Plant Cell Culture

Julian Coleman, David Evans, Anne Kearns

• Publisher: Taylor & Francis
• Year: 2020
• ISBN:
  9781000144710
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Botany
• Index: Biological Sciences

Plant cell culture is an essential methodology in plant sciences, with numerous variant techniques depending on the cell type and organism. Plant Cell Culture provides the reader with a concise overview of these techniques, including basic plant biology for cell culture, basic sterile technique and media preparation, specific techniques for various plant cell and tissue types including applications, tissue culture in agriculture, horticulture and forestry and culture for genetic engineering and biotechnology. This book will be an essential addition to any plant science laboratory's bookshelf.

Chapter 1

An introduction to plant cell and tissue culture

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1. Plant cell and tissue culture

Plant cell and tissue culture is the cultivation of plant cells, tissues and organs under aseptic conditions in controlled environments. Whilst at one time the techniques of plant cell and tissue culture were the preserve of specialists in the field, culture methods are now well established and widely used in many areas of research and commercial plant science.

The technology of plant cell and tissue culture (sometimes termed in vitro culture) is based on a variety of methods that range from the isolation and culture of protoplasts (naked plant cells) to the regeneration and clonal propagation of entire plants. The development of plant and tissue culture as a fundamental science was closely linked with the discovery and characterization of the plant hormones, and has facilitated our understanding of plant growth and development. Furthermore, the ability to grow plant cells and tissues in culture and to control their development forms the basis of many practical applications in agriculture, horticulture and industrial chemistry and is a prerequisite for plant genetic engineering.

1.1 Early experiments in plant tissue culture

It has long been known that many plants show the ability to regenerate tissues and organs and to reproduce vegetatively. Over the centuries, horticulturists have exploited this remarkable regenerative ability by using cuttings to propagate new plants with desirable traits. The date of the first successful plant propagation in horticulture is not known, but by the end of the 19th century such techniques had become quite sophisticated. In contrast, the birth of plant tissue culture can be accurately dated. The first experiments were undertaken in Graz, Austria, by Gottlieb Haberlandt between 1898 and 1902 and preceded the beginnings of animal cell culture by about 10 years. Haberlandt succeeded in maintaining isolated leaf cells alive for extended periods but the cells failed to divide because the simple nutrient media he used lacked the necessary plant hormones. In the early part of the 20th century, progress in growing excised plant tissues in culture continued with the development of sterile working methods and the discovery of the need for B vitamins and auxins for tissue growth. The first successful experiments to maintain growth and cell division in plant cell culture were those of White (1934), who established isolated tomato roots in aseptic culture. White’s medium was simple, containing only sucrose, minerals and a yeast extract, which supplied vitamins. It became clear that under these culture conditions, excised organs like roots were capable of synthesizing the hormones necessary to maintain cell division.

1.2 Developments in culture media

Growing interest in plant tissue culture followed White’s experiments. It was discovered that growth and cell division of tissues isolated from a variety of plants could be stimulated by additions to the medium. The first such addition was the plant hormone auxin; later coconut milk (the liquid endosperm of the coconut, also known as coconut water) was used. In the late 1930s, Gautheret and Nobecourt were the first to establish reliable protocols for growing masses of undifferentiated plant tissue; first from willow (Salix)and later from carrot (Daucus carota). These masses of undifferentiated cells, termed callus, could be re-cultured repeatedly on media containing the plant growth substance (hormone) auxin. At the same time, White, who was also studying callus formation, achieved production of a tobacco (Nicotiana tobacum)callus that did not require auxin for growth.

1.3 Embryogenesis in culture

In the 1950s, the work of Skoog and his colleagues on the media requirements for tobacco tissue culture led to many important advances. Included were the discovery of the plant growth substance kinetin, a cytokinin, and the development of an important tissue culture medium, the Murashige and Skoog or MS medium (Murashige and Skoog, 1962).

In a classic paper, Skoog and Miller (1957) demonstrated that by manipulating the auxin to cytokinin balance in the medium, they could control the differentiation of roots and shoots from tobacco pith callus. High concentrations of auxin promoted rooting and high concentrations of cytokinin supported shoot formation, whereas at equal concentrations the tissue remained as callus. This process of organ development is known as organogenesis.

Another technical breakthrough came when Steward et al. (1958) used nutrient media enriched with coconut milk to regenerate somatic embryos from callus clumps and cell suspensions of carrot. This process was called somatic embryogenesis (embryos generated from somatic cells) and the somatic embryos formed by this process were fully viable and could be grown to mature plants, capable of flowering and setting seed.

The techniques of in vitro regeneration of whole plants by organogenesis stepwise via individual organs (e.g. the differentiation of shoot followed by root) and by somatic embryogenesis are important strategies for clonal propagation that have found application in agriculture and horticulture. Furthermore, regeneration is also an essential component of plant genetic engineering, allowing the production of clones of genetically modified plants.

