JPH0227982A - Tissue culture of perfume plant - Google Patents

Abstract

PURPOSE:To efficiently produce a highly essential oil-containing cultured product by irradiating calluses or adventitious buds derived from the cultured product of a perfume plant belonging to the genus Viola with light of 10000-30000 luxes. CONSTITUTION:A perfume plant belonging to the genus Viola is cultured and calluses or adventitious buds are differentiated and derived from the cultured product. When the calluses or adventitious buds are multiplied, the calluses or buds are irradiated with light of 10000-30000 luxes during the producing and culturing period thereof to produce a perfume component. The perfume plant belonging to the genus Viola includes concretely Viola grypoceras, Viola orientalis, Viola blandaeformis, Viola obtusa, etc. The light of the light source is required to contain at least light having wavelengths of 300-700nm and commercially available fluorescent lamps, white lamps, etc., can be used.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention effectively produces fragrance components by irradiating light during the production and culture period when culturing plant Mi fabric obtained from fragrance plants. A group of plants that made it possible to
Concerning the Saiyo method.

[Conventional technology]

A method is known in which the tissue of a plant belonging to the genus Viola is cultured, the culture is induced to differentiate into a tissue mass, and the essential oil components produced and accumulated in the tissue mass are obtained. (Unexamined Japanese Patent Publication Showa 6l-
78393) L. It was very difficult to obtain a culture with a higher content of essential oil components than the explanted plant.

[Problem to be solved by the invention]

An object of the present invention is to effectively obtain a culture with a high essential oil content by irradiating light during the production and culturing period when culturing plant tissues obtained from fragrance plants.

[Means to solve the problem]

The present invention provides a method for culturing a fragrance plant belonging to the genus Viola, inducing differentiation of callus or adventitious buds from the culture, and then propagating and culturing the callus or adventitious buds.
This is a tissue culture method for perfume plants characterized by producing perfume ingredients by irradiating light of 00 to 30,000 Lux during the production culture period.

The plants to which the present invention is applied include Viola grypoceras +
Viola orientalis (V, orienta)
lis), Viola plandeformis (V
, blandaeformis).

Viola obtusa (V, obtusa), Viola violacea (V, violacea) +
Viola minor (V, m1nor), Viola japonica (V.

japanica) * Viola ovato-obloBa (V, ovato-obloBa), Viola rossica (V, rossica), Viola tricolor (V.

tricolor), viola odosoda (V,
Examples include aromatic plants belonging to the genus Viola such as P. odorata).

The light source used in the present invention may be any light source containing light with a wavelength of 300 to 700 nm, and examples thereof include commercially available fluorescent lamps, white lamps, metal halide lamps, sodium lamps, and the like.

The method of the present invention will be explained below.

After thoroughly washing a fragrant plant belonging to the genus Viola under running water, cutting it into leaves, stems, and roots, briefly soaking it in ethanol or benzalkonium chloride, washing it with water, and sterilizing it by soaking it in sodium hypochlorite. wti! Rinse with water and place in a test tube or Erlenmeyer flask containing medium. The medium to be used is not particularly limited as long as it is commonly used in plant tissue culture. For example, Linsmeyer Skoog, Murashige Skoog 9 B5. Examples include media such as Ninch & Niche and White. When actually culturing, these basic media are mixed with sugars, vitamins, and if necessary, growth regulators.

Examples of growth regulators include indole-3-acetic acid H (I
AA), IA F-Lou 3-@acid (IBA).

These include auxins such as α-naphthalene vinegar #I (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), and cytokinins such as kinetin (Kn) and benzyladenine (BA). Used alone or in appropriate combinations. In such a medium, 3000 Lux
If the culture is continued at about 22 to 30° C. under moderate light irradiation, callus and adventitious buds will develop from the culture in about 1 to 8 weeks.

At this time, the quality (color, form) and quantity of callus and adventitious buds mainly depend on the amount of the growth regulators, oak, cylindrical, and cytokinins, and the plant tissues subjected to &Il$1 culture. These conditions are shown for plants belonging to the genus. Easy to obtain callus and adventitious buds, especially lowering auxin level to 1
0-? When the amount is 10-''M, green callus is produced, which grows quickly, and it is easy to obtain callus with a good smell containing desirable components by the selection described below.

The induced callus is divided into small chunks and further cultured in an agar medium or liquid medium under light irradiation of about 1500 Lux. (A callus mass cultured on an agar medium is called a stock. In liquid culture, a callus in one capacity such as a flask is called a stock.) The callus of each stock was sensory evaluated for odor, and the original We selected strains that could be confused by the odor of one or more of the main components contained in the plants, and after further culturing and evaluating the components through zero-instrument analysis that re-manipulated the selection, we found that the selected strains were We were able to obtain a strain in which more than one main odor component of the original plant was recognized. In addition, when subculturing the selected strain in liquid, hormones may or may not be added to the medium, as strains containing the same growth and flavor components as in the hormone-added medium will appear even in the hormone-free medium.

In addition, the induced adventitious buds can be easily subcultured by dividing or sectioning them on an agar medium and continuing the culture at 22 to 30° C. under 1500 Lux light irradiation. When these adventitious buds were also evaluated for their composition by instrumental analysis,
More than one main odor component of the original plant was recognized.

