IN-VITRO PROPAGATION OF SELECTED CASSAVA (Manihot 
esculenta CRANTZ) CULTIVARS USING MULTIPLE SHOOT 
INDUCTION AND SOMATIC EMBRYOGENESIS.
Kenneth Ellis Danso B.Sc (Hons). Dip. Ed.
A dissertation presented to the University of Ghana 
in partial fulfilment of the requirements for the award 
of degree of Master of Philosophy
November, 1997. ■
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DECLARATION
I, the undersigned candidate do hereby declare that work reported in this thesis is my 
original work and also wish to state that the work has not be presented to any other 
university for the award of any degree by me.
Signed..^..............................
Kenneth Ellis Danso 
(CANDIDATE)
Signed.....
Dr.
( SUPERVISOR)
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DEDICATION
This work is dedicated to the GLORY of GOD and to Charity, Cobby and Kofi whose 
prayer support enabled me to complete this work.
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ACKNOWLEDGEMENT
I sincerely do acknowledge the dedicated supervision of this work by Dr. Elizabeth 
Acheampong of Botany Department, University of Ghana. Her helpful suggestions and 
guidance saw me through this work.
My sincere thanks also go to Dr. H.M. Amoatey, the Head of Department of Plant and 
Soil Sciences, Biotechnology and Nuclear Agriculture Research Institute (BNARI), 
Ghana Atomic Energy Commission, Kwabenya for his helpful suggestions and 
guidance.
I also wish to thank Prof. R. G. F. Viser and Dr. C. J. J. M. Raemakers of Plant Breeding 
Department, Wageningen Agricultural University, The Netherlands for their helpful 
suggestions on work done on somatic embryo germination.
The helpful suggestions offered by Prof. Oteng Yeboah, Dr. Enu Kwesi, Prof. G.T. 
Odamtten and all the lecturers of the Department of Botany, University of Ghana is duly 
acknowledged.
My gratitude also goes to all the staff of the Tissue Culture Unit of the Botany 
Department of the University of Ghana, Legon my staff colleagues at the Department of 
Plant and Soil Sciences of BNARI/GAEC, and Mr. Kojo Gyampiah Montford and Dr. 
Josephine Nketsia-Tabiri for their assistance on the use of the computer for graphical 
representation and statistical analysis.
I finally wish to thank the Government of Ghana and the International Atomic Energy 
Agency for their financial support during the period of research.
May the Lord God Almighty bless them all.
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I A b s e is ic  a c id  
ABA...................... J,
ACMV...................... .African Cassava Mosaic Virus
AgDP.......................Agriculture gross domestic product
BA........................... Benzylaminopurine
Cl AT........................ Centro International de Agriculture Tropical
Dicamba.................. 3,6-dichloro-2-methoxybenzoic acid
ESCaPP................... Ecologically Sustainable Cassava Plant Protection
2,4-D........................2,4-dichlorophenoxy acetic acid
DGIS........................Directorate General for International Corporation
FAO......................... Food and Agricultural Organisation of the United Nations
GA3..........................Gibberellic acid
NAA......................... a-Naphthalene acetic acid
MS........................... Murashige and Skoog basal medium plus Gamborg B5 vitamins
Picloram...................4-amino-3,5,6-trichloropicolinic acid
RAPD.......................Random Amplified Polymorphic DNA
RFLP........................Restriction Fragment Length Polymorphism
TDZ (Thidiazuron) N-phenyl-N(123)-thiadiazolyl urea.
LIST OF ABBREVIATIONS
v
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TABLE OF CONTENTS
TITLE PAGE i
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
LIST OF ABBREVIATIONS V
TABLE OF CONTENTS vii
LIST OF TABLES xi
LIST OF FIGURES xii
LIST OF PLATES xiii
ABSTRACT xiv
CHAPTER ONE 1
1.0 INTRODUCTION AND LITERATURE REVIEW 1
1.1. Origin and cultivation of cassava 1
1.2. Utilisation of cassava 2
1.3. Yield of cassava 3
1.4. Cassava diseases 4
1.5. Germplasm collection and selection 5
1.6. Propagation and regeneration 6
1 .6.1 . Traditional methods of propagation 6
1.6.2. In-vitro regeneration of cassava 6
1.6.2.1. Meristem culture 6
1.6.2.2. Multiple shoot induction 7
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1.6.3. Organogenesis 0
1.6.4. Somatic embryogenesis 9
1.6.4.1. Origin and uses 9
1.6.4.2. Primary embryogenesis in cassava 10
1.6.4.3. Cyclic embryogenesis 11
1.6.4.4. Cyclic embryogenesis in cassava 12
1.6.4.5. Germination of somatic embryos 12
1.6.4.6. Somatic embryo germination in cassava 13
1.7. Objectives 14
CHAPTER TWO 16
2.0 MATERIALS AND METHODS 16
2.1. Experimental layout 16
2.2. Germplasm collection and propagation 16
2.3. Selection of cultivars 17
2.4 Source of explant 17
2.5. Media preparation and sterilisation 18
2.6. Sterilisation of explants 18
2.7. Culture procedures and incubation 18
2.8. Experimental procedures 20
2.8.1. Plant regeneration 20
2.8.2. Plant regeneration from apical meristem of cassava 20
2.8.3. Induction of multiple shoots from apical meristems 21
2.8.4. Induction of multiple shoots from single bud cuttings 21
2.8.5. Induction of somatic embryos from young leaf lobes of (greenhouse
vii
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grown and in vitro plants) 22
2.8.6. Desiccation of somatic embryos 23
2.8.7. Effect of ABA pre-treatment on embryo germination 24
2.8.8. Effect of cytokinins and auxins on embryo germination 24
2.8.9. Relation between moisture loss and BA on embryo germination 24
2.8.10. Effect of prewashing of embryos on germination 25
2.8.1. Influence of light/dark regimes on germination of desiccated embryos 25
2.8.12. Effect of different light intensities on embryo germination 25
2.8.13. Influence of different light regimes on embryo germination 26
2.8.14. Effect of desiccation in complete darkness on embryo germination 26
2.8.15. Evaluation 26
2.8.16. Statistical analysis 26
CHAPTER THREE 27
3.0 RESULTS 27
3.1. Germplasm collection 27
3.2. Severity of ACMV disease on cassava cultivars 28
3.3. Effect of medium A on apical meristem of cassava cultivars 32
3.4. Effect of medium B on apical meristem of cassava cultivars 34
3.5. Effect of BA on induction of multiple shoots from apical meristems 38
3.6. Effect of BA on induction of multiple shoots from single bud cuttings 39
3.7. Effect of 2,4-D on induction of somatic embryos from greenhouse
grown cassava 43
3.8. Induction of somatic embryos from leaf lobes, apical meristems and stipules 47
3.9 Induction of somatic embryos from leaf lobes of in-vitro cassava plantlets 48
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3.9.1. Embryo germination 56
3.9.2. Effect of different desiccation methods on embryo germination 56
3.9.3. Effect of ABA pre-treatment on embryo germination 58
3.9.4 Influence of auxins and cytokinins on embryo germination 61
3.9.5. Relation between moisture loss and BA on embryo germination 65
3.9.6. Effect of embryo pre-washing of somatic embryos on
germination 67
3.9.7. Influence of light/dark regimes on root and normal plant
development from somatic embryos 70
3.9.8. Influence of different light intensities on embryo germination 75
3.9.9. Effect of different light regimes on somatic embryo germination 77
3.9.10. Germination of embryos desiccated in complete darkness or light 79 
CHAPTER FOUR 81
4.0 DISCUSSION AND CONCLUSION 81
4.1 Germplasm collection 81
4.2 Meristem culture 82
4.3. Multiple shoot induction 84
4.4. Primary somatic embryogenesis 85
4.5. Germination of somatic embryos 88
SUMMARY 94
REFERENCES 95
APPENDIX -106
ix
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LIST OF TABLES
Table 1. Composition of Murashige and Skoog (1962) basal salts and
Gamborg B5 vitamins.
Table 2. Cassava landraces collected from farmers during the survey.
Table 3. Effect of Medium A on apical meristem of cassava at four weeks.
Table 4. Effect of Medium B on apical meristem of cassava at 4 weeks.
Table 5. Effect of BA on induction on multiple shoots and buds from apical
meristems of M. Col 22 and Biafra.
Table 6. Effect of BA on induction of multiple shoots from nodal cuttings
of cassava cultivars.
Table 7. Mean number of buds produced by cassava cultivars at 
28 days of culture.
Table 8. Effect of 2,4-D on leaf lobes cultures of different cassava 
cultivars from the greenhouse
Table 9. Effect of 2,4-D on leaf lobe cultures of greenhouse grown cassava.
Table 10. Effect of 16 mg/l 2,4-D on callus formation from various 
explants after 21 days of culture in the dark.
Table 11. Effect of 2,4-D on callus production from leaf lobes explants of 
five cassava cultivars.
Table 12. Effect of different desiccation methods on somatic embryo 
germination
Table 13a. Effect of ABA pre-treatment ( without desiccation) on embryo 
germination
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Table 13b. Effect of ABA pre-treatment ( with desiccation) on embryo 
germination
Table 14. Influence of different growth regulators on shoot and root formation 
of Adira 4 somatic embryos
Table 15a. Effect of prewashing and BA concentrations on germination 
of Gading embryos ( without desiccation).
Table 15b. Effect of prewashing and BA concentrations on desiccated somatic 
embryos of Gading
Table 16. Effect of desiccation in complete darkness and light on germination 
of Adira 4 on somatic embryos
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LIST OF FIGURES
Figure 1. Percentage of callus which formed somatic embryos after 21 days of 
culture in maturation medium in light
Figure 2. Percentage of callus cultures which formed somatic embryos after 
21 days of culture on maturation medium in light
Figure 3. Effect of moisture loss and different BA concentrations on 
germination of somatic embryos of Gading and Adira 4.
Figure 4. Influence of light/dark conditions on germination of NAA-
and 2,4-D-induced somatic embryos of TMS 90853, Gading, Line 11 
and Adira 4.
Figure 5. Influence of light/dark conditions on root formation of NAA- and
2,4-D-induced somatic embryos of TMS 90853, Gading, Line 11 
and Adira 4.
Figure 6. Effect of different light intensities on root, shoot
and plant germination in Adira 4 somatic embryos.
Figure 7. The effect of different light regimes on germination 
of Adira 4 somatic embryos
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Plate 1. Biafra cultivar showing symptoms of brown leaf spot disease 30
Plate 2. Bosomnsia cultivar showing symptoms of ACMV disease 33
Plate 3. In-vitro plantlets of Santom showing symptoms of ACMV
disease on leaf 37
Plate 4. Single bud cutting of Biafra showing multiple shoots 41
Plate 5. Leaf lobe cultures of Santom showing calli with roots 48
Plate 6. Callus of Santom with matured somatic embryos 54
Plate 7. Callus of Bosomnsia with matured somatic embryos 54
Plate 8. Calli of Bosomnsia showing malformed somatic embryos and
foliose structures. 57
Plate 9. Germinated somatic embryos of Adira 4on MS medium with
on 4 mg/l kinetin 63
Plate 10. Germinated somatic embryo of Adira 4 on MS medium with
1 mg/l BA. 64
Plate 11. Germinated somatic embryos Adira 4 cultured on 0.1 mg/l
BA in dark 74
Plate 12. Germinated somatic embryos of Adira 4 cultured on
1 mg/l BA in dark 74
LIST OF PLATES.
O'
r
S..
xiii
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ABSTRACT
The study showed that farmers cultivate different cassva cultivars based on popularity, 
duration to maturity and tolerance to African Cassava Mosaic Virus (ACMV) disease. All 
the five selected local were propagated in vitro using meristem explants on Murashige 
and Skoog (1962) basal salts and Gamborg B5 vitamins modified with 
benzylaminopurine (BA) (0.0 - 0.15 mg/I). There was profuse callus formation in all the 
cultivars. The optimal BA concentration for shoot proliferation in this medium was 0.10 
mg/l BA. With reduced NAA and (GA3) concentrations (0.02 and 0.04 mg/l respectively) 
in the MS medium 0.05 mg/l was optimum and resulted in 100% and 46% shoot 
regeneration in Bosomnsia ans Santom respectively compared to 37% and 0% in the 
previous treatment.
In M. Col 22 multiple shoots were produced from apical meristems as well as single 
nodal cutting explants. In the local cultivars multiple shoots were produced from nodal 
cuttings alone. The number of shoots produced by nodal cuttings in M. Col 22 were 
comparatively higher than the local cultivars at all the BA levels in the medium. In all, the 
number of shoots produced was dependent on the BA concentration in the medium.
Leaf lobe explants of both greenhouse and in vitro plantlets developed embryogenic calli 
on MS amended with 0.0-16 mg/l 2,4-D on a step one-induction medium. However, 
calli formation was depended on the type of explants. Calli formation from young leaf 
lobe and apical meristem explants was significantly higher than stipule explants. On 
transfer to a Step two BA amended maturation medium, embryogenic calli derived from 
in vitro plantlets only formed matured somatic embryos.
Embryo formation was depended on the concentration of the auxin in the induction 
medium and the cassava cultivar. Somatic embryo formation was higher on a medium 
with 16 mg/l 2,4-D. Santom produced the highest percentage of embryos (25%) among 
all the cultivars tested. Embryogenic calli which did not form somatic embryos formed 
foliose structures and/ or roots which also depended on the concentration of 2,4-D in the 
induction medium.
xiv
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NAA induced somatic embryos required desiccation to stimulate normal germination in 
all the four cultivars studied. The best desiccation procedure was the petri dish method. 
The desiccated embryos required a medium supplemented with BA and kinetin for 
germination . Incubation of cultures in darkness increased frequency of germination and 
also reduced germination period to 14 days when compared to cultures incubated in 
light under growth room conditions. The development of the seedling was dependent on 
the concentration of BA as well as the dark/light photoperiod of the incubation period. 
Cultures on medium with 0.1 mg/l BA and incubated in the dark produced normal single 
shoots whereas most cultures on 1 mg/l in the dark produced multiple shoots.
Germination of somatic embryos was dependent on the level of moisture loss and the 
BA concentration in the medium. At optimal moisture loss (40%) higher frequency of 
germination was achieved on a medium with lower concentration of BA (0.1 mg/l). At 
lower moisture loss 1 mg/l BA was needed for higher germination in Gading and Adira 4.
Wrapping of cultures with aluminium foil to achieve desiccation in complete darkness did 
not enhance embryo germination. Low light intensity (64 lux) resulted in optimum 
germination. Light regimes had significant effect on seedling morphology but not on 
frequency of germination
2,4-D-induced embryos showed a different response from NAA-induced embryos. In 
both NAA- and 2,4-D - induced somatic embryos desiccation followed by incubation in 
the dark stimulated root formation than shoot.
At a concentration of 1 mg/l, ABA shoot formation only in somatic embryos of Adira 4; 
while at 40 mg/l, it had an inhibitory effect on the germination of these embryos.
Prewashing of somatic embryos with water for 24 hours enhanced whole plant 
germination on MS medium modified with BA.
xv
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CHAPTER ONE
1.0 INTRODUCTION AND LITERATURE REVIEW
1.1 ORIGIN AND CULTIVATION OF CASSAVA
Cassava (Manihot esculents Crantz) is a perennial woody shrub which belongs to the family 
Euphorbiaceae. The genus Manihot consists of at least 98 species (Roggers and Appan, 
1973). The centres of diversity are Central Brazil, Northeastern Brazil, Southwestern Mexico 
and Eastern Bolivia (Naser, 1987). Cassava was introduced into West Africa from South 
America in the 16th century and into East Africa in the 18th century (Byrne, 1984). The crop is 
now widely cultivated in Africa between latitudes 30 degrees north and south of the equator. It 
has been reported that the crop is grown in 39 African countries. Although the crop is grown 
in every country in sub-Saharan Africa, cultivation is concentrated in the humid tropics.
A number of agricultural advantages have made the cassava crop of utmost importance for 
food security in Africa. First, productivity of cassava in terms of calories per unit land area per 
unit time is significantly higher than that of other staple food crops. The crop has high 
biological efficiency as food producer possibly due to its structural engineering considerations: 
the edible part of the tuberous roots lies beneath the ground and does not have to be 
supported by the plant in the form of heavy stems and branches (Byrne, 1984; Cock, 1985). 
Also the qjpp is productive even on poor soils and under adverse climatic conditions where 
other crops fail. Typically, cassava is the only crop able to grow on eroded or marginal lands 
including those abandoned as a result of failed attempts to grow other crops. Growth of 
cassava requires low input of man hours compared to other crops. Furthermore, the roots can 
be left in the ground without harvesting for up to two or three years until needed (DGIS, 1991). 
Thus, the certainty of obtaining some yield even under most adverse conditions and the
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possibility of maintaining continuity of supply throughout the year make this root crop a basic 
component of the farming system in many areas of Africa south of the Sahara (Nweke et al, 
1994). It has further reported that famine rarely occurs in areas where cassava is widely 
grown, since it provides a staple base to the food production system (DGIS, 1991). This 
indicates that cassava has a potential for eliminating food crises and famine.
1.2 UTILISATION OF CASSAVA
Despite its low protein content (1.0%; Purseglove, 1968) cassava is mainly utilised for human 
consumption in Africa . It provides a source of carbohydrate for 450-500 million people (Cock, 
1982; FAO, 1993) ranking ninth on the world scale. In the developing countries it ranks fourth 
on the list of major food crops after rice, wheat and maize (FAO, 1989). The tuberous root 
can be used as an industrial source of starch. Cassava starch is used in the food industry in 
many preparations , including glucose production, confectionery and baking products (IITA, 
1990). In the livestock industry also, cassava is used in feed rations or as animal feed 
concentrate. It is estimated that about 65 percent of world production is used for human 
consumption, 19 percent for cattle feed, 6 percent for industrial starch and 10 percent are lost 
(Cock, 1985).
Cassava is the most widely grown crop in rural areas of Ghana. It is cultivated either as a 
monocrop or intercrop. Al-Hassan (1990) reported that cassava production in Ghana grew 
from 2.225 million tonnes in 1985 to 3.3 million tonnes in 1988, an average growth rate of 14 
percent. The per capita consumption is high (148 kg/yr) compared to that of plantain at 83 
kg/yr (Annor-Frempong, 1994). According to the Ministry of Agriculture (MOA, 1990), the 
contribution of cassava to the Agriculture Gross Domestic Product (AgDP) is 22 and is the
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highest for any crop grown in the country. This high cultivation rate is attributed to the fact that 
cassava is cheap to produce, affordable and is utilised in many diets across the country. 
However, there are constraints to full utilisation of the crop; the tuberous root deteriorates 
after harvest, and has poor quality and quantity of proteins (Byrne, 1984). Cassava is also 
known to be cyanophoric, containing cyanogenic glucosides, limanarin and lotaustralin which 
liberate toxic cyanogenic compounds when the tissues are crushed (Roca, 1984; Obigbesan, 
1994). Intake of improperly processed cassava leads to neurological disorder (Cock, 1985) 
and can also contribute to the aetiology of goitre and parpareis which are endemic in several 
African countries (Annon, 1990). Bokanga et al., (1990) have however reported that 
submerged fermentation of cassava and the efficient processing procedures (grinding, 
grating, and pounding) are effective in removing limanarin.
