In Vitro Cell.Dev.Biol.
—Plant
DOI 10.1007/s11627-010-9327-2
INVITED REVIEW
Use of biotechnologies for the conservation of plant
biodiversity
Florent Engelmann
Received: 12 April 2010 / Accepted: 14 October 2010 / Editor: P. Lakshmanan
# The Society for In Vitro Biology 2010
Abstract In vitro techniques are very useful for conserving
plant biodiversity, including (a) genetic resources of
recalcitrant seed and vegetatively propagated species, (b)
rare and endangered plant species and (c) biotechnology
products such as elite genotypes and genetically engineered
material. Explants from recalcitrant seed and vegetatively
propagated species can be efficiently collected under field
conditions using in vitro techniques. In vitro culture
techniques ensure the production and rapid multiplication
of disease-free material. Medium-term conservation is
achieved by reducing growth of plant material, thus
increasing intervals between subcultures. For long-term
conservation, cryopreservation (liquid nitrogen, −196°C)
allows storing plant material without modification or
alteration for extended periods, protected from contamina-
tions and with limited maintenance. Slow growth storage
protocols are routinely employed for a large number of
species, including numerous endangered plants, from
temperate and tropical origin. Cryopreservation is well
advanced for vegetatively propagated species, and techni-
ques are ready for large-scale experimentation in an
increasing number of cases. Research is much less
advanced for recalcitrant species due to their seed character-
istics, viz., very high sensitivity to desiccation, structural
complexity and heterogeneity in terms of developmental
F. Engelmann (*)
IRD, UMR DIAPC,
911 Avenue Agropolis, BP 64501,
34032 Montpellier cedex 5, France
e-mail: florent.engelmann@ird.fr
F. Engelmann
Bioversity International,
Via dei Tre Denari 472/a,
00057 Maccarese (Fiumicino), Rome, Italy
stage and water content at maturity. However, various
technical approaches should be explored to develop cryopres-
ervation techniques for a larger number of recalcitrant seed
species. A range of analytical techniques are available, which
allow understanding physical and biological processes taking
place in explants during cryopreservation. These techniques
are extremely useful to assist in the development of
cryopreservation protocols. In comparison with crop species,
only limited research has been performed on cryopreservation
of rare and endangered species. Even though routine use of
cryopreservation is still limited, an increasing number of
examples where cryopreservation is used on a large scale can
be found both in genebanks for crops and in botanical gardens
for endangered species.
Keywords In vitro collecting . Slow growth storage .
Cryopreservation . Germplasm conservation . Crops .
Rare and endangered species
Introduction
A large number of crop species have seeds, which are
termed orthodox, i.e. that can be dehydrated down to low
water contents and can thus be stored at low temperature
for extended periods (Roberts 1973). There are three main
categories of plant species for which conservation in seed
form is problematic. First, some plants such as banana and
plantain do not produce seeds and are thus propagated
vegetatively. Second, some species such as potato or
sugarcane include both sterile genotypes and genotypes
which produce orthodox seeds. However, these seeds are
generally highly heterozygous and are thus of limited
interest for the conservation of particular genotypes. These
species are thus mainly maintained as clones. Third,
 ENGELMANN
numerous fruit and forest tree species, especially from
tropical origin, produce recalcitrant seeds, i.e. seeds that
cannot be dried to sufficiently low moisture level to allow
their storage at low temperature (Roberts 1973). There is
also a large number of species, termed intermediate (Ellis et
al. 1990, 1991) for which conservation in seed form is still
problematic. The traditional ex situ conservation method for
these categories of plant species is in the form of field
collections. Conservation in the field presents major draw-
backs, which limit its efficacy and threaten the safety of
plant genetic resources conserved in this way.
