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AN EFFICIENT METHOD FOR IN VITRO CLONAL
PROPAGATION OF Stevia rebaudiana Bertoni
ACCESSION MS007
BY
ZANNAT URBI
A thesis submitted in fulfilment of the requirement for the
degree of Master of Science (Biotechnology)
Kulliyyah of Science
International Islamic University Malaysia
APRIL 2016
ii
ABSTRACT
Stevia rebaudiana, commonly known as Stevia, is an economically important
sweetening medicinal herb species from the family Asteraceae. Although this plant is
native to Paraguay, many countries including Malaysia have shown interest to
cultivate this plant in large commercial-scale. The accession MS007 produced more
axillary branching, high leaf number with big sizes which implies good quality
sweetening compounds. To overcome conventional propagation method, in vitro
clonal propagation is a potential alternative to ensure regular supply of planting
material. Therefore, this study aims to explore the possibility of mass producing S.
rebaudiana accession MS007 using in vitro clonal propagation technique with two
major purposes i.e., to establish an effective surface sterilization protocol for shoot
tips and nodal explants of Stevia and to develop multiple shoot induction and plant
regeneration. Shoot tips and nodal explants collected from the field were sterilized
with 5% NaOCl for 10 minutes with 0.2% carbendazim (fungicide) pre-treatment
demonstrated least contamination 16.67% (shoot tips) and 18.89% (nodes). After
sterilization, explants were inoculated on MS medium with different concentrations of
cytokinins, BAP (0.5-3 mg/L) and Kn (0.5-3 mg/L) either in singly or in combination,
and combination with auxin NAA (0.5 mg/L) for shoot induction. The highest
frequency (85.19% for shoot tips and 86.67% for nodes) of multiple shoot
regeneration with maximum number of shoots (14.30 shoots/explant for shoot tips and
12.77 shoots/explant for nodes) was noticed on MS medium supplemented with 1.0
mg/L BAP. The proliferated healthy shoots (>2 cm) were dissected out from in vitro
shoot cluster and cultured on MS medium for root induction either on half strength or
full strength MS medium fortified with different concentrations of auxins IBA, NAA
and IAA (0.5, 1.0, and 1.5 mg/L). Highest frequency of rooting (92.22% and 91.11%)
with highest number of roots (14.92 and 13.82 roots/explant) and length (4.29 and
3.09 cm) from shoot tips and nodes, respectively was noticed on half-strength MS
medium augmented with 0.5 mg/L IBA. Finally, the rooted plantlets were successfully
transferred into plastic cups containing peat moss and subsequently established in the
field and it was noticed that 86.67% of plantlets survived which is phenotypically
similar to the parental mother plant. This present research offers a reliable method for
clonal propagation of Stevia (MS007) for large scale production.
iii
خلاصة البحثABSTRACT IN ARABIC
ة (، المعروفة باسم ستيفيا، هي من النباتات الطبية التي لها قيمStevia rebaudianaإن ستيفيا ريبواديانا )(. فعلى الرغم من أن منشأ هذه النباتات هي الباراغواي، Asteraceaeإقتصادية هامة. وهي من عائلة استراسيا )
فقد أظهر العديد من البلدان، بما في ذلك ماليزيا، اهتماماً كبيراً لزراعة هذا النبات على نطاق تجاري واسع. تنتج أوراق بأحجام كبيرة، مما يعني أنها تحوي مركبات مُحليَة تفرعات إبطية وعدد من MS007هذا النبات المنمية
in vitro clonalعالية الجودة. للاستغناء عن طرق التكاثر التقليدية، يعتبر التكاثر النسيلي في المختبر )
propagation بديلًا جيداً لضمان توفير إمدادات منتظمة من المزروعات. لذلك، تنوي هذه الدراسة إلى )( باستخدام تقنية التكاثر MS007من المنمية C. rebaudianaإمكانية الانتاج هذا النبات )نبتة استكشاف
