IN VITRO MICROPROPAGATION AND MOLECULAR STUDIES OF ABUTILON SEPALUM HUS. & BAQ. AND PULICARIA BOISSIERI HOOK. F. (ENDEMIC PLANTS OF PAKISTAN) SHAZIA MANSURI Thesis submitted for the fulfilment of the degree of DOCTOR OF PHILOSOPHY DEPARTMENT OF BOTANY, UNIVERSITY OF KARACHI, KARACHI-75270, PAKISTAN 2016 ii Dedication It is an immense pleasure for me to present this thesis to my beloved parents Mr.& Mrs.Muhammad Farooq Mansuri iii ACKNOWLEDGEMENT All the commendations to ALLAH Subhanahu wa ta'ala for all His countless blessings and provision of courage in every part of my life and to accomplish my Ph.D. Countless Durood to beloved Holy Prophet Hazrat Muhammad (Salal-laho-alaihe-wa’aalehe-Wassallam), who enlightened conscience with the essence of faith in Allah. It is a great pleasure for me to pay my regards to those who made this thesis possible. I am immensely thankful to my supervisor Prof. Dr. Anjum Perveen (Director, Centre for Plant Conservation), for her guidance, co-operation and the facilities provided throughout my thesis. Her knowledge and vision of investigation gave me inspiration to complete my research. I am deeply obliged to my co supervisor Prof. Dr. Saifullah Khan (Professor, Department of Agriculture, University of Karachi) for his enormous support, thoughtful intelligence, encouragement and motivation which inspires and helped me in several occasions during the course of my research. It is honour to me to thank Prof. Dr. Ehtesham ul Haq, Chairman, Department of Botany, University of Karachi for all his encouragements, devoted instructions, help and provision of Lab facility of the department. I also want to thank Prof. Dr. Syed Afrozuddin Ahmed, Dean, Faculty of Science for providing funds for the research. I want to acknowledge Prof. Dr. Irtifaq Ali (Prof. Emeritus, KIBGE), and Prof. Dr. Muhammad Qaiser (Vice Chancellor, University of Karachi) for their support during my studies. My cordial appreciation goes to Dr. Sadia Gilani, Assistant Professor KIBGE, University of Karachi, for all her support, advice and cooperation during my thesis especially in protein analysis and electrophoresis. I would like to oblige to my friends Mrs. Tabassum Sabir, Ms. Kehkashan Kazmi, Ms. Huma Rao, and all Lab seniors and juniors from the Biotechnolgy wing (ICCBS, University of Karachi) for their support and help throughout my research. I am thankful to lab technicians Mr. Imran, Mr. Zafar and Mr. Abdul Ghaffar for their cooperation during my research. My sincere thanks and deeply regards to all my friends specially Dr. Muneeba Nadir and Ms. Shaista Zeb for all their encouragements, support, help and cooperation during my research and my daily life I am also thankful to Dr. Nadir for his support and services in completion of my research. Without them my work would not have been easily possible. I am thankful and pay my heartily gratitude to my parents Mr.& Mrs Muhammad Farooq Mansuri, My brothers (Fahad Mansuri, Fahim Mansuri and Fazal Mansuri), Bhabhis and my naughtiest, cutest and adorable nephew Hamza Fahad Mansuri for all their love, support and efforts during my research and in my life. SHAZIA MANSURI iv CONTENTS Content Page No. SUMMARY xvi Chapter 1: In Vitro Micropropagation and Molecular Studies of Abutilon sepalum Hus. & Baq. And Pulicaria boissieri Hook.f. (Endemic plants of Pakistan) 1.1 Introduction 1-17 1.1.1 Plant Biodiversity 1-2 1.1.2 Endemism 2-6 1.1.3 Conservation 6-11 1.1.4 Molecular Studies 11-15 1.1.5 Region of research 15 1.1.6 Selection of Endemic Plants 15-16 1.2 Materials and Methods 18-31 1.2.1 In Vitro Culture 18-23 1.2.2 Molecular Assessment 23-31 1.2.2.1 Protein Analysis 23-26 1.2.2.2 Genetic Diversity on the Basis of Molecular Traits 26-31 1.3 Results and Observations 32-92 1.3.1 Abutilon sepalum Hus. & Baq. 32-66 1.3.1.1 Conservation of plant by micropropagation 36-55 (a) Initiation 36 v (b) Multiplication 36 (c) Rooting 46 (d) Acclimatization 46 Content Page No. (e) Callus formation of Abutilon sepalum 46 (f) Shoot regeneration from callus 46-47 1.3.1.2 Molecular Analysis 56-66 (a) SDS PAGE 56 (b) DNA Polymorphism among Abutilon species 57-66 1.3.2 Pulicaria boissieri Hook.f. 67-92 1.3.2.1 Plant conservation by micropropagation 71-86 (a) Initiation 71 (b) Multiplication 71 (c) Rooting 71 (d) Acclimatization 72 (e) Callus formation 72 (f) Callus regeneration 72 1.3.2.2 Molecular Analysis 87-92 (a) SDS PAGE 87 (b) DNA Polymorphism among Pulicaria species 88-92 vi 1.4 Discussion 93-103 1.4.1 Abutilon sepalum Hus. & Baq. 94-99 (a) Conservation by Micropropagation 