KARYOMORPHOLOGICAL STUDIES IN TREE LEGUMES
OGUNTUASE OLUWASEUN A.
(B.SC PLANT SCIENCE)
MATRIC. NUMBER 146574
A literature review in the department of BOTANY AND MICROBIOLOGY, Faculty of
Sciences.
Submitted to the School of Postgraduate Studies, University of Ibadan, in partial fulfilment
of the requirement for the award of the degree of MASTER OF SCIENCE
UNIVERSITY OF IBADAN.
AUGUST, 2010.
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CERTIFICATION
This is to certify that the literature review of this thesis and the subsequent preparation of this
thesis by Oguntuase Oluwaseun Anthony, Matriculation number 146574 were carried out under
my supervision and that to the best of my knowledge this work has not been presented elsewhere
for the award of any degree.
.....................................................................
Dr. A. I. Adesoye
(B. Sc, M. Sc, PhD. (Ibadan))
(Supervisor)
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DEDICATION
I dedicate this research work to God the Father, the Almighty; Jesus Christ, the only Son of God
who is in being with the Father; and to the Holy Spirit, the Lord, the Giver of life, who with the
Father and the Son is adored and glorified.
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ACKNOWLEDGEMENT
I wish to thank God for the possibility and attainment of this Master’s programme and this
literature review. May His kingdom come.
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TABLE OF CONTENT
TITLE PAGE..............................................................................................................................i
CERTIFICATION ....................................................................................................................ii
DEDICATION............................................................................................................................iii
ACKNOWLEDGEMENT..........................................................................................................iv
TABLE OF CONTENTS...........................................................................................................v
LIST OF TABLES......................................................................................................................ix
LIST OF FIGURES....................................................................................................................x
ABSTRACT................................................................................................................................xi
CHAPTER ONE
1.0
INTRODUCTION...........................................................................................................1
1.1 Legume.........................................................................................................................1
1.2 Classification of Legumes.............................................................................................1
1.3 Tree Legumes................................................................................................................2
1.4 Importance of Tree Legumes.........................................................................................3
1.4.1 Degradation of Natural Systems.....................................................................4
1.4.2 Soil Reclamation and Erosion Control...........................................................4
1.4.3 Tree Legumes as Forage for Animals.............................................................5
1.4.4 Tree Legumes for Fuelwood...........................................................................6
1.4.5 Tree Legumes As Planted Forage In Cropping and Grazing Systems ...........8
5
1.4.6 Tree Legumes as Nutrition Supplements........................................................9
1.4.6 Tree Legumes as Nutrition Supplements........................................................9
1.11 Objectives of the Review.............................................................................................9
CHAPTER TWO
2.1 Introduction to Karyomorphology..........................................................................................11
2.2 Karyotypes and Morphology...................................................................................................11
2.3 Karyotypes and Karyomorphological Studies ........................................................................11
2.3.1 Plants in General.......................................................................................................11
2.3.2 In Legumes................................................................................................................12
2.4 Techniques Used in Karyomorphological Studies...................................................................13
2.4.1 The Conventional Method........................................................................................14
Plant Material.........................................................................................................14
Pretreatment..........................................................................................................14
Fixation..................................................................................................................15
Hydrolysis..............................................................................................................15
Staining..................................................................................................................15
2.5 Some New and Modern Methods...........................................................................................16
2.5.1 Flow Cytometry Method..........................................................................................16
6
2.6 Depiction of Karyotypes..........................................................................................................17
2.6.1 Classic Karyotype Cytogenetics...............................................................................18
2.6.2 Spectral Karyotype (SKY Technique)......................................................................18
2.6.3 Digital Karyotyping..................................................................................................19
2.7 Methods Used In the Analysis of Karyotypes Studies...........................................................19
2.8 Parameters Observed In Karyomorphological Studies............................................................20
CHAPTER THREE
3.0 Applications of Karyomorphological Studies..........................................................................22
3.1 Determination of Ploidy Level.....................................................................................22
3.1.1 Ploidy............................................................................................................22
3.1.2 Haplo-diploidy..............................................................................................22
3.1.3 Endopolyploidy.............................................................................................22
3.1.5 Chromosomal Polymorphism.......................................................................23
3.2 Other Uses of Karyomorphological Studies................................................................24
CHAPTER FOUR
4.0 Review of Karyomorphological Studies In Some Leguminous Trees.......................26
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CHAPTER FIVE
5.0
Conclusion.........................................................................................................................34
5.1 Chromosome Studies In Tree Legume........................................................................34
5.2 Ploidy Level In Tree Legume......................................................................................34
REFERENCES.............................................................................................................................36
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LIST OF TABLES
1. Chromosome Number and Ploidy Level of Some Major Tree Legumes.......................26
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ABSTRACT
Worldwide, chromosomes have been counted for many tree legumes. As very few are known for
some genus there have been some extensive knowledge in some others. 169 tree legumes were
reviewed among legume trees which include Abrus (1), Albizzia (2), Alhagi (1), Astragalus (1),
Bauhinia (3), Cajanus (11), Dalbergia (10), Erythrina (1), Gleditsia (2), Laburnum (2), Mimosa
(2), Prosopis (11), Robinia (1), Senna (1), Sesbania (1), Tamarindus (1), Acacia (60), Cassia
(11), Leucaena (11), Lonchocarpus (46). The chromosome numbers and the ploidy level were
majorly reviewed among other information documented at KEW Database on these genera. It
was observed that most sampled tree legume have their chromosome number n = 13. The ploidy
level ranges from 2 to 8. Constant chromosome number of 22 was noted in the entire
Lonchocarpus trees assessed. It was also observed also that genera Prosopis and Cassia have the
same chromosome number of 28. Likewise Abrus, Cajanus and Lonchocarpus have the same
haploid number of chromosomes as n = 22. The genera Astragalus, Robinia, Senna, Sesbania,
Erythrina, Gleditsia, Laburnum, Abrus, Albizzia, Alhagi, and Bauhinia are under-represented to
justify the constancy or differences of the chromosome numbers and ploidy levels. Between the
genera Cassia and Astragalus with the highest and lowest chromosome numbers respectively,
there are intermediate genera to bridge the gap of relatedness among the tree legume. The types
of chromosome in relation to the position of the centromere in the tree legumes have no major
documentation like their chromosome numbers and ploidy levels. Other data that were not
commonly documented for these tree legumes include the relative sizes of their chromosomes
and the number and positions of their satellite (if any). Also not listed is the degree and
distribution of heterochromatic regions of these chromosomes.
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CHAPTER ONE
1.0 INTRODUCTION
1.1 LEGUME
A legume in botanical writing is a plant in the family Fabaceae (or Leguminosae), or a
fruit of these specific plants. A legume fruit is a simple dry fruit that develops from a simple
carpel and usually dehisces (opens along a seam) on two sides. A common name for this type of
fruit is a pod, although "pod" is also applied to a few other fruit types, such as vanilla and radish
(http://en.wikipedia.org/wiki/Legume). The Fabaceae, or legumes, constitute the third largest
family of flowering plants, comprising more than 650 to 700 genera and 18,000 to 20,000
species (Polhill and Raven, 1981; Doyle and Luckow, 2003; Lewis et al., 2005). The legumes
are, in fact, an old family. The history of legumes is tied in closely with that of human
civilization, appearing early in Asia, the Americas (the common Phaseolus bean in several
varieties), and Europe (broad beans) by 6,000 BC, where they became a staple, essential for
supplementing protein where there was not enough meat. For reference, the common ancestor of
soybean and pea, estimated at approximately 54 million years (Lavin et al., 2005), predates by
10 million years the earliest complete primate fossil, Darwinius masillae (Franzen et al., 2009).
