universiti putra malaysia in vitro plant ...pembiakan secara in vitro dan pencirian molekul tanaman...

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)


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


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


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



3.5 Rooting



3.5.3 Treatments 50



3.6 Acclimitization

3.6.1 Plantlets material 52

3.6.2 Acclimitization media 53

3.6.3 Treatments 53



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



4.2.2 Pre culture preparation 73

4.2.3 Nutrient stock solution 73


4.2.5 Media preparation 74

4.2.6 Culture conditions 74


4.3 Callus induction


4.3.2 Treatments 76


4.4 Subculture

4.4.1 Callus subculture 77


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



5.2.2 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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