KR20160048456A - Optimal medium composition for tissue culture of Chrysanthemum morifolium 'Baekma' and tissue culture method using the same - Google Patents

Abstract

The present invention relates to an optimal medium composition for culturing a tissue of Dendranthema grandiflorum, Baekma, and a method for culturing a tissue using the same. According to the present invention, by using a medium composition for inducing a bud of Dendranthema grandiflorum, Baekma, a medium composition for inducing new shoot formation, and a method for reproduction of a plant of Dendranthema grandiflorum, a plant of Dendranthema grandiflorum can be mass produced from a leaf explant of Dendranthema grandiflorum, Baekma, and a new variety of a plant of Dendranthema grandiflorum to which a functional gene is introduced, can be mass produced stably in a short period of time.

Description

TECHNICAL FIELD The present invention relates to an optimal medium composition for tissue culture of chrysanthemum 'White Horse' and a tissue culture method using the same.

The present invention relates to an optimal culture composition for tissue culture of chrysanthemum 'white horse' and a tissue culture method using the same. More particularly, the present invention relates to a method for culturing a chrysanthemum 'white horse' And inducing elongation and rooting of the shoot to differentiate into a plant, a method of regenerating a chrysanthemum plant, a method of inducing buds from the chrysanthemum leaf segment, And to a medium composition for inducing shoot formation from the bud.

Chrysanthemum morifolium ) is one of the three major crops in the world and domestic, along with roses and carnations. The cultivation area of cut flower chrysanthemum in Korea is 527 ha, which corresponds to about 30% of total cut flower cultivation area (MIFAFF 2012). However, since most of the domestic chrysanthemums cultivated in Korea are cultivated and exported from overseas, a considerable amount of royalties are paid each year, so it is urgent to cultivate new varieties in Korea.

Chrysanthemum 'White Horse' is a cultivar cultivated in 2004 by the National Institute of Horticultural Science. It is a bright white variety that naturally blooms in late September. It is possible to cultivate all over the year with temperature control, and the number of petals is about 250. There is no core phenomenon and the middle part of the flower shows light green at the early flowering time (Shin et al., 2005, Kor, J. Breed, 119-120). Since the introduction of Chrysanthemum 'white horse' as a farmer in 2007, the domestic cultivation area has been gradually expanding due to its high preference in both domestic and overseas markets, and exports to Japan are expected to increase in the future. However, untreated control, premature aging, and abnormal color development due to inadequate management are present, and measures are needed to solve these problems. Therefore, biotechnology breeding technology through transformation and molecular breeding is needed as a way to alleviate the advantages of 'white horse', an excellent cut flower, and to improve problems. For biotechnological breeding through transformation, establishment of an efficient plant regeneration system based on plant tissue culture should be preceded, and plant regeneration technology through organogenesis can be applied not only to micro-breeding but also to mutation breeding.

Plant reproduction of chrysanthemum was successfully accomplished through the formation of organs from leaves, stems, petals and other somatic embryos. The formation of shoot organ of chrysanthemum is greatly influenced by factors such as the type and condition of the slices, the type and concentration of plant growth regulator and media additives, medium composition, and pH. Especially, plant growth regulators play an important role in shoot organ formation. The ratio and concentration of auxin and cytokinin have a great influence on shoot organ formation. Although various studies on the regeneration of chrysanthemum have been successful in various cultivars, there have been few reports on the regeneration of chrysanthemum 'white horse'.

Therefore, this study was conducted to investigate the proper conditions of culture medium and culture period including plant growth regulator and AgNO ₃ to induce shoot organogenesis from cultivated leaf slices of domestic chrysanthemum 'White horse' And to establish an efficient plant regeneration system.

Korean Patent No. 1136150 discloses a method for transforming chrysanthemum using a petal fragment of chrysanthemum and a transformed chrysanthemum, and Korean Patent No. 1300658 discloses a method for transforming chrysanthemum And a complex stress tolerant chrysanthemum plant produced by the above method. However, as described in the present invention, the optimal culture medium for tissue culture for mass production of chrysanthemum 'white horse' plants and a method of culturing tissue using the same have not been disclosed.

