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Conservation In vitro of threatened plants—Progress in the past decade

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In Vitro Cell. Dev. Biol.—Plant 42:206–214, May– June 2006 DOI: 10.1079/IVP2006769
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CONSERVATION IN VITRO OF THREATENED PLANTS – PROGRESS IN THE PAST DECADE

VISWAMBHARAN SARASAN*, RYAN CRIPPS, MARGARET M. RAMSAY, CAROLINE ATHERTON, MONICA MC MICHEN,
GRACE PRENDERGAST, AND JENNIFER K. ROWNTREE

Micropropagation Unit, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK

(Received 22 August 2005; accepted 13 March 2006; editor P. Lakshmanan)

Summary
In vitro techniques have found increasing use in the conservation of threatened plants in recent years and this trend is
likely to continue as more species face risk of extinction. The Micropropagation Unit at Royal Botanic Gardens, Kew, UK
(RBG Kew) has an extensive collection of in vitro plants including many threatened species from throughout the world. The
long history of the unit and the range of plants cultured have enabled considerable expertise to be amassed in identifying
the problems and developing experimental strategies for propagation and conservation of threatened plants. While a large
body of knowledge is available on the in vitro culture of plants, there are limited publications relating to threatened plant
conservation. This review highlights the progress in in vitro culture and conservation of threatened plants in the past
decade (1995 – 2005) and suggests future research directions. Works on non-threatened plants are also included wherever
methods have applications in rare plant conservation. Recalcitrant plant materials collected from the wild or ex situ
collections are difficult to grow in culture. Different methods of sterilization and other treatments to establish clean
material for culture initiation are reviewed. Application of different culture methods for multiplication, and use of
unconventional materials for rooting and transplantation are reviewed. As the available plant material for culture initiation
is scarce and in many cases associated with inherent problems such as low viability and endogenous contamination,
reliable protocols on multiplication, rooting, and storage methods are very important. In this context, photoautotrophic
micropropagation has the potential for development as a routine method for the in vitro conservation of endangered plants.
Long-term storage of material in culture is challenging and the potential applications of cryopreservation are significant in
this area. Future conservation biotechnology research and its applications must be aimed at conserving highly threatened,
mainly endemic, plants from conservation hotspots.
Key words: threatened plants; sterilization; in vitro conservation; recalcitrant taxa; autotrophic micropropagation;
cryopreservation.

Introduction
The world’s biodiversity is declining at an unprecedented rate.
During the period 1996–2004, a total of 8321 plant species were
added to the The International Union for the Conservation of Nature
and Natural Resources (IUCN) Red List of Threatened Species
(IUCN, 2004). The main purpose of the IUCN Red List is to
catalogue and highlight those taxa that are facing a higher risk of
global extinction (i.e. those listed as critically endangered,
endangered, and vulnerable). During this period, there was also
an increase of over 60% in the number of plants recorded as
critically endangered. This is alarming and immediate conservation
measures are required to safeguard many of these species.
A plant biodiversity hotspot is an area featuring an exceptional
concentration of endemic species and undergoing significant habitat
loss. A hotspot must contain at least 0.5% or 1500 of the world’s
estimated 300 000 plant species as endemics. Previous work has
estimated that 44% of all plant species are found within 25
biodiversity hotspots (Myers et al., 2000). Sixteen of these are
*Author to whom correspondence should be addressed: Email v.sarasan@
kew.org
situated in the tropics. This means that the majority of hotspots are
located in developing countries, where threats to their survival are
greatest and conservation resources most scarce (Myers et al.,
2000). Tropical Andes, Sundaland, Mediterranean Basin, and
Madagascar (representing Madagascar, Mauritius, Reunion,
Seychelles, and Comores) are the major hotspots. Concentrating a
large proportion of conservation support within these hotspots would
significantly reduce the mass extinction of species that is currently
occurring (Myers et al., 2000).
