JPH05103664A - Method for cell fusion of saffron - Google Patents

Abstract

PURPOSE:To obtain the subject fused cell capable of efficiently producing a coloring matter for a colorant, etc., used in foods by converting plural saffron cells having different physical properties into protoplasts, carrying out cell fusion and creating a genetic modified strain, having a high proliferation rate and producing a large amount of the coloring matter. CONSTITUTION:The exodermis on a bulb of a saffron having a flower bud elongating to about 10cm is peeled and discolored parts are removed. The bulb is washed with flowing water and then successively treated with 10% benzalkonium chloride, 70% ethanol and 5% sodium hypochlorite and a caliculus is taken out of the flower bud and excised to collect a pistil, which is subsequently inoculated into a culture medium. Naphthaleneacetic acid, benzylarginine, etc., are then added to collect a cell redifferentiated from a callus. A cell having a high proliferation rate and a cell capable of producing a large amount of a coloring matter as two or more kinds of cells having different characteristics are respectively selected from the cells and treated with an enzymic solution such as cellulase and converted into protoplasts. Suspensions of both the protoplasts are mixed and a cell fusion agent such as polyethylene glycol is added to culture the fused protoplasts. Thereby, the objective fused cell is obtained.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for cell fusion of saffron. More specifically, the present invention relates to a method for efficiently obtaining a pigment by cell fusion of protoplasts of two or more kinds of saffron having different characteristics such as saffron cells having a high proliferation rate and saffron cells having a large amount of pigment production.

[0002]

2. Description of the Related Art Saffron is a perennial herbaceous crocus sativa L. (Crocus Sativus L.),
The dried stigma is used as a crude drug. Spain,
In France, Italy and Japan, it is produced in Hiroshima, Kagawa, Oita and Iwate prefectures. As a component, it contains about 2% of carotenoid pigment, its component is crocin (glycoside of crocetin), and the bitter glycoside picrocrocin (Picroc).
rocin) about 2% and essential oil 0.4-1.3%. The main component of essential oil is safranal (aroma component peculiar to crude drugs).

This saffron is used as a spice, a coloring agent for foods, or as a medicinal agent, but since it is a dried product of messive, it can be taken in a small amount as a natural product.
The unit price is also very high. In addition, natural products are difficult to obtain with a certain quality due to climate change. Therefore, various tissue cultures have been tried.

For example, Japanese Patent Application Laid-Open No. 62-275617 discloses Crocus Sativas L.A. That is, there is disclosed a method for growing, expanding and maturing the pistil of saffron or an organ corresponding thereto with cytokinin or an artificial culture medium containing cytokinin and auxin. Cytokinin has long been known as a substance that induces cell division in cultured plant cells, and it is also known that in combination with auxin, callus induction and cell proliferation are promoted.

Japanese Patent Laid-Open No. 63-109788 discloses Crocus Sativas L.A. Of the female organ of Escherichia coli was cultured and cultured in an artificial culture medium containing cytokinin, and selected from the group consisting of safranal, picrocrocin and their analogs, crocin, crocetin and their analogs.
It is disclosed to produce the above substances. But,
It has long been known that saffron pistil contains these substances.

Japanese Unexamined Patent Publication (Kokai) No. 63-160580 discloses that callus of a crocus plant is cultured in an MS medium containing plant hormones. It has long been well known to use plant hormones such as auxin and cytokinin for culturing plant tissue, and Murashige-Skoog medium is also a commonly used medium.

In Japanese Patent Laid-Open No. 63-258574, the stigma, style, ovary, ovule and petal of saffron are extracted, and the cut tissue is placed in LS medium or B5 medium containing cytokinin and auxin. Transplanting and subculturing are disclosed. It has been practiced for a long time to extract plant parts, cut and culture, and plant hormones such as cytokinin and auxin are usually used, and Rinsmeier-Skoog medium, Gamborg's medium and the like are also usually used.

Testing any of these methods does not significantly increase the growth rate of the active ingredient of saffron, and does not dramatically increase the production of the active ingredient. These methods belong to the so-called callus induction method, which is obtained by cutting a part of a plant and culturing it under appropriate conditions. There is nothing related to cell fusion of saffron protoplasts.

[0009]

DISCLOSURE OF THE INVENTION The object of the present invention is suitable for adding genetic modification in order to obtain a strain of saffron which can efficiently obtain the pigments of crocin and crocetin, which are one of the active ingredients of saffron. To provide a method for cell fusion of protoplasts.

