Pollen Cryopreservation Protocols

Foreword Preface Contents Contributors Chapter 1: Pollen Cryopreservation: Advances and Prospects 1 Introduction and Relevance of Plant Cryopreservation 2 Historical Aspects of Plant Cryopreservation 3 Pollen Cryopreservation 3.1 Why Pollen Cryopreservation? 3.2 Pollen Cryobanks 3.3 Pollen Cryopreservation: Advantages and Disadvantages 3.4 Requirements and Practical Considerations in Pollen Cryopreservation 3.5 Uses and Applications 4 Present Status of Plant and Pollen Cryopreservation 5 Prospects and Way Forward References Chapter 2: Pollen Cryobanking-Implications in Genetic Conservation and Plant Breeding 1 Introduction 1.1 Germplasm Conservation 1.2 Increasing Breeding Efficiency 1.3 Exchange of Pollen (Nuclear Genetic Diversity) 1.4 Integration of Conventional Breeding Methods with Modern Biotechnological Practices 1.5 Genetic Amelioration 2 Uses of Cryopreserved Pollen in Production Fields 3 Pollen Morphology and Pollen Classification 4 Pollen Cryopreservation Protocol 4.1 Pollen collection Methods 4.2 Pollen Viability Testing 4.2.1 Staining Techniques 4.2.2 In Vivo Pollen Germination 4.2.3 In Vivo Pollen Germination and Pollen Tube Growth 4.3 Moisture Content Determination 4.4 Pollen Desiccation 4.5 Pollen Cryopreservation 4.6 Pollen Rehydration 4.7 Post-cryopreservation Viability and Fertility Assessment 5 Factors Affecting Pollen Viability and Cryopreservation 6 Advantages of Cryopreserving Pollen 7 Limitations of Pollen Cryopreservation 8 Need for Protection and Grant of IPR to Cryopreserved Pollen 9 Way Forward References Chapter 3: Establishment of a Pollen Cryobank and General Procedures of Pollen Cryopreservation 1 Introduction 2 Infrastructure Facilities 3 Cryo-equipments Required 3.1 Laboratory Equipments and Accessories Required 3.2 Storage Containers 4 Cryotransit and Shipment 5 Cost of Cryobanking 6 Potential Hazards/Threats in Cryostorage 7 Basic Safety Measures 8 Pollen Cryopreservation: General Procedures 8.1 Pollen Collection 8.2 Pollen Viability Assessment 8.3 Pollen Cryopreservation 8.4 Field Pollinations with Cryopreserved Pollen for Fertility Assessment 9 Pollen Cryobank at ICAR-IIHR, Bengaluru 10 Case Studies on Successful Pollen Cryopreservation in Species of Economical Importance 10.1 Orchid Pollen Cryopreservation 10.2 Tomato Pollen Cryopreservation 10.3 Apple Pollen Cryopreservation 11 Conclusion and Way Forward References Part I: Fruits Chapter 4: Pollen Cryopreservation Protocol in Annona Species 1 Introduction 2 Materials 2.1 Plant Material for Pollen 2.2 Required Laboratory Equipments 2.3 General Laboratory Equipments 2.4 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.1.1 Inventory 3.1.2 Anthesis in Annonaceous Species 3.1.3 Bagging of Flower and Pollen Collection 3.2 Dehydration of Pollen Grains 3.3 Immersion of the Pollen Grains in Liquid Nitrogen 3.4 Retrieval 3.5 Precautions 3.6 Viability Testing In Vitro 3.6.1 Inventory 3.6.2 Preparation of Pollen Germination Medium 3.6.3 In Vitro Pollen Germination 3.6.4 Precautions 3.7 In Vivo Fertility Test 3.7.1 Inventory 3.7.2 Procedure 3.7.3 Precautions 3.8 SEM Analysis of Fresh and Cryopreserved Pollen 3.8.1 Inventory 3.8.2 Procedure 4 Notes 4.1 To Be Taken Care Of References Chapter 5: Pollen Cryopreservation in Aonla 1 Introduction 2 Materials 2.1 Collection of Aonla Pollen 2.2 In Vitro Germination of Pollen Grains 2.3 Cryopreservation of Pollen in Liquid Nitrogen 2.4 In Vivo Fertility Test 3 Methods 3.1 Collection of Aonla Pollen 3.2 Pollen Cryopreservation in Aonla 3.3 Pollen Germination in Aonla 3.3.1 Preparation of Culture Medium 3.3.2 In Vitro Germination of Pollen Grains 3.4 In Vivo Viability Testing of Aonla Cryostored Pollen 3.5 Scanning Electron Microscopy Analysis 4 Conclusion References Chapter 6: Cryopreservation of Pollen Grains of Carica papaya and Different Vasconcellea Species 1 Introduction 2 Materials 2.1 Plant Material 2.2 Laboratory Equipment Required to Carry Out Experiments 2.3 General Laboratory Supplies 2.4 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.1.1 Inventory 3.1.2 Time of Anthesis 3.1.3 Flower Bagging and Collection 3.1.4 Pollen Collection from Anthers 3.2 Cryopreservation 3.3 Retrieval 3.4 Precautions 3.5 In Vitro Viability Testing 3.5.1 Inventory 3.5.2 Medium Preparation 3.5.3 In Vitro Pollen Germination 3.5.4 Precautions 3.6 In Vivo Test for Fertility 3.6.1 Inventory 3.6.2 Procedure 3.6.3 Fruit and Seed Set 3.6.4 Precautions 3.7 SEM Analysis of Cryopreserved Pollen 3.7.1 Inventory 3.7.2 Procedure 4 Notes References Chapter 7: Cryopreservation of Pollen Grains of Citrus and Other Aurantioideae 1 Introduction 2 Materials 2.1 Collection 2.2 Dehydration 2.3 Storage in Liquid Nitrogen 2.4 Thawing 2.5 Rehydration 2.6 In Vitro Germination 2.7 In Vivo Viability Testing 3 Methods 3.1 Collection 3.2 Dehydration 3.3 Storage in Liquid Nitrogen 3.4 Thawing 3.5 Rehydration 3.6 Acetocarmine Staining 3.7 In Vitro Germination 3.8 In Vivo Viability Testing 4 Notes References Chapter 8: Pollen Cryopreservation Protocol for Dragon Fruit 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.2 Dehydration of Pollen 3.3 Preparation of Pollen Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains 3.6 Post-Cryopreservation Viability Assessment 3.7 Scanning Electron Microscopy Analysis 3.8 Retrieval and Post-Storage Fertility Assessment 4 Notes: Precautions, Tips to Get Good Quality Pollen and In Vitro Germination References Chapter 9: Cryopreservation of Grape Pollen (Vitis Species) 1 Introduction 2 Material 2.1 Plant Material 2.2 Laboratory Equipment Required to Carry Out Experiments 2.3 General Laboratory Supplies 2.4 Chemicals and Solutions 2.5 Inventory 2.5.1 Pollen Collection 2.5.2 In Vivo Fertility Assessment Fresh and Cryopreserved Pollen 2.5.3 In Vitro Viability Testing 3 Methods 3.1 Time of Anthesis 3.2 Flower Bagging and Pollen Collection 3.3 Cryopreservation 3.3.1 Precautions 3.3.2 Procedure of Retrieval and Post-storage Fertility Assessment 3.4 In Vitro Viability Testing 3.4.1 Medium Preparation 3.4.2 Procedure for In Vitro Pollen Viability Assessment 3.4.3 Precautions 3.5 Procedure of In Vivo Fertility of Cryopreserved Pollen 3.5.1 Fruit and Seed Set 3.5.2 Precautions 4 Notes References Chapter 10: Pollen Cryopreservation in Jackfruit (Artocarpus heterophyllus) for Crop Improvement 1 Introduction 2 Materials 2.1 Laboratory Equipments Required to Carry Out Experiments 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.1.1 Inventory 3.1.2 Procedure 3.1.3 Precautions 3.2 Dehydration and Determination of Moisture Content of Pollen 3.2.1 Inventory 3.2.2 Procedure 3.2.3 Precautions 3.3 Cryostorage 3.3.1 Inventory 3.3.2 Cryopreservation 3.3.3 Precautions 3.4 Thawing and Retrieval 3.5 Pollen Germination Medium 3.5.1 Inventory 3.5.2 Preparation of Pollen Germination Medium 3.5.3 Precautions 3.6 In Vitro Germination Test 3.6.1 Inventory 3.6.2 Hanging Drop Technique 