JPH07155081A - Production of seed and seedling of dwarfed eustoma resselianum g. don - Google Patents

Abstract

PURPOSE:To provide a method for efficiently producing a seed and a seedling of dwarfed Eustoma russelianum G. Don having the same character by using an adventitious embryo. CONSTITUTION:This method for producing a seed and a seedling of dwarfed Eustoma russelianum G. Don is to carry out a callus-forming step (A) for infecting the Eustoma russelianum G. Don with Agrobacterium rhizogenes holding an Ri plasmid or a microorganism, derived therefrom and having a recombinant vector in which a DNA fragment having genetic information about the dwarfing of the Eustoma russelianum G. Don is integrated, culturing the resultant tissue fragment of a hairy root in a solid culture medium containing a phytohormone in the dark and forming a callus and a plant body-forming step (B) for culturing the resultant callus in a solid culture medium, containing 0.5-1.5% gelling agent and free of the phytohormone or containing naphthaleneacetic acid (NAA) as the phytohormone and benzyladenine or kinetin, forming an adventitious embryo and regenerating a plant body from the adventitious embryo.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing dwarfed Turkish ginkgo seedlings, which has an Agrobacterium rhizogene carrying a Ri plasmid.
s; hereinafter also referred to as "AR bacterium") or Turkey infected with a microorganism which is derived from this microorganism and has a recombinant vector into which a DNA fragment having the gene information involved in dwarfing of Turkish ginkgo is incorporated. The present invention relates to a method for producing dwarfed Turkish ginkgo seedlings from Gigyo.

[0002]

BACKGROUND OF THE INVENTION To obtain a large number of plants having a single genetic trait in a short period of time by culturing cells or tissue pieces of one plant and reproducing / growing seedlings of the plant from this. Since it is possible to do so, it is advantageous not only for breed improvement but also for stable market supply of seeds and seedlings. As a method for obtaining seedlings from cell culture of such a plant, plant cells obtained from the plant, forming adventitious buds from tissue pieces, a method of producing a regenerated plant from the adventitious buds, and plant cells, A method is known in which an adventitious embryo is obtained from a tissue piece via an embryogenic callus and a regenerated plant is produced from this adventitious embryo.

[0003] Conventionally, in the case of Turkey ginkgo, the former method has been adopted to reproduce and produce seedlings of plants from cells or tissue pieces.
A method of obtaining seedlings by culturing tissue pieces such as stems and roots in a medium containing benzyladenine to form adventitious buds, and rooting the adventitious buds in a plant hormone-free medium [plant tissue culture, Volume 5 Pages 96-97 (1987): Plant Celtish and Organ Culture (Pl
ant Cell Tissue and Organ Culture), Vol. 8, No. 24
Pp. 9-253 (1987)], AR bacterium carrying Ri plasmid or a DNA fragment derived from this bacterium and having dwarfing genetic information of Turkish ginkgo is incorporated into a vector, and the obtained recombinant vector is used as a microorganism. And then infecting the turkish gully plant, the plant cells or tissue pieces obtained from this plant are cultured, and the dwarfed turkish gulberry (dwarfed Method for producing seedlings (Turkey)
192156) is known.

Further, a method in which hairy roots obtained by infecting a plant with AR fungus are cultured in a plant hormone-free medium to obtain adventitious buds, and the adventitious buds are cultivated to obtain regenerated plants. The gardening July issue, separate volume, Bioholty, pages 21 to 23 (1991), published by Seibundo Shinkosha) is known.

However, heretofore, a method for efficiently producing seedlings by the latter method of obtaining an adventitious embryo from a plant tissue of Turkish Gourd and regenerating a plant from this adventitious embryo has not been known.

In other plants, application examples of a method for producing seedlings of a plant using an adventitious embryo are known. For example,
Stem of wasabi radish stems and tissue pieces cut from leaves were sterilized and placed on MS solid medium containing benzyladenine and 2,4-dichlorophenoxyacetic acid (hereinafter referred to as "2,3-PA") to induce callus, The obtained callus was treated with benzyladenine and 2,
After the cells are released in MS liquid medium containing 4-PA to give suspended cells, the suspended cells are filtered and sorted to a uniform particle size, and then the sorted plant cells are cultured in a plant hormone-free MS liquid medium. To obtain a uniform somatic embryo, and a method for regenerating a plant from this somatic embryo (Japanese Patent Laid-Open No. 2-182124)
Are known.