Although species conservation is achieved most effectively
through the management of wild populations and natural habitats
(in situ conservation), ex situ techniques can be used to complement
in situ methods and, in some instances, may be the only option for
some species (Maunder et al., 1998; Ramsay et al., 2000). The
importance of ex situ conservation has gained international
recognition with its inclusion in Article 9 of the Convention on
Biological Diversity (CBD) (Glowka et al., 1994) and in Target 8 of
the Global Strategy for Plant Conservation (www.bgci.org.uk/files/7/
0/global_strategy.pdf). The world’s 1600 botanic gardens and
arboreta cultivate an estimated 80 000 or more than one-third of the
world’s flowering plants (UNEP, 1995). Many of these species were
not collected specifically for conservation purposes and do not

206
 CONSERVATION IN VITRO OF THREATENED PLANTS
necessarily contain a large amount of genetic diversity. However
their cultivation, the increased establishment and maintenance of
seed banks and use of in vitro techniques have enabled botanic
gardens to contribute to the successful conservation of a number of
threatened plants (Maunder et al., 2001; Ramanatha Rao and
Hodgkin, 2002; Pritchard et al., 2004).
In vitro techniques have been found to be useful in the
propagation of a large number of threatened plants (AmoMarco and
Lledo, 1996; Dhar et al., 2000) and the Micropropagation Unit at
RBG Kew has been involved in the propagation and maintenance of
more than 3000 plant taxa, from all over the world, for over 30 yr.
Our facility holds representatives of almost all major taxonomic
groups of plants, including bryophytes, ferns, grasses, orchids,
carnivorous plants, succulents, palms, geophytes, shrubs, and
woody plants. Most of the taxa held are threatened, many have an
IUCN rating, some are classified as extinct in the wild and a
significant proportion is from major conservation hotspots. The in
vitro collection at RBG Kew has a dual purpose; the propagation of
plants for the living collections of the garden and the rearing of
plants specifically for conservation. Madagascan and UK orchids, as
well as UK threatened bryophytes are currently propagated in vitro
to support specific conservation projects.
Other botanic gardens and research groups are also involved
in the in vitro conservation of plants. The following information
was collected from the respective websites of these institutes.
The laboratory at Kings Park & Botanic Garden, Perth, Australia
(http://www.kpbg.wa.gov.au) specializes in the conservation of
threatened flora of Western Australia and has developed
techniques for the micropropagation of over 200 species
representing 33 families of Australian plants. Researchers at
Mount Annan Botanic Gardens, New South Wales, Australia
(http://www.rbgsyd.nsw.gov.au/mount_annan_botanic_garden) have
micropropagated 17 threatened Grevillea species. The Plant
Conservation Research Group at University of Abertay, UK is
applying micropropagation and other conventional propagation
techniques for the conservation of Scottish native plants (http://
scieng.tay.ac.uk/plant/). The in vitro conservation of c. 30 species
of threatened American plants is currently being investigated at
Cincinnati Zoo and Botanic Garden, Ohio, USA. (http://www.
cincyzoo.org) in collaboration with eight other institutions. The
Tropical Botanic Gardens and Research Institute in Kerala, India
has studied the in vitro conservation of 46 medicinal plants and
30 orchids from the conservation hotspot of Western Ghats
(www.tbgri.org). A laboratory for the in vitro conservation of
tropical bryophytes has recently been established at the
Universidad de Magallanes, Punta Arenas, Chile (J. G. Duckett,
personal communication).
Although the application of in vitro methods for threatened plant
conservation is gaining momentum, the use of conserved germplasm
in further research and dissemination of this knowledge through
publication in international journals is not encouraging. For
example, every year about a quarter of the articles published in
leading genetic resources and plant breeding/improvement journals
such as Crop Science, Euphytica, Plant Breeding, and Theoretical
and Applied Genetics were on the collection, conservation, and
diversity of genetic resources of crop plants (Dudnik et al., 2001).
However, published articles on similar studies of threatened plants
in international journals are limited and a direct comparison is
difficult.
207
The role of in vitro methods in the conservation of threatened
plants has been previously reviewed (Fay, 1992; Benson, 1999) and
this paper aims to highlight important developments in the past
decade. Developments of techniques using both threatened and
non-threatened plants, which may have conservation applications,
are discussed. Relevant examples concerning the initiation of plant
tissue into culture, multiplication, rooting, weaning, storage
(including cryopreservation), and future research direction, are
presented.
Collection and Culture Initiation
The process of initiation of plant material into culture usually
occurs in the laboratory after collection in the field. However,
techniques have been developed for ‘in vitro collection’ or initiation
directly in the field (Pence, 2005). Use of in vitro collecting has
been reported for crop plants (Engelmann, 2002; Silvana Alvarenga
et al., 2002), and for the collection of buds and leaves of threatened
plants (Pence et al., 2002). There is great potential for its wider
application in threatened plant conservation (Pence, 2005). In vitro
collection or initiation of plant material in the field allows
establishment of cultures of seeds with short viability periods or
vegetative material that is difficult to transport because of the
remoteness of the source plant. Specialized collecting techniques
and media are, therefore, necessary to transport such plant material.
