JPH07123879A - Method for regenerating plant body by forming embryoid of bent grass - Google Patents

Abstract

PURPOSE:To regenerate a plant body of bent grass according to the formation of an embryoid by inducing an E callus from an explant of the bent grass, proliferating only the E callus in a liquid proliferating culture medium, inducing an embryoid in a liquid or a solid culture medium and providing the plant body. CONSTITUTION:This method for regenerating a plant body is to bed an explant of bent grass (a full ripe seed, an unripe embryo, a panicle, etc.) on a callus- inducing culture medium, inducing the callus, sorting out an opaque and compact E callus having the redifferentiating and the embryogenetic abilities, proliferating only the E callus in a liquid proliferating culture medium, culturing the proliferated callus in a liquid or a solid embryoid-inducing culture medium, germinating or rooting the formed embryoid in a liquid or a solid seedling formation culture medium and forming the plant body.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for regenerating a plant by forming an embryoid body of bentgrass, and more particularly to a method for regenerating an embryoid body of bentgrass using a plant tissue culture technique.

[0002]

2. Description of the Related Art Bentgrass is mainly distributed in the market as seeds, and seeds are produced in Europe and the United States where a vast collection field can be secured. In Japan, such seeds are imported and seeds are sown at golf courses or the like to obtain turf.

Since bentgrass is used in a wide area such as a golf course, a huge amount of seeds are required. Therefore, in order to produce seeds, not only a vast field is required, but also a large labor force is required. In view of such circumstances, there is a strong demand for a method for producing a large amount of embryoid bodies in place of seeds in a short period of time by utilizing biotechnology which has made remarkable progress in recent years. The prior art relating to the tissue culture technology of bentgrass is shown below.

A report on plant regeneration from callus of bentgrass is described in J. V. Krans et al. (J. V. Kra
ns, V.N. T. Henning, K .; C. Torr
es, Crop Sci. , Vol 22 p119
3-1197 (1982)), C.I. Blanche et al. (C. Blanche, JV Krans, G. et al.
E. Coats, Crop Sci. , Vol 26
p1245-1248 (1986)) and basic researches have been conducted. Zhong et al. (HZ
Hong, C.I. Srinivasan, Maria
m B. Sticklen Plant Cell
Pep. Rep. vol 10 , p453-456 (1
991)) as an auxin, DICAMBA (2,
6-dichloro-O-anisic acid)
Successful callus induction. As for the efficient induction of E callus by the addition of L-proline, see Asano et al. (Yoshito Asano, Calidas Shetty, Vol. 44 (Annex 1) p2.
0 (1991)). The mutation of bentgrass regenerated from callus has been made by Kunitake et al. (Kunitake Hisato, Nakajima Hisame, Mori Kinya, Tanaka Masanobu, Zukuri 42 (Annex 2) p110 (1992)).

[0005]

However, there has been no report on the formation of plants from E callus of bentgrass in a liquid medium, and efficient embryoid body production and its formation were difficult. On the other hand, plant tissue culture technology that has advanced in recent years has established a breeding technology for bentgrass, which enables cell fusion and transformation.

However, the new varieties produced by these new technologies are practically unusable unless they go through vegetative propagation due to the small number of individuals. In particular, many new varieties created by new technology have problems with fertility and it is difficult to produce seeds. Under such circumstances, development of a proliferation technique by tissue culture has been earnestly desired.

The present invention has been made in view of the above problems, and provides a method for efficiently reproducing bentgrass by applying a growth technique of tissue culture to the formation of an embryoid body of bentgrass. That is the purpose.

[0008]

The present invention relates to a method relating to efficient embryoid body production of bentgrass, in which E callus is induced from an explant, and in particular, only E callus is proliferated in a liquid medium, Inducing embryoid bodies by changing the sugar concentration and auxin concentration that are carbon sources in the medium,
The present invention relates to a method for regenerating a plant body by forming an embryoid body of bentgrass so as to regenerate the plant body.

The bentgrass according to the present invention is a plant within the genus Nucus, and is characterized by creeping bentgrass ( Agrostis sto
lonifla L. , Agrostis pal
ustris Huds. ) Colonial bentgrass ( Agrostis tenu )
is Sibt. ) Red top ( Agrostis alba L.,
Agrostis gigantea Roth. ) Velvet bent glass ( Agrostis cani )
na L. ) Etc. and their hybrids, or hybrids or transformed plants obtained by cell fusion.

