tolerance is a complex trait, governed by several
physiological and biochemical parameters and these
In India, available fodder for stock is estimated to be 40‒
parameters greatly influence the normal growth and
50% below requirements, and this scenario is gradually
development of plants (Zhu 2000). Salt tolerance of any
worsening due to the concomitant decrease in grass
individual species is demonstrated as the ability to
coverage and increase in livestock population (Indian
maintain an optimal physiological and biochemical
Council of Agricultural Research 2009). Global climate
equilibrium under NaCl treatment (Sairam and Tyagi
change in the last decade has been correlated with changes
2004). Ashraf and Harris (2004) suggested different
in the productivity of forage grasses and is likely to have
biomarkers as indicators of salinity tolerance, including
a detrimental effect on the overall grass coverage in the
soluble sugars, proteins, amino acids, ammonium
long term (Abberton et al. 2008). A huge proportion of
compounds, polyamines, polyols, antioxidants and
ATPases.
land in the country is classified as wasteland due to the
In the present study however, 6 biochemical markers,
problems of soil salinity, alkalinity and waterlogging. The
viz . relative water content (RWC), proline and soluble
selection of grass germplasm for salinity tolerance is
sugar concentrations, membrane lipid peroxidation
critical for more efficient utilization of these degraded
(malondialdehyde, MDA), electrolyte leakage (EL) and
lands by establishing stress-tolerant grasses in non-arable
H2O2 concentration were selected for use in screening for
marginal areas (Ashraf 2006). Species that are relatively
salinity tolerance of the selected grasses. Increase in leaf
salt-tolerant show greater endurance and adaptability
RWC in the halophyte Atriplex nummularia with
among the native species (Squires 2015). Therefore there
increasing salinity indicated an efficient mechanism to
is an urgent need to: identify salt-tolerant traits in wild
adjust cell cytosol osmotically (Araújo et al. 2006).
forage grasses; evaluate their potential for enhancing the
Accumulation of osmolytes like proline, soluble sugars
productivity of grasslands in their native habitats; and
and glycine betaine and elevated levels of antioxidative
utilize them for the rejuvenation of grasslands and
enzymes play a vital role in conferring salt tolerance in
croplands with reduced or lost productivity.
grasses (Roy and Chakraborty 2014). Accumulation of
Abiotic stresses, in particular water and salinity stress,
glycine betaine in Cynodon and Spartina, proline in
play a major role in disrupting the growth and
Paspalum and myo-inositol in Porteresia has been found
development of grasses including cereals (Tester and
to confer salinity tolerance (Wyn Jones and Storey 1981;
Bacic 2005). Salinity limits plant growth and productivity
Marcum and Murdoch 1994; Sengupta et al. 2008).
through the toxic effects of Na+ and Cl- ions, which leads
Accumulation
of
proline,
fructans
and soluble
to ionic imbalances, osmotic and oxidative stress (Munns
carbohydrates was also correlated with salinity tolerance
and Tester 2008). Native grasses, however, show variable
in salt-tolerant cultivars of wheat (Kafi et al. 2003). MDA
degrees of NaCl tolerance, especially those belonging to
concentration has been proposed as an indicator of
the subfamilies Panicoideae and Chloridoideae (Bromham
oxidative damage and a lesser accumulation of the same
and Bennett 2014; Roy and Chakraborty 2014). Salinity
in root tissues was employed for screening the salt-
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
Salinity tolerance of some forage grasses in India 131
tolerant genotypes of Cenchrus ciliaris (Castelli et al.
acclimatize for 48 hours in the growth chamber, with a
2009). Electrolyte leakage as an indicator of cell
standard temperature of 20‒25 °C, RH 65‒70% and 16 h
membrane stability of durum wheat cultivars under
photoperiod. Following acclimatization, 2 groups of plants
osmotic stress was demonstrated, with level of electrolyte
were grown in NaCl treatments of 100 and 200 mM for 9
leakage being inversely related to degree of salt tolerance
days, while the third group remained as control and the
of cultivars (Bajji et al. 2002).
effects of NaCl on the plants in terms of several biomarkers
In addition to the characterization of 12 forage grasses
after 3, 6 and 9 days of treatment were analyzed.
that are widely grazed by and fed to livestock in the eastern
Three individual samplings from 3 different locations
parts of the Terai-Duar grasslands by observing the
(Figure 1) were completed for each grass and the results
changes in 6 biomarkers for salinity tolerance, the objective
were expressed as mean ± SD for all parameters analyzed.
of our study was to evaluate the salt-tolerance potential of
For grasses with broad leaves like Thysanolaena and
those grasses by using a rapid screening technique where
Arundo, 3 plants were taken per sampling site, whereas
the inherent tolerance of saline conditions was assessed as
for grasses with small narrow leaves, 5‒6 plants were
a precursor to selective propagation in varied environ-
taken per sampling site.
mentally challenged wastelands.
Salt sensitivity index (SSI)
Materials and Methods
The youngest healthy fully expanded leaves from the
Study area and plant materials
plants were briefly washed in deionized water and 1 cm
Twelve native grasses were collected from the different
diameter leaf discs were finely cut and floated in a 5 ml
regions of the eastern part of the Terai-Duar grasslands
solution of NaCl (100 and 200 mM) for 96 hours. Leaf
(88.22‒89.66° E, 26.45‒26.86° N; Figure 1). These
discs floated in sterile dH2O served as the experimental
grasses are widely grazed by livestock and harvested by
control for the bioassay (Fan et al. 1997). The effects of
local people for feeding to domestic animals, viz . Arundo
salt treatment on leaf discs were assessed by observing the
donax L. of the subfamily Arundinoideae; Axonopus
phenotypic changes and the extent of NaCl effect in terms
compressus (Sw.) P. Beauv., Capillipedium assimile
of SSI, which was quantified by estimating the
(Steud.) A. Camus, Chrysopogon aciculatus (Retz.) Trin.,
chlorophyll concentration in NaCl-treated and control
Digitaria ciliaris (Retz.) Koeler, Arundinella bengalensis
sets. Briefly, the leaf discs were crushed in 80% acetone
(Spreng.) Druce, Imperata cylindrica (L.) Raeusch.,
and the absorbance was recorded in a UV-VIS
Oplismenus burmanni (Retz.) P. Beauv., Setaria pumila
spectrophotometer at 645 and 663 nm and the chlorophyll
(Poir.) Roem. & Schult. and Thysanolaena latifolia
concentration was calculated using Arnon’s formulae
(Roxb. ex Hornem.) Honda of the subfamily Panicoideae;
(Arnon 1949). SSI values were then calculated at 100 and
and Cynodon dactylon (L.) Pers. and Eragrostis amabilis
200 mM NaCl as the percent decrease in chlorophyll
(L.) Wight & Arn. of the subfamily Chloridoideae. In the
concentration of the NaCl treatment in comparison with
subsequent text only the generic names are used.
