1. 1
CHAPTER ONE
1.0 INTRODUCTION
1.1 GENERAL DESCRIPTION
Yam is the common name for some plant species in the genus Dioscorea
(family Dioscoreaceae) that forms edible tubers. These are perennial
herbaceous vines cultivated for the consumption of their starchy tubers in
Africa, Asia, Latin American, the Caribbean and Oceania. There are many
cultivars of yam. Yam tubers can grow up to 1.5 meters (4.9 feet) in length and
weigh up to 70 kilograms (154 lb.) and 3 to 6 inches high. Yams are monocots
related to lilies and grasses (Dumont and Vernier, 2000).
Yam is a monocotyledonous angiosperm, which belongs to the order Liliflorae,
family Dioscoreaceae, and genus Dioscorea. It is considered to be among the
most primitive of the angiosperms and contains over 600 species, of which only
about ten are considered as edible (Purseglove, 1988). Cultivated species
include D. alata, D. cayenensis, D. rotundata, D. esculenta, D. bulbifera, D.
nummularia, D. pentaphylla, D. hispida, D. trifida and D. dumetorum. The
range of D. alata cultivars was estimated to be not less than 85 in New
Caledonia (Coursey, 1967). However, the diversity of cultivars has been
reduced due to the progressive urbanization (Coursey, 1967). D. alata species
are never found in the wild and may have developed from crosses and

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domestication from D. hamiltonii or D. persimilis (Purseglove, 1972). In
contrast, D. bulbifera is common in the wild in Asia and Africa and the different
forms of D. bulbifera were named as separate species by some authors
(Coursey, 1967). Morphological characteristics of the genus Dioscorea were
first used for classification and divided the genus into six different sections
(Alexander and Coursey, 1969). However, the morphological characters do not
allow the distinction between species such as D. cayenensis and D. rotundata,
and also between cultivars. D. rotundata may in fact be a subspecies of D.
cayenensis or alternatively may have originated from D. praehensilis (Coursey,
1967).
1.2 CLASSIFICATION
Kingdom: Plantae
(Unranked): Angiosperms
(Unranked): Monocots
Order: Dioscoreales
Family: Dioscoreaceae
Genus: Dioscorea

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1.3 CULTIVARS OF YAM
There are many cultivars of yam but for the sake of this research we will be
focusing on these three:
 Dioscorea alata – common name - water yam
 Dioscorea dumentorum – common name (Ibo) - ona
 Dioscorea rotundata– common name – white yam
Dioscorea alata L
Vigorously twining herbaceous vine, from massive underground tuber. Stems to
10 m (30ft) or more in length, freely branching above; internodes square in
cross section, with corners compressed into “wings,” these often red-purple
tinged. Aerial tubers (bulbils) formed in leaf axils (not as freely as in D.
bulbifera), elongate, to 10 cm (4 in) x 3 cm (1.2 in), with rough, bumpy
surfaces. Leaves long petioled, opposite (often with only 1 leaf persistent);
blades to 20 cm (8 in) or more long, narrowly heart shaped, with basal lobes
often angular. Flowers small, occasional, male and female arising from leaf
axils on separate plants (i.e., a dioecious species), male flowers in panicles to 30
cm (1ft) long, female flowers in smaller spikes. Fruit a 3-parted capsule; seeds
winged (Florida Exotic Pest Plant Council, 2014)

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Dioscorea dumentorum(Kunth.) Pax
Trifoliate yam (Dioscorea dumentorum) is known by various names including
three leaved yam, bitter yam and cluster yam. The tuber skin is coarse; one plant
usually produced a cluster of tubers. The starch granules are smaller, more
soluble and more digestible than those of the other yam species. The tubers are
rich in protein (9.6%), and reasonably balanced in essential amino acid
(chemical score of 0.94) compared to white yam (Effect of drying method and
variety on functional properties of Trifoliate yam, 2014).
Dioscorea rotundataPoir.
The white yam is a native to Africa. Its tuber is roughly cylindrical in shape, the
skin is smooth and brown and the flesh usually white and firm. It has a shorter
period of vegetation and also a longer dormancy. They are large plants; the
vines can be as long as 10 to 12 meters (33 to 39ft). The tubers most often
weigh about 2.5 to 5 kilograms (5.5 to 11.0lb) each but can weigh as much as
25 kilograms (55lb) (Yam Wikipedia, 2014).
1.4 JUSTIFICATION
Chromosome number data remain valuable today in systematics. Knowing
which plant are polyploids and which are diploids allows for a better selection
of individuals to be included in phylogenetic-DNA sequence studies.
Cytogeographic studies are useful in understanding distribution patterns and can

5. 5
help explain morphological studies. While flow cytometry can result in large
sample sizes and eliminates the need to collect buds for meiotic counts or
transplanting for mitotic counts. It is critical that initial work begin with plants
whose chromosome numbers have been determined by traditional means of
squashing and counting. Once ploidy levels are known, then non-counting
methods for determining ploidy level can be accurately applied. Also,
traditional methods of squashing and counting allow the researcher to detect
chromosomal rearrangement, e.g. unequal translocation or dysploid, reductions
in chromosome number with no change in the amount of DNA. These are not
revealed by flow cytometry method (John, 2013).
One can infer quite a lot about plant sexual and asexual reproduction from
chromosome numbers. Odd number ploidy is often associated with meiotic
disharmony in pollen and ovule production (i.e. it is difficult to have uniformly
haploid gametophytes). Chromosome numbers are sometimes associated with
the amount of genetic material in the cells, and with genetic locid, phenotypic
plasticity and adaptability to stresses. One can infer a great deal, but then
observations and experiments should follow (Peter, 2013).
Feulgen densitometry, image cytometry, and flow cytometry (FCM) are among
the cytometric techniques which have played a significant role in plant
taxonomy, biosystematics, and ecology in determining chromosomal and ploidy
level data (Suda et al., 2006). The merits of FCM lie in its simplicity and speed,