[Detailed Description of the Invention]

(1) Induction of Callus Examples of bentgrass explants used for induction of callus include mature seeds, immature embryos, panicles, root runner growth points, and anthers. These explants are sterilized using a 70% ethyl alcohol aqueous solution and a sodium hypochlorite aqueous solution. The sterilized explant is placed on a callus induction medium to induce callus. These operations are preferably performed under aseptic conditions.

Specifically, the following medium components can be shown for induction. That is, carbon sources (mainly sugars), plant growth substances (plant hormones), inorganic salts, osmotic pressure regulators,
It contains amino acids, vitamins, buffers and the like, and gelling gum or agar or the like is added as a gelling agent in the solid medium.

Typical media include MS medium, LS medium, N6 medium, B5 medium, R2 medium, AA medium, KM-
8p medium etc. are illustrated.

An important substance for inducing callus of bentgrass is a plant growth substance, especially auxin. Specifically, 1 to 20 mg / l of auxin and 0
The addition of ~ 5 mg / l cytokinin is effective.
However, the concentration varies depending on the type of plant growth substance and the explant.

Callus is induced by placing the explant on the above medium and continuing the culture in the dark. The callus is roughly classified into those having a redifferentiation ability and those having no redifferentiation ability. Further, among callus having redifferentiation ability, a callus having a milky white and compact embryogenic ability is called E callus.

After callus formation, at the time of subculture, only this E callus is selected and transplanted to a new medium,
It becomes possible to suppress the growth of NE callus (callus other than E callus). Further, by adding L-proline or thiazolidine-4-carboxylic acid, which is an analog of proline, to the medium, it becomes possible to suppress the growth of NE callus.

(2) Growth of Callus E-callus can be grown by culturing the induced E-callus in a liquid growth medium. As the growth liquid medium, conventionally known carbon sources (mainly sugars), plant growth substances (plant hormones), inorganic salts, osmotic pressure regulators, amino acids, vitamins, buffers and the like are added. Typical mediums include MS medium, N6 medium, B5 medium,
AA medium, R2 medium, KM-8p medium and the like can be mentioned.

As the plant growth substance, it is effective to add 1 to 20 mg / l of auxin and 0 to 5 mg / l of cytokinin, and the appropriate concentration varies depending on each substance. Examples of the osmotic pressure adjusting agent include mannitol and sorbitol, and the addition of 2 to 5% (W / V) is preferable. The addition of sorbitol is particularly effective.

Examples of the carbon source include sucrose and glucose, and the addition of 1 to 5% (W / V) is preferable.

The amino acids include L-glutamic acid,
L-aspartic acid, L-proline, L-arginine,
Addition of glycine or the like is preferred. Particularly, by using L-proline and diazolidine-4-carboxylic acid, which is an analog of proline, in combination, E callus can be efficiently selected. These amino acids are preferably filtered and sterilized when used as a medium.

Further, by removing the nitrate-state and ammonia-state nitrogen contained in the inorganic salts and substituting the organic nitrogen from the amino acid, the suspension-cultured cells are finely divided,
Moreover, cells suitable for growth can be obtained.

As a method for culturing E callus in a liquid medium, the induced E callus is placed in a culture flask, the medium is added, and the medium is added in the dark at 50 to 150 rpm and 15 to 30 ° C.
Perform culture by agitation.

The medium is replaced every 5 to 10 days, at which time the E callus is lined with a mesh. This decomposes the large E callus and promotes growth.

E callus that has been passaged 5 to 10 times or more is screened with a nylon mesh or the like to separate the E callus. This removes NE callus of 100 μm or less and 1000 μm or more so that only E callus can grow. E callus contaminated with NE callus has a growth rate of NE callus higher than that of E callus. Therefore, if the operation of selecting E callus is neglected, not only NE callus grows, but also E callus becomes NE callus. Will be promoted.

The E callus thus grown is divided into separate flasks so that the fresh weight of E callus is 100 to 500 mg per 100 ml flask.

When a culture container other than the culture flask is used, that is, when a jar fermenter or the like is used, aeration with an air lift and agitation with a spinner are required in addition to the above operations. Especially when the container is large, ventilation of gas such as air is very effective,
In this case, the ventilation rate is preferably 50 to 800 ml / min. In addition, stirring with a spinner is preferably low speed,
~ 70 rpm is preferred. When the stirring speed is increased, damage to E callus and the like increases.