3.6.3 Incubation 3.6.4 Precautions 3.7 Staining and Scoring of Pollen 3.7.1 Inventory 3.7.2 Staining 3.7.3 Scoring 3.7.4 Precautions 3.8 In Vivo Test for Fertility by Field Pollination 3.8.1 Inventory 3.8.2 Procedure 3.8.3 Fruit Set and Seed Set 3.8.4 Precautions 3.8.5 Scanning Electron Microscopy Analysis 4 Notes References Chapter 11: Pollen Cryopreservation in Jamun (Syzygium cuminii Skeels) 1 Introduction 2 Materials 2.1 Collection 2.2 Viability Tests In Vitro 2.3 Cryostorage/Cryopreservation (Storage in LN) 2.4 Viability Tests In Vitro 3 Methods 3.1 Collection 4 Viability Testing 4.1 Media Preparation for Pollen Germination 4.2 In Vitro Germination 4.3 Incubation 4.4 Staining 4.5 Scoring of Pollen Cultures 5 Storage 6 Retrieval and Rewarming 7 In Vivo Test for Fertility 7.1 Breeder´s Crossing Kit 7.2 Procedure 8 Notes References Chapter 12: Pollen Cryopreservation in Passiflora Species 1 Introduction 2 Materials 2.1 Laboratory Equipments Required to Carry Out Experiments 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 2.4 Collection 2.5 Dehydration and Determination of Moisture Content of Pollen 2.6 Viability Tests In Vitro 2.7 Cryopreservation (Storage in LN) 2.8 Viability Tests In Vitro 2.9 In Vivo Fertility Test 3 Methods 3.1 Collection 3.2 Dehydration 3.3 Medium Preparation for Pollen Germination 3.4 Pollen Viability and In Vitro Germination 3.5 Differential Staining Techniques 3.6 In Vitro Pollen Germination 3.7 Incubation 3.8 Scoring of Pollen Cultures 3.9 Cryopreservation 3.10 Retrieval and Rewarming 3.11 In Vivo Test for Fertility 4 Notes References Chapter 13: Pollen Cryopreservation Protocol for Poncirus trifoliata 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.2 Dehydration of Pollen Grains 3.3 Preparation of Pollen Germination Medium 3.4 Viability Assessment 3.5 Cryopreservation of Pollen Grains 3.6 Fertility Assessment 3.7 Seed Germination 4 Notes: Precautions, Tips to Get Good Quality Pollen and In Vitro Germination References Chapter 14: Cryopreservation of Pollen Grains of Different Psidium Species 1 Introduction 2 Materials 2.1 Plant Material 2.2 Laboratory Equipments Required to Carry Out Experiments 2.3 General Laboratory Supplies 2.4 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.1.1 Inventory 3.1.2 Anthesis in Psidium Species 3.1.3 Flower Bagging and Collection 3.1.4 Pollen Collection From Anthers 3.2 Cryopreservation 3.3 Retrieval 3.4 Precautions 3.5 In Vitro Viability Testing 3.5.1 Inventory 3.5.2 Medium Preparation 3.5.3 In Vitro Pollen Germination 3.5.4 Precautions 3.6 In Vivo Test for Fertility 3.6.1 Inventory 3.6.2 Procedure 3.6.3 Fruit and Seed Set 3.6.4 Precautions 3.7 SEM Analysis of Fresh and Cryopreserved Pollen 3.7.1 Inventory 3.7.2 Procedure 4 Notes References Chapter 15: Cryopreservation of Pomegranate (Punica granatum L.) Pollen 1 Introduction 2 Materials 2.1 Plant Material 2.2 Required Laboratory Equipments 2.3 General Laboratory Supplies 2.4 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.1.1 Inventory 3.1.2 Anthesis Time 3.1.3 Flower Bagging and Pollen Collection 3.2 Pre-storage Viability Assessment 3.2.1 Inventory 3.2.2 In Vitro Pollen Germination 3.2.3 Precautions 3.3 Processing and Cryopreservation 3.4 Retrieval and Post-storage Viability Assessment 3.4.1 Precautions 3.5 In Vivo Viability of Cryopreserved Pollen of Pomegranate 3.5.1 Inventory 3.5.2 Procedure 3.5.3 Fruit and Seed Set 3.5.4 Precautions 4 Notes References Chapter 16: Pollen Cryopreservation in Mango 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.2 Extraction of Pollen 3.3 Dehydration of Pollen 3.4 Preparation of Pollen Germination Medium 3.5 Initial Viability Assessment 3.6 Cryopreservation 3.7 Scanning Electron Microscopy Analysis 3.8 Retrieval and Post-storage Viability Assessment 3.9 Fertility Assessment 4 Notes References Part II: Vegetables Chapter 17: Pollen Cryopreservation in Ash Gourd (Benincasa hispida (Thunb.) Cogn.) 1 Introduction 2 Materials 2.1 Required Laboratory Equipments 2.2 General Laboratory Equipments 2.3 Chemicals and Solutions 3 Method 3.1 Pollen Collection 3.2 Pollen Drying or Desiccation 3.3 Pollen Viability Assessment in vitro 3.3.1 Brewbaker and Kwack´s Medium (100 mL) Stock Solution Preparation 3.3.2 Preparation of Pollen Germination Medium 3.3.3 Procedure For Viability Assessment of Fresh Pollen 3.4 Pollen Cryopreservation 3.5 Retrieval and Post-storage Viability/Fertility Assessment 3.5.1 Viability Assessment of Cryopreserved Pollen 3.5.2 Field Pollination 4 Notes References Chapter 18: Pollen Cryopreservation in Bitter Gourd 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Pollen Viability Test 2.3 Dehydration and Packaging of Pollen 2.4 Immersion of Pollen Grains in Liquid Nitrogen 2.5 In Vitro Germination of Pollen 2.6 In Vivo Fertility Test 3 Methods 3.1 Collection of Anthers with Pollen Grains 3.2 Collection of the Pollen 3.3 Dehydration of the Pollen 3.4 Preparation of the Pollen Germination Medium 3.5 In Vitro Germination of the Pollen 3.6 Cryopreservation of Pollen Grains in Liquid Nitrogen 3.7 In Vivo Viability Testing 3.8 Scanning Electron Microscopy Analysis 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 19: Pollen Cryopreservation in Bottle Gourd for Breeding and Conservation 1 Introduction 2 Checklist for In Vitro Pollen Germination 2.1 Pollen Germination Medium Composition 2.2 Pollen Collection 2.3 Pollen Desiccation 2.4 Viability Assessment 3 Procedure 3.1 Cryopreservation 3.2 Procedure 3.3 Retrieval and Post-Storage Fertility Assessment 3.4 Procedure 4 The Following Points Should Be Noted References Chapter 20: Cryopreservation of Eggplant (Solanum melongena L.) Pollen for Utilization in Crop Improvement 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Dehydration and Packaging of Pollen Grains 2.3 Immersion of Pollen Grains in Liquid Nitrogen 2.4 In Vitro Germination of Pollen Grains 2.5 In Vivo Viability Test 3 Methods 3.1 Collection of Anthers Pollen 3.2 Collection of the Pollen Grains 3.3 Dehydration of the Pollen Grains 3.4 Cryopreservation of Pollen Grains 3.5 In Vitro Germination of the Pollen Grains 3.5.1 Preparation of Nutrient Medium [21] 3.5.2 Preparation for In Vitro Germination 3.5.3 Viability Test 3.6 In Vivo Fertility Testing 4 Notes References Chapter 21: Cryopreservation of Pollen Grains of Hot Pepper 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Dehydration and Packaging of Pollen Grains 2.3 Immersion of Pollen Grains in Liquid Nitrogen 2.4 In Vitro Germination of Pollen Grains 2.5 In Vivo Viability Test 3 Methods 3.1 Collection of Anthers with Pollen Grains 3.2 Collection of the Pollen Grains 3.3 Dehydration of the Pollen Grains 3.4 Cryopreservation of Pollen Grains 3.5 In Vitro Germination of the Pollen Grains 3.6 Fertility Assessment of the Pollen Grains (see Note 10) 4 Notes References Chapter 22: Cryopreservation of Muskmelon (Cucumis melo) Pollen for Exploring Breeding