That early small primate lived in a paratropical rain forest already richly populated by
leguminous trees (Engelhardt, 1922). Legumes range from tiny herbs to giant trees, dominating
many tropical rainforests. Economically, legumes represent the second most important family of
crop plants after Poaceae (grass family), accounting for approximately 27% of the world's crop
production. Legumes cover 180,000,000 ha and 12-15% of the arable land in the world (Graham
and Vance, 2003).
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1.2 CLASSIFICATION OF LEGUMES
The legumes have traditionally been placed into the three subfamilies: the Papilionoideae,
with approximately 70% of species; the Mimosoideae, with approximately 15%; and the
remainder in the Caesalpinoideae—though this last subfamily is now known to be comprised of a
collection of early diverging legume taxa. Recent results indicate that the Caesalpinoideae is
actually paraphyletic. (Doyle and Luckow, 2003; Lewis et al., 2005). According to Young et al.,
2003, most cultivated legumes are found within the Papilionoideae, the subfamily with largest
total number of genera. This subfamily contains nearly all economically important crop legumes,
including soybean (Glycine max), peanut (Arachis hypogaea), mungbean (Vigna radiata),
chickpea (Cicer arietinum), lentil (Lens culinaris), common bean (Phaseolus vulgaris), pea
(Pisum sativum), and alfalfa (Medicago sativa). With the notable exception of peanut, all these
important crop legumes of agronomic interest fall into two Papilionoid clades, namely, Galegoid
(including the genera Melilotus, Trifolium, Medicago, Pisum, Vicia, Lotus, Cicer and Lens) and
Phaseoloid (including the genera Phaseolus, Vigna, Glycine and Cajanus), which are often
referred to as cool season and tropical season legumes, respectively. Doyle and Luckow, 2003,
and Lewis et al., 2005 also reported that the four large subdivisions in the Papilionoideae are the
galegoid, millettioid, dalbergioid, and genistoid clades. Despite their close phylogenetic
relationships, crop legumes differ in their base chromosome number and ploidy level (Table I).
Nevertheless, earlier studies indicated that members of the Papilionoideae subfamily
exhibited extensive genome conservation based on comparative genetic mapping (Weeden et al.,
1992; Menancio-Hautea et al., 1993). The papilionoid subfamily includes most crop legumes and
the major model legume species, and thus is the taxonomic space across which much of legume
comparative genomics and translational genomics will take place (Cannon et al., 2009). The
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papilionoid origin is dated at approximately 59 million years (Lavin et al., 2005). The galegoid
clade contains the robinioid clade, with birdsfoot trefoil and several allied forage and tree
legumes (including Sesbania, and Robinia, e.g. the black locust tree). Of course, other legume
genera are important to people — forest and lumber trees in Mimosoideae, such as Acacia and
Albizia; other forage crops and shade trees in the Papilionoideae, such as Robinia (locust bean);
and finally, important ornamental, timber and even food plants in Caesalpinoideae, such as
Poinciana (flame tree) and Tamarindus (the source of tamarind fruit).
1.3 TREE LEGUMES
Until recently, according to Gutteridge and Shelton (1994), tree legumes were largely
neglected by researchers because their utilisation and management fell between the disciplines of
forestry and pasture agronomy. They are now receiving increased research attention because of
their multipurpose value and some distinctive features which set them apart from herbaceous
legumes. Tree legumes can be regarded as truly multipurpose trees for agriculture in respect of
their special characteristics which include
1. They are usually long-lived and low maintenance, and therefore enhance the
sustainability of farming systems,
2. They provide high quality forage for feeding of livestock,
3. They stabilise sloping lands against erosion because of their deep-rooted habit,
4. They supply nitrogen-rich mulch for cropping systems,
5. They can be used to colonise and rehabilitate adverse environments, e.g. saline or arid
locations,
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6. They provide a source of timber and firewood for either domestic or industrial use,
7. They are used in farming systems as living fences, as shade trees for plantation crops, and
as living trellises for climbing crops, and
8. They are a source of fruit and vegetables for human consumption.
1.4 IMPORTANCE OF TREE LEGUMES
1.4.1 Degradation of Natural Systems
Unfortunately, due in large part to over-exploitation by people and livestock, valuable
tree and shrub resources over vast areas in arid and semiarid regions have been destroyed in the
last few decades. In these areas, it is important that management practices are adopted which
foster the wise use of diminishing tree and shrub resources. Livestock access should be
restricted, and pruning and harvesting of products performed on a rotational basis, to ensure time
for regeneration. In some cases, the presence of tree legumes has contributed to the degradation
of the landscape. Animals can be maintained long after the loss of palatable perennial grass
species due to drought or overgrazing, by feeding the foliage of hardy tree species. Vast areas of
South-western Queensland and East Africa have been degraded in this way.
1.4.2 Soil Reclamation and Erosion Control
The restoration and maintenance of soil fertility is a basic and critical environmental
problem. It is especially serious in tropical and subtropical regions where many soils lack plant
nutrients and organic matter and intense rainfall erodes vulnerable top soil. The nitrogen fixing
ability of tree legumes allows them to grow on difficult sites subject to erosion, low fertility or
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other adverse soil conditions. Once established, they can create conditions favourable for the
growth of other species leading to a balanced plant ecosystem. Tree legumes are a good source
of organic matter for green manure. Their dry foliage contains 2.5-5.5% nitrogen and leaf
material incorporated into the soil improves fertility, moisture and nutrient retention and general
filth. At the same time, by improving soil structure, erosion can be retarded. The extensive root
systems of tree legumes enable them to adapt to steeply sloping sites unsuited to conventional
cropping or grazing thus stabilising the sites from erosion and providing a measure of production
which would not otherwise exist. The Sloping Agricultural Land Technology (SALT) developed
in the Philippines (Tacio et al., 1987) is a prime example of the use of tree legumes in
substantially reducing soil erosion and restoring moderately degraded hilly lands to a profitable
farming system.
1.4.3 Tree Legumes as Forage for Animals
Browse has been defined as the leaves, shoots and sprouts including tender twigs and
stems of woody plants which are cropped to a varying extent by domestic and wild animals. It
should be extended to include the fruit, pods and seeds which provide valuable feed, especially if
the tree is deciduous. Trees and shrubs have provided valuable forage to man's herbivorous
animals probably since the time of their domestication (Robinson 1985). At least 75% of the
shrubs and trees of Africa serve as browse plants and many of these are leguminous (Skerman
1977). McKell (1980) pointed out that shrubs and trees are the most visible plant forms in many
landscapes, yet have been neglected in most scientific research. Much research effort has
concentrated on methods for their eradication. In some arid and semiarid climates, livestock
would not exist without browse species to supply feed. Many tree legume species have evolved
in semiarid regions alongside herbivorous animals and therefore have developed means of
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protection against browsing or grazing. Among the protective devices are thorns, toxins, fibrous
foliage and height of tree crowns (Brewbaker 1986). Thorns characterise many woody legumes
and are particularly prevalent on juvenile plants. Toxins are of two general types, those which
deter feeding and those which poison the animal.
The use of naturally occurring browse species is a vital component of livestock
production systems in many regions of the world. In the Sahelian savannahs in Africa from
Senegal to the Sudan, Faidherbia albida is a native leguminous species which is extremely
important both in providing forage for livestock and in enhancing soil fertility for crops.
