JP2001245543A - Artificial seed - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an artificial seed having a high preservation stability, and rapidly germinating when seeded in a soil. SOLUTION: This artificial seed is composed of a seamless soft capsule having an adventitious embryo, an adventitious bud, a multiple shoot, a shoot apex, a growing point, a protocorm-like body, an adventitious root, a capillary root or the like included in the most inner layer, a hardened oil used as an inner coat layer for covering the most inner layer, and an outer layer constituted of a biodegradable outer cover layer made of a gelatin, a polysaccharide or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an encapsulated artificial seed containing a regenerable plant cell tissue which has high storage stability and which germinates quickly upon sowing in soil. More specifically, the present invention relates to an artificial seed comprising a seamless soft capsule having a structure of three or more layers.

[0002]

2. Description of the Related Art Conventionally, attempts have been made to convert regenerated plant cell tissues (hereinafter, sometimes referred to simply as "cell tissues") into artificial seeds. Generally, cell tissues are dispersed in a solution of polysaccharide, polymerizable low-molecular or cross-linkable polymer,
It has been practiced to suspend and gelate this to cover the cell tissue in the gel and to form it into beads, plates, rods or fibers to form artificial seeds.

By the way, artificial seeds need to function as seeds, and can be stored for at least three months in a dry place without special refrigeration equipment, such as a barn or a warehouse of a farmer, and when seeded on soil, they can take several days. Germination is required.

[0004] However, as described above, artificial seeds containing cell tissues in a gel are not resistant to drying at room temperature, and must be sown within 4 days after production. In order to prevent this, it was not possible to store for one month unless refrigerated or stored in liquid.

[0005] In order to solve such a drying problem,
Attempts have been made to cover the surface of the gel with paraffin, wax, or the like. Although the storage stability is improved by this attempt, the paraffin or the wax is stable and hardly decomposed, so that there is a problem that it does not germinate even when seeded in soil. Therefore, at the time of sowing, there is an inconvenience that a hole must be formed one by one, and it cannot be a practical artificial seed.

[0006]

SUMMARY OF THE INVENTION As described above,
Actually, artificial seeds that have reached the practical level have not yet been obtained, and can be stored for at least 3 months in a dry place without special refrigeration equipment, and when seeded on the soil, can germinate in a few days Is required. Once such artificial seeds are obtained, they can be applied to a wide variety of plants in practice, and combined with plant cell tissue culture technology, cloning technology and virus-free technology, can be used for agriculture, forestry, horticulture, flowers, etc. Application becomes possible.

[0007]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have found that the inner shell of a hollow seamless soft capsule containing regenerable plant cell tissues (cell tissues) is obtained. Was used as a hardened oil, the outer film was composed of biodegradable gels (proteins and polysaccharides), biodegradable plastics, and the like, and the surface was dried to produce artificial seeds. With this configuration, the supply of oxygen into the capsule is restricted, and the cell tissue can survive in a kind of dormant state in the capsule, and the evaporation of water is suppressed, and the capsule is stored at room temperature for 2 months or more. It was found that it is possible. Furthermore, by sowing the capsules in the soil, the hardened oil of the outer coat and the inner coat is decomposed by the action of water and microorganisms in the soil, increasing the permeability of oxygen, regenerating and leading to germination. As a result, the present invention has been completed.

[0008] The present invention provides an artificial seed comprising a seamless soft capsule having three or more layers of an innermost layer, an endothelial layer covering the innermost layer, and an outer layer covering the innermost layer. Is an artificial membrane that contains regenerable plant cell tissue (cell tissue), the endothelial layer is composed of an inner coat composed mainly of hardened oil, and the outer layer is a biodegradable outer coat. For seeds.

[0009] In a preferred embodiment, the outer coating is dried.

In a preferred embodiment, the regenerated plant cell tissue is an adventitious embryo, an adventitious bud, a multibud, a shoot apex,
It is selected from the group consisting of vegetative point, protoctoid body, adventitious root, and hairy root.

In a preferred embodiment, the inner coating is a hardened oil which is solid at normal temperature and is biodegradable.

