JPS5876085A - Tissue culture of lichen - Google Patents

Abstract

PURPOSE:To promote the symbiotic effect and activate the growth in the tissue culture of lichen, by culturing the undifferentiated symbiont derived from the tissue of lichen in the atmosphere of air enriched with CO2 under light irradiation. CONSTITUTION:A lichen (e.g. AKASARUOGASE belonging to Usneaceae family, Lecanorales order) is washed with deionized aseptic water, cut to a proper size with a sterilized knife to obtain a small piece containing both green algae and fungi. The piece is placed on a solid medium and cultured at a constant temperature conditions between 0 and 40 deg.C in a light place to obtain undifferentiated symbiont, which is transplanted in a medium containing nutrious source, carbon source, organic materials such as various natural extracts, etc., and cultured at a constant temperature between 0 and 40 deg.C under the irradiation of light of 500-10,000 lux in an atmosphere of air containing 0.1-5vol% CO2 gas.

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 skin (the outermost tissue of the lichen that protects it, made up of hyphae that gather together and fuse with each other).
Algal layer (a tissue in which hyphae surround and hold the algae that make up the lichen body), pith II (a tissue in which hyphae are loosely intertwined and form the basis of the lichen body), pseudoroot (from the lower cortex layer) It can be seen that the lichen has protruded and differentiated into a tissue that fixes the lichen to the substrate (however, in some cases, the pseudoroot does not arise from the lower cortical layer).

In addition to slow growth, lichens are susceptible to constraints from the natural environment such as season, climate, temperature, and latitude, as well as from the human environment such as sulfur dioxide concentration and smoke concentration, making it extremely difficult to cultivate them naturally. Not successful. 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 the wild.

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., does not require much labor for collection, and can be produced systematically on an industrial scale.

The present inventors had previously succeeded in culturing lichen tissue by inducing and culturing undifferentiated symbionts from lichen tissue, but as a result of further research, they were able to cultivate the symbiont under light irradiation. We have discovered that by carrying out the process 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 Patent Laid-Open No. 54-527
87 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 present invention relates to a lichen tissue culture method characterized by culturing differentiated symbionts in an air atmosphere enriched with carbon dioxide gas under irradiation with light.

The method of the present invention is a universal method that can be applied to various lichens. That is, the method of the present invention can be generally applied to each of the following lichen families: Teloscysteaceae, Centipedeaceae,
Sumipogotaceae, Spermaceae, Antibacteriaceae, Prunustidae, Candlemotaceae, Chasipugitaceae, Aconitidae, Botonicaceae, Itagaceae, Spermaceae, Sperminaceae, Asteraceae, Asteroptaceae, Prunustaceae, Asterotiliaceae, Asteroptaceae, Laminaceae, Laminaceae, Coralinaceae, Chrysophyllidae, Hetupolaceae, Iwanolidae, Lichinaceae, Chrysophyllidae, Chipsagolaceae, Chrysophyllidae, Chrysophyllidae, Coralinaceae, Coralinaceae, Coralolinaceae, Albainaceae, Leopardinae, Ivoshigolaceae , Spermaceae, Nisesanaegidae, Bosigoniaceae, Ceratofunaceae, Broomraceae, Matsutakeaceae, etc.

Here, the term "undifferentiated symbiont" refers to a system that does not have the characteristic differentiation structure of lichens, but exhibits a symbiotic effect between lichen algae and lichen tubes, and includes at least one algae cell and at least one algae cell. 1
A system consisting of individual bacterial cells.

Furthermore, the term "symbiotic effect" refers to a synergistic effect that works between lichen algae and lichen tubes to promote 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 obtained by deriving from lichen as a raw material. Taking as an example the Lecanola spp., which belongs to the family Luogaceae, specific operating procedures for inducing and culturing the undifferentiated symbionts 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. In this case, it is necessary that the small piece includes both a ring part and a picture part. This small piece is placed on an appropriate medium, for example, a solid medium such as Murashige-Skoog medium, and
Culture is carried out under constant temperature conditions of ~40C, usually in the light. Through such culture, undifferentiated symbionts are formed from the surface of the lichen body on the oral part for 3 weeks.

