JPH0430734A - Formation of adventitious root of thyme - Google Patents

Abstract

PURPOSE:To efficiently form and derive adventitious roots from tissue pieces of thyme, by using a medium containing a specific amount of cytokinin and a specific amount of auxin. CONSTITUTION:Adventitious roots are formed from tissue pieces of thyme, by using a medium containing 10<-5>-10<-8>M cytokinin (preferably benzyladenine, kinetin or zeatin) and 10<-4>-10<-8>M auxin (preferably indoleacetic acid, 2,4- dichlorophenoxyacetic acid or naphthaleneacetic acid).

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for efficiently forming (or inducing) adventitious roots from thyme tissue pieces.

[Conventional technology]

Herbal plants are widely used as plants with medicinal effects, such as crude drugs, herbal teas, and bath additives. Among them, thyme contains many useful essential oil components such as thymol, and is said to be a therapeutic agent for headaches, sore throats, colds, swelling caused by gout and rheumatism, sciatica, leprosy, epilepsy, etc. The medicinal properties of herb are actually proven ("Enjoyment of Herbs JA," by Hattfield).

On the other hand, plant tissue culture has come to be practiced industrially as a method for obtaining large quantities of plant materials with stable quality. For example, in Japanese Patent Application Laid-Open No. 1-218582, sage, lahendar, rosemary, spearmint,
A method has been shown in which a plant callus such as lemon palm is cultured in a nutrient medium containing auxin and cytokinin, and then ethanol is added to obtain a large amount of plant essential oil components from the callus.

[Problem to be solved by the invention]

However, at present, tissue culture technology for mass production of the above-mentioned useful components from thyme has hardly been established, and production technology using adventitious roots, which are generally said to be prone to accumulating secondary metabolites, has not been established. do not have.

It is generally said that when auxins are added to the culture medium, adventitious roots are more easily formed from plant tissue pieces.
In the case of thyme, the problem is that the roots obtained are thick and short, and the growth is slow.

Therefore, an object of the present invention is to provide a tissue culture method for inducing adventitious roots from thyme tissue pieces.

[Means to solve the problem]

In order to solve the above problems, the present invention provides cytokinins 10-s to 10-8M and auxins 10-4M.
The gist is a method for forming adventitious roots of thyme from tissue pieces of thyme using a medium containing ~10-@M.

Thymus (Thymus vulgaris L) is
It is a perennial plant belonging to the Lamiaceae family, and plants of any season may be used. Any part of the thyme tissue may be used, but for example, the part of the stem between the leaves, the part of the stem containing the leaves, etc. are used.

In the present invention, a medium containing, for example, inorganic components, carbon sources, and plant hormones as essential components, and further containing vitamins and amino acids as necessary, is used.

The medium used must contain cytokinins and auxins, which are plant hormones. If cytokinins are present but auxins are not present, rooting will not occur and only callus will form. In addition, as mentioned above, roots will form even if auxins are included and cytokinins are not included, but thicker and shorter roots will be formed and growth will be faster than when both are used together at the specific concentrations listed below. It's also slow.

Examples of cytokinins include benzyladenine, kinetin, zeatin, etc.
One or more may be used, but are not limited to those listed here. Examples of auxins include indole acetic acid, 2.4 dichlorophenoxy acetic acid (2,4-D
), naphthalene acetic acid, etc., any one or 2 L) may be used, but are not limited to those listed here.

The concentration of cytokinins in the medium is 10-s to 10-8
M, preferably 10-6 to 10-'M. The concentration of auxins in the medium is 10-4 to 10-@M, preferably 10-5 to 10-'M. If the concentration of cytokinins in the medium exceeds the upper limit of the above range, cells may turn brown and die, and if it is below the lower type, rooting may not occur. The same applies when the concentration of auxins is outside the above range.

Inorganic components of the medium include, for example, inorganic salts containing elements such as nitrogen, phosphorus, potassium, sodium, calcium, magnesium, iron, manganese, zinc, molybdenum, copper, cobalt, sulfur, boron, chlorine, and iodine. can. Specifically, for example, potassium nitrate, sodium nitrate, ammonium nitrate, potassium chloride, calcium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, ammonium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, sulfuric acid. manganese, copper sulfate,
Zinc sulfate, sodium molybdate, potassium iodide,
Examples include compounds such as boric acid and cobalt chloride.

Examples of carbon sources in the medium include carbohydrates such as sucrose and derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol. Examples of vitamins in the medium include biotin, thiamine, pyridoxine, Examples include pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid, inositol, nicotinic acid, and nicotinamide.

Examples of amino acids in the medium include glycine,
Examples include alanine, glutamic acid, cysteine and phenylalanine.

The medium usually contains the inorganic component from about 10-'M to about 0.1M and the carbon source from about 10-'M to about 50g/l.
g/A', the above vitamins from about 0.1 ■/l to about 150 ■/I! , 0 to about 1 g of the amino acid/
1 and plant hormones at the above-mentioned concentrations. If each of these components exceeds the upper limit of the above-mentioned range, the cells may turn brown and die, while if the content is less than the lower limit or is zero, the growth of the cells may deteriorate. However, the types and amounts of components other than the plant hormones are not particularly limited.

