TISSUE CULTURE AND BIOLOGICAL ACTIVITIES OF GARDENIA JASMINOIDES ELLIS REZA FARZINEBRAHIMI

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1 TISSUE CULTURE AND BIOLOGICAL ACTIVITIES OF GARDENIA JASMINOIDES ELLIS REZA FARZINEBRAHIMI DISSERTATION SUBMITTED IN FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE INSTITUTE OF BIOLOGICAL SCIENCES FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR 2012

2 I dedicate this thesis to my deceased father, who taught me the best kind of knowledge and my lovely mother for her love and measureless support.

3 UNIVERSITI MALAYA ORIGINAL LITERARY WORK DECLARATION Name of Candidate: Reza Farzinebrahimi (Passport No: U ) Registration/Matric No: SGR Name of Degree: Master of Science Title of Dissertation: Tissue Culture and Biological Activities of Gardenia jasminoides Ellis Field of Study: Plant Biotechnology I do solemnly and sincerely declare that: (1) I am the sole author of this Work. (2) This work is original. (3) Any use of any work in which copyright exists was done by way of fair dealing and for permitted purposes and any excerpt or extract from, or reference to or reproduction of any copyright work has been disclosed expressly and sufficiently and the title of the work and its authorship have been acknowledged in this Work. (4) I do not have any actual knowledge nor ought I reasonably to know that the making of this work constitutes an infringement of any copyright work. (5) I hereby assign all and every rights in the copyright to this Work to the University of Malaya (UM), who henceforth shall be owner of the copyright in this Work and that any reproduction or use in any form or by any means whatsoever is prohibited without the written consent of UM having been first had and obtained. (6) I am fully aware that if in the course of making this Work I have infringed any copyright whether intentionally or otherwise, I may be subject to legal action or any other action as may be determined by UM. Candidate s Signature Date: Subscribed and solemnly declared before, Witness s Signature Name: Rosna Mat Taha Designation: Professor Date: i

4 Abstrak Gardenia jasminoides Ellis adalah sejenis tumbuhan renek malar hijau dari keluarga Rubiacea. Spesies ini adalah salah satu tumbuhan ubatan dan terkenal dalam perubatan tradisional Cina. Buah Gardenia jasminoides Ellis mengandungi antioksidan yang tinggi, sementara pigmen tumbuhan ini digunakan dalam industri makanan. Kajian ini dijalankan untuk saringan aktiviti antibakteria dan antioksidan menggunakan ekstrak in vivo dan in vitro, juga untuk mendapatkan kaedah propagasi in vitro tumbuhan Gardenia jasminoides yang efisien dengan menggunakan pelbagai media kultur tisu yang ditambah dengan hormon auksin dan sitokinin pada kepekatan yang berbeza. Bagi tujuan ini, eksplan daun daripada G. jasminoides telah dikultur pada media MS dan WPM yang mengandungi 3% sukrosa dan hormon auksin (NAA, IBA, IAA, 2-4, D) serta sitokinin (TDZ & KN) pada kepekatan yang berbeza. Kepekatan hormon yang digunakan adalah pada julat 0-5 mg l -1 untuk mendapatkan kalus, serta pembentukan pucuk dan akar. Selepas 4 minggu kalus telah terbentuk, pada kedua-dua jenis media yang telah ditambah dengan pelbagai hormon pada kepekatan yang berbeza. Hasil kajian menunjukkan bahawa 100% kalus telah terhasil pada MS dan WPM yang ditambah dengan 2, 2.5 dan 3 mg l -1 hormon NAA. Tiada perbezaan yang signifikan dalam pembentukan kalus yang diperhatikan di antara pelbagai jenis auksin pada media WPM. Sebaliknya, media MS menunjukkan perbezaan yang signifikan pada hormon IAA dibandingkan dengan auksin yang lain. Selain itu, Media MS yang ditambah dengan hormon Kinetin dan TDZ menunjukkan perbezaan dari segi statistik berbanding dengan media WPM. ii

