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MICROPROPAGATION of Curcuma Aeruginosa and Curcuma Heyneana USING AERATED CULTURE SYSTEM NOVIANTI SUWITOSARI UNIVERSITI SAINS MA

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MICROPROPAGATION of Curcuma Aeruginosa and Curcuma Heyneana USING AERATED CULTURE SYSTEM NOVIANTI SUWITOSARI UNIVERSITI SAINS MA MICROPROPAGATION OF Curcuma aeruginosa AND Curcuma heyneana USING AERATED CULTURE SYSTEM NOVIANTI SUWITOSARI UNIVERSITI SAINS MALAYSIA 2012 MICROPROPAGATION OF Curcuma aeruginosa AND Curcuma heyneana USING AERATED CULTURE SYSTEM by NOVIANTI SUWITOSARI Thesis submitted in fulfillment of the Requirements for the degree of Master of Science June 2012 ACKNOWLEDGEMENTS First of all I would like to thank Allah S.W.T for giving me the opportunities, patience and passions to complete my post-graduate (MSc) study at School of Biological Sciences, Universiti Sains Malaysia. My foremost thanks and gratitude to my supervisor, Professor Chan Lai Keng from the School of Biological Sciences, Universiti Sains Malaysia, Penang who has given me all the support and guidance throughout my study. My appreciation also goes to my co-supervisor, Dr. Amir Hamzah Bin Ahmad Ghazali from the School of Biological Sciences, Universiti Sains Malaysia, Penang. My special thanks to my field supervisor, Professor Gunawan Indrayanto from the Faculty of Pharmacy, Airlangga University, Indonesia for his assistance and advice in the chemical analysis. I wish to thank the Dean of Biological Sciences and Dean of Institute of Higher Learning (IPS) of Universiti Sains Malaysia for allowing me to pursue my higher degree. I would like to thank the Postgraduate Research Grant Scheme of USM (USM-RU-PGRS 1001/PBIOLOGI/832021) for funding my research. I must also thank all the staff and lab assistants from School of Biological Sciences, USM and Research Unit staff from Faculty of Pharmacy, UNAIR, especially, Mbak Yanti and Mas Gunarso for assisting me in LC-MS/MS analysis. My sincere thanks to all my lab-mates at The Plant Tissue and Cell Culture Laboratory, especially Chee Leng, mama Zainah, kak Rafidah and Christine for their invaluable advice. Thank you to Fung Hui, Lay Pin and Vun Hui for the friendship and happy moments. I would like to thank Shu Ying, Suganthi, Nisa, Salmee, Eugene, Hasmah, Kiah Yann, Nadia, Song Jin, Taufiq, Laleh, Fatemeh, Mr. Ahmed, ii Li See, E Shuen, King Wey and Samantha for their company. Also, thanks to Dr. Ning and Dr. Arvind for their assistance and advice. Special thanks to my best friends (Herlina and Yostia) for standing by me through the hard times. Thank you to Patricia for the ‘Ganbatte Oneechan’ always. Thanks to ‘Derong’ and ‘Chaeyoung’ for their support and the ‘JYJ’ craziness which makes me forgets all my difficult and tiring moments. I am also grateful to my family for their support and encouragement they had given me. Mama, you were always there for me, pushing me and guiding me always to succeed. I want to thank you for what you have done. Hopefully, I can give back to you. Mom, you always was the perfect fan. My beloved Dad, I knew you always support me even you were not here. I really miss you and always love you Dad. My little brother and sister who did all they could to support me, thank you so much. Last but not least, many thanks to my big family (grandmother, budhe Ban, Mas Bambang, Mbak Yani and all my cousins) who have showed their love and support so far. Always keep the faith and do your best until the end. NOVIANTI SUWITOSARI iii TABLE OF CONTENTS Title Page Acknowledgement ii Table of Contents iv List of Tables viii List of Figures ix List of Plates x List of Abbreviations xii Abstrak xiii Abstract xv 1. Introduction 1 2. Literature Review 5 2.1 The Curcuma species 5 2.1.1 Curcuma aeruginosa 6 2.1.2 Curcuma heyneana 7 2.2. Secondary Metabolites 8 2.3 Plant Tissue Culture 11 2.3.1 History and developments 11 2.3.2 Micropropagation 12 2.3.2.1 Establishment of aseptic cultures 12 2.3.2.2 Shoot multiplication 13 2.3.2.3 Rooting 15 2.3.2.4 Acclimatization 16 iv 2.3.3 Mass propagation using aerated culture system 17 2.4 Chemical analysis and antioxidant activities 19 3. Materials and Methods 22 3.1 Micropropagation of Curcuma aeruginosa and Curcuma 22 heyneana 3.1.1 Establishment of aseptic plant materials 22 3.1.1.1 Rhizomatous bud explants 22 3.1.1.2 In vitro seedlings 23 3.1.2 Shoot multiplication 23 3.1.2.1 Shoot multiplication using MS medium 23 supplemented with 2 mg/l BA and 0.5 mg/l NAA 3.1.2.2 Effect of solid and liquid medium on shoot 23 proliferation 3.1.2.3 Effect of incised and intact whole shoot on shoot 24 proliferation 3.1.3 Rooting of micro-shoots 25 3.1.4 Acclimatization of in vitro plantlets 25 3.2 Micro rhizome