ESTABLISHMENT OF AXENIC CULTURE AND CALLUS INDUCTION OF SYNSEPALUM DULCIFICUM DANIEL (MIRACLE BERRY) TAN HONG WEE Bachelor of Science with Honours (Plant Resource Science and Management) 2015 ESTABLISHMENT OF AXENIC CULTURE AND CALLUS INDUCTION OF SYNSEPALUM DULCIFICUM DANIEL (MIRACLE BERRY) TAN HONG WEE This project is submitted in partial fulfillment of the requirements for the Degree of Bachelor of Science with Honours (Plant Resource Science and Management) Faculty of Resource Science & Technology UNIVERSITI MALAYSIA SARAWAK 2015 APPROVAL SHEET Name of candidate : Tan Hong Wee Title of dissertation: Establishment of Axenic Culture and Callus Induction of Synsepalum dulcificum Daniel (Miracle Berry) (Prof Dr. Hamsawi Sani) Supervisor (Dr. Rebicca Edward) Coordinator Plant Resource Science and Management Programme Department of Plant Science and Environmental Ecology Faculty of Resource Science and Technology DECLARATION I declare that this thesis entitle “Establishment of Axenic Culture and Callus Induction of Synsepalum dulcifiucm Daniel (Miracle Berry) is the result of my own research except as cited in the references. The thesis has not been accepted for any degree and is not concurrently submitted in candidature of any other degree. Signature: Name : TAN HONG WEE Date : 15th June 2015 ACKNOWLEDGEMENT First of all, I would like to express my thanks to Prof Dr. Hamsawi Sani for his comments, guidance and advice in the preparation of this Final Year Project (FYP) report. I would also like to express grateful to my family members especially my father, mother and my brother who had always encouraged and contributes plenty of information for the writing up of this thesis report. Thanks to my friends and colleagues for their patience and cooperation during the entire thesis writing process and during the whole experimentation process. Finally, I would also like to thank all lecturers in the Department of Plant Science Resource and Management of Faculty Resource Science and Technology (FRST) Universiti Malaysia Sarawak (UNIMAS) for the materials and facilities that had provided to me in fulfilment of this thesis report. Establishment of Axenic Culture and Callus Induction of Synsepalum dulcificum Daniel Tan Hong Wee Plant Resource and Management Programme Faculty of Science and Technology Universiti Malaysia Sarawak ABSTRACT Synsepalum dulcificum is a slow growing shrub from family Sapotaceae. The fruits contain high medicinal values which aid in chronic diseases due to the presence of active component Miraculin, a taste modifying properties. The current research emphasizes on the axenic cultures establishment and callus induction of Synsepalum dulcificum from leaf and seed explants. The sterilized leaf explants were obtained from both 25% Clorox® solution (Sodium hypochlorite) which produced 91.7% axenic cultures and 25% CaOCI solution (Calcium hypochlorite) that produced 94.4% axenic cultures with 20 minutes exposure time upon addition of 1% Benomyl solution respectively. The sterilized leaf explants were cultured onto Murashige & Skoog (MS) media supplemented with various concentrations of 2,4- Dichlorophenoxyacetic Acid (2,4-D), and Picloram. No callus was induced despite some of the leaf explants remained viable after certain period of time and most of the leaf explants results in browning conditions whereas callus was induced from seeds supplemented with 2.0 mg/L of 2,4-D after 3 weeks of culturing. The poor callus induction from leaves was highly due to the presence of high poly-phenolic compounds within the miracle berry plants. Future work should utilize different PGR‟s at different concentrations and also the use of different explants. Key words: Synsepalum dulcificum, Miraculin, Axenic Culture, Callus Culture, Plant Growth Regulators (PGR) ABSTRAK Synsepalum dulcificum adalah sejenis pokok renek yang berasal dari keluarga Sapotaceae. Tumbuhan ini mengandungi nilai perubatan yang tinggi dengan adanya komponen aktif dalam Pokok Ajaib yakni Miraculin yang membantu dalam penyakit kronik dan turut berfungsi sebagai agen pengubahsuai rasa. Penyelidikan ini menekankan aspek penghasilan kultur arsenik dan pembentukan kalus dari bahagian daun dan biji Synsepalum dulcificum sebagai sumber eksplan. Daun eksplan yang steril terhasil dari penambahan 1% Benomyl dalam kedua-dua kepekatan larutan iaitu 25% Clorox® (Natrium hipoklorit) yang menghasilkan 91.7% aresenik dan 25% CaOCI (Kalsium hipoklorit) yang menhasilkan 94.4% arsenik dengan pendedahan masa selama 20 minit. Daun eksplan yang steril dikulturkan dalam media Murashige & Skoog (MS) yang ditambah dengan variasi kandungan kepekatan 2,4-Dichlorophenoxyacetic acid (2,4-D), dan Picloram. Walaubagaimanapun, tiada kalus terbentuk walaupun sebahagian daun eksplan masih dalam keadaan sihat tetapi kebanyakkan eksplan berubah