KR102408810B1 - Production method having high improved productivity for tissue cultured seedlings of Larix kaempferi - Google Patents

Abstract

The present invention discloses a method for producing a Japanese larch tissue-cultured seedling with significantly improved productivity through a process for promoting germination and re-differentiation of somatic embryos and a process for promoting the growth of domesticated seedlings.
The production method of Japanese oak tissue cultured seedlings according to the present invention can be efficiently applied to a mass production system of Japanese oak tissue cultured seedlings for forestry. In addition, the Japanese larch seedlings according to the present invention are healthy and domesticated seedlings with good growth, and thus have high utility for mass afforestation.

Description

The production method of Japanese larch tissue culture seedlings with significantly improved productivity {Production method having high improved productivity for tissue cultured seedlings of Larix kaempferi}

The present invention relates to a production method of Japanese oak tissue-cultured seedlings with significantly improved productivity, and more particularly, Japanese oak tissue cultured seedlings with significantly improved productivity through a process for promoting germination and redifferentiation of somatic embryos and a process for promoting growth of domesticated seedlings It relates to a method for producing cultured seedlings.

Japanese larch [ Larix kaempferi (Lamb.) Carriere; Larch] is a deciduous coniferous tree that was first introduced from Japan in 1904, and is one of the major lumber tree species in Korea because it grows quickly, has a straight trunk, and can produce a lot of wood within a short period of time. However, due to the irregular cycle of harvest and harvest, it is difficult to supply and supply seedlings for afforestation.

As an alternative to solving these difficulties, a propagation method through somatic cell culture of Japanese Larch using tissue culture technology was developed (Patent Registration No. 10-0720338). However, in the method, the efficiency of somatic embryo germination and redifferentiation is low, and due to the problems that the growth from the redifferentiated germinal body to the purified seedling of the plant is not performed well when the environment is acclimatized in artificial soil, the mass production efficiency of Japanese oak tissue culture seedlings It is very low, so it is difficult to put it into practical use.

Therefore, by developing a process for promoting somatic embryo germination and re-differentiation and a process for promoting the growth of domesticated seedlings, the production method of Japanese oak tissue-cultured seedlings with significantly improved productivity that can be used as a mass production system for Japanese oak tissue-cultured seedlings for plantation in mountainous areas This is an urgent need.

Accordingly, as a result of continuous research to meet the needs of the prior art, the present inventors combined the process of germination and redifferentiation of Japanese oak somatic embryos through drying treatment under specific conditions and the purification process of germinating Japanese oaks under specific conditions. In this case, it was confirmed that the productivity of Japanese oak seedlings can be significantly improved, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a method for producing a Japanese larch tissue culture seedling with significantly improved productivity.

Another object of the present invention is to provide a Japanese larch seedling produced by the above method.

In order to achieve the above object of the present invention, the present invention

i) preparing Japanese larch somatic embryos;

ii) placing a filter paper on the germination medium and then culturing and culturing the Japanese Larch somatic embryo to germinate;

iii) culturing by transferring the germination medium on a germination medium without filter paper to obtain a redifferentiated seedling; and

iv) transplanting the seedlings into artificial soil in an acclimatization container maintained at 80 to 90% humidity, and culturing them with Hyponex to obtain healthy domesticated seedlings; providing a method for producing Japanese larch tissue culture seedlings do.

The production method of the present invention may further include, if necessary, the step of propagating by transplanting the domesticated seedlings into soil after step iv).

Step i) Preparation of Japanese larch somatic cells

In the present invention, as the Japanese L. somatic cell embryo, a J. L. somatic cell line obtained by inducing embryogenic cells from the Japanese L. L. tissue may be used.

Induction of embryogenic cells and somatic embryos from Japanese Larch tissue can be performed by a conventional method.

In the present invention, the Japanese larch tissue is a seed embryo, a cotyledon, an internode section, etc., and preferably a seed embryo.

In the present invention, the Japanese larch somatic embryo is preferably a cotyledon stage somatic embryo, but is not limited thereto.

