IE67530B1 - A storage process for vegetable embryos - Google Patents

Abstract

In order to ensure the preservation of plant embryos for long periods, the embryos are pretreated in a medium containing an osmotic pressure agent before they are deep-frozen at a temperature of -15/-40 DEG C.

Description

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 multiplication 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 meristems 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 to be kept alive.

There are various known processes for the low-temperature storage of embryos. * One of these processes comprises the steps of preculturing the embryos on a medium enriched with sorbitol, proline or mannitol, impregnation at O’C in the culture medium containing added dimethyl sulfoxide and/or glycerol, induced slow freezing to -40 °C and then rapid freezing in liquid nitrogen. The resumption of embryogenesis after defrosting necessitates the addition of 2, 4-dichlorophenoxyacetic 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 cryotube in liquid nitrogen.

In another known process, somatic embryos are precultured on a medium to which at least 2.5% dimethyl sulfoxide is gradually added and are then frozen slowly to -100°C and then rapidly in liquid nitrogen. The resumption of embryogenesis is facilitated by culture on a medium containing 0.1 mg l'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-dichlorophenoxyacetic 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 embryogenesis and of increasing the risks of secondary embryogenesis.

To obviate the disadvantages of the prior art, the present invention seeks to provide a process for storing 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 ambryogenesis or callogenesis. & 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 -15°C to -40°C.

The embryos frozen at -15 to -40°C 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 (-15/40°C) 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 embryogen.esis is harmful to industrial distribution of the product ** because unwanted multigerm seeds can be obtained.

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 maybe 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 suspension 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 aggregates 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 Preferred embryos are in the initial development when they are between 150 and 600 μη in size because they are more stable to freezing at this stage. These sizes correspond to the heart (150-300 μχη) or torpedo (300-600 gm) 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 of development, stages of their if it does not directly damage the embryos during freezing, can induce destructive recrystallisation during defrosting.

In the process according to the invention, the embryos are o 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 substances which are capable of penetrating the tissues and providing a good osmotic pressure to obtain correct dehydration 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 contains 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 calls and to increase their stability to freezing. Nor should the pretreatment last too long because otherwise the stability of the embryos to freezing may diminish, as reflected in a reduction in the number of embryos still alive after defrosting when the pretreatment exceeds 36 hours. In the event 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 protective effect of the osmotic pressure agent during long pretreatments 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 24°C, under medium lighting, for example of 150 to 250 lux.

The pretreated embryos are cooled and then frozen to a temperature of -15°C to -40°C. They are preferably frozen to a temperature of approximately -20 °C. The freezing process may be carried out by transferring the embryos with the pretreatment medium to crvotubes 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 1°C per minute, for the temperature range from -5°C to -40°C in order to promote the subsequent resumption of growth of the embryos. For cooling from ambient temperature to a temperature of -6°C, the cooling rate may be more rapid, for example between 1 and 5°C per minute.

The embryos frozen at a temperature in the range from -15°C to -40°C 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 refrigerant. This is because it seems that prolonged storage at -15/-40°C is incompatible with the resumption of growth and hence the viability of the embryos after defrosting. It seems that the temperature of -15/-40°C is too high to ensure complete stoppage of the metabolism of the embryo. If prolonged storage is required, freezing to -15/=40°C 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 -15/40°C 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 -196°C, 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 encapsulated with artificial or natural polymers, for example of the alginate type, to obtain artificial seeds.

The embryos may be defrosted, for example, by immersion of the cryotubes in a water bath at 40°C for 2 minutes. 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-containing substrate, such as sucrose, in a concentration of 1 to 10 gl'1 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 callogenesis or adventitious embvrogenesis.

