JPH0446320B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for obtaining essential oil components from adventitious roots obtained by culturing plant tissues and inducing differentiation, and more specifically, by culturing the tissues of plants belonging to the genus Iris (hereinafter abbreviated as plants according to the present invention). , relates to a method for producing essential oil, which involves inducing differentiation of adventitious roots from a culture and obtaining essential oil components produced and accumulated in the adventitious roots. Essential oil components are usually obtained by solvent extraction from whole plants or flowers of plants grown from seeds sown in the soil or by vegetative propagation of plant parts such as bulb cuttings.
It is obtained by methods such as steam distillation, and the essential oil components are used in high-end cosmetics, cosmetics, fragrances for soaps, etc.
Furthermore, it is widely used as a food flavor. However, when cultivating plants and obtaining essential oil components from them, there are constraints based on the localization of vegetation distribution due to temperature, climate, rainfall, soil quality, etc., and plant growth also varies greatly depending on weather conditions from year to year. There were problems such as it being extremely difficult to produce a certain amount of a specific essential oil in a planned manner. As a method to solve this problem, a method has been proposed in which essential oil is produced from the amorphous culture obtained by culturing plant tissue, so-called callus. However, it has been found that such a method is completely impractical because the active ingredients of essential oil in the callus are almost completely eliminated. (Reference, Japanese Society of Agricultural Chemistry Showa
In view of the current situation in the production of essential oils, the present inventors have continued intensive research, and as a result of culturing the tissue of the plant according to the present invention. The present invention was completed based on the discovery that essential oil active ingredients similar to those of the naturally cultivated original plant are produced and accumulated in adventitious roots derived from culture. An object of the present invention is to provide a method for producing essential oil easily and with high yield from adventitious roots that have been cultured and differentiated from plant tissue under a completely controlled environment without being influenced by the natural environment. be. Another object of the present invention is to provide a method for culturing plant tissue, inducing differentiation of adventitious roots from the culture in a short period of time with good reproducibility, and obtaining essential oil components from the adventitious roots at any time. Further objects and advantages will become apparent from the description below. The above object is achieved by culturing tissue of a plant belonging to the genus Iris, inducing adventitious roots from the culture, and obtaining essential oil components produced and accumulated in the adventitious roots. As used herein, the term "adventitious root" refers to a tissue with the shape of a root or rhizome that is derived from cultured plant tissue or secondarily derived dedifferentiated cells. In the plant according to the present invention, essential oil components that are not substantially contained in the callus obtained by culturing the tissue are produced and accumulated in the adventitious roots. The fact that such essential oil components are produced and accumulated in adventitious roots is something that the present inventors have newly discovered. Obtaining the ingredients is the most important point of the present invention. Plants to which the present invention is applied include Iris nertschin skia, I. sibirica, and I. sibirica.
ensata), Irispuseudoacorus (I.
pseudoacorus) Irisra evigata (I.
laevigata), I. japonica, I. gracilipes, I. setosa, I. rossii, I. tectorum, I.
pallida), I.florentina (I.florentina),
I. germanica, I. versicolor and I. caroliniana, among others Iris pallida, I. florentina, I. germanica, With Iris Versicolor, and Iris Caroliana,
Good results were obtained. An example of culturing such plant tissues is as follows. For example, after washing the above plants thoroughly under running water, the leaves,
Cut into stems and roots, sterilize with ethanol, benzalkonium chloride, sodium hypochlorite, etc.
Subsequently, it is washed with sterile water and placed in a test tube or Erlenmeyer flask containing a medium. The medium to be used is not particularly limited as long as it is commonly used in plant tissue culture, and examples include Linsmeyer Scoob, Murashige Scoob, B5, Nitsuchi & Nitsuchi,
Examples include media such as 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 (IAA) and indole-3-acetic acid (IAA).
Acetic acid (IBA), α-naphthalene acetic acid (NAA), 2,
Examples include auxins such as 4-dichlorophenoxyacetic acid (2,4-D), and cytokinins such as kinetin (Kn) and 6-benzyladenine (6-BA), which may be used alone or in combination as appropriate. used together. If the culture is continued in such a medium at about 22 to 30°C, adventitious roots will differentiate from the culture in about 1 to 4 weeks. At this time, the amount of adventitious roots generated depends mainly on the amount of the growth regulators, oosins and cytokinins, and the tissue used for culture. For example, when using stems as culture tissue,
It is recommended to use 10 ^-5 to 10 ^-6 M auxin and cytokinin at a concentration below 10 ^-6 M, or if rhizomes are used, use only 10 ^-5 to ^{10 -6} M auxin. It was effective. In some cases, when stems with buds were used, callus formation preceded adventitious root differentiation, and then adventitious roots differentiated. When the redifferentiated adventitious roots were cut out and planted in a medium, they branched from the base and became many adventitious roots, which themselves were elongated and enlarged. When the adventitious roots obtained in this way are stored dry for 2 years, they contain the same essential oil components as the rhizomes of the original plants that were also stored dry for 2 years. Essential oil components can be easily obtained by commonly used known methods, such as extraction using oil and steam distillation. The present invention will be specifically explained below with reference to Examples. In the examples, the proliferation rate of adventitious roots, the sensory evaluation of aroma, and the analysis of essential oil components were performed by the following methods. (1) Multiplication rate of adventitious roots The fresh weight of adventitious roots at the time of sub-planting and the fresh weight after 3 weeks of culture were weighed and calculated using the following formula. Proliferation rate of adventitious roots (double) = Fresh weight after 3 weeks of culture (g) / Fresh weight of adventitious roots at time of sub-planting (g) (2) Sensory evaluation of aroma Crush the dry stored adventitious roots with your fingers to remove the original plant ( The aroma was determined by smell using the rhizomes as a control. The evaluation was conducted by 20 experts and the average value was used as the sensory evaluation. The evaluation criteria are as follows. 1 No odor of essential oil components peculiar to the original plant 2 〃 〃 Slightly present 3 〃 〃 Yes 4 〃 〃 Strong 5 〃 〃 Very strong (3) Analysis of essential oil components Using gas chromatography and mass spectrometry, standard substances were determined. The main components were identified from the retention time and mass spectrum agreement with The amount of the component was determined from the height of the peak in the chromatogram and expressed as a percentage of the total amount of essential oil shown in the chromatogram. The essential oil was extracted from adventitious roots that had been dry-stored for 2 years using methylene chloride as a solvent and using the conventional method of crushing, salting out, centrifugation, separating the solvent layer, and concentrating. Measurement conditions: - (1) Gas chromatography Model: Shimadzu GC-6AM gas chromatograph Column: FFAP coating fused silica
Capillary column (0.25 mm x 50 m) Injection temperature: 250°C Open temperature: 70 to 180°C (heating 2°C/min) _N2 flow rate: 1 ml/min Sp/9 ratio: 1/150 (2) Mass spectrometry model :JEOL JMS-D300 mass spectrometer (JEOL
(Equipped with LGC20K and JMA-2000 data system) Ionization voltage: 70eV Ion source temperature: 200℃ Examples 1 to 2 Cut out the upper part of the stem (close to the bud) with buds of Iris pallida that has been thoroughly washed with water and surface with 70% alcohol. After sterilization, the samples were sterilized with a 0.1% penzalkonium chloride aqueous solution for 10 minutes and then with a 1% sodium hypochlorite aqueous solution for 15 minutes. After completely removing the drug with sterile distilled water, the tissue was cut into small pieces and placed in a culture medium. A Rinsmeyer Skoog medium containing 10 ^-6 M of indolebutyric acid (IBA) and 10 ^-6 M of 6-benzyladenine (6-BA), 3% sucrose, and 1% agar, adjusted to pH 5.7. A medium was used. 25℃
When cultured in a relatively dark place with 300 to 800 lux during the day and darkness at night, callus appeared after 3 to 4 weeks. Furthermore, the culture was continued for several days and the development of adventitious roots was observed. Cut out adventitious roots from the base and use IBA10 ^-5 M, 6
When transferred to a Linsmeyer-Skoog medium containing -BA10 ^-8 M, adventitious roots were derived from the base and multiplied. On the other hand, callus is IBA10 _-5 M, 6-BA10 ^-5 M
When transferred to Rinsmeyer-Skoog medium, growth improved. The obtained adventitious roots were evaluated after dry storage for 2 years, and the results are shown in Example 1. On the other hand, the calli evaluated immediately after culture are shown in Comparative Example 2, and the calli that were dry stored for 2 years are shown in Comparative Example 1. 1 in Table 1. We also evaluated the rhizomes of the original plants that had been dried and stored for 2 years, and compared them to Comparative Example 3.
And so. Furthermore, the rhizomes (so-called bulbs) of Iris florentina were washed with water and then punched out into a cylindrical shape with a cork bowler centering on the budding point (or rooting point), sterilized in the same manner as above, washed with sterile water, and cut into a cylindrical shape approximately 5 mm wide. The cut disk-shaped sections were placed in the medium. 4 as a medium
- adding 5 x 10 ^-6 M of fluorophenoxyacetic acid;
A white medium containing 3% sucrose and 1% agar and adjusted to pH 5.7 was used. When cultured under the same culture conditions as above, adventitious roots began to appear after 3-4 weeks. When these adventitious roots were transferred to the same medium, they branched from the base and multiplied. The obtained adventitious roots were dried and stored for two years and then evaluated, and the results are shown in Table 1 as Example 2. In addition, the rhizomes of the original plants were similarly dried for two years and evaluated. The results are also shown in Table 1 as Comparative Example 4.

