JPH0987102A - Freezing and preserving of plant cell and thawing of the frozen plant cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the freezing and preserving methods of plant cell, high in the survival rate of the plant cell on the thawing of the frozen plant cell and useful for the preservation of the species of the plant. SOLUTION: The characteristic of the method for freezing and preserving a plant cell comprises using sucrose, glycerol, ethylene glycol and dimethylsulfoxide(DMSO) as freezing-protecting agents, or using a modified freezing process by cooling the plant cell from the ordinary temperature to -20 deg.C at a cooling rate of 0.1-0.5 deg.C/min, cooling the plant cell from -20 deg.C to -80 deg.C at a cooling rate of 0.3-1.0 deg.C/min and subsequently quickly freezing the cooled plant cell in nitrogen liquid. Further, this invention relates to the method for quickly thawing the frozen flower at a thawing rate of 35-42 deg.C/min.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for cryopreserving and thawing plant cells, which has a high survival rate during freeze-thawing and is useful for preservation of plant species.

[0002]

2. Description of the Related Art Semi-permanent preservation of gene sources of useful plants such as agricultural plants is a raw material for the development of new varieties.
It is also important to prevent the extinction of rare species.

For this reason, various plant preservation studies have been conducted. Among varieties of plants that produce seeds, those that can be preserved for a long period of time should be preserved. Frozen storage of
It is optimal because there is no genetic change or reduction in differentiation ability that can occur during passage preservation.

[0004]

However, in the conventional cryopreservation method, the survival rate after thawing was about 40% at the maximum, and the yield was poor.

Therefore, an object of the present invention is to provide a cryopreservation method and a thawing method which have a high survival rate when thawing.

[0006]

Means for Solving the Problems In view of such circumstances, the present inventors have conducted diligent research and found that a plant cell is frozen using a cryoprotective agent containing a specific component as shown below, or a frozen treatment is performed. The inventors have found that the survival rate can be significantly improved by devising a method of lowering the temperature of (1) and freezing plant cells or rapidly thawing frozen plant cells at 35 to 42 ° C, and thus completed the present invention.

That is, the present invention relates to a plant cell containing sucrose,
It is intended to provide a method for cryopreserving plant cells, which comprises freezing in the presence of a cryoprotective agent containing glycerin, ethylene glycol and dimethyl sulfoxide.

From room temperature to -20 ° C., 0.1 to
Cooling at a cooling rate of 0.5 ° C / min, -20 ° C to -80 ° C
The present invention provides a method for cryopreserving plant cells, characterized by cooling at a cooling rate of 0.3 to 1.0 ° C / min, and then rapidly freezing in liquid nitrogen.

Further, the present invention provides a method for thawing plant cells, which comprises thawing frozen plant cells rapidly at 35 to 42 ° C.

Furthermore, the present invention provides a cryoprotective agent for plant cells containing sucrose, glycerin, ethylene glycol and dimethyl sulfoxide.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The plant cell plant to which the method of the present invention can be applied may be a herbaceous plant or a woody plant. This includes, for example, agricultural plants, medicinal plants, and ornamental plants, and among them, it is particularly useful for cells of plants that do not produce seeds or are difficult to produce.

The plant cells to which the method of the present invention can be applied include plant cells isolated by a conventional method, cells grown after cell culture after isolation, plant cells protoplastized by a conventional method, and cell fusion. Examples include fused cells obtained by the method described above.

The cryoprotective agent used in the present invention contains sucrose, glycerin, ethylene glycol and dimethylsulfoxide (DMSO). Each component contains 30 to 50% by weight of sucrose before freezing (hereinafter,
Simply indicated by "%"), glycerin 20-40%, ethylene glycol 10-20%, DMSO 10-20.
Those contained in the range of% are preferable. Further, the cryoprotective agent may contain other components such as medium components.
Although it has been known that each component used in the cryoprotective agent of the present invention has a cryoprotective action, it is not possible to significantly improve the effect by combining these four kinds and combining the four kinds. Not known at all.

To carry out the freeze treatment of plant cells for the purpose of cryopreservation using the cryoprotective agent of the present invention, first, the plant cells are incubated in the cryoprotective agent at around 0 ° C., and intracellular water is added. Replace most of the with a protectant. The substitution is preferably carried out under the condition that the cryoprotective agent is stored at 80% or more, preferably 90% or more. Incubation time is suitably 10 minutes to several hours.

Thus, the plant cells treated with the cryoprotective agent of the present invention may be directly subjected to quick freezing with liquid nitrogen, but by combining the freezing method of the present invention described below, Further satisfactory results can be obtained.

The freezing method of the present invention is a method of freezing while lowering the temperature at once without controlling the temperature at once, and first, at a temperature of 0 ° C to -20 ° C from the start of cooling, Cooling at a cooling rate of 0.5 ° C./min, then at −20 ° C. to −80 ° C., cooling at a cooling rate of 0.3 to 1.0 ° C./min, then in liquid nitrogen at −198 ° C. By freezing rapidly with, it is possible to prevent ice crystallization of intracellular water. After quick freezing, it can be stored semipermanently by storing it in liquid nitrogen.

The thawing method of the present invention is a method of rapidly thawing at 35 to 42 ° C. The thawing temperature is preferably 38 to 40 ° C, particularly preferably around 38 ° C.

When the conventional thawing method, that is, when the temperature is gradually raised, ice crystals are formed on the way and the cells are often damaged, the rapid thawing in lukewarm water at 35 to 42 ° C. The formation of ice crystals can be prevented, and damage to frozen cells can be prevented.

The frozen plant cells used in the thawing method of the present invention may be cells frozen by a conventional method, but if the cells frozen by the cryopreservation method of the present invention are thawed by this thawing method, the Survival rate is improved.