the untreated leaf discs using the following formula:
Chlorophyll conc. of NaCl-treated leaf discs
Experimental design and NaCl treatment
SSI =
x 100
Chlorophyll conc. of untreated leaf discs
A rapid screening protocol was implemented for the
differentiation of salt-tolerance potential of the forage
Biochemical markers for assessment of NaCl tolerance
grasses. The grasses were collected from their natural
habitats and placed in small flasks containing 0.1X
For an alternative screening of grasses for their salt-tolerant
Hoagland solution with their roots intact, before being
attributes, 6 different biochemical parameters were chosen,
transferred to the plant growth chamber in the laboratory of
viz. relative water content (RWC), proline and soluble
the Department of Botany, University of North Bengal,
sugar concentrations, membrane lipid peroxidation
Siliguri. Before NaCl treatment, the roots were gently
(malondialdehyde, MDA), electrolyte leakage (EL) and
washed with sterile dH
H
2O to remove any mud and then
2O2 concentration. For these experiments, the first 3 fully
again transferred to conical flasks containing 0.1X
expanded leaves from the top of each grass subjected to the
Hoagland solution. The plants were then allowed to
various growth solutions were collected.
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
132 S. Roy and U. Chakraborty
Figure 1. Geographical location of the Terai-Duar grasslands and the sampling area. Sampling area (enlarged view) with major locations from which the forage grasses were collected.
Relative water content. RWC was measured following the
Total sugar. Soluble sugar in leaves was extracted in 95%
protocol of Barr and Weatherley (1962). Briefly, fresh
ethanol following the method of Harborne (1973).
leaf samples from control and different treatment sets
Anthrone reagent was used to estimate total sugar
were weighed to obtain fresh weight (FW). The samples
following the method of Plummer (1978). Briefly, 4 ml of
were then immediately hydrated to full turgidity for 4 h,
anthrone reagent was added to 1 ml test solution and kept
dried of surface moisture and weighed to obtain fully
over boiling water bath for 10 min, after which the
absorbance was taken at 620 nm. Total sugar was finally
turgid weight (TW). Samples were then oven-dried at 80
calculated using a standard curve of D-glucose.
°C for 24 h and weighed to determine dry weight (DW).
RWC was calculated by the following equation:
Membrane lipid peroxidation. Membrane lipid peroxi-
dation was measured in terms of concentration of malon-
RWC (%) = [(FW - DW) / (TW - DW)] × 100
dialdehyde (MDA) produced by the thiobarbituric acid
(TBA) reaction, following the method of Heath and
Proline. Extraction and estimation of proline were done
Packer (1968). Leaves were homogenized in 0.1% (w/v)
by the method of Bates et al. (1973). Leaf tissue was
trichloroacetic acid (TCA) and estimation was done with
homogenized in 3% sulfosalicylic acid. Ninhydrin
0.5% (w/v) TBA in 20% TCA. The absorbance of the
reagent was used for the estimation of proline in the
reaction mixture was determined at 532 and 600 nm and
extract, which was separated in a separating funnel using
the MDA content was calculated using an extinction
toluene, prior to recording the absorbance at 520 nm.
coefficient of 155 mM/cm.
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
Salinity tolerance of some forage grasses in India 133
Electrolyte leakage. Electrolyte leakage (EL) was
( Oplismenus). SSIs of grasses determined by leaf disc
measured as described by Lutts et al. (1996). Leaves were
assay and represented in terms of % decrease in
washed thoroughly with deionized water and placed in
chlorophyll concentration in the leaf discs floated in 100
culture tubes containing 10 ml of deionised water on a
mM and 200 mM NaCl solutions relative to the control
rotary shaker for 24 h. Subsequently, the electrical
sets, i . e. leaf discs kept in sterile dH2O, are shown in Table
conductivity of the solution (L
1. At 100 mM NaCl, the senescence assay indicated that
t) was determined and the
samples were then autoclaved at 120 °C for 20 min and
Setaria, Thysanolaena, Imperata and Cynodon were least
cooled to room temperature before determining the final
affected with SSI values of 0.45‒7.36. At the same time,
electrical conductivity (L
Capillipedium, Axonopus and Arundinella were much
0). EL was calculated as follows:
more sensitive (SSI values of 24.20‒18.37). However, at
Electrolyte leakage (%) = (L
200 mM NaCl, Imperata, Digitaria and Cynodon were
t / L0) × 100
least affected by salt concentration (SSI values of 6.59‒
H2O2 concentration. The extraction and estimation of
15.00). Interestingly, Thysanolaena and Setaria were
H2O2 were done by the method given by Jana and
more affected by 200 mM NaCl, showing marked
Choudhuri (1981) with slight modification. Leaf tissue
increases in SSI values (23.38 and 57.98, respectively).
was homogenized in 50 mM phosphate buffer (pH 6.5)
Capillipedium showed the highest sensitivity to both 100
and mixed with 0.1% titanium sulphate in 20% (v/v)
and 200 mM NaCl with SSI values of 24.20 and 61.93,
H2SO4 and centrifuged at 6,000 rpm for 15 min.
respectively. This result was also reciprocated by the
Absorbance was measured at 410 nm and H2O2
phenotypical changes in the leaf discs floated in NaCl
concentration was measured using the extinction
solutions, which can be clearly observed in Figure 2.
coefficient of 0.28 µmol/cm.
