Morpho-Genetic Variation Analysis and in Vitro Propagation of Rosa Centifolia
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MORPHO-GENETIC VARIATION ANALYSIS AND IN VITRO PROPAGATION OF ROSA CENTIFOLIA By GULZAR AKHTAR M. Sc. (Hons.) Horticulture 2003-ag-1849 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN HORTICULTURE INSTITUTE OF HORTICULTURAL SCIENCES, FACULTY OF AGRICULTURE, UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN 2014 In The Name Of The Most Beneficent The Most Merciful Declaration I hereby declare that the contents of the thesis “Morpho-Genetic Variation analysis and in vitro propagation of Rosa centifolia” are the product of my own research and no part has been occupied from any published sources (except the references, standard mathematical or genetic model/ equations/formula/ protocol etc). I further declare that this work has not been submitted for the award of any other diploma/degree. The university may take action if the information provided is found inaccurate at any stage. In case of any default, the scholar will be proceeded against as per HEC plagiarism policy. ________________ GULZAR AKHTAR 2003-ag-1849 M.Sc. (Hons) Horticulture To The Controller of Examinations, University of Agriculture, Faisalabad. “We, the members of supervisory committee, certify that the contents and form of thesis submitted by Mr. Gulzar Akhtar, 2003-ag-1849, have been found satisfactory and recommend that it be processed for evaluation, by the External Examiner(s) for the award of degree” Supervisory Committee 1. Chairman _______________________ (Dr. Muhammad Aslam Khan) 2. Member _______________________ (Dr. Muhammad Jafar Jaskani) 3. Member ________________________ (Dr. Muhammad Ashfaq) This humble effort is Dedicated To My Father and Mother By virtue of whose prayers I have been able to reach at this position ACKNOWLEDGEMENTS All humble praise and thanks for ALMIGHTY ALLAH who is the most kindly Merciful and ﺳﻠّﻢ و ﻟﮭ آ و ﻋﻠﯿﮭ ) (Durood e Pak on the Holy Prophet, Muhammad (Peace Be Upon Him .who is a Mercy and source of guidance for all humanity ( ا یﺻﻞ I have been indebted in the research work and preparation of this thesis to my supervisor, Dr. Muhammad Aslam Khan, Professor, Institute of Horticultural Sciences (IHS), whose kindness and academic experience have been invaluable to me. I am extremely grateful to Dr. Muhammad Jafar Jaskani, Professor IHS and Dr. Muhammad Ashfaq, Professor, Department of Plant Breeding and Genetics who always kept an eye on my work as member of my supervisory committee. I would like to thank Dr. Adnan Younis, Dr. Atif Riaz and Dr. Iftikhar Ahmad Assistant Professor HIS, for their help and guidance throughout my research work and thesis write-up. I also need to express my deeply-felt thanks to Dr. Muhammad Aslam Pervez, Director IHS and Dr. M. Qasim, Professor IHS, Professor IHS, for their warm encouragement and thoughtful guidance. How can I forget my Co-supervisor, Prof. Dr. David H. Byrne and Natalie Anderson (Research Associate) for their guidance and cooperation during my work on Microsatellites at Department of Horticultural Sciences, Texas A & M University, Texas, USA. I also acknowledge with thanks financial support from The Higher Education Commission (HEC) of Pakistan for giving me opportunity of Indigenous 5000 Fellowship scholarship and funds for International Research Support Initiative Program (IRSIP) to conduct research at Texas A & M University, Texas, USA as a visiting scientist. The informal support and encouragement of many friends has been indispensable, and I would like particularly to acknowledge the contribution of Mr. Ahsan Akram, Dr. Muhammad Nadeem, Mr. Yasir Sajad, Mr. Asim Mehmood, Dr. Abdul Kareem, Mr. Faheem Nawaz, Mr. Manan Shah, Mr. Adnan Bukhari, Mr. Muhammad Rashid Abbasi, Mr. Imran Muaavia, Mr. Muhammad Shafique, Mr. Ockert, Tim Hartman, Suheb Mohammed, Qianni Dong, Mr. Jake, Sabeeh Ahmad and (Texas A&M University, College Station, TX). My father, mother and Taya jaan have been a constant source of support, emotional, moral and of course financial and this thesis would certainly not have existed without them. It is thanks to my uncle who encouraged and supported me to get into PhD but he left me forever when I was doing my research work in Texas A & M University, USA. May Allah keeps his soul in peace (Ameen). I pay my respect to my beloved brothers Shahzad Akhtar, Shahbaz Akhtar, Waqar Akhtar, Sadam Hussian my cusion Muhammad Nadeem, Muhammad Waseem, Muhammad Kaleem my loving, sincere and well wisher sister for the sacred prayers and endless kind