ABSTRACT
This study examined the effects of different concentrations of EMS on seed germination,growth and yield of three varieties of Capsicum chinense. The accessions were collected from different localities (oriugba, ubani market and isi gate) in Abia State. The study adopted a Randomized Complete Block Design (RCBD) with three replicates. The three varieties of Capsicum chinense were exposed to different EMS concentrations (0.1%, 0.3%, 0.5%, 0.7% and 0.9% v/v) and 5hrs exposure time, non treated seed were used as a comparative control. After treatment for 5hrs, the seed were grown in planting bags and were watered every day. The germination percentage were recorded, to determine the effects of EMS dose for the three varieties by subjecting the data collected to linear regression on the basis of their( growth parameters) plant height , number of leaves, number of branches, stem girth, leaf area were taken at every three weeks interval (3,6,and 9 Weeks after transplant) and yield parameters ( numbers of days to first fruits flowering, number of fruit per plant, number of seeds and weight of fresh fruit) was also recorded. Data were subjected to Analysis of Variance (ANOVA) and Least significant difference (LSD) was obtained at P<0.05. The results obtained from germination percentage showed that the germination of seeds was highest at the control (0.0%) and reduced among the treated plants. This reduction was observed to be concentration dependent. The LD50 values of 0.8%, 0.5% and 0.5% (v/v) EMS dosage was identified as an optimal doses for the three varieties of pepper plants. The results of different EMS concentrations on growth parameters of Capsicum chinense at 3,6 and 9 weeks after transplant indicated that there were significant difference (P<0.05) between the means of the treated plant when compared with control plants. Data obtained showed that treatment range from 0.1% to 0.5% EMS improved the genetic variability of the three varieties of pepper plants at morphological levels. The yield performances on the pepper varieties significantly improved the yield characters induced by the mutagen. The results of the analysis of variance showed that the plants treated with 0.1% EMS and 0.3% EMS enhanced the yield of this species. Molecular analysis was carried out using ISSR marker (Intersimple sequence repeat). Ten ISSR markers and rbcL primer was combined for the sequencing analysis and showed variability among the treated plants compared with the control. The ten ISSR marker detected 30 polymorphic alleles. The number of alleles for the ISSR loci ranged between 2, 3 and 4, with an average of 2.7 allele’s locus -1.The polymorphic information content were characterized in terms of their similarity to the records deposited in a Gene bank database. The types of sequences present in the polymorphic bands reflected the arrangements of the pepper varieties genome. Shifting the seasonal timing of reproduction is a major goal of plant breeding efforts to produce novel varieties that are better adapted to local environments. Hence lower EMS concentration positively improved the growth and yield characters of this study.
TABLE OF
CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table
of contents vi
List
of tables xi
List
of figures xii
List
of plates xiii
Abstract xiv
CHAPTER 1:
INTRODUCTION 1
1.1 Background of
Information 1
1.2 Statement of
Problem 3
1.3 Justification
of the study 4
1.4 Objectives of
the Study 4
CHAPTER 2: LITERATURE REVIEW 6
2.1 General
Information 6
2.2 Geographical
Origin and Climatic Requirements 7
2.2.1 Climatic
Requirements 8
2.2.2 Cultivation
Requirements 9
2.3 Cytogenetic of
Pepper 10
2. 4 Medicinal Benefits
and Other Uses of Habanero Pepper 12
2.5 Mutagenesis 13
2.5. 1 Spontaneous
Mutagenesis 15
2.5.2 Induced
Mutagenesis 16
2.6 Effects of
Chemical Mutagenesis 16
2.6.1 Assessment of the genetical variability of Ethyl methane
sulphonate (EMS) 18
2.6.2 Alkylating
Agents 19
2.6.3
Physiochemical properties as they mutagenicity of alkylating agents 20
2.6.3.1
Methylating and Ethylating Agents 20
2.6.3.2 Number of
Functional Group 21
2.6.3.3 Solubility
in Lipids 21
2.6.3.4 Charge of
Molecules 21
2.6.3.5 pH and
Buffers 22
2.6.3.6
Temperature 23
2.7 Dose
Determination and Mutational Loads 23
2.7.1
Concentration 24
2.7.2 Duration of
