JPH062053B2 - Plant tissue culture method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a tissue culture method for efficiently producing arbutin using callus or tumor tissue of Catharanthus roseus.

[Prior Art] The conventional arbutin production method is a synthetic method (Arther D.Jaret
t-corp of Delaware USP3201385) and Japanese Urushi (Ar)
ctostaphylos uva-ursi), cowberry (Vaccinium vitis)
-idaea) and other natural plants.

[Problems to be Solved by the Invention] The synthetic method is very complicated because it comprises four steps of 1) acetylation of glucose, 2) addition of benzyl ether, 3) deacetylation, and 4) debenzylation. As for the extraction method, the arbutin content of natural oak and cowberry is 5.0-7.5% and 4.0-7.0% of the dry weight, respectively.
%, And a large amount of lead is used during extraction. The method of using lead is a heavy metal such as lead, which is a heavy metal, as a method of producing a substance or its raw material for use in medicines, agricultural chemicals, cosmetic additives, food additives, etc. which are directly ingested or contacted by the human body. There is a risk of contamination, and there are difficulties in processing and discarding waste liquid containing used heavy metals, which is unsuitable.

As a means for solving the problems of these conventional techniques, the present inventors have studied a method of collecting arbutin by tissue culture of plant callus or tumor tissue, and as a result, a large amount of arbutin is added by adding hydroquinone to the medium. We found that it accumulates in culture and filed a patent application earlier.
However, this method also has a drawback in that the conversion efficiency is lowered and the yield is lowered as a result of increasing the addition amount of hydroquinone as a substrate for the purpose of increasing the yield.

[Means for Solving the Problems] In view of the above circumstances, the present inventors have earnestly conducted studies on a tissue culture method for obtaining arbutin at a high yield, and as a result, the callus or tumor tissue of a plant used for producing arbutin It was found that the above object can be achieved by substituting a certain amount or less of hydroquinone into the medium when subculturing is carried out, and based on this finding, the present invention was completed.
That is, the present invention provides a plant tissue culture method for inducing a culture excellent in arbutin-producing ability by subculturing callus or tumor tissue of Catharanthus roseus in a medium containing 1 mM or less of hydroquinone.

Hereinafter, the present invention will be described in detail.

First, cell groups or tissue pieces such as roots, hypocotyls, cotyledons, roots of mature plants, stems, leaves, petioles, flowers, and pollen of the periwinkle plant (young plant) are used as a starting material, and this is treated by an ordinary method. Callus is induced by culturing in a medium supplemented with or cytokinin. In this case, callus is induced although there is a difference in difficulty regardless of the cell group or tissue of any plant organ used as the material. The medium to be used is usually Murashige-Skoog medium mixed with agar, but not limited to this, White, Gamborg, Nitsch.Heller.Schenk-Hildebr
Any medium such as andt, Nitsch-Nitsch, Kohlenbach-Schmidt may be used. Of course, callus can be induced even in a liquid medium containing no agar. Generally, auxin is required for callus induction, but 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthalene acetic acid (NAA),
2,4,5-trichlorophenoxyacetic acid (2,4,5-T),
Any such as indole acetic acid (IAA) may be added.
Further, as cytokinin, any of zeatin, 6-benzyladenine, kinetin, ribosylzeatin, isopentenyladenine and the like may be added. The concentration of auxin added is in the range of 10 ^-7 M to 10 ^-5 M, and the concentration of cytokinin is also in the range of 10 ^-8 M to 10 ^-4 M. The callus thus induced is subcultured by shaking culture in a liquid medium containing no agar. Of course, callus grows and grows even in a medium containing agar. In liquid shaking culture, it is always shaken in a rotary shaker or reciprocating shaker for aeration. The rotation speed may be any value in the range of 50 rpm to 150 rpm, but about 110 rpm is preferable. Light may or may not be irradiated during the culture. The culture temperature is 20 ° C to 30 ° C, preferably 27 ° C. Callus 1 a week
Subculture into new medium and subculture. 1 subculture
It is carried out in a medium containing hydroquinone of mM or less. Since callus cannot grow when the concentration of hydroquinone is too high, it is cultivated in a medium having a concentration of several steps of 0.01 mM to 1 mM, and the callus grown in the medium having the highest concentration among them is selected and planted. When such an operation is repeated, the resistance of callus to hydroquinone gradually increases.

