Abiotic Stress and Its Impact on Protein Concentration or

American Journal of Biology and Life Sciences 2016; 4(3): 16-23

http://www.openscienceonline.com/journal/ajbls

ISSN: 2381-3784 (Print); ISSN: 2381-3792 (Online)

Abiotic Stress and Its Impact on Protein Concentration or Polymorphism of Gloriosa superba Plant

Dharmendra Singh1, *

, Manish Mishra2, Anirudha Singh Yadav

1

1Department of Botany, Govt. M. V. M., Barkatullah University, Bhopal, India 2Department of Ecosystem Management, IIFM, Bhopal, India

Email address

[email protected] (D. Singh) *Corresponding author

To cite this article Dharmendra Singh, Manish Mishra, Anirudha Singh Yadav. Abiotic Stress and Its Impact on Protein Concentration or Polymorphism of

Gloriosa superba Plant. American Journal of Biology and Life Sciences. Vol. 4, No. 3, 2016, pp. 16-23.

Received: March 29, 2016; Accepted: April 11, 2016; Published: June 15, 2016

Abstract

Biotic and abiotic stresses exert a considerable influence on the production of several secondary metabolites in plants.

Temperature, pH and light are one of the important abiotic stress that affected survival, growth, reproduction and geographic

distribution of crop plants. Biochemical characterization of species and their genetic association and polymorphism within the

related species based on morphological data is becoming difficult because these morphological traits are highly influenced by the

environment. Proteins play significant biological function in human as well in the plants. In this study, G. superba cultured in MS

medium under abiotic stress. To study the effect of stresses on protein concentration, protein extracted and quantified by Lowry

method. Total levels of protein were found to be varied in plants grow under different temperature. Maximum 11.308 µg/100mg

was observed in plant cultured at 25°C temperature and minimum amount was 4.791 µg/100mg at 35°C temperature. pH and

photoperiod does not exist more difference in protein concentrations. To investigate the molecular weight of proteins, a standard

protein marker was used. Electrophoresis of proteins has been successfully used for the characterization of different taxonomic,

evolutionary and genetic relationship studies. In the present study, the electrophoratic banding profile of total soluble proteins of

G. superba plant cultured under different abiotic stress exhibited presence versus absence type of polymorphism. The present

investigation of SDS denatured proteins showed differences in a number of bands, bandwidth and intensity and exhibited genetic

diversity between all variants. This study is very important for researcher, who is working in the field of climate change. By this

research we can conserve endangered plant species in challenging environment and can enhance important plant chemical

constitute under stress conditions.

Keywords

Abiotic Stress, Protein Concentration, Protein Polymorphism, Micropropagation, Gloriosa superba

1. Introduction

Recently, there has been an increased interest in

understanding the mechanism of plant acclimatization to

environmental stresses. Plants take action to an undesirable

ecosystem by changing their morphology, physiology, and

biochemistry. Some of the adaptations to stress may include

the changes in both the nature and levels of their genetic

resources. Biotic and abiotic stresses exert a considerable

influence on the production of several secondary metabolites

in plants [1]. Temperature, pH and light are one of the

important abiotic stress that affected survival, growth,

reproduction and geographic distribution of crop plants. Each

plant species have evolved a degree of stress tolerance.

Variation tolerance level can be genetically determined, which

reduces its metabolic activity. The following this process

many biochemical and physiological changes occur, including:

increase levels of sugars, soluble proteins, antioxidant

enzymes, proline, chlorophyll fluorescence, the appearance of

17 Dharmendra Singh et al.: Abiotic Stress and Its Impact on Protein Concentration or Polymorphism of Gloriosa superba Plant

new isoforms of protein and membrane lipid composition

changes that which called cold accumulation [2]. Plant tissue

cultures are exposed to stresses and stress combinations that

they may not have encountered in nature in their long

evolution. It is a remarkable reflection on the plasticity of the

plant genome that it can decipher and respond to novel in vitro

stresses. Today various tissue culture techniques are used to

enhance yield of secondary metabolites by triggering the

stress response like using Elicitors, Precursors and

Biotransformation, Change in environment conditions,

Change in medium constituents etc [3]. Biochemical

characterization of species and their genetic association and

polymorphism within the related species based on

morphological data is becoming difficult because these

morphological traits are highly influenced by environment [4].