Treatments 25
2.7.3 Properties
of Chemical Mutagens that Influence the effect of Treatments 26
2.8 Modifying
Factors 27
2.8.1 Pre Soaking 27
2.8.2 Metallic
ions 28
2.8.3 Carrier
Agents 28
2.8.4 After
Treatments 29
2.9 Methods of Pre
and Post Treatment in Chemical Mutagenesis 30
2.9.1 Pre
Treatments soaking 30
2.9.2 Infusion of
the mutagens 32
2.9.3 Post
Treatments Drying 34
2.9.4 Methods of
Drying 35
2.9.5 Post
Treatments Washing 36
2.9.6 Post
Treatments Application of Chemical Mutagen 36
2.9.7 Handling and
Disposal of Chemical Mutagens 37
2.9.7.1 Disposal
of Chemical Mutagens 38
2.10 Effects of
Mutagens on Crop Improvements 39
2.11 Economics
Impacts of Mutational Breeding 40
2. 11.1 Economics
Importance and Potentials in Poverty Reduction 42
2.11.2 Past
Achievements 42
2.11.3 Some
Highlight of Mutant Varieties in the World 43
2.11.3.1 Genetics
Enhancement of Rice 43
2.11.3.2
Developing Drought and Salinity Tolerance in Wheat Crop 44
2. 11.3.3
Enhancing Lodging in Barley Crop 45
2.11.3.4
Developing Early Maturity Varieties of Peanuts 45
2.11.3.5 High
Yielding and Wilt Disease Resistant Chickpea Mutant 45
2.11.3.6
Ornamental Plants 46
2.12 Limitations
and Advantages of Mutagenesis as a Plant Breeding Technique 47
2.12.1 Limitation
of Mutagenesis 47
2. 12.2 Advantages
of Mutagenesis 48
CHAPTER 3: MATERIALS AND METHODS 49
3.1 Study Area 49
3.1.1 Collection
of Plant Material 49
3.1.2 Seed viability
test 49
3.1.3 Seed
Treatments 50
3.1.4 Experimental
Design 51
3.1.5 Data
Collection 51
3.1.5.1 Recording
of Growth Parameters 51
3.1.5.2 Recording
of Yield Parameters 53
3.1.6 Statistical
Analysis 53
3.2 Polymerase
chain reaction (PCR) for Intersimple Sequence Repeat (ISSR)
Analyses 54
3.2.1 Genomic DNA
Extraction from the Pepper Samples 54
3.2.2 Agarose Gel
Electrophoresis 55
3.2.3 Dilution of
DNA for PCR 56
3.2.4 DNA Amplification 56
3.2.5 Sequencing 56
3.2.6 Data
Analysis 57
3.3 Ethical
Consideration/ Issues 57
CHAPTER 4: RESULTS AND DISCUSSIONS 59
4.1 Results 59
4.1.1 Effects of
different concentration of EMS on germination percentage 59
4.1.2 Effects of
different concentration of EMS on LD50 61
4.1.3 Effects of
different concentration of EMS on plant height 64
4.1.4 Effects of
different concentration of EMS on number of leaves 66
4.1.5 Effects of
different concentration of EMS on number of branches 68
4.1.6 Effects of
different concentration of EMS on stem girth 70
4.1.7 Effects of
different concentration of EMS on leaf area 72
4.2 Yield
parameters 74
4.2.1 Effects of different concentration of
EMS on number of days to first
flowering 74
4.2.2 Effects of different concentration of
EMS on number of fruits 76
4.2.3 Effects of different concentration of
EMS on weight of fresh fruits 78
4.2.4 Effects of different concentration of EMS
on number of seeds 80
4.3 Molecular
analysis 82
4.3.1 Genetic
polymorphism and banding pattern among the EMS concentration
of pepper varieties 82
4.3.2 Percentage
polymorphism of the primer and Genetic relatedness/similarities
between the three EMS treated pepper
varieties using Dendrogram analysis 90.
4.3.3 Mutational
analysis on sequenced EMS treated and untreated (control) on
the three varieties of pepper. 94
4.4 Discussion 98
CHAPTER 5: CONCLUSION AND RECOMMENDATIONS 104
5.1 Conclusion 104
5.2
Recommendations 105
REFERENCES
APPENDIXES
LIST OF TABLES
Table 4.1: Effects
of different concentration of EMS on germination percentage 60
Table 4.2: Effects
of different concentration of EMS on plant height 65
Table 4.3: Effects
of different concentration of EMS on number of leaves 67
Table 4.4 :Effects
of different concentration of EMS on number of branches 69
Table 4.5: Effects
of different concentration of EMS on stem girth 71
Table 4.6: Effects
of different concentration of EMS on leaf area 73
Table 4.7: Effects
of different concentration of EMS on number of days to first flowering 75
Table 4.8: Effects
of different concentration of EMS on number of fruits 77
Table 4.9: Effects
of different concentration of EMS on weight of fresh fruits 79
Table 4.10:
Effects of different concentration of EMS on number of seeds 81
Table 4.11:
Scoring sheet for statistical analysis 89
Table 4.12: ISSR
marker used and their polymorphic information content 92
Table 4.13:
Tabular description of mutational types occurred at the sequence on
gene bank database 96.