Specifically, the medium used for culturing the thyme tissue pieces of the present invention may be a conventionally known culture medium used for plant tissue culture, such as Murashige-Skoog's medium or Linsmeyer-Skoog's medium. Add the carbon source and plant hormones described above to a medium, White's medium, Gamborg's B-5 medium, Niche Niche medium, etc.
Furthermore, a medium prepared by adding the above-mentioned vitamins and amino acids as necessary can be exemplified, but in this invention, a medium prepared using a Murashige-Skoog medium is particularly desirable.

In this invention, the medium is used as a solid medium containing, for example, 0.1 to 1% by weight of a polysaccharide such as agar. If the polysaccharide exceeds the upper limit of this range, the cells may wither and die, and if it falls below the lower limit, the medium may not solidify.

[For production]

Adventitious roots of thyme are formed by using a medium containing cytokinins and auxins at the above-described specific concentrations as a medium for culturing thyme tissue pieces.

Moreover, they are thinner, longer, and multiply faster than when auxins are used and cytokinins are not used.

〔Example〕

Specific examples and comparative examples of the present invention are shown below, but the present invention is not limited to the following examples.

Example 1 Benzyl adenine 10-
A solid medium of Murashigeskoog medium [containing 3% by weight of sucrose and 0.3% by weight of vegetable gum (gellan gum)] containing 10% M of naphthalene acetic acid was prepared, and this medium was mixed with thyme stems (leaves and The part between the leaves or the part containing the leaves (1 to 2 cm in length) was placed on a bed and cultured at 25° C. for 16 hours in the dark for 15 days. As a result, more adventitious roots were formed than in all tissue pieces.

Gellan gum 3000 μ/ltr is added to this medium to prepare a solid medium.

Example 2 - Instead of the Murashigeskoog medium used in Example 1, B-
Culture was carried out under the same conditions as in Example I using a solid medium of 5 medium [containing 0.3% by weight of vegetable gum (gellan gum)]. As a result, more adventitious roots were formed in more than 90% of the tissue pieces.

3,000 μl/l of gellan gum is added to this medium to prepare a solid medium.

Example 3 - In a 100-capacity culture dish, kinetin 101M, 2.
White medium containing 4-D and 10-'M gold [contains 3% by weight of sucrose and 0.3% by weight of vegetable gum (gellan gum)]
A solid medium was prepared and cultured under the same conditions as in Example 1. As a result, more adventitious roots were formed than in more than 90% of the tissue pieces.

3,000 μl/l of gellan gum is added to this medium to prepare a solid medium.

Example 4 - The plant hormone composition in Example 3 was changed to kinetin 104M.
, naphthaleneacetic acid 10-'×3M and cultured under the same conditions, a large number of adventitious roots were formed from all the tissue pieces.

Example 5 As a result of culturing under the same conditions as in Example 3 using a medium using Murashige-Skoog medium instead of the White medium used in Example 3, a large number of adventitious roots were formed from all tissue pieces. .

Example 6 Zeatin 10 as a cytokinin in Example 1
4M and 10-'M indoleacetic acid as the auxin. As a result, a large number of adventitious roots were formed from more than 90% of the tissue pieces.

Example 7 In Example 1, the concentration of cytokinins was changed to 10-@M.
A comparative example 1 in which a large number of adventitious roots were formed from 70% or more of the tissue pieces as a result of the same procedure as in Example 1 except that the concentration of auxins was 10-@M. As a result of culturing under the same conditions with the composition of hensyl adenine at 10-'M and 2.4-D at 10-'M, a little callus formation occurred compared to all the tissue pieces, but it soon turned brown. Oops.

Comparative Example 2 The plant hormone composition in Example 3 was changed to kinetin 10-'
As a result of culturing under the same conditions with M and indole acetic acid at 10-8 M, all tissue pieces turned brown and no adventitious roots were formed.

- Comparative Example 3 - In Example 1, the concentration of cytokinins was 10-8M.
As a result of carrying out the same procedure as in Example 1 except that the concentration of auxin and auxin was 101M, all the tissue pieces turned brown and died.

In addition, adventitious roots cultured using a medium containing only auxins and no itokainins as plant hormones,
Compared to the adventitious roots formed in Examples 1 to 7 above, they are thicker, shorter, and grow slower.

〔Effect of the invention〕

As described above, the method for forming adventitious roots of thyme according to the present invention can easily root thyme tissue pieces. Furthermore, according to the present invention, adventitious roots can be formed better than when using a medium containing only auxins as plant hormones. This is expected to yield a large amount of useful material from thyme.

Agent Patent Attorney Takehiko Matsumoto Procedural Amendment (Spontaneous 1゜2゜3゜4゜1) Name of Invention Patent Application No. 02-139379 Relation to the case of the person amending the method of forming indefinite roots of time Patent Applicant Address 1048 Kadoma, Kadoma City, Osaka Name (583) Matsushita Electric Works Co., Ltd.

Claims (1)

[Scope of Claims] 1. Thyme in which adventitious roots are formed from thyme tissue pieces using a medium containing 10^-^5 to 10^-^8M of cytokinins and 10^-^4 to 10^-^8M of auxins. adventitious root formation method. 2 The cytokinin is at least one selected from the group consisting of benzyladenine, kinetin, and zeatin, and the auxin is indoleacetic acid, 2,4-
The method for forming adventitious roots of thyme according to claim 1, wherein at least one root is selected from the group consisting of dichlorophenoxyacetic acid and naphthaleneacetic acid.