JPH0823806A - Method for multiplicative culture of plant belonging to genus tulipa - Google Patents

Abstract

PURPOSE:To multiply a large amount of tulips, to practically use breeding such as gene transfer or cell fusion and to provide a method for forming an adventitious bud capable of inducing and proliferating a callus which can be differentiated into an adventitious bud and obtaining the adventitious bud from the callus. CONSTITUTION:In a culrure method for forming the adventitous bud, tissue in a bud of a plant belonging to the genus Tulipa is cultured to induce/ proliferate a callus capable of forming an adventitious bud and the callus is cultured. In the method for multiplying a plant of the genus Tulipa, the induction/proliferation of the callus is carried out at 23-32 deg.C and the formation of the adventitious bud is done at 11-22 deg.C. This method can form a large amount of adventitious buds and small bulbs and higher utility in cell breeding and practical production of seed and seedling than a conventional method can be attained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for inducing and growing callus capable of adventitious shoot differentiation of tulip plants and a method for mass-proliferating tulip plants utilizing a plant tissue culture technique by adventitious bud formation from the callus. .

[0002]

2. Description of the Related Art In recent years, the development of tissue culture technology has increased the possibility of commercialization by mass growth in many plants. Also in tulip, a technique suggesting the possibility of mass growth has been reported. When a tulip is to be propagated by tissue culture, for example, a method of directly inducing and differentiating adventitious shoots from a tulip plant tissue piece to proliferate (Acta Horticulturae, Vol.
109, 263 to 270, 1980), but the adventitious bud formation efficiency is low, and in view of the proliferation efficiency, it has not yet reached a commercial mass proliferation technique.

Further, as an example in which a relatively large amount of adventitious buds are obtained, a method of vertically dividing adventitious buds induced by culture from flower stem tissue and culturing to induce and propagate adventitious buds (Acta Horticulturae, Vo
l. 325, 441 to 446, 1992) and a long diameter of 2 m
m Adventitious buds by culturing adventitious buds (Acta Horticulturae, Vo
l. 266, 83-90, 1990, JP-A-5-192
No. 053), but it is not suitable as a commercial mass-propagation technique.

In addition, a report of callus culture from scaly tissue (Hokkaido University of Education Bulletin, 33, 49-65, 1)
982, J. Kor. Soc. Hort. Sci. , V
ol. 23 (4), 348-356, 1982),
Callus induction is possible, but there are no reports of inducing and proliferating callus capable of adventitious bud differentiation. As described above, there has been no report up to now to induce and propagate callus capable of adventitious bud differentiation from plant tissue of tulip and further form adventitious bud from the callus.

[0005]

[Problems to be Solved by the Invention] As an application of plant tissue culture technology to breeding, many plants are subjected to cell selection in the introduction of genes derived from animals / plants and microorganisms, induction of mutation / selection, and cell fusion. ing. Tulips are also desired to breed using these techniques, and in order to use these breeding methods, dedifferentiated cell mass (callus) and cells for obtaining protoplasts are required. Furthermore, it is also necessary to establish a technique for a plant regeneration method from these cells.
However, the above-mentioned breeding method is not used in tulips at present because the induction / proliferation of callus capable of plant redifferentiation and the regeneration of plant from callus are not performed. Therefore, in order to carry out the above breeding method, it is desired to establish a technique for inducing / proliferating callus capable of regenerating a plant from a tulip plant tissue and a method for regenerating a plant from the callus. At the same time, this technique can obtain a large amount of tulip plants on a commercial scale in a short period of time, and is greatly desired as a technique for a method for growing and cultivating tulip plants.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has conducted a method for inducing and proliferating adventitious shoot-differentiating callus by culturing tulip flower bud tissue. We have found a method to form adventitious shoots from callus. Further, by using the adventitious bud, a large amount of tulip small bulbs can be produced by a known method.

That is, the present invention is as described below.
Induction of adventitious callus of tulip plant
The present invention relates to a growth method and a large-scale growth method of Tulip plants using a plant tissue culture technique by forming adventitious shoots from the callus.