النسيلي في المختبر، و ذلك بهدفين رئيسين، هما: أولًا إنشاء بروتوكول فعال لتعقيم سطوح فسائل ومزدرعات المزدرعات التي تم جمعها من الحقل بواسطة الستيفيا؛ وثانياً تطوير الفسائل وتجديد النباتات. تم تعقيم الفسائل و
% كاربيندازيم )مضاد فطريات(. 0.2دقائق مع المعالجة المسبقة بــ 10% هيبوكلوريت الصوديوم لمدة 5% )العقد(. بعد التعقيم، تم تلقيح الفسائل الزراعية 18.89% )الفسائل( و 16.67التعقيم أظهر تلوثاً أقل بنسبة
ملغ / 3 - 0.5و ) BAPملغ / لتر( 3 - 0.5لى تراكيز مختلفة من السيتوكينين، و )الغذائي ع MSفي وسط مغ / لتر( لحث نمو الفسائل. أعلى تردد 0.5) NAA، إما منفردة أو مجمعة، والجمع مع أوكسين Kn لتر(
مزدرع 14.30٪ للعقد( للتجدد المتعدد للفسائل مع أكبر عدد ممكن من الفسائل )86،67٪ للفسائل و85.19) 1.0الغذائي المدعم بــ MSوسيط فسيلة للعقد( لوحظ على / مزدرع 12.77فسيلة لأطراف الفسائل و /
سم( من شبكة الفسائل المنماة في المختبر ومن ثم 2. تم تقطيع الفسائل المكثرة الجيدة )<BAPملغ / لتر من وسطة أو قوة عالية من الوسيط المدعم بتراكيز غذائي لحث نمو الجذور، إما على قوة مت MSتنبيتها على وسيط
ذي القوة MSمغ / لتر(. في وسيط 1.5، و 1.0، و 0.5) IAAو IBA ،NAAمختلفة من أوكسينات %( 91.11و %92.22تمت ملاحظة أعلى معدلات التجذر ) IBAمغ / لتر من 0.5المتوسطة المدعم بــ
سم( من أطراف الفسائل 3.09و 4.29فسيلة( ، وطول ) جذر / 13.82و 14.92، وأعلى عدد للجذور )والعقد ، كلا على حده. و أخيراً، تم نقل الشتلات المتجذرة بنجاح في أكواب بلاستيكية احتوت على الجفت
٪ من الشتلات واصلت النمو مما شابهت ظاهرياً النبتة الأم. 86.67موس ووضعت بعد ذلك في الحقل ولوحظ أن ( للإنتاج بكميات كبيرة.MS007ث طريقة يمكن الاعتماد عليها للتكثير النسيلي للستيفيا )يقدم هذا البح
iv
APPROVAL PAGE
I certify that I have supervised and read this study and that in my opinion, it conforms
to acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a thesis for the degree of Master of Science (Biotechnology).
…………………………………..
Zarina Zainuddin
Supervisor
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
thesis for the degree of Master of Science (Biotechnology).
…………………………………..
Zaima Azira Zainal Abidin
Internal Examiner
…………………………………..
S. Sreeramanan
External Examiner
This thesis was submitted to the Department of Biotechnology and is accepted as a
fulfilment of the requirement for the degree of Master of Science (Biotechnology).
…………………………………..
Suhaila Bt. Mohd Omar
Head, Department of
Biotechnology
This thesis was submitted to the Kulliyyah of Science and is accepted as a fulfilment
of the requirement for the degree of Master of Science (Biotechnology).
…………………………………..
Kamaruzzaman Bin Yunus
Dean, Kulliyyah of Science
v
DECLARATION
I hereby declare that this thesis is the result of my own investigations, except where
otherwise stated. I also declare that it has not been previously or concurrently
submitted as a whole for any other degrees at IIUM or other institutions.
Zannat Urbi
Signature ........................................................... Date .........................................
vi
INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA
DECLARATION OF COPYRIGHT AND AFFIRMATION OF
FAIR USE OF UNPUBLISHED RESEARCH
AN EFFICIENT METHOD FOR IN VITRO CLONAL
PROPAGATION OF Stevia rebaudiana Bertoni ACCESSION MS007
I declare that the copyright holders of this thesis are jointly owned by the student and
IIUM.
Copyright © 2016 Zannat Urbi and International Islamic University Malaysia. All rights reserved.
No part of this unpublished research may be reproduced, stored in a retrieval system,
or transmitted, in any form or by any means, electronic, mechanical, photocopying,
recording or otherwise without prior written permission of the copyright holder
except as provided below
1. Any material contained in or derived from this unpublished research may
be used by others in their writing with due acknowledgement.
