JPH0433435B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for culturing lichen tissue, and more particularly to a method for culturing undifferentiated symbionts derived from lichen tissue.

Lichens are symbionts made up of certain fungi and algae, and are a group of plants that occupy a botanically unique position. When observed under a microscope, the internal structure is found to be a cortical layer (the outermost tissue of the lichen that protects it, made up of hyphae that aggregate and fuse with each other), an algal layer (a tissue that protects the lichen), and an algal layer (which protects the lichen). (a tissue in which hyphae surround and hold the constituent algae), pith (a tissue in which hyphae are loosely intertwined and form the basis of the lichen), pseudoroot (a tissue that protrudes from the lower cortex and forms the base of the lichen) (However, in some cases, pseudoroots do not arise from the lower cortical layer.)

In addition to being slow-growing, lichens are seasonal and
Natural cultivation is extremely difficult and has not been successful because it is subject to constraints from the natural environment such as climate, temperature, and latitude, as well as from the human environment such as sulfur dioxide concentration and smoke concentration. Furthermore, although many lichens have similar external shapes, they often have completely different components, and it is extremely difficult to collect large quantities of the same species from nature.

Recently, research on plant tissue culture has been progressing as a method for producing plant components. Plant tissue culture grows at a much faster rate than natural plants, which grow on a yearly or monthly basis, making it possible to produce desired ingredients in a short period of time. Also, unlike natural cultivation, it is not affected by weather etc.
Collection does not require much labor, and planned production on an industrial scale is possible.

The present inventors had previously succeeded in culturing lichen tissue by inducing undifferentiated symbionts from lichen tissue and culturing them. We have discovered that by carrying out irradiation in an air atmosphere enriched with carbon dioxide gas, a symbiotic effect can be significantly expressed and growth can be activated. It has been known that plant cells containing chlorophyll can be cultured under light irradiation in an atmosphere enriched with carbon dioxide gas (Japanese Unexamined Patent Application Publication No. 1989-1999).
52787 publication specification). However, all of these cultures targeted only cells with chlorophyll, and did not target complex systems of cells with and without chlorophyll, especially symbiotic systems. Therefore, the above discovery by the present inventors cannot be predicted from conventional knowledge.

The gist of the present invention resides in a lichen tissue culture method characterized by culturing undifferentiated symbionts induced from lichen tissue under light irradiation in an air atmosphere enriched with carbon dioxide gas.

The method of the present invention is a universal method that can be applied to various lichen plants. That is, the method of the present invention can be generally applied to each of the following lichen families: Teloscystetheceae, Centipedeaceae, Spermaceae, Araceae, Antigraceae, Prunusaceae, Candleraceae, Porphyllidae, Aconitidae, Prunustaceae, Prunustaceae, Prunustaceae,
Spermaceae, Asteroidaceae, Helitorifoliaceae, Asteroidaceae, Asterotiliaceae, Asteroidaceae, Asteroidaceae, Asteroidaceae, Cicariaceae, Blackstripaceae, Hesperaceae, Ilanaceae, Lichinaceae, Mojigollaceae, Prunusaceae, Asteroidaceae, Antrumaceae , Corallaceae, Corallaceae, Corallaceae, Pingolaceae, Lycodontaceae, Ilocaceae, Corallaceae, Locustraceae, Asteraceae, Acolyceae, Alocataceae, Matsutakeceae, etc.

Here, "undifferentiated symbiont" is a system that does not have the characteristic differentiation structure of lichen plants, but exhibits a symbiotic effect between lichen algae and lichen fungi, and includes at least one algae cell and at least one algae cell. A system consisting of bacterial cells.

Furthermore, the term "symbiotic effect" refers to a synergistic effect between lichen algae and lichen fungi that promotes the growth of both and the production of metabolites. It is considered to be the movement of trace amounts of physiologically active substances.

The undifferentiated symbiont of lichen used in the present invention is
It is obtained by deriving from lichen plants. Taking as an example the Physcomitrella sinensis belonging to the order Lecanora and the family Ceraminaceae, specific operating procedures for inducing and culturing the undifferentiated symbiont are as follows.

