WO2006095439A1

WO2006095439A1 - Ultra-low temperature storage technique for cultured plant cells

Info

Publication number: WO2006095439A1
Application number: PCT/JP2005/004363
Authority: WO
Inventors: Daisuke Shibata; Yoichi Ogawa; Hideyuki Suzuki; Nozomu Sakurai; Kumiko Mori
Original assignee: Kazusa Dna Research Institute Foundation
Priority date: 2005-03-11
Filing date: 2005-03-11
Publication date: 2006-09-14

Abstract

Ultra-low temperature storage is performed by employing pre-freezing technique using a cryoprotective solution comprising glycerin, sucrose and proline blended in a suitable manner. By using this method, procedures for the treatment of a cultured plant cell line (collection of cells by centrifugation/addition of a cryoprotective solution to the cells and resuspension of the cells in the solution/dispensation of the resuspended solution into Cryogenic vials) can be continued to operate at ordinary temperature for at least 120 minutes. Moreover, by using the cryoprotective solution in the cryoprotection treatment, ultra-low temperature storage of cultured plat cells can be achieved conveniently using an inexpensive, readily available cryogenic preservative container or EPS tube rack without using any expensive program freezer. The ultra-low temperature storage method using the cryoprotective solution is applicable to a wide range of plant species and a wide range of cultured cell lines.

Description

Ultra low temperature storage technology for plant cultured cells

Technical field

[0001] The present invention relates to a technique for cryogenic storage of plant cultured cells.

Background art

[0002] Plant cultured cells are undifferentiated and uniform cell aggregates, and have been established from various plant species so far. Since these cultured cells are grown in a sterile medium in an artificial environment, it is easy to conduct experiments under various conditions, including cell cycle, nutrient transfer, defense reaction against pests, metabolites. It is widely used in various plant studies such as biosynthesis. Currently, the BY-2 strain of tobacco (Nagata, et al: Int Rev Cytol 132: 1-30, 1992) is the most widely used for such studies.

Cultured cells can be isolated from Arabidopsis thaliana, whose genome base sequence is completely deciphered in model plants in plant research, and are expected to be used as new standard culture cells in plant research. The T87 strain (Axelos, et al: Plant Physiol Biochem 30: 123-128, 1992; Non-patent document 1) established from Arabidopsis thaliana seedlings is one example. Due to its superior features such as the ability to isolate and regenerate protoplasts, it has begun to be used for various plant studies. However, in order to maintain cultured cells, it is indispensable to continue transplantation to a fresh medium at regular intervals of several days to several weeks, that is, to perform passage. The labor, cost, and equipment required for this work are tremendous, and there is always a risk of loss of the cultured cell line due to contamination with bacteria and improper operation.

[0003] One means for avoiding the drawbacks associated with maintaining plant cultured cells as described above is a so-called ultra-low temperature storage method, in which cells are stored at extremely low temperatures using liquid nitrogen or the like. Research on ultra-low temperature storage of plants was started in the 1960s, and various methods corresponding to various plant species and cultured cell lines have been established to date. The established methods are the pre-freezing method (Withers and King: Cryo-Lett 1: 213-220, 1980) and the vitrification method (Uragami, et al: Plant Cell Rep 8: 418-421, 1989). There are two types of special machines. However, there are drawbacks such as the need for a vessel, complicated operation, and limitations on applicable plant species and cultured cell lines.

[0004] Ultra low temperature preservation method of cultured Arabidopsis cell is described by Ford (Plant Cell Rep 8: 534-537,

1990; Non-patent document 2), Ribeiro, et al. (Plant Sci 115: 115-121, 1996; Non-patent document 3), Menges and Murray (Plant J 37: 635-644, 2004; Non-patent document 4) Are reported by the preliminary freezing method. The pre-freezing method is a method in which a target plant tissue / cell is gently cooled at a constant speed, that is, pre-freezing, before storage at an ultra-low temperature. Pre-freezing induces freezing of water outside the cell (ice nucleation) and intracellular dehydration associated with its growth, avoiding physical and physiological damage to cells due to intracellular freezing, and storage at ultra-low temperatures. Is possible. However, pre-freezing must be performed while maintaining an essentially constant cooling rate, and the use of an expensive dedicated program freezer is essential to achieve this. Therefore, ultra-low temperature storage can only be performed at a limited research institution with a program freezer.

In addition, before and during pre-freezing, it is essential to treat the cells with agents collectively called frost protection agents. Commonly used freeze protection agents are dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, etc., but many of these freeze protection agents are extremely toxic to plant cells. In the process of using strong anti-frost protection agents, processing under low temperature (generally on ice), mild addition of chemicals, mixing, and strict processing time! / Can be forced to perform very complicated operations.