production 25 3.3 Propagation of Curcuma aeruginosa and Curcuma heyneana 26 using aerated culture system 3.3.1 Set up of aerated culture vessel 26 3.3.2 Determination of suitable inoculum size 26 3.3.3 Propagation in aerated culture vessel 27 3.3.3.1 Effect of aerated culture vessel sizes on 27 propagation of C. aeruginosa 3.3.3.2 Propagation of C. aeruginosa using combination 29 of aerated culture system and MS basal gelled medium 3.4 Chemical analysis 29 v 3.4.1 Qualitative analysis using Thin Layer Chromatography 29 (TLC) 3.4.1.1 Materials and preparation of plant extracts 29 3.4.1.2 Thin Layer Chromatography (TLC) analysis 30 3.4.2 Qualitative analysis using High Performance Liquid 30 Chromatography-Mass Spectrometry (LC-MS/MS) 3.4.2.1 Materials and preparation of plant extracts 30 3.4.2.2 High Performance Liquid Chromatography-Mass 31 Spectrometry (LC-MS/MS) analysis 4. Results 32 4.1. Micropropagation of Curcuma aeruginosa and Curcuma 32 heyneana 4.1.1 Establishment of aseptic plant materials 32 4.1.1.1 Rhizomatous bud explants 32 4.1.1.2 In vitro seedlings 32 4.1.2 Shoot multiplication 33 4.1.2.1 Shoot multiplication using MS medium 33 supplemented with 2 mg/l BA and 0.5 mg/l NAA 4.1.2.2 Effect of solid and liquid medium on shoot 33 proliferation 4.1.2.3 Effect of incised and intact whole shoot on shoot 37 proliferation 4.1.3 Rooting of micro-shoots 37 4.1.4 Acclimatization of in vitro plantlets 38 4.2 Micro rhizome production 38 4.3 Propagation of Curcuma aeruginosa and Curcuma heyneana 44 using aerated culture system 4.3.1 Set up of aerated culture vessel 44 4.3.2 Determination of suitable inoculum size 44 vi 4.3.3 Propagation in aerated culture vessel 45 4.3.3.1 Effect of aerated culture vessel sizes on 45 propagation of C. aeruginosa 4.3.3.2 Propagation of C. aeruginosa using combination 46 of aerated culture system and MS basal gelled medium 4.4 Chemical analysis 49 4.4.1 Qualitative analysis using Thin Layer Chromatography 49 (TLC) 4.4.2 Qualitative analysis using High Performance Liquid 50 Chromatography-Mass Spectrometry (LC-MS/MS) 5. Discussion 54 5.1 Micropropagation of Curcuma aeruginosa and Curcuma 54 heyneana 5.2 Micro rhizome production 58 5.3 Propagation of Curcuma aeruginosa and Curcuma heyneana 58 using aerated culture system 5.4 Chemical analysis 61 6. Conclusion 64 6.1 Conclusion of study 64 6.2 Suggestions for future research 64 References 65 Publication List 77 vii LIST OF TABLES Page Table 4.1 Establishment of aseptic bud explants and their 34 survival percentage of C. heyneana after immersion in 70% ethanol for 10 minutes followed by Clorox® surface-sterilization method Table 4.2 Shoot multiplication of Curcuma aeruginosa and 36 Curcuma heyneana on gelled and liquid MS medium supplemented with 2 mg/l BA and 0.5 mg/l NAA. Table 4.3 Rooting percentage of C. aeruginosa and C. 40 heyneana on MS basic medium and MS medium supplemented with NAA (1, 2 and 3 mg/l) Table 4.4 The survival percentage of in vitro plantlets of C. 40 aeruginosa and C. heyneana after acclimatization for two weeks in three different soil mixtures Table 4.5 Shoot multiplication of Curcuma aeruginosa and 47 Curcuma heyneana using 4-10 half shoots as inoculum size Table 4.6 Shoot multiplication of Curcuma aeruginosa and 47 Curcuma heyneana using 10-40 half shoots as inoculum size Table 4.7 Shoot multiplication of Curcuma aeruginosa and 48 Curcuma heyneana in aerated culture vessel Table 4.8 Shoot formation of Curcuma aeruginosa using 48 combination of aerated system and MS basal gelled medium with different culture duration (weeks) viii LIST OF FIGURES Page Fig. 4.1 Multiple shoot formation of Curcuma aeruginosa and 36 Curcuma heyneana on MS medium supplemented with 2 mg/l BA and 0.5 mg/l NAA Fig. 4.2 Effect of shoot incision on shoot proliferation of C. 39 heyneana and C. aeruginosa in liquid MS medium supplemented with 2 mg/l BA and 0.5 mg/l NAA after four weeks of culture Fig. 4.3 Micro rhizome production of C. aeruginosa and C. 43 heyneana in the soil mixture of top soil: sand: organic soil (1:1:1) after 90 days of acclimatized Fig. 4.4 TIC scan (A) and Spectrum results (B) of 53 curcuminoids standard by LC-MS/MS. (1) Mass spectra of bisdemethoxycurcumin. (2) Mass spectra of demethoxycurcumin. (3) Mass spectra of curcumin Fig. 4.5 TIC scan of Curcuma aeruginosa extract (A) and 53 Curcuma heyneana extract (B) by LC-MS/MS ix LIST OF PLATES Plate Plate 3.1 Set up of aerated culture vessel (1 L) in the culture 28 room Plate 4.1 The aseptic buds of Curcuma heyneana (A) and 35 Curcuma aeruginosa (B) Plate 4.2 The bud-derived seedlings of Curcuma heyneana (A) 35 and Curcuma aeruginosa (B) Plate 4.3 The multiple shoots formed from half shoot explants 39 of C.
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