Table 4.1 4: Blast
results showing identity to already established sequence on
gene bank database. 97
LIST OF FIGURES
Figure 4.1:
Effects of different concentrations of EMS on lethal dose (LD50) showing germination percentage plotted
against EMS concentrations of Datil pepper 62.
Figure 4.2:
Effects of different concentrations of EMS on lethal dose (LD50) showing germination percentage plotted
against EMS concentrations of Ose Ibeku 62.
Figure 4.3: Effects of different concentrations of
EMS on lethal dose (LD50) showing
germination percentage plotted against EMS concentrations of Ose Nsukka 63
Figure 4.4:
ISSR –based dendogram showing genetic relatedness/ similarities between the mutated three species of capsicum chinense during M1 generation obtained by UPMGA 93
Figure 4.5: rbcL
sequence of three pepper species with their parents in M1 generation. 95
LIST OF PLATES
Plate 4.1: Gel
image showing amplification within the range of 50bp to 150bp 83
Plate 4.2: Gel
image showing amplification within the range of 150bp to 600bp 84
Plate 4. 3: Gel
image showing amplification within the range of 200bp to 700bp 84
Plate 4.4: Gel
image showing amplification within the range of 300bp to 350bp 85
Plate 4.5: Gel
image showing amplification within the range of 300bp to 766bp 86
Plate 4.6: Gel
image showing amplification within the range of 350bp to 916bp 86
Plate 4.7: Gel
image showing amplification within the range of 400bp to 500bp 87
Plate 4.8: Gel
image showing amplification within the range of 400bp to 700bp 87
Plate 4.9: Gel
image showing amplification within the range of 400bp to 766bp 88
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND INFORMATION
One
of the major global active ingredients in ointment, nasal sprays and pain
relief drugs is the capsaicin. Capsaicin an alkaloid in Datil pepper, Aji dulce
(Ose Ibeku) and habanero chile (Ose Nsukka) that makes them bioactive plants is
used as analgesic in dermal patches, nasal spray and topical ointments etc,
which makes pepper one of the most globally important commercially
produced crops and are annually grown as vegetable crops in tropical and
subtropical regions because of its pungency and high nutritional value. It is
becoming increasingly popular among consumers, with industrial applications
also rising worldwide to match
with the rapid growth of population in the world, because of its
nutritional values and medicinal needs around the world therefore, there is
need to increase Habanero pepper
production (The Royal Society, 2009; Fattori et al., 2016; Wargent and Joradan, 2013; Oladosu et al.,2011 ). Average world
production and cultivated area of dry, red, yellow and green peppers are
estimated at 3.9, 4.0 and 34.5 million tons for about 60% of the global pepper
production in 2016, Habanero Pepper is the most
widely used spice and condiment in the world and is greatly priced for its
pungency and adding special flavor to many cuisines throughout the world.
Edible pepper contains the basic
nutritional components such as essential minerals, amino acids and vitamins.
However, the demand of ever- increasing population cannot attend low production
of pepper. To meet this ever-increasing Industrial demand, the exploitation of
non-conventional/induced mutagenesis seeds has become inevitable (Bado et al., 2015). Such explorations may
assuage the problems of food security, agricultural development,
self-dependence and enhancement of the economy of developing countries. In
other regions of the world, Pepper production has grown steadily, accounting
for ~60% of global production in 2016 and improved yields have been the major
driver of pepper production across most regions.
Nutritionally,
Habanero Pepper have been reported to be rich in minerals and vitamins, containing
important amino acid, anti-oxidants such as vitamin B5. The mineral and vitamin
contents are comparable with those in other Lycopersicon
family like potato and tomato. It is rich in mineral quality, vitamin and
antioxidant carotene lycopene contents (Ahloowalia et al., 2004;
Materska and Perucka, 2005). Cultivations of this plant have been reported in
many countries like Indian, Portuguese, Europe, etc. down to West Africa like
Nigeria especially in Northern Nigeria were Datil pepper are grown and sold to
other parts of the country and also to the Eastern Nigeria especially in
Umuahia North, Abia State among the Ibeku people. Aji dulce is known as Ose Ibeku popularly
cultivated for their native dishes because of its flavor and Habanero chile is known as Ose Nsukka popularly cultivated by Nsukka people as
economic crop in Enugu State
(Usman,
2016). The major constraint in the development of improved varieties is the
limited genetic variability results (Irfaq and Nawab, 2003). There are many
techniques for breeding plants; mutation breeding is one of such techniques
applied for crop improvement.