Prosopis species provide forage for the sheep and cattle industries of the arid subtropical plains
of Brazil, Argentina, Uruguay and Northern Chile. Prosopis chilensis contributes regular cattle
feed in northwest Argentina and central Chile while P. tamarugo, a native of Chile's northern
plateau, is the only tree that survives on the arid salt flats producing the only available forage,
timber and fuelwood in that region. In South-western Queensland and northern New South
Wales, mulga (Acacia aneura) occurs naturally often in monospecific stands and is used as a
drought reserve for grazing sheep. Under natural conditions, a large proportion of the foliage of
tree species will be out of reach of grazing animals so utilisation can be manipulated by cutting
or lopping to make it available when needed. Sometimes natural leaf fall through senescence is
an important day-to-day component of the diet of some grazing animals. In Africa, goats thrive
on the leaf fall of Acacia melliflora (Dougall and Bogden 1958).
1.4.4 Tree Legumes for Fuelwood
In many of these intensive cropping areas, tree legumes are planted not only for their
forage but also for firewood, green manure and other uses. It has been estimated (Eckholm 1975)
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that at least half the timber cut in the world is used as a fuel for cooking and heating.
Approximately 2 billion people derive at least 90% of their energy requirements from wood and
charcoal while a further 1.5 billion meet at least 50% of their requirements this way. This
essential resource, however, is seriously threatened. If the pace of tree planting around the world
is not greatly accelerated, at least 500 million people will be without fuelwood for their
minimum cooking and heating needs by the end of the century. Tree legumes offer a partial
solution to the fuelwood crisis. Of the 88 species recommended for fuelwood production by the
National Academy of Sciences (1980, 1983), almost half were tree legumes.
Tree legumes also meet many of the characteristics which are considered desirable in
fuelwood species which include:
1. Rapid growth,
2. Nitrogen fixing ability,
3. Ease of establishment,
4. Ability to coppice,
5. Wood of high calorific value,
6. Wood which burns without sparks or toxic smoke,
7. Ability to grow well in a wide range of environments including difficult sites,
8. Multipurpose nature.
Tree legumes have been used successfully in sustained fuelwood production systems. In the early
1920s, in the Paliparan area of the Philippines, Leucaena leucocephala was planted over a large
area of unproductive Imperata cylindrica grassland. Since then it has yielded on average 20 m3
of fuelwood per hectare per year and is still the main source of fuel for the city of Laguna (NAS,
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1980) although recent damage by the Leucaena psyllid has reduced the production of wood.
Other tree legumes that are highly regarded as fuelwood species include Acacia auriculiformis,
A. saligna, A. senegal, A. tortilis, Calliandra calothyrsus, Cassia siamea, Pithocellobium dulce
and Prosopis spp.
1.4.5 Tree Legumes As Planted Forage In Cropping and Grazing Systems
As well as naturally occurring stands, tree legumes are often planted specifically for
forage both in extensive grazing systems and in association with crops. In many of the more
intensive agricultural areas of Asia and Africa, where livestock are raised in small numbers by
smallholder farmers, tree legumes are planted as 'forage banks' on unused land along field
borders or fence lines, on rice paddy bunds or in home gardens. These areas are usually
harvested under a 'cut-and-carry' system and are a principal source of high quality forage used to
supplement low quality roughages such as crop residues. Productivity from these areas can be
quite high. In the Batangas region of the Philippines, a 2 ha area of Leucaena leucocephala
grown in association with the fruit tree Anona squamosa was able to supply the forage
requirements of 20 growing cattle over a 6 month period (Moog 1985). At Ibadan in Nigeria,
Reynolds and Atta-Krah (1986) suggested that the surplus foliage produced over a year from 1
ha of Leucaena leucocephala and Gliricidia sepium planted at 4 m intervals in an alley cropping
system could be used as a supplement to provide half the daily forage requirements for 29 goats.
In the more extensive grazing areas of Australia, southern Africa and South America, tree
legumes are increasingly being planted in association with improved grasses to increase carrying
capacity and productivity of grazing cattle. In central Queensland, over 20,000 ha have been
sown to Leucaena leucocephala in the past 10 years. The Leucaena is sown in wide spaced rows
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4-10 m apart and an improved grass such as green panic (Panicum maximum var. trichoglume),
Rhodes grass (Chloris gayana), buffer grass (Cenchrus ciliaris) or signal grass (Brachiaria
decumbens) sown between the Leucaena rows. A high stocking rate (up to 3-4 animals/ha) and
liveweight gain (up to 1 kg/head/day) can be achieved with this system. A record liveweight gain
of 1,442 kg/ha for cattle grazing a grass/legume pasture was achieved on an irrigated
leucaena/pangola grass mixture in the Ord River District of north Western Australia (Jones
1986). Other tree legume species that are being investigated for use in extensive grazing systems
include Calliandra calothyrsus, Albizia chinensis, Cajanus cajan, Gliricidia sepium and
Sesbania sesban.
1.4.6 Tree Legumes as Nutrition Supplements
On a worldwide basis, legumes contribute about one-third of humankind's protein intake.
Legumes also accumulate natural products (secondary metabolites) such as isoflavonoids that are
beneficial to human health through anticancer and other health-promoting activities (Dixon and
Sumner, 2003). The nutritional quality of tree legumes varies from excellent (Leucaena
leucocephala) to quite poor (most Australian Acacia species). Poor quality can be due to tannins
which reduce the digestibility of both herbage and protein. The presence of tannins is often
evident as brownish, reddish tinges in juvenile growth. Another reason for poor quality is that
some species have phyllodes (expanded and flattened leaf petioles) instead of compound pinnate
or bipinnate leaves which are very high in fibre and therefore of low digestibility, e.g. the
Australian acacias. Forage from tree legumes is often used as a buffer to overcome feed gaps that
arise from seasonal fluctuations in the productivity of other feed sources. For example, grasses
and other herbs may die when upper soil layers lose their moisture but the deep-rooted trees
exploit moisture at depth and continue to grow. During the dry season or in times of drought,
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trees provide green forage rich in protein, minerals and vitamins while the herbaceous cover
provides only poor quality straw.
Tree legumes have an important role in many agricultural production systems throughout
the world as they can be used in a multitude of ways. The aim of this review, therefore, is to look
at the genetic status of these multipurpose trees as documented by several researchers through
their karyomorphological studies. This review also looked into how these karyomorphological
studies are being carried out, some notable results among the legume tree, and the areas of
application of karyomorphological data.
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CHAPTER TWO
2.1 INTRODUCTION TO KARYOMORPHOLOGY
This is the combine study of the karyotype and chromosome morphology of organisms. As
suggested by Mhinana et al., 2010, these studies could be used as an additional tool in the
classification of organism.
2.2 KARYOTYPES AND MORPHOLOGY
A karyotype is the number and appearance of chromosomes in the nucleus of a eukaryote cell
(White, 1973; Stebbins, 1950). The term is also used for the complete set of chromosomes in a
species, or an individual organism. Karyotypes describe the number of chromosomes, and what
they look like under a light microscope. Attention is paid to their length, the position of the
centromeres, any differences between the sex chromosomes, and any other physical
characteristics (King et al., 2006).
Morphology is a branch of bioscience dealing with the study of the form and structure of
organisms and their specific structural features (http://en.wikipedia.org/wiki/morphology).
Therefore juxtaposing with the knowledge of karyotye, karyomorphological studies focuses on
the morphological features of chromosomes. This reveals chromosomes that are metacentric,
submetacentric, intercentric, chromocenter, proximal, interstitial, acrocentric, telocentric types.
This is majorly determined by the position of their centromere.
