by naveed ahmad

OPTIMIZATION OF CALLUS, CELL SUSPENSION AND ADVENTITIOUS

ROOT CULTURES FOR THE PRODUCTION OF ACTIVE

COMPONENTS IN STEVIA REBAUDIANA

BY

NAVEED AHMAD

A dissertation submitted to The University of Agriculture Peshawar, in partial fulfillment of the

requirements for the degree of

DOCTOR OF PHILOSOPHY IN AGRICULTURE

(HORTICULTURE)

DEPARTMENT OF HORTICULTURE

FACULTY OF CROP PRODUCTION SCIENCES

THE UNIVERSITY OF AGRICULTURE

PESHAWAR-PAKISTAN

JANUARY, 2017


ROOT CULTURES FOR THE PRODUCTION OF ACTIVE

COMPONENTS IN STEVIA REBAUDIANA

BY

NAVEED AHMAD

A dissertation submitted to The University of Agriculture Peshawar, in partial fulfillment of the

requirements for the degree of

DOCTOR OF PHILOSOPHY IN AGRICULTURE

(HORTICULTURE) Approved By:

______________________ Chairman Supervisory Committee Prof. Dr. Abdur Rab

______________________ Co-Supervisor for Research (University of Swat)

Dr. Nisar Ahmad

Assistant Professor

______________________ Member (Major Field)

Dr. Muhammad Sajid

Assistant Professor

_____________________ Member (Minor Field) Prof. Dr. Razi Uddin

______________________ Chairman & Convener Board of Studies

Prof. Dr. Noor-ul-Amin

______________________ Dean Faculty of Crop Production Sciences Prof. Dr. Muhammad Jamal Khan

______________________ Director Advanced Studies & Research Prof. Dr. Muhammad Jamal Khan

DEPARTMENT OF HORTICULTURE

FACULTY OF CROP PRODUCTION SCIENCES

THE UNIVERSITY OF AGRICULTURE

PESHAWAR-PAKISTAN

JANUARY, 2017

DEDICATION

This dissertation is dedicated to my Parents, Brothers and Sisters, without their

tremendous inspiration and encouragement it wasn’t possible. I love you Mom and

Dad, You gave me your wisdom to know when to turn away and when to charge

ahead, you taught me how to live right, to be gentle, to live day by day, to be

patient and forgiving, to hope and to pray, to be proud of who I am and giving me

the strength to always strive for better. You are my rock and foundation. You're my

angel in the darkness, keeping the way ahead bright. You sheltered me all through

the years, calmed my worries and my fears. Thanks to you, my hopes and dreams can

never grow dim. “Allah took two pair of Angel wings, gentle loving hands, eyes

that only see the good, a heart that understands, a smile to encourage, love that

never ends. He wrapped it up with tender care and called this gift as "PARENTS".”

NAVEED AHMAD

TABLE OF CONTENTS

Chapter No. Title Page No.

List of Tables ................................................................................................... i-v

List of Abbreviations ....................................................................................... vi

Acknowledgments ........................................................................................ vii

Abstract ........................................................................................................... viii-x

CHAPTER-I Introduction…………………………………………………………………… 1-5

CHAPTER-II

Review of Literature…………………………………………………………..

6-20

CHAPTER-III

Light-induced biochemical variations in secondary metabolites production

and antioxidant activity in callus cultures of Stevia rebaudiana (Bert.) …....

21-36 CHAPTER-IV

Sucrose-induced osmotic stress improved biomass and production of

antioxidant secondary metabolites in callus, cell suspension and adventitious

root cultures of Stevia rebaudiana (Bert.)…………………………………….

37-81

CHAPTER-V

The influence of pH on the development of callus, cell suspension and

adventitious root cultures and production of Steviol glycosides in Stevia

rebaudiana (Bert.) ……………………………………………………………

82-124

CHAPTER-VI

The effect of inoculum size on biomass, phenolics and flavonoids,

antioxidant activity and bioactive compounds in callus, cell suspension and

adventitious root cultures of Stevia rebaudiana (Bert.)……………………….

125-166

CHAPTER-VII

General Summary, Conclusion and Recommendations ……………………...

167-175

Literature Cited………………………………………………………………..

176-234

i

LIST OF FIGURES

Fig. No. Title Page No.

3.1: Effect of different spectral lights on callus morphological features in S. rebaudiana (a)

red light induced callus (b) blue light (c) yellow light (d) green light and (e) control white

light. .............................................................................................................................................. 28

3.2: Spectral lights induced variation in callogenic frequency (%) from leaf explants in S.

rebaudiana. ................................................................................................................................... 28

3.3: Spectral lights induced variation in biomass accumulation during growth kinetics of

callus cultures. .............................................................................................................................. 30

3.4: Fresh weight, dry weight and extractive values of callus cultures exposed to different

spectral lights.. .............................................................................................................................. 30

3.5. Effect of different spectral lights on total phenolic and flavonoid content in callus

cultures of S. rebaudiana.. ............................................................................................................ 32

3.6: Correlation of total phenolics content with antioxidant activities in callus cultures of S.

rebaudiana.. .................................................................................................................................. 33

3.7: Correlation of total flavonoids content with antioxidant activities in callus cultures of S.

rebaudiana.. .................................................................................................................................. 34

4.1. Effect of sucrose concentrations (a; 05 g l-1), (b; 10 g l-1), (c; 15 g l-1), (d; 20 g l-1), (e; 25

g l-1), (f; 30 g l-1), (g; 35 g l-1), (h; 40 g l-1), (i; 45 g l-1) and (j; 50 g l-1) on callus

proliferation of Stevia rebaudiana. ............................................................................................ 49


g l-1), (f; 30 g l-1), (g; 35 g l-1), (h; 40 g l-1), (i; 45 g l-1) and (j; 50 g l-1) on development of

cell suspension cultures of Stevia rebaudiana .............................................................................. 50


g l-1), (f; 30 g l-1), (g; 35 g l-1), (h; 40 g l-1), (i; 45 g l-1) and (j; 50 g l-1) on establishment of

adventitious root cultures of Stevia rebaudiana ........................................................................... 51

4.4. Sucrose induced osmotic stress (05-50 g l-1) variations in biomass accumulation during

growth kinetics (period 30 days; interval 03 days) of callus cultures of Stevia rebaudiana. ....... 52


growth kinetics (period 30 days; interval 03 days) of cell cultures of Stevia rebaudiana ............ 52


growth kinetics (period 30 days; interval 03 days) of adventitious root cultures of Stevia

rebaudiana. ................................................................................................................................... 53

ii

4.7. Effect of sucrose induced osmotic stress on fresh and dry weight (g l-1) of callus cultures

of Stevia rebaudiana. ................................................................................................................... 56

4.8. Effect of sucrose induced osmotic stress on fresh and dry weight (g l-1) of cell suspension

cultures of Stevia rebaudiana. ..................................................................................................... 57

4.9. Effect of sucrose induced osmotic stress on fresh and dry weight (g l-1) of callus culture

of Stevia rebaudiana. ................................................................................................................... 58

4.10. Effect of sucrose induced osmotic stress on accumulation of total phenolic content (mg/g-

DW) in callus, cell suspension and adventitious root cultures of Stevia rebaudiana. ................. 62

4.11. Effect of sucrose induced osmotic stress on accumulation of total flavonoids content

(mg/g-DW) in callus, cell suspension and adventitious root cultures of Stevia rebaudiana.

...................................................................................................................................................... 63

4.12. Effect of sucrose concentration on antioxidant activities (%) in callus, cell suspension

and adventitious root culture of Stevia rebaudiana. .................................................................... 66

4.13. Correlation of total phenolic and flavonoids content with antioxidant activities in callus

cultures of Stevia rebaudiana. ...................................................................................................... 68

4.14. Correlation of total phenolic and flavonoids content with antioxidant activities in cell

suspension cultures of Stevia rebaudiana. .................................................................................... 69

4.15. Correlation of total phenolic and flavonoids content with antioxidant activities in

adventitious root cultures of Stevia rebaudiana. .......................................................................... 69

4.16. Effect of sucrose concentration on stevioside, rebaudioside and dulcoside in callus culture

of Stevia rebaudiana. .................................................................................................................... 74

4.17. Effect of sucrose concentration on stevioside, rebaudioside and dulcoside in cell culture

of Stevia rebaudiana ..................................................................................................................... 75

4.18. Effect of sucrose concentration on stevioside, rebaudioside and dulcoside in adventitious

root culture of Stevia rebaudiana ................................................................................................. 76

5.1. pH levels (a; 5.1, b; 5.2, c; 5.3, d; 5.4, e; 5.5, f; 5.6, g; 5.7, h; 5.8, i; 5.9, j; 6.0) induced

variations in callus cultures of Stevia rebaudiana. ....................................................................... 94


variations in cell suspension cultures of Stevia rebaudiana. ....................................................... 95


variations in adventitious root cultures of Stevia rebaudiana. ..................................................... 96

5.4. Effect of pH levels (5.1-6.0) on biomass accumulation during growth kinetics of callus


iii

5.5. Effect of pH levels (5.1-6.0) on biomass accumulation during growth kinetics of cell


5.6. Effect of pH levels (5.1-6.0) on biomass accumulation during growth kinetics of


5.7. Effect of pH levels on fresh and dry weight (g l-1) of callus culture of Stevia rebaudiana. ........ 101

5.8. Effect of pH levels on fresh and dry weight (g l-1) of cell suspension culture of Stevia

rebaudiana. .................................................................................................................................. 101

5.9. Effect of pH levels on fresh and dry weight (g l-1) of adventitious root culture of Stevia

rebaudiana. .................................................................................................................................. 102

5.10. Various pH levels induced variations in total phenolics content (mg/g-DW) accumulation

in callus, cell suspension and adventitious root culture of Stevia rebaudiana. ............................ 105

5.11. Various pH levels induced variations in total flavonoids content (mg/g-DW)

accumulation in callus, cell suspension and adventitious root culture of Stevia

rebaudiana. ................................................................................................................................... 108

5.12. Various pH levels induced variations in antioxidant activities in callus, cell suspension

and adventitious root culture of Stevia rebaudiana. .................................................................... 111







5.16. Effect of various pH levels on stevioside, rebaudioside and dulcoside contents in callus

culture of Stevia rebaudiana. ........................................................................................................ 118

5.17. Effect of various pH levels on stevioside, rebaudioside and dulcoside contents in cell

suspension culture of Stevia rebaudiana. .................................................................................... 119

5.18. Effect of various pH levels on stevioside, rebaudioside and dulcoside contents in

adventitious root culture of Stevia rebaudiana. ............................................................................ 120

6.1. Effect of inoculum size (a) 0.5 g, (b) 1.0 g, (c) 1.5 g and (d) 2.0 g on proliferation of

callus cultures of Stevia rebaudiana. ........................................................................................... 136

6.2. Effect of inoculum size (a) 0.5 g, (b) 1.0 g, (c) 1.5 g and (d) 2.0 g on establishment of cell

suspension cultures of Stevia rebaudiana. ................................................................................... 137

iv

6.3. Effect of inoculum size (a) 0.5 g, (b) 1.0 g, (c) 1.5 g and (d) 2.0 g on establishment of

adventitious root cultures of Stevia rebaudiana. ......................................................................... 138

6.4. Effect of inoculum size on biomass accumulation during growth kinetics of callus


6.5. Effect of inoculum size on biomass accumulation during growth kinetics of cell


6.6. Effect of inoculum size on biomass accumulation during growth kinetics of adventitious

root cultures of Stevia rebaudiana.. .............................................................................................. 140

6.7. Effect of inoculum size on fresh and dry weight (g l-1) of callus culture of Stevia

rebaudiana. ................................................................................................................................... 142

6.8. Effect of sucrose induce osmotic stress condition on fresh and dry weight (g l-1) of cell

suspension culture of Stevia rebaudiana. .................................................................................... 142

6.9. Effect of sucrose induce osmotic stress condition on fresh and dry weight (gl-1) of

adventitious root culture of Stevia rebaudiana. ........................................................................... 143

6.10. Effect of inoculum size on accumulation of total phenolics content (mg/g-DW) in callus,

cell suspension and adventitious root culture of Stevia rebaudiana. ........................................... 146

6.11. Effect of inoculum size on accumulation of total flavonoids content (mg/g-DW) in callus,

cell suspension and adventitious root culture of Stevia rebaudiana. ............................................ 149

6.12. Effect of inoculum size on antioxidant activities (%) in callus, cell suspension and

adventitious root culture of Stevia rebaudiana. ........................................................................... 152







6.16. Effect of inoculum size on stevioside, rebaudioside and dulcoside contents in callus

culture of Stevia rebaudiana. ........................................................................................................ 160

6.17. Effect of inoculum size on stevioside, rebaudioside and dulcoside contents in cell

suspension culture of Stevia rebaudiana. ..................................................................................... 161

6.18. Effect of inoculum size on stevioside, rebaudioside and dulcoside contents in adventitious

root culture of Stevia rebaudiana. ................................................................................................ 162

v

LIST OF ABBREVIATIONS

2, 4-D 2, 4-dichlorophenoxy acetic acid

BA 6-benzyle adenine

BAP Benzyl aminopurine

CAT Catalase

CF Conversion factor

CRD Completely randomized design

DPPH 2, 2-diphenyl-1-picrylhydrazyl

DRSA DPPH-radical scavenging activity

DW Dry weight

FW Fresh weight

g l-1 Gram per litre

GAE Gallic acid equivalents

HPLC High performance liquid chromatography

IAA Indole 3-acetic acid

IBA Indole 3-butyric acid

Kn Kinetin

mg l-1 Milligram per litre

MS Murashige and Skoog

NAA Naphthalene acetic acid

PGRs Plant growth regulators

POD Peroxide dismutase

PTC Plant tissue culture

RE Rutin equivalent

ROS Reactive oxygen species

RPA Reducing power assay

SE Standard errors

SGs Steviol glycosides

SOD Super oxide dismutase

Stevia rebaudiana S. rebaudiana

TAC Total antioxidant capacity

TFC Total flavonoids content

TPC Total phenolics content

UV Ultra violet

vi

ACKNOWLEDGMENTS

I feel an honor to express cordial gratitude to my Advisor Prof. Dr. Abdur Rab for

his guidance, sincere cooperation, execution and subsequent completion of this work, I

must say his moral support at every critical moment, during this arduous business, was

instrumental, in egging me on, to surge and forward complete this long overdue task. It is

hard to find words of appropriate dimensions to express gratitude to my worthy advisor.

In short, he is the exact translation of my parent’s prayers.

I would feel incomplete if I do not mention the consistent support of Prof. Dr.

Noor-ul-Amin, Chairman Department of Horticulture, The University of Agriculture

Peshawar for his keen interest, useful suggestions, consistent encouragement, and

friendly behavior throughout the course of study. I also express heartfelt and highly

indebted gratitude to Prof. Dr. Razi Uddin, Department of Plant Breeding and Genetics,

The University of Agriculture Peshawar for assisting throughout my research work. I

would like to extend my thanks to Dr. Zafar Iqbal and Muhammad Nauman for his

sincere support in chemical analysis throughout my research work.

I also would like to thank my co-supervisor, Dr. Nisar Ahmad for the patient

guidance, encouragement and advice, he has provided throughout my time as his student.

I have been extremely lucky to have a supervisor who cared so much about my work, and

who responded to my questions and queries so promptly. I would also like to thank all the

members of staff especially Prof. Dr. Abdul Mateen Khattak, Dr. Gohar Ayub and Dr.

Muhammad Sajid for their continued support and encouragement.

I sincerely and cordially pay humble and heartedly thanks to my affectionate

parents, brothers and sisters for their moral and financial support and encouraging

attitude throughout my studies. Heartfelt thanks are also extended to Supdt. Arshad

Parvez, Mr. Imran Ullah and Lab. Assistants, Fazal Mahmood, Ashraf Ali, Faizan

Mehmood and Sami Ullah for their kind support.

Last but not the least, I consecrate my sincere thanks to my loving friends and in

particular my dear students for their worthy support, sharing concerns and floating ideas

during my research work.

NAVEED AHMAD

vii


ROOT CULTURES FOR THE PRODUCTION OF ACTIVE COMPONENTS

IN STEVIA REBAUDIANA

Naveed Ahmad and Abdur Rab

Department of Horticulture

Faculty of Crop Production Sciences

The University of Agriculture

Peshawar-Pakistan

January, 2017

ABSTRACT

Stevia rebaudiana Bertoni is an important plant known for antidiabetic steviol glycosides and

several other bioactive compounds. Owing to the importance of Stevia plant and demand of

natural secondary metabolites, the current research was conducted at Plant Tissue Culture Lab.,

Departmnet of Plant Breeding and Genetics, The University of Agriculture Peshawar, during the

year of 2014-15. The objectives of the study were to optimize/evaluate the effect of sucrose (05,

10, 15, 20, 25, 30, 35, 40, 45 and 50 g l-1), pH (5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0) and

inoculum sizes (0.5, 1.0, 1.5 and 2.0 g) on biomass yield and valuable secondary metabolites

accumulation in callus, cell and adventitious root cultures. Preliminary experiments were also

carried out to optimize the effect of various monochromatic spectral lights (white, blue, green,

yellow and red) on callus culture growth and secondary metabolites production. To check the

effect of sucrose, media pH and inoculum sizes on cultures productivity, research work was

conducted in Complete Randomized Design (CRD). Each culture was planned for a period of 30

days and 3 days intervals were kept to develop growth curves. The control light (16/8 hr) resulted

in the maximum callogenic response (92.73%) along with the accumulation of maximum biomass

(5.78 g l-1) during prolong log phase at 18th day of culture. Blue light was found the most effective

for the production of total phenolics content (TPC), total flavonoids content (TFC) along with total

antioxidant capicity (TAC) (102.32 µg/g-DW) (22.07 µg/g-DW) and (11.63 µg/g DW),

respectively. On the other hand, reducing power assay (RPA; 0.71 Fe (II) g -1 DW) and DPPH-

radical scavenging activity (DRSA; 80%) were considerably enhanced as a result of green and red

lights, respectively. Sucrose concentrations also affected the biomass accumulation, growth

kinetics and secondary metabolites production. The lag phase of 9 days was followed by log phase

till 27th day of culture was recorded in callus cultures on 05, 10, 15 and 20 g l-1 sucrose. The rest of

the cultures did not show a lag phase. All cultures, except a few displayed nonviability after 27

days of inoculation. The cultures initiated in media having 20, 25 and 30 g l-1 sucrose were found

in stationary phase after 27 days of log phase. Similarly, cell cultures grown on 05-30 g l-1 sucrose

concentrations displayed relatively shorter lag phase of 3 days as compared to 12 days lag phase in

cultures with 35-50 g l-1 sucrose concentrations. The Lag phase in each cell culture was preceded

by log phase till 18th day. Most cultures showed a stationery phases with or without decline phases.

However, growth curve of adventitious root cultures was characterized with direct log phase till

18th day. The lag phase of 15 days was observed in cultures developed in media having 5 and 10 g

l-1 sucrose, followed by very short log phase. The fresh and dry biomass of callus, cell suspension

and adventitious root cultures was significantly affected by sucrose concentrations. The highest

fresh and dry biomass (142.38 g l-1and 11.71 g l-1) in callus culture was with 40 g l-1 and 50 g l-1

sucrose, respectively. However, the maximum total phenolics content (TPC; 124.20 mg/g-DW),

total flavonoids content (TFC; 49.36 mg/g-DW), rebaudioside contents (6.56 mg/g-DW) and

antioxidant activity (92.82 %) in callus cultures was observed at sucrose concentration of 30 g l-1.

By contrast, the highest stevioside (42.34 mg/g-DW) and rebaudioside (22.67 mg/g-DW) contents

viii

were recorded in callus culture grown at 15 and 20 g l-1 sucrose, respectively. In cell suspension

culture, media having 20 g l-1 sucrose resulted in the maximum fresh (97.71 g l-1) and dry (8.57 g l-

1) but the highest TPC (139.20 mg/g-DW) and TFC (41.46 mg/g-DW) was at 40 g l-1 sucrose. The

highest antioxidant activity (83.87%) was observed at 30 g l-1 sucrose. While the stevioside content

(40.32 mg/g-DW) was the maximum on media supplemented with 10 g l-1 sucrose, the highest

rebaudioside (27.64 mg/g-DW) and dulcoside (6.43 mg/g-DW) contents were observed with 20 g

l-1 sucrose. In case of adventitious root culture, the maximum fresh (175.43 g l-1) and dry (11.14 g

l-1) biomass was accumulated in cultures having 50 g l-1 sucrose but the highest TPC (155.00

mg/g-DW) and TFC (94.78 mg/g-DW) were recorded with 30 g l-1 sucrose. While the highest

antioxidant activity (94.43 %) was recorded in culture, established in media augmented with 20 g l-

1 sucrose. The stevioside (73.97 mg/g-DW) and rebaudioside (24.57 mg/g-DW) content were the

highest in media containing 10 g l-1 sucrose. By contrast, the dulcoside content (12.24 mg/g-DW)

was the maximum at 40 g l-1 sucrose. It is suggested that sucrose concentration modulates biomass

and metabolites of interest in callus, cell suspension and adventitious root cultures of Stevia

rebaudiana. The media pH significantly influenced fresh and dry biomass of callus, cell

suspension and adventitious root cultures. The biomass accumulation revealed a short lag phase of

3 days in callus cultures on media pH 4.0, 5.9 and 6.0; and cell suspension culture on pH 5.6 and

5.7; while the adventitious roots culture expressed a lag phase of 3 days on media pH 5.5-6.0. The

log phase was followed by decline phases in callus and cell suspension cultures. However, root

growth was not restricted in cultures having pH 5.2 and 5.4 even after 27 days of the culture. pH

5.6 was optimized for the maximum fresh (130.57 g l-1) and dry biomass (12.10 g l-1) of callus

cultures. However, the highest TPC (43.38 mg/g-DW), TFC (37.55 mg/g-DW) and antioxidant

activities (87.68 %) in callus cultures were recorded on media pH 5.6. Media pH 5.6 was also

found optimum for the maximum stevioside (62.20 mg/g-DW) and rebaudioside (22.79 mg/g-

DW), while 5.1 for dulcoside (5.92 mg/g-DW) production in callus cultures. Similarly, the

maximum fresh and dry biomass (85.81 g l-1; 8.84 g l-1) of cell suspensions were observed on

media pH 5.6 and 5.5, respectively. The highest TPC (72.13 mg/g-DW), TFC (57.32 mg/g-DW),

DRSA (93.99%), rebaudioside (7.01 mg/g-DW) and dulcoside (4.72 mg/g-DW) contents were

observed in cell suspension cultures established in media having pH 5.8. However, stevioside

contents were induced to their maximal level (41.47 mg/g-DW) at pH level 5.2 in cell culture. In

contrast, the highest fresh (112.86 g l-1) and dry (8.29 g l-1) biomass were accumulated in

adventitious root culture on media pH 6.0. The maximum TPC (70.06 mg/g-DW), TFC (50.19

mg/g-DW), DRSA (92.67 %) and dulcoside contents (2.57 mg/g-DW) in adventitious root cultures

were recorded on media pH 5.8. However, the highest stevioside (79.48 mg/g-DW) and

rebaudioside (13.10 mg/g-DW) contents in adventitious root cultures were accumulated at 5.1

media pH. Various inoculum sizes also had significant influence on growth kinetics, biomass and

secondary metabolites production in callus, cell suspension and adventitious root cultures.

Relatively short lag phase of 3 days in callus cultures, while elongated lag phases from day 3rd to

12th day of the cultures developed from inoculum sizes (0.5-2.0g) was recorded. However,

adventitious root cultures did not display lag phases. An increase in biomass with elongated log

phases from day 3rd to 27th day of the culture was observed in callus cultures. Among all inoculum

sizes, 2.0 g started sudden increased in biomass accumulation up to 15 days and increments in

growth was further continued till 27th day of culture. Log phase was followed by sudden decline

phase without having any stationery phase in all cultures. Similarly, the highest fresh and dry

biomass (112.29 g l-1; 7.71 g l-1) in callus cultures was observed at 2.0 g inoculum. However,

cultures developed from smaller inoculum (0.5 g) resulted the maximum TPC (28.54 mg/g-DW),

TFC (24.78 mg/g-DW), DRSA (77.57 %), stevioside (43.89 mg/g-DW) and rebaudioside (36.54

mg/g-DW) contents in callus cultures, while the highest dulcoside contents (2.57 mg/g-DW) were

observed at 1.0 g inoculum. On the other hand, Cell suspension also accumulated the maximum

fresh (102.71 g l-1) and dry biomass (5.38 g l-1) at 1.5 g inoculum. Moreover, the highest TPC

(45.36 mg/g-DW), TFC (36.50 mg/g-DW), stevioside (59.89 mg/g-DW), rebaudioside (24.41

ix

mg/g-DW) and dulcoside (1.85 mg/g-DW) contents were found in cell cultures established from

0.5 g inoculum. However, the maximum DRSA (78.30%) was found in cell cultures having initial

inoculum size 2.0 g. Similarly, adventitious root cultures accumulated the maximum fresh biomass

(106.86 g l-1), dry biomass (5.05 g l-1) and dulcoside contents (0.71 mg/g-DW) at 1.5 g inoculum.

Inoculum size 2.0 g was optimized for the maximum TPC (41.46 mg/g-DW), TFC (33.44 mg/g-

DW) and DRSA (98.82 %). However, stevioside (64.75 mg/g-DW) and rebaudioside (29.67 mg/g-

DW) contents were significantly increased to their maximal level using initial inoculum size (1.0

g). Herein, we concluded that the utilization of various colored spectral lights, concentrations, pH

levels and inoculum sizes are promising strategies for enhanced biomass yield and secondary

metabolites production in callus, cell suspension and adventitious root cultures of Stevia

rebaudiana.

1

CHAPTER-I

INTRODUCTION

Stevia (Stevia rebaudiana Bertoni) is a potent medicinal plant of the family Asteraceae

that is, mainly, grown in tropical and subtropical regions of the world (Sreedhar et al.,

2008; Yadav et al., 2011). Stevia rebaudiana (S. rebaudiana) originated from Paraguay

and Brazil, but is cultivated almost round the world as a natural alternative to

commercially available sugar (Ahmad et al., 2011). The other members of the family

Asteraceae having sweetness potential within this genus, including S. lemmonii, S.

dianthoidea, S. viscida, S. bertholdii, S. crenata, S. micrantha, S. serrata, S. enigmatica,

S. anisostemma and S. eupatoria (Carakostas et al., 2008). S. rebaudiana ranks 1st in

sweetness in the genus Stevia (Yadav et al., 2011).

S. rebaudiana was botanically classified for the first time by a Swiss Botanist, Moises

Santiago Bertoni in early 1899. Even before classification, he had already explored the

importance of its sweetness and other health properties (Barriocanal et al., 2008). In

addition, he had also made efforts to extract the water soluble sweet contents of Stevia.

However, Dr. Rebaudi, a Paraguayan Chemist, for the first time identified and isolated

two important sweetening agents (stevioside and rebaudioside) from Stevia leaves,

stevioside being the most efficient and attractive one. In light of his significant

contributions regarding Stevia plant, it was named as Stevia rebaudiana Bertoni (Gupta

et al., 2013).

Stevia plant is a perennial herb but is grown as an annual plant especially in areas where

temperature is low. For optimum vegetative and reproductive growth, it requires warm

climate with temperature ranging from 15 to 30°C and sufficient rainfall. Stevia plant

performs better in well aerated moist soils. The plant height may reach up to 100 cm

(Chan et al., 2000). Typically, Stevia is a subtropical plant (Madan et al., 2010) with a

critical photoperiod of 12-13 hours. However, the sensitivity to photoperiod may vary

extensively (Valio and Rocha, 1966; Zaidan et al., 1980).

2

Stevia is diploid having 11 pairs of chromosomes (Frederico et al., 1996). Self-

incompatibility has been reported based on daillel cross with eight parental plants.

However, there might be 0-0.5% selfing and 0.7-68.7% outcrossing (Katayama et al.,

1976).

The Stevia is given different names such as candy leaf, sweet leaf, honey leaf and honey

yerba, due to its sweetness (Madan et al., 2010). The Stevia plant is cultivated in various

countries including Indonesia, China, Japan, Korea, Mexico, Malaysia, United Kingdom

and South America due to its demand in food and pharmaceutical industries (Ahmad et

al., 2011; Dey et al., 2013). The importance of Stevia plant is due to its steviol glycosides

content that comprise of diterpene compounds such as steviosides, rebaudioside (Reb. A-

F) and dulcoside etc. (Ahmad et al., 2011; Reis et al., 2011; Mathur and Shekhawat,

2012). The steviol glycosides are known for their non-mutagenic, nontoxic, low caloric

properties; and stability during storage and high temperature (Liu et al., 2010). The

steviol glycosides are 300 times sweeter than commercial sugar (Bondarev and

Reshetnyak, 2003; Dey et al., 2013) and may, therefore, offer as a natural source of sugar

to patients on non-carbohydrate diets. It controls blood pressure and sucrose level in mild

hypertension and Type II diabetic patients (Hsieh et al., 2003).

The stevioside, being non-caloric in nature, is highly recommended substitute of sugar in

diabetic, hypoglycemic obesity, cardiovascular diseases and dental problems (Lailerd et

al., 2004). The Stevia extracts also has antimicrobial effects against a variety of

pathogens (Tomita et al., 1997). Besides its medicinal values, steviosides containing

compounds have been utilized in food industries as additives for various purposes such as

bakery products, ice creams, juices, cold drinks, tee, coffee, and several other beverages

(Komissarenko and Bublik, 1994). The S. rebaudiana plant also accumulates several

other economically and chemically valuable phytochemicals such as phenolics, alkaloids,

essential oils, vitamins, sterols and hydroxycinamic acids in addition to its sweet tasting

stevioside contents (Komissarenko et al., 1994). The accumulation of these phyto-

chemicals has been reported in leaves, stem, flower, seed and even in roots (Bondarev et

al., 2003).

3

The S. rebaudiana plant can be propagated through sexual and asexual means. The sexual

propagation, through seed has not been successful due to poor germination that is

attributed to low fertility or self-incompatibility (Goettemoeller and Ching, 1999; Maiti

and Purohit, 2008). Besides, poor germination, sexual propagation is not desirable

because the seedlings produced are not true to type that leads to great variation in growth

and important qualitative attributes (Tamura et al., 1984; Nakamura and Tamura, 1985).

The S. rebaudiana plant is, generally, propagated through cuttings, which require large

number of mother plants, optimum seasonal conditions, more space and longer duration

to produce Stevia plants in bulk (Sakaguchi and Kan, 1982; Sivaram and Mukundan,

2003).

Due to the increased demand for steviol glycosides and the problems associated with

conventional propagation of S. rebaudiana, the use of in vitro culture could be a viable

alternative. Such biotechnological approaches like micropropagation, cell suspension

culture, anther culture, pollen culture, adventitious root culture, and even germplasm

conservation can minimize such problems (Borroto et al., 2008). Such approaches can be

used for mass population and biosynthesis of important secondary metabolites in a short

span of time without any seasonal limitation (Ahmad et al., 2014). There are numerous

examples of tissue culture, based on micropropagation and phytochemical productions

from S. rebaudiana leaves, but very limited information is available on optimization of

callus and establishment of cell and adventitious root cultures of S. rebaudiana

(Yamazaki and Flores, 1991).

Callus, an unorganized mass of cells, has the potential to synthesize important secondary

metabolites in the same way as by the intact organs in plant. It is an essential material for

plant cell culture. Cell suspension culture can be developed by dispersing cells of friable

callus in an agitated liquid media. These dispersed cells are totipotent in nature and are

able to carry all the characteristics associated with the intact plant organs (Allan, 1996).

Rate of cell multiplication in suspension culture is more rapid than in callus culture,

offering more opportunities for mass production of totipotent cells (Philips et al., 1995)

and biosynthesis of secondary metabolites in larger quantities (Phillipson, 1990). Hence,

it is a reliable method for rapid cell division, biomass and secondary metabolites

4

accumulation (Elio et al., 2004; Dixon et al., 2005). Cell suspension culture is, therefore,

considered as one of the advanced biotechnological approaches for enhanced

accumulation of valuable secondary metabolites, which are either difficult to synthesize

or developed in limited amounts in wild species (Kolewe et al., 2008).

Among various plant tissue culture techniques, adventitious root culture is another

prominent approach for biomass production and accumulation of medicinally important

bioactive compounds (Wang et al., 2013). The adventitious roots develop from unusual

non-embryonic points such as leaf, stem, and shoots (Esau, 1977; Barlow, 1986). The

adventitious root culture is not only an attractive method for accumulation of biomass

and secondary metabolites on large scale but also has exceeded expectations in plant

propagation industry (Ford et al., 2001). This approach allows rapid culture and

biosynthesis of metabolites of interest in large quantity in a natural way (Murthy et al.,

2008). Thus, the adventitious root culture may help in accumulating comparable amount

or even higher quantities of secondary metabolites in a short span of time and round the

year than wild or in vivo cultivated plants (Giri and Narasu, 2000). Therefore, such

techniques provide unique opportunities for the biosynthesis of pharmaceutically

important natural metabolites on large scale without field cultivation. Furthermore, it has

been also an efficient tool for asexual propagation and germplasm conservation (Borroto

et al., 2008; Bernabe- Antonio et al., 2010).

Keeping in view the importance of S. rebaudiana, as medicinal plant in food and

pharmaceutical industries; and the difficulties in sexual and asexual propagation, the

present research was initiated to attempt the in vitro cultures of S. rebaudiana. These

modern in vitro culture approaches are not only cost effective with respect to time, space

and land cultivation but also overcome the drawbacks of conventional approaches. The

current study could, thus, be a milestone in this regard and will help to meet demands of

food and pharmaceutical industries for steviol glycosides and several other desirable

secondary metabolites in S. rebaudiana.

5

Objectives:

Establishment of callus culture from leaf explant

Optimization of cell suspension culture from callus cultures

Production of steviosides thorough cell suspension culture

Optimization of adventitious root culture for steviosides production

Determination of other secondary metabolites production

6

CHAPTER-II

REVIEW OF LITERATURE

The medicinal plants have been utilized in a variety of pharmaceutical, cosmetics, food,

and dietary products. Majority of these pharmaceutical products are still in used and

regarded as traditional and safe therapy. No valuable alternatives have been found to have

the efficacy and pharmacological properties like the natural pharmaceuticals (Balandrin

and Klocke, 1988). Many important and active constituents of medicinal plants have been

chemically identified and used as isolated compound for the treatment of various kinds of

ailments. Such chemicals include morphine (pain killer), scopolamine (travel sickness),

caffeine (stimulant), berberine (psoriasis), capsaicin (rheumatic pains), quinine

(antimalarial), papaverine (phosphordiesterase inhibitor), reserpine (antihypertensive),

pilocarpine (glaucoma), codeine (antitussive), galanthamine (acetylecholine esterase

inhibitor), ajmaline (antirrhythmic), yohimbine (aphrodisiac) and various types of cardiac

glycosides (heart insufficiency) etc. (Wink et al., 2005).

The pharmaceutical industries are in constant search of such potent plants that are rich in

compounds having antimicrobial, antibiotic, antioxidant, anticarcinogenic, and antiallergic

characteristics. The identification and isolation of such compounds will enable the

researchers and industries to design innovative chemical models and novel pharmaceutical

products in future (Rajeswara et al., 2012). However, the demand for food and other

biological products with the increasing population of the world exerts an extreme pressure

on the available agricultural land. Therefore, it is important to manage the available

cultivable land effectively and find out the novel means for the production of these

biological products. Thus, the utilization of modern approaches to meet the increasing

demand for food and other pharmaceutical products is of prime importance (Rao and

Ravishankar, 2002).

In addition, conservation of the available plant genetic resources is important because of

the indiscriminate usage and exploitation of the available resources for food and drugs

which are the root causes of extinction of plant species (Driscoll and Lindenmayer,

2012). The biotechnological tools are prerequisites in biodiversity conservation for the

7

sustainability of these natural resources. Furthermore, the evaluation of the valuable

phytochemicals in recent times increased the interest of the scientist to alter the metabolic

pathways of the plants for maximum production of these secondary metabolites (Bohidar

et al., 2013).

Biotechnological approaches have been proven as efficient alternatives to traditional

cultivation for the production of secondary metabolites. Plant tissue culture is the

foundation of biotechnological approaches such as micro propagation, transgenic

engineering, germplasm conservation, secondary metabolite production, micro grafting;

and development of somatic clones, embryos, and hybrids (Lynch, 1999; Yadav et al.,

2012). Additionally, the plant tissue culture techniques act as tools to conserve the

endangered plants (Lynch, 1999; Yadav et al., 2012). Similarly, the callus, plant cell and

adventitious root cultures are potent alternatives to traditional agriculture for the

synthesis of commercially important secondary compounds without any limitation of

natural harvest and high cost association (Wilson and Roberts, 2012).

Plant tissue culture research: An overview

Plant tissue culture refers to the techniques of culturing of plant cells, tissues, or organ in

a chemically defined nutritive solution (medium) under sterile and controlled

environment. Historically, the development of this technology is inherent with the

discovery of plant cell, and cell theory postulated by Schleiden (1838) and Schwann

(1839), who implicitly hypothesized that each cell has the potential of autonomy and is

totipotent, popularly known as totipotency. Haberlandt (1902), a German Botanist, for the

first time practically employed the concept of totipotency by growing cells in a sterile in

vitro condition. The plant tissue culture research was further advanced with the discovery

and purification of plant growth regulators that enabled the researchers to control the

growth and developmental process of plant cell, tissue, and organs. All the credits were

named by the relentless efforts of White (1937, 1943) and Bergmann (1960), who

cultured tobacco hybrid for callus and shoot induction. Skoog (1944) further designed

experiments to confirm and extend the findings of White. Furthermore, the efforts of

8

Skoog and Miller (1957) made it possible to identify the role of chemical interaction of

components in controlling growth and developmental processes.