In addition, tissues and cells to be stored at ultra-low temperatures are pre-cultured in a medium supplemented with plant hormones or high-concentration sugars or under low temperature to obtain a physiological / physical state that can withstand frost damage protection and subsequent pre-freezing. It is necessary to keep. These series of drawbacks are major barriers that make it difficult to achieve large-scale, high-throughput, and ultra-low temperature storage in a wider range of research institutes, which will be required in future plant research.

[0005] In order to compensate for the disadvantages of the pre-freezing method, it is commercially available for use with frost damage protective agents with relatively low phytotoxicity and for animal cells! Alternative use of simple cryopreservation containers has also been reported. The scope of application of these methods is limited to limited species of the genus Citrus. Therefore, in this study, we first used Arabidopsis cultured cell strain T87 as a model plant material. We attempted to establish an ultra-low-temperature storage method that can be applied to simple, large-scale and high-throughput applications. In addition, established methods were introduced into various plant cell lines and their broad applicability was confirmed.

Non-Patent Document 1: Axelos M, Curie C, Mazzolini L, Bardet C, Lescure B (1992) A protocol for gene expression in Arabidopsis thaliana protoplasts isolated from cell suspension cultures. Plant Physiol Biochem 30: 123—128

Non-Special Terms 2: Ford KG (1990) Plant regeneration from Arabidopsis thaliana protoplasts. Plant Cell Rep 8: 534—537

Non-Patent Document 3: Ribeiro RCS, Jekkel Z, Mulligan BJ, Cocking EC, Power JB, Davey MR, Lynch PT (1996) Regeneration of fertile plants from cryopreserved cell suspension of Araoidopsis thaliana (L.) Heynh. Plant Sci 115: 115 —121

Non-Special Reference 4: Menges M, Murray J AH (2004) Cryopreservation of transformed and wild-type Arabidopsis and tobacco cell suspension cultures. Plant J 37: 635—644 Disclosure of the Invention

Problems to be solved by the invention

In the cryopreservation of plant cell culture in the pre-freezing method, the major drawback that prevents the large-scale 'high-throughput' is the complexity of the frost damage protection process. Commonly used frost damage prevention solutions contain DMSO, ethylene glycol, or propylene glycol, but these frost damage protection agents are highly toxic to cells with high penetration into cells. (Fuller: Cryo-Lett 25: 375-388, 2004) o This is a low temperature treatment (generally on ice), a gentle addition of the drug, mixing, and a set processing time. It is a factor that requires extremely complicated operations such as strict adherence.In addition, ultra-low temperature storage by the pre-freezing method requires the use of a program freezer, which is an expensive dedicated device, and this is the reason for most research institutions. This is the reason why the ultra-low temperature storage cannot be carried out.

Furthermore, the ultra-low temperature preservation methods established so far have a problem that applicable plant species and cultured cell lines are limited. Means for solving the problem

[0007] First, the present inventors combined glycerin, sucrose, and proline, which are frost damage protective agents known to be usable at room temperature because of their low penetration into cells and low toxicity. We tried to develop a frozen frost damage prevention solution and a frost damage prevention treatment method using it. Therefore, the main feature of the present invention is that the pre-freezing method is performed using a frost damage prevention solution appropriately mixed with glycerin, sucrose, and proline.

In addition, the present inventors can perform a pre-freezing method without using a program freezer if a storage container such as an expanded polystyrene (EPS) tube rack is used, and achieve a high cell viability of 95%. I found out that I can do it. Therefore, the main feature of the present invention is that the pre-freezing method is performed without using the product freezer.

Furthermore, the present inventors have found that the pre-freezing method using the frost damage prevention solution of the present invention can be applied to various plant cultured cells. Therefore, the main feature of the present invention is that a pre-freezing method is performed on various plant species and cultured cell lines using a frost damage prevention solution appropriately mixed with glycerin, sucrose, and proline.

[0008] More specifically, the present invention relates to the invention described in the following (1) to (16).

(1) A frost damage prevention solution for plant cells containing the following (a) — (c):

(a) 1 3M glycerin,

(b) 0.2-0.8M sucrose, and

(c) 0.4—400 mM proline.

(2) The frost damage prevention solution according to (1), comprising the following (a) — (c).

(a) 2M glycerin,

(b) 0.4M sucrose, and

(c) 8.7—86.9 mM proline.

(3) The frost damage prevention solution according to (1), wherein the plant cells are cultured Arabidopsis thaliana cells.

(4) The frost damage prevention solution according to (3), wherein the Arabidopsis cultured cells are T87 strain.

(5) Use of the frost damage prevention solution of (1) in a cryogenic storage method.