Effect of NaCl on biochemical markers for analysis of
Hierarchical cluster analysis
salinity tolerance
For cluster analysis of the grasses for their NaCl
Relative water content. Leaf RWC values were found to
tolerance, the data for fold change values of RWC,
decrease in all grasses with both increase in NaCl
proline, soluble sugar, MDA, EL and H2O2 after NaCl
concentration and duration of treatment (Table 2). The
treatments for 3, 6 and 9 days with respect to the control
fold change values of RWC in plants subjected to 100 and
sets were taken. Hierarchical cluster analysis was
200 mM NaCl in comparison with the control sets
performed using the CLUSTER 3.0 program by the
revealed the smallest changes in Cynodon and Imperata
uncentered matrix and complete linkage method
and the largest changes in Chrysopogon and Digitaria
following the protocol of de Hoon et al. (2004). The
(Figure 3a).
resulting tree figure was displayed using the software
Proline concentration. Proline concentration in fresh
package, Java Treeview, as described by Chan et al.
untreated leaves varied from 11.6 µg/g ( Chrysopogon)
(2012).
and 12.4 µg/g ( Setaria) to 63.1 µg/g ( Imperata) and 64.5
µg/g ( Digitaria). During the first 3 days of NaCl treatment
Statistical analysis
(100 and 200 mM), proline concentration in fresh tissue
All experiments were repeated with sampling from 3
increased with increase in NaCl concentration in all
different locations (n = 3) for each species. Species and
grasses except Axonopus, where levels of proline declined
treatment means were statistically analyzed using Least
(Table 3; Figure 3b). The largest increases (on a
Significant Difference (P≤0.05) for a completely
percentage basis) were recorded in Cynodon, Arundinella
randomized design.
and Imperata. Similarly after 6 and 9 days of treatment,
proline concentrations increased as NaCl concentration
Results
increased in all grasses except Axonopus, Chrysopogon,
Thysanolaena and Oplismenus, where concentrations
Salt sensitivity index (SSI) of grasses
declined with increasing NaCl concentration. The largest
percentage increases in proline concentration were
Chlorophyll concentration in fresh untreated leaves
observed in Cynodon and Arundinella (1.8‒3-fold
varied from 0.72 mg/g ( Capillipedium) to 1.45 mg/g
increase).
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
134 S. Roy and U. Chakraborty
Table 1. Chlorophyll concentration in detached leaf discs of grasses dipped in 0, 100 and 200 mM NaCl solutions and salt sensitivity index expressed as relative % decrease of chlorophyll concentration of detached leaves at 100 and 200 mM NaCl.
Grass
Chlorophyll concentration
Salt sensitivity index
(mg/g fresh weight of tissue, fwt)
(% decrease in chlorophyll conc.)
Concentration of NaCl (mM/L)
Concentration of NaCl (mM/L)
0
100
200
100
200
Arundo
1.22 ± 0.21
1.00 ± 0.07
0.75 ± 0.05
18.37
38.11
Axonopus
1.00 ± 0.12
0.78 ± 0.04
0.60 ± 0.01
21.72
39.94
Capillipedium
0.72 ± 0.09
0.55 ± 0.02
0.27 ± 0.01
24.20
61.93
Chrysopogon
0.78 ± 0.11
0.70 ± 0.04
0.53 ± 0.02
10.29
32.49
Cynodon
1.17 ± 0.22
1.09 ± 0.08
1.00 ± 0.03
7.36
15.00
Digitaria
0.91 ± 0.08
0.8 ± 0.04
0.78 ± 0.03
11.86
14.11
Arundinella
1.29 ± 0.08
1.02 ± 0.08
0.71 ± 0.05
21.33
44.58
Eragrostis
0.94 ± 0.07
0.82 ± 0.07
0.65 ± 0.01
12.61
30.33
Imperata
1.35 ± 0.14
1.28 ± 0.11
1.26 ± 0.08
5.67
6.59
Oplismenus
1.45 ± 0.17
1.22 ± 0.15
1.12 ± 0.12
15.82
22.35
Setaria
0.80 ± 0.11
0.80 ± 0.04
0.33 ± 0.01
0.45
57.98
Thysanolaena
0.81 ± 0.08
0.80 ± 0.02
0.62 ± 0.02
1.52
23.38
Values for chlorophyll concentration are mean ± SD (n = 3). Greater values of salt sensitivity index denote greater sensitivity or susceptibility to NaCl, whereas lower values denote lesser sensitivity.
Figure 2. Leaf disc senescence bioassay: Phenotypic changes observed as chlorophyll bleaching occurs in response to 0, 100 and 200 mM NaCl treatment (left to right) after 96 h. (a) Arundo; (b) Axonopus; (c) Capillipedium; (d) Chrysopogon; (e) Cynodon; (f) Digitaria; (g) Arundinella; (h) Eragrostis; (i) Imperata; (j) Oplismenus; (k) Setaria; and (l) Thysanolaena.
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
Salinity tolerance of some forage grasses in India 135
Table 2. Relative water content (%) of grasses under treatment of 0, 100 and 200 mM NaCl solutions for 3, 6 and 9 days.
