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Micropropagation and Acclimatization of Aloe Polyphylla and Platycerium Bifurcatum

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Micropropagation and Acclimatization of Aloe Polyphylla and Platycerium Bifurcatum Micropropagation and acclimatization of Aloe polyphylla and Platycerium bifurcatum BY JUDE CHINEDU CHUKWUJEKWU submitted in fulfilment of the requirements for the degree of (Master of science) in the Research Centre for Plant Growth and Development, School of Botany and Zoology, Faculty of Science and Agriculture. University of Natal, Pietermaritzburg June, 2001 PREFACE With great pleasure, I wish to declare that this thesis, submitted for the degree of Master of Science in the Research Centre for Plant Growth and Development, School of Botany and Zoology, University of Natal, Pietermaritzburg, except where the work of others is acknowledged, is the result of my own investigation. JUDE CHINEDU CHUKWUJEKWU JUNE 2001 We certify that the above statement is correct. ~ ~ ............. .. PROFES RJ VAN STADEN (SUPERVISOR) ................. A·~ . MISS CW FENNELL (CO-SUPERVISOR) PUBLICATIONS FROM THIS THESIS CHUKWUJEKWU JC, FENNELL CW and VAN STADEN J (2001) Micropropagation and acclimatization of Aloe polyphyJla (In preparation). CONFERENCE CONTRIBUTIONS FROM THIS THESIS CHUKWUJEKWU JC, FENNELL CW and VAN STADEN J (2000) Micropropagation and acclimatization ofAloe polyphyJla (paper). Second Annual Research Centre Meeting, University of Natal, Pietermaritzburg. 11 ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Professor J. Van Staden (supervisor) and Miss CW Fennell (co-supervisor) fortheir guidance during this investigation and in the preparation of this manuscript. To Or. Mia Abrie, thank you for taking me through tissue culture for the first time. It was really great. To my parents and Dr. CI Chukwujekwu, thank you for your encouragement and financial support throughout my years of study. To every member of the Research Centre for Plant Growth and Development, especially Cathy Ford, Georgina Arthur and Noxwenda Makunga, for your ever available assistance. You were all a source of inspiration to me. Lastly, but not the least, to Almighty God, whom by his grace made it possible for me to successfully complete this work. I give you all the glory and honour. 111 ABSTRACT Shoot cultures of Aloe polyphylla were initiated from young shoot explants of in vitro grown plants. The basal medium was MS medium (MURASHIGE and SKOOG, 1962), supplemented with 100 mgl-1myo-inositol, and 30 gl-1 sucrose. Agar (0.8 %) was used as the gelling agent. Different cytokinins, singly or in combination with auxins (IBA and NAA), were tested for shoot proliferation activity. All the cytokinins tested (kinetin, zeatin, iP, and BA) gave a good shoot proliferation response. The optimal concentrations for shoot proliferation of each 1 of the cytokinins tested were: zeatin (0.5 mgl-1), kinetin (1.5 mgl-1), iP (1.0 mgl- ) and BA (1.5 mgl-1). In combination with auxins, the optimal combinations were 1 kinetin/NAA (2.0/0.1 mgl-1), kinetinllBA (1.5/1.0 mgl-1), zeatinllBA (1.0/0.5 mgl- ), zeatin/NAA (1.0/1.0 mgl-1), BA/IBA (1.0/1.0 mgl-1), BA/NAA (1.5/0.1 mgl-1). Although it gave the highest number of shoots per explant, BA was responsible for hyperhydricity. Temperature and sucrose also influenced shoot proliferation. The optimal temperature was 25°C, while 30 gl-1 was the optimal concentration of sucrose for shoot proliferation. Plants rooted well in plant growth regulator-free MS medium. Amongst the potting mixtures tested, soil: sand: vermiculite (1:1:1 v/v) was the best with 98 % plantlet survival. In the second part of this project, Platycerium bifurcatum cultures were established using leaf explants. The basal medium was MS medium (MURASHIGE and SKOOG, 1962), supplemented with 100 mgl-1 myo-inositol and 30 gl-1 sucrose. For bud initiation, 1.0 mgl-1BA was used, while 0.8 % agar was used as the gelling agent. Three different strengths of MS medium (full, half, and one-quarter strength) without plant growth regulators were tested for further bud growth and development. Half-strength MS proved to be the best for further IV bud growth and development. Rooting was best achieved in one-quarter strength MS medium without plant growth regulators. In vitro grown plantlets were successfully acclimatized using peat as the potting medium. v CONTENTS PREFACE i PUBLICATIONS FROM THIS THESiS ii CONFERENCE CONTRIBUTIONS FROM THIS THESIS ii ACKNOWLEDGEMENTS iii ABSTRACT iv I • CONTENTS Vl LIST OF ABBREVIATIONS USED ix LIST OF TABLES x LIST OF FIGURES xi LISTOF PLATES xv 1.Generalliterature review (Introduction) 1.1 Literature review of the genus Aloe 1 ,- ------- 1.2 Horticultural and other uses of Aloe. 4 1.3 Conservation 5 1.4 Conventional propagation .............................. .. 5 1.5 Review of tissue culture of the genus Aloe. ................ .. 6 1.5.1 Shoot -tip and meristem culture. .................. .. 7 1.5.2 Callus culture ................................ .. 11 1.5.3 Other in vitro work ............................ .. 