AGRONOMY, BREEDING AND CYTOGENETIC

FACTORS AFFECTING GROWTH OF

Stevia rebaudiana Bertoni

BY

RAJI AKINTUNDE ABDULLATEEF

A thesis submitted in fulfilment of the requirement for the

degree of Doctor of Philosophy (Bioscience)

Kulliyyah of Science

International Islamic University Malaysia

DECEMBER 2013

ii

ABSTRACT

Stevia rebaudiana bertoni is known for its production of sweet glycosides, an

alternative to sugar due to its zero calorie and strong health and dietary implications. The cultivation of stevia in this country is hampered by few factors. First, stevia

produces flowers at early stage under Malaysia photoperiod condition, thus leading to

poor leaf development where the leaves are the most needed for sweetener extraction. In addition, stevia accessions do not grow well under local condition, and there has

been no breeding work to develop suitable cultivars. Moreover, no suitable vegetative propagation techniques have been established, leading to high production cost. Also

seed germination is very low making production of plant materials difficult and

expensive. Lastly, among the sweet glycosides, rebaudioside A is more preferred, but the content is low compared to stevioside in natural varieties. Thus, the project was

undertaken to study the agronomy, breeding and cytogenetic factors affecting growth of Stevia rebaudiana Bertoni under Malaysian condition. Firstly, agronomic studies

were conducted on the effects of stem cutting types, position and IBA on rooting.

YAS-MS012 cuttings treated with IBA concentration of 2.3 mM performed best. Under light treatment condition, YAS-MS012 treated with red light showed highest

rooting quality and IBA treatment may not be necessary. Pollen viability test using cotton blue-in-lactophenol and germinability test using boric acid in collected

accessions showed high pollen viability range from 87 to 93 %. The pollen

germinability ranged from 62 to 74 %. Boric acid concentration of 0.25 mg/ml was optimal. Seed viability using 1% tetrazolium chloride was found to be 68.67 %;

whereas germinability using red and white lights was found to be 41 and 31 % respectively. New technique improved seed germination to 67.33 %, than the initial

technique, which germinated a maximum of 41 % seeds. ANOVA showed there are

significant differences between treatments at p < 0.05. Utilization of Trigona was highly effective in increasing the total number of seed production, specifically on

black seeds. Day light extension using white, yellow, and red lights delayed flowering period and promoted vegetative yield in S. rebaudiana in comparison to control. Red

light had the highest influence (p < 0.05). Pruning increased plant height, number of

branches and number of leaves in S. rebaudiana (p < 0.05). The collected accessions were found to be genetically divergent where homogeneity test (p < 0.05) clustered

them into four groups. Breeding was also carried out to develop stevia hybrids. Mutation using gamma irradiation showed that the LD50 was 55 grays. The number of

germinated seeds was different in respect of doses. Genetic studies showed four types

of chlorophyll mutants were induced. They are albina, chlorina, xantha and xantha-viridis. Frequency and spectrum analysis revealed chlorina has the highest occurrence

of 33.03%. Tetraploidy induction using colchicine concentrations of 0.2 to 0.5 mM was performed. The concentration of 0.2% (w/v) was optimal in raising new

tetraploids. However, HPLC analysis showed a reduced rebA and stevioside content

in tetraploids, as compared to the diploid. Trigona increased cross-pollination between tetraploids and diploids. New triploid, RAAMBO/02, was developed. This research

led to classical novelties, such as plant materials: (i) Tetraploid RAAMBO/01, (ii) Triploid RAAMBO/02, (iii) M1 RAAMBO/03, and (iv) M2 RAAMBO/04. Several

prototypes were also developed (i) red light box for rhizogenesis, (ii) M2 seed dome for seed germination, and (iii) Trigona breeding net house for agronomy and breeding

research. The current research also improved several experimental protocols: (i)

rhizogenesis in stem cuttings, (ii) seed germination, and (iii) cytogenetic in stevia.

iii

iv

APPROVAL PAGE

The thesis of Raji Akintunde Abdullateef has been approved by the following:

________________________________ Zarina Zainuddin

Supervisor

________________________________

Mohamad bin Osman Co-Supervisor

________________________________ Mohamed Ismail Abdul Karim

Internal Examiner

________________________________

Ghizan bin Saleh

External Examiner

________________________________

Faiz Ahmed Mohamed EL-Faki Chairman

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.

