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Stratagies for the Production of Chemically Consistent Plantlets of Silybum Marianum L

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Stratagies for the Production of Chemically Consistent Plantlets of Silybum Marianum L STRATAGIES FOR THE PRODUCTION OF CHEMICALLY CONSISTENT PLANTLETS OF SILYBUM MARIANUM L. By MUBARAK ALI KHAN DEPARTMENT OF BIOTECHNOLOGY QUAID-I-AZAM UNIVERSITY ISLAMABAD, PAKISTAN 2015 Strategies for the Production of Chemically Consistent Plantlets of Silybum marianum L. Thesis submitted to The Department of Biotechnology Quaid-i-Azam University Islamabad In the partial fulfillment of the requirements for the degree of Doctor of Philosophy In BIOTECHNOLOGY BY MUBARAK ALI KHAN Supervised by Dr. BILAL HAIDER ABBASI Department of Biotechnology Quaid-i-Azam University, Islamabad, Pakistan 2015 DECLARATION The whole of the experimental work included in this thesis was carried out by me in the Plant Cell Culture Laboratory, Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan and in Mass spectrometry laboratory, The University of Sheffield United Kingdom. The findings and conclusions are of my own investigation with discussion of my supervisor Dr. Bilal Haider Abbasi. No part of this work has been presented for any other degree. MUBARAK ALI KHAN ACKNOWLEDGEMENTS The successful completion of this journey is due to the combined efforts of many people. Ph.D is a long and complicated journey. During this journey, I was helped by many and I would like to show my gratitude to all of them. This work would not have been finished without their help. Primary, I would greatly appreciate my supervisor Dr. Bilal Haider Abbasi, Assistant Professor, Department of Biotechnology, Quaid-i-Azam University, Islamabad for his dynamic supervision. It is his confidence, imbibing attitude, splendid discussions and endless endeavors through which i have gained significant experience. My special thanks are due to Prof. Zabta Khan Shinwari, Chairman Department of Biotechnology, Quaid-i-Azam University Islamabad, for his immense concern throughout my Ph.D period. I wish to express my deep gratitude and respect to all my teachers whose knowledge and guidance enabled me to attain this target. I feel pleasure in acknowledging the nice company of my lab fellows Mohammad Ali, Mohammad Adil, Nisar Ahmad and Ahmad Zada. I will always remember the nice company of my friends Abid Jan, Naseer Ali Shah, Akhtar Nadhaman, Mutiullah, Nazifullah, Zia Mashwani, Khalid Khattak, Zahidullah, Naveed Alam, Asad Zia, Ikramullah, Tariq Khan, Amaan and Ishtiaq Hussain. I must acknowledge my debt to Prof. Mike Burrell, Department of Animal and Plant Sciences, The University of Sheffield, United Kingdom for providing me an opportunity to carry out part of my research project in his mass spectrometry laboratory. His constructive comments, guidance and great co-operation played a fundamental role for carrying out this epic work. I am also thankful to technical staff members Bob Keen and Heather Walker for their support and assistance during my stay in The University of Sheffield. This project was supported by Higher Education Commission of Pakistan and I acknowledge this institution for providing me Indigenous and Overseas “IRSIP” scholarship for my research in Pakistan and United Kingdom. Deepest regards and gratitude to my brothers Dr. Sartaj Wali Khan and Dr. Abdul Ali Khan for their support. My all the four sisters prayer for my success, I am also thankful to them. My infinite thank goes to my wife Huma Ali for her moral support and tolerance in this long and hard journey. Lots of love to my kids Saad Ali Khan and Mohammad Ali Khan for making me smile whenever I met them. I must say that all the happiness in my life is because of my beloved father Prof. Muhammad Iqbal and my ever caring and sweetest mother. Their constant prayers and appreciations always kept me satisfied. Mubarak Ali Khan i I Dedicate This Thesis To My Parents, My Wife & My Kids! ii TABLE OF CONTENTS ACKNOWLEDGEMENTS.……………………………………………………….. i DEDICATION……………………………………………………………………… ii LIST OF FIGURES………………………………………………………………… vii LIST OF TABLES ……………………………………………………………….. x LIST OF ABBREVIATIONS .……………………………………………………. xi SUMMARY………………………………………………………………………….. xii 1. GENERAL INTRODUCTION………………………………………………….... 1 1.1 SILYBUM MARIANUM L: AN IMPORTANT MEDICINAL PLANT ………… 1 1.1.1 Silymarin the medicinally active ingredient ………………………………… 2 1.1.2 Conventional propagation methods …………………………………………. 5 1.1.3 Chemical inconsistencies in Silybum products ……………………………… 6 1.1.4 Plant in vitro techniques for mass propagation ……………………………… 7 1.1.4.1 In vitro seed germination ………………………………………………… 7 1.1.4.2 Explant type and choice …………………………………………………. 8 1.1.4.3 In vitro Regeneration …………………………………………….............. 9 1.1.4.3.1 Shoot organogenesis …………………………………………………. 