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UNIVERSITI PUTRA MALAYSIA
NURASHIKIN BINTI KEMAT
FP 2012 32
IN VITRO PLANT REGENERATION AND RAPD ASSESSMENT OF SOMACLONAL VARIATIONS IN SAFED MUSLI (Chlorophytum
borivilianum Santapau & R.R.Fernandes)
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IN VITRO PLANT REGENERATION AND RAPD ASSESSMENT OF SOMACLONAL VARIATIONS IN
SAFED MUSLI (Chlorophytum borivilianum Santapau & R.R.Fernandes)
NURASHIKIN BINTI KEMAT
MASTER OF SCIENCE
UNIVERSITI PUTRA MALAYSIA
2012
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This thesis is dedicated to:
My dear mother, Norainon binti Arshad and my dear father, Kemat bin Adnan for their encouragement throughout my study,
my beloved husband, Sharul Nizam bin Ahmad for his sacrifies, understanding and commitment throughout my study
and my lovely son, Raes Zafran bin Sahrul Nizam for making my life more meaningful
words cannot express alone my gratitude to people above for their endless and boundless love,
and most of all for their ever continuous do’a for my life.
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Abstract of the thesis presented to the Senate of Universiti Putra Malaysia in fulfillment
of the requirements for the degree of Master of Science
IN VITRO PLANT REGENERATION AND RAPD ASSESSMENT OF SOMACLONAL
VARIATIONS IN SAFED MUSLI (Chlorophytum borivilianum Santapau &
R.R.Fernandes)
By
NURASHIKIN BINTI KEMAT
May 2012
Chairman : Associate Professor Mihdzar Abdul Kadir, PhD
Faculty : Agriculture
Safed musli (Chlorophytum borivilianum), is a traditional medicinal plant which belongs
to liliaceae family and has an increasing demand due to its wide range of uses. It has an
aphrodisiac property and form an important ingredient of herbal tonics prescribed in the
Ayurvedic systems of medicine. Due to its medicinal properties and high demand in
herbal industry, Safed musli has been introduced in Malaysia by Felda in 2008 under
Felda Herbal Corporation Sdn. Bhd. The major constraint in the cultivation of Safed
musli is long tuber dormancy at about 6 -7 months, so it’s only one crop per year can be
grown. Besides, the other constraints include shortage of quality planting material, lower
tuber multiplication rate and impossible to obtain true to type and disease free plants
either from seed tuber or seed. A study was carried out using in vitro propagation and
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molecular characterization of Safed musli. In vitro culture has shown the advantage
over the conventional methods of vegetative production as it ensures rapid rate of
multiplication and disease free abundant plantlets. Young shoot buds of Safed musli
were collected and sterilized. 100% of clean culture and plant survived in sterilization
treatment with 70% alcohol (6 minutes), 50% Sodium hypochlorite (20 minutes), 0.1%
aqueous mercuric chloride (15 minutes). Mass production of shoots was achieved on
MS medium supplemented with 3 mg/L BAP which produced the highest mean number
of shoots (18.9) and shoot length (6.0) cm. MS medium supplemented with 1 mg/L IBA
which produced the highest mean number of roots (52.4) and root length (6.6) cm. In
acclimatization, plantlets on media containing vermiculite : organic matter (1:1) showed
the highest percentage of survival plants (83%) and mean number of shoot length
(18.69) cm. Callus of Safed musli were induced from young shoot bud on MS medium
containing 5 mg/L 2,4-D with the highest percentage of callus formation (66.66%) and
mean weight of callus (10.65) g. For shoot regeneration, the highest percentage
(66.67%) and the highest mean number of shoots per vial (9.6) was obtained on MS
medium with 0.5 mg/L BAP . The RAPD assessment of somaclonal variations among
Safed musli plantlet was performed. Out of 90 bands, 55 were polymorphic bands and
35 were monomorphic bands ranging between 115.43bp – 3388.24bp. RAPD marker
effectively recognized the genetic difference among the in vitro shoots and the mother
plant. From this study, it can improve the production efficiency and cost effectiveness in
Safed musli.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
REGENERASI TANAMAN IN VITRO DAN PENILAIAN RAPD UNTUK VARIASI
SOMAKLONAL PADA SAFED MUSLI (Chlorophytum borivilianum Santapau &
R.R.Fernandes)
Oleh
NURASHIKIN BINTI KEMAT
Mei 2012
Pengerusi : Profesor Madya Mihdzar Abdul Kadir, PhD
Faculti : Pertanian
Safed musli (Chlorophytum borivilianum), merupakan tumbuhan ubatan tradisional yang
tergolong dalam keluarga liliaceae dan kini mempunyai permintaan yang semakin tinggi
disebabkan oleh pelbagai kegunaannya. Ia mempunyai ciri-ciri aphrodisiac dan
merupakan ramuan tonik herba yang penting di dalam sistem perubatan Ayurveda.