2.3 KARYOTYPES AND KARYOMORPHOLOGICAL STUDIES
2.3.1 Plants in General – Chromosomes were first observed in plant cells by Karl Wilhelm von
Nägeli in 1842. Their behavior in animal (salamander) cells was described by Walther
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Flemming, the discoverer of mitosis, in 1882. The name was coined by another German
anatomist, von Waldeyer in 1888. The next stage took place after the development of genetics in
the early 20th century, when it was appreciated that the set of chromosomes (the karyotype) was
the carrier of the genes. Levitsky (1924, 1931) seems to have been the first to define the
karyotype as the phenotypic appearance of the somatic chromosomes, in contrast to their genic
contents. The subsequent history of the concept can be followed in the works of Darlington
(1939) and White (1973a; 1973b).
The study of whole sets of chromosomes is sometimes known as karyology. The
chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a
karyogram or idiogram: in pairs, ordered by size and position of centromere for chromosomes of
the same size. The basic number of chromosomes in the somatic cells of an individual or a
species is called the somatic number and is designated 2n. In normal diploid organisms,
autosomal chromosomes are present in two copies. There may, or may not, be sex chromosomes.
Polyploid cells have multiple copies of chromosomes and haploid cells have single copies.
2.3.2 In Legumes – The chromosome maps of some legumes such as soybean (Glycine max),
Lotus (Lotus japonicus), and red clover (Trifolium pratense) have been reviewed by Ohmido et
al., 2007. Papilionoideae contains the majority of pulse crops such as pea (Pisum sativum, 2n =
14, 5000 Mb), alfalfa (Medicago sativa, 2n =16, 1600 Mb), and soybean (Glycine max (L.)
Merr., 2n = 40, 1100 Mb). These species, Glycine max and Lotus japonicus, have relatively small
chromosomes and therefore are difficult to exploit for chromosome studies. Nevertheless, the
identification of individual chromosomes became feasible, and chromosome maps have been
developed applying image analysis and fluorescence in-situ hybridization. For Lotus japonicus,
e.g. detailed chromosome maps have been developed using the information of genetic linkage
22
maps. For legume species with large chromosomes, such as Vicia faba (2n = 12) and Pisum
sativum (2n =14), it is possible to use ordinary karyotyping and/or banding methods for
chromosome identification (Ohmido et al., 2007). A comprehensive survey of the molecular and
cytogical features of the chromosome complement was provided for V. faba based on FISH and
various Giemsa and fluorescence banding patterns (Fuchs et al., 1998a).
In the case of legumes with small chromosomes, identification of individual
chromosomes and their centromeric positions is difficult, especially after condensating
pretreatment with colchicine, 8-hydroxyquinoline or cold water. The Chromosome Image
Analyzing System (CHIAS) for small chromosomes makes use of distinct stainability along
mitotic prometaphase chromosomes, due to uneven condensation, a feature specific to small
plant chromosomes (Fukui & Iijima 1991). The density profiles at the center line of both
chromatids (mid-rib line) of prometaphase chromosomes allowed establishment of the first
chromosome maps of several legumes with small chromosomes (Yanagisawa et al., 1991; Ito et
al., 2001; Sato et al., 2005).
2.4 TECHNIQUES USED IN KARYOMORPHOLOGICAL STUDIES
2.4.1 The Conventional Method
Many different methods of making squash preparations for chromosome studies have been
described. These involve the application of various chemical and physical agents for the
pretreatment and fixation of the dividing cells. A good technique should permit the chromosomes
to become well spread during the squashing, and, at the same time, the fixation should bring out
the details of chromosome morphology as distinctly as possible. Well spread chromosomes with
23
a clear morphology are essential for karyotype studies and chromosome measurements
(Östergren and Heneen, 2009). For other species, various types of pretreatment may be more
useful. The pretreatment to be used depends not only on the species studied but also on
the special aspect of chromosome cytology in which the investigator is interested. When it is
essential to get the chromosomes well separated during the squashing, it is desirable to
have them strongly contracted. On the other hand, when more interest is focused on
morphological landmarks, such as satellites and secondary constrictions, it is often useful to
have the chromosomes more weakly contracted (Östergren and Heneen, 2009).
2.4.2 Plant materials
Chromosome observation is usually carried out in cells of the root tips (Lv et al., 2009). Some
preparation is made from the shoot tips of some other plants. Some of these plants are cultured in
water in a greenhouse (Lv et al., 2009), Petri dishes (Mansion and Zeltner, 2004), experimental
pots or in the field.
2.4.3 Pretreatment
Pre-treatment of cells helps to arrest the dividing cells at metaphase by interfaring with the
formation of spindle fibre. This treatment does not really kill the cell but limits the completion of
the cell division. The root tips are mostly collected from young plants in the morning and pretreated with 0.002 mol/L 8-hydroxyquinoline solution from few minutes to several hours
depending on the type of plant used. Other pretreatment agents used include oxyquinoline (Tjio
and Levan, 1950), alpha bromonaphthalene (Schmuck and Kostoff, 1935), Colchicine (O’Mara,
1939), paradichlorobenzene (Meyers, 1945) and cold water (Hill and Myers, 1945). Besides
inducing an increased contraction of the chromosomes, oxyquinoline causes an inactivation of
24
the spindle and an increase in the viscosity of the cytoplasm (Tjio and Levan, 1950;
Stalfelt, 1950).
2.4.4 Fixation
This treatment helps to kill the cell in such a way that all the stages of division, in which the cell
is undergoing, remain as they are. The most common fixating agent is Carnoy’s solution. It is
prepared using ethanol and acetic acid in ratio 3 to 1 respectively. The treatment time for fixation
also vary depending on the plant used.
2.4.5 Hydrolysis
This is the stage at which the plant parts used for chromosome studies are softened for easy
maceration or squashing. The time and temperature for hydrolysis is mostly determined by the
researcher in relation to the plants under investigation. Mostly prepared concentration for
hydrolysis is 1N of Hydrochloric acid as recently used by Tabur and Oney (2009) among many
other researchers.
2.4.6 Staining
This is among the major stages for chromosome studies because it helps to differentiate the
chromosomes from their immediate cell environment. Stains used for chromosome studies
include Feulgen (Tabur and Oney, 2009), Aceto-carmine (Gill and Obembe, 1991; Medina and
Conagin, 1964), Carbol Fuchsin solution (Li, 1982), Giemsa technique (Guerra, 1983),
Leucobasic fushin (Badr and Gasim, 1992).
25
2.5 SOME NEW AND MODERN METHODS
Chromosome squashes are time-consuming and are rapidly becoming a lost art (Goldblatt 2007).
Fortunately, with recent refinements in the accuracy and cost efficiency of flow cytometry,
researchers now initiate investigations into chromosome differences to detect intraspecific
cytotype variation (Murray and Young, 2001; Kron et al., 2007).
2.6 DEPICTION OF KARYOTYPES
According to Gustashaw (1991), Cytogenetics employs several techniques to visualize different
aspects of chromosomes
1. G-banding is obtained with Giemsa stain following digestion of chromosomes with
trypsin. It yields a series of lightly and darkly stained bands - the dark regions tend to be
heterochromatic, late-replicating and AT rich. The light regions tend to be euchromatic,
early-replicating and GC rich. This method will normally produce 300-400 bands in a
normal, human genome.
2. R-banding is the reverse of G-banding (the R stands for "reverse"). The dark regions are
euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic
(thymine-adenine rich regions).
3. C-banding: Giemsa binds to constitutive heterochromatin, so it stains centromeres.
4. Q-banding is a fluorescent pattern obtained using quinacrine for staining. The pattern of
bands is very similar to that seen in G-banding.
5. T-banding: visualize telomeres.
26
6. Silver staining: Silver nitrate stains the nucleolar organization region-associated protein.
This yields a dark region where the silver is deposited, denoting the activity of rRNA
genes within the NOR.
