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TITLE
DECLARATION
CERTIFICATE
DEDICATION
CONTENTS
Preface
ACKNOWLEDGEMENT
ABBREVIATIONS
Part I - In vivo Screening of Solanum Species
1.1 Introduction
1.1.1 Aims and objectives
1.1.2 Source plants
1.1.2.1 Solanum aculeatissimum, Jacq.
1.1.2.2 Solanum incanum, Linn.
1.1.2.3 Solanum indicum, Linn.
1.1.2.4 Solanum macrocarpon, Linn.
1.1.2.5 Solanum nigrum, Linn.
1.1.2.6 Solanum torvum, Sw.
1.1.2.7 Solanum trilobatum, L.
1.1.2.8 Solanum viarum, Dunal.
1.1.2.9 Solanum wendlandii, Hook. f.
1.1.2.10 Solanum wrightii, Benth.
1.2 Review of Literature
1.2.1 Solasodine and allied compounds - chemical aspects
1.2.2 Solasodine and pharmaceutical industry
1.2.3 Screening of Solanum for glycoalkaloids
1.2.4 Agriculture of Solanum species
1.2.5 Biogenesis and dynamics of solasodine accumulation in plants
1.2.6 Methods of estimation of solasodine
1.3 Materials and Methods
1.3.1 Materials
1.3.1.1 Collection and maintenance of source plants
1.3.1.2 Preparation of plant material for chemical analyses
1.3.1.3 Chemicals
1.3.1.4 Glassware
1.3. 1.5 Instruments
1.3.2 Methods
1.3.2.1 Spectrophotometry
1.3.2.1.1 Extraction and estimation
1.3.2.1.2 Experimental standardization
1.3.2.1.3 Calculation of solasodine content
1.3.2.2 High performance liquid chromatography (HPLC)
1.3.2.2.1 Eluent system
1.3.2.2.2 Extraction and estimation
1.3.2.2.3 Experimental standardization
1.3.2.2.4 Calculation of solasodine content
1.3.2.3 Data collection and presentation
1.3.2.4 Photographs
1.4 Results
1.4.1 Standardisation of analytical procedure and confirmation of solasodine
1.4.2 Solasodine content in Solanum species
1.4.3 Distribution of solasodine in various organs
1.4.4 Solasodine level assayed by HPLC
1.4.5 Comparison of values obtained by colorimetry and HPLC
1.5 Discussion
1.5.1 Specificity of colorimetry/spectrophotometric assay
1.5.2 Solanum species as promising sources
1.5.3 Solasodine in Solanum species
1.5.4 Distribution of solasodine in plant parts
1.5.4.1 Solasodine in stem
1.5.4.2 Solasodine in leaf
1.5.4.3 Solasodine in root
1.5.4.4 Solasodine in fruit
1.5.5 Change in location
1.6 Illustrations
Part II - Establishment of In vitro Systems
2.1 Introduction
2.1.1 Aims and objectives
2.2 Review of Literature
2.2.1 Callogenesis
2.2.2 Cell suspension cultures
2.2.2.1 Callus cell aggregates
2.2.3 Root cultures
2.2.4 Regeneration in Solanum species
2.2.4.1 Rooting
2.2.5 Somatic embryogenesis
2.2.5.1 TDZ and somatic embryo genesis
2.2.5.2 Maturation and germination of somatic embryos
2.3 Materials and Methods
2.3.1 Materials
2.3.1.1 Chemicals
2.3.1.2 Glassware
2.3.1.3 Accessories
2.3.1.4 Instruments
2.3.1.5 Media
2.3.1.6 Sterilization of media, glassware, accessories
2.3.1.7 Source of explant
2.3.1.8 Surface sterilization of explant
2.3.1.9 Inoculation and incubation
2.3.2 Methods
2.3.2.1 Callus culture
2.3.2.2 Cell suspension culture
2.3.2.3 Root culture
2.3.2.4 Regeneration studies
2.3.2.4.1 Selection of PGR
2.3.2.4.2 Optimization of PGR concentration
2.3.2.4.3 Rooting and hardening
2.3.2.4.4 Maturation and germination of somatic embryos
2.3.2.5 Subculture
2.3.2.6 Internal structure
2.3.2.7 Statistical analysis
2.3.2.8 Photography
2.4 Results.
2.4.1 Callus cultures
2.4.1.1 Effect of 2, 4-D on callogenesis