1.3 YIELD OF CASSAVA
Cassava has high potential yield of 90 tonnes per hectare per year (Cock, 1985). Yields of 
more than 80 tonnes per hectare per year have been obtained under experimental conditions 
(Hershey, 1993) and yields of 50 to 60 tonnes have been obtained by farmers cultivating 
cassava as first crop on newly cleared lands. However the average cassava yield (8.8 t/ha) in 
the world is low and it is even lower (6.4 t/ha) in Africa (Cock, 1985).
In Ghana estimates on cassava production show that the average yield for the period 
between 1987-1990 is 7.8 t/ha (MOA, 1991). Ezumah (1988) reported that farmers who use 
improved technologies have the potential for achieving over 30 t/ha. The current lower yield in 
Ghana is mainly due to low adoption of improved technologies in the cultivation of cassava 
including lack of improved genotypes or varieties thereby forcing farmers to rely on 
susceptible genotypes for planting which are easily affected by diseases and pests.
3
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1.4 CASSAVA DISEASES
The most notable diseases which affect cassava are African Cassava Mosaic Virus (ACMV; 
disease, Cassava Bacterial Blight disease (CBB) and Cercospora leaf spot. CBB is the mosl 
widespread bacterial disease of cassava. The causal organism is Xanthomonas campestris 
pathovar manihotis. In Africa it was first reported in Madagascar in 1946 (IITA, 1990) and now 
occurs in all cassava-growing areas. The symptoms of the disease include water soaked leal 
spot, stem die-back and vascular necrosis. Severe attack results in rapid defoliation of the 
plant. Estimates of yield losses vary from 20 to 100% depending on the cultivar (IITA, 1990.
Cassava brown leaf spot caused by Cercosporidium henningsi is the most important 
cercospora leaf spot disease. Symptoms which are brownish are restricted to upper and lower 
leaf surfaces of older leaves. It has been estimated that the disease can caused about 20% 
yield loss in cassava (Theberge, 1985).
The African Cassava Mosaic Virus (ACMV) disease is the most widespread disease of 
cassava in Tropical Africa. It is transmitted by the whitefly vector Bemisia tabaci (Gennadius) 
which is prevalent in many parts of Africa (Annon, 1990). The etiology of ACMV has been 
widely documented in West Africa (Hahn et at., 1989; Fauquette and Fargette, 1990) and 
also in East Africa (Thresh et al., 1994).
Symptoms of ACMV disease include a characteristic light green, yellow or white patches, 
irregularly distributed. The chlorotic areas may be only small flecks or spots or they may cover 
the entire leaf surface. The mottling is sometimes accompanied by leaf deformation and a 
general stunting of the plant.
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Estimates of yield reduction per plant from ACMV infection generally range from 24% in 
highly resistant genotypes to 75% in highly susceptible varieties (Terry and Hahn, 1980; 
Fargette et al., 1985). Bocks and Woods (1983) have estimated that in highly susceptible 
clones yield losses of up to 95 % can occur. In East Africa , Seif (1982) showed that the mean 
yield loss of several Kenyan varieties was 57.7 % and disease incidence and yield were 
highly correlated.
The use of cassava stakes infected with the disease and pests enhance their rapid spread. In 
Ghana there is lack of good, healthy planting materials. Hence the prevalence of the disease. 
The control of ACMV disease can be achieved either through sanitation or the use of 
resistant/tolerant varieties obtained through breeding.
1.5 GERMPLASM COLLECTION AND SELECTION
Cassava and its wild relatives show an enormous genetic variation and there is a large 
number of cultivars. Africa abounds in wild varieties of cassava. African farmers cultivate 
over 2000 varieties of the crop (Iweke, et al., 1994) and in Ghana farmers cultivate about 78 
varieties (Annor-Frempong, 1994). The extensive and complete collection of local germplasm 
of cassava in Africa is important because native varieties have usually been selected over 
long periods for adaptability and palatability.
These varieties differ in their morphology, time-to-maturity and the type of food for which they 
are used. Besides these local clones carry genes for adaptation to local conditions especially 
tolerance to drought, diseases and pests and therefore serve as base material for cassava 
breeding.
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Selection of desired genotypes is a critical step in plant breeding (Ahloowalia, 1995). 
Germplasm collections made from local farmers so as to select for cultivars with desirable 
traits is highly recommended. This would be used as base material for crop improvement and 
breeding using advance biotechnological techniques. For maintenance of these selected 
genotypes an efficient regeneration system is a necessary prerequisite.
1.6 PROPAGATION AND REGENERATION OF CASSAVA
Two methods of propagating can be distinguished. Cassava may be propagated by stem 
cutting which is the traditional method and by the tissue culture (in-vitro) method.
1.6.1 TRADITIONAL METHOD OF PROPAGATION
True seeds of cassava have hardly been used by local farmers for propagation of the crop 
due to its high level of heterogeneity which is a result of genetic segregation. They rely on 
cuttings prepared from matured stakes. This traditional method of propagation is slow and 
also has an inherent problem of disease transfer from one vegetative generation to the next 
because very often the cuttings carry pests and diseases ( example, ACMV). The method 
also poses an enormous limitation during maintenance and international transfer of 
germplasm.
1.6.2 IN-VITRO REGENERATION
1.6.2.1 Meristem Culture
In-vitro culture of cassava is almost always accomplished by vegetative propagation of stem 
and bud cuttings. For production of disease-free planting materials, either meristem culture or 
a combination of meristem culture and thermotherapy are employed. These in-vitro
6
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techniques result in non-adventitious shoot regeneration where existing meristems are 
allowed to regenerate into plants.
The first in-vitro procedure for cassava regeneration using meristem culture was described by 
Kartha et al. (1974) and a year later by Liu (1975). The procedure employed the culture of 
meristematic dome from shoot tips on a medium supplemented with naphthalene acetic acid 
(NAA), benzylaminopurine (BA), gibberelic acid (GA3) and 20 g/l sucrose.
The meristem culture was later applied to the production of disease-free plants (Kartha and 
Gamborg, 1975). In cases where viruses are difficult to eliminate by meristem culture alone, a 
combination of thermotherapy and meristem culture has proved beneficial. Kaiser and 
Teemba (1979) combined meristem culture with thermotherapy to eradicate cassava mosaic 
diseases (CMD) and another “virus-like” pathogen from several East African cassava varieties 
successfully. Kartha and Gamborg (1975) further reported that after heat treatment, relatively 
larger meristem tips (0.8 mm) could be cultured. It is believed that the higher temperature 
either restricts multiplication of the virus or enhances vegetative growth, resulting in the 
production of virus-free shoots.
1.6.2.2 Multiple shoot induction
Healthy planting materials can also be produced in large numbers by induction of multiple 
shoots from either single nodes or apical meristems. Smith et al. (1986) induced multiple 
shoots from shoot tips and single buds on a medium supplemented with 0.5 mg/l NAA and 0- 
0.5 mg/l BAP. On this medium, between 4.2 and 18 nodes were produced within three weeks. 
Roca (1984) has reported that the number of apical nodes produced depends on the 
concentration of BA in the medium.
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However, these techniques require several tissue culture steps ( mechanical isolation of 
cuttings, rooting of cuttings and hardening of plants ) which makes the procedure labour 
intensive.
1.6.3 ORGANOGENESIS
Organogenesis refers to the process in which a unipolar structure is formed from 
multicellular origin (Reinert et al., 1977; Thorpe, 1990; Terzi and Loschiavo, 1990). The 
unipolar structure is either a shoot or a root and is physically connected to the tissue of origin. 
Plant regeneration through organogenesis can occur either directly or indirectly. Direct 
organogenesis involves the emergence of adventitious organs directly from the explant 
without callus phase. In contrasts, adventitious organs are produced through a callus phase in 
indirect organogenesis.
The first successful attempts of organogenesis in cassava were reported by Tilquin (1979) 
and Shahin and Shephard (1980). However, they were not repeatable by others.
A high concentration of auxin and low concentration of cytokinin promotes callusing. Tisserat 
et al (1979) reported that callus production on an agar medium could also be induced by the 
inclusion of 2,4-D alone. The cellular composition of the callus is heterogeneous (Evans et al., 
1981). Meristematically active cells in the organogenic callus are the precursors of root and 
shoot organs.
8
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1.6.4 SOMATIC EMBRYOGENESIS
1.6.4.1 Origin and uses
Somatic embryogenesis is the development of embryos from somatic cells through a series of 
developmental stages similar to zygotic embryogenesis. The embryo produced is a bipolar 
structure with no vascular connection to the parental tissue (Tulucke, 1987).
Production of somatic embryos may occur either directly or indirectly (Sharp et al., 1980). 
Direct embryogenesis involves the production of embryos without an intervening callus 
phase. During indirect embryogenesis, callus formation precedes embryo development ( 
Carman, 1990; Sharp et al., 1980; Wann , 1988).The somatic cell which gives rise to the 
embryo is called embryogenic cell. Even though every living cell is totipotent only a limited 
number of cells from explants give rise to structures that exhibit embryogenesis. Embryo 
induction from cells is dependent on the culture environment, such as hormonal balance, 
sugars, amino acids, salt concentration and the growth room condition (Vasil and Vasil, 1981; 
Franklin and Dixon, 1994). The methods for achieving appropriate conditions for embryo 
induction are still the main concern in regeneration research. In different plant species 
somatic embryogenesis is achieved on media with different inductive growth regulator(s). In 
monocots, embryogenesis is exclusively induced by an auxin supplemented medium whereas 
in the dicotyledonous species growth regulator-free, cytokinin and cytokinin plus auxin 
supplemented media are used (Raemakers, 1993). Synthetic auxins such as Picloram, 
Dicamba or 2,4-D are frequently used in the dicots.
Somatic embryogenesis has some advantages over other regeneration methods. Plants 
produced by somatic embryogenesis have taproots while in shoot regeneration adventitious 
roots are produced (Raemakers et al., 1995b). Also in somatic embryogenesis it is possible
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to produce a bipolar structure bearing both root and shoot apices in one step. The system 
also enhances large scale clonal propagation compared to vegetative propagation. More 
recently somatic embryogenesis has been interfaced with gene transformation methods to 
obtain transgenic cassava (Schopke et al., 1996).
1.6.4.2 Primary embryogenesis in cassava.
Somatic embryogenesis was defined by Haccius (1978) as a non sexual developmental 
process which produces a bipolar embryo which has its own vascular tissue. In cassava 
somatic embryogenesis was first reported by Stamp and Henshaw (1982). Since then, 
several groups of workers have described it as an efficient regeneration system in the crop ( 
Szabados et al., 1987; Mathews et al., 1993; Raemakers et al., 1993a). In the original 
method, embryos were induced on cotyledons, embryo axis or zygotic embryos using a two- 
step procedure . Induction of embryos on young leaf lobes have also been achieved (Stamp 
and Henshaw, 1987a; Szabados et al., 1987; Raemakers et al., 1993abc). In step one, 
embryos were induced on MS medium supplemented with 20 g/l sucrose and 16 mg/l 2,4-D. 
In step two, embryos were transferred to MS medium supplemented with 20 g/l sucrose, 0.1 
mg/l 2,4-D and 0.1 mg/l BA for further maturation of embryos. The efficiency of primary 
embryogenesis was however very low. Mathews et al., (1993) improved embryogenic 
frequency by transferring 2,4-D-induced globular embryos attached to the explant to a 
hormone-free medium supplemented with 0.5% charcoal after 15 days of induction. This 
procedure increased the number of embryos from 0.4 to 3.4 per initial leaf lobe.
In another method, Raemakers et al., (1993b) treated donor plants with liquid 2,4-D two days 
before leaf lobes were isolated for culture for embryo production. In this method 9.4 mature 
embryos were produced per leaf explant of the genotype M. Col 22 compared with 3.5 mature
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Although exogenous application of auxin has a positive effect on embryo induction, 
continuous exposure of somatic embryos to auxin has detrimental effect on the development 
of the apical meristem (Halperin and Witherel, 1965) which result in poor plant regeneration. 
Parrot et al., (1991) reported that continued exposure of somatic embryos to auxin lock them 
into a given developmental phase and establish cultures undergoing repetitive 
embryogenesis. In soybean, it has been shown that one week exposure of explant tissue to 
auxin was necessary for embryo production and that increasingly longer exposure resulted in 
decreased conversion frequency (Parrot et al., 1991).
Desiccation is also another characteristic of zygotic embryogenesis and can also play a role in 
germination of somatic embryos. It influences developmental processes and triggers the 
germination of embryos (Rosenberg and Rhine, 1986). Desiccation causes changes in tugor 
pressure, membrane permeability and levels of endogenous absissic acid (ABA) which in turn 
induce expression of specific genes involved in plant development (Oishi and Bewley, 1990). 
Gray (1987) has reported that in Vitis longii, 20% of somatic embryos that were desiccated 
germinated as opposed to 5% in non-desiccated embryos. Similarly, in spruce 50% of the 
desiccated embryos germinated whereas in the non-desiccated embryos only 5% germinated 
(Roberts etal., 1990).
1.6.4.6 Somatic embryo germination in cassava.
Two main methods have been described for shoot conversion in cassava. In the first method 
Stamp and Henshaw (1987a) regenerated cassava plants from mature 2,4-D-induced 
embryos by culturing them in a basal medium supplemented with 0.1 mg/l BA plus 0.01 mg/l
2,4-D. Raemakers et al., (1993b) improved shoot conversion by increasing the BA 
concentration to 1.0 mg/l in the basal medium. Depending on the genotype, 10% (M. Col
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1505) to 70% (M. Col 22 ) of the embryos developed into plants. However, the process of 
embryo germination was long and plants were initially malformed and lacked roots. 
Adventitious roots were formed after transfer of plants to growth regulator-free medium. 
Mathews et al., (1993) further improved embryo germination by culturing desiccated mature 
embryos on a hormone-free medium. With this method, 85% of M.Col 1505 embryos induced 
on 2,4-D medium germinated into plants.
1.7 OBJECTIVES
The current significant changes of cassava utilisation in the world, due to international market 
development and domestic demand will put pressure on the cassava breeder to supply the 
desired cultivars in a short time. To achieve this goal, breeding programmes in cassava 
should emphasize on the use of novel biotechnological techniques for genetic modification of 
the crop. An efficient regeneration system is therefore a necessary prerequisite for production 
of transgenic cassava. The most reliable regeneration system for cassava, thus far, has been 
through somatic embryogenesis and cyclic embryogenesis (Stamp and Henshaw, 1987a,b; 
Szabados et al., 1987; Raemakers, 1993). The multiplication of virus-free planting materials 
would be greatly increased, if an efficient regeneration system such as somatic 
embryogenesis is developed for cassava in the producing countries. Although somatic 
embryos and plantlets formation have been obtained from Latin-American clones (Stamp and 
Henshaw, 1982, 1987a,b; Szabados et al., 1987), this is yet not possible with African clones 
(Ng, 1992). Secondly, in-vitro methods used to produce healthy planting materials of cassava 
have not been employed in the local genotypes cultivated by local farmers.
The objective of this study was to select for cassava cultivars with desired agronomic traits 
from local farmers and to regenerate the selected cultivars in vitro using meristem culture,
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multiple shoot Induction and somatic embryogenesis. Other specific objectives include i. 
studies on embryogenic competence of selected local cassava cultivars ii. comparison of 
multiple shoot induction in Latin-American cultivar M. Col 22 and local cultivars and iii. high 
frequency germination of mature somatic embryos of African cultivars and Latin-American 
cultivars.
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CHAPTER TWO
2.0 MATERIALS AND METHODS
2.1 EXPERIMENTAL LAYOUT.
Work described in this thesis is divided into two phases. The first phase involved germplasm 
collection of cassava (Manihot esculenta Crantz) from local farmers in Dodowa, Kasoa, 
Kwabenya and North Taifa. Selected cultivars were then propagated in-vitro using meristem 
culture, multiple shoot induction and somatic embryogenesis. These set of experiments were 
conducted at the Botany Department of the University of Ghana. Experiments in the second 
phase were conducted at the Plant Breeding Department, Wageningen Agricultural 
University, The Netherlands.
The second phase involved studies on germination of mature somatic embryos using 
selected cultivars from Nigeria (TMS 90853), Zimbabwe (Line 11), Indonesia (Adira 4 and 
Gading) and CIAT, Columbia (M. Col 22).
2.2 GERMPLASM COLLECTION AND PROPAGATION
Twenty-five farmers from Dodowa, Kwabenya, Kasoa and North Taifa were interviewed using 
a structured questionnaire for the collection of qualitative information on cassava cultivars 
planted, duration to maturity, incidence of diseases, utilisation of tubers and yield of cultivars 
(Appendix I). Ten-centimetre long cuttings were prepared from stakes of each cultivar and 
were planted in black polythene bags (15 cm diameter) filled with topsoil. The pots were kept 
in the greenhouse at the Botany Department, University of Ghana. The temperature in the 
greenhouse was 28-35°C in normal daylight. The pots were watered every other day with
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tapwater. The cuttings sprouted within seven days of planting. After 28, days apical shoot 
tips were harvested and used for in-vitro culture.
The severity of ACMV on the local cultivars was scored using ESCaPP Survey Software, 
IITA Benin Station, (Appendix ii) in order to determine their susceptibility to ACMV. Six 
month-old cassava plants on the farm were used for the score test. Shoot tips of 25 plants of 
each cultivar were selected at random and scored for ACMV by examination of the shoot tip.
2.3 SELECTION OF CULTIVARS.
Based on popularity, duration to maturity (early bulking), yield and susceptibility to ACMV, five 
local cultivars namely, Ankrah , Bosomnsia, Biafra , Santom and Afisiafi were selected for 
this studies on multiple shoot induction and somatic embryogenesis. The cultivars Ankrah and 
Santom were collected from farmers at Dodowa whilst Bosomnsia and Biafra were collected 
from Kwabenya. Afisiafi is an introduction from the International Institute of Tropical 
Agriculture (IITA).
For studies on somatic embryo germination, the cassava cultivars: Line 11 (provided by 
University of Zimbabwe), TMS 90853 (provided by IITA), Adira 4 and Gading (provided by 
Central Research Institute for food Crops, Indonesia) and M. Col 22 (provided by CIAT, 
Columbia) were used.
2.4 SOURCE OF EXPLANTS
Four-week old shoot tips harvested from greenhouse grown cuttings were used as source 
explants for in vitro studies (Phase 1). Meristematic tissues (0.2 mm) were dissected from the 
apical shoot tips for culture. Mature embryos of Line 11, Adira 4, TMS 90853, and Gading 
used for studies on embryo germination (Phase 2) were provided by the Plant Breeding 
Department, Wageningen Agricultural University, The Netherlands.
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2.5 MEDIA PREPARATION AND STERILISATION
Culture media were prepared from Murashige and Skoog basal salts (1962) supplemented 
with Gamborg B5 vitamins (Gamborg et al, 1968) formulated by Sigma Chemical Company, 
USA (MS). The composition of the MS medium is shown in Table 1. The MS medium was 
supplemented with sucrose and growth regulators as required. For solid media, MS was 
solidified with 7 g/l agar (oxoid). The pH of the media was adjusted to 5.7 prior to autoclaving. 
Media were dispensed into sterilised testtubes mm in diameter). Each testtube contained 
10 ml of the culture medium. Petri dishes contained 5 ml of the media.
Culture media, testtubes, distilled water, forceps and scapel blades were sterilised by 
autoclaving for 15 minutes at 121°C. Petri dishes were sterilised in an oven ( Gallenkamp 
Oven, 300 Plus Series) at a temperature of 160°C for 24 hours. Growth regulators were filter- 
sterilised using 0.22 |am millipore filter sterilizer and were added to autoclaved MS under the 
laminar air flowhood.
