Biologicul Journal of the Linncun Society ( 1991) , 43: 3 1-42
4. In-vitro conservation
LYNDSEY A. WITHERS
International Board for Plant Genetic Resources (IBPGR), c/o FAO, 142 Via delle
Sette Chiese, 00145 Rome, Itab
In-vitro (tissue culture) techniques offer ways of overcoming serious problems in the conservation of
crop genetic resources. These primarily involve the use of slow growth and cryopreservation in
liquid nitrogen to store germplasm, but there are also important applications in other areas,
including germplasm collecting, multiplication and exchange. Slow growth techniques for mediumterm storage of cultures are relatively well developed and in-vitro active gene bank establishment is
feasible. Cryopreservation for long-term storage is possible for some materials but, in general,
requires further research and development. Among the aspects to be examined are the behaviour of
different culture systems when exposed to ultralow temperatures, crop-specific requirements and the
genetic stability of stored material.
KEY WORDS:-In-vitro
storage - crop genetic resources - cryopreservation - genetic stability.
CONTENTS
Introduction . . . . . . .
Methods of in-vitro storage
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Slow growth . . . . . .
Cryopreservation . . . . .
In-vitro conservation systems . . .
Complementary conservation strategies .
References
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INTRODUCTION
The storage of seeds under conditions of low moisture and low humidity meets
the conservation needs of most crop plants. Seeds that can survive under these
conditions for long periods of time are termed ‘orthodox’. There are, however,
two important categories of problem crop: recalcitrant seed-producing species
and vegetatively propagated crops whose conservation needs are not met in this
way. Examples of recalcitrant seed-producing crops are coconut, cacao,
rambutan, jackfruit and mango. Their seeds are typically large and fleshy. They
lack a natural dormancy mechanism and deteriorate rapidly, usually within
days or weeks of collecting.
Vegetatively propagated crops include a number of important staple foods in
developing countries, such as the root and tuber crops cassava, potato, yam, taro
and other aroids. Dessert and cooking bananas (Musa spp.) and several fruits
including citrus, apple, pear and Prunus spp. are also normally conserved
vegetatively in the form of clones. In some cases this is because of sterility. In
other cases it is to conserve gene combinations which would be lost by conversion
to seed.
0024-4066/9 1/050031
+ 12 $03.00/0
31
0 1991 The Linnean Society of London
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L. A. WITHERS
Problem crops are traditionally conserved in field gene banks. Whilst offering
a relatively simple solution to immediate conservation needs, field gene banks
present economic and practical drawbacks. They are expensive to maintain and
they are open to environmental threats which can result in the loss of valuable
germplasm to disease, fire or other natural disasters. Most importantly, they do
not offer safe long-term storage conditions comparable to base collections in seed
gene banks.
In-vitro (tissue culture) conservation has been proposed as a safer alternative
to the field gene bank (De Langhe, 1984; Withers, 1982, 1984, 1989). The
establishment of collections of miniature plants maintained under carefully
controlled conditions is conceptually simple but is not without its own problems.
In-vitro culture originated as a means of rapid clonal propagation, using
nutritional and environmental conditions conducive to rapid growth. Under
these conditions, cultures require transfer to fresh medium every few days or
weeks. At each transfer event, there is a risk of contamination with microbial
organisms. The transfer operation itself is relatively labour-intensive and
vigilance is needed to maintain adequately controlled culture conditions over
long periods of time. Therefore, in-vitro culture, in its conventional form, could
simply be seen as a parallel, yet more exacting, laboratory version of the field
gene bank.
When the phenomenon of genetic instability generated in vitro, so-called
‘somaclonal variation’ (Scowcroft, 1984), is taken into account, it could be
argued that in-vitro conservation might be more, not less, risky than the field
gene bank. The use of highly organized shoot or embryo cultures greatly reduces
the risk of somaclonal variation. Nevertheless, other measures must be taken to
minimize the overall level of risk and also of inputs to in-vitro culture to make i t
acceptable as a satisfactory alternative to the field gene bank.
