JPH01153087A - Method for tissue culture of marigold - Google Patents

Abstract

PURPOSE:To obtain a culture capable of exhibiting especially powerful nematocidal activity, by inducing marigold in a special culture medium into calli and propagating the resultant calli in a culture medium containing auxin in a low concentration. CONSTITUTION:Tissual pieces collected from leaves, stems, roots buds, etc., of marigold are sterilized by a conventional method and induction of calli is then carried out in a callusing culture medium containing auxin or further cytokinin as a phytohormone. A culture medium containing the auxin in a concentration as low as 0.01-1mg/l and in the absence of cytokinin or in a concentration thereof as low as <=3mg/l is used as a culture medium for successively cultivating the marigold induced into the calli.

Description

[Detailed Description of the Invention] Industrial Application Field This invention relates to a method for culturing marigold fibers, and the culture obtained by the method of the present invention has particularly excellent insecticidal properties against nematodes. It shows.

BACKGROUND OF THE INVENTION Marigold is a plant of the Asteraceae family, and is known to contain α-terchenyl represented by the following formula and its analogues, which have nematicidal activity. [For example, Reculee des Trapacs Chimique des Bayes-Passes (Recu
eille des Travelaux Chimiq
ue des Pays-Bas) Volume 77 1004
Pages 1009 (1958) and Volume 79, 382
Pages 390-390 (1959)) Methods for chemically synthesizing α-terchenyl have also been studied (eg, JP-A-52-118462), but have not yet been put to practical use.

Problems that the invention aims to solve The method of planting marigolds directly in fields to reduce the density of nematodes in the soil is widely known, and compared to chemicals, there is no concern about residual toxicity and the effect is long-lasting. It has the advantage of being long-lasting, but on the other hand, it is difficult to grow vegetables in fields planted with marigolds at the same time, so they are forced to fallow or reduce production during that time.

Means for Solving the Problems In view of the above circumstances, the present inventors have conducted various experiments and research on methods for mass-producing marigolds by tissue culture. After inducing callus with a callus-forming medium, auxin was added at 0.01 mg/l.
It has been discovered that a culture exhibiting particularly strong nematicidal activity can be produced by growing in a growth medium regulated at a low concentration of 1 mg/l to 1 mg/l.

Typical marigolds (T.
Agetes genus) is a French marigold (Tag
etes patura), African marigold (Tagetes erecta), etc., and in the present invention, tissue pieces collected from the leaves, stems, roots, lily pads, etc. of these marigolds are sterilized by a conventional method, and then treated as a plant hormone. Callus induction is performed in a callus-forming medium containing auxin or auxin and cytokinin.

Note that typical auxins suitable for carrying out the present invention include indoleacetic acid, naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, etc., and typical cytokinins include kinetin, benzyladenine, zeatin, etc.

The callus formation medium is a basic medium such as Murashige-Skoog's medium, Linsmeyer-Skoog's medium, Gamborg's medium, Nirachie's medium, etc., and an appropriate amount of carbon sources such as sucrose and glucose in addition to the above-mentioned plant hormones. For example, 1 to 5% by weight of sucrose and 0.5 to 1.0% by weight of agar to Murashige-Skoog's basic medium are suitable.
, naphthalene acetic acid as auxin from 0.01 to 20 m
g/R, benzyladenine as cytokinin 0
For medium added in the range of ~20 mg/l, 2 to 3
Good callus formation is observed after a week of culture. The conditions for callus culture are the same as for normal plant tissue culture, and the temperature is 15-35°C, preferably 20-30°C.
°C and pH range from 4 to 8, preferably from 5 to 6.

The callus-induced marigolds are subsequently grown in a growth medium containing the same auxin or auxin and cytokinin as described above, together with other additives, but in the practice of the present invention, a culture with particularly strong nematicidal activity is used. In order to obtain this product, the amount of auxin added in the growth medium was set to a low concentration of 0.01 mg/1 to 1 mg/l, and cytokinin was either absent or at 3 m
The concentration should be as low as g71 or less.

When the amount of auxin and cytokinin added to the growth medium exceeds a predetermined amount, the efficacy of the nematicide obtained by extracting the culture is significantly reduced.

In addition, as a method of propagation culture, either static culture using a solid medium containing agar or shaking culture using a liquid medium excluding agar can be used.

In addition, in order to produce a nematicide from marigolds cultured by the method of the present invention, after drying the cultured marigolds, extraction is performed using an organic solvent such as n-hexane, acetonitrile, acetonitrile, etc. The organic solvent may be removed from the extract.

The method of the present invention will be specifically explained below using Examples and Comparative Examples.

