EST- Panicum maximum

F: CCCGAGGCGATCCGATTCGTT

63–53/58

R: TACGCCGACGACGAGGACGA

3- (AT)13

EST- Panicum virgatum

F: TCCAGATGACTCCCAGGAAC

50–40/45

R: TCATCACTCGATTCCTCAAGC

4- (GT)38

Genomic- Panicum virgatum

F: GCAACCATGACAAGAAGCAT

63–53/58

R: ATACAAACCGGGGTGCTAAG

5- (CGT)n

EST- Eragrostis curvula

F: TCTCCAACACGCCACGAC

63–53/58

R: CAATCCACTACAAGAAACCAC

SSR 1 and 2 (Ebina et al. 2007); 3 (Tobias et al. 2006); 4 (Wang et al. 2011); 5 (Cervigni et al. 2008).

TD/Tm: Touchdown/Annealing temperature; F: forward primer; R: reverse primer.

controls (no DNA template) were also included. Poly-

removed from the roots, placed in a drop of 2% hema-

merase chain reactions (PCR) were carried out in an MJ

toxylin with 2% ferric citrate used as mordant, and

Research Thermal Cycler. The optimum annealing

squashed (modified from Núñez 1968). Cells with fully

temperature (Tm) was determined for each locus (Table

contracted and well spread metaphase chromosomes were

2). By touchdown PCR (TD), the annealing temperature

photographed using a digital camera.

was decreased to 1 °C starting with 5 °C over the set

annealing temperature. Initial denaturation step of 95 °C

Data analyses

for 3 min was followed by 10 touchdown cycles of 94 °C

for 30 sec, touchdown annealing temperature for 30 sec

Average number of filled and empty seeds, germination

and 72 °C for 45 sec. PCR products were subsequently

percentages at day 7, time at which 50% of seeds had

amplified for 34 cycles at 94 °C for 30 sec, annealing

germinated (T50) and survival of 15- and 40-day-old

temperature for 30 sec, and 72 °C for 45 sec with a final

seedlings were recorded for self- and open-pollinated

extension at 72 °C for 20 min. Amplifications were

conditions. The mean values obtained for the 2 forms of

initially checked on 1% agarose gels. PCR products were

pollination were compared through one-tailed Student’s t-

analyzed on 6% denaturing polyacrylamide gel, with a

test for paired samples, since a higher number of filled

TBE 1x electrophoresis buffer at 50W for 1 h and 45 min

seeds was expected in cross-pollination. Analysis of

to 2 h. Bands were visualized by silver staining and

variance (ANOVA) for the mean number of seeds was

scanned.

performed and accessions were compared by Fisher’s

LSD tests. Statistical analyses were performed using

Chromosome number

Infostat software (Di Rienzo et al. 2008).

Based on the number of seeds, germination and

To confirm the chromosome number of P. coloratum var.

survival data per panicle, probability of seed production,

makarikariense, 8 different plants were evaluated. One plant

and probability of seedling survival for self- or cross-

from each accession was chosen at random and considered

pollinated conditions were estimated by using the

representative of each accession (DF, UCB, MR, BR, ER

and CM), while 2 plants were selected from IFF.

conditional probability and Bayes Theorem (Quinn and

Root tips of 3-month-old plants were pretreated with

Keough 2002). The probability of seedling survival (SS)

8-hydroxyquinoline (0.002 ml/g) for 5 h. The roots were

was calculated as: P(SS) = P(SS/SPs)*P(SPs) +

fixed in a freshly prepared mixture of ethanol:glacial

P(SS/SPo)*P(SPo), where P(SS/SPS) and P(SS/SPO) are

acetic acid (3:1 v/v) for 48 h at room temperature and then

the conditional probabilities of seedling survival from

placed in 70% ethanol at 4 °C for several weeks. Treated

seeds produced under self- and open-pollinated condi-

roots were put in 95, 70 and 40% ethanol and distilled

tions, respectively; P(SPs) and P(SPo) are the probabil-

water for 15 min each, hydrolyzed in 1 N HC1 at 60 °C

ities of seed production under self- and open-pollination;

for 8 min, transferred to distilled water for 2 min and

and P(SS) is the probability of seedling survival. The

stained in leuco-basic fuchsin for 1 h at room temperature

conditional probability of finding a plant given that it was

in the dark. The 3–4 mm deeply stained root tips were

produced under self- or open-pollination was calculated

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

122 L.V. Armando, M.A. Tomás, A.F. Garayalde and A.D. Carrera

as: P(SPs/SS) = P(SS/SPs)*P(SPs)/P(SS) and P(SPo/SS)

per panicle was highly variable among plants, registering

= P(SS/SPo)*P(SPo)/P(SS), respectively.

