KR100815436B1 - Method for mass propagation of oplopanax elatus nakai using root-derived somatic embryo - Google Patents

Abstract

A method for mass-producing an Oplopanax elatus Nakai plantlet is provided to establish an in vitro mass-production system of the Oplopanax elatus Nakai, thereby mass-propagating the Oplopanax elatus Nakai with useful medicinal effects but hard to be bred. A method for mass-producing an Oplopanax elatus Nakai plantlet comprises the steps of: (a) explanting a tissue segment of Oplopanax elatus Nakai roots in an MS culture medium to induce directly somatic embryos; (b) culturing the induced somatic embryos in a liquid culture medium or a solid culture medium to propagate them; (c) culturing the propagated somatic embryos in a liquid culture medium or a solid culture medium having a pH of 5.0-5.75 to redifferentiate a plantlet; and (d) acclimatizing the redifferentiated plantlet in a soil where bed soil and sand are mixed in a ratio of 1:1.

Description

Method for Mass Propagation of Oplopanax elatus Nakai Using Root-derived Somatic embryo}

Figure 1 shows the embryogenic callus formation rate in the medium containing 2,4-D and TDZ combination plant growth regulator using the leaf, stem, root tissue section

Figure 2 shows the embryogenic callus formation rate in the medium containing NAA and TDZ combination plant growth regulator using the tissue-specific sections,

Figure 3 shows the callus formation of the leaves, stems, roots in the medium containing NAA and TDZ combination plant growth regulator,

Figure 4 shows the growth rate of callus in the medium containing embryogenic callus 2,4-D by the method of the present invention,

Figure 5 shows the somatic embryo formed according to the presence or absence of plant growth regulators by suspension culture of the root tissue of the present invention,

Figure 6 shows the somatic embryo formed by suspension culture of the root tissue according to the type and concentration of the medium of the present invention,

Figure 7 shows the development of somatic embryos in suspension culture in accordance with the type and concentration of the medium of the present invention,

8 is a diagram showing a plant grown by suspension culture of embryogenic cells of the present invention in a medium having a different inorganic salt concentration,

9 is a diagram showing a plant grown by suspension culture of the embryogenic cells of the present invention in a medium containing a polyamine,

Figure 10 shows the induced shoot formation rate by suspension culture of embryogenic cells by varying the pH of the present invention,

Figure 11 shows the development of somatic embryos formed by suspending the root tissue of the elder tree of the present invention,

Figure 12 shows the re-differentiation according to the sucrose concentration of the seedlings obtained by culturing the germinated somatic cell embryo of the present invention in a solid medium,

Figure 13 shows the plants survived after transplanting the plants re-differentiated through the soil purification process of the present invention,

Figure 14 shows the growth characteristics of the ground portion of the plants that survived after transplanting the plants re-differentiated through the soil purification process of the present invention,

Figure 15 shows the growth characteristics of the underground part of the plants that survived after transplanting the plants re-differentiated through the soil purification process of the present invention.

The present invention relates to a method for mass-producing a seedling tree seedling using somatic embryos derived from tissues, and more specifically, using somatic embryos derived from embryogenic callus derived from the seedling plant tissues or directly derived from plant roots. The present invention relates to a mass production method of seedling seedlings, which are grown in large quantities and then re-divided and transplanted into soil.

Oplopanax elatus Nakai is a perennial deciduous broad-leaved shrub belonging to the genus Elm and Elm. The distribution is in a very narrow range and is known to be distributed in small amounts within three borders of Korea, China, and Russia (Wang et al., 1989). The elm tree is mainly used for dried roots and stems, and it has been reported that the roots and stems contain many saponins, volatile oils, flavonones, anthraquinones and fatty acids. It is reported that there is no significant difference between the components of root and stem (Wang et al., 2004).