The present invention has been made in view of the above-mentioned needs, and the present inventors have found that buds are formed when leaf cut pieces of chrysanthemum 'white horse' were cultured in 11 different media with different concentrations of plant growth regulator and AgNO ₃ The development of shoots was not carried out and two - step experiments were carried out using media with different plant growth regulator composition to induce shoot formation after eye induction. That is, the leaf fragment of chrysanthemum 'white horse' was cultivated in the bud induction medium of the chrysanthemum 'white horse' of the present invention to effectively induce the development of the eye. In the shoot induction medium of the present invention, Efficient seedlings were efficiently regenerated, and the present inventors completed the present invention by confirming that chrysanthemum 'white horse' can be rapidly mass produced through the chrysanthemum tissue culture method of the present invention.

In order to solve the above problems, the present invention includes a step of inducing buds from chrysanthemum leaf sections, inducing shoot formation from the eyes, and inducing elongation and rooting of the shoots to differentiate into plants A method for regenerating a chrysanthemum plant is provided.

The present invention also provides a chrysanthemum plant regenerated by the above method.

The present invention also provides a medium composition for guiding buds from the chrysanthemum leaf section and a medium composition for inducing shoot formation from the buds.

Using the medium composition for guiding bud growth of chrysanthemum 'white horse' and the medium composition for inducing shoot formation, and the method for regenerating chrysanthemum plant, the chrysanthemum plant can be mass produced within 14 weeks from the leaf segment of chrysanthemum 'white horse' Therefore, it is expected that a new variety chrysanthemum plant having environmental resistance tolerance or functional gene introduced through this method can be mass-produced stably in a short time.

Fig. 1 shows a plant regeneration process through shoot organ formation from a leaf segment of chrysanthemum 'white horse'. (A, D) mother culture and inoculated leaves. (B, E) callus and eyes (bud) formed after 4 weeks cultivation of leaf slices on MS medium supplemented with 0.2 mg / L of BA, 1.0 mg / L of IAA and 1.0 mg / L of 2,4-D. (C, F) BA 1.0 mg / L, IBA 1.5 mg / L and AgNO ₃ 5.0 mg / L has four weeks on an MS medium supplemented shoot formation from the body cultured leaf segment (eight main culture). (G) Shoot development after 8 weeks of culture (total 16 weeks culture). (H) Roots of shoots regenerated in MS medium. (I) Purified plants in the greenhouse. Scale bar: 1 cm.
Fig. 2 shows the results of a comparison of shoot regeneration from a leaf segment of chrysanthemum "White horse" according to various culture periods on an eye-forming medium. The leaf slices were cultured for 1 to 4 weeks on an eye-induced medium consisting of MS medium containing 0.2 mg / L of BA, 1.0 mg / L of IAA and 1.0 mg / L of 2,4-D, and the plant growth regulator-free (PGR free MS medium, MS medium containing BA 1.0 mg / L, IBA 1.5 mg / L and AgNO ₃ 5.0 mg / L or BA 1.0 mg / L, NAA 1.0 mg / L and AgNO ₃ 5.0 mg / MS medium, and then cultured for 4-7 weeks.

In order to achieve the object of the present invention,

1) Chrysanthemum leaves were cultured on MS medium supplemented with BA (6-benzyladenine), IAA (indoleacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) Inducing;

2) transferring the induced eye to MS medium supplemented with BA (6-benzyladenine), IBA (indole-3-butyric acid) and AgNO ₃ , and culturing under normal conditions to induce shoot formation; And

3) Separating the shoots from the leaf slices and transferring them to MS medium to induce kidney and rooting and differentiating them into plants. The present invention provides a method for regenerating chrysanthemum plants using a leaf blade of chrysanthemum.

In a method according to an embodiment of the present invention, the chrysanthemum may be a 'white horse' variety but is not limited thereto. The white horse breed is the main standard breed of chrysanthemum breeding which is known to be difficult to regenerate through in-flight culture. In the present invention, an efficient plant regeneration technique is accomplished through the formation of shoot organs from the leaf segment of white horse.

The chrysanthemum plant regeneration method using the chrysanthemum leaf fragment according to the present invention is characterized in that, in step 1), the leaves of chrysanthemum are divided into BA (6-benzyladenine), IAA (indoleacetic acid) and 2,4- acid in an MS medium to induce buds.