[Means for Solving the Problems]

As a result of intensive studies to solve the above-mentioned problems, the present inventor converted saffron cells into protoplasts and fused the cells to produce fused cells having high pigment productivity and high proliferation rate. It was found that the intended purpose can be achieved by selective culturing of, and the present invention was completed.

That is, the present invention provides (1) a method for cell fusion of saffron, which comprises protoplasting cells of two or more kinds of saffron having different characteristics and cell fusion.
(2) A feature of saffron is that at least a cell with a high proliferation rate and a cell with a large amount of pigment production are made into protoplasts, the protoplast suspensions of both are mixed, a cell fusion agent is added, and the fused protoplasts are cultured. (3) The method for cell fusion of saffron, (3) the method for cell fusion of saffron according to the above (1) or (2), which comprises performing two-step enzyme treatment when producing protoplasts, (4) cell-fused protoplasts The cell fusion method according to any one of (1), (2), and (3) above, which comprises culturing in a state of being embedded in agarose.

First, a method for preparing saffron protoplasts will be described. The cells used may be cultured or cut out from a plant. When using the one cut out from the plant, a saffron slice sterilized by a conventional method can be used, and when using the cultured one, for example, the method of Japanese Patent Application No. 3-179053 by the present applicant can also be used.

The medium used for this is not particularly limited, and Murashige and Skoog medium,
White medium, Gamborg et al. Medium, Nitsch medium, Heller medium,
Schenk and Hildebrandt medium, Nitsch and Nitsch medium, Knop medium, Rinsmeier Skoog (L
insmaier and Skoog).

If necessary, cytokinins and auxins known as plant growth regulators may be added to these media. Examples of cytokinins include benzyladenine, zeatin, kinetin, and the like, and when these are added, 0.01 to 2 is added when added.
Add 0 ppm. Examples of the auxin include naphthalene acetic acid, indole acetic acid, 2,4-dichlorophenoxyacetic acid, etc. When these are added, 0.01 to 20 ppm is added.

If necessary, other media agents can be added. Also, the culture is carried out at a temperature of 20 to 25 ° C. in the dark.

As a result, cells cut out from the plant or cultured cells are used, but it is particularly preferable to use messy tissue redifferentiated from saffron callus. As the method, among the cells cultured by the above-mentioned culture method, the messy tissue redifferentiated from saffron callus may be selected.

To adjust the osmotic pressure, glucose, saccharose, mannitol, sorbitol, ficoll, pacol, etc. are added in an amount of 0.3 to 0.7 mol, and the pH is 4.5 to.
A predetermined enzyme is allowed to act in the solution adjusted to 6.5.
As enzymes, the cell wall lysing enzymes cellulase, dolicerase, macerozyme, pectolyase, etc.
5 to 2.0%, preferably 2 or more kinds in total 0.1
The temperature is 6.0%, the temperature is 25 to 35 ° C., and the working time varies depending on the conditions, but the working time is 1 to 5 hours.

Particularly in the first step, the enzyme treatment with the cell-adhesive lytic enzyme is carried out as usual, but in the second step, the enzyme treatment with the mixed enzyme of the cell-adhesive lytic enzyme and the cell wall lytic enzyme is carried out. Processing will increase the chance of getting protoplasts. In this case, the enzyme treatment in the first step is a cell-adhesive-dissolving enzyme such as 0.05 to 2.0% each of lyserase and pectolyase as an enzyme, the temperature is 25 to 35 ° C, and the action time varies depending on the conditions.
Let it work for 3 hours. After that, as a second step of enzyme treatment, 0.05 to 0.05% of each of dolicerase and pectinase was added.
2.0%, 0.1% cell wall lysing enzyme such as cellulase
The reaction time is 2.0%, the temperature is 25 to 35 ° C., and the operation time is 1 to 3 hours although it varies depending on the conditions. This increases the probability that protoplasts will be obtained by mixing the cell wall lysing enzyme also in the second step. Further, by performing mesh filtration before the second step treatment, cells or fragments thereof that are not suitable for plast can be removed. In the method of obtaining protoplasts from mesophyll tissue such as tobacco, the enzyme treatment is performed in two steps due to the histological properties of mesophyll tissue. That is, first, the substance that has fused cells to each other is lysed with pectinase or the like to isolate the cells, and then the cell wall is lysed with cellulase or the like to obtain protoplasts. This method has been applied to the isolation of protoplasts of mesophyll cells other than tobacco, but it is difficult for some plants to become protoplasts, and it is necessary to find the optimal conditions for protoplast isolation for individual materials. There is. On the other hand, in the protoplast formation of cultured cells, the difficulty of protoplast formation is controlled by the origin of the cultured cells, cell line and culture conditions, and changes depending on the combination of medium composition and plant growth substance.
The messivels obtained from the saffron cultured cells were found to have strong epidermal cells and a considerably thickened cell wall between cells. Then, as a result of preparing a mixture of enzyme solutions capable of protoplastizing both epidermal cells and cells inside the epidermis, good results were obtained.