Grass
Concentration of NaCl (mM/L) and duration of treatment
3 days1
6 days2
9 days3
0
100
200
0
100
200
0
100
200
Arundo
85.2 ±1.1
80.6 ±2.1
78.5 ±1.1
84.1 ±0.9
77.5 ±0.6
71.2 ±1.4
84.6 ±1.2
70.2 ±0.8
66.5 ±2.2
Axonopus
84.5 ±1.2
78.6 ±2.3
74.1 ±1.7
83.2 ±1.5
76.5 ±0.7
74.2 ±0.9
83.2 ±1.3
73.1 ±1.1
68.6 ±0.4
Capillipedium
85.2 ±1.4
77.3 ±1.8
76.5 ±1.3
86.6 ±1.2
75.4 ±1.2
72.3 ±0.8
85.5 ±2.1
75.5 ±1.1
70.1 ±0.9
Chrysopogon
82.1 ±0.9
74.3 ±1.2
72.1 ±2.3
81.8 ±0.8
73.2 ±1.5
69.4 ±0.6
82.6 ±2.2
66.5 ±1.5
60.7 ±1.1
Cynodon
91.5 ±0.8
89.6 ±1.5
87.2 ±2.5
90.2 ±1.3
87.2 ±1.1
82.9 ±1.8
90.7 ±1.2
84.2 ±1.7
81.5 ±0.8
Digitaria
84.1 ±1.2
78.6 ±2.4
74.5 ±1.2
83.9 ±2.1
77.2 ±0.8
68.9 ±0.9
85.8 ±1.5
74.3 ±1.3
61.2 ±0.6
Arundinella
80.1 ±2.1
75.5 ±1.2
72.5 ±2.5
81.5 ±2.3
73.2 ±1.2
70.8 ±0.7
80.6 ±1.5
70.4 ±2.1
65.4 ±1.4
Eragrostis
85.1 ±1.9
81.2 ±1.1
79.6 ±2.6
83.2 ±1.8
76.7 ±1.6
72.1 ±1.2
84.1 ±1.8
71.2 ±2.4
63.1 ±0.7
Imperata
82.5 ±1.4
80.2 ±0.9
78.2 ±1.6
81.9 ±0.9
79.2 ±1.8
77.6 ±1.8
80.5 ±0.9
76.1 ±1.5
75.9 ±0.9
Oplismenus
87.3 ±0.8
80.5 ±0.9
77.6 ±1.4
86.5 ±1.4
78.2 ±0.8
74.6 ±1.9
85.9 ±1.1
76.1 ±0.8
72.3 ±1.1
Setaria
82.4 ±0.6
77.5 ±1.2
74.1 ±0.7
80.5 ±1.2
74.1 ±1.1
70.6 ±0.3
80.5 ±2.1
71.1 ±0.6
62.3 ±1.3
Thysanolaena
86.5 ±1.1
80.5 ±1.7
76.2 ±1.2
87.1 ±2.2
74.5 ±0.7
70.2 ±1.6
85.2 ±1.5
70.7 ±1.3
64.2 ±1.8
1LSD (P≤0.05) Species = 2.23; Treatment = 1.12. 2LSD (P≤0.05) Species = 3.41; Treatment = 1.7. 3LSD (P≤0.05) Species = 5.19; Treatment = 2.59. Values represent mean ± SD, where n = 3.
Figure 3. Fold change values of the biochemical markers in grasses subjected to NaCl stress. (a) Relative water content; (b) Proline concentration; (c) Soluble sugar concentration; (d) MDA concentration; (e) Electrolyte leakage; and (f) H2O2 concentration. 3D, 6D
and 9D represent the duration of exposure to NaCl solutions (days) and 100 and 200 represent the concentrations of NaCl (mM/L).
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
Table 3. Proline concentration (µg/g fwt) in grasses under treatments of 0, 100 and 200 mM NaCl solutions for 3, 6 and 9 days.
Grass
Concentration of NaCl (mM/L) and duration of treatment
3 days1
6 days2
9 days3
0
100
200
0
100
200
0
100
200
Arundo
40.5 ±0.8
60.8 ±0.3
98.3 ±0.1
45.2 ±0.4
57.9 ±0.5
78.2 ±0.9
42.5 ±0.2
55.2 ±0.4
78.4 ±1.6
Axonopus
32.3 ±0.7
29.8 ±0.1
27.4 ±0.7
30.2 ±0.3
26.5 ±0.2
20.7 ±0.1
30.5 ±0.3
19.7 ±0.1
8.2 ±0.1
Capillipedium
39.3 ±0.7
45.2 ±0.2
51.3 ±0.7
35.9 ±0.4
60.4 ±0.6
55.2 ±0.8
36.6 ±0.1
64.2 ±0.2
56.1 ±1.1
Chrysopogon
12.2 ±0.2
15.3 ±0.2
17.8 ±0.1
11.9 ±0.1
10.8 ±0.1
7.1 ±0.5
10.6 ±0.7
8.8 ±0.4
3.5 ±0.2
Cynodon
48.1 ±1.2
145.3 ±2.1 215.1 ±2.5
50.1 ±0.7
160.2 ±1.5 210.8 ±2.3
45.8 ±0.2
100.3 ±1.3 178.2 ±1.4
Digitaria
65.3 ±1.1
78.9 ±1.7
95.6 ±1.5
63.2 ±1.1
80.6 ±1.1
90.7 ±1.5
66.1 ±0.2
82.6 ±1.4
85.1 ±0.9
Arundinella
30.5 ±0.8
70.1 ±1.1
75.2 ±1.5
34.2 ±0.6
86.1 ±0.9
102.5 ±1.6
32.1 ±0.1
107.1 ±1.5
90.2 ±1.3
Eragrostis
40.5 ±0.7
51.2 ±0.9
68.7 ±1.1
42.5 ±0.6
65.4 ±0.8
79.8 ±0.9
44.4 ±0.2
86.5 ±1.5
97.3 ±1.5
Imperata
63.3 ±0.9
83.5 ±1.1
120.2 ±0.9
60.7 ±0.1
85.2 ±0.9
125.3 ±1.3
65.4 ±0.4
96.9 ±1.6
132.1 ±1.1
Oplismenus
23.1 ±0.5
25.6 ±0.7
27.1 ±0.5
22.7 ±0.3
20.1 ±0.5
23.5 ±0.3
20.9 ±0.1
17.6 ±0.4
15.2 ±0.5
Setaria
12.2 ±0.1
15.5 ±0.3
20.8 ±0.4
14.3 ±0.3
32.1 ±0.2
34.5 ±0.1
10.6 ±0.7
27.6 ±0.4
26.7 ±0.1
Thysanolaena
25.6 ±0.3
30.8 ±0.4
41.1 ±0.8
23.2 ±0.2
28.7 ±0.3
26.2 ±0.5
20.2 ±0.6
17.8 ±0.7
12.5 ±0.1
1LSD (P≤0.05) Species = 40.82; Treatment = 20.41. 2LSD (P≤0.05) Species = 41.82; Treatment = 20.91. 3LSD (P≤0.05) Species
= 40.15; Treatment = 20.07. Values represent Mean ± SD, where n = 3.
