MORPHOGENESIS IN PLANT TISSUE CULTURES

MORPHOGENESIS IN PLANT TISSUE CULTURES

Edited by

WOONG-YOUNG SOH

and

SANT S. BHOJWANI

Springer-Science+Business Media, B.V.

A C.I.P. Catalogue record for this book is available from the Library of Congress .

ISBN 978-90-481-5206-3 ISBN 978-94-015-9253-6 (eBook)DOI 10.1007/978-94-015-9253-6

Printed on acid-free paper

All Rights Reserved

© 1999 Springer Science+Business Media Dordrecht

Originally published by Kluwer Academic Publishers in 1999.

Softcover reprint ofthe hardcover 1st edition 1999

No part of the material protected by this copyright notice may be reproduced orutilized in any form or by any means, electronic or mechan ical,including photocopying, recording or by any information storage andretrieval system, without written permission from the copyright owner.

Contents

INTRODUCTION xi

PART I BASIC STUDIES

CHAPTER 1. DIFFERENTIATION OF VASCULAR ELEMENTS IN TISSUE CULTURE 3

1 INTRODUCTION 3 2 SYSTEMS OF STUDY 4 3 MARKERS FOR VASCULAR DIFFERENTIATION 11 4 ANALYSIS OF TRANSCRIPTIONAL

REGULATION OF VASCULAR GENE EXPRESSION 23

5 SIGNALS 24 6 CONCLUSIONS 27 7 ACKNOWLEDGEMENTS 27 8 REFERENCES 28

CHAPTER 2. PLANT REGENERATION FROM CULTURED PROTOPLASTS 37

1 INTRODUCTION 37 2 REGENERATION PROCESS IN PROTOPLAST

CULTURE 39 3 PLANT REGENERATION VIA ORGANOGENESIS 48 4 PLANT REGENERATION FROM PROTOPLAST

VIA EMBRYOGENESIS 53 5 GENTIC VARIATIONS IN PROTOPLAST-

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DERIVED PLANTS 56 6 REFERENCES 58

CHAPTER 3. MORPHOGENESIS IN HAPLOID CELL CULTURES 71

1 INTRODUCTION 71 2 ORIGIN OF ANDROGENIC HAPLOIDS 72 3 FACTORS AFFECTING ANDROGENESIS 79 4 CONCLUDING REMARKS 87 5 REFERENCES 87

CHAPTER 4. LIGHT AND ELECTRON MICROSCOPIC STUDIES OF SOMA TIC EMBRYOGENESIS IN SPRUCE 95

1 INTRODUCTION 95 2 SPRUCE SOMATIC EMBRYOGENESIS 96 3 IMMATURE SOMATIC EMBRYOS 97 4 MATURING SOMATIC EMBRYOS 101 5 PROTOPLASTS FROM IMMATURE SPRUCE

SOMATIC EMBRYOS 104 6 ACKNOWLEDGEMENTS III 7 REFERENCES III

CHAPTER 5. PHYSIOLOGICAL AND MORPHOLOGICAL ASPECTS OF SOMATIC EMBRYOGENESIS 115

1 INTRODUCTION 115 2 DEVELOPEMENT OF SOMATIC EMBRYOS 116 3 FORMATION OF EMBRYOGENIC CELL

CLUSTERS FROM SINGLE CELL 123 4 DEVELOPMENT OF FUCUS ZYGOTES AS A

MODEL FOR THE ESTABLISHMENT OF POLARITY DURING EMBRYOGENESIS 127

5 REFERENCES 129 CHAPTER 6. DEVELOPMENTAL AND STRUCTURAL

ASPECTS OF ROOT ORGANOGENESIS 133 1 INTRODUCTION 133 2 ROOT PRIMORDIUM DIFFERENTIATION 135 3 ORGAN CULTURE SYSTEM 144 4 TISSUE CULTURE SYSTEM 149 5 VASCULAR DIFFERENTIATION IN ROOT

PRIMORDIUM 156 6 ROOT EMERGENCE AND GROWTH 158 7 REFERENCES 161

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CHAPTER 7. SHOOT MORPHOGENESIS: STRUCTURE, PHYSIOLOGY, BIOCHEMISTRY AND MOLECULAR BIOLOGY 171