First, the lichen body of A. chinensis is thoroughly washed with deionized sterile water, and then cut into small pieces with a sterile scalpel to an appropriate size. At this time, it is necessary that the small pieces contain both algae and fungus parts. This small piece is placed on a suitable medium, for example, a solid medium such as Murashigeskoog medium, and cultured under constant temperature conditions of 0 to 40°C, usually in a light place. Through such culture, undifferentiated symbionts are formed from the lichen surface within 3 weeks.

The obtained undifferentiated symbionts are transplanted into a new medium with an appropriate composition, under a constant temperature of 0 to 40°C, preferably 20 to 35°C, under light irradiation of usually 500 to 10,000 lux, and under carbon dioxide gas. Culture is carried out in an air atmosphere enriched with In particular, it is preferable to continuously supply air containing carbon dioxide gas into the culture vessel in order to make the growth of undifferentiated symbionts more active. The concentration of carbon dioxide in the air atmosphere is preferably in the range of 0.1 to 5% (by volume), particularly 0.5 to 2%, in order to make the growth of the undifferentiated symbiont more active. As a light source, the sun, light, fluorescent lamp, incandescent lamp, mercury lamp, etc. can be used. When light irradiation is not performed, substantially no algae growth is usually observed. Irradiation may be continuous or may be cycled every few hours.

Cultivation may be carried out by any method such as shaking culture, static culture, or stirring culture. Natural or synthetic, organic or inorganic media can be used as the culture medium. For example, it may be based on various known inorganic synthetic mediums, to which organic substances such as nutrients, carbon sources, and various naturally extracted substances may be added within a range that does not interfere with the symbiotic effect. Representative examples of the inorganic synthetic base include White base, Hildebrand base, Linsmeyer-Skoog base, Murashiges-Skoog base, and the like. In addition, those with improved compositions can also be used. The above nutritional sources include thiamine hydrochloride,
Vitamins such as pyridoxine hydrochloride and nicotinic acid, amino acids such as chrysin and asparagine, and hexahydric alcohols such as inosyte and sorbitol may be used. As the carbon source, carbonized products (sucrose, glucose, maltose, etc.), organic acids (acetic acid, etc.), and alcohols (methanol, glycerin, etc.) can be used. Examples of the above-mentioned various natural extracts include casein hydrolyzate, coconut milk, yeast extract, and malt extract, which may be used alone or in appropriate combinations.

According to the method of the present invention, lichen tissue grows rapidly, making it possible to industrially produce lichen components from the tissue.

Next, the present invention will be specifically explained with reference to Examples.

Example 1 A red-spotted grass belonging to the order Lecanolae and family Slowfishes collected in Kyoto City was cut into small pieces of about 1 cm in length, which were thoroughly washed with water and then washed by immersing them several times in sterile distilled water in a sterile box. do. The small pieces of lichen of P. elegans thus obtained are placed aseptically on a synthetic agar medium having the following composition. As a base, thiamine hydrochloride 0.1ppm and pyridoxine hydrochloride are added to Murashige Skoog's inorganic salt base.
0.5ppm, nicotinic acid 0.5ppm, glycine 2ppm,
Add 100 ppm of isositol to adjust the pH to 6.0, add 1.0% agar W/V, and use a medium that has been sterilized in the usual manner.

A small piece of A. chinensis placed in such a medium is cultured at a culture temperature of 25° C. under light irradiation of 2000 lux. Green, undifferentiated symbionts appear in the third week.

Approximately 1 g (fresh weight) of the obtained undifferentiated symbionts was transplanted into the medium and incubated at 25°C and 2000 lux while continuously blowing sterilized air containing 1% carbon dioxide at a rate of 5 ml/min. Continue culturing for 2 months under light irradiation. After 2 months, approximately 7 g (fresh weight) of undifferentiated symbionts were obtained.