Total sugar concentration. Concentration of sugars in
Membrane lipid peroxidation. MDA concentration in
untreated fresh leaves varied from 16.1 mg/g
untreated fresh leaves varied from 2.2 mM/g
( Capillipedium) to 56.9 mg/g ( Eragrostis). Changes in
( Chrysopogon) to 11.9 mM/g ( Arundo). Concentrations
concentration followed no consistent pattern across the
showed a consistent pattern, increasing across all
various grasses subjected to NaCl treatments (Table 4;
concentrations and durations of NaCl treatment in all
Figure 3c), with some showing decreases while a few
grasses with greater responses to increasing concen-
showed increases. Those showing greatest decreases
were Capillipedium (69% decrease) and Oplismenus
tration than to increasing duration of exposure (Table 5;
(45% decrease), with most of the grass species showing
Figure 3d). After 9 days, greatest increases in MDA
little change in sugar concentration over the 9 days, even
concentration occurred in Chrysopogon (5-fold),
at 200 mM NaCl.
Capillipedium (3-fold) and Axonopus (2.4-fold).
Table 4. Soluble sugar concentration (mg/g fwt) in grasses under treatments of 0, 100 and 200 mM NaCl solutions for 3, 6 and 9 days.
Grass
Concentration of NaCl (mM/L) and duration of treatment
3 days1
6 days2
9 days3
0
100
200
0
100
200
0
100
200
Arundo
35.2 ±0.7 33.1 ±0.3 36.7 ±0.1 34.1 ±0.2 30.2 ±0.1 28.9 ±0.1
33.9 ±0.1 31.1 ±0.1 24.6 ±0.1
Axonopus
50.1 ±1.5 48.9 ±1.5 52.1 ±0.6 47.8 ±1.4 46.8 ±0.8 45.1 ±1.2
47.5 ±0.9 44.3 ±1.2 40.1 ±1.1
Capillipedium 15.6 ±0.2 14.9 ±0.1 16.5 ±0.2 16.1 ±0.1 15.1 ±0.1 13.4 ±0.3
16.7 ±0.1 10.9 ±0.1
5.4 ±0.1
Chrysopogon
32.1 ±0.1 34.4 ±0.4 36.7 ±0.3 30.5 ±0.2 33.1 ±0.2 31.6 ±0.2
30.9 ±0.2 31.5 ±0.2 27.8 ±0.1
Cynodon
40.1 ±0.1 42.1 ±1.4 45.3 ±0.2 41.8 ±0.5 43.2 ±0.8 46.3 ±1.4
40.5 ±0.9 42.6 ±1.1 44.9 ±1.2
Digitaria
35.4 ±0.2 40.1 ±0.6 44.3 ±1.4 34.6 ±0.3 43.2 ±1.3 47.6 ±1.6
36.1 ±0.2 40.5 ±1.3 35.5 ±0.2
Arundinella
29.8 ±0.1 28.6 ±0.1 36.5 ±0.5 27.6 ±0.1 31.5 ±0.2 38.7 ±0.2
30.5 ±0.2 34.2 ±0.3 29.9 ±0.1
Eragrostis
56.1 ±1.1 60.3 ±0.7 62.3 ±0.7 57.8 ±1.3 60.5 ±1.5 61.4 ±0.2
56.8 ±0.6 61.3 ±0.5 63.3 ±0.3
Imperata
33.2 ±0.9 36.1 ±0.5 35.3 ±0.2 30.8 ±0.2 36.6 ±0.4 40.9 ±1.5
33.3 ±0.3 34.5 ±0.6 35.7 ±0.7
Oplismenus
40.5 ±0.2 34.5 ±0.3 31.2 ±0.3 43.2 ±0.5 30.6 ±0.2 28.7 ±0.2
41.9 ±1.4 26.7 ±0.3 23.2 ±0.2
Setaria
49.2 ±1.1 46.5 ±1.2 45.5 ±1.5 47.8 ±1.2 44.4 ±1.1 46.5 ±0.3
47.7 ±0.5 44.3 ±0.3 41.1 ±1.2
Thysanolaena
36.5 ±0.5 34.2 ±0.3 31.3 ±0.1 35.5 ±0.2 33.3 ±0.2 29.8 ±0.3
37.7 ±0.2 30.1 ±0.2 28.9 ±0.3
1LSD (P≤0.05) Species = 4.96; Treatment = 2.48. 2LSD (P≤0.05) Species = 7.24; Treatment = 3.62. 3LSD (P≤0.05) Species = 6.92; Treatment = 3.46. Values represent Mean ± SD, where n = 3.
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
Salinity tolerance of some forage grasses in India 137
Table 5. MDA concentration (mM MDA/g fwt) of grasses under treatments of 0, 100 and 200 mM NaCl solutions for 3, 6 and 9 days.