Possibilities 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Dehydration and Packaging of Pollen Grains 2.3 Cryopreservation of Pollen Grains 2.4 In Vitro Germination of Pollen Grains 2.5 In Vivo Fertility Test 3 Methods 3.1 Collection of Flowers with Pollen Grains 3.2 Extraction of the Pollen Grains 3.3 Dehydration of the Pollen Grains 3.4 Preparation of the Pollen Germination Medium 3.5 In Vitro Germination of the Pollen Grains 3.6 In Vivo Fertility Testing 4 Notes References Chapter 23: Onion (Allium cepa L.) Pollen Cryopreservation Protocol 1 Introduction 2 Materials 2.1 Onion Pollen Grain Collection 2.2 Dehydration and Packaging 2.3 Immersion of Pollen Grains 2.4 In Vitro Pollen Grain Germination 2.5 In Vivo Fertility Test 3 Methods 3.1 Collection of Anthers from Umbel 3.2 Collection of the Pollen Grains from Anther 3.3 Dehydration of Onion Pollen Grains 3.4 Cryopreservation of Pollen Grains 3.5 In Vitro Pollen Grain Germination 3.5.1 Preparation of Germination Medium 3.5.2 Preparation for Pollen Germination Test 3.5.3 Viability Test 3.6 In Vivo Fertility Test 4 Notes References Chapter 24: Cryopreservation of Okra (Abelmoschus esculentus L.) Pollen 1 Introduction 2 Materials 2.1 Plant Material 2.2 Collection of Pollen 2.3 In Vitro Pollen Germination Medium 2.4 Pollen Viability Assessment 2.5 Pollen Moisture Content Estimation 2.6 Desiccation of Pollen 2.7 Cryopreservation 3 Methods 3.1 Collection of Pollen 3.2 In Vitro Pollen Germination Medium 3.3 Pollen Germination (Sitting Drop Method) 3.4 Fresh Pollen Moisture Content Estimation 3.5 Desiccation of Pollen 3.6 Determination of Optimum Desiccation Period to Attain Optimum Moisture Content for Cryopreservation 3.7 Cryopreservation 3.8 Retrieval and Regrowth of Cryopreserved Pollen (see Note 13) 3.9 Fertility Assessment in Field (Fig. 3) 4 Notes References Chapter 25: Protocol for Pollen Viability and Cryopreservation of Pumpkin (Cucurbita moschata Duchesne ex Poir) 1 Introduction 2 Materials 2.1 Collection of Pollen 2.2 Dehydration of Pollen 2.3 Cryopreservation of Pollen in Liquid Nitrogen 2.4 In Vitro Germination of Pollen 3 Methods 3.1 Collection of Flowers 3.2 Pollen Extraction 3.3 Dehydration of Pollen 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen in Liquid Nitrogen 3.6 Post-cryopreservation Viability and Fertility Assessment 3.7 Scanning Electron Microscopy Analysis References Chapter 26: Pollen Cryopreservation in Ridge Gourd (Luffa acutangula (Roxb.) L.) 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Viability Test 2.3 Dehydration and Packaging of Pollen Grains 2.4 Immersion of Pollen Grains in Liquid Nitrogen 2.5 In Vitro Germination of Pollen Grains 2.6 In Vivo Fertility Test 3 Methods 3.1 Collection of Flowers with Pollen Grains 3.2 Extraction of the Pollen Grains 3.3 Dehydration of the Pollen Grains 3.4 Preparation of the Pollen Germination Medium 3.5 In Vitro Germination of the Pollen Grains 3.6 In Vivo Testing References Chapter 27: Pollen Cryopreservation Protocol for Wild Species of Solanum 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Pollen Collection and Dehydration 3.2 Preparation of Pollen Medium 3.3 Viability Assessment 3.4 Cryopreservation 3.5 Retrieval and Post-storage Fertility Assessment 3.6 Scanning Electron Microscopy Analysis 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 28: Feasibility Studies on Pollen Cryopreservation in Spine Gourd (Momordica dioica Roxb.) 1 Introduction 2 Materials 2.1 Collection of Pollen 2.2 Dehydration of Pollen 2.3 Cryopreservation of Pollen in Liquid Nitrogen 2.4 In Vitro Germination of Pollen 3 Methods 3.1 Collection of Flowers 3.2 Pollen Extraction 3.3 Dehydration of Pollen 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen in Liquid Nitrogen 3.6 Post-cryopreservation Viability and Fertility Assessments 3.7 Scanning Electron Microscopy Analysis 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 29: Cryopreservation Protocol for Tomato (Solanum lycopersicum L.) and its wild relatives Pollen for Utilization in Cr... 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Dehydration and Packaging of Pollen Grains 2.3 Immersion of Pollen Grains in Liquid Nitrogen 2.4 In Vitro Germination of Pollen Grains 2.5 Fertility Assessment of Pollen Grains 3 Methods 3.1 Collection of Anthers with Pollen Grains 3.2 Collection of the Pollen Grains 3.3 Dehydration of the Pollen Grains 3.4 Cryopreservation of Pollen Grains 3.5 In Vitro Germination of the Pollen Grains 3.6 Fertility Assessment of Pollen Grains 3.7 Scanning Electron Microscopy Analysis 4 Notes References Chapter 30: Pollen Cryopreservation Protocol for Teasel Guard (Momordica subangulata subsp. Renigera) 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Dehydration of Pollen Grains 2.3 Cryopreservation of Pollen Grains in Liquid Nitrogen 2.4 In Vitro Germination of Pollen Grains 3 Methods 3.1 Collection of Flowers 3.2 Pollen Extraction 3.3 Dehydration of Pollen 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains in Liquid Nitrogen 3.6 Post-cryopreservation Viability and Fertility Assessments 3.7 Scanning Electron Microscopy Analysis 4 Notes: Precautions, Tips to Get Good Quality Pollen and In Vitro Germination References Chapter 31: Cryopreservation of Watermelon Pollen to Explore New Breeding Possibilities 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Dehydration and Packaging of Pollen Grains 2.3 Cryopreservation of Pollen Grains 2.4 In Vitro Germination of Pollen Grains 2.5 In Vivo Fertility Test 3 Methods 3.1 Collection of Anthers 3.2 Extraction of the Pollen Grains 3.3 Dehydration of the Pollen Grains 3.4 Preparation of the Pollen Germination Medium 3.5 In Vitro Germination of the Pollen Grains 3.6 In Vivo Fertility Testing 4 Notes References Part III: Ornamentals Chapter 32: Pollen Cryopreservation Protocol for Gladiolus 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.2 Dehydration of Pollen Grains 3.3 Preparation of Pollen Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains 3.6 Post-cryopreservation Viability Assessments 3.7 Fertility Assessment 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 33: Pollen Cryopreservation Protocol for Gloriosa superba L. 