Morphological and Molecular Characterization, Assessment of
Nutritional Composition and Micropropagation of Cocoyam
(Xanthosoma sagittifolium (L.) Schott) from Ethiopia
Eyasu Wada Wachamo
Addis Ababa University
Addis Ababa, Ethiopia
June, 2018
Morphological and Molecular Characterization, Assessment of
Nutritional Composition and Micropropagation of Cocoyam
(Xanthosoma sagittifolium (L.) Schott) from Ethiopia
A Dissertation Submitted to
the Department of Microbial, Cellular and Molecular Biology, Addis
Ababa University, in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy in Biology (Applied Genetics)
By
Eyasu Wada Wachamo
Addis Ababa University
Addis Ababa, Ethiopia
June, 2018
Declaration
I, the undersigned, declare that this Dissertation is my original work and its composition has
never been submitted elsewhere for any other award. All sources of materials used for the
Dissertation have been duly acknowledged.
Name: Eyasu Wada Wachamo Signature: ___________________ Date: _______________
Acknowledgments
First and foremost, I am highly indebted to express my deepest and genuine appreciation to my
supervisors: Dr. Tileye Feyissa, Dr. Kassahun Tesfaye and Prof. Zemede Asfaw for their excellent
guiding, follow-up and support. Next, I would like to owe my special thanks to Dr. Kifle Dagne and
Asfaw Kifle (PhD prospective graduate) for their special pieces of support. I would also like to thank
Prof. Daniel Potter from Plant Science Department, University of California-Davis (UCDavis), USA
and Prof. Dr. Birgit Gemeinholzer and Prof. Dr. Volker Wessmann from Justus Liebig University
Giessen (JLU), Germany for inviting me to their Molecular Biology Laboratories as a visiting scholar.
I extend my deepest acknowledgments to different institutions that have provided financial support:
Department of Microbial, Cellular and Molecular Biology and Graduate Program Office of Addis
Ababa University (SIDA project), Wolaita Sodo University and The German Academic Exchange
Service (DAAD). I am thankful to Areka Agricultural Research Center, Department of Plant Sciences
of UCDavis and Systematic Botany Group of JLU for making the laboratories accessible for this study.
I am grateful to the Ethiopian Institute of Biodiversity for giving me permits to export X. sagittifolium
leaf samples to UCDavis and JLU for molecular analyses. My unreserved thanks go to farmers for
providing information of the crop. I have many thanks to driver Mr. Beyene Dilbore and field and
laboratory technical assistances: Mr. Abeje Yilma, Mr. Wolde Pilto, Mr. Sied Mohammed, Mrs.
Eyerusalem Natinael, Ms. Gloria Diaz-Britz, Mrs. Hela Krufzick, Mrs. Sabine Mutz and Mr. Peter
Seum for providing unreserved technical help. Many other persons also stretched their hands for help
during this research work. Although I cannot mention all of their names, Prof. Hoysign Won, Mr.
Genene Gezahegn, Mr. Girma, Mrs. Tigist Markos, Mrs. Christena Muller, Mr. Teshome Matusala and
Mr. Micheal Mears can be mentioned as their representatives. Finally, my deepest thanks go to my
wife Meseret Gano, my children Fitsum Eyasu and Keleul Eyasu, and my nieces Suna Esayas and
Konjit Minota for their patience and encouragement at all times of this study period.
May God Bless You All!
i
Dedication
This Dissertaion is dedicated to my mother Asase Anebo and my late father Wada Wachamo
for giving me the foundation of learning that they never enjoyed themselves. The efforts that
they made for my education are in my mind. I have been able to appreciate the value of
learning.
ii
Table of Contents
page
Acknowledgments ............................................................................................................................... i
Dedication .......................................................................................................................................... ii
Table of contents……………………………………………………………………………………iii
List of Tables.................................................................................................................................... vii
List of Figures ................................................................................................................................. viii
List of Appendices ............................................................................................................................ ix
List of Acronyms and Abbreviations ................................................................................................. x
Abstract ............................................................................................................................................. xi
Chapter One ..................................................................................................................................... 1
General Introduction ....................................................................................................................... 1
1.1 Background .............................................................................................................................. 1
1.2 Statement of the problem ......................................................................................................... 4
1.3 Research questions, hypotheses and objectives ....................................................................... 5
1.3.1
Research questions ..................................................................................................... 5
1.3.2 Hypotheses ........................................................................................................................ 5
1.3.3 Objectives of the study .......................................................................................................... 6
1.4 Organization of the dissertation ............................................................................................... 7
Chapter Two ..................................................................................................................................... 8
Literature Review............................................................................................................................. 8
2.1 Origin and distribution of cocoyam ......................................................................................... 8
2.2 Taxonomic description of cocoyam ......................................................................................... 8
2.3 Etymology and common names ............................................................................................... 9
2.4 Botanical description of cocoyam .......................................................................................... 10
2.5 Importance and use values of cocoyam.................................................................................. 10
2.6 Nutritional profile of cocoyam ............................................................................................... 11
2.7 Growth condition and cultivation of cocoyam ....................................................................... 12
2.8 Cocoyam breeding and tissue culture..................................................................................... 13
2.9 Assessment of genetic diversity ............................................................................................. 14
2.9.1 Morphological markers ................................................................................................... 15
2.9.2 Biochemical Markers....................................................................................................... 16
2.9.3 Molecular Markers .......................................................................................................... 17
2.10 Molecular marker techniques ............................................................................................... 18
2.11 State of knowledge on cocoyam genetic diversity ............................................................... 22
iii
Table of contents continued
Chapter Three ................................................................................................................................ 24
Farmers’ Knowledge and Perception of the Constraints, Trait Preferences, Uses and
Management Practices of Cocoyam (Xanthosoma sagittifolium (L.) Schott) in Ethiopia ........ 24
Abstract ........................................................................................................................................... 24
3.1 Introduction ............................................................................................................................ 25
3.2 Materials and methods ........................................................................................................... 27
3.2.1 Description of the study area ........................................................................................... 27
3.2.2 Sampling frame and informant selection ......................................................................... 27
3.2.3 Ethical consideration ....................................................................................................... 28
3.2.4 Data collection and analysis ............................................................................................ 28
3.3 Results .................................................................................................................................... 30
3.3.1 Age of farmers and their experiences in cocoyam cultivation ........................................ 30
3.3.2 Distribution and cultivation of cocoyam in the study area .............................................. 30
3.3.3 Local names of cocoyam and meanings .......................................................................... 32
3.3.4 Cocoyam introduction into the study areas and tuber sources for the first time garden
cultivation ................................................................................................................................. 33
3.3.5 The farmers’ planting material and cropping system ...................................................... 34
3.3.6 Land preparation, planting and harvesting of cocoyam................................................... 34
3.3.7 The local uses of cocoyam .............................................................................................. 35
3.3.8 Farmers’ perceptions of cocoyam characters and uses .................................................... 36
3.4 Discussion .............................................................................................................................. 37
Chapter Four .................................................................................................................................. 41
Morphological Traits Based Genetic Diversity in Cocoyam (Xanthosoma sagittifolium (L.)
Schott) from Ethiopia .................................................................................................................... 41
Abstract ........................................................................................................................................... 41
4.1 Introduction ............................................................................................................................ 42
4.2 Materials and methods ........................................................................................................... 43
4.2.1 Germplasm collection ...................................................................................................... 43
4.2.2 Description of the experimental site ................................................................................ 43
4.2.3 Experimental design and crop management .................................................................... 46
4.2.4 Morphological traits and data collection ......................................................................... 46
4.2.5 Data analysis.................................................................................................................... 47
4.3 Results .................................................................................................................................... 48
4.3.1 Qualitative traits .............................................................................................................. 48
4.3.2 Descriptive statistical parameters and variance ............................................................... 50
4.3.3 Genotypic and phenotypic variances, coefficients of variations and heritability ............ 51
iv
Table of Contents continued
4.3.4 Principal components and clustering of accessions ......................................................... 52
4.4 Discussion .............................................................................................................................. 56
Chapter Five ................................................................................................................................... 60
Assessment of Genetic Diversity and Differentiation of Cocoyam (Xanthosoma sagittifolium
(L.) Schott) from Ethiopia Based on SSR Markers ..................................................................... 60
Abstract ........................................................................................................................................... 60
5. 1 Introduction ........................................................................................................................... 61
5.2 Materials and methods ........................................................................................................... 63
5.2.1 Plant materials ................................................................................................................. 63
5.2.2 DNA extraction ............................................................................................................... 63
5.2.3 SSR primers and PCR amplification ............................................................................... 64
5.2.4 Data analysis.................................................................................................................... 66
5.3 Results .................................................................................................................................... 67
5.3.1 Genetic diversity of cocoyam as revealed by SSR markers ............................................ 67
5.3.2 Genetic diversity within populations and within morphotypes ....................................... 68
5.3.3 Genetic differentiation ..................................................................................................... 69
5.3.4 Cluster analysis and population structure ........................................................................ 70
5.4 Discussion .............................................................................................................................. 74
Chapter Six ..................................................................................................................................... 76
AFLP Fingerprinting for Assessment of Genetic Diversity and Differentiation of Cocoyam
(Xanthosoma sagittifolium (L.) Schott........................................................................................... 76
Abstract ........................................................................................................................................... 76
6.1 Introduction ............................................................................................................................ 77
6.2 Materials and methods ........................................................................................................... 79
6.2.1 Plant materials ................................................................................................................. 79
6.2.2 DNA extraction ............................................................................................................... 80
6.2.3 AFLP analysis ................................................................................................................. 80
6.2.4 Data scoring and analyses ............................................................................................... 81
6.3 Results .................................................................................................................................... 83
6.3.1 Genetic diversity of cocoyam as revealed by AFLP markers.......................................... 83
6.3.2 Genetic differentiation and cluster analysis..................................................................... 84
6.4 Discussion .............................................................................................................................. 89
Chapter Seven................................................................................................................................. 92
Proximate, Mineral and Antinutrient Contents of Cocoyam (Xanthosoma sagittifolium (L.)
Schott) from Ethiopia .................................................................................................................... 92
Abstract ........................................................................................................................................... 92
v
Table of Contents continued
7.1 Introduction ............................................................................................................................ 93
7.2 Materials and methods ........................................................................................................... 95
7.2.1 Sample collection ............................................................................................................ 95
7.2.2 Preparation of cocoyam flour .......................................................................................... 95
7.2.3 Determination of proximate composition ........................................................................ 96
7.2.4 Determination of mineral content .................................................................................... 99
7.2.5 Analysis of antinutritional factors ................................................................................... 99
7.2.6 Statistical analysis ......................................................................................................... 100
7.3 Results .................................................................................................................................. 101
7.3.1 Proximate composition .................................................................................................. 101
7.3.2 Mineral composition and antinutritional factors ........................................................... 101
7.4 Discussion ............................................................................................................................ 102
Chapter Eight ............................................................................................................................... 107
Micropropagation of Cocoyam (Xanthosoma sagittifolium (L.) Schott) from Shoot Tip ....... 107
Abstract ......................................................................................................................................... 107
8.1 Introduction .......................................................................................................................... 108
8.2 Materials and methods ......................................................................................................... 109
8.2.1 Preparation of donor plant and stock solutions ............................................................. 109
8.2.2 Culture medium preparation .......................................................................................... 110
8.2.3 Surface sterilization of explant ...................................................................................... 110
8.2.4 Culture condition ........................................................................................................... 111
8.2.5 Shoot initiation .............................................................................................................. 111
8.2.6 Shoot multiplication ...................................................................................................... 112
8.2.7 Rooting and acclimatization .......................................................................................... 112
8.2.8 Experimental design and data analysis .......................................................................... 113
8.3 Results .................................................................................................................................. 113
8.3.1 Shoot initiation .............................................................................................................. 113
8.3.2 Shoot Multiplication ...................................................................................................... 114
8.3.3 Rooting and acclimatization .......................................................................................... 119
8.4 Discussion ............................................................................................................................ 121
Chapter Nine................................................................................................................................. 124
Synthesis, Conclusion and Recommendations ........................................................................... 124
9.1 Synthesis .............................................................................................................................. 124
9.2 Conclusion............................................................................................................................ 129
9.3 Recommendations ................................................................................................................ 130
References ..................................................................................................................................... 131
vi
List of Tables
Page
Table 2.1 Comparison of different characteristics of molecular markers……....................22
Table 3.1 Study areas and number of respondents replied to interview guide………...…28
Table 3.2 Age of respondents and number of years of cocoyam cultivation........................30
Table 3.3 Source of cocoyam clones used for the first time planting in gardens......………34
Table 3.4 Characters/uses of cocoyam and farmers’ preference….…...……………...…...37
Table 4.1 List of cocoyam accessions with accession code, collection sites, coordinates
(latitude and longitude), altitude and color of accessions………………………...44
Table 4.2 Frequency distribution of 16 qualitative traits of cocoyam............….........…….48
Table 4.3 Basic statistics of 13 quantitative traits of cocoyam…........................…...…….50
Table 4.4 Summary of mean squares of 13 quantitative traits of cocoyam…..................…51
Table 4.5 Genetic parameters of 13 quantitative traits of cocoyam….........................……52
Table 4.6 Eigen value, proportion of variability and the first 3 PCs of cocoyam….............53
Table 4.7 Cluster means of 13 quantitative traits of cocoyam…......…......................…….56
Table 5.1 Primers sequences used for amplification of microsatellite markers........…...…65
Table 5.2 Genetic diversity parameters by 11 SSR loci in 100 cocoyam accessions...........67
Table 5.3 Genetic diversity within populations and morphotypes using 11 SSR loci….….68
Table 5.4 F-statistics for 11 SSR loci across populations and between morphotype...........69
Table 5.5 Summary of AMOVA for five cocoyam populations based on SSR marker….70
Table 6.1 Genetic diversity statistics of cocoyam based on AFLP data……...….………84
Table 6.2 Summary of AMOVA for populations and morphotypes based on AFLP….......85
Table 7.1 Proximate composition of green- and purple- cocoyams…........................…...101
Table 7.2 Mineral contents and antinutritional factors of cocoyam…………………….102
Table 8.1 Compositions of plant growth regulators for shoot multiplication (mg/l) ……112
Table 8.2 Effect BAP on shoot initiation of cocoyams from shoot tip...............................114
Table 8.3 Mean square of in vitro induced shoot parameters cocoyam on MS media
supplemented with different PGRs...….......................................................….115
Table 8.4 Effects of different concentrations of plant growth regulators on shoot
multiplication of cocoyam……...…………………….…………....……….…117
Table 8.5 Mean square of in vitro induced root number and length of cocoyam
on MS medium supplemented with different concentrations of PGRs………...119
Table 8.6 Effect of IBA and NAA on root induction of green- and purple-cocoyams....120
vii
List of Figures
page
Fig. 3.1 Map of Ethiopia showing the location of the study area.....................…......…......27
Fig 3.2 Cocoyam (Xanthosoma sagittifolium) plants from Ethiopia……...…..........……..31
Fig 3.3 Cocoyam plants: at homegarden (a), in natural ecosystem (b)in the shade of
coffee plants (c) and as ornamental in the urban centers….……...………….......31
Fig 3.4 Local uses of cocoyam in the study zones based on the interview
of 10 key informants per zone ………….…......……………...……………...…...35
Fig 4.1 Qualitative morphological traits of cocoyam…...……...…...……………...…......49
Fig 4.2 Score plot of 100 cocoyam accessions based on 13 quantitative traits……........…53
Fig 4.3 Cluster analysis showing the relationship among 100 cocoyam
accessions based on 13 quantitative traits.............…...………………............…...55
Fig 5.1 A two-dimensional plot of the Principal Coordinate Analysis (PCoA)
of 100 cocoyam accessions based on SSR data………......……......……….......…70
Fig 5.2 Neighbor joining (NJ) tree showing the relationships among
100 cocoyam accessions generated from SSR data…….…………………………71
Fig 5.3 Bayesian model-based clustering STRUCTURE analysis
as inferred at k = 2 based on SSR data...…………....………………………..........72
Fig 6.1 Representation of the first two coordinates obtained from the PCoA
of 78 cocoyam accessions based on AFLP data…...............................................…86
Fig 6.2 Neighbor-Joining (NJ) tree representing clustering of cocoyam
accessions generated from AFLP data................................................................…87
Fig 6.3 Bayesian model-based clustering STRUCTURE analysis as inferred at
K = 3 based on AFLP data…………………….……..........………….………...…88
Fig 8.1 Shoot tip explants for shoot initiation…………………………………………...111
Fig. 8.2 Shoot initiation from the cocoyam shoot-tip cultured on
MS medium supplemented with 2.0 mg/l BAP………….........……………...….114
Fig 8.3 Green- and purple- cocoyam on different multiplication media............................118
Fig 8.4 In vitro rooting of cocoyam shoots on MS medium containing 2.0 mg/l IBA…...120
Fig 8.4 Micropropagated plants after two weeks of acclimatization in greenhouse..........121
viii
List of Appendices
Page
Appendix 1 Passport data and semi-structured interview guide for cocoyam
(Xanthosoma sagittifolium (L.) Schott) study in Ethiopia……………….147
Appendix 2
Selected morphological descriptors used to characterize cocoyam
(Xanthosoma sagittifolium) grown in Ethiopia…………………………151
Appendix 3
Mean performances of 13 quantitative traits of 100 cocoyam accessions
(65 green- and 35 purple-cocoyam morphotypes………………………152
Appendix 4
Nutrient composition and concentration of MS basal medium………….154
Appendix 5
Effects of different concentrations of PGRs on shoot multiplication
of green- and purple- cocoyam (comparative analysis)……………........155
ix
List of Acronyms and Abbreviations
AFLP
Amplified Fragment Length Polymorphism
AMOVA
Analysis of molecular variance
AOAC
Association of Official Analytical Chemists
BAP
6-benzyl aminopurine
FAM
6-Carboxyfluorescein
FAO
Food and Agricultural Organization
Fst
Genetic differentiation among populations
GCV
Genotypic coefficient of variation
GLM
General linear model
Gst
Genetic differentiation among populations
h2b
Broad sense heritability
He
Expected heterozygosity/Nei’s gene diversity
HEX
Hexachloro-fluorescein
Ho
Observed heterozygosity
I
Shanon information index
IBA
Indol-3-butyric acid
IBPGR
International Board for Plant Genetic Resource
ISSR
Inter simple sequence repeats
Kn
Kinetin
MSE
Mean square of error
MSG
Mean squares for genotypes
NAA
α-naphthalene acetic acid
Na
Number of alleles
Ne
Number of effective allele
NJ
Neighbor joining
PCA
Principal component analysis
PCoA
Principal coordinate analysis
PCV
phenotypic coefficient of variation
RAPD
Randomly amplified polymorphic DNA
RFLP
Restriction fragment length polymorphism
SNP
Single nucleotide polymorphism
SSR
Simple sequence repeats
x
Abstract
Morphological and Molecular Characterization, Assessment of Nutritional Composition and
Micropropagation of Cocoyam (Xanthosoma sagittifolium (L.) Schott) from Ethiopia
Eyasu Wada Wachamo, PhD Dissertation
Addis Ababa University, June 2018
Cocoyam (Xanthosoma sagittifolium (L.) Schott) is a tuberous root crop in the Araceae family. It is an exotic crop to Ethiopia
that was introduced fairly recently but has spread widely and already become part of the agricultural and food systems of the
people, wherein tuber and root crops play an important role as sources of food. However, cocoyam has not been given research
attention commensurate to its importance as it is a neglected crop by research and development community. This study was
conducted to characterize cocoyam diversity at morphological and molecular levels through documenting farmers’
knowledge, perceptions and management practices; determining nutritional composition; and developing a micropropagation
protocol for this neglected crop. The present ethnobotanical survey results showed that the crop is given different local names
by farmers and that it is locally used for food, fodder, medicine and other purposes. Furthermore, the results showed that the
uses of cocoyam as a food crop and fodder are the most preferred traits as perceived by the farmers while hardness, sour taste,
unpleasant smell and low market demand were the major constraits for cocoyam production. Green- and purple-colored
cocoyam plants were observed during our survey. The field study helped to distinguish two classes of qualitative traits for
petiole color, lamina orientation, color of veins on leaf surfaces, position of cormel apex and shape of cormels. Analysis of
variance (ANOVA) revealed significant variation in 11(84.6%) of the 13 studied quantitative traits. Principal Component
Analysis (PCA) reduced the 13 quantitative traits to 3 Principal components (PCs) with the eigen values >1, which explained
69.2% of the observed variations. In the genetic diversity analysis using 11 SSR markers, a total of 36 alleles were detected
(mean 3.273). High SSR marker diversity was detected within populations (average Ho = 0.503 and He = 0.443) and when
all collections were considered as single population (Ho: 0.508, He: 0.566). Supporting these results, genetic diversity analysis
using AFLP markers revealed high Nei’s gene diversity (He) within populations (He = 0.349) and at the entire collection
level (He = 0.389). SSR markers revealed strong genetic differentiation among populations and between green and purple
cocoyam morphotypes by Fst values 0.196 and 0.463, respectively. However, unlike SSR markers, AFLP marker-based
analysis showed low genetic differentiation among populations (Gst = 0.072) as well as between green and purple cocoyam
morphotypes (Gst = 0.024). The nutritional composition analysis showed that both the green- and purple-cocoyam
morphotypes can provide nutrient-rich products, albeit slight differences in the quantities of proximate, minerals and
antinutritional factors. A micropropagation protocol was successfully developed in which the green- and purple-cocoyam
shoot tip explants were best initiated on Murashige and Skoog (MS) medium containing 2.0 mg/l BAP, best multiplied on
MS medium containing 2.5 mg/l BAP and 0.5 mg/l NAA and the best IBA concentration for rooting was MS medium
supplemented with 2.0 mg/l IBA. Overall, a lot of useful indigenous knowledge exists within the farming communities in the
rural areas, but cocoyam is poorly studied and underutilized crop in spite of its nutritional value & its potential as food crop.
The findings of this study are very important to enhance the future use of cocoyam in the country. Collaborative research
intervention involving the development of varieties, making available high quality planting material for farmers & promoting
value chains and market opportunities are valuable for sustainable use of the exiting diversity & to safeguard the potential
end users of cocoyam in the country.
Keywords: AFLP markers, genetic diversity, indigenous knowledge, in vitro propagation, morphological trait, neglected
crop, SSR markers
xi
Chapter One
General Introduction
1.1 Background
Edible aroids play a significant role in the livelihood of millions of relatively poor people in
the developing countries (Sarma et al., 2016). They are potential crops for food security,
income generation and nutritional enhancement at household level. They have a greater
ability to produce more energy per hectare per day and produce satisfactorily under adverse
conditions where other crops fail (Onwueme, 1999). Cocoyam (Xanthosoma sagittifolium
(L.) Schott) is one of the edible aroids in the Araceae family. It is the 6th most important root
and tuber crop in the world, after potato (Solanum tuberosum L.), cassava (Manihot
esculenta Crantz), sweet potato (Ipomoea batatas (L.) (Lam.), yam (Dioscorea spp.) and
taro (Colocasia esculenta (L.) Schott) (Bown, 2000).
Cocoyam is native to the northern part of South America (Giacometti and Leon, 1994). It is
widely naturalized in the Caribbean, Africa, Asia and Islands in the Pacific Ocean (Ponce,
2010). Nowadays, Africa is the major producer with the West and Central parts of the
continent (Nigeria, Ghana, Cameroon) contributing over 60% of the total production
(Onyeka, 2014; Owusu-darko et al., 2014). It was introduced into Eastern Africa through
Western Africa initially coming from tropical America (Maundu et al., 2009). Thus, it has
been known to be exotic to Ethiopia but there is no clear information on when and how it
was introduced into the country. However, cocoyam has widely spread and is now part of
the local food system of the people, wherein the root and tuber crops play an important role
in food security.
It grows even in poor soils and under dry conditions that are too difficult for cultivation of
other tuberous root crops at the Malo area of Gamo-Gofa zone (Fujimoto, 2009). It was
1
ranked second next to ensete (Ensete verticosum) (Welw.) Cheesman) among the top 10
most preferred plants around Bonga city in Kefa based on use values (Zemede Asfaw,
2001a). Amsalu Nebiyu and Tesfaye Awas (2006) alluded the presence of a considerable
amount of cocoyam gene pool in south and southwest Ethiopia in farmers’ fields and
homegardens.
Many developing countries experience great difficulty in sustaining the conservation and
genetic improvement of neglected and underutilized root and tuber crops, mostly aroids
(Lebot et al., 2005). Most of the neglected and underutilized crops are being conserved by
the elders or left to grow on their own (Matthews, 2002). Cocoyam is one of the neglected
and underutilized root and tuber crops (Doungous et al., 2015). A large pool of germplasm
is being lost due to lack of knowledge on its importance. These losses pose a threat to
germplasm conservation which determines the future use of genetic resources (Onwueme,
1999).
A proper analysis of the genetic variation and relationships between accessions is important
to understand the genetic variability and its potential use; estimate any possible loss of
genetic diversity; offer evidence of the evolutionary forces shaping the genetic diversity and
help to choose priority genotypes for conservation (Smith and Duvick, 1989). A prerequisite
for any genetic improvement programme is the knowledge of the extent of genetic variation
present among accessions and genetic distance among all closely related species with which
hybrids could be produced (Beeching et al., 1993). This can be achieved through assessment
of genetic diversity using genetic markers. Methods for detecting and analyzing genetic
diversity have gradually progressed from Mendelian analyses of discrete morphological
traits, to statistical characterization of continuously varying quantitative characters, to
2
electrophoretic assays of biochemical variants and to molecular examinations of DNA
variation (Zhang et al., 1993).
Cocoyam is valuable because most parts of the plant are edible (Lebot, 2009; Vanker and
Slaats, 2013). Cocoyam is mainly used as food and the plant parts are also used as
fodder/feed and medicine, including its use as antipoisonous agents against tarantula,
scorpion and snake bites (Boakye et al., 2018). The usefulness of food composition data at
the level of the genetic resource (i.e. taxonomic level below species) is becoming
increasingly acknowledged. Research has been providing data to confirm the nutritional
superiority of some neglected and underutilized crops and their wild relatives over other
more extensively utilized crops (Burlingame et al., 2009). Data on nutritional composition
would help to assess the value of neglected and underutilized varieties and encourage their
sustainable use as well as coming up with a database of nutrient rich plant species that will
help in planning nutritional intervention programmes (Grivetti and Ogle, 2000).
Cocoyam is mostly produced through vegetative multiplication which is constrained by
many obstacles including the dormancy period in which the initial materials for conventional
propagation undergo for approximately five weeks. The conventional method for
propagating cocoyam is not adequate to meet the demand because the propagules (materials
used as seeds) of cocoyam such as the cormels or their fragments are used for food (Wilson,
1984). Cocoyam is usually preserved under field condition which is risky since diseases or
natural catastrophes can cause the loss of genetic resources. In vitro storage of plants under
minimal growth conditions is a suitable alternative to rely on field collections (Caula et al.,
2008).
3
1.2 Statement of the problem
Cocoyam can play a considerable role in addressing food security as its tubers and leaves
serve as food source. However, research on aroid in general and cocoyam in particular is
rare. Cocoyam is a neglected and underutilized crop. The use potential, genetic diversity,
nutritional composition, in vitro propagation capacities and the likes of cocoyam grown in
Ethiopia have not been fully examined. There was no known research that was carried out
to document the farmers’ knowledge and perceptions of cocoyam. The farmers’ indigenous
knowledge of the agromorphological traits, uses and management of cocoyam exists largely
within the farming communities in the rural areas. The Ethiopian farmers who cultivate
cocoyam are the main owners of knowledge about the crop. There was a need to compile
data on farmers’ knowledge and perceptions of agromorphological traits, uses and
management of cocoyam for effective usage and conservation of the crop. Information on
genetic diversity and nutritional composition of cocoyam is scarce in Ethiopia. It is
important to assess the genetic diversity and population genetic structure, and nutritional
composition of Ethiopian cocoyam and to compile and disseminate the results. Cocoyam is
propagated vegetatively from corms, cormels and their fragments which are not adequate to
meet the demands for seed material. Leaving cocoyam propagules under field conditions for
an extended period poses risk from diseases, pests and natural catastrophes to the genetic
resources. Tissue culture techniques are reliable methods for production of planting
materials with the highest rates of multiplication. Currently, there is lack of tissue culture
protocol for cocoyam genotypes in Ethiopia. There was a need to develop an efficient
micropropagation protocol that can contribute to promote faster and massive production of
cocoyam.
4
1.3 Research questions, hypotheses and objectives
1.3.1 Research questions
This study addressed the following broad research questions:
1. What is the knowledge held by Ethiopian cocoyam cultivators on the agromorphology,
use and management of the crop?
2. What is the extent of genetic diversity among cocoyam accessions growing in Ethiopia?
3. What amount of proximate, minerals and antinutritional factors are found in cocoyam
growing in Ethiopia?
4. Can efficient in vitro propagation protocol be developed for Ethiopian cocoyam?
1.3.2 Hypotheses
The following null hypotheses have been tested.
1. Ethiopian cocoyam farmers have no experiential/customary knowledge on cocoyam
crop cultivated in their areas.
2. There is no genetic diversity within and among cocoyam accessions growing in
Ethiopia.
3. Cocoyam growing in Ethiopia do not have acceptable proximate and mineral contents
and antinutritional factors.
4. Cocoyam growing in Ethiopia cannot be propagated via in vitro culture.
5
1.3.3 Objectives of the study
General objective
The general objective of this study was to characterize cocoyam accessions collected from
various locations in southern and southwestern Ethiopia at morphological and molecular
levels through documenting farmers’ knowledge and perceptions of the crop, assessing
nutritional composition and developing a micropropagation protocol for cocoyam from
Ethiopia.
Specific objectives
The specific objectives of this study were to:
document farmers’ knowledge and perceptions of the constraints, trait preferences, uses
and management practices of cocoyam to guide production, variety design and
development and commercialisation of the crop in Ethiopia;
characterize cocoyam accessions based on morphological traits and select farmerpreferred and high performing genotypes;
examine the genetic diversity and differentiation of cocoyam accessions using SSR
markers to complement morphological data;
assess the genetic diversity and population structure of cocoyam accessions from
Ethiopia using AFLP markers to complement data collected based on SSR markers and
morphological traits;
determine proximate, minerals and antinutritional factors of green- and purple cocoyams
growing in Ethiopia;
establish micro-propagation technique using shoot tip explants derived from green- and
purple- cocoyams grown in Ethiopia and to recommend optimum composition of plant
growth regulators.
6
1.4 Organization of the dissertation
This dissertation is organized as follows:
Chapter One presents the general introduction of the dissertation which includes the
background, statement of the problem and objectives of the study. Chapter Two presents the
literature review on cocoyam origin, distribution, taxonomy, botany, importance and use
values, growth condition and cultivation, breeding and tissue culture and genetic diversity
studies. Chapter Three presents farmers’ knowledge, perceptions and management practices
of cocoyam based on the results of the semi structure interview conducted, together with the
analyses and the discussion made. This was published in the African Journal of Agricultural
Research 12 (35): 2681-2691(2017). Chapter Four presents the work on the genetic
diversity, differentiation and population structure of cocoyam from Ethiopia based on
morphological traits and molecular markers (SSR and AFLP), of which the AFLP molecular
markers-based study part is submitted for possible publication. Chapters Five and Six,
respectively, present the nutritional compositions and micropropagation experiment
conducted to develop protocol for massive and faster production of green and purple
cocoyam morphotypes growing in Ethiopia. These chapters are prepared as manuscripts.
Chapter Seven is the part that presents the synthesis bringing together all the findings from
the various aspect followed by conclusion and recommendations.
7
Chapter Two
Literature Review
2.1 Origin and distribution of cocoyam
Cocoyam (Xanthosoma sagittifolium (L.) Schott) is likely to have been domesticated and
cultivated in the northern part of South America from very ancient times (Giacometti and
Leon, 1994). Since recent times, it has been widely spread throughout the tropical world.
The main areas of distribution of the crop include the Caribbean (Cuba, Dominican
Republic, Puerto Rico, West Indies), USA (Florida, Hawaii), West Africa (Ghana, Nigeria,
Cameroon, Togo) and tropical Asia (Indonesia, Malaysia, the South Pacific Islands) (Ponce,
2010). It was introduced to Central and West Africa between the 16th and 17th centuries and
has become a subsistence crop in West African countries, which are the major cocoyam
producers of the world (Bown, 2000). It was introduced into East Africa through Western
Africa and is popular in Tanzania and common in Uganda (Maundu et al., 2009). In
Ethiopia, it is largely unknown or considered synonymously with Colocasia esculenta (L.)
Schott), which is locally known as taro or
GODERE
(Simone, 1992). There is no clear
information on how and when cocoyam was introduced into the country. It was not
mentioned in the Flora of Ethiopia wherein the tribes of the Araceae are described (Reidl,
1997). The species was found during an ethnobotanical survey of homegrdens and annotated
as a new record for the Ethiopian Flora (Zemede Asfaw, 2001a). However, the crop was
missed out in volume eight of the Ethiopian Flora, which is supposed to have a checklist of
all the plants (wild and cultivated) found in Ethiopia (Hedberg et al., 2009).
2.2 Taxonomic description of cocoyam
The genus Xanthosoma belongs to the Colocasioideae subfamily, tribe Caladieae of the
family Araceae. The basic chromosome number for the Xanthosoma sagittifolium is n=13
8
(Mayo et al., 1997). Based on the color of underground stem (corm), the color and shape of
off shoots (cormels) and the color of petiole and leaf, some authors allocate the cultivated
species of the genus Xanthosoma into X. atrovirens K. Koch & C.D. Bouche, X. violaceum
Schott (X. nigrum, Stellfeld), X. caracu K. Koch & C.D. Bouche and X. sagittifolium (L.)
Schott and X. mafaffa (L.) Schott (Wilson, 1984; Manner, 2011). Others allocate the
cultivated species of the genus only into two main species (X. sagittifolium (L.) Schott and
X. violaceum Schott) (Salazar et al., 1985; Monge et al., 1987; Bown, 2000, Weaver, 2000;
Sarma et al., 2016). However, these species share some characterstics and viable fruit
formation have been reported (Oghenekome et al., 1992; Onokpise, 1992; Tambong et al.,
1992), suggesting that the two may be varieties of the same species.
In general, the division of the genus Xanthosoma into species has been difficult (Saborio,
2007). There is much confusion, discrepancies and uncertainties regarding the taxonomy of
Xanthosoma at the species level (Giacometti and Leon, 1994). Various names have been
used synonymously (http://www.theplantlist.org; Govaerts et al., 2002; Lim, 2015), the
same plant being given more than one species name (O’Hair and Maynard, 2003). Thus, the
name of Xanthosoma sagittifolium (L.) Schott has usually been given to the most cultivated
members of this genus (Giacometti and Leon 1994; Govaerts et al., 2002). In the course of
this study, we refer to the accepted species status of X. sagittifolium in the Araceae family
(Govaerts et al., 2002) and in The Plant List (http://www.theplantlist.org), which does not
tackle taxonomy in further detail.