In the last three decades, tissue culture has been employed for micro propagation,

germplasm conservation, somatic embryogenesis, organogenesis, cell suspension culture,

adventitious root culture and synthetic seed development of commercially important

crops like sugar cane, potato, forest plants, medicinal plants and other horticultural plants

(Akin-Idowu et al., 2009; Abul-Soad and Mahdi, 2010; Verma et al., 2012). Plant tissue

culture is flourishing with multi-directional growth and good economic turnover. A

number of commercially important crops including banana, strawberries, potato, olive,

sugarcane, and various medicinal plants have been propagated through plant tissue

culture (Garcia-Gonzales et al., 2010; Hossain et al., 2013). Hence, it is a significant tool

for the mass propagation of several crops and plant secondary metabolites production,

and has become a powerful technique at industrial level (Singh and Shetty, 2011).

Micropropagation of medicinal plants

Micro propagation is an efficient approach for the clonal propagation of large number of

elite plants irrespective of the season and increased production of valuable secondary

metabolites (Hossain et al., 2013). The clonal propagation of numerous commercially

valuable plants such as fruits, vegetables, medicinal and forest plants has been

successfully attempted through micro propagation (Rout et al., 2006; Cordeiro et al.,

2012; Nguyen et al., 2013). Besides clonal propagation, genetic modifications for

enhanced secondary metabolites are also possible among the clones by causing

somaclonal variation. Somaclonal variation may be genetic or epigenetic depending on

the source of mutagenesis (Rout et al., 2000). The plants can be propagated directly or

indirectly from explant by micro propagation depending upon the interest of the

researcher. However, for clonal propagation, the most reliable method is meristem culture

without callus induction, resulting numerous shoots (Murashige, 1974), having unique

characteristics to scale up (Takayama and Misawa, 1981).

9

While, the addition of plant growth regulators with optimum concentrations to chemically

defined media enables the scientists to control the growth of in vitro plantlets, however,

the optimum concentration of these regulators are genotype dependent (Jana and

Shekhawat, 2010). Therefore, it is important to optimize the plant growth regulators, their

concentrations, proper explant, suitable inoculums size, media composition, and physical

factors of the growth chamber (Narayanswamy, 1977; Guo et al., 2012; Nguyen et al.,

2013).

The Stevia rebaudiana plant is recently domesticated in various countries for its naturally

sweet constituents and other secondary metabolite production. In Asian countries, Japan

was the first to trade the steviosides as a natural zero caloric sweetening agent to food and

pharmaceutical industries in purified form. Several other Asian countries like China,

Malaysia, Thailand, and South Korea adopted Stevia rebaudiana cultivation. The Stevia

rebaudiana is also grown in different states of America and European countries

(Midmore and Rank, 2001).

Due to the commercial importance and increasing demand of Stevia rebaudiana crop for

its stevioside and other secondary compounds, micropropagation is a viable approach for

the large-scale production (Yadav et al., 2011). There has been considerable work on

micro-propagation of this crop. Sivaram and Mukundan (2003) used leaf and stem as

explant for callus induction. Auxiliary shoot development and proliferation has been

reported by using apical and nodal segments as explant (Patel and Shah, 2009; Sairkar et

al., 2009; Kalpana et al., 2010, Singh et al., 2012). Micro-propagation of this plant has

also been successfully attempted by inoculated leaf, nodal and intermodal portions

(Uddin et al., 2006).

Different plant growth regulators (PGRs) for callogenesis, shoot, root induction are used

for micro propagation of Stevia rebaudiana. According to Ahmad et al. (2011), who used

flower as an explant, quality callus was developed with addition of 2 mg l-1 BAP and 2,

4-D each in MS-Medium. However, the best shooting response was observed with callus

culture in medium having 2 mg l-1 IBA. By contrast, Aamir et al. (2010) reported the

superior shooting response on callus incubated in medium supplemented with 1 mg l-1

10

BAP. Similarly, work has been done by Ali et al. (2010) for investigating callogenic

response of Stevia rebaudiana leaf, nodal and intermodal explants to various

concentrations of 2, 4-D. Among all concentrations, leaf explants were found superior for

callus induction on medium supplemented with 2, 4-D (3 mg l-1) as compared to callus

induced from nodal and intermodal explants inoculated in 3 mg l-1 (2, 4-D) and 1 mg l-1

(BAP) supplemented medium. Moreover, high callogenic and regeneration responses of

Stevia rebaudiana were found with combination of both cytokinins (BAP) and auxin

(NAA) at 2 mg l-1 each in MS-Medium (Patel and Shah, 2009). Successful results are

also obtained with addition of BAP (1 mg l-1) and NAA (1.5 mg l-1) to MS media for

shoot multiplication and root induction, respectively of Stevia plant (Hossain et al.,

2008). Ahmed et al. (2007) reported that the addition of Kn (0.5 mg l-1) and BAP (1.5 mg

l-1) both in combination and IBA alone were more effective for shoot proliferation and in

vitro rooting, respectively.

Plant cell suspension culture

In vitro propagation of plant cell and tissue has emerged as an important technique for the

synthesis of commercially valuable bioactive compounds (Mustafa et al., 2011). Early

efforts were based on the analogy of plant cells to microbial population in culture.

Specialized units were designed to propagate considerable amount of cells in dissociated

form (Short et al., 1969; Wilson et al., 1971). Further research reports claimed the

cloning of cells in suspension (Zenk et al., 1977) or callus culture (Akasu et al., 1976)

resulting in higher bioactive compounds than the entire individual plant that could be

further increased substantially (Zenk, 1978). Further investigations of the Staba (1982)

encouraged the idea of cell suspension culture for the production of biologically active

compounds, who reported that more than 30 compounds synthesized in suspension that

were greater or equal in amount to the whole plant. Shortly afterwards, the National

Aeronautics and Space Administration (NASA) started supporting research in the field of

plant cell cultures for regenerative life support systems (Sajc et al., 2000). In parallel to

these findings, Japan started a collaborative effort among the industries and researchers

group for commercial application of this technology for mass production of plant based

bio agents. In early efforts, several companies in Japan exploited cell suspension culture

11

for proliferation of tobacco cells as a raw material for cigarette (Japan Tobacco Inc.) and

Panax ginseng cells for pharmaceutical purpose (Meiji Seika). Nitto Denko Company in

Japan also used this technology for obtaining ginseng cells on commercial level.

Likewise, further efforts by other groups (Ajinomoto, Nippon Shin Yaku) attempted to

accumulate alkaloids, steroids and many other compounds in high concentration in

suspension culture (Misawa, 1994). The techniques of cell culture were also adopted by

another firm (Phyton Gesellschaft fur Bioechnik mbH) in Japan as an alternate source for

taxol and taxenes accumulation in culture in higher levels than extracted from stem bark

(Dornenburg and Knorr, 1994). Hence, the techniques of cell culture have been widely

used in basic researches to improve exploitation at industrial level. It has been also

exploited for the production of several other valuable medicinal constituents including

shikonin (Lin and Wu, 2002), berberine (Vanisree et al., 2004), camptothecin (Lorence et

al., 2004) and hypericins (Kirakosyan et al., 2004) from Lithospermum erythrorhizon,

Coptis japonica, Camptotheca acuminate and Hypericum perforatum plants, respectively.

Biotechnological approaches, like cell suspension culture for production of different

secondary metabolites offer many advantages over conventional propagation such as

provision of controlled aseptic conditions (Rao and Ravishankar, 2002) that ensure

consistent supply of such products on large scale with uniform quality, independent of

any topographical location (Vanisree et al., 2004). In spite of all preventive

measurements, chances of natural catastrophes and pathogen attacks might be there. It is

therefore, important to optimize economically viable protocols for convenient production

of commercially important biologically active compounds from culture cells (Aijaz et al.,

2011).

Cell suspension culture productivity

Many strategies have been made to optimize the protocols for accumulating important

bioactive compounds on commercial level (Wilson and Roberts, 2012). Culture

productivity is crucial for the practical implementation of cell suspension culture

techniques for synthesizing secondary constituents. However, careful selection of cells

and conducive cultural environment is essential for the production of valuable

metabolites in higher concentration (Tan et al., 2010).

12

Plant secondary metabolites are organ specific in majority of the plants. Therefore, the

choice of explant containing substantial amount of the desired compounds for callus

culture and to obtain quality cell culture containing high quantity of metabolites is

important. Emphasis is given to alter and stimulate the biosynthetic pathways of

inoculated cells for commercial exploitation of metabolites (Roberts, 2007). In this

regard, several approaches have been adopted to improve the accumulation of such

compounds in culture (Namdeo, 2007). Providing stress condition by manipulating

physical factors of the culture environment (Zare et al., 2010), changes in medium

composition (Karwasara and Dixit, 2012; Praveen and Murthy, 2013), careful selection of

desirable cell for optimum culture productivity (Tan et al., 2010), introducing the

precursor (Sinlaparaya et al., 2007) or elicitor (Huang et al., 2013) are some of the

common techniques to enhance the production of metabolites of interest. Similarly, a

number of physical and chemical agents like composition of the medium, combination

and concentration of certain constituents and type of plant growth regulators, medium

pH, aeration, photoperiod, light intensity are found effective in stimulation for the

accumulation of bioactive compounds (Lee and Shuler, 2000; Nagella and Murthy, 2010;

Trejo-Espino et al., 2011).

The manipulation of physical and chemical parameters in culture is the most fundamental

approaches to stimulate the biosynthetic pathways and increasing the concentration of

useful metabolites in cultured cells. Previously, cell culture of various plants has been

established for production of valuable compounds in high quantity and quality. For

example, cell culture of Coleus bluemei, Nicotiana tabacum, Coptis japonica, Echium

italicum, Nothapodytes nimmoniana and Panax ginseng have been established for

rosmarinic acid, ubiquinone-10, berberin, shikonin, camptothresin and ginsenosides

production, respectively (Ulbrich et al., 1985; Fontanel and Tabata, 1987; Matsubara et

al., 1989; Zare et al., 2010; Karwasara and Dixit, 2012; Huang et al., 2013).

However, these biotechnological approaches have not been fully investigated to optimize

callus and suspension culture of Stevia rebaudiana for diterpenoid glycoside and other

secondary metabolites production. In addition, the available reports are highly

contradictory. The findings of Nabeta et al. (1976) and Suzuki et al. (1976) did not

13

confirm the accumulation of glycosides in cultured cells of Stevia rebaudiana both in

clustered (callus) and detached form (suspension). Whereas, the results of Striedner et al.

(1991) confirmed the presence of the highest concentration (0.4%) of cell dry weight, after

incubation period of 49 days in medium containing 100 g l-1 sucrose. Afterwards, the

research findings of Bondarev et al. (2001) also supported the availability of glycosides in

suspension culture, with the maximum (103 mg. g dry weight-1) steviosides on 14th day of

inoculation at the last stage of exponential phase. However, there is limited information

regarding culture establishment and maintenance for stevioside, rebaudioside, dulcoside

and other secondary metabolites accumulation. The current study is an attempt to develop

protocols for the establishment and maintenance of cell suspension culture of Stevia

rebaudiana with optimized concentrations of PGRs and other media components for

substantial cell growth and metabolites accumulation.

Adventitious root culture

The adventitious roots develop from the non-embryonic points or from the aerial portion

of the plant. The adventitious roots respond to chemically defined medium having

specific phytohormones and have potential of important plant base bioactive compounds

accumulation in in vitro conditions (Murthy et al., 2008). Adventitious roots are true to

type of their parental plants and grow at optimal rate as well as synthesize important

metabolites in in vitro conditions (Dubrovsky and Rost, 2003; Goel et al., 2009). Thus,

this approach has been applied for the production of pharmaceutically valuable

compounds like anthraquinone obtained from Morinda citrifolia and Rubia tinctorum

plants (Sato et al., 1997; Baque et al., 2010). Similarly, Kevers et al. (1999) accumulated

higher amount of ginsenosides than obtained from the whole Panax ginseng plant using

adventitious root culture. The quantity and quality of such compounds depend on the

plant species, stage, plant part, environment, and nutritional status of the soil. Besides this

obstruction, the imprudent and continuous harvest of such plants for exploitation of

metabolites causes a serious problem of extinction of the plants from their natural habitat

(Abdullah et al., 2000; Chattopadhyay et al., 2002).

14

It has been reported that adventitious root culture is more efficient than the cell

suspension culture for the production of important metabolites (Vijaya et al., 2010) and

adventitious root culture is regarded as the most potent and efficient method for the

production of plant secondary metabolites (Choi et al., 2000). Saifullah et al. (2008) also

reported enhanced biomass growth and production of these bioactive products in

adventitious root culture. Yet, it is essential to optimize the protocol (Holobiuc and

Blindu, 2006; Cui et al., 2010).

Role of auxin in adventitious root culture development

The auxin is an important phytohormone that regulates callus induction, cell

differentiation, proliferation and accumulation of bioactive compounds in in vitro culture.

Generally, auxin type, combinations, and concentrations are critical for morphogenetic

responses and cell growth promotion or inhibition. Several synthetic auxins such as 2, 4-

D, NAA, IBA have been used in plant tissue culture media for various growth and

morphogenetic responses (Hasan et al., 2014). It has been reported that auxin controls the

development and proliferation of adventitious roots, either exogenic or endogenic in

nature (Bellamine et al., 1998; Baque et al., 2009) and the application of anti-auxin

compounds results in complete inhibition of root growth and development (Blakesley

and Chaldecott, 1997; Sabatini et al., 1999). The responses of adventitious roots growth

and development depend on specific auxin in culture medium and vary from species

(Praveen et al., 2009). Several plants including Psoralea coryfolia, Labisia pumila and

Echinacea angustifolia induced adventitious roots on addition of IBA to media rather

than IAA and NAA. By contrast, superior adventitious roots of Antirrhinum majus and

Helianthus annuus were induced with IAA supplementation to culture (Atkinson et al.,

1991; Vesperinas, 1998; Wu et al., 2007; Baskaran and Jayabalan, 2009; Hasan et al.,

2014). Similarly, significant induction of adventitious roots were caused by NAA in

Eurycoma longifolia (Hussein et al., 2012), Cornus mas (Thakur and Karnosky, 2007)

and Ulmus parvifolia (Durkovic and Bukovska, 2009). Comparative study of different

auxins supplementation to medium for adventitious root development of Centella asiatica

was also conducted by Ling et al. (2009) and reported that IBA is more efficient as it is

more stable with lower toxic level. Similarly, Qaddoury and Amss (2004) stated that IBA

15

is more efficient due to its unique quality of stability and higher potential of adventitious

roots stimulation and promotion than NAA and IAA. Moreover, the conjugation rate of

IBA is very low and has been widely used for adventitious root development of several

plants (Pyrus communis, Vitis vinifera, Malus pumila and Olea europaea) over a longer

period of time (Krisantini et al., 2006). However, Zolman et al. (2000) further described

that root induction and promotion vary due to various kinds of auxins in medium and are

highly dependent on differences in uptake and its metabolism. Since, roots are very

sensitive to auxin concentration, therefore, optimize level of each auxin is critical.

Addition of higher levels of auxins alters the natural regeneration pathways by inducing

degraded compounds in plants. As a result, inhibition of roots occurs at higher levels and

at lower levels roots are very responsive to initiation and development (Hussein et al.,

2012).

The role of physical factors on culture development and secondary metabolism

Plants are exposed to a variety of physical and chemical stresses, that adversely affect the

plant growth and development; and accumulation of bioactive compounds. Fluctuation in

temperature, photoperiod; salinity, acidity, alkalinity, water logging and drought

conditions are some of the common stresses (Ahmad et al., 2008; 2010a; 2010b) that

cause variations in physiological, morphological, biochemical and cellular features of the

plants (Ahmad et al., 2012). Thus, several physical and chemical factors such as

temperature, photoperiod, pH, humidity, sucrose concentrations and agitation have been

evaluated for plant secondary metabolites (Lee and Shuler, 2000; Nagella and Murthy,

2010; Trejo-Espino et al., 2011).

The role of pH in plant growth and secondary metabolites production

Plants in natural conditions are exposed to a diverse soil pH that needs to be optimized

for the maximum biomass and plant secondary metabolites production. Abbasi et al.

(2007a) reported that Echinacea species are widely adoptable to 5.9-8.0 soil pH levels.

However, Galambosi (2004) documented that Echinacea responded well in neutral pH.

Furthermore, Zheng et al. (2006) evaluated that Echinacea pallida favors lime rich soil

for best crop. Likewise, soil pH, media pH also has dominant role in plants growth,

16

development and metabolites production in in vitro culture. Like, Ahmadian et al. (2013)

reported that it is required to optimize the pH for plant morphogenesis. Similarly,

secondary metabolites production of the plant can also be enhanced by optimizing the pH

levels in in vitro culture (Eilert, 1987; Barz et al., 1988). The adjustment of desirable pH

level is critical for nutrients uptake, gelling of media and activities of phytohormone

(Hussain et al., 2012). So far, in majority of the protocols developed for in vitro

propagation, the pH is adjusted at 5.6-5.8 as a standard (Abbasi et al., 2007a). Similarly,

Hussain et al. (2012) emphasized on pH (5.4-5.8) adjustment for in vitro culture. Several

other researchers focused on the pH of cell and adventitious root culture. In this context,

Gorret et al. (2004) monitored the pH of Elaeis guineensis culture in bioreactor and a

significant decline in medium pH 5.6 to 5.4 was observed in initial 7 days of culture that

finally declined to 4.4 after 25 days. It is believed that nutrients uptake especially

ammonium results in the decline in pH at initial few days of culture (Do and Cormier,

1991; Lee and Shuler, 2000). Similarly, cell lysis in bioreactor culture acidifies the

medium and consequently affects the nutrients level (Ruffoni et al., 2009). Tautorus et al.

(1992) stated that adjusting pH level is an effective tool to control the growth stages of

culture. However, many experiments have been conducted without monitoring the pH of

the medium (Hahn et al., 1997; Lee and Shuler, 2000).

The role of chemical factors on culture development and secondary metabolism

The accumulation of secondary metabolites productions is also controlled by chemical

factors. Several chemical such as plant growth regulators, sucrose, nutrients and other

media components act as elicitors and influence culture growth and metabolites

production (Lee and Shuler, 2000; Trejo-Espino et al., 2011). Thus, the optimization of

media composition and physical environments are critical for culture productivity.

Several studies are available about the accumulation of valuable secondary compounds in

optimized in vitro cultures rather than intact plants. The production of different

metabolites such as, ubiquinone-10 (Fontanel and Tabata, 1987;), berberin (Ulbrich et al.,

1985), rosmarinic acid (Matsubara et al., 1989), shikonin (Zare et al., 2010) and

camptothesin (Karwasara and Dixit, 2012) in optimized in vitro culture of Nicotiana

17

tabacum, Coptis japonica, Coleus bluemei, Echium italicum and Nothapodytes

nimmoniana, respectively have been evaluated.

Sucrose induced osmotic stress

Sucrose is one of the readily available sources of carbon, energy and osmotic potential.

The energy generated from sucrose is utilized in all developmental processes (Gibson,

2000). Sucrose is known to influence gene expression, developmental signals and

improves plant immune system (Morkunas et al., 2005; Wind et al., 2010). Sucrose as an

osmotic agent, regulate the uptake of various components in culture medium and water

absorption (Ahmadian et al., 2013).

In vitro plants require continuous supply of carbohydrates for survival. Sucrose is the

vital source of carbohydrates utilized in plant tissue culture media. In both liquefied and

solidified types of media, 30 g l-1 sucrose is added (Murashige and Skoog, 1962). During

cell suspension culture of Catharanthus roseus, 5 days of lag phase was recorded in

which hydrolysis of sucrose took place (Smart et al., 1984). Ruffoni et al. (2009) also

investigated that most of the sucrose was exhausted in first week by suspension culture of

alfalfa. The sucrose concentration depends on the developmental stage of culture and

increasing sucrose concentration to 40 g l-1 in basal medium resulted 100 % rooting in

rose cultivars (Davies, 1980). Similarly, Hyndaman et al. (1982) also recorded the

maximum roots length of in vitro plants with higher sucrose concentration in the medium.

In Rosa species increased root length was recorded with increasing concentrations of

sucrose (Pati et al., 2005). Sucrose also has a dominant role in culture growth and

metabolites production and the accumulation of bioactive compounds is higher in culture

with optimized level of sucrose and other physical and chemical culture conditions

(Kittipongpatana et al., 1998; Putalun et al., 2006). Medium supplemented with 5 percent

sucrose developed a sound hairy root culture of Pueraria candollei with more iso-

flavonoid production but an inhibitory response was observed with increasing

concentration (Udomsuk et al., 2009). Media having 8% sucrose was considered optimal

for in vitro culture of two potato (Solanum tuberosum) cultivars, due to less duration of

tuber induction, with more quantity of micro tubers per explant (Aslam and Iqbal, 2010).

18

Polyphenol contents of different elite plant species were also significantly varied with

various concentrations of sucrose in in vitro culture (Ferri et al., 2011; Ali et al., 2013).

Importance of polyphenolics

The demand for plant based antioxidants has increased rapidly (Joo et al., 2010).

Polyphenols and flavonoids are leading antioxidants with high consistency and efficiency

among the plant based antioxidants. Due to high efficacy as natural antioxidants,

polyphenols represent the principal group among other plant based antioxidants like

ascorbic acid (Vitamin C), Vitamin E and carotenoids (Harborne, 2001). Cai et al. (2003)

extensively studied 112 medicinally important plant species for antioxidant activities and

the major antioxidant compounds like tannins, anthocyanin, flavonols and caffeic acids

were reported. In vitro culture has the potential to produce plant based phenolic

compounds using shikimic pathway through benzoic and carboxylic acids. At present,

several polyphenols including gallotannins, lignans, caffeic acids, tannins and stilbenes,

are derived from in vitro cultures of plants (Matkowski, 2006-2008). There has been an

inverse relation in lipid peroxidation, age linked diseases, cancer and cell aging with

flavonoids enrich diets and decrease of such diseases occur due to redox capacity of

flavonoids (Ferreira et al., 2010). Plants and in vitro culture adopts phenylalanine

pathways (PAL) for the synthesis of phenols and flavonoids. The most active biological

flavonoids include anthocyanin, proanthocyanidins and flavanols. Besides this, several

other biologically active flavonoids like catechins, anthocyanidins, flavones and

flavanons also exist (Matkowski et al., 2008).

Exploitation of in vitro cultures for antioxidants production

The oxidative breakdown of carbohydrates in respiration process provides energy for

various developmental processes. However, this biological combustion process also

results in production of harmful reactive oxygen species (ROS), causing damages to

lipids, proteins and DNA. As the number of the ROS increases, the body remains in

oxidative stress conditions (Dudonne et al., 2009). Generally, the body contains both

oxidants and antioxidants at proper proportionate. However, an imbalance in favor of

ROS causes oxidative stress resulting in chronic diseases (Halliwell, 1994). Therefore,

proper balance between oxidants and antioxidants is fundamental for running normal

19

biological system (Tiwari, 2001). Such chronic diseases can be minimized by utilization

of specific plants with plenty of antioxidant constituents (Halliwell, 2007). Phenolic

compounds are one of the most abundant antioxidants (Blokhina et al., 2003; Scalbert et

al., 2005) that may improve the defense mechanism at cellular level by protecting the

biomolecules from oxidative damages (Evans and Halliwell, 2001). Thus, naturally

occurring antioxidants are being used as an alternative to synthetic materials that could

have toxic effect (Kahl and Kappus, 1993). According to Dudonne et al. (2009) naturally

occurring antioxidants are effective as pharmaceutical products in controlling cellular

damages in the body. Most of medicinal plants are rich sources of natural antioxidants,

required to scavenge free radicals. Since, the free radicals are detrimental to human

health causing cardiac problems, eyesight diseases, inflammatory ailments, paralysis and

dementia (Alzheimer’s) diseases (Rehman et al., 2014). The consumption of natural

antioxidants could help in such problems (Yang et al., 2001; Sun et al., 2002).

In vitro cultures are reliable and efficient approaches for the synthesis of natural

antioxidants, irrespective of seasonal variation and climatic factors. These antioxidants

can be easily quantified and isolated in purified form from cultures and used to minimize

the oxidation of biomolecules (Ahmad et al., 2010a). Khalaf et al. (2009) also reported

that damages caused by free radicals, produced as a result of biological combustion in

respiration, can be reduced by antioxidants due to their free radicals scavenging activities.

The phenolic compounds produced in cultures have strong tendency towards antioxidant

activities (Ali et al., 2013). Several analytical methods has been used to quantify

antioxidant potential including DPPH (2, 2-diphenyl-1-picrylhydrazyl) scavenging

activity, ferric reducing antioxidant potential (FRAP), oxygen radical absorption capacity

(ORAC) and ABTSB (2, 2-azinobis-3- ethylbenzothiazoline-6-sulphonic acid) (Dudonne

et al., 2009). However, among all these assays, DPPH activities are the preferred method

of quantifying the antioxidant activities. Being a simple method, having high efficacy in

determining activities of more lot of sample in a shortest possible time. Additionally,

DDPH assay is not only designed for only specific antioxidant compounds but can be

applied to determine the overall antioxidant activities of sample (Ahmad et al., 2010a;

2011).

20

Steviol glycoside in Stevia plants

Analytical methods designed for the discovery of plant secondary metabolites and the

development of commercial products of such metabolites are of prime importance

(Johnson et al., 2011). The isolation of biologically active components from herbal

extracts is a tedious and lengthy process. The traditional methods of studying natural

products include the fractionation of a complex mixture, separation and isolation of the

individual components using liquid chromatography and structure elucidation using

various spectroscopic methods (Hota, 2010). Several studies have been carried out to

isolate and quantify the steviol glycosides in Stevia by using different analytical

approaches. Stevia rebaudiana leaves were analyzed by Nikolova-Damyanova et al.

(1994) for stevioside and rebaudioside a contents, using high performance liquid

chromatography (HPLC) and silica gel thin layer chromatography (TLC).

Chromatographic techniques are one of the simplest and cheapest methods for detecting

plant constituents because these methods are easy to run, reproducible and require little

equipments (Hota, 2010). Several reports have been published about detection and

quantification of steviol glycoside by using HPLC. The chromatographic fingerprints can

be visualized and kept as an electronic image. Makapugay et al. (1984) isolated eight

steviol glycosides by using amino phase column eluted in a linear gradient mode with UV

detection. Similarly, Kitada et al. (1989) quantified four different kinds of glycosides in

food harvests by using analogues approaches. Attempts were also made by Nishiyama et

al. (1992) for the quantification of stevioside contents in Stevia leaves through HPLC and

spectrophotometry (near-infrared reflectance). Similarly, Ahmed et al. (1980) designed a

novel approach, involving pre-column, to separate two important steviol glycosides in

Stevia plant. Furthermore, p-bromophenacyl bromide was used to esterify these identified

compounds and successful separation through C18 column. While comprehensive study

about the chemical structure of organic compounds available in samples were obtained by

employing 1H NMR spectroscopy (Patra, 2012). Inamake et al. (2010), used analytical

approaches to identify and isolate stevioside, an important steviol glycoside of Stevia

plant, which was further studied structurally through HPLC, FTIR, TLC and NMR

techniques.

21

CHAPTER-III

Light-induced biochemical variations in secondary metabolites

production and antioxidant activity in callus cultures of Stevia

rebaudiana (Bert.)


Abstract

Stevia rebaudiana (S. rebaudiana) is an important specie with a worldwide medicinal and

commercial uses. Light is one of the major elicitor of morphogenic and biochemical

responses. The effect of various spectral lights on biomass accumulation and secondary

metabolites production in callus cultures of S. rebaudiana was investigated. Leaf explants

were cultured on Murashige and Skoog (MS) media and exposed to various spectral

lights. Growth promoters i.e. 6-benzyle adenine (BA) and 2, 4-dichlorophenoxy acetic

acid (2, 4-D; 2.0 mg l-1) was used for callus induction. The white light (16/8 hr) resulted

in the maximum callogenic response (92.73%) and biomass accumulation (5.78 g l-1)

during a prolong log phase at 18th day of growth kinetics. Cultures grown under blue light

had high total phenolics content (TPC; 102.32 µg/g DW), total flavonoids content (TFC;

22.07 µg/g DW) and total antioxidant capacity (TAC; 11.63 µg/g DW). On the contrary,

the green and red lights improved reducing power assay (RPA; 0.71 Fe (II) g -1 DW) and

DPPH-radical scavenging activity (DRSA; 80%). Herein, we concluded that the

utilization of colored lights is a promising strategy for enhanced production of

antioxidant secondary metabolites in callus cultures of S. rebaudiana.

22

INTRODUCTION

Stevia rebaudiana is one of the most important members of the genus Stevia of Asteracea

family, grown throughout the world (Sreedhar et al., 2008). Since long time, it has been

used in Paraguay and Brazil as herbal remedy for the treatment of heartburn and was

named as “yerba mate” (Ahmad et al., 2011).

The leaves of S. rebaudiana accumulate steviosides, which add 300 times more

sweetness than normal sugar (Singh and Rao, 2005; Hwang, 2006). The stevioside

content of S. rebaudiana leaves is one of the sweetest compounds present in larger

quantities and is commonly used in various commercial products (Dey et al., 2013). The

steviosides are considered as zero caloric sweetener because, unlike to glucose, there are

no receptors for this compound. Therefore, steviosides are very useful for diabetic

patients and also helpful in weight reduction.

Different in vitro cultures techniques have been exploited for steviosides production

(Aman et al., 2013; Dey et al., 2013; Khalil et al., 2015) and Callus culture is found

superior than micropropagation for accumulation of secondary metabolites. However, the

biosynthetic pathways of secondary metabolites are markedly influenced by various

elicitors. The addition of such elicitors to culture media may modulate the production of

secondary metabolites. The abiotic and biotic stresses alter the accumulation of bioactive

compounds in higher quantities as compared to naturally growing plants (Sivanandhan et

al., 2012). Among various elicitors, light quality/quantity influence plant development,

morphogenetic responses, and synthesis of valuable bioactive compound (Tariq et al.,

2014). The light plays a key role in primary and secondary metabolism and various plant

developmental processes (Liu et al., 2006; Shohael et al., 2006; Abbasi et al., 2007b). It

has been suggested that light sources directly stimulated the production of important

secondary metabolites including anthocyanins, artemisinin, caffeic acid derivatives and

flavonoids (Kreuzaler and Hahlbrock, 1973; Zhong et al., 1991; Liu et al., 2002; Abbasi

et al., 2007b).

23

Callus culture of Dioscorea deltoidea, exposed to light of selective wavelength with

appropriate intensity accumulated diosgenin contents in higher amount (Evans et al.,

1983). However, the physiological and morphological responses of plants towards light

quality depend upon the plant species (Ali and Abbasi, 2014). Similarly, morphological

and biochemical varaitions were observed in Artemisia absinthium callus with the

exposure to various spectral lights (Tariq et al., 2014). The inhibitory effects of light on

nicotine and shikonin production were also reported (Tabata et al., 1974). Since light

regulates secondary metabolites, it may have synergistic or antagonistic effects on

secondary metabolites (Kim et al., 1988).

Medicinal plants synthesize various bioactive compounds under specific conditions. The

plant based compounds are gaining more attention as potential nutraceuticals,

pharmaceuticals and food additives (Fazal et al., 2014). Plants under stress conditions

either release antioxidative enzymes or phenolics and flavonoids as defense system

(Hong et al., 2008). Plant polyphenols represent the principal group of natural

antioxidants among various classes of secondary metabolites that are considered to be

more valuable as compared to carotenoids and vitamins (Matkowski, 2008). Plant based

flavonoids are well reputed for their antioxidant properties due to their redox capacity. It

has also been observed that a flavonoids rich diet decreases lipid peroxidation, cell aging

and cancer (Ferreira et al., 2008), by scavenging toxic reactive oxygen species (ROS)

(Ahmad et al., 2014). Thus, such compounds are used in pharmacological activities like

antioxidant, anti-carcinogenic, to cure cardiovascular diseases and promote immune

system (Lai and Singh, 2006). In vitro cultures are amongst the best options for

production of antioxidant compounds (Ahmad et al., 2013) that minimize the oxidation

process (Ahmad et al., 2010).

There exists very limited information about the effect of various monochromatic lights on

callus culture establishement and secondary metabolites accumulation in Stevia

rebaudiana. Therefore, the current study was designed to find out suitable light of

selective waavelength for the enhanced proliferation and optimum biactive compounds

production in callus culture of Stevia rebaudiana.

24

MATERIALS AND METHODS

Leaf explants collection and sterilization

Young fresh leaves were harvested from S. rebaudiana plants, grown in the Ground and

Garden (G&G) Nursery, Department of Horticulture, The University of Agriculture

Peshawar (UAP). Freshly harvested leaves were washed out and dipped in double

distilled water to maintain its viability. Prior to inocultation, these leaves were surface

sterilized by using 70% ethanol and 0.2% mercuric chloride for a period of 1 and 2

minutes, respectively according to the protocols followed by Aman et al. (2014). After

surface sterilization, leaves were gently rinsed with autoclaved distilled water for dilution

of mercuric chloride and ethanol contents on their surfaces and thereafter, placed on

sterilized filter papers to remove execess moisure contents.

Establishment of callus cultures under different colored lights

Leaves of uniform sizes (3-4 mm2) were used as explants for culturing on MS (Murashige

and Skoog, 1962) media containing optimized concentrations of BA (2.0 mg l-1) and 2, 4-

D (2.0 mg l-1) for callus induction as reported by Aman et al. (2013). Similarly, media

witout PGRs was kept as control. Media was further concentrated with 30 and 8 %

sucrose and agar as gelling agent, respectively. Prior to the addition of agar, media pH

was adjusted to 5.8 through pH meter and finally media were sterilized by using an

electric utoclave at 121°C for 25 min. The effect of various spectral lights i.e. green (40W

Litex; 480-670 nm), yellow (36 W, Philips Ltd.; 530-780 nm), blue (220 V; 50 Hz,

Keliang Ltd.; 380-560 nm) and red (25 W, BINXIANG; 610-715 nm) lights were

evaluated. White light (fluorescent; 20 W, Toshiba FL20T9D/19; 380-780 nm; ~40-50

µmol m-2 s-1) with 16/8 photoperiod was kept as control. All cultured flasks were then

transfered to culture room having temperature 25±1°C. Each treatment was divided into

three independent experiments. Each experiment was designed according to Completely

Randomized Design (CRD). After 30 days of callus establishment, the averages were

randomly recorded using each replication as % callus induction.

25

Callus growth kinetics and biomass accumulation

Growth kinetics curve was ploted for rapidly growing callus biomass for a period of 30

days with 03 days interval against each colored light. After one month (30 days) of

culture period, fresh weight (FW) was determined after harvesting from media and then

oven dried at 50 ˚C for determination of dry weight (DW) (Fig. 4). Fresh and dry weights

of calli were expressed in gram/litre (g l-1).

Analytical methods

Oven dried calli developed under various monochromatic lights were used for extract

preparation. For this purpose, calli of each treatment was well powdered by using pestle

and morter. Extract was further used for determination of total phenolics content (TPC)

according to the protocol followed by Ahmad et al. (2014). For quantification of TPC,

0.03 ml of each extracted sample along with 0.1 ml Folin-Ciocalteus Reagent (FCR) was

taken and mixed with 2.55 ml autoclaved distilled water, prior to incubation (10,000 rpm;

15 min). Centrifuged samples were then subjected to dark condition for a period of 30

minutes. The supernatant of each centrifuged sample was taken and filtered through 45

µm membrane filter paper. Standard curve was established by using Gallic acid (Sigma;

1.0-10 mg/ml). The absorbance of each callus extract and gallic acid was monitored at

760 nm. Results were expressed as gallic acid equivalent (GAE mg/g DW) of callus.

Similarly, total flavonoids content (TFC) in each calli obtained from various

monchromatic lights were determined according to the method followed by Ahmad et al.

(2014). Sample extract of 0.25 ml was added with 0.075 ml AlCl3 (5% w/v) and 0.5 ml

NaOH. Mixture was further diluted with 1.25 ml sterile distilled water and centrifuged

for 14 min at 10,000 rpm, prior to incubation in dark for 30 min. After incubation,

samples were analysed for TFC at 510 nm with a UV-visible spectrophotometer. Rutin

(Sigma; 1.0-10 mg/ml) was used for establishment of standard calibration curve. The

total flavonoids content was expressed as rutin equivalent (RE; mg/g-DW) of callus.

DPPH-radical scavenging activity (DRSA) in each callus treated with different

monochromatic lights was found out according to the mothod followed by Ahmad et al.

(2010). Briefly, 5 mg extract of each callus was dissolved in 20 ml methanol (HPLC

26

grade). The DPPH solution was prepared by taking 0.25 mg DPPH powder in 20 ml

methanol. The DPPH solution was diluted four times. Afterward, 1.0 ml of each callus

methanolic solution was mixed with 2.0 ml of DPPH solution. The mixture was

incubated in dark for 30 min to scavenge maximum radicals. After incubation, the

absorbance of the solution was monitored on 517 nm at room temperature by using a UV-

visible spectrophotometer (Shimadzu-1650PC, Japan). The DRSA in each sample was

calculated as percentage of DPPH discoloration using the following equation;

DRSA (%) = 100 × (1 – AP/AD)

Where AP represents absorbance of shoots extract at 517 nm and AD is the absorbance of

the DPPH solution without extract.

For the determination of total antioxidant capacity (TAC), protocol of Pia-tczak et al.

(2014) was used and expressed as ascorbic acid milligram equivalent per gram of DW.

Similarly, the method of Pulido et al. (2000) was used for quantification of reducing

power assay (RPA) in each callus developed in response to various lights. The RPS was

calculated against known value of FRAP, ferrous sulphate and the calibration curve was

established from 0-2000 µM concentrations and was expressed in µmol Fe (II) g -1 of

DW.

Statistical analysis

Analysis of replicated values for each attribute of each sample, standard errors (± SE),

and their corresponding least significant difference (LSD) were carried out by using

Statistix software (8.1 versions) while Origin Lab (8.5) software was used for graphical

presentation.

