Plant Protoplasts

Protoplast isolation and culture; Regeneration of plants; Protoplasts for studies of cell organelles; Protoplast fusion, Plant protoplasts and the cell cycle; Isolated protoplasts as laboratory tools in the study of cell wall deposition; Cytodifferentiation; The use of protoplasts for studies on membrane transport in plants; Protoplasts in virology; Plant protoplasts and genetic variation; Plant mutant isolation via protoplasts; Hybridization by somatic cell fusion; Plant protoplast transformation by Agrobacterium in relation to plant biotechnology.

The present book thus emphasizes the importance of plant protoplasts for fundamental research. This book is intended to be used b senior undergraduates, graduate students, and research scientists in plant biology.

Isolated plant protoplasts are 'naked' cells that have had their cell wall removed either by mechanical action or by enzymic digestion. As a result of wall removal the only barrier that exists between the cell protoplasm and the external environment is the plasma membrane. The removal of the cell wall has drastic osmotic consequences for the isolated cell. Rather like animal cells, the isolated protoplasts must be maintained in an osmotically balanced (isotonic) nutrient medium or they will burst. As will be seen later, the isolation of plant protoplasts is not a particularly new event; as early as the 1890s scientists were isolating protoplasts mechanically and studying effects such as protoplast streaming. What has brought protoplast technology to the forefront of plant biology is the potential application of these structures in non-traditional plant improvement methods. Extreme care must be exercised by the reader, however, in be lieving some of the claims that have been put forward for the use of protoplasts in improvement programmes. In the 20 years since the first experiments in routine enzyme isolation of large numbers of protoplasts no real improvement has been obtained in a commercial crop plant. This does not mean that improvement is impossible; it simply indicates that it may be a longer term objective than some people would like to admit.

In continuation of Volumes 8, 9, 22, and 23, this new volume deals with the regeneration of plants from isolated protoplasts and genetic transformation in various species of Actinidia, Allocasuarina, Anthurium, Antirrhinum, Asparagus, Beta, Brassica, Carica, Casuarina, Cyphomandra, Eucalyptus, Ipomoea, Larix, Limonium, Liriodendron, Malus, Musa, Physcomitrella, Physalis, Picea, Rosa, Tagetes, Triticum, and Ulmus. These studies reflect the far-reaching implications of protoplast technology in genetic engineering of plants. The book contains a wealth of useful information for advanced students, teachers, and researchers in the field of plant tissue culture, molecular biology, genetic engineering, plant breeding, and general biotechnology.

The idea for the p~esent book arose from a 3-day seminar which I organized in March 1984 for young research workers in plant physiology. Participants came from several universities of the French-speaking part of Switzerland and speakers from Basel, Mtinchen, Nottingham, Perpignan, Regensburg, Sheffield, Toulouse, Yale, ZUrich ... and Lausannne. The theme of the seminar was chosen from the range of research fields of our Institute. Meanwhile, feeling it was important to bear in mind that some of our hearers were not specialists in the chosen topic, I wished to pro vide a subject that would be of scientific, methodological and epistemologi cal interest. The critical analysis of the structural and functional characteristics of plant protoplasts exactly met these criteria. There exists ample material for discussion of the techniques of protoplast preparation, methods used in morphological, biological and biochemical studies, and for the comparison of protoplasts with the cells from which they are obtained.

Isolated protoplasts are a unique tool for genetic manipulation of plants. Since the discovery of a method for the enzymatic isolation of pro-· toplasts by Professor E. C. Cocking in 1960, tremendous progress has been made in this very fascinating area of research. I have witnessed the struggle in the 1960's and early 1970's, when obtaining a clean prepara tion of protoplasts was considered an achievement. I also shared the pioneering excitement and enthusiasm in this field during the 2nd Inter national Congress of Plant Tissue Culture held at Strasbourg in 1970, where Dr. I. Thkebe of Japan presented his work on the induction of division in tobacco protoplasts. This was followed by my participation in three international conferences devoted to plant protoplasts held in 1972 in Salamanca (Spain) and Versailles (France), and then in 1975 in Nottingham (England). The enthusiasm shown by plant scientists at these meetings was ample proof of the bright future of protoplast technology, and it became evident that protoplasts would playa major role in plant biotechnology, especially in genetic engineering. Since then we have never looked back, and now the methods for isolation, fusion, and culture, as well as regeneration of somatic hybrids, have become routine laboratory procedures for most plant species. Currently the focus is on cereal and tree protoplasts. In order to bring about any genetic manipulation through fusion, in corporation of DNA, and transformation, the regeneration of the entire plant through manipulation of protoplasts is a prerequisite.

In continuation of Volumes 8 and 9 (1989) on in vitro manipulation of plant protoplasts, this new volume deals with the regeneration of plants from protoplasts and genetic transformation in various species of Agrostis, Arabidopsis, Atropa, Brassica, Catharanthus, Datura, Cucumis, Daucus, Digitalis, Duboisia, Eustoma, Festuca, Helianthus, Hordeum, Kalanchoe, Linum, Lobelia, Lolium, Lotus, Lycium, Lycopersicum, Mentha, Nicotiana, Pelargonium, Pisum, Pyrus, Salvia, Scopolia, and Solanum.These studies reflect the far reaching implications of protoplast technologyin genetic engineering of plants. They are of special interest to researchers in the field of plant tissue culture, molecular biology, genetic engineering, and plant breeding.

Proceedings of the 7th International Protoplast Symposium, Wageningen, The Netherlands, December 6-11, 1987