[0007] Also, explants of asparagus plants are cultured in a liquid medium containing auxin and cytokinin under light irradiation to form callus, which is then cultured in a liquid medium free of plant hormones to give an adventitious embryo. After forming the somatic embryo, the adventitious embryo thus obtained is suspended and dispersed by adding 10% by weight or less of a gelling agent to a liquid medium, which is released onto a solid medium and cultured to obtain a plant body. Method (JP-A-4-22
No. 8016) is also known.

However, a method for obtaining a regenerated plant body from cells or tissue pieces of such another plant body through an adventitious embryo is such that the adventitious embryo is directly applied to the production of seedlings of dwarfed Turkish ginkgo. Therefore, there was a problem that the plant body was not regenerated at all or the regeneration rate was extremely low, which was not practical.

[0009]

SUMMARY OF THE INVENTION The present invention is intended to solve the above problems of the prior art and is derived from an AR bacterium carrying a Ri plasmid or the microorganism and is involved in the dwarfing of Turkish ginkgo. Efficient dwarf turkish seedlings with the same trait by regenerating plants from adventitious turkeys infected with a microorganism carrying a recombinant vector incorporating a DNA fragment containing The purpose is to provide a method for mass production.

[0010]

SUMMARY OF THE INVENTION The method for producing dwarfed Turkish ginkgo seedlings according to the present invention comprises an AR bacterium carrying a Ri plasmid or a DNA fragment derived from the microorganism and having genetic information involved in dwarfing of Turkish dwarf. Hairy roots were collected from Turkish ginkgo that was infected with a microorganism carrying the recombinant vector, and the obtained hairy root tissue pieces were cultured in the dark on a solid medium containing plant hormones. Callus formation step (A) in which callus is formed, and the callus contains 0.5 to 1.5% gelling agent and is free of plant hormones, or naphthalene acetic acid as a plant hormone, and benzyl. A plant regenerating step (B) of culturing under light irradiation on a solid medium containing adenine or kinetin to form an adventitious embryo, and regenerating a plant from the adventitious embryo. The features.

According to the method for producing dwarf turkish gypsum seedlings according to the present invention, a hairy root tissue piece obtained from a plant infected with the above-mentioned AR fungus is darkened in a solid medium containing a plant hormone. Cultivated under to form callus, which is then containing 0.5-1.5% by weight of gelling agent and is plant hormone free or naphthalene acetic acid as plant hormone and benzyladenine or Since a plant body is regenerated (step B) from an adventitious embryo cultured under light irradiation in a solid medium containing kinetin, a plant body having a uniform trait can be efficiently obtained.

[0012]

DETAILED DESCRIPTION OF THE INVENTION The method for producing a seedling of dwarf Turkish ginger according to the present invention will be described in more detail below.

The method for producing a dwarfed turkish seed and seedling according to the present invention is a tulip tufted hair obtained by infecting an AR bacterium carrying a Ri plasmid or a microorganism carrying a specific recombinant vector derived therefrom. A tissue piece is obtained from the roots, and then a callus formation step (A) of forming a callus from the tissue piece under specific conditions, and the obtained callus is cultured on a specific fixed medium under specific conditions. A plant regenerating step (B) of regenerating a plant from the formed somatic embryo.

The steps (A) and (B) will be described in detail below. Callus forming step (A ) In the callus forming step (A ) of the present invention, hairy roots obtained by infecting Turkish cypress with an AR bacterium carrying the Ri plasmid or a microorganism carrying the specific recombinant vector derived therefrom are used. Used as a raw material, callus is formed from the hairy root cells or tissue section under specific conditions.

Turkey is Eustoma
) Genus plant, in the present invention, Eustoma (Eustoma
) The raw material can be prepared using any variety belonging to the genus.

[0016] These AR bacteria to infect Turkey must be those possessing the Ri plasmid. Specifically, for example, NIAES1724 strain (MAFF03-01724 strain stored by the Institute for Biological Resources, Ministry of Agriculture, Forestry and Fisheries), NI.
AES1725 strain, C8 strain, D6 strain, H4 strain, Y1 strain,
A5 strain, A13 strain, A4 strain, ATCC15834 strain and the like can be exemplified, and a set derived from an AR bacterium carrying a Ri plasmid and incorporating a DNA fragment having genetic information involved in dwarfing of Turkish ginkgo It may be a microorganism carrying a replacement vector. Examples of such transformed microorganisms include E. coli.

