ABSTRACT
The present study was carried out to investigate the effect of x-ray doses on the germination, survival, growth, yield, proximate, mineral, vitamin compositions and DNA sequence characterization of the fluted pumpkin (Telfairia occidentalis Hook F.) in M1 and M2 generations. The treatments include 0.00 mGy, 6.75 mGy, 10.08 mGy, 14.08 mGy and 18.75mGy. The experiment was set up in a randomized complete block design (RCBD) with five replications. Treated seeds were planted and data obtained in both generations revealed a reduction in germination and survival percentages with increase in x-ray doses. The result showed that there was no significant difference (P>0.05) in germination percentage during M1 generation whereas in M2 generation, germination percentage was highly different when compared to the control. Observation on the survival percentages also revealed a highly significant difference between the treated plants when compared to the control in M1 and M2 generations. The result of the analysis of variance showed that the effect of the mutagen doses on the leaf area during M1 and M2 generation was not significantly different (P>0.05) when compared to the control. The effect of the x-ray doses on the number of leaves per plant, number of branches and vine length was highly significant (P<0.05). Data obtained on the yield parameters showed that the mutagen significantly enhanced the yield of fluted pumpkin. The result showed that treatment 14.08 mGy significant enhanced all the yield traits studied. The proximate composition of fluted pumpkin was affected by the mutagen treatments during M1 and M2 generations. Crude protein, moisture and carbohydrate contents showed a significant decrease effect (P<0.05) in both generations whereas crude fibre, ash and fat contents were not significantly different but showed increase greater than the control at different treatment concentration levels. Data obtained on the mineral composition showed that the effect of the mutagen doses on the calcium, phosphorus, iron and nitrogen compositions showed significant difference when compared to the control plants during M1 and M2 generations. The result obtained on the vitamin content of the plant showed that there was a gradual reduction and increase at varying treatment concentrations in the vitamins studied during M1 and M2 generations. The aligned DNA sequence of the treated and untreated sequences reveals all kinds of SNP mutation in the form of indels and nucleotide substitutions. However; the indels were the most occurred. A total of 17 indels were recorded with 5 of cytidine and 4 thymidine, adenosine and guanosine respectively. Cytidine, which had the highest indel occurrence were seen at positions 385, 460, 506, 545 and 551. Thymidine indel which had a total of 4 occurrences were at positions 46, 393, 417 and 472 while 4 guanosine indel were seen at positions 360, 339, 453 and 522 respectively. Adenosine indel were seen at positions 371, 423, 529, and 540. Both the transversion and transition occurred in this study and all were in the ratio 4:1 except for G: T transversion and A:G transition at positions 495 and 87 respectively which both had a ratio of 1:1. A total of 7 transversion were recorded among which were the A:C at position 411, A:T at positions 88, 124, and 352, G:C at position 349 and G:T at positions 474 and 495. While only 4 transitions were recorded which were T:C at location 402 and A:G in positions 87, 435 and 530. These variations reveal the effectiveness of physical mutagens. The observed field performance and variation in physiological traits, proximate, mineral and vitamin levels designed by the DNA polymorphism as induced by different doses of x-ray irradiation reveals the effectiveness of physical mutagens. The variation at different treatment levels is linked to more of genetic effects to environmental as this can serve as a useful selection tool. It is therefore recommended that treatment 14.08 mGy of x-ray should be employed in mutation breeding of fluted pumpkin for improved agronomic traits.