[Table] From Table 1, adventitious roots obtained by tissue culture from stems of Iris pallida according to the present invention (Example 1)
A clear difference was observed in sensory evaluation compared to callus (Comparative Examples 1 and 2), and an essential oil having almost the same composition as the rhizome of the original plant (Comparative Example 3) was obtained. Furthermore, it can be seen that the adventitious roots obtained from the rhizomes of Iris florentina (Example 2) also yielded essential oils having substantially the same composition as the rhizomes of the original plant (Comparative Example 4). Example 3 The rhizome of Iris germanica was used as the plant, and the material was prepared and sterilized in the same manner as in Example 2, followed by B5 containing 5×10 ^-6 M of indole acetic acid (IAA), 3% sucrose, and 1% agar. After 3 weeks when placed on the culture medium,
Adventitious roots were produced. Adventitious roots grown in the same manner as in Example 2 were dried and stored for two years and then evaluated. The results are shown in Table 2 as Example 3. In addition, the rhizomes of the original plants were similarly dried for two years and evaluated. It is also shown in Table 2 as Comparative Example 5. As is clear from Table 2, the adventitious roots obtained from the rhizomes of Iris germanica according to the present invention (Example 3) yield essential oil with approximately the same composition as the rhizomes of the original plant (Comparative Example 5). I can see that it was done.

