Tài liệu Plant tissue culture engineering - S. DUTTA GUPTA - YASUOMI IBARAKI

Plant tissue culture engineering (469 pages)


FOCUS ON BIOTECHNOLOGY
Volume 6

Series Editors
MARCEL HOFMAN
Centre for Veterinary and Agrochemical Research, Tervuren, Belgium

JOZEF ANNÉ
Rega Institute, University of Leuven, Belgium


Volume Editors
S. DUTTA GUPTA
Department of Agricultural and Food Engineering,
Kharagpur, India
Indian Institute of Technology,

YASUOMI IBARAKI
Department of Biological Science,
Yamaguchi University,

Yamaguchi, Japan

COLOPHON
Focus on Biotechnology is an open-ended series of reference volumes produced by
Springer in co-operation with the Branche Belge de la Société de Chimie Industrielle
a.s.b.l.
The initiative has been taken in conjunction with the Ninth European Congress on
Biotechnology. ECB9 has been supported by the Commission of the European
Communities, the General Directorate for Technology, Research and Energy of the
Wallonia Region, Belgium and J. Chabert, Minister for Economy of the Brussels Capital
Region.

FOREWORD
It is my privilege to contribute the foreword for this unique volume entitled: “Plant
Tissue Culture Engineering,” edited by S. Dutta Gupta and Y. Ibaraki. While there have
been a number of volumes published regarding the basic methods and applications of
plant tissue and cell culture technologies, and even considerable attention provided to
bioreactor design, relatively little attention has been afforded to the engineering
principles that have emerged as critical contributions to the commercial applications of
plant biotechnologies. This volume, “Plant Tissue Culture Engineering,” signals a
turning point: the recognition that this specialized field of plant science must be
integrated with engineering principles in order to develop efficient, cost effective, and
large scale applications of these technologies.
I am most impressed with the organization of this volume, and the extensive list of
chapters contributed by expert authors from around the world who are leading the
emergence of this interdisciplinary enterprise. The editors are to be commended for
their skilful crafting of this important volume. The first two parts provide the basic
information that is relevant to the field as a whole, the following two parts elaborate on
these principles, and the last part elaborates on specific technologies or applications.
Part 1 deals with machine vision, which comprises the fundamental engineering
tools needed for automation and feedback controls. This section includes four chapters
focusing on different applications of computerized image analysis used to monitor
photosynthetic capacity of micropropagated plants, reporter gene expression, quality of
micropropagated or regenerated plants and their sorting into classes, and quality of cell
culture proliferation. Some readers might be surprised by the use of this topic area to
lead off the volume, because many plant scientists may think of the image analysis tools
as merely incidental components for the operation of the bioreactors. The editors
properly focus this introductory section on the software that makes the real differences
in hardware performance and which permits automation and efficiency.
As expected the larger section of the volume, Part 2 covers Bioreactor Technologythe
hardware that supports the technology. This section includes eight chapters
addressing various applications of bioreactors for micropropagation, bioproduction of
proteins, and hairy root culture for production of medicinal compounds. Various
engineering designs are discussed, along with their benefits for different applications,
including airlift, thin-film, nutrient mist, temporary immersion, and wave bioreactors.
These chapters include discussion of key bioprocess control points and how they are
handled in various bioreactor designs, including issues of aeration, oxygen transport,
nutrient transfer, shear stress, mass/energy balances, medium flow, light, etc.
Part 3 covers more specific issues related to Mechanized Micropropagation. The two
chapters in this section address the economic considerations of automated
micropropagation systems as related to different types of tissue proliferation, and the
use of robotics to facilitate separation of propagules and reduce labour costs. Part 4,
Engineering Cultural Environment, has six chapters elaborating on engineering issues
related to closed systems, aeration, culture medium gel hardness, dissolved oxygen,
vi
photoautotrophic micropropagation and temperature distribution inside the culture
vessel.
The last part (Part 5) includes four chapters that discuss specific applications in
Electrophysiology, Ultrasonics, and Cryogenics. Benefits have been found in the use of
both electrostimulation and ultrasonics for manipulation of plant regeneration.