2.3 Etymology and common names
The etymology of the genus Xanthosoma comes from the Greek Xanthos (yellow) and soma,
somatos (body) refers to the yellow color of the stem tissue present in several species (Mayo
et al., 1997). Many common names have been listed for the genus Xanthosoma in different
9
publications (Morton, 1972; Giacometti and Leon, 1994; Lim, 2015). In English, for
example, Xanthosoma species are known as cocoyam, new cocoyam, tannia, yautia,
American taro, arrowleaf, elephant’s ear, malanga (Lebot, 2009; Quero-Garcia et al., 2010;
Lim, 2015). Xanthosoma sagittifolium (L.) Schott) and (C. esculenta (L.) Schott) frequently
share the common terms cocoyam and taro (Shewry, 2003; Manner, 2011).
2.4 Botanical description of cocoyam
Cocoyam is an herbaceous monocotyledonous crop up to 2 m tall with long petioles
(Ramawat and Merillon, 2014). There are several leaves with large sagittate or hastate (nonpeltate) blades arising directly from the corm (Mayo et al., 1997). The leaves have a
marginal vein and two large basal lobes. Cocoyam has unisexual flowers, with the female
flowers located at the base of a spadix and the male flowers at the top. Between the pistillate
and the staminate flowers, sterile flowers are located. The inflorescence is protogynous,
pistillate flowers are normally receptive 2 to 4 days before pollen shed (Kay, 1987).
Cocoyam can be distinguished morphologically from two other related aroids, mainly taro
(C. esculenta (L.) Schott) and giant taro (Alocasia acuminata Schott), by the place where
the petiole meets the leaf and the angle between the petiole and leaf blade. In cocoyam, the
petiole attachment is at the margin of the leaf between the lobes. In taro, the petiole
attachment is peltate or around the middle of the leaf (Manner, 2011). In cocoyam, mature
leaves are angled about 30º off the petiole but for giant taro, the leaf blade and the petiole
are in the same plane (Mayo et al., 1997).
2.5 Importance and use values of cocoyam
Cocoyam and taro are widely consumed, very ancient aroids included in more than 400
million people’s diets. They play very important roles in the livelihood of rural farmers who
often use the crops as alternative source of their daily calories (Onyeka, 2014). Cocoyam
10
has become much more important worldwide (Mwenye, 2009). In many tropical areas, it
has overtaken taro as the main edible aroid and has been replacing taro because of its better
yield (Bown, 2000). In Pacific Island, cocoyam is often grown because of the higher yield,
larger tuber size and taste and hence it is increasingly being adopted by Island peoples
(Manner, 2011). Cocoyam is valued as a superior species compared with taro because of its
flavor and texture in most Latin America countries (Giacometti and Leon, 1994). Around
Bonga city in Kefa, Ethiopia, cocoyam was ranked among the most preferred plants based
on the use values, cultural significance and other reasons (Zemede Asfaw, 2001a). Cocoyam
is valuable because most parts of the plant can be used for food (Lebot, 2009). It is mainly
cultivated for its starchy cormels, but young leaves can be used as a green vegetable and an
important source of proteins and vitamins. The central or primary tube (the corm) is acrid
and not used for human consumption in most edible species of cocoyam (Lebot, 2009;
Manner, 2011), rather mostly used to feed animals. Cocoyam is also used for traditional
medicine (Manner, 2011).
2.6 Nutritional profile of cocoyam
Cocoyam is postulated to have superior nutritional value over other major root and tuber
staples of West Africa, especially in terms of their protein digestibility and mineral
composition (Calcium, Phosphorous and Magnesium) (Lim, 2016).The nutritional
composition of cocoyam per 100g edible portion was reported by Kay (1987) as energy 556
kJ/100 g, moisture 70–77 %, protein 1.3–3.7 %, fat 0.2–0.4 %, carbohydrate 17–26 %, fibre
0.6–1.9 %, starch 17–34.5 %, ash 0.6–1.3 %, Ca 20 mg, Fe 1 mg, thiamine 1.1 mg, riboflavin
0.03 mg, niacin 0.0005 mg and ascorbic acid 6–10 mg. The mean values of the proximate
composition of cocoyam, red flesh corms and white flesh corms from Ghana were reported
by Sefa-Dedeh (2004), respectively, as moisture 57.63–77.41% g and 54.46–71.97%;
protein 3.94–4.09% and 4.92–5.50%; fat 0.43–0.74% and 0.28–0.58%; ash 2.68–3.93% and
11
1.98–3.29%, fibre 1.16–1.77% 1.11–1.72%. The protein, fat, ash and fibre contents
decreased in the cooked samples (Akpan and Umoh, 2004). The mineral contents of raw
cocoyam corm based on analyses conducted in Nigeria were reported as per 100 g edible
portion: Na 66 mg, K 525 mg, Mg 46.6 mg, C 18.64 mg, Fe 0.42 mg, P 70.4 gm Zn 0.4 mg
and Mn 0.63 mg. Both white- and red-fleshed corms provide good sources of Na, K, Mg and
Ca (Owuamanam et al., 2010). Mineral elements analysis showed general decreases in the
cooked samples (Akpan and Umoh, 2004). Akpan and Umoh (2004) found no significant
differences between the oven-dried and solar-dried samples. However, drum drying reduced
the oxalate levels by approximately 50% to average levels and a general reduction in the
level of antinutrients was observed after heat treatments. There was an improvement in food
quality with respect to the antinutrients but with decreased values of the desired nutrients.
However, there was also a general reduction in the undesirable properties of the cocoyam
such as the acridity factors caused by crystal of oxalate when the cocoyam corms were
cooked (Akpan and Umoh, 2004).
2.7 Growth condition and cultivation of cocoyam
Cocoyam was domesticated in the New World and was under cultivation in tropical America
since the Pre-Columbian times (Purseglove, 1972). It is adapted to grow in great variety of
substrates and habitats ranging from full sun to deep shaded areas beneath the canopy of
natural forests (Manner, 2011). It grows in a wide range of soils except in hard clays or pure
sands. It tolerates drier soils, but does not tolerate waterlogged soils (Kay, 1987; Bown,
2000). It produces the best yield on moist, mulched and well-drained soil with pH of 5.56.5 (Jackson, 2008). Although cocoyam is a lowland crop, it grows well in upland situations
with well distributed rain fall (Manner, 2011). It can be grown under a mean annual
temperature of 24°C with variations ranging from 13 to 29°C and in areas where the annual
rainfall is 1,000-2,000 mm (Kay, 1987). Yellowing of older leaves indicates maturity.
12
Harvest usually occurs during the dry season after 9-12 months, but cormels can be
harvested after 6-7 months of growth. In water stressed conditions, leaves may die but corms
can continue to grow as a perennial crop (Jackson, 2008). Corms can remain viable
underground and survive through unfavorable environmental conditions (Castro, 2006).
Cocoyam is propagated vegetatively from corm sets, headsets or cormels (Jackson, 2008).
The portion of the corm or cormels is placed in the furrows and covered with a 5 to 7 cm
layer of soil. Root formation takes place immediately after planting followed by rapid
growth of the shoot (Castro, 2006). Corm set produces a quicker and higher yielding crop
while headsets are the lowest yielding (Kay, 1987). To facilitate the growth, the soil needs
to be well ploughed and furrowed. Plantation is just before the start of the wet season.
However, in areas where rainfall is 2,000-3,000 mm and if an irrigation system is available,
planting can be made throughout the year (Wilson, 1984).
2.8 Cocoyam breeding and tissue culture
Flowers are rare in most cocoyam varieties and non-existent in some. When flowers occur
in some varieties, they are protogynous (Jackson, 2008), the pistillated flowers are normally
receptive 2 to 4 days before pollen is shed (Wilson, 1984). This makes the use of classical
breeding methods difficult, but treatments were successfully applied to induce flowering
with artificial pollination techniques. Induction of flowering, hand pollination and
production of seed and seedlings has become possible at experimental settings (Wilson,
1979; Goenaga and Hepperly, 1990). A spray of gibberellic acid on the tissue culture derived
cocoyam plants has provided a high number of inflorescences as well as pollen quantity
(Onokpise, 1992). Cocoyam genotypes were combined through artificial crossings but no
new improved varieties were reported (Castro, 2006).
13
In Ethiopia, propagation of cocoyam is entirely convenstional from tuber fragments, which
was constrained by scarcity of planting materials (Thehifet Solomon, 2010). Preserving
cocoyam under field condition is risky since diseases or natural catastrophes can cause the
loss of genetic resources (Caula et al., 2008). Tissue culture technique will open up many
possibilities for sustainable production and improvement of many crops. The technique is
important in achieving rapid clonal multiplication, recovery of pathogen free plants and
germplasm conservation (Larkins and Scowcroft, 1981). It has given the possibility to
generate new genotypes through somaclonal variation and a variety of morphological
changes were observed in callus derived cocoyam plants (Gupta, 1985). The flowering of
tissue culture derived cocoyam occurred 20-30 days earlier than reported for non-tissue
culture derived plants (Onokpise, 1992). Induction of tetraploid was successfully achieved
by incorporating colchicine in tissue culture medium (Esnard et al., 1993). Cormels from
meristem derived cocoyam grew faster and the tuberization started earlier in comparison
with conventionally propagated cocoyam (Castro, 2006). In vitro protocol was investigated
for callus induction and regeneration of cocoyam, offering an opportunity for culturing virus
free propagules and undertaking genetic engineering research (Paul and Bari, 2007).
Temporary immersion system was reported to be an alternative micropropagation technique
to obtain high quality planting material in cocoyam (Pino et al., 2012; Niemenak et al.,
2013).
2.9 Assessment of genetic diversity
Plant genetic resources comprise a diversity of genetic material contained in traditional
varieties, modern cultivars and crop wild relatives and other wild species. Genetic diversity
provides options to develop new and more productive crops that are resistant to pests and
diseases and adaptive to changing environments (Rao, 2004). Measurement and
characterization of genetic diversity have always been a primary concern in population
14
genetic studies (Zhang et al., 1993). Understanding the genetic variation within and among
populations is crucial for the effective conservation, management and efficient utilization of
plant genetic resources. An adequate knowledge of existing genetic diversity in plant
population is of fundamental interest for basic science and applied aspects. The ability to
identify the genetic diversity is indispensable to effective management and utilization of
genetic resources (Mondini et al., 2009). Thus, genetic resource conservation activities
require characterization of the germplasm (Karp et al., 1997), which is performed using
genetic markers (Mondini et al., 2009).
The genetic markers are broadly categorized into three major types: morphological markers,
biochemical markers (or isozyme markers) and DNA or molecular markers (Collard et al.,
2005).
2.9.1 Morphological markers
Genetic diversity is assessed by measuring variation in qualitative morphological traits such
as texture, color, growth habit or quantitative traits like yield potential, height, size, weight,
etc. The morphological traits have their basis in genetic alterations that lead to visible
differences in the phenotype (Rao, 2004). The morphological characterization does not
require expensive technology. This approach has, however, certain limitations: large tracts
of land are often required for the experiments; highly heritable traits often show little
variation; the genetic information provided by morphological characters is often limited and
expression of quantitative traits is subjected to strong environmental influence because only
a small fraction of genes code for traits is manifested in observable phenotypes (Karp et al.,
1997; Mondini et al., 2009). The use of morphological markers alone, therefore,
immediately excludes analysis of those portions of the genome containing non-coding
sequences, which in plants can often account for more than 90% of the complete DNA
15
sequence. Thus, using morphological traits alone is often undesirable although cannot be
replaced by any of the biochemical or molecular techniques (Smith and Smith, 1989). The
results of biochemical or molecular studies should be considered as complementary to
morphological characterization (Karp et al., 1997).
2.9.2 Biochemical Markers
Biochemical markers reveal differences between seed storage proteins or enzymes encoded
by different alleles at one gene locus (allozymes) or more gene loci (isozymes) (Rao, 2004).
Allozymes are variant proteins produced by allelic forms of the same locus. On the other
hand, isozymes are different molecular forms in which proteins may exist with the same
enzymatic specificity (Markert and Moller, 1959). The more general term for allozymes is
isozymes, and refers to any variant form of an enzyme, whereas allozyme implies a genetic
basis for the variant form. Isozymes can be differentiated by their relative migration speed
during gel electrophoresis due to the amino acid charge differences (White et al., 2007).
The major advantages of biochemical markers consist in assessing co-dominance, ease of
use and it is a fast method which requires small amounts of biological material. They allow
large numbers of samples to be analyzed. The technique is comparatively inexpensive yet
powerful method of measuring allele frequencies for specific genes within and among
populations, in genetic relatedness studies, mating system estimations and genetic diversity
assessments (Mondini et al., 2009). The major disadvantages of biochemical markers are
the limited number and are influenced by environmental factors or developmental stage
(Winter and Kahl, 1995). There are only few isozyme systems per species with
corresponding markers; the enzymatic loci represent only a small and not random part of the
genome (the expressed part). Therefore, the observed variability may not be representative
of the entire genome. They are unable to detect low levels of variations. Comparisons of
16
samples from different species, loci and laboratories are problematic since they are affected
by extraction methodology, plant tissue and developmental stage and environmental factors
(Mondini et al., 2009).
2.9.3 Molecular Markers
Molecular markers are the most widely used type of genetic markers. Their analyses
comprise a large variety of DNA markers which arise from different classes of DNA
mutations such as substitution, insertions, deletions and errors in replication of tandemly
repeated DNA (Paterson, 1996). The molecular markers are selectively neutral because they
are usually located in non-coding regions of DNA. They are unlimited in number and are
not affected by environmental factors and/or the developmental stage of the plant. They are
detectable in all stages of plant growth (Winter and Kahl, 1995).
Molecular markers have numerous applications in plant genetics such as assessing the level
of genetic diversity within germplasm and cultivar identity. They offer great opportunity for
improving the efficiency of conventional plant breeding by carrying out selection not
directly on the trait of interest but on molecular markers linked to that trait (Mohan et al.,
1997). They are powerful diagnostic tools used to detect DNA polymorphism both at the
specific loci and whole genome level (Laurentin, 2009).
All types of molecular marker assays have different properties. An ideal molecular marker
should possess the following features: (1) be polymorphic and evenly distributed throughout
the genome; (2) provide adequate resolution of genetic differences; (3) generate multiple,
independent and reliable markers; (4) be simple, quick and inexpensive; (5) need small
amounts of tissue and DNA samples; (6) have linkage to distinct phenotypes and (7) require
no prior information about the genome of an organism. Nevertheless, no molecular marker
presents all the listed advantages. Thus, the most important criteria to determine the type of
17
molecular marker should be informativeness and ease of genotyping for the specific crop
system (Powell et al., 1996; Agarwal et al., 2008).
2.10 Molecular marker techniques
The basic molecular markers techniques have been broadly categorized into DNA-DNA
hybridization-based techniques and polymerase chain reaction (PCR) based techniques. The
property of complementary base pairing allowed for DNA-DNA hybridization-based
techniques to be developed whereby small pieces of DNA could be used as probes to reveal
polymorphisms in the sequences homologous to the probe. PCR technique enables the
production of a large amount of a specific DNA sequence without cloning, starting with just
a few molecules of the target sequence (White et al., 2007).
i. DNA-DNA hybridization-based technique
Restriction fragment length polymorphism (RFLP) is first class DNA-DNA hybridizationbased markers and used for detecting variation at the DNA sequence level (Botstein et al.,
1980). In RFLP technique, DNA polymorphism is detected by hybridizing a chemically
labeled DNA probe to a Southern blot of DNA digested by restriction endonucleases,
resulting in differential DNA fragment profile. The differential profile is generated due to
nucleotide substitutions or DNA rearrangements like insertion or deletion (Agarwal et al.,
2008). The RFLP markers are highly polymorphic, codominantly inherited and highly
reproducible; exist throughout the genome, high heritability and locus specific. The RFLP
based method provides an opportunity to simultaneously screening of numerous samples.
However, the technique is time-consuming, involves expensive reagents, requires large
quantities of high quality DNA and prior sequence information for probe generation. These
limitations led to the conceptualization of a new set of less technically complex methods
known as PCR based techniques (Mondini et al., 2009).
18
ii. The PCR based techniques
Random Amplified Polymorphic DNA (RAPD): The RAPD technique uses a short single
primer (usually 10 bases) to amplify anonymous stretches of DNA under specific PCR
condition. RAPD provides a more arbitrary sample of the genome and can detect unlimited
number of loci. The number of amplified fragments depends on the distribution and number
of annealing sites throughout the genome (Marwal et al., 2014). There is no specific target
DNA, so each particular primer adheres to the template DNA randomly. As a result, the
nature of the obtained products is unknown. RAPD analysis shows the difference in the
pattern of bands amplified from genetically distinct individuals behaves as Mendelian
genetic markers. The most limiting properties of RAPD molecular marker technique are low
reproducibility, dominance inheritance and homology. Although these problems were
raised, RAPDs have been widely used for studies on taxon identification, hybridization and
population genetic structure studies (Semagn et al., 2006).
Amplified Fragment Length Polymorphism (AFLP): The AFLP is DNA fingerprinting
technique that combines the power of the RFLP with the flexibility of PCR based technique
by ligating primer recognition sequences (adaptors) to the restricted DNA (Lynch and
Walsh, 1998). This technique involves the following steps: (1) restriction of the DNA and
ligation of oligonucleotide adapters; (2) Pre-selective amplification and selective
amplification of sets of restriction fragments and (3) gel analysis of the amplified fragments.
The AFLP technique is a powerful DNA fingerprinting technique applicable to any organism
without the need of prior sequence information (Vuylsteke et al., 2007). It has a capacity to
simultaneously screen representative DNA regions distributed throughout the genome
(Semagn et al., 2006). The technique is highly reproducible and sensitive for detecting
polymorphism. The molecular basis of AFLP polymorphism can be a nucleotide
19
polymorphism in the restriction sites or selection nucleotides adjacent to the restriction sites.
In addition, deletions, insertions and rearrangements affecting the presence or size of
restriction fragments can result in detectable polymorphism (Paun and Schonswetter, 2012).
The major advantage of the technology is in the high marker density which allows scoring
of a large number of markers in a given population. The frequency with which AFLP
markers are detected depends on the sequence polymorphism between the tested DNA
samples (Somers, 2004). It has been employed for a variety of applications such as to assess
genetic diversity within species or among closely related species, to infer population level
phylogenies and biogeographic pattern, to generate genetic maps and to determine
relatedness among cultivars (Somers, 2004; Paun and Schonswetter, 2012).
Inter Simple Sequence Repeats (ISSR): The ISSR technique is a PCR based multilocus
marker system that employs oligonucleotide primers homologous to microsatellites. PCR
product obtained only if simple sequence repeats (SSRs) are found in opposite orientation
within a PCR amplification distance, with flanking sequence matching the oligonucleotides
(Tomar et al., 2010). It is semi-arbitrary markers amplified by PCR in the presence of one
primer. It does not require genome sequence information (Meyer, 1993). It is a fast,
inexpensive genotyping technique based on variation in the regions between SSRs. It is
highly polymorphic but dominant marker. The method has a wide range of uses including
genetic fingerprinting, gene tagging, clonal variation detection, cultivar identification,
phylogenetic analysis and assessment of hybridization (Abdel-Mawgood, 2012). Like
RAPD markers, ISSR markers are quick and easy to handle, but they seem to have the
reproducibility of SSR markers because of the longer length of primers (Bornet and
Branchard, 2001).
20
Simple Sequence Repeats (SSR): The SSRs also called microsatellites are short tandem
repeats, their length being 1 to 10 bp, most typically, 2-3 bp. The number of repeated units
varying widely among organisms as high as 40 copies of the repeated unit (Lopez-Flores
and Garrido-Ramos, 2012). When DNA is being replicated, errors occur in the process and
extra sets of these repeated sequences are added to the strand. Over time, these repeated
sequences vary in length between one cultivar to another. The DNA sequence flanking SSRs
are known to be conserved within species and sometimes in the higher taxa. The DNA
sequence flanking SSRs have been used to design suitable primers for amplification of SSR
loci. Differences in length of the PCR product from different genotypes reveal the SSR
polymorphism. The length difference attributed to the variation in the number of repeat units
of a particular locus possibly caused by slippage during DNA replication (Tomar et al.,
2010).
SSR markers are the most widely applied class of molecular markers used in genetic studies
with applications in many fields of genetics including genetic conservation, population
genetics, molecular breeding and paternity testing. This range of applications is due to the
fact that they are co-dominant and multi-allelic, highly reproducible and have high
resolution (Oliveira et al., 2006).
Single nucleotide polymorphism (SNP): The SNPs are the most abundant class of DNA
markers. They are single base pair positions in genomic DNA at which different sequence
alternatives (alleles) exist in normal individuals in some populations, wherein the least
frequent allele has an abundance of at least 1% or greater (Brookes, 1999). SNPs are less
mutable as compared to other markers, particularly microsatellites. The low rates of
recurrent mutation make them evolutionarily stable. SNPs are a novel class of DNA markers
that recently became highly preferred in genomic studies. They occur more frequently than
21
any other type of marker and are very near to or even within the gene of interest. They have
tremendous applications and prospects in crop genetics. They are excellent markers for
studying complex genetic traits and for understanding the genomic evolution. They can be
used in association studies, tagging of economic important genes, genotyping, diversity
analysis and evaluation among the species (Jehan and Lakhanpaul, 2006). The molecular
marker techniques differ from each other with respect to important features (Table 2.1).
Table 2.1 Comparison of different characteristics of molecular markers
Molecular marker
RFLP RAPD
AFLP
ISSR
SSR
SNP
Degree of polymorphism
High
medium medium medium medium
high
Locus specific
Yes
no
no
no
no
yes
Dominance (D)/ co-dominance (C)
C
D
D
D
C
C
Reproducibility
high
low
high
high
high
high
Abundance
high
high
high
high
medium
high
Sequence information required
yes
no
no
no
yes
yes
Quantity of DNA required
high
low
medium
low
low
Low
Amenable to automation
no
yes
yes
yes
yes
yes
Technical requirement
high
low
medium
low
medium medium
Cost per assay
high
low
medium
low
medium medium
RFLP - restriction fragment length polymorphism; RAPD - random amplified polymorphic DNA; AFLP
- amplified fragment length polymorphism, ISSR - inter simple sequence repeats; SSR - simple sequence
repeat; SNP - single nucleotide polymorphism (compiled from Oliveira et al., 2006; Agarwal et al.,
2008; Henry, 2008 and Mondini et al., 2009)
Characteristics
2.11 State of knowledge on cocoyam genetic diversity
Morphological, biochemical and molecular based genetic markers have been used for
genetic diversity analysis of cocoyam accessions. Historical and morphological evidences
were used to clarify the diagnostic features of cocoyam species growing in Nigeria
(Mbouobda et al., 2007). The multiple component analyses of morphological parameters
were used to distinguish taro and cocoyam genotypes in Malawi (Mwenye, 2009).
Morphological parameters which were used to estimate genetic parameters for yield and its
components in cocoyam genotypes from Bangladesh showed the presence of significant
differences with wide ranges of variation among the genotypes (Paul and Bari, 2012). The
22
authors stated that the genotypic variances for most of the characters were remarkably higher
than their corresponding environmental variances. In Ethiopia, the genetic diversity of
cocoyam collections was studied using multivariate analysis at Jimma Agricultural Research
Center based on 16 quantitative traits (Solomon Fantaw et al., 2014a). The authors stated
that the existing diversity among the cocoyam genotypes can give an opportunity for genetic
improvement for desirable characters.
Xanthosoma species from different locations in Indonesia were characterized using the
morphological parameters and isozyme markers. The results showed that the correlation
between morphological data and data from esterase and glutamate were very good (0.97 and
0.94), but there were no obvious differences among the samples from different locations of
the country (Nurmiyati et al., 2009).
RAPD analysis of cocoyam has revealed that very little genetic variation exists within the
USDA-ARS collections in Florida (Schnell et al., 1999). But in Ghana, 70 cocoyam
accessions analyzed with RAPD markers showed significantly higher polymorphisms (Offei
et al., 2004). The AFLP analysis was carried out to assess the status of species of Caladium
versus Xanthosoma showed that AFLP can distinguish between the different species by their
unique and different banding patterns (Loh et al., 2000). RAPD analyses, - (CA) 8RYmicrosatellite repeat unit, chloroplast- (trnR/Q 01; trnL 03/04) and mitochondrial-specific
(NAD 4.2/4.3; rps14/COB) primer pairs revealed 4 species of Xanthosoma among the
cocoyam accessions from Jamaica (Brown and Asemota, 2009). SSR markers identified by
Cathebras et al. (2014) gave variable degrees of heterozygosity, observed at levels ranging
from 0.00 to 0.97. A set of six chosen long terminal repeat (LTR) primers yielded 92%
polymorphic bands across 20 cocoyam accessions (Doungous et al., 2015).
23
Chapter Three
Farmers’ Knowledge and Perception of the Constraints, Trait
Preferences, Uses and Management Practices of Cocoyam
(Xanthosoma sagittifolium (L.) Schott) in Ethiopia
Abstract
Cocoyam (Xanthosoma sagittifolium (L.) Schott) is one of the tuberous root crops in the
monocotyledonous family known as Araceae and has been grown in Ethiopia for a good
number of years. Less research attention is given to cocoyam as it is one of the neglected and
underutilized crops in Ethiopia. There was no concrete ethnobotanical research that has been
carried out to document indigenous knowledge held by Ethiopian farmers cultivating and
using cocoyam. In this study, a survey was conducted to assess the state of cocoyam in
Ethiopia based on farmers’ knowledge and perceptions. Purposive sampling technique was
used to select 50 farmers from five zones. During our survey, two morphotypes (green
colored and purple colored) were observed. Different local names were given to farmerrecognized types of the crop. The most commonly encountered local names were KENI ZHANG,
CUBI ZHANG, SUDAN KIDO
and SAMUNA BOINA.
The naming systems were, in most cases, followed
by the local name given to taro (Colocasia esculenta (L.) Schott), as seen in the cases of
ZHANG
and BOINA. The local term GODERE or taro was also used for both species. Cocoyam is
locally used for food (100%), fodder (60%) and other purposes such as medicine and organic
fertilizer. Farmers use local methods in the preparation of cormels for food and medicine.
Corms were the preferred planting materials for Ethiopian farmers. The farmers’ preference
to the crop was related mainly to serving as emergency food and as fodder whereas hard
texture, low market demand, sour taste and unpleasant smell of cocoyam were traits disliked
by farmers. In this study, useful comprehesive knowledge about cocoyam in Ethiopia was
documented and this helped to sharply focus on the morphological and molecular traits, the
nutritional compositions and the micropropagation aspects. The quality and productivity of
cocoyam need to be improved based on farmer preferred attributes to ensure dissemination
of the useful aspects and enhance its sustainable production in Ethiopia.
Keywords: Cocoyam, ethnobotany, field survey, indigenous knowledge
24
3.1 Introduction
Cocoyam (Xanthosoma sagittifolium (L.) Schott) is an herbaceous tuberous root crop that
belongs to the monocotyledons in the Araceae family. Cocoyam is likely to have been
domesticated in the northern part of South America where it was cultivated from very
ancient times (Giacometti and Leon, 1994). It is widely cultivated in tropical America,
Africa, Asia, Caribbean and other parts of the tropics mainly by small-scale farmers (Bown,
2000). It is unknown to many people in east Africa (Raemaekers, 2001). In Ethiopia,
cocoyam is largely known synonymously with taro (Colocasia esculenta (L.) Schott), which
is known to have been grown in Ethiopia since immemorial times (Simone, 1992). Cocoyam
was not mentioned in the Flora of Ethiopia wherein the tribes of the Araceae are described
(Reidl, 1997). Later in 2001, it was listed among the crop species cultivated in Ethiopian
homegradens and annotated as a new record for the Ethiopian Flora (Zemede Asfaw, 2001a).
But other reports mentioned that cocoyam accessions had been introduced into Ethiopia
early in 1980s (Fujimoto, 2009). Surprisingly, however, the crop was again missed out in
volume eight of the Ethiopian Flora (Hedberg et al., 2009), which is supposed to have a
checklist of all the plants (wild and cultivated) found in Ethiopia.
Cocoyam has spread widely and has become an important part of the agricultural and food
systems of indigenous communities in southern, southwestern and western parts of Ethiopia,
where root and tuber crops are part of the local food systems of the people. It was ranked by
farmers second among the top 10 most preferred plants around Bonga city, in Kefa, based
on the use values, adaptability, cultural significance and other reasons (Zemede Asfaw,
2001b). It grows even in poor soils and under dry conditions that are too difficult for
cultivation of other tuberous root crops. It diffused mainly into the lowest settlements (below
1000 m.a.s.l) (Fujimoto, 2009).
25
Despite its increasing importance, limited research efforts have been directed to cocoyam.
The preservation and use of cocoyam is far less unlike other root crop genetic resources
(Matthews, 2002). The loss of germplasm has become the major challenge to its production
and poses a threat to germplasm conservation (Onwueme, 1999). Most of the research on
neglected and underutilized crops are being conserved by the elders and/or are being left to
grow on their own. Hence, these are being lost due to lack of knowledge on their importance
(Matthews, 2002). Ethnobotanical study can be important in genetic resource conservation
and application in crop improvement. Early advances in ethnobotany provided us with
utilitarian benefit of plants and on that basis, plants were classified. Today, such
documentation is essential for the conservation of earth’s vast biological resources (Osawaru
and Ogwu, 2015). Knowledge on different qualities that affect the use, preparation and
consumption is important to plant breeders because it is critical for the acceptance of new
cultivars by consumers (Matthews, 2002).
Research on cocoyam has been scarce in Ethiopia except a few attempts initiated at
agricultural research centers to collect and maintain its germplasm. Ethiopian farmers hold
enormous indigenous knowledge of the crop that they cultivate. They are the main owners
of knowledge about the uses, cultivation practices and management of the crop. No concrete
work to date has looked at the cocoyam grown in the country in commensurate with its
importance to smallholder farmers. The indigenous knowledge of farmers on cocoyam has
remained largely within the domain of farmers’ knowledge in the rural areas. The main aim
of this study was, therefore, to collect the knowledge that is available with the farmers on
cocoyam to retrieve the knowledge held by the farming communities who cared to manage
and use this neglected and underutilized crop species.
26
3.2 Materials and methods
3.2.1 Description of the study area
The Federal Democratic Republic of Ethiopia is composed of regional states. The regions
are organized into zones which are cluster of woredas. Kebeles are the smallest
administrative units within the Woredas. The study area covered the cocoyam belt between
latitudes 06°20.301’ N and 07°25.213’ N and longitudes 035°29.829’ E and 037°47.173’ E.
Farms were located at altitudes from 1132 to 2319 meters above sea level (Fig. 3.1; Table
3.1).
Fig. 3.1 Map of Ethiopia showing the location of the study area. Map was prepared
with ArcGIS Desktop (ArcGIS Desktop 10.2.2., Esri)
3.2.2 Sampling frame and informant selection
Farmers’ knowledge and perceptions of cocoyam was collected from 10 Woredas of five
zones (Table 3.1) involving 50 informants, following the steps in purposive sampling
technique outlined by Tongco (2007). Purposive sampling technique is a type of nonprobability sampling that is most effective when one needs to describe a phenomenon and
to generate data with knowledgeable informants (Tongco, 2007). To gather data from
27
knowledgeable and reliable informants, two Woredas in which cocoyam grows at a larger
extent were purposefully selected from each of five zones by consulting zonal agricultural
offices. Five farmer-experts who have rich experience in growing cocoyam were
purposefully selected from each woreda with the assistance of leaders of the farmers’
associations and plant experts of the farmers’ associations.
Table 3.1 Study areas and number of respondents replied to interview guide
Zone
Bench-Maji
Kefa
Dawuro
Wolaita
Gamo-Gofa
Woreda
South-Bench
North-Bench
Chena
Gimbo
Tocha
Loma
Kindo-Koysha
Humbo
Qucha
Demba-Gofa
Total
No of respondents
M
F
3
2
3
2
4
1
3
2
4
1
4
1
2
3
3
2
4
1
4
1
34 (68%)
16 (32%)
Altitude range
(masl)
1312-1594
1288-2070
1800-2136
1405-1745
1245-1535
1910-2319
1132-1315
1746-1925
1204-1692
1189-1816
1132-2319
masl-meters above sea level
3.2.3 Ethical consideration
Before collecting farmers’ perception of agromorphological traits, how they manage and use
cocoyam and collection of plant materials, informants were informed about the purpose of
the research and its benefits clearly underlining the fact that the results will be used for
academic purposes and that no commercial interest will be attached to it. When the farmers
assertively said that this research is useful and agree to provide the required information on
their own will, they were interviewed.
3.2.4 Data collection and analysis
The selected farmers were interviewed (see plate below) using semi-structured interview
guide (Appendix 1). As indicated by Albuquerque et al. (2014) in semi-structured interview,
the questions are partially established by the researcher but largely flexible and more
28
attention is to be paid to issues that arise during the interview. Semi-structured interviews
give interviewees the possibility to express their point of view. It was determined to be the
most appropriate in order to gather information for qualitative research, creating the
opportunity for interviewees to discuss topics that may have been dismissed and might be
of importance to them. The selected farmers were encouraged to express information in the
way they knew and perceived cocoyam through experience. Farmers were told to be free to
tell all what they know about cocoyam on their own accord using their native languages
although they were being interrupted abruptly when they deviate from the main topic of the
interview. To collect information on precise themes, the following factors were mainly
considered while recording the data (1) The cocoyam farming experience of farmers; (2) the
introduction time of cocoyam to the farmers’ locality and its origin in garden; (3) the local
name given to cocoyam and its meaning; (4) whether Ethiopian farmers distinguish cocoyam
from taro and if they do, how they do it; (5) the general distribution of cocoyam in the study
area; (6) local uses of cocoyam; (7) the size of land allotted for cocoyam cultivation; (8)
farmers’ preferred and/or disliked traits of cocoyam; (9) the farmers’ planting materials of
cocoyam and the cultivation methods; (10) the time course of planting and harvesting and
(11) methods that farmers have adopted for conservation of cocoyam. The resulting data
were entered into excel spreadsheet and descriptive statistical analysis was made. The
resulting values expressed as percentage.
Plate: Interview being carried out with farmers (Photo by Eyasu Wada, 2014)
29
3.3 Results
3.3.1 Age of farmers and their experiences in cocoyam cultivation
The age of the farmers who responded to the interview ranged from 20 to 83 years. The
respondents had lived in their respective area at least for 15 years. A total of 26%, 30%, 36%
and 8% of the respondents had grown the crop for 10 or less years, 11-20 years, 21-30 years
and for more than 30 years, respectively. Seventy-four percent of the respondents had been
involved in cocoyam farming activities for more than 10 years (Table 3.2).
Table 3.2 Age of respondents and number of years of cocoyam cultivation
Variables
Zone
Bench-Maji
20-30
4
Age (year)
31-40
41-50
6
-
51-60
-
>61
-
Farming experience (year)
1-10
11-20
21-30
>31
3
4
3
-
Kefa
1
5
2
2
-
2
4
3
1
Dawuro
-
5
2
1
2
2
2
6
-
Wolaita
-
3
3
1
3
4
3
2
1
Gamo-Gofa
2
2
2
1
3
2
2
4
2
7 (14) *
21(42)
9 (18)
5 (10)
8(16)
13(26)
15 (30)
18 (36)
4 (8)
Total
*numbers in parenthesis indicate the percentage values
3.3.2 Distribution and cultivation of cocoyam in the study area
During this study, only the green colored cocoyam was observed in Bench-Maji and Kefa
zones while both green- and purple- colored cocoyams (Fig. 3.2) were observed in Dawuro,
Wolaita and Gamo-Gofa zones. In these zones, purple cocoyam was seen more frequently
than the green cocoyam. According to the farmers, cocoyam cultivation has been increasing
in their localities due to its re-emerging ability from under buried corms.
