Tissue Culture Techniques

Plant Tissue Culture Techniques and Experiments is a manual that contains laboratory exercises about the demonstration of the methods and different plant materials used in plant tissue culture. It provides an overview on the plant cell culture techniques and plant material options in selecting the explant source. This book starts by discussing the proper setup of a tissue culture laboratory and the selection of the culture medium. It then explains the determination of an explant which is the ultimate goal of the cell culture project. The explant is a piece of plant tissue that is used in tissue culture. Furthermore, the book discusses topics about callus induction, regeneration and morphogenesis process, and haploid plants from anther and pollen culture. The meristem culture for virus-free plants and in vitro propagation for commercial propagation of ornamentals are also explained in this manual. The book also provides topics and exercises on the protoplast isolation and fusion and agrobacterium-mediated transformation of plants. This manual is intended for college students, both graduate and undergraduate, who study chemistry, plant anatomy, and plant physiology.

ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xl II INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I STERILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Aseptic Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Physical manipulations • Use of the sterile cabinet (hood) Sterilization Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Heat • Radiation • Toxic gas • Filtration • Antibiotics Quality Control of Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Routine labeling Suggested Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 vi CONTENTS ROUTINE CELL CULTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Feeding Schedules and Media Components . . . . . . . . . . . . . . . . . . . . 29 General properties of media and salt solutions • water as a reagent· Establishingfeeding schedules Subcultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Solutions and methods for adherent cells • Common enzyme solutions • Inoculating (seeding) the cultures Cell Enumeration and Cell Viability . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Hemocytometer • Particle counter • Cell viability Putting Routine Methods to Work . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Normal cell growth characteristics Detecting and Disposing of Contamination . . . . . . . . . . . . . . . . . . . . 66 Bacteria and fUngi • Fungi • Mycoplasma • Viruses • Dealing with contamination Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Inadequate cell growth • Recurrent contamination • When to call your vendor Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Biological hazards • Chemical hazards Suggested Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Problem Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 EXPERIMENTS IN CULTURE . . . . . . . . . . . . . . . . . . . . . . . . . . 91 II Alterations of the Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Serum • Treatments of serum • Plasma-derived serum • Serum-free and low-protein media Substrata. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Coatingplasticware with solutions • Alterations with polymers • Using cells to coat the plasticware • Culturing cells on microcarriers Altering the Environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Temperature changes • Gaseous changes Problem Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 CONTENTS vii PRIMARY CELL CULTURE. . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Dissection • Enzymatic dissociation methods • Nonenzymatic isolation • Purification of cell suspensions • Consideringyield and survival Chatacterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Plant Tissue Culture, Third Edition builds on the classroom tested, audience proven manual that has guided users through successful plant culturing A.tumefaciens mediated transformation, infusion technology, the latest information on media components and preparation, and regeneration and morphogenesis along with new exercises and diagrams provide current information and examples. The included experiments demonstrate major concepts and can be conducted with a variety of plant material that are readily available throughout the year. This book provides a diverse learning experience and is appropriate for both university students and plant scientists. Provides new exercises demonstrating tobacco leaf infiltration to observe transient expression of proteins and subcellular location of the protein, and information on development of a customized protocol for protoplast isolation for other experimental systems Includes detailed drawings that complement both introductions and experiments Guides reader from lab setup to supplies, stock solution and media preparation, explant selection and disinfestations, and experimental observations and measurement Provides the latest techniques and media information, including A. tumefaciens mediated transformation and infusion technology Fully updated literature

This book was written for those individuals who are concerned about the techniques and practices of plant cell cultures for horticultural crops. It was designed to serve as a text and reference for students and professionals in ornamental horticulture, fruit and vegetable crop pro duction, botany, forestry, and other areas of plant science. Research during the last twenty-five years in the area of plant tissue culture has led to many developments and changes in this field. Al though the techniques involved in the manipulation of plant tissue culture are now relatively straightforward, the presentation of these techniques in a short volume for the beginner in the field is generally unavailable. In addition to describing the techniques for establishment and manipulation of specific species, several chapters in this book also provide a brief, general review of important cultural parameters. Spe cific protocols and laboratory procedures may also be found in the appendix. I hope that this presentation of information will be helpful to those individuals wanting to apply plant tissue culture techniques for horticultural crops.

Tissue Culture: Methods and Applications presents an overview of the procedures for working with cells in culture and for using them in a wide variety of scientific disciplines. The book discusses primary tissue dissociation; the preparation of primary cultures; cell harvesting; and replicate culture methods. The text also describes protocols on single cell isolations and cloning; perfusion and mass culture techniques; cell propagation on miscellaneous culture supports; and the evaluation of culture dynamics. The recent techniques facilitating microscopic observation of cells; cell hybridization; and virus propagation and assay are also encompassed. The book further tackles the production of hormones and intercellular substances; the diagnosis and understanding of disease; as well as quality control measures. Scientists and professionals interested in methodology per se will find the book invaluable.

It is a pleasure to contribute the foreword to Introduction to Cell and Tissue Culture: The ory and Techniques by Mather and Roberts. Despite the occasional appearance of thought ful works devoted to elementary or advanced cell culture methodology, a place remains for a comprehensive and definitive volume that can be used to advantage by both the novice and the expert in the field. In this book, Mather and Roberts present the relevant method ology within a conceptual framework of cell biology, genetics, nutrition, endocrinology, and physiology that renders technical cell culture information in a comprehensive, logical for mat. This allows topics to be presented with an emphasis on troubleshooting problems from a basis of understanding the underlying theory. The material is presented in a way that is adaptable to student use in formal courses; it also should be functional when used on a daily basis by professional cell culturists in a- demia and industry. The volume includes references to relevant Internet sites and other use ful sources of information. In addition to the fundamentals, attention is also given to mod ern applications and approaches to cell culture derivation, medium formulation, culture scale-up, and biotechnology, presented by scientists who are pioneers in these areas. With this volume, it should be possible to establish and maintain a cell culture laboratory devot ed to any of the many disciplines to which cell culture methodology is applicable.

Concise introduction to a major technique of cell biology laboratories for those new to the field.

I started insect cell culture work in 1962, when T. D. C. Grace reported the first establishment of invertebrate continuous cell lines. He obtained grow ing cells from pupal ovaries of the emperor gum moth, Antheraea euca lypti. At that time, I was trying to obtain growing cells from leafhoppers. Grace's method could not be applied directly to my culture because of the differences in species, the size of the insects, and the tissue to be cul tured. The vertebrate tissue culture methods gave me some ideas for pre paring cultures from leafhoppers, but those could not be used directly either. There were no textbooks and no manuals for invertebrate tissue culture, so I had to develop a method by myself. First, I considered what type and what size of vessels are suitable for insect tissue culture. Also, I had to look for suitable materials to construct the culture vessels. Sec ond, I had to examine various culture media, especially growth-promot ing substances, such as sera. Then I had to improve culture media by trial and error. The procedure to set up a primary culture was also a problem. How could I sterilize materials? How could I remove tissues from a tiny insect? How many tissues should I pool in order to set up one culture? I had to find out the answers. Naturally, it took a lot of time.