Plant Tissue Culture Concepts and Laboratory Exercises

Robert N. Trigiano

• Publisher: CRC Press
• Year: 2018
• Print ISBN: 9781138705814
• eBook ISBN: 9781351424035
• Topic: Sciences biologiques
• Subtopic: Biologie

Alternating between topic discussions and hands-on laboratory experiments that range from the in vitro flowering of roses to tissue culture of ferns, Plant Tissue Culture Concepts and Laboratory Exercises, Second Edition, addresses the most current principles and methods in plant tissue culture research. The editors use the expertise of some of the top researchers and educators in plant biotechnology to furnish students, instructors and researchers with a broad consideration of the field. Divided into eight major parts, the text covers everything from the history of plant tissue culture and basic methods to propagation techniques, crop improvement procedures, specialized applications and nutrition of callus cultures. New topic discussions and laboratory exercises in the Second Edition include ""Micropropagation of Dieffenbachia,"" ""Micropropagation and in vitro flowering of rose,"" ""Propagation from nonmeristematic tissue-organogenesis,"" ""Variation in culture"" and ""Tissue culture of ferns.""It is the book's extensive laboratory exercises that provide a hands-on approach in illustrating various topics of discussion, featuring step-by-step procedures, anticipated results, and a list of materials needed. What's more, editors Trigiano and Gray go beyond mere basic principles of plant tissue culture by including chapters on genetic transformation techniques, and photographic methods and statistical analysis of data. In all, Plant Tissue Culture Concepts and Laboratory Exercises, Second Edition, is a veritable harvest of information for the continued study and research in plant tissue culture science.

Part I

Introduction

chapter one

Introduction to plant tissue culture

Dennis J. Gray and Robert N. Trigiano

The purpose of this chapter is to provide an initial focus to Plant Tissue Culture Concepts and Laboratory Exercises and to begin the process of defining terms and ideas unique to this subject area. Keeping in mind that the mission of the entire book is to introduce, define, and provide training, we will use this “introduction” primarily to orient the reader on the book’s structure and to highlight information that is discussed in depth in subsequent chapters. We have taken a “minimalist” approach to development of definitions and use of terminology for the simple reason that this serves, in part, the role of an introductory textbook; in our experience, the specific meaning of many terms varies depending on whom is asked and it is often impossible to arrive at a globally satisfactory definition. Rather than confuse the beginning student (and ourselves) with “verbal gymnastics,” we chose to simplify terminology. This results in the emphasis being placed on assimilation of key concepts and allows the student to become aware of the richness and natural controversy that involves terminology of such an emerging field after having acquired preliminary background knowledge and confidence. An example of our minimal approach to terminology can be seen in the very title of the book — “… plant tissue culture. …” To be painstakingly exact, the title should have been “… plant cell, tissue, organ, and organ system culture. …” However, we achieved the desired effect of announcing the subject of the book without being tedious or confusing. This same approach represents our goal throughout the text.

Meant to serve as a primary text for both introductory and advanced courses, Plant Tissue Culture Concepts and Laboratory Exercises furnishes instructors and students alike with a broad consideration of the field — providing historical perspectives, discussing state-of-the-art techniques and methodologies, and looking forward to future advances and applications. It also presents many useful protocols and procedures and, thus, should serve as a valuable reference. The book is intentionally written to be rather informal — it provides the reader with a minimum number of references but does not sacrifice essential information or accuracy. Broad-topic chapters are authored by specialists with considerable experience in the field and are supported by one or more laboratory exercises illustrating central concepts of the topic. Collectively, the laboratory exercises are exceptionally diverse in nature, providing something for everyone — from beginning to advanced students. Importantly, the authors have successfully completed the exercises many times, often with either tissue culture classes or in their own research laboratories. A unique feature of all the laboratory exercises is that the authors have provided in general terms what results should be expected from each of the experiments. At the end of each exercise, there are a series of questions designed to provoke individual thought and critical examination of the experiment and results. Our intentions are that instructors will not attempt to do all the experiments, but rather select one or two for each concept that serves the needs and interests of their particular class. Thus, exercises for different crop types, such as grasses, ornamentals, trees, and vegetables, are variously provided to allow tailoring of a class to the department or discipline in which it is taught. For an advanced class, different experiments may be assigned among resourceful students. More advanced experiments following the general or beginning class exercises are embedded within some of the laboratory chapters.

The laboratory projects are executed with many different agronomic and horticulturally important plants including chrysanthemum, orchardgrass, syngonium, ajuga, petunia, watermelon, peanut, and lilac, all of which are easy to grow, can be ordered from common sources for nominal fees, or can be purchased at the local market. This range of plants is substantially broader than that used in any previous text on the subject and should stimulate interest in students from many botanical and agricultural disciplines.