METHODS OF IN-VITRO STORAGE
Slow growth
There are very few cases in which cultures grow sufficiently slowly for their
normal growth pattern to be suitable for storage. This is because mass
propagation culture conditions are normally designed to give a relatively rapid
rate of production of, for example, new shoots from which plantlets can be
produced. Accordingly, it is necessary to modify the culture conditions for
storage.
A common approach is to reduce the temperature at which cultures are
maintained. Most temperate species are grown in vitro at a temperature in the
range of 20°C to 25°C. Tropical plants tend to favour a temperature slightly
higher, for example between 25°C and 30°C. To reduce the rate of growth and
thereby extend the interval between transfer to fresh medium sufficiently for
convenient storage it is necessary to reduce the culture temperature to between
6°C and 10°C for temperate material, and to between 15°C and 25°C for
tropical material. Typically, this will extend the subculturing interval to a
period of between 1 and 2 years (e.g. Banerjee & De Langhe, 1985; Roca,
1985). Slow growth storage at a reduced temperature is simple. It need not
involve any modifications to culture procedures other than the use of an
IN-VITRO CONSERVATION
33
alternative culture environment, but it may be necessary to adjust carefully the
culture temperature within the anticipated range and monitor cultures with care
during the subculturing interval to detect deterioration in particularly sensitive
genotypes.
A definite disadvantage of slow growth storage at a reduced temperature is
that it necessitates the provision of an additional controlled environment cabinet
or room normally maintained below ambient temperatures. In laboratories with
limited resources and in tropical climates this may be a requirement that is
difficult to achieve. As an alternative, cultures may be maintained on a modified
medium. Additives to the culture medium such as osmotic inhibitors (mannitol,
sorbitol), natural or synthetic hormonal inhibitors (abscisic acid, Alar, CCC) or
modifications to the culture medium to influence the growth rate in other ways,
can be very effective alternatives to growth at low temperatures (see Withers,
1987a).
A dilemma presented by the slow growth approach to storage is that cultures
are by definition placed under conditions of stress, which may have serious
detrimental effects upon their health, upon their ability to re-establish in the
field when transferred from culture, and upon clonal uniformity where there is a
risk of genetic instability, the frequency of which might be amplified by selection.
Attempts have been made to reduce the detrimental effects of growth
inhibiting treatment whilst retaining the desired retardation of growth by the
combination of more than one stress factor, such as combining a reduced
temperature with the application of an osmotic inhibitor. In some cases this has
proved beneficial, in others it has proved even more detrimental than a single
stress (Wanas, Callow & Withers, 1986). Research is still needed to develop slow
growth storage methods that are reproducible, that are widely applicable among
different crops and among genotypes of a crop, and which can be readily
adopted in different geographical locations. Despite these cautions, it is
encouraging to note that slow growth storage is in use for a number of root and
tuber crops (potato, cassava, yam, sweet potato, aroids), for Musa and for
temperate fruits including apple, pear and strawberry (see Withers, 1990).
Although most efforts to develop slow growth storage methods have
concentrated upon the use of a reduced temperature or retardant additives to
the culture medium, some other approaches are worthy of note. These include
mineral oil overlay, reduced oxygen tension, and defoliation of shoots (see
Withers, 1987a). However, in no case have studies been carried out upon a wide
range of material and it is unlikely that any of them would prove more generally
suitable than the currently preferred approaches.
It is important to note that slow growth storage has been attempted for both
unorganized cultures, in the form of callus, and organized cultures in the form of
shoots, but has only proved satisfactory for the latter. Callus cultures may
survive in slow growth storage and retain certain desirable characters, but there
is evidence that they may be physiologically impaired, for example showing
reduced secondary product synthesis and/or a reduced rate of growth when
returned to normal culture conditions (Hiraoka & Kodama, 1984; Withers,
1986, 1987a). It is not clear whether the changes observed in the limited number
of studies carried out are transient or permanent. I n any case, they signal
caution for the use of slow growth storage for unorganized cultures which are
intrinsically prone to risks of somaclonal variation.