The chrysanthemum leaf intervertebral body may have a size of 8 to 10 mm ² , but the present invention is not limited thereto. The concentration of BA may be 0.1 to 0.3 mg / L, preferably 0.2 mg / L. The concentration of IAA may be 0.8-1.2 mg / L, preferably 1.0 mg / L. The concentration of 2,4-D may be 0.8-1.2 mg / L, preferably 1.0 mg / L. The pH of the culture medium can be adjusted to 5.6 to 6.0, preferably to pH 5.8. The incubation period may be 3 to 4 weeks, preferably 4 weeks.

Chrysanthemum plant reproduction method using a chrysanthemum leaf segment according to the present invention 2) a step, which the the eyes derived from body chrysanthemum leaf segment BA (6-benzyladenine), IBA (indole-3-butyric acid) and AgNO ₃ was added MS medium and cultured under light conditions to induce shoot formation.

The concentration of BA may be 0.8 to 1.2 mg / L, preferably 1.0 mg / L. The concentration of IBA may be 1.0 to 2.0 mg / L, preferably 1.5 mg / L. The concentration of AgNO ₃ may be 4.0 to 6.0 mg / L, preferably 5.0 mg / L. The pH of the culture medium can be adjusted to 5.6 to 6.0, preferably to pH 5.8. The incubation period may be 4 to 5 weeks, preferably 4 weeks.

In the method of regenerating chrysanthemum plants using the chrysanthemum leaf section of the present invention, in step 3), the shoots formed are separated from leaf slices and transferred to MS medium to induce elongation and rooting and differentiate into plants. The pH of the culture medium can be adjusted to 5.6 to 6.0, preferably to pH 5.8.

Therefore, the chrysanthemum plant regeneration method using the chrysanthemum leaf section of the present invention is preferably,

1) Leaf slices of chrysanthemum were cultured on MS medium supplemented with 0.1 ~ 0.3 mg / L of BA, 0.8 ~ 1.2 mg / L of IAA and 0.8 ~ 1.2 mg / L of 2,4-D, ;

2) The induced eye was transferred to MS medium supplemented with 0.8 to 1.2 mg / L of BA, 1.0 to 2.0 mg / L of IBA and 4.0 to 6.0 mg / L of AgNO ₃ and cultured under light conditions to induce shoot formation ; And

3) The shoots thus formed are separated from leaf slices and transferred to MS medium to induce kidney and rooting to differentiate into plants.

More preferably,

1) Chrysanthemum leaves were cultured on MS medium supplemented with 0.1-0.3 mg / L of BA, 0.8-1.2 mg / L of IAA and 0.8-1.2 mg / L of 2,4-D at a temperature of 25 ± 2 ℃ for 3 ~ Culturing for 4 weeks under dark conditions to induce buds;

2) The induced eye was transferred to an MS medium supplemented with 0.8 to 1.2 mg / L of BA, 1.0 to 2.0 mg / L of IBA and 4.0 to 6.0 mg / L of AgNO _3, and then incubated at 25 ± 2 ° C for 4 to 5 weeks Lt; / RTI > to induce shoot formation; And

3) Separation of the shoots formed from the leaf slices and transferring them to MS medium to induce kidney and rooting to differentiate into plants, but the present invention is not limited thereto.

The present invention also provides a chrysanthemum plant regenerated by the above method. Preferably, the chrysanthemum is a 'white horse' variety.

In addition, the present invention relates to a method for producing a leaf of a chrysanthemum "White Horse" comprising an MS medium supplemented with 0.1 to 0.3 mg / L of BA, 0.8 to 1.2 mg / L of IAA and 0.8 to 1.2 mg / Thereby providing a bud-inducing medium composition from the fragment.

In addition, the present invention provides a method for producing a chrysanthemum 'white horse' leaf segment containing an MS medium supplemented with 0.8 to 1.2 mg / L of BA, 1.0 to 2.0 mg / L of IBA and 4.0 to 6.0 mg / L of AgNO ₃ as an active ingredient And a culture medium for inducing shoot formation from the formed bud.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Plant material and shoot cultivation

Leaf slices obtained from in vitro shoot cultivation of Chrysanthemum morifolium 'white horse' (FIG. 1A) were used as material for plant regeneration experiment. The medium was prepared by adding MS salts and vitamins (Murashige and Skoog 1962), sucrose 30 g / L and agar 8 g / L, and adjusting the pH to 5.8. Cultivation was carried out in a culture room at 25 ± 2 ° C under fluorescent light at 32 μmol · m ^-2 · s ^-1 and 16/8 h light period, and subcultured at 4-6 week intervals.