Most preferably, in the present invention, as the somatic cell embryo of L. L. J. L. stem cell embryo, 16.0 mg/L ABA, 54 g/L maltose, and 0.8% gelite are added to LM (Litvay Medium) medium. Cotyledon-stage somatic embryos obtained by culturing for 7-8 weeks in

Step ii) somatic embryo germination

After placing the filter paper on the germination medium, the Japanese larch somatic embryos are dented and cultured to germinate.

At this stage, the germination medium uses solid LM (Litvay Medium) with 2~4% sucrose and 0.2~0.4% gelite added, and most preferably LM with 3% sucrose and 0.3% gelite added. Use the medium.

In the present invention, it is preferable to use a quantitative filter paper as the filter paper.

In this step, the culture of somatic embryos is maintained at 25±1° C. for 15 to 17 hours a day, and 50 to 70 μmol·m ^-2 ·s ^-1 lighting is maintained.

The period of the culture is preferably 2 to 4 weeks, more preferably 2 to 3 weeks, and most preferably 3 weeks.

When dried with filter paper and cultured under the same conditions as above, the germination rate of Japanese larch somatic embryos was significantly increased (FIG. 1b).

Step iii) Redifferentiation

The germination body from step ii) is transferred to a germination medium without filter paper, cultured and redifferentiated to obtain a seedling.

The germination medium uses a solid LM medium to which 2-4% sucrose and 0.2-0.4% gelite are added, and most preferably, an LM medium supplemented with 3% sucrose and 0.3% gelite is used.

In this step, the culture of somatic embryos is maintained at 25±1° C. for 15 to 17 hours a day, and 50 to 70 μmol·m ^-2 ·s ^-1 lighting is maintained. Preferably, it is cultured under conditions in which illumination is maintained within the range of 60 μmol·m ^-2 ·s ^-1 for 16 hours a day at 25°C.

The period of the culture is preferably 4 to 8 weeks, and most preferably 6 weeks.

When cultured under the same conditions as described above, the germination of Japanese larch (FIG. 3A) proceeded well with re-differentiation and grew into a seedling with properly developed stems and roots (FIG. 3B).

Step iv) Purification

The seedlings from step iii) are transplanted into artificial soil in an acclimatization container maintained at 80-90% humidity, treated with Hyponex and cultured to obtain purified seedlings.

The purification container can be used without particular limitation as long as it can maintain a humidity of 80 to 90%.

The artificial soil may be a mixture of vermiculite, peat moss and perlite, preferably vermiculite: peat moss: perlite mixed in a ratio of 1:1: 1 (v:v:v).

In this step, Hyponex treatment is diluted with 1,000-fold solution and treated with watering and foliar spraying once a week. Hyponex treatment can be performed 4 to 8 times.

In this step, the culture is carried out at 25±1° C. for 15 to 17 hours a day, and 50 to 70 μmol·m ^-2 ·s ^-1 lighting is maintained.

The period of the culture is preferably 4 to 8 weeks, and most preferably 6 weeks.

When cultured under the same conditions as described above, the Japanese larch seedlings were well acclimatized to the environment and became healthy and acclimated seedlings with good growth (FIG. 5).

Step v) Soil Transplantation

If necessary, the acclimated seedlings from step iv) are transplanted into soil and propagated.

According to another object of the present invention, the present invention provides a Japanese larch seedling produced by the above method.

The Japanese larch seedlings according to the present invention are healthy and domesticated seedlings with good growth, so they are suitable for mass afforestation.

According to the production method of Japanese oak tissue-cultured seedlings according to the present invention, the germination rate of Japanese maple somatic embryos is significantly increased, redifferentiation is well progressed, and stems and roots are properly developed, and the environment It can be efficiently applied as a mass production system for Japanese oak tissue cultured seedlings for mountain afforestation as it is possible to obtain wholesome domesticated seedlings with good growth through good acclimatization.

In addition, the Japanese larch seedlings according to the present invention can be utilized for mass afforestation because they are healthy and domesticated seedlings with good growth.

