JPS6374421A - Tissue culture of plant - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a culture method in plant tissue culture. More particularly, the present invention relates to a method for culturing plant cells, organs or tissue pieces on a support floating on the surface of a liquid medium.

[Prior art and problems]

Conventionally, two types of media have been used in plant tissue culture: liquid or solid. In the former case, the culture is carried out in a culture solution supplied with oxygen by shaking and aeration, and in the latter case, the culture is carried out on a medium solidified by adding agar or the like. The suitable medium format differs depending on the plant species to be cultured or the culture site, and many plants grow more vigorously on solid media. On the other hand, when a solid medium is used, it is difficult to add or replenish medium components as the medium is consumed if the culture continues for a long period of time. For this purpose - the culture is removed from the old medium in which the medium components have been consumed and reduced.

In addition, transplantation to a new solid medium that has been appropriately supplemented with medium components is performed.

Depending on the plant species or part to be cultured, it may be necessary to change the medium composition during cultivation, but in this case, with solid media, the culture can be transferred to a culture container containing a medium with a new composition. We will deal with it. On the other hand, with a liquid medium, medium components can be easily replenished, added, and replaced, so operations such as transplantation can be omitted.

However, in the case of liquid media, there is a problem of WI element supply, and methods such as rotational culture, swirling culture, horizontal shaking culture, or air aeration culture have been taken to solve this problem, but the culture requires operating equipment. It is also possible that differentiation or growth may be inhibited due to physical force being applied to the plant pieces to be cultured.

By the way, Web6r and Lark are theoretical
aland Applied Genetics)
55.81 (1979), as a method for culturing Datura datura using a-body S substrate, a urethane support was placed on a screen made of SUS, and a membrane filter placed on the support was used to culture callus of Datura datura etc. The cell colonies are cultured. According to this method, it is easy to exchange the medium or transplant the culture.

This method has the disadvantage that it is impossible to supply nutrients to the culture unless the amount of culture solution is always maintained at a constant level.

Also, Japanese Patent Publication No. 49-16300, Public Utility Model No. 56
No. 117671 and No. 57-201160 disclose a hydroponic cultivation method for plants using floating panels with floor cotton.

There are no known examples of its application to aseptic tissue culture of very small objects such as organs or tissue pieces.

[Purpose of the invention]

The present inventors have recognized that conventionally known plant tissue culture methods have the various problems described above, and have found that they can solve these problems and simplify the culture operation.

A new type of plant tissue culture that can promote the growth and differentiation of plant species that tend to be inhibited in liquid culture, such as single differentiation, and can therefore be more efficiently cultured than conventional methods. We considered the method.

[Summary of the invention]

As a result, they found that the above object could be achieved by employing the following method, and completed the present invention.

That is, according to the first method of the present invention, plant cells, organs, or tissue pieces are placed on a floating support on a liquid medium via a polysaccharide-containing solid culture medium, and then callus and tissue pieces are aseptically grown. Provided is a method for culturing plant tissue, which comprises newly forming and propagating plant bodies. According to the second aspect of the present invention, plant cells, organs, or tissue pieces.

There is provided a method for culturing plant tissue, which is characterized in that the tissue is placed on a water-absorbing material on a floating support on a liquid medium, and then callus and plant bodies are newly formed and propagated in an aseptic manner.

[Detailed description of the invention]

The tissue culture of the present invention is carried out using a liquid medium, and specifically, the liquid medium is a medium conventionally used for tissue culture of plants.

Example, tLJ, A Rashige Skoog ('62) CM
Linsmeyer-Skoog (RM-1965) CLtnsma
Ier & Skoog) medium.

White (63) (whttθ) medium, Gamborg (
For example, the culture medium may be prepared by adding a carbon source and a plant hormone to the E-5 medium (Gamborg), the M-9 medium (Sampaki), etc., and further adding vitamins and amino acids as necessary. Regarding the composition of the conventionally known culture medium mentioned above, for example, see [New Plant Tissue Culture JP] by Otsuchi, Nakajima and Furuya.
386-P391, breakfast bookstore.

Described in 1979.

There are no particular restrictions on the plants to which the tissue culture method of the present invention can be applied, and any plant that has been conventionally cultured can be tissue cultured using the method of the present invention. is possible. In the present invention, examples of the plants include plants belonging to the genus Lili such as gun lily, cuckold lily, squash lily, tiger lily, mountain lily, and plants such as strawberries and carnations.

In the present invention, tissue culture of a plant is performed using cells of the plant.

It can be performed using organs or tissue pieces.

Specifically, the tissue pieces and organs include shoot tips and stems.

Examples include leaves, flower seeds, bulbs, phosphorus flakes, roots or other plant tissues and organs cut into small pieces, which are usually treated with sodium hypochlorite, Used after sterilization with ethyl alcohol or flame.

