UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD

COLLEGE OF AGRICULTURE, DHARWAD - 580005

Programme of Research on

Physiology of salinity stress tolerance in guinea grass (Megathyrsus maximus) varieties.

1. Name of the student Miss. APOORVA B. KAWADIKAI

2. I.D. No. & Year of Admission PGS17AGR7441, 2017-18

3. Degree programme M.Sc.(Agri.) Crop Physiology

4. College College of Agriculture, Dharwad

5. The tentative title of the thesis Physiology of salinity tolerance in Guinea grass
(Megathyrsus maximus(Jacq.)varieties

1.Introduction and scope of this study

Guinea grass, Megathyrsus maximus(Jacq.) is one of the productive grasses which is

valuable for pasture, green fodder, hay and silage. The average yield is about 18-29 tons/ha.
(Fernandis et al 2013). It can be combined with the herbaceous legume for increased
nutritional benefits in animals. Guinea grass is free from significant pest or diseases. It is a
fast-growing bulky grass and provides rapid ground cover. Hence it helps in prevention of
soil erosion too.

Today India faces an acute fodder shortage to the tune of 63.5%. (Jitendra, 2016) that
has left farmers and life stock vulnerable. The cost of green fodder increased by three times
between 2011-2016. The amount of milk produced depends on the amount of green fodder
the animal consumes and simultaneously it brings down the cost of milk production by
cutting the cost of concentrate feed. The area under fodder crop cultivation is very less, and
the animal population is high. There are no enough fodder to feed the cattle leading to
distress sales, farm suicides and disturbances in farming communities especially in drought-
hit states like UP, Maharashtra, Telangana and Karnataka.

Fertile and productive lands in India are preferred for the cultivation of food crops, cash crops
and vegetables. Such productive lands are not available for fodder crops cultivation. Instead,
waste and unproductive lands are preferred for the fodder crop cultivation in India. India has -
17.29- million ha of uncultivable land, and saline lands occupy 5.95 million ha (Kumar,
2005). Saline soils can be utilised for fodder crop cultivation to meet the growing demands of
fodder.

Plants require physiologically tuned in mechanisms to grow under saline condition.

Halophytes are naturally adapted to grow under saline environments and other crops tolerate
or resist salinity tolerance through morphological, physiological, anatomical and molecular
means. Among the monocots, Rhodes grass is considered as a tolerant grass to salinity due to
the presence of salt glands that excrete excess salts (Kobayishi and Masaoka,2008). However,
the yield reported for Rhodes grass on an average is 20 ton /ha (Arshad, 2015 and Mohamad
Osman et al, 2013) and is very low compared to Guinea grass yield.

Guinea grass is a nutritious grass fodder grown in all parts of India. Over the years of
forage development throughout the world, Scientists selected bred for high yielding guinea
grass varieties. Indian Grassland and fodder research Institute, has >200 accessions of guinea
grass. Several varieties like BG1, BG2, BG4, DGG1 etc. were released for cultivation all
over India. However, none of these varieties was tested for its potential to yield greenfodder
under saline conditions. Hence this study will be conducted to understand the adaptability of
guinea grass varieties under saline conditions.

Green foliage and green stems are the economic part of any forage plant. The abiotic
stress of any kind induces senescence in crops, and the forage crops are no exception to it.
Accelerated senescence under stress is an escape strategy in plants to avoid the unfavourable
condition (Sade et al., 2018). In perennial grasses, senescence can remove the green foliage
which is the economically relevant part. Reactive oxygen species (ROS ) produced during
any stress causes oxidation of proteins, inactivation of enzymes disruption of membranes
(Jajic et al., 2015) leading to senescence and death of plants. The induction of antioxidant
defence enzymes can protect against this damage. Hence for this study, we hypothesise that
varieties which have higher antioxidant gene and enzyme activity will have a lower level of
senescence and survive under higher salinity stress and yield on par with plants under non-
saline conditions.

2. Objectives of this investigation :

1) To screen varieties of guinea grass for salinity tolerance.

2) To establish a relationship between senescence and antioxidants at gene and

enzyme level in guinea grass under salinity stress.

