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
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
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,
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/
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;
Russell Jessup for their helpful comments on a very early
Schultze-Kraft R. 2005. Tropical Forages: An interactive
version of the manuscript.
selection tool. CSIRO, DPI&F (Qld), CIAT and ILRI,
Brisbane, Australia. www.tropicalforages.info/
References
Di Rienzo JA; Casanoves F; Balzarini MG; Gonzalez L;
Tablada M; Robledo CW. 2008. InfoStat, Versión 2008,
Armando LV; Carrera AD; Tomás MA. 2013. Collection and
Grupo InfoStat, FCA, Universidad Nacional de Córdoba,
morphological characterization of Panicum coloratum L. in
Argentina. www.infostat.com.ar
Argentina. Genetic Resources and Crop Evolution 60:1737–
Ebina M; Kouki K; Tsuruta S; Akashi R; Yamamoto T;
1747. DOI: 10.1007/s10722-013-9982-3
Takahara M; Inafuku M; Okumura K; Nakagawa H;
Armando LV; Tomás MA; Garayalde AF; Carrera AD. 2015.
Nakajima K. 2007. Genetic relationship estimation in
Assessing the genetic diversity of Panicum coloratum var.
guineagrass ( Panicum maximum Jacq.) assessed on the basis
makarikariense using agro-morphological traits and
of simple sequence repeat markers. Grassland Science
microsatellite-based markers. Annals of Applied Biology
53:155–164. DOI: 10.1111/j.1744-697X.2007.00086.x
167:373–386. DOI: 10.1111/aab.12234
Eckert CG. 1994. Inbreeding depression and the evolutionary
Barnett FL; Carver RF. 1967. Meiosis and pollen stainability in
advantage of outbreeding. In: Goldman CA, ed. Tested
switchgrass, Panicum virgatum L. Crop Science 7:301–304.
studies for laboratory teaching. Volume 15. Proceedings of
DOI: 10.2135/cropsci1967.0011183X000700040005x
the 15th Workshop/Conference of the Association for
Biology Laboratory Education (ABLE), 8-12 June 1993,
Barrett SCH; Harder LD. 1996. Ecology and evolution of plant
University of Toronto, Canada. p. 215–238. goo.gl/WGoC8k
mating. Trends in Ecology and Evolution 11:73–79. DOI:
Edwards K; Johnstone C; Thompson C. 1991. A simple and
rapid method for the preparation of plant genomic DNA for
Barrios C; Armando L; Berone G; Tomás A. 2010. Seed yield
PCR analysis. Nucleic Acids Research 19:1349. DOI:
components and yield per plant in populations of Panicum
coloratum
L.
var.
makarikariense
Goossens.
7th
Giordano MC; Berone GD; Tomás MA. 2013. Selection by seed
International Herbage Seed Conference, Dallas, TX, USA,
weight improves traits related to seedling establishment in
11–13 April 2010. p. 152–158. https://goo.gl/UNQy6w
Panicum coloratum L. var. makarikariense. Plant Breeding
Baumann U; Juttner J; Bian X; Langridge P. 2000. Self-
132:620–624. DOI: 10.1111/pbr.12106
incompatibility in the grasses. Annals of Botany 85:203–
Hamoud MA; Haroun SA; Macleod RD; Richards AJ. 1994.
209. DOI: 10.1006/anbo.1999.1056
Boelt B; Studer B. 2010. Breeding for grass seed yield. In:
Cytological relationships of selected species of Panicum L.
Boller B; Posselt UK; Veronesi F, eds. Fodder crops and
Biologia Plantarum 36:37–45. DOI: 10.1007/BF02921265
amenity grasses. Handbook of Plant Breeding 5. Springer
Huff DR; Peakall R; Smouse PE. 1993. RAPD variation within
New York, USA. p. 161–174. DOI: 10.1007/978-1-4419-
and among natural populations of outcrossing buffalograss
[ Buchlöe dactyloides (Nutt.) Engelm.]. Theoretical and
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
128 L.V. Armando, M.A. Tomás, A.F. Garayalde and A.D. Carrera
Applied Genetics 86:927–934. DOI: 10.1007/BF00211043
analysis for biologists. Cambridge University Press, UK and
Hutchison DJ; Bashaw EC. 1964. Cytology and reproduction of
New York, USA.