Raji Akintunde Abdullateef

Signature…………………………. Date..……………………

vi

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF

FAIR USE OF UNPUBLISHED RESEARCH

Copyright ©2013 Raji Akintunde Abdullateef. All rights reserved.

AGRONOMY, BREEDING AND CYTOGENETIC FACTORS

AFFECTING GROWTH OF Stevia rebaudiana Bertoni

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 the prior written permission of the copyright holder except as provided below.

1. Any material contained in or derived from this unpublished research may

only 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 retrieval system and supply copies of this unpublished research if requested by other universities

and research libraries.

Affirmed by Raji Akintunde Abdullateef.

______________________ ______________________

Signature Date

vii

This project is dedicated to the beloved Prophet and Messenger of Allah (S.W.T.),

Muhammad Mustapha (S.A.W.), his household, companions and the generality of the

Muslim faithfuls and strugglers throughout the world.

viii

ACKNOWLEDGEMENTS

All praise is due to Allah (S.W.T.) the creator and the nourisher. May His endless

blessings be showered on the noble soul of my beloved, the best of creatures, the messenger and prophet of Allah, Muhammad Al Mustapha (S.A.W.), his family and

Sahabah (Ameen).

I sincerely appreciate and show gratitude to my competent and ever dynamic

supervisor, Professor Dr. Mohamad bn Osman for his dedications and constructive criticisms that helped shape this research towards the attainment of its goal. May the

almighty Allah be your strength always. Jazakallahu khairah kathiirah, Ameen. Similarly, to Dr. Zarina binti Zainuddin who replaced my previous supervisor,

JazakiLlahu khairah. May your morale and knowledge never get dampened, and I pray

that Allah grants your requests. I am grateful to you.

I would like to express my gratitude to the dean, Prof. Dr. Kamaruzzaman bin Yuunus and the deputy dean postgraduate studies, Prof. Dr. Ahmed Jalal Khan

Chowdhury for their moral and kind supports. I am also thankful to the dean and staff

of CPS for granting me the niche area and president scholarships, Jazakummullahu khairah.

I show gratitude to Allah for the gentle soul of Ameer, Muhammad Ali

Olukade (late), may he be granted Jannatul Firdaos, ameen. I pray for His guidance on

Mallam Al Ameen Abdul Wahab and the entire Muslim Ummah, may we all succeed in our endeavours.

Much thanks and appreciations to my beloved father, Mallam Abdul Azeez

(Late) may he be pardoned. To my beloved mother, Mallama Wasilat Raji, and my

wives, Hajia Hassanat Bint Jimoh and Hajia Ameenat Bint Yuusuf, may all never lack Allah’s mercies ameen. Lastly, I thanked my beloved children: Mu’minat,

Muhammad, Rukayyah, Sakeenah, Maryaam, Ni’mat, Abdullah, and Abdur Rahman (and Aishat, Nurudeen and Rahmat). May all be Allah’s vicegerents insha Allah,

ameen. Wa akhiru da’wana anil HamduliLLAHI Rabbil A’lamiin.

ix

TABLE OF CONTENTS

Abstract .................................................................................................................... ii

Abstract in Arabic .................................................................................................... iii Approval page .......................................................................................................... iv

Declaration ............................................................................................................... v Declaration of Copyright ......................................................................................... vi

Dedication ................................................................................................................ vii

Acknowledgements .................................................................................................. viii List of tables ............................................................................................................. xiii

List of figures ........................................................................................................... xiv List of symbols/Abbreviations ................................................................................. xviii

CHAPTER ONE: INTRODUCTION .................................................................. 1 1.1 Background of the Study........................................................................ 1

1.1.1 Classification ................................................................................ 2 1.1.2 Plant description ........................................................................... 3

1.1.3 Cultivation .................................................................................... 3

1.1.4 Uses .............................................................................................. 4 1.2 Statements of the problem...................................................................... 5