.
3. Review of literature:-

3.1. Morphological parameters

Global climate change causes an increase in saline areas and salinity stress is becoming
one of the ruinous stresses faced by plants limiting plant growth and productivity. Soil
salinity can affect the plant morphological characters, especially in crop plants. When turf
grasses were irrigated with sea water having 72 ds/m EC, shoot growth decreased and the
% decrease was genotypic specific (Uddin et al 2011). The shoot fresh weight and dry
weight decreased in Paspalum and Bermuda grass with an increase in NaCl salinity
(Pessarakali and Touchane 2006). Among the turf grasses Paspalum vaginatum, Zoysia
matrella, Paspalum vaginatum local), Cynodon dactylon, Eremochloa ophiuroides),
Axonopus compressus studied for salinity stress, Paspalum vaginatum were always found
to have less decrease in shoot weight when growth under salinity conditions implying the
salinity tolerance varies in plants belonging to different genus(Uddin et al 2009). In
Miscanthus biomass yield was reduced by 50% at 10.65 dS m-1 NaCl compared to no
stress control (Stavridou, 2017). Some genus of grasses can survive and grow well under
salinity stress whereas some are sensitive to salinity in terms of biomass reduction.

Leaves are the organ that intercepts radiation and houses the photosynthetic machinery in
plants. Reduction in leaf growth is an earliest visible symptoms of salinity stress. Leaf
area decreased in five hybrids of canola with salt treatment in comparison with control
(Bacarin et al 2011). Leaf firing or leaf drying in turf grasses increased with increasing
salinity and was observed to be species dependent (Uddin et al 2011). Leaf lengths of
ornamental grasses Briza media, Deschampsia cespitosa, Koeleria glauca, Sesleria
caerulea and Sorghastrum nutans decreased significantly when irrigated with water
containing NaCl (Hensehke 2016). Percentage of green tissue in Buffalo grass cultivars
declined steadily with the increase in salinity(Zhang et al 2012). These studies reveal that
photosynthetic leaf surface area is very sensitive to salinity stress causing a significant
reduction in leaf area.

A tolerance criterion commonly used in salinity studies is the salinity level that results in
50% shoots dry weight reduction relative to the control (Uddin et al 2011). Hence stem
dry weight observation is of great importance when evaluating genotypes for salinity
 tolerance. The plant dry weight reduced with increased NaCl concentration (Bacarin et
al.2011) in canola. The above ground biomass in Paspalum and Bermuda grass was
affected more severely than the shoot length and root length as the exposure time to salt
stress progressed and Bermudagrass was more severely affected than Paspalum under
any levels of NaCl applications (Pessarkli and Touchene, 2006). Turfgrass species also
recorded 50 % shoot growth reduction at salinity ranging from 18-36 ds/m (Uddin et al
2011). The variations in morphological parameters due to varying levels of salinity
indicate the importance of measuring these parameters in any salinity related
experiments. Hence it is essential to study the morphological parameters to understand
and visualize the effect of salinity stress in the grass.

