In vitro techniques for the improvement of growth and secondary metabolite production in Eucomis autumnalis subspecies autumnalis By Nqobile Andile Masondo (B.Sc Hons University of KwaZulu-Natal, South Africa) Submitted in fulfilment of the requirements for the degree of Master of Science Research Centre for Plant Growth and Development School of Life Sciences College of Agriculture, Engineering and Science University of KwaZulu-Natal Pietermaritzburg, South Africa March, 2014 i Table of Contents College of Agriculture, Engineering and Science Declaration 1 - Plagiarism ...................................... v Student Declaration ................................................................................................................................... vi Declaration by Supervisors...................................................................................................................... vii Publications from this Thesis ................................................................................................................. viii Conference Contribution from This thesis .............................................................................................. ix College of Agriculture, Engineering and Science Declaration 2 - Publications ................................... x Acknowledgements ................................................................................................................................... xi List of Figures ........................................................................................................................................... xii List of Tables .......................................................................................................................................... xviii List of Abbreviations ................................................................................................................................ xx Abstract .................................................................................................................................................... xxii Chapter 1: General introduction .................................................................................. 1 1.1. Importance of plants in African Traditional Medicine ........................................................... 1 1.2. Application of micropropagation in conservation of medicinal plants ................................... 2 1.3. Therapeutic value of secondary metabolites in plants ......................................................... 4 1.4. Eucomis autumnalis subspecies autumnalis ....................................................................... 5 1.4. Aims and objectives ............................................................................................................ 6 1.5. General overview of the thesis ............................................................................................ 6 Chapter 2: Pharmacological potential and conservation prospects of the genus Eucomis (Hyacinthaceae) endemic to southern Africa .............................................. 8 2.1. Introduction ......................................................................................................................... 8 2.2. Distribution and general morphology of Eucomis species .................................................... 9 2.2.1. Distribution ................................................................................................................... 9 2.2.2. General morphology ....................................................................................................12 2.3. Horticultural potential of Eucomis .......................................................................................14 2.4. Documented uses in African traditional medicine ...............................................................15 2.5. Phytochemistry and pharmacology of Eucomis species .....................................................16 2.5.1 Phytochemistry .............................................................................................................17 2.5.2. Pharmacology .............................................................................................................18 2.5.2.1. Anti-inflammatory screening .................................................................................18 2.5.2.2 Antimicrobial screening ..........................................................................................23 i 2.5.2.3. Other pharmacological properties .........................................................................27 2.6. Safety and toxicity of Eucomis ............................................................................................29 2.7. Conservation status ...........................................................................................................30 2.7.1. Conventional propagation ............................................................................................31 2.7.1.1. Response of Eucomis to conventional propagation ..............................................31 2.7.2. Micropropagation of Eucomis ......................................................................................35 2.7.2.1. Effect of plant growth regulators on the micropropagation of Eucomis ..................36 2.8. Conclusions .......................................................................................................................37 Chapter 3: Influence of gelling agents, explant source and plant growth regulators in micropropagated Eucomis autumnalis subspecies autumnalis ......................... 39 3.1. Introduction ........................................................................................................................39 3.2. Materials and methods .......................................................................................................41 3.2.1. Sources of plant growth regulators ..............................................................................41 3.2.2. Explant source, decontamination regime and culture initiation .....................................42 3.2.3. In vitro shoot proliferation using different gelling agents, explant source and plant growth regulators ..................................................................................................................44 3.2.4. Preparation of extracts for phytochemical quantification ..............................................45 3.2.4.1. Determination of iridoid content ............................................................................46 3.2.4.2. Determination of condensed tannin content ..........................................................46 3.2.4.3. Determination of flavonoid content .......................................................................47 3.2.4.4. Determination of total phenolic content .................................................................47 3.2.5. Data analysis ...............................................................................................................48 3.3. Results and discussion.......................................................................................................48 3.3.1. Explant decontamination and regeneration frequency .................................................48 3.3.2. Effect of gelling agents on shoot proliferation ..............................................................50 3.3.3. Effect of explant source on shoot proliferation .............................................................55 3.3.4. Effect of plant growth regulators on shoot proliferation ................................................56 3.3.5. Effect of gelling agents on secondary metabolite content ............................................57 3.3.6. Effect of explant source on secondary metabolite content ...........................................60 3.3.7. Effect of plant growth regulators on secondary metabolite content ..............................60 3.4. Concluding remarks ...........................................................................................................61 ii Chapter 4: The role of plant growth regulators on growth, phytochemical content and antioxidant activity of Eucomis autumnalis subspecies autumnalis .............. 63 4. 1. Introduction .......................................................................................................................63 4.2. Materials and methods .......................................................................................................65 4.2.1. Plant growth regulators and explant source .................................................................65 4.2.2. In vitro shoot proliferation using different cytokinins .....................................................66 4.2.3. In vitro shoot proliferation using different cytokinins and varying concentrations of α- naphthalene acetic acid .........................................................................................................66 4.2.4. Acclimatization of in vitro-derived Eucomis autumnalis subspecies autumnalis ...........67 4.2.5. Phytochemical evaluation of in vitro and greenhouse-acclimatized Eucomis autumnalis subspecies autumnalis ..........................................................................................................68 4.2.6. Antioxidant evaluation of in vitro and greenhouse-acclimatized Eucomis autumnalis subspecies autumnalis ..........................................................................................................68