KR101925316B1 - Medium for callus induction or callus proliferation of Acer takesimense and a method for callus induction or callus proliferation using thereof - Google Patents

Abstract

The present invention provides a callus inducing or proliferating medium of Acer takesimense . The present inventors have established culture conditions suitable for massive tissue culture of island maple by inducing callus from maple trees and revealing culture conditions suitable for callus proliferation. As a result, it is possible to mass-produce island maple trees which are difficult to mass-produce due to their regional characteristics.

Description

[0001] The present invention relates to a callus inducing or proliferation medium for the callus induction or proliferation using an islet of the present invention, and a callus induction or proliferation method using the same,

The present invention relates to a medium for inducing or proliferating callus of maple and an induction or propagation method of callus using the same.

Ulleungdo is a volcanic island located on the East Sea between the Korean Peninsula and Japan. It has a geographical location separated from the inland, and has warm and humid climates. Ulleungdo's botanical gardens are separated from the Korean Peninsula and separated into Ulleungdo's botanical gardens, and there are 750 species of plants such as jelly, juniper, and camellia (Lee et al., 2007). Ulleungdo's native plants have high functional activity such as antioxidant and anti-inflammatory activity, and thus have high value as functional materials that can be used variously in the field of cosmetics and pharmaceuticals (Kim et al., 2013).

Acer takesimense is a plant native to Ulleungdo. It has various flavonoid contents such as flavonols and flavones (Chang and Giannasi, 1991). However, island maple trees have a great difficulty in mass production because of their local characteristics that they are native to the islands.

Plant cells and tissue cultures have been undergoing various studies as effective production methods in the mass production of active materials of plants. Tissue culture has the advantage of selecting genetically superior individuals and continuously delivering uniform fuel without affecting soil, climate, etc. (Roberts 2007, Dagla 2012, Negi and Saxena 2011).

Therefore, in the present invention, a culture condition suitable for callus induction and callus growth from island maple is established.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to establish culture conditions suitable for massive tissue culture of Acer takesimense with high flavonoid content. As a result, the present inventors have found that callus induction or proliferation effect varies depending on the kind and concentration of a plant growth regulator, the kind of a medium, and a medium supplement, and as a result, culture conditions suitable for induction or proliferation of an island maple . In the present invention, the survival rate of callus, callus induction rate, and adventitious root induction rate in each culture condition were measured to confirm culture conditions optimized for callus induction and proliferation, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide callus induction medium of Acer takesimense .

Another object of the present invention is to provide a callus proliferation medium of Acer takesimense .

It is another object of the present invention to provide a method for inducing callus of Acer takesimense .

It is another object of the present invention to provide a method of propagating callus of Acer takesimense .

Another object of the present invention is to provide acer isimense plant.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a method of inducing callus induction of Acer takesimense comprising Murashige and Skoog (MS) as a basic medium and containing sucrose, gelrite and a plant growth regulator callus induction medium.

According to another aspect of the present invention, the present invention provides a method for promoting callus growth of Acer takesimense comprising Murashige and Skoog (MS) as a basic medium and containing sucrose, gelrite and a plant growth regulator callus proliferation medium.

The inventors of the present invention have conducted intensive studies to establish culture conditions suitable for massive tissue culture of Acer takesimense having a high content of flavonoids. As a result, they have found that callus induction or proliferation can be induced depending on the type and concentration of plant growth regulator, As a result, culture conditions suitable for the callus growth of the island maple were established. In the present invention, the culture conditions optimized for callus induction and proliferation were determined by measuring the callus survival rate, callus induction rate, and adventitious root induction rate in each culturing condition.

The Acer takesimense used in the present invention can be obtained by direct cultivation / harvesting of a specific plant of Ulleungdo Island. According to the present invention, the germination rate of island maple seeds was 16%, and the rate of in vitro shoot formation through the primary culture was 44%. The primary culture conditions of the present invention followed the primary culture conditions of the research papers published by the present inventors in 2015 (Park JA, Park BJ, Kim AH, Park SY, Paek KY (2015) Compound Production from In Vitro Culture of Vitis flexuosa Plantlets. Hort. Environ. Biotechnol. 56 (3): 358-365).