3.2. Physiological parameters

Physiological parameters like photosynthesis, fluorescence, Membrane stability index,
Relative water content are good indicators of plant salinity tolerance. The net
photosynthesis rate (Pn) and stomatal conductance (gs) decreased rapidly with increasing
of salinity-alkalinity stress in Maize. The intercellular CO2 concentration (Ci) decreased
at low levels of stress and vice versa at high-stress levels. The maximal efficiency of
photosystem II photochemistry (FV/FM) decreased only at high-stress levels implying
that stomatal limitation is the main reason of decreased photosynthesis rate at low-stress
levels, and non-stomatal limitation, i.e. decreased photosynthesis activity in PSII leads to
decreased photosynthesis rate at high-stress level (Deng et al 2010). In wheat under
greenhouse conditions, under control, 8,12 and 16 Ec, chlorophyll fluorescence related
parameters like the efficiency of photosystem II from light adopted leaves (ΦPSII),
Photochemical quenching (qP), the quantum yield of dark-adapted leaves (fv/fm) and
non-photochemical quenching (NPQ) were affected. Nonphotochemical quenching by
salinity caused a decrease in shoot dry matter (Abdeshahian et al 2010). According to
Kafi, 2009 although photosynthesis in wheat was well correlated with stomatal
conductance, wheat genotypes recorded differential response to Fv/fm and respiration
rate, in different salt concentrations, as growth stage, time of the day and duration of salt
exposure. In C4 estuarine grasses, growth under salinity did not affect the maximum
intrinsic efficiency of photosystem II, suggesting salinity had no effect on photosynthesis
through inactivation of PSII reaction centres (Maricle et al, 2007). In estuarine C4
grasses, even when net rates of CO2 uptake (PN) was saturated at ~500 μmol(quantum)
m–2 s–1, gross rates of O2 evolution (JO2) continued to increase up to full sunlight,
indicating that carbon assimilation was not tightly coupled to photochemistry in these
halophytic species implying an alternative electron flow under high irradiance in these
halophytes for dissipating excess energy (Maricle et al 2007). The inhibition of
photosynthesis in seashore paspalum and centipedegrass under salinity stress was due to
both stomatal closure and non-stomatal factor (Liu et al, 2011). In a rocket, under salinity
stress, photosynthetic rate (Pn) decreased at 100 mmol/L NaCl and a further increase of
salt concentrations did not affect photosynthesis rate (Hnilickovanet et.al, 2017). In
poaceous halophytes C4 Spartina alterniflora and C3 Phragmites australis maximal net
assimilation rates of appeared with 400 and 100mmol L-1 NaCl, respectively. Although
there was no significant change in maximum quantum yield of photosynthesis in C 3 and
C4 grasses, the instant quantum yield of PSII decreased in C3 grass whereas it increased in
the C4 grass. This may be due to fact that the electron transfer from photosystem and the
energy use efficiency being limited in the C3 P. australis grown under high salinity(Zhou
et al 2015). Whereas photosynthesis was reduced to only a smaller extent in Miscanthus a
bioenergy crop, under salinity (Stavridou et al 2017), Thus, Chlorophyll fluorescence
indicates the degree of nonstomatal limitations of photosynthesis under saline stress in
planta.

Salinity stress significantly decreased RWC in all the turfgrass species (Zulkaliph et.al
2017). Salinity stress reduced relative water content (RWC) also in five Coleus species
(Kotagori and Kolluru, 2017), and in a rocket plant (Hnilickovanet et.al 2017). In
turfgrass species s salinity increased, RWC decreased. However, salinity decreased RWC
only after 24 dsm-1 compared to control in turfgrass species (Uddin et. al., 2009)

Chlorophyll content in the leaves determines the stay green quality in any grass species.
Loss of chlorophyll can be correlated to senescence. Chlorophyll loss due to abiotic stress
leads to premature leaf senescence in plants altering source-sink relationship (Sade et al
2017). During senescence, the Chlorophyll a/b ratio gives a valuable indication of the
underlying process of chloroplast degradation (Bresson et al 2018) wherein Chlorophyll
a is linked to energy processing centres of the photosystems (PS) and Chlorophyll b is
considered an accessory pigment that transfers light energy to Chlorophyll a. Senescence
can affect the Chlorophyll a/b ratio differently depending on the species ( Bresson et al
2018). In Arabidopsis, an increase in Chlorophyll a/b ratio was observed during darkness
induced senescence and in late senescence stage in Barley. Whereas, a decrease was
 observed in Phaseolus vulgaris and Acer pseudoplatanus (Bresson et al 2018). A strong
positive correlation (R2 =0.93 ) and the statistically significant relationship was observed
between SPAD-502 values and total chlorophyll content under salinity stress in wheat
(Shah et al 2017). In an experiment conducted fodder grasses at varying levels of salinity
ranging from 0-50 ds/m-1), it was observed that the amounts of both chlorophyll a and b
increased with the increase in salinity suggesting that the chlorophyll was not more active
or efficient during photosynthesis and amount of chlorophyll b was more than chlorophyll
a (Varshney and Baijal,1977). In turfgrass species, salinity up to 24dSm−1 did not affect
chlorophyll-a content. However different species recorded loss of chlorophyll b content at
low levels of too high levels of salinity (Uddin et al 2009). Zahra et.al. (2017) conducted
an experiment to evaluate and compare pistachio based on physiological parameters
under salinity stress. It was observed that there was a reduction in chlorophyll a,
chlorophyll b, total chlorophyll and carotenoids content at high saline concentration due
to cultivar, stress and their interactions.
3.3. Biochemical parameters

Plant Stress tolerance is positively related to the antioxidant defence systems in

plants. Plant antioxidant systems consist of various antioxidant enzymes like catalase,
peroxidase and superoxide dismutase and metabolites like Ascorbate and glutathione.
Antioxidant capacities protect the plants from oxidative damage caused by different stress.
Hence an understanding of the level of expression of antioxidant enzyme system is empirical
to understand the stress mitigation strategies and senescence induced due to abiotic stress.

