the legumes worldwide
(Bayuelo-Jiménez et al., 2002; Wang et al., 2003). This
problem is more severe in arid and semi-arid regions,
and legume plants already face or in the near future
will face a notable impact of salt stress in these regions
(Graham and Vance, 2003). C. ternatea L. is
commonly considered as low to moderate salt tolerant
(Keating et al., 1986; Naidu and Harwood, 1997). In
comparison, L. sativus L. has been reported to tolerate
a wide range of biotic and abiotic stresses (Vaz Patto
et al., 2006), and is commonly known as moderate
salt-tolerant (Campbell, 1997). Thus, there remains the
possibility of developing genotypes with improved salt
tolerance if appropriate parents and breeding
techniques are adopted. However, low level of genetic
variability in cultivated varieties and poor
understanding of physiological parameters on its
tolerance to environmental stresses are the two prime
hindrances for fully exploitation of the remarkable
potentiality of these two crops through conventional
breeding methods. In grass pea, extensive researches
are now going on to augment its high nutritional quality
with low seed toxin (β-N-oxalyl L α, β-diamino
propionic acid) content in improved lines through the
creation of additional variability by induced
mutagenesis and biotechnological approaches, and
number of high yielding lines with low seed toxin level
have been reported (Asthana, 1995;Talukdar et al.,
2001). On the other hand, development of a rapid in
vitro micropropagation technique and introduction of C.
ternatea L. as a new and promising pasture legume
crop in Australia are related to enormous potentiality of
this medicinal herb in tropical regions (Hackney et al.,
2007; Pandeya et al., 2010). Despite the importance
for their cultivation in rapidly increasing saline areas,
detail investigation has not been undertaken to study
the salinity tolerance of these two crops.
Induced mutagenesis is a powerful tool to create
additional variability in crop plants. Recently, a number
of valuable mutant lines and different cytogenetic
stocks including aneuploids, tetraploids and
translocation lines have been developed in grass pea
through induced mutagenesis which can be used as
effective tools to explore the underlying genetic
mechanisms of various biotic and abiotic stress
responses of this crop (Vaz Patto et al., 2006; Talukdar
and Biswas 2007; Talukdar, 2008, 2009b, 2010a, b,
2011b). But, no such reports are available in Clitoria.
Recently, mutagenesis has been successfully induced
through gamma ray treatment in seeds of Clitoria in
present author’s laboratory, and preliminary
investigation revealed variation in response to salinity
between Clitoria and grass pea seedlings. The
objective of the present study is to assess the salt
stress responses based on different morpho-
physiological and biochemical parameters at seedling
stage of C. ternatea L. and L. sativus L and to identify
salt tolerant mutants in these two crops.
MATERIALS AND METHODS
Plant materials and induction of mutation
Fresh and healthy seeds of C. ternatea L. var. CAZR I466, 752,
and IGFR 12-1 and L. sativus var. RLS-188, Pusa-90-2 and
WBK-CB-14 were irradiated each with 100, 150, 200, 250, 300
and 350 Gy doses of gamma rays to induce mutation. Treated
seeds were sown treatment-wise during March 2006 for Clitoria
and October 2006 for grass pea to grow M1 generation in
separate plots with 30 and 50 cm uniform distances between
plants and rows, respectively. Untreated plants were used as
control. Seeds of individual M1 plants in all the treatments along
with control were separately harvested and sown in different
rows in a randomised block design to raise M2 progenies. M2
seeds were used to test the effect of salinity at seedling stage of
the two crops.
Experiment set-up
Dry, healthy and uniform-sized 20 M2 seeds genotype-1
treatment-1 were surface sterilized in 70% ethanol for 2 min,
rinsed twice in deionized water and then placed on water-
moistened filter papers in 9 cm diameter Petri dishes in an
incubator at 25°C with 12-h light (ISTA, 2008). Germinated
seeds were immediately transferred to twelve inches earthen
pots containing a mixture of fine soil, vermiculite and farm yard
manure (1:1:1). Each pot was labeled with accession number,
treatment dose and planting date. Seedlings were thinned to two
per pot after emergence and watered evenly for their uniform
growth until 7 days after first emergence. Initially, pots were kept
away from direct sunlight but with good light intensity, and
watered very carefully to keep the soil moist but not wet. Once
the seedlings reached 10 cm in height, the pots were kept under
normal field conditions with mean day/night temperature
32°C/27°C, 12-h photoperiod and relative humidity of 75±7% for
Clitoria and 27°C/20°C, 10-h photoperiod and relative humidity
of 77±5% for grass pea at climatic condition of lower Indo-
Gangetic plain during March and October, respectively. The salt
treatment commenced on 15-day-old seedlings. The control
plants from each of the six varieties were irrigated with distilled