30
a
b
Fig. 3.2 Cocoyam (Xanthosoma sagittifolium) plants from Ethiopia: green cocoyam, CUBI ZHANG,
from Bench-Maji Zone, South-Bench Woreda (a) and purple cocoyam, SAMUNA BOINA from Wolaita
Zone, Humbo Woreda (b) (Photo by Eyasu Wada, 2014).
In all the areas, cocoyam is cultivated in the form of smallholders. Farmers mainly rely on
rainfall for cocoyam cultivation. Most farmers (94%) cultivate cocoyam at homegarden
patches as a backyard garden crop that grows closely associated with the houses. Cocoyam
is also found in the natural ecosystem and around road sides as well as in the shade of other
plants or as an ornamental plant in the urban centers (Fig. 3.3).
a
b
d
c
Fig. 3.3 Cocoyam plants: at homegarden (a), in natural ecosystems (b) in the shade of coffee
plants (c) and as ornamental in the urban centers (photo by Eyasu Wada, 2014).
31
3.3.3 Local names of cocoyam and meanings
In different ethno-linguistic communities living in the surveyed areas of Ethiopia, different
local names are used for X. sagittifolium. The naming systems are, in most cases, followed
by the local name given to taro (C. esculenta). Most respondents (70%) consider cocoyam
as a variety of taro. They distinguish it from taro mainly by leaf pigmentation and size, shape
of cormels and size of corms.
The local names KENI ZHANG and CUBI ZHANG were used for cocoyam in the Bench-Maji Zone.
The majority (80%) of the respondents from Bench-Maji zone relate the terms KENI or CUBI
as the crop was introduced from Kenya or from Cuba, respectively. The term ZHANG is used
for taro in Bench language. The terms
GOCHELI KIDO
and
SUDAN KIDO
were used to refer to
cocoyam by the farmers of Kefa zone. According to the respondents, the term SUDAN is given
to cocoyam to indicate that the crop was introduced into Kefa zone from Sudan.
The local names TEPIYA BOINA, SAMUNA BOINA, GUDETA andAGARFAwere used for cocoyam in the
Dawuro zone.
BOINA is
a local term used for taro. Farmers use the term
TEPIYA BOINAfor
the
green cocoyam and consider that the green cocoyam had been introduced into their areas
from Tepi area of Bench-Maji Zone. The term SAMUNA meaning soap in Dawuroto language
was given to purple cocoyam due to its cormel which has the smell of soap when cooked. In
Dawuro area, prefix
ZO’O
meaning red was used for purple cocoyam to distinguish it from
the green cocoyam.
The local names SAMUNA BOINA, DAWURO BOINA, FARANJA BOINA, TONNEKA and BADADIYAwere used
for cocoyam in Wolaita zone. The meaning of the term
Dawuro zone. The term
DAWURO BOINAis
SAMUNA
is similar to that given in
used for cocoyam in Kindo-Koysha Woreda of
Wolaita zone, which is bounded by Dawuro zone indicating that cocoyam was introduced
into Kindo-Koysha Woreda of Wolaita zone from Dawuro zone. Farmers’ use the term
32
FARANJA BOINA, which
is to mean ‘foreign taro’ for cocoyam to indicate that it is an introduced
crop. According to respondents the term TONNEKA is used for purple cocoyam because it has
a sour nature when eaten. The term BADADIYAis used for green cocoyam to indicate its giant
size.
SAMUNA BOINA, TONNEKA
and
BADADIYAare
local terms given to cocoyam in Gamo-Gofa
zone. The meanings of these terms are similar to those explained in Dawuro and Wolaita
zones as the languages spokem in these zones belong to Omotic language family.
3.3.4 Cocoyam introduction into the study areas and tuber sources for the first time
garden cultivation
The majority (84%) of the farmers did not remember the time when cocoyam was introduced
into their areas. Farmers in South-Bench Woreda of the Bench-Maji Zone remembered that
the crop was introduced into their areas in mid 1970s by Cubans, who came to Ethiopia to
build micro dams after the 1974/1975 major drought. Some farmers of Kefa Zone recall
that cocoyam was introduced into their areas two to three years before the fall of the Derg
regime (previous governance) in 1991. According to some respondents from Tocha and
Loma Woredas of Dawuro Zone, cocoyam was introduced during the settlement program
(1986). According to 34 (68%) farmers, cocoyam tubers for garden cultivation comes from
market, nearby area, neighbourhood, family and relatives but 16 (32%) did not remember
the origin of cocoyam in their gardens for the first time (Table 3.3).
33
Table 3.3 Source of cocoyam clones used for the first time planting in gardens (numbers in the
table indicate the number of respondents)
Study area
Zone
Bench-Maji
Kefa
Dawuro
Wolaita
Gamo-Gofa
Total
Source of cocoyam clones used for the first time palnting
Nearby
Family and
Not
Woreda
Market
Neighbor
zone
relative
remember
South-Bench
2
1
2
North-Bench
2
1
2
Chena
2
1
2
Gimbo
2
2
1
Tocha
2
3
Loma
1
1
3
Kindo-Koysha
2
2
1
Humbo
1
1
1
Qucha
2
2
1
Demba-Gofa
1
2
1
1
4 (8 %) 5 (10%) 15 (30%)
10 (20%)
16 (32%)
34 (68%)
3.3.5 The farmers’ planting material and cropping system
Fifty percent of farmers use corm and headsets for cocoyam propagation. The remaining
half use corm and cormels as planting material. Farmers prefer using corms and headsets for
cocoyam propagation because cormels are used for consumption while the corms have no
food value. In the study areas, cocoyam is cultivated in a mono and mixed cropping system.
Thirty percent, 30% and 40% of the respondents cultivate cocoyam by mono, mixed and
both cropping systems, respectively. When cocoyam grows in mixed cropping system, it
grows mainly mixed with taro (Colocasia esculenta (L.) Schott), enset (Ensete ventricosum
(Welw.) Cheesman), banana (Musa spp.) or coffee (Coffea arabica L.). Farmers prefer to
cultivate cocoyam mixed with coffee (Fig 3.3c) because cocoyam is shade tolerant and its
leaves serve as organic fertilizer for coffee when detached from the plant.
3.3.6 Land preparation, planting and harvesting of cocoyam
The time of land preparation and planting time of cocoyam varies in different zones. In
Bench-Maji zone, the land is prepared in May and planting is mainly from June to July. In
Dawuro, Wolaita and Gamo-Gofa zones, land preparation is from late November to January
34
and planting takes place from February to March at the onset of the rainy season. According
to the respondents, piecemeal way of harvesting (harvesting cormels leaving the mother
plant in the place as a perennial crop) take place begging from 7 months after plantation.
The crop can be harvested 12 months after plantation. During harvesting, farmers use local
methods, digging around the plant and applying force to uproot the crop.
3.3.7 The local uses of cocoyam
Respondents (100%) use cocoyam for food although there is difference in the use of
cocoyam for food, the part of cocoyam used for food and the mode of preparation from one
zone to another. Six and 3 respondents from Bench-Maji and Kefa zones, respectively,
responded that leaves of cocoyam serve as organic fertilizer for coffee when leaves are
detached from the plant (Fig. 3.4). Farmers of Dawuro, Wolaita and Gamo-Gofa zones,
where purple cocoyam was observed, responded that the purple cocoyam has less food value
when other crops are available. According to farmers of these zones, purple cocoyam serves
only as emergency food and mainly eaten by those people who are at low economic status
and are food deficient. The purple cocoyam is classified by the respondents to be a nonpreferred food crop because of its sour taste and unpleasant smell.
Number of respondents
60
50
50
Food
40
20
10
Fodder
30
30
Medicine
107
6
10
6 3
10
54
10
64
10
6
10
2
9
Leaves as organic
fertilizer
0
Zones
Fig. 3.4 Local uses of cocoyam in the study zones based on the interview of 10 key informants
per zone. The number of respondents is shown by vertical line as well as on the top of bars
35
Mode of preparation when used for food: In all of the surveyed areas, cocoyam cormles
are eaten after cooking. The cocoyam shoots (young leaves) are used for food by preparing
as cabbage (cooked leafy vegetable) and/or mixing with cabbage in Bench-Maji Zone.
Respondents from Dawuro zone indicated that only those people who are traditionally
considered belonging to the lower social stratum eat the leaves of cocoyam as cooked leafy
vegetable.
Cocoyam as a medicinal plant: A total of 10 farmers from Dawuro, Wolaita and GamoGofa zones responded that purple cocoyam is considered to have medicinal value (Fig. 3.4).
According to these farmers, purple cocoyam is used to treat WULAWUSHIYA (related with
hepatitis), BARQA (postpatrum deperssion) and GERGEDA (related with rheumatoid arthritis).
WULAWUSHIYA
is a general term for yellow eye, for disease that affects liver or disease that
burns urinary tract and symptomized by the presence of blood in urine. BARQA is a type of
mood disorder associated with childbirth. To relieve from the disorder, a woman who gave
birth use the purple cocoyam cormels after a course of preparation. According to respondents
the meal is prepared by cooking using pot, peeled, grinded, mixed with butter and spices
such as garlic (Allium sativum L.), black cumin seeds (Nigella sativa L.) and onion (Allium
cepa L.). The respondents mentioned that the leaf of purple cocoyam is used to treat GERIGEDA
which is generalized pain in joints. Farmers who use purple cocoyam to treat GERIGEDA
mentioned that the pain feeling areas of the body are rubbed with young cocoyam leaves.
3.3.8 Farmers’ perceptions of cocoyam characters and uses
Cocoyam was preferred by all farmers as it is serving as food crop (Table 3.4). Cocoyam
was also a preferred crop by respondents due to its leaf edibility in addition to cormels and
piecemeal way of harvesting (possibility of harvesting cormels by leaving mother plant in
place), short cooking time and possibility of cormels to be roasted on hot stone. However,
36
farmers dislike cocoyam due to its sour taste and unpleasant smell (purple cocoyam),
inedibility of the corm and low market demand (Table 3.4).
Table 3.4 Characters/uses of cocoyam and farmers’ preference
No of respondent from each Zone
Preferred characters/uses
Serve as food
Serve as fodder
Leaves serve as fertilizer
Short cooking time
Piecemeal (harvesting cormels,
leaving the plant in place)
Young leaves edible
Medicinal
High yield
Disliked characters/uses
Sour taste, unpleasant smell
and continuously eating could
be irritable
Corm inedibility
Hard texture to eat
Not appetizing
Low market demand
Bench-Maji Kefa
10
10
7
6
9
6
10
8
Dawuro
10
5
2
-
Wolaita
10
4
2
-
Gamo-Gofa
10
6
4
-
Total
respondents
No
%
50
100
28
56
23
46
18
36
9
10
4
4
3
4
4
-
4
-
2
2
17
10
10
9
34
20
20
18
6
5
8
10
8
37
74
10
6
2
10
10
5
1
10
4
6
4
10
5
8
10
5
7
10
39
32
7
50
78
64
14
100
3.4 Discussion
In Ethiopia, different local names are used for X. sagittifolium. The meanings of these names
were linked either with the area of collection or the crop’s particular trait such as the growth
condition. Similar study conducted in the Edo State, Nigeria, indicated that the local people
distinguish cocoyam local types by area of collection (Osawaru and Ogwu, 2015). Various
local names have also been used for X. sagittifolium worldwide (Giacometti and Leon, 1994;
Mayo et al., 1997; Raemaekers, 2001; Lebot, 2009; Quero-Garcia et al., 2010). In this study,
it was found that most of local names were followed by local names given to taro (C.
esculenta). The Ethiopian farmers consider cocoyam as a variety of taro. According to
Maundu et al. (2009), Xanthosoma and Colocasia frequently share the local names taro and
cocoyam.
37
We found that the term taro or GODERE (Amharic term) is used for both aroids (Colocasia and
Xanthosoma). However, the local farmers distinguish the two crops and give them different
local names. In most literature, cocoyam (X. sagittifolium) is discussed jointly with the taro
(C. esculenta) (Onwueme, 1999). Morton (1972) noticed that the familiarity of Xanthosoma
had been burdened by highly localized vernacular names and hence she proposed the general
adoption of the euphonious and appetizing term, cocoyam, as a collective trade name for
Xanthosoma species. However, the term cocoyam has been used not only for Xanthosoma
rather it has been used for both Xanthosoma and Colocasia (Lebot, 2009; Owusu-Darko et
al., 2014, Osawaru and Ogwu, 2015). In many parts of Asia and Pacific, the term tannia
which is a modification or qualification of the term taro has been used for Xanthosoma.
Onwueme (1999) wrote in her book Cocoyam Cultivation in Asia and Pacific, taro (C.
esculenta) should not be confused with the related aroid Xanthosoma species.
Cocoyam was introduced into the surveyed areas later than taro, as recalled by farmers who
grow both crops. Amsalu Nebiyu et al. (2008) reported that cocoyam accessions which were
collected from Ethiopia and introduced from abroad since 1978 were maintained at Jimma
Agricultural Research Center, southwest Ethiopia. Another report mentioned that cocoyam
entered the Malo area of Gamo-Gofa Zone in southwestern Ethiopia in the 1980s (Fujimoto,
2009). The majority (84%) of farmers did not remember the time when cocoyam was
introduced into their areas despite the assertion by some farmers from Benchi-Maji and Kefa
zones that cocoyam was introduced into their areas in 1970s. Cocoyam was widely
distributed in the surveyed areas, growing mainly in the homegarden patches. Thus, the
circumstantial evidences force us to believe that this crop might have much longer history
in Ethiopia. During this study, farmers responded that cocoyam cultivation status has been
increasing in their areas since they have known the crop. Cocoyam has expanded fast into
new areas in western Africa since its introduction in the 16th or 17th century and its
38
importance is increasing since then (Maundu et al., 2009). A survey in Ethiopia indicated
that cocoyam grows even in poor soils and under dry conditions (Fujimoto, 2009). Due to
the related factors such as better yield, more robust and drought tolerance, cocoyam has
become an important food for over 400 million people and has become the main edible aroid
in many tropical areas (Giacometti and Leon, 1994; Matthews, 2002; Lebot 2009; Maundu
et al., 2009). Farmers mentioned that cocoyam cultivation is increasing in their areas due to
its re-emerging ability from under buried corm whenever it gets rain. They adopted
harvesting cormels leaving the plant in place as perennial crop for germplasm conservation.
It was noticed that the traditional seed exchange systems are the major way of seed supply
in the surveyed areas.
In the study areas, cocoyam is cultivated in a mono and mixed cropping system. Farmers
who cultivate cocoyam in mixed cropping system indicated that cocoyam is shade tolerant
and it could serve as organic fertilizer when the leaves fall off. The farmers’ response is in
line with the research reports. Lebot (2009) pointed out that cocoyam tolerates a certain level
of shade. Mazhar (2000) noted that mixed cropping of cocoyam with other crops is crucial
to improve soil fertility.
During this study, the respondents mentioned that cocoyam cormels are used for human
consumption after cooking or roasting while the corm is not used for human consumption.
In concordant with farmers’ response, it was mentioned in publications that domestication
history of cocoyam was based on processes such as roasting and cooking tubers. The usable
parts in cocoyam are the subterranean tuberous off shoots known as cormels and the main
corm is usually acrid and is not eaten (Giacometti and Leon, 1994) or it is only eaten when
no other food is available, during and after cyclones in some Pacific Islands (Lebot, 2009).
This indicates the traditional way of preparation of cocoyam for food has transferred to
39
Ethiopia with the crop. According to farmers, the cormels of the purple cocoyam can be
eaten only at the time of food emergency. Farmers in Dawuro, Wolaita and Gamo-Gofa
Zones responded that purple cocoyam provides medicinal values. According to Nzietchueng
(1988), some of the species of genus Xanthosoma such as X. auriculatum, X. helleborifolium,
X. mexicanum, X. pentaphyllum and X. robustum are used as medicinal plants. Since there
is dearth of information on the taxonomy of species, the purple cocoyam may be related to
either of these species which are used as medicinal plant or the purple cocoyam growing in
the surveyed areas may be related to X. sagittifolium variety growing in Pacific Islands,
which are only eaten when there is shortage of other food crops (Lebot, 2009).
Farmers from Bench-Maji zone responded to the interview explained that the young leaves
of cocoyam are eaten after cooking in addition to cormels. Previously Amsalu Nebiyu et al.
(2008) reported that young leaves of the cocoyam are eaten in southwestern Ethiopia. In the
literature, it was mentioned that young leaves of some cocoyam cultivars can be used as a
vegetable and can be an important source of proteins and vitamins (Giacometti and Leon,
1994; Lebot, 2009). Cocoyam was originally introduced to Africa for their cormels, but their
leaves are also used as a vegetable (Maundu et al., 2009). Traits of cocoyam that farmers
prefer include serving as emergency food, fodder, young leaves edibility (Benchi-Maji
Zone). Most of the farmers’ preferred traits of cocoyam were traits that were also preferred
by Nigerian farmers (Osawaru and Ogwu, 2015). In another way around, in Ethiopia,
cocoyam traits such as sour taste, unpleasant smell, irritability with continuously eating,
corm inedibility, hard texture to eat, non-appetizing nature have been hindering its potential
to be a major crop as perceived by farmers. The farmers need to get these disliked traits
improved. This study has shown that the farmers have useful knowledge on
agromorphological traits and uses of cocoyam.
40
Chapter Four
Morphological Traits Based Genetic Diversity in Cocoyam
(Xanthosoma sagittifolium (L.) Schott) from Ethiopia
Abstract
Cocoyam (Xanthosoma sagittifolium (L.) Schott) has been cultivated since fairly recent years in
Ethiopia. Its genetic diversity has not been fully studied to date. A field study was conducted to
assess the genetic diversity among cocoyam accessions from Ethiopia based on morphological traits.
A total of 100 cocoyam accessions (65 green- and 35 purple-colored) were evaluated using a 10*10
simple lattice square design. Sixteen qualitative and 13 quantitative traits were studied. Two classes
of qualitative traits were observed for petiole color, lamina orientation, color of the leaf surfaces,
color of veins on leaf surface, position of cormel apex and shape of cormels. Analysis of variance
(ANOVA) revealed significant variation in 11 (84.6%) of the 13 studied quantitative traits such as
plant height, petiole length, lamina length, lamina width, circumference of pseudostem, number of
cormles/plant, cormel diameter, cormel fresh weight/plant, corm length, corm diameter and corm
fresh weight/plant. The estimates of genetic variance components and coefficient of variations
showed that the δ2p and PCV were higher than the δ2g and GCV all of the 13 studied quantitative
traits. However, the difference between PCV and GCV was small for 9 (69.2%) of the studied traits
and there was high h2b% estimate for 6 (46.2%) of studied quantitative traits such as lamina length,
lamina width, circumference of pseudo-stem, number of cormels/plant, corm diameter and corm
fresh weight per plant, suggesting the environmental effect on these traits observed to be low. PCA
reduced the original 13 characters in the experiment to 3 PCs, with the eigen values >1 which
explained 69.2% of the observed variations among accessions, with PC1 has 40.02% of the total
variation, PC2 and PC3, respectively, contributed, 16.10% and 12.9% of the total variation. The
traits that loaded highly for PC1 were lamina length, lamina width, circumference of pseudo-stem
and corm diameter, for PC2 were plant height, number of cormels/plant, corm length and corm fresh
weight per plant and for PC3 were petiole length, number of cormels per plant, cormel length and
cormel fresh weight per plant. Score plot has effective separated 35 purple cocoyam morphotypes
from 65 green cocoyam morphotypes. Cluster analysis grouped the accessions into four clusters,
irrespective of the collection sites. The study identified qualitative and quantitative traits that will
help researchers in recommending best traits for morphological identification of cocoyam
germplasms. The accessions should be tested in more than one environment to select elite genotypes
for future utilization of cocoyam in Ethiopia.
Keywords: Cocoyam, genetic diversity, morphological traits
41
4.1 Introduction
Collecting, characterizing and evaluating the genetic diversity of crop plants is very
important to identify genetically diverse accessions for efficient and effective usage,
conservation and improvement. Morphological traits are conventional tools to analyze the
genetic diversity by measuring the variation in qualitative morphological traits such as
texture, color, growth habit and/or quantitative morphological traits like yield potential,
height, size, weight, stress tolerance, etc. (Sinha and Kumaravadivel, 2016). The use of
morphological traits to estimate genetic diversity is the most common approach to estimate
the relationship between accessions. It is also an essential step for effective utilization of
germplasms because it offers a useful approach for assessing the extent of diversity
(Koorneef and Stam, 2001). Determination of the relevant morphological traits to describe
the genetic diversity is very important in the cases of limited financial and human resource
so that the least relevant traits can be eliminated (Lima et al., 2012). International Board for
Plant Genetic Resource (IBPGR, 1989) prepared standard format that encourages the
collection of data on accessions, characterization and preliminary evaluation of
Xanthosoma. The morphological descriptors provide information underlying the
conclusions on the genetic variability of cocoyam accessions.
Cocoyam (Xanthosoma sagittifolium (L.) Schott) is a perennial crop grown in the humid
tropics and subtropics, but for practical purposes it is harvested after 9-12 months after
plantation (Bown, 2000) although the time from planting to harvest can vary with genotypes
and method of cultivation (Castro, 2006). In different countries, genetic diversity studies of
cocoyam have been conducted using morphological traits such as plant height, the leaf
lamina characters, corm shape, maturity period, growth habit, corm flesh color and taste,
etc. (Mbouobda et al., 2007; Mwenye, 2009; Nurmiyati et al., 2009; Solomon Fantaw et al.,
2014a, b). Amsalu Nebiyu and Tesfaye Awas (2006) stated that there is considerable amount
42
of cocoyam gene pool in south and southwest Ethiopia in farmers’ fields and home-gardens.
However, it is a neglected crop by research and development community. Its genetic
diversity has not been fully researched in Ethiopia. The germplasms in the farmers’ field are
the major hope for use, conservation and improvement of cocoyam. Thus, there is a need to
characterize the available cocoyam accessions for genetic diversity. A better understanding
of genetic diversity will facilitate its usage, conservation and improvement.
To assess the genetic diversity of crop plants based on morphological traits, researches have
used statistical methods such as basic statistical parameters (Opoku-Agyeman et al., 2004;
Mandal et al., 2013) and multivariate analysis (Okpul et al., 2004; Tewodros Mulualem et
al., 2013; Solomon Fantaw et al., 2014a). In this study, the genetic diversity of 100 cocoyam
accessions collected from Ethiopia have been assessed based on morphological traits by
using basic statistical parameters and multivariate analyses (principal components and
cluster analysis).
4.2 Materials and methods
4.2.1 Germplasm collection
One hundred cocoyam cormels were collected from 16 woreda (Table 4.1). The individual
samples (accessions) were collected from sites at least 5 km apart unless they were clearly
distinguished morphologically by leaf/petiole color difference (green- and purple-colored).
4.2.2 Description of the experimental site
The field evaluation was carried out at Areka Agricultural Research Center, which is located
in the Southren Nations Nationalities and Peoples Region in Wolaita zone, 303 km
southwest of Addis Ababa, Ethiopia, at an altitude of 1750-1830 (masl), 07°19’N latitude
and 37°08’E longitude. The site is situated in the warm sub-humid lowlands (SH2) major
agroecology, which is tepid to cool-sub-humid mid highlands. The average annual rain fall
43
of the study area was 1520 mm, which occurs in two seasons in the year. The first short rain
season is Belg, which is from February to May and the second main rainy season Mehir
which occurs from June to October. The average maximum and minimum temperature of
Areka area are 25.4 oC and 13.4oC, respectively.
Table 4.1 List of cocoyam accessions with accession code, collection sites (Zone, Woreda and
Kebele), coordinate (latitude and longitude), altitude and color of accessions
Accession
Code*
BS/2014/Xs1
BS/2014/Xs2
BS/2014/Xs3
BS/2014/Xs4
BS/2014/Xs5
BS/2014/ Xs5
BS/2014/Xs7
BN/2014/ Xs8
BN /2014/Xs9
BN/2014/Xs10
BN/2014/Xs11
BN/2014/Xs12
BN/2014/Xs13
BN/2014/Xs14
KC/2014/Xs15
KC/2014/Xs16
KC/2014/Xs17
KC/2014/Xs18
KC/2014/Xs19
KG/2014/Xs20
KG/2014/Xs21
KG/2014/Xs22
KG/2014/Xs23
KG/2014/Xs24
JS/2014/Xs25**
JS/2014/Xs26**
DT/2014/Xs27
DT/2014/Xs28
DT/2014/Xs29
DT/2014/Xs30
DT/2014/Xs31
DT/2014/Xs32
DT/2014/Xs33
DM/2014/Xs34
DM/2014/Xs35
DM/2014/Xs36
DL/2014/Xs37
DL/2014/Xs38
DL/2014/Xs39
DL/2014/Xs40
DL/2014/Xs41
DL/2014/Xs42
DL/2014/Xs43
DL/2014/Xs44
DL/2014/Xs45
Zone
Woreda
Kebele
Bench-Maji
Bench-Maji
Bench-Maji
Bench-Maji
Bench-Maji
Bench-Maji
Bench-Maji
Bench-Maji
Bench-Maji
Bench-Maji
Bench-Maji
Bench-Maji
Bench-Maji
Bench-Maji
Kefa
Kefa
Kefa
Kefa
Kefa
Kefa
Kefa
Kefa
Kefa
Kefa
Jimma
Jimma
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
Dawuro
South-Bench
South-Bench
South-Bench
South-Bench
South-Bench
South-Bench
South-Bench
North-Bench
North-Bench
North-Bench
North-Bench
North-Bench
North-Bench
North-Bench
Chana
Chana
Chana
Chana
Chana
Gimbo
Gimbo
Gimbo
Gimbo
Gimbo
Sheba-Sumbo
Sheba-Sumbo
Tocha
Tocha
Tocha
Tocha
Tocha
Tocha
Tocha
Maraka
Maraka
Maraka
Loma
Loma
Loma
Loma
Loma
Loma
Loma
Loma
Loma
Kokin
Debrework-01
Jenehu
Kite
Kite
Kite
Kite
Fanika
Fanika
Woshiken
Woshiken
Temenja-Yazh
Temenja-Yazh
Wacha
Wacha
Daha
Daha
Woreta
Woreta
Jakaraba
Ufudo
Ufudo
Shomba-Kichibe
Shomba-Kichibe
Kesh
Jebiye
Gorika-Doma
Gorika-Doma
Wara-Wori
Wara-Wori
Gorika-Dama
Gorika-Dama
Warma-Galcha
Shaba
Shina-Gaburi
Tercha-02
Gasa-Chare
Gasa-Chare
Gasa-Chare
Gasa-Chare
Tulama
Tulama
Tulama
Elaa-Bacho
Elaa-Bacho
44
Latitude Longitude
(⁰N)
(⁰E)
7.078
35.687
6.891
35.703
7.023
35.651
7.040
35.606
7.134
35.542
6.939
35.714
6.939
35.714
7.129
35.571
7.129
35.520
7.184
35.790
7.350
35.791
7.210
35.791
35.782
7.136
7.137
35.781
7.373
35.919
7.455
36.200
7.439
36.261
7.437
36.006
7.544
36.194
7.531
36.518
7.607
36.224
7.593
36.268
7.652
36.487
7.476
36.518
7.586
36.675
7.719
36.768
37.079
7.272
37.080
7.272
7.268
37.165
7.354
37.071
7.289
37.186
7.333
37.016
7.246
37.259
7.351
37.326
7.294
37.312
7.323
37.296
7.188
37.522
7.124
37.533
7.124
37.533
7.074
37.438
7.054
37.345
7.037
37.338
7.085
37.364
7.160
37.426
7.170
37.426
Altitude Color of No of
(masl) accession accessions
1594
Green
1581
Green
1403
Green
1345
Green
1312
Green
1349
Green
1349
Green
14
1297
Green
1288
Green
1404
Green
1404
Green
1481
Green
2070
Green
2070
Green
2136
Green
1993
Green
1913
Green
1810
Green
1800
Green
1620
Green
1745
Green
12
1715
Green
1445
Green
1405
Green
1350
Green
1030
Green
Green
1535
Green
1538
1357
Green
1497
Green
1520
Green
1418
Green
1498
Green
1245
Green
1290
Green
1327
Green
2129
Green
2106
Green
2106
Purple
28
2278
Green
2319
Green
2299
Green
2253
Purple
1910
Green
1910
Purple
Table 4.1 continued
DB/2014/Xs46
Dawuro
Bosa-Gena
Lala-Ambe
7.038
37.435
1523
Purple
DB/2014/Xs47
Dawuro
Bosa-Gena
Lala-Ambe
7.029
37.435
1523
Green
DB/2014/Xs48
Dawuro
Bosa-Gena
Deneba
7.028
37.461
1221
Green
DB/2014/Xs49
Dawuro
Bosa-Gena
Deneba
7.029
37.462
1221
Purple
DB/2014/Xs50
Dawuro
Bosa-Gena
Zima
7.113
37.494
1222
Green
DB/2014/Xs51
Dawuro
Bosa-Gena
Zima
7.113
37.494
1222
Purple
DB/2014/Xs52
Dawuro
Bosa-Gena
Sere-Beta
7.267
37.428
1733
Green
DB/2014/Xs53
Dawuro
Bosa-Gena
Sere-Beta
7.014
37.425
1816
Purple
DB/2014/Xs54
Dawuro
Bosa-Gena
Sere-Beta
7.269
37.425
1236
Green
DB/2014/Xs55
Wolaita
Kindo-Koysha
Bayana
6.943
37.664
1132
Purple
WK/2014/Xs56
Wolaita
Kindo-Koysha
Fagena-Mata
7.005
37.503
1160
Purple
WK/2014/Xs57
Wolaita
Kindo-Koysha
Fagena-Mata
7.002
37.505
1158
Green
WK/2014/Xs58
Wolaita
Kindo-Koysha
Fagena-Mata
7.077
37.712
1156
Purple
WK/2014/Xs59
Wolaita
Kindo-Koysha
Fagena-Mata
7.078
37.714
1156
Green
Wolaita
Kindo-Koysha
Bale-01
37.660
1315
Purple
WK/2014/Xs60
7.115
WK/2014/Xs61
Wolaita
Kindo-Koysha
Bale-02
7.115
37.660
1315
Green
WH/2014/Xs62
Wolaita
Humbo
Gututo-Larena
6.803
37.785
1849
Purple
WH/2014/Xs63
Wolaita
Humbo
Gututo-Larena
7.015
38.027
1852
Green
WH/2014/Xs64
Wolaita
Humbo
Gututo-Larena
6.803
37.785
1849
Purple
WH/2014/Xs65
Wolaita
Humbo
Gututo-Larena
6.858
37.942
1785
Green
WH/2014/Xs66
Wolaita
Humbo
Gututo-Larena
6.858
37.942
1785
Purple
WH/2014/Xs67
Wolaita
Humbo
Bosa-Wanche
6.799
37.831
1755
Purple
WH/2014/Xs68
Wolaita
Humbo
Bosa-Wanche
6.826
37.831
1746
Purple
WH/2014/Xs69
Wolaita
Humbo
Demba-Koyisha
6.768
37.767
1925
Purple
WS/2014/Xs70
Wolaita
Sodo-Zuriya
Humbo-Larena
7.004
37.939
1836
Purple
WS/2014/Xs71
Wolaita
Sodo-Zuriya
Wareza-Esho
7.085
37.782
1890
Purple
WS/2014/Xs72
Wolaita
Sodo-Zuriya
Wareza-Esho
7.085
37.782
1890
Green
WS/2014/Xs73
Wolaita
Sodo-Zuriya Gurumo-Woyde
7.051
37.891
1960
Green
Wolaita
Sodo-Zuriya
Wareza-Lasho
1969
Green
37.931
WS/2014/Xs74
7.085
Wolaita
Sodo-Zuriya
Wareza-Lasho
1969
Purple
37.931
WS/2014/Xs75
7.085
WS/2014/Xs76
Wolaita
Sodo-Zuriya
Damota-Waja
7.134
37.883
1940
Purple
WS/2014/Xs77
Wolaita
Sodo-Zuriya
Damota-Waja
7.134
37.883
1940
Green
WB/2014/Xs78
Wolaita
Boloso-Sore
Dola
6.187
37.798
1796
Purple
WB/2014/Xs79
Wolaita
Boloso-Sore
Dola
6.268
37.923
1827
Purple
WB/2014/Xs80
Wolaita
Boloso-Sore Gurumo-Koysha
7.087
37.976
1914
Green
WB/2014/Xs81
Wolaita
Boloso-Sore
Gunnuno-01
7.146
37.875
2052
Purple
WB/2014/Xs82
Wolaita
Boloso-Sore
Gunnuno-02
7.119
37.892
2042
Purple
GQ/2014/Xs83
Gamo-Gofa
Qucha
Dana Sefera II
6.697
37.678
1692
Green
GQ/2014/Xs84
Gamo-Gofa
Qucha
Dana Sefera II
6.697
37.678
1692
Purple
GQ/2014/Xs85
Gamo-Gofa
Qucha
Basa
6.578
37.675
1390
Green
GQ/2014/Xs86
Gamo-Gofa
Qucha
Basa
6.578
37.675
1390
Purple
GQ/2014/Xs87
Gamo-Gofa
Qucha
Basa
6.578
37.672
1393
Purple
GQ/2014/Xs88
Gamo-Gofa
Qucha
Selamber-03
6.583
37.461
1413
Green
GQ/2014/Xs89
Gamo-Gofa
Qucha
Selamber-04
6.583
37.461
1413
Purple
GQ/2014/Xs90
Gamo-Gofa
Qucha
Selamber-01
6.639
37.704
1364
Green
GQ/2014/Xs91
Gamo-Gofa
Qucha
Selamber-02
6.639
37.704
1364
Purple
GQ/2014/Xs92
Gamo-Gofa
Qucha
Morka
6.706
37.667
1325
Purple
GQ/2014/Xs93
Gamo-Gofa
Qucha
Morka
6.448
37.375
1314
Purple
Gamo-Gofa
Qucha
Morka
1314
Green
GQ/2014/Xs94
6.448
37.375
Gamo-Gofa
Daramalo
Dita
1204
Purple
GD/2014/Xs95
6.676
37.508
GD/2014/Xs96
Gamo-Gofa
Demba-Gofa
Dorga
6.458
36.983
1192
Purple
GD/2014/Xs97
Gamo-Gofa
Demba-Gofa
Dorga
6.464
37.073
1816
Green
GD/2014/Xs98
Gamo-Gofa
Demba-Gofa
Dorga
6.417
37.056
1189
Green
GD/2014/Xs99
Gamo-Gofa
Demba-Gofa
Boreda
6.486
36.947
1306
Purple
GD/2014/Xs100 Gamo-Gofa
Demba-Gofa
Boreda
6.486
36.947
1306
Green
*In the accession code the first two letters stand for the zone and woreda followed by the year of collection, Xs for species
name and serial number of accessions; **accessions collected from Sheba-Sumbo woreda of Jimma zone, which is close
to Kefa zone. Thus, these accessions were considered with Kefa population for genetic diversity study
45
28
18
4.2.3 Experimental design and crop management
In 2015/16 cropping season, 100 cocoyam accessions were planted in February 16, 2015 for
establishment purpose. Similar sized cormels were harvested at the end of 12th months after
plantation. In 2016/17 cropping season, the 100 cocoyam accessions were planted using a
10*10 simple lattice square design in well-drained, loose soil on flat ground in February 16,
2016. Five cormels of an accession were planted in a single row plot of 3 m, spaced 0.6 m
between plants and 0.75 m between rows. Weeding was conducted as required to keep plots
weed free.