values from 0 to 72 (CV = 149%) for self-pollinated

In the progeny test, offspring’s DNA fingerprints were

panicles and from 14 to 795 (CV = 61%) for open-

individually compared with the maternal pattern. The

pollinated panicles (Figure 2). Mean number of filled

occurrence of a sexual reproduction event was defined as

seeds differed significantly among accessions for both

when progeny DNA fingerprints revealed a deviation

self- and open-pollination methods (Figure 3). According

from the maternal profile. Each band was considered as

to Fisherś LSD test, the commercial variety (cv.

an independent locus, and polymorphic bands were

Bambatsi) had the highest mean number of filled seeds

scored visually as either absent (0) or present (1) for each

under open-pollination, while the lowest number was

of the 45 plants. Only those bands consistently scored

from accession BR (Figure 3).

were considered for analysis. By combining the markers,

Regarding progeny performance, the seed germination

unique profiles were obtained for each individual. Genetic

percentages were above 80% and together with T50 no

diversity in the progeny was estimated using the total

significant differences were observed in filled seeds

number of alleles, number of alleles shared with female

obtained from both self- and open-pollinated conditions

parents (maternal alleles) or deriving from male parents

(Table 3). In all the plants evaluated T50 values showed

(paternal alleles), and percentage of polymorphic loci

that at least half of the seeds (range 50–97%) had

(%P).

germinated by the 3rd day after the trial started, except for

The genetic dissimilarity between the maternal parents

one plant where T50 extended to the 5th day (data not

and their progeny was analyzed using a genetic binary

shown). The survival of both self- and open-pollinated

distance (GD) according to Huff et al. (1993). An

seedlings decreased over time, but the survival of seedlings

UPGMA cluster was obtained from GD matrix. Analyses

derived from open-pollination was significantly higher

of molecular data were performed using GenAlEx 6,

(P<0.001) than those obtained from selfing at both 15 and

Genetic Analysis in Excel (Peakall and Smouse 2012) and

40 days of age. In particular, survival of 40-day-old

Infostat programs (Di Rienzo et al. 2008).

seedlings from self-pollinated panicles was much lower

than that from out-crossing (4.8 vs. 35.7%) (Table 3).

Results

Estimates of total seed production and seedling survival

of plants from accessions of var. makarikariense are shown

Effect of pollination method on progeny number and

in Table 4, differentiating those obtained from the different

seedling survival

methods of pollination. Of the total number of filled seeds

produced per panicle, the probability of them being

The mean number of filled seeds per panicle under open-

produced by open-pollination was greater (92%) than by

pollination was considerably higher than those produced

self-pollination (8%). The estimated survival at 40 days of

under self-pollination (P<0.001, Table 3). In contrast, the

a seedling from a seed obtained by self-pollination was

mean number of empty seeds (empty perfect florets

lower (6%) than the survival of a seedling coming from a

and/or caryopses unable to germinate) did not differ

seed obtained by open-pollination (32%). In addition, the

significantly between the 2 forms of pollination (P =

estimated probability of plant survival for this period of

0.1518, Table 3). In general, the number of filled seeds

time, independently of whether it came from self- or open-

Table 3. Comparison of the average number of filled (n° Fs) and empty (n° Es) seeds per panicle, seed germination percentage (% G), time needed for seeds to reach 50% germination (T50) and seedling survival percentage (% SS) of 15- and 40-day-old seedlings, between self-pollinated and open-pollinated panicles, using the student t-test in P. coloratum var. makarikariense.

n° Fs

n° Es

% G

T50

% SS 15

% SS 40

n

32

32

6

6

6

6

Mean Self

10.9

176.1

88.5

80.1

38.1

4.78

Mean Open

279.2

207.6

81.7

85.6

77.6

35.73

Mean difference

-268.3

-31.5

6.83

-5.48

-39. 5

-31.0

T

-9.13

-1.05

0.91

-1.94

-10.15

-5.80

P

<0.0001

0.1518

0.7980

0.0551

0.0001

0.0011

n: number of genotypes evaluated.

T: Student t-values. P: p value of student t-test.

