JPH0648949B2 - Method of forming somatic embryo of cyclamen - Google Patents

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to a method for forming somatic embryos from cyclamen bud tissue.

[0002]

2. Description of the Related Art Cyclamen has been cultivated so far by collecting seeds, sowing and raising seedlings. In this conventional method, since cyclamen individuals are crossed during the seed production process, important traits such as flower color and flower type are not fixed, and the same strain as the parent strain cannot be stably obtained.

In order to solve such a problem, in recent years,
A tissue culture method using various tissues such as the anther base, leaf axis, cotyledon, and ovule of cyclamen has been proposed (Japanese Patent Publication No.
No. 53895, JP-A-63-226215,
JP-A-2-92221 and JP-A-2-245127.
Japanese Patent Laid-Open No. 3-244328). However, these tissue culture methods usually cause mutations in the process of callus dedifferentiation, and the percentage of adventitious buds that die is high due to the long culture period of adventitious buds.

On the other hand, embryogenic callus obtained by culturing cyclamen tissue
callus; which means a callus having the ability to form somatic embryos) is known to form somatic embryos. As an example, for example, a method of using leaf pieces of an adult leaf [(Plant Tissue Culture Letters)
(Plan Tissue Cult. Lett.)
Volume 8 (No. 2) Page 121-Page 123 199
1 year, (Horticulture magazine No. 58, Supplement 1, page 574 19
1989)] and a method using a root piece (Horticultural Society magazine, Volume 60, Supplement 1, 445 to 446, 1991). These methods use specific varieties, leaves, and specific tissue pieces of roots as test materials, and the number of somatic embryos formed is small.

[0005]

Therefore, in place of the conventional technique, there is a demand for a method of forming an adventitious embryo which has a high adventitious embryo formation rate and is capable of obtaining seedlings having the same trait.

The present invention is to provide a method for forming a somatic embryo of cyclamen that meets these needs.

[0007]

[Means for Solving the Problems] The present inventors have conducted extensive studies in order to achieve these objects. As a result, we found a method of forming somatic embryos of cyclamen that has a high rate of adventitious embryo formation by forming somatic embryos from embryogenic callus formed from cyclamen bud tissue and that can give seedlings of the same trait. It was We have also found that this method can be applied to various varieties of cyclamen.

Therefore, the gist of the present invention is that cyclamen bud tissue is cultured to obtain an embryogenic callus, and the callus is then cultured to form an adventitious embryo. It is in the method of forming somatic embryos.

Next, the method of the present invention will be described. The cyclamen used in the present invention are plants of the genus Cyclamen, and examples of varieties include Victoria, burger, pure white, pastel, and mini cyclamen railac rose, but are not limited to these varieties.

The bud of cyclamen used in the present invention has a diameter of 4 mm.
A bud having a length of -40 mm is sufficient, and is not limited by the maturity of the tissue in the bud or the growth stage.

Examples of the bud tissue used in the present invention include anthers, anther bases, styles, ovary walls, ovaries and the like, with the anther bases being preferred.

The cyclamen bud tissue may be sterilized only on the surface of the bud, but it is preferable to further sterilize the inside of the bud. To sterilize only the buds, the buds are in an ethanol solution of about 70% -80% for 10 seconds, then in a 0.2% -5% solution of sodium hypochlorite, preferably a 1% solution for 2-3 minutes.
Immersion may be performed for 0 minutes, preferably 10 minutes. Aseptically peel the buds that have been sterilized in this way to cut out the petals, and further remove approximately 7 parts of the exposed bud tissue.
After dipping in a 0% to 80% ethanol solution for 10 seconds, and then in a sodium hypochlorite solution of about 0.2% to 5%, preferably 0.5%, for 2 minutes to 30 minutes, preferably 10 minutes, It may be washed with sterile water.

The medium used for forming the somatic embryo of the present invention includes, for example, Murashige-Skoog medium (hereinafter referred to as "MS medium").
Abbreviated), Gumborg's B5 medium, white medium, niche-niche medium, N6 medium and the like for plant tissue culture. Regarding these known medium compositions, for example, Takeuchi, Nakajima, and Furuya “New Plant Tissue Culture” No. 38.
6 to 391 (1979, published by Asakura Shoten). As these media, the concentrations of the inorganic components may be used as they are or diluted to ⅓ or ½, and MS medium is particularly preferable.

[0014] Plant hormones for use in the present invention, as auxin, indoleacetic acid, alpha-naphthalene acetic acid, indole butyric acid, (hereinafter referred to as "2,4-D") 2,4-dichlorophenoxyacetic acid, 3,6 Dichloromethoxybenzoic acid, 4-amino-3,5,6-trichloropicolinic acid and the like can be mentioned. In addition, examples of cytokinins include benzyladenine, kinetin, zeatin, and the like.

As these plant hormones, auxins and cytokinins may be used in combination, or auxins may be used alone, but preferably 2,4-D of 0.2 mg /
1 to 10.0 mg / l and kinetin 0.01 mg / l
˜1.0 mg / l, more preferably 2,4-D 1.
It is preferable to add 0 to 4.0 mg / l and kinetin at 0.1 mg / l before use.