2. IIUM or its library will have the right to make and transmit copies (print
or electronic) for institutional and academic purposes.
3. The IIUM library will have the right to make, store in a retrieved system
and supply copies of this unpublished research if requested by other
universities and research libraries.
By signing this form, I acknowledged that I have read and understand the IIUM
Intellectual Property Right and Commercialization policy.
Affirmed by Zannat Urbi
……..…………………….. ………………………..
Signature Date
vii
ACKNOWLEDGEMENTS
First and above all, I praise almighty ALLAH (Subhanu Wa Ta’ala), the lord of
creation, the most compassionate, the most merciful, the most greatest, the ruler of the
day of judgment, the creator and cherisher of the world, for providing me this
opportunity and granting me the capability to proceed successfully. This thesis
appears in its current form due to the assistance and guidance of several people. I
would therefore like to offer my sincere thanks to all of them.
It is honour for me to express my deep indebtedness and my sincere
appreciation and deepest gratitude to my supervisor, Assistant Professor Dr. Zarina
Zainuddin for her guidance and supervision to complete this thesis. I have learned lot
of valuable things from her which motivated me in research area especially she helped
me to think how to question thoughts and express ideas. Her patience and support
helped me in all the time of research and writing of this thesis. I will be really grateful
forever for her support, cooperation, encouragement and invaluable comments. I can
just say thanks for everything and may Allah give her all the best in return.
I want to give gratitude to Assistant Professor Dr. Ing Chia Phang and
Assistant Professor Dr. Zaima Azira Zainal Abidin. Both of them gave me valuable
suggestions in my proposal defence and pre-viva presentation, which help much to
improve my thesis. I would like to thank Professor Dr. Ahmed Jalal Khan Chowdhury
for his inspiring suggestions during my study.
I am also grateful to all of my friends and all laboratory staffs who helped me
in different issue and also for their cordial behave in friendship.
I would like to express my deepest and special thanks to my beloved husband
Md. Sanower Hossain because of his mental support, encouragement, sharing
knowledge and idea during my study period which helped me very much for a
successful completion of my thesis. I owe my utmost gratitude to my family members,
parents, brother and sister, for their endless love, spiritual support all through my life.
Moreover, their unwavering faith in me has been a source of constant
inspiration for me. Finally, I just want to say that, may almighty ALLAH (Subhanu
Wa Ta’ala) blessed all who helped me and give them the best rewards.
viii
TABLE OF CONTENTS
Abstract .................................................................................................................... ii Abstract in Arabic .................................................................................................... iii Approval Page .......................................................................................................... iv
Declaration ............................................................................................................... v Copyright ................................................................................................................. vi Acknowledgements .................................................................................................. vii List of Tables ........................................................................................................... x List of Figures .......................................................................................................... xii
List of Abbreviations ............................................................................................... xiv List of Symbols ........................................................................................................ xv
CHAPTER ONE: INTRODUCTION .................................................................. 1 1.1 Background of the Study ........................................................................ 1 1.2 Problems Statement and Justification ..................................................... 3 1.3 Hypothesis of the Study .......................................................................... 5
1.4 Objectives of the Study ........................................................................... 5 1.4.1 General Objective ......................................................................... 5
1.4.2 Specific Objectives ....................................................................... 5
CHAPTER TWO: LITERATURE REVIEW ..................................................... 6 2.1 Plant Descriptions ................................................................................... 6 2.2 Propagation ............................................................................................. 7
2.3 Chemical Compounds ............................................................................. 9 2.4 Importance of Stevia ............................................................................... 11
2.5 Plant Tissue Culture ................................................................................ 12 2.5.1 In vitro Clonal Propagation ........................................................... 13
