Plant Cell Culture and Innovation

Plant Cell Culture and Innovation

Ameyatma Mahajan

Plant Cell Culture and Innovation

Ameyatma Mahajan

ISBN - 9789361522024

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Preface

Plants have created a large chemical cornucopia to sustain their sessile lifestyles. Man has been using this natural resource since the Neolithic times and is now using plant-derived chemicals for a variety of applications. Plant cell culture has become a unique process in biotechnology that many researchers have been interested in because it can produce products that bacteria or animal cells cannot produce. Plant cells are the only sources of alkaloids and anthocyanins. Biotransformation of biological substances such as terpenoids or steroids is possible by the use of plant cells. Proteins can also be generated using plants, in particular genetically modified plants. Plant cell culture is a series of techniques required to expand and multiply cells and tissues using nutrient solutions in an aseptic and regulated environment. This technology investigates conditions that facilitate cell division and genetic reprogramming in vitro. Plant cell cultures also provide an enticing route for the production of highly valuable plant-derived products, such as flavors, fragrances, alkaloids, pigments and pharmaceuticals that are costly for chemical and pharmaceutical synthesis.

Plant cell cultures have been studied as a possible source of secondary metabolites for rival extraction processes based on whole plant material for more than 30 years. To date, however, progress has been minimal. Many problems remain unanswered, with a particular need for enhanced cell culture efficiency and streamlined and enhanced process technology. Metabolite engineering could give new opportunities to increase production in cultured cells. The relation between the transcription of secondary metabolism and the differentiation program has been shown to exist, and the frequency of certain secondary metabolism pathways can depend on general or primary growth. Many plant secondary metabolites are commercially valuable and are used as pharmaceuticals in medicine. However, for commercial utilization, the production of secondary metabolites by plant cell cultures must be economically viable and easy to scale up. This book contains six chapters; chapter 1: plant cell culture: significance, types, and media; chapter 2: an overview of micropropagation techniques; chapter 3: callus culture and suspension culture; chapter 4: in vitro plant propagation methods; chapter 5: embryo and endosperm culture; chapter 6: somatic embryogenesis and artificial seed production. This book will serve as a valuable tool for students as well as others dealing with the field.

