JPH062055B2 - Method for producing saffron stigma-like tissue - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Industrial Field of the Invention The present invention efficiently processes saffron containing components useful as coloring agents, flavors, bittering agents, etc. by tissue culture of the styles and ovaries constituting the pistil. It relates to a method of producing.

Conventional technology Saffron pistil is composed of stigma, style and ovary from the upper part.The stigma is crimson in color and contains useful ingredients such as medicinal and aroma components in addition to saffron pigment. I'm out.

Conventionally, the saffron stigma containing such useful components has been collected from its pistil by cultivating saffron to bloom.

Usually, even if you grow large saffron bulbs of about 300,
Since only about 6 flowers arrive, it divides into 3 and only about 18 stamens can be collected. If you try to take 1 kg of pistil stigma, the required amount of saffron bulb is about 50.
It can be as much as 0 kg. Therefore, the production of stigma stigma of saffron requires a vast cultivation area. Moreover, natural cultivation and production takes time, is greatly affected by the weather, and saffron is an extremely unwilling plant, so it is difficult to cultivate and produce pistil stigma efficiently. Even the stigma stigma of saffron is very expensive.

In recent years, generally, for the purpose of producing natural pigments, studies on pigment production by tissue culture of plants have been widely conducted, and saffron has also been proposed for tissue culture in a medium containing a compound having a hormone action. However, it is not always satisfactory.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The present invention relates to tissue culture, particularly in tissue culture using a medium containing benzyladenine and α-naphthalene acetic acid,
It was made for the purpose of providing a method for efficiently producing stigma-like stigma-like tissue.

Means for Solving the ProblemsThe inventors of the present invention include benzyladenine and α-naphthalene acetic acid in Murashige-Skoog medium in tissue culture of saffron style and ovary, and the concentration of these compounds and the inclusion of both. It was found that the production amount unexpectedly increases by adjusting the ratio within the specific range, and the present invention has been completed based on this finding.

That is, the present invention, when the saffron style and or ovary or both in tissue culture in Murashige-Skoog medium containing benzyladenine and α-naphthalene acetic acid,
Provided is a method for producing a saffron stigma-like tissue, which comprises adjusting the concentration of benzyladenine to around 10 ppm and keeping the concentration of α-naphthalene acetic acid below the concentration of benzyladenine.

As described above, the useful component of saffron is contained in the part of the stigma that composes the pistil of saffron in the case of natural production, but according to the present invention, the stigma and the ovary, which are parts (organs) different from the stigma originally. The stigma-like tissue can be efficiently produced by culturing the part of the stigma, and useful components of saffron such as pigment can be efficiently collected from this stigma-like tissue.

The present invention relates to a specific substance 2 belonging to a so-called plant hormone.
Using Muratige-Skoog medium containing seeds, that is, benzyladenine and α-naphthaleneacetic acid together, and adjusting the content and content of these tissues when culturing specific organs of saffron in the light or in the dark As a result, it is characterized in that colored stigma-like tissue is efficiently proliferated.

In the method of the present invention, saffron styles and / or ovaries are used, and the saffron style style ovaries are different from the colored stigmas that are usually used for production of saffron pigments and medicinal purposes. Means the organ of.

When tissue culture of saffron style or organs other than ovary, such as stigma and flower stem, is performed, callus is produced, but colored stigma-like tissue cannot be produced.

In the present invention, the part of the style and / or the ovary is taken out before the flowering of saffron, that is, in the state of lightning, and this is subjected to tissue culture according to a usual method to produce and grow stigma-like tissue. It is difficult to efficiently produce stigma-like tissue if the same operation is performed after flowering of saffron.

To describe a typical example of the stigma-like tissue obtained in the present invention, the shape is very similar to that of the stigma of the saffron pistil, and the color depends on the content of the saffron pigment produced.
The color changes from yellow to red.

In the method of the present invention, the medium is Murashige Scoog (Mu
rashige-Skoog) medium is used. In the present invention, benzyladenine and α-naphthalene acetic acid are added to this basal medium for culturing.

Verdyl adenine contained in the medium in the method of the present invention is a substance belonging to the plant hormone cytokinins, and α-naphthalene acetic acid is a substance belonging to the plant hormone auxins. In the method of the present invention, a medium in which these two specific substances coexist is used, and the concentration of benzyladenine in the medium is set to about 10 ppm, and α
-It is necessary to adjust the concentration of naphthalene acetic acid below the concentration of benzyladenine.

When the concentration of benzyladenine is considerably lower than 10 ppm, for example, 5 ppm, or when it is considerably high, for example, 15 ppm, the amount of stigma-like tissue, that is, saffron pigment, is significantly reduced. In addition, when the concentration of α-naphthalene acetic acid is higher than that of benzyladenine, the production amount also decreases.

In the method of the present invention, by significantly increasing the concentration of benzyladenine in the medium as compared with the usual case, the production and growth of stigma-like tissue is further promoted, and the combined use of α-naphthalene acetic acid further improves the efficiency. Has been done.

In the method of the present invention, other medium such as Lismeier-Skoog medium or Gamborg B5 medium may be used instead of Murashige-Skoog medium, or other substance belonging to cytokinins such as kinetin may be used instead of benzyladenine. Moreover, even if other substances belonging to the auxins such as indole-3-acetic acid are used instead of α-naphthalene acetic acid in the present invention, such a result cannot be obtained.

The Murashige-Skoog medium may appropriately contain other substances as long as the object of the present invention is not impaired.

The tissue culture of the method of the present invention may be, for example, a static culture method using a solid medium or a shaking culture method using a liquid culture. As a solid medium, for example, agar is about 0.
A Murashige-Skoog medium containing 8% by weight is used. The culture may be in the light or in the dark. Culture temperature is 15 ~
It is carried out at 30 ° C, preferably in the range of 20 to 30 ° C.