6. 6
the small amount of tissue sample required the use of various types of plant
tissues: leaves, stems, roots, sepals, petals and seeds in FCM assays. This
provides the possibility of extensively exploring rare and endangered plant
species with no risk of population destruction (Sgorbati et al., 2004). Through
FCM, ploidy level at various spatial scales, interactions among cytotypes, and
evolutionary processes in diploid- polyploid sympatric populations can also be
reliably assessed (Baack, 2004; Husband and Sabara, 2004). Moreover, FCM
holds great potential in reshaping former taxonomic concepts and facilitating
robust classification based on cytotype characteristics (Rosenbaumova et al.,
2004). Thus, the application of molecular cytogenetics to the species of
Dioscorea under study will greatly improve an understanding of chromosome
structure and karyotype variation within the species (Suda et al., 2006).
Chromosome observation is necessary to clarify the structure, function,
organization and evolution of yam genomes. However, the determination of
ploidy level in yam somatic cells by chromosome counting is limited by the
polyploid nature of the crop, dot-like nature of chromosomes and small volume
of mitotic cells. These characteristics hinder the preparation of distinct and well-
spread chromosomes visible in a single focal plane (Staudt, 1989). A simple,
rapid and reliable procedure is needed to determine the chromosome number of
meristematic regions of yam root tips (Dansi et al., 2001). Furthermore, an
understanding of the ploidy and chromosome status in plants generated from
anther, ovary and callus cultures, or cell fusion for the identification

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of haploids, heterokaryons or doubled haploid genotypes is imperative in
augmenting plant breeding efforts to develop new genotypes.
1.5 OBJECTIVES
The main objective of this work is to investigate and confirm the chromosome
numbers of these three species of yam. Specifically the study intend to:
1. Determine the ploidy levels of these species
2. Evaluate the chromosome numbers of D. alata, rotundata and
dumentorum.

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CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 ORIGIN
Yams (Dioscorea species) are annual or perennial tuber-bearing and climbing
plants belonging to the family of Dioscoreaceae. Some species of yam
originated from Africa before spreading to other parts of the world while some
originated from Asia and have spread to Africa (Hahn et al., 1987). Today,
yams are grown widely throughout the tropics and they have a large biological
diversity including more than 600 species worldwide (Burkill, 1960; Coursey,
1967) but only six are widely cultivated in West and Central Africa. These
cultivated species are D. alata, D. bulbifera, D. dumetorum (Pax), D. esculenta
(Lour), and D. cayenensis (Lamk) and D. rotundata (Poir).
In spite of its importance, the little that has been written about the origin of D.
alata is mostly speculation. That great numbers of varieties are known from
India to the islands of the South Pacific, many of which are quite local in
distribution, suggests that the species was domesticated and widely distributed
very early. Furthermore, the occurrence of distinct but closely related varieties
on somewhat isolated islands suggests that it has continued to evolve in areas
where it has been introduced. De Candolle (1886) hypothesized an Indo-
Malayan center of origin for D. alata, based on the widespread varieties found

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there. Burkill (1917), a superb student of the yam, placed the origin of the
species in Southeast Asia and attempted to trace its distribution throughout the
world and gradual decline in importance as cassava, sweet potatoes, and
potatoes displaced it. The existence in Burma of two closely related wild
species, D. hamiltonii (Hook) and D. persimilis (Prain and Burk), suggested that
D. alata had been selected from these or from their hybrids. Both are
characterized by long, deeply buried tubers that superficially resemble some
cultivated but inferior varieties of D. alata. Burkill (1917) hypothesized that
deep tubers evolved as a protection from wild pigs and that human selection
resulted in the short-tubered, compact varieties. For varieties with upward-
curving tubers, which eventually push their way out of the soil, he hypothesized
another human selection, since this growth habit makes harvesting easy. There
is little evidence elsewhere to support Burkill’s hypothesis. Wild species related
to D. alata are also found in Papua New Guinea. Moreover, the variation in D.
alata is enormous, and some varieties are unique to the region. Indonesia also
has extremely diverse varieties that must have existed for centuries. Relying on
what is known about historical movements of people in Southeast Asia and on
the scanty information about distribution and variation in yams, Alexander and
Coursey (1969) placed the origin of D. alata in an area lying between the
distribution of D. hamiltonii (East India and West Burma) and that of D.
persimilis (Indochina). The difficulty of their theory is that cultivated races in
this area (roughly Burma) do not include many examples of the short-tubered,

10. 10
compact types found in Indonesia (especially Celebes) and Papua New Guinea.
It is highly likely that simple or primitive forms of an initial species, perhaps
well distributed, were selected locally to give rise to the principal varietal types.
This could have happened independently on widely separated islands.
Interchange and hybridizations increased variation. Inspection of varieties from
worldwide sources suggests that at least two distinct species were involved in
the evolution of D. alata, in one of which the stem was not winged.
2.2 GEOGRAPHICAL DISTRIBUTION
Dioscorea alata is a plant of the hot humid Tropics; it seldom occurs where
cool temperatures or dry periods prevail during the growing season. There is no
single area, however, where D. alata is the chief starchy food; it is almost
always utilized as one of several farinaceous crops (other yams, cassava, sweet
potatoes, and aroids) that are used to some extent interchangeably (Onwueme,
1984). In Southeast Asia, D. alata is grown chiefly as a dooryard crop. D. alata
varieties are found chiefly in the Caribbean, the north shores of South America,
and in scattered locations throughout Central America. In localized areas and
certain islands, especially Trinidad and Barbados, D. alata is of considerable
economic importance. Isolation has undoubtedly played an important role in
establishing varieties, for the principal varieties differ from island to island
throughout the Caribbean (Coursey, 1967). In South and Central America, D.
alata is hardly known and poorly distributed except for isolated, usually coastal

