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 Materials
2.1 Collection
2.2 Determination of Moisture Content
2.3 Cryostorage
2.4 Viability Assessment
2.4.1 Culture Medium
2.4.2 Hydration/Rehydration Conditions
2.4.3 Germination In Vitro
3 Methods
3.1 Collection
3.2 Moisture Content
3.3 Storage
3.4 Retrieval and Rewarming
3.5 Viability Assessment
3.5.1 Medium Preparation
3.5.2 Hydration/Rehydration Conditions
3.5.3 In Vitro Germination
3.5.4 Scoring Pollen Viability
4 Notes
References
Chapter 50: Cryopreservation of Coconut and Arecanut Pollen
1 Coconut
2 Materials and Methods
2.1 Male Flower Extraction
2.2 Drying of Male Flowers
2.3 Pollen Germination Studies
2.4 Cryopreservation
2.5 Male Flower collection and Extraction of Pollen
2.6 Dehydration of Pollen
2.7 Assessment of Pollen Viability Before Storage in Liquid Nitrogen
2.8 Pollen Germination Procedure
2.9 Cryopreservation Procedure
2.10 Retrieval of Cryostored Pollen
2.11 Assessment Fertility of Cryopreserved Pollen
2.11.1 Collection and Preparation of Pollen
3 Arecanut
3.1 Materials and Methods
3.2 Collection
3.3 Desiccation, Extraction, and Determination of Moisture Content of Pollen
3.4 Viability Tests In Vitro and Documentation
3.5 Cryopreservation and Rewarming
3.6 Fertility Assessment
4 Conclusion
References
Part VI: Root Crop
Chapter 51: Pollen Cryopreservation in Cassava
1 Introduction
2 Materials
2.1 Laboratory Equipment
2.2 General Laboratory Supplies
2.3 Chemicals and Solutions
3 Methods
3.1 Collection of Male Flowers
3.2 Immersion and Storage of Male Flowers with Pollen Grains in Liquid Nitrogen
3.3 Preparation of Medium for In Vitro Germination of Pollen
3.4 Pollen Viability Test Through In Vitro Germination
3.5 Pollen Viability Test Through Acetocarmine Staining Assay
3.6 Evaluation of Pollen Fertility by Fruit and Seed Set Test
4 Notes
References
Part VII: Forest Species
Chapter 52: Pollen Cryopreservation Protocol for Neolamarckia cadamba (Roxb.) Miq
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 Grains
3.5 Cryopreservation of Pollen Grains
3.6 Post-Cryopreservation Viability Assessments
4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination
References
Chapter 53: Pollen Cryopreservation of Coniferous Serbian Spruce (Picea omorika/Panč./Purkyne) and Deciduous Pedunculate Oak (...
1 Introduction
2 Materials
2.1 Collection of Pollen Grains
2.2 Laboratory Equipment and Materials
3 Method
3.1 Viability of Pollen Laboratory Equipment, Material, and Reagents
3.2 Pollen Viability Analysis
3.3 In Vitro Test on Nutrient Medium: Pollen Germination and Pollen Germination (Pollen Tube Length)
4 Notes
References
Chapter 54: Pollen Cryopreservation Protocol for Couroupita guianensis Aubl.
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
3.6 Post-Cryopreservation Viability Assessment
4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination
References
Chapter 55: Pollen Cryopreservation Protocol for Gmelina arborea
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
4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination
References
Chapter 56: Pollen Cryopreservation in Moringa concanensis for Crop Improvement
1 Introduction
2 Materials
2.1 Laboratory Equipment 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 Scanning Electron Microscopy Analysis
4 Notes
References
Chapter 57: Cryopreservation of Sandalwood (Santalum album L.) Pollen
1 Introduction
2 Materials Required
2.1 Collection of Pollen Grains
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 Collection and Dehydration of Anthers
3.3 Preparation of Pollen Medium
3.4 Viability Assessment of Pollen
3.5 Cryopreservation of Pollen
3.6 Post-Cryopreservation Viability and Fertility Assessment
4 Notes: Precautions, Dos and Don´ts to Get Good-Quality Pollen and In Vitro Germination
References
Chapter 58: Cryopreservation of Pollen for Long-Term Storage in Teak (Tectona grandis)
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 Anthers
3.2 Preparation of Pollen Medium
3.3 Viability Assessment of Pollen
3.4 Cryopreservation of Anthers in Liquid Nitrogen
3.5 Post-Cryopreservation Viability Assessment
3.6 Scanning Electron Microscopy Analysis
4 Notes: Precautions, Tips to Get Good-Quality Pollen and In Vitro Germination
References
Index