Under the above culture conditions, E callus can be efficiently propagated. The amount of growth depends on the growth container, conditions, etc., but increases 10 to 50 times in one month. (3) Induction of embryoid bodies By culturing the grown callus in an embryoid body-inducing medium, it becomes possible to form embryoid bodies. The term “embryoid body” as used herein refers to a tissue having a redifferentiation ability and having an incomplete shape with respect to an adventitious embryo.

The embryoid body-inducing medium is based on a growth liquid medium of E callus, on which carbon sources, plant growth substances (plant hormones), and inorganic salts (mainly nitrogen sources) are changed. . Specifically, the concentration of sucrose or glucose, which is a carbon source, is changed from 1 to 5% to 3 to 10% to reduce the concentration of auxin as a plant growth substance (plant hormone), and the nitrogen source of inorganic salts (nitric acid) State and ammonia nitrogen) are added. This embryoid body induction medium may be carried out in liquid or solid form. When solid, the use of gellan gum or agar as the gelling agent is desirable.

As a method for culturing E callus in an embryoid body-inducing medium, in the case of a liquid medium, the liquid growth medium is removed, and the embryoid body-inducing medium is added, followed by darkness (or low light), 50
Incubate at 150 rpm and 15 to 30 ° C.

The medium is replaced every 5 to 15 days,
At that time, it is necessary to remove floating NE callus having a small size (specifically, less than 100 μm) with a mesh or the like. If NE callus does not exist, this operation is unnecessary.

Since NE callus is easily browned during culture,
It is desirable to select only NE callus in the growth medium in advance.

By continuing the culture in the embryoid body-inducing medium for 2 to 6 weeks, embryoid bodies are formed on the surface of E callus. The appearance of this embryoid body is smooth, glossy, and white. Also, when the cross section is observed with a microscope, development of the epidermal system is recognized. Addition of abscisic acid (ABA) is effective for maturation of this embryoid body.

(4) Formation of plant body In this step, a plant body is formed by promoting germination and rooting in the seedling medium from the embryoid body induced by the embryoid medium. The seedling medium used here includes carbon sources, plant growth substances (plant hormones), inorganic salts, osmotic pressure regulators,
It can be prepared by adding amino acids, vitamins, buffers and the like. Typical media include MS media and N
6 medium, B5 medium, LS medium and the like.

Further, the addition of a casein hydrolyzate or the like gives more preferable results. Examples of the carbon source include sucrose and glucose, and specifically, addition of 1 to 4% is preferable. Examples of the osmotic pressure adjusting agent include mannitol and sorbitol, and the addition of 1 to 4% is preferable.

As a plant growth substance, it is efficient to use auxin and cytokinin together, and the concentration of addition thereof varies depending on the type. Especially by the addition of cytokinin,
Germination is promoted. Nitrate and ammonia nitrogen contained in inorganic salts are effective as nitrogen sources,
Vitamins, as a trace element, give favorable results to plant formation.

The culture was carried out by completely removing the embryoid body induction medium, adding a seedling medium, and illuminating under low light at 10 to 80 rpm.
Perform at 15-30 ° C. After 3 to 5 weeks of culturing, germination is observed from the embryoid body and further rooting is observed.

Since aeration is important at this stage, it is preferable to reduce the amount of medium in the culture flask. Also, in jar fermenters, agitation is stopped or slowed to damage embryoid bodies and increase aeration.

When the culture has turned green, it is preferable to aerate air containing a large amount of carbon dioxide as an aeration component. As a result, the greening of the seedlings progresses, while the seedlings become tough and the subsequent acclimation process becomes smooth. Further, if the aeration amount is decreased, vitrification occurs in the seedlings, resulting in poor acclimation.

For seedling on a solid medium, gellan gum, agar or the like is added at a high concentration as a gelling agent to a medium having the same components as the liquid, and the medium is solidified. Specifically, 0.5-1.0% gellan gum or 1.0-2.
0% agar is preferred.

By placing the embryoid bodies formed in the embryoid body-inducing medium on the solid medium, seedlings can be easily formed. Specific culture conditions include a temperature of 15 to 3
The temperature is 0 ° C., the illuminance is 100 to 5000 lux, and the photoperiod is 16 hours.

Vitrification can be suppressed by forming seedlings on a solid medium, but some albino may be mixed in the regenerated plant.