Grass
Concentration of NaCl (mM/L) and duration of treatment
3 days1
6 days2
9 days3
0
100
200
0
100
200
0
100
200
Arundo
12.1 ±0.1 14.2 ±0.8 17.3 ±0.4
11.3 ±0.2 18.2 ±0.8 21.6 ±0.3
12.3 ±0.1 26.1 ±0.6 32.3 ±0.2
Axonopus
10.1 ±0.1 16.2 ±0.2 23.1 ±0.2
10.6 ±0.4 23.1 ±0.4 34.2 ±0.3
11.1 ±0.3 25.6 ±0.4 37.6 ±0.4
Capillipedium
5.6 ±0.2
10.1 ±0.3 16.7 ±0.1
5.7 ±0.1
11.1 ±0.1 17.6 ±0.2
4.9 ±0.3
13.2 ±0.6 19.8 ±0.6
Chrysopogon
2.2 ±0.7
4.3 ±0.1
7.8 ±0.1
2.1 ±0.5
5.4 ±0.2
10.5 ±0.8
2.2 ±0.1
9.8 ±0.1
13.2 ±0.2
Cynodon
10.2 ±0.6 13.2 ±0.2 15.6 ±0.2
10.5 ±0.1 14.1 ±0.1 16.4 ±0.1
11.2 ±0.1 13.9 ±0.2 16.5 ±0.4
Digitaria
3.5 ±0.4
5.1 ±0.1
7.2 ±0.7
4.1 ±0.9
6.2 ±0.7
8.1 ±0.6
3.7 ±0.9
6.7 ±0.3
9.5 ±0.8
Arundinella
4.8 ±0.8
5.1 ±0.1
6.7 ±0.1
4.5 ±0.1
6.7 ±0.1
8.8 ±0.2
4.1 ±0.8
7.5 ±0.7
8.5 ±0.9
Eragrostis
8.6 ±0.6
9.1 ±0.1
10.7 ±0.6
8.1 ±0.4
9.7 ±0.3
11.8 ±0.4
8.8 ±0.5
10.1 ±0.1 13.4 ±0.6
Imperata
5.4 ±0.3
6.5 ±0.1
7.8 ±0.5
4.8 ±0.8
7.1 ±0.2
8.9 ±0.3
5.1 ±0.2
7.7 ±0.3
10.1 ±0.2
Oplismenus
9.8 ±0.5
17.1 ±0.2 21.3 ±0.7
9.5 ±0.1
18.6 ±0.1 22.5 ±0.1
10.1 ±0.2 21.3 ±0.3 25.4 ±0.4
Setaria
10.1 ±0.3 12.1 ±0.3 15.4 ±0.3
9.7 ±0.2
14.3 ±0.2 20.5 ±0.1
10.2 ±0.2 17.3 ±0.7 23.7 ±0.4
Thysanolaena
3.1 ±0.6
5.6 ±0.2
8.7 ±0.4
3.4 ±0.3
4.9 ±0.6
10.7 ±0.3
3.6 ±0.5
10.8 ±0.6 13.2 ±0.1
1LSD (P≤0.05) Species = 3.34; Treatment = 1.67. 2LSD (P≤0.05) Species = 5.07; Treatment = 2.53. 3LSD (P≤0.05) Species =
6.25; Treatment = 3.12. Values represent Mean ± SD, where n = 3.
Electrolyte leakage. Electrolyte leakage levels in
across all concentrations of and durations of exposure to
untreated fresh leaves varied from 5.1% ( Arundinella) to
NaCl solutions for all grasses (Table 7; Figure 3f). The
15.5% ( Setaria) and increased across all concentrations
most
responsive
grasses
were
Chrysopogon,
and durations of NaCl treatment in all grasses (Table 6;
Capillipedium and Arundo, while the least responsive
Figure 3e). Arundo and Capillipedium showed the
were Cynodon and Imperata.
greatest increases in electrolyte leakage with exposure to
NaCl treatment with a much greater response to
Hierarchical cluster analysis for the evaluation of NaCl
increasing concentration (80‒90%) than to duration of
tolerance
exposure (10‒24%). The lowest responses occurred with
Based on the variable effects of NaCl treatment on
Cynodon and Imperata.
biochemical parameters, the grasses were grouped
H2O2 concentration. Concentrations of H2O2 in untreated
according to their NaCl tolerance through hierarchical
fresh leaves ranged from 2.4 µmol/g ( Chrysopogon) to
cluster analysis, where the fold change values of all
11.8 µmol/g ( Digitaria and Thysanolaena) and increased
parameters were taken into consideration (Figures 3a‒3f).
Table 6. Electrolyte leakage (%) of grasses under treatments of 0, 100 and 200 mM NaCl solutions for 3, 6 and 9 days.
Grass
Concentration of NaCl (mM/L) and duration of treatment
3 days1
6 days2
9 days3
0
100
200
0
100
200
0
100
200
Arundo
14.1 ±0.5 21.2 ±0.1 25.4 ±0.5
14.1 ±0.6 23.1 ±0.3 24.9 ±0.2
14.3 ±0.2 22.9 ±0.4 26.7 ±0.3
Axonopus
10.1 ±0.7 12.2 ±0.3 14.3 ±0.3
10.3 ±0.3 13.4 ±0.2 15.6 ±0.3
10.6 ±0.3 14.3 ±0.6 17.2 ±0.3
Capillipedium
8.7 ±0.1
14.3 ±0.5 16.7 ±0.6
9.1 ±0.3
15.1 ±0.3 17.8 ±0.3
8.8 ±0.3
16.2 ±0.6 18.6 ±0.3
Chrysopogon
5.2 ±0.5
6.7 ±0.3
8.1 ±0.3
6.1 ±0.5
8.2 ±0.4
9.7 ±0.3
5.5 ±0.3
7.8 ±0.3
10.6 ±0.2
Cynodon
11.9 ±0.9 12.1 ±0.4 13.2 ±0.5
12.2 ±0.4 13.2 ±0.5 13.9 ±0.3
10.8 ±0.4 12.9 ±0.4 14.3 ±0.4
Digitaria
11.2 ±0.8 13.4 ±0.3 14.5 ±0.3
10.7 ±0.7 14.5 ±0.5 15.6 ±0.3
11.1 ±0.4 15.2 ±0.2 17.2 ±0.3
Arundinella
5.2 ±0.6
6.2 ±0.2
6.5 ±0.2
5.1 ±0.2
6.1 ±0.3
6.7 ±0.1
4.9 ±0.3
5.5 ±0.3
7.1 ±0.3
Eragrostis
10.1 ±0.9 13.1 ±0.2 14.5 ±0.3
10.4 ±0.3 14.2 ±0.4 15.4 ±0.2
10.6 ±0.3 14.5 ±0.2 16.7 ±0.4
Imperata
14.3 ±1.1 16.1 ±0.3 16.5 ±0.3
14.5 ±0.4 15.8 ±0.3 17.2 ±0.3
14.9 ±0.4 18.8 ±0.3 19.7 ±0.3
Oplismenus
13.4 ±0.7 16.1 ±0.4 17.2 ±0.4
13.1 ±0.3 16.8 ±0.2 18.1 ±0.4
13.4 ±0.2 17.5 ±0.2 19.2 ±0.6
Setaria
15.1 ±0.8 17.2 ±0.2 19.3 ±0.4
15.4 ±0.2 18.9 ±0.4 21.3 ±0.4
16.1 ±0.3 24.3 ±0.3 26.7 ±0.5
Thysanolaena
7.6 ±0.6
8.1 ±0.1
9.7 ±0.5
7.3 ±0.3
9.5 ±0.3
11.2 ±0.3
7.8 ±0.4
10.1 ±0.4 13.4 ±0.4
1LSD (P≤0.05) Species = 2.6; Treatment = 1.3. 2LSD (P≤0.05) Species = 2.53; Treatment = 1.27. 3LSD (P≤0.05) Species = 2.82; Treatment = 1.41. Values represent Mean ± SD, where n = 3.