2.5.2 Factors Influencing Plant Tissue Culture ...................................... 15 2.5.2.1 Selection of Explants ........................................................ 15
2.5.2.2 Microbial Contamination .................................................. 16 2.5.2.3 Hyperhydricity (vitrification) and Shoot tip Necrosis ...... 18 2.5.2.4 Growth Medium ............................................................... 19
2.5.2.5 Plant Growth Regulators .................................................. 21 2.5.2.6 Environmental Factors ...................................................... 24
2.5.3 Micropropagation of Stevia rebaudiana ....................................... 25 2.5.3.1 Establishment of Aseptic Culture of Stevia rebaudiana ... 25 2.5.3.2 Nodal Explants Culture of Stevia rebaudiana ................... 27 2.5.3.3 Shoot tip Culture ............................................................... 30 2.5.3.4 Leaf Culture ...................................................................... 31
CHAPTER THREE: MATERIALS AND METHODS ..................................... 32 3.1 Mother Plant Preparation ........................................................................ 32 3.2 Stock Solution and Media Preparation ................................................... 33
3.2.1 MS Stock Solution Preparation ..................................................... 33 3.2.2 Plant Growth Regulator Stock Solution Preparation .................... 33 3.2.3 Culture Media Preparation ............................................................ 34
ix
3.3 Sterilization of Working Bench, Instruments, Culture Vessels,
Media and Plant Growth Regulator ........................................................ 35 3.4 Surface Sterilization of Explants and Culture Initiation ......................... 35
3.5 Shoot Induction and Multiplication ........................................................ 37 3.6 Rooting ................................................................................................... 39 3.7 Acclimatization ....................................................................................... 40 3.8 Culture Conditions .................................................................................. 40 3.9 Experimental Designs and Statistical Data Analysis .............................. 40
CHAPTER FOUR: RESULTS AND DISCUSSION .......................................... 42 4.1 Aseptic Establishment of Stevia explants ............................................... 43 4.2 Shoot Induction ....................................................................................... 49 4.3 Root Induction ........................................................................................ 82
4.4 Acclimatization ....................................................................................... 98
CHAPTER FIVE: CONCLUSIONS AND FUTURE DIRECTION ................. 101
REFERENCES ....................................................................................................... 103
APPENDIX ............................................................................................................. 122
x
LIST OF TABLES
Table No. Page No.
3.1 Different treatments for surface sterilization of Stevia shoot tips and
nodes. 37
3.2 Different concentrations of PGRs for shoot multiplication. 38
4.1 Effect of different type of disinfectants with different concentrations
and variation of exposure of time on inoculated shoot tips and node
explants of Stevia. All results have shown in percentage. 47
4.2 Effect of different concentrations of BAP on shoot bud induction
from shoot tips and nodal explants of Stevia 53
4.3 Effect of different concentrations of Kn on shoot bud induction from
shoot tips and nodal explants of Stevia. 58
4.4 Effect of 0.5 mg/L Kn in combination with different concentrations
of BAP on shoot bud induction from shoot tips and nodal explants of
Stevia 63
4.5 Effect of 0.5 mg/L BAP in combination with different concentrations
of Kn on shoot bud induction from shoot tips and nodal explants of
Stevia 66
4.6 Effect of 0.5 mg/L NAA in combination with different
concentrations of BAP on shoot bud induction from shoot tip and
nodal explants of Stevia 69
4.7 Effect of 0.5 mg/L NAA in combination with different
concentrations of Kn on shoot bud induction from shoot tips and
nodal explants of Stevia. 73
4.8 In vitro derived shoots from shoot tip and nodal explants were
cultured on MS medium enriched by 1.0 mg/L BAP and subcultured
onto the same medium for large scale propagation. 81
4.9 Effect of different concentrations of IBA in full strength MS media
on in vitro rooting of elongated shoots of Stevia. 86
4.10 Effect of different concentrations of IBA in half strength MS media
on in vitro rooting of elongated shoots of Stevia. 88
4.11 Effect of different concentrations of NAA in full strength MS media
on in vitro rooting of elongated shoots of Stevia. 90
xi
4.12 Effect of different concentrations of NAA in half strength MS media
on in vitro rooting of elongated shoots of Stevia. 92
4.13 Effect of different concentrations of IAA in full strength MS media
on in vitro rooting of elongated shoots of Stevia. 94
4.14 Effect of different concentrations of IAA in half strength MS media
on in vitro rooting of elongated shoots of Stevia. 96
xii
LIST OF FIGURES
Figure No. Page No.
