chew tiong dar - universiti putra malaysiapsasir.upm.edu.my/48685/1/fbsb 2012 57r.pdf · copyright...

UNIVERSITI PUTRA MALAYSIA

CHEW TIONG DAR

FBSB 2012 57

DIRECT SHOOT REGENERATION OF Hibiscus rosa-sinensis L. CV. ‘BRILLIANT RED’

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DIRECT SHOOT REGENERATION OF

Hibiscus rosa-sinensis L. CV. ‘BRILLIANT RED’

CHEW TIONG DAR

MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2012

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By

CHEW TIONG DAR

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Master of Science

September 2012

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Abstract of this thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Master of Science



By

CHEW TIONG DAR

September 2012

Chair: Janna Ong Abdullah, PhD

Faculty: Biotechnology and Biomolecular Sciences

Hibiscus rosa-sinensis cv. ‘Brilliant Red’, a five-petal, single-layered, and bright red

perennial herb, is the national flower of Malaysia. For more than 50 years the local

people recognise the status and sovereignty of this plant from the perspective of

nationality, and even self-esteem. After decades of advancement in plant tissue culture

field, propagation of Hibiscus spp. via in vitro method is starting to pick up its pace. So

far, no report has been published to generate H. rosa-sinensis with fragrant flowers or

flowers with longer life span. Under this context, plant tissue culture is a good means to

produce large quantity of H. rosa-sinensis rapidly to cater for the market needs and pave

ways for genetic manipulation of the plant for desired traits. A medium optimised for

‘Brilliant Red’ is yet to be formulated. Thus, this study reports on the effects

of modifying the Murashige & Skoog (MS) medium in facilitating direct shoot

regeneration from nodal explants of H. rosa-sinensis L. cv. ‘Brilliant Red’. The

sterilisation conditions for 6 types of explants (basal leaf, leaf blade, leaf midrib, node,

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internode, and shoot tip) harvested from an open field were initially compared. The

optimised sterilisation conditions for the explants were found to be 30% Clorox-15min

exposure, 15% Clorox-30min exposure, 35% Clorox-15min exposure, 40% Clorox-

20min exposure, 10% Clorox-15min exposure, and 5% Clorox-40min exposure for the

basal leaf, leaf blade, leaf midrib, node, internode and shoot tip, respectively. Using the

optimised sterilisation conditions as mentioned, the survival-contamination percentages

for each explant type were as follows: basal leaf: 86%, 0%; leaf blade: 99%, 0%; leaf

midrib: 91%, 2%; node: 94.5%, 2%; internode: 97%, 0%; and shoot tip: 60%, 0%. In the

direct shoot regeneration study using the nodal explants, MS medium containing 40 g/L

sucrose, 0.3% (w/v) activated charcoal, and supplementations with myo-inositol,

thiamine and nicotinic acid (Concentration: MS vitamin standard) were found to be

suitable. The in vitro shoot survival rate was 30% with a mean leaf numbers of 2.7±0.45

produced, and a mean leaf length of 1.71±0.46 cm achieved after 5 weeks of culture on

the modified medium. Callus induction from nodal explant could be performed by using

20 μM IBA, with callusing rate at 91.3±1.8% after 5 weeks of culture. Further

histological study on the calli revealed that no embryogenic cell was found. Overall,

shoot could be induced directly from H. rosa-sinensis cv. ‘Brilliant Red’ nodal explant

based on the above formulation. More effort should be put on the indirect shoot

regeneration. However, shoots maintenance required further refinement of the

formulated MS medium. This study also reveals that callus induction could be an

alternative way to produce H. rosa sinensis L. cv. ‘Brilliant Red’ in vitro plants.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia

sebagai memenuhi keperluan untuk ijazah Sarjana Sains



Oleh

CHEW TIONG DAR

September 2012

Pengerusi: Janna Ong Abdullah, PhD

Fakulti: Bioteknologi dan Sains Biomolekul

Hibiscus rosa-sinensis L. cv. ‘Brilliant Red’ (bunga raya) adalah sejenis tumbuhan

