KR20210087175A - Production method having high improved productivity for tissue cultured seedlings of Larix kaempferi - Google Patents

Abstract

Disclosed in the present invention is a method for producing tissue cultured seedling of Larix kaempferi having highly improved productivity through a somatic embryo germination and redifferentiation acceleration process and an acclimatized seedling growth acceleration process. The method for producing tissue cultured seedling of Larix kaempferi according to the present invention can be efficiently applied to a mass production system of tissue cultured seedling of Larix kaempferi for forest plantation. Also, the seedling of Larix kaempferi according to the present invention is strong acclimated seedling with good growth, thereby being highly used for forest plantation.

Description

TECHNICAL FIELD [0002] 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 maple tissue with significantly improved productivity through a process for promoting germination and re-differentiation 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 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, it is difficult to supply and supply seedlings for afforestation due to the irregular cycle of harvest and harvest.

As an alternative to solving these difficulties, a propagation method through the culture of Japanese larch somatic cells 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 the mass production efficiency of Japanese larch tissue culture seedlings due to the problems that