ADDIS ABABA UNIVERSITY COLLEGE OF NATURAL AND COMPUTATIONAL SCIENCES DEPARTMENT OF ZOOLOGICAL SCIENCES GENERAL BIOLOGY PROGRAM The Effects of Culture Conditions on In vitro Propagation of Korarima (Aframomum corrorima (Braun) P.C.M. Jansen) By Abdela Zeinu Mohammed Addis Ababa, Ethiopia June, 2020 1 ADDIS ABABA UNIVERSITY SCHOOL OF GRADUATE STUDIES The Effects of Culture Conditions on In vitro Propagation of Korarima (Aframomum corrorima (Braun) P.C.M. Jansen) By Abdela Zeinu Mohammed Supervised By; Tileye Feyissa (PhD, Associate Professor) A Thesis Presented to School of Graduate Studies, Addis Ababa University, in Partial Fulfillment of the Requirements for the Degree of Master of Science in Biology (Biotechnology stream) Addis Ababa, Ethiopia June, 2020 i ACKNOWLEDGEMENTS I express my genuine and heartfelt gratitude to my advisor Tileye Feyissa (PhD, Associate Professor) for his constructive technical and practical guidance that encouraged me to be strong enough to accomplish this work. Without his inestimable efforts, this paper may not be substantial, he thought me what it means to be dedicated research as the result of his consistent useful suggestions and critical comments significantly improved the preparation of this paper. My sincere gratitude also goes to the Institute of Biotechnology, Addis Ababa University, College of Natural Science for gave me the opportunity to join the program and finish my study. In addition to this my great thanks goes to Mr. Muluken Birara for his technical assistance that enabled me to do my entire practical works at plant tissue culture laboratory. I express my deep gratitude to Addis Ababa city administration office of educational bureau for allowing me to join Addis Ababa University for this study and financial support to pursue my study. I am indebted to Mr. Agegnehu Wasse for his technical comments regarding to experimental designs and statistical analyses. Words cannot express my appreciation to my beloved colleagues Mr. Abrham Sissay, Andualem Manaye and Girma Gebrehiwot for their consistent support, inspiration, thoughtful guidance and suggestions throughout the completion of this research work. Finally heartfelt thanks to my family for their support and encouragement in many ways throughout the course of my study. I am very grateful to all people and institution that are not mentioned here but who contributed in any way for the success of this thesis. ii TABLE OF CONTENTS page ACKNOWLEDGEMENTS………………………………………………………………ii LIST OF TABLES……………………………………………………………………..…vi LIST OF FIGURES………………………………………………………………………vii LIST OF ABBREVIATIONS………………………………………………………...…viii ABSTRACT.........................................................................................................................ix 1.INTRODUCTION …………………………………………………….………………..1 2. LITERATURE REVIEW………………………………………….………………..…3 2.1. Origin and Geographic Distribution of Korarima….…………………………….….3 2.2. Botanical Description of Korarima………………..………………………………...3 2.3. Growth, Development& Ecological Requirements of Korarima …………....……...3 2.4. Morphological Description of Korarima ……….…………………………………...4 2.5. Shade Requirement and Management ………………………………………………5 2.6. Harvest and Processing of Korarima….…………………...…………………...……5 2.7. Chemical Components of Korarima Essential Oils.…………………………………6 2.8. Importance of Korarima Oils………………….……………………………………..6 2.9. Uses of Korarima in Ethiopia..……………..…………….……………………….…7 2.10. Propagation Methods of Korarima………………………….…...…………………8 2.10.1. Propagation of korarima by rhizomes/clumps..…….…………….…………...8 2.10.2. Propagation of korarima by seeds……………………………..……………....9 2.11. Plant Tissue Culture Techniques and its Application……………….……………..9 2.12. Environmental Control of In Vitro Cultured Plants………………………….…...11 2.13. The Components of Plant Tissue Culture Media…….…………….……………...12 2.14. Liquid Culture Media…………………………………..………………………….12 2.15. Acclimatization of In Vitro Rooted Korarima Plantlets……..…………………….13 iii 3.OBJECTIVES OF THE STUDY……………………………………………………...14 3.1. General Objective………………………………………………………………….14 3.2. Specific Objectives………………………………………………………………...14 4. MATERIALS AND METHODS……………………………………………………...15 4.1. Plant Material Source and Experimental Area…...………………………………...15 4.2. Preparation of Stock Solutions and Growth Media……………...…………………15 4.2.1. MS Stock Solution Preparation………………………………………………...15 4.2.2. Preparation of PGR Stock Solution……………………………………………15 4.2.3. Liquid Culture Media Preparation……………………………………………..15 4.3. Seed Surface Sterilization and In vitro Seed Germination of Korarima………...…16 4.4. Shoot Initiation…………………………………………………………..…………16 4.5. Shoot Multiplication……………...……………………………………...…………16 4.6. Rooting……………………..