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.2 Dehydration of Pollen 3.3 Preparation of Pollen Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains 3.6 Scanning Electron Microscopy Analysis 3.7 Post-cryopreservation Viability Assessments 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 34: Pollen Cryopreservation in Marigold (Tagetes spp.) 1 Introduction 2 Materials 2.1 Plant Material 2.2 Checklist for In vitro Pollen Germination 2.3 Pollen Germination Medium Composition (Brewbaker and Kwack Medium) 3 Methods 3.1 Pollen Collection 3.2 Pollen Desiccation 3.3 Brewbaker and Kwack Medium (100 mL) Stock Solution Preparation 3.4 Viability Assessment 3.5 Cryopreservation 3.6 Retrieval and Post-storage Fertility Assessment 4 Notes References Chapter 35: Pollinia Cryopreservation of Selected Orchid Species of Western Ghats, India 1 Introduction 2 Materials 2.1 Collection 2.2 Dehydration If Required and Determination of Moisture Content of Pollen 2.3 Viability Tests In Vitro 2.4 Cryopreservation (Storage in LN) 2.5 Thawing and Rewarmig 2.6 Fertility Tests If Any in In Vivo Controlled Field Pollination 3 Methods 3.1 Collection 3.2 Dehydration/Controlled Hydration/Drying 3.3 Precautions 4 Viability Testing 4.1 Inventory 4.2 Medium Preparation for Pollen Germination 4.3 Precautions 4.4 In Vitro Germination 4.4.1 Sitting Drop Method (Calanthe masuca, Cymbidium bicolor, C. ensifolium, D. ovatum, E. cullenii, R. retusa) 4.4.2 In Vivo Method (A. praemorsa, V. spathulata, V. wightii) 4.5 Incubation 4.5.1 In Vitro Method 4.5.2 In Vivo Method 4.6 Staining 4.6.1 In Vitro Germination 4.6.2 In Vivo Germination 4.7 Scoring of Pollen Cultures 4.8 General Precautions 5 Storage 5.1 Inventory 5.2 Procedure 5.3 Precautions 6 Retrieval and Re-warming 6.1 Procedure 6.2 Precautions 7 In Vivo Test for Fertility/Field Pollination 7.1 Inventory 7.2 Breeder Crossing Kit 7.3 Procedure 7.4 Fruit Set and Seed Set Test 7.5 In Vivo Germination and Pollen Tube Growth 7.6 Precautions 8 Notes References Chapter 36: Pollen Cryopreservation Protocol for Tuberose (Polianthes tuberosa L.) 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Flowers 3.2 Collection of Pollen 3.3 Dehydration of Pollen 3.4 Viability Assessment of the Pollen 3.5 Cryopreservation of Pollen in Liquid Nitrogen 3.6 Post-cryopreservation Viability and Fertility Assessments References Chapter 37: Pollen Cryopreservation Protocol for Rose 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.2 Dehydration of Pollen Grains 3.3 Preparation of Pollen Medium 3.4 Viability Assessment 3.5 Cryopreservation (Storage in LN) 3.6 Fertility Assessment 3.7 Scanning Electron Microscopy Analysis References Part IV: Medicinal Plants Chapter 38: Pollen Cryopreservation in Aloe vera 1 Introduction 2 Materials 2.1 Laboratory Equipments 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.2 Dehydration of Pollen 3.3 Preparation of Pollen germination Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen 3.6 Post-Cryopreservation Viability Assessment 3.7 Scanning Electron Microscopy Analysis 4 Notes: Precautions, Tips to Get Good Quality Pollen and In Vitro Germination References Chapter 39: Pollen Cryopreservation Protocol for Cayratia pedata 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen Grains 3.2 Dehydration of Pollen 3.3 Preparation of Pollen Medium 3.4 Initial Viability Assessment 3.5 Cryopreservation of Pollen 3.6 Post-Cryopreservation Viability Assessment 4 Notes: Precautions, Tips to Get Good Quality Pollen and In Vitro Germination References Chapter 40: Pollen Cryopreservation in Celastrus paniculatus 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Pollen Collection and Dehydration 3.2 Preparation of Pollen Germination Medium 3.3 Viability Assessment of Pollen 3.4 Cryopreservation 3.5 Scanning Electron Microscopy Analysis 3.6 Post Storage Viability and Fertility Assessment Through Field Pollination 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 41: Pollen Cryopreservation Protocol for Decalepis arayalpathra (Apocyanaceae) 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen Grains 3.2 Dehydration of Pollen Grains 3.3 Preparation of Pollen Germination Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains 3.6 Post-Cryopreservation Viability Assessment 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 42: Pollinia Cryopreservation Protocol for Decalepis hamiltonii Wight & Arn 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollinia 3.2 Dehydration of Pollinia 3.3 Preparation of Pollen Germination Medium 3.4 Viability Assessment of Pollinia 3.5 Cryopreservation of Pollinia 3.6 Post-Cryopreservation Viability Assessment 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 43: Cryopreservation and In Vitro Pollen Germination Protocol in Embelia ribes Burm. f 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 Chemicals and Solutions 3 Methods 3.1 Pollen Collection and Preparation for Cryopreservation 3.2 Cryopreservation 3.3 Pollen Viability 3.4 In Vitro Germination 3.5 Post-Cryopreservation Viability Assessment 4 Notes References Chapter 44: Pollinia Cryopreservation Protocol for Holostemma ada-kodien 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection and Dehydration of Pollen Grains 3.2 Preparation of Pollen Germination Medium 3.3 Viability Assessment of Pollen 3.4 Estimation of Pollen Germination 3.5 Cryopreservation of Pollen Grains 3.6 Post-Cryopreservation Viability Assessment 3.7 Fertility Assessment 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 45: Pollen Cryopreservation Protocol for Oroxylum indicum 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.2 Pollen Moisture Determination 3.3 Dehydration of Pollen Grains 3.4 Preparation of Pollen Medium 3.5 In Vitro Viability Assessment 3.6 Staining and Scoring of Pollen (Fig. 4) 3.7 Cryopreservation of Pollen Grains 3.8 Scanning Electron Microscopy Analysis 3.9 Post-Cryopreservation Viability Assessment 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 46: Pollen Cryopreservation Protocol for Salacia chinensis Linn 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.2 Dehydration of Pollen 3.3 Preparation of Pollen Germination Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains 3.6 Post-Cryopreservation Viability Assessment 3.7 Scanning Electron Microscopy Analysis 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 47: Pollen Cryopreservation Protocol For Saraca asoca (Roxb.) De Wilde 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen Grains 3.2 Dehydration of Pollen Grains 3.3 Preparation of Pollen Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains 3.6 Post-Cryopreservation Viability Assessment 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 48: Pollen Cryopreservation in Stevia rebaudiana Bertoni 1 Introduction 2 Material and Methods 2.1 Plant Material 2.2 Laboratory Equipment Required to Carry Out Experiments 2.3 General Laboratory Supplies 2.4 Chemicals and Solutions Required for Viability Assessment 2.5 Collection of Pollen 2.5.1 Procedure for Pollen Collection 2.6 In Vitro Pollen Germination Medium 2.6.1 Brewbaker and Kwack Medium (100 mL) Stock Solution Preparation 2.6.2 Preparation of Pollen Germination Media 2.7 Pollen Germination (Hanging Drop Method ) 2.8 Fresh Pollen Moisture Content Estimation 2.9 Desiccation of Pollen 2.10 Cryopreservation 2.11 Retrieval and Regrowth of Cryopreserved Pollen 2.12 Fertility Assessment in Field 2.13 Scanning Electron Microscopy Analysis 3 Notes References Part V: Plantation Crops Chapter 49: Methods for Cryopreserving of Date Palm Pollen 1 Introduction 2 Foreword Preface Contents Contributors Chapter 1: Pollen Cryopreservation: Advances and Prospects 1 Introduction and Relevance of Plant Cryopreservation 2 Historical Aspects of Plant Cryopreservation 3 Pollen Cryopreservation 3.1 Why Pollen Cryopreservation? 