Electrostimulation may be a useful tool for directing signal transduction within and
between cells in culture. Ultrasound has also applications in monitoring tissue quality,
such as state of hyperhydricity. Finally the application of engineering principles has
improved techniques and hardware used for long-term cryopreservation of plant stock
materials.
Readers of this volume will find a unique collection of chapters that will focus our
attention on the interface of plant biotechnologies and engineering technologies. I look
forward to the stimulation this volume will bring to our colleagues and to this emerging
field of research and development!
Gregory C. Phillips, Ph. D.
Dean, College of Agriculture
Arkansas State University

PREFACE
Plant tissue culture has now emerged as one of the major components of plant
biotechnology. This field of experimental botany begins its journey with the concept of
‘cellular totipotency’ for demonstration of plant morphogenesis. Decades of research in
plant tissue culture has passed through many challenges, created new dreams and
resulted in landmark achievements. Considerable progress has been made with regard to
the improvement of media formulations and techniques of cell, tissue, organ, and
protoplast culture. Such advancement in cultural methodology led many recalcitrant
plants amenable to in vitro regeneration and to the development of haploids, somatic
hybrids and pathogen free plants. Tissue culture methods have also been employed to
study the basic aspects of plant growth, metabolism, differentiation and morphogenesis
and provide ideal opportunity to manipulate these processes.
Recent development of in vitro techniques has demonstrated its application in rapid
clonal propagation, regeneration and multiplication of genetically manipulated superior
clones, production of secondary metabolites and ex-situ conservation of valuable
germplasms. This has been possible not only due to the refinements of cultural practices
and applications of cutting-edge areas of molecular biology but also due to the judicious
inclusion of engineering principles and methods to the system. In the present scenario,
inclusion of engineering principles and methods has transformed the fundamental in
vitro techniques into commercially viable technologies. Apart from the
commercialization of plant tissue culture, engineering aspects have also made it
possible to improve the regeneration of plants and techniques of cryopreservation.
Strategies evolved utilize the disciplines of chemical, mechanical, electrical, cryogenics,
and computer science and engineering.
In the years to come, the application of plant tissue culture for various
biotechnological purposes will increasingly depend on the adoption of engineering
principles and better understanding of their interacting factors with biological system.
The present volume provides a cohesive presentation of the engineering principles and
methods which have formed the keystones in practical applications of plant tissue
culture, describes how application of engineering methods have led to major advances
in commercial tissue culture as well as in understanding fundamentals of
morphogenesis and cryopreservation, and focuses directions of future research, as we
envisage them. We hope the volume will bridge the gap between conventional plant
tissue culturists and engineers of various disciplines.
A diverse team of researchers, technologists and engineers describe in lucid manner
how various engineering disciplines contribute to the improvement of plant tissue
culture techniques and transform it to a technology. The volume includes twenty four
chapters presenting the current status, state of the art, strength and weaknesses of the
strategy applicable to the in vitro system covering the aspects of machine vision,
bioreactor technology, mechanized micropropagation, engineering cultural environment
and physical aspects of plant tissue engineering. The contributory chapters are written
by international experts who are pioneers, and have made significant contributions to
viii
this emerging interdisciplinary enterprise. We are indebted to the chapter contributors
for their kind support and co-operation. Our deepest appreciation goes to Professor G.C.
Phillips for sparing his valuable time for writing the Foreword. We are grateful to
Professor Marcel Hofman, the series editor, ‘Focus on Biotechnology’ for his critical
review and suggestions during the preparation of this volume.
Our thanks are also due to Dr. Rina Dutta Gupta for her efforts in checking the
drafts and suggesting invaluable clarifications. We are also thankful to Mr. V.S.S.
Prasad for his help during the preparation of camera ready version. Finally, many thanks
to Springer for their keen interest in bringing out this volume in time with quality work.