Table of Contents

1 Plant Cell Culture: Significance, Types, and Media 1

1.1 Plant Cell Culture: An Overview 2

1.1.1 Historical prospect of Cell Culture 3

1.2 Cell Culture - Basics, Techniques and Media 4

1.2.1 Morphology 5

1.3 Significance of Cell Culture 5

1.4 Types of Cell Culture 6

1.4.1 Cell Culture Media 8

1.5 Cell Culture Protocol 10

1.5.1 Protocols for Cell Culture Preparation 11

1.6 Applications of Cell Culture 12

1.6.1 Seed Culture 12

1.6.2 Meristem Culture 13

1.6.3 Callus Culture 13

1.6.4 Bud Culture 13

1.6.5 Anther Culture 13

1.6.6 Cell Suspension Culture 13

1.6.7 Micropropagation and Tissue Culture 14

1.7 Applications of Plant Cell and Tissue Culture 14

1.8 Plant Cell Cultures for the Production of Recombinant Proteins 19

1.9 Applications of Plant Tissue Culture in Forestry 20

1.10 Plant Cell Culture Strategies for the Production of Natural Products (NPs) 25

1.10.1 Plant Cell Cultures 27

1.10.2 Hairy Root Cultures 28

1.10.3 Cell Line Selection for Enhanced NP Production 29

1.10.4 Culture Condition Optimization 30

1.10.5 Elicitation 32

1.10.6 Immobilization of Plant Cells 32

1.10.7 Permeabilisation, Secretion and Extraction of NPs 33

1.10.8 Cambial Meristematic Cells 35

1.10.9 Scale-Up Strategies for NP Production 36

1.11 Exercise 38

2 Micropropagation - An Overview of its Techniques 39

2.1 Micropropagation: Definition and Purpose 39

2.1.1 Purpose of Micropropagation 40

2.1.2 Methods of Micropropagation 42

2.2 Steps of Micropropagation 43

2.3 Advantages And Disadvantages Of Micropropagation

With Respect To Commercialization 56

2.3.1 Advantages Of Micropropagation 56

2.3.2 Disadvantages Of Micropropagation 57

2.3.3 Key Points of Micropropagation 58

2.4 Factors Affecting Micropropagation 59

2.5 Micropropagation Techniques 60

2.5.1 Micropropagation by Axillary and Apical Buds 60

2.5.2 Micropropagation by Adventitious Shoots 61

2.6 Methods of Micro-Propagation 62

2.6.1 Meristem Culture 62

2.6.2 Callus Culture 63

2.6.3 Suspension Culture 63

2.6.4 Embryo Culture 64

2.6.5 Protoplast Culture 64

2.7 Difference Between Tissue Culture And Micropropagation 64

2.8 Application Of Tissue Culture To Different Fields 66

2.8.1 Major Agricultural Applications 67

2.8.2 Application Of Tissue Culture To Forestry 69

2.8.3 Application of Tissue Culture to Horticulture 70

2.9 Limitations of Micropropagation 72

2.10 Exercise 73

3 Callus Culture and Suspension Culture 74

3.1 Plants Callus 74

3.1.1 Historical Preview 76

3.1.2 Dedifferentiation and Callus Formation 77

3.1.3 Totipotency and Somatic Embryogenesis 81

3.2 Callus Culture 89

3.2.1 Meaning of Callus Culture 90

3.2.2 Materials 91

3.2.3 Subculture 91

3.2.4 Nutrient Medium of Callus Culture 91

3.2.5 Brief History of Callus Culture 92

3.2.6 Principles of Callus Culture 93

3.2.7 Protocol of Callus Culture 94

3.2.8 Nutrient Medium of Callus Culture 96

3.2.9 Methods of Callus Culture 97

3.2.10 Nature of Callus Tissue 100

3.2.11 Measuring Growth in Callus Cultures 101

3.2.12 Significance of Callus Culture 101

3.2.13 Biotechnology Applications of Plant Callus Cultures 102

3.3 Cell Suspension Culture 105

3.3.1 Brief History 106

3.3.2 Principle of Cell Suspension Culture 106

3.3.3 Characteristics of Suspension Culture 107

3.3.4 Protocol 108

3.3.5 Different Categories of Cell Suspension Culture 110

3.3.6 Importance of Cell Suspension Culture 111

3.3.7 Growth Measurements 112

3.3.8 Production Of Secondary Metabolites From Cell Suspension Cultures 114

3.3.9 Adherent Cell Culture vs. Suspension Cell Culture 116

3.4 Exercise 117

4 In Vitro Plant Propagation Methods 118

4.1 Technique for Plant In Vitro Culture 119

4.1.1 Micropropagation 119

4.1.2 Somatic Cell Genetics 119

4.1.3 Transgenic Plants 121

4.2 In Vitro Plant Tissue Culture: Means for Production of

Biological Active Compounds 121

4.2.1 Production Of Biologically Active Compounds 123

4.2.2 Plants Employed For The Production Of

Biologically Active Compounds 126

4.2.3 Methods Of Plant Tissue Culture 127

4.2.4 Improvements In Traditional Culturing Techniques 127

4.2.5 Alternatives To In Vitro Plant Culture 132

4.2.6 Strategies For The Expression Of Plant Biologically

Active Compounds 135

4.2.7 Perspectives For Plant Production Of Bioactive Molecules 137