5 Eksplan daun juga telah dikultur pada media MS dan media WPM yang ditambah dengan kombinasi semua auksin dengan TDZ dan Kn, tetapi tiada pembentukan pucuk diperhatikan. Pengakaran telah mula terbentuk pada kedua-dua media yang ditambah dengan pelbagai auksin selepas minggu kelima. Media MS yang ditambah dengan (1.5 dan 2 mg l -1 ) NAA telah menunjukkan respons yang lebih tinggi untuk pembentukan akar (14.8 dan 13.4 cm) dan IAA (4.5 dan 5 mg l -1 ) yang telah ditambah pada media WPM telah menunjukkan pembentukan akar terpanjang (18.3 dan 18.7cm). Analisis data menunjukkan perbezaan yang signifikan dalam media WPM ditambah dengan IAA, NAA, dan IBA masing-masing. Walau bagaimanapun, media MS menunjukkan perbezaan signifikan apabila ditambah dengan hormon NAA berbanding hormon IAA dan IBA. Kalus segar telah terbentuk pada media MS yang ditambah dengan NAA (3 mg l -1 ) dan Kn(5 mg l -1 ), masing-masing dengan berat 34.23g dan 3.39 g. Di samping itu, kalus basah yang terbentuk pada media WPM yang ditambah dengan NAA (2.5 mg l - 1 ) dan Kn (4 mg l -1 ) mempunyai berat basah 30.04g dan 3.78g. Lebih banyak kalus terbentuk daripada eksplan daun Gardenia jasminoides Ellis pada kedua-dua media MS dan media WPM yang ditambah dengan hormon NAA dan IBA pada kepekatan yang berbeza berbanding hormon auksin yang lain. Kalus yang terbentuk pada kedua-dua media MS dan WPM yang ditambah dengan hormon NAA menunjukkan aktiviti antibakteria terhadap Escherichia coli dan Bacillus cereus, iaitu bakteria gram negatif dan gram positif. Ekstrak in vivo dan in vitro yang lain tidak menunjukkan zon antibakteria terhadap semua bakteria yang diuji. Data yang dianalisis dari kajian ini telah menunjukkan potensi antioksidan pada tisu kalus in vitro yang menggunakan NAA, IBA, TDZ, dan Kn, dan kajian ini telah iii

6 menunjukkan bahawa aktiviti antioksidan kalus mempunyai perbezaan ketara dibandingkan dengan tumbuhan in vivo. iv

7 Abstract In order to establish an efficient in vitro propagation protocol for Gardenia jasminoides Ellis, Murashige and Skoog (MS) and woody plant medium (WPM) with different concentrations and types of auxin and cytokinin were used. The leaf explants of G. jasminoides were cultured on MS and WPM medium containing 3% sucrose and different concentrations of NAA, IBA, IAA, 2,4-D as auxin and TDZ & Kn as cytokinin with range of 0-5 mg l -1 in order to obtain callus, shoot and root formation. After 2 weeks, the callus was formed on both types of media supplemented with various hormones at different concentrations. The results showed that 100% callus was formed on MS and WPM supplemented with 2, 2.5 and 3 mg l -1 of NAA. No significant difference in callus formation was observed between various auxins on WPM media, in contrast, MS media showed significant differences between IAA to other auxins. In addition, Kinetin and TDZ supplemented to MS media showed statistical differences compare to WPM media. When leaf explants were cultured on MS and WPM media supplemented with a combination of all used auxins with TDZ and Kn, no shoot formation was observed. Rooting started on both media supplemented with various auxins after the fifth weeks. MS medium supplemented with (1.5 and 2 mg l -1 ) NAA showed higher response for root length (14.8 and 13.4 cm) and IAA (4.5 and 5 mg l -1 ) supplemented to WPM media showed the longest root length (18.3 and 18.7 cm), respectively. Data analyses showed significant differences in WPM medium supplemented with IAA, NAA and IBA, respectively. However, MS media showed statistical differences when supplemented with NAA compare to IAA and IBA. v