S. Dutta Gupta
Y. Ibaraki
Kharagpur/Yamaguchi, January 2005
Preface


TABLE OF CONTENTS
FOREWORD v
PREFACE . vii
TABLE OF CONTENTS 1
PART 1 13
MACHINE VISION 13
Evaluation of photosynthetic capacity in micropropagated plants by image
analysis . 15
Yasuomi Ibaraki 15
1. Introduction . 15
2. Basics of chlorophyll fluorescence . 16
3. Imaging of chlorophyll fluorescence for micropropagated plants 18
3.1. Chlorophyll fluorescence in in vitro cultured plants 18
3.2. Imaging of chlorophyll fluorescence . 21
3.3. Imaging of chlorophyll fluorescence in micropropagated plants 22
4. Techniques for image-analysis-based evaluation of photosynthetic capacity 25
5. Estimation of light distribution inside culture vessels 26
5.1. Understanding light distribution in culture vessels 26
5.2. Estimation of light distribution within culture vessels 26
6. Concluding remarks 27
References . 28
Monitoring gene expression in plant tissues . 31
John J. Finer, Summer L. Beck, Marco T. Buenrostro-Nava, Yu-Tseh Chi and
Peter P. Ling 31
1. Introduction . 31
2. DNA delivery 32
2.1. Particle bombardment 32
2.2. Agrobacterium 33
3. Transient and stable transgene expression 33
4. Green fluorescent protein 34
4.1. GFP as a reporter gene . 34
4.2. GFP image analysis 35
4.3. Quantification of the green fluorescence protein in vivo . 36
5. Development of a robotic GFP image acquisition system 37
5.1. Overview 37
5.2. Robotics platform . 37
5.3. Hood modifications 39
5.4. Microscope and camera 40
5.5. Light source and microscope optics . 40
6. Automated image analysis 41
6.1. Image registration . 41
6.2. Quantification of GFP 43
2
7. Conclusions . 43
Acknowledgements . 44
References . 44
Applications and potentials of artificial neural networks in plant tissue
culture 47
V.S.S. Prasad and S. Dutta Gupta . 47
1. Introduction . 47
2. Artificial neural networks 48
2.1. Structure of ANN . 48
2.2. Working principle and properties of ANN . 49
2.2.1. Computational property of a node . 49
2.2.2. Training mechanisms of ANN 51
2.3. Types of artificial neural networks 51
2.3.1. Classification and clustering models . 51
2.3.2. Association models . 52
2.3.3. Optimization models . 52
2.3.4. Radial basis function networks (RBFN) . 52
2.4. Basic strategy for network modelling 52
2.4.1. Database 52
2.4.2. Selection of network structure 53
2.4.2.1. Number of input nodes . 54
2.4.2.2. Number of hidden units 54
2.4.2.3. Learning algorithm . 54
2.4.3. Training and validation of the network . 55
3. Applications of ANN in plant tissue culture systems . 56
3.1. In vitro growth simulation of alfalfa 56
3.2. Classification of plant somatic embryos
3.3. Estimation of biomass of plant cell cultures 58
3.4. Simulation of temperature distribution inside a plant culture vessel . 59
3.5. Estimation of length of in vitro shoots . 61
3.6. Clustering of in vitro regenerated plantlets into groups . 61
65
Acknowledgement . 66
References . 66
Evaluation of plant suspension cultures by texture analysis . 69
69
1. Introduction . 69
2. Microscopic and macroscopic image uses in plant cell suspension culture . 69
3. Texture analysis for macroscopic images of cell suspensions 71
3.1. Texture features 71
3.2. Texture analysis for biological objects 72
3.3. Texture analysis for cell suspension culture 73
3.4. Considerations for application of texture analysis . 73
4. Evaluation of embryogenic potential of cultures by texture analysis . 73
4.1. Evaluation of embryogenic potential of cultures . 73
4.2. Texture analysis based evaluation of embryogenic potential 74
Table of Contents
58
Yasuomi Ibaraki
4. Conclusions and future prospects