1.4 Early experiments with suspension cultures

The possibility of culturing individual plant cells in liquid media, and thus allowing the application of standard microbiological methods to their study, was one of the objectives of early work on plant cell cultures. However, it was not until the middle of the 1950s that the successful establishment of suspension cultures was achieved in carrot (Nobecourt, 1955). Cells separated from callus into liquid media and kept constantly agitated were shown to grow and divide to form small aggregates of cells suspended in the medium. Later, in the 1960s, Steward et al. (1964) and Vasil and Hildebrandt (1965) demonstrated that fully viable somatic embryos could be regenerated from individual single cells grown in culture. The results of these experiments were taken as proof of totipotency in plants — that a single somatic cell can regenerate to form an entire organism. Totipotency is an unusual property of many plant cells and presumably results from the remarkable plasticity of development shown by plants.

2. Industrial-scale plant cell culture

With the development of protocols for the establishment of plant suspension cultures, there was great interest in exploiting in vitro cultures grown on a large scale in bioreactors for the production of speciality organic compounds (e.g. pharmaceuticals) that are plant products. This production system for plant compounds was seen as an advantageous alternative to their extraction from whole plants or their chemical synthesis. For a variety of reasons, this technology was less successful than the equivalent fermentation systems for yeast and industrial-scale culture systems for animal cells. Plant cell cultures grow slowly and are easily damaged by pressure effects in the aeration and agitation required to maintain the cultures. In addition, high-value plant compounds are often the products of complex pathways of secondary metabolism that are actively expressed in differentiated organs or tissues and inactive in undifferentiated cells. In a developing field of research, attempts are being made to overcome the differentiation problem by using either organ cultures like hairy roots (Chapter 10) or by using transgenic cell suspensions that constitutively express genes for the critical enzymes. Recently, the production of high-value compounds (e.g. human therapeutic proteins) in transgenic plants growing in the field without the problems of complex culture cycles and the maintenance of aseptic conditions has attracted considerable attention.

3. Plant tissue culture, plant breeding and crop improvement

Plant cells from which the cell walls have been removed are termed protoplasts (Chapter 2). Techniques using enzymatic degradation of cell walls to obtain large numbers of protoplasts were first devised in the 1960s by Cocking and co-workers. In suitable media, protoplasts can synthesize new cell walls, divide to form small cell colonies and ultimately regenerate whole plants. Protoplasts brought into close contact can be induced to fuse by the application of certain agents. The fusion of protoplasts from different species is used to produce so-called somatic hybrids (sometimes termed cybrids). Somatic hybridization circumvents naturally occurring incompatibility mechanisms that prevent the formation of new species through sexual crosses. This technique has been used in plant breeding to hybridize sexually incompatible species. Perhaps the best-known example of protoplast fusion is the somatic hybrid of potato and tomato, the ‘pomato’ that was first created in the late 1970s (Melchers et al., 1978). Protoplasts have also proved very useful in genetic transformation, particularly for cereals, and can be generated from intact plant material (leaves, roots) or from suspension cultures.

Tissue culture techniques were also explored as a means of improving crop productivity. These included: the identification of beneficial mutations in clonal lines produced by tissue culture (somaclonal variation, see Chapters 3 and 12), and artificial mutagenesis and the production of disease free plants by meristem tip culture (see Chapter 3).

4. Plant tissue culture and plant genetic engineering

Development of plant tissue culture as a technique in its own right was augmented in the 1980s by the exciting prospect of deliberately and specifically modifying the characteristics of a plant by genetic manipulation. The description of the mechanism of infection of plants by the soil organism Agrobacterium tumefaciens (Chapter 4) was followed by its modification to deliver foreign genes. The first demonstrations of stable plant transformation were then made by both American and European scientists (Horsch et al., 1984; Zambryski et al., 1983). Development of standardized methods of plant transformation followed, using A. tumefaciens or Agrobacterium rhizogenes, or direct methods like microinjection, electroporation or particle bombardment (Chapter 4). Methods were suitable first for herbaceous dicot plants like tobacco (Nicotiana tabacum), petunia (Petunia hybrida)and tomato (Lycopersicon esculentum)(e.g. Horsch et al., 1985), later for woody dicots and lastly, monocots (e.g. Gasser and Fraley, 1989).

It has already been indicated that the techniques of tissue culture are essential tools for plant genetic modification. Subsequent chapters will describe the production of protoplasts, callus and other materials for transformation and their use to generat...

Table of contents

• Cover
• Half Title
• Forthcoming Titles
• Title Page
• Copyright Page
• Table of Contents
• Abbreviations
• Preface
• Chapter 1 An introduction to plant cell and tissue culture
• Chapter 2 Basic plant biology for cell culture
• Chapter 3 Tissue culture in agriculture, horticulture and forestry
• Chapter 4 Tissue culture in genetic engineering and biotechnology
• Chapter 5 Culture facilities, sterile technique and media preparation
• Chapter 6 Callus cultures
• Chapter 7 Cell suspension cultures
• Chapter 8 Protoplast culture
• Chapter 9 Haploid cultures
• Chapter 10 Organ and embryo culture
• Chapter 11 Regeneration of plants and micropropagation
• Chapter 12 Somaclonal variation
• Chapter 13 Bacterial culture in the plant cell culture laboratory
• Chapter 14 Industrial uses of plant cell culture
• Chapter 15 Prospects and future challenges
• Suppliers of chemicals, apparatus and cell culture products
• Glossary
• Index