4.3 A New Temporary Immersion Bioreactor System for Micropropagation 138

4.3.1 Micropropagation in Semi-Solid Medium 139

4.3.2 Culture in Liquid Media 139

4.3.3 Methods 140

4.4 Micropropagation for Endangered Plant Species 152

4.4.1 Materials 156

4.4.2 Methods for Micropropagation of Taxus 158

4.4.3 Microporopogation of Chinese Aloe 159

4.5 Materials 163

4.5.1 Methods 163

4.6 Exercise 169

5 Embryo and Endosperm Culture 170

5.1 Embryo Culture 171

5.1.1 What is Embryo Culture? 171

5.1.2 Different Categories of Embryo-Culture and

their Objectives 172

5.1.3 Principles of Embryo Culture 174

5.1.4 Protocol for Embryo Culture 176

5.1.5 Precocious Germination Embryo in Culture 177

5.1.6 Types of Embryo Culture 179

5.1.7 Culture Technique for Embryo Rescue 180

5.1.8 Embryo-Endosperm Transplant 180

5.1.9 Nutritional Requirements of Embryo Cultures 181

5.1.10 Composition of the Medium 182

5.1.11 Applications of Embryo Culture 182

5.1.12 Importance of Embryo Culture In Relation to

Biological Knowledge 184

5.1.13 Seed Dormancy and Embryo Culture 191

5.1.14 Other Applications 192

5.1.15 Techniques of Embryo Culture 193

5.1.16 Role of Suspensor in Embryo Culture 196

5.1.17 Protocol for Embryo Culture 196

5.2 Endosperm Culture 197

5.2.1 The Endosperm: A Mediator of Communication

between the Embryo and its Environment 198

5.2.2 What Is Endosperm Made Of In Plants? 201

5.2.3 The Functions Of The Endosperm During Seed Germination 201

5.2.4 The Role of Endosperm in Seed Development 202

5.2.5 Gene Expression and Transcriptomes in the Endosperm 206

5.2.6 Transcriptional Regulators in the Endosperm 208

5.2.7 Types of Endosperms 208

5.2.8 Various Types of Endosperm of Flowering Plants 209

5.2.9 Fate of Endosperm 211

5.2.10 Factors Controlling Callus Proliferation And Plant Regeneration 212

5.2.11 Shoot Regeneration 214

5.2.12 Histology 216

5.2.13 Cytology 218

5.2.14 Endosperm Culture: A Novel Method For Triploid

Plant Production 220

5.3 Exercise 220

6 Somatic Embryogenesis and Artificial Seed

Production: Principle, Aspects and Applications 221

6.1 Somatic Embryogenesis. An Overview 221

6.1.1 What Is Somatic Embryogenesis? 224

6.1.2 What is Embryo genic Potential? 225

6.1.3 Brief Historical Background 225

6.1.4 Principles of Somatic Embryogenesis 226

6.1.5 Protocols for Inducing Somatic Embryogenesis

in Culture 230

6.1.6 Importance of Somatic Embryogenesis 232

6.1.7 Types of Somatic Embryogenesis 233

6.1.8 Process of Somatic Embryogenesis 234

6.1.9 Applications of Somatic Embryogenesis 240

6.1.10 Application of Somatic Embryogenesis in Woody Plants 244

6.1.11 Plant Regeneration By Somatic Embryogenesis 255

6.1.12 Factors Affecting the Embryogenesis 255

6.2 In Vitro Somatic Embryogenesis 257

6.2.1 Organogenesis Versus Embryogenesis 259

6.2.2 Embryo Maturation And Germination 259

6.2.3 Secondary Somatic Embryogenesis 260

6.2.4 Synchronization Of Embryo Development 261

6.3 Production Of Synthetic Seeds Or Artificial Seed 261

6.3.1 Steps followed for Making Artificial Seeds 263

6.3.2 Disadvantages of Artificial Seeds 264

6.4 Similarities and Differences between Organogenesis and

Somatic Embryogenesis 265

6.4.1 What is Organogenesis? 265

6.4.2 What is Somatic Embryogenesis? 266

6.5 Exercise 269

Glossary 270

References 273

Index 276

Chapter 1. Plant Cell Culture Significance, Types, and Media

Plant cell cultures provide an attractive route to obtain highly valuable plant-derived products, such as flavors, fragrances, alkaloids, pigments and pharmaceuticals that are expensive to synthesize chemically and that naturally occur only at low concentrations. Recently, there have been some significant successes in the production of valuable plant second metabolites, but in most cases, the yields of secondary metabolites are too low for commercial production. Plant cell and tissue culture techniques involve the culture of cells, protoplasts, tissues, and organs isolated aseptically on a defined sterile medium under controlled environmental conditions.