Proteins play significant biological function in human as well

in the plants. These can be analyzed from seeds and other parts

of plants such as leaves and stems. Protein synthesis occurs in

leaves and green stems and is mobilized into seeds or fruits.

Proteins have been assayed from almost all parts of plants

including leaves, nodules, stem, fruit and seed. Proteins have

been extracted, identified and analyzed by using different

analytical techniques. At present, extracellular and

intracellular proteins have been isolated and quantitatively

estimated by spectrophotometrically [5].

G. superba L., a member of the Liliaceae family is a

perennial tuberous climbing herb that distributed in the

tropical and subtropical region of India. G. superba L. is

among some of the modern important medicinal plants, which

actually facing local extinction due to climate change.

Different parts of the plant have a wide variety of uses,

especially within an Indian traditional medicine of the time in

immemorial. Tubers and seeds of the G. superba L. are an

expensive export commodity. All parts of the plant contain

colchicines and related alkaloids [6]. Due to the medicinal

value, this plant collected from the wild and used as raw

material for large-scale medicinal industries, leading to over

exploiting condition, proved to be 95% endangered medicinal

plant becomes endangered plant species and included in the

red data book [7]. The purpose of the present study was to

contribute a better understanding of the genetic responses of G.

superba plant to different abiotic stress. We investigated the

influence of three types of abiotic stress on the contents of

proteins to analyze changes in the protein pattern under stress

condition using SDS-PAGE gel electrophoresis.

2. Materials & Methods

2.1. Sample Collection and Preparation

Different parts (Seed, tuber and whole plant) of G. superba

Linn. were used as explants in the present study (Fig. 2). The

plant was collected from the northern forest range of Betul

district of Madhya Pradesh (Fig. 1). Seeds and tubers were

washed thoroughly in running tap water to remove the

superficial dust and washed with 2% (v/v) tween 20 for 10 min

then washed well with distilled water. Then the seed surface

was sterilized with 0.1% mercuric chloride (HgCl2) for 5 min.

and tubers for 2 minute. After pretreatment explants were

dipped in 70% ethanol for 30 seconds. This was followed by

rinsing the explants with double distilled sterile water thrice.

North forest division of Betul (Madhya Pradesh)

Source: Department of Remote Sensing, Madhya Council of Science & Technology, Bhopal.

American Journal of Biology and Life Sciences 2016; 4(3): 16-23 18

Fig. 1. Location of survey site in Madhya Pradesh, India.

Fig. 2. Tubers and Seeds of G. superba plant.

2.2. Micropropagation in Different Stress

Condition

For this purpose, explants cultured on Murashige-Skoog

(MS, 1962) [8] medium containing sucrose 30%, coconut

water 15% and 0.8% agar (PTC grade, Hi media, India) and

supplemented with different concentrations and combinations

of 6-Banzylaminopurine (0.5-1.5 mg/l) and Gibberellic acid

(0.5-1.0 mg/l) for seed germination and BAP (0.5-2.5 mg/l),

Indole -3-acetic acid (0.5 mg/l) or Kinetin (0.5 mg/l) for shoot

proliferation and multiplication. For the rooting different

concentration of Indole-3-butyric acid (0.5-2.5 mg/l) and

1-Naphthaleneacetic acid (0.5 mg/l) were used. The pH of

medium was adjusted to 5.6-5.8 using 0.1 N NaOH or 0.1 N

HCl before autoclaving (121°C, 20 min). The cultured plants

were exposed to abiotic stress created artificially by applying

different light period (12, 14 and 16 hrs), different pH (6, 7 and

8.5) and different temperature (25, 30 and 35°c). On the

medium in the presence of stressful factors cultivated plants

observed for 1 month. After 1 month all plants were harvested

to study the effect of abiotic stress on concentration of protein.

2.3. Extraction of Protein

For the extraction of proteins, 100mg of G. superba plants

were taken, crushed and grounded into a fine powder in a

mortar and pestle with 100mM 1.5M Tris HCL (pH 7.2). The

aliquot was centrifuged at 12,000 rpm for 10 minutes at room

temperature. The pellet was discarded and extracted crude

proteins were recovered as clear supernatant, transferred into

new 1.5 ml eppendorf tubes and stored at -20oC for

electrophoresis.

