Note: Descriptions are shown in the official language in which they were submitted.
2013821
This invention relates to a process for storing
somatic or zygotic vegetable embryos.
Numerous species may be stored at low temperatures in
the form of cell suspensions, calluses or even meristems.
Low-temperature storage of embryos is justified in
many cases, for example for regulating the production of
plantlets where it is seasonal or for maintaining a clonal
line.
The low-temperature storage of embryos affords the
possibility of temporarily stopping the development of the
embryos, the time required for their transport to the seed
bed or for their storage and the possibility of creating
banks of genotypes to avoid progressive depreciation of the
genetic patrimony.
Somatic embryos have certain advantages for the multi-
plication of plants. They emanate in principle from a
single cell and give genetically uniform plants. From the
beginning of their formation, somatic embryos have a
bipolar structure: they have the two stem and root meris-
tems necessary to produce a plant. Accordingly, somatic
embryogenesis appears an interesting alternative for the
propagation of plants: it could be used for the rapid
multiplication of species that are expensive to produce or
of high-performance individuals emanating from in vitro
cultures or of transformed plants that are difficult to
handle by sexing for example.
The problems involved in the storage of embryos are
complex. In particular, the two meristematic poles capable
of ensuring the resumption of elongation and organogenesis
of the root and stem have tobe kept alive.
There are various known processes for the low-tempera-
ture storage of embryos.
One of these processes comprises the steps of precul-
turing the embryos on a medium enriched with sorbitol,
proline or mannitol, impregnation at 0C in the culture
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medium containing added dimethyl sulfoxide and/or glycerol,
induced slow freezing to -40C and then rapid freezing in
liquid nitrogen. The resumption of embryogenesis after
defrosting necessitates the addition of 2,4-dichlorophen-
oxyacetic acid to the culture medium.
Another process, which is applicable to somatic
embryos cultured on an agar medium, comprises the steps of
preculturing the embryos for one week on a sucrose-enriched
medium and direct freezing of the embryos placed dry in a cryo-
tube in liquid nitrogen.
In another known process, somatic embryos are precul-
tured on a medium to which at least 2.5% dimethyl sulfoxide
is gradually added and are then frozen slowly to -100C and
then rapidly in liquid nitrogen. The resumption of embryo-
genesis is facilitated by culture on a medium containing
0.1 mg 1~1 2,4-dichlorophenoxyacetic acid.
The function of the cryoprotective agents, such as
dimethyl sulfoxide or glycerol, is to prevent the formation
of ice during freezing. However, the use of substances
such as these can have certain disadvantages. On the one
hand, they are not generally readily sublimable and cannot
therefore participate in a subsequent lyophilization
treatment; on the other hand, they can be cytotoxic.
The function of the auxins, such as 2,4-dichlorophen-
oxyacetic acid, is to keep the cells in the neutral state
during callogenesis and to promote the resumption of cell
proliferation after defrosting of the embryos. Now,
stopping the development of the embryos by freezing often
induces abnormal resumption of their growth. The auxins
are capable of promoting the formation of adventitious
embryos by cell division during the resumption of embryo-
genesis and of increasing the risks of secondary embryo-
genesis.
To obviate the disadvantages of the prior art, the
present invention seeks to provide a process for storing
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2013821
somatic or zygotic vegetable embryos which enables the
development of the embryos to be stopped by freezing and
subsequently resumed without the appearance of other forms
of morphogenesis, such as secondary embryogenesis or callo-
genesis.
To this end, the process according to the invention is
characterized in that
- the embryos are pretreated on a pretreatment medium
containing an osmotic pressure agent, after which
- the pretreated embryos are cooled and frozen to a
temperature of -15C to -40C.
The embryos frozen at -15 to -40C may be stored at
that temperature or may be subsequently immersed in a
liquid refrigerant and stored at a lower temperature.
One advantage of this process is that it avoids the
use of toxic and/or non-sublimable cryoprotective agents.