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L. A. WITHERS
The likely future prospects for slow growth storage are that its routine use will
become widespread for the genetic conservation of root and tuber crops,
temperate fruits and also, in some situations where stock cultures need to be
maintained, for mass propagation, such as in the ornamental/floriculture and
forestry industries. There is an insufficient knowledge base, though, to
recommend slow growth storage even for shoot cultures for other than the shortto medium-term (say 10-15 years). Therefore, there are two respects in which a
satisfactory alternative is needed: for the long-term storage of organized cultures,
particularly shoots, and for the storage in the short-, medium- and long-term for
all other types of culture.
Since the problematic feature of slow growth storage is the combination of
culture under stress and potential selection leading to deterioration and loss of
clonal homogeneity, a satisfactory alternative storage method should involve the
suspension of growth. The obvious candidate is cqapreservation (freezepreservation in liquid nitrogen a t - 196OC).
Cryopreservation
This approach to storage has a relatively long history in microbiology and
animal cell culture. It is in routine use for the maintenance of type cultures of
these materials and also for the storage of semen and embryos in the livestock
industry and human medicine (see Ashwood-Smith & Farrant, 1980; Fuller,
1987).
Less attention has been given to the development of cryopreservation methods
for plant material (Withers, 1987a, 1990). Routine methods have only begun to
emerge during the last 10 years; even these do not necessarily have wide
applicability. This situation is largely the result of a lack of adequate attention
combined with the great complexity and heterogeneity of the types of material
that are presented for in-vitro storage in plant research. Higher plant culture
systems vary enormously in size, complexity, culture requirements and responses
to freezing and thawing. It is, therefore, impossible to generalize about the
cryopreservation conditions appropriate for each type of culture system or the
progress achieved in their development.
The most widely studied system, the cell suspension culture, has shown the
most favourable response to cryopreservation. This is partly a reflection of the
level of attention given but the relative simplicity of structure and the
homogeneity of cell cultures are also important factors. Since 1980 it has been
possible to offer a routine cryopreservation method for cell suspension cultures.
The method reported by Withers & King (1980) and Withers ( 1989b, 1990) has
been shown to be very widely applicable and this method, or slight variations of
it, are used in several laboratories for routine culture storage.
The cryopreservation procedure can be broken down into a number of stages:
pregrowth, cryoprotection, cooling, storage, thawing, post-thaw treatment and
recovery growth. In the case of cell suspension cultures the following treatments
are recommended.
Pregrowth: cells should be a t the exponential phase of growth in which they
are at a small size, have relatively small vacuoles and a low percentage water
content. It may be advantageous to passage the cells through a medium
IN-VITRO CONSERVATION
35
containing mannitol, sorbitol or proline or during the pregrowth phase to
increase freeze-tolerance. This is in part due to a reduction in cell size. Small cell
aggregates have a higher freeze-tolerance than large cell aggregates. Therefore,
culture conditions which reduce the mean aggregate size are desirable. Where
this cannot easily be carried out, cell aggregates can be fractionated by physical
treatments.
Cryoprotection: in a few exceptional cases, a single cryoprotectant (usually
dimethyl sulphoxide-DMSO) is effective. However, a cryoprotectant mixture
consisting of DMSO, glycerol (each at 0.5 M) and a third component such as
sucrose, proline, mannitol or sorbitol (at 1 M), is usually far more effective. In all
cases, cryoprotectants are more effective when prepared in culture medium
rather than in water. The pH of the mixture should be adjusted to that of the
standard culture medium, filter sterilized, chilled, and then applied to the cell
suspension culture. The cryoprotectant and cells are mixed thoroughly and left
to incubate for approximately 1 hour. The cryoprotected cells are then dispensed
into sterile ampoules made, for example, of polypropylene.
Cooling: slow cooling is necessary to enable the process of protective
dehydration to occur. The extracellular medium freezes first and this causes
extraction of water from the cell, thereby reducing the amount of intracellular
water which could produce ice damage when freezing eventually takes place. In
the case of cell suspension cultures, an effective dehydrating procedure is to
freeze at a rate of 1°C per minute to approximately - 35"C, followed by holding
at that temperature for approximately 40 minutes. Once protective dehydration
has been achieved, the ampoules containing the cells should be transferred
rapidly to liquid nitrogen. Slow cooling can be carried out in a purpose-built
controlled freezing unit or in inexpensive, improvised equipment (Withers,
1989b; Withers & King, 1980).