As used herein, the term " plant growth regulator " means an organic compound that is not a nutrient but a substance that inhibits, promotes, or regulates plant physiological processes in a small amount. Plant growth regulators include, for example, auxin, cytokinin, gibberellin, ethylene, and abscisic acid. Among these, auxin is known to be mainly involved in root formation, and 2,4-dichlorophenoxyacetic acid (2,4-D), -naphthaleneacetic acid (NAA) and indoleacetic acid (IAA) In the case of 2,4-D, it generally affects the cell growth of auxin in general. Cytochinin is an essential growth regulator for plant cell cultures together with auxin such as 6-furfurylamino purine, 6-BAP (6-benzylamino purine), zeatin and 2-ip. Among them, kinetin is a substance isolated from sperm DNA of high pressure sterilized sardine. It is known that kinetin is highly effective in promoting mitosis and cell division in tissue culture of tobacco callus. Japanese Laid-Open Patent Publication No. 1988-245668 discloses a plant growth effect of gibberellin.

The callus induction medium of Acer takesimense of the present invention is characterized by containing MS (Murashige and Skoog) as a basic medium and sucrose, gelrite and a plant growth regulator do.

According to one embodiment of the present invention, the plant growth regulator contained in the callus inducing medium of the present invention is 2,4-dichloro-phenoxyacetic acid (2,4-D), alpha-naphthaleneacetic acid (NAA) Benzyl adenine (BA). According to another embodiment of the present invention, the callus inducing medium of the present invention is a plant growth regulator comprising 2,4-dichloro-phenoxyacetic acid (2,4-D), alpha-naphthaleneacetic acid (NAA) (BA) can be contained in concentrations of 0.2-0.8 mg / L, 0.2-0.8 mg / L and 0.05-0.15 mg / L, respectively. According to a specific embodiment of the present invention, the callus inducing medium of the present invention contains 2,4-dichloro-phenoxyacetic acid (2,4-D), alpha-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) at concentrations of 0.5 mg / L, 0.5 mg / L and 0.1 mg / L, respectively.

According to one embodiment of the present invention, sucrose contained in the callus inducing medium of the present invention may be contained at a concentration of 25-33 g / L and gelite at 1.8-2.8 g / L. According to a particular embodiment of the present invention, the callus inducing medium of the present invention comprises 30 g / L of sucrose, 2.3 g / L of gelrite, and a plant growth regulator.

According to one embodiment of the present invention, the callus inducing medium is 2,4-dichloro-phenoxyacetic acid (2,4-D), alpha-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) 0.2-0.8 mg / L and 0.05-0.15 mg / L, respectively.

According to the present invention, the callus inducing medium of the present invention is prepared by adding sucrose, gelite and plant growth regulator to MS (Murashige and Skoog, 1962), adjusting the HP to 5.8, Lt; 0 > C for 20-30 minutes).

On the other hand, the callus proliferation medium of Acer takesimense of the present invention contains MS (Murashige and Skoog) as a basic medium and includes sucrose, gelrite and a plant growth regulator .

According to one embodiment of the present invention, the plant growth regulator contained in the callus propagation medium of the present invention is 2,4-dichloro-phenoxyacetic acid (2,4-D). According to another embodiment of the present invention, the callus inducing medium of the present invention may contain 2,4-dichloro-phenoxyacetic acid (2,4-D) as a plant growth regulator at a concentration of 0.5-1.5 mg / L . According to another embodiment of the present invention, the callus inducing medium of the present invention may contain 2,4-dichloro-phenoxyacetic acid (2,4-D) at a concentration of 1 mg / L as a plant growth regulator.

According to one embodiment of the present invention, sucrose contained in the medium for callus proliferation of the present invention may be contained at a concentration of 25-33 g / L, gelite at 1.8-2.8 g / L. According to a particular embodiment of the present invention, the callus propagation medium of the present invention comprises 30 g / L of sucrose, 2.3 g / L of gelrite and a plant growth regulator.

The callus growth medium of the present invention may further include citric acid or ascorbic acid in order to improve callus growth rate. As in the following examples, the citric acid or ascorbic acid treatment group had a high friable callus induction rate and a low browning rate. On the other hand, the inositol and casein hydrolyzate treatment groups had high browning rates of 28% and 36%, respectively, and were not suitable for callus growth (see Table 6).

