JPH0474983B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field of the Invention The present invention relates to an artificial seed which can be sown in soil and grown in the same manner as natural seeds by encapsulating somatic embryos of plants.

Prior art and its problems In recent years, as a method for obtaining clones of superior plants,
Tissue culture methods have become widely adopted.
In particular, the callus culture method, in which callus cells that have been dedifferentiated are multiplied and then redifferentiated to regenerate the original plant, has attracted attention as a promising method for obtaining an almost unlimited number of clones, and is being actively implemented. ing. However, in this callus culture method, callus cells do not grow if they are directly sown in culture soil, so they are transferred to a regeneration medium, then transferred to a rooting medium, grown to seedlings, and then transplanted to soil. In addition to requiring a considerable amount of effort to replant, it requires large cultivation equipment, a vast sterile space, and special land to acclimatize the young plants grown in the artificial medium to natural soil. necessary,
It is difficult to carry out industrial mass production. In addition, when somatic embryos are once formed from callus cells, the somatic embryos are placed on an agar medium, grown to seedlings, and then transplanted into soil, which causes the same problem.

Features of the Invention The present inventors have conducted intensive research to improve the above situation, eliminate the trouble of transplanting, and efficiently regenerate plants through callus culture.
Somatic embryos of plants, together with nutrients, antibacterial agents, and a water-insoluble water-absorbing polymer substance that absorbs and retains water tens of times its own weight, at 1×10 ^-16 cc・cm/cm ²・sec・cmHg
By coating them with a water-soluble polymer substance with oxygen permeability, they can be sown directly into the soil and grown in the same way as natural seeds. No hassle,
The present inventors have discovered that plant regeneration can be performed efficiently and easily without the need for large or special cultivation equipment or land, leading to the present invention.

The present invention will be explained in more detail below.

Structure of the Invention The artificial seed of the present invention is made by coating a somatic embryo of a plant with a water-soluble polymer substance, and the somatic embryo includes callus cells, protoplasts, anthers, pollen,
Those derived from nucifera, ovary, ovule, etc. are preferable, and this allows reproduction and growth of clones of the original plant in which superior traits are maintained from superior plants with desired properties. Here, any plant that forms a somatic embryo can be used; examples include vegetables such as carrots, pumpkins, asparagus, celery, and parsley, and flowers such as poinsettia, tagara, cycad, and petijunia. , herbal medicines such as Korean ginseng, Indian snakewood, belladonna, and Korean daisies, grains such as barley and corn,
Examples include trees such as navel orange and buchan. The present invention can also be applied to new plants obtained by cell fusion, and the artificial seeds of the present invention are particularly effective for varieties that do not produce seeds and new plants.

According to the present invention, the somatic embryo of the above-mentioned plant is coated with nutrients, an antibacterial agent, and a water-absorbing polymeric substance, so that a plant can be grown from the somatic embryo by sowing it in soil.

Here, nutrients are nutrients such as sugars, inorganic salts, and vitamins necessary to grow somatic embryos and regenerate plants from somatic embryos. For example, Murashige-Skoog
Mediums used in normal tissue culture, such as medium and White medium, can be used. Also, if necessary, 2 or 4
Auxins such as -D, cytokinins such as kinetin, gibberellins, and other plant hormones can be added.

In addition, antibacterial agents are necessary to keep the inside of the covering sterile and prevent the growth of mold and bacteria in order to protect the somatic embryo, and according to the studies of the present inventors, antibacterial agents are If somatic embryos are coated without addition, the somatic embryos will not grow sufficiently. In this case, the antibacterial agent may be any agent as long as it does not act on somatic embryos; examples include organic copper agents such as copper 8-oxyquinoline; antibiotics such as penicillin, streptomycin, and chloramphenicol; and zineb. , maneb, thiadiazines, thiurams, and other organic sulfur agents; TPN,
Examples include captan, vinclozolin, pracimidone, and benzazole, and one or more of these can be used in combination.

