JPH01240183A - Culture atmosphere regulator - Google Patents

Abstract

PURPOSE:To enable promotion of growth rate of seedlings and remarkable shortening of culture period, by hermetically sealing a culture atmosphere regulator consisting of a gaseous carbon dioxide substitution type disoxidizer coexisting together with a culture vessel containing plant tissues bedded therein. CONSTITUTION:A gaseous carbon dioxide substitution type disoxidizer, containing an iron based oxygen absorber, such as reduced iron powder or ferrous sulfate, hygroscopic or deliquescent alkaline earth metal salt, such as calcium chloride, a carbonate and/or hydrogencarbonate, such as sodium carbonate, and a solid substance, such as benzoic acid, reactive with water to assume acidity as essential components and capable of absorbing oxygen and generating gaseous carbon dioxide is prepared to provide a culture atmosphere regulator. The resultant regulator, together with a growth point cut at the top of a stem and a culture vessel used for germ-free culture in a culture medium, etc., are then hermetically sealed to exist together in a light-transmitting and air- impermeable vessel, such as polyvinylidene chloride-coated film to culture the plant tissues. Thereby, growth of the culture seedlings is promoted to remarkably shorten the culture period. Equipment cost is also reduced.

Description

Detailed Description of the Invention A. Object of the Invention (Field of Industrial Application) The present invention relates to a culture atmosphere conditioning agent and a culture atmosphere regulator used in a plant tissue culture method in which tissue is removed from a living plant and grown and differentiated in a medium. This is related to plant tissue culture methods using drugs.
It is widely used by general farmers as well as in the seed and seedling industry and seedling raising industry.

(Prior technology) The plant tissue culture method, in which tissue is removed from a living plant and grown and differentiated in an artificially prepared medium, has already been put into practical use, and most of the garden plants such as carnations and orchids are grown by tissue culture. being produced.

The most typical type of plant tissue culture is called growing point culture (or shoot tip culture), in which the growing point at the tip of the stem is cut out.
This is grown by aseptic culture in a medium containing nutrients such as inorganic salts, sugars, and plant hormones. Next, it is transferred to a medium that encourages rooting, and once roots appear, it is planted in soil and grown.

Since tissue culture allows the production of large numbers of seedlings in a short period of time, it has been put into practical use for mass-growing seedlings of crops that are difficult to grow.

In addition, since there are no viruses that inhibit plant growth at the growing point, it has the advantage of producing healthy, high-quality plants, and has been put to practical use in growing strawberries and Japanese potatoes.

Currently, plant tissue culture is performed not only by culturing organs such as roots, stems, leaves, buds, and bulbs, but also by culturing cells (callus), decay, and embryos. Also, attempts have been made to digest the cell walls of plants with enzymes, culture protoplasts in a free state, and breed new plants by fusion of somatic cells.

As described above, plant tissue culture is not only already in practical use, but is also an agricultural technology that will become increasingly important in the future.

(Problems to be Solved by the Invention) Current problems in the above plant tissue culture include the following (1) to (5).

(1) Cultivation requires a long period of time ranging from several months to several years.

(2) The survival rate during acclimation transplantation of cultured bacteria is low, at less than 50%.

(3) There is a lot of simple manual work, and labor costs are high.

(4) Utility expenses such as lighting, heating and cooling, and expenses for consumables such as culture media and nutritional drugs are high.

(5) Equipment costs such as incubators and lighting equipment are high.

All of these problems (1) to (5) increase the production cost of tissue culture bacteria.

In view of the above circumstances, the present inventors conducted various studies with the aim of finding a simple culture method that accelerates the growth rate of seedlings and significantly shortens the culture period.

[Structure of the Invention] (Means for Solving the Problems) The present inventors have developed an explant explant cut from a plant in a plant tissue culture in which tissue cut from a living plant is grown and differentiated in a medium. When the foliage-differentiated individuals obtained by culturing are grown by photosynthesis in a medium, the foliage-differentiated individuals are placed in an incubator containing a carbon dioxide gas-substituting oxygen scavenger that absorbs oxygen and generates carbon dioxide gas. The present invention was completed by discovering that culture is promoted when an atmosphere conditioner is present.