Until now, most activities on ex situ conservation of
plant biodiversity have focussed on crop species. However,
conservation of wild, rare and endangered plant species has
also become an issue of concern. Indeed, as highlighted by
Sarasan et al. (2006), the world’s biodiversity is declining
at an unprecedented rate. During the period 1996–2004, a
total of 8,321 plant species have been added to the Red List
of Threatened Species (IUCN 2004), and the number of
plants recorded as critically endangered has increased by
60%. In the case of wild species, the traditional conserva-
tion approach is in situ conservation. However, it is now
recognised that ex situ techniques can be efficiently used to
complement in situ methods, and they may represent the
only option for conserving certain highly endangered and
rare species (Ramsay et al. 2000). It is therefore of
paramount importance to develop techniques ensuring
optimal storage and rapid multiplication of such species.
Botanic gardens play a very important role in ex situ
conservation of plant biodiversity. UNEP (1995) estimated
that botanic gardens conserve more than one third of the
world’s flowering plants, among which Botanic Gardens
Conservation International identified more than 15,000
threatened species (http://www.bgci.org/ourwork/1977/).
Botanic gardens and agricultural genebanks should be seen
as playing a complementary role for the conservation of
plant biodiversity (Engels and Engelmann 1998).
The development of biotechnology leads to the produc-
tion 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.
In the light of the problems presented by the different
categories of plant species outlined above, it is not
surprising that efforts have been made to improve the
quality and security of conservation offered by field
genebanks and botanic gardens and to understand and
overcome seed recalcitrance to make seed storage more
widely available. However, it is clear that alternative
approaches to genetic conservation are needed for these
problem materials, and since the early 1970s, attention has
turned to the possibilities offered by biotechnology,
specifically in vitro or tissue cultures.
Tissue culture techniques are of great interest for the
collecting, multiplication and storage of plant germplasm
(Engelmann 1991; Bunn et al. 2007). Tissue culture
systems allow propagating plant material with high multi-
plication rates in an aseptic environment. Virus-free plants
can be obtained through meristem culture in combination
with thermotherapy, thus ensuring the production of disease-
free stocks and simplifying quarantine procedures for the
international exchange of germplasm. The miniaturization of
explants allows reducing space requirements and conse-
quently labour costs for the maintenance of germplasm
collections. In vitro propagation protocols have been
established for several thousand plant species (George
1996), including numerous rare and endangered species
(Fay 1992; Sarasan et al. 2006).
Different in vitro conservation methods are employed,
depending on the storage duration requested. For short- and
medium-term storage, the aim is to reduce growth and to
increase the intervals between subcultures. For long-term
storage, cryopreservation, i.e. storage at ultra-low temper-
ature, usually that of liquid nitrogen (−196°C), is the only
current method. 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,
requiring very limited maintenance. In vitro collecting, slow
growth and cryopreservation techniques are described and
analysed in the sections below.
Applications of Biotechnologies for Conservation
In vitro collecting. Collectors are faced with various
problems when collecting germplasm of recalcitrant seed
and vegetatively propagated plant species. Collecting
missions often require travelling for relatively long periods
in remote areas. It is thus necessary to keep the material
collected in good state for some d/wk before it can be
placed in optimal growth or storage conditions. There are
thus great risks that recalcitrant seeds either germinate or
deteriorate before they are brought back to the genebank or
botanic garden. In addition, many recalcitrant seeds have a
sheer weight and bulk, which is a source of problems in
terms of volume of material to handle and which induces
additional costs, if an adequate sample of the population is
to be collected. With vegetatively propagated species, the
material collected will consist of stakes, pieces of budwood,
tubers, corms or suckers. Not only will most of these
explants not be adapted to survival once excised from the
 BIOTECHNOLOGIES FOR CONSERVING BIODIVERSITY
parent plant but they will also present health risks due to
their vegetative nature and contamination with soil-borne
pathogens (Withers 1987). Difficulties can also be encoun-
tered when collecting germplasm of orthodox seed-
producing species. Even with careful planning of the time
of the collecting mission, there might be no or little seed
available for all or part of the germplasm to be collected, or
seeds might not be at the optimal developmental stage, shed
from the plant or eaten by grazing animals (Guarino et al.
1995). These problems can be overcome if it is realized that
the seed is not the only material which can be collected:
Zygotic embryos or vegetative tissues such as pieces of
budwood, shoots, apices or even leaf discs can be sampled,
transported and grown successfully if placed under ade-
quate conditions.