Plant in vitro technologies is used mainly for the regeneration of organs or somatic embryos for propagation, virus elimination, the transformation and generation of transgenic plants for improvement of plant traits, and for germplasm storage. Additionally, tissue cultures and cell suspension cultures under controlled conditions – especially defined medium composition – are valuable as model systems for basic studies of cell physiology and metabolism. The ability to control metabolic pathways of in vitro cultured cells and tissues also resulted in a major practical application, i.e., the production of secondary metabolites – phytochemicals for the food and pharmaceutical industries. Propagation in tissue culture (micropropagation) is used to develop high-quality pathogen-free plants, selected genotypes, or transformed cloned plants. Propagation in tissue culture depends on cell competence and totipotency, that is, the ability of plant cells to re-express their genetic potential, to undergo dedifferentiation and redifferentiation, and to regenerate new plants.

This chapter introduces the basics of plant cell culture and discusses the techniques, which utilize the ability of plant cells to be cultured. All of these techniques have an agricultural application and are being used throughout the world to improve agricultural productivity.

1.1 Plant cell culture: an overview

Plant cell culture is a unique process in biotechnology, which has interested many researchers because it can produce products that bacteria or animal cells cannot produce. Plant cells are the sole producers of alkaloids and anthocyanins. The biotransformation of biological compounds such as terpenoids or steroids is possible using plant cells. Proteins can also be produced using plants, particularly using genetically engineered plants, and there is little risk of human infection of proteins by viruses or pathogens compared to other methods such as production using animal cells or microorganisms. In addition, plant cell culture will enable the preparation of artificial seeds for seedling production, increasing the potential of producing various kinds of useful plants in field cultivation in agriculture. Artificial seeds provide genetically homogeneous plants and help enable easily controlled agriculture or plantation.

However, plant cell culture is costly because of the slow proliferation rate and small productivity. The features of plant cells have been described in published literature. The use of plant cell culture needs to compete with production by intact plants cultivated in fields. It is often cheaper and easier to produce useful substances by intact plants than by cell culture. Careful, economical assessments are, therefore, always necessary for the application of plant cell culture. Another drawback of plant cell culture is gene instability during culture. Unless effective procedures to stabilize the genes of the cultured cells are developed, the commercial application of plant cell culture may still be limited.

Bioreactor studies for plant cell culture have been reported since the mid-20th century. There are a very large number of publications on plant cell cultures toward the end of the century. The types of bioreactors and their behavior are summarized. Because of the above-described serious drawbacks of plant cell culture, however, the number of articles on their application seems to have recently decreased.

Several devices have been proposed to realize efficient production by the use of plant cell culture.

Plant cells are sensitive to the environment compared with microorganisms and are easily damaged by stresses such as shear stress. Reactor design to avoid fluid dynamic damage is salient, and several devices have been proposed for this purpose. Often, feedback inhibition by products is observed and simultaneous cultivation with separation of products is desirable in this case.

Therefore, culture with product separation has been studied by many researchers. Immobilization is also useful to avoid fluid dynamic stress if products can be obtained without destroying cells. Continuous operation with product separation is possible by using immobilized plant cells. In many cases, mass transfer is not a limiting process in the secondary metabolite production by callus cells, although it often has a strong effect on hairy roots. Immobilization is a useful method in the application of plant cell culture using callus cells. By immobilizing plant cells, the continuous operation becomes possible and fluid dynamic stresses can be avoided.

