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"

DEDICATION
To JEHOVAH God Almighty be all the Glory
This work is dedicated to my guardians and family for their immeasurable
support, inspiration, guidance, love and prayers.

ACKNOWLEDGEMENT
I am grateful, first and foremost to Jehovah God Almighty, for His Divine
Grace, favour and sustenance to complete this work. I would like to express
my deep gratitude and appreciation to my supervisors DR. HM AMOATEY,
Head of Department of Nuclear Agriculture and Radiation Processing and
PROF. GYP KLU, former Head of Department of Nuclear Agriculture and
Radiation Processing of the Graduate School of Nuclear and Allied Sciences,
University of Ghana (Atomic campus) for their valuable contributions,
guidance, patience, encouragement and constructive suggestions towards the
success of this work.

I would also like to acknowledge Dr. DK Asare, Manager of Nuclear


Agriculture Research Centre of the Biotechnology and Nuclear Agriculture
Research Institute (BNARI)-GAEC for his support and valuable contributions
toward the acquisition of land and accessions for this study. I would like to
thank Dr. G Amenorpe, Mr. AS Appiah, Mr. EE Quartey, Mr. HA Doku, Mr.
S Laar, of BNARI and all the Research Scientists and Technologists at the
Radiation Technology Centre of the Ghana Atomic Energy Commission
especially, Mr. JK Apatey, Mr. WSK Agbemavor, EA Ayeh for their support
and suggestions during the mucilage analyses. I am also indebted to Dr. MA
Yeboah of the West African Centre for Crop Improvement-Legon for his
suggestions and support, and to all my colleagues who through diverse ways
helped me to come out with this work.

"

"

I am also grateful to Mr. DG Achel, E Achoribo, S Donkor, and S


Agbenyegah of the Applied Radiation Biology Centre of the Ghana Atomic
Energy Commission for their assistance and guidance during the antioxidant
analyses. My heartfelt appreciation also goes to Dr. H Dzahini-Obiatey, Mr.
Ossae and Mr. C Osei-Aryee of the Cocoa Research Institute of Ghana, CRIG
for making their laboratories available for the research work. Finally, I am
most thankful to Dr. MMAA Elmosallamy and Dr. Olaf Werner (Heidelberg)
of

the

Laboratory

of

Molecular Systematics,

Conservation of Bryophytes,

Phylogeography

and

Botany Department, Faculty of Biology,

University of Murcia, Spain for providing primers and making their


laboratories available for the molecular characterisation.

"

"

TABLE OF CONTENT

CHAPTER ONE0..1
INTRODUCTION........1
1.1 GENERAL INTRODUCTION0......1
1.1.1 PROBLEM STATEMENT......02
1.1.2 JUSTIFICATION AND RELEVANCE OF THE STUDY03
1.1.3 OBJECTIVES OF THE RESEARCH.06
REFERENCES0..007

CHAPTER TWO......8
LITERATURE REVIEW.8
2.1 ORIGIN, DISTRIBUTION AND UTILISATION0008
2.2 TAXONOMIC CLASSIFICATION AND BOTANY0010
2.3 GENETICS AND CYTOLOGY...11
2.4 SOIL, CLIMATIC AND AGRONOMIC REQUIREMENTS......14
2.5 GROWTH AND DEVELOPMENT..00014
2.6 POLLINATION AND FERTILISATION..0016
2.7 RATOONING..017
2.8 NUTRIENT CONTENTS AND HEALTH BENEFITS...18
2.8.1 Biochemical and nutritional composition18
2.8.2 Nutritional healing properties0019
2.8.3 Bioactive properties019
"

"

2.9 VARIABILITY AND POTENTIAL OF OKRA GERMPLASM....19


2.10 CHARACTERISATION OF OKRA.......21
2.10.1 Morphological Characterisation of Okra00023
2.10.2 Molecular Characterisation of Okra0024
2.11 OKRA GERMPLASM CONSERVATION....26
2.12 CURRENT PRODUCTION CONSTRAINTS OF OKRA IN WEST
AFRICA.......................................................................................................29
2.12.1 Lack of Improved Genotypes 29
2.12.2 Pest and Diseases ..30
2.12.3 Weed Competition.32
2.13 STATUS OF BIOTECHNOLOGICAL INTERVENTION IN OKRA
IMPROVEMENT032
REFERENCES...35
"

CHAPTER THREE...057
MORPHOLOGICAL CHARACTERISATION OF 29 ACCESSIONS OF
OKRA (Abelmoschus spp L.)..00057
3.1 INTRODUCTION00057
3.1.1 Objectives of the Study58
3.2 MATERIALS AND METHODS59
3.2.1 Experimental site .59
3.2.2 Weather Conditions .59
3.2.3 Germplasm Assembly 0000059
3.2.4 Land Preparation .60
3.2.5 Experimental Design ...60
"

"

3.2.6 Field Management Practices61


3.2.7 Seed Sowing 61
3.2.8 Data Collection61
3.2.9 Data Analyses..63
3.3 RESULTS000"64
3.3.1 Morphological Traits of dry pods of Accessions 64
3.3.2: Variations in morphological characteristics of some of the
accessions in the field. ..67
3.3.3 Variability in Quantitative Traits among Accessions . 69
3.3.4 Variability in Qualitative Traits of Accession 73
3.3.5 Genetic Relationship among Accessions using Quantitative and
Qualitative Traits. 79
3.3.6 Correlation Coefficients of 13 Quantitative Traits. 80
3.3.7 Principal Components Analysis for Quantitative Traits . 83
3.6 DISCUSSION0085
3.6.1 Variation in Quantitative and Qualitative Traits . 85
3.6.2 Diversity in Qualitative Morpho-Agronomic Traits86
3.6.3 Genetic Relationship among Accessions based on Cluster Analysis
. 89
3.6.4 Correlations among Quantitative Characters ..91
3.6.5 Contribution of Traits to Genetic Diversity via Principal Component
Analysis 000092
3.7 CONCLUSION94
REFERENCES096
"
"
"

"

CHAPTER FOUR00107
MOLECULAR CHARACTERISATION OF 29 ACCESSIONS OF OKRA
(Abelmoschus spp L. Moench) USING INTER-SIMPLE SEQUENCE
REPEATS (ISSR) MARKER00107
4.1 INTRODUCTION000107
4.1.1 Objective of the study112
4.2 MATERIALS AND METHODS113
4.2.1 Experimental Site ..113
4.2.2 DNA Isolation ...113
4.2.3 Preparation of Cocktail for PCR. ..114
4.2.4 Amplification 114
4.2.5 Preparation of Polyacrylamide Gel (PAGE) . 115
4.2.6 Electrophoresis ..115
4.2.7 Data Collection and Analysis 116
4.3 RESULTS00117
4.3.1 PCR Amplification Products .117
4.3.2 Cluster Analysis based on six ISSR primers . 118
4.4 DISCUSSION120
4.4.1 Cluster Analysis 120
4.4.2 Genetic Diversity/polymorphism among Accessions as Revealed by
ISSR Primers . 121
4.5 CONCLUSIONS0122
4.6 RECOMMENDATIONS0123
REFERENCES000125

"

"

CHAPTER FIVE135
TOTAL

FLAVONOID,

PHENOLIC

CONTENTS

AND

ANTIOXIDANT000135
5.1 INTRODUCTION000135
5.1.1 ESTIMATION OF ANTIOXIDANT SCAVENGING ACTIVITY
139
5.1.2 Objective of study .140
5.2 MATERIALS AND METHODS00140
5.2.1 Experimental site...140
5.2.2 Sample preparation141
5.2.3 DPPH Radical Scavenging Assay . 142
5.2.4 Total phenolic content ...143
5.2.5 Total flavonoid content .144
5.2.6 Statistical analyses of data.144
5.3 RESULTS00145
5.3.1 DPPH radical scavenging assay 145
5.3.2 Total flavonoid and phenolic contents of okra accessions 146
5.4 DISCUSSION149
5.4.1 Total flavonoid and phenolic contents of ethanoic and aqueous
extracts of Accessions of Abelmoschus spp ........................................... 149
5.4.2 Total antioxidants activity of 25 accessions of Abelmoschus spp 151
5.5 CONCLUSION00152
5.6 RECOMMENDATIONS153
REFERENCES.154

"

"

CHAPTER SIX000164
DETERMINATION OF ESSENTIAL MINERAL ELEMENTS AND
MUCILAGE IN SOME ACCESSIONS OF OKRA (Abelmoschus spp)164
6.1 INTRODUCTION000164
6.1.1 Okra Production in the world 164
6.1.2 Patronage and Consumption of okra . 165
6.1.3 Proximate composition of okra . 166
6.1.4 Essential mineral elements in okra167
6.1.5 Significance of mineral elements to human ..168
6.1.6 Mucilage172
6.1.7 The Instrumental Neutron Activation Analysis (INAA) Method..174
6.1.8 Objective of the study175
6.2 MATERIALS AND METHODS175
6.2.1 Experimental site...175
6.2.2 Technique for Analysis of Mineral Elements176
6.2.3 Sample preparation for elemental analysis176
6.2.4 Sample irradiation and counting177
6.2.5 Sample preparation and extraction of mucilage 177
6.2.6 Determination of mucilage contents . 178
6.2.7 Data analyses 179
6.3 RESULTS00179
6.3.1 Concentrations of Nine Essential Elements in Accessions of
Abelmoshus spp L. . 179
6.3.2: Association of Essential Mineral Elements. 183
6.3.3 Maximum viscosity of mucilage of Abelmoschus spp L. . 184
"

"

6.4 DISCUSSION0186
6.4.1 ASSESSMENT OF ESSENTIAL MINERAL ELEMENTS IN
OKRA ACCESSIONS ...186
6.4.2 Intensity of Association between Mineral Elements . 188
6.4.3 Mucilage content of Abelmoschus spp L. . 190
6.5 CONCLUSIONS192
REFERENCES..194

CHAPTER SEVEN207
GENERAL CONCLUSIONS AND RECOMMENDATIONS000207
7.1 CONCLUSIONS207
7.2 RECOMMENDATIONS.209
8.0 APPENDICES..00211

"

"

LIST OF TABLES"

Table 2.1: Potential of recombination breeding involving two


Abelmoschus spp000013"
Table 3.1: Okra accessions used in the study0.60"
Table 3.2: Variability in Quantitative Traits among 29 accessions of
Okra.70"
Table 3.3: Morphological Characterisation of Qualitative Traits of
29 Accessions of Okra.00073"
Table 3.4: Correlation Coefficients among 13 Quantitative Traits of
Abelmoschus spp082"
Table 3.5: Principal components analyses showing the contribution
(factor scores) of 13 quantitative characters among the 29 Okra
accessions, Eigen values and Percentage total variance accounted
for by five principal components *.84"
Table 5.1: Okra accessions used in the study141"
Table 5.2: Free radical scavenging potential of 25 accessions of
okra000146"
Table 5.3: Contents of total flavonoids (g/g/QE) and phenolics
(g/g/GAE) in accessions of okra.....00148"
Table 6.1: Estimated production of okra in selected geographical
regions and countries (metric tonnes)000164"

"

"

Table 6.2: Concentration of Nine Mineral Elements in the


Accessions of Okra in mg/kg*00181"
Table 6.3: Correlations among nine Mineral Elements in 22
accessions of Abelmoschus spp*..183"
Table 6.4: Maximum Viscosity of mucilage of 21 Accessions of
Okra00185"

LIST OF FIGURES
Figure 2.1: Stages in the growth and development of okra plant (Valeriana,
4233+015
Plate 1: Photographs of dried fruits of okra accessions..63
Plate 2: Photographs of accessions with fruits0..66
Figure 3.3: A Dendrogram Showing Genetic Relationships among 29
Accessions based on Quantitative and Qualitative Traits using Coefficient of
Euclidean, complete Linked Similarity Matrix.079
Figure 4.1(a): Banding pattern of Genetic diversity studies on okra accessions
numbered 1-15 with CA4 primer.0117
Figure 4.1(b): Banding pattern of Genetic diversity studies on okra accessions
numbered 16-29 with CA4 primer.000000117
Figure 4.2: A dendrogram showing genetic relationship revealed by six
primers among the accessions based on dice coefficient using single link
similarity matrice method000..............................119

"

"

LIST OF ABBREVIATIONS
"

spp

species

et al

and others

NARP

National Agricultural Research Programme

IBPGR

International Board for Plant Genetic Resources

IPGRI

International Plant Genetic Resources Institute

CIP

International Potato Centre

BSc.

Bachelor of Science

MSc.

Master of Science

MPhil.

Master of Philosophy

AVRDC

Asian Vegetable Research and Development Centre

PGRRI

Plant Genetic Resources Research Institute

CSIR

Council for Scientific and Industrial Research

VEPEAG

Vegetable Exporters Association of Ghana

DNA

Deoxyribonucleic Acid

RNA

Ribonucleic Acid

ISSR

Inter-Simple Sequence Repeats

AFLP

Amplified Fragment Length Polymorphism

EST

Expressed Sequence Tag

SRAP

Sequence Related Amplified Polymorphism

QTL

Quantitative Traits Loci

SSR

Simple Sequence Repeat

MAS

Marker Assisted Selection

RAPD

Random Amplified Polymorphic DNA

RFLP

Restriction Fragment Length Polymorphism

SNP

Single Nucleotide Polymorphism

"

"

PCR

Polymerase Chain Reaction

CTAB

Cetyltrimethyl Ammonium Bromide

TEMED

N, N-bismethylene Acrylamide

APS

Ammonium per sulphate

PAGE

Polyacrylamide Gel Electrophoresis

AMOVA

Analysis of Molecular Variance

ITS

Internal Transcribed Spacer sequence

ECHO

Educational Concerns for Hunger Organisation

WAT

West African Taxon

FAO

Food and Agriculture Organisation

UNESCO
USDA
NIHORT

United Nations Educational, Scientific and Cultural Organisation


The United States Development Agency
National Horticultural Research Institute

Basic chromosome number

subsp

sub species

IPM

Integrated Pest Management

FAOSTAT

Food and Agriculture Orgapkucvkqpu"Uvcvkuvkecn"Fcvcdcug

PCA

Principal Components Analysis

SLCA

Single Link Cluster Analysis

YVMV

Yellow Vein Mosaic Virus

NAA

g-Nathalene Acetic Acid

IAA

Indole-3-Acetic Acid

IBA

Indole-3-Butyric Acid

TDZ

Thidizuron

BAP

6-aminobenzylpurine

CP

Coat Protein

NAC

Nuclear Agriculture Centre

"

"

BNARI

Biotechnology and Nuclear Agriculture Research Institute

RAMSRI

Radiological and Medical Sciences Research Institute

GHARR-I

Ghana Research Reactor one

ARBC

Applied Radiation Biology Centre

RTC

Radiation Technology Centre

GAEC

Ghana Atomic Energy Commission

HPGe

High-Purified Germanium

INAA

Instrumental Neutron Activation Analysis

MCA

Multi-Channel Analyser

KW

kilowatt

KeV

kilo electron volts

ANOVA

Analysis of Variance

RCBD

Randomised Complete Block Design

CRD

Completely Randomised Design

DMRT

Fwpecpu"ownvkrng"tcpig"vguvu

LoS

Level of significance

Lsd

Least significance difference

GS

Genetic similarity index

CIRAD

Centre for International Research in Agricultural Development

MT

Metric tonnes

UV

Ultra violet

ROS

Reactive oxygen species

SOD

superoxide dismutase

ORAC

Oxygen Radical Absorbance Capacity

FRAP

Ferric Reducing/Antioxidant Power

DPPH

Diphenylpicryl-Hydrazyl

LDL

Low-Density Lipoprotein

"

"

TRAP

Total Antioxidant Parameter

TAA

Total Antioxidant Activity

TPC

Total Phenolics Content

TFC

Total Flavonoid Content

dH20

Distilled water

FCR

Folin-Cicalteu reagents

GAE

Gallic acid equivalent

QE

Quercetin equivalent

AOA

Antioxidant Activity

R2

Coefficient of regression

SD

Standard deviation

HPLC

High Performance Liquid Chromatography

TLC

Thin Layer Chromatography

LCMS

Liquid Chromatography Mass Spectrometry

USB

Universal Serial Bus

Metre (s)

cm

Centimetre (s)

mg/g

milligram per gram

ug/g

microgram per gram

bu

Brabender units

rpm

revolution (s) per minute

RDI

Recommended Daily Intake

"

"

ABSTRACT
A series of investigations were carried out to determine the genetic variability
within 29 accessions of okra (Abelmoschus spp L. Moench) through
characterisation using morphological, biochemical, nutritional and molecular
markers. The goal was to obtain information on key traits of okra germplasm
relevant to breeders and other researchers towards improvement of the crop.
Twenty six (26) indigenous (landraces) and three (3) exotic accessions of
okra were collected from eight regions of Ghana and their morpho-agronomic
traits were evaluated under field conditions at the Biotechnology and Nuclear
Agriculture

Research

Institute (BNARI) research

fields

using the

International Plant Genetic Research Institute (IPGRI) descriptor list for okra.
The 29 accessions exhibited significant variation in all but two quantitative
traits studied. Block coefficients of variation were extremely low, implying
that results obtained are reliable and repeatable over replications. The 29
accessions were grouped into two major clusters and subsequently into five
sub-clusters based on both quantitative and qualitative characters studied. The
association between pairs of quantitative yield traits in the okra landraces
revealed that flowering and fruiting parameters had significant (P = 0.01)
positive associations. Factor scores of 12 characters contributed substantially
to total genetic variation among the 29 okra accessions studied. The pattern of
clustering did not show distinct association between morpho-agronomic
characters and geographic origin of the collections. The output of the
Principal Components Analysis (PCA) revealed that different characters
contributed differently to total genetic variation. The means of maximum
viscosity values for mucilage extracted from the fruits ranged from 53.0 366.8bu, with three accessions; DKA (366.8bu), Yeji-Local (329bu) and
Amanfrom (316.8bu) recording very high values whilst Cape (53.0bu) had
the least maximum viscosity value. There was low level of polymorphism
detected among all accessions using inter-simple sequence repeat (ISSR)
markers. The accessions, Atomic and Akrave were detected to have
"

originated from a common ancestry. While there was high variability among
Okra accessions for the amounts of flavonoids, phenolics and total
antioxidant activity in the fresh fruits and the quantities were generally high
making okra a good source of natural antioxidants.

Ethanol extraction

yielded better antioxidant activity than aqueous (water) solvent. The


accession, Agric short fruit recorded the highest total flavonoid content (TFC)
of 5159.2112.90g/g/QE while Cs-Legon had the lowest TFC of
2003.692.55g/g/QE in the ethanol extract. On the other hand, KortebortorASR registered the highest total phenolic content of (63.223.95g/g/GAE)
while Volta had the lowest TPC of 6.820.09g/g/GAE in the aqueous
extract.

Debo

and

Kortebortor-ASR

recorded

the

highest

(25.835.30g/g/GAE) and lowest (8.00.37g/g/GAE) Total Phenolics


Content in the ethanol extract respectively. Nine essential mineral elements
(sodium, magnesium, potassium, calcium, bromine, chlorine, copper,
aluminium and manganese) were detected among all accessions using
Instrumental Neutron Activation Analysis (INAA). There was significant
variation in concentrations of these elements found in fresh fruits of the
accessions. There were strong positive associations between five pairs of
elements contained in the fruits of the accessions of okra.

"

CHAPTER ONE
INTRODUCTION
1.1 GENERAL INTRODUCTION
Okra (Abelmoschus spp L.) also known as okro, is a tropical vegetable crop,
grown throughout Africa (Schippers, 2000; Norman, 1992; Sinnadurai, 1992;
Kochhar, 1986), Asia and the Americas (Siemonsma and Kouame, 2004;
Tindall, 1983). It is a semi-fibrous annual herb (Bennett-Lartey and OtengYeboah, 2008; Oyelade et al., 2003; Rice et al., 1990), and belongs to the
family Malvaceae (Rice et al., 1990; Tindall, 1983). The plant is robust and
erect, ranging between 1 to 2m in height, with simple leaves which are
alternate and palmately veined. The flowers are regular and solitary, with
superior ovaries and numerous stamens. It is widely cultivated for its
immature fruits and seeds.

Mucilage occurs in various parts of the plant, usually in relatively small


quantities, and is frequently associated with other substances, such as tannins
(Woolfe et al., 1977). The most common sources are the root, bark and seed,
but they are also found in the flower, leaf, and cell wall (Kumar et al., 2010).
Any biological functions of mucilage within the plant are still being explored,
but they may aid in water storage, decrease diffusion in plants, aid in seed
dispersal and germination and act as a membrane thickener and food reserve.

The name "okra", most often used in the United States and the Philippines, is
of West African origin. Okra is often known as Lady's Fingers outside the
United States. In Bantu language, okra is called kingombo, quiabo in
Portuguese, quimbomb or guigamb in Spanish, gumbo in French, bhinde or
bhendi in India. It is called Molondrn in the Dominican Republic, and
"

"aj" in Panama (AVRDC, 2010). In Ghana, it is called nkuruma in Akan


and hgvktk in Ewe (National Academies Press, 2006). It is successfully grown
across the forest and grassland zones under rain fed conditions. It is usually
inter-cropped with pepper, tomato, yam, cassava or other crops by
subsistence farmers (Norman, 1992).

1.1.1 PROBLEM STATEMENT"


Notwithstanding the potential of the crop, there are few improved varieties
for cultivation by okra farmers in Ghana. Moreso, there are few reports of any
attempt by breeders at improving the vegetable in terms of developing core
collections for breeding towards improved yield or quality. Varieties under
cultivation, over the years, in various regions across the country are mainly
landraces. These landraces are however, highly susceptible to biotic and
abiotic stresses such as diseases, pests and nematode (Oppong-Sekyere et al.,
2012; Oppong-Sekyere, 2011; Sinnadurai, 1992). Currently, a number of
exotic varieties are available locally. These are meant for production to meet
specific export markets.

India, Costa Rica, Nigeria and Ghana are among the major producers of the
crop (FAOSTAT, 2012; NARP, 1993). The Brong Ahafo, Northern, Volta,
Greater Accra and Central regions are the bulk producers in Ghana (NARP,
1993).

The immature green pods and fresh leaves are used locally as

potherbs. Locally, they are mainly employed as a boiled vegetable, they can
be stir-fried, battered and deep-fat fried, microwaved, steamed, baked,
grilled, blanched and processed as a frozen (plain or breaded), pickled, or
canned product (Oppong-Sekyere et al., 2012). Dried fruits of okra, as slices
or in powder form are often stored and used in stews and soups. The fresh
okra fruits are high in vitamins A and C; and in calcium (NARP, 1993).

"

"

Intra as well as interspecific variations do exist in okra in different phytogeographic areas of Ghana (Anonymous, 2010; Ahiakpa, 2009). However,
like most tropical crops, very little research attention has been devoted to this
crop, particularly in Ghana (Ahiakpa, 2009). Duplications are masked by the
use of local names. Previous works are limited to the use of morphological
traits in cultivar identification and characterisation (Oppong-Sekyere, 2011;
Essilfie et al., 2010; Bennett-Lartey and Oteng-Yeboah, 2008). Hence, there
is the need to also carry out biochemical, nutritional and molecular
characterisation to fully describe the available germplasm of okra (Ahiakpa,
2009; Lester et al., 1990). This information will consequently enhance rapid
improvement of the crop.

"1.1.2

JUSTIFICATION AND RELEVANCE OF THE STUDY

Germplasm are strategic resources essential to both national and global


agricultural food security (IBPGR, 1991). The value of germplasm depends
not only on the number of accessions, but also upon the diversity present
within it (Ahiakpa, 2009; Ren et al., 1995). Characterisation and
quantification of genetic diversity has long been a major goal in evolutionary
biology which make available essential information on genetic diversity
within and among closely related crop varieties for breeders to make a
rational use of plant genetic resources. Diversity based on phenotypic and
agro-morphological characters are usually variable with environments and
evaluation of traits requires growing the plants to full maturity prior to
identification (Aladele et al., 2008). The almost absence of readily available
information on agronomic traits and characters of okra germplasm for
breeders and researchers is a limitation to most okra improvement
programmes especially in the West African sub-region (IITA, 2001).

"

The rapid development of biotechnology allows easy analysis of large


number of loci distributed throughout the genome of plants (Aladele, 2005).
Molecular markers for instance, have proven to be powerful tools in the
assessment of genetic variation and in elucidation of genetic relationships
within and among species (Chakravarthi and Naravaneni, 2006).

However, diversity in traditional local cultivars is much greater than


improved and released varieties (Ahiakpa, 2009). Okra has considerable area
under cultivation in Africa and Asia with huge socio-economic potential.
Collection of accessions helps to assess the diversity that exists among these
accessions and to select resources of choice for okra improvement and
research.

More importantly, increase in food demand and the need for

continuous breeding programme, to come out with new or improved crops to


satisfy the needs of society, requires characterisation to determine their
specific attributes.

In addition, plant genetic resources are conserved for future use. Using them
is only possible if their characteristics or attributes are known in detail and
their utilisation visualised (Jaramillo and Baena, 2000). Such knowledge and
visualisation can be achieved only through the study of the morphological,
structural, and functional attributes of germplasm, as the carriers of all the
hereditary characteristics of any species.
These methodologies (molecular and biochemical) help locate genes of
interest with greater accuracy but do not evaluate the effect of the
environment on the expression of those genes (Westman and Kresovich,
1997). Accordingly, they do not replace but complement morpho-agronomic
characterisation and evaluation (IPGRI, 2004; IPGRI and CIP, 2003a&b).

"

"

The local landraces in Ghana have long maturity periods and short
harvesting duration. They are of poor nutritional quality, non-standard in
shape, colour and size, making them unfit for the Ghanaian okra vegetable
export market (Oppong-Sekyere, 2011). This has a consequential impact of
causing a tremendous reduction in the per capita income of the nation. It is
therefore important that plant breeders develop improved varieties of okra,
for adoption by Ghanaian vegetable farmers and for the export market.
Varieties that are perennial in growth habit and at the same time combine
higher yields and early maturity with longer harvest duration and also
resistant to diseases and pests would be ideal for the okra vegetable industry
in Ghana (Oppong-Sekyere et al., 2012).

Such varieties must also possess improved fruit size, shape and colour,
quality characteristics, very much desired in the okra export market (Boateng,
2011.per comm.). Currently, this vegetable is a non-traditional export crop in
Ghana but interestingly, only exotic varieties meet export specifications."
Vjgtghqtg." vjgtg" ku" vjg" need to develop local varieties to expand production
base. The first step towards attaining this objective involves collection and
thorough characterisation of available germplasm of the vegetable.

Collection, characterisation and genetic diversity studies are important first


steps, among others, for the amelioration of the crop in Ghana thereby
providing the foundation on which to enhance its potential and utilisation.
Identification and description of the genetic variability available in
germplasm collections are the bases of long-term plans to control genetic
erosion; they are also a preliminary requirement for the exploitation of all
useful traits of the crop (Oka, 1991). To harness and utilise useful traits in
okra genetic resources, it is essential to assemble, characterise, evaluate and
conserve them so as to enhance and facilitate their utilisation in any
"

improvement programme of the crop (Ahiakpa, 2009; Lester et al., 1990; and
Charrier, 1983).

1.1.3 OBJECTIVES OF THE RESEARCH


The overall objective of this work was to determine the genetic variability
inherent in twenty-nine (29) local accessions of okra through characterisation,
using morphological, biochemical, nutritional and molecular traits.
The specific objectives of the research are;
1. To assess phenotypic and molecular diversity among a collection of okra;
2. To determine variability in production of mucilage and its association
with other traits;
3. To generate a genetic relationship using morphological and molecular data
via dendrogram;
4. To determine variation in mineral contents of the fruits and assess antioxidant
scavenging activity in the various accessions of okra;
5. To determine any duplicates among the accessions and also identify superior
ones with desirable characteristics for further breeding work.

"

"

REFERENCES
References for CHAPTER ONE are merged with those for CHAPTER
TWO.

"

CHAPTER TWO
LITERATURE REVIEW
2.1 ORIGIN, DISTRIBUTION AND UTILISATION
Okra (Abelmoschus spp L.) is a member of the family Malvaceae same as
cotton. It is of African origin (ECHO, 2003; Purseglove, 1987), discovered
around Ethiopia during the 12th century B.C and was cultivated by the
ancient Egyptians (Purseglove, 1987). Currently, the crop is grown as a
popular vegetable throughout the tropical and sub-tropical regions of the
world (Kumar et al., 2010).

Hamon and van Sloten (1989) classified the cultivation areas of okra into four
climatic zones. These include; desert (village or oasis cultivation), sahelian
(north of latitude 12 oN), savannah (between latitudes 8 oN and 12 oN) and
rain forest climatic zones. Abelmoschus esculentus is primarily distributed
throughout the intermediate savannah zone between the rain forest and the
arid sahel. The species is less frequently found in the rain forest zone but is,
on the other hand fairly well represented in the sahel zone. On the contrary,
the West African Taxon (WAT), A. caillei does not occur in the sahelian zone
since it has a long life cycle and usually requires abundant, continuous
rainfall. Hamon and van Sloten, (1989) stated that since a natural interspecific
hybrid of the two cultivated species occurs in the central part of Sudan, A.
caillei is possibly more widely distributed than currently known.

In addition to its usefulness as a vegetable crop, okra fruit is useful


medicinally, in curing ulcer and suppressing the pains and effects of
haemorrhoid. The mucilage has been used as a plasma replacement or blood
volume expander (Siemonsma and Kouame, 2004; Vickery and Vickery,
1979). Reports from research in China revealed that an alcohol extract from
"

"

the leaves of okra can eliminate oxygen free radicals, alleviate renal tubularinterstitial diseases, improve renal function and reduce proteinuria
(Siemonsma and Kouame, 2004).

