In vitro studies on growth and morphogenesis of some potential medicinal plants through tissue culture DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF iRas^ter of ^fjilosiopbp 31n ^ iBotanp ^ /•# L By SHEIKH ALTAF HUSSAIN DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH -202002 (INDIA) 2014 2 4 NOV 201i DS4373 m f^>^a(pra6le &ra4ferUA -ccHTK^^O^ ^i <Prof. (<Dr.)!MofiamanufAnts Plant Biotechnlogy Lab., PhD(Luck.), FISPM, FISG, FBS Department of Botany Former Chairman & Program Coordinator Aljgarh Muslim University DST-FISTII (2011-16) Aligarh-202002, U.P. (India) UGC-DRSI (2009-14) E-mail: [email protected] OBT-HRD (2009-14 Cell: +91-9837305566 DST-FISTI (2006-10) . (Set.6A,2oA^ Visiting Professor, KSU, Riyadh Dated: P. Maheshwari Medal Awardee Certificate Certified that the dissertation entitled "In vitro studies on growth and morphogenesis of some potential medicinal plants through tissue culture" submitted to the Aligarh Muslim University, Aligarh, in fulfillment of the requirement for the award of Master of Philosophy, embodies the original research work carried out by Mr. Sheikh Altaf Hussain under my guidance and supervision. The work has not been submitted elsewhere in part or full for the award of any other degree of this or any other university. (ProfessorJAoham^^FAms) ^ - "^^^--"^ap^fvfsor ^ X ) 4 Tel. Off: 0571-27020]6: 2700920, 21, 22 (Ext. 3308). Home: 07. Rainbow Roofs-I. Anoopshahar Road. Aligarh -202 122. UP (India) CONTENTS Acknowledgements Abbreviations CHAPTER 1: INTRODUCTION 1-10 1.1. Micropropagation 1.2. Micropropagation ofR. tetraphylla and R.serpentina 1.3. Classification of ^. tetraphylla 1.3.1 Distribution 1.3.2 Botanical description 1.3.3 Characteristics and constituents 1.3.4 Medicinal importance 1.4. Classification of/?, serpentina 1.4.1 Distribution 1.4.2 Botanical description 1.4.3 Medicinal importance 1.5 Objectives CHAPTER 2: REVIEW OF LITERATURE 11-18 2.1 Historical background 2.2 Micropropagation 2.3 Explant type 2.4 Plant growth regulators 2.5 Subculturing 2.6 Rooting 2.7 Hardening and Acclimatization CHAPTER 3: MATERIALS AND METHODS 19-25 3.1 Plant material and explant sources 3.2 Surface sterilization of explants 3.3 Establishment and Preparation of aseptic explants 3.4 Culture media 3.5 Preparation of stock solution 3.6 Growth regulator and carbon source 3.7 Adjustment of pH and gelling of medium 3.8 Sterilization of glasswares and instruments 3.9 Sterilization of laminar airflow hood 3.10 Inoculation 3.11 Incubation 3.12 Subculturing 3.13 Rooting 3.14 Hardening and Acclimatization 3.15 Chemical and glasswares 3.16 Statistical analysis CHAPTER 4: RESULTS (with Tables & Figures) 26-36 4.1 Direct shoot regeneration of/?, tetraphylla 4.1.1. Effect of Cytokinins 4.1.2. Combined effect of Cytokinins and auxin 4.1.3. Effect of thidiazuron (TDZ) 4.1.4. Combined effect of TDZ and BA 4.1.5. Effect of metatopolin 4.1.6. Rooting of regenerated shoots 4.1.7. Acclimatization 4.2 Direct shoot regeneration in R. serpentina 4.2.1. Combined effect of cytokinin and auxin 4.2.2. Rooting 4.2.3. Acclimatization CHAPTERS: DISCUSSION 37-42 5.1. Direct shoot regeneration in R. tetraphylla 5.2. In vitro Rooting in regenerated shoots of R. tetraphylla 5.3. Direct shoot regeneration in R. serpentina. 5.4 In vitro Rooting in regenerated shoots of R. serpentina 5.5 Hardening and Acclimatization CHAPTER 6: SUMMARY AND CONCLUSIONS 43-44 REFERENCES 45-60 (praise 6e to JiClali, tfie cHerisHer ancf tHe sustainer of the worCd, 'Who Bestowed on me the divine guidance, enough courage and patience to successfuCCy compete thu worH^ It 6rings me coCossaC pCeasure while placing on record my sincere gratitude and reverence to my esteemed supervisor (prof. ((Dr.) 'Mohammad J/lnis for his unparaCleC and sincere guidance, ^en interest, incessant encouragement, strong motivation, constructive criticism and idea oriented discussion, which has enabled me to accomplish this wor^ It is my proud privilege to express my sense of than^ulness to (prof. Tiroz Mohammad, Chairman (Department of (Botany, JUMV for providing necessary infrastructure and technical facilities during my wor^ I deem it a privilege to ey^ress my sincere gratitude to (Dr. Jlnwar Shazad(Jlssistant (professor) and (Dr. ^aseem Jlhmad ('Young scientist 0)31) for all time support, encouragement, motivation and suggestions all through my wor^ I am highly appreciative of the support, encouragement and fruitful suggestions expended all through the course 6y my seniors and laB-mates especially (Dr. J^n({ita varsheny (Young scientist (DST), ^r. S^igar Tatima, Ms. jAfshan Jiaaz, Ms. (Rjuphi 'Xaz, Saad (Bin Javid, Md (Rafique Jlhmed, Ms. Jifsheen Shaid, J^aushad jAlam, Jlness Jlhmad and Ms. Jltjumund Shaeen. JL to^n of appreciation will always remain for (Dr. JVaseem JLhmadfor his support cooperation in creating a congenial, amicadle and friendly atmosphere in the la6. M this inexplicahk moment of joy, I deem it a privilege to thanl^ all