2.4.1.1.1 S. trilobatum
2.4.1.1.2 S. wendlandii
2.4.1.2 Effect of NAA on callogenesis
2.4.1.2.1 S. trilobatum
2.4.1.2.2 S. wendlandii
2.4.1.3 Effect of IAA on callogenesis
2.4.1.3.1 S. trilobatum
2.4.1.3.2 S. wendlandii
2.4.1.4 Growth patterns of calli
2.4.1.4.1 S. trilobatum
2.4.1.4.2 S. wendlandii
2.4.2 Cell suspension cultures
2.4.2.1 Establishment of primary suspension cultures
2.4.2.2 Selection of medium
2.4.2.2.1 S. trilobatum
2.4.2.2.1.1 Formation of.CCA in S.trilobatum cell suspension cultures
2.4.2.2.2 S. wendlandii
2.4.2.3 Growth curve
2.4.2.3.1 S. trilobatum
2.4.2.3.2 S. wendlandii
2.4.3 Root cultures
2.4.3.1 Rhizogenesis
2.4.3.2 Selection of medium for root culture
2.4.3.2.1 S. trilobatum
2.4.3.2.2 S. wendlandii
2.4.4 Regeneration studies
2.4.4.1 Regeneration by organogenesis in S. trilobatum
2.4.4.1.1 Selection of PGR combination
2.4.4.1.2 Optimization of PGR concentration
2.4.4.1.2.1 Direct caulogenesis
2.4.4.1.2.2 indirect caulogenesis
2.4.4.1.3 Rooting
2.4.4.1.4 Hardening
2.4.4.2 Regeneration by somatic embryogenesis in S. wendlandii
2.4.4.2.1 Selection of PGR combination
2.4.4.2.2 Optimization of PGR concentration
2.4.4.2.3 Maturation of somatic embryos
2.4.4.2.4 Germination of somatic embryos
2.5 Discussion
2.5.1 Basal medium
2.5.2 Role of auxins on callogenesis
2.5.3 Cell suspension cultures
2.5.3.1 Callus cell aggregates
2.5.4 Root cultures
2.5.4.1 Medium
2.5.4.2 Growth
2.5.4.3 Light and dark
2.5.4.4 Role of ammonium ions
2.5.4.5 Auxins
2.5.5 Morphogenetic response of S. trilobatum and S. wendlandii
2.5.5.1 Direct and indirect caulogenesis in S. trilobatum
2.5.5.1.1 Role of 2iP
2.5.5.1.2 Role of IAA
2.5.5.1.3 Vitrescence and albinism
2.5.5.1.4 Rooting
2.5.5.1.5 Hardening
2.5.5.2 Somatic embryogenesis in S. wendlandii
2.5.5.2.1 Role of TDZ and 2, 4-D in embryogenesis
2.5.5.2.2 Maturation and germination of somatic embryos
2.6 Illustrations
Part III - Evaluation and Enhancement of Solasodine in Cultures
3.1 Introduction
3.1.1 The scenario
3.1.2 Problems
3.1.3 The solution
3.1.4 The present study
3.1.5 Aims and objectives
3.2 Review of Literature
3.2.1 Factors affecting solasodine production
3.2.1.1 Light and dark
3.2.1.2 Growth phase
3.2.1.3 Medium composition
3.2.1.4 Supplements
3.2.1.5 Precursor
3.2.1.6 Growth regulators
3.2.1.7 Explant source
3.2.1.8 Elicitors
3.2.1.9 Transformation
3.2.1.10 lmmobilization
3.2.1.11 Morphological differentiation
3.2.2 Solasodine cultures as a source of valuable chemicals other than solasodine
3.2.3 Production of solasodine in different in vitro systems
3.2.3.1 Callus cultures
3.2.3.2 Suspension cultures
3.2.3.3 Root cultures
3.2.3.4 Shoot culture systems
3.2.4 Solasodine levels obtained in in vitro cultures
3.3 Materials and Methods
3.3.1 Materials
3.3.1.1 Chemicals
3.3.1.2 Glassware
3.3.1.3 Accessories
3.3.1.4 Instruments
3.3.1.5 Cultures
3.3.1.6 Surface sterilization
3.3.2 Methods
3.3.2.1 Thin layer chromatography (TLC)
3.3.2.2 Estimation of protein
3.3.2.3 Estimation of solasodine
3.3.2.4 Experiments to evaluate and enhance solasodine in in vitro conditions
3.3.2.4.1 Callus system
3.3.2.4.1.1 Effect of light and dark on calli
3.3.2.4.1.2 Effect of medium parameters
3.3.2.4.1.3 Effect of supplements
3.3.2.4.1.4 Effect of precursor (cholesterol)
3.3.2.4.1.5 Effect of gibberellic acid (GA3) and abscisic acid (ABA)