herba tahunan di mana bunganya mempunyai lima kelopak berwarna merah. Ia

merupakan bunga kebangsaan Malaysia. Sejak 50 tahun dahulu, penduduk tempatan

menganggap tumbuhan ini sebagai lambang kedaulatan dan kekuatan jati diri. Kemajuan

dalam bidang tisu kultur tumbuhan melalui keadah mikro-pembiakan Hibiscus spp telah

mula berkembang sejak beberapa dekad dahulu. Setakat ini, masih tiada laporan tentang

penghasilan bunga raya wangian dan pokok bunga raya yang berbunga dengan jangka

hayat panjang. Dalam konteks ini, kaedah tisu kultur tumbuhan merupakan satu kaedah

yang baik untuk menghasilkan bunga raya secara besar-besaran supaya dapat memenuhi

keperluan pasaran dan juga membolehkan manipulasi genetiknya untuk mendapatkan

sifat-sifat yang diingini. Memandangkan penyediaan media untuk ’Brilliant red’ yang

optimum masih belum diformulasikan, oleh itu, kajian ini melaporkan kesan

pengubahsuaian media Murashige & Skoog (MS) dalam membantu percambahan

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semula pucuk secara langsung daripada tunas sisi H. rosa-sinensis cv. ‘Brilliant Red’.

Perbandingan dibuat bagi 6 jenis steril eksplan (daun basal, bilah daun, midrib daun,

tunas sisi, internod, hujung pucuk) yang dipetik dari tempat terbuka. Didapati bahawa

keadaan pensterilan yang optimum untuk eksplan ialah pendedahan 30% Clorox-15min,

15% Clorox-30min, 35% Clorox-15min, 40% Clorox-20min, 10% Clorox-15min, dan

5% Clorox-40min untuk daun basal, bilah daun, midrib daun, tunas sisi, internod, dan

hujung pucuk. Melalui kaedah pensterilan optimum yang telah dinyatakan, peratusan

kelangsungan hidup-kontaminasi untuk setiap eksplan adalah seperti berikut: daun basal:

86%, 0%; daun bilah: 99%, 0%; daun midrib: 91%, 2%; tunas sisi: 94.5%, 2%; internod:

97%, 0%; dan hujung pucuk: 60%, 0%. Dalam percambahan semula pucuk secara

langsung menggunakan tunas sisi, MS media yang mengandungi 40 g/L sukrosa, 0.3%

(w/v) arang aktif, dan penambahan dengan myo-inositol, thiamin, dan asid nikotinik

adalah sesuai. Peratus hidup bagi pucuk adalah 30% dengan purata bilangan daun

sebanyak 2.68±0.45, dan purata panjang daun ialah 1.71±0.46 cm selepas 5 minggu

dikultur dalam medium yang telah diubah suai. Induksi kalus boleh dilakukan dengan

menggunakan 20 μM IBA, dengan pembentukan kalus sebanyak 91.3±1.8% selepas

dikultur selama 5 minggu. Kajian histologi yang lebih mendalam terhadap kalus

menunjukkan bahawa tiada embriogenik sel dijumpai. Kesimpulannya, pertumbuhan

pucuk boleh dirangsang secara langsung dari tunas sisi H. rosa-sinensis L. cv. ‘Brilliant

Red’ berdasarkan formulasi di atas. Walau bagaimanapun, pengekalan pucuk

memerlukan optimisasi lagi terhadap formulasi ini. Kajian ini juga menunjukkan

bahawa cara induksi kalus berkemampuan untuk menghasilkan H. rosa-sinensis L. cv.

‘Brilliant Red’.

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ACKNOWLEDGEMENT

“A long journey will has its end eventually no matter how arduous it was; a magnificent

feast will has its dispersal finally no matter how glorious it was”. This sentence is

correct for many cases, yet it is just partially true for my project, because the completion

of my project is not the end but just one of the milestones along the journey towards

new world.

Millions thanks to my supervisor Dr. Janna Ong Abdullah for her invaluable lesson,

enthusiastic encouragement, and incessant guidance in helping me to complete this

project. Her unceasing endeavor in doing every thing always impresses me greatly. Also,

I would like to express my ineffable gratitude to Dr. Parameswari Namasivayam and Dr.

Siti Habsah. Their assistance was on time during the most critical period of my work.