It is known that ginseng tree is similar to ginseng, and the name of Chinese medicine is Jain ginseng (刺 人蔘), which is mainly used to help cure cardiovascular diseases, diabetes, customs, and to stimulate the central nervous system, nervous breakdown, hypotension, mental It has been shown to be effective in schizophrenia (Zhang et al., 1994), and volatile oils are known to be effective in convulsive antagonism, anti-aging, anti-inflammatory and antibacterial activity. Organic germanium, known to contain trace amounts of ginseng, ganoderma lucidum, and garlic, was extracted from the cedar, which has been reported to have surprising effects on diseases known as incurable diseases such as cancer and chronic adult diseases. It began to lose (Lee, 2004).

It is known to use asexual reproductive methods such as dispensing, burying, and folding because the seedlings have a low seed loss rate and even seed germination is very difficult (Wang et al., 1989). However, in asexual reproduction, comprehensive factors such as incompatibility due to infection of microorganisms, incompatibility of lumber and grafting, and differences according to age of grafting used as materials make the reproduction difficult and distribution area narrow and ecological colony There is a small back disadvantage. In addition, native wild elm tree is endangered by artificial indiscriminate overfishing, and is designated and managed as a second-class protector in Korea, China and Russia.

Therefore, the present inventors conducted a study for breeding the elm tree, which is difficult to obtain seeds, and confirmed that the somatic embryo derived from the elm tree tissue was able to multiply, thereby completing the present invention.

The present invention has been made in accordance with the above-described demands, and an object of the present invention is to provide a method for mass production of elm elm, which has useful pharmacological effects but is difficult to breed by establishing an in-flight mass production system of endangered elm seedlings. I would like to.

The present invention for achieving the above object relates to a mass production method of the seedling tree seedlings using somatic embryos derived from embryogenic callus derived from plant tissues, characterized in that it comprises the following steps.

(B) incubating the tissue sections of the Cabbage in vitro culture on MS solid medium to which plant growth regulators including TDZ are added and incubating the embryogenic callus;

(B) inducing somatic embryos by incubating the cultured embryogenic callus in a liquid medium and culturing;

증식 propagating the induced somatic cell culture in a liquid medium or a solid medium;

배양 re-differentiating seedlings by culturing the proliferated somatic cell medium in a liquid medium or a solid medium;

를 purifying the regenerated seedlings;

The medium in the first step of the present invention is characterized in that the MS medium to which 2,4-D or NAA is additionally added as a plant growth regulator.

The medium in the first step of the present invention is characterized in that it comprises a sucrose 10 to 30 mg / L level as a carbon source.

In addition, the present invention relates to a method for mass-producing a seedling tree seedling using somatic embryos directly derived from plant roots, characterized in that it comprises the following steps.

(B) directing somatic embryos directly by culturing and incubating the tissue sections of the roots of the elder tree in MS medium;

증식 multiplying the thus obtained somatic cell culture in a liquid medium or a solid medium;

배양 re-differentiating seedlings by culturing the proliferated somatic cell medium in a liquid medium or a solid medium;

를 purifying the regenerated seedlings;

The medium in the re-differentiation step of the present invention is characterized in that 1/3 MS medium or 1/4 MS medium added with sucrose at a level of 1% to 3% (w / v), the culture is 16 ℃ 25 It is characterized in that it is carried out in time light conditions and 8 hours dark conditions.

In addition, the pH of the medium in the regeneration step of the present invention is characterized in that the range of 5.0 to 5.75.

The purifying step of the present invention is characterized in that the soil and sand is carried out in a soil mixed with a composition of 1: 1.

Hereinafter, the configuration of the present invention will be described in more detail with reference to preferred embodiments, but these embodiments are only for illustrating the present invention specifically, the scope of the present invention is not limited by these examples, The scope should be defined only by what is stated in the claims.

Example 1 Induction of Embryonic Cells from Tissues of Elder Tree

Tissue culture material was used on leaves, roots and stem tissues of seedlings of the elder tree, and was densified in MS solid medium by varying the concentrations of 2,4-D and NAA in the plant growth regulator TDZ. The medium was used by completely dissolving 3% sugar (sucrose) based on MS medium and then treated with plant growth regulators. At this time, the growth regulator was added and then adjusted to pH 5.7 and agar (plant agar) was added 1.0%. Each medium was dispensed by 50ml in 100 × 40㎜ Petri dish, which was autoclaved for 15 minutes under conditions of 121 ℃, 1.5 atm and solidified with a solid medium.