1a is a photograph showing the germination state of Japanese larch somatic embryos cultured in the dried condition according to the present invention.
Figure 1b is a graph showing the germination rate of Japanese larch somatic embryos cultured in the dried conditions according to the present invention (LK-1, LK-2).
Figure 2a is a graph showing the moisture content according to the culture period of Japanese larch somatic embryos cultured in the dried condition according to the present invention.
Figure 2b is a photograph showing the anatomical characteristics of Japanese larch somatic embryos cultured in the dried conditions according to the present invention.
Figure 3 is a photograph showing the redifferentiated plant according to the present invention.
4 is an acclimatization container (right) according to the acclimatization conditions of the present invention, a photograph showing an acclimated seedling transplanted into artificial soil in the acclimatization container and cultured.
5A and 5B are photographs showing domesticated seedlings (Example 2) cultured according to the present invention.

Hereinafter, the configuration and effect of the present invention will be described in more detail with reference to specific examples in order to help the understanding of the present invention. However, the following examples are merely illustrative in order to understand the present invention more clearly, and the scope of the present invention is not limited by the following examples.

Example 1: Germination analysis according to drying treatment of Japanese Larch somatic embryos

As the somatic embryos of Japanese oaks, two lines (LK-1, LK-2; provided by the Clone Development Lab of the National Institute of Forest Science) were prepared as test materials. cotyledon stage somatic embryo).

A solid LM medium (Duchefa, Netherlands) supplemented with 3% sucrose and 0.3% gelite was prepared as a germination medium, and filter paper (quantitative filter paper, Advantec 55mm) was placed on it, and the cotyledon stage somatic culture medium prepared above was placed on it. After planting, culture for 1 week, 2 weeks, 3 weeks, and 4 weeks, respectively, in a culture environment maintained with illumination (cold white fluorescent lamp, 60 μmol·m ^-2 ·s ^-1 ) at a temperature of 25±1°C for 16 hours a day. Then, it was transferred to the same new germination medium without filter paper and cultured for a total incubation period of 9 weeks (Example 1).

For comparison, as in the prior art, after placing the prepared cotyledon stage somatic embryos on the germination medium, which is a solid LM medium containing 3% sucrose and 0.3% gelite, without filter paper, the temperature for 9 weeks It was cultured in a culture environment maintained with illumination (cold white fluorescent lamp, 60 μmol·m ⁻² ·s ⁻¹ ) at 25±1° C. for 16 hours a day (Comparative Example 1), and the resulting photograph is shown in FIG. 1A.

In addition, the germination rate (germination body production rate) of Japanese larch somatic cells of Example 1 and Comparative Example 1 for each drying treatment period (1 week to 4 weeks) was calculated, and the results are shown in FIG. 1B .

As shown in FIG. 1A , in Example 1 according to the present invention, which was dried with a filter paper, it was observed that Japanese L. somatic cell embryos germinated and the papillae developed normally, but in Comparative Example 1, most of Japanese L. somatic cell embryos Like this callus, it was dedifferentiated, so germination did not work properly, and normal development of the rhizome was not observed.

As shown in Fig. 1b, the germination rate of Japanese larch somatic embryos dried and cultured using filter paper was significantly improved, and showed a significant difference depending on the drying treatment period.

Specifically, the germination rates of LK-1 and LK-2 cultured without drying treatment of Comparative Example 1 were only 7.69% and 14.25%, respectively, but 2 ~ The germination rate of LK-1 and LK-2 somatic cell lines LK-1 and LK-2 cultured after drying for 4 weeks was improved to about 30 to 60% (about 3 to 4 times). In particular, the germination rates of LK-1 and LK-2 somatic cell lines LK-1 and LK-2, which were dried and cultured for 3 weeks using filter paper, were the highest at 59.4% (about 8 times) and 43.5% (about 3.1 times), respectively.

From the above results, it can be seen that the drying treatment using filter paper has a significant effect in promoting the germination of Japanese Larch somatic embryos.

Example 2: Analysis of water content according to drying treatment of Japanese larch somatic embryos

In order to investigate whether the drying treatment using the filter paper of Japanese larch somatic embryos according to the present invention is effective, the change in water content according to the culture period was comparatively analyzed.