2.6 STERILISATION OF EXPLANTS.
Shoot tips were trimmed of older leaves and washed thoroughly in running tap water. They 
were then transferred to sterilised GA-7 flasks under the laminar air flow hood (Dalton, 
Ogawa Seiki Co. Ltd). Explants were washed again with two changes of sterilised distilled 
water, immersed in 70% alcohol for 1 minute and rinsed with three changes of sterilised 
distilled water.
2.7 CULTURE PROCEDURES AND INCUBATION.
In-vitro procedures involving sterilisation of plant tissues, growth regulators, dissection of 
meristems and leaf lobes, inoculation and subculture were done under the laminar air
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flowhood. All cultures were incubated in a growth room at a temperature of 27+1 °C and 12 
hour photoperiod and light intensity of 3,500 lux. The light was provided by fluorescent tubes.
Table 1. Composition of Murashige and Skoog (1962) basal salts and Gamborg 
B5 vitamins (MS).
Macro salts mg/l mM
CaCI2. 2H20 332.02 2.99
KH2 P 0 4  170.00 1.25
KN 0 3  1900.00 18.79
MgS0„.7H20  370.00 1.50
NH4 NO3  1650.00 20.61
Micro Salts
CoCI2 .6H20  0.025 0.11
CuS0 4 .5H20  0.025 0.10
H3BO3 6.20 0.10
Kl 0.83 5.00
MnS0 4 H20  16.90 0.10
Na2M o0 4 2H20  0.25 1.03
ZnS0 4 .7H20  8.60 29.91
FeNaEDTA 36.70 0.10
B5 Vitamins
myo-inositol 100.00 0.56
nicotinic acid 1 . 0 0  8 . 1 2
pyridoxine HCI 1.00 4.86
thiamine HCI 10.00 29.65
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2.8 EXPERIMENTAL PROCEDURES
2.8.1 Plant Regeneration
Three methods of plant regeneration were used. These are meristem culture, multiple shoot 
induction and somatic embryogenesis.
2.8.2 Plant regeneration from apical meristem of cassava.
The effect of different concentrations of BA on shoot induction from apical meristem was 
studied in Bosomnsia, Ankrah, Santom, Biafra , Bodwease bosomnsia and Afisiafi using two 
media formulations, Medium A and Medium B as indicated below. Four-week old shoots were 
harvested from greenhouse grown cuttings. Explants were derived from four week old shoot 
sterilised as described in section 2.6. The meristem (0.2-0.4 mm) was dissected under a 
stereomicroscope and were cultured on 10 ml of Medium A or Medium B.
Medium A
Murashige and Skoog (1962) basal salts and vitamins (full strength)
Naphthalene acetic acid (NAA) 0.1 mg/l
Gibberelic acid (GA3) 0.1 mg/l
Sucrose 30 g/l
Agar 7 g/l
Medium B
Murashige and Skoog (1962) basal salts and vitamins (full strength)
Naphthalene acetic acid (NAA) 0.02 mg/l
Gibberelic acid (GA3) 0.04 mg/l
Sucrose 30 g/l
Agar 7 g/l
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Both Medium A and B were supplemented with the following BA concentrations, 0.0 (control),
0.05, 0.10, 0.15 or 1.0 mg/l BA. Medium B was formulated to reduce the callusing effect of 
NAA in the medium. Media were adjusted to pH 5.7 before autoclaving (see 2.4). Cultures 
were incubated in a growth room at 27 ± 1°C. The cultures were observed daily for organ 
formation.
Shoots were defined as organs with distinct nodes, internodes and at least one primary leaf. 
Shoots were transferred to a hormone-free medium after six weeks of culture for further 
growth. Plantlets obtained thereafter were subcultured onto hormone-free medium every four 
weeks using nodal cuttings.
The leaves of thirty six plantlets of each cultivar were examined in-vitro for ACMV symptoms 
after 28 days of culture on a hormone-free medium.
2.8.3 Induction of Multiple shoots from apical meristems.
Induction of multiple shoots from apical meristems were compared in Latin-American cultivar 
M. Col 22 and the Ghanaian cultivar Biafra. Apical meristems were cultured on MS medium 
supplemented with 20 g/l sucrose, 0.1 mg/l NAA, 0.1 mg/l GA3 and 2.0, 1.0, 0.1 or 0.0 
(control) mg/l BA. For each treatment, 10 ml of this medium were dispensed into a test tube 
which formed an experimental unit. One apical meristem was inoculated in a test tube. Each 
experimental treatment was replicated six times and arranged in a completely randomised 
design. The number of shoots and buds developed at four weeks was recorded.
2.8.4 Induction of multiple shoots from single bud cuttings.
To enhance the multiplication rate of plantlets multiple shoots were induced in Ankrah, 
Bosomnsia, Biafra and M. Col 22. Single nodal explants were prepared from four weeks
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old in-vitro plantlets and cultured on 10 ml of medium. Each experimental unit was replicated 
four times and arranged in a completely randomised design. The number of shoots which 
sprouted per node as well as the number of buds per shoot were recorded.
2.8.5 Induction of somatic embryos from young leaf lobes of green house 
grown and in vitro plants.
Primary embryos were produced using a two-step procedure described by Stamp and 
Henshaw (1982) and Raemakers et al. (1993a,b). Four weeks old shoot tips from 
greenhouse-grown cassava and two weeks old in vitro plantlets were used. Shoot tips were 
sterilised as described in section 2.6. Young leaf lobes (0.5-2 mm) were dissected from the 
shoot tips using a stereomicroscope in the laminar air flowhood. The leaf lobe explants ( 
from greenhouse grown cassava and in vitro plantlets) were cultured on MS medium 
supplemented with adenine sulphate(80 mg/l), sucrose (20 mg/l), agar (7 g/l) and 2,4-D at a 
concentration of 16, 8,4 or 0 mg/l (control). The explants were cultured in petri dishes with 
their adaxial surfaces in contact with the medium and were incubated in the dark (Step one).
After 24 days of culture, the number of explants which formed embryogenic calli were 
recorded and transferred onto MS medium supplemented with 0.1 mg/l BA (Step two) and 
incubated in the light. Each experimental treatment was replicated five times with 5 explants 
per treatment in a completely randomised design. The number of somatic embryos, foliose 
structures, roots and shoots produced were recorded.
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In another experiment, young leaf lobes, apical meristems and stipules were dissected from 
four weeks old greenhouse grown cassava and cultured on MS medium supplemented with 
16 mg/l 2,4-D and incubated as described above.
2.8.6 Desiccation of somatic embryos.
Mature NAA-induced embryos were subjected to various desiccation treatments for a period 
of seven days using the following procedures:
i. Partial desiccation using petri dish. Mature embryos of Adira 4 (8g) induced in liquid 
MS medium were transferred to sterile Whatmann 3 mm filter paper for one hour to absorb 
water from the embryos under the laminar air flowhood. Four grammes (4g) of these embryos 
were transferred to sterile petri dishes, weighed, sealed with parafilm and kept in the growth 
room for seven days. The petri dishes were changed every other day to remove condensed 
moisture. The percentage moisture loss through desiccation was calculated from the ratio 
between the weight of the embryos after and before desiccation, multiplied by 100. 
Desiccated embryos were cultured on solid MS medium supplemented with 1.0 mg/l BA. 
Cultures were incubated in the light. The number of embryos which germinated was recorded.
ii. Desiccation using glycerol. Four grams of mature embryos of Adira 4 were 
transferred to petri dishes and sealed with parafilm. The petri dishes were then placed in 
desiccators containing 200 ml of glycerol to control the internal humidity for seven days. The 
desiccated embryos were reweighed to determine the moisture loss before culture on solid 
MS medium with 1 mg/l BA.
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iii. Desiccation by using sugar and other solutions. Four grains of matured embryos 
were transferred to solid or liquid MS medium supplemented with 0-60 g/l sucrose or liquid 
MS supplemented with 0-10 g/l mannitol. The control was water supplemented with 0-60 mg/l 
sucrose. After seven days half of each treatment was cultured on MS medium supplemented 
with 1.0 mg/l BA while the other half was desiccated using petri dish method for further seven 
days before they were cultured for germination.
2.8.7 Effect of ABA pre-treatment on embryo germination.
Four grams of mature embryos of Adira 4 were transferred to 300 ml culture jars containing 
MS medium supplemented with 0, 1 or 40 mg/l abscissic acid. The jars were incubated on a 
gyrator at 120 rpm in the growth room. After seven days half of the embryos of each 
treatment were cultured on solid MS medium supplemented with 1.0 mg/l BA for germination 
while the other half was desiccated for further seven days before cultured.
2.8.8 The effect of cytokinins and auxins on embryo germination.
The effects of three cytokinins Thidiazuron (TDZ), Benzylaminopurine (BA) and Kinetin as 
well as two auxins (NAA and IBA) on germination of somatic embryos were studied. Mature 
NAA liquid-induced somatic embryos of Adira 4 were desiccated using the petri dish method 
(Section 2.8.6) before culture on solid MS medium supplemented with 20 g/l sucrose as 
control or solid MS medium supplemented with various concentrations of cytokinins and 
auxins. Cultures were all incubated in the light.
2.8.9 Relation between moisture loss and BA on embryo germination
The relationship between the degree of moisture loss and BA on embryo germination was 
studied using mature embryos of Adira 4 and Gading. Four grams of mature embryos were
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transferred to sterile petri dishes and sealed with parafilm. The petri dishes were then placed 
in 2L jars filled with 50 ml of various concentrations of sodium chloride solution. After seven 
days the amount of moisture loss was determined and the embryos which lost 20, 40 or 75 
percent moisture were cultured for germination on MS medium supplemented with 0.1 mg/l
2.8.10 Effect of prewashing of embryos on germination.
Four grams of mature embryos of Gading were transferred to 300 ml culture jars each 
containing 200 ml of sterile distilled water. The jars were incubated on a gyrator at 120 rpm. 
After 1, 5, 10, or 14 days half the embryos were cultured for germination while the other half 
were petri dish desiccated for a further one week before they were cultured for germination.
2.8.11 Influence of light/dark regimes on germination of desiccated embryos.
Mature NAA- or 2,4-D-induced embryos of TMS 90853, Line 11, Adira 4 and Gading were 
desiccated using the petri dish method. The moisture loss was determined as previously 
described for seven days. Desiccated embryos were cultured for germination on MS amended 
with 0.1, 1.0, or 4.0 mg/l BA including control (0 mg/l). After seven days one-half of the 
cultures were incubated in a dark cardbox while the remaining half were incubated under 
growth room light conditions of 3,500 lux. Cultures were evaluated after 14 or 28 days .
2.8.12 Effect of different light intensities on embryo germination.
Four grams of NAA induced embryos of Adira 4 were transferred to sterile petri dishes and 
desiccated using the petri dish method. After seven days the desiccated embryos were 
cultured on MS medium supplemented with various concentrations of BA. The cultured 
embryos were incubated in different boxes covered with transparent polyethylene sheets to
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control the light intensities. The light intensities used were 0 6,4, 64, 640, or 6400 lux. The 
light intensity in each box was determined using a light meter.
2.8.13 Influence of different light regimes on embryo germination.
Four grams of matured embryos of Adira 4 were transferred to sterile petri dishes and 
incubated in white, red, blue, far red or dark cabinets.
2.8.14 Effect of desiccation in complete darkness on embryo germination.
Four grams of matured embryos of Adira 4 were transferred to sterile petri dishes, wrapped in 
aluminium foil to prevent entry of light or placed in the light (L) for 7 days. At the end of the 7 
days, half of the embryos wrapped with aluminium foil were cultured for germination in the 
dark while the other half was incubated in light. Embryos desiccated in light were also cultured 
and incubated in the light.
2.8.15 Evaluation
Embryos cultured in the dark/light experiment were evaluated for germination after 14 and 28 
days and all other experiments after 28 days. The embryos were evaluated for presence or 
absence of organs, the morphology of the root whether thin or thick, taproots or adventitious. 
Also the morphology of the shoot was determined, whether thin or thick, single or multiple. An 
embryo is said to be germinated when it had both shoot and root. A shoot was present when 
an internode has appeared with at least one primary leaf.
2.8.16 STATISTICAL ANALYSIS
Statistical analysis, where applicable, was performed using Statistical Graphics Corporation 
Software.
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CHAPTER THREE
3.0 RESULTS
3.1 GERMPLASM COLLECTION.
Germplasm collections of cassava were made from local farmers in Dodowa, Kwabenya and 
North Taifa in the Greater Accra Region in order to identify cultivars which have desired 
agronomic traits using a structured questionnaire (Appendix 1). These collections will serve 
as basis for improvement of the crop.
The survey indicates that farmers distinguish cassava genotypes by local names which are 
often descriptive of the physical characteristics of the plant. The name may also describe the 
original source of the genotype such as the place from which it was introduced for the first 
time. Twelve (12) descriptive names of cassava were recorded including two IITA introduced 
cultivars (Table 2). The name of the cultivar may vary from village to village. For example 
Bosomnsia is associated with the village where the farmers collected it from. Hence 
Bodwease bosomnsia and Kuntunse bosomnsia refer to the same variety collected from 
Bodwease and Kuntunse, respectively.
Almost all the farmers cultivate Bosomnsia either as single cultivar or with other cultivars on 
the same farm. Sixty (60%) of the farmers interviewed cultivate Ankrah and Biafra. The 
remaining cultivars are cultivated on a minor scale.
The cultivars collected show different morphological features. For instance, the colour of the 
petiole is either red or green. A petiole is considered red if it has any red spot on the petiole,
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otherwise green. All the cultivars collected had red petiole except Afisiafi which had green 
petioles.
The leaves of cassava are either described as broad or narrow. All the cultivars collected had 
broad leaves. However, the leaves of Biafra, Bankye tumtum and Ankrah were much broader 
than the other cultivars.
For the cultivars collected duration to maturity varied from 6-18 months (Table 2). For 
instance farmers indicated that Bosomnsia matures at six months after planting compared to 
18 months in Saakwanyah, Bankye tumtum and Ankrah.
Farmers interviewed also indicated that the yield obtained in Bosomnsia is lower compared 
to Bodwease bosomnsia, Ankrah and Biafra.
3.2 Severity of ACMV disease on cassava cultivars
Symptoms of two main types of cassava diseases were identified on the leaves of cultivars 
collected. These are Brown leaf spot (Plate 1) and ACMV disease (Plate 2). Severity of 
ACMV symptoms was scored for five of the most frequently cultivated cultivars. Results are 
as shown in Table 2.
Symptoms of ACMV disease observed on the farms are light green or chlorotic patterns on 
leaves, wrinkled or distorted leaf surface (Plate 2) and reduction of leaf size. Symptoms of 
ACMV disease were observed in all the cultivars in every village. The severity of the 
symptoms however varied from cultivar to cultivar. Among the cultivars scored ACMV was 
found to be most conspicuous and severe in Bosomnsia compared to the other cultivars 
Table 2. Severity was least in Bodwease bosomnsia and Biafra. In Bosomnsia the symptoms 
were observed on both older and younger leaves whereas in Biafra and Bodwease
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bosomnsia, symptoms were observed only on younger leaves. For Ankrah and Biafra the 
symptoms are not conspicuous at some locations. Farmers indicated that symptoms of 
ACMV appear four (4) weeks after shoots have sprouted in Bosomnsia and Ankrah 
compared to six (6) weeks after sprouting in Bodwease bosomnsia and Biafra.
Field observations also showed that brown leaf spot affected only older leaves of cultivars 
studied in this work. In addition cultivars with conspicuous symptoms of brown leaf spot 
express very low symptoms of ACMV disease.
Table 2. Cassava landraces collected from farmers during the survey.
Cultivars maturity period 
(months)
Yield ACMV Score Remarks
Ankrah 18 high 2 Has good fufu making qualities
Bosomnsia 6 medium 4 used for making fufu, ampesi
Bodwease 9 high 1 used for making fufu, gari, ampesi
bosomnsia
Biafra 12 high 2 used for making gari, agblema
Santom 9 medium 2 has sweet taste, good for ampesi
Afisiafi1 12 high 2 good for making gari, agblema
Bankye broni 12 medium used for making fufu, gari
Bankye tumtum 18 high used for making gari, agblema
Katawire 18 medium used for making gari, agblema
Koasekohwe 9 medium used for making fufu, ampesi
Saakwanyah 18 medium used for making gari
Kuntunse 9 high used for making gari, agblema
bosomnsia
Gblomaduade1 - high used for making gari, agblema
Note 1: IITA introduced cultivar.
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Plate 1. Biafra cultivar showing symptoms of brown leaf spot.
30
/ y - x  01
UA
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3.3 Effect of Medium A on apical meristem of cassava cultivars.
In this experiment, apical meristems were cultured on MS medium amended with 0.1 mg/l 
NAA, 0.1 mg/l BA and 0.1 mg/l GA3( Medium A) .Theresults are as indicated in Table 3.
Explanted meristems became chlorophyllous within seven (7) days and after 14 days formed 
creamy callus. Explants cultured on a hormone-free medium did not form callus. The size of 
the callus was dependent on the concentration of BA in the medium. In all the cultivars callus 
formation was high in MS medium supplemented with high BA but decreased at the controls. 
Callus formation was however low in Santom explants.
In Bankye tumtum there was no shoot development at any of the BA levels. Only shoot-like 
structures developed when explants were cultured on 0.10 mg/l BA. These shoot-like 
structures had no distinct nodes or internodes and could also not develop into functional 
shoots when transferred to hormone-free medium.
In general, shoot development by Biafra and Bodwease bosomnsia was comparatively higher 
than Ankrah, Santom and Bosomnsia. The optimal treatment for shoot development was 
0.15 mg/l BA for Ankrah and Bodwease bosomnsia whereas for Santom and Biafra the 
optimal treatment was 0.10 mg/l. The highest percentage number of shoots (75%) occurred 
on Biafra followed by Bodwease bosomnsia (74%), Santom (50%) and Ankrah (33%). Most 
explants which did not form shoots developed into shoot-like structures. These shoot-like 
structures did not developed into functional shoots when transferred onto hormone-free 
medium. Shoots were transferred to a hormone-free medium after 28 days of culture for 
further in vitro studies.
32
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Table 3. Effect of Medium A on apical meristems of cassava cultivars at four weeks.
BA ( mg/l) No. of Callus Roots (%) Shoots Shoot-like
Cuitivar explantsa (%) structures (%)
0.00 8 _ 0 0 (0) 0 (0)
Ankrah 0.05 8 ++ 0 2(25) 0 (0)
0.10 10 +++ 0 0 (0) 3 (30)
0.15 15 +++ 0 5 (33) 3 (20)
0.00 8 - 0 0 (0) 0 (0)
Bosomnsia 0.05 8 +++ 0 3 (37) 1 (13)
0.10 10 +++ 0 2 (20) 3 (30)
0.15 11 +++ 0 3 (27) 3 (27)
0.00 8 - 0 0 (0) 0 (0)
Santom 0.05 8 + 0 0 (0) 0 (0)
0.10 10 ++ 0 5 (50) 1 (10)
0.15* - - - - -
0.00 12 - 0 0 (0) 0 (0)
Biafra 0.05 12 ++ 0 7 (58) 2 (17)
0.10 12 +++ 0 9 (75) 3 25)
0.15 11 +++ 0 8 (73) 2 18)
0.00 9 - 0 0 (0) 0 (0)
Bodwease 0.05 7 +++ 0 5(71) 2 (28)
bosomnsia 0.10 7 +++ 0 4 (57) 0 (0)
0.15 19 +++ 0 14 (74) 4 (21)
0.00 8 - 0 0 (0) 0 (0)
Bankye 0.05 12 ++ 0 0 (0) 0 (0)
tumtum 0.10 11 +++ 0 0 (0) 2 (18)
0.15 10 +++ 0 0 (0) 0 (0)
Note: - no callus + low callus ++ moderate +++ high callus
a: Number recorded after contaminated cultures had been discarded, 
b: Figures in parenthesis indicate percentages.
