Introduction

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)

136 S. Roy and U. Chakraborty

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)

140 S. Roy and U. Chakraborty

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

References

tolerance in rice cultivars (Chunthaburee et al. 2016). In

Abberton MT; Marshall AH; Humphreys MW; Macduff JH;

the present study we utilized the relative fold change

Collins RP; Marley CL. 2008. Genetic improvement of

values of all the parameters in forming clusters. Based on

forage species to reduce the environmental impact of

the variations of the physiological parameters, all grasses

temperate livestock grazing systems. Advances in

were grouped according to their NaCl tolerance that could

Agronomy 98:311–355. DOI: 10.1016/S0065-2113(08)

be interpreted with the aid of the fold change values

00206-X

denoted by colored bars. The relationships between

Araújo SAM de; Silveira JAG; Almeida TD; Rocha IMA;

the physiological parameters themselves was also

Morais DL; Viegas RA. 2006. Salinity tolerance of

illustrated in the cluster analysis. The grasses were clearly

halophyte Atriplex nummularia L. grown under increasing

divided into 2 groups - a susceptible group ( Axonopus,

NaCl levels. Revista Brasileira de Engenharia Agrícola e

Chrysopogon, Oplismenus and Thysanolaena) and a

Ambiental 10:848–854. DOI: 10.1590/S1415-43662006000

relatively salt-tolerant group containing the remaining

400010

Arnon DI. 1949. Copper enzymes in isolated chloroplasts.

grasses. Critical analysis of the second group revealed 3

Polyphenoxidase in Beta vulgaris. Plant Physiology 24:1–

subgroups of less tolerant ( Arundo and Capillipedium),

15. DOI: 10.1104/pp.24.1.1

moderately tolerant ( Arundinella and Setaria) and

Ashraf M. 2006. Tolerance of some potential forage grasses

tolerant grasses ( Digitaria, Cynodon, Eragrostis and

from arid regions of Pakistan to salinity and drought. In:

Imperata). These results are in accordance with the

Öztürk M; Waisel Y; Khan MA; Görk G, eds. Biosaline

findings of other workers who reported the use of some of

agriculture and salinity tolerance in plants. Birkhäuser

these and other related, tolerant grasses for the

Verlag, Basel, Switzerland. p. 15–27. DOI: 10.1007/3-

reclamation and utilization of saline soils and increased

7643-7610-4_2

forage production (Kaffka 2001; Weber and Hanks 2006).

Ashraf M; Harris PJC. 2004. Potential biochemical indicators

Based on the results of hierarchical clustering, we

of salinity tolerance in plants. Plant Science 166:3–16. DOI:

conclude that Imperata cylindrica, Eragrostis amabilis,

10.1016/j.plantsci.2003.10.024

Bajji M; Kinet J-M; Lutts S. 2002. The use of the electrolyte

Cynodon dactylon and Digitaria ciliaris were relatively

leakage method for assessing cell membrane stability as a

salt-tolerant. SSI values individually pointed towards the

water stress tolerance test in durum wheat. Plant Growth

superior salt-tolerance of Imperata, Digitaria and

Regulation 36:61–70. DOI: 10.1023/A:1014732714549

Cynodon, whereas proline concentration indicated

Barr HD; Weatherley PE. 1962. A re-examination of the

marked tolerance in Cynodon, Arundinella, Imperata,

relative turgidity technique for estimating water deficit in

Eragrostis and Setaria. If we consider the MDA

leaves. Australian Journal of Biological Sciences 15:413–

concentrations, Cynodon, Arundinella, Imperata and

428. DOI: 10.1071/BI9620413

Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)

Salinity tolerance of some forage grasses in India 141

Bates LS; Waldren RP; Teare ID. 1973. Rapid estimation of

Lanza Castelli S; Grunberg K; Muñoz N; Griffa S; Lopez

free proline for drought stress determination. Plant and Soil

Colomba E; Ribotta A; Biderbost E; Luna C. 2009.

39:205–207. DOI: 10.1007/BF00018060

Oxidative damage and antioxidant defenses as potential

Bhaskaran S; Savithramma DL. 2011. Co-expression of

indicators of salt-tolerant Cenchrus ciliaris L. genotypes.

