See

discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/248134206

Plant cryopreservation: Progress and

prospects. In Vitro Cell Dev Biol Plant 40: 427433
ARTICLE in IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY - PLANT SEPTEMBER 2004
Impact Factor: 1.16 DOI: 10.1079/IVP2004541

CITATIONS

143

1 AUTHOR:
F. Engelmann
Institute of Research for Development
210 PUBLICATIONS 1,903 CITATIONS
SEE PROFILE

Available from: F. Engelmann

Retrieved on: 25 August 2015

In Vitro Cell. Dev. Biol.Plant 40:427433, SeptemberOctober 2004

q 2004 Society for In Vitro Biology
1054-5476/04 $18.00+0.00

DOI: 10.1079/IVP2004541

PLANT CRYOPRESERVATION: PROGRESS AND PROSPECTS

FLORENT ENGELMANN*

Cirad, Station de Roujol, 97170 Petit-Bourg, Guadeloupe, French West Indies

(Received 12 August 2003; accepted 4 February 2004; editor J. Aitken)

Summary
Cryopreservation (liquid nitrogen, 21968C) represents the only safe and cost-effective option for long-term conservation
of germplasm of non-orthodox seed species, vegetatively propagated species, and of biotechnology products. Classical
cryopreservation techniques, which are based on freeze-induced dehydration, are mainly employed for freezing
undifferentiated cultures and apices of cold-tolerant species. New cryopreservation techniques, which are based on
vitrification of internal solutes, are successfully employed with all explant types, including cell suspensions and calluses,
apices, and somatic and zygotic embryos of temperate and tropical species. The development of cryopreservation protocols
is significantly more advanced for vegetatively propagated species than for recalcitrant seed species. Even though its
routine use is still limited, there are a growing number of examples where cryopreservation is employed on a large scale for
different types of materials, including seeds with orthodox and intermediate storage behaviour, dormant buds, pollen,
biotechnology products, and apices sampled from in vitro plantlets of vegetatively propagated species. Cryopreservation
can also be employed for uses other than germplasm conservation, such as cryoselection, i.e., the selection through
freezing of samples with special properties, or cryotherapy, i.e., the elimination of viruses from infected plants through
apex cryopreservation. Because of its high potential, it is expected that cryopreservation will become more frequently
employed for long-term conservation of plant genetic resources.
Key words: cryopreservation techniques; genetic resources; large-scale application; long-term storage; genebank; nonorthodox seed species; vegetatively propagated species.

Introduction

efficacy and threaten the safety of plant genetic resources conserved

in this way. Indeed, collections remain exposed to natural disasters,
attacks by pests and pathogens, and moreover, labor costs and the
requirement for technical personnel are very high. In addition,
distribution and exchange from field genebanks is difficult because
of the vegetative nature of the material and the greater risks of
disease transfer from country to country (Engelmann, 1997b).
The development of biotechnology has led to the production of a
new category of germplasm including clones obtained from elite
genotypes, cell lines with special attributes, and genetically
transformed material (Engelmann, 1992). This new germplasm is
often of high added-value and very difficult to produce. The
development of efficient techniques to ensure its safe conservation
is therefore of paramount importance.
Cryopreservation, i.e., the storage of biological material at ultralow temperature, usually that of liquid nitrogen (2 1968C), is the
only technique currently available to ensure the safe and costefficient long-term conservation of these different types of
germplasm. At this temperature, all cellular divisions and metabolic
processes are stopped. The plant material can thus be stored without
alteration or modification for a theoretically unlimited period of
time. Moreover, cultures are stored in a small volume, protected
from contamination, and require a very limited maintenance.
In this paper, we present a brief overview of the various
cryopreservation techniques available, of the achievements made

Most agricultural crops have orthodox seeds, i.e., seeds that can
be dehydrated down to low moisture contents and can thus be stored
at low temperature for extended periods (Roberts, 1973). However,
conservation in seed form is problematic for three main categories
of plant species. The first category includes crops such as banana
and plantain, which do not produce seeds and are thus vegetatively
propagated. The second category comprises species such as potato
or sugarcane, which have both sterile genotypes and genotypes
which produce orthodox seeds. However, the seeds are generally
highly heterozygous and are thus of limited interest for conserving
particular genotypes. These species are thus mainly maintained by
means of vegetative propagation. The third category consists of fruit
and forest tree species, especially from tropical origin, which
produce recalcitrant seeds, i.e., seeds that cannot be dried to
sufficiently low moisture level to permit their storage at low
temperature (Roberts, 1973). Conservation in seed form is also still
problematic for a large number of species, termed intermediate
(Ellis et al., 1990). Genetic resources of all these species are
traditionally conserved ex situ as whole plants in field collections.
Field conservation presents major drawbacks which limit its
*Author to whom correspondence should be addressed: Email florent.
engelmann@cirad.fr