(6) The use according to (5), wherein the cryoprotective solution contains the following (a) — (c).

(a) 2M glycerin, (b) 0.4M sucrose, and

(c) 8.7—86.9 mM proline.

(7) Plant cell freezing method by the following steps (A) — (D):

(A) a step of suspending plant cells in the anti-frost damage solution of (1),

(B) The step of putting the small container containing the suspension of step (A) into a storage container,

(C) the step of placing the storage container of step (B) into a freezer, and

(D) The process of removing the storage container from the freezer after a predetermined time.

(8) The freezing method according to (7), wherein the plant cell is a callus.

(9) The freezing method according to (7), wherein the storage container in step (B) is a simple cryopreservation container for animal cells.

(10) The freezing method according to (7), wherein the storage container of step (B) is an expanded polystyrene tube rack.

(11) The freezing method of (7), wherein the temperature in the freezer of step (C) is 20 40 ° C.

(12) The freezing method according to (7), wherein the predetermined time in step (D) is 4 to 10 hours.

(13) The freezing method according to (7), comprising a step of shaking the suspension for 60 minutes after step (A), and further, a predetermined time in step (D) is 6 to 8 hours.

(14) The freezing method according to (7), wherein the plant cell in step (A) is derived from any of Arabidopsis thaliana, carrot, Miyakodasa, tobacco, and rice.

(15) The freezing method according to (7), wherein the plant cell in step (A) is derived from either Arabidopsis thaliana CoH) or tobacco BY-2.

(16) A plant cell culture frozen by the freezing method of (7).

[0009] Aspects of the present invention are as follows, for example.

Suspend plant cells in a maintenance medium containing 2M glycerin, 0.4M sucrose, and 86.9mM proline, and dispense to the nozzle. Place the vial containing the cell suspension in a polystyrene foam black and place the entire rack in a freezer at 30 ° C. After 6 hours, remove the rack from the freezer. Remove the vial from the rack and immediately place it in an aluminum cane. Immediately immerse it directly in liquid nitrogen for rapid cooling and store it in liquid nitrogen or a freezer below 80 ° C. The invention's effect

[0010] Ultra low temperature storage by the pre-freezing method cools cells at a constant cooling rate (generally 1 ° C / min). This method induces extracellular freezing and intracellular freezing and dehydration associated with the growth of ice crystals, and avoids physical and physiological damage caused by intracellular freezing. is there. In order to effectively freeze and dehydrate the cells during pre-freezing and to withstand the low temperature exposure, the cells must be treated with a solution containing anti-frost protection agents. . Commonly used frost damage protection solutions are extremely toxic to cells, such as DMSO, ethylene glycol, and propylene glycol, and contain frost damage protection agents, so strict treatment at low temperatures is required. So far, the ability to protect against frost damage with relatively low phytotoxicity has been reported. The scope of its application is limited to Citrus sinensis embryonic heart cells.

The present inventors have developed a frost damage prevention solution appropriately formulated with glycerin, sucrose, and proline, and have demonstrated that a high survival rate can be achieved in many plant cultured cells. It was found that using the developed frost damage prevention solution, frost damage protection treatment can be performed at room temperature, and that the survival rate is not affected even if treatment is performed for 0 minutes to at least 120 minutes. These characteristics indicate that the frost damage prevention treatment using the solution of the present invention is very suitable for large-scale high-throughput storage of ultra-low temperature storage.

In addition, the use of commercially available cryopreservation containers or EPS tube racks for pre-freezing as an alternative to program freezers eliminates the need for the introduction of very expensive, dedicated functions and ineffective prod- ule freezers. This facility enables ultra-low temperature storage of cultured plant cells. Alternative use of simple cryopreservation containers that are commercially available for animal cells has also been reported by Engelmann, et al. (Cryo-Lett 15: 53-58, 1994), but its scope of application is Citrus As shown here, the survival rate is clearly lower compared to the case of using EPS tube racks that are cheaper, easier to obtain, and capable of processing multiple specimens. Pre-freezing conditions are also limited.

However, most of the existing cryopreservation methods for plant cultured cells require the use of cells that have been pre-cultured at low temperatures or in a medium supplemented with sugar or plant hormones. If used, all of the cultured cells provided this time could be stored at an ultra-low temperature without pre-culture. As described above, the series of technologies related to ultra-low temperature storage of plant culture cells developed this time is rich in novelty and yet It can be said that various viewpoints are also superior compared to technology.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] The frost damage prevention solution of the present invention contains 1 to 3M glycerin. Preferably, the anti-frost protection solution of the present invention contains 1.5-2.5M glycerin. More preferably, the anti-frost damage solution of the present invention contains 2M glycerin.