2.1 Stevia rebaudiana plant Accession MS007. 7
2.2 Chemical structures of (a) stevioside and (b) rebaudioside A. 10
2.3 Stages of in vitro clonal propagation. 15
3.1 Stem cutting of Stevia rebaudiana (MS007). 33
3.2 Explants ready to inoculate in MS medium after surface sterilization. 37
4.1 A representative photo of effect of different surface sterilization
treatments on shoot tips of Stevia rebaudiana. 46
4.2 Culture initiation and shoot bud breaks of shoot tips and nodes of
Stevia on MS medium without plant growth hormone. 50
4.3 Representative photos of shoot multiplication from shoot tips of
Stevia. 54
4.4 Representative photos of shoot multiplication from nodes of Stevia. 55
4.5 Representative photos of shoot multiplication from shoot tips of
Stevia. 59
4.6 Representative photos of shoot multiplication from nodes of Stevia. 60
4.7 Representative photos of shoot induction from shoot tips and nodes. 64
4.8 Representative photos of shoot induction from shoot tips and nodes. 70
4.9 Representative photos of shoot induction from shoot tips. 74
4.10 Representative photos of subculture for shoot multiplication of
Stevia rebaudiana from shoot tip and node. 80
4.11 Representative photos of rooting in in vitro regenerated shoots of
Stevia. 83
4.12 Representative photos of rooting in in vitro regenerated shoots of
Stevia. 87
4.13 Representative photos of rooting in in vitro regenerated shoots of
Stevia. 91
xiii
4.14 Representative photos of rooting in in vitro regenerated shoots of
Stevia. 95
4.15 Representative photos of acclimatization of in vitro regenerated
plants of Stevia. 100
xiv
LIST OF ABBREVIATIONS
2, 4-D 2, 4-dichlorophenoxyacetic acid
2-iP N6-(2-isopentyl) adenine
BAP 6-benzyl aminopurin
DNA Deoxyribonucleic acid
EtOH Ethanol
HCl Hydrochloric Acid
HgCl2 Mercuric chloride
H2O Water
IAA Indole-3-acetic acid
IBA Indole-3-butyric acid
ISSR Intersimple sequence repeat
Kn Kinetin
MARDI Malaysian Agricultural Research and Development Institute
MS Murashige and Skoog’s medium
MS007 MARDI Stevia Accession 007
NAA 1-naphthaleneacetic acid
NaOCl Sodium Hypochlorite
NaOH Sodium Hydroxide
PGR Plant Growth Regulator
RAPD Random amplified polymorphic DNA
TDZ Thidiazuron
xv
LIST OF SYMBOLS
% Percent
> More than
≤ Equal or less than
≥ Equal or more than
°C Degree Celsius
µM Micromole
cm Centimetre
mg Milligram
mL Millilitre
mm Millimetre
N Normal
1
CHAPTER ONE
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
The association between obesity and type 2 diabetes are becoming increasingly
prevalent worldwide, and both are correlated at increased risk for a number of
deleterious health conditions such as heart disease and cancer (Anton et al., 2010;
Gallagher & LeRoith, 2015). The consumption of high sugar sweetened beverages
has been demonstrated to contribute to an increase risk factors of metabolic disorders
(Anton et al., 2010). As alternatives to cane sugar (sucrose), different types of
sweetener compounds including natural sweetener (honey, maple syrup, coconut palm
sugar and molasses) and artificial sweetener (aspartame, acesulfame K, saccharin,
sucralose and neotame) are available in the market (Inglett, 1976; Weihrauch & Diehl,
2004). Natural sweetening compound is considered comparatively safe than artificial
sweetener because natural sweeteners have been reported to be free from side effects
or health problems such as headache, skin eruptions, muscle dysfunction, depression,
weight gain, liver and kidney effects, multiple sclerosis and blurred vision caused by
artificial sweetener (Saad et al., 2014). Although different plants based natural
sweeteners are beneficial in certain ways, most of them contain calories (Kim &
Kinghorn, 2002). Since consumption of natural sweetener with calories still possesses
increase risk factors of metabolic disorders, zero-calorie natural sweetener, which
have no toxicity effect on human health would be a good alternative.