1.3 Justification ............................................................................................ 6 1.4 Significance of the study ........................................................................ 6

1.5 Research questions and hypotheses ....................................................... 7

1.5.1 Research questions: ...................................................................... 7 1.5.2 Research hypotheses: ................................................................... 7

1.6 Objectives of the study ........................................................................... 8 1.6.1 Main objective:............................................................................. 8

CHAPTER TWO: LITERATURE REVIEW ..................................................... 10 2.1 Agronomy .............................................................................................. 10

2.1.1 Propagation .................................................................................. 10 2.1.2 Photoperiod effects on flowering ................................................. 17

2.2 Breeding studies ..................................................................................... 20

2.2.1 Stevia genetic resources ............................................................... 20 2.2.2 Hybridization by stingless bees.................................................... 21

2.2.3 Stingless bees in malaysia ............................................................ 23 2.2.4 Polyploid breeding ....................................................................... 26

2.2.5 Mutation breeding ........................................................................ 29

2.2.6 Sweeteners.................................................................................... 34 2.2.7 Glycoside contents ....................................................................... 39

2.2.8 HPLC assay for identification and quantification of glycosides.. 41 2.3 Cytogenetics of stevia ............................................................................ 42

CHAPTER THREE: MATERIALS AND METHODS...................................... 46

3.1 Agronomic studies ................................................................................. 46

3.1.1 Plant propagation ......................................................................... 46

x

3.1.2 Agronomic field techniques ......................................................... 59 3.2 Breeding Studies .................................................................................... 61

3.2.1 Germplasm evaluation ................................................................. 61

3.2.2 Mutation breeding by gamma irradiation..................................... 62 3.2.3 Tetraploidy induction using colchicine ........................................ 65

3.2.4 Prototype on the development of Trigona for hybridization studies.................................................................................................... 73

3.3 Statistic analysis ..................................................................................... 75

CHAPTER FOUR: RESULTS ............................................................................. 76

4.1 Agronomic Studies ................................................................................. 76 4.1.1 Effects of stem cutting types, position and IBA on rooting ......... 76

4.1.2 Effects of light on the rooting of YAS-MS012 treated with

2.3 mM IBA .......................................................................................... 81 4.1.3 Efficacies of red and white light boxes on rooting ...................... 83

4.1.4 Pollen viability ............................................................................. 86 4.1.5 Pollen Germination ...................................................................... 89

4.1.6 Seed viability................................................................................ 92

4.1.7 Seed germination under different light wavelengths ................... 94 4.1.8 Effects of Trigona on seed formation .......................................... 103

4.1.9 Effects of photoperiod treatments on plant growth ...................... 112 4.2 Breeding Studies .................................................................................... 120

4.2.1 Germplasm Evaluation ................................................................. 120

4.2.2 Mutation breeding by gamma irradiation..................................... 124 4.2.3 Tetraploidy induction using colchicine ........................................ 140

4.2.4 Development of Trigona for hybridization studies ...................... 169

CHAPTER FIVE: DISCUSSION ......................................................................... 175

CHAPTER SIX: CONCLUSION ......................................................................... 191

Future Studies............................................................................................... 195

BIBLIOGRAPHY .................................................................................................. 196 Appendix 1: Effects of stem cuttings and IBA on rooting in MS012 and MS007 .. 214

Appendix 2: ANOVA on day of root emergent, number, length, and width of roots.......................................................................................................................... 215

Appendix 3: Effects of lights and iba on root sprouting. ......................................... 216

Appendix 3: Effects of lights and iba on root sprouting .......................................... 217 Appendix 4: ANOVA on data collected from irradiation boxes. ............................ 218

Appendix 5: Post hoc on efficacy of light boxes on rooting in stem cuttings. ........ 219 Appendix 6: Percentage of pollen viability in three accessions............................... 220

Appendix 7: Percentage pollen viability in ten accessions. ..................................... 220 Appendix 8: Analysis of variance on pollen viability studies in ten accessions. .... 220