13 1.6 Literature review of the genus Platycerium ............... .. 13 1.7 Horticultural and other uses of Platycerium ............... .. 14 1.8 Conventional propagation 14 1.9 Review of tissue culture of the genus Platycerium 16 1.9.1 Shoot - tip and meristem culture. ................ .. 16 1.9.2 Callus culture 16 VI 2. Shoot Culture of Aloe polyphylla 2.1 Introduction. ....................................... .. 22 2.1.1 Objectives 22 2.2 Materials and methods ............................... .. 23 2.2.1 Decontamination procedures and aseptic techniques. .. 23 2.2.2 Explant source. .............................. .. 24 2.2.3 Media and supplements 24 2.2.4 Environmental conditions 27 2.2.5 Acclimatization .............................. .. 28 2.3 Results and Discussion 29 2.3.1 Decontamination procedures. ................... .. 29 2.3.2 Media and supplements 29 2.3.2.1 Effects of plant growth regulators on regeneration 29 2.3.2.2 Effects of sucrose on regeneration 42 2.3.3 Temperature 45 2.3.3.1 Effects of temperature on shoot regeneration 45 2.3.4. Acclimatization 46 2.3.5 Conclusions. ................................ .. 48 3. Tissue culture of Platycerium bifurcatum 3.1 Introduction 50 3.1.1 Objectives 50 3.2 Material and methods. ............................... .. 51 3.2.1 Decontamination procedures and aseptic techniques .. 51 3.2.2 Explant source 51 3.2.3 Media and supplements ....................... .. 52 3.2.4 Environmental conditions 52 3.2.5 Acclimatization .............................. .. 52 3.3 Results and Discussion 52 vu 3.3.1 Decontamination procedures. ................... .. 52 3.3.2 Media and supplements ....................... .. 54 3.3.3 Acclimatization 55 3.3.4 Conclusions 56 4. General Conclusions 57 References 59 V111 LIST OF ABBREVIATIONS USED BA Benzyladenine iP isopentenyladenine K Kinetin Z Zeatin IBA indole-3-butyric acid NAA ex: - naphthaleneacetic acid IX LIST OF TABLES Page Table 1 8 Summary of in vitro work on Aloe species. Table 2 17 Summary of in vitro work on Platycerium species. Table 3 26 Different constituents of the basal medium used throughout this study as described by Murashige and Skoog (1962) (MS). Table 4 49 Mean number of roots produced by Aloe polyphylla at different strengths of MS medium. Table 5 56 Survival percentage of Platycerium bifurcatum plantlets obtained with different potting mixtures. x LIST OF FIGURES Page Figure 1 3 The morphology of Aloe polyphylla (A) showing the clockwise arrangement ofthe leaves, (B) the counterclockwise arrangement of leaves and a flowering shoot with the arrangement of flowers at the tip of the inflorescence. Figure2 15 The morphology ofPlatycerium bifurcatum showing its typical forked fertile leaves that provide its excellent ornamental qualities. Figure3 31 Effect of various concentrations of kinetin on shoot and root proliferation of Aloe polyphylla in vitro. Figure4 31 Effect of various concentrations of BA on shoot and root proliferation of Aloe polyphylla in vitro. Figure5 34 Effect of various concentrations of zeatin on shoot and root proliferation of Aloe polyphylla in vitro. Figure6 34 Effect of various concentrations of iP on shoot and root proliferation of Aloe polyphylla in vitro. Xl Figure7 36 Effect of various combinations of kinetin/IBA on shoot proliferation of Aloe polyphylla in vitro. FigureS 36 Effect of various combinations of kinetin/NAA on shoot proliferation of Aloe polyphylla in vitro. Figure9 37 Effect of various combinations of zeatin/IBA on shoot proliferation of Aloe polyphylla in vitro. Figure 10 37 Effect of various combinations of zeatin/NAA on shoot proliferation of Aloe polyphylla in vitro. Figure 11 39 Effect of various combinations of BA/I BA on shoot proliferation of Aloe polyphylla in vitro. Figure 12 39 Effect ofvarious combinations of BAlNAA on shoot proliferation ofAloepolyphylla in vitro. Figure 13 44 Effect of various concentrations of sucrose on shoot proliferation of Aloe polyphylla in vitro. Xli Figure 14 44 Effect of various temperatures on shoot proliferation of Aloe polyphylla in vitro. Xlll LIST OF PLATES Page Plate 1 , 32 Multiple, stunted and light green shoots obtained after 5 weeks oftreating cultures with 3.0 mgl-1 of BA. Plate 2 32 Multiple shoot production with 1.5 mgl-1 kinetin in the culture medium. Plate3 32 Callus production at the base of an explant with 3.0 mgl-1 kinetin in the basal medium. Plate4 35 Multiple shoot production with cytokinins tested. (A) 0.5 mgl-1 zeatin, (B) 1.0 mgl-1 iP. PlateS 35 Multiple shoot production obtained with different combinations of kinetin and auxin 1 in the basal medium. (A) kinetin/NAA (2.0/0.1 mgl- ), (B) kinetin/lBA (1.5/1.0 mgl­ 1). Plate6 38 Multiple shoot production obtained with different combinations ofzeatin and auxin 1 in the basal medium. (A) zeatin/NAA (1.0/1.0 mgl- ), (B) zeatin/IBA (1.0/0.5 mgl-1). Plate7 38 Multiple shoot production obtained with different combinations of BA and auxin XlV 1 1 in the basal medium. (A) BA/NAA (1.5/0.1 mgl- ), (B) BA/IBA (1.0/1.0 mgl- ). PlateS 43 Effect of sucrose on shoot proliferation of Aloe polyphyJla in vitro.
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