27

RESULTS AND DISCUSSION

Effect of different colored lights on callogenic frequency

Elicitation has been one of the most efficient strategies to improve in vitro culture

development and production of desirable secondary metabolites (Wang et al., 2004; Ali

and Abbasi, 2014). Light is one of the important elicitors that play a key role in

photosynthesis, plant architectural development and plant morphogenesis (Kim et al.,

2006). Practically, fluorescent tubes are the major source of light energy for in vitro

cultures development (Tariq et al., 2014). Selective wavelength and optimum intensity of

light stimulate the production of important secondary metabolites in various cultures of

medicinal plants (Ellis and Roberts, 1980; Senger, 1987). Previous studies confirmed that

light quality directly affects morphological and physiological responses depending upon

plant species (Ali and Abbasi, 2014; Tariq et al., 2014). In this study, leaf explants were

placed on MS-media augmented with the combination of 2, 4-D and BA (2.0 mg l-1).

These cultured flasks were then kept under different colored monochromatic lights for

callogenic response. The control light produced optimum callogenic response (92.73%)

than other colored lights. The yellow light induced 88.34% callogenesis followed by blue

(76.4%) and green (75.12%) lights. However, the red light was found less effective in

callus induction (64.34%) from leaf explants of S. rebaudiana (Fig. 1 and 2). The current

data are in agreement with the reports of Tariq et al. (2014) that white light enhanced

callus development (90%) from leaf explants of Artemisia absinthium L. Ali and Abbasi

(2014) also observed higher biomass accumulation under white light in cell suspension

culture of Artemisia absinthium L. Efficient development of callus culture under white

light condition could be due to the provision of optimum energy as compared to other

colored lights. Therefore, it is suggested that white light could be the most suitable option

establishment of callus culture in S. rebaudiana as compared to other colored lights.

28

Fig.1: Effect of different spectral lights on callus morphological features in S. rebaudiana

(a) red light induced callus (b) blue light (c) yellow light (d) green light and (e) control

white light.

Fig. 2: Spectral lights induced variation in callogenic frequency (%) from leaf explants in

S. rebaudiana. Bars with common alphabets are nonsignificant at P ≤ 0.05.

Red Blue Yellow Green White

40

50

60

70

80

90

100

110

Light treatments

Cal

lus

induct

ion (

%)

a

ab

bb

c

b c e d a

29

Callus growth kinetics and biomass accumulation under different monochromatic

lights

The callus biomass accumulation was recorded for a period of 30 days with 3 days

interval under the influence of various monochromatic lights (Fig. 4). All callus cultures

developed under various colored lights experienced shorter lag phases. However, most of

the colored lights along with white light from day 3 started an elongated log phases till

18th day of the culture. During log phases, a period of day 12 to 18 was found the most

critical for the maximum biomass accumuation. However, from day 21 to day 30, decline

in biomass accumulation was observed in all applied colored lights. Among various

monochromatic lights tested, the maximum biomass accumulation (2.71 g l-1) was

displayed by red light during log phase (day 18) of growth kinetics (Fig. 4). However, the

control white light has shown 2 folds increase in biomass accumulation (5.78 g l-1) than

red light at day 18 of growth kinetics (Fig. 4). Furthermore, blue (2.02 g l-1), yellow

(1.95) and green (1.83 g l-1) lights accumulate significantly similar biomass but

comparatively lower than control white light. The current results showed that white light

is more effective for callogenesis and biomass accumulation than colored lights. We did

not found specific reports on the effect of colored lights on biomass accumulation in

callus cultures of S. rebaudiana. However, Tariq et al. (2014) observed positive response

of white light on callogenesis than other colored lights in Artemisia absinthium.

Similarly, Ali and Abbasi (2014) documented that cell culture grown under white lights

have shown maximum biomass accumulation than colored lights in Artemisia

absinthium. Moreover, various studies confirmed that these responses vary considerably

depending upon plant species and light quality (Shohael et al., 2006; Khan et al., 2013).

30

Fig. 3: Spectral lights induced variation in biomass accumulation during growth kinetics

of callus cultures.

Fig. 4: Fresh weight, dry weight and extractive values of callus cultures exposed to

different spectral lights. Mean values (± S.E) with common alphabets are nonsignificant

at P ≤ 0.05.

0

1

2

3

4

5

6 Yellow

Blue

Red

Green

White

Gro

wth

kin

etic

s (F

W-g

/l)

30272421181512963

Culture period (days)

0

Green Yellow Blue Red White

0

10

20

30

cc

b b

b

a

a

cc

Extr

act

call

us

wei

ght

(FW

-g l

-1)

Dry

cal

lus

wei

ght

(FW

-g l

-1)

FW

DW

EW

Fre

sh c

allu

s w

eight

(FW

-g l

-1)

5

a

b

c

c cc

Light treatments

0

1

2

3

4

5

0

1

2

3

4

31

Total phenolics and flavonoids accumulation

In this study, we evaluated the effect of different colored lights on total phenolics and

total flavonoids accumulation in callus cultures of S. rebaudiana. Callus cultures grown

under blue lights enhanced the accumulation of TPC (102.32 µg/g-DW) as compared to

control (33.27 µg/g-DW). Similarly, TFC were also positivly encourged by blue light and

the highest TFC (22.07 µg/g-DW) were found in callus culture obtained in blue light

condition as compared to other colored lights (Fig. 5). However, callus cultures in

response to green colored light yielded TPC (58.12 µg/g-DW) and TFC (12.26 µg/g-DW)

followed by yellow and red lights. Overall, TPC was founnd addiction of blue, green,

yellow, red and white colore light, respectively (Fig. 5). TFC also followed the similar

increasing trend like TPC contents, which positively suggests a strong correlation with

TPC production. Plants adopt various defence mechanisms as a result of various biotic

and abiotic stress conditions (Tan et al., 2004) by releasing TPC and TFC as a strong

antioxidant agents (Ali et al., 2006). Light is one of the important elicitors which has

direct influence on morphological and biochemical features of invitro cultures (Ahmad et

al., 2013). The effect of colored lights on secondary metabolites production in callus

cultures of S. rebaudiana is little known. However, the effect of colored lights on

secondary metabolites production is widely reported in many medicinal plant species (Ali

and Abbasi, 2014; Tariq et al., 2014). Tariq et al. (2014) reported that callus cultures of

Artemisia absinthium maintained under white light accumulated maximum content of

phenolics and flavonoids than colored lights. Ali and Abbasi (2014) also documented that

white light enhanced total phenolics and total flavonoids content in cell cultures of

Artemisia absinthium. The variation in data may be due plant species and the exposure

time to colored lights. The transformation efficiency of secondary metabolites also

depends on light quality. It may be possible that blue light enhanced the transformation

efficiency to produce higher quantities of phenolics and flavonoids in current study.

32

Fig. 5. Effect of different spectral lights on total phenolic and flavonoid content in callus

cultures of S. rebaudiana. Mean values (± S.E) with common alphabets are

nonsignificant at P ≤ 0.05.

Phenolics content and its correlation with antioxidant activities

Here, we observed a strong correlation of phenolics and flavonoids accumulation with

antioxidant activities. As we discussed earlier that blue light enhanced phenolics and

flavonoids content (Fig. 6-7). Similarly, the blue light enhanced TAC (11.63 µg/g DW)

as compared to control (Fig. 6). Contrary, green light enhanced reducing power assay

(RPA; 0.71 Fe (II) g -1 DW) as compared to other treatments. The DRSA and TAC have

shown maximum dependency on phenolics and flavonoids accumulation (Fig. 6-7). It

means that maximum antioxidant activities in callus cultures are due to phenolics

accumulation. The red lights also influenced the DRSA (80%) as compared to control.

These results suggest that blue and red lights are very effective for accumulation of

secondary metabolites in callus cultures of S. rebaudiana. Up to some extent phenolics

and flavonoids showed a positive correlation with antioxidant activities. Many available

reports indicated a significant correlation of phenolics production and antioxidant

activities in various medicinal plants (Ali et al., 2007; Al-Khateeb et al., 2012; Diwan et

al., 2012; Amid et al., 2013).

33

Fig. 6: Correlation of total phenolics content with antioxidant activities in callus cultures

of S. rebaudiana. Mean values (± S.E) with common alphabets are nonsignificant at P ≤

0.05.


-20

0

20

40

60

80

100

-20

0

20

40

60

80

100

Light treatments

aaaa

c

bc

Pow

er r

educi

ng a

ssay

(µ

mol

Fe(

II)/

g-D

W)

Tota

l phen

oli

cs c

onte

nt

(µg/g

-DW

)

TPC

PRA

a

b

bc

a

c

0

20

40

60

80

100

120

0

20

40

60

80

100

120

b bab

a

c

Tota

l an

tioxid

ant

capac

ity (

µg/g

-DW

)

Tota

l phen

oli

cs c

onte

nt

(µg/g

-DW

)

TPC

TAC a

b

bcc

ab

b

0

20

40

60

80

100

120

0

20

40

60

80

100

120

b

c

DP

PH

rad

ical

sca

ven

gin

g a

ctiv

ity (

%)

Tota

l phen

oli

cs c

onte

nt

(µg/g

-DW

)

TPC

DRSAa

b

bcc

aab

b

c

a

34

Fig. 7: Correlation of total flavonoids content with antioxidant activities in callus cultures

of S. rebaudiana. Mean values (± S.E) with common alphabets are nonsignificant at P ≤

0.05.

c


0

5

10

15

20

0

5

10

15

20

Light treatments

aaa

Pow

er r

educi

ng a

ssay

(µ

mol

Fe(

II)/

g-D

W)

Tota

l fl

avonoid

s co

nte

nt

(µg/g

-DW

)

TFC

RPA

a

b

c c

d

ab

-10

0

10

20

30

40

50

60

70

80

-10

0

10

20

30

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DP

PH

rad

ical

sca

ven

gin

g a

ctiv

ity (

%)

c

Tota

l fl

avonoid

s co

nte

nt

(µg/g

-DW

)

TFC

DRSA

a

b

cd

aab

bbc

c

a

0

5

10

15

20

25

0

5

10

15

20

25

c

b

Tota

l an

tioxid

ant

capac

ity (

µg/g

-DW

)

Tota

l fl

avonoid

s co

nte

nt

(µg/g

-DW

)

TFC

TACa

b

cc

d

a

b

bc

b

35

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary

Stevia rebaudiana belongs to the family Asteraceae and has a long history of

ethnomedicinal uses. Stevia rebaudiana was grown in Ground and Garden Nursery,

Department of Horticulture, The University of Agriculture Peshawar during the year of

2014. Fresh leaves from these plants were selected to study the effect of various spectral

lights (green, 480-670 nm; yellow, 530-780 nm; blue, 380-560 nm and red, 610-715 nm)

on callogenic frequency, total phenolics and flavonoids content along with antioxidant

activities. Fresh sterilized leaves were cultured on Murashige and Skoog (MS) media

augmented with BA (2.0 mg l-1) and 2, 4-D (2.0 mg l-1) for callus induction. However,

white fluorescent tube lights (380-780 nm) with 16/8 h photoperiod and light intensity

ranges from ~40-50 µmol m-2 s-1 were used as control. The MS-media was further

supplemented with 30 g l-1 sucrose and 8 g l-1 agar as solidifying agent. The pH of the

media was adjusted to 5.8 by using pH meter, prior to autoclaving. The experiment was

designed in Completely Randomized Design (CRD).

Calli induced under various mono chromatic lights were observed with significant

variation in biomass accumulation and secondary metabolites production. The control

light (white) was found with maximum callogenic response (92.73%) followed by yellow

light (88.34%), blue (76.4%) and green (75.12%) lights. However, the red light was

found with poor callogenic response (64.34%).

Each culture was characterized with lag, log and decline phases. Callus induced under

white light accumulated the highest biomass (5.78 g l-1) at day 18 of growth kinetics.

Similarly, callus developed under red light accumulated (2.71 g l-1) during log phase (day

18) of culture. Blue (2.02 g l-1), yellow (1.95) and green (1.83 g l-1) lights accumulated

significantly similar biomass but comparatively lower than control white light.

Callus cultures developed in blue light condition were observed with maximum

accumulation of TPC (102.32 µg/g-DW) and TFC (22.07 µg/g-DW) as compared to

control (33.27 µg/g-DW) and other colored lights. Moreover, blue light also enhanced

36

TAC (11.63 µg/g-DW). However, cultures maintained under green light produced 58.12

µg/g-DW of TPC and 12.26 µg/g-DW of TFC followed by yellow and red lights.

Contrary, the highest reducing power assay (RPA; 0.71 Fe (II) g -1 DW) was observed in

calli established under green light as compared to other treatments.

Conclusions

The application of colored lights was found as an effective strategy to enhance

biomass accumulation and production of bioactive compounds.

In this study, control white light improved callogenic frequency than other

colored lights. The control was followed by yellow, blue and green lights.

During growth kinetics, the red light enhanced biomass accumulation but was

lower than control cultures.

However, the blue light improved phenolics and flavonoid contents. The TPC

showed a linear correlation with TFC and total antioxidant capacity. However,

green and red lights enhanced reducing power assay and DRSA.

Recommendations

These results suggest that the application of colored lights is a promising

approach to enhance the production of antioxidant secondary metabolites.

Moreover, white light should be used for improved callogenic frequency.

Callus culture should be established under blue light for the accumulation of

maximum phenolics and flavonoid contents than control.

Similarly, for the highest reducing power assay and DPPH-radical scavenging

activity green colored light should be used.

37

CHAPTER IV

Sucrose-induced osmotic stress improved biomass and production of

antioxidant secondary metabolites in callus, cell suspension and

adventitious root cultures of Stevia rebaudiana (Bert.)


Abstract

Stevia rebaudiana is an important medicinal plant known for anti-diabetic activity, and

nontoxic naturally sweet agents i.e. steviol glycosides. Callus, cell suspension and

adventitious root cultures were established using 05, 10, 15, 20, 25, 30, 35, 40, 45 and 50 g l-1

sucrose concentrations to analyse its influence on the biomass accumulation and secondary

metabolites production. For growth kinetics, a curve was established for accumulated biomass

of each treatment with 03 days interval for a period of 30 days. The biomass accumulation in

callus culture was found highly dependent on sucrose concentrations. Lag phase of 9 days

followed by log phase till 27th day of culture was found in callus cultures proliferated in 05,

10, 15 and 20 g l-1 sucrose. The rest of the cultures did not experience any lag phase. All

cultures, except a few (20-30 g l-1) displayed nonviability after 27 days of inoculation.

Similarly, cell cultures induced by various concentrations of sucrose (05-30 g l-1) displayed

relatively shorter lag phase of 3 days as compared to cultures having sucrose (35-50 g l-1) with

lag phase of 12 days of growth kinetics. Lag phase in each cell culture was preceded by log

phase till 18 days of growth kinetics. Stationery phases were found in most of the cultures

with or without decline phases. However, growth curves of adventitious root cultures at 20-50

g l-1 sucrose were characterized with direct log phase till 18 days of culturing. Lag phase of 15

days was observed in cultures developed in media having 5 and 10 g l-1 sucrose, followed by

very short log phase. Sucrose concentrations (05-50 g l-1) significantly influenced the fresh

and dry biomass (g) of callus, cell suspension and adventitious root cultures. The callus

accumulated significantly high fresh and dry biomass (142.38 g l-1, 11.71 g l-1) with 40 g l-1

and 50 g l-1 sucrose, respectively. However, the maximum total phenolics content (TPC;

124.20 mg/g-DW), total flavonoids content (TFC; 49.36 mg/g-DW), dulcoside contents (6.56

mg/g-DW) and DPPH-radical scavenging activity (DRSA; 92.82 %) in callus cultures were

observed at sucrose concentration of 30 g l-1. By contrast, the highest stevioside (42.34 mg/g-

DW) and rebaudioside (22.67 mg/g-DW) contents were recorded in callus culture grown at 10

and 20 g l-1 sucrose, respectively. In cell culture, liquid media having 20 g l-1 sucrose resulted

in the maximum fresh (97.71 g l-1) and dry (8.57 g l-1) biomasses but the highest TPC (139.20

mg/g-DW) and TFC (41.46 mg/g-DW) at 40 g l-1 sucrose. The DRSA (83.87%) and stevioside

contents (42.23 mg/g-DW) were observed at 30 g l-1 sucrose. The highest rebaudioside (27.64

mg/g-DW) and dulcoside (6.43 mg/g-DW) contents were observed with 20 g l-1 sucrose. In

case of adventitious root culture, the maximum fresh (175.43 g l-1) and dry (11.14 g l-1)

biomass was accumulated in cultures having the highest sucrose concentration (50 g l-1) but

the highest TPC (155.00 mg/g-DW) and TFC (94.78 mg/g-DW) were recorded with 30 g l-1

sucrose. While the highest DRSA (94.43 %) was recorded in culture, established in media

augmented with 20 g l-1 sucrose. The stevioside (73.97 mg/g-DW) and rebaudioside (24.57

mg/g-DW) content were the highest in media containing 10 g l-1 sucrose. The dulcoside

content (12.24 mg/g-DW) was the maximum at 40 g l-1 sucrose. It is suggested that sucrose

concentration modulates biomass and metabolites of interest in callus, cell suspension and

adventitious root cultures of Stevia rebaudiana.

38

INTRODUCTION

Stevia rebaudiana belongs to the family Asteraceae, and is well known sweet herb due to

its sweet steviol glycosides (SG) content. The sweet SG contents such as stevioside,

rebaudioside and dulcoside are 300 to 400 sweeter than sugar cane and beet sugar

(Ahmad et al., 2011; Reis et al., 2011; Mathur et al., 2012). In SG group, the stevioside

and rebaudioside are famous for their heat stability, calorie free, and non-toxic properties

(Dey et al., 2013). The stevioside and rebaudioside are, therefore, attractive sugar

substitutes especially for obese and diabetic patients. The stevioside and rebaudioside

have been approved by FDA as a dietary supplement in many countries including

America, Europe and Asia (Bondarev et al., 2003). Besides its sweet taste, Stevia plant is

also known for antimicrobial, antioxidant, antidiabetic and anticarcinogenic properties

(Ahmad et al., 2011; Dey et al., 2013). The quantity and quality of these compounds

depend on plant species, growth stage, plant part, environmental and nutritional status of

the soil (Abdullah et al., 2000; Chattopadhyay et al., 2002). The biotic and abiotic

stresses also alter the biological pathways resulting in poor yield of such compounds

(Sivanandhan et al., 2012).

The conventional methods of propagation are limited by environmental fluctuations, poor

cultural practices, pests and diseases that also minimize the metabolites of interest

(Ahmad et al., 2014). There has been an increasing interest in various plant tissue culture

approaches to increase the synthesis of sweet tasting agents of therapeutic importance

(Dey et al., 2013; Aman et al., 2013). Among these techniques, callus, cell and

adventitious root cultures are the most reliable and effective in vitro methods for

enhanced production of biomass and secondary metabolites as compared to micro and in

vivo propagation. The in vitro techniques are more effective in enhancing the specified

bioactive compounds, which are, otherwise, available in trace amount in natural growing

plants (Ali et al., 2013).

In vitro culture techniques such as callus, cell suspension and adventitious root cultures

have the potentials to synthesize commercially valuable bioactive compounds on large

scale (Mustafa et al., 2011). Callus culture has been widely exploited for production of

39

such useful bioactive compounds (Meratan et al., 2009). However, for effective callus

induction, optimization of appropriate plant growth regulators, explant and other physical

and chemical factors are important (Ananthi et al., 2011). The cell culture of various

medicinal plants are used for the efficient production of secondary metabolites such as

ginsenosides, shikonin, berberine and several others for various purposes (Baque et al.,

2012; Bourgaud et al., 2001; Mulabagal and Tsay, 2004; Sivakumar et al., 2011; Wu et

al., 2007). Cell culture not only ensures speedy cell growth but also guarantees the

continuous supply of targeted bioactive compounds (Rao and Ravishankar, 2002).

Additionally, the cell culture may also help in understanding biosynthetic pathways of

such phytochemicals (Meratan et al., 2009).

Like callus and cell culture, adventitious roots have also been exploited for increased

production of secondary metabolites (Dubrovsky and Rost, 2003; Goel et al., 2009).

Several pharmaceutically important compounds like anthraquinone have been obtained

from Morinda citrifolia and Rubia tinctorum (Sato et al., 1997; Baque et al., 2010).

Similarly, Kevers et al. (1999) established adventitious root culture of Panax ginseng in

bioreactor for production of ginsenosides.

The in vitro culture techniques such as callus, cell suspension and adventitious root

cultures yield more bioactive compounds, especially with biotic and abiotic elicitors

(Eilert, 1987; Barz et al., 1988). Among various kind of elicitors, light, temperature,

humidity, pH and sucrose have been reported for enhanced production of useful bioactive

compounds in various in vitro cultures of medicinal plants (Tariq et al., 2014).

Sucrose is the primary source of energy, required for various metabolic processes in

intact plants as well as in in vitro cultures (Fazal et al., 2014). Sucrose not only provides

energy but also acts as an effective source of carbon for structural development of cell,

tissues and organs (Calamar and Klerk, 2002). Among the various carbohydrates, it is the

most abundant in phloem of higher plants and regulates gene expression (Morkunas et

al., 2005; Wind et al., 2010). Furthermore, sucrose acts as an osmotic agent and regulates

the absorption of water and other nutrients from the medium (Cui et al., 2010). The

plants, in response to mild stresses, may generate non-toxic free radicals and other

40

reactive species such as phenol and flavonoids to trigger the defense system (Lee et al.,

2006; Baque et al., 2012; Ahmad et al., 2013a; Ahmad et al., 2014). The sucrose induced

antioxidants and other secondary metabolites production in various cultures of medicinal

plants have been reported (Ferri et al., 2011; Ali et al., 2013).

Despite, extensive work on in vivo and micropropagation of Stevia rebaudiana, limited

information is available on callus, cell suspension and adventitious root culture of this

plant. Similarly, limited information is available to understand the effect of various

cultural conditions on development of callus, cell suspension and adventitious root

cultures for efficient production of bioactive compounds in Stevia rebaudiana. Therefore,

the current study was designed to investigate the effect of sucrose concentration on

callus; cell and adventitious root culture growth (growth kinetics, fresh and dry weight),

production of important secondary metabolites (phenolics, flavonoids, stevioside,

rebaudioside and dulcoside contents) as well as antioxidant activities.

41


The research work was conducted at Plant Tissue Culture Lab, Department of Plant

Breeding and Genetics, The University of Agriculture Peshawar, during the year of

2014/15, while qualitative analysis of the resultant samples were carried out in Centre for

Biotechnology and Microbiology (CB&M), University of Swat.

To develop reliable protocols, the effect of sucrose on callus, cell and adventitious root

culture was screened for efficient culture establishment and production of important

secondary metabolites. For this purpose, the research work was divided into three (03)

experiments. Each experiment was carried out in completely randomized design (CRD).

The detail of the treatments and analytical approaches is given as below;

Establishment of callus cultures from leaf explants

Black coated seeds of Stevia rebaudiana were collected from plants grown in Ground and

Garden Nursery, Department of Horticulture, The University of Agriculture Peshawar.

These seeds were subjected to sterilization prior to inoculation on Murashige and Skoog

(MS) medium (Murashige and Skoog, 1962). The MS medium was supplemented with 30

g l-1 sucrose as carbon source and solidified with 8.0 g l-1 agar. The pH of the medium

was adjusted to 5.8 through weak acid or base by using pH meter. The medium was

finally autoclaved at 121 °C for 15 minutes. Seeds were surface decontaminated

according to the protocol of Aman et al. (2013). Sterilized seeds were inoculated in flasks

(100 ml) containing MS media without plant growth regulators. The cultured flasks were

placed in growth chamber under controlled conditions for plantlets development. After 30

days of seed germination, leaf explants were collected for callus development. In the

previous experiment, field grown plants were used for callus development that resulted in

poor response due to their differentiation and cultural conditions. Here for callus

development, MS media was augmented with auxin or cytokinin alone, or synergistic

combination of auxin and cytokinine and were tested. In preliminary experiments,

different concentrations of NAA (0.5, 1.0, 1.5, 2.0 mg l−1) and IBA (0.5, 1.0, 1.5, 2.0 mg

l−1) were found least effective for callus development. MS medium without plant growth

regulators was used as control. However, different concentrations of 2, 4-D (0.5, 1.0, 1.5,

42

2.0 mg l−1) and BA (0.5, 1.0, 1.5, 2.0 mg l−1) induced callus from leaf explants but was

slower as compared to synergistic combination of 2, 4-D and BA. Among different

combinations, 2, 4-D (2.0 mg l−1) and BA (0.5 mg l−1) produced 92.3% calli from leaf

explants. While other combinations of 2, 4-D (0.5, 1.5, 2.0 mg l−1) with BA (0.5 mg l−1)

produced callus ranging from 75 to 84%. Therefore, the combination of 2, 4-D (2.0 mg

l−1) and BA (0.5 mg l−1) were selected for further studies on callus culture. Different

sucrose concentrations (05, 10, 15, 20, 25, 30, 35, 40, 45 and 50 g l-1) were tested for

callus biomass accumulation and production of antioxidant secondary metabolites. The

growth kinetics of proliferating calli was determined with three days interval for a period

of 30 days. After 30 days of callus cultures development, fresh and dry biomass were

determined and the dried materials were used for determination of phenolics, flavonoids,

antioxidant activity, stevioside, rebaudioside and dulcoside contents.

Establishment of cell suspension culture in Stevia rebaudiana

To establish cell suspension culture, 40 days old whitish granular calli was inoculated in

Erlenmeyer flasks (500 ml) containing MS media fortified with various concentration of

2, 4-D (0.5, 1.0, 1.5, 2.0 mg l−1) in combination with BA (0.5, 1.0, 1.5, 2.0 mg l−1). The

culture was placed on rotary shaker with 120 adjusted rpm at 25 °C in dark condition for

14 days, in order to establish cell suspension culture. Among different combinations, 2,

4-D (1.0 mg l−1) and BA (0.5 mg l−1) was found the most effective treatment for cell

suspension culture development. Therefore, the same combination of 2, 4-D (1.0 mg l−1)

and BA (0.5 mg l−1) was used for further development of cell suspension culture from

whitish granular calli as inoculum. MS medium without plant growth regulators was used

as control. A known amount of friable calli was used as inoculum for subsequent

experiment. Subsequent experiments were carried out in Erlenmeyer flasks (100 ml)

containing MS media (50 ml) supplemented with different sucrose concentrations (05,

10, 15, 20, 25, 30, 35, 40, 45, 50 g l−1). Each experiment was carried out in Completely

Randomized Design (CRD) and treatments repeated 3 times during each experiment. In

order to test the effect of sucrose induced osmotic stress on culture development, all the

cultured flasks were placed on orbital shaker at 120 rpm for a period of 30 days in dark.

The data regarding growth kinetics was determined at 3 days intervals. Further, fresh and

43

dry biomass was determined and the dried cells were used for the determination of

antioxidant secondary metabolites and steviol glycosides.

Establishment of adventitious root culture in Stevia rebaudiana

Fresh viable seeds were collected and germinated in vitro by using the method of Aman

et al. (2013) for the development of stock plantlets. Roots were collected from in vitro

germinated seedlings after 30 days of germination. Collected roots were then cultured in

Erlenmeyer flasks containing half and full strength liquid MS media supplemented with

different concentrations of IBA (0.5, 1.0, 1.5, 2.0 mg l−1) or NAA (0.5, 1.0, 1.5, 2.0 mg

l−1) or combination of IBA and NAA. The cultured flasks were placed on orbital shaker

(120 rpm, 25 °C) in dark condition for a period of 30 days. Half and full strength liquid

media containing combination of IBA and NAA was found the least effective for root

development. Similarly, various concentrations of IBA alone in half and full strength

liquid media did not produce roots. However, about 90% root development response was

observed in half MS medium containing 0.5 mg l−1 NAA, while other NAA treatments

induced small callus and then started roots development. Therefore, 0.5 mg l−1 NAA was

used for further experiments and stock adventitious root development. MS medium

without plant growth regulators was used as control. In order to test the effect of different

sucrose concentrations (5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 g l-1) on adventitious roots

development, a known amount of inoculum roots were inoculated in liquid MS media

supplemented with 0.5 mg l−1 NAA and different sucrose concentration and placed on

orbital shaker in dark for a period of 30 days. Optimization of adventitious root culture

was carried out in Completely Randomized Design (CRD) and each treatment was

repeated 3 times. The growth kinetics was determined at 3 days interval for a period of 30

days. Fresh biomass (FB), dry biomass (DB), antioxidant activity, phenolics, flavonoids

and active compounds were determined in adventitious roots of Stevia rebaudiana.

Development of growth curve

A growth curve was developed for the rapidly growing calli, cells and adventitious root

cultures in response to different sucrose concentration. The growth kinetics of calli, cells

and adventitious root cultures were determined for 30 days period with 3 days interval.

44

The lag, log and stationary phases were determined for fresh accumulated biomass of

calli, cells and adventitious roots from established growth curve. These tissues were then

used for the determination of fresh and dry biomasses.

Fresh and dry biomass determination

Fresh calli, cells and adventitious roots were collected from flasks after 30 days of

inoculation. These in vitro cultures product were rinsed with sterile distilled water to

remove media particles. The excess water from these cultures were removed by using

autoclaved filter paper and used for fresh weight determination. These cultures were then

placed in oven at 50 ˚C for 24 hours and, finally, the dry weight was determined.

Extract preparation

The oven-dried materials were grinded using fruit juicer and pestle and mortar. Well-

powdered samples of 20 mg were taken in a test tube and 20 ml ethanol was added.

Samples were kept for one week in refrigerator with periodic shaking in order to dissolve

maximum metabolites. Finally, the samples were centrifuged at 5000 rpm for 15 minutes.

The supernatant was used for the assessment of total phenolic content, total flavonoid

content, antioxidant activity, stevioside, rebaudioside and dulcoside contents.

Determination of total phenolic content

Calli, cells and adventitious root cultures extract was used for the determination of total

phenolic content (TPC) according to the recent method of Ahmad et al. 2014. During

phenolics determination, a mixture containing 0.1 ml Folin-Ciocalteus reagent (2N), 0.03

ml of sample extract and 2.55 ml sterile distilled water was incubated for 30 minutes in

dark to avoid oxidation. The same procedure was repeated independently for calli, cells

and adventitious root culture extracts. The absorbance reading of the resulted samples

was taken at 760 nm through UV visible spectrophotometer (Shimadzu-1650; Japan). For

plotting calibration curve, gallic acid (Sigma; 1.0-10 mg/ml; R2 = 0.9878) was used.

Finally total phenolic content were obtained by using the following equation, expressed

as gallic acid equivalents (GAE)/g of dry weight.

45

Total phenolic content (%) = (AS-AB) / (CF × DF)

Where AS is the absorbance of the sample and AB is absorbance of blank. CF is the

conversion factor from standard curve and DF is the dilution factor.

Determination of total flavonoids content

For estimation of total flavonoids content (TFC), ethanol extract of the treated samples

(0.25 ml) was added to test tube containing 1.25 ml sterile distilled water, AlCl3 (0.075

ml; 5% w/v) and NaOH (0.5 ml; 1M) according to the recent method of Ahmad et al.

(2014). Mixtures were centrifuged for 14 minutes at 14,000 rpm and finally incubated for

10 mint. Furthermore, 45 μm membrane were used to filter the resultant mixtures and the

absorbance was taken through spectrophotometer (UV-Visible; Shimadzu-1650; Japan) at

510 nm. Rutin (Sigma; 1.0-10 mg/ml; R2 = 0.9866) was used for plotting standard

calibration curve. The total flavonoid content was expressed as rutin equivalent (RE)

mg/g-DW of extracts.

DPPH-radical scavenging activity

DPPH-radical scavenging activity (DRSA) was monitored according to the method of

Ahmad et al. (2010). Ethanol extracts of the treated samples (1.0 ml) was mixed with

DPPH free radical solution (2.0 ml) and incubated for 30 minutes in fully dark

conditions. Absorbance was recorded at 517 nm by using visible spectrophotometer

(Shimadzu-1650PC, Japan). Finally, the radical quenching activity was calculated as

percentage of DPPH discoloration using the following equation;

DRSA (%) = 100 × (1 – AE/AD)

Where AE represents absorbance of extract at 517 nm and AD is the absorbance of the

DPPH solution without tissue extract.

46

Quantification of steviol glycosides in different in vitro cultures

The method of Aman et al. (2013) was used to determine stevioside, rebaudioside and

dulcoside contents in calli, cells and adventitious root cultures of Stevia rebaudiana. For

quantification of stevioside, rebaudioside and dulcoside contents, Perkin-Elmer HPLC

system (USA) was used with quaternary pump, solvent vacuum degasser, C18 column

(ODS) with 150 × 4.6 mm, 5 μm particle size, a variable wavelength detector, and an

auto sampler with a 10-μl injection loop. In mobile phase, 25% HPLC grade water and

75% acetonitrile was used as solution A and B, respectively. During quantification,

volume of 10 µl was used to inject at 1.0 ml min-1 flow rate. Steviol glycoside standard

containing stevioside, rebaudioside and dulcoside, purchased from Sigma (USA)

laboratories was run at first for standardization of retention time of each content.

Stevioside, rebaudioside and dulcoside contents were identified in each sample of calli,

cells and adventitious root cultures by comparing retention times of samples with

standard. Quantified amount of stevioside, rebaudioside and dulcoside were expressed in

mg/g of dry weight (DW).

Statistical Analysis

Analysis of replicated values, standard errors (± SE), and least significant difference

(LSD) were carried out by using Statistix software (8.1 versions) and Origin Lab (8.5)

software was used for graphical presentation.

47


Effect of sucrose induced osmotic stress on growth kinetics of growing cultures

The effect of sucrose induced osmotic stress (05-50 g l-1) on growth of callus, cell

suspension and adventitious root cultures of Stevia rebaudiana was evaluated (Fig. 1, 2

and 3). Biomass accumulation in callus, cell suspension and adventitious root cultures

was found highly dependent on sucrose concentrations. Some of the callus cultures

exposed to 05, 10, 15 and 20 g l-1 showed lag phase of 9 days followed by log phase up to

27 days. Other cultures exposed to (25-50 g l-1) sucrose induced osmotic stress had a

prolong log phases of 27 days without any lag phase. All cultures except few displayed

nonviability after 27 days of inoculation. Cultures initiated in media having 20, 25 and 30

g l-1 sucrose were found to be in stationary phase after 27 days of log phase (Fig. 4). The

highest callus biomass (141.44 g l-1) was observed in cultures augmented with 45 g l-1

sucrose at 27th day of culture period. Furthermore, cultures having sucrose (40 g l-1)

showed almost similar result for biomass accumulation (141.03 g l-1) after 24 days of

culturing (Fig. 1a). The growth kinetics of cell cultures exposed to various concentrations

of sucrose (05-30 g l-1) displayed relatively shorter lag phase of 3 days as compared to

cultures having sucrose (35 -50 g l-1) with lag phase of 12 days. The lag phase in each

cell culture was preceded by log phase till 18 days. The stationery phase was observed in

most of the cultures with or without the decline phase (Fig. 5). The maximum biomass

accumulation (97.18 g l-1) in cell suspension culture was recorded on day 18 of log phase

induced by 20 g l -1 sucrose in culture media (Fig. 5). However, growth curve of

adventitious root cultures was characterized with direct log phase till 18 days of

culturing. Lag phase of 15 days was observed in cultures developed in media having 05

and 10 g l-1 sucrose, followed by very short log phase. Among these cultures, the highest

sucrose augmentation (50 g l-1) resulted in the maximum biomass accumulation (174.42 g

l-1), followed by a stationary phase up to 24 days of culturing followed by a decline in

growth. The log phase in cultures induced by 40 and 45 g l-1 sucrose were followed by

stationery phase from day 18 to day 24 of the culture period. In rest of the cultures, log

phase was followed by decline phase (Fig. 6).

48

The callus growth is, generally, inhibited by higher osmotic stress, that ultimately reduces

the biomass yield. By contrast, lower sucrose concentration is undesirable for callus

biomass accumulation (Parveena and Veeresham, 2014). Thus, an optimum sucrose

augmentation into the media is required to speed up cell division, which ultimately

produced enhanced fresh biomass (Gurel and Gulsen, 1998). The current findings of cell

suspension culture are partially supported by the findings of See et al. (2011) who found

that sucrose at higher concentration (60 g l-1) reduced cell hydration in Melastoma

malabathricum suspension culture. However, sucrose (15 g l-1) supplementation was

found to be enough for the growth of suspended cells and provision of energy as a carbon

source, which is required for the metabolism of cells. Similarly, Sato et al. (1996) also

found that addition of sucrose (>30 g l-1) in the cell suspension culture of strawberry

resulted in cell growth reduction due to increase in osmotic potential or medium

viscosity, that lead to the inhibition of nutrient uptake. High biomass accumulation in

liquid cultures of adventitious roots in the current investigation is supported by the

findings of Cui et al. (2010) who reported enhanced biomass accumulation with

enhanced sucrose levels (30 and 50 g l-1) in Hypericum perforatum adventitious root

cultures. Additionally, the addition of higher sucrose concentrations (30 and 50 g l-1) into

liquid media resulted in elevated biomass accumulation in liquid cultures of Echinacea

angustifolia. The increased biomass of adventitious roots might be due greater demand

for sucrose at the differentiation phase for structural integrity and growth (Tremblay and

Tremblay, 1995).

49

Fig. 1. Effect of sucrose concentrations (a; 05 g l-1), (b; 10 g l-1), (c; 15 g l-1), (d; 20 g l-1),

(e; 25 g l-1), (f; 30 g l-1), (g; 35 g l-1), (h; 40 g l-1), (i; 45 g l-1) and (j; 50 g l-1) on callus

proliferation of Stevia rebaudiana.

a b

d e f

h

j

i g

c

50


(e; 25 g l-1), (f; 30 g l-1), (g; 35 g l-1), (h; 40 g l-1), (i; 45 g l-1) and (j; 50 g l-1) on

development of cell suspension cultures of Stevia rebaudiana.

g h

c b

e f

a

d

i

j

51


(e; 25 g l-1), (f; 30 g l-1), (g; 35 g l-1), (h; 40 g l-1), (i; 45 g l-1) and (j; 50 g l-1) on

establishment of adventitious root cultures of Stevia rebaudiana.

a b c

f e d

g h i

j

52

Fig. 4. Sucrose induced osmotic stress (05-50 g l-1) variations in biomass accumulation

during growth kinetics (period 30 days; interval 03 days) of callus cultures of Stevia

rebaudiana.


during growth kinetics (period 30 days; interval 03 days) of cell cultures of Stevia

rebaudiana.