Origin Example 1 and Comparative Example 1 French marigold (variety name: Bolero) after germination 2
The cotyledons of sterile seedlings that have been aged for a week are cut into approximately 5 mm square pieces and placed in a Murashige-Skoog basic medium containing 3% by weight of Shoto, 0.8% by weight of agar, and 0.1 mg of naphthalene acetic acid.
42. 0.1 mg/l of benzyladenine was added, and the mixture was placed on a callus-forming medium sterilized by a conventional method. 25 in this state
Calli induction was carried out under continuous illumination while kept at a temperature of 20°C, and after 1 month, the culture was subcultured into a growth medium with a composition excluding benzyladenine from the callus formation medium, and cultured again under the same conditions for 1 month to allow growth. I did it. Thereafter, the cells were passaged twice at one-month intervals for propagation.

The culture thus obtained was a dark green, hard callus, and showed an approximately 15-fold increase in fresh weight due to each growth.

The marigold culture cultured according to this example was dried at room temperature under aseptic conditions, lightly crushed, and n/μ of the dried culture was dried.
- Hexane was mixed at a ratio of 50 ml, extraction was performed for 30 minutes, and solid matter was filtered off to obtain an extract.

A nematicidal test was conducted using the extract thus obtained.

For the tl nematode test, Pratylen nematodes
chus penetrans) and Caenorhabditis elegans (Caenorhabditis elegans).
(hereinafter abbreviated as C, elegans) was used.

Pratylenchus pen
The test using (etrans) is as follows. That is, 50 μl each of the extract, its diluted solution, and pure n-hexane as a control were dropped onto separate slide glasses and air-dried, and then 50 μl of each of the extract and its diluted solution, as well as pure n-hexane as a control, were dropped onto separate slide glasses and air-dried.
enetrans) collected in deionized water using the Bellman method, 50 μl of each solution (there are 20 to 40 nematodes in this) was dropped, and the slide glass was placed in a Petri dish that had been made into a greenhouse and incubated at 25°C. After standing in a lighted incubator for 8 hours, microscopic observation was performed to determine whether the nematodes were alive or dead.

The test using C. elegans is roughly the same; 20μβ of each of the extract and its diluted solution and pure n-hexane as a control were dropped onto separate glass slides, air-dried, and E. coli was added onto the slide glass in NG medium. Transfer 20 to 100 C. elegans cultured using C. elegans as bait, and add 30μ of NG medium on top! In addition, the slide glass was placed in a petri dish that was used as a greenhouse, and after being allowed to stand for 8 hours in a lighted incubator at 25°C, microscopic observation was performed to determine whether the nematodes were alive or dead.

As a comparative example, C. elegans was used for extracts and diluted solutions obtained by drying the roots of French marigold (variety name: Polero) by natural cultivation methods and performing the same extraction process as above. A nematicidal test was conducted.

These test results are shown in Table 1, and the marigold extract cultured by the method of the present invention has
Strong nematicidal activity comparable to that obtained from naturally cultivated roots was observed.

Table 1 (Survival rate: 2%) Furthermore, as a result of analyzing the extracts of this example and comparative example by high performance liquid chromatography using α-terchenyl manufactured by Fluka, it was found that the extracts of this example had a survival rate of 0. 5μg/
le1. The extract of the comparative example contained 0.4 μg and 7 ml of α-terchenyl.

Comparative Examples 2 and 3 In Example 1, French marigold cotyledons were added to Murashige-Skoog's basal medium with 3% by weight of ShotJAi.
Callus was induced using a callus-forming medium containing 0.8% by weight agar, 3 mg/1 naphthaleneacetic acid, and 3 mg/l benzyladenine, and the same culture treatment was performed using a growth medium containing the same components and concentrations as the callus medium. The culture was extracted in the same manner as described above, and a nematicidal test was conducted against the nematodes. (Comparative Example 2) In addition, the same culture was carried out using 3 tsg/l of naphthalene acetic acid and 10 mg/l of benzyladenine in the callus formation medium and growth medium, and the extract of the culture thus obtained was used to detect C. elegans. A nematocidal test was conducted against. (Comparative Example 3) These test results are as shown in Table 2. ) Examples 2 to 4 and Comparative Example 4 French marigold (variety name: Polero) after germination 2
Collect the cotyledons (Example 2) that have passed for 7 weeks according to the usual method.
After sterilizing with 0% ethanol and antiformin solution, wash thoroughly with sterilized purified water and use this as Murashige-Skoog's basic medium. Sugar 3% by weight, agar 0.8% by weight, naphthalene acetic acid 1ag/j! was added and placed in a sterilized callus-forming medium using a conventional method to induce callus, and callus was also induced using the same conditions for the marigold roots (Example 3) and buds (Example 4). .