Most of the plants formed of liquid or solid are germinated or rooted, and the seedlings are also strong. After a short period of acclimatization, it is possible to go outdoors. The seedling survival rate is 90% or more. However, seedlings that have undergone vitrification or seedlings with incomplete rooting require some time for acclimation.

[0041]

EXAMPLES Example 1 [Induction of explants and callus] Gleeping bentgrass cultivar Pencloth ( Agrorsis stolonif)
ela L. ) And a colonial bentgrass variety Astoria ( Agrostis tenuis Sibt
h. The ripened seeds in 1) were sterilized with a 70% aqueous solution of ethyl alcohol and an aqueous solution of sodium hypochlorite, washed with sterile water several times, and placed in a callus induction medium under aseptic conditions.

The callus induction medium used here is as follows.

MS medium, 3% sucrose, 2-5 mg
/ L 2,4-D or Picloram, 0-0.1
mg / l KIN, 0.2-1.0% gellan gum or 0.8-2.0% agar, pH 5.5-5.8 Culture conditions were 25 ° C and dark conditions. From the callus formed after 1-2 months, only E callus was cut out and subcultured in the E callus passage medium. As a result, E-Callus was selected. The E callus subculture medium is as follows.

E callus passage medium MS medium, inorganic salts and vitamins: 3% sucrose, 2
~ 5 mg / l or Picloram, 2,4-D, 0
~ 0.1 mg / l KIN, 1-20 mM L-proline, 1-5 mM (L-thiazolidine-4-carboxylic acid), 0.2-1.0% gellan gum or 0.8-2.
0% agar, pH 5.5 to 5.8 [growth of callus in liquid medium] 1 E callus induced
In a 00 ml or 300 ml culture flask, add 20 to 100 ml of growth liquid medium, and in the dark to 50 to 15
Culture was performed at 0 rpm and 25 ° C. The growth liquid medium is as follows.

Callus Growth Medium AA medium, inorganic salts: 1-3% sucrose, 1-4% sorbitol, 2-5 mg / l, 2,4-D or Pi
chloram or DICAMBA 1.15 g / l L-proline, 0.876 g / l L
-Glutamic acid, 0.266 g / l L-aspartic acid, 0.174 g / l L-arginine, 7.5 mg /
l glycine, 2.95 g / l potassium chloride, 1-5
mM L-thioproline (thiazolidine-4-carboxylic acid), 100-500 mg / l casein hydrolyzate,
5-10 mM MES, pH 5.5-5.8 Since the liquid medium contains amino acids, it is desirable to perform sterilization by filtration, but high temperature and high pressure sterilization in an autoclave is also possible. The medium was exchanged every one week, and at that time, the grain size of E callus was made uniform by lining with a stainless mesh or the like. The E callus that had been subcultured in the growth liquid medium 5 to 10 times or more was sieved with a nylon mesh or the like.

The selected E callus is 100 to 1000 μm.
Callus of smaller or larger size was removed. As a result, unnecessary NE callus can be removed and the growth of E callus can be promoted. The proliferated E callus was divided into a new culture flask at the time of subculture so that a constant amount of E callus in the flask could be cultured.

[Induction of Embryoid Body] The proliferated E callus was subcultured in an embryoid body induction medium to induce an embryoid body. The embryoid body induction medium is as follows.

MS medium, inorganic salts and vitamins: 3 to 7
% Sucrose, 1-4% sorbitol, 1.15 g /
1 L-proline, 0.876 g / l L-glutamic acid, 0.266 g / l L-aspartic acid, 0.17
4 g / l L-alginic acid, 1-5 mM L-thioproline (thiazolidine-4-carboxylic acid), 100-10
00 mg / l casein hydrolyzate, 5-10 mM M
ES, pH 5.5 to 5.8 Culturing was performed in the dark at 50 to 150 rpm and a temperature of 25 ° C. The subculture was performed every 7 to 14 days, at which time NE callus having a small particle size was removed. After 2 to 6 weeks of culture, embryoid bodies were formed on the surface of E callus. The formed embryoid body is
It was white and shiny and the development of the epidermal system was observed under the microscope. The size of the embryoid body was 200 to 1000 μm, which was a tissue having a shape obviously different from that of E callus.

[Formation of plant] A plant was formed by subculturing the induced embryoid body in a seedling medium. The seedling medium is as follows.