3.2 Pollen Cryobanks 3.3 Pollen Cryopreservation: Advantages and Disadvantages 3.4 Requirements and Practical Considerations in Pollen Cryopreservation 3.5 Uses and Applications 4 Present Status of Plant and Pollen Cryopreservation 5 Prospects and Way Forward References Chapter 2: Pollen Cryobanking-Implications in Genetic Conservation and Plant Breeding 1 Introduction 1.1 Germplasm Conservation 1.2 Increasing Breeding Efficiency 1.3 Exchange of Pollen (Nuclear Genetic Diversity) 1.4 Integration of Conventional Breeding Methods with Modern Biotechnological Practices 1.5 Genetic Amelioration 2 Uses of Cryopreserved Pollen in Production Fields 3 Pollen Morphology and Pollen Classification 4 Pollen Cryopreservation Protocol 4.1 Pollen collection Methods 4.2 Pollen Viability Testing 4.2.1 Staining Techniques 4.2.2 In Vivo Pollen Germination 4.2.3 In Vivo Pollen Germination and Pollen Tube Growth 4.3 Moisture Content Determination 4.4 Pollen Desiccation 4.5 Pollen Cryopreservation 4.6 Pollen Rehydration 4.7 Post-cryopreservation Viability and Fertility Assessment 5 Factors Affecting Pollen Viability and Cryopreservation 6 Advantages of Cryopreserving Pollen 7 Limitations of Pollen Cryopreservation 8 Need for Protection and Grant of IPR to Cryopreserved Pollen 9 Way Forward References Chapter 3: Establishment of a Pollen Cryobank and General Procedures of Pollen Cryopreservation 1 Introduction 2 Infrastructure Facilities 3 Cryo-equipments Required 3.1 Laboratory Equipments and Accessories Required 3.2 Storage Containers 4 Cryotransit and Shipment 5 Cost of Cryobanking 6 Potential Hazards/Threats in Cryostorage 7 Basic Safety Measures 8 Pollen Cryopreservation: General Procedures 8.1 Pollen Collection 8.2 Pollen Viability Assessment 8.3 Pollen Cryopreservation 8.4 Field Pollinations with Cryopreserved Pollen for Fertility Assessment 9 Pollen Cryobank at ICAR-IIHR, Bengaluru 10 Case Studies on Successful Pollen Cryopreservation in Species of Economical Importance 10.1 Orchid Pollen Cryopreservation 10.2 Tomato Pollen Cryopreservation 10.3 Apple Pollen Cryopreservation 11 Conclusion and Way Forward References Part I: Fruits Chapter 4: Pollen Cryopreservation Protocol in Annona Species 1 Introduction 2 Materials 2.1 Plant Material for Pollen 2.2 Required Laboratory Equipments 2.3 General Laboratory Equipments 2.4 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.1.1 Inventory 3.1.2 Anthesis in Annonaceous Species 3.1.3 Bagging of Flower and Pollen Collection 3.2 Dehydration of Pollen Grains 3.3 Immersion of the Pollen Grains in Liquid Nitrogen 3.4 Retrieval 3.5 Precautions 3.6 Viability Testing In Vitro 3.6.1 Inventory 3.6.2 Preparation of Pollen Germination Medium 3.6.3 In Vitro Pollen Germination 3.6.4 Precautions 3.7 In Vivo Fertility Test 3.7.1 Inventory 3.7.2 Procedure 3.7.3 Precautions 3.8 SEM Analysis of Fresh and Cryopreserved Pollen 3.8.1 Inventory 3.8.2 Procedure 4 Notes 4.1 To Be Taken Care Of References Chapter 5: Pollen Cryopreservation in Aonla 1 Introduction 2 Materials 2.1 Collection of Aonla Pollen 2.2 In Vitro Germination of Pollen Grains 2.3 Cryopreservation of Pollen in Liquid Nitrogen 2.4 In Vivo Fertility Test 3 Methods 3.1 Collection of Aonla Pollen 3.2 Pollen Cryopreservation in Aonla 3.3 Pollen Germination in Aonla 3.3.1 Preparation of Culture Medium 3.3.2 In Vitro Germination of Pollen Grains 3.4 In Vivo Viability Testing of Aonla Cryostored Pollen 3.5 Scanning Electron Microscopy Analysis 4 Conclusion References Chapter 6: Cryopreservation of Pollen Grains of Carica papaya and Different Vasconcellea Species 1 Introduction 2 Materials 2.1 Plant Material 2.2 Laboratory Equipment Required to Carry Out Experiments 2.3 General Laboratory Supplies 2.4 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.1.1 Inventory 3.1.2 Time of Anthesis 3.1.3 Flower Bagging and Collection 3.1.4 Pollen Collection from Anthers 3.2 Cryopreservation 3.3 Retrieval 3.4 Precautions 3.5 In Vitro Viability Testing 3.5.1 Inventory 3.5.2 Medium Preparation 3.5.3 In Vitro Pollen Germination 3.5.4 Precautions 3.6 In Vivo Test for Fertility 3.6.1 Inventory 3.6.2 Procedure 3.6.3 Fruit and Seed Set 3.6.4 Precautions 3.7 SEM Analysis of Cryopreserved Pollen 3.7.1 Inventory 3.7.2 Procedure 4 Notes References Chapter 7: Cryopreservation of Pollen Grains of Citrus and Other Aurantioideae 1 Introduction 2 Materials 2.1 Collection 2.2 Dehydration 2.3 Storage in Liquid Nitrogen 2.4 Thawing 2.5 Rehydration 2.6 In Vitro Germination 2.7 In Vivo Viability Testing 3 Methods 3.1 Collection 3.2 Dehydration 3.3 Storage in Liquid Nitrogen 3.4 Thawing 3.5 Rehydration 3.6 Acetocarmine Staining 3.7 In Vitro Germination 3.8 In Vivo Viability Testing 4 Notes References Chapter 8: Pollen Cryopreservation Protocol for Dragon Fruit 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.2 Dehydration of Pollen 3.3 Preparation of Pollen Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains 3.6 Post-Cryopreservation Viability Assessment 3.7 Scanning Electron Microscopy Analysis 3.8 Retrieval and Post-Storage Fertility Assessment 4 Notes: Precautions, Tips to Get Good Quality Pollen and In Vitro Germination References Chapter 9: Cryopreservation of Grape Pollen (Vitis Species) 1 Introduction 2 Material 2.1 Plant Material 2.2 Laboratory Equipment Required to Carry Out Experiments 2.3 General Laboratory Supplies 2.4 Chemicals and Solutions 2.5 Inventory 2.5.1 Pollen Collection 2.5.2 In Vivo Fertility Assessment Fresh and Cryopreserved Pollen 2.5.3 In Vitro Viability Testing 3 Methods 3.1 Time of Anthesis 3.2 Flower Bagging and Pollen Collection 3.3 Cryopreservation 3.3.1 Precautions 3.3.2 Procedure of Retrieval and Post-storage Fertility Assessment 3.4 In Vitro Viability Testing 3.4.1 Medium Preparation 3.4.2 Procedure for In Vitro Pollen Viability Assessment 3.4.3 Precautions 3.5 Procedure of In Vivo Fertility of Cryopreserved Pollen 3.5.1 Fruit and Seed Set 3.5.2 Precautions 4 Notes References Chapter 10: Pollen Cryopreservation in Jackfruit (Artocarpus heterophyllus) for Crop Improvement 1 Introduction 2 Materials 2.1 Laboratory Equipments Required to Carry Out Experiments 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.1.1 Inventory 3.1.2 Procedure 3.1.3 Precautions 3.2 Dehydration and Determination of Moisture Content of Pollen 3.2.1 Inventory 3.2.2 Procedure 3.2.3 Precautions 3.3 Cryostorage 3.3.1 Inventory 3.3.2 Cryopreservation 3.3.3 Precautions 3.4 Thawing and Retrieval 3.5 Pollen Germination Medium 3.5.1 Inventory 3.5.2 Preparation of Pollen Germination Medium 3.5.3 Precautions 3.6 In Vitro Germination Test 3.6.1 Inventory 3.6.2 Hanging Drop Technique 3.6.3 Incubation 3.6.4 Precautions 3.7 Staining and Scoring of Pollen 3.7.1 Inventory 3.7.2 Staining 