ABSTRACT
Induced mutation is one of the prime methods used for the improvement of the agronomic traits of crops. The aim of this research was to evaluate the effect of different levels of X-ray radiation on the growth components and to detect DNA polymorphism in two accessions of Bambara groundnut (Vigna subterranea (L.) Verdc.). Seeds of Bambara groundnut accessions were arranged into five groups each into a paper envelope containing 50 seeds each. The first groups served as the control (untreated) whereas the other groups were bombarded with different doses of X-ray (0, 5, 10, 15 and 20 MGy) respectively. The experiment was set up in a randomized complete block design (RCBD) with three replicates. Seeds were sown and monitored for germination at 21 days after sowing. Growth parameters investigated include germination, plant height, number of leaves, number of branches and leaf area. Growth parameters were recorded at 2, 4, 6, 8 and 10 Weeks After planting (WAP). The results obtained on germination percentage showed a gradual reduction as the doses increased. Higher doses reduced seed germination in the two accessions. The result on the plant height ranged from 25 cm to 30 cm. the lowest irradiation dose 5 MGy enhanced the plant height in the two Bambara accessions whereas the highest dose significant caused a decrease in the plant height. Also, there was a higher leaf number with plants that received the lowest dose of X-ray in the Bambara accessions. In the Red accession, 5 MGy had 96 leaf numbers whereas 114 were counted in the Milk-color accession at 10 WAP. The result obtained on the number of branches showed an increase in the number of branches at 5 MGy. In the Red accession, the highest number of branches (34) was counted under 5 MGy followed by 10 MGy (32) and then the control. However, in the milk-color accession, the maximum number of branches (33) was observed under the control followed by 5 MGy (31). Similarly, the result obtained on the leaf area was positively influenced by the lowest irradiation dose 5 MGy in the two Bambara accessions studied. Irradiation dose of 20 MGy significantly reduced the plant leaf area. Five (5) primers used in this study showed DNA polymorphism. Primer OPB 02, OPB 08, OPB 10 and OPB 20 were 100 % polymorphic while primer OPB 14 was 90 % polymorphic. A total of 59 bands were detected, 58 were polymorphic whereas 01 was monomorphic. The DNA banding patterns observed in the gel electrophoresis revealed the presence or absence of different bands with variations in their intensities as a result of the x ray exposures. Based on Jaccard’s coefficient of similarity values, the maximum similarity value (0.833) was observed between 5 MGy and 10 MGy in the milk color accession whereas the Red accession recorded similarity value of (0.883) between 10 MGy and 15 MGy. The result has revealed that X-ray changed the banding patterns of the DNA of the mutated seeds when compared with the controls, hence can be adopted in mutation breeding of Bambara groundnut. From the results of this study, exposing seeds of the two Bambara accessions to 5 MGy of X-ray enhanced the vegetative growth of the plants and could be employed in mutation breeding for improved agronomic qualities. This result has further confirmed the use of RAPD marker in the identification of mutagenic treated plants in mutation breeding.
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification
iii
Dedication iv
Acknowledgements
v
Table of
Contents vi
List of
Tables ix
List of
Figures x
List of
Plates xi
Abstract xii
CHAPTER 1: INTRODUCTION 1
1.1 Background of the Study 1
1.2 Statement of Problem 5
1.3 Objectives of the Study 6
CHAPTER 2: LITERATURE REVIEW 7
2.1 Botany of the Plant 7
2.2 Origin and Distribution of Bambara
Groundnut 8
2.3 Morphological and Physiological
Characteristics of Bambara
Groundnut 9
2.3.1 Morphological characteristic 9
2.3.2 Physiological characteristic 10
2.3.2.1 Photoperiod 11
2.3.3.2 Drought tolerance 12
2.3.3.3 Flowering and maturity 13
