JPH064027B2 - Safflower yellow pigment producing tissue culture method - Google Patents

Description

The present invention relates to a method for producing a tissue culture containing a large amount of safflower yellow pigment by culturing.

More specifically, the present invention relates to a method for producing a significant amount of safflower callus containing a yellow pigment by using safflower flower buds.

In general, safflower is a plant of the chrysanthemum family that is often cultivated in the Akita region, and the flowers harvested contain not only the beautiful red pigment (cartamine) but also the yellow pigment, and because it also contains other medicinal herbs, it is dried. The flowers are made as tea. The red pigment extracted from flowers is sold as a scarlet dye and as a natural lipstick. The red pigment is also mixed with a yellow pigment.

In order to mass-produce useful safflower yellow pigments, it is sufficient to cultivate safflower itself in large quantities and to separate only the yellow pigments from the flowers, but it takes time for the safflower flowers to bloom, and There is a problem that the quality of the item depends on the weather, and the price is high.

Therefore, it is conceivable to produce such a useful safflower yellow pigment using undifferentiated cell groups (callus), but heretofore, there have been only examples of producing safflower yellow pigment by callus culture. I can't.

The present inventors have conducted intensive studies on callus culture of safflower in order to mass produce safflower yellow pigment,
By culturing cells or cell groups separated from safflower flower buds, they succeeded in producing a large amount of yellow-colored callus.

The present invention, in safflower yellow pigment-producing tissue culture,
It is a yellow pigment-producing tissue culture method for safflower characterized by using flower buds of safflower.

The safflower flower bud used in the present invention refers to an undifferentiated or just-differentiated young tissue that occurs in the axillary leaves of the leaves that are below the buds on the apex of the safflower plant that have grown and have buds on the apices. This has the ability to become a flower by itself when the top buds are gone.

Cells or cells isolated from safflower flower buds can be grown first on agar and then in liquid medium to obtain large amounts of callus containing yellow pigment.

An auxin acting substance may be added to both the agar medium and the liquid medium used in the present invention. Naphthalene acetic acid (NAA) is a good auxin agent with a concentration of 1
It may be about 0 ^-4 to 10 ^-8 M.

Regarding the other components of the medium, an ordinary plant tissue culture medium may be used, and there is no particular limitation.

That is, examples of carbon sources and energy sources include carbohydrates such as sucrose, derivatives thereof, organic acids such as fatty acids, primary alcohols such as ethanol, amino acids such as aspartic acid, and the like, and calcium chloride, magnesium sulfate, and sulfuric acid as inorganic salts. Examples include iron and calcium dihydrogen phosphate.

Also, as the nitrogen source, ammonium ions, nitrate ions,
Examples include nitrogen-containing compounds such as amino acids and protein cleavage products such as peptone.

Specifically as the liquid medium, Murashige-Skoog's medium,
There are White's medium, Gamborg's medium and these modified media. In addition, when cytokinins such as kinetin, zeatin and benzyladenine are allowed to coexist in a liquid medium at a concentration of 0.1 to 100 µM, particularly 10 ^-6 to 10 ^-7 M, it is favorable for the generation of callus growth pigment. . If necessary, nutrients such as yeast extract, malt extract, tomato juice, casamino acid, coconut milk and vitamin mixture may be added.

As the solid medium, it is sufficient to add about 0.8% agar to the above liquid medium.

The following methods are suitable examples of the tissue culture method of the present invention. That is, cells or cell groups of flower buds of safflower are aseptically collected, and the concentration of nitrogen, phosphorus, and potassium in the medium of Murashige-Skoog is halved, and NAA is 10 ^-6 M, benzyladenine (BA). Place on agar medium at 10 ^-6 M and
Cultivate the cells or cell groups at 5 ° C for about 7 to 30 days to callus. When the callus thus obtained is subcultured, the production rate gradually increases and a stable callus is obtained.
The callus is added to a liquid medium having the same composition or reduced NH ₄ ⁺ ions, and cultivated by swirling.

In the culture of the present invention, light is not always necessary, and the culture temperature is preferably 10 to 35 ° C, particularly around 25 ° C.

The callus turns yellow due to liquid culture that has undergone solid culture,
It can be seen that a large amount of yellow pigment is formed.

To extract the yellow pigment from the callus, the conventional method for extracting the pigment of safflower can be used.

In the present invention, callus containing a yellow pigment could be produced by performing tissue culture using safflower flower bud cells or cell groups.

Next, examples of the present invention will be shown. The respective compositions of the A medium and the B medium used here are shown in Table 1 below.

Example 1. When the flower buds of safflower slightly expanded, a large number of cell groups were aseptically separated.

Separately, a solid medium prepared by adding agar to the instep medium shown in Table 1 was prepared, and the safflower flower blast cell group was dispersed in the solid medium at 25 ° C.
After culturing for 20 days, many calli were obtained.

Next, 130 ml of Otsu's medium shown in Table 1 was placed in a 100 ml Erlenmeyer flask and sterilized at 120 ° C. for 10 minutes.

After culturing, callus was collected by filtration, air-dried for 24 hours and dried callus 9 g /
A culture solution was obtained, which was ground, extracted with ethanol, and ethanol was evaporated to obtain 40 mg / g dry callus of yellow pigment.

Example 2. When the flower buds of safflower were still indistinguishable from the appearance, the place was cut and many small cell groups were aseptically separated.

Separately, the above-mentioned small cell group was dispersed in a solid medium prepared by adding agar to the instep medium shown in Table 1 and cultured at 25 ° C for 15 days to obtain a large number of callus.

Next, 30 ml of B-culture medium shown in Table 1 was placed in a 100 ml Erlenmeyer flask and sterilized at 120 ° C. for 10 minutes.

After culturing, callus was collected by filtration, air-dried for 24 hours and dried callus 8 g /
A culture broth was obtained, which was ground, extracted with ethanol, and the ethanol was evaporated to give 50 mg / g dry callus of yellow pigment.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References Y, Yamamoto et al, “Theor, Appl, Genet”, 61 p. 113- (1982) T, Yamakawa et al, "Agric, Biol, Chem", 47 (1983) P. 997-1001

Claims (1)

[Claims] 1. A method for cultivating yellow pigment-producing tissues of safflower, which comprises using flower buds of safflower in the culture of safflower yellow pigment-producing tissue.