Oleh kerana ciri-ciri perubatan dan permintaan yang tinggi dalam industri herba,
tanaman Safed musli telah diperkenalkan di Malaysia oleh Felda pada tahun 2008 di
bawah Felda Herbal Corporation Sdn. Bhd. Masalah utama dalam penanaman Safed
musli ialah tempoh dormansi pada umbisi yang panjang iaitu 6-7 bulan jadi hanya satu
pusingan tanaman sahaja dalam tempoh setahun. Antara masalah yang lain ialah
kekurangan bahan tanaman yang berkualiti, kadar penggandaan umbisi yang lebih
rendah dan masalah untuk mendapatkan kesaaman genetik sama seperti tanaman
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induk dan bebas dari penyakit adalah sukar. Kajian telah dijalankan menggunakan
pembiakan secara in vitro dan pencirian molekul tanaman Safed musli. Kultur in vitro
telah menunjukkan kelebihan dalam pertumbuhan anak pokok, dengan penggandaaan
yang tinggi dan menghasilkan tanaman bebas penyakit berbanding dengan kaedah
penanaman konvensional. Pucuk muda Safed musli telah diambil dan steril. 100%
kultur bersih dan pucuk tumbuh dalam rawatan pensterilan dengan menggunakan
rawatan alkohol 70% (6 minit), 50% Natrium hipoklorit (20 minit), 0.1% larutan merkuri
klorida (15 minit). Pengeluaran pucuk secara besar-besaran diperolehi pada medium
MS yang ditambah dengan 3 mg/L BAP yang menghasilkan purata bilangan pucuk
(18.9) dan panjang pucuk tertinggi (6.0) cm. MS Medium yang ditambah dengan 1
mg/L IBA menghasilkan purata bilangan akar (52.4) dan panjang akar (6.6) cm.
Penyesuaian, anak pokok pada media yang mengandungi vermiculite: bahan organik
(1:1) menunjukkan peratusan (83%) dan purata bilangan pucuk tertinggi (18.69) cm.
Kalus Safed musli terhasil pada medium Murashige dan Skoog (MS) yang ditambah
dengan 5 mg/L 2,4-D dan menghasilkan peratusan pembentukan kalus (66.66%) dan
purata berat kalus tertinggi (10.65) g. Regenerasi pucuk daripada kalus diperolehi pada
medium MS yang ditambah dengan 0.5 mg/L dengan peratusan penghasilan pucuk
(66.67%), purata bilangan pucuk tertinggi (9.6) per vial. Penilaian RAPD untuk variasi
somaklonal di kalangan anak pokok Safed musli telah dilakukan. Daripada 90 jalur yang
diperolehi, 55 adalah jalur polimorfik dan 35 jalur monomorfik di antara 115.43 bp –
3388.24 bp. RAPD mengesan perbezaan genetik di antara pucuk in vitro dan induk
tanaman (mother plant). Daripada kajian yang dilakukan ini, kecekapan pengeluaran
dan keberkesanan kos bagi tanaman Safed musli dapat ditingkatkan
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ACKNOWLEDGEMENT
First and foremost, praise to Almighty Allah (SWT) the most Benevolent, Merciful and
Compassionate, for giving me the utmost strength, patience and guidance to have this
work completed.