The various chapters necessarily assume that the student has a good understanding of botany and botanical terminology. As such, it is recommended that companion plant anatomy, genetics, morphology, and physiological textbooks be available as needed in order to provide access to basic botanical knowledge.

In addition to this introductory section, the textbook is divided into the following five primary parts: History of Plant Tissue Culture, Supporting Methodologies, Propagation Techniques, Crop Improvement Techniques, and Special Topics. Each section combines related facets of plant tissue culture and includes one to several concept chapters, usually with accompanying laboratory exercises.

Part II, “History of Plant Tissue Culture,” is an abbreviated account of how the field developed from an early theoretical base to the highly technical discipline that exists today. This treatment is somewhat unique because it documents research progress as modulated by significant world events. In addition to recording the people, places, and dates for pertinent discoveries, we feel it is interesting for students to see the challenges encountered by researchers that result in the often uneven pace of research. We end this chapter on a contemporary “hot” topic by discussing progress being made with genetically engineered plants in light of society’s reaction, much of which information is gleaned from news reports. Beyond a past historical account, we hope that this chapter will provide a “snapshot” of the controversial present.

Part III, “Supporting Methodologies,” begins the process of teaching tissue culture methodology. Chapter three covers several key topics that must be assimilated to accomplish subsequent laboratory exercises. It describes basic equipment needed and discusses the nature of nutrient culture medium and provides formulas for the most commonly used types. Chapter three also discusses various methods to prepare medium, and provides complete and logical examples of how to make solutions and dilutions and to accomplish sterile culture work. An experienced teacher with laboratory resources and methodologies already in place may choose to pay less attention to this chapter, whereas the instructor of a newly established course will find it indispensable. Chapter four is meant to bring the student into first contact with actual “tissue culture” by demonstrating the essential need for specific nutrients to grow “callus cultures.” “Callus” is probably the most common term used to describe tissue cultures, but it only pertains to a certain type of unorganized tissue. As previously mentioned, a common term like “callus” can be defined in several, often conflicting, ways depending on opinion, but for here we consider a callus to be an unorganized tumor-like growth of cells. Part III ends with three chapters designed to emphasize common methods to visualize and document studies (histology and microscopy/photography, respectively) and to quantify responses (statistical analysis) of tissue culture in research.

Part IV, “Propagation Techniques,” encompasses the essential foundation of plant tissue culture. In this section the three types of commonly used culture regeneration systems are introduced, then discussed and illustrated in depth.

We begin Part IV by discussing “Propagation from Preexisting Meristems” (Chapter eight), a process that is more commonly termed “micropropagation.” Students tend to particularly enjoy some of the exercises in this section because they culminate in “house plants” that can be taken home and reared. Micropropagation is the simplest and most commercially useful tissue culture method. The tissue culture industry uses micropropagation almost exclusively for ornamental plant production. This is reflected by the range of ornamental plants used in the laboratory exercises. However, a unique exception to micropropagation of ornamental plants is the exercise on tissue culture production of potato (Chapter eleven); tissue culture-derived microtubers now are commonly used to establish potato fields. Other points of interest in this section are given in Chapter ten, which demonstrates the exact methodology used by industry to produce Dieffenbachia, and Chapter thirteen, which shows the production of rose flowers in culture.

The second propagation system to be discussed in Part IV is “Organogenesis” (Chapter fourteen). Organogenesis is the development of organs, typically shoots and/or roots, from cells and tissues that would not normally form them. The term “adventitious” has also been used to describe the plant parts formed by the process of organogenesis. Shoot organogenesis is another means of propagating plants. While not used much in commercial production, organogenesis is used extensively in genetic engineering as a means to produce plants from genetically altered cells. In Chapter fourteen, both the theory and developmental sequences of how cells are induced to follow such a developmental pathway are discussed. This chapter is followed by laboratory exercises that show the induction of shoots from leaves of chrysanthemum (Chapter fifteen), watermelon (Chapter sixteen), Torenia (Chapter seventeen), and petunia (Chapter eighteen).