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

Pollination mode and progeny of Panicum coloratum 123

Figure 2. Number of filled seeds produced per panicle under self-pollination and open-pollination in 32 individuals of P. coloratum var. makarikariense.

Figure 3. Mean number of filled seeds produced per panicle under self-pollination and open-pollination in accessions of P. coloratum var. makarikariense. In each type of pollination, different letters indicate significant differences between accessions (P<0.001) using Fisher’s LSD tests and bars represent standard errors of means (lower case letters refer to open-pollinated and upper case to self-pollinated).

Table 4. Probabilities of seed production (SP) under self- (S) or open-pollination (O) and seedling survival (SS) in P. coloratum var.

makarikariense.

Pollination

Probabilities

UCB

MR

BR

ER

CM

IFF

Mean

Self

p(SPS)

0.29

0.09

0.09

0.11

0.03

0.05

0.08

p(SS/SPS)

0.13

0.00

0.00

0.03

0.00

0.10

0.06

p(SPS/SS)

0.15

0.00

0.00

0.01

0.00

0.02

0.03

Open

p(SPO)

0.71

0.91

0.91

0.89

0.97

0.95

0.92

p(SS/SPO)

0.30

0.23

0.36

0.53

0.17

0.37

0.32

p(SPO/SS)

0.85

1.00

1.00

0.99

1.00

0.98

0.97

-

p(SS)

0.25

0.21

0.32

0.48

0.16

0.35

0.30

p(SS/SPS) and p(SS/SPO): conditional probability of seedling survival given that seed production was under self- or open-pollination, respectively.

p(SPs/SS) and p(SPO/SS): conditional probability of finding a plant given that it was produced under self- or open-pollination, respectively.

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

124 L.V. Armando, M.A. Tomás, A.F. Garayalde and A.D. Carrera

Figure 4. Percentage of SSR alleles attributed to female ( ) and male ( ) parents in progenies of P. coloratum var. makarikariense from 3 accessions (names are detailed in Table 1).

pollination, was 30%. Combining data, the estimated prob-

maternal and paternal alleles could be recognized in

ability of finding a plant obtained by open-pollination was

individual progeny (Table 5). On average, 85% (range

significantly higher (97%) than those produced by self-

67–100%) of individual progeny presented alleles not

pollination (3%).

found in the female parent (Figure 4). Additionally,

offspring were not identical with their maternal patterns

Effects of pollination method on genetic variability and

but they were closely grouped in the dendrogram

contrast with female parents

according to families (Figure 5). Moreover, the 3 female

plants were genetically distinct.

Progeny from 3 female parents were analyzed with 5 SSR

markers. Offspring plants were classified as identical

Chromosome numbers in the P. coloratum var.

when patterns were the same as the female parents at all

makarikariense collection

the evaluated SSR loci or distinct when any band

differences between female parent and progeny were

In all mitotic cells observed, 36 chromosomes were

observed. The SSR analysis showed values of percent

counted at metaphase. This number remained stable for

polymorphic loci (%P) over 50% in progenies, and

all 8 P. coloratum var. makarikariense plants evaluated.

Table 5. Genetic diversity in 3 progeny of P. coloratum var. makarikariense assessed by 5 simple sequence repeat markers (SSR).

Female parent

N° progeny

Total alleles

Maternal alleles

Paternal alleles

%P

UCB3

15

22

14

8

50.0

ER1

15

23

12

11

56.3

IF10

12

25

16

9

62.5

%P: percentage of polymorphic SSR loci in progeny.

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

Pollination mode and progeny of Panicum coloratum 125

Figure 5. Unweighted pair-group method with arithmetical average (UPGMA) dendrogram based on the GD SSR matrix of 3

female parents (F) and their progeny (P) in families of P. coloratum var. makarikariense. The female plants are underlined.