The anti-frost damage solution of the present invention contains 0.2-0.8M sucrose. Preferably, the anti-frost protection solution of the present invention contains 0.3-0.6M sucrose. More preferably, the frost damage prevention solution of the present invention contains 0.4 M sucrose.

The anti-frost damage solution of the present invention contains 0.4-400 mM proline. Preferably, the anti-frost damage solution of the present invention contains 0.8-200 mM proline. More preferably, the frost protection solution of the present invention contains 8.7-86.9 mM proline.

[0012] The ultra-low temperature storage method is a method for storing genetic resources and the like under an ultra-low temperature. The ultra-low temperature generally means about 25 ° C or less, preferably about 80 ° C or less. The lower limit of the ultra-low temperature is not particularly limited, but is generally about 196 ° C.

The pre-freezing method is an ultra-low-temperature storage method in which pre-freezing is performed and then stored at an ultra-low temperature. Pre-freezing is a method in which a target plant tissue 'cell is gently cooled and frozen before being stored at an ultra-low temperature. Say to do.

A freezing protection agent is a substance used to protect against the effects of freezing, and is added to the medium mainly to prevent the formation of large ice crystals in the cells. DMSO, ethylene glycol, propylene glycol, etc. are known as commonly used frost protection agents.

A frost damage prevention solution is a medium with frost damage prevention agent added. When a cultured cell callus is suspended in the frost damage protection solution, it is added to the medium with a frost damage protection agent suspended. In comparison, frost damage is reduced. Although the reduction rate of frost damage can be measured from various indicators, cell viability is generally used as an indicator. Cell viability can be measured by the Regrowth Test method of Ishikawa, et al. (Plant Sci 107: 83-93, 1995).

The program freezer refers to a freezer that can control the temperature decrease rate. In the present invention, a small container containing a suspension of cultured cells or callus refers to a sealable container made of polypropylene or glass. The capacity of the small container is not particularly limited. Preferably, the volume is about 0.1 to about 10 mL, more preferably about 1.0 to about 5.0 mL.

The suspension of cultured cells or callus may be prepared in a small container that need not be prepared prior to being placed in the small container. In other words, the cultured cell or callus suspension is put in a small container in advance, and the cultured cell or callus solution to be suspended is placed in the small container in advance, and then the cultured cell or callus is suspended later. Callus may be added and suspended in a small container.

In the present invention, the storage container refers to a container that can store a plurality of small containers. The number that can be stored is not particularly limited, but is preferably 16 or more, and more preferably 32 or more. Because the storage container needs to accurately and gently convey the temperature in the freezer to the small container, the storage container and the small container must be in contact. The contact area between the storage container and the small container is preferably 50% or more of the surface area of the small container, and more preferably 75% or more of the surface area of the small container. The whole or part of the storage container must be filled with a material with low thermal conductivity. The part is preferably 50% or more of the volume of the preservation solution, more preferably 80% or more, and further preferably 90% or more. Substances with low thermal conductivity are those with a thermal conductivity of 0.5 W / m / K or less, and include, for example, isopropyl alcohol and EPS. The space inside the freezer and outside the storage container must not touch the small container. That is, when the temperature in the freezer is transferred to the small container, the whole or part of the storage container must be directly or indirectly mediated by a low thermal conductivity substance. Among commercially available products, Mr. Frosty "(Nalgene), a simple cryopreservation container for animal cells, can be used as a storage container, but the number of small containers that can be stored is limited. EPS tube racks such as HS4283 (Heathrow Scientific) and SD-14 (Marem) can be used.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples. It should be noted that all prior art documents cited in this specification are incorporated herein by reference.

Example 1 Plant material cultivation

1. Arabidopsis suspension culture cell line T87

A suspension cultured cell strain T87 (Axelos, et al: Plant Physiol Biochem 30: 123-128, 1992) derived from Arabidopsis thaliana (L.) Heynh. Ecotype Columbia seedlings was used. Suspension cultured cells were obtained from mJPL3 medium [stock A '(Axelos, et al.) Modified from JPL medium (Jouanneau and Pe'aud-Lenoe "' l: Physiol Plant 20: 834-850, 1967) used in the original report. al, 1992), lZ triple concentration MS inorganic salt (Murashige and Skoog: Physiol Plant 15: 473-497, 1962), MS vitamin, O. lg / L casein coconut hydrolyzate (casamino acid), 15 g / L sucrose , 1 μΜ Naphthalene acetic acid (NAA), 2.5 mM

Using 2- (N-morpholino) ethanesulfonic acid (MES), pH 5.8], swirling shaking culture at 120 rpm was performed at 22 ° C under a 24-hour light period. Subculture is performed every 14 days by filtering suspension culture cells through a stainless steel mesh with a pore size of 500 μm, collecting cells with a nylon mesh with a pore size of 50 μm, and 300 mL of cells with a wet weight of 0.5 g (g WW). Transplanted into lOOmL medium in a volumetric flask. In addition, transformed T87 callus, which was introduced in the mJPL3 medium solidified with 3g / L gellan gum, which had been transfected by the agrobatterium method, was also tested.