4.2.4 Morphological traits and data collection
Twenty-nine morphological traits were selected from International Board for Plant Genetic
Resources Descriptors for Xanthosoma (IBPGR, 1989) (Appendix 2). The qualitative data
were scored for qualitative traits such as plant growth habit, petiole attachment, petiole color
(upper 2/3rd), petiole color (lower 1/3rd), color of edge of petiole, lamina orientation, leaf
marigin color, leaf shape, color of upper leaf surface, color of lower leaf surface, position of
cormel apex, shape of cormels, color of cormel apex and fresh cormel color at the 7th month
after date of plantation, from the middle individual clone by cross-checking other clones
when needed. The Munsell Plant Tissue Color Chart (Wild and Voigt, 1977) was used to
discriminate colors. The quantitative data were recorded from the middle three plants of
each replication, leaving the two plants grown as boarder plants. The aboveground
quantitative traits such as overall plant height (cm), petiole length (cm), petiole sheath length
(cm), circumference of pseudo-stem (cm), lamina length and lamina width were measured
at the end of 7th months after plantation. The underground quantitative traits such as the
number of cormels/plant, cormel length (cm), cormel diameter (cm), cormel fresh
weight/plant (kg), corm length (cm), corm diameter (cm) and corm fresh weight/plant (kg)
were recorded at the end of 11th months after date of plantation.
46
4.2.5 Data analysis
Frequency distribution of qualitative traits and basic statistical parameters based on quantitative
morphological traits were calculated using SPSS version 23 (IBM SPSS, 2015). Analysis of
variance was computed by PROC GLM procedure of SAS 9.3 (SAS, 2011) because the relative
efficiency of the lattice design compared with CRBD was low (less than 105%). Total genotypic
and phenotypic variances were calculated following Singh and Chaudhary (1979) by using the
expectations as: Genotypic variance (δ2g) = (MSG-MSE)/r, where MSG - mean squares for
accessions, MSE-mean square of error and r is replication. Phenotypic variance (δ2p) = δ2g +
δ2e, where δ2g - genotypic variance component, δ2e Foreinenvironmental variance, which was
MSE. The genotypic and phenotypic coefficient of variations were estimated by the method as
suggested by Singh and Chaudhary (1979) using the formulas: Genotypic coefficient of variation
(GCV%) = √δ2g/ x *100 and phenotypic coefficient of variation (PCV%) = √δ2𝑝/ x *100,
where x is the mean value of the particular trait of interest. Heritability in broad sense (h2b%)
was estimated as a ratio of genotypic variance to phenotypic variance (Falconer, 1981), i.e.,
heritability in broad sense (h2b %) = δ2g/δ2p*100; where δ2g genotypic variance and δ2p
phenotypic variance. Genetic advance and genetic advance as % of mean were estimated by the
formula described by Johnson et al. (1955) as follows: Genetic advance (GA) = δ2g/δ2p*k*δp,
where δ2g is genotypic variance, δ2p is phenotypic variance, δp is standard deviation of
phenotypic variance and k is the selection differential at a particular selection intensity, i.e., 2.06,
suggested by Falconer (1981) at 5% selection intensity. Genetic advance as percentage of mean
(GA %) = GA/ x *100, where x is the mean of a trait. The quantitative traits were also subjected
to multivariate analysis such as principal component analysis (PCA) and cluster analysis. The
PCs based on correlation matrix was calculated using Minitab 17.1 (Minitab, 2013) and the PCs
with Eigen values >1.0 were selected to define the morphological trait variation among
accessions. Cluster analysis was conducted by employing average linkage clustering strategy of
the observation. Variables were standardized to a common scale by subtracting the means and
47
dividing by the standard deviation. The number of cluster was determined by following the steps
recommended by Minitab 17.1 (Minitab, 2013). The correlation between clusters was computed
using generalized Euclidean distance and the dendrogram showing the Euclidean distance
between clusters was constructed by plotting the results of cluster analysis using Minitab 17.1
(Minitab, 2013).
4.3 Results
4.3.1 Qualitative traits
Of 16 qualitative traits, 9 discriminated 100 cocoyam accessions into two-character states
but 7 characters did not discriminate the accessions.
Table 4.2 Frequency distribution of 16 qualitative traits of cocoyam
No
1
Plant character
Plant growth habit
Character state
Acaulescent
2
Petiole attachment
Non-peltate
3
Petiole color (upper 2/3rd)
4
Petiole color (lower 1/3rd)
5
Color of edge of petiole sheath
6
Lamina orientation
7
8
Leaf shape
Leaf margin color
9
Color of upper leaf surface
10
Color of lower leaf surface
11
Color of veins on upper leaf surface
12
Color of veins on lower leaf surface
13
Position of color apex
14
Shape of cormels
15
16
Color of cormel apex
Internal color of cormels
Green
Purple
Green streaked with purple
Purple
The same as the rest of petiole and sheath
Purple
One plane - apex down (Droopy)
3-dimentional (cup-shaped)
Sagitate basal lobes
Purple edge
Medium green
Dark green
Light green
Purplish green
Lighter green than lamina
Same as color as lamina
Purple
Underground
Above ground and under ground
Globose
Ovate
Red
White streaked with purple
%*
100
10
0
65
35
65
35
35
65
35
65
100
100
65
35
65
35
100
65
35
65
35
65
35
100
100
*100% indicates the character common to both morphotypes, 65% character specific to green morphotypes
and 35% character specific to purple morphotypes
48
Two classes of petiole color (upper 2/3rd), petiole color (lower 1/3rd), color of edge of petiole
sheath, lamina orientation, color of upper leaf surface, color of lower leaf surface, color of
veins on lower leaf surface, position of color apex and shape of cormels existed in the
accessions. Qualitative traits such as plant growth habit, petiole attachment, leaf shape, leaf
margin color, color of veins on upper leaf surface, color of cormel apex and fresh cormel
color did not discriminate the accessions included in this study (Table 4.2 and Fig. 4.1).
a
b
c
d
e
f
g
h
i
Fig. 4.1 Qualitative morphological traits of cocoyam: (a) Acaulescent plant growth habit (nonpeltate petiole attachment indicated); (b) Petiole color -upper 2/3rd (green/purple); (c) Petiole color - lower
1/3rd (green streak/purple); (d) Color of edge of petiole sheath; (e) Lamina orientation (cup shaped/droopy);
(f) Color of upper leaf surface (medium green/dark green; (g) Color of lower leaf surface and veins on lower
leaf surface; (h) Shape of cormels (globose/ovate); (i) color of cormel apex (red) and internal color of cormels
(white streaked with purple).
49
4.3.2 Descriptive statistical parameters and variance
Descriptive statistics such as mean, standard deviation (SD), minimum and maximum values
and coefficient of variation (CV%) are summarized in Table 4.3. Plant height (cm) ranged
from 57.58 to 84.83 (mean: 71.49), petiole length (cm) ranged from 42.75 to 67.83 (mean:
55.22), petiole sheath length (cm) ranged from 25.50 to 43.92 (Mean: 31.24), lamina length
(cm) ranged from 29.69 to 45.25 (mean: 37.77), lamina width ranged from 17.28 to 25.47
(mean: 22.02), the circumference of pseudo-stem (cm) ranged from 21.42 to 32.42 (Mean:
28.83). The mean values of underground quantitative traits such as the number of
cormels/plant, cormel length (cm), cormel diameter (cm), cormel fresh weight/plant (kg),
corm length (cm), corm diameter (cm) and corm fresh weight/plant (kg), respectively, were
10.05, 9.05, 3.26, 1.28, 11.94, 5.70 and 1.18. Maximum SD (5.63) corresponded to plant
height and minimum SD (0.31) corresponded to cormel fresh weight/plant (kg). The
coefficient of variation (CV%) varied from 7.65% for lamina width to 29.10% for corm fresh
weight/plant. Analysis of variance (ANOVA) revealed significant variation in 11(84.6%) of
the 13 studied quantitative traits (Table 4.4).
Table 4.3 Basic statistics of 13 quantitative traits of cocoyam
Quantitative traits
Plant height (cm)
Petiole length (cm)
Petiole sheath length (cm)
Lamina length (cm)
Lamina width (cm)
Circumference of pseudo-stem (cm)
Number of cormels/plant
Cormel length (cm)
Cormel diameter (cm)
Cormel fresh weight/plant (kg)
Corm length (cm)
Corm diameter (cm)
Corm fresh weight/plant(kg)
Mean
71.49
55.22
31.24
37.77
22.02
28.83
10.05
9.05
3.26
1.28
11.94
5.70
1.18
SD
5.63
4.82
3.46
3.20
1.68
2.78
1.81
1.09
0.46
0.31
2.11
0.77
0.34
50
Minimum Maximum
57.58
84.83
42.75
67.83
25.50
43.92
29.69
45.25
17.28
25.47
21.42
34.42
5.92
15.33
6.58
11.83
2.25
4.21
0.68
2.42
3.49
16.92
3.49
7.73
0.53
2.30
CV%
7.87
8.72
11.08
8.48
7.65
9.66
17.98
12.00
13.97
23.79
17.71
13.52
29.10
Table 4.4 Summary of mean squares of 13 quantitative traits of cocoyam
Quantitative traits
Rep (df =1)
Mean squares
Accessions (df =99) Error (df =81)
Plant height (cm)
100.30
82.46*
54.46
Petiole length (cm)
294.64
49.57*
31.66
Petiole sheath length (cm)
333.62
28.16
21.48
Lamina length (cm)
20.09
16.73***
8.10
Lamina width (cm)
0.74
4.96***
2.41
128.27
14.02***
5.72
Number of cormels/plant
5.01
7.40**
3.91
Cormel length (cm)
0.02
2.17
2.30
Cormel diameter (cm)
1.12
0.29**
0.17
Cormel fresh weight/plant (kg)
1.71
0.23**
0.13
102.84
7.65**
4.38
Corm diameter (cm)
2.95
1.06***
0.45
Corm fresh weight/plant (kg)
1.98
0.24***
0.12
Circumference of pseudo-stem (cm)
Corm length (cm)
df - degree of freedom, *, ** and *** significant at p= 0.05, 0.01 and 0.001, respectively
4.3.3 Genotypic and phenotypic variances, coefficients of variations and heritability
For each of the evaluated quantitative traits, genetic parameters including genotypic and
phenotypic variance component and their coefficients of variation, broad sense heritability,
genetic advance and genetic advance as % of mean are summarized in Table 4.5. Across the
traits studied, δ2g ranged from -0.07 for cormel length to 14.00 for plant height. The lowest
δ2p (0.18) was for corm and cormel fresh weights/plant (kg) and the highest δ2p (68.46) was
for plant height. The GCV values were ranged from the below 0% for cormel length to
20.34% for corm fresh weight/plant (kg). PCV ranged from 8.72% for lamina width to
35.59% for corm fresh weight/plant. The heritability estimates ranged from below 0% (2.91) for cormel length to 42.05% for circumference of pseudo-stem. Genetic advance as %
of mean ranged from -0.99% for cormel length to 24.44% for corm fresh weight/plant (kg).
51
Table 4.5 Genetic parameters of 13 quantitative traits of cocoyam
Quantitative traits
Plant height (cm)
Petiole length (cm)
Petiole sheath length (cm)
Lamina length (cm)
Lamina width (cm)
Circumference of pseudo-stem (cm)
Number of cormels/plant
Cormel length (cm)
Cormel diameter (cm)
Cormel fresh weight/plant (kg)
Corm length (cm)
Corm diameter (cm)
Corm fresh weight/plant (kg)
δ 2g
δ 2p
GCV
(%)
PCV
(%)
14.00
8.95
3.34
4.32
1.28
4.15
1.75
-0.07
0.06
0.05
1.80
0.31
0.06
68.46
40.61
24.82
12.42
3.69
9.87
5.66
2.24
0.23
0.18
6.02
0.76
0.18
5.24
5.42
5.86
5.51
5.13
7.07
13.13
7.36
17.19
10.72
9.82
20.34
11.59
11.54
15.94
9.32
8.72
10.89
23.68
16.57
14.72
32.81
20.52
15.26
35.59
h2b
(%)
GA
GA as %
of mean
20.45
22.04
13.46
34.76
34.60
42.05
30.92
-2.91
26.09
27.78
27.18
40.40
33.33
3.48
2.89
1.38
2.52
1.37
2.72
1.52
-0.09
0.26
0.24
1.37
0.72
0.29
4.88
5.24
4.42
6.67
6.21
9.43
15.08
-0.99
7.91
18.78
11.49
12.70
24.44
δ2g - genotypic variance; δ2p - phenotypic variance; GCV - genotypic coefficient of variation; PCV phenotypic coefficient of variation; h2b broad sense heritability; GA - genetic advance
4.3.4 Principal components and clustering of accessions
The PCA was conducted to determine traits that most strongly contribute to the total
variation. The analysis reduced the original 13 characters in the experiment to 3 PCs, with
the Eigen values >1 which explained 69.2% of the observed variations among the accessions
(Table 4.6). PC1 accounted for 40.2% of the total variation. The morphological traits that
loaded highly for PC1 were lamina length (0.38), lamina width (0.34), circumference of
pseudo-stem (0.35) and corm diameter (0.35). PC2 accounted for 16.1% of the total variation
and the traits with the greatest weight on this component were plant height (-0.50), number
of cormels/plant (-0.35), corm length (0.34) and corm fresh weight per plant (0.34). PC3
contributed 12.9 % of the total variation and mainly related to petiole length (0.36), number
of cormels/plant (-0.49), cormel length (-0.37) and cormel fresh weight/plant (0.50). Score
plot of first component (horizontal axis) and second component (vertical axis) was drawn to
study grouping of accessions. The first component was effective in separating purple
cocoyam morphotypes from green cocoyam morphotypes in which most of purple cocoyam
morphotypes were clustered together (Fig. 4.2).
52
Table 4.6 Eigen value, proportion of variability and the first 3 PCs of cocoyam
Quantitative traits
Eigen value
Proportion of variance (%)
Cumulative variance (%)
Plant height (cm)
Petiole length (cm)
Petiole sheath length (cm)
Lamina Length (cm)
Lamina width (cm)
Circumference of pseudo-stem (cm)
Number of cormels/plant
Cormel length (cm)
Cormel diameter (cm)
Cormel fresh weight/plant (kg)
Corm length (cm)
Corm diameter (cm)
Corm fresh weight/plant (kg)
PC1
5.22
40.2
40.2
0.22
0.31
0.29
0.38
0.34
0.35
0.09
0.08
0.27
0.20
0.28
0.35
0.27
PC2
2.09
16.1
56.3
-0.50
-0.24
-0.28
0.07
-0.03
0.05
-0.35
-0.32
0.27
-0.20
0.34
0.20
0.34
PC3
1.67
12.9
69.2
0.25
0.36
0.33
0.03
-0.03
0.11
-0.49
-0.37
-0.18
-0.50
0.01
-0.20
-0.07
Fig. 4.2 Score plot of 100 cocoyam accessions based on 13 quantitative traits.
The numbes stand for the serial numbers of the accession code as defined in Table 4.1. The accessions 1-14, 15-26, 27-54,
55-82 and 83-100 were from Bench-maji, Kefa, Dawuro, Wolaita and Gamo-Gofa zones, respectively.
53
The cluster analysis based on the mean values of 13 quantitative traits grouped the 100
cocoyam accessions into 4 clusters (Fig. 4.3). The clustering means of accessions based on
13 quantitative traits is presented in Table 4.7. Cluster four, C-IV (blue), was the largest
cluster composed of 41 (41%) of accessions and the least compacted cluster than all other
clusters, having the largest within cluster sum of squares. Accessions in this cluster were
characterized by the highest mean value for number of cormels per plant (Table 4.1.7).
Cluster three, C-III (purple), was the second larger cluster contained 27 (27%) of the total
accessions. Of the 27 accessions grouped in this cluster, 25 (92.3%) were purple cocoyam
morphotypes. Cluster two, C-II (red), included the smallest number of accessions (11%) than
all other clusters and it is a more compact cluster, which has the smallest within cluster sum
of squares. Accessions in this cluster were characterized by the highest mean values for plant
height, petiole length, lamina length, lamina width, circumference of pseudo-stem and corm
length. Cluster one, C-I (green), contained 21 (21%) genotypes, which were characterized
by the lowest mean values for most of the traits studied (Table 4.7). The cluster analysis
showed that most of the accessions collected from different zones, woredas or kebeles were
clustered together.
54
30
98
65
24
20
100
90
59
50
61
48
60
80
54
34
47
35
25
15
38
13
97
87
76
70
82
75
11
6
8
83
5
53
91
81
67
99
78
45
68
93
84
64
96
55
51
89
71
66
86
95
92
46
43
58
69
56
79
33
10
21
41
12
42
23
14
63
9
73
28
29
77
44
22
19
16
52
18
4
37
57
27
40
88
31
3
26
72
94
7
85
74
49
36
39
32
17
62
2
1
C-I
C-II
C-III
C-IV
12.21
4.07
8.14
0.00
Euclidean distance
Fig. 4.3 Cluster analysis showing the relationship among 100 cocoyam accessions based on
13 quantitative traits. The numbers stand for the serial numbers of the accession code as defined in Table 4.1. The
accessions 1-14, 15-26, 27-54, 55-82 and 83-100 were from Bench-maji, Kefa, Dawuro, Wolaita and Gamo-Gofa zones,
respectively.
55
Table 4.7 Cluster means of 13 quantitative traits of cocoyam
Traits
Plant height (cm)
Petiole length (cm)
Petiole sheath length(cm)
Lamina length (cm)
Lamina width (cm)
Circumference pseudo-stem (cm)
Number of cormels per plant
Cormel length (cm)
Cormel diameter (cm)
Cormel fresh weight per plant (kg)
Corm length (cm)
Corm diameter (cm)
Corm fresh weight per plant (kg)
Within cluster sum of squares
C-I
67.26
50.16
38.44
34.50
20.33
26.00
9.19
9.03
2.91
1.02
10.27
5.00
0.92
167.63
Means values of cluster
C-II
C-III
80.00
67.60
63.16
55.15
37.86
30.80
41.13
39.71
23.89
22.63
31.69
30.42
10.32
9.28
9.61
8.60
3.59
3.68
1.43
1.31
12.30
14.08
6.19
6.31
1.29
1.51
117.39
192.94
C-IV
73.93
55.51
31.21
37.28
22.14
28.48
10.93
9.21
3.08
1.35
11.28
5.55
1.07
304.46
4.4 Discussion
In this study, out of 16 qualitative traits, 9 discriminated cocoyam accessions included in
this study into two groups while 7 qualitative traits did not show differences among
accessions. Mbouobda et al. (2007) reported a list of five qualitative descriptors while
evaluating cocoyam in Cameroon. Opoku-Agyeman et al. (2004) reported 15 qualitative
descriptors while evaluating cocoyam germplasms in Ghana. Among non-discriminatory
qualitative traits, leaf margin color, color of cormel apex and internal color of cormels,
however, showed polymorphism among cocoyam accessions from Ghana (Opoku-Agyeman
et al., 2004). Inability of seven traits to discriminate our collections may be due to cocoyam
accessions included in this study might be less diverse for the traits than the cocoyam
accessions characterized by Opoku-Agyeman et al. (2004) in Ghana. Thirty-five (35%) of
the accessions were purple colored (in generic term), having purple petiole, dark green color
of upper leaf surface and purplish green color of lower leaf surface. Sixty- five (65%) of the
accessions were green, having green petiole color (upper 2/3rd), green streaked with purple
petiole color (lower 1/3rd), green color of leaf surface. Green- and purple-colored cocoyam
56
plants were observed in the farmers’ field collected for this study. Farmers identify the green
and purple cocoyam by different local names (Chapter 3). Diversity in leaf color is
considered to be most important as it is frequently used to characterize plant germplasms
and to distinguish them visually (Mandal et al., 2013).
Basic statistical parameters based on quantitative morphological traits showed high
differences between the maximum and minimum values for plant height (57.58 cm, 84.83
cm), petiole length (42.75 cm, 67.83 cm) and petiole sheath length (19.17 cm, 48.67 cm).
CV% value for 11 (84.6%) of the studied traits were <20%. According to Mohammadi and
Talebi (2015) CV% <20% is considered to be good, indicating the accuracy of conducted
experiments. In this study, 84.6% of the quantitative traits showed significant variation
among the accessions, supporting the study of Solomon Fantaw et al. (2014b) that reported
significant variation for all of the 16 studied quantitative traits. Villavicencio et al. (2016)
reported that the morphological traits such as lamina width, lamina length, petiole length,
fresh weight of cormels per plant and corm size manifested significant variation among the
cocoyam accessions. All of these traits also showed significant variation in this study. The
underground traits, except cormel length, showed significant variation. Solomon Fantaw et
al. (2014a) reported that cocoyam accessions from Ethiopia were significantly discriminated
by underground traits. The analysis of 50 taro accessions from Nagaland revealed maximum
contribution of underground traits such as corm girth, corm yield per plant and number of
cormels towards genetic divergence (Mezhii et al., 2015).
The estimates of genetic variance components and coefficient of variations showed that the
δ2p and PCV were higher than δ2g and GCV, respectively, suggesting the presence of
environmental influence on the traits studied. However, the differences between the PCV
and the GCV were low for 9 (69.3%) of the 13 studied traits, implying that the environmental
effect on these traits observed to be low. The PCV and GCV values greater than 20% are
57
regarded as high, values between 10-20% are regarded as medium and less than 10% are
classified as low (Deshmukh et al., 1986). In our assessment, the GCV was high for only
corm fresh weight per plant and the PCV were low for lamina length and lamina width.
The broad sense heritability (h2b) could be classified as high (>30%), medium (10-30%) and
low (<10%) (Bhateria et al., 2006). In our assessment, the h2b estimates were high for 6
(46.2%) of studied quantitative traits such as lamina length, lamina width, circumference of
pseudo-stem, number of cormels/plant, corm diameter and corm fresh weight per plant,
suggesting the environmental effect on these traits observed to be low. Due to higher h2b
estimates, greater benefits from clonal selection might be expected for these traits. The h2b
and GA% were negative for cormel length. Normally, heritability values range from 0-1 but
this is not always the case since the experimental error could lead to estimates outside the
stated range (Conner and Hartl, 2004). Negative h2b for cormel length with negative GA%
may indicate that cormel length is mostly influenced by environment, suggesting that limited
benefit might be expected from selection of cormel length for cocoyam improvement.
PCA is often conducted to build a new set of orthogonal coordinate axes and to find out the
relative significance of classification traits (Shinwari et al., 2014). There are no dealings to
discover the worth of a coefficient but that is eigen vector (Düzyaman, 2005). The overall
observations for the 3 PCs, with the eigen values >1 for 13 quantitative traits explained
69.2% of the observed variations among 100 cocoyam accessions. This indicated that the
traits within these 3 PCs exhibited great influence on the phenotype of the accessions and
can be used for selection among accessions. Solomon Fantaw et al. (2014a) reported 4 PCs
which explained 70.5% of the total variation present among 64 cocoyam accessions at
Jimma, Ethiopia.
58
The quantitative morphological traits which had loaded relatively higher to the 3 PCs were
plant height, petiole length, lamina length, lamina width, circumference of pseudo-stem,
number of cormels per plant, cormel length, cormel fresh weight/ plant, corm diameter and
corm fresh weight per plant. These quantitative traits and 9 qualitative traits that categorized
the cocoyam accessions into two groups will help the researchers in recommending best
traits for morphological identification of cocoyam germplasms. Top coefficients for traits
designate the relatedness of traits to the PC axes (Sneath and Sokal, 1973). Score plot
effectively separated purple cocoyam morphotypes from green cocoyam morphotypes.
Supporting the PC analysis, cluster analysis based on Euclidean distance had grouped most
of the purple cocoyam morphotypes in distinct cluster (cluster III). This may suggest the
existence of genetic difference between the two groups of cocoyam morphotypes.
Most of the accessions collected from different zones, districts or kebeles were clustered
together, suggesting that cocoyam accessions might have been transported across localities.
Similar study conducted by Solomon Fantaw et al. (2014a) showed that most of the cocoyam
accessions from different districts and villages of Ethiopia were clustered together. In
Ghana, 70 cocoyam accessions collected from different geographical regions clustered
together (Offei et al., 2004). Similarly, Opoku-Agyeman (2004) reported that 78 cocoyam
accessions from 7 regions of Ghana were clustered into eight different groups, irrespective
of the collection sites. The study identified morphological traits for morphological
identification of cocoyam accessions. The accessions should be tested in more than one
environment to select elite genotypes for future utilization of cocoyam in Ethiopia.
59
Chapter Five
Assessment of Genetic Diversity and Differentiation of Cocoyam (Xanthosoma
sagittifolium (L.) Schott) from Ethiopia Based on SSR Markers
Abstract
Cocoyam (Xanthosoma sagittifolium (L.) Schott) is originated in the tropical America and
nowadays it is distributed throughout the tropical world. It is cultivated in southern, southwest and
western parts of Ethiopia, for its food and feed values. There is no information available on
molecular diversity of cocoyam growing in the country. This study was carried out to examine the
genetic diversity and differentiation of 100 cocoyam accessions (65 green colored and 35 purple
colored) using 11 polymorphic microsatellite loci. A total of 36 alleles were detected with an
average of 3.273 alleles per locus with range between 2 and 5 alleles. The average Ho value across
populations was 0. 503. The Ho values of green morphotypes, purple morphotypes and when all
collections were considered as single population were 0.466, 0.577 and 0.508, respectively. The
average He values across populations was 0.443. The He values of green morphotypes, purple
morphotypes and when all collection were considered as single were 0.291, 0. 345 and 0.566,
respectively. These high values of He imply that our collections contained genetically diverse
cocoyam accessions with high levels of heterozygosity. The greater Ho than He for most of the
loci (54.5%) may suggest high rates of vegetative propagation. Based on Nei’s F-Statistics for
overall loci showed strong differentiation among populations (mean Fst = 0.196). Very strong
differentiation was observed between green and purple cocoyam morphotypes (mean Fst = 0.463).
The analysis of molecular variance showed that the variation among population explained 14%
while the variation from among individuals within populations and within individuals,
respectively, explained 18% and 68% of the total variation. Dendrogram based on Nei’s standard
genetic distance grouped the green morphotypes together while the purple cocoyam morphotypes
occupied separate position within the dendrogram. Two clusters, which were detected by using
model-based Bayesian clustering, were fully associated with two cocoyam morphotypes. A high
level of genetic diversity within population, within morphotypes as well as at entire collection level
implies genetically diverse cocoyam accessions probably with multiple lineage have been growing
in Ethiopia, and which should be considered as a good opportunity for future use and conservation
of cocoyam in the country.
Keywords: Cocoyam, genetic diversity, SSR markers
60
5. 1 Introduction
Cocoyam (Xanthosoma sagittifolium (L.) Schott) is one of tuberous root crops. It was ranked
as 6th among root and tuber crops in cultivation and production (Bown, 2000). It originated
and first domesticated in tropical America. Nowadays, it is widely cultivated in many
tropical countries for its edible tubers and leaves (Ramawat and Merillon, 2014). It has high
acceptance and serve as food security crop, mainly for small scale farmers (Osawaru and
Ogwu, 2015).
Cocoyam is exotic crop to Ethiopia although it is not clear when and how it was introduced
into the country. The existence of cocoyam gene pool in south, southwest and western parts
of the country was reported by different authors (Zemede Asfaw, 2001a, b; Amsalu Nebiyu
and Tesfaye Awas, 2006; Amsalu Nebiyu et al., 2008; Fujimoto, 2009; Solomon Fantaw et
al., 2014a, b; Feleke Woldeyes et al., 2016), but not received thoughtful research effort.
The knowledge of genetic diversity result from analysis and characterization of accessions
allows evaluation of genetic variability, which is a fundamental element to determine
conservation plans and breeding strategies (Mwenye, 2009). Morphological markers-based
characterization of cocoyam accessions from Ethiopia was undertaken by Solomon Fantaw
et al. (2014a) and Chapter 4 of this study. The genetic information provided by
morphological traits is, however, often limited and expression of quantitative traits is
subjected to strong environmental influence (Karp et al., 1997; Mondini et al., 2009). The
use of morphological markers alone immediately excludes analysis of those portions of the
genome containing non-coding sequences, which in plants can often account for more than
90% of the genome.
Molecular markers have several advantages over morphological markers (Kordrostami and
Rahimi, 2002) although they cannot completely replace morphological traits-based
61
characterization. Thus, complementary to morphological characterization, the use of
molecular markers-based characterization has been increasing important as it suggests more
specific results (Karp et al., 1997). Molecular markers do not affect the phenotype of the
trait of interest because they are located only near or linked to genes controlling the trait, but
they can be used as signs or tags when they are located in loci, where they control specific
traits within the organism (Ishikawa et al., 1989). They could reveal polymorphism in a
DNA sequence or the presence or absence of a particular DNA sequence at a particular site
in the genome.
Molecular marker-based techniques such as random amplified polymorphic DNA (RAPD)
(Schnell et al., 1999; Offei et al., 2004), amplified fragment length polymorphisms (AFLPs)
(Loh et al., 2000), chloroplast and mitochondrial-specific primers (Brown and Asemota,
2009), simple sequence repeats (SSRs) (Cathebras et al., 2014), retrotransposon based
molecular markers (Doungous et al., 2015) and inter simple sequence repeats (ISSR)
(Sepúlveda-Nieto et al., 2017) have been used to characterize cocoyam accessions from
different countries. Genetic diversity of cocoyam accessions from Ethiopia has not been
reported using molecular markers. In order to enhance effective usage and conservation of
this neglected and underutilized crop in the country, it is important to study its genetic
diversity in detail. In this study, therefore, the genetic diversity of cocoyam in Ethiopia was
assessed by using SSR markers to generate data that will be beneficial in usage and
conservation of cocoyam in the country.
62
5.2 Materials and methods
5.2.1 Plant materials
One hundred cocoyam tubers were collected from 5 zones (Bench-Maji, Kefa, Dawuro,
Wolaita and Gamo-Gofa). Twelve to twenty-eight accessions were sampled from each zone.
The individual accessions were collected from at least 5 km apart unless they were clearly
distinguished by leaf and petiole color difference (65 green- and 35 purple-colored) (Table
4.1). The collected accessions were planted at a common garden at Areka Agricultural
Research Center, 303 km southwest of Addis Ababa, Ethiopia. Young leaf samples of 100
cocoyam accessions were collected by using silica gel (Bio lab). In the assumption that
accessions might have been shared within zones more frequently than among zones, those
cocoyam accessions collected from the same zone were considered as one population for
genetic diversity analysis.
5.2.2 DNA extraction
Total genomic DNA was extracted by using DNeasy plant min kit (QIAGEN), briefly as
follows: The dried leaf sample (~20 mg) was grinded using liquid nitrogen. Cell lysate buffer
(400 µl) and RNase A (4 µl) were added and mixed by vortexing. The mixture was incubated
for 30 minutes in dry heat shock by continuously mixing at 300 rpm. After adding
neutralization buffer (130 µl), the sample was incubated on ice for 10 minutes and
centrifuged for 5 minutes at 14,000 rpm. The lysate was pipetted into QIAGEN shredder
spin column and centrifuged for 2 minutes at 14,000 rpm. The flow-through was transferred
to a new tube without disturbing the pellet. Then, washing buffer (1.5 * volume of the flow
through) was added, mixed by pipetting and transferred to a DNeasy mini spin column and
centrifuged for 1 minute at 8,000 rpm. The flow-through was discarded, and the spin column
was placed into a new 2 ml collection tube. For final washing, another washing buffer (500
µl) was added and centrifuged for 1 minute at 8,000 rpm. The spin column was removed
63
carefully and transferred to a new 1.5 ml microcentrifuge tube. Finally, elution buffer (100
µl) was added and incubated for 5 minutes at room temperature and then centrifuged for 1
minute at 8,000 rpm. The genomic DNA was checked by 2% agarose gel and measured by
Nanodrop 2000 (Thermo Fisher).
5.2.3 SSR primers and PCR amplification
Seventeen microsatellite primer pairs identified by Cathebras et al. (2014) for X.
sagittifolium were tested with 5 randomly selected samples. Twelve primer pairs, which
gave an intense band on an agarose gel were selected for subsequent analysis (Table 5.1).
The forward primer of each of the 12 pairs was labeled either with 6-FAM (6Carboxyfluorescein) or Hexachloro-fluorescein (HEX). The markers were paired into 6 PCR
sets with each set containing a 6-FAM labeled primer pair and a HEX labeled primer pair.
These labeled primers were used to test 15 samples in a pre-analysis. PCR was performed
in a total volume of 20 μl containing 2µl of 10x PCR buffer (15 mM MgCl2 included), 0.4
µl dNTP mix (10 mM each), 0.5 µl (16 pmol/μl) of each of the forward and reverse SSR
primers, 2 µl genomic DNA (20 ng/µl), 0.2 μl Taq polymerase and 14.4 μl ddH2O.
Amplification was performed in ABI 2720 Thermocyler. After initial denaturation at 95°C
for 2 minutes, 30 cycles of denaturation at 94°C for 30 seconds, annealing at 52°C for 45
seconds, extension at 72°C for 1 minute followed with a final extension of 72°C for 10
minutes were used.
After verification of PCR amplification through 1% agarose gel electrophoresis, capillary
electrophoresis was run on the same basic principles. The aliquots of PCR product (1
µl/well) were mixed with formamide (12 µl/well) and GeneScan™ 400HD ROX™ Size
Standard (0.4 µl/well) in a 96 plate. The contents were denatured using a Boekel dry bath,
which was heated to 95°C for 5 minutes and then the samples were quenched at -20°C for 5
64
minutes and then electrophoresed on an ABI 3130XL Genetic Analyzer (Applied
Biosytems). The number and sizes of electrophoresed DNA fragments were identified using
GeneMapper version 4.0 (Currie-Fraser et al., 2010), which calculates allele sizes at each
microsatellite locus compared to a size standard (HD400 ROX). Data were collected,
evaluated and edited by using GeneMapper and exported to a Microsoft Excel spreadsheet.