water, while others were subjected to salinity stress by watering
them with 130 mM NaCl supplemented distilled water (300 ml
water in each pot), respectively, thrice in a week. Five pots (two
plants pot-1) genotype-1 were arranged in a randomized complete
block design with three replications of each treatment (radiation
dose). Salt concentration was regularly checked by measuring
electrical conductivity with a conductivimeter (Systronics M-308,
Kolkata, India), and evapotranspirational losses were
compensated with deionized water. Based on overall plant
growth and yield, six best-performing mutants- two in C. ternatea
L. (300 Gy, CAZR-1466 and 250 Gy, IGFR 12-1) and four in L.
sativus L. (one each from variety RLS-188, 200 Gy, and PUSA-
90-2, 300 Gy and rest two from WBK-CB-14, 350 Gy) was
selected for further morphological and biochemical study in the
next growing season (M3 generation). The mutants selected in
Clitoria were tentatively designated as CR1 and CR2, while
those from Lathyrus varieties were named as LR1, LR2, LR3
and LR4, respectively.
Assessment of growth parameters of selected mutants (M3)
To assess the effect of salt treatment, ten seedlings mutant-
1
along with un-mutagenised mother variety were subjected to
130 mM NaCl treatment exactly in the same procedure followed
in previous M2 generation. The treated variety was designated as
negative control (NC), whereas mutant plants given only distilled
water were served as positive control (PC). Growth performance
of the selected mutant plants along with PC and NC was
assessed by measuring the following parameters, plant height
(cm), number of primary branches /plant, leaves /branch, leaflets
/leaf, leaf injury level, root and shoot dry weight (g)/ plant after 25
days of commencement of salt treatment. To determine dry
weights, after harvesting, plants were separated into roots and
shoots. Roots were washed in tap water and rinsed in de-ionised
water. Plant materials were dried at 65°C for 48 h and weighed.
For ascertaining level of leaf injury, individual seedling was rated
on a scale of 0 to 4, based on the following salt injury gradation
of Chen et al. (2007) with some modifications for grass pea and
Clitoria, 0: No salt injury (100% survival with no damage); 1: Mild
salt injury, indicated by small area (approximately 1/5) of leaflet
apex and margin turning brownish yellow; 2: Moderate salt
injury, indicated by ½ of the leaflet turning whitish-yellow; 3:
Severe salt injury, when over 80% of total leaflet area turned
whitish-yellow and very thin, and 4: Extreme injury, when leaflets
became necrotic, crinkled, finally fell off and the plant ultimately
died. Leaflets, borne on first formed primary branches, were
considered for visual scorings of the trait.
Estimation of proline
Leaf proline content was estimated according to the method of
Bates et al., (1973) from fully expanded leaf samples collected
from first formed primary branches on 17th stress day of
treatment.
Estimation of Na+ and K+ contents
Fully expanded leaves of control and salt-treated plants were
analysed for total Na+ and K+ contents following the method of
Kumar and Sharma (1989). The oven-dried leaf (0.2 g) was
ground to fine powder and transferred to a digestion flask (50 ml)
containing acid mixture (3 ml) of concentrated H2SO4 and HClO4
in the ratio of 9:1 (v/v). The flask was heated gently over a hot
Talukdar 3621
plate for 10 to 12 min until the solution became colorless. The
cooled digest was then diluted by adding double distilled water
and volume was made up as required. The estimation of Na+
and K+ contents in acid extracts was carried out using an atomic
absorption spectrophotometer (Perkin Elmer, Analyst-100).
Estimation of relative water content (RWC)
Leaf RWC was estimated according to the method of Whetherley
(1950). The turgid weight of 1 g of fresh leaf sample was
determined by keeping it in water for 4 h. Dry weight (DW) was
measured by drying the same sample in a hot air oven (80°C)
until constant weight was achieved. RWC was expressed in
percentage following the formula, RWC (%) = [(FW-DW) /
(Turgid weight-DW)] × 100.
Estimation of chlorophyll and carotenoids contents
Leaf chlorophyll and carotenoid contents were determined by the
method of Lichtenthaler (1987). Leaf tissue (50 mg) was
homogenized in 10 ml chilled acetone (80%). The homogenate
was centrifuged at 4000 g for 12 min. Absorbance of the
supernatant was recorded at 663, 647 and 470 nm for
chlorophyll a, chlorophyll b and carotenoids, respectively. The
contents were expressed as mg chlorophyll or carotenoids g-1
FW.
Estimation of ascorbic acid (AA)
Ascorbic acid content will be estimated following the methods of
Mukherjee and Choudhari (1983). Five hundred milligrams of
leaf tissue was homogenized in 10 ml of 6% trichloroacetic acid.
Four ml of extract was mixed with 2 ml of 2%
dinitrophenylhydrazine in acidic medium followed by the addition
of 1 drop of 10% thiourea in 70% ethanol. The mixture was
boiled for 15 min in water bath and after cooling to room
temperature, 5 ml of 80% v/v H2SO4 was added in an ice bath
(0°C). The absorbance will be recorded at 530 nm.
Concentration of AA will be determined from a standard curve
plotted with known concentration of ascorbic acid.
Lipid peroxidation
Lipid peroxidation rates were determined by measuring the
malondialdehyde equivalents following the method of Hodges et
al. (1999). About 0.5 g of fresh leaf tissue was homogenized in a
mortar with 80% ethanol. The homogenate was centrifuged at
3000 ×g for 12 min at 4°C. The pellet was extracted twice with
the same solvent. The supernatants were pooled and 1 ml of this
sample was added to a test tube with an equal volume of either
the solution comprised of 20% TCA and 0.01% butylated
hydroxy toluene (BHT) or solution of 20% TCA, 0.01% BHT and
0.65% TBA. Samples were heated at 95°C for 25 min and
cooled to room temperature. Absorbance was measured at 440,