Appendix 9: Post hoc analysis on percentage pollen viability in ten accessions. .... 221

Appendix 10: Percentage of pollen germinability with boric acid in three accessions. ................................................................................................................ 221

Appendix 11: Percentage pollen germinability with boric acid in three accessions 222 Appendix 12: Percentage pollen germinability with boric acid in ten accessions. .. 223

xi

Appendix 13: ANOVA on pollen germinabiity with boric acid in ten accessions. . 224 Appendix 14: Post hoc on pollen germinability with boric acid 0.025 g/ml in ten

accessions. ................................................................................................................ 225

Appendix 15: Percentage seeds viability with triphenyltetrazolium chloride (TTC)................................................................................................................................... 226

Appendix 16: Period and percentage of seed germination under light treatments. . 226 Appendix 17: ANOVA on period and percentage of seed germination under light

treatments. ................................................................................................................ 227

Appendix 18: Evaluations on other seed germination parameters with effects of light treatments......................................................................................................... 227

Appendix 19: Comparisons on seed germination techniques .................................. 228 Appendix 20: Effects of different techniques on seed germination in stevia .......... 228

Appendix 21: Effects of Trigona pollinating agents on seed formation .................. 229

Appendix 22: ANOVA on effect of Trigona on seed formation. ............................ 230 Appendix 23: Effects of Trigona on seed formation. .............................................. 231

Appendix 24: Morphological evaluated data on photoperiod experimets ............... 232 Appendix 25: ANOVA on morphological data collected on photoperiod experiment.

.................................................................................................................................. 233

Appendix 26: Effects of photoperiod extension on morphological characters . ..... 234 Appendix 27: Effects of pruning on morphological characters. .............................. 235

Appendix 28: Accession descriptions and Global Positioning System (GPS) ....... 236 Appendix 29: Morphological characters of collected stevia under natural

environment. ............................................................................................................ 237

Appendix 30: Homogeneity of variance using plant height marker in collected stevia ........................................................................................................................ 238

Appendix 31: Number of germinated seeds with gamma treatment 0-2000 Gry. .. 239 Appendix 32: ANOVA on number of germinated seeds with gamma 0-2000 Gry. 240

Appendix 33: Height of seedlings with gamma irradiation treatment 0-100 Gry. .. 240

Appendix 34: ANOVA on height of seedlings with gamma treatment 0-100 Gry. 241 Appendix 35: Height of seedlings with gamma treatments 45, 55 and 65 Gry. ...... 241

Appendix 36: ANOVA on height of seedlings with gamma 45, 55 and 65 Gry. ... 242 Appendix 37: Morphological characters evaluated on matured M2 plants .............. 243

Appendix 38: ANOVA on morphological and reproductive characters in M2 of stevia.

.................................................................................................................................. 244 Appendix 39: Response to concentrations of colchicine in MS007 and MS012. ... 245

Appendix 40: ANOVA on response of stevia MS007 and MS012 to colchicine. . 245 Appendix 41: Post hoc comparisons on plants survival with colchicines treatments.

.................................................................................................................................. 246

Appendix 42: Morphological traits in tetraploid and diploid plants ........................ 247 Appendix 42: Morphological traits in tetraploid and diploid plants ........................ 248

Appendix 43: ANOVA on six morphological traits in both tetraploid and diploid. 249 Appendix 44: Post hoc on morphological traits in tetraploids and diploids. ........... 250

Appendix 44: Post hoc on morphological traits in tetraploids and diploids. ........... 250 Appendix 45: Cytological and cytogenetic traits tetraploids and diploids .............. 252

Appendix 46: ANOVA on five cytological traits in tetraploids and diploids of stevia

.................................................................................................................................. 253 Appendix 47: Post hoc analysis on cytological traits. ............................................. 254

Appendix 47: Post hoc analysis on cytological traits .............................................. 255 Appendix 48: Morphological evaluations on triploid and diploid varieties ........... 255

xii

Appendix 49: Cytological and cytogenetic evaluations on triploid and diploid plants ........................................................................................................................ 256

Appendix 50: Novelties from the research .............................................................. 256

AWARDS AND PUBLICATIONS ......................................................................... 260

xiii

LIST OF TABLES

Table No. Page No.