Discussion

production per panicle among genotypes from different

accessions in the collection (Barrios et al. 2010). These

There is an increasing need for a better understanding of

accessions are also distinctive in other morphological

the reproductive biology in P. coloratum, given that this

characters, both vegetative and reproductive (Giordano et

is an extremely important prerequisite to develop breed-

al. 2013; Armando et al. 2013; 2015). In addition, seed

ing strategies and also for germplasm management and

yield is a complex character and additional variability

conservation purposes. In this study, we sought to

arises as seed set is the culmination of a series of process-

determine the preponderant mode of reproduction of one

es at canopy level including radiation interception, bio-

collection of the var. makarikariense used for breeding

mass production and partitioning. Therefore, variability

purposes both by evaluating seed production in open and

among individuals may be the result of both genetic

forced-to-inbreed panicles and by a progeny test using

variation and the integration of these different genetic

SSR markers. The evaluated plants in this study were

backgrounds with the multiple environmental influences

selected to cover the variability present in the germplasm

(Boelt and Studer 2010). The considerable differences in

collection at INTA EEA Rafaela, which comprises

seed production we observed among plants and

accessions collected within semi-arid temperate mesic

accessions suggest a promising scenario for selection to

and subtropical zones, both grazed and non-grazed areas,

increase seed production by means of augmenting the

and a commercial variety. Although we analyzed only 3

number of seeds per panicle.

panicles per accession, our results corroborate previously

The results from seed production, germination and

reported studies pointing out extensive variability in seed

seedling survival assessments in var. makarikariense

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

126 L.V. Armando, M.A. Tomás, A.F. Garayalde and A.D. Carrera

indicated that open-pollination is by far the most frequent

Progeny without clear paternal contribution were

form of pollination in the reproductive biology of this

probably derived from self-pollination or open-

variety. A similar behavior is suggested for var.

pollination involving parents with the same molecular

coloratum based on a small sample of 3 plants (data not

pattern, although this value may be reduced with an

shown). Additionally, as only a few panicles produced

increased number of markers. In addition, the level of

good quality seeds by self-pollination, a limited number

genomic DNA polymorphism of the analyzed progeny

of plants could be analyzed. However, the results from the

suggests sexuality and genetic recombination, and

combined probabilities clearly showed that progeny from

provides additional evidence that P. coloratum is mainly

selfing make low to no contribution to the population over

an allogamous species.

time since a mean of only 6% of the plants obtained from

All our analyses accumulated evidence pointing to

self-pollinated seeds survived. Although seeds obtained

P. coloratum var. makarikariense as a species with

under self-pollination were not weighed, they were

mainly sexual reproduction that depends primarily on

visibly much smaller than and had a different color (white

open-pollination to obtain viable offspring. All var.

vs. brown) from those produced by open-pollination,

makarikariense plants evaluated in this study had 36

which could explain the lower vigor of the seedlings

chromosomes, and the same number was observed in 3

(Tomás et al. 2007). The fact that we did obtain seeds

plants of var. coloratum (data not shown). Considering a

from selfing demonstrates the existence of some degree

basic number of x = 9 (Hamoud et al. 1994), these plants

of self-compatibility. Burson and Young (1983), using

are tetraploids. This ploidy level is one of the most

fluorescence microscopy, demonstrated that, when var.

frequently reported for P. coloratum (Hutchison and

coloratum was self-pollinated, 90% of the pollen germi-

Bashaw 1964; Pritchard and De Lacy 1974). Apomixis

nated within minutes after the pollen grain came in

occurs throughout the plant kingdom and is always

contact with the stigma, but only 2% of the pollen tubes

associated with polyploidy as was reported in Panicum

actually grew into the ovary and entered the micropyle

maximum and Paspalum notatum (Warmke 1954; Quarin

within 1 hour after pollination, suggesting active self-

et al. 2001). However, sexuality also occurs at the 4x and

incompatibility mechanisms. A gametophytic S-Z in-

6x ploidy levels as in Panicum virgatum and Brachiaria

compatibility system has been found in related species

humidicola (Barnett and Carver 1967; Pagliarini et al.

(Martinez-Reyna and Vogel 2002) and is common for

2012). The natural distribution of diploid and tetraploid

most of Poaceae (Baumann et al. 2000). In the majority of

levels in P. coloratum at its center of origin appears to

the grasses this system is not absolute, and some seeds

occur throughout central and South Africa, while the

may be obtained from self-fertilization. Further, selfed

hexaploid level was confined to East Africa (Pritchard

progeny of highly heterozygous genotypes could result in

and De Lacy 1974).

fitness reduction due to inbreeding (Eckert 1994), a point

Knowledge of the chromosome number and ploidy

that we cannot prove with only one generation of selfing.