All cultures were performed at a temperature of 25°C under continuous illumination.
After one month, the culture was subcultured into a growth medium with the same composition as above,
Furthermore, growth was performed for 1 month under the same culture conditions, and
The cultures thus obtained, which were propagated twice at intervals of months, had a large number of short roots differentiated into yellow-green to brown calluses.

The marigolds cultured in this way were dried at room temperature under sterile conditions, lightly crushed, and the n-
Extraction was carried out for 30 minutes by adding hexane, solid matter was filtered out to form an extract, and as a result of analysis by high performance liquid chromatography, the extract in each example was 0.04μ
g 7 ml of α-terchenyl.

In addition, for comparison, synthesized α-terchenyl (Flu
ka) in n-hexane solution at 0.0411g/r
Samples were prepared by adding 1.0 ml of the above extracts, and a nematicidal test was conducted together with each of the above-mentioned extracts. (Comparative Example 4) These test results are shown in Table 3. Marigold tissue cultured from any tissue site has the same nematocidal properties, and α-terchenyl It was found that the extract from the marigold culture had clearly stronger nematicidal activity than the extract containing synthetic α-terchenyl, even if the concentration of α-terchenyl was the same.

Examples 5 and 6 French marigold [variety name: Bolero (Example 5)
] and African marigolds [cultivar name: Niorange Hawaii (Example 6)] two weeks after germination, cotyledons were collected, sterilized with ethanol and antiformin solution, washed with sterile purified water, and mixed with Murashige Skoog. Add 3% by weight of sucrose, 0.8% by weight of agar, 1mg/l of naphthaleneacetic acid, and 3mg/l of benzyladenine to the basic medium of
Callus was induced by placing the cells on a sterilized callus-forming medium using a conventional method. Both callus-forming cultures were performed at a temperature of 25°C under continuous illumination, and after one month, the resulting cultures were subcultured into a growth medium with the same composition as above, and further grown for one month under the same culture conditions. . Thereafter, the cells were grown again for one month using the same composition of medium and under the same conditions.

All of the cultures thus obtained were yellow-green callus, and each growth showed an approximately 20-fold increase in fresh weight.

Bring the cultured marigold culture to room temperature.

After drying under sterile conditions and crushing lightly, 50ml per Ig
Mix with n-hexane in a proportion of , extract for 30 minutes,
The solid matter was filtered off to obtain an extract.

Using the extract obtained in this way and its diluted solution,
A nematicidal test was conducted in the same manner as in Example 1 above.

The test results are shown in Table 4, and the extracts obtained from the cultures of both French marigold and African marigold exhibited excellent nematocidal properties.

Table 4 (Survival rate 2%) Examples 7 and 8 French marigold (variety name: Bolero) after germination 2
Collect week-old cotyledons, sterilize them with 70% ethanol and antiformin solution according to the conventional method, wash thoroughly with sterile purified water, and add 3% by weight of ShigL, 0.8% by weight of agar to Murashige-Skoog's basic medium. Indole acetic acid ltag/l
, a medium containing 1 mg/l of benzyladenine and sterilized by a conventional method (Example 7) and a Murashige-Skoog basic medium containing 3 wt. The cells were placed in a medium (Example 8) which had been sterilized using a conventional method to induce callus.

Both cultures were carried out at a temperature of 25°C under continuous illumination, and after one month, the resulting cultures were subcultured into a medium with the same composition as above, and propagated for another month under the same culture conditions, and then for one month. A yellow-green callus was obtained by propagation through two passages at intervals.

The marigold culture cultivated in this way is grown at room temperature.
After drying under sterile conditions and crushing lightly, 50+/1g
! of n-hexane was added to perform extraction for 30 minutes, and the solid matter was filtered off to obtain an extract. A nematicidal test was conducted in the same manner as shown in Example 1 using the above extract and its diluted solution.

The test results are shown in Table 5, and the extracts of these cultures were found to have excellent nematocidal properties, regardless of the types of auxin and cytokinin.

Claims (3)

[Claims] (1) In marigold tissue culture, callus was induced with a callus-forming medium containing auxin or auxin and cytokinin, and then auxin was added at 0.01 m
A tissue culture method for marigold, which is characterized by growing marigold in a growth medium regulated at a low concentration of g/l to 1 mg/l. (2) The method according to claim (1), wherein the type of marigold is French marigold. (3) The method according to claim (1), wherein the type of marigold is African marigold.