ASSESSMENT OF DIVERSITY AND IN VITRO RESPONSES IN PINUS ROXBURGHII FROM INDIAN PROVENANCES A THESIS SUBMITTED TO THE UNIVERSITY OF PUNE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY By VARSHA PARASHARAMI RESEARCH GUIDE DR. (MRS.) S.R. THENGANE PLANT TISSUE CULTURE DIVISION, NATIONAL CHEMICAL LABORATORY PUNE-411008 JANUARY, 2011 Dedicated to My Mother ii CONTENTS Page No ACKNOWLEDGEMENTS v CERTIFICATE vi DECLARATION vii KEY TO ABBREVIATIONS viii ABSTRACT x‐xviii CHAPTER 1: INTRODUCTION 1‐39 1.1 Introduction 1.2 Diversity in Chir Pine 1.3 In Vitro studies 1.4 Micrografting technique in Pines CHAPTER 2: MATERIALS AND METHODS 40‐59 2.1 Introduction 2.2 Genetic Diversity Studies 2.3 Plant Tissue Culture 2.4 Micrografting technique ASSESSMENT OF DIVERSITY IN PINUS ROXBURGHII FROM DIFFERENT PROVENANCES USING ISSR MARKERS 60‐120 3.1 Introduction 3.2 Materials and Methods 3.3 Results 3.4 Discussion 3.5 Conclusions of the Diversity Analysis in P. roxburghii CHAPTER 4: IN VITRO RESPONSES IN PINUS ROXBURGHII USING MATURE SHOOTS FROM DIFFERENT PROVENANCES 123‐171 4.1 Introduction 4.2 Materials & Methods 4.3 Results 4.4 Discussion CHAPTER 5: THE MORPHOGENENTIC RESPONSE AND DEVELOPMENT OF A MICROPROPAGATION PROTOCOL FOR P.ROXBURGHII USING MATURE ZYGOTIC EMBRYOS(MZE) 172‐210 iii 5.1 Introduction 5.2 Materials & Methods 5.3 Results 5.4 Discussion CHAPTER 6: DEVELOPMENT OF IN VITRO AND IN VIVO MICROGRAFTING TECHNIQUE IN P.ROXBURGHII 211‐233 6.1 Introduction 6.2 Material & Methods 6.3 Results 6.4 Discussion SUMMARY AND CONCLUSIONS 234‐241 BIBLIOGRAPHY 242‐266 iv ACKNOWLEDGEMENT I would like to take this opportunity to express my gratitude to all those who have helped in their own ways in the successful completion of this Ph.D. thesis. I would like to express my deep sense of gratitude to my research guide, Dr. (Mrs.) S.R. Thengane, Plant Tissue Culture Division, National Chemical Laboratory, Pune, for her excellent guidance, constant encouragement, sincere advice and unstinted support during my Ph.D. work. Her guidance and motivation abilities goaded me into working harder and putting in my best. I am grateful to Dr. B.M. Khan, Head, Plant Tissue Culture Division, National Chemical Laboratory for his constant support. I take this opportunity to thank Prof. R.J. Thengane, Department of Botany, University of Pune, for the useful suggestions in the improvement of this work. My special thanks to my colleagues Dr. S.K. Rawal, Dr C. H Phadke, Dr. D.C Agrawal, Dr. U.J. Mehta, Dr. D.K. Kulkarni, Dr. A. V. Prabhune, Dr S Pundle, Dr C.K. John, Dr. M.M. Jana, Mrs S. V. Kendurkar and Mrs M. V Shirgurkar. I would also like to thank Dr. N. Kadoo, Dr.V.S.S. Prasad, Dr. B. Panda, Dr. K. Pawar, Dr.N.Singh, Dr. R. Pinjori, Dr.K.Dhotkar, Dr.S Deodhar, Ms V Dhawande, Mr Shivraj , Ms Poonam,Ms Rita, Ms Archana, Mr. Suganthval, Mr. D. Trivadi, Mr. Anandh, Mr. Usman Attar, Ms Richa and Ms Sujata for their timely help during my research work. I would like to acknowledge the help provided by the Maharashtra Forest Department during Gureghar research plot collection. Acknowledgements are also due to the Forest department of Himachal Pradesh, West Bengal and Andhra Pradesh for sanctioning due permissions. My heartfelt thanks to Dr. P. S Ahuja, Director IHBT and Dr. Anil Sood, Head, Biotechnology Department, IHBT, Palampur for all the help rendered to me during plant material collection. I am thankful to Dr Madhu Sharma and Mr Om Prakash, IHBT Palampur for their timely help. It is a pleasure to offer my thanks to my family and friends and all the well wishers who have supported me throughout. Finally, I would like to thank former Director Dr. Sivaram, present Director Dr. S. Pal and distinguished scientist Dr. B. D. Kulkarni, National Chemical Laboratory, for allowing me to submit my work in the form of a thesis. Date: 17.1.2011 (Mrs.Varsha Parasharami) Place: Pune v CERTIFICATE This is to certify that the work incorporated in the thesis, “Assessment of Diversity and In vitro Responses in Pinus roxburghii from Indian Provenances” submitted by Mrs. Varsha Parasharami, for the Degree of Doctor of Philosophy, was carried out by the candidate under my supervision in Plant Tissue Culture Division, National Chemical Laboratory, Pune – 411008, India. Such material as has been obtained from other sources has been duly acknowledged in the thesis. Dr. (Mrs.) S.R. Thengane (Research Supervisor) vi DECLARATION BY THE CANDIDATE I declare that the thesis entitled “Assessment of Diversity and In vitro Responses in Pinus roxburghii from Indian Provenances” Submitted by me for the degree of Doctor of Philosophy is the record of work carried out by me under the guidance of Dr. (Mrs.) S.R. Thengane and has not formed the basis for the award of any degree, diploma, associateship, fellowship, titles in this or any other university or other institute of higher learning. I further declare that the material obtained from other sources has been duly acknowledged in the thesis. 