[0020]

EFFECTS OF THE INVENTION According to the method for cryopreserving plant cells and the method for thawing the plant cells of the present invention, the survival rate of the plant cells upon thawing is high, and it is useful as a method for preserving seeds.

[0021]

EXAMPLES The present invention will be described in more detail below with reference to examples.

Example 1 1. Collection and isolation of rice cells From rice (Oryza sativa L. cv), Toyonen Waseda, 50 million stone and Norin No. 8 were selected as samples.
These seeds were sterilized, and AA agar medium (glutamine 876 mg / L, asparagine 266 mg / L, arginine 174 mg / L, glycine 7.5 mg / L, respectively) was sterilized.
2,4-D 1 mg / L, kinetin 0.2 mg / L, G
A3 0.1 mg / L, sucrose 30 g / L, agar 8
Then, callus was induced from the scutellum at 25 ° C. in the dark. The induced callus was subcultured every month in the same medium, and the proliferated callus was treated with AA liquid medium (glutamine 876 mg / L, asparagine 266 mg).
/ L, arginine 174mg / L, glycine 7.5mg
/ L, 2,4-D 1 mg / L, kinetin 0.2 mg /
L, GA3 0.1 mg / L, sucrose 20 g / L, pH
5.7) and established a suspension culture cell line. The suspension culture cells were passaged every two weeks.

2. Incubation step with a normal protective agent 1. AA containing 1.2M sucrose was added to the cultured cells isolated in
The cells were precultured in the medium for 17 hours in the dark. Then, incubate on ice (0 ° C.) for 10 minutes. Then, as a cryoprotectant, AA liquid medium (pH 5.7) containing 30% glycerin and 1.2 M sucrose was added so that the glycerin concentration became 5% as a whole, and further 1% on ice (0 ° C).
I kept it for hours.

3. Freezing Step (Temperature Control Method) After the incubation step, the sample was placed in an ampoule, and then, a 0.1.
Cool to -20 ° C at a speed of 1-0.5 ° C / min,
Next, it was cooled to −80 ° C. at a speed of 0.3 to 1.0 ° C./min, and then frozen and stored in liquid nitrogen.

4. Use of protective agent of the present invention (vitrification method) AA containing 1.2M sucrose was added to the cultured cells isolated in
It was pre-cultured in the liquid medium for 17 hours in the dark. Then
Incubate on ice (0 ° C) for 10 minutes, then
The cryoprotective agent of the present invention (AA medium containing 1.2 M sucrose, 30% glycerin, 15% ethylene glycol, 15% DMSO) was added so that the protective agent had a concentration of 50%, and the mixture was incubated on ice for 5 minutes, Further, thereafter, a cryoprotective agent was added so as to have a concentration of 85%, and then a protective agent was sequentially added so that the protective agent had a concentration of 98.5%. This was left to stand on ice for 20 minutes, then sealed in an ampoule, rapidly frozen in liquid nitrogen, and stored frozen in liquid nitrogen.

[5] Thawing step By each method, the cryopreserved ampoule encapsulating the cells was rapidly thawed in a warm water bath at 38 to 40 ° C.

6. Measurement of the survival rate of thawed cells The survival rate of cultured cells after thawing was measured by the TTC method, which is determined from the activity of dehydrogenase present in the membrane system. That is, the thawed cells contained 0.05M phosphate buffer (pH: 0.6% 2,3,5-triphenyltetrazolium chloride).
7.4) 3 ml and 100 mM Na succinate solution 0.65 ml
Was added, the mixture was degassed for 5 minutes, and incubated at 25 ° C. in the dark for 17 hours. The product formazan was extracted with 95% ethanol, centrifuged at 2000 × g for 3 minutes, the absorbance of the obtained supernatant at 530 nm was determined, and the percentage of the ratio to the control was taken as the survival rate. The results are shown in Table 1.

[0028]

[Table 1]

7. 4. Reculture of thawed cells The cells thawed in (3) were directly washed and placed on a double filter paper on AA agar medium and left overnight. The next day, the agar medium was replaced with a new one, and the cells were further cultured at 25 ° C. in the dark for 3 days. After replacing the agar medium with a new one again and culturing for 1 week, only the cells were transplanted to AA agar medium, cultivated, and turned into callus. The growth was good.

8. Redifferentiation of thawed cells 7. Callus regrown in N6 agar for regeneration (3%
Sucrose, 0.8% agar, 0.3ppm NAA, 0.5ppm
It was succeeded in forming a green spot having an embryogenic ability by transplanting to BAP (including BAP) and culturing. Furthermore, a regenerated individual was successfully obtained by transplanting into a new medium for regeneration and culturing for 20 days.

Claims (6)

[Claims] 1. A method of cryopreserving plant cells, which comprises freezing the plant cells in the presence of a cryoprotective agent containing sucrose, glycerin, ethylene glycol and dimethylsulfoxide. 2. From room temperature to -20 ° C., 0.1 to 0.5.
A plant characterized by being cooled at a cooling rate of C / min, from -20 ° C to -80 ° C at a cooling rate of 0.3-1.0 ° C / min, and then rapidly frozen in liquid nitrogen. Method for cryopreserving cells. 3. The cryopreservation method according to claim 2, which comprises cooling and freezing in the presence of the cryoprotective agent according to claim 1. 4. The cryopreservation method according to claim 1, wherein the plant cells are rice cultured cells. 5. A cryoprotective agent for plant cells, which comprises sucrose, glycerin, ethylene glycol and dimethylsulfoxide. 6. A method for thawing plant cells, which comprises rapidly thawing frozen plant cells at 35 to 42 ° C.