Table 5.1 Primers sequences used for amplification of microsatellite markers
Duplex
A
Locus name
mXsCIR05
Primer sequence (5 ′-3 ′)
Repeat motif
(CA)8 (CACA)3
6-FAM-CGCATTATTAACGAATATC
TCATCTATGGCTATCACCT
mXsCIR07
B
mXsCIR10
(TG)7 (AG)19
(AG)22
HEX -GGACTGGGAGTCTGAGTAG
C
mXsCIR11
mXsCIR24
(AG)22
(TG)10 (GA)16
(AG)23
mXsCIR12
(TC)17 (TTC)7
6-FAM-ATGTCTGTAGTGGCCTAGT
Forward
(AG)15 (GAA)6
Forward
AATTTGCTCTGTCATTGTG
Reverse
6-FAM-AATTCTTAGCAGCATTGTTA
Forward
CATTCGTATCAACTTCCTTT
Reverse
HEX- AATTTGAAGTGAAACGATCA
6-FAM-TACATTTCCATTGCCATC
AAATTAAAGAGGGAGACAG
HEX-TGCATGAATTGAAGAAAT
AACAAAGAGTCTCACCACAT
E
mXsCIR19
mXsCIR21
(AC)8 (AC)24 (AC)8
(AG)30
mXsCIR16
mXsCIR28
(AG)15
(GA)9
Forward
Reverse
Forward
Reverse
Forward
Reverse
6-FAM -AACTTGTGTATCCTACATCC
Forward
GCGTGGTTTATGTGTATCTT
Reverse
HEX-CTTAACCTTGTCAGCCTCT
GAGCGGTATAACAATTCATC
F
Reverse
HEX-CGTGAGAAACACCTGAATTA
(TCCC)3 (TTCTTG)3
mXsCIR27
Forward
Reverse
TCCTGTCATCAGAATTGTA
D
Reverse
CCTTTCCCCTCACTATAAA
AATTAAGTTGGGTGGTAGAT
mXsCIR22
Primer
direction
Forward
Forward
Reverse
6-FAM-CTTATTGATGCCGAGAATAC
Forward
TTCCTCACAATATGTTCTCAT
Reverse
HEX- ACAGAAGTTGACATGGAGAG
Forward
AATGTTAAAGAGCAAAAGGA
Reverse
65
5.2.4 Data analysis
The number of alleles (Na), the number of effective allele (Ne) (Kimura and Crow, 1964),
Shanon information index (I) (Lewontin, 1972), observed (Ho) and expected (He)
heterozygosity within individual populations, within green morphotype, within purple
morphotype and at entire collection level were generated using the program POPGENE
version 1.32 (Yeh et al., 1999). The genetic differentiation among populations as well as
between green- and purple-morphotypes were estimated locus by locus by using the software
GenAlEx version 6.503 (Peakall and Smouse, 2006), in terms of Nei’s F-statistics (Fst) (Nei,
1987). AMOVA was performed to evaluate the partitioning of molecular variation within
and among populations by using the same program, by performing a significance tests using
999 permutations. The genetic clustering was assessed by performing PCoA. A neighbor
joining tree, depicting relationships among individuals was constructed by using neighbor
joining algorithm implemented in the computer program PHYLIP version 3.6 (Felsenstein,
2005) using Nei’s genetic distance (Nei et al., 1983) based on the frequencies generated by
using MSA version 4.05 (Daniel et al., 2003). The genetic structure was determined by using
the program STRUCTURE version 2.2.4 (Pritchard et al. 2000) based on Bayesian
clustering approach (Evanno et al., 2005) to infer the most likely number of gene pools and
the extent of genetic admixture among populations. The program was run 10 times for each
K, starting from K=1 to K=20 with an initial length of 100,000 burn-in periods followed by
200,000 Markov Chain Monte Carlo (MCMC) repeats for K. The analysis was carried out
using an admixture model with allele frequencies correlated among populations and the
parameter of individual admixture alpha set to be the same values for all clusters with a
uniform prior.
66
5.3 Results
5.3.1 Genetic diversity of cocoyam as revealed by SSR markers
Among a total of 12 SSR loci, 11 (91.7%) showed polymorphism. One locus (locus
mXsCIR28), which showed monomorphic fragments in all analyzed samples, was not used
for further analyses. A total of 36 alleles were detected. The Na at each locus ranged from 2
to 5 with mean of 3.273. The Ne per locus ranged from 1.412 (locus mXsCIR07 to 3.759
(locus mXsCIR22) with mean Ne of 2.516. The observed heterozygosity (Ho) ranged from
0.000 (loci mXsCIR19, mXsCIR16 and mXsCIR24) to 1.000 (loci mXsCIR22 and
mXsCIR27) with mean Ho of 0.508. The expected heterozygosity (He) ranged from 0.294
(locus mXsCIR07) to 0.738 (locus mXsCIR22) with mean He of 0.566 (Table 5.2). Six SSR
markers (54.5%) presented Ho values higher than the He values when all collections are
considered as single population.
Table 5.2 Genetic diversity parameters shown by 11 polymorphic loci in 100 cocoyam
accessions
Locus name
mXsCIR05
Na
4
Ne
3.499
I
1.304
Ho
0.800
He
0.720
mXsCIR07
2
1.412
0.468
0.355
0.294
mXsCIR19
2
1.851
0.652
0.000
0.462
mXsCIR10
4
2.810
1.087
0.905
0.648
mXsCIR21
4
2.498
1.098
0.372
0.603
mXsCIR22
4
3.759
1.354
1.000
0.738
mXsCIR11
4
2.158
0.969
0.650
0.539
mXsCIR24
2
1.821
0.643
0.000
0.453
mXsCIR12
3
2.368
0.972
0.505
0.581
mXsCIR27
5
3.689
1.364
1.000
0.733
mXsCIR16
2
1.807
0.639
0.000
0.449
Mean
3.273
2.516
0.959
0.508
0.566
St Dev
1.103
0.823
0.318
0.395
0.142
Na - number of different alleles, Ne - effective number of alleles, I - Shannon information index, Ho
- observed heterozygosity and He - expected heterozygosity
67
5.3.2 Genetic diversity within populations and within morphotypes
The observed number of alleles (Na), effective number of alleles (Ne), observed
heterozygosity (Ho) and expected heterozygosity (He) and Shannon information index (I)
across populations are presented in Table 5.3. The average Na for individual populations
varied from 1.727 (Bench-Maji, Kefa) to 3.091 (Gamo-Gofa) and the average Ne for
individual populations varied from 1.560 (Kefa) to 2.660 (Gamo-Gofa), with mean Na =
2.455 and Ne = 2.091, cross-populations. The Ho values ranged from 0.446 (Bench-Maji) to
0.540 (Gamo-Gofa). The He values ranged from 0.267 (Bench-Maji) to 0.608 (Gamo-Gofa).
The Shannon diversity index ranged from 0.391 (Bench-Maji) to 0.990 (Gamo-Gofa). The
genetic diversity analysis conducted by grouping green and purple morphotypes separately
showed that green morphotype had lower mean Na =1.909, Ne = 1.594, Ho = 0.466, He =
0.291 and I = 0.439 than purple cocoyam morphotype, which had mean Na = 2.273, Ne =
2.516, Ho = 0.508, He = 0.3447 and I = 0.5263 (Table 5.3).
Table 5.3 Genetic diversity within populations (A) and within morphotypes (B) using 11
microsatellite loci
Population
A
N
Na
Ne
Ho
He
I
Bench-Maji
14
1.727
1.631
0.446
0.267
0.391
Kefa
12
1.727
1.560
0.515
0.289
0.399
Dawuro
28
2.818
2.093
0.492
0.476
0.787
Wolaita
28
2.909
2.513
0.520
0.574
0.935
Gamo-Gofa
18
3.091
2.660
0.540
0.608
0.990
Average across populations
20
2.455
2.091
0.503
0.443
0.700
Green cocoyam
65
1.909
1.594
0.466
0.291
0.439
Purple cocoyam
35
2.272
1.770
0.577
0.345
0.526
Single population
100
3.273
2.516
0.508
0.566
0.959
B
N - number of accessions; Na - number of different alleles; Ne - effective number of alleles; Ho observed heterozygosity; He - expected heterozygosity; I - Shannon information index
68
5.3.3 Genetic differentiation
Paired comparisons based on locus by locus analysis for 11 polymorphic microsatellite loci
among populations revealed that Fst ranged from 0.080 at locus mXsCIR10 to 0.418 at locus
mXsCIR21. Most of the loci have indicated strong genetic differentiation among
populations as well as between green- and purple-morphotypes. The Fst values between
green- and purple-morphotypes ranged from 0.208 at locus mXsCIR21 to 1.000 at loci
mXsCIR19, mXsCIR24, i.e., at these two loci, green- and purple-morphotypes showed
complete differentiation. This shows that alleles at these loci were private either to green or
to purple morphotype. Average F-statistics over all loci showed that genetic differentiation
among five populations and morphotypes were strong and very strong as revealed by mean
Fst values of 0.196 and 0.463, respectively (Table 5.4).
Table 5.4 F-statistics for 11 polymorphic loci across populations and between morphotypes
mXsCIR05
Fis
-0.358
Fit
-0.177
Fst
0.134
Fis
-0.621
Green and purple
morphotypes
Fit
Fst
-0.040
0.363
mXsCIR07
-0.377
-0.176
0.146
-1.000
-0.333
0.333
mXsCIR19
1.000
1.000
0.343
N/A
1.000
1.000
mXsCIR10
-0.597
-0.469
0.080
-0.770
-0.334
0.247
mXsCIR21
0.048
0.446
0.418
0.043
0.242
0.208
mXsCIR22
-0.570
-0.415
0.099
-0.982
-0.333
0.327
mXsCIR11
-0.405
-0.209
0.140
-0.561
-0.105
0.292
mXsCIR24
1.000
1.000
0.362
N/A
1.000
1.000
mXsCIR12
-0.081
0.101
0.169
-0.714
0.047
0.444
mXsCIR27
-0.586
-0.410
0.111
-0.969
-0.326
0.326
mXsCIR16
1.000
1.000
0.339
1.000
1.000
0.943
Mean F statistics
over all loci
-0.171
0.0790
0.196
-0.664
0.105
0.463
Locus name
Five cocoyam populations
Fis - Fixation index among individuals within population or color groups, Fit- fixation
index among individuals within total data set, Fst- genetic differentiation among
populations or between green and purple morphotypes
69
The partitioning of molecular variance (AMOVA) showed that the highest genetic
variability (68%) existed within individuals and the lowest variability (14%) existed among
populations (Table 5.5).
Table 5.5 Summary of AMOVA for five cocoyam populations based on SSR markers
df
Sum of
squares
Estimated
variance
% of
variation
Fst
P
value
Among Pops
4
94.046
0.510
14
0.143
0.001
Among Indivs
95
349.994
0.637
18
Within Indivs
100
241.000
2.410
68
Total
199
685.040
3.558
Source of variation
5.3.4 Cluster analysis and population structure
The PCoA indicated that the first coordinate fully separated green morphotype from purple
morphotype. Cocoyam accessions collected from Bench-Maji and Kefa zones were
separated by the first coordinate while those from Dawuro, Wolaita and Gamo-Gofa Zones
were not separated by both coordinates (Fig. 5.1). When the collection data is referred, only
green morphotype were observed and collected from Bench-Maji and Kefa zones while both
green and purple morphotypes were collected from the other zones (Table 4.1). Individual
genotype clustering is presented in the neighbor joining (NJ) tree based on Nei’s genetic
distance. The clustering fully separated green morphotype from the purple morphotype (Fig.
5.2), supporting PCoA.
70
A
Coord. 2 (15.19%)
By
Leaf and petiole color
Green
Purple
Coord. 1 (54.05%)
B
Populations
Bench-Maji
Kefa
Dawuro
Coord. 2
Wolaita
Gamo-Gofa
Coord. 1
Fig. 5.1 A two-dimensional plot of the Principal Coordinate Analysis (PCoA) of 100
cocoyam accessions based on SSR data based on leaf/petiole color (A) and populations (B)
71
Fig. 5.2 Neighbor Joinings tree showing the relationships among 100 cocoyam accessions.
The tree was based on Nei’s genetic distance (Nei et al., 1983) using 11 microsatellite markers. Green- and purple-cocoyam
morphotypes, represented by green and purple colors. Xs and the numbers, respectively stand for the species name and serial numbers
of the accession code as defined in Table 4.1. The accessions Xs001-Xs014, Xs015-Xs026, Xs027-Xs054, Xs055-Xs082 Wolaita and
Xs083 - Xs100 were from Bench-maji, Kefa, Dawuro, Wolaita and Gamo-Gofa zones, respectively.
72
To measure genetic structure of population and degree of admixture, the STRUCTURE
algorithm was applied. The highest log-likelihood score was obtained for K=2 (Fig. 5.3a).
Population structure detected by using the STRUCTURE software confirmed the results of
PCoA and the neighbor joining (NJ), clustering green-colored cocoyam accessions from all
of 5 populations in one group (Fig. 5.3b). The clusters identified for K=2 were 100%
associated with the green and purple morphotypes, i.e., 65 green morphotype were assigned
to one cluster and 35 purple morphotype were assigned to another cluster (Fig. 5.3c), where
the genetic admixture was insignificant.
Fig. 5.3 Bayesian model-based clustering STRUCTURE analysis as inferred at K = 2 based
on SSR data. The relationship between ΔK and K showing the highest peak at K = 2 (A); Each individual
accession is epresented by a vertical line divided into colored segments (B). Membership of each individual
within green- and purple cocoyam (C). Among the 20 runs carried out for each K, graphical representation of the
highest ∆K at K=2 estimate was shown.
73
5.4 Discussion
The results of this study revealed that the majority (91.7%) of the SSR loci were
polymorphic within 100 cocoyam accessions. In related study, Doungous et al. (2015)
reported 88% to 97% polymorphic loci across 20 cocoyam accessions from Cameroon using
retrotransposon molecular markers. The values of genetic diversity indices were high within
populations, within green morphotype, within purple morphotype and at entire collection
level. For example, He values across populations, within green morphotype and within
purple morphotype were 0.443, 0.291, 0.345, respectively, all of which were high compared
to that of Ambrosina bassii L. (Araceae, Ambrosineae), where mean He was 0.263 (Geraci
et al., 2009) and Anthurium crenatum (L.) Kunth (Araceae) (He = 0.167) (Acosta-Mercado
et al., 2002). The mean He (0.566) detected when all collections were considered as single
population was also greater than the average He (0.456) detected in taro (C. esculenta)
accessions by using microsatellite loci (Wansha et al., 2011). The high He values detected
in this study imply that our collections contained genetically diverse cocoyam accessions.
The average observed heterozygosity across populations as well as within green morphotype
and within purple morphotype were higher than the respective average expected
heterozygosity values. Six SSR markers (54.5%) presented Ho values higher than He values
when all collections were considered as single population. The result was in agreement with
the report of Cathebras et al. (2014), suggesting high rates of vegetative propagation in
cocoyam. According to Bisognin (2011), vegetative propagation enables to fix high levels
of heterozygosity. The progenies of vegetatively propagated crops are clones, which are
genotypically similar to the parental genotypes (Simmond, 1962).
The mean values of Fst between green- and purple- cocoyam morphotype showed very
strong differentiation demonstrated by Fst value of 0.463. According to Wright (1965), the
genetic differentiation is low, moderate, strong and very strong when Fst value is between
74
0-0.05, 0.05-0.15, 0.15-0.25 and >0.25, respectively. The mean value of Fis was negative
and also negative for most of the loci, implying that there is less effect of outcrossing among
individuals within populations. This is may be due to rarely flowering nature of cocoyam.
The analysis of molecular variance (AMOVA) showed that the variation among populations
explained 14% while the variation from among individuals within populations and within
individuals explained 18% and 68% of the total variation, respectively. This indicates that
highest percentages of variation which can be used to improve cocoyam for traits of interest
were found within individuals.
PCoA, NJ tree based on Nei’s genetic distance and population genetic structure analyses
resulted in clusters that were associated with green and purple morphotype, which can easily
be differentiated by their leaf color (green or purple). This indicates that the significant
fraction of the SSR markers between morphologically distinct cocoyams. Over all, there is
high genetic diversity within populations, within green morphotype, within purple
morphotype as well as when all collections were considered as single population. Very
strong genetic differentiation was detected between green and purple morphotypes. This
indicates that the vegetative propagation from stem underground structures (Kay, 1987),
may resulted in a fixed SSR markers genetic structure and cocoyam grown in Ethiopia may
have remained genetically unchanged with high heterozygosity. A high level of genetic
diversity implies geneticaly diverse cocoyam accessions probably with multiple lineage
have been growing in Ethiopia, and which should be considered as a good opportunity for
future use and conservation of cocoyam in the country.
75
Chapter Six
AFLP Fingerprinting for Assessment of Genetic Diversity and
Differentiation of Cocoyam (Xanthosoma sagittifolium (L.) Schott
Abstract
Cocoyam (Xanthosoma sagittifolium (L.) Schott) is an exotic crop to Ethiopia. Less research
attention was given to cocoyam as it is a neglected and underutilized crop. To estimate the
genetic diversity and population structure, 78 cocoyam accessions (54 green colored and 24
purple colored) were analyzed using AFLP markers. Three pairs of AFLP primers resulted in
478 scorable bands and 99.2% of polymorphic loci. The mean Nei’s gene diversity (He) across
populations was 0.349 the green morphotype showing 0.385 whereas purple morphotype
showing 0.351. Considering all collections as a single population, the expected heterozygosity
(He) was 0.389. Low level of genetic differentiation was detected among populations by the
Nei’s Gst = 0.072 as well as between green and purple morphotypes by the Nei’s Gst = 0.024.
ANOVA indicated 4% and 3% of genetic variation existed among populations and between
green and purple morphotype, respectively. These low genetic differentiation results were
further confirmed by PCoA, NJ clustering and Bayesian Model based STRUCTURE. Taking
together, this AFLP markers-based study results showed a high level of genetic diversity within
population, within green morphotype, within purple morphotype as well as when all collections
were considered as single population, implying that genetically diverse cocoyams have been
growing in Ethiopia. Low level genetic differentiation detected among populations indicates that
there might have been movement of germplasm across the locations, possibly by farmer-tofarmer planting materials exchange. Cocoyam management and conservation strategies should
concentrate on maintaining the existing high diversity within each population for sustainable
utilization of the crop in Ethiopia.
Keywords: AFLP, Cocoyam, genetic diversity, genetic differentiation, X. sagittifolium
76
6.1 Introduction
The genetic diversity of cultivated plant species is the result of evolution and human
intervention. Hereby, multiple factors shape the genetic structures of cultivated plants
(Oyama et al., 2006), including genetic drift, migration, mutation and selection (Wright
1978). The ultimate goal for the conservation of genetic resources is to guarantee
sustainability of genetic diversity (Kingston et al., 2004; Magbagbeola et al., 2010; Dansi et
al., 2012). Thus, it is important to examine the genetic diversity of crop plants for
conservation and for inferences in plant breeding programs (Lopes et al., 2014; Larranaga
et al., 2017).
Cocoyam, tania, tannia, arrowleaf, elephant's ear, malanga, marron, taye or tayove are some
of the globally used vernacular names to define a group of cultivated plants in tropical and
subtropical regions of the world that belong to the genus Xanthosoma (Araceae). The name
X. sagittifolium (L.) Schott has usually been given to the most cultivated members, either
for their ornamental use or for food source (tubers and leaves), of this genus (Giacometti
and Leon 1994; Govaerts et al., 2002). Various taxonomic names have also been used
synonymously (Lim, 2015; http://www.theplantlist.org) as X. sagittifolium is one of the
neglected and understudied cultivated plant species (Doungous et al., 2015). There are
discrepancies and uncertainties regarding the taxonomy even at the species level (Castro
2006; Giacometti and Leon 1994). In the course of this study, we refer to the accepted
species status of X. sagittifolium in the Araceae Family (Govaerts et al., 2002) and in The
Plant List (http://www.theplantlist.org).
Cocoyam is one of the oldest cultivated aroid in the world. There are several adaptations
that allow the species to survive and spread. It grows in a variety of substrates and habitats.
It tolerates drier, but not waterlogged soils (Kay, 1987; Bown, 2000) and grows in full sun
77
or deeply shaded areas under the canopy of natural forests (Manner 2011). Although
cocoyam is a lowland plant, it also grows in the highlands, e.g. in humid, tropical climate
up to 1500 m.a.s.l (Manner, 2011).
Cocoyam is a perennial plant, but for practical purposes, most often cultivated as an annual
crop. Harvest usually occurs during the dry season, 9-12 months after plantation (Lebot,
2009). At the end of the growing season and in water stressed conditions, leaves may die
and shoots may wither completely (Onwueme and Charles, 1994; Jackson, 2008; Ramawat
and Merillon, 2014). Cocoyam accessions seldom flower. When flowering occurs, the
flowers are monoecious within a compound inflorescence. The female flowers are at the
base of the spadix and the male flowers are above. Sterile flowers are located between the
pistillate and staminate flowers. The inflorescence is protogynous; the stigma is normally
receptive two to four days before pollen shed (Kay, 1987). Thus, cocoyam rarely set sexual
seeds and hence it is mainly propagated vegetatively from corm sets, headsets or cormels
(Jackson, 2008).
Cocoyam is believed to have originated from northern South America, spread to the
Caribbean and Mesoamerica, and was subsequently introduced elsewhere into Africa, Asia
and the Pacific. It has become a subsistence crop in West African countries (Ghana, Nigeria,
Cameroon), which are by now the major cocoyam producers of the world (Giacometti and
Leon, 1994). Cocoyam was introduced into East Africa through Western Africa and is
popular in Tanzania and common in Uganda (Maundu et al., 2009). It is exotic crop to
Ethiopia, but there is no clear information on how and when it was introduced into the
country. The species is not mentioned in the Flora of Ethiopia (Reidl, 1997; Hedberg et al.,
2009). According to Amsalu Nebiyu and Tesfaye Awas (2006) there are a considerable
amount of cocoyam gene pool in south and southwest Ethiopia in farmers’ fields and home-
78
gardens. Cocoyam has become an important part of agricultural and food system of the
indigenous communities in the country (Amsalu Nebiyu et al., 2008; Fujimoto, 2009; Feleke
Woldeyes et al., 2016).
Genetic diversity of cocoyam has been investigated by using Randomly Amplified
Polymorphic DNA (RAPD) in Florida (Schnell et al., 1999) and Ghana (Offei et al., 2004).
Doungous et al. (2015) used Inter-Retrotransposon Amplified Polymorphism (IRAP) to
discriminate between cocoyam and taro. Brown and Asemota (2009) and Cathebras et al.
(2014) applied microsatellite markers for the same question. Loh et al. (2000) applied
Amplified Fragment Length Polymorphism (AFLP) to determine the intergeneric
relationships between Caladium, Xanthosoma and other closely related genera within
Araceae. Different marker systems are currently available for assessmet genetic diversity
among populations. According to Donald (1994), AFLP is the most efficient marker to
identify individual genotypes in a highly clonal species. It has the potential to increase the
throughput of marker data production in organisms by allowing scoring of a large number
of characters in a given population (Vuylsteke et al., 2008). In this study, we have used
AFLP markers in order to achieve comprehensive information on the genetic diversity and
differentiation of the Ethiopian cocoyam.
6.2 Materials and methods
6.2.1 Plant materials
A total of 82 X. sagittifolium tubers, representing four populations were collected from
Bench-Maji (14), Kefa (12), Dawuro (28) and Wolaita zones (28) of Ethiopia (Table 4.1).
Tubers of individual X. sagittifolium samples (accessions) that could be clearly distinguished
by an observable morphological trait (color difference) were recorded as green and purple
morphotypes. The collected tubers were planted in a common garden and young leaf samples
were collected by using silica gel (Bio lab). The dried leaf samples were stored at room
79
temperature until further processing. The accessions that were collected from the same zone
were considered as one population, assuming that clones are more likely shared within zones
than among zones.
6.2.2 DNA extraction
Total genomic DNA was extracted using QIAGEN DNeasy plant min kit according to the
manufacturer’s instructions. The quality of DNA was checked on a 2% agarose gel. DNA
concentration was assessed by using Nanodrop 2000.
6.2.3 AFLP analysis
The AFLP assay was performed according to a modified protocol of Vos et al. (1995).
Genomic DNA was diluted to 30 ng/µl with ddH2O and restricted using two different
restriction enzymes: a frequent cutter (Tru1I) and a rare cutter (EcoRI) (Fermentas). The
reaction volume was 25 µl in total (9.8 µl ddH2O, 5 µl 10x Buffer Tango 2x Fermentas, 0.10
µl of 10 U/µl EcoRI, 0.10 µl of 10 U/µl Tru1I and 10 µl DNA). The mixture was incubated
at 37°C for 1 h followed by at 65°C for another 1 h. Double stranded adapters were ligated
to the restriction sites by adding 3 µl 10 mM ATP (Thermo Scientific), 0.5 µl 10x T4 DNA
ligase buffer (Fermentas), 0.5 µl EA +/- (Eco +/-) (5 pmol) (MWG Biotech), 0.5 µl MA +/(Mse +/-) (50 pmol) MWG-Biotech and 0.5 µl T4 DNA ligase (~2 U/µl) (Fermentas). The
mixture was incubated at 22°C for 1 hr and 75°C for 10 min to inactivate the enzyme
activities.
Two consecutive PCR amplifications were carried out with primers contained first one (+1)
then three (+3) selective nucleotides at their 3`ends. The pre-PCR was carried out in 25 µl
total reaction volume which contained 17.36 µl ddH2O, 2.5 µl 10x dream Taq DNA
polymerase buffer (20 mM MgCl2 included), 2.5 µl dNTP-Mix (10 mM each), 0.5 µl 5 µM
primer EpA (E01) MWG Biotech, 0.5 µl 5 µM primer MpC (M02) MWG Biotech, 0.14 µl
80
dream Taq DNA polymerase and 1.5 µl restriction-ligation product. After initial
denaturation at 94°C for 3 minutes, 30 cycles were repeated at 94°C for 30 seconds, 56°C
for 1 min and 72°C for 1 min, and a final extension at 72°C for 7 min. Then, the PCR product
was run on 2% control agarose gel. The pre-PCR products were diluted 1:10 by using
ddH2O. Selective PCR amplification was carried out with three primer combinations (E32AAC/M51-CCA, E35-ACA/M60-CTC, E40-AGC/M54-CCT) MWG Biotech. The sel-PCR
reaction contained 8.53 µl ddH2O, 1.28 µl 10x dream Taq DNA polymerase buffer (20 mM
MgCl2 included), 0.28 µl dNTP Mix (10 mM each), 0.42 µl (1 µM) primer EpANN (MWG
Biotech), 0.42 µl (5 µM) primer MpCNN (MWG Biotech), 0.07 µl dream Taq DNA
polymerase and 1.8 µl pre-PCR product. The touchdown PCR program was run after
adjusting the initial denaturation temperature at 94°C for 3 minutes, 30 cycles at 94°C for
30 seconds, 55°C for 1 min, 72°C for 1 min were repeated by reducing the primer annealing
temperature by 0.7°C each and a final primers extension of 72°C for 7 minutes. After
verification of PCR amplification through 1% agarose gel electrophoresis, sel-PCR products
were separated by capillary electrophoresis (ABI Genescan/Applied Biosystems), which
was done at LGC-Forensics/Cologne laboratory.
6.2.4 Data scoring and analyses
AFLP fragments (bands) were scored by using Genographer version 2.1.4 (Banks and
Benham, 2008). Only distinct and well resolved AFLP bands in the size range of 40-520 bp
were scored and a binary data matrix was constructed. POPGENE version 1.32 (Yeh et al.,
1999) was used to calculate genetic diversities, which include the number of polymorphic
loci (No. of PPL), the percentage of polymorphic loci (% PPL), the observed number of
alleles (Na) and the effective number of alleles (Ne) (Kimura and Krow, 1964), Nei’s gene
diversity (He) (Nei, 1973) in individual populations and the Shannon’s information index of
diversity (I) (Lewontin, 1972). The coefficients of total population gene diversity (Ht), the
81
mean gene differentiation among populations (Gst) and within populations (Hs) were
estimated using the same program in terms of Nei’s F-statistics in subdivided populations.
Analysis of molecular variance (AMOVA) was performed to evaluate the genetic variation
within and among populations by using the software GenAlEx version 6.503 (Peakall and
Smouse, 2006), performing significance tests using 999 permutations. The AMOVA
technique was also used to determine the partitioning of molecular variation between and
within green and purple morphotypes by using the same program. Potential correlations
between genetic and geographic distances among individuals were estimated by using
GenAlEx version 6.503 (Peakall and Smouse, 2006).
The genetic clustering was assessed by performing principal coordinate analysis (PCoA) by
using the GenAlEx version 6.503 (Peakall and Smouse 2006). A neighbor joining tree was
constructed by using neighbor joining algorithm (Saitou and Nei 1987) to examine the
genetic relationships at individual level by SplitTree4 version 4.14.5 (Huson and Bryant,
2006). The genetic structure was also determined by using the program STRUCTURE
version 2.2.4 (Pritchard et al., 2000) based on Bayesian and admixture models with a burnin of 100,000 followed by a run-length of 200,000 Markov Chain Monte Carlo (MCMC)
iterations for K. The analysis included 20 independent interactions and K-values ranging
from 1 to 10 to determine the number of genetic clusters based on the criterion proposed by
Evanno et al. (2005). Error rate of AFLP data was estimated using 12.8% of replicated
samples according to Pompanon et al. (2005) because quantifying genotyping error rates is
an essential component of an AFLP analysis.
82
6.3 Results
6.3.1 Genetic diversity of cocoyam as revealed by AFLP markers
From 82 cocoyam accessions belonging to four populations were, four accessions (accession
codes 23, 36,49 and 73) were removed from analysis due to lab letdown. Thus, AFLP
analysis was performed on 78 cocoyam accessions consist of 54 green and 24 purple
morphotypes using three different sets of AFLP primer combinations. A total of 478 scorable
bands were produced, of which, 474 (99.16%) were polymorphic. The amount and
percentage of polymorphic loci (PPL) within populations ranged from 407 (85.15%)
(Bench-Maji cocoyam population) to 461 (96.44%) (Dawuro and Wolaita cocoyam
populations), with an average of 434.50 (90.90%) (Table 6.1). The highest Na (1.964) and
Ne (1.649) exhibited by Dawuro cocoyam population while the lowest number of Na (1.852)
and Ne (1.576) were observed in Bench-Maji cocoyam population. The green cocoyam
morphotypes had slightly higher Na (1.985) and Ne (1.681) than purple cocoyam
morphotypes which had Na = 1.941 and Ne = 1.628. Estimates of Nei’s gene diversity in
individual populations showed high values within each population with the highest diversity
in Dawuro cocoyam population (He = 0.368) and the lowest diversity in Bench-Maji
cocoyam population (He=0.327), with an average He = 0.349. The Nei’s gene diversity
estimate in green- and in purple-cocoyam morphotypes were high within each group, He =
0.385 and 0.351, respectively. Considering all collections as a single population, Nei gene
diversity was also high (He = 0.389). The estimates of Shannon’s information index showed
values that are in similar trend with Nei’s gene diversity (Table 6.1).
83
Table 6.1 Genetic diversity statistics based on AFLP data for cocoyam populations from Ethiopia:
grouped according to the collection zones (A) and leaf and petiole color difference (B)
Population
A
N
No. of PPL
% PPL
Na
Ne
He
I
Bench-Maji
14
407
85.15
1.852
1.576
0.327
0.480
Kefa
11
409
85.56
1.856
1.602
0.338
0.493
Dawuro
26
461
96.44
1.964
1.649
0.368
0.541
Wolaita
27
461
96.44
1.964
1.639
0.363
0.534
19.5
434.50
90.90
1.909
1.617
0.349
0.512
Green cocoyam
54
471
98.54
1.985
1.681
0.385
0.564
Purple cocoyam
24
450
94.14
1.941
1.618
0.351
0.518
Single population
78
474
99.16
1.992
1.666
0.389
0.557
Average across pops
B
N – number of accessions, No. of PPL- number of polymorphic loci, % PPL - percentage of polymorphic
loci, Na - observed number of alleles, Ne - effective number of alleles. He - Nei’s gene diversity, I Shannon’s information index
6.3.2 Genetic differentiation and cluster analysis
Analysis of genetic variation among and within populations in terms of Nei's (1987) Fstatistics in subdivided populations showed that the total population genetic diversity (Ht)
was 0.378+0.02 and the mean genetic differentiation within populations (Hs) was
0.349+0.01. The mean genetic differentiation (Gst) among populations was 0.072. The Gst
between green- and purple-cocoyam morphotypes was 0.024. The analysis of molecular
variance (AMOVA) indicated little genetic differentiation among populations revealed by
only 4.0% while most of the genetic differentiation (96.0%) found within populations. Only
3.0% of the total genetic variability explained the differentiation between green- and purplecocoyam morphotypes (Table 6.2). Correlation between genetic distance and geographic
distance among individuals was insignificant (y=-0.0082x + 168.57, R2 = 0.0053, p>0.05).
84
Table 6.2 Summary of AMOVA for populations: grouped according to the collection zones
(A) and morphotypes: grouped by leaf and petiole color difference (B)
Source of Variation
A
Among populations
df
SS
Est. Var. % of Var.
3
432.953
3.397
4
Within populations
74
5993.80
80.988
96
Total
77
6426.077
84.385
100
B
Between morphotypes
1
163.077
2.428
3
Within morphotypes
76
6263.000
82.408
97
Total
77
6426.077
84.835
100
PhiPT
P value
0.040
0.001
0.029
0.001
df - degrees of freedom, SS - sum of squares, Est.Var. - estimated variance, % of Var. - percentage of variation
The two-dimensional plot of the PCoA represented 5.66% and 5.19% (in total 10.85%) of
the detected variation revealing that most of the accessions, independent of origin or leaf
color, cluster together whereas two groups consisted of two (accessions codes 34, 77) and
five (accessions codes 29, 42, 50, 59, 61) green cocoyam accessions from Dawuro and
Wolaita zones feature comparatively distinct patterns (Fig. 6.1). The PCoA analysis
supported the genetic differentiation results, indicating that the genetic structure of cocoyam
accessions included in this study was low. Clustering analysis also depicted a clear
admixture of populations, regardless of the source populations and color groups (Fig. 6.2).
85
A
55
62
By leaf and petiole
color
22
purple
72 69
54
74 58 40 6
32
17
71 37
64 63 10 19
60 2156
38
43
11 79
35
66 1
224
81
14
57
20
70
31
53 30
8065
51 9
76
4 16 48
45
39 67 3 5
8
68
15
1246 7 4426
18
75
25 27
13
28
4152
33
47
78
Coord. 2 (5.19)
82
Green
77
34
59
50 29
61 42
Coord. 1 (5.66%)
B
Populations
55
62
78
Coord. 2 (5.19%)
82
22
Bench-Maji
72
54
74 58 40 6
17
71
64 63 10
2169
60
19
81 56
79
43 32
1
11
35
24
66
37
2
14
57
70
31
53
65
30
80
20
51 9
76
48
16
4
45
25
6875 39
13 38
28
15
67 333546
7
27
26
12 4744
18
Kefa
Dawuro
Wolaita
8
4152
77
34
59
50 29
61 42
Coord. 1 (5.66%)
Fig. 6.1 Representation of the first two coordinates obtained from the PCoA of 78 cocoyam
accessions based on AFLP data based on leaf/petiole color (A) and populations (B)
86
Fig. 6.2 Neighbor-Joining (NJ) tree representing clustering of cocoyam accessions generated from AFLP data.
The tree was constructed by using neighbor joining algorithm implemented in the SplitTree4 version 4.14.5. The 78 accessions
were clustered regardless of the source populations. Green and purple colors, respectively, indicate green- and purple-cocoyam
morphotypes. The numbers stand for the serial numbers of the accession code as defined in Table 4.1. The accessions 1-4, 15-26, 27-54
and 55-82 were from Bench-maji, Kefa, Dawuro and Wolaita zones, respectively. Four accessions (23, 36, 49 and 73) were missing due
to lab letdown.