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
138 S. Roy and U. Chakraborty
Table 7. H2O2 concentration (µmol/g fwt) in grasses under treatment with 0, 100 and 200 mM NaCl solutions for 3, 6 and 9 days.
Grass
Concentration of NaCl (mM/L) and duration of treatment
3 days1
6 days2
9 days3
0
100
200
0
100
200
0
100
200
Arundo
6.5 ±0.1
11.2 ±0.2 13.1 ±0.3
6.6 ±0.4
12.3 ±0.2 15.6 ±0.1
6.7 ±0.3
15.4 ±0.3 18.7 ±0.5
Axonopus
7.2 ±0.3
10.2 ±0.3 14.5 ±0.2
8.1 ±0.1
14.3 ±0.2 17.8 ±0.2
8.3 ±0.1
16.7 ±0.1 21.3 ±0.3
Capillipedium
4.5 ±0.2
5.4 ±0.4
8.7 ±0.2
4.1 ±0.2
6.7 ±0.1
10.9 ±0.3
4.8 ±0.3
8.8 ±0.1
15.4 ±0.5
Chrysopogon
2.5 ±0.8
4.1 ±0.2
7.2 ±0.4
2.1 ±0.3
5.3 ±0.2
9.3 ±0.4
2.7 ±0.1
7.4 ±0.5
11.2 ±0.2
Cynodon
10.1 ±0.7 11.2 ±0.2 13.2 ±0.5
9.7 ±0.4
14.5 ±0.3 17.6 ±0.3 10.3 ±0.4 13.2 ±0.5 18.1 ±0.3
Digitaria
12.1 ±0.8 15.4 ±0.3 17.8 ±0.3 11.7 ±0.5 17.1 ±0.4 23.1 ±0.5 11.9 ±0.1 20.1 ±0.4 24.3 ±0.2
Arundinella
6.8 ±0.5
7.6 ±0.5
8.9 ±0.2
6.6 ±0.6
9.9 ±0.4
11.7 ±0.4
6.5 ±0.4
11.7 ±0.4 15.3 ±0.5
Eragrostis
4.5 ±0.4
4.7 ±0.2
6.7 ±0.2
4.1 ±0.3
6.2 ±0.5
8.4 ±0.1
4.3 ±0.2
7.6 ±0.3
10.9 ±0.2
Imperata
8.7 ±0.3
9.1 ±0.7
10.3 ±0.3
8.2 ±0.3
10.3 ±0.3 13.4 ±0.3
8.6 ±0.4
12.9 ±0.5 15.2 ±0.3
Oplismenus
11.3 ±0.4 14.3 ±0.2 18.7 ±0.3 10.9 ±0.2 16.5 ±0.2 21.8 ±0.1 11.1 ±0.1 17.6 ±0.3 20.1 ±0.5
Setaria
8.5 ±0.5
9.1 ±0.3
9.8 ±0.2
8.1 ±0.3
14.3 ±0.2 17.6 ±0.2
7.9 ±0.8
15.1 ±0.2 18.9 ±0.3
Thysanolaena
11.1 ±0.9 14.5 ±0.2 17.6 ±0.1 12.2 ±0.6 15.2 ±0.3 18.1 ±0.3 12.1 ±0.7 21.5 ±0.3 23.8 ±0.1
1LSD (P≤0.05) Species = 2.1; Treatment = 1.05. 2LSD (P≤0.05) Species = 2.19; Treatment = 1.09. 3LSD (P≤0.05) Species =
2.42; Treatment = 1.21. Values represent Mean ± SD, where n = 3.
Figure 4. Hierarchical cluster analysis of the grasses using the fold change values of relative water content (RWC); proline concentration (PRO); soluble sugar concentration (SUG); membrane lipid peroxidation (malondialdehyde, MDA); electrolyte leakage (EL); and H2O2 concentration after NaCl treatments (100 mM and 200 mM) for 3, 6 and 9 days. Resulting tree figure was displayed using Java Treeview after hierarchical cluster analysis through CLUSTER 3.0. The color grids in the cluster analysis represent the relative fold change values (-3 to +3 shown by different colors) of the specific biochemical markers for each of the individual grasses. For the analysis of salt tolerance, the greenness of the grids for biomarkers like MDA, EL and H2O2 and redness for RWC, PRO and SUG was considered; which means a species for which the grids are more reddish for RWC, PRO and SUG and less greenish for MDA, EL and H2O2 could be considered the most tolerant of all. However, this was easily recognized in the cluster analysis due to grouping of the studied species on the basis of their responses to biochemical markers.
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
Salinity tolerance of some forage grasses in India 139
The ranges of fold change values in the clusters are
technique to screen out the potential salt-tolerant forage
represented by the colored bars. Results suggested the
grasses.
probable interrelations among biochemical parameters
The 6 biomarkers we selected to analyze the salt-
subjected to NaCl stress and variable salt tolerance
tolerance potential of the forage grasses, namely relative
between all grass genera.
water content (RWC), proline and soluble sugar
Based on their salt sensitivities, the grasses formed 2
concentrations, membrane lipid peroxidation, electrolyte
distinct groups (Figure 4). One group was comprised of
leakage and H2O2 concentration, proved useful in
Axonopus, Chrysopogon, Oplismenus and Thysanolaena.
indicating differences between species in ability to
The remaining grasses with varying response patterns to
tolerate saline conditions both simply and rapidly.