1 INTRODUCTION 171 2 SHOOT MORPHOGENESIS 172 3 CELLULAR AND MOLECULAR ASPECTS

DURING ORGANIZED DEVELOPMENT 195 4 CONCLUSIONS 203 5 ACKNOWLEDGMENTS 204 6 REFERENCES 204

CHAPTER 8. FLORAL AND VEGETATIVE DIFFERENTIATION IN VITRO AND IN VIVO 215

1 INTRODUCTION 215 2 IN VIVO SYS1EMS: LIMITATIONS IN

MORPHOGENETIC CHANGES 216 3 IN VITRO SYS1EMS: PROGRAMMABLE

DIFFERENTIATION OF AND VEGETATIVE MERIS1EMS DIRECTLY FROM CELL LAYERS AND NOT FROM PRE-EXISTING MERIS1EMS 223

4 GENERAL CONCLUSIONS 229 5 ACKNOWLEDGEMENTS 230 6 REFERENCES 230

CHAPTER 9. DEVELOPMENTAL AND STRUCTURAL PATTERNS OF IN VITRO PLANTS 235

1 INTRODUCTION 235 2 PATHWAYS IN DEVELOPMENTAL EVENTS IN

VITRO 237 3 ABNORMAL MORPHOGENETIC PAT'IERNS 239 4 DEVELOPMENTAL ABERRATION IN

SOMATIC EMBRYOGENESIS 239 5 PRECOCIOUS GERMINATION AND

HYPERHYDRICITY IN SOMATIC EMBRYOS 241 6 HYPERHYDRICITY IN DEVELOPING SHOOTS 242 7 EMBRYO AND ORGAN MORPHOGENESIS IN

LIQUID CULTURES 244 8 CONCLUSION 248 9 REFERENCES 249

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CHAPTER 10. MORPHOGENESIS IN CELL AND TISSUE CULTURES 255

1 INTRODUCTION 255 2 HISTORICAL BACKGROUND 256 3 ETHYLENE BIOSYNTHESIS AND REGULATION 257 4 ETHYLENE ACTION 260 5 EFFECT OF ETHYLENE 262 6 FACTORS AFFECTING CULTURE RESPONSE IN

RELATION TO ETHYLENE 277 7 PA BIOSYNTHESIS AND REGULATION 281 8 EFFECT OF PAS AND ITS RELATION WITH

ETHYLENE 286 9 CONCLUSIONS 289

10 REFERENCES 290

CHAPTER 11. REGULATION OF MORPHOGENESIS BY BACTERIAL AUXIN AND CYTOKININ BIOSYNTHESIS TRANS GENES 305

1 INTRODUCTION 305 2 EFFECTS OF AUXIN AND CYTOKININ 306 3 CONCLUDING REMARKS 321 4 REFERENCES 321

PART II APPLICATIONS

CHAPTER 12. IN VITRO INDUCED HAPLOIDS IN PLANT GENETICS AND BREEDING 329

1 INTRODUCTION 329 2 ORIGIN OF HAPLOIDS 330 3 GENETICS OF HAPLOIDS 340 4 APPLICATIONS OF IN VITRO INDUCED

HAPLOIDS 350 5 CONCLUSION 356 6 REFERENCES 357

CHAPTER 13. SOMATIC HYBRIDIZATION FOR PLANT IMPROVEMENT 363

1 INTRODUCTION 363 2 CURRENT STA1E OF PROTOPLAST FUSION

1ECHNOLOGY 364 3 PROGRESS USING SOMATIC HYBRIDIZATION

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FOR PLANT IMPROVEMENT 376 4 FUTURE PERSPECTIVE 400 5 REFERENCES 405

CHAPTER 14. GERMINATION OF SYNTHETIC SEEDS 419 1 INTRODUCTION 419 2 ARTIFICIAL SEEDS AND THE DELIVERY

PROCESS 420 3 CELLULOSE ACETATE MINI-PLUGS 424 4 PHYTOPROTECTION OF CARROT SOMATIC

EMBRYOS 435 5 CONCLUSION 439 6 ACKNOWLEDGMENTS 440 7 REFERENCES 440

CHAPTER 15. MORPHOGENESIS IN MICRO-PROPAGATION 443

1 INTRODUCTION 443 2 PLANT FACTORS 444 3 THE CULTURE MEDIUM 448 4 THE CULTURE ENVIRONMENT 453 5 ACKNOWLEDGEMENTS 458 6 REFERENCES 458

