JP2619546B2 - Method for preparing somatic embryos of Podfilm plants - Google Patents

Description

The present invention relates to a method for preparing an adventitious embryo of a plant of the genus Podophyllum. More specifically, the present invention relates to a method for efficiently preparing a somatic embryo of a plant of the genus Podophyllum from cells of a plant of the genus Podophyllum by a plant tissue culture method.

[Conventional technology]

Somatic embryos derived from plant cells, as a material for mass propagation for the purpose of efficient breeding and cultivation of the plant,
It also has industrial value as a major component of artificial seeds. Further, it has value as a raw material for tissue culture for industrial production of useful components of the plant. A perennial plant of the genus Podophllum has been prized as a foliage plant in the past, but in recent years the plant contains substances useful in the field of medicine and cosmetics, such as podophyllotoxin compounds and flavonoid compounds. It became clear that the industrial value was increased. However, the self-cultivation of Podofilm plants is extremely limited, and the cultivation of such plants is limited by the growth rate, etc., and there has been a problem in the stable acquisition of the plants and useful components of the plants. . As one method of solving this problem, breeding of the plant using an adventitious embryo of a genus Podofilm, supply of the plant by cultivation, and supply of the plant body by cultivation, and a useful component obtained by tissue culture of the plant using the adventitious embryo as a raw material Is considered.

However, in general, it is extremely difficult to obtain a somatic embryo of a plant with good reproducibility, and at present, there are only reports of somatic embryo induction for a limited number of plant species. Furthermore, since the induction of somatic embryos is generally performed on a solid medium, it takes much time to induce somatic embryos, and it takes much time to harvest somatic embryos, which hinders industrial use of somatic embryos.

Until now, there has been no known example of somatic embryo induction in Podofilm genus plants irrespective of solid culture or liquid culture.

Several methods have been proposed for obtaining and producing adventitious embryos as a means for growing useful plants and providing raw materials for tissue culture as described above. For example, a method of inducing somatic embryos to differentiate by adding and growing a plant growth regulator such as auxin to undifferentiated cells of a plant, and a method for culturing undifferentiated cells without using a plant growth regulator. A method for inducing somatic embryos by changing the osmotic pressure of a medium is known. However, these methods are also not applicable to all plants, and the difficulty of inducing somatic embryos, specific conditions vary depending on individual plants, and conditions for one plant cannot be applied to other plants as they are. Therefore, there has been a need for a method for inducing a somatic embryo applicable to a plant of the genus Podofilm.

[Problems to be solved by the invention]

Accordingly, an object of the present invention is to provide a method capable of industrially preparing a somatic embryo of a plant of the genus Podofilm with high efficiency.

[Problems to be solved by the invention]

The above object is to prepare a somatic embryo of a plant belonging to the genus Podophyllum in a method for preparing an adventitious embryo of a plant belonging to the genus Podophyllum by culturing cultured cells of the plant belonging to the genus Podofilm under a sugar concentration of 2% by weight or less, and then culturing the cells under a condition where the sugar concentration exceeds 2% by weight. The somatic embryo is induced by performing the method, and the somatic embryo is harvested.

[Specific explanation]

According to the present invention, cultured cells of a plant of the genus Podophyllum are cultured under conditions of a sugar concentration of 2% by weight or less, and then cultured under conditions of a sugar concentration of over 2% by weight to induce somatic embryos of the plant. And optionally, harvesting the somatic embryo.
(yllum) genus plant.

The plant belonging to the genus Podofilm used in the method of the present invention is, for example, Podophyllum pertatum (Podophyllu).
m petatum), plants belonging to the genus Podofilm such as P. film, P.enodi, P. film hexanedrum, P. hexandrum, P. pleianthum, P. versipelle, and their subgenus. Species, varieties and the like can be suitably used.

The method of the present invention includes a step of culturing cells of a plant belonging to the genus Podofilm, and the cells are usually used in the form of callus or liquid cultured cells. Callus can be obtained according to a conventional method. That is, the leaves of the genus Podofilm,
Each part such as stem and root is thoroughly washed with water, sterilized with an aqueous solution of ethyl alcohol, an aqueous solution of sodium hypochlorite, an aqueous solution of benzalkonium chloride, and then washed with sterile water. The resulting sterilized plant is placed on a culture medium in a test tube, flask, petri dish, or the like and cultured to obtain a cell or tissue of the plant. As the medium to be used at this time, any medium that is generally used for plant tissue culture can be used without any problem. Examples of such media include Murashige-Skoog's medium, Camborg's B5 medium, niche & niche medium as basic medium,
A medium obtained by adding vitamins and a plant growth regulator to a white medium or the like can be suitably used, and is not limited to the above-described one. Examples of the direct growth regulator include indole-
Examples include auxins such as 3-acetic acid, α-naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid, and cytokinins such as kinetin and 6-benzyladenine. These are used alone or in combination with other components. By continuing culturing at 20-30 ° C in the above medium, usually 1
-Callus of the target plant of the genus Podofilm can be obtained in 4 weeks. By culturing this callus in a solid medium or a liquid medium, liquid culture cells of plants of the genus Podofilm can be easily obtained.

The culture in the present invention means cultivation of the cells of the genus Podofilm on a solid medium and culture in a liquid medium as described above. The solid medium means that the above-mentioned cells are placed on a hydrogel containing a medium component described below and cultured. As examples of the hydrogel, agar, gellan cam, and the like are suitably used. The liquid culture is a culture condition in a state in which some or all of the cells of the genus Podofilm obtained as described above are in contact with a liquid-phase medium. This includes a culture tank, a reciprocating or rotary shaken Erlenmeyer flask, and a culture method using a paper wick method.