(1) A method for cultivating a tulip genus plant, which comprises culturing a flower bud tissue of a tulip genus plant to induce and grow callus capable of adventitious bud differentiation. (2) The method for cultivating a tulip plant according to (1) above, wherein the callus induction / proliferation is performed under a temperature condition of 23 to 32 ° C. (3) A method for cultivating a tulip genus plant, which comprises culturing a tissue in a flower bud of a tulip genus plant and forming adventitious buds from the obtained callus capable of adventitious bud differentiation. (4) The method for cultivating a Tulip plant according to (3) above, wherein the adventitious bud formation is performed under the temperature condition of 11 to 22 ° C. (5) A method for proliferating and cultivating a tulip genus plant, which comprises culturing a tissue in a flower bud of a tulip genus plant to induce / proliferate a callus capable of adventitious bud differentiation, and then culturing the callus to form adventitious buds . (6) In producing a bulb of a tulip plant using an adventitious bud, an adventitious bud formed from an adventitious bud-differentiable callus obtained by culturing a flower bud tissue of a tulip plant is used. A method for producing a plant of a tulip plant.

The present invention will be described in detail below. 1) Plant used for inducing callus capable of adventitious bud differentiation The tulip used in the method of the present invention refers to a plant of the genus Tulip, for example, Paul Richter and Mary Widow belonging to the Triumph cultivar group, and the Darwin hybrid cultivar group. Tulipa tarda belonging to Apeldorn and Oxford Elite varieties and other species (wild species)
a), Thuripa Hagery (T. hager
i), T. Turkestanika
stanica) and the like.

2) Plant tissue used for the induction of callus capable of adventitious bud differentiation Tulip buds used in the method of the present invention are 5 to 3
It is a flower bud with a length of 0 mm, and the flower bud is in the bulb and is immature in the growth stage. Examples of the tissue in the flower bud include anthers, anther bases, styles, ovary walls, flower thread bases, ovaries, and the like, more preferably ovary.

3) Induction and Propagation of Adventitious Differentiable Callus A callus induction / proliferation medium for inducing and proliferating adventitious differentiable callus from plant tissue contains at least inorganic salts, plant hormones and saccharides as essential components. And vitamins,
Amino acids and casein hydrolyzate are added as necessary. Specifically, a basic medium conventionally used for tissue culture of plants, for example, Murashige Scoog (M
urashige &Skoo's medium (MS medium),
Linsmeier & Skoo
oo's medium (LS medium), Gamborg (Gambo)
rg) B5 medium or the like can be used. The composition and the like of these conventionally known media are described in, for example, “Plant Tissue Culture” by Harada and Komine, p390-391, Science and Engineering, 198.
Listed in 4 years.

Examples of saccharides in the medium include shoe cloth, glucose and the like, and the concentration thereof is preferably 0.1 to 6%, particularly preferably 1 to 3%. Examples of plant hormones include auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthaleneacetic acid (N
AA) or the like can be used. As the cytokinin, benzyladenine (BA), 6-furfurylaminopurine (kinetin), or the like can be used, and auxin and cytokinin may be used in combination, or auxin may be used alone. The concentration of auxin added was 0.
It is preferable to use 1 to 20 μmol / l, and 1 to 1
0 μmol / l is particularly preferred. Cytokinin may be added at a concentration of 10 μmol / l or less, preferably not added or at a concentration of 3 μmol / l or less.

Examples of amino acids include L-proline and glutamine, and it is preferable to add them at a concentration of 0.1 to 10 mM. Casein hydrolyzate is 10-500p
It is preferable to add it at a concentration of pm.

As the gelling agent for preparing the solid medium, agar, gellite and the like can be exemplified. The concentration of these is 0.7 to 0.9% for agar and 0.15 to 0 for gellite. 0.25% is preferable. Next, in order to induce callus capable of adventitious bud differentiation from plant tissue, the above-described induction medium is first prepared, and sterilized plant tissue is placed. The sterilization of the plant tissue can be carried out using ethanol, sodium hypochlorite solution or the like according to a conventional method.

In induced callus growth, adventitious shoot-differentiable callus, which is yellow and has high cytoplasmic density
And callus that is not adventitious bud differentiation (this is white and has a low cytoplasmic density), and a callus that is incapable of adventitious bud differentiation is selected and propagated. Passage of adventitious callus capable of differentiating in a growth medium (hereinafter referred to as callus) is performed every week.

Induction and growth of callus is preferably carried out under the temperature condition of 23 to 32 ° C, particularly preferably about 25 ° C.

The culture light is irradiated with white light (for example,
Although it is possible to induce and grow callus even when performed at 100 to 2,000 lux), better results can be obtained by culturing in the dark.

