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INTERNSHIP REPORT "STUDY OF EVALUATION OF PLANT GENETIC RESOURCES

IN COWPEA"

Research · September 2017

DOI: 10.13140/RG.2.2.14959.18081

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 INTERNSHIP REPORT
NATIONAL AGRICULTURE RESEARCH CENTRE (NARC) ISLAMABAD
PLANT GENETIC RESOURCE INSTITUTE (PGRI)

Submitted By:
Muhammad Hunan Faiz
Registration No:
796-FBAS/BSBT/S16

Date of Submission:
25.09.2017
Topic:
“STUDY OF EVALUATION OF PLANT GENETIC RESOURCES
IN COWPEA”
“Seek knowledge from the cradle to the grave”

(Muhammad SAW)
 DEDICATIAON

Every challenging work needs self efforts as well as guidance of

elder especially those who are very close to heart.

I dedicate this humble effort to my sweet and loving

Father and Mother

Whose affection, love, encouragement and prays of day and night
make me able to get such success and honor.

Along with hard working and respected teacher.

A trillions thanks goes to my

Brothers and sisters

For walking with me when i needed support, for

walking ahead me when i needed guidance, for walking
behind me when i needed someone to watch my back.
 CERTIFICATION
It is certified that Mr. Muhammad Hunan Faiz S/O Faiz Muhammad, student of
Department of Bioinformatics and Biotechnology, International Islamic University Islamabad
has completed his internship on topic “EVALUATION OF PLANT GENETIC RESOURCES
IN COWPEA” at Evaluation Lab of Plant Genetic Resources Institute, National Agricultural
Research Center Islamabad,”

Supervisor at NARC:

Dr Muhammad Asif Javaid,

Principal Scientific Officer (PSO)

Plant Genetic Resources Institute (PGRI)

National Agricultural Research Centre, Islamabad.

Director PGRI:

Dr. Abdul Ghafoor,

Plant Genetic Resources Institute

National Agricultural Research Centre, Islamabad.

Head of Department:

Dr. Asif Mir

Dept. of Bioinformatics and Biotechnology
International Islamic University Islamabad.
 ACKNOWLEDGEMENT
All worships and praises are only due to the Lord of creation who Himself invited the
mankind, in particular the believers, to ponder over the phenomenon of the infinite cosmos. I
offer my humblest thanks and countless salutations to the Holy Prophet Muhammad(PBUH), the
beacon of enlightenment, the fountain of knowledge, the messenger of peace ,biggest benefactor
of the mankind ever had and forever torch of guidance for humanity.

A word of thanks goes to Dr Abdul Ghafoor Director PGRI, NARC, Islamabad and Dr
Muhammad Asif Mir, Chairman of Department Bioinformatics and Biotechnology IIUI for
giving me opportunity to fulfil my dream.

First of all my deepest thanks and gratitude goes to Dr. Kashif Ilyas (SSO PGRI,
NARC Islamabad) for their inspiration and great efforts to explain things clearly and simply. I
am also grateful for proper regulation and enthusiasm for uphill struggle by lab fellows including
Wisal Mehmood, Muhammad Kamran, Muhammad Aashar Tanveer, Shahab Bashir, Ali
Hassan, Ayesha Tahir, Yusra Riaz, Maimoona Bibi, Madam Arusa, Farah Javaid, Alvira
Zafar and Isba whose inspiration guided me to reach at this level. I feel great pleasure and
honor in expressing my heartiest and sincerest thanks to them for their friendly behavior and
time to time help during my lab work.

A big share of thanks goes to my affectionate and loving Brother (Engineer Hafiz
Muhammad Irfan Faiz) for his encouragement, inspirations, prayers and sacred emotions of
sincerity.

At last but not least, I pay my heartiest thanks to my sweet encouraging parents, loving
Brothers and Sisters who is always with me either I am right or wrong and for their moral and
financial support. They always scarified themselves for me so that I may entirely devote myself
to studies and encouraged me whenever I was demoralized during my academic career. No
words can really express the feelings that I have for my beloved Parents, Brothers and Sister.