Another advantage is that it avoids the use of auxins in
the culture media used before freezing and after defrosting
during the resumption of growth. A further advantage of
the process is that it provides for storage at a readily
accessible low (-15/40C) temperature (for example
in a domestic freezer) without any need for expensive
equipment.
Another advantage of the invention is that it provides
a storage process which can be carried out quickly and
easily.
Another advantage is that it provides for subsequent
lyophilization of the embryos.
The storage process according to the invention makes
it possible to obtain embryos which show normal growth
resumption with no need for growth promoters and which have
no adventitious proliferation, resulting in improved qual-
ity of the seeds produced because secondary embryogenesis
is harmful to industrial distribution of the product
because unwanted multigerm seeds can be obtained.
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2013821
The embryos used in the present invention may be of
any species and various origins. For example, the embryos
may be carrot embryos.
The embryos may be somatic embryos or zygotic embryos.
The somatic embryos may be obtained from indifferentiated
cell suspensions. In this case, seeds of a hybrid parent for
example may be aseptically germinated. The hypocotyls may
be cut and then placed on a culture medium containing
auxins. The calluses obtained may then be dissociated in
a liquid culture medium. This gives an indifferentiated cell suspen-
sion of which the cells, after several subcultures, may be
transferred to a culture medium. After about ten days, the
cell suspension may be filtered so that only cell aggre-
gates of the required size are retained. These aggregates
may be cultured for a few days on an auxin-free culture
medium to induce formation of the embryos.
The zygotic embryos may be obtained by sampling by
dissection of the seeds at the mature or slightly immature
stage.
The somatic and zygotic embryos obtained may be
classified according to their stage of development.
Preferred embryos are in the initial stages of their
development when they are between 150 and 600 ~m in size
because they are more stable to freezing at this stage.
These sizes correspond to the heart (150-300 ~m) or torpedo
(300-600 ~m) stages of the development of somatic embryos.
The embryos obtained are then pretreated on a culture
medium called the pretreatment medium. This medium may be
a typical medium in the field of embryo culture, such as a
Murashige and Skoog medium for example, to which an osmotic
pressure agent is added and to which certain organic
substances, such as vitamin Bl, nicotinic acid or adenine,
may also be added.
It is necessary in low-temperature storage processes
to prevent the formation of intracellular ice which, even
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if it does not directly damage the embryos during freezing,
can induce destructive recrystallization during defrosting.
In the process according to the invention, the embryos are
partly dehydrated by pretreatment on a medium containing an
osmotic pressure agent to reduce any damage they may
suffer.
This agent may be selected by the expert from substan-
ces which are capable of penetrating the tissues and pro-
viding a good osmotic pressure to obtain correct dehydra-
tion of the cell without influencing membranal permeability.
In addition, this agent must not be in any way toxic to the
embryos. This agent may be a sugar, such as sucrose or
trealose, or any other known substance which is capable of
performing the same functions.
The concentration of osmotic pressure agent in the
pretreatment medium should not be too low in order to
ensure correct dehydration of the cell. Neither should it
be too high so as not to damage the embryos or prevent the
resumption of their growth after defrosting.
In one preferred embodiment of the storage process
according to the invention, the pretreatment medium con-
tains sucrose in a concentration of from 75 to 190 gl~1 and
preferably in a concentration of 100 to 150 gl~1.
It has been found that this pretreatment alone,
followed by a freezing step to the temperature indicated,
enables the desired objective to be achieved in a simple,
effective and reliable manner.
The pretreatment may last 30 seconds to 36 hours. The
pretreatment should not be too short in order to allow the
osmotic pressure agent partly to dehydrate the cells and to
increase their stability to freezing. Nor should the pre-
treatment last too lonq because otherwise the stability of
the embryos to freezing may diminish, as reflected in a re-
duction in the number of embryos still alive after defrost-
ing when the pretreatment e~ceeds 36 hours. In the event
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of prolonged pretreatment, the embryo might possibly
undergo morphological changes which could reduce its
stability to freezing due to the fact that the embryo
continues to grow and reaches a stage less compatible with
freezing. On the other hand, the reduction in the protec-
tive effect of the osmotic pressure agent during long pre-
treatments could also be explained by adaptation of the
embryo to the pretreatment medium which would result in a
reduction in the effect of the osmotic pressure.
