JPH0251598B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing, by culturing, a tissue culture containing a significant amount of yellow pigment of safflower. More specifically, the present invention relates to a method for producing a significant amount of safflower callus containing yellow pigment by culturing safflower cells in two stages, ie solid culture and liquid culture. In general, safflower is a plant of the Chrysanthemum family that is commonly cultivated in the Akita region.The flowers that are harvested contain a beautiful red pigment (carthamine) as well as a yellow pigment, as well as other Chinese herbal medicinal ingredients. The flowers are prized as tea. In addition, the red pigment extracted from the flower is sold as red dye and as a natural lipstick. This red pigment is also mixed with a yellow pigment. Since the yellow pigment contained in safflower is extremely useful as a natural pigment, the present inventors
In order to mass-produce yellow safflower pigment, we conducted intensive research on safflower callus culture and found that by lowering the concentration of specific components in the liquid culture medium, safflower cells were cultured in two stages: solid culture and liquid culture. They succeeded in producing large quantities of yellow-colored callus. The present invention involves culturing safflower cells or cell groups in a solid medium, and culturing the resulting callus in a solid medium in which at least ammonium ions are eliminated and the concentration of naphthalene acetic acid is reduced to 1/10 or less. This is a method for producing yellow pigment from safflower, which is characterized by culturing in a liquid medium containing: Safflower cells or cell groups used in the present invention are collected from growing points, but can also be collected from flower buds. The term "flower bud" as used herein refers to a young tissue that is undifferentiated or is about to differentiate, and is formed in the axil of a leaf that is lower than the bud at the top after a safflower plant has grown and has a bud at the top. This has the ability to turn into a flower by itself when the top bud dies. In the present invention, safflower cells or cell groups are first cultured in a solid medium, and then a liquid medium containing at least ammonium ions among the solid medium components is eliminated and the concentration of naphthalene acetic acid is reduced to 1/10 or less. It is cultivated to produce yellow-colored callus. Furthermore, in the present invention, it is also possible to carry out solid culture, then liquid culture, and then repeat liquid culture in order to further increase the size of the callus. In this case, it is also possible to use a liquid medium containing solid medium components in which the concentrations of other components are further reduced. As the culture medium used in the present invention, the usual Murashige
A medium used for plant tissue culture such as Skoog, White, and Gamborg is used, and the inorganic components used here include nitrogen, phosphorus, potassium,
Calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine, cobalt, etc. Specifically, potassium nitrate, sodium nitrate, calcium nitrate, monopotassium phosphate, and disodium phosphate. , potassium chloride, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate,
Examples include manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, and cobalt chloride. Examples of carbon sources include hydrocarbons such as sucrose, derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol. Plant hormones include auxins such as indoleacetic acid (IAA), naphthaleneacetic acid (NAA), p-chlorophenoxyisobutyric acid, and 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, and zeatin. Examples include cytokinins such as , dihydrozeatin, and benzyladenine. Examples of vitamins include biotin, thiamine (vitamin B ₁ ), pyridoxine (vitamin B ₆ ), pantothenic acid, ascorbic acid (vitamin C), inositol, and nicotinic acid. Examples of amino acids include glycine, alanine, glutamine, and cysteine. Regarding the component composition of the liquid medium in the present invention, it is necessary to eliminate at least ammonium ions among the components of the solid medium and reduce the concentration of naphthalene acetic acid to 1/10 or less. In a liquid medium, other components that lower the concentration compared to a solid medium are preferably at least one component selected from inorganic components, plant hormones, vitamins, and amino acids. Among these, inorganic components such as nitrate ions, phosphate ions, potassium ions, calcium ions, iron ions, manganese ions, cobalt ions, iodine ions, sodium ions, chloride ions, cytokinins, At least one component selected from vitamins and amino acids is suitable. In addition, for naphthalene acetic acid, solid medium,
Required for both liquid media, but not for solid media.
When containing about 10 ^-5 M, the liquid medium contains 10 ^-6 ~
It is necessary to reduce the concentration to about 10 ^-9 . Further, as a solid medium, it is sufficient to add about 0.8% agar to a liquid medium containing various components. Preferred examples of the tissue culture method of the present invention include the following methods. That is, safflower cells or cell groups are placed on a solid medium and cultured at 10 to 35° C. for about 7 to 30 days to form calluses from the cells or cell groups. When the callus thus obtained is subcultured, the production rate gradually increases and stable callus is obtained. This callus is added to a liquid medium containing components in which at least ammonium ions have been removed from the solid medium and the concentration of naphthalene acetic acid has been reduced to 1/10 or less, and cultured by rotation. In the culture of the present invention, light is not necessarily required, and the culture temperature is preferably 10 to 35°C, particularly around 25°C. The callus that has been cultured in liquid after solid-state culture turns yellow, and it can be seen that a large amount of yellow pigment is produced. Yellow pigment can be extracted from callus by the same method as conventionally used for extracting safflower pigment. Next, examples of the present invention will be shown, and the modified Murashige-Skoog medium used here is A medium,
Each composition of the Otsu medium is shown in Table 1 below.

【table】

[Table] Example 1 On the 60th day after sowing safflower, when the flower buds had slightly swollen, a large number of cell groups were separated aseptically. Separately, a solid medium prepared by adding agar to the A medium shown in Table 1 was prepared, and safflower flower bud cells were dispersed therein and cultured at 25°C for 20 days to obtain a large number of calli. Next, 30 ml of Otsu medium 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 placed therein and cultured with rotation at 25°C for 14 days. After culturing, the callus was collected by filtration and air-dried for 24 hours to obtain 9 g of dry callus/culture solution, which was ground, extracted with ethanol, and 40 mg of yellow pigment/g of dry callus was obtained by evaporating the ethanol. Ta. Example 2 When a safflower flower bud was still in a state where it could not be determined visually, the safflower bud was cut and a large number of small cell groups were separated aseptically. Separately, the above small cell groups were dispersed in a solid medium prepared by adding agar to the A medium shown in Table 1, and cultured at 25°C for 15 days to obtain a large number of calli. Next, 30 ml of Otsu medium 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 placed therein and cultured with rotation at 25°C for 15 days. After culturing, the callus was collected by filtration and air-dried for 24 hours to obtain 8 g of dry callus/culture solution, which was ground, extracted with ethanol, and the ethanol was evaporated to remove the yellow pigment.
50 mg/g dry callus was obtained. Example 3 When a safflower flower bud was still in a state that could not be determined visually, the safflower bud was cut and a large number of small cell groups were separated aseptically. Separately, the above small cell groups were dispersed in a solid medium prepared by adding agar to the A medium shown in Table 1, and cultured at 25°C for 15 days to obtain a large number of calli. Next, 30 ml of Otsu medium 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 placed therein and cultured with rotation at 25°C for 15 days. Furthermore, the callus obtained here was added to 30 ml of Otsu medium shown in Table 1 in a 100 ml Erlenmeyer flask, and cultured with rotation at 25° C. for 14 days. After culturing, the callus was collected by filtration and air-dried for 24 hours to obtain 14 g of dry callus/culture solution, which was ground, extracted with ethanol, and the ethanol was evaporated to obtain 65 mg/g of dry callus with yellow pigment. Ta.

Claims (1)

[Claims] 1. Cells or cell groups of safflower are cultured in a solid medium, and the resulting callus is prepared by removing at least ammonium ions among the components of the solid medium and containing components in which the concentration of naphthalene acetic acid is reduced to 1/10 or less. A method for producing yellow pigment from safflower, which is characterized by culturing in a liquid medium.