Pennisetum glaucum vacuolar Na+/H+ antiporter and

Flora - Morphology, Distribution, Functional Ecology of

Arabidopsis H+-pyrophosphatase enhances salt tolerance in

Plants 205:622–626. DOI: 10.1016/j.flora.2010.04.004

transgenic tomato. Journal of Experimental Botany

Lutts S; Kinet JM; Bouharmont J. 1996. NaCl-induced

62:5561–5570. DOI: 10.1093/jxb/err237

senescence in leaves of rice ( Oryza sativa L.) cultivars

Bromham L; Bennett TH. 2014. Salt tolerance evolves more

differing in salinity resistance. Annals of Botany 78:389–

frequently in C4 grass lineages. Journal of Evolutionary

398. DOI: 10.1006/anbo.1996.0134

Biology 27:653–659. DOI: 10.1111/jeb.12320

Marcum KB; Murdoch CL. 1994. Salinity tolerance

Chan Z; Bigelow PJ; Loescher W; Grumet R. 2012.

mechanisms of six C

Comparison of salt stress resistance genes in transgenic

4 turfgrasses. Journal of the American

Arabidopsis thaliana indicates that extent of transcriptomic

Society of Horticultural Science 119:779–784. http://

change may not predict secondary phenotypic or fitness

journal.ashspublications.org/content/119/4/779.short

effects. Plant Biotechnology Journal 10:284–300. DOI:

Miller G; Suzuki N; Ciftci-Yilmaz S; Mittler R. 2010. Reactive

10.1111/j.1467-7652.2011.00661.x

oxygen species homeostasis and signaling during drought

Chunthaburee S; Dongsansuk A; Sanitchon J; Pattanagul W;

and salinity stresses. Plant, Cell & Environment 33:453–

Theerakulpisut P. 2016. Physiological and biochemical

467. DOI: 10.1111/j.1365-3040.2009.02041.x

parameters for evaluation and clustering of rice cultivars

Mostafa EM; Tammam AA. 2012. The oxidative stress caused

differing in salt tolerance at seedling stage. Saudi Journal of

by NaCl in Azolla caroliniana is mitigated by nitrate.

Biological Sciences 23:467–477. DOI: 10.1016/j.sjbs.

Journal of Plant Interaction 7:356–366. DOI: 10.1080/

2015.05.013

17429145.2011.628452

de Hoon MJL; Imoto S; Nolan J; Miyano S. 2004. Open source

Munns R; Tester M. 2008. Mechanisms of salinity tolerance.

clustering software. Bioinformatics 20:1453–1454. DOI:

Annual Review of Plant Biology 59:651–681. DOI:

10.1093/bioinformatics/bth078

10.1146/annurev.arplant.59.032607.092911

Fan L; Zheng S; Wang X. 1997. Antisense suppression of

Nedjimi B. 2011. Is salinity tolerance related to osmolytes

phospholipase D alpha retards abscisic acid- and ethylene-

accumulation in Lygeum spartum L. seedlings? Journal of

promoted senescence of postharvest Arabidopsis leaves. The

the Saudi Society of Agricultural Sciences 10:81–87. DOI:

Plant Cell Online 9:2183–2196. DOI: 10.1105/tpc.9.12.2183

10.1016/j.jssas.2011.03.002

Harborne JB. 1973. Phytochemical methods. Chapman and

Plummer DT. 1978. An introduction to plant biochemistry. Tata

Hall, London, UK. DOI: 10.1007/978-94-009-5921-7

McGraw Hill Publications, New Delhi, India.

Hayat S; Hayat Q; Alyemeni MN; Wani AS; Pichtel J; Ahmad

A. 2012. Role of proline under changing environments.

Rout NP; Tripathi SB; Shaw BP. 1997. Effect of salinity on

Plant

Signaling

&

Behavior

7:1456–1466.

DOI:

chlorophyll and proline contents in three aquatic

10.4161/psb.21949

macrophytes. Biologia Plantarum 40:453–458. DOI:

Heath RL; Packer L. 1968. Photoperoxidation in isolated

10.1023/A:1001186502386

chloroplasts. I. Kinetics and stoichiometry of fatty acid

Roy S; Chakraborty U. 2014. Salt tolerance mechanisms in Salt

peroxidation. Archives of Biochemistry and Biophysics

Tolerant Grasses (STGs) and their prospects in cereal crop

125:189-198. DOI: 10.1016/0003-9861(68)90654-1

improvement. Botanical Studies 55:31. DOI: 10.1186/1999-

Indian Council of Agricultural Research. 2009. Forage crops

3110-55-31

and grasses. In: Handbook of Agriculture. ICAR, New

Sairam RK; Tyagi A. 2004. Physiology and molecular biology

Delhi, India. p. 1353–1417.

of salinity stress tolerance in plants. Current Science

Jana S; Choudhuri MA. 1981. Glycolate metabolism of three

86:407–421. http://www.jstor.org/stable/24108735

submersed aquatic angiosperms: Effect of heavy metals.