Contamination, of both exogenous and endogenous origin, is a
major obstacle for in vitro culture of plants. It is particularly
important when dealing with threatened taxa for which the source
material is often limited and usually located in the wild. Plant
material is routinely initiated into axenic culture via a variety of
sterilization procedures. These vary considerably from single-step
to more complex protocols and utilize a huge variety of chemicals
depending on the nature of tissue used. Sodium dichloroisocyanu-
rate (SDICN) has proved to be a reliable sterilizing agent (Parkinson
et al., 1996; Niedz and Bausher, 2002) and has been used
effectively at RBG Kew to initiate plants from many taxonomic
groups into axenic culture (Table 1). Phytotoxicity of the compound
is low and appears to act preferentially on old leaves and cut
surfaces, making it ideal for use where plant material is limited
(Parkinson et al., 1996).
Contamination from internal sources can be potentially serious in
culture as many plants harbor endophytic bacteria or fungi. Pence
(1996) reported the reduction of endophytic contamination to a
workable level by the incorporation of fungicides and antibacterial
compounds in the culture medium. In addition, the isothiozolone
biocide Plant Preservative Mixture (PPM) has been successfully
used at concentrations between 0.5 and 4 ml l21 for a range of plant
species at RBG Kew. Watt et al. (2003) reported control of
endogenous bacteria in Eucalyptus species by spraying long,
leafless stems with 70% (v/v) ethanol and the inclusion of 1 g l21
calcium hypochlorite in the first culture medium. Submersion of
plant material in low concentrations of SDICN for long periods of
time has also been reported to control endophytic contamination
(Niedz and Bausher, 2002). A similar approach, using very low
concentrations of sodium hypochlorite, has been used successfully
to eliminate contamination in aquatic plants collected from muddy
ponds and highly polluted waterways (Prakash Lakshmanan,
personal communication).
208 SARASAN ET AL.
TABLE 1
RANGE OF TAXA SUCCESSFULLY INITIATED INTO AXENIC CULTURE AT ROYAL BOTANIC GARDEN, KEW (RBG KEW) USING SODIUM
DICHLOROISOCYANURATEw (SDICN)
Plant category Taxa
Bryophyte Orthodontium gracile
Bryophyte Lunularia cruciata
Bryophyte Micromitrium tenerum
Succulent plant Aloe calcairophila
Palm Hyophorbe lagenicaulis
Bulbous plant Paramongia weberbaurii
Tree species Ramosmania rodriguesii
Hairy perennial Cylindrocline lorencei
Cactus Blossfeldia liliputana
Aroid Amorphophallus titanum
w
Concentration used equals 0.5% w/v SDICN unless otherwise stated.
x
0.1% w/v SDICN.
y
1% w/v SDICN.
z
Percentages not available.
Tissue and medium browning is a major barrier for successful
culture initiation. Successful introduction to culture has been
hampered by exudation of toxic materials from cut surfaces of the
explant in a large number of threatened plants at RBG Kew.
However, this problem has been successfully controlled in many
plant species with medium additives such as activated charcoal
(e.g. Pelargonium sp.) and PVP (e.g. Aloe sp.) (RBG Kew,
unpublished results). Lethal browning of explants of the critically
endangered Western Australian shrub Symonanthus bancroftii was
prevented by pretreatment with an antioxidant solution of potassium
citrate/citric acid [0.1% (w/v) in a 4:1 ratio] (Panaia et al., 2000).
Similarly, excessive browning and necrosis of young shoots of the
endemic tree Calophyllum apetalum was prevented by transferring
nodes of 6 – 8-wk-old shoots to fresh medium twice weekly (Nair and
Seeni, 2003). With this treatment, however, 1 –2-wk-old fresh
growth of C. apetalum did not survive the culture cycle as browning
could not be controlled. Pretreating explants with dimethyl
sulfoxide (DMSO) was found to be effective in controlling medium
darkening and eventual callus formation in Mussaenda philippica
(Bhattacharya and Dey, 1998). Benson et al. (2000) found reduced
hyperhydricity in clones of seed-derived explants of Primula scotica
by frequent subculturing and growth on plant-growth-regulator
(PGR)-free medium.