3.7.3 Scoring 3.7.4 Precautions 3.8 In Vivo Test for Fertility by Field Pollination 3.8.1 Inventory 3.8.2 Procedure 3.8.3 Fruit Set and Seed Set 3.8.4 Precautions 3.8.5 Scanning Electron Microscopy Analysis 4 Notes References Chapter 11: Pollen Cryopreservation in Jamun (Syzygium cuminii Skeels) 1 Introduction 2 Materials 2.1 Collection 2.2 Viability Tests In Vitro 2.3 Cryostorage/Cryopreservation (Storage in LN) 2.4 Viability Tests In Vitro 3 Methods 3.1 Collection 4 Viability Testing 4.1 Media Preparation for Pollen Germination 4.2 In Vitro Germination 4.3 Incubation 4.4 Staining 4.5 Scoring of Pollen Cultures 5 Storage 6 Retrieval and Rewarming 7 In Vivo Test for Fertility 7.1 Breeder ́s Crossing Kit 7.2 Procedure 8 Notes References Chapter 12: Pollen Cryopreservation in Passiflora Species 1 Introduction 2 Materials 2.1 Laboratory Equipments Required to Carry Out Experiments 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 2.4 Collection 2.5 Dehydration and Determination of Moisture Content of Pollen 2.6 Viability Tests In Vitro 2.7 Cryopreservation (Storage in LN) 2.8 Viability Tests In Vitro 2.9 In Vivo Fertility Test 3 Methods 3.1 Collection 3.2 Dehydration 3.3 Medium Preparation for Pollen Germination 3.4 Pollen Viability and In Vitro Germination 3.5 Differential Staining Techniques 3.6 In Vitro Pollen Germination 3.7 Incubation 3.8 Scoring of Pollen Cultures 3.9 Cryopreservation 3.10 Retrieval and Rewarming 3.11 In Vivo Test for Fertility 4 Notes References Chapter 13: Pollen Cryopreservation Protocol for Poncirus trifoliata 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.2 Dehydration of Pollen Grains 3.3 Preparation of Pollen Germination Medium 3.4 Viability Assessment 3.5 Cryopreservation of Pollen Grains 3.6 Fertility Assessment 3.7 Seed Germination 4 Notes: Precautions, Tips to Get Good Quality Pollen and In Vitro Germination References Chapter 14: Cryopreservation of Pollen Grains of Different Psidium Species 1 Introduction 2 Materials 2.1 Plant Material 2.2 Laboratory Equipments Required to Carry Out Experiments 2.3 General Laboratory Supplies 2.4 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.1.1 Inventory 3.1.2 Anthesis in Psidium Species 3.1.3 Flower Bagging and Collection 3.1.4 Pollen Collection From Anthers 3.2 Cryopreservation 3.3 Retrieval 3.4 Precautions 3.5 In Vitro Viability Testing 3.5.1 Inventory 3.5.2 Medium Preparation 3.5.3 In Vitro Pollen Germination 3.5.4 Precautions 3.6 In Vivo Test for Fertility 3.6.1 Inventory 3.6.2 Procedure 3.6.3 Fruit and Seed Set 3.6.4 Precautions 3.7 SEM Analysis of Fresh and Cryopreserved Pollen 3.7.1 Inventory 3.7.2 Procedure 4 Notes References Chapter 15: Cryopreservation of Pomegranate (Punica granatum L.) Pollen 1 Introduction 2 Materials 2.1 Plant Material 2.2 Required Laboratory Equipments 2.3 General Laboratory Supplies 2.4 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.1.1 Inventory 3.1.2 Anthesis Time 3.1.3 Flower Bagging and Pollen Collection 3.2 Pre-storage Viability Assessment 3.2.1 Inventory 3.2.2 In Vitro Pollen Germination 3.2.3 Precautions 3.3 Processing and Cryopreservation 3.4 Retrieval and Post-storage Viability Assessment 3.4.1 Precautions 3.5 In Vivo Viability of Cryopreserved Pollen of Pomegranate 3.5.1 Inventory 3.5.2 Procedure 3.5.3 Fruit and Seed Set 3.5.4 Precautions 4 Notes References Chapter 16: Pollen Cryopreservation in Mango 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.2 Extraction of Pollen 3.3 Dehydration of Pollen 3.4 Preparation of Pollen Germination Medium 3.5 Initial Viability Assessment 3.6 Cryopreservation 3.7 Scanning Electron Microscopy Analysis 3.8 Retrieval and Post-storage Viability Assessment 3.9 Fertility Assessment 4 Notes References Part II: Vegetables Chapter 17: Pollen Cryopreservation in Ash Gourd (Benincasa hispida (Thunb.) Cogn.) 1 Introduction 2 Materials 2.1 Required Laboratory Equipments 2.2 General Laboratory Equipments 2.3 Chemicals and Solutions 3 Method 3.1 Pollen Collection 3.2 Pollen Drying or Desiccation 3.3 Pollen Viability Assessment in vitro 3.3.1 Brewbaker and Kwack ́s Medium (100 mL) Stock Solution Preparation 3.3.2 Preparation of Pollen Germination Medium 3.3.3 Procedure For Viability Assessment of Fresh Pollen 3.4 Pollen Cryopreservation 3.5 Retrieval and Post-storage Viability/Fertility Assessment 3.5.1 Viability Assessment of Cryopreserved Pollen 3.5.2 Field Pollination 4 Notes References Chapter 18: Pollen Cryopreservation in Bitter Gourd 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Pollen Viability Test 2.3 Dehydration and Packaging of Pollen 2.4 Immersion of Pollen Grains in Liquid Nitrogen 2.5 In Vitro Germination of Pollen 2.6 In Vivo Fertility Test 3 Methods 3.1 Collection of Anthers with Pollen Grains 3.2 Collection of the Pollen 3.3 Dehydration of the Pollen 3.4 Preparation of the Pollen Germination Medium 3.5 In Vitro Germination of the Pollen 3.6 Cryopreservation of Pollen Grains in Liquid Nitrogen 3.7 In Vivo Viability Testing 3.8 Scanning Electron Microscopy Analysis 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 19: Pollen Cryopreservation in Bottle Gourd for Breeding and Conservation 1 Introduction 2 Checklist for In Vitro Pollen Germination 2.1 Pollen Germination Medium Composition 2.2 Pollen Collection 2.3 Pollen Desiccation 2.4 Viability Assessment 3 Procedure 3.1 Cryopreservation 3.2 Procedure 3.3 Retrieval and Post-Storage Fertility Assessment 3.4 Procedure 4 The Following Points Should Be Noted References Chapter 20: Cryopreservation of Eggplant (Solanum melongena L.) Pollen for Utilization in Crop Improvement 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Dehydration and Packaging of Pollen Grains 2.3 Immersion of Pollen Grains in Liquid Nitrogen 2.4 In Vitro Germination of Pollen Grains 2.5 In Vivo Viability Test 3 Methods 3.1 Collection of Anthers Pollen 3.2 Collection of the Pollen Grains 3.3 Dehydration of the Pollen Grains 3.4 Cryopreservation of Pollen Grains 3.5 In Vitro Germination of the Pollen Grains 3.5.1 Preparation of Nutrient Medium [21] 3.5.2 Preparation for In Vitro Germination 3.5.3 Viability Test 3.6 In Vivo Fertility Testing 4 Notes References Chapter 21: Cryopreservation of Pollen Grains of Hot Pepper 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Dehydration and Packaging of Pollen Grains 2.3 Immersion of Pollen Grains in Liquid Nitrogen 2.4 In Vitro Germination of Pollen Grains 2.5 In Vivo Viability Test 3 Methods 3.1 Collection of Anthers with Pollen Grains 3.2 Collection of the Pollen Grains 3.3 Dehydration of the Pollen Grains 3.4 Cryopreservation of Pollen Grains 3.5 In Vitro Germination of the Pollen Grains 3.6 Fertility Assessment of the Pollen Grains (see Note 10) 4 Notes References Chapter 22: Cryopreservation of Muskmelon (Cucumis melo) Pollen for Exploring Breeding Possibilities 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Dehydration and Packaging of Pollen