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during growth kinetics (period 30 days; interval 03 days) of adventitious root cultures of

Stevia rebaudiana.

Effect of sucrose induced osmotic stress on fresh biomass accumulation

Sucrose induced osmotic stresses (05-50 g l-1) significantly influenced the fresh biomass

accumulation in callus, cell suspension and adventitious root cultures of Stevia

rebaudiana. During callus culture, the fresh biomass accumulation was gradually

increased with increasing sucrose concentrations (05-40 g l-1). However, further increase

in sucrose concentration (45-50 g l-1) resulted in reduction of fresh biomass

accumulation. The highest accumulation of fresh callus biomass (142.38 g l-1) was

recorded at 40 g l-1 sucrose. By contrast, the least fresh biomass (60.10 g l-1) was

accumulated in cultures with 05 g l-1 of sucrose (Fig. 7). In cell suspension cultures,

lower osmotic stress of sucrose (05-20 g l-1) enhanced the fresh biomass of cells that

declined gradually with increasing sucrose stress (25-50 g l-1) (Fig. 8). Liquid media

having sucrose (20 g l-1) resulted in maximum fresh biomass (97.71 g l-1) and the highest

sucrose stress (50 g l-1) caused reduction of fresh biomass accumulation (25.43 g l-1) in

cell cultures (Fig. 8). Similar to callus cultures, the addition of different sucrose

concentrations (5-50 g l-1) into the liquid media enhanced fresh biomass of adventitious

0

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54

root culture. The maximum fresh biomass (175.43 g l-1) was accumulated in root cultures

having the highest sucrose concentration (50 g l-1), while the minimum fresh biomass

(37.71 g l-1) was noted for medium having sucrose at a level of 5 g l-1 (Fig. 9).

Carbon as an external source of energy is needed for cell metabolism and plant

development (Mello et al., 2001). Several carbon sources (sucrose, fructose and maltose)

are available for quick release of energy, needed for various metabolic processes of the

plants. Among these carbon sources, sucrose is more efficient in aseptic cultures

development (Shahnewaz and Bari, 2004; Jayaraman et al., 2015). Sucrose is a key factor

and its specific concentration directs the induction and growth of in vitro cultures and

production of desired compounds (Gurel and Gulsen, 1998; Gibson, 2000). Therefore, an

optimum concentration of sucrose is required for the provision of external energy, needed

for the division of cells and differentiation of tissues without having an adverse effect on

organ formation (Stavarek et al., 1980). High sucrose concentration induces an osmotic

stress which inhibits callus growth and ultimately causes reduction in biomass yield

(Parveena and Veeresham, 2014). The current results of higher callus biomass are in line

with the findings of Parveena and Veersham (2014), who noted increased biomass

accumulation with the addition of higher sucrose concentrations (30 and 40 g l-1) to

culture medium. Similarly, Evan et al. (1976) reported the highest growth in callus of

soybean, when 40 g l-1 of sucrose was added into the culture medium. The biomass yield

may decrease significantly with lower or higher than optimum sucrose concentration

(Alkhateeb et al., 2008). Similarly, Kaul and Sabharwal (1970) found reduction in callus

growth of tobacco by using higher concentration of sucrose (60 or 80 g l-1) in culture

medium. However, enhanced growth of callus was found with the addition of lower

concentration (4 %) of sucrose into the media (Saika et al., 2013). The enhanced fresh

biomass of callus could be due to the reason that higher sucrose addition into the media

elevates the process of cell division that increases the fresh biomass accumulation (Gurel

and Gulsen, 1998). In contrast, cells growth was not supported by higher concentration of

sucrose (25-50 g l-1) in cell suspension culture of Stevia in our study. See et al. (2011)

also observed that higher sucrose concentration (60 g l-1) resulted reduction in biomass

yield of suspended cells of Melastoma malbathricum. Similarly, Sato et al. (1996) also

55

observed poor growth of strawberry cells with addition of higher sucrose concentration

into the liquid media. Recent findings of Fazal et al. (2016) are very close to our findings,

who found high fresh biomass yield of suspended cells with the provision of optimum

sucrose concentrations (20 and 25 g l-1) in suspension cultures of Prunella vulgaris.

Further, the reduction in biomass yield of suspended cells at higher concentrations (25-50

g l-1) may be due to cell dehydration, which decrease the cell and tissue growth and

proliferation (Jalil et al., 2015). The maximum biomass accumulation in adventitious root

cultures at 50 g l-1 sucrose was supported by the findings of Cui et al. (2010) who noted

enhanced biomass accumulation at higher sucrose levels (50 g l-1) in adventitious root

cultures of Hypericum perforatum and Echinacea angustifolia.

Effect of different sucrose concentrations on dry biomass of various cultures

Dry biomass (g l-1) of calli, cells and adventitious roots were significantly influenced by

varied sucrose concentrations (05-50 g l-1). The incremental increase in sucrose levels in

the callus culture gradually enhanced the dry biomass of callus. The maximum dry

biomass (11.71 g l-1) was observed for 50 g l-1 sucrose concentration in contrast to the

least dry biomass (2.67 g l-1) in callus cultures of Stevia with the least concentration (05 g

l-1) (Fig. 7). In contrast to callus cultures, a gradual increase in dry biomass of suspended

cells was noted with sucrose stress (05-20 g l-1) but further increase in sucrose

concentrations (25-50 g l-1) resulted in a gradual reduction in dry biomass accumulation.

The maximum dry biomass (8.57 g l-1) was observed when 20 g l-1 sucrose was added to

the culture media, while minimum dry biomass (4.57 g l-1) of cells was noted for liquid

media having 5 g l-1 of sucrose (Fig. 8). Like callus cultures, similar trend of enhanced

biomass accumulation with increasing sucrose levels (05-50 g l-1) was also noted in

adventitious root cultures. The highest dry biomass (11.14 g l-1) was observed for 50 g l-1

of sucrose, while the lowest dry biomass (2.86 g l-1) was being observed for 05 g l-1 of

sucrose in culture media (Fig. 9).

The sucrose concentrations in the culture media greatly influence fresh and dry biomass

of callus cultures. Increasing sucrose concentration from 30 g l-1 to 90 g l-1, significantly

enhanced dry weight of callus tissue but reduced fresh biomass accumulation

(Gerdakaneh et al., 2010). Sucrose at an optimum concentration influences the uptake of

56

inorganic ions from the nutrient media; therefore, optimization of sucrose concentration

is needed for efficient uptake of nutrients (Gamborg et al., 1974). Furthermore, its

optimum concentration is also necessary because sucrose helps differentiation and growth

of cells and tissues (Gibson, 2000). Sucrose besides a carbon source, also acts as osmotic

stress agent when used above certain level (Mehta et al., 2000; Kim and Kim, 2002).

Sometimes, the stress conditions may improve the biomass accumulation of callus tissues

(Kishore and Dange, 1990; Juhasz et al., 1997). The current findings are in partial

agreement with the findings of Ambrosio and Melo (2004), who found the highest dry

weight at 30 and 45 g l-1 sucrose concentration. The enhanced dry biomass accumulation

of callus may be attributed to sucrose stress (Gerdakaneh et al., 2010). The reduction in

dry biomass with relatively higher sucrose stress (25-50 g l-1) could be due to the

inhibition of nutrient uptake and chlorophyll synthesis at above the optimum

concentrations (Edelman and Hanson, 1972; Sato et al., 1996).

Fig. 7. Effect of sucrose induced osmotic stress on fresh and dry biomass (g l-1) of callus

cultures of Stevia rebaudiana. Mean values (± S.E) with common alphabets are


40

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aa

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cc

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57

Fig. 8. Effect of sucrose induced osmotic stress on fresh and dry weight (g l-1) of cell

suspension cultures of Stevia rebaudiana. Mean values (± S.E) with common alphabets

are nonsignificant at P ≤ 0.05.

Fig. 9. Effect of sucrose induced osmotic stress on fresh and dry weight (g l-1) of

adventitious root cultures of Stevia rebaudiana. Mean values (± S.E) with common

alphabets are nonsignificant at P ≤ 0.05.

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58

Effect of differential sucrose concentrations on phenolics content

Considerable variations were observed in accumulation of total phenolic contents (TPC)

in callus, cell suspension and adventitious root cultures of Stevia in response to varying

concentrations of sucrose. Generally, a linear increase in all three cultures was found with

increasing sucrose concentration in the medium up to a specific level but further increase

significantly reduced the production of phenolic content.

The maximum TPC (124.20 mg/g-DW) was accumulated by callus cultures established in

medium having 30 g l-1 sucrose but decreased significantly with increasing or decreasing

sucrose levels. Among all the tested sucrose levels, the minimum TPC (42.60 mg/g-DW)

was observed in callus cultures developed in medium supplemented with 05 g l-1 sucrose

(Fig. 10a). Likewise, cell suspension cultures accumulated more TPC (139.20 mg/g-DW)

at higher sucrose level (40 g l-1) as compared to callus culture but lower sucrose

concentration (05 g l-1) in cell culture yielded poor TPC (51.34 mg/g- DW) (Fig.10b).

Total phenolic contents in adventitious root cultures of stevia were also found to be

sucrose concentration dependent. The highest total phenolic content (155.00 mg/g DW)

was recorded in adventitious roots cultured in medium having 30 g l-1 sucrose. However,

linear decrease in TPC was observed with further increase in sucrose levels in culture

medium. The least TPC (17.77 mg/g DW) was observed in roots obtained from medium

added with 05 g l-1 sucrose. However, considerable increase in TPC was recorded with

the increasing concentrations of sucrose up to 30 g l-1 sucrose (Fig. 10c).

Cultured cells in optimized conditions have higher potential to accumulate important

metabolites like polyphenol than intact plants (Trejo-Espino et al., 2011). In this regard,

fluctuation of in vitro culture conditions, media compositions like nutrients and sucrose

has a fundamental role in culture productivity (Lee and Shuler, 2000). Generally, it is

essential to incorporate carbohydrates as a carbon and energy source in culture medium

for several metabolic processes and synthesis of important phytochemicals in plant cell,

tissue and organ culture (George, 1993; Karhu, 1997; Du Toit et al., 2004). Addition of

carbohydrates also influences the expression of different genes responsible for various

developmental processes in plants (Koch, 1996). Besides metabolism and developmental

process, carbohydrates also act in signaling to regulate division and cell differentiation

processes (Sherson et al., 2003; Rolland et al., 2006). Thus, sucrose in culture medium

59

has been regarded as the most potent and energetic carbon source in plant cell, tissue and

organ culture for accumulation of important phytochemicals (Wu and Zhong, 1999). The

current study revealed that callus, cell suspension and adventitious root cultures

accumulated more phenolic content with the increasing sucrose concentration in culture

medium. Accumulation of higher secondary metabolites with the increasing levels of

sucrose in callus culture of Catharanthus roseus has been reported by Zhao et al. (2001).

However, Zhang and Zhong (1997), reported that incremental or intermittent supplication

of sucrose to the culture medium has more pronounced effect on culture growth and

biosynthesis of secondary metabolites in several plants than increasing concentration at

initial stages. On the other hand, majority of plant cell cultures were reported to produce

the highest metabolites with primary sucrose levels. Previous reports of Cui et al. (2010)

are in agreement with our findings, who found the highest phenolic content in Hypericum

perforatum root culture having 30 and 50 g l-1 sucrose concentrations. However, findings

of Wu et al. (2006) are in contrast with our data and in partial agreement with Cui et al.

(2010). As both research groups quantified higher phenolic content at 50 g l-1 sucrose

concentration, whereas, Wu et al. (2006) reported higher amount of phenolic content at

90 g l-1 sucrose than lower sucrose levels. These findings suggest that sucrose induce

osmotic stresses depend upon plant species and culture type to synthesize various

metabolites of interest. Varying responses of plant and culture types for accumulating

secondary metabolites like phenolics against sucrose induce stresses may be due to

adoptive strategy to cope with the stress (Tan et al., 2004) involving biosynthesis of

valuable metabolites like phenols and flavonoids and other active compounds (Ali et al.,

2006). Induced phenolics in response to varied concentration of sucrose in sweet potato

(Ipomoea batatas), parsley (Petroselinum crispum) and petunia have also been reported

(Hahlbrock et al., 1971; Ranjeva et al., 1975; Solfanelli et al., 2006). Similarly, several

reports have been cited the accumulation of phenolics in callus cultures of many

pharmaceutically important medicinal plants (Schmeda-Hirschmann et al., 2005; Naz et

al., 2008; Giri et al., 2012). Similarly, cell culture of nine different species of Artemisia

and Silybum was found to be more productive for accumulating phenolic metabolites

(Riedel et al., 2010). Bhakuni et al. (2001) also reported phenolics and other important

metabolites in in vitro cultures of Artemisia annua. While, the accumulation of phenolic

content in callus, cell and adventitious root cultures for accumulation of phenolics have

been extensively studied in other medicinal plants (Bhakuni et al., 2001; Morkunas et al.,

60

2005; Riedel et al., 2010; Ferri et al., 2011; Ali et al., 2013), limited research work has

been conducted on optimization of sucrose concentration for the production of phenolic

content in callus, cell and adventitious root cultures of Stevia.

Effect of sucrose induced osmotic stress on flavonoids production

Total flavonoids content (TFC) was found to be sucrose dependent in calli, cell suspension

and adventitious root cultures of Stevia. An increasing trend in flavonoids production was

observed with increasing sucrose concentration in culture media.

The addition of 30 g l-1 sucrose significantly induced TFC (49.36 mg/g-DW) at maximal

level in callus cultures. However, cultures developed in media having 15, 20, 25, 35 and 40

g l-1 sucrose, yielded statistically similar amount of TFC as that of 30 g l-1 sucrose in

culture medium. Interestingly, callus culture developed in media containing above and

below 15-40 g l-1 sucrose did not maintain sufficient amount of TFC and the lowest

quantity of TFC (23.20 mg/g-DW) was found in calli obtained from media concentrated

with 05 g l-1 sucrose (Fig. 11a). Similarly, in cell suspension culture, sucrose dependent

production of flavonoid content was observed. The least sucrose content (05 g l-1) inhibited

flavonoids yield in cell suspension culture resulting in the minimum TFC (17.28 mg/g-

DW). As concentration of sucrose in culture media was increased from 05 to 10 g l-1, two-

fold increase in TFC (34.55 mg/g-DW) was observed. Further increase resulted in linear

increase in TFC accumulation, however, the maximum TFC (41.46 mg/g-DW) biosynthesis

was observed in cells cultured in medium having 40 g l-1 sucrose. However, the flavonoids

production was statistically similar cell suspension culture developed in media having 25-

50 g l-1 sucrose concentrations (Fig. 11b). By contrast, adventitious root culture did not

show statistical similarity for optimum accumulation of total flavonoid content. The

maximum total flavonoid content (94.78 mg/g-DW) was accumulated in roots, cultured in

medium concentrated with 30 g l-1 sucrose. Further increase or decrease of sucrose

concentration form 30 g l-1 significantly inhibited flavonoids production in Stevia

adventitious roots and the minimum total flavonoids content (23.20 mg/g-DW) was

recorded in adventitious roots, cultured in medium having 05 g l-1 sucrose (Fig. 11c).

Plant growth and metabolic activities depend on carbohydrates. Besides an energy source,

it regulates the expression of number of genes involved in plant growth and development

(Koch, 1996). Among carbohydrates, the sucrose has been reported as the most active one

61

in regulating vital processes like nitrogen assimilation, photosynthetic activities,

metabolites accumulation, respiration and linked genes modulation (Jang et al., 1997).

Moreover, sucrose helps in the defense mechanisms especially in various stress conditions

(Gazzarrini and McCourt, 2003; Gibson, 2004). Generally, genes involved in biosynthetic

pathway of flavonoids are very specific to plant, tissue and culture types (Dixon and Paiva,

1995; Ferri et al., 2009). Besides, endogenous signals like phytohormones and external

stimulus like biotic and biotic stresses, irradiations, light and sucrose are also known to

elicit the transduction of flavonoids linked genes (Tsukaya et al., 1991; Dixon and Paiva,

1995; Leyva et al., 1995; Mol et al., 1996; Laura et al., 2007; Ferri et al., 2009).

Comparative response was observed in our study that sucrose induced osmotic stress

enhanced the production of flavonoids in callus, cell and adventitious root cultures of

Stevia. Sucrose induces the biosynthesis of flavonoids in callus, cell and adventitious root

cultures. It may be due to the fact that flavonoids associated gene is sugar specific

(Solfanelli et al., 2006). Previously, Wong et al. (1974) and Thimann et al. (1950) also

stated that sugar mediates accumulation of flavonoid content by altering its main pathway.

The findings of our study are consistent with the previous studies regarding the

accumulation of the maximum flavonoids in several medicinally important plant cultures

with high concentrations of sucrose (Antognoni et al., 2007; Andreazza et al., 2009; Tan et

al., 2010). The induced flavonoids such as anthocyanin has also been recorded in

Arabidopsis, cultured in sucrose augmented medium (Ohto et al., 2001). There was a clear

association of flavonoids with sucrose concentration. The flavonoids production was low at

lower concentration but increased with the increasing levels of sucrose (30-50 g l-1) in all

cultures. However, declined in flavonoids concentration in callus and adventitious root

cultures was observed in current study. These findings are in consistency with the previous

work of Ferri et al. (2011), who reported that increasing sucrose concentration increases the

biosynthesis of flavonoid content of berries and cell culture of Vitis vinifera. The sugar

induced flavonoids accumulation in Vitis vinifera berries and cell culture might be due to

the enhanced expression of flavonoids interlinked with sugar signaling (Boss et al., 1996).

Similar trend of high flavonoids accumulation has also been reported with further

concentrating the medium with carbon source (Wan et al., 2015). Similarly, the findings of

Xiaohua et al. (2011), who observed the accumulation of rutin contents in buckwheat at 50

g l-1 sucrose, followed by decrease with 70 g l-1 sucrose concentrated medium are

comparable to the trend observed in this study.

62

20

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160c

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Sucrose concentrations (g l-1

)

05 10 15 20 25 30 35 40 45 50

Fig. 10. Effect of sucrose induced osmotic stress on accumulation of total phenolic

content (mg/g-DW) in callus, cell suspension and adventitious root cultures of Stevia

rebaudiana. Mean values (± S.E) with common alphabets are nonsignificant at P ≤ 0.05.

63

20

30

40

50 adcd

abca ababababc

bc

c

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20

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f

a-ea-d

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To

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/g-D

W)

0

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efg

c

a

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j


)

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Fig. 11. Effect of sucrose induced osmotic stress on accumulation of total flavonoids

content (mg/g-DW) in callus, cell suspension and adventitious root cultures of Stevia


64

Effect of sucrose concentrations on DPPH-radical scavenging activity

The addition of sucrose in culture media affected the DPPH-radical scavenging activities

in callus, cell suspension and adventitious root cultures of Stevia rebaudiana. Generally,

sucrose dependent DPPH-radical scavenging activities were observed in callus, cell

suspension and adventitious root cultures of Stevia rebaudiana. Among different sucrose

concentrations in culture media, lower concentration (05 g l-1) showed poor DPPH-

radical scavenging activity (DRSA; 34.31 %). However, increasing sucrose concentration

caused considerable increase in DRSA and the highest DRSA (92.82 %) was estimated in

callus cultures developed in media added with 30 g l-1 sucrose. Further increase of

sucrose concentration did not induce significant variation in DPPH-radical scavenging

activities (Fig. 12a). In cell suspension culture, there was the least potential (33.28%) to

scavenge free radicals (DPPH) at lower sucrose concentration (05 g l-1). However,

increase in sucrose concentration considerably enhanced DRSA. Among all

concentrations tested, cells cultured in media added with 30 g l-1 sucrose were found with

the maximum DRSA (83.87%). However, sucrose concentrations (25-45 g l-1) can be

regarded as the optimum levels for inducing DRSA in Stevia rebaudiana cell suspension

culture, because the activities at 25-45 g l-1 sucrose were statistically at par with each

other (Fig. 12b). The DRSA in adventitious roots showed slightly different growth

pattern against various sucrose concentrations in culture media. The DRSA (46.55 %)

observed in adventitious roots, developed in culture medium having 05 g l-1 sucrose were

increased with increase in sucrose concentration in culture media and was the highest

(94.43 %) in roots established in media augmented with 20 g l-1 sucrose. However,

further increase of sucrose did not enhance DRSA up to 40 g l-1 but beyond 40 g l-1,

DRSA tended to increase significantly once again (Fig. 12c).

Most of medicinal plants are rich sources of natural antioxidants, which scavenge toxic

free radicals, detrimental to human health causing cardiac problems, eyesight diseases,

inflammation, paralysis and dementia diseases (Yang et al., 2001; Sun et al., 2002;

Rehman et al., 2014). The Stevia rebaudiana plant has enormous antioxidant activity

(Kim et al., 2011; Tadhani et al., 2007). The results of this study confirmed the presence

of antioxidant activities in callus, cell and adventitious root cultures. Furthermore, the

potential of sucrose concentration to augment the antioxidant activity in all cultures was

65

also established. The steviol glycosides of Stevia plant contribute to the antioxidant

activities (Shukla et al., 2012; Criado et al., 2014), beside phenolics with antioxidant

activities (Abou-Arab et al., 2010; Lemus-Mondaca et al., 2012; Barba et al., 2014).

Several studies have been confirmed the correlation of polyphenol with antioxidant

activities of Stevia (Kim et al., 2011; Tadhani et al., 2007; Rao et al., 2014).

Furthermore, correlation between phenolics and antioxidant properties of Stevia has been

evaluated by Shukla et al. (2009), and later on reported by Zayova et al. (2013) and

observed that Stevia ethanol extracts have more antioxidant potential than Vitamin C.

The results of this study support the positive correlation of phenolic and flavonoid

contents with antioxidant activity in callus and cell suspension cultures of Stevia. As both

phenolic and flavonoid contents varied considerably with sucrose induced stress, the

addition, of high sucrose concentration in culture medium resulted in greater

accumulation of total phenolic and flavonoid contents in callus and cell suspension.

Similarly, greater DRSA was recorded with raising sucrose concentration in callus, cell

and even adventitious root cultures. These results are consistent with the previous studies

to accumulate the maximum flavonoid and phenolic contents in several medicinally

important plant cultures (Fu et al., 2005; Antognoni et al., 2007; Andreazza et al., 2009;

Tan et al., 2010). However, there are several other low molecular weight constituents

including vitamins, proteins and peptides having antioxidant activities (Foyer et al., 1995;

Park et al., 2005; Durak et al., 2013).

66

30

40

50

60

70

80

90

100


)

cc

dfe

dc

aabb

g

50454035302520151005

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DP

PH

-rad

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scav

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gin

g a

cti

vit

y (

%)

bbab

aaaabb

cd

e

20

40

60

80

100 a

a a a a a

b c

d e

f

Fig. 12. Effect of sucrose concentration on DPPH-radical scavenging activity (%) in

callus, cell suspension and adventitious root culture of Stevia rebaudiana. Mean values (±

S.E) with common alphabets are nonsignificant at P ≤ 0.05.

67

Correlation of phenolics and flavonoids with DPPH-radical scavenging activity

The addition of sucrose significantly increased the phenolics, flavonoids and DPPH-

radical scavenging activity (DRSA) in callus, cell suspension and adventitious roots

cultures of Stevia rebaudiana (Fig. 10, 11 and 12). In callus cultures, the DRSA was

found to be dependent on phenolics and flavonoids production. Significantly, higher

amount of phenolics (124.20 mg/g-DW) and flavonoids (49.36 mg/g-DW) content were

recorded with medium containing 30 g l-1 sucrose, with the maximum DRSA (92.82%).

However, further increase in sucrose resulted in no significant increase in DRSA but both

TPC and TFC contents were significantly decreased (Fig. 13). The DRSA in cell culture

was found to be strictly dependent on flavonoids content as compared to phenolics

content. Because significant higher profile of total flavonoids contents were found in cell

cultures, established in media having (20-50 g l-1) sucrose. Meanwhile, cell cultures

developed in media added with 25-45 g l-1 sucrose also exhibited statistically higher

antioxidant potentials. Although positive correlation was noticed between total phenolics

content and DRSA in cell culture at 40 g l-1 sucrose. As cell culture also accumulated

higher amount of total phenolics content (139.20 mg/g-DW) at 40 g l-1 sucrose resulted in

high antioxidant potentials (Fig. 14). In contrast to callus and cell culture, antioxidant

activity in adventitious root cultures was found independent of phenolics and flavonoids

content. The maximum quantity of total phenolics (155.00 mg/g-DW) and flavonoids

(94.78 mg/g-DW) was in cultures developed at media supplemented with 30g l-1 sucrose.

On the other hand, enhanced DRSA (94.43 %) was observed in adventitious root cultures

developed in media added with 20 g l-1 sucrose (Fig. 15).

The anti-oxidizing components of the plants help to detoxify free radical effects (Ahmad

et al., 2010). The role of antioxidants has been cited in literature against stressful

conditions (Cozzi et al., 1997). In vitro cultures have the ability to synthesize such

valuable compounds. In vitro cultures have been investigated for antioxidant potentials

(Shukla, 2009) and has been attributed to the presence of polyphenols in Stevia (Bidchol

et al., 2011) and other plants (Jayasinghe et al., 2003; Ali et al., 2006; Kim et al., 2006;

Ali et al., 2007; Al Khateeb et al., 2012; Amid et al., 2013). A positive correlation among

antioxidant activities/DRSA and polyphenolics in callus and cell suspension cultures was

68

observed in this study. These results are further supported by the findings of

Canadanovic-Brunet et al. (2005), who reported positive correlation between phenolics

and flavonoids with antioxidant potentials of Artemisia plants. Similarly, Bajpai et al.

(2005) also suggested a positive correlation between TPC and antioxidant potential in a

number of medicinal plants. Recently, in vitro cultures of several other plants have been

evaluated for antioxidant potentials in relation to phenolics content. Giri et al. (2012)

reported phenolics dependent antioxidant activities in callus culture of Habenaria

edgeworthii. Similarly, Diwan et al. (2012) observed positive correlation between

antioxidant activities and TPC in cell suspension culture of Ruta graveolens. However,

antioxidant activity in adventitious root cultures was independent of phenolics and

flavonoids content. This might be due the fact that beside phenolics and flavonoids

several other non-enzymatic antioxidant compounds such as proteins, vitamins and

antioxidant enzymes (super oxide dismutase, per oxide dismutase etc.), contribute to the

antioxidant activities in plants (Head, 1996; Halliwell, 1998).

Fig. 13. Correlation of total phenolic and flavonoids content with DPPH-radical

scavenging activity in callus cultures of Stevia rebaudiana. Mean values (± S.E) with

common alphabets are nonsignificant at P ≤ 0.05.

20

40

60

80

100

aaaaa

b

abdcdabcabababcbc

c

gf

de

DPPH

TFC

TPC

20

40

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cd

e

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a

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DP

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-rad

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)

Tot

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lavo

noid

con

tent

(m

g/g-

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)

Tot

al p

heno

lic

cont

ent (

mg/

g-D

W)

05 10 15 20 25 30 35 40 45 50


)

69


scavenging activity in cell suspension cultures of Stevia rebaudiana. Mean values (± S.E)

with common alphabets are nonsignificant at P ≤ 0.05.


scavenging activity in adventitious root cultures of Stevia rebaudiana. Mean values (±


20

40

60

80bab

aaa

e

a-ea-da-daababcc-ede

DPPH

TFC

TPC

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abb

cd

e

f

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ddde

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on

oid

co

nte

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(mg

/g-D

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To

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enoli

c co

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(mg

/g-D

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-rad

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ven

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)


)

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)

c

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e

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cba

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i

Tot

al f

lavo

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tent

(m

g/g-

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)

Tot

al p

heno

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(mg/

g-D

W)

05 10 15 20 25 30 35 40 45 50

70

Effect of sucrose induced osmotic stress on steviol glycosides production

Stevia plant is famous for its sweet contents commonly known as steviol glycosides. The

leading compounds in steviol glycosides are stevioside, rebaudioside and dulcoside. In

this study, callus, cell suspension and adventitious root cultures were evaluated against

various sucrose concentrations. Significantly different responses of each culture for each

active compound were found with varying sucrose concentrations in culture media.

Callus cultures exposed to various sucrose concentrations resulted in variations in the

production of stevioside, rebaudioside and dulcoside content. Callus cultures treated with

05 g l-1 sucrose yielded the least stevioside (21.17 mg/g-DW), rebaudioside (6.23 mg/g-

DW) and dulcoside (0.01 mg/g-DW). The stevioside content was the maximum (42.34

mg/g-DW) in callus culture supplemented with 10 g l-1 sucrose. Statistically similar

amount of stevioside (41.89 mg/g-DW) and (41.45 mg/g-DW) was also observed in

callus cultures at 15 and 20 g l-1 sucrose, respectively, in culture media. Further increase

in sucrose concentration in culture media significantly reduced the stevioside

accumulation in callus cultures (Fig. 16a). On the other hand, callus culture accumulated

considerably higher amount of rebaudioside content (22.67 mg/g-DW) in media

concentrated with 20 g l-1 sucrose that was statistically at par with the rebaudioside

content (21.43 mg/g-DW) in callus culture medium having 25 g l-1 sucrose (Fig. 16b).

Conversely, slightly higher sucrose concentration (30 g l-1) significantly increased the

dulcoside content (6.56 mg/g-DW) in callus cultures. However, significant decline was

observed with more statistical similarities in other cultures (Fig. 16c).

Accumulation of stevioside, rebaudioside and dulcoside did not follow the same pattern

against sucrose levels in cell suspension culture. Whereas, the least concentrations of

sucrose (05 g l-1) resulted in the least stevioside content (20.16 mg/g-DW) in cell

suspension culture and 2-fold increase in stevioside content (40.32 mg/g-DW) was

observed in the media supplemented with 10 g l-1 sucrose. However, further increase of

sucrose concentration significantly reduced the quantity of steviosides in cell cultures but

again a sudden increase in stevioside content (42.23 mg/g-DW) was observed by using 30

g l-1 sucrose in culture media (Fig. 17a). Similarly, rebaudioside content in cell

suspension cultures were significantly influenced by sucrose induce stresses. The

rebaudioside was not inhibited by lower sucrose concentrations in cell cultures like other

71

compounds but as the concentrations were increased from 25 g l-1, the rebaudioside

contents were significantly reduced. The lowest rebaudioside content (7.97 mg/g-DW)

was recorded at the highest sucrose concentration (50 g l-1). In contrast, the highest

rebaudioside content (27.64 mg/g-DW) was calculated in cells cultured in medium

fortified with 20 g l-1 sucrose (Fig. 17b). Likewise, sucrose levels also significantly

affected dulcoside contents in cell suspension cultures. The maximum dulcoside content

(6.43 mg/g-DW) were found in cells cultured in medium having 20 g l-1 sucrose, while

further increase or decreases drastically reduced the accumulation of dulcoside contents

in cell cultures, while 40, 45 and 50 g l-1 sucrose concentrations were not able to

synthesize dulcoside contents (Fig. 17c).

Like callus and cell suspension cultures, the adventitious root cultures also showed

considerable variations in biosynthesis of steviol glycosides (stevioside, rebaudioside and

dulcoside) exposed to various sucrose concentrations in culture media. Almost similar

pattern for both stevioside and rebaudioside accumulation in adventitious root cultures of

Stevia against various sucrose levels was observed. The maximum accumulation of

stevioside (73.97 mg/g-DW) and rebaudioside contents (24.57 mg/g-DW) was found in

roots taken from media containing 10 g l-1 sucrose. Both contents were significantly

reduced with further increase in sucrose concentration in the media. By contrast, the

minimum stevioside (25.58 mg/g-DW) and rebaudioside (10.02 mg/g-DW) contents were

found in adventitious roots obtained from media supplemented with 20 g l-1 sucrose.

However, again a linear increase was observed in stevioside and rebaudioside with

further increase of sucrose in culture media up to 40 g l-1 followed by a gradual decrease

at 45 and 50 g l-1 sucrose (Fig. 18 a and b). In case of dulcoside content in adventitious

root culture of Stevia, the minimum (0.10 mg/g-DW) was recorded in roots in media

having 05 g l-1 sucrose, while the maximum dulcoside content (12.24 mg/g-DW) were

obtained at 40 g l-1 sucrose (Fig. 18c).

The pharmaceutical importance of medicinal plants mainly depends on accumulation of

valuable secondary metabolites (Selmar and Kleinwächter, 2013). It is therefore,

important to understand and optimize the role of stress induce elicitors like sucrose, pH,

light and several others in biosynthesis of these medicinally important compounds (Zhao

et al., 2005; Vasconsuelo and Boland, 2007). Sucrose has been reported as a key elicitor

in regulation of secondary metabolites of various medicinal plants (Fowler, 1983). In the

72

current findings, it was also observed that steviol glycoside (stevioside, rebaudioside,

dulcoside) contents varied considerably with sucrose concentrations in the media. There

is little information on the enzymes encoded by various genes, which are actively

involved in biosynthetic pathway of steviol glycoside (Richman et al., 1999; Brandle and

Telmer, 2007; Yadav and Guleria, 2011). Generally, sucrose mediates the release of

energy and alters metabolic process in plants and in vitro cultures by regulating gene

expression (Koch, 1996). According to Guleria et al. (2011), exogenous application of

sucrose increased the expression of genes, involved in biosynthetic pathway of steviol

glycoside. However, optimize concentration has key importance because slight increase

or decrease from the optimum level adversely affect the whole metabolic processes.

Responses of plant cell, tissue and organ cultures to varying sucrose concentrations has

been attributed to the fact that plants anatomical, morphological, physiological features

and genes expression levels are highly dependent on sucrose concentration (Koch, 1996;

Loreti et al., 2001). Similarly, the response of several medicinal plants was evaluated in

various sucrose concentrated media that resulted in the maximum accumulation of

alkaloids within a certain limit of sucrose concentrations (Vazquez-Flota et al., 1994).

The results of this study are in line with the findings of Paiva and Janick (1983), who

observed a linear increase in secondary metabolites (alkaloids, anthocyanin, fats) with the

increasing sucrose levels in embryo culture of Theobroma cacao. Likewise, biosynthesis

of rosmarinic acid was also enhanced in cell culture of Coleus blumei with elevated

sucrose levels in culture media (Misawa, 1985). Furthermore, Knobloch and Berlin

(1980) also optimized 8% sucrose concentration among tested range of sucrose (4-12%,

w/v) for biosynthesis of alkaloids in cell culture of Catharanthus roseus in higher

amounts. Similarly, several fold increase was observed in synthesis of specific alkaloids

in cells of Eschscholtzia californica cultured in suspension with 8% sucrose (Berlin et al.,

1983). Accumulation of anthocyanin was also induced in cell culture of grapes (Vitis

vinifera) with sucrose induced osmotic stresses (Do and Cormier, 1990). Results of

Sakamoto et al. (1993) are also in conformity to findings, who recorded more

anthocyanin contents in cells, cultured in media having 3% sucrose. However, further

increase in sucrose did not encourage anthocyanin yield in cell culture of Aralia cordata.

Generally, metabolites biosynthesis are tissue and culture specific, showing different

responses to varying concentrations of sucrose. In this regard, we obtained the maximum

concentration of stevioside and rebaudioside at 10 g l-1 sucrose in adventitious root

73

culture of Stevia rebaudiana. However, dulcoside contents were favored by 40 g l-1

sucrose in cultured medium. Although, there is no report available on the effect of

sucrose on secondary metabolites accumulation in adventitious root culture of of Stevia

but in hairy root culture of Withania somnifera withanolide A was enhanced at 4%

sucrose in culture media (Lulu et al., 2015). Reports are also available on glycyrrhizin

biosynthesis on higher levels in hairy roots of Glycyrrhiza inflata cultured in media

supplemented with 6% sucrose (w/v) (Wongwicha et al., 2011).

74

0

1

2

3

4

5

6

7

ccc

a

bb

cccc

Dulc

osid

e c

onte

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-DW

)


)

05 10 15 20 25 30 35 40 45 50

c

10

20

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h

f

ede

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abaa

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vio

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) a

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bcab

a

d

e

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Reb

au

dio

sid

e c

on

ten

t (m

g/g

-DW

) b

Fig. 16. Effect of sucrose concentration on stevioside, rebaudioside and dulcoside in

callus culture of Stevia rebaudiana. Mean values (± S.E) with common alphabets are


75

0

10

20

30

40

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ddd

b

a

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lco

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ten

t (m

g/g

-DW

) c


)

05 10 15 20 25 30 35 40 45 50

Fig. 17. Effect of sucrose concentration on stevioside, rebaudioside and dulcoside in cell

culture of Stevia rebaudiana. Mean values (± S.E) with common alphabets are


76

0

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30

40

50

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70

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fe

bde

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) a

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f

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c

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hh

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lco

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on

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-DW

)


)

05 10 15 20 25 30 35 40 45 50

c

Fig. 18. Effect of sucrose concentration on stevioside, rebaudioside and dulcoside in

adventitious root culture of Stevia rebaudiana. Mean values (± S.E) with common


77


The research work entitled “sucrose-induced osmotic stress improved biomass and

production of antioxidant secondary metabolites in callus, cell suspension and

adventitious root cultures of Stevia rebaudiana (Bert.) was conducted at Plant Tissue

Culture Lab., Department of Plant Breeding and Genetics, The University of Agriculture

Peshawar, during the year of 2014-15. The research work was aimed to evaluate the

effect of sucrose concentration on callus; cell suspension and adventitious root cultures

growth (growth kinetics, fresh and dry biomasses), production of important secondary

metabolites (phenolics, flavonoids, stevioside, rebaudioside and dulcoside contents) as

well as antioxidant activities. For this purpose, sterilized leaf explants were placed on

Murashige and Skoog (MS) medium supplemented with PGRs (2, 4-D, 2.0 mg l-1; BA,

0.5 mg l-1) and sucrose (30 g l-1) for callus induction. The established friable calli was

then inoculated in medium without gelling agent supplemented with 2, 4-D (1.0 mg l−1)

and BA (0.5 mg l−1) for development of cell suspension culture. For establishment of

adventitious root cultures, newly collected seeds of Stevia were in vitro germinated and

roots were shifted to half MS liquid medium containing 0.5 mg l−1 NAA. Each culture

was exposed to varying concentrations of sucrose (05, 10, 15, 20, 25, 30, 35, 40, 45 and

50 g l−1) according to Complete Randomized Design (CRD). During growth kinetics, a

growth curve was established for accumulated biomass for each treatment with 3 days

interval for a period of 30 days.

