JPH05260870A - Regeneration of young plant body of cyclamen - Google Patents

Abstract

PURPOSE:To enable the regeneration of the subject young plant body in high efficiency from an adventitious embryo produced by preparing an embryogenic callus by the tissue culture of cyclamen, culturing the callus in a liquid and further culturing in a medium containing gibberellin, auxin, etc. CONSTITUTION:A tissue such as anther base part of cyclamen is cultured to obtain an embryogenic callus. The callus is transplanted to a liquid medium containing sucrose, kinetin, etc., cultured at 25 deg.C in dark place under shaking at a rotational speed of 100rpm and subjected to subculture in the same medium composition every 14 days after transplantation. The cultured callus is sieved with a sieve having a mesh opening of 1mm to collect callus having a size of <=1mm. The collected callus is cultured in a medium containing only gibberellin or gibberellin and auxin and/or cytokinin at 25 deg.C in dark place for 60 days to form an adventitious embryo. The embryo is subjected to sprouting and rooting to regenerate a young plant body of cyclamen in high efficiency.

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 Related Art Up to now, embryogenic callus (embryo) obtained by culturing tissue of a cyclamen plant
genetic callus (meaning callus having somatic embryogenesis) 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 (Plan Tis
suture Culture Letters) Volume 8 (N
o. 2) Pages 121-123 (1991), Horticultural 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 always 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 culture and culturing it in a medium containing gibberellin as an essential component to form an adventitious embryo, and thereby regenerating a young plant.

[0004]

DISCLOSURE OF THE INVENTION 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 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 the plant and can obtain seedlings with the same trait.

Therefore, the gist of the present invention is to carry out a liquid culture of the embryogenic callus obtained by culturing the tissue of cyclamen, the callus containing gibberellin alone or a medium containing gibberellin and auxin or / and cytokinin. And a step of regenerating a somatic embryo from the somatic embryo after culturing the somatic embryo, the method for regenerating a cyclamen young plant body.

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, lylacrose, but are not limited to these varieties.

As the embryogenic callus used in the present invention, those obtained as follows may be used.

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 on a solid medium of 5.8 and 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 culture 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 concentration of the inorganic component may be used as it is or diluted to 1/3 or 1/2, and MS medium is particularly preferable.

Gibberellin used in the present invention is 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, but 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 carried out 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,
Adjust the pH to 5.8 and sterilize by autoclave to prepare the medium.

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 carried out 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
Then, the medium was prepared. 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 by autoclave to prepare a medium.

20 to 1000, preferably 50 to 100, of the embryogenic callus obtained 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 somatic embryos are further cultivated on the same medium for 20 to 60 days, preferably 30 days after the development of the somatic embryo, so that the somatic embryo normally germinates and roots to give a seedling. 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, etc. 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) containing 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-sized stainless steel 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 to which 30 g / l of sucrose and 5 g / l of gellite were added, and GA ₃ , NAA, and BA were added to a predetermined concentration to give a solid medium (pH 5). .8)
100 petri dishes were transplanted to each petri dish (diameter 90 mm). When this was cultured at 25 ° C. in the dark for 60 days, adventitious embryos were formed 30 days after transplantation, and 60 days after transplantation, germination and rooting from adventitious embryos gave seedlings.

The investigation was carried out 60 days after 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 with 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]

EFFECTS OF THE INVENTION According to the present invention, the regeneration rate of seedlings from somatic embryos is higher than that of the conventional method for regenerating seedlings from somatic embryos of cyclamen, 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, wherein the callus is cultured in a medium containing gibberellin alone or gibberellin and auxin or / and cytokinin to form adventitious embryos. And a step of regenerating a plantlet from the somatic embryo. [0001]