532 and 600 nm. Lipid peroxidation rate equivalents (nmol
malondialdehyde ml-1) were calculated by using the formulae of
Hodges et al. (1999).
Hydrogen peroxide estimation
The H2 O2 content of fully matured leaves was colorimetrically
3622 J. Med. Plant. Res.
measured as described by Mukherjee and Choudhuri (1983) by
extracting leaf samples in cold acetone and mixing of an aliquot
(3 ml) of the extracted solution with 1 ml of 0.1% titanium dioxide
in 20% (v:v) H2SO4. After centrifugation at 6,000 g for 15 min,
the intensity of yellow colour of the supernatant was measured at
415 nm. The concentration of H2O2 was calculated from a
standard curve plotted within the range of 100 to 1000 nM H2O2.
Electrolyte leakage
Electrolyte leakage (EL) was assayed by measuring the ions
leaching from leaf tissue into deionised water (Dionisio-Sese and
Tobita, 1998). Fresh leaf samples (100 mg) were cut into small
pieces (about 5 mm segments) and placed in test tubes
containing 10 ml deionised water. Tubes were kept in a water
bath at 32°C for 2 h. After incubation, electrical conductivity
(EC1) of the bathing solution was recorded with an electrical
conductivity meter (Systronics M-308, Kolkata, India). The
samples were then autoclaved at 121°C for 20 min to completely
kill the tissues and release all electrolytes. Samples were then
cooled to 25°C and final electrical conductivity (EC2) was
determined. The EL was expressed as a percentage by the
formula, EL (%) = (EC1) /(EC2) ×100
Antioxidant enzyme assays
Superoxide dismutase
Plant material was homogenized in 50 ml phosphate buffer, pH
7.0, containing 1% PVP. The homogenate was filtered and then
centrifuged in a refrigerated centrifuge at 15,000×g for 20 min.
The resultant supernatant was used as source of enzyme. The
extraction was performed at 4°C. The activity of superoxide
dismutase (SOD, EC 1.15.1.1) was determined by the nitro-blue
tetrazolium (NBT) photochemical assay method as described by
Beyer and Fridovich (1987). The reaction mixture (3 ml) contains
50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 µM
NBT, 0.1 mM EDTA, 2 µM riboflavin and 0.1 ml of enzyme
extract.
The test tubes with reaction mixture were shaken carefully and
kept for 25 min under 30 cm below a light source of 30 W
fluorescent lamps. A parallel control was run where buffer was
used instead of sample. The reaction was then stopped by
switching off the lights, and the tubes were covered with black
cloth. The absorbance of the solution was measured at 560 nm
in a UV-Vis spectrophotometer. A non-irradiated complete
reaction mixture served as a blank. SOD activity was expressed
as Enzyme unit mg-1 protein. One unit of SOD was defined as
the amount of protein causing a 50% NBT photoreduction.
Ascorbate peroxidase
100 mg of plant samples were homogenized in 1 ml of 50 mM
phosphate buffer (pH 7.8) containing 5 mM ascorbate, 5 mM
DTT, 5 mM EDTA, 100 mM NaCl and 2% (w/v) PVP. The
homogenized material was centrifuged at 15,000×g for 15 min at
4°C. Ascorbate peroxidase (APX, EC1.11.1.11) activity was
assayed following the method of Nakano and Asada (1981). The
hydrogen peroxide-dependent oxidation of ascorbate was
followed by a decrease in the absorbance at 290 nm with
extinction constant 2.8 mM-1 cm-1. The enzyme activity was
expressed as change in absorbance units, mg-1 protein.
Statistical analysis
The results are presented as mean values ± standard errors
(SE). Statistical significance between mean values was
measured by simple ‘t-test’. A probability of p<0.05, 0.01 was
considered significant.
RESULTS AND DISCUSSION
Effects of salinity on plant growth
In comparison to PC plants, significant reduction in
plant height, primary branches/plant, leaves/ branch,
leaflets/ leaf, respectively was exhibited by LR1 mutant
under salinity stress condition. Mutant CR1 also
manifested decrease in mean values of plant height
and number of primary branches, but the reduction
was not significant for leaf and leaflet number under
salt stress (Table 1). The CR2 mutant showed
marginal decrease in these four traits. Among the
Lathyrus mutants, increase in mean value of above
four parameters was observed in both LR3 and LR4
mutants in different magnitudes, whereas LR2 showed
non-significant variation for the traits as compared to
PC plants (Table 2). Both shoot and root dry weight (g)
decreased significantly in CR1, while marginal
reduction for both the traits was estimated for CR2
plants. Mean value of shoot dry weight varied
marginally in LR1, LR2 and LR3 mutants, but it
increased significantly in LR4 mutant. Root dry weight
decreased in all the four mutants, but significantly only
in LR1 under salinity stress. Drastic reduction in mean
of all the six growth parameters was observed in NC
plants, showing much higher rate of reduction than the
mutant lines under NaCl treatment (Tables 1 and 2).
Munns (2005) described dry weight of plants as one
of the realistic criteria in determining salt responses in
plants. An increase in height, primary branches and
leaf number might be attributed to higher shoot dry
weight in LR3 and LR4 plants. Therefore, high
tolerance was found associated with high total plant
dry weight, which was increased by positive
contribution from other components, least affected by
salt induced injury in tolerant lines. In grass pea,
superior performance of dwf1 and dwf2 dwarf mutant
lines at 170 mM NaCl treatment was indicated by their
normal plant dry weight, while low plant biomass
accumulation in the third dwarf line, dwf3, was
associated with symptoms of increased salt sensitivity
(Talukdar, 2011b). Reduction in plant dry weight under
salt stress was reported in different varieties of grass
pea (Mahdavi and Sanavy, 2007) and Phaseolus
(Bayuelo-Jime´nez et al., 2002). Interestingly, root
development was more sensitive to salt treatment than
shoot development in the present mutant lines, and the
result was in accordance with earlier reports in grass
 Talukdar 3623