It should be apparent to those skilled in the art that the components of the callus inducing / proliferating medium of the present invention may contain substances that directly or indirectly affect the growth of the plant, in addition to the components described above. For example, a high concentration of carbon sources (sucrose, lactose, fructose, glucose, etc.) in the medium may be added to increase the final volume productivity of the high-concentration cell culture and the secondary metabolite.

According to another aspect of the invention there is provided the island maple (Acer The present invention also provides a method for inducing callus induction of an islet maple tree comprising culturing a cut section of a plant of the present invention in a callus inducing medium of the present invention as described above.

According to another aspect of the invention there is provided the island maple (Acer The present invention also provides a method for propagating callus of an island maple, comprising culturing the callus derived from a plant of the present invention in a callus growth medium of the present invention as described above.

The callus induction or propagation method of the present invention is a method of using the callus induction or propagation medium of the present invention described above, wherein the description common to the above-mentioned medium is omitted in order to avoid the excessive complexity of the present specification.

According to another aspect of the invention there is provided the island maple (Acer Taking a fraction of a plant-derived plantlet in the medium of claim 1 to induce a callus; Growing the callus by culturing the callus in the medium of claim 5; Culturing the callus to induce a shoot; And culturing the shoots in a rooting medium to produce a roots-shaped article . The present invention also provides a method for producing an Acer takesimense plant.

Acer maple of the present invention takesimense ) is a method of using the callus induction / propagation medium and the callus induction / propagation method of the present invention described above, wherein the common content in relation to the medium or method is to avoid the excessive complexity of the present invention, The description is omitted.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a callus inducing or proliferating medium of Acer takesimense .

(b) The inventors of the present invention have established culture conditions suitable for massive tissue culture of island maple by inducing callus from maple of island and revealing culture conditions suitable for callus proliferation.

(c) This makes mass production of island maple trees difficult to mass-produce due to their regional characteristics.

Figure 1 shows in vitro seeding and primary culture results of the maple seeds of the islets. (A: maple seeds of the island, B: germination, C: maple trees cultivated outdoors, D: in-flight culture seedlings)
Figure 2 shows the callus induction results of the Island maple. A. 2,4-D 1 mg / L; B. 2,4-D 0.5 mg / L + NAA 0.5 mg / L; C. 2,4-D 0.5 mg / L + NAA 0.5 mg / L + BA 0.5 mg / L.
Figure 3 shows the effect of plant growth regulators on the growth of islet maple callus. A. PGR free; B. 2,4-D 1 mg / L; C. NAA 1 mg / L; D. 2,4-D 0.5 mg / L + NAA 0.5 mg / L + Kinetin 0.5 mg / L. The red circle of B represents the friable callus.
Fig. 4 shows the results of callus propagation of the maple tree according to the kind of the medium. A. MS; B. WPM; C. B5; D. SH
Fig. 5 shows the results of the callus propagation of the island maple with additives (addctives). A. Citric acid; B. inositol; C. ascorbic acid; D. Casein hydrolyzate.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Methods

Plant material

 Mature seed of the maple tree was used as the experimental material. Seed removed from seed coat was immersed in 70% ethanol for 30 seconds, and the surface of seed was sterilized in 2% (w / v) NaOCl for 15 minutes. Surface sterilized seeds were washed three times with sterilized water. The surface-sterilized seeds were germinated on a MS medium supplemented with 30 g / L sucrose. In addition, primary plants of the maple trees cultivated in the greenhouse were collected and primary cultures were carried out. The seedlings collected from the greenhouse were sterilized in the same manner as the seed disinfection process and subjected to MS medium supplemented with 0.1 mg / L of 6-benzyladenine (BA) and 30 g / L of sucrose.