Following an expert meeting organised by IBPGR in
1984 and sponsorship of various research programmes,
simple and efficient in vitro collecting techniques have been
developed for different materials including embryos or
vegetative tissues of various species including crops such as
coconut or cacao, as well as wild and endangered species
(Pence et al. 2002a, b). The critical points to consider for
the development of in vitro collecting techniques have been
synthesized and analysed by Withers (1995).
Slow Growth Storage
Growth reduction is generally achieved by modifying the
environmental conditions and/or the culture medium. The
most widely applied technique is temperature reduction,
which can be combined with a decrease in light intensity or
culture in the dark. Tropical species are often cold-sensitive
and have to be stored at higher temperatures, which depend
on the cold sensitivity of the species. Musa in vitro plants
can be stored at 15°C without transfer for up to 15 mo
(Banerjee and De Langhe 1985). Other tropical species
such as cassava are much more cold-sensitive since cassava
shoot cultures have to be conserved at temperatures higher
than 20°C (Roca et al. 1984). Modifications of the culture
medium can include dilution of mineral elements, reduction
of sugar concentration, changes in the nature and/or
concentration of growth regulators and addition of osmoti-
cally active compounds.
Numerous parameters influence the efficiency of in vitro
slow growth storage protocols including the type of explants,
their physiological state when entering storage, the type of
culture vessel, its volume and the volume as well as the type
of closure of the culture vessel (Engelmann 1991).
In vitro slow growth storage techniques are being
routinely used for medium-term conservation of numerous
species, both from temperate and tropical origin, including
crop plants, e.g. potato, Musa, yam, cassava (Ashmore
1997; Razdan and Cocking 1997; Engelmann 1999) and
rare and endangered species (Fay 1992; Sarasan et al.
2006). However, if in vitro conservation appears as a simple
and practical option for long-term conservation of numerous
species and has obvious wide medium-term applications, its
implementation still needs customizing to any new material,
continuous inputs are required and long-term questions
remain as regards the genetic stability of the stored material.
Moreover, it is not always possible to apply one single
protocol for conserving genetically diverse material. As an
example, a storage experiment performed with an in vitro
collection of African coffee germplasm including 21
diversity groups revealed a large variability in the response
of the diversity groups to the storage conditions (Dussert et
al. 1997a). Some groups showed high genetic erosion during
storage whilst others did not show any erosion. Technical
guidelines have been published recently (Reed et al. 2004),
which provide guidance to researchers and genebank and
botanic garden managers for the establishment and manage-
ment of in vitro germplasm collections.
Cryopreservation
Cryopreservation is the only technique currently available
to ensure the safe and cost-efficient long-term conservation
of the germplasm of problem species. In this section, we
briefly describe the various cryopreservation techniques
available, summarize the achievements made and problems
faced with vegetatively propagated and recalcitrant species
and present the current utilization of cryopreservation for
plant material.
Cryopreservation Techniques
Some materials, such as orthodox seeds or dormant buds,
display natural dehydration processes and can be cryopre-
served without any pretreatment. However, most of the
experimental systems employed in cryopreservation (cell
suspensions, calluses, shoot tips, embryos) contain high
amounts of cellular 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 the damages caused by
the crystallization of intracellular water into ice (Mazur
1984). The techniques employed and the physical mecha-
nisms upon which they are based are different in classical and
new cryopreservation techniques (Withers and Engelmann
1998). Classical techniques involve freeze-induced dehydra-
tion, whereas new techniques are based on vitrification.
Vitrification can be defined as the transition of water directly
 ENGELMANN
from the liquid phase into an amorphous phase or glass,
whilst avoiding the formation of crystalline ice (Fahy et al.
1984).
Classical cryopreservation techniques. Classical cryopres-
ervation techniques involve slow cooling down to a defined
prefreezing temperature, followed by rapid immersion in
liquid nitrogen. With temperature reduction during slow
cooling, the 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
freeze-induced dehydration can incur different damaging
events due to concentration of intracellular salts and
changes in the cell membrane (Meryman et al. 1977).
Rewarming should be as rapid as possible to avoid the
phenomenon of recrystallization in which ice melts and
reforms at a thermodynamically favourable, larger and
more damaging crystal size (Mazur 1984).