Okra is a storehouse of valuable nutrients. Compared to other fleshy fruit


vegetables (Solanum melongena and Lycopersicon esculentum), okra is
particularly rich in calcium and ascorbic acid (Siemonsma and Kouame,
2004). It is low in calories and is fat-free (National Academies Press, 2006;
Siemonsma and Kouame, 2004; Tindall, 1983).

In West Africa, the plant is cultivated as a vegetable crop and the leaves, buds
and flowers are often eaten (Siemonsma and Kouame, 2004; Tindall, 1983).
The fruits may be dried, stored and powdered for use in soups in the dry
season when fresh fruits are scarce (Oppong-Sekyere, 2011; Siemonsma and
Kouame, 2004; Oyolu, 1977). The immature fruits are used as boiled or fried
vegetable (Tindall, 1983). The roots can be used to cure syphilis (FAO,
1988). The mucilage in pods (a glutinous substance) is used in thickening
soups and stews (National Academies Press, 2006; Woolfe et al., 1977). The
matured seeds contain about 20% of edible oil (Tindall, 1983) and can be
used for biodiesel production (Anwar et al., 2010).

Okra seeds are reportedly used as substitutes or additives in feed compounds


(Purseglove, 1974), as substitute for coffee in Turkey (Yildiz et al., 2005), in
the preparation of okra seed meal (flour) and a number of baked products
(Martin and Roberts, 1990). Industrially, okra mucilage is used to glace
papers and also useful in confectionery among other uses as bioabsorbent
(Kumar et al., 2010; Norman, 1992).

"

"

"2.2

TAXONOMIC CLASSIFICATION AND BOTANY

Okra (Abelmoschus spp L.) belongs to the family Malvaceae, genus


Abelmoschus and the species esculentus (Kumar et al., 2010; IBPGR, 1991).
Previous classification placed okra in the genus Hibiscus; section
Abelmoschus in the family Malvaceae (Joshi and Hardas, 1956; Vavilov,
1926). The section Abelmoschus was subsequently proposed to be raised to
the rank of distinct genus (Anonymous, 2010; Medikus, 1787). The wider use
of Abelmoschus was subsequently accepted in taxonomic and contemporary
literature (Joshi and Hardas, 1953). This genus is distinguished from the
genus Hibiscus by the characteristics of a spathulate calyx, with five short
teeth, connate to the corolla and caduceus after flowering (Kumar et al., 2010;
Terrell and Winters, 1974; Kundu and Biswas, 1973).

Okra is an upright annual, herbaceous plant with a hibiscus-like flower. It is


a direct-sown vegetable with duration of 90-100 days and a deep taproot
system. Its stem is semi-woody and sometimes pigmented with a green or
reddish tinge of colour. It is erect, variable in branching, with many short
branches that are connected to the thick semi-woody stem. The stem attains
heights from 3m in dwarf varieties to 7 or 8m in others (Anonymous, 2010).
The stems bear leaves that are lobed and are generally hairy, some reaching
up to 12cm in length. Leaves are cordate (heart-shaped), simple, usually
palmately 3-7 lobed and veined. They are subtended by a pair of narrow
stipules. The okra leaf is dark green in colour and resembles a maple leaf
(Kumar et al., 2010).
Flowers are borne vertically only on the orthotropic axis every two or three
days. The flower is axillary and solitary, borne on a peduncle 2.0 2.5cm
long within the leaf axil (Valeriana, 2011). The flowers are large about 2cm
in diameter, with five white to yellow petals bearing a red or purple spot at
the base of each petal and endure only for a day. The flowers are almost
"

"

always bisexual and actinomorphic (Kumar et al., 2010). The perianth


consists of 5 valvate, distinct or basally connate sepals and 5 distinct petals
that are usually basally adnate to the androecium.

The androecium consists of numerous monadelphous stamens with apically


divergent filaments bearing one-celled anthers. The gynoecium is a single
compound pistil of two-many carpels, an equal number of styles or style
branches, and a superior ovary with two-many locules, each bearing one or
more ovules. The calyx is completely fused to form a protective case for the
floral bud and splits into lobes when the bud opens (Valeriana, 2011). The
calyx, corolla and stamens are fused together at the base and fall off as one
piece after anthesis. Each blossom develops a small green pod. It grows
rapidly into long (10-30cm) and narrow (1-4cm) pod with a tip that is either
pointed like a beak or blunt. The elongated, conical pod is generally ribbed
and consists of five cavities containing ovules (Chandra and Bhatnagar, 1975)
and spineless in cultivated types (Anonymous, 2010). It is normally yellowish
green to green, but sometimes purple or white. The pods are harvested while
still tender and immature (Kumar et al., 2010; National Academies Press,
2006). The okra fruit houses numerous oval-shaped whitish seeds, which turn
dark

green

to

dark

brown

upon

maturity

(http://www.wiu.edu/altcrops/okra.htm). Improved types have fewer spines


on the pods than landraces and wild types.

2.3 GENETICS AND CYTOLOGY


Different authors have variably reported the chromosome number (2n) of
Abelmoschus esculentus L. (Moench). The most frequently observed somatic
chromosome number is 2n=130, although Datta and Naug (1968) suggested
that the numbers 2n=72, 108, 120, 132 and 144 are in regular series of
"

"

polyploids with n = 12. This makes the existing taxonomical classifications at


the species level in the genus Abelmoschus quite unsatisfactory. There was an
ongoing detailed cytogenetical study on Asian okra and related species,
which has been reported to have provided more evidence of the existence of
amphidiploids in the genus (Siemonsma, 1982a).

The nomenclature of Abelmoschus spp with varied chromosome numbers of


both cultivated and wild species of Abelmoschus genus as distinctly reported
involved most species of the genus. Chromosome number of 2n = 72 have
been reported in Abelmoschus moschatus for both cultivated and wild type
(Chevalier, 1940; Ford, 1938). van Borssum-Waalkes (1966) observed 2n =
130-1388 in A. manihot subsp tetraphyllus var tetraphyllus for wild species,
Siemonsma (1982a&b) reported 2n = 138 in A.manihot subsp tetraphyllus
var pungens for wild type; Hamon and Yapo (1986) observed 2n = 66-144
for Abelmoschus esculentus (cultivated type). Again, Pal et al. (1952)
recorded 2n = 72-78 in A. ficulneus (wild), and 2n = 38 in A. angulosus
(wild), 2n = 185-198 in Abelmoschus caillei (cultivated), and 2n = 58 in
Abelmoschus tuberculatus (wild type) were recorded by Singh et al. (1975),
Joshi et al. (1974), and Kuwada (1957) respectively. From all this, okra can
be regarded as a polytypic complex (Singh and Bhatnagar, 1975) that exhibits
both high polyploidy and hybridity of which the parental wild species is yet
to be determined.

Aladele et al. (2008) collected 93 accessions of okra comprising of 50 West


African genotypes (Abelmoschus caillei) and 43 Asian genotypes (A.
esculentus) and assessed for genetic distinctiveness and relationship using
randomly amplified polymorphic DNA (RAPD), and concluded that all the
thirteen primers used revealed clear distinction between the two genotypes.
More diversity among the Asian genotypes; possibly due to the fact that they
"

"

were originally collected from six different countries in the region. Six
duplicate accessions were discovered while accession TOT7444 distinguished
itself from the other two okra species, an indication that it might belong to a
different species. This recent study at molecular level emphasises the need for
a deeper study into the variable polymorphism at chromosomal level in the
genus Abelmoschus.

Kumar et al. (2010) examined the possible outcome of a recombination of


these species and their possible contrasting characters after a cross between
Asian genotype and the West African genotype. The table below details their
findings and observations.

Table 2.1:

Potential

of

recombination breeding

involving

two

Abelmoschus spp"L""
Species
A. esculentus (common okra)
95% cultivated area

Cytogenetics
Amphidiploid (2n=130-140): A.
tuberculatus or A. ficulneus (2n-5860) x unknown?

Contrasting Traits
Poor adaptation in humid zone,
more susceptible to biotic
stresses, less vigorous, short life
cycle (suitable for short rainy
season areas), usually day neutral,
cultivated in both rainy (rain fed)
and dry (irrigated) season

A. caillei (West African okra)


5% cultivated area

Amphipolyploid (2n = 196-200): A.


esculentus (2n=130-140) x A.
manihot (2n = 60-68)

Better adaptation in humid zone,


tolerant/ resistant to biotic
stresses, more vigorous, longer
life cycle, mostly photoperiod
sensitive, cultivated mainly in dry
season.

Source: Kumar et al. (2010).

"

Diversity in pod shape, size and flowering behaviour account for most of the
variation between the genotypes of West and Central African origin
(Duzyaman, 1997) and scope for further gain in pod yield per plant is limited
because of low phenotypic and genotypic variability (Ariyo, 1990a). To break
the yield barrier in existing genotypes of common okra (A. esculentus) and
breed for different market types, a hybridisation-based breeding strategy
would be desirable.

2.4 SOIL, CLIMATIC AND AGRONOMIC REQUIREMENTS


Okra is a warm season crop, which grows best between minimum and
maximum mean temperatures of 18C and 35C, respectively and optimum
temperature range from 21C to 30C (Martin, 1982). It can be grown on a
wide range of soil types under good drainage. It is intolerant, to wet and
poorly drained and acidic soils, but will tolerate a soil pH range from 6.0 to
7.5 (Incalcaterra and Curatolo, 1997). Optimum soil temperature for seed
germination is between 24C-32C, with germination taking place in 5-14
days (Hamon and Nairot, 1991).

Short day length stimulates flowering of most cultivars (Martin et al., 1979).
Flowering begins at a very early stage of growth at day lengths of less than 11
hrs; under long days, the flower buds tend to abort (Chauhan, 1972). Okra
requires adequate soil moisture throughout its growing period for optimum
growth and yield (Norman, 1992).

2.5 GROWTH AND DEVELOPMENT


Okra is a stout, erect annual herb that grows to 4.0m tall, bearing spirally
arranged leaves with leaf blades up to 50cm in diameter and more or less
"

"

deeply 3-5- and 7-lobed (Incalcaterra and Curatolo, 1997). Proper


understanding of the growth stages and development of okra is very essential
in the development of cultivar and integrated pest management (IPM)
strategies (Valeriana, 2011). Stages in the life cycle of okra plant may be
divided into four: seed stage, seedling stage, vegetative stage and
reproductive stage.

(a)Seeding stage

(c) Vegetative stage

(b) Seedling stage

(d) Reproductive stage

Figure 2.1: Stages in the growth and development of okra plant (Valeriana, 2011)
(a) Seeds are numerous, gray to black in colour and about 3-6 mm in diameter. Seeds germinate about 5-7 days
after sowing.

(b) This is between one to two weeks from seed germination. Seedlings have at least 3-4 leaves with a height
approximately 12-18cm.

"

(c) This occurs three to four weeks from seed germination. Leaves are bigger and the plant has more than eight
leaves. Length of stem between leaves is longer. The leaves are spreading and spirally arranged.

(d) Plants start to flower at five weeks from seed germination. Yellow solitary flowers are in the leaf axils. Okra
usually flowers within 40-90 days after sowing. Flower opens in the morning. Fruits or pods are green,
cylindrical to pyramidal capsule 5-35cm long and 1-5cm in diameter.

It takes about one month from anthesis to fruit maturity. Mature green pods
turn brown and dry. On the seed crop, vegetative growth stops soon after
anthesis, all assimilates are partitioned to the reproductive parts of the plant
(Kumar et al., 2010). Flower initiation and flowering are delayed at higher
temperatures, indicating a positive correlation between temperature and
number of vegetative internodes (Norman, 1992).
"
"2.6

POLLINATION AND FERTILISATION

Flower bud initiation, flowering, anthesis and stigma receptivity are


influenced by genotype and climatic factors like temperature and humidity
(Anonymous, 2010). From studies made on six okra varieties, Sulikeri and
Swamy Rao (1972) concluded that flower buds are initiated at 22-26 days and
the first flower opened 41-48 days after sowing. Once initiated, flowering
continues for 40-60 days. Anthers dehisce prior to flower opening, and hence
self-pollination may occur at anthesis. The dehiscence of anthers is transverse
and complete dehiscence occurs in 5-10 minutes (Shalaby, 1972). Pollen
fertility is maximum between an hour before and an hour after opening of the
flower. Flowers open only once in the morning and close after pollination on
the same day. The corolla withers the next morning (Datta and Naug, 1968).

Okra has perfect flowers, thus male and female reproductive parts are in the
same flower and are self-pollinating. If okra flowers are bagged to exclude
"

"

pollinators, 100% of the flowers will set seed. It has been demonstrated
experimentally that there is no significant difference in fruit set under openpollinated, self-pollinated (by bagging alone) and self-pollinated (hand
pollination of bagged flowers) plants, indicating that it is primarily a selfpollinated crop (Valeriana, 2011; Datta and Naug, 1968).

Inbreeding depression which is well pronounced in cross-pollinated crops


has not been reported in okra (Shalaby, 1972). Although insects are
unnecessary for pollination and fertilisation in okra, the flowers are very
attractive to bees and the plants are sometimes cross-pollinated. Crosspollination up to the extent of 4-19% (Choudhury and Choomsai, 1970) with
maximum of 42.2% (Mitidieri and Vencovsky, 1974) has been reported. The
extent of cross-pollination in a particular place will depend upon the cultivar,
competitive flora, insect population and season, among other factors
(Chandra and Bhatnagar, 1975).

2.7 RATOONING
Some farmers practise ratooning when seeds are scarce and when the buying
period is extended. Instead of establishing a new crop, farmers cut the main
stem of the first crop. They then apply herbicides to kill the weeds before
applying fertiliser and irrigating (Schippers, 2000). Some farmers, however,
apply sodium nitrate to enhance flowering. It takes less than a month to
harvest from the ratoon crop (Valeriana, 2011). However, pods from ratoon
crop are of less quality and fewer than the former crop (Camciuc et al., 1996).
This is so because there would be more branches per plant to provide the
nutrients.

"

Incidence of more larvae of fruit worm and cutworm (Agrotis spp.) has been
monitored from ratoon crops. The larvae of these pests come from the first
crop and they pupate in the soil (Anonymous, 2010). Ratoon crops do not
require tillage and therefore soil is not disturbed. The duration of time from
pupa to adult, egg laying and hatching of eggs into larvae coincides with that
time the ratoon crop is producing more leaves and flower (Siemonsma and
Kouame, 2004). Besides the larvae, other insect pests will transfer to the
ratoon crops when they have no more food from the first crop in the
neighbouring fields (Midmore et al., 2005).

2.8 NUTRIENT CONTENTS AND HEALTH BENEFITS


In the African context, okra has bggp"ecnngf"cu"c"rgthgev"xknncigtu"xgigvcdng"
because of its robust nature, usable dietary fibre and distinct seed protein
balanced in both lysine and tryptophan as well as amino acids it provides to
diet unlike cereals and pulses (Kumar et al., 2010; National Academies Press,
2006). The fruit contains 88% moisture. It is low in saturated fat, and very
low in cholesterol and sodium. It is also a good source of protein, niacin, iron,
phosphorus, zinc, and copper (Norman, 1992), and a very good source of
dietary fibre, vitamin A, vitamin C, vitamin K, thiamine, riboflavin, folate,
calcium, magnesium, potassium and manganese (Lamont, 1999; Tindall,
1983).

2.8.1 Biochemical and nutritional composition


Abelmoschus esculentus is known to be rich in polyphenolic compounds
(Arapitsas, 2008), protein and fat (Oyelade et al., 2003), oil and gossypol
(Maganha et al., 2010; Martin et al., 1979), fat and fibre (Rao, 1985),
carbohydrate (Woolfe et al., 1977), ash, phosphorous, and iron (El-Nahry et

"

al., 1978), calcium and iron (Savello et al., 1980), which are integral to health
in humans (Kumar et al., 2010).

2.8.2 Nutritional healing properties


Okra has been clinically proven to be involved in alkaline reaction, soothes
irritated membrane of the intestinal tract, lowering blood sugar, heal burn and
any kind of skin rashes (Bansal, 2002). Its mucilaginous texture soaks up
unhealthy cholesterol, toxin and mucous, waste and clean them from the
intestinal tract, acts as laxative that can heal ulcer and may reduce acid reflux
(Inda, 2011), promote good cardiovascular and gastrointestinal health,
confers antioxidant and anticancer effects to the consumer (Collins, 2010).

2.8.3 Bioactive properties


The richness of bioactive compounds of Abelmoschus are said to match that
of typical medicinal plants. Activities of the biochemical compounds
identified in the plant include antioxidative effect (Adelakun et al., 2009;
Adetuyi,

2008),

mucilagenous

effect

(Ameena

et

al.,

2010),

anticomplementary and hypoglycaemic activity (Tomoda et al., 1990),


hypoglycaemic effect (Zhenzhong and Hongjun, 2010), antimicrobial effect
(Lengsfeld et al., 2004), anticancer (Dan and Gu, 2010), antiproliferative and
proapoptotic actions (Vayssade et al., 2010). These are required in medicine
to counter or manage diabetes (Dan and Gu, 2010).

2.9 VARIABILITY AND POTENTIAL OF OKRA GERMPLASM


There are about 2,283 reported accessions of okra in the world (OppongSekyere, 2011; Charrier and Siemonsma, 1984; 1991; Hammon and van
Slotten, 1989); of which 2,029 of these are from the African continent with
"

"

1,769 from West Africa (Oppong-Sekyere, 2011; Hammon and van Slotten,
1989). Okra, therefore, is far more prominent in West Africa than in other
parts of the world (Omonhinmin and Osawaru, 2005). The genetic diversity
of okra is clearly shown by the wide range of morphological characteristics
displayed by the taxon in different ecogeographical, edaphic and
environmental conditions (Omonhinmin and Osawaru, 2005).

Okra, (Abelmoschus esculentus L. Moench) can be found in nearly every


market in Africa. In Ghana, it is the fourth most popular vegetable after
tomato, pepper, and garden egg (Oppong-Sekyere, 2011; Sinnadurai, 1973).
One can find okra in a fresh state in almost all markets in Ghana, during the
rainy season and in a dehydrated form during the dry season, particularly in
Northern Ghana due to its strong commercial value for resource poor women
farmers and its vital role in the diet of inhabitants of cities and villages. In
2005, world production of okra was at 730 metric tonnes (FAOSTAT, 2005),
and Ghana was ranked 15th coqpiuv"vjg"yqtnfu"nctiguv"rtqfwegtu"yjkej"ycu"
valued at 100,000 metric tonnes (FAOSTAT, 2005). In 2009 alone, the total
production of okra from Ghana was 71,350 metric tonnes, yielding the nation
45,627 US dollars (FAOSTAT, 2011).

In agriculture, the importance of okra cannot be overemphasised. It is


vjgtghqtg"rqrwnctn{"tghgttgf"vq"cu"c"rqygtjqwug"qh"xcnwcdng"pwvtkgpvu0"Vjku"
vegetable contains pepsin E3 (Williams, 1960) which provides bowelcleansing action and tissue-healing properties (Standard Process, 2009;
Charvan, 1991).

In addition, pepsin E3 in okra provides healing through stimulating elevated


serum levels of calcium and promoting phagocytosis, the process in which
unwanted microorganism and debris are execreted from the body. It contains
"

"

allantoin, which has been demonstrated to be clinically beneficial to healthy


epithelial tissues and stimulating immune function (Standard Process, 2009).
The mucilage from okra coats in the various tissues, provides lubrication as
well as cooling to relieve gastrointestinal discomfort (Charvan, 1991).

There exists a wide genetic diversity among cultivated species of the genus
Abelmoschus (Omonhinmin and Osawaru, 2005; Bisht et al., 1995) and are
highest amongst species found in countries such as Turkey and India (Bisht et
al., 1995). Porter et al., (1974) reported that large morphological variability
abounds in the tropics suggesting adequate research into the germplasm
structure for development of hybrids with specific ecological adaptation.
Variability is more prominent in days to flowering, plant height and various
fruit characteristics among okra germplasm. Hence, these traits could be
important in differentiating varieties of A. esculentus (Ariyo and Odulaja,
1991). Within species variation among 30 West African genotypes were
found to be considerably large based on phenotypic assessment (Ariyo,
1993). Gulsen et al. (2007) however stated that, the West African sub region
being the second largest producer of okra may have considerable level of
genetic diversity as in many other important crop species, which calls for
thorough research investment to fully exploit these potentials in the crop.

2.10 CHARACTERISATION OF OKRA


Crop improvement through breeding is dependent on the availability of
genetic variability in the species and how easily this variability could be fixed
in genotypes (Kiran Patro and Ravisankar, 2004; Ariyo, 1990a). Heritable
traits or characters of plant germplasm are studied precisely during
characterisation; where sometimes, characterisation data and morphoagronomic evaluation are insufficient in establishing distinctiveness between
"

"

species or between accessions. In these cases, genome characteristics such as


the karyotype, chromosome number, and ploidy level may be studied. The
genome itself can be studied directly, using biochemical isoenzymes
(Simpson and Withers, 1986) and molecular markers such as simple sequence
repeat (SSR); restriction fragment length polymorphism (RFLP), randomly
amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR),
sequence-related amplified polymorphism (SRAP), expressed sequence tag
(EST), and quantitative traits loci (QTLs) (Kiran Patro and Ravisankar, 2004;
Ariyo, 1990b).

Genetic diversity has been reported in the West African and South Asian
accessions of okra (Bisht et al., 1995; Ariyo, 1993). A good understanding of
genetic variability in the different characters of okra would be a useful tool in
the genetic improvement of the crop. This will enhance the identification of
useful genes and their behaviour as an aid in hybridisation programmes. The
characterisation of a range of genetic variability among genotypes is pivotal
to the maintenance and further acquisition of germplasm resources even as
accessions from diverse origins are needed as parent stocks for the
development of improved varieties (Aremu et al., 2007).

Knowledge of genetic diversity and relationships among okra germplasm


may play significant role in breeding programmes for biotic and abiotic
stresses of okra. High degree of morphological variation has been reported in
previous studies on West African okra (Omonhinmin and Osawaru, 2005).
Many local cultivars occur in Africa and Asia, and differ from one another in
growth habit (branching or non-branching, large or dwarf, late or early, hairy
or glabrous, light green, dark green or red) and fruit characteristics (upright or
pendulous, slender or wide, with 5 to 10 ridges). Pod shapes range from
round to ridged and short to long. The plant and pods may have small spines
"

"

on their surfaces, creating allergies in some people (Splittstoessor, 1990).


Comprehending the genetic structure and germplasm diversity of okra being
kept in gene banks all over the world will bring valuable information for okra
breeding programmes.

Characterisation of a sample is a careful delineation of special characteristics


that are heritable, scorable, and expressible consistently in all environments
(Rubenstein and Heisey, 2003). Since most of the traits recorded during
characterisation are those that are visible, the person responsible for
managing the germplasm material is best placed to carry out the work of
documenting these characteristics (De Vicente et al., 2005). Many of the
characteristics that are recorded on individual accessions can serve as
diagnostic descriptors for accessions. Such diagnostic characters help
genebank curators keep track of an accession and check for the genetic
integrity over a number of years of conservation (Rubenstein and Heisey,
4225+0" Cickp." fguetkrvqtu" nkuvu" ctg" c" xkvcn" vqqn" hqt" gpuwtkpi" ucog" ncpiwcig"
and standards during documenting characteristics of conserved species (De
Vicente et al., 2005; Rubenstein and Heisey, 2003).

"2.10.1

Morphological Characterisation of Okra

For the improvement of the yield and other desirable characters, a knowledge
of the magnitude of variation in the available genotypes, the relation of
characters with yield, extent of environmental influences on these and the
heritability of the materials are essential (Saifullah and Rabbani, 2009).
Morphological characterisation is the most dominant in okra germplasm.
These include recent assessment of genetic diversity in a collection of 22
Ghanaian okra germplasm (Abelmoschus spp L.) using morphological
markers (Oppong-Sekyere, 2011); morphological classification of genetic
"

"

diversity in 29 accessions of cultivated okra, Abelmoschus esculentus L.


Moench using principal component analysis (PCA) and single linkage cluster
analysis (SLCA) in Nigeria (Nwangburuka et al., 2011),

morphological

distinctiveness and metroglyph analysis of fifty accessions of West African


okra (Abelmoschus caillei) (Aladele, 2009 ), multivariate analysis of
morphological and agronomic characteristics in 30 accessions of

West

African okra (Abelmoschus caillei) (Ariyo, 1993), evaluation and


characterisation of 120 accessions of okra (Abelmoschus esculentus L.
Moench.) genotypes in Bangladesh (Saifullah and Rabbani, 2009). Hamon
and van Sloten (1989) published a comprehensive report on their years of
research on the different species within the genus Abelmoschus. Findings
from these studies are however limited as they are environmentally
dependent.

2.10.2 Molecular Characterisation of Okra


Studies using molecular markers in okra are lagging behind the other major
crop species (Aladele et al., 2008). The only study reported is by Martinello
et al. (2001) using randomly amplified polymorphic DNA (RAPD) marker
and sequence-related amplified polymorphism (SRAP) marker by Gulsen et
al. (2007). Collecting DNA marker data to determine whether phenotypically
similar cultivars are genetically similar is of great interest in okra breeding
strategies (Duzyaman, 2005).

Kresovich et al. (1992) showed that molecular markers are of great value in
genetic resource management as quick, cost-effective and reliable methods
for identification, measurement of variation and determination of similarity at
the intra-specific level. Reports on marker development in okra are very
scanty and have been mostly limited to characterisation of cultivars.
"

"

Barazani et al. (2003) emphasised the importance of combining molecular


markers with morphological markers by researchers. Martinello et al. (2001)
reported an agreement between clustering patterns obtained from
morphological traits and molecular markers in Abelmoschus spp. Ninety-three
accessions of common (A. esculentus) and West African (A. caillei) were
molecularly characterised and distinguished using random amplified
polymorphic DNA (RAPD) markers (Aladele et al., 2008).

Marker aided selection (MAS) for various traits in okra germplasm in Turkey
has been suggested using sequence-related amplified polymorphism (SRAP)
(Gulsen et al., 2007). Recently, twenty okra accessions from Burkina Faso
were analysed using 16 primers designed to amplify SSR regions of
Medicago truncatula. Two accessions were found distinct from the other 18,
based on the presence of an unique 440 bp fragment generated primer MT-27
and also based on presence of hairs on fruits and delayed maturity of these
two accessions (Sawadogo et al., 2009).

Among wide relatives of okra, Abelmoschus angulosus showed complete


resistance to yellow vein mosaic virus (YVMV) and powdery mildew
disease. A. ficulneus and A. moschatus accompany a high degree of resistance
only to powdery mildew and these germplasm can be potential genetic
resources in breeding okra for YVMV and powdery mildew resistance,
following a marker-assisted characterisation of disease resistance variation in
some wild okra accessions in Sri Lanka (Samarajeewa and Rathnayaha,
2004).

"

"

2.11 OKRA GERMPLASM CONSERVATION


More than 46 institutions in different countries worldwide possess about
11,000 accessions of cultivated okra and wild related species (IBPGR, 1991,
1990). Major institutions holdings are more than 100 accessions. Plant
breeders cannot develop a new variety without the use of genetic material
with some levels of variation. The role of genetic resources in the
improvement and development of cultivated plants has been well recognised
(Tigerstedt, 1994). Characterisation of collected germplasm is indispensable
in any plant breeding programme. It helps breeders in selecting suitable
parents for crossing experiments to develop new varieties (Frankel, 1984;
1976; Hartwig, 1972).

