Plant Protoplasts and Genetic Engineering I

460 Pages·1989·34.121 MB·English

by  Y. P. S. Bajaj (auth.), Professor Dr. Y. P. S. Bajaj (eds.)

Volumes alread! published
Volume 1: "frees I (1986)
Volume 2: Crops I (1986)
Volume 3: Potato (1987)
Volume 4: Medicinal and Aromatic Plants I (1988)
Volume 5: "frees II (1988)
Volume 6: Crops II (1988)
Volume 7: Medicinal and Aromatic Plants II (1989)
Volume 8: Plant Protoplasts and Genetic Engineering I (1989)
Volumes in preparation
Volume 9: Plant Protoplasts and Genetic Engineering II (1989)
Volume 10: Legumes and Oilseed Crops I
Volume 11: Somaclonal Variation in Crop Improvement
Volume 12: Haploids in Crop Improvement
Volume 13: Wheat
Volume 14: Rice
Biotechnology in
Agriculture and Forestry 8
Plant Protoplasts
and Genetic Engineering I
Edited by Y. P. S. Bajaj
With 152 Figures
Springer-Verlag Berlin Heidelberg New York
London Paris Tokyo Hong Kong
Professor Dr. Y. P. S. BAJAJ
A-137
New Friends Colony
New Delhi 110065, India
ISBN-13: 978-3-642-73616-2 e-ISBN-13: 978-3-642-73614-8
DOl: 10.1007/978-3-642-73614-8
Plant protoplasts and genetic engineering 1 edited by Y. P. S. Bajaj. p. cm. - (Biotechnology in
agriculture and forestry; 8 - ) Includes bibliographies and index. 1. Plant protoplasts. 2. Plant genetic
engineering. I. Bajaj, Y.P.S., 1936- . II. Series. QK725.P578 1989 581.87'3 - dc 1988-31197
This work is subject to copyright. All rights are reserved, whether the whole or part of the material
is concerned, specifically the rights of translation, reprinting, re-use of illustrations, recitation, broad
casting, reproduction on microfilms or in other ways, and storage in data banks. Duplication of this
publication or parts thereof is only permitted under the provisions of the German Copyright Law of
September 9, 1965, in its version of June 24, 1985, and a copyright fee must always be paid. Violations
fall under the prosecution act of the German Copyright Law.
© Springer-Verlag Berlin Heidelberg 1989
Softcover reprint of the hardcover 1s t 1989
The use of registered names, trademarks, etc. in this publication does not imply, even in the absence
of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
'JYpesetting: K+V Fotosatz GmbH, Beerfelden.
Offsetprinting and bookbinding: Konrad 1riltsch, Graphischer Betrieb, Wllrzburg.
2131/3145-543210 - Printed on acid-free paper
Dedicated to my sister Nirmal
Preface
Isolated protoplasts are a unique tool for genetic manipulation of plants.
Since the discovery of a method for the enzymatic isolation of pro-·
toplasts by Professor E. C. Cocking in 1960, tremendous progress has
been made in this very fascinating area of research. I have witnessed the
struggle in the 1960's and early 1970's, when obtaining a clean prepara
tion of protoplasts was considered an achievement. I also shared the
pioneering excitement and enthusiasm in this field during the 2nd Inter
national Congress of Plant Tissue Culture held at Strasbourg in 1970,
where Dr. I. Thkebe of Japan presented his work on the induction of
division in tobacco protoplasts. This was followed by my participation
in three international conferences devoted to plant protoplasts held in
1972 in Salamanca (Spain) and Versailles (France), and then in 1975 in
Nottingham (England). The enthusiasm shown by plant scientists at
these meetings was ample proof of the bright future of protoplast
technology, and it became evident that protoplasts would playa major
role in plant biotechnology, especially in genetic engineering. Since then
we have never looked back, and now the methods for isolation, fusion,
and culture, as well as regeneration of somatic hybrids, have become
routine laboratory procedures for most plant species. Currently the focus
is on cereal and tree protoplasts.
In order to bring about any genetic manipulation through fusion, in
corporation of DNA, and transformation, the regeneration of the entire
plant through manipulation of protoplasts is a prerequisite. Today we are
at the threshold of the breakthrough in genetic engineering through
manipulation of protoplasts. This volume on Plant Protoplasts and
Genetic Engineering I is the first in the series, containing 28 chapters
dealing with the isolation, fusion, culture, immobilization, and cryopre
servation of protoplasts, and the regeneration of somatic hybrid and
cybrid plants. The technology and literature on the regeneration of com
plete plants from isolated protoplasts of rice, potato, citrus, soybean,
linseed, cabbage, chicory, lettuce, butterbur, orchids, and some forest
tree species are discussed. Various methods of fusion of protoplasts are
also reviewed, and the work on the regeneration of interspecific and in
tergeneric somatic hybrids in Lycopersicon, Petunia, Nicotiana,
Hyoscyamus, Solanum, Citrus, Brassica, Medicago, and Trifolium is
discussed. In the next volume, Plant Protoplasts and Genetic Engineer
ing II, some of the newer techniques such as microinjection, electrofu-
VIII Preface
sion, flow cytometry, uptake of organelles, and viruses are described and
the work on recombinant DNA technology for direct gene
transferlAgrobacterium-mediated transformation in potato, rice, maize,
cotton, tobacco, Brassica, poplars, Vinca, Hyoscyamus etc. is reviewed.
These studies reflect the far-reaching impact of protoplast alterations for
agriculture and forest biotechnology. The greatest use of these recent ad
vances in protoplast technology is likely to be made for the induction of
genetic variability and the development of new genotypes for specific
plant improvement programs.
New Delhi, March 1989 Y. P. S. BAJAJ
Series Editor
Contents
Section I Isolation Culture and Preservation of Protoplasts
1.1 Recent Advances in the Isolation and Culture of Protoplasts
and Their Implications in Crop Improvement
Y. P. S. BAJAJ
1. Introduction ......................................... 3
2. Enzymes ............................................ 5
3. Viability ............................................ 6
4. Agarose Plating ...................................... 6
5. Regeneration of Plants from Protoplasts ................ 8
6. Culture of Haploid Protoplasts ........................ 10
