Plant Cryopreservation: A Practical Guide

533 Pages·2008·6.708 MB·English

by  Barbara M. Reed (auth.), Barbara M. Reed (eds.)

Plant Cryopreservation: A Practical Guide
Plant Cryopreservation:
A Practical Guide
Editor
Barbara M. Reed
USDA-ARS National Clonal Germplasm Repository
Corvallis, OR, USA
Editor
Barbara M. Reed
USDA-ARS National Clonal
Germplasm Repository
33447 Peoria Road
Corvallis, OR 97333-2512
U SA
ISBN 978-0-387-72275-7 e-ISBN 978-0-387-72276-4
Library of Congress Control Number: 2007935104

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This book is dedicated to my late parents, Mary and Howard Baltensper-
ger. Their longstanding belief that goals should be viewed as “when”
rather than “if” made many of my dreams possible. It is also dedicated to
my husband for his constant support and encouragement.
Preface
The alarming loss of plant biodiversity both in nature and within agricul-
tural systems has led the plant biology community to look for alternatives
to in situ conservation. Although cryopreservation by itself is not a pana-
cea for the global loss of biodiversity, it is a useful tool for long-term
maintenance of select plant germplasm. The development of plant cryopre-
servation techniques for cell cultures in 1968 has led us now, 40 years
later, to the stage where cryopreservation of organized tissues is a reality.
I came to the field of cryobiology through the need to conserve crop
germplasm for future generations. At the time I began the field was still
young and the techniques were being applied mostly to unorganized tissues
and cells. The first applications of cryopreservation to organized tissues by
Dr. Kutty Kartha and Dr. Akira Sakai showed the promise of the technique
for the storage of plant diversity. With this encouragement I started toward
my goal of storing the unique and invaluable plant germplasm at the
USDA Agricultural Research Service’s National Clonal Germplasm Reposi-
tory, Corvallis, Oregon. I started my studies with the initial guidance of Dr.
Bernard Finkle and picked up tips along the way from Dr. Lyndsey Withers,
Dr. Akira Sakai and Dr. Jean Dereuddre.
While long-term storage of clonally propagated plants or those with re-
calcitrant seeds was once a dream, that dream became a reality by the mid
1990s. Cryopreserved collections, now located in several countries around
the world, are a testament to the utility of cryopreservation. Now the chal-
lenge is to expand the utility of these techniques by making them available
to laboratories that do not specialize in cryopreservation, but rather wish to
use it as a safe backup for valuable plant materials.
The availability of well-tested and widely-used protocols makes the
development of a book of this type possible. The first cryogenic tech-
nique, “controlled rate cooling” (also called slow cooling and two-step
cooling), was the only available protocol for many years. This tech-
nique is very successful for a wide range of plant materials and is
widely used for callus and suspension cell cultures. It is also easily
applicable to the shoot tips of temperate plants. With the aid of a pro-
grammable freezer, relatively large amounts of plant material can be
stored at one time with little technical input. At the end of the 1980s the
vii
viii Preface
development of vitrification techniques provided a second approach that
is applicable even to tropical plants. Several techniques were developed,
but the development of Plant Vitrification Solution number 2 (PVS2) by
Dr. Akira Sakai, led to the wide use of vitrification for plant tissues. Soon
thereafter encapsulation dehydration was developed in the laboratory of
Dr. Jean Dereuddre. This technique is also widely used and highly success-
ful for a wide range of plants. Modifications of all these techniques are
available as well. With the wide choice of techniques available it should be
possible to store most types of plants.
The choice of a technique for storing a particular plant should be based
on several factors. Laboratories that wish to store an occasional plant or a
tropical plant will choose vitrification or encapsulation-dehydration tech-
niques. These protocols require little more than a standard tissue culture
laboratory. The techniques can be adapted to the plant material with a few
simple experiments. Facilities with large amounts of temperate plant materi-
als may wish to use controlled rate cooling to more efficiently store larger
quantities of plants at one time.
This book was developed to aid in the use of cryopreservation tech-
niques throughout the world, for the conservation of all forms of plant biodi-
versity. It is hoped that this volume will provide the step-by-step instructions
needed to transfer cryopreservation technology to general plant biology
laboratories that might make use of these protocols to store important plant
materials. Often, published techniques are difficult to interpret and apply
in a laboratory that is not familiar with cryopreservation. The protocols
presented in this volume were tested on a range of genotypes and should
be suitable for storing additional materials. By using the complete and
tested protocols presented here, laboratories will have a starting point and
may only need to make slight modifications before storing their valuable
plant materials.
This volume was written for those active in cryobiology, and also for
those who are not cryobiologists, but in need of a long-term storage
method. The volume is divided into two parts. The first section introduces
the reader to cryopreservation and the main techniques used. The second
combines literature reviews of plant groups with defined step-by-step
protocols. It is hoped that these techniques will be directly useable by the
scientific community.