33
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Following the experiment on Medium A, it was thought that excessive callus formation 
caused by high concentration of NAA and BA may have hinder shoot and root development. 
The hypothesis was tested by culturing apical meristems on MS supplemented with reduced 
concentrations of NAA (0.02 mg/l) and GA3 (0.04 mg/l), while varying the concentration of 
BA. This constituted medium B.
The explants became chlorophyllous after seven days followed by callus formation and shoot 
regeneration within fourteen days. However, callus formation was moderate compared to 
Medium A and in Santom and Afisiafi most of the meristem explants did not form callus. The 
results are presented in Table 4.
In general, shoot regeneration was enhanced in Medium B compared to Medium A.
In all the cultivars, explants on a hormone-free medium became chlorophyllous but did not 
develop into shoots or roots.
For Bosomnsia, Santom and Afisiafi the optimal concentration of BA for shoot formation was 
0.05 mg/l. At this optimal concentration, all the explants of Bosomnsia formed shoots 
compared to only 27% in Medium A. Similarly, in Afisiafi and Santom 90 and 46%, 
respectively, formed shoots. For Ankrah and Biafra, the optimal concentration was 0.15 mg/l 
BA and shoot formation was 90% and 85% respectively. In Medium A, only 33% of Ankrah 
meristems cultured on the same BA treatment were able to form shoots.
3.4 Effect of Medium B on apical meristem of cassava cultivars.
34
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Multiple shoots were observed in Ankrah and Santom explants cultured on 0.15 mg/l BA 
compared to single shoots in Medium A. In Ankrah 26% of the cultures developed multiple 
shoots with a range of 2-4 shoots per meristem explant. In Santom, only one meristem 
explant developed three shoots.
Fifty five percent (55%) of Ankrah cultures on 0.15 mg/l BA developed thick roots after 4 
weeks. In Bosomnsia and Santom, 38 and 6% respectively developed thick roots.
Cultures were transferred to a hormone-free medium and root development occurred within 
seven (7) days in all the cultivars.
Symptoms of ACMV disease were observed on 25% of the plantlets of Santom (Plate 3) 
suggesting that meristem culture alone could not eradicate the virus. This observation was 
not found on the remaining cultivars. The ACMV symptoms were not observed in the other 
cultivars.
35
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Table 4. Effect of Medium Bon apical meristems of cassava cultivars at 4 weeks.
Cultivar BA mg/l No. of Explants Callus Roots3 (%) Shoots (%)
0.00 12 0 (0) 0 (0)
Ankrah 0.05 14 + 0 (0) 8 (57)
0.15 20 ++ 11 (55) 17 (85)
1.00 15 +++ 0 (0) 12 (80)
0.00 14 - 0 (0) 0 (0)
Bosomnsia 0.05 10 ++ 0 (0) 10 (100)
0.15 13 ++ 5 (38) 11 (85)
1.00 10 +++ 0 (0) 5 (50)
0.00 12 - 0 (0) 0 (0)
Santom 0.05 13 + 0 (0) 6 (46)
0.15 18 + 1 (6) 7 (39)
1.00 11 + 0 (0) 2 (18)
0.00 12 - 0 (0) 0 (0)
Biafra 0.05 12 ++ 0 (0) 7 (58)
0.15 10 +++ 0 (0) 9 (90)
1.00 12 +++ 0 (0) 9 (75)
0.00 12 - 0 (0) 0 (0)
Afisiafi 0.05 10 + 0 (0) 9 (90)
0.15 10 + 0 (0) 7 (70)
1.00 11 ++ 0 (0) 6 (5)
Note : - no callus, + low callus formation, ++ moderate callus, +++ high callus formation 
a: Number recorded after contaminated cultures had been discarded, 
b: figures in bracket indicate percentages
36
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Plate 3. In vitro plantlet of Santom showing symptoms of ACMV disease on leaf 
(arrowed)
37
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Apical meristems of M. Col. 22 and Biafra were induced to produce multiple shoots on an MS 
medium supplemented with 0.1 mg/l GA3, 0.1 mg/l NAA and varying concentrations of BA 
(0.0-2.0 mg/l). The objective of this experiment was to compare multiple shoot induction in a 
Latin-American cultivar (M. Col. 22) and a Ghanaian cultivar (Biafra). Table 5 shows the 
number of shoots or buds produced by the two cultivars. The two cultivars were cultured at 
different times.
In M. Col. 22, there was shoot induction on a hormone-free medium (control) as well as the 
treated media whereas in Biafra shoot induction occurred only on hormone-amended media. 
The number of apical shoots or buds produced by the two cultivars differed significantly (P< 
0.05) Appendix 3a, b. Also BA had a positive effect on the induction of multiple shoots from 
apical meristem of M. Col. 22 but not Biafra.
The number of apical shoots produced in M. Col. 22 correlated highly (r = 0.9) with the 
concentration of BA in the medium. Statistical analysis of individual data showed significant 
difference in the number of shoots produced between the controls and the BA treated 
media at P< 0.05 in both cultivars (Table 5, Appendix 4a). The highest number of shoots (7.0) 
was produced on a medium supplemented with 2.0 mg/l BA.
The highest number of buds (19) in M. COI. 22 was produced on a medium supplemented 
with 2.0 mg/l BA but this was not significantly different from the controls (Table 5, Appendix
3.5 Effect of BA on induction of multiple shoots from apical meristem.
38
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In Biafra, single shoots were produced irrespective of BA concentration in the medium. Also 
the number of buds produced in the BA-amended media did not differ significantly at P^0.05 
(Appendix 5, Table 5).
Table 5. Effect of BA on induction of multiple shoots and buds from apical 
meristems of M. Col. 22 and Biafra.
BA mg/l Mean number of apical shoots and buds
___________M. Col 22  Biafra
Apical shoots Buds Apical shoots Buds
0.0 1.0 a 9 a 0.0 a 0.0 a
0.1 3.0 ab 15 a 1.0 b 10.0 b
1.0 4.8 ab 16 a 1.0 b 9.0 b
2.0 7.0 c 19 a 1.0 b 12.0 b
Note: Numbers in a column followed by different letters are significantly different (P<0.05). 
M. Col 22 and Biafra were cultured at different times.
3.6 Effect of BA on induction of multiple shoots from single bud cuttings.
Single nodal explants were cultured on MS medium supplemented with 0.0-2.0 mg/l BA. The 
mean number of shoots and buds produced by the nodal cuttings are presented in Tables 6 
and 7 respectively. The results indicate that BA induced multiple shoots from buds in all the 
cultivars tested (Plate 4) but the number of shoots produced was dependent on the
39
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concentration of BA in the medium and the cultivar. Analysis of Variance (ANOVA) showed 
highly significant differences (P^0.05) between the number of shoots produced by the 
Ghanaian cultivars (Ankrah, Bosomnsia and Biafra) and the Latin American cultivar (M. Col. 
22) and also between the BA treatments (Appendix 6a).
In all the cultivars except Biafra, the optimum concentration of BA for highest number of 
shoot production was 0.1 mg/l BA. At this concentration, M. Col 22 produced 6.4 which was 
the highest whilst the local cultivar Ankrah produced 3.1 which was the lowest. Thus the later 
showed higher potential for multiple shoot production. The number of shoots produced was 
significantly different from the controls.
Statistical analysis did not show significant differences between the number of buds produced 
between the cultivars (Appendix 6b). Bud production in M. Col 22 was lowest on the control 
medium. However, Ankrah showed the highest bud formation on the same medium. On the 
whole, bud production on BA media was higher in M. Col 22 than in the African cultivars.
40
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Plate 4. Single nodal cutting of Biafra showing multiple shoots. 
Note five shoots from one explant.
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Table 6. Effect of BA on induction of multiple shoots from nodal cuttings of cassava 
cultivars.
BA mg/l Mean number of shoots produced by cultivars3
M. Col 22 Ankrah Bosomnsia Biafra
0.00 1.0 a 1.0 a 1.0 a 1 .0 a
0.02 - 1.5 ab 2.1 ab 1.6 a
0.10 6.4 c 3.1 c 3.8 c 2.0 a
1.00 3.9 b 2.8 be 3.3 ab 4.2 b
2.00 5.0 be * * *
Note a: * no shoots were produced at the time records were taken.
b: Numbers in a column followed by different letters are significantly different (P<0.05).
Table 7. Mean number of buds produced by cassava cultivars at 28 days of culture.
BA mg/l Mean number of buds produced by cultivars3
M. Col. 22 Ankrah Bosomnsia Biafra
0.00 4.9 a 9.1 a 5.1 a 7.8 a
0.02 - 6.3 a 7.9 ab 5.7 a
0.10 16.0 b 7.1 a 11.4 c 5.9 a
1.00 13.0 b 6.0 a 7.8 ab 8.8 a
2.00 13.0 b * * *
Note a: * no shoots were produced due to excessive callus formation.
b: Numbers in a column followed by different letters are significantly different (P<0.05).
42
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3.7 Effect of 2 ,4 -D on induction of somatic embryos from greenhouse-grown 
cassava.
Young leaf lobes of greenhouse-grown four cassava cultivars were cultured on MS medium 
supplemented with 2,4-D in the dark (induction medium, step one). After 21 days, the cultures 
were transferred to MS medium amended with 0.1 mg/l BA (maturation medium, step two) to 
study their embryogenic competence.
Within five days of culture on the induction medium (step 1) the explants developed 
abundant callus. However, on a hormone-free medium (control), only swellings were 
observed at the cut surfaces of the leaf lobes explants. Examination of the calli seven days 
later showed that they contained embryogenic tissue indicated by numerous globular 
structures. The calli were compact and creamy . When the data for percentage number of 
leaf lobes that callused were analysed, significant differences were detected between the 
cultivars and also between the 2,4-D treatments (Appendix 7, Table 8). Callus formation 
was significantly higher in Santom than from the remaining cultivars. Callus formation was low 
in Ankrah but not significantly different from Biafra and Bosomnsia.
The data for calli formation were also analysed separately for Ankrah, Bosomnsia and Biafra 
(Appendix 8a,b and c) and in each cultivar, there were significant differences between the 
controls and 2,4-D concentrations used (Table 9). For Ankrah, calli formation on explants 
cultured on 4 and 8 mg/l 2,4-D differed significantly. At 16 mg/l 2,4-D, all explants of the 
tested cultivars developed calli and in Santom all leaf explants formed calli independent of
2,4-D concentration in the induction (step one) medium.
43
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Table 8. Effect of 2,4-D on callus formation ofleaf lobes cultures
of different cassava cultivars from the greenhouse.
Cultivar Percentage calli formation ± SE
Ankrah 81.50 ±5.95 a
Biafra 90.89 ±4.18 ab
Bosomnsia 91.25 ± 2.95 ab
Santom 100.00 ± 0.00 c
Note a: Figures are means of four replicates.
b: Numbers in a column followed by different letters are significantly 
different (P<0.05).
Table 9. Effect of 2,4-D on callus formation of leaf lobe cultures of 
greenhouse grown cassava.
2,4-D concentration (mg/l Percentage calli formation
Ankrah Bosomnsia Biafra Santom
0 0.0 a 0.0 a 0.0 a 0.0 a
4 55.0 ± 2.04 b 92.0 ± 4.83 b 87.6 + 4.29 b 100.0 ± 0.00 b
8 89.5 ±4.11 c 81.8 ± 10.57 b 85.0 ± 5.93 b 100.0 ± 0.00 b
16 100.0 ± 0.00 c 100.0+ 0.00 b 100.0± 0.00 b 100.0 + 0.00 b
Note a: Figures are means of four replicates.
b: Numbers in a column followed by different letters are significantly different (P<0.05).
44
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Within 14 days of culture on the maturation medium, 36% and 4% of Santom and AFI calli 
respectively developed adventitious roots (Plate 5). The roots were initially white but became 
chlorophyllous after 28 days.
Within 21 days of culture on the maturation medium embryogenic tissues were overgrown by 
calli and consequently none could grow into a mature somatic embryo. Foliose structures 
however frequently developed on some calli indicating that leaf lobes used as explants were 
’ morphologically competent. After 28 days calli became brownish and senesced.
45
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Plate 5. Leaf lobe cultures of Santom showing calli with roots (arrowed).
46 \v
’ "-I, 1 ' t '  V  \fHV. •4,
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3.8 Induction of somatic embryos from leaf lobes, apical meristems and 
stipules from greenhouse plants.
In this experiment, young leaf lobes, apical meristems and stipules of greenhouse-grown 
cassava were cultured on 16 mg/l 2,4-D in order to identify other sources of explants for 
primary embryo production in cassava. Responses of explants following a 21-day incubation 
inthe dark on 16 mg/l 2,4-D are summarised in Table 10.
Within 15 days of culture on the induction medium, prolific callus formation occurred with 
globular structures appearing on apical meristem explants and on the adaxial surfaces of the 
leaf lobe explants of all the cultivars tested. Calli developed by apical meristem explants 
were friable, brownish and large in size compared to compact, creamy and small size calli 
developed by leaf lobe explants. Stipule explants developed swellings at the cut surfaces and 
only Santom and Afisiafi explants could form compact and creamy calli which were 
embryogenic in nature. Calli were however smaller in size compared to those obtained from 
explants of leaf lobes and apical meristems.
Statistical analysis showed that callus formation differed significantly (P<0.05) between 
explants (Appendix 9). All explants from apical meristems developed calli independent of 
the cultivar (Table 10, Appendix 10a and b). In Bosomnsia and Santom, the differences 
between leaf lobe and meristem explants were significant whereas for Ankrah, Biafra and 
Afisiafi they were not. Stipule explants of Santom developed the highest percentage calli 
(74%) but this was not found to be significantly different from the leaf lobe explants of the 
same cultivar.
When embryogenic calli were transferred to the maturation medium (step two) growth of calli 
continued without embryo maturation. Only one callus out of the 12 cultures developed
47
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cotyledonary stage embryo from apical meristem of Bosomnsia after 14 days of culture on the 
maturation medium.
There was no organ development in any of the cultures except in Santom, where one leaf 
lobe explant developed thick adventitious roots.
Table 10. Effect of 16 mg/l 2,4-D on callus formation from various explants
after 21 days culture in the dark.
Explants Percentage calli formation
Ankrah Bosomnsia Biafra Santom Afisiafi
Stipules 
Leaf lobes 
Meristem
0 ± 0.0 a 
94 + 4.0 b 
100 + 0.0 b
52 ±3.7 a 
72 ±13.9 ab 
100 ± 0.0 c
0 ± 0.0 a 
100 ± 0.0 b 
100 ± 0.0 b
74 ± 8.1 a 
94 ± 4.0 ab 
100 ± 0.0 c
10 +10.0 a 
100 + 0.0 b 
100 + 0.0 b
Note: Letters in a column followed by different letters are significantly different (P<0.05)
Figures are means of 5 replicates.
3.9 Induction of somatic embryos from leaf lobes of in vitro cassava plantlets.
Leaf lobes of all cultivars formed white creamy callus with globular structures at the adaxial 
surfaces within 15 days of culture on induction medium. Statistical analysis showed 
significant differences ( P ^ 0.05) between the cultivars and 2,4-D concentrations (Appendix 
11). However, analysis of individual data did not show significant differences between 2,4-D 
concentrations (Table 11). Leaf lobe explants cultured on 8 mg/l 2,4-D formed the highest 
percentage callus in all the tested cultivars but these were not significantly different from
48
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explants cultured on 4 or 16 mg/l 2,4-D (Table 11). On a hormone-free medium (control), 
leaf lobes formed swollen structures which did not result in callus formation. Results were 
therefore not included in the statistical analysis shown in Appendix 11 and Table 11.
For maturation of embryos, it was essential that embryogenic calli with globular embryos were 
transferred to MS supplemented with 0.1 mg/l BA and incubated in the light. After 21 days of 
transfer to the maturation medium globular embryos of Santom, Bosomnsia and Biafra 
cultured on 8 or 16 mg/l 2,4-D matured into embryos. During maturation, globular embryos 
passed through cotyledonary and torpedo stages before they matured (Plate 6,7). Figure 1 
shows percentage calli with matured somatic embryos after 21 days of incubation in light. 
Most of the globular embryos were overgrown by callus. In some cases callus developed 
foliose structures (Plate 8). The number of embryos formed were generally low. Also, most of 
the embryos isolated were malformed (Plate 8 ) except in Santom where all the embryos 
formed were normal (Plate 6).
Table 11. Effect of 2,4-D on callus production from leaf lobes explants in five cassava 
cultivars.
2,4-D (mg/l)1 Ankrah Bosomnsia Biafra Santom Afisiafi
4 90.75 + 9.2 a 85.07 ± 5.6a 76.77 + 10.1a 100.00 + 0.0a 65.17 + 3.7a
8 97.00 ± 3.0 a 100.00 ± 0.0a 92.75 ± 7.2 a 100.00 ± 0.0a 92.20 + 4.8a
16 93. 75 ± 6.2 a 97.00 ± 3.0a 91.65 ±8.3 a 100.00 ± 0.0a 89.40 ± 3.7a
Note 1: No callus formation at control treatments. Figures are means of four replicates. 
Figures in a column followed by the same letter are not significant.
49
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The highest number of matured embryos was isolated from Santom (25%) when cultured on 
16 mg/l 2,4-D (Figure 1). On this same treatment, only 9% embryogenic calli of Biafra 
matured into somatic embryos.
Bosomnsia, Biafra and Santom produced 18.6, 6.3 and 14.2% matured embryos respectively 
on a medium supplemented with 8 mg/l 2,4-D (Figure 1). Ankrah could form mature embryos. 
Mostly, single embryos were obtained with one embryo per callus.
50
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Pe
rc
en
ta
ge
 
ca
lli
 w
ith
 
so
m
at
ic
 
em
br
yo
s 
(%
)
35 j  
30 - 
25
2,4-D concentration (mg/l) ' ■  4 e  8 mi6
20
Ankrah B. nsia Biafra Santom Afisiafi
Cultivar
Figure 1. Percentage of callus which formed somatic embryos 
after 21 days of culture in maturation mediumin light. 
B. nsia = Bosomnsia. '
51
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Root formation was also promoted within 14 days of transfer to the maturation medium but 
was dependent on the cultivar and the concentration of 2,4-D on the induction medium. 
Figure 2 shows root formation from calli 21 days after transfer to maturation medium. 
Generally, root induction decreased with increased concentration of 2,4-D in the induction 
medium. With the exception of Bosomnsia, 4 mg/l 2,4-D enhanced root development in all 
the cultivars. Ankrah developed the highest percentage of roots (63.5%) followed by Afisiafi 
(27.6%) and Bosomnsia (28.2%). In Bosomnsia, the highest percentage of roots was 
induced on 8 mg/l 2,4-D. Morphologically, the roots were thin and adventitious in nature (Plate 
9) with two to four roots per callus.