[0012] In another preferred embodiment, the outer coat is a biodegradable outer coat selected from the group consisting of proteins, polysaccharides, and biodegradable plastics.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The artificial seed of the present invention is an artificial seed comprising a seamless soft capsule having three or more layers of an innermost layer, an inner layer covering the innermost layer, and an outer layer covering the inner layer. is there. FIG. 1 shows a schematic cross-sectional view of the artificial seed of the present invention. FIG. 1 is a diagram of a three-layer structure. The innermost layer contains the somatic embryo, a cellular tissue. The innermost layer is filled with a liquid component or a gel containing a medium component and a growth regulator necessary for maintaining the life of the somatic embryo. The endothelial layer is composed of an inner film containing a hardened oil as a main component (shown as an inner film (hardened oil film) in the figure).
And this hardened oil film prevents evaporation of moisture and also suppresses permeation of oxygen. The outer layer is shown as an outer film (gelatin film) in FIG. 1, and maintains physical strength and suppresses oxygen permeation.

In the description of various academic documents and patent documents, single-sphere beads of calcium alginate gel are sometimes referred to as capsules, but such capsules will be apparent in later production methods. Thus, it is completely different from the seamless soft capsule of the present invention.
Furthermore, the artificial seed of the present invention preferably has a dried surface and is different from a wet alginic acid gel.

As the cell tissue contained in the innermost layer used in the present invention, adventitious embryos, adventitious buds, multibuds, shoot apex, growing point, protochmoid body, adventitious root, hairy root and the like are preferably used. . A virus-free tissue may be used.

In order to maintain the life of the cell tissue, for example, water, physiological saline, a buffer, a culture solution, or a liquid substance containing a component necessary for maintaining the regeneration (germination) activity of the cell tissue or It is preferable to suspend cell tissues in a gel-like substance (hereinafter collectively referred to as a medium). The medium is a medium suitable for a desired plant, and is a medium usually used by those skilled in the art, for example, Murashige Skoog (Mur
ash & Skoog) (1962:
Hereinafter, the medium will be referred to as “MS medium”) or a modified medium thereof, but is not limited thereto.
Further, plant hormones, coconut milk, casein hydrolyzate, yeast extract, and the like used in ordinary culture may be added together. Further, when the inner coat disintegrates after seeding and it takes time to germinate and is damaged by microorganisms, an antibacterial substance may be added to suppress the growth of microorganisms.

As the inner film, a fat or oil containing a hardened oil as a main component is preferably used. The phrase “having a hardened oil as a main component” means a case including only a hardened oil or a material obtained by mixing a hardened oil with another fat or the like so as to have desired properties. The preferred inner coating is a hardened oil that is solid at room temperature and biodegradable. The term “hardened oil that is solid at ordinary temperature” refers to a hardened oil having a melting point of about 20 ° C. or higher. The melting point may be 30 ° C. or higher, 40 ° C. or higher, or 50 ° C. or higher. Melting point is 20-50
° C hardened oil is preferably used. What kind of hardened oil is selected may be determined in consideration of storage temperature, seeding time, and the like.

Examples of the hardened oil include triglycerides and diglycerides of medium-chain fatty acids, such as butter, margarine, shortening and cocoa butter.
It is not limited to these.

It is important that the inner coating is water-resistant not only for forming a capsule of an aqueous substance, but also for preventing the evaporation of water in the innermost layer containing cell tissues.

The term "biodegradable or microbial degradable" means that when seeded on soil, it is degraded by microorganisms or other organisms.
Or it means being assimilated. Decomposition or assimilation of the hardened oil breaks down the inner coat and removes obstacles when it appears outside the capsule, increases oxygen permeability, and allows cell tissues to wake up from dormancy, differentiate and proliferate Environment.

The outer coat is preferably swellable with water and biodegradable or microbial degradable, from the viewpoint of obstructing cell tissues from differentiation, multiplication and germination outside the capsule. Examples of the biodegradable or microbial degradable outer film include proteins, polysaccharides, biodegradable plastics, and the like. These can be used alone or in combination of two or more.

The proteins include, but are not limited to, gelatin, collagen and the like. These can be used alone or in combination of two or more.