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 preparation of somatic embryos 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 I’1 sucrose and 0.1 mg I’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 24 °C under 200 lux lighting 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 μχη in size are retained. These small aggregates represent a proembrvonic 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 600 μια 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 (coloration 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 coloration 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 NH4NO3 mg I'1 1650 CaCl2 » 2H2O 440 MgSO4 - 7H20 370 kno3 1900 kh2po4 170 Microelements CoCl2 0.025 CuSO4 · 5Hz0 0.025 FeS04 · 7H20 27.8 Na2 - EDTA 37.3 MnS04 · 4H2O 22.3 KI 0.83 Na2Mo04 0.25 ZnSO4 « 7Hz0 10.6 H3BO3 6.2 Other elements Nicotinic acid 5 Thiamine (vit. BJ 5 Adenine 2 + osmotic pressure agent (pretreatment medium) or assimilable carbon-containing substrates (culture medium) Example 1 Somatic carrot embryos at the torpedo stage, average size 550 μη, obtained as described above, are placed in Petri dishes on a liquid Murashige and Skoog pretreatment medium 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 20°C under 200 lux lighting. They are then transferred to cryotubes containing 1.8 ml pretreatment medium. The crvotubes are placed in a domestic freezer in which their contents are cooled at a rate of 1°C/minute to -6°C and then at a rate of 0.4°C/minute from -6°C to -20°C.

After 24 hours at -20°C, the crvotubes are separated into two groups. The first group is defrosted by immersion of the frozen cryotubes in a water bath at 40°C for 2 minutes. The second group is immersed in liquid nitrogen at -196°C 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 remove the pretreatment medium and placed for 10 days on a liquid Murashige and Skoog culture medium containing 5 gl1 sucrose and free from auxins. They are then transferred to solid culture medium having the same composition.

Control embryos which have not been pretreated or frozen ara placed on the same culture media.

After culture for 10 days on solid medium, the capacity of the embryos to regenerate carrot plantlets is compared.

Among the control embryos, 46% are capable of regenerating a plantlet. For the embryos frozen to »20°C and to -196°C, the regeneration rates are 44% and 43%, respectively.

The capacity of the embryos to regenerate plantlets is thus not affected by freezing providing they have been pretreated.

Example 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 Mm, the other population consists of embryos at the torpedo stage with an average size of 500 Mm.

Five samples (A, 3, C, D and control) each containing approximately 100 embryos are taken from each population of * embryos.

Samples Ah, ,¾ 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 containing 135 gl'1 sucrose for 1 hour at 20 °C under 2 00 lux lighting.

Samples Ah, Bj,, At and 3t are then cooled to -20°C at a rate of approximately 0.5°C/minute. They are then kept at -20°C for 24 hours» Samples Ah and At are defrosted while samples Bh and 3t are immersed in liquid nitrogen at -196°C where they remain for 1 hour before being defrosted.

For comparison, samples Ch and Ct are directly immersed in liquid nitrogen at -196°C after the pretreatment. They are defrosted after 1 hour. For comparison, samples Dj, and Dt are cooled to -20°C at a rate of approximately 0.5°C/minute 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 (%) Sample Comparison Stage A 3 'c ~ --— D Control Heart 88 71 0 0 90 Torpedo 84 67 0 0 91 It can be seen that, in the absence o f pretreatment, the embryos frozen to -20°C (D) do not survive whereas impregnation for one hour in the pretreatment medium p. ensures the survival of almost all the embryos at the heart and torpedo stage frozen to -20°C (A).

Likewise, the embryos do not survive direct immersion in liquid nitrogen, even after pretreatment for one hour (C) .

The embryos frozen to -196°C after pretreatment and (i freezing to -20°C (3) have a very satisfactory viability rate.

Accordingly, if it is desired to store the embryos at -196°C, they have to be subjected to the phases of pretreatment and freezing to - 20°C.

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 3 and also the control samples have developed to the plantlet stage with a clearly differentiated root point and chiorophyIlian cotyledonary apex.

These embryos show no sign of callogenesis or secondary 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 gl1 sucrose for different periods at 20°C under 200 lux lighting.

These embryos are then cooled to -20"C at a rate of approximately 0.5’C/minute. After 24 hours at -20‘C, some of the embryos are immersed in liquid nitrogen and kept there for 1 hour.