The lack of published methods for in vitro propagation of related
taxa and the limited amount of experimental plant material make
the choice and development of initial culture medium for threatened
plants somewhat arbitrary (Krogstrup et al., 2005). Once initiated
into axenic culture, growth and multiplication in vitro can remain
problematic, requiring the development of novel treatments. In the
initial stage, slow growth of cultures has been observed in some
critically endangered tree species such as Medusagyne oppositifolia
at RBG Kew.
Most tropical orchids germinate readily in axenic culture (Seaton
and Ramsay, 2005); however, many temperate orchids do not and
techniques such as mycorrhiza-assisted germination (Rasmussen,
2002) have been developed for such species. The majority of this
work involves terrestrial species (Batty et al., 2001; Stewart and
Zettler, 2002; Esitken et al., 2005), but investigations with
Immersion
Material time (min) Survival (%)
Leafy gametophore 2 –z
Gemmaex 2 –z
Sporophytey 3 –z
Axillary buds 20 69
Large seed 40 100
Thin seed 40 89
Leaf 90 85
Stem piece 70 71
Microscopic seeds 20 100
Large seed 45 100
epiphytic species are increasing (Zettler et al., 1998). Mycorrhizal
specificity and performance were compared in Tolumina variegata
and Ionopsis urticularioides, two phylogenically related orchids from
Puerto Rico. Symbiotic seed germination experiments showed that
T. variegata establishes its association with several fungal
symbionts while I. urticularioides was more specific (Otero et al.,
2004). In addition to the work on natural symbionts, fungal isolates
from non-orchid sources have been shown to stimulate seed
germination (Salman et al., 2002). At RBG Kew, problems of orchid
seed dormancy have been overcome by germinating seeds from
immature capsules. These plants have been used for reintroduction
and cryopreservation. Over 250 orchid genera have been
successfully propagated at RBG Kew including species from
Angraecum, Paphiopedilum, Phragmipedium, and Cypripedium,
many of which are threatened in the wild.
Culture Maintenance and Multiplication
Once successfully initiated into culture, induction, and
multiplication of shoots can be difficult for some species,
particularly woody plants. Many plant species have very specific
in vitro requirements for multiplication and, therefore, substantial
variation in culture medium formulations exists. In general, mineral
salt requirement varies from one plant group to the other. Similarly,
specific growth hormones or supplements greatly enhance
regeneration and growth in many cases. Cultures can be maintained
using liquid or solid medium under a variety of conditions,
depending on the purpose of the collection and individual culture
requirements of the plant. The following section lists different
supplements and methods used for the successful culture of some
threatened plants.
Multiplication and rooting of the endangered tree Ginkgo biloba
was aided by the addition of endosperm extract obtained from
mature seeds of the same species (Tommasi and Scaramuzzi, 2004).
Maximum shoot multiplication in an endangered Chinese aloe was
achieved with the inclusion of 6-benzyladenine (BA),
a-naphthalene acetic acid (NAA), and 0.6 g l21 polyvinylpyrroli-
done (PVP) in the medium (Liao et al., 2004). Phloroglucinol in the
 CONSERVATION IN VITRO OF THREATENED PLANTS
culture medium increased shoot multiplication of an endemic
Spanish threatened plant Minuartia valentina (Ibanez and
Amo-Marco, 1998). Kodja et al. (1997) reported improved green
shoot formation from callus cultures of Tabernaemontana
persicariaefolia, a threatened species endemic to Mascarene
Islands, in the presence of difluoromethylornithine (DFMO), a
metabolic inhibitor of polyamine biosynthesis.
The threatened cycad Ceratozamia euryphyllidia was multiplied
by somatic embryogenesis (Chavez et al., 1998) using mature leaf
explants. Further growth of germinated embryos occurred on a
medium containing 0.5% activated charcoal devoid of PGRs.
Cultures of several cycads retained their embryogenic competence
for more than 11 yr in culture (Litz et al., 2004). Research into this
group of plants is very important as c. 52% of cycads were
categorized as threatened by IUCN in 2004.
Somatic embryos have been induced directly from longitudinally
cut seedling explants in the critically endangered bottle palm,
Hyophorbe lagenicaulis (Sarasan et al., 2002). This method could be
used for the rescue of one of the world’s rarest palms, Hyophorbe
amaricaulis, of which only a single individual in Mauritius remains.