level of the germplasm is necessary for developing an

Brown and Emery (1958) observed typical sexual 8-

efficient strategy of preservation of this promising forage

nucleate embryo sacs in 82 ovules of var. makarikariense

species and is crucial for use in the P. coloratum breeding

and var. coloratum plants indicating both varieties

program. The ploidy level of this species may partially

reproduced sexually . Hutchison and Bashaw (1964) also

explain the high level of genetic variability in the species

observed 8-nucleate embryo sacs in both varieties but

and the highly polymorphic progeny. New genetic

about 2% of the older ovules had large vacuolated cells,

combinations seem to be obtained relatively easily

which appeared similar to multiple embryo sacs. They

through segregation and recombination. This fact may

considered this as possible evidence of apospory, but

represent an interesting means to increase germplasm

developed embryos were not observed in these cells,

variability and may be an important factor to consider

which dispelled the possibility of apomixis in this species.

when releasing new materials resulting from selection and

The high amount of variation expressed in open-

breeding. In addition, the mode of reproduction in a given

pollinated makarikariense progeny rules out the

species must be clear to the plant breeder to accomplish

possibility of apomictic reproduction in the species. In our

crop improvement. Knowledge about how a plant

study, molecular progeny tests revealed that an average of

reproduces naturally helps the breeder to predict the

85% of offspring possessed paternal alleles; this is

behavior under field conditions and to establish the most

indicative of cross-pollination and 100% of them were

appropriate selection method, which markedly differs

genetically distinguishable from the maternal genotype.

between self- and cross-pollinated crops. Knowledge of

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

Pollination mode and progeny of Panicum coloratum 127

breeding systems is also of benefit for germplasm banks.

Bogdan AV. 1977. Tropical pasture and fodder plants (grasses

Based on our results, the number of individuals destined

and legumes). Tropical Agriculture Series, Longman Group,

for seed increase should be large enough to include the

London, UK.

Brown WV; Emery WHP. 1958. Apomixis in the Gramineae:

original variability in genetic diversity. Moreover,

Panicoideae. American Journal of Botany 45:253–263.

isolation distances need to be taken into account in order

www.jstor.org/stable/2439258

to prevent gene flow among accessions and thus preserve

Burson BL; Young BA. 1983. Pollen-pistil interactions and

the genetic identity of each accession.

interspecific-incompatibility among Panicum antidotale, P.

coloratum, and P. deustum. Euphytica 32:397–405. DOI:

Acknowledgments

10.1007/BF00021448

Cervigni G; Paniego N; Diaz M; Selva JP; Zappacosta D;

Zanazzi D; Landerreche I; Martelotto, L; Felitti S; Pessino

A fellowship for L.V. Armando from the National

S; Spangenberg G; Echenique V. 2008. Expressed sequence

Research Council of Argentina (CONICET) is

tag analysis and development of gene associated markers in

acknowledged. The authors thank M. Pisani, B. Tolozano,

a near-isogenic plant system of Eragrostis curvula. Plant

C. Barrios, M. Giordano, N. Dreher and M. Maina for

Molecular Biology 67:1–10. DOI: 10.1007/s11103-007-

their help in the field assay. We also thank M. Poverene,

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S. Ureta and M. Sartor for their contribution in the

Chistiakov DA; Hellemans B; Volckaert FAM. 2006.

cytogenetic study. Financial support was provided by

Microsatellites and their genomic distribution, evolution,

National Institute of Agricultural Technology (INTA

function and applications: A review with special reference

Rafaela) PNPA 1126072 and the Agencia Nacional de

to fish genetics. Aquaculture 255:1–29. DOI 10.1016/

j.aquaculture.2005.11.031

Promoción Científica y Tecnológica (ANPCyT, PICT

Cook BG; Pengelly BC; Brown SD; Donnelly JL; Eagles DA;

2011-2188). We are grateful to Drs. Byron Burson and

Franco MA; Hanson J; Mullen BF; Partridge IJ; Peters M;

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(Received for publication 30 December 2016; accepted 16 August 2017; published 30 September 2017)

© 2017

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

Tropical Grasslands-Forrajes Tropicales (2017) Vol. 5(3):129–142 129

DOI: 10.17138/TGFT(5)129-142

Research Paper

Screening of salt-tolerance potential of some native forage grasses

from the eastern part of Terai-Duar grasslands in India

Evaluación de la tolerancia a la sal de algunas gramíneas forrajeras nativas

de la parte oriental de los Terai-Duar Grasslands en la India

SWARNENDU ROY1,2 AND USHA CHAKRABORTY1

1 Plant Biochemistry Laboratory, Department of Botany, University of North Bengal, Siliguri, Darjeeling, West Bengal,

India. www.nbu.ac.in

2 Department of Botany, Kurseong College, Dow Hill Road, Kurseong, Darjeeling, West Bengal, India.

www.kurseongcollege.net

Presently: Molecular & Analytical Biochemistry Laboratory, Department of Botany, University of Gour Banga,