The callus and adventitious buds obtained by the above method are grown in an air lift culture tank or large jar fermenter for 10 days to 1 hour.
When performing production culture (scale-up culture) for several months, preferably two weeks, 10,000 to 30,000 L
Irradiates light of ux.

Continuous irradiation is preferable for this light irradiation, but the effect of the present invention can also be obtained by intermittent irradiation.

Thereafter, essential oil components are obtained from these callus and adventitious buds by a commonly used known method such as extraction using a solvent such as petroleum ether or steam distillation.

The absolute obtained by this light irradiation contained noedienal and noedienol, which are aroma components unique to the Viola genus, in a content 1.5 to 3 times higher than that of the commercially available absolute. Note that the term "commercially available absolute" in this specification refers to an absolute manufactured by Chalabo Co., Ltd. that has a high content of the above-mentioned fragrance components.

Conventionally, production culture has been carried out under light irradiation of several thousand Lux, but production culture under light irradiation with strong light intensity as in the present invention has not been carried out so far, and by this, a culture with high essential oil content can be obtained. It is very important that this is possible.

〔Example〕

The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto.

Example-1 After washing Viola odorata tissue mass with running water, the leaves were momentarily immersed in ethanol, then washed with water, sterilized by immersing them in sodium hypochlorite, and then washed with sterile water, and MS medium ( Hormone concentration: 2.4
-Dlo-・M, BA 10-'M) was placed in an Erlenmeyer flask. Cultivation was continued in this medium at 28° C. under 3000 Lux light irradiation to obtain callus for 2 months. This callus was cultured in MS medium (hormone concentration) for 2 months in liquid culture, and a stable callus was obtained in Hormones. This callus has the shape of a bunch of grapes. By separating the clusters one by one during subculturing, we were able to obtain stable and uniform callus.Furthermore, during production culture (scale-up culture), the clusters were separated. As a result, the proliferation rate was improved compared to the conventional method.

The above callus was removed using a 21 air lift for 1500
The cells were continuously irradiated with light of ~30,000 Lux and subjected to liquid enrichment for 2 weeks. After that, flavor ingredients are extracted from the culture.
Extraction was performed using a commonly used petroleum ether extraction method to obtain an absolute, which was analyzed by gas chromatography. The results are shown in Table 1.

Note that gas chromatography was performed under the following measurement conditions.

Model: Shimadzu GC-15A Column: PIE020M capillary column (0,2
mm x 50 mm) Injection temperature = 250°C Oven temperature 260°C - 200°C 3°C/min Carrier gas: Helm 1/min
n Detector: FID Viola Odosoda's tomb, prey is 10,000 to 30
By irradiating with 000 Lux of light, the content of nonajenal and nonajenal was significantly higher than that of a commercially available product, indicating that the fragrance ingredient could be effectively produced.

〔Effect of the invention〕

Since the present invention is configured as described above, it produces the effects described below.

When culturing a plant tissue obtained from a fragrance plant belonging to the genus Viola, it is possible to effectively produce fragrance ingredients by irradiating light of 10,000 to 30,000 Lux during the production culture period.

Table 1 Procedural amendment (voluntary) 1. Display of the case 1988 Patent Application No. 175539 2 Name of the invention Tissue culture method for aromatic plants 3 Person making the amendment Relationship to the case Patent applicant address 5-17-4 Sumida, Sumida-ku, Tokyo 534
5-90-6, Tomobuchi-cho, Bejima-ku, Osaka City, Contents of the amendment (1) From line 13 to line 14 of paragraph 4 of the specification, it says 1 under light irradiation of about '3000 Lux, 71
Light irradiation of about 5000 Lux for 6 to 24 hours and light period maintenance for 8 to 0 hours are performed alternately, and corrected to 1.

(2) Under light irradiation of about f1500 Lux from page 5, line 10 to line 11 of the specification, the value 1 is changed to r16
Light irradiation of about 1500 to 5000 Lux for ~24 hours and light period maintenance for 8 to 0 hours are alternately performed, and approximately 22 to 30
Correct it to 1 in °C.

(3) r2 from page 6 line 6 to line 7 of the specification
2-30℃ under 1500 Lux light irradiation,
'16~241111fol (7) 1500~5000
Light irradiation of about Lux and light period maintenance for 8 to 0 hours are performed alternately, and correction is made to 1 at approximately 22 to 30°C.

(4) In page 6, line 12 of the specification, insert 1 at r approximately 22 to 30° C. next to 1 in t in the fermenter.

(5) On page 6, line 13 of the specification, 1 for r2 weeks is corrected to ``1 for about 2 weeks.

(6) In page 8, line 18 of the specification, continuously irradiate with r and insert 1 next to 1 at 28°C.

(7) In the 8th line of page 9 of the specification, r50kaku1 is corrected to r50m1.

that's all

Claims (1)

[Claims] When culturing a fragrance plant belonging to the genus Viola, inducing differentiation of callus or adventitious buds from the culture, and then propagating and culturing the callus or adventitious buds, 10,000 to 300
A tissue culture method for fragrance plants, characterized in that a fragrance ingredient is produced by irradiating light of 0.00 Lux during a production culture period.