427

428

ENGELMANN

and problems faced with cryopreservation of vegetatively

propagated and recalcitrant species, and of the current utilization
of cryopreservation for long-term storage of plant germplasm.
Cryopreservation Techniques
Some materials, such as orthodox seeds or dormant buds, display
natural dehydration processes and can be cryopreserved without
any pretreatment. However, most of the experimental systems
employed in cryopreservation (cell suspensions, calluses, shoot tips,
embryos, etc.) contain high amounts of cellular free water and are
thus extremely sensitive to freezing injury since most of them are
not inherently freezing-tolerant. Cells have thus to be dehydrated
artificially to protect them from damage caused by crystallization of
intracellular water into ice (Mazur, 1984). The techniques employed
and the physical mechanisms upon which they are based are
different in classical and new cryopreservation techniques (Withers
and Engelmann, 1998). Classical techniques involve freeze-induced
dehydration, whereas new techniques are based on vitrification.
Vitrification can be defined as the transition of water directly from
the liquid phase into an amorphous phase or glass, while avoiding
the formation of crystalline ice (Fahy et al., 1984).
Classical Cryopreservation Techniques
Classical cryopreservation techniques involve slow cooling down
to a defined prefreezing temperature, followed by rapid immersion
in liquid nitrogen. With temperature reduction during slow cooling,
cells and the external medium initially supercool, followed by ice
formation in the medium (Mazur, 1984). The cell membrane acts as
a physical barrier and prevents the ice from seeding the cell interior
and the cells remain unfrozen but supercooled. As the temperature
is further decreased, an increasing amount of the extracellular
solution is converted into ice, thus resulting in the concentration of
intracellular solutes. Since cells remain supercooled and their
aqueous vapour pressure exceeds that of the frozen external
compartment, cells equilibrate by loss of water to external ice.
Depending upon the rate of cooling and the prefreezing
temperature, different amounts of water will leave the cell before
the intracellular contents solidify. In optimal conditions, most or all
intracellular freezable water is removed, thus reducing or avoiding
detrimental intracellular ice formation upon subsequent immersion
of the specimen in liquid nitrogen. However, too intense freezeinduced dehydration can incur different damaging events due to
concentration of intracellular salts and changes in the cell
membrane (Meryman et al., 1977). Thawing should be as rapid as
possible to avoid the phenomenon of recrystallization in which ice
melts and reforms at a thermodynamically favorable, larger and
more damaging crystal size (Mazur, 1984).
Classical freezing procedures include the following successive
steps: pregrowth of samples, cryoprotection, slow cooling (0.5
2.08C min21) to a determined prefreezing temperature (usually
around 2 408C), rapid immersion of samples in liquid nitrogen,
storage, rapid thawing, and recovery. Classical techniques are
generally operationally complex since they require the use of
sophisticated and expensive programmable freezers. In some cases,
their use can be avoided by performing the slow freezing step with a
domestic or laboratory freezer (Kartha and Engelmann, 1994;
Engelmann, 1997b).

Classical cryopreservation techniques have been successfully

applied to undifferentiated culture systems such as cell suspensions
and calluses (Kartha and Engelmann, 1994; Withers and
Engelmann, 1998). In the case of differentiated structures, these
techniques can be employed for freezing apices of cold-tolerant
species (Reed and Chang, 1997).
New Cryopreservation Techniques
In vitrification-based procedures, cell dehydration is performed
prior to freezing by exposure of samples to concentrated
cryoprotective media and/or air desiccation. This is followed by
rapid cooling. As a result, all factors that affect intracellular ice
formation are avoided. Glass transitions (changes in the structural
conformation of the glass) during cooling and rewarming have been
recorded using thermal analysis (Sakai et al., 1990). Vitrificationbased procedures offer practical advantages in comparison to
classical freezing techniques. Like ultra-rapid freezing (above), they
are more appropriate for complex organs (shoot tips, embryos) which
contain a variety of cell types, each with unique requirements under
conditions of freeze-induced dehydration. By precluding ice
formation in the system, vitrification-based procedures are
operationally less complex than classical ones (e.g., they do not
require the use of controlled freezers) and have greater potential for
broad applicability, requiring only minor modifications for different
cell types (Engelmann, 1997b).
A common feature to all these new protocols is that the critical
step to achieve survival is the dehydration step, and not the freezing
step, as in classical protocols. Therefore, if samples to be frozen are
amenable to desiccation down to sufficiently low water contents
(which vary depending on the procedure employed and the type and
characteristics of the propagule to be frozen) with no or little
decrease in survival in comparison to non-dehydrated controls, no
or limited further drop in survival is generally observed after
cryopreservation (Engelmann, 1997b).
Seven different vitrification-based procedures can be identified:
(1) encapsulation dehydration; (2) vitrification; (3) encapsulation
vitrification; (4) dehydration; (5) pregrowth; (6) pregrowth
dehydration; and (7) droplet freezing.
Encapsulation dehydration. The encapsulation dehydration
procedure is based on the technology developed for the production
of artificial seeds. Explants are encapsulated in alginate beads,
pregrown in liquid medium enriched with sucrose for 1 7 d,
partially desiccated in the air current of a laminar airflow cabinet or
with silica gel to a water content around 20% (fresh weight basis),
then frozen rapidly (Dereuddre et al., 1991). Survival is high and
growth recovery of cryopreserved samples is generally rapid and
direct, without callus formation. A modified protocol has been
proposed by Sakai et al. (2000) in which encapsulation and
pregrowth in medium with sucrose and glycerol are performed
simultaneously. This technique has been applied to apices of
numerous species from temperate and tropical origin as well as to
cell suspensions and somatic embryos of several species
(Engelmann, 1997b; Engelmann and Takagi, 2000).
Vitrification. Vitrification includes the following steps: preculture of samples on medium enriched with cryoprotective
substances, treatment with a loading solution (e.g., a mixture of
2 M glycerol and 0.4 M sucrose; Matsumoto et al., 1994),
dehydration with a highly concentrated vitrification solution such