3.1: Evaluations on environmental conditions in light chambers. 50

4.1: Effects of different light and IBA treatment on rooting in stem

cuttings 81

4.2: Efficacy of light irradiation boxes on rooting in stem cuttings.

Different alphabets 84

4.3: Description of the chlorophyll mutant characters observed in the M2 seedlings 132

4.4: Frequency and spectrum of chlorophyll mutants in seedlings of M2 generation. 133

4.5: Morphological evaluations on mutant M2 seedlings 134

4.6: HPLC analysis on the standards for rebaudioside A and stevioside. 164

4.7: Concentration of rebaudioside A and stevioside in diploid and

tetraploid MS012 165

4.8: Quantity of glycoside extracts based on leaf yield per 30 plants 168

xiv

LIST OF FIGURES

Figure No. Page No.

3.1: Micro-cutting/mist-chamber device for rooting 46

3.2: Stem cuttins from apical shoots 47

3.3: Chart flow of procedures involved in rooting of stem micro cuttings. 48

3.4: Prototype red and white light irradiation boxes for rhizogenesis in 51

3.5: Seed germination chambers. 55

3.6: New seed germination chamber. 57

3.7: The extraction of glycosides from dry leaves. 71

3.8: Purification of crude glycoside 72

4.1: Effects of stem cutting types, position, and IBA on day of rooting in stevia. 78

4.2: Effects of stem cutting types, position, and IBA on number of roots in stevia. 79

4.3: Stem cuttings rooted in mist chamber. 80

4.4: Rooting in stem cuttings due to different lights and IBA. 82

4.5: Rooted stem cuttings by light irradiation boxes and sunlight. 85

4.6: Percentage of pollen viability in three accessions of stevia MS007- Mardi stevia 007; MS 012- Mardi stevia 012 and SBK- Souk bukhari. 87

4.7: Viable and non-viable pollens of MS012 in cotton blue in lactophenol

observed under 200x magnification 87

4.8: Percentage of pollen viability in ten accessions of stevia. MS007-

Mardi stevia 007 MS 012- Mardi stevia 012, SBK- Souk bukhari, Aziz, Rawang, Bangi, Matan Pagar, Taman Pertanian, Mergong and

Lnagat. 88

4.9: Percentage of pollen germinability in three accessions stevia MS007- Mardi stevia 007, MS 012- Mardi stevia 012, SBK- Souk bukhari. 90

4.10: Germinated and non- germinated pollens of accession MS012 observed under 1000x magnification. 90

xv

4.11: Percentage of pollen germinability in ten accessions of stevia MS007- Mardi stevia 007, MS 012- Mardi stevia 012, SBK- Souk bukhari,

Aziz, Rawang, Bangi, Matan Pagar, Taman Pertanian, Mergong and

Lnagat. 91

4.12: Seed viability tests with tetrazolium chloride (TTC) 93

4.13: Percentage seed viability of stevia accession MS012. 93

4.14: Effects of light on period and number of germinated seeds. 96

4.15: Stages in seed germination processes in stevia. 97

4.16: Effects of light on seed germination characters. 99

4.17: Innovated technique for seed germination Enhancement 101

4.18: Effects of varying techniques on germination rate in seed of stevia. 102

4.19: Effects of varying techniques on germination percentage in seed of

stevia. 102

4.20: Effect of Trigona on total number of seed production. 104

4.21: Effect of Trigona on number of black seeds formation. 104

4.22: Effect of Trigona on number of tan seed formation. 106

4.23: Two varieties of Trigona utilized for flower pollination. 108

4.24: Trigona assisted, non-assisted and field pollinations. 109

4.25: Photomicrograph on Trigona insect showing adhering pollens. 100x magnification. 110

4.26: Harvested seeds from Trigona assisted, non-assisted and field pollinations. 111