2.4. Quantification of Protein

The concentration of the extracted protein samples was

determined using the Lowry’s method (1951) using different

samples against the control [9]. The relative concentrations of

all samples were calculated using the formula from the BSA

standard chart.


2.5. Polymorphism of Protein

Protein samples were mixed with 4X gel loading dye to

make its final concentration of 1X in mixture and was heated

at 95°C in water bath for 10 min prior to loading. Protein

sample (100 µg) was loaded in each lane. Protein molecular

weight marker (Bangalore Genei, India) was used as reference.

Protein samples were electrophoresed at 8 V/cm for about 4 h

at constant current. Preparative gels were visualized by

staining with Coomassie Brilliant Blue R-250 [10].

2.6. Statistic Analysis

The Similarity Index was also calculated with each primer

and a matrix was developed. Finally, all matrices were

analyzed as an average. The graphic phenogram of the genetic

relatedness among the four accessions was produced by means

of UPGMA {Unweighed pair group method with arithmetic

average} cluster analysis of averages similarity index [11].

3. Results

3.1. Micropropagation of G. superba Under

Abiotic Stress

Plant tissue cultures are exposed to stresses and stress

combinations that they may not have encountered in nature in

their long evolution. It is a remarkable reflection on the

plasticity of the plant genome that it can decipher and respond

to novel in vitro stresses. Today various tissue culture

techniques are used to enhance yield of secondary metabolites

by trigger stress response. Cultured plants of G. superba

exposed in different abiotic stress condition to maintained in

vitro. Various stress condition influences not only growth of

plants but also their secondary metabolites and biomolecules.

In present study tissue cultured plants of G. superba shows

different growth pattern and survival rate (Fig. 3). All

survivals further used to study the effect of abiotic stess on

biomolecules (proteins).

Fig. 3. Invitro cultured plants of G. superba.

3.2. Quantification of Protein

The protein concentration found to vary in G. superba plant

which was cultured under various physical stress conditions

(pH, Temperature and Light period) in the laboratory. In the

present investigation, G. superba was evaluated quantitatively

for the analysis of total soluble protein. The results of protein

quantification are represented in Table-1, 2 and 3 according to

abiotic stress. Total levels of protein were found to be varied in

plants grow under different temperature. Maximum 11.308

µg/100mg was observed in plant cultured at 25°C temperature

and minimum amount was 4.791 µg/100mg at 35°C

temperature. pH and photoperiod does not exist more

difference in protein concentrations. Bovine Serum Albumin

was used as standard (Fig. 4).

Fig. 4. Standard Graph of Bovine Serum Albumin.


Table 1. Effect of temperature on protein concentration of G. superba.

Sample ID Variant Absorbance at

660 nm

Concentration

(µg/100mg)

1 25°c .407 11.308

2 30°c .277 7.386

3 35°c .191 4.791

Table 2. Effect of pH on protein concentration of G. superba.


660 nm

Concentration

(µg/100mg)

1 6pH .253 6.662

2 7pH .362 9.950

3 8.5pH .186 4.640

Table 3. Effect of light period on protein concentration of G. superba.


660 nm

Concentration

(µg/100mg)

1 12hrs .302 8.140

2 16hrs .310 8.381

3 18hrs .355 9.739

3.3. Protein Polymorphism

The SDS-PAGE of storage proteins is a method to

investigate protein patterns and classify plant varieties

because these proteins are highly preserved. The observations

of this study showed a large variation in the number of protein

bands among the studied accessions (Fig. 5). To investigate

the molecular weight of proteins, a standard protein marker

was used. From the electrophoretic studies, it was observed

that the plant grows in different temperature revealed 11

protein bands, pH with 6 bands and Light period 10 bands.

Fig. 5. Electrophoresis Image of protein run on SDS PAGE gel electrophoresis.

Lane 1, 2 and 3 represents the banding pattern of protein under different temperature (25°C, 30°c, 35°C) whereas lane 4, 5 and 6 show under different pH (6,7 and

8.5) and lane 7,8 9 exhibit bands of protein affected photoperiod stress (12, 16, 18 hrs ), M represents the standard marker of medium range.

Table 4. Jaccard’s similarity matrix of accessions of temperature, pH and photoperiod of G. superba plant.