7. Protoplast Culture and Somaclonal Variation ............ 11
8. Immobilization of Protoplasts ......................... 12
9. Cryopreservation of Protoplasts ........................ 14
10. Conclusions and Prospects ............................ 15
References .............................................. 16
1.2 Enzymes for the Isolation of Protoplasts
S. ISHII (With 1 Figure)
1. Introduction .......................................... 23
2. Cell Wall Compositions ................................ 24
3. Enzymes that are Effective for Isolating Protoplasts from
Gramineae Tissues .................................... 25
4. Comparison of Enzymes Between Monocots and Dicots that
are Effective for Isolating Protoplasts .................... 28
5. Purification Procedures of Enzymes ..................... 29
6. Protoplasts Isolated by Purified Enzymes ................ 30
7. Summary and Conclusion .............................. 31
References .............................................. 32
1.3 Viability of Plant Protoplasts
P. B. GAHAN (With 6 Figures)
1. Introduction .......................................... 34
2. Methodology ......................................... 34
3. Efficacy of the Methods ............................... 43
4. Summary ............................................ 46
References .............................................. 47
x
Contents
1.4 Agarose Plating of Protoplasts and Its Applications
J. J. M. DONS and C. M. COLIJN-HooYMANS (With 2 Figures)
1. Introduction .......................................... 50
2. Embedding of Protoplasts in Agarose ............... ~ . . . . 50
3. Methods for the Culture of Agarose-Embedded Protoplasts 53
4. A Survey of Literature on the Application of Agarose
Plating in Protoplast Culture ........................... 56
5. Special Applications of Agarose Culture ................. 59
6. Concluding Remarks .................................. 60
References .............................................. 61
1.5 Immobilization of Plant Protoplasts
H. SCHNABL and U. ZIMMERMANN (With 11 Figures)
1. Introduction ......................................... 63
2. Immobilization in Gel Matrices ........................ 64
3. Entrapment of Cell Protoplasts in Different Matrices ..... 70
4. Viability of Immobilized Cells and Protoplasts ........... 72
5. Applications of Immobilized Plant Cells and Protoplasts .. 79
6. Biotransformation .................................... 87
7. Biosynthesis ......................................... 87
8. Product Release ...................................... 89
9. Conclusions ......................................... 92
10. Protocol for the Immobilization of Protoplasts .......... 92
References .............................................. 93
1.6 Cryopreservation of Plant Protoplasts
Y.P.S. BAJAJ (With 2 Figures)
1. Introduction .......................................... 97
2. Significance of Cryopreservation of Protoplasts ........... 98
3. Review of Work ....................................... 98
4. Isolation, Freezing and Culture of Protoplasts ............ 99
5. Survival of Somatic Hybrid Protoplasts .................. 103
6. Summary and Conclusions ............................. 105
References .............................................. 105
Section n Regeneration of Plants from Protoplasts
11.1 Regeneration of Plants from Rice Protoplasts
J. KYOZUKA, K. SHIMAMOIO and H. OOURA (With 6 Figures)
1. Introduction .......................................... 109
2. Isolation of Protoplasts ................................ 111
3. Culture of Protoplasts ................................. 112
4. Regeneration of Plants ................................. 113
5. Somaclonal Variation .................................. 115
Contents XI
6. Agronomic Thlits of Protoplast-Derived Plants..... ....... 118
7. Conclusions and Prospects ............................. 121
8. Protocol ............................................. 121
References .............................................. 122
11.2 Isolation and Culture of Potato Protoplasts
M. ZUBA and H. BINDING (With 2 Figures)
1. Introduction .......................................... 124
2. Isolation of Protoplasts ................................ 127
3. Culture of Protoplasts ................................. 129
4. Regeneration of Plants and the Characters of Regenerated
Plants ............................................... 130
5. Fusion of Protoplasts .................................. 135
6. Conclusions and Prospects ............................. 137
7. Protocols ............................................. 137
References .............................................. 142
11.3 Isolation and Culture of Citrus Protoplasts
A. VARDI and E. GAWN (With 5 Figures)
1. General Account ...................................... 147
2. Isolation of Protoplasts ................................ 152
3. Protoplast Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
4. Embryogenesis ........................................ 155
5. Regeneration of Plants. . . ....... ........ . . . . . . ......... 155
6. Fusion of Protoplast ................................... 156
7. Conclusions and Prospects ............................. 156
8. Protocol ............................................. 157
References .............................................. 158
11.4 Regeneration of Plants from Linum Protoplasts
M.N. BARAKAT and E.C. COCKING (With 6 Figures)
1. Economic Importance and Breeding Objectives in Linum ... 160
2. Source Material for Protoplast Isolation ................. 161
3. Protoplast Isolation ................................... 161
4. Protoplast Culture ..................................... 165
5. Plant Regeneration from Protoplast-Derived Callus ........ 167
6. Protoplast Fusion ..................................... 170
7. Conclusions .......................................... 170
8. Protocol ............................................. 170
References .............................................. 171
11.5 Isolation and Culture of Glycine Protoplasts
C.A. NEWELL and H. T. Luu (With 2 Figures)
1. Introduction .......................................... 173
2. Isolation of Protoplasts ................................ 179