JP2019023178A - Freezing preservation method of plant seedling - Google Patents

Abstract

To provide a low-cost, high-safety, high-versatility freezing preservation technique of plant seedling without using a freezer or a cryogenic-temperature apparatus having a high-level control mechanism or liquid.SOLUTION: In a freezing preservation method of plant seedling, a plant seedling is frozen and preserved alive by spraying or coating ice nucleation-active bacteria that induce freezing at high temperature on the plant seedling. In the freezing preservation method of plant seedling, freezing is progressed at the speed of 10°C/minutes or more, and a frozen seedling is slowly thawed naturally at normal temperature. In the freezing preservation method of plant seedling, the ice nucleation-active bacteria contain P.fluorescens, P.syringae, P.viridiflava or the like which belongs to Pseudomonas or E.ananas, E.hervicola, E.stewartii which belongs to Erwinia, or X.campestris which belongs to Xanthomonas or the like, and furthermore bacterial liquid or extract liquid obtained by cloning the bacteria-derived genes is used.SELECTED DRAWING: None

Description

Detailed Description of the Invention

  The present invention relates to a technique for freezing plant seedlings and thawing them with activity.

Refrigeration techniques for maintaining freshness have recently been evolving day by day, with rapid freezing methods and low freezing temperatures being common.
However, it is extremely difficult to freeze and thaw plant seedlings in the growing season without damage. In particular, it is necessary to use liquid nitrogen or the like for the storage temperature. Unlike animal embryos, it was almost impossible to revive after thawing by ordinary methods.
Efficient dehydration is indispensable for freezing cells without damage, and it is important to vitrify so that ice crystals are not formed in the cells. In cultured cells or bacteria of animals and plants, carbohydrates, DMSO and the like have been used for a long time, and they are treated in the liquid for a certain period of time and rapidly frozen in liquid nitrogen.

  In recent years, ice nucleus active bacteria (INA) have been found from the surface of tea leaves. It has been found that this bacterium produces a protein that becomes an ice nucleus, provides a nucleus for freezing regardless of whether the cell is alive or dead, and freezes water at a high temperature of −2 ° C. (Reference 1). In addition, separation of almost proteins, isolation and analysis of genes (ina group) (References 2 and 3), and cloning into E. coli-based plasmids have already been completed.

Reference 1

S. W. Linda Annual Rev. Plant Ptology 21: 363 (1982)

Reference 2

R. L. Green and G.G. J. et al. Warren Nature 317: 6465 (1985)

Reference 3

G. J. et al. Green, L.M. Gorotto and P.M. Wolver Nucleic Acids Res. 144: 8047 (1986)

  An object of the present invention is to stably store frozen plant seedlings in an ordinary freezer (around −20 ° C.), and to restore and maintain the cells with good reproducibility even after thawing after storage.

As a result of intensive studies to achieve the above object, the present inventor has found that the above problems can be solved by treating plant seedlings with an ice nucleation active bacterial suspension.
The present invention is based on this finding.
1. It is a technology for cryopreservation without impairing the physiological activity of plant seedlings in the growth period, and it is applied or sprayed with ice nucleus active bacteria or crushed liquid containing nonionic surfactant, or protein having the activity, A method for preserving plant seedlings, which is frozen and stored,
2. The method for preserving in plants according to 1, wherein the freezing proceeds at a rate of 10 ° C / min or more,
3.1 The method for preserving plant seedlings according to 1, wherein the plant seedlings frozen by the method according to 3.1 are naturally thawed slowly at room temperature,
4). P. cerevisiae active bacteria belong to Pseudomonas. fluorescens, P.M. syringae, P.M. viridiflava, etc. or belonging to Erwinia. ananas, E .; hervicola, E .; stewartii, or X. belonging to Xanthomonas. a bacterial solution obtained by cloning a gene derived from these bacteria, or an extract, of which 10 ⁶ / mL or more of cells remain alive or at the same concentration. 3. Each of 1 to 3, characterized by using a crushed liquid obtained by crushing a turbid liquid with pressure, polishing or ultrasonic waves, or a bacterial suspension deprived of physiological activity by treatment with ultraviolet rays, γ rays, or the like. Storage method of plant seedlings,
Is to provide.

  Cultivation of ice nuclei bacteria is easy and safe, can be carried out by simple operations such as spraying or coating the target plant seedlings, and does not require a highly controlled freezer and is generally used There are many advantages such as being economical because it can be used.

Examples and Comparative Examples

  Next, examples and comparative examples of the present invention will be described. In addition, the Example shown below is for making an understanding of this invention easy, and this invention is not restrict | limited to these Examples. That is, other examples or modifications based on the technical idea of the present invention are naturally included in the present invention.

Examination of vitrification solution Vitrification solution is essential for cryopreservation of higher animal embryos, etc., and this treatment increases the survival rate of embryos. The present inventor first examined whether it acts effectively in plants from the analogy of animal cell storage technology. A wide variety of vitrification solutions have been proposed and used in practice, but what was tried was 1) 20% ethylene glycol (EG), 2) 30% Ficoll 70-0.5M sucrose-20 % EG-20% DMSO, 3) 15% DMSO-20% sorbitol, 4) 30% glycerol-15% EG-15% DMSO-14% sucrose. Since both were hypertonic solutions, even after immersion or application, they were dehydrated, wilted and died after a few minutes. Attempts were made to dilute the vitrification solution to 1/10 with similar results. Therefore, it has been found that a technique similar to that for animal embryos cannot be used.