Further, in order to obtain such turkish pilus-like roots, for example, AR bacteria such as Agrobacterium.
A method based on the method described in Biohorti Vol. 7, pp. 24 to 25, using Rhizogenes NIAES1724 strain can be adopted. That is, specifically, the hairy root tissue piece used as a raw material in the present invention can be prepared by the following method.

First, after sterilizing the leaf blades of Turkish ginkgo biloba, AR pieces are inoculated into the leaf pieces by immersing the tissue pieces cut from the leaf blades in the AR bacterium suspension. The AR bacterium suspension is prepared by culturing AR bacterium in a known medium under predetermined conditions. Then, the obtained tissue piece was placed on an MS solid medium containing no plant hormone and cultured for a predetermined period,
When transplanted to MS solid medium containing antibiotics and further cultured for several weeks, hairy roots develop from the cut surface of the leaf piece.

The hairy roots thus generated are excised and collected, and then cultivated in MS solid medium containing antibiotics for a few more weeks to eliminate AR bacterium. The hairy root from which the AR bacterium has been sterilized is cut again so that its tip is included, and if desired, it is further cultivated in a MS solid medium for a predetermined period of time to be elongated.

In the callus forming step (A) of the present invention, the hairy root tissue pieces thus obtained are cultured in the dark on a solid medium containing plant hormones to form callus.

Such a solid medium can be prepared by adding a carbon source and a plant hormone to a liquid medium, adjusting the pH, and further adding a gelling agent to solidify.

The liquid medium used to prepare the solid culture is
It is an aqueous solution containing inorganic salts containing elements such as nitrogen, phosphorus, potassium, magnesium, calcium, manganese, copper, zinc, boron, molybdenum and iron necessary for growing plants, and has been widely used for culturing plant bodies. Any of those used may be used. Specifically, for example, Murashige-Skoog medium (hereinafter referred to as "MS medium"), Gamborg's B5
Medium, white medium, niche-niche medium, N6 medium and the like can be mentioned. These media may be used as they are, or may be diluted with water to ⅓ to ½. Of these, MS medium is particularly preferable.

Examples of the carbon source added to the medium include sugars such as sucrose and glucose, of which sucrose is preferred. The concentration of these carbon sources is appropriately selected depending on the type of sugar and the like, but is, for example, 10 g / liter to 60
It is desirable to use it at a concentration of g / liter, preferably 30 g / liter.

The plant hormones added to the medium include indole-3-acetic acid (IAA), 4-chloroindoleacetic acid (4-CI-IAA) and 2,4-dichlorophenoxyacetic acid (2,4-PA). , 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T), naphthaleneacetic acid (NAA), indoleacetic acid (IB-A) and 2,3,6-trichlorobenzoic acid (2,
3,6-T) and other auxins; if desired, cytokinins such as zeatin, 2iP, ribosylzeatin, kinetin, benzyladenine (BA), 8-azakinetin, and diphenylurea can be used in combination.

In the present invention, such a plant hormone is
The callus is appropriately selected and used in combination, and of these, 2,4-PA is preferably used as the auxin and is preferably used in combination with BA or kinetin.

The addition amount of the plant hormone is selected according to the plant hormone and the kind thereof. For example, when 2,4-PA and BA or kinetin are used in combination, 4-PA is 0.1 mg / liter to 1
0 mg / liter, preferably 0.5 mg / liter to 4 mg
/ L, more preferably 1 mg / L, preferably BA or kinetin,
It is desirable to use each in a concentration of 0.01 mg / liter to 1 mg / liter, preferably 0.05 mg / liter to 0.5 mg / liter, and more preferably 0.1 mg / liter.

As the gelling agent for solidifying the medium, agar, agarose, gellite (gellan gum)
And the like, and particularly gellite is preferable.
The amount of such a gelling agent used is appropriately selected according to the type of the gelling agent, and is used, for example, in an amount of 0.1 to 10 g / liter, preferably 2 to 5 g / liter, and particularly preferably 3 g / liter. Is desirable.

The solid medium is prepared by adding the above carbon source and plant hormones to a liquid medium, adjusting the pH to a desired pH, specifically pH 4 to 7, and then adding a gelling agent to solidify the medium. It is prepared by

The solid medium thus prepared is preferably sterilized by an autoclave or the like before use.
If desired, various vitamins, amino acids, and other natural products may be added to the solid medium.

