TABLE OF CONTENTS

Acknowledgement i-ii

List of Tables iii-iv

List of Figures v

List of Abbreviations vi-vii

Abstract viii-ix

Preface x-xi

CHAPTER 1 INTRODUCTION 1-14

1.1 Medicinal plant and their importance

1.2 Production of secondary metabolites by organ culture

1.3 Liquid Culture systems

1.3.1 Physical state of culture medium

1.3.2 Factors affecting growth in liquid cultures

1.3.3 Morphogenesis in liquid cultures

1.4 Bioreactor for biomass and secondary metabolite production

1.4.1 Types of bioreactors for plant propagation

1.4.2 Aseptic condition and control of microbial contamination

1.4.3 Secondary metabolite production in organ culture using bioreactor

systems

1.5 Memory enhancing herbs

1.6 Objectives of Study

CHAPTER 2 REVIEW OF LITERATURE 15-37

2.1 Bacopa monnieri (L.) Wettst

2.1.1 Classification, Distribution and Habitat

2.1.2 Botanical features

2.1.3 Propagation and Cultivars

2.1.4 Phytochemistry

2.1.5 Therapeutic Applications

2.1.6 Pharmacological studies

2.1.7 Substituents and Adulterants

2.1.8 Ayurvedic preparations

2.1.9 Field Performance studies

2.2 Tissue culture studies in B. monnieri

2.2.1 Micropropagation leading to plant regeneration

2.2.2 Studies on micropropagation/regeneration in Liquid medium

2.2.3 Production of bacosides from in vitro cultures

2.2.4 Genetic transformations studies

2.2.5 Elicitation studies

2.2.6 Molecular studies

CHAPTER 3 MATERIALS AND METHODS 38-53

3.1 Collection and maintenance of source plant

3.2 Qualitative and Quantitative determination of bacoside

3.2.1 Extraction of bacosides (sample preparation)

3.2.2 Preparation of standard bacoside (mixture of A3 and A2)

3.2.3 Thin Layer Chromatography

3.2.4 High Performance Liquid Chromatography

3.2.4.1 Mobile phase preparation

3.2.4.2 Chromatographic conditions

3.2.4.3 Standard curve and assay

3.3 Annual variation in bacoside content in net house plants

3.4 Quantitative determination of bacoside in various ex vitro plant

parts

3.5 Initiation of axenic shoot cultures in vitro

3.5.1 Explant selection and sterilization

3.5.2 Culture vessels, instruments and chemicals

3.5.3 Preparation of stock solutions of nutrients

3.5.4 Preparation of stock solutions of growth regulators (GRs)

3.5.5 Inoculation and Incubation conditions

3.6 Data recording

3.7 Establishment of cell and shoot culture of high bacoside yielding

lines as production vehicle

3.7.1 Effect of different basal culture media

3.7.2 Qualitative and quantitative analysis of bacoside content in in vitro

regenerated mother culture

3.7.3 Effect of plant growth regulators on culture growth and bacoside

production in vitro

3.7.4 Optimization of process conditions for Synthetic seed production for

efficient plant recovery

3.7.5 Growth pattern and bacoside production studies in agar cultures of B.

monnieri

3.8 Biochemical approaches to enhance in vitro bacoside production –

through elicitation and media manipulations approaches

(optimization)

3.8.1 Effect of Carbon, Nitrogen and Phosphate concentration (CNP ratio)

3.8.2 Effect of depletion of major and minor mineral salt stocks of MS

medium

3.8.3 Effect of MS culture medium salt strength

3.8.4 Effect of abiotic and biotic elicitors on biomass and bacoside

production

3.8.4.1 Abiotic elicitation

3.8.4.2 Biotic elicitation

3.8.4.2a Isolation of microbial culture associated with in vitro shoot culture

3.8.4.2b 16S rRNA sequence analysis

3.8.4.2c Growth pattern of bacterial culture

3.8.4.2d Preparation of Biotic elicitor

3.8.4.3 Addition of abiotic and biotic elicitor

3.9 Development of bench scale process for production of bacoside

using bioreactor approach

3.9.1 Production dynamic of biomass and bacoside in liquid medium under

static and agitated conditions

3.9.2 In vitro shoot biomass and bacoside production in agar versus non-

aerated liquid medium with different support systems

3.9.3 Feasibility studies of various bench scale bioreactor systems for shoot

biomass and bacoside production

3.9.3.1 Agitated Shake Flask (SF, 1L)

3.9.3.2 Growtek® (GK, 1L)

3.9.3.3 Glass Bottle Bioreactor (GBB, 1L)

3.9.3.4 Balloon Type Bubble Bioreactor (BTBB, 2L)

3.9.3.5 Bench Top Air Lift Bioreactor (ALB, 5L)

3.9.3.6 Medium analysis for various bioreactor systems

3.10 Statistical analysis of Data

3.11 Photography

CHAPTER 4 OBSERVATIONS AND RESULTS 54-97

4.1 Annual variation in bacoside content in net house plants

4.2 Quantitative determination of bacoside in various ex vitro plant parts

4.3 Effect of different basal culture media

4.4 Quantitative analysis of bacoside content in in vitro regenerated tissue culture

lines

4.5 Effect of plant growth regulators on culture growth and bacoside production in

vitro

Effect of cytokinins (BAP, KN, TDZ)

Effect of auxins (2,4-D, IBA)

Synergistic effect of cytokinins and auxins

4.6 Optimization of process conditions for synseed production for efficient plant

recovery

Plantlet regeneration and acclimatization

4.7 Growth pattern and bacoside production in cultures grown on agar solidified

MS medium

4.8 Effect of Carbon (C), Nitrogen (N) and Phosphate (P) concentration (CNP

ratio)

4.9 Effect of depletion of major and minor mineral salt stocks of MS medium

4.10 Effect of MS culture medium salt strength

4.11 Effect of abiotic and biotic elicitors on biomass and bacoside production

4.11.1 Abiotic elicitation

Effect of Copper sulphate (CuSO4)

Effect of Salicyclic acid (SA)

Effect of Jasmonic acid (JA)

4.11.2 Biotic elicitation

Morphological charactersistics of isolated microbial culture

Molecular analysis of bacterial isolate

Growth pattern of the microbial culture

Elicitation with Achromobacter xylosoxidans at late exponential

phase

4.12 Production dynamics of shoot biomass and bacoside in liquid medium under

static and agitated conditions

4.13 In vitro shoot biomass and bacoside production in agar versus non-aerated

liquid medium with different support systems

4.14 Feasibility studies of various bench scale systems for shoot biomass and

bacoside production

4.14.1 Medium analysis

CHAPTER 5 DISCUSSION 98-109

CHAPTER 6 CONCLUSIONS 110-111

CHAPTER 7 REFERENCES 112-146

List of Research Publications 147