8 Fresh grown callus on MS medium supplemented with NAA (3 mg l -1 ) and Kn (5 mg l -1 ) were g and 3.39 g respectively. In addition, g and 3.78 g were the fresh weight of the callus, cultured on WPM media supplemented with (2.5 mg l -1 ) NAA and (4 mg l -1 ) Kn. A high amount of callus was formed from leaf explants of G. jasminoides Ellis on both MS and WPM media supplemented with different concentrations of NAA and IBA. Extracts from callus grown on both MS and WPM supplemented with NAA showed antibacterial activity against Escherichia coli (gram-negative) and Bacillus cereus (gram-positive). The other extracts from in vivo and in vitro calluses showed no antibacterial zone against the rest of the bacteria tested. The in vitro-grown callus derived using NAA, IBA, TDZ, and Kn disclosed the antioxidant activity of the callus extract which is significantly different from the intact plant. vi

9 ACKNOWLEDGEMENTS Even if there was my full dedication to the accomplishment of this thesis work, but there is the hand of GOD, many people s and organizations behind. First, I would like to thank the Almighty GOD for the strength he has given me throughout the thesis work. My deepest gratitude will go to my respected Supervisor, Prof. Dr. Rosna Mat Taha for her guidance, patience, unconditional support, valuable comments and positive approach throughout the thesis work, without her help, this work could not be completed. I acknowledge the Dean of institute postgraduate study, Dean of Faculty of science, Head of Institute of Biological Sciences for allowing me to use all the chemicals and equipment for my study and financial support during my study period. I am very grateful to my friends in laboratory, Arash Khorasani, Arash Rafat, Imdadul, Norhayati, Azani, Anis, Nooraini, Azura, Sharifah, Hashimah, Fatima, Zarifah and all peoples at tissue culture laboratory second floor and fourth floor of block B at ISB building in University of Malaya, without their absolutely kind help, the completion of this work was not possible. I also wish to express my gratefulness to my beloved Bokhi, Leila and Mozaffar for their support. I am greatly indebted to my families, my lovely son Kasra and my faithful wife Zahra, who encouraged and helped me throughout the study period and always believe in me and encourage me to find my own path through life, and support me along the path, thank you but I know this word does not express what you are. The last but not the least, I pay my enthusiastic feelings to my mother again, for her love, affection, encouragement and prayers for my success and without their moral vi

10 and financial support, I could not have completed my research work who supported me during these years and have always been my motivation and inspiration I do not know what to say but just THANK YOU. To the academic staff and administrators of ISB in University of Malaya, the year and all those people whom I failed to mention their names, thank you very much. vii

11 Table of Contents ORIGINAL LITERARY WORK DECLARATION... I ABSTRAK... II ABSTRACT... V ACKNOWLEDGEMENTS... VI LIST OF ABBREVIATIONS... VI INTRODUCTION... IX 1.1 INTRODUCTION OBJECTIVES OF STUDY... 6 LITERATURE REVIEW BOTANICAL CHARACTERISTICS OF GARDENIA JASMINOIDES BENEFITS OF GARDENIA JASMINOIDES ELLIS BIOTECHNOLOGY TISSUE CULTURE TECHNIQUE REVIEW OF TISSUE CULTURE In Vitro Microropagation of Medicinal and Aromatic Plants REVIEW OF PGR S EFFECT ON PLANT Explant Sterilization SECONDARY METABOLITES Secondary Metabolites in Plants Antioxidant Compounds ANTIOXIDANT ACTIVITY SCREENING METHODS a. Total Phenolic Content (TPC) b. Total Flavonoid (TF) c. Free Radical Scavening Assay d. Superoxide Anion Radical Scavenging (SO) Assay e. Xanthine Oxidase Method REVIEW OF PREVIOUS EXPERIMENTS ANTIBACTERIAL ACTIVITY METHODS AND REVIEW ON ANTIBACTERIAL ACTIVITY ASSAY CELL SUSPENSION CULTURES AND SOMATIC EMBRYOGENESIS DOUBLE STAINING TEST MATERIALS AND METHODS INCUBATION CONDITIONS Basic Processes in Tissue Culture Basic Organization of Laboratory General Laboratory Area Culture Area Aseptic Transfer Area Chamber Sterilization Steps PREPARING ETHANOL SOLUTION Production Stage PREPARATION MS MEDIA WPM MEDIUM PREPARATION METHOD HORMONE STOCK PREPARATION PLANT MATERIALS STERILIZATION TECHNIQUES INOCULATION SECONDARY METABOLITES ASSAY viii