Storage: it is not possible to improvise adequate storage conditions and
cryopreserved material must be held at a suitably low temperature in a vacuuminsulated refrigerator.
Warming: this is normally carried out rapidly, to avoid any risk of ice damage
by recrystallization, by agitating the ampoules in a container of sterile warm
water at approximately 40°C.As soon as the last visible ice has disappeared from
the ampoules, they should be transferred from the warm water bath to avoid
overheating.
Post-thaw treatment: freshly thawed cells are extremely susceptible to injury,
particularly by deplasmolysis. If transferred directly to liquid medium the cells
may suffer severe loss of viability and/or delayed recovery growth; therefore they
should be layered on to a plate of semi-solid medium.
Recovery growth: after a few days on semi-solid medium, the cells will have
reabsorbed much of the liquid in which they were suspended. Any remaining
medium containing cryoprotectants can carefully be pipetted out of the dish and
the cells left for a further period of days to weeks to continue recovery growth.
Once growth is clearly established, the cells can be transferred to liquid culture
medium and the normal subculturing cycle resumed.
A number of studies have been carried out to determine the stability of
cryopreserved cell suspension cultures and the evidence obtained is
overwhelmingly in favour of the retention of stability through a freeze-thaw
cycle (see Withers, 1986, 1987a, 1990). Characters tested include antimetabolite
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L. A. WITHERS
resistance, secondary product synthesis (qualitative and quantitative aspects),
morphogenetic potential and growth parameters.
No truly long-term experiments have been carried out upon cryopreserved cell
suspension cultures to determine whether there is any threat to stability with
time in storage. However, precedents from other biological systems would
suggest that cryopreservation is a secure method of storage, the only problems
likely to be encountered relating to exposure to background radiation. It is
possible to take appropriate precautions against radiation damage, by the use of
radioprotectant chemicals and by adequate shielding. Radiation damage is
thought to be mediated by free radicals. This type of damage is widespread in
various areas of pathology. Both natural and artificial means of avoiding free
radical injury may need to be taken into consideration in many aspects of the
broader subject of in-vitro culture as well as storage itself (Benson, 1990; Benson
& Noronha-Dutra, 1988).
As the cell suspension culture is not normally the system of choice for
conservation work, the widest application of cell cryopreservation is likely to be
in the secondary product industry and in some aspects of the application of
biotechnology to crop improvement. However, progress in the development of
embryogenic cell suspension culture systems may change that picture (see
below). Investigations, particularly with gymnosperms, would suggest that the
embryogenic suspension culture may be an ideal system from the point of
totipotency and amenability to cryopreservation (Gupta, Durzan & Finkle,
1987; Kartha el al., 1988).
Nevertheless, until embryogenesis becomes a more widespread phenomenon
among the species of interest from the point of view of in-vitro conservation of
crop genetic resources, the shoot-culture system will be favoured for conservation
work. It is unfortunate, therefore, that shoot cultures have proved much more
difficult to cryopreserve than cells. This difference is in no way surprising, as
revealed by an examination of the anatomy of a shoot culture. A shoot is
considerably larger than a typical cell aggregate and contains many different cell
types. The effective functioning of the shoot depends upon the maintenance of
intercellular connections and the retention of viability within a large proportion
of the cells. As there is a relationship between cell size, structure, tissue size and
freezing requirements, it is not surprising that i t is extremely difficult to
determine a set of cryopreservation conditions that are suitable for all of the
different cell types within a shoot and for a structure of the dimensions of a shoot.
Shoots can suffer massive structural damage as a result of freezing and
thawing. Theoretically, only the shoot apex needs to survive for it to be possible
for the entire shoot to regenerate. However, frequently the only surviving regions
of a shoot-tip are areas of the leaf primordia; thus adventitious regeneration is
common among cryopreserved shoots (Haskins & Kartha, 1980; Withers,
Benson & Martin, 1988). The amount of structural damage may also lead to a
failure to regenerate under normal culture conditions. Recovery growth of
severly damaged shoots on a culture medium containing growth regulators may
permit regeneration from a few surviving cells. However, this would then
normally take place via a callus phase with associated risks of genetic instability.