87
The STUCTURE analysis based on the ∆K method revealed a clear maximum ∆K at K=3
in which accessions were classified into three clusters (Fig. 6.3). These results showed that
the cocoyam accessions from four populations (Bench-Maji, Kefa, Dawuro and Wolaita)
were clustered into three groups. This result supports the lack of association between genetic
and geographic distance that was revealed by Mantle test.
A
B
Fig. 6.3 Bayesian model-based clustering STRUCTURE analysis as inferred at K = 3 based on AFLP
data. The relationship between ΔK and K showing the highest peak at K = 3; The genotype of each individual
accession is represented by a vertical line divided into colored segments, the lengths of which indicate the
proportions of the genome attributed to the inferred clusters (B). Among the 20 runs carried out for each K,
graphical representation of the highest ∆K at K=3 estimate is shown.
88
6.4 Discussion
This study reports genetic diversity of Ethiopian cocoyam accessions by using AFLP
markers. The three AFLP primer combinations resulted in 478 bands, of which 99.16% were
polymorphic. This value is comparable to other finding by Mwenye et al. (2016) who
detected 93% polymorphism by using eight AFLP primer combinations. Doungous et al.
(2015) reported 92.0% polymorphic bands by using inter-retrotransposon amplified
polymorphism (IRAP) markers to characterize white, red and yellow cocoyam accessions in
Cameroon. Schnell et al. (1999) identified only 17.5% polymorphic bands by using RAPD
on 18 cocoyam accessions from Florida. The analysis of eight ISSR molecular markers
showed low polymorphism (4.19%) among cocoyam accessions from Brazil (SepúlvedaNieto et al., 2017). The high level of polymorphism detected in our analysis suggests that
the AFLP primer combinations, used in this study, were highly discriminatory, revealing the
presence of higher genetic diversity of cocoyam accessions than the previously studied
accessions in Florida (Schnell et al., 1999) and in Brazil (Sepúlveda-Nieto et al., 2017).
The results of genetic diversity analysis of cocoyam detected by this study were higher than
the report by Offie et al. (2004), who studied cocoyam accessions from the eastern and Volta
region of Ghana by means of RAPD, where the mean Na, Ne and He were 1.99, 1.54 and
0.319, respectively. The average He value across populations, within green cocoyam
morphotypes, within purple cocoyam morphotypes and when all collections were considered
as single population were higher than the average He value (0.014) reported by SepúlvedaNieto et al. (2017) from Brazil by using ISSR molecular markers. The Shannon information
indices (I) detected by this study were also higher than the I (0.49), reported by Doungous
et al. (2015) who studied 20 cocoyam accessions by using IRAP markers. The genetic
diversity detected in this study was also higher than the allozyme variation (mean He = 0.26)
in Ambrosina bassii L. (Araceae) (Geraci et al., 2009) and Anthurium crenatum (L.) Kunth
89
(Araceae) (mean He = 0.23) (Acosta-Mercado, 2002). The high genetic diversity suggests
that our collection contained genetically diverse accessions.
The detected high genetic diversity in the present study supports earlier findings based on
morphological traits, which revealed the presence of diversity among cocoyam accessions
in the country (Solomon Fantaw et al., 2014a). Since cocoyam is exotic to Ethiopia, the high
genetic diversity may indicate that there might have been multiple introductions of cocoyam
accessions into the country, but this needs to be investigated. Many introduced plant species
had high level of diverse propagules through multiple introduction events (Rollins et al.,
2013). The predominantly vegetative propagation of cocoyam (McKey et al., 2010) supports
the fixation of high levels of diversity (Bisognin, 2011). The high genetic diversity detected
in this study should be considered as positive opportunity for effective usage and
conservation of cocoyam accessions. According to Herron and Freeman (2014), genetic
diversity holds a key to the ability of species to persist through changing environments.
The results of this study rejected our assumption to find a specific pattern in cocoyam
accessions by using AFLP markers due to the result revealed by the AMOVA, cluster
analysis and Nei’s genetic differentiation (Gst = 0.072 among populations). The most likely
reason for that may be the exchange of accessions across locations. The low genetic
differentiation (Gst = 0.024) between green and purple cocoyam morpthotypes furthermore
indicates that the green and purple morphotypes may have hybridized in the past. This
finding is in concordance to the ones reported by Mwenye et al. (2016) using AFLP markers
in that cocoyam accessions were clustered together. Doungous et al. (2015) studied the
genetic diversity of cocoyam accessions by using IRAP markers which could not
discriminate between different morphotypes (white tuber flesh color and red tuber flesh
color). Some sweet potato (Ipomoea batatas L.) accessions with different flesh colors were
90
assigned to the same cluster by population structure analysis, whereas some accessions with
the same flesh color were assigned to different clusters based on ISSR markers (Kai et al.,
2014).
The low levels of genetic differentiation detected among populations as well as between
green and purple morphotypes by AFLP markers are not in agreement with the results from
SSR markers. Some inconsistencies have been observed between the results (genetic
differentiation, cluster analysis and genetic structure). Contrasting patterns are commonly
found when different markers are used to detect genetic structure. The correlations of RAPD
marker data with those obtained using RFLP, AFLP and SSR marker systems were lower in
soybean (Powell et al., 1996). AFLP displayed no correspondence with RAPD and ISSR in
cashew (Anacardium occidentale L.) (Archak et al., 2003). The dendrogram obtained with
SSR markers was less similar to that obtained with AFLP markers in olive (Olea europaea
L.) (Belaj et al., 2003). Among the molecular markers (RAPD, RFLP, AFLP and SSR) used
to estimate genetic distance of tropical maize inbred lines, AFLP and RFLP gave the most
correlated results than others (Garcia et al., 2004). In conclusion, AFLP fingerprinting has
revealed high genetic diversity but low differentiation of Ethiopian cocoyam accessions.
Conservation and management plans of cocoyam in the country should concentrate on the
existing high levels of genetic diversity within each population as well as when all
collections were considered as single population.
91
Chapter Seven
Proximate, Mineral and Antinutrient Contents of Cocoyam
(Xanthosoma sagittifolium (L.) Schott) from Ethiopia
Abstract
Cocoyam (Xanthosoma sagittifolium (L.) Schott) is an important food crop especially in the tropics
and subtropics. Its cormels and leaves are eaten after cooking in the rural areas in Ethiopia. There is
lack of information on the nutritional composition of cocoyam grown in the country. In this study,
cormels of green- and purple- cocoyams were analyzed to determine proximate and mineral contents
and antinutritional factors. The moisture contents (%) of green- and purple- cocoyams were 61.91
and 63.53, respectively. Crude protein (10.10%) and fiber (2.66%) contents of purple cocoyam were
significantly higher than crude protein (8.48%) and fiber (2.14%) contents of green cocoyam. Fat
contents (%) of the green and purple cocoyam were 0.85 and 0.22, respectively. Ash content of green
cocoyam (3.25%) was significantly higher than the ash content of purple cocoyam (2.27%). The
carbohydrate contents (%) and gross energy values (Kcal/100g) of green- and purple- cocoyam,
respectively, were 85.36 and 378.47 and 84.76 and 380.27, showing that cocoyam grown in Ethiopia
can be a good source of energy. Mineral contents (mg/100g) of green cocoyam were determined as
Fe (8.20), Zn (3.07), Cu (1.04), Mg (78.77), Mn (2.48), P (120.93), Na (29.22), K (1085.70) and Ca
(56.57) while purple cocoyam had Fe (9.88), Zn (3.12), Cu (1.14), Mg (82.00), Mn (3.74), P
(129.87), Na (24.33), K (1223.30) and Ca (44.90). The antinutritional factors (mg/100g) of purple
cocoyam (187.57 phytate and 156.1 tannin) were significantly higher than that of green cocoyam
(167.76 phytate and 139.62 tannin). This study provided important information about the nutritional
composition of cocoyam from Ethiopia, which can help to develop cocoyam food products and to
promote production and utilization of cocoyam by encouraging its sustainable use. More detailed
analyses including processing and sensory testing are suggested for further investigation.
Key Words: Ethiopia, proximate, minerals, antinutritional factors
92
7.1 Introduction
Humankind has used over 7000 edible plant species, at one time or another. Research,
however, has concentrated on a few crops to meet the food and industrial needs. Over 50%
of humankind’s requirements for calories and protein are met by just three crops (maize,
wheat and rice) and 95% of the world’s food energy needs are provided by just about 35
crop plant species. Many plant species with a considerable importance for food security are
categorized under neglected and underutilized crops. Most of these crops are particularly
useful in marginal lands where they have been selected to withstand stress conditions and
where they contribute to sustainable production with low inputs (GFUS, 2009). Some
researchers have provided data to confirm the nutritional superiority of neglected and
underutilized crops and their wild varieties over other more extensively utilized crops. Root
and tuber crops are staple foods in many countries and are considered a good and
inexpensive source of energy and carbohydrate in the diets (Burlingame et al., 2009).
Aroids are grouped with the neglected and underutilized crops which over the years have
received little research attention (Adelekan, 2012) although they are important tuberous root
crops playing a significant role in the livelihood of millions of relatively poor people in
developing countries (Sarma et al., 2016). The most important food aroids are from tribes
Colocasieae and Caladieae, i.e., taro (Colocasia) and cocoyam (Xanthosoma). They are
often considered jointly and many developing countries depend on these aroids as a source
of carbohydrates and they are important food for more than 400 million people around the
world (Bown, 2000).
Cocoyam (Xanthosoma sagittifolium (L.) Schott has overtaken taro (Colocasia esculenta
(L.) Schott as the main edible aroid in many tropical areas (Mayo et al., 1997; Matthews,
2002). Cocoyam is reported to have superior nutritional value over major root and tuber
93
staples of, especially in terms of their protein digestibility and mineral composition (Boakye
et al., 2018). It is nutritionally superior to taro both in terms of proximate and mineral
contents (Ndabikunze et al., 2011). It plays major role in the lives of many as a food security
crop, mainly for smallholder farmers. It occupies an important place in the diet of many
tropical countries.
Cormels of cocoyam are boiled, baked or partly boiled and fried in oil before consumption
(Kay, 1987). The corms are peeled, dried and ground to flour for pastry that can be stuffed
with meat or other fillings (Lim, 2015). The young leaves can be boiled and used as
vegetable similar to spinach (Mayo et al., 1997; Ramawat and Merillon, 2014). It was found
to be superior to barley or sorghum as a substrate for brewing beer due to its high
carbohydrate content (71-78 %) compared to barley (65%) and sorghum (70-73%) (Onwuka
and Eneh, 1996).
In Ethiopia, cocoyam is expanding to new areas, growing even in poor soils and under dry
conditions (Fujimoto, 2009; Feleke Woldeyes et al., 2016). The cultivation of cocoyam is
increasing in the country. The cormels are eaten by cooking using pots or roasting using
stones. The young leaves of green cocoyam are edible in some areas of southwestern parts
of Ethiopia. Nutritional composition of roots and tubers reported to vary from place to place
depending on the difference in climate, habitat, soil type, crop variety (genetic background)
and other factors (FAO, 1998). Studies on varieties of cassava, sweet potato and yam showed
that there are great differences in nutrient content within species, and that some varieties can
provide a substantial contribution to nutritional requirements, not only for energy but also
for protein and micronutrients (Burlingame et al., 2009). The data on the nutritional
composition of cocoyam is much less than that for other root and tuber crops. There is dearth
of information on nutritional composition of cocoyam grown in Ethiopia. There is a need to
94
analyze, compile and disseminate data on the nutritional composition of cocoyam. The main
aim of this study was, therefore, to determine proximate composition (moisture, crude
protein, crude fiber, crude fat, total ash, total carbohydrate and gross energy), minerals such
as Iron (Fe), Zinc (Zn), Copper (Cu), Magnesium (Mg), Manganese (Mn), Phosphorus (P),
Sodium (Na), Potassium (K) and Calcium (Ca), and antinutritional factors (phytate and
tannin) of green- and purple- cocoyam grown in Ethiopia and to compare the difference.
7.2 Materials and methods
7.2.1 Sample collection
Fresh cormels (small, middle and large sizes) that were not attacked by pests and which were
not damaged during harvesting were selected from green-and purple-cocoyam, after 9
months of plantation.
7.2.2 Preparation of cocoyam flour
Cormels of three size groups (small, medium and large) were carefully selected from greenand purple- cocoyam (one accession from each) for purpose of including the size groups.
The selected samples were washed using running tap water. Then hand peeled using stainless
steel knife, washed and sliced to uniform thickness (~ 5 mm). The slices were blanched in
hot water (80°C) for 5 minutes followed by immediate cooling in cold water in order to
inactivate enzymes that may cause browning. The slices were placed on a stainless-steel tray
and dried overnight in a dry oven at 105°C. The dried cormels chips were grinded using
mortar and pestle to convert into flour. Then the flour was filled in polyethylene bags, packed
and kept in desiccators until analyzed for contents of proximate, mineral and antinutritional
factors.
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7.2.3 Determination of proximate composition
Proximate composition (total moisture content, crude protein, crude fat, crude fiber, total
ash, total carbohydrate and gross energy values) of the two types of cocoyams were
determined by the following methods.
Determination of moisture content: Moisture content (%) was determined in an oven
drying methods at 105+5°C according to the procedure described in Association of Official
Analytical Chemists (AOAC, 2005). Five grams of each fresh sample was accurately
weighed in triplicate and placed in a pre-weighed aluminum dish and dried in an oven at
105+5°C till the constant weight of dry matter was obtained. The moisture content in the
sample was determined as:
Moisture (%) =
𝑊𝑡.𝑜𝑓 𝑓𝑟𝑒𝑠ℎ 𝑠𝑎𝑚𝑝𝑙𝑒−𝑊𝑡.𝑜𝑓 𝑑𝑟𝑖𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒
𝑊𝑡.𝑜𝑓 𝑓𝑟𝑒𝑠ℎ 𝑠𝑎𝑚𝑝𝑙𝑒
∗ 100
Determination of crude protein: The powdered cormel samples were analyzed for crude
protein content according to the Kjeldahl’s method described in the Association of Official
Analytical Chemists (AOAC, 2005).
Protein digestion: Five grams of the sample was weighed in an ash less filter paper and put
into 250 ml digestion flask. Then 3 g of a catalytic mixture, tablet (75 g of CuSO4 and 0.7
g of K2SO4) and 15 ml of 98% H2SO4 were added into a digestion flask. The whole mixture
was subjected to heating in a digestion chamber until transparent residue (clear light green)
content was obtained. Then, it was allowed to cool. After cooling, the digest was transferred
into a 100 ml volumetric flask and made up to the mark (100 ml) with distilled water and
then distilled using distillation apparatus.
Protein distillation: Before use, the distillation apparatus was steamed for 15 min. After
which, 100 ml conical flask containing 20 ml of 40% boric acid and 2 or 3 drops of Tashiro’s
indicator was placed under the distillation apparatus with its out let tubes inserted into the
96
conical flask. The digest was washed down with distilled water followed by addition of 3-4
drops of phenolphthalein and 20 ml of 40% (w/v) NaOH solution. The distillation was
continued until about 25 ml of distillate was trapped into the boric acid plus indicator
solution changed from red to light grey, showing that all the ammonia liberated had been
trapped. That means the digest in the condenser was steamed through until enough ammonia
gas captured by the boric acid.
Titration: The solution in the receiving flask was titrated with 0.1 mM HCl to a brown
color. After titration the % of nitrogen was calculated as:
Nitrogen (%) =
(𝑉𝑠−𝑉𝐵)∗𝑚𝑀 𝐻𝐶𝑙∗0.014008
𝑊𝑡.𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
∗ 100, where Vs = Volume (ml) of HCl required to
titrate sample; VB = Volume (ml) of acid required to titrate the blank; mM acid= Molarity
of acid; W=Weight of sample (g). Then, percentage of crude protein in the sample was
calculated from the % nitrogen as: % crude protein = % N x F, where, F (conversion factor)
is equivalent to 6.25 (AOAC, 2005). A blank was run through along with the sample and
triplicate analysis was conducted for samples.
Determination of crude fiber: Six gram of powdered sample (E) was taken into 50 ml tube
and 2.5 ml of alpha-amylase was added and incubated at room temperature for 10 min. Then,
60 ml of a mixture composed of 700 ml 70% acetic acid, 100 ml 65% nitric acid and 20 g
trichloroacetic acid was added. Digestion was undertaken in 250 ml flask by heating at
200°C by continuous string at 500 rpm for 30 min. Then after cooling on ice, filtrated with
vacuum filtration on dry filter paper with known mass (Mf) by using distilled water until the
filtrate became neutral. The residue on the filter paper was washed with 10 ml ethanol for 3
times and 10 ml acetone for 2 times to dissolve organic constituent. Then after transferring
the dried residue with the filter paper into pre-weighted crucible, the residue was oven dried
at 105⁰C overnight to drive off moisture. The oven dried crucible containing the residue and
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filter paper was cooled in a desiccator and weighted (M1). The residue and filter paper were
burned first in Bunsen burner and then 550°C. The crucible containing white and grey ash
(free of carbonaceous material) was cooled in a desiccator and weighted to obtain M2.
The % of crude fiber was calculated as: Crude fiber (%) =
(𝑀1−𝑀𝑓)−𝑀2
𝐸
∗ 100
Determination of crude fat: The crude fat in the powdered samples was determined by
automated Soxhlet extraction method (AOAC, 2005). After weighting the dried flask
containing sand to constant weight, 15 g of homogenized samples were measured by using
filter paper of known mass and placed in extraction flask. The dried flasks (250 ml) were
weighed correspondingly and filled with 150 ml of petroleum ether. The extraction thimbles
were plugged tightly with cotton wool and run for 2 h. The extraction chamber continuously
filled with the sample there by extracting the fat. When the optimum sensor reached, the
magnetic valve was opened and the samples were washed with freshly filled solvent
(petroleum ether). Finally, the solvent was recovered by collection in solvent tank. The fat
was collected in filter paper. and the extract was gently evaporated to dryness. The remaining
petroleum ether was removed by sonication. The extraction flask containing crude fat in the
filter paper was dried in 105°C to constant weight. The % fat in the sample was calculated
using the formula:
Fat (%) =
𝑊𝑡.𝑜𝑓 𝑓𝑙𝑎𝑠𝑘 𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑖𝑛𝑔 𝑡ℎ𝑒 𝑐𝑟𝑢𝑑𝑒 𝑓𝑎𝑡 𝑖𝑛 𝑓𝑖𝑙𝑡𝑒𝑟 𝑝𝑎𝑝𝑒𝑟−𝑊𝑡. 𝑜𝑓 𝑓𝑙𝑎𝑠𝑘 𝑝𝑙𝑢𝑠 𝑓𝑖𝑙𝑡𝑒𝑟 𝑝𝑎𝑝𝑒𝑟
𝑊𝑡.𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
∗ 100
Determination of total ash content: A crucible was dried at 550°C for 30 min and cooled
down in a desiccator for 1 hr. The weight of crucible was measured (M1). Five grams of
powdered sample was added in the dried crucible and the crucible containing sample was
measured (M2). Then the sample was burned by using Bunsen burner until the steam off and
then in oven at 550°C for 5 h. Ash is an inorganic residue remaining after the material has
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been completely burnt. The crucible containing ash was cooled in a desiccator and then reweighted (M3) (AOAC, 2005).
The % of ash contents in the cocoyam sample was calculated as: Ash (%) =
𝑀3−𝑀1
𝑀2−𝑀1
∗ 100.
Determination of total carbohydrate: Total carbohydrate content was calculated adding
the total values of crude protein, crude fat, crude fiber and total ash contents of the sample
and subtracting it from 100%.
Total carbohydrate (%) = 100 - (% crude fiber + % crude protein + % crude fat + % ash).
Determination of energy value: Gross energy value (Kcal/100g) of the samples was
determined by multiplying the protein content by 4, carbohydrate content by 4 and fat
content by 9 (AOAC, 2005).
Energy value = (Crude protein × 4) + (total carbohydrate × 4) + (crude fat × 9).
7.2.4 Determination of mineral content
Iron, Zinc, Copper, Magnesium, Manganese, Sodium and Potassium and Calcium
were determined according to the standard method of AOAC (2005) using an Atomic
Absorption Spectrophotometer (Varian SAA-20 Plus). Ashing of the samples was
followed by digestion and absorption. Phosphorus was determined by AAS method
of AOAC (1984).
7.2.5 Analysis of antinutritional factors
Determination of Phytate: The phytate contents of green- and purple-cocoyam were
determined according to method described by Latta and Eskin (1980). Dried sample of
cocoyam flour (0.1 g) was extracted with 10 ml 2.4% HCl for 1 h at room temperature and
centrifuged at 3000 rpm for 30 min. The clear supernatant was used for the phytate
estimation. One ml of Wade reagent (0.03% solution of FeC13.6H2O containing 0.3%
sulfosalicylic acid in water) was added to 3 ml of the sample solution and the mixture was
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centrifuged. The absorbance at 500 nm was measured using UV-VIS spectrophotometer.
The phytate concentration was calculated from the difference between the absorbance of the
control (3 ml of water + 1 m1 Wade reagent) and that of assayed sample and expressed as
mg/100g.
Determination of Tannin: Tannin contents of green- and purple-cocoyam were determined
using the method of Burns (1971). Cocoyam flour (0.25 g) was weighed in a screw capped
test tube and 10 ml of 1% HCl in methanol was added to each test tube containing the
samples. Then the tubes were put on mechanical shaker for 24 h at room temperature. After
24 h of shaking, the tubes were centrifuged at 1000 rpm for 5 minutes. One milliliter of the
clear supernatant was taken and mixed with 5 ml of vanillin-HCl reagent in another test tube
and this mixture was allowed to stand for 20 minutes to complete the reaction. After 20
minutes, the absorbance was read at 500 nm using spectrophotometer. The tannin
concentration was calculated from the difference between the absorbance of control and that
of the sample and expressed as mg/100g.
7.2.6 Statistical analysis
A comparative statistics and comparative analysis was conducted to present the difference
in proximate composition (moisture, crude protein, crude fiber, crude fat, total ash, total
carbohydrate and gross energy values), mineral contents (Ca, K, Na, Mg, Mn, Cu, Fe Zn and
P) and antinutritional factors (phytate and tannin) in green- and purple-cocoyam samples.
The analyses were performed using SPSS version 23 (IBM SPSS, 2015). Differences in
means at p < 0.05 were considered significant.
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7.3 Results
7.3.1 Proximate composition
The results of proximate analysis showed that the crude protein (10.10%), crude fiber
(2.66%) and gross energy value (380.27 kcal/100g) of purple cocoyam were significantly
higher than the crude protein (8.48%), crude fiber (2.14%) contents and gross energy value
(378.47 kcal/100g) of the green cocoyam whereas the green cocoyam had showed
significantly higher total ash content (3.25 %) than the total ash content (2.27%) of purple
cocoyam. Moisture (61.19%), fat (0.85 %) and total carbohydrate (85.36%) contents of
green cocoyam did not differ (p>0.05) from the moisture (63.53%), total carbohydrate
(84.76%) and fat (0.22%) contents of purple cocoyam (Table 7.1).
Table 7.1 Proximate composition of green- and purple- cocoyams
proximate
Moisture (%)
Crude protein (%)
Crude fiber (%)
Crude fat (%)
Total ash (%)
Total carbohydrate (%)
Energy value (kcal/100g)
Green cocoyam
Purple cocoyam
Significance
61.91+2.50
8.48+0.36
2.14+0.12
0.85+0.60
3.25+0.09
85.36+0.49
378.47+0.71
63.53+1.10
10.10+0.18
2.66+0.09
0.22+0.10
2.27+0.07
84.76+0.38
380.27+0.43
0.372
*
**
0.211
**
0.195
*
Data presented are independent sample t-test. Values are means of triplicates analysis ± standard
deviations. Value in level of significance are **p < 0.01, *P<0.05and mean values with p > 0.05
are not significantly different and their respective p-values are shown.
7.3.2 Mineral composition and antinutritional factors
Nine different minerals and two antinutritional factors were analyzed for their concentration
in dry weight basis (mg/100g). The green cocoyam had Fe (8.20), Zn (3.07), Cu (1.04), Mg
(78.77), Mn (2.48), P (120.93), Na (29.22), K (1085.70) and Ca (56.57) while purple
cocoyam had Fe (9.88), Zn (3.12), Cu (1.14), Mg (82.00), Mn (3.74), P (129.87), Na (24.33),
K (1223.30) and Ca (44.90). The result of the comparative analysis showed that Mg, Mn, P,
Na, K and Ca contents of green cocoyam were significantly higher than Mg, Mn, P, Na, K
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and Ca contents of purple cocoyam. The Fe, Zn and Cu contents of green cocoyam were not
significantly different from the Fe, Zn and Cu contents of the purple cocoyam. Significant
quantities of antinutritional factors namely: phytate and tannin were found in both cocoyam
morphotypes. High antnutritional factors were determined, where the phytate (187.57) and
tannin (156.11) contents (mg/100g) of purple cocoyam were significantly higher (P<0.001)
than the phytate (167.76) and tannin (139.62) contents of green cocoyam (Table 7.2).
Table 7.2 Mineral contents and antinutritional factors of green- and purple-cocoyams
Minerals (mg/100g)
Fe
Zn
Cu
Mg
Mn
P
Na
K
Ca
Antinutritional factor
Phytate
Tannin
Green cocoyam
8.20+0.6
3.07+0.10
1.04+0.08
78.77+0.67
2.48+0.19
120.93+2.07
29.22+1.44
1085.70+32.1
56.57+1.50
167.76+2.82
139.62+0.97
Purple cocoyam
9.88+1.97
3.12+0.12
1.14+0.05
82.00+1.04
3.74+0.06
129.87+2.06
24.33+0.82
1223.30+28.70
44.90+1.81
187.57+0.55
156.11+2.35
Significance
0.305
0.582
0.183
*
**
*
*
*
**
***
***
Data presented are independent sample t-test. Values are means of triplicate analysis ± standard
deviations. Value in level of significance are ***p< 0.001, **p < 0.01, *p < 0.05 and mean values
with p > 0.05 are not significantly different and their respective p-values are shown.
7.4 Discussion
The proximate and mineral contents and antinutritional factors (phytate and tannin) of greenand purple- cocoyam were investigated. The moisture content of the green cocoyam was
61.91% and that of the purple cocoyam was 63.53%. These values were lower than moisture
content of cocoyam (70–77%) reported by Kay (1987), but the values were close to the
report of Plucknett (1984) in which the mean moisture content of 37 cocoyam samples was
67.1% and within the range of moisture contents of cocoyam from Ghana (Sefa-Dedeh,
2004). The moisture contents of cocoyam reported from Tanzania and Uganda (Ndabikunze
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et al., 2011) and Nigeria (Eddy et al., 2012) were 68.41% and 68.28%, respectively. The
relative low moisture content determined in this study can be helpful for the storage of
cocoyam cormels at ambient temperature.
The crude protein content of green cocoyam (8.48%) and purple cocoyam (10.10%)
determined by this study were higher than mean protein contents (1.55%) of 37 samples
from Tanzania and Uganda (Plucknett, 1984) and later reported by Ndabikunze et al. (2011)
from the same countries as 4.75%. The crude protein contents were also higher than the
crude protein contents of white (5.1%), yellow (5.2%) and red (5.4%) cocoyams from
Cameroon (Onokpise et al., 1999 and the Assam State of India (2.42%) (Sarma et al., 2016).
Relatively, higher mean protein contents of cocoyam determined in the study may be due to
cormel samples (not corms) were used for protein determination. The results were
comparable with tuber proteins of some cocoyam accessions from Cameroon (up to 9.4%)
(Onokpise et al., 1999), protein contents of Ede Uhie (8.08%) and Ede Ocha (8.74%)
varieties of cocoyam cormles from Nigeria (Owuamanam et al., 2010) and protein content
of taro varieties (10.49 to 12.13%) from Ethiopia (Melese Temesgen, 2017). The present
study results were also in agreement with a review conducted by Shewry (2003) and reported
that plant tubers contain protein up to 10%. According to Onwueme (1978), cocoyam
(Colocasia and Xanthosoma) has relatively high protein content compared to other tuberous
root crops such as cassava, yam, potato and sweet potato. According to Akpan and Umoh
(2004), the protein content of cocoyam was slightly superior to taro. This study results
showed that both green- and purple- cocoyams are rich in proteins, which can be considered
as good opportunity since it is mainly consumed by resource poor farmers.
The crude fiber contents of green cocoyam (2.14%) and purple cocoyam (2.66%) determined
in this study were lower than the minimum crude fiber content (2.80%) of two related aroids
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(Colocasia and Xanthosoma) from Assam State of India (Sarma et al., 2016). The crude
fiber contents were, however, higher than the range of crude fiber contents of cocoyam (0.61.9%) reported by Kay (1987), (0.1%) reported by Plucknett (1984) and (0.88%) reported
by Akpan and Umoh (2004) and within the range of the crude fiber content (1.11-3.00%) of
cocoyam reported by Sefa-Dedeh and Kofi-Agyir (2004). The purple cocoyam had
significantly higher crude fiber content (2.66%) than the crude fiber content (2.14%) of
green cocoyam (p<0.01). In contrast to this, Sarma et al. (2016) reported the crude fiber
content of purple cocoyam (2.80%) was lower than the crude fiber content (3.50%) of dark
green leaved cocoyam. The present study result showed that fiber contents in green- and
purple- cocoyam grown in Ethiopia can be effective and useful source of fiber. The
differences in the fibers contents could be attributed to the genotype difference.
Comparative analysis showed that the crude fat contents of green cocoyam (0.85%) was not
significantly different from the fat content of purple cocoyam (0.22%) (p>0.05). The values
were low and within the range of crude fat contents (0.2-0.4%) reported by Kay (1987) and
also relatively comparable with the average crude fat contents of cocoyam reported by
Akpan and Umoh (2004) (0.93%) and Ndabikunze et al. (2011) (0.43%), indicating
cocoyam is a low-fat crop. Thus, it can be said to be preferred food crop that can contribute
less to the health problems related with excess fat intake.
The total ash content of green cocoyam (3.25%) was significantly higher than the total ash
content of purple cocoyam (2.27%) (p<0.001). The values were comparable with the total
ash content of Ede ofe (3.00%) and Ede ocha (2.45%) cocoyam cultivars from Nigeria
(Owuamanam et al., 2010), but lower than the total ash content of white-fleshed (5%),
yellow-fleshed (4.6%) and red-fleshed (4.5%) cocoyam varieties reported latter by
Ihediohanma et al. (2014) from the same country. The values were also lower than the total
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ash contents of cocoyam (3.51%) grown along the Lake Victoria Basin in Tanzania and
Uganda (Ndabikunze et al., 2011). The differences could be the influence of the
environments in which cocoyams were grown. The ash content can reflect the minerals
content, which varies with the locality or soil type (Onyeike et al., 1995).
The total carbohydrate contents of green cocoyam (85.36%) and purple cocoyam (84.76%)
were comparable with total carbohydrate content (85.65%) of released taro variety from
Ethiopia (Boloso I) (Adane Tilahun et al., 2013), but higher than the average carbohydrate
content (72.53 to 75.49 %) of different taro varieties from Ethiopia (Melese Temesgen,
2017). Gross energy values (kcal/100g) of green cocoyam (378.47) and purple cocoyam
(380.27) were relatively higher than the gross energy values (kcal/100g) of Boloso I, which
varied from 370 to 374 kcal/100g (Adane Tilahun et al., 2013). The carbohydrate contents
and gross energy values indicate that cocoyam growing in Ethiopia is one of carbohydrate
rich foods in supplying high energy.
The minerals such as Fe, Zn, Cu, Mn, Na and K contents (mg/100g) of green- and purplecocoyams were higher than the Fe, Zn, Cu, Mn, Na and K content of cocoyam grown along
the Lake Victoria Basin in Tanzania and Uganda (Ndabikunze et al., 2011). The Mg, P and
Ca (mg/100g) contents of cocoyam were lower than Mg (90.62), P (207.50) and Ca (110.17)
contents of cocoyam reported by Ndabikunze et al. (2011). The Cu content of green
cocoyam (1.04) and purple cocoyam (1.14) were higher than the Cu contents of whitefleshed cocoyam (0.52), red-fleshed cocoyam (0.78) and taro (0.78) cultivars reported by
Njoku and Ohia (2007). The present study showed that both green- and purple- cocoyams
are rich in minerals. According to Njoku and Ohia (2007), consumption of nutrient rich
foods such as cocoyam helps the body to utilize protein, carbohydrates and other nutrients.
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High phytate concentrations (mg/100g) observed in green cocoyam (167.76) and purple
cocoyam (187.57). Phytate has been recognized as an anti-nutrient due to its adverse effects
because it lowers the availability of many minerals such as copper, iron and zinc (Sarkiyayi
and Agar, 2010). The tannin level (mg/100 g) of green- and purple- cocoyams were 139.62
and 156.11, respectively. These tannin contents were higher than the tannin content of taroBoloso I reported by Adane Tilahun et al. (2013). It is known to exert negative effect on the
bioavailability of proteins and minerals (Ramakrishna et al., 2006). Processing methods
such as boiling, fermentation and roasting can significantly reduce antinutritional factors
(phytate and tannin) to low level (Ramakrishna et al., 2006; Adane Tilahun et al. 2013;
Melese Temesgen, 2017; Habtamu Azene and Tesfahun Molla, 2017). These processing
methods, therefore, need to be tested for the reduction of antinutritional contents of cocoyam
grown in Ethiopia. These processing methods, therefore, need to be tested for the level of
reduction of antinutritional factors of cocoyam grown in Ethiopia. This study provided
important information about the nutritional composition of cocoyam, which can help to
develop cocoyam food products and to promote production and utilization of cocoyam by
encouraging its sustainable use. More detailed analyses including processing and sensory
testing are suggested for further investigation.
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Chapter Eight
Micropropagation of Cocoyam (Xanthosoma sagittifolium (L.) Schott)
from Shoot Tip Explants
Abstract
Cocoyam (Xanthosoma sagittifolium (L.) Schott) is one of the main edible aroid in many tropical
areas of the world. It contributes to the food security of households in the rural communities of south
and southwestern parts of Ethiopia. Its propagation is entirely traditional from tuber fragments, which
was constrained by scarcity of planting materials. This study was carried out to develop in vitro
propagation protocol of this species using shoot tip explants. MS medium supplemented with
different concentrations of plant growth regulators (BAP with Kn, BAP with NAA, and BAP with
NAA and Kn) were used. The shoots were transferred to MS medium supplemented with various
concentrations of auxin (IBA or NAA) for rooting. Factorial experiments were conducted in a
completely randomized design with PGRs as one factor and genotype as another factor. Each
treatment was replicated six times. Shoots were best initiated on MS medium supplemented with 2.0
mg/l BAP. The maximum mean number of shoots per explant, 4.56 for green cocoyam and 4.83 for
purple cocoyam, were recorded on MS medium supplemented with 2.5 mg/l BAP in combination
with 0.5 mg/l NAA. The maximum mean leaf number per explant, 4.67 for green cocoyam and 4.94
for purple cocoyam, were recorded on MS medium supplemented with 1.0 mg/l BAP in combination
with 0.25 mg/l Kn. The longest shoots, 3.92 cm for green cocoyam and 4.36 cm for purple cocoyam,
were recorded on MS medium supplemented with 5.0 mg/l BAP in combination with 1.0 mg/l Kn
and 0.5 mg/l NAA. This result was, however, not significantly different from the shoot length
measured on MS medium supplemented with 2.5 mg/l BAP in combination with 0.5 mg/l NAA. The
highest mean root number per shoot (6.00) for green cocoyam and (5.83) for purple cocoyam were
obtained on MS medium containing 2.0 mg/l IBA. The longest roots, 5.94 cm for green cocoyam and
6.44 cm for purple cocoyam, were also recorded on the same medium. Thus, maximum cocoyam
shoots per explant could be obtained by initiating culture on MS medium containing 2.5 mg/l BAP
in combination with 0.5 mg/l NAA and inducing roots on MS medium containing 2.0 mg/l IBA. This
protocol can be used for propagation of large number of disease free planting materials within short
period of time.