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables xii
List of Figures xiii
Abstract xiv
CHAPTER 1: INTRODUCTION 1
1.1 Background
of the Study 1
1.1 Statement
of the Problem 2
1.3 Justification
of the Study 3
1.4 Objectives
of the Study 4
CHAPTER 2: LITERATURE REVIEW 5
2.1 Botanical Classification of Telfairia occidentalis 5
2.2 Description of the Plant 5
2.3 Structure of the Plant 6
2.4 Nutritional
Composition 7
2.5 Cultivation 8
2.6 Storage 8
2.7 Pest and Pathogens 9
2.8 Uses Fluted Pumpkin 9
2.8.1 Anti-microbial activities 9
2.8.2 Anti-diabetic activity 9
2.8.3 Treatment of infertility 10
2.8.4 Hematological properties 10
2.8.5 Anti-malaria properties 10
2.9 Crop Improvement through Mutagenesis 10
2.10 Types of Mutagens 13
2.11 Consequences of Genetic
Alteration in Plant Morphology 14
2.12 Business Interests of Mutation Breeding 16
2.13 X-ray Irradiation 18
2.14 X-ray Unit of Measures and Exposure 20
2.15 Measurement of Agronomic
Response of Plants Exposed to
X-ray
Radiation 20
CHAPTER
3: MATERIALS AND METHODS 22
3.1 Study Area 22
3.2 Collection of Research Material 22
3.3 Seed Treatments 22
3.4 Land Preparation and Planting Dates 22
3.5 Experimental Design 23
3.6 Data Collection 23
3.6.1 Germination percentage 23
3.6.2 Survival percentage 23
3.7 Data on Growth Parameters 23
3.7.1 Vine length per treatment 23
3.7.2 Number of secondary branches per treatment 23
3.7.3 Number of leaves per vine per treatment 24
3.7.4 Leaf area per treatment 24
3.8 Data on Yield Parameters 24
3.8.1 Size of pods per treatment 24
3.8.2
Number of pods per treatment 24
3.8.3 Number
of seeds per pod per treatment 24
3.9 Molecular Assay 24
3.9.1
DNA extraction 24
3.9.2 Extraction of buffer components 25
3.9.3 DNA
quantification 25
3.9.4 SNP-PCR gene amplification 26
3.9.4.1 PCR Mix component 26
3.9.4.2 Primer sequences 26
3.9.4.3 Cycling
conditions 26
3.9.4.4 Electrophoresis for PCR 27
3.9.4.5 Tracing of DNA polymorphism
along sequenced regions 27
3.10 Proximate Analysis 27
3.10.1 Crude protein percentage 27
3.10.2 Crude fibre percentage 28
3.10.3 Fat percentage 29
3.10.4 Ash content 29
3.10.5
Moisture content percentage 30
3.10.6
Carbohydrate percentage 30
3.11 Mineral Analysis 31
3.11.1
Calcium
determination 31
3.11.2
Phosphorus determination 32
3.11.3 Determination of total nitrogen (Kjeldahl
method) 33
3.11.4 Iron determination by orthophenantroline
method 33
3.12 Vitamin Analysis 33
3.12.1 Determination of vitamins 34
3.12.2 Riboflavin (vit B2) determination
(Okwu and Ndu.,2005) 34
3.12.3 Vitamin B3 (niacin) content
determination (Okwu and Ndu., 2005) 35
3.12.4 Determination
of vitamin C (Okwu and Ndu., 2005) 36
3.13 Field Maintenance 36
3.14 Harvest 37
3.15 Data Analysis 37
CHAPTER
4: RESULTS AND DISCUSSION 38
4.1 Results 38
4.1.1 Effect of x-ray doses on the germination
percentage during
M1 and M2
generations 40
4.1.2 Effect
of x-ray doses on survival percentage in M1 and M2
generations 41
4.1.3 Effect x-ray does on the growth parameters
of fluted pumpkin during
M1 and M2
generations 42
4.1.3.1.
Leaf area 42
4.1.3.2
Number of branches per vine 43
4.1.3.3.
Number of leaves per vine 44
4.1.3.4.
Vine length 45
4.1.4 Effect different of x-ray doses on the yield
parameters of fluted pumpkin
during M1 and M2
generations 47
4.1.4.1 Number
of pods per plant 47
4.1.4.2 Number
of pods per plant 48
4.1.4.3.
Pod diameter 49
4.1.4.4.
Pod length 50
4.1.4.5.