2.6.1 Classic Karyotype Cytogenetics
In the "classic" (depicted) karyotype, a dye, often Giemsa (G-banding), less frequently
Quinacrine, is used to stain bands on the chromosomes. Giemsa is specific for the phosphate
groups of DNA. Quinacrine binds to the adenine-thymine-rich regions. Each chromosome has a
characteristic banding pattern that helps to identify them; both chromosomes in a pair will have
the same banding pattern. Karyotypes are arranged with the short arm of the chromosome on top,
and the long arm on the bottom. Some karyotypes call the short and long arms p and q,
respectively. In addition, the differently stained regions and sub-regions are given numerical
designations from proximal to distal on the chromosome arms (Shaffer and Tommerup, 2005).
2.6.2 Spectral Karyotype (SKY Technique)
Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all
the pairs of chromosomes in an organism in different colors. Fluorescently labeled probes for
each chromosome are made by labeling chromosome-specific DNA with different fluorophores.
Because there are a limited number of spectrally-distinct fluorophores, a combinatorial labeling
method is used to generate many different colors. Spectral differences generated by
combinatorial labeling are captured and analyzed by using an interferometer attached to a
fluorescence microscope. Image processing software then assigns a pseudo color to each
spectrally different combination, allowing the visualization of the individually colored
chromosomes (Schröck et al., 1996) This technique is used to identify structural chromosome
27
aberrations in cancer cells and other disease conditions when Giemsa banding or other
techniques are not accurate enough.
2.6.3 Digital Karyotyping
Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.
Short sequences of DNA from specific loci all over the genome are isolated and enumerated
(Wang et al., 2002). This is method is also known as virtual karyotyping.
2.7 METHODS USED IN THE ANALYSIS OF KARYOTYPES STUDIES
For karyotypes studies, idiograms are constructed for each standard karyotypes. The
measurements of the short and long chromosome arms, and the absolute chromosome length is
usually performed on an Amplival microscope at a very high magnification (<2000) (Pavlova
and Tosheva, 2004). The karyotype asymmetry can easily be described using the method of
Romero Zarco (1986). The latter is expressed by two numerical parameters A1
(intrachromosomal asymmetry index) and A2 (interchromosomal asymmetry index). Karyotype
asymmetry for the relations between the chromosome arms has been estimated for every sample
using the following equation:
A1
= 1- (Sbi / Bi)
n
where
A1 – intrachromosomal asymmetry index, ranging from zero to one;
n – number of homologous chromosome pairs;
bi − average length for short arms in every homologous chromosome pair;
Bi − average length for long arms in every homologous chromosome pair.
28
Karyotype asymmetry due to relations between sizes of different chromosomes has been
estimated using Pearson’s dispersion coefficient
A2 = S /ξ
S − standard deviation
where
ξ − the mean of chromosome length for each sample.
The interchromosomal asymmetry index A2 is independent from the chromosome size,
chromosome number and has no units. This method is appropriate when there are only slight
differences in the karyotype asymmetry. The variations in chromosome size and type of a given
species observed in natural conditions, and the changes in the structural type of chromosomes in
the evolutionary process indicate that the karyotype is most susceptible to the environmental
factors resulting in genotype changes. The karyotype symmetry is a reflection of the mutation
process (Pavlova and Tosheva, 2004).
2.8 PARAMETERS OBSERVED IN KARYOMORPHOLOGICAL STUDIES
Some six different characteristics of karyotypes are usually observed and compared according to
Stebbins (1971)
1. Differences in absolute sizes of chromosomes. Chromosomes can vary in absolute size by
as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba
29
(legumes), both have six pairs of chromosomes (n=6) yet V. faba chromosomes are many
times larger. This feature probably reflects different amounts of DNA duplication.
2. Differences in the position of centromeres. This is brought about by translocations.
3. Differences in relative size of chromosomes can only be caused by segmental interchange
of unequal lengths.
4. Differences in basic number of chromosomes may occur due to successive unequal
translocations which finally remove all the essential genetic material from a chromosome,
permitting its loss without penalty to the organism (the dislocation hypothesis).
5. Differences in number and position of satellites, which (when they occur) are small
bodies attached to a chromosome by a thin thread.
6. Differences in degree and distribution of heterochromatic regions. Heterochromatin stains
darker than euchromatin, indicating tighter packing, and mainly consists of genetically
inactive repetitive DNA sequences.
A full account of a karyotype may therefore include the number, type, shape and banding of the
chromosomes, as well as other cytogenetic information. According to White (1973b), variation is
often found:
1. Between the sexes
2. Between the germ-line and soma (between gametes and the rest of the body)
3. Between members of a population (chromosome polymorphism)
4. Geographical variation between races
5. Mosaics or otherwise abnormal individuals.
30
CHAPTER THREE
3.0 APPLICATIONS OF KARYOMORPHOLOGICAL STUDIES
3.1 DETERMINATION OF PLOIDY LEVEL
3.1.1 Ploidy is the number of complete sets of chromosomes in a cell. Polyploidy, where there
are more than two sets of homologous chromosomes in the cells, occurs mainly in plants. It has
been of major significance in plant evolution according to Stebbins (Stebbins, 1940, 1950;
Comai, 2005; Adams and Wendel, 2005). The proportion of flowering plants which are
polyploid was estimated by Stebbins to be 30-35%, but in grasses the average is much higher,
about 70% (Stebbins, 1970). Polyploidy in lower plants (ferns, horsetails and psilotales) is also
common, and some species of ferns have reached levels of polyploidy far in excess of the highest
levels known in flowering plants. Polyploidy in animals is much less common, but it has been
significant in some groups (Gregory and Mable, 2005). Polyploid series in related species which
consist entirely of multiples of a single basic number are known as euploid.
3.1.2 Haplo-diploidy is where one sex is diploid, and the other haploid. It is a common
arrangement in the Hymenoptera, and in some other groups.
3.1.3 Endopolyploidy occurs when in adult differentiated tissues the cells have ceased to divide
by mitosis, but the nuclei contain more than the original somatic number of chromosomes
(White, 1973c). In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting'
nucleus undergo reduplication, the daughter chromosomes separating from each other inside an
intact nuclear membrane according to Lilly and Duronio (2005). In many instances,
endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly
counted). The cells do not always contain exact multiples (powers of two), which is why the
31
simple definition 'an increase in the number of chromosome sets caused by replication without
cell division' is not quite accurate. This process (especially studied in insects and some higher
plants such as maize) may be a developmental strategy for increasing the productivity of tissues
which are highly active in biosynthesis (Edgar and Orr-Weaver, 2001). The phenomenon occurs
sporadically throughout the eukaryote kingdom from protozoa to man; it is diverse and complex,
and serves differentiation and morphogenesis in many ways (Nagl, 1978).
3.1.4 Aneuploidy is the condition in which the chromosome number in the cells is not the typical
number for the species. This would give rise to a chromosome abnormality such as an extra
chromosome or one or more chromosomes lost. Abnormalities in chromosome number usually
cause a defect in development. Down syndrome and Turner syndrome are examples of this.
Aneuploidy may also occcur within a group of closely related species. Classic examples in plants
are the genus Crepis, where the gametic (= haploid) numbers form the series x = 3, 4, 5, 6, and 7;
and Crocus, where every number from x = 3 to x = 15 is represented by at least one species.
Evidence of various kinds shows that that trends of evolution have gone in different directions in
different groups (Stebbins and Ledley, 1972). Closer to home, the great apes have 24x2
chromosomes whereas humans have 23x2. Human chromosome 2 was formed by a merger of
ancestral chromosomes, reducing the number (Ijdo et al., 1991).