There is no particular limitation on the timing of switching the callus subcultured in liquid culture to the subculture in a medium containing hydroquinone, but it is faster if the callus is not induced after the callus is induced. Is preferred because it obtains

At least 3 subcultures in medium containing hydroquinone
After continuing for more than a month, when 10 mM or less of hydroquinone is added again between the third and tenth days after the planting, a large amount of arbutin is immediately produced in the medium.

EXAMPLES Next, the present invention will be described in more detail with reference to examples. The present invention is not limited to these.

Example 1 Murashige-Skoog medium containing 2.2 × 10 ^-6 M of 2,4-D as auxins and containing no agar (KC, the same applies hereinafter)
Each 180 ml was dispensed into a 500 ml Erlenmeyer flask and sterilized by an autoclave.

The callus of periwinkle used in the experiment was treated as follows. The culture conditions were all 27 ° C. and no light irradiation, and in the case of shaking culture, it was performed at 110 rpm using a rotary shaking culture device (Iwashiya Kagaku). That is, the callus derived from the stem of Catharanthus roseus by the conventional method was used for hydroquinone (Mitsui Petrochemical, the same applies hereinafter) 0.02 mM, 0.2 m
When transplanted to Murashige-Skoog medium containing M, 0.5 mM and 1 mM respectively, it did not grow in a medium containing 0.5 mM or more of hydroquinone. Since the callus grew in the experimental group of 0.2 mM or less, the callus of the 0.2 mM group was subcultured again several times in a medium containing 0.2 mM hydroquinone. The callus was transplanted to a medium containing 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1.0 mM hydroquinone, respectively. Since callus grew slowly in the experimental section of 0.6 mM or less, this callus was subcultured for one year or more in a medium containing 0.6 mM hydroquinone. 2g of callus after such treatment
A 20 ml cell suspension containing (raw weight) was placed in an autoclave-sterilized 500 ml Kolben. On the 5th day of culture, 88.1 mg of hydroquinone was added to the medium as an aqueous solution.

As a comparative example, callus subcultured in a medium containing no hydroquinone was prepared, and 10 other cultures were carried out under the same conditions as in Example. On the 6th day, culture solution was added to Toyo Roshi filter paper No. 2
Suction filtration was performed with and the residue was thoroughly washed with pure water. 200m residue
1 Transfer to an eggplant-shaped flask, add 100 ml of pure water, and add 3
The mixture was homogenized with a Hiscotron (manufactured by Nisson Rika Kagaku Kikai Seisakusho) for 1 minute, and extracted with hot water at 100 ° C for 2 hours on a hot water bath. This was centrifuged to stock the supernatant, which was applied to a silica gel column (Wako gel C-300, Wako Pure Chemical Industries, Ltd.), and mixed eluate (chloroform: methanol: water = 30: 1).
Elution at 0: 1) and evaporation of the solvent gave 205 mg of arbutin as crystals. In the comparative example, it was 120 mg.

Examples 2 and 3 and Comparative Examples 2 and 3 The culture was performed under the same conditions as in Example 1 except that the medium of Rinsmeier-Skoog (manufactured by Kyokuto Pharmaceutical Co., Ltd.) was used. On the 6th day of culture, hydroquinone (110 mg (Example 2), 132 mg)
(Example 3) was added, and on the 7th day, extraction was performed as in Example 1 and quantification was performed by high performance liquid chromatography (manufactured by JASCO Corporation). 5% methanol (ph2.1) is used as the solvent, and the flow rate is
1.5 ml / min. The detection wavelength was 230 nm. The column is an ODS column (Senshu Scientific, SSC-OD
S-161) was used. As a result, the yields of arbutin were 259 mg (Example 2) and 306 mg (Example 3). On the other hand, the yields of 101 mg (Comparative Example 2) and 69 mg (Comparative Example 3) were 101 mg (Comparative Example 2) and Comparative Example using callus subcultured without hydroquinone treatment.

Table 1 shows the arbutin production and the conversion rates of Examples 1 to 3 and Comparative Examples 1 to 3. However, the values are average values of the total amount produced in 10 flasks.

The data obtained by instrumental analysis of arbutin produced according to the present invention were consistent with those of commercially available arbutin (manufactured by Sigma) in each spectrum analysis of ultraviolet absorption, infrared absorption and ¹³ C nuclear magnetic resonance.

Claims (1)

[Claims] 1. A plant tissue culture method for inducing a culture excellent in arbutin-producing ability by subculturing callus or tumor tissue of Catharanthus roseus L. in a medium supplemented with 1 mM or less of hydroquinone.