4) Induction of adventitious buds from callus Adventitious bud induction medium for inducing adventitious buds from callus contains at least inorganic salts, plant hormones and sugars as essential components, and requires vitamins, amino acids and casein hydrolysates. It is added according to. Specifically, the above MS medium, L
S medium, Gamborg's B5 medium and white (Wh
ite) and the like. Usually, the inorganic salts excluding the inorganic nitrogen source are preferably used at a concentration of 1/8 to 2 times, and particularly preferably at a concentration of 1/4 to 1 times. No addition or 3 mM of calcium salt
The following are preferred.

As the inorganic nitrogen source, nitrate nitrogen and ammonia nitrogen can be supplied, but it is preferable to use nitrate nitrogen alone or a combination of nitrate nitrogen and ammonia nitrogen, and the total nitrogen concentration is 1 mM to 30 mM.
Is preferred. At that time, the concentration of nitrate nitrogen is 1 mM to 30 m
M is preferable, and the ammonia nitrogen concentration is preferably no addition or 20 mM or less, particularly preferably no addition or 10 mM or less. At this time, the ammonia / nitric acid ratio is preferably 0 or more and 2 or less, and particularly preferably 0 or more and 1 or less.

Examples of saccharides in the medium include shoe cloth, glucose and the like, and the concentration thereof is preferably 0.1 to 6%, particularly preferably 0.5 to 2%. The plant hormone is a combination of auxin and cytokinin. Examples of auxins include 2,4-D, NAA, indole-3-acetic acid (IAA), and the like.
Auxin is 0.1-10 μmol / l, preferably 1
B as cytokinin at a concentration of ~ 3 μmol / l
A, kinetin, 2-isopentenyl adenine (2i
P) and the like can be exemplified, and 0.1 to 10 μmol /
It may be added at a concentration of 1, preferably 1 to 10 μmol / l.

Examples of amino acids include L-proline and glutamine, and it is preferable to add them at a concentration of 0.1 to 10 mM. Casein hydrolyzate is 10-500p
It is preferable to add it at a concentration of pm.

As the gelling agent for preparing the solid medium, agar, gellite and the like can be exemplified. The concentration of these is 0.7 to 1.5% for agar and 0.2 to 0 for gellite. 0.6% is preferable.

The adventitious bud-inducing culture is preferably carried out under a temperature condition of 11 to 22 ° C., particularly preferably about 18 ° C.

The culture light is irradiated with white light (for example,
Adventitious buds can be induced even at 100 to 2,000 lux), but better results can be obtained by culturing in the dark from the start of culturing until the adventitious buds are induced. .

Next, bulb induction is performed by low temperature treatment,
The adventitious shoots (length 1 to
It is necessary to grow 2 mm) to shoot (30 mm or more). Shoots are formed even in the adventitious bud-inducing medium, but the adventitious buds are cultured in the shoot-forming medium in order to further enhance the shoot-forming efficiency. The shoot forming medium may use the same medium composition as that shown in the above step 4) in the step of inducing adventitious shoots from callus, except for plant hormone conditions. The plant hormone conditions for shoot formation are auxin and cytokinin addition, As an auxin,
NAA, IAA, etc. can be exemplified, and auxin is 0.01 to 3 μmol / l, preferably 0.1 to 1 μm.
BA, kinetin, 2iP and the like can be exemplified as the cytokinin at a concentration of ol / l, and the cytokinin is 0.01 to 3 μmol / l, preferably 0.1 to 1
Add at a concentration of μmol / l. Then, culture is performed at 18 ° C. under illumination (for example, 1,000 lux, 16-hour light period) to obtain shoots.

The obtained shoot is, for example, as described above (Act
a Horticulturae, Vol. 266,8
3 to 90, 1990), low temperature treatment or gibberellin (GA ₃ ) treatment at 4 ° C. and culturing can be performed to form small bulbs. The small bulb becomes a flowerable bulb under natural or forcing conditions.

[0027]

EXAMPLES Next, the method of the present invention will be specifically described with reference to Examples and Comparative Examples, but the method of the present invention is not limited to these examples.