PLANT CRYOPRESERVATION

as the glycerol-based PVS2 solution (Sakai et al., 1990) which has

a molarity of 7.8 M, rapid freezing and thawing, removal of
cryoprotectants and recovery. In order to reduce the toxicity of the
vitrification solution, a modified vitrification protocol has been
developed recently in which apices are first treated with
half-strength vitrification solution, then with a full-strength one
(Matsumoto and Sakai, 2003). This procedure has been developed
for apices, cell suspensions, embryogenic tissues, and somatic
embryos of numerous species (Cyr, 2000; Sakai, 2000; Sakai et al.,
2002).
Encapsulation vitrification. Encapsulation vitrification is a
combination of encapsulation dehydration and vitrification procedures, where samples are encapsulated in alginate beads, then
subjected to freezing by vitrification. It has been applied to apices
of an increasing number of species (Sakai, 2000; Sakai et al., 2002).
Dehydration. Dehydration is the simplest procedure since it
consists of dehydrating explants, then freezing them rapidly by
direct immersion in liquid nitrogen. This technique is mainly used
with zygotic embryos or embryonic axes extracted from seeds. It has
been applied to embryos of a large number of recalcitrant and
intermediate species (Engelmann, 1997a, 2000). Desiccation is
usually performed in the air current of a laminar airflow cabinet, but
more precise and reproducible dehydration conditions are achieved
by using a flow of sterile compressed air or silica gel. Ultra-rapid
drying in a stream of compressed dry air (a process called flash
drying developed by Prof. Berjaks group in South Africa) allows
freezing of samples with a relatively high water content, thus
reducing desiccation injury (Berjak et al., 1989). Optimal survival
is generally obtained when samples are frozen with a water content
between 10 and 20% (fresh weight basis).
Pregrowth. The pregrowth technique consists of cultivating
samples in the presence of cryoprotectants, then freezing them
rapidly by direct immersion in liquid nitrogen. The pregrowth
technique has been developed for Musa meristematic cultures
(Panis et al., 2002).
Pregrowth dehydration. In a pregrowth dehydration procedure, explants are pregrown in the presence of cryoprotectants,
dehydrated under the laminar airflow cabinet or with silica gel, and
then frozen rapidly. This method has been applied notably to
asparagus stem segments, oil palm polyembryonic cultures, and
coconut zygotic embryos (Uragami et al., 1990; Assy-Bah and
Engelmann, 1992; Dumet et al., 1993).
Droplet freezing. The droplet-freezing technique has presently
been applied to potato (Schafer-Menuhr, 1996), asparagus (MixWagner et al., 2000), and apple apices (Zhao et al., 1999). Apices
are pretreated with liquid cryoprotective medium, then placed on an
aluminum foil in minute droplets of cryoprotectant and frozen
slowly (apple) or rapidly (potato) in liquid nitrogen.
Cryopreservation of Vegetatively Propagated and
Recalcitrant Seed Species
Vegetatively Propagated Species
Several review papers have been published recently, which
provide lists of species which have been successfully cryopreserved
(Engelmann, 1997a, b; Cyr, 2000; Engelmann and Takagi, 2000;
Sakai et al., 2002). For vegetatively propagated species,
cryopreservation has a wide applicability both in terms of species

429

coverage, since protocols have been successfully established for

root and tubers, fruit trees, ornamentals, forestry and plantation
crops, from temperate and tropical origin, and in terms of numbers
of genotypes/varieties within a given species. With a few exceptions
(e.g., potato, pear, mulberry), vitrification-based protocols have
been employed. It is also interesting to note that in many cases
different protocols can be employed for a given species and produce
comparable results. Survival is generally high to very high and up to
100% survival could be achieved in some cases, e.g., Allium, yam,
potato, and conifers. Regeneration is rapid and direct, and callusing
is observed only in cases where the technique is not optimized.
Different reasons can be mentioned to explain these positive
results (Engelmann, 2000). The meristematic zone of apices, from
which organized growth originates, is composed of a relatively
homogeneous population of small, actively dividing cells, with few
small vacuoles and a high nucleo-cytoplasmic ratio. Cells of somatic
embryogenic tissues display the same characteristics. These
characteristics make such cells more likely to withstand desiccation
than highly vacuolated and differentiated cells. No ice formation
takes place in vitrification-based procedures, thus allowing direct,
organized regrowth of frozen explants. Other reasons for the good
results obtained are linked with tissue culture protocols. Many
vegetatively propagated species successfully cryopreserved until
now are cultivated crops, often of great commercial importance, for
which cultural practices, including in vitro propagation, are well
established. In addition, in vitro material is synchronized by the
tissue culture and pregrowth procedures. Relatively homogeneous
samples in terms of size, cellular composition, physiological state,
and growth response are employed for freezing, thus increasing the
chances of positive and uniform response to treatments. Finally,
vitrification-based procedures allow the use of samples of relatively
large size (shoot tips of 0.5 3 mm) which can regrow directly
without difficulty.
Freezing techniques are now operational for large-scale
experimentation and commercialization with an increasing number
of vegetatively propagated plants. In view of the wide range of
efficient and operationally simple techniques available, any
vegetatively propagated species should be amenable to cryopreservation, provided that the tissue culture protocol is sufficiently
operational for this species.

Recalcitrant Seed Species

Several review papers have been published in recent years which
present extensive lists of plant species whose embryos and/or
embryonic axes have been successfully cryopreserved (e.g., Kartha
and Engelmann, 1994; Engelmann et al., 1995; Pence, 1995;
Engelmann, 1997a, b). This might lead to the conclusion that
freezing of embryos is a routine procedure applicable to numerous
species, whatever their storage characteristics. However, careful
examination of the species mentioned in these papers reveals that
only a limited number of truly recalcitrant seed species are in fact
included (Engelmann, 1999). This is because research in this area
is recent and addressed by very few teams worldwide and because
recalcitrance is a dynamic concept which involves research on the
biology of species and improvement in classical storage procedures.
Some species previously classified as recalcitrant have thus been
moved to the intermediate or even sub-orthodox categories and