That is, the present invention absorbs oxygen in the atmosphere when culturing tissue cut from a living body of a plant by photosynthesis in a medium,
A culture atmosphere conditioning agent comprising a carbon dioxide gas-substituting oxygen scavenger that generates carbon dioxide gas to create an atmosphere that promotes culture, and
1i! When culturing tissue by photosynthesis in a medium, the culture vessel in which the tissue is placed is made of a light-permeable and non-air permeable culture medium along with a culture atmosphere conditioning agent consisting of a carbon dioxide gas displacement type oxygen scavenger that absorbs oxygen and generates carbon dioxide gas. The present invention relates to a method for culturing plant tissue, which is characterized by culturing plant tissues in a container in a sealed manner.

O Culture Atmosphere Conditioner As the culture atmosphere conditioner used in the present invention, various carbon dioxide gas displacement type oxygen scavengers that absorb oxygen and generate carbon dioxide gas can be used.9 For example, ascorbic acid and its salts absorb oxygen. At the same time, it is possible to use a type of agent that decomposes itself and generates carbon dioxide as the main ingredient, or a type that contains both an oxygen absorber and a carbon dioxide gas generating agent.

For the present invention, the latter type, in which an oxygen absorber and a carbon dioxide gas generating agent coexist, is preferable because the amount of oxygen absorption and the amount of carbon dioxide gas generated can be arbitrarily changed by changing the blending ratio of the two.

An example of such a drug is shown below.

A drug consisting of the following components (A) to (D).

(A) Iron-based oxygen absorber (B) Hygroscopic or deliquescent alkaline earth metal salt (C) Carbonate and/or hydrogen carbonate (D) Solid substance that becomes acidic when acting with water (A) In the present invention, it is preferable that in addition to the components (D), a slightly water-soluble powder filler is mixed as a diluent to improve the mutual miscibility and dispersibility of each component.

The above composition will be explained in more detail.

The iron-based oxygen absorbent, which is the component (A), is an iron-based substance that absorbs oxygen by reacting with oxygen in the air and being oxidized. For example, it mainly contains one or more types selected from the following groups. Includes:

(2) Iron powders such as reduced iron powder, electrolytic iron powder or atomized iron powder, and partial oxides of the iron powders.

■ For example, ferrous compounds consisting of ferrous sulfate, ferrous oxide, ferrous hydroxide, etc.

■For example, iron complex oxides such as triiron tetroxide.

■For example, iron compounds consisting of iron carbide, iron sulfide, iron carbonyl, iron silicon, etc.

(2) A metal halide and/or water and/or poorly water-soluble filler added to the iron powder and/or iron compound of (1) to (2) above.

It is particularly preferable that these iron-based oxygen absorbers have a particle size of less than 80 mesh.

Next, as the deliquescent or hygroscopic alkaline earth metal salt which is the component (B), for example, the following can be used. Namely, calcium chloride, magnesium chloride, beryllium chloride, strontium chloride, calcium nitrate, magnesium nitrate, beryllium nitrate, calcium bromide, magnesium bromide, beryllium bromide, strontium bromide, calcium iodide, magnesium iodide, strontium iodide. , beryllium fluoride, magnesium chloride ammonium chloride, magnesium chloride double salts such as magnesium chloride potassium chloride, etc., which have as a main component one or more alkaline earth metal salts are preferably used.

The above alkaline earth metal salts can be used in the form of anhydrous salts or various hydrated salts.

There are no particular restrictions on the carbonate and/or hydrogen carbonate that is component (C), and examples include lithium carbonate, sodium carbonate, potassium carbonate, magnesium carbonate, calcium carbonate, barium carbonate, lithium hydrogen carbonate, sodium hydrogen carbonate, Alkali metal or alkaline earth metal carbonates, bicarbonates, and ammonium carbonate salts such as potassium bicarbonate.

Ammonium salts such as ammonium hydrogen carbonate, carbonates of transition metals such as ferrous carbonate, cobalt carbonate, nickel carbonate,
It mainly contains one or more types selected from double salts such as KMgH(CO3)z・4H20 and others.

(D) A solid substance that becomes acidic when it interacts with water, which is an ingredient, is a normally solid substance that becomes acidic when it chemically and/or physically interacts with water vapor in the atmosphere inside the package or container. For example, those mainly containing one or more selected from the following group may be mentioned.