Grains 2.3 Cryopreservation of Pollen Grains 2.4 In Vitro Germination of Pollen Grains 2.5 In Vivo Fertility Test 3 Methods 3.1 Collection of Flowers with Pollen Grains 3.2 Extraction of the Pollen Grains 3.3 Dehydration of the Pollen Grains 3.4 Preparation of the Pollen Germination Medium 3.5 In Vitro Germination of the Pollen Grains 3.6 In Vivo Fertility Testing 4 Notes References Chapter 23: Onion (Allium cepa L.) Pollen Cryopreservation Protocol 1 Introduction 2 Materials 2.1 Onion Pollen Grain Collection 2.2 Dehydration and Packaging 2.3 Immersion of Pollen Grains 2.4 In Vitro Pollen Grain Germination 2.5 In Vivo Fertility Test 3 Methods 3.1 Collection of Anthers from Umbel 3.2 Collection of the Pollen Grains from Anther 3.3 Dehydration of Onion Pollen Grains 3.4 Cryopreservation of Pollen Grains 3.5 In Vitro Pollen Grain Germination 3.5.1 Preparation of Germination Medium 3.5.2 Preparation for Pollen Germination Test 3.5.3 Viability Test 3.6 In Vivo Fertility Test 4 Notes References Chapter 24: Cryopreservation of Okra (Abelmoschus esculentus L.) Pollen 1 Introduction 2 Materials 2.1 Plant Material 2.2 Collection of Pollen 2.3 In Vitro Pollen Germination Medium 2.4 Pollen Viability Assessment 2.5 Pollen Moisture Content Estimation 2.6 Desiccation of Pollen 2.7 Cryopreservation 3 Methods 3.1 Collection of Pollen 3.2 In Vitro Pollen Germination Medium 3.3 Pollen Germination (Sitting Drop Method) 3.4 Fresh Pollen Moisture Content Estimation 3.5 Desiccation of Pollen 3.6 Determination of Optimum Desiccation Period to Attain Optimum Moisture Content for Cryopreservation 3.7 Cryopreservation 3.8 Retrieval and Regrowth of Cryopreserved Pollen (see Note 13) 3.9 Fertility Assessment in Field (Fig. 3) 4 Notes References Chapter 25: Protocol for Pollen Viability and Cryopreservation of Pumpkin (Cucurbita moschata Duchesne ex Poir) 1 Introduction 2 Materials 2.1 Collection of Pollen 2.2 Dehydration of Pollen 2.3 Cryopreservation of Pollen in Liquid Nitrogen 2.4 In Vitro Germination of Pollen 3 Methods 3.1 Collection of Flowers 3.2 Pollen Extraction 3.3 Dehydration of Pollen 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen in Liquid Nitrogen 3.6 Post-cryopreservation Viability and Fertility Assessment 3.7 Scanning Electron Microscopy Analysis References Chapter 26: Pollen Cryopreservation in Ridge Gourd (Luffa acutangula (Roxb.) L.) 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Viability Test 2.3 Dehydration and Packaging of Pollen Grains 2.4 Immersion of Pollen Grains in Liquid Nitrogen 2.5 In Vitro Germination of Pollen Grains 2.6 In Vivo Fertility Test 3 Methods 3.1 Collection of Flowers with Pollen Grains 3.2 Extraction of the Pollen Grains 3.3 Dehydration of the Pollen Grains 3.4 Preparation of the Pollen Germination Medium 3.5 In Vitro Germination of the Pollen Grains 3.6 In Vivo Testing References Chapter 27: Pollen Cryopreservation Protocol for Wild Species of Solanum 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Pollen Collection and Dehydration 3.2 Preparation of Pollen Medium 3.3 Viability Assessment 3.4 Cryopreservation 3.5 Retrieval and Post-storage Fertility Assessment 3.6 Scanning Electron Microscopy Analysis 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 28: Feasibility Studies on Pollen Cryopreservation in Spine Gourd (Momordica dioica Roxb.) 1 Introduction 2 Materials 2.1 Collection of Pollen 2.2 Dehydration of Pollen 2.3 Cryopreservation of Pollen in Liquid Nitrogen 2.4 In Vitro Germination of Pollen 3 Methods 3.1 Collection of Flowers 3.2 Pollen Extraction 3.3 Dehydration of Pollen 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen in Liquid Nitrogen 3.6 Post-cryopreservation Viability and Fertility Assessments 3.7 Scanning Electron Microscopy Analysis 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 29: Cryopreservation Protocol for Tomato (Solanum lycopersicum L.) and its wild relatives Pollen for Utilization in Cr... 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Dehydration and Packaging of Pollen Grains 2.3 Immersion of Pollen Grains in Liquid Nitrogen 2.4 In Vitro Germination of Pollen Grains 2.5 Fertility Assessment of Pollen Grains 3 Methods 3.1 Collection of Anthers with Pollen Grains 3.2 Collection of the Pollen Grains 3.3 Dehydration of the Pollen Grains 3.4 Cryopreservation of Pollen Grains 3.5 In Vitro Germination of the Pollen Grains 3.6 Fertility Assessment of Pollen Grains 3.7 Scanning Electron Microscopy Analysis 4 Notes References Chapter 30: Pollen Cryopreservation Protocol for Teasel Guard (Momordica subangulata subsp. Renigera) 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Dehydration of Pollen Grains 2.3 Cryopreservation of Pollen Grains in Liquid Nitrogen 2.4 In Vitro Germination of Pollen Grains 3 Methods 3.1 Collection of Flowers 3.2 Pollen Extraction 3.3 Dehydration of Pollen 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains in Liquid Nitrogen 3.6 Post-cryopreservation Viability and Fertility Assessments 3.7 Scanning Electron Microscopy Analysis 4 Notes: Precautions, Tips to Get Good Quality Pollen and In Vitro Germination References Chapter 31: Cryopreservation of Watermelon Pollen to Explore New Breeding Possibilities 1 Introduction 2 Materials 2.1 Collection of Pollen Grains 2.2 Dehydration and Packaging of Pollen Grains 2.3 Cryopreservation of Pollen Grains 2.4 In Vitro Germination of Pollen Grains 2.5 In Vivo Fertility Test 3 Methods 3.1 Collection of Anthers 3.2 Extraction of the Pollen Grains 3.3 Dehydration of the Pollen Grains 3.4 Preparation of the Pollen Germination Medium 3.5 In Vitro Germination of the Pollen Grains 3.6 In Vivo Fertility Testing 4 Notes References Part III: Ornamentals Chapter 32: Pollen Cryopreservation Protocol for Gladiolus 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.2 Dehydration of Pollen Grains 3.3 Preparation of Pollen Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains 3.6 Post-cryopreservation Viability Assessments 3.7 Fertility Assessment 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 33: Pollen Cryopreservation Protocol for Gloriosa superba L. 