Development of a Protocol for the Proliferation of In Vitro Axillary Buds in Avocado (Persea americana) cv. ‘Edranol’ Faatimah Mansoor School of Animal, Plant and Environmental Sciences University of the Witwatersrand, Johannesburg, South Africa A Dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree Master of Science Johannesburg, 2018 DECLARATION I, Faatimah Mansoor (Student number: 453883), am a student registered for the degree of MSc (Dissertation) in the academic year 2018. I hereby declare the following: I am aware that plagiarism (the use of someone else’s work without their permission and /or without acknowledging the original source) is wrong. I confirm that the work submitted for assessment for the above degree is my own unaided work except where I have explicitly indicated otherwise. I have followed the required conventions in referencing the thoughts and ideas of others. I understand that the University of the Witwatersrand may take disciplinary action against me if there is a belief that this is not my own unaided work or that I have failed to acknowledge the source of the ideas or words in my writing. Signature: _________________________ Date: ________________________ i ABSTRACT Seed recalcitrance in avocado (Persea americana) has meant that avocado genetic material cannot be conserved in orthodox seed banks. Thus, biotechnological approaches have been considered for the long-term conservation of this species’ genetic material, through the cryopreservation of tissue culture-generated axillary buds. A study was conducted to develop a system for the proliferation of in vitro avocado cv. ‘Edranol’ axillary buds for the purpose of cryopreservation. Experiments were conducted to optimise avocado mother plant establishment and pretreatment. It was determined that potting soil mixes comprising of either 1:1:1 pine bark, perlite, river sand or 1:1:1 peat, perlite, river sand were suitable to culture healthy avocado mother plant seedlings. With these soil mixes approximately 2 shoots per plant developed after 11 weeks of transplanting and between 2.9 ± 0.31 and 3.37 ± 0.32 secondary shoots were produced after 5 months. Additionally, the mother plants produced well extended shoots (7.30 ± 1.29 cm; 8.77 ± 1.39 cm) with a sufficient number of axillary buds (7.75 ± 0.39; 6.33 ± 0.53), which were subsequently used as nodal explants. After surface decontamination, the establishment of an aseptic culture in vitro was successfully achieved. Six semi-solid tissue culture media were tested for the proliferation of in vitro axillary buds. Four media comprised of half (½) and full strength Murashige and Skoog (MS) medium (Murashige and Skoog, 1962), with either 0.5 or 1mg/l 6-Benzylaminopurine (BAP). Two media were based on the P. indica medium as proposed by Nel et al. (1983), and comprised of half strength MS macronutrients, full strength MS micronutrients, 2mg/l BAP and 1mg/l GA3. All media were supplemented with 3g/l Gelrite and 30g/l sucrose at pH 5.6-5.8. Physiological measurements were taken six weeks after establishment, the first, the second and the third subculture. Tissue browning, death and contamination were observed in explants cultured on the media containing 0.5mg/l BAP, suggesting that this concentration of BAP was not suitable for cv. ‘Edranol’. Additionally, hyperhydricity appeared to be associated with the media containing ½ MS, which could be attributed to mineral deficiencies. Overall, there was no significant difference in the number of shoots and axillary buds developed across all the media tested, suggesting that endogenous auxin levels were higher than the concentration of cytokinin used in the media tested. In support of this, strong apical dominance and callus formation was observed. An increase in tissue browning, death and hyperhydricity on all the media tested, ii coupled with a decrease in shoot length, suggested a decline in the vigour of explants in vitro. 