■For example, benzoic acid, erythorbic acid, caproic acid, glutamic acid, cinnamic acid, succinic acid, salicylic acid, oxalic acid, tartaric acid, sorbic acid, nicotinic acid, lactic acid, fumaric acid,
Organic acids such as folic acid, malic acid, and citric acid.

■For example, inorganic acids such as boric acid.

■For example, anhydrous acids such as phosphoric anhydride (phosphorus pentoxide) and sulfuric anhydride (sulfur dioxide), which become acidic through a chemical reaction with water vapor.

■Inorganic or organic acid salts of transition metals that become acidic when hydrolyzed. For example, iron, copper/L/nickel t14+zinc, titanium, manganese.

Halogenates, oxyacids, and organic acid salts of metals such as aluminum, tin, antimony, lead, and bismuth, more specifically, for example, iron chloride, cobalt chloride, nickel chloride, copper chloride, zinc chloride, and manganese chloride. , aluminum chloride, antimony chloride, titanium chloride, aluminum sulfate, iron sulfate, antimony sulfate, tin sulfate, titanium sulfate, manganese sulfate.

Aluminum nitrate, cobalt nitrate, iron nitrate, copper nitrate, nickel nitrate, nickel perchlorate, iron bromide.

Examples include copper bromide, nickel bromide, iron iodide, cobalt acetate, and the like.

In addition to the essential components (A) to (D) above, examples of poorly water-soluble powder fillers that can be added to improve the miscibility and dispersibility of these essential components include activated carbon, diatomaceous earth, and acidic fillers. Powders of inorganic or organic substances that are sparingly soluble in water, such as clay, silica, perlite, zeolite, and cellulose, can be used.

The blending ratio of the components (A) to (D) is not particularly limited and can be varied widely depending on specific conditions such as the type of plant to be cultured. Further, by using an iron-based oxygen absorbent in combination with the above-mentioned culture atmosphere conditioner, it is possible to vary the blending ratio of the iron-based oxygen absorbent in the culture atmosphere conditioner over a wider range.

The culture atmosphere conditioner containing the components (A) to (D) as essential components can be used in the method of the present invention in any form. For example, the individual components may be simply mixed in powder form (or may be formed into pellets, beads, rods, blocks, sheets, etc.). When forming into various shapes, Known excipients and lubricants such as carboxymethyl cellulose and polyvinyl alcohol can be used.

Moreover, the above-mentioned components (A) to (D) can also be used by being packaged in an air-permeable packaging material. Breathable packaging materials include paper, microporous synthetic resin membranes, nonwoven fabrics, and other breathable materials alone, and/or breathable packaging materials laminated with one or more synthetic resin breathable materials. is used. Such synthetic resin breathable materials include perforated synthetic resin films, microporous membranes,
Non-woven fabric etc. are used.

○Cultivation method The culture atmosphere conditioner of the present invention can be used in cultivation, such as by cutting off the growing point at the tip of the stem and culturing it aseptically in a medium containing nutrients such as inorganic salts, sugars, and plant hormones. All you have to do is place them together in a light-permeable, air-impermeable container together with the culture vessel in which they will be grown, and with such a simple method,
Cultivation of plant tissue is significantly promoted.

The material for the airtight container used to house the culture vessel and culture atmosphere conditioning agent may be any light-transmissive, non-breathable material, including synthetic resin film and synthetic resin film. Containers made of one or more types selected from the group consisting of sheets, synthetic resin molded containers, and various inorganic glasses can be used.

For example, a bag made of a synthetic resin film as exemplified below is used.

That is, a non-breathable film made of a laminate of one or more plastic films such as polypropylene, polyester, polyamide, polyvinyl alcohol copolymer, polyvinyl chloride, vinyl chloride/vinylidene chloride copolymer, polyethylene, etc. is used. be able to.

Desirably, a composite film containing a polyvinylidene chloride coated film, an EVAL vinylon film, and a polyvinylon film having an oxygen permeability of 100 ral/II2.atII+.24 hr or less can be used.

The culture method using the culture atmosphere conditioner of the present invention accelerates the growth rate of leaf-differentiated individuals compared to conventional methods, significantly shortens the culture period, and improves the acclimatization and transplantation of cultured seedlings. This has the excellent effect of significantly increasing the survival rate.