In contrast, the propagation of the endangered Aloe polyphylla was
achieved via callus-mediated shoot regeneration (Ramsay and
Gratton, 2000; Abrie and van Staden, 2001). Callus-mediated
regeneration from juvenile explants was also used as a method for
rapid multiplication of the critically endangered tree Syzygium
travancoricum (Anand et al., 1999) and the threatened tree Maclura
tinctoria (Gomes et al., 2003).
The majority of orchid micropropagation at RBG Kew is based on
in vitro seed germination to maintain genetic diversity, with a few
investigations producing protocorm-like bodies (PLBs) from somatic
or meristematic regions to provide material for molecular studies. In
the past decades, the majority of orchid micropropagation research
has concentrated on vegetative propagation such as mericloning.
Progress made in orchids on production of PLBs in horticulturally
important genera such as Dendrobium, Cymbidium, and Phalae-
nopsis have wider applicability to threatened taxa. PLBs have the
advantage that they can be produced from almost any part of the
plant and are usually formed directly on the tissue rather than
through a callus stage. In Phalaenopsis, PLB formation can be
induced by maltose and sorbitol (Tokuhara and Mii, 2003). The
polyamine putrescine has also been found to be effective at
inducing PLB production in orchids (Saiprasad et al., 2004), while
methyl jasmonate was reported to increase PLB formation from
shoot cultures of Cymbidium species (Shimasaki et al., 2003). Maize
extract has been shown to induce high frequency plantlet
regeneration from PLBs in Doritaenopsis sp. (Rahman et al.,
2004). The addition of sucrose and jasmonic acid to orchid media
has been shown to induce tuber formation in the terrestrial orchid
Pterostylis sanguinea from southwestern Australia (Debeljak et al.,
2002).
Liquid culture systems have proved to be highly efficient for the
regeneration of a number of species. The bromeliad Cryptanthus
sinuosus was successfully propagated in liquid Murashige and
Skoog (MS) medium without the addition of growth regulators
(Arrabal et al., 2002), and the rare Rhododendron maddenii was
micropropagated from cotyledonary nodal segments of 7-wk-old
seedlings in liquid Anderson medium, with 7 mg l21 (34.4 mM) 2-
isopentyladenine (2iP) added for maximum multiplication (Kumar
et al., 2004). Bioreactor cultures established from adventitious roots
209
of Eleutherococcus koreanus in 1/3-strength MS medium
supplemented with 60 g l21 sucrose allowed harvesting of
adventitious roots, embryos and plants 12 wk after initiation (Park
et al., 2005). Bioreactor culture systems have also been utilized for
the mass production of the moss Physcomitrella patens in Knops
medium (Hohe and Reski, 2002). Bioreactor technology may prove
to be a useful tool for mass production of orchid PLBs (Young et al.,
2000). Mass multiplication in liquid cultures and extraction of
active principles in vitro have been used in threatened species of
Eleutherococcus, reducing pressure on the wild population (Choi
et al., 2002). Arnica chamissonis was confirmed as a pharmaceutical
substitute for the medicinal plant A. montana. A plantation was
developed using micropropagated plants of A. chamissonis for the
commercial production of medicinal compounds (Cassells et al.,
1999). Producing plants in large numbers for reintroduction into the
wild (Misic et al., 2005) and long-term maintenance of cultures
without losing regeneration capacity as noticed in cycads (Litz et al.,
2004) are integral parts of in vitro conservation.
Rooting and Weaning
Successful rooting and weaning of plants are fundamental to the
in vitro culture of threatened species for collection and
conservation. Once rooted and weaned, plants are normally used
for re-establishment programs or for botanic garden living
collections.
Rooting can be particularly problematic for in vitro-reared woody
and recalcitrant taxa, requiring novel approaches, and methods.
Sarasan (2003) reported the use of supporting materials such as
Florialite and Sorbarods to improve rooting and the quality of roots
produced from the critically endangered tree Trochetiopsis ebenus.
The addition of activated charcoal in the medium and the use of
Suncaps to seal containers were shown to improve rooting in an
endemic Mexican cactus Ariocarpus kotschoubeyanus (Moebius-
Goldammer et al., 2003). Rooting in these plants was improved
primarily due to the increased gaseous exchange.
