JP3414420B2 - Carrier for artificial seed, artificial seed and method for producing artificial seed - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to a carrier suitable for artificial seeds,
In particular, the present invention relates to a carrier for artificial seed, which has excellent transport and storage stability and is capable of controlling the germination time, an artificial seed using the same, and a method for producing the artificial seed.

[0002]

2. Description of the Related Art In recent years, the technology for growing or regenerating plant bodies has been dramatically improved, and as such techniques, plant bodies such as leaves, stems, roots, petals, anthers (pollens) or young plants are used. A variety of techniques have been developed, including a technique for directly growing the plant, and a technique for isolating and culturing protoplasts from these plants to regenerate and grow callus or hairy root. In addition, gene transfer, cell fusion technology and the like have been introduced with the aim of breeding plants that are more suitable for the purpose. Utilizing such technology, the road to industrial application such as mass breeding of excellent seedlings and breeding of useful varieties is being opened.

However, even excellent seedlings developed by using these techniques have serious problems in terms of storage and transportation in order to distribute them as seedlings. Therefore, development of artificial seeds suitable for storage and transportation as compared with seedlings has been actively conducted in recent years.

A plant tissue suitable for artificial seeds is generally "a meristematic tissue capable of differentiating into a complete plant",
In many cases, somatic embryos and somatic buds are used. The conventional methods for producing artificial seeds include the following.

[0005] The most commonly used method is a method of encapsulating somatic embryos, adventitious buds and the like with a gel such as alginic acid. That is, somatic embryos are dispersed in an aqueous solution of sodium alginate, and droplets of the aqueous solution of sodium alginate in which the somatic embryos are embedded are contacted with a concentrated calcium chloride aqueous solution to form a calcium alginate gel by an ion exchange reaction, and artificial It is a method for producing seeds.

In this method, the calcium alginate gel containing the adventitious embryos and adventitious buds contains a large amount of water and therefore plays a role of protecting adventitious embryos and adventitious buds that are vulnerable to drought. In the case of the majority of plants, adventitious embryos and adventitious buds do not have vegetative storage organs.
The calcium alginate gel, which contains a large amount of nutrients, plays its role.

As described above, gels such as calcium alginate play important roles such as protection of somatic embryos and adventitious shoots and nutritional supply, but on the other hand, this gel also has serious drawbacks as described below. .

That is, since calcium alginate gel has low mechanical strength, it is difficult to transport and store artificial seeds. Also, adventitious embryos, adventitious buds, etc. are different from seeds, and when encapsulated with nutrient-containing calcium alginate gel, etc., they continue to develop without dormancy and premature germination tends to occur. That is, it is almost impossible to control the germination time. This is a decisive problem when industrially used as artificial seeds.

Furthermore, when transplanted to the soil as artificial seeds, the nutrient-rich calcium alginate gel becomes a suitable medium for the propagation of bacteria, mold and the like, and there is a risk of spoiling somatic embryos and somatic buds. . In addition, various problems have been pointed out that calcium alginate gels containing somatic embryos and somatic buds interfere with germination.

[0010] As described above, the conventional carrier for artificial seeds has a large problem left unsolved.

[0011]

An object of the present invention is to provide a carrier for artificial seeds, which has excellent transport and storage stability and can control the germination time, an artificial seed using the carrier, and a method for producing the artificial seed. To provide.

[0012]

Means for Solving the Problems As a result of earnest research, the present inventor has found that a specific property which is completely opposite to that of a normal gel, that is, a sol state (liquid state) which is reversibly exhibited at a temperature lower than a sol-gel transition temperature. It has been found that using a polymer as a carrier for artificial seeds is extremely effective in solving the above-mentioned problems.

The carrier for artificial seeds of the present invention is based on the above findings. More specifically, it contains a polymer compound having a hydrophobic portion and a hydrophilic portion in the molecule and having a sol-gel transition temperature, and , And exhibits a liquid state (sol state) reversibly at a temperature lower than the sol-gel transition temperature.

According to the present invention, it further comprises a carrier and a plant meristem retained in the carrier, wherein the carrier has a hydrophobic portion and a hydrophilic portion in the molecule, and has a sol-gel transition temperature. Provided is an artificial seed, which comprises a polymer compound having: and wherein the carrier reversibly exhibits a liquid state (sol state) at a temperature lower than the sol-gel transition temperature.