ROS overaccumulation was observed in salt-sensitive barnyard grass compared to

tolerant ones (Abogdallah 2010). Under salt stress, the SOD activity significantly increased in
all genotypes of barnyard grass but there were no significant differences in the activity
between the green and chlorotic leaves except in the wild-type where it was lower in the
chlorotic leaves. Similarly, POD activity also increased in the chlorotic and green leaves of
the stressed plant when compared to the control. However, the activity was higher in green
than in chlorotic leaves one (Abogdallah 2010). Catalase enzyme activity was significantly
lower in unstressed plants compared to saline stressed plants of Barnyard grass. The activity
was more in chlorotic leaf compared to green leaf. This study in Barnyard grass implies that
there is an overall increase in ROS activity in sensitive genotypes and peroxidase and catalase
activity was highest in senescing leaves (Abbogdallah, 2010). Under salinity stress, salt
tolerant rice cultivars exhibited much lower lipid peroxidation, maintained elevated levels of
reduced ascorbic acid and showed increased activities of the APX and SOD enzymes system
during seedling stage and reproductive stage (Moradi and Ismail,2007). Kurup et al (2011)
conducted an experiment to evaluate the antioxidant potential of turfgrass species which was
used for urban land scaping in the United Arabian Emirates. They found that initially there
was an increase in POD activity but there was a gradual reduction upon an increase in salinity
level. Saline H2O irrigation brought a significant effect on antioxidant enzyme system which
imparts oxidative stress tolerance in turfgrass species.

Salt tolerance was attributed to induced antioxidant genes leading to the stimulation of
antioxidant enzymes and protection during salt stress in Perennial ryegrass (Hu et al 2012).In
miscanthus grass, a bioenergy crop, strong induction of the osmoprotectant, proline was
observed under increasing salinity. Whereas there was no significant increase in
malondialdehyde content under increasing salinity in miscanthus (Stavirdou,2017). Salt
tolerance studies in rice suggest that that the increase in proline concentration may not be
associated with salinity tolerance (Moradi and Ismail,2007)

Zulkaliph et.al (2017) observed an increase in the Na+ concentration and decrease in
K+ concentration with salinity stress in turfgrass spp. The tolerance in Kentucky bluegrass
spp. was due to increased accumulation of Na+ to K+ ratio (Qian et al, 2001). Na+ in the leaf
accumulated in salinity stressed plant which led to necrosis of the leaves (Parvaiz. et.al
2008).
In pistachio, increase in salinity concentration caused an increase in proline and total
soluble sugars. The Na+ concentration in plant organ was proportional to NaCl content in the
medium. There was a decrease in Fe and Pi content. The tolerant variety showed better
growth by accumulating more osmolytes, less toxic Na ion and lower Na+/K+ ratio in shoots
(Raheshneshan et. al.2017). Zan et. al., (2011) conducted an experiment to determine the
effect of salinity stress on photosynthetic productivity. They found that the photosynthetic
rate, stomatal conductance and K+ content reduced significantly following increased NaCl
concentration. There was an increase in Na+ Content in leaf increase in NaCl concentration.
Zan et. al., (2011) conducted an experiment to determine the effect of salinity stress on
biochemical studies. They concluded by saying that there was a significant accumulation of
lipid peroxidation and proline content in leaves which helped in osmotic adjustment and
protection of membrane stability during severe salt stress. These character of the plants
helped to differentiate the oat grass as salinity sensitive and salinity tolerant.