The obtained undifferentiated symbionts are transplanted into a new medium with an appropriate composition, and enriched with carbon dioxide under a constant temperature of 0 to 40 C, preferably 20 to 35 C, under light irradiation of usually 500 to 10,000 lux. Cultivation is carried out in a cool air atmosphere. 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 0.1 to 5% (by volume), especially 0.5 to 2%.
% range is desirable in order to make the growth of undifferentiated symbionts 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 performed by any method such as shaking culture, static culture, or stirring culture. As the culture medium, natural or synthetic, organic or inorganic media are used. For example, it may be based on various known inorganic synthetic media, to which organic substances such as nutrients, carbon sources, and various naturally extracted substances are added within a range that does not interfere with the symbiotic effect. Representative examples of the above-mentioned inorganic synthetic medium include White's medium, Hildebrand medium, Linsmeyer-Skoog medium, Murashige-Skoog medium, and the like. In addition, these media with improved compositions can also be used. As the above-mentioned nutritional source, vitamins such as thiamine hydrochloride, pyridoxine hydrochloride, and nicotinic acid, amino acids such as chrysin and asparagine, hexavalent acetate such as inosit and sorbitol, alcohol, and the like may be used. Examples of the carbon sources include carbohydrates (sucrose, glucose, maltose, etc.), organic acids (acetic acid, etc.), alcohols (methanol, glycerin, etc.), casein hydrolyzate, coconut milk, yeast extract, malt extract, etc. They 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 Lecanora, which belongs to the family Luogaceae, collected in Kyoto City, Kyoto Prefecture, was cut into small pieces of about 1 cm in length.
After washing thoroughly with water, the page is immersed several times in sterile distilled water in a sterile box for washing. The small pieces of lichen obtained in this manner are placed aseptically on a synthetic agar medium having the following composition. The medium was Murashige-Skoog's inorganic salt medium containing 0.1 ppm of thiamine hydrochloride, 0.5 ppm of pyridoxine hydrochloride, 0.5 ppm of nicotinic acid,
A medium is used which has been sterilized in a conventional manner by adding 2 ppm of glycine and 100 ppm of isositol to adjust the pH to 6.0, and adding 1.0% W/V agar.

A small piece of P. chinensis placed on such a medium is cultured at a culture temperature of 25 r under light irradiation of 2000 lux.

Green, undifferentiated symbionts appear in the third week.

Approximately tg (fresh weight) of the obtained undifferentiated symbionts were transplanted into the above medium, and sterilized air containing 1% carbon dioxide gas was added to the medium at a rate of 5 d/mi.
25C12000 while blowing continuously at a rate of n.
Cultivation is continued for 2 months under light irradiation of Lutucus. After 2 months, an undifferentiated symbiont of approximately 71 fresh weight) was obtained.

Example 2 A moss moss belonging to the family Lecanoraformes, which was collected in Kyoto City, Kyoto Prefecture, was cut into small pieces of about 0.5 d in width, and after being thoroughly washed with water, they were placed in a sterile box in sterile distilled water. Soak and wash several times.

The thus obtained pieces of lichen of the moss moss are placed aseptically on a synthetic agar medium having the following composition. The medium was Murashige-Skoog's inorganic salt medium containing 0.1 ppm of thiamine hydrochloride and 0.5 p of pyridoxine hydrochloride.
pm, nicotinic acid 0.5 ppm, glycine 2 ppms
A medium is used which is adjusted to pl (6.0) by adding 100 ppm of isocyto-#, and which is sterilized by adding 1.0% W/V agar and sterilized in a conventional manner.

A small piece of Prunus moss placed on such a medium is cultured under light irradiation at a culture temperature of 25 C and 11,000 lux.

Green, undifferentiated symbionts appear in the third week.

Approximately 19 (fresh weight) of the obtained undifferentiated symbionts were transplanted into the medium, and 5 t of sterilized air containing 1.5% carbon dioxide was added to the medium.
While blowing continuously at the rate of nl/min,
Cultivation is continued for 2 months under light irradiation of 25C, 1000 lux. After 2 months, undifferentiated symbionts weighing approximately 6.9 (fresh weight) were obtained.