430

ENGELMANN

stored using classical or new storage techniques (Engelmann,

2000).
In comparison to the results obtained with vegetatively
propagated species, research is still at a very preliminary stage
for recalcitrant seeds. The desiccation technique is mainly
employed for freezing embryos and embryonic axes. Survival is
extremely variable, regeneration is frequently restricted to callusing
or incomplete development of plantlets, and the number of
accessions tested per species is generally very low. A number of
reasons explain the limited development of cryopreservation for
recalcitrant seed species (Engelmann, 2000). A huge number of
species have recalcitrant or suspected recalcitrant seeds and the
majority are wild species. As a consequence, little or nothing is
known of their seed biology, and even less on seed storage behavior.
In cases where information on seed storage behaviour is available,
tissue culture protocols, including inoculation in vitro, germination
and growth of plantlets, propagation and acclimatization which are
needed for regrowth of embryos and embryonic axes after freezing,
are often non-existent or not fully operational (Engelmann, 1999).
Seeds and embryos of recalcitrant species also display very large
variations in moisture content and maturity stage between
provenances, between and among seed lots, as well as between
successive harvests, thus making their cryopreservation difficult.
Seeds of many species are too large to be frozen directly and
embryos or embryonic axes are thus successfully employed for
cryopreservation. However, embryos are often of very complex
tissue composition which display differential sensitivity to
desiccation and freezing, the root pole seeming more resistant
than the shoot pole. In some species, embryos are extremely
sensitive to desiccation and even a minor reduction in their
moisture content down to levels much too high to obtain survival
after freezing leads to irreparable structural damage, as observed
notably with cacao (Chandel et al., 1995). Finally, embryos of some
species are too large to envisage using them for cryopreservation,
and seeds of some species do not contain well-defined embryos.
Various options can be considered for improving storage of nonorthodox seeds. With some species, very precisely controlled
desiccation (e.g., using saturated salt solutions) and cooling
conditions may allow freezing of whole seeds, as demonstrated
recently with various coffee species (Dussert et al., 1997). There is
scope for technical improvements in the current cryopreservation
protocols for embryos and embryonic axes. Pregrowth on media
containing cryoprotective substances may confer increased
tolerance to the tissues for further desiccation and reduce the
heterogeneity of the material. Flash drying followed by ultra-rapid
freezing has also been very effective for cryopreservation of several
species (Berjak et al., 1989; Wesley-Smith et al., 1992). Other
cryopreservation techniques, including pregrowth desiccation,
encapsulation dehydration, vitrification, and encapsulation vitrification which have seldom been employed so far, should be
investigated (Engelmann, 2000). Finally, selecting embryos at the
right developmental stage is of critical importance for the success of
any cryopreservation experiment (Engelmann et al., 1995).
However, in these cases, basic protocols for disinfection,
inoculation in vitro, germination of embryos or embryonic axes,
plantlet development, and possibly limited propagation will have to
be established prior to any cryopreservation experiment.
With species for which attempts to freeze whole embryos or
embryonic axes have proven unsuccessful, it has been suggested

using shoot apices sampled from embryos, adventitious buds, or

somatic embryos induced from the embryonic tissues (Pence, 1995).
This might be the only solution for species which do not have welldefined embryos; however, this will require more sophisticated
tissue culture procedures to be developed and mastered.

Large-scale Utilization of Cryopreservation for Germplasm

Conservation
Even though its routine use is still limited, there are a growing
number of examples where cryopreservation is employed on a large
scale for different types of materials, which are, or are not, tolerant
to dehydration.
In the case of orthodox seed species, cryopreservation is used
mainly for storing seeds with limited longevity and of rare or
endangered species (Pritchard, 1995; Gonzales-Benito et al., 1999;
Pence, 1999). The National Center for Genetic Resources
Preservation (NCGRP, Fort Collins, CO) conserves 37 654
accessions (a total of over 360 629 seeds) over the vapors of liquid
nitrogen (Towill et al., 1998). The National Bureau for Plant Genetic
Resources (NBPGR, New Delhi, India) conserves 1200 accessions
from 50 different species, consisting mainly of endangered
medicinal plants (Mandal, 2000). This technique is also used in
several botanic gardens. Over 110 accessions of rare or threatened
species are stored under cryopreservation at the Perth Royal
Botanic Garden in Australia (Touchell and Dixon, 1994) and the
Cincinnati Botanic Garden in the USA conserves seeds of rare and
endangered native species in liquid nitrogen (Pence, 1991).
Cryopreservation is also applied to intermediate seeds which are
tolerant to freezing. In Catie (Tropical Agricultural Research and
Higher Education Center, Costa Rica), seeds of 80 accessions of
Coffea arabica, which constitute the core collection of the field
collection, have been stored in liquid nitrogen after controlled
dehydration and freezing (Dussert et al., 1997, 1999). In France,
under the framework of a national grape genetic resources
conservation project, seeds of several hundred accessions are
being cryopreserved after partial desiccation (Dussert, personal
communication).
In the case of dormant buds, the 2200 accessions of the US apple
germplasm field collection are duplicated under cryopreservation
(Forsline et al., 1999). In Japan, the mulberry field collection
maintained at the National Institute of Agrobiological Resources
(NIAR, Yamagata) includes a total of 756 accessions, 420 of which
have been cryopreserved (Niino, 1995) and freezing of the whole
collections will be completed within 3 yr. Dormant buds of over 300
European elm accessions are conserved in liquid nitrogen by Afocel
(Nangis, France) and research is under way in France (IRD,
Montpellier) and the USA (NCGRP, Fort Collins) on freezing of
grape dormant buds for germplasm conservation.
Breeders routinely store pollen in liquid nitrogen in the
framework of their improvement programs (Towill and Walters,
2000). Pollen, which is an interesting material for genetic resource
conservation of various species, is stored by several institutes. In
India, the NBPGR conserves cryopreserved pollen of 65 accessions
belonging to different species (Mandal, 2000) and the Indian
Institute for Horticultural Research (IIHR, Bangalore) conserves
pollen of 600 accessions belonging to 40 species from 15 different
families, some of which have been stored for over 15 yr (Ganeshan