Ghane et. al., (2014) conducted an experiment to check the biochemical response of
Niger cultivar under salinity stress. They said that the Na+ increased in both cultivars with an
increase in salt concentration. The K+ content was decreased with respect to salt
concentration when compared to control plants. Osmolytes like proline glycine betaine, TSS
content was increased in salt stress condition. There was a severe damage to membrane lipids
due to salinity. The enzyme SOD activity was increased initially but then there was a
reduction as an increase in salinity level. The APX activity was increased but the catalase
enzyme activity was decreased under different salt concentration.

4. Detailed Programme of Research

4.1 Experimental detail:

Crop Guinea grass

Genotype BG1, BG2, DGG-1, BG4, C0-3, Rivers dale
Treatment Contol, 4EC, 8EC, 12EC
Location Southern Regional Research Station, Indian
Grassland and Fodder Research Institute
Dharwad
Replication 3
Design Pot culture in Factorial RBD

4.2. Preparation of saline soil:

As procurement of natural saline soils with varying salinity and natural saline areas near the
site of the experiment was unavailable, artificial saline soils were prepared based on the
USDA Agriculture handbook no 60. Containers which can hold 60 kg of soil was used for
each treatment. The containers were without any drainage holes to ensure retention of salts in
the soils and not to be washed away due to irrigation. The soil salinity created based on a
percentage of salt ratio was allowed to uniformly distribute in the soil in the container for
more than 45 days by subjecting it to repeated cycles of irrigation and evaporation.

The desired treatments were 0, 4, 8,12 Ece. A combination of salts of NaCl, Na2SO4, MgCl2,
and CaSO4, in the salt ratio: 13:7:1:2 respectively was used to prepare saline soils. The soil
used for the experimental purpose was dried properly without any moisture content and its
initial Ece was checked. This existing soil Ece was subtracted from the desired treatment Ece
and it was used for calculating the salt required for creating the salinity. The dried soil was
weighed and filled in the container. The percentage of salt required to create the required
salinity in Ece for 1080 kg soil was calculated after converting the ECe into ppm and then
from PPm to percentage.

(* There were 18 containers containing 60 kg of soil for 4 ECe treatment (six varieties and 3
replications, 6 x3 =18 The soil required for 18 containers is 1080 kg)

Amount of salts(Kg) required for 1080 Kg soil =(((Ece x 640)/10000)x 1080 kg))/100) kg
=A

The percentage of salts for desired Ece in the ratio13:7:1:2 was calculated

% of salts= (salt ratio X Kg of salt(A)) / (total salt ratio)

The salt was mixed in the soil and 60 kg soil was filled in containers. The soil in the container
containing added salts was irrigated every alternate day with the known amount of water for
almost 60 days for the salt to disperse well in the soil. The salts added will dissolve and
disperse in the soil due to repeated irrigation and drying. After 45 days of repeated irrigating
and evaporation or before planting, soil Ece was measured.

The individual salt weight for 1080 kg of soil used in this experiment is shown in the table

kg of salt /1080 Kg soil

EC (ds/m) NaCl Na2SO4 MgCl2 CaSO4
0 0 0 0
0 (control)
1.56 0.84 0.12 0.24
4
3.12 1.68 0.24 0.48
8
4.29 2.3 0.33 0.652
12**
** The soil used for creating 12 Ece had already 1 Ece in the soil. Hence the calculation for salts was made for
11 Ece. Whereas soil used for other treatments recorded negligible Ece. Hence calculations were made for 4 and
8 Ece only
4.3. Observations to be recorded

4.2.1. Morphological observations:-

i. Plant height
ii. No of tillers at 30-45 days of interval
iii.Leaf area at 30-45 days of interval
iv. Fresh weight at harvest
v. Dry weight after harvest
vi. Regeneration

4.3.2. Physiological parameters

i. Photosynthesis rate
ii. Light utilization efficiency
iii. RWC
iv. Fluorescence
v. Senescence

4.3.3. Biochemical and molecular studies

i. The activity of Super oxide dismutase
ii. The activity of peroxidase
iii. The activity of catalase
iv. Chlorophyll content and stability index
v. K –Na ratio in leaves.
vi Isoenzyme pattern
vii gene expression studies

4.3.4. Anatomical observations

i. Presence of salt glands

5. Materials and methods:

5.1.Morphological observation:-

5.1.1. Plant height:

Plant height will be measured from the base of the plant to the tip of the terminal leaf at 30-
45 days of interval and expressed in cm.
5.1.2. Fresh weight:-
The plants will be harvested by leaving 10 cm of the stem. Leaves and stem will be separated.
Fresh weight of leaf and stem will be weighed separately and expressed as g plant-1. The
observation will be taken at 45 days interval.