……………………………………............................17 4.7. Acclimatization and Transfer of Plantlets to Soil……….…………………………18 4.8. Experimental Design and Statistical Data Analysis……………….………………18 5. RESULTS………………………………………………………………………………19 5.1. In vitro Seed Germination and Culture Initiation of Korarima……….……………19 5.2. Effect of Different Sucrose Concentrations on Shoot Multiplication………...……20 5.3. Effect of Different Salt Strength MS Media on Shoot Formation of Korarima……21 5.4. Effect of pH on Shoot Multiplication ..……………………………………………22 5.5. Effect of Different Sucrose Concentrations on Rooting……………………………23 5.6. Effect of Different MS Salt Strength on Root Formation……..……………………25 5.7. Effect of Different pH on Root Formation…….…………………………………..26 5.8. Acclimatization of Plants………………………..………………………………….27 6. DISCUSSION…………………………………………………………………………..29 iv 6.1. In vitro Seed Germination and Culture Initiation of Korarima…………………….29 6.2. Effect of Different Sucrose Concentrations on Shoot Multiplication…..………….29 6.3. Effect of Different MS Salt Strength on Shoot Multiplication…………..…………30 6.4. Effect of Different pH on Shoot Multiplication.………………….…..…….……...31 6.5. Effect of Different Sucrose Concentrations on Rooting……………………………32 6.6. Effect of Different MS Salt Strength media on Rooting…………...………………33 6.7. Effect of Different pH on Rooting…………………………………………………34 6.8. Acclimatization of Korarima Plantlets…………………………..…………………34 7.CONCLUSION…………………………………………………………………………35 8.RECOMMENDATIONS……………….…………………………………….………..36 REFERENCES………………………………..………………………………….………37 APPENDIX…..………………………………………………..…….……………………44 v LIST OF TABLES Page Table 1. Effect of different sucrose concentrations on shoot multiplication…………21 Table 2. Effect of different MS salt strength on shoot multiplication………………..22 Table 3.Effect of different pH ranges on shoot multiplication……………………….23 Table 4.Effect of different sucrose concentrations on root induction………………...25 Table 5.Effect of different MS salt strength on root induction……………………….26 Table 6.Effect of different pH on root induction…………………….………………..27 vi LIST OF FIGURES Page Figure 1. Germination of korarima Seeds ............................................................................. 19 Figure 2. Culture initiation korarima explants ..................................................................... 19 Figure 3.Shoot multiplication of korarima with different sucrose concentrations ................ 21 Figure 4.Shoot multiplications on MS medium containing different salt strength, .............. 22 Figure 5.Shoot multiplications on MS medium containing different pH levels ................... 24 Figure 6.Root induction of korarima in different concentrations of sucrose ........................ 25 Figure 7. Root inductions on MS medium containing different salt strength ....................... 26 Figure 8.Root inductions on MS medium at different pH levels .......................................... 27 Figure 9. Acclimatization of in vitro rooted microshoots of A. corrorima ........................... 28 vii LIST OF ABBREVIATIONS ANOVA Analysis of Variance BAP 6-Benzyl Amino Purine CRD Complete Randomized Design DMRT Duncan‟s Multiple Range Test EDTA Ethylene Di-amine Tetra acetic Acid EARI Ethiopian Agricultural Research Institute ESC Ethiopian Sugar Corporation GA3 Gibberillic Acid LSD Least Significance Difference MS Murashige and Skoog NAA α-Naphthalene Acetic Acid PGRs Plant Growth Regulators SPSS Statistical Package for Social Science viii ABSTRACT Aframomum corrorima is one of the important spice and medicinal plants of the family Zingiberaceae which can be propagated from its seed and rhizome. Conventionally, propagation of A.corrorima by rhizome causes for genetic erosion and it is a time consuming process. Hence, plant tissue culture has played a very important role to clone the endangered plant species with desirable traits urgently. The present study was conducted to investigate the effects of culture conditions on in vitro propagation of A.corrorima using MS liquid media. Surface sterilization of seeds in 70% ethanol for 3 minutes then followed by dipping in 25% sodium hypochlorite for 25 minutes gave the best disinfected seeds and the percentages of seed germination and shoot initiation obtained on MS media were 93.5% and 96.25% ,respectively. In the sucrose containing MS media, 3% gave the highest number of shoots (3.10±0.15), leaves (8.77±0.13), roots (9.03±0.72) and shoot length (3.39±0.63 cm) per explant/shoot. Out of different pH levels, pH 5.8 gave the best result regarding the number of shoots (3.20±0.13), leaves (11.40±0.27) and length of shoots (4.08±0.13cm) and root number (9.03±0.72) of korarima. In another MS liquid culture media experiment, maximum shoot number (3.03 ±0.21) and shoot length (3.17 ± 0.12 cm) were recorded in