Plant cell cultures have been known for decades to have a density effect, such that suspension cells cultured at a low density did not divide even if provided with additional hormones or nutrients. However, if condition media from a high-density culture is added to low-density cultured cells, the latter is able to divide by sensing some kinds of division-promoting factor released from the former. It was thus deduced that a density factor may be secreted by the suspension culture cells, which provides a bioassay for its isolation.

1.1.1 Historical prospect of Cell Culture

The origin of plant cell culture derived from an interest in determining how cells would behave when isolated from the whole plant. In the wild, certain plants are capable of regeneration from small pieces of severed tissue, for example, dandelions proliferate from isolated roots and Begonia plantlets directly from leaf tissue. These observations aroused an interest in the plasticity of plant development and the potential for cell development if removed from the ‘control’ of the whole plant. In 1902, Gottlieb Haberlandt spoke of his vision for cell biology in the future: To my knowledge, no systematically organized attempts to culture isolated vegetative cells from higher plants in simple nutrient solutions have been made. Yet, the results of such culture experiments should give some interesting insight into the properties and potentialities which the cell, as an elementary organism, possesses. Moreover, it would provide information about the inter-relationships and complementary influences to which cells within the whole multicellular organism are exposed. Haberlandt was never able to induce cell division in vitro, but in 1934, White cultured tomato roots on a basic medium of inorganic salts, sucrose and yeast extract. In the same year, Gautheret found that the cambial tissue of Salix capraea and Populus Alba could proliferate for several months once aseptically isolated, but growth was limited. In 1939, as a result of recognizing the importance of B vitamins and the auxin, indole-3- acetic acid, Gautheret reported on the unlimited growth of a cell culture of carrot, which resulted in the production of viable callus. From this early work developed the concept of plant cell culture, enabling scientists through the manipulation of plant cells to develop techniques that would be of great benefit to a range of industries, such as agriculture and the pharmaceutical industry. Plant cell culture is a generic description used to describe the growth of microbe-free plant material in an aseptic (sterile) environment, such as a sterilized nutrient medium in a test tube. From this early work in the 20th-century, plant cell culture has developed far beyond what was first thought possible, and with the introduction of genetically modified organisms agriculture [see also - Nitrogen fixation biotechnology], we see the ultimate in the manipulation of plant cell culture. During the development of this area, numerous techniques have been established which have, in many cases, resulted in practical application. These techniques can be categorized according to whether they can be used for propagation, improvement, conservation and utilization of plant germplasm.

1.2 Cell Culture - Basics, Techniques and Media

Essentially, cell culture involves the distribution of cells in an artificial environment (in vitro), which is composed of the necessary nutrients, ideal temperature, gases, pH and humidity to allow the cells to grow and proliferate.

In vivo - When the study involves living biological entities within the organism.

In vitro - When the study is conducted using biological entities (cells, tissue etc.) that have been isolated from their natural biological environment. E.g., tissue or cells isolated from the liver or kidney.

Whereas pieces of tissue can be put in the appropriate culture to produce cells that can then be used for culture (explant culture), cells from tissues (soft tissue) can be obtained through enzymatic reactions. Such enzymes as trypsin and proname are used to break down the tissue and release the desired cells.

When cells have been obtained directly from the organism/animal tissue (or even plant tissue) through enzymatic or mechanical techniques, such cells are referred to as primary cells. However, cells that continue to proliferate indefinitely (after the first subculture) under special conditions are referred to as cell lines.

These particular cells tend to have been passaged for a long period of time, which causes them to acquire homogenous (similar) genotypic and phenotypical traits.

1.2.1 Morphology

Based on their appearance, cells in culture can be categorized in to three main groups:

• Fibroblastic - This includes cells that tend to be bipolar/multipolar with elongated shapes. These cells are attached to the substrate as they grow.