Muhammad Hunan Faiz

Chapter 3 Materials and Methods

Table of Contents
INTERNSHIP REPORT ............................................................................................................1

NATIONAL AGRICULTURE RESEARCH CENTRE (NARC) ISLAMABAD ................................1

PLANT GENETIC RESOURCE INSTITUTE (PGRI) ...................................................................1

CERTIFICATION ......................................................................................................................4

ACKNOWLEDGEMENT ........................................................................................................5

INTRODUCTION .....................................................................................................................8

Introduction of Internship Program: .........................................................................................8

Introduction to NARC .............................................................................................................8

Mandate: .....................................................................................................................................9

Plant Genetic Resources Institute (PGRI): ................................................................................. 10

Plant Exploration Laboratory: ............................................................................................... 11

Gene bank and Seed Preservation Laboratory: ..................................................................... 11

In Vitro Preservation:.............................................................................................................. 12

Germplasm Evaluation: .......................................................................................................... 12

Plant Introduction and seed health lab: ................................................................................. 12

Abstract ................................................................................................................................... 13

Key Words: ............................................................................................................................. 13

Plant Introduction ................................................................................................................... 13

Vigna unguiculata: ................................................................................................................ 13

Genetic diversity ................................................................................................................... 15

Objectives of the study: ........................................................................................................... 15

Introduction to SDS-PAGE .................................................................................................... 16

Review of Literature ............................................................................................................... 17

Materials and Methods ........................................................................................................... 19

Chapter 3 Materials and Methods

Preparation of Buffers for SDS PAGE ................................................................................... 20

A) Protein Extraction Buffer: ................................................................................................ 20

B) Electrode Buffer Solution: ................................................................................................ 21

STAINING / DE-STAINING SOLUTIONS FOR SDS PAGE: ............................................ 21

SOLUTIONS FOR ELECTROPHORESIS: .............................................................................. 21

Chemicals for Solution C: ..................................................................................................... 22

Separation and stacking Gel: .................................................................................................. 22

A) Separation Gel:................................................................................................................. 22

B) Stacking Gel ..................................................................................................................... 22

SOME OF THE PICTURES OF DIFFERENT INSTRUMENTS USED IN LAB .............. 23

Results and Discussion ............................................................................................................ 25

Dandogram ............................................................................................................................... 28

Preparation of Seed Sample .................................................................................................... 29

Preparation of Electrophoresis Gel ........................................................................................ 29

Pouring the separation Gel: .................................................................................................... 30

Preparation Of 12.25% Separation Gel: ................................................................................ 30

Pouring the Stacking Gel: ....................................................................................................... 30

Preparation of Stacking Gel: .................................................................................................. 31

Electrophoresis ........................................................................................................................ 31

Staining Procedure: ................................................................................................................ 32

Destaining Procedure: ............................................................................................................. 32

De-staining Solution: ............................................................................................................... 32

NOTES .................................................................................................................................... 32

References................................................................................................................................ 33
Chapter 3 Materials and Methods

INTRODUCTION

Introduction of Internship Program:

Internship is a program during which a student gains practical experience related to
his/her specified field of study.
Department Bioinformatics and Biotechnology (IIUI) provides opportunities of
internship for BS students in some research institute/organization during their study. Internship
program not only helps the students to select field of interest before entering the practical field
but also in gaining practical skills and techniques, which are beneficial for them in future career.
Therefore internship has an important place in the degree program.
I have keen observation for practical work more than theoretical work that’s why I joined
Plant Genetic Resource Institute (PGRI), National Agriculture Research Centre (NARC),
Islamabad as an internee. During this internship program, I learnt lot of skills and techniques
practically, which are very beneficial and will be useful for my future career when I’ll put step in
the practical field.
Some techniques which I have learnt are: Preparation of chemical solutions, SDS-
PAGE analysis, PCR analysis, Gel preparation, Gel electrophoresis, Gel documentation,

Introduction to NARC

National Agricultural Research Centre (NARC), Islamabad established in 1984, is the

largest research Centre of the Pakistan Agricultural Research Council (PARC). NARC, with a
Chapter 3 Materials and Methods

total land area of approximately 1400 acres, is located six kilometers south-east of Islamabad
near Rawal Lake. Physical facilities in term of experimental fields, laboratories, green houses,
gene bank, library/ documentation, auditorium, machinery & lab equipment repair workshops,
stores, hostels, cafeteria, audio visual studios, are also available at NARC also.

Vision:
Key Elements of the Strategy to Address Future Challenges are:
 Research planning and prioritization as a bottom up initiative
 Promote participatory approach for research
 Diversify agriculture with focus on bio-technology, horticulture, livestock, fisheries and
agro-forestry
 Post-harvest technology, value addition, agri-business
 Promote eco-friendly and resource conservation technologies
 Conservation and effective use of natural resources.