The culture atmosphere conditioning agent of the present invention can be applied to all methods of culturing plant fibers, such as culturing organs such as shoot apices, roots, stems, leaves, and bulbs, as well as culturing cells (callus), leopards, embryos, etc. Applicable to tissue culture from

It can also be applied to culturing protoplasts obtained by enzymatically digesting plant cell walls and culturing cells formed by fusing different types of protoplasts.

(Function) The growth rate of cultured seedlings can only be accelerated by sealing the culture vessel and coexisting with a culture atmosphere conditioning agent consisting of a carbon dioxide gas displacement type oxygen scavenger that absorbs oxygen and generates carbon dioxide gas inside the vessel. Although it is not clear why the survival rate of cultured seedlings during transplantation after acclimatization is significantly higher, it is presumed as follows.

That is, the photorespiration of the plantlets is suppressed due to the decrease in oxygen concentration in the culture vessel, and as a result, the effect of reducing the amount of photosynthesis due to photorespiration is alleviated. On the other hand, it is presumed that an increase in carbon dioxide gas concentration increases the photosynthetic rate of plantlets, and that both factors combine to promote the growth of cultured seedlings.

In addition, the reason why the survival rate of cultured seedlings is high when transplanted after acclimatization is because photosynthesis is actively carried out as mentioned above, and organs such as leaves, stomata, and roots are sufficiently developed. It is presumed that they have become more resistant to environmental stress since then.

[Example 1] The present invention will be described in more detail below using Examples and Comparative Examples.

Examples 1 and 2. Comparative Example 1 About 0.3 mm of the shoot apex was cut from the tip of the runner of a strawberry (variety: Kobun Wase) seedling, placed in a medium in a test tube, and cultured. MS medium was used as the medium. 25°C1 illuminance 2
After culturing for 3 weeks at 0001 ux with a photoperiod of 18 hours, individuals with stem and leaf differentiation were obtained.

The following culture treatments were performed on this stem-and-leaf differentiated individual.

That is, 10 culture vessels (test W) containing stem and leaf differentiated individuals were placed in KON (vinylidene chloride coated rolled nylon).
High gas barrier of 18μ/PE (1 polyethylene) 40μ
(MIJ degree: 8 cc/Bo ・24Hr -
Atm), put it in a transparent plastic Rv film bag (inner volume: 51cm) and add it to a culture atmosphere conditioning agent (oxygen absorption capacity 200cc, carbon dioxide generation Noh crab 20
One (Example 1) and two (Example 2) 0cc) were sealed together with a heat sealer. Continuing, 25
The cells were cultured at 1°C, 2000 lux, and 18 hour photoperiod. After 40 days, the growth status of the cultured individuals of each culture method was observed, and the results shown in Table 1 were obtained. Growth and rooting of the cultured individuals in Examples 1 and 2 were better than in the untreated plot (Comparative Example 1), which was not treated with a culture atmosphere conditioner consisting of a carbon dioxide displacement type oxygen scavenger.

Example 3.4. and Comparative Example 2 The same tests as in Example 1.2 and Comparative Example 1 were conducted except that the strawberry variety was Reihong, and the results shown in Table 2 were obtained. The growth-promoting effect of treatment with a culture atmosphere conditioner consisting of a carbon dioxide gas-substituted oxygen scavenger was also observed in Reihong.

Example 5. 6. 7 and Comparative Example 3 About 0.5 mm of the shoot apex of the chinensis was cut off, placed in a culture medium in a test tube, and cultured. The medium used was LS medium.

The plants were cultured for 4 weeks at 25° C., 20,001 ux of illumination, and 18 hours of photoperiod to obtain individuals with stem and leaf differentiation.

This stem-and-leaf differentiated individual was treated with a culture atmosphere conditioner consisting of a carbon dioxide gas-substituting oxygen scavenger as follows. That is, 10 culture vessels (test tubes) containing stem-and-leaf differentiated individuals were KON
(PVC rolled nylon) 18μ/
High gas barrier properties of PE (l-lyethylene) 40μ (Oxygen permeability: 8 cc/rrf ・24Hr-at
m) transparent plastic laminated film bag (inner volume:
5I) and 5 culture atmosphere conditioners (oxygen absorption capacity = 50 cc, carbon dioxide generation capacity 37 cc) consisting of a carbon dioxide gas displacement type oxygen scavenger mainly composed of iron powder and sodium bicarbonate (Example 5) 10 (Example 6) and 20 pieces (Example 7) were sealed with a heat sealer. Subsequently, the cells were cultured at 25° C., 2000 lux, and 18 hours photoperiod. After 45 days, the growth status of the cultured individuals according to each culture method was observed, and the results shown in Table 3 were obtained. Examples 5-
In No. 7, the cultured individuals had good growth and rooting.