4.27: Effects of photoperiod extension with different lights on

morphological paraameters. 114

4.30: Pruning in Stevia. 119

4.32: collected accessions and young seedlings of Stevia rebaudiana. 123

4.33: Accessions arranged in rows on the field, in the Kulliyyah of Science,

IIUM Kuantan. 123

4.34: The subset groups among ten accessions of Stevia rebaudiana. 124

4.35: Effects of gamma irradiation dose (0 – 2000) on seed germination. 125

xvi

4.36: Stages involved in mutation induction from seed to M1 plants. 127

4.37: Simple linear regression curve on heights of seedlings due to gamma

irradiation dose (0 -100 grays). 128

4.38: Effects of gamma irradiation dose 45, 55 and 65 on heights in M1

seedlings. 130

4.40: Varying morphological mutations in the M2 population of stevia. 135

4.41: M2 plants of stevia on the field. 135

4.42: Morphological characters in M2 generation of stevia. 138

4.42: Morphological characters in M2 generation of stevia. 139

4.43: Percentage survival in MS007 and MS012 treated with colchicine. 141

4.45: Size of leaves in tetraploid and diploid accessions of stevia MS007- Mardi Stevia 007, MS012- Mardi Stevia 012, SBK- Souk Bukhari,

Aziz, Rawang, Bangi, Matan Pagar, Taman Pertanian, Mergong and

Langat. 144

4.46: Stem girth in tetraploid and diploid accessions of stevia MS007-

Mardi Stevia 007, MS 012- Mardi Stevia 012, SBK- Souk Bukhari, Aziz, Rawang, Bangi, Matan Pagar, Taman Pertanian, Mergong And

Langat. 145

4.47: Plants and leaves of tetraploid and diploid plants. 146

4.50: Number of germinal pore in tetraploid and diploid accessions of stevia

MS007- Mardi stevia 007, MS 012- Mardi stevia 012, SBK- Souk bukhari, Aziz, Rawang, Bangi, Matan Pagar, Taman Pertanian,

Mergong and Langat. 150

4.51: Stomata size in tetraploid and diploid plants at 200 x (10,000 µm field of view). 151

4.52: Stomata size in tetraploid and diploid plants at 1000X (10,000 µm field of view). 152

4.53: Pollens in polyploid and diploid at 200x (10,000 µm field of view). 154

4.54: Labeled micrograph of a typical pollen in tetraploid at 1000X (10,000 µm field of view). 155

4.55: Pollens at 1000X (10,000 µm field of view) under photomicrography. 155

4.56: Germinal pores in pollen of diploid and tetraploid at 200x (10,000 µm

field of view) further magnified by lens. 156

xvii

4.57: Chromosomes at different stages in meiosis of tetraploids and diploids. 158

4.58: Chromosomal behaviours at different stages of cell divisions in

mitosis of tetraploids and diploids. 162

4.62: HPLC chromatogram for tetraploid MS012. Chromatogram for

tetraploid stevia MS012. 167

4.64: Cytological Characters in Triploid And diploid plants of stevia. 172

4.65: Triploid and diploid plants of stevia on the field. 173

4.66: Stomata of triploid and diploid plants. 173

4.67: Chromosomal behaviours at different stages in mitosis of triploid

plants. 174

xviii

LIST OF SYMBOLS/ABBREVIATIONS

µm Micrometer

TTC Tetrazolium chloride

YAS Young apical shoot

OAS Old apical shoot

NAS Non apical shoot

∑ Summation

oC Temperature

RH Humidity

Lx light intensity

LD50

GPS

Lethal dose

Global Positioning System

1

CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Stevia rebaudiana Bertoni, also known as sweet leaf, or sugar leaf, is one of the 950

genera of the family Compositae (Asteraceae) and constitutes about 154 species of

herbs and shrubs (Soejarto et al., 1982, 1983). The species is native to Paraguay in

Southern America (Mark, 2009) and one of the only two that produce sweet steviol

glycosides (Madan et al., 2010).

Stevia was first brought to the attention of Europeans in 1887 when M.S.

Bertoni learned of its unique properties from the Paraguayan Indians and Mestizos

(Lewis, 1992). He later described the plant and gave it scientific nomenclature in 1905

in honour of Paraguayan chemist Dr. Rebaudi; hence the name ‘Stevia rebaudiana

Bertoni’.