(Example 1) Oxford elite of tulip was used as a test species. Immature ovary tissues with flower buds in bulbs were cut into 2 mm square pieces, placed in the following callus induction medium containing various concentrations of auxin and cytokinin, and placed at 25 ° C. in the dark for 2 days.
When the cells were cultured for a week and subcultured in the same medium every week, callus formation was observed after 3 weeks, and the culture was continued until the 5th week. Note that 2,4-D was used as the auxin and BA was used as the cytokinin at various concentrations to be added to the medium. Table 1 shows the results of the influence of plant hormones on the callus formation rate (ratio of tissue pieces on which callus was formed per total tissue piece) at the time of callus induction. As a result, callus formation was observed in all combinations of plant hormones. Among them, the high callus formation rate of 70% or more is that 2,4-D is 3 to 10 μm.
The concentration was ol / l and the concentration of BA was 1 to 3 μmol / l. [Callus induction medium] MS medium containing 2% sucrose, 1 mM L-proline, 100 ppm casein hydrolyzate, phytohormones, pH 5.8, and 0.2% gellite.

[0029]

[Table 1]

Example 2 Tulip Paul Richter was used as a test species. Cut out immature filamentous tissue at the growth stage with flower buds in bulbs to a size of 1 mm square,
When placed in the following callus induction medium containing various concentrations of auxin and cytokinin, cultured at 25 ° C. in the dark for 2 weeks, and subcultured in the same medium every week, 4
Callus formation was observed after a week, and the cells were cultured until the 6th week. NA as an auxin added to the medium
A and kinetin were used as cytokinins at various concentrations. The results of the effect of plant hormones on the callus formation rate during callus induction are shown in Table 2.
As a result, callus formation was observed in all combinations of plant hormones. Among them, the callus formation rate is 36%
NAA had a concentration of 10 μmol / l and kinetin had a concentration of 1 μmol / l. [Callus induction medium] MS medium containing 2% sucrose, 1 mM L-proline, 100 ppm casein hydrolyzate, phytohormones, pH 5.8, and 0.2% gellite.

[0031]

[Table 2]

Example 3 Tulip merry widow was used as a test species. Immature ovary tissue in the growth stage with flower buds in bulbs was cut into 2 mm square size, placed on the following callus induction / growth medium, and cultured at 25 ° C. in the dark for 2 weeks. When the cells were subcultured every one week in the above medium, callus was formed after 2 weeks. Then, the induced callus is removed from the plant tissue piece, and a callus having a fresh weight of 50 mg is placed on the following callus induction / growth medium,
Subculture was performed 4 times every week. The culturing temperature at that time was set to 11, 18, 23, 25, 32 ° C., the culturing was carried out in the dark, and the fresh weight of the callus was measured 4 weeks after the start of the culturing to measure the callus growth rate (after 4 weeks with respect to the bed callus The raw weight multiple of callus) was calculated.

The results of the influence of the culture temperature at the passage of callus on the growth rate of callus are shown in Table 3. As a result, callus was proliferated under all temperature conditions, and the callus proliferation rate was preferably 5 times or more, which was good.
° C. More preferably, at 25 ° C., the callus growth rate at that time was 7 times as much as 350 mg after 4 weeks of culture with respect to the fresh weight of the bed callus of 50 mg. [Callus induction / growth medium] 2% shoe cloth, 1 mM
L-proline, 100 ppm casein hydrolyzate, 2,
4-D 10 μmol / l, kinetin 1 μmol / l
1, MS medium containing pH 5.8 and 0.2% gellite.

[0034]

[Table 3]

(Example 4) The culture temperature in callus induction / proliferation was set to 25 ° C, and conditions other than the culture temperature were used in Example 3
The callus was obtained by culturing in the same manner as in. The callus was placed on the following adventitious bud induction medium containing various concentrations of auxin and cytokinin, and cultured at 18 ° C. in the dark.
7 weeks after the start of culture, the adventitious bud formation rate (the ratio of the number of adventitious callus masses to the total number of callus masses) and the number of adventitious shoot differentiation (1
The average number of adventitious buds differentiated per callus mass) was calculated.

The effects of plant hormones on adventitious bud formation from callus are shown in Table 4. As a result, adventitious bud formation was observed in all treatments, and the adventitious bud formation rate was 75%, which was particularly good.
Was 3 μmol / l and BA was 3 μmol / l. [Adventitious bud induction medium] 1% sucrose, 1 mM glutamine, 100 ppm casein hydrolyzate, plant hormone,
An MS-modified medium containing pH 5.8 and 0.7% agar (nitrate nitrogen concentration 11.5 mM, ammonia nitrogen not added, calcium chloride salt not added).

