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Establishment of in Vitro Haploids of Tea (Camellia Spp) and Their Potential for the Production of Secondary Metabolites

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Establishment of in Vitro Haploids of Tea (Camellia Spp) and Their Potential for the Production of Secondary Metabolites ESTABLISHMENT OF IN VITRO HAPLOIDS OF TEA (CAMELLIA SPP) AND THEIR POTENTIAL FOR THE PRODUCTION OF SECONDARY METABOLITES THESIS SUBMITTED TO IIT GUWAHATI FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 2013 VIJAY KUMAR MISHRA DEPARTMENT OF BIOTECHNOLOGY INDIAN INSTITUTE OF TECHNOLOGY GUWAHATI GUWAHATI-781039 ASSAM, INDIA ------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------- I have great pleasure to dedicate this thesis to my parents, brother, sister and my revered teachers. -------------------------------------------------------------------------------------------- TH-1176_07610612 INDIAN INSTITUTE OF TECHNOLOGY GUWAHATI Department of Biotechnology STATEMENT I do, hereby, declare that the matter embodied in this thesis is the result of investigations carried out by me in the Department of Biotechnology, Indian Institute of Technology Guwahati, India, under the supervision of Dr Rakhi Chaturvedi. In keeping with the general practice of reporting scientific observations, due acknowledgements have been made wherever the work described is based on the findings of other investigators. Date: June 24, 2013 Vijay Kumar Mishra TH-1176_07610612 INDIAN INSTITUTE OF TECHNOLOGY GUWAHATI Department of Biotechnology CERTIFICATE It is certified that the work described in this thesis entitled “Establishment of in vitro haploids of Tea (Camellia spp) and their potential for the production of secondary metabolites” by Vijay Kumar Mishra for the award of degree of Doctor of Philosophy is an authentic record of the results obtained from the research work carried out under my supervision in the Department of Biotechnology, Indian Institute of Technology Guwahati, India, and this work has not been submitted elsewhere for a degree. Date: June 24, 2013 Dr Rakhi Chaturvedi Associate Professor (Thesis Supervisor) Department of Biotechnology IIT Guwahati Guwahati-781039 Assam, India TH-1176_07610612 ACKNOWLEDGEMENTS -------------------------------------------------------------------------------------------- First and foremost I am grateful to THE ALMIGHTY “GOD” for granting me an opportunity to prove myself. Then I would like to extend my gratitude towards Dr Rakhi Chaturvedi, my supervisor, an amazing mentor who believed in me and my potential to get success. She helped me in the process of learning haploid production, and was very patient while I was not getting regeneration from cultures. She taught me how to ask questions and express my ideas. She showed me different ways to approach a research problem and the need to be persistent to accomplish any goal. She is one of the rare advisors that students dream that they will find. She was always there to help me with my minutest problems, inside or outside the lab. Without her support, I could not have done what I was able to do. I owe all my success to her. My gratitude goes to my doctoral committee members, chairperson, Dr. V.V. Dasu; members, Prof. A. Goyal and Dr. U. Bora, for their constructive criticism and suggestions, which helped me to improve my work pertaining to PhD thesis. I owe my thanks to the Department of Biotechnology, IIT Guwahati and CIF for providing me the necessary facilities to fulfill my PhD thesis objectives. I would like to extend my thanks to Tea Research association (TRA), Tocklai, Jorhat, for providing explant materials. I gratefully acknowledge all the faculty members for their support and guidance. I am thankful to all the technical staff especially, Mr Sharan, Mr Nurul, Ms Anitha, Ms Prarthana and Ms Rashmi and non teaching staff of the department for their affection and cooperation. I also would like to thank Department of Biotechnology (DBT) Government of India and IIT Guwahati for providing financial assistance which made this study possible. TH-1176_07610612 It was pleasure to work with my research lab members Dr Priyanka Srivastava, Dr Mithilesh Singh, Miss Rashmi, Ms Radhika, Ms Ruchira and Ms Zannat. Thanks to them for their suggestions, time, help in practical things and kindness throughout my PhD, this is an unforgettable experience. Special praises to my friends for sharing their knowledge, experience, support and providing a welcome diversion from the critical situations during my PhD, whenever I needed. I must acknowledge all my friends for their love, encouragement and support. My special thanks and appreciation goes to my parents as well as my brother and sister for their blessings, love, patience, support and understanding throughout my studies. Vijay Kumar Mishra June 24, 2013 TH-1176_07610612 ABSTRACT -------------------------------------------------------------------------------------------- The tea plant is a tropical, evergreen plant of the family Theaceae and genus Camellia. It is one of the most economically important beverage crops in the world. Chinese were the first to use tea as medicinal drink, later as beverage and have been doing so far for more than 3000 years. The original home or ‘the primary centre of origin’ of tea was South-East Asia. The cultivated taxa of tea comprise three main types, which are mainly differentiated on the basis of their leaf size. Assam type has the biggest leaf size while China type has the smallest. The Cambod type has leaf in between Assam and China types. Tea plant is a highly cross pollinated and genetically complex species. The genus Camellia includes more than 325 species which indicates genetic instability and high out-breeding nature of the genus. There are 600 cultivated varieties worldwide with unique traits, such as high caffeine content, drought tolerance, blister blight disease tolerant etc. In spite of having valued properties, improvement of tea by conventional methods is very laborious and time consuming and owing to its highly heterozygous nature and long reproductive cycle. In this context, it is noteworthy that, studies utilizing gametophytic cells are in infancy, in this tree species. In vitro haploid production from gametophytic cells enables the establishment of completely homozygous lines in a shortened time frame compared to conventional methods and has many potential applications in plant improvement to establish inbred lines rapidly and to observe recessive traits. Therefore, the aim of the present study was to establish in vitro androgenic lines of tea from anthers. Androgenic haploids were produced by anthers, cultured at early-to-late uninucleate stage of pollen. Haploid development occurred via callusing of microspores. TV21 and TV19 cultivars were regenerated and developed to haploid plants. Further, the androgenic lines were checked for the production of medicinally important compounds, such as (+)-Catechin, (-)-Epicatechin, (-)-Epigallocatechin gallate, Caffeine and Theophylline. These compounds are observed to be present in maximum amount in young leaves of parent plants, followed by in vitro embryos and the least in calli. The presence of compounds has been confirmed by chromatographic and spectroscopic techniques. Antioxidant activity assays of in vitro androgenic cultures were also investigated and found that hot water extract (80ºC for 20 min.) shows maximum activity. Batch kinetics TH-1176_07610612 i study in cell suspension cultures of TV21 cultivar was also performed and found that the production of (+)-Catechin, (-)-Epicatechin, (-)-Epigallocatechin gallate, Caffeine and Theophylline was observed to be growth associated and increased with an increase in fresh weight of the cells. The thesis is divided into five chapters. Chapter 1, introduces and reviews all the major contributions and studies perform on tea until now, with regard to tissue culture, secondary metabolite production and antioxidant activity in tea. Chapter 2, contains all the protocols and methodologies used for the present work. Chapter 3, presents the results obtained in the current investigation. The tables and graphs are included within the text while all the figures have been compiled at the end of the thesis, in the form of plates. Inferences drawn from the results are discussed in chapter 4, in the light of other reports available on related aspects. Chapter 5, throws light on the major highlights of the present work and its future scope. This is followed by the appendix that mentions the taxonomic classification of the plants studied. The thesis concludes with the list of bibliography and visible research output in terms of peer-reviewed journal publications, book chapters and conference proceedings. TH-1176_07610612 ii TABLE OF CONTENTS ---------------------------------------------------------------------------------------------------- ABSTRACT i-ii CONTENTS iii LIST OF FIGURES v LIST OF TABLES vi LIST OF GRAPHS vii LIST OF ABBREVIATIONS viii CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW 1-38 1.1. HISTORICAL IMPORTANCE, ORIGIN AND DISTRIBUTION 1 1.2. BOTANICAL ASPECTS 3 1.3. ECONOMIC IMPORTANCE, HEALTH AND OTHER BENEFITS 5 1.4. CONVENTIONAL BREEDING APPROACH IN TEA 8 1.5. TISSUE CULTURE STUDIES 10 1.5.1 Micropropagation 10 1.5.2. Somatic embryogenesis 21 1.5.3. Haploid production 29 1.6. SECONDARY METABOLITES 32 1.7. OBJECTIVES OF THE PRESENT STUDY 37 CHAPTER 2: MATERIALS AND METHODS 39-56 2.1. MATERIALS 39 2.2. METHODS 41 CHAPTER 3: RESULTS 57-96 3.1. ANTHER CULTURE 57 3.1.1 Callus induction 58 3.1.2. Callus multiplication and maintenance 67 3.1.3. Regeneration from anther callus 69 3.1.4. Embryo maturation, germination and plantlets development 73 3.1.5. Ploidy
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