JPH11266726A - Freeze preservation and defrosting of seaweed gametophyte - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject method capable of increasing a survivability of a seaweed gametophyte after freeze-preserved and defrosted and preventing a reduction of survivability after cultured by suspending a gametophyte isolated from a seaweed in a specific freezing medium, subjecting it to a preparatory freeze for dehydrofreezing and immediately immersing it in a liquid nitrogen for quick-freeze. SOLUTION: This method for freeze preservation of a seaweed gametophyte comprises; suspending a gametophyte isolated from a seaweed belonging to e.g. the genera Eisenia, Laminaria or Kjellmaniella gyrata Miyabe in a freezing medium obtained by dissolving 10% of ethylene glycol and 10% of proline into 100% of seawater, preparatorily freezing it to -20 to -60 deg.C at the rate of <=1 deg.C/min. for dehydrofreezing, and immediately immersing it in a liquid nitrogen for quick-freezing. The seaweed gametophyte in freeze preservation is pref. defrosted by stirring it in a water bath at 40 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cryopreservation method for immersing seaweeds, particularly seaweeds belonging to the genus Laminariaceae (Eisenia), in liquid nitrogen (-196 ° C.) for quick freezing.

[0002]

2. Description of the Related Art With the development of biotechnology, human varieties of various organisms can be manipulated at the genetic stage, and the preservation of genes expressing the diversity of organisms has become essential. For this reason, a seed storage facility equipped with a cold storage for storing seeds has been created as a gene bank for agricultural products that propagate seeds. For crops that do not produce seeds or that grow by vegetative propagation, methods for preserving their genes are being developed. As for livestock, sperm and eggs of excellent races have been successfully cryopreserved, contributing to the spread of excellent varieties of livestock.

[0003] Regarding marine algae, in the early stage of aquaculture, seeds and seedlings are produced from wild parents in various places, and they are raised and harvested. However, as aquaculture has progressed and excellent varieties have been developed and used, the same seeds and seedlings have been cultivated in various places, and the disappearance of wild species is often regarded as a problem. future,
In order for the algae aquaculture industry to continue to develop, preservation of not only excellent varieties but also wild species has become an important issue.

[0004] In the case of algae, the growth environment is sometimes called the sea, and there is no stage in the life history that is resistant to adverse environments such as high temperature, low temperature, and dryness corresponding to the seeds of higher plants. Therefore, research on cryopreservation of seaweed has not been advanced. Therefore, conventional storage and maintenance of seaweed have been performed by subculture.

However, a great deal of labor is required for subculture.
It is time-consuming, expensive, and performed by humans, so that the risk of contamination by other algae is high during the subculture. For this reason, it is urgently necessary to develop a cryopreservation method for preserving algae genes in order to stably maintain the gene for a long time.

[0006] In the case of cryopreservation, liquid nitrogen is only replenished at regular intervals, no labor is required, and there is no mixing of other algae. In addition, it is said that it is genetically stable even when stored for a long period of time.

In general, when cells and tissues are cryopreserved, many two-stage freezing methods are used. The two-stage freezing method is a method in which cells are freeze-dehydrated by slowly pre-freezing to a certain temperature, and then rapidly cooled to liquid nitrogen temperature to vitrify the water in the cells.

[0008] Freeze-dehydration means that dehydration occurs due to a difference in osmotic pressure between the inside and outside of a cell during pre-freezing, and the pre-freezing temperature and the freezing medium are deeply involved in the degree of cell dehydration. Since the degree of dehydration has a profound effect on the survival rate after thawing, selection of a lyotoxic medium and a pre-freezing temperature is an important issue in order to use frozen cells and tissues in a live state after thawing.

[0009] Thus, the expectation for the cryopreservation of marine algae has become very large. However, regarding the cryopreservation of marine algae, there have been reports on susabinori, asakusanori, ogonori, and aonori. Only.

[0010] Although there is a report on the cryopreservation of seaweed of the order Laminaria to which Alame belongs, this cryopreservation method evaluates the survival rate immediately after thawing. In the case of brown algae, it is said that survival after thawing and culturing decreases. Therefore, the evaluation of the cryopreservation method for the seaweed must be examined not only immediately after thawing but also after thawing.