3
5. Concluding remarks 77
References . 77
PART 2 81
BIOREACTOR TECHNOLOGY . 81
Bioengineering aspects of bioreactor application in plant propagation 83
Shinsaku Takayama and Motomu Akita . 83
1. Introduction . 83
2. Advantages of the use of bioreactor in plant propagation 84
3. Agar culture vs. liquid culture . 85
4. Transition from shake culture to bioreactor culture 85
5. Types of bioreactors for plant propagation . 86
6. Preparation of propagules for inoculation to bioreactor . 87
7. Characteristics of bioreactor for plant propagation . 88
7.1. Fundamental configuration of bioreactor . 88
7.2. Aeration and medium flow characteristics . 90
7.2.1. Medium flow characteristics . 90
7.2.2. Medium mixing . 91
7.2.3. Oxygen demand and oxygen supply . 92
7.3. Light illumination and transmittance . 93
8. Examples of bioreactor application in plant propagation . 95
9. Aseptic condition and control of microbial contamination . 95
10. Scale-up to large bioreactor . 96
10.1. Propagation of Stevia shoots in 500 L bioreactor 96
10.2. Safe inoculation of plant organs into bioreactor 98
11. Prospects 98
References . 98
Agitated, thin-films of liquid media for efficient micropropagation 101
Jeffrey Adelberg 101
1. Introduction . 101
2. Heterotrophic growth and nutrient use 102
2.1. Solutes in semi-solid agar 102
2.2. Solutes in stationary liquids . 103
2.3. Sugar in shaker flasks and bioreactors . 105
3. Efficiency in process . 108
3.1. Shoot morphology for cutting and transfer process . 108
3.2. Space utilization on culture shelf . 109
3.3. Plant quality 109
4. Vessel and facility design 110
4.1. Pre-existing or custom designed vessel . 110
4.2. Size and shape 111
4.3. Closures and ports 112
4.4. Biotic contaminants 113
4.5. Light and heat . 113
5. Concluding remarks 115
Disclaimer . 115
References . 115
Table of Contents
4
Design, development, and applications of mist bioreactors for
micropropagation and hairy root culture . 119
Melissa J. Towler, Yoojeong Kim, Barbara E. Wyslouzil,
Melanie J. Correll, and Pamela J. Weathers . 119
1. Introduction . 119
2. Mist reactor configurations . 120
3. Mist reactors for micropropagation . 122
4. Mist reactors for hairy root culture . 125
5. Mist deposition modelling . 128
6. Conclusions . 130
Acknowledgements . 131
References . 131
Bioreactor engineering for recombinant protein production using
plant cell suspension culture . 135
Wei Wen Su . 135
1. Introduction . 135
2. Culture characteristics . 136
2.1. Cell morphology, degree of aggregation, and culture rheology 137
2.2. Foaming and wall growth . 140
2.3. Shear sensitivity . 141
2.4. Growth rate, oxygen demand, and metabolic heat loads . 145
3. Characteristics of recombinant protein expression . 146
4. Bioreactor design and operation 148
4.1. Bioreactor operating strategies . 148
4.2. Bioreactor configurations and impeller design 151
4.3. Advances in process monitoring 153
5. Future directions 154
Acknowledgements . 155
References . 155
Types and designs of bioreactors for hairy root culture . 161
Yong-Eui Choi, Yoon-Soo Kim and Kee-Yoeup Paek . 161
1. Introduction . 161
2. Advantage of hairy root cultures . 162
3. Induction of hairy roots . 162
4. Large-scale culture of hairy roots . 163
4.1. Stirred tank reactor . 164
4.2. Airlift bioreactors . 164
4.3. Bubble column reactor . 165
4.4. Liquid-dispersed bioreactor . 165
5. Commercial production of Panax ginseng roots via balloon
type bioreactor . 166
Acknowledgements . 169
References . 169
Oxygen transport in plant tissue culture systems 173
Wayne R. Curtis and Amalie L. Tuerk 173
Introduction . 173
Table of Contents
1.
