JP3396262B2 - Tissue cultivation method for Taxus plant and method for producing taxane-type diterpene by the tissue cultivation method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for tissue culture of Taxus plants and a method for producing taxane-type diterpenes by the tissue culture.

[0002]

2. Description of the Related Art Taxus (Taxus brevifolia NUTT), which is a Taxus of the Taxus family, is expected to be a therapeutic drug for ovarian cancer, breast cancer, lung cancer and the like.
ol) is included. Currently, taxol is collected from natural or cultivated plants, but yew plants are slow growing plants that take more than 10 years to grow to a height of 20 cm above the ground, and taxol can be easily collected in large amounts. Is hard to get. If taxane or a precursor of taxol, taxane-type diterpene such as baccatin III, can be synthesized using tissue culture, a large amount of taxol can be easily obtained without cutting trees, which is advantageous. Is. However, the growth rate of cultured cells of Taxus genus plants is extremely slow, which is a large barrier to the production of taxane-type diterpenes such as taxol using the cultured cells. For the conventional tissue culture method of Taxus plants, refer to Taxus
brevifolia NUTT) Patented tissue culture of cultured cells [US Pate
nt: 5019504]. Although it is stated in this patent that cultured cells grow 5 to 10 times in 3 to 4 weeks,
Insufficient for industrial production.

[0003]

An object of the present invention is to improve the productivity of taxane-type diterpenes using tissue culture by increasing the growth rate of cultured cells of Taxus plants.

[0004]

As a result of earnest studies, the present inventors have found that when tissue culture is performed by adding oxalic acid or oxalate to a tissue culture medium of a Taxus plant, the growth rate of cultured cells is improved. The inventors have found that they have done so and have completed the present invention. That is, the present invention is a tissue culture method for Taxus plants, which comprises culturing Taxus plants in a tissue culture medium containing oxalic acid or oxalate. The present invention also provides a method for producing taxane-type diterpenes, which comprises recovering taxane-type diterpenes from the culture.

Examples of Taxus species used for tissue culture in the method of the present invention include Taxus taxus (Taxus
baccata LINN), yew (T. cuspidata SIEB.et ZUC
C), Karaboku (T. cuspidata SIEB.et ZUCC var. Nana
REHDER), Taihei Yew (T. brevifolia NUTT),
Canadian yew (T. canadiensis MARSH) etc. can be mentioned. The taxane-type diterpene in the present invention is not particularly limited as long as it is a diterpene having a taxane skeleton, and examples thereof include taxol, baccatin III, cephalomannine, 10-deacetylbaccatin and the like.

Tissue culture of the plant can be carried out by a conventionally known method except that oxalic acid or oxalate is added according to the present invention. Conventionally, no example of using oxalic acid or oxalate as a medium additive in tissue culture of taxane-type diterpene-producing plants has been reported,
It was unexpected that the tissue culture method of the present invention using oxalic acid or oxalate improves the growth rate of Taxus plants.

Examples of the oxalic acid or oxalate include alkali metal salts of oxalic acid such as sodium oxalate and potassium oxalate, alkaline earth metal salts of oxalic acid such as magnesium oxalate and calcium oxalate, and ammonium oxalate, in addition to oxalic acid.
The concentration of oxalic acid or oxalate in the medium needs to be 0.1 mM or more, and among them, it is particularly preferable for the method of the present invention to adjust the concentration of oxalic acid to the range of 0.5 mM to 10 mM.

The medium used in the present invention contains ordinary medium components in addition to the oxalic acid or oxalate. Inorganic components and carbon sources are generally used as such components, and plant hormones and vitamins can be added thereto, and amino acids can be added as necessary. As the carbon source, disaccharides such as sucrose, maltose and lactose, monosaccharides such as glucose, fructose and galactose, starch, or a mixture of two or more of these sugar sources in an appropriate ratio can be used.

Examples of the inorganic component include phosphorus, nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine and cobalt. These components are, for example, Potassium nitrate, sodium nitrate, calcium nitrate,
Potassium chloride, potassium monohydrogen phosphate, potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, It can be added as a compound such as molybdenum trioxide, potassium iodide, or cobalt chloride.

Examples of plant hormones include auxins such as indoleacetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin,
Cytokinins such as zeatin and dihydrozeatin are used. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6),
Pantothenic acid, inositol, nicotinic acid, etc. are used. As the amino acids, for example, glycine, phenylalanine, leucine, glutamine, cysteine and the like can be added. Generally, each of the above components contains about 0.1 μM of an inorganic component.
To 100 mM, carbon source about 1 to about 30 g / l, plant hormones about 0.01 to about 10 μM, vitamins and amino acids at concentrations of about 0.1 to about 100 mg / l, respectively.

The medium used for tissue culture of the present invention is a conventionally known medium used for tissue culture of plants, for example, Murashige Scoog (1962) [Murashig
e & Skoog] medium, Rinsmeier Scoog (1965)
[Linsmaier Skoog] medium, Woody Plant Medium (1981) [Woody Plant Medium] medium, Gamborg B-5 medium, Mitsui M-
9 can be exemplified by a medium prepared by adding the above-mentioned plant hormone to the medium and the like and further adding the above-mentioned carbon source, vitamins, amino acids, etc., if necessary. Among them, the Woody Plant Medium medium is particularly preferable. A medium prepared by using In the present invention, any of a liquid medium and a solid medium containing 0.1 to 1% of agar, gellan gum or the like can be used, but the liquid medium is usually preferable.