Examination of freezing rate using ice nucleus active bacteria The inventor has found that plant seedlings can be cryopreserved with only ice nucleus active bacteria. However, since the freezing rate was also observed to have a significant effect on plant seedling freezing, the freezing rate was examined next. For the examination of the freezing rate, a digital thermometer in which time and temperature are recorded simultaneously and a freezer capable of temperature adjustment were used.
Apply ice broth active bacteria culture solution (10 ⁹ / mL) to the morning glory seedlings (those with true leaves) with a brush and lightly wipe them with tissue paper on a plastic tray. (1) Styrofoam box (thickness 1 cm) (2), 2 cm thick styrofoam box (3), doubled 1 cm thick styrofoam (4), put in freezer (−20 ° C.) After 1 hour, the expanded polystyrene box was taken out of the freezer and left at room temperature (24 ° C.), and the result was confirmed. The results are shown in Table 1. As a result, the one frozen as it was had a freezing rate of about 11 ° C./min, and the seedlings were safely stored. From this, it was found that the ideal freezing rate is about 10 ° C./min, and this can be realized by setting the freezing temperature to −20 ° C.
[Table 1]
In addition, when applying with a brush, the water-soluble bacterial solution may be rebound due to the cuticle layer on the leaf surface. An attempt was made to mix a nonionic surfactant (such as Tween 80) that does not affect the bacterial solution so that this can be applied uniformly. When 0.5 to 1% Tween 80 was used, it could be applied without spots, and it was not uneven after thawing, and the reproducibility was high.

Examination of thawing speed The next important factor in cryopreservation is the thawing speed. In the case of animal embryos, it is recommended to thaw instantly. In the case of plant seedlings, we examined how. The morning glory seedlings treated in the same manner as in Example 2 were taken out of the foamed polystyrene box and immediately placed in a thermostat of 30 ° C. (1), placed at room temperature (2), placed in the foamed polystyrene box, A comparison was made on (3). As a result, leaf thaw occurred in both the product thawed with a thermostat and the product thawed at room temperature, and safe storage was not possible. On the other hand, it was well preserved when placed in a box at room temperature.
Therefore, it has been found that when thawing, it can be safely stored by thawing slowly rather than thawing rapidly.

Next, the frozen storage temperature was examined. It is known that ice nuclei active bacteria form ice nuclei at −2 ° C. and freeze at an approximate temperature. We examined whether seedlings frozen at near temperature can be stored for a long time at lower temperatures. Although the freezer generally used is maintained at -20 ° C, it is -5 ° C, -10 ° C, -20 ° C, -40 ° C, -60 ° C, -80 ° C using a temperature-controllable freezer. After being stored all day and night at each of the storage temperatures, thawing as shown in Example 3 (3) was performed, and the storage state of morning glory seedlings was observed.
As a result, regardless of the storage temperature, all could be stored safely and there was no difference in frozen storage. It was shown that seedlings once frozen can be stored at any temperature.

Long-term shelf life Long-term shelf life was examined. The morning glory seedlings were processed and stored by the method shown in Example 2. The storage temperature was set to −20 ° C. in a general freezer and stored for about one month. Thawing was also performed as in Example 3 (3), and the preservation state of the seedlings was observed. However, it was carried out with both ice nucleation active bacteria as they were and those that were ground and crushed.
As a result, it was shown that seedlings can be stored for about one month at -20 ° C.

Effect of the invention

The plant seedling freezing preservation method of the present invention can use the storage temperature (about −20 ° C.) of a general freezer, is easy to operate and does not require any special technique, and further can be used for ice nucleation active bacteria. Cultivation is as easy as general bacteria, and it is economical because a concentration of 10 ⁹ / mL can be secured by overnight culture. In addition, although ice nucleation active bacteria are not pathogenic, even if they are not used as they are, they have the same activity even in extracts that are completely destroyed by pressure, polishing, or ultrasonic waves, ultraviolet rays, or γ rays, and safety is further increased. Moreover, since it is thought that it can be applied to plants other than plant seedlings, it is highly versatile.

[Table 1] Examination of freezing rate This is a table comparing the preservation state of plant seedlings when the freezing temperatures are different.

Claims (4)

It is a technology for cryopreservation without impairing the physiological activity of plant seedlings in the growth period, and it is applied or sprayed with ice nucleus active bacteria or crushed liquid containing nonionic surfactant, or protein having the activity, A method for preserving plant seedlings, which is frozen and stored The method for preserving in plants according to claim 1, wherein the freezing is carried out at a rate of 10 ° C / min or more. The method for preserving plant seedlings according to claim 1 or 2, wherein the plant seedlings frozen by the method according to claim 1 are naturally thawed slowly at room temperature. P. cerevisiae active bacteria belong to Pseudomonas. fluorescens, P.M. syringae, P.M. viridiflava, etc. or belonging to Erwinia. ananas, E .; hervicola, E .; stewartii, or X. belonging to Xanthomonas. a bacterial solution obtained by cloning a gene derived from these bacteria, or an extract, of which 10 ⁶ / mL or more of cells remain alive or at the same concentration. The pulverized liquid obtained by crushing the turbid liquid with pressure, polishing or ultrasonic waves, or the bacterial suspension deprived of physiological activity by treatment with ultraviolet rays, γ rays or the like is used, respectively. Preservation method for plant seedlings