In general, we are presented with a potential trade-off between quality and
quantity, either a small number of high quality regenerants can be obtained, or
a larger number of low quality regenerants. In the context of genetic
IN-VITRO CONSERVATION
37
conservation the choice would be for the former, although efforts should clearly
be made to improve cryopreservation conditions to achieve a high frequency of
recovery.
It is not possible as yet to recommend a routine cryopreservation method for
shoot cultures. Considerable variations in treatments are reported at all stages,
from pregrowth to recovery growth (Withers, 1987a, 1990). However, some
important points should be made, particularly those which demonstrate how
differently shoots must be treated from, for example, cell suspension cultures. A
shoot culture is far too large to survive freezing intact. Therefore, the first stage
of the procedure is dissection to reduce the shoot down to the apical dome and a
few leaf primordia. This dissection is followed by a period of pregrowth recovery,
on either a standard medium, or medium supplemented with, for example,
DMSO, mannitol or sorbitol. DMSO appears to be the most effective
cryoprotectant for shoots and there is no evidence for the superiority of
cryoprotectant mixtures over a single compound prepared in culture medium.
There are examples of successful cryopreservation using a wide range of slow
freezing conditions, rapid freezing and ultra-rapid freezing. However, rapid
thawing is almost invariably quoted for shoot cultures. Post-thaw washing is
included in some treatments and not others, and widely differing recovery media
have been used. Cold-hardening treatments have been used to improve the
freeze-tolerance of shoot cultures (Reed, 1989). Their use derives from the
observations that resistance to freezing in buds of some species varies with season
(Tyler, Stushnoff & Gusta, 1988). It appears to be possible to stimulate such
seasonal changes in vilro. Examples of the successful cryopreservation of shoots
are accumulating. However, it is clear that the rate of progress in the
development and application of cryopreservation procedures for such specimens
has not been as dramatic as for cell suspension cultures.
Two possible ways forward may be offered. Firstly, it may be possible to
develop cryopreservation procedures that accommodate the different needs of
the various cell-types within a shoot and the demands of cryopreserving
relatively large structures. One such approach might be ‘vitrification’. This
involves loading tissues with extremely high levels of cryoprotectant mixtures.
These would normally cause toxicity and therefore have to be added with care at
relatively low temperatures. The loaded tissues are then quenched, i.e. frozen
very rapidly in liquid nitrogen. Neither protective dehydration nor ice
crystallization take place, the water in the tissues passing directly into a ‘glassy’
state. Thawing must be carried out extremely rapidly and cryoprotectants must
be removed carefully. There is accumulating evidence that vitrification, already
proved successful with animal tissues, may be applicable to plant material
(Langis el af., 1989; Uragami et af., 1989).
A second approach might be to look for an alternative culture system that is
highly organized and, therefore, likely to carry a low risk of somaclonal
variation, but which has a structure that is more amenable to cryopreservation.
These characteristics would point again to the somatic embryo, the value of
which has already been indicated above. Somatic embryogenesis is an extremely
promising system offering mass propagation combined with clonal fidelity.
Somatic embryogenesis has been demonstrated in diverse explants including
both zygotic embryos and somatic tissues.
Seeds and zygotic embryos of many orthodox species have been shown to be
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L. A. WITHERS
susceptible to storage in liquid nitrogen (Stanwood, 1985; Withers, 1987b). The
potential for cryopreservation of recalcitrant seeds and embryos is also promising
(e.g. Chin, Krishnapillay & Alang, 1988). However, the embryo and even the
embryonic axis of some recalcitrant seeds is very large and outside the range
likely to survive cryopreservation without serious structural damage. This
problem could be overcome either by using immature embryos or by inducing
secondary somatic embryogenesis from an original zygotic embryo.
When the maternal genotype is the target for conservation, problems can be
encountered in achieving embryogenesis in mature tissues of woody species. In
such cases, i t may be possible to induce embryogenesis from nucellar tissues
which are juvenile in their behaviour yet can be obtained from plants that are
sufficiently mature to demonstrate their desirable characteristics (Litz, 1987).