Sengupta S; Patra B; Ray S; Majumder AL. 2008. Inositol

Aquatic Botany 11:67–77. DOI: 10.1016/0304-3770(81)

methyl transferase from a halophytic wild rice, Porteresia

90047-4

coarctata Roxb. (Tateoka): Regulation of pinitol synthesis

Kaffka S. 2001. Salt tolerant forages for the reuse of saline

under abiotic stress. Plant, Cell & Environment 31:1442–

drainage water. In: Proceedings of 31st California Alfalfa

1459. DOI: 10.1111/j.1365-3040.2008.01850.x

and Forage Symposium, University of California, Davis,

Shi H; Wang Y; Cheng Z; Ye T; Chan Z. 2012. Analysis of

CA, USA. https://goo.gl/JQowRC

natural variation in bermudagrass ( Cynodon dactylon)

Kafi M; Stewart WS; Borland AM. 2003. Carbohydrate and

reveals physiological responses underlying drought

proline contents in leaves, roots, and apices of salt-tolerant

tolerance. PLoS One 7(12):e53422. DOI: 10.1371/journal.

and salt-sensitive wheat cultivars. Russian Journal of Plant

Physiology 50:155–162. DOI: 10.1023/A: 1022956727141

pone.0053422

Karsensky J; Jonak C. 2012. Drought, salt, and temperature

Squires VR. 2015. Intake and nutritive value of some salt-

stress-induced metabolic rearrangements and regulatory

tolerant fodder grasses and shrubs for livestock: Selected

networks. Journal of Experimental Botany 63:1593–1608.

examples from across the globe. In: El Shaer HM; Squires

DOI: 10.1093/jxb/err460

VR, eds. Halophytic and salt-tolerant feedstuffs: Impacts on

Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)

142 S. Roy and U. Chakraborty

nutrition, physiology and reproduction of livestock. CRC

Wyn Jones RG; Storey R. 1981. Betaines. In: Paleg LG;

Press, Boca Raton, FL, USA. p. 218–246. DOI: 10.1201

Aspinall D, eds. Physiology and biochemistry of drought

/b19862-16

resistance in plants. Academic Press, Adelaide, Australia. p.

Tada Y; Komatsubara S; Kurusu T. 2014. Growth and

171–204.

physiological adaptation of whole plants and cultured cells

Yadav NS; Shukla PS; Jha A; Agarwal PK; Jha B. 2012. The

from a halophyte turf grass under salt stress. AoB Plants

SbSOS1 gene from the extreme halophyte Salicornia

6:plu041. DOI: 10.1093/aobpla/plu041

brachiata enhances Na+ loading in xylem and confers salt

Tester M; Bacic A. 2005. Abiotic stress tolerance in grasses.

tolerance in transgenic tobacco. BMC Plant Biology 12:188.

DOI: 10.1186/1471-2229-12-188

From model plants to crop plants. Plant Physiology

Zhu J-K. 2000. Genetic analysis of plant salt tolerance using

137:791–793. DOI: 10.1104/pp.104.900138

Arabidopsis.

Plant

Physiology

124:941–948.

DOI:

Thomas JC; de Armond RL; Bohnert HJ. 1992. Influence of

10.1104/pp.124.3.941

NaCl on growth, proline, and phosphoenolpyruvate

Ziaf K; Amjad M; Pervez MA; Iqbal Q; Rajwana IA; Ayyub M.

carboxylase levels in Mesembryanthemum crystallinum

2009. Evaluation of different growth and physiological

suspension cultures. Plant Physiology 98:626–631. DOI:

traits as indices of salt tolerance in hot pepper ( Capsicum

10.1104/pp.98.2.626

annuum L.). Pakistan Journal of Botany 41:1797–1809.

Weber DJ; Hanks J. 2006. Salt tolerant plants from the Great

http://www.pakbs.org/pjbot/PDFs/41(4)/PJB41(4)1797.pdf

Basin region of the United States. In: Khan MA; Weber DJ,

Zulkaliph NA; Juraimi AS; Uddin MK; Ismail MR; Ahmad-

eds. Ecophysiology of high salinity tolerant plants. Tasks for

Hamdani MS; Nahar UA. 2013. Screening of potential salt

Vegetation Science, Vol. 40. Springer, Dordrecht, The

tolerant turfgrass species in Peninsular Malaysia. Australian

Netherlands. p. 69–106. DOI: 10.1007/1-4020-4018-0_5

Journal of Crop Science 7:1571–1581. https://goo.gl/c2iVJX

(Received for publication 25 August 2016; accepted 3 June 2017; published 30 September 2017)

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