431

PLANT CRYOPRESERVATION

and Rajashekaran, 2000). In the USA, the NCGRP conserves pollen

of 13 pear cultivars and 24 Pyrus species (Reed et al., 2000).
Cryopreservation is also applied to biotechnology products. Over
1000 callus strains of species of pharmaceutical interest are stored
at 21968C in the UK (Spencer, 1999) as well as several thousand
conifer embryogenic cell lines employed in large-scale clonal
planting programs in Canada (Cyr, 2000). In France, cryopreservation is systematically employed for storing all the new embryogenic
cell lines of coffee and cacao produced by the Biotechnology
Laboratory of the Nestle Company (Florin et al., 1999) and the
banana lines produced in Vitropic, a private tissue culture
laboratory. Polyembryonic cultures of around 80 oil palm
accessions have been cryopreserved and stored at IRD (Montpellier,
France) (Dumet, 1994).
Finally, cryopreservation is being applied in genebanks for longterm storage of genetic resources of vegetatively propagated species,
using apices sampled from in vitro plantlets. Potato cryopreservation is currently the most advanced, since 519 old potato varieties
are cryostored in Germany at the Institute for Crop and Grassland
Science in Braunschweig (Mix-Wagner et al., 2003) and over 200
accessions at the International Potato Center (CIP, Lima, Peru)
(Golmirzaie and Panta, 2000). A duplicate of around 100 accessions
of the Pyrus field collection of the US National Clonal Germplasm
Repository (NCGR, Corvallis, OR) is cryostored at NCGR, with
another duplicate at the NCGRP in Fort Collins (Reed et al., 2000).
At the INIBAP (International Network for the Improvement of
Banana and Plantain) Transit Centre (ITC), based at KU Leuven,
Belgium, 100 accessions of the international Musa germplasm
collection have already been cryopreserved. Cryopreservation of the
remaining 1036 accessions of the collection is under way (Panis,
personal communication). Large-scale testing of cryopreservation
protocols is also performed notably with cassava and strawberry
(Roca et al., 2000; Matsumoto, personal communication).
Large-scale utilization of cryopreservation implies scaling-up of
the amount of material to be handled and stored, from one or a few
genotypes in the laboratory to several tens, hundreds, or even
thousands in the cryobank, which requires the establishment of
specific procedures for their management. With this aim,
probabilistic tools have been developed recently to assist genebank
curators in the establishment and management of cryopreserved
germplasm collections (Dussert et al., 2003).
Cryopreservation imposes a series of stresses to plant material,
which is susceptible to induced modifications in cryopreserved
cultures and regenerated plants. It is thus important to verify that
the genetic stability of cryopreserved material is not altered before
routinely using this technique for long-term conservation of plant
germplasm. The number of papers published on this aspect has been
increasing recently (see, e.g., Dixit et al., 2003; Gagliardi et al.,
2003; Zhai et al., 2003). No modification has been observed at the
phenotypical, biochemical, chromosomal, or molecular level which
could be attributed to cryopreservation (Engelmann, 1997b). The
few studies comparing the vegetative and floral development in the
field of plants originating from control and cryopreserved material
performed with oil palm (Engelmann, 1991), potato (Mix-Wagner
et al., 2003), sugarcane (Gonzalez Arnao, 1996), and banana (Cote
et al., 2000) did not reveal any differences in the characters studied.
Studies performed on the cost of cryopreservation are still
fragmentary, but the first estimates tend to confirm the interest of this
technique also from a financial perspective. Hummer and Reed

(2000) indicate that, at the NCGR, the annual maintenance cost of

one temperate fruit tree accession is US$77 in the field, US$23 under
in vitro slow growth storage, and only US$1 under cryopreservation,
to which US$50 60 should be added once for cryopreserving this
accession. Roca (personal communication) evaluates the annual
maintenance cost of CIATs (International Center for Tropical
Agriculture, Cali, Colombia) cassava collection, which includes
5000 accessions, at around US$5000 under cryopreservation,
against US$30 000 under in vitro slow growth storage.
Additional Uses of Cryopreservation
Cryopreservation is also employed for uses other than germplasm
conservation. One such use is cryoselection, i.e., the selection of
samples with special properties in the frozen population. For
example, when lavender cell suspensions were submitted to
successive freeze thaw cycles, the number of colonies recovered
from cryopreserved cells increased with the number of freeze thaw
cycles (Watanabe et al., 1985). However, no modifications were
noted in the biosynthetic capacities of cryopreserved cells,
suggesting a change in population structure rather than genetic
change. A similar observation was made by Bercetche et al. (1990)
who noted that cryopreserved Picea abies embryogenic calluses
recovered faster than non-frozen controls. For these authors, nonembryogenic tissues were killed by freezing, thus leading to the
production of a more homogeneous population, with a higher
embryogenic potential. This suggests that cryopreservation could be
used as a tool to rejuvenate cultures when their proliferation
capacities are decreasing. In the case of wheat, Kendall et al.
(1990) were able to regenerate plants with increased cold tolerance
from calluses submitted to repeated freeze thaw cycles. A different
protein pattern was associated with these cold-tolerant plants.
More recently, cryopreservation has been used for cryotherapy,
i.e., for eliminating viruses from infected plants, as a substitute or
complementary to classical virus eradication techniques such as
meristem culture and cryotherapy. After cryopreservation of plum
shoot tips sampled from in vitro plantlets infected by the plum pox
virus, 50% of plants obtained were virus-free against only 20%
through meristem culture (Brison et al., 1997). With infected
banana meristematic cultures, Helliot et al. (2002) obtained 30%
and 90% virus eradication after cryopreservation, for cucumber
mosaic virus (CMV) and banana streak virus (BSV), respectively.
Finally, Wang et al. (2003) have shown that cryopreservation led to
97% elimination of grape virus A, against only 12% by meristem
culture. Cryotherapy is thus based on selective cell destruction by
cryopreservation. The differentiated cells of apices which contain
viruses also have a high water content; they are killed by the
formation of ice crystals during freezing. By contrast, meristematic
cells, the multiplication of which leads to growth of apices, have a
more concentrated cytoplasm and withstand freezing.
Finally, in large-scale propagation programs of conifers based on
somatic embryogenesis, the ability to maintain donor tissue juvenility
through cryopreservation represents an immeasurable advantage
over propagation programs based on rooted cuttings (Cyr, 2000).
Conclusion
Even though cryopreservation is still routinely employed in a
limited number of cases only, the development of the new