As the polysaccharide, a polysaccharide that forms a gel is preferably used. Such polysaccharides include agar,
Examples include, but are not limited to, carrageenan, gum arabic, gellan gum, xanthan gum, pectin, alginic acid, and the like. These can be used alone or in combination of two or more.

Examples of biodegradable plastics include, but are not limited to, polylactic acid, polyhydroxybutyric acid, and mixtures thereof. These can be used alone or in combination of two or more.

If necessary, the properties of the film can be changed by adding sugar, sugar alcohol, polyhydric alcohol, pullulan, chitosan or the like to the biodegradable substance.

A particularly preferred film is gelatin which has an increased oxygen barrier property after drying.

The encapsulated artificial seed of the present invention can have a structure of four or more layers as required, and various properties can be imparted to the film by selecting a material.
Further, the capsule surface may be coated with a drug or the like.

The artificial seed comprising the seamless soft capsule of the present invention is produced by a method commonly used by those skilled in the art when producing a seamless soft capsule. In the case of producing a seamless soft capsule having a three-layer structure, it is most preferable to produce it by a dropping method using a triple nozzle. A method for producing a seamless sobut capsule is described in, for example, Bioscience and Industry, Vol. 58, no.
7, p31-34 (2000). Since this method for producing seamless sobuto capsules is already used in industrial mass production, mass production of the seamless soft encapsulated artificial seeds of the present invention is easy.

Specifically, for example, an adventitious embryo of an appropriate size is obtained from a cell tissue (eg, an adventitious embryo) created in a liquid medium or solid medium, suspended in the medium, and the contents of the capsule (innermost layer) A hardened oil that is solid at room temperature is used as the inner film, and the outer film is converted into a solution (for example, a 22% gelatin solution) of the biodegradable substance having an appropriate concentration using a triple tube nozzle seamless soft capsule manufacturing machine. Granulate by the medium drop method. At this time, the pump is adjusted so that the adventitious embryos of the content liquid enter one by one. Capsule droplet formation and solidification are performed in coagulated oil to form a three-layer capsule. After decoagulating the coagulated oil from the obtained capsules, the resulting capsules are dried by a drum to obtain artificial seeds of dried encapsulated somatic embryos. Drying is performed at a low temperature so that the cell tissue does not die.

The particle size of the obtained encapsulated artificial seed varies depending on the size of the tissue cells, but is from 1 mm to 12 mm.
It is. More preferably, it is 3 mm to 10 mm. The number of cell tissues to be enclosed in the capsule depends on the size of the tissue, but it can be from one to several cells. Therefore, considering the regeneration rate (germination rate), a property suitable as an artificial seed is provided. It is good to enclose the number that can do it. Depending on the plant,
Preferably, it is 1 to 4.

The thus-obtained artificial seed of the present invention can be stored at room temperature in a dry barn or the like for 3 months or more while maintaining its germination ability, and is particularly stored in a refrigerator or low-temperature water. do not have to. If stored in a refrigerated storage at 10 ° C or lower, long-term storage of 6 months or more is possible.

The encapsulated artificial seed of the present invention is sown in the soil and sprayed with water to swell the gelatin layer if the outer coat is gelatin, and is decomposed by the action of soil microorganisms, and then hardened. Oil is also broken down by the action of microorganisms. At that time, it is considered that oxygen permeability increases, awakens from a dormant state, and shifts to a germinating state.

[0033]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited to these examples.

Example 1: Artificial Seeds Encapsulating Carrot Adventitious Embryos Carrot cultured cells and adventitious embryos were produced using carrot roots in "plant cell tissue culture" (1979, edited by Harada and Komamine, Science and Engineering) The method was carried out according to the method described on pages 91-104. Adventitious embryos produced in liquid medium were
Sorting was performed using nylon meshes of 0 μm and 850 μm to obtain somatic embryos having a size of 500 μm to 850 μm.
The obtained somatic embryo had a shape ranging from a heart type to a torpedo type. These adventitious embryos were suspended in an MS medium containing no hormone and containing 0.2% of chitosan to obtain the contents of the capsule.

