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Chapter 1 General Introduction MICROPROPAGATION AND MEDICINAL PROPERTIES OF BARLERIA GREENII AND HUERNIA HYSTRIX BY STEPHEN OLUWASEUN AMOO (M.Sc. OBAFEMI AWOLOWO UNIVERSITY, NIGERIA) Submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Research Centre for Plant Growth and Development School of Biological and Conservation Sciences University of KwaZulu-Natal, Pietermaritzburg November 2009 TABLE OF CONTENTS STUDENT DECLARATION ................................................................................... vii DECLARATION BY SUPERVISORS ................................................................... viii FACULTY OF SCIENCE & AGRICULTURE DECLARATION 1 - PLAGIARISM.... ix FACULTY OF SCIENCE & AGRICULTURE DECLARATION 2 - PUBLICATIONS x ACKNOWLEDGEMENTS ..................................................................................... xii LIST OF FIGURES ............................................................................................... xiii LIST OF TABLES .................................................................................................xvii LIST OF ABBREVIATIONS .................................................................................. xix ABSTRACT….. ....................................................................................................xxii Chapter 1 General introduction ........................................................................ 1 1.1 Use of plants in horticulture and traditional medicine .......................... 1 1.2 The need for conservation of plant species ........................................... 1 1.3 Distribution, morphology, uses and conservation status of the studied plant species ............................................................................... 3 1.3.1 Barleria greenii .................................................................................... 3 1.3.2. Huernia hystrix..................................................................................... 5 1.4 Value of tissue culture ............................................................................. 7 1.5 Aims and objectives ................................................................................. 8 1.6 General overview of the thesis ................................................................ 8 Chapter 2 Literature review ............................................................................ 10 2.1 Micropropagation ................................................................................... 10 2.1.1 Introduction ........................................................................................ 10 2.1.2 Stage 0: Selection and preparation of mother plants ......................... 10 2.1.3 Stage I: Initiation and establishment of aseptic culture ...................... 11 ii 2.1.4 Stage II: Proliferation or multiplication of propagules ......................... 15 2.1.5 Stage III: Preparation of propagules for transfer to soil ..................... 17 2.1.6 Stage IV: In vivo rooting and acclimatization for soil establishment... 19 2.1.7 Effects of auxins and cytokinins on plant regeneration in micropropagation ............................................................................. 22 2.1.8 Environmental factors affecting micropropagation ............................. 29 2.1.8.1 Light ............................................................................................... 29 2.1.8.2 Temperature ................................................................................... 31 2.1.9 Tissue culture of the families: Acanthaceae and Asclepiadaceae ..... 32 2.2 Pharmacological and phytochemical investigation of plant extracts 36 2.2.1 Introduction ........................................................................................ 36 2.2.2 Antimicrobial activity .......................................................................... 37 2.2.3 Anti-inflammatory activity ................................................................... 39 2.2.4 Acetylcholinesterase inhibition .......................................................... 42 2.2.5 Antioxidant activity ............................................................................. 43 2.2.6 Phytochemical property ..................................................................... 45 Chapter 3 In vitro propagation of Barleria greenii ........................................ 48 3.1 Introduction ............................................................................................ 48 3.2 Materials and methods ........................................................................... 49 3.2.1 Explant decontamination, selection and bulking ................................ 49 3.2.2 Effects of BA and NAA on shoot multiplication .................................. 50 3.2.3 Effects of types and concentrations of cytokinins on shoot multiplication .................................................................................... 50 3.2.4 Effects of photoperiod on shoot multiplication ................................... 51 3.2.5 In vitro rooting of regenerated shoots ................................................ 51 3.2.6 Ex vitro rooting and acclimatization ................................................... 52 3.2.7 Data analyses .................................................................................... 52 iii 3.3 Results and discussion ......................................................................... 53 3.3.1 Explant decontamination ................................................................... 54 3.3.2 Effects of BA and NAA on shoot multiplication .................................. 55 3.3.3 Effects of types and concentrations of cytokinins on shoot multiplication .................................................................................... 58 3.3.4 Effects of photoperiod on shoot multiplication ................................... 62 3.3.5 In vitro rooting of regenerated shoots ................................................ 63 3.3.6 Ex vitro rooting and acclimatization ................................................... 65 Chapter 4 In vitro propagation of Huernia hystrix ........................................ 67 4.1 Introduction ............................................................................................ 67 4.2 Materials and methods ........................................................................... 69 4.2.1 Source material, decontamination and bulking of explants ................ 69 4.2.2 Effects of BA and NAA on shoot multiplication .................................. 69 4.2.3 Effects of temperature and photoperiod on shoot multiplication ........ 70 4.2.4 Determination of titratable acidity ...................................................... 70 4.2.5 Effects of culture vessel size on shoot multiplication ......................... 71 4.2.6 Indirect organogenesis ...................................................................... 71 4.2.7 Rooting and acclimatization ............................................................... 71 4.2.8 Data analyses .................................................................................... 72 4.3 Results and discussion ......................................................................... 72 4.3.1 Explant decontamination ................................................................... 72 4.3.2 Shoot and root organogenesis .......................................................... 74 4.3.3 Effects of temperature and photoperiod on shoot multiplication ........ 77 4.3.4 Titratable acidity ................................................................................ 82 4.3.5 Effects of culture vessel size on shoot multiplication ......................... 84 4.3.6 Indirect organogenesis ...................................................................... 86 4.3.7 Rooting and acclimatization ............................................................... 87 iv Chapter 5 Pharmacological and phytochemical evaluation of Barleria species and Huernia hystrix ......................................................... 90 5.1 Introduction ............................................................................................ 90 5.2 Materials and methods ........................................................................... 91 5.2.1 Collection of plant materials .............................................................. 91 5.2.2 Pharmacological evaluation ............................................................... 92 5.2.2.1 Preparation of extracts ................................................................... 92 5.2.2.2 Antibacterial activity ....................................................................... 92 5.2.2.3 Antifungal activity ........................................................................... 93 5.2.2.4 Anti-inflammatory activity ............................................................... 94 5.2.2.5 Acetylcholinesterase (AChE) inhibition........................................... 95 5.2.2.6 Antioxidant activity ......................................................................... 96 5.2.2.6.1 DPPH radical-scavenging activity ............................................. 96 5.2.2.6.2 Ferric reducing power activity .................................................... 97 5.2.2.6.3 β-Carotene linoleic acid assay .................................................. 97 5.2.3 Phytochemical evaluation .................................................................. 98 5.2.3.1 Preparation of extracts
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