432

ENGELMANN

vitrification-based freezing techniques has made its application to a

broad range of species possible. A significant advantage of these
new techniques is their operational simplicity, since they will be
mainly applied in developing tropical countries where the largest
part of genetic resources of problem species is located. For many
vegetatively propagated species, cryopreservation techniques are
sufficiently advanced to envisage their immediate utilization for
large-scale experimentation in genebanks. Research is much less
advanced for recalcitrant seed species. This is due to the large
number of mainly wild species, with very different characteristics,
which fall within this category, and to the comparatively limited
level of research activities aiming at improving the conservation of
these species. However, there are various technical approaches to
explore to improve the efficiency and increase the applicability of
cryopreservation techniques to recalcitrant species. In addition,
research is actively performed by various groups worldwide to
improve the knowledge of biological mechanisms underlying seed
recalcitrance. It is hoped that new findings on critical issues such as
understanding and control of desiccation sensitivity will contribute
significantly to the development of improved cryopreservation
techniques for recalcitrant seed species. A very positive
development in the establishment of cryopreservation protocols is
that an increasing number of researchers make systematic use of
analytical tools (e.g., histo-cytology, differential scanning calorimetry) which provide a scientific basis to the success/failure of an
experimental treatment. Such a rational and scientific approach
should greatly facilitate the establishment of freezing protocols,
especially for problem species.
It is important to stress, however, that cryopreservation is not
seen as a replacement for conventional ex situ approaches (Withers
and Engelmann, 1998). Cryopreservation offers genebank curators
an additional tool to allow them to improve the conservation of
germplasm collections placed under their responsibility. In
conclusion, it can be realistically expected that in the coming
years, cryopreservation will become more frequently employed for
long-term conservation of plant genetic resources.
References
Assy-Bah, B.; Engelmann, F. Cryopreservation of mature embryos of coconut
(Cocos nucifera L.) and subsequent regeneration of plantlets.
CryoLetters 13:117126; 1992.
Bercetche, J.; Galerne, M.; Dereuddre, J. Efficient regeneration of plantlets
from embryogenic callus of Picea abies (L.) Karst after freezing in
liquid nitrogen. C. R. Acad. Sci. Paris Ser. 3 310:357 363; 1990.
Berjak, P.; Farrant, J. M.; Mycock, D. J.; Pammenter, N. W. Homoiohydrous
(recalcitrant) seeds: the enigma of their desiccation sensitivity and
the state of water in axes of Landolphia kirkii Dyer. Planta
186:249261; 1989.
Brison, M.; de Boucaud, M. T.; Pierronnet, A.; Dosba, F. Effect of
cryopreservation on the sanitary state of a cv. of Prunus rootstock
experimentally infected with Plum Pox Potyvirus. Plant Sci.
123:189196; 1997.
Chandel, K. P. S.; Chaudhury, R.; Radhamani, J.; Malik, S. K. Desiccation
and freezing sensitivity in recalcitrant seeds of tea, cocoa and
jackfruit. Ann. Bot. 76:443450; 1995.
Cote, F. X.; Goue, O.; Domergue, R.; Panis, B.; Jenny, C. In-field behavior of
banana plants (Musa AA sp.) obtained after regeneration of
cryopreserved embryogenic cell suspensions. CryoLetters 21:1924;
2000.
Cyr, D. R. Cryopreservation: roles in clonal propagation and germplasm
conservation of conifers. In: Engelmann, F.; Takagi, H., eds.
Cryopreservation of tropical plant germplasm current research

progress and applications. Tsukuba: JIRCAS; Rome: IPGRI;

2000:261268.
Dereuddre, J.; Hassen, M.; Blandin, S.; Kaminski, M. Resistance of alginatecoated somatic embryos of carrot (Daucus carota L.) to desiccation
and freezing in liquid nitrogen: 2. thermal analysis. CryoLetters
12:135148; 1991.
Dixit, S.; Mandal, B. B.; Ahuja, S.; Srivastava, P. S. Genetic stability
assessment of plants regenerated from cryopreserved embryogenic
tissues of Dioscorea bulbifera L. using RAPD, biochemical and
morphological analysis. CryoLetters 24:7784; 2003.
Dumet, D. Cryoconservation des massifs dembryons somatiques de palmier
a` huile (Elaeis guineensis Jacq.) par deshydratation-vitrification.
Etude du role du saccharose pendant le pretraitement. PhD thesis,
Universite Paris 6, France; 1994.
Dumet, D.; Engelmann, F.; Chabrillange, N.; Duval, Y. Cryopreservation of
oil palm (Elaeis guineensis Jacq.) somatic embryos involving a
desiccation step. Plant Cell Rep. 12:352355; 1993.
Dussert, S.; Chabrillange, N.; Engelmann, F.; Anthony, F.; Hamon, S.
Cryopreservation of coffee (Coffea arabica L.) seeds: importance of
the precooling temperature. CryoLetters 18:269276; 1997.
Dussert, S.; Chabrillange, N.; Vasquez, N. Cryopreservation of seeds for
long-term conservation of coffee germplasm and elite varieties:
successful application at CATIE. In: Proc. 18th Int. Conf. on Coffee
Science (ASIC99), Helsinki, August 26, 1999.
Dussert, S.; Engelmann, F.; Noirot, M. Development of probalistic tools to
assist in the establishment and management of cryopreserved plant
germplasm collections. CryoLetters 24:149160; 2003.
Ellis, R. E.; Hong, T.; Roberts, E. H. An intermediate category of seed
storage behaviour?. I. coffee. J. Exp. Bot. 41:11671174; 1990.
Engelmann, F. In vitro conservation of tropical plant germplasm a review.
Euphytica 57:227243; 1991.
Engelmann, F. Cryopreservation of embryos. In: Dattee, Y.; Dumas, C.;
Gallais, A., eds. Reproductive biology and plant breeding. Berlin:
Springer Verlag; 1992:281290.
Engelmann, F. Importance of desiccation for the cryopreservation of
recalcitrant seed and vegetatively propagated species. Plant Genet.
Res. Newslett. 112:918; 1997a.
Engelmann, F. In vitro conservation methods. In: Ford-Lloyd, B. V.;
Newburry, J. H.; Callow, J. A., eds. Biotechnology and plant genetic
resources:
conservation
and
use.
Wallingford:
CABI;
1997b:119162.
Engelmann, F. Alternative methods for the storage of recalcitrant seeds an
update. In: Marzalina, M.; Khoo, K. C.; Tsan, F. Y.; Krishnapillay,
B., eds. IUFRO Seed Symposium 1998 Recalcitrant Seeds, Kuala
Lumpur, Malaysia, October 12 15, 1998. Kuala Lumpur: FRIM;
1999:159170.
Engelmann, F. Importance of cryopreservation for the conservation of plant
genetic resources. In: Engelmann, F.; Takagi, H., eds. Cryopreservation of tropical plant germplasm current research progress and
applications. Tsukuba: JIRCAS; Rome: IPGRI; 2000:8 20.
Engelmann, F.; Dumet, D.; Chabrillange, N.; Abdelnour-Esquivel, A.; AssyBah, B.; Dereuddre, J.; Duval, Y. Factors affecting the
cryopreservation of coffee, coconut and oil palm embryos. Plant
Genet. Res. Newslett. 103:2731; 1995.
Engelmann, F.; Takagi, H. Cryopreservation of tropical plant germplasm
current research progress and applications. Tsukuba: JIRCAS;
Rome: IPGRI; 2000.
Fahy, G. M.; MacFarlane, D. R.; Angell, C. A.; Meryman, H. T. Vitrification
as an approach to cryopreservation. Cryobiology 21:407426; 1984.
Florin, B.; Brulard, E.; Lepage, B. Establishment of a cryopreserved coffee
germplasm bank. In: Abstracts Cryo99, World Congress of
Cryobiology, Marseilles, France, July 1215, 1999:167.
Forsline, P. L.; McFerson, J. R.; Lamboy, W. F.; Towill, L. E. Development
of base and active collections of Malus germplasm with
cryopreserved dormant buds. Acta Hort. 484:7578; 1999.
Gagliardi, R. F.; Pacheco, G. P.; Carneiro, L. A.; Valls, J. F. M.;
Vieira, M. L. C.; Mansur, E. Cryopreservation of Arachis species by
vitrification of in vitro grown shoot apices and genetic stability of
recovered plants. CryoLetters 24:103 110; 2003.
Ganeshan, S.; Rajashekaran, P. E. Current status of pollen cryopreservation
research: relevance to tropical agriculture. In: Engelmann, F.; Takagi,
H., eds. Cryopreservation of tropical plant germplasm current