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pockets. A few varieties were introduced in Brazil years ago, and recently
others have been brought in. Introduced D. alata competes in some areas with
introduced African species (Lea, 1966).
The Yam Belt, where the majority of African yams are found, extends from the
Ivory Coast to Cameroon, a distance of about 3,200 kilometers (Coursey, 1987).
Yams are usually not found near the seacoast, but their production begins within
a few to about 160 kilometers from the coast and continues for one to several
hundred kilometers inland. This is an upland region, often of rolling hills, where
rainfall is regular enough through one or more periods to permit the plants to
mature. Little is known of the prehistorically distribution of yams outside the
Yam Belt. The impression frequently given is that African yams do not grow
outside this belt. However, while not as likely to be a staple food, yams are
cultivated at least as far north as Senegal, in quantity in Sierra Leone, and to a
sufficiently great extent in the Congo. The West African yams occur in limited
areas in Tanzania and possibly other regions of East Africa where climatic
conditions are suitable. Outside Africa, the West African yams are very
sporadically distributed, and their occurrence in particular regions appears to be
due to historical accident. In the Caribbean, for example, the pattern of
cultivation is not rational; whereas very good cultivars of D. rotundata are
important in Jamaica, Puerto Rico, and the French West Indies, they are
virtually unknown on other islands (Candolle, 1886). The African yams are

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seldom seen in Southeast Asia or on the islands of the Pacific except in New
Caledonia, where they were introduced by the French. In several areas of Brazil
they have also become important. Wide distribution of the West African yams
appears desirable. Because of their adaptability to areas somewhat drier than
those where D. alata is grown and because of the earliness of most cultivars, the
African species, especially D. rotundata, should be able to fill a special niche
where other species of yams are less successful (Alexander and Coursey, 1969).
2.3 TAXONOMY OF PLANT
The genus name Dioscorea was chosen by Linné in honour of the Greek medico
and herbalist Dioscorides, who lived in the first century AC (Sprecher von
Bernegg, 1923). Plants of the genus Dioscorea are angiosperms that were most
times and are presently taxed to belong to the monocotyledon plant class
Liliopsida, the subclass Liliidae that comprises the orders Asparagales,
Orchidales, Pandanales, Liliales and Dioscoreales. The order Dioscoreales is
characterized by some dicotyledonous features, i.e. reticulate-veining, stalked
net-nerving leaves, circular arranged vascular bundles in the stem cross-section
and lateral position of the pistil (Purseglove, 1988).
The order Dioscoreales comprises three plant families, namely the
Dioscoreaceae, called the yam plants, the Trilliaceae and the Smilacaceae.
Among the otherwise tropical plants in the family of Dioscoreaceae, Tamus
communis is the only representative in temperate regions (Brenner, 1912).

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Beside dicotyledonous features already mentioned, representatives of the
Dioscoreaceae plant family show a second delayed cotyledon, which renders
the family interesting for the discussion of possible phylogenetic relations
between mono- and dicotyledonous plants, even if the traditional division of the
angiosperms in mono- and dicotyledonous plants was formally given up with
the introduction of the Magnoliopsida as distal class of the angiosperms (Frohne
& Jensen, 1998). In the southern United States the name yam is used for sweet
potato (Ipomoea batatas, L. Poir) and elsewhere for the edible tubers of aroids
(Frohne & Jensen, 1998; Purseglove, 1988).
More generally and in the present investigations the term yam is confined to
plants of the genus Dioscorea. This genus was widely reported as comprising
around 600 species (Burkill, 1960). More recent reports count approximately
200 species, distributed throughout the tropics and subtropics (Ayensu, 1972).
The genus Dioscorea is subdivided into five sections. The section
Enantiophyllum comprises most of the economically important yam species (D.
alata, D. cayenensis, D. rotundata) as well as two species of minor importance
(D. opposita, D. japonica) and is characterized by vines twining to the right
(dextrorsely). Species of the sections Lasiophyton (D. dumetorum, D. hispida),
Opsophyton (D. bulbifera), Combihum (D. esculenta) and Macro-gynodium
(D. trifida) twine to the left (Burkill, 1960).

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D. dumetorum is one of the six species of yam cultivated in Nigeria. It is an
important food security crop and is mostly consumed in West Africa. It
originated in tropical Africa and occurs in both wild and cultivated forms. Its
cultivation is mainly in West and Central Africa especially Nigeria and
Cameroon (Sefa-Dedeh and Afoakwa, 2002). It is easily identifiable by its
trifoliate compound leaves that twine clockwise unlike most other yams of
economic importance. The local names in Nigeria are: Kosanrogo in Hausa,
Ona in Ibo and Esuru in Yoruba. Other common names are three-leaved
(trifoliate) yam, bitter yam, cluster yam and sweet yam in Cameroon. In Yoruba
tribe of Nigeria, the wild type is called Esuru-Igbo or Gudugudu. It is the most
nutritious of the commonly cultivated yam species. It is a good source of
carbohydrate, protein, vitamins and minerals when compared with other
common species of yam (Alozie et al., 2010). The amino acid profile of the yam
has been reported to be quite balanced in essential amino acids with slight
deficiency in sulphur containing amino acids and lysine as the most limiting
(Alozie et al., 2009).
2.4 AGRONOMY CHARACTERISTICS OF YAM
Yam is a plant of the tropical climates and does not tolerate frosty conditions
(Coursey, 1967). Temperatures below 20 °C impede the growth of the plant
which needs temperatures between 25 and 30 °C to develop normally. Light
intensity is known to affect growth and tuber formation. Short days between 10