I would like to express my most sincere gratitude and deepest appreciation to Assosiate
Professor Dr. Mihdzar Abdul Kadir, chairman of my supervisory committee, for his
dedicated efforts, supports invaluable advice and intellectual guidance during the
accomplishment of this research work. I greatly appreciate all the help he availed to me
while pursuing my studies. I would like to thank my supervisory committee members,
Dr. Nur Ashikin Psyquay Abdullah for the help, assistance and constructive comments
throughout the period of this study.
I am very grateful to Felda Herbal Corporation Sdn. Bhd. for the source of planting
materials and Yayasan Felda, Malaysia for the financial support through research grant.
Besides, thanks to Department of Agriculture Technology, Faculty of Agriculture,
Universiti Putra, Malaysia for the facilities and many thanks to UPM staffs from
Agrotechnology Laboratory and Department of Agriculture Technology, UPM for their
help and kindness during my study. Appreciations also go to all my lab mates for their
warm friendship, advice and help during the period of study especially to Innaka Ageng
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Rineksane, Mahmood Danae, Shida Othman and Farshad Ashraff for their advice,
kindness and help with my works.
Special thanks to my beloved family, my mother and father, for their love and
encouragement over the years to help me reach this point. I am especially grateful to
my husband, Sharul Nizam Ahmad for his understanding, encouragement and
wholehearted support during the period of study. Not to forget to my love son, Raes
Zafran who made my life complete and more enjoyable.
Finally, I pray that I shall be a good steward of this honor.
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I certify that a Thesis Examination Committee has met on 21st May 2012 to conduct the final examination of Nurashikin Binti Kemat on her thesis entitled “In vitro Plant
Regeneration and RAPD Assessment of Somaclonal Variations in Safed musli
(Chlorophytum borivilianum)” in accordance wih the Universities and University College Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Master Science.
Member of the Thesis Examination Committee were as follows:
Mahmud b. Tengku Muda Mohamed, PhD Professor Faculty of Agriculture University Putra Malaysia (Chairman) Datin Siti Nor Akmar binti Abdullah, PhD Associate Professor Faculty of Agriculture University Putra Malaysia (Internal Examiner) Faridah binti Qamaruz Zaman, PhD Associate Professor Faculty of Science University Putra Malaysia (Internal Examiner) Ahmad Tarmizi b. Hashim, PhD Abbc Biology Division Malaysian Palm Oil Board Malaysia (External Examiner)
SEOW HENG FONG, PhD
Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia
Date:
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Master Science. The members of the Supervisory Committee were as follows: Mihdzar Adbul Kadir, PhD Assosiate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Nur Ashikin Psyquay Abdullah, PhD Lecturer Faculty of Agriculture Universiti Putra Malaysia (Member) BUJANG BIN KIM HUAT, PhD
Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date:
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DECLARATION I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or any other institution. NURASHIKIN BINTI KEMAT Date: 21 May 2012
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TABLE OF CONTENTS
Page
DEDICATION ii ABSTRACT iii ABSTRAK v ACKNOWLEDGEMENTS vii APPROVAL ix DECLARATION xi LIST OF TABLES xvii LIST OF FIGURES xix LIST OF ABBREVIATIONS xxi CHAPTER
1 INTRODUCTION
1.1 Background 1
1.2 Objectives 4
1.3 Significance of the study 4
2 LITERATURE REVIEW