Mandate:
Strategic Research on

• National and Provincial Priorities

• Emerging Challenges in Agriculture

Services to Provincial System in

• Conservation and Supply of Germplasm

• Agricultural Informatics

• Human Resource Development

Facilities

• Research Labs 58

• National Gene Bank 01

• National Library of Agricultural Sciences 01

• National Herbarium 01

• Grain Quality Testing Lab 01

Chapter 3 Materials and Methods

• Auditorium 01

• Hostels 03

• Workshops 04

• Total Area: 565 ha

• Farm Area 465 ha

• Infrastructure 100 ha

Plant Genetic Resources Institute (PGRI):

Plant Genetic Resources Institute of NARC, Islamabad has a mandate to explore,
conserve evaluate and document the plant biodiversity of Pakistan for benefit of our future
generation. The institute not only provides vital support to national crop improvement programs
in the form of required germplasm resources of cultivated crops and their wild relatives, The
institute has linkage with national crop coordinators, Agriculture research institutes/Universities,
and other NGOs working for biodiversity conservation. The institute was established in 1993
with the financial and technical assistance from Japan International Cooperation Agency
(JICA).
Chapter 3 Materials and Methods

PGRI comprises of six laboratories

 Plant Exploration

 Gene bank and Seed Preservation

 In Vitro Preservation

 Germplasm Evaluation

 Plant Introduction and Seed Health

 Data Management

Plant Exploration Laboratory:

Plant exploration laboratory is the avenue to germplasm, for crop improvement that
cannot be obtained by exchange. The spread of improve varieties has resulted in the loss of
indigenous crop genetic diversity.

The current situation has compelled us to collect and conserve the existing agro-
biodiversity in the country before it disappears forever. The plant exploration Lab organizes 3-4
plant collecting expeditions every year to collect the targeted plant species. The main emphases
are to collect major crops and their wild relatives as these species are under threats.

Gene bank and Seed Preservation Laboratory:

The genetic conservation of germplasm aims at preserving the genetic integrity of the
accessions to avoid the loss of certain genotypes in collection. The active collections are
maintained at 10c to meet the immediate demand of researchers and plant breeders on their
request, while the same stock are kept at 5 degree centigrade as midterm storage. This seed stock
act as backup to active collections Currently 23050 accessions of more than 150 crop plant
species are available for distribution to research institutes and public. Plant germplasm
catalogues in collaboration with documentation laboratory are prepared and distributed to the end
users. Seed stock in gene bank is periodically subjected to germination tests for their viability
and vigor. Studies are also conducted to find out more appropriate storage conditions for
conservation of germplasm .Research on the seed storage aims at the enhancement in the seed
longevity in the gene bank.
Chapter 3 Materials and Methods

In Vitro Preservation:
All crops do not retain their viability upon drying therefore for these crops Recalcitrant (a
different conservation technique) has to be adopted. Similarly for vegetative propagated crops
which do not produce seeds or whose seeds cannot be used for crop production, in-vitro
conservation is suitable and widely used.

Germplasm Evaluation:
The Germplasm collection and preservation is meaningless if not evaluated properly
evaluated for the traits of interest. Almost 95% of the germplasm of cereals and legumes have
been evaluated for various agro-morphological and genetic traits. Detailed evaluation against
biotic and abiotic stresses is being carried out and will remain the major future thrust. SDS-
PAGE, Isozyme, and DNA analysis was used to evaluate genetic diversity in different crop
species.

Plant Introduction and seed health lab:

The plant introduction and seed health labs are engaged with the equistion of exotic germplasm,
and indexing the health status of material stored in gene bank. Introduced germplasm as well as
the conserved seed stocks are examined for contamination by pathogens and pests. Indexing the
health status helps to avoid the spread the pathogens in new geographical regions. In
collaboration with crop commodity program, the lab is also engaged in identifying the sources of
resistance for various pathogens like pea seed borne mosaic virus.
Chapter 3 Materials and Methods

Abstract
The experiment was conducted at the evaluation Lab of Plant Genetic Resources Institute
(PGRI), National Agriculture Research Centre, Islamabad. Total seed storage protein based
variation was studied among 18 genotypes of Vigna unguiculata by using SDS-PAGE. Seeds were
obtained from Gene bank of PGRI NARC Islamabad. Seed storage protein was extracted from the
ground seeds of each accession using the standard lab protocol by addition of different solutions in
different concentrations. The diverse germplasm from different areas of Pakistan was evaluated and
maximum polymorphism was noted in protein bands. Maximum protein bands (09) were noted in
each accession.