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to 11hr promote tuber formation, while days longer than 12hr promote vine
growth. This is usually the reason why yam vines are staked to ensure
maximum interception of light by the leaves to promote yield (Coursey, 1982;
Okezie, 1987). An annual rainfall of about 1000 mm spread over five to six
months and deep, fertile, friable, and well-drained soils are ideal for yam
cultivation (IITA, 2009). Most food yams give the highest yields in areas where
long rainy seasons prevail. Yam is also able to survive long dry periods, though
yields are reduced considerably.
Traditionally, yams are propagated vegetatively from whole tubers (seed yams),
large tuber pieces (sets) or from minisetts (Otoo et al., 1985). The growth of
yam starts with a sprout from the post dormant tuber (Passam, 1977; Onwueme,
1984). According to Craufurd et al., (2001) and Sobulo (1972), yams exhibit a
sigmoidal growth pattern common to most annual plants. A period of slow
growth during establishment is followed by a phase of rapid exponential growth
as the canopy reaches maximum area and, finally, growth rates decline as the
canopy senesces. Maturity has not been well defined in yam even though it is
traditionally measured by the dryness of vines (Okoli, 1980). Osagie and Opute
(1981) also reported that the physiological status of yam tuber at harvest may
influence its storage period and food quality characteristics.

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2.5 PLANT MORPHOLOGY
Yam has an annual vegetative system composed of a root apparatus (some
extend throughout the upper layers of the soil, others consist of root hairs), a
stem apparatus, and a foliar apparatus. The adventitious roots arising from the
base of the stem absorb mineral nutrients, and water (Orkwor et al., 1998). The
yam stem is usually a thin twining vine allowing the plants to climb. It is
frequently winged and commonly spiny. Several species have deep striations in
their stem, some contain anthocyanins, and others have large thorns. The
direction of the twining is used as a taxonomic feature. The leaves are petiolate
(except for D. dumetorum, D. hispid and, D. pentaphylla, which have trifoliate
leaves, and hairs on their stems), and have an arrangement either opposite or
alternate with axillary buds (Frohne and Jensen, 1998). The reproductive system
consists of sexual components, and a male or female inflorescence (Frohne and
Jensen, 1998). The genus Dioscorea is dioeceous with an extremely irregular
production of male and female flowers pollinated by insects (Ayensu, 1972).
The seed is flat, has a wing-like structure, and usually goes through a dormancy
period of three to four months before germination can occur. As flowering is
rare, yams are vegetatively propagated using the basal nodal region, the tuber,
and the bulbils (Ayensu, 1972). The tuber, the economically important part of
the yam plant, is rich in carbohydrates, and contains modest amounts of mineral
matter (calcium, iron), vitamin B, vitamin C and crude fibre (Brenner, 1912).

17. 17
Plants usually produce a single annual tuber, which is 20 to 40 cm long and
weighs from two to several dozen kilograms, depending on cultivar, and
growing conditions. The body can be elongated or spherical with a white,
yellow or purple flesh. The bulbils, which are a characteristic of D. alata, are
produced on the leaf axis, and weigh about 20 to 100 g. By contrast, D.
bulbifera produces only aerial tubers, which can weigh up to one kg (Burkil,
1960).
D. alata is a glabrous vine that climbs by twining to the right. The length of the
vine varies from 2 to 30 meters or more in primitive forms. The fleshy stems are
characterized by wings, typically membranous and often ruffled. In some
varieties the wings are reduced or almost absent, but the stems may be quite
thorny, in which cases the thorns occur in straight lines representing the wing;
the habit is associated with two distinct tuber types and with a distinct leaf
shape (Burkil, 1917). In rather primitive varieties with well-developed wings,
the wings of the thickened basal stem are often modified into thick, blunt thorns
quite different from the thorns of the Feo type. Wings are believed to help the
stem grasp smooth objects in twining. The leaves are often quite large, although
those of the better varieties may be smaller. They are commonly opposite on the
stem, but in a few varieties and on the young vigorous stem of most varieties,
they are alternate. The leaves are glabrous, with a slight bluish bloom in a few
varieties. Many varietal differences can be distinguished. The shape of the leaf

18. 18
is determined principally by the width of the sinus between the leaf lobes
(Coursey, 1967). Varieties differ in the folding and undulation of leaves, in the
thickness of the lamina, in green color intensity, and in anthocyanin coloration.
The wings of the petiole of the leaf are usually enlarged at the stem, producing a
characteristic appearance. Stipules are rarely present. The flowers of D. alata
are produced during the last third of the growing season, when tubers are
forming. Not all varieties flower, and some flower sporadically, perhaps in
response to seasonal variations (Coursey, 1967). It is unknown whether
flowering can be induced by changing environment. Male and female flowers
are borne on different plants. The male flowers are small (1-2 millimeters in
diameter) and are produced in crowded panicles that originate in the axils of the
leaves and the tips of the branches. The sessile flowers range from green to
white. All six stamens are well developed. In many varieties, the male flowers
do not open or open such a small amount that pollinating insects cannot enter
(IITA, 2009). The pollen of most varieties, as seen under the microscope, is
malformed or aborted, but a few varieties, especially of the wild or primitive
type, appear to be functionally fertile. The female flowers are borne on long
racemes from the axils of the leaves. The flowers are characterized by a
prominent trilocular inferior ovary from 8 to 15 centimeters long. The capsules,
which develop rapidly after pollination, reach 20 to 30 millimeters in length and
they mature, dry, and split along the sutures of the wings to release two seeds
from each of the three locules (Sprecher von Bernegg, 1929). Few capsules are