In the tissue culture of the present invention, tissue pieces or cells such as roots, growth points, leaves, stems, seeds, pollens, anthers, sepals, etc. of the above plants, or these are treated with the above medium or another conventional medium. Cultured cells or tissue obtained by culturing can be used. The cultured tissue includes shoots and adventitious roots obtained by differentiating from tissue or callus. When these tissues or cells are tissue-cultured using the medium of the present invention to which the oxalic acid or oxalate is added, it is observed that the growth rate is improved as compared with the case of no addition. Taxane-type diterpenes can be separated from these tissues or cells by extraction with an organic solvent such as methanol.

A preferred example of the tissue culture of the present invention is the following method. First, a plant of a plant belonging to the genus Taxus, for example, roots, growing points, leaves, stems, after sterilizing plant pieces collected from seeds, etc., placed on a solid medium of Woody Plant Medium hardened with gellan gum,
A part of the tissue piece is callused at 10 to 35 ° C. for about 14 to 60 days. When the callus thus obtained is subcultured, the growth rate gradually increases and a stable callus is obtained. The term "stabilized callus" as used herein refers to one in which a part of the callus does not differentiate into shoots or roots during culture and retains the state of the callus and has a uniform cell growth rate.

The stabilized callus is transferred to a liquid medium suitable for growth, for example, a liquid medium of Woody Plant Medium and allowed to grow. The growth rate is further increased in the liquid medium. In the present invention, the stabilized callus or cells constituting the callus is cultured in a solid medium or liquid medium containing oxalic acid or oxalate.

The culture temperature in the tissue culture of the present invention is preferably about 10 to about 35 ° C., particularly about 23 to 28 ° C., because the growth rate is high. The culture period is preferably 14 to 42 days. When a liquid medium is used in the method of the present invention, the cultured tissue or cultured cells are separated from the medium by a method such as decantation or filtration after the culture is completed, and the target taxane-type diterpene is extracted from this with an organic solvent. And the like.

[0016]

EXPERIMENTAL EXAMPLES Hereinafter, the present invention will be described in more detail with reference to experimental examples, but the scope of the present invention is not limited to these experimental examples. Experimental Example 1 A solid medium of Woody Plant Medium (gellan gum 0.25% by weight) to which naphthalene acetic acid was added at a concentration of 10 ^-5 M was sterilized in advance with a 2% antiformin solution or a 70% ethanol solution. Placed a part of the stem of the treated yew (Taxus baccata LINN),
It was cultured at 25 ° C. in the dark in the dark to obtain Taxus callus. Next, 0.1 g of this callus (fresh weight) was transferred to a φ18 mm well containing 1 ml of Woody Plant Medium liquid medium to which the above components were added at the same concentration, and swirling culture (amplitude 25 mm, 12 mm) on a rotary shaker.
0 rpm) and planted every 28 days to accelerate the growth rate of the callus.

0.5 g of the cultured cells thus obtained
(Fresh weight) was transferred to a 20 ml Erlenmeyer flask containing 5 ml of Woody Plant Medium liquid medium to which the above components were added at the same concentration and oxalic acid was added so that the final concentration was as shown in Table 1. And cultured at 25 ° C. for 28 days with shaking. The amount of dried cells in the medium at the start of culture was 2.8 g / l.

After the completion of the culture, the Taxus vulgaris cultured cells were collected by filtration, lyophilized, and the dry weight thereof was measured to determine the growth weight of the cultured cells per 1 L (liter) of the liquid medium. The taxane-type diterpene was extracted from the obtained dry callus with methanol or the like, and the taxol yield was measured by comparative quantification with the standard taxol using high performance liquid chromatography. The results are shown in Table 1.

[0019]

[Table 1] [ ^*) : Content was calculated by total production (in cells + medium) / cultured cell growth. ]

Experimental Example 2 The procedure of Experimental Example 1 was repeated, except that sodium oxalate was added in place of oxalic acid so that the final concentration was as shown in Table 2. The results are shown in Table 2.

[0021]

[Table 2] [ ^*) : Content was calculated by total production (in cells + medium) / cultured cell growth. ]

Industrial Applicability According to the method of the present invention, it is possible to improve the growth rate of cultured cells of Taxus plants and improve the productivity of taxane-type diterpenes using tissue culture.

Front Page Continuation (56) References US Patent 5319112 (US, A) US Patent 5470866 (US, A) US Patent 5019504 (US, A) International Publication 92/132 (WO, A1) Journal of Organic Chemistry (1987) ), Vol. 57, No. 17, p. 3745-3952 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12P 17/00 A61K 35/78 C12N 5/00 CA (STN) BIOSIS / MEDLINE / WPID S (STN)

Claims (4)

(57) [Claims] 1. A method of culturing a Taxus plant, which comprises culturing a Taxus plant in a tissue culture medium containing oxalic acid or an oxalate. 2. A taxane-type diterpene, which comprises recovering a taxane-type diterpene from a culture medium of a tissue culture medium containing oxalic acid or an oxalate, and culturing cultured cells or tissue of a Taxus plant. Method. 3. The method according to claim 1, wherein the oxalate salt is at least one oxalate salt selected from alkali metal salts, alkaline earth metal salts and ammonium salts. 4. The concentration of oxalic acid or oxalate is 0.1 mM or more and 10 mM or less.
The method described in.