Main description:

The long-term storage and maintenance of viable plant cells and organs is an area of active concern across the range of pure and applied plant sciences. In academic, government and commercial laboratories, the extended storage of propagules of one sort or another, with maximum protection of the genome from mutation and altered expression, is often a very necessary activity that can draw heavily on resources and effort. However, preservation per se is typically not an activity in its own right, but a facilitating technology that is part of a larger programme of work. Consequently, there are many laboratories that do not have the benefit of a specialist in storage technology, and have to delegate the responsibility to individuals, or teams, who are faced with a daunting learning curve. To maximise the chances of success, in the shortest possible time and with minimum losses, these researchers need sources of reference that are au thoritative and soundly based in practical experience.

Contents:

1 Introduction to the in Vitro Preservation of Plant Cells, Tissues and Organs.- 1.1 Storage Techniques.- 1.2 Principles of Cryogenic Storage.- References.- 2 Minimal Growth Storage.- 2.1 Growth at Reduced Temperature.- 2.2 The Addition of Inhibitory Growth Regulators.- 2.3 The Addition of Osmotic Regulators.- 2.4 Combined Treatment.- References.- 3 Cryopreservation of Protoplast, Suspension and Callus Cultures.- 3.1 General Practical Considerations.- 3.2 Protoplast Cryopreservation by Slow Cooling.- 3.3 Cryopreservation of Cell Suspension Cultures by Slow Cooling.- 3.4 Cryopreservation of Callus Cultures by Slow Cooling.- References.- 4 Cryopreservation of Excised Meristems, Shoot Tips and Somatic Embryos.- 4.1 Meristem/Shoot-Tip Cryopreservation by Slow Cooling.- 4.2 Meristem/Shoot-Tip Cryopreservation by Rapid Cooling.- 4.3 Cryopreservation of Somatic Embryos by Dry Freezing.- 4.4 Cryopreservation of Somatic Embryos by Rapid Cooling.- 4.5 Cryopreservation of Somatic Embryos Following Dessication.- References.- 5 Storage of Free Pollen, Pollen Embryos and the Zygotic Embryos of Seed by Cryopreservation and Freeze Drying.- 5.1 Freezing Drying of Pollen.- 5.2 Pollen Cryopreservation.- 5.3 Cryopreservation of Pollen Embryos.- 5.4 Cryopreservation of Excised Zygotic Embryos.- References.- 6 Conservation of Algae.- 6.1 Serial Subculture.- 6.2 Cryopreservation.- References.- 7 Cryopreservation by Vitrification.- 7.1 Vitrification of Shoot Tips.- 7.2 Encapsulation/Dehydration Method.- 7.3 Vitrification of Cells and Protoplasts.- 7.4 Further Experiments.- References.- 8 Biochemical and Molecular Methods for Assessing Damage, Recovery and Stability in Cryopreserved Plant Germplasm.- A Biochemical Methods.- 8.1 Non-Destructive Volatile Hydrocarbon Monitoring of Freezing Damage.- 8.2 Colorimetric Detection of Malondialdehyde (MDA).- 8.3 Fluorimetric Detection of Malondialdehyde (MDA).- 8.4 Fluorimetric Detection of Schiffs bases.- 8.5 Colorimetric Determination of Sulphydryl Group (SH) Status.- 8.6 Determination of Viability Using Fluorescein Diacetate Vital Staining.- 8.7 Triphenyl Tetrazolium Chloride Colorimetric Assay for Viability.- B Molecular Methods.- 8.8 Mini DNA Extraction Procedure (CTAB Method).- 8.9 Purification of DNA Extracts.- 8.10 DNA Microassay.- 8.11 Restriction Enzyme Digestion.- 8.12 Agarose Gel Electrophoresis.- 8.13 Southern Blotting.- 8.14 Hybridisation Conditions.- 8.15 Biotin DNA Labelling Procedures.- 8.16 Calibration of Biotin-Labelled Probes.- 8.17 Slot Blot Analysis of Biotin-Labelled DNA.- 8.18 Colorimetric Detection of Biotin-Labelled DNA.- 8.19 Chemiluminescent Detection of the Ribosomal RNA Genes.- 8.20 Analysis of Results.- 8.21 Sensitivity of the Detection Procedures.- References.