Lewis (1992) reported that it was long known to the Guarani Indians of the

Paraguayan highlands who called it caá-êhê, meaning sweet herb. The leaves were

used either to sweeten maté or as a general sweetening agent.

Sumida (1968) began a large effort aimed at establishing stevia as a crop in

Japan. Since then, stevia has been introduced as a crop in many countries including

Brazil, Korea, Mexico, United States, Indonesia, Tanzania and Canada (Donalisio et

al., 1982, Goenadi, 1983, Shock, 1982, Saxena and Ming, 1988, Brandle and Rosa,

1992, Fors, 1995).

Since 1970s, stevia extracts have been widely used in a number of countries as

a sugar substitute. Stevia usage in the United States was limited because the Food and

2

Drug Administration (FDA) did not allow its use as a sweetener in food processing

and manufacturing claiming that it is not a safe food additive and that it has

implications for health. So in 1991, FDA banned stevia. However, after several proofs

from scientists and petitions for pro stevia industries, FDA now allows the sale of

stevia, but only as a nutritional supplement (Mark, 2009).

Stevia is considered as a good substitute of sugar. Diabetic patients can take

herbal powder of stevia available in market places as it lowers the sugar level, giving

the sweet taste better than sugar. Stevia produce sweet glycosidic compounds which

are non-caloric and the sweet compounds pass through the digestive process of the

body without chemically breaking down, thus making stevia a safe substance for

consumption for diabetic patients who need to reduce the sugar content of their blood

(Strauss, 1995).

From most of the previous work, stevia has been reported to have no adverse

effect on humans (Brandle and Rosa, 1992). The leaves could be eaten fresh or dried

and it could be boiled in tea to release the sweetener. It has been used for centuries by

the Guarani Indians of Paraguay, where the plant originated from, as sweeteners for

mate tea (Goettemoeller and Ching, 1999).

1.1.1 Classification

The classification of this plant is as shown below:

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Compositales

Family : Compositae

Genus : Stevia

Species: rebaudiana

3

1.1.2 Plant Description

Stevia exists as both herbs and shrubs (Robert, 2010). The plant, under cultivation can

reach up to 1 m or more in height (Shock, 1982). It possesses a widespread root

system and strong stems producing small, elliptic leaves. The leaves are sessile,

alternately arranged in lanceolate to oblancoelate, ovate in shape and serrated above

the middle edge. Trichome structures are found on the leaf surface and sometimes on

stems (depending on varieties) are of two distinct sizes: large (4-5 µm), and small (2.5

µm) (Shaffert and Chetobar 1994). The tiny white florets are perfect, borne in small

corymbs of 2–6 florets. Corymbs are arranged in loose panicles (Goettemoeller and

Ching, 1999). Oddone (1997) considered stevia as self-incompatible, insect pollinated

and tan seeds as non-fertile. Seeds are contained in elongated achenes, about 3 mm in

length. Each achene has an average of 20 persistent pappus bristles.

1.1.3 Cultivation

Stevia is now cultivated in most parts of the world. Being a sugar substitute and seeing

the percentage of diabetes patients in the world, this herb has gained popularity

widely. It is commercially cultivated in many parts of Brazil, Paraguay, Uruguay,

Central America, Thailand, India and China. In China, stevia is cultivated in large

scale. In 2009, extracts from stevia output amounted to about 4,000 tons (Research

Report on China's Stevia Extract Industry, 2011). Eighty percent of the global supply

volume comes from China, which are exported to Malaysia, Mexico, the U.S.A.,

Japan and Hong Kong (Chen, 2011).

Stevia prefers moist, sandy and loamy soil with high organic matter and

adequate drainage. It tolerates a wide range of soil pH. It also prefers partial shade

with considerate summer sunshine (Rhonda, 2004).

4

Stevia is grown as a perennial in subtropical regions including the United

States, but must be grown as an annual in mid to high latitude regions, where longer

days favor leaf yield and stevioside contents (Goettemoeller and Ching, 1999).

