Available online at http://www.urpjournals.

com

International Journal of Research in Plant Science

Universal Research Publications. All rights reserved

ISSN 2249-9717
Original Article
STUDY THE EFFECT OF SALT STRESS ON ANTIOXIDANT ACTIVITY INMillet Sps.
RazaMazahir, Shukla Anshul Kumar, Fatima Tatheer.
R& D Center, AMA Herbal Laboratories (P) Ltd, Lucknow, India
Email-m.raza@amaherbal.com, anshul.shukla@amaherbal.in, tatheer@amaherbal.in
Received 27 February 2015; accepted 26 March 2015
Abstract
The present investigation was made to study the effect of salt stress on the antioxidant activity of the seedlings of species
of Millet. The seedlings of millet species (Panicummiliaceum) were grown hydroponically. Part of
Panicummiliaceumtreated with salt (NaCl5gm for 35 days) and the remaining was untreated (Control plant). The upper
limit for the survival of the seedlings wasupto 600 mMNaCl. The maximum amount of fresh and dry weight was recorded
at 600 mMNaCl concentration. Beyond 600mMNaCl, the growth parameters reduced drastically.The proline content
increased with the increasing concentration of the salt.The antioxidant enzymes such as catalase (CAT), peroxidase (POX),
superoxide dismutase (SOD)and polyphenol oxidase (PPO), ascorbate oxidase increased up to the optimum level in saline
condition when compare to control plant and beyond these levels the contents decreased marginally.
© 2015 Universal Research Publications. All rights reserved
Key words: NaCl, salinity, halophytes, Organic constituents, antioxidant enzymes.

and physiological responses in plants, and thus affects

INTRODUCTION
Millets are the most important staple food for the millions
of people in the semi-arid tropics of Asia and Africa. India
is the largest producer of many kinds of millets, which are
often referred to as coarse cereals. Millets grown in India
are sorghum (Jowar), pearl millet (Bajra) and small millets,
which include finger millet (Eleusinecoracana), kodo
millet (Paspalumscrobiculatum), fox tail millet (Setaria
italic), little millet (Panicumsumatrense), proso millet
(Panicummiliaceum) and barnyard millet
(Echinocloafrumentosa). Ragi is an important cereal crop
widely cultivated in African and Asian countries. It is
grown for grain and forage. In northern India grains are
used mostly in the form of chapaties. In South India grains
are used in many preparations like cakes, puddings, sweets
etc. Ragi grain contains 9.2% protein, 1.29% fat, 76.3%
carbohydrates, 2.2% minerals, 3.9% ash and 0.33%
calcium. Besides vitamin A and B, phosphorus is also
present in smaller quantities. It is good for green fodder and
for hay making. The grains can be stored for 8-10 years
and, thus, it is an important famine food.
Salinity stress is one of the most significant limiting factors
in agricultural crop productivity (Boyer, 1982). Salinity and
drought are among the major stresses that adversely affect
plant growth and crop productivity. These constraints
remain the primary causes of crop losses worldwide,
reducing average yields by more than 50% (Boyer, 1982;
Wang et al., 2003). Salt stress alters various biochemical
almost all plant processes including photosynthesis, growth
and development (Iqbal et al., 2006).Plants that can survive
on high concentrations of salt in the rhizosphere and grow
well are called halophytes. Salinization of agricultural soils
is a worldwide concern especially inirrigated lards. Saline
soil is characterized by the presence of toxic levels of
sodium and its chlorides and sulphates. Salinity tolerance is
defined as the ability of plants to continuously grow under
salt stress conditions (Munns’ 2002). Another major factor
of salt tolerance mechanisms is the ability of plant cells to
adjust osmotically and to accumulate organic solutes
(proteins, sugar, amino acids, etc.). The accumulation of
these compounds is not only important for cell
osmoregulation but also for the protection of subcellular
structure (Munns’ 2002) and maintenance of protein
structures. Many of the physiological adaptations of plants
to saline conditions are similar to the adaptations shown by
plants to desiccation stress and it has been suggested that
plants showing drought resistance would also exhibit
salinity tolerance. However, in some halophytes, the salt
tolerance mechanisms are not sufficient for tolerance of
drought or frost. The present study was made to investigate
the effect of salt stress on antioxidant activity of Millet
species.
MATERIALS AND METHODS
Plant Material and Treatment
Healthy seedlings of prosomilletwere up rooted without
damaging the root system and brought to the laboratory in