12 EXTRACT PREPARATION ANTIBACTERIAL ACTIVITY A. MEDIA PREPARATION B. Equipment Sterilization C. Preparation of bacteria D. Nutrient Broth or NB medium Preparation E. Antibacterial Assay ANTIOXIDANT ASSAY A. REAGENT PREPARATION B. ASSAY PROTOCOL C. STANDARD PREPARATION D. PERFORMING THE ASSAY E. CALCULATIONS RESULTS AND DISCUSSION INITIATION OF CALLUS AND MAINTENANCE Callus Formation in MS Medium Callus Formation in WPM Media Comparison of WPM and MS Media Supplemented with Various Concentrations of NAA for Callus Formation Comparison of WPM and MS Media Supplemented with Various Concentrations of IBA for Callus Formation Comparison of WPM and MS Media Supplemented with Various Concentrations of IAA for Callus Formation Comparison of WPM and MS Media Supplemented with Various Concentrations of 2, 4- D for Callus Formation ROOT ELONGATION STUDY A. MS medium B. WPM Medium Comparison of MS and WPM Media Supplemented With NAA on Root Formation Comparison of MS and WPM Media Supplemented With IAA for Root Formation Comparison of MS and WPM Media Supplemented with IBA On Root Formation FRESH AND DRY WEIGHT OF CALLUS ANTIBACTERIAL ASSAY ANTIOXIDANT ASSAY CONCLUSION INVESTIGATION OF MEDIA EFFECTS ON CALLUS FORMATION INVESTIGATION OF HORMONES EFFECTS ON ROOT INDUCTION SCREENING OF ANTIOXIDANT ACTIVITY BETWEEN INTACT PLANT AND DIFFERENT CALLUS GROWN ON MS AND WPM SCREENING OF ANTIBACTERIAL ACTIVITY DIFFERENT CALLUS GROWN ON MS AND WPM AND INTACT PLANT RECOMMENDATIONS FOR FUTURE WORK BIBLIOGRAPHY ix

13 LIST OF FIGURES FIGURE 2.1: LEAF AND FLOWER OF GARDENIA JASMINOIDES ELLIS... 8 FIGURE 2.2: DRY AND FRESH FRUIT OF GARDENIA JASMINOIDES ELLIS... 9 Figure 2.3: Structure of genioposide (R-Glucose) and genipin (R-H)...10 FIGURE 3.1: A TYPICAL LAYOUT OF SOD STANDARDS AND SAMPLES FIGURE 4.1: GREENISH AND YELLOWISH CALLUS FROM 1 MG L -1 TDZ AND 1.5 MG L -1 IBA AFTER 5 WEEKS FIGURE 4.2: EARLY STAGE EMBRYOS AFTER DOUBLE STAINING, EMBRYONAL HEADS STAINED RED (ACETOCARMINE) AND SUSPENSORS STAINED BLUE (EVAN S BLUE) Figure 4.3: Percentage of callus formation of G. jasminoides in MS medium supplemented with differrent type of auxin at various concentration 58 Figure 4.4: Percentage of callus formation on MS medium supplemented with different types of cytokinin at various concentrations.60 Figure 4.5: Callus induction and root formation from leaf explant of G. jasminoides Ellis on MS medium supplemented with 1.5 mg l -1 NAA after 2.5months 60 Figure 4.6: Percentage of callus formation in WPM medium supplemented with different type of auxin at various concentrations..62 Figure 4.7: Callus induction and root formation from leaf explant of G. jasminoides Ellis on MS medium supplemented with 1.5 mg l -1 NAA after 2.5 months 62 Figure 4.8: Percentage of callus formation on WPM medium supplemented with different types of cytokinin at various concentrations 63 Figure 4.9: A comparison between WPM and MS media supplemented with NAA for callus formation 64 Figure 4.10: A comparison between WPM and MS media supplemented with IBA for callus formation..66 Figure 4.11: Callus formation from leaf explant in WPM supplemented with 2.5 mg l-1 IBA after 4 months.. 67 Figure 4.12: A comparison between WPM and MS media supplemented with IAA for callus formation 68 Figure 4.13: A comparison between WPM and MS media supplemented with 2, 4-D for callus formation 70 FIGURE 4.14: ROOT ELONGATION ON MS MEDIUM.71 Figure 4.15: Rooting formation in MS medium supplemented with 2 mg l -1 NAA after 6 months.72 Figure 4.16: Root elongation on WPM medium...73 Figure 4.17: Rooting in WPM supplemented with 1 mg l -1 IAA one week after the second subculture Figure 4.18: hoot eioneation in id meeia s ooiementee tith 2 mg l -1 NAA after 4 months from leaf explant of G. jasminoides Ellis...75 Figure 4.19: A Comparison between MS and WPM media supplemented with various concentrations of NAA for root elongation..76 Figure 4.20: A Comparison between MS and WPM media supplemented with various concentrations of IAA for root elongation.78 Figure 4.21: Rooting in WPM supplemented with 2 mg l -1 IAA 4 weeks after the second subculture of leaf explant of G. jasminoides Ellis..78 Figure 4.22: Root elongation in WPM medium supplemented with 2 mg l -1 IBA after 4 months Figure 4.23: Rooting in WPM medium supplemented with 2 mg l -1 IBA 3 weeks after the second sub culture Figure 4.24: A Comparison between MS and WPM media supplemented with various concentrations of IBA for root eolngatiln Figure 4.25: Fresh weight of callus in MS medium supplemented with various types of hormones and ieeerent concentrations. 81 FIGURE 4.26: DRY WEIGHT OF CALLUS IN MS MEDIUM SUPPLEMENTED WITH VARIOUS TYPE OF HORMONES IN TNRRFhFND CONCENTRATIONS..84 Figure 4.27: Fresh weight of callus in WPM medium supplemented with various type of hormones and eifferent concentrations.. 84 Figure 4.28: Dry weight of callus in WPM medium supplemented with various type of hormones in 85...erent eoneentrationseiff x