17th January, 2011 (Mrs.Varsha Parasharami) vii viii P.roxburghii Plantation along the road. ix ABSTRACT x ABSTRACT Rationale of the study: Pinus roxburghii Sargent, commonly known as ‘chir pine’ is the most important pine among the six indigenous pine species of Indian subcontinent and amongst the dominant species of the subtropical pine forests of India. The species has wide ecological and economic value. During phytosociological studies of this species in lesser Himalayan and Hindu Kush ranges it was observed that forests are unstable and degraded and would be vanished if not maintained properly (Siddique et.al., 2009). But in Nepal this species comprises 17% of total forest area. There it is widely planted by individual farmers and in government-managed forests and community forests (Jackson, 1994). In Nepal P.roxburghii has outnumbered all other species in afforestration programs in its natural zone of occurrence (Gauli et.al., 2009). Chir Pine is much valued for its timber and oleoresin. It occurs in the monsoon belt of outer Himalaya, from Northeastern part of Pakistan to Arunachal Pradesh in India at elevations varying from 450 to 2300 msl. The forest type of this species comes under – The Himalayan subtropical pine forests which are the largest in the Indo: Pacific regions (www.worldwildlife.org). It is found distributed over a long strip of 3,200 km between latitudes 260 N to 360 N and longitudes 710E to 930 E. The species is economically very important and is used for variety of purposes viz., timber for house construction, fuel wood extraction, charcoal formation, resin tapping, needles for fuel briquetting, cattle bedding and manufacturing organic manures etc (Ghidiyal et.al., 2009). As rightly said every part of the Chir pine from the needle to the bark can be used for commercial purposes. An understanding of the degree of diversity within and between the populations of a species is required to support the continuously rising plantation programs and to provide information for the conservation of the species’ genetic resources since geographically separated populations are expected to have different genetic compositions (Sharma et.al., 2002). Genetic diversity is of prime importance for the species’ persistence (Wang and Szmidt, 2001), since the evolutionary xi adaptive potential of populations depends on genetic variation patterns (Siregar, 2000, Finkeldey and Hattemer, 2007). Within each tree species, amount and distribution of genetic variation is crucial for long term stability of forest ecosystems (Sharma, et.al., 2002). Genetic structures of numerous plant populations have been altered by humans for many generations (Finkeldey and Hattemer, 2007).This applies particularly to the tree species used for the establishment of plantations. Genetic structures of plantations are assumed to strongly differ from natural strands. They exhibit reduced levels of genetic variations and, hence, reduced adaptive potentials in comparison to natural strands (Pande et.al., 2004). Maintenance of variation within the species is essential for their long-term survival and fitness. The genetic variation helps the trees sustain in different geographical terrain and climatic conditions. A good understanding of level and distribution of genetic variability within the species is vital for their effective conservation and utility. This study can help us in developing strategies for their conservation. Hence, there is a need to study the existing variation within the species. On account of the destruction of forests due to human activities the need for conservation as well as reintroduction of this species becomes important, the knowledge of genetic diversity within this species covering Himalayan subtropical forests as well as diversity in provenance trial plantations is essential. xii Fig 1: Distances between the locations for provenance trials. All Distances in Km by shortest road route. Website used for finding distance - http://www.mapxl.com/path-finder/map_routing.phtml We have not come across any genetic diversity reports in Indian provenances hence genetic diversity studies from both native populations from Himachal Pradesh – Palampur (PA) and West Bengal – Darjeeling (DJ) provenances and also from Systematic provenance trial location from east and west side of Southern states of Indian subcontinent covering Andhra Pradesh-Rajahmundry (RJ)- Meredumilli location and Maharashtra – Panchagani-Gureghar (GG) provenance trial plots was undertaken during these studies. For Pinus roxburghiii, these four locations cover 4 corners of Indian provenances (Fig- 1 where distances are indicated as shortest road distances.) Deforestation due to human activity, accompanied by depletion of forest tree genetic resources, is occurring in many parts of the world. Thus there is an urgent need to conserve forest ecosystems for both environmental and economic reasons (Fenning and Gershenzon 2002). Micropropagation is one of the alternatives to do so.