In the callus-forming step (A) of the present invention, a tissue piece obtained from the hairy root is placed on such a solid medium and cultured in the dark, whereby the callus is removed from the hairy root. To form.

The term "callus" as used herein means an embryogenic callus having a somatic embryogenesis ability in which the cells are relatively small and the cytoplasm is clogged. Cultivation of such hairy root tissue pieces is usually performed at 15 ° C to 3 ° C.
0 ° C, preferably 20-28 ° C, more preferably 25
At a temperature of ℃, usually 20 to 90 days, preferably 30 to 6
It will be held for 0 days.

Plant Regeneration Step (B) In the plant regeneration step (B) of the present invention, the callus obtained in the callus formation step (A) is treated on a specific fixed medium.
The somatic embryo is formed by culturing under light irradiation, and then the plant body is regenerated from the somatic embryo.

The solid medium used in the plant regeneration step (B) is free of plant hormones, or contains NAA in combination with benzyladenine or kinetin as plant hormones, and a specific amount of gelling. Contains agents.

When NAA is used as a plant hormone in combination with BA or kinetin, NAA is used.
Is used in an amount of 0.005 mg / liter to 0.1 mg / liter, preferably 0.01 mg / liter to 0.05 mg / liter, and more preferably 0.01 mg / liter. At this time, BA is 0.0
1 mg / liter to 1 mg / liter, preferably 0.1 mg
The amount of kinetin is 0.01 mg / liter to 0.5 mg / liter, more preferably 0.1 mg / liter.
/ Liter to 1 mg / liter, preferably 0.1 mg / liter to 0.5 mg / liter, more preferably 0.1
It is preferably used in an amount of mg / l.

Specific examples of the gelling agent include starch, cellulose, starch / acrylic acid copolymer, cellulose / acrylic acid copolymer, cellulose / acrylic acid copolymer, polyacrylic acid crosslinked product, agar, Examples include agarose.

In the present invention, such a gelling agent is added to the solid medium in an amount of 5 to 15 g / liter (0.5 to 1.5%), preferably 8 g / liter to 12 g / liter (0.8 to
1.2%), more preferably 10 g / liter (1
%).

In such a solid medium, a carbon source and, if desired, a plant hormone in the above combination are added to a liquid medium,
Desired pH, specifically pH 4-7, preferably pH
It is prepared by adjusting the pH to 5.6 to 6, more preferably pH 5.8, and then adding a gelling agent to the above-mentioned predetermined amount to solidify.

As the carbon source and the liquid medium, the same carbon source and the same liquid medium as in the above callus forming step (A) can be used in the same amounts. The solid medium thus prepared is preferably sterilized by an autoclave or the like before use. If desired, various vitamins, amino acids, and other natural products may be added to the solid medium.

On the solid medium thus prepared,
The callus obtained in the callus forming step (A) is placed. Such callus is placed on a solid medium by dividing the callus into callus blocks having a desired diameter, usually 0.5 to 1 cm in diameter, or using a mesh to obtain a desired size, usually a diameter of 0. It can be carried out by selecting callus having 1 to 1 mm, preferably 0.25 to 0.5 mm, and placing this on a solid medium.

In the plant regeneration step (B), the callus placed on the solid medium is cultured under light irradiation. Light irradiation,
5 to 20 hours a day, preferably 16 hours, 100
It is desirable to carry out at 0 to 10000 lux, preferably 4000 lux.

When cultured for 60 days under such light irradiation conditions, adventitious embryos are formed from the callus, and then normal germination and rooting occur from the adventitious embryos to obtain plants.

The regenerated seedlings thus obtained are
The seeds may be supplied as they are, but if desired, they may be further grown in the plant growing step (C). In this plant growing step (C), specifically, a plant suitable for supplying seedlings by transplanting seedlings into a pot filled with soil, cultivating in a greenhouse to grow and acclimate Raise your body.

The Turkish ginkgo seedlings obtained by the above-described method for producing dwarfed Turkish gyogi seedlings according to the present invention lead to flowering when grown in soil, but the Ri plasmid possessed by Agrobacterium rhizogenes Since the gene involved in the above dwarfing has been introduced, features such as shortened plant height, clogged internodes, and increased number of branches are exhibited.