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produced, however, even when male and female plants are grown together.
Moreover, the developing capsules often contain only a single seed, and this
frequently aborts before maturity. Thus, as in the case of males, most female
plants are sterile. Only newly germinated seeds have true roots; the roots of
developing tubers are strictly adventitious, the tuber being node less and
originating from the stem. The principal roots arise from what is sometimes
called the primary nodal complex, or crown, of the plant, near the surface of
the ground, but in some varieties, a variable number of roots arise from the body
of the tuber itself. When stems touch moist soil they often develop adventitious
roots and may even develop large tubers (Craufurd et al., 2001). The tubers of
D. alata are extremely variable, and the number of possible forms almost
endless. Plants normally bear only one tuber, but two to five are common, even
on varieties typically producing one tuber. These are normally located vertically
in the soil and usually descend. Unusual varieties are known in which, after
growing down awhile, the tip of the tuber begins to grow upward, producing a
long, thin, U-shaped tuber. Tubers often enlarge as they descend to form a
pyramid, but they may also be constricted later in a spindle shape. Tubers of
some varieties are extremely smooth, while those of others are extremely
irregular. Shape is complicated by tuber branching, which may occur during
early or late growth. There may be few or many branches, either the same size
as the main tuber or smaller, and the branches may themselves continue to
branch (Craufurd et al., 2001). Some of the most unusual shapes are found

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among the New Guinea yams, in which the tuber can occur as a series of
vertical intersecting plants. The tubers are renewed annually. A tuber produced
one year gives rise in the next to a shoot, the growth of which is usually
preceded by the appearance of the primary nodal complex from which roots,
new tuber, and stem arise. The new tuber remains extremely small during the
initial months of growth, but develops rapidly near the end of the growing
season. Many varieties also produce single or multiple aerial tubers in the axils
of the leaves at the same time underground tubers are being formed (Onwueme,
1984). Aerial tubers often resemble underground tubers in shape and branching
habit. When they touch damp soil, they grow into it rapidly. In this way a single
plant can give rise to many small, rooted tubers in a short time. Aerial tubers
continue to be produced until foliage dies back at the end of the growing season.
All are edible, but the smaller ones are immature and inconvenient to prepare
for cooking. Varieties are distinct with respect to flesh color (IITA, 2009).
Anthocyanins color some flesh purple, usually irregularly. The flesh of varieties
with no anthocyanin varies from white or cream to yellow. Unidentified
carotenoids are among the pigments. Purple-fleshed varieties are favored over
white in the Philippine Islands because of the agreeable color that they lend to
desserts. Nevertheless, anthocyanins have no nutritional value, and in the
opinion of many persons, distract from the appearance of the cooked tuber. Both
smooth- and coarse-textured varieties are found. Coarse tuber texture, which
appears curd-like, is attributable to bundles of storage tissue in a somewhat

21. 21
clear matrix. When a tuber is cut, the flesh exudes mucilaginous substances,
now known to be glycoproteins, which can discolor the surface, slowly or
rapidly, upon oxidation. Exposed to air, cuts are healed by the formation of
layers of dead, dry cells. There is no evidence of tuber in deposition in this
species, but dried cells effectively reduce moisture loss and fungal penetration
(Ayensu, 1972).
D. rotundata is a vigorous vine that climbs by twining to the right. Their foliage
may be entirely glabrous, and it may have a purplish waxy bloom. Spines are
very common, especially on the lower and larger stems. The stems may be
somewhat striated vertically, and the diameter varies among varieties. The
length of the vine varies, but heights of 10 meters are easily accomplished. The
leaves are extremely varied. Their length varies from 4 to 20 centimeters
(Orkwor et al., 1998). Shapes are ovate, cordate, or almost orbicular. The form
of the leaf is also influenced by the degree of folding, reflexing, cupping,
undulation of the margins, and the position on the plant. In addition, leaves vary
in number of veins, in presence and size of lobes, in the size of the sinus
between lobes, in rugosity, and in intensity of color. These traits, or
combinations of traits, are useful in identifying varieties. Leaf veins and stems
contain variable amounts of anthocyanin, but in contrast to D. alata, the two
African species are never brightly colored by anthocyanin. Anthocyanin
coloration is a varietal trait (Sadik and Okereke, 1975a). The petioles are

22. 22
usually enlarged at the two extremes and may be striated. The flowers of D.
rotundata are usually produced early during the vegetative cycle. In Ibadan,
Nigeria, most D. rotundata cultivars usually start flowering in June. The
percentage of flowering plants in a yam field is low, and among plants that
flower, there is a preponderance of male over female plants. D. rotundata
usually produces male and female flowers on separate plants (IITA, 2009).
Sadik and Okereke (1975a) reported plants that produce male, female, or
complete flowers on the same plant. The male flowers of D. rotundata are 1 to 3
millimeters in diameter, sessile, and borne on spikes subtended by small bracts.
The perianth is slightly connate at the base and consists of three light-green
sepals and a corolla of three light-yellow petals. The androecium consists of two
whorls of three stamens each. Anthers are basifixed on short, slightly connate
filaments and are composed of two thecae dehiscing in extrorse and introrse
fashion. The highly vacuolated pollen grains are small and sticky, which makes
pollination by wind impossible. Female flowers are seen infrequently in D.
rotundata, a consequence of the complex determining mechanism that results in
more males than females. Female flowers occur on axillary spikes and are about
0.5 centimeter long. The perianth consists of two whorls of three sepals and
three petals, which are lobed over the ovary. The pistil is single, with a
trifurcated stigma. Two to three staminodes are sometimes present and are
located peripherally to the style. The ovary is inferior and trilocular, with each
locule containing two ovules. Upon maturation, the perianth dries out while the