S1 S2 S3 S4 S5 S6 S7 S8 S9

S1 1.000

S2 0.500 1.000

S3 0.220 0.000 1.000

S4 0.440 0.545 0.250 1.000

S5 0.220 0.181 0.250 0.250 1.000

S6 0.220 0.545 0.250 0.500 0.500 1.000

S7 0.440 0.363 0.250 0.250 0.000 0.000 1.000

S8 0.000 0.363 0.000 0.250 0.000 0.250 0.250 1.000

S9 0.000 0.000 0.000 0.000 0.000 0.000 0.571 0.857 1.000

Where; S1 is plant cultured under 25°C temp., S2 under 30°C, S3 under 35°C, S4 under 6pH, S5 under 7pH, S6 under 8.5 pH, S7 Under 12Hrs photoperiod, S8

under 16hrs, S9 under 18hrs.


Fig. 6. Dendrogram of Protein Polymorphism of all variants.

Dendrogram showing Protein polymorphism in protein

content of G. superba cultured under different physical stress

condition. The dendrogram based on Similarity Index (SI)

showed distinct separation of the collected from different

stress condition (Fig. 6). The dendrogram separated the

different physical stress condition from 3 different physical

stresses based on the genetic diversity (GD). The basic root

node has a main leaf group S-7 and S-6 group showing

neighbor joining with S-7. The S-4 again split out into two

root node, lower root node splits into two root branches S-2

and S-3 and upper root node again splitting in S-5 and S-9,

The S-9 again splitting into S-1 and S-8. The polymorphism

observed was considered to be reasonably high. A total of 27

polypeptide bands were observed in our study having

molecular weights in the range of 19 to 94 kDa. Jaccards

similarity coefficient was obtained in the range of 0.00 to 1

(Table 4).

The average root distance observed in samples of different

physical stress was 0.426 with a variation of 0.022 and a pair

distance with average 0.261 and average pair variation of

0.043 SSQ (sum of squares) was found to be 0.4255.

According to average variation of different physical condition

ei. Temperature, pH and light respectively, were found the

SSQ value 0.994, 0.0416 and 0.154 respectively. These SSQ

values of data are very important for statistical analysis of

variants. The highest value represents more distance between

variants.

4. Discussion

In the present study, Gloriosa superba L. was preferred as

an experimental plant because of its importance in natural

medicine. This glorious herb was found in abundance once

upon a time in the forest. Due to its marvelous medicinal

properties, human activities and environmental pollution, the

plant was callously exploited from forest, leads to depletion of

the species and has become endangered. This evoked use to

conserve this plant through in vitro techniques.

Modern biotechnology methods are qualitatively new tool

for direct study of the structural and functional organization of

the genetic material, as well as to assess the impact of stress on

the plant and study the mechanisms of cell and tissue tolerance

in vitro [12].

Abiotic stress factors are the main limitation to plant growth

and yield in agriculture. Abiotic stress leads to a series of

morphological, physiological, biochemical, and molecular

changes that adversely affect plant growth and productivity.

During the course of its evolution, plants have developed

mechanisms to cope with and adapt to different types of

abiotic and biotic stress. Plants face adverse environmental

conditions by regulating specific sets of genes in response to

stress signals, which vary depending on factors such as the

severity of stress conditions, other environmental factors, and

the plant species [13]. Abiotic stresses exert a profound effect

on the viability, production, growth and morphology of plants.

Hence, these stresses have been used to crop improvement

through genetic engineering [14].