Stevia rebaudiana, commonly known as Stevia, is a commercially important
high non-caloric sweetening herb and also possesses many potential medicinal
2
properties such as antiviral (Takahashi et al., 2001), antihyperglycemic (Jeppesen et
al., 2002), anti-inflammatory (Boonkaewwan et al., 2006), gastroprotective (Shiozaki
et al., 2006), bactericidal (Brusick, 2008), antioxidant (Tadhani et al., 2007; Rao,
2013) activity. It is a perennial herbaceous medicinal plant belongs to the Asteraceae
family and this family is one of the large families containing about 950 genera and
more than 20,000 recognized species. Stevia rebaudiana is one of the 300 species of
the genus Stevia (Soejarto et al., 1983; Soejarto, 2002). Leaves of this plant produce
zero-calorie ent-kaurene diterpene glycosides (namely stevioside and rebaudiosides) a
non-nutritive, high-potency sweetener which is 300 times sweeter than sucrose
(Soejarto et al., 1983; Singh & Rao, 2005). Stevia extract and its bioactive compounds
such as stevioside and rebaudioside A are being used as substitute of sugar, which did
not show any toxicity effects on human health (Megeji et al., 2005; Carakostas et al.,
2008; Lemus-Mondaca et al., 2012). For example, Japan, USA and European
countries use Stevia as food additives and supplements (Stones, 2011; Lucas, 2011).
Therefore, Stevia would be a good alternative source of high-potency
sweetener for the growing natural food market in the future. For these reasons, many
countries including Malaysia have shown interest in its commercial cultivation and
research activities for the last two decades due to obesity problem as well as diabetics
and to fill up the shortage of sugar. Moreover, Malaysians consume a lot of sugar in
their daily food intake and to satisfy local demand Malaysia depends largely on
imported sugar. This unhealthy diet habit may be partially responsible for the
alarming rise in diabetic and obesity cases (Arpita et al., 2011; Lee and Hussein,
2012). The prevalence of diabetes is increasing in alarming rate from 6.3% in 1986 to
20.8% in 2011 among Malaysian adults above the age of 30 years old (Lee and
3
Hussein, 2012), and the prevalence might be reduced by using Stevia instead of sugar
(Arpita et al., 2011).
Stevia propagation through conventional way is difficult to carry out and
hardly can meet the commercial demand. Due to the commercial importance and
extensive use of Stevia, there is a need to develop rapid and reliable methods of in
vitro propagation of local variety/accession which allow propagation of large numbers
of plant in a relatively short period with no seasonal barrier. Using in vitro
propagation or tissue culture technique, complete new plantlet can be produced in a
nutrient culture medium from a single explant following several stages namely explant
collection, sterilization, shoot multiplication, root induction and acclimatization
(Thiyagarajan & Venkatachalam, 2012; Abdul Razak et al. 2014). It is possible to
regenerate plant in completely true to type like mother plants by using shoot tip and
nodal explants. Shoot tips and nodes are the most desirable explants to be used and
their usages minimize genetic variation (George & Debergh, 2008; Bhojwani &
Dantu, 2013).
1.2 PROBLEMS STATEMENT AND JUSTIFICATION
Generally, the common propagation method of stevia is through seed and stem
cuttings, but there are problems associated with these techniques, where seed
germination is very poor and stem cutting is time consuming. Stevia plant has
heterozygous and self-incompatibility natures which lead to the lack of fertilization.
Furthermore, the seeds have low vigorosity and propagation activity which do not
allow the production of homogenous population (Ramesh et al., 2006; Anbazhagan et
al., 2010). Due to the problems, variation occurs in sweetening level and composition
4
of chemical compound produced by Stevia (Tamura et al., 1984; Ramesh et al., 2006;
Anbazhagan et al., 2010; Abdullateef et al., 2015).