Key Words: SDS, Vigna unguiculata, Vigna, Cowpea,

Plant Introduction

Vigna unguiculata:
The cowpea is grain with highly digestible protein and carbohydrates. The genus Vigna is
relatively large pan-tropical genus, which contain mostly species on Africa. The name cowpea
was originated from the fact that this crop is a greater and major source of hay for cow in the
southeastern United States and in other parts of the world. Regardless of it’s precise origin,
cowpea was probably cultivated by 7000 to 6000 BC and arrived in India about 4000 years ago
with the grain species. India appears to be secondary centre of genetic diversity. Considerable
divergence occurred in chickpea soon after it’s cultivation began. It is believed that cowpea was
used as a fodder crop for cattle prior to domestication for human consumption. The original
forms were probably spreading, short-day plants that readily scattered their seeds. Pods
dehiscence and seed dormancy were most probably lost quickly in conjunction with
domestication. Upright, day-neutral types may have first emerged after introgression with local
wild relatives in the rain forests of Africa. The fodder and green vegetable were probably
developed after the crop arrived in Asia. About the middle of the sixteenth century they were
introduced into Europe and came into America by the means of West Indies the latter part of the
seventeenth century. They were probably first brought to the United State in the Early part of
 Chapter 3 Materials and Methods

eighteenth century. Cowpea grown extensively except where the climate is cold and the soil wet
during the summer. Although cowpeas are warm-temperature loving plant, they are grown to
considerable extent in New York, Ohio, Indiana, Missouri, Kanas and Lowa. The domesticated
species are divided in the two groups Asiatic and African given in table in Table 1.1
This classification is the morphological base. Africa is major country producing the cowpea
and this crop is very important crop with significant role in low agricultural areas of Africa. All
parts of the cowpea are useful and useable.

Sr. No. Category Scientific Name Sr. No. Category Scientific Name
1 Category Scientific Name 8 Sub Class Rosidae
2 Common Name Cowpea 9 Order Fabales
3 Scientific Name Vigna unguiculata 10 Family Fabaceae
4 Kingdom Plantae 11 Sub Family Fabiodae
5 Sub Kingdom Tracheobionta 12 Genus Vigna
6 Super Division Spermatophyta 13 Specie unguiculata
7 Division Magnoliophyta Sub Class Rosidae

The cowpea is most important legume in all over the world and also in Pakistan because
the following table shows that all parts of cowpea.

Sr. Plant Part Advantage and beneficial use

No.
1 Young leaves Vegetable & Fodder for livestock
2 Young Pods Vegetable
3 Seed Also as vegetable
4 Fresh Pods Vegetable
5 Dry Pods Vegetable
6 Roots Nitrogen Fixation.

Sr. No. Asiatic Sr. No. African

1 Mung Bean (Vigna radiate L. ) 1 Bambara groundnut (Vigna subterranean L.)
2 Urd Bean (Vigna mungo L.) 2 Cowpea (Vigna unguiculata L.)
3 Moth Bean (Vigna aconitifolia L.)
4 Adzuki Bean (Vigna angularis L.)
5 Rice Bean (Vigna umbellate L.)
Chapter 3 Materials and Methods

Genetic diversity
The genetic diversity promotes the improvement in the genetic characters in cowpea. The
genetic diversity data is collected of fist says to flowering, flower colours which are different
with each other in different accessions as white, purple, yellow, purplish white and yellowish
white. Moreover the pod length, plant height that matters the fodder quantity and seeds per pod
are variable in each accession.
Plant genetic diversity is a useful character in plants that can be transmitted genetically
from parents to offspring. The sources of tremendous variation in plants support all other forms
of life on land. Plant genetic diversity covers a wide range, at both the evolutionary and
ecological level. Ecologically the variation ranges from the natural ecosystems and traditional
low-input agriculture to modern, intensive production systems. At the crop, evolutionary level, it
covers a wide range of diversity from wild ancestors to modern cultivars. The resulting diversity
in plants has been the basis for providing food and satisfying other human needs for millennia.
There are two ways of studying and evaluating genetic diversity (Anon; 2008).
 Morphological characterization of given accessions
 Biochemical evaluation of given accessions.

In morphological characterization we analyze the characters of our crop in the field.

Biochemical markers such as DNA provide more accurate assessment regarding genetic diversity
in germplasm collection. Many lines can be characterized in a short period through this
approach. In addition, the data reflect genetic variability more precisely, as molecular markers
are direct gene products not influenced by the environment.

Objectives of the study:

 The present study was conducted to Evaluate Vigna unguiculata germplasm present in
the PGRI Gene bank for genetic diversity.
 The present study was conducted to Study the SDS-PAGE technique for its potential as
an efficient tool for the study of genetic diversity in crop germplasm for seed storage
genetic diversity.
 The present study was conducted to Selection of desirable genotype breeding program.
Chapter 3 Materials and Methods

 The study of the genetic diversity in Vigna unguiculata for germplasm accessions from
different areas of Pakistan on the basis of variation in seed genetic diversity.
 The present study was conducted to Identification of genetic diversity occurring in
qualitative and quantitative traits of Vigna unguiculata.