Table 1. Assessment of 12 growth parameters in positive control (PC), negative control (NC) and two selected mutant lines (CR1
and CR2) of C. ternatea L. at harvest (25 days after commencement of 130 mM NaCl treatment on 15-days-old seedlings).

+
Traits PC CR1 CR2 NC
Plant height (cm) 49.9±0.11 33.6±0.09* 41.9±0.10 22.4±0.15**
Primary branches plant-1 8.8±0.21 6.0±0.13* 8.2±0.09 2.9±0.10**
-1
Leaves branch 5.7±0.19 4.9±0.11 5.5±0.10 3.0±0.16*
-1
Leaflets leaf 6.3±0.20 5.9±0.04 6.0±0.05 2.3±0.02**
Shoot dry weight (g) 0.097±0.31 0.077±0.22* 0.089±0.18 0.041±0.24**
Root dry weight (g) 0.10±0.21 0.072±0.15* 0.098±0.18 0.031±0.17**
Leaf injury level 0 1 0 3
Chlorophyll a (mg g-1fr.wt.) 4.34±0.56 3.89±0.34* 4.29±0.29 1.79±0.31**
-1
Chlorophyll b (mg g fr.wt.) 1.70±0.49 0.94±0.25* 1.73±0.28 0.75±0.30*
-1
Carotenoids (mg g fr.wt.) 1.22±0.18 1.09±0.25 1.30±0.17 0.79±0.23*
Na+ content (mg g-1 fr. wt.) 2.39±0.30 2.89±0.12 2.41±0.17 5.01±0.27*
K+ content (mg g-1 fr. wt.) 3.43±0.24 3.51±0.20 4.67±0.14* 2.22±0.17*
+ Values are means ± standard error (n = 10); * and ** significant from PC at 5 and 1% levels, respectively .