2. Shrub organ behavior from leaves

Experiments were conducted to determine the conditions suitable for regeneration of shoots from leaves obtained from in vitro cultured shoots with different growth media and culture periods depending on the plant growth regulator and AgNO ₃ concentration. After fully developed leaves were cut from the upper part of the in-flight plant cultured for 4-6 weeks, the sections were cut into 4 sides with 8-10 mm ² size including the main stem, so that the anti-axis surface reached the medium (Fig. 1D ). All cultures were carried out at 25 ± 2 ° C and under light conditions of 32 μmol · m ^-2 · s ^-1 , 16/8 h photoperiod.

3. Plant growth regulators and AgNO ₃ Induction of shoot organogenesis in single medium supplemented with

To determine the effects of plant growth regulators and AgNO ₃ on the formation of shoot organisms from leaf slices, BA (6-benzyladenine) was added to MS basal medium (MS salts and vitamins, sucrose 30 g / L and agar 8 g / 0.5 mg / L or 1.0 mg / L of indole-3-butyric acid (IBA), 1.0 mg / L or 1.0 mg / L of α-naphthaleneacetic acid, L or 2.0 mg / L, 2,4-D (2,4-dichlorophenoxyacetic acid) 1.0 mg / L or 2.0 mg / L and AgNO ₃ 5.0 mg / L, 10.0 mg / L or 15.0 mg / The experiment was performed in combination (Table 1). Leaf slices were streaked on the medium and cultured for 4 weeks under dark condition. Then, buds were induced and shoot formation was induced by incubation.

4. Induction of shoot bud formation by culture medium and culture period of 2 stage medium for Bud induction and shoot formation

Experiments were carried out using a two-step culture medium for bud induction and shoot formation from leaf slices. In the eye induction, 0.2 mg / L of BA, 1.0 mg / L of IAA and 1.0 mg / L of 2,4-D were added to the MS base medium (MS salts and vitamins, sucrose 30 g / L and agar 8 g / Medium supplemented with 1.0 mg / L of BA, 1.5 mg / L of IBA, 5.0 mg / L of AgNO ₃ , 1.0 mg / L of BA, 1.0 mg / L of NAA and AgNO ₃ 5.0 mg / L. The leaf slices were dipped in an eye-induced medium, and the slices were transferred to three different shoot-forming media at intervals of 1-4 weeks while culturing under dark conditions. After incubation for 4 weeks in the dark for 4 weeks, the shoot-forming medium in which the slices were streaked was transferred to healthy condition and cultured for 4 weeks (Table 2).

5. Experimental Section and Investigation

All treatments were repeated 5 times per treatment with 6 replicates per culture dish, one at a time, and placed in a completely random manner. After 8 weeks of culture, the survival rate, callus formation rate, eye formation rate, number of eyes per cut slice, shoot formation rate and number of shoots per cut slice were examined. Respectively.

6. Rooting and rooting

Extracted shoots of at least 1 cm in length were separated from the reconstructed shoots and transferred to shoot culture medium (MS salts, vitamins, sucrose 30 g / L, agar 8 g / L) to induce kidney and rooting. Successfully roots were taken out of the cabin, and the medium adhered to the roots was removed. Then, the roots were transferred to a 128-well plug tray containing the culture soil (Dukshim, Kwonwoo Bio) to induce activation in the long day condition (16 / 8h) . After 2 weeks, the plant was placed in a 12 cm diameter flowerpot containing the culture soil and cultivated in a greenhouse.

Example  1. Leaves From the fragment Shinshu  Organ formation

In order to induce plant regeneration through organ formation from leaf blade of Chrysanthemum 'white horse', experiments were carried out with different culture media and incubation periods including plant growth regulator and AgNO ₃ . AgNO ₃ is a potential inhibitor of ethylene action. After 2-3 weeks of cultivation, callus bearing a pale yellow color began to form mainly along the cut surface in the leaf slice cultured in the dark condition by denting on the shoot organ formation medium. After 4 weeks of culture, the cells were cultured under light conditions, and it was confirmed that buds were formed after 6 weeks of culture (FIG. 1B, E).