The encapsulation was carried out using a triglyceride hardened oil having a melting point of 32 ° C. (Pharmasol B-115, manufactured by Nippon Oil & Fats Co., Ltd.) and an outer coating as a 22% gelatin solution. By a dropping method in liquid. The pump was adjusted so that the adventitious embryos of the content liquid entered one by one. Capsule droplet formation and solidification were performed in coagulated oil to give a three-layered capsule.
Coagulated oil was deoiled from the obtained raw capsules, followed by drum drying to obtain artificial seeds of dried encapsulated somatic embryos.
The particle size after drying was 7 mm.

On the other hand, a carrot somatic embryo embedded with alginate gel beads (Comparative Example 1) was prepared as a comparative example. That is, 3% (w / v) of somatic embryos produced and selected in Example 1 were selected.
Suspended in MS medium containing sodium alginate, 50m
Calcium alginate gel beads (particle diameter: about 5 mm) in which the adventitious embryo was embedded were prepared by dropping the mixture into a calcium chloride solution of M. This is the same as that conventionally reported as an artificial seed.

Preservation and Germination Test The artificial seed (Example 1) in which the adventitious embryo was encapsulated and the gel beads (Comparative Example 1) in which the adventitious embryo was embedded with calcium alginate gel beads were placed in a thermostat at 20 ° C. and 65% humidity. For 3 months. On the third day of storage, the calcium alginate gel beads are quite dry, the beads are smaller,
In one week, the adventitious embryo also became dry and dried. On the other hand, the encapsulated artificial seed did not show any change in appearance even after one week.

Immediately after the preparation of artificial seeds, one month, two months,
And after three months, each pot was placed at a depth of 2 cm from the topsoil in a pot containing soil from a field that had not been subjected to soil sterilization.
The seeds were sown in 00 grains and watered. After seeding, place in a 25 ° C. incubator at 3000 Lux for 16 days per day.
The plants were sprinkled once every two days while being illuminated for hours to grow. Table 1 shows the results of counting the number of germinations after 3 weeks.

[0039]

[Table 1]

As shown in Table 1, the germination rate immediately after the preparation was
Example 1 and Comparative Example 1 were almost the same. However, when stored for one month or longer, no germination occurred in Comparative Example 1, which is a conventional artificial seed. On the other hand, in the encapsulated artificial seed of the present invention, a high germination rate of 68% was observed even after storage for 3 months.

From these results, it was shown that the encapsulated somatic embryo of the present invention is far superior in storage stability as an artificial seed to the conventional alginate gel beads embedded somatic embryo type artificial seed.

Example 2: Artificial seeds encapsulating strawberry leaf buds A shoot apical tissue extracted from cultivated strawberries was treated with 10 g / l of sucrose and 2 g of gellan gum as a gelling agent.
/ L, and cultured in a dark place at 25 ° C for 14 days, and then transferred to an MS liquid medium containing 0.2 mg / l of benzyladenine and 10 g / l of sucrose.
While irradiating at 2000Lux for 16 hours per day,
Rotational shaking culture was performed at 150 rpm. After culturing for 30 days, the obtained leaf buds were selected using nylon meshes having an opening of 1 mm and 1.7 mm to obtain leaf buds having a size of 1 to 1.7 mm. The obtained leaf buds were suspended in a hormone-free MS liquid medium and used as the contents of the encapsulation.

The encapsulation was carried out in the same manner as in Example 1 except that the flow rate of the pump for feeding the leaf bud suspension was adjusted. The resulting capsules were dried to obtain artificial seeds of encapsulated strawberry leaf buds. The particle size after drying was 8 mm.

On the other hand, for comparison, strawberry leaf buds embedded in alginate gel beads were prepared (Comparative Example 2). Using the strawberry leaf buds produced and selected in Example 2, strawberry leaf buds were embedded in the same manner as Comparative Example 1 to prepare alginate-potassium-lucidium gel beads (particle diameter: about 5 mm). This is the same as that conventionally reported as an artificial seed.

[0045] Strawberry leaf bud artificial seed obtained (Example 2)
The strawberry leaf buds embedded with alginate gel beads (Comparative Example 2) were preserved and germinated in the same manner as in Example 1. Table 2 shows the results.