9 1.1.4.3.2 Somatic embryogenesis ……………………………………………… 10 1.1.4.3.3 Role of Auxins and Cytokinins in S. marianum regeneration……….. 10 1.1.4.3.4 Thidiazuron a multifaceted plant growth regulator ………………….. 11 1.1.4.3.5 Dark-light cycling and in vitro culture …………………………......... 11 1.1.4.3.6 Root organogenesis ………………………………………………... 12 1.1.4.3.7 Acclimatization and ex vitro transfer ……………............................. 12 1.1.5 In vitro cultures and biochemical charaterization ………………………...... 15 1.1.5.1 DPPH free radical scavenging activity ………………………………… 15 1.1.5.2 In vitro cultures and anti-oxidative enzyme activities …………………. 16 1.1.5.3 PAL and silymarin biosynthesis………………………………………... 17 1.1.5.4 In vitro cultures and quality control …………………………………….. 19 1.1.5.5 Analysis of metabolic changes during in vitro morphogenesis ………… 20 1.1.6 Objectives …………………………………………………………………..... 22 iii 2. EFFECTS OF LIGHT REGIMES ON IN-VITRO SEED GERMINATION AND 23 SILYMARIN CONTENT IN SILYBUM MARIANUM L………………………….. 2.1 ABSTRACT……………………………………………………………………...... 23 2.2 INTRODUCTION………………………………………………............................ 24 2.3 MATERIALS AND METHODS………………………………………………......……………………... 25 2.3.1 Seed germination………………………………………………………………. 25 2.3.2 Analytical methods…………………………………………………………... 26 2.3.2.1 Antioxidant enzyme activities……………………………………………… 26 2.3.2.2 Assay of PAL activity………....................................................................... 27 2.3.2.3 DPPH- Free radical-scavenging activity (FRSA)………………………….. 27 2.3.2.4 Silymarin extraction and HPLC analysis…………………………………… 28 2.3.2.5 Statistical analysis……………………………………………………………….. 28 2.4 RESULTS AND DISCUSSION………………………………………………………… 29 2.4.1 Seed germination frequency and growth parameters ………………………….. 29 2.4.2 Antioxidant enzymes activity…………………………………………………… 36 2.4.3 Silymarin content, phenylalanine ammonia lyase activity and radical scavenging 38 activity……………………………………………………………………………………. 3. THIDIAZURON-INDUCED REGENERATION AND SILYMARIN CONTENT 41 IN SILYBUM MARIANUM L…………………………………………… 3.1 ABSTRACT……………………………………………………………………....... 41 3.2 INTRODUCTION………………………………………………………………….. 42 3.3 MATERIALS AND METHODS…………………………………………………… 43 3.3.1 Micro propagation………………………………………………………………. 43 3.3.1.1 Seed germination……………………………………………………………. 43 3.3.1.2 Plant growth regulators for regeneration……………………………………. 43 3.3.1.3 Silymarin assesment by HPLC ………………………………....................... 44 3.3.1 Statistical analysis………………………………………………………………. 44 3.4 RESULTS AND DISCUSSION……………………........................................................ 45 3.4.1 Callus induction frequency …………………………………………………….. 45 3.4.2 Shoot organogenesis …………………………………………………………… 47 3.4.3 Rooting and acclimatization …………………………………………………… 52 iv 3.4.4 Silymarin content ………………………………………………………………. 53 4. ESTABLISHMENT OF AN EFFICIENT ROTOCOL FOR SOMATICE MBRYOGENESIS IN SILYBUM MARIANUM L…………………. 56 4.1 ABSTRACT……………………………………………………………………..... 56 4.2 INTRODUCTION………………………………………………………………… 57 4.3 MATERIAL AND METHODS…………………………………………………… 58 4.3.1 Plant material………………………………………………………………….. 58 4.3.1.2 Establishment of embryogenic callus……………………………………… 58 4.3.1.3 Development of somatic embryos…………………………………………. 58 4.3.1.4 Germination of somatic embryos into plantlets……………………………. 59 4.3.1.5 Histological study…………………………………………......................... 59 4.3.2 Statistical Analysis……………………………………………………………. 60 4.4 RESULTS AND DISCUSSION………………………………………………….. 61 4.4.1 Induction of embryogenic callus……………………………………………… 61 4.4.2 Somatic embryo development……………………………………………………. 62 4.4.3 Embryo germination and conversion into plantlet……………………………. 65 5. TEMPORAL VARIATIONS IN METABOLITE PROFILES AT DIFFERENT GROWTH PHASES DURING SOMATIC EMBRYOGENESIS OF SILYBUM 68 MARIANUM L……………………………………………………………………….. 5.1 ABSTRACT…………………………………………………………………….... 68 5.2 INTRODUCTION……………………………………………………………….. 69 5.3 MATERIAL AND METHODS………………………………………………….. 69 5.3.1 Plant samples…………………………………………………………………. 69 5.3.2 Biochemical characterization............................................................................ 70 5.3.3 Metabolite profiling………………………………………………………….. 70 5.3.3.1 Extraction of Metabolites………………………………………………….. 70 5.3.3.2 Electrospray ionisation time of flight mass spectrometry (ESI-TOF MS)… 70 5.3.3.3 Data processing……………………………………………………………. 71 5.3.3.4 Putative metabolite identification …………………………………………. 72 5.3.4 Statistical Analysis…………………………………………………………… 72 5.4 RESULTS AND DISCUSSION…………………………………………………. 72 v 5.4.1Phenylalanine ammonia lyase activity, antioxidant potential 72 and silymarin content…………………………………………………………. 5.4.2 Metabolite profiling…………………………………………………………… 74 5.4.2.1 Mass spectra…………………………………………………………………. 74 5.4.2.2 Principal Component Analysis (PCA)………………………………………. 77 5.4.2.3 Evaluation of Putatively known metabolites ……………………………….. 82 5.4.2.3.1 Bin masses………………………………………………………………… 82 5.4.2.3.2
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