In general, the plant regeneration protocol of chrysanthemum shows that shoot shoots are induced in the shoot organ formation medium, and then regenerated shoots are separated, and then the shoots are obtained from the kidney and root induction medium, and then the seedlings are allowed to undergo normal planting. Successful results have been reported in many studies on the induction of shoot organ formation on the basis of the combination of auxin and cytokinin in various genotypes, since the shoot induction stage plays an important role in determining the efficiency of the whole process in such plant regeneration. In addition, the in vitro culture of plants shows a considerable difference according to the genotype. It is known that even in the case of chrysanthemum which is relatively easy to regenerate by in vitro culture, there is a large difference in the shoot formation reaction depending on the genotype. In previous studies, shoot regeneration was induced in 6 different varieties of chrysanthemum, but the regeneration rate was about 20.0% in 3 varieties, but not in 2 varieties (Song et al., 2011, Plant Cell Tiss. Org. Cult., 107: 295-304).

Example 2. Plant growth regulator and AgNO ₃ Induction of shoot organogenesis in single medium supplemented with

MS medium supplemented with plant growth regulator and AgNO ₃ was added to 11 different mediums and cultured for 8 weeks. Then, the reaction from the cut pieces was examined. As a result, The overall survival rate of the interspecies was high, and the callus formation rate was over 70.0% in all treatments (Table 1). There was a significant difference between the treatments in terms of the eye formation rate and the number of eyes per slice, and the rate of eye formation was higher than that in the treatment with 2,4-D as auxin (60.0%).

Among the all treatments, the best results were obtained with 96.7% of eyes and 4.1 of eyes per slice in the treatment of addition of BA 0.2 mg / L, IAA 1.0 mg / L and 2,4-D 1.0 mg / L to MS base medium (Table 1). However, despite the formation of numerous eyes in all treatments, they did not develop into shoots, indicating that it is difficult to induce shoot formation from leaf slices in single medium treated with plant growth regulators and AgNO ₃ .

Table 1. Chrysanthemum 'White Horse' Leaf fragments from the body of the affected plant growth regulators on shoot organogenesis and AgNO ₃ (cultured for 8 weeks)

* ^z 4 weeks incubation and 4 weeks culture

* ^y Mean ± standard error

In the present invention, in the treatment of plant growth regulators of 11 combinations of oxyne and cytokinin, chrysanthemum 'white horse' induced snowing from the leaf segment, but snow did not develop into shoots. In addition, the rate of snow formation was significantly different depending on the plant growth regulator composition. Low concentrations of cytokinin (BA 0.2 mg / L) and high concentration of auxin (IAA 1.0 mg / L, 2,4-D 1.0 mg / The highest induction rate was 96.7% in the supplemented medium. This suggests that a higher concentration of auxin is required than that of cytokinin and that the treatment with IAA and 2,4-D is effective in the induction of the eye from the leaf segment in the white horse, It was judged that a separate step was needed for development.

Example  3. Eye induction and Shinshu  A two-stage medium composition for formation During the incubation period  Following Shinshu  Induction of organ formation

In order to investigate the composition of the different two-stage medium for eye induction and shoot formation and the optimal conditions for the incubation period, the eye-induced medium (BA 0.2 mg / L, IAA 1.0 mg / L and 2,4-D 1.0 mg / L MS medium), leaf cultures were cultured for 1-4 weeks to induce eye formation. Three kinds of shoot formation medium (no plant growth regulator, BA 1.0 mg / L, IBA 1.5 mg / L and AgNO ₃ 5.0 mg / L or BA 1.0 mg / L, NAA 1.0 mg / L and AgNO ₃ 5.0 mg / L), and the shoots were cultured for 4-7 weeks to induce shoot formation. Regardless of the culture period in the eye - induced medium, the rate of eye formation was low in the transplanted body without the plant growth regulator, and no shoot formation occurred. After the cultivation in the eye - induced medium, the number of eyes and the number of eyes per slice increased in the treatment group transferred to the shoot growth medium containing the plant growth regulator. After 4 weeks of incubation in the eye induction medium, when the sections were transferred to a medium supplemented with 1.0 mg / L of BA, 1.5 mg / L of IBA and 5.0 mg / L of AgNO ₃ to the MS base medium, the oocyte formation rate was 100.0% The best results were obtained with 4.2 eyes (Table 2). In addition, shoot formation from the eyes was achieved when cultured in the eye-induced medium for 3 weeks or more and then transferred to the shoot-forming medium supplemented with the plant growth regulator (Fig. 1C, F). Comparing the incubation period in the eye - induced medium, shoot growth rate and shoot number per shoot increased regardless of the composition of plant growth regulator in the cultivation for 4 weeks compared to 3 weeks. Among all the treatments, the slices were incubated for 4 weeks in an eye-induced medium, followed by incubation in a basal medium supplemented with 1.0 mg / L of BA, 1.5 mg / L of IBA and 5.0 mg / L of AgNO ₃ for 4 weeks The highest shoot growth rate was 50.0% and the number of shoots per slice was 1.6 (Table 2, Fig. 2).