[0046]

[Table 2]

As shown in Table 2, the germination rate immediately after preparation was almost the same in Example 2 and Comparative Example 2. When stored for one month or longer, no germination occurred in Comparative Example 2 which was a conventional type artificial seed, but germination was observed at a high rate with the encapsulated artificial seed of the present invention, and germination of 60% or more even after three months. The rate was maintained.

From these results, it is clear that the storage stability of the encapsulated artificial seed of the present invention is far superior to that of the conventional artificial seed of the type in which alginate gel beads are embedded.

Example 3 Artificial Seeds Encapsulating the Protocomb-like Body of Phalaenopsis Protocomb-like bodies formed from the flower stem axillary buds of a white-flowered yellow lip of phalaenopsis were selected using nylon meshes with 1.5 mm and 3 mm openings. , 1.5 to 3 mm in size. The obtained protocomb-like body was subjected to hyponex fertilizer (N / P / K = 6.5: 6: 1).
9) The suspension was suspended in a medium having a pH of 5.6 containing 6 g / l, sucrose 15 g / l, and 0.2% chitosan to obtain the contents of a triple capsule.

The encapsulation was carried out in the same manner as in Example 1 except that the pump for feeding the plutocomb-like body suspension was adjusted so that one protocomb was included in one capsule. The resulting capsules were dried to obtain dried and encapsulated artificial seeds of a protocorm-like body of phalaenopsis. The particle size after drying was 9 mm.

After storing these artificial seeds in a dark place at 15 ° C. for 2 months, 100 seeds were sowed at a depth of 2 cm from the topsoil into a pot containing soil in a field not subjected to soil sterilization, and watered. After seeding, put in a 25 ° C incubator and
At 0 Lux, while lighting for 16 hours per day,
Sprinkled once every two days and allowed to grow. When the number of germinations after 2 months was indicated, 63 out of 100 germinated.

Examples 4 to 9 Adventitious embryos, apical buds or adventitious roots of plants shown in Table 3 were produced, encapsulated in the same manner as in Example 1, dried, and dried at 20 ° C. and 65% humidity for 3 months. After preservation in a thermostat, each 100 seeds were sown in a pot as in Example 1, allowed to grow for 1 to 3 months in the same manner as in Example 1, and germinated. Table 3 shows the results.

[0053]

[Table 3]

The results in Table 3 also show that the encapsulated artificial seed of the present invention has excellent storage stability.

[0055]

Industrial Applicability The encapsulated artificial seed containing the regenerated plant cell tissue of the present invention has high storage stability in a dry state at room temperature, and when seeded on soil, the capsule film swells due to microbial action. An artificial seed that collapses and rapidly germinates can be provided. Combined with plant cell tissue culture technology, cloning technology and virus-free technology,
It can be widely applied to agriculture, forestry, horticulture, flowers, etc.

[Brief description of the drawings]

FIG. 1 is a schematic sectional view of an artificial seed of the present invention.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Yoshimasa Kamaguchi 1-30, Tamazuki, Chuo-ku, Osaka City, Osaka Prefecture Inside Morishita Jintan Co., Ltd. (72) Hideki Sunohara 1-1-1, Tamazuki, Chuo-ku, Osaka City, Osaka No. 30 Morishita Nittan Co., Ltd.

Claims (5)

[Claims] 1. An artificial seed comprising a seamless soft capsule having three or more layers of an innermost layer, an endothelial layer covering the innermost layer, and an outer layer covering the innermost layer, wherein the innermost layer comprises: An artificial seed comprising regenerated plant cell tissue, wherein the endothelial layer is constituted by an inner coat mainly composed of hardened oil, and the outer layer is an outer coat having biodegradability. 2. The method of claim 1, wherein the outer coating is dried.
2. The artificial seed according to item 1. 3. The re-differentiable plant cell tissue is selected from the group consisting of adventitious embryos, adventitious buds, multiple shoots, shoot apex, vegetative point, protomic body, adventitious root, and hairy root.
The artificial seed according to claim 1 or 2. 4. The artificial seed according to claim 1, wherein the inner coating is a hardened oil that is solid at normal temperature and is biodegradable. 5. The artificial skin according to claim 1, wherein the outer skin is a biodegradable outer skin selected from the group consisting of proteins, polysaccharides, and biodegradable plastics. seed.