5
2. Intraphase transport . 175
2.1. Oxygen transport in the gas phase . 175
2.2. Oxygen transport in the liquid phase . 176
2.3. Oxygen transport in solid (tissue) phase 177
3. Interphase transport . 179
3.1. Oxygen transport across the gas-liquid interface . 179
3.2. Oxygen transport across the gas-solid interface 179
3.3. Oxygen transport across the solid-liquid interface 180
4.2. Experimental observation of oxygen limitation . 182
4.3. Characterization of oxygen mass transfer 182
5. Conclusions . 185
Acknowledgements . 185
References . 185
Temporary immersion bioreactor . 187
F. Afreen 187
1. Introduction . 187
2. Requirement of aeration in bioreactor: mass oxygen transfer 188
3. Temporary immersion bioreactor 189
3.1. Definition and historical overview . 189
3.2. Design of a temporary immersion bioreactor 189
3.3. Advantages of temporary immersion bioreactor 190
3.4. Scaling up of the system: temporary root zone immersion bioreactor 191
3.5. Design of the temporary root zone immersion bioreactor . 191
3.6. Case study – photoautotrophic micropropagation of coffee 193
3.7. Advantages of the system . 198
4. Conclusions . 199
References . 200
Design and use of the wave bioreactor for plant cell culture 203
Regine Eibl and Dieter Eibl 203
1. Introduction . 203
2. Background . 204
2.1. Disposable bioreactor types for in vitro plant cultures 204
2.2. The wave: types and specification . 206
3. Design and engineering aspects of the wave . 209
3.1. Bag design 209
3.2. Hydrodynamic characterisation . 210
3.3. Oxygen transport efficiency . 217
4. Cultivation of plant cell and tissue cultures in the wave . 217
4.1. General information . 217
4.2. Cultivation of suspension cultures . 220
4.3. Cultivation of hairy roots . 222
4.4. Cultivation of embryogenic cultures 223
liquid culture 181
4. Example: oxygen transport during seed germination in aseptic liquid
4.1. The experimental system used for aseptic germination of seeds in
culture . 181
Table of Contents
6
5. Conclusions . 224
Acknowledgements . 224
References . 224
PART 3 229
MECHANIZED MICROPROPAGATION 229
Integrating automation technologies with commercial micropropagation . 231
Carolyn J. Sluis 231
1. Introduction . 231
2. Biological parameters 232
2.1. The plant’s growth form affects mechanized handling 232
2.2. Microbial contaminants hinder scale-up 235
3. Physical parameters . 236
3.1. Culture vessels 237
3.2. Physical orientation of explants for subculture or singulation . 237
3.3. Gas phase of the culture vessel impacts automation 238
4. Economic parameters 238
4.1. Baseline cost models 238
4.2. Economics of operator-assist strategies . 241
4.3. Organization of the approach to rooting: in vitro or ex vitro . 241
4.4. Economics of new technologies . 242
5. Business parameters 242
5.1. Volumes per cultivar 243
5.2. Seasons . 244
5.3. Cost reduction targets . 244
6. Political parameters . 246
7. Conclusions . 247
Acknowledgements .