To survive under such conditions, plants have evolved

convoluted mechanisms to perceive external signals, allowing

optimal response to abiotic stress. Responses to abiotic

stresses occur at all levels of the organization. Furthermore,

plant acclimation to a particular abiotic stress condition

requires a specific response that is linked to the precise

environmental conditions that the plant encounters. Thus,

molecular, biochemical and physiological processes set in

motion by a specific stress condition might differ from those


activated by a slightly different composition of environmental

parameters [15]. Gisela Jansen et al., 2012 also reported the

effect of abiotic stress (pH) on the alkaloid content of Lupinus

angustifolius [16]. Their Results clearly show that the alkaloid

content is significantly influenced by the soil pH, but

genotypic differences regarding the reaction to different pH

values in the soil were observed. Temperature strongly

influences metabolic activity and plant ontology and high

temperatures can induce premature leaf senescence. Several

studies have examined the effects of increased temperatures

on secondary metabolite production of plants. Chan et al.,

reported that Melastoma malabathricum cell cultures

incubated at a lower temperature range (20 ± 2°C) grew better

and had higher anthocyanin production than those grown at 26

± 2°C and 29 ± 2°C. Optimum temperature (25°C) maximizes

the anthocyanin yield as demonstrated in cell cultures of

Perilla frutescens and strawberry [17]. It is well known that

light is a physical factor which can affect the metabolite

production. A positive correlation between increasing light

intensity and levels of phenolics has been reported. Arakawa

studied the effect of UV light on anthocyanin accumulation in

light colored sweet cherry [18]. In the present investigation,

protein content in different experiment quantified by Lowery

method. The ability to easily and reliably quantified the total

protein content in samples is paramount to many biological

assays. Although the Lowry protein assay was first published

in 1951, several improvements, not the least of which is the

reduction in assay volume, have increased sensitivity of the

assay.

The concentration of proteins present in different samples is

compared with the help of graph above. Table 1 results

indicate a positive effect of temperature using various

concentrations of total protein of G. superba plants. It appears

from the data that there was a general increase in protein

content that corresponded with the optimum temperature of

25˚C. Pandey, M. and Chikara, S.K. 2014 found similar results

on their study in vitro regeneration and effect of abiotic stress

on physiology and biochemical content of Stevia rebaudiana

‘Bertoni’ [19].

In the present study, G. superba cultured under different

abiotic stress were used to protein profile analysis. To

investigate the molecular weight of proteins, a standard

protein marker was used. Electrophoresis of proteins has been

successfully used for the characterization of different

taxonomic, evolutionary and genetic relationship studies. In

the present study, the electrophoratic banding profile of total

soluble proteins of G. superba plant cultured under different

abiotic stress exhibited presence versus absence type of

polymorphism. The present investigation of SDS denatured

proteins showed differences in a number of bands, bandwidth

and intensity. Aparadh, V.T. et al., 2012 compared the banding

pattern of seeds and leaf proteins of cleome species by SDS

page [20]. According to the results of the SDS-PAGE, the

overall pattern of storage-proteins showed the diversity of all

cultivars of G. superba. Genetic diversity in different plant

species has been carried out by using electrophoretic patterns

of total seed proteins as revealed by SDS-PAGE of seed

storage protein [21]. Protein profiling revealed significant

inter-specific genetic diversity or genotype specific bands,

with some cultivars exhibiting remarkable polymorphic and

unique bands, that can be evaluated further “tags” for these

cultivars [22]. The polymorphism observed was considered to

be reasonably high. These proteins may be much important in

crop improvement programs through breeding and genetic

engineering. Protein profiling at seed level to assist in the

early detection of species at seed level [23].

5. Conclusion

It can be concluded that abiotic stress condition is highly

responsible not only for population decline, but also for

protein polymorphism based diversity of G. superba plant. G.

superba plant, which cultured under different abiotic

condition showed variation in protein banding. Protein

variation also confirmed by quantification method. In stress

conditions, temperature was highly responsible for protein

polymorphism. Biodiversity is important for human

civilization. Global warming is a threat before the existing

biodiversity of this earth. The effect of climate change was the

most important factor in population decline of many plant

species. Abiotic stress is the primary cause of plant loss

worldwide. Therefore, resolutions from plant biotechnology

discussions aiming at overcoming severe environmental

stresses need to be quickly and fully implemented, with

intensive molecular assisted genetic engineering. We have

made great progress in understanding the responses of plants

to abiotic stress. There are inherent physical, morphological

and molecular limitations to the plant’s ability to respond to

stress. The present study will be important in evaluation,

identification and characterization of germplasm on the basis

of genetic variation in proteins under abiotic stress.

Endangered plants can be conserved by understanding the

effect of their surrounding environment on plants and can be

developed, new species by the modification in genomes

according to climate.

Acknowledgment

Authors are thankful to Principal, Govt. M.V.M., Bhopal for

providing research permission at your center and also thankful

to the Director, CMBT, Bhopal for their guidance and

encouragement.

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Abiotic Stress and Its Impact on Protein Concentration or