23. 23
ovary continues its development into a capsule, which opens vertically and
releases up to six seeds with thin leathery wings that help in seed dispersal. The
tubers of both species are much less variable than those of D. alata. They are
most commonly cylindrical and seldom branched. On encountering an obstacle
a tuber may develop a foot, lumps, or appendages. The tuber skin, being thick
and corrugated, provides good protection in storage. The size of the tuber varies
from less than 200 grams in wild types to about 25 kilograms in cultivated
varieties. The flesh of the D. rotundata tuber is white and because of the
presence of vascular bundles, around which starch deposits are formed, appears
curd like. The flesh of D. cayenensis is usually yellow and smoother than the
flesh of D. rotundata. Aerial tubers are seldom produced but may be stimulated
by accidental or intentional girdling of the stem (Sadik and Okereke, 1975).
2.6 PEST, DISEASES AND CONTROL
Weeds can be serious competitors with yams. While hand-weeding is the most
common practice, pre-emergence spraying with atrazine or ametryn will control
weeds until plants have sprouted; subsequently paraquat carefully applied with a
shielded spray may be used. In due course the foliage should become thick
enough to cover the ground and eliminate weeds, especially when the vines are
unstaked. Pests: yam beetles of several species are important, especially in
Africa: these include the greater yam beetle (Heteroligusmeles), the lesser yam
beetle (H. appius), also Heteronychuslicas, Prionoryctes rufopiceus, P.

24. 24
caniculus and Lilioceris spp. These attack the tuber setts and may prevent
sprouting. Dusting the plant setts with 2% aldrin or 0.5% gamma-HCH will
normally prevent attack. The termite, Amitermes evancifer, is occasionally a
serious pest of yam tubers in Africa. Yam scale (Aspidiella hartii) attacks stored
yams in Africa, Asia and the Pacific. Several species of nematodes attack yams.
The yam nematode, Scutellonema bradys, is widely distributed in both Old and
New World tropics and causes ‘dry rot’ of the tubers. Diseases include
anthracnose (caused by Glomerella cingulate), which produces black necrotic
lesions on leaves and stems, and can kill the plant by attacking the terminal bud,
and leaf spot, caused by various species of Cercospora, Colletotrichum, and
Phyllosticta. Control involves sanitation by removal of crop debris, and
fungicide treatment: maneb, benomyl, zineb and mancozeb have all been
reported to give reasonably good results (Noon, 1978).
2.7 CYTOLOGY
Chromosomes were first seen by C. Nägeli in 1842, and named in 1888 by W.
Waldeyer. Walther Flemming studied and documented the behavior of
chromosomes during cell division, a process he termed mitosis. We will
perform experiments similar to these early scientists. Cell division is especially
rapid in the growing root tips of sprouting seeds. The ability to count
chromosomes is among the valuable tools used by plant breeders and
cytogeneticists. This exercise is difficult with yams (Dioscorea spp.) since the

25. 25
chromosomes are small, dot-like; frequently having only a few cell divisions
visible in a single root tip (Dansi et al., 2000)
Generally, it can be said to be a thread-like structure of nucleic acids and protein
found in the nucleus of most living cells, carrying genetic information in the
forms of genes. Each chromosome is made up of DNA tightly coiled many
times around proteins called histones that support its structure. Chromosomes
are not visible in the cell’s nucleus – not even under a microscope – when the
cell is not dividing. However, the DNA that makes up chromosomes becomes
more tightly packed during cell division and is then visible under a microscope
(Allison, 2007). Each chromosome has a constriction point called the
centromere, which divides the chromosome into two sections, or “arms”. The
short arm of the chromosome is labeled the “p arm”. The long arm of the
chromosome is labeled the “q arm”. The location of the centromere on each
chromosome gives the chromosome its characteristic shape, and can be used to
help describe the location of specific genes (Allison, 2007). Numerous counts
have been made of the chromosomes ofD. alata. All numbers reported, whether
based on pollen-mother- cell or root-tip observations, are based on multiples of
10. The occurrence of 2n numbers such as 30, 50, or 70 can only be accounted
for through the hybridization of varieties with different numbers of sets of 10
chromosomes (Miĕge, 1954). Thus, polyploidy is normal in the species and
probably accounts for its high sterility. Variations in chromosomal number

26. 26
within the cells of a single variety have been reported. Such variation can be
fixed by asexual propagation and in fact probably accounts for the occurrence of
sibling varieties—that is, varieties that differ only in minor details. Odd
chromosomenumbers due to the presence of one or two extra chromosomes are
uncommon, and such counts are not reliable. Aneuploidy, when it occurs, would
contribute to the sterility of the species. Extra chromosomes have been
attributed to a sex-determining mechanism. There is, however, no concrete
evidence to suggest an XO sex-determining system (Martin, 1960).
Furthermore, Martin (1960) has shown how polypoidy can explain the unusual
sex ratios found in most species. Males predominate over females in most
controlled crosses, in predictable ratios associated with numbers of sex-
controlling chromosomes present. As previously noted, male and female
flowers are usually sterile, and in varietal collections seeds are seldom
produced. No efforts to breed the wing-stemmed yam have been reported in the
literature. It is difficult to escape the conclusion that existing varieties are very
old and perhaps have diverged from their progenitor varieties by somatic
mutation. Nevertheless, a sexual system must have been present at one time to
have produced the variation exhibited by this species. Primitive man
presumably practiced selection and gradually produced the fine varieties now
known. It appears that this process of improvement is no longer possible.