52
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Pe
rc
en
ta
ge
 
ro
ot 
fo
rm
at
io
n 
(%
)
Ankrah B.nsia Biafra Santom Afisiafi
Figure 2. Percentage of callus which formed roots 
after 21 days on maturation
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Plate 6. Callus of Santom with matured somatic embryo (arrowed).
Plate 7. Callus of Santom with matured somatic embryo (a). Note foliose structures (b) 
at the base of embryo.
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Plate 8. Callus of Bosomnsia with malformed somatic embryos (a) and foliose structures (b
55
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3.9.1 Embryo germination
The use of somatic embryogenesis as an efficient system of propagation will be fully realised 
if somatic embryos regenerate into plants. Germination of mature somatic embryos were 
studied using Adira 4, Gading, Line 11 and TMS 90853 .
3.9.2 The effect of different desiccation methods on embryo germination.
Different methods of desiccation were used to stimulate germination of NAA-induced somatic 
embryos of Adira 4. As initial experiments have indicated that sugar solutions above 20 g/l 
have adverse effect on germination of somatic embryos, optimal concentrations only were 
recorded in Table 12. After 21 days in culture non-desiccated embryos did not germinate into 
complete plants shoots and roots. Only 17% and 3% of the cultured embryos developed into 
either roots only or shoots only respectively.
Embryos desiccated using the petri dish method lost 40% of their fresh weight as moisture 
while those desiccated using glycerol lost 55%. In these two treatments, 88% of cultured 
embryos germinated into complete plants with roots and shoots. Only 4% of the embryos 
developed roots only.
In the desiccation method using sugar solutions and mannitol, the moisture loss could not be 
determined exactly. At lower concentrations of sugars, the embryos continue to grow, 
indicated by an increase in fresh weight. When higher concentrations of sugars were used 
embryo growth ceased and the cotyledons became yellowish. The highest percentages of 
shoot formation (with or without roots) were found in the treatments without or with relatively 
lower sugar concentrations (10 or 20g/l). In these optimal treatments, shoot formation was
56
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higher than in non-desiccated embryos (control) but lower than petri dish desiccated embryos. 
The highest shoot formation (68%) among the optimal treatments occurred on liquid MS 
medium with 20 g/l sucrose with further one week desiccation. Most of the shoots formed 
were malformed and did not possess roots.
Table 12. Effect of different desiccation methods on somatic embryo germination. 
(Optimal concentrations only indicated).
Method of desiccation
No.of SEs 
cultures
Roots 
only1 (%)
Shoots 
only (%)
Germination
(%)
without desiccation (control) 30 5 (17) 1 (3) 0 (0)
petri dish 17 0 (0) 2 (12) 15 (88)
desiccator + glycerol 25 1 (4) 1 (4) 22 (88)
Solid MS + sucrose (20 mg/l) 78 2 (3) 27 (35) 5 (6)
liquidMS + sucrose (20mg/l) - desiccation 19 0 (0) 11 (55) 0 (0)
liquidMS + sucrose (20mg/l)+ desiccation 19 0 (0) 13 (68) 3 (16)
liquidMS -desiccation 15 0 (0) 6 (40) 1 (7)
liquid MS + desiccation 19 1 (5) 12 (63) 1 (5)
water + sucrose (30 mg/l) - desiccation 20 0 (0) 5 (25) 1 (5)
water + desiccation 19 1 (5) 1 (5) 17(89)
Note 1: Figures in bracket indicate percentages. Ses = somatic embryos.
Embryos were cultured for 28 days on MS supplemented with 1 mg/l BA and 
incubated in the light for germination.
57
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3,9.3 Effect of ABA pre-treatment on embryo germination.
The effect of abscissic acid (ABA) on the germination of NAA-induced somatic embryos of 
Adira 4 was studied. Mature NAA-induced somatic embryos transferred to different 
concentrations of ABA (0-40 mg/l) were either cultured directly or desiccated before culturing 
on MS medium with 0.0-4.0 mg/l BA. After 7 days the cotyledons of the embryo grew very 
large with deep green coloration. With increased concentration of ABA (40 mg/l) there was no 
growth and the embryos turned yellowish. The results are as indicated in Table 13a.
Embryos pre-treated with ABA but without desiccation did not stimulate germination. Only 
13% of the embryos pre-treated with 1 mg/l ABA germinated into shoots ( without roots) 
when cultured on 0.1 mg/l BA.
Embryos which had been pre-treated with ABA and desiccated showed higher organ 
development than non-desiccated pre-treated embryos. With desiccation, germination and 
organ (root) development generally were higher in the control than in the ABA treated 
cultures (Table 13b). However, shoot development was comparatively higher when embryos 
were pre-treated with 1 mg/l ABA (Table 13b)
High ABA concentration of 40 mg/l inhibited both shoot or root formation. Only 7% showed 
shoot development on 0.5 and 1.0 mg/l BA compared with 13 and 28% in the control.
58
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Table 13a. Effect of ABA pre-treatment (without desiccation) on embryo germination.
ABA mg/l BA mg/l No. of SEs 
cultured.
Roots only(%) Shoots only (%) Germination
(%)
0.0 16 0 1 (6) 0
0.1 15 0 2 (13) 0
0.0 0.5 23 0 5 (22) 0
1 .0. 13 0 0 (0) 0
4.0 22 0 6 (27) 0
0.0 15 0 0 (0) 0
0.1 16 0 2 (13) 0
1.0 0.5 - - - -
1 .0. 16 0 0 (0) 0
4.0 15 0 1 (7) 0
0.0 15 0 0 (0) 0
0.1 15 0 0 (0) 0
40 0.5 16 0 0 (0) 0
1 .0. 15 0 0 (0) 0
4.0 15 0 0 (0) 0
Note: Figures in parenthesis indicate percentages.
Embryos were pre-treated with different concentrations of ABA for seven days and 
cultured for germination on 0-4 mg/l BA.
Ses = somatic embryos.
59
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Table 13b. Effect of ABA pre-treatment (with desiccation) on embryo germination.
ABA mg/l BA mg/l No. of SEs 
cultured
Roots only (%) Shoots only
(%)
Germination (%)
0.0 15 3 (20) 0 (0) 3 (20)
0.1 16 5 (31) 3 (19) 5 (31)
0.0 0.5 15 5 (33) 2 (13) 6 (40)
1.0 18 2 (11 ) 5 (28) 9 (50)
4.0 15 4 (27) 1 (7) 5 (33)
0.0 15 0 (0) 0 (0) 0 (0)
0.1 16 0 (0) 3 (19) 0 (0)
1.0 0.5 15 7 (7) 10 (67) 1 (7)
1.0 15 1 (7) 2 (13) 3 (20)
4.0 15 0 (0) 7 (47) 0 (0)
0.0 15 0 (0) 0 (0) 0 (0)
0.1 15 0 (0) 0 (0) 0 (0)
40 0.5 15 0 (0) 1 (7) 0 (0)
1.0 15 0 (0) 1 (7) 0 (0)
4.0 15 0 (0) 0 (0) 0 (0)
Note: Figures in bracket indicate percentages
Embryos were pre-treated with ABA for seven days and desiccated for a further one 
week before culture for germination.
Ses = Somatic embryos.
60
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3.9.4 Influence of auxins and cytokinins on embryo germination
Table 14 shows the effect of auxins and cytokinins on somatic embryo germination. The 
embryos used in this experiment lost 20% moisture from their fresh weight. None of the 
embryos cultured on hormone-free medium could form shoots and only s % formed roots .
The auxins IBA and NAA stimulated root formation with NAA being more effective. More than 
55% of the embryos formed roots at all the NAA levels tested. At 4.0 mg/l NAA 82% of the 
embryos formed roots. Thirteen (13%) and 18% of the embryos developed into whole plants 
when cultured on 0.1 and 4.0 mg/l NAA. The roots were numerous and thin.
All the cytokinins tested stimulated higher shoot formation compared to the auxins. The 
highest percentage of shoot development was observed on the BA supplemented media. The 
optimal BA concentration was 1.0 mg/l. At this concentration, 67% of the cultured embryos 
developed shoots without roots and 10% germinated into plants with roots. The morphology 
of the roots and shoots of plants was dependent on the concentration of BA in the medium. 
Embryos cultured on 1.0 or 4.0 mg/l BA formed thick roots. The shoots stout (had short 
internodes) with many branched (Plate 10).
With kinetin, germination increased with higher concentrations. On a medium supplemented 
with 4 mg/l kinetin, 36% of the embryos germinated and 18% formed only shoots. 
Morphologically, the roots were all thin and normal while the shoots had more than one 
apical meristem each (Plate 9).
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Thiadizuron (TDZ) gave the lowest percentage germination; at all the concentrations tested 
not more than 12% germinated into complete plants.
Table 14. Influence of different growth regulators on shoot and root formation of Adira 
4 somatic embryos.
Growth regulator concentration No of SEs Roots only (%) Shoots only Germination
(mg/l) cultured (%) (%)
Control 0.0 20 1 (5) 0 (0) 0 (0)
0.1 25 2 (8) 2 (8) 3 (12)
Thiadiazuron 1.0 24 1 (4) 1 (4) 2 (8)
(TDZ) 4.0 23 4 (17) 3 (13) 1 (4)
0.5 24 1 (4) 0 (0) 2 (8)
Kinetin 1.0 20 0 (0) 1 (5) 1 (5)
4.0 22 0 (0) 4 (18) 8 (36)
0.5 15 0 (0) 6 (40) 2 (13)
Benzyladenine 1.0 10 1 (10) 14 (67) 3 (10)
(BA) 4.0 22 0 (0) 7 (32) 2 (13)
0.1 15 9 (60) 0 (0) 2 (13)
Napthalene 1.0 20 11 (55) 0 (0) 0 (0)
acetic acid(NAA) 4.0 11 7 (64) 0 (0) 2 (18)
1.0 20 3(15) 0 (0) 0 (0)
Indole butyric 5.0 10 3 (30) 0 (0) 0 (0)
acid (I BA) 10.0 15 0 (0) 0 (0) 0 (0)
Note 1: Figures in bracket indicate percentages. 
SEs = Somatic embryos.
62
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Plate 9. Germinated somatic embryos of Adira 4 on MS medium with 4 mg/l kinetin. 
Note the slender stems and thin roots.
63
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Plate 10. Germinated somatic embryos of Adira 4 on MS medium with 1 mg/l BA. 
Note thick roots and multiple shoots (ft Z*s).
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3.9.5 Relation between moisture loss and BA on embryo germination.
Figure 3 shows the relationship between moisture loss and BA on embryo germination in 
Gading (A,B,C) and Adira 4 (D,E,F). In both cultivars, embryo germination was dependent on 
moisture loss and the concentration of BA in the cultured medium (Figure 3). Embryos with a 
moisture loss of 20% did not germinate into complete plants when cultured on a hormone-free 
medium (Figure 3A,D). Three percent of the Gading somatic embryos developed only roots 
while 11% developed only shoots (Figure 3A). For Adira 4 somatic embryos, 17% developed 
roots (Figure 3D). Somatic embryos of Gading with a moisture loss of 20% gave the highest 
percentage (31%) shoot development on a medium with 1 mg/l BA (Figure 3A). For Adira 4, 
the highest was on a medium with 4 mg/l BA (Figure 3D).
In both cultivars, embryos with a moisture loss of 40% gave a higher percentage of complete 
plant germination (Figure 3B,E). In Gading, formation of roots was comparably higher at all 
the BA treatments than when embryos lost 20% moisture. The highest germination of 
somatic embryos 72% and 82% in both Gading and Adira 4 respectively occurred on 0.1 
mg/l BA (Figure 3B,E).
In Gading, shoot development at 75% moisture loss was comparatively higher on all BA 
treated media than when embryos had lost 20% moisture. In Adira 4, complete plant 
germination from embryos with moisture loss of 75% was not comparatively better than when 
embryos had lost 20% of their moisture (Figure 3F and 3D).
65
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% 
ge
rm
in
at
io
n
Gading
■  roots S  shoots ■germination
A 20%
0.1 1 
BA concentration
0.1 1 
BA concentration
o3
C 75%
0.1 1 
BA concentration
100 — 
80 4 
60 -  
40 
20 
0
Adira 4
D 20%
0 0.1 4
BA concentration
0.1
BA concentration
0.1
BA concentration
Figure 3. Effect of moisture loss and different BA concentrations on germination of somatic embryos of Gading (A,B,C) and Adira 4 
(D,E,F)
6 6
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3.9.6 Effect of prewashing of somatic embryos on germination.
In the earlier experiments, somatic embryo germination into complete plants was below 
90%. This experiment was conducted with the aim of improving the frequency of somatic 
embryo germination. Embryos of Gading were therefore transferred to sterile distilled water 
for periods of 1 , 5 , 10 or 14 days and then cultured directly or desiccated for a further one 
week before culture. The results obtained are presented in (Table 15a).
Embryos washed for one or five days without further desiccation did not enhance germination 
into whole plants nor development of roots only. Shoot formation only was stimulated at day 
10 and 14. The highest shoot formation ( 29%) occurred at 14 days of washing on a 
medium supplemented with 1.0 mg/l BA.
Root and shoot formation were comparatively higher when washed embryos were 
desiccated. On hormone-free media, root formation increased with period of washing. The 
highest percentage (67%) of root development occurred in the treatment that involved 10 
days of washing. Root formation on media supplemented with various concentrations of BA, 
in contrast, decreased with the days of washing.
Germination of embryos into whole plants did not follow any obvious trend but was higher 
when embryos were washed for 1 day (Table 15b). At one or five days of washing, the 
optimal BA concentration needed for whole plant development was 0.1 mg/l BA whereas at 10 
and 14 days, it was 1.0 mg/l. The highest percentage germination (47%) occurred at day 1 
on a medium supplemented with 0.1 mg/l BA and the lowest at day 14.
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Table 15a. Effect of prewashing and BA concentrations on germination of Gading
somatic embryos ( without desiccation).
Period of BA (mg/l) No. of SEs Roots only Shoots only Germination
Washing (days) cultured (%) (%) (%)
0.0 20 0 (0) 0 (0) 0 (0)
1 0.1 26 0 (0) 3 (12) 1 (4)
1.0 26 0 (0) 2 (8) 0 (0)
4.0 30 0 (0) 2 (7) 0 (0)
0.0 20 0 (0) 0 (0) 0 (0)
5 0.1 19 0 (0) 0 (0) 0 (0)
10 20 1 (5) 1 (5) 0 (0)
4.0 21 0 (0) 0 (0) 0 (0)
0.0 31 0 (0) 1 (3) 0 (0)
10 0.1 28 0 (0) 2 (7) 6 (21)
1.0 31 1 (3) 6 (19) 0 (0)
4.0 28 0 (0) 2 (7) 1 (4)
0.0 22 0 (0) 0 (0) 0 (0)
14 0.1 26 2 (8) 1 (5) 2 (8)
1.0 24 0 (0) 7 (29) 1 (4)
4.0 24 0 (0) 3 (13) 1 (4)
Note: Figures in brackets indicate percentages. 
SEs = somatic embryos.
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Table 15b. Effect of prewashing and BA concentrations on germination of
desiccated somatic embryos of Gading.
Period of BA No. of SEs Roots Shoots only Germination
Washing (days) (mg/l) cultured only(%) (%) (%)
0.0 36 0 (0) 0 (0) 0 (0)
1 0.1 36 11 (31) 1 (3) 17 (47)
1.0 48 14 (29) 9 (19) 18 (38)
4.0 28 4 (14) 5 (18) 12 (43)
0.0 27 6 (22) 0 (0) 0 (0)
5 0.1 27 8 (30) 0 (0) 4 (15)
1.0 36 11 (31) 7 (19) 2 (6)
4.0 28 0 (0) 3 (10) 0 (0)
0.0 30 20 (67) 3 (10) 0 (0)
10 0.1 31 5 (16) 6 (19) 6 (19)
1.0 23 4 (17) 3 (13) 9 (39)
4.0 39 4 (10) 15 (38) 4 (10)
0.0 31 20 (65) 6 (19) 0 (0)
14 0.1 26 5 (19) 2 (6) 2 (8)
1.0 24 2 (8) 8 (33) 1 (4)
4.0 32 0 (0) 14 (44) 1 (4)
Note: Figures in bracket indicate percentages 
SEs = somatic embryos
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3.9.7 Influence of light/dark regimes on plantlet development from somatic 
embryos.
Desiccated NAA-induced embryos of TMS 90853 (Figure 4A), Gading (Figure 4B) and Line 
11 (Figure 4C) germinated at higher frequencies than 2,4-D-induced embryos of the same 
cultivars (Figure 4 A,B and C). This was observed independent of the light/dark conditions 
and the concentration of BA used in the germination medium. For Adira 4 (Figure 4D) only 
NAA-induced embryos cultured on 0 and 0.1 mg/l BA germinated at a higher frequency than
2,4-D-induced embryos. On a medium with 1 and 4 mg/l BA, there were no differences 
(Figure 4D).
In all four genotypes, NAA-induced embryos cultured in the dark showed the highest 
percentage germination with roots in a medium supplemented with 0 or 0.1 mg/l BA and this 
was higher than in the light. Under dark conditions, root development occurred within 4 days 
followed by the elongation and the curvature of the hypocotyl and finally the opening of the 
hypocotylar hook after 10 days. The stems of the shoots were slender with long internodes. 
Embryos cultured on control or 0.1 mg/l BA formed single shoots mostly (Plate 11) whereas 
those cultured on 1 mg/l BA formed mostly two or more shoots (Plate 12). The shoots were 
etiolated but de-etiolation occurred seven days after transfer to the light.
Germination of 2,4-D-induced embryos was low in TMS 90853 (Figure 4A), Gading (Figure 
4B) and Line 11 (Figure 4C) when cultured under both light and dark conditions. In Adira 4, 
40% to 50% of the embryos cultured in the light and on a medium with 1 or 4 mg/l BA 
germinated. This was higher than comparable treatments in the dark.
Desiccation of 2,4-D-induced embryos had a clear effect on root development in all 
genotypes. The type of roots appeared four to seven days after culture and they were
70
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dependent on the light/dark conditions; predominantly adventitious in light and taproots in the 
dark. Also there was comparatively higher root formation in 2,4-D-induced than NAA- 
induced somatic embryos incubated in the dark than light in all cultivars (Figure 5). This 
observation was more profound in Line 11 embryos.
71
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TMS 90853
100
BA concentration (mg/l)
Gading
100
v?o'
c0
‘-Mroc
1
tuO)
1 4 0 0.1 1
BA concentration (mg/l)
BA concentration (mg/l)
Adira 4
100
90
80
70
60
50
40
30
20
10
0
NAA
1
I Dark EH Light :
2,4-D
0 0.1 1 4 0 0.1
BA concentration (mg/l)
Figure i f  Influence of light/dark conditions on germination of NAA-and 2,4-D-induced somatic embryos of TMS 90853, 
Gading, Line 11 and Adira 4.