Okra has huge potential for enhancing livelihoods in urban and rural areas
and to several stakeholders (Kumar et al., 2010; National Academies Press,
2006). It offers a possible route to prosperity for small-scale and large-scale
producers alike and all those involved in the okra value chain, including
women producers and traders. Both pod skin (mesocarp) and seeds are
excellent sources of zinc (80 mg/g) (Cook et al., 2000; Glew, 1997). Okra
seed is mainly composed of oligomeric catechins (2.5mg/g of seeds) and
flavonol derivatives (3.4 mg/g of seeds), while the mesocarp is mainly
composed of hydroxycinnamic and quercetin derivatives (0.2 and 0.3mg/g of
skins). Pods and seeds are rich in phenolic compounds with important
biological properties like quartering derivatives, catechin oligomers and
hydroxycinnamic derivatives (Arapitsas, 2008). These properties, along with
the high content of carbohydrates, proteins, glycol-protein, and other dietary
elements enhance the importance of this foodstuff in human diet (Arapitsas,
2008; Manach et al., 2005).
In addition, fresh okra pods are the most important vegetable source for
viscous fibre, an important dietary component to lower cholesterol (Kendall
"

"

and Jenkins, 2004). Okra seed oil has potential hypocholesterolemic effect
(Rao, 1985; Ramu, 1976). The potential for wide cultivation of okra for
edible oil as well as for cake is very high (Rao, 1985). Okra seed flour could
also be used to fortify cereal flour (Adelakun et al., 2009). Okra mucilage has
potential for use as food, non-food products, and medicine (Ndjouenkeu et
al., 1996; BeMiller et al., 1993). Okra mucilage is used in Asian medicine as
a protective food additive against irritating and inflammatory gastric diseases.
Researchers worldwide are looking for already characterised available
germplasm of the plant to explore all these enumerated potentials and exploit
them fully to the benefit of society (Lengsfeld et al., 2004).
"

Okra offers many production possibilities; however, there are limited studies
conducted on its germplasm due to limited resources devoted to the species
by national and international research institutes. Additionally, recent research
goals are geared towards fast maturing types well suited to tropical heat, and
humidity conditions and require adequate knowledge of available germplasm
of the plant to facilitate full exploitation of these potentials (Kumar et al.,
2010). Most crop geneticists agree that enrichment of the cultivated gene pool
will be necessary to meet the challenges that lie ahead (Tester and Langridge,
2010).
"

Qmtcu" rqvgpvkcn" hqt" kpfwuvtkcn" wug" cpf" eqpvtkdwvkqp" vq" gpjcpeg" nkxgnkjqqfu"
cannot be underestimated. Okra offers many production possibilities;
however, there are limited studies conducted on okra biology and production
due to limited resources devoted to the species by national and international
research programmes. With huge prospects for emerging agro-industry
worldwide, demand of the plant for specific use such as oil, mucilage
material, paper making material, bioabsorbent, pharmaceuticals and its other
untapped potentials for industrial use, makes it imperative for breeding

"

"

towards a specific usage since existing varieties are specific-bred for


consumption.
"

Fast maturing types are well suited to tropical heat, humidity and also to dry
(rain-fed) and hot (Sudano-Sahelian) conditions. Pods contain high amounts
of usable dietary fibre and they are often dried, stored and consumed as
soup/sauce much like a staple food. Half a cup of the cooked pods (fresh)
provides about 10% of the recommended levels of vitamin B6, folic acid and
vitamins A and C (Camciuc et al., 1996) and is sought after in food
processing industries.
"

The seed (usually consumed with pods) protein is distinct from both cereals
and legumes. Because it can easily be dried and stored for long periods
(unlike perishable vegetables), producers and processors are better able to add
value and take advantage of seasonal fluctuations in price. Besides pod yield,
the foliage and stems can weigh up to 27 tons per hectare of biomass, which
is likely to become useful with fuel prices increasing globally and new
technologies promising efficient conversion to liquid fuels. It is worth noting
that okra stems generate considerable heat without sparks, excessive smoke,
or bad odours. The potential for non-vegetable use include paper pulp, like its
close relative kenaf, oil seed, sacks and ropes, plasma replacer and
suspending agent in medicine (National Academies Press, 2006).
"

Okra as a multi-purpose crop is said to house hemicellulose, cellulose and


lignin in all parts with outstanding rheological properties, which are
employed for cosmetics, dyes and glues in France. Okra oil, a potential
source of palmitic acid is an important raw material for soaps, esters and
plasticisers and improves the quality of soybean oil, which has limitations as
a shortening as it only contains around 11% palmitic acid. In addition, the
linoleic acid, are separated and utilised for producing dyes, plastics and resins
"

"

and are in extremely high demand in industries (Camciuc et al., 1996).


Germplasm needs to be gathered up not only in Africa but also in Asia and
other regions that know the crop to open the way for improving the
compositional value of the crop for the various separate products and
functional utilities. Varieties bred, for instance, for fibre, biomass, oil,
protein, mucilage (type and yield), colour, and ornamental use, improved
yields, cultivation conditions, nutritional value, and nutraceuticals are among
facets of the vegetable unexplored (Camciuc et al., 1996; Martin, 1982).

2.12 CURRENT PRODUCTION CONSTRAINTS OF OKRA IN WEST


AFRICA
The West and Central African region accounts for more than 75% of okra
produced in Africa, but the average productivity in the region is extremely
low (2.5 t/ha) compared to East (6.2 t/ha) and North Africa (8.8 t/ha)
(FAOSTAT, 2006). Nigeria is the largest producer (1,039,000 t) followed by
Eqvg"fKxqktg."Ijcpc"cpf"qvjgtu"*HCQUVCV."422:+0"Kpugev"rguvu"kphguvcvkqp"
is one of the most limiting factors to increasing yield potential of okra in this
part of the world. The crop is prone to damage by various insects, fungi,
nematodes and viruses, although there is wide variability in their degree of
infestation. Some of the important insects are fruit and shoot borers (Earias
spp), aphids (Aphididae), white flies (Bemisia tabaci), and ants (Formicidae)
(Midmore et al., 2005). The crop is also susceptible to the attack of many
diseases affecting leaves, flowers and fruits. Viruses especially yellow vein
mosaic virus (YVMV) causes the most serious diseases of okra.
"

2.12.1 Lack of Improved Genotypes


Lack of improved or suitable genotypes, compounded by okra leaf curl
(particularly in Sub-Saharan Africa), nematode infestation (in West Africa),
"

"

and photoperiod sensitivity and insensitivity are among the most devastating
factors that militate against okra production in the West African sub-region.
Lack of improved genotypes has however been identified as the most
pressing challenge that requires prioritised attention as a first step towards
improving the crop in the sub-region (AVRDC, 2010).

There are few varieties across the sub-region, well-adapted to the respective
geographical climates but are generally low yielding, have long maturity
periods yet short harvesting periods and often with narrow genetic base
(Schippers, 2000). Most of these are also highly susceptible to pest and
disease attacks, highly sensitive to photoperiodic fluctuations, and have nonuniform pod shape and size, varied nutritional constituents, undesirable
features (spines) for handling and unwholesome for export. This problem can
be solved through thorough and comprehensive germplasm screening,
evaluation, purification and promotion of better and superior adapted lines
that meet market types (specific market demands such as mucilage, balanced
nutritional composition) and preferences, high yielding with shorter maturity
and longer harvesting durations and generally adaptable to the sub-regional
agro-climatic and ecological conditions.
"

2.12.2 Pest and Diseases


Okra is plagued by several species of insect pests and infected by a number of
diseases from seedling to harvesting. Economic losses depend on the degree
of damage, pest density, environmental conditions, stage of growth and the
plant part damaged by the pest (Valeriana, 2011; Splittstoessor, 1990).

Stem borers (Earias spp) damage shoots at early vegetative stage. The plants
then develop branches to compensate for the damage by stem borers. Unlike
"

"

other crops such as eggplant, development of more branches in okra is


disadvantageous. Pods from branches are less and smaller. Fruit worm, stem
and pod borer feed on flowers and bore inside the pods thereby damaging the
flowers and no pods are developed. Cutworm (Agrotis spp) usually feeds on
the leaves (Lamont, 1999; Martin, 1982). Damage by cutworm also greatly
affects the photosynthetic ability of the plant and drastically limits effective
partitioning of assimilates.

Damping off at seedling stage can cause tremendous losses unless most of the
seeds sown are treated with fungicides. Leafhoppers, aphids and whiteflies
attack at seedling to early vegetative stage and transmit the yellow vein
mosaic virus. Infected plants produce poor quality pods. Rapid increase in
leafhopper population during the dry season causes hopperburn in okra
especially when there is no source of yellow vein mosaic virus in
neighbouring fields. Leaves turn red and eventually dry up due to feeding by
high density of sucking insects at the vegetative stage predisposing them to
leaf curl and defoliation (De Lannoy, 2001).

Cercospora leaf spot and powdery mildew (Oidium spp) are two fungal
diseases from the late vegetative to the reproductive stage. The two are fungal
infections and spread rapidly on the field due to crowded and overlapping
broad leaves of plants. Besides wind, spreading fungal spores to plants,
people harvesting daily and passing along okra rows are also responsible for
the widespread infection in the field. Farmers remove and drop the old yellow
leaves with Cercospora leaf spot to reduce infection but usually these leaves
are not properly disposed off and the sources remain on the field (Norman,
1992).

"

"

2.12.3 Weed Competition


Weeds compete with okra for nutrients, space, sunshine and water as in many
other crops. They also serve as alternate host for pests and diseases in okra.
Weeds are difficult to manage because they have several means of
propagation such as by seed, rhizomes, tubers or stem. They produce many
small seeds that can be carried by the wind, animals and by farm tools.

The most devastating group of weeds to okra are stargrass (Cynodon


dactylon), etqyu" hqqv" itcuu" *Eleusine indica), the common okra sedge,
purple nut edge (Cyperus rotundus) (Norman, 1992). The broad leaf weeds of
okra are the giant pigweed (Trianthema portulacastrum); spiny amaranth
(Amaranthus spinosus); morning glory (Ipomoea triloba); Ageratum
conyzoides, Synedrella nodiflora and Cleome rutidosperma. Trianthema for
instance is known to harbour small larvae that feed on okra (Tindall, 1983).

However, herbicide application is considered the easiest and least expensive


method of weed control (Lamont, 1999). Application of broad-spectrum
herbicides is known to cause death of okra seedlings (from herbicide drift).
Phytotoxicity always happens in the okra field due to over reliance on
herbicides for weed control.

2.13 STATUS OF BIOTECHNOLOGICAL INTERVENTION IN


OKRA IMPROVEMENT
The last two decades or so have witnessed unprecedented technological
advances in biological sciences. The techniques have provided hitherto
unthinkable approaches for harnessing genes of interest from across
biological systems even from those separated by sexual incompatibilities, for

"

"

developing superior genotypes of different crops (Chadha and Choudhary,


2007).

Some of the tools and the techniques of biotechnology presently being


employed for improvement of horticultural crops such as okra include;
micropropagation of elite genotypes and rootstocks, molecular, primarily
DNA based diagnostics of plant pathogens and developing pathogen-free
planting material, ovule and embryo rescue for distant hybridisation,
development of cyto-plasmic male sterility system for hybrid seed production
especially in vegetable and flower crops.

In addition, molecular breeding based on marker assisted selection for both


qualitatively and quantitatively inherited traits especially for yield and abiotic
stress tolerance, search for novel alleles and genetic engineering for enhanced
shelf life in vegetables, fruits and flowers, improvement of nutritional quality
including efficient bio-fertilisers and tolerance to biotic and abiotic stresses
(Chadha and Choudhary, 2007).
Genetic improvement programmes however, have made little progress on
okra development due to the fact that okra continues to be regarded as a
marginal species. Certain sources of resistance have been identified
particularly to viral diseases, Yellow Vein Mosaic virus being major disease
of okra, attempts are being made to incorporate specific genes such as coat
protein (CP) gene and antisense RNA gene for elevated viral resistance
(Anonymous, 2010).

Shoot and fruit borers (Earias spp) are the most destructive pests in okra
crop. Efforts have been made to develop insect resistant okra varieties by
incorporating cry1Ac gene in okra from a bacterium mainly Bacillus
"

"

thuringiensis, commonly known as Bt okra. The Bt okra developed by M/s


Maharashtra Hybrid Seeds Company Limited containing cry1Ac gene (Event
OE-17A) was under safety evaluation and confined field trials a couple of
years ago (Anonymous, 2010).

A viable protocol has been developed for indirect shoot organogenesis in


okra, a leaf disc and hypocotyl explants were tested for different
eqodkpcvkqpu"qh" g-nathalene acetic acid (NAA), indole-3-acetic acid (IAA),
indole-3-butyric acid (IBA), thidizuron (TDZ) and 6-aminobenzylpurine
(BAP). Morphogenic callus induction was recorded at highest frequency from
hypocotyl explants by culturing in MS medium supplemented with 2.0 mg L1

NAA and 0.5 mg L-1 TDZ. Highest percentage shoots regeneration and

means number of callus regeneration per mass callus was obtained at 2.0 mg
L-1 BAP and 0.1 mg L-1 IBA, root regeneration was observed at 1.5 mg L-1
NAA. Eighty percent (80%) of the regenerated plantlets survived and showed
new leaves development under ex vitro condition (Anisuzzaman et al., 2008).
This protocol would be useful for creation of somaclonal variation and
enhancing utilisation of transgenic approaches for varietal improvement of
okra.

Gamma radiation was successfully applied to induce a mutation that


conferred yellow vein mosaic virus (YVMD) resistance in the okra variety
Qmwtc" kp" Vjckncpf0" Cnvjqwij" u{orvqou" ygtg" ugxgtg." vjg" oclqtkv{" qh" vjg"
mutant individuals yielded plump, greener pods and developed YVMD
symptoms later than the susceptible variety. Progenies of this mutant line
without YVMD have also been screened further for selection of superior
agronomic and quality related traits and also to obtain uniform YVMD
resistant okra lines for production in Kanchanaburi district of Thailand
through Tissue culture procedures (Phadvibulya et al., 2009).
"

"

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Aremu, C.O., Adebayo, M.A., Ariyo, O.J., Adewale, B.B. (2007).
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Woolfe, M. L., Chaplin, M. F. and Otchere, G. (1977). Studies on the


mucilages extracted from okra fruits (Hibiscus esculentus L.) and baobab
leaves (Adansonia digitata L.). Journal of the Science of Food and
Agriculture, 28(6): 519-529.

Yildiz, M. U., Sedat, C., Musa, O., Haydar, H. (2005). A study on some
Physico-chemical properties of Turkey okra (Hibiscus esculentus L.) seeds.
Journal of Food Engineering, 68: 7378.

Zhenzhong, X. and Hongjun, S. (2010). Effects of okra capsule combined


with

valsartan

in

treatment

of

early

diabetic

nephropathy

with

microalbuminuria. Modern Journal of Integrated Traditional Chinese and


Western Medicine, pp. 3.

"

"

CHAPTER THREE

MORPHOLOGICAL CHARACTERISATION OF 29 ACCESSIONS


OF OKRA (Abelmoschus spp L.).

3.1 INTRODUCTION"
The primary objectives of okra germplasm characterisation have generally
been to identify high yielding genotypes with resistance to yellow vein mosaic
virus (YVMV), fruit borer (Spodoptera spp.), jassid (Cicadellidae) and higher
vitamin C content in the species that can be utilised for the improvement of
the crop (Nwangburuka et al., 2011; Bisht and Bhat, 2006). Morphological
markers are the traditionally accepted and proven tools as a first step, among a
host of other techniques, for characterisation of plant germplasm. They
constitute the most readily available technique. Thus, published descriptor
lists are readily available for most major crop species including okra. The
technique can be carried out in situ (on-farm), it is relatively inexpensive, easy
to carry out with little skills and minimum facility requirements for use
(Hoogendijk and Williams, 2001).

Conversely, characterisation based on phenotypic traits is not easily


reproducible particularly, since they are influenced by environmental
variations (Staub et al., 1996). In addition, it requires a large tract of land
and/or greenhouse space in which to grow large populations of plants, it is
labour intensive and difficult to manage (Ahiakpa, 2009; Vogel et al., 1996).
However, they have remained useful as a highly recommended first step to be
undertaken prior to more in-depth biochemical or molecular studies in okra
germplasm (Oppong-Sekyere, 2011; Smith and Smith, 1992).
"

Descriptors are tools used to describe or provide information on accessions


(Charrier et al., 1997). They provide detailed and standardised evaluation data
for accessions of crops. Numerous characters recorded on individual
accessions can serve as diagnostic descriptors for accessions (Rubenstein and
Heisey, 2003). Such diagnostic characters help genebank curators keep track
of an accession and check for genetic integrity over a number of years of
conservation (Rubenstein and Heisey, 2003). Descriptor lists ensure
uniformity of standards and use of a common language in documenting
characteristics of conserved species (De Vicente et al., 2005; Rubenstein and
Heisey, 2003). Potential value of germplasm is hugely dependent on the
efficiency of techniques designed to differentiate among accessions and
detailed study of individual traits (De Vicente et al., 2005; Engels and Visser,
2003; Rubenstein and Heisey, 2003).

A typical content of a crop descriptor is a trait name, an identifying code or


number, an interpretation (definition), a descriptor state or values or a range of
values. Descriptors could be a permanent part of the system (cannot be
changed) or are created and modified (Hamon and Nairot, 1991; Charrier,
1984). A few modifications were done on the descriptor for this particular
work to suit conditions on the field and enhance data collection within the
resources available.

3.1.1 Objectives of the Study


The objective of the study was to characterise the 29 accessions of okra
(Abelmoschus spp L.) assembled from the eight geographical regions of
Ghana using morphological markers, so as to remove any duplicates among
the accessions and to identify superior ones with desirable characteristics for
further breeding work.
"

3.2 MATERIALS AND METHODS


3.2.1 Experimental site
The experiment was conducted at the Nuclear Agriculture Centre (NAC) of
the Biotechnology and Nuclear Agriculture Research Institute (BNARI),
Ghana Atomic Energy Commission (GAEC). The Research Field of BNARI
is located at latitude 0540 "N and longitude 0 13 W at an elevation of 76m
above sea level within the coastal savannah agro-ecological zone. The soil at
the site is the Nyigbenya-Haatso series, which is a typically well-drained
savannah

Ochrosol

(Ferric

Acrisol)

derived

from quartzite

Schist

(FAO/UNESCO, 1994).

3.2.2 Weather Conditions


The maximum and minimum average temperature for the period (July, 2011January, 2012) of the experiment was 31.12oC and 23.20oC, respectively. The
mean annual rainfall was 220mm. Average relative humidity, sunshine and
wind speed for the same period were 42.30, 194.35 w/m2 and 752.92 m/s,
respectively (Local Weather Station Data, 2012).

3.2.3 Germplasm Assembly


Thirty (30) accessions were assembled from eight different regions of the
country. These are captured in Table 3.1.

"

Table 3.1 Okra accessions used in the study


Location

Accession

Greater Accra region

Labadi, Asontem-GAR, Atomic, Cs-Legon, Legon


fingers, Clemson spineless, Indiana, Volta

Ashanti region

Asontem-ASR, Agric type I, Debo, Asante type II,


Kortebortor-ASR, Agric short fruit

Central region

Cape

Eastern region

Amanfrom, Asontem-ER, DKA, Akwaduso*

Brong-Ahafo region

Yeji-Local, Asontem nv, Asontem-BAR,


Kortebortor-BAR, Nkran nkuruma

Western region

Juaboso

Upper East region

Mamolega, Wune mana, Mapelega

Volta region

Akrave, Kpeve

*Accession failed to germinate.

3.2.4 Land Preparation


A total land area of 60m x 33m was acquired and cleared; all stumps were
removed and ploughed to a fine tilth for planting.

3.2.5 Experimental Design


An experimental plot size of 58m x 31.8m was pegged and marked out, within
the prepared land leaving the rest of it as a periphery. Approximately 2.0m
was created around the plot to serve as a control for pests and to ease
movement around the field. The Randomised Complete Block Design was
used with four replications, each replicate measured 30m x 12.5m, and
separated by a distance of 2m from the other with 30 subplots within a block.
Each subplot had a dimension of 3.5m x2.5m and spaced from one another by
1m.

All subplots within the various blocks were randomly assigned by

drawing lots to avoid bias and each plot was well labelled.
"

3.2.6 Field Management Practices


Thirty (30) okra accessions from eight regions of Ghana were planted on a
32m x 59m size of land. Twenty nine (29) out of the thirty (30) accessions
were however available for the experiment as seeds of one accession were not
viable and failed to germinate.
After germination, an area of 3m x 2m within each subplot was marked out as
a point of reference for data collection and all plants that fell outside this
range were treated as border plants. Data were taken on eight plants within
each subplot.

No fertiliser was applied, but weeds were controlled and other agronomic and
management practices were carried out. Weeding was done fortnightly. The
rainfall pattern during this period was quite consistent and regular hence there
was no need for supplementary water to support plant growth and
development.

3.2.7 Seed Sowing


Seeds of the various accessions were sown on 19th July, 2011 after a heavy
downpour of rainfall that facilitated uptake of water by the seeds and boosted
germination. The seeds were sown at a depth of 2cm, at a spacing of 0.70m x
0.50m within and between rows with three to four seeds per hole and thinned
to two per hill after germination.

3.2.8 Data Collection


Data were collected using the International Plant Genetic Resources Institute
(IPGRI, 1991) Descriptor List for okra. Data were taken on 35 characters,

"

which include the following parameters and were grouped into four growth
stages of the plant;

(a) Vegetative characters: general aspect of the plant, branching type (BRT),
stem pubescence, stem colour, leaf shape, leaf colour and total number of
leaves per plant (NLPP). Data was taken on these characters prior to first
fruiting of all accessions ensuring that all accessions within each block
receive uniform treatment. Five plants were randomly selected for quantitative
traits such as number of leaves and this was done after each flowering. Data
on qualitative traits were taken based on confirmation from each block.
(b) Inflorescence characters: number of epicalyx segments (NES), shape of
epicalyx segments (SES), persistence of epicalyx segments (PES), petal
colour, and colouration of petal base. These characters were recorded during
the flowering stage of each accession in all blocks. This was done on five
plants out of the eight plants.
(c) Reproductive characters: days to 50% germination (FPGer), maximum
plant height (cm) (MPH), stem diameter at the base (mm) (STB), maximum
number of internodes (MNI), days to 50% flowering (FPFl), first flowering
node (FFN), first fruit-producing node (FFPN), total production of pods, and
number of pods per plant (NPPP). A tape measure was employed in taking
plant height at the fruiting stage for five data plants. Stem diameter at the base
was taken at maturity level of the accessions using a pair Vernier Callipers.
(d) Fruit characters: position of fruit on main stem (PFMS), fruit colour, fruit
length at maturity, length of peduncle, fruit shape, number ridges per fruit
(NRPP), fruit pubescence and number of days to 50% fruiting (FPFr).
Position of fruit on main stem was determined on five data plants prior to
harvesting of fruits. Measuring rule was used for measuring length of pods
"

and peduncle of pods after harvest. Number of ridges was done by counting
the quantity of ridgeline or natural striations through the fruit and then coded
accordingly. This was done on five pods of each accession. Data on fruit
pubescence was taken by a visual assessment of hairiness or smoothness of
rqfu"cpf"c"rtcevkecn"hggn"qp"jctxguvgf"htguj"htwkvu0
(e) Seed characters: shape of seed, aspect of the surface, average number of
seeds per pod (ASPD), weight of 1000 seeds (g), (TSwt). All seed characters
were measured after harvest of the accessions. Harvested pods were further
dried and seeds extracted and then dried separately to reduce moisture content
to an appreciable level. Seeds per pod were counted for each accession; one
thousand (1000) seeds per accession were weighed using a digital scale.

3.2.9 Data Analyses


Collected data were analysed using Analysis of Variance (ANOVA) and
Duncan Multiple Range Test (DMRT) for mean separation. Data were also
analysed using regression and correlation analysis to determine whether one
trait has variable effect on the other and the degree of association among the
accessions.

Furthermore, the principal component analysis was employed to assess the


percentage contribution of each trait to total genetic variability among the
accessions. Cluster analysis based on similarity matrices was also employed
to obtain a dendrogram. Genstat Statistical Software Programme (Payne et al.,
2007), Microsoft Excel Software, and Statgraphics Plus XV.I Software (2010)
were used for all the data analyses.

"

3.3 RESULTS
3.3.1 Morphological Traits of dry pods of Accessions
The photographs below show phenotypic characteristics of fruits of
the various accessions at maturity.

DKA

Labadi

Yeji-Local

Asontem nv.

Cs-Legon

Mapelega

Plate.2: Photographs of dried fruits of okra accessions

"

"

Cape

Volta

Juaboso

Debo

Akrave

Kpeve

Asontem-ER

Legon fingers

Figure.2: contf

"

"

Asontem-BAR

Asante type II

Amanfrom

Agric short fruit

Asontem-ASR

Atomic

Agric type I

Kortebortor-BAR

Asontem-GAR

Plate.2: eqpvd

"

"

3.3.2: Variations in morphological characteristics of some of the


accessions in the field."

"
Plate 1: Variation in Vegetative and Reproductive Characteristics of some Accessions of Okra.

"

"

Rncvg"3"*Eqpvf+<

"

"

3.3.3 Variability in Quantitative Traits among Accessions


Table 3.3 shows phenotypic variability of thirteen quantitative traits among
the accessions of Abelmoschus spp. The accessions exhibited significant
variation with respect to the thirteen quantitative characters. Taking plant
height for instance, Nkran Nkuruma recorded the highest of 162.93cm whilst
Clemson Spineless recorded the least of 41.43cm. The accessions, Akrave
and Atomic however were not significantly different from Clemson
Spineless. Nkran Nkuruma was not significantly different from AsontemBAR, Asante type II, and Asontem-GAR.

In addition, Kortebortor-BAR had the thickest stem diameter, but this was not
significantly different from Amanfrom. Asontem-ASR had the thinnest stem
diameter but was not significantly different from Cape, Debo and Clemson
Spineless.

"

Table 3.2: Variability in Quantitative Traits among 29 accessions of Okra.

DESIGNATION

MPH

MNI

FFN

NLPP

ASPD

STB

NPPP

FPGer

FFPN

FPFl

FPFr

TSwt

APP

Nkran Nkuruma

170.78a

19.00a

23.00a

20.00de

18.25r

9.98c

23.50ab

53.50ghi

10.75bc

50.00e

6.00j

69.43b

40.50e

128.53

18.00

14.50

jk

9.85

17.50

de

51.25

mn

ab

47.00

8.00

53.92

11.00o

116.33

bc

18.00

16.00

hij

8.15

gh

16.25

de

53.00

ijk

47.00

9.50

50.81

21.50lm

113.40

cd

18.00

16.00

12.50

lm

9.00

14.25

ef

52.00

klm

39.00

8.00

64.41

22.00l

103.95

cde

16.78

34.00

9.55

de

18.25

cd

78.00

39.00

62.14

37.25f

102.53

def

15.02

13.75

kl

5.93

op

18.25

cd

53.75

gh

38.50

59.03

52.50b

18.50

13.75

kl

25.25

92.00

82.00

53.83

57.50a

46.71r

44.50c

Asontem-BAR
Asante type II
Asontem-GAR
Yeji-Local
Cape
Kortebortor-

99.28

ef

13.00
9.00
8.00

l
e

11.75

19.00

21.25

pq

22.75

op

41.25

63.00

43.00

28.5

lmn

11.03

11.75
9.25
8.0

fghij

8.25
9.5

cdefg

fghij

cdefg

9.75

bcdef

10.00

11.75

cd

8.00

BAR
Juaboso

94.75efg

17.50d

18.00c

26.75c

46.25c

8.10hi

14.50ef

53.30hij

8.00fghij

47.00g

10.25ef

Labadi

92.55efgh

18.00c

11.00i

18.50ef

37.00f

8.78f

90.08fghi

13.67j

10.25j

17.00fgh

37.00f

6.38 lm

7.50hi

51.00mn

7.75ghij

40.00n

13.00b

46.71r

28.25h

17.00de

51.00mn

7.75ghij

44.00k

12.00c

52.45l

26.00ij

LoS

**

**

**

**

**

**

**

**

**

**

**

**

**

BE

ns

ns

ns

ns

ns

**

ns

ns

**

ns

ns

ns

ns

TCV (%)

11.9

1.1

1.5

6 .1

3.6

2.6

18.0

2.1

16.7

1.2

1.3

0.5

3.8

BCV (%)

1.3

0.3

0.3

1.7

0.5

1.0

2.9

0.2

6.3

0.42

0.3

0.1

1.0

ns kpfkecvgu"pqp"ukipkhkecpeg"cv"vjg"r2027"ngxgn.","kpfkecvgu"ukipkhkecpeg"cv"vjg"r2027"ngxgn"cpf",,"kpfkecvgu"jkij"ukipkhkecpeg"cv"r2023"ngxgn0"NqU"?"ngxgn"qh"
significance, BE = block efficiency, TCV = treatment co-efficient of variation, BCV = block co-efficient of variation and Mean represent average of the
individual characters measured for all accessions under consideration. NV = Northern Version, MPH = Maximum plant height (cm), MNI = Maximum number
of internodes, FFN = First flowering node, NLPP = Number of leaves per plant, ASPD = Average seeds per pod, STB = Stem diameter at base (mm), NPPP =
Number of pods per plant, FPGer = 50% Germination, FFPN = First fruit producing node, FPFl = 50%Flowering, FPFr = 50%Fruiting, TSwt = 1000 seed
weight (g), APP = Average pods per plant.

"

Tcdng"504"*Eqpvf+<"
DESIGNATION

MPH

MNI

FFN

NLPP

ASPD

STB

NPPP

FPGer

FFPN

FPFl

FPFr

TSwt

Asontem-ASR

89.75fghi

15.87g

9.00l

13.75kl

33.00h

5.25q

15.5de

55.00f

8.00fghij

42.00l

8.00i

41.32w

15.75n

Asontem NV.

83.70ghij

17.00e

17.00d

21.00d

46.00c

7.55k

23.25ab

51.25mn

7.0 0ijk

41.00m

12.00c

63.54e

58.25a

Amanfrom

82.85ghij

17.87c

13.00g

26.00c

51.00b

10.53b

21.50bc

89.00c

13.50a

71.50d

10.50e

56.02i

30.50g

17.00

11.00

15.75

ij

31.25

jk

47.00

12.00

33.92

51.50b

18.93

7.00

18.75

ef

31.75

hi

47.00

11.50

53.24

28.00h

18.00

9.00

27.75

mn

47.00

12.00

58.98

42.25d

17.00

10.00

29.00

lm

32.00

10.00

51.87

21.50lm

16.00

11.00

37.50

45.00

10.00

74.95

25.00jk

16.46

7.00

20.00

45.00

10.00

67.37

12.00o

14.37

7.00

37.00

40.00

10.00

56.33

hi

21.50lm

Volta
Debo
Agric type I
Indiana
Legon fingers
Mamolega
Cs- Legon

79.5

hijk

77.7

ijkll

76.05
71.0

jk

jklmn

72.63

jklm

62.13

nop

64.75

lmno

11.0

mn

12.50

lm

29.00

12.75

20.75

6.55

5.93

op

8.10

hi

7.85

ij

6.35

lmn

6.15

mno

8.40

17.50

de

15.25

de

10.00

ghi

16.25

de

17.75

de

8.25

ghi

17.00

de

53.00

ijk

10.00

51.25

mn

jk

54.25

fg

39.25

51.00

mn

49.00

51.5

lmn

6.50

10.25

bcde

bcd

8.80

defghi

8.80

defghi

8.80

defghi

7.00

ijk

APP

LoS

**

**

**

**

**

**

**

**

**

**

**

**

**

BE

ns

ns

ns

ns

ns

**

ns

ns

**

ns

ns

ns

ns

TCV (%)

11.9

1.1

1.5

6 .1

3.6

2.6

18.0

2.1

16.7

1.2

1.3

0.5

3.8

BCV (%)

1.3

0.3

0.3

1.7

0.5

1.0

2.9

0.2

6.3

0.42

0.3

0.1

1.0

ns kpfkecvgu" pqp" ukipkhkecpeg" cv" vjg" r2027" ngxgn." ," kpfkecvgu" ukipkhkecpeg" cv" vjg" r2027" ngxgn" cpf" ,," kpfkecvgu" jkij" ukipkhkecpeg" cv" r2023" ngxgn0" NqU" ?" ngxgn" qh"
significance, BE = block efficiency, TCV = treatment co-efficient of variation, BCV = block co-efficient of variation and Mean represent average of the individual
characters measured for all accessions under consideration. NV = Northern Version, MPH = Maximum plant height (cm), MNI = Maximum number of internodes, FFN
= First flowering node, NLPP = Number of leaves per plant, ASPD = Average seeds per pod, STB = Stem diameter at base (mm), NPPP = Number of pods per plant,
FPGer = 50% Germination, FFPN = First fruit producing node, FPFl = 50%Flowering, FPFr = 50%Fruiting, TSwt = 1000 seed weight (g), APP = Average pods per plant.