NaCl solutions formed the second group and were
While RWC of any plant always decreases with the
classified into 3 subgroups: Arundo and Capillipedium;
increase in NaCl concentration, a lower decrease in RWC
Arundinella and Setaria; and Digitaria, Cynodon,
is a valuable marker in the selection of salt-tolerant
Eragrostis and Imperata.
species (Ziaf et al. 2009). In our study, lowest decreases
in RWC were observed in Cynodon, Eragrostis and
Discussion
Imperata across all concentrations and durations of NaCl
treatments, identifying them as salt-tolerant species. In
This rapid screening for salinity tolerance in the forage
contrast, accumulation of proline and soluble sugars is
grasses has been attempted as a simple method of
considered to be positively correlated with salinity
identifying the most salt-tolerant grasses for introduction
tolerance (Karsensky and Jonak 2012; Hayat et al. 2012).
into areas with increasing soil salinity and decreasing
Accumulation of higher levels of proline has been
productivity. Previously, Zulkaliph et al. (2013) in their
reported
in
the
halophytes,
Mesembryanthemum
studies with turfgrasses ranked the different species of
crystallinum and Sporobolus virginicus when compared
grasses for salinity tolerance on the basis of shoot and root
with the glycophytes carrot and rice (Thomas et al. 1992;
growth, leaf firing, i.e. yellowing of leaves resulting from
Tada et al. 2014). In the present study, apart from
cell death due to osmotic imbalances, turf color and turf
Axonopus, Chrysopogon and Oplismenus, proline accu-
quality. We estimated salinity tolerance of the grasses
mulation increased in all grasses subjected to NaCl
primarily by a salt sensitivity index (SSI), determined by
treatment. We also observed that soluble sugar accumu-
evaluating the effects of NaCl solutions on leaf discs over
lation decreased in Arundo, Axonopus, Capillipedium,
96 hours. This type of bioassay has been used previously
Oplismenus, Setaria and Thysanolaena across all
in several transgenesis experiments to evaluate the
concentrations of NaCl and durations of exposure. In
tolerances of transgenic plants relative to the wild type
contrast, accumulation of soluble sugars increased in
plants from which they were bioengineered (Bhaskaran
Digitaria, Imperata and Arundinella subjected to NaCl
and Savithramma 2011; Yadav et al. 2012).
treatments for 3, 6 and 9 days. Nedjimi (2011) also
The amount of chlorophyll leached out from the leaf
correlated the accumulation of greater amounts of soluble
discs into the NaCl solution was used as an indicator of
sugars in the forage grass Lygeum spartum with osmotic
the effect of NaCl on leaf tissues. The decrease in
adjustment and protection of membrane stability that
chlorophyll concentration in plants subjected to NaCl
conferred salinity tolerance.
treatment has been inversely correlated with salinity
Increase in malondialdehyde (MDA) concentration,
tolerance. For instance, the decrease in Chlorophyll a:
an indication of lipid peroxidation, is considered
Chlorophyll b ratio in salt-tolerant Najas graminea was
unfavorable for plant health, and plants, which show
lower than in Hydrilla verticillata and Najas indica (Rout
little increase in MDA concentration when exposed to
et al. 1997). In the present study, we quantified the
NaCl, are considered to be salt-tolerant (Miller et al.
amount of chlorophyll in the leaf discs in both control and
2010). Marked increases in MDA concentration were
treatment sets and the values were used to reciprocate the
observed in Axonopus, Capillipedium, Chrysopogon and
sensitivity of grasses towards NaCl treatment. Greater salt
Thysanolaena, following exposure to salt. However,
sentivity index values denoted greater susceptibility of the
minimal increase was observed in Cynodon and
grasses towards NaCl. Overall, the results of the bioassay
Eragrostis across all concentrations and durations of
indicated that among the grasses tested, Imperata,
treatment.
Cynodon and Digitaria could be considered as less
Similarly, low electrolyte leakage (EL) and limited
sensitive or resistant on the basis of SSI values at 100 and
increase in H2O2 concentration in response to NaCl
200 mM NaCl. SSI therefore presents an easy and rapid
treatment are also considered as markers of the salt
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
tolerance of plants (Mostafa and Tammam 2012).
Eragrostis could be considered salt-tolerant. Thus, while
Accumulation of H2O2 in plants interferes with the normal
individual biochemical markers provide good indications
biochemical processes inside plants. In the present study,
of the degree of salt tolerance of a species, cluster
EL in all grasses increased with the increase in NaCl
analysis, which incorporates the results with several
concentration and duration of treatment. Least EL was
biomarkers, provides a much more reliable indication.
observed in Cynodon, Imperata and Arundinella, which
However, SSI values can provide an easy and rapid tool
could be considered salt-tolerant species in comparison
for the screening of salt tolerance. Based on our screening
with the other grasses. The high increases in H
results, we consider that the selective propagation of the
2O2
concentration
observed
in
Arundo,
Axonopus,
most salt-tolerant species could be utilized for the
Capillipedium and Chrysopogon indicate that these
rejuvenation of native grasslands and also for the
species can be considered susceptible to salination on the
reclamation of salinity infested wastelands.
basis of this trait. Comparatively, low increases in H
2O2
concentration observed in Imperata, Setaria and Cynodon
Acknowledgments
indicate that they can be considered salt-tolerant.
The authors are grateful to CSIR, New Delhi (Award No.
Finally, hierarchical cluster analysis using the software
09/285(0046)/2008-EMR-I) and UGC, ERO, Kolkata
CLUSTER 3.0 was used to represent the inter-relations
(Minor Research Project No. PSW-80/12-13) for the
among the physiological parameters and to align the
financial support which enabled the carrying out of this
grasses on the basis of their salinity tolerance as a similar
work.
type of hierarchical cluster analysis has been performed
to evaluate the natural variation in drought tolerance in
bermuda grass (Shi et al. 2012) and the variation in salt
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(Received for publication 25 August 2016; accepted 3 June 2017; published 30 September 2017)
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