Introduction to SDS-PAGE
SDS-PAGE is a reliable, stable, cheaper, and best option for the study of seed protein
because seed proteins are not sensitive to environmental effects. Banding pattern has been used
for the identification cultivars. Seed storage protein is convenient tool for estimation of genetic
diversity in cultivated cowpea. However literature on the SDS-PAGE on Vigna species for
genetic diversity is limited. Variation in proteins exhibited by SDS-PAGE can distinguish
between different cultivars of a crop. Identification of Vigna unguiculata based on SDS-PAGE
is useful to sort out desirable entries for various characters, which can be used as parents in
hybridization programmed..

In Sodium Dodecyl sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

separations, migration is determined by molecular weight. Sodium Dodecyl sulphate (SDS) is an
anionic detergent that denatures proteins by wrapping the hydrophobic tail around the
polypeptide backbone. For almost all proteins, SDS binds at a ratio of approximately 1.4g SDS
per gram of protein, thus conferring a net negative charge to the polypeptide in proportion to its
length. The SDS also disrupts hydrogen bonds, blocks hydrophobic interactions, and
substantially unfolds the protein molecules, minimizing differences in molecular form by
eliminating the tertiary and secondary structures. The proteins can be totally unfolded when a
reducing agent is employed. The SDS denatured and reduced polypeptides are flexible rods with
uniform negative charge per unit length. Thus, because molecular weight is essentially a linear
function of peptide chain length, in sieving gels the proteins separate by molecular weight. There
are two types of buffer systems used in protein gel electrophoresis: Continuous and
discontinuous
Chapter 3 Materials and Methods

Review of Literature
Cowpea is among those crops which are used and cultivated for the dual purpose fodder and
vegetable. The cowpea is tropical and arid areas crop (Fana Sylla Ba et al.2003). Cowpea is
leguminous crop with great importance as fodder and grain. Africa is contributing about 64% of
world annual production of cowpea. African major role and contribution highlights it’s really a
great production of cowpea. Cowpea is used as human food, but in addition to its useful for soil
as a fertilizer, Cowpea is symbiotically nitrogen fixing crop. Cowpea is used as food and
vegetable. Cowpea is mostly resistant to diseases, insect, pest and weeds. (Getachew Mekonnen
et al. 2008). World population increment is the main reason for the food and agricultural land
shortage and this issue is being world headache. With estimation in next half century million of
people will suffer the unavailability of food and they would have no access to food and food
products qualitatively and quantitatively. Different challenges i.e. land shrinking, lethargic yield,
seasonal flooding, pest and climatic deviations. Scientists and scientific staff is being interested
in improving the genetic and environmental adaptations, these adaptations may be changed in
genetic level.(Mochida and Shinozaki, 2010).

The portentous contents in the seed of cowpea rely in between 23-32 % and fat content
with a great level of fibre that check the heart diseases, in addition to this property, the reduction
of lipoprotein with do diversity (Phillips et all; 2003) are present in cow pea. Blood glucose
level is raised up with consumption of cow pea, cow pea is low digestible legume that help in
lowering diabetes (Phillips et all; 2003). The estimation of cow pea production is world widely is
about 4.5 million metric ton. The total land used for the agricultural purpose of cowpea is about
12-14 million hectors. West and central Africa is contributing a greater portion of cowpea at
global level is about 70%. Other world countries like Brazil, Nigeria and Nigar are producing the
major portion and becoming the largest producers in world. (Singh et al; 2002).cowpea is self
pollinating crop which is encouraged by the arrangement of the floral parts. The cowpea is has
second most importance in Nigeria. The cowpea competes with equal quantity of meat and fishes
in proteinous contents. Cowpea has null value of metabolites and toxins that cause the
carcinogenic diseases in body. It inhibit the effect of metabolites and toxins with the
consumption of any part of the plant ( Ige et al; 2011 ). The cowpea is dicotyledonous crop. FAO
suggested the world production about 2.27 million tons in 2002. Nigeria was producing the
Chapter 3 Materials and Methods

greater portion of world production of cowpea about 0.85 million tons at that time. The people of
developing countries are fully dependant on the cultivation of cowpea (Islam et al; 2006 ). The
cowpea is the crop that is great source of protein source for poor man. Cowpea is considered as
good health nutritional crop for human beings and livestock. Cowpea is easily available,
markedly easily available legume, no expensive and cheaper legume. It is available in low price
legume in comparatively to all that legumes, meat, egg, fishes and vegetables that have starch,
carbohydrates, and fat and proteins contents (Agbogidi et al; 2012). Cowpea has the ability to
bear the drought condition because it is the crop of tropical areas. The African countries are
taking a good interest in cowpea production as Africa is contributing at larger level in the world.
The African region is producing up to 95% of world population.(FAO; 2012) Nigeria is
contributing at second larger level in the world after Africa. Brazil is also showing its interest in
the production of cowpea. The Brazilian production of cowpea is about0.822 million metric tons
with average production of 525 kg per hector. The world cowpea production has increased
during last 30 years from 1970s to 2000s up to 5 million metric tons from 1.3 million metric
tons.( Ousmane Boukar et al; 2012).