Table 2. Assessment of growth performance of positive control (PC), negative control (NC) and four selected mutant lines (LR1, LR2,
LR3 and LR4) of L. sativus L. at harvest (25 days after commencement of 130 mM NaCl treatment on 15-day-old seedlings).

Traits+ PC LR1 LR2 LR3 LR4 NC

X1 37.3±0.22 27.8±0.19* 33.9±0.22 38.2±0.11 40.2±0.09 21.2±0.19
X2 7.5±0.22 5.3±0.17* 7.0±0.09 7.8±0.03 8.8±0.11* 3.9±0.19**
X3 5.5±0.15 4.2±0.11* 5.2±0.17 6.3±0.12* 7.0±0.03* 2.5±0.11**
X4 6.0±0.12 4.7±0.08* 5.7±0.09 6.2±0.02 5.8±0.10 3.1±0.20**
X5 0.082±0.23 0.077±0.15 0.080±0.19 0.085±0.13 0.094±0.25* 0.037±0.19**
X6 0.094±0.31 0.076±0.22* 0.086±0.32 0.083±0.18 0.090±0.20 0.026±0.22**
X7 0 1 0 0 0 3
X8 4.98±0.69 3.34±0.22* 4.74±0.31 5.21±0.18* 5.18±0.19* 1.89±0.30**
X9 1.67±0.54 0.91±0.14* 1.58±0.21 1.89±0.69* 2.02±0.25* 0.79±0.33**
X10 1.17±0.29 0.67±0.20* 1.10±0.22 1.29±0.21 1.45±0.09* 0.70±0.31*
X11 1.89±0.40 2.71±0.32* 2.19±0.27 2.10±0.16 2.05±0.17 4.35±0.29**
X12 4.30±0.34 4.39±0.25 4.81±0.17* 4.92±0.22* 5.15±0.09* 2.09±0.27*