[0044]

EFFECTS OF THE INVENTION According to the method for producing dwarfed Turkish ginkgo seedlings according to the present invention, the hairy root tissue pieces obtained from the plant infected with the AR bacterium are treated with a solid medium containing a plant hormone. Culturing in the dark to form callus, which is then containing 0.5-1.5% by weight of gelling agent,
The plant body is regenerated from an adventitious embryo cultured under light irradiation in a solid medium that is free of plant hormones or contains NAA as a plant hormone and benzyladenine or kinetin. It is possible to efficiently obtain the plants that the plant has.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0046]

[Example 1] (A) Callus formation step Seeds of Turkish ginkgo (variety: purple peak) were sown and cultivated in a greenhouse. A tissue piece was cut from the leaf blade of the obtained Turkish ginkgo plant, and the obtained tissue piece was immersed in an ethanol solution of about 70 to 80% for 10 seconds, and then immersed in a solution of about 1% sodium hypochlorite for 20 minutes. 1 more after sterilization
The section was cm square.

The slices of the leaf blades were taken from L medium (polypeptone 1%, yeast extract 0.5%, sodium chloride 0.5%,
The AR bacterium was inoculated by immersing it in the AR bacterium suspension obtained by culturing at 28 ° C. for about 12 hours in glucose (0.1%).

The slice ingested with AR bacterium was placed on MS solid medium containing no plant hormone [solid medium containing inorganic salt of a predetermined concentration of MS solid medium, sucrose 30 g / liter and gellite 3 g / liter]. After culturing for 3 days, the cells were transplanted to an MS solid medium supplemented with the antibiotic carbenicillin (500 mg / liter) and cultured for 4 to 6 weeks,
Hairy roots were generated from the cut surface of the leaf blade section.

The hairy roots obtained were cut off, and carbenicillin (500 mg / liter) was added to the solidified MS medium to give 4 cells.
AR culture was eradicated by culturing for a week. Then, the hairy root was cut so as to include the root tip, and further cut with MS solid medium.
The culture was carried out for 0 days to elongate the hairy roots.

These hairy roots were cut into 1 cm-long fragments using a scalpel under sterile conditions, and prepared in a petri dish having a diameter of 90 mm to prepare a callus-forming solid medium (an inorganic salt of a predetermined concentration in the MS solid medium, sucrose). 30 g / liter, gellite 3 g / liter, 2,4-PA 1 mg / liter and BA 0.1
Solid medium containing mg / l: pH 5.8), 1
Five pieces were placed on each petri dish.

Hairy root tissue pieces placed on a solid medium for callus formation were cultured at 25 ° C. in the dark for 60 days (culture start 3
On day 0, the solid medium was changed to a new solid medium having the same composition and subcultured, whereby yellow embryogenic callus having somatic embryo formation ability was formed.

Table 1 shows the callus formation rate (%) determined according to the following equation.

[0053]

[Equation 1]

(B) Plant Regeneration Step 1 g of the callus obtained in the step A was passed through a stainless steel sieve having a hole diameter of 0.5 mm to separate callus lumps having a diameter of 0.5 mm or less.

0.5 g of the callus thus obtained was added to 100 ml of a liquid medium containing 30 g / l of sucrose and an inorganic salt having a predetermined concentration in MS medium to obtain a suspension. Predetermined concentration of inorganic salt in MS medium, sucrose 30 g / liter, gellite 10 g
Solid medium for plant regeneration (pH 5.
8), sucrose 30 g / liter, gellite 10 g /
In addition to liters, NAA and BA at the concentrations shown in Table 1
2 kinds of solid medium for regenerating plant (pH 5.8), 30 g / l of sucrose, 10 g / l of gellite, and solid medium for regenerating plant containing NAA and kinetin at the concentrations shown in Table 1. Two kinds (pH 5.8) were prepared in a petri dish (90 mm in diameter), and the above-mentioned callus suspension was placed on each of these 5 kinds of solid medium for plant regeneration, 1 ml per petri dish.

The placed callus was cultured for 30 days under the conditions of a temperature of 25 ° C., a light period (4000 lux) of 16 hours per day, and a dark period of 8 hours for 30 days. Adventitious embryos were formed from the callus in each solid medium. After further culturing for 30 days, seedlings germinated and rooted from the somatic embryo were obtained.

The plant regeneration rate (%) determined by the following formula is shown in Table 1.

[0058]

[Equation 2]

(C) Seedling growing step 5 seedlings obtained from 5 kinds of solid medium for regeneration in step B
A plastic pot of 500 ml capacity (diameter 6c) filled with 100 ml of vermiculite.
m) transplanted.