72
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Pe
rc
en
ta
ge
 
ro
ot 
de
ve
lo
pm
en
t 
(%
) 
j 
Pe
rc
en
ta
ge
 
ro
ot 
de
ve
lo
pm
en
t 
(%
)
100
Line 11 ■ Dark 11 Light
NAA 2,4-D
1 4 0 0.1
BA concentration (mg/l)
Adira 4 ■ Dark H  Light
2,4-D
1 4 0 0.1 1
BA concentration (mg/l)
Figure 5. Influence of light/dark conditions on root formation of NAA- and 2,4-D - induced somatic embryos of TMS 90853, 
Gading, Line 11 and Adira 4
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Plate 11. Germinated somatic embryos of Adira 4 cultured on 0.1 mg/l BA in the dark. 
Note single shoots emerging from embryos ( *  1*$.
Plate 12. Germinated somatic embryos of Adira 4 cultured on 0.1 mg/l BA in the dark. 
Note multiple shoots emerging from embryos
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3.9.8 Influence of different light intensities on embryo germination.
Since germination of some seeds are sensitive to light intensities, it was suggested that 
germination of somatic embryos might also be influenced by light intensities. To test this 
assumption, NAA-induced embryos of Adira 4 were desiccated for 7 days and cultured on 
media supplemented with 0-4 mg/l BA. The cultures were incubated at different light 
intensities as indicated in Figure 6.
The optimum light intensity for whole plant germination was 64 lux and complete plant 
formation was higher at all the BA levels investigated. At 64 lux, the optimal BA 
concentration for germination was 1.0 mg/l and percentage germination was 73%. 
Morphologically, the shoots were all etiolated. However, they became green 7 days after 
transfer to growth room light conditions.
The formation of shoot only was generally low at all the light intensities compared to either the 
formation of root only or the formation of root with shoot.
When compared to the control (0 lux), root formation at all the light intensities was high when 
the embryos were cultured on a hormone-free medium. At 4 mg/l BA, no root only 
development was observed at all the light intensities except 64 lux. The highest percentage 
(65%) of root only formation occurred at a light intensity of 6410 lux when embryos were 
cultured on a hormone- free medium.
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% 
ge
rm
in
at
io
n 
% 
ge
rm
in
at
io
n
■  ROOTS S SHOOTS ■  GERMINATION!
0.1 1 4
BA concentration (mg/l)
0.1 1 4
BA concentration (mg/l)
0.1 1 4
BA concentration (mg/l)
0 0.1 1 4
BA concentration (mg/l)
, I 100
c 80 -
.2
ire 1 
£  | 60 -
E
a i 40 -1 O)
20 -
1 0 -
6410 lux
L
0 0.1 1 4
BA concentration (mg/l)
Figure 6. Effect of different lightintensities dn root, shoot and plant germination in Adira 4 somatic embryos.
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Four grams of matured somatic embryos of Adira 4 were transferred to sterile petri dishes 
and incubated in white, red, blue, far red and dark cabinets for 7 days and the results 
obtained are as shown in Figure 7.
In white and blue light there was no germination of embryos into roots, shoots nor complete 
plants on a hormone free - medium. Only seven (7) percent of the embryos incubated under 
blue light developed roots. In the hormone-supplemented media, germination of embryos into 
complete plants was below 20% under both light regimes. The optimal BA concentration for 
germination was 1 mg/l under both light regimes.
Cultures incubated under red, far red and dark cabinets showed higher germination 
compared to blue and white light. The highest percentage germination (40%) of embryos into 
complete plants was observed under dark conditions on 1.0 mg/l BA medium. Under far red 
light conditions, however, the highest germination (37%) was observed when embryos were 
cultured on 0.1 mg/l BA amended medium. The development of shoots only was higher in far 
red and dark regimes than red light.
Morphologically, embryos incubated under far red and dark regimes produced plantlets 
which were normal, single and etiolated.
3.9.9 Effect of different light regimes on somatic embryo germination.
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□  roots 0 shoots ■germ ination
Blue red
50
c  400
1 3 0
c
1 20
o
?10ss
0 J
0.10 0.1 1 
BA concentration (mg/l) BA concentration (mg/l)
white
------- H
0 0.1 1 
BA concentration (mg/l)
50 f a r r e d
BA concentration (mg/l)
Dark
50 T
c
0 0.1 1
C BA concentration (mg/l) OS.
Figure 7. Effect of different light regimes on root, shoot ana plant germination of Adira 4 somatic embryos.
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3.9.10 Germination of embryos desiccated in complete darkness or light.
Mature NAA-induced somatic embryos of Adira 4 desiccated in complete darkness (CD) for 7 
days were cultured for germination and incubated in complete darkness and in the light. The 
results are presented in the Table 16.
Desiccation of embryos in the light (L) followed by culture in complete darkness (CD) 
enhanced formation of roots as well as whole plant germination. Whole plant germination was 
high (46%) on a medium with 0.1 mg/l BA whereas the highest roots only formation (61%) 
occurred on a medium with 1.0 mg/l BA. There was no shoot only formation except on a 
medium with 0.1 mg/l BA.
When embryos were desiccated in complete darkness (CD) followed by culture in the light (L) 
whole plant germination decreased (Table 16). Only 13% of the embryos germinated on a 
medium with 1.0 mg/l BA.
Desiccation of embryos in complete darkness followed by culture in complete darkness had 
a negative effect on shoots only formation. In this treatment the highest number of shoots 
only was 5% and was obtained when embryos were cultured on a hormone-free medium. 
When embryos were cultured on BA-supplemented medium only 3% of the embryos formed 
shoot on a 1 mg/l BA-amended medium. Also, the highest whole plant germination was 
20% and this occurred on 0.1 mg/l BA amended medium. On a medium with 4.0 mg/l BA 
there was neither root nor shoot development. The highest percentage (28%) of root only 
formation occurred when embryos were cultured on a medium amended with 1.0 mg/l BA.
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Table 16. Effect of desiccation in complete darkness and light on germination
of Adder 4 somatic embryos.
BA (mg/l) Desiccation
treatment
Incubation
treatment
No.of
SEs
Roots only
(%)
Shoots 
only (%)
Germination
(%)
0.0 CD CD 20 3 (15) 1 (5) 0 (0)
0.1 CD CD 30 5 (17) 0 (0) 6 (20)
1.0 CD CD 29 8 (28) 1 (3) 3 (10)
4.0 CD CD 31 2 (6) 0 (0) 0 (0)
0.0 CD L 28 0 (0) 1 (4) 0 (0)
0.1 CD L 27 4 (15) 3 (11) 0 (0)
1.0 CD L 32 6 (19) 3 (9) 4 (13)
4.0 CD L 32 1 (3) 0 (0) 0 (0)
0.0 L CD 35 11 (33) 0 (0) 5 (14)
0.1 L CD 35 10 (28) 3 (9) 16 (46)
1.0 L CD 36 22 (61) 0 (0) 2 (6)
4.0 L CD 31 0 (0) 0 (0) 0 (0)
Note: Figures in parenthesis indicate percentages. 
CD = complete darkness; L = light
80
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CHAPTER FOUR
4.0 DISCUSSION AND CONCLUSION
4.1 Germplasm collection.
The germplasm survey indicated that farmers in the Dodowa, Kwabenya, Kasoa and North 
Taifa cultivate cassava cultivars principally based on early bulking, yield and the use to 
which they are put. Consequently, a wide range of cassava with tolerance to ACMV are 
cultivated. In this study, over 12 descriptive names of cassava cultivars with different 
morphological traits were identified. This result agrees with findings by Annor-Frimpong 
(1994) who reported that farmers in the Central and Western regions of Ghana cultivate 
different cultivars. Iweke et al., (1994) collected over 1200 descriptive names of cassava from 
East and West African farmers in their Collaborative Study of Cassava in Africa (COSCA) 
project suggesting that farmers in various locations of Africa cultivate more than one cassava 
cultivar. They however pointed out that such descriptive names cannot be used for botanical 
classification because the names vary from village to village. In this study, it was observed 
that the cultivar Bosomnsia had different names associated with the villages of origin. A 
more detailed study using molecular markers such as Restriction Fragment Length 
Polymorphism (RFLP) or Random Amplified Polymorphic DNA (RAPD) may be used to 
ascertain their relationships and botanical classification.
The results obtained in this study show that ACMV and brown leaf spot disease were the 
most common disease of the cultivars collected. The results further show that ACMV disease 
affects all the cultivars popularly cultivated by farmers in Ghana. Hahn et al. (1989) has 
reported that ACMV is the most widespread and economically important disease in West
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Africa and can cause up to 95% yield loss in most susceptible cultivars. It has also been 
reported that the disease is caused by three related geminiviruses which occur in West Africa, 
East and Southern Africa and India (Hahn et al., 1989). Its spread may be due to lack of 
resistant cultivars. Intensive breeding programmes should be initiated by research institutions 
and the universities to produce resistant cultivars for farmers as ACMV has serious effect on 
yield. Secondly, a wider survey covering all the cassava growing regions should be 
conducted in order to identify landraces which are tolerant to the disease and can serve as 
bases for the improvement of the crop.
4.2 Meristem culture
In this study, Medium B with 0.5 or 0.15 mg/l BA and a lower concentration NAA (0.02 mg/l) 
gave much higher shoot formation except in Santom than Medium A with the higher 
concentration of NAA. (0.1 mg/l ). There was 100% shoot formation in Bosomnsia when 
Medium B was used but most of them had no roots. The results obtained in Medium B was in 
agreement with that of Adejara and Coutts (1981) who obtained 100% shoot formation in all 
their tested cassava cultivars cultured on 0.02-0.2 mg/l BA in combination with 0.02 mg/l 
NAA. Work done at CIAT with over 300 cultivars has shown that shoots do not form roots on 
a medium amended with 0.01-0.1 mg/l BA and 0.02-0.04 mg/l NAA and 0.04 mg/l GA3. 
(Raemakers et al., 1997). A second step is therefore necessary for rooting of the shoots thus 
making the procedure more labour intensive. This is similar to observation in this study 
whereby on Medium B high percentage of the shoot formed had no roots.
The high concentration of NAA in Medium A caused high callus formation. According to 
Raemakers et al., (1997) NAA at concentrations higher than 0.02 mg/l decreased shoot
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formation but increased callus formation. Excessive callus formation during shoot formation 
can cause somaclonal variation. Therefore, this medium cannot be used for clonal 
multiplication. However, for breeding purposes somaclonal variation may result in the 
production of desirable mutants.
Visual observation of the plantlets produced from the meristem culture showed conspicuous 
symptoms of ACMV on some leaves of Santom cultures despite the fact that cultures were 
derived from meristem explants. Meristem culture technology has been used to free 
vegetatively propagated crops from viruses (Hartmann et al., 1990). Kartha (1981) reported 
that potato, sweet potato, yam and cassava have been freed of viral pathogens by meristem 
culture. This is in contrast with the observations made in Santom in this study. However, the 
observation made on Santom is similar to the report of Mellor and Stace-Smith (1977) that 
potato leaf roll virus (PLRV) and virus Y (PVY) could not be eliminated by meristem culture 
alone. Virus elimination by meristem culture depends on the size of the meristem cultured and 
host -virus combinations ( concentration of virus) (Kartha, 1981). High concentration of the 
virus in the stake of Santom could possibly explain its presence in the in vitro plantlets. It has 
been suggested that in cases where viruses are difficult to eliminate by meristem culture 
alone, a combination of thermotherapy and meristem culture has proved beneficial. Kartha 
and Gamborg (1975) reported that mosaic disease of cassava and seed-borne mosaic virus 
in pea were eliminated in 60% and 90% frequency respectively by culturing meristems not 
exceeding 0.4 mm in length whereas 100% mosaic-disease-free plants were regenerated 
from cassava meristems grown at 35°C for 30 days prior to meristem excision. Kaiser and 
Teemba (1979) also reported that donor explants of F 29 cassava cultivars treated to high 
temperatures (35-40 °C) freed them from brown streak virus in East Africa. In Canada,
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meristem culture was combined with heat therapy to collect a virus-free germplasm of potato 
(Wright, 1988).
Enzyme-linked immunosorbent assay (ELISA) could be an effective method to detect the 
presence or absence of the ACMV virus in the remaining cultivars.
4.3 Multiple shoot induction
Both in vivo and in vitro cassava exhibit strong apical dominance. Breaking of this apical 
dominance results in the production of multiple shoots. In M.Col 22 multiple shoots were 
produced from apical meristems as well as nodal cuttings and the number of shoots produced 
depended on the BA concentration in the culture medium. In Ankrah, Biafra and Bosomnsia 
multiple shoots were produced from single nodal cuttings alone. The observation in this study 
is similar to the work of Roca (1984) and Smith et al., (1986). Roca (1984) produced multiple 
shoots from nodal cuttings of cassava on a medium amended with only 1.0 mg/l BA. 
According to his report about 20 shoots and nodal cuttings were harvested every three 
weeks from the rosette multiple shoot culture but the number decreased with time. For 
rooting the harvested nodes were cultured in a liquid medium supplemented with 0.05 mg/l 
BA, 0.04 mg/l GA3 and 0.02 mg/l NAA Smith et al., (1982) also produced multiple shoots 
from single bud cuttings and between 2-6 apical shoots and 4-19 buds were produced 
depending on the cultivar. The results of this study and the above observations shows that 
multiple shoot production is dependent on BA concentration in the medium as well as the 
cassava cultivar.
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Leaf lobe explants from from greenhouse and in vitro plantlets produced embryogenic calli on 
2,4-D-amended step one-induction medium. However, only embryogenic calli from in vitro 
plantlets produced matured somatic embryos on step two maturation medium. Embryogenic 
tissues obtained from in vitro leaf lobes matured into somatic embryos on a second-step 
maturation medium while those from greenhouse grown explants did not, indicating that the 
source of donor explant plays a great role in embryo production.
The failure of the greenhouse derived embryogenic calli to produce somatic embryos may 
have been caused by the rigorous sterilisation procedure applied to greenhouse grown 
explants. According to Raemakers et al., (1993b), rigorous sterilisation procedures applied to 
donor explants had damaging effects on the tissues and subsequent somatic embryo 
production. It is also possible that the growth conditions of the donor plants were 
unfavourable for subsequent somatic embryogenesis.
There was excessive callus production when embryogenic calli derived from leaf lobes of both 
greenhouse grown cassava and in vitro cassava plantlets were transferred to the second step 
medium which contained BA. According to Raemakers et al. (1997) excessive callusing tend 
to have a negative effect on further development of embryogenic tissue. It was explained that 
during callusing the embryogenic tissue itself can proliferate into callus. Also, many of the 
embryogenic tissue may become arrested by the proliferating callus. This means that only a 
fraction of the globular embryos in the embryogenic calli would develop into torpedo - shaped 
embryos.
4.4 Primary somatic embryogenesis
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Matured embryos isolated from the explants were few in number. In most cultures, only one 
embryo per callus was obtained. Such low frequency of embryo production has been 
reported by many researchers. Finer (1995) has also observed that somatic embryo 
formation from cotyledon explants in soybean and maize occurred at very low frequency. In 
cassava, Raemakers (1993) reported that embryo production in eight Nigerian genotypes was 
very low and varied from 0.1 to 1.1 embryo per explant. Excessive callusing in the maturation 
medium (step two) could possibly explain the low frequency of embryo production in this 
study.
Results of this study seem to suggest that embryo production was dependent on the cassava 
cultivar and 2,4-D concentration in the step one medium. Of five local cultivars tested, four 
formed somatic embryos. Afisiafi did not produce somatic embryos on any of the media 
tested. Somatic embryogenic potential has been reported to vary from species to species 
and between varieties. Szabados et al. (1987), found wide variation among 15 cassava 
cultivars tested for embryogenic capacity using immature leaf lobes as explants. It was 
observed that M. cercopiaefolia and M. aesculofolia did not form somatic embryos whereas 
M. Ven and M. Col. 1505 formed between 60-70% somatic embryos when cultured on the 
same medium. Mroginsky and Scocchi (1993) have also reported that only two of eleven 
Argentinean genotypes formed somatic embryos.
Results obtained in this study show that frequency of embryo formation was dependent on the 
concentration of auxin in the induction medium as well as the cultivar (Figure 1). In Biafra and 
Santom somatic embryo production was higher on a medium with 16 mg/l 2,4-D than 8 mg/l. 
The observation made in this study is similar to that of Raemakers et al., (1993). It was 
reported in their study that on a higher concentration of 2,4-D (8 mg/l), 49% of leaf lobe
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explants of M. Col 22 formed embryos compared to 15 % on a lower concentration of 2,4-D (4 
mg/l).
This study showed that the type of explant used for embryo induction influenced calli 
formation and subsequent somatic embryogenesis. A comparison of leaf lobe, apical 
meristem and stipule explant showed that apical meristem formed the highest embryogenic 
calli on all the auxin concentrations tested in the induction medium.
Foliose structures were observed on calli derived from both in vitro and greenhouse grown 
leaf lobe explants indicating that they are morphogenetically competent for somatic embryo 
production. This observation is similar to that of Raemakers et al., (1993 a) who obtained 
foliose structures from embryogenic calli derived from leaf lobe explants of M. Col 22. They 
explained that the development of the foliose structures were favoured by low 2,4-D 
concentrations and short duration of explants on the induction medium. According to Stamp 
and Henshaw (1987a) foliose structures are caused by partial expression of morphogenetic 
competence.
Root development occurred when calli derived from in vitro explants were transferred to the 
step two medium. However, root development was high on calli derived from low 2,4-D (4 
mg/l) medium (Figure 1). Results obtained in this study is similar with observation made by 
Krikoran et al. (1987) who found out that formation of somatic embryos and adventitious 
organs ensues when the auxin is removed. It has further been reported that although high 
concentrations of 2,4-D are considered herbicidal, the auxin is able to cause cell division 
Krikoran etal. (1987).
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4.5 Germination of somatic embryos.
The result of this study showed that, of the various desiccation methods used, the highest 
somatic embryo germination was obtained in the petri dish method , desiccator plus glycerol 
method and then water plus desiccation method. Because of the simplicity of the petri dish 
method, it was considered the best and was therefore used in all the subsequent 
experiments.
Results of this study show that both NAA- and 2,4-D-induced somatic embryos require 
desiccation for normal germination ( section 3.9.2). This observation is similar to the 
observation made by Finer (1995) that desiccation broke embryo dormancy and greatly 
enhanced germination of soybean somatic embryos. Kermode (1990) suggested that in vivo 
desiccation terminates the developmental process of embryo formation and activates a switch 
from maturation to germination. It has also been reported that desiccation causes changes in 
tugor pressure, membrane permeability and levels of endogenous ABA which in turn induces 
the expression of specific genes involved in plant development (Parrot et al., 1991). However, 
it was argued that the somatic embryos had to be developmentally mature before the 
desiccation treatment could be effective. Mathews et al., (1993) have also reported that 
desiccation improves germination of matured somatic embryos of cassava cultivar M. Col 
1505.
The result obtained in this study show that BA and Kinetin were necessary to stimulate 
somatic embryo germination. Among the cytokinins tested, BA enhanced shoot formation in 
somatic embryos than TDZ and Kinetin. It was also observed in this study that BA stimulated 
both taproot and shoot formation. Of kinetin-supplemented medium, highest percentage of
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shoot formation and full germination occurred at 4 mg/l which was the highest level employed. 
This suggest that its effects should be investigated at higher concentrations.
No shoot only formation occurred in the auxin amended media. Instead there was high root 
formation in the NAA and IBA media (Table 14). These cultures, however, did not form shoots 
on subsequent transfer to hormone-free media.