CHAPTER 16. CELL DIFFERENTIATION AND SECONDARY METABOLITE PRODUCTION 463

1 THE CHALLENGE 463 2 THE GOALS 465 3 THE APPROACH 465 4 DEVELOPMENTAL PHYSIOLOGY AND

PRODUCT FORMATION 466 5 STRESS PHYSIOLOGY AND PRODUCT

FORMATION 483 6 ST ABILITYN ARIABILITY OF PRODUCT

FORMATION 486 7 MOLECULAR BIOLOGY OF PRODUCT

FORMATION 487 8 CONCLUDING REMARKS 490 9 REFERENCES 492

INDEX 503

Introduction

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Morphogenesis, or developmental morphology, is a dynamic process! and a fascinating field of investigation. Since the beginning of this century plant morphologists (Eames, 1961), anatomists (Eames and Macdaniels, 1947) and embryologists (Maheshwari, 1950) have studied the processes of development and differentiation by observing whole plants and their histological preparations and have generated a wealth of information on the structural changes associated with various developmental stages in the life cycle of a large number of plant species. Around 1940 plant morphogenesis became an experimental field when plant physiologists initiated studies on the effect of treatments, such as application of physiologically active compounds or growth regulators, irradiation, exposure to different day­length, temperature and injury, on morphological and structural changes. During the past two decades geneticists and molecular biologists have become interested in plant morphogenesis and extensive work is being done to understand the regulation of gene expression during morphogenesis and how the products of genetic information control the developmental processess.

All sexually reproducing organisms begin as a single-celled zygote, which by growth and cell divisions gives rise to the complex body of the mature organism. Since the divisions involved are mitotic, every cell in the plant body of an organism should have the same genetic material as in the zygote. Yet the organism comprises a vast array of cell, tissue and organ types which are structurally and fimctionally very different. This process of differentiation of genetically identical cells into various different types of tissues has intrigued biologists for a very long time.

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As early as 1868, Hofmeister, in his publication "General Morphology of Growing Things" had raised a very basic question, as to how during growth does the observed form or structure of an organism comes to be as it is and what factors are involved. A similar question was asked by Haberlandt in his famous address to the German Academy in 1902. The classic experiments of Vochting on polarity in cuttings, carried out in 1878, had clearly demonstrated that all cells along the stem length are capable of forming shoots as well as roots but it was Haberlandt who introduced the concept of cellular trotipotency and suggested that terminally differentiated plant cells, as long as they contain the normal complement of chromosomes, should be capable of regenerating whole plants. He made a novel approach to prove this hypothesis in which single cells were isolated from a highly differentiated tissue of the plant body and cultivated in nutrient solution. Although Haberlandt did not succeed in his experiments due to technical problems at that time, his ideas attracted many scientists to pursue this line of investigation, and in 1939 ability of plant cells for unlimited growth was demonstrated by Gautheret, Nobecourt and White, independently. Further progress in the cultivation of plant cells, tissues and organs on artificial medium, under controlled environmental conditions, gave birth to a large number of aseptic techniques, collectively called Plant Tissue Culture. These laboratory techniques have proved to be a powerful approach to manipulate morphogenesis under highly controlled conditions and to understand the role of various extrinsic factors and correlative effects of various tissues in morphogenesis. The three important aspects of morphogenesis to which significant contributions have been made with the aid of plant tissue culture techniques are: (1) cytodifferetiation, (2) organogenic differentiation and (3) somatic embryogenesis. These studies are not only of academic interest but of considerable practical importance.

1 CYTODIFFERENTIATION

In vitro tracheary element differentiation has long been used to study cytodifferentiation. A variety of experimental systems, such as internodes, storage tissues, suspension cultures, single cells and protoplasts, have been used in these studies. One of the most significant advances was the optimization of the Zinnia mesophyll cell system by Fukuda and Komamine (1980) and Church and Galston (see Church, 1993). In this case 50-60% of the cells directly (without a cell division) and fairly synchronously differentiate into tracheary elements within 72 h (Church, 1993).

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2 ORGANOGENIC DIFFERENTIATION

As early as 1939, White (l939b) had reported controlled differentiation of shoots from tissue cultures of tobacco. In 1957 Skoog and Miller put forth the concept of hormonal control of organ formation, according to which root and shoot differentiation is a function of the auxin-cytokinin ratio; relatively higher concentration of cytokinin promotes shoot formation and relatively higher auxin concentration favours root formation. This concept proved to of wide applicability. In 1965 it was shown for the first time that full plants can be obtained starting from a single isolated cell (Vasil and Hildebrandt, 1965). By emperically manipulating the medium and other environmental factors it has been possible to induce shoot differentiation from explants, calli, cells and/or protoplasts of over 1000 species (Thorpe, 1993). It is now well established that plant regeneration can be achieved from all living cells irrespective of their nature of specialization and ploidy level. In 1964 Guha and Maheshwari first time reported organogenic differentiation from haploid microspores. The technique of anther and isolated microspore culture to produce androgenic haploid plants is now well established. Even the triploid endosperm cells have been shown to be totipotent (Johri and Bhojwani, 1965).