However, when using aseptically cultivated plants, the above sterilization operation is not necessary. Furthermore, in the case of propagating seedlings of disease-free and virus-free plants, the tissue near the growing point, the above-mentioned tissue pieces of the plant obtained from the tissue near the growing point, etc. can be used as culture materials. Cells that can be used in the plant tissue culture of the present invention are undifferentiated amorphous cells obtained by culturing the tissue pieces by a known method.

According to the first method of the present invention, plant cells, organs or tissue pieces are placed on a polysaccharide-containing solid culture medium located on a floating support on a liquid medium;
In the method of the invention, tissue is cultured aseptically by placing the tissue on a water-absorbing material placed on a floating support on a liquid medium. First, the floating support will be described in detail. The floating support according to the present invention can be made of any material as long as it does not interfere with tissue culture and can float on the liquid medium without contaminating the medium. In principle, it can be used, but in tissue culture, the medium contains sugar and other organic substances, and the culture system needs to be kept sterile for a long period of time, so it is recommended to use autoclave sterilization as a floating support. It is preferable that the material and shape be such that it can be sterilized. Specifically, the material for such a floating support is polypropylene fiber (
Toughnel■), bamboo, styrofoam, urethane foam,
Synthetic organic materials such as 14-methyl-1-pentene polymers,
Examples include natural materials such as balsa, such as bamboo and wood chips.

In the first method of the present invention, tissue culture is performed by placing plant cells, organs, or tissue pieces on the floating support via a polysaccharide-containing solid culture medium. The saccharide-containing solid culture medium can be placed directly on the floating support (method A), or the polysaccharide-containing solid culture medium can be placed on the floating support via a water-absorbing material that absorbs water from the liquid medium. (Method B) can also be adopted. These methods will be explained in detail below.

Specifically, the following method can be used as method A. As a floating support according to the present invention.

A polysaccharide-containing solid culture medium is placed on top of the polysaccharide-containing solid culture medium by using a medium having a large number of holes of an appropriate size and arbitrary shape, and the polysaccharide-containing solid culture medium is suspended and supported so that the bottom surface is in contact with the liquid medium. An example of this method is to place the plant on the body and then place plant cells, organs, or tissue pieces on this polysaccharide-containing solid culture medium for tissue culture (see FIG. 1).

As the method B above, the following method can be shown. An absorbent material is placed on the floating support so that the absorbent material absorbs water from the liquid medium.Next, a polysaccharide-containing solid culture medium is placed on top of the water absorbent material, and plants are grown on the culture medium. cell,
An example is a method of culturing tissues by placing organs or tissue pieces on a bed. Specifically, the water-absorbing material in this case is made of water-absorbing materials such as bleached cotton, P paper, natural fibers such as cellulose, and synthetic or semi-synthetic fibers such as cellulose derivatives such as nitrocellulose and cellulose acetylacetate. Various shapes such as sheets, cloths, fibers, etc. of arbitrary shapes and appropriate thicknesses can be mentioned. The cellulose derivative is used as a porous membrane filter.
It may also be used as a water-absorbing material. In method B of the present invention, the water-absorbing material is placed on the floating support so as to wrap it around the support, the liquid medium is absorbed by the water-absorbing material, and the polysaccharide-containing solid culture medium is placed on the water-absorbing material. It's dark.

The polysaccharide-containing solid culture medium according to the present invention refers to the aforementioned liquid medium to which a solidifying agent such as agar, gelatin, and/or thermocoagulable protein, which solidifies the medium, is usually added in an amount of 11r, 5 to 3%. In the first aspect of the present invention, the solid medium is formed into a suitably shaped plate and placed directly on the floating support as in method A above.

Alternatively, tissue culture is performed by placing plant cells, organs, or tissue pieces on this polysaccharide-containing solid culture medium by placing it on the polysaccharide-containing solid culture medium by placing it on the polysaccharide-containing solid culture medium by placing it on the polysaccharide-containing solid culture medium as in method B above.

When the first method of the present invention is adopted, the calli and plants obtained by tissue culture have excellent shapes and have sufficient and stable growth.

According to the second invention method of the present invention, plant cells.

The organ or tissue piece is placed on a water-absorbing material on a floating support on a liquid medium, and new callus and plant bodies are formed and grown aseptically.

The floating support and water absorbing material in this case are the same as those used in the first invention described above. The tissue culture method according to the second method of the invention is the culture method (B) in the tissue culture method according to the first method of the invention, in which the water-absorbing material is not placed on the water-absorbing material. The method is the same as 03) except that plant cells, organs, or tissue pieces are placed directly on top of the cellulose and cultured. A concrete example of this is shown in FIG. The method of the present invention has the advantage that medium components can be easily replenished, added, or replaced as in the case of liquid culture.

In the present invention, tissue culture is performed in a manner that allows it to be performed aseptically. The culture tank for carrying out the tissue culture of the present invention is covered by an appropriate method as shown in FIG. 1, for example, to prevent contamination with various bacteria. Furthermore, in the present invention, culture can be performed by aerating an oxygen-containing gas into the liquid medium as necessary.