Corvallis, Oregon Barbara M. Reed
April 2007
Acknowledgements
The Editor gratefully acknowledges the support and cooperation of the
chapter authors and the many protocol contributors. I am also appreciative
of the staff of the National Clonal Germplasm Repository for their assis-
tance in keeping the laboratory running while I was occupied with this pro-
ject. I especially acknowledge the invaluable assistance of my husband
Ralph, for his final reading of the chapters and protocols. Without his
eagle eye and scientific insight, many errors and inconsistencies might
have slipped by unnoticed.
ix
Contents
Preface...................................................................................................................vii
Section I: Introduction to Cryopreservation.......................................................1
1. Cryopreservation—Practical Considerations.................................................3
Barbara M. Reed
1.1 Introduction....................................................................................................3
1.2 Planning for a Liquid Nitrogen Stored Collection.........................................4
1.2.1 Plant Materials......................................................................................4
1.2.2 Storage Records....................................................................................5
1.2.3 Storage Form.........................................................................................7
1.2.4 How Many to Store...............................................................................7
1.2.5 Protocol Testing....................................................................................8
1.2.6 Storage Controls...................................................................................9
1.2.7 Recovery...............................................................................................9
1.3 Standard Protocols.......................................................................................10
1.4 Conclusions..................................................................................................11
References..........................................................................................................11
2. Cryopreservation Theory................................................................................15
Erica E. Benson
2.1 Introduction..................................................................................................15
2.2 The Biological Chemistry of Water.............................................................15
2.3 Thermal Properties of Water and Ice...........................................................17
2.3.1 Ice Nucleation.....................................................................................17
2.3.2 Controlled Rate Cooling.....................................................................20
2.4 Vitrification: The Glassy State....................................................................21
2.5 Water State Transitions and Instabilities.....................................................22
2.6 Cryoprotection.............................................................................................23
2.6.1 Biophysical Aspects of Natural Freezing Tolerance..........................23
2.6.2 Aspects of Natural Freeze Tolerance.................................................24
2.6.3 Artificial Cryoprotection....................................................................25
2.7 New and Retrospective Insights in Cryoprotection.....................................27
References..........................................................................................................30
xi
xii Contents
3. Development of PVS-Based Vitrification and Encapsulation-Vitrification
Protocols............................................................................................................33
Akira Sakai, Dai Hirai and Takao Niino
3.1 Introduction..................................................................................................33
3.2 The Concept of Vitrification........................................................................34
3.3 Vitrification Procedure................................................................................35
3.4 Preconditioning............................................................................................38
3.5 Preculture.....................................................................................................41
3.5.1 Herbaceous Plants...............................................................................41
3.5.2 Woody Plants......................................................................................41
3.5.3 Tropical Plants....................................................................................43
3.6 Osmoprotection (Loading Treatment).........................................................43
3.7 Exposure Time to PVS2..............................................................................44
3.8 Regrowth......................................................................................................46
3.9 Encapsulation Vitrification..........................................................................47
3.10 A Personal View of the Development of PVS2 by Akira Sakai...............48
3.10.1 The Ultimate Goal—Storing Tropical Plants.................................48
3.10.2 The Core of the Technology Development.....................................48
3.10.3 Success with Vitrification of Tropical Plants.................................50
3.10.4 Fifteen Years of Research...............................................................50
References..........................................................................................................51
4. Development of Encapsulation Dehydration.................................................59
Florent Engelmann, Maria-Teresa Gonzalez Arnao, Yongjie Wu
and Roosevelt Escobar
4.1 Introduction..................................................................................................59
4.2 Successive Steps of the Protocol..................................................................62