"

Vcdng"504"*Eqpvf+<"
DESIGNATION

MPH

MNI

FFN

NLPP

ASPD

STB

NPPP

FPGer

FFPN

FPFl

FPFr

TSwt

APP

Mapelega

62.40nop

13.16k

10.25j

18.00fg

38.00f

6.08no

6.50i

47.25p

8.00fghij

32.00r

10.00f

56.41h

20.25m

Wune mana

62.13nop

13.00k

6.00o

16.75ghi

12.25s

6.30lmn

6.50i

59.00e

4.25l

49.00f

10.00f

50.23o

7.00p

15.00

43.73

52.50b

12.00

48.12

27.00hi

44.86

15.00n

52.75

22.50l

45.50s

24.25k

DKA
Agric short fruit
Kpeve
Kortebortor-

60.70

nopq

59.00

opqr

56.68

pqrs

48.50

qrs

2.00

17.00

12.32

11.23

17.00

6.00

8.00

7.00

21.50

13.75

kl

17.25

fghi

16.50

ghi

31.50

ijk

23.75

35.00

30.00

kl

8.90

7.80

jk

7.55

9.80

cd

10.25
8.00

ghi

11.25
6.70

gh

fg

hi

125.00

52.50

jkl

55.00

50.75

7.50

hijk

7.00

ijk

9.00

defghi

8.00

fghij

115.00

46.50

36.75

12.00

42.00

12.00

49.00f

9.00h

ASR
Atomic
Akrave
Clemson

48.40qrs
47.05

rs

44.38

16.00g
19.00

15.00

15.00f
9.00

8.00

16.00hij
27.00

10.25

27.25n
31.75

ij

18.25

6.25mn
9.30

5.75

16.50de
15.00

de

14.75

def

53.75gh
89.75

42.75

5.50kl
7.50

hijk

8.50

efghij

80.00

37.00

10.00
8.00

48.63

21.75lm

43.24

16.50n

spineless
LoS

**

**

**

**

**

**

**

**

**

**

**

**

**

BE

ns

ns

ns

ns

ns

**

ns

ns

**

ns

ns

ns

ns

TCV (%)

11.9

1.1

1.5

6 .1

3.6

2.6

18.0

2.1

16.7

1.2

1.3

0.5

3.8

BCV (%)

1.3

0.3

0.3

1.7

0.5

1.0

2.9

0.2

6.3

0.42

0.3

0.1

1.0

ns kpfkecvgu" pqp" ukipkhkecpeg" cv" vjg" r2027" ngxgn." ," kpfkecvgu" ukipkhkecpeg" cv" vjg" r2027" ngxgn" cpf" ,," kpfkecvgu" jkij" ukipkhkecpeg" cv" r2023" ngxgn0" NqU" ?" ngxgn" qh"
significance, BE = block efficiency, TCV = treatment co-efficient of variation, BCV = block co-efficient of variation and Mean represent average of the individual
characters measured for all accessions under consideration. NV = Northern Version, MPH = Maximum plant height (cm), MNI = Maximum number of internodes, FFN
= First flowering node, NLPP = Number of leaves per plant, ASPD = Average seeds per pod, STB = Stem diameter at base (mm), NPPP = Number of pods per plant,
FPGer = 50% Germination, FFPN = First fruit producing node, FPFl = 50%Flowering, FPFr = 50%Fruiting, TSwt = 1000 seed weight (g), APP = Average pods per plant.

"

3.3.4 Variability in Qualitative Traits of Accession


Table 3.3: Morphological Characterisation of Qualitative Traits of 29 Accessions of Okra.
TRAITS AND DESCRIPTION
Accession

PES

SES

NES

RCPB

Cs-Legon

Persistent

Lanceolate

KortebortorASR
Mapelega

Persistent

Lanceolate

Persistent

Triangular

Kpeve

Linear

Yeji-Local

Nonpersistent
Persistent

Amanfrom

Persistent

Lanceolate

Cape

Persistent

Triangular

AsontemASR
Mamolega

Persistent

Triangular

Persistent

Lanceolate

Atomic

Nonpersistent

Lanceolate

More
than 10
More
than 10
More
than 10
From 5
to 7
From 5
to 7
More
than 10
More
than 10
More
than 10
From 5
to 7
From 8
to 10

Inside
only
Inside
only
Inside
only
Inside
only
Inside
only
Both
sides
Inside
only
Inside
only
Both
sides
Inside
only

Triangular

Petal
colour
Cream

Fruit
pubescence
Downy

Cream

Prickly

Cream

Cream

Slightly
rough
Slightly
rough
Prickly

Yellow

Downy

Cream

Cream

Slightly
rough
Slightly
rough
Prickly

Cream

Downy

Cream

Cream

Fruit
colour
Yellowish
green
Green with
red patches
Yellowish
green
Green

PFMS
Erect

Stem
pubescence
Slight

Erect

Slight

Horizontal

Conspicuous

None
(smooth)
None
(smooth)
5 to 10

Pendulous

Slight

5 to 10

Pendulous

Slight

Pendulous

Slight

None
(smooth)
None
(smooth)
5 to 10
None
(smooth)
8 to 10

None
(smooth)

Green with
red patches
Yellowish
green
Green with
red patches
Green

Erect

Conspicuous

Erect

Slight

Red

Pendulous

Conspicuous

Green

Erect

Slight

NRPPD

Fruit
shape
1
7
8

1
7

NES= Number of Epicalyx Segment; RCPB= Red Colouration of Petal Base; PES=Persistence of Epicalyx Segment; SES=Shape of Epicalyx Segment; PFMS=Position
of Fruit on Main Stem; NRPPD=Number of Ridges per Pod.

"

Vcdng"505"*Eqpvf+"
TRAITS AND DESCRIPTION
Accession

PES

SES

NES

RCPB

Asontem-ER

Persistent

Triangular

Juaboso

Persistent

Lanceolate

Debo

Nonpersistent
Persistent

Triangular

Agric short
fruit
Asontem nv

Persistent

Triangular

Inside
only
Inside
only
Inside
only
Inside
only
Both sides

Persistent

Lanceolate

Labadi

Persistent

Lanceolate

Indiana

Persistent

Lanceolate

Asante type II

Persistent

Lanceolate

Legon fingers

Persistent

Lanceolate

More than
10
More
than10
From 7 to
10
More than
10
More than
10
From 7 to
10
More than
10
More than
10
From 8 to
10
More than
10

Volta

Triangular

Petal
colour
Cream
Cream
Cream
Cream
Cream

Both sides

Cream

Inside
only
Both sides

Cream

Inside
only
Inside
only

Cream

Cream

Cream

Fruit
pubescence
Slightly
rough
Slightly
rough
Slightly
rough
Prickly
Slightly
rough
Slightly
rough
Prickly
Slightly
rough
Slightly
rough
Downy

Fruit
colour
Yellowish
green
Green

PFMS

NRPPD

Horizontal

Stem
pubescence
Slight

5 to 10

Fruit
shape
2

Erect

Slight

5 to 10

Green

Pendulous

Slight

5 to 10

10

Green with
red patches
Green

Pendulous

Slight

8 to 10

Pendulous

Conspicuous

5 to 10

12

Green

Erect

Slight

5 to 10

14

Yellowish
green
Yellowish
green
Green

Horizontal

Slight

8 to 10

Horizontal

Glabrous

8 to 10

Erect

Glabrous

5 to 10

Erect

Conspicuous

None
(smooth)

Yellowish
green

NES= Number of Epicalyx Segment; RCPB= Red Colouration of Petal Base; PES=Persistence of Epicalyx Segment; SES=Shape of Epicalyx Segment; PFMS=Position
of Fruit on Main Stem; NRPPD=Number of Ridges per Pod.

"

Vcdng"505"*Eqpvf+
TRAITS AND DESCRIPTION
Accession

PES

Clemson

Non-

Spineless

persistent

Wune mana

Persistent

Agric type I

Kortebortor-

Persistent
Persistent

SES

From 7

Both

to 10

sides

Linear

More

Both

than 10

sides

From 8

Inside

to 10

only

Lanceolate
Lanceolate

Persistent

Triangular

Persistent

Lanceolate

nkuruma
AsontemBAR
DKA

Asontem-

Persistent
Persistent

Lanceolate
Lanceolate

GAR
Akrave

Persistent

RCPB

Linear

BAR
Nkran

NES

Linear

More

Both

than 10

sides

From 8

Inside

to 10

only

More

Inside

than 10

only

More

Both

than 10

sides

More

Inside

than 10

only

From 7

Inside

to 10

only

Petal

Fruit

Fruit

colour

pubescence

colour

PFMS

Stem

NRPPD

Cream

Slightly

Green

Erect

Slight

8 to 10

Green

Erect

Conspicuous

5 to 7

Erect

Conspicuous

5 to 7

Pendulous

Conspicuous

5 to 7

Horizontal

Slight

5 to 7

Erect

Slight

None

pubescence

Fruit
shape

rough
Cream

Slightly
rough

Cream
Cream
Cream
Cream
Cream

Slightly

Yellowish

rough

green

Slightly

Yellowish

rough

green

Slightly

Yellowish

rough

green

Slightly

Yellowish

rough

green

Downy

Green with

(smooth)
Horizontal

Glabrous

8 to 10

Green

Erect

Conspicuous

5 to 7

11

Green with

Pendulous

Glabrous

5 to 10

11

red patches
Cream

Slightly
rough

Cream

Downy

red patches

NES= Number of Epicalyx Segment; RCPB= Red Colouration of Petal Base; PES=Persistence of Epicalyx Segment; SES=Shape of Epicalyx Segment; PFMS=Position
of Fruit on Main Stem; NRPPD=Number of Ridges per Pod.

"

Vcdng"505"*Eqpvf+"
TRAITS AND DESCRIPTION
Accession

Asontem-ER

Juaboso

Debo

PES
Persistent
Persistent
Non-

SES
Triangular
Lanceolate
Triangular

persistent
Volta

Agric short

Persistent
Persistent

Triangular
Triangular

RCPB

More than

Inside

10

only

More

Inside

than10

only

From 7 to

Inside

10

only

More than

Inside

10

only

More than

Both sides

Petal

Fruit

Fruit

colour

pubescence

colour

Cream

Slightly

Yellowish

rough

green

Slightly

Cream

Persistent

Lanceolate

From 7 to

Cream

Indiana

Persistent
Persistent

Lanceolate
Lanceolate

Cream

Persistent

Lanceolate

II
Legon
fingers

Persistent

Lanceolate

NRPPD

pubescence

Fruit
shape

Horizontal

Slight

5 to 10

Green

Erect

Slight

5 to 10

Slightly

Green

Pendulous

Slight

5 to 10

10

Prickly

Green with

Pendulous

Slight

8 to 10

Green

Pendulous

Conspicuous

5 to 10

12

Green

Erect

Slight

5 to 10

14

Yellowish

Horizontal

Slight

8 to 10

Horizontal

Glabrous

8 to 10

red patches
Cream

Slightly
rough

Both sides

Cream

Slightly
rough

More than

Inside

10

only

More than

Both sides

Cream

From 8 to

Inside

10

only

More than

Inside

10

only

Prickly

green
Cream

10
Asante type

Stem

rough

10
Labadi

PFMS

rough

10

fruit
Asontem nv

NES

Cream

Slightly

Yellowish

rough

green

Slightly

Green

Erect

Glabrous

5 to 10

Yellowish

Erect

Conspicuous

None

rough
Cream

Downy

green

Pms 1-9=plant maturity on a scale of 1-9; nv= northern version.

"

(smooth)

Vcdng"505"*Eqpvf+"
TRAITS AND DESCRIPTION
Fruit
peduncle

Leaf
colour

Round

Aspect of
seed
surface
Downy

1 to 3cm

Mixed

Reniform

Downy

1 to 3cm

Green with
red veins
Green

Early

Erect

Reniform

Downy

1 to 3cm

Green

Late

Mixed

Round

Glabrous

1 to 3cm

Green

Early

Erect

Round

Glabrous

Green

Strong

Late

Medium

Round

Downy

More
than 3cm
1 to 3cm

Mixed

Stocky

Medium

Midseason

Erect

Round

Glabrous

1 to 3cm

Green

10

Curved

Medium

Early

Procumbent

Reniform

Glabrous

Green

Medium

11

Mixed axes

Midseason

Procumbent

Reniform

Downy

Mixed

Straight

Orthotropic
stem only
Medium

More
than 3cm
1 to 3cm

Midseason

Medium

Round

Glabrous

Accession

Pod
length

Pod
diameter

Leaf
shape

Pod axis
type

Branching
type

PMS 1-9

General
plant type

Seed
shape

Asontem nv.

Very
short
Very
long
Very
long

Mixed

Mixed axes

Medium

Midseason

Medium

Big

Mixed axes

Medium

Late

Medium

10

Straight

Orthotropic
stem only

Asante type II

Medium

Mixed

10

Stocky

Legon fingers

Short

Medium

Stocky

Orthotropic
stem only
Medium

Akrave

Short

Big

Curved

Atomic

Short

Small

Clemson
spineless
Wune mana

Long

Medium

Medium

Agric type I

Medium

Labadi
Indiana

Pms 1-9=plant maturity on a scale of 1-9; nv= northern version

"

More
than 3cm

Green with
red veins
Green with
red veins

Vcdng"505"*Eqpvf+"
TRAITS AND DESCRIPTION
Accession

Pod length

Pod

Leaf

Pod axis

Branching

diameter

shape

type

type

PMS 1-9

General

Seed shape

plant type

Aspect of

Fruit

seed surface

peduncle

Leaf colour

Asontem-BAR

Medium

Mixed

Stocky

Mixed

Midseason

Erect

Round

Downy

1 to 3cm

Green with

DKA

Medium

Mixed

Mixed

Strong

Very late

Mixed

Reniform

Downy

More than

Green with

3cm

red veins

Asontem-GAR

Medium

Big

Stocky

Mixed

Late

Erect

Reniform

Glabrous

More than

Mixed

red veins
axes

3cm
Kortebortor-

Medium

Medium

Straight

Strong

Very late

Erect

Reniform

Downy

More than

Green

3cm

BAR
Nkran nkuruma

Mixed

Big

Mixed

Medium

Very late

Mixed

Reniform

Glabrous

Debo

Medium

Medium

Stocky

Strong

Late

Medium

Reniform

Downy

1 to 3cm

Volta

Long

Medium

Green

Curved

Medium

Early

Erect

Round

Glabrous

1 to 3cm

Green

Agric short fruit

Medium

Medium

Straight

Medium

Early

Medium

Reniform

Glabrous

1 to 3cm

Green

Juaboso

Medium

Medium

Mixed

Medium

Midseason

Erect

Reniform

Glabrous

1 to 3cm

Green

axes

axes

Pms 1-9=plant maturity on a scale of 1-9; nv= northern version

"

More than

Green with

3cm

red veins

"

"

3.3.6 Correlation Coefficients of 13 Quantitative Traits.


Table 3.4 depicts the associations among thirteen quantitative traits of the
various okra accessions. The trait fifty percent (50%) germination was not
statistically significant to all the other quantitative traits. However, it was
negatively correlated with eleven traits namely 50% flowering, 50% fruiting,
maximum plant height, maximum number of internode, first-flowering node,
number of leaves per plant, 1000-seed weight, average number of seeds per
pod, stem diameter at the base, number of pods per plant and first fruitproducing node in descending order of their strength and significance. It was
only positively correlated with average number of pods per plant, which is a
yield-determining character.

Fifty percent (50%) flowering was positively correlated with traits such as
50% fruiting, maximum number of internode, first-flowering node, number
of leaves per plant, stem diameter at the base, number of pods per plant but
negatively correlated with all the other traits. The association of 50%
flowering with all other traits whether negative or positive were not
statistically significant except with 50% fruiting.

On the other hand, 50% fruiting was strongly correlated with number of
leaves per plant and stem diameter at the base as well as positively correlated
with other traits such as first-flowering node, average number of seeds per
pod, number of pods per plant and first fruit-producing node. Nevertheless, it
was negatively correlated with characters such as the maximum plant height,
maximum number of internodes, 1000-seed weight and average number of
pods per plant. Maximum plant height was correlated positively with all
other traits except 50% germination, 50% fruiting and 50% flowering.
"

"

"

However, it was significantly correlated with only first flowering nodes (r =


0.62).

Interestingly, maximum number of internode, first flowering node, number of


leaves per plant, number of pods per plant, 1000-seed weight, average
number of seeds per pod, stem diameter at the base, average number of pods
per plant, number of pods per plant and first fruit-producing node were all
positively correlated with one another except the association between 1000seed weight and first fruit-producing node. However, statistically significant
association was recorded between first flowering node and average number
of pods per plant (r = 0.71) as well as number of pods per plants (r = 0.68).
Statistically significant relationship was also recorded for average number of
seeds per pods and number of leaves per pod (r = 0.57), average number of
pods per plant and average number of seeds per pods (r = 0.60) and finally
between average number of pods per plant and number of pods per plant (r =
0.64).

"

Table 3.4: Correlations among 13 Quantitative Traits of Abelmoschus spp L.


Trait

FPGer

FPFl

FPFr

MPH

MNI

FFN

NLPP

TSwt

ASPPD

STB

APP

NPP

FPGer
FPFl

-0.24

FPFr

-0.19

0.95**

MPH

-0.03

-0.05

-0.03

MNI

-0.08

0.05

-0.03

0.42

FFN

-0.13

0.05

0.01

0.62*

0.53*

NLPP

-0.22

0.43

0.57*

0.12

0.12

0.15

TSwt

-0.07

-0.17

-0.13

0.39

0.18

0.30

0.29

ASPPD

-0.03

-0.13

0.08

0.34

0.14

0.36

0.57*

0.30

STB

-0.03

0.44

0.50*

0.36

0.33

0.37

0.46

0.16

0.24

APP

0.11

-0.10

-0.05

0.45

0.42

0.71**

0.26

0.21

0.60*

NPPP

-0.28

0.14

0.14

0.44

0.47

0.68**

0.41

0.24

0.46

0.29

0.64*

FFPN

-0.02

-0.05

0.01

0.14

0.27

0.20

0.00

-0.06

0.29

0.14

0.10

*Significant at P = 0.5

"

0.22

0.30

FFPN

3.3.7 Principal Components Analysis for Quantitative Traits

Table 3.5 displays principal components analysis (PCA) of the 13 quantitative


traits, showing the factor scores of each character among the 29 okra accessions,
eigen values and percentage total variance accounted for by five principal
components (PCs).

The five PCs accounted for about 78.51% of total variance with the first
principal component (PC1) recording the highest (32.44%). The second, third,
fourth and fifth principal components (PC2, PC3, PC4 and PC5) accounted for
19.78%, 9.68%, 8.45% and 8.15% respectively. The Eigen values shows the
relative discriminating power of the principal axes with (4.22) for axis 1 and as
low as (1.06) for axis 5. PC1, which accounted for the highest proportion
(32.44%) of total variation mostly correlated with number of pods per plant, first
flowering node, average pods per plant, maximum plant height, average seeds
per pod, maximum number of internode, stem diameter at the base, number of
leaves per plant and 1000-seed weight.
Average pods per plant, maximum plant height, 50% germination, first
flowering node and 1000-seed weight, 50% fruiting, 50% flowering; number of
leaves per plant, stem diameter at the base and maximum number of internodes
were mostly correlated with PC2.

The third principal component (PC3) was positively correlated with maximum
number of internodes, first fruit-producing node, first flowering node, 50%
flowering but negatively correlated with number of leaves per plant, 1000-seed
weight, average seeds per pods, average pods per plant. First fruit-producing
"

node, average seeds per pods, 50% germination, and 1000-seed weight made
substantial contribution to PC4. Finally, 50% germination, first fruit-producing
node and stem diameter at the base substantially contributed to PC5.

Table 3.5: Principal components analysis showing the contribution (factor scores) of 13
quantitative characters among the 29 accessions of Okra, Eigen values and Percentage
total variance accounted for by five principal components *.
CHARACTER

PC1

PC2

PC3

PC4

-0.44768

-0.41751

PC5
-0.02082
-0.08673

1000-Seed weight

0.203819*

0.153724*

50% Flowering

0.111969

-0.56337

0.184005*

-0.07344

50% Fruiting

0.14188

-0.57211

0.02661

0.07007

-0.10149

50% Germination

-0.09528

0.17753*

-0.05589

0.36608*

-0.78663

Average pods per plant

0.35933*

0.21348*

-0.05857

0.14673

-0.14057

Average seeds per pod

0.318568*

0.09009

-0.43818

0.41697*

0.11012

First flowering node

0.389668*

0.16413*

0.22046*

-0.16218

-0.04067

First fruit-producing node

0.14729

0.08145

0.34392*

0.59379*

0.27169*

Maximum number of internode

0.29276*

0.11885

0.41954*

-0.14524

-0.04057

Maximum plant height

0.32356*

0.1805*

0.08216

-0.26811

-0.20185

Number of leaves per plant

0.28419*

-0.3141

-0.44695

0.11124

0.07890

Number of pods per plant

0.39654*

0.0471

0.09077

0.03341

0.26771*

Stem diameter at the base

0.29013*

-0.2554

0.09383

-0.02465

-0.36787

Eigen value

4.22

2.57

1.26

1.09

1.06

% Variance

32.44

19.78

9.68

8.45

8.15

Cumulative % Variance

32.44

52.23

61.90

70.35

78.51

* Values bolded and asterisked made substantial contribution to total variance in the respective axes. Maximum
and least discriminating power (eigen value), maximum and least percentage variance and maximum cumulative
percentage variance values are bolded.

"

3.6 DISCUSSION
3.6.1 Variation in Quantitative and Qualitative Traits
The 29 accessions of okra exhibited significant variation in all but two
quantitative traits studied. Treatment coefficients of variation were low for all
traits. Block coefficients of variation were extremely low but high for stem
diameter at the base and first fruit-producing node, implying that results
obtained are reliable and repeatable over replications. Number of pods per plant,
first fruit-producing node and maximum plant height recorded the highest
treatment coefficients of variation but these were within ranges obtained by
Gomez and Gomez (1984) and less than what was obtained by Ogunbayo
(2005). On the other hand, all the accessions showed similarity in some
qualitative traits and variability in other qualitative traits. The highly significant
differences observed for most characters investigated are an indication of their
genetic diversity (Amoatey, 1987).

Analysis of variance showed that the genotypes under study differed


significantly among all the characters. This finding is in consonance with reports
by Subramanian and Subbaraman (2010) working on maize, Akotkar et al.
(2010), and Mehta et al. (2006), Dhankar and Dhankar (2002), Panda and Singh
(1997), Patil et al. (1996) all working on okra. It, however, contrasts with
results obtained by Moukoumbi et al. (2011), Akinwale et al. (2011), Belete
(2011), Karuri et al. (2010), Saifullah and Rabbani (2009), Aytac and Kinaci
(2009), Hien et al. (2007), Campos et al. (2005) working on Asian rice (Oryza
sativa L.), mustard (Brassica carinata A. Brun), sweet potato (Ipomea batatas

"

"
"

L.), rapeseed (Brassica napus L.), Asian okra (Abelmoschus esculentus L.) and
mandarin (Citrus spp) respectively.

Again, height of plants in this study varied significantly among the okra
accessions studied (Table 3.2). Nkran Nkuruma recorded a mean height of
170.78cm followed by Asontem-BAR, Asante type II, Asontem-GAR, YejiLocal, and Cape recording mean maximum plant height of 128.53cm, 116.33cm,
113.40cm, 103.95cm, and 102.53cm respectively. On the other hand, the
accessions; Kpeve, Kortebortor-ASR, Atomic, Akrave and Clemson spineless
recorded shortest mean plant height of 56.68cm, 48.50cm, 48.40cm, 47.05cm
and 44.38cm respectively. Height at flowering and fruiting (final height) are of
particular interest for breeding programmes because tall, thin stems increase rate
of lodging near harvesting time (Doku, 2011) and this could culminate in loss of
dry matter and a subsequent decrease in fruit yield. This is in consonance with
reports by Doku (2011), Akinyele and Oseikita (2006) and Myanmar (1995)
who worked on rice and okra respectively.

3.6.2 Diversity in Qualitative Morpho-Agronomic Traits


Results for days to maturity show hqwt" ocvwtkv{" fwtcvkqpu<" gctn{" *" 62" fc{u+."
midseason (41-65 days), late (66-90 days) and very late (91-125 days), (Table
3.2). Differences in duration to maturity can be attributed to the duration of the
vegetative stage of the accessions. This is because the length of both
reproductive and edible stages of any okra variety, under a given environment,
has a more or less fixed duration of thirty days whilst the vegetative stage varies

"

"

with end of seed formation. Therefore, the longer the duration of the vegetative
stage, the longer the life cycle of the plant and vice versa (Doku, 2011).

Generally, all the okra accessions recorded high germination percentages.


Earliness in okra is determined by the number of days from sowing to 50% fullbloom. This character recorded a wide variation among the okra accessions
studied. The early maturing plant types for instance, could be selected for areas
with short rainy seasons in rain fed ecologies. Such genotypes will also be
suitable in areas where farmers grow a second crop to take advantage of residual
water after harvesting the early crop. This is supported by results obtained by
Oppong-Sekyere et al. (2012) and Nwangburuka et al. (2011).

Results obtained in this study show that the okra accessions exhibited varying
degrees of fruit pubescence spanning from prickly, slightly rough (smooth),
downy to little hairs on fruits but with the majority having smooth fruits. This
result contrasts those of Bish et al. (1995) and Thomas (1991) who found downy
type of fruit pubescence to be the most pronounced, followed by slightly rough
while prickly fruits was the least in the okra accessions they studied. This could
dg"cp"kpfkecvkqp"qh"rtghgtgpeg"qh"Ijcpckcp"hctogtu"hqt"uoqqvj"htwkv"v{rgu"cpf"
thus, discarded the hairy types (Oppong-Sekyere, 2011).

Petal colour was either cream or yellow with golden yellow flowers (cream)
recording the highest. This is contrary to observation made by Myanmar (1995)
who found petal colour to be 100% yellow for all 40 okra accessions examined.
Akinyele and Akinlosotu (1991) also found similar results in their okra research.
"

Variations observed in this study were conspicuous for position of fruit on main
stem that was erect, horizontal or pendulous. Most of the accessions had their
fruits with erect position on main stem as against pendulous and horizontal
positioning of fruits on main stem of the accessions. This is because different
genotypes have the tendency of exhibiting different growth habits, whether as a
result of selection or a natural adaption mechanism. This is similar to an
observation made by Hanson (2005) working on tomato. Yellowish green was
the most predominant fruit colour while green and green with red patches were
least observed among the accessions. Similar observation was found with leaf
colour. These results are similar to those found by Oppong-Sekyere (2011) and
Myanmar (1995) in okra.

Ariyo (1993) indicated that the pattern of genetic variation observed in


characters studied in West African okra suggests a lot of out crossing among the
taxon. The considerable morphological variation observed in the characters of
the accessions studied could perhaps, be attributed to the preponderance of outcrossing among these different accessions (Oppong-Sekyere, 2011; Lim and
Chai, 2007; Adeniji, 2003). There were also intense variation in number of
branches per plant, number of seeds per pod (fruit), total number of leaves per
plant, stem pubescence, fruit shape, type of pod axis, branching type, seed
shape, fruit peduncle and fruit length. These agree with results found in okra
morphological diversity studies by Khanorkar and Kathiria (2010), and Mishra
and Chhonkar (1979).

"

3.6.3 Genetic Relationship among Accessions based on Cluster Analysis


The 29 accessions of okra were grouped into two major clusters and
subsequently into five sub-clusters based on both quantitative and qualitative
characters studied. The four accessions, Clemson spineless, Agric short fruit,
Debo and Kpeve were placed in cluster one while cluster two contains the rest of
the accessions with six sub-clusters. These four accessions had in common the
following characteristics; long pod length, medium type of branching, reniform
seed shape, glabrous aspect of seed surface, green leaf colour, creamy petal
colour, position of their fruits on main stem being pendulous, slight stem
pubescence, number of ridges per pod ranges from 5 to 10, and green stem
pubescence. The above results reveal that most of the genotypes of cultivated
okra (A. esculentus) under study were highly variable considering individual
character but considering constellation of characters, collectively they belong to
the same group. This is in line with what was reported by Akotkar et al. (2010),
and Martin et al. (1981).