27. 27
Chromosome numbers of Dioscorea are based on multiples of 9 and 10, the
latter being the most common. Miège (1954) has postulated a third basic
number of 12. Odd numbers of chromosomes deviating from these multiples
have been reported and indeed could partly account for the sterility of some
species, but such numbers are hard to verify. The chromosomes of Dioscorea
are particularly small, and determination methods, very tedious. Chromosomes
of the African species have been counted by Miège (1954). Races of D.
rotundata and D. cayenensis have either 36 or 54 chromosomes. These numbers
correspond to tetraploid and hexaploid numbers, respectively. In the Ivory
Coast, the 36 chromosome races are found in the north and the 54 chromosome
types in the south. In the 36 chromosome races, meiosis is normal, which
suggests that the species is a very old tetraploid. Occasional reproductive
failures and sterilities in open pollinations and controlled crosses could be
associated with hexaploid numbers. The study of chromosome numbers has not
been sufficiently extensive too well characterize the two species. Wider study
could shed light on their origins and their relationships. However, this field of
study has been neglected in recent years. The production of seed by female
plants is not common but is enhanced by planting several varieties together.
Seedlings have been grown by a number of different investigators in what might
be described as preliminary breeding attempts (Lawton and Lawton, 1967).

28. 28
The seedlings are quite tender and may require 2 years to produce a suitably
large tuber. Breaking of the normal dormancy period, which inhibits
germination during the dry season, may make possible a 1 year seed-to-seed
cycle. Efforts to breed D. rotundata have been begun by Sadik and Okereke
(1975) at the International Institute of Tropical Agriculture in Ibadan, Nigeria.
Three generations of plants, about 40,000, have been raised from seeds
produced by natural crossing. These plants flower readily and cross with ease;
moreover, they exhibit a wide spectrum of genetic diversity in respect to disease
resistance, degree of flowering and fruiting, and many morphological features.
The use of seeds has increased the percentage of flowering offspring twofold
over plants grown from tuber cuttings, and the percentage of female plants and
the numbers of flowers and fruits per plant have also increased. Of interest is
the production of monoecious plants, which could provide self-pollination
capabilities for breeding. In addition, a number of plants have been dwarf or
semi dwarf, ranging in height from 30 to 70 centimeters. These could be
important in that such plants do not require staking. Characteristics of
importance in breeding include tuber shape and size, disease resistance, and
eating and pounding quality (Sadik and Okereke, 1975).

29. 29
CHAPTER THREE
3.0 MATERIALS AND METHOD
3.1 MATERIALS
Watch glass, specimen bottles, razor blades, cover slips, microscope slides,
immersion oil, forceps, specimen root tips, planting container, concentrated
HCL, ethanoic acid, FLP orcein stain, microscope, thermometer, filter papers,
beakers, distilled water, measuring cylinder, and hot plate.
3.2 REAGENTS AND CHEMICALS PREPARATION
3.2.1 8- HYDROXYQUINOLINE SOLUTION
Roots tips were pretreated with 8-hydroxyquinoline which was prepared by
dissolving 0.029 of 8-hydroxyquinoline salt in 100ml of distilled water. This
causes contraction and improves spreading of chromosomes. Heating of the
mixture which allows the salt to dissolve and is filtered with aid of funnel, filter
paper and conical flask.
3.2.2 CARNOY’S FLUID SOLUTION
Carnoy’s fluid was prepared in the ratio of 3:1 which is 75% of ethanol and
25% of glacial acetic acid. The fluid further disintegrates the cell wall of the
nucleus.

30. 30
3.2.3 70% ETHANOL
Using measuring cylinder, 70ml of ethanol and 30ml of distilled water were
used to prepare 70% ethanol. The root tips were stored in this solution after
pretreatment for whenever they are needed.
3.2.4 5% AND 18% CONCENTRATION OF HYDROCHLORIC ACID
5ml of conc. HCL and 95ml of distilled water make up for 5% concentration of
HCL while 18ml of conc. HCL and 82ml of distilled water make up for 18%
concentration of HCL. This solution helps to further disintegrate and soften the
root tips because they can be hard to squash after pretreatment.
3.3 METHODS
3.3.1 COLLECTION OF PLANT MATERIAL
Dioscorea alata and rotundata seed yams were gotten from Eke awka market,
Awka and Dioscorea dumentorum seed yams were gotten from Nimo market,
Nimo all in Anambra State. They were planted in a good loamy soil. The roots
served as specimens for cytology of roots.
3.3.2 PLANTING AND HARVESTING
Viable dioscorea alata, dumentorum and rotundata seed yams were planted for
a period of 14 days and afterwards placed in transparent plastic containers

31. 31
carrying water to enable evident existence of root tips for the practical. These
were harvested after the roots are evident enough.
3.3.3 PRE TREATMENT
The active roots were washed of adhering soil particles with clean water and cut
from the tip about 1 – 2cm into 0.002M 8-hydroxyqunoline solution where they
were allowed to stay for 4 hours in order to increase the percentage of cells at
metaphase stage by inhibiting spindle formation. Due to the hardness of yam
root tip during squashing, it was extended to 6 hours.
3.3.4 FIXATION
The roots were transferred with the aid of a sterilized forceps into Carnoy’s
fluid. They were kept in the fluid for 24hrs.
3.3.5 HYDROLYSIS
The specimens were transferred into 5% and 18% HCL (acid added to water for
safety) and contained in sample bottles and allowed to stay about 5 minutes
with and without heating. This process was carried out in order to soften the
root tissues and loosen the cement substances between cells and allow good
spread of cells during squashing.