Classical freezing procedures include the following suc-
cessive steps: pregrowth of samples, cryoprotection, slow
cooling (0.5–2.0°C/min) to a determined prefreezing temper-
ature (usually around −40°C), 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).
Classical cryopreservation techniques have been suc-
cessfully applied to undifferentiated culture systems such as
cell suspensions and calluses (Kartha and Engelmann 1994;
Withers and Engelmann 1998) and apices of cold-tolerant
species (Reed and Uchendu 2008).
New cryopreservation techniques. In vitrification-based pro-
cedures, 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 confor-
mation of the glass) during cooling and rewarming have been
recorded with various materials using thermal analysis (Sakai
et al. 1990; Dereuddre et al. 1991; Niino et al. 1992).
Vitrification-based procedures offer practical advantages in
comparison to classical freezing techniques. Like ultra-rapid
freezing, 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: (a) encapsulation–dehydration, (b) a procedure
actually termed vitrification, (c) encapsulation–vitrification,
(d) dehydration, (e) pregrowth, (f) pregrowth–dehydration
and (g) droplet–vitrification.
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 to 7 d, partially
desiccated in the air current of a laminar air flow cabinet or
with silica gel to a water content around 20% (fresh weight
basis), then frozen rapidly. Survival is high and growth
recovery of cryopreserved samples is generally rapid and
direct, without callus formation. This technique has been
applied to apices of numerous species from temperate and of
tropical origin as well as to cell suspensions and somatic
embryos of several species (Gonzalez-Arnao and Engelmann
2006; Engelmann et al. 2008).
Vitrification involves treatment of samples with cryo-
protective substances, dehydration with highly concentrated
vitrification solutions, rapid cooling and rewarming, re-
moval of cryoprotectants and recovery. This procedure has
been developed for apices, cell suspensions and somatic of
numerous different species (Sakai and Engelmann 2007;
Sakai et al. 2008).
Encapsulation–vitrification is a combination of encapsu-
lation–dehydration and vitrification procedures, in which
samples are encapsulated in alginate beads, then subjected
 BIOTECHNOLOGIES FOR CONSERVING BIODIVERSITY
to freezing by vitrification. It has been applied to apices of
an increasing number of species (Sakai and Engelmann
2007; Sakai et al. 2008).
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).
Desiccation is usually performed in the air current of a laminar
airflow cabinet, but more precise and reproducible dehydra-
tion 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 Berjak’s group in South Africa) allows freezing 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
comprised between 10% and 20% (fresh weight basis).
The pregrowth technique consists of cultivating samples
in the presence of cryoprotectants, then freezing them
rapidly by direct immersion in liquid nitrogen. The pre-
growth technique has been developed for Musa meriste-
matic cultures (Panis et al. 2002).
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 somatic embryos and
coconut zygotic embryos (Uragami et al. 1990; Assy-Bah
and Engelmann 1992; Dumet et al. 1993).
Droplet–vitrification is the latest technique developed
(Sakai and Engelmann 2007). The number of species to
which it has been successfully applied is increasing
steadily. Apices are pretreated with vitrification solution,
then placed on an aluminium foil in minute droplets of
vitrification solution and frozen rapidly in liquid nitrogen.
Cryopreservation of Vegetatively Propagated
and Recalcitrant Seed Species
Vegetatively propagated species. A number of publications
provide lists of species which have been successfully
cryopreserved (Engelmann 1997a, b; Engelmann and Takagi
2000; Reed 2008). For vegetatively propagated species,
cryopreservation has a wide applicability both in terms of
species coverage, since protocols have been successfully
established for root and tubers, fruit trees, ornamentals and
plantation crops, both from temperate and tropical origin and
in terms of numbers of genotypes/varieties within a given
species. With a few exceptions, 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 and potato.
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. The meristematic zone of apices, from which
organised growth originates, is composed of a relatively
homogenous population of small, actively dividing cells, with
little vacuoles and a high nucleocytoplasmic ratio. These
characteristics make them more susceptible to withstand
desiccation than highly vacuolated and differentiated cells.