Furthermore, water-absorbing polymeric substances include water-insoluble polymeric substances that absorb and retain water tens to hundreds of times or more than a thousand times their own weight, such as crosslinked polyethylene oxide, crosslinked polyvinylpyrrolidone, and sulfonated polyethylene. Crosslinked product, starch (meth)
Saponified acrylonitrile graft copolymer, hydrolyzed crosslinked polyacrylamide, self-crosslinked polyacrylate, crosslinked starch-(meth)acrylate copolymer, polyvinyl alcohol crosslinked product, vinyl ester-unsaturated carboxylic acid ( saponified ester) copolymer, saponified ethylene-vinyl ester-unsaturated carboxylic acid (ester) copolymer, saponified hydroxyalkyl acrylate-acrylamide copolymer, etc. A mixture of two or more species may be used. Furthermore, copolymers of these polymeric substances can also be suitably used. These polymeric substances are imparted with water insolubility and the ability to absorb and retain extremely large amounts of water by crosslinking and polymerizing water-soluble polymerizable monomers. For example, water-insoluble water-absorbing polymeric substances that are commercially available as materials for improving the urine and blood absorption ability of disposable diapers and sanitary products are preferably used.

In the present invention, it is necessary to blend a water-absorbing polymer substance in this way to impart a water-retention function to the capsule, and according to the studies of the present inventors, artificial seeds without blending a water-absorbing polymer substance can be produced. When planted in soil, they did not germinate, and upon examination, it was found that the water in the capsules had evaporated and the capsules became small and deflated. However, the capsules retain water due to the combination of water-absorbing polymeric substances. It has been found that germination occurs well when functions are provided.

Here, nutrients, antibacterial agents,
The amount of the water-absorbing polymer substance to be mixed differs depending on the type of these and the type of somatic embryo, but the amount of antibacterial agent added to the liquid medium usually used for tissue culture is 50 to 50%.
It is preferable to mix 200 ppm and 0.5 to 20% by weight of a water-absorbing polymer substance.

The artificial seeds of the present invention are produced by mixing somatic embryos of plants with the above-mentioned components, and then using natural water-soluble polymer substances such as carrageenan, fur-cerelan, and xanthan gum, and synthetic water-soluble polymer substances such as acrylamide and acrylic acid. The somatic embryo is coated with a water-soluble polymeric substance, which protects the somatic embryo from the external environment. Examples of water-soluble polymer substances include water-soluble polymer substances that have gas permeability. Here, gas permeability refers to oxygen permeability, and oxygen permeability is 1×10 ^-16 1×10 ^-16 cc・
By coating with a water-soluble polymeric substance having an oxygen permeability of cm/cm ² ·sec ·cmHg or more, the usefulness of the artificial seeds, such as their germination rate, is increased. Examples of such water-soluble polymer substances include alginic acid, gelatin, acrylamide, carrageenan, agar, etc. These can be used alone or in combination of two or more, or CMC,
It can also be used in combination with other polymeric substances such as gum arabic, polyvinyl alcohol, and polyethylene glycol, and plasticizers such as sorbitol and glycerin. In addition, there are various methods for coating somatic embryos with such water-soluble polymer substances, such as coacervation method, interfacial polymerization method, and submerged drying method. An in-liquid curing method with a small amount of curing can be suitably employed.

Although the method for producing artificial seeds of the present invention is not necessarily limited to this, for example, callus cells are grown in a callus culture tank, and components such as an antibacterial agent and a water-absorbing polymer substance are mixed therein. , a method such as preparing an aqueous solution of a coating substrate for protecting callus cells from the external environment and mixing both solutions with a solution for coating production (for example, a calcium chloride solution when sodium alginate is used as the coating substrate). It is possible to manufacture artificial seeds by coating with

Effects of the Invention As explained above, the artificial seeds of the present invention have the same functions as natural seeds by coating the somatic embryo of a plant with a water-soluble polymer substance together with nutrients, an antibacterial agent, and a water-absorbing polymer substance. It can be sown directly into the soil and grown, making it possible to regenerate plants efficiently and easily without the complicated replanting operations and vast sterile spaces required in conventional callus culture. It is effective for maintaining the superior traits of superior plants and for cloning.