This was acclimated for 10 days in a acclimation room kept at a temperature of 25 ° C. and a humidity of 80% to obtain seedlings. The seedlings obtained in the step C were transplanted to a 1 liter capacity plastic pot (diameter 15 cm) filled with soil, cultivated in a greenhouse for 4 months, allowed to flower, and the plant height of 50 plants was measured. The average value was calculated.

The results obtained are shown in Table 1.

[0062]

[Examples 2 to 5] Callus formation, seedling regeneration and seedling growth were carried out in the same manner as in Example 1 except that the turkish ginkgo was changed to the variety shown in Table 1, and seedlings were obtained. Similarly, these seedlings were raised, flowered, and the plant height was measured (Example 2: Azuma cherry tree, Example 3:
Azuma snow, Example 4: Azuma galaxy, Example 5: Azuma wave).

The obtained results are shown in Table 1.

[0064]

[Comparative Example 1] In the plant regeneration step (B), 24 per day
Example 1 except that the plants were regenerated in the dark period of time.
The plant regeneration rate was determined in the same manner as in.

The results obtained are shown in Table 1.

[0066]

[Comparative Example 2] Same as Example except that in the plant regeneration step (B), 5 types of solid medium for plant regeneration were prepared by changing the gellite concentration to 3 g / liter (0.3%). Then, the plant regeneration rate was determined.

The results obtained are shown in Table 1.

[0068]

[Comparative Example 3] 30 g / sucrose in the plant regeneration step (B)
Liter, gellite 10 g / liter, and NAA, BA or kinetin at the concentrations shown in Table 1 as plant hormones alone were added to the solid medium for plant regeneration (pH
5.8) was prepared in a petri dish (diameter 90 mm) and these 4 types (NAA: 2 types, BA: 1 type, kinetin: 1) were prepared.
The plant regeneration rate was determined in the same manner as in Example 1 except that 1 ml of the above-mentioned callus suspension was placed on each solid medium for plant regeneration of (Seed).

The results obtained are shown in Table 1.

[0070]

[Comparative Example 4] Seeds of 5 kinds (purple peak, Azuma cherry tree, Azuma snow, Azuma galaxy, Azuma wave) were placed in a 1 liter plastic pot (15 cm in diameter) filled with soil. The seeds were sown, cultivated in a greenhouse for about 7 months, flowered, and the plant height was measured.

The results obtained are shown in Table 1. The dwarfing rates of Examples 1 to 5 shown in Table 1 were calculated based on the plant height of the plants of each variety obtained here.

[0072]

[Table 1]

[0073]

[Table 2]

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Claims (5)

[Claims] 1. Infection with Agrobacterium rhizogenes harboring a Ri plasmid or a microorganism harboring a recombinant vector derived from the microorganism and having a DNA fragment having a genetic information relating to dwarfing of Turkish ginkgo is incorporated therein. Callus formation step (A) in which hairy roots are collected from the turkeys that have been allowed to grow, and the obtained hairy root tissue pieces are cultured in the dark on a solid medium containing plant hormones to form callus. , And said callus on a solid medium containing 0.5-1.5% gelling agent and free of plant hormones or containing naphthalene acetic acid as a plant hormone and benzyladenine or kinetin. A dwarf turkish which comprises a plant regenerating step (B) of culturing under light irradiation to form an adventitious embryo and regenerating a young plant from the adventitious embryo. Today's seedling production method. 2. The seedling growing step (C) for growing and acclimatizing the seedlings obtained in the plant regeneration step (B) to form seedlings is included. Of dwarfing Turkish ginkgo seedlings. 3. The solid medium used in the callus-forming step (A), wherein the plant hormone is 0.1 mg / liter to 10 mg / liter of 2,4-dichlorophenoxyacetic acid,
The method for producing a seedling of a dwarf turkish ginger according to claim 1 or 2, which comprises 0.01 mg / liter to 1 mg / liter of benzyladenine or kinetin. 4. The solid medium used in the plant regeneration step (B) contains 0.005 mg / liter to 0.1 mg / liter of naphthalene acetic acid as a plant hormone and 0.0.
4. 1 to 1 mg / liter of benzyladenine or kinetin are contained.
9. The method for producing a dwarf turkish seedling according to claim 1. 5. In the plant regenerating step (B), the light irradiation is performed for 5 hours to 20 hours per day, and 1000 to
The method for producing a dwarf turkish seedling according to any one of claims 1 to 4, wherein the method is performed at 10,000 lux.