Various sucrose concentrations (05-50 g l-1) in culture media significantly influenced

biomass accumulation and secondary metabolites production in callus, cell suspension

and adventitious root cultures of Stevia. Among various sucrose concentrations, callus

accumulated significantly higher amount of fresh (142.38 g l-1) and dry (11.71 g l-1)

biomass in media having 40 g l-1 and 50 g l-1 sucrose, respectively. However, lower

sucrose concentration (05 g l-1) in culture media resulted in minimum fresh and dry

biomass (60.10 g l-1; 2.67 g l-1) in callus cultures of Stevia. Similarly, among different

sucrose concentrations (05-50 g l-1), 30 g l-1 sucrose was found the optimum for the

production of maximum total phenolics content (TPC; 124.20 mg/g-DW), total

flavonoids content (TFC; 49.36 mg/g-DW), rebaudioside contents (6.56 mg/g-DW) and

78

DPPH-radical scavenging activity (DRSA; 92.82 %) in callus cultures. However,

significantly higher amount of stevioside (42.34 mg/g-DW) and rebaudioside (22.67

mg/g-DW) contents were observed in callus cultures at 15 and 20 g l-1 sucrose,

respectively. On the other hand, callus cultures, established in media having 05 g l-1

accumulated significantly lower amount of TPC (42.60 mg/g-DW), TFC (23.20 mg/g-

DW), DRSA (34.31 %), stevioside (21.17 mg/g-DW), rebaudioside (6.23 mg/g-DW) and

dulcoside (0.01 mg/g-DW) contents.

In cell suspension cultures addition of 20 g l-1 sucrose resulted in maximum fresh (97.71

g l-1) and dry (8.57 g l-1) biomass accumulation, while minimum fresh (25.43 g l-1) and

dry biomass (4.57 g l-1) were observed at 50 g l-1 and 05 g l-1 sucrose, respectively.

However, the highest TPC and TFC (139.20 mg/g-DW; 41.46 mg/g-DW) in cell cultures

were accumulated at 40 g l-1 sucrose, while 05 g l-1 sucrose resulted the least TPC (51.34

mg/g- DW), TFC (17.28 mg/g-DW) and DRSA (33.28%). However, cells cultured in

media having 30 g l-1 sucrose were found with the maximum DRSA (83.87%). Lower

concentrations of sucrose (05 g l-1) also strictly inhibited biosynthesis of stevioside (20.16

mg/g-DW) in cell suspension culture. Increase in stevioside content (40.32 mg/g-DW)

was noticed as the media was supplemented with 10 g l-1 sucrose. However, further

increase of sucrose concentration significantly reduced steviosides in cell cultures but

again a sudden increase in stevioside content (42.23 mg/g-DW) was observed at 30 g l-1

sucrose. On the other hand, the lowest rebaudioside content (7.97 mg/g-DW) was

quantified at 50 g l-1 sucrose. In contrast, the highest rebaudioside (27.64 mg/g-DW) and

dulcoside contents (6.43 mg/g-DW) were observed at 20 g l-1 sucrose. However, 40, 45

and 50 g l-1 sucrose concentrations were not able to synthesize dulcoside contents in

cultured cells.

Similarly, in adventitious root cultures, 50 g l-1 sucrose was optimized for maximum

fresh (175.43 g l-1) and dry (11.14 g l-1) biomass. However, minimum fresh and dry

biomass (37.71 g l-1; 2.86 g l-1) was observed at 05 g l-1 sucrose in culture media.

Similarly, 30 g l-1 sucrose was optimized for the highest TPC (155.00 mg/g-DW) and

TFC (94.78 mg/g-DW) in adventitious roots. The lowest TPC (17.77 mg/g-DW), TFC

(23.20 mg/g-DW) and DRSA (46.55 %) was observed at 05 g l-1 sucrose. However, the

79

highest DRSA (94.43 %) was noted at 20 g l-1 sucrose. Moreover, maximum stevioside

(73.97 mg/g-DW) and rebaudioside (24.57 mg/g-DW) contents were found in roots at 10

g l-1 sucrose. Both contents were significantly reduced in further concentrated media and

the minimum stevioside (25.58 mg/g-DW) and rebaudioside (10.02 mg/g-DW) contents

were found with 20 g l-1 sucrose. However, minimum dulcoside content (0.10 mg/g-DW)

was recorded in roots in media having 05 g l-1 sucrose, while maximum dulcoside content

(12.24 mg/g-DW) was obtained at 40 g l-1 sucrose.

Conclusions

In conclusion, it was found that addition of 2.0 mg l-1 2, 4-D and 0.5 mg l-1 BA

was found superior for callus induction from leaf explant and proliferation on

Murashige and Skoog (MS) media. Similarly, liquid media having 2, 4-D (1.0 mg

l−1) and BA (0.5 mg l−1) was found to be more effective for cell suspension

culture development. Half MS liquid media augmented with 0.5 mg l−1 NAA was

proven to be the best for adventitious root culture development.

Furthermore, addition of sucrose at various concentrations acted as effective

elicitors for inducing significant variations in biomass accumulation as well as

targeted secondary metabolites in callus, cell suspension and adventitious root

cultures of Stevia rebaudiana.

Addition of sucrose at the rate of 40 and 50 g l-1 was found optimum for higher

accumulation of fresh (142.38 g l-1) and dry (11.71 g l-1) biomasses, respectively

in callus cultures. However, liquid media having 20 g l-1 sucrose resulted in

maximum fresh (97.71 g l-1) and dry (8.57 g l-1) biomass in suspension culture. In

contrast, adventitious root cultures favored higher concentration of sucrose (50 g

l-1) for the maximum accumulation of fresh (175.43 g l-1) and dry (11.14 g l-1)

biomasses.

Similarly, callus and adventitious root cultures yielded significantly higher

amount of TPC (124.20 mg/g-DW; 155.00 mg/g-DW) and TFC (49.36 mg/g-DW;

94.78 mg/g-DW), respectively as a result of 30 g l-1 sucrose in cultures media.

However, cell suspension culture accumulated maximum TPC (139.20 mg/g-DW)

and TFC (41.46 mg/g-DW) at 40 g l-1 sucrose in culture media. Moreover,

80

addition of 30 g l-1 sucrose was found to be superior for enhanced antioxidant

activities in callus (92.82 %) and cell suspension (83.87%) cultures. However,

adventitious root culture developed in media having 20 g l-1 sucrose exhibited the

highest antioxidant potential (94.43 %).

Significantly higher amount of stevioside (42.34 mg/g-DW) content in callus

culture was observed with the addition of 10 g l-1 sucrose in culture media. On the

other hand, cell suspension and adventitious root cultures accumulated

considerably higher quantities of stevioside contents (42.23 mg/g-DW; 73.97

mg/g-DW) at 30 and 10 g l-1 sucrose, respectively. Callus, cell suspension and

adventitious root cultures accumulated higher amount of rebaudioside contents

(22.67 mg/g-DW), (27.64 mg/g-DW), (24.57 mg/g-DW) at 20, and 10 g l-1 sucrose,

respectively. Similarly, the highest amount of dulcoside contents in callus (22.67

mg/g-DW) and cell suspension (6.43 mg/g-DW) was found at 20 g l-1 sucrose,

while in adventitious root cultures (12.24 mg/g-DW), addition of 40 g l-1 was

found optimum sucrose level.

Recommendations

On the basis of above conclusion, it is recommended that media should be

supplemented with 2, 4-D (2.0 mg l−1) and BA (0.5 mg l−1) for efficient callus,

cell suspension (2, 4-D, 1.0 mg l−1; BA 0.5 mg l−1) and adventitious root (0.5 mg

l−1 NAA; half MS media) cultures development.

Similarly, 40 and 50 g l-1 sucrose should be added to culture media for the

maximum fresh and dry biomass yield in callus cultures. Furthermore, for

optimum fresh and dry biomass accumulation in cell suspension and adventitious

root cultures, liquid media should be concentrated with 20 g l-1 and 50 g l-1

sucrose, respectively.

For maximum TPC and TFC accumulation, 30 g l-1 sucrose is recommended as an

optimum level in callus and adventitious root cultures, while 40 g l-1 sucrose

should be used in liquid media for higher TPC and TFC production in cell

suspension culture.

81

Similarly, 30 g l-1 sucrose should be used in callus and adventitious root cultures,

while 20 g l-1 in cell suspension culture media for the highest antioxidant

potentials.

Callus culture needs 10 g l-1 sucrose, while cell suspension and adventitious root

cultures require 30 and 10 g l-1 sucrose, respectively to obtain maximum

stevioside content. Similarly, 30, 20 and 10 g l-1 sucrose should be added to

culture media for larger quantities of rebaudioside contents in callus, cell

suspension and adventitious root culture, respectively. Addition of 20 g l-1 sucrose

is also recommended for enhanced accumulation of dulcoside in callus and cell

suspension cultures. However, for enhanced dulcoside production, 20 g l-1 sucrose

is an optimum dose in adventitious root cultures.

82

CHAPTER-V

The influence of pH on the development of callus, cell suspension and

adventitious root cultures and production of Steviol glycosides in Stevia

rebaudiana (Bert.)


Abstract

In vitro grown cultures require an optimum pH level for biomass accumulation and

production of secondary metabolites. For this purpose; calli, cell suspension and

adventitious root cultures of Stevia rebaudiana were established to evaluate the effect of

pH levels (5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0) on culture growth and

secondary metabolites. Varying media pH significantly influenced fresh as well dry

biomass of callus, cell suspension and adventitious root cultures. The biomass

accumulation was determined at 3 days interval for a period of 30 days. Shorter lag phase

of 3 days was observed for callus cultures, developed in media having pH 4.0, 5.9 and 6.0.

However, the rest of the cultures skipped the lag phase and followed prolonged log phases

of 27 days. Similarly, cell suspension cultures displayed shorter lag phase of 3 days on

media adjusted with pH 5.6 and 5.7. Relatively longer lag phase of 9 days was adopted by

culture developed in media with 5.8 pH level. Lag phase in all three cultures was followed

by exponential phase till day 27 of the growth study. Remaining cell cultures started

growth and entered into log phases (27 days) without any lag phases. Relatively shorter

lag phases of 3 days were found in adventitious roots, developed at various media and pH

levels. Media pH 5.1 displayed longer lag phase of 18 days with poor biomass

accumulation. Roots obtained from media having pH 5.4 and 5.2 did not adopt lag phases.

In almost all cultures log phases were followed by decline phases. However, root growth

was not restricted in cultures having pH 5.2 and 5.4 even after 27 days of the culture. The

highest fresh (130.57 g l-1) and dry biomasses (12.10 g l-1) of callus cultures were

observed at 5.6 pH. Similarly, fresh (85.81 g l-1) and dry (8.84 g l-1) biomasses of cell

suspension cultures were found maximum at 5.6 and 5.7 pH levels, respectively. However,

adventitious root cultures favored high media pH (6.0) among tested levels for

accumulation of maximum fresh (112.86 g l-1) and dry biomass (8.29 g l-1). Similarly, the

accumulation of total phenolic content (TPC) in all three cultures was also significantly

influenced by changing the pH of the media. The maximum TPC in callus (43.38 mg/g-

DW), cell suspension (72.13 mg/g-DW) and adventitious root cultures (70.06 mg/g-DW)

was observed on media having 5.8 pH level. Significantly higher amount of total

flavonoid content in callus (37.55 mg/g-DW), cell suspension (57.32 mg/g-DW) and

adventitious root cultures (50.19 mg/g-DW) was observed by using low acidic level (pH

5.8). Furthermore, all tested cultures favored 5.8 pH level and also showed maximum

antioxidant activities in callus (87.68 %), cell suspension (93.99 %) and adventitious root

cultures (92.67 %) as compared to other pH levels. Medium pH 5.6 was found optimum

for stevioside production (62.20 mg/g-DW) in callus cultures. On the other hand, cell

suspension culture yielded maximum amount of stevioside (41.47 mg/g-DW) at initial

medium pH 5.2. Similarly, maximum content of stevioside (79.48 mg/g-DW) was

83

observed in culture developed on medium having initial medium pH (5.1). Rebaudioside

content was also influenced by initial medium pH and significantly higher amount of

rebaudioside content (22.79 mg/g-DW) was found in callus cultures inoculated in medium

having pH level 5.6. Moreover, the highest amount of rebaudioside, contents was

quantified in cell suspension (7.01 mg/g-DW) and adventitious root cultures (13.10 mg/g-

DW) established at pH level 5.8 and 5.1, respectively. Callus culture favored low pH

level (5.1) for accumulation of dulcoside content (5.92 mg/g-DW). However, dulcoside

contents in cell suspension (4.72 mg/g-DW) and adventitious root cultures (2.57 mg/g-

DW) were found in higher quantities at pH level 5.8. This study will help in understanding

the role of pH on the development of callus, cell suspension and adventitious root cultures

in Stevia rebaudiana for biomass accumulation and commercially important metabolites

production on commercial basis.

84

INTRODUCTION

The presence of various bioactive compounds in different parts of the medicinal plants is

gaining attention as cure for various diseases. The demand of plants having the potential

to synthesize natural sweeteners is increasing day by day as additive in food and

pharmaceutical products. The natural sweeteners are low caloric and are natural

substitutes of sucrose, which are commonly used in dietary products. Stevia rebaudiana

is one of the potent perennial herbs of Asteraceae family, commonly known as sweet leaf

for its natural sweeteners (Savita et al., 2004; Lemus-Mandaca et al., 2012). It is

originated in Paraguay and Brazil but, nowadays, Stevia is commercially grown in

various countries of Asia, Europe and America (Brandle et al., 1998). The natural sweet

contents of Stevia plants are diterpene glycosides consisting of stevioside, being the most

abundant and rebaudioside A, B, C etc. Stevia plant is also a potent source of other

important health promoting agents, and food components like essential and non-essential

amino acids, carbohydrates, minerals, polyphenol and flavonoid contents (Madan et al.,

2010).

The presence of pharmaceutically valuable natural, non-caloric sweetening agents has

increased demand of Stevia plant. However, the commercial cultivation of this plant is

limited by poor seed germination, and poor response to asexual propagation through stem

cutting (Debnath et al., 2006; Taware et al., 2010). Therefore, in vitro propagation is an

alternative technique for the clonal production of Stevia plant for commercial usage

(Sairkar et al., 2009). Meanwhile, in vitro clonal multiplication of Stevia provides an

opportunity to propagate it from leaf (Ali et al., 2010; Preethi et al., 2011a, b), nodal,

inter-nodal (Uddin et al.. 2006; Ahmed et al., 2007; Verma et al., 2008; Sairkar et al.,

2009) and shoot tip explants throughout the year (Anbazhagan et al., 2010; Das et al.,

2011). However, the increasing world population and decrease in the available cultivable

land, requires the use of modern techniques for crop improvement (Rao and Ravishankar,

2002). The increasing demand for the commercially important bioactive compounds that

are used in preparation of medicines, food and dietary products, has increased the interest

of the researchers in medicinal plants and methods are explored to accelerate the

85

synthesis of commercially valuable plant secondary metabolites (Wilson and Roberts,

2012).

The in vitro culture (callus, cell and adventitious root cultures) have been used for

uniform and quality production of medicinally important bio-active compounds. The

callus, an unorganized, undifferentiated mass of cells, is produced by dedifferentiation of

explant cell with the help of plant growth regulators (Bhojwani and Razdan, 1996).

Callus culture can be used to synthesize and release important secondary metabolites

(Fowler et al., 1993). Similarly, in vitro cell suspension is another convenient and

efficient method for the production of plant secondary metabolites on commercial basis

(Roberts, 2007). Besides callus and cell culture, adventitious root culture is another

important biotechnological approach for the quick proliferation of the culture material

and commercial production of secondary metabolites (Yu et al., 2005). Adventitious

roots, induced in medium having appropriate plant growth regulators have been widely

exploited for industrial purposes (Murthy et al., 2008).

However, the biosynthetic pathways of secondary metabolites are markedly influenced

by various factors like light, humidity, nutrients, medium pH, etc. Mostly, biotic and

abiotic stresses stimulate the accumulation of these bioactive metabolites in higher

quantities (Eilert, 1987; Barz et al., 1988; Sivanandhan et al., 2012). The regular and

reliable production of plant secondary metabolites in callus, cell suspension and

adventitious root cultures are markedly influenced by different kinds of elicitors. Such

elicitations are widely studied in cell culture of Panax ginseng, Uncaria tomentosa and

Artemisia absinthium (Huang et al., 2013; Ali and Abbasi, 2014). Elicitation is being one

of the most effective strategies to alter the biosynthesis of these phyto-based products

(Wang et al., 2004; Ali et al., 2014).

Among the different elicitors, pH is one of the most influential media components for the

growth, development and secondary metabolites production in cell, tissue and organ

cultures (Williams et al., 1990). Efficient growth, development and metabolites

production of different plants require specific optimal pH. The optimal pH of the culture

medium also varies with different morphogenetic phases like root and shoot induction

86

(Ostrolucka et al., 2004). An undesirable pH of the medium may result in abnormalities

in growth and morphogenetic responses (Gurel and Gulsen, 1998; Laukkanen et al.,

2000). While the pH of the media, generally, changes due to autoclaving and heat

sterilization. The initial pH adjustment strongly regulates the nutrients uptake and

metabolic activities of in vitro cultures (Rossi-Hassani and Zryd, 1995). Furthermore, the

cellular pH adjustment, cellular growth, gene expression and transcription are remarkably

regulated by the medium pH (Lager et al., 2010).

Keeping in view the importance of valuable secondary metabolites and in vitro culture

techniques, the aim of the current study was to evaluate the relationship of culture

biomass, total phenol and flavonoids content; antioxidant potentials and steviol

glycosides in response to various pH levels in callus, cell suspension and adventitious

root cultures of Stevia rebaudiana.

87


Seed collection and explant selection

Fresh viable seeds of Stevia rebaudiana were collected from plants grown in Ground and

Garden Nursery, Department of Horticulture, The University of Agriculture Peshawar.

Two types of seeds were observed during seed collection. One is whitish color and the

other was black color seed. The black-coated seeds were selected for germination while

the whitish seeds were discarded due to incomplete or missing embryo. Freshly harvested

seeds were surface sterilized prior to inoculation according to the method of Ahmad et al.

(2014). Surface sterilized seeds were cultured on Murashige and Skoog (1962) medium

without any plant growth regulators (PGRs). However, it was further concentrated with

30 g l-1 and 7-8 g l-1 agar as gelling agent. Media pH was adjusted at 5.5-5.8 and sterilized

through autoclaving at 121 °C for 20 min providing 15 psi pressure. All in vitro cultured

seeds were placed in 16/8 h photoperiod for one month. After seed germination, different

explants were selected according to the requirements of experiments.

Application of different pH levels for callus development

The leaf explants of in vitro germinated seedlings were used for callus development. The

leaf explants were placed on MS medium augmented with 2, 4-D (2.0 mg l−1) and BA

(0.5 mg l−1) for callus induction. Induced calli was further used as inoculum for further

experiments. The same procedure was used for media sterilization and growth conditions

as discussed earlier. For optimization of desirable pH levels for culture growth and

secondary metabolites production, callus cultures were exposed to various pH levels (5.1,

5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0). For this purpose each culture was treated as

an individual experiment, arranged in completely randomized design. All cultured flasks

were placed in growth chamber with light intensity of 40 m m-2 s-1 under 16/8 h

photoperiod for one month. In order to investigate the effects of various pH levels on

biomass accumulation, a define growth curve was established for each treatment. Data

regarding growth kinetics was recorded at 3 days interval for a period of 30 days. Fresh

calli was harvested after 30 days and used for fresh and dry weight determination. The

calli treated with different pH levels was also used for different activities.

88

Application of different pH levels for cell culture development

Friable and whitish soft callus was selected for establishment of cell suspension culture

(stock) in liquid media for further use. Friable calli was shifted to liquid MS media

augmented with 2, 4-D (1.0 mg l−1) and BA (0.5 mg l−1). The pH of the media was

adjusted as T1 (5.1), T2 (5.2), T3 (5.3), T4 (5.4), T5 (5.5), T6 (5.6), T7 (5.7), T8 (5.8), T9

(5.9) and T10 (6.0). The cultures were placed on orbital shaker at 120 rpm for 30 days

period. The cultures were placed in dark room under controlled conditions for the

development of cell suspension cultures. To test the effects of various pH levels on fresh

biomass accumulation, the growth curve was established for rapidly growing cells. The

biomass accumulation was determined with 3 days interval for a period of 30 days. The

growth curve was divided into lag, log and decline phases on the basis of visual

observation from growth curve of each treatment. Further, the fresh cells were harvested

for different activities and determination of fresh and dry biomass.

Application of different pH levels for adventitious root development

The roots from one-month-old in vitro germinated Stevia plants were collected and

inoculated in half strength MS medium (1962) having 0.5 g l-1 NAA without

incorporating agar as solidifying agent. Cultured flasks were placed in dark on orbital

rotary shaker for development of stock adventitious root culture for 15 days. After stock

root development, a known amount of inoculum roots were transferred to flasks

containing liquid MS media and 0.5 g l-1 NAA. The pH of the media was adjusted as T1

(5.1), T2 (5.2), T3 (5.3), T4 (5.4), T5 (5.5), T6 (5.6), T7 (5.7), T8 (5.8), T9 (5.9) and T10

(6.0). The flasks were placed on orbital shaker for 30 days to accumulate maximum fresh

biomass. The fresh biomass was determined at 3 days intervals. The data collected was

plotted for the development of growth kinetics. The growth curve was further divided

into lag, log and decline phases in order to check the maximum accumulation phases. The

adventitious root cultures were further used for dry biomass accumulation, antioxidant

activity, phenolics and flavonoid content as well as steviol glycosides production.

89

Estimation of fresh and dry biomass of different in vitro cultures

The fresh callus, suspended cells and adventitious roots were collected form culture

media, respectively and washed separately with sterile distal water and placed on

Whatman filter paper for removing media particles and extra water. Afterwards, samples

were weighed for fresh biomass accumulation. In order to estimate dry biomass, samples

from each culture was oven dried (50 ˚C) for 24 hours and finally the dry weight was

measured. Both fresh and dry biomass was expressed in gram per liter.

Quantification of total phenolic and flavonoid contents in different in vitro culture

The oven dried samples of each culture was properly grinded to prepare extract. Well

ground sample (10 mg) was taken to dissolve in 10 ml ethanol. Mixture was placed in

refrigerator for one week with periodic shaking. Ethanolic samples were used for

centrifugation (14,000; 15 min). The supernatant was collected from centrifuged samples

for quantification of total phenolic content (TPC) and total flavonoid content (TFC) by

the method used by Ahmad et al. (2014). Shortly, for quantification of TPC, 0.1 ml of

(2N) Folin Ciocalteus reagent (FCR) was added to 0.03 ml ethanolic extract. The samples

extract having FCR was further diluted by adding 2.55 ml sterilized distilled water. The

mixtures were again centrifuged at 10,000 rpm for 15 minutes and filtered using 45 μm

membrane. The filtered samples were added to cuvette for taking absorbance at 760 nm

through UV visible spectrophotometer (Shimadzu-1650; Japan).

Gallic acid (Sigma; 1.0-10 mg/ml; R2 = 0.9878) was used for plotting standard

calibration curve. Results as Gallic acid equivalent (GAE) mg/g of DW were calculated

from % TPC by using the following equation.

Total phenolic content (%) = 100 × (AS-AB)/ (CF × DF)



The total flavonoids content was also determined according to the protocol of Ahmad et

al. (2014). Briefly, 0.25 ml centrifuged ethanolic extract and 0.075 ml AlCl3 (5%, w/v)

90

was taken and dissolved in 1.25 ml sterile distilled water. Thereafter, 1M NaOH (0.5)

was added to the mixture and centrifuged for 15 minutes at 10, 000 rpm prior to

incubation for 10 min. Absorbance was recorded at 510 nm by using ultra violet (UV)

visible spectrophotometer (Shimadzu-1650PC, Japan). Rutin (Sigma; 1.0-10 mg/ml; R2

= 0.9866) was used for plotting standard calibration curve. The total flavonoid content

was expressed as rutin equivalent (RE) mg/g-DW of the extracts.

Determination of DPPH radical scavenging activity

DPPH-radical scavenging activity (antioxidant activity) was investigated according to the

method followed by Ahmad et al. (2010). Incubated ethanolic extracts of the treated

samples (1.0 ml) was mixed with DPPH free radical solution (2 ml) and incubated for 30

minutes in fully dark condition. Absorbance was recorded at 517 nm by using ultra violet

(UV) visible spectrophotometer (Shimadzu-1650PC, Japan).

Finally, the radical scavenging activity was calculated as percentage of DPPH

discoloration using the following equation;

DRSA (%) = 100 × (1 – AP/AD)

Where AP represents absorbance of extract at 517 nm and AD is the absorbance of the

DPPH solution without tissue extract

Quantification of steviosides in in different in vitro cultures

In callus, cell and adventitious root cultures, quantification of all the three important

components (stevioside, rebaudioside and dulcoside) were investigated according to the

protocol followed by Aman et al. (2013). Perkin-Elmer HPLC system (USA) was used

with quaternary pump, solvent vacuum degasser, C18 column (ODS) with 150 × 4.6 mm,

5 μm particle size, a variable wavelength detector, and an auto sampler with a 10-μl

injection loop. Solution A (25%; HPLC grade water) and B (75%; acetonitrile) was used

in mobile phase. Flow rate was maintained at 1.0 ml min-1 of injected sample (10 µl).

Prior to quantification, standard (Sigma; USA) containing stevioside, rebaudioside and

dulcoside was injected to standardize the retention time for all the three compounds.

91

Quantification of treated samples was finalized by comparing the retention time with the

standard one. Quantified amount of stevioside, rebaudioside and dulcosides were

expressed as mg/g of dry weight (DW).





92


Effect of various pH levels on growth kinetics of multiple cultures

The effect of various media pH levels on callus, cell suspension and adventitious root

culture growth of Stevia was evaluated (Fig 1, 2 and 3). During growth kinetics, a short

lag phase of 3 days was observed for callus cultures, developed in media having pH 4.0,

5.9 and 6.0. However, the rest of the cultures skipped the lag phase and followed prolong

log phases of 27 days. The maximum biomass accumulation (130 g l-1) was recorded for

callus, established with medium pH of 5.6 on day 27 of growth period which was

followed by stationery phase (Fig. 4). Similarly, cell suspension cultures displayed

shorter lag phase of 3 days on media adjusted with pH 5.6 and 5.7. Relatively longer lag

phase of 9 days was observed in culture developed in media with 5.8 pH level. Lag phase

in all three cultures was followed by exponential phase till day 27 of the growth study.

The remaining cell cultures started growth and entered into log phases (27 days) without

any lag phases. The highest accumulation of cell biomass (85.56 g l-1) was recorded on

day 27 in medium adjusted at pH 5.6, followed by a steady decline in growth. Cells in all

cultures did not maintain their growth after day 27 and entered the decline phase

afterward (Fig. 5). Relatively shorter lag phases of 3 days were observed in adventitious

roots, developed at various media pH levels (5.5-6.0). By contrast, media pH 5.1 resulted

in longer lag phase of 18 days with poor biomass accumulation. Roots obtained from

media having pH 5.4 and 5.2 did not show a lag phase. In almost all cultures log phases

were followed by decline phases. However, root growth showed no decline phase even

after 27 days in cultures having pH 5.2 and 5.4. But the highest biomass of adventitious

roots (112.5 g l-1) was observed in log phase after 27 days of inoculation in the medium

having 6.0 pH level (Fig. 6).

The pH of the media influences nutrient uptake, hormonal and enzymatic activities and

hence the growth and development of culture (Bhatia and Ashwath, 2005). The biomass

varied considerably with the pH levels. The pH dependent variation in biomass

accumulation in multiple cultures of several other plants has also been reported earlier.

Iercan and Nedelea (2012) obtained the maximum biomass accumulation in callus culture

of grapevine at medium pH 6.0. Similarly, the maximum rate of regeneration in almond

93

was recorded at medium pH 5.9 (Tabachnik and Kester, 1977). According to Jalil et al.

(2015) the optimum pH for efficient biomass accumulation of the culture ranges between

5.5 to 6.0 and further increase in medium pH may inhibit the growth due to poor

availability of NO3- and micronutrients (Owens et al., 2005). Similarly, Wolf and Chin

(1986) observed a significant decrease in the growth at high pH. The growth of callus,

cell suspension and adventitious root cultures in the present study was inhibited by low

medium pH (Borkowska, 1996), probably due to the poor availability of sugar and

ammonia (Martin and Rose, 1976). Moreover, the highest biomass was accumulated on

day 27 of growth kinetics in current study. While there has been no study on the effect of

pH on callus, cell suspension and adventitious root cultures growth kinetics of Stevia.

However, biomass accumulation in cell culture of Pyrus communis L, Daucus carota and

Capsicum frutescens is significantly affected by media pH, osmolarity and conductivity

(Ryu et al., 1990; Madhusudhan et al., 1995). However, the optimum pH level may vary

with different plant species and culture type. The maximum biomass accumulation also

depends on the specie and tissue under study. For example, Soltani et al. (2015) observed

the maximum cell biomass yield of Cordyceps militaris on day 21 of the culture period.

By contrast, the highest biomass accumulation of adventitious roots occurred after 25

days of culture in Chlorophytum borivilianum (Bathoju and Giri, 2012).

94

Fig. 1. pH levels (a; 5.1, b; 5.2, c; 5.3, d; 5.4, e; 5.5, f; 5.6, g; 5.7, h; 5.8, i; 5.9, j; 6.0)

induced variations in callus cultures of Stevia rebaudiana.

a b c

d e f

g h i

j

95

a b c

d e f

g h i

j

Fig. 2. pH levels (a; 5.1, b; 5.2, c; 5.3, d; 5.4, e; 5.5, f; 5.6, g; 5.7, h; 5.8, i; 5.9, j; 6.0)

induced variations in cell suspension cultures of Stevia rebaudiana.

96

Fig. 3. The influence of pH levels on adventitious root cultures of Stevia rebaudiana.

(a; 5.1, b; 5.2, c; 5.3, d; 5.4, e; 5.5, f; 5.6, g; 5.7, h; 5.8, i; 5.9, j; 6.0).

a

b c

d e f

g h i

j

97

Fig. 4. Effect of pH levels (5.1-6.0) on biomass accumulation during growth kinetics of

callus cultures of Stevia rebaudiana.


cell suspension cultures of Stevia rebaudiana.

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Effect of various pH levels on fresh biomass accumulation

Various pH levels significantly influenced the fresh biomass of callus, cell suspension

and adventitious roots cultures of Stevia. The callus biomass was increased with

increasing the initial medium pH from 5.1 to 5.6, but further increase in pH led to decline

in fresh biomass accumulation. The maximum fresh biomass (130.57 g l-1) was obtained

when the medium pH was 5.6, while the minimum fresh biomass (77.43 g l-1) was

observed at the lowest pH (5.1) (Fig. 7). Similarly, the fresh biomass accumulation in cell

suspension cultures was increased steadily with medium pH from 5.1 to 5.6, but further

increase in pH caused a drastic decrease in fresh biomass accumulation. Thus, the

maximum fresh biomass (85.81 g l-1) was recorded with the medium pH 5.6, that

declined to the least (55.14 g l-1) at the medium pH 6.0 (Fig. 8). By contrast, fresh

biomass accumulation of adventitious root culture was the minimum (62.19 g l-1) at

medium pH 5.1, that increased with increasing pH levels and was the maximum (112.86

g l-1) with medium pH 6 (Fig. 9).

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The increase or decrease in medium pH from the optimum level not only cause reduction

in nutrients availability, but also has significant effect on hormones and enzymatic

activities (Bhatia and Ashwath, 2005). Thus, the fresh biomass accumulation in callus,

cell suspension and adventitious root cultures of Stevia was altered by different pH

levels. The current results indicated that callus biomass was significantly affected by

initial medium pH and the highest fresh weight of callus was observed in medium having

pH 5.6. While there has been no study to understand the effect of medium pH on callus,

cell suspension and adventitious root culture of Stevia, but several other plants have been

extensively studied in this regard. The maximum biomass accumulation was recorded

with pH 5.9 in Almond and 6.0 in grapevine callus cultures (Tabachnik and Kester, 1977;

Iercan and Nedelea, 2012). Similarly, Bhatia and Ashwath (2005) also observed

significant variation in culture growth as a result of varying pH levels, and found the

highest biomass accumulation at below 6.5 pH. Recently, Jalil et al. (2015) also stated

that medium pH (5.5-6.0) significantly induced culture growth. However, decline in

culture growth was observed at medium pH 6.5 (Wolfe and Chin, 1986). Borkowska

(1996) reported that the growth of Vaccinium corymbosum is optimal at 5.0 pH and

ceased at 3.0 pH. At high pH, the growth retarded because of the poor micronutrients

availability such as, iron and manganese (Owens et al., 2005). The deficiencies of these

micronutrients influence several metabolic processes and enzymatic activities (Wetzel

1983; Riemer 1984). However, it is observed that fresh biomass accumulation of

adventitious root culture increased to the maximum pH level (6.0) in this study.

Effect of various pH levels on dry biomass accumulation

The dry biomass of callus, cells and adventitious roots of Stevia was significantly

affected by various pH levels (5.1–6.0). The callus and cell suspension cultures almost

responded in similar manner to different initial medium pH. The dry biomass of callus

was increased with increasing medium pH from 5.1 to 5.6, but decreased with further

increase in pH. The maximum dry biomass (12.10 g l-1) was accumulated with medium

pH 5.6 and the minimum (5.90 g l-1) was recorded in the medium having lower pH (5.1)

(Fig. 7). Similarly, cell suspension culture accumulated higher dry biomass at wide range

of media pH (5.1-5.7). Dry biomass of cell cultures recorded at this pH range were

100

statistically at par with the maximum dry biomass (8.84 g l-1) accumulated at pH (5.5).

Further increase or decrease of media pH significantly reduced the dry biomass of the

cultures. The cell culture developed in medium with 5.1 pH had the least (5.43 g l-1) dry

biomass (Fig. 8). The adventitious root cultures showed an increasing trend with

increasing pH levels from 5.1 to 6.0. In contrast to callus and cell suspension cultures,

adventitious root culture accumulated the maximum dry biomass (8.29 g l-1) at the

highest medium pH 6.0 among all tested levels (5.1-6.0). The least dry biomass

accumulation (2.29 g l-1) was observed in medium having 5.1 pH (Fig. 9).

The wild plants are exposed to a diverse soil pH that influences the biomass

accumulation. The media pH has dominant role in in vitro cultures establishment. The

medium pH above or below optimum levels influenced the growth and development of in

vitro plants (El-Zefzafy et al., 2015). In this study, it is found that cultures accumulated

dry biomass at specified range of medium pH and either increase or decrease from the

optimum range decrease the dry mass accumulation. The pH level of the medium is

critical for in vitro cultures development because it affects the synthesis of important

bioactive constituents (Ahmadian et al., 2013). Thus, it is required to optimize the pH for

vigorous culture growth (Hussain et al., 2012). The optimum media pH is critical for

nutrients uptake, gelling of media and activities of enzymes and phytohormone (Gorret et

al., 2004; Thorpe et al., 2008; Hussain et al., 2012). The uptake of ammonia (NH4+) is

greater in the medium having 5.5 pH (Thorpe et al., 2008) and medium with high pH

(6.0) has a negative influence on the dry biomass of in vitro seedlings (Finn et al., 1991;

Ostrolucka et al., 2010). The decline in growth of cultures below or above optimum pH

levels of callus, cell suspension and adventitious root culture of Stevia might be due to

the fact that, at low pH, cultures poorly uptake nitrogen (NO3) as well as utilize energy to

maintain a proper interior physiological pH (Martin and Rose, 1976). The uptake of

several nutrients such as nitrogen, iron and phosphorous is enzymes dependent and

deviation from optimum pH may alter the activities of these enzymes (Moog and

Bruggemann, 1994; Poonnachit and Darnell, 2004; George et al., 2008). The medium pH

not only influences the uptake of nutrients but also regulates absorption of sucrose in

culture media (Martin and Rose, 1976). Sucrose besides a source of energy in the form of

carbon, also acts as an osmotic stress agent (Kim and Kim, 2002), which resultantly

enhance dry biomass of the cultures (Kishore and Dange, 1990; Juhasz et al., 1997).

101

Fig. 7. Effect of pH levels on fresh and dry weight (g l-1) of callus culture of Stevia


Fig. 8. Effect of pH levels on fresh and dry weight (g l-1) of cell suspension culture of

Stevia rebaudiana. Mean values (± S.E) with common alphabets are nonsignificant at P ≤

0.05.

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102

Fig. 9. Effect of pH levels on fresh and dry weight (g l-1) of adventitious root culture of

Stevia rebaudiana. Mean values (± S.E) with common alphabets are nonsignificant at P ≤

0.05.