3.1.5 Chromosomal Polymorphism
According to Ford (1975), some animal species are polymorphic for chromosome fusions or
dissociations. When this happens, the chromosome number is variable from one individual to
another. Well-researched examples are the ladybird beetle Chilocorus stigma, some mantids of
the genus Ameles, the European shrew Sorex araneus. There is some evidence from the case of
32
the mollusc Thais lapillus (the dog whelk) on the Brittany coast, that the two chromosome
morphs are adapted to different habitats (White, 1973d).
Chromosome observation has been used in many plants to identify chromosomal
ploidy (Nathewet et al., 2007; Kazuo and Maltide, 1993; Nankui and Paul, 2003; Li et al.,
2008; Ramon and Manuel, 2004; Brutovska et al., 2000; Agnieszka et al., 2006; Kazuo et al.,
2000). Knowledge of the chromosomal ploidies of germplasms do provide theoretical basis
for
crossbreeding (Lv et al., 2009). Polyploid formation is associated with extensive
chromosome restructuring (Ahn and Tanksley, 1993; Reinisch et al., 1994; Song et al., 1995a,
1995b; Lagercrantz and Lydiate, 1996; Brubaker et al., 1999) and possibly with retrotransposon
amplification (Matzke and Matzke, 1998; Zhao et al., 1998). Identification of different polyploid
populations can be used to establish seed-quarantine zones, in opposition to seed-transfer zones,
as insurance that incompatible cytotypes will not be mixed (Severns and Liston, 2008).
3.2 Other Uses of Karyomorphological Studies
Karyotype concept has been extensively used in characterizing and distinguishing
chromosomes of various organisms (Ayşe and Haşim, 2005). Future investigation into the
patterns of variation of intraspecific chromosome number will provide a safeguard for
conservation of rare plants in an age of restoration, contribute important information to a
relatively neglected topic in plant population and landscape genetics (Levin, 2002; Halverson et
al., 2008), and enhance understanding of plant speciation (Soltis et al., 2007).
A karyological study is significant in determining taxonomical position of organisms
(mosses) and it seems to be effective for analyzing Antarctic mosses which are usually sterile
(Kanda and Okada, 1993). Thus, the information on the karyomorphology is useful for generic
33
comparisons and might be of use to support the distinctness of species (Kazuo and Hiroshe,
2000). Karyotypes can also be used for many purposes; such as, to study chromosomal
aberrations, cellular function, and to gather information about past evolutionary events.
Karyomorphological studies in plant populations usually show similarities or differences in their
chromosomal morphology. It also provides additional data on the variation pattern within
populations for the solution of taxonomical problems and successful attempts for selection.
34
CHAPTER FOUR
4.0 RESULT OF KARYOMORPHOLOGICAL STUDIES IN SOME LEGUMINOUS
TREES
Worldwide, chromosomes have been counted for many tree legumes. As very few are
known for some genus there have been some extensive knowledge in some others. Only 26
species of Sophora have had their chromosome numbers reported (Stiefkens et al., 2001).
Several chromosome numbers reported for this genus include: n = 9, 18, 11, and 2n = 18, 28, 36,
16, 32, 22, 54, in decreasing order of frequency in each case (cf. Federov, 1974; Goldblatt,
1981a,b,c, 1984, 1988, 1990, 1991, 1994, 1996, 1998). In Table 1 below, the genera that were
reviewed among legume trees include Abrus (1), Albizzia (2), Alhagi (1), Astragalus (1),
Bauhinia (3), Cajanus (11), Dalbergia (10), Erythrina (1), Gleditsia (2), Laburnum (2), Mimosa
(2), Prosopis (11), Robinia (1), Senna (1), Sesbania (1), Tamarindus (1), Acacia (60), Cassia
(11), Leucaena (11), Lonchocarpus (46). These are 169 tree legumes documented at KEW
Datadase (http://www.rbdkew.org.uk/cval/homepage.html).
TABLE 1: CHROMOSOME NUMBER AND PLOIDY LEVEL OF SOME MAJOR TREE
LEGUMES
Genus
Species
Abrus
precatorious
Albizzia
amara
Albizzia
lucida
Alhagi
Authority
Chromosome
no.
22
Ploidy
level
2
Boiv.
26
Benth.
Reference
Life
cycle
Angiosperm
group
2
Al-Turki et al.,
(2000)
Ohri, 1996
P
Eudicot
26
2
Ohri, 1996
Eudicot
camelorum
16 + 2B
2
Astragalus
abyssinicus
16
2
Bauhinia
Bauhinia
tomentosa
purpurea
L.
L.
28
28
2
2
Bauhinia
hookeri
26
Cajanus
cajan
(F.Muell.)
Pedley
(L.) Millsp.
Al-Turki et al.,
(2000)
Al-Turki et al.,
(2000)
Ohri, 2002
P
Ohri
and P
Kumar, 1986
Ohri, 2002
P
22
2
35
P
Bennett
and P
Smith, 1976
Eudicot
Eudicot
Eudicot
Eudicot
Cajanus
Cajanus
Cajanus
goensis
scarabaeoides
volubilis
22
22
22
2
2
2
Ohri, 1996
Ohri, 1996
Ohri, 1996
P
B
P
Eudicot
Eudicot
Eudicot
22
2
Ohri, 1996
P
Eudicot
22
2
Ohri, 1996
B
Eudicot
22
2
Ohri, 1996
P
Eudicot
22
2
Ohri, 1996
P
Eudicot
22
2
Eudicot
22
2
22
2
Ohri and Singh, B
2002
Ohri and Singh, A
2002
Ohri and Singh, P
2002
lanceolaria
Dalz.
(L.) Thou.
(Blanco)
Blanco
(W. & A.)
Maesen
(Benth.)
Maesen
(Haines)
Maesen
(F.Muell.)
Maesen
(Benth.)
Maesen
(Wight.
&
Am.) Maesen
(Benth.
ex
Bak.)
Maesen
Lin. f.
Cajanus
lineatus
Cajanus
platycarpus
Cajanus
cajanifolius
Cajanus
acutifolius
Cajanus
mollis
Cajanus
albicans
Cajanus
sericeus
Dalbergia
20
2
P
Eudicot
Dalbergia
sissoo
Roxb.
20
2
P
Eudicot
Dalbergia
latifolia
Roxb.
20
2
P
Eudicot
Dalbergia
sissoides
Grah.
20
2
P
Eudicot
Dalbergia
volubilis
Roxb.
20
2
P
Eudicot
Dalbergia
horrida
Dennst.
20
2
P
Eudicot
Dalbergia
paniculata
Roxb.
20
2
P
Eudicot
Dalbergia
rubiginosa
Roxb.
20
2
P
Eudicot
Dalbergia
malabarica
Prain.
20
2
P
Eudicot
Dalbergia
melanoxylon
Guill & Perr.
20
2
P
Eudicot
Erythrina
Gleditsia
suberosa
ferox
Roxb.
Desf.
42
28
2
2
P
P
Eudicot
Eudicot
Gleditsia
sinensis
Lam.
28
2
P
Eudicot
Laburnum
anagyroides
Med.