Example 2 Prunus moss belonging to the family Prunus in the order Lecanora was cut into small pieces of approximately 0.5 cm ² in size, which were collected in Kyoto City, were thoroughly washed with water, and then immersed several times in sterile distilled water in a sterile box. Wash with water. The thus obtained pieces of lichen of the moss moss are placed aseptically on a synthetic agar medium having the following composition. As a base, Murashige Skoog's inorganic salt base includes thiamine hydrochloride 0.1ppm, pyridoxine hydrochloride 0.5ppm, nicotinic acid 0.5ppm, and glycine.
2 ppm and isositol 100 ppm to adjust the pH to 6.0, add 1.0% W/V agar, and use a medium that has been sterilized in the usual manner.

A small piece of Prunus moss placed in such a medium is cultured at a culture temperature of 25° C. under light irradiation of 1000 lux. Green, undifferentiated symbionts appear in the third week.

Approximately 1 g (fresh weight) of the obtained undifferentiated symbionts was transplanted into the medium and incubated at 25°C and 1000 lux while continuously blowing sterilized air containing 1.5% carbon dioxide at a rate of 5 ml/min. Continue culturing for 2 months under light irradiation. After 2 months, approximately 6 g (fresh weight) of undifferentiated symbionts were obtained.

Example 3 A lizardfish belonging to the order Lecanolae and the family Aridaceae collected in Kyoto City was cut into small pieces of about 1 cm in length, which were thoroughly washed with water and then immersed several times in sterile distilled water in a sterile box. Wash with water.
The thus obtained pieces of lichen of C. lichen are placed aseptically on a synthetic agar medium having the following composition. As a base, thiamine hydrochloride 0.1ppm is added to Murashige Skoog's inorganic salt base.
Pyridoxine hydrochloride 0.5ppm, nicotinic acid
0.5ppm, glycine 2ppm, isositol 100ppm
, adjust the pH to 6.0, add 1.0% W/V agar, and use a medium that has been sterilized as usual.

A small piece of C. chinensis placed on such a medium is cultured at a culture temperature of 25° C. under light irradiation of 2000 lux. Green, undifferentiated symbionts appear in the third week.

Approximately 1 g (fresh weight) of the obtained undifferentiated symbionts was transplanted into the medium and incubated at 25°C and 2000 lux while continuously blowing sterilized air containing 1% carbon dioxide at a rate of 5 ml/min. Continue culturing for 2 months under light irradiation. After 2 months, approximately 7 g (fresh weight) of undifferentiated symbionts were obtained.

Example 4 Prickly pear moss, which belongs to the order Lecanora and family Centipedeaceae, was collected in Hirakata City and cut into small pieces of about 0.5 cm ² in size, which were thoroughly washed with water and placed in sterile distilled water in a sterile box. Soak twice and wash. The thus obtained pieces of lichen of P. chinensis are placed aseptically on a synthetic agar medium having the following composition. As a base, 0.1 ppm of thiamin hydrochloride, 0.5 ppm of pyridoxine hydrochloride, 0.5 ppm of nicotinic acid, 2 ppm of glycine, and 100 ppm of isositol were added to Murashige Skoog's inorganic salt base to adjust the pH to 6.0, and 1.0% agar was added. Use a double base that has been sterilized in the usual manner by adding W/.

A small piece of Prickly Pear moss placed in such a medium is cultured at a culture temperature of 25°C under light irradiation of 1000 lux. Green, undifferentiated symbionts appear in the third week.

1 g (fresh weight) of the obtained undifferentiated symbionts was transplanted into the medium and exposed to 1000 lux light at 25°C while continuously blowing sterilized air containing 1.5% carbon dioxide at a rate of 5 ml/min. Continue culturing for 2 months under irradiation. After 2 months, approximately 6 g (fresh weight) of undifferentiated symbionts were obtained.

Claims (1)

[Scope of Claims] 1. Undifferentiated symbionts formed by culturing small pieces containing both bacterial cells and algal cells collected from the same individual of a natural lichen on a solid medium are exposed to carbon dioxide gas under light irradiation. A method for propagating lichen tissue, which comprises culturing in an air atmosphere enriched with lichen tissue. 2. The method according to item 1, wherein the light irradiation is performed at an illuminance of 500 to 10,000 lux. 3 Carbon dioxide concentration in the air atmosphere is 0.1-5%
(capacity). 4. The method according to item 1, wherein the culture is carried out while continuously supplying air containing carbon dioxide gas in the incubator.