In order to maintain or grow a culture of a Podofilm plant in a viable state for a long period of time, the presence of more than 2% by weight of sugar is generally suitable, but the present invention relates to a cell of a Podofilm plant such as a callus or a liquid culture cell. Temporarily
In addition, it is characterized in that somatic embryos are induced by leaving the cells in a sugar-starved state to the extent that the survival of the cells is maintained. In order to maintain such starvation, the sugar concentration may be reduced to 2% or less, preferably 1.5% by weight, more preferably 1% by weight or less, for example 0.5% by weight. It is also possible to reduce the sugar concentration to 0% or a low concentration close thereto, but in this case, if the culture period is long, for example, more than 10 weeks, plant cells are killed, which is not preferable.

The culture of the cultured cells used in the present invention at a sugar concentration of 2% by weight or less preferably uses Murashige-Skoog's medium, Camborg's B5 medium, Niche &Niche's medium, or a modified medium thereof as a basic medium. be able to. In addition, casein hydrolyzate or coconut milk can be used as a nutrient source. A plant growth regulator is not used, or auxin and cytokinin can be suitably used alone or in combination at a concentration of 3 mM or less, respectively. The culturing period in this step varies depending on the sugar concentration and the like in this step, but is usually preferably 2 days to 10 weeks.

According to the method of the present invention, a culture cultured in a medium having a low sugar concentration is then cultured in a medium having a normal sugar concentration. The sugar concentration in this case may be a concentration exceeding 2% by weight. This concentration is preferably at least 2.5% by weight, for example about 3% by weight.
It is.

Culture of cultured cells in a medium having a sugar concentration of more than 2% by weight of the cultured cells used in the present invention is preferably performed using Murashige-Skoog's medium, Gamborg's B5 medium, Niche &Niche's medium or a modified medium thereof as a basic medium. Can be used. In particular, supplementation with magnesium ions has the positive consequence of accelerating the induction of negative embryos. Casein hydrolysis or coconut milk can also be used as a nutrient source. A plant growth regulator is not used, or auxin and cytokinin can be suitably used singly or in a mixture at a concentration of 3 mM or less, respectively. The cultivation period required for this step is usually from 2 days to 5 weeks, and as this period elapses, the desired adventitious embryo of the plant belonging to the genus Podofilm is induced into the culture.

As the culture conditions that can be suitably used as common culture conditions through the above two steps, a culture temperature of 10 to 30 ° C. and illumination under both bright and dark conditions can be mentioned. Further, sucrose, glucose, and galactose can be used as sugars.

The somatic embryo thus obtained can be used as it is as a material for the next culture step. Alternatively, if the culture is performed on a solid medium, it can be easily harvested using tweezers or the like. If the culture is liquid culture, it can be easily harvested by an operation such as filtration.

By using the following invention, it has become possible to produce an adventitious embryo of a plant of the genus Podofilm that is industrially valuable without being affected by external factors such as the natural environment and difficulty in obtaining it.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to the examples.

Example 1 (Callus formation) 70% of rhizomes of Podofilm pertatum thoroughly washed with water
Sterilized with alcohol for 2 minutes. Next, after sterilizing with 1% sodium hypochlorite for 1 minute, the plate was sufficiently washed with sterilized water. The sterilized rhizome is cut into pieces of about 5 mm in an aseptic atmosphere, and a Murashige Skoog medium (sucrose 3
% By weight), α-naphthalene acetic acid 1 mg / L, kinetin
1% with 0.2mg / L and casein hydrolyzate 500mg / L
When the medium was placed on an agar medium having a high concentration and left still in a dark place for 5 weeks, callus was generated. This callus was subcultured several times.

(Low sugar concentration treatment) Next, this callus was transferred to a 300 ml flask containing 100 ml of a liquid medium in which 500 mg / L of casein hydrolyzate was added to a Murashige-Skoog medium having a sucrose concentration of 0.5%, in a dark place for 5 weeks. Rotary shaking culture was performed at 25 ° C. and a shaking speed of 130 rpm.

(Formation of somatic embryos) Thereafter, the culture was placed in Murashige-Skoog medium having a sucrose concentration of 3% by weight, α-naphthaleneacetic acid 1 mg / L, kinetin 0.0
Liquid medium 1 containing 2 g / L, casein hydrolyzate 500 mg / L
Transfer to a 300 ml flask containing 00 ml and place in dark place for 2 weeks
Rotary shaking culture was performed at 130 ° C. and a shaking speed of 130 rpm. 30% by weight of the cells in the culture flask were somatic embryos.

Comparative Example 1 The same method as in Example 1 was performed. However, a medium having a sucrose concentration of 3% by weight and the same other composition was used as the medium in the low concentration treatment stage. In this case, the callus continued to grow, and a large amount of callus was obtained, but no somatic embryos were obtained.

Comparative Example 2 The same method as in Example 1 was performed. However, as the medium at the stage of adventitious embryo formation, a medium having a sucrose concentration of 0% instead of 3% by weight was used. In this case, not only the adventitious embryo was not formed, but also the callus was killed.

Claims (1)

(57) [Claims] [Claim 1] After culturing cells of a plant of the genus Podophyllum under the condition of a sugar concentration of 2% by weight or less, a sugar concentration of 2% by weight or less is obtained.
A method for preparing a somatic embryo of a plant belonging to the genus Podofilm, wherein the somatic embryo of the plant is induced by culturing the plant in an amount exceeding% by weight.