JPH0755112B2 - Cyclamen seedling regeneration method - Google Patents

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to a method for regenerating a young plant from a cyclamen somatic embryo.

[0002]

2. Description of the Prior Art Embryogenic callus (embryo) obtained by culturing tissue of a cyclamen plant
genetic callus (meaning callus capable of forming somatic embryos) is cultured in a solid medium to form somatic embryos, and then cytokinin and auxin are added thereto, or a medium without addition of these plant hormones is used, A method for regenerating somatic embryos into seedlings is known [Plant
Tissue Culture Letters (Plant Is
suture Culture Letters) Volume 8 (N
o. 2) 121st to 123rd pages (1991), Horticulture Magazine No. 58 Separate Volume 1, 574 (1989), Journal of the Horticultural Society Volume 60, Separate Volume 1, 445-446 (1
991)]. However, these methods have a low regeneration rate of seedlings from somatic embryos and are not necessarily satisfactory as methods for obtaining plants from somatic embryos of cyclamen.

On the other hand, it is also known that plant hormones such as gibberellin are used in the process of culturing a plant from the somatic embryo of medicinal carrot (Japanese Patent Application Laid-Open No. 62-201520). ). However,
There is no known method for culturing Embryogenic callus of a cyclamen plant in a liquid and culturing the same in a medium containing gibberellin as an essential component to form an adventitious embryo, and thereby regenerating a young plant.

[0004]

The present invention is a method for regenerating a cyclamen seedling which has a high regeneration rate of the somatic embryo from an adventitious embryo and is capable of obtaining seedlings having the same trait, instead of the conventional technique. The purpose is to provide.

[0005]

[Means for Solving the Problems] The present inventors have conducted extensive studies in order to achieve these objects. As a result, the embryogenic callus obtained by liquid-culturing cyclamen tissue was used to efficiently form adventitious embryos in a medium containing gibberellin as an essential component, and by regenerating the somatic embryos, somatic embryos were regenerated. We have found a method for regenerating cyclamen seedlings that has a high regeneration rate of seedlings from A.

[0006] Therefore, the gist of the present invention is to carry out a liquid culture of embryogenic callus obtained by culturing cyclamen tissue, in which the callus is a medium containing gibberellin alone or gibberellin and auxin and / or cytokinin. And a step of regenerating a somatic embryo from the somatic embryo after culturing the somatic embryo to produce a somatic embryo.

Next, the method of the present invention will be described.

The cyclamen used in the present invention are plants of the genus Cyclamen, and varieties thereof include Victoria, burger, pure white, pastel, and lylacrose, but are not limited to these varieties.

The embryogenic callus used in the present invention may be one obtained as follows.

First, a plant of cyclamen, such as leaf blades,
Tissue pieces are collected from petioles, anthers, roots, flower stems, etc., and immersed in a 70% ethanol solution for 10 seconds, and then in a sodium hypochlorite solution having an effective chlorine concentration of about 1% for 20 minutes for sterilization treatment. . Then, this tissue is taken out, the sodium hypochlorite adhering to the surface thereof is washed three times with sterilized water, and then cut under aseptic conditions to obtain small pieces. This is Murashige-Skoog medium (hereinafter abbreviated as "MS medium")
Sucrose 50 g / l (liter), 2,4-dichlorophenoxyacetic acid (hereinafter abbreviated as "2,4-D") 4 mg /
1 and kinetin 0.1 mg / l were added to adjust pH
The tissue piece obtained above is placed in a solid medium containing 5.8 g of gellite (3 g / l). When this is cultured at 25 ° C. in the dark, embryogenic callus can be obtained 90 days after being placed.

The medium used in the present invention is, for example, MS
Mediums for culturing plant tissues, such as medium, B5 medium of Gamborg, White medium, Niche-Niche medium, N6 medium and the like can be mentioned. These known medium compositions are described, for example, in "New Plant Tissue Culture" by Takeuchi and Furuya, pages 386 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.

The gibberellins used in the present invention are GA ₁ , G
A ₃ , GA _{7 and the} like are known, and any of these may be used in the present invention, but GA ₃ is particularly suitable.

As auxins, indole acetic acid, α
-Naphthalene acetic acid (hereinafter abbreviated as "NAA"), indole butyric acid, 2,4-D, 3,6-dichloromethoxybenzoic acid, 4-amino-3,5,6-trichloropicolinic acid and the like can be mentioned.

Examples of cytokinins include benzyladenine (hereinafter abbreviated as "BA"), kinetin, zeatin and the like.

Examples of the carbon source for the medium used in the present invention include saccharides such as sucrose and glucose, and sucrose is preferably used.

The pH of the medium used in the present invention is 4.0 to 7.
Adjust to 0, preferably 5.8.

Next, a method for regenerating a young plant from a somatic embryo of cyclamen will be described.

The step of liquid-culturing the embryogenic callus in the present invention is performed as follows.