Panicum coloratum and related species. Crop Science
Quinn J. 1998. Ecological aspects of sex expression in grasses.
4:151–153. DOI: 10.2135/cropsci1964.0011183x0004000
In: Cheplick GP, ed. Population biology of grasses.
Cambridge University Press, UK and New York, USA.
Liu L; Wu Y. 2012. Development of a genome-wide multiple
Stebbins GL. 1950. Variation and evolution in plants. Columbia
duplex-SSR protocol and its applications for the identi-
University Press, New York, USA.
fication of selfed progeny in switchgrass. BMC Genomics
Tischler CR; Young BA. 1987. Development and character-
13:522. DOI: 10.1186/1471-2164-13-522
istics of a kleingrass population with reduced post-harvest
Maina M; Armando LV; Tomás MA. 2017. Understanding
seed dormancy. Crop Science 27:1238–1241. DOI:
shattering process: Differences in seeds characteristics
10.2135/cropsci1987.0011183x002700060030x
shattered at different times in Panicum coloratum var.
Tischler CR; Ocumpaugh WR. 2004. Kleingrass, blue panic
makarikariense. 9th Conference of the International
and vine mesquite. In: Moser LE; Burson BL; Sollenberger
Herbage Seed Group, Pergamino, Argentina, October 2017.
LE,
eds.
Warm-season
(C4)
grasses.
Agronomy
Martinez-Reyna JM; Vogel KP. 2002. Incompatibility systems
Monograph. American Society of Agronomy, Madison, WI,
in switchgrass. Crop Science 42:1800–1805. DOI:
USA. p. 623–649. DOI: 10.2134/agronmonogr45.c18
Tobias CM; Hayden DM; Twigg P; Sarath G. 2006. Genic
Núñez O. 1968. An acetic-haematoxylin squash method for
microsatellite markers derived from EST sequences of
small chromosomes. Caryologia 21:115–119. DOI:
switchgrass ( Panicum virgatum L.). Molecular Ecology
10.1080/00087114.1968.10796290
Notes 6:185–187. DOI: 10.1111/j.1471-8286.2006.01187.x
Pagliarini MS; Valle CB do; Vieira MLC. 2012. Meiotic
Tomás A; Berone G; Pisani M; Ribotta; Biderbost E. 2007.
behavior in intra- and interspecific sexual and somatic
Relación entre peso de semillas, poder germinativo y
polyploid hybrids of some tropical species. In: Swan A, ed.
emergencia de plántulas en clones de Panicum coloratum L.
Meiosis ‒ molecular mechanisms and cytogenetic diversity.
Revista Argentina de Producción Animal 27(supl. 1):205‒
INTECH Open Access Publisher. p. 331-348. DOI:
Tomás A; Giordano M; Pisani M. 2015. Effect of increasing
Peakall R; Smouse PE. 2012. GenAlEx 6.5: Genetic analysis in
temperatures on germination of two varieties of Panicum
Excel. Population genetic software for teaching and
coloratum. 8th Conference of the International Herbage
research – an update. Bioinformatics 28:2537–2539. DOI:
Seed Group, Lanzhou, Gansu, China, 21-30 June 2015.
Wang Y; Samuels T; Wu Y. 2011. Development of 1,030
Pritchard AJ; De Lacy IH. 1974. The cytology, breeding system
genomic SSR markers in switchgrass. Theoretical and
and flowering behaviour of Panicum coloratum. Australian
Applied Genetics 122:677–686. DOI: 10.1007/s00122-010-
Journal of Botany 22:57–66. DOI: 10.1071/BT9740057
Quarin CL; Espinoza F; Martinez EJ; Pessino SC; Bovo OA.
Warmke H. 1954. Apomixis in Panicum maximum. American
2001. A rise of ploidy level induces the expression of
Journal of Botany 41(1):5–11. DOI: 10.2307/2438575
apomixis in Paspalum notatum. Sexual Plant Reproduction
Young BA. 1986. A source of resistance to seed shattering in
13:243–249. DOI: 10.1007/s004970100070
kleingrass, Panicum coloratum L. Euphytica 35:687–694.
Quinn GP; Keough MJ. 2002. Experimental design and data
(Received for publication 30 December 2016; accepted 16 August 2017; published 30 September 2017)
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Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
Tropical Grasslands-Forrajes Tropicales (2017) Vol. 5(3):129–142 129
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.
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.
Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)
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)
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