As mentioned earlier, no ice formation takes place in
vitrification-based procedures, thus avoiding the extensive
damage caused by ice crystals which are formed during
classical procedures. The whole meristem is generally
preserved when vitrification-based techniques are employed,
thus allowing direct, organised regrowth. By contrast,
classical procedures often lead to destruction of large zones
of the meristems, and callusing only or transitory callusing is
often observed before organised regrowth starts.
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 micropropagation, are
well established. In addition, in vitro material is “synchro-
nized” by the tissue culture, and pregrowth procedures and
relatively homogenous 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 using samples of
relatively large size (shoot tips of 0.5 to 2–3 mm), which
can regrow directly without any difficulty.
Cryopreservation techniques are now operational for
large-scale experimentation in an increasing number of
cases. 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. Some publications present ex-
tensive lists of plant species whose embryos and/or
embryonic axes have been successfully cryopreserved
(Kartha and Engelmann 1994; Pence 1995; Engelmann et
al. 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
 ENGELMANN
mentioned in these papers reveals that only a limited
number of truly recalcitrant seed species is in fact included.
This is because research in this area is recent and addressed
by very few teams worldwide and because recalcitrance is a
dynamic concept which evolves with research on the
biology of species and improvement in classical storage
procedures. Some species previously classified as recalci-
trant have thus been moved to the intermediate or even sub-
orthodox categories and 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
(Normah and Makeen 2008). Survival is extremely variable,
regeneration frequently restricted to callusing or incomplete
development of plants and the number of accessions tested
per species generally very low. There are a number of
reasons to explain the current limited development of
cryopreservation for recalcitrant seed species (Engelmann
2000). First of all, there is a huge number of species with
recalcitrant or suspectedly recalcitrant seeds, and they are
wild species in their large majority. As a consequence, little
or nothing is known on the biology and all the more so on
the seed storage behaviour of many of these species. In
cases where some information on seed storage behaviour is
available, tissue culture protocols, including inoculation in
vitro, germination and growth of plants, propagation and
acclimatization which are needed for regrowth of embryos
and embryonic axes after freezing, are often non-existent or
not fully operational. Seeds and embryos of recalcitrant
species also display very important variations in moisture
content and maturity stage between provenances, between
and among seed lots, as well as between successive
harvests, which make their cryopreservation difficult.
Seeds of many species are of too large dimensions 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 displays 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 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.
There are various options to consider for improving
storage of non-orthodox seeds. With some species, very
precisely controlled desiccation (e.g. using saturated salt
solutions) and cooling conditions may allow to freeze
whole seeds, as demonstrated recently with various coffee
species (Dussert et al. 1997b). There is scope for technical
improvements in the current cryopreservation protocols for
embryos and embryonic axes. Pregrowth on media con-
taining cryoprotective substances may confer the tissues’
increased tolerance to further desiccation and to 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 techni-
ques, including pregrowth–desiccation, encapsulation–
dehydration and vitrification, which seldom have been
employed so far with recalcitrant species, should be
experimented on (Pence 1995; 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, plant develop-
ment 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 to use shoot apices sampled on the
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 well-defined
embryos; however, this will require that more sophisticated
tissue culture procedures be developed and mastered.
Cryopreservation of Rare and Endangered Species
The number of publications on cryopreservation of rare and
endangered species is still relatively limited. However,
freezing protocols have been developed for various higher
plants (Bunn et al. 2007), including orchids (Hirano et al.
2006), as well as for bryophytes and ferns (Pence 2008)
using the techniques described in the above sections, which
were applied to different explant types.
A classical protocol, involving treatment with DMSO
followed by slow cooling, was successful for freezing seeds
of 68 native Western Australian species out of the 90
species tested (Touchell and Dixon 1993). Desiccation has
been employed for freezing seeds of rare temperate orchids
by direct immersion in liquid nitrogen (Nikishina et al.
2007). Several authors have used the desiccation technique
for freezing seeds of endangered, rare, ancient and wild
Citrus species (Malik and Chaudhury 2006; Lambardi et al.