Pod weight 51
4.1.5 Effect of different x-ray doses on the
proximate composition of fluted
pumpkin during M1 and M2
generations 52
4.1.5.1 Crude protein 52
4.1.5.2. Crude fiber 53
4.1.5.3. Fat 53
4.1.5.4. Ash 54
4.1.5.5. Moisture content 54
4.1.5.6. Carbohydrate percentage 55
4.1.6 Effect of different x-ray doses with mineral
composition of fluted
pumpkin during M1 and M2
generations 56
4.1.6.1 Calcium level 56
4.1.6.2 Phosphorus level 56
4.1.6.3. Iron 57
4.1.6.4. Nitrogen 57
4.1.7 Effect of different x-ray doses on the
vitamin content during M1 and M2
generations 58
4.1.7.1. Thiamin (vitamin B1) 58
4.1.7.2. Riboflavin (vitamin B2) 59
4.1.7.3. Niacin (vitamin B3) 59
4.1.7.4. Vitamin C. 60
4.1.8. Effects of different doses of x-ray
irradiation on the DNA sequence
of fluted
pumpkin 61
4.1.8.1 Sequence alignment of the treated samples 62
4.1.8.2 The positions of single nucleotide
polymorphisms (SNP’s) mutations
along the ribulose bisphosphate
carboxylase (RBCL) sequence regions 63
4.1.8.3 Influence of mutation along the amplified
sequence 64
4.2 Discussion 66
4.2.1 Effect of x-ray doses on the germination and
survival percentages
of fluted pumpkin 66
4.2.2 Effect of X-ray doses on the growth yield
parameters, and proximate
composition during m1
and m2 generations 68
CHAPTER
5: CONCLUSION AND RECOMMENDATIONS 70
5.1 Conclusion 70
5.2 Recommendations 71
References 72
Appendices 80
LIST OF TABLES
PAGE
4.1: The positions of single
nucleotide polymorphisms (SNP’s) mutations
Along the ribulose
bisphosphate carboxylase (rbcl) sequence regions. 64
4.2: Influence of the mutations
along the amplified sequence as investigated
in the treated samples
and examined with a phylogenetic analysis 66
LIST OF FIGURES
PAGE
2.1: Fluted pumpkin guard 6
2.2: Fluted pumpkin leaves 7
4.1: Research
study area, two weeks after planting 38
4.2: Research study area, six weeks after
planting 39
4.3 Research study area, thirty-three weeks
after planting 39
4.4: Effect of X-ray doses on the germination
percentage 41
4.5: Effect of X-ray doses on the survival
percentage 42
4.6 Effect of different x-ray doses on the
leaf area 43
4.7: Effect of different x-ray doses on the
number of branches produced
per vine 44
4.8: Effect
of different x-ray doses on the number of leaves produced
per vine 45
4.9: Effect
of different x-ray doses on the vine length 46
4.10: Effect
of different x-ray doses on the number of pods produced
per plant 48
4.11: Effect
of different x-ray doses on the number of seeds produced
per pod 49
4.12: Effect
of different x-ray doses on the pod diameter of fluted pumpkin 50
4.13: Effect
of different x-ray doses on the pod length of fluted pumpkin 51
4.14 Effect
of different x-ray doses on the pod weight of fluted pumpkin 52
4.15 Effect
of different x-ray doses on the proximate levels of fluted pumpkin 55
4.16: Effect
of different x-ray doses on the mineral levels of fluted pumpkin 58
4.17: Effect
of different x-ray doses on the vitamin levels of fluted pumpkin 60
4.18 The DNA amplification result 61
4.19: Sequence
alignment of treated samples 63
4.20: A
dendogram representing the morphological variation among the
different treatments 65
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
The essence of natural evolution (land race) and induced
mutation has been the notable sources for competitive and improved plant stock
for farmers and plant breeders. Despite the fact that nature has been playing
its role in evolving desirable crop species, the constraint of time cannot be
overlooked in the speed of producing results. Induced mutation technique is
used to breach the vacuum created by nature. This have been found to
successfully create variability and produce new desirable mutants which are
multiplied and put to use after vigorous selection and breeding to
homozygosity. The frequency of induced mutation is 10-3 gametes against 10-6
gametes in the case of spontaneous mutation and this has made the induced
mutation preferable to spontaneous mutation (Muller, 1927). Due to its
applications in agriculture, medicine, pharmaceutical usage, and numerous
technical innovations, findings on the fundamental interaction of ionizing
radiation with biological processes has made a significant contribution to
human society. Between the initial absorption of energy and the most relevant
target, the water molecule, which is ubiquitous in organisms, the prevalence of
such biological systems to ionizing radiation resuscitates a lot of physical
and chemical reactions. The fundamental process of excitation and ionization
produces the ionized water molecule (H2O+) as well as the radicals H+ and OH-.