Effect of various pH levels on phenolics production in cultures in vitro

The accumulation of total phenolics content (TPC) in callus, cell suspension and

adventitious root cultures was significantly influenced by media pH. Generally, among

all the three cultures, callus cultures produced the minimum phenolics content (42.32-

43.38 mg/g-DW), followed by adventitious root culture (67.52-70.06 mg/g-DW).

However, cell culture accumulated comparatively higher (70.70 to 72.13 mg/g-DW) total

phenolics content (Fig. 10).

In callus cultures, the maximum TPC (43.38 mg/g-DW) was recorded with media pH 5.8,

which was comparable with pH 5.6 to 6.0. On the other hand, the TPC was decreased

linearly with decreasing the medium pH and was (42.32 mg/g-DW) in callus cultured on

medium pH 5.1 (Fig. 10a). By contrast, the cell cultures accumulated higher TPC at

lower pH levels. While the maximum TPC (72.13 mg/g-DW) was recorded with medium

pH 5.8, but it was statistically at par with the TPC accumulated in cells inoculated in

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adventi

tous

cult

ure

(g l

-1)

5.35.2

pH levels

5.1 5.65.55.4 5.7 6.05.95.8

2

3

4

5

6

7

8

9

10

11

12

13

aaaab

bc

c

dde

eff

a

bcc

d

e

fg

hi

103

media of lower pH (5.2-5.7). On the other hand, further increase in initial medium pH

from 5.8 significantly reduced the accumulation of TPC and the minimum TPC (70.70

mg/g-DW) was observed in culture media having pH 6.0 (Fig. 10b). The adventitious

root culture synthesized higher content of phenolics (70.06 mg/g-DW) in media having

5.8 pH but further rise in medium pH did not significantly reduced TPC like in cell

culture. Furthermore, increase in medium pH was statistically similar with the

accumulation of TPC (69.99 mg/g-DW) and (69.85 mg/g-DW) in adventitious roots,

developed in media having pH 5.9 and 6.0, respectively. The minimum TPC (67.50

mg/g-DW) in adventitious root cultures of Stevia was recorded with medium pH 5.1 (Fig

10c).

Secondary metabolites play a fundamental role in plant body and are available

biologically inactive or active forms (Ncube et al., 2008). The phenols, flavonoids,

tannins, stevioside, rebaudioside and other glycoside contents are metabolites of

medicinal importance found in Stevia (Sheeja and Beena, 2015; Ahmad et al., 2016). The

polyphenols are synthesized in response to stress condition through phenyl propanoid

pathway (Hahlbrock and Scheel, 1989). The accumulation and biological activities of

polyphenols are dependent on temperature, photoperiod, humidity, medium pH, plant

growth regulators and nutrients uptake (Bano et al., 2003). The medium pH regulates the

availability and uptake of nutrients and accumulation of polyphenols (Thorpe et al.,

2008). The ammonium is available on high medium pH but low pH reduces availability

of nitrogen in the form of nitrate (Martin and Rose, 1976). Consequently, nitrogen form

and availability influences cell, tissue and organ growth as well as accumulation of

phenolics (Pissarra et al., 1988). The results of the current study suggest that all three

cultures (callus, cell and adventitious roots) accumulated significantly higher amount of

total phenolic content at higher medium pH. Since, these enzymes activities depend on

the pH and each have their own optimum pH level at which they are in active state

(Singh, 2005). The increase phenolic compounds at higher medium pH in callus, cell

suspension and adventitious root cultures may be due to the enhanced activities of related

enzymes especially L-phenylalanine ammonia lyase (PAL), a major enzyme, in the

biosynthetic pathway of phenolic compounds (Ramanad and Lal, 2004). The activities of

104

L-phenylalanine ammonia lyase enzyme decrease at poor availability or deficiency of

ammonium that decrease the accumulation of phenolics in cultures (Laukkanen et al.,

1997). The nitrate is, generally, available at lower medium pH, and correlation between

nitrate concentration and PAL activities have been established (Hahlbrock et al., 1974).

Generally, an increased polyphenols content have been observed with increased PAL

activities in cell suspension cultures of Acer pseudoplatanus (Westcott and Henshaw,

1976). Since, the polyphenols and other secondary metabolites have very strong anti-

bacterial potential and the enhanced amount of these metabolites in callus developed at

pH 6.0, resulted in inhibitory zone development (Ivancajic et al., 2010; Riedel et al.,

2012). The pH can act as an elicitor, that enhances the accumulation of important

compounds in plant cell, tissue and organ cultures (Dicosmo and Misawa, 1985; Sudha

and Ravishankar, 2003; Karuppusamy, 2009), by influencing nutrients availability and

uptake (Thorpe et al., 2008). The nutrient deficiency stress may, in turn, promote the

accumulation of phenolics (Chalker-Scott and Fnchigami, 1989). Poor availability of

important primary nutrients like nitrogen and phosphorus has direct impact on

biosynthesis of phenylpropanoids (Dixon and Paiva, 1995). Besides nitrogen and

phosphorus, deficient concentration of sulfur, potassium, manganese and iron also has an

impact on release of phenolics in cell, tissue and organ cultures (Chalker-Scott and

Fnchigami, 1989). The work of several other scientists also supports our results indirectly

who postulated their hypothesis that plant releases phenolics and other metabolites in

defense when the plant tissue or organ realize stresses at cellular levels (Edreva et al.,

2000; Winkel-Shirley, 2001). Several other scientists also has shown correlation among

impact of medium pH, nutrients availability, polyphenols and other important metabolites

accumulation in various cultures (Chalker-Scott and Fnchigami, 1989; Rajendra et al.,

1992; Bongue-Bartelsman and Phillips, 1995; Seigler, 1998; Dixon and Paiva, 1995;

Tuteja and Mahajan, 2007). The alteration of important factors like pH and nutrients are

key sources to regulate the basic metabolic secondary metabolite pathways for efficient

accumulation of phenolics and other metabolites (Stafford et al., 1986; Misawa, 1985).

105

41

42

43

44

a

abcd

aabab

abcbcd

cdedede

e

70

71

72

73

b

d

c

aababababababbc

Tota

l phenoli

c c

onte

nt

(mg/g

-DW

)

60

62

64

66

68

70

72

74

76 c

aaab

dcdbc

dee

6.0

pH levels

5.65.55.4 5.95.85.75.35.25.1

Fig. 10. Various pH levels induced variations in total phenolics content (mg/g-DW)



106

Effect of various pH levels on flavonoids production in cultures in vitro

The flavonoids are important compounds with numerous pharmaceutical properties. The

pH levels of the medium influenced the flavonoid content significantly in callus, cell

suspension and adventitious root cultures of Stevia. Among all three cultures, cell

cultures accumulated higher amount of flavonoid contents than callus and adventitious

root cultures.

The total flavonoids content (TFC; 37.55 mg/g-DW) was observed in callus culture with

media pH 5.8, which was followed by 37.17 and 37.36 mg/g-DW in callus cultured at pH

5.6 and 5.7, respectively. The minimum TFC (32.68 mg/g-DW) was observed at medium

pH of 5.1. The overall results showed that callus cultures accumulated more flavonoids

with the increasing pH levels of the media up to 5.8. However, further increase in media

pH did not maintain similar increasing trend in flavonoids accumulation (Fig. 11a). In

contrast to callus cultures, the cell cultures yielded significantly higher amount of TFC

(57.32 mg/g-DW) at acidic pH (5.1) and low acidic pH (5.8). However, there were no

significant differences in flavonoids content of cell cultures at media pH 5.1, 5.8, 5.9 and

6.0. Increasing the media pH from 5.1 and decrease from 5.8 resulted in significantly

lower total flavonoids content with the minimum TFC (50.80 mg/g-DW) recorded in cell

suspension cultures on medium of 5.6 pH (Fig. 11b). The adventitious root culture

synthesized considerable amount of TFC (50.19 mg/g-DW) at media pH 5.8 that was

statistically similar to TFC (49.81 mg/g-DW) in culture on media pH 5.7. Either increase

or decrease of media pH from the optimum range significantly reduced TFC production.

However, culture developed in media with minimum pH (5.1) resulted in the least

flavonoid content (44.84 mg/g-DW) (Fig. 11c).

The flavonoids are important metabolites in Stevia leaves and callus cultures, but little

information is available on the effect of medium pH on flavonoids production in callus,

cell suspension and adventitious root cultures of Stevia (Ahmad et al., 2014; Chavasco et

al., 2014). Generally, the plants accumulate secondary metabolites and activate enzymes

to cope with stress conditions (Hahlbrock and Scheel, 1989). Since medium pH alters

metabolic pathways of secondary metabolites production (Karuppusamy, 2009). The

107

various pH levels resulted in significant variation in flavonoids contents in callus, cell

and adventitious root cultures of Stevia that is in conformity to Ali et al. (2013) who

reported significant variations in bioactive compounds production. The pH may alter such

biosynthetic pathways due to selective uptake of nutrients at various pH levels (Thorpe et

al., 2008). The flavonoids synthesis is dependent on nitrogen in the form of ammonium

and nitrates. At low medium pH, the uptake of ammonium is reduced (Martin and Rose,

1976) but it enhances nitrate availability (Martin and Rose, 1976) that ultimately

enhances the production of flavonoid (Pissarra et al., 1988). The data is supported by the

observation that alteration in pH influence the upregulation of biochemical pathways and

activation of genes involve in flavonoids biosynthesis (Zhang et al., 2014). Since, various

kind of flavonoids have their own biosynthetic pathways depending upon cultivar and

culture type, therefore, variations in responses to pH are commonly observed (Henry-

Kirk et al., 2012). It was noticed that almost all cultures accumulated greater amount of

flavonoid at slightly higher pH among the tested levels. Therefore, these findings are in

line with the earlier reports of high accumulation of flavonoids more specifically

flavonols under high pH (Gutha et al., 2010; Fraser et al., 2013).

108

30

31

32

33

34

35

36

37

38

39

40

a

d

c

aaab

c

d

e

f

42

45

48

51

54

57

60

63 b

aaa

b

dc

bbb

a

Tota

l fl

avonoid

conte

nt

(mg/g

-DW

)

40

42

44

46

48

50

52

54 c

g

e

aabccdde

fg

h

6.0

pH levels

5.65.55.4 5.95.85.75.35.25.1

Fig. 11. Various pH levels induced variations in total flavonoids content (mg/g-DW)



109

The pH levels and DPPH-radical scavenging activity in different cultures of Stevia

Media pH is one of the important factors for culture growth and production of

metabolites. The various pH levels, under study, significantly affected the DPPH-radical

scavenging activity in callus, cell suspension and adventitious root cultures of Stevia. The

overall results suggest that cell and adventitious root culture has more DPPH-radical

scavenging activities as compared to callus cultures.

The lowest DPPH-radical scavenging activity (DRSA; 66.13 %) was recorded in callus

grown in media with 5.1 pH that increased with increasing media pH so that the

maximum DRSA (87.68 %) was recorded in callus cultured on medium pH 5.8. The

antioxidant potentials of callus cultured in media with 5.7 and 5.6 pH was 87.46 and

87.24 %, respectively but further increase of media pH caused significant reduction in

DRSA (Fig. 12a). The cell culture also followed similar increasing trend in DRSA like

callus cultures with raising media pH. The cell cultures developed in media with 5.1 pH

had the minimum DRSA (71.85 %) that increased to the maximum (93.99 %) in cells

with media pH 5.8 and either increase or decrease pH from 5.8 significantly reduced the

DRSA (Fig. 12b). The influence of media pH on DRSA of adventitious roots of Stevia

revealed the minimum DRSA (75.81 %) in Stevia adventitious roots with medium pH

5.1, which was increased with increasing pH of media to 91.94 and 92.67 % at media pH

5.7 and 5.8, respectively (Fig. 12c).

The acidic environment of the medium is characterized by high hydrogen ions that affect

the plant growth and metabolites production (Schubert et al., 1990; Koyama et al., 2001;

Kochian et al., 2004). It is suggested that access H+ ions induce reactive oxygen species

(ROS) causing the release of free radicals of hydrogen peroxide and superoxide (Shi et

al., 2006) that result in oxidative stress (Schubert et al., 1990). The plants, as defense

mechanism, activate a number of antioxidant enzymes like CAT (Catalase), SOD (Super

oxide dismutase) and POD (Peroxidase) etc. (Asada, 1999; Mittler, 2002; Yin et al.,

2003). It has been investigated that plant cell, tissues and organs in stress produce

reactive oxygen species (ROS) (Pandhair and Sekhon, 2006; Maeda et al., 2008; Ahmad

et al., 2010), causing severe damages to cellular membrane structures through lipid

110

peroxidation in abiotic stress situations (Pandhair and Sekhon, 2006; Shi et al., 2006;

Chen et al., 2013). The higher activities of these enzymes counteract such oxidative

damages (Chen et al., 2013). Thus, all the cultures were exposed to acidic conditions

caused significant variations in antioxidant activities. Besides higher accumulation of

ROS and H2O2, lower pH reduces antioxidant potentials of SOD and CAT as has been

reported in various crops (Mead, 1976; Shi et al., 2006). In this study, callus, cell and

adventitious root cultures accumulated less total phenolics (TP) and flavonoids content

(FC), at lower pH that increased significantly at higher pH levels. As antioxidant

activities are the result of both enzymatic and non-enzymatic factors like phenols and

flavonoids (Ahmad et al., 2014). Therefore, it is suggested that higher antioxidant

activities with increasing media pH might be due to higher antioxidant enzymes activities

or accumulation of phenolics and flavonoids. Since, phenol and flavonoids counteract

oxidative damages (Pieta et al., 1998; Middleton et al., 2000). The effect of phenols on

antioxidant activities has been established in callus, cell and adventitious root cultures of

different medicinal plants (Jayasinghe et al., 2003; Canadanovic-Brunet et al., 2005; Ali

et al., 2006; Kim et al., 2006; Ali et al., 2007; Sengul et al., 2009; Bidchol et al., 2011;

Diwan et al., 2012; Giri et al., 2012). Several reports are also available that confirm the

correlation between phenol and flavonoids content with the antioxidant potentials of

various plants (Canadanovic-Brunet et al., 2005; Wong et al., 2006; Lopes-Lutz et al.,

2008; Mahmoudi et al., 2009; Asghar et al., 2011; Craciunescu et al., 2012).

111

65

70

75

80

85

90

95 a

cbaaac

d

e

f

g

55

60

65

70

75

80

85

90

95

100

bcbad

e

fg

hi

j

D

PP

H-r

adic

al

scavengin

g a

cti

vit

y (

%)

75

80

85

90

95

c

g

f

aabbccdcddef

h

6.0

pH levels

5.65.55.4 5.95.85.75.35.25.1

Fig. 12. Various pH levels induced variations in DPPH-radical scavenging activity in

callus, cell suspension and adventitious root culture of Stevia rebaudiana. Mean values (±


112

Correlation of total phenolics and flavonoids content with DPPH-radical scavenging

activity

The DPPH-radical scavenging activities in callus, cell suspension and adventitious root

cultured at various medium pH was dependent on total phenolics and flavonoids content.

All three cultures callus, cell suspension and adventitious roots accumulated higher

amount of phenolics (43.38 mg/g-DW), (72.13 mg/g-DW), (70.06 mg/g-DW) and

flavonoids (37.55 mg/g-DW), (57.32 mg/g-DW), (50.19 mg/g-DW), respectively at

media pH 5.8. Increasing or decreasing the pH of media from 5.8 significantly decreased

the phenolics and flavonoids content. Meanwhile, considerable decrease in DPPH-radical

scavenging activity in callus, cell suspension and adventitious root cultures was observed

with respect to decrease in phenolics and flavonoids content (Fig. 13, 14 and 15).

The oxidative stress develops an imbalance between highly reactive free radicals and

anti-oxidizing system that consequently causes cellular damages and even death of the

cells (De-Leo et al., 1998; Delibas et al., 2002; Maier and Chan, 2002; Veurink et al.,

2003; Berr et al., 2004; Saito et al., 2005). The oxidative stress conditions accelerate the

release of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Bourdel-

Marchasson et al., 2001; Ozcankaya et al., 2002; Apelt et al., 2004), that are toxic effect

to lipids, DNA, proteins and several other macromolecules (Bourdel-Marchasson et al.,

2001; Meydani, 2001). Generally, plants have their own antioxidant system to detoxify

the negative effects of these highly reactive free radicals through enzymatic and non-

enzymatic reactions (Panter and Scott, 1991; Bourdel-Marchasson et al., 2001; Rinaldi et

al., 2003; Berr et al., 2004). Thus, the health of plant and survival is associated with

enzymatic antioxidant (SOD, POD, and CAT) and non-enzymatic systems (phenols,

flavonoids, vitamins) especially in oxidative stress conditions (Sigalov and Stern, 1998;

Mates et al., 1999; Meydani, 2001; Berr et al., 2004). Phenolics and flavonoids, as non-

enzymatic antioxidants, are the first line of defense (Skerget et al., 2005). A direct

relationship between antioxidant activities with phenol and flavonoids content is

observed in the current study and support the findings of Li et al. (2006), who stated that

phenol and flavonoids are directly involved in most of the plant antioxidant activities due

to their remarkable free radical quenching potentials (Arts and Hollman, 2005;

113

41.2

41.6

42.0

42.4

42.8

43.2

43.6

To

tal

flav

on

oid

co

nte

nt

(mg

/g-D

W)

TPC

TFC

To

tal

ph

en

oli

c c

on

ten

t (m

g/g

-DW

)

32

33

34

35

36

37

38

39

40

d

c

aaab

c

d

e

f

abcdab

aab

abcbcd

cdedede

e

60

65

70

75

80

85

90

95

cbaaac

d

e

f

g

6.0

DP

PH

-rad

ical

scav

en

gin

g a

cti

vit

y (

%)

pH levels

5.65.55.4 5.95.85.75.35.25.1

Maisuthisakul et al., 2007). Such a positive correlation between antioxidant activities and

polyphenolic compounds has been reported in a number of plants (Lee et al., 2003; Wong

et al., 2006; Maisuthisakul et al., 2008).


scavenging activity in callus cultures of Stevia rebaudiana. Mean values (± S.E) with


114

70.6

70.8

71.0

71.2

71.4

71.6

71.8

72.0

72.2

72.4

a

Tota

l phenoli

c c

onte

nt

(mg/g

-DW

)

Tota

l fl

avonoid

conte

nt

(mg/g

-DW

)

TPC

TFC

d

aababababab

bc

aaa

b

d

c

bbb

a

50

52

54

56

58

60

62

64

55

60

65

70

75

80

85

90

95

100

6.0

DP

PH

-radic

al

scavengin

g a

cti

vit

y (

%)

pH levels

5.65.55.4 5.95.85.75.35.25.1

cbad

e

fg

hi

j




115

40

45

50

55

60

65

70

75

80

40

45

50

55

60

65

70

75

80

geaabccddefg

h

aaabdcdbcdee

Tota

l phenoli

c c

onte

nt

(m

g/g

-D

W)

Tota

l phenoli

c c

onte

nt

(m

g/g

-D

W) TPC

TFC

75

80

85

90

95

100

g

f

aabbccdcddef

h

6.0

DP

PH

-radic

al

scavengin

g a

cti

vit

y (

%)

pH levels

5.65.55.4 5.95.85.75.35.25.1


scavenging activities in adventitious root cultures of Stevia rebaudiana. Mean values (±


116

Various pH levels in relation to steviol glycosides production

In vitro grown cultures require an optimum initial medium pH for biomass accumulation

and secondary metabolites production. Initial medium pH not only influences nutrients

availability but also regulate the enzymatic and hormonal activities. The effect of initial

medium pH on accumulation of active compounds (stevioside, rebaudioside and

dulcoside) in various cultures (callus, cell and adventitious root culture) of Stevia was

investigated. Each culture synthesized significantly different amount of stevioside,

rebaudioside and dulcoside content at various initial medium pH.

The stevioside (62.20 mg/g-DW) and rebaudioside contents (22.79 mg/g-DW) were

significantly higher in callus cultures grown on medium with initial pH 5.6. By contrast,

the dulcoside contents were the maximum (5.92 mg/g-DW) at medium pH of 5.1 that was

statistically at par with 5.85 mg/g-DW recorded at initial medium pH (5.6) (Fig. 16).

In cell suspension cultures, the maximum amount of stevioside (41.47 mg/g-DW) was

estimated at pH level 5.2 but increasing or decreasing the pH of medium reduced

stevioside contents. Among all initial medium pH levels tested, the minimum stevioside

contents (17.60 mg/g-DW) were noted at higher pH (6.0). On the other hand,

rebaudioside and dulcoside content showed similar production pattern in cell cultures.

The highest rebaudioside (7.01 mg/g-DW) and dulcoside (4.72 mg/g-DW) content were

recorded in cell cultures established at medium pH (5.8) that declined in medium pH

below or above 5.8. The least amount of rebaudioside and dulcoside was found between

initial medium pH (5.4) and pH (5.1) (Fig. 17).

In contrast to callus and cell cultures, the stevioside and rebaudioside production of

adventitious root cultures were favored by low initial medium pH with the maximum

stevioside (79.48 mg/g-DW) and rebaudioside (13.10 mg/g-DW) contents in culture

developed at medium pH of 5.1. By contrast, cultures established at initial medium pH

5.6 and 6.0, respectively had less rebaudioside (1.33 mg/g-DW) and dulcoside (0.30

mg/g-DW) contents. The medium pH 5.8 was found the optimum for high dulcoside

content (2.57 mg/g-DW) that declined with increasing or decreasing the medium pH so

that no dulcoside contents were detected at medium pH (5.1) (Fig. 18).

117

The plants are exposed to a diverse soil pH that may alter the biomass accumulation and

plant secondary metabolites production. In vitro cultures also require an optimum pH for

uniform production of secondary metabolites for optimum nutrients availability,

enzymatic activities and hormonal regulations (Naik et al., 2010; Hussain et al., 2012).

The secondary metabolites are synthesized in response to biotic and abiotic stresses (Aziz

et al., 2008). The media pH has been used as an elicitor for enhanced accumulation of

secondary metabolites in plant cell, tissue and organ cultures of valuable medicinal plants

(Dicosmo and Misawa, 1985; Dixon and Paiva, 1995; Sudha and Ravishankar, 2003;

Karuppusamy, 2009). As physical and chemical conditions of the culture predominantly

influence biosynthetic pathways of these metabolites (Dixon and Paiva, 1995). The

stressed plants energy is converted for secondary metabolites biosynthesis rather than

growth (Saenz-Carbonell et al., 1993; Seigler, 1998). Alteration of the media pH and

alter nutrients availability are effective methods to alter metabolic pathways of secondary

metabolites production (Misawa, 1985; Stafford et al., 1986; Eilert, 1987; Barz et al.,

1988; Bano et al., 2003; Gorret et al., 2004; Thorpe et al., 2008). In the current

experiment, the maximum stevioside accumulation was observed in cell suspension and

adventitious root cultures at lower pH of the medium. While, rebaudioside and dulcoside

contents were slightly higher at pH levels 5.1 to 6.0. Overall, all the cultures (callus, cell

and adventitious roots) were developed in acidic medium. The increased concentrations

of stevioside at lower pH in cell and adventitious root cultures may be due to the

availability and uptake of nitrogen at low pH as compared to ammonium available at

higher pH levels (Ikeda et al., 1977; Nakagawa et al., 1984; Bohm and Rink, 1988;

Fujita, 1988). The results indicated that each secondary metabolite has their own specific

biosynthetic pathways and pH sensitivity (Rajendra et al., 1992; Dixon and Paiva, 1995).

In this experiment the metabolites accumulated in higher amounts at more acidic pH in

callus, cell suspension and adventitious root cultures of stevia. Similarly, Lakshmi and

Sridevi, (2009) reported greater amount of withanolide A contents in adventitious root

cultures of Withania somnifera plant at acidic pH (5.5).

118

0

1

2

3

4

5

6

7

d

e

f

d

a

b

c

e

f

a

6.0

Dulc

osid

e c

onte

nt

(mg/g

-DW

)

pH levels

5.65.55.4 5.95.85.75.35.25.1

c

0

10

20

30

40

50

60

70

bc

d

b

a

e

hgf

i

Ste

vio

sid

e c

on

ten

t (m

g/g

-DW

)

a

0

5

10

15

20

25

30

e

f

g

e

ab

dddc

Reb

au

dio

sid

e c

on

ten

t (m

g/g

-DW

) b

Fig. 16. Effect of various pH levels on stevioside, rebaudioside and dulcoside contents in



119

0

1

2

3

4

5

f

c

a

b

d

effff

6.0

Du

lco

sid

e c

on

ten

t (m

g/g

-DW

)

pH levels

5.65.55.4 5.95.85.75.35.25.1

c

0

5

10

15

20

25

30

35

40

45

ihgfgfe

d

b

a

cS

tev

iosid

e c

on

ten

t (m

g/g

-DW

)

a

0

1

2

3

4

5

6

7

e

d

aabb

e

ffff

Reb

au

dio

sid

e c

on

ten

t (m

g/g

-DW

)

b


cell suspension culture of Stevia rebaudiana. Mean values (± S.E) with common


120

0.0

0.5

1.0

1.5

2.0

2.5

3.0

f

c

a

b

ddee

f

gg

Dulc

osid

e c

onte

nt

(m

g/g

-D

W)

6.0

pH levels

5.65.55.4 5.95.85.75.35.25.1

c

0

10

20

30

40

50

60

70

80

90

d

f

ghh

f

e

c

b

a

Ste

vio

sid

e c

onte

nt

(m

g/g

-D

W)

a

0

2

4

6

8

10

12

14

h

gf

ff

e

d

cb

a

Re

baudio

sid

e c

onte

nt (m

g/g

-DW

) b




121


Calli, cell suspension and adventitious root cultures of Stevia rebaudiana were

established to evaluate the effect of pH levels on culture growth and desirable metabolites

production. For this purpose, fresh viable seeds of Stevia rebaudiana were collected from

plants grown in Ground and Garden Nursery, Department of Horticulture, The University

of Agriculture Peshawar. Freshly harvested seeds were surface sterilized prior to

inoculation according to the recent method of Ahmad et al. (2014). Surface sterilized

seeds were cultured on Murashige and Skoog (MS, 1962) medium without any plant

growth regulators (PGRs). Prior to adjusting media pH (5.5-5.8), 30 g l-1 sucrose and 7-8

g l-1 agar was added to culture media and was autoclaved (121 °C; 20 min; 15 psi).

Freshly collected surface sterilized seeds were cultured on autoclaved media and kept for

a period of one month providing 16/8 photoperiod. Leaves of in vitro seed derived

plantlets were used as explants for callus development on Murashige and Skoog (MS)

media fortified with 2.0 mg l-1 2, 4-D and 0.5 mg l-1 BAP. Established calli was further

used for callus cultures. Similarly, whitish friable callus induced from leaf explant was

transferred as inoculum for establishment of cell suspension culture to liquid media

having 2, 4-D (1.0 mg l−1) and BA (0.5 mg l−1). For the development of adventitious root

cultures, roots obtained from in vitro germinated seedlings were shifted to flasks having

half MS liquid media augmented with 0.5 mg l−1 NAA. Different media with varying pH

levels (5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0) were prepared to optimize

suitable pH level for callus, cell suspension and adventitious root cultures growth and

secondary metabolites production. To evaluate the effect of various pH levels on fresh

biomass accumulation of each culture, the growth curve with 3 days interval for a period

of 30 days was established for rapidly growing cells. Further, the biomass of each culture

was used for different calculation of fresh and dry biomass along with secondary

metabolites production.

In callus cultures, shorter lag phase of 3 days was observed at media pH 4.0, 5.9 and 6.0.

However, rest of the cultures skip lag phase and followed prolong log phases of 27 days.

Similarly, cell suspension cultures displayed shorter lag phase of 3 days on media

adjusted with pH 5.6 and 5.7. Relatively longer lag phase of 9 days was adopted by

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culture developed in media with 5.8 pH level. Remaining cell cultures among tested

levels (5.1-6.0) started growth and entered into log phases (27 days) without any lag

phases. In adventitious root cultures, shorter lag phases of 3 days were found at media pH

5.5-6.0. Media pH 5.1 displayed longer lag phase of 18 days with poor biomass

accumulation. Roots obtained from media having pH 5.4 and 5.2 did not adopt lag

phases. In almost all cultures log phases were followed by decline phases. However, root

growth was not restricted in cultures having pH 5.2 and 5.4 even after 27 days of the

culture.

Callus culture fresh biomass was considerably increased with increasing medium initial

pH (5.1–5.6) among tested levels (5.1–6.0). The highest fresh and dry biomasses (130.57

g l-1; 12.10 g l-1) were accumulated at media pH (5.6), while the lowest (77.43 g l-1; 5.90

g l-1) were obtained at minimum pH (5.1). Similarly, callus cultures resulted maximum

total phenolics content (TPC; 43.38 mg/g-DW), total flavonoids content (TFC; 37.55

mg/g-DW) and DPPH-radical scavenging activity (DRSA; 87.68 %) at pH level 5.8.

However, lower medium pH (5.1) resulted in minimum TPC (42.32 mg/g-DW), TFC

(32.68 mg/g-DW) and DRSA (66.13 %) in callus cultures. Positive correlation of

antioxidant activities with TPC and TFC was also found in callus culture. The highest

stevioside (62.20 mg/g-DW) and rebaudioside (22.79 mg/g-DW) contents were found in

callus cultures grown on medium having initial pH 5.6. However, dulcoside contents

were found maximum (5.92 mg/g-DW) at medium pH of 5.1.

Cell suspension cultures developed in media having various pH levels (5.1-6.0) also

experienced considerable variations in culture growth and secondary metabolites

accumulation. The maximum fresh and dry biomasses (85.81 g l-1; 8.84 g l-1) were found

at media pH 5.6 and 5.5, respectively. However, cell suspension cultures at pH 6.0 and

5.1 resulted in minimum fresh biomass (55.14 g l-1) and dry biomass (5.43 g l-1).

Similarly, media pH (5.8) was optimized for the maximum TPC (72.13 mg/g-DW), TFC

(57.32 mg/g-DW) and DRSA (93.99 %) in cell cultures. However, the minimum TPC

(70.70 mg/g-DW), TFC (50.80 mg/g-DW) and DRSA (71.85 %) were observed at media

pH 6.0, 5.6 and 5.1, respectively.

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Moreover, the highest stevioside content (41.47 mg/g-DW) was estimated at pH level 5.2,

while the lowest (17.60 mg/g-DW) was noted at higher pH level (6.0). On the other hand,

rebaudioside and dulcoside content showed similar production pattern in cell cultures.

The highest amount of rebaudioside (7.01 mg/g-DW) and dulcoside (4.72 mg/g-DW)

content were quantified in cell cultures at medium pH 5.8.

Adventitious root culture was also found dependent on media pH levels for optimum

biomass and secondary metabolites production. Adventitious root culture accumulated

considerably higher amount of fresh (112.86 g l-1) and dry (8.29 g l-1) at pH 6.0 than other

levels, however, lower fresh (62.19 g l-1) and dry (2.29 g l-1) biomasses were observed at

pH 5.1. Adventitious root cultures also favored higher media pH (5.8) for the maximum

accumulation of TPC (70.06 mg/g-DW), TFC (50.19 mg/g-DW) and DRSA (92.67 %).

However, cultures at pH 5.1, showed minimum TPC (67.50 mg/g-DW), TFC (44.84

mg/g-DW) and DRSA (75.81 %). In contrast, maximum quantities of stevioside (79.48

mg/g-DW) and rebaudioside (13.10 mg/g-DW) contents were observed pH 5.1. However,

cultures at pH 5.6 and 6.0 were observed with poor stevioside (13.77 mg/g-DW) and

rebaudioside (1.33 mg/g-DW), respectively. In case of dulcoside contents, 5.8 pH levels

was found the optimum for higher amount of dulcoside content (2.57 mg/g-DW) but at

pH 5.1, dulcoside contents were not detected.

Conclusions

Considerable variations were observed in biomass accumulation and secondary

metabolites production in response to various media pH (5.1-6.0) in callus, cell

suspension and adventitious root cultures of Stevia.

Most of the growth kinetics was characterized with lag and log phases. Few of the

peak points in all cultures displayed relatively shorter stationery phases followed

by decline phases.

Media pH 5.6 was optimized for the maximum accumulation of fresh (130.57 g l-1)

and dry biomasses (12.10 g l-1) in callus and cell suspension cultures (85.81 g l-1;

8.84 g l-1). On the other hand, adventitious root cultures favored high media pH

(6.0) for accumulation of maximum fresh (112.86 g l-1) and dry biomasses (8.29 g

l-1).

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Similarly, significant higher amount of TPC, TFC and DRSA in callus (43.38

mg/g-DW; 37.55 mg/g-DW; 87.68 %), cell suspension (72.13 mg/g-DW; 57.32

mg/g-DW; 93.99 %) and adventitious root cultures (70.06 mg/g-DW; 50.19 mg/g-

DW; 92.67 %) were recorded at pH 5.8.

Moreover, the highest stevioside (62.20 mg/g-DW) and rebaudioside (22.79

mg/g-DW) contents were observed in callus culture at pH 5.6. While callus

culture favored low pH (5.1) for the maximum accumulation of dulcoside content

(5.92 mg/g-DW). On the other hand, cell suspension culture yielded maximum

amount of stevioside (41.47 mg/g-DW) at pH (5.2); rebaudioside (7.01 mg/g-

DW) and dulcoside (4.72 mg/g-DW) contents at media pH 5.8. In addition,

adventitious root culture favored media pH (5.1) for maximum biosynthesis of

stevioside (79.48 mg/g-DW) and rebaudioside (13.10 mg/g-DW) contents.

However, dulcoside contents in adventitious root cultures (2.57 mg/g-DW) were

found in higher quantities at pH level 5.8.

Recommendations

The following recommendations regarding biomass yield and secondary

metabolites production in callus, cell suspension and adventitious root cultures

were derived from the above conclusions.

Media pH should be adjusted to 5.6 for callus as well as cell suspension cultures

biomass, while 6.0 for adventitious root culture to obtain maximum fresh and dry

biomasses.

Similarly, 5.8 should be adjusted as an optimum media pH for enhanced TPC and

DRSA in callus, cell suspension and adventitious root cultures.

For maximum stevioside and rebaudioside contents in callus culture, media pH

should be adjusted at 5.6 and 5.1 for dulcoside contents. Similarly, cell suspension

culture requires 5.2 as an optimum level for biosynthesis of stevioside and 5.8 for

rebaudioside and dulcoside contents. Low media pH (5.1) is recommended for the

production of stevioside as well as rebaudioside and pH 5.8 for dulcoside contents

in larger quantities in adventitious root cultures.

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CHAPTER-VI

The effect of inoculum size on biomass, phenolics and flavonoids,

antioxidant activity and bioactive compounds in callus, cell suspension

and adventitious root cultures of Stevia rebaudiana (Bert.)


Abstract

In vitro propagation of plant cell, tissue and organ has been an imperative technique for

the synthesis of commercially valuable bioactive compounds. In this regard, many

strategies have been made for optimization of culture conditions for substantial

accumulation of important bioactive compounds on commercial level. Proper selection

and use of appropriate inoculum size has influential role on culture growth and secondary

metabolites production. Since no work has been found on the effect of inoculum size on

callus, cell suspension and adventitious root cultures of Stevia rebaudiana. In our study,

an effort has been made to investigate the effect of initial inoculum size on callus, cell

and adventitious root cultures growth and secondary metabolites production. Cultures

were established using various initial inoculum sizes (0.5 g, 1.0 g, 1.5 g and 2.0 g). Callus

culture was established on Murashige and Skoog (MS) media supplemented with 2.0 mg

l-1 2, 4-D and 0.5 mg l-1 NAA. Cell culture was established by using friable calli in liquid

media having 2, 4-D (1.0 mg l−1) and BA (0.5 mg l−1). Roots were collected from in vitro

plantlets and transferred to half MS liquid media having 0.5 mg l-1 NAA for

establishment of adventitious root culture. Growth kinetics, fresh and dry biomass of

callus, cell suspension and adventitious root cultures were positively encouraged with the

increasing inoculum size (0.5–2.0 g). Among these, callus culture was characterized with

relatively shorter lag phase of 3 days of the inoculation for all inoculum sizes. An

increase in biomass with elongated log phases from day 3rd to 27th day of the culture was

observed in callus cultures. Among all inoculum sizes, 2.0 g started sudden increased in

biomass accumulation up to 15 days and increments in growth was further continued till

27th day of culture. Log phase was followed by sudden decline phase without having any

stationery phase in all cultures. Similarly, cell culture developed from various inoculum

sizes was characterized by an elongated lag phase started from day 3 to 12 days of the

culture. Lag phase was subsequently followed by a long log phase (12-27 days duration).

Cell culture did not experience stationary phase and after 27th day of culture, decline in

growth was occurred. Adventitious root cultures did not displayed lag phases. Sudden

increased in growth curve was found at early stage (day 3) of log phases, which was

continued till 27 days of culture. After 27th day of culture, decline was occurred in

biomass accumulation in all cultures initiated from various inoculum sizes. Meanwhile,

the highest fresh (112.29 g l-1) and dry biomass (7.71 g l-1) was accumulated when the

nutrient medium was inoculated with 2.0 g inoculum, whereas, the callus developed from

minimum inoculum size (0.5 g) resulted the accumulation of the least fresh (69.81 g l-1)

and dry biomass (3.43 g l-1). Similarly, inoculum size (1.5 g) was optimized for

accumulation of fresh (102.71 g l-1) and dry biomass (5.38 g l-1) of cell cultures. Lower

inoculum size (0.5 g) in culture media resulted minimum fresh (70.19 g l-1) and dry (2.86

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g l-1) biomass of cells. On the other hand, the maximum amount of fresh (106.86 g l-1)

and dry (5.05 g l-1) biomass was accumulated in the liquid media when it was inoculated

with 1.5 g inoculums. On the other hand, adventitious root culture established from

smaller inoculum size (0.5 g) resulted in poor fresh (70.57 g l-1) and dry biomass (2.29 g

l-1) accumulation. Results also showed that initial inoculum size not only influenced fresh

and dry biomass of the cultures but also significantly induced desirable secondary

metabolites production. Among various tested inoculum sizes, 0.5 g was proven to be the

best initial inoculum size for maximum production of total phenolic content (TPC; 28.54

mg/g-DW), total flavonoid content (TFC; 24.78 mg/g-DW) along with more DPPH-

radicals scavenging activities (DRSA; 77.57 %) in callus cultures. Similarly, callus

culture developed from lower initial inoculum size (0.5 g) also yielded higher amount of

stevioside (43.89 mg/g-DW) and rebaudioside (36.54 mg/g-DW) contents. Whereas,

dulcoside content (2.57 mg/g-DW) was found in higher amount in callus culture

established from initial inoculum size (1.0 g). For cell suspension culture, initial

inoculum size (0.5 g) was also regarded as an optimum for accumulation of maximum

TPC (45.36 mg/g-DW), TFC (36.50 mg/g-DW), stevioside (59.89 mg/g-DW),

rebaudioside (24.41 mg/g-DW) and dulcoside content (1.85 mg/g-DW). In contrast, cell

culture did not show higher potential of free radical scavenging (72.73 %) at lower initial

inoculum size (0.5 g). In comparison to callus culture, cell culture was found with

enhanced free radical scavenging potential (78.30 %) at high inoculum sizes (2.0 g).

