JPS6254796A - Production of refined oil - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method of inducing differentiation of plants having a novel essential oil composition using tissue culture and obtaining essential oil components from such plants.

[Conventional technology] Traditionally, natural essential oils have been produced by extracting them from cultivated or wild plants, or by creating large numbers of clones of existing fragrance plants using tissue culture technology and obtaining them from the culture (Japanese Patent Laid-Open No. 5
9-213393) is known.

In addition, improvements in the quality of natural essential oils and increases in oil production have been achieved by creating new varieties through classical hybridization of plants.

[Problems to be solved by the invention] Breeding through crossbreeding takes a long time, spanning from several years to several decades, and because it is usually carried out outdoors, there are many problems such as temperature, climate, rainfall, soil quality, pests, etc. What is fundamentally required is a plant that is adapted to the constraints of the soil and climate, rather than its natural ability to produce essential oil under the influence of soil and climate. Therefore,
Even if the plants are of high quality or produce a large amount of oil, it is quite possible that they have not been selected.

[Means for Solving the Problems] In view of the current situation, the present inventors have conducted intensive research and found that the tissue of the plant according to the invention can be grown under an artificially controlled environment. It was discovered that by culturing and causing mutations in the culture, a plant of better quality or containing more essential oil than the original plant can be cultivated, and based on this knowledge, the present invention was accomplished.

The method of the present invention involves culturing plant tissue, genetically mutating the culture, inducing differentiation of adventitious shoots and roots from the mutated culture, and extracting essential oil components produced and accumulated in the plant body. It consists of things.

At that time, it is of course possible to extract essential oil even if the roots remain as adventitious buds without differentiation. Furthermore, if the plant body in which adventitious buds and roots have been induced to differentiate is adapted to the outdoor environment, it is of course possible to cultivate it outdoors and extract essential oil from the cultivated plant body.

The present invention relates to Pelargon iu.
m), Geranium, Lavandula, Menta
It can be applied to plants belonging to the genus Salvia 5calrea, Pogostemon, and Salvia 5calrea. The method for culturing such plant tissue is as follows.

Callus can be induced by culturing the above-mentioned plants in a medium supplemented with auxin or cytokinin in a conventional manner using cell groups or tissue pieces such as roots, stems, leaves, petioles, flowers, and pollen as starting materials. Any medium that is commonly used in plant tissue culture can be used. For example, agar mixed with a culture medium such as Murashige and Skoog (MS), Linsmeyer and Skoog (LS), White, B5, Niche, Sidenk and Hildebrand, Niche and Niche, Korenbatscha and Schmidt is used.

At this time, callus can be induced even in a liquid medium that does not contain agar. Additionally, auxin is generally required for callus induction, but 2,4-dichlorophenoxyacetic acid (2,4
-D), α-naphthalene acetic acid, indole-3-acetic acid (
(hereinafter abbreviated as IAA), indole-3-butyric acid, 2,4.
Any such as 5-trichlorophenoxyacetic acid may be added. Further, as for the cytokinin, any of zeatin, 6-benzyladenine (hereinafter abbreviated as BA), kinetin (hereinafter abbreviated as KT), ribosylzeatin, isopentenyladenine, etc. may be added. The concentration of auxin added is in the range of 10-714 to 10-5M, and the concentration of cytokinin is also in the range of 10-8M to 10-4. Light irradiation is not necessary for callus induction. When such treatment is performed, callus usually occurs within a few days to 1 month.

The resulting callus may be cultured for an additional month or more, or at the beginning of callus formation, it may be immersed in a 0.1χ to 0.5χ ethyl methanesulfonic acid solution for 30 minutes to 3 hours, and then the callus may be treated with 1 to 10 μg/ml of cytokinin and 0 auxin. ~2μg/
Transfer to agar medium containing ml. In such a medium 1
2-24 hours, 5000-20. ．． When culturing is performed by alternating light irradiation at 000 lux and darkness for 12 to 0 hours, adventitious buds will differentiate after about 2 weeks. Auxin in such adventitious buds is 10μg/m! When transplanted into a medium containing the following, rooting can be seen after a few days. At this time, some plants will root even in a medium that does not contain any auxin. The regenerated plant body obtained in this way contains essential oil components, and those that have the same essential oil components as the original plant,
Various types of original plants were observed, including those containing unidentified components that cannot be detected by gas chromatography, and those that produce the same components as the original plants but with completely different abundance ratios.