References . 248
Machine vision and robotics for the separation and regeneration of plant
tissue cultures . 253
Paul H. Heinemann and Paul N. Walker . 253
1. Introduction . 253
253
3. Robotic system component considerations . 254
3.1. Plant growth systems for robotic separation 255
3.1.1. Nodes . 255
3.1.2. Clumps 255
3.2. An experimental shoot identification system for shoot clumps . 256
3.2.1. Shoot identification using the Arc method . 257
3.2.2. Shoot identification using the Hough transform method 259
3.2.3. Testing the Hough transform 263
3.3. Robotic mechanisms for shoot separation . 264
3.3.1. Manual separation device 264
3.3.2. Automated separation device 265
3.3.3. Single image versus real-time imaging for shoot separation 268
3.3.4. Shoot re-growth . 269
Table of Contents
248
2. Examples of automation and robotics .
7
3.3.5. Cycle time . 270
3.3.6. Commercial layout 270
References . 271
PART 4 273
ENGINEERING CULTURAL ENVIRONMENT . 273
Closed systems for high quality transplants using minimum resources 275
T. Kozai . 275
1. Introduction . 275
2. Why transplant production systems? 276
3. Why closed systems? 278
4. Commercialization of closed transplant production systems 280
5. General features of high quality transplants . 280
6. Sun light vs. use of lamps as light source in transplant production 282
7. Closed plant production system 284
7.1. Definition . 284
7.2. Main components . 284
7.3. Characteristics of main components of the closed system . 285
7.4. Equipments and facilities: a comparison . 285
7.5. Features of the closed system vs. greenhouse 286
7.6. Equality in Initial investment . 290
7.7. Reduction in costs for transportation and labour . 291
7.8. Uniformity and precise control of microenvironment . 292
7.9. Growth, development and uniformity of transplants . 293
8. Value-added transplant production in the closed system 293
8.1. Tomato (Lycopersicon esculentum Mill.) 294
8.2. Spinach (Spinacia oleracea) 295
8.3. Sweet potato (Ipomoea batatas L. (Lam.)) 295
8.4. Pansy (Viola x wittrockiana Gams.) . 297
8.5. Grafted transplants . 297
8.6. Vegetable transplants for field cultivation . 298
9. Increased productivity to that of the greenhouse 299
10. Costs for heating, cooling, ventilation and CO2 enrichment . 300
10.1. Heating cost 300
10.2. Cooling load and electricity consumption . 301
10.3. Cooling cost 301
10.4. Electricity consumption . 303
10.5. Electricity cost is 1-5% of sales price of transplants . 303
10.6. Relative humidity . 304
10.7. Par utilization efficiency 304
10.8. Low ventilation cost . 305
10.9. CO2 cost is negligibly small . 305
10.10. Water requirement for irrigation 306
10.11. Disinfection of the closed system is easy . 307
10.12. Simpler environmental control unit . 307
10.13. Easier production management 308
10.14. The closed system is environment friendly 308
Table of Contents
8
10.15. The closed system is safer 309
11. Conclusion . 310
Acknowledgement . 311
References . 311
Aeration in plant tissue culture 313
S.M.A. Zobayed 313
1. Introduction . 313
2. Principles of aeration in tissue culture vessel . 314
2.1. Aeration by bulk flow 317
2.2. Aeration by diffusion . 319
2.3. Humidity-induced convection in a tissue culture vessel
2.4. Aeration by venturi-induced convection 325
2.5. Forced aeration by mass flow 326
3. Conclusions . 326
References . 327
Tissue culture gel firmness: measurement and effects on growth 329
Stewart I. Cameron 329
1. Introduction . 329
2. Measurement of gel hardness 330
3. Gel hardness and pH . 333
4. The dynamics of syneresis 334
5. Conclusion . 335
References . 336
Effects of dissolved oxygen concentration on somatic embryogenesis . 339
Kenji Kurata and Teruaki Shimazu . 339
1. Introduction . 339
2. Relationship between DO concentration and somatic embryogenesis . 341
2.1. Culture system and DO concentration variations 341
2.2. Time course of the number of somatic embryos 342
2.3. Relationship between somatic embryogenesis and oxygen
3. Dynamic control of DO concentration to regulate torpedo-stage embryos 347
3.1. The method of dynamic DO control 347
3.2. Results of dynamic DO control 351
4. Conclusions . 352
References . 352
A commercialized photoautotrophic micropropagation system . 355
T. Kozai and Y. Xiao . 355
1. Introduction . 355
2. Photoautotrophic micropropagation 356
2.1. Summary of our previous work 356
3. The PAM (photoautotrophic micropropagation) system and its
357
3.1. System configuration 357
3.2. Multi-shelf unit . 358
3.3. Culture vessel unit 360
concentration . 346
components
3.4. Forced ventilation unit for supplying CO2-enriched air . 360
3.5. Lighting unit . 362
3.6. Sterilization 362
4. Plantlet growth, production costs and sales price . 362
4.1 Calla lily plantlet growth . 362
4.2. China fir plantlet growth 365
4.3. Percent survival during acclimatization ex vitro 366
4.4. Production cost of calla lily plantlets: A case study 367
4.4.1. Production cost per acclimatized plantlet . 368
4.4.2. Cost, labour and electricity consumption for multiplication
4.4.3. Sales price of in vitro and ex vitro acclimatized plantlets 370
5. Conclusions . 370
Acknowledgement . 370
References . 370
Intelligent inverse analysis for temperature distribution in a plant culture
vessel . 373
H. Murase, T. Okayama, and Suroso 373
1. Introduction . 373
2. Theoretical backgrounds . 375
3. Methodology . 378
3.1. Finite element neural network inverse technique algorithm 378
3.2. Finite element formulation . 379
3.3. Finite element model 380
3.4. Neural network structure 381
3.5. Neural network training . 381
3.6. Optimization of temperature distribution inside the culture vessel . 382
3.6.1. Genetic algorithm flowchart . 382
3.6.2. Objective function . 383
3.6.3. Genetic reproduction . 383
3.7. Temperature distribution measurement 386
3.7.1. Equipment development for temperature distribution
386
3.7.2. Temperature distribution data . 388
4. Example of solution 388
4.1. Coefficient of convective heat transfer 388
4.2. Verification of the calculated coefficient of convective heat transfer . 390
4.3. Optimum values of air velocity and bottom temperature 391
References . 394
PART 5 395
PHYSICAL ASPECTS OF PLANT TISSUE ENGINEERING . 395
Electrical control of plant morphogenesis 397
Cog lniceanu Gina Carmen 397
1. Introduction . 397
2. Endogenous electric currents as control mechanisms in plant development 397
3. Electrostimulation of in vitro plant development . 400
or rooting 368
measurement
5. Potential applications of the electric manipulation in plant biotechnology 410
References . 411
417
Victor Gaba, K. Kathiravan, S. Amutha, Sima Singer, Xia Xiaodi and G.