2007; Hamilton et al. 2009). Encapsulation–dehydration has
been used notably for cryopreserving protocorms of Celisos-
toma areitinum, a rare Thai orchid (Maneerattanarungroj et
 BIOTECHNOLOGIES FOR CONSERVING BIODIVERSITY
al. 2007), and shoot tips of the endemic endangered plant
Centaurium rigualii (Gonzalez-Benito and Perez 1997).
Turner et al. (2001) and Tanaka et al. (2009) report
successful cryopreservation of shoot tips of endangered
Australian and Japanese species, respectively, using the
vitrification technique. The droplet–vitrification technique
has been used for freezing shoot tips of wild potatoes
(Yoon et al. 2007) and of wild relatives of Diospyros (Niu et
al. 2009).
Large-Scale Utilization of Cryopreservation
for Germplasm Conservation
Even though its routine use is still limited, there is a
growing number of genebanks and botanic gardens 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. The National Center for Genetic
Resources Preservation (NCGRP; Fort Collins, CO) con-
serves 43,400 accessions over the vapours of liquid
nitrogen (Walters 2010, personal communication). The
National Bureau for Plant Genetic Resources (NBPGR;
New Delhi, India) stores 1,200 accessions from 50 different
species, consisting mainly of endangered medicinal plants
(Mandal 2000). This technique is also used in several
botanic gardens. More than 110 accessions of rare or
threatened species are stored under cryopreservation at the
Kings Park and Botanic Garden in Perth, Australia
(Touchell and Dixon 1994; see also http://www.bgpa.wa.
gov.au/). In the USA, the Center for Conservation and
Research of Endangered Wildlife at the Cincinnati Zoo and
Botanical Garden conserves several cryopreserved collec-
tions, including collections of seeds of regional endangered
species (Pence 1991), of endangered plant tissues and of
spores and tissues of Bryophytes and Pterydophytes (http://
www.cincinnatizoo.org/earth/CREW/frozengarden.html).
Cryopreservation is also applied to intermediate seeds
which are tolerant to freezing. Cryopreserved collections of
coffee seeds are being established in Tropical Agricultural
Research and Higher Education Center (CATIE; Cañas,
Guanacaste, Costa Rica) and in IRD (Montpellier, France),
using a protocol including after controlled dehydration and
freezing (Dussert and Engelmann 2006). In France, in the
framework of a national grape genetic resources conserva-
tion project, seeds of several hundred accessions are being
cryopreserved after partial desiccation (Dussert 2010,
personal communication).
In the case of dormant buds, the 2,200 accessions of the
US apple germplasm field collection are duplicated under
cryopreservation (Forsline et al. 1999), as is the case for the
420 accessions of the mulberry field collection maintained
at the National Institute of Agrobiological Resources
(Yamagata, Japan; Niino 1995). Dormant buds of more
than 440 European elm accessions are conserved in liquid
nitrogen by Afocel (Bordeaux Nangis, France; Harvengt et
al. 2004), and research is under way in France (IRD) and
the USA (NCGRP) for grape germplasm conservation.
Breeders routinely store pollen in liquid nitrogen in the
framework of their improvement programmes (Towill and
Walters 2000). Pollen, which is an interesting material for
genetic resource conservation of various species, is stored
with this aim 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 (Bangalore, India) conserves
pollen of 600 accessions belonging to 40 species from 15
different families, some of which have been stored for more
than 15 yr (Ganeshan and Rajashekaran 2000). In the USA,
the NCGRP conserves pollen of 13 pear cultivars and 24
Pyrus species (Reed et al. 2000). In China, pollen of more
than 700 accessions of traditional Chinese flower species is
conserved under cryopreservation (Li et al. 2009).
Cryopreservation is also applied to biotechnology prod-
ucts. More than 1,000 callus strains of species of pharma-
ceutical interest are stored at −196°C in the UK (Spencer
1999), as well as several thousand conifer embryogenic cell
lines employed in large-scale clonal planting programmes
in Canada (Cyr 2000). In France, cryopreservation is
systematically employed for storing all the new embryo-
genic cell lines of coffee and cacao produced by the
Biotechnology Laboratory of the Nestlé Company located
in Notre Dame d’Oé, France (Florin et al. 1999) and the
banana lines produced in Vitropic, a private tissue-culture
laboratory based in Saint-Mathieu de Tréviers, France.