2.1 Chlorophytum 5
2.2 Botany of Safed musli (Chlorophytum borivilianum) 6
2.3 Uses and commercial importance of Safed musli 8
2.4 In vitro propagation system 9
2.4.1 Organogenesis 11
2.4.2 Callus 12
2.5 Controlling factors in micropropagation 13
2.5.1 Plant growth regulators (PGRs) 14
2.5.1.1 Auxins 16
2.5.1.2 Cytokinins 17
2.5.1.3 Amino acids as supplement 19
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2.5.2 Explant types 20
2.5.3 Medium used 21
2.6 Sterilization technique in plant micropropagation 21
2.6.1 Microbial contamination in tissue culture 22
2.6.2 Explants surface sterilization 23
2.6.2.1 Hypoclorite solution 24
2.6.2.2 Alcohol 24
2.6.2.3 Mercuric chloride 25
2.7 Somaclonal variation 26
2.8 Type of DNA markers 27
2.8.1 Molecular markers 29
2.8.2 Principles of RAPD 32
2.8.3 Application of RAPD 35
2.8.4 Advantage of RAPD 36
2.8.5 Limitations of RAPD 36
2.8.6 RAPD in Chlophytum spp. 38
3 IN VITRO PLANTLET REGENERATION OF SAFED MUSLI (Chlorophytum borivilianum) THROUGH SHOOT AND ROOT INDUCTION
3.1 Introduction 39
3.2 Material and methods
3.1.1 Location of study 42
3.1.2 Pre culture preparation 42
3.1.3 Nutrient stock solution 43
3.1.4 Media preparation 43
3.1.5 Explants material 44
3.1.6 Culture conditions 44
3.2 Surface sterilization
3.2.1 Treatments 45
3.2.2 Parameters measurement 46
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3.2.3 Experimental design and statistical analysis 46
3.3 Shoot induction
3.4.1 Explant material 47
3.4.2 Stock solution of PGRs 47
3.4.3 Treatments 48
3.4.4 Parameters measurement 49
3.4.5 Experimental design and statistical analysis 49
3.5 Rooting
3.5.1 Explant material 50
3.5.2 Stock solution of PGRs 50
3.5.3 Treatments 50
3.5.4 Parameters measurement 51
3.5.5 Experimental design and statistical analysis 52
3.6 Acclimitization
3.6.1 Plantlets material 52
3.6.2 Acclimitization media 53
3.6.3 Treatments 53
3.6.4 Parameters measurement 54
3.6.5 Experimental design and statistical analysis 54
3.7 Results and discussion
3.7.1 Surface sterilization 55
3.7.2 Shoot induction 59
3.7.3 Rooting 64
3.7.4 Acclimitization 66
3.8 Conclusion 69
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4 IN VITRO PLANTLET REGENERATION OF SAFED MUSLI (Chlorophytum
borivilianum) THROUGH CALLUS
4.1 Introduction 71
4.2 Material and methods
4.2.1 Location of study 73
4.2.2 Pre culture preparation 73
4.2.3 Nutrient stock solution 73
4.2.4 Stock solution of PGRs 74
4.2.5 Media preparation 74
4.2.6 Culture conditions 74
4.2.7 Experimental design and statistical analysis 75
4.3 Callus induction
4.3.1 Explant material 75
4.3.2 Treatments 76
4.3.3 Parameters measurement 77
4.4 Subculture
4.4.1 Callus subculture 77
4.4.2 Parameters measurement 78
4.5 Shoot regeneration
4.5.1 Treatments 78
4.5.2 Parameter measurement 79
4.6 Result and discussion
4.6.1 Callus induction 80
4.6.2 Callus subculture 84
4.6.3 Shoot regeneration from callus 87
4.7 Conclusion 91
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5 DETECTION OF SOMACLONAL VARIATIONS IN SAFED MUSLI BY USING
RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD)
5.1 Introduction 92
5.2 Material and methods
5.2.1 Location of study 94
5.2.2 Plant materials, sample preparation and primers tested 94
5.2.3 DNA extraction 97
5.2.4 DNA quantification 98
5.2.5 RAPD – PCR 98
5.2.6 DNA and PCR products electrophoresis 99
5.2.7 Gel scoring 100
5.2.8 Data analysis 101
5.3 Results and discussion
5.3.1 DNA quantification 101
5.3.2 Primers screening 102
5.3.3 RAPD analysis 104
5.4 Conclusion 107
6 CONCLUSION AND FUTURE DIRECTION 111
REFERENCES 114
APPENDICES 134
BIODATA OF STUDENT 139
LIST OF PUBLICATION 140
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