2.4 Environmental Requirements for Bambara
Groundnut Climate 14
2.4.1 Soil 14
2.4.2 Cultural practices 14
2.4.3 Cultural practices 15
2.4.3.1 Sowing date 15
2.4.3.2 Land preparation 16
2.4.3.3
Spacing and seed rate 16
2.4.3.4 Fertilization 16
2.4.3.5
Weed control 17
2.4.3.6
Pests and diseases 17
2.4.3.7
Harvesting 18
2.5 Ecology, Importance and Uses of Bambara
Groundnut 19
2.6 Genetic Studies on Bambara Groundnut 21
2.7 DNA
Fingerprinting in Plants 23
2.7.1 DNA isolation 24
2.7.2 DNA quantification and quality assessment 24
2.7.3 Polymerase chain reaction (PCR) 25
2.7.4 Steps involved in PCR 25
2.7.5 Restriction fragment length polymorphisms
(RFLPs) 26
2.7.6 Randomly amplified polymorphic DNAs (RAPDs) 28
2.7.7 Amplified length polymorphisms (AFLPs) 30
2.8 Applications of DNA
Markers 31
2.9 Induced Mutation Studies in Bambara
Groundnut 32
CHAPTER 3: MATERIALS AND METHODS 34
3.1
Study Area 34
3.2 Collection of Plant Materials 34
3.3 Irradiation of Plant Materials 34
3.4 Experimental Design 34
3.5 Data Collection 35
3.6 Germination Percentage 35
3.7 Plant Height (cm) 35
3.8 Number of Leaves 35
3.9 Number of Branch 35
3.10 Leaf Area (cm2) 35
3.11 Genomic DNA Extraction 35
3.12 DNA Electrophoresis 36
3.13 Dilution of DNA for PCR 37
3.14 PCR Reaction Mix 37
3.15 Gel Electrophoresis 37
3.16 Statistical Analysis 37
CHAPTER 4: RESULTS AND DISCUSSION 38
4.1
Effect of Different Levels of X- Ray
Irradiation on Seed Germination 38
4.2 Effect of Different X-Ray Irradiation
Levels on Plant Height (cm) of
Two Bambara Groundnut Accessions 40
4.3 Effect of Different X-Ray Irradiation
Levels on Number of Leaves
on Two Accessions of Bambara
Groundnut During Growth 43
4.4 Effect of Different X-Ray Irradiation
Levels on the Number of Branches
During Growth 45
4.5 Effect of Different X-Ray Irradiation
Levels on the Leaf Area (cm2) at
Different Week Intervals 47
4.6 RAPD Primers Used, Their Sequences and
Fragment Size 49
4.7 Number of Polymorphic, Monomorphic Bands
and Percentage
Polymorphism 50
4.8 Similarity Coefficient 58
4.9 Discussion 62
4.9.1 Effect of x-ray radiation on germination
percentage 62
4.9.2 Effect of x-irradiation on growth parameters 62
4.9.3 DNA polymorphism 64
CHAPTER 5: CONCLUSION
AND RECOMMENDATIONS 66
5.1 Conclusion 66
5.2
Recommendations 66
References 68
Appendices 77
LIST OF
TABLES
4.1: Effect of
different X-ray irradiation levels on Seed Germination 38
4.2: Effect
of different X-ray irradiation levels on plant height (cm) 41
4.3: Effect
of different X-ray irradiation levels on Number of leaves 44
4.4: Effect
of different X-ray irradiation levels on number of branches 46
4.5: Effect of
X-ray doses on leaf area (cm2) per plant 48
4.6: List of the
five RAPD primers, their sequences and fragment size 49
4.7: Fragment size
and percentage polymorphism 50
LIST OF FIGURES
1: Dendrogram illustrating genetic
relationship among the Bambara accessions 57
LIST OF PLATES
1: Electrophoresis gel for RAPD primer OPB
02 51
2: Electrophoresis gel for RAPD primer OPB
08 51
3: Electrophoresis gel for RAPD primer OPB
10 52
4: Electrophoresis gel for RAPD primer OP
B 14 52
5: Electrophoresis gel for RAPD primer OPB
20 53
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF
THE STUDY
Mutagenesis
is defined as a process whereby sudden heritable changes occur in the genetic
constitution of an organism. These changes are not caused by genetic
segregation or genetic recombination, but induced by chemical, physical or
biological agents. Three types of mutagenesis are known Roychowdhury and Tah
(2013), These are induced mutagenesis, which occurs as a result of exposing
plant materials to radiation (gamma rays, X-rays, ion beam, etc.) or treatment
with chemical mutagens; site-directed mutagenesis, which is the process of
inducing a mutation at a defined site in a DNA molecule; and insertion
mutagenesis, which occur as a result of DNA insertions, either through genetic
transformation and insertion of T-DNA or activation of transposable elements
(Kharkwal and Shu, 2009; Forster and Shu 2013).