The finding in this study is in agreement with the report of Ammirato (1983) that cytokinins are 
known to organise the shoot apex during early maturation of somatic embryos. It has also 
been shown that cytokinins are needed to break dormancy in poorly germinating somatic 
embryos (Gray and Puhit, 1991). The high root formation in the auxin media observed in this 
study agrees with the report of Krikoran et al. (1987) that high auxin levels leads to root 
formation during organogenesis.
Observation made in this study shows that the BA concentration needed for somatic embryo 
germination was dependent on moisture loss (Figure 3). Of the moisture loss levels used (20, 
40 and 75%), 40% was found to be the best for embryo germination in both Gading and Adira
4. At this optimal moisture loss, 0.1 mg/l BA gave the highest percentage of germination. 
Although germination occurred at higher BA concentrations, the developing plantlets had 
thick stems with multiple shoots.
Germination of NAA-induced somatic embryos was found to be faster in the dark than in the 
light. In the dark, full plant germination occurred within 14 days whereas in the light 
germination occurred within 28 days. Also, cultures incubated in the dark enhanced higher
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root development than in the light (Figure 5). Furthermore, this study showed that BA 
concentration needed in the dark for germination was lower than in the light. In the dark, 0.1 
mg/l BA gave the highest percentage germination but in light 1 mg/l was needed. 
Morphologically, developing seedlings from somatic embryos cultured on 0.1 mg/l in the dark 
had single shoots whereas embryos on 1 mg/l BA in dark had multiple shoots (Plate 11 and
For 2,4-D-induced somatic embryos, dark condition stimulated higher germination in only one 
cultivar (Figure 4). The results obtained in this study are in contrast with the work of Mathew 
et. al. (1993) and Raemakers et al. (1993b,c) who found that embryos formed in liquid media 
amended with 8 mg/l 2,4-D did not posses root meristem. On culturing these embryos 
without desiccation on BA medium, the embryos formed shoots but there was no root 
formation. The result of this study indicate that the desiccation process may have triggered 
the development of the root meristem and subsequent outgrowth of the roots. The contrasting 
results between this study and that of Mathews et al (1993) and Raemakers (1993 b,c) could 
be explained by the way the embryos were induced. The induction of embryos using 2,4-D 
in this study differed from that of Mathews et al., (1993). In the later study, 2,4-D exposure 
time was shorter, the concentration was lower and for maturation the embryos were 
transferred to charcoal-amended medium. Secondly, in this study, the embryos were obtained 
in liquid medium whereas in Mathews method the embryos were induced from solid medium. 
Aithough no direct evidence is available, the culture system in this study may have favoured 
higher germination of NAA - induced somatic embryos than 2,4-D induced embryos. Their 
culture system on the other hand, may have activated the root meristem of 2,4-D-induced 
embryos and subsequent whole plant germination. Furthermore, the charcoal incorporated in 
their culture medium may have removed the inhibitory effect of 2,4-D on apical meristem of
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somatic embryos. According to Parrot et al., (1991) charcoal removes excess auxins which 
are detrimental to apical meristem organisation in somatic embryos.
In this study, the embryo germination process lasted 4 weeks. Thus, the seedlings were 
produced within four weeks of a new cycle. The shorter in vitro culture period is 
advantageous. Especially as it may lead to few changes in the phenotypic characteristics of 
the resultant plants. In the procedure described by Mathews et al., (1993), embryo 
germination process lasted 10 weeks.
For direct delivery of somatic embryos to the field through synthetic seed development, 
taproot formation will be desirable as it will enhance quick seedling establishment. But for the 
farmer, single taproot formation will be disadvantageous because the yield of cassava tuber 
will be lower since it is dependent on the number of roots formed. To use synthetic seed 
therefore will require further research to produce somatic embryos which are morphologically 
similar to their zygotic counterparts and also to determine optimal desiccation and dark period 
required for germination.
When a range of various light intensities of 0, 6.4, 64, 641 and 6410 lux were used, 64 lux 
was found to enhance embryo germination. Different light intensities have been reported to 
influence adventitious root and shoot formation in stem cuttings (George and Sherington, 
1984).
Different light regimes were found to affect somatic embryo germination. In this study red light 
was found to enhance germination of somatic embryos whereas blue light was inhibitory. This 
observation is similar to the observation of Henrington and Mcpherson (1993) who reported
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that shoot proliferation of Prunus insita was high when cuttings were exposed to continuous 
red light. According to Norton et al., (1988) blue light inhibited shoot proliferation of Spiraea 
nipponica. It has been explained that red light appears to have either the same effect as 
cytokinin or it reinforces cytokinin in the medium. Leshem et al. (1988) have reported that light 
regime may be used to reduce high concentration of exogenous growth regulator such as BA 
in vitro which causes vitrification and bushiness in ex vitro developed plants.
Desiccation of somatic embryos in light followed by incubation in darkness enhanced 
germination of somatic embryos (Table 16). This results may seem to suggest that somatic 
embryo germination may not be under phytochrome control. Further research is, however, 
needed to verify this inference.
Abscissic acid has been shown to improve desiccation tolerance in seeds. It is also known to 
mobilise storage proteins which are needed for germination in zygotic seeds (Parrot et al., 
1991). It has also been shown that in somatic embryogenesis ABA prevents precocious 
germination (Krikorian et al., 1987) Etienne et al., (1993), showed that ABA improves 
germination of somatic embryos In the rubber plant (Heavea brasiliensis). However, in this 
study ABA was found to decrease somatic embryo germination. The reason for this 
contrasting result might be due to the high concentration of ABA applied to the mature 
embryos. Secondly, the methods used for Heavea brasiliensis are different from what have 
been described in this thesis. Etiene et al., (1993) incorporated ABA in the maturation 
medium. In this report, already matured somatic embryos were transferred to ABA-amended 
medium for a further one week before culture for germination. This procedure might have had 
detrimental effect on organisation of shoot and root apices resulting in poor germination. 
Their culture system mimics what happens during zygotic seed development and may have
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prepared the somatic embryos for desiccation as ABA induces desiccation tolerance and 
brings about normal desiccation (Krikorian et al., 1987).
Washing of embryos for one day followed by desiccation was found to stimulate germination 
of Gading somatic embryo. Also root development of somatic embryos on a hormone-free 
media increased with the period of washing. The observation made in this study could be 
explained by the fact that the washing of the somatic embryos with water removed excess 
auxins absorbed by the embryos in the induction medium which were detrimental to the 
development of the apical meristem. It has been reported that in soybean longer exposure of 
somatic embryos to auxin in the induction medium decreased the frequency of germination of 
somatic embryos (Parrot et al. 1991).
The ever-growing demand for cassava products on the international market has resulted in a 
trend towards more efficient methods of propagation. Induction of multiple shoots and somatic 
embryogenesis has the potential to speed up propagation of elite cassava and provide 
significant economic advantage to the cassava industry.
r\ &
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SUMMARY
Farmers in Ghana cultivate cassava cultivarsbased on the following criteria: early bulking, 
yield and potential use. ACMV and brown leaf spot disease are the common diseases of the 
cultivars collected. ACMV disease affects all the cassava cultivars popularly cultivated by 
farmers in Ghana.
High concentration of NAA in a medium caused high callus formation.
Meristem culture alone does not free cassava cultivars example, Santom from viruses 
Multiple shoot production is dependent on BA concentration in the medium as well as the 
cultivar of cassava.
Apical meristem formed the highest embryogenic calli on all auxin concentrations tested in the 
induction medium when compared to leaf lobe and stipule as explants.
NAA- and 2,4-D induced embryos require desiccation for normal germination. BA 
concentration needed in the dark for germination of somatic embryos was lower than in the 
light (0.1 mg/l BA in the dark compared to 1.0 mg/l in the light).
The study has also indicated that for field synthetic seed development, further research will be 
needed to produce somatic embryos which are morphologically similar to their zygotic 
counterparts and also to determine optimum desiccation and dark period required for 
germination.
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REFERENCES
Ahloowalia, B.S. (1995). In vitro selection of mutants. 14th IAEA/FAO International Training 
Course on Advances in Plant Mutation Techniques. 18th April-26th May, 1995.
Vienna, Austria, pp 1-3.
Adejara, G.O. and Coutts, H.A. (1981). Eradication of cassava mosaic disease from Nigerian 
cassava clones by meristem culture. Plant Cell Tissue and Organ Culture. 1: 25-32.
Al-Hassan, R.M. (1990). Cassava in the economy of Ghana. In: Iweke, I. F., Lynam, J. and 
Coffi, P. Status of data in major producing countries in Africa: Cameroun, Cote 
d’Ivoire, Ghana, Tanzania, Uganda and Zaire
Ammirato, P.V (1983). Embryogenesis. In: Evans D.A., Sharp, W.R., Ammirato P.V. and 
Yamada, Y. (eds) Handbook of Plant Cell Culture Vol 1 pp 82-123. Macmillan, New 
York.
Annor-Frempong, C. (1994). A survey of cassava cultivation practices in Ghana. In: Ofori, F. 
and Hahn S.K. (eds) Tropical Root Crops in a developing economy. Proceedings of 
the ninth symposium of the international society for tropical root crops, 20-26th 
October, 1991. Accra, Ghana, pp
Baker, C.M. and Wetzetein H.E. (1992). Somatic embryogenesis and plant regeneration from 
leaflets of peanuts (Arachis hypogea). Plant Cell Report. 11:122-125.
Bai, K.B.V. (1987). Recent advances in cassava genetics and cytogenetics. In: Hershey, C.H. 
(eds) Cassava breeding: a multidisplinary review, CIAT Columbia, pp 35-49.
Bewley, D.D. and Black, M. (1985). Seed development and maturation. In: Seed physiology of 
development and germination. Plenum Press. New York, pp 22-88.
Bocks, K.K. and Woods, R.D. (1983). The aetiology of African Cassava Mosaic Virus disease. 
Plant disease 67:994-995.
95
University of Ghana                              http://ugspace.ug.edu.gh
Bokanga, M , O’ Hair SK, Narayanan, K.R. and Steinkrans K.H. (1990). Cyanide detoxification 
and nutritional changes during cassava (Manihot esculenta, Crantz) fermentation. In: 
Howler, R.H. (ed) Proceedings of 8th Sympossium of the International Society for 
Tropical Root Crops, 30th October-15th November, 1988, Bangokok, Thailand, pp 
385-392
Byrne, D. (1984). Breeding Cassava. In Janick J (ed) Plant Breeding Reviews, 2: 73-113.
Carman, J.G. (1990). Embryogenic cells in plant tissues: occurrence and behaviour. In Vitro 
Cellular Developmental Biology 26:746-753.
Cock, J.H. (1982). Cassava: a basic energy source in the tropics. Science 218: 755-762.
Cock, J.H. (1985). Cassava: New Potential for a neglected crop. Wetview Press, Boulderland, 
London, p 191.
Crouch, M.L. (1982). Non-zygotic embryos of Brassica napus L. contain embryo specific 
proteins. Planta 156: 520-524.
DGIS (1991). Cassava and Biotechnology. Proceedings of workshop held in Amsterdam, 21- 
23 March, 1990. The Hague:Directorate General for International Co-operation, p 4.
Durham, R.E and Parrot W.A. (1992). Repetitive somatic embryogenesis from peanuts 
cultivars in liquid medium. Plant Cell reports 11: 122-125.
Eskes, A.B, Varga, A, Staritsky, G. and Bruinsma, J. (1984). Callus growth and rooting of 
cassava stem segments cultured in vitro. Acta Botanica Neerlandica. 23:315-320.
Etienne, H., Montoro, P., Michaux-Ferriere, N. and Carron, M. P. (1993). Effects of
desiccation, medium osmolarity and abscissic acid on the maturation of Heavea 
brasiliensis somatic embryos. Journal of Experimental Botany, 44 (267):1613-1619.
96
University of Ghana                              http://ugspace.ug.edu.gh
Evans, D.A., Sharp, W.R. and Flick, C.E. (1981). Growth and behaviour of cell cultures: 
embryogenesis and organogenesis. In Thorpe, T.A. (ed) Plant Tissue Culture:
Methods and Applications in Agriculture. Proceedings of UNESCO Symposium, Saulo 
Paulo. Academic Press, New York, pp 45-113.
Ezumah, H.C. (1988). Cassava-based cropping system research collaboration. Cassava 
based cropping system research. Vol. 1. IITA, Ibadan.
FAO, (1989). Food and Agricultural Organisation of the United Nations. Production yearbook, 
1988. Rome.
FAO (1993). Food and Agriculture Organisation of United Nations. Production yearbook 1992, 
Rome, pp 281.
Fargette, D., Fauquet, C. and Thouvenel, J.C. (1985). Field studies on the spread of African 
mosaic virus. Annals of Applied Biology: 106: 285-295.
Fauquet, C. and Fargette, D. (1990). African Cassava Mosaic Virus: Etiology, epidemiology 
and control. Plant Disease 74 (6): 404-441.
Finer, J.J. (1995). Direct somatic embryogenesis. In: Gamborg, O.L. and Phillips, G.C (eds.) 
Plant Cell, Tissue and Organ Culture. Fundamental methods. Springer, Berlin pp 90- 
99.
Franche, C., Bogusz, D., Schokpe, C., Faquet, C. and Beechy, R.N. (1991). Transient gene 
expression in cassava using high velocity microprojectiles. Plant Molecular 
Biology 17:493-498.
Franklin, C.l. and Dixon, R.A. (1994). Initiation and maintenance of callus and cell suspension 
cultures. In: Dixon, R.A and Gonzales, R.A. (eds). Plant Cell Culture. A practical 
approach. Second Edition. IRL Press.,Oxford University Press, pp 1-22.
University of Ghana                              http://ugspace.ug.edu.gh
Gamborg, O.L., Miller, R.A. and Ojima, K. (1968). Nutrient requirements of suspension culture 
of soybean root cells. Experimental Cell Res. 50: 151-158.
George, E.F. and Sherrington, P.D. (1984). Plant propagation by tissue culture. Handbook 
and directory of commercial laboratories. Exegetics Ltd. England, pp 141-158.
Gray, D.J. (1987). Quiescence in monocotyldonous and dicotyledonous somatic embryos 
induced by dehydration. Hortscience 22: 810-814.
Gray, D.J. and Puhit, A (1991). Somatic embryogenesis and development of synthetic seed 
technology. Critical Reviews in Plant Sciences 10: 33-61
Haccius, B. (1978). Question of unicellular origin of non-zygotic embryos in callus cultures. 
Phytomorphology. 28:74-81
Hahn, S.K., John, C., Isoba, G., and Ikotun, T. (1989). Resistant breeding of root and tuber 
crops at the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. Crop 
Protection 8: 147-168.
Halperin, W. and Witherel, D.F. (1965). Ontogeny of adventitious embryos in wild carrot. 
Science 147: 756-8.
Hartmann, H.T., Kester, T.T. and Davies, T.T. (1990) Plant propagation, 5th edition, Prentice 
Hall, Englewood Cliffs, N.J.
Herrington, E .and McPherson, J.C. (1993). Light quality growth promotion of Spiraea 
nipponica: the influence of low photon fluence rate and transfer time to a higher 
fluence rate. Plant Cell, Tissue and Organ Culture 32: 161-167.
Hershey, C.H. (1993). Cassava (Manihot esculenta Crantz) In: Kalloo, G. and Bergh, B.O.
(eds). Genetic improvement of vegetable crops. Pergamon Press. Oxford-New york pp 
669-691.
98
University of Ghana                              http://ugspace.ug.edu.gh
IITA, (1990) Cassava in Tropical Africa : a reference manual IITA, 1990. pp 60-89.
Iweke, F.l. Poison, R. and Strauss, J. (1994). Cassava production trends in Africa. In: Ofori,
F. and Hahn, S.K. (eds). Tropical Root Crops in a developing economy. Proceedings 
of the ninth symposium of the international society for tropical root crops, 20-26th 
October, 1991. Accra, Ghana.
Kaiser, W.J. and Teemba, L.R (1979) Use of Tissue Culture and Thermotherapy to free East 
African cultivars of African Cassava Mosaic and Brown streak disease. Plant Science 
Reporter 63: 780-784.
Kartha, K.K. (1981). Tissue Culture techniques for virus elimination and germplasm
preservation. ln:Rachie, K.O. and Lyman, J.M. (eds), Genetic engineering for crop 
improvement. Working papers, Rockefeller Foundation, pp 123-134.
Kartha, K.K., Gamborg, O.L. Constabel, F. and Shylnk ,J.P. (1974). Regeneration of cassava 
plants from apical meristems. Plant Science Lett. 2: 107-113.
Kartha, K.K. and Gamborg O.L. (1975). Elimination of Cassava Mosaic Virus disease by 
meristems. Plant Science Lett. 107-113.
Kermode, A.R. (1990). Regulatory mechanism involved in the transition from seed 
development to germination. CRC Crit. Rev. Plant Sci. 9: 155-195.
Komatsuda, T. and Ohyma, K. (1988). Genotypes of high competence for somatic
embryogenesis and plant regeneration in Soybean max. Theoretical and Applied 
Genetics 75: 695-700.
Krikorian, A.D., Kelly, K. and Smith, D. L. (1987). Hormones in plant tissue culture and
micropropagation. In: Peter, J. D. (Ed). Plant Hormones and their role in plant growth 
and development. Martinus Nijhoff Publishers, Dordrect, pp 593-613.
Leshem, B., Shaley, D.P and Izhar, S. (1988). Cytokinin as an inducer of vitrification in melon.
Ann. Bot. 61: 255-260.
99
University of Ghana                              http://ugspace.ug.edu.gh
Liu, M.C. (1975). The in-vitro induction and regeneration of cassava plant from apical 
meristem. Taiwan Sugar 22: 171-177.
Mathews, H, Schokpe, C, Carcamo, R, Chavarriaga, P, Fauquet, C and Beachy, R.N. (1993). 
Improvement of somatic embryogenesis and Plant regeneration in cassava. Plant Cell 
Report. 12: 328-333.
Mellor, F.C., and Stace-Smith, R. (1977). Virus-free potatoes by tissue culture. In J. Reinert 
and Y.P.S. Bajaj (eds.), Applied and fundamental aspects of plant cell, tissue and 
organ culture. Springer-Verlag, Berlin pp. 616-635.
Merkle, S.A. and Wiecko, A.T. (1990). Somatic embryogenesis in three Magnolia species. 
Journal of American Horticultural Science. 115: 858-860.
MOA (1990). Ministry of Agriculture. Ghana medium term agricultural development
programme: an agenda for sustained agricultural growth and development (1991- 
2000) MOA, Accra.
Mroginsky, L.E. and Scocchi, a. (1993). Somatic embryogenesis of Argentine cassava
varieties. In: Roca, W.M. and Thro, A.M. (eds.). Proceedings of the First International 
Scientific Meeting of the Cassava Biotechnology Network. Carte-agena, Columbia, 25- 
28 August, 1992. Cali, Columbia: Centro International de Agricultura Tropical, 1993.
Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassay with 
tobacco cultures. Physiologum Plantarum. 15: 473-494.
Nasser, N.M.A. (1978). Conservation of genetic resources of cassava (Manihot escuienta): 
determination of wild species localisation with emphasis on possible origin. Econ. Bot. 
311-320.
100
University of Ghana                              http://ugspace.ug.edu.gh
Ng, S.Y.C. (1992). Tissue Culture of root and tuber crops at IITA. In: Thottapilly, G., Monti 
L.M, Mohan Raj D.R. and More A.W. (eds), Biotechnology: Enhancing research on 
Tropical Crops in Africa, pp 135-141.