48
4
Hiramath et al.,
2001
Hiramath et al.,
2001
Hiramath et al.,
2001
Hiramath et al.,
2001
Hiramath et al.,
2001
Hiramath et al.,
2001
Hiramath et al.,
2001
Hiramath et al.,
2001
Hiramath et al.,
2001
Hiramath et al.,
2001
Ohri, 1996
Ohri
and
Kumar, 1986
Ohri
and
Kumar, 1986
Olszewska and
Osiecka, 1984
P
Eudicot
36
Eudicot
Eudicot
Laburnum
alpinum
Mimosa
pudica
Mimosa
invisa
Prosopis
Prosopis
Prosopis
Prosopis
Prosopis
Prosopis
Prosopis
Prosopis
Prosopis
flexusa
alba
lampa
pallida
glandulosa
siliquas
juliflora
juliflora
chilensis
Prosopis
chilensis
Prosopis
Robinia
cineraria
pseudoacacia
Senna
alexandrina
Sesbania
rostrata
Tamarindus
Acacia
Acacia
Acacia
Acacia
Acacia
Acacia
Acacia
Acacia
Acacia
Acacia
Acacia
indica
modesta
catechu
seyal
albida
sieberana
nubica
implexa
mearnsii
sophorae
polycantha
heterophylla
Acacia
falcata
Acacia
bivenosa
Acacia
raddiana
Brecht
Persl
& 48
4
52
4
Mart
26
2
DC.
Griseb.
Willd.
Willd.
Torr.
(Willd.)
(Swartz) DC.
DC.
(Molina)
Stuntz.
(Molina)
Stuntz.
(L.) Druce
L.
28
28
28
28
28
28
28
52
28
56
Bremek.
Oberm
L.
Wall.
Willd.
(Del.)
(Del.)
DC.
Benth.
Benth.
Willd.
Benth.
Willd.
P
Eudicot
P
Eudicot
B-P
Eudicot
2
2
2
2
2
2
2
4
2
Olszewska and
Osiecka, 1984
Bennett et al.,
1998
Bennett et al.,
1998
Bukhari, 1997
Bukhari, 1997
Bukhari, 1997
Bukhari, 1997
Bukhari, 1997
Bukhari, 1997
Bukhari, 1997
Ohri, 1996
Bukhari, 1997
P
P
P
P
P
P
P
P
P
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
4
Bukhari, 1997
P
Eudicot
Ohri, 2002
P
Olszewska and P
Osiecka, 1984
Al-Turki et al.,
(2000)
Eudicot
Eudicot
Arumuganathan
and Earle, 1991
Ohri, 2002
Ohri, 1996
Ohri, 1996
Bukhari, 1997
Bukhari, 1997
Bukhari, 1997
Bukhari, 1997
Bukhari, 1997
Bukhari, 1997
Bukhari, 1997
Bukhari, 1997
Marie
and
Brown, 1993
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
-
Eudicot
P
P
P
P
P
P
P
P
P
P
P
-
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
52
20
2
28
2
& 12
2
26
26
26
104
26
26
56
26
26
26
52
26
2
2
2
8
2
2
4
2
2
2
4
2
26
2
DC.
26
2
Savi
104
8
37
Acacia
caffra
26
2
Bukhari, 1997
P
Eudicot
senegal
(Thunb.)
Willd.
Willd.
Acacia
26
2
P
Eudicot
Acacia
tortilis
Hayne
78
6
P
Eudicot
Acacia
crassa
Pedley
26
2
P
Eudicot
Acacia
orites
Pedley
26
2
P
Eudicot
Acacia
raddiana
78
6
P
Eudicot
Acacia
nilotica
(Savi.)
Brenan
Delile
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Bukhari, 1997
26
2
P
Eudicot
Acacia
salicina
Lindl.
26
2
P
Eudicot
Acacia
gerrardi
Benth.
52
4
P
Eudicot
Acacia
mellifera
26
2
P
Eudicot
Acacia
tortilis
52
4
Bukhari, 1997
P
Eudicot
Acacia
tortilis
104
8
Bukhari, 1997
P
Eudicot
Acacia
mangium
(Vahal)
Benth.
(Forssk.)
Hayne
(Forssk.)
Hayne
Willd.
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Bukhari, 1997
26
2
P
Eudicot
Acacia
dealbata
Link.
26
2
P
Eudicot
Acacia
dealbata
Link.
39
3
P
Eudicot
Acacia
dealbata
Link.
52
4
P
Eudicot
Acacia
ligulata
A.Cunn.
26
2
P
Eudicot
Acacia
suberosa
A.Cunn.
26
2
P
Eudicot
Acacia
victoriae
Benth.
26
2
P
Eudicot
Acacia
falciformis
DC.
26
2
P
Eudicot
Acacia
irrorata
Sieber
26
2
P
Eudicot
Acacia
melanoxylon
R.Br.
26
2
P
Eudicot
Acacia
farnesiana
Willd.
52
4
Blakesley et al.,
2002
Blakesley et al.,
2002
Blakesley et al.,
2002
Blakesley et al.,
2002
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
P
Eudicot
38
Acacia
bancrofti
Maiden
26
2
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
4
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Bukhari, 1997
Acacia
pycnantha
Benth.
26
2
Acacia
leucophloea
Willd.
52
4
Acacia
hylonoma
L.Pedley
26
2
Acacia
maidenii
F.Muell.
26
2
Acacia
concurrens
Pedley
26
2
Acacia
nilotica
52
Acacia
Acacia
drepanolobiu
m
nilotica
Acacia
verek
Acacia
longispicata
(L.) Willd.
ex Del.
Harm.
ex
Sios.
(L.) Willd.
ex Del.
Guill.
&
Perr.
Benth.
P
Eudicot
52
4
Bukhari, 1997
P
Eudicot
104
8
Bukhari, 1997
P
Eudicot
26
2
P
Eudicot
26
2
P
Eudicot
F.Muell.
26
2
P
Eudicot
A.Cunn.
26
2
P
Eudicot
Acacia
auriculaeform
is
podalyriifolia
A.Cunn.
26
2
P
Eudicot
Acacia
leiocalyx
(Domin)
Pedley
26
2
P
Eudicot
Acacia
simsii
26
2
P
Eudicot
Acacia
greggii
26
2
P
Eudicot
Acacia
fimbriata
26
2
P
Eudicot
Acacia
leptocarpa
26
2
P
Eudicot
Acacia
holosericea
52
4
P
Eudicot
Acacia
crassicarpa
26
2
P
Eudicot
Acacia
aulacocarpa
26
2
P
Eudicot
Acacia
iteaphylla
26
2
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Mukherjee and
Sharma, 1995
Acacia
cincinnata
Acacia
P
Eudicot
A.Gray,
P.L.Wright
A.Cunn. ex
G.Don
A.Cunn. ex
Benth.
A.Cunn. ex
G.Don
A.Cunn. ex
Benth.
A.Cunn. ex
Benth.
F.Muell. ex
Benth.
39
Cassia
Cassia
Cassia
Cassia
Cassia
Cassia
Cassia
Cassia
tora
absus
fistula
biflora
siamea
glauca
auriculata
occidentalis
L.
L.
L.
L.
Lam.
Lam.
L.
L.
26
28
28
28
28
28
28
28
2
2
2
2
2
2
2
2
Ohri et al., 1986
Ohri et al., 1986
Ohri et al., 1986
Ohri et al., 1986
Ohri et al., 1986
Ohri et al., 1986
Ohri et al., 1986
Ohri et al., 1986
A
A
P
P
P
P
P
A
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Eudicot
Cassia
excelsa
Schrad.
28
2
Ohri et al., 1986 P
Eudicot
Cassia
australis
ex 28
2
Ohri et al., 1986 P
Eudicot
Cassia
angustifolia
Reinw.
Steud.
Vahl.
28
4
-
Eudicot
Leucaena
retusa
Benth.
56
2
P
Eudicot
Leucaena
lanceolata
S.Watson
56
2
P
Eudicot
Leucaena
greggii
S.Watson
56
2
P
Eudicot
Leucaena
confertiflora
Zárate
112
4
P
Eudicot
Leucaena
shannonii
J.D.Smith
52
2
P
Eudicot
Leucaena
trichodes
52
2
P
Eudicot
Leucaena
diversifolia
56
2
P
Eudicot
Leucaena
diversifolia
104
4
P
Eudicot
Leucaena
pulverulenta
56
2
P
Eudicot
Leucaena
esculenta
56
2
P
Eudicot
Leucaena
esculenta
112
4
Palomino et al., P
1995
Eudicot
Lonchocarpus xuul
(C.E.Hughes
) C.E.Hughes
(Schltdl.)