[0011]

The gametophytes of seaweeds such as alame can be frozen and stored for a long time, and the survival rate after thawing is high.
There is a need for a cryopreservation method in which the survival rate does not decrease much after culturing.

[0012]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have repeatedly studied the freezing medium and the pre-freezing temperature using female female gametophytes. We have found a cryopreservation method that has a high rate, minimizes the decrease in the survival rate after culture, and can be stored for a long time.

[0013] The method for cryopreserving a gametophyte of seaweed according to claim 1 comprises:
Gametophytes isolated from seaweed are suspended in a freezing medium containing 10% ethylene glycol and 10% proline dissolved in 100% seawater, pre-frozen and freeze-dehydrated, and then immediately immersed in liquid nitrogen for rapid freezing. It is characterized by making it.

[0014] The method for cryopreserving gametophytes of seaweed according to claim 2 comprises:
2. The method according to claim 1, wherein the pre-freezing is performed at a cooling rate of 1 ° C./minute or less to -20 ° C. to -60 ° C.

[0015] The method for cryopreserving gametophytes of seaweed according to claim 3 comprises:
2. The method according to claim 1, wherein the preliminary freezing is performed at a cooling rate of 1 ° C./min or less to −40 ° C.

The method for cryopreserving gametophytes of seaweed according to claim 4 is characterized in that, in the cryopreservation method according to any of the above claims, gametophytes of seaweed of the genus Alame, Laminaria or Trolocomb are cryopreserved.

Further, the method of thawing a cryopreserved seaweed gametophyte of the present invention is characterized in that the seaweed gametophyte cryopreserved by the method of claim 1, claim 2, claim 3 or claim 4
It is characterized in that it is stirred and thawed in a water bath at ℃.

In order to complete the present invention, the conditions under which gametophytes can be stably frozen and stored for a long period were examined. Specifically, male and female gametophytes are isolated from mature alame, each of which is placed in a vial tube resistant to low humidity, suspended in a freezing medium containing a cryoprotectant, and cooled at a cooling rate of 1 ° C or less per minute. After pre-freezing to a certain temperature, it is immersed in liquid nitrogen for quick freezing. The conditions of the freezing medium and the temperature of pre-freezing at this time were examined under different conditions.

As frost protection agents, dimethyl sulfoxide (hereinafter referred to as DMSO), dextran, polyvinylpyrrolidone (hereinafter referred to as PVP), proline, glycerin, ethylene glycol, propylene glycol, sorbitol, mannitol, glucose, sucrose and the like are used. Although possible, the combination of ethylene glycol and proline was the most effective, and the freezing medium in which 10% ethylene glycol and 10% proline were combined with 100% seawater was the most effective. Table 1 below uses Alame female gametophytes,
9 is a table showing effects when various freezing media are used.

[0020]

【table 1】

As a result of examining the pre-freezing temperature, 1 minute
It was possible to survive when cooled from -20 ° C to -60 ° C at a cooling rate of not more than 0 ° C. Particularly preferred is −40 ° C.
, Indicating a high survival rate.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described using preferred embodiments. The present invention is not limited to these embodiments.

[Examples] (1) Cryopreservation method Alame (Eisenia bicyclis) collected in Omaezaki, Shizuoka Prefecture
Gametophyte (female; MGEB-001 strain, male; MGEB-002)
Co., Ltd.). The shredded gametes are cultured for 2 weeks, and the gametes and the base solution (100 ml) are placed in a vial tube (capacity: 2 ml).
% HEPES dissolved in seawater to 0.01M) 0.75m
l was suspended and cooled with ice in ice water. To this, the same amount of the freezing medium dissolved twice or in combination with various frost protection agents at twice the concentration which had been previously ice-cooled, and gradually added over 15 minutes,
They were equilibrated for 45 minutes. In this embodiment, the freezing medium is 100
% Seawater with 10% ethylene glycol and 10% proline dissolved.

Using a simple pre-freezing device or a program freezer, this is cooled at a cooling rate of 1 ° C./min or less to -20 ° C. to -60 ° C.
After pre-freezing to ℃, it was immediately immersed in liquid nitrogen and rapidly frozen.