PLANT CRYOPRESERVATION
research progress and applications. Tsukuba: JIRCAS; Rome: IPGRI;
2000:360 365.
Golmirzaie, A.; Panta, A. Advances in potato cryopreservation at the
International Potato Center, Peru. In: Engelmann, F.; Takagi, H.,
eds. Cryopreservation of tropical plant germplasm current research
progress and applications. Tsukuba: JIRCAS; Rome: IPGRI;
2000:250 254.
Gonzalez Arnao, M. T. Desarollo de una tecnica para la crioconservacion de
meristemos apicales de cana de azucar. Tesis presentada en opcion
al grado de Doctor en ciencias tecnicas, Centro Nacional de
Investigaciones Cientificas, La Habana, Cuba; 1996.
Gonzalez-Benito, M. E.; Martin, C.; Iriondo, J. M. Conservation of rare and
endangered plants endemic to Spain. In: Benson, E. E., ed. Plant
conservation biotechnology. London: Taylor & Francis;
1999:251 264.
Helliot, B.; Panis, B.; Poumay, Y.; Swennen, R.; Lepoivre, P.; Frison, E.
Cryopreservation for the elimination of cucumber mosaic and banana
streak viruses from banana (Musa spp.). Plant Cell Rep.
20:1117 1122; 2002.
Hummer, K. E.; Reed, B. M. Establishment and operation of a temperate
clonal field genebank. In: Engelmann, F. Management of field and in
vitro germplasm collections. Rome: IPGRI; 2000:2931.
Kartha, K. K.; Engelmann, F. Cryopreservation and germplasm storage. In:
Vasil, I. K.; Thorpe, T. A., eds. Plant cell and tissue culture.
Dordrecht: Kluwer; 1994:195230.
Kendall, E. J.; Qureshi, J. A.; Kartha, K. K.; Leung, N.; Chevrier, N.;
Caswell, K.; Chen, T. H. H. Regeneration of freezing tolerant spring
wheat (Triticum aestivum L.) plants from cryoselected callus. Plant
Physiol. 94:17561762; 1990.
Mandal, B. B. Cryopreservation research in India: current status and future
perspectives. In: Engelmann, F.; Takagi, H., eds. Cryopreservation of
tropical plant germplasm current research progress and
applications. Tsukuba: JIRCAS; Rome: IPGRI; 2000:282286.
Matsumoto, T.; Sakai, A. Cryopreservation of axillary shoots of in vitro grown
grape (Vitis) by a two-step vitrification protocol. Euphytica
131:299 304; 2003.
Matsumoto, T.; Sakai, A.; Yamada, K. Cryopreservation of in vitro grown
apical meristems of wasabi (Wasabia japonica) by vitrification and
subsequent high plant regeneration. Plant Cell Rep. 13:442446;
1994.
Mazur, P. Freezing of living cells: mechanisms and applications. Am.
J. Physiol. Cell Physiol. 247:C125C142; 1984.
Meryman, H. T.; Williams, R. J.; Douglas, M. S. J. Freezing injury from
solution effects and its prevention by natural or artificial
cryoprotection. Cryobiology 14:287302; 1977.
Mix-Wagner, G.; Conner, A. J.; Cross, R. J. Survival and recovery of
asparagus shoot tips after cryopreservation using the droplet
method. NZ J. Crop Hort. Sci. 28:283 287; 2000.
Mix-Wagner, G.; Schumacher, H. M.; Cross, R. J. Recovery of potato apices
after several years of storage in liquid nitrogen. CryoLetters
24:3341; 2003.
Niino, T. Cryopreservation of germplasm of mulberry. In: Bajaj, Y. P. S., ed.
Biotechnology in agriculture and forestry, vol. 32. Cryopreservation
of plant germplasm I. Berlin: Springer Verlag; 1995:102113.
Panis, B.; Strosse, H.; Van den Henda, S.; Swennen, R. Sucrose preculture to
simplify cryopreservation of banana meristem cultures. CryoLetters
23:375384; 2002.
Pence, V. C. Cryopreservation of seeds of Ohio native plants and related
species. Seed Sci. Technol. 19:235 251; 1991.
Pence, V. C. Cryopreservation of recalcitrant seeds. In: Bajaj, Y. P. S., ed.
Biotechnology in agriculture and forestry, vol. 32. Cryopreservation
of plant germplasm I. Berlin: Springer Verlag; 1995:29 52.
Pence, V. C. The application of biotechnology for the conservation of
endangered plants. In: Benson, E. E., ed. Plant conservation
biotechnology. London: Taylor & Francis; 1999:227250.
Pritchard, H. W. Cryopreservation of seeds. In: Day, J. G.; McLellan, M. R.,
eds. Methods in molecular biology, vol. 38. Cryopreservation and
freeze-drying protocols. Totowa, NJ: Humana Press; 1995:133144.
Reed, B. M.; Chang, Y. Medium- and long-term storage of in vitro cultures of
temperate fruit and nut crops. In: Razdan, M. K.; Cocking, E. C., eds.