[0037]

[Table 4]

(Example 5) The callus growth temperature was set to 25 ° C, which was the best callus growth result in Example 3, and Example 3 was used.
Culture was carried out in the same manner as above to induce and grow callus. Callus after 4 weeks of passage was transplanted to the following adventitious bud induction medium,
The culture temperature was set to 11, 18, 22, 25 and 32 ° C., the culture was performed in the dark, and the adventitious bud formation rate and the adventitious bud differentiation number were counted 7 weeks after the start of the culture.

The results that the culture temperature at the time of inducing adventitious buds affects the adventitious bud formation rate are shown in Table 5. As a result, adventitious buds were formed under all temperature conditions, and the adventitious bud formation rate was preferably 50% or more at 11, 18, and 22 ° C.
More preferably at 18 ° C, the adventitious bud formation rate at that time is 72
%, The number of adventitious bud differentiation was 13.7. [Adventitious bud induction medium] 1% shoe cloth, 1 mM L-proline, 100 ppm casein hydrolyzate, NAA 3
μmol / l, BA 3 μmol / l, pH 5.8,
MS modification containing 0.7% agar (nitrate nitrogen concentration 11.5
mM, ammonia nitrogen concentration 2 mM, calcium chloride salt 0.1 mM) medium.

[0040]

[Table 5]

(Example 6) Wild tulip tulipa tarda (T. tarda) was used as a test seed. Take out the flower buds with a length of 5-10 mm from the bulb,
Add this to 70% ethanol solution for 10 seconds, then 1%
It was immersed in the sodium hypochlorite solution of 10 minutes and washed with sterilized water. The ovary tissue was aseptically cut out from this flower bud and used as a test material. This ovary tissue was cut into a size of 1 mm square, placed on the following callus induction / growth medium, cultured for 2 weeks, and subcultured once a week in the same medium as described above. Been formed. Then, the induced callus was removed from the plant tissue and placed in the following callus induction / growth medium, and subcultured every week. The culture temperature for callus induction / proliferation was 25 ° C., and the culture was performed in the dark. [Callus induction / growth medium] 2% shoe cloth, 1 mM glutamine, 100 ppm casein hydrolyzate, 2,4-
D 10 μmol / l, BA 1 μmol / l, pH
5.8 MS medium with 0.2% gellite.

After the callus subcultured every week was subcultured for 7 weeks, the callus was transplanted to the following adventitious bud induction medium and cultured at 18 ° C. in the dark. Adventitious buds were formed after 7 weeks. . [Adventitious bud induction medium] 1% shoe cloth, 1 mM glutamine, 100 ppm casein hydrolyzate, IAA 3 μm
ol / l, 2iP 3 μmol / l, pH 5.8, 0.
MS modification including 7% agar (nitrate nitrogen concentration 11.5m
M, no ammonia nitrogen added, no calcium chloride salt added) medium.

The obtained adventitious buds (1 to 2 mm in length) were transplanted to the shoot forming medium described below, and the temperature was set at 18 ° C. at 1,000.
When it was cultured under looks for 16 hours under illumination, shoots elongated to about 30 mm were obtained at 4 weeks. [Shoot formation medium] 1% shoe cloth, 1 mM proline, 100 ppm casein hydrolyzate, IAA 0.3
μmol / l, BA 0.3 μmol / l, pH 5.
8 、 MS modification containing 0.8% agar (nitrate nitrogen concentration 1
1.5 mM, ammonia nitrogen concentration 2 mM) medium.

The obtained shoots were subjected to a low temperature treatment for 10 weeks under illumination at 4 ° C. and 1,000 lux for 16 hours, and then 6% shoe cloth was added to reduce the inorganic salt concentration to 1/2. Then, by culturing for 30 days under illumination at 25 ° C. and 1,000 lux for 16 hours, small bulbs were formed at the shoot base.

As a result, the callus formation rate at the 4th week from the start of the culture was 96%, the callus proliferation rate in the 7-week growth culture was 42.9 times, and the adventitious bud formation was obtained 7 weeks after the transplantation to the adventitious bud induction medium. The rate is 76%, and the number of shoots formed after 4 weeks from transplantation into shoot forming medium, in other words, about 150 days after the start of culture (the total number of shoots formed per piece of ovary tissue for one bulb) is 1220, The finally obtained bulb differentiation number (the number of bulbs differentiated per piece of ovary tissue for one bulb) was about 250 days after the start of culture, and 620 small bulbs were obtained.