The first sub-cluster and last two sub-sub clusters exhibited higher inter-cluster
distance and accordingly could be utilised for obtaining heterobeltiosis. The
accessions Cs-Legon, Mamolega, and Wune mana, which appear to be the most
diverse, may be potentially useful as sources of variable characters in okra
improvement programme, as affirmed by Irwin et al. (1998) that closely related
accessions are normally located within 8090% similarity. Crosses between
accessions with similarity indices of 80100% may not be desirable; but the
potential for successful crossing of unrelated varieties may produce an array of
genotypes from which useful agronomic types may be selected (Gulsen et al.,
2007).
"

Cs-Legon was the most diverse and isolated in a cluster. This may be explained
by its unique characteristics of persistent epicalyx segment, lanceolate shape of
epicalyx segment, creamy petal colour, downy fruit pubescence, yellowish green
fruit colour, erect position of pods on main stem, slight stem pubescence,
smooth pods, medium pod length and diameter, stocky pod axis, round seed
shape, green leaf colour and prolific yielding potential.

Mamolega and Wune mana were placed in sub-cluster five. This coincides with
the geographical origin of the accessions. The two accessions were collected
from the same geographical location (Upper East Region) and possess similar
morpho-characteristics (Belete, 2011), as a reflection of adaptation to similar
environmental conditions or related ancestry.

Asontem-ER and Volta had the highest genetic similarity index (GS) of 94.1%,
followed by Asante type II and Agric type I (91.5%), Cape and Asontem-ASR
(91%), Kpeve and Debo (90.6%), and Kortebortor-ASR and Asontem-BAR
(90.4%) all separated in cluster two except Kpeve and Debo which were
separated in cluster one into a different sub-cluster. There were however, no
duplicates among the accessions collected. For accessions to be taken as
genetically identical, their genetic similarity index (GS) should be equal to or
greater than 95% (Andersson et al., 2007). In this study, the maximum genetic
similarity was 94.1% recorded between Asontem-ER and Volta (as the closest
pair of accession).
The large size of the accessions from a wide range of geographical areas is very
essential for genetic distance estimation (Nei, 1978). Broad range of genetic
similarity indices and clustering of accessions provides useful variability in okra
"

germplasm collections for genetic conservationists and plant breeders for


improvement programmes (Torkpo et al., 2006; Singh et al., 1974). Except for
cluster five (which contains only two members), the pattern of clustering did not
show distinct association between morpho-agronomic characters and geographic
origin of the collections studied. Some other workers, Hien et al. (2007), Hanson
(2005), Martin et al. (1981) made a similar observation.

3.6.4 Correlations among Quantitative Characters


Correlations are measures of the intensity of association between traits (Steel
and Torrie, 1984). Selection for a single character may increase chances for all
traits that are positively correlated but declines for characters that are negatively
correlated. The association between pairs of quantitative yield traits in the okra
landraces studied saw flowering and fruiting parameters (50% flowering, 50%
fruiting, average number of pods per plant, first-flowering node, and first
fruiting node), maximum plant height and maximum number of internodes
recording significant (P = 0.01) positive associations. This corroborates findings
of several researchers (Hazra and Basu, 2000; Kaul et al., 1978; Singh and
Singh, 1977) and suggests that component breeding would be very effective
(that is, when there is positive association of major yield characters) as found in
this study. Weak correlation between average number of seeds per pod and first
fruit-producing node (r = 0.29), as found in this study is inconsistent with results
obtained by Akinwale et al. (2011), Ogunbayo et al. (2005) and Ariyo et al.
(1987) where the correlations between the two traits were r = 0.58 and r = 0.546,
respectively.

"

Additionally, plant height is strongly and positively correlated to first flowering


node, average number of pods per plant and maximum number of internode.
This may be explained by accumulation and partitioning of photosynthates to
these characters. This is consistent with findings of Verma (1993), Perdosa
(1983) and Ariyo et al. (1987) that plant height is controlled by genetic factors
and is closely associated with number of flowering node, average fruits per plant
and number of internodes. There was a strong positive correlation among
number of pods per plant, and maximum plant height with 1000-seed weight.
Adeniji and Aremu (2007) also reported strong positive correlation between
maximum plant height and 1000-seed weight, suggesting its relevance for
genetic improvement in seeds of okra. In addition, the negative correlation
between 50% flowering and average number of pods per plant(r = - 0.10)
suggests late flowering could provide more pods per plant for the landraces.
Adeniji and Aremu (2007) and Mehta et al. (2006) reported negative correlation
between these two characters and recommended selecting for the West African
okra (A. caillei) (late flowering) accessions for high yield of pods.

3.6.5 Contribution of Traits to Genetic Diversity via Principal Component


Analysis
These characters; 1000-seed weight, 50% flowering, 50% germination, average
pods per plant, average seeds per pod, first flowering node, first fruit-producing
node, maximum number of internodes, maximum plant height, number of leaves
per plant, total number of pods per plant, and stem diameter at the base were
mostly correlated with the first, second, third, fourth and fifth principal
components (PC1, PC2, PC3 PC4 and PC5) of the Principal Components Axes.
"

Thus, factor scores of these 12 characters indicate their substantial contribution


to total genetic variation among the 29 okra accessions studied. This finding is
similar to that of Doku (2011) and Ogunbayo et al. (2005), in which factor
scores of nine and twelve characters for rice respectively accounted for variance
among accessions and were mostly correlated with PC1, PC2, PC3 and PC4 of the
Principal Components Axes.

The output of the PCA revealed that different characters contributed differently
to the total variation. The mean contributions of maximum plant height, 50%
germination, average seeds per pod, average pods per plant, first flowering node,
first fruit-producing node, maximum number of internodes, number of leaves
per plant, number of pods per plant were relatively high in the principal axes.
This observation confirmed the individual contributions of these traits to the
variations observed in the 29 okra accessions as well as seed yield in all the
accessions. This agrees with the report of Ogunbodede (1997), Ariyo, and
Odulaja (1991). It further suggests that selection for seed yield must take into
consideration these traits. This corroborates the report of Aremu et al. (2007)
working with cowpea and Olika et al. (2011) working with coffee.

The first four principal axes accounted for over 70% of total variation among the
13 characters describing the accessions. These characters can be employed in
differentiating okra accessions (Nwangburuka et al., 2011; Clifford and
Stephenson, 1975). The total contribution of the five principal component axes
of this study was higher (78.51%) than observations made by other workers
(Nwangburuka et al., 2011; Moukoumbi et al., 2011; Campos et al., 2005;
Ogunbayo et al., 2005) where the principal component axes contributed 64.32%,
"

66.37%, 76.62% and 64.5% variation, respectively. In this present study, all the
eigen values were lower than what was observed by Nwangburuka et al. (2011).
First fruit-producing node and number of leaves per plant were found to have
contributed positively and significantly to total genetic variance in this study,
confirming a similar observation by Nwangburuka et al. (2011). The factor
scores of the 12 characters imply that, high priorities should be given to these
genetic traits, if selection is to be made for a future breeding programme.

3.7 CONCLUSION
The following conclusions can be drawn from the current study:
I.

The 29 accessions of okra exhibited great variability with respect to the


morpho-agronomic characters studied.

II.

III.

No duplicate was identified per the morphological traits studied.

Cs-Legon was the most diverse with outstanding traits such as smooth
pod shape, early fruiting, uniform-sized, medium height, prolific fruiting
potential, straight and stocky stem, and smooth pod shape (no spines).
Mamolega and Wune mana were also identified as most diverse and distantly
related accessions and could be utilised as parent stocks to introgress the
desirable traits inherent in them into the exportable cultivar Indiana, and as
sources of variability for future breeding work.

"

IV.

The output of the PCA revealed that different characters contributed

differently to total genetic variation.

"

"
"

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"

CHAPTER FOUR

MOLECULAR CHARACTERISATION OF 29 ACCESSIONS OF OKRA


(Abelmoschus spp L. Moench) USING INTER-SIMPLE SEQUENCE
REPEATS (ISSR) MARKER.

4.1 INTRODUCTION"
The most significant breakthrough in agricultural biotechnology is the
understanding of the structure of genomes and the genetic mechanisms behind
economically important traits. Plant genomics also known as plant molecular
biology is now able to provide information on the identity, location, impact and
functions of genes controlling traits, which can be identified, catalogued and
mapped as single gene markers in countless species of higher plants (Jonah et
al., 2011). Molecular markers are identifiable DNA sequences, found at specific
loci (locations) within the genome and associated with inheritance of a trait or
linked gene (FAO, 2004). Thottappilly et al. (2000) defined molecular markers
as naturally occurring polymorphism, which include proteins and nucleic acids
that are detectably different. Thus, molecular DNA markers are individual genes
flanking within a defined close interval in the genome of an organism.

Genetic analysis by molecular markers can lead to the determination of the


actual genetic distances among species, populations or individuals, and reveals
patterns of evolutionary relationships. Molecular markers are useful tools for
characterising genetic diversity of germplasm collections (Andersson et al.,
2007; Viard et al., 2002; Tsumura et al., 1996). Molecular markers are
"

frequently associated with specific gene and cevu" cu" c" ukip" rquvu" vq" wughwn"
genes and such markers when very tightly linked to genes of interest, can be
exploited to select indirectly for desirable alleles and this represents the simplest
form of marker- assisted selection (MAS) (Hoisington et al., 2002).

Molecular markers can be employed to verify genetic diversity assessed by


phenotypic characterisation or as an alternative procedure alone (Sawadogo et
al., 2009; Schoen and Brown, 1995). The integrated use of morphological,
agronomic, biochemical and molecular evaluations of genetic resources of
different types of traits provide complementary information and deepens
understanding of the level of polymorphism within germplasm (Singh et al.,
2002; Gupta et al., 1999; Diwan and Cregan, 1997).

There are several molecular marker techniques. These include restriction


fragment length polymorphisms (RFLP), which was the first molecular marker
(Botstein et al., 1980) generated for genome analysis and mapping. There are
also those based on polymerase chain reaction (PCR) technology such as
random amplified polymorphic DNA (RAPDs) (Lynch and Milligan, 1994;
Williams et al., 1990; Welsh and McClelland, 1990), inter- simple sequence
repeats (ISSR) (Zietkiewicz et al., 1994), simple sequence repeats (SSR) or
microsatellites (Zietkiewicz et al., 1994), single nucleotide polymorphisms
(SNPs) (Choi et al., 2007; Nasu et al., 2002; Kruglyak and Nickerson, 2001),
microarrays

(Lindroos

et

al., 2002) and

amplified

fragment

length

polymorphisms (AFLP) (Karp et al., 1997; Vos et al., 1995). The different
genetic markers vary with respect to important features such as genomic
"

distribution, level of polymorphism detected, locus specificity, reproducibility,


as well as technical and financial investments (Yeboah et al., 2007).
The choice of the most appropriate technique depends on given pre-conditions
such as technical and financial facilities, expertise, time constraints, presumed
level of polymorphism and the specific objective of application (Park et al.,
2003; Viard et al., 2002; Richards and Sutherland, 1994). Therefore, none of the
techniques available is superior to all others for a broad range of applications,
but the key question is which marker to be used in a specific situation (Yeboah
et al., 2007).
Okra (Abelmoschus spp L. Moench) is one of the few vegetables found in many
homes of Ghanaians and is gradually becoming a delicacy of most consumers in
the country and abroad. It is the fourth most popularly patronised vegetables in
Ghana as results of its vitamin, mineral and mucilage contents. In 2005 alone,
world production of okra was estimated at 73,000 metric tonnes (MTs),
(FAOSTAT, 2005), Ghana recording 11,3084MTs. In 2008, world production
was 6395570 MTs but Ghana produced only 108000 MTs and in 2010, total
estimated global production was 6917062 MTs with Ghana recording
82500MTs. India is the largest producer worldwide followed by Nigeria and
Sudan (FAOSTAT, 2012).

Vq"rtqxkfg"pwvtkvkqpcnn{"dcncpegf"hqqf"hqt"vjg" yqtnfu"vggokpi" rqrwncvkqp"cpf"


relieve the intense pressure on land use and natural resources, plant species used
as food must be diversified (Kumar et al., 2010; Hughes, 2009). Omonhinmin
and Osawaru (2005) and Bisht et al. (1995) all reported the existence of broad
"

"
"

genetic diversity and polymorphism within the cultivated species of the genus
Abelmoschus. West Africa alone holds 1,769 of the 2,283 accessions reported
worldwide. Variability is more pronounced particularly in plant height, days to
flowering and fruiting, tolerance to the yellow vein mosaic virus, yielding
potential and a host of other fruit characteristics among okra germplasm (Gulsen
et al., 2007; Ariyo and Odulaja, 1991). On the contrary, there is little research
investment towards improving the crop in the sub-region though being the
second largest producer globally.
"

Okra is put to diverse uses by different end users. This is because every part of
this crop seems to offer some useful purpose or the other. Varieties are needed
as sources of protein, fibre, biomass, oil, mucilage (type and yield), colour,
medicinal and ornamental use (National Academies Press, 2006). Traditional
farmers often engage in selection for different purposes and eventually mixed
them up. Okra in the hands of these traditional farmers diversifies into vast
number of cultivars (landraces) adapted to various environments (De Lannoy,
2001). However, with the emerging status of okra as an export crop, there is
therefore the need for standardisation of traits inherent in these landraces to meet
specific end uses and markets.

Although, there has been considerable selection and breeding of okra, it has
been limited to production of immature pods. The rest of the fantastic genetic
diversity within this species is untapped, or even unexplored. Molecular
characterisation is the ultimate tool to exploit the genetic variability (intra and
interspecific variations) in this crop from different phyto-geographic areas; this
will open the way for improving the compositional value of the crop, improving
"

"

yields, cultivation conditions, nutritional value, and nutraceuticals (National


Academies Press, 2006).
The Inter-Simple Sequence Repeat (ISSR) marker was developed by
Zietkiewicz et al. (1994) based on the amplification of a single primer
eqpvckpkpi"c"oketqucvgnnkvg"eqtg"ugswgpeg"cpejqtgf"cv"vjg"7"qt"5"gpf"d{"c"ugv"
of 2-4 purine or pyrimidine residues, offering a high degree of reproducibility
with the detection of a rich level of polymorphism in a relatively simple
procedure. It has been widely utilised in assessment of genetic diversity and
cultivar identification (Bhattacharya et al., 2010). ISSRs have been successfully
used to estimate the extent of genetic polymorphism at inter and intra specific
level in a wide range of crop species which include melon (Yildiz et al., 2011),
rice (Reddy et al., 2002), wheat (Nagaoka and Ogihara, 1997), finger millet
(Reddy et al., 2002), soybean (van Toai et al., 2001; Tohme et al., 1996), sweet
potato (van der Voort et al., 1997), plantago (Reddy et al., 2002), and cucumber
(Yeboah et al., 2007). ISSR exploits the abundance of SSRs to generate complex
banding profiles that vary within and between species. Each band corresponds to
a DNA sequence delimited by two inverted microsatellites.

ISSR markers are quick and easy to handle, but the longer length of their
primers make them seem to have the reproducibility of SSR markers. ISSR
primers contain sequences complementary to SSR motif with 1-3 base anchors
cv" gkvjgt" vjg" 5" qt" 7" gpf." dcug" rqukvkqp" ykvjkp" vjg" cpejqt" cpf" oc{" jcxg" cp{"
nucleotide other than that needed to continue the repeat sequence ensuring that
the primer binds exclusively to one end of the targeted SSR and not in the
middle. Numerous amplification products are generated by ISSRs, which
"

consists mainly of regions between neighbouring and inverted SSR sites


(Zietkiewicz et al., 1994).
Zietkiewicz et al. (1994) were the first to use ISSR to differentiate between
closely related genotypes but it has since been applied to cultivar identification
(Jackson and Ram, 2003; Charters et al., 1996; Kantey et al., 1995), to detection
of gene flow (Ferriol et al., 2003), and introgression of desired traits into
breeding lines (Allainguillaume et al., 1997) and for genetic mapping of
agronomically important traits (QTLs) (Kojima et al., 1998). Akagi et al. (1997),
first demonstrated the utility of ISSR markers for tagging agronomically
important genes by identifying tight linkage with a nuclear restorer gene in rice.
ISSR markers are highly informative, repeatable, dominant and repeated
sequences are abundant throughout eukaryotic genome. They anneal in several
regions giving a complex amplification pattern in which fragments are often
polymorphic among different individuals, hence the choice of this marker for
this work.

4.1.1 Objective of the study


The objective of employing ISSR marker was to assess the extent of genetic
variation and polymorphism within the 29 okra collections under study.

"

4.2 MATERIALS AND METHODS


4.2.1 Experimental Site
The experiment was undertaken at the Laboratory of Molecular Systematics,
Phylogeography and Conservation of Bryophytes, Department of Botany,
Faculty of Biology, Murcia University, Spain. The work was carried out from
February 2012 to May 2012.

4.2.2 DNA Isolation


Seeds of each accession were sown and their fresh leaves were then harvested
three weeks after germination, oven dried at a temperature of 20C. They were
then packaged, well labelled and airlifted to Murcia University, Spain. CTAB or
detergent method according to Doyle and Doyle (1990) was employed to isolate
DNA from all accessions. Oven dried fresh leaves of each accession (200mg)
ygtg"ygkijgf"cpf"qhh"nqcfgf"kpvq"3722" n"*307on+"oketqhwig"vwdg"cpf"ncdgnngf0"
Each accession was grounded to a fine powder by means of liquid nitrogen and
pestle. Thousand micro litres (1000 n+" qh CTAB buffer was added to each
sample preparation (due to the slim and high viscosity of the samples). The
tubes containing the CTAB and plant extract mixture were incubated for 30
minutes at 60C.
The samples were further incubated at room temperature for 5minutes. Eight
hundred micro litres (:22" n) of chloroform AIA (24:1) was then added. The
samples were then centrifuged at 1200 rpm for 10 minutes and the supernatant
of each accession sample was recoupinated into a new 1.5ml microfuge tube and
labelled.
"

Gcej"ucorng"ycu"vjgp"ycujgf"cickp"ykvj"822" n"ejnqtqhqto"CKC"cpf"urkppgf"cv"
1300 rpm for 10 minutes. Sample supernatant of each was then pipetted into a
new 1.5ml microfuge tube. The DNA of each sample was then precipitated with
vjg"wug"qh"822 n"kuqrtqrcpqn"cpf"gcej"oketqhwig"vwdg"ycu"vjgp"igpvn{"kpxgtvgf0"
The samples were then stored at -20C for 30 minutes. Afterwards, each sample
was then spinned at 1300 rpm for 15 minutes. The supernatant of each sample
was carefully eluted out to leave the DNA pellet, and allowed to dry. The pellet
was reuwurgpfgf"kp"322 n"VG"buffer and stored at -20 C for later use.

4.2.3 Preparation of Cocktail for PCR.


RET" cornkhkecvkqp" tgcevkqp" xqnwog" qh" 47" n" ycu" ugv" wr" ugrcrately for each
accession containing MgCl2 at a concentration of 1.5mM, 0.2mM each of dATP,
dCTP, dGTP and dTTP (Fermentas, USA), 0.4 units of Taq polymerase (Green
Iq"Vcs="Rtqogic."WUC+."407 n"qh"32z"RET"dwhhgt."206oO"qh"gcej"hqtyctf"cpf"
reverse primer anf"207 n"qh"igpqoke"FPC0"Ukz"*8+"fkhhgtgpv"rtkogtu"ygtg"wugf"
for the study.

4.2.4 Amplification
The amplification was done with thermal cycler from BIO-RAD. The
temperature cycles were, a first denaturation step of 5 min at 94C, followed by
35 cycles of 94C for 30s, 55C for 30s and 72C for 1min followed by a final
extension at 72C for 7minutes. The complete reactions were then held at 4C
until electrophoresis. The amplicons were verified for amplification by
electrophoresis on 1.5% agarose gel.
"

4.2.5 Preparation of Polyacrylamide Gel (PAGE)


The PAGE solution was prepared using urea, TBE and 30% acrylamide
solution (30% acrylamide bis acrylamide (37.5: 1) and 40% acrylamide bis
acrylamide (19:1). Ten percent (10%) of the page solution was prepared. PAGE
was then cast using the 10 % prepared PAGE solution, 10% ammonia per
uwnrjcvg" *203i" qh" cooqpkwo" rgt" uwnrjcvg" fkuuqnxgf" kp" 3222 n" qh" fkuvknngf"
water) and TEMED. Ammonium per sulphate (APS) and TEMED act as
catalyst and oxidising agent. For each gel cast, 40ml of the PAGE solution,
47 n"qh"VGOGF"cpf"55 n"qh"vjg"32'"CRU"ygtg"wugf0"
The glass on which the gel was to be cast was cleansed with ethanol. Brine
saline was spread on it and then washed with ethanol, rinsed with distilled water
and dried with a tissue. The prepared glass was then fastened to the Perspex
glass containing the comb via clips ensuring that the string that could prevent
the leakage of the solution was in 113 place. Prepared PAGE solution was
immediately eluted into the set plate. After 45 minutes, the clips were taken off
and the PAGE bounded to the glass was carefully removed from the Perspex
glass.

4.2.6 Electrophoresis
The PAGE was warmed in the electrophoresis tank at 250volts for 15minutes to
ensure the removal of any excessive sanv" kp" vjg" ign0" Chvgtyctfu." 407 n" qh" vjg"
cornkeqpu" qh" vjg" xctkqwu" ceeguukqpu" ygtg" vjqtqwijn{" okzgf" ykvj" 4 n" nqcfkpi"
dye and loaded into the wells of the gel. 50bp DNA ladder (marker) was also
loaded in one well to estimate the molecular size of the amplicons. It was then
ran in 1X TBE for 1hour; first 40mins at 100volts to remove the running
"

amplicons from the wells and set it running and the remaining 80minutes at
140volts to enable amplicons move fast and prevent the fading of bands.
Afterwards, it was then stained in 400ml of 0.05% silver stain via orbital shaker
for 1hour and then photographed using BioDoc-it gel documentation system
(http.www.molecularinfo.com/MTM/C/C1/C1-1.html).

4.2.7 Data Collection and Analysis


The amplified ISSR DNA bands representing different alleles were scored as
different genotypes. Binary scoring system was used in which 0 when band is
absent and 1 when present. PCO3 was used to calculate genetic distance using
the binary data; the topology based on the genetic distance, and was under the
assumption that the generated bands are independent. Distance matrix was
generated using dice similarity index.
Phylip package was used to calculate the neighbour joining (NJ) tree
(neighbor.exe) and the extension .tre was added to the "outtree" file in order to
be visualised by treegraph2 and saved in Newick format, where the tree was
imported to mega5 in order to visualise the tree into the topology form. A
hierarchical analysis of molecular variance (AMOVA) of the okra populations
computed to partition genetic diversity within and among regions was done.
Further testing was used to evaluate the origin of used samples through parental
and maternal lines; in which two regions, ITS (nuclear sequence) and trnL
regions (chloroplast sequence) of the accessions, Atomic and Akrave were
sequenced.

"

4.3.2 Cluster Analysis based on six ISSR primers


Figure 4.3 displays the maximum genetic relationship as revealed by six ISSR
primers based on dice coefficient using single link similarity matrix method.
PCO3 was used to calculate genetic distance; the topology based on the genetic
distance, and was under the assumption that the generated bands are
independent, which is a non-spatial descriptive method. Distance matrix was
generated using dice similarity index.
Phylip package was used to calculate the neighbour joining (NJ) tree
(neighbor.exe) and the extension (.tre) was added to the "outtree" file in order to
be visualised by treegraph2 and saved in Newick format, where the tree was
imported to mega5 in order to visualise the tree into the topology form. Two
groups was clearly differentiated, group1 in red and group 2 in Blue.

The dendrogram revealed two major clusters at a genetic or similarity distance


of 96% for all the accessions studied. The first major cluster contains Asante
type II, Yeji-Local, Atomic and Indiana at 91.7% similarity index. However, all
the other 25 accessions started to separate at 100% similarity distance and
further formed a single entry cluster. The closest resemblance was observed
between Volta and Asontem-ER at a similarity index of 98.9%. Furthermore, the
accessions Asante type II, Yeji-Local, Atomic and Indiana were widely
separated.

"

4.4 DISCUSSION
4.4.1 Cluster Analysis
Assessment of the extent of relatedness among potential parents forms the
foundation for selection of genetically distant and diverse parents in any
breeding programme. The accessions Asante type II, Yeji-Local, Atomic and
Indiana were contained in cluster entry 1 whilst the remaining 25 accession were
all grouped into cluster entry 2. This could be explained by their outstanding
characteristics (relatively early fruiting, uniform in shape, moderate level of
mucilage, medium in height, glabrous stem, and moderate length of peduncle),
as these accessions were collected from different geographical areas. Asante
type II from the Brong-Ahafo region and Yeji-Local (forest savanna belt),
Atomic from the greater Accra region (coastal savanna zone) and Indiana from
India (template climatic region).

Indiana however was the most diverse with a genetic similarity index of 96.1%.
However, the wide variation between Indiana and the rest of the populations
indicates the usefulness of each population, particularly Asante type II, YejiLocal, Atomic populations which were most diverse, as a valuable genetic
resource for selection of superior genotypes. Indiana is an exotic cultivar and is
not well adapted to the local conditions but possess appreciable desired qualities
(slim and narrow in shape and size, smooth, yellowish green in colour, early
fruiting, short peduncle, moderate level of mucilage and less bulky in weight)
for the export market. Asante types II, Yeji-Local, Atomic are all indigenous
cultivars, well adapted to local conditions and very hardy to both biotic and
abiotic stresses yet non-uniform in shape and size. The desirable traits in Indiana
"

could be introgressed into any of these three accessions to improve them for
both export market and local consumption.

"4.4.2

Genetic Diversity/polymorphism among Accessions as Revealed by

ISSR Primers
The genetic relationships among individual accessions or groups are further
elucidated by the dendrogram generated via the ISSR primers. Results obtained
from the dendrogram reveal two major clusters at a genetic or similarity distance
of 96% for all the accessions studied. All 25 accessions in group II could be
considered as possible duplicates with genetic similarity index of 100%
(Andersson et al., 2007). This suggests that these accessions are genetically
closely related or they have a common ancestry descent (Kalivas et al., 2011).
This contrasts with findings by Pasqualone et al. (2012), Yao et al. (2008),
Nwangburuka et al. (2011), Aladele et al. (2008), Aladele (2005) who concluded
that there is high genetic diversity among Glycine max, Glycyrrhiza uralensis, A.
esculentus and A. caillei species. Few of the ISSR primers however, were able
to amplify more than one band per genotype; hence, less residual heterogeneity
could be suspected within the lines (Ogunbayo et al., 2005).

The low level of polymorphism detected with the six ISSR primers among the
accessions could be attributed to the dominant nature and low level of locus
specificity associated with ISSR primers. ISSRs are dominant primers and hence
almost unlikely to detect specific loci within genomes, thus unable to detect
heterozygous alleles within genomes of organisms.

"

Nonetheless, another dendrogram generated from scores for both quantitative


and qualitative morpho-agronomic traits identified all accessions as separate
entities with no duplications, and discriminated at a genetic distance of 54.3%.
This contrasts with results from the dendrogram produced from the ISSR
markers (Figure 4.2) which conversely identified duplications in the collection.

In addition, two regions, ITS and trnL regions of two accessions (Akrave and
Atomic) were sequenced. The sequenced ITS regions were chaotic and
multicopies indications were found, demonstrating a multiple origin of the
genomic DNA of the individual accession (hybrid). Therefore the trnL intron
(maternal DNA), which is more conserved than the ITS, is a non-functional unit
(intron) of the gene. This indicates that, they are the same, which means that
these two okra accessions originated from the same maternal line (no different
maternal lines) or common source.

4.5 CONCLUSIONS
The use of the six ISSR primers yielded low level of polymorphism among the
29 accessions of okra.
a)

25 accessions were identified as possible duplicates

b)

Indiana was the most distantly distinct accession and stand as a unique

source of genes for improvement programmes

"

c)

Yeji-Local, Asante type II and Atomic were also detected as unique

genotypes
d)

Akrave and Atomic were identified to have originated from the same

ancestry descent.

4.6 RECOMMENDATIONS
For a co-dominant marker to be employed in any characterisation work, the
sequence of the genome of the plant must be deciphered and known. To the best
of my knowledge, the genome of okra has not yet been sequenced hence no SSR
primers are available now to conduct a comprehensive and thorough genetic
diversity study at the gene level. Sequencing calls for huge financial and
infrastructural investments, therefore I recommend the following;

a)

International and national institutions concerned with promoting and

maximising the potentials of unexploited and underutilised crops like okra


should collaborate to invest in a project towards sequencing of the genome of
this versatile vegetable since this cannot be done by a single entity.
b)

Researchers in Ghana could also explore and introgress the desirable

qualities of Indiana, Yeji-Local, Asante type II and Atomic into landraces that
are well adapted to the local agro ecological conditions but lack these qualities
(narrow shape and size, smooth pod pubescence, yellowish green in colour,
early fruiting, short peduncle, moderate level of mucilage, persistent epicalyx
segments, medium pod length and less bulky in weight).

"

"
"

c)

In addition, collections was done from eight regions of Ghana, this should

be extended to other regions for thorough characterisation of okra germplasm


and for further conservation.
d)

Genes linked to agronomically important traits in okra should be

genetically mapped through Quantitative Traits Loci (QTLs) to serve as a


baseline data platform for researchers and breeders.