The combination of somatic embryogenesis, artificial seed technology and
cryopreservation is considered to be a potentially fruitful new approach to the
genetic conservation of problem subjects including both root and tuber crops
and other clonally propagated material through to recalcitrant seed-producing
species. Attempts have been made to develop artificial seed technology as an aid
to mass propagation (Redenbaugh el al., 1986). The production of artificial seeds
involves encapsulating somatic embryos in a supporting medium. Some of the
stresses experienced in the desiccation that accompanies encapsulation are
similar to those involved in cryopreservation. Thus, it is possible that treatments
to enhance survival in artificial seed production might also increase tolerance of
cryopreservation. A further link between the two technologies is the fact that
long-term storage of artificial seeds is problematic; the introduction of a
cryopreservation stage between artificial seed production and delivery would
offer considerable flexibility in handling.
IN-VITRO CONSERVAI‘ION SYS‘I’EhlS
The main prerequisite of conservation is satisfactory storage and, in parallel to
the seed gene bank, there is a need for both ‘active’ and ‘base’ in-vitro storage
technologies. However, this is only part of the picture. Conservation begins with
the acquisition of germplasm and ends with the germplasm’s being made
available for utilization. Additional important stages are disease indexing,
disease eradication, quarantine (if necessary), propagation, stability monitoring,
and distribution. In-vitro techniques have a part to play at all stages of the
conservation process (Withers, 1989). Some of the most practical applications
relate to germplasm acquisition and movement.
Collecting germplasm in the field can present problems. Suitable material is
not always available at the time of a plant collecting mission. Seed may be
poorly formed, it may be immature, and i t may have been removed by grazing
animals. Vegetative material and recalcitrant seeds may deteriorate in transit
back to the gene bank. Therefore, it would be useful to be able to develop a
collecting method that enabled advantage to be taken of any available material
in the field and for this material to be kept in good condition until i t could be
processed further. An additional practical problem in collecting is the weight
and bulk of material in examples such as coconut. This can limit the amount of
germplasm that the collector can gather and thereby limit the adequacy of
sampling of populations during the collecting mission.
IN-VITRO CONSERVATION
39
In 1983 the International Board for Plant Genetic Resources (IBPGR) held a
small working group meeting to examine the possibility of applying the
principles of in-vitro propagation under field conditions and away from
sophisticated laboratory facilities (IBPGR, 1984). Since that time a number of
investigations have been carried out to demonstrate the feasibility and flexibility
of this approach to collecting (Withers, 1 9 8 7 ~ )More
.
than ten different crops
have been studied, with procedures being developed for material as diverse as
budwood in cacao (Yidana, Withers & Ivins, 1987), axillary buds in forage
grasses (Ruredzo & Hanson, unpublished observations) and zygotic embryos in
coconut (Assy Bah, Durand-Gasselin & Pannetier, 1987). In two cases at least
(cotton: Altman el al., 1990, and coconut: Luntungan & Tahardi, personal
communication), techniques have been applied successfully on actual collecting
missions.
In-vitro collecting will, of course, only be completely successful if the collected
material can be processed and transferred to a gene bank. In the case of cacao
there are problems in that plant regeneration from explants is problematic.
However, the possibility of micrografting shoots from collected budwood appears
to be promising (Villalobos, personal communication). In coconut, embryo
culture is more or less routine but we are still presented with a situation where
one collected embryo only produces one plantlet at most, and the embryos are
not amenable to in-vitro storage. In a current project IBPGR, (Centre de
Coopkration Internationale en Recherche Agronomique pour le
Diveloppement) CIRAD (France) and (Institut de Recherche pour les Huiles et
Olkagineaux) I R H O (CBte d’Ivoire, France) are attempting to develop
cryopreservation techniques which could be applied to immature zygotic
embryos and, in due course, multiple somatic embryos. This would provide a far
more satisfactory destination for collected material.
In-vitro technology can offer alternative and improved techniques over those
traditionally used for disease indexing and disease eradication. In principle, an
entirely contained indexing, eradication and quarantine procedure could be
developed to add greatly to the safety and efficiency of germplasm introduction.