The third propagation system discussed in Part IV is “Nonzygotic Embryogenesis” (Chapter nineteen). Nonzygotic embryogenesis is a broadly defined term meant to cover all instances where embryogenesis occurs outside of the normal developmental pathway found in the seed. One type of nonzygotic embryogenesis is termed somatic embryogenesis. This is a unique phenomenon exhibited by vascular plants, in which somatic (nonsexual) cells are induced to behave like zygotes. Such induced cells begin a complex, genetically programmed series of divisions and eventual differentiation to form an embryo that is more or less identical to a zygotic embryo. This type of propagation system is important since the embryos develop from single cells, which can be genetically engineered, and are complete individuals that are capable of germinating directly into plants. Thus, potentially, somatic embryogenesis also represents an efficient propagation system. Chapter nineteen discusses the developmental processes and significance of nonzygotic embryogenesis. Laboratory exercises illustrate nonzygotic embryogenesis for a range of crop types, including a grass (orchardgrass) (Chapter twenty), a vegetable (cantaloupe) (Chapter twenty-one), an agronomic crop (peanut) (Chapter twenty-two), a flowering ornamental plant (cineraria) (Chapter twenty-three), and both angiosperm (yellow poplar) and gymnosperm (white spruce) tree species (Chapters twenty-four and twenty-five, respectively).

In Part V, “Crop Improvement Techniques,” the aforementioned propagation techniques are integrated with other methodologies in order to modify and manipulate germplasm. Chapter twenty-six discusses the use of plant protoplasts. Protoplasts are plant cells from which the cell wall has been enzymatically removed, making the cells amenable to cell fusion and other methods of germplasm manipulation. Two laboratory exercises concerning tobacco and potato (Chapter twenty-seven) and chrysanthemum and orchardgrass (Chapter twenty-eight) follow. Chapter twenty-nine details the use of haploid culture in plant improvement. Haploid cultures usually are derived from microspore mother cells and result in cells, tissues, and plants with half the normal somatic cell chromosome number. Such plants are of great use in genetic studies and breeding, since all of the recessive genes are expressed and by doubling the haploid plants back to the diploid ploidy level, dihaploid plants are produced, which are completely homozygous. True homozygous plants are time consuming and often impossible to produce by conventional breeding. Chapter thirty is a laboratory exercise detailing haploid plant production from the microspores contained in tobacco anthers.

Concept Chapter thirty-one discusses genetic transformation (also known as genetic engineering), which is a current hot topic in agriculture. Transformation wherein genes from unrelated organisms can be integrated into plants without sexual reproduction, resulting in “transgenic plants,” is the most significant application for plant tissue culture when considering its impact on humankind (see Part II). Two laboratory exercises discuss transformation of tobacco and carrot (Chapter thirty-two) and chrysanthemum (Chapter thirty-three) using Agrobacterium tumefaciens, nature’s own and the original genetic engineer. This soil-inhabiting bacterium is ubiquitous and strains infect a wide range of host plants including angiosperms (monocots and dicots), gymnosperms, and ferns. Agrobacterium causes a tumor-like proliferation of cells, hence the common name of the disease is “crown gall,” by transferring some of its plasmid (T_i DNA to the host cell. Researchers have cleverly learned how to disarm (can no longer cause disease) Agrobacterium by deleting the genes that cause tumor growth. They can substitute genes that we want to transfer to our host species. Among these useful genes are those for disease resistance, herbicide tolerance, flower color, and fruit ripening. Chapter thirty-four describes the construction of a device for particle bombardment of plants, an alternate method of transformation. In this method, DNA is coated onto small tungsten or gold particles and literally shot into the plant cells.

Chapter thirty-five describes the use of cryopreservation of germplasm and Chapter thirty-six is a laboratory exercise illustrating its use. Cryopreservation is an efficient means of safeguarding valuable plant germplasm by freezing all metabolic activity. Cryopreserved cells and tissue can be kept for extended periods of time without mutations occurring or any physiological decline. Concept Chapter thirty-seven describes the production of secondary products by plant cells in culture. This subject is somewhat unique in the context of previous topics, because the end product is not a regenerated plant, but rather a chemical. Use of plants as biofactories to produce complex pharmaceuticals is of great interest, particularly due to its potential application in health care. In Chapter thirty-eight, the methods for production of an intensely colored pigment from Ajuga cell cultures are described. A final topic Part V is that of in vitro plant pathology. This topic (Chapter thirty-nine) is introduced to demonstrate the use of in vitro systems to mimic whole plants in the development of disease symptoms — overall, a convenient means of studying hostpathogen interactions.

Part VI, “Special Topics,” is a bit of a “catch-all” section where we placed topics that we considered important enough to warrant inclusion in the book, but which did not quite fit in the other sections. Chapter forty discusses reasons that genet...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• Part I: Introduction
• Part II: History of plant tissue culture
• Part III: Supporting methodologies
• Part IV: Propagation techniques
• Part V: Crop improvement techniques
• Part VI: Special topics
• Part VII: Appendix
• Part VIII: Index