"

"

"
"

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"

"

polymerase

chain

reaction

"
"

CHAPTER FIVE
"

TOTAL FLAVONOID, PHENOLIC CONTENTS AND ANTIOXIDANT


SCAVENGING ACTIVITY IN 25 ACCESSIONS OF OKRA
"

5.1 INTRODUCTION
Antioxidants are powerful free radical scavengers in the body, while free
radicals are highly reactive chemical substances such as peroxide, hydroxyl
radical and singlet oxygen that travel around in the body and cause damage to
body cells (Alia et al., 2003). Many human degenerative diseases have been
ascribed to free radical damages in the body and numerous studies have been
undertaken on how to delay or prevent onset of these diseases (Oboh, 2005).

Radicals are chemical species with one or more unpaired electrons, and free
radicals are radicals that have moved out of the immediate molecular
environment of their generation. There are several endogenous sources of
oxidants in the body: reduction of molecular oxygen in mitochondria during
cellular respiration takes place in sequential steps (Halliwell et al., 1995),
yielding the radical by-products superoxide (O2), hydroxyl (OH ), and hydrogen
peroxide (H2O2); degradation of fatty acids and other molecules in peroxisomes
produce H2O2; phagocytosis results in an oxidative burst of nitric oxide (NO ),
which also reacts with superoxide to produce the oxidising and nitrating species
peroxynitrite (ONOO-) (Atta and Choudhary, 2001; Frei, 1994).

"

"

"
"

Free radicals and oxidants are noted to trigger lipid peroxidation, as well as the
oxidation of proteins and DNA, causing extensive damage to body cells.
Oxidative stress resulting from an imbalance of oxidising species and natural
antioxidants in the body has been identified to contribute to aging, cell
crqrvquku."cpf"ugxgtg"fkugcugu"uwej"cu"ecpegt."Rctmkpuqpu"fkugcug."Cn|jgkogtu"
disease, and even cardiovascular disorders (Long et al., 2007; Atawordi, 2005;
Kassab et al., 2003; Favier, 2003). Epidemiological studies and intervention
trials on prevention of cancer and cardiovascular disease in people taking
antioxidant supplements indicate that, dietary intake of antioxidants can help
scavenge free radicals and oxidants and protect the body against diseases (Frei,
1994). It is also proven that most of the dietary antioxidants have low or
minimal toxicity and intake can be increased without adverse effects.

Phenolic and polyphenolic compounds constitute the major class of natural


antioxidants present in plants, foods, and beverages. An antioxidant is defined as
food substance which when present at low concentrations, converse to those of
an oxidisable substrate, substantially decreases or precludes the adverse effects
of reactive species in normal physiological functions in the body (Halliwell et
al., 1995).

Polyphenols are one of the most copious and widely distributed groups of
secondary metabolites in plants with over 8000 phenolic structures known
(Atawordi et al., 2005; Soobrattee et al., 2005). Naturally, these polyphenols
span from simple molecules of phenolic acids, phenylpropanoids, and
flavonoids to completely polymerised lignins, melanins, and tannins (Kassab et
al., 2003; Giasson et al., 2002). Polyphenols confer a host of biological
"

"

"
"

repercussions

or

effects

such

as

anti-inflammatory,

antiallergic,

hepatoprotective, antiviral, antithrombotic, antibacterial, vasodilatory and


anticarcinogenic actions to human (Middleton Jr. et al., 2000) and most of these
biological effects are directly ascribed to free radical scavenging and antioxidant
activities (Soobrattee et al., 2005).

Free radical damage is one of the most prominent causes of devastating diseases
that are responsible for killing millions of people in the world, such as
cardiovascular infections, which can manifest as heart attacks and cancer (Amic
et al., 2003; Chaudire and Ferrai-Iliou, 1999). Several research on antioxidants
in biological systems have confirmed the neutralising effects on oxidative stress
that predispose lethal diseases such as atherosclerosis, diabetes mellitus, chronic
inflammation, neurodegenerative disorders and cancerous tumours and thus,
generating keen interest in assessment of antioxidant potentials of consumable
food compounds (Karadag et al., 2009). Free radicals naturally occur in the
body as a result of chemical reactions during normal cellular processes such as
during conversion of food into energy in the body (Oboh and Rocha, 2006).

Antioxidant nutrients in foods soak up all the excess energy of free radicals,
converting them into harmless molecules or waste products that can be
eliminated (Oboh, 2005). Antioxidants are powerful substances that can
neutralise free radicals before they damage the body cells (Oboh and Rocha,
2006). Many phenolics such as flavonoids possess stronger and higher
antioxidant capacities than even ascorbic acid (vitamin C) and tocopherols
(vitamin E). Flavonoids could protect membrane lipids from oxidation. Major
sources of flavonoids are vegetables and fruits (Cai et al., 2004). Current
"

"

"
"

growing interest in antioxidants lies in their capability to preclude and avert


deleterious effects of free radicals in the body and deterioration of fats and other
food constituents (Apea-Bah et al., 2009).

Mechanisms of antioxidant action include serving as


(a) physical barriers to prevent the generation of reactive oxygen species (ROS)
or access of ROS to vital biological sites such as cell membranes, UV filters;
(b) chemical traps/sinks that absorb energy and electrons to quench ROS;
(c) catalytic system capable of neutralising or diverting ROS, for instance
superoxide dismutase (SOD), catalase and glutathione (Chaudire and FerrariIliou, 1999);
(d) binding or inactivation of metal ions to prevent generation of ROS, eg.
ferritin, ceruloplasmin, catechins; and
(e) chain breaking antioxidants that scavenge and destroy ROS, eg. ascorbic
acid, tocopherols, flavonoids, and uric acid (Atawordi, 2005; Benzie, 2003).
"

Okra has been describgf"cu"c" uvqtgjqwug"qh"pwvtkgpvu."pgctn{" jcnh" qh" yjkej"ku"


soluble fibre in the form of gums and pectins and its soluble fibre helps to lower
serum cholesterol, reducing the risk of heart diseases (Amic et al., 2003;
Schippers, 2000). The other half is insoluble fibre which helps to keep the
intestinal tract healthy (Jeff, 2002). There is however, a dearth of information on
antioxidant-phytoconstituents present in okra.

"

"

5.1.1 ESTIMATION OF ANTIOXIDANT SCAVENGING ACTIVITY


Antioxidants can be measured by methods such as oxygen radical absorbance
capacity (ORAC) assay (Huag et al., 2005; Ou et al., 2001), diphenylpicrylhydrazyl (DPPH) assay (Botchway et al., 2007), ferric reducing/antioxidant
power (FRAP) (Karadag et al., 2009), deoxyribose assay (Adom and Liu, 2005),
quantification of products formed during peroxidation (Chumark et al., 2008),
low-density lipoprotein (LDL) oxidation (Gheldoff and Engeseth, 2002) and
total antioxidant parameter (TRAP) (Huag et al., 2005).
However, Diphenylpicryl-Hydrazyl (DPPH) assay (Botchway et al., 2007), was
employed in the study, because, it is technically simple, rapid and inexpensive
method to measure and determine antioxidant activity of foods, which employs a
stable 2, 2-diphenyl- 1-picrylhydrazyl (DPPH) radical. The DPPH free radical
contains an odd electron, which gives it a strong maximum absorption at 517 nm
and is purple coloured. The colour turns from purple to yellow as the molar
absorptivity of the DPPH radical at 517 nm declines from 9660 to 1640 when its
odd electron pairs with hydrogen from a free radical scavenging antioxidant to
form the reduced DPPH-H. The resulting decolourisation is stoichiometric with
respect to captured number of electrons (Prakash et al., 2010).
Antioxidant compounds may be soluble in water, lipid, insoluble, or bound to
cell walls. Hence, extraction efficiency is an essential factor in quantification of
antioxidant activity of foods. DPPH is widely used to test the ability of
compounds to act as free radical scavengers or hydrogen donors, and to evaluate
antioxidant activity of foods. It has also been used to quantify antioxidants in
complex biological systems in recent years (Brand-Williams et al., 1995).
"

"
"
"

The DPPH method can be used for solid or liquid samples and is not restricted
or specific to any particular antioxidant component, but applies to the overall
antioxidant capacity of a sample. A measure of total antioxidant capacity helps
understand the functional properties of foods. Antioxidant activity has been
expressed in various ways including percentage of the reagent used, oxidation
inhibition rate and so on. An easier way to present antioxidant activity of foods
would be to reference a common reference standard. One common reference
standard, (S)-(-)-6 hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid,
also known as Trolox, serves this purpose (Karadag et al., 2009; Brand-Williams
et al., 1995).
"

5.1.2 Objective of study


The objective of the study was to determine the total antioxidant scavenging
activity, total phenolics and total flavonoid contents of 25 accessions of okra
(Abelmoschus spp L.).
"

5.2 MATERIALS AND METHODS


5.2.1 Experimental site
Okra samples used in this experiment were planted on a 32m x 59m plot of land
with a randomised complete block design at the research fields of the
Biotechnology and Nuclear Agriculture Research Institute (BNARI). Four
accessions were not available at the time of the experiment as they fruited very
early and were out of bloom to obtain fresh fruit. The laboratory study was

"

"

"
"

carried out at the Applied Radiation Biology Centre of the Radiological and
Medical Sciences Research Institute (RAMSRI).
"

5.2.2 Sample preparation


Whole fruits were harvested at edible maturity stage and lyophilised (freezedried) at the Ghana Research Reactor one (GHARR1) of the Ghana Atomic
Energy Commission (GAEC) and subsequently homogenised in a stainless steel
blender to obtain a powdery form.
Two grammes (2g) of the powdered okra were poured into centrifuge tubes.
Thirty millilitres (30ml) of distilled water was added and stirred on a mechanical
agitator for 2hours. This was topped up with 20ml of distilled water and agitated
for additional 2hours, making a total volume of 50mls.The supernatant was then
stored and kept in the freezer till needed.

Table 5.1: Okra accessions used in the study


Source

Accession

Greater Accra region

Labadi, Asontem- GAR, Atomic, Cs-Legon, Legon

Ashanti region

Asontem-ASR, Agric type I, Debo, Asante type II,

fingers, Clemson spineless, Indiana, Volta


Kortebortor-ASR, Agric short fruit
Central region

Cape

Eastern region

Asontem-ER

Brong-Ahafo region

Yeji-Local, Asontem nv, Asontem-BAR, KortebortorBAR, Nkran nkuruma

Western region

Juaboso

Upper East region

Mamolega, Wune mana, Mapelega

"

"

5.2.4 Total phenolic content


Total phenolic content (TPC) were determined according to the Folin-Ciocalteau
method (Kujala et al., 2000; Singleton et al., 1999), using gallic acid as a
standard. The samples were dissolved in 5 ml of distilled water (50:50, v/v).
Fifty microlitres (72 n) of the extract was mixed with 3ml of distilled water
(dH2Q+" cpf" 472 n" qh" Folin-Ciocalteau reagents (FCR). The mixtures were
allowed to stand for 5 minutes."cpf"vjgp"972 n"qh 20% Na2CO3 was added. After
incubation of the resulting reaction mixture for 30 minutes at room temperature
absorbance values were measured spectrophotometrically at 760nm using a UVVIS Spectrophotometer (Shimadzu Corporation, 1201, Kyoto, Japan). All
determinations were carried out in triplicate. A calibration curve was derived
using the following dilution regimes; 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml
and 1.0mg/ml from a stock solution of 1000mg/ml Gallic acid dissolved in
water. Fifty microlitres (72 l) each of these solutions were treated in same
manner as the samples and a calibration linear regression equation established as
y = 1.094x-225:."*yjgtg"z"ku" i1i"ICG+."T2 = 0.995, where R is the coefficient
of the regression line, to explain the model. The total phenolic content in each
extract were expressed as gallic acid equivalent (GAE) in g per gram sample
using the formula by Achoribo et al. (2012);
C = c v / m
Where; C = total phenolic content in mg/g okra extract, in GAE.
c = the concentration of gallic acid established from the calibration curve in mg/g.
v = the volume of okra extract in micro litres.
m = the weight of okra extract in grams.

"

"
"

5.2.5 Total flavonoid content


Aluminium chloride colourimetric method was employed for the determination
of flavonoids (Zhishen et al., 1999). Five hundred microlitres (722 n) of extract
ycu" okzgf" ykvj" 3722 n" qh" ;;0;'" gvjcpqn" *GvQJ+." 322 n" qh" 3" O" rqvcuukwo"
cegvcvg." 322 n" qh" 32'" cnwokpwo" ejnqtkfg" cpf" 5222 n" qh" fkuvknngf" ycver. The
resulting mixtures were incubated for 30 minutes at room temperature and
corresponding absorbance measured at 415 nm. All determinations were carried
out in triplicates. A standard calibration curve was constructed using quercetin
standard solutions qh"407 i1on."7 i1on."907 i1on."32 i1on and 12.5 i1on0 Five
hundred microlitres (722 n+"qh each standard was treated in the same manner as
the samples above and calibration linear regression equation of y = 0.043x was
obtained."*yjgtg"z"?" i"rgt"Swgtegvkp+. R2 = 0.973, where R is the coefficient of
the regression line. Total flavonoid content (TFC) was expressed as g of
quercetin equivalents (QE) /g of extract according to the formula by Chang et al.
(2002):
TFC = (c df v) / w
Where; c = concentration obtained from the standard curve,
df = dilution factor
v = volume of stock solution
w = weight of okra extract used in the experiment.

5.2.6 Statistical analyses of data


Microsoft

Excel, (ver. 2007) was used to

collate results of the

spectrophotometric analysis. They were evaluated for statistical significance


with one-way analysis of variance (ANOVA) and means separated by the
"

"

Fwpecpu" ownvkrng" tcpig" vguvu" *Uvcvitcrjkeu" Egpvwtkqp" ZXK." xgtukqp" 3803033."


USA) and expressed as the Mean SD (standard deviation of the mean) upon
three independent analyses. A p-value of 0.05 or less was considered as
statistically significant.

5.3 RESULTS
5.3.1 DPPH radical scavenging assay
Table 5.2 shows concentration of each extract (ethanol and aqueous) against
their percentage inhibition of the DPPH radical scavenger for all 25 accessions
of okra. The coefficient of regression (R2) of the logarithmic trend line was 0.94,
which implies that 94% of the variability in DPPH can be ascribed to the
concentration of the extracts. The analysis of variance (ANOVA) showed that,
concentration significantly (p = 0.05) affected percent inhibition. From the mean
eqorctkuqp."wukpi"vjg" Fwpecpu"ownvkrng"tcpig" vguv"*FOTV+."Citke"ujqtv"htwkv"
(3705.81g/ml), Asontem-GAR (506.49g/ml) gave the highest and lowest
significant DPHH inhibition for ethanol extract compared to the other
concentrations.

Again, the accessions, Kortebortor-ASR (56.33%) and Cape (14.27%) gave the
highest and lowest percent DPHH inhibition for the aqueous extract,
respectively. On solvent extraction basis, ethanol extract recorded a higher total
mean (1417.8613.1319g/ml) of antioxidant activity (AOA) compared to the
aqueous extraction which recorded an AOA value of 1022.1050.22g/ml. This
implies that, ethanol is a better solvent for assessing antioxidant activity in okra
compared to water.
"

"
"

Table 5.2: Free radical scavenging potential of 25 accessions of okra


Ethanol Extract

Aqueous Extract

Accession
Asontem-GAR
Asontem-ASR
Asante Type II

%Inhibition
16.74
18.53
32.38

Concentration
506.490.00j
665.020.13ij
1012.001.11fgh

%Inhibition
29.13
27.13
22.06

Concentration
906.690.00efgh
992.432.22efg
678.330.00hijklm

Asontem-ER

21.68

732.71.22hij

23.05

1074.27821.62

Wune Mana
Labadi
Agric Type I

54.060
31.97
27.70

3030.3869.31b
941.631.05fghi
1183.440.76def

48.47
17.94
17.43

2660.700.00b
514.140.00lmn
726.921.52ghijkl

Clemson Spineless
Volta
Agric Short Fruit

45.27
32.14
46.24

1885.32732.38c
1440.871.59de
3705.810.00a

21.65
23.95
25.94

877.830.00efghij
1060.731.59de
2039.890.00c

Debo'
Juaboso
Kortebortor-BAR

51.12
35.01
46.12

3812.160.00a
1206.490.61def
2001.2212.19c

50.84
23.47
20.96

3791.252.61a
796.161.22fghijk
883.920.00efghi

Legon Fingers
Indiana
Asontem-BAR
Mamolega
Nkran Nkuruma

34.89
55.97
44.80
51.15
28.99

1001.851.02fgh
1829.58483.00c
1195.87506.53def
1483.520.00d
763.450.94hij

21.51
24.90
23.71
15.09
18.18

605.542.03klmn
795.550.00fghijk
618.990.00ijklmn
415.5111.18n
467.970.94mn

Atomic
Cape'
Mapelega
Kortebortor-ASR

23.16
25.56
29.81
28.22

942.2213.17fghi
1135.490.00efg
766.310.91hij
643.360.41ij

23.84
14.27
30.43
56.33

971.482.93fg
612.194.77jklmn
782.770.00ghijkl
1309.570.81d

Yeji-Local
Cs-Legon
Asontem N V.

47.21
29.02
22.89

2050.140.15c
805.700.00ghij
705.491.66hij

16.60
25.39
28.01

690.341.52hijklm
700.900.98hijklm
870.660.55efghij

de

sd=standard deviation, mean with same letters in a column are not statistically differepv"*r2027+"
from each other ceeqtfkpi"vq"Fwpecpu"ownvkrng"tcpig"vguvu0
"

5.3.2 Total flavonoid and phenolic contents of okra accessions


Total flavonoid contents of the okra extracts measured by the Aluminium
chloride colourimetric method in terms of quercetin equivalent (QE) ranged
from 871.573.84g/g/QE (Cs-Legon) to 5159.2112.90g/g/QE (Agric short
"

"

"
"

fruit) and 122.482.69g/g/QE (Yeji-Local) to 2003.692.55g/g/QE (CsLegon) for the ethanol and aqueous extracts respectively.

The

mean

total

flavonoid

content

(TFC)

for

ethanol

extract

(2288.2027.75g/g/QE) was higher than that of the aqueous extract


(686.176.17g/g/QE) indicating that ethanol is a better solvent for extracting
total flavonoids from okra fruits than aqueous solvent.
"

In addition, there were statistically significant differences (p = 0.05, Table 5.3)


in TFCs recorded for both the ethanol and aqueous extracts of the accessions,
indicating variability with respect to TFCs among the different accessions under
study.

Phenolic contents measured by the Folin Cicalteu reagents in terms of gallic acid
equivalent

(GAE)

ranged

from

25.835.30g/g/GAE

(Debo)

to

8.010.37g/g/GAE (Kortebortor-ASR) for ethanol extract, while for aqueous


extract; Kortebortor-ASR recorded the highest (63.223.95g/g/GAE) with
Volta recording the lowest phenolic content (6.820.09g/g/GAE).

The

average

total

phenolic

content

(TPC)

for

ethanol

extract

(12.991.75g/g/GAE) was lower than the aqueous extract (21.090.99


g/g/GAE), demonstrating that water is a better solvent for extracting total
phenolics in okra pods than ethanol. Statistically significant differences (p =
0.05, Table 4.3) were recorded in both the ethanol and aqueous extracts of the
accessions.
"

"

"
"

Table 5.3: Contents of total flavonoids (g/g/QE) and phenolics (g/g/GAE)


in 25 accessions of okra
Flavonoids
Accession

Phenolics

Asontem-GAR

Concentration
(eth)
2264.980.00gh

Concentration
(aq)
492.899.56k

Concentration
(eth)
11.381.77defghi

Concentration
(aq)
20.940.00fg

Asontem-ASR

2338.878.31gh

493.0814.74k

13.182.30cdef

22.460.64ef

Asante Type II

1339.722.12n

465.122.16l

10.211.84efghi

19.661.39g

hi

efghi

19.760.87g

Asontem-ER

2235.140.89

Wune Mana

4980.501.23b

1449.241.28c

18.192.25b

8.580.84l

Labadi

1958.800.00jk

379.290.96n

9.020.28ghi

19.740.11g

Agric Type I

2420.543.36g

345.521.89o

12.3172.21cdefg

28.211.92d

330.210.94

532.772.11

10.631.20

9.691.17

fghi

27.082.00d

Clemson Spineless

1288.0692.91

Volta
Agric Short Fruit

3204.582.34e
5159.2112.90a

400.222.16m
487.6714.92k

14.592.54cd
24.124.64a

6.820.09l
39.460.15b

Debo'
Juaboso

3387.60499.67d
2086.821.55ij

1402.502.38d
648.163.00g

25.835.30a
11.791.47defgh

29.070.00d
20.161.93fg

Kortebortor-BAR

1515.503.88n

279.7233.92p

15.761.71bc

23.621.33e

efghi

16.920.61hi
19.970.00fg
16.380.00ij
14.251.17jk
16.540.36ij

Legon Fingers
Indiana
Asontem-BAR
Mamolega
Nkran Nkuruma

3601.420.65
1609.011.45mn
1517.550.60n
1181.910.45n
1940.7715.19 jkl

693.073.84
750.8328.82e
485.243.39k
517.912.51j
480.623.88kl

9.890.61
10.510.63efghi
8.551.01hi
12.231.26cdefg
8.890.54ghi

Atomic

988.532.15o

582.501.92i

13.422.22cde

19.560.24gh

Cape'
Mapelega
Kortebortor-ASR

3077.1811.14ef
1874.811.78kl
1774.3322.10lm

179.984.04q
1579.183.84b
1443.880.00c

10.183.27efghi
9.070.53ghi
8.010.37i

12.991.22k
32.275.40c
63.223.95a

Yeji-Local

1595.612.24n

122.482.69r

18.263.03b

13.370.75k

Cs-Legon
Asontem N V.

871.573.84

2993.192.93

2003.692.55
f

608.396.68

10.760.89

efghi

7.970.00l

18.350.79

8.330.00l

sd=standard deviation, mean with same letters in a column are not statistically difhgtgpv" *r2027+"
from each other ceeqtfkpi"vq"Fwpecpu"ownvkrng"tcpig"vguvu0, aq = aqueous, eth = ethanol.

"

"

5.4 DISCUSSION
5.4.1 Total flavonoid and phenolic contents of ethanoic and aqueous
extracts of Accessions of Abelmoschus spp
The mean total flavonoid contents (TFCs) were (2288.2027.75g/g/QE) for
ethanol extract and (686.176.17g/g/QE) for the aqueous extracts. The
antioxidant activity and total flavonoid contents were generally moderate,
affirming that ethanoic extraction method was more efficient. This also indicates
that, flavonoids in pods of okra are more soluble in organic solvents such as
ethanol

than

in

water.

The

highest

total

flavonoid

content

(5159.2112.90g/g/QE) was recorded by the accession, Agric short fruit while


Cs-Legon registered the lowest TFC of 2003.692.55g/g/QE in the ethanol
extract. This is however inconsistent with findings of Khomsug et al. (2010),
where he obtained 10.750.02mg/100g and 142.480.02mg/100g for pulped
okra and okra seeds respectively. Adelakun et al. (2009) obtained
32.5432.42mg/g for blanched okra seeds, 48.30.00mg/g for raw okra seeds,
51.28mg/g for soaked okra seeds who worked on okra seed flour in Nigeria and
Thailand. They both concluded that okra contain small amount of flavonoid.

This finding is however in consonance with findings of Achel et al. (2012)


working with ten leafy vegetable extracts in Ghana, Achoribo et al. (2012)
working on Bissap (Hibiscus sabdariffa) extract; Tibiri et al. (2010) working on
Entada africana organ extracts; Veerapur et al. (2007) working with Ficus
racemosa stem bark extracts; Pourmorad et al. (2006) with extracts of Iranian
medicinal plants; Silva et al. (2002) working with structure relationships of

"

flavonoids in ten plants; Chang et al. (2002) who also worked on total flavonoid
in propolis.
Specifically, flavonoids have variously been reported to substantially influence
antioxidant properties in mucilagenous vegetables (Mariani et al., 2008; Firuzi
et al., 2005; Choi et al., 2002; Rice-Evans et al., 1996).

On the other hand, average total phenolic contents were 21.090.99g/g/GAE


for aqueous and 12.991.75g/g/GAE ethanol extracts respectively. In spite of
the comparatively low yield for both extracts, the antioxidant activity and total
phenolic contents of aqueous extract for all the accessions were appreciably
good suggesting that the water extraction method was more efficient. The
highest

total

phenolic

content

was

registered

by

Kortebortor-ASR

(63.223.95g/g/GAE) while Volta had the lowest TPC of 6.820.09g/g/GAE


in the aqueous extract. Comparatively, Debo (25.835.30g/g/GAE) and
Kortebortor-ASR (8.00.37g/g/GAE) recorded the highest and lowest TPCs in
the ethanol extract respectively, attesting to the fact that phenolic compounds in
okra are more soluble in water than in organic solvents such as ethanol, and
hence corroborating the report of Owusu-Ansah (2010) working on Moringa
oleifera in Ghana.

The total phenolic composition of the accessions of okra compares well with
common fruits and vegetables noted for their relatively high phenolic
constituents such as cranberries (52.722.15mg/g), apple (29.630.64mg/g),
strawberries

(16.000.12mg/g),

pineapple

(9.430.15mg/g),

banana

(9.040.32mg/g), lemon (8.190.35mg/g), orange (8.120.11mg/g), pear


(7.060.16mg/g), and grape (4.960.26mg/g) (Weng et al., 2005; USDA, 1998).
"

A number of authors have reported a strong correlation between antioxidant


activities of plant extracts and their phenolic compounds content (AbdelHameed, 2009; Arcan and Yemenicioglu, 2009; Almela et al., 2006). From the
analysis of variance, statistically significant differences were observed among
the 25 accessions of okra in terms of total phenolic contents. This may be
attributed to the type of accession, and other polyphenols present that may not
have been determined in this experiment. Thus, TPCs may have been
determined for the accessions without identification of their distinct individual
molecules and other related compounds.

5.4.2 Total antioxidants activity of 25 accessions of Abelmoschus spp


Total mean percent inhibition of DPPH radical for ethanol extract was 35.27%
and 30.73% for the aqueous extract of the accessions. Indiana registered the
highest percent DPPH inhibition of 55.97% with a correspondent antioxidant
activity of 1829.58438.00mg/g, while Asontem-GAR (16.74%) gave the lowest
percent DPPH inhibition and the least antioxidant activity (506.490.00mg/g) in
the ethanol extract. Similarly, the accessions Kortebortor-ASR (56.33%,
1309.570.81mg/g) and Cape (14.27%, 612.194.77mg/g) recorded the highest
and lowest percent DPPH inhibition and antioxidant activity from the aqueous
extract, respectively.
The extracts of the okra accessions showed promising free radical scavenging
potential of DPPH in a dose or concentration dependent fashion. This
demonstrates that the ethanol extracts of the accessions better reduce or more
markedly inhibit the free radical DPPH than the aqueous extracts. This also
"

suggests that compounds present in the okra accessions responsible for


scavenging the free DPPH radicals have high solubility in ethanol than in water.
This is consistent with results of Chakrabortthy and Ghorpade (2010), Apea-Bah
et al. (2009) and Wu et al. (2009) working with Abutilon indicum L. sweet stem
extracts, gambir (Uncaria gambir), Pigeon pea (Cajanus cajan L.) leaves,
respectively.

The relatively high radical scavenging impact on the DPPH radicals may be
attributed to high flavonoid and phenolic contents in the extracts of the
accessions (Maltas and Yildiz, 2012; Maltas et al., 2011). There was however, a
discrepancy observed between the ethanol and aqueous extracts, in that, not all
accessions with high TFC(s) or TPC(s) necessarily gave a corresponding higher
scavenging activity, corroborating earlier reports of Owusu-Ansah (2010) for
Moringa, Quartey (2010) for tomatoes and Prior et al. (2005) for dietary
supplements.

5.5 CONCLUSION
The high carcinogenicity nature of synthetic antioxidants has enkindled
considerable interest in finding naturally occurring antioxidants for use in foods,
cosmetics or medicinal materials as possible substitutes for the synthetics (Sasaki
et al., 2002). High antioxidant foods have greater potential to reduce free radicals
in the body than do low antioxidant foods. It is therefore, imperative to ascertain
the antioxidant content of foods such as okra, in addition to knowing the basic
nutritional information such as the vitamin, fibre, fat, mineral and protein
contents (Prakash et al., 2010).
"

From the results above, it can be concluded that;


I.

Okra possesses appreciably high amounts of total flavonoids, moderate


content of phenolics and a good source of natural antioxidants, which will inure
to the health of consumers.

II.

There was high variability with respect to TFCs, TPCs and total

antioxidant activity in the various accessions used in the study.


III.

Ethanol as an extraction solvent may yield higher total antioxidants

activity in okra than in water. Breeders can also explore the possibility of
breeding for high phenolic and antioxidants containing varieties. The strong free
radical scavenging activity of Abelmoschus spp requires further studies for the
isolation and identification of other bioactive compounds inherent in this
species.