Propagation of stevia is usually by stem cuttings, which root easily, but require high

labour inputs. Poor seed germination (10%) possibly due to self-incompatibility is one

of the factors limiting large-scale cultivation (Goettemoeller and Ching, 1999).

Leaves of stevia are dehydrated in sunshine or heaters and if they are eaten

fresh, it gives a bitter taste. Dry leaves are further powdered to be used as a sweetener.

Leaves are preferably collected in autumn being a transition season between summer

and winter when leaves fall naturally. Dead leaves and black leaf spots diseases in

stevia are caused by Septoria and Alternaria species, fungi that live in soil (Morita et

al., 2009).

1.1.4 Uses

In China, Korea, Brazil, Paraguay, Japan and many other countries, stevia and its

stevioside extract are used as a tabletop sweetener because they have zero calories,

zero carbohydrates and zero fats. (Mark, 2009). Being an alternative to sugar, it does

not have harmful effects on humans (Goettemoeller and Ching, 1999) and can be use

in tea, chocolate, and soft drinks. Stevia is added to ice cream and beverages to lower

the calorie level and to give a natural taste. Additionally, drinking stevia tea or stevia

enhanced-teas helped to reduce a person’s desire for tobacco, alcoholic beverages, as

well as the desire for sweets and fatty foods.

Stevia is also used for cavity prevention. In China, for example, stevia is used

as an ingredient in certain toothpaste and mouthwash. Stevia and stevioside have been

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shown to inhibit the growth and reproduction of bacteria that are responsible for tooth

decay, thus making it an important ingredient in toothpaste (Misra et al., 2011).

Stevia benefits diabetic patients due to its low calories (Strauss, 1995). As

herbal medicines, stevia stimulates alertness and counter fatigue, facilitates digestion

and gastrointestinal functions, nourishes the liver, pancreas, and spleen, helps the

body sustain a feeling of vitality and well-being and is also used for body weight loss

(Rhonda, 2004). Stevia has been extensively used to control high blood pressure

(Strauss, 1995). Recent medical research has shown promising effect of stevia in

treating obesity (Park and Cha, 2010).

Other properties of stevia include anti-hypertensive (Chan et al., 2000), anti-

hyperglycemic (Jeppesen et al., 2002) and antioxidant (Xi et al., 1998).

1.2 STATEMENTS OF THE PROBLEM

(1) Stevia produces flowers at early stage under short day length Malaysia condition.

When flowers are formed the leaf development is impaired and this is a disadvantage

because the leaves are the most needed part of the plant for sweetener extraction.

(2) Stevia accessions in Malaysia are exotic and do not grow well under local

condition, and there has been little or no breeding work and breeding technique to

develop suitable varieties under local environment.

(3) No suitable vegetative propagation techniques have been established under local

environment, leading to high production cost.

(4) Seed germination is very low making production of plant materials difficult and

expensive.

(5) Among the sweet glycosides, rebaudioside A is preferred, but the content is low

compared to stevioside in natural varieties.

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1.3 JUSTIFICATION

(1) Stevia helps to control blood glucose levels. Consequently, it has been used as a

non-sugar sweetener in food and drinks and as remedy for diabetes (Leung, 1996).

(2) New varieties, which are insensitive to photoperiod would produce high leaf yield

for sweetener extraction.

(3) Effective production techniques would enhance vegetative and seed propagation

for large-scale cultivation.

(4) Recent medical research has shown that stevia has promising effects in treating

obesity (Park and Cha, 2010), this would help people with obesity problem to manage

their health.

(5) Stevia has the potential as a new emerging crop for Malaysia.

1.4 SIGNIFICANCE OF THE STUDY

New protocols on stevia agronomy and breeding programs would facilitate raising

stevia varieties, enhance better production technology, and increase in vegetative yield

under local environmental condition. High leaf yield in new varieties implies high

quantity of extractable sweet stevia glycosides. With promising potential and technical

knowhow, plans for stevia industry and business could be drawn and initiated in

Malaysia.

Stevia glycosides help to control glucose levels; consequently, it can be used

as a non-sugar sweetener for food and drinks and as remedy for diabetes.