Protoplast Technology

This detailed volume collects protocols from scientists who are actively engaged in developing or using protoplast technology. The book begins with chapters that focus on basic protoplast techniques and their utilities, such as protocols on protoplast isolation, transfection, and regeneration, as well as examples of how to use protoplasts for genome editing and gene function analysis in a number of major crop or model plant species. It continues by exploring protoplast automation, large scale functional genomics, and synthetic biology. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Protoplast Technology: Methods and Protocols aims to inspire the new generation of researchers to further improve their protocols and apply this technology to accelerate the field of plant genomic study.

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.

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.

1 John H. Dodds The culture offragmen ts of plant tissue is not a particularly new science, in fact as long ago as 1893 Rechinger (1893) described the formation of callus on isolated fragments of stems and roots. The culture of plant tissues in vitro on a nutrient medium was performed by Haberlandt (1902), however, his attempts were unsuccessful because he chose too simple a medium that lacked critical growth factors. Over the last fifty years there has been a surge of development in plant tissue culture techniques and a host of techniques are now avail able (Dodds and Roberts, 1982). The major areas are as follows. Callus Culture Callus is a rather ill-dermed material. but is usually described as an un organised proliferating mass of tissue. Although callus cultures have a great deal of potential in the biotechnological aspects of tissue culture, i.e. secondary product formation, they are not very suitable for plant propagation. The key reason for their unsuitability is that genetic aber rations occur during mitotic divisions in callus growth (D'amato.l965). The aberrations can be of a major type, such as aneuploidy or endo reduplication. It follows therefore that the genetic status of the re generated plants is different from that of the parent type. In general terms this genetic instability is undesirable, but there are occasions when a callus stage can be purposely included to diversify the genetic base of the crop.

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.

Plant Protoplasts covers the techniques involved with, and uses of, protoplast technology. The book discusses isolation, fusion, and culture of higher plant protoplasts, lower plant protoplasts, and blue-green algal protoplast. The text also describes the production of haploid protoplasts from developing pollen grains; the use of protoplasts in mutant selection schemes, and the development of protoplast systems for use with monocotyledonous plants. The book will be invaluable to plant technologists, botanists, biochemists, research workers as well as advanced students interested in gaining a background knowledge of the field.

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 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.