Embryogenic cultures of around 80 oil palm accessions
have been cryopreserved and stored at IRD (Dumet 1994).
Finally, cryopreservation is being applied in genebanks
for long-term storage of genetic resources of vegetatively
propagated species, using apices sampled from in vitro
plants. The plant for which its development is the most
advanced is potato, since more than 1,000 old potato
varieties are cryostored in Gatersleben, Germany at the
Leibnitz Institute of Plant Genetics and Crop Plant
Research (Keller et al. 2005, 2006) and more than 200
accessions at the International Potato Center (Lima, Peru;
Golmirzaie and Panta 2000). A duplicate of around 100
accessions of the Pyrus field collection National Clonal
Germplasm Repository (NCGR; Corvallis, OR) is cryo-
stored at NCGR, with another duplicate at the NCGRP
(Reed et al. 2000). In Korea, two cryopreserved collections
of Allium have been established, which comprise a total of
more than 800 accessions (Kim et al. 2009). Finally,
 ENGELMANN
cryopreserved collections are under development for long-
term storage of tropical plants: 630 banana accessions have
been cryopreserved at the INIBAP International Transit
Center, Leuven, Belgium (Panis et al. 2007) and 540 cassava
accessions at the International Center for Tropical Agricul-
ture (CIAT; Cali, Colombia; Gonzalez-Arnao et al. 2008).
The large-scale utilization of cryopreservation implies a
scaling up of material to handle and to store 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. In 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 the plant
material, which are susceptible of inducing modifications in
cryopreserved cultures and regenerated plants. It is thus
necessary to verify that the genetic stability of the
cryopreserved material is not altered before routinely using
this technique for the long-term conservation of plant genetic
resources. There is no report of modifications at the
phenotypical, biochemical, chromosomal or molecular level
which could be attributed to cryopreservation (Engelmann
1997b, 2004). Recent studies comparing the vegetative and
floral development in the field of plants originating from
control and cryopreserved material performed with several
species including oil palm (Konan et al. 2007), potato
(Mix-Wagner et al. 2003), sugarcane (Gonzalez-Arnao
1996) and banana (Côte et al. 2000) did not reveal any
differences in the characters studied.
The few studies performed on the cost of cryopreserva-
tion confirm the interest of this technique from a financial
perspective, in view of its low utilization cost. Hummer and
Reed (2000) indicate that, at the NCGR, the annual 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 (per-
sonal communication) evaluates the annual maintenance
cost of CIAT’s cassava collection, which includes 5,000
accessions, at around US $5,000 under cryopreservation,
against US $30,000 under in vitro slow growth storage.
More recently, a detailed study compared the costs of
maintaining one of the world’s largest coffee field collec-
tions with those of establishing a coffee cryocollection at
CATIE in Costa Rica (Dulloo et al. 2009). The results
indicate that cryopreservation costs less (in perpetuity per
accession) than conservation in field genebanks. A com-
parative analysis of the costs of both methods showed that
the more accessions there are in cryopreservation storage,
the lower the per accession cost. In addition to cost, the
study examined the advantages of cryopreservation over
field collection and showed that for species that are difficult
to conserve using seeds and that can only be conserved as
live plants, cryopreservation may be the method of choice
for long-term conservation of genetic diversity.
Additional Uses of Cryopreservation
Recently, cryopreservation has been used for cryotherapy, i.e.
for eliminating viruses from infected plants, as a substitute or
in complement to classical virus eradication techniques such
as meristem culture and cryotherapy (Wang et al. 2008). In
cryotherapy, plant pathogens such as viruses, phytoplasmas
and bacteria are eradicated from shoot tips by exposing them
briefly to liquid nitrogen. Uneven distribution of viruses and
obligate vasculature-limited microbes in shoot tips allows
elimination of the infected cells by injuring them with the
cryotreatment and regeneration of healthy shoots from the
surviving pathogen-free meristematic cells. Thermotherapy
followed by cryotherapy of shoot tips can be used to enhance
virus eradication. Cryotherapy of shoot tips is easy to
implement. It allows treatment of large numbers of samples
and results in a high frequency of pathogen-free regenerants.
