Micropropagation of Hazelnut (Corylus Species) by Jyoti A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Plant Agriculture Guelph, Ontario, Canada © Jyoti, August, 2013 ABSTRACT MICROPROPAGATION OF HAZELNUT (CORYLUS SP.) Jyoti Advisor: University of Guelph, 2013 Dr. Praveen Saxena An efficient micropropagation protocol for large scale production of hazelnut plants is required for consistent supply of elite germplasm to support the growing industry in Ontario. The main focus of current research was to develop a bioreactor based micropropagation protocol for hazelnut multiplication. An integrated approach was developed to increase the multiplication rate by optimizing the nutrient medium supplements and culture technology using a temporary immersion bioreactor system (TIS). As compared to conventional semi-solid and liquid based culture system, the Liquid LabTM temporary immersion bioreactor system (LIS) showed a significant enhancement in the number of shoots (3.3 shoots/explant), shoot height, leaf area and chlorophyll content in micropropagated plantlets. Antioxidant supplements such as ascorbic acid and melatonin along with iron (460 µM FeEDDHA) significantly increased the shoot proliferation (5.5 shoots/explant). However, a much higher shoot proliferation (10.9 shoots/explant) was observed with the addition of aspirin (10 µM) in the presence of BA (17.6 µM). Among several hazelnut cultivars, HF-16 had the highest multiplication rate followed by Geneva and Epsilon. Medium supplemented with 10 µM IBA was the best for rooting of microshoots of HF-16 and the plantlets acclimatized in the green house with 80% survival rate. iii ACKNOWLEDGEMENTS Sincere appreciation to my advisor, Dr. Praveen Saxena for all the guidance and support he has provided throughout my thesis. His valuable inputs have instilled the confidence, enthusiasm and perseverance that were essential in this academic pursuit. I would like to thank my advisory committee: Dr. Gopinadhan Paliyath, Dr. Jayasankar Subramanian, and Dr. Adam Dale for their helpful suggestions. My deepest appreciation to Dr. Mukund Shukla for providing me with direction, technical training, and encouragement. Special thanks to Dr. Max Jones for valuable editing advice during thesis preparation. I express my gratitude to Shuping, Robert, Abhishek, Dr. Sherif Sherif and Dr. Vikramjit Bajwa with whom I have shared the laboratory and laughs over the years. I acknowledge Dr. Krishnaraj Tiwari (Raj) and Renu for their friendship, support, motivation, and exchange of knowledge and skills. I owe a great deal of gratitude to Ramany, Valsala and Neeta for giving me home like environment away from home. I am indebted to my parents and family for their endless support and constant encouragement without which I could not have completed this dissertation. I would also like to thank Ontario Centers of Excellence and Ontario Ministry of Agriculture, Food and Rural Affairs for their financial support. iv TABLE OF CONTENTS Sl. No. Title Page I Abstract II Acknowledgements iii III Table of Contents iv X List of Tables vii XI List of Figures viii XII List of Appendices xii XIII List of Abbreviations xiii Chapter 1: Introduction and Review of Literature 1 1.1 Introduction 1 1.2 Hazelnut based products 1 1.3 Hazelnut production 2 1.4 Taxonomy 3 1.5 Tree characteristics 4 1.6 Hazelnut in North America 5 1.7 Climatic conditions 5 1.8 Vegetative propagation of hazelnut 6 1.8.1 Layering 7 1.8.2 Grafting 7 1.8.3 Shoot cuttings 8 1.9 Problems of hazelnut propagation in Ontario 9 1.10 Need for an efficient method of propagation 10 1.11 Micropropagation 10 1.12 Micropropagation of hazelnut 11 1.12.1 Culture initiation and establishment and explant source 11 1.12.2 Problems associated with initiation 12 1.12.2 Multiplication phase 15 1.12.2.1 Medium composition 15 1.12.2.2 Carbon source 18 1.12.2.3 Plant growth regulators (PGR) 19 1.12.3 Rooting 21 1.12.4 Physical state of medium 23 1.13 Bioreactor 24 1.13.1 Temporary Immersion System (TIS) 29 1.14 Other factors affecting micropropagation 32 1.14.1 Phenolic compounds 32 1.14.2 Endogenous level of phytohormones 33 1.14.3 Salicylic and acetylsalicylic acid 33 1.15 Hypotheses 35 1.16 Objectives 36 2.0 Chapter 2: Growth optimization of hazelnut culture in Liquid LabTM Rocker temporary immersion bioreactor system 38 Abstract 38 2.1 Introduction 39 v 2.2 Materials and Methods 41 2.2.1 Plant Material and culture establishment 41 2.2.2 Effect of iron supplements on growth of Corylus avellana L. x 42 Corylus americana M. cv. Geneva 2.2.2.1 Iron detection in leaves 43 2.2.2.2 Quantification of total iron