At this time, the leaves, roots, and stem tissues of the seedling tree seedlings grown in the culture chamber were used to form somatic embryogenic callus, which was cut into 5 mm × 5 mm size and wound on the medium used in each experiment. After 5 weeks, the roots produced the most embryogenic callus in the NAA combination hormone, followed by stem and leaf. In 2,4-D, stems had higher rates of embryogenic callus production than roots (see FIGS. 1 and 2). However, both NAA and 2,4-D combination hormones showed better roots in both embryonic callus (embryogenic callus) and leaf than stem and stem. Comparing 2,4-D and NAA, NAA was not only more productive but also more abundant. In addition, it was found that the concentration of embryonic callus was 100% in the combination hormone NAA 0.5mg / L, and the optimal concentration was found. The concentration of TDZ in the NAA 0.5mg / L was 0.05 mg / L. When the leaves (see Fig. 3A) and the stem (see Fig. 3B) were cultured, somatic embryonic callus was hardly formed and non- somatic embryonic callus was formed intermittently, but the root tissue actively formed embryogenic callus. It could be confirmed (see FIG. 3C).

In the formation of somatic embryogenic callus with stem and root tissues of the elder tree, the concentrations of MS medium, SH medium, and WPM medium were densified in order to confirm the regeneration rate according to the concentration of the inorganic salt. After 3% sugar was completely dissolved in the medium, the medium was added to a medium containing 0.5 mg / l NAA and 0.05 mg / l TDZ. At the concentration of stem regeneration, minus WPM medium, WPM medium and SH medium showed similar levels, and MS medium was effective. When the roots were used as materials, MS, SH, and WPM were found to be effective, and MS medium was identified as the most suitable medium for regeneration of stems and roots (Table 1).

[Table 1] Effect of Media Type and Salt Concentration on Embryonic Callus Formation of Stem and Root Tissues (5-week Culture)

badge stem Root Transplant population Embryonic callus formation Transplant population Embryonic callus formation SH 10 4.0 ± 0.8 ^{† BC ‡} 10 6.3 ± 0.5 ^CD 1 / 2SH 10 3.0 ± 0.0 ^CD 10 7.3 ± 0.9 ^BC 1 / 3SH 10 4.6 ± 0.5 ^BC 10 1.0 ± 0.8 ^F 1 / 4SH 10 5.0 ± 0.8 ^B 10 0.0 ± 0.0 ^F MS 10 8.6 ± 0.9 ^A 10 9.7 ± 0.5 ^A 1 / 2MS 10 7.3 ± 0.5 ^A 10 5.6 ± 0.5 ^D 1 / 3MS 10 5.0 ± 0.8 ^B 10 8.3 ± 0.9 ^AB 1 / 4MS 10 1.0 ± 0.8 ^DE 10 6.7 ± 0.5 ^CD WPM 10 7.7 ± 1.2 ^A 10 8.0 ± 0.8 ^B 1 / 2WPM 10 4.7 ± 1.2 ^B 10 3.0 ± 0.8 ^E 1 / 3WPM 10 1.0 ± 0.8 ^E 10 0.0 ± 0.0 ^F 1 / 4WPM 10 4.3 ± 0.5 ^BC 10 0.7 ± 0.5 ^F

^† ; Data represent mean ± SE of three independent experiments.

^‡ ; Within columns, means followed by the same letters are not significantly different at ρ = 0.05 according to Duncan's multiple range test.

In the formation of somatic embryogenic callus with stem and root tissues of the elm, the concentrations of carbon sources Dextrine, d-fructose, d-glucose, and sucrose were determined to determine the regeneration rate according to the concentration of carbon source. After completely dissolving the carbon source on the basis of MS medium, the medium was added to a medium containing 0.5 mg / l NAA and 0.05 mg / l TDZ. The incidence of embryogenic callus after 5 weeks of incubation by the type and concentration of carbon source was inefficient for d-fructose and inhibited dextrine and d-glucose. The proper carbon source was confirmed to be sucrose, sucrose also did not re-differentiate at high concentration of 50mg / l at 10, 30mg / l was found to be appropriate (Table 2).