Adventitious root culture accumulated total phenolics content in contrast manner to the

callus and cell suspension culture. Adventitious root culture accumulated significantly

higher amount of TPC (41.46 mg/g-DW), TFC (33.44 mg/g-DW) as well as higher

potential (98.82 %) for scavenging free radicals at more condensed (2.0 g) initial

inoculum size. However, stevioside (64.75 mg/g-DW) and rebaudioside (29.67 mg/g-

DW) contents were significantly increased to their maximal level using initial inoculum

size (1.0 g). On the other hand high dulcoside contents (0.71 mg/g-DW) were found in

cultures developed from 1.5 g initial inoculum size.

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INTRODUCTION

In recent years, there has been greater emphasis on plants based bioactive compounds, of

therapeutic value, as a natural dietary supplement (Uikey et al., 2010). The secondary

metabolites have enormous biological potentials like antimicrobial, anticarcinogenic,

antitumor and antiallergic (Huang et al., 2013). The plant secondary metabolites are

derivatives of primary metabolites and are specific to plant species and organs. The

secondary metabolites are not directly involved in growth and metabolic activities of the

plant but help in survival of the plants in many stress conditions like oxidative damages,

pathogenic infections, climatic factors, saline and drought stresses (Ahmad et al., 2014)

and could be source for novel drugs and cosmetic products (Joo et al., 2010).

A major group of phytochemicals includes glycosides, terpenoids, alkaloids, polyphenols

and flavonoids. The polyphenols rank the largest group with antioxidant potentials

(Ahmad et al., 2013). Polyphenols, such as lignins, stilbenes, ellegitannins, derivatives of

caffeic acids and many more are effective candidates to strengthen body defense system,

regulate enzyme activities and scavenge harmful free radicals (Matkowski, 2006; Joo et

al., 2010; Ahmad et al., 2013). Besides polyphenols, the flavonoids have strong free

radical scavenging activities (Amid et al., 2011). Being friendly to human health, there

has been increased demand for such natural metabolites as compared to synthetic

compounds (Matkowski, 2008).

Most of these valuable bioactive compounds are naturally synthesized in plants but their

potential is limited due to environmental conditions and seasonal limitations (Ali et al.,

2013; Ahmad et al., 2013). Thus, in vitro culture (plant, cell, tissue and organ culture) has

been used to synthesize the desired bioactive compounds (Kolewe et al., 2008). The in

vitro conditions can be modified to enhance the accumulation of useful metabolites

(Baque et al., 2012). Thus, the in vitro culture techniques can be used for the commercial

production of phytoconstituents (Murthy et al., 2008). Among different in vitro culture

techniques, callus, cell suspension and adventitious root cultures are reliable methods to

study and optimize cultural conditions for biomass accumulation and commercial

production of secondary metabolites (Rao and Ravishankar, 2002). The callus culture is

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an efficient alternative to micro and conventional method of propagation for the

production of secondary metabolites (Aman et al., 2013; Dey et al., 2013; Khalil et al.,

2015). However, the plant cell culture may offers a unique opportunity over solid culture

due to complete immersion of cells in suspension that provides more chances of nutrients,

plant growth regulators and vitamins uptake (Soomro and Memon, 2007). In addition, the

plant cell culture is also desirable substitute for such plants that has generally long

cultivation cycle or hard to propagate (Hippolyte et al., 1992). In cell suspension culture,

cells have short growth cycle with rapid division rate due to physical manipulation of

cultural conditions, which enables the culture to synthesize novel secondary metabolites

in larger quantities (Mulabagal and Tsay, 2004). The adventitious root culture is another

promising approach for large scale production of valuable bioactive compounds (Wang et

al., 2013), and can be easily up-scale to bioreactor (Baque et al., 2013). Thus,

adventitious root culture has been widely exploited for the synthesis of commercially

important phytoconstituents (Cui et al., 2010).

The biosynthetic pathways of bioactive compounds are markedly altered by exposure of

cultures to different stress conditions that may enhance the production of commercially

important metabolites (Sivanandhan et al., 2012). The inoculum size affects cell growth

and development (Franklin and Dixon, 1994). The inoculum size has direct impact on

biological condition of the in vitro culture as well as cell-to-cell and cell to medium

interaction and, therefore, on biomass accumulation and secondary metabolites

production (Tanaka, 1981). It has been reported that initial inoculum size have a

stimulating effect on accumulation of valuable compounds in root culture of ginseng

(Jeong et al., 2009), saponin content in root culture of Talinum paniculatum (Manuhara et

al., 2012), 20-hydroxyecdysone in cell culture of Vitex glabrata (Sinlaparaya et al.,

2007), gallic acid production in suspension culture of Acer ginnala (Jun-Ge et al., 2006),

withanolide A-B accumulation in adventitious root culture of Withania somnifera

(Sivanandhan et al., 2012).

The Stevia is a perennial sweet herb (Savita et al., 2004) that is a natural alternative

source of calorie-free sweetener (Ahmad et al., 2011). The leaves of Stevia produce

Diterpene glycosides (stevioside and rebaudiosides) (Yadav et al., 2011). The food-

129

derived antioxidants, such as vitamins and phenolic phytochemicals are known to

function as chemo preventive agents against oxidative damages (Kim et al., 2011). The

dry extract of Stevia leaves also contains several other bioactive compounds like

flavonoids, alkaloids, water-soluble chlorophylls, xanthophylls, hydroxycinnamic acids

etc. (Komissarenko et al., 1994).

It is desirable to evaluate the antioxidant capacities of Stevia rebaudiana propagated in

different ways. The in vitro culture techniques such as callus, cell suspension and

adventitious root cultures have the added benefits of enhancing the accumulation of

secondary metabolites production. Therefore, the present study was aimed to evaluate the

effect of inoculum size on culture growth and synthesis of bioactive compounds in callus,

cell suspension and adventitious root cultures of Stevia rebaudiana.

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Selection of inoculum size for callus development

Freshly harvested seeds of Stevia rebaudiana were cultured in vitro for 30 days on PGRs

free MS media for the development of plantlets. After 30 days of seed germination, the

leaf explants were used for callus development and the roots were used for adventitious

root developments. The leaves of in vitro plantlets were used as explants for callus

development. The excised leaves from in vitro seed derived plantlets were inoculated on

Murashige and Skoog, (1962) medium (MS) supplemented with 2, 4-D (2.0 mg l−1) and

BA (0.5 mg l−1). To optimize inoculum size for efficient callus proliferation and

secondary metabolites production, 30 days old callus of various sizes was inoculated on

MS medium (sucrose 30 g l-1; agar 8 g l-1) containing a similar set of PGRs. The culture

media was autoclaved (121 °C for 20 min) before callus inoculation. All the cultures

were placed in growth room at 25 ±2°C. For determination of optimize inoculum size, an

experiment was design in Complete Randomized Design (CRD), using various inoculum

sizes as T1 (0.5 g), T2 (1.0 g), T3 (1.5 g) and T4 (2.0 g).

Selection of inoculum size for cell suspension culture development

For development of cell suspension culture, 30 days old proliferated callus was sub

cultured on MS media and maintained in dark conditions to develop soft and whitish

friable callus, that was used for inoculum size (T1; 0.5 g, T2; 1.0 g, T3; 1.5 g and T4; 2.0

g), to determine the suitable size for culture growth and secondary metabolites

accumulation. For this purpose, the experiment was designed in Complete Randomized

Design (CRD) with 3 repeats. The cultures were placed on orbital rotary shakers for one

month in dark conditions. The growth of the treated cultures was recorded periodically

for growth kinetics. All the cultures were evaluated for fresh and dry biomass, total

phenolics and flavonoids content, DPPH scavenging activity, stevioside, rebaudioside

and dulcoside contents.

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Selection of inoculum size for adventitious root culture development

The roots were harvested from in vitro seed-derived plantlets after one month. The

harvested roots were transferred to agar free median fortified with 30 g l-1 sucrose and

already optimized level of NAA (0.5 g l-1) for culture development. The root culture was

placed on orbital rotary shaker in dark for a period of 30 days for stock culture

development. Adventitious roots from stock culture as inoculum (T1; 0.5 g, T2; 1.0 g,

T3; 1.5 g and T4; 2.0 g) were tested to optimized suitable inoculum size for culture

growth, fresh and dry biomass, phenolics, flavonoids, antioxidant potential and steviol

glycoside (stevioside, rebaudioside and dulcoside) production.

Growth kinetics and biomass accumulation of different in vitro cultures

Periodic data was recorded for estimation of growth kinetics at 3 days interval for 30

days. The growth curve was plotted for callus, cell suspension and adventitious root

cultures in response to the initial inoculum sizes. For calculation of fresh and dry

biomasses, all cultures were separated from cultured media and gently washed with

sterile distilled water. The rinsed cultures were placed on Whatman filter paper to extract

extra water and the fresh weight was recorded. Likewise, for determination of dry weight,

fresh biomass of callus, cells and adventitious root cultures were oven dried (50 ˚C for 24

hours) and the dry biomass was calculated. Both fresh and dry biomasses were presented

in g l-1.

Analytical methods

The extract was prepared from oven-dried fine powder by mixing 10 mg in 10 ml of

ethanol. The mixture was stored for one week with periodic shaking prior to

centrifugation for 15 minutes at 10,000 rpm. The supernatant was taken from centrifuged

samples for further sample preparation for TPC, TFC and DRSA determination. The

method of Ahmad et al. (2014) was used for sample preparation and determination of

TPC and TFC. For quantification of TPC, (0.1 ml; 2N). Folin Ciocalteus reagent was

added to 0.03 ml ethanolic extract and 2.55 ml sterile distilled water. Prior to incubation

(10 min), mixtures were centrifuged for 14 minutes at 10, 000 rpm. Finally, 45 μm

132

membrane was used to filter the resultant mixtures and the absorbance was taken through

spectrophotometer (UV-Visible; Shimadzu-1650; Japan) at 760 nm.

Gallic acid (Sigma; 1.0-10 mg/ml; R2 = 0.9878) was used for plotting standard

calibration curve. Results as Gallic acid equivalent (GAE) mg/g of dry weight (DW) were

obtained from TPC by using the following equation.

% total phenolic content = 100 × (AS-AB)/ (CF × DF)



Ethanolic extract (0.25 ml), sterile distilled water (1.25 ml), (5%; 0.075) AlCl3 and NaOH

(0.5ml) were mixed for TFC determination. The mixtures were centrifuged for 10

minutes at 10,000 rpm and finally incubated for 10 min in dark. Furthermore, 45-μm

membrane was used to filter the resultant mixtures and the absorbance was taken through

spectrophotometer (UV-Visible; Shimadzu-1650; Japan) at 510 nm. Rutin (Sigma; 1.0-10

mg/ml; R2 = 0.9866) was used for plotting standard calibration curve. The total flavonoid

content was expressed as rutin equivalent (RE) mg/g-DW of extracts.

DPPH-radical scavenging activity was investigated according to the method of Ahmad et

al. (2010). The incubated ethanolic extracts of the treated samples (1.0 ml) was mixed

with DPPH free radical solution (2 ml) and incubated for 30 minutes in fully dark

condition. The absorbance was recorded at 517 nm by using ultra violet (UV) visible

spectrophotometer (Shimadzu-1650PC, Japan). Finally, the radical scavenging activity

was calculated as percentage of DPPH discoloration using the following equation;

DRSA (%) = 100 × (1 – AP/AD)

Where AP represents absorbance of extract at 517 nm and AD is the absorbance of the

DPPH solution without tissue extract

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Quantification of steviol glycosides in different in vitro cultures

Steviol glycosides mainly stevioside, rebaudioside and dulcoside contents were

determined in callus, cell and adventitious root cultures according to the method of Aman

et al. (2013). Perkin-Elmer HPLC system (USA) was used for this purpose with

quaternary pump, solvent vacuum degasser, C18 column (ODS) with 150 × 4.6 mm, 5 μm

particle size, a variable wavelength detector, and an auto sampler with a 10 μl injection

loop. In mobile phase, 25% HPLC grade water and 75% acetonitrile was used as solution

A and B respectively. During quantification, volume of 10 µl was injected at 1.0 ml min-1

flow rate. Steviol glycoside standard containing stevioside, rebaudioside and dulcoside,

(Sigma, USA) was run at first for standardization of retention time of each. Stevioside,

rebaudioside and dulcoside contents were identified in each sample of callus, cell and

adventitious root culture by comparing retention times of samples with standard. The

stevioside, rebaudioside and dulcosides were quantified and expressed mg/g of dry

weight (DW).





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Effect of inoculum size on growth kinetics of different Stevia cultures

The growth pattern of callus, cell suspension and adventitious root cultures for various

initial inoculum sizes was investigated in current study (Fig 1, 2 and 3). The callus

culture was characterized with relatively shorter lag phase of 3 days of the inoculation for

all inoculum sizes. An increase in biomass with elongated log phases from day 3rd to 27th

day of the culture was observed in callus cultures. Among all inoculum sizes, 2.0 g

started sudden increased in biomass accumulation up to 15 days and the incremental

increase in growth continued till 27th day of culture. The inoculum size of 2.0 g,

incubated for callus development, also accumulated the maximum biomass (111.97 g l-1)

at the end of log phases (day 27) of growth kinetics. Log phase was followed by sudden

decline phase without having any stationery phase in all cultures (Fig. 4). Similarly, cell

culture developed from various inoculum sizes was characterized by an elongated lag

phase started from day 3 to 12 days of the culture. The lag phase was subsequently

followed by a long log phase (12-27 days duration) with the highest biomass yield

(102.50 g l-1) from initial inoculum size of 1.5 g. Culture did not experience stationary

phase and after 27th day of culture and decline in growth was observed (Fig. 5). The

adventitious root cultures did not displayed lag phases but a sudden increased in growth

curve was found at early stage (day 3) of log phases, which continued till 27 days of

culture. Among all inoculum sizes, 1.5 g resulted in the maximum biomass (107.25 g l-1)

during the log phases of the growth curve. After 27th day of culture, a decline was

observed in biomass accumulation in all cultures initiated from various inoculum sizes

(Fig. 6).

The callus, cell suspension and adventitious root cultures displayed a sigmoidal growth

pattern during growth kinetics study. Both callus and cell cultures showed lag phases but

adventitious root culture was found without lag phase. After 27 days of growth kinetics, a

decline phases was observed. Such sigmoidal growth pattern adopted by all three cultures

in this study are in agreement with the growth kinetics studies in callus, cell suspension

and root cultures of other medicinal plants (Karam et al., 2003; Sujanya et al., 2008;

Kolewe et al., 2010). Moreover, it was also observed during growth kinetics study that

callus, cell suspension and adventitious root cultures accumulated the maximum

biomasses at an elevated inoculum sizes. However, in cell and adventitious root cultures

135

further increase in inoculum size from 1.5 g was not supported by culture media to attain

the maximum biomass. Similarly, use of lower initial inoculum size was also found not

satisfactory for desirable biomass accumulation. Basically, use of proper inoculum size is

an important factor for cell metabolism in cultures in vitro (Yang et al., 2009) because it

encourages biomass of the culture and helps in metabolites accumulation (Wang et al.,

1997; Yang et al., 2009). Several fold increase in cell biomass of Perilla frutescens was

also recorded with high initial inoculum density (Zhong and Yoshida, 1995). These

results suggest that use of optimum inoculum size is critical for culture growth

(Schlatmann et al., 1994). The accumulation of poor biomass in cultures developed from

small size inoculum could be due to the availability of water, nutrients and other medium

compositions in excess amount, which have negative impact on the growth and survival

of the cells (Yusuf et al., 2012). On the other hand, culture developed form higher than

optimum inoculum size has antagonistic effect on biomass yield of the culture. It is

observed that cell and adventitious root cultures accumulated higher biomasses at 1.5 g

inoculum. However, use of 2.0 g inoculum was not able to develop the highest biomass.

Reduction in biomass yield due to heavy inoculum size might be due to reduction of

oxygen and nutrients concentrations in culture medium (Contin et al., 1998; Blackhall et

al., 1999). The cultures did not maintain growth pattern after 27 days of the culture and

decline in growth was noticed. During log phases, cell growth and proliferation rate is

very high (Smith, 2000) and the accelerated growth of the culture, deplete nutrients

resulting the decline phase. Besides this, several other factors such as toxic metabolites

from cultured cells, lack of oxygen, drying of gelled media or concentration of

evaporation of liquid media could have negative impact on cell growth during the decline

phase. Hence, it is best to carry subculture before entering into decline phase of culture

(Yeoman and Macleod, 1977; Smith, 2000; Karam et al., 2003).

136

a b

c d

Fig. 1. Effect of inoculum sizes (a) 0.5 g, (b) 1.0 g, (c) 1.5 g and (d) 2.0 g on proliferation

of callus cultures of Stevia rebaudiana.

137

a b

c d

Fig. 2. Effect of inoculum sizes (a) 0.5 g, (b) 1.0 g, (c) 1.5 g and (d) 2.0 g on

establishment of cell suspension cultures of Stevia rebaudiana.

138

Fig. 3. Effect of inoculum sizes (a) 0.5 g, (b) 1.0 g, (c) 1.5 g and (d) 2.0 g on

establishment of adventitious root cultures of Stevia rebaudiana.

a b

c d

139

Fig. 4. Effect of inoculum size on biomass accumulation during growth kinetics of callus

cultures of Stevia rebaudiana.

Fig. 5. Effect of inoculum size on biomass accumulation during growth kinetics of cell

suspension cultures of Stevia rebaudiana.

10

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Fig. 6. Effect of inoculum size on biomass accumulation during growth kinetics of


Effect of inoculum size on fresh and dry biomass accumulation in Stevia cultures

The inoculum size influenced the fresh and dry biomass of callus, cell suspension and

adventitious root cultures. In callus culture, fresh and dry biomass was encouraged by the

increasing inoculum sizes (0.5–2.0 g). The highest fresh (112.29 g l-1) and dry biomass

(7.71 g l-1) was accumulated when the nutrient medium was inoculated with 2.0 g

inoculum, whereas, the least fresh biomass (69.81 g l-1) and dry biomass (3.43 g l-1)

developed from the minimum inoculum size (0.5 g) (Fig. 7). Similarly, the fresh and dry

biomass of cell culture increased with increasing inoculum size (0.5-1.5 g). The inoculum

size (1.5 g) resulted in the maximum accumulation of fresh (102.71 g l-1) and dry biomass

(5.38 g l-1) of cell cultures. The lower inoculum size (0.5 g) in culture media resulted in

the minimum fresh (70.19 g l-1) and dry (2.86 g l-1) biomass accumulation (Fig. 8). On the

other hand, increasing trend in fresh biomass of adventitious root culture was observed

with increasing inoculum size up to certain level (0.5–1.5 g), while further increase in

inoculums size lead to a decline in the accumulation. The maximum fresh (106.86 g l-1)

and dry (5.05 g l-1) biomass was accumulated in the liquid media when it was inoculated

10

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30

40

50

60

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0.5 g

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1.5 g

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03 181512 21 302724

141

with 1.5 g inoculums. On the other hand, adventitious root culture established from

smaller inoculum size (0.5 g) resulted in poor fresh (70.57 g l-1) and dry biomass (2.29 g

l-1) accumulation in adventitious roots of Stevia (Fig. 9).

The size of inoculum has crucial role in culture growth and development. Below a

particular size, the proliferation of inoculums does not occur (Baque et al., 2012). An

appropriate inoculum size leads to the optimum growth of cells (Kanokwaree et al., 1997).

The inoculum size in culture medium counteracts for space, oxygen and medium

compositions. Generally, medium conditioning is used to overcome demand for medium

composition by the enlarge biomass of the inoculum (Jeong et al., 2009; Lee and Shuler,

2000). In this study, fresh and dry biomasses in callus, cell suspension and adventitious

root cultures were significantly induced by culturing relatively larger inoculums sizes as

compared to the lower ones. Similar results of cell development were observed in

cultures of Panax ginseng (Akalezi et al., 1999). Wu et al. (2006) also reported that a

lower quantity of root biomass accumulation was due to smaller inoculum size and 10–20

g inoculums size increased the growth remarkably. Relatively large portion of tissue used

in culture enhances opportunity of gaining a living culture due to the presence of

maximum numbers of cells (Yeoman, 1973). Zhang and Zhong (2009) also optimized

comparatively larger inoculum size for the enhanced biomass yield of Panax notoginseng

in cell cultures. Zhong and Yoshida (1995) also revealed that the dry biomass of cell

culture was found the highest at higher initial inoculum size. Yann et al. (2012) also

found that when the least inoculums size 0.25 g of Artemisia annua L. was tested, the

growth rate was found higher, however, it acquired extra time to gain utmost growth as

compared to the other inoculums sizes and because of this long duration the chances of

contamination were increased and recommended comparatively larger inoculum size (0.5

g).

142

Fig. 7. Effect of inoculum size on fresh and dry biomass (g l-1) of callus culture of Stevia


Fig. 8. Effect of inoculum size on fresh and dry biomass (g l-1) of cell suspension culture

of Stevia rebaudiana. Mean values (± S.E) with common alphabets are nonsignificant at

P ≤ 0.05.

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a

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143

Fig. 9. Effect of inoculum size on fresh and dry biomass (g l-1) of adventitious root

culture of Stevia rebaudiana. Mean values (± S.E) with common alphabets are


Effect of inoculum size on phenolics production in different Stevia cultures

Significant relation has been found among inoculum size and phenolics accumulation in

callus, cell suspension and adventitious root cultures. In callus cultures, the highest total

phenolics content (TPC; 28.54 mg/g-DW) was estimated at inoculum size of 0.5 g that

was comparable to the total phenolics content (28.28 mg/g-DW) with initial size of

inoculum 1.0 g in callus cultures. As the inoculum size increased from 1.0 g to 1.5 g and

2.0 g, a considerable decrease in TPC (21.53 mg/g-DW) and (16.31 mg/g-DW) was

found (Fig. 10a). Similarly, cell suspension culture yielded the highest amount of TPC

(45.36 mg/g-DW), by using initial inoculum size of 0.5 g but no statistical differences in

total phenolics content (42.80 mg/g-DW) were there by using inoculum size of 1.0 g.

However, there was significant decreased in TPC (40.29 mg/g-DW; 38.71 mg/g-DW) in

cell cultures developed from slightly heavy initial inoculum sizes (1.5 and 2.0 g),

respectively (Fig. 10b). Adventitious root cultures accumulated phenolics content in

contrasting manner to the callus cultures. In adventitious root cultures, lower quantity of

60

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144

initial inoculum (0.5 g) resulted the minimum total phenolics content (38.60 mg/g-DW),

that increased with increasing inoculum size accumulation of phenolics. The initial

inoculum size (1.0 g) yielded 39.27 mg/g-DW of TPC that was statistically similar to

phenolics (39.65 mg/g-DW) accumulated at initial inoculum sizes of 1.5 g. The

maximum total phenolic content (41.46 mg/g-DW) was recorded in culture developed

from inoculum size of 2.0 g (Fig. 10c).

The proper inoculum size is essential for culture growth and secondary metabolites

production (Akalezi et al., 1999; Zhao et al., 2001; Zhang et al., 2002; Lee et al., 2006).

Since no work has been found on the effect of inoculum size on culture growth and

secondary metabolites production in Stevia. However, a few reports are available in this

context in several other plants. The plants accumulate secondary metabolites in stressed

conditions probably due to the activation of several kinds of enzyme (Wang et al., 2010).

Generally, plants utilize phenylpropanoids as a precursor in phenols biosynthetic

pathway. Additionally, phenylalanine ammonia lyase (PAL) enzyme activities are critical

in regulating phenyl propanoids pathway (Winkel-Shirley, 2002). Enhanced activities of

PAL are the first committed steps to accelerate the chain of phenyl propanoid pathway

subsequent reactions for efficient accumulation of phenol compounds (Winkel-Shirley,

2001). According the results, callus and cell suspension cultures were proven to be more

responsive to lower initial inoculum size for phenol production. By contrast, adventitious

root culture accumulated higher amount of total phenolics content at high initial inoculum

size. Since both callus and adventitious roots have their own anatomical features due to

which both responded in different manners. Previously, it has been reported that

secondary metabolites in most of the plants are tissue and organ specific (Ali et al., 2013;

Ahmad et al., 2013). Moreover, level of expression and activities of enzymes in various

plant tissues have different responses to biotic and abiotic stress (Zhao et al., 2005; Wang

et al., 2010). The enhanced phenolics in callus cultures with smaller inoculum sizes could

be attributed to greater availability of nutrients, plant growth regulators, water and other

media components. In this experiment, callus culture was induced with addition of 2, 4-D

and BAP in culture media. Therefore, the maximum accumulation of TPC in callus

culture as a result of lower inoculum size could be due to sufficient availability of 2, 4-D

as compared to higher inoculum size as auxin such as 2, 4-D enhances PAL activities and

consequently increase the accumulation of TPC (Khandaker et al., 2012; Tariq et al.,

2014). These results are also in agreement with the findings of (Ozeki and Komamine,

145

1985; Lee and Shuler, 2000), who observed that low initial inoculum size in cultures

enhanced the accumulation of phenolics. By contrast, the adventitious root cultures of

Stevia accumulated higher quantities of total phenolics content at higher initial inoculum.

Generally, it is a well-known fact that cells in cultures competes for space, water,

nutrients and other resources (Yusuf et al., 2012), thus the use of higher amount of initial

inoculum size in adventitious root culture develops a stress condition (Jacinda et al.,

2008). Moreover, the morphological differentiation and biochemical processes are often

linked in tissue cultures. For many plant species, the synthetic capacity of

dedifferentiated cells is lower than that of the fully differentiated tissues, both

quantitatively and qualitatively (Wu et al., 2003). Chemical gradients in differentiated

tissue complex or cell aggregates encourage the synthesis of secondary metabolites. The

morphological differentiation of the cell is manipulated by changing the biological,

chemical and physical factors (Edahiro and Seki, 2006). Besides this, intact plants and in

vitro cultures have their own physiological, morphological, biochemical and molecular

mechanisms for growth and secondary metabolites production that could be induced with

stress conditions like nutrients deficiencies (Vance et al., 2003). As in this study,

adventitious root cultures accumulated the highest amount of TPC at higher inoculum

size as compared to the lower one. In case of higher inoculum size, there is more

competition for nutrients and can lead to nutrient stress conditions (Yeoman, 1973; Yusuf

et al., 2012). In this context, several studies have been conducted to enhance the

accumulation of important plant metabolites with altering strength of the medium

(Buitelaar and Tramper, 1992). Ruiz et al. (2003) reported increased in phenolics

production in low nutrients culture environment. Cui et al. (2010) also induced phenolics

and flavonoids in adventitious root cultures of Hypericum perforatum L. by changing

strength of the culture medium from full to half and MS/4. More specifically, Mg and Ca

in deficit amount in culture medium enhanced accumulation of phenolic content in

various species of Digitalis (Sahin et al., 2013).

146

10

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30a

c

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aa

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Tot

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heno

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cont

ent (

mg/

g-D

W)

25

30

35

40

ca

bbc

c

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Inoculum size (g)

0.5 2.01.5

Fig. 10. Effect of inoculum size on accumulation of total phenolics content (mg/g-DW)

in callus, cell suspension and adventitious root culture of Stevia rebaudiana. Mean values

(± S.E) with common alphabets are nonsignificant at P ≤ 0.05.

147

Effect of inoculum size on flavonoids production in different Stevia cultures

The inoculum size significantly affected the total flavonoids content in callus, cell

suspension and adventitious root cultures of Stevia. In callus and cell cultures, the small

inoculum size resulted in greater flavonoids, which were higher at larger inoculum size in

adventitious root culture.

The total flavonoid content (24.78 mg/g-DW) was the highest in callus cultures having

initial inoculum size of 0.5 g, and declined with further, increased in initial inoculum size

to 23.50, 21.53, and 16.31 mg/g-DW with 1.0, 1.5, and 2.0 g, respectively (Fig. 11a).

The total flavonoids content of cell cultures also varied with the initial inoculum size.

The cell cultures developed from initial inoculum 0.5 g, yielded more flavonoids (36.50

mg/g-DW) but further increase in initial inoculum sizes resulted in no increase in

flavonoids synthesis, rather a significant decline (32.87 mg/g-DW) with 2.0 g inoculum

size (Fig. 11b). Thus, callus and cell cultures at small size of initial inoculum

accumulated significantly higher amount of flavonoids. However, the adventitious root

cultures produced the maximum flavonoids (33.44 mg/g-DW) with large inoculum size.

Decreasing inoculum size to 1.5, 1.0 and 0.5 g decreased the flavonoids content to 32.20,

31.82 and 31.43 mg/g-DW, respectively (Fig.11c).

The flavonoids are one of the major low molecular weight secondary metabolites, which

have a key role in plant growth, development and survival in stress conditions (Harbone

and Williams 2000). The selection of proper inoculum size is essential for culture growth

and secondary metabolites production (Wang et al., 1997; Akalezi et al., 1999; Zhao et

al., 2001; Zhang et al., 2002; Lee et al., 2006; Jayaraman and Mohamed, 2015). In this

study, callus and cell suspension cultures accumulated more flavonoids from smaller

inoculum size that could be due to the proper availability of nutrients, plant growth

regulators, water and other media components. This argument is supported by the

observation that supplementing the media with plant hormones, water, nutrients and

dissolved gases in sufficient amount increase the PAL activities and flavonoids

production (Khandaker et al., 2012; Tariq et al., 2014). The high phenolic and flavonoid

contents with low initial inoculum size in cultures has also been reported by several

148

researchers (Ozeki and Komamine, 1985; Lee and Shuler, 2000). However, in contrast to

our results, Yang et al. (2009) optimized protocols for the maximum accumulation of

flavonoids and concluded that cell cultures yielded more total flavonoids content at large

initial inoculum size. However, further increase in initial inoculum reduced accumulation

of total flavonoids content. Similarly, significantly higher amount of flavonoids were

achieved from in cell suspension culture established from higher inoculum size (Tan et

al., 2013). The contrast variations in flavonoids accumulation in cell suspension culture

of various plants in response to various inoculum size could be due to the differences in

genetic makup of the plants. However, in this study, the adventitious root cultures yielded

more flavonoids at high initial inoculum size. Previously, several other scientists have

reported high flavonoids (anthocyanin) at higher initial inoculum size in strawberry in

vitro cultures (Sakurai et al., 1996). Increase in total flavonoids content in culture in vitro

might be due to the fact that cells in culture compete more vigorously for medium

compositions (Jacinda et al., 2008). Several workers have been reported that condensed

and large initial inoculum sizes established cultures have potential to accumulate

secondary metabolites at high rate as compared to lower inoculum size (Paek et al.,

2001).

149

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34

36c

a

bb

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Inoculum size (g)

0.5 2.01.5

Fig. 11. Effect of inoculum size on accumulation of total flavonoids content (mg/g-DW)

in callus, cell suspension and adventitious root culture of Stevia rebaudiana. Mean values

(± S.E) with common alphabets are nonsignificant at P ≤ 0.05.

150

Effect of inoculum size on DPPH-radical scavenging activity in different Stevia

cultures

The results indicated that callus, cell suspension and adventitious root cultures

established from various initial inoculum sizes showed significant variations in DPPH-

radical scavenging activity (DRSA).

The callus culture established from 0.5 g initial inoculum size had the highest DRSA

(77.57 %). However, significant reduction in DRSA (66.13 %), (61.44 %) and (39.00 %)

was observed with the increasing inoculum size 1.0 g, 1.5 g and 2.0 g, respectively for

culture development (Fig. 12a). However, cell cultures showed the lowest DRSA (72.73

%) at 0.5 g inoculum size. In comparison to callus culture, cell culture was found with

enhanced free radical scavenging potential by increasing the size of initial inoculum.

Further increase significantly induced DRSA in cell culture with respect to lower initial

inoculum size (0.5 g) but all DRSA (77.42 %), (77.57 %) and (78.30 %) at high inoculum

sizes (1.0 g), (1.5 g) and (2.0 g), respectively were found statistically at par among each

other (Fig. 12b). Adventitious root cultures resulted higher potential (98.82 %) for

scavenging free radicals at more condensed (2.0 g) initial inoculum size. Rest of the

initial tested inoculum sizes showed significantly similar potential. However, the

minimum (93.99 %) was observed in cultures established from initial inoculum sizes (1.0

g) and (0.5 g) (Fig. 12c).

Beneficial properties of antioxidant compounds have been increased the interest of the

scientists (Kahkonen et al., 1999; Robards et al., 1999). These antioxidant compounds

available in plants are health friendly and have been approved as a dietary supplement

(Seong et al., 2004). A number of agronomic, horticultural and medicinal plants are

potent sources of antioxidant activities (Bravo, 1998). The antioxidant activities of the

plants are due to development of vitamins, essential oils, polyphenols and flavonoids,

which are important to protect cellular damages in oxidative stress conditions (Mazid et

al., 2011). Under in vitro condition, success of plant cell, tissue and organ culture

depends upon media compositions, nature of the culture, explant type and size of the

inoculum (explant). As the inoculums are incubated on culture medium, they face stress

conditions. To compete, plants not only accumulate certain secondary metabolites but

151

also enhance the activities of antioxidant enzymes like superoxide dismutase (SOD),

catalase, peroxidases etc. (Foyer et al., 1997). In our current study, callus, cell and

adventitious root cultures of Stevia also showed free radical scavenging activities. In all

three cultures, antioxidant activities were varied significantly as a result of culturing

initial inoculums of various sizes. The presences of antioxidant potentials in all three

cultures further justify previous statements that in vitro cultures are in stress from the

very beginning. In such conditions, biological tissues develop several biochemical

responses to encourage synthesis of antioxidant constituents or increased antioxidant

enzymatic activities (Ramotar et al., 1998). Since, it has been reported that most of the

antioxidant activities are mainly due phenols and flavonoids (Shadidi and Nazck, 1995;

Hollman et al., 1996). In callus cultures, antioxidant potentials were maximum at lower

initial inoculum sizes. The current study described that callus culture also yielded

maximum amount of total phenolics content by using lower initial inoculum size. Thus,

our result has direct correlation with results obtained for total phenolics production.

Several other studies conducted on various plant cultures, also revealed that low initial

inoculum size in cultures enhanced the production of phenolic contents and resultantly

maximum antioxidant activities were observed (Lee and Shuler, 2000). In contrast to

callus cultures, cell and adventitious root cultures showed significant higher amount of

antioxidant activities at higher initial inoculum size. The enhanced antioxidant activities

in cell culture might be due to the fact that cells in cultures compete for space, water,

nutrients and other resources, thus the use of higher amount of initial inoculum has

greater demand for media components as compared to the lower ones. In this regard, Yu

et al. (1998) reported significant variation in free radical scavenging potentials due to

several nutrients (K, Fe, Zn, Mg, Cu, B) deficiencies. Besides this, significant increase in

antioxidant activities as a result deficiencies in phosphorous (Ferreria et al., 2008) and

magnesium (Tewari et al., 2006) has been reported. Deficient amount of some nutrients

trigger expression of genes that cause synthesis of various secondary metabolites like

phenols, flavonoids, vitamins and induce various biological processes (Quiroga et al.,

2000). Improved accumulation of vitamin c as a natural antioxidant was also observed in

Mg deficient environment (Tewari et al., 2006). Nutrients deprive conditions also trigger

antioxidant enzyme activities (Cakmak and Marschner, 1988).

152

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80 a

d

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sca

veng

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acti

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(%

)

80

85

90

95

100

c

a

bbb

1.0

Inoculum size (g)

0.5 2.01.5

Fig. 12. Effect of inoculum size on DPPH-radical scavenging activities (%) in callus, cell

suspension and adventitious root culture of Stevia rebaudiana. Mean values (± S.E) with


153

Correlation of DPPH-radical scavenging activity with phenolics and flavonoids

The DPPH-radical scavenging activity in callus and adventitious root cultures generally,

depended on total phenolics and flavonoids contents. However, DPPH-radical scavenging

activity in cell cultures showed slightly different response. The results demonstrated that

the initial low inoculum size in callus and cell suspension cultures yielded higher

phenolics (28.54 mg/g-DW) and flavonoids (24.78 mg/g-DW), respectively and had the

maximum DPPH-radical scavenging activity (77.57 %) as compared to callus culture

from larger initial inoculum sizes (Fig. 13). By contrast, the cell culture had higher

phenolics (45.36 mg/g-DW) and flavonoids (36.50 mg/g-DW) contents but lower DPPH-

radical scavenging activity at initial inoculum size of 0.5 g. The phenolics and flavonoids

contents decreased with increasing inoculum size but the DPPH-radical scavenging

activities increased significantly. Thus, the maximum DPPH-radical scavenging activity

of cell culture (78.30 %) with 2.0 g inoculum size was independent of total phenolics and

flavonoids content (Fig. 14). In adventitious root culture, however, the DPPH-radical

scavenging activity was clearly depended on total phenolics and flavonoids. At lower

initial inoculum size, adventitious root culture yielded low total phenolics (38.60 mg/g-

DW) and flavonoids (31.43 mg/g-DW) content and had lower DPPH-radical scavenging

activity (93.99 %). However, a linear increased was observed in phenol (41.46 mg/g-

DW), flavonoids (33.44 mg/g-DW) as well as DPPH-radical scavenging activity with

increasing inoculum size so it was the highest (98.82 %) at the highest inoculum size (2.0

g) (Fig. 15).