[Example 1] After sterilizing the surface of a commercially available enlarged root of carrot (Daucas carota) with a 1/10 diluted sodium hypochlorite solution, it was washed twice with sterile water, and its pith was aseptically cut out. Contains 4-D (1 mg/)
The cells were placed on a Murashige-Skoog agar medium and callus was induced at 25°C.

Approximately 1 g of induced callus without 2,4-D
The cells were each transplanted into 5 bottles of Murashige-Skoog's liquid medium (50 ml in a 200 ml Kolben beaker) and cultured with shaking at 25° C. for 2 weeks to induce somatic embryos. Hereinafter, this culture solution will be referred to as a somatic embryo culture solution.

Antibacterial agents (copper 8-oxyquinoline 10 mg, penicillin 5 mg), absorbent polymer substance (0.5 g of a water-insoluble cross-linked polyacrylate that can absorb and retain approximately 200 times its own weight of water) are added to the above somatic embryo culture solution. were added and mixed.
As a comparative example, we also prepared a sample that did not contain either an antibacterial agent or an absorbent polymer substance.

Separately, prepare a 1.5% sodium alginate aqueous solution, drop the previously prepared somatic embryo culture solution into the inner tube of a double orifice tube into the 10% calcium chloride solution by passing the sodium alginate aqueous solution into the outer tube, and add the alginic acid A calcium coating was prepared. After the coated material had sufficiently hardened, it was taken out and those containing somatic embryos were selected and used as artificial seeds.

This artificial seed was sown on vermiculite and 1
Water was given once a day and the plants were left at 25°C under continuous lighting for 24 hours. Those containing both an antibacterial agent and an absorbent polymeric substance broke their film in about 10 days, and grew steadily thereafter. On the other hand, those that did not contain antibacterial agents were contaminated with microorganisms and died. In addition, in the case of those containing no absorbable polymeric substance, the coating material shriveled up in about 3 days, and no somatic embryos were observed to grow.

[Example 2] Callus was induced from shoots of asparagus (Asparagus officinalis L.). This callus
The embryos were transferred to Murashige-Skoog liquid medium, and somatic embryos were induced by shaking culture.

An antibacterial agent (streptomycin 1.0 mg) was added and mixed to 50 ml of the above somatic embryo culture solution. Furthermore, K-
Carrageenan was dissolved in physiological saline to a concentration of 7%,
An absorbent polymer material (a water-insoluble starch-acrylonitrile graft copolymer saponified product capable of absorbing and retaining about 100 times its own weight of water) was swollen in this to a concentration of 1.5%.

The somatic embryo culture solution and K-carrageenan solution were mixed 1:1 at a temperature of 37°C, and the mixture was heated to a temperature of 15°C.
A coated product containing K-carrageenan as a film was prepared by dropping it onto a 0.1M KC solution. After sufficiently curing, those containing somatic embryos were selected and used as artificial seeds.

This artificial seed was sown on vermiculite and 1
When watered once a day and left at 25°C under continuous lighting, the film broke down in about two weeks and grew steadily.

[Example 3] Antibacterial agents: 5 mg of zineb, 5 mg of chloramphenicol
Artificial seeds prepared in the same manner as in Example 1 except that the amount was changed to mg gave the same results as in Example 1.

Claims (1)

[Claims] 1 Somatic embryos of plants together with nutrients, antibacterial agents, and a water-insoluble water-absorbing polymer substance that absorbs and retains water tens of times its own weight at 1×10 ^-16 cc・cm/cm ²・sec・cm
An artificial seed coated with a water-soluble polymer substance having an oxygen permeability higher than Hg.