Table 2. Effects of medium composition and incubation period on the formation of shoot organ from chrysanthemum "White horse" leaf slice (8 weeks after culture)

* ^z 4 weeks incubation and 4 weeks culture

* ^y Mean ± standard error

Example 4. Growth of shoots and shoots

When shoots were formed from the leaf intercalation through the eye induction stage and shoots were formed in the shoot formation medium and cultured for 8 weeks without subculture, the shoots were elongated and some shoots were rooted (Fig. 1G). When the elongated shoots of more than 1 cm were separated from the sectioned shoots and cultured in the shoot cultivation medium, roots began to develop from 1 week after the incubation, and after 2 weeks, the elongated shoots and roots of the shoots were obtained 1H). It has been confirmed that after a 2 cm-long purifying process, the microorganisms in the cabin were transplanted into soil, and the microorganisms were successfully activated (Fig. 1I). When the plant was planted in a greenhouse with planted potted plants, the plant exhibited a normal phenotype.

Claims (12)

1) Chrysanthemum leaves were cultured on MS medium supplemented with BA (6-benzyladenine), IAA (indoleacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) Inducing;
2) transferring the induced eye to MS medium supplemented with BA (6-benzyladenine), IBA (indole-3-butyric acid) and AgNO ₃ , and culturing under normal conditions to induce shoot formation; And
3) Separating the shoots from the leaf slices and transferring them to MS medium to induce kidney and rooting and differentiate into plants. The method for regenerating chrysanthemum plants using the leaves of chrysanthemum. [3] The method according to claim 1, wherein the concentration of BA used in the step 1) is 0.1 to 0.3 mg / L. [2] The method according to claim 1, wherein the IAA concentration used for eye induction in step 1) is 0.8 to 1.2 mg / L. [2] The method according to claim 1, wherein the 2,4-D concentration used for eye induction in step 1) is 0.8 to 1.2 mg / L. [2] The method according to claim 1, wherein the BA concentration used in the step 2) is 0.8 to 1.2 mg / L. [2] The method according to claim 1, wherein the IBA concentration used in the step 2) is 1.0 to 2.0 mg / L. [2] The method according to claim 1, wherein the concentration of AgNO ₃ used in step 2) is 4.0 to 6.0 mg / L. The method according to claim 1,
1) Leaf slices of chrysanthemum were cultured on MS medium supplemented with 0.1 ~ 0.3 mg / L of BA, 0.8 ~ 1.2 mg / L of IAA and 0.8 ~ 1.2 mg / L of 2,4-D, ;
2) The induced eye was transferred to MS medium supplemented with 0.8 to 1.2 mg / L of BA, 1.0 to 2.0 mg / L of IBA and 4.0 to 6.0 mg / L of AgNO ₃ and cultured under light conditions to induce shoot formation ; And
3) Separating the shoots from the leaf slices and transferring them to MS medium to induce kidney and rooting to differentiate into plants. The method for regenerating chrysanthemum plants according to claim 1, 9. The method according to claim 8, wherein the chrysanthemum is a 'white horse'. 10. A chrysanthemum plant regenerated by the method of any one of claims 1 to 9. The extract from the leaves of the chrysanthemum 'white horse', containing the MS medium supplemented with 0.1 to 0.3 mg / L of BA, 0.8 to 1.2 mg / L of IAA and 0.8 to 1.2 mg / (bud). (Bud) of leaves of chrysanthemum 'white horse' containing an MS medium supplemented with 0.8 to 1.2 mg / L of BA, 1.0 to 2.0 mg / L of IBA and 4.0 to 6.0 mg / L of AgNO ₃ as an active ingredient, To form a shoot formation.

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