Norton, C.R., Norton, M.E., Herrington, E. and Philips, D. (1988). Light quality and light pipes 
in the micropropagation of woody ornamental plants. In: von Hetig W-U and Gruber,
G. (eds) International sympossium on propagation of ornamental plants. Acta Hort. 26: 
413-416.
Obigbessan, G.O. (1994). Issues of cassava cyanide: Nigeria’s experience. In: Ofori, F. And 
Hahn, S.K. (eds) Tropical Root Crops in a developing economy. Proceedings of the 
ninth sympossium of the international society for tropical root crops, 20-26th October, 
1991. Accra, Ghana, pp 233-236.
Oishi, M.Y. and Bewley, J.D. (1990). Distinction between the responses of developing maize 
kernels to flouridone and desiccation in relation to germinability, a- amylase activity 
and abscissic acid content. Plant Physiol. 94: 592-598.
Parrot, W.A., Merkle, S.A. and Williams, E.G. (1991). Somatic embryogenesis: Potential for 
use in propagation and gene transfer systems. In: Murray, D.R. (ed) Advanced 
methods in plant breeding and biotechnology. CAB Int. Wallingford, pp 158-200.
Purseglove, J.W. (1968). Tropical Crops: Dicotyledons. Longman Group Ltd. pp 172-185.
Raemakers, C.J.J.M, Bessember, J., Staristy, G., Jacobsen, E. and Visser, R.G.F. (1993a). 
Induction, germination and shoot development of somatic embryos in cassava. Plant 
Cell, Tissue and Organ Culture 33:151-156.
Raemakers, C.J.J.M, Amati, M., J., Staristy, G., Jacobsen, E. and Visser, R.G.F. (1993b). 
Cyclic embryogenesis in cassava. Annals of Botany 71:289-294.
101
University of Ghana                              http://ugspace.ug.edu.gh
Raemakers, C.J.J.M, Schamakers, C.M., Jacobsen, E. and Visser, R.G.F. (1993c).
Improvement of Cyclic embryogenesis of cassava (Manihot esculenta Crantz). Plant 
Cell Reports 12:2269-229.
Raemakers, C.J.J.M, Jacobsen, E. and Visser, R.G.F. (1995). Secondary somatic 
embryogenesis and application in plant breeding. Euphytica. 81:93-107
Raemakers, C.J.J.M, Sofiari, E., Kanju, E, Jacobsen, E. and Visser, R.G.F. and van
Lammeren A.M.M. (1995b). NAA induced somatic embryogenesis in cassava. In: 
Roca W.M., and Thro, A.M. (eds) Proceedings of the second international Scientific 
meeting of the Cassava Biotechnology Network, Bogor, Indonesia.
Raemakers, C.J.J.M, Sofiari, E., Kanju, E, Jacobsen, E. and Visser, R.G.F. and van
Lammeren A.M.M. (1996). Production of transgenic cassava (Manihot esculenta 
Crantz) plants by particle bombardment using luciferase activity as selection marker. 
Molecular Breeding, Kluwer Academic Publishers, Belgium, pp 1-11.
Raemakers, C.J.J.M., Visser, R. G. F. and Jacobsen, E. (1997). Micropropagation of cassava. 
(Manihot esculenta Crantz). In Y.P.S. Bajaj (ed). Biotechnology in Agriculture and 
forestry, High-tech and micropropagation, Springer-Verlag Berlin. 39:77-102.
Raemakers C.J.J.M. (1993). Primary and cyclic embryogenesis in cassava (Manihot 
esculrenta Crantz). Ph.D Thesis, Agricultural University, Wageningen, The 
Netherlands.
Reinert J, Bajaj, Y.P.S and Zbell, B. (1977). Aspects of organogenesis and embryogenesis.
In: Street E.D. (ed) Plant tissue and cell culture. University of Columbia Press,
Berkley, pp 389-429.
Roberts, D.R., Lazarroff, W.R. and Webster, F.B. (1990). Interaction between maturation and 
relative humidity treatments and their effects on germination of Stika spruce somatic 
embryos. Journal of Plant Physiology 138: 1-6.
102
University of Ghana                              http://ugspace.ug.edu.gh
Roca, W.M. (1984). Cassava. In: Sharp, W.R., Evans W.A., Ammirato, P.V. and Yamada
(eds) Handbook of plant cell culture, Vol 2, Crop species. Macmillan Publishing, New 
York pp 269-301.
Roggers, D.J. and Appan, S.G. (1973). Manihot and Manihotoides (Euphorbiaceae). Flora 
Neotropica Monograph 13. Hafner press, New York.
Rosenberg, L.A and Rhine, R.W. (1986). Moisture loss, a prerequisite for seedling growth in 
seeds of soybean (Glycine max L. Merr). Journal of Experimental Botany 37:1663- 
1674.
Schopke, C., Taylor, N., Carcamo, R., Konan, N.K., Marmey, P., Henshaw, G.G., Beachy, 
R.N., Fauquet, C. (19$»6). Regeneration of transgenic cassava plants (Manihot 
esculenta Crantz) from microbombarded embryogenic suspension cultures.
Nature Biotechnology 14: 731-735.
Seif, A.A. (1982). Effect of Cassava Mosaic Virus on yield of cassava. Plant Diseases. 
66:661-662.
Seitz, Kris M.H. and Bingham E.T. (1988) Interactions of highly regenerative genotypes of 
alfafa (Medicago sativa) and tissue culture protocols. In-vitro cell and developmental 
Biology pp. 84
Shahin, E.A. and Shephard, J.F. (1980). Cassava mesophyll protoplasm: isolation, 
proliferation and shoot formation. Plant Sci. Lett 17:459-465.
Sharp W.R., Sohndahl M.R., Evans, A.E., Caldas L.A. and Maraffa, S.B. (1980). The 
physiology of in vitro asexual embryogenesis. Horticultural Reviews 2: 268-310.
Slack, S.A. and Tufford, L. A. (1995). Meristem culture for disease elimination. In: Gamborg,
G.L. and Philips G.C. (eds). Plant cell, tissue and organ culture. Fundamental 
methods, Springer-Verlag Berlin, pp 11-128.
o \
University of Ghana                              http://ugspace.ug.edu.gh
Smith, M.K., Biggs, B. J. And Scott, K.J. (1986). In vitro propagation of cassava (Manihot 
esculenta Crantz). Plant Cell, Tissue and Organ Culture 6:221-229.
Stamp, J.A and Henshaw, G.G. (1982). Somatic embryogenesis in cassava. Z. 
Pflanzenphysiol 105: 183-187.
Stamp, J.A. and Henshaw G.G. (1987a). Somatic embryogenesis from clonal leaf tissue of 
cassava. Annals of Bot. 59: 445-450.
Stamp, J.A. and Henshaw G.G. (1987b). Secondary somatic embryogenesis and plant 
regeneration in cassava. Plant Cell Tissue and Organ cultural 0: 227-233.
Szabados, L., Hoyos, R. And Roca, W. (1987). In vitro somatic embryogenesis and plant 
regeneration of cassava. Plant Cell Reports. 6:248-251.
Terry, E.R. and Hahn, S.K. (1980). The effect of cassava mosaic disease on growth and yield 
of a local and improved variety of cassava. Tropical Science 34: 26-421
Terzi, M. and Loschiavo, F. (1990) Somatic embryogenesis. In Bhojawani, S.S. (ed)
Development in Crop Science, Plant Tissue Culture: Applications and limitations pp 1- 
33.
Theberge, R.L. (1985). Common African pests and diseases of cassava, yam, sweet potato 
and cocoyam. IITA Handbook, pp 14.
Thomas, E.T. and Wenzel, G. (1975) Embryogenesis from microspores of Brssica napus: 
Zeitscrif fur Pflanzenzuchtung 74:77-81.
Thorpe, T.A. (1990). The current status of plant tissue culture. In Bhojawani, S.S. (ed)
Development in Crop Science, Plant Tissue Culture: Applications and limitations pp 1- 
33.
104
University of Ghana                              http://ugspace.ug.edu.gh
Thresh, J.M., Fargette, D. and Otim-Nape, G.W. (1994). Effect of African Cassava Mosaic 
geminivirus on yield of cassava. Tropical Science 34:26-42.
Tiliquin, J.P. (1979). Plant regeneration from stem callus of cassava. Canadian Journal of 
Botany 57: 1761-1763.
Tisserat, B., Esan, E.B., and Murashige, T., (1979). Somatic embryogenesis in Angiosperms. 
Horticultural Review 1:1-77.
Tulucke, W. (1987). Somatic embryogenesis in woody perennials. In: Bonga, J.M. and 
Durzan, D.J. (eds) Cell and tissue culture in forestry, Vol 2, specific principles and 
methods: growth and development, Martinus Nyhoff Publishers, Dordrecht, Boston, 
Lancaster, pp 61-91.
Umbeck, P., Johnson, G. Barton, K. and Swain, W. (1987). Genetically transformed cotton 
(Gossypium hirstum) plants. Bio/technology 5: 263-266.
Vasil, V., and Vasil, I. K. (1981). Somatic embryogenesis and plant regeneration from
suspension cultures of Pearl Millet (Pennisetum americanum). Annals of Botany 47: 
669-678.
Wann, S.R. (1988). Somatic embryogenesis in woody species. Horticultural Review 10: 153- 
189.
William , E.G. and Maheswaran, G. (1986). Somatic embryogenesis: factors influencing
coordinated behaviour of cells as an embryogenic group. Annals of Botany 57: 443- 
462.
Wright, N.S (1988). Assembly, quality control and use of potato cultivar collection rendered 
virus-free by heat therapy and tissue culture. Am Potato Journal, 65:181-198.
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APPENDIX
Appendix 1 
GERMPLASM COLLECTION QUESTIONAIRE
1. Name of farmer (optional).........................................................................
2. Location of farm  .................................................................................
3. Local names of cassava cultivated.........................................................
4. Type of farm I. Peasant/subsistence  ii. Government.
5. Source of variety/cultivar .......................................................................
6. Region of origin........................................................................................
7. Country of origin......................................................................................
8. Morphological features
i. colour of stem..................................................
ii. colour of petiole................................................
iii. number of leaflets.............................................
iv. leaf arrangement..............................................
v. leaf shape........................................................
vi. branching habits a. no branching b. branching
vii. Does your cultivar flower a. yes b. No
viii. Colour of petal..................................................
9. Diseases
i. Which diseases affect your cultivar?...............................................
ii. Where are the symptoms noticed?..................................................
jjj. Describe the symptoms briefly.........................................................
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iv. At what stages are the symptoms first noticed?
a. 1 month after shooting
b. 2-4 months after shooting
c. 4-6 months after shooting
d. 6-10 months after shooting
10. The variety/cultivar is good for making a. fufu b. ampesi. c. gari d. agbelema 
e. konkonte f. others...............................
11. How long does your cultivar take to mature?
a. 6 months b. 9 months c. 12 months d. 18 months
e. 24 months
12. The yield per hectare is a. high b. medium c. low.
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ACMV INCIDENCE AND SEVERITY SCORING SHEET
( C ESCaPP Survey Software, IITA, Benin Station. 
Village..............................................
Name/I.D no. of................................................ Crop age.
Name/I.D. no. of researcher:......................  Cultivars.................
Appendix 2
Date:......................................   Field size.
Plant
sample
No. of shoot tips per ACMV symptom score 1-5 Total no. of 
shoot tips
Average 
score 
per tip
ACMV 
score / 
whole plant
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24 !
25
Total i
Mean
S.D.
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Appendix 3a
ANOVA for the effect of cultivar and BA on induction of multiple shoots from apical meristem 
of M. Col 22 and Biafra.
Source of variation SS d.f. MS F-ratio Sig.level (P<0.05)
Main effects 210.18750 9 23.35417 5.451 0.0001
Cultivars 123.52083 (1) 123.52083 28.833 0.0000
Replicates 7.60417 (5) 1.52083 0.355 0.8758
BA concentration 79.06250 (3) 26.35417 6.152 0.0016
Residual 162.79167 38 4.2839912
Total (Corr) 372.97917 47
Appendix 3b
ANOVA for the effect of cultivar and BA on induction of multiple shoots from single bud 
cuttings of M. Col 22 and Biafra.
Source of variation SS d.f. MS F-ratio Sig.Level (P<0.05)
Main effects 1464.1875 9 162.68750 7.429 0.0000
Cultivars 595.0208 (1) 595.02083 27.172 0.0000
Replicates 80.4375 (5) 16.08750 0.735 0.6021
BA concentration 788.7292 (3) 262.90972 12.006 0.0000
Residual 832.12500 38 21.898026
Total (Corr.) 2296.3125 47
109
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Appendix 4a
ANOVA for the effect of BA on induction of multiple shoots from apical meristem M. Col 22 .
Source of variation SS d.f. MS F-ratio Sig. level (P<0.05)
Main effects 133.3333 8 16.666667 2.240 0.0848
Replicates 15.20833 (5) 3.041667 0.409 0.8353
BA concentration 118.12500 (3) 39.375000 5.291 0.0109
Residual 111.62500 15 7.4416667
Total (Corr.) 244.95833 23
Appendix 4b
ANOVA for the effect of BA on the number of buds produced by apical meristem of M. Col
Source of variation SS d.f. MS F-ratio Sig. level (P<0.05)
Main effects 154.83333 8 68.229167 1.904 0.1344
Replicates 251.83333 (5) 50.366667 1.406 0.2780
BA concentration 294.00000 (3) 98.000000 2.735 0.0803
Residual 537.50000 15 35.833333
Total (Corr.) 1083.3333 23
110
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Appendix 5
ANOVA for the effect of BA on the number of buds produced by apical meristem of Biafra
Source of variation SS d.f. MS F-ratio Sig. level (P^0.05)
Main effects 542.33333 8 67.79167 13.446 1.0000
Replicates 25.20833 (5) 5.04167 1.000 1.0000
BA concentration 517.12500 (3) 172.37500 34.190 1.0000
Residual 75.625000 15 5.0416667
Total (Corr.) 617.95833 23
111
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ANOVA for the effect of cultivar and BA on induction of shoots from single bud cuttings of M. 
Col 22, Ankrah, B. nsia and Biafra.
Appendix 6a
Source of variation SS d.f. MS F-ratio Sig. Level (P<0.05)
Main effects 116.16473 9 12.907192 9.995 0.0000
Replicates 5.28512 (3) 1.761706 1.364 0.2635
BA concentration 71.03637 (3) 23.678789 18.337 0.0000
Cultivar 39.84324 (3) 13.281081 10.285 0.0000
Residual 69.84324 54 1.2913238
Total (Corr.) 185.89621 63
Appendix 6b
ANOVA for the effect of cultivar and BA on induction of buds from single bud cuttings of M. 
Col 22, Ankrah, Bosomnsia and Biafra.
Source of variation SS d.f. MS F-ratio Sig. Level (P<0.05)
Main effects 322.61975 9 35.846639 3.886 0.0008
Cultivar 233.67598 (3) 77.891993 8.444 0.0001
Replicate 21.90617 (3) 7.302056 0.792 0.5039
BA concentration 67.03760 (3) 22.345868 2.422 0.0758
Residual 498.11595 54 9.2243694
Total (Corr.) 185.89621 63
112
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Appendix 7
ANOVA for the effect of cultivar and 2,4-D concentration on calli formation from leaf 
lobes of greenhouse grown cassava.
Source of variation Sum of squares d.f. MS F-ratio Sig. level (PS0.05)
Main effects 4600.0608 8 575.0076 4.001 .0015
Cultivars 2055.5056 (3) 685.1685 4.767 .0063
Replicate 328.9225 (3) 109.6408 0.763 .5218
2,4-D conc. 2215.6329 (2) 1107.8165 7.708 .0015
Residual 5605.1440 39 143.72164
Total (Corr.) 10205.205 47
113
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ANOVA for the effect of 2,4-D concentration on calli formation of greenhouse grown cassava
%
(Ankrah) cultivar.
Appendix 8a
Source of variation SS d.f. MS F-ratio Sig. level (P<0.09)
Main effects 24474.000 6 4079.0000 262.691 0.0000
Replicates 113.250 (3) 37.7500 2.431 0.1321
2,4- D conc. 24360.750 (3) 8120.2500 522.950 0.0000
Residual 139.75000 9
15.527778
Total (Corr) 24613.750 15
Appendix 8b
ANOVA for the effect of 2,4-D concentration on calli formation of greenhouse grown cassava 
(Bosomnsia) cultivar.
Source of variation SS d.f. MS F-ratio Sig. level (P<0.05)
Main effects 25778.219 6 4296.3698 25.658 0.0000
Replicates 115.672 (3) 38.5573 0.230 0.8730
2,4- D conc. 25662.547 (3) 8554.1823 51.086 0.0000
Residual 1507.0156 9 167.44618
Total (Corr) 27285.234 15
114
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ANOVA for the effect of 2,4-D concentration on calli formation of greenhouse grown cassava 
(Biafra) cultivar.
Appendix 8c
Source of variation SS d.f. MS F-ratio Sig. level (P^0.05)
Main effects 25604.434 6 4267.4056 114.510 0.0000
Replicates 308.467 (3) 102.8223 2.759 0.1039
2,4- D conc. 25295.967 (3) 8431.9890 226.260 0.0000
Residual 335.40062 9 37.266736
Total (Corr) 25939.834 15
115
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ANOVA for the effect of explant and cultivar on calli formation leaf lobes, apical 
meristem and stipules.
Appendix 9
Source of variation SS d.f. MS F-ratio Sig. level (P<0.05
Main effects 86080.000 10 8608.000 20.169 0.0000
Cultivar 5821.333 (4) 1455.333 3.410 0.0137
Explant 79690.667 (2) 39845.333 93.360 0.0000
Replicate 568.000 (4) 142.000 0.333 0.8550
Residual 27314.667 64 426.79167
Total (Corr) 113394.67 74
116
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ANOVA for the effect of 2,4-D on calli formation from leaf lobe, apical meristem and stipules 
of Santom.
Appendix 10a.
Source of variation SS d.f. MS F-ratio Sig. level (P<0.05)
Main effects 2746.6667 6 457.77778 4.905 0.0216
Explant 1853.3333 (2) 926.66667 9.929 0.0068
Replicate 893.3333 (4) 223.33333 2.393 0.1366
Residual 746.66667 8 93.333333
Total (Corr) 3493.3333 14
Appendix 10b.
ANOVA for the effect of 2,4-D on calli formation from leaf lobe, apical meristem and stipules
of Bosomnsia
Source of variation SS d.f. MS F-ratio Sig. level (P<0.05)
Main effects 7120.0000 6 1186.6667 3.327 0.0600
Explant 5813.3333 (2) 2906.66667 8.150 0.0117
Replicate 1306.6667 (4) 326.6667 0.916 0.4994
Residual 2853.3333 8 356.6667
Total (Corr) 9973.3333 14
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Appendix 11.
ANOVA for the effect of cultivar and 2,4-D concentration on calli formation in-vitro explants.
Source of variation SS d.f. MS F-ratio Sig. level (P<0.05)
Main effects 4804.6558 9 533.85065 3.347 0.0028
Cultivar 2274.4157 (4) 568.60392 3.565 0.0123
Replicate 621.4978 (3) 207.16594 1.299 0.2851
2,4-D concentration 1908.74234 (2) 954.37117 5.984 0.0047
Residual 7973.9940 50 159.47988
Total (Corr) 12778.650 59
118
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