Induction of callus

The cotyledon of the seedlings germinated in the cabin was cut into a size of 5 x 5 mm, and the callus was induced in the medium. The callus induction medium was prepared by adding 30 g / L of sucrose, 2.3 g / L of gelrite and a plant growth regulator to MS (Murashige and Skoog, 1962), adjusting the HP to 5.8, Lt; / RTI > and sterilized in a Petri dish. The plant growth regulators are 2,4-dichloro-phenoxyacetic acid (2,4-D) and 2,4-D, alpha-naphthaleneacetic acid (NAA), 2,4-D, NAA and thidiazuron , TDZ). The leaves of the maple leaves were repeatedly plucked five times per petri dish. Four weeks later, survival rate, callus induction rate, adventitious roots induction rate and number of adventitious roots were examined.

Effects of Plant Growth Regulators (PGRs) on callus proliferation

To determine the type and concentration of plant growth regulators suitable for the callus growth of island maple trees, 30 g / L sucrose and 2.3 g / L gellite were added to MS medium and 2,4-D, NAA, 6- Benzyladenine (BA) and kinetin were added to the medium alone or mixed (Table 1). The callus was repeated 4 times per petri dish for 9 weeks, and cultured for 3 weeks in a dark condition incubator. After 3 weeks, friable callus induction, callus diameter, browning rate, and adventitious root induction were investigated.

Plant Growth Regulators (PGRs) Treatment for Callus Growth of Island Maple NO PGRs (mg / L) 2,4-D NAA BA Kinetin One 0 0 0 0 2 1.0 0 0 0 3 2.0 0 0 0 4 1.0 0 0.1 0 5 1.0 0 0 0.1 6 0 1.0 0 0 7 0 2.0 0 0 8 0 2.0 0.1 0 9 0 2.0 0 0.1 10 0.5 0.5 0.1 0 11 0.5 0.5 0 0.1

Effect of media type on callus proliferation

MS, Murashige and Skoog, WPM, B5, and SH were used for the selection of medium suitable for the callus growth of the island maple. All media were supplemented with 1 mg / L 2,4-D, 30 g / L sucrose and 2.3 g / L gellite, and the callus was plated five times per petri dish and repeated five times. After 3 weeks, friable callus induction, callus diameter, browning rate, and adventitious root induction were investigated.

Effect of Additives in Media on the Growth of Callus

Citric acid, ascorbic acid, casein hydrosylate and inositol were each treated with 1 g / L in the medium to improve the callus growth rate. The medium used was MS medium supplemented with 1 mg / L of 2,4-D, 30 g / L of sucrose and 2.3 g / L of gelite. The callus was plated 5 times per petri dish and was repeated 5 times. The callus was cultured for 3 weeks under the arm culture condition. After 3 weeks, friable callus induction, callus diameter, browning rate, and adventitious root induction were investigated.

Experiment result

In-flight sowing and initial culture

As a result of sterilization of the maple seeds and seeding in the cabin, 18 seeds were contaminated out of 37 seeds and 19 seeds survived. Of these, only six seeds germinated and developed into a seedling plant (Table 2 and Fig. 1). Therefore, the germination rate of island maple seeds was 16%.

As a result of preincubating the shoots of the maple trees cultivated in the greenhouse, 15 of the 25 interspecies survived and the shoots differentiated in 11 interspecies. Therefore, the incidence of in vitro shoots was 44%.

In vitro seeding and primary culture results of Acer takesimense process Inoculation
(No. of explant) survival Shoot formation
(Shoot formation) Count % Count % Seeding 37 19 51 6 16 Invitation culture
(Primary culture) 25 15 60 11 44

Induction of callus

To investigate the effect of plant growth regulator type and concentration on the callus induction efficiency from cotyledon sections of island maple trees, 2,4-D, NAA and BA were treated singly or in combination. As a result, the survival rate and callus induction rate of the slices were the highest at 100.00% and 95.83% in the mixed treatment of 2,4-D, NAA, and BA, respectively (Table 3). In addition, the callus volume induced by 2,4-D, NAA, and BA mixed treatment was the largest (Fig. 2). On the other hand, 2,4-D and 2,4-D and BA combination treatments have lower callus induction rate and higher adventitious root incidence than 2,4-D, NAA and BA combination treatments, I did.