Examples of the carbon source of the medium used in the method of the present invention include sugars such as sucrose and glucose. Sucrose is 20 g / l to 120 g / l, preferably 30 g / l.
It is preferable to use it at a concentration of -60 g / l.

The pH of the medium is adjusted to 4.0 to 7.0, preferably 5.8 before use.

The medium used in the method of the present invention may be solid or liquid, but solid medium is preferred. Agar, agarose, gellite, gellan gum, carrageenan, etc. are used as the gelling agent.
It is better to use it after adding%.

Next, a method for forming a cyclamen somatic embryo using MS medium will be described.

First, 50 g / l of sucrose and 1.0 to 4.0 mg / l of 2,4-D were added to a medium having an inorganic salt composition of MS medium.
Kinetin was added at 0.1 mg / l to adjust the pH to 5.8,
Gelrite (2 g / l) was added and sterilized by an autoclave to prepare a medium.

A piece of bud tissue is placed on the medium thus prepared. The sterilized bud tissue may be cut out either from the anther base only or from the whole anther.

The cyclamen somatic embryos of the present invention are preferably cultured in the dark. In that case, the culture temperature is
15 ° C to 30 ° C, preferably 25 ° C.

[0023]

[Test Example] Next, an example of the present invention will be described. Example 1 A cyclamen anther structure was obtained as follows. That is,
10.0 from the flowering part of cyclamen (variety: Victoria)
Pick a bud with a length of mm to 20.0 mm,
% Ethanol solution for 10 seconds and then a 1% sodium hypochlorite solution for 10 seconds. Aseptically remove the sepals and petals from this bud, and soak the barely exposed bud tissue in a 70% ethanol solution for 10 seconds, and then in a 0.5% sodium hypochlorite solution for 10 minutes, It was washed with sterile water. Anther tissue including anther and anther base was cut out from this bud tissue and used as a test material.

The tissue piece thus obtained was sucrose 5
%, Gellite 0.2% and the concentration of 2, shown in Table 1,
MS medium containing 4-D and kinetin (pH 5.8)
Was placed in a petri dish (diameter 90 mm) and placed on this.

Then, subculture was carried out at a culture temperature of 25 ° C. in the dark every 30 days after plating with the same medium composition as above.
Callus was formed after 30 days of culture, and yellow embryogenic callus was selected from this. After 90 days of culturing, a yellowish white granular somatic embryo of about 0.5 mm was formed on the surface of the callus.

The number of embryogenic callus formed after 30 days of culture and the total number of adventitious embryos formed after 90 days of culture were examined, and the number of adventitious embryos per piece of anther tissue and the callus formation rate (% ) Was asked. The result is
The results are shown in Table 1.

[0027]

[Equation 1]

Comparative Example 1 Cyclamen leaf pieces were prepared by immersing a mature leaf of a flowering strain of cyclamen (variety: Victoria) in a 70% ethanol solution for 10 seconds and then in a 1% sodium hypochlorite solution for 20 minutes, followed by sterilized water. Washed with. This adult leaf was cut into about 1 cm square and used as a test material. The operation thereafter is the first embodiment
It was carried out according to. The results are shown in Table 1.

Comparative Example 2 A cyclamen root piece was obtained as follows. That is, cyclamen (variety: Victoria) seeds were immersed in a 70% ethanol solution for 10 seconds, then in a 1% sodium hypochlorite solution for 20 minutes, and then washed with sterile water. The seeds were placed in a medium (pH 5.8) containing 3% sucrose and 0.5% gellite in an MS medium with an inorganic salt concentration of 1/2, and after 90 days of culture, the roots of the seedlings were about 5 mm. Cut out into
This was used as the test material. The following operations were performed according to Example 1. The results are shown in Table 1.

[0030]

[Table 1] Note 1) EC is an abbreviation for embryogenic callus.

Example 2 As a variety of cyclamen, 10.0 mm to 20.0 mm buds of each flowering strain of pastel, mini cyclamen lylacrose, pure white and burger were used, sterilized according to Example 1 and cultured. . The medium is MS solid medium (pH: 5%, gellite 0.2%, 2,4-D 1.0 mg / l, kinetin 0.1 mg / l (pH
5.8) was used.

The investigation was carried out according to Example 1, and the number of embryogenic callus formed from anther tissue and the total number of adventitious embryos were examined, and the number of adventitious embryos per piece of anther tissue and according to Example 1 were examined. Then, the callus formation rate was obtained. The results are shown in Table 2.

[0033]

[Table 2] Note 1) EC is an abbreviation for embryogenic callus.

[0034]

EFFECTS OF THE INVENTION According to the present invention, somatic embryos can be efficiently formed from the anther tissue of cyclamen, and the number of somatic embryos is remarkably large, as compared with the conventional method for forming somatic embryos using leaf pieces or root pieces.

Therefore, the method of the present invention can be effectively used for mass production of seedlings of cyclamen having the same trait.

Claims (1)

[Claims] 1. A method for forming a somatic embryo of cyclamen, which comprises culturing a cyclamen bud tissue to obtain an embryogenic callus, and then culturing the callus to form an somatic embryo. [0001]