Protoplast Fusion

Although only about 3 years have passed since the preparation of the original manuscript of this book for the Russian edition, the number of successful experiments on somatic hybridization of higher plants has doubled. Although the main inferences of the first edition still remain in force, most of them have received conclusive experimental support and, moreover, some new con­ clusions have been drawn. It can be expected that these inferences and conclusions will constitute a more or less durable foundation for somatic cell genetics of higher plants. We thus hope this book will also remain useful over the next years, in spite of the rapid progress of experiments and the increase in the number of scientific reports in this field. Though it might appear strange to an uninvolved observer, the principal progress in hybridization of somatic cells of higher plants has been due to plant physiologists (who entered the field by elaborating methods and techniques for plant cell and, later, for isolated protoplast culture) rather than plant geneticists. However, further qualitative improvement in this field is inconceivable without the instillation of genetic ideology and the strict logic of genetic experiments. The main purpose ofthis book is the attempt to organize the available experimental data in terms and cate­ gories of genetic analysis. For this reason, this book lays no claim on being a comprehensive treatise on somatic hybridization.

1 Introduction.- 2 Techniques of Parasexual Hybridization.- 2.1 Culture Techniques.- 2.1.1 Culture of Isolated Plant Protoplasts.- 2.1.2 Culture of Single Cells and Protoplasts.- 2.2 Isolated Protoplast Stage and Plant Cell Engineering.- 2.2.1 Protoplast Fusion.- 2.2.2 Cell Reconstruction Involving Fusion of Subprotoplasts.- 2.2.3 Cell Modification Through Uptake of Isolated Cell Organelles by Plant Protoplasts.- 2.3 Problems of Genetic Variability.- 2.3.1 Genetic Variability Associated with in Vitro Manipulation of Plant Cells.- 2.3.2 Genetic Changes in Cells Induced During the Stage of Isolated Protoplasts.- 3 Protoplast Fusion and Parasexual Hybridization of Higher Plants.- 3.1 Perspectives.- 3.1.1 Terminology.- 3.1.2 History of Parasexual Hybridization of Higher Plants.- 3.2 Methods of Screening for Parasexual Hybrids.- 3.2.1 Introduction.- 3.2.2 Genetic Complementation.- 3.2.3 Physiological Complementation.- 3.2.4 Restoration of Growth Capacity in Hybrids Upon Fusion of Inactivated Cells.- 3.2.5 Mechanical Isolation.- 3.2.6 Physical Enrichment.- 3.3 Frequency of Formation of Parasexual Hybrids.- 3.4 Protoplast Fusion and the Bypassing of Sexual Barriers.- 3.5 Analysis of the Parasexual Hybridization Process and the Genetic Composition of Plant Forms Arising from Protoplast Fusion.- 3.5.1 General Guidelines.- 3.5.2 Genetic Methods of Analysis.- 3.5.2.1 Hybrid Crosses.- 3.5.2.2 Cloning.- 3.5.2.3 Cytogenetic Studies.- 3.5.2.4 Cytophotometric Measurement of Nuclear DNA Content.- 3.5.3 Biochemical Analysis.- 3.5.3.1 Multiple Molecular Forms of Enzymes.- 3.5.3.2 Analysis of Fraction I Protein.- 3.5.3.3 Restriction Endonuclease Analysis of Chloroplast and Mitochondrial DNA.- 3.5.3.4 Molecular Hybridization of Nucleic Acids.- 3.5.3.5 Analysis of Low Molecular Weight Substances.- 4 Transmission Genetics of Parasexual Hybridization in Closely Related Crosses.- 4.1 General Considerations.- 4.1.1 Perspectives and Philosophy of Analysis of Transmission Genetics.- 4.1.2 Initial Experiments on Transmission Genetics of Parasexual Hybrids.- 4.1.3 Hybridization of More Than Two Parental Cells.- 4.2 Fate of Extranuclear Genetic Determinants.- 4.2.1 Perspectives.- 4.2.2 Protoplast Fusion and the Formation of Cytoplasmic Heterozygotes.- 4.2.3 Mitotic Segregation of Plasmagenes and the Sorting Out of Parental Genotypes.- 4.2.4 Recombination of Plasmagenes.- 4.3 Fate of Nuclear Genes in the Process of Somatic Hybridization.- 4.3.1 Segregation of Nuclei.- 4.3.2 Chromosome Sets of Hybrid Cells.- 4.4 Transmission Genetics in Hybridization Systems Utilizing Parental Cell Inactivation.- 4.4.1 Consideration of Animal and Bacterial Systems.- 4.4.2 Treatment with Iodoacetate.- 4.4.3 Irradiation.- 4.5 Genetic Diversity of Hybrid Plants Obtained by Protoplast Fusion.- 5 Protoplast Fusion and Hybridization of Distantly Related Plant Species.- 5.1 Introduction.- 5.2 Initial Stages of Culture of Hybrid Cells of Distantly Related Plant Species.- 5.3 Interfamily Cell Hybrids of Higher Plants.- 5.4 Intertribal Hybrids of Higher Plants.- 5.4.1 Hybridization of Cruciferae Species.- 5.4.2 Hybridization of Distantly Related Members of the Solanaceae and Other Families.- 5.4.3 General Conclusions of Results Obtained with Intertribal Hybrids of Higher Plants.- 5.5 Intergeneric Hybrids.- 5.6 Influence of Physical and Chemical Factors on the Fate of Genetic Material in Distantly Related Species Combinations.- 5.6.1 Introduction.- 5.6.2 Irradiation and the Induction of Genetic Asymmetry.- 5.6.3 Induction of Mitotic Chromosome Segregation by Chemical Agents.- 6 Use of Somatic Hybridization.- 6.1 Genetic Analysis Using Parasexual Hybridization.- 6.1.1 Introduction and Perspective.- 6.1.2 Analysis of the Nature of Inherited Traits.- 6.1.3 Analysis of Nuclear Genes.- 6.1.3.1 Resistance to 5-Methyltryptophan.- 6.1.3.2 Resistance to S-(2-Aminoethyl)-L-Cysteine.- 6.1.3.3 Resistance to Cycloheximide.- 6.1.3.4 Resistance to Azetidine-2-Carboxylate.- 6.1.3.5 Additional Complementation Studies.- 6.1.4 Cosegregation of Extranuclear Genes.- 6.1.5 Analysis of Mitotic Cycle Mechanisms.- 6.1.6 Analysis of the Mechanisms of Differentiation and Morphogenesis.- 6.2 Practical Use of Somatic Cell Hybridization.- 6.2.1 Introduction.- 6.2.2 Bypassing of Incompatibility in Interspecific Hybridization.- 6.2.3 Reconstruction of Cytoplasmic Genomes.- 6.2.4 Transfer of Cytoplasmic Genomes and Cytoplasmic Male Sterility.- 7 Conclusion.- References.