my friends especially Manzoor (Raiees %han, Jiddil(Rashid, Imran Mustafa Mali^ Jl6. Latif "Wani, Sheilih Mohammad jAsif Kjianday Tanveer, Mir (Rayees, (Pervaiz J4hmad, SheiUJi (Rameez Jlli and(Bilaljlhmadfor their incessant motivation, encouragement and support throughout the wor^ I extend my special than^ to (Dr Manzoor J^hmadfor his sincere help, advice and support during the wor^ I feeC -pCeasure to pay su6Cime oSeisance ancCgratitude to my reverecf ancC Bethvecf parents, ivdose e^cjuisite Benedictions, unconditionaC Cove and affection, unfCincHing care and support, prayers and 6(essings right from tHe cradCe ignited my confidence to exceC academicaCCy andfufiCC my wisfies. (Dedicating this wor^ to my deCoved parents, I apohgize to them for Searing with aCC the faiCures in commitment on my part, arising out of Susy worli^schedufe during my studies. 94.y heartfeCt devotion is due to my sisters who extended uCtimate cooperation and every possiSCe support aCC through my studies. I may 6e ungratefuC without than^ng aCCmy relatives for their affection, support and encouragement throughout my studies. Last 5ut not the [east, I wish to offer my than^ to aCCthose names which couhdnot 6e incCudedhut wiCCaCways hefondCy rememhered. (Sheikh ACtaf^Hussain) 11 ABBREVIATIONS 2-iP 2-isopentyl adenine 2, 4-D 2, 4-dichlorophenyoxy acetic acid BA 6-benzyladenine CH Casein hydrolysate cm Centimeter DDW Double distilled water EDTA Ethylene diamine tetra acetic acid g Gram GA3 Gibberellic acid CM Coconut milk h Hour HCl Hydrochloric acid lAA Indole-3-acetic acid IBA Indole-3-butyric acid Kin Kinetin mT metatopolin |iM Micromolar mg Milligram mg 1 • Milligram per liter min Minute ml Milliliter mM Millimolar MS Murashige and Skoog's medium N Normality NAA a-napthalene acetic acid NaOH Sodium hydroxide PABA Para- amino benzoic acid PGRs Plant growth regulators TDZ Thidiazuron UV Ultraviolet v/v Volume by volume w/v Weight by volume % Percentage C Degree celsius ^/udftet^ - / 1^ Introuction Introduction Biodiversity is the store house of species richness and serves as the cushion against the potentially dangerous environmental changes and economic reforms (Yadav et ui. 2012). It encompasses ail the biological entities that occurs due to their dynamic interactions among different levels of integration within the living world, in an ecosystem and in their habitat. Biodiversity of plants is collectively known as plant genetic resources, and serves as the major biological basis of the world food securit> and in all means they support the livelihoods of every life form on earth (Alexander ei ai. 2011). India with wide spectrum of phytogeographic and agro-climatic zones is blessed with rich diversity of plants. Plants form the key element in maintaining the biodiversity on this planet Earth. Plants are endowed with the unique capacity to convert solar energy into chemical energy and synthesize thousands of chemical substances which are very important to our lives. India has very rich plant diversity and is one of the 12 mega biodiversity hot spot centers of the world with 4"^ rank in Asia. India represents about 12% of the world s recorded flora with 49,000 plant species, among them 20,000 species belongs to higher plants, one third of it being endemic and 1000 species being medicinally important (Krishnan et al., 2011; Singh et ai, 2003). At least 40% of the world economy and 80% of the needs of the poor people are derived from biological resources, in addition, the richer the diversity of life, greater the opportunity for medicinal discoveries, economic development and adaptive responses to such new challenges as climate change. Since the beginning of human civilization, medicinal plants have been used virtual!) in all social cultures for its therapeutic value. Nature has been a source of medicinal assets from prehistoric times and an impressive number of modem and valuable drugs have been isolated from these natural sources. Many of these isolations were based on the uses of agents in herbal medicine. The plant-based traditional medicine system continues to play an essential role in health care, with about 80% of the world s inhabitants relying mainly on traditional, largely on herbal medicine for their primar) health care. The ancient texts like Rig veda, and Atharva Veda mention the use of several plants as medicine. The books on ayurvedic medicine such as Charaka Samhita and Susruta Samhita refer to the use of more than 700 herbs (Jain, 1968). Introtiction According to WHO, 80% of world population and 65% of Indian rural population uses traditional form of medicine to meet their primary health care needs (Jain and Singh. 2010), because they are easily available, non narcotic and also having no side effects. India is called the botanical garden of the world because it houses the largest number of the medicinal plants and acts as the gold mine, serves as the well recorded and most practiced knowledge of herbal medicine.