JPH064026B2 - Red pigment producing tissue culture method of safflower - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing by culture a tissue culture containing a large amount of safflower red pigment.

More specifically, the present invention relates to a method for producing a large amount of safflower callus containing a red 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 are dried because they contain beautiful red pigments (cartamine), yellow pigments, etc. Flowers are often used as tea. The red pigment extracted from flowers is sold as a scarlet dye and as a natural lipstick.

Large quantities of safflower itself can be cultivated in order to mass-produce useful safflower red pigments, but it takes time for the safflower flowers to bloom, and the quality of the flowers depends on the weather. However, the price is high.

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

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

The present invention, in safflower red pigment-producing tissue culture,
A method for producing a red pigment producing tissue of safflower, which is 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.

A large amount of callus containing a red pigment can be obtained by growing cells or cell groups isolated from safflower flower buds first on an agar medium and then on a liquid medium.

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 a source of asphyxiation, ammonium ions, nitrate ions,
Examples thereof include nitrogen-containing compounds such as amino acids and degradation products of proteins 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 other media, 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 good for 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 red due to liquid culture that has undergone solid culture,
It can be seen that a large amount of caltamine red pigment is produced.

Extraction of cartamine from callus may be performed by the same method as that conventionally used for extracting safflower red pigment.

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

Next, examples of the present invention will be shown, and the respective compositions of A medium, Otsume medium 1 and Otsume medium 2 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 0.8% agar to the instep medium of Table 1 was prepared, and the safflower flower blast cell group was dispersed therein,
The cells were cultured at 25 ° C for 20 days to obtain a large number of callus.

Next, 30 ml of Otsu's medium 1 shown in Table 1 was placed in a 100 ml Erlenmeyer flask and sterilized at 120 ° C. for 10 minutes, 0.7 g of the above callus was added, and the mixture was cultivated at 25 ° C. for 14 days by swirling.

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

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 0.8% 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 Otsu's medium 2 shown in Table 1 was placed in a 100 ml Erlenmeyer flask and sterilized at 120 ° C. for 10 minutes, 0.7 g of the above callus was added thereto, and the cells were cultivated at 25 ° C. for 15 days by swirling.

After culturing, callus was collected by filtration, air-dried for 24 hours and dried callus 8 g /
lCulture liquid was obtained, ground, and extracted with ethanol,
The ethanol was evaporated to give 50 mg / g dry callus of the red pigment carthamine.

 ─────────────────────────────────────────────────── ─── 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 culturing safflower red pigment-producing tissue, which comprises using flower buds of safflower in safflower red pigment-producing tissue culture.