The
ability of man to deliberately induce mutations in plants originated directly
from the discoveries of X-rays by Roentgen in 1895; radioactivity by Becquerel
in 1896; and radioactive elements by Marie and Pierre Curie in 1898. These
achievements gave rise to the Nobel Prize for Physics awarded to Roentgen in
1901 and to Becquerel, Marie and Pierre Curie in 1903.
Ionizing
radiations are the most commonly used physical mutagens (Mba et al., 2012). These radiations
constitute parts of the electromagnetic (EM) spectrum which on account of their
relatively high energy levels, are capable of dislodging electrons from the
nuclear orbits of the atoms that they impact upon. The impacted atoms therefore
become ions hence the term ionizing radiation. These ionizing components of the
EM include cosmic, gamma (γ) and X-rays. Ultra violet (UV) light are
non-ionizing, this to some extent is capable of penetrating the tissues and has
been applied in inducing mutations. The mutagenicity of UV derives from its
ability to react with DNA and other biological molecules as its wavelengths are
preferentially absorbed by bases in DNA molecules and by the aromatic amino
acids of proteins. In addition to the ionizing radiations, other commonly used
physical mutagens are the high energy ionizing particles- alpha (α), beta (β)
particles and neutrons. The damages caused by these mutagens range from changes
at the DNA level (breaking the chemical bonds in DNA molecules, deleting or
adding nucleotides and by substituting one nucleotide for the other) to gross
chromosomal breakages and rearrangements.
Acquaah
(2006) pointed out the importance of applying radiation at the proper dose, a
factor that depends on radiation intensity and duration of exposure. Grays (Gy)
are the measurement unit of radiation dose. For the sterilization of food
products, processors typically use rates as high as 10 kGy. In the mutagenic
treatment of plant material, doses can range from as low as 2 Gy for cell
cultures or leaf tissues, to as high as 700 Gy for seed material (Ahloowalia
and Maluszynski, 2001). The exposure may be chronic (continuous low dose
administered for a long period) or acute (high dose over a short period). The
quality of mutation (proportion of useful mutations) is not necessarily
positively correlated with dose rate. It is common knowledge that a high dose
does not necessarily yield the best results.
The
dose of a mutagen that achieves the optimum mutation frequency with the least
possible unintended damage is regarded as the optimal dose for induced
mutagenesis (Mba et al., 2010). For physical mutagens, the dose is estimated
by carrying out tests of radiosensitivity- a term described as a relative
measure that gives an indication of the quantity of recognizable effects of
radiation exposure on the irradiated subject (Van Harten (1998). Its predictive
value, therefore, guides the researcher in the choice of optimal exposure
dosage depending on the plant materials and the desired outcome.
Traditionally,
to induce mutations in crops, planting materials are exposed to physical and
chemical mutagenic agents. Mutagenesis can be carried out with all types of
planting materials. For example, whole plants, usually seedlings, and in vitro cultured cells. However, the
most commonly used plant material is seed (Wani et al., 2014). Multiple forms of plant propagules, such as bulbs,
tubers, corms and rhizomes and more recently, the induction of mutations in
vegetatively propagated plants is becoming more efficient as scientists take
advantage of totipotency (ability of a single cell to divide and produce all of
the differentiated cells in an organism to regenerate into whole plants) using
single cells and other forms of in vitro
cultured plant tissues (Mba, 2013). The starting materials for the induction of
mutations are vegetative cuttings, scions, or in vitro cultured tissues like leaf and stem explants, anthers,
calli, cell cultures, microspores, ovules, protoplasts, etc. Gametes, usually
inside the inflorescences, are also targeted for mutagenic treatments through
immersion of spikes, tassels, etc., (Wani et
al., 2014). Whereas chemical mutagens are preferably used to induce point
mutations, physical mutagens induce gross lesions, such as chromosomal abberations
or rearrangements (Kharkwal, 2009).
It
is important to note that the frequency and types of mutations are direct
results of the dosage and rate of exposure or administration of the mutagen
rather than its type( Mba,2013) In the end, the choice of a mutagen will be
based more often than not on the particular researcher's circumstances, such as
safety of usage, ease of use, availability of the mutagens, effectiveness in
inducing certain genetic alterations, suitable tissue, cost and available infrastructure
among other factors.