2. Arabidopsis suspension culture cells Co 卜 0 strain

The suspension culture cell (Mathur, et al: Plant J 13: 707-716, 1998) derived from seedling roots of AraDidopsis thaliana (L.) Heynh j ecotype Columbia Concentration B5 vitamin (Gamborg, et al: Exp Cell Res 50: 151-158, 1968), lmg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g / L sucrose, 2.5 mM MES , PH 5.8], and rotating shaking culture at 120 ° C / min at 22 ° C in the dark. For subculture, every 7 days, 30 mL suspension culture cells were transferred to 70 mL maintenance medium in a 300 mL flask.

3. Tobacco suspension culture cell BY-2

Tobacco (Nicotiana tabacum L. cv. Bright Yellow 2) suspension culture cell BY-2 (Nagata, et al: Int Rev Cytol 132: 1-30, 1992) is modified with modified LS medium (Nagata, et al. 1992). ) And swirling shaking culture at 130 ° C / min in the dark at 27 ° C. For subculture, 1.5 mL of suspension culture cells were transferred to lOOmL medium in a 300 mL flask every 7 days. 4. Rice suspension culture cells

Suspension cultured cells (Hayakawa, et al: Plant Cell Physiol 31: 1071-1077, 1990) derived from rice (Oryza sativa L. cv. Sasanishiki) embryos are R-2 medium containing lmg / L 2,4-D. (Ohira, et al: Plant Cell Physiol 14: 1113-1121, 1973) was subjected to swirl shaking culture at 25 ° C in the dark at 120 rpm. Subculture is performed every 14 days after filtering through a stainless steel mesh with a pore size of 500 μm, collecting cells with a nylon mesh with a pore size of 50 m, and placing a 0.5 g (g WW) wet cell in a 300 mL flask. Transplanted to the medium.

5. Carrot suspension culture cells

Carrot (Daucus carota L.) suspension culture cells were swirled and shaken at 120 ° C / min in the dark at 25 ° C using R-2 medium containing lmg / L 2,4-D. . Subcultures were transferred every 7 days to 80 mL of medium containing 20 mL of suspension culture cells in a 300 mL flask.

6. Miyakogusa suspension culture cells

Suspension culture cells of Jacobsa (Lotus japonicus (Regel) Larsen ecotype Gifu accession no.B- 129) are maintained medium (MS inorganic salt, B5 vitamin, lmg / L 2,4-D, 0. lmg / L kinetin). , 30g / L sucrose, 2.5mM MES, pH 5.8) was subjected to rotary shaking culture at 120 ° C / min in the dark at 25 ° C. In subculture, cells were collected with a nylon mesh with a pore size of 1000 m every 3 weeks and transplanted to a lOOmL medium containing wet lg (g W.W.) cells in a 300 mL flask.

Example 2

Ultra low temperature storage

1. Freezing damage protection treatment of suspension culture cells

15 mL of suspension culture cells in the early logarithmic growth phase and the stationary phase (Arabidopsis Cd-Ο · Tobacco BY-2 'Carrot 4 days after passage, Arabidopsis Τ87 · Rice 7 days after passage, Miyakogusa 12 days after passage) Dispense into centrifuge tubes and centrifuge at 200 xg for 1 minute at room temperature. After removing the supernatant with decantation or aspiration, the recovered cultured cells are resuspended with 9 times the amount of anti-frost damage solution (2M glycerin, 0.4M sucrose, 0-86.9mM proline), and the cell density is 10 % Cell suspension was prepared. The frost damage prevention solution was prepared by dissolving each component in distilled water or the maintenance medium of each cultured cell. Gently suspend the suspension at approximately 45 rpm Incubated for 0-120 minutes at room temperature with reciprocal shaking to prevent frost damage. The cryopreservation-treated cell suspension was dispensed in 0.5 mL aliquots into ultra-low temperature storage vials (1.2 mL cryovials).

2. Callus freeze protection

Callus in the early logarithmic growth phase and stationary phase were transplanted into cryovials previously dispensed with a freezing protection solution so that the cell density was about 10%. The frost damage protection solution used was the same composition as that for suspension culture cells, and the cells were transplanted using a microspatula.