Furthermore, when the adventitious buds before rooting are transplanted to an LS agar medium containing 2 Mg/ml of BA and 1 Mg/ml of IAA, a large number of adventitious buds with the same morphology will differentiate and proliferate. No matter which buds of this culture are analyzed, they match the essential oil components contained in the leaves when the plants are rooted. However, the amount of essential oil per fresh weight is often increased by rooting and increasing the size of the plant.

Examples] Next, the present invention will be explained in detail by giving examples.

The quality of essential oil components in the adventitious buds obtained according to the present invention was evaluated using sensory evaluation under the conditions shown below, gas chromatography, and gas chromatography/mass spectrometry.

-L-" [Noh Jl 5] Crush several milligrams of the sample with your fingertips to determine the odor. The evaluation was made by a panel of 10 experts who gave ratings based on the following criteria and took the average as the evaluation value.

a. There is no similar odor compared to the mother plant.
・・・・・・・・・1 Somewhat...
There are 2...
・・・・・・・・・・・・・・・・・・・・・・・・
...3 It's quite strong...
・・・・・・4 Very strong・・・・・・・・・・・・－・
・・・・・・5b, There is no new odor compared to the mother plant・・・・・・・・・・・・・・・・・・・・・・・・
・・・・・・・・・1 Somewhat...
There are 2...
・・・・・・・・・・・・・・・・・・・・・・・・
...3 It's quite strong...
・・・・・・4 Very strong・・・・・・・・・・・・
... Sampling 2 to 10 mg of a 5% sample and weighing it accurately.

The sample is inserted into the sample tube attached to the analyzer shown below and attached to the system. Next, increase the sample tube temperature to 2
Components that volatilize from the sample by heating to 00 to 250° C. are collected in Tenax GC (manufactured by AKZO, Netherlands). The collected components are reheated (220° C.) and introduced into a gas chromatography analysis column for component analysis.

(1) Component collection conditions and gas chromatography sample amount: 2 to 10 mg Sample tube temperature: 200 to 250°C Component collection: H23030-6O/min to collect 5 ml of components; 220°C, 4 ml of gas chromatography model: Yukawa Hewlett-Packard 58800C attached purge and trap device 7675 type column: 5% phenylmethyl silicone fused silicon capillary force ram 0.21 mm x 50 m polyethylene glycol 20M fused silicon capillary force ram 0.21 mm x 50 m (different column simultaneous analysis method) Column temperature: 50°C/m1n -30°C (held for 4 min) -m-→30°C5°C/min (held for 4 min) -m-→210°C (constant) Carrier gas: He 1. Oml/rain splitless method%
Formula %: (Fully automatic from component collection to analysis completion) (2
) Gas chromatography/mass spectrometry and gas chromatography/infrared spectroscopy Gas chromatography: Mass spectrometer with the same conditions as in (1): Model: Hitachi To80 Ionization voltage: 20 eV Ion source temperature: 200°C Infrared spectrometer: Digilab FTS-15C light pipe temperature 200°C When a new component not contained in conventional essential oils was confirmed as a result of component analysis, the aroma of the component was examined using the following method to search for a component with aroma.

a. Collection of aroma The aroma components volatilized from several milligrams of the sample are collected in Tenax GC (manufactured by AKZO, Netherlands) by the method described above.

b. Desorption of components and aroma evaluation Desorption temperature: 220°C 4m1n Gas chromatography model: Shimadzu 7A GC column: 5% FFAP/Chromosorb W 80-100 mesh inner diameter 3mm, length 2m C column outlet is branched into two directions, detector and GC system I took him outside and put a sniffer on him. ) Column change rate: 80℃→240℃5℃/min Carrier gas: He 50ml/mln (divided 1:1 at the column outlet) Detector: ID While performing normal analysis under the above conditions, recorder At the same time as the separated peaks were recorded, the odor was determined using a sniffing port led outside the GC system. As a result of the determination, components that had a useful aroma were designated as unknown components.

Combining the results of sensory evaluation and analysis of essential oil composition (qualitative and calculation of essential oil production), a final comprehensive evaluation was performed by a panel of 10 experts on fragrance according to the following criteria.