According to the present invention, it further comprises a support having a recess, a carrier retained in the recess, and a plant meristem tissue retained in the carrier, wherein the carrier is hydrophobic in the molecule. Characterized by comprising a polymer compound having a sol-gel transition temperature and having a hydrophilic portion and a hydrophilic portion, and wherein the carrier reversibly exhibits a liquid state (sol state) at a temperature lower than the sol-gel transition temperature. Artificial seeds are provided.

According to the present invention, a polymer compound having a hydrophobic portion and a hydrophilic portion in the molecule and having a sol-gel transition temperature is further contained, and the liquid is reversibly liquid at a temperature lower than the sol-gel transition temperature. The carrier showing the state (sol state),
An artificial seed characterized in that the carrier is mixed with a predetermined medium at a temperature lower than the sol-gel transition temperature of the carrier, and plant meristem is mixed in the mixture to bring the mixture into a gel state at a temperature higher than the sol-gel transition temperature. A manufacturing method is provided.

The above-mentioned carrier for artificial seeds of the present invention is in a gelled state at a temperature during storage and transportation of artificial seeds (a temperature higher than the sol-gel transition temperature) while it is transplanted to soil. Since it can be changed to be water-soluble by setting the temperature lower than the transition temperature, it can be easily removed by washing with water or the like. In addition, by combining the carrier for artificial seeds of the present invention with a germination inhibitor, it becomes possible to control the germination time of artificial seeds.

In the present invention, the hydrophilic portion of the polymer compound is necessary so that it becomes water-soluble at a temperature lower than the sol-gel transition temperature. The hydrophobic portion of the polymer is necessary to maintain the gelled state at the temperature during storage and transportation (higher than the sol-gel transition temperature). In other words, bonds between the hydrophobic moieties are necessary for the formation of cross-linking points in the gel.

Since the hydrophobic bond has a property that its binding force becomes stronger as the temperature rises, the strength of the crosslink and the crosslink density increase as the temperature rises, and the carrier of the present invention is in a water-soluble state ( It changes from a sol state) to a gelled state.
Further, since the temperature dependence of the hydrophobic binding force is reversible, this sol-gel change also occurs reversibly.

On the other hand, the hydrophilic portion in the above-mentioned polymer compound is imparted with the water solubility necessary for the gelation or encapsulation of plant meristems as described above, and when it is transplanted to soil (sol-gel). It is necessary to change the compound to a sol state (at a temperature lower than the transition temperature) and to impart water solubility for washing removal (dissolution removal), and at the same time during transportation or storage (higher than the sol-gel transition temperature). It is also necessary to prevent the compound from coagulating and precipitating due to excessive increase in hydrophobic binding force and to form a hydrogel state.

The present invention will be described in more detail below.

(Sol-gel transition temperature) In the present invention,
The "sol state", "gel state" and "sol-gel transition temperature" are defined as follows. For the definition, refer to (Polymer Journal, 18 (5), 411-416 (1
986)).

1 mL of a polymer solution (a polymer solution to be used as a carrier) is placed in a test tube having an inner diameter of 1 cm and kept in a water bath at a predetermined temperature (constant temperature) for 12 hours. After that, when the test tube is turned upside down and the solution / air interface (meniscus) is deformed by its own weight (including the case where the solution flows out), the polymer solution is Defined as "sol state". On the other hand, when the solution / air interface (meniscus) is not deformed by the weight of the solution even when the test tube is turned upside down, the solution is defined as "gel state" at the predetermined temperature.

On the other hand, when the above "predetermined temperature" is gradually increased (for example, in increments of 1 ° C) to obtain the temperature at which the "sol state" transitions to the "gel state", the transition temperature obtained by this is calculated. It is defined as "sol-gel transition temperature" (at this time, the "predetermined temperature" may be lowered in steps of 1 ° C, for example, and the temperature at which the "gel state" transitions to the "sol state" may be determined).