pea (Talukdar 2011b), chickpea (Ashraf and Waheed,
1993; Dua, 1997; Tejera et al., 2006) and in bean
(Ashraf and Rasul, 1988).
Effect of salinity on leaf injury
Among the mutants, CR1 and LR1 manifested level 1
injury level on leaflets, while CR2, LR2, LR3 and LR4
showed complete absence (level 0) of leaf injury under
salt treatment (Tables 1 and 2). Marking of injury was
also absent (level 0) in leaflets of PC seedlings, but
severe injury was exhibited on leaflets of NC plants as
level 3. Initially, the injury was marked by small
chlorotic spots on leaflet tips, but as treatment
progressed further, large areas in lamina became
yellow. The lower leaflets in NC plants also showed
marking of necrosis at later stages of growth. Leaf
injury has been considered as one of the important salt
response traits in plants (Munns, 2005). Among the
five levels (0 to 4) standardized on the basis of visual
scoring of leaf injury under salt stress, level 3 injury
was manifested by NC plants, indicating highest level
of susceptibility of these plants to salt treatment. The
level 1 injury in the leaves of CR1 and LR1 mutants
suggested lower level of salt sensitivity, while absence
of injury (level 0) on leaves of CR2, LR2, LR3 and LR4
genotypes as per PC level indicated their better
tolerance to salt stress. The cause of leaf injury was
attributed to salt load exceeding the capability of leaf
3624 J. Med. Plant. Res.
cells to compartmentalize salts in the vacuole. Salts
would then build up rapidly either in the cytoplasm
inhibiting enzyme activity or in the cell walls resulting in
dehydration of the cell (Munns, 2005).
Salt stress effect on leaf proline content
Among the parameters responding to salt treatment,
rapid accumulation of free proline content is one of the
significant events in plants. In the present material,
compared with PC plants, endogenous free proline
content increased at highest level in LR4, and it was
followed by CR2, LR3 and LR3 plants, whereas its
accumulation decreased significantly in NC plants
(Figure 1A). However, the exact role of proline in
abiotic stress tolerance is being debated. A positive
correlation between proline over accumulation and
increasing salinity/drought tolerance has also been
found in different crop plants including transgenics that
were engineered for overproduction of proline (Kavi,
1989; Anoop and Gupta, 2003). Increase in proline
content under NaCl stress was also reported in Pisum
sativum (Ahmad et al., 2008) and Phaseolus aureus
(Misra and Gupta, 2005). In contrast, proline
accumulation in plants was regarded by some workers
as a symptom of stress in less-salinity-tolerance
species, and its contribution to osmotic adjustment was
found negligible as compared with K+ (Wang et al.,
2004). In the present study, higher accumulation of
proline was estimated in those genotypes which
manifested tolerance phenotypes for other traits also.
Further study, however, is needed to ascertain its
exact role in salt-induced Lathyrus and Clitoria
genotypes.
Effect of salinity on Na+ and K+ and relative water
content (%)
NaCl-induced salinity exhibited highest accumulation
+ +
of Na and reduction of K concentration in leaves of
NC plants for both Clitoria and Lathyrus mutants.
Among the six mutants, Na+ accumulation was as per
with PC plants in five mutants except LR1 where its
content increased significantly under salt treatment. All
the mutant showed an increase in K+ content but the
change was significant in CR2, LR2, LR3 and LR4
mutant plants (Tables 1 and 2). The K+/ Na+ ratio
increased significantly over PC plants in CR2 and LR4
mutants, but decreased significantly in NC plants for
both crops (Figure 1B).
As compared to control, significant decrease in
RWC% was recorded in NC plants (1.3-fold) and in
CR1 (1.2-fold) mutants under salt stress (Figure 1C).
Marginal decline was recorded in rest of the
genotypes.
+ +
Equilibrium of cellular Na and K content is
absolutely essential in imparting salinity tolerance in
plants. Excessive accumulation of Na+ and Cl- in the
leaves has been considered highly harmful for normal
metabolism of plants, and tolerant genotypes has the
capacity of successful salt exclusion (Greenway and
Munns, 1980; Yeo and Flowers, 1986; Munns, 2005).
The K+: Na+ ratio has been used as a discriminating
factor between tolerant and sensitive genotypes with
greater capacity of former to block or reduce the
uptake or exclude the excess amount of Na+ and
+
associated increase in K content (Munns, 2005). In
the present material, significant increase in K+ content
and marginal variation in Na+ level, in comparison to
PC, might be one of the reasons for better plant growth
in CR2, LR2, LR3 and LR4 plants. High accumulation
of Na+ and decrease in K+ content has led to lowest
K+:Na+ ratio, rendering NC plants highly susceptible to
salt treatment. Leaf RWC content followed the same
trend.
Chlorophyll, carotenoids contents and ascorbate
pool
Salinity has significant effect on the chlorophyll a and b
content. As compared to PC plants, mean value
decreased significantly in CR1 and LR1 seedlings but
increased in LR3 and LR4 under salinity stress (Tables
1 and 2). The change, however, was not significant in
case of CR2 and LR2 mutants. Among the 10
genotypes, the ratio of chlorophyll a/b was highest
(4.13) in CR1and lowest (2.38) in NC-Clitoria (Figure
1D). The change in carotenoid content was not
significant in the mutant plants except LR1where it
reduced and in LR4 where its content enhanced
significantly under salt stress. The NC plants exhibited
lowest mean value for both chlorophyll and
carotenopid contents.
Ascorbate content was increased by nearly 2-folds in
the leaves of LR4 plants. Significant increase was also
estimated in LR3, LR2 and CR2 plants (Figure 1). Its
content, however, decreased in other genotypes, but
the value was significant in NC plants under salinity
stress (Figure 1E).
Photosynthetic pigment contents-chlorophyll a, b and
carotenoids were also affected by NaCl treatment in
different magnitudes. Among the ten genotypes, LR4
and LR3 exhibited higher level of these three pigment
components, while CR2 and LR2 maintained level of
PC plants. In contrast, reduction of chlorophyll
contents in NC, CR1 and LR1 plants was, presumably,
due to the increased activity of chlorophyllase enzyme
or due to the disruption of ultrastructure of pigment-
protein complex by ion toxicity (Saha et al., 2010).