The pretreatment is preferably carried out at ambient
temperature, i.e. at approximately 18 to 24C, under medium
lighting, for example of 150 to 250 lux.
The pretreated embryos are cooled and then frozen to
a temperature of -15C to -40C. They are preferably
frozen tc a temperature of approximately -20C. The
freezing process may be carried out by transferring the
embryos with the pretreatment medium to cryotubes and
placing the cryotubes in a domestic freezer for example.
The cooling rate applied during freezing influences the
speed at which ice forms and the degree of dehydration of
the embryos at the moment ice forms. The cooling rate is
preferably moderate, of the order of 0.1 to 1C per minute,
for the temperature range from -6C to -40OC in order to
promote the subsequent resumption of growth of the embryos.
For cooling from ambient temperature to a temperature of
-6C, the cooling rate may be more rapid, for example
between 1 and 5C per minute.
The embryos frozen at a temperature in the range from
-15C to -40C may be stored as such for at least 1 month.
For longer storage times, the embryos are preferably stored
at a lower temperature, for example in a liquid refriger-
ant. This is because it seems that prolonged storage at
15/-40C is incompatible with the resumption of growth and
hence the viability of the embryos after defrosting. It
seems that the temperature of -15/-40C is too high to
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ensure complete stoppage of the metabolism of the embryo.
If prolonged storage is required, freezing to -15/-40C
should be considered as a first step which may be followed
by freezing in a liquid refrigerant at a lower temperature.
In this case, the embryos are preferably kept at -15/-
40C for a certain period, of the order of 18 to 24 h, so
that freezing is complete.
The embryos may then be directly immersed in a liquid
refrigerant, for example in a bath of methanol at -70~C or
in a bath of liquid nitrogen at -196C, or cooled to a very
low temperature by any other means.
The frozen embryos may then be stored for long periods
of up to several years. The frozen embryos may then be en-
capsulated with artificial or natural polymers, for example
of the alginate type, to obtain artificial seeds.
The embryos may be defrosted, for example, by immer-
sion of the cryotubes in a water bath at 40C for 2 min-
utes. The tubes may be withdrawn from the water bath
before the ice melts completely to avoid an over-rapid rise
in temperature. After complete defrosting, the embryos may
be freed from the pretreatment medium by simple washing,
the washed embryos may be placed on a culture medium
typical in the field of embryo culture, such as a Murashige
and Skoog medium for example.
This medium may contain an assimilable carbon-contain-
ing substrate, such as sucrose, in a concentration of 1 to
10 gl~l for example.
This culture medium is remarkable in the fact that it
is free from auxins.
After this reculture, the embryos resume a development
comparable with that of non-frozen embryos.
Survival after freezing and reculture is reflected in
bipolar growth of the embryos. The embryo effectively
retains the integrity of its structure during subsequent
development with no other form of morphogenesis, such as
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callogenesis or adventitious embyrogenesis.
Accordingly, the storage process according to the
invention on the one hand provides for survival of the
embryos with their morphological integrity intact and on
the other hand enables the embryos effectively to maintain
their capacity to regenerate a plantlet.
The present invention is illustrated in more detail by
the following Examples. These Examples are preceded by an
example of the conventional preparation of somatic embryos,
by the description of a viability test and by Table 1 which
gives the composition of a preferred pretreatment and
culture medium.