Keywords: Explant, in vitro propagation, shoot tip, X. sagittifolium
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8.1 Introduction
Cocoyam (Xanthosoma sagittifolium) is one of the main edible aroid in many tropical areas
of the world. The usable parts are the cormles and young leaves (Giacometti and Leon,
1994). It is contributing to the food security of households in the rural communities of south
and southwestern parts of Ethiopia. However, its cultivation is constrained by scarcity of
planting materials because the seeds of cocoyam such as cormels or their fragments are used
for food. Preserving cocoyam under field condition is risky since diseases or natural
catastrophes can cause the loss of genetic resources. In vitro propagation technique is a
possible solution to the major problems of vegetative propagated plants associated with
pathogen dissemination and subsequent loss of vigor and productivity (Paul and Bari, 2007),
health and quality (Ko et al., 2008; Vilchez et al., 2009) and planting material scarcity
(Tsehifet Solomon et al., 2010). It is an efficient method to propagate good quality materials
that can substantially improve production rate (Caula et al., 2008).
Micropropagation involves using smaller propagules and a substantially faster
multiplication rate than the conventional field multiplication methods (Sama et al., 2012).
Its objective is to propagate plants as clones. Plant cells, tissues or organs can be cloned, i.e.,
produced in large numbers where all the individuals have the same genetic constitution as
the stock parent although irregularities sometimes occur, resulting in somaclonal variation
(George et al., 2008).
Production of a large number of plants of selected germplasms is the main prerequisite for
establishment of plantations, for ex-situ conservation and improvement of plants (Siddique
and Anis, 2009). Multiplication of many clonally propagated species in a short time is among
many advantages of micropropagation systems (Panell, 1984). There are reports that have
generated information to use for in vitro propagation of cocoyam through refining culture
108
media and plant growth regulator (PGR) compositions. Shoots were induced from cormel
axillary bud meristems on MS media supplemented with different combinations of auxins
(IAA, 2,4-D and NAA) and cytokinins (BAP and Kn) (Paul and Bari, 2007). Cocoyam was
propagated in vitro from shoot tip through the use of BAP and Thidiazuron (TDZ) (Sama et
al., 2012) and in temporary immersion bioreactor using sucrose as a determinant factor
(Niemenak et al., 2013).
The ideal concentration and combination of PGRs required for micropropagation differ from
species to species, genotype to genotype and explant source (Obidiegwu, 2015). According
to Gomes et al. (2010), the composition of PGRs need to be established accurately to achieve
the effective rates of multiplication. A study has been carried out to compare the
conventional and in vitro propagation methods in order to increase planting material of
cocoyam in Ethiopia (Tsehifet Solomon et al., 2010). The in vitro propagation protocol was,
however, not adequately developed for routine micropropagation. The situation justifies
looking for efficient micropropagation protocol in order to achieve higher benefits in the
production of cocoyam in Ethiopia. Shoot tip culture is a useful and an expanding alternative
to obtain large numbers of propagules rapidly. This study was aimed to develop in vitro
propagation protocol for cocoyam using shoot tip explants.
8.2 Materials and methods
8.2.1 Preparation of donor plant and stock solutions
Donor plant preparation: Cormels of green and purple cocoyam genotypes were planted
in polyethylene pots containing sterile forest soil and allowed to sprout in greenhouse at
average temperature of 25°C under natural photoperiod.
MS stock preparation: Murashinge and Skoog (MS) (1962) medium with its full macro
and micro nutrients, vitamins compositions, sucrose and agar were used as the basic
109
components of the medium. All components of MS stock solution were prepared by
weighing and dissolving the powder in double distilled water.
Plant growth regulators stock preparation: The stock solutions of PGRs (BAP, NAA, Kn
and IBA: Indol-3-butyric acid) were prepared by weighing and dissolving the powder in
double distilled water at a concentration of 1.0 mg/ml by dissolving in 3-4 drops of 1N
NaOH for auxins and 1N HCl for cytokinins.
8.2.2 Culture medium preparation
Culture medium was prepared by taking proper amount of MS stock solutions and 3% (w/v)
sucrose. PGR was added as treatment and pH was adjusted to 5.8 using 1N NaOH or 1N
HCl. Finally, 0.6% (w/v) agar was added and dissolved by micro-oven. The medium was
sterilized by autoclaving at a temperature of 121°C with a pressure 105 Kpa for 15 min. For
shoot initiation, shoot multiplication and rooting, 40 ml of the medium was poured into baby
jar culture vessel.
8.2.3 Surface sterilization of explant
After six weeks of planting in greenhouse, sprouts were collected. The roots were removed
and the tuber was scrubbed with a brush, 0.5% sodium hypochlorite and dish soap under tap
water. The lower portion of the corm was cut off, leaving 3 cm and the leaf stalk was
trimmed to 2.0 to 3.0 cm. The explant was gently scrubbed with toothbrush using double
distilled water, rinsed with double distilled water followed by washing in 70% ethanol for
five min and washed three to four times with double distilled water. Then, the explant was
placed in a clean beaker containing a solution of 1.5% sodium hypochlorite made with
double distilled water and two drops of Tween-20 and the beaker was placed on a magnetic
stir bar plate and stir gently for 45 min. The solution was poured off, the explant was rinsed
with double distilled water and taken into the laminar flow hood. The outer leaves were
110
removed until inner cleaner section appears and the explant was trimmed to 1.0 cm, rinsed
three to four times with sterile double distilled water, 70% ethanol for 30 seconds, 1%
sodium hypochlorite for 1 min and sterile double distilled for three to four times.
8.2.4 Culture condition
Green- and purple-cocoyams were equally treated while all exogenous factors were held
constant. Each culture vessel and its cap were flamed prior to closing and sealed with a strip
of parafilm. The vessels were clearly labelled with the medium code, date of inoculation,
name of cocoyam (Green or Purple). Three explants per culture vessel and six replications
per treatment were used for shoot initiation, shoot multiplication and root induction
experiments. Cultures were incubated and monitored in growth room of 12 h photoperiod
under light intensity of 40 µmole m-2 s-1 provided by cool white fluorescent lamps at a
temperature of 25+2°C.
8.2.5 Shoot initiation
Shoot tip (1.0 cm) comprising the apical meristem with some leaf primordia and some corm
tissue at the base was used for shoot initiation (Fig. 8.1). The explants were cultured on MS
medium supplemented with different concentrations of BAP (0.0, 0.5, 2.0, 3.0, 4.0 and 5.0
mg/l). The number of initiated shoots was recorded beginning from fourth day of culture
and expressed as percentage of shoot initiation. The number of shoots per explant, number
of leaves per explant and shoot length were recorded after two weeks of culture initiation to
select the best shoot initiation medium.
B
A
Fig. 8.1 Shoot tip explant for shoot initiation: During surface sterilization (A); ready for culture (B)
111
8.2.6 Shoot multiplication
To screen for an optimal shoot multiplication medium, the initiated explants were cultured
on 20 different PGRs concentrations including PGRs free MS medium as control. Five
different combination of BAP with Kn, nine different combination of BAP with NAA and
five different combinations of BAP with NAA and Kn were used (Table 8.1). The number
of shoots per explant, number of leaves per explant and shoot length were recorded after
three subsequent cultures at two-week interval.
Table 8.1 Compositions of plant growth regulators for shoot multiplication (mg/l)
BAP
0
0.5
1.0
1.5
2.5
5.0
1.0
1.5
2.5
5.0
+
NAA
0
0.25
0.25
0.25
0.25
0.25
0.5
0.5
0.5
0.5
BAP
0
0.5
1.0
1.5
2.5
5.0
+
Kn
0
0.10
0.25
0.50
0.75
1.0
BAP
0
0.5
2.5
5.0
2.5
5.0
+ NAA
0
0.25
0.25
0.25
0.5
0.5
+ Kn
0
1.0
1.0
1.0
1.0
1.0
8.2.7 Rooting and acclimatization
Before conducting the rooting experiment, in vitro derived shoots were transferred onto a
fresh PGRs free MS medium for two weeks in order to avoid any carry over effect of PGRs.
Then, the shoots were cultured on MS medium supplemented with different concentrations
of IBA (0.25, 0.5, 1.0, 2.0 and 3.0 mg/l) or NAA (0.25, 0.5, 1.0, 2.0 and 3.0 mg/l) for root
induction. PGRs free MS medium was used as a control. The percentage of root forming
shoots, number of roots per shoot and root length were recorded after three weeks of culture.
After being in rooting medium for three weeks, the rooted shoots were removed from the
culture vessel and the roots were washed in double distilled water. Sixty plantlets (30 plantlets
112
of each of green and purple cocoyams) were planted in forest soil, coffee husk and sand in the
ratio of 2:1:1, respectively. All plantlets were covered with a plastic cloche for three days
before being left open in greenhouse. The number of survived plants was recorded after two
weeks of acclimatization.
8.2.8 Experimental design and data analysis
The experiments were laid in a completely randomized factorial design with PGRs as one
factor and genotypes (green- and purple-cocoyams) as another factor. Statistical analysis was
carried out using Minitab 17.1 (2013). Treatment separation was performed using Tukey mean
comparison test at a probability level of p < 0.05.
8.3 Results
8.3.1 Shoot initiation
The shoot tip explants responded to different BAP concentrations and turned green beginning
from four days of culture (Fig. 8.2). All explants (100%) induced shoots on MS medium
supplemented with 2.0 mg/l BAP. The lowest percentage shoot induction (84.19%) and
(83.56%) was observed on PGRs free MS medium for green- and purple- cocoyam,
respectively. The number of shoots per explant produced on PGRs free MS medium and the
media containing different concentrations of BAP were not significantly different. However,
among five different concentration of BAP used for shoot initiation, relatively higher shoot
and leaf numbers per explant and shoot length were produced on the MS medium containing
2.0 mg/l BAP. The maximum mean number of shoots per explant (2.44+0.47 and 2.50+0.25),
leaves per explant (3.11+0.13 and 3.41+0.43) and shoot length (2.17+0.15 and 2.33 + 0.16
cm) were produced, respectively, for green-and purple-cocoyam on the medium containing
2.0 mg/l BAP (Table 8.2).
113
Table 8.2 Effect BAP on shoot initiation of green- and purple- cocoyams from shoot tip
Green cocoyam
Shoot initiation (%)
BAP concentration (mg/l)
0.0
1.0
84.19
2.0
84.19
a
2.00+0.42
3.0
100.00
a
2.44+0.47
4.0
94.44
a
2.22+0.20
5.0
94.44
a
1.89+0.25
89.30
a
1.55+0.07 a
Mean shoot no/explant
1.50+0.21
Mean leaf no/explant
1.56+0.18 b
2.06+0.17 ab
3.11+0.13 a
2.78+0.28 a
2.67+0.19 a
2.89+0.20 a
Mean shoot length (cm)
1.53+0.21a
2.08+0.15a
2.17+0.15a
2.11+0.24a
1.78+0.44 a
1.92+0.21 a
100.00
94.90
94.90
89.38
Purple cocoyam
Shoot initiation (%)
83.56
89.30
Mean shoot no/explant
1.94 +0.25a
2.22+0.16 a
2.50+0.25a
2.28+0.16a
2.22+0.13 a
1.83+0.17 a
Mean leaf no/explant
1.86+0.10b
2.36+0.07ab
3.41+0.43a
3.08+0.20a
2.97+0.09a
3.1+0.18a
Mean shoot length (cm)
1.72+0.41a
2.08+0.18a
2.33+0.16a
2.17+0.20a
1.94+0.10a
1.83+0.28a
Data are given as means ± standard error of means (SE); Means that do not share a letter in superscript in the same
row are significantly different at p < 0.05 by Tukey mean comparison
Fig. 8.2 Shoot initiation from cocoyam shoot-tip cultured on MS medium supplemented with 2.0 mg/l
BAP: After 4 days of culture (A), After 7 days of culture (B) and After two weeks of culture (C)
8.3.2 Shoot Multiplication
The effects of genotypes (green- and purple-cocoyam) and PGRs on shoot multiplication of
cocoyam are displayed in Table 8.3. The mean numbers of shoots and leaves per explant
were not differentially influenced by genotypes. Genotypes had significant effect on the
shoot length. PGRs had highly significant effect (p<0.001) on the number of shoots per
explant, leaf numbers per explant and shoot length.
114
Table 8.3 Mean square of in vitro induced shoot parameters of genoytpes (green- and
purple- cocoyam) on MS medium supplemented with different PGRsa
Shoot multiplication parameters/explant
Shoot number
Leaf number
Shoot length
1.820
0.669
4.630**
Treatment
df
Genotypes
1
PGRs
19
5.650***
4.672***
3.126***
Error
219
0.567
0.513
0.406
Total
239
a
PGS-Plant growth regulators: BAP + NAA, BAP + Kn and BAP + NAA + Kn. ** and *** indicate
the mean square values of shoot multiplication parameters which were significant at, 0.01 and 0.001
probability level, respectively.
Among the different combinations of BAP with Kn, BAP with NAA and BAP with Kn and
NAA, the media containing BAP in combinations with NAA provided relatively higher
mean number of shoots per explant than the media containing BAP in combination with Kn
and BAP in combination with Kn and NAA (Table 8.4). Maximum mean number of shoot
per explant (4.56 + 0.35) and (4.83 + 0.26) was obtained on MS medium containing 2.5 mg/l
BAP in combination with 0.5 mg/l NAA for green- and purple- cocoyam, respectively. The
next highest number of shoots per explant (4.39 + 0.44) and (4.50 + 0.41) was produced on
MS medium containing 5.0 mg/l BAP and 1.0 mg/l Kn for green- and purple- cocoyam,
respectively.
The media containing BAP in combinations with Kn provided relatively higher mean
number of leaves per explant than the media containing BAP + NAA and BAP + NAA +
Kn (Table 8.4). Maximum mean number of leaves per explant (4.67 + 0.41) and (4.94 +
0.50) was obtained on MS medium containing 1.0 mg/l BAP in combination with 0.25 mg/l
Kn for green- and purple- cocoyams, respectively. Statistically the same number of leaves
per explant was produced on the MS medium containing 2.5 mg/l BAP + 0.5 mg/l NAA, 0.5
mg/l BAP + 0.1 mg/l Kn or 5.0 mg/l BAP + 1.0 mg/l Kn for both cocoyam genotypes.
115
Among the different combinations of BAP with Kn, BAP with NAA and BAP with Kn and
NAA, the media containing BAP + NAA + Kn provided relatively longer shoots for both greenand purple-cocoyams than the media containing BAP + Kn and BAP + NAA (Table 8.4). The
longest shoots, 3.92 + 0.40 cm and 4.36 + 0.46 cm, were obtained on MS medium containing
5.0 mg/l BAP + 0.5 mg/l NAA + 1.0 mg/l Kn for green- and purple- cocoyam, respectively.
Statistically, similar result could be achieved on MS media supplemented with 5.0 mg/l BAP +
1.0 mg/l, 2.5 mg/l BAP + 0.5 mg/l NAA, 5.0 mg/l BAP + 1.0 mg/l Kn + 0.25 mg/l NAA and or
5.0 mg/l BAP 1.0 mg/l Kn + 0.5 mg/l NAA for both green- and purple cocoyams. In general,
shoots obtained in the medium containing 2.5 mg/l BAP + 0.5 mg/l NAA, looks more
comparable with the medium composition (Fig. 8.3).
116
Table 8.4 Effects of different concentrations of plant growth regulators on shoot multiplication of green-and purple-cocoyams
Combinations PGRs* (mg/l)
BAP
Kn
NAA
Mean shoot number/explant +SE
Green cocoyam
Purple cocoyam
Mean leaf number/explant +SE
Green cocoyam
Purple cocoyam
Mean shoot length (cm) + SE
Green cocoyam
Purple cocoyam
0
0.5
1.0
1.5
2.5
5.0
0
0.10
0.25
0.50
0.75
1.0
0
0
0
0
0
0
2.17+0.33c
2.33+0.21c
2.28+0.23c
2.44+0.50c
2.17+0.28c
4.39+0.44ab
2.33+0.00bc
2.50+0.42bc
3.78+0.44ab
2.39+0.25bc
2.72+0.25bc
4.50+0.41a
2.33+0.17e
3.67+0.44abcd
4.67+0.41a
3.11+0.11bcde
3.33+0.19bcde
3.83+0.42ab
2.72+0.10bc
4.28+0.41ab
4.94+0.50a
3.06+0.34bc
3.00+0.27bc
4.11+0.49ab
1.69+0.16c
1.75+0.16c
1.78+0.24c
2.39+0.34bc
2.28+0.44bc
2.72+0.21abc
2.08+0.18c
2.22+0.14c
2.33+0.10c
2.50+0.11bc
2.67+0.20bc
3.06+0.57abc
0.5
1.0
1.5
2.5
5.0
1.0
1.5
2.5
5.0
0
0
0
0
0
0
0
0
0
0.25
0.25
0.25
0.25
0.25
0.5
0.5
0.5
0.5
2.17+0.31c
2.23+0.25c
2.39+0.23c
3.17+0.07abc
2.17+0.11c
3.50+0.27abc
3.22+0.38abc
4.56+0.35a
2.44+0.27c
2.11+0.31c
2.61+0.30bc
2.67+0.27bc
2.89+0.17bc
2.39+0.13bc
2.28+0.22bc
3.56+0.41abc
4.83+0.26a
3.44+0.20abc
2.89+0.19bcde
2.78+0.16bcde
2.72+0.13bcde
2.78+0.17bcde
2.44+0.43de
2.94+0.26bcde
2.72+0.20bcde
3.72+0.18abc
2.94+0.18bcde
2.50+0.11bc
2.72+0.17bc
3.11+0.18bc
2.94+0.48bc
2.83+0.14bc
3.22+0.31abc
3.22+0.11abc
3.83+0.45abc
2.89+0.68bc
2.00+0.16bc
2.67+0.05bc
2.44+0.11bc
2.33+0.09bc
2.03+0.10bc
2.50+0.17bc
2.89+0.44bc
2.89+0.13abc
2.39+0.37bc
2.33+0.28c
2.58+0.13bc
2.75+0.11bc
2.22+0.31c
2.11+0.13c
2.44+0.11bc
2.47+0.22bc
3.78+0.78ab
2.67+0.46bc
0.5
2.5
5.0
2.5
5.0
1.0
1.0
1.0
1.0
1.0
0.25
0.25
0.25
0.5
0.5
2.11+0.21c
2.11+0.25c
2.44+0.35c
3.00+0.22bc
2.24+0.08c
2.39+0.14bc
2.17+0.21bc
2.11+0.22c
3.39+0.16abc
2.22+0.22bc
2.44+0.25de
2.28+0.11e
2.56+0.16cde
3.28+0.07bcde
3.28+0.29bcde
2.11+0.17c
2.22+0.22c
3.22+0.46abc
3.06+0.23bc
2.94+0.15bc
2.17+0.15bc
2.72+0.23abc
2.69+0.13abc
3.19+0.25ab
3.92+0.40a
2.61+0.16bc
2.53+0.11bc
3.14+0.39abc
3.44+0.10abc
4.36+0.46a
*PGRs- Plant growth regulators; BAP: 6-Benzylaminopurine; NAA: α-naphthalene acetic acid; Kn: Kinetin. Data are given as means ± standard error of means
(SE); means that do not share a letter in superscript within a column are significantly different at p < 0.05 by Tukey pairwise comparison.
117
Fig. 8.3 Green- and purple-cocoyams on different multiplication media: (A) 2.5 mg/l BAP + 0.5 NAA; (B)1.0 mg/l BAP + 0.25 mg/l Kn; (C) 5.0 mg/l BAP
+ 1.0 mg/l Kn + 0.5 mg/l NAA; (D) control (without growth regulator)
118
8.3.3 Rooting and acclimatization
The influence of genotypes (green- and purple-cocoyams) on the production of roots and
root length was not significant. The number of roots per explant and root length were
influenced by different concentration of PGRs (IBA or NAA) (Table 8.5).
Table 8.5 Mean square of in vitro induced root number and length of cocoyam genotypes (greenand purple-cocoyams) on MS medium supplemented with different concentrations of PGRs (IBA or
NAA) a
Root parameters/shoot
root number
root length
Treatment
df
Genotypes
1
2.45
3.08
PGRs
10
24.61***
25.85***
Error
120
1.658
2.130
Total
131
PGRs - Plant growth regulators; IBA- Indole-3-butyric acid; NAA- α-naphthalene acetic acid; *** indicates
the mean square values of number of roots per explant and root length which were significant at 0.001
probability level
a
Maximum of 93.3% shoots formed roots on MS medium containing 2.0 mg/l IBA followed
by 86.7% on MS medium containing 2.0 mg/l NAA for both green- and purple- cocoyams.
Maximum mean number of roots, 6.00 + 0.74 and 5.83+0.49 for green- and purplecocoyams, respectively, were recorded on MS medium supplemented with 2.0 mg/l IBA.
Among the tested concentrations, relatively less number of roots was obtained from IBA
and NAA at lower concentration (0.25 mg/l) and NAA at higher concentration (3.0 mg/l).
The longest roots (4.06+0.11 and 6.44+0.0.99 cm) were produced on medium supplemented
with 1.0 mg/l IBA and 2.0 mg/l IBA for green- and purple- cocoyams, respectively (Table
8.6). The roots formed in 2.0 mg/l IBA were shown in Fig. 8.4. Upon acclimatization, 23
(76.7%) of green cocoyam and 25 (83.3%) of purple cocoyam survived. The
micropropagated cocoyam after two weeks of acclimatization in greenhouse are shown in
Fig. 8.5.
119
Table 8.6 Effect of IBA and NAA on root induction of green- and purple-cocoyams
IBA
NAA
Root forming shoots (%)
Green
Purple
cocoyam
cocoyam
43.3
33.3
No. of roots/shoot + SE
Green
Purple
cocoyam
cocoyam
b
0.94+0.34
0.56+0.07c
Root length (cm) + SE
Green
Purple
cocoyam
cocoyam
c
0.61+0.22
0.39+0.10b
(mg/l)
(mg/l)
0
0
0.25
0
66.7
53.3
1.28+0.25b
1.28+0.30bc
2.00+0.48bc
1.67+0.37b
0.50
0
76.8
76.8
2.11+0.85b
2.94+0.67b
2.75+0.26b
2.33+0.29b
1.0
0
83.3
83.3
2.72+0.94b
3.28+0.67b
4.06+0.11ab
3.00+0.45b
2.0
0
93.3
93.3
6.00+0.74a
5.83+0.49a
5.94+0.71a
6.44+0.99 a
3.0
0
76.8
76.8
2.39+0.50b
3.28+0.66b
3.94+0.75ab
2.83+0.84b
0
0.25
33.3
23.3
1.00+0.37b
0.50+0.28c
1.61+0.61bc
1.22+0.50b
0
0.50
73.3
60.0
2.06+0.13b
2.00+0.23bc
1.94+0.42bc
1.89+0.21b
0
1.0
76.8
70.8
2.22+0.32b
3.06+0.26b
2.22+0.42bc
2.17+0.45b
0
2.0
86.7
86.7
2.56+0.62b
3.33+0.38b
3.00+0.60bc
2.50+0.58b
0
3.0
70.0
53.3
1.67+0.12b
1.89+0.76c
2.17+0.64bc
2.44+1.11b
Means that do not share a letter in superscript within a column are significantly different at p = 0.05 by Tukey
pairwise comparison. Data are given as means ± standard error of means (SE)
Fig. 8.4 In vitro rooting of cocoyam shoots on MS medium containing 2.0 mg/l IBA: Green cocoyam (A) and
Purple cocoyam (B), the number of roots per plantlet was counted and the root length was measured (cm) (C)
120
Fig. 8.5 Micropropagated plants after two weeks of acclimatization in the greenhouse
8.4 Discussion
In this study, MS medium containing different concentrations of BAP (1.0, 2.0, 3.0, 4.0 and
5.0 mg/l) resulted in shoot formation in the first culture. The percentage of shoot formation
achieved by this study ranged from 83.56% to 100%, which were better achievement than
that achieved by Castro (2006) who obtained the highest percentage (83%) initiated shoots
of cocoyam from Nicaragua in MS medium containing 0.5 mg/l BAP and 1.0 mg/l IAA.
Paul and Bari (2007) achieved the highest percentage of initiated explants from Bangladesh
cocoyam (65%) using 0.5 mg/l BAP and 1.0 mg/l IAA. Wokabi (2012) achieved 100% shoot
initiation by supplying BAP and IAA, respectively, in concentrations of 2.0 + 0.5, 4.0 + 1.0,
6.0 + 2.0 and 8.0 + 3.0 mg/l. No significant difference in number of shoots per explant and
shoot length was observed on MS medium containing different concentrations of BAP. In
similar study, Onwubiko et al. (2012) reported that five different concentrations (0.25, 0.5
0.75, 1.0 and 1.25 mg/l) BAP did not differentially influence number of shoots per explant
and shoot length of two cocoyam varieties from Nigeria. However, Vilchez et al. (2009)
reported that the use of 2.0, 3.0 and 4.0 mg/l BAP improved the quality of cocoyam shoots
in comparison to PGRs free medium. In present study, all explants (100%) induced shoots
on MS medium containing 2.0 mg/l BAP, showing that cocoyam shoots can be better
initiated by adding BAP alone in MS medium. Analogues to this, Vilchez (2009) reported
121
the decisive role of incorporation of 2.0 mg/l BAP in culture medium for shoot initiation of
cocoyam. Thus, the MS medium containing 2.0 mg/l BAP found to be optimum for in vitro
shoot initiation as this concentration resulted in 100% initiated quality shoots, which looks
more comfortable with the medium composition and turned green within 4 days of culture.
When the initiated shoots were transferred to MS medium containing different
concentrations of PGRs, significant difference were observed in terms of number of shoots
and leaves per explant and shoot length. Maximum mean number of shoots per explant (4.56
for green cocoyam and 4.83 for purple cocoyam) was obtained on MS medium containing
2.5 mg/l BAP and 0.5 mg/l NAA. Paul and Bari (2007) obtained maximum mean number of
cocoyam shoots per explant (1.00) in MS media supplemented with different concentrations
of auxins and cytokinins. Sama et al. (2012) achieved the average number of cocoyam shoots
per explant (9.7) using 4.5 mg/l BAP. Tsehifet Solomon et al. (2010) reported 1.5 mg/l BA
in combination with 0.5 mg/l IAA was the best concentrations for cocoyam shoot
multiplication. The differences in shoot multiplication performance among these studies can
be attributed to variations in genotype, type and concentration of culture media compositions
and environmental factors such as CO2 enrichment and light intensity. Among the tested
PGRs, the longest shoots were obtained on MS medium supplemented with 5.0 mg/l BAP
+1.0 mg/l Kn + 0.5 mg/l NAA. Statistically the same length of shoots but better quality was
obtained on MS medium containing 2.5 mg/l BAP and 0.5 mg/l NAA. This indicates the
importance of using 2.5 mg/l BAP and 0.5 mg/l NAA on the MS medium for cocoyam shoot
multiplication as far as its objective is to obtain highest possible shoot numbers with best
length in order to facilitate its management during acclimatization.
The number of roots per shoot and root length were influence by the different concentrations
of IBA and NAA. The highest rooting percentage (93.3) was obtained on MS medium
122
containing 2.0 mg/l IBA. Highest number of roots per shoot and the longest roots were
obtained on MS medium supplemented with 2.0 mg/l IBA. Paul and Bari (2007) achieved
the highest number of cocoyam roots per shoot (7.9) and root length (6.9 cm) on MS medium
supplemented with 0.4 mg/l IAA. Relatively less number of roots was obtained on PGRs
free MS medium.
Acclimatization of in vitro rooted plantlets was successful, where 76.7% of green cocoyam
and 83.3% of purple cocoyam were survived, which were, however, low achievement
compared to the reports of Onokpise et al. (1992) and Onokpise et al. (1999) who obtained
100% survival with acclimatization studies. The relative lower survival percentage observed
in this study could be due to the plantlets were kept under a plastic cloche only for three
days or due to these cocoyam genotype may be different from the cocoyam genotypes
acclimatized by Onokpise et al. (1992) and Onokpise et al. (1999). Genotype can influence
the ability of regenerated plants to withstand the ex vitro growing conditions (Hazarika et
al., 2006). Regarding the green- and purple- cocoyams, no considerable difference was
observed for the shoot multiplication and root induction parameters and acclimatization
success. This indicates any perceived difference was due to random error and the genetic
variation that existed between the two may be limited to be differentially influenced by the
media compositions. In conclusion, the present study results showed that cocoyam (X.
sagittifoulium) growing in Ethiopia could be propagated in vitro by incorporating
appropriate concentration of PGR on MS medium for the phases of shoot initiation (2.0 mg/l
BAP), shoot multiplication (2.5 mg/l BAP + 0.5 mg/l NAA) and root induction (2.0 mg/l
IBA). This protocol can be used to supplement the farmers’ conventional methods of
propagation of cocoyam in the country. Further work is suggested to evaluate the cormel
yields of the multiplied cocoyam plants on field and to investigate growth condition of
cocoyam for ex situ conservation.
123
Chapter Nine
Synthesis, Conclusion and Recommendations
9.1 Synthesis
This study employed the techniques of ethnobotanical documentation and genetic
characterization using morphological descriptors and molecular markers. The proximate and
mineral contents and antinutritional factors were also analyzed along with the development
of a micropropagation protocol. Thus, the farmers’ knowledge on cocoyam was
complemented with the study of genetic diversity using morphological and molecular
markers and determination of key nutritional compositions. The problems that farmers face
regarding the piecemeal process of cocoyam propagation was taken to the laboratory and a
micropropagation technique was developed. The latter could be extended to farmers in due
course to promote faster and massive production of cocoyam. Thus, the study on the one
hand addressed the knowledge gap that existed in cocoyam, particularly the paucity of
scientific data, and on the other hand addressed problems that farmers described in relation
to the food quality and those concerned with developing a means for easy propagation of the
crop.
The field survey helped to document farmers’ knowledge and perceptions of
agromorphological traits, uses and management of cocoyam and interpretation in scientific
terms. Though cocoyam (X. sagittifolium) was considered by farmers as a variety of taro (C.
esculenta), it is a distinct species in a different genus within the same Araceae family.
Different local names (KENI ZHANG, CUBI ZHANG, GOCHELI KIDO, SUDAN KIDO,
BOINA, GUDETA, AGARFA, DAWURO BOINA, FARANJA BOINA, TONNEKA, BADADIY
TEPIYA BOINA, SAMUNA
and FARANJA BOINA) are in
use for X. sagittifolium in the surveyed areas further affirming its distinctiveness although
the term
GODERE
(Amharic term) has been used for both crops (which is likely more related
124
to the similarities in growth patterns and mode of utilization). Numerous local names are
used for X. sagittifolium worldwide (Giacometti and Leon, 1994; Mayo et al., 1997; QueroGarcia et al., 2010; Lim, 2015). A multiplicity of names for a given crop usually indicates
the usefulness and popularity of the crop among produces and end users. Farmers in the study
area also helped with the attempts made in this study towards the reconstruction of the history
of the crop in Ethiopia by recalling that cocoyam was introduced into their areas later than
taro. They affirmed that it is one of the crops introduced fairly recently into the Ethiopian
farming systems. However, the large majority (84%) of farmers, understandably, did not
remember the time when cocoyam was introduced into their areas despite the assertion by
some farmers from Benchi-Maji and Kefa zones that cocoyam was introduced into their areas
in the 1970s. During our survey, we observed that cocoyam was widely distributed in the
surveyed areas, growing mainly in the homegarden patches. It is also commonly found in
natural ecosystems as escapes from cultivation, on roadsides, in river valleys and as
ornamentals mainly in urban centers. This may indicate that cocoyam might have a longer
history in Ethiopia.
The knowledge of genetic diversity of plants is a fundamental element to enhance the
classification of germplasm, determine conservation plans for existing germplasm and for
development and introduction of new varieties. Morphological traits (16 qualitative and 13
quantitative), and SSR and AFLP molecular genetic markers provided insight into a broader
context for identifying the genetic diversity and differentiation of cocoyam accessions from
Ethiopia. The majority (84.6%) of the quantitative morphological traits showed significant
variation among accessions. Both SSR and AFLP molecular markers revealed high genetic
diversity across populations, within green morphotype, within purple morphotype as well as
when all collections were considered as single population. Solomon Fantaw et al. (2014b)
characterized 64 cocoyam accessions on the bases of 16 quantitative traits and reported
125
significant variation among all of the studied quantitative traits. For reason that the exact date
of cocoyam introduction into Ethiopia and who introduced it cannot be unequivocally spelt
out, the high genetic diversity can fairly indicate that introduction of cocoyam into the
country has been made multiple times, through multiple routes and probably by multiple
agents. Many introduced plant species have high level of diverse propagules due to multiple
introduction events (Rollins et al., 2013).
The extent of genetic differentiation and clustering is of great interest in a number of
biological fields including evolution, conservation, breeding, and ethnobotany in general
including anthropology (Hedrick, 2005). Of 16 qualitative morphological traits, 9
discriminated the cocoyam accessions into two groups. The quantitative morphological traits
and SSR markers-based clustering analyses effectively separated the purple cocoyam
morphotype from green cocoyam morphotype. The clustering pattern matches with greenand purple-colored cocoyam morphotypes that were observed during field studies. Farmers
also identify the two by different local names. In the Qucha Woreda of the Gamo-Gofa Zone,
for example, the term TONNEKA is used for purple cocoyam because it has a sour taste whereas
the term BADADIYA is used for green cocoyam to indicate its giant size. The color difference
could be considered as important for easy identification of cocoyam. Cluster and population
structure analyses revealed that the ocoyam accessions that were collected from different
zones, districts and kebeles were grouped regardless of the collection sites, indicating that
there might have been movement of germplasm between the locations, possibly by farmerto-farmer planting materials exchange. The fact that as many as 32% of the interviewed
farmers did not know how cocoyam was introduced into their garden for the first time also
indicates that the crop may be established from cormels and fragments dropped while
transporting, irrespective of conscious human planting. A recent article by Carl Zimmer
(April 12, 2018) that appeared in the Science column of the New York Times showed that
126
the
sweet
potato
has
travelled
a
long
distance
(www.nytimes.com/2018/04/12/science/sweet-potato-pacific-dna.html).
all
by
Cocoyam
itself
is
probably traveling to some distance within Ethiopia without human involvement and its
introduction into some gardens could be accidental.