(2) Measurement of Survival Rate The vial tube stored for one day or more was rapidly thawed in a water bath at 40 ° C. with vigorous stirring. 40 ℃
The reason for thawing is to thaw as quickly as possible. If the thawing speed is low, the time for cells to come into contact with the surrounding high-concentration medium will be prolonged. This is because dehydration is likely to occur.

After a little ice was left in the vial tube, the vial was transferred to ice water, thawed, and gradually diluted with ice-cooled seawater over 45 minutes to remove the frost protection agent. Immediately after thawing and until one week after culturing, gametes were stained with erythrosine, and live and dead cells were counted and determined under a microscope.

FIG. 1 is a graph comparing the survival rates of female female gametes with different pre-freezing temperatures. According to this, it can be seen that a high survival rate can be obtained particularly when pre-freezing to around -40 ° C.

FIG. 2 shows a cryopreservation at a pre-freezing temperature of -40 ° C.
It is a figure showing the result of having investigated the survival rate from immediately after thawing to after a short period of culture. The survival rate of Alame gametophytes is immediately after thawing,
The ratio was 62.0% for females and 52.6% for males. After culturing, the survival rate began to decrease, and on the fourth day of culture, the minimum was 31.1% for females and 27.2% for males. It should be noted that the survival rate increased after the fourth day, which is considered to be due to the fact that viable cells that divide and proliferate more than dead cells.

FIG. 3 is a diagram showing the result of examining the relationship between the storage period in liquid nitrogen and the survival rate. As can be seen from this figure, no decrease in the survival rate was observed even when the storage period exceeded 200 days.

As described above, when the present invention is stored frozen by using the present invention, the survival rate is high even during long-term storage, and the decrease in the survival rate when cultured after thawing can be reduced.

In the above embodiment, the cryopreservation of the gametophytes of Alame was described as an example. However, the gametophytes of seaweeds of the genus Laminaria and the genus Trolocomb can be frozen and preserved in the same manner.

[0032]

As described above, according to the present invention, the survival rate after thawing is higher than that of the conventional cryopreservation method, the decrease in the survival rate is suppressed even in the subsequent culture, and the cryopreservation is carried out for a long time. be able to. Therefore, it can be thawed when necessary and cultured. Such a cryopreservation method is extremely useful as a technique for stably preserving valuable genetic resources,
It is useful for academic research of seaweed and for the development of seaweed industry.

[Brief description of the drawings]

FIG. 1 is a view showing the result of examining the relationship between a pre-freezing temperature and a survival rate.

FIG. 2 is a diagram showing the results of examining the survival rate from immediately after thawing to after a short period of culturing, by preserving frozen at a preliminary freezing temperature of −40 ° C.

FIG. 3 is a diagram showing a result of examining a relationship between a storage period in liquid nitrogen and a survival rate.

 ──────────────────────────────────────────────────の Continuing from the front page (72) Inventor Nao Saga 3-20-1, Orito, Shimizu-shi, Shizuoka Pref.

Claims (5)

[Claims] 1. A gametophyte isolated from seaweed is suspended in a freezing medium in which 10% of ethylene glycol and 10% of proline are dissolved in 100% of seawater, preliminarily frozen and dehydrated, and immediately thereafter, liquid nitrogen is added. A method for cryopreserving a gametophyte of seaweed, characterized by immersing in freezing and rapidly freezing. 2. The cryopreservation method of seaweed gametophytes according to claim 1, wherein the preliminary freezing is carried out at a cooling rate of 1 ° C./min or less at a temperature of −20 ° C. to −60 ° C. 3. Preliminary freezing is performed at a cooling rate of 1 ° C. or less per minute to -40 ° C.
Method of cryopreservation of seaweed gametophytes. 4. The method for cryopreserving seaweed gametophytes according to claim 1, wherein gametophytes of seaweeds of the genus Alame, Laminaria or Trolocomb are cryopreserved. 5. The seaweed gametophyte cryopreserved by the method according to claim 1, 2, 3, or 4,
A method for thawing a cryopreserved seaweed gametophyte, which comprises stirring and thawing in a water bath at ℃.