Pollen grains are everywhere - in the air, in the water, in soil and in the food we eat. Pollen has direct relevance in agriculture, horticulture, plant breeding, crop improvement and biotechnology. They are further of use for monitoring cytotoxic effects - by herbicides, pesticides and pollutants - testing for allergic reactions, and for basic studies on gene expression, research on differentiation and polarity. Detailed instruction of the standard techniques, which have all been tested and improvised by the authors, are given, such as collection and storage of pollen, pollen culture, germination, tests for viability, incompatibility and isolation of protoplasts. Introduced by an explanation of the principles involved, the step-by-step protocols are complemented by personal notes and precautions, specifying the reagents used and various appendices on basic and specific requirements for laboratory exercises on pollen.
1 Introduction.- 2 Pollen Collection.- 3 In Vitro Germination Methods.- Controlled Hydration/Prehydration.- Culture Medium.- Fixation of Pollen Cultures.- Scoring of Pollen Cultures.- General Precautions.- Methods.- Exercise 1 Hanging Drop Culture.- Exercise 2 Sitting Drop Culture.- Exercise 3 Suspension Culture.- Exercise 4 Surface Culture.- Exercise 5 Estimation of Pollen Tube Growth by the Alcian Blue Method.- 4 Pollen Germination and Pollen Tube Growth in Vitro.- Exercise 6 Effects of Boron and Calcium on Pollen Germination and Pollen Tube Growth.- Exercise 7 Leaching of Pollen Substances in Vitro.- Exercise 8 Regulation of pH by Pollen Leachate.- Exercise 9 Role of Transcription and Translation in Pollen Germination and Pollen Tube Growth.- 5 Tests for Pollen Viability.- Exercise 10 In Vitro Germination Test.- Exercise 11 Tetrazolium Test.- Exercise 12 Fluorochromatic Reaction (FCR) Test.- Exercise 13 Fruit Set and Seed Set Test.- Exercise 14 In Vivo Germination and Pollen Tube Growth.- 6 Pollen Storage.- Exercise 15 Effects of Temperature and RH on Viability of Stored Pollen.- Exercise 16 Storage of Pollen Grains in Organic Solvents.- 7 Pollen-Pistil Interaction.- Exercise 17 Cytochemical Localization of Esterases on Stigma Surface.- Exercise 18 Aniline Blue Fluorescence Method to Study Pollen Germination and Pollen Tube Growth in the Pistil.- Exercise 19 Multiple Staining for Localizing Pollen Tubes in the Pistil.- Exercise 20 Semi-Vivo Technique to Study Pollen Germination and Pollen Tube Growth.- Exercise 21 Use of DNA Fluorochromes for Studies on Pollen Grain and Pollen Tube Nuclei.- Exercise 22 Controlled Pollinations.- 8 Self-Incompatibility.- Exercise 23 Confirmation of Self-Incompatibility.- Exercise 24 Studies on Heteromorphic Self-Incompatibility.- Exercise 25 Bud Pollination and Delayed Pollination to Overcome Self-Incompatibility.- Exercise 26 In Vitro Placental Pollination and Fertilization.- 9 Isolation of Protoplasts.- Exercise 27 Isolation of Microspore Protoplasts.- Exercise 28 Isolation of Protoplasts from Pollen Grains.- Exercise 29 Isolation of Sperm Cells (Male Gametes).- Exercise 30 Isolation of Embryo Sacs and Protoplasts from Embryo Sacs.- 10 Appendices.- Appendix 1 Basic Requirements for Laboratory Exercises.- Appendix 2 Suitable Plant Systems.- Appendix 3 Pollen Morphology.- Appendix 4 Pollen Counts.- Appendix 5 Methods to Obtain Constant Relative Humidity.- Appendix 6 Principles of Fluorescence Microscopy.- Appendix 7 Commonly Used Fluorochromes, and Their Excitation and Fluorescence Ranges.- Appendix 8 Filter Combinations Commonly Used for Different Excitation Ranges.- Appendix 9 Basic Statistical Analyses.- References.