SPRINGER LAB MANUAL Springer-Verlag Berlin Heidelberg GmbH o. L. Gamborg G. C. Phillips (Eds.) Plant Cell, Tissue and Organ Culture Fundamental Methods With 86 Figures Springer Professor Or. 0LUF L. GAMBORG University of California, Oavis 1404 Solana Orive Belmont, CA 94002-3654 USA Professor Or. GREGORY C. PHILLIPS New Mexico State University Molecular Biology Program Department of Agronomy and Horticulture Box 30001, Mail Stop 3Q Las Cruces, NM 88003-0003 USA Library of Congress Cataloging-in-Publication Data Plant cell, tissue, and organ culrure : fundamental methods / O. L. Gamborg, G. C. Phillips (eds.). p. em. - (Springer lab manual) Includes bibliographical references and index. ISBN 978-3-642-48974-7 ISBN 978-3-642-79048-5 (eBook) DOI 10.1007/978-3-642-79048-5 1. Plant cell eulture-Laboratory manuals. 2. Plant tissue eulrure-Laboratory manuals. 3. Plant micropropagation-Laboratory manuals. I. Gamborg, O. L. (Oluf L.), 1934- . II. Phillips, Gregory C. III. Series. QK725.P5571995 581'.0724-de20 The material is eoncerned, speeifically the rights of translation, reprinting reuse of illustrations, recitation, broadcasting, reproduetion on microfilm or in any other way, and storage in data banks. Duplication of this pu blieation or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permissions for use must always be obtained from Springer-Verlag Berlin Heidelberg GmbH. Violations are liable for prosecution under the German Copyright Law. © Springer-Verlag Berlin Heidelberg 1995 Originally published by Springer-Verlag Berlin Heidelberg New York in 1995 The editors dedicate this volume to all the mentors, colleagues, and students with whom they have learned. We thank each ofy ou for your patience, encouragement and devotion. Preface A wide variety of technologies in tissue culture, genetic manipulation, and mo lecular biology have been developed for an increasing number of plant species. Jointly, these laboratory-based technologies are referred to as plant biotechnol ogy. They have emerged as practical procedures for enhancing the efficiency of all branches of plant production and improvement, as well as contributing to the study of basic aspects of plant science. There is general agreement that plant biotechnology will have a major impact on all aspects of cropping industries from the producers to the users. Plant biotechnology has direct application in genetic improvement and production of plants. The application of micropropagation in plant production has become a multibillion dollar industry for nurseries and commercial centers world-wide. Micropropagation is used in the production of plants which are free of viruses and other pathogens, and for the rapid multiplication of vegetatively propagated species. The fundamental basis for the applications of plant biotechnology is the capability of plant cells and organs to develop into complete plants, which can be grown to maturity. The methods of culturing meristems or shoot tips, or devel oping somatic embryogenic lines, are used extensively as procedures in micropropagation. The increasing availability of useful, defined genes as recom binant DNA molecules, the refinement of methods to introduce the genes into targeted plant cells, as well as advances in regenerating plants from a variety of crop plants, have resulted in the production of a large number of transgenic plants. The results have been plants with potential for improved disease resis tance, insect resistance, new flower colors, and higher product quality. Geneti cally improved crops developed with the assistance of plant biotechnology will have a major impact on the production and marketing of food and industrial products. Because of the rapid advances in plant biotechnology, there is an increasing need for training and developing skills in the techniques required for modern plant research as well as for practical applications. The present volume will provide effective access to acquiring practical skills and experience in a wide range of the fundamental techniques of plant biotechnology. The unique feature of this volume is the fact that it is comprehensive and self-contained. All the information and procedures needed to carry out the methods in the chapters are found in the volume. VIII Preface The topics begin with the setting up and management of a functionallabora tory unit and the fundamental procedures of media preparation and sterile techniques. The following chapters present the basic procedures used for plant propagation and regeneration from explant tissues and from cultured cells. There is a special chapter illustrating the more recent technologies of produc tion, preservation, and uses of somatic embryogenesis as propagules. The third major section deals with specialized culture systems including meristem culture for virus elimination, embryo culture for wide hybridization and other applica tions, and anther and microspore cultures for haploid plant production. A sepa rate section is devoted to protoplast procedures and methods of transformation and production of transgenic plants. The last section of the volume covers many of the analytical methods which are used frequently in conjunction with plant cell and tissue culture. These methods include histology, electron microscopy, and immunofluorescence tech niques used for analyzing