[Table 2] Effects of Carbon Source Types and Concentrations on Embryonic Callus Formation of Stem and Root Tissues (5-week Culture)

Carbon source concentration (mg / ℓ) stem Root Transplant population Embryonic callus formation Transplant population Embryonic callus formation Dexrine 10 10 4.3 ± 0.6 ^{† BC ‡} 10 2.0 ± 0.0 ^C 30 10 1.0 ± 1.0 ^EF 10 0.3 ± 0.6 ^D 50 10 1.0 ± 1.0 ^EF 10 0.3 ± 0.6 ^D D-glucose 10 10 1.6 ± 0.6 ^DEF 10 0.3 ± 0.6 ^D 30 10 4.3 ± 0.6 ^BC 10 2.7 ± 0.6 ^C 50 10 1.7 ± 0.6 ^DEF 10 4.0 ± 1.0 ^B D-fructose 10 10 6.3 ± 1.6 ^A 10 1.0 ± 1.0 ^D 30 10 2.0 ± 0.0 ^CDE 10 0.0 ± 0.0 ^D 50 10 0.0 ± 0.0 ^F 10 0.0 ± 0.0 ^D sucrose 10 10 3.7 ± 1.2 ^CD 10 10.0 ± 0.0 ^A 30 10 5.0 ± 1.7 ^AB 10 9.7 ± 0.6 ^A 50 10 3.3 ± 0.6 ^BCD 10 0.0 ± 0.0 ^D

^† ; Data represent mean ± SE of three independent experiments.

^‡ ; Within columns, means followed by the same letters are not significantly different at ρ = 0.05 according to Duncan's multiple range test.

For the growth of embryonic callus, experiments were carried out using 2,4-D, a kind of auxin, at concentrations of 1 mg / l, 2 mg / l, 3 mg / l, and 4 mg / l. Auxin has a characteristic of promoting cell division, so it is not easily decomposed in the medium in this experiment and has a long-lasting property, and 2,4-D, which is commonly used in daily life, was selected and used in the experiment. Callus proliferation was shown to be most effective in the medium in which 2,4-D 1 mg / L was added to 1/2 MS medium, followed by the addition of 2,4-D 2 mg / L in 1/2 MS medium. It was shown to be effective in the medium, and callus did not proliferate in the 1/2 MS medium without the growth regulator added (see FIG. 4). Embryonic callus grown in 2,4-D-added medium is transferred to a hormone-free medium to induce mature somatic embryos.

Example 2 Somatic Embryogenesis Directly from the Roots of Mulberry

To find out how direct somatic embryogenesis using root tissues of Rhizome japonicus appeared in liquid culture, medium with 0.5A / mg of plant growth regulator and 0.05mg / L of TDZ and medium without addition of plant growth regulator 100 ml was dispensed into each 250 ml Erlenmeyer flask, and the root tissue cut into 0.5 mm was toothed. As a result, good callus formation was observed in the medium to which NAA 0.5 mg / L + TDZ 0.05 mg / L was added (see FIG. 5A). However, callus formation under other conditions including this turned brown again one month later (see FIG. 5B). Specifically, two months later, embryogenic cells and direct somatic embryos were observed in MS medium without hormones (see FIG. 5C).

In order to confirm the regeneration rate according to the concentration of the inorganic salt in the direct somatic embryogenesis using the root tissue, liquid culture was performed by varying the type of medium and the concentration of the inorganic salt. Plant growth regulators were not added and sugar was added 1.0% to completely dissolve. The pH was adjusted to 5.7, 100 mL was dispensed into a 250 mL Erlenmeyer flask, and the root tissue cut to 0.5 mm was healed into 5 sections. As a result, it was found that somatic embryos were formed well in MS medium, 1/2 SH medium, and 1/3 WPM medium (see Table 3 and FIG. 6). More specifically, the development and maturation of active secondary folds were confirmed in 8-week MS medium, and the generation of most secondary folds was confirmed in 1/2 SH medium, but immature cells, and somatic embryos in 1/3 WPM medium. The maturation and germination of them could be confirmed (see FIG. 7). Therefore, MS medium was evaluated as the most effective in the somatic embryo's organic phase in the development and maturation of the secondary embryo.