32. 32
3.3.6 STAINING AND SQUASHING
Using forceps, the hydrolyzed roots were mounted on clean sterile slide with the
unwanted tissues discarded leaving behind the whiter portion onto which about
two drops of FLP orcein was added and squashed using a biro tip. A clean and
sterile cover slip was gently laid on top of the specimen. The slide was placed
between a large filter paper placed on a smooth but hard surface and thumb
pressed to blot out excess stain as well as spread out cells. Care was taken to
avoid lateral movement of the cover slip. The slide was then mounted onto the
microscope for viewing at X1000 magnification using oil immersion. Scroll up,
down and sideways to view and take pictures with a camera when visible
chromosomes are seen and are at their mitotic stages (interphase, metaphase,
anaphase and telophase). Manual method of counting of visible chromosomes
was used.
3.4 HOW TO COUNT CHROMOSOMES
The rule of thumb
The number of chromosome = count the number of functional centromere.
The number of DNA molecule = count the number of chromatids.
Rule 2
Number of chromatid = one i.e. number of DNA molecule = one.

33. 33
Number of chromatid = two i.e. number of DNA molecule = two.
These methods were repeated more than 15 times so as to get a clear and good
picture that has very visible chromosome number.

34. 34
CHAPTER FOUR
4.0 RESULT
The three species of yam, Dioscorea rotundata, alata and D. dumetorum were
evaluated and found to have chromosomenumbers ranging from 20 to 50. The
genomic number of chromosomes of the three species are a multiple of the basic
chromosomenumber, 10 (fig 1 – 3).
For the species of yam researched on, it was discovered that in their diploid
level, Dioscorea dumentorum is a pentaploid with the chromosomenumber of
5n = 50, Dioscorea rotundata is a tetraploid with the chromosomenumber of
4n = 40 while Dioscorea alata is a diploid with the chromosomenumber of 2n
= 20. Based on conventional chromosome, the chromosomewas determined as
x = 10 for the various species studied (Figure 1 – 6).
Figure 1.chromosomes of D. dumentorum.

35. 35
Figure 2.chromosomes of D. rotundata
Figure 3.chromosomes of D. alata

36. 36
Different stages of mitosis were observed in the evaluation of the species.
Figure 4. mitotic chromosomes in the root tip cells of D. dumentorum showing
prophase(a), metaphase(b), anaphase(c) and telophase(d) stages of mitosis.
Figure 5. prophase(a), metaphase(b) and telophase(c) stages of mitosis of D.
rotundata
a b c d
a b
c

37. 37
Figure 6. mitotic chromosomes of D. alatashowing the interph ase(a),
prophase(b), metaphase(c), anaphase(d) and telophase(e) stages of mitosis.
a b c d e

38. 38
CHAPTER FIVE
5.0 DISCUSSION AND CONCLUSION
5.1 DISCUSSION
A basic chromosome number, x = 10 was reported by Dansi et al., (2000a).
Generally, the dot-like and clumping nature of the chromosomes made counting
difficult. In yams, the occurrence of one or two extra chromosomes in cells of
individual genotypes is not rare (Gamiette et al., 1999; Dansi et al., 2000b).
However, the presence of the extra chromosomes is often attributed to the B-
chromosomes or satellites which are sometimes as large as the chromosomes
themselves as opposed to aneuploidy (Dansi et al., 2000a). The B-
chromosomes, which may be involved in directing non-disjunction of
chromatids during cell division, are dispensable and extra to the basic A-
chromosome set (Hasterock et al., 2002). The results were also in agreement
with the ploidy results obtained from FCM, which indicated that FCM was a
reliable technique for rapid determination of ploidy level in yams (Dansi et al.,
2000a; Norman, 2010). Both flower buds and root tips are vital materials in
karyology for the determination of chromosome numbers by the conventional
counting method. Roottips method is a quicker means of locating chromosomes
in cells in mitosis. Chromosomes are not pairing providing the opportunity to
count each one individually. Wang et al., (2010) adduced that variability in
chromosome size is possibly influenced by meiosis consequently leading to

39. 39
non-independent assortment of chromosomes. Their findings suggested that
insertions and deletions may influence chromosome segregation patterns. The
flower bud method was not used in this experiment due to the lack of flower set
among species studied. The technique also make counting more difficult due to
the pairing of the dot-like and the clumping of yam chromosomes compared to
other plant species.
In the course of this research, it was discovered that the root tips of D.
rotundata are hardened by different concentration of HCL (5% and 18%) and
the hardness of these root tips break cover slips while squashing so it is advised
to hydrolyze before fixing in Carnoy’s fluid.
5.2 CONCLUSION
An adequate knowledge of the chromosome/ ploidy nature of yam is needed for
its effective utilization in yam breeding programme. Since yam chromosome
counting technique appears to be difficult in accessing the ploidy level of the
crop, flow cytometry offers a relatively easy and adequate means of such
evaluation. It is believed that the information generated from this study would
provide guidance in a yam improvement programme both in terms of selection
of initial breeding material and choice of breeding methods. The development
of flowering species and the exploration of chromosome size and content form
part of future research to improve the agriculture sector in Nigeria.

40. 40
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APPENDIX