Panaia et al. (2000) observed that paclobutrazol (PBZ) improved
desiccation tolerance and survival rate of micropropagated critically
endangered S. bancroftii. Recently, Sarasan et al. (2002) reported
that in vitro germinated seedlings of H. lagenicaulis could be
successfully hardened-off by treating with PBZ and by improving air
exchange. Combining ex vitro rooting and hardening into a single
step reduced the cost and time required to wean the rare Australian
perennial herb Stackhousia tryonii (Bhatia et al., 2002) and the
threatened rhoeophytic woody plant Rotula aquatica (Martin, 2003).
Orchids raised in vitro have been successfully reintroduced into the
wild including Anacamptis laxiflora (Wood and Ramsay, 2004),
Cypripedium calceolus (Ramsay and Stewart, 1998) and Vanda
spathulata (Decruse et al., 2003). Similar trials are currently
underway at RBG Kew to establish protocols for the weaning of
bryophytes.
Addition of a carbon source is still a primary requirement in
conventional micropropagation systems for most plants, although it
is unnecessary for most bryophytes (Duckett et al., 2004). However,
in vitro culture without a carbon source has added benefits and been
used in a number of plants in the past decade. Zobayed et al. (2004)
reviewed the progress achieved in autotrophic micropropagation
systems in economically important plants with special reference to
the development and utilization of large culture vessels. They
210 SARASAN ET AL.
discussed choice of supporting medium, ventilation system,
planting density, vessel volume, sterilization procedure of large
culture vessels, and problems of achieving plant uniformity. In
comparison with plantlets produced by conventional systems, those
produced by photoautotrophic systems with a sugar-free medium
showed, in many cases, better growth, higher quality, lower
contamination rate in vitro, and higher percentage survival ex vitro
(Aitken-Christie et al., 1995; Zobayed et al., 2004). Application of
forced ventilation coupled with carbon dioxide enrichment and
increased light intensity can be effective in achieving a high-
efficiency autotrophic system. Forced ventilation allows the removal
of ethylene from culture vessels, which improved the growth of slow-
growing plants, especially recalcitrant species. Contamination
control, difficulty in rooting and weaning can all be managed
effectively if easy-to-apply methods for forced ventilation and
carbon dioxide enrichment are available. The relatively simple
growing conditions in autotrophic systems may also contribute to a
reduction in somaclonal variation. Thus, if good multiplication rates
can be achieved, this method should be explored to generate quality
plants for reintroduction and long-term conservation of threatened
species.
Storage of Cultures
The long-term maintenance of in vitro plant collections can be
expensive, requiring high labor input, and controlled growth
conditions. Also, long-term in vitro maintenance may not be
desirable to maintain genetic fidelity of the species. Increasingly,
methods are being sought for the space-efficient long-term storage
of plant collections. This is clearly illustrated by the development of
seed banks, cryopreservation, and slow-growth storage using
different propagules, including synthetic seeds. The following
section focuses on recent developments in these fields.
The banking of vegetative material, cell cultures, seeds, spores,
and DNA at ultra-low temperature, at or near the temperature of
liquid nitrogen (LN; 21968C), provides a safe and cost-effective
method for the long-term storage of genetic resources. The potential
value of cryopreservation as a conservation tool has been extolled
by many authors (Stacey et al., 1999; Engelmann and Dussert,
2000; Engelmann, 2004), and it is currently the only available
technique for the safe long-term storage of species with recalcitrant
seeds or those that are vegetatively propagated (Engelmann and
Dussert, 2000).
Cryopreservation can be used on a wide range of vegetative
material such as shoot-tips, axillary buds, embryonic axes,
bryophyte and fern gametophytic material, somatic embryos, callus,
and suspension cultures. These potential explant sources permit
many possible applications for this technique. Indeed, cryopre-
servation is being used for the routine banking of germplasm in
some taxa (Stacey et al., 1999). Two key advances in methodology
have made this possible; the development of vitrification using
PVS2 solution (Sakai et al., 1990) and the encapsulation –
dehydration technique (Fabre and Dereuddre, 1990). Plant
vitrification solution 2 (PVS2) is a highly concentrated cryoprotec-
tive solution containing glycerol, ethylene glycol, and DMSO. This
solution allows cells to dehydrate sufficiently, permitting vitrifica-
tion during freezing, and subsequent steps in cryopreservation.
Encapsulation– dehydration involves encapsulation of plant
material in a protective matrix and its subsequent controlled
dehydration. Advantages of encapsulation include easier manipu-
lation of delicate tissues, and direct protection during dehydration
and thawing processes (Paul et al., 2000).