Example of the ~re~aration of somatic embrvos
An indifferentiated cell suspension of carrot cells (Daucus carota
L.) is subcultured every 12 days (1 gram biomass to 100 ml
medium) on a Murashige and Skoog liquid culture medium
having the composition shown in Table 1, to which 20 g 11
sucrose and 0.1 mg 1-~ 2,4-dichlorophenoxyacetic acid have
been added. All handling is carried out under aseptic
conditions beneath a laminar flow hood. The suspension is
placed on a stirrer making an eccentric gyratory movement
of 100 r.p.m. and is cultured at 24C under 200 lux light-
ing with a photoperiod of 16 hours.
After culture for 8 to 10 days, the cell suspension is
filtered so that only cell aggregates between 50 and 180 ~m
in size are retained. These small aggregates represent a
proembryonic stage of the embryos which will continue their
development to the heart, torpedo and plantlet stages. The
aggregates are washed and placed on a Murashige and Skoog
medium containing no 2,4-dichlorophenoxyacetic acid in a
quantity of approximately 1.5 x 103 aggregates per ml
medium.
After culture for 10 days, embryos have formed. The
suspension is filtered so that only embryos between 150 and
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600 ~m in size are retained.
Viability test
A quick and simple viability test has been developed
to evaluate the viability rate of the embryos after freez-
ing.
Among the various criteria which may be used to
evaluate the viability rate of the embryos,
- the increase in the size of the embryos and
- the appearance of a chlorophyllian coloration
are particularly appropriate.
These criteria may be evaluated in various ways, for
example by visual counting or by biochemical tests (color-
ation test for example).
Under the principle of this test, the defrosted
embryos are placed on a liquid culture medium. After
culture for 10 days, the number of embryos which have
increased in size and show signs of chlorophyllian colora-
tion is recorded. The ratio between this number and the
total number of embryos present enables the viability rate
of the embryos to be determined.
The embryos may then be placed on a solid culture
medium having the same composition as the preceding liquid
medium so that they may continue their development to the
plantlet stage.
After culture for 10 days on this solid medium, the
regeneration rate is determined as the ratio between the
number of embryos which have developed to the plantlet
stage and the total number of embryos.
Table 1
Composition of the Murashige and Skoog medium (pH 5.8 - 6)
Macroelements mg 11
NH4N03 1650
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20~2~
CaCl2 2H2
MgSO4 7H20
KNO3 1900
KH2PO4 170
Microelements
CoCl2 0.025
CuS04 5H20 O. 025
FeSO4 7H2O 27.8
Na2 - EDTA 37-3
MnSO4 4H2O 22.3
KI 0.83
Na2MoO4 0.25
znSO4 7H2O 10.6
H3BO3 6.2
Other elements
Nicotinic acid 5
Thiamine (vit. B1)
Adenine 2
+ osmotic pressure agent (pretreatment medium) or
assimilable carbon-containing substrates (culture
medium)
Exam~le 1
Somatic carrot embryos at the torpedo stage, average
size 550 ~m, obtained as described above, are placed in
Petri dishes on a liquid Murashige and Skoog pretreatment
30medium containing 135 gl~1 sucrose and free from auxins in
a quantity of approximately 100 embryos to 10 ml medium.
These embryos are pretreated for 1 hour at 20C under
200 lux lighting. They are then transferred to cryotubes
containing 1.8 ml pretreatment medium. The cryotubes are
35placed in a domestic freezer in which their contents are
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11
cooled at a rate of 1C/minute to -6C and then at a rate
of 0.4C/minute from -6OC to -20C.
After 24 hours at -20C, the cryotubes are separated
into two groups. The first group is defrosted by immersion
of the frozen cryotubes in a water bath at 40C for 2 min-
utes. The second group is immersed in liquid nitrogen at
-196C where it remains for 1 hour before being defrosted
in the same way as the first group.
After total defrosting, the embryos are washed to re-
move the pretreatment medium and placed for 10 days on a
liquid Murashige and Skoog culture medium containing 5
gll sucrose and free from auxins. They are then trans-
ferred to solid culture medium having the same composition.