14 Figure 4.29: A comparison between dry and fresh weight of callus in WPM and MS media supplemented tith eifferent hormones 85 Rie re 4.30: Nnhibition zone in tetraeyeiine as a eontroi Figure 4.31: Inhibition zone of in vitro extract grown on MS medium supplemented with NAA against E. coli 87 Figure 4.32: A comparison of SOD activity between in vivo (leaf of G. jasminoides Ellis) and in vitro from eifferent meeia ane vario s PGh s 90 List of Tables Table 2.1: Secondary Metabolites by Plant Cell Cultures...28 Table 3.1: Plant Growth Regulators Concentration Conversions and Chemical Specifications..43 Table 3.2: Superoxide Dismutase standards...52 Table 4.3: Inhibition zone from 100 g l -1 in vivo and in vitro leaf extracts of Gardenia jasminoides Ellis against the four pathogenic bacteria 88 xi

15 LIST OF ABBREVIATIONS 2, 4-D 2, 4-Dichlorophenoxyacetic acid 2iP ANOVA ACC 6-Gamma, gamma-dimethyl allylamino purine Analysis of variance 1- aminocylopropane-1- carboxylic acid B BAP C Ca CFU cm Cu DMCT DPPH DTPA Fe g Hcl IAA IBA K Kn Boron 6-Benzylaminopurine Degree of Celsius Calcium Colonies-Forming Units Centimeter Copper Duncan s Multiple Comparison Test 2, 2-diphenyl-1-picrylhydrazil Diethylenetriaminepentaacetic acid Iron gram Hydrochloride acid Indole-3-Acetic Acid Indole-3-Butric Acid Potassium Kinetin vi

16 L LS nm Mg mg mg l -1 MH Mn MS mm ml N Na NAA P PBS ph psi PGR SOD UV V/V WPM Litter Linsmaier & skoog medium Nano meter Magnesium Milligram Milligram per liter Mueller Hinton broth Manganese Murashige and Skoog Medium Millimeter Milliliter Nitrogen Sodium Naphthalene acetic acid Phosphate Phosphate buffer solution Hydrogen ion concentration Per Square Inch Plant Growth Regulator Superoxide Dismutase Ultra Wave Volume per volume Woody Plant Media vii

17 W/V TDZ Zn μg μl Weight per Volume Thidiazuron Zinc Microgram Micro liter viii