In the case of culturing in Murashige-Scoog's medium at 25 ° C. in the dark, production of stigma-like tissue is usually observed after 1 to 2 months. The stigma-like tissue thus produced is subsequently cultured in solid or liquid medium in the dark or in the light to grow the stigma-like tissue.

In order to extract the saffron pigment from the colored stigma-like tissue grown by the method of the present invention, the stigma-like tissue is used as it is or is ground and extracted with a solvent such as water, hydrous ethanol, hydrous propylene glycol or the like at room temperature or under heating. do it.

The ultraviolet and visible absorption spectra of the yellow colored liquid obtained by extracting the stigma tissue obtained in the present invention with water were compared with the absorption spectrum of the aqueous saffron dye solution extracted from the natural stigma stigma, and obtained in the present invention. It was confirmed that the obtained pigment was equivalent to the natural saffron pigment.

EFFECTS OF THE INVENTION According to the present invention, it is possible to obtain a large amount of pigment in a short period of time regardless of the season, as compared with a method of obtaining saffron pigment by performing natural cultivation. The stigma-like tissue obtained in the present invention and the pigment obtained by extraction from the stigma-like tissue can be used for coloring foods, cosmetics, etc., or as a raw material for medicinal purposes.

EXAMPLES Next, the present invention will be described in more detail with reference to examples.

Example 1 Benzyl adenine and α were added to a Murashige-Skoog medium.
-Naphthalene acetic acid was added so as to have the amounts shown in Table 1, and agar powder was added thereto at 0.8% by weight, and sterilized in an autoclave at 120 ° C for 20 minutes. The obtained solution was dispensed into a plastic petri dish having a diameter of 55 mm and cooled and solidified to prepare a medium.

On the other hand, before the flowering of saffron, when it is still in the bud, the pistil part is collected, immersed in a 0.5 wt% sodium hypochlorite aqueous solution for 10 minutes to sterilize it, and then washed with water, and then the ovary part is cut out. Was placed in the above medium.

When this was statically cultivated in the dark at 25 ° C, production of stigma-like tissue was observed about 6 weeks after culturing, depending on the amounts of benzyladenine and α-naphthalene acetic acid, and
After a week, the tissue had grown and had become fairly reddish, producing considerable saffron pigment.

Regarding the production amount of stigma-like tissue, the pigment concentration and the production amount of saffron pigment, the results shown in Table 1, Table 2 and Table 3 were obtained.

Note (1) BA: benzyladenine α-NAA: α-naphthalene acetic acid Note (2) Apparent production of stigma-like tissue containing saffron pigment: An amount equivalent to one stigma portion of natural saffron containing saffron pigment 10, the non-quantitative one was set as 0, and the gap was proportionally divided to be a scale showing 11 levels of quantity, and saffron explants (tissue pieces cut from the ovary part of natural saffron, The stigma-like tissue was produced by culturing the tissue under a specific condition.) The equivalent of the stigma-like tissue containing the saffron dye generated per piece was visually determined, and the value was applied to the above scale.

Note (1) BA: benzyladenine α-NAA: α-naphthalene acetic acid Note (2) Apparent pigment concentration of stigma-like tissue containing saffron dye: The color density of the stigma portion of natural saffron containing saffron pigment is 10 Colorlessness is set to 0, and the interval is divided into 11 steps so as to be proportional to the concentration of saffron pigment, and used as a scale to indicate the color intensity, and the color intensity of stigma-like tissue containing saffron pigment generated by tissue culture is determined. It is judged visually and applied to the above scale to give a numerical value.

Note (1) BA: benzyladenine α-NAA: α-naphthalene acetic acid Note (2) Amount of apparent saffron pigment per saffron tissue explanted shoulder: 1 part of stigma containing natural saffron pigment The amount corresponding to the amount of saffron pigment contained in was 100 and the amount that was not quantitatively was 0, and the amount was proportionally divided and used as a scale showing 11 levels, which was generated per saffron explant by tissue culture. The production amount of the saffron dye is visually determined with reference to Tables 1 and 2 and applied to the above scale to give a numerical value.

As is clear from these results, when the concentration of benzyladenine is around 10 ppm, the production amount of stigma-like tissue and the production amount of saffron pigment are maximized when the concentration of α-naphthalene acetic acid is below the concentration of benzyladenine. Become.

Example 2 Instead of culturing the ovary part of saffron in Example 1, an experiment was conducted under the same conditions using a style part collected from a bud, and similar results were obtained.

Comparative Example 1 In Example 1, indole-3-acetic acid was used instead of α-naphthalene acetic acid, and when the culture was attempted in the same manner as in Example 1, the organ differentiation did not occur, and the saffron dye was Did not generate.

Comparative Example 2 When an experiment was conducted in the same manner as in Example 1 except that benzyladenine was used alone, only a small amount of stigma-like tissue was produced, and the amount of saffron pigment produced was compared to that in Example 1. And obviously there were few.

Comparative Example 3 When an experiment was conducted in the same manner as in Example 1 except that α-naphthalene acetic acid was used alone in Example 1, stigma-like tissue could not be produced.

Claims (1)

[Claims] 1. When culturing saffron style and / or ovary or both in tissue culture in Murashige-Skoog medium containing benzyladenine and α-naphthaleneacetic acid, the concentration of benzyladenine is adjusted to around 10 ppm, and α
-A method for producing saffron stigma-like tissue, characterized in that the concentration of naphthalene acetic acid is kept below the concentration of benzyladenine.