Chain reaction which produce free radicals (H+ and e-aq) that are trapped
occurs due to the consequences of ionization on living tissues (Esnault et al., 2010). These radicals have the
potential to destroy or change essential parts of plant cells, as well as
physiological and biochemical processes that are important for surviving of
organism.
X-ray and gamma which are mutagenic sources have been used to induce
plant traits such as nutritional attributes, disease resistance, yields, and
height (Iwo et al., 2013). It allows
for the induction of the desired characteristics that are either not exhibited
naturally or lost during the evolutionary passage. Previous studies (Akpaniwo et al., 2015) revealed that the seed of
fluted pumpkin when exposed to high doses of X-rays reduced germination and
survival rates of pumpkin. Also, (Al-Enezi et
al. 2012) revealed significant retardation in seed germination and survival
rate in date palm. Hameed et al.
(2008) and Al-Salhi et al. (2004) has
found that high exposure to gamma rays disrupt protein synthesis, leaf gas
exchange, hormonal balance, and activities of enzymes in seeds. The process of
Induced mutation has long been used to improve cultivars, and it has proven to
be a powerful tool for expanding the resources in genetics, particularly for
odd plants, and for selecting mutants with favorable agronomic features (Taheri
et al., 2014). Induced mutation of
about 170 different plant species have given rise to above 2700 improved
varieties (Luo et al., 2013). Induced
mutations increase DNA polymorphism, which contributes to genetic diversity
(Dhakshanamoorthy et al., 2010). Maize
(Rustikawati et al., 2012), faba bean
(Mejri et al., 2012), Curcuma alismatifolia Gagnep (Taheri et al., 2014), and Acorus calamus
(Taheri et al, 2014) are only a few
examples of plants.
1.2 STATEMENT OF PROBLEM
Ensuring that crop
production meets the anticipated population increase to more than 9 billion by
2050 is an astounding challenge for the plant scientist/breeder as reported by
the United Nations department of Economic and Social Affairs Population Division
(UNDESAPD). This objective is confronting generally on the account that the
numerical mean rate of crop production increase is only 1.3 % per year, and it
cannot keep tread with population growth. Breeding by the process of mutation
is one of the plant breeding approaches used to create the genetic variety in
yield giving traits and to boost crop output, according to Ahloowalia et al. (2000). Induced mutation,
primarily using x-ray irradiation, has been acknowledged as a useful addition
to traditional breeding in crop development. The genetic variability from
mutagenesis complements that from germplasm collection as well as from the
crossing and the demand for improved varieties of cowpea that integrate good
quality, high yield and disease resistance can be achieved through initiation
of gainful mutation in pumpkin through X-ray.
1.3 JUSTIFICATION OF RESEARCH
With the recent global food crisis occasioned by the scarce
supply of food crops, especially vegetables, which includes fluted Pumpkin,
there is an urgent need to increase its production through adequate research,
bearing in mind the high economic benefits with regards to nutrition and
medicine. Improving agricultural sustainability while lowering its
environmental impact with the intent of meeting the expanding global
population's food need is a major challenge facing humanity (Edmondson et al., 2014). Following the lofty
nutritional and economic importance of fluted pumpkin, its production level has
always been below demand. The major reason for this has been traced to non-availability
of genetically modified varieties for planting; hence farmers have resorted to
the use of landraces. Differentiation among T.
occidentalis species is low and improved varieties are non-existent (Fayeum
and Odiyi, 2012). Hence, crop genetic modification is critical to increasing
the yield of this valuable and important vegetable.
There is limited research information on the impact of x-ray on
the genetic sequence, agronomic performance, proximate, vitamin and mineral
concentration on fluted pumpkin.
1.4 OBJECTIVES OF THE STUDY
The objectives of this research
are as follows;
1.
To evaluate the impact of different x-ray doses on the
germination and survival percentages of fluted pumpkin during the M1 and the M2
generations.
2.
To determine x-ray
irradiation impacts on germination, growth and yield parameters in fluted
pumpkin at the M1 and the M2 generations.
3.
To investigate the impact of x-ray treatment on the
proximate, vitamin and mineral concentration in T. occidentalis M1 and M2 generations.
4.
To determine an economic profitable x-ray treatment dose
in fluted pumpkin.
5.
To investigate the influence of X-ray irradiation on the
genetic sequence in the M1 and the M2 generations.
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