Example 8 A piece of Lecanora japonica belonging to the family Lecanolaidae, collected in Kyoto City, Kyoto Prefecture, was cut into small pieces of about 1 m in length, which were thoroughly washed with water and then placed in sterile distilled water in a sterile box. Soak and wash several times. The small pieces of lichen of C. variegata obtained in this manner are placed aseptically on a synthetic agar medium having the following composition. As a medium,
Thiamine hydrochloride 0.0% in Murashige-Skoog's inorganic salt medium.
lppm, pyridoxine hydrochloride 0.5 ppm, nicotinic acid 0.5 ppm, glycy 2 ppm, isositol i
o.

Adjust the pH to 6.0 by adding ppm, and add agar 1.0% W/
Use a medium that has been sterilized in the usual manner by adding ①.

A small piece of C. chinensis placed on such a medium is cultured under light irradiation at a culture temperature of 25 C and 12,000 lux. At the 8th week mark, green undifferentiated symbionts appear.

Approximately 1.9 (fresh weight) of the obtained undifferentiated symbionts were transplanted into the medium, and sterilized air containing 1% carbon dioxide gas was added to the medium at 5 d/m.
While blowing continuously at a rate of 25C, 200
Continue culturing for 2 months under light irradiation at 0 lux. After 2 months, undifferentiated symbionts weighing approximately 7.9 (fresh weight) were obtained.

Example 4 Prickly pear moss, which belongs to the family Centipedeidae of the order Lecanora and collected in Hirakata City, Osaka Prefecture, was cut into small pieces of about 0.5 mm in width, thoroughly washed with water, and then subjected to sterile distillation in a sterile box. Wash by soaking in water several times.

The thus obtained pieces of lichen of P. aeruginosa are placed aseptically on a synthetic agar medium having the following composition.

The medium was Murashige-Skoog's inorganic salt medium containing 0.1 ppm of thiamine hydrochloride and 0.5 ppm of pyridoxine hydrochloride.
ppm, nicotinic acid 0.5 ppm, glycine 2 ppm
, 100 ppm of insitol was added to adjust the pH to 6.0, 1.0% w/v agar was added, and the medium was sterilized in the usual manner.

A small piece of P. chinensis placed on such a medium is cultured under light irradiation at a culture temperature of 25 C and 11,000 lux. At the 8th week mark, green undifferentiated symbionts appear.

Approximately 1.9 (fresh weight) of the obtained undifferentiated symbionts were transplanted into the medium, and 5 m of sterilized air containing 1.5% carbon dioxide was added to the medium.
While blowing continuously at a rate of l/min, 25C,
Cultivation is continued for 2 months under light irradiation of 1000 lux.

After 2 months, an undifferentiated symbiont of approximately 61 fresh weight) was obtained.

1.Display of the incident 1981 Patent Application No. 172605 2. Name of the invention Lichen tissue culture method Representative Masao Suzuki 5. Date of amendment order: Voluntary 6. Subject of correction Detailed description of the invention in the specification 7. Contents of correction The following points will be corrected in the statement.

(1) Corrected "Kyoto City, Kyoto Prefecture" to "Kyoto City" on page 8, line 10, page 9, line 13, and page 10, line 16.

(2) Page 11, line 19 "Hirakata City, Osaka Prefecture" has been corrected to "Hirakata City."

that's all

Claims (1)

[Scope of Claims] 1. A method for culturing lichen tissue, which comprises culturing undifferentiated symbionts derived from lichen tissue in an air atmosphere enriched with carbon dioxide gas under irradiation with light. 2. The method according to item 1, wherein the light irradiation is performed at an illuminance of 500 to 10,000 lux. 3. The method according to item 1, wherein the carbon dioxide concentration in the air atmosphere is 0.1 to 5% (by volume). 4. The method according to item 1, wherein the culture is carried out while continuously supplying air containing carbon dioxide gas during the culture.