Cowpea in Pakistan is not considered as so much as the oldest crop that was grown with
other crops as crop mixture with sorghum, barley, maize and sugarcane due to different reasons
as to enhance the digestibility and viability, as well as the protein and nutritional value. In
Pakistan cowpea is planted for both irrigated and rain fed areas. This study was just performed to
examine and study the high level of green fodder with high productive cultivars of cowpea with
best character of yield and pest control (Y. Ali et al; 2004.). Cowpea is used also as fodder after
the fruit collection in Pakistan for cattle.

The morphological study was conducted at NARC Islamabad during two crop years (2003-
04) . this study was conducted to examine the effect of weed control methods on the yield and
yielding components in cowpea. Different weed controlling methods were performed
comparatively to point out most efficient method for controlling the weeding in the cowpea crop.
The two methods appeared as most efficient to control the weeding in cowpea crop which were
Chemical weed controlling at the stage when plant grew up to it’s 2-3 leaves and the other is the
hand weeding after the 50 days of sowing. These methods increased the yield up 68% in
production. The economical analysis was also improved with these two weed controlling
Chapter 3 Materials and Methods

methods as compared to the rest of all others methods of weed controlling in cowpea crop.(M
Riaz Chattha et al; 2007). Pakistan is concentrating now at the production of the pales and
specially the genera Vigna.

Materials and Methods

Eighteen accessions of cowpea (Vigna unguiculata L.) were obtained from Plant Genetic
Resources Program, National Agricultural Research Centre, Islamabad, Pakistan for
investigation of genetic diversity for total seed proteins using SDS-PAGE. For extraction of
proteins, mortar and pestle was used to crush and grind seeds. Ten mg seed flour was placed into
1.5 ml micro tube. To extract proteins, protein, 400 μl extraction buffer was added to the flour as
an extraction liquid and mixed thoroughly in Apendrof tube with a small glass rod. Extraction
buffer was constituted of 0.5M Tris HCl (pH 8.0), 0.2% SDS, 5M Urea, and 1% 2-
mercaptoethanol. Bromophenol blue was added to extraction buffer as a dye to point out the
movement of protein in the gel. To purify extraction, the homogenate samples were mixed
thoroughly by vortexing and centrifuged at 12,000 rpm for 5 minutes at room temperature (RT).
Extracted crude proteins were found as clear supernatant and transferred into new 1.5 ml
Apendrof tubes and stored at -20o C until gel electrophoresis.SDS-PAGE of total seed proteins
was performed in polyacrylamide slab gels in discontinuous buffer system. Vertical gel slabs
were prepared in a glass sandwich tightened by a set of plastic clips lined with a band of foamed
silicon rubber. Separating gels consisted of 20% by weight acrylamide and 0.135% by weight
N.N-methylene-bis acrylamide in 0.5M Tris-HCl buffer (pH 8.8) with 0.27% SDS. The gels
were made for genetic Variation in Rapeseed Germplasm Using Total Seed Proteins Profile. The
gel is polymerized chemically by adding 15μl by volume of TEMED (Tetramethylethylene-
diamine) and 10% APS (ammonium persulphate). Stacking gels contained 30% acrylamide and
0.8% N.N-methylene-bis-acrylamide in 0.25M Tris-HCl buffer (pH 6.8) containing 0.2% SDS.
Polymerization of the stacking gel was made chemically in the same way as for the separation
gels. Electrode buffer contained Tris-glycine (9.0g Tris-HCl and 43.2g glycine per 3 liters buffer
solution at pH8.9) with 3.0g SDS (0.1%). Eight micro liters (μl) of protein supernatant and 2 μl
of Bromophenol blue which served as tracking dye were applied into the stacking gel sample
wells with the help of a micropipette.
Chapter 3 Materials and Methods

Electrophoresis was conducted at 150 V for approximately 2 hour until Bromophenol

blue marker went to bottom of gel. After electrophoresis, the gel is stained in staining solution
for 2 days. After staining, de-staining is done and the bands become visible. Gel is packed in
plastic sheet and scanned by scanner after de-staining.