Difficulties related to excision and regeneration of small
meristems are largely circumvented. To date, severe pathogens
in banana (Musa spp.), Citrus spp., grapevine (Vitis vinifera),
Prunus spp., raspberry (Rubus idaeus), potato (Solanum
tuberosum) and sweet potato (Ipomoea batatas) have been
eradicated using cryotherapy. These pathogens include nine
viruses (banana streak virus, cucumber mosaic virus, grape-
vine virus A, plum pox virus, potato leaf roll virus, potato virus
Y, raspberry bushy dwarf virus, sweet potato feathery mottle
virus and sweet potato chlorotic stunt virus), a sweet potato
little leaf phytoplasma and a bacterium, Huanglongbing.
Cryopreservation: Progress and Prospects
Even though cryopreservation is still routinely employed in
a limited number of cases, the development of the new
vitrification-based freezing techniques has made its appli-
cation to a broad range of species possible (Engelmann
2004). An important 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.
Another one is their broad applicability, which is of
particular relevance to conservation of wild species, for
which large amounts of genetic diversity need to be
conserved. For many vegetatively propagated species,
cryopreservation techniques are sufficiently advanced to
envisage their immediate utilization for large-scale applica-
tion, and the number of cases where it is used routinely is
increasing steadily. Research is much less advanced for
 BIOTECHNOLOGIES FOR CONSERVING BIODIVERSITY
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 to increase the applicability of cryopreservation
techniques to recalcitrant species. In addition, research is
actively performed by various groups in universities,
research institutes, botanic gardens and genebanks world-
wide to improve 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. In this regard, it is interesting to
mention that an EU-funded COST (European Cooperation
in the field of Scientific and Technical Research) Action
(COST 871: Cryopreservation of crop species in Europe)
has been initiated recently. This Action aims notably at
improving fundamental knowledge about cryoprotection
through the determination of physicobiochemical changes
associated with tolerance towards cryopreservation and at
developing and applying new cryopreservation protocols.
For additional information, see http://www.biw.kuleuven.
be/dtp/tro/cost871/Home.htm. It can thus be realistically
expected that in the coming years, our understanding of the
biological mechanisms involved in cryopreservation will
increase and that cryopreservation will become more
frequently employed for the long-term conservation of
plant genetic resources.
Conclusion
In this paper, we have presented the new possibilities
provided by biotechnologies for improving ex situ conser-
vation of plant biodiversity in genebanks and botanic
gardens. During recent years, dramatic progress has been
made with the development of new conservation techniques
for non-orthodox and vegetatively propagated species,
especially in the area of cryopreservation, and the current
ex situ conservation concepts should be modified accord-
ingly to accommodate these technological advances.
It is now well recognised that an appropriate conserva-
tion strategy for a particular plant genepool requires a
holistic approach, combining the different ex situ and in situ
conservation techniques available in a complementary
manner. In situ and ex situ methods, including a range of
techniques for the latter, are options available for the
different genepool elements. Selection of the appropriate
methods should be based on a range of criteria, including
the biological nature of the species in question, practicality
and feasibility of the particular methods chosen (which
depend on the availability of the necessary infrastructures),
as well as the cost-effectiveness and security afforded by their
application. As already mentioned in this paper, the comple-
mentarity of genebank and botanic conservation should be
fully recognised and capitalized upon to optimize plant
biodiversity conservation. Considerations of complementarity
with respect to the efficiency and cost-effectiveness of the
various conservation methods chosen are also important. In
many instances, the development of appropriate complemen-
tary conservation strategies will still require further research to
define the criteria, refine the methods and test their application
for a range of genepools and situations. In this context, it is
important to stress that the new, efficient in vitro conservation
techniques developed are not seen as replacements for
conventional ex situ approaches. They offer genebank and
botanic garden curators additional tools to allow them to
improve the conservation of germplasm collections placed
under their responsibility.
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