1MS + 1mg/l BAP was selected as the most appropriate medium for the initiation of cv. ‘Edranol’ cultures, producing between 3.2 ± 0.2 and 4.9 ± 0.5 axillary buds per explant. However, hyperhydricity, browning and death were observed in explants cultured on this medium. Overall, the in vitro axillary bud explants did not behave predictably or uniformly. Thus, the system was not optimised, indicating that further study is needed for the mass multiplication of axillary buds to be used for the cryo-conservation of avocado genetic material. It is recommended that future experiments will be needed to further test tissue culture media, with a focus on the optimisation of the nutrient and plant growth regulator concentrations. Additionally, the recalcitrance of explants to the in vitro environment may have been influenced by the physiological state of the mother plants, indicating that research should be focused on the effect which the mother plants may have on the endogenous responses of the in vitro explants. iii Dedication: This dissertation is dedicated to my Beloved Parents and Brothers ACKNOWLEDGEMENTS In the name of Allah (God), the Beneficent, the Merciful To my Supervisor Prof. David Mycock and Co-supervisor Dr. Kershree Padayachee: Thank you for the constant support that you have given me throughout this journey. The completion of my work has not been easy; I am grateful for the kindness, patience and understanding that you have shown me. Additionally, I thank you for giving me the opportunity to attend conferences and allowing me to be a part of the courses that you teach. I give abundant thanks to my support system: my Family and closest friend Yakira Bahadur. I wish to acknowledge Miss Refilwe Kai for being a caring friend and a lifesaver. Special thanks are given to Mr. Bruce Patterson and Mr. Bongi Hlalukane for their assistance. I acknowledge AGM Sawmill Depot for sponsoring the pine bark which was used in this study. For providing funding for this research, I would like to acknowledge the University of the Witwatersrand, the National Research Foundation (NRF) and TATA. I also acknowledge the Jamiatul Ulama South Africa and the Islamic Aid Fund Azhad for their assistance. iv CONTENTS PAGE DECLARATION i ABSTRACT ii ACKNOWLEDGEMENTS iv LIST OF FIGURES x LIST OF TABLES xii LIST OF SYMBOLS AND NOMENCLATURE xv STRUCTURE OF THIS DISSERTATION xvi 1 CHAPTER ONE - General Introduction 1 1.1.1 Background 1 1.1.2 Avocado the fruit crop species 1 1.1.2.1 Distribution of avocado 2 1.1.2.2 Varieties of avocado 2 1.1.2.3 Uses of avocado 3 1.1.2.4 The economic importance of avocado 3 1.1.3 Propagation of avocado and germplasm conservation 3 1.1.3.1 Propagation by seed 4 1.1.3.2 Seed storage 4 1.1.3.3 Grafting 4 1.1.3.4 In vitro propagation 5 1.1.3.5 Storage of in vitro tissues 7 1.1.4 Understanding plant responses to the in vitro environment 8 1.1.5 Rationale 8 1.1.6 Aims and objectives of the study 8 2 CHAPTER TWO - Initial Tissue Culture Experiments 9 2.1 INTRODUCTION 9 2.1.1 The micropropagation of avocado (P. americana) 9 2.1.1.1 Stage 0. Mother plant selection and preparation 9 2.1.1.1.1 Explant source / starting material 9 2.1.1.1.2 Primary explant type 10 2.1.1.2 Stage 1. Establishing an aseptic culture 10 v 2.1.1.3 Stage 2. Multiplication of suitable propagules 11 2.1.1.3.1 Basal medium 11 2.1.1.3.2 Plant growth regulators 12 2.1.1.3.3 Media texture 12 2.1.2 Some physiological responses of avocado explants to the in vitro environment 21 2.1.2.1 Browning 21 2.1.2.2 Hyperhydricity 21 2.1.2.3 Callus 22 2.1.3 Aims and objectives 22 2.2 MATERIALS AND METHODS 22 2.2.1 Experimental design and protocols 22 2.2.1.1 Stage 0. Mother plant establishment and preparation 22 2.2.1.2 Stage 1. Establishment of an aseptic culture - Surface decontamination 24 2.2.1.3 Stage 2. In vitro establishment and multiplication 25 2.2.1.4 Physiological measurements 25 2.2.1.5 Subculture 27 2.2.1.6 Sample numbers 28 2.2.2 Data analysis 28 2.2.3 Imagery 28 2.3 RESULTS 28 2.3.1 Stage 0. Mother plant establishment and preparation 29 2.3.2 Stage 1. Establishment of an aseptic culture 29 2.3.3 Stage 2. In vitro establishment and multiplication 31 2.3.3.1 Tissue browning 31 2.3.3.2 Percentage death 33 2.3.3.3 Hyperhydricity 35 2.3.3.4 Number of shoots 37 2.3.3.5 Number of axillary buds 39 2.3.3.6 Shoot length 41 2.3.3.7 Callus formation 43 2.4 DISCUSSION 45 2.4.1 Stage 0. Mother plant establishment and pre-treatment 45 2.4.2 Stage 1. Establishment of an aseptic culture 46 vi 2.4.2.1 Contamination 46 2.4.3 Stage 2. In vitro establishment and multiplication 46 2.4.3.1 Tissue browning and death 47 2.4.3.2 Hyperhydricity 49 2.4.3.3 The number of shoots and axillary buds 49 2.4.3.4 Shoot length 51 2.4.3.5 Callus formation 52 2.4.4 Conclusion and recommendations 53 3 CHAPTER THREE - Avocado Mother Plant Establishment and Preparation 54 3.1 INTRODUCTION 54 3.1.1 Stage 0 preparations 54 3.1.2 Avocado root rot 54 3.1.3 Containers and soil for avocado propagation 54 3.1.4 Soil characteristics 55 3.1.4.1 Physical characteristics 55 3.1.4.2 Chemical characteristics 57 3.1.5 Avocado soil mixes 57 3.1.6 Irrigation 59 3.1.7 Vegetative growth in avocado 61 3.1.8 Motivation 61 3.1.9 Aims and objectives 61 3.2 MATERIALS AND METHODS 62 3.2.1 Experimental design and protocols 62 3.2.1.1 Stage 0.