3. Pre-freezing using program freezer

Immediately place the cryovial containing the cryopreserved cell suspension in the chamber of the program freezer, cool to 35 ° C at a cooling rate of 0.5 ° C / min, and then continue at 35 ° C. 0—pre-frozen by holding for 60 minutes. The pre-frozen cells were immediately attached to an aluminum cane, immediately immersed in liquid nitrogen and rapidly cooled, and stored in liquid nitrogen or in a freezer at a very low temperature.

4. Pre-freezing without using program freezer

Cryovials containing cryoprotective cell suspensions are immediately stored in a simple cryopreservation container (Cryo 1 ° C Freezing Container “Mr. Frosty”; Nalgene) or commercially available EPS Tu Black (HS4283; Heathrow Scientific or SD-14; Marem) was cooled in a freezer at -30 ° C or 80 ° C for 11-8 hours and pre-frozen. The pre-frozen cells were immediately attached to an aluminum cane, immediately immersed in liquid nitrogen for quick cooling, and stored at low temperature in liquid nitrogen or freezer.

5. Re-thawing and re-growth of cryopreserved cells

The cryopreserved cultured cells were immersed in a 35 ° C water bath together with the cryovial and rapidly warmed and thawed by incubating for 2 minutes while shaking at about 180 rpm. The thawed cultured cells were collected in an undiluted 'unwashed suspension by aspiration with a micropipette and dropped directly onto the regrowth medium. As the re-growth medium, a medium used for subculture was solidified with 7 g / L agar and two filter papers were laid. The medium containing the collected cells was incubated in the same culture environment as the subculture. After 1 day of incubation, the cultured cells The sample was transplanted to a medium with the same composition together with one sheet of filter paper, and statically cultured in the same culture environment as the cultured cells tested. The survival rate was measured according to the Regrowth Test method of Ishikawa, et al. (Plant Sci 107: 83-93, 1995).

Example 3

Development of protection treatment using freezing protection solution II

First, ultra-low temperature preservation of Arabidopsis cultured cell strain T87, which was subjected to frost damage prevention treatment using a frost damage prevention solution prepared by adding proline at various concentrations to solutions of 2M glycerin and 0.4M sucrose, was performed. A program freezer was used for pre-freezing, and it was cooled from room temperature (25 ° C) to -35 ° C at a cooling rate of 0.5 ° C / min. The addition of proline was effective at increasing the survival rate from 87 mM (Fig. 1). When using a freezing protection solution supplemented with 86.9 mM proline, the survival rate is as high as about 70%, which is about 1.8 times that of the solution containing no proline, which only contains glycerin and sucrose. Improved.

In addition, this frost damage protection solution can be used and treated at room temperature, that is, in a general laboratory environment, and the effect of the frost damage protection treatment method on the survival rate is clearly low. (Figure 2). When frost damage protection treatment is performed using a solution containing frost damage protection agents such as DMSO, ethylene glycol, propylene glycol, etc., it is possible to carry out the treatment for a specified period of time precisely while shaking the cell suspension. Indispensable, in the frost damage prevention treatment using the frost damage prevention solution of the present invention, the treatment while shaking and the treatment time contributed only to the improvement of the survival rate. On the other hand, in the frost damage protection treatment in a stationary state without shaking, even if treatment was performed from 0 minutes to at least 120 minutes, there was no clear change in survival rate (Fig. 2). ).

In other words, it consists only of glycerin, sucrose, and proline, and is highly toxic and does not contain frost protection agents such as DMSO, ethylene glycol, and propylene glycol! The plant culture cell line to be processed (recovery of cells by centrifugation, addition of anti-freezing protection solution 'dispensing into resuspension one cryovial) can be continuously operated at room temperature for at least 120 minutes. I mean. This ultra-low temperature preservation method can be applied to cells cultured on a solidified medium, that is, callus and cultured cells into which a gene has been introduced. As described above, the frost damage of the present invention It was shown that frost damage protection treatment for the protection solution and the pre-freezing method using it is a technology that can greatly contribute to the large-scale 'high-throughput storage of plant cryogenic cells.

Example 4

Program freezer

Next, the present inventors tried to develop a pre-freezing method using the cryoprotective solution of Example 3 without using a program freezer.

