UNIVERSITI TEKNOLOGI MARA
IN VITRO MUTATION BREEDING ON Musa acuminata cv. Berangan
USING GAMMA RADIATION
AISYAH ATHIRAH BINTI HASIM
Thesis submitted in partial fulfillment of the requirements for the degree of
Master of Science (Plant Biotechnology)
Faculty of Plantation and Agrotechnology
January 2022
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ABSTRACT
The traditional breeding mode is poor solely due to the lack of species variability, infertility and polyploidy state of the banana cultivar. The application of in vitro cultivation is aimed at providing continuous banana supply in a highly efficient and systematic manner. In fact, mutation is also considered as an effective strategy to improve the performance of the in vitro culture since it is a potent alternative to invent novel varieties for banana improvement. Hence, induced mutagenesis via gamma ray in combination with in vitro tissue culture through micro cross section culture procedure was conducted to evaluate the effects of gamma radiation on the growth and development of the in vitro plantlet as well as to identify the genetic variations of the irradiated plant. The stem of the tissue cultured banana plantlet was used as the source of explant in this study. The plantlet was cut into half (2 cm) and exposed to gamma ray with different dosages at 0 Gy (control), 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, 60 Gy and 80 Gy. The radiosensitivity test was conducted to determine Lethal Dose (LD50) and the result obtained was at 37 Gy. Highest percentage of survival rate was recorded in 20 Gy (23.33%) while the lowest was in 80 Gy (0.33%). The morphological study on the growth of in vitro radiated plantlets revealed that 25 Gy induced stimulant effects in terms of fresh weight (13.63± 4.77), number of shoot emerged (5.56 ± 0.98)and plant height (6.34± 0.24) over non-radiated banana explants on in vitro culture condition. Interestingly, the mutants of 25 Gy showed the best growth performance in all morphological traits when transferred to ambient condition. Next, molecular analysis was performed using PCR based RAPD marker to assess the genetic variation of the radiated plantlets. Based on the observation, RAPD molecular analysis generated 81.16% of genetic polymorphism, a high reposition incidence in the DNA sequence of irradiated banana. Several primers namely OPA7, OPA11, OPA14, OPC01 and OPD16 displayed unique banding profile ranged from 200 bp to 1500 bp. Thus, this study contributed to the development of gamma irradiated banana using in vitro culture system for future breeding of new varieties of banana and the DNA polymorphism revealed by RAPD study can be interpreted to offer a reliable molecular marker for the identification of modifications in gamma treated plants.
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ACKNOWLEDGEMENT
I would like to express my sincere gratitude to Assoc. Prof. Ts. Dr. Shamsiah Abdullah for taking me under her guidance, offering valuable suggestions and assisted in financial and administrative issues. I also want to convey my immense appreciation to Dr. Sobri Hussien, my co-supervisor for this project, for giving me the opportunity to use the gamma facility in Agensi Nuklear Malaysia (ANM). I am also highly grateful to Encik Shuhaimi from ANM for his excellent technical support and guidance in handling the BIOBEAM gamma irradiation device to irradiate my samples.
I am also very thankful to my friends at the Plant Tissue Culture Laboratory, Faculty of Plantation and Agrotechnology, UiTM Kampus Puncak Alam for the friendliness, helpfulness and lively atmosphere. Special thanks to my lovely lab mates who always supported and cheered me up whenever I faced some issues with my work.
Last but not least, I would like to dedicate my special gratitude to my parents for their endless encouragement, both emotionally and financially, and also my family members for their endless support throughout the project. It would not be an easy journey to accomplish the project without them.
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TABLE OF CONTENTS
Page CONFIRMATION BY PANEL OF EXAMINERS ii
AUTHOR’S DECLARATION iii
ABSTRACT iv
ACKNOLEDGEMENT v
TABLE OF CONTENTS vi
LIST OF TABLES ix
LIST OF FIGURES x
LIST OF PLATES xi
LIST OF ABBREVIATIONS xii
LIST OF NOMENCLATURE xiii
CHAPTER ONE: INTRODUCTION 1
1.1 Research Background 1
1.2 Problem Statement 3
1.3 Objectives 4
1.4 Research Question 4
1.5 Significance of Study 4
1.6 Scope of Study 5
1.7 Limitation of Study 5
CHAPTER TWO: LITERATURE REVIEW 7
2.1 Morphology of Banana (Musa Spp.) 7
2.1.1 Centre of Origin 8
2.1.2 Cultivation of Banana in Malaysia 8
2.2 Plant Tissue Culture 10
2.2.1 Stages of Plant Tissue Culture 10
2.2.1.1 Culture Initiation Phase (M1V1) 10
2.2.1.2 Multiplication Phase (M1V2) 11
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2.2.1.3 Root Formation Phase (M1V3) 11
2.2.1.4 Acclimatization Phase (M1V4) 11
2.2.2 Tissue Culture of Musa Spp. 11
2.2.3 Micro - Cross Section Cultivation System 12
2.3 Mutation Breeding 13
2.3.1 Gamma Radiation as Physical Mutagen 14 2.3.2 The Pros and Cons of Physical and Chemical Mutagen 16
2.4 Molecular Marker 19
2.4.1 PCR Based Molecular Marker 20
2.4.2 Rapid Amplified Polymorphic DNA (RAPD) 21
CHAPTER THREE: RESEARCH METHODOLOGY 24
3.1 Plant Materials and Experimental Location 24
3.2 Experimental Design 24
3.3 In Vitro Tissue Culture of Berangan Banana (Musa acuminata cv. Berangan)
25 3.3.1 Establishment of Micro Cross Section and Gamma Radiation
Treatment
25 3.3.1.1 Micro Cross Section Initiation Culture 25
3.3.1.2 Plantlet Regeneration 26
3.4 Data Collection 26
3.5 Identification of Mutant Variation Using Molecular Marker 27
3.5.1 Isolation of DNA 27
3.5.2 Quantification of DNA 28
3.6 Molecular Analysis 29
3.6.1 Polymerase Chain Reaction (PCR) Using RAPD Marker 29 3.6.2 Gel Electrophoresis for Polymorphism Analysis 30 3.6.3 Band Scoring and Phylogenetic Analysis 31
3.7 Statistical Analysis 32
CHAPTER FOUR: RESULTS AND DISCUSSIONS 33 4.1 Effects of Mutation Induction on Radiosensitivity
and Post - Radiation Recovery on Berangan Banana
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