There are excellent precedents for the routine application of in-vitro techniques
for germplasm exchange, e.g. the distribution of potato cultures from the
International Potato Centre (CIP) Headquarters in Peru, the distribution of
several crops including Mum and Dioscorea (yam) from (International Institute
of Tropical Agriculture) IITA in Nigeria and the distribution of cassava from
(Centro Internacional de Agricultura Tropical) CIAT in Colombia (Roca el al.,
1984; Withers, 1989).
The groundwork is laid for the adoption of an entirely in-vitro-based
conservation scheme for crops which can be propagated in vilro. As yet, only
strawberry can be said to have had adequate attention and success at all stages
for the scheme to be complete. This crop is not of the highest priority for in-vitro
conservation but it is possible to quote other examples in which most
components are in place. These include potato and cassava, in which the main
deficiencies would be in development, refinement and application of
cryopreservation technology. Other less well-developed examples include Musa,
which is also deficient in aspects of base storage through cryopreservation and
which presents problems in relation to genetic stability during clonal
propagation (see Vuylsteke, 1989).
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L. A. WITHERS
Because of the relatively well-developed state of in-vitro conservation
technologies for cassava, it has been chosen as the subject for a collaborative
study between IBPGR and CIAT to test standards for an in-vitro active gene
bank involving storage by slow growth (Chavez, Roca & Williams, 1987). It is
important to emphasize that a collection of in-vitro plantlets is not necessarily a
gene bank. Rigorous standards must be applied to ensure that the in-vitro gene
bank gives sufficient coverage of genotypes, and sufficient security and
accessibility of the germplasm. Thus, the pilot in-vitro active gene bank project
includes attention to both isozyme and molecular technology for
characterization of genotypes and detection of genetic instability. The potential
interaction between the generation of instability and selection under slow growth
conditions indicates that careful attention must be given to the ‘family tree’
developed during the propagation procedure and to control of the numbers of
cultures maintained with minimal risk of selecting for abnormal genotypes.
This project, now in its final stages, will result in the production of guidelines
for in-vitro gene bank management for cassava and other crops. The guidelines
will address levels of replication, subculturing procedures, frequency of
monitoring and approaches to monitoring. There will also be associated software
to assist with the management tasks.
COMPLEMENTARY CONSERVATION STRATEGIES
The comprehensive, safe and accessible conservation of crop gene pools is the
primary aim of genetic resources efforts. In this endeavour, attention should be
given to the different conservation methodologies available and the part that
they can play to complement each other.
The components of a complementary conservation scheme would include
storage in the field gene bank and in-vitro, seed storage, pollen storage, in-situ
conservation, and in due course perhaps, the storage of DNA sequences. No
single technology holds all of the answers and no single technology is suitable for
all situations. Furthermore, it is likely that there will be a shift in emphasis
between the different storage modes with time, as technical improvements come
on stream, as confidence is built in the application of the newer approaches, and
as users’ needs change.
The conservation of clonally propagated crops will involve a strong emphasis
on the storage of vegetative material but when seed production is possible this
too should be recognized as a valuable means of conserving genes if not
genotypes. In-vitro conservation should be selected for the clones because it is
able to offer advantages over more traditional technologies, not simply because it
is possible and ‘modern’. It should be used if and when i t is more efficient, less
costly and safer than the field gene bank, and in circumstances when it is the
most effective way of duplicating a collection. These advantages need to be
balanced against risks of instability (which are not, of course, zero in the field),
the need for relatively sophisticated laboratory facilities and technical input, and
the fact that evaluation studies cannot be carried out to any meaningful extent
in vitro.
Finally, it is important to realize that we are not always forced to make
absolute choices; conservation is very much the ‘art of the possible’. We should
draw upon available technologies and use them where they are efficient, at the
IN-VITRO CONSERVATION
41
same time ensuring a flow of information to those who are in a position to further
develop new and improved approaches. Using an idealized complementary
conservation strategy as a basis for identifying technological deficiencies is a
sound way of determining research objectives and priorities. The application of
technological developments necessitates attention to training and technology
transfer. Flow of technology and flow of information are, therefore, equally
important components of the collective conservation effort.
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