International Journal of Research in Plant Science 2015; 5(2): 17-20

17
polythene bags. Uniform sized and healthy seedlings after a
through wash in the tap water were planted individually in
polythene bags. The polythene bags filled with
homogenous mixture of garden soil. The seedlings were
irrigated with tap water and allowed to establish well. After
establishment in polythene bags for 35 days.The treatment
constituted control, 100, 200, 300, 400, 500 mM and
600mMNaCl concentration. At higher concentrations,
seedlings mortality occurred within a week after the salt
treatment. The experimental plants thereafter, maintained
upto 600mM NaCl. Salt solutions were prepared with NaCl
(Laboratory Grade, Thomas baker, India). The treatments
were continued until the plants received the required
concentrations of the salt, after this all the plants were
irrigated with tap water. The experimental yard was roofed
with transparent polythene sheet at the height of 3 m from
the ground in order to protect the plants from rain.
Sampling for various studies was taken on the 35 day after
NaCl treatment.
Estimation of Superoxide Dismutase (SOD) activity
The assay of superoxide dismutase was done according to
the procedure of (Das K et. al., 2000).In this method, 1.4ml
aliquots of the reaction mixture (comprising 1.11 ml of 50
mM phosphate buffer of pH 7.4, 0.075 ml of 20 mM L-
Methionine, 0.04ml of 1% (v/v) Triton X 100, 0.075 ml of
10 mM Hydroxylamine hydrochloride and 0.1ml of 50 mM
EDTA) was added to 100 ul of the sample extract and
incubated at 30ºC for 5 minutes. 80 ul of 50 mM riboflavin
was then added and the tubes were exposed for 10 min to
200 W-Philips fluorescent lamps. After the exposure time,
1ml of Greiss reagent (mixture of equal volume of 1%
sulphanilamide in 5% phosphoric acid) was added and the
absorbance of the color formed was measured at 543 nm.
One unit of enzyme activity was measured as the amount of
SOD capable of inhibiting 50% of nitrite formation under
assay conditions.
Estimation of Catalase
Catalase activity was assayed by the method of (Sinha A. K,
1972). The enzyme extract (0.5 ml) was added to the
reaction mixture containing 1ml of 0.01 M phosphate
buffer (pH 7.0), 0.5 ml of 0.2 M H2O2, 0.4 ml H2O and
incubated for different time period. The reaction was
terminated by the addition of 2 ml of acid reagent
(dichromate/acetic acid mixture) which was prepared by
mixing 5% potassium dichromate glacial acetic acid (1:3 by
volume). To the control, the enzyme was added after the
addition of acid reagent. All the tubes were heated for 10
minutes and the absorbance was read at 610 nm. Catalase
activity was expressed in terms of μ moles of H2O2
consumed/min/mg protein.
Estimation of Peroxidase activity
The assay was carried out by the method of (Addy and
Goodman, 1972).The reaction mixture consisted of 3ml of
buffered pyrogallol [0.05 M pyrogallol in 0.1 M phosphate
buffer (pH 7.0)] and 0.5 ml of 1% H2O2. To this added 0.1
ml enzyme extract and O.D. change was measured at 430
nm for every 30 seconds for 2 minutes. The peroxidase
activity was calculated using an extinction coefficient of
oxidized pyrogallol (4.5 liters/mol).
Estimation of Ascorbate Oxidase
Based on that of (Vines and Oberbacher, 1965)in which the
decrease of absorbance due to oxidation of Ascorbate by
AO (Ascorbate Oxidase) is measured at 265nm and 25°C.
Unit Definition
That amount of enzyme catalyzing the oxidation of 1
micromole Ascorbate per minute at 25°C.
Reagents
0.1M Phosphate / EDTA Buffer pH5.6
Substrate [0.005M Ascorbic Acid]
Record apparent increase of absorbance at 265nm for ± 4
minutes - check temperature in cells.
Estimation of proline
Proline was assayed according to the method Bates et al.
(1973). Five hundred mg of plant tissue was homogenized
in 10 ml of 3 per cent aqueous sulphosalicylic acid. The
homogenate was filtered through Whatmann No. 42 filter
paper. Two ml of acid ninhydrin (1.25 g ninhydrin in 30 ml
of glacial acetic acid and 20 ml of 6 M phosphoric acid)
and 2 ml of glacial acetic acid in a test tube was heated for
an hour at 100°C. The reaction mixture was extracted with
4 ml of toluene and mixed vigorously by using a vortex
mixture for 15-20 seconds. The chromophore containing
toluene was aspirated from the aqueous phase. The
absorbance of the toluene layer was measured in a
spectrophotometer at 520 nm using toluene as blank.
Statistical analysis
Each treatment was analyzed with at least five replicates
and a standard deviation (SD) was calculated data are
expressed in mean ± SD of five replicates.
RESULTS AND DISCUSSION
1. Effect of SOD Activity:
There was a considerable increase in the SOD activity
at the time of germination (6 days) and after 35 days in salt