cell and tissue development during culture, immunoabsorbent methods for certifying virus-free plants, and the use of chro mosome staining procedures and random amplified polymorphic DNA markers for characterization of cultured cells and regenerated plants. In the Appendices we provide lists of media compositions, lists of frequently used compounds with molecular weights, a list of suppliers with addresses, answers to the study ques tions found in each chapter, as well as an extensive glossary of terms. The design of the chapters, and of the entire volume, will meet the needs of research technicians, students, and trainees, as well as being of great value to professional scientists, research leaders, managers, and teachers, who mayor may not have had prior experience in plant biotechnology. Las Cruces, April 1995 OLUF L. GAMBORG GREGORY C. PHILLIPS Orientation to Using the Volume The present volume is designed to serve a variety of purposes. The major use will be as an excellent book of instruction, for information and training to gain hands-on experience and skills in the technologies. Another major use will be as a versatile resource for technical information in the field of plant biotechnology. The design of each chapter will facilitate training and education as well as provide a guide for research applications. The introduction provides a general guide to the applications of the particular technology and a brief explanation of how the technology works, followed by a statement on objectives and goals of the chapter. Lists of equipment, apparatus, reagents, and materials which are needed in order to carry out the technology are then presented. Prior to listing the procedures, there is a section describing the preparations of media, solu tions, plant materials, and other items. The chapter then details the procedures. The style for the protocols makes it very easy to follow, both with respect to the language and to minimize errors. Each chapter then shows examples of the results which can be expected, and how data can be presented. Throughout the chapter there are notes with precau tions and suggestions, but at the end of the chapter there is also a section on solving the most frequently encountered problems. Keeping in mind the use of the volume for training and education, each chapter has a few study questions designed to test the comprehension of the content by anyone using the book and for use in group discussions. The answers to the questions are presented in Appendix C. The book is designed with the possibility of being used as the principal text for a complete course in plant biotechnology. For example, Chapters 1 through 16 or some subset of these chapters could form the basis for an intensive semester long or year-long course. Alternatively, a smaller number of chapters can be chosen for a semester or for a series of courses which would be appropriate for beginners, followed by other chapters for more advanced scientists and for special assignments. For example, Chapters 2 and 3, along with 4 and/or 5, could provide the basis for a brief beginner course, along with Chapter 1 as a reading assignment. Chapters 6, 7, 8, and 9 could be an advanced assignment. Chapters 12 or 13 would be other examples of an advanced assignment. A special assignment could be Chapters 10 and 21, which deal with the use of producing virus-free plants by meristem culture and how to assay the plants with ELISA tests. Another special assignment could be Chapters 15 and/or 16, which deal with the production of transgenic plants, followed by Chapter 22 as X Orientation to Using the Volume an advanced assignment which would be used to provide evidence of stable integration of the foreign gene into the genome of the transgenic plant. A special assignment to learn the specialized techniques presented in any of the Chapters 17 through 20 could be combined with any of the Chapters 5 through 8 or 14 to provide the necessary plant materials. A student who has mastered the technologies presented in this volume should be well prepared to conduct basic plant research, participate in crop genetic improvement programs, or enter the plant production industry in any role requiring a working knowledge of plant biotechnology. Note to Readers and Users of the Protocols in this Volume The editors would be grateful for any comments on the usefulness of the infor mation contained in the volume. We also would appreciate very much to receive comments on possible errors and the success, or lack of success, in the use of any of the protocols. Any suggestions you may have on the improvement of any part of the volume would be helpful. For example, please tell us of any changes you made in any of the steps of the procedures that improved the results. Such feedback information would be invaluable in any future revisions of the volume. Disclaimer In many cases authors have cited the use of equipment and materials with specific brands and trade names from specific manufacturors of those items. This method of citation is not meant to provide an endorsement for the item to the exclusion of other equivalent sources. The editors asked the authors to provide specific examples of items used in their laboratories so that the results of the exercises could be more easily reproduced by the novice learning the techniques.