Influence of Plant Growth Regulators (PGRs) on Induced Maple Callus Induction PGRs (mg / L) Survival rate (%) Callus induction rate (%) Root induction rate (%) Adiposity number (ea) 2,4-D NAA BA One 0 0 95.29 + - 2.59 59.63 + - 12.83 58.23 ± 9.06 2.73 ± 0.66 0.5 0.5 0 78.33 + - 9.87 11.67 + - 6.60 51.67 + - 10.38 5.74 ± 1.47 0.5 0.5 0.1 100.00 ± 0.00 95.83 + - 3.90 25.35 + - 11.68 3.16 ± 0.86

Effects of Plant Growth Regulators on the Growth of Callus

A variety of plant growth regulators were treated at different concentrations to determine the type and concentration of plant growth regulators suitable for the growth of calli of the Island maple (Table 4). As a result, the friable callus induction rate of 36.0% was highest in the 2,4-D 1 mg / L treatment group, and the callus diameter was 1.08 cm in diameter. In addition, the browning rate was 8.33%, which is the lowest among the treatment groups. On the other hand, the most treated group had a white callus color, but the callus was hard and the adventitious roots were not formed. The treated group in which 2,4-D of auxin and NAA and the BA or kinetin of cytokinins were mixed showed a high browning rate and was not suitable for callus proliferation (FIG. 3).

Effect of Plant Growth Regulators (PGRs) on Callus Propagation in Island Maple Plant growth regulator (mg / L) Friable callus induction rate (%)
Callus diameter (cm)
Browning rate (%)
Root induction rate (%)
2,4-D NAA BA Kinetin 0 0 0 0 0.00 ± 0.00 0.63 + - 0.23 33.33 + - 22.68 0.00 ± 0.00 1.0 0 0 0 36.11 ± 14.61 1.08 ± 0.11 8.33 + - 5.32 0.00 ± 0.00 2.0 0 0 0 2.78 ± 2.78 0.93 + 0.03 16.67 ± 3.21 8.33 + - 2.78 1.0 0 0.1 0 22.22 ± 12.00 0.85 ± 0.05 8.33 + - 2.78 0.00 ± 0.00 1.0 0 0 0.1 2.78 ± 2.78 0.85 ± 0.05 11.11 + - 4.54 0.00 ± 0.00 0 1.0 0 0 0.00 ± 0.00 0.86 ± 0.05 8.33 + - 2.78 8.33 + - 5.32 0 2.0 0 0 0.25 0.25 0.90 + - 0.05 16.67 ± 7.12 13.89 ± 5.32 0 2.0 0.1 0 27.78 ± 13.98 0.89 + 0.03 16.67 + - 7.17 8.33 + - 5.31 0 2.0 0 0.1 13.89 ± 5.32 0.80 + 0.09 8.33 + - 11.11 13.89 ± 13.89 0 0.5 0.1 0 11.11 + - 7.86 0.68 ± 0.01 22.22 + - 11.11 0.00 ± 0.00 0 0.5 0 0.1 5.56 ± 3.21 0.83 0.06 41.67 8.33 11.11 + - 4.54

Effect of media type on callus proliferation

To investigate the optimum culture media suitable for callus growth, various media were cultured and cultured for 3 weeks. As a result, friable callus was generated in MS medium, and the rate of browning and adventitia formation were 16% and 12%, respectively, which were lower than those of the other treatment groups. On the other hand, the WPM medium had a 52% higher adventitious formation rate and a lowest callus diameter of 0.86 cm. In addition, the B5 medium had the highest browning rate of 52% and was not suitable for callus culture (FIG. 4).

Influence of Medium on Islet Callus Propagation in Island process Friable callus induction rate (%) Callus diameter (cm) Browning rate (%) Adrenaline induction rate
(%) MS 4.00 + - 4.00 1.13 + 0.04 16.00 + - 7.48 12.00 ± 8.00 WPM 8.00 ± 4.90 0.86 + 0.04 36.00 + - 7.48 52.00 ± 33.56 B5 8.00 ± 4.90 1.16 + 0.04 52.00 ± 1 2.00 12.00 ± 8.00 SH 0 1.10 + 0.03 28.00 ± 4.90 20.00 ± 6.32

Effect of Additives in Media on the Growth of Callus

To improve callus growth rate, citric acid, ascorbic acid, casein hydrosylate and inositol were each treated with 1 g / L of MS medium for 3 weeks. As a result, the friable callus induction ratio and callus diameter were the highest in the citric acid-treated group, which was 28% and 1.45 cm, respectively (Table 6). In addition, browning rate and adventitious roots induction rate were the lowest, 8% and 16%, respectively, and were suitable for callus proliferation (FIG. 5). Ascorbic acid treatment group had higher friable callus induction rate and lower browning rate than citric acid treatment group. In the inositol and casein hydrolyzate treatment groups, the browning rates were as high as 28% and 36%, respectively, and were not suitable for callus growth.