Malda, West Bengal, India. www.ugb.ac.in

Abstract

The salt tolerance of 12 native forage grasses from the eastern part of Terai-Duar grasslands was assessed using a rapid

method of leaf disc senescence bioassay. Samples of these grasses were grown in untreated water as well as 100 and 200

mM NaCl solutions for periods of 3, 6 and 9 days. Discs of fresh leaf were then placed in untreated water as well as in

100 and 200 mM NaCl solutions for 96 hours. Quantitative effects were measured as the effects on chlorophyll

concentration in leaves in response to exposure to the varying solutions. From these results, the salt sensitivity index

(SSI) of the individual grasses was determined. The SSI values indicated that Imperata cylindrica, Digitaria ciliaris and Cynodon dactylon were most salt-tolerant of all grasses tested. Further characterization of the grasses was done by observing the changes in 6 biomarkers for salinity tolerance: relative water content, total sugar concentration, proline

concentration, electrolyte leakage, membrane lipid peroxidation and H2O2 concentration following exposure to 100 and

200 mM NaCl concentrations for 3, 6 and 9 days. Finally, hierarchical cluster analysis using the software CLUSTER 3.0

was used to represent the inter-relations among the physiological parameters and to group the grasses on the basis of

their salinity tolerance. The overall results indicated that Imperata cylindrica, Eragrostis amabilis, Cynodon dactylon and Digitaria ciliaris were potentially salt-tolerant grasses and should be planted on saline areas to verify our results.

On the other hand, Axonopus compressus, Chrysopogon aciculatus, Oplismenus burmanni and Thysanolaena latifolia were found to be highly salt-sensitive and would be unsuitable for use in saline areas.

Keywords : Biomarkers, hierarchical cluster analysis, leaf disc senescence bioassay, salinity tolerance.

Resumen

En la University of North Bengal, Siliguri, India, utilizando a nivel de laboratorio un método rápido de bioensayo de

senescencia de discos foliares, fue evaluada la tolerancia a salinidad de 12 gramíneas forrajeras nativas de la parte oriental

de los Terai-Duar Grasslands en la India nororiental. Las gramíneas fueron cultivadas tanto en agua no tratada como en

soluciones de 100 y 200 mM NaCl durante 3, 6 y 9 días. Después se colocaron discos de hoja fresca tanto en agua no

tratada como en soluciones de 100 y 200 mM NaCl durante 96 horas. Los efectos cuantitativos se midieron como la

___________

Correspondence: Usha Chakraborty, Plant Biochemistry Labora-

tory, Department of Botany, University of North Bengal, Siliguri -

734013, Darjeeling, West Bengal, India.

Email: ucnbu2012@gmail.com

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

130 S. Roy and U. Chakraborty

concentración de clorofila en las hojas en respuesta a la exposición a las diversas soluciones. Los resultados, con base

en un índice de sensibilidad a la sal, mostraron que Imperata cylindrica, Digitaria ciliaris y Cynodon dactylon fueron las gramíneas más tolerantes a la salinidad. Además se realizó una caracterización de las gramíneas mediante la

determinación de los cambios en 6 biomarcadores para la tolerancia a la salinidad: contenido relativo de agua;

concentración de azúcar total; concentración de prolina; pérdida de electrolitos; peroxidación lipídica de membrana; y

concentración de H2O2 después de la exposición a concentraciones de 100 y 200 mM NaCl durante 3, 6 y 9 días. El

análisis de conglomerados jerárquicos utilizando el software CLUSTER 3.0 para representar las interrelaciones entre los

parámetros fisiológicos y agrupar las gramíneas sobre la base de su tolerancia a la salinidad mostró que, en general, que

Imperata cylindrica, Eragrostis amabilis, Cynodon dactylon y Digitaria ciliaris fueron gramíneas potencialmente tolerantes a la sal que deberían ser cultivadas en suelos salinos para verificar nuestros resultados. Por otra parte, Axonopus compressus, Chrysopogon aciculatus, Oplismenus burmanni y Thysanolaena latifolia resultaron ser altamente sensibles

a la sal y no son especies apropiadas para uso en áreas salinas.

Palabras clave : Análisis de conglomerados jerárquicos, bioensayo de senescencia de discos foliares, biomarcadores,

tolerancia a la salinidad.

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