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.2 Dehydration of Pollen 3.3 Preparation of Pollen Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains 3.6 Scanning Electron Microscopy Analysis 3.7 Post-cryopreservation Viability Assessments 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 34: Pollen Cryopreservation in Marigold (Tagetes spp.) 1 Introduction 2 Materials 2.1 Plant Material 2.2 Checklist for In vitro Pollen Germination 2.3 Pollen Germination Medium Composition (Brewbaker and Kwack Medium) 3 Methods 3.1 Pollen Collection 3.2 Pollen Desiccation 3.3 Brewbaker and Kwack Medium (100 mL) Stock Solution Preparation 3.4 Viability Assessment 3.5 Cryopreservation 3.6 Retrieval and Post-storage Fertility Assessment 4 Notes References Chapter 35: Pollinia Cryopreservation of Selected Orchid Species of Western Ghats, India 1 Introduction 2 Materials 2.1 Collection 2.2 Dehydration If Required and Determination of Moisture Content of Pollen 2.3 Viability Tests In Vitro 2.4 Cryopreservation (Storage in LN) 2.5 Thawing and Rewarmig 2.6 Fertility Tests If Any in In Vivo Controlled Field Pollination 3 Methods 3.1 Collection 3.2 Dehydration/Controlled Hydration/Drying 3.3 Precautions 4 Viability Testing 4.1 Inventory 4.2 Medium Preparation for Pollen Germination 4.3 Precautions 4.4 In Vitro Germination 4.4.1 Sitting Drop Method (Calanthe masuca, Cymbidium bicolor, C. ensifolium, D. ovatum, E. cullenii, R. retusa) 4.4.2 In Vivo Method (A. praemorsa, V. spathulata, V. wightii) 4.5 Incubation 4.5.1 In Vitro Method 4.5.2 In Vivo Method 4.6 Staining 4.6.1 In Vitro Germination 4.6.2 In Vivo Germination 4.7 Scoring of Pollen Cultures 4.8 General Precautions 5 Storage 5.1 Inventory 5.2 Procedure 5.3 Precautions 6 Retrieval and Re-warming 6.1 Procedure 6.2 Precautions 7 In Vivo Test for Fertility/Field Pollination 7.1 Inventory 7.2 Breeder Crossing Kit 7.3 Procedure 7.4 Fruit Set and Seed Set Test 7.5 In Vivo Germination and Pollen Tube Growth 7.6 Precautions 8 Notes References Chapter 36: Pollen Cryopreservation Protocol for Tuberose (Polianthes tuberosa L.) 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Flowers 3.2 Collection of Pollen 3.3 Dehydration of Pollen 3.4 Viability Assessment of the Pollen 3.5 Cryopreservation of Pollen in Liquid Nitrogen 3.6 Post-cryopreservation Viability and Fertility Assessments References Chapter 37: Pollen Cryopreservation Protocol for Rose 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.2 Dehydration of Pollen Grains 3.3 Preparation of Pollen Medium 3.4 Viability Assessment 3.5 Cryopreservation (Storage in LN) 3.6 Fertility Assessment 3.7 Scanning Electron Microscopy Analysis References Part IV: Medicinal Plants Chapter 38: Pollen Cryopreservation in Aloe vera 1 Introduction 2 Materials 2.1 Laboratory Equipments 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.2 Dehydration of Pollen 3.3 Preparation of Pollen germination Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen 3.6 Post-Cryopreservation Viability Assessment 3.7 Scanning Electron Microscopy Analysis 4 Notes: Precautions, Tips to Get Good Quality Pollen and In Vitro Germination References Chapter 39: Pollen Cryopreservation Protocol for Cayratia pedata 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen Grains 3.2 Dehydration of Pollen 3.3 Preparation of Pollen Medium 3.4 Initial Viability Assessment 3.5 Cryopreservation of Pollen 3.6 Post-Cryopreservation Viability Assessment 4 Notes: Precautions, Tips to Get Good Quality Pollen and In Vitro Germination References Chapter 40: Pollen Cryopreservation in Celastrus paniculatus 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Pollen Collection and Dehydration 3.2 Preparation of Pollen Germination Medium 3.3 Viability Assessment of Pollen 3.4 Cryopreservation 3.5 Scanning Electron Microscopy Analysis 3.6 Post Storage Viability and Fertility Assessment Through Field Pollination 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 41: Pollen Cryopreservation Protocol for Decalepis arayalpathra (Apocyanaceae) 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen Grains 3.2 Dehydration of Pollen Grains 3.3 Preparation of Pollen Germination Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains 3.6 Post-Cryopreservation Viability Assessment 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 42: Pollinia Cryopreservation Protocol for Decalepis hamiltonii Wight & Arn 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollinia 3.2 Dehydration of Pollinia 3.3 Preparation of Pollen Germination Medium 3.4 Viability Assessment of Pollinia 3.5 Cryopreservation of Pollinia 3.6 Post-Cryopreservation Viability Assessment 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 43: Cryopreservation and In Vitro Pollen Germination Protocol in Embelia ribes Burm. f 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 Chemicals and Solutions 3 Methods 3.1 Pollen Collection and Preparation for Cryopreservation 3.2 Cryopreservation 3.3 Pollen Viability 3.4 In Vitro Germination 3.5 Post-Cryopreservation Viability Assessment 4 Notes References Chapter 44: Pollinia Cryopreservation Protocol for Holostemma ada-kodien 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection and Dehydration of Pollen Grains 3.2 Preparation of Pollen Germination Medium 3.3 Viability Assessment of Pollen 3.4 Estimation of Pollen Germination 3.5 Cryopreservation of Pollen Grains 3.6 Post-Cryopreservation Viability Assessment 3.7 Fertility Assessment 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 45: Pollen Cryopreservation Protocol for Oroxylum indicum 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Pollen Collection 3.2 Pollen Moisture Determination 3.3 Dehydration of Pollen Grains 3.4 Preparation of Pollen Medium 3.5 In Vitro Viability Assessment 3.6 Staining and Scoring of Pollen (Fig. 4) 3.7 Cryopreservation of Pollen Grains 3.8 Scanning Electron Microscopy Analysis 3.9 Post-Cryopreservation Viability Assessment 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 46: Pollen Cryopreservation Protocol for Salacia chinensis Linn 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen 3.2 Dehydration of Pollen 3.3 Preparation of Pollen Germination Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains 3.6 Post-Cryopreservation Viability Assessment 3.7 Scanning Electron Microscopy Analysis 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 47: Pollen Cryopreservation Protocol For Saraca asoca (Roxb.) De Wilde 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 General Laboratory Supplies 2.3 Chemicals and Solutions 3 Methods 3.1 Collection of Pollen Grains 3.2 Dehydration of Pollen Grains 3.3 Preparation of Pollen Medium 3.4 Viability Assessment of Pollen 3.5 Cryopreservation of Pollen Grains 3.6 Post-Cryopreservation Viability Assessment 4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination References Chapter 48: Pollen Cryopreservation in Stevia rebaudiana Bertoni 1 Introduction 2 Material and Methods 2.1 Plant Material 2.2 Laboratory Equipment Required to Carry Out Experiments 2.3 General Laboratory Supplies 2.4 Chemicals and Solutions Required for Viability Assessment 2.5 Collection of Pollen 2.5.1 Procedure for Pollen Collection 2.6 In Vitro Pollen Germination Medium 2.6.1 Brewbaker and Kwack Medium (100 mL) Stock Solution Preparation 2.6.2 Preparation of Pollen Germination Media 2.7 Pollen Germination (Hanging Drop Method ) 2.8 Fresh Pollen Moisture Content Estimation 2.9 Desiccation of Pollen 2.10 Cryopreservation 2.11 Retrieval and Regrowth of Cryopreserved Pollen 2.12 Fertility Assessment in Field 2.13 Scanning Electron Microscopy Analysis 3 Notes References Part V: Plantation Crops Chapter 49: Methods for Cryopreserving of Date Palm Pollen 1 Introduction