Due to inherent differences between taxa it is difficult to define a
single cryopreservation protocol with a wide application. It is
necessary to consider the ecological origin and nature of each
species to be treated (e.g. whether it is desiccation and/or freezing
tolerant and the type, size, condition, and culture of the explant) to
identify potentially useful cryopreservation methods (Turner et al.,
2000; Baek et al., 2003; Towill et al., 2004). Preculture with
abscisic acid (Pence, 2000; Bian et al., 2002; Burch and Wilkinson,
2002), specific sugars, and sugar alcohols (Suzuki et al., 1998;
Turner et al., 2001a, d; Schulte and Reski, 2004), cytokinins
(Turner et al., 2000, 2001c), low levels of DMSO (Decruse et al.,
1999; Sharma and Sharma, 2003; Schulte and Reski, 2004),
reduced levels of NH4þ and NO32 (Decruse et al., 2004) and low
temperature (Sharma and Sharma, 2003) have all been shown to
improve dehydration tolerance, cryoprotection, and/or recovery of
plants of threatened species.
The use of 0.75 M sucrose during the freezing protocol is well
documented and has been used successfully in a number of species
(González-Benito and Pérez, 1997; Decruse et al., 1999; Pence,
2001). Preculture of the endangered Australian plants Anigozanthus
and Conostylis, with high levels of glycerol significantly improved
survival rates (Turner et al., 2001b). Additional research on
Anigozanthus viridis revealed that the polyols, glycerol, sorbitol, and
inositol, resulted in higher survival rates than the sugars, sucrose,
glucose, trehalose, and raffinose, when used at the same osmolarity
(0.4 M), or at the equivalent concentration of total hydroxyl groups
(Turner et al., 2001a).
Whilst the use of PVS2 provides a good method for vitrification,
one of its primary components, DMSO, can be toxic. Turner et al.
(2001d) showed that it is possible to substitute DMSO with
propylene glycol during cryopreservation. Wilkinson et al. (1998)
presented an interesting modification to the encapsulation –
dehydration method, where shoot tips of the endangered species
Cosmos atrosanguineus were encapsulated on alginate-covered
paper strips to allow easier transfer of the samples and permit better
positioning of the explant. This method has been used successfully
for the cryopreservation of protonemata from the endangered
bryophyte Ditrichum cornubicum (Burch and Wilkinson, 2002) and
is currently being used for the cryopreservation of a wide range of
bryophyte species at RBG Kew.
Traditionally, samples are frozen at a controlled rate or plunged
directly into liquid nitrogen. They are recovered normally by
thawing either at ambient temperature or at 408C. Leunufna and
Keller (2003) presented an alternative method in which they used
the droplet method to cryopreserve apical buds. They found that
freezing apical meristems in droplets of cryoprotective solution or
PVS2 on small aluminum foil pieces increased survival and
regrowth in comparison to the normal vitrification protocol. This
success was, in part, attributed to more rapid and uniform heat
distribution in the small drops. Dumet et al. (2002) thawed
encapsulated Perlargonium meristems by dropping them directly
into 0.75 M sucrose-enriched liquid medium at 208C for 1 min,
yielding significantly higher survival rates than thawing at 408C or
at ambient temperature. Success was attributed to a reduced
thawing rate and possible additional osmoprotection during the
process.
 CONSERVATION IN VITRO OF THREATENED PLANTS
Slow-growth cultures. Plant tissue cultures can be stored for the
medium-term by maintaining them in a slow growth state (Mandal
et al., 2000; Negri et al., 2000). Iriondo and Perez (1996) stored
shoots of the endangered Spanish plant Centaurium rigualii at 58C
with a 16-h photoperiod, achieving a survival rate of 90% after 3 yr.
The orchid Ipsea malabarica, endemic to Western Ghats, India was
maintained successfully in a state of slow growth for 20 mo. on 1/2-
strength MS medium, without the addition of sucrose or PGRs
(Martin and Pradeep, 2003).
Production and storage of synthetic seeds via the encapsulation
of somatic embryos and other propagules is another option
(Standardi and Piccioni, 1998). Mandal et al. (2000) successfully
prepared synthetic seeds from axillary buds of different species of
basil (Ocimum spp.) for storage. Encapsulated protocorms of orchids
Agrostophyllus myrianthum, Cymbidium longifolium, Phaius
tankervillae, and Renanthera imschootiana were stored at 48C and
showed 70% viability after more than 6 mo. (Devi et al., 1998).