Effect of additives (addictives) on callus proliferation of island maple process Friable callus induction rate (%) Callus diameter (cm) Browning rate (%) Adrenaline induction rate
(%) Citric acid 28.00 ± 13.56 1.45 + 0.04 8.00 ± 4.90 16 Inositol 8.00 ± 4.90 1.39 + 0.02 28.00 ± 4.90 4.00 + - 4.00 Ascorbic acid 12.00 ± 8.00 1.16 + 0.04 14.14 ± 6.32 0 Casein hydrolyzate 4.00 + - 4.00 0.96 + - 0.05 36.00 ± 11.67 28.00 ± 13.56

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

references

Chang, C. S., & Giannasi, D. E. (1991). Foliar flavonoids of Acer sect. Palmata series. Systematic Botany, 225-241.

Dagla, H. R. (2012). Plant tissue culture. Resonance, 17 (8), 759-767.

Lee, J., H., Cho, H., J., Lee, B., C., Oh, S. H., & Bae, K. H. (2007). Forest vegetation types and growth characteristics of Seongin-bong in Ulleung Island, Korea. Korean Journal of Agricultural and Forest Meteorology, 9 (1), 37-48.

Negi, D., & Saxena, S. (2011). PLANT TISSUE CULTURE. In Vitro Cell. Dev. Biol., Plant, 47, 604-610.

Roberts, S. C. (2007). Production and engineering of terpenoids in plant cell culture. Nature chemical biology, 3 (7), 387-395.

Kim, Hyun Joon, Dong Joon, Koo Jae Jung, Choi Kyung, Park Kwang Woo, Kang Shin Ho, ... & Lee Hyung Jae. (2013). Anti - Inflammatory Effects of Folk Plant Extracts from Ulleungdo. Korean J. Plant Res., 26 (2), 169-177.

Claims (12)

As callus induction medium,
The medium was prepared by using MS (Murashige and Skoog) as a basic medium and containing sucrose, gelrite and a plant growth regulator,
The sucrose is contained at a concentration of 25-33 g / L, the gelrite is contained at a concentration of 1.8-2.8 g / L,
The plant growth regulator may be selected from the group consisting of 0.2-0.8 mg / L 2,4-dichloro-phenoxyacetic acid (2,4-D), 0.2-0.8 mg / L alpha-naphthaleneacetic acid (NAA) Callus induction medium of Acer takesimense, a mixture of L 6-benzyladenine (BA).
delete delete delete As callus proliferation medium,
The above-
MS (Murashige and Skoog) as the basic medium, sucrose, gelrite and plant growth regulator,
The sucrose is contained at a concentration of 25-33 g / L, the gelrite is contained at a concentration of 1.8-2.8 g / L,
The plant growth regulator is 2,4-dichloro-phenoxyacetic acid (2,4-D)
The 2,4-D is a callus proliferation medium of Acer takesimense, which is contained at a concentration of 0.5 to 1.5 mg / L.
delete delete delete The culture medium according to claim 5, wherein the culture medium further comprises citric acid or ascorbic acid.
Acer maple Claims : What is claimed is: 1. A method for inducing callus induction in an island maple comprising cultivating a piece of a plant of the order Takeda plant, Takeshima plant, in a medium of claim 1.
Acer maple Claims : What is claimed is: 1. A callus propagation method of an islet maple comprising culturing a callus derived from a plant of the order Takeda plant,
Acer maple Taking a fraction of a plant-derived plantlet in the medium of claim 1 to induce a callus;
Growing the callus by culturing the callus in the medium of claim 5;
Culturing the callus to induce a shoot; And
Culturing the shoots in a rooting medium to produce roots;
Island maple ( Acer takesimense) method of the plant.

Images