First, it is marketed for ultra-low temperature storage of animal cells! An attempt was made to use "Mr. Frosty" (Nalgene), a simple freezing storage container, as a substitute for the program freezer. As the test material, Arabidopsis thaliana cultured cell strain T87 was used, and the above-mentioned frost damage protection treatment was performed. In other words, the cultured cells collected by centrifugation are resuspended in a freezing protection solution containing 2 M glycerin, 0.4 M sucrose, and 0.87 mM prolinker, and the cell suspension is divided into cryovials without shaking. Noted. Place the cryovial containing the cell suspension in a simple freezing storage container and pre-freeze it in the freezer at 80 ° C specified by the manufacturer. (Figure 3. Pre-freezing for 2 hours showed the highest survival rate, but it was about 30%, which was lower than that of the program freezer. Pre-freezing for more than 3 hours) It was speculated that the plant cells were not resistant to excessive cooling at 80 ° C, as the viability was clearly reduced when this was done, so pre-freezing in a -30 ° C freezer Attempts were made to survive after ultra-low-temperature storage until 2 hours of pre-freezing, but clear survival was observed from 3 hours of pre-freezing, with a high survival rate of about 60% over 6 hours. As you can see, glycerin It was shown that commercially available simple cryopreservation containers for animal cells, such as Nalgene products, can be used for ultra-low temperature storage of plant culture cells by using a frost damage protection solution that also contains sugar, sucrose, and prolinker.

However, commercially available cryopreservation containers have a limited number of nozzles (often a dozen), and Nalgene products are used only 5 times, as well as the need for harmful isopropyl alcohol. Problems remain, such as the need to replace with a new one each time. Therefore, it is cheaper and easier to obtain expanded polystyrene (EPS), so-called expanded polystyrene. We tried to use a tube rack made of steel as a substitute for the program freezer. Two commercially available products capable of accommodating 50 cryovials, HS4283 (Heathrow Scientific) and SD-14 (Marem) were provided as EPS tube racks. Frost damage prevention treatment was performed in the same manner as a commercial cryopreservation container, and preliminary freezing was performed in a freezer at 30 ° C. When the EPS tube rack was used for pre-freezing, both products were found to survive after ultra-low temperature storage for 2 hours from pre-freezing, and it was clear that the survival rate was about 70% after 4 hours. (Figure 3b). This survival rate is remarkably higher than when a commercially available simple cryopreservation container is used for pre-freezing, and it is the same as when pre-freezing is performed under optimum conditions using a program freezer. It was. It was also found that even if the preliminary freezing time was extended to at least 8 hours, the survival rate was not decreased, and it was not necessary to strictly observe the preliminary freezing time as long as the treatment was performed for a certain time or longer. . As described above, by using a frost damage prevention solution consisting of glycerin, sucrose, and proline for frost damage prevention treatment, an inexpensive and easy-to-use EPS tube rack can be used instead of the program freezer, that is, an expensive program freezer is used. It has been shown that plant cultured cells can be stored at ultra-low temperatures without the need to do so.

Example 5

Pre-freezing method without using program freezer to various cultured cell lines] ^ Applying simple cryogenic storage method without using program freezer as described above to ultra-cold storage of a wider range of plant species' cultured cell lines The scope of application of this method was investigated. As cultured cells, Arabidopsis thaliana CoH) strain, carrot, Miyakodasa, tobacco BY-2 strain and five different plant species of rice were used. The frost damage protection solution was composed of 2M glycerin, 0.4M sucrose, and 0.87mM proline, and was not subjected to frost damage protection treatment (incubation) (0 minutes) or 60 minutes with shaking. Pre-freezing was performed in a freezer at 30 ° C using a commercially available simple cryopreservation container (Nalgene) or EPS tube rack (Heathrow Scientific).

For each of the five lines tested, frost damage protection treatment (incubation) is not necessary, but the type of storage container and the appropriate minimum pre-freezing time are different, but for all cultured cells, Survival was observed (Figure 4). Its survival rate is Arabidopsis NA CoH) and tobacco BY-2 strains are extremely high, reaching 95%, and even the lowest Miyakogusa is 50% (cLellan, et ai: aintenance or algae ana protozoa, in Maintenance of Microorganisms and Cultured Cells "(Kirs op BE and Doyle A eds.) Adademic Press, London, pp.183-208, 1991). It is clear that the highest viability can be obtained in all the cultured cell lines tested, regardless of the type of storage container, when the shaking force is applied for 60 minutes and preliminary freezing is performed for at least 6 hours. It became a trap.

As shown above, using the ultra-low temperature storage method developed this time, that is, using a freezing protection solution such as glycerin, sucrose, and prolinker, a commercially available simple cryopreservation container or EPS tube is used as a pre-freeze as an alternative to the program freezer. It was shown that the technique used in the above can be applied to a wide range of plant species that have been difficult in the past.

