JPS6321471B2 - - Google Patents

Description

[Detailed description of the invention] Technical field: The present invention relates to medicinal carrots (Panax ginseng CA
Meyer) Tissue culture cultivation method, in particular, by culturing a part of the living tissue of medicinal carrot plants, which have been prized as a useful herbal medicine since ancient times, by culturing the tissue culture in a unique improved medium. The present invention relates to a method for producing at a high rate a medicinal carrot tissue culture containing a large amount of the same medicinal components as medicinal carrots, such as crude saponin and crude sapogenin.

Prior Art: The roots of medicinal carrots, such as Panax ginseng, Chikusetsu ginseng, American ginseng, Sanchi ginseng, etc., are prized as useful herbal medicines and are still widely used today. Medicinal properties include tonicity, longevity, sedation,
It has been shown to have stimulant and diuretic effects. Saponin and sapogenin are the main medicinal components of crude drugs obtained from medicinal carrots. Saponins extracted from medicinal carrots contain many component groups, Ro,
It includes Ra, Rb, Rc, Rd, Re, Rf, Rg and Rh, but it is Rb and Rg that have the central medicinal effect. Rb
is said to have a sedative effect, and Rg is said to have an stimulatory effect. Currently, there are almost no medicinal carrots in the wild, and they are cultivated. Cultivation is very difficult, requiring special considerations such as shading and using well-drained land at high altitudes with cool summers. Once cultivated, it is impossible to continue cultivating in the same place for 20 to 50 years.
Moreover, it takes 4 to 7 years to harvest. For these reasons, they have become very expensive.

For industrial tissue culture of medicinal carrot tissue culture, it is desirable to obtain a high yield using a medium with as simple a composition as possible. In order to increase the growth rate of tissue culture and improve the yield of tissue culture in plant tissue culture, conventional techniques such as MS
Medium (Murashige and Skoog's medium), White's medium, Gantheret's medium, Tulecke's medium,
Morel's medium, Linsmaier-Skoog,
An ordinary basic medium for plant tissue culture, such as Hildebrandt's medium, is used, to which casein decomposition products, soybean flour, cornstarch liquor, and vitamins are added. However, these methods may complicate the medium composition of the basic culture medium used conventionally, or may alter the properties of the painstakingly selected medicinal carrot tissue culture and suppress the production of medicinal ingredients. do not have.

OBJECT OF THE INVENTION: An object of the present invention is to provide a culture method for culturing medicinal carrot tissue culture with high yield using a medium with a relatively simple composition. Another object of the present invention is to provide a method for culturing a medicinal carrot tissue culture containing a large amount of crude saponin and crude sapogenin, which are the same medicinal ingredients as natural medicinal carrots.

Summary of the invention: The method for culturing medicinal carrot tissue culture of the present invention comprises:
Modified MS containing substantially no ammonium nitrate from tissue culture obtained from raw tissue of medicinal carrots
The above purpose can be achieved by culturing in a medium. "Substantially not containing ammonium nitrate" means that ammonium nitrate is actively removed from the prescription as an MS medium, but does not mean that even trace amounts of ammonium nitrate contained as impurities derived from other ingredients are removed.

Example: The present invention will be explained in detail below based on examples.

Conventional MS containing 2% and 3% by weight of sucrose and 0.165% by weight of ammonium nitrate
Modification that practically removes ammonium nitrate from the culture medium (does not contain ammonium nitrate from the beginning)
150 ml of MS medium was placed in a 300 ml Erlenmeyer flask and sterilized under high pressure. 8 to 10 g of medicinal carrot tissue culture previously cultured on MS solid medium containing 0.9% agar was inoculated into the above modified MS medium, and shake cultured at 25°C for 5 weeks using a reciprocating shaker at 90 strokes per minute. did. The dry weight of the tissue culture obtained (the weight obtained by drying it to a constant weight in a warm air dryer at 50°C) was measured and the results obtained were analyzed using conventional MS.
It is shown in the figure together with a comparative example using a culture medium. As is clear from the figure, when cultured in a modified MS medium that does not substantially contain ammonium nitrate, the yield of medicinal carrot tissue culture was increased by about 40 to 60% in terms of dry weight, regardless of the level of sucrose concentration.
This was observed throughout the entire culture period. The growth inhibitory effect of ammonium nitrate is similar to that of other Gamborg.
Although it was also observed in media, it was noticeable in MS media, which is easy to prepare.

The following method was used to obtain tissue culture from medicinal carrots used in Examples of the present invention. After thoroughly washing medicinal carrots with water, divide them into stems and roots, cut into large pieces, sterilize them with a disinfectant such as filtrate of salad powder, and then rinse well with sterilized water. The tissue pieces are then cut aseptically into appropriate sizes (for example, 0.5 to 1.0 cm) and placed on an agar medium. As the medium contained in this agar medium, a known plant tissue culture medium comprising various inorganic salts, vitamins and sugars may be used. As growth regulators, auxins include β-indoleacetic acid (IAA), α-naphthaleneacetic acid (NAA), 2.4
-Dichlorophenoxyacetic acid (2.4-D) and, as cytokinins, kinetin and gibberellin are added individually or in combination. By doing so, callus formation (dedifferentiation) can be achieved. Callus formation can be efficiently performed by adding coconut milk, yeast, casein hydrolyzate (casamino acid), etc. alone or in combination. Callus formation begins around the first week by multiplying the cells of the plant tissue piece under conditions of 23 to 28° C. in the dark and forms a callus in about 4 weeks. However, the medicinal carrot callus obtained in this way does not contain medicinal ingredients such as saponin and sapogenin. This is because the callus has not redifferentiated.

In order to redifferentiate medicinal carrot callus, this was combined with 2 ppm of indolebutyric acid and cytokinin.
Place on MS medium containing 0.1 ppm and illuminate with white light at 100 °C.
Irradiate at ~2000 lux. The callus redifferentiates. Similarly, strain selection is performed to obtain a tissue culture containing crude saponin and crude sapogenin, the medicinal principal components, similar to those of natural medicinal carrots. The crude saponin content of this strain was approximately 7 to 10% by dry weight.

Effects of the invention: According to the present invention, by using a modified MS medium that does not substantially contain ammonium nitrate, crude saponin, which has the same medicinal properties as natural medicinal carrots, can be replaced with medicinal carrot tissue culture containing crude sapogenin. The yield was approximately 40~40% of that obtained by conventional MS medium containing ammonium nitrate (0.165% by weight).
It can be increased by about 60%. Needless to say, the present invention does not negate the addition of conventionally used casein decomposition products, soybean flour, cornstarch liquor, waste molasses, vitamins, and the like.

[Brief explanation of the drawing]

The figure is a graph showing the yield of medicinal carrot tissue culture when using MS medium modified with ammonium nitrate.

Claims (1)

[Claims] 1. A method for culturing medicinal carrot tissue culture in which a tissue culture obtained from living tissue of medicinal carrot is cultured in a modified MS medium that does not substantially contain ammonium nitrate.