behavior. May Allah bless my parents and family members with good health and prosperity. Gulzar Akhtar C O N T E N T S Chapter Title Page 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 7 2.1 Morphological Analysis 7 2.2 Molecular Analysis 9 2.3 In vitro propagation 11 3 MATERIALS AND METHODS 18 3.1 Phenotypic plasticity among the genotypes of Rosa 18 centifolia 3.1.1 Climate of Pakistan 18 3.1.2 Areas selected for morphological study 18 3.1.3 Layout of experiment 21 3.1.3.1 Genotypes of Rosa centifolia from Pakistan 21 3.1.3.2 Genotypes of Rosa centifolia from USA 21 3.1.4 Morphological Parameters 21 3.1.4.1 Plant parameters 22 3.1.4.2 Leaf parameters 23 3.1.4.3 Flower parameters 25 3.1.5 Statistical Analysis 26 3.2 Genetic diversity among genotypes of Rosa centifolia 27 3.2.1 Plant material from Pakistan 27 3.2.2 Plant material from USA 27 3.2.3 Rosa damascena samples 27 3.2.4 Primers 29 3.2.5 DNA extraction protocol 29 3.2.6 DNA Quantification 30 3.2.7 PCR (Polymerase Chain Reaction) Amplification 30 3.2.8 Gel electrophoresis 30 3.2.9 Data analysis 31 3.3 IN VITRO PROPAGATION OF ROSA CENTIFOLIA 31 3.3.1 Plant Material 31 3.3.2 Media 31 3.3.3 Preparation of stock solutions 31 3.3.4 Stock solutions for growth regulator 32 3.3.5 Preparation of media 32 3.3.6 Sterilization of explant 33 3.3.7 Inoculation 33 3.3.8 Sub culture 34 3.3.9 Hardening 34 3.3.10 Collection of Data 34 3.3.10.1 Number of days for shoot initiation 34 3.3.10.2 Number of shoots 34 3.3.10.3 Number of leaves per shoot 34 3.3.10.4 Shoot length 34 3.3.10.5 Number of days to initiate roots 35 3.3.10.6 Number of roots 35 3.3.10.7 Root length 35 3.3.11 Data analysis 35 4 RESULTS 36 4.1 Phenotypic plasticity among the genotypes of Rosa 36 centifolia 4.1.1 Cluster analysis for different morphological characters of 36 genotypes of Rosa centifolia from Pakistan 4.1.2 Principle component analysis for different morphological 36 characters of genotypes of Rosa centifolia 4.1.3 Correlation between morphological parameters of Rosa 36 centifolia genotypes from Pakistan 4.1.4 Cluster analysis for different morphological characters of 40 different varieties of Rosa centifolia from USA 4.1.5 Principle component analysis for different morphological 40 characters of different varieties of Rosa centifolia from USA 4.1.6 Correlation between morphological parameters of Rosa 40 centifolia varieties from USA 4.1.7 Cluster analysis for different morphological characters of 44 different varieties of Rosa centifolia from Pakistan and USA 4.2 Genetic diversity among genotypes of Rosa centifolia 48 4.2.1 Genetic analysis of Rosa centifolia from Pakistan 48 4.2.2 Genetic analysis of Rosa centifolia from Pakistan and USA 53 4.2.3 Genetic analysis of Rosa centifolia and Rosa damascena from 59 Pakistan, USA and Iran 4.3 In vitro propagation of Rosa centifolia through axillary 64 buds 4.3.1 Number of days to initiate shoots 64 4.3.2 Number of shoots 64 4.3.3 Shoot length 65 4.3.4 Number of leaves 65 4.3.5 Number of days to induce roots 65 4.3.6 Number of roots 65 4.3.7 Root length 65 5 DISCUSSION 75 5.1 Phenotypic plasticity among the genotypes of Rosa 75 centifolia 5.2 Genetic diversity among genotypes of Rosa centifolia 77 5.2.1 Genetic analysis of different genotypes of Rosa centifolia from 77 Pakistan 5.2.2 Genetic analysis of different genotypes of Rosa centifolia from 78 Pakistan and USA 5.2.3 Genetic analysis of different genotypes of Rosa centifolia and 79 Rosa damascena from Pakistan, USA and Iran 5.3 In vitro propagation of Rosa centifolia through axillary 80 buds 5.3.1 In vitro shoot formation of Rosa centifolia 80 5.3.2 In vitro root induction of Rosa centifolia 82 SUMMARY 84 LITARATURE CITED 87 List of Tables No. Title Page 3.1 Different site of data collection Punjab, Pakistan 19 3.2 Analysis of water samples collected from different sites of Punjab, 19 Pakistan 3.3 Analysis of soil samples collected from different sites of Punjab, 19 Pakistan 3.4 Roses genotypes used for SSR analysis 28 3.5 List of primers 29 3.6 Ingredients of MS media 32 3.7 Treatments for shoot formation 33 3.8 Treatments for root formation 33 4.1 Correlation between morphological parameters of Rosa centifolia 39 genotypes from Pakistan 4.2 Correlation between morphological parameters of Rosa centifolia 43 genotypes from USA 4.3 Grouping of 24 genotypes of Rosa centifolia on the bases of 53 microsatellite markers 4.4 Grouping of 10 genotypes of Rosa centifolia on the bases of 59 microsatellite markers 4.5 Grouping of 14 genotypes of Rosa centifolia and Rosa damascena 63 on the bases of microsatellite markers List of Figures No.