Table 3 Regeneration of Somatic Embryos According to Medium Composition in Liquid Culture (8-week Culture)

badge Somatic Embryogenesis MS 2 - ^＊ One ++++ 1/2 + 1/3 + 1/4 ++ 1/8 ++ SH 2 + One - 1/2 - 1/3 +++ 1/4 + 1/8 ++ WPM 2 - One + 1/2 ++++ 1/3 ++ 1/4 + 1/8 -

^＊ -; none +; 5 ~ 20 somatic embryos per flask

++; 20-40 somatic embryos per flask +++; 40 ~ 60 somatic embryos per flask

++++; More than 60 somatic embryos per flask

Example 3 Plant Regeneration from Somatic Embryos

To determine the effective rate of regeneration into plants, embryonic cells selected from 60 mesh sieves were selected from MS medium, 1/2 MS medium, 1/3 MS medium, 1/4 MS medium, and 1/8 MS medium, respectively. 100 ml of 1% (w / v) sucrose was added to a 250 ml Erlenmeyer flask in which 100 ml of each was inoculated at a level of 20 mg per flask, and the shaker was adjusted to 135 rpm to adjust the light conditions at 25 ° C. for 16 hours. After 8 hours of incubation, the germination of somatic embryos started 4 weeks after inoculation, and the complete maturation and regeneration of the somatic embryos was confirmed after 8 weeks. In addition, the development and maturation of the secondary fold was observed in MS medium, it was confirmed that the germination almost in 1/2 MS medium and 1/3 MS medium (see Fig. 8).

After a treatment on 1/3 MS medium by GA _3, spermidine, spermine concentrations to assess the effects of GA ₃ and polyamine acids in the germination of somatic embryos, GA ₃ is 0.01㎎ / ℓ, 0.1㎎ / ℓ, 2 At all treatment concentrations of mg / l, germination of somatic embryos began to be actively started 3 weeks after inoculation of embryonic cells of 60 mesh or less (see FIG. 9). After 3 weeks of inoculation, putrescine was somatic embryo in polyamines. The development and maturation of the spermidine could not be observed, and the development and germination of spermidine at 0.01 mg / l and spermine at 2 mg / l could be observed (see FIG. 10). Therefore, preferably, a medium treated with GA ₃ in 1/3 MS medium was evaluated to be effective for germination of somatic embryos.

The pH of the medium not only absorbs growth regulators and other nutrients, but also affects the availability of water by changing the properties of the colloids, which in turn affects the differentiation and development of the culture. As a result of investigating the shoot formation rate by changing the pH of the culture medium, it showed the best shoot formation rate at 80% at pH 5.75, and showed a relatively good shoot formation rate at 40% at pH 5 and pH 7. At 4 and pH 6, shoot formation was not practical (see Figure 11). As a result, the pH range of the preferred medium was evaluated to be 5.0 to 5.75.

The degree of adaptation of the somatic cell germination in liquid medium, ie, plant regeneration according to MS concentration, formed both roots and shoots in 1/8 MS medium during the first month, but almost no elongation after 3 months. No root differentiation and only roots in the form of side roots were considered to be unsuitable for regeneration medium. Meanwhile, in the 1/3 MS medium and the 1/4 MS medium, root formation was actively formed after shoot formation to form a root root, which was evaluated as a preferable medium for regeneration (Table 4 and Table 5).

