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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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DIRECT SHOOT REGENERATION OF
Hibiscus rosa-sinensis L. CV. ‘BRILLIANT RED’
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
DIRECT SHOOT REGENERATION OF
Hibiscus rosa-sinensis L. CV. ‘BRILLIANT RED’
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
DIRECT SHOOT REGENERATION OF
Hibiscus rosa-sinensis L. CV. ‘BRILLIANT RED’
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
Faculty Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Noor Azmi Shaharuddin, PhD
Faculty Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(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
Universiti Putra Malaysia
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
Faculty Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Parameswari Namasivayam, PhD
Associate Professor
Faculty Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(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
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 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