Preparation of Buffers for SDS PAGE

A) Protein Extraction Buffer:

Chemicals Amount

Tris 0.6g

Sodium Dodecyl Sulphate (SDS) 0.2g

Urea-solubilise and denature proteins 30.3g

Distilled water 70ml

HCL Adjust pH 8

2-Mercaptoethanol 1ml

Bromophenol blue (BPB) Little bit

Buffer solution stored in a refrigerator Total volume of 100ml

Chapter 3 Materials and Methods

B) Electrode Buffer Solution:

Chemicals Amount

Tris 3g

Glycine 14.4g

SDS 1.25g

Distilled water 1000ml

STAINING / DE-STAINING SOLUTIONS FOR SDS PAGE:

Chemicals Staining Solution De-staining Solution

Methanol 440ml 200ml

Acetic acid 60ml 50ml

Distilled water 500ml 750ml

Coomassie Brilliant Blue (CBB) 2.25g _

SOLUTIONS FOR ELECTROPHORESIS:

Chemicals Solution A Solution B

Tris 36.3g 5.98g

SDS 0.4g 0.4g

Distilled water 70ml 80ml

HCL (Conc.) Adjusted to pH 8.8 Adjusted to pH 7

Chapter 3 Materials and Methods

Chemicals for Solution C:

Acrylamide 30g

Bis-acrylamide 0.8g

Distilled water 100ml

10% APS

Ammonium Per Sulphate (APS) 0.1g

Distilled water 1ml

Separation and stacking Gel:

A) Separation Gel:
Separation Gel 12.25%
Solution A 5ml
Solution C 7.5ml
10% APS 200µl
Distilled water 7.5ml
Temed 40µl

B) Stacking Gel

Stacking Gel 4.5%

Solution B 2.5ml
Solution C 1.5ml
10% APS 70µl
Distilled water 6ml
Tamed 50µl
Chapter 3 Materials and Methods

SOME OF THE PICTURES OF DIFFERENT INSTRUMENTS

USED IN LAB

Gel running apparatus Incubator

Drying Oven
Fume Hood

Autoclave
Distillery
Chapter 3 Materials and Methods

Centrifuge Machine Weighing Machine

Micro pipits
Vertex Machine

Magnetic stirrer Shakers

Chapter 4 Results and Discussion

Results and Discussion

The experiment was conducted in the Evaluation Lab of Institute of Plant Genetic
Resources institute (PGRI), National Agricultural Research Centre (NARC), Islamabad. Plant
Experimental material germplasm comprised of 17 Vigna unguiculata Accessions were sourced
from gene bank of PGRI, NARC Islamabad and is given below:

List of Accessions of Cowpea Vigna unguiculata L for Biochemical Evaluation SDS-PAGE

Sr. No Accession No. Sr. No Accession No.

1 14649 10 14680

2 14671 11 14681

3 14672 12 14682

4 14674 13 14683

5 14675 14 14684

6 14676 15 14685

7 14677 16 14689

8 14678 17 14692

9 14679
Chapter 4 Results and Discussion

Fig.1. protein based variation among V. unguiculata;Line represents accessions 14649,

14671,14672, 14674, 14675, 14676, 14677, 14678, 14679.
Chapter 4 Results and Discussion

Fig.2. protein based variation among V. unguiculata;Line represents accessions 14680,

14681,14682, 14683,14684, 14685, 14689, 14692.
Chapter 4 Results and Discussion

Dandogram
The 142 accession were planted on 05-07-2017 that were observed in the field and data of these
accessions were noted and then further proceeded by cluster analysis of 17 accessions Cluster
analysis of 17 accessions for seed storage proteins using unweighted pair-group average method

14649
14671
14672
14674
14676
14677
14678
14683
14680
14675
14685
14692
14689
14679
14681
14682
14684

0.0 0.5 1.0 1.5 2.0 2.5

Linkage Distance

Figure: Cluster analysis of 17 accessions for seed storage proteins using unweighted pair-group
average method.
Chapter 4 Results and Discussion

17 accessions were picked randomly out 142 total accessions for biochemical evaluation,
after the completion of the SDS we found fallowing data of protein bands on the gels which are
on the basis of their molecular weight. The gels have bands which show similarities and
differences among the members of a same cowpea germplasm taken from different geographical
areas of Pakistan. We make dandogram, using (cluster analysis) through unweigted pair group
average method. Dandogram based on electrophoresis data make various protein
fractions clusters. It divides the whole genotype in to two clusters at 1.0 linkage distance as
evident from the dandogram figure 4.1. Constructed for the similarity coefficient cluster 01
contain 10 members including the accessions ,14649, 14671, 14672, 14674, 14676, 14677,
14678, 14681, 14682, and 14684 1 are similar to each other. From the above given scenario we
can summarize that one cluster is similar but there are still enough accessions that didn’t make
any cluster which shows genetic diversity. We hope that if these accessions which shows
diversity are further processed and analysed through protein ladder technique etc, they would
prove helpful to us in the future breeding programs in order to make such a crop which is more
resistant to diseases and more resistant to biotic and abiotic factors.