Table 4 Plant Regeneration Patterns According to MS Medium Composition (After 1 Month of Culture)

badge Transplant population Survival Population * Survival rate (%) % Differentiation New planting number * Root Water * 2MS 12 1.67 ± 0.58 13.89 5.56 1.33 ± 0.58 ^{D †} 1.00 ± 1.00 ^D 1MS 12 2.33 ± 0.58 19.44 2.78 2.33 ± 0.58 ^D 0.33 ± 0.58 ^D 1 / 2MS 12 7.33 ± 0.58 61.11 50.00 7.00 ± 0.00 ^C 6.33 ± 1.15 ^C 1 / 3MS 12 7.00 ± 1.00 58.33 47.22 7.00 ± 1.15 ^C 5.67 ± 1.15 ^C 1 / 4MS 12 9.33 ± 1.15 77.78 69.44 9.00 ± 1.15 ^B 9.00 ± 1.00 ^B 1 / 5MS 12 9.33 ± 1.15 77.78 58.33 7.00 ± 0.58 ^C 9.33 ± 1.15 ^B 1 / 8MS 12 12.00 ± 0.00 100.00 94.44 11.67 ± 0.58 ^A 11.67 ± 0.58 ^A

* Each value is the mean of at least three independent experiments ± SD.

^† ; Within columns, means followed by the same letters are not significantly different at ρ = 0.05 according to Duncan's multiple range test.

Table 5 Plant Regeneration Patterns According to MS Medium Composition (After 3 Months of Culture)

badge Transplant population Survival population Extra long (mm) Length (mm) 2MS 10 0 ± 0.0 ^† 0.0 ± 0.0 ^{D ‡} 0.0 ± 0.0 ^C 1MS 10 1 ± 0.0 0.6 ± 0.6 ^BC 0.5 ± 0.1 ^C 1 / 2MS 10 7.5 ± 0.5 0.8 ± 0.2 ^AB 1.9 ± 0.9 ^B 1 / 3MS 10 10 ± 0.0 0.9 ± 0.4 ^A 3.0 ± 1.7 ^A 1 / 4MS 10 8.5 ± 0.0 0.8 ± 0.5 ^AB 3.0 ± 2.1 ^A 1 / 5MS 10 10 ± 0.0 0.7 ± 0.4 ^ABC 2.3 ± 2.2 ^BA 1 / 8MS 10 10 ± 0.0 0.6 ± 0.2 ^C 1.8 ± 1.3 ^B

^† ; Each value represents the mean ± SD

^‡ ; Within columns, means followed by the same letters are not significantly different at ρ = 0.05 according to Duncan's multiple range test.

In order to examine the regeneration patterns of plants according to the sucrose concentration of the medium, the regeneration characteristics of the germination stage grown through the somatic embryo development in the liquid medium in the medium prepared by varying the sucrose concentration in the basal medium of 1/3 MS Was investigated. Plant regeneration according to sucrose concentration (w / v) of the medium showed active regeneration at 3% in observations after 1 month of culture, and similar levels were good after 3 months (see FIG. 12B). At 0.5% level, shoot formation was active for the first two weeks, but as a result, root formation was not good (see FIG. 12C). At the 5% level, almost whitening occurred (see FIG. 12A), and browning was observed at the 0% level (see FIG. 12D). Overall, differentiation into seedlings in the sucrose concentration range of 1% to 3% was evaluated as good (Table 6, Table 7).

TABLE 6 Plant Regeneration Patterns According to Sucrose Concentration of Medium (After 1 Month of Culture)

Sucrose Concentration (%) Transplant population Survival Population * Survival rate (%) % Differentiation New planting number * Root Water * 5 12 1.00 ± 1.00 5.56 2.78 0.67 ± 0.58 ^{D †} 0.33 ± 0.58 ^C 3 12 10.67 ± 0.58 88.89 83.33 10.33 ± 0.58 ^A 10.33 ± 0.58 ^A One 12 9.33 ± 0.58 77.78 77.78 9.33 ± 0.58 ^B 9.33 ± 0.58 ^A 0.5 12 8.33 ± 0.58 69.44 22.22 8.33 ± 0.58 ^B 3.00 ± 1.00 ^B 0.1 12 4.00 ± 0.00 33.33 2.78 4.00 ± 0.00 ^C 0.67 ± 0.58 ^C 0 12 0.33 ± 0.58 2.78 0.00 0.33 ± 0.58 ^D 0.00 ± 0.00 ^C

* Each value is the mean of at least three independent experiments ± SD.

^† ; Within columns, means followed by the same letters are not significantly different at ρ = 0.05 according to Duncan's multiple range test.