However, relatively higher reduction in chl b content
as compared to chl a led to an increase in chl a/b ratio
in CR1 plants, and this can be explained by the fact
that degradation of chl b under salt stress begins with
its conversion to chl a, as reported in mung bean
(Saha et al., 2010). In plants, carotenoids and
ascorbate play a primary role as a non-enzymatic
antioxidant defense in combating stress by quenching
reactive oxygen species (ROS). Elevated levels of
these two components in leaves of NaCl-treated CR2,
LR2, LR3 and LR4 seedlings indicated their better free
radical scavenging capacity. A positive correlation
between ascorbate contents and anti-oxidant defense
enzymes, particularly peroxidase has recently been
established (Diaz-Vivancos et al., 2010).
Effect of salinity on lipid peroxidation, tissue H2O2
content and EL%
Cell membrane stability is often related to salt
tolerance in plants, and the conductance measurement
of electrolyte leakage from leaf cells is usually used as
an indicator of membrane damage in salt-treated
plants. Change in cell membrane integrity is closely
linked with extensive membrane lipid peroxidation in
plants including legumes (Hernández et al., 2000).
Reactive oxygen species, namely superoxide radicals
induces the degradation of phospholipids and the
resulting polyunsaturated fatty acids released by such
breakdown are then peroxidised (Kellogg and
Fridovich, 1975). In the present study, enhanced rate
of lipid peroxidation was recorded as indicated by
gradually increasing malondialdehyde (MDA) contents
in seedlings of both Clitoria and Lathyrus mutant and
NC plants exposed to salinity. The MDA content
increased significantly in leaves of NC, CR1 and LR1
plants, and the increase was the highest in NC
seedlings (Figure 1F). Non-significant variation in MDA
content was, however, estimated for the rest of the
genotypes. Similar trend was recorded in tissue
hydrogen peroxide content in the 10 genotypes under
salt treatment (Figure 1G). This result was supported
by the significant increase in percentage of electrolyte
leakage (EL%) in leaves of NC plants under salt
treatment. It was followed by CR1, LR1 and LR2
plants. Electrolyte leakage was increased by 3.1 to 7.8
folds in NC plants, 1.6-fold in CR1, 5.3-fold in LR1 and
2.7-fold in LR2 mutants (Figure 1H). The result
strongly indicated enhanced rate of membrane lipid
peroxidation in salt-sensitive NC and CR1 as well as
LR1 mutants resulting in much higher level of H2O2
content and electrolyte leakage in leaves in
comparison to relatively tolerant genotypes of CR2 in
Clitoria and LR4 as well as LR3 in L. sativus. The
result is in accordance with earlier findings in legumes
Talukdar 3625
like pea, cowpea, mungbean (Hernάndez et al., 2000;
Ahmad et al., 2008; Maia et al., 2010; Saha et al.,
2010;) and other crops (Dionisio-Sese and Tobita,
1998; Sairam and Srivastava, 2002; Hossain et al.,
2006).
Salinity-induced response of two antioxidant
enzymes activities
Under salt treatment, leaf SOD activity increased
significantly in CR1 (1.7-fold), CR2 (1.8-fold), LR1 (1.7-
fold), LR2 (2.5-fold), LR3 (2.7-fold) and LR4 (2.9-fold)
plants (Figure 1I), but reduced significantly in NC
plants (Figure 1I). The activity of the H2O2-
decomposing enzyme APX was significantly higher in
CR2 (1.9-fold) mutants of C. ternatea, while among
four mutants of L. sativus L., highest increase in
enzyme activity was estimated in leaves of LR4 (3.2-
fold) under salt stress. It was closely followed by LR3
(2.9-fold) and LR2 (2.0-fold) plants (Figure 1J).
Significant decrease in APX activity, however, was
recorded in salt-treated NC, CR1 and LR1 plants
(Figure 1J). The regulation of cellular redox state is a
crucial factor when a plant experiences environmental
stresses like salinity (Apel and Hirt, 2004).
The production of different classes of reactive
oxygen species (ROS) due to enhanced leakage of
electron to oxygen in mitochondria and an increase in
the H2O2 content in chloroplasts and their export to
cytosol under salt stress conditions trigger the increase
in anti-oxidant enzyme activities in plants. Superoxide
dismutase (SOD) and ascorbate peroxidase constitute
the first line of defense along with other non-enzymatic
primary components (Ahmad et al., 2008; Hernάndez
et al., 2000). SOD catalyzes the chemical conversion
of superoxide radicals into hydrogen peroxide, which is
in turn metabolized by the action of peroxidases. In the
present study, SOD activity increased significantly in
all the mutant lines, but decreased APX activity in CR1
and LR1 plants might have rendered these two mutant
lines less effective in scavenging the H2O2 radicals,
leading to the significant accumulation of H2O2 in
tissues. In contrast, high SOD level coupled with
relatively higher APX activity in CR2, LR3 and LR4
seedlings helped them to remove H2O2 properly. A
slight decrease in APX activity as compared with SOD
level in LR2 plants might be responsible for
accumulation of H2O2 level, close to significant level
and significant EL% under salt stress. The decrease of
both SOD and APX activity led to NC plants highly
susceptible to salinity treatment, as supported by other
parameters in the present study. Increase in SOD and
APX activity in relatively salt tolerant genotypes was
also reported in different crop plants including legumes
(Ahmad et al., 2008; Hernάndez et al., 2000;
3626 J. Med. Plant. Res.