Control embryos which have not been pretreated or
frozen are placed on the same culture media.
After culture for 10 days on solid medium, the capac-
ity of the embryos to regenerate carrot plantlets is compared.
Among the control embryos, 46% are capable Gf regener-
ating a plantlet. For the embryos frozen to -20C and to
-196C, the regeneration rates are 44% and 43%, respec-
tively.
The capacity of the embryos to regenerate plantlets is
thus not affected by freezing providing they have been
pretreated.
Exam~le 2 -
Two populations of somatic Daucus carota L. carrot
embryos are selected.
One population consists of embryos at the heart stage
with an average size of 300 ~m, the other population
consists of embryos at the torpedo stage with an average
size of 500 ~m.
Five samples (A, B, C, D and control) each containing
approximately 100 embryos are taken from each population of
embryos.
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12
Samples Ah, ~ and Ch of embryos at the heart stage and
samples At, Bt and Ct of embryos at the torpedo stage are
pretreated on a Murashige and Skoog culture medium contain-
ing 135 gl~l sucrose for 1 hour at 20C under 200 lux
lighting.
Samples Ah, ~, At and Bt are then cooled to -20C at a
rate of approximately 0.5C/minute. They are then kept at
-20C for 24 hours. Samples Ah and At are defrosted while
samples ~ and Bt are immersed in liquid nitrogen at -196C
where they remain for 1 hour before being defrosted.
For comparison, samples Ch and Ct are directly immersed
in liquid nitrogen at -196C after the pretreatment. They
are defrosted after 1 hour. For comparison, samples Dh and
Dt are cooled to -20C at a rate of approximately 0.5C/min-
ute without being pretreated. They are defrosted after 24
hours.
The heart and torpedo control samples are neither
pretreated nor frozen.
The various embryo samples are then recultured on a
Murashige and Skoog liquid medium containing 5 gl~1 sucrose.
After culture for 10 days, the following results are
obtained:
Viability rate of the embryos t%)
Sample
Comparison
Stage A B ~ Control
Heart 88 71 0 0 90
Torpedo 84 67 0 0 91
It can be seen that, in the absence of pretreatment,
the embryos frozen to -20C tD) do not survive whereas
impregnation for one hour in the pretreatment medium
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13
ensures the survival of almost all the embryos at the heart
and torpedo stage frozen to -20C (A).
Likewise, the embryos do not survive direct immersion in
liquid nitrogen, even after pretreatment for one hour (C).
The embryos frozen to -196C after pretreatment and
freezing to -20C (B) have a very satisfactory viability
rate.
Accordingly, if it is desired to store the embryos at
-196C, they have to be subjected to the phases of pre-
treatment and freezing to - 20C.
The embryos are then placed on a solid culture medium
having the same composition as the liquid medium.
After culture for 10 days, the majority of the embryos
of samples A and B and also the control samples have devel-
oped to the plantlet stage with a clearly differentiated
root point and chlorophyllian cotyledonary apex.
These embryos show no sign of callogenesis or secon-
dary embryogenesis.
Example 3
Somatic carrot embryos at the heart stage and at the
torpedo stage are pretreated on a Murashige and Skoog
liquid culture medium containing 135 gl~~ sucrose for
different periods at 20C under 200 lux lighting.
These embryos are then cooled to -20C at a rate of
approximately 0.5C/minute. After 24 hours at -20C, some
of the embryos are immersed in liquid nitrogen and kept
there for 1 hour.
After defrosting, the embryos are placed on a Nurash-
ige and Skoog liquid culture medium containing 5 gl'
sucrose.
Control embryos which have not been pretreated or
frozen are also cultured. The viability rate of the
embryos is determined after culture for 10 days.
The following results are obtained:
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14
Viability rate of embryos at the heart stage (%)
Pretreatment time
Comparison
30 min. lh 24h 48h 72h
Embryos frozen to -20C 89 88 87 56 40
Embryos frozen to 196C 73 72 68 42 32
The control embryos have a viability rate of 90%.