3.3.2.4.1.6 Effect of individual auxins
3.3.2.4.1.7 Explant source
3.3.2.4.2 Cell suspension system
3.3.2.4.2.1 Growth pattern
3.3.2.4.2.2 Effect of gibberellic acid (GA3) and abscisic acid (ABA)
3.3.2.4.2.3 Effect of elicitor (xanthan)
3.3.2.4.3 Root culture system
3.3.2.4.3.1 Effect of physical and chemical conditions on solasodine production
3.3.2.4.3.2 Effect of gibberellic acid (GA3) and abscisic acid (ABA)
3.3.2.4.4 Differentiating systems
3.3.2.5 Statistical methods
3.3.2.6 Photography
3.4 Results
3.4.1 Confirmation of solasodine in callus tissue
3.4.2 Evaluation and enhancement of solasodine in callus culture system
3.4.2.1 Effect of light
3.4.2.1.1 S. trilobatum
3.4.2.1.2 S. wendlandii
3.4.2.2 Effect of dark
3.4.2.2.1 S. trilobatum
3.4.2.2.2 S. wendlandii
3.4.2.3 Kinetics of solasodine accumulation in callus
3.4.2.3.1 S. trilobatum
3.4.2.3.2 S. wendlandii
3.4.2.4 Effect of medium parameters
3.4.2.4.1 S. trilobatum
3.4.2.4.2 S. wendlandii
3.4.2.5 Effect of supplements
3.4.2.5.1 S. trilobatum
3.4.2.5.2 S. wendlandii
3.4.2.6 Effect of cholesterol
3.4.2.6.1 S. trilobatum
3.4.2.6.2 S. wendlandii
3.4.2.7 Effect of gibberellic acid (GA3) and abscisic acid (ABA)
3.4.2.7.1 S. trilobatum
3.4.2.7.2 S. wendlandii
3.4.2.8 Effect of individual auxins
3.4.2.9 Explant source
3.4.2.9.1 S. trilobatum
3.4.2.9.2 S. wendlandii
3.4.2.10 Relationship of biomass, protein and solasodine content
3.4.2.10.1 S. trilobatum
3.4.2.10.2 S. wendlandii
3.4.3 Evaluation and enhancement of solasodine in suspension culture system
3.4.3.1 Growth and solasodine
3.4.3.1.1 S. trilobatum
3.4.3.1.2 S. wendlandii
3.4.3.2 Effect of GA3 and ABA on cell suspension cultures
3.4.3.2.1 S. trilobatum
3.4.3.2.2 S. wendlandii
3.4.3.3 Effect of elicitor on cell suspension cultures
3.4.3.3.1 S. trilobatum
3.4.3.3.2 S. wendlandii
3.4.4 Evaluation and enhancement of solasodine in root culture system
3.4.4.1 Effect of growth in light / dark and ammonium nitrate in solasodine production
3.4.4.1.1 S. trilobatum
3.4.4.1.2 S. wendlandii
3.4.4.2 Effect of GA3 and ABA on root cultures
3.4.4.2.1 S. trilobatum
3.4.4.2.2 S. wendlandii
3.4.5 Solasodine accumulation in different in vitro systems
3.4.5.1 Callus and suspension culture systems (undifferentiated cultures)
3.4.5.2 Root cultures
3.4.5.3 Differentiating systems
3.5 Discussion
3.5.1 Callus culture system
3.5.1.1 Effect of light and dark
3.5.1.2 Effect of growth phase
3.5.1.3 Effect of medium composition
3.5.1.4 Effect of supplements
3.5.1.5 Effect of cholesterol
3.5.1.6 Effect of individual auxins
3.5.1.7 Effect of explant source
3.5.1.8 Relation between biomass and solasodine production
3.5.1.9 Relation between protein and solasodine production
3.5.2 Cell suspension culture system
3.5.2.1 Suspension culture and solasodine production
3.5.2.2 Callus cell aggregates (CCA)
3.5.2.3 Effect of elicitor
3.5.3 Root culture system
3.5.3.1 Effect of physical and chemical conditions on solasodine production in root culture system
3.5.4 Effect of gibberellic acid and abscisic acid on solasodine production of callus, suspension and root cultures
3.5.5 Effect of differentiation
3.5.6 Undifferentiated systems vs. differentiated systems
3.5.7 Conclusion
3.5.8 Scope of future studies
3.6 Illustrations
Summary
References