TABLE 7 Plant Regeneration Patterns According to Sucrose Concentration of Medium (After 3 Months of Culture)

Sucrose Concentration (%) Transplant population Survival population Extra long (mm) Length (mm) 5 10 1 ± 0.0 ^† 0.3 ± 0.0 ^{B ‡} 0.2 ± 0.0 ^D 3 10 9 ± 1.0 0.9 ± 0.5 ^A 2.5 ± 1.7 ^B One 10 9 ± 0.0 0.8 ± 0.4 ^A 2.0 ± 1.3 ^BC 0.5 10 7.5 ± 0.5 10.0 ± 1.3 ^A 1.4 ± 1.0 ^C 0.1 10 0.5 ± 0.5 0.8 ± 0.0 ^A 2.0 ± 0.0 ^BC 0 10 1 ± 0.0 1.05 ± 0.0 ^A 3.2 ± 0.1 ^A

^† ; Each value represents the mean ± SD

^‡ ; Within columns, means followed by the same letters are not significantly different at ρ = 0.05 according to Duncan's multiple range test.

Example 4 Purification of Seedlings

Somatic embryos in the germination stage grown through somatic embryo development in the liquid medium of Example 3 were transferred to a solid medium containing 1% (w / v) sucrose in a 1/3 MS basic medium and incubated for 3 months. Purification was performed. Adapted in a growth chamber of 16 hours of light conditions and 8 hours of dark conditions at 25 ° C. for 2 weeks, and grown for 2 weeks in a greenhouse treated with 50% shading film for 2 weeks. The purity was 100%, and the relatively high purity was 85% in the 20 mm seedlings. However, the survival rate of the plants was generally high, but the state of the purified plants was very good for the seedlings of 30 mm or more, but poor for the seedlings of 20 mm or less (see FIG. 13).

In order to determine the purifying efficiency according to soil conditioning, in-flight cultured plants were grown for 5 months with different composition of the purified soil, and then the height, leaf width, and leaf length were examined, and rooting degree was evaluated by examining root diameter and length. Leaf width and leaf length of the purified plants showed similar levels, and the grass length was high in soils in which sand and pearlite were combined with soil and soils + vermiculite + pearlite respectively (see FIG. 14). The root diameters of the purified plants were low in soils with top soil + pearlite and soils with top soil + vermiculite + pearlite, and rooting was good in terms of root diameter and length under soil + sand soil conditions. . Taken together, the most preferable composition of the purified soil is to mix the soil with sand in a ratio of 1: 1.

Overestimated (see FIG. 15).

As described above, the mass production method of cedar seedlings using somatic embryos derived from embryonic callus derived from plant tissues or somatic embryos directly derived from plant roots is effective for endangered plant species of cedar seedlings. In-flight volume production was evaluated.

In addition, the mass production method of the seedlings of young elm tree seedlings using somatic embryos derived from embryonic callus derived from plant tissues or somatic embryos directly derived from plant roots is evaluated as an effective off-site preservation means of difficult elm. It was expected that this method could provide a useful method to realize the useful pharmacological effects of cedar.

Claims (4)

In the method of producing a large number of seedlings using somatic embryos directly derived from the plant roots of the elm tree, (1) directing somatic embryos by directing and incubating the tissue sections of the roots of the elder tree in MS medium; (2) culturing the thus obtained somatic cell culture in a liquid medium or a solid medium; (3) re-differentiating the seedlings by culturing the proliferated somatic cell in a liquid medium or a solid medium in the range of pH 5.0 to 5.75; (4) purifying the regenerated seedlings; Mass production method of seedling seedlings, characterized in that comprises a. According to claim 1, wherein the medium in the re-differentiation step is characterized in that the 1/3 MS medium or 1/4 MS medium added with sucrose at a level of 1% to 3% (w / v), in the re-differentiation step Cultivation of the method of mass production of seedling seedlings, characterized in that carried out at 25 ℃ 16 hours light conditions and 8 hours dark conditions. delete The method of claim 1, wherein the purifying step is mass production method of seedling seedlings, characterized in that the soil is mixed with soil 1: 1 composition.

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