433

Conservation of plant genetic resources in vitro, vol. 1. General

aspects. Enfield: Science Publishers; 1997:67105.
Reed, B. M.; DeNoma, J.; Chang, Y. Application of cryopreservation
protocols at a clonal genebank. In: Engelmann, F.; Takagi, H., eds.
Cryopreservation of tropical plant germplasm current research
progress and applications. Tsukuba: JIRCAS; Rome: IPGRI;
2000:246249.
Roberts, H. F. Predicting the viability of seeds. Seed Sci. Technol.
1:499 514; 1973.
Roca, W.; Debouck, D.; Escobar, R.; Mafla, G. Cryopreservation and cassava
germplasm conservation at CIAT. In: Engelmann, F.; Takagi, H., eds.
Cryopreservation of tropical plant germplasm current research
progress and applications. Tsukuba: JIRCAS; Rome: IPGRI;
2000:273279.
Sakai, A. Development of cryopreservation techniques. In: Engelmann, F.;
Takagi, H., eds. Cryopreservation of tropical plant germplasm
current research progress and applications. Tsukuba: JIRCAS;
Rome: IPGRI; 2000:17.
Sakai, A.; Kobayashi, S.; Oiyama, I. Cryopreservation of nucellar cells of
navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by
vitrification. Plant Cell Rep. 9:30 33; 1990.
Sakai, A.; Matsumoto, T.; Hirai, D.; Charoensub, R. Survival of tropical
apices cooled to 21968C by vitrification. In: Li, P. H.; Palva, E. T.,
eds. Plant cold hardiness, gene regulation and genetic engineering.
New York: Kluwer Academic/Plenum Publishers; 2002:109119.
Sakai, A.; Matsumoto, T.; Hirai, D.; Niino, T. Newly developed
encapsulation dehydration protocol for plant cryopreservation.
CryoLetters 21:5362; 2000.
Schafer-Menuhr, A. Refinement of cryopreservation techniques for potato.
Final Report for the period 1 Sept. 199131 Aug. 1996; Rome:
IPGRI; 1996.
Spencer, M. The challenges of developing cryopreservation strategies to suit
the requirements of a large industrial in vitro plant cell collection. In:
Abstracts Cryo99, World Congress of Cryobiology, Marseilles,
France, July 1215, 1999:245.
Touchell, D. H.; Dixon, K. W. Cryopreservation for seedbanking of
Australian species. Ann. Bot. 40:541 546; 1994.
Towill, L. E.; Eberhart, S.; Roos, E. Cryopreservation at the USDA-ARS
national seed storage laboratory. In: Abstracts Int. Workshop on
Cryopreservation of Tropical Plant Germplasm current research
progress and applications, Tsukuba, Japan, October 2023, 1998.
Towill, L. E.; Walters, C. Cryopreservation of pollen. In: Engelmann, F.;
Takagi, H., eds. Cryopreservation of tropical plant germplasm
current research progress and applications. Tsukuba: JIRCAS;
Rome: IPGRI; 2000:115129.
Uragami, A.; Sakai, A.; Magai, M. Cryopreservation of dried axillary buds
from plantlets of Asparagus officinalis L. grown in vitro. Plant Cell
Rep. 9:328331; 1990.
Wang, Q.; Mawassi, M.; Li, P.; Gafny, R.; Sela, I.; Tanne, E. Elimination of
grapevine virus A (GVA) by cryopreservation of in vitro-grown shoot
tips of Vitis vinifera L. Plant Sci. 165:321327; 2003.
Watanabe, K.; Yamada, Y.; Ueno, S.; Mitsuda, H. Change of freezing
resistance and retention of metabolic and differentiation potentials in
cultured green Lavandula vera cells which survived repeated freeze
thaw procedures. Agr. Biol. Chem. Tokyo 49:17271731; 1985.
Wesley-Smith, J.; Vertucci, C. W.; Berjak, P.; Pammenter, N. W.; Crane;, J.
Cryopreservation of desiccation-sensitive axes of Camellia sinensis in
relation to dehydration, freezing rate and the thermal properties of
tissue water. J. Plant Physiol. 140:596604; 1992.
Withers, L. A.; Engelmann, F. In vitro conservation of plant genetic
resources. In: Altman, A., ed. Biotechnology in agriculture. New
York: Marcel Dekker Inc.; 1998:5788.
Zhai, Z.; Wu, Y.; Huang, X.; Chen, R.; Zhao, Y.; Engelmann, F. Assessments
of genetic stability of grape and kiwi in vitro plantlets regenerated
from cryopreserved shoot tips using RAPD. CryoLetters
24:315322; 2003.
Zhao, Y.; Wu, Y.; Engelmann, F.; Zhou, M.; Chen, S. Cryopreservation of
apple in vitro shoot tips by the droplet freezing method. CryoLetters
20:109112; 1999.