Industrial applicability

[0019] In ultra-low temperature storage by the prefreezing method using the frost damage protection solution of the present invention, treatment of the cultured plant cell line (recovered cells by centrifugation, addition of the frost damage protection solution to the resuspended cryovial) Dispensing) can be continuously operated at room temperature for at least 120 minutes. In addition, by using the frost damage prevention solution of the present invention for frost damage protection treatment, it is possible to easily cultivate plants using a cryopreservation container or an EPS tube rack that is inexpensive and easily available without using an expensive program freezer. Allows ultra-low temperature storage of cells. Furthermore, the method disclosed in the present invention can be applied to a wide range of plant species and cultured cell lines.

Brief Description of Drawings

[0020] FIG. 1 is a graph showing the effect of proline addition to a frost damage prevention solution on the cryogenic storage of Arabidopsis cultured cell strain T87 (Example 3). T87 cells were suspended in frost damage protection solution (2M glycerin, 0.4M sucrose, 0-86.9 mM proline), and then pre-frozen to 35 ° C using a program freezer at a cooling rate of 0.5 ° C / min.

FIG. 2 shows the effect of frost damage protection treatment time on the cryogenic storage of Arabidopsis cultured cell strain T87 (Example 3). Suspend T87 cells in anti-frost damage solution and leave or shake. Protected against frost damage for 0-120 minutes with shaking. After the frost damage prevention treatment, it was pre-frozen to 35 ° C at a cooling rate of 0.5 ° C / min using a program freezer.

FIG. 3 is a diagram showing ultra-low temperature preservation of Arabidopsis cultured cell strain T87 without using a program freezer (Example 4). In (a), cryopreservation container 〃 Mr. Frosty ”(Nalgene) was used and pre-frozen for 18 hours at 30 ° C or 80 ° C. In (b), expanded polystyrene (EPS) tube rack (HS4283 And SD-14) and pre-frozen at -30 ° C for 1-8 hours.

FIG. 4 is a diagram showing application of the cryogenic storage method without using a program freezer to various cultured cell lines (Example 5). (A), (b) Arabidopsis cultured cells Cd-0 strain, (c), (d) carrot cultured cells, (, (1) Miyagigusa Gifo cultured cells, (g), (h) tobacco cultured cells Rice cultured cells were used for BY-2 strains, (i) and (j), and each cultured cell was suspended in frost damage protection solution and subjected to frost damage protection treatment for 0 minutes or 60 minutes with shaking. After that, (a), (c), (e), (g), (i) use a cryopreservation container "Mr. Frosty" (Nalgene), and (b), (d), (1), (H), (j) are pre-frozen for 2-6 hours at 30 ° C using EPS tube rack HS4283 (Marem).

Claims

The scope of the claims

[I] Frost protection solution for plant cells containing the following (a) — (c):

(a) 1 3M glycerin,

(b) 0.2-0.8M sucrose, and

(c) 0.4—400 mM proline.

[2] The frost damage prevention solution according to claim 1, comprising the following (a) — (c):

(a) 2M glycerin,

(b) 0.4M sucrose, and

(c) 8.7—86.9 mM proline.

[3] The frost damage prevention solution according to claim 1, wherein the plant cell is a cultured cell of Arabidopsis thaliana.

[4] The frost damage prevention solution according to claim 3, wherein the cultured cell of Arabidopsis thaliana is T87 strain.

[5] Use of the frost damage prevention solution according to claim 1 in a cryogenic storage method.

[6] The use according to claim 5, wherein the cryoprotective solution comprises the following (a) — (c):

(a) 2M glycerin,

(b) 0.4M sucrose, and

(c) 8.7—86.9 mM proline.

[7] Method of freezing plant cells by the following steps (A) — (D):

(A) a step of suspending plant cells in the frost damage prevention solution according to claim 1,

(C) the step of placing the storage container of step (B) into a freezer, and

8. The freezing method according to claim 7, wherein the plant cell is callus.

[9] The freezing method according to claim 7, wherein the storage container in the step (B) is a simple cryopreservation container for animal cells.

[10] The freezing method according to claim 7, wherein the storage container in the step (B) is an expanded polystyrene tube rack.

[II] The freezing method according to claim 7, wherein the temperature in the freezer of step (C) is 20 40 ° C.

[12] The method of freezing according to claim 7, wherein the predetermined time in step (D) is 4 to 10 hours. Law.

[13] The freezing method according to claim 7, further comprising a step of shaking the suspension for 60 minutes after step (A), and further, the time specified in step (D) is 6 to 8 hours .

[14] The freezing method according to claim 7, wherein the plant cell strength in the step (A) is derived from any one of Arabidopsis thaliana, carrot, Miyakodasa, tobacco, and rice.

[15] The freezing method according to claim 7, wherein the method is derived from either plant cell force Arabidopsis CoH) or tobacco BY-2 strain in step (A).

16. A culture of plant cells frozen by the freezing method according to claim 7.