【table】

[Table] Example 4 The rhizomes of iris versicolor were prepared and sterilized in the same manner as in Example 2, and placed in a culture medium. As a medium, a Murashigeskube medium containing 3% sucrose and 1% agar to which 10 ^-5 M of naphthalene acetic acid was added and adjusted to pH 5.7 was used. After 3 weeks of culturing under the same conditions as in Example 1, adventitious roots were produced. When these adventitious roots were transferred to the same medium, they branched from the base and multiplied. The obtained adventitious roots were stored dry for 2 years (2.8 kg), and after steam distillation, a yellow-brown concrete-like distillate was obtained (1.22 g). The result was subjected to sensory evaluation by an expert along with a concrete-like distillate similarly obtained from the rhizome of the original plant, and the results were as follows. It has the same aroma as the original plant rhizome (stored for 2 years)
20/20 Smells similar to 〃
0/20 It smells different from 〃 0/20 Example 5 Iris caroliana stems are used as the plant, 2,4-D10 ^-6 M is added as a medium, 3% sucrose and 1%
When Nitsuchi & Nitsuchi medium (PH5.7) containing agar was used, only adventitious roots were produced after 3 to 4 weeks. Adventitious roots grown in the same manner as in Example 4 were stored and dried for two years (2.0 kg), and after steam distillation, a yellow-brown concrete-like distillate was obtained (2.15 g). As in Example 4, the concrete-like distillate from the rhizome of the original plant that had been dry-stored for two years was subjected to sensory evaluation by experts, and the results were as follows. It has the same aroma as the original plant rhizome (stored for 2 years)
19/20 Smells similar to 〃
1/20 It smells different from 〃 0/20

Claims (1)

[Scope of Claims] 1. A method for producing essential oil, which comprises culturing tissue of a plant belonging to the genus Iris, inducing differentiation of adventitious roots from the culture, and obtaining essential oil components produced and accumulated in the adventitious roots. 2. The method for producing essential oil according to claim 1, wherein the plant belonging to the genus Iris is Iris pallida, Iris florentina, Iris germanica, Iris versicolor, or Iris caroliana.