4.2.1 Physiological Status of the Plant Material........................................62
4.2.2 Preconditioning.................................................................................63
4.2.3 Preculture..........................................................................................63
4.2.4 Encapsulation....................................................................................64
4.2.5 Osmoprotection................................................................................64
4.2.6 Dehydration......................................................................................65
4.2.7 Cryopreservation..............................................................................65
4.2.8 Rewarming........................................................................................66
4.2.9 Recovery...........................................................................................66
4.2.10 Viability Assessment......................................................................67
4.3 Current Development and use of Encapsulation Dehydration....................67
4.4 Genetic Stability..........................................................................................68
4.5 Conclusions.................................................................................................68
References..........................................................................................................68
5. Controlled Rate Cooling..................................................................................77
Barbara M. Reed and Esther Uchendu
5.1 Basic concepts of controlled rate cooling....................................................77
5.2 Preconditioning............................................................................................78
Contents x i i i
5.2.1 Cold Acclimation................................................................................78
5.2.2 In Vitro Culture...................................................................................80
5.2.3 Abscisic Acid......................................................................................80
5.3 Preculture.....................................................................................................82
5.4 Cryoprotection.............................................................................................83
5.4.1 Single Chemicals................................................................................83
5.4.2 Chemical Combinations......................................................................84
5.5 Cooling.........................................................................................................84
5.6 Warming.......................................................................................................85
5.7 Recovery and Viability Assessment............................................................86
5.8 Summary of the Basic Protocol...................................................................87
References..........................................................................................................88
Section II. Cryopreservation Protocols for Working Laboratories................93
6. Cryopreservation of Algae...............................................................................95
John G. Day and Keith Harding
6.1 Algae............................................................................................................95
6.2 Physiological Status.....................................................................................96
6.3 Preconditioning............................................................................................96
6.4 Preculture.....................................................................................................97
6.5 Cryoprotection.............................................................................................98
6.6 Cryopreservation..........................................................................................99
6.6.1 Rewarming........................................................................................100
6.6.2 Recovery...........................................................................................100
6.6.3 Viability Assessment........................................................................101
References........................................................................................................102
6.7 Protocols.....................................................................................................104
6.7.1 Passive Cooling Two-Step Methods.................................................104
6.7.2 Controlled Rate Cooling Protocols...................................................108
6.7.3 Encapsulation Dehydration Method.................................................112
6.7.4 Viability Assessments used for Microalgae and Cyanobacteria......114
7. Cryopreservation of Bryophytes and Ferns................................................117
Valerie C. Pence
7.1 Introduction................................................................................................117
7.2 Bryophyte Gametophytes...........................................................................118
7.3 Fern Gametophytes....................................................................................120
7.4 Fern Spores................................................................................................121
7.5 Fern Sporophytes and Fern Allies..............................................................122
7.6 The Future..................................................................................................122
References........................................................................................................124
7.7 Protocols.....................................................................................................126
7.7.1 Bryophyte Gametophyte Cryopreservation—Mr. Frosty.................126
7.7.2 Cryopreservation of Bryophyte Gametophytes—
Three Drying Protocols....................................................................128