The present survey results showed that both green- and purple-colored cocoyams are mainly
used for food and fodder/feed while the purple-colored cocoyam is also used as medicine to
treat WULAWUSHIYA (related with hepatitis), BARQA (postpartum depression) and GERGEDA
(related with rheumatoid arthritis). However, there is a paucity of data on the nutritional
compositions of cocoyam growing in the country. To address this lack of information,
proximate and mineral contents and antinutritional factors of green- and purple-colored
cocoyams were determined. The analysis showed that both morphotypes of cocoyam can
provide nutrient-rich products, with slight differences in the quantities of proximate and
minerals contents. However, high antinutritional factors (phytate and tannin) were
determined from both morphotypes of cocoyam with significantly higher quantities in purple
morphotype, supporting the farmers’ response that stated as purple cocoyam has unpleasant
smell compared to green cocoyam and that they give the local name SAMUNA BOINA meaning
soap taro in Dawuroto, Wolaitato and Gofato languages for purple cocoyam due to its
cormel has the smell of soap when cooked. Although cocoyam was appreciated by all
respondents for its food values (serving as emergency food), the farmers disliked traits and
high antinutritional factors would affect its acceptance because that could impose some
adverse effects on its end users. Studies have shown that processing methods such as boiling,
fermentation and roasting can significantly reduce contents of antinutritional factors of taro
(C. esculenta) from Ethiopia (Adane Tilahun et al. 2013; Melese Temesgen, 2017; Habtamu
Azene and Tesfahun Molla, 2017). Thus, the processing methods should to be tested for the
127
level of reduction of antinutritional contents of cocoyam in order to obtain healthful and
comfortable cocoyam products.
Traditional propagation method of cocoyam was unable to ensure dissemination of its useful
aspects, enhance sustainable production and conservation of cocoyam in Ethiopia. The
production of a large number of plants of selected germplasm is the prerequisite for
establishment of plantations, ex-situ conservation and improvement (Siddique and Anis,
2009). A micropropagation protocol was developed for green- and purple- cocoyams
growing in the country, by initiating shoot tip explants on MS medium containing 2.0 mg/l
BAP, multiplying using MS medium supplemented with 2.5 mg/l BAP and 0.5 mg/l NAA
and inducing roots on MS medium containing 2.0 mg/l IBA. This could help to promote
faster and massive production of cocoyam to address the problems that farmers face
regarding the piecemeal process of cocoyam propagation and for possible wider
applications.
128
9.2 Conclusion
A lot of useful indigenous knowledge on agromorphological traits, uses and management of
cocoyam was documented from cocoyam growing areas of Ethiopia. We found that
cocoyam is synonymously considered with taro (C. esculenta). Two morphotypes of
cocoyam were perceived by farmers, which were also distinguished by morphological traits
and SSR markers. The study identified qualitative and quantitative traits that will help
researchers in recommending traits for morphological identification of cocoyam
germplasms. The study revealed high level of genetic diversity, implying genetically diverse
cocoyam accessions have been cultivated in the country. This indicates that there might have
been multiple introductions of cocoyam into the country although this needs to be
investigated. SSR marker-based study revealed that the purple cocoyam was strongly
separated from the green cocoyam while the AFLP marker-based study correlated cocoyam
accesions neither to geographic locations (populations) nor to morphotypes. This
inconsistent result could be attributed to the important features that SSR and AFLP markers
differ from each other. The nutritional composition analysis showed that both green- and
purple- cocoyams can provide nutrient-rich products although the high antinutritional
factors would affect its acceptance. The micropropagation protocol established in this study
can be used to supplement the farmers’ conventional methods of propagation of cocoyam.
The cocoyam growing in Ethiopia can be considered as a crop which can play a significant
role in alleviating the household food insecurity and periodic food shortages existing in some
families inhabiting cocoyam growing areas.
129
9.3 Recommendations
Based on the findings of this study, the following recommendations are drawn:
It has been the farmers who have managed cocoyam to meet their needs. This study is one
among the very few pioneer cocoyam researches undertaken in Ethiopia. It did not cover all
cocoyam growing areas of the country despite the considerable efforts that have been made to
address various aspects. Thus, the future efforts should focus on developing core collections
from a wider possible cocoyam growing areas of the country. The cocoyam genotypes should
be tested in more than one environment to select elite genotypes for future utilization and
conservation.
The high genetic diversity, detected in this study, should be considered a good opportunity for
effective usage and conservation of cocoyam. All applicable conservation strategies should be
sought to maintain the existing high genetic diversity of cocoyam accessions in Ethiopia.
Since some inconsistency has been observed between the results of SSR and AFLP markers
regarding clustering of green- and purple-colored cocoyam morphotypes, the results should be
interpreted with a degree of caution for a reasonable assessment of true genetic situation of
cocoyam in Ethiopia. Full taxonomic study of cocoyam is recommended in order to facilitate
investigation of the relationships between its gene pool in Ethiopia and elsewhere in the world
including other species of the genus Xanthosoma.
More detailed nutritional analyses including processing and sensory testing are suggested for
further study.
Further work is suggested to evaluate the cormel yields of the tissue culture derived cocoyam
plants at a field and to investigate minimum growth condition for in vitro storage.
The existing uses of cocoyam is limited in view of its potential uses. Cocoyam is poorly
studied and underutilized crop in spite of its nutritional value and its potential as food crop.
Thus, collaborative research intervention involving the development of varieties, making
available high quality planting materials for farmers and promoting value chains and market
opportunities are valuable for sustainable use of the exisisting diversity and to safeguard the
potential end users of cocoyam in the country.
130
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Apendix 1 Passport data and semi-structured interview guide for cocoyam (Xanthosoma
sagittifolium (L.) Schott) study in Ethiopia
School of Graduate Studies, Addis Ababa University
Passport data
1. Acquisition date -----------------------------------------------------------------------------2. Accession code (include the first letters of Zone and Woreda, year of collection Xs and
number) (e.g. BS/2014/Xs1 to refer to an accession from Bench-Maji Zone, SouthBench Woreda in 2014 and Xs was given to refer Xanthosoma sagittifolium and that was
following by number 1 to refer accession number 1---------------------3. Scientific name: Genus----------------species----------------- subspecies or variety--4. Collector name ------------------------------------------------------------------------------5. Country of collection------------------------------------------------------------------------6. Donor name ----------------------------------------------------------------------------------7. Ethnic group of the individual and/or farmer donating the accession ----------------8. Local name/vernacular name/ of the crop------------------------------- Language: --- Is there any special meaning linked to the name? Yes --------------------No----- If yes, what is the meaning of the vernacular name? ------------------------------9. Collection source (home-garden, outfield farm, road side, wild habitat (forest), village
market, institution, other) ---------------------------------------------------------10. If from farm, status of plantation (backyard garden-------- smallholding (<5 ha) ---midsize holding (5-10 ha) ------------------------larger size holding (>10 ha) --------11. General distribution of the accessions in the area (rare, limited, widely distributed but
scattered small populations, extensive stands of large population) --------------12. Growing conditions: substrate (wet land (flooded), low land (not flooded), swamp,
upland (add approximate slope) -----------------------------------------------------------13. Growth conditions: canopy (deep shade, partial sun, full sun) -----------------------14. Type of material received (corm, cormel, leaf) -----------------------------------------15. Type of maintenance: (vegetative, tissue culture, seeds) ------------------------------16. Number of plants sampled -----------------------------------------------------------------17. Location of collection site:
Kebele--------------- District --------------Zone ------------------Region ----------------Altitude ------------------------ latitude -------------------------- longitude---------------18. Institution where herbarium specimen will be deposited--------------------------------
148
Semi-structured interview guide
1. Sex and age of each farmer responded to the interview
Male --------female --------Age--------. For how long had you lived in this area? ---For
how long had you grown cocoyam? -------------------------------------------------2. Description of cocoyam germplasm by farmers
Do you remember the year when this crop (cocoyam) was introduced into your area? ------------------------------------------------------------------------------------------ Do you remember from where (from whom) you obtained cocoyam for the first time?
Yes/No If yes, from where (from whom) ----------------------------------------- Are taro and cocoyam the same or different crops? ------------------------------------ How do you differentiate cocoyam from taro? ------------------------------------------ How many types of cocoyam do you know/grow? ------------------------------------- If two or more, how do you differentiate them ------------------------------------------ Did you observe flower of cocoyam? Yes------------------------No---------------------If yes,
at which month of cultivation? ----------------------------------------------------Under what conditions? --------------------------------------------------------------------3. Present cultivation status: at the time of collection (increasing, decreasing, static, noncultivated); why ------------------------------------------------------------------------4. Local uses of cocoyam
For what purposes do you use cocoyam? No use------------ Food-------------------------Medicinal (witchcraft)------------ Fodder------------ Other (specify) ------------- If the crop is used for food, Edible: corm------------ cormel------------ petiole------leaves-----------other-------------------------------------------------------------------------What is the
mode/method of preparation? ----------------------------------------------- If cocoyam is used for medicinal purpose, what is the mode of preparation,
administration and for the treatment of which disease/s is it used for? --------------5. Farmers’ preferred traits of cocoyam
Is there a preferred trait of cocoyam? Yes------------------------ No --------------------If yes,
would you mention some? --------------------------------------------------------- Is there any aspect of cocoyam that you hate, and that you like to see improved?
Yes----- No ------. If yes would you mention some? ------------------------------------6. Farmers’ planting material and cropping system
149
What part of the crop do you use for planting? Corm -------cormel-------other------which
one of these is preferred? ---------------------------why -------------------------- How would you cultivate cocoyam? Crop by irrigation ------------By rain-fed------ Do you use fertilizer? Yes--------------------------------No------------------------------- How do you crop cocoyam?
Mono
crop
--------------------------------------------
Cocoyam mixed with other crops----------------------------------. If mixed with other crop,
with which crop and what is your reason? ---------------------------------------- Any antagonism with any other crop? Yes ---------------------------No------------If yes,
which crop(s) and how? ---------------------------------------------------------------7. Time course of land preparation, planting and harvesting of cocoyam
What is the time course that you prepare land for cocoyam planting? --------------- What is the time course that you plant cocoyam? --------------------------------------- What is the time course that cocoyam takes to be harvested? -------------------------8. Methods that farmers have adopted for cocoyam germplasm conservation
9. Fresh corms buried on farm -----------------Fresh cormels buried on farm------------Fresh
corms stored at home ---------------------- Fresh cormels stored at home-------Others
(specify) ------------------------------------------------------------------------------10. Type of herbarium specimen prepared (leaf, inflorescence, tuber/stem) -------------
150
Appendix 2 Selected morphological descriptors used to characterize cocoyam (Xanthosoma sagittifolium) grown in Ethiopia
S. N
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1
2
3
4
5
6
7
8
9
10
11
12
13
Qualitative character
Plant growth habit
Petiole attachment
Petiole color (upper 2/3rd)
Petiole color (lower 1/3rd)
Color of edge of petiole sheath
Lamina orientation
Leaf margin color
Leaf shape
Color of upper leaf surface
Color of lower leaf surface
Color of veins on upper leaf surface
Color of veins on lower leaf surface
Position of cormel apex
Shape of cormels
Color of cormel apex
Flesh cormel color
Quantitative trait
Over all plant height (cm
Petiole length (cm)
Petiole sheath length (cm
Leaf length (cm)
Leaf width (cm)
Circumference of the above ground stem (cm)
Number of cormels
Cormel length (cm)
Cormel diameter (cm
Cormel fresh weight per plant (kg)
Corm length (cm)
Corm diameter (cm)
Corm fresh weight (kg)
Character state
Acaulescent
2. Erect above ground stem
3. Reclining aboveground stem
Peltate
2. Subpeltate
3. Non peltate
Light green
2. Green
3. Red/Purple
4. Green streaked with red/purple
Light green
2 Green
3. Red/Purple
4. Green streaked with red/purple
The same as the rest of petiole and sheath
3. Lighter than the rest of petiole and sheath
Darker than the rest of petiole and sheath
4. Pink/Red/Purple
One plan - apex up (Erect)
2. One plane - apex down (Droopy)
3. 3-dimentional (cup shaped)
Green to the edge 2. Clear to the edge
3. Purple/Red edge
4. Pale yellow/creamy edge
No basal lobes
3. Hastate (basal lobes flared)
4. Sagittate. basal lobes <1/8th the length of leaf
Sagittate. basal lobes >1/8th -1/4th the length of leaf 5. Sagittate basal lobes >1/4th the length of leaf
Light green
2. Medium green
3. Dark green
4. Redish/ Purplish green
5. Other
Light green
2. Medium green
3. Dark green
4. Redish/ Purplish green
5. Other
Same as color as lamina
3. Lighter green than lamina
Darker green than lamina
4. Red/Purple
Same as color as lamina 2. Darker green than lamina
3. Lighter green than lamina
4. Red/Purple
Aboveground
2. Underground
3. Both
Globose
2. Ovate
3. Cylindrical
4. Elliptical 5. Mixed (state which of these---)
White
2. Pink
3. Red
White
2. Yellow
3. Orange 3. Pink or pale red
4. Purple 5. other
Remak
Measured from ground level to the top of plant
Length of the longest petiole from the basal zone of the plant to the point of leaf attachment
Length of the sheath of longest petiole from the beginning to the end to sheath
The length of the leaf was measured
The width of the leaf was measured
The circumference of pseudostem just above ground measured
The cormels produced by a plant counted
The length of large. medium and small sized cormels measured
The diameter of large. medium and small sized cormels measured
The weight of cormels per plant weighed
The length of corm measured
The diameter of corm measured
The fresh weight of corm per plant was weighed
1.
1.
1.
1.
1.
2.
1.
1.
1.
2.
1.
1.
1.
2.
1.
1.
1.
1.
1.
151
Appendix 3 Mean performances of 13 quantitative traits of 100 cocoyam accessions (65 green- 35 purple morphotypes)
from two replications by accession
Accession
BS/2014/Xs1
BS/2014/Xs2
BS/2014/Xs3
BS/2014/Xs4
BS/2014/Xs5
BS/2014/ Xs5
BS/2014/Xs7
BN/2014/ Xs8
BN /2014/Xs9
BN/2014/Xs10
BN/2014/Xs11
BN/2014/Xs12
BN/2014/Xs13
BN/2014/Xs14
KC/2014/Xs15
KC/2014/Xs16
KC/2014/Xs17
KC/2014/Xs18
KC/2014/Xs19
KG/2014/Xs20
KG/2014/Xs21
KG/2014/Xs22
KG/2014/Xs23
KG/2014/Xs24
JS/2014/Xs25
JS/2014/Xs26
DT/2014/Xs27
DT/2014/Xs28
DT/2014/Xs29
DT/2014/Xs30
DT/2014/Xs31
DT/2014/Xs32
DT/2014/Xs33
DM/2014/Xs34
DM/2014/Xs35
DM/2014/Xs36
DL/2014/Xs37
DL/2014/Xs38
DL/2014/Xs39
DL/2014/Xs40
DL/2014/Xs41
DL/2014/Xs42
DL/2014/Xs43
DL/2014/Xs44
DL/2014/Xs45
DB/2014/Xs47
DB/2014/Xs48
DB/2014/Xs48
DB/2014/Xs49
DB/2014/Xs50
DB/2014/Xs51
DB/2014/Xs52
DB/2014/Xs53
DB/2014/Xs54
DB/2014/Xs55
WK/2014/Xs56
WK/2014/Xs57
WK/2014/Xs58
WK/2014/Xs59
WK/2014/Xs60
WK/2014/Xs61
WH/2014/Xs62
WH/2014/Xs63
Morphotype
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Green
Purple
Green
Green
Green
Purple
Green
Purple
Purple
Green
Green
Purple
Green
Purple
Green
Purple
Green
Purple
Purple
Green
Purple
Green
Purple
Green
Purple
Green
PH
78.25
78.08
81.33
72.42
83.58
84.83
72.33
80.92
74.42
75.25
79.08
67.92
66.83
73.67
68.17
66.25
75.58
72.83
74.08
68.17
66.08
70.50
75.33
65.00
74.67
71.33
76.25
69.42
77.17
74.25
75.50
77.00
67.17
68.42
67.75
71.67
74.00
70.67
77.42
77.83
66.08
73.00
66.58
69.25
69.33
64.75
68.50
68.33
71.59
71.92
67.09
73.83
65.75
70.25
64.75
71.33
75.50
62.59
62.33
62.00
69.08
74.59
75.67
PL
58.67
62.00
63.33
56.33
64.58
67.42
54.17
53.83
55.50
55.83
65.58
52.25
52.58
53.42
48.92
51.50
55.25
53.17
54.33
50.17
50.00
56.92
58.58
45.92
56.33
52.58
55.67
51.75
58.58
51.92
57.83
58.50
50.25
49.33
56.50
55.08
55.33
48.33
60.58
56.25
51.58
54.75
54.58
52.17
59.33
53.08
50.92
49.33
53.83
50.08
55.50
54.42
58.92
53.33
52.83
54.33
56.33
51.08
44.67
49.83
48.08
57.92
58.67
PSL
31.08
33.92
33.83
29.50
39.25
41.42
30.42
36.08
32.67
34.33
38.75
28.50
28.75
30.67
28.33
27.92
33.33
29.92
30.17
28.50
26.25
31.25
33.92
30.33
31.17
30.25
30.50
29.67
36.17
30.58
33.08
34.83
28.08
27.33
30.58
31.58
29.42
29.58
32.92
33.83
27.92
31.08
30.75
29.83
30.33
29.83
28.58
27.75
29.75
27.92
29.00
30.42
38.33
25.50
30.67
29.42
27.58
29.42
26.25
26.17
27.92
33.08
32.17
LL
42.78
41.56
36.42
35.06
43.17
36.81
38.92
40.25
35.86
36.14
39.56
35.78
32.97
33.19
31.11
35.47
38.06
33.69
38.11
29.69
36.25
36.69
33.33
30.53
35.11
39.08
38.17
34.44
35.03
37.17
36.06
37.47
35.47
36.89
35.00
39.64
40.28
34.06
38.61
39.25
35.06
34.33
40.89
38.11
43.11
40.28
36.08
37.17
38.17
35.75
38.31
35.53
45.25
35.47
37.50
38.89
38.14
35.67
37.92
33.72
36.53
40.06
35.94
LW
24.89
23.67
21.31
19.67
24.11
21.50
23.08
22.89
20.53
23.22
22.56
20.89
20.31
19.94
19.14
20.19
22.75
20.81
19.92
17.28
20.47
21.03
20.00
18.42
20.86
22.97
23.36
22.39
20.44
21.58
21.22
23.06
22.14
21.89
19.97
23.06
24.50
20.33
22.58
23.33
19.78
20.75
22.69
22.14
23.11
22.50
20.33
23.56
23.39
21.86
22.83
21.03
24.25
20.94
22.06
22.33
23.08
20.53
22.25
19.92
22.47
25.47
21.42
152
CAGS
32.25
31.75
29.83
25.75
32.33
33.00
28.08
33.82
24.17
27.50
30.92
28.08
26.17
25.17
25.08
27.25
28.83
27.58
29.08
25.42
28.00
27.00
26.33
23.50
27.92
29.08
29.00
25.92
27.33
28.75
33.33
31.75
27.67
26.67
27.58
29.50
31.50
26.83
29.83
31.33
25.33
27.92
28.25
27.67
32.25
34.42
28.83
27.58
29.08
25.83
31.25
26.83
30.42
27.08
27.75
29.25
29.83
27.58
28.00
25.00
29.00
30.92
25.75
NC
12.83
11.83
10.25
11.83
15.33
9.58
12.08
13.33
10.08
8.25
11.33
7.33
10.33
9.92
9.75
12.67
11.75
11.17
12.67
5.92
12.67
11.08
9.75
8.83
9.08
13.00
9.25
11.58
13.17
6.75
10.75
11.42
8.42
8.92
10.17
10.83
11.25
10.17
11.33
9.50
8.83
6.92
8.75
11.42
7.58
11.08
9.42
9.67
9.50
8.42
8.92
10.42
8.67
8.17
8.58
9.67
9.58
8.92
8.67
10.17
9.17
11.25
9.17
CL
9.42
9.00
9.08
9.00
9.75
11.18
10.83
10.58
8.92
9.08
8.92
7.08
8.33
9.83
10.33
9.50
10.67
10.33
9.50
6.58
6.75
8.83
7.42
7.17
10.25
11.83
9.67
9.75
8.58
8.42
8.42
10.00
8.33
8.08
9.42
8.83
9.50
8.58
9.50
9.08
7.58
8.17
9.50
8.17
7.42
8.83
10.33
9.33
9.50
9.75
8.42
9.08
7.17
7.92
8.08
10.25
9.42
10.67
10.50
8.75
9.17
8.25
9.00
CD
3.38
3.10
2.56
2.85
2.93
3.10
3.04
3.98
3.48
2.25
3.23
2.68
2.41
2.96
2.59
3.20
3.35
3.09
3.22
2.69
2.69
2.86
2.64
2.42
2.80
3.00
2.79
2.78
3.11
2.61
3.05
3.16
3.81
3.18
2.79
3.32
2.76
2.39
3.79
3.17
2.60
3.50
3.46
3.16
3.53
3.25
2.62
3.02
3.68
2.90
3.18
3.25
3.23
2.87
3.68
3.79
3.03
3.90
2.88
3.48
2.94
3.36
2.96
CFW
1.90
1.40
1.25
1.33
1.88
1.28
1.85
2.43
1.38
0.90
1.10
1.08
1.15
1.35
1.13
1.35
1.45
1.48
1.30
0.68
0.98
1.45
1.13
0.88
1.03
1.60
1.00
1.75
1.48
0.90
0.90
1.18
1.40
0.90
0.83
1.18
1.15
1.35
1.38
1.30
1.13
0.95
1.95
1.58
1.00
1.25
0.75
0.80
1.38
0.90
1.00
1.43
0.95
1.10
1.20
1.08
1.08
1.25
0.93
1.13
1.28
1.63
1.23
CrL
11.83
12.08
11.50
12.33
13.17
13.50
11.42
11.33
10.33
10.50
12.92
11.33
9.33
10.25
10.83
13.08
12.50
11.83
11.71
7.42
10.83
10.50
12.58
9.92
12.00
10.00
11.92
9.75
10.50
6.08
12.00
11.92
11.50
10.92
11.58
10.92
13.17
10.67
11.42
8.42
9.00
9.83
13.25
10.00
16.58
13.75
12.75
10.50
12.92
8.75
14.08
11.33
13.08
10.92
15.92
11.17
11.33
13.92
8.92
11.92
9.67
13.83
10.33
CrD
6.21
5.73
5.24
5.58
6.96
6.31
5.75
6.64
5.38
4.79
6.27
4.66
5.08
5.23
5.73
5.49
5.88
5.49
5.59
3.49
5.39
5.81
5.18
4.48
5.35
5.70
4.96
4.79
6.12
3.81
5.93
6.15
6.28
5.98
5.12
6.01
6.05
5.27
5.98
5.38
4.29
5.16
6.46
5.78
6.48
5.53
5.54
5.00
6.37
4.41
6.10
5.65
5.31
5.63
5.70
6.97
5.98
6.17
5.70
5.33
4.90
5.76
5.48
CrFw
1.25
1.20
0.93
1.25
1.23
0.98
1.03
1.63
0.98
2.10
1.73
0.88
0.65
0.88
0.90
0.95
1.18
1.10
0.78
0.58
1.15
0.90
0.98
1.03
0.83
1.53
0.83
1.15
1.00
0.53
1.05
1.13
0.98
1.20
0.90
0.85
0.83
1.23
1.63
0.90
1.00
0.90
1.73
1.05
2.30
1.68
0.90
1.18
1.23
0.83
1.05
1.33
1.50
1.08
1.15
1.68
0.78
1.48
0.88
1.23
1.10
1.00
1.05
Appendix 3 Continued
WH/2014/Xs64
Purple
70.67 56.58 31.67 40.39 22.50 28.17
8.67
8.33 3.66
1.10 14.50 5.66
1.28
WH/2014/Xs65
Green
63.00 50.08 28.83 32.58 18.11 24.50
9.75
8.08 3.03
1.13 11.25 3.78
0.75
WH/2014/Xs66
Purple
67.08 55.08 33.50 37.42 20.92 30.25 12.58 8.58 4.21
1.55 16.08 6.43
1.35
WH/2014/Xs67
Purple
67.67 54.75 30.75 40.89 22.67 31.08
9.58
8.42 3.54
1.15 14.50 6.95
1.85
WH/2014/Xs68
Purple
57.58 42.75 25.50 38.22 21.83 31.83 11.67 8.75 3.80
1.48 12.42 6.22
1.65
WH/2014/Xs69
Purple
71.42 55.58 31.83 38.83 21.14 28.17
9.00
9.25 4.11
1.20 12.75 6.15
1.65
WS/2014/Xs70
Purple
78.25 63.50 36.33 42.33 24.39 31.33
8.92
9.50 3.93
1.48 12.00 7.28
1.68
WS/2014/Xs71
Purple
65.92 55.58 28.50 37.64 22.44 32.92 11.67 8.58 3.72
1.73 13.92 5.99
1.65
WS/2014/Xs72
Green
71.50 51.58 31.58 40.75 24.19 30.25 13.50 11.25 3.14
1.75 10.08 5.68
0.85
WS/2014/Xs73
Green
75.67 53.50 33.50 36.22 23.50 24.50 12.92 8.42 3.13
1.63
8.92
5.50
1.08
WS/2014/Xs74
Green
75.08 55.17 31.00 38.06 23.33 27.92 10.00 10.00 3.53
1.48 10.67 5.33
1.08
WS/2014/Xs75
Purple
74.75 64.25 36.08 39.28 22.33 30.08
8.67
8.92 3.38
1.15 13.17 5.40
1.05
WS/2014/Xs76
Purple
80.17 66.50 43.92 43.33 23.69 30.08
8.33
9.50 3.95
1.20 14.17 6.51
1.63
WS/2014/Xs77
Green
76.08 53.25 29.83 38.44 22.17 27.75 11.33 8.50 3.26
1.50 11.17 5.63
0.93
WB/2014/Xs78
Purple
72.50 59.75 32.75 40.44 22.67 30.92
8.00
8.92 3.93
1.15 16.33 6.90
1.70
WB/2014/Xs79
Purple
68.58 48.08 28.67 37.25 23.67 28.17
8.25
9.33 4.21
1.35 12.25 6.82
1.45
WB/2014/Xs80
Green
70.92 55.83 27.42 36.86 21.44 24.92
9.25
9.83 3.53
1.05 12.50 6.07
1.08
WB/2014/Xs81
Purple
71.08 54.92 34.08 42.03 23.58 30.75
9.08
7.25 3.48
1.55 16.50 6.93
1.80
WB/2014/Xs82
Purple
76.92 59.42 36.58 39.89 22.08 32.58
8.25
8.17 3.61
0.98 13.17 5.95
1.23
GQ/2014/Xs83
Green
83.42 63.58 35.92 42.42 24.00 30.33 12.92 9.67 3.48
2.00
9.25
4.76
1.05
GQ/2014/Xs84
Purple
69.33 58.83 30.33 40.39 23.42 27.92 10.67 8.08 3.54
1.30 13.50 5.33
1.38
GQ/2014/Xs85
Green
79.67 60.25 32.67 38.50 23.28 28.83 12.58 10.42 3.48
1.50 16.92 5.78
0.88
GQ/2014/Xs86
Purple
68.92 54.92 28.83 40.97 23.75 32.50
9.75 10.17 4.05
1.10 15.25 6.30
1.35
GQ/2014/Xs87
Purple
82.50 67.83 40.50 42.39 23.75 34.00
9.08
8.42 3.94
1.15 13.83 6.74
1.28
GQ/2014/Xs88
Green
79.92 54.33 29.75 36.42 22.67 31.92
9.75
8.25 2.73
1.03 11.08 4.89
0.95
GQ/2014/Xs89
Purple
68.34 58.75 30.33 38.22 21.67 32.08 10.17 8.58 3.73
1.40 13.50 6.67
1.25
GQ/2014/Xs90
Green
57.67 52.17 31.50 36.06 19.19 23.17
9.75 10.75 3.77
1.23 10.83 4.90
0.68
GQ/2014/Xs91
Purple
65.00 55.75 31.67 44.97 25.22 31.67
7.67
7.08 3.55
1.80 16.67 7.73
1.70
GQ/2014/Xs92
Purple
65.00 51.83 30.25 40.31 23.50 32.92
8.58
7.42 3.48
1.35 14.33 5.98
1.40
GQ/2014/Xs93
Purple
68.67 60.08 33.83 38.61 22.86 27.83
9.17
9.08 3.49
1.15 13.83 6.39
1.38
GQ/2014/Xs94
Green
71.92 54.58 29.58 39.42 22.44 27.75 12.67 11.17 3.41
1.73 10.50 5.66
1.23
GD/2014/Xs95
Purple
69.83 57.25 30.58 41.36 23.69 33.08
8.58
8.50 3.67
1.65 12.42 5.64
1.48
GD/2014/Xs96
Purple
67.56 58.75 28.25 35.06 20.08 28.83
9.00
7.67 3.61
1.23 13.33 6.17
1.15
GD/2014/Xs97
Green
75.58 58.25 31.67 43.00 24.83 30.08
7.75 11.08 3.98
1.10
8.75
5.28
0.75
GD/2014/Xs98
Green
61.08 49.25 28.67 32.58 18.67 22.50
6.83
8.50 2.83
1.03
8.33
3.75
0.75
GD/2014/Xs99
Purple
70.92 59.83 34.50 43.83 22.92 33.42
8.00
9.42 3.88
1.13 14.83 7.08
1.85
GD/2014/Xs100
Green
63.50 49.25 25.50 31.22 18.56 21.42 13.92 9.50 3.33
1.38 10.58 5.39
1.05
Grand mean
71.49 55.22 31.25 37.25 22.02 28.83 10.05 9.05 3.26
1.28 11.94 5.70
1.18
PH= plant height, PL= petiole length, PSL=petiole sheath length, LL=lamina length, LW=lamina width, CAGS = circumference of above ground
pseudo-stem, NC= number of cormels per plant, CL=cormel length, CD=cormel diameter, CFW=cormel fresh weight per plant, CrL= corm
length, CrD=corm diameter, CrFW= corm fresh weight per plant.
153
Appendix 4 Nutrient composition and concentration of MS basal medium
1. Salts
A. Macro nutrient
1. NH4NO3
2. KNO3
3. Cacl2.2H2O
4. MgSO4.7H2O
5. KH2PO4
mg/l
1650
g/l
1.65
10 x
16.5 gm
1900
1.9
19 gm
440
370
170
0.44
0.37
0.17
4.4 gm
3.7 gm
1.7 gm
B. Micro nutrient
1. H3BO3
2. MnSO4.4H2O
3. ZnSO4.7H2O
4. KI
5. Na2MoO4.2H2O*
6. CoCl2.6H2O**
7. CuSO4.5H2O**
2. MS Vitamins
1. Thiamine (HCl)
2. Niacine
3. Pyrodoxine(HCl)
4. Glycine
5. Myo-inositol
mg/l
6.2
22.3
8.6
0.83
0.25
0.025
0.025
mg/l
0.1
0.5
0.5
2.0
Fresh added
3. Growth regulators
4. Iron-EDTA Na salt
??? in required amount
Fresh added
Fresh added
Fresh added
5. Sucrose
6. Agar
g/l
1000x
0.006
6.2 gm
0.022
22.3 gm
0.0086
8.6gm
0.00083
0.83 gm
0.00025
0.25 gm
0.000025 0.025 gm
0.000025
0.25gm
in 100ml
100mg
0.1gm
100mg
0.1gm
100mg
0.1gm
100mg
0.1gm
100mg/l
0.1g/l
Preparation remark
Prepared in 500 ml double distilled water and
50 ml was drawn for 1litre medium (because
in 500 ml it was already became 20x),
i.e.,1000 ml/20 =50 ml
Prepare in 250 ml double distilled water and
25ml was drawn for 1-liter medium (because
in 250 ml it was already became 40x), i.e.,
1000/40 = 25 ml
Prepared in 1 liter double distilled water and
1ml was drawn for 1-liter medium, i.e.,
1000/1000 = 1ml
To prepare 1-liter medium draw
0.1 ml
0.5 ml
0.5 ml
2 ml
40mg/l =0.04g/l
30 g/l
6 g/l
5.8
7. pH
Stock solution required for one-liter medium
Stock
10 x
20 x
40 x
100 x
200 x
400 x
800 x
1000 x
ml required for one-liter medium
100ml/litre
50 ml/litre
25 ml/litre
10ml/litre
5ml/litre
2.5 ml/litre
1.25ml/litre
1ml/litre
154
Appendix 5 Effects of different concentrations of PGRs on shoot multiplication of green- and
purple- cocoyams (comparative analysis)
BAP
Kn
NAA
(mg/l)
(mg/l)
(mg/l)
Shoot numbers/explant
Leaf numbers/explant
Shoot length (cm)
G
P
p-value
G
P
p-value
G
P
p-value
0
0
0
2.17 2.33
0.636
2.33
2.72
0.088
1.69 2.08
0.233
0.5
0.1
0
2.33 2.50
0.732
3.67
4.28
0.339
1.75 2.22
0.053
1.0
0.25
0
2.28 3.78
0.020
4.67
4.94
0.701
1.78 2.33
0.127
1.5
0.50
0
2.44 2.39
0.924
3.11
3.06
0.884
2.39 2.50
0.766
2.5
0.75
0
2.17 2.72
0.143
3.33
3.00
0.347
2.28 2.67
0.451
5.0
1.0
0
4.39 4.50
0.850
3.83
4.11
0.680
2.72 3.06
0.597
0.5
0
0.25 2.17 2.11
0.892
2.89
2.50
0.112
2.00 2.33
0.327
1.0
0
0.25 2.33 2.61
0.483
2.78
2.72
0.835
2.67 2.58
0.404
1.5
0
0.25 2.39 2.67
0.459
2.72
3.11
0.124
2.44 2.75
0.075
2.5
0
0.25 3.17 2.89
0.363
2.78
2.94
0.751
2.33 2.22
0.743
5.0
0
0.25 2.17 2.39
0.238
2.44
2.83
0.223
2.03 2.11
0.777
1.0
0
0.5
3.50 2.28
0.006
2.94
3.22
0.510
2.50 2.44
0.793
1.5
0
0.5
3.22 3.56
0.556
2.72
3.22
0.065
2.89 2.47
0.155
2.5
0
0.5
4.56 4.83
0.541
3.72
3.83
0.825
2.89 3.78
0.024
5.0
0
0.5
2.44 3.44
0.024
2.94
2.89
0.940
2.39 2.67
0.549
0.5
1.0
0.25 2.11 2.39
0.470
2.44
2.11
0.132
2.17 2.61
0.072
2.5
1.0
0.25 2.11 2.17
0.869
2.28
2.22
0.843
2.72 2.53
0.476
5.0
1.0
0.25 2.43 2.11
0.463
2.56
3.22
0.212
2.69 3.14
0.331
2.5
1.0
0.5
2.00 3.39
0.151
3.28
3.06
0.203
3.19 3.44
0.394
5.0
1.0
0.5
2.24 2.22
0.940
3.28
2.94
0.200
3.92 4.36
0.486
Independent sample t-test. P-values are shown for shoot multiplication parameters of green (G)- and purple
(P)- cocoyam. Mean values with p > 0.05 are not significantly different their and respective p-values are
shown
155
Eyasu Wada