• Epithelial - Epithelial-like cells attain a polygonal shape with regular dimensions. Although they tend to grow in discrete patches, these cells also grow attached to the substrate.

• Lymphoblast - These cells are usually spherical in shape and do not attach to the surface of the substrate. As a result, they are grown in suspension.

1.3 Significance of Cell Culture

Cell culture is an important technique in both cellular and molecular biology, given that it provides the best platform for studying the normal physiology and biochemistry of cells. A cell is the basic structural, functional and biological unit of all living things.

In order to understand an organism or given tissues, it is important to understand how its cells work. Through cell culture, this becomes possible, especially due to the fact the primary cells resemble the parental cells from the organism/tissue.

Whatever is learned about the cells in vitro is representative of what is happening to the organism/tissue. This makes cell culture significantly important for vaccine development, screening (drugs etc.) and diagnosis of given diseases/conditions.

Given that different types of cells require different environments for proliferation, there are different types of media used for cultures, such as serum-free media and serum-containing media, among others.

Once the right requirements have been provided, the cells will increase in numbers and may form colonies, which can then be easily seen and identified. However, all this requires that the purpose of the procedure be understood.

Having a good understanding of what the procedure is meant to achieve, it becomes easier to prepare the culture with the right components. By understanding what the procedure is aimed for, the researcher will know whether to prepare a selective media (which allows for specific cells to grow) or differential media (allowing for different types of cells to grow).

Cell culture is a process where cells (animal or plant cells) are removed from the organism and introduced in to an artificial environment with favorable conditions for growth. This allows for researchers to study and learn more about the cells.

1.4 Types of cell culture

There are three major types of cell culture, which include:

• Primary cell culture

• Secondary cell culture, and

• Cell line

Here, we shall focus on primary cell culture.

There are two types of primary cells:

Adherent cells - Also referred to as anchorage-dependent cells, these are the type of cells that require attachment for growth. Adherent cells are immobile and obtained from such organs as the kidney.

Suspension cells - These are the type of cells that do not require attachment in order to grow. They are therefore also referred to as anchorage-independent cells and include such cells as lymphocytes found in the blood system.

In primary cell culture, cells obtained from such parental tissues (living tissues) as the liver and kidney are introduced into suitable media for growth. Once the cells have been obtained, they can either be cultured as explants culture, suspension or monolayer.

* In primary cell culture, the cells must have been obtained from the parental/living tissue. That is, they are not from another culture process.

Before the cells are cultured, they are first subjected to enzymatic treatment for dissociation. However, has to be for a minimal amount of time to avoid damaging or killing the cells. Once single cells are obtained, they are then appropriately cultured in media to allow those to grow (divide) are reach the desired numbers.

Initially, the culture tends to be heterogeneous in that it’s composed of different types of cells obtained from the tissue. Although this can be maintained through the in vitro process (in a culture in a suitable media), this would only be for a limited period of time.

Through the transformation process, the primary cells may be used for a long period of time, changing the culture over time. These cells are referred to as continuous cell lines.

However, primary cells are typically preferred over continuous cell lines because of the fact that they are more similar (physiologically) to in vivo cells (cells from the living tissue). In addition, continuous cell lines may undergo certain changes (phenotypic and genotypic changes), which would result in discrepancies during analysis. As such, they cannot be used to determine what is happening to the in vivo cells. It’s for this reason that primary cells are preferred.

Given that the primary cells significantly resemble the cells obtained from living tissue, they are important for research purposes in that they can be used to study their functions, metabolic regulations, cell physiology, development, defects and conditions affecting the tissue of interest.

Also, they are used for such purposes as vaccine production, genetic engineering drug screening as well as toxicity testing and prenatal diagnosis, among others.

1.4.1 Cell Culture Media

In cell culture techniques, cells (or tissues) are removed from a plant or an animal and introduced into a new, artificial environment that can support their proliferation (survival and growth).

Some