(Example 7) The callus induction / proliferation and adventitious bud induction were always performed at 18 ° C, and the culture was carried out in the same manner as in Example 6 except for the culture temperature conditions. Got a shoot, a bulb. As a result, the callus formation rate was 54%, the callus growth rate was 7.4 times, the adventitious bud formation rate was 78%, and the shoot formation number was 205.
The number of bulb differentiations finally obtained was 120.

Example 8 Callus induction / proliferation and adventitious bud culture were carried out in the same manner as in Example 6 except that the culture temperature was always 25 ° C. Got a shoot, a bulb. As a result, the callus formation rate was 90%, the callus growth rate was 62.6 times, the adventitious bud formation rate was 36%, and the shoot formation number was 430.
The number of bulb differentiations obtained in the book and finally was 139.

(Example 9) Tulip wild variety Tulipa hagueri (T. hageri) was used as a test species,
Culture was performed in the same manner as in Example 6 except for the callus induction / proliferation medium and adventitious bud induction medium shown below. [Callus induction / growth medium] 2% shoe cloth, 1 mM glutamine, 100 ppm casein hydrolyzate, 2,4-
D MS medium containing 10 μmol / l, pH 5.8, 0.2% gellite.

[Adventitious bud induction medium] 1% shoe cloth, 1
mM glutamine, 100 ppm casein hydrolyzate, N
AA 3 μmol / l, BA 3 μmol / l, pH
5.8, MS modified medium containing 0.7% agar (nitrate nitrogen concentration 11.5 mM, ammonia nitrogen not added, calcium chloride salt not added) medium.

Adventitious shoots were induced after 7 weeks of callus propagation. As a result, the number of shoots obtained from 5 explants was 1020 in 150 days. The shoot thus obtained was stored at 4 ° C. for 10 minutes.
Small bulbs were formed by low temperature treatment for a week and culturing. As a result, 520 small bulbs were obtained 250 days after the start of the test.

(Comparative Example 1) Tulip wild-type Thulipa hagueri (T. hageri) was used as a test species for culturing. Immature flower stalks at the stage of growth with flower buds in bulbs were cultured for 60 days in MS medium supplemented with both NAA and BA at 1 mg / l to induce whitish light green granular adventitious buds with a diameter of 1 mm to 2 mm. The adventitious buds were cut into 3 mm blocks. As a result of subculture for 60 days in a growth medium, 80 adventitious sprouts of 5 explants were obtained. Further, the adventitious buds thus obtained were transplanted to a shoot regeneration medium and cultured for 30 days.
Got 560 shots in 0 days. In this way,
The obtained shoots were subjected to low temperature treatment at 4 ° C. for 10 weeks and cultured to form small bulbs. As a result, only 315 small bulbs were obtained 250 days after the start of the test.
Compared with the method of the present invention in Example 9, the number of adventitious bud formation and the number of bulb formation were clearly lower in the method not via callus.

[0052]

INDUSTRIAL APPLICABILITY The method of the present invention makes it possible to induce and proliferate adventitious shoot-differentiating callus derived from tulip flower bud tissue, and to induce adventitious shoots from the callus. Mass production and protoplast fusion that are commercially available by allowing plant redifferentiation from callus,
A gene transfer such as electroporation or a breeding method such as mutation can be used.

Claims (6)

[Claims] 1. A method for cultivating a tulip plant, which comprises culturing a tissue in a flower bud of a tulip plant to induce / proliferate callus capable of adventitious bud differentiation. 2. The method for cultivating a tulip plant according to claim 1, wherein the culturing is performed under a temperature condition of 23 to 32 ° C. during the callus induction / proliferation. 3. A method for culturing a tulip plant, which comprises culturing a tissue in a flower bud of a tulip plant and forming adventitious buds from the callus capable of adventitious bud differentiation. 4. The method of cultivating a tulip plant according to claim 3, wherein the adventitious bud is cultured under a temperature condition of 11 to 22 ° C. 5. Proliferation of a tulip plant characterized by culturing an inflorescence tissue of a tulip plant to induce / proliferate adventitious shoot-differentiable callus, and then culturing the callus to form adventitious shoots. Culture method. 6. An adventitious bud formed from an adventitious bud-differentiable callus obtained by culturing a flower bud tissue of a tulip genus plant in producing a bulb of a tulip genus plant using an adventitious bud, A method for producing a plant body of a tulip plant characterized by the above.