Seeds
treated with mutagenic agents give rise to chimeric plants. Chimeric plants
produce both mutant and non-mutant seeds. Usually, this can be problematic;
however, one needs to plant more seeds to find the desired mutants. As long as
an efficient screening method is in place, this should produce no significant
pitfalls. Mutagenic treatment of seed is by far the most popular method in
mutation breeding programs. Mutagenic treatment of seeds and other parts of the
plant remains a useful tool for isolating the desired variants and developing
resistance to biotic and abiotic stresses in various crops because of its
relative simplicity and low cost. Exposing plant materials to mutagens enhances
the chance of isolating unique genetic material (Mba, 2013).
Bambara
groundnut, (Vigna subterranea (L.)
Verdc.) (syn. Voandzeia subterranea (L.),
is a known pulse which originated
from African continent. The common name appears to be derived from the place “The upper valley of Bambara” in the
region of North Eastern Nigeria
(Rassel 1960, Hepper 1963, Begemann 1988). The crop ranks third among the grain legume crops of Africa in terms
of production and consumption after
groundnut and cowpea. Nigeria is a major
producer of the crop. Seed yields in Africa average 650–850 kg/ha. It is also cultivated to a limited
extent in South Africa and United States.
The
world production is estimated at 330,000 tons annually, of which about half is
produced in West Africa. The main producing countries are, Nigeria, Ghana,
Chad, Niger, Togo, Benin, Asia, parts of Northern Australia, South and Central
America. Basically, Bambara groundnut is cultivated in almost all countries
South of Sahara in Africa.
Bambara
can be eaten fresh or grilled while still immature. They are boiled when the
seeds become matured and hard. In some African countries, the fresh pods are
boiled with salt and pepper and eaten as a snack. In eastern Africa they are
used to make a soup. Seeds are pulverized into flour and used as bread flour in
Zambia. The dried seeds are pounded into paste and used in the preparation of
various fried or steamed products such as Akara and Moi–Moi in Nigeria. The
plant is also used as animal fodder as its leaves are reported to be rich in
Nitrogen and Phosphorus and therefore suitable for animal grazing (Rassel et al., 1960).
1.2 STATEMENT
OF PROBLEM
Africans are faced with problems
that arise from malnutrition and famine. Semi-arid Africa has conditions (high
temperatures and low rainfall) that are do not favor many crops to sustain the
growing human population. Most countries that are found in famine ravaged
regions depend on food donated from United Nations (UN) or other organizations
for survival. At the regional level, Sub-Saharan Africa including Nigeria had
the highest proportion of undernourished and malnourished from 1997-1999 at 34
%. Asia and Pacific (excluding China) follows at 20 %, the Latin American and
the Caribbean with 11 %, and the Near East and North Africa region at 9% (www.worldhunger.org/articles/global/ray.htm).
According
to the Food and Agricultural Organization (FAO) of the United Nations, 850
million people worldwide were malnourished in 1999-2005 and the number of
malnourished people has increased. According to Jean Ziegler (the UN special
rapporteur on the Right to Food for 2002 to March 2008), mortality due to
malnutrition accounted for 58 % of the total mortality in 2006
(http://en.wikipedia.org/wiki /Malnutrition).
Bambara
groundnut has the potential to contribute to the food security of resource and
nutritionally poor communities throughout the semi-arid and tropical regions of
Nigeria. This research seeks to identify characteristics of Bambara groundnut genotypes
that enhance productivity, nutritional quality and tolerance to environmental
stresses as these might contribute significantly to food security, agricultural
diversification and income generation. If a country’s climate is unpredictable,
crop failure and livestock mortalities are usually the consequence. Despite all
the efforts being made, the unpredictable weather in the country continues to
take its toll on the agricultural sector in Nigeria. Therefore, it is important
for the Nigerian government to find ways to improve food production starting
with crops that are adapted to our environment, and that contain required
dietary constituents to avoid starvation and loss of human life. Although, a
number of studies have been done on induced mutagenesis in many pulses, limited
works has been carried out in this underexploited but highly nutritive crop.
Hence, the aim of this study will be to establish the effect of X-ray doses,
some growth parameters and molecular changes in the two varieties of Bambara groundnut.
1.3 OBJECTIVES
OF THE STUDY
The overall objective of this study
is on mutagenic effect of X-ray irradiation on some growth parameters and
molecular changes in Bambara Groundnut. The specific objectives are to;
●
To determine the effect of different levels of
X-ray on the germinability.
●
Investigate the effect of X-ray doses on the
growth components.
●
Carry out an RAPD analysis on the DNA of the
plant.
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