In the carrier of the present invention, the sol-gel transition temperature is higher than 0 ° C. and preferably 60 ° C. or lower, more preferably 4 ° C. or higher and 50 ° C. or lower (particularly 4 ° C. or higher and 40 ° C.).
The following is preferable from the viewpoint of preventing thermal damage to plant meristems. When the artificial seeds are stored at room temperature (about 25 ° C), the sol-gel transition temperature is higher than 0 ° C.
The temperature is preferably 3 ° C or lower, more preferably 4 ° C or higher and 23 ° C.
The following is preferable. The polymer compound having such a suitable sol-gel transition temperature can be easily selected from the specific compounds described below according to the above-mentioned screening method (sol-gel transition temperature measuring method).

In the method for producing artificial seeds of the present invention,
The above-mentioned sol-gel transition temperature (a ° C) is the temperature at the time of storage and transportation of artificial seeds (b ° C) and the temperature at the time of mixing plant meristems during the production of artificial seeds (or the dissolution of gel during transplantation into soil). Removal temperature) (c ° C.). That is, it is necessary that there is a relation of b>a> c among the three temperatures a ° C., b ° C. and c ° C. More specifically, (ba) is preferably 1 to 40 ° C, more preferably 2 to 30 ° C, and (ac) is 1 to 40 ° C, further 2 to 30 ° C. Is preferred.

(Polymer compound) The suitable sol-gel transition temperature as described above is, for example, the composition of the hydrophobic portion and the hydrophilic portion in the polymer compound used for the carrier of the present invention, and the degree of hydrophobicity and hydrophilicity. , Molecular weight, etc., respectively.

Specific examples of the polymer compound containing such a hydrophobic portion and a hydrophilic portion include, for example, polyalkylene oxide block copolymers represented by block copolymers of polypropylene oxide and polyethylene oxide; Etherified cellulose such as methyl cellulose and hydroxypropyl cellulose; chitosan derivative (KRHolme, et al, Macromolecules, 24 , 3828)
(1991)); Pullulan derivative (Degashira, Polymer
Preprints, Japan, 19 , 936, (1990)) and other modified polysaccharides; poly (N-substituted) (meth) acrylamide derivatives, polyvinyl alcohol partial acetic acid, polyvinyl methyl ether, and other temperature-sensitive polymers and water-soluble polymers Conjugates with polymer compounds (eg Takehisa Matsuda et al., Polyme
r Preprints. Japan 39 (8), 2559, (199)
0)) and the like, but the invention is not limited thereto.

The concentration of the above-mentioned polymer compound in the present invention varies depending on the sol-gel transition temperature to be set and the like, but when used in a mixture with a predetermined medium as described later, the concentration is generally 0.1 to 30 wt%. , Further 1-20
A concentration of wt% is preferable.

(Medium) In the present invention, the medium or culture medium to be used in combination with the above-described polymer compound is a known medium or culture medium for plant tissue culture which does not substantially contain agar (for example, Murashige). (Skoog medium)
Can be used without particular limitation.

(Artificial Seed) In the first preferred embodiment of the present invention, as shown in the schematic sectional view of FIG. 1, the artificial seed comprises the carrier of the present invention containing the above-mentioned predetermined polymer ( Capsule 1 containing desired medium as needed)
And the plant meristem 2 retained in the capsule 1. The carrier containing the polymer having the above-mentioned properties,
Since a gel state is formed at a temperature higher than the sol-gel transition temperature, the method described below is used in the present invention.
It is possible to form a capsule-shaped artificial seed as shown in FIG.

FIG. 2 shows a schematic side sectional view of a second preferred embodiment of the artificial seed of the present invention. In this embodiment, the artificial seed has the carrier 4 of the present invention as described above (including a desired medium if necessary) held in the recess 3a of the support 3 having the recess 3a, and further the carrier 4 The plant meristem 5 is held therein. Drying prevention of such artificial seeds,
From the viewpoint of protection and the like, it is preferable to seal or seal the open side of the support 3 (at least the recess 3a) with the seal member 6. In the artificial seed of the second aspect, the carrier 4 itself does not necessarily have the shape-retaining property.