5.6 RECOMMENDATIONS
I.

Biochemical screening for secondary metabolites and specific individual

compounds such as saponins, and their structures conferring the antioxidants


activity via high performance liquid chromatography (HPLC), thin layer
chromatography (TLC) and liquid chromatography mass spectrometry (LCMS)
facilities ought to be undertaken to fully exploit the biochemical wealth of this
vegetable to human health.
II. Mariani et al. (2008); Firuzi et al. (2005); Choi et al. (2002); Rice-Evans et al.
(1996) have all noted that flavonoids substantially influence antioxidant
properties in mucilagenous vegetables. The relation between antioxidants and
mucilage and their effects on or interference with the nutritional composition of
Abelmoschus spp should be investigated.
"

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"

"
"

CHAPTER SIX
"

DETERMINATION OF ESSENTIAL MINERAL ELEMENTS AND


MUCILAGE IN SOME ACCESSIONS OF OKRA (Abelmoschus spp L).
"

6.1 INTRODUCTION
6.1.1 Okra Production in the world
Okra, (Abelmoschus spp L.), is prominently featured in nearly every market in
Africa. In Ghana, it is the fourth most popularly patronised vegetable after
tomatoes, pepper and garden eggs (Oppong-Sekyere, 2011; Lamont Jr., 1999;
Sinnadurai, 1973). The world okra production, as of 2010, was estimated at 6.9
million tonnes with Asia leading the production by 75.92% followed by Africa
(23.41%) and the Americas (0.54%) (Table 6.1) (FAOSTAT, 2012). On the
whole, India is the leading producer of okra with over 70% of the total world
production, followed by Nigeria, Pakistan, Sudan, Ghana, Egypt and Iraq
(FAOSTAT, 2012; Gulsen et al., 2007).
"
Table 6.1: Estimated production of okra in selected geographical regions and countries (metric
tonnes)
Year

World

Africa

Asia

Americas

Ghana

Nigeria

Sudan

India

USA

2010

6917062

1619525

5251415

37122

82500

955600

256000

4803000

8500

2009

6570901

1524558

4985571

51612

71350

826170

252000

4528000

9825

2008

6400222

1722399

4611226

57534

89731

1039000

249000

4179000

9673

2007

6533044

1970326

4495866

58352

108000

1280000

216950

4070000

10000

2006

6191495

1669583

4447523

62839

105000

1000000

168000

3974600

9500

Source: FAOSTAT, (2012).

"

"

6.1.2 Patronage and Consumption of okra


Okra is usually consumed fresh in sauces and soups. It is used by the growing
fast food, hotel and restaurant industry in Asia, the Americas, Africa and other
parts of the globe (Lamont Jr., 1999; Tyler et al., 1989), just like any other
vegetable. Okra is consumed almost daily and traded by a broad range of market
participants worldwide.

In Ghana, okra is traded in the fresh state in almost all markets during the rainy
season and in a dehydrated form during the dry season. It is particularly found in
Northern Ghana due to its tremendous commercial potential for resourceconstrained women farmers and its vital importance as a component of various
recipes (Oppong-Sekyere et al., 2012; Torkpo et al., 2006). The pods, which
possess unique flavour and texture, are boiled and added to soups, or sliced and
fried. They may be used alone or mixed with other vegetables in local
preparations.

Mucilage from fruits of okra is known to be a good thickening agent for gravy.
In West Africa, young pods are thinly unkegf" vq" rtgrctg" qmtc" uqwr." c" rgthgev"
partner of hwhw" *Mwoct" et al., 2010; National Academies Press, 2006). Gums
stay intact inside the pods when sliced and dried and remain useful for
flavouring and thickening foods. Okra is sometimes used in place of eggplant in
many recipes in some parts of Asia (Baytop, 1984). The seeds of mature pods
are used for animal feed and for production of edible oil (Oyelade et al., 2003;
Oyenuga, 1969; Busson, 1963).

"

"
"

In West Africa, okra pods are often sliced, sun dried, and ground into powder
for use in the lean season (hungry time) that hits each year just before the new
harvest (Akintoye et al., 2011; Kumar et al., 2010; National Academies Press,
2006). Young immature okra pods can be consumed in different forms
(Ndunguru and Rajabu, 2004), and in variety of cuisines in Asia and Africa. In
the Caribbean islands, okra is cooked up and eaten as soup, often with fish
(www.whfoods.com/genpage.php?pfriendly=1&tname=foodspice&dbid=128).
The pods are generally cut into small circular sections and the immature leaves
are utilised in local cuisines in Nigeria (Ayodele, 1993), and also eaten raw in
salads

in

Papua

New

Guinea,

the

Caribbean

and

Malaysia

(www.whfoods.com/genpage.php?pfriendly=1&tname=foodspice&dbid=128).
In Turkey, the pods are strung out to dry for use in winter and the dried seeds are
used as substitutes for coffee (Calisir et al., 2005; Moekchantuk and Kumar,
2004). The leaf buds and flowers are also edible (Doijode, 2001). Locally, okra
is commonly used in soups and stews. It is also used as a sieving agent in
dtgykpi"qh"rkvq"*cp"kpfkigpous wine) in northern Ghana.

6.1.3 Proximate composition of okra


The nutritional composition of ripe seed of okra has been reported to contain
edible oil (20%), calcium (92mg/100mg), vitamin C (25mg/100g), 2.0% protein,
vitamin A (0.2mg/100g) (Dilruba et al., 2009; Arapitsas, 2008; Mays et al.,
2007; Gopalan et al., 2007; Owolarafe and Shotonde, 2004; Lamont, 1999).
Approximately, the edible portion of the fruit contains 88.6 g of water, 36 kcal
energy, 2.10 g protein, 8.20 g carbohydrate, 0.20 g fat, 1.70 g fibre, together
with small amounts of -carotene (3:7022" i), riboflavin (0.08 mg), thiamine
(0.04 mg), niacin (0.60 mg), ascorbic acid (47.00) (Kolawole et al., 2011;
"

"

Saifullah and Rabbani, 2009; Kahlon et al., 2007). Okra is also a good source of
vitamin B, some essential minerals (Lamont, 1999), rhamnose (22%),
galacturonic acid (27%) and amino acids (11%) (Benchasri, 2012a).

The composition of okra leaf per 100 g edible portion is: 81.50 g water, 56.00
kcal energy, 4.40 g protein, fat 0.60 g, carbohydrate11.30 g, fibre 2.10 g,
ascorbic acid 59.00 mg, -carotene 5:7022" i, thiamine 0.25 mg, riboflavin 2.80
mg, niacin 0.20 mg (Benchasri, 2012a; Varmudy, 2011; Gopalan et al., 2007).
Carbohydrates are mainly present in the form of mucilage (Kumar et al., 2009;
Liu et al., 2005).

6.1.4 Essential mineral elements in okra


Essential mineral elements are elements that are indispensable to the human
body and yet cannot be produced by the body. These essential elements in the
body can be categorised into two main classes; namely macro elements or major
minerals, and microelements or the trace or minor minerals. Macro elements are
elements required in appreciably high or large quantity. Examples include
Calcium (Ca), Potassium (K), Magnesium (Mg), Phosphorus (P). Micro or trace
elements on the other hand are required in minute quantities. Zinc (Zn), Iron
(Fe), Copper (Cu), Manganese (Mn), Chromium (Cr), Selenium (Se), Iodine (I),
and Boron (B) are all examples of essential trace or microelements.
However, K, Na, Mg and Ca are the principal elements in the pods of okra
(Kumar et al., 2010). Presence of Fe, Zn, Mn and Ni has also been reported
(Benchasri, 2012a; Saifullah and Rabbani, 2009; Moyin-Jesu, 2007; Kahlon et
al., 2007). The pod skin (mesocarp) and seeds are excellent sources of zinc (80
"

"
"

mg/g) (Cook et al., 2000; Glew, 1997). The leaves are also known to contain Ca,
P, and Fe (Benchasri, 2012a&b; Varmudy, 2011; Gopalan et al., 2007).
6.1.5 Significance of mineral elements to human
Manganese helps the body form connective tissues, bones, blood clotting
factors, and sex hormones. It also plays a role in fat and carbohydrate
metabolism, calcium absorption, and blood sugar regulation. Manganese is also
integral to normal brain and nerve function. In addition, it is a component of the
antioxidant enzyme superoxide dismutase (SOD), which helps combat free
radicals

in

the

body

(http://www.umm.edu/altmed/articles/manganese-

000314.htm#ixzz1zoHTO27T). Okra has been identified as one of the rich


natural sources of Mn (Ismail et al., 2011). Reported daily dietary intake is 25mg per day (Namlk and Yavuz, 2006; Institute of Medicine, 2001; Dietary
Reference, 1991).

Okra pod contains appreciably high amount of calcium, approximately 90.00


mg/100g (Benchasri, 2012a; Kahlon et al., 2007). Adults aged 19 and older
require 1,000 mg of calcium each day. Calcium is required for vascular
contraction and vasodilation, muscle function, nerve transmission, intracellular
signalling and hormonal secretion, though less than 1% of total body calcium is
needed to support these critical metabolic functions (National Academies Press,
2010). It is also important in blood clotting process. Calcium deficiency is
associated with rickets in young children, osteoporosis and osteomalacia
(softening

of

bones)

in

adults

and

(http://ods.od.nih.gov/factsheets/Calcium-HealthProfessional).

"

"

older

people

Magnesium facilitates protein synthesis, muscle contraction, and breakdown of


glucose into energy. Fifty to sixty percent of magnesium is stored in the bones.
A magnesium deficiency can lead to muscle weakness and soreness. Okra is a
good source of magnesium and helpful for reducing the severity of asthma,
lessening the frequency of migraine and headaches, lowering high blood
pressure,

and

reducing

the

risk

of

heart

attack

and

stroke

(http://www.whfoods.com/genpage.php?pfriendly=1&tname=foodspice&dbid=1
28). The daily requirement of magnesium for children between the ages of 1-10
spans from 150 to 250 mg, 350 mg for males between the ages of 11 and 14
years. Males between the ages of 15 and 18 require 400 mg while all females
require 300mg. Pregnant women and nursing mothers however, require 450 mg
of

Mg

(http://www.rawfoodexplained.com/minerals/the-minerals-in-the-

body.html).

Copper combines with certain proteins to produce enzymes that act as catalysts
to help a number of body functions. It is also involved in the transformation of
melanin for pigmentation of the skin and helps to form cross-links in collagen
and elastin and thereby maintain and repair connective tissues. This is especially
important for the heart and arteries (Dietary Reference, 1991). Research
suggests that copper deficiency is one factor leading to an increased risk of
developing coronary heart disease. Copper, along with iron, helps in the
formation of red blood cells. It also helps in keeping the blood vessels, nerves,
immune

system,

and

bones

healthy

(http://www.copper.org/consumers/health/papers/cu_health_uk/cu_health_uk.ht
ml). It helps iron-rich foods to form red haemoglobin in the blood. In fact, it is
essential for the normal healthy growth and reproduction of all higher animals.
"

"
"

The daily requirement of copper for both males and females is 0.4-1.2 mg/day
(Dietary Reference, 1991).

Fresh edible portion (100g) of okra contains approximately 20mg of potassium.


Average daily requirement of potassium is 2000mg (2g) for both males and
females (Dietary Reference, 1991) and for children, between the ages of 2 to 9
require 1400mg to 1600mg. Potassium is crucial to heart function and plays a
key role in skeletal and smooth muscle contraction, facilitating normal digestive
and muscular functions. It also assists in the regulation of the acid-base balance
and in protein synthesis from amino acids as well as in carbohydrate metabolism
(WHO, 2003; USDA, 2002). It is also required in the body for the proper
functioning of all cells, tissues, and organs in the human body. It is also an
electrolyte, a substance that conducts electricity in the body, along with sodium,
chloride, calcium, and magnesium.

Chlorine (Cl) exists primarily as chloride anion that interacts with cations such
as sodium to produce salt (table salt / sodium chloride), and with hydrogen to
produce stomach acid (hydrochloric acid). It is an essential nutrient in human
diet and is necessary for healthy nervous and digestive systems. It is required for
protein digestion (pepsin), Vitamin B12 absorption (intrinsic factor), and
absorption of metallic minerals (http://www.acu-cell.com/fcl.html).

Sodium is one of the essential elements required in an appropriate amount in


daily diet to regulate blood pressure and the nervous system for transmitting
signalling pathway of the body to stabilise water in our cells. It is an important
"

"

"
"

extracellular cation to stabilise extracellular fluid. Low levels of Na can lead to


confusion and seizures (Sagnella et al., 1989). The body responds to increased
Na by expanding the space for extracellular fluid and thus, inducing thirst.
Recommended daily intake of Na is 2400-5175mg/day (Dietary reference, 1991;
Sagnella et al., 1989). Vegetables including okra (Benchasri, 2012a) and fruits
possess considerable amount of Na (Ismail et al., 2011). After renal filtration,
excess Na is excreted from the body. Na and K are responsible for maintenance
of total body fluid required in acid-base equilibrium as well as osmotic
regulation in the body (Ismail et al., 2011; Dietary reference, 1991).

Bromine is important for the pituitary gland, whose hormones profoundly


influence

brain

functioning

and

the

production

of

sex

hormones

(http://www.cancernaturaltherapyfoundation.org/health-articlesview.php?id=12). It has also been associated with anti-seizure properties, and it


is an effective trace mineral in the treatment of hyperthyroid conditions
(http://www.acu-cell.com/br.html).

Aluminium is present in only minute amount in plant and animal tissues and it is
commonly ingested in foods and in medicines. It is found in the lungs, kidneys,
brain, liver, and thyroid. The daily intake of aluminium may range from 10-110
mg, but most of this is eliminated from the body through faeces, urine and some
in sweat. More aluminium is stored, particularly in the bones when there is
decreased kidney function (http://www.healthy.net/scr/article.aspx?Id=1958).
Recommended daily dietary intake of Al is 2.0mg/kg/day (Dietary Reference,
1991).

"

"

"
"

6.1.6 Mucilage
Mucilages are water-soluble polysaccharides inherent in several plant species
and in some microorganisms (National Academies Press, 2006; Woolfe et al.,
1977). Mostly plants belonging to the Malvaceae family and other related
species such as baobab (Adansonia digitata L.), cotton (Gossypium spp),
ambrette (Abelmoschus moschatus Medik.), kenaf (Hibiscus cannabinus),
plantago (Plantago psyllium), aloe (Aloea vera), cocoa (Theobroma cacao),
kapok (Ceiba pentandra), roselle (Hibiscus sabdariffa) and okra (Abelmoschus
spp) are known for their considerable mucilage content (Ahmad et al., 2009;
Cvancara, 2001). Mucilage in okra can however be found in almost all parts of
the crop (Kumar et al., 2010; National Academies Press, 2006). The immature
pods, seeds, leaves, stems, barks, roots, flowers and even the cell walls of the
fruits are noted to harbour considerable amount of mucilage (Sengkhamparn et
al., 2009; Woolfe et al., 1977).

The mucilage and gums in plants are known to aid in water storage, decrease
diffusion in the plant, aid in seed dispersal and germination, and act as a
membrane thickener and food reserve. Plant gums and mucilage, in particular,
have generated tremendous interest due to their varied pharmaceutical
applications, among other uses as diluents, bio-binders, tablet disintegrants,
protective colloids in suspensions, thickeners in oral liquids, gelling agents in
gels, gum substitute, effluents in rheological engineering and bases in
suppository (Okoye et al., 2009; Tomoda et al., 1987; Woolfe et al., 1977).

"

"

The rheological behaviour of okra mucilage is pseudoplastic (Gurbuz et al.,


2003), and can be used to enhance viscosity and stability in many food products,
for example, as stabiliser in ice cream, sauce and salad dressing (Nussinovitch,
1997), thus making it an ideal substitute for otherwise costly synthetic and semisynthetic excipients. In Ghana and some parts of West Africa, mucilage is used
to impart desired slimy consistency to local soups and stews.

Mucilage from okra also contains appreciable levels of protein, carbohydrate,


neutral sugars, minerals and other complex polysaccharides (Ndjouenkeu et al.,
1996; Woolfe et al., 1977). Its viscosity decreases markedly with high
temperature, but this attribute does not prevent its use for culinary preparations.
Some consumers prefer varieties with high mucilage content for thickening
soups and stews (Siemonsma and Kouame, 2004), while others see the mucilage
as a nuisance; hence, prefer low mucilage containing varieties for culinary
preparations or in cuisines. The slimy consistency facilitates swallowing and
transport of macerated food via the oesophagus by lubricating the peristaltic
valve, relieves gastrointestinal discomfort, and prevents constipation (Martin
and Ruberte, 1998).
The mucilage found in okra has been associated with anticancer, antimicrobial,
hypoglycaemic, anti-ulcer activities (Pieroni et al., 2005; Gurbuz et al., 2003), as
well as the ability to bind cholesterol and bile acid carrying toxins by filtering
the liver (Shui and Lai Peng, 2004; Tomoda et al., 1987). Okra mucilage is an
acidic polysaccharide composed of galacturonic acid, rhamnose, and glucose
(1.3: 1.0: 0.1). It attains maximum viscosity at neutral pH, and tends to break
down under intense heat (Haydar et al., 2011; Sengkhamparn et al., 2009;
Woolfe et al., 1977).
"

"
"

In recent years, chemical modification of natural macromolecules, especially


polysaccharides, has received considerable attention (Ameena et al., 2010).
Polysaccharides are unique raw materials as they are from renewable sources,
widely and readily available in many countries, inexpensive, stable, hydrophilic
and modifiable biopolymers (Mishra et al., 2008). They offer tremendous
potential for development of alternate materials (Ameena et al., 2010; Singh et
al., 2009; Kumar et al., 2009; Okieimen, 2003; Chauhan et al., 2002).
"

6.1.7 The Instrumental Neutron Activation Analysis (INAA) Method


Instrumental Neutron Activation Analysis (INAA) is one of the most sensitive
analytical techniques used for quantitative multi-element analysis of major,
minor, and trace elements in samples from almost every conceivable field of
scientific or technical interest. The technique of INAA measures the total
amount of an element present in sample matrices without regard to chemical or
physical

form

and

without

any

pre-treatment

of

the

sample

(www.ne.ncsu.edu/nrp/naa.html#ag).
For certain elements, INAA offers sensitivities that are superior to those possible
by other techniques; approximately parts per billion or better. In addition to the
elemental and isotopic analysis of samples, INAA permits the analysis of nonradioactive tracers introduced into biological, chemical, and/or industrial
processes for process identification and optimisation. It is non-destructive and
therefore, does not suffer from the errors associated with yield determinations; it
has very high sensitivities for most of the elements that can be determined by
INAA - most detection limits span from ~0.05 to ~50 ppm ( 1 ppb for some
high-purity materials). It is highly precise and accurate; permits the analysis of
"

"

samples ranging in volume from 0.1 ml to 20 ml, and in mass from ~0.001 gram
to 10 grams depending on sample density. Samples for INAA can be solids,
liquids, gases, mixtures, and suspensions, hence the choice of this technique for
this study.

6.1.8 Objective of the study


The objective of the study was to assess the composition of essential mineral
elements and the level of mucilage in the various accessions of okra. The
specific objectives were:
i.

To investigate the mineral element composition of fresh fruits (pods) of


some accessions of okra (Abelmoschus spp)

ii.

To determine any association between pairs of essential minerals found in

the fruits of okra


iii.

To assess the amount of mucilage in fresh fruits of the various accessions

of okra

6.2 MATERIALS AND METHODS


6.2.1 Experimental site
The experiment was conducted at the Ghana Research Reactor 1 (GHARR-1),
the Nuclear Agriculture Centre (NAC) and the Radiation Technology Centre
(RTC) of the Ghana Atomic Energy Commission, Kwabenya (Accra), Ghana.
The laboratory study of mucilage was done at the physico-chemical laboratory
of the Radiation Technology Centre (RTC).
"

"
"

6.2.2 Technique for Analysis of Mineral Elements


Instrumental Neutron Activation Analysis (INAA) was employed for the
determination of variability in composition of mineral elements concentration in
the pods of 22 okra accessions. Pods of the remaining accessions were not
available for analysis, as the plants were late flowering/fruiting.

6.2.3 Sample preparation for elemental analysis


Five (5) pods of each of the 22 accessions were sliced into several pieces, and
put into plastic bowls, and lyophilised (freeze-dried) for 48hrs in a CHRIST
FREEZE DRYER before blending into fine powder. Samples were pulverised
and put into clean plastic containers and shaken thoroughly to ensure uniformity
before weighing. Two hundred milligram (200mg) of each sample (accession)
was weighed into small polyethylene vials, labelled and hot sealed. The
polyethylene capsules of diameter 1.2cm and height 2.35cm containing the
samples were in turn, each put into a bigger polyethylene capsule of diameter
1.6cm and height 5.5cm (Rabbit capsule). Thus, doubly encapsulated and
smoothly heat-sealed with a soldering rod. Standard Reference material 1547
Peach Leaves from the National Institute of Technology and 1547 Citrus Leaves
and blanks were equally prepared in the same manner as the test samples. Each
sample was then irradiated by means of the Ghana Nuclear Research Reactor
(GHARR-1) for 1 minute to detect short radionuclides. This was immediately
followed by quantification of the samples.
"
"
"
"

"

"

6.2.4 Sample irradiation and counting


Samples and controls were irradiated in the Ghana Research Reactor (GHARR1) at the Ghana Atomic Energy Commission, operating at 15 KW at a thermal
flux of 51011 n.cm -2.s-1. Samples were transferred into irradiation sites via
pneumatic transfer system at a pressure of 0.6 Mpa. The irradiation was
categorised according to the half-life of the element of interest. For, 27Mg, 66Cu,
and 38Cl samples were irradiated for two minutes and counted for ten minutes.
For medium lived radionuclides like K, Na, samples were irradiated for one hour
and delayed for 24hrs with ten minutes counting. After the irradiation,
radioactivity measurements of induced radionuclide were performed by a PCbasef" -ray spectrometry set-up. This consisted of an n-type HPGe detector
coupled to a computer based multi-channel analyser (MCA) via electronic
modules.

The relative efficiency of the detector is 25% with energy resolution of 1.8 keV
cv"c" -ray energy of 1332keV of

60

Co. Through appropriate choice of cooling-

vkog."fgvgevqtu"fgcf"vkog"ycu"eqpvtqnngf"vq"dg"nguu"vjcp"32'0" Identification of
-ray of product radionuclide was done through the energies and quantitative
analysis of the concentration was achieved wukpi" vjg" -ray spectrum analysis
software, ORTEC MEASTRO-32.

6.2.5 Sample preparation and extraction of mucilage


Fresh okra fruits were harvested at edible maturity stage in the early morning
hours of each day of the experiment. The peduncles and other undesirable

"

"
"

structures on the harvested pods were cut or peeled off and then washed off any
debris.
Three hundred and fifty grammes (350g) of freshly harvested pods of okra were
thoroughly washed, sliced and chopped. Four hundred and fifty millilitres
(450ml) of distilled water was added and blended for 30 seconds. This was
topped up to 900ml of distilled water and then filtered using a muslin cloth to
extract the mucilage. Three hundred and fifty millilitres (350ml) of distilled
water was added to rinse and also to prevent suspended colloidal solid seepage
and speed up to ease the release of the mucilage from the supernatant.

6.2.6 Determination of mucilage contents


The extracted mucilage was analysed using the Brabender Viscograph-E.,
(USB). This instrument is able to give measurements of viscosity at different
temperatures, during both heating and cooling at different times of running. Four
hundred and fifty grammes (450g) of the extracted sample was measured into
Erlenmeyer flask and transferred to the measuring bowl of the instrument. Based
on the moisture content of the sample, the equipment corrects the moisture to
zero and automatically calculates what quantity in terms of weight of the sample
to be taken and the volume of water to be added to the sample to form a
unwtt{0"
The instrument has a measuring range of 700cm/g. It was programmed to run at
three different temperature regimes. The tests were programmed to start at 50oC
with heating at the rate of 5oC per minute up to a temperature of 95 oC. This
temperature was held constant for 15 minutes, then cooled at the rate 5oC to a
temperature of 55oC and held constant for another 15 minutes. During the test,
"

"

the spindles were programmed to revolve at a speed of 75 revolutions per


minute (rpm). The resistance encountered by the spindles within the measuring
bowl was electronically conveyed to the measuring head, which was plotted
electronically into the software interface. The result was generated electronically
on user interface in the form of a graph and summarised, tabulated values for
viscosities and temperatures at various times.

6.2.7 Data analyses


The statistical design employed was the completely randomised design (CRD)
and replicated three times for each accession. Microsoft Excel, (ver. 2007) was
used to collate results of the Brabender readings. They were evaluated for
statistical significance with one-way analysis of variance (ANOVA) and means
ugrctcvgf" d{" vjg" Fwpecpu" ownvkrng" tcpig" vguv" *Uvcvitcrjkeu" Egpvwtkqp" ZXK."
version 16.1.11, USA, 2010) and expressed as the Mean upon three independent
analyses. A p-value of 0.05 or less was considered as statistically significant.

6.3 RESULTS
6.3.1 Concentrations of Nine Essential Elements in Accessions of
Abelmoshus spp L.
Concentrations of five essential macro elements (Na, Mg, Ca, K and Cl), three
micro elements (Al, Mn and Cu) and one trace element (Br) detected by
instrumental neutron activation analysis (INAA) in 22 okra accessions are
shown in Table 6.2. Asontem-BAR registered the highest content (61.529.23
mg/kg) of Mg while Asontem NV had the least (40.096.01 mg/kg) Mg
"

"
"

concentration. Similarly, the accession Nkran Nkuruma registered 97.4314.61


mg/kg of K with Indiana recording the least (13.422.01 mg/kg) of K. AsontemGAR recorded the highest Ca content of 96.2114.43 mg/kg; while Yeji-Local
however registered the least (10.121.52 mg/kg) Ca content. Ca concentration
for all the accessions was high compared to all the other macro elements.

In addition, the elements Al, Na, Mg, Br, Cu and Cl detected using the technique
of Instrumental Neutron Activation Analysis (INAA), varied considerably
among all accessions under study. Mamolega recorded the highest concentration
(778.511.60 mg/kg) of Na whilst Asontem-ASR registered the least
concentration (16.322.45 mg/kg) of Na. Most of the accessions recorded fairly
high concentration of Na. Mamolega also recorded the highest Al concentration
(77.4112.31 mg/kg) while the least Al concentration was recorded by Indiana
(31.164.67 mg/kg). On the other hand, Indiana registered the highest Mn
concentration (36.355.45 mg/kg) with the least registered by Kortebortor-ASR
(11.002.49 mg/kg). Similarly, Cape gave the highest Cu concentration
(424.563.67 mg/kg), while Kortebortor-ASR once again, recorded the lowest
(85.2612.79 mg/kg).

With the exceptions of the accessions Asontem-BAR, Labadi, Juaboso, and


Atomic, the concentrations of Cu could not be detected for the rest of the
accessions as the levels were below the detection limit (0.01mg/kg) of the INAA
for copper. Finally, no single accession recorded the highest concentration for
all the elements analysed though some accessions such as Asontem-BAR which
recorded the highest concentrations of Mg and Cl, also recorded appreciably
high
"

concentration

of

other
"

elements

analysed.

"
"

Table 6.2: Concentration of Nine Mineral Elements in the Accessions of Okra in mg/kg*
Accession
Asontem-ASR

Macro Elements
Mg
54.758.21

Micro Elements
Ca

Cl

Na

Br

23.803.57

89.2613.39

61.539.23

16.322.45

15.542.33

Al
33.805.41

Mn
15.562.93

Cu
< 0.01

Asontem NV

40.096.01

16.042.41

91.9813.79

81.3912.20

190.128.52

29.964.49

47.967.67

15.682.99

< 0.01

Agric Type I

45.096.76

27.214.08

82.2512.34

54.818.22

189.028.24

20.013.00

32.845.29

14.592.95

< 0.01

Asontem-BAR

61.529.23*

19.812.97

11.131.67

88.1913.23*

191.528.25

31.644.75

37.175.95

14.452.77

186.527.98

Atomic

48.537.28

16.972.55

76.9111.54

36.845.53

362.654.39

51.867.78*

37.345.94

16.473.12

165.624.84

43.966.59

76.4311.46*

73.0910.96

51.377.71

221.4133.21

41.626.24

51.398.17*

17.073.12

< 0.01

Cape

54.708.21

19.482.92

11.711.76

10.851.63

385.657.16*

36.205.43

38.086.13

25.214.84*

424.563.67*

Asante Type II

50.107.52

19.072.86

90.9313.64

71.2810.69

279.340.99

25.653.85

37.766.04

17.073.28

< 0.01

Juaboso

59.598.94

19.622.94

11.691.75

83.4412.52

218.332.75

31.424.72

35.265.64

24.014.29

263.439.51

Labadi

55.768.36

23.143.47

10.631.59

85.5212.83

316.7847.52

27.434.11

41.396.62

20.483.63

276.441.46

46.867.03

21.143.17

94.2314.13*

43.016.45

170.025.50

36.105.42

36.535.88

11.002.49

85.2612.79

Mean

49.997.49

32.064.81

64.319.64

64.739.71

31942.97

34.415.25

42.456.77

17.83.33

63.729.56

CV (%)

23.01

17.33

187.74

269.75

6.14

15.73

60.55

193.13

31.97

RDI

320-420

2000

500-1200

100-500

2400-5175

2-5

2-5

0.4-1.2

Clemson
Spineless

KortebortorASR

"
*Values bolded and asterisked refers to the highest concentration for a particular element; Bolded values represents the lowest concentration for a particular
element. refers to standard error of the mineral element concentration in the accession. CV = Coefficient of Variation; RDI = recommended daily intake:
(Source = Dietary Reference, 1991).