〔Effect of the invention〕

When the plant tissue culture method according to the present invention is used, the growth of cells and organs 1 tissue pieces can be maintained at the same or higher rate than when cultured using conventional culture methods, and at the same time as or better than when cultured in liquid culture. It is possible to maintain further growth, and to easily replenish, add, or replace medium components in the same way as in liquid culture, which greatly simplifies the cultivation process, making plant tissue culture more efficient than conventional methods. You can do well and multiply. In particular, when the first invention method using the polysaccharide-containing solid culture medium according to the present invention is adopted, the calli and plants obtained by culturing have better shapes, and the growth is more sufficient and stable. .

〔Example〕

Hereinafter, the structure and effects of the present invention will be specifically explained using Examples.

Example 1 Foamed 4-methyl-
As shown in Figure 1, a thin agar layer was placed within the frame of a floating support made of 1-pentene polymer (TPX), and this layer contained 4% sucrose and 0.1 mg/l of naphthalene acetic acid. Sterile Murashige-Skoog composition with a pH of 5.7 (1
Float the agar layer on 10 DJ of a culture solution (composition shown in Table 1) of 962) so that the bottom surface of the agar layer is in contact with the culture solution.
Phosphorous pieces of sterile virus-free small bulbs obtained by shoot apical culture of gun lily were placed on this agar and cultured for 50 days at 25°C in the dark.
~3 small bulbs with excellent shape and good growth were differentiated. The small bulbs were then allowed to grow into healthy explants.

Example 2 The same procedure as in Example 1 was carried out, except that instead of the gun lily scale section, a cut lily scale section was used. As a result, after 50 days of culture, we found that from 1 to 3 pieces of scale
~Excellent formation of 4 shapes Example 3 Gun lily scale sections were treated with naphthalene acetic acid 0.1 mg/g
, benzyl azotin o, o 1 mg/l, sucrose 4%,
Calli obtained by culturing on a solid medium containing 0.8% agar were cultured for 60 days under the same culture conditions as in Example 1. Approximately 300 calli of excellent shape were grown from 1 g of callus. Good small bulbs were differentiated.

Example 4 The same procedure as in Example 1 was carried out except that a sterilized stem section of gladiolus with a thickness of 3 to 4 mm was used instead of the scale section of gun lily. As a result, two to three well-shaped, well-grown globules were differentiated from one stem section after 50 days of culture.

Example 5 The same procedure as in Example 1 was carried out except that a lateral bud section of a one-bulb iris was used instead of a scale section of a gun lily. As a result, 5 to 6 well-grown adventitious buds were differentiated from one lateral bud section after 90 days of culture.

Example 6 In Example 1, one leaf primordium was used instead of the gun lily scale section.
~Using two carnation shoot apical tissues, and using Glutooth 4 instead of the Murashige-Skoog culture medium.
The procedure was carried out in the same manner as in Example 1, except that a culture solution of Holley-Baker composition (19 (53)) containing 11.0 mg// of naphthalene i[l] was used. As a result, 2 to 3 G Healthy seedlings were obtained that had grown rapidly.

Example 7 In Example 1, strawberry cormorants were used instead of gun lily scale sections, and the hormone conditions of the culture solution were changed to naphthalene vinegar u1o.
The same procedure as in Example 1 was carried out except that M, Henzyl Adeno 10M was used. the result.

Stable callus with good growth was formed after 40 days of culture.

Example 8 In Example 1, instead of the scale sections of gun lily, callus 1. Using 5g (live weight),
In addition, the same procedure as in Example 1 was carried out except that a culture solution having the same composition as the medium in which callus formation was induced was used instead of the Murashige-Skoog composition. As a result, it grew to 15 g (live weight) after 15 days of culture.

Example 9 Using the same culture solution and gun lily scale pieces used in Example 1, a floating support whose outer periphery was covered with dry cotton as shown in Figure 2 was placed on top of the culture solution. Float the tip of the scale section, expose it, place it on cotton, and incubate at 25℃ in the dark for 50 minutes.
After culturing for days.

One to two bulblets were differentiated per scale section.

[Brief explanation of the drawing]

FIGS. 1 and 2 are diagrams showing an example of the plant tissue culture method according to the present invention.

Claims (3)

[Claims] (1) Plant cells, organs, or tissue pieces are placed on a floating support on a liquid medium via a polysaccharide-containing solid culture medium, and new callus and plant bodies are formed and grown aseptically. A method for culturing plant tissue, characterized by: (2) A patent characterized in that the powder support has holes of an appropriate size, and tissue culture is performed by placing a polysaccharide-containing solid culture medium located above the hole in such a manner that the bottom surface of the solid culture medium is in contact with a liquid medium. The method according to claim 1. (3) Plant cells, organs, or tissue pieces are placed on a water-absorbing material on a floating support on a liquid medium, and new callus and plant bodies are formed and grown aseptically. Characteristic plant tissue culture method.