2% sucrose was added to the medium of inorganic salt composition of MS medium.
0 g / l to 120 g / l, preferably 30 g to 60 g /
1, and added 2,4-D 0.2 mg / l to 10 mg /
1, preferably 1 mg / l, kinetin 0.01 mg
/ ~ 1 mg / l, preferably 0.1 mg / l added,
The pH is adjusted to 5.8, and the medium is prepared by sterilizing with an autoclave.

The embryogenic callus obtained above is transplanted into the liquid medium thus prepared. This one
5 ° C to 30 ° C, preferably 25 ° C in a dark place, rotation speed 50
Embryogenic callus is grown by shaking culture at rpm-200 rpm, preferably 100 rpm. In this case, the embryogenic callus may be subcultured under the same medium and under the same conditions. The embryogenic callus thus cultured and propagated for about 30 days can be used for somatic embryogenesis.

The steps of somatic embryogenesis and seedling regeneration in the present invention are performed as follows.

20 g / l to 120 g of sucrose in MS medium
/ L, preferably 30 g / l was added, and gibberellin was added to 0.1 g / l.
02 mg / l to 2 mg / l, preferably 0.2 mg / l
The medium was prepared by adding. Auxin and cytokinin can be used in combination in this medium.
A is 0.01 mg / l to 10 mg / l, preferably 0.
01 mg / l, BA 0.01 mg / l to 10 mg /
1, preferably 0.1 mg / l is added to adjust the pH to 5.8 and gellite is added in an amount of 1 g / l to 10 g / l, preferably 5
Add g / l and sterilize in an autoclave to prepare a medium.

20 to 1000, preferably 50 to 100, of the embryogenic callus obtained as described above is placed on the medium thus obtained. In this case, the size of the callus is 0.02 mm to 2 mm, preferably 0.25.
It is preferable to use it after sorting it into mm to 1 mm.

Embryogenic callus of such a size, in the dark at 15 ℃ ~ 30 ℃, preferably 25 ℃,
Adventitious embryos can be obtained by culturing for 20 to 60 days after implantation, preferably 30 days after implantation.

These adventitious embryos are further cultivated on the same medium for 20 to 60 days, preferably 30 days after the development of the adventitious embryos, so that the adventitious embryos normally germinate and root to give seedlings. Can be played.

The medium for the step of regenerating a somatic embryo from an adventitious embryo may be a medium containing the above-mentioned plant hormone used for somatic embryo formation, or may be a medium from which the plant hormone is removed.

Agar, agarose, gellite, gellan gum, carrageenan and the like are used as the gelling agent for the solid medium used for the regeneration of the somatic embryo seedling of the present invention.
It is preferable to add 0.1 to 2% of gellite before use.

[0028]

EXAMPLES Examples of the present invention will be described below.

Example 1 Embryogenic callus was obtained by culturing the anther base of cyclamen (variety: Victoria). This callus is about 50m
g in MS medium, sucrose 30 g / l, 2,4-D 1 mg /
Erlenmeyer flask (100 ml containing 30 ml of liquid medium (pH 5.8) supplemented with 0.1 mg / l of kinetin)
(ml volume). This at 25 ℃, in the dark, rotation speed 1
The culture was performed with shaking at 00 rpm. When subcultured with the same medium composition every 14 days after transplantation, about 1 g 60 days after transplantation
The embryogenic callus was obtained.

1 g of this callus was screened with a 1 mm stainless sieve to select callus having a size of 1 mm or less.

0.8 g of the embryogenic callus thus selected was added to MS medium with 30 g / l of sucrose and 5 g / l of gellite, and GA ₃ , NAA, and BA were further added to give a predetermined concentration in a solid medium (pH 5). .8)
100 petri dishes were transplanted into each petri dish (diameter 90 mm). When this was cultured at 25 ° C. in the dark for 60 days, an adventitious embryo was formed 30 days after the transplantation, and 60 days after the transplantation, germination and rooting from the adventitious embryo gave a seedling.

The investigation was carried out 60 days after the transplantation, and the regeneration rate (%) of the seedlings from the somatic embryo was calculated by the following formula.

The results are shown in Table 1.

[Equation 1]

[0034]

Comparative Example Embryogenic callus of cyclamen (variety: Victoria) was liquid-cultured according to Example 1 and 0.8 g of the selected callus was added to MS medium and 30 g of sucrose.
/ L, Gellite 5g / l added, NAA, BA
Solid medium (pH 5.
It was transplanted to a petri dish (diameter 90 mm) containing 8). This was cultured at 25 ° C. in the dark for 60 days.

The results are shown in Table 1.

[0036]

[Table 1]

[0037]

INDUSTRIAL APPLICABILITY According to the present invention, as compared with the conventional method for regenerating seedlings from somatic embryos of cyclamen, the rate of regenerating seedlings from somatic embryos is good and many seedlings can be obtained. Therefore, the method of the present invention can be effectively used for mass production of cyclamen seedlings having the same trait.

Claims (1)

[Claims] 1. A step of liquid-culturing embryogenic callus obtained by culturing cyclamen tissue, the callus being cultured in a medium containing gibberellin alone or gibberellin and auxin or / and cytokinin to form adventitious embryos. And a step of regenerating a young plant from the somatic embryo, the method for regenerating a young plant of cyclamen. [0001]