"

"

"
"

Table 6.2 *Eqpvf+*:

"
Macro Elements

Accession

Mg

Micro Elements

Ca

Na

Br

Al

Mn

Cu

46.276.94

521.3278.20

18.452.77

31.164.67

36.355.45*

< 0.01

88.3813.26

72.5410.88

223.433.51

34.755.21

70.1111.15

17.273.51

< 0.01

90.6213.59

65.089.76

570.785.61

34.675.20

43.576.93

15.502.88

< 0.01

52.897.93

336.4250.46

53.187.98

34.695.59

15.892.86

< 0.01

90.913.63

95.4814.32*

778.511.60*

104.615.69*

77.4112.31*

18.033.55

< 0.01

89.4913.42

58.068.71

171.125.67

22.673.40

37.896.06

13.882.58

< 0.01

96.2114.43

93.7814.07

590.488.56

25.723.86

35.965.75

20.353.81

< 0.01

18.312.75

72.5210.87

62.119.32

277.041.55

38.425.76

42.506.80

13.312.64

< 0.01

92. 1413.82

10.341.55

69.1710.37

205.2630.79

21.562.23

42.726.88

15.532.92

< 0.01

13.422.01

68.3910.26

Cl

Indiana

44.576.69

Wune mana

50.967.64

23.333.49

Legon Fingers

48.957.34

15.322.30

Nkran

51.277.69

97.4314.61*

84.7912.71

Mamolega

50.917.64

40.796.12

Kortebortor-

51.477.72

20.093.01

Asontem-GAR

47.597.14

17.882.68

Asontem-ER

42.956.44

Agric

48.577.29

Nkuruma

BAR

Short

Fruit
Yeji-Local

55.718.36

65.479.82

10.121.52

78.8511.83

387.8258.17

46.787.02

50.217.98

22.774.08

< 0.01

Volta

45.946.89

18.452.77

68.6310.29

61.109.17

415.2662.29

28.654.30

38.116.10

11.632.61

< 0.01

Mean

49.997.49

32.064.81

64.319.64

64.739.71

31942.97

34.415.25

42.456.77

17.83.33

63.729.56

CV (%)

23.01

17.33

187.74

269.75

6.14

15.73

60.55

193.13

31.97

RDI

320-420

2000

500-1200

100-500

2400-5175

2-5

2-5

0.4-1.2

"
*Values bolded and asterisked refers to the highest concentration for a particular element; Bolded values represents the lowest concentration for a particular
element. refers to standard error of the mineral element concentration in the accession. CV = Coefficient of Variation; RDI = recommended daily intake:
(Source

"

Dietary

"

Reference,

1991).

"6.3.2:

Association of Essential Mineral Elements.

Table 6.3 shows the association between pairs of elements in the accessions
analysed. Aluminium showed strong association with Bromine and moderate
association with four other elements but was negatively associated with
manganese, copper and magnesium. Magnesium showed strong association with
copper. Manganese exhibited moderate association with copper, sodium and
magnesium but was weakly or negatively correlated with bromine, potassium,
calcium and chlorine. Copper registered a strong positive association with
magnesium but weakly correlated with the rest of the mineral elements detected
among the accessions. Bromine gave a moderate positive association with four
other elements. Strong negative association existed between magnesium and
calcium but the same element exhibited a weak positive association with
potassium and chlorine. There was no correlation between potassium and
chlorine but weak negative correlation with calcium. Calcium recorded a very
weak negative association with chlorine.
Table 6.3: Correlations among Nine Mineral Elements in 22 accessions of Abelmoschus spp*.
Mineral
Element

Al

Mn

Cu

Na

Br

Mg

Ca

Al
Mn

-0.086

Cu

-0.219

Na

0.344

0.371

Br

0.662*

-0.018

0.011

0.589

Mg

-0.042

0.158

0.565

-0.105

0.041

0.173

-0.082

-0.258

-0.053

0.270

0.012

Ca

0.128

-0.342

-0.646

0.115

0.069

-0.615

-0.210

Cl

0.376

-0.098

-0.276

0.178

0.226

0.000

0.291
-0.064

* p = 0.05

"

0.168

-0.057

Cl

"
"

6.3.3 Maximum viscosity of mucilage of Abelmoschus spp L.


The results of maximum viscosity of fresh fruits of twenty-one (21) accessions
of okra are presented in Table 6.4. Accessions were observed to be significantly
different from each other at significant difference level using the Duncan
Multiple Range Test (DMRT) for the mean separation. DKA had the maximum
mean value of 366.8 bu. The lowest viscosity value was registered by Cape
(53.00 bu). It can be observed that, most accessions from the Brong Ahafo
region (semi-equatorial climatic zone) and all accessions from the Volta region
(coastal equatorial climatic zone) recorded higher viscosity values. Most of the
accessions with late maturity duration and moderate yielding potential registered
correspondingly higher viscosity values, thus higher mucilage content. These
accessions exhibited marked plant height.

"

"

Table 6.4: Maximum Viscosity of mucilage of 21 Accessions of Okra


Accession

Mean maximum viscosity (bu)

DKA

366.75a*

Yeji-Local

329.00ab

Amanfrom

316.75abc

Asontem NV.

284.75bcd

Kortebortor-BAR

269.75bcd

Atomic

247.75cde

Akrave

246.25cde

Asontem-ER

221.75def

Nkran Nkuruma

182.00efg

Asontem-ASR

165.50fgh

Juaboso

162.25fghi

Legon fingers

160.75fghi

Mapelega

148.00ghij

Volta

133.00ghijk

Agric short fruit

116.00ghijkl

Cs-Legon

103.75hijkl

Labadi

97.25hijkl

Asontem-GAR

91.75ijkl

Asante type II

88.25jkl

Asontem-BAR

64.25kl

Ecrg

53.00l*

Mean

183.26101.19

CV (%)

55.22

*Accession with highest mean maximum viscosity is bolded and underlined and
accession with lowest mean maximum viscosity is bolded. = standard deviation of
mean; bu = Brabender unit.

"

6.4 DISCUSSION
6.4.1 ASSESSMENT OF ESSENTIAL MINERAL ELEMENTS IN OKRA
ACCESSIONS
Determination of the mineral element composition of the okra accessions brings
to light the extent of genetic variations among the accessions. There was high
variability among the okra accessions for concentrations of all five essential
macro elements in the edible fruits as detected using INAA. Concentration of
sodium recorded ranged from 778.511.60 mg/kg to 16.322.45 mg/kg for
Mamolega and Asontem-ASR respectively. The Report by Kumar et al. (2010)
that okra pods contain appreciable levels of Na is consistent with the result of
this study. The high concentration of Na as revealed in this study could also be
explained by the nutrient status and anthropogenic activities on the soils at the
experimental site.

Potassium concentration on the other hand was equally appreciable for most of
the accessions. Concentration values recorded in this study were above what was
reported by Benchasri (2012a) in okra and Adotey et al. (2009) in tomato.
Indiana recorded the lowest of 13.422.01 mg/kg; while Nkran Nkuruma
registered the highest concentration of 97.4314.61 mg/kg. The recommended
daily

intake

for

potassium

is

200mg/kg

(http://www.lenntech.com/recommended-daily-intake.htm). This means that


100g of okra consumed a day would supply less than 300 mg of potassium.
Nkran Nkuruma could provide substantial contribution to attaining the RDI for
K as consumption of 100g would yield about 700 mg.

"

"
"

Values obtained for magnesium in this study spanned from 40.096.01mg/kg


(Asontem NV) to 61.529.23 mg/kg (Asontem-BAR). The concentrations of Mg
recorded in this study exceeded what was reported in guava (1.91 mg/100 gm)
and mint (2.89 mg/100 gm) (Ismail et al., 2011) which are noted for their
content of essential mineral elements. Recommended daily dietary intake of
magnesium ranges from 400-420 mg/day (Whitney et al., 1990; Pennington et
al., 1986). Adotey et al. (2009) and Zaichick (2002) reported 0.16g/kg in tomato.
The values reported by these researchers are inconsistent with what is reported
in this study for Mg concentration among the okra accessions.

Calcium is one of the predominant essential elements in foods. Values recorded


for Ca in this study ranged from 10.121.52 mg/kg to 96.2114.43mg/m. All the
accessions recorded very low amounts of calcium compared to the
recommended daily allowance of 500-1200mg/kg (Oduro et al., 2008;
http://nutritiondata.self.com/facts/cereal-grains-and-pasta/5707/2).
Soil nutritional conditions could have accounted partly for the variations in Ca
concentrations among the accessions. Accessions with appreciable concentration
could be developed for supplementation of other sources of Ca to meet the
recommended dietary intake in okra. Concentrations of chlorine were
remarkably higher for most accessions. This could be attributed to influence of
soil nutritional conditions.

Concentrations of all the micro elements (Al, Cu, Mn and Br) detected were
considerably high. Quartey (2010) and Adotey et al. (2009) reported seemingly
high values for these elements in tomato, concurring concentrations of these
elements in this study. Al contents recorded for all accessions were
"

"

"
"

comparatively higher than clover (23.6 mg/kg), oats (7.3 mg/kg), bean (5.1
mg/kg), green lentil (6.6 mg/kg), spinach (12.6 mg/kg), corn (11.90 mg/kg), red
lentil (3.7 mg/kg) and rice (8.4 mg/kg) as reported by Hicsonmez et al. (2012).
The significant variable concentrations of Al in all accessions may partly be
explained by the genetics of the individual accessions and the influence of the
soil at the experimental field. Children, women of reproductive age and pregnant
women are most vulnerable to micro nutrient deficiency and anaemia (Ghana
Demographic Health Survey, 2004); hence, diets for this group of persons ought
to be rich in micro nutrients. Okra could serve as source of Al, Mn and other
essential micro nutrients in the diets of children and pregnant women in Ghana.
Copper was however not detected for most accessions, this could be ascribed to
low prevalence level of Cu in these accessions.

6.4.2 Intensity of Association between Mineral Elements


Fifty two percent (52.78%) strong or positive correlations was observed between
pairs of the elements detected in the 22 accessions of okra. Strong associations
exist between copper and magnesium (r = 0.565) and between sodium and
bromine (r = 0.589). The strongest correlation was between aluminium and
bromine (r = 0.662). This suggests that it may be possible for the breeder to
simultaneously select for higher concentrations of these pairs of elements in a
breeding programme involving biofortification. Copper and magnesium were
highly prevalent in Cape and Asontem-BAR, sodium and bromine were equally
high in concentration in Mamolega and Atomic. These elements recorded strong
correlation, hence could be considered as potential candidates for future
breeding work.

"

"

"
"

Mamolega, Yeji-Local, Asontem-BAR, Atomic, Asontem-GAR, Cape were high


yielding, and more slimy (Table 3.2, Chapter 3) but with non-uniform fruit
shape, size and late maturing. Mamolega for instance possesses relatively high
concentration of Al, Na, Br, Ca and Cl (Table 6.2), Yeji-Local and AsontemBAR recorded high concentration of Mg while Cape registered good
concentrations of Mn, Cu, Na. Atomic had moderate concentration of Br, Ca, Na
and Cu. Asontem-GAR however recorded high concentrations of Ca, Cl and fair
concentration of Na. Clemson spineless, Indiana and Legon fingers were early
maturing with uniform fruit size, shape, and optimum mucilage content
(sliminess). Clemson spineless was high in Al and K. Indiana recorded the
highest concentration of Mn while Legon fingers registered fair concentrations
of Na, Mg, and Ca. Therefore, selecting Mamolega for instance, for high content
of Na imply simultaneously selecting for high content of Br and vice-versa.

Historically, international breeding efforts in okra have been oriented towards


intensive cultivation with high production in a short period (early maturity) and
wide adaptation (photoperiod insensitivity, resistance to insects and diseases)
(Benchasri, 2012b); however, the importance of the quality of food source for
reducing micro nutrient deficiencies has come into the limelight of late. There is
now greater recognition of the global prevalence of many forms of micro
nutrient malnutrition (Kennedy et al., 2006). Hence, enhancing nutritional
quality through diet selection holds greatest prospects of success in the near
future. Okra has no intraspecific barrier to crossability. Okra is a good candidate
for inclusion in many diets by virtue of the high concentration of several mineral
elements in its edible fruits. Superior lines such as Mamolega, Yeji-Local,
Asontem-BAR, Clemson spineless, Cape, Atomic, Asontem-GAR, Indiana and
"

"

"
"

Legon fingers may be selected for future hybridisation work aimed at further
improvement in mineral element constituents alongside fresh fruit yield and
other desirable traits.

6.4.3 Mucilage content of Abelmoschus spp L.


The means of maximum viscosity values obtained ranged from 53.0 - 366.8 bu
(Brabender units). DKA (366.8 bu), Yeji-Local (329 bu), Amanfrom (316.8 bu),
Asontem NV (284.8 bu) and Kortebortor-BAR (269.8 bu) were among
accessions with high value whilst Cape (53.0 bu) had the least maximum
viscosity value. This implies that those with higher values could be used to
provide sliming ability of okra fruit. The higher the mucilage content of okra
fruit, the higher the slimming ability and viscosity (Thanatcha and Pranee, 2011;
Woolfe et al., 1977). This is consistent with an earlier work by Woolfe et al.
(1977), who reported that higher concentration of mucilage solution led to
higher viscosity, and Thanatcha and Pranee (2011) working with guar gum and
xanthan gum. From the analysis of variance, there was statistically significant
variability in the viscosity of the mucilage of the various accessions. This may
be attributed to the type of accession and its genetics.

Research indicates that cultivars belonging to the West African Taxon (WAT)
mostly A. caillei are high in mucilage content (National Academies Press, 2006).
DKA (366.8 bu), Yeji-Local (329 bu), Amanfrom (316.8 bu), Asontem NV
(284.8 bu) and Kortebortor-BAR (269.8 bu) met all the requirements of
Abelmoschus caillei. Stev. (WAT) in terms of their morphological
characteristics. These accessions were hardy and perennial in nature with stout
"

"

stems and flattened broad leaves (Table 3.3). They flowered very late and
produced slimy pods, which were non-uniform in shape and size (Table 3.3),
compared to the other accessions. These accessions could be employed as source
of genes for breeding okra with higher mucilage content (Kumar et al., 2010).

These accessions with high level of mucilage were generally high yielding, late
maturing with erect growth habit (Table 3.2; chapter 3). Accessions from the
Volta, Ashanti and Brong Ahafo regions recorded equally high viscosity with
moderate level of mucilage. These may be attributed to the feeding habits of
inhabitants of these regions and their preference for slimy varieties. The people
of the Volta region and certain tribes (Ewe(s), Krobo(s), the Brong(s),
Dagarti(s), Frafra(s)) in Ghana are known to patronise okra varieties with
variable level of sliminess with correspondent high yielding potential. Hence,
the accessions may have been deliberately selected for by farmers of these
regions to meet preference (s) of consumers. The Bulk of okra from Ghana is
produced from the Brong Ahafo, Volta, Northern, Greater Accra and Central
regions (Torkpo et al., 2006; NARP, 1993); however, farmers cultivate different
varieties to meet local market demands in these regions. Some local consumers
prefer slimier varieties for their preparations while others prefer less slimy
varieties. In addition, growth habits of these varieties (accessions) could have
kphqtogf" hctogtu" ugngevkqp" kp" vjgug" tgikqpu" cu" oquv" qh" vjgug" ceeguukqpu" ykvj"
high viscosity (mucilage) values exhibited erectile growth habit, hardy, stocky
stem and medium to tall plant height. These accessions (DKA, Yeji-Local,
Amanfrom, Asontem NV, and Kortebortor-BAR) were less influenced by the dry
period and even flowered when the rainfall pattern became erratic (November-

"

"
"

2011 to January-2012) and may be selected for further improvement for high
sliminess (viscosity) and for cultivation in areas with erratic rainfall patterns.

On the other hand, accessions with low sliminess (mucilage) were generally
early maturing, short in height, exhibited branched growth habits with short
harvesting duration (single harvest). Greater Accra and other coastal regions in
Ghana have a fluctuating rainfall pattern, which probably dictated hctogtu"
choice in these regions for the type of okra varieties cultivated (accessions).
Cape, Asontem-BAR, Asante type II, Asontem-GAR, Labadi, and Juaboso may
be selected for further improvement as varieties with low mucilage (sliminess)
content.

6.5 CONCLUSIONS
a)

Nine essential mineral elements were detected among the accessions with

varying prevalence levels indicating high level of genetic variability among the
accessions with respect to concentrations of these elements in their edible fruits.
b)

Concentrations of Al, Mn, Br (micro elements) in all accessions were

above their recommended daily dietary intake (RDI); therefore, excess intake of
these varieties could pose health risk to consumers. However, concentrations of
major elements such as Na, Mg, K, Ca and Cl were below their RDI (s), hence
intake of these varieties ought to be increased in order to meet RDI(s) for these
essential mineral elements by consumers.
c)

Indiana, Clemson spineless, Legon fingers, Cape exhibited early fruiting

potential, uniform pod shape and size with minimum level of sliminess also

"

"

recorded correspondent optimum concentrations of Al, K, Mn, Cu, Na, Mg, and
Ca.
d)

Mamolega, Yeji-Local, Asontem-BAR, Atomic, Asontem-GAR, Cape

may be selected as parents for future breeding work with increased and/or
optimum level of essential elements (K, Na, Ca, Mg, Cl), high mucilage
(sliminess), with good yield potential.
e)

The strong positive associations existing between most pairs of elements

contained in the accessions of okra will be key in future breeding work


involving biofortification, as selecting for one element will imply simultaneous
inclusion of another essential element.
f)

The means of maximum viscosity ranged from 53.0 - 366.8 bu for all

accessions studied.
g)

DKA, Amanfrom, Asontem NV, Yeji-Local and Kortebortor-BAR were

high in mucilage contents.


h)

There was marked significant statistical variations in contents of mucilage

among all accessions under study.


i)

Accessions belonging to the West African Taxon recorded appreciably

high amounts of mucilage compared to those belonging to the Asian taxon (A.
esculentus).

"

"
"

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"

"

"
"

CHAPTER SEVEN
"

GENERAL CONCLUSIONS AND RECOMMENDATIONS


"

7.1 CONCLUSIONS
Immense genetic variations have been observed in okra (Abelmoschus spp L.)
particularly the West African type, Abelmoschus caillei but ironically no serious
breeding effort have yet been committed to exploit and harness the proven
genetic richness to advance the improvement of the crop. Previous international
efforts have been limited to intensive cultivation with increased production base
in a short period (early maturing) and resistance to insects and diseases. There is
still enormous scope for cultivar improvement in Africa for the commercial
sector where hardy, robust, high to moderate mucilage and nutrient content,
long-lived types are required. Germplasm characterisation of 29 indigenous
cultivars (accessions) was done to augment efforts towards improvement of the
crop in Ghana. The following conclusions can be drawn from this study:
1.

(a) The morpho-agronomic characters studied exhibited intense

variability among all 29 accessions of okra.


(b) Morphologically, there was no duplicate detected among the accessions
studied (dendrogram).
(c) The most diverse and distantly related accessions with outstanding traits
were Mamolega, Wune mana and Cs-Legon and could be potential sources of
variability for future breeding work.
(d) Moderate to strong association exists between pairs of quantitative yield
traits in the okra landraces studied, specifically flowering and fruiting
"

"

"
"

parameters such as 50% flowering, 50% fruiting, average number of pods per
plant, first-flowering node, and first fruiting node, maximum plant height and
maximum number of internodes.
(e) Different characters contributed differently to overall genetic variability
among the accessions (PCA) under study.
2

(a) The ISSR primers yielded low level of polymorphism among the 29

accessions; 25 accessions however, were identified as possible duplicates


(b) Indiana, Yeji-Local, Asante type II and Atomic were detected as distinct
unique genotypes and could be unique sources of genes for improvement
programmes.
*e+"Cvqoke"cpf"Cmtcxg"ygtg"identified to have a common ancestry.

(a) There was high variability in the amounts and levels of total

flavonoids, total phenolics and total antioxidant activity, making okra a good
source of natural antioxidants.
(b) Ethanol extraction solvent yielded better antioxidant activity than aqueous
(water) solvent.
(c) Mucilage levels were highly variable in all accessions. DKA, Amanfrom,
Asontem NV, Yeji-Local and Kortebortor-BAR recorded high mucilage content.
(d) Nine essential mineral elements were detected among the accessions with
varying concentrations among the accessions.
(e) There were strong positive associations existing between most pairs of
elements contained in the accessions of okra.

"

"

"
"

7.2 RECOMMENDATIONS
For a co-dominant marker to be employed in any characterisation work, the
sequence of the genome of the plant must be deciphered and known. To the
best of my knowledge, the genome of okra has not yet been sequenced
hence no SSR primers are available now to conduct a comprehensive and
thorough genetic diversity study at the gene level. Sequencing calls for
huge financial and infrastructural investments, therefore I recommend the
following;

(a)

International and national institutions concerned with promoting and

maximising the potentials of unexploited and underutilised crops like okra


should collaborate to invest in a project towards sequencing of the genome of
this versatile vegetable since this cannot be undertaken by a single entity.
(b)

Researchers in Ghana could explore and introgress the desirable qualities

of Indiana, Yeji-Local, Asante type II and Atomic into landraces that are well
adapted to the local agro ecological conditions but lack these qualities (narrow
shape and size, smooth pod pubescence, yellowish green in colour, early
fruiting, short peduncle, moderate level of mucilage, persistent epicalyx
segments, medium pod length and less bulky in weight).
(c)

Amanfrom, Indiana, Volta, Clemson spineless, Agric short fruit, Yeji-

Local, Asante type II, Cs-Legon, Mamolega, and Atomic were the topmost
accessions with desirable qualities and could be utilised for developing
commercial variety for the export market in Ghana.
(d)

In addition, collections was done from eight regions of Ghana, this

should be extended to other regions and base collections in the genebank of the

"

"

"
"

Plant Genetic Resources Research Institute (PGRRI) at Bunso, should be


thoroughly characterised and for further conservation.
(e)

Genes linked to agronomically important traits in okra should be

genetically mapped through Quantitative Traits Loci (QTLs) to serve as a


baseline data platform for researchers and breeders.
(f)

Biochemical screening for secondary metabolites and specific individual

compounds such as saponins, and their structures conferring the antioxidants


activity via high performance liquid chromatography (HPLC), thin layer
chromatography (TLC) and liquid chromatography mass spectrometry (LCMS)
facilities ought to be undertaken to fully exploit the biochemical wealth of this
vegetable to human health.
(g)

Several authors have noted that flavonoids substantially influence

antioxidant properties in mucilagenous vegetables. The relation between


antioxidants and mucilage and their effects on or interference with the
nutritional composition of Abelmoschus spp should be investigated.
(h)

Protein, elemental constituents, crude fibre, total carbohydrate and other

nutritional compositions should be assessed in both the leaves and the pods.

"

"

"
"

8.0 APPENDICES
"

Appendix 8.1: Passport data used for Collection of Okra Germplasm

Accession Identifiers

(1). Accession number (2) Species


number (3)Varietal name (4) Fqpqtu"
name (5) Fqpqtu"pwodgt"

Collection Descriptors

(1) Collection number (2) Common


name (3) Local name/vernacular name
(4) Ethnic group (5) Scientific name (6)
Collection date (7) Locality of collection
(8) Country of collection (9)
Province/state (10) District/township
(11) Collection site (12) Altitude (13)
Latitude

Meteorological data of collection site

(1) Average annual rainfall (2) Average


annual maximum temperature (3)
Average annual minimum temperature

Source of collection

(1) Local uses (2) Local cultivation


methods
(3) Time of planting (4) Time of
harvesting (5) Associated crops (6)
Agronomic score (7) Habitat

Source: IPGRI, 1991.

"

"

"
"

Appendix 8.2: Analysis of Variance for Quantitative Morphological


Traits

Appendix 8. 2. 1: Variate: 50% GERMINATION (FPGer)


Source

d.f.

BLOCK

s.s.

m.s.

0.17365

0.04341

v.r.

F pr.

0.70

0.595

ACCESSION

28

449.11176

16.03971 258.29

Residual

83

5.15425

0.06210

Total

115

454.43966

3.95165

< 0.001

Appendix 8.2.2: Variate: 50% FLOWERING (FPFl)


Source

d.f.

s.s.

m.s.

BLOCK

19.78448

4.94612

ACCESSION

28

Residual

83

3.51632

0.04237

Total

115

33475.19828

291.08868

33451.89748

1194.71062

v.r.
116.75

F pr.
< 0.001

28200.23

< 0.001

v.r.

F pr.

Appendix 8.2.3: Variate: 1000 seed weight (g) (TSwt)


Source of variation

d.f.

BLOCK stratum

s.s.

m.s.

0.15411

0.05137

0.88

335.45909

5742.80

ACCESSION

28

9392.85464

Residual

84

4.90676

"

0.05841
"

<.001

"
"

Total

115

9397.91552

Appendix 8.2.4: Variate: AVERAGE NUMBER OF PODS PER PLANT


(APP)
Source of variation

d.f.

s.s.

m.s.

v.r.

BLOCK stratum

F pr.

7.379

2.460

1.94

ACCESSION

28

23832.690

851.167

670.58 <.001

Residual

84

106.621

1.269

Total

115

23946.690

Appendix 8.2.5: Variate: 50% FRUITING (FPFr)


Source of variation

d.f.

s.s.

m.s.

v.r.

BLOCK stratum

1.4483

0.4828

0.80

TREATMENT

28

37505.4483

1339.4803

2225.77

Residual

84

50.5517

0.6018

Total

115

37557.4483

F pr.

<.001

Appendix 8.2.6: Variate: MAXIMUM PLANT HEIGHT (MPH)


Source of variation

d.f

s.s.

m.s.

v.r.

BLOCK stratum

97.50

32.50

0.35

TREATMENT

28

87351.43

3119.69

Residual

84

7884.71

93.87

Total

115

95333.64

"
"

"

33.24

F pr.

<.001

"
"

Appendix 8.2.7: Variate: MAXIMUM NUMBER OF INTERNODE (MNI)


Source of variation

d.f.

s.s.

m.s.

BLOCK stratum

0.16524

0.05508

ACCESSION

28

567.00834

20.25030

Residual

84

2.62686

0.03127

Total

115

569.80044

v.r.

F pr.

1.76
647.55 <.001

Appendix 8.2.8: Variate: FIRST FLOWERING NODE (FFN)


Source of variation

d.f.

BLOCK stratum

s.s.

m.s.

0.09483

v.r.

0.03161

1.23
2881.03

ACCESSION

28

2069.70690

73.91810

Residual

84

2.15517

0.02566

Total

115

2071.95690

F pr.

<.001

"
Appendix 8.2.9: Variate: TOTAL NUMBER OF LEAVES PER PLANT
(NLPP)
Source of variation

d.f.

s.s.

BLOCK stratum

7.931

m.s.
2.644

2.13
107.89

ACCESSION

28

3742.759

133.670

Residual

84

104.069

1.239

115

3854.759

Total

"

"

v.r

F pr.

<.001

"
"

Appendix 8.2.10: Variate: AVERAGE NUMBER OF SEED PER POD (ASPD)


Source of variation

d.f.

s.s.

m.s.

BLOCK stratum

2.716

0.905

0.64
336.51

TREATMENT

28

13239.966

472.856

Residual

84

118.034

1.405

Total

115

v.r.

F pr.

<.001

13360.716

Appendix 8.2.11: Variate: STEM DIAMETER AT BASE (cm) (STB).


Source of variation

d.f.

BLOCK stratum

TREATMENT

s.s.

m.s.

v.r.

F pr.

0.50828

0.16943

4.17

28

296.86828

10.60244

261.04

Residual

84

3.41172

Total

115

300.78828

<.001

0.04062

Appendix 8.2.12: Variate: NUMBER OF PODS PER PLANT (NPPP)


Source of variation

d.f.

s.s.

BLOCK stratum

15.931

5.310

TREATMENT

28

3070.052

109.645

Residual

84

598.569

7.126

115

3684.552

Total

"

m.s.

"

v.r.

F pr.

0.75
15.39

<.001

"
"

Appendix 8.2.13: Variate: FIRST FRUIT-PRODUCING NODE (FFPN)


Source of variation

d.f.

s.s.

BLOCK stratum

24.276

8.092

4.13

28

380.310

13.583

6.93

164.724

1.961

TREATMENT
Residual

84

Total

115

"

569.310

"

m.s.

v.r

F pr

<.001

"
"

"""""""""""""Appendix 8.4.1: Sequences of six ISSR primers used in the study"


Primer

Sequence

C4

GAGAGAGAGAGAGAGYT

O4

GAGAGAGAGAGAGAGAYA

O9

ATGATGATGATGATG

O11

AGAGAGAGAGAGG

O12

GTGGTGGTGGTGGTG

UBC888

BDBCACACACACACACA

"

Appendix 8.4.2: Banding pattern of Genetic Diversity Studies on Accessions of Okra


(ISSR)
Code

Accession

Code

Accession

1=ATM

Atomic

16=KBR

Kortebortor-BAR

2=AKV

Akrave

17=IDA

Indiana

3=YJL

Yeji-local

18=DBO

Debo

4=AT2

Asante type II

19=ASF

Agric short fruit

5=CSP

Clemson spineless

20=AAR

Asontem-ASR

6=NKN

Nkran nkuruma

21=LBI

Labadi

7=CSL

Cs-Legon

22=MLA

Mapelega

8=ABR

Asontem-BAR

23=WMA

Wune mana

9=KPV

Kpeve

24=MLA

Mamolega

10=JBO

Juaboso

25=CPE

Cape

11=LGF

Legon fingers

26=KAR

Kortebortor-ASR

12=AGR

Asontem-GAR

27=VTA

Volta

13=ANV

Asontem nv.

28=AER

Asontem-ER

14=AT1

Agric type I

29=DKA

15=AFM

Amanfrom

DKA

Figure 4.1(a&b).

"

"

"
"

Appendix 8.5: Analysis of variance for mucilage content.

Appendix 8.5.1: Variate: Maximum viscosity


Source of variation

d.f.

s.s.

m.s.

v.r.

Replication stratum

1799.

600.

0.22

Accession

20

687709.

34385.

12.86

Residual

60

160430.

2674.

Total

83

849938.

"

"

F pr.

<.001

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