Viability rate of embryos at the torpedo stage (%)
Pretreatment time
Comparison
30 min. lh 24h 48h 72h
Embryos frozen to -20C 85 84 82 56 34
Embryos frozen to -196C 78 77 78 46 36
The control embryos have a viability rate of 91%.
It can be seen that the viability rate after culture
for 10 days is virtually identical for a pretreatment time
of 30 min., 1 h or 24 h both for embryos at the heart stage
and for embryos at the torpedo stage.
By contrast, beyond 24 h, the viability rate decreases
progressively with increasing pretreatment time. This rate
is no more than about 35% after a pretreatment time of 72
h. Accordingly, the resistance of the embryos to freezing
diminishes in the event of prolonged pretreatments.
Exam~le 4
Somatic carrot embryos at the torpedo stage are
pretreated for various times on a Murashige and Skoog
liquid medium containing 135 gl~l sucrose at 20C under 200
lux lighting.
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2013821
These embryos are cooled and frozen to -20C at a rate
of approximately 0.5C/minute.
After 72 hours at -20C, the embryos are defrosted and
placed on a Nurashige and Skoog liquid culture medium
containing 5 gl~1 sucrose.
Control embryos which have not been pretreated or
frozen are also cultured.
The viability rate of the embryos is determined after
culture for 10 days. -
The following results are obtained:
Pretreatment 1 15 30 45 60 Control
time (min.)
Viability rate (%) 86 85 73 68 83 80
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It can be seen that the viability rate of the embryos
after freezing to -20C is good for all these pretreatment
times. The stability of the embryos to freezing is thus
acquired very rapidly.
Exam~le 5
Somatic carrot embryos at the torpedo stage with an
average size of 550 ,~m are pretreated and frozen to -20C.
Some of the embryos are then immersed in liquid nitrogen by
the method described in Example 1.
The embryos are stored at these temperatures for
various times. They are then defrosted and placed on a
Murashige and Skoog liquid culture medium containing 5
gl~1 sucrose. The viability rate of the embryos is deter-
mined as a function of their storage time at -20~C or
-196C.
The control embryos which have not been pretreated or
frozen have a viability rate of 72%.
The following results are obtained:
For the embryos frozen to -20~C:
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16
Storage time 24h 1 months 2 months
Viability rate (%) 64 69 0
For the embryos frozen to -196C:
Storage time 24h 1 months 2 months
Viability rate (%) 66 67 67
The average viability rate after freezing to -20~C or
to -196C is approximately 67% for storage times of one day
or less. This rate remains unchanged after storage for one
month at -20C or at -196C. After storage for 2 months,
there are no survivors among the embryos stored at -20C
whereas, for the embryos stored at -ls60c, the viability
rate is still the same.
Example 6
Somatic carrot embryos at the torpedo stage with an
average size of 500 ~m are pretreated for 1 hour at 20C
under 200 lux lighting on Murashige and Skoog liquid media
having a sucrose concentration varying from 35 gl~1 to 205
gl-l .
The embryos are then cooled and frozen to -20C.
After 1 hour at -20C, the embryos are defrosted and placed
on a Murashige and Skoog liquid culture medium containing
5 gl~1 sucrose.
Control embryos which have not been pretreated or
frozen are also cultured.
The viability rate of the embryos is determined after
culture for 10 days as a function of the sucrose concentra-
tion of the pretreatment medium.
The following results are obtained:
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Sucrose (gl~1) 35 68 100 135 170 205 Control
Viability rate (%) 22 41 77 82 65 14 88
The viability rate of the embryos pretreated on a
medium containing 135 gl~1 sucrose is comparable with that
of the control embryos.
This rate is still fairly high for a sucrose concen-
tration of 100 or 170 gl~1.
The viability of the embryos is affected when the
sucrose concentration becomes too high (205 gl~1) or too low
(35 gl1),
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