Plant mutation
breeding is neither a novel topic nor a novel technique. Since the early 20th
century, mutation breeding has been applied to both plants and animals. Plant mutation breeding has been accepted as
a great technique for increasing the genetic diversity of plants; more
importantly, commercially grown crops. Unlike traditional breeding approaches,
mutation breeding is more effective and less time-consuming. As science
advances, mutation breeding approaches have developed significantly over the
past few decades. Most approaches to mutation breeding rely on mutagenic
agents, which are responsible for the creation of mutations in plant genetic
material. These mutagenic agents have been in use for many decades now and
proven to be an integral part of plant mutation breeding as it creates mutation
at a much faster rate than a spontaneous mutation processes.
Induced
mutagenesis is one of the most important approaches for broadening crop genetic
variability to overcome the limitations associated with a narrow genetic basis
(Asif and Khalil- Ansari, 2019). Induced mutants not only serve as an important
functional genomic tool, but additionally, as intermediate material in crop
breeding (Henry et al., 2014). Induced
mutations have played a significant role in meeting challenges relating to the
world food and nutritional security by way of mutant germplasm enhancement and
utilization for the development of improved varieties in several crops.
Different mutagens are applied on
seeds to increase yield. Among them, ethyl methane sulphonate (H3SO2OC2H5)
(EMS) is a potent chemical mutagen used to induce mutational variability on
plants to increase yield and ameliorate the hunger problem of the world. It is
more effective than other chemical and physical mutagens (Bhat et al., 2009; Wani et al., 2012). It is
pertinent to mention that in spite of the numerous works that have been done on
pepper plants, most of them are still understudied.
1.2 STATEMENT
OF PROBLEM
In today’s rapidly challenging
climates, ever-increasing human population growth and increased competition to
source for bioactive plants to solve industrial needs, the production for spicy
and high quality vegetables to meet increasing demands presents an enormous challenge.
There is much dependence on Pepper (Capsicum
chinense ) which has led to the tremendous increase in the market price.
Pepper research in terms of mutational breeding in Nigeria is still at a low
level compared to other Solanaceous
vegetable crops such as eggplants and farmers still cultivate the traditional
varieties (Akerberg and Hagberg, 1963). In large parts of Sub Saharan Africa
particularly Nigeria, small holder agricultural production has remained
consistently low and food security is catastrophically poor (Kraft et al., 2014).
Induced mutations are known to
enhance the genetic variability of crops and spontaneous mutations facilitate the
development of improved varieties at a swiftless rate (Pathirana, 2011). High
demand for pepper for improved varieties with high yield and morphological
changes can be achieved through induction of beneficial mutation in traditional
landrace of Capsicum through the use
of chemical mutagens and there has not be much work done on our indigenous
crops (pepper plants) using this methods.
1.3
JUSTIFICATION OF THE STUDY
Mutagenic methods have contributed
immensely to the development of genetically improved crop varieties. Their
methods continue to enrich the crop germ-plasm base by evolving genetically superior
varieties for cultivation. Existing germ-plasm may not be adequate to meet the
capsaicin needs of an era-increasing human population, estimated to swell to 9
billion by 2050 (Mohan and Suprasanna, 2011; Burke et al., 2009).
Further increase in agricultural
productivity, equitability and in an environmentally sustainable manner, with
the face of limiting resource, is a challenging task. The use of induced
mutations have played pivotal role in the improvement of superior plant
varieties (Ahloowalia and Malsuzynski, 2001; Jain, 2005).Despite the numerous
uses and nutritional values of Habanero Pepper, the plant has received little mutagenic research attention,
hence, the need for this study.
1.4
OBJECTIVES OF THE STUDY
The broad objective of this study is
to examine the mutagenic effect of EMS on the morphology, yield and some
molecular attributes of three pepper varieties.
The Specific objectives of this study
are as follows;
· To
Study the optimal concentration of EMS suitable for mutation of pepper;
· To
investigate the effect of different concentrations of EMS on the growth and
yield parameters of Pepper in M1 generations.
· To
assess the genetical variability of pepper plants at the morphological levels.
· Carryout
ISSR-PCR assay of the mutated plants in M1 to detect genetic
changes.
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