warna menjadi perang manakala kalus terbentuk dari biji yang dikulturkan dalam 2.0 mg/L kepekatan 2,4-D. Kekangan pembentukan kalus daripada bahagian daun adalah disebabkan faktor utama iaitu kandungan kimia poli-phenolik yang tinggi dalam pokok ajaib. Kajian yang akan datang seharusnya menggunakan pelbagai PGR’s dalam kepekatan berbeza dan juga jenis eksplan yang lain. Kata kunci: Synsepalum dulcificum, Miraculin, Kultur Arsenik, Kultur kalus, Hormon Pertumbuhan Tumbuhan TABLE OF CONTENTS TITLE PAGE I APPROVAL SHEET II DECLARATION III ACKNOWLEDGEMENT IV ABSTRACT/ABSTRAK V TABLE OF CONTENTS VI APPENDICES VII LIST OF FIGURES VIII LIST OF TABLES IX LIST OF ABBREVIATIONS X 1.0 INTRODUCTION 1 2.0 LITERATURE REVIEW 2.1 Botanical Description of Synsepalum dulcificum 4 2.2 History of Synsepalum dulcificum 6 2.3 Habitat and Distribution of Synsepalum dulcificum 7 2.4 Chemical Constituents of Miracle Berry 10 2.5 Economic Importance of Synsepalum dulcificum 12 2.5.1 Diabetic Health Benefits 12 2.5.2 Chemotherapy Health Benefits 13 2.5.3 Weight Loss Benefits 13 2.6 Growth of Synsepalum dulcificum 14 2.7 Plant Tissue Culture as an Alternative Propagation Method 15 2.8 Plant Tissue Culture and Sterilizing Agent 16 2.9 Plant Tissue Culture and Plant Growth Regulators 18 3.0 MATERIALS AND METHODS 3.1 Plant Tissue Culture Medium 20 3.1.1 Preparation of Murashige & Skoog (MS) Stock Solution 21 3.1.2 Preparation of Stock Packs for Used Medium 21 3.2 Establishment of Axenic Culture 22 3.2.1 Effect of Different Clorox ® (Sodium Hypochlorite) Concentrations 23 and Exposure Time 3.2.2 Effect of Different Calcium Hypochlorite (CaOCI) Concentrations 24 and Exposure Time 3.3 Induction of Callus Culture from Leaf Explants 25 3.3.1 Effect of 2,4-Dichlorophenoxyacetic (2,4-D) on Callus Induction 26 3.3.2 Effect of Picloram on Callus Induction 27 3.3.3 Effect of 2,4-Dichlorophenoxyacetic (2,4-D) in Activated Charcoal 28 on Callus Induction 3.4 Induction of Callus from Seed Culture 29 3.4.1 Effect of 2,4-Dichlorophenoxyacetic (2,4-D) on Seed Culture 30 4.0 RESULTS AND DISCUSSIONS 4.1 Establishment of Axenic Culture 4.1.1 Effect of Different Clorox® Concentrations and Exposure Time 32 4.1.2 Effect of Different CaOCI Concentrations and Exposure Time 36 4.2 Induction of Callus Culture from Leaf Explants 4.2.1 Effect of 2,4-Dichlorophenoxyacetic (2,4-D) on Callus Induction 39 4.2.2 Effect of Picloram on Callus Induction 44 4.2.3 Effect of 2,4-Dichlorophenoxyacetic (2,4-D) in Activated Charcoal 50 on Callus Induction 4.3 Effect of 2,4-Dichlorophenoxyacetic (2,4-D) on Seed Culture 54 4.4 Factors Contributing to Inhibition of Callus Formation 57 4.4.1 Browning of Culture Materials 57 4.4.2 Age of Explants 59 4.4.3 Effect on the Presence of Contaminants on Explant Culture 61 5.0 CONCLUSIONS AND RECOMMENDATIONS 63 6.0 REFERENCES 65 APPENDICES 73 APPENDICES Appendix Page A Murashige and Skoog (1962), MS medium composition 73 B Results of Surface Sterilization from leaf explants Synsepalum 74 dulcificum Daniel (Miracle Berry) C Statistical Analysis of Clorox® and CaOCI Solution 76 D Anova Analysis of Clorox® and CaOCI Solution 77 LIST OF FIGURES Figure Page 1 Geographical range habitat of Synseplaum dulcificum in Tropical 7 West Africa Region 2 Map showing Benin within the Dahomey gap encircled 8 3 Map showing the current suitable habitat distribution of Synsepalum 8 dulcificum. 4 Seed Culture supplemented in 0.0 mg/L of 2,4-D for callus induction 31 Seed Culture supplemented in 2.0 mg/L of 2,4-D for callus induction 31 Seed Culture supplemented in 3.0 mg/L of 2,4-D for callus induction 31 Seed Culture supplemented in 5.0 mg/L of 2,4-D for callus induction 31 5 (a) Contamination occurred on Day 2 in 20% Clorox Concentration 34 (b) Fungi contamination in 25% Clorox Concentration on Day 3. 34 (c) Slight damaged leaf explants in 30% Clorox Concentration after 34 14 days. 6 The effect of Clorox® solutions with 3 different treatments on the 35 percentage of axenic cultures 7 (a) Contamination in 20% CaOCI Concentration at Day 4. 37 (b) Contamination in 25% CaOCI Concentration at Day 5. 37 (c) Contamination in 30% CaOCI Concentration at Day 6. 37 8 The effect of 3 different treatments concentrations of CaOCI 38 solutions on the percentage of axenic cultures. 9 (a) Leaf explants after 4 weeks culturing on 0.0 mg/L of 2,4-D. 40 (b) Leaf explants after 6 weeks culturing on 1.0 mg/L of 2,4-D. 40 (c) Leaf explants after 8 weeks culturing on 3.0 mg/L of 2,4-D. 40 (d) Leaf explants after 8 weeks culturing on 5.0 mg/L of 2,4-D. 40 (e) Leaf explants after 6 weeks culturing on 1.0 mg/L of 2,4-D under 43 light condition for 4 days. (f) Leaf explants after 6 weeks culturing on 3.0 mg/L of 2,4-D under 43 light condition for 4 days. (g) Leaf explants after 6 weeks culturing on 5.0 mg/L of 2,4-D under 43 light condition for 4 days.