Proline-1content (µ mol
6
Proline content (µmol
4
g-1 FW)
g FW)
2
0
PC

PC
CR1

LR1
CR2**

LR2**
LR3**
LR4**
NC*

NC**
(A) (B)
100

Chlorophyll a/b ratio

80 5
RWC %

60 4
3
40 2
20 1
0
0

CR2

LR1
LR2
LR3
LR4
PC

NC

PC

NC
CR1**
PC

PC
LR1
LR2
LR3
LR4
CR1*
CR2
NC**

NC**
(C) (D)
g-1DDW)
W)

MDA content (µM g FW)

80 MDAcontent (µmol g -1FW)
5
-1

-1
ol g

60 4
(µmmol

3
40
scorbate (µ

2
Ascorbate

20 1
0 0
PC

PC
CR1
CR2**
NC**

LR1
LR2**
LR3**
LR4**
NC**
A

PC

NC**

PC
LR1*

NC**
CR2

LR2
LR3
LR4
CR1*

C. ternatea L. L. sativus L. C. ternatea L. L. sativus L.

(E) (F)
Electrolyte leakage %
H2O2 content (µM g-1

6 100
80
4 60
FW)

40
2
20
0 0
PC

PC

LR2*
CR1**

NC**

LR1**

NC**
CR2

LR3
LR4
PC

PC

LR2
LR3
LR4
CR1*

LR1*
CR2
NC**

NC**

(G) (H)
SOD (U mg-1 protein)

protein
tein

800 1200
1000
pro

600
(Ugmg-1

800
-1

400 600
(Um

200 400
APXAPX

200
0 0
PC

PC
CR1**
CR2**
NC**

LR1**
LR2**
LR3**
LR4**
NC**

PC

PC
CR2**
NC**

LR2**
LR3**
LR4**
NC**
CR1

LR1

C. ternatea L. L. sativus L. C. ternatea L. L. sativus L.

(I) (J)

Figure 1. Assessment of different leaf biochemical components and antioxidant activities in six selected
induced mutant lines of C. ternatea L. (CR1 and CR2) and L. sativus L. (LR1, LR2, LR3 and LR4) under
130 mM NaCl treatment; PC-positive control (untreated), NC-negative control (treated). (A) Proline
content; (B) K+/Na+ ratio; (C) Relative water content (RWC) %; (D) Chlorophyll a/b ratio; (E) Ascorbate
content; (F) MDA content; (G) Hydrogen peroxide (H2O2) content; (H) Electrolyte leakage (EL%); (I)
Superoxide dismutase, and (J) Ascorbate peroxidase activity. Unit of measurement was given within
bracket in each case. Data were expressed as the mean of 10 independent values (n = 10); * and **
mean values are significantly different from PC value at 5 and 1% level, respectively.

Maia et al., 2010; Sairam and Srivastava, 2002;
Hossain et al., 2006).
Based on the performance of six mutants in their
responses to 22 parameters under salt stress in the
present study, it was concluded that LR4 exhibited
highest tolerance to 130 mM NaCl treatment, and it
was closely followed by CR2, LR3 and LR2 mutants.
The CR1 and LR1 showed moderate tolerance, while
NC plants exhibited highest susceptibility to NaCl-
induced salinity stress at seedling stage.
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