Preparation of Seed Sample

2 seeds of each accession of Cowpea are taken, crushed and grinded in mortar and pestle.
10mg (0.01g) seed flour is weighed by an electronic balance and put into 1.5 ml micro tube.
After each sample weighing mortar and pestle are cleaned with great care so that there should not
be even a single particle of last seed flour. To extract proteins from flour, 400µl of the protein
extraction buffer is put into the micro tube and mixed well by the test tube mixer (vortex). This
sample is preserved in a freezer (- 4ºC). The 0.01 gm seed was optimum for best results.

Preparation of Electrophoresis Gel

Glass plates used for electrophoresis were cleaned up from internal side with 80%
Ethanol and Kim wipe. Gaskets were sealed on glass plates with spacer; it was kept in mind that
gaskets should not overlap with spacer of plates. Sets of glass plates were fixed with double clips
Chapter 4 Results and Discussion

and marked 2cm from the top. To make sure that there is no leakage; glass plate set ups were
filled with water and placed for some time.

Pouring the separation Gel:

Separation Gel are used for the preparation gel for protein extraction. We can make
different percent separation gel according to our desire. ( e.g. 7.5%, 12%, 20%, 15% etc) but
12.25% separation gel works great that’s why we mostly used 12.5% separation gel.

Preparation Of 12.25% Separation Gel:

For preparation of 12.25% separation Gel, the following solution/chemicals are required.

Solution A 5ml

Solution C 7.5ml

10% APS 400ul

Distilled water 7.5ml

Tamed 60ul

After preparation of separation gel I filled Electrophoresis plate about 90% and waited for about
30 minutes to make the gel or to solidify.

Pouring the Stacking Gel:

Stacking gel is also used for the preparation of gel for protein extraction. This gel play
important rule in preparation of gel because it firstly load the sample and then that sample run in
separation gel.
Chapter 4 Results and Discussion

Preparation of Stacking Gel:

Due the mixture of the following solution/substances the stacking gel we can make.

Solution B 2.5ml

Solution C (30%) 1.5ml

10% APS 70ul

Distilled water 6ml

Tamed 50ul

After preparation stacking gel, the remaining 10% space is filled by adding stacking gel.
Insert the comb straight on down, then pour a little more stacking gel on the sides of the comb to
fully seal it. Remove any bubbles from underneath the comb, if possible, by moving the comb
gently from side to side so the bubbles get into the space in between and float up. The stacking
gel should polymerize in 20 to 30 minutes.

Electrophoresis
Electrophoresis procedure was carried out using slab type SDS-PAGE with 12.5%
polyacrylamide gel. The molecular weight of dissociated proteins was estimated by using
molecular weight standard proteins “MW-SDS-70 Kit”. Electrode buffer solution was put into
the bottom pool of the apparatus. Gel plates were placed in the apparatus, here again air bubble
formation was avoided. Electrode buffer solution was also put into the top pool of the apparatus;
wells formed by combs were washed by syringe. Seed samples were centrifuged at 13,000 rpm
for 08 minutes, 8µl of supernatant was put into wells with the help of micropipette. Protein
molecular weight marker was put in first well of each glass plate. The numbering of seed
samples and wells were noted to avoid repetition. The apparatus was connected with +ve (red)
and –ve (black) electrodes of power supply. The voltage of apparatus was kept constant at 130 V
and apparatus was left until a blue line of BPB came at the bottom of the gel plates. The 130 V
current was good for optimum protein separation.
Chapter 4 Results and Discussion

Staining Procedure:
Once the bands of samples reached to the bottom of the separation gel then i removed the Gel
and kept that in plate as well as add staining solution for over night.

By the mixing of the following chemicals/substance we make the staining solution.

Methanol 440 ml
Acetic Acid 60 ml
Distilled water 500 ml
Co-massive Brilliant Blue (CBB) * R250 2.25g
Total volume of 1litre
Solution was stirred for 30 minutes and then filtered, stored at room temperature. *CBB is a
protein staining dye.

Destaining Procedure:
After over night of staining the next step is Destaining. For Destaining process, removed the
staining solution from gel and washed gently with tab water. After washing the gels with tab
water the Destaining solution was added to that gel for overnight. The chemical composition of
the Destaining solution is given as follows in table.

De-staining Solution:
Methanol 200 ml

Acetic Acid 50 ml

Distilled water 750 ml

Total volume 1 litter, Stored at room temperature

NOTES: After Destaining process there was result.

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