Thus in vitro clonal propagation could be an ideal technology to overcome the
problems caused by conventional propagation. In vitro clonal propagation is a
promising method of rapidly producing numerous, uniform plants that are free of
microbial contamination. This propagation procedure has been used successfully in
many plant species including the Asteraceae family such as Artemisia pallens (Nathar
& Yatoo, 2014), Gynura procumbens (Keng et al., 2009), Echinacea angustifolia
(Lucchesini et al., 2009) and Silphium perfoliatum (Tomaszewska-Sowa & Figas,
2011). Due to high popularity and demand, Stevia has becomes one of the important
commercial targets for in vitro propagation, where this culture technique can be
utilized for the large-scale production of this plant. Previously some in vitro works
have been established for the production of Stevia plant from different type of
explants including shoot tip, node, leaf and seed (Das et al., 2011; Thiyagarajan &
Venkatachalam, 2012; Pande & Gupta, 2013). However, in Malaysia, the in vitro
work on Stevia is very limited. Moreover, there was no plant tissue culture
experiment established with the Stevia accession MS007 Since in vitro propagation of
plants is also dependent on the variety of a species, thus, the present study was
attempted with Stevia of accession MS007, a local accession to determine a suitable
protocol and to find out the effect of different types of growth regulators on shoot
induction, multiplication and rooting of Stevia. The accession MS007 has been
chosen in this study because of its high morphological characteristics including, more
branching of axillary buds, higher number of leaves, bigger leaf sizes. The Stevia
sweetening compound stay more in the leaves compared to other parts of the plant.
Hence, leaf size is one of the important characteristics with larger leaf weight which
5
may implies higher quantity of sweetening content in the leaves (Tan et al. 2008;
Othman et al. 2015). It is hoped that the findings of this present study will allow the
mass propagation of disease free Stevia plants in a shorter time and low cost through
tissue culture technique.
1.3 HYPOTHESIS OF THE STUDY
An efficient in vitro clonal propagation technique for Stevia rebaudiana will be
achieved using shoot tip and nodal explants in different types and concentrations of
plant growth regulators. There are differences in terms of growth response of
explants, shoot and root number as well as length towards different types and
concentrations of growth regulators.
1.4 OBJECTIVES OF THE STUDY
1.4.1 General Objective
Development of an efficient in vitro clonal propagation technique of Stevia
rebaudiana using shoot tip and nodal explants.
1.4.2 Specific Objectives
i. To standardize a suitable sterilization method for Stevia rebaudiana using
shoot tip and nodal explants.
ii. To develop an efficient in vitro clonal propagation method for rapid
production of Stevia rebaudiana plantlet by using different plant growth
regulators (PGRs) through shoot induction and multiplication, rooting and
subsequent establishment in soil following acclimatization.
6
CHAPTER TWO
LITERATURE REVIEW
2.1 PLANT DESCRIPTIONS
Stevia rebaudiana commonly called as Stevia is a perennial shrub, 65-120 cm
tall with brittle stems and extensive root system (Ramesh et al., 2006). The stem
holds leaves which are sessile and arranged in opposite direction while the shape of
leaves is lanceolate to oblancoelate and they are serrated above the middle. The
trichome structures of leaf surface can be two distinct sizes; 4–5 µm (large) and 2.5
µm (small). The flowers are white and small in size with an irregular cyme
arrangement (Figure 2.1). Stevia is considered as self-incompatible and insect
pollinated plant which produces achene type seeds with feathery pappus (Brandle et
al., 1998). Stevia responds well to rich soil with adequate water and high organic
matter. It also prefers a hot and humid environment and can tolerate a wide range of
pH (5-7.5) (Singh & Rao, 2005; Ramesh et al., 2006). Although Stevia is a rare plant
in its natural habitat, it is widely cultivated in a wide range of soils with an adequate
drainage and consistent supply of moisture (Shock, 1982; Ramesh et al., 2006).
Stevia is native to Paraguay and also available in the neighbouring parts of
Brazil and Argentina (Soejarto, 2002). Today cultivation of Stevia has spread all over
the world, including America, Canada, Asia and Europe (Lemus-Mondaca et al.,
2012). Different Stevia accessions were available also across different locations in
Malaysia. The accessions were named either after the areas where they were collected
or after the collector. The Accession MS007 of Stevia has useful genetic traits, larger
leaf sizes (14.05 cm) with significant number of leaves and more axillary branches (6
7
branches/plant) (Abdullateef & Osman, 2011). There are few countries commercially
cultivating Stevia, including China, Taiwan, Thailand, Korea, Japan, India and
Malaysia (Thiyagarajan & Venkatachalam, 2012) with China as the top most Stevia
producer. In 2009, China's Stevia extract output was about 4,000 tons which
accounted for 80% of the global supply volume (Research Report on China's Stevia
Extract Industry, 2011).