content in plantlets 44 2.2.3 Comparison of LIS and semi-solid system for growth of various 44 hazelnut cultivars 2.2.3.1 Chlorophyll estimation 45 2.3 Results 46 2.3.1 Plant material and culture establishment 46 2.3.2 Effect of iron supplements on growth of Corylus avellana L. x 46 Corylus americana M. cv. Geneva 2.2.2.1 Iron detection in leaves 48 2.2.2.2 Quantification of total iron content in plantlets 52 2.3.3 Comparison of LIS and semi-solid system for growth of various 52 hazelnut cultivars 2.4 Discussion 54 Conclusions 66 3.0 Chapter 3: Effect of various additives on the growth 67 performance of hazelnut plantlets in Liquid LabTM Rocker temporary immersion bioreactor system Abstract 67 3.1 Introduction 68 3.2 Materials and Methods 70 3.2.1 Plant material and culture conditions 70 3.2.2 Chlorophyll content 71 3.3 Results 72 3.4 Discussion 75 Conclusions 85 4.0 Chapter 4: Effect of salicylic acid and acetylsalicylic acid on 86 the growth and proliferation of hazelnut plantlets in vitro Abstract 86 4.1 Introduction 87 4.2 Materials and Methods 89 4.3 Results 92 4.3.1 Effect of acetyl salicylic acid in micropropagation of hazelnut 92 cultivar, HF-16 4.3.2 Effect of salicylic acid (SA) in micropropagation of hazelnut 93 cultivar, HF-16 4.4 Discussion 98 Conclusions 107 5.0 Chapter 5: Effect of various carbon sources and antiauxins on 108 hazelnut and; root induction at different concentrations of IBA vi Abstract 108 5.1 Introduction 109 5.2 Materials and Methods 112 5.2.1 Effect of various carbon sources on shoot development 112 5.2.2 Effect of antiauxins on hazelnut growth performance 112 5.2.3 Effect of IBA on root development 113 5.2.4 Culture conditions 114 5.3. Results 115 5.3.1 Effect of various carbon sources on shoot development 115 5.3.2 Effect of antiauxins on hazelnut growth performance 115 5.3.3 Effect of IBA on root development 117 5.4 Discussion 124 Conclusions 129 Chapter 6: Summary and General Conclusions 131 References 137 Appendices 169 vii LIST OF TABLES Sl. No. Title Page 4.1 Different media types used to evaluate the effect of Aspirin and 90 Salicylic acid on the growth parameters of hazelnut cultivar HF-16 viii LIST OF FIGURES Sl. No. Title Page Fig. 2.1 Bud initiation from nodal explants of Corylus avellana L. x Corylus 47 americana M. cv. Geneva on the DKW basal semi-solid medium with antibiotic on (a) day 1 (b) day 21 and (c) day 28 of culturing. Fig. 2.2 Bud initiation from nodal explants of Corylus avellana L. x Corylus 47 americana M. cv. Geneva on DKW basal semi-solid medium on (a) day 1 and (b) day 21 of culturing. Fig. 2.3 Effect of various concentrations of FeEDDHA alone (MDKW medium) or 49 in combination with constant amount of FeSO4 (121.58 µM; CDKW medium) on nodal explants of Corylus avellana L. x Corylus americana M. cv. Geneva on (a) shoot height, (b) number of shoots and (c) number of nodes after eight weeks of culture. Columns with same letter indicate no significant difference using Tukey's test (P<0.05). Fig. 2.4 Iron detection in the leaf tissue of Corylus avellana L. x Corylus 50 americana M. cv. Geneva grown at different concentrations of iron supplements using Perls' Prussian blue/Diaminobenzidine. Arrows indicating iron pigments after staining and pictures were taken at same magnification level. Fig. 2.5 Iron content in whole plant tissue of Corylus avellana L. x Corylus 53 americana M. cv. Geneva plantlets grown in medium containing different concentrations of iron source provided by constant amount of FeSO4 (121.58 µM) + varying FeEDDHA (Blue line) and in plantlets grown in varying concentrations of FeEDDHA (Red line). Fig. 2.6 Liquid Lab™ temporary immersion system used for the micropropagation 53 of hazelnut. Fig. 2.7 Comparison of (a) Number of shoots (b) Number of nodes (c) Shoot height 55 (d) Multiplication rate (e) Leaf area (f) Chlorophyll a and (g) Chlorophyll b of hazelnut cultivars, Epsilon, Jefferson, Geneva and HF-16 in LIS supplemented with 460 µM of FeEDDHA in basal DKW medium after 8 weeks of culturing. Columns with the same letter indicate no significant difference (P<0.05). Fig. 2.8 Growth performance of hazelnut cultivars on semi-solid medium after 57 eight weeks of culture (a) Geneva (b) HF-16 (c) Epsilon and (d) Jefferson on DKW medium containing BA (17.6 µM) and GA3 (0.29 µM) and supplemented with 460 µM of FeEDDHA in basal DKW medium. ix Fig. 2.9 Growth performance of hazelnut cultivars in LIS after eight weeks of 57 culture (a) Geneva, (b) Jefferson (c) Epsilon and (d) HF-16 on DKW medium containing BA (17.6 µM) and GA3 (0.29 µM) and supplemented with 460 µM of FeEDDHA in basal DKW medium.