The oxidative stress release reactive oxygen species (ROS), that damages the macro

molecules (Ragavendran et al., 2012), resultantly destabilize cellular structures and

functions (Lee et al., 2007). The plant contains an antioxidant system to prevent the

formation of ROS. Such antioxidant system may be enzymatic such as super oxide

dismutase (SOD), peroxidase and catalase (Hakiman and Maziah, 2009) and non-

enzymatic like phenols, flavonoids, ascorbic acid and glutathione (Gout et al., 2001;

Johnson et al., 2003). Generally, in vitro cultures the antioxidant activities are dependent

on phenolics and flavonoids content (Roby et al., 2013). While, a positive correlation in

phenolics and flavonoids has been suggested (Ali and Abbasi, 2014; Tariq et al., 2014),

154

14

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18

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22

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26

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TPC

TFC

16

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26

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Tota

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avonoid

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(mg/g

-DW

)

Tota

l phen

oli

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onte

nt

(mg/g

-DW

)

30

40

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70

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d

c

b

a

1.0

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PH

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sca

ven

gin

g a

ctiv

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%)

Inoculum size (g)

0.5 2.01.5

such a dependence was observed in callus and adventitious root culture. By contrast, in

cell suspension culture, the antioxidant activity was the highest despite low phenolics and

flavonoids. Since, the antioxidant activities in cell cultures seemed less dependent on

phenolics and flavonoids, rather the enzymatic factor to detoxify the effect of ROS.

Several enzymes such as superoxide dismutase (SOD) and Catalase have been known to

detoxify superoxide and hydrogen peroxide radicals (Scandalios, 1987; Kusvuran et al.,

2012). Besides the enzymatic antioxidant system, vitamins such as E and C have also

been shown to have strong anti-oxidizing activities (Kayang, 2007).


scavenging activities in callus cultures of Stevia rebaudiana. Mean values (± S.E) with


155

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/g-D

W)

TPC

TFC

To

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ph

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oli

c c

on

ten

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g/g

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31

32

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%)

Inoculum size (g)

0.5 2.01.5


scavenging activities in adventitious root cultures of Stevia rebaudiana. Mean values (±


157

Effect of inoculum size on steviol glycosides production

Steviol glycosides such as stevioside, rebaudioside and dulcoside were significantly

varied in callus, cell suspension and adventitious root cultures, established from various

inoculum sizes.

Callus culture developed from smaller inoculum size (0.5 g) yielded higher amount of

stevioside (43.89 mg/g-DW) and rebaudioside (36.54 mg/g-DW) contents. However,

dulcoside contents (2.57 mg/g-DW) were found in higher amount in callus cultures

established from initial inoculum size (1.0 g). Significant reduction in all three contents

was observed as initial inoculum size was further increased up to 1.5 g. But again

significant increase in stevioside (33.55 mg/g-DW), rebaudioside (26.33 mg/g-DW) and

dulcoside (1.09 mg/g-DW) contents were found with further incremental increase of

initial inoculum size from 1.5 g to 2.0 g (Fig. 16 a, b and c).

Cell cultures also accumulated significantly higher contents of stevioside (59.89 mg/g-

DW), rebaudioside (24.41 mg/g-DW) and dulcoside (1.85 mg/g-DW) at 0.5 g initial

inoculum size. Further, increase of initial inoculum size did not maintain that level of

steviol glycoside contents and the minimum stevioside (25.26 mg/g-DW), rebaudioside

(8.49 mg/g-DW) and dulcoside (0.00 mg/g-DW) contents were observed at 2.0 g of initial

inoculum size (Fig. 17 a, b and c).

Slight contrast results were noted in adventitious root culture of Stevia in obtaining

stevioside, rebaudioside and dulcoside contents. The smaller inoculum size (0.5 g)

yielded lower amount of stevioside (32.37 mg/g-DW), rebaudioside (14.84 mg/g-DW)

and dulcoside (0.11 mg/g-DW) contents. However, stevioside (64.75 mg/g-DW) and

rebaudioside (29.67 mg/g-DW) contents were significantly increased to their maximal

level using initial inoculum size (1.0 g). Both contents were significantly reduced with

further increase of initial inoculum size. On the other hand high dulcoside contents (0.71

mg/g-DW) were found in culture developed from 1.5 g initial inoculum size (Fig. 18 a, b

and c).

158

Inoculum size has an important role in in vitro culture growth and development

(Kanokwaree et al., 1997). To ensure higher production of secondary metabolites in

culture in vitro, it is important to optimize appropriate inoculum size as it has been

evaluated for in vitro cultures of several plants for efficient accumulation of secondary

metabolites (Dornenburg and Knorr, 1995; Akalezi et al., 1999; Zhao et al., 2001; Zhang

et al., 2002; Lee et al., 2006; Jeong et al., 2009; Praveen and Murthy, 2010). Till to date,

not even single report has been cited on the effect of inoculum size on secondary

metabolites accumulation in Stevia rebaudiana in vitro cultures. However, research has

been conducted on several other important medicinal plants in this context. Our current

findings suggested that like in vitro cultures of other plants, biosynthesis of secondary

metabolites in Stevia cultures were also considerably influenced by various inoculum

sizes. Lower inoculum size (0.5-1.0 g) yielded significantly higher amount of stevioside,

rebaudioside and dulcoside contents in callus, cell suspension and adventitious root

cultures. However, all three cultures established from heavy inoculums (1.5-2.0 g) among

tested sizes, accumulated lesser contents of stevioside and rebaudioside except dulcoside

in adventitious root culture. Results of Contin et al. (1998) are in harmony with our

current findings, who observed enhanced accumulation of artemisinin in Artemisia annua

at smaller size inoculum and cultures developed from comparatively higher initial

inoculum yielded lesser amount of artemisinin contents. Decline in such important

metabolites in Stevia cultures at high initial inoculum might be due to the competition of

cultured cell, tissue or organ for nutrients, oxygen, plant growth regulators and other

medium compositions (Henshaw et al, 1966; Contin et al., 1998; Blackhall et al., 1999).

However, in contrast to Stevia cultures (callus, cell suspension and adventitious root), in

vitro cultures of several other plants yielded comparatively higher amount of

corresponding secondary metabolites. Furthermore, according to our results, mainly

stevioside and rebaudioside contents were raised at 2.0 g inoculum size. This indicated

the fact that like other plants, in vitro cultures of Stevia plant tends to accumulate higher

amount of metabolites in stress condition due to higher inoculum size. A similar

phenomenon was found by Jeong et al. (2009) in cell culture of Panax ginseng. Wu et al.

(2006) also observed enhanced accumulation of plant base valuable compounds in

cultures in vitro comparatively established from larger initial inoculum. Besides these,

159

cell culture of Catharanthus roseus also required higher initial inoculum size for the

maximum production of ajmalicine (Lee and Shuler, 2000). Enhanced biosynthesis of

anthocyanin in cell culture of Perilla frutescens was recorded at higher initial inoculum

size (Zhong and Yoshida 1995). Several other reports are available in support that

inoculum size has influential role in secondary metabolites production in various in vitro

cultures of several medicinal plants. Sakurai et al. (1996) also mentioned in their report

that anthocyanin contents were significantly improved in cell culture of strawberry with

the use of various initial inoculum sizes. The stimulatory effects of initial inoculum size

on accumulation of valuable compounds have been reported in cell culture of ginseng for

ginsinoside production (Jeong et al., 2009), saponin contents in root culture of Talinum

Paniculatum (Manuhara et al., 2012), 20-hydroxyecdysone in cell culture of Vitex

glabrata (Sinlaparaya et al., 2007), gallic acid production in suspension culture of Acer

ginnala Maxim (Jun-Ge et al., 2006), withanolide A-B accumulation in adventitious root

culture of Withania somnifera (Sivanandhan et al,. 2012).

160

0.0

0.5

1.0

1.5

2.0

2.5

3.0

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Fig. 16. Effect of inoculum size on stevioside, rebaudioside and dulcoside contents in



161

0

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iosid

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)

b

Fig. 17. Effect of inoculum size on stevioside, rebaudioside and dulcoside contents in cell

suspension culture of Stevia rebaudiana. Mean values (± S.E) with common alphabets

are nonsignificant at P ≤ 0.05.

162

0.0

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Fig. 18. Effect of inoculum size on stevioside, rebaudioside and dulcoside contents in



163


Optimization of appropriate inoculum size is important for higher production of

secondary metabolites in in vitro culture. Therefore, the current research work entitled

“The effect of inoculum size on biomass, phenolics and flavonoids, antioxidant activity

and bioactive compounds in callus, cell suspension and adventitious root cultures of

Stevia rebaudiana (Bert.)” was planned in 2014. For this purpose; callus, cell suspension

and adventitious root cultures were developed using inoculums of various sizes (0.5, 1.0,

1.5 and 2.0 g). Each culture was treated as a single experiment, designed in Complete

Randomized method. A growth curve (30 days; 03 days interval) was developed for the

rapidly growing calli, cells and adventitious root cultures, to evaluate the growth pattern

of each culture against various inoculum sizes. Along with fresh and dry weight, samples

from each treatment of each experiment were further subjected to analyze total phenolics

content (TPC), total flavonoids content (TFC), DPPH-radical scavenging activity

(DRSA) and its correlation with TPC and TFC. HPLC analysis was used for

determination of stevioside, rebaudioside and dulcoside contents.

Callus culture growth and metabolites accumulation were found dependent on initial

inoculum size. Relatively shorter lag phase of 3 days of the inoculation for all inoculum

sizes (0.5-2.0 g) was observed in callus culture. Lag phase was followed by an elongated

log phase from day 3rd to 27th day of the culture. Decline in growth was occurred after 27

day of culture in all inoculum sizes. Similarly, fresh and dry weights were also positively

encouraged by the increasing inoculum sizes (0.5–2.0 g). Comparatively, cultures

initiated from higher inoculum size (2.0 g) resulted in the highest fresh (112.29 g l-1) and

dry (7.71 g l-1) biomass. Whereas, callus developed from lower inoculum size (0.5 g)

resulted the accumulation of the poor fresh (69.81 g l-1) and dry (3.43 g l-1) biomasses. In

contrast, callus culture developed from lower initial inoculum size (0.5 g) yielded higher

amount of TPC (28.54 mg/g-DW), TFC (24.78 mg/g-DW), stevioside (43.89 mg/g-DW)

and rebaudioside (36.54 mg/g-DW) contents with pronounced DRSA (77.57 %).

However, dulcoside contents (2.57 mg/g-DW) were found in higher amount in callus

culture, established from initial 1.0 g inoculum size. Use of comparatively higher initial

164

inoculum sizes did not encourage TPC, TFC and DRSA along with stevioside,

rebaudioside and dulcoside contents.

However, in cell suspension culture, an elongated lag phase started from day 3 to day 12

of the culture was subsequently followed by a long log phase (12-27 days duration). All

cultures did not experience stationary phases and after 27th day of culture, decline in

growth was occurred. Similarly, fresh and dry weight of cell cultures increased with

increasing inoculum size (0.5-1.5 g). Initial inoculum size 1.5 g was found to be the

optimum for maximum accumulation of fresh (102.71 g l-1) and dry biomass (5.38 g l-1)

of cell cultures. However, minimum fresh (70.19 g l-1) and dry (2.86 g l-1) biomass were

accumulated in cultures developed from lower inoculum size (0.5 g). In contrast, cell

suspension culture yielded the highest amount of TPC (45.36 mg/g-DW) and TFC (36.50

mg/g-DW) at 0.5 g inoculum size. However, the least TPC (38.71 mg/g-DW) and TFC

(32.87 mg/g-DW) were observed at 2.0 g inoculum size. On the other hand, cell culture

exhibited the maximum DRSA (78.30%) at 2.0 g initial inoculum as compared DRSA

(72.73 %) at lower initial inoculum size (0.5 g). on the other hand, the highest stevioside

(59.89 mg/g-DW), rebaudioside (24.41 mg/g-DW) and dulcoside (1.85 mg/g-DW) contents were

recorded at 0.5 g initial inoculum size. While, cell suspension cultures produced lesser amount of

stevioside (25.26 mg/g-DW), rebaudioside (8.49 mg/g-DW) and dulcoside (0.00 mg/g-DW)

contents at 2.0 g of initial inoculum size.

Adventitious root cultures developed from various initial inoculum size (0.5-2.0 g) did

not display lag phase and an increase in growth curve was found at early stage (day 3) as

log phase of the culture that was continued till 27 day of culture period followed by

decline phase. Similarly, adventitious root cultures established from various inoculum

sizes (0.5-2.0 g) were found with significant variations in fresh and dry biomass

accumulation along with secondary metabolites production. Inoculum size 1.5 g was

regarded to be the best for the accumulation of maximum fresh (106.86 g l-1) and dry

(5.05 g l-1) biomasses in root cultures. On the other hand, adventitious root culture

established from smaller inoculum sizes (0.5 g) resulted in poor fresh (70.57 g l-1) and

dry biomass (2.29 g l-1). However, the maximum TPC (41.46 mg/g-DW), TFC (33.44

mg/g-DW) and DRSA (98.82 %) were recorded at 2.0 g inoculum size. The lower

165

inoculum (0.5 g) resulted in minimum TPC (38.60 mg/g-DW), TFC (31.43 mg/g-DW)

and DRSA (93.99 %). Similarly, small inoculum size (0.5 g) yielded poor amount of stevioside

(32.37 mg/g-DW), rebaudioside (14.84 mg/g-DW) and dulcoside (0.11 mg/g-DW) contents.

However, stevioside (64.75 mg/g-DW) and rebaudioside (29.67 mg/g-DW) contents were

significantly increased to their maximal level using initial 1.0 g as initial inoculum size. On the

other hand the highest dulcoside contents (0.71 mg/g-DW) were found in culture developed from

1.5 g initial inoculum size.

Conclusions

Our current results suggested that growth kinetics, fresh and dry biomass of

callus, cell suspension and adventitious root cultures were positively encouraged

with the increasing inoculum size (0.5–2.0 g). Growth kinetics of each culture

was characterized with lag, log and decline phase. Meanwhile, the highest fresh

(112.29 g l-1) and dry biomass (7.71 g l-1) in callus culture was accumulated when

the nutrient medium was inoculated with 2.0 g inoculum. Similarly, inoculum size

(1.5 g) was optimized for accumulation of fresh and dry biomasses (102.71 g l-1;

5.38 g l-1) in cell suspension and adventitious root (106.86 g l-1; 5.05 g l-1)

cultures.

It was also concluded that initial inoculum size not only influenced fresh and dry

biomass of the cultures but also significantly induced the production of desirable

secondary metabolites. Among various tested inoculum sizes, 0.5 g was proven to

be the best initial inoculum size for maximum production of TPC (28.54 mg/g-

DW), TFC (24.78 mg/g-DW), stevioside (43.89 mg/g-DW) and rebaudioside

(36.54 mg/g-DW) contents along with higher DRSA (77.57 %) in callus cultures.

Whereas, dulcoside content (2.57 mg/g-DW) was found in higher amount in

callus culture established from initial inoculum size (1.0 g).

In cell suspension culture, initial inoculum size (0.5 g) was also regarded as an

optimum for accumulation of maximum TPC (45.36 mg/g-DW), TFC (36.50

mg/g-DW), stevioside (59.89 mg/g-DW), rebaudioside (24.41 mg/g-DW) and

dulcoside (1.85 mg/g-DW) contents. While the highest DRSA (78.30%) were

found at 2.0 g inoculum size.

166

Adventitious root culture accumulated significantly higher amount of TPC (41.46

mg/g-DW), TFC (33.44 mg/g-DW) as well as DRSA (98.82 %) at 2.0 g initial

inoculum size. However, stevioside (64.75 mg/g-DW) and rebaudioside (29.67

mg/g-DW) contents were significantly increased to their maximal level using

initial inoculum size (1.0 g). On the other hand high dulcoside contents (0.71

mg/g-DW) were found in cultures developed from 1.5 g initial inoculum size.

Recommendations

The following recommendations could be made on the basis of above

conclusions.

Higher inoculum size, more specifically 2.0 g for callus culture, 1.5 g for cell

suspension and adventitious root cultures should be used to obtain maximum

fresh and dry biomasses.

Lower inoculum size (0.5 g) is recommended to obtain maximum TPC, TFC,

DRSA, stevioside and rebaudioside contents in callus cultures. However, to

obtain more dulcoside content, inoculum size 1.0 g should be used.

Similarly, cell suspension culture should be developed from lower inoculum size

(0.5 g) to obtain TPC, TFC, stevioside, rebaudioside and dulcoside contents in

higher amounts.

Using higher inoculum size (2.0) in adventitious root culture is best approach to

accumulate TPC, TFC and antioxidant potential in higher quantities. However,

adventitious root culture should be developed by using initial inoculum size (1.0

g) for the maximum accumulation of stevioside and rebaudioside contents, while

1.5 g is best to use for dulcoside production in root culture.

167

GENERAL SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Stevia botanically named as Stevia rebaudiana Bertoni ranks on top in genus Stevia due

to its sweet taste. Sweet taste of Stevia is mainly due to stevioside and rebaudioside

contents. These sweet tasting agents are considered to be 300 to 400 times sweeter than

commercial sugar and are regarded as natural substitute for diabetic and obese patients.

Keeping in view, the commercial importance of steviol glycoside, this study was aimed

to optimize reliable protocols for callus, cell suspension and adventitious root culture for

the production of important secondary metabolites. The research work was conducted at

Plant Tissue Culture Lab., Department of Plant Breeding and Genetics, The University of

Agriculture Peshawar., during the year of 2014/15. For the development of reliable

protocols, the effect of sucrose (05, 10, 15, 20, 25, 30, 35, 40, 45 and 50 g l-1), pH (5.1,

5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0) and inoculum size (0.5, 1.0, 1.5 and 2.0 g) on

callus, cell and adventitious root culture was screened out for efficient culture

establishment and important secondary metabolites production. In preliminary

experiment, callus culture was also exposed to various spectral lights such as green,

yellow, blue and red lights. While, white light was kept as control. For this purpose, over

all research study was divided into 3 main experiments. Each main experiment was

further subdivided into sub experiments.

Complete randomized design (CRD) was used throughout optimization process, to study

quantitative and qualitative parameters, more in scientific way. For this purpose, a growth

curve was developed for the rapidly growing calli, cells and adventitious root culture in

response to different spectral lights, sucrose concentrations, media pH and inoculum

sizes. The growth kinetics of calli, cells and adventitious root cultures were determined

for 30 days period with 3 days interval. The lag, log and stationary phases were

determined for fresh accumulated biomass of calli, cells and adventitious roots from

established growth curve. These tissues were then used for the determination of fresh and

dry weight. Oven dried samples of each experiment was further subjected to analyze total

phenolics content (TPC), total flavonoids content (TFC), DPPH-antioxidant activities

(DRSA) and its correlation with TPC and TFC. HPLC analyses were used for

determination of stevioside, rebaudioside and dulcoside contents.

168

Among various spectral lights, control white light improved callogenic frequency than

other colored lights. However, the blue light enhanced phenolics and flavonoid contents

in callus culture. The TPC showed a linear correlation with TFC and total antioxidant

capacity. However, green and red lights enhanced reducing power assay and DRSA.

Sucrose induced osmotic stresses (05-50 g l-1), pH levels (5.1-6.0) and various inoculum

sizes (0.5-2.0 g) significantly influenced fresh and dry biomass (g) and production of

secondary metabolites in callus culture of Stevia rebaudiana. Among various sucrose

concentrations, callus accumulated significantly higher amount of fresh and dry biomass

(142.38 g l-1, 11.71 g l-1) in media having 40 g l-1 and 50 g l-1 sucrose, respectively.

Among different sucrose concentrations (5.0-50 g l-1), 30 g l-1 sucrose was found to be

the optimum for the production of maximum total phenolics content (TPC; 124.20 mg/g-

DW), total flavonoids content (TFC; 49.36 mg/g-DW), rebaudioside contents (6.56 mg/g-

DW) and DRSA (92.82 %) in callus cultures. However, significantly higher amount of

stevioside (42.34 mg/g-DW) and rebaudioside (22.67 mg/g-DW) contents were

accumulated in callus culture treated with 15 and 20 g l-1 sucrose, respectively.

Similarly, among various pH levels, fresh (130.57 g l-1) and dry biomass (12.10 g l-1) of

callus cultures was found maximum at 5.6 pH level. Likewise, callus culture favored

media pH 5.8 for the maximum accumulation of TPC (43.38 mg/g-DW), TFC (37.55

mg/g-DW) and DRSA (87.68 %). However, media pH 5.6 was also found optimum for

stevioside (62.20 mg/g-DW) and rebaudioside (22.79 mg/g-DW) production in callus

cultures. On the other hand, the highest amount of dulcoside content (5.92 mg/g-DW) in

callus culture was observed at low pH level (5.1).

In callus culture, fresh and dry weight was also positively encouraged by the increasing

inoculum sizes (0.5–2.0 g). The highest fresh and dry biomass (112.29 g l-1; 7.71 g l-1)

was accumulated as a result of 2.0 g inoculum, whereas, callus developed from minimum

inoculum size (0.5 g) resulted the accumulation of the least fresh (69.81 g l-1) and dry

(3.43 g l-1) biomasses. In contrast, callus culture developed from lower initial inoculum

size (0.5 g) yielded higher amount of TPC (28.54 mg/g-DW), TFC (24.78 mg/g-DW),

stevioside (43.89 mg/g-DW) and rebaudioside (36.54 mg/g-DW) along with DRSA

169

(77.57 %). However, dulcoside contents (2.57 mg/g-DW) were found in higher amount in

callus culture, established from initial 1.0 g inoculum size. Use of comparatively higher

initial inoculum sizes did not encouraged TPC, TFC, free radical scavenging potentials,

stevioside, rebaudioside and dulcoside contents.

Biomass yield and secondary metabolites production were also considerably varied in cell

suspension culture with various sucrose induce osmotic stress conditions (05-50 g l-1), pH

levels (5.1-6.0) and various inoculum sizes (0.5-2.0 g). Lower sucrose induce stress

conditions (5-20 g l-1) enhanced the fresh biomass of cells, while higher sucrose stress (25-

50 g l-1) gradually reduced the accumulation of fresh biomass of suspended cells. Among

various sucrose levels, liquid media having 20 g l-1 sucrose resulted in maximum fresh

(97.71 g l-1) and dry (8.57 g l-1) biomass of suspended cells while the highest sucrose stress

(50 g l-1) significantly reduced fresh biomass accumulation (25.43 g l-1). In contrast,

minimum dry biomass (4.57 g l-1) of cells was noted for liquid media having 5 g l-1 of

sucrose. Furthermore, cell suspension cultures accumulated more TPC and TFC (139.20

mg/g-DW; 41.46 mg/g-DW) at higher sucrose level (40 g l-1) but lower sucrose

concentration (5 g l-1) in cell culture yielded poor TPC (51.34 mg/g- DW) and TFC (17.28

mg/g-DW). Similarly, there was minimum potential (33.28%) to scavenge free radicals

(DPPH) in cell suspension culture at lower sucrose concentration (05 g l-1). However,

further incremental increase of sucrose concentration considerably enhanced DRS

activities. Among all concentrations tested, cells cultured in media having 30 g l-1 sucrose

were found with the maximum DRSA (83.87%). Lower concentrations of sucrose (05 g l-1)

also strictly inhibited biosynthesis of stevioside (20.16 mg/g-DW) in cell suspension

culture. Increase in stevioside content (40.32 mg/g-DW) was noticed as the media was

supplemented with 10 g l-1 sucrose. However, further increase of sucrose concentration

significantly reduced the quantity of steviosides in cell cultures but again a sudden increase

in stevioside content (42.23 mg/g-DW) was observed by using 30 g l-1 sucrose in culture

media. On the other hand, the lowest rebaudioside content (7.97 mg/g-DW) was quantified

at the most sucrose concentrated medium (50 g l-1) among all tested levels. In contrast, the

highest rebaudioside (27.64 mg/g-DW) and dulcoside contents (6.43 mg/g-DW) were

calculated in cells, cultured in medium fortified with 20 g l-1 sucrose. However, 40, 45 and

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50 g l-1 sucrose concentrations were not able to synthesize dulcoside contents in cultured

cells.

Considerable variations were also observed in cell suspension culture biomass yield and

secondary metabolites production as a result of adjusting media pH at various levels (5.1-

6.0). Among all tested pH levels, the maximum fresh and dry biomass (85.81 g l-1; 8.84 g

l-1) were found when the medium pH was kept as 5.6 and 5.5, respectively. However, the

lowest fresh biomass (55.14 g l-1) was resulted at high medium pH (6.0). In contrast, cell

culture developed in medium having 5.1 pH levels produced poor dry biomass (5.43 g l-1).

In case of secondary metabolites production, cell cultures developed in medium having

5.8 pH, yielded maximum TPC (72.13 mg/g-DW) and TFC (57.32 mg/g-DW) along with

93.99 % DRSA. The minimum TPC (70.70 mg/g-DW) and TFC (50.80 mg/g-DW) was

recorded in cell suspension cultures, established in medium having 6.0 and 5.6 pH levels,

respectively. Similarly, cell cultures developed in media with 5.1 pH level disclosed the

minimum DRSA (71.85 %). Moreover, the highest amount of stevioside (41.47 mg/g-

DW) was estimated at pH level 5.2, while the minimum stevioside contents (17.60 mg/g-

DW) were noted at higher pH level (6.0). On the other hand, rebaudioside and dulcoside

content showed similar production pattern in cell cultures. The highest amount of

rebaudioside (7.01 mg/g-DW) and dulcoside (4.72 mg/g-DW) content were quantified in

cell cultures established at initial medium pH (5.8). Statistically, the least amount of

rebaudioside and dulcoside was found between initial medium pH (5.4) and pH (5.1).

Similarly, fresh and dry weight of cell culture was increased with increasing inoculum

size (0.5-1.5 g). Inoculum size (1.5 g) was optimized for maximum accumulation of fresh

(102.71 g l-1) and dry biomass (5.38 g l-1) of cell cultures. Lower inoculum size (0.5 g) in

culture media resulted minimum fresh (70.19 g l-1) and dry (2.86 g l-1) biomass

accumulation. Similarly, cell suspension culture yielded the highest amount of TPC

(45.36 mg/g-DW) and TFC (36.50 mg/g-DW), by using initial inoculum size of 0.5 g.

However, there was significant decreased in TPC (38.71 mg/g-DW and TFC (32.87

mg/g-DW) in cell suspension cultures developed from high initial inoculum size (2.0 g).

In contrast, cell cultures did not show higher potential for free radicals scavenging (72.73

%) at lower initial inoculum size (0.5 g). In comparison, cell culture was found with

171

enhanced free radical scavenging potential (78.30%) at 2.0 g initial inoculum. Cell

cultures also accumulated significantly higher contents of stevioside (59.89 mg/g-DW),

rebaudioside (24.41 mg/g-DW) and dulcoside (1.85 mg/g-DW) at 0.5 g initial inoculum

size. However, cell suspension cultures produced lesser amount of stevioside (25.26

mg/g-DW), rebaudioside (8.49 mg/g-DW) and dulcoside (0.00 mg/g-DW) contents at 2.0

g of initial inoculum size.

Addition of differential sucrose concentrations (05-50 g l-1) into the liquid media also

significantly induced fresh and dry biomass along with accumulation of secondary

metabolites in adventitious root culture. The maximum fresh (175.43 g l-1) and dry (11.14

g l-1) biomass was accumulated in root cultures having the highest sucrose concentration

(50 g l-1), while the minimum fresh and dry biomass (37.71 g l-1; 2.86 g l-1) was being

observed for 5 g l-1 of sucrose in culture media. The highest TPC (155.00 mg/g-DW) and

TFC (94.78 mg/g-DW) was determined in adventitious roots, cultured in media having 30

g l-1 sucrose. The lowest TPC (17.77 mg/g- DW) and TFC (23.20 mg/g-DW) was

observed in roots obtained from media added with 5 g l-1 sucrose. However, noticeable

amount of DRSA (94.43 %) was recorded in roots established in media augmented with

20 g l-1 sucrose. In contrast, lower DRSA (46.55 %) were found in adventitious roots,

developed in culture medium having 5 g l-1 sucrose. Adventitious root cultures also

showed considerable variations in biosynthesis of steviol glycosides (stevioside,

rebaudioside and dulcoside) exposed to various sucrose concentrations in culture media.

The maximum accumulation of stevioside (73.97 mg/g-DW) and rebaudioside (24.57

mg/g-DW) contents was found in roots taken from media containing 10 g l-1 sucrose.

Both contents were significantly reduced in further concentrated media and the minimum

stevioside (25.58 mg/g-DW) and rebaudioside (10.02 mg/g-DW) contents were found in

adventitious roots obtained from media supplemented with 20 g l-1 sucrose. In case of

dulcoside content in adventitious root culture of stevia, minimum content (0.10 mg/g-

DW) was recorded in roots in media having 5 g l-1 sucrose, while maximum dulcoside

content (12.24 mg/g-DW) were obtained at 40 g l-1 sucrose.

172

Various pH levels (5.1-6.0) as elicitors also significantly influenced fresh and dry

biomass as well as secondary metabolites production in adventitious root culture of

Stevia. Adventitious root culture favored high initial medium pH (6.0) for the highest

fresh (112.86 g l-1) and dry (8.29 g l-1) biomass accumulation. Poor amount of fresh

(62.19 g l-1) and dry (2.29 g l-1) biomass of adventitious roots was observed at minimum

pH level (5.1). On the other hand, adventitious root culture also synthesized higher TPC

and TFC (70.06 mg/g-DW; 50.19 mg/g-DW) in media having 5.8 pH. Root culture with

same pH (5.8) also exhibited maximum DRSA (92.67 %). However, culture developed in

media with minimum pH (5.1), accumulated poor amount TPC (67.50 mg/g-DW) and

TFC (44.84 mg/g-DW) along with DRSA (75.81 %). In contrast, adventitious root

cultures were found in favor of low initial medium pH for stevioside and rebaudioside

production. The maximum quantities of stevioside (79.48 mg/g-DW) and rebaudioside

(13.10 mg/g-DW) contents were observed in culture developed in medium at initial

medium pH of 5.1. However, cultures established at initial medium pH (5.6) and (6.0)

were less successful to accumulate rebaudioside and dulcoside contents respectively. In

case of dulcoside contents, 5.8 pH levels was found optimum for higher amount of

dulcoside content but as medium pH was turned towards low or high acidic range, the

reduction was observed in dulcoside production. Even at initial medium pH (5.1),

dulcoside contents were not detected.

Similarly, adventitious root cultures established from various inoculum sizes (0.5-2.0 g)

were found with significant variations in fresh and dry biomass accumulation along with

secondary metabolites production. Among various adventitious root cultures, the

maximum amount of fresh (106.86 g l-1) and dry (5.05 g l-1) weight was accumulated in

the liquid media when it was inoculated with 1.5 g inoculums. On the other hand,

adventitious root cultures established from smaller inoculum size (0.5 g) resulted in poor

fresh (70.57 g l-1) and dry biomass (2.29 g l-1) accumulation in adventitious roots of

Stevia. However, adventitious root cultures accumulated secondary metabolites in

contrast manner to fresh and dry biomass yield. The maximum TPC (41.46 mg/g-DW),

TFC (33.44 mg/g-DW) and DRSA (98.82 %) were recorded in cultures, developed from

inoculum size of 2.0 g. However, lower quantity of initial inoculum (0.5 g) resulted

173

minimum TPC (38.60 mg/g-DW), TFC (31.43 mg/g-DW) and DRSA (93.99 %). Slight

contrast results were noted in adventitious root culture of Stevia in obtaining stevioside,

rebaudioside and dulcoside contents. Lower initial inoculum size (0.5 g) yielded poor

amount of stevioside (32.37 mg/g-DW), rebaudioside (14.84 mg/g-DW) and dulcoside

(0.11 mg/g-DW) contents. However, stevioside (64.75 mg/g-DW) and rebaudioside

(29.67 mg/g-DW) contents were significantly increased to their maximal level using

initial inoculum size (1.0 g). On the other hand high dulcoside contents (0.71 mg/g-DW)

were found in culture developed from 1.5 g initial inoculum size.

Conclusions

It was concluded from overall results that the establishment of callus, cell

suspension and adventitious root cultures of Stevia are the most promising

approaches for efficient accumulation of biomass and secondary metabolites

production.

Among various elicitors, the application of colored lights, various sucrose

concentrations (05-50 g l-1), media pH (5.1-6.0) and inoculum sizes (0.5-2.0 g) are

important elicitation strategies to enhance biomass accumulation and production

of bioactive compounds.

For establishment of callus, cell suspension and adventitious root cultures of

Stevia, addition of 2, 4-D and NAA (2 mg l-1; 0.5 mg l-1) were optimized for

efficient callus induction, 2, 4-D and NAA (1 mg l-1; 0.5 mg l-1) for cell

suspension and NAA (0.5 mg l−1) in half strength media for adventitious root

cultures.

For maximum biomass accumulation in callus culture, sucrose at the rate of 40-50

g l-1, media pH 5.6 and inoculum size 2.0 g was optimized under white light

condition. Similarly, addition of 20 g l-1 sucrose, media pH 5.6 and inoculum size

1.5 g was found to be the optimum for higher biomass yield in cell suspension

culture. On the other hand, adventitious root cultures favored higher concentration

of sucrose (50 g l-1), media pH (6.0) and inoculum size (1.5 g) for maximum fresh

and dry biomasses.

174

Furthermore, callus culture accumulated significantly higher amount of TPC and

TFC as a result of 30 g l-1, pH (5.8) and inoculum size along with blue light

exposure. Moreover, cell suspension culture synthesized TPC and TFC in larger

amount in response to (40 g l-1), media pH (5.8) and inoculum size (0.5 g).

Similarly, adventitious root cultures yielded more TPC and TFC with the addition

of (30 g l-1), media pH (5.8) and inoculum size (0.5 g).

Moreover, addition of 30 g l-1 sucrose was found to be superior for enhanced

DRSA in callus (92.82 %) and cell suspension (83.87%) cultures. However,

adventitious root culture developed in media having 20 g l-1 sucrose exhibited the

highest DRSA (94.43 %).

Moreover, optimized condition for callus culture to accumulate maximum

stevioside (15 g l-1; pH 5.6; inoculum 0.5 g), rebaudioside (20 g l-1; pH 5.6;

inoculum 0.5 g) and dulcoside (20 g l-1; pH 5.1; inoculum 1.0 g) were found. On

the other hand cell suspension cultures accumulated more stevioside, rebaudioside

and dulcoside contents as a result of (10 g l-1; pH 5.8; inoculum 0.5 g), (20 g l-1;

pH 5.8; inoculum 0.5 g) and (20 g l-1; pH 5.8; inoculum 0.5 g), respectively in

culture media. Similarly, more optimized conditions (10 g l-1 sucrose; pH 5.1;

inoculum 1.0 g) for stevioside and rebaudioside contents, while (40 g l-1 sucrose;

pH 5.8; inoculum 1.5 g) for dulcoside contents were observed.

Recommendations

Over all study showed that various spectral lights, sucrose concentrations, media

pH and inoculum sizes positively regulated growth and secondary metabolites

accumulation in callus, cell suspension and adventitious root cultures and could

be used as an active elicitor.

Media should be supplied with 2, 4-D and NAA (2 mg l-1; 0.5 mg l-1) for efficient

callus induction, 2, 4-D and NAA (1 mg l-1; 0.5 mg l-1) for cell suspension culture

and NAA (0.5 mg l−1) in half strength media for adventitious root cultures

development.

Similarly, sucrose at the rate of 40-50 g l-1, media pH (5.6) and inoculum size (2.0

g) under white light condition are recommended for enhanced biomass yield of

callus culture. On the other hand, media should be supplied with 20 g l-1 sucrose,

175

media pH (5.6) and inoculum size (1.5 g) for higher biomass yield in cell

suspension culture. However, sucrose (50 g l-1), media pH (6.0) and inoculum size

(1.5 g) should be used for maximum biomass production in adventitious root

cultures.

Moreover, production media should be supplied with 30 g l-1, pH (5.8) and

inoculum size along with blue light exposure for maximum TPC and TFC

accumulation in callus cultures.

Moreover, to synthesis TPC and TFC in larger amount in cell suspension cultures,

production media should be used having 40 g l-1 sucrose, media pH (5.8) and

inoculum size (0.5 g). Similarly, for more TPC and TFC adventitious root cultures

media should be characterized with 30 g l-1 sucrose, media pH (5.8) and inoculum

size (0.5 g).

Moreover, optimized condition (20 g l-1 sucrose; pH 5.6; inoculum 0.5 g), (20 g l-1

sucrose; pH 5.1; inoculum 1.0 g) and (20 g l-1 sucrose; pH 5.1; inoculum 1.0 g)

should be used for maximum stevioside, rebaudioside and dulcoside production in

callus culture of Stevia.

While for maximum stevioside, rebaudioside and dulcoside contents in cell

suspension culture of Stevia, culture conditions (10 g l-1; pH 5.8; inoculum 0.5 g),

(20 g l-1; pH 5.8; inoculum 0.5 g) and (20 g l-1; pH 5.8; inoculum 0.5 g),

respectively should be developed. Similarly, the use of more optimized conditions

(10 g l-1 sucrose; pH 5.1; inoculum 1.0 g) for stevioside and rebaudioside

contents, while (40 g l-1 sucrose; pH 5.8; inoculum 1.5 g) for dulcoside contents in

adventitious root cultures is recommended.

176

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