5.1.3. Dry weight:-

The dry weights of oven dried stems and leaves will be recorded at harvest and expressed as
g plant-1.

5.1.4. No.of tillers:-

No of tillers at 30-45 days of the interval of tillers each plant will be counted.

5.2. Physiological observations:

5.2.1. Bio-physical observations (IRGA)
The index leaf (third leaf from top) will be selected to measure net photosynthetic
rate, transpiration rate, fluorescence, light utilization efficiency and stomatal conductance by
using IRGA (Infrared gas analyser) of LICOR 6400-40 XT Portable Photosynthesis System.
5.2.2. Relative water content:-
It is a measurement of leaf hydration status relative to its maximal water holding
capacity at full turgidity. Leaf punches were collected from the third leaf from the top of the
primary branch of each genotype, and are floated in water for 6 hours and allowed to gain
turgidity. Turgid weights are recorded and dried in hot air oven at 80°C to a constant weight
to record dry weight. RWC is estimated and expressed in per cent using the following
formula.

Fresh weight – Dry weight

RWC (%) = ______________________ × 100
Turgid weight - Dry weigh

5.2.3. Senescence
Whole plant senescence due to stress observed in annual plants is not observed in
Perennial plants. The perennial plants tend to keep the upper leaves green and senescence
may occur in 4, 5th 6th or 7 the leaves in a tiller based on the total number of leaves.
Senescence will be recorded using SPAD readings in all the leaves in a tiller. On an average
35-40 tillers, reading will be taken an average of readings will be taken. These spad readings
were plotted on the y-axis with leaf position on the x-axis. The difference in the SPAD values
of lower leaves indicates the level of senescence.

5.2.4. The membrane injury index:

The membrane injury index (%) will be measured according to Suvillian test (1972)

Procedure:

Five leaf discs of the test samples will be taken in a test tube with 10 ml of distilled water.
The test tubes will be covered and kept at room temperature for one hour. Then the test tubes
will be subjected to two high temperatures i.e. 45ºC for 30 minutes and 100ºC for 10
minutes in a water bath and autoclave respectively. The respective EC will be recorded at
room temperature (ECa), after exposer to 45ºC (ECb) and 100ºC (ECc). Membrane injury
index will be calculated by using the following formula.
ECb - ECa
Membrane injury index (%) = ------------------× 100
ECc

5.2.5. SPAD value:

SPAD (Single Photoelectric Analyzing Device) is a simple diagnostic tool that gives the
chlorophyll content and greenness in leaves in terms of SPAD values.

5.3. Anatomical observations:

5.3.1. Presence of salt glands

Salt glands are highly specialized organs which consist of several cells that accumulate salts
and often burst to release their content. Located in the slight depression of leaf epidermis and
covered by the cuticle predominantly excrete cl-, Na+ .salts glands or any salt secreting cells
will be observed using scanning electron microscope. (Pathan et. al., 2009)

5.4. Biochemical and molecular observations:

5.4.1. Assay of super oxide dismutase:

SOD was assayed according to the method of Kakkar et al., (1984). The assay of SOD is
based on the inhibition of the formation of NADH-phenazine methosulphate-
nitrobluetetrazolium formazon. The colour formed at the end of the reaction can be extracted
into butanol and measured at 560nm. One unit of enzyme activity is defined as the amount of
enzyme that gave 50%inhibition of NBT reduction in one minute.

5.4.2. Assay of catalase:

Catalase activity was assayed following method of Luck (1974). The UV absorption of
hydrogen peroxide can be measured at 240nm, whose absorbance decreases when degraded
by the enzyme catalase.from the decrease in absorbance, the activity can be calculated. One
unit was calculated as the amount of enzyme required to decrease the absorbance at 240nm
by 0.05 unit.