94-97 (b) Protein analysis by electrophoresis 97-98 (c) DNA Electrophoresis 98-99 1.4.2 Pulicaria boissieri 99-103 Content Page No. (a) Conservation by Tissue culture 99-101 (b) Fragmentation of Protein by SDS PAGE 101-102 (c) Investigation of DNA Electrophoresis by RAPD Markers 102-103 Chaper 2: Pharmacognostic Assessment 2.1 Introduction 104-106 2.2 Materials and Methods 107-111 2.2.1 Macroscopic or Organoleptic Assessment 107 2.2.2 Physicochemical Analysis 107-109 2.2.3 Fluorescence Analysis 109 2.2.4 Phytochemical Analysis 109-111 2.3 Results and Observations 112-122 2.3.1 Abutilon sepalum Hus & Baq. 112-117 (a) Morphology or Organoleptic Assessment 112 vii (b) Physicochemical parameters 112 (c) Fluorescence Analysis 112 (d) Phytochemical Testing 112 2.3.2 Pulicaria boissieri Hook. f. 118-122 (a) Morphology or Organoleptic Assessment 118 (b) Physicochemical parameters 118 (c) Fluorescence Analysis 118 (d) Phytochemical Testing 118 2.4 Discussion 123-126 Content Page No. 2.4.1 Abutilon sepalum Hus.& baq. 123-124 (a) Morphological Attributes 123 (b) Physicochemical Assessment 123 (c) Qualitative Phytochemical Analysis 124 (d) Quantitative Phytochemical Analysis 124 2.4.2 Pulicaria boissieri Hook.f. 124-126 (a) Morphology 124 (b) Physicochemical Assessment 125 (c) Qualitative Phytochemical Assessment 125 (d) Quantitative Phytochemical Analysis 126 viii Conclusion 126-127 References 128-158 Appendices I MS Media Composition 159 II PBS (Phosphate Buffer Saline Composition) 160 III DNA Extraction Buffer 160 IV Washing buffer 161 V TE buffer or Resuspension buffer 161 VI TBE Buffer or Running Buffer 5x 161 VII TBE Buffer or Running Buffer 1x 162 VIII List of Abbreviations 163-164 IX List of Publications 165 LIST OF FIGURES S. No. Title Page No 1 Herbarium sheet of Abutilon sepalum Hus. & Baq. submitted to 19 KUH 2 Herbarium sheet of Pulicaria boissieri Hook.f. submitted to KUH 20 3 Thermal cycler for PCR 28 4 A twig of Abutilon sepalum 33 5 Competition of A. sepalum with other species for survival 35 6 Striking of Insects to Abutilon sepalum 35 ix 7 Initiation of Abutilon sepalum Hus. & Baq. On medium ½ MS + 0.1 39 % Charcoal 8 Germination of seeds on ½ MS medium + 0.06 % charcoal 39 9 Effect of BAP on number of Shoots of Abutilon sepalum 40 10 Effect of BAP on Shoot Length of A. sepalum 40 11 Multiplication of Abutilon sepalum in 1mg/L BAP 41 12 Multiple shoot formation of A. sepalum in medium containing 1 mg/L 41 BAP 13 Effect of Kinetin on number of shoots of A. sepalum 43 14 Effect of Kinetin on shoot length of A. sepalum 42 15 Multiplication of A. sepalum in 1mg/L Kinetin 44 16 Comparison of BAP and KIN on number of shoots of A. sepalum 45 17 Comparison of BAP and KIN on shoot length of A. sepalum 45 18 Development of roots of A. sepalum in ½ MS medium containing 49 0.5 mg/L NAA S. No. Title Page No 19 Rooting of A. sepalum in ½ MS medium supplemented with 0.5 mg/L 49 NAA + 0.06% charcoal 20 Comparison of various phytohormones of root development of 50 Abutilon sepalum 21 Acclimatization of Abutilon sepalum 51 x 22 Initiation of A. sepalum for callus formation 54 23 Callus formation of Abutilon sepalum in medium containing 2 mg/L 54 2,4D 24 Callus formation of Abutilon sepalum in MS medium in addition with 55 1mg/L BAP 25 Regeneration of Shoots from Callus of Abutilon sepalum containing 55 MS medium with 1mg/L BAP + 0.1 NAA 26 Polymorphic bands of Protein of 6 species of Abutilon on SDS 56 PAGE 27 Bands of DNA by RAPD primer OPA 1 of Abutilon species 59 28 Different bands by RAPD primer OPA 4 of Abutilon species 59 29 Several bands by primer OPA 10 of Abutilon species 60 30 Bands formed by OPB 4 of Abutilon species 60 31 Polymorphic bands by OPB 7 of Abutilon species 61 32 Bands developed by primer OPL 1 of Abutilon species 61 33 Different bands development by primer LC 87 of Abutilon species 62 34 Bands formed by primer LC 90 of Abutilon species 62 35 Various bands by primer LC 77 of Abutilon species 63 36 Bands produced by primer LC 83 of Abutilon species 63 S. No. Title Page No 37 Different bands by primer LC 76 of Abutilon species 64 xi 38 Phylogenetic tree of Abutilon species 65 39 Study area Pulicaria boissieri Hook.f. 68 40 Morphology of Pulicaria boissieri Hook.f. 68 41 Human induced intervention in the study area 70 42 Initiation of P. boissieri in medium containing 1 mg/L BAP 74 43 Initiation of P. boissieri in medium containing 1 mg/L KIN 74 44 Effect of BAP and KIN on number of shoots developed in P. 75 boissieri 45 Effect of BAP and KIN on number of leaves developed in P.