Figure 2.1 Stevia rebaudiana plant Accession MS007: (a) aerial part, (b) flower, and
(c) seed. Bar = 1 cm.
2.2 PROPAGATION
Self-incompatibility and seed dormancy complicate breeding of Stevia. This plant
produces very small size seeds (about 3 mm length; 1000 seeds with weight of 0.3-1.0
g) with poor quality (Ramesh et al., 2006; Yadav et al., 2011). Some varieties
produce pale or clear coloured infertile seeds. Germination and establishment of
Stevia from seed is often poor and sometimes unsuccessful. Therefore, to increase
(c)
(b) (a)
8
pollination and seed fertility, outcrossing and allogamy are highly encouraged.
Outcrossing and allogamy can produce viable seed. A high density of bees (3-4 hives
per hectare) can be used in pollination purposes which increased good seed
production. Harvesting of immature seed may also contribute to poor germination
(Midmore & Rank, 2002) where seed needs to be collected frequently from mother
plant and stored at 4°C for 6-12 month to support germination (Rank &, Midmore,
2006). There is also great difference shown between greenhouse and field collected
seed germination i.e., 90% and 34%, respectively (Yadav et al., 2011).
Stevia is heterozygous plants in nature thus the variation among species is
high. This plant does not produce true to type plant and constant re-selection for type
is required in mother-seed plots (Ramesh et al. 2006). Normally this plant does not
propagate in cold weather. It prefers summer season and for specific region it needs
to wait for suitable time of propagation (Rank & Midmore, 2006). Since seed culture
gave low germination percentage (36.30%) and slow development of seedlings, which
takes 45 to 60 days to reach a suitable size to transplant in the field thus Stevia is
propagated by stem cutting (Goettemoeller & Ching, 1999; Yadav et al., 2011). Stem
cuttings give roots easily but the cultivation procedure is time consuming and required
high labour input. There are limited numbers of stem propagation occurred if they are
planted in the field directly. Stem cutting types and positions have great influence on
rooting and post-rooting growth and development (Abdullateef & Osman, 2012).
Generally, the top part of the main shoot with four internodes gave the successful
results of rooting (Yadav et al., 2011). Furthermore, stem cutting propagation is also
limited by the less number of individuals obtained from single plant. The problems
associated with seeds and stem cuttings are major limiting factors for large scale
cultivation of Stevia for commercial usage (Thiyagarajan & Venkatachalam, 2012).
9
Therefore, in vitro clonal propagation using a small part of plant as explants such as
shoot tips, nodes, hypocotyl and leaves can be applied for large scale production
within a short period, which is a suitable alternative method of plant propagation.
Moreover, micropropagation techniques regenerated genetically more stable plants
through shoot tips and nodes culture (George & Debergh, 2008).
2.3 CHEMICAL COMPOUNDS
Stevia contains highly sweetening and flavouring compounds which increased its
economic and commercial importance. The chemical constituents are mainly
extracted from the leaves and to a lesser extent; some of them can be found in flowers,
stems, seeds and roots. Around 34 sweet diterpene steviol glycosides have been
identified together with eight isomers and glycosylated forms of oxidised steviol
derivatives (Ceunen & Geuns, 2013). Among the steviol glycosides, stevioside is the
main sweetener, found at 4–13% of dry leaves, while other important compounds are
known as rebaudioside A (2–4%), rebaudioside B, C (1–2%), rebaudioside D, E (2%),
rebaudioside F (2.7%) and steviolbioside and dulcoside A (0.3%-0.9%) (Ahmed &
Dobberstein, 1982; Kennelly, 2002; Midmore & Rank, 2002; Staratt et al., 2002).
Impure form of stevioside and an ent-kaurene diterpene diglycoside were first
isolated at the beginning of twentieth century, but its final structure was only
elucidated about 60 years later (Mosettig & Nes, 1955; Kinghorn, 2002). The second
important sweetener rebaudioside A (1.4% of dry leaves) as well as rebaudioside B
(0.04% of dry leaves) were isolated by Tanaka and his co-workers in 1970 (Kohda et
al., 1976). Later, six further less abundant sweetened steviol glycosides were isolated
from Stevia, namely rebaudioside C, D, E (Sakamoto et al., 1977), dulcoside A, B