5.4.3. Assay of peroxidase:

The method proposed by Reddy et al. (1995)was adopted for assaying the activity of
peroxidase. In the presence of the hydrogen donor pyrogallol, peroxidase converts hydrogen
peroxide to water and oxygen .The oxidation of pyrogallol to a coloured product called
purpurogallin can be followed spectrophotometrically at 430 nm. One unit of peroxidase is
defined as the change in absorbance /minute at 430nm.

5.4.4. Isoenzyme identification:

Antioxidant isoenzyme will be analysed on native PAGE with 5 per cent of stacking
gel and 10 per cent of running a gel with an equal amount of soluble protein per lane
(Lamelli, 1970).

SOD activity in the gels will be visualised. Gels will be incubated in 50 mM sodium
phosphate buffer (pH 7.8) containing 2.5 mM NBT for 20 min. In dark at room temperature
and then washed 3 times with ice-cold distilled water followed by soaking gels in 28µM
riboflavin and 28mM tetramethyl ethylenediamine (TEMED) in 36 Mm sodium phosphate
buffer (7.8) for another 20 min. Finally, gels will be incubated at 50 Mm sodium phosphate
buffer (pH 7.8) containing 0.1 mm EDTA with gentle agitation and exposed to cool white
fluorescent lamps (40µM m-2s-1) until the enzymes appeared as colourless bands in a purple
background.

For the staining of peroxidase isoforms, the gel will be incubated in the dark reaction
solution containing 25mM sodium buffer (pH 7) for 15 min. The gell will be immersed again
in a freshly prepared solution containing 18mM guiacol and 25 mm hydrogen peroxide in
25mM sodium phosphate buffer (Ph 7) until the peroxidase activity containing brown band
visualized clearly.
5.4.5. Estimation of Na+ to k+ ratio :

5.4.5.1. Estimation of potassium in leaf :

Potassium content in leaf was determined by flame Photometer method (Jackson, 1973).
The digested extract was used for determination of K content in leaf by Flame Photometer
using the standard curve and expressed as total K (%).

Total K % = R× dilution factor/10000

R = Flame Photometer reading.

5.4.5.2. Estimation of sodium in leaf :

The sodium content in leaf sample was determined by Flame photometer method (Jackson,
1973). The digested extract was used for determination of Na content in leaf by Flame
Photometer using the standard curve and expressed as total Na (%).

Total Na % = R× dilution factor /10000

R = Flame Photometer reading

5.4.6 Gene expression studies

Gene expression studies will be conducted using total RNA using specific primers at Specific
temperatures through single tube RT-PCR.

6. The Special features of this investigation:

This study will elucidate physiological mechanism involved in salinity tolerance in guinea
grass varieties and correlate senescence with the activity of antioxidants. If correlated, the
antioxidant activity can be used as a physiological marker for selecting stay green Guinea
grass genotypes under stress condition.

7. Statistical analysis

The data obtained from the experiment will be subjected to statistical analysis
using Randomized Complete Block Design (RCBD). Interpretation of the data will be
carried out in accordance with Gomez and Gomez (1984). The levels of significance
used in the ‘F’ and‘t’ test was P=0.05. The critical difference values will be calculated
 wherever the ‘f’ test values are significant. The treatment means will be compared by
applying Duncan’s multiple range test (DMRT).

The mean values of treatments subjected to Duncan’s Multiples Range Test

(DMRT) using the corresponding error mean sum of squares and degrees of freedom
values at five per cent probability under MSTATC programme.

8. Time schedule of activities in proposed research work

2018-19
June July Aug. Sep. Oct . Nov. Dec. Jan . Feb. Mar. April
Activity 1 *
Activity 2 * * *
Activity 3 * *
Activity 4 * * * * * * *
Activity 5 *
Activity 6 * *

Activity 1 : Transplanting.
Activity 2 : Morphological parameters and senescence.
Activity 3 : Physiological parameters.
Activity 4 : Biochemical analysis and gene expression studies.
Activity 5 : Complilation and analysis of data.
Activity 6 : Thesis writing, corrections, typing, circulation and submission.

9. References

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reactive oxygen species in the salt tolerant mutants of barnyard grass and their
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Arshad, I., 2015, Performance of Different Rhodes Grass Varieties under the Agro-Climatic
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Physiol., 23:245-253.
Breson, J., Bieker, S., Riester, L., Doll, J., Zentgraff, U., 2018, A guidline for leaf senescence
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