Specifically, filter paper was placed on the germination medium, which is a solid LM medium containing 3% sucrose and 0.3% gelite, and the cotyledon stage LK-1 and LK-2 somatic cell lines LK-1 and LK-2 were placed thereon, followed by a temperature of 25 It was cultured for 4 weeks in a culture environment maintained with illumination (cold white fluorescent lamp, 60 μmol·m ^-2 ·s ^-1 ) at ±1° C. for 16 hours a day (Example 1), and water content analysis equipment (SARTORIUS MA150) was used to measure the water content per 1 g of fresh Japanese larch somatic embryo on days 3, 7, 10, 14, 21, and 28 during the incubation period, and the results are shown in FIG. 2A.

For comparison, as in the prior art, after placing the prepared cotyledon stage somatic embryos on the germination medium, which is a solid LM medium containing 3% sucrose and 0.3% gelite, on which no filter paper is placed, the temperature for 4 weeks It was cultured in a culture environment maintained with illumination (cold white fluorescent lamp, 60 μmol·m ^-2 ·s ^-1 ) at 25±1° C. for 16 hours a day (Comparative Example 1), and Japanese larch somatic cells were cultured in the same manner as above. The moisture content per 1 g of fresh weight was measured, and the results are shown in FIG. 2A.

In addition, the anatomical photos taken with an optical microscope of the germinal bodies of Example 1 and Comparative Example 1 before culturing and after culturing for one week are shown in FIG. 2B, respectively.

As shown in FIG. 2A , in Example 1 according to the present invention, the somatic cell embryo water content of the somatic cell line LK-1 was 54.59% after 14 days of culture, 53.04% after 21 days, and 48.73% after 28 days after 2 weeks of culture. It gradually decreased according to the culture period, and the somatic embryo water content of the somatic cell line LK-2 also started to gradually decrease from the second week of culture, and decreased to 64.87% after 21 days of culture and 59.80% after 28 days.

According to the above results, it can be confirmed that Japanese larch somatic embryos cultured for 2 to 4 weeks dry treatment according to the present invention have a moisture content suitable for germination.

Conversely, in Comparative Example 1, the water content of the somatic embryos of the somatic cell lines LK-1 and LK-2 was 60.48% and 58.74% after 7 days of culture, 69.04% and 70.62% after 21 days of culture, and 75.43% after 28 days of culture, respectively. % and 77.06%, which continued to increase after 1 week of culture. Therefore, it can be confirmed that when cultured in the prior art without drying treatment, the somatic cell embryo of Japanese Larch has an excessive moisture content and thus germination is not performed well.

As shown in FIG. 2B , in the somatic embryo of Example 1, it can be confirmed that germination was achieved due to the normal development of the papilla after culturing. In contrast, the somatic embryo of Comparative Example 1 was dedifferentiated after culturing, so that the sprout was not properly developed. It can be seen that it was not

As a result, it can be seen that the drying-treated culture conditions as described above according to the present invention have an effect of remarkably enhancing the germination rate of Japanese larch somatic embryos.

Example 3: Germination body redifferentiation

In Example 1, the germinal bodies of Japanese larch somatic embryos dried and cultured using filter paper for 3 weeks were placed on a new germination medium without filter paper (solid LM medium with 3% sucrose and 0.3% gelite added). After implantation, redifferentiated seedlings were obtained by culturing for 6 weeks in a culture environment maintained at a temperature of 25±1° C. and illuminated for 16 hours a day (cold white fluorescent lamp, 60 μmol·m ^-2 ·s ^-1 ), and the A photograph of the resulting plant body is shown in FIG. 3 .

As shown in FIG. 3, when cultured after drying treatment using filter paper for a certain period of time according to the present invention and culturing again without drying treatment, the Japanese larch germination proceeds well with re-differentiation so that the stem and roots are properly developed. It can be confirmed that it has grown as a seedling (stem length 0.3-0.6 cm, root length 3.1-3.6 cm).