3 SOMATIC EMBRYOGENESIS

In 1958 an important contribution was made to the field of experimental morphogenesis when Steward (working in the USA) and Reinert (working in Germany) observed the differentiation of embryos in somatic cell cultures of carrot. The development and appearance of these somatic embryos were comparable to the zygotic embryos of this plant. These reports attracted the attention of several developmental botanists because until that time it was believed that embryo formation is the monopoly of the zygote and a few other ovular cells, and embryo development requires a special environment available only inside the female gametophyte or embryo sac. During the last three decades somatic embryogenesis has dominated the research activities in the field of plant morphogenesis not only because of its curiosities but because of its potential role in basic and applied aspects of plant sciences. Until recently, plant embryogenesis had been studied intensively to describe morphological and anatomical changes that characterize embryo development in different species (Natesh and Rau, 1984; Raghavan, 1986; Bhojwani and Bhatnagar, 1999); almost nothing was known about the molecular and genetic control of embryogenesis because of the practical

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problems of inaccesibility to the zygotic embryo which develops under several cell layers. The progress in the field of somatic embryogenesis during the last two decades has been such that in systems such as carrot (Terzi et al., 1985) gram quantities of practically pure fractions of embryos (histologically and biochemically similar to zygotic embryos) at different developmental stages can be obtained. It has, therefore, now become possible to study genetic and molecular control of morphogenesis during embryogenesis. Using this system about 21 "embryo specific" or "embryo enhanced" genes have been cloned from somatic embryos (Zimmerman, 1993). Investigations related to cellular and molecular aspects of organogenesis and somatic embryogenesis would not only increase our knowledge and understanding of the processes involved but is also expected to enhance our capacity to increase the level of regeneration in presently recalcitrant systems.

4 PRACTICAL IMPORTANCE

The value of in vitro morphogenesis as a commercially viable procedure for plant propagation and as an essential step in genetic improvement of crops through the modem methods of biotechnology has considerably promoted research in this area. Mass production of genetically identical plants by regeneration from vegetative tissues via organogenesis or somatic embryogenesis is of considerable interest to the horticulturists and foresters. Regeneration via embryogenesis is of particular interest as embryos are bipolar structures bearing both root and shoot meristems and may allow certain degree of automation during production and their field planting. Several groups are investigating large scale production of somatic embryos in bioreactors and their encapsulation to produce artificial seeds to facilitate their mechanized planting in the field.

Regeneration of haploid plants from pollen grains has become a popular technique in plant breeding as it enables to achieve homozygosity in a single step, as against several years required to achieve it by the traditional method of recurrent selfing. Androgenesis is also serving as a novel sourse of useful genetic variability (Gametoclonal variation). Plant regeneration from protoplasts, cells and tissue explants is extremely important in hamesing the potential values of somatic hybridization and genetic engineering in crop improvement.

Thus, in vitro morphogenesis is of considerable importance in basic and applied areas of growth and differentiation. However, most of the tissue culture books published during the last two decades (over 100) mainly deal

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with the basic aseptic techniques and their practical applications with very little or no emphasis on morphogenesis. The only book which has discussed in vitro morphogenesis to some extent is "Plant Tissue Culture and Morphogenesis", written in 1964 by Butenko. On the other hand, the book entitled "Plant Morphogenesis", by Sinnot (1960), only deals with in vivo morphogenesis. The substantial advances made in the field of in vitro morphogenesis during the last 25 years was the driving force to bring out this volume on Morphogenesis in Tissue Cultures. It covers all aspects of morphogenesis at cellular, tissue and organ levels. This book includes 16 chapters written by some internationally renowned scientists with considerable experience in their fields. Each chapter discusses upto-date literature in the field, and the text is amply supported by tables and illustrations. The chapters have been classified into two sections, one dealing with articles on the basic aspects and the other on applied aspects of morphogenesis. We feel that the students and teachers of plant sciences would find this book useful.

We are indeed very grateful to the contributors for their ready co­operation without which it would have not been possible to bring out this volume. We would also like to thank Professor Duck Yee Cho, Department of Biology, WooSuk University, Chonbuk and our students Mrs. Sil Yoon, Ee Y oub Kim, Jong Cheun Lee, Hak Soo Kim, Ki Shik Moh and Jae Dong Lee, and Misses. Byung Sook Kang, Ae Ryeong Cho, Eun Kyung Lee, Sun Ah Joo, Bo Rib Jang, and Jung Soon Shin at the Department of Biological Sciences, Chonbuk National University, Korea for their help in various ways in the preparation of this book for press.

Woong -Young Soh Sant S. Bhojwani

Editors