Using the above-mentioned method, for example, cells with a high proliferation rate and cells with a large amount of pigment production are converted into protoplasts. The obtained protoplasts were purified by filtration and centrifugation with a nylon mesh, and the density was adjusted to 1 × 10 ³ to 1 with a liquid adjusted to the same osmotic pressure and pH as when protoplasted.
Resuspend at × 10 ⁴ . Mix both protoplast suspensions and let stand for 5-30 minutes. A cell fusion agent is added to this mixed solution, gently stirred for 1 to 5 minutes, and allowed to stand for 5 to 30 minutes. Polyethylene glycol (molecular weight 300 to 6000) polyvinyl alcohol or the like is used as the cell fusion agent at a concentration of 10 to 50%, although it varies depending on the type.

Thereafter, the cell fusion agent is removed by diluting the solution 10 to 50 times with a solution adjusted to the same osmotic pressure as that used for protoplast formation and adjusted to a higher pH, that is, pH 9 to 11 to remove the cell fusion agent. Let stand for 30 minutes. The fused cells are cultured. In this case, the liquid medium contains 0.3 to 0.
Protoplasts are embedded in a medium added to 7% so as to be embedded and cultured. Saffron protoplasts do not grow as colonies unless cultured in such a state of being embedded in agarose gel. For example, the suspension of protoplasts is sucked up with a pipette, then pierced into the medium of an agarose gel, and then the contents are extruded to embed the protoplasts in the gel.

Agarose may be commercially available, preferably low melting point agarose. Agarose is [C ₁₂ H ₁₄ O
_It is a polysaccharide represented by the formula of ₅ (OH) ₄ ] _n , and is almost exclusively composed of D-galactose and 3,6-anhydro-L-galactose, and its abundance ratio is 1: 1. The medium used for this is not particularly limited, and Murashige and Skoog medium, White (White)
Medium, Gamborg et al. Medium, Nitsch (Nit
sch medium, Heller medium, Schenk and Hildebrandt medium, Nitsch and Nitsch medium, Knop
Medium, Linsmaier and Sko
og) medium and the like.

If necessary, cytokinins and auxins known as plant production regulators may be added to these media. Examples of cytokinins include benzyladenine, zeatin, kinetin, and the like, and when these are added, 0.01 to 2 is added when added.
Add 0 ppm. Examples of the auxin include naphthalene acetic acid, indole acetic acid, 2,4-dichlorophenoxyacetic acid, etc. When these are added, 0.01 to 20 ppm is added.

If necessary, other media agents can be added. Also, the culture is carried out at a temperature of 20 to 25 ° C. in the dark.

[0024]

The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the invention thereto. (Example 1) a, Selection of cells After removing the discolored portion by peeling off the outer skin of a saffron bulb with a flower bud extending to about 10 cm, the cell was washed with running water for 3 hours. 10
% Benzalkonium chloride for 3-4 seconds, 70% ethanol for 1-2 seconds, 5% sodium hypochlorite for 20-40 minutes. After that, it was performed aseptically. The flower buds were cut off leaving the bulb portion and washed 3 times with sterile water. Then, the buds were taken out from the flower buds, and the buds were incised and the messies were taken out in a state where the styles were long and inoculated into the medium. The medium is Murashige
And and Skoog's medium was adjusted to 4% sucrose, 0.9% agar powder, and pH 5.8. To this, 10 ppm of naphthalene acetic acid and 1 ppm of benzyladenine were added.

B, Protoplast formation The saffron callus cultured under these conditions was treated with dolcerase 1.
The mixture was shaken in an enzyme solution of 0%, pectinlyase 0.1%, mannitol 0.6 mol, pH 5.5 at 30 ° C. for 1 hour, filtered through a 200 messi nylon mesh, and the cells remaining on the mesh were lyserase. 1.5%, pectinase 0.05%, cellulase 0.5%, mannitol 0.6 mol, pH 5.5 in an enzyme solution at 30 ° C. for 2 hours 30
After allowing it to act for a minute, it was filtered through a 200 mesh mesh nylon, and the portion passing through the mesh was centrifuged at 600 rpm to obtain protoplasts. This is density 5 × 10 ⁴ ce
It was suspended in an aqueous solution of 0.6 mol mannitol so that the concentration became ll / ml. This was carried out for cells with a fast growth rate and cells with a large amount of dye production, and the protoplasts were mixed.