Table 1 Table 2 Table 3-1) Fresh type, stem leaves + roots *3) Number of epiphytes per plant Example 8, Comparative Example 4 The growth point is Q from the top of the chrysanthemum shoot, and the size is about 3 mm. It was cut out, placed on a culture medium in a test tube, and cultured.

MS medium was used as the medium. 25°C9 illuminance 20001u
x, 18-hour photoperiod for 3 weeks to obtain shoot- and leaf-differentiated individuals.

This stem-and-leaf differentiated individual was treated with a culture atmosphere conditioning agent as follows. That is, eight incubators (test tubes) containing shoot-and-leaf differentiated individuals were heated to 18μ/PE (18μ/PE), 40μ high gas J~l3V-property (oxygen permeability). : 8cc/nf・
2411r/atm) transparent bra, Utsuk laminated fin B
Place one culture atmosphere conditioner (oxygen absorption capacity: 200 cc, carbon dioxide generation capacity: 200 cc) consisting of a carbon dioxide gas displacement type oxygen scavenger whose main components are iron powder and bicarbonate of soda, in a mug bag (inner volume: 41 cm). (Example 8) was sealed with a heat sealer. Continuing, 25°C1 illuminance 20
00 lux and 18 hour photoperiod. After 21 days, the growth status of the cultured individuals according to each culture method was observed, and the results shown in Table 4 were obtained. Compared to the untreated area (Comparative Example 4), which was not treated with a culture atmosphere conditioner consisting of a carbon dioxide gas displacement type oxygen scavenger, the growth and growth of cultured individuals in Example 8 was
Rooting was good.

Example 9, Comparative Example 5 The growing point of orchid (cymbidium) is 0°3 mm from the shoot tip.
They were cut into pieces of a certain size, placed on a medium in a test tube, and cultured. Hyvonex solid medium was used as the medium. 25
A protocomb-like body (hereinafter abbreviated as PLB) was obtained by culturing for 5 weeks at 1 °C, 20,001 ux illuminance, and 18 hour photoperiod. This PLB was transferred to Hyponex liquid medium and cultured with shaking for one month. The PLBs that had grown in clumps were divided and placed on the Hyponex solid medium again, and the culture vessel containing the divided PLBs was treated with a culture atmosphere conditioning agent in the same manner as in the example. After 21 days, the growth status of the cultured individuals of each culture method was observed, and the results shown in Table 5 were obtained. Growth and rooting of the cultured individuals in Example 9 were better than in the untreated plot (Comparative Example 5), which was not treated with a culture atmosphere conditioner consisting of a carbon dioxide gas displacement type oxygen scavenger.

Table 4 Table 5 C [Effects of the Invention] As detailed above, according to the culture method using the culture atmosphere conditioner made of the carbon dioxide gas displacement type oxygen scavenger of the present invention, the culture is faster than the conventional method. In addition to promoting the growth of seedlings and significantly shortening the cultivation period, there are significant benefits such as lower equipment costs due to the ease of creating a sterile system and lower drug costs due to the lower sucrose concentration in the medium. Economic effects can be obtained.

In addition, the unexpected effect that the survival rate of cultured seedlings when transplanted after acclimatization is significantly high can be obtained.

Claims (1)

[Scope of Claims] 1. The present invention is characterized by absorbing oxygen in the atmosphere when culturing tissue cut from a living body of a plant by photosynthesis in a medium, and generating carbon dioxide gas to create an atmosphere that promotes culture. A culture atmosphere conditioning agent consisting of a carbon dioxide gas displacement type oxygen scavenger. 2. When culturing tissue cut from a living body of a plant by photosynthesis in a medium, the culture vessel in which the tissue is placed is treated with a culture atmosphere conditioning agent consisting of a carbon dioxide gas displacement type oxygen scavenger that absorbs oxygen and generates carbon dioxide gas. A method for cultivating plant tissue, characterized by culturing plant tissue in a sealed container that is light-transmissive and air-impermeable.