On the other hands, I would like to convey my gratitude to the lab and department

assistants who allowed me to use the research facilities and devices, which had greatly

facilitated this project: Encik Hussein Jirangon, Puan Sharipah Samah, Puan Rosna binti

Angsor, and Cik Siti Nordiana Abu Bakar.

Besides, I would also like to thank my fellow friends for the assistances they gave

especially Miss Kok Sau Yee, Mr. Oii Chai Theam and Puan Siti Nurhadis who

repeatedly taught and advised me throughout my work.

Wish all of you be blessed always with good health. Thanks!

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APPROVAL

I certify that a Thesis Examination Committee has met on (28th September 2012) to

conduct the final examination of (Chew Tiong Dar) on his thesis entitled “Direct Shoot

Regeneration of Hibiscus rosa-sinensis L. cv. ‘Brilliant Red’” in accordance with the

Universities and University Colleges 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 degree of Master of Science.

Members of the Thesis Examination Committee were as follows:

Wan Suhainis Saad, PhD

Faculty Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Umi Kalsom Md. Shah, PhD

Associate Professor



(Internal Examiner)

Noor Azmi Shaharuddin, PhD



(Internal Examiner)

Tee Chong Siang, PhD

Department of Biological Science, Faculty of Science

Universiti Tunku Abdul Rahman

Malaysia.

(External Examiner)

___________________________________

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies


Date:

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APPROVAL

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of requirement for the degree of Master of Science. The members

of the Supervisory Committee were as follows:

Janna Ong Abdullah, PhD

Associate Professor



(Chairman)

Parameswari Namasivayam, PhD

Associate Professor



(Member)

Siti Habsah Roowi, PhD

PT Lengkuk Technology

Felda Biotechnology Centre

Nilai

(Member)

___________________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


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 previously, and is not

concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other

institution.

___________________

CHEW TIONG DAR

Date: 28 September 2012

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK iv

ACKNOWLEDGEMENTS vii

APPROVAL viii

DECLARATION ix

LIST OF TABLES xii

LIST OF FIGURES xiii

LIST OF ABBREVIATIONS xiv

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW

2.1 Hibiscus rosa-sinensis L. cv. ‘Brilliant Red’

2.1.1 Taxonomy 3

2.1.2 Background 4

2.1.3 Physical Appearances 5

2.1.4 Uses of Hibiscus 6

2.2 Plant Tissue Culture 7

2.2.1 Tissue Culture Media 9

2.2.2 Gelling Agents 13

2.2.3 Activated Charcoal 14

2.2.4 pH of Media 15

2.2.5 Explant Source 17

2.2.6 Contamination and Sterilisation 18

2.2.7 Rooting 20

2.3 Plant Growth Regulator (PGR) 21

2.3.1 Auxin 22

2.3.2 Cytokinin 23

3 MATERIALS AND METHODS/METHODOLOGY 25

3.1 Culture Media 25

3.1.1 Media used for surface sterilisation study

25

3.1.2 Media used for culturing explants in the MS medium

modification study 26

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3.1.3 Murashige & Skoog Medium Supplemented with

PGRs

26

3.2 Surface Sterilisation of Explants 27

3.3 Direct Shoot Regeneration 29

3.4 Calculations for Mean Percentage of Survival,

Contamination, and Regeneration 30

3.5 Histological Study Using Induced Callus 31

3.6 Statistical Analysis 32

4 RESULTS AND DISCUSSION

4.1 Surface Sterilisation 33

4.2 Direct Shoot Regeneration 46

4.3 Possible Ways to Overcome Chlorosis, Shoot Tip 81

Necrosis , and Leaf Abscission

4.4 Histological Study Using Induced Callus 84

5 SUMMARY, CONCLUSION AND

RECOMMENDATIONS FOR FUTURE RESEARCH 87

REFERENCES 89

APPENDICES

Appendix A: Composition of Plant Tissue Culture Media Used in 102

in This Research

Appendix B: List of Various Hibiscus spp. with Optimal PGR(s) in 103

Shoot Induction Appendix C: Appearances of Surviving Leaf Explants 105 Appendix D: Statistical Analysis 108

BIODATA OF STUDENT 113

LIST OF PUBLICATION 114

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