Somaclonal variation
Continued subculturing, treatment with PGR and other media
additives, and intervening callus phase can cause genetic changes
in plants and lead to progenies with altered characteristics, which is
commonly referred to as somaclonal variation. Somaclonal variation
is unpredictable in nature and can be both heritable (genetic) and
non-heritable (epigenetic) (Jain, 2001). Genetic variation can
include cytological and chromosomal abnormalities in addition to
point mutations (Harding, 1999). It is a problem for all in vitro-
grown plants, but is of particular concern for conservation
collections, where production of clonal material is desired for re-
establishment programs. In an attempt to avoid mutations, Edson
et al. (1996) minimized application of PGR in the multiplication
protocol for an endangered North American perennial endemic
Hackelia venusta.
A number of screening methods to detect somaclonal variation in
plants is available. These include phenotype analysis, flow cytometry,
isozyme systems, and the use of molecular markers. Flow cytometry
techniques are widely used to track changes in ploidy levels and DNA
content, and its application has been demonstrated in Cymbidium
(Fukai et al., 2002). Somaclonal variation has been studied using
seven isozyme systems in the endangered Spanish plant C. rigualii
(Iriondo and Perez, 1996). In general, genetic fingerprinting
techniques based on polymerase chain reaction (PCR) and restriction
fragment length polymorphism (RFLP)-based screening techniques
are applied to rare and threatened species (Chase and Fay, 1997).
PCR-based techniques are also suitable for in vitro material of
threatened plants that is normally only maintained in relatively small
quantities. Wilkinson et al. (2003) used AFLPe for screening
cryopreserved clonal shoot material of the endangered C.
atrosanguineus. Methods such as these are more sensitive and
reproducible than phenotypic, karyological, or isozyme evaluation.
Whilst not recommended for conservation purposes, development
of somaclones with superior characteristics has the potential for the
production of medicinal compounds and thereby to reduce pressure
on wild populations. However, it must be ensured that somaclones
are used only in cultivation for multiplication for sustainable use
and not introduced into conditions where they may cross-pollinate
with wild populations. As yet the authors do not know of any
published research on the development of somaclones for improving
211
genetic diversity of endangered plants. But a well-monitored
program of generating populations with increased genetic variability
may yield beneficial results.
Conclusions
The use of in vitro techniques in germplasm conservation is
increasing, but there is great potential for further development. In
vitro culture allows for the provision of plant material for DNA
analyses, autecological studies, and commercial uses. Efficient
propagation protocols for threatened plants allow for the continuous
supply of large quantities of material with medicinal or food value
as demonstrated by Cassells et al. (1999) and Choi et al. (2002).
The development of successful storage methods enables the
establishment of extensive basal collections, with representative
genetic diversity. These collections include many species
threatened with imminent habitat loss (Ramsay and Dixon, 2003).
In addition, the culture of plants under axenic conditions reduces
the restrictions on quarantine requirements and international
movement of material. Collections of critically endangered plants
need to be maintained in simple media formulations. Endangered
plants could be maintained in culture or at ultra-low temperatures
through carefully managed procedures. Use of PGRs, which are
potential stimulators of somaclonal variation, need to be minimized
in plants with long life cycles, and robust screening methods should
be in place to eliminate undesirable somaclones.
The intrinsic value of plant biodiversity must be recognized and
its conservation prioritized. Areas of particular interest are the
development of in vitro collection techniques and sterilization
techniques, novel rooting and weaning procedures. Progress in the
cryopreservation of recalcitrant plants and the development of more
universal protocols is desirable. Prolonging the culture vigor and
avoiding somaclonal variation through autotrophic micropropaga-
tion should be considered, at least for woody/recalcitrant species, as
they often need prolonged and complex culture maintenance
conditions.
A large proportion of the critically endangered plants are in
developing countries. Increasingly, botanic gardens in developing
countries are establishing their own in vitro culture facilities and
this will facilitate the rescue and recovery of most threatened plants
in the future. Networking and exchanging information and material
between in vitro facilities in botanic gardens and other research
centers will be an important step for conservation programs to be
successful. Furthermore, the dissemination of practically useful in
vitro protocols is necessary to promote the continued development
and application of conservation techniques. Scientists and
conservationists need to work together more closely to develop
conservation programs for plants on the verge of extinction.
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