Ananthakrishnan 417
417
2. The generation of ultrasound 418
3. Mechanisms of action of ultrasound . 419
4. Sonication-assisted DNA transformation 420
5. Sonication-assisted Agrobacterium-mediated transformation 420
6. Stimulation of regeneration by sonication 421
422
8. Fractionation of somatic embryos . 423
9. Secondary product synthesis . 423
10. Ultrasound and control of micro-organisms . 423
11. Conclusions . 424
Acknowledgements . 424
References . 424
Mikio Fukuhara, S. Dutta Gupta and Limi Okushima 427
1. Introduction . 427
2. Theoretical considerations and system description . 428
3. Case studies on possible ultrasonic diagnosis of plant leaves 430
3.1. Ultrasonic testing of tea leaves for plant maturity . 430
3.1.1. Wave velocity and dynamic modulus for leaf tissue development 431
3.1.2. Dynamic viscosity and imaginary parts in complex waves 432
3.2. Ultrasonic diagnosis of rice leaves . 434
3.3. Acoustic characteristics of in vitro regenerated leaves of gladiolus 435
4. Conclusions . 438
Acknowledgement . 438
References . 438
Physical and engineering perspectives of in vitro plant cryopreservation . 441
Erica E. Benson, Jason Johnston, Jayanthi Muthusamy and Keith Harding 441
1. Introduction . 441
2. The properties of liquid nitrogen and cryosafety 442
3. Physics of ice . 443
3.1. Water’s liquid and ice morphologies . 444
3.1.1. Making snowflakes: a multiplicity of ice families 445
4. Cryoprotection, cryodestruction and cryopreservation . 447
4.1. Physical perspectives of ultra rapid and droplet freezing 448
waves
Acoustic characteristics of plant leaves using ultrasonic transmission
plantlets
4.2. Effects of electric pulses treatment on tissue fragments or entire
The uses of ultrasound in plant tissue culture
4.1. Effects of electric pulses treatment on plant protoplasts .
7. Summary of transformation and morphogenic responses to ultrasound .
systems .
1. Introduction .
4. High-voltage, short-duration electric pulses interaction with in vitro
4.2. Controlled rate or slow cooling 450
4.3. Vitrification 451
5. Cryoengineering: technology and equipment . 451
5.1. Cryoengineering for cryogenic storage 451
5.1.1. Controlled rate freezers . 452
5.1.2. Cryogenic storage and shipment . 455
5.1.3. Sample safety, security and identification 456
6. Cryomicroscopy 456
6.1. Nuclear imaging in cryogenic systems 458
7. Thermal analysis . 459
7.1. Principles and applications . 460
7.1.1. DSC and the optimisation of cryopreservation protocols . 462
7.1.2. A DSC study comparing cryopreserved tropical and temperate
7.1.2.1. Using thermal analysis to optimise cryoprotective strategies 468
8. Cryoengineering futures 470
Acknowledgements . 473
References . 474
INDEX . 477