C, Cell fusion Polyethylene glycol 1540 was added to this solution at 50%, lightly stirred for 5 minutes, and allowed to stand for 25 minutes.
Then, an aqueous solution of 0.6 mol mannitol pH 10.5 was added 10 times, and the mixture was left for 30 minutes.

D, Cultivation method Saccharose 4%, 2,4-dichlorophenoxyacetic acid 0.5 ppm and kinetin 0.1 ppm were added to an MS medium at pH 5.
Preparation of 8, agarose 0.6% was added, and after heating and stirring, the mixture was dispensed into a glass petri dish and solidified. A 0.6 mol mannitol aqueous suspension of protoplast was sucked up by a glass tube, stabbed into the gel, the contents were extruded, and saffron protoplast was embedded in an agarose gel. This was sealed with parafilm and cultured at 23 ° C. in the dark for 2 months.

(Example 2) After removing the discolored portion by peeling off the outer skin of a saffron bulb with flower buds stretched to about 10 cm, it was washed with running water for 3 hours. 3 to 10% benzalkonium chloride
-4 seconds, 70% ethanol for 1-2 seconds, 5% sodium hypochlorite for 20-40 minutes. After that, it was performed aseptically. The flower buds were cut off leaving the bulb portion and washed 3 times with sterilized water. Then, the buds were taken out from the flower buds, and the buds were incised and the messies were taken out in a state where the styles were long and inoculated into the medium.
The medium was Murashige and Skoo's medium adjusted to 4% sucrose, 0.9% agar powder, and PH 5.8. Naphthaleneacetic acid (5 ppm) and benzyladenine (0.5 ppm) were added to this, and the redifferentiated messiebe was collected from the callus.

B, Protoplast formation Saffron messive cultivated under these conditions was treated with cellulase 1.0.
%, Dolicerase 2.0%, macerozyme 0.5%, mannitol 0.6 mol, pH 5.8 in an enzyme solution at 30 ° C.
Shake for 1 hour, confirm with a microscope, filter with a 200-mesh nylon mesh, and then pass 600 parts through the mesh.
Centrifugation was performed at rpm to obtain protoplasts. This was suspended in a 0.6 mol mannitol aqueous solution so that the density was 5 × 10 ⁴ cells / ml. The above procedure was carried out for cells having a high proliferation rate and cells producing a large amount of dye, and the protoplasts were mixed.

C, Cell fusion Polyethylene glycol 1540 was added to this solution at 50%, lightly stirred for 5 minutes, and allowed to stand for 25 minutes.
Then, an aqueous solution of 0.6 mol mannitol pH 10.5 was added 10 times, and the mixture was left for 30 minutes.

D, Cultivation method Cultivation was carried out using the culture method of Example 1.

Example 3 a, Cell Selection Saffron cells were obtained using the cell selection method of Example 2. b, Protoplastization The method of protoplastization of Example 1 was used for protoplastization. c, Cell fusion Culture was performed using the cell fusion of Example 1. c, Culture method Culture was performed using the culture method of Example 1.

Comparative Example 1 The mixed protoplasts of Example 1 were cultured without cell fusion. (Comparative Example 2) According to the culture method of Example 1, the cells were cultured without being embedded in agarose.

The results of the culture are shown in Table 1.

[Table 1]

[0035]

[Effects of the Invention] Protoplasts of cells having a high proliferation rate and protoplasts of cells having a large amount of pigment production, such as protoplasts having different characteristics, are fused to each other and cultured to obtain genetically modified protoplasts. It is possible to make a saffron strain having both of them, and it is possible to efficiently produce a pigment which is one of the active ingredients of saffron.

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Claims (4)

[Claims] 1. A method for cell fusion of saffron, which comprises protoplasting cells of two or more kinds of saffron having different characteristics and cell fusion. 2. A method in which at least a fast-growing cell of saffron and a cell that produces a large amount of pigment are protoplasts, and a protoplast suspension of both is mixed, a cell fusion agent is added, and the fused protoplasts are cultured. A method for cell fusion of saffron, which comprises: 3. A two-step enzyme treatment in which, during the production of protoplasts, an enzyme treatment with a cell-adhesive lytic enzyme is carried out in the first step, and an enzyme treatment with a mixed enzyme of a cell-adhesive lytic enzyme and a cell wall lytic enzyme is carried out in the second step. The method for cell fusion of saffron according to claim 1 or 2, wherein 4. The cell fusion method according to claim 1, wherein the cell-fused protoplasts are cultured in a state of being embedded in agarose.