Seedling medium 1/2 MS (inorganic salts and vitamins) or 1/2 N6,1
~ 3% sucrose, 1-3% sorbitol, 0.5 ~
2.0 mg / l NAA or 2,4-D, 0-1.0
mg / l KIN or BAP or zeatin, 500
~ 2000mg / l Casein hydrolyzate, 1-3mM
The MES culture was performed at an illuminance of 1000 to 8000 lux, a 16-hour photoperiod, and a temperature of 25 ° C. or higher. After 2-4 weeks of culture,
Germination and rooting were observed from the embryoid body, and greening of the seedlings was observed. When the seedling turned green, air containing carbon dioxide was aerated to improve the efficiency of plant formation.

The rooted seedlings were planted on a nursery sheet and acclimated in a humidified greenhouse. After that, the seedlings were transplanted to the field, and the seedlings survived almost 100%.

[Proliferation rate and number of seedlings] About 1 g of E callus was charged and cultured for 2 months to obtain 10 E callus.
It has become possible to grow to 0-300 g. In addition, when this E callus was seeded, the number of seedlings was 1000 to 100.
The number is 00, and this number slightly varies depending on the selection condition of E callus. That is, in a line contaminated with NE callus, both the number of embryoid bodies and the number of seedlings decrease.

Example 2 [Explant and Callus Induction] Creeping bentgrass varieties Pencloth ( Agrostis stolonify)
la L. After thoroughly rinsing the plant of (1) with water, it was sterilized with 70% aqueous solution of ethyl alcohol with sodium hypochlorite aqueous solution, and then washed several times with sterilized water, and the growth point of the runner part (hofuku) was placed on the callus induction medium. .

The callus induction medium used is the same as in Example 1. The culture conditions were darkness and a temperature of 25 ° C.

After 1 to 2 months of culturing, only E callus was cut out from the formed callus, placed on an E callus passage medium, and cultured under the same conditions. As a result, only E callus was selected and propagated.

[Callus Growth in Liquid Medium] Using the induced E callus, a suspension (suspension cultured cells) of E callus having a uniform particle size was prepared in the same manner as in Example 1. For preparation, a lining operation and sieving with a mesh were performed. This suspension 100
-1000 mg (fresh weight) was put in a 500 ml-5 liter jar fermenter, and the temperature was 25 ° C. for 2-6 weeks, while performing spinner agitation in the dark at 10-60 rpm.
The culture was performed with aeration of 50 to 500 ml / min.
The growth liquid medium used is the same as in Example 1.

The important point here is that the callus to be charged to the jar fermenter consists of only E callus and is completely selected. Should it be contaminated with NE callus, the growth of NE callus will suppress the growth of E callus in the jar fermenter, resulting in the growth of NE callus. When the culture is carried out for a long period of time, the culture is oxidized by aeration, and therefore it is necessary to maintain the pH by supplying an alkaline substance such as potassium hydroxide or sodium hydroxide. Also, the use of a buffer is effective in stabilizing the pH.

[Induction of embryoid bodies] E callus grown in a jar fermenter was subcultured to an embryoid body induction medium to induce embryoid bodies. Culture is performed in the dark at a temperature of 25 ° C. and 0 to 60 rpm.
Spinner agitation and 50-500 ml / min aeration were used. The embryoid body induction medium is the same as in Example 1. Culture 2
After 6 weeks, embryoid bodies were formed on the surface of E callus. This embryoid body is white and shiny, showing development of the epidermal system,
The size was 200-1000 μm.

[Formation of Plant] The induced embryoid body was subcultured in a seedling medium to form a plant. The seedling medium is the same as in Example 1. The illuminance of the culture is 1000 to 10,000 l
ux, 16 hours photoperiod, temperature 25 ° C, 50-500 ml /
The ventilation was set to min.

After 2 to 4 weeks of culture, the embryoid bodies germinated and rooted and turned green. At this point, air containing 0-10% carbon dioxide was aerated. Comparing the presence or absence of aeration, no seedlings were formed in the non-aerated area, whereas 100 to 500 seedlings were formed per 1 g of E callus by performing aeration of 50 to 500 ml / min. Sata These seedlings could be transplanted to the field after acclimation. [Proliferation Rate of E Callus and Number of Seedlings] About 1 g of E callus was placed in a 1 liter jar fermenter and cultured for 2 months, whereby it became possible to grow E callus to 100 to 500 g. The number of seedlings was 500 to 20,000, and the growth rate could be improved by selecting E callus and examining the aeration conditions.