(Support) As for the support 3, the material is not particularly limited as long as it has a shape-retaining property such that the carrier gel 4 can be retained in the recess 3a, but in terms of weight and strength. Considering the above, it is preferably made of plastic. The shape (planar shape, cross-sectional shape) and size of the recess 3a of the support 3 are not particularly limited as long as the carrier gel 4 can be retained in the recess 3a. When the recess 3a has a circular planar shape, the recess 3a has an inner diameter of 2 mm to 2 cm (more preferably 5 mm) from the viewpoint of transportability as an artificial seed.
.About.1 cm), and the recess 3a
The depth is preferably about 2 mm to 1 cm (further, 5 mm to 1 cm). It is preferable that the support 3 has a plurality of recesses 3a from the viewpoint of efficient production of artificial seeds, but the number of the recesses 3a is about 2 to 100 (more preferably 10 to 10).
It is preferably about 100).

(Seal Member) The seal member 6 is not particularly limited in its shape and the like as long as it can seal the concave portion 3a of the support 3 to the extent that the carrier gel 4 and the like can be prevented from drying. It is preferable to have a shape of. In addition, its material is preferably one that has the ability to exchange oxygen, carbon dioxide, etc. necessary for the survival of plant meristems (adventitious embryos, adventitious buds, etc.), and at the same time has low water vapor permeability necessary to prevent drying. Thus, for example, a polyolefin resin (polyethylene, polypropylene, etc.) is preferable. The seal member 6 can be bonded to the support 3 by a known means such as heat sealing or an adhesive.

The artificial seed of the present invention of the embodiment shown in FIG.
From the viewpoint of prevention of drying, prevention of bacterial infection, convenience of transportation and storage, it is preferably used from the embodiment of FIG.

(Plant meristem) In the present invention, "a meristem capable of differentiating into a complete plant" can be generally used as a plant meristem. As such meristems, for example, adventitious embryos and / or adventitious buds are preferably used.

(Germination Inhibitor) A germination inhibitor means a substance having an action of inhibiting or delaying the germination of the meristematic tissue retained in the carrier when added to the carrier at a predetermined concentration.

The germination inhibitors used in combination with the carrier of the present invention typically include, but are not limited to, abscisic acid, transcinnamic acid, caffeic acid, ferulic acid, coumarin, scopoletin. Not a thing. Although these (molar) concentrations differ depending on the germination inhibitor used, they are generally 5 × 10 ^{−3 to} 5 × 1.
It is preferably about 0 ^-7 M, more preferably about 10 ^-4 to 10 ^-6 M.

(Manufacturing Method of Artificial Seed) A preferred example of a method of manufacturing an artificial seed using the polymer compound as described above will be described below.

First, the polymer compound is uniformly dissolved in a medium containing desired nutrients at a temperature lower than the sol-gel transition temperature of the polymer compound. Next, plant meristems such as adventitious embryos and adventitious buds are mixed and dispersed at a temperature in the medium solution of the polymer compound. Next, this mixture is gelled. This gelation is most conveniently carried out by collecting a desired amount of the polymer solution containing the plant meristem and then heating it in a medium heated to a temperature higher than the sol-gel transition temperature. It can be added dropwise to.
In this case, the polymer solution rapidly gels and artificial seeds (the embodiment of FIG. 1) are formed.

The above polymer compound solution containing plant meristems is added at a temperature lower than the sol-gel transition temperature to a large amount of liquid paraffin that has been cooled to a temperature lower than the sol-gel transition temperature, and the suspension is stirred to form granular particles. By forming the droplets of the polymer compound in paraffin and then making the temperature higher than the sol-gel transition temperature, the polymer compound can be rapidly gelated. Next, in order to recover the particles of the gel from the liquid paraffin, a medium heated to a temperature higher than the sol-gel transition temperature is added to the gel particle suspension, and centrifugation is performed at a temperature higher than the sol-gel transition temperature, whereby liquid paraffin is added to the upper layer. The layer is separated into the lower layer and the medium suspension layer of the gel particles, which makes it possible to separate the artificial seed from the liquid paraffin.

However, in the above method, the probability of producing gel particles free of plant meristems such as adventitious embryos and adventitious buds is generally not always low, so it is preferable to separately provide a step of selecting those gel particles. An example of a manufacturing method that eliminates the need for such a sorting step will be described below.

A board or plate (support) having a large number of wells (recesses) having a desired size and shape is previously cooled to a temperature lower than the sol-gel transition temperature,
A desired amount of the above-mentioned medium solution of the polymer compound is injected into the well at a temperature lower than the sol-gel transition temperature.