After defrosting, the embryos are placed on a Murashige and Skoog liquid culture medium containing 5 gl'1 sucrose.

Control embryos which have not been pretreated or υ frozen are also cultured. The viability rate of the embryos is determined after culture for 10 days.

The following results ar® obtained: Viability rate of embryos at the heart stage (%) Pretreatment time Comparison min. lh 24h 48h 72h Embryos frozen to -20 °C 89 88 87 56 40 Embryos frozen to -196°C 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 min. lh 24h 48h 72h Embryos frozen to -20°C 85 84 82 56 34 Embryos frozen to -196°C 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., l 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.

Example 4 Somatic carrot embryos at the torpedo stage are pretreated for various times on a Murashige and Skoog liquid medium containing 135 gl*1 sucrose at 20°C under 200 lux lighting.

These embryos are cooled and frozen to ·20°C at a rate of approximately 0.5’C/minute.

After 72 hours at -20°C, the embryos are defrosted and placed on a Murashige and Skoog liquid culture medium containing 5 gl’1 sucrose. v 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 It can be seen that the viability rate of the embryos after freezing to -20°C is good for all these pretreatment times. The stability of the embryos to freezing is thus acquired vary rapidly.

Example 5 Somatic carrot embryos at the torpedo stage with an average size of 550 μπι are pretreated and frozen to -20°C.

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 determined as a function of their storage time at -20°C or -3,96 °C» The control embryos which have not been pretreated or frozen have a viability rate of 72%. ι The following results are obtained: For the embryos frozen to =20°C: Storage time 24h 1 months 2 months Viability rate (%) 64 69 0 For the embryos frozen Storage time to -196°C: 24h 1 months 2 months Viability rate (%) 66 67 67 The average viability rate after freezing to -20°C or to -196°C is approximately 67% for storage times of one day or less. This rate remains unchanged after storage for one month at 20°C or at -196°C. After storage for 2 months, there are no survivors among the embryos stored at -20°C whereas, for the embryos stored at -196°C, the viability rate is still the same.

Example 6 Somatic carrot embryos at the torpedo stage with an average size of 500 μπι are pretreated for I hour at 20°C under 200 lux lighting on Murashige and Skoog liquid media having a sucrose concentration varying from 35 gl1 to 205 gl’1.

The embryos are then cooled and frozen to -20°C. After 1 hour at 20°C, 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 concentration of the pretreatment medium.

The following results are obtained: 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 concentration of 100 or 170 gl'1.

The viability of the embryos is affected when the sucrose concentration becomes too high (2 05 gl1) or too low (35 gl'1) .

Claims (11)

1. A storage process for vegetable embryos, characterized in that the embryos are pretreated on a culture medium containing an osmotic pressure agent and, after pretreatment, are cooled and frozen to a temperature in the range from -15°C to —40 °C»

2. A process as claimed in claim 1, characterized in that the osmotic pressure agent is sucrose.

3. A process as claimed in claim 2, characterized in that the pretreatment medium contains the sucrose in a concentration of 75 to 190 gl’ 1 .

4. A process as claimed in claim 1, characterized in that the embryos frozen to -15/-40°C are immersed in a liquid refrigerant.

5. A process as claimed in claim 1, characterized in that the pretreatment is carried out for 30 seconds to 36 hours.

6. A process as claimed in claim 1, characterized in that the cooling rate between -6°C and -40°C is between 0.1 and 1°C/minute.

7. A process as claimed in claim 1, characterized in that the pretreatment medium is a Murashige and Skoog medium to which an osmotic pressure agent is added.

8. The application of the process claimed in claim 1 to the production of plantlets, characterized in that the frozen embryos are defrosted and washed to remove the pretreatment medium and the washed embryos are cultured on a culture medium for development into plantlets.

9. The application of the process claimed in claim 1 to the production of artificial seeds, characterized in that the frozen embryos are encapsulated in artificial or natural polymers.

10. Artificial seeds obtained in accordance with claim 9.

11. A process as claimed in Claim 1 for storing vegetable embryos substantially as hereinbefore described by way of Example.