Benth.
(Schltdl.)
Benth.
(Schltdl.)
Benth.
(Mociño &
Sessé
ex
A.DC.)
Benth.
(Mociño &
Sessé
ex
A.DC.)
Benth.
Lundell
Bahl
and
Srivastava,
1987
Hartman et al.,
2000
Palomino et al.,
1995
Hartman et al.,
2000
Palomino et al.,
1995
Hartman et al.,
2000
Hartman et al.,
2000
Palomino et al.,
1995
Hartman et al.,
2000
Hartman et al.,
2000
Palomino et al.,
1995
22
2
Eudicot
Lonchocarpus mutans
M.Sousa
22
2
Palomino and P
Sousa, 2000
Palomino and P
Sousa, 2000
40
Eudicot
Lonchocarpus rugosus
Benth.
22
2
Lonchocarpus obovatus
Benth.
22
2
Lonchocarpus punctatus
Kunth
22
2
Lonchocarpus hintonii
Sandw.
22
2
Lonchocarpus morenoi
M.Sousa
22
2
Lonchocarpus chiangii
M.Sousa
22
2
Lonchocarpus cruentus
Lundell
22
2
Lonchocarpus hughesii
M.Sousa
22
2
Lonchocarpus caudatus
Pittier
22
2
Lonchocarpus epigaeus
M.Sousa
22
2
Lonchocarpus castilloi
Standl.
22
2
Lonchocarpus hermannii
M.Sousa
22
2
Lonchocarpus parviflorus
Benth.
22
2
Lonchocarpus agyrotrichus
Harms
22
2
Lonchocarpus andrieuxii
M.Sousa
22
2
Lonchocarpus eriophyllus
Benth.
22
2
Lonchocarpus macrocarpus
Benth.
22
2
Lonchocarpus lanceolatus
Benth.
22
2
Lonchocarpus emarginatus
Pittier
22
2
Lonchocarpus spectabilis
Hermann
22
2
Lonchocarpus constrictus
Pittier
22
2
Lonchocarpus hidalgensis
Lundell
22
2
Lonchocarpus schubertiae
M.Sousa
22
2
41
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
Palomino and
Sousa, 2000
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
P
Eudicot
Lonchocarpus atropurpureus
Benth.
22
2
Palomino and P
Eudicot
Sousa, 2000
Lonchocarpus guatemalensis Benth.
22
2
Palomino and P
Eudicot
Sousa, 2000
Lonchocarpus phaseolifolius Benth.
22
2
Palomino and P
Eudicot
Sousa, 2000
Lonchocarpus eriocarinalis
Micheli
22
2
Palomino and P
Eudicot
Sousa, 2000
Lonchocarpus salvadorensis Pittier
22
2
Palomino and P
Eudicot
Sousa, 2000
Lonchocarpus luteomaculatu Pittier
22
2
Palomino and P
Eudicot
s
Sousa, 2000
Lonchocarpus santarosanus
Donn. Sm.
22
2
Palomino and P
Eudicot
Sousa, 2000
Lonchocarpus minimiflorus
Donn. Sm.
22
2
Palomino and P
Eudicot
Sousa, 2000
Lonchocarpus chavelasii
M.Sousa,
22
2
Palomino and P
Eudicot
ined.
Sousa, 2000
Lonchocarpus martinezii
M.Sousa,
22
2
Palomino and P
Eudicot
ined.
Sousa, 2000
Lonchocarpus molinae
Standl.
& 22
2
Palomino and P
Eudicot
L.O.Wms.
Sousa, 2000
Lonchocarpus torresiorum
M.Sousa,
22
2
Palomino and P
Eudicot
ined.
Sousa, 2000
Lonchocarpus schiedeanus
(Schldl.)
22
2
Palomino and P
Eudicot
Harms
Sousa, 2000
Lonchocarpus angusticarpus M.Sousa,
22
2
Palomino and P
Eudicot
ined.
Sousa, 2000
Lonchocarpus balsensis
M.Sousa & 22
2
Palomino and P
Eudicot
J.C.Soto
Sousa, 2000
Lonchocarpus acuminatus
(Schldl.)
22
2
Palomino and P
Eudicot
M.Sousa
Sousa, 2000
Lonchocarpus sanctuari
Standl.
& 22
2
Palomino and P
Eudicot
L.O.Wms.
Sousa, 2000
Lonchocarpus peninsularis
(J.D.
Sm.) 22
2
Palomino and P
Eudicot
Pittier
Sousa, 2000
Lonchocarpus huetamoensis M.Sousa & 22
2
Palomino and P
Eudicot
J.C.Soto
Sousa, 2000
Lonchocarpus phlebophyllus Standl.
& 22
2
Palomino and P
Eudicot
Steyerm.
Sousa, 2000
Lonchocarpus longipeduncul M.Sousa & 22
2
Palomino and P
Eudicot
atus
J.C.Soto
Sousa, 2000
Data were from the Plant DNA C-values Database query results (http://www.rbdkew.org.uk/cval/homepage.html).
42
CHAPTER FIVE
5.0 CONCLUSION
5.1 CHROMOSOME STUDIES IN TREE LEGUME
It was observed that most sampled tree legume have their chromosome number n = 13.
The ploidy level ranges from 2 to 8. It was observed that there is constant chromosome number
of 22 in the entire Lonchocarpus trees assessed with their ploidy level at 2. Cassia and Leucaena
show similaties in their chromosome number though with little variation in their ploidy levels. It
was noted that they both have their chromosome number n = 14 with very few of them having
their chromosome number n = 13. The few species of Cassia, with n = 13, show similarity with
the genus Acacia that have haploid chromosome numbers of n = 13.
It was observed also that Prosopis and Cassia has the same chromosome number of 28.
Likewise Abrus, Cajanus and Lonchocarpus have same haploid number of chromosomes as n =
22. The similarities or closeness observed in theses tree legume indicates the closeness of these
genera. Between the genera Cassia with the highest and Astragalus with the lowest chromosome
numbers, there are intermediate genera to bridge the gap of relatedness among the tree legume.
5.2 PLOIDY LEVEL IN TREE LEGUME
It was also observed within the genera of the tree legume that as there are variations
within same genus of tree legumes, there is constancy in the chromosome number within another
where there are no multiples of single basic number. Some genera with different ploidy level in
their chromosomes include Mimosa, Leucaena, Acacia, Prosopis and Cassia. These genera are
euploid. Some genera with constancy in their chromosome ploidy level include Cajanus,
Lonchocarpus and Dalbergia. The genera Astragalus, Robinia, Senna, Sesbania, Erythrina,
43
Gleditsia, Laburnum, Abrus, Albizzia, Alhagi, and Bauhinia are under-represented to justify the
constancy or differences of the chromosome numbers and ploidy levels. From the Table 1, only
one species in the genera Cassia and Prosopis show different ploidy level which consist entirely
of multiples of their single basic number.
It was also observed that in the majority of literatures consulted for the Table 1, only the
chromosome number and the ploidy level of the tree legumes were recorded. This could indicate
that the type of chromosomes in relation to the position of the centromere has no major
documentation. Other data that were not commonly documented for these tree legumes include
the relative sizes of their chromosomes and the number and positions of their satellite (if any).
Also not listed is the degree and distribution of heterochromatic regions of these chromosomes.
44
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