International Journal of Research in Plant Science 2015; 5(2): 17-20

18
Fig.1. Showing SOD activity at the time of germination Fig.3.1 Showing Peroxidase activity at the time of
after (6days) germination (6days).

Fig.1.2 Showing SOD activity after (35days) treated plants Fig.3.2 Showing Peroxidase activity after 35 days.
in compare to normal plants.

Fig.2.1 Showing CAT activity at the time of germination Fig.4.1 Showing Ascorbate oxidase activity at the time of
(6days). germination (6days)

Fig.2.2 Showing CAT activity after 35 days. Fig 4.2 Showing Ascorbate oxidase activity after 35 days.

International Journal of Research in Plant Science 2015; 5(2): 17-20

19

Fig 5.1 Showing proline activity at the time of germination
(6days)
Fig 5.2 Showing proline activity after 35 days.
2. Effect of Catalase activity
There was a considerable increase in the catalase activity at
the time of germination (6 days) and after 35 days in salt
treated plants in compare to normal plants.
3. Effect of Peroxidase activity
There was a considerable increase in the peroxidase activity
at the time of germination (6 days) and after 35 days in salt
treated plants in compare to normal plants.
4. Effect of Ascorbate oxidase activity
There was a considerable increase in the Ascorbate oxidase
activity at the time of germination (6 days) and after 35
days in salt treated plants in compare to normal plants.
Source of support: Nil; Conflict of interest: None declared
International Journal of Research in Plant Science 2015; 5(2): 17-20
20
5. Effect onProline Content.
There was considerable increase in Proline content at the
time of germination (6 days) and after 35 days in salt
treated plants in compare to normal plants.
CONCLUSION
In the present investigation, the effect of different
concentration of sodium chloride on growth, organic
components and activities of certain key enzymes of
Panicummiliaceumhas been studied. The Panicu-
-mmiliaceum could survive a wide range of 100-600mM
NaCl concentration. The upper limit for survival of this
species to NaCl salinity was 600 mM. However, favorable
growth response by the seedlings was confined to
400mMNaCl.
REFERENCE
1. Boyer, J.S., 1982. Plant productivity and environment,
Science, 218: 443-448.
2. Wang, W., B. Vinocur, A. Altman, 2003. Plant
response to drought, salinity and extreme temperatures:
towards genetic engineering for stress tolerance. Plana,
218: 1-14.
3. Iqbal, M., M. Ashraf, A. Jamil, S. Rehman, 2006. Does
seed priming induce changes in the levels of some
endogenous plant hormones in hexaploid wheat plants
under salt stress.J. Integrative Plant Biol., 48: 181-189
J. Exp. Bot., 59: 1315-1326.
4. Munns, R. 2002. Comparative physiology of salt and
water stress. Plant Cell Environ.,25: 239-250.
5. Bates, L.S., R.P. Waldren and I.D. Teare, 1973. Rapid
determination of the free proline in water stress
studies. Plant Soil, 38: 205-208.
6. Das, K; Samanta, L and Chainy, GBN (2000), “A
modified spectrophotometric assay of superoxide
dismutase using nitrite formation by superoxide
radicals”, Ind. J. Biochem, 37, 201-204
7. Addy and Goodman, Ries, S., V. Wert, D. O'Leary and
M. Nair. (1972)). 9-β-L (+)- adenosine: a new naturally
occurring plant growth substance elicited by
triacontanol in rice. Plant GrowthRegul., 9: 263-273.
8. Sinha A. K, (1972). Plant resistance to environmental
stress. CurrOpin Biotech 9:214–1181.
9. Vines, HM and Oberbacher, MF (1965), “Response of
oxidation and phosphorylation in citrus mitochondria
to arsenate”, Nature, 206, 319-320.