O: 8 or more out of 10 people said it was better than the mother stock △: 5 to 7 people out of 10 said it was better than the mother stock ×: 4 or less out of 10 people said it was better than the mother stock Examples 1 to 4 Pelargonium graveolens
umgraveo 1ens), and after covering both ends with melted paraffin, it was sterilized by immersing it in 70% ethyl alcohol for 1 minute and then in 1% aqueous sodium hypochlorite solution for 15 minutes. Next, the petiole from which the drug has been completely removed with sterile pure water is cut into approximately 5 mm length under sterile conditions.
．． 4-D lppm and KT 0. The cells were planted on an LS agar medium (0°9χ concentration, same hereinafter) containing lppm. 27
When cultured in the dark at ℃, callus appeared after about one month. When callus began to appear, the culture was immersed in 0.2% ethyl methanesulfonic acid solution at 27°C for 1 hour. Then B
LS containing 2 μg/ml of A and 1 μg/ml of IAA
The cells were transplanted onto an agar medium, and cultured at 27°C for 12 hours under 7000 lux white light illumination and 12 hours in the dark (hereinafter referred to as photoculture). After two weeks, a large number of green tissues were differentiated from the callus. Many of the plants either died or grew into amorphous green tissues, but some developed adventitious buds. When 86 such adventitious buds were selected and transplanted to an LS agar medium containing 1 μg/ml of IAA, rooting was observed in 68 plants. The essential oil in the leaves of such regenerated plants was analyzed.

Table 1 shows the analysis results. However, * in the table represents a new component that is not found in the conventional (mother plant) geranium component, and TP
0-26.0 (min): Top note component, TR
26,1-38,0 (min): middle note component, T
R38,1-60,0 (min): Shows the GC peak area ratio (%) confirmed in the base note component region.

(The following is a blank space) Examples 5 to 9 Pelargonium denticulatum (Pelargonium denticulatum)
Cut off the leaves of iun denticulatum).
Callus was visited in the same manner as in Examples 1-4. After continuing to culture such callus for another 2 months, 2 μg/ml of BA was added.
1 μg/ml of +■AA was transferred to an LS agar medium, and photoculture was performed at 27°C. The adventitious buds that appeared were sequentially treated in Example 1
Rooting was performed in the same manner as in 4. In this way, 56 seedlings were obtained. Examples 5 to 9 are shown in Table 2. However, * in the table represents a new component that is not found in the conventional (mother stock) geranium component, TP 0-26.0 (min): Top note component, TR26,1-38,0 (min): Middle note component, TR38.1 to 60.0 (min): Indicates the GC peak area ratio (χ) confirmed in the base note component region.

(Left below) Examples 10 to 13 Patch lily (Pogostemon cablin)
Calli were induced using the stems of Benth, ) in the same manner as in Examples 1 to 4, except that the medium was LS medium. After mutation treatment in the same manner as in Examples 1 to 4, KT 1 μg/
ml and LS agar medium containing 11 μg/ml of IAAO was photocultured at 27° C. for 2 months. The adventitious buds that appeared were rooted in the same manner as in Examples 1 to 4, and 58 seedlings were obtained in this way. Examples 10 to 13 are shown in Table 3. However, * in the table represents a new component that is not found in the conventional (mother strain) Patchyoly component, and TRO ~ 33.0 (min)
Nidotsubu Notre: middle note component, TR33,1-
QO, O (min): indicates the GC peak area ratio (χ) confirmed in the base note component region.

(Margin below)

Claims (7)

[Claims] (1) A feature of culturing plant tissue, genetically mutating the culture, inducing differentiation of adventitious buds and roots from the mutated culture, and extracting essential oil components produced and accumulated in the plant body. A method for producing essential oils. (2) The plant is of the genus Pelorgonium.
The method for producing essential oil according to claim (1), which belongs to item m). (3) The method for producing essential oil according to claim (1), wherein the plant belongs to the genus Patchouli (Pogostemon). (4) The method for producing an essential oil according to claim (1), wherein the method for mutating the cultured tissue involves culturing it as a callus for one month or more. (5) The method of mutating cultured tissue is 0.1 to 0.5%
The method for producing essential oil according to claim (1), wherein the essential oil is treated with ethyl methanesulfonic acid. (6) The method for inducing differentiation of adventitious buds is cytokinin 1~
The method for producing essential oil according to claim (1), which uses a medium containing 10 μg/ml of auxin and 0 to 2 μg/ml of auxin. (7) The method of rooting from adventitious shoots is auxin 0-10.
Claim No. 1 (1) using a medium containing μg/ml
) The method for producing essential oils described in section 2.