Example 3 In Example 1, the medium used for the formation of plants was compared with liquid medium and solid medium. In the case of a solid medium, the medium used was liquid seedling medium supplemented with 0.2-10% gellan gum or 0.8-2.0% agar. In the case of liquid, the number of seedlings is 40 to 1 for 1 g of embryoid body.
The number was 00, and in the case of a solid, it was 20 to 80. In addition, among the plants formed, the number of albino plants was 3 to 10 in the case of solids, whereas it was not in the case of liquids.

Example 4 It was examined to selectively grow E callus by adding an amino acid such as L-proline to the E callus growth medium of Example 1.

The medium used was the E callus growth medium of Example 1 except that L-proline and thiazolidine-4-carboxylic acid were not added, and 1-10 mM L-proline and 0-5 mM thiazolidine-4-carboxylic acid were added. The plot was set up and the growth amount of E callus was compared. The culture conditions were in accordance with the method shown in Example 1.

As a result of culturing 1 g of E callus for 1 month,
In the L-proline- and thiazolidine-4-carboxylic acid-free group, callus grew to 20 to 30 g, but NE callus grew a lot, and as a result of microscopic observation, it was revealed that most cells were NE callus. became.

On the other hand, in the L-proline- and thiazolidine-4-carboxylic acid-added groups, the cultured cells were dense tissues and formed of E callus, although the growth amount did not change.

Also L-proline and thiazolidine-4
-Callus grown in the carboxylic acid-free group and the carboxylic acid-containing group were allowed to form embryoid bodies according to Example 1 to be seedlings.
The number of seedlings at this time was 100 to 300 in the non-addition group, whereas it was 1000 to 4000 in the addition group.

The abbreviations of the culture media used in Examples 1 to 4 are as follows.

2,4,5-T: 2,4,5-tric
hlorophenoxyacetic acid 2,4-D: 2,4-dichlorophenox
yacetic acid NAA: α-napht
hylacetic acid Picloram: 4-amino-3,5,6-t
Richoropicolinic acid DICAMBA: 2,6-dichloro-2-m
Ethoxybenzoic acid MES: 2- (N-morpholino) etha
nsulfonicacid, monohydrate KIN: 6-furfurylaminopurin
e BAP: 6-benzylaminopurine

[0069]

INDUSTRIAL APPLICABILITY As described above, the present invention induces E callus from an explant of bentgrass, grows only E callus in a liquid growth medium, and transforms E callus into embryoid bodies in a liquid or solid medium. It was induced to obtain a plant from the embryoid body in a liquid or solid medium. Therefore, according to the present invention, it becomes possible to efficiently regenerate a bentgrass plant by applying a growth technique by tissue culture.

Claims (7)

[Claims] 1. A method of inducing and growing embryoid bodies from callus derived from bentgrass explants, comprising: a. Inducing E callus from bentgrass explants; b. Growing only E callus in a liquid growth medium, c. Inducing embryoid bodies in liquid or solid medium from said E callus, d. A method for regenerating a plant by forming embryoid bodies of bentgrass, comprising the steps of obtaining a plant from the embryoid body in a liquid or solid medium. 2. The method for regenerating a plant by forming embryoid bodies of bentgrass according to claim 1, wherein the steps of E callus proliferation, embryoid body induction and plant formation are performed in a liquid medium. 3. The liquid medium comprises inorganic salts, vitamins,
The method for regenerating a plant by forming embryoid bodies of bentgrass according to claim 1, which comprises a carbon source, an amino acid, an auxin, and a cytokinin. 4. The method according to claim 1, wherein the induction of the embryoid body in a liquid or solid medium is carried out by changing the concentration of sugar constituting the carbon source and the concentration of auxin. A method for regenerating a plant by forming embryoid bodies of bentgrass. 5. The method for regenerating a plant by forming embryoid bodies of bentgrass according to claim 1, wherein the nitrogen source in the growth medium is composed of amino acids as well as inorganic salts. 6. The size of the E callus is 50 to 1000.
The method for regenerating a plant by forming an embryoid body of bentgrass according to claim 1, wherein the plant body has a diameter of μm. 7. The auxin added to the liquid medium is
2,4-dichlorophenoxyacetic acid, 4-amino-3,
4,6-Trichloropicolinic acid, 2,6-Dichloro-O
-Anisic acid, naphthalene acetic acid, indole acetic acid, 2,
The method for regenerating a plant by forming embryoid bodies of bentgrass according to claim 1, which comprises one or more auxins of 4,5-trichlorophenoxyacetic acid.