Example 4: Production of purified seedlings through acclimatization of seedlings

The Japanese larch seedlings (3-4 cm) from Example 3 were transplanted into artificial soil [vermiculite: peat moss: perlite = 1: 1: 1 (v: v: v)] contained in an acclimatization container (left of Fig. 4) . After transplantation, irrigate sufficiently and cover with the acclimatization vessel cover (Fig. 4 right) to maintain the humidity in the acclimatization vessel at 80%~90%, and the acclimatization chamber culture environment is illuminated at a temperature of 25±1℃ for 16 hours a day (cold white fluorescent lamp). , 60 μmol·m ^-2 ·s ^-1 ) and cultured for 8 weeks. In addition, in order to promote the growth of seedlings in the acclimatization container, Hyponex (NPK 7-10-6) was diluted with a 1,000-fold solution and cultured by irrigation and foliar spraying once a week. Example 2).

For comparison, as in the prior art, the Japanese larch seedlings (3~4 cm) from Example 3 were placed in artificial soil [vermiculite: peat moss: perlite = 1: 1: 1 (v: v: v) in a acclimatization container. )] after transplantation (left side of Fig. 4), it was sufficiently watered and covered with a acclimatization container cover (Fig. 4 right side). The culture environment in the acclimatization chamber was maintained for 8 weeks while maintaining the same as above. Hyponex (NPK 7-10-6) was not added to the culture by irrigation and foliar spraying using tap water, and the results are shown in Table 1 and FIGS. 5A and 5B (Comparative Example 2).

somatic embryo
line survival rate
(%) seedlings Pure flower seedlings (8 weeks later) stem length
(cm) root length
(cm) stem length
(cm) root length
(cm) Comparative Example 2 LK-1 85.7 0.4 3.5 0.9 11.9 LK-2 61.3 0.3 3.2 0.6 7.1 Example 2 LK-1 91.2 0.4 3.1 2.4 12.6 LK-2 78.1 0.6 3.6 1.5 9.5

As shown in Table 1, in the case of Example 2, which was acclimatized to the acclimatization conditions according to the present invention, the acclimatization rates of Japanese larch seedlings were measured to be 91.2% and 78.1%, respectively, for somatic embryo lines LK-1 and LK-2, , it can be seen that compared to the case of Comparative Example 2 (85.7% and 61.3%), it was significantly improved. In addition, the stem and root lengths of the domesticated seedlings of Example 2 according to the present invention were also measured to be 2.4 cm, 1.5 cm, 12.6 cm, and 9.5 cm on average, respectively, and in Comparative Example 2 (0.9 cm and 0.6 cm, 11.9 cm and 7.1 cm) ), it can be seen that the growth is significantly better.

As shown in FIGS. 5A and 5B , it can be confirmed that the domesticated seedlings of Example 2 according to the present invention had better overall growth than those of Comparative Example 2, and thus grew into healthy purified seedlings.

Therefore, it can be seen that, according to the acclimatization conditions according to the present invention as described above, it is possible to produce healthy Japanese larch seedlings that can be used for afforestation because of good growth during acclimatization.

Claims (12)

i) preparing Japanese larch somatic embryos;
ii) placing a filter paper on the germination medium and then fertilizing the Japanese larch somatic embryos and culturing them for 3 weeks to germinate;
iii) transferring the germinated body to a germination medium without filter paper, culturing for 4 to 8 weeks, and re-differentiating to obtain a seedling; and
iv) transplanting the seedlings into artificial soil in an acclimatization container maintained at 80-90% humidity, diluting Hyponex 1,000-fold, irrigating or foliar spraying once a week, and culturing to obtain purified seedlings; including,
The germination medium is a solid LM medium to which 2-4% sucrose and 0.2-0.4% gelite are added,
The culturing is carried out under conditions in which lighting is maintained within the range of 50-70 μmol·m ^-2 ·s ^-1 for 15-17 hours a day at 24-26° C., a method for producing Japanese larch tissue culture seedlings.
delete delete delete The method according to claim 1, wherein the filter paper is a quantitative filter paper.
delete delete The method according to claim 1, wherein the somatic embryo is a cotyledon stage somatic cell embryo.
The method according to claim 1, wherein the artificial soil is a mixture of vermiculite, peat moss and perlite in a ratio of 1:1:1 (v:v:v).
delete The method according to claim 1, wherein the culture period in step iv) is 4 to 8 weeks.
A Japanese larch seedling produced by the method according to claim 1.

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