Next, 1 plant meristem such as adventitious embryo or adventitious bud is
Then, the cells are inserted into the well and embedded in the polymer medium solution in the well. Next, the temperature of the board is made higher than the sol-gel transition temperature to gelate the polymer medium solution, whereby artificial seeds containing the plant meristem (aspect of FIG. 2) are produced in the wells.

The artificial seeds encapsulated in the wells by the above method are very suitable for storage and transportation in terms of mechanical strength and prevention of drying. Also, by lowering the temperature of the well below the sol-gel transition temperature during transplanting to soil,
The gel can be dissolved and removed, and the plant meristem encapsulated inside can be easily taken out.

On the other hand, as the agent for controlling the germination time of artificial seeds of the present invention, as described above, germination inhibitors such as abscisic acid, trans-cinnamic acid, caffeic acid, ferulic acid, coumarin and scopoletin, or a combination of these substances is used. Can be used. In order to add these germination-inhibiting substances to the carrier gel, these germination-inhibiting substances may be added in advance to the medium solution of the above-mentioned polymer compound at a desired concentration and uniformly dissolved. Manufacture of artificial seeds containing these germination-inhibiting substances in gel can be carried out by the same method as described above.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is determined by the description of the scope of claims and is not limited by the following examples. Absent.

[0048]

Example 1 10 g of Pluronic F-127 (manufactured by Asahi Denka Co., Ltd.), which is a block copolymer of polypropylene oxide and polyethylene oxide, was dissolved in 30 mL of dry chloroform, and hexamethylene diisocyanate was coexisted in the presence of phosphorus pentoxide. 0.13 g was added, and the mixture was reacted under reflux of boiling point for 6 hours.

After removing the solvent under reduced pressure, the residue was dissolved in distilled water and subjected to ultrafiltration dialysis using an ultrafiltration membrane having a molecular weight cutoff of about 100,000 to recover an aqueous solution containing only the high molecular weight polymer.
The obtained aqueous solution was freeze-dried to obtain Pluronic F-127.
A polymer was obtained.

The polymer obtained as described above was cooled with ice to 10
It was dissolved in distilled water at a concentration of wt%. When this aqueous solution is gradually heated, the viscosity gradually increases from 21 ° C,
It completely solidified at about 27 ° C. and changed into a gel state. When the gel was cooled, it turned into an aqueous solution at about 21 ° C. This change was repeatedly observed reversibly.

On the other hand, carrot somatic embryos were prepared by the following method. The carrot root was thoroughly washed and immersed in ethanol for a few seconds, and then 15
It was soaked for a minute and sterilized. Next, using a cork borer with an inner diameter of about 5 mm, extract the internal tissue from the sterilized root, cut it into a thickness of about 5 mm with a razor, and cut it into 2,4
Murashi to which -D (2,4-dichlorophenoxyacetic acid, manufactured by Wako Pure Chemical Industries, Ltd.) was added at a concentration of 0.5 mg / L.
It was inoculated on an agar medium of ge-Skoog medium (Wako Pure Chemical Industries, Ltd.). When cultured at 28 ° C. for 3 weeks in this state, nodular callus was formed.

Next, the callus is cut into an appropriate size,
Carrot somatic embryos were obtained by culturing in Murashige-Skoog medium (agar-free) containing no 2,4-D at 28 ° C. for 2 weeks.

5 g of the Pluronic F-127 polymer obtained by the above method was added to 45 mL of Murashige-Skoog medium.
It was dissolved under ice-cooling and sterilized by autoclaving. The solution was dispensed into each well (internal diameter: about 7 mm, depth: about 8 mm) of a 96-well microtest plate (manufactured by Nippon Becton & Deckson Co., Ltd.) in an amount of about 0.3 mL under ice cooling. Next, the carrot somatic embryo obtained by the above method is transplanted and embedded in a medium solution of the above-mentioned polymer compound in each well, and then the temperature is raised to 29 ° C. to gel the polymer compound solution. Let 29 in this state
After culturing at 3 ° C. for 3 weeks and germination was observed, the well was ice-cooled and the gel was changed to a solution state again, and the carrot germinated tissue could be easily recovered.

Example 2 A 10 wt% Murashige-Skoog medium solution of Pluronic F-127 polymer was prepared in the same manner as in Example 1,
Further, abscisic acid (Wako Pure Chemical Industries, Ltd.) was added to the solution at a concentration of 10 ⁻⁵ M. This abscisic acid-containing medium was dispensed into each well of a 96-well microtest plate as in Example 1.

Next, a carrot somatic embryo induced by the same method as in Example 1 was transplanted and embedded in the above well, and gelated by raising the temperature to 29 ° C. In this state, the cells were cultured at 29 ° C. for 1 month, but the germination observed in Example 1 was not observed at all.

[0056] After one month culture, the gel was ice-cold the well is completely dissolved, it can ^retrieve from readily wells-embedded somatic embryos.

The adventitious embryo thus taken out was washed twice with Murashige-Skoog medium, acclimated and transplanted into soil.
Germination was observed after 2 weeks.

[0058]

INDUSTRIAL APPLICABILITY As described above, in the present invention, a gel (containing a germination inhibitor, for example) is brought into a sol state by lowering the temperature below the sol-gel transition temperature of the polymer immediately before transplanting artificial seeds. It can be changed and completely removed by washing with water. In the case of a conventional calcium alginate gel, in order to dissolve the gel, a harmful calcium chelating agent such as EDTA (ethylenediaminetetraacetic acid) must be used, and somatic embryos, adventitious buds and other plants Virtually complete removal of the gel was not possible due to possible damage to the meristem.

Furthermore, in the present invention, since the gel can be dissolved and removed immediately before transplantation, it is possible to remove the germination-inhibiting substance which may be contained in the gel, and at the same time, the problems of the conventional gel can be solved. The problems such as the germination-inhibiting effect (mechanical germination-inhibiting effect) of the existing gels, or the spoilage of plant meristems such as adventitious embryos and adventitious buds due to bacteria and mold propagating in the gel can be solved.

Therefore, according to the present invention, there are provided an artificial seed carrier and an artificial seed which are not only excellent in stability during transportation and storage but also capable of easily removing a gel body by lowering the temperature below the sol-gel transition temperature. Provided. Furthermore, by incorporating a germination-inhibiting substance into the carrier of the present invention, artificial seeds whose germination timing can be controlled can be easily obtained.

[Brief description of drawings]

FIG. 1 is a schematic cross-sectional view showing one embodiment of the artificial seed of the present invention.

FIG. 2 is a schematic side sectional view showing another embodiment of the artificial seed of the present invention.

[Explanation of symbols]

1 Capsule carrier 2 plant meristem 3 support 4 carriers 5 plant meristems 6 Seal member

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Claims (7)

(57) [Claims] 1. A polymer compound having a hydrophobic portion and a hydrophilic portion in the molecule and having a sol-gel transition temperature,
A carrier for artificial seeds, which reversibly exhibits a liquid state (sol state) at a temperature lower than the sol-gel transition temperature. 2. The carrier for artificial seeds according to claim 1, wherein the sol-gel transition temperature is higher than 0 ° C. and 60 ° C. or lower. 3. The method according to claim 1, which contains a germination inhibitor.
The carrier for artificial seeds according to. 4. A polymer compound comprising a carrier and a plant meristem retained in the carrier, wherein the carrier has a hydrophobic portion and a hydrophilic portion in the molecule and has a sol-gel transition temperature. An artificial seed comprising the carrier and reversibly exhibiting a liquid state (sol state) at a temperature lower than the sol-gel transition temperature. 5. A support having a recess, a carrier retained in the recess, and a plant meristem retained in the carrier, wherein the carrier has a hydrophobic portion and a hydrophilic portion in the molecule. An artificial seed comprising a polymer compound having a sol-gel transition temperature, and wherein the carrier reversibly exhibits a liquid state (sol state) at a temperature lower than the sol-gel transition temperature. 6. The artificial seed according to claim 5, wherein the support has a plurality of recesses. 7. A polymer compound having a hydrophobic portion and a hydrophilic portion in the molecule and having a sol-gel transition temperature,
A carrier exhibiting a reversible liquid state (sol state) at a temperature lower than the sol-gel transition temperature is mixed with a predetermined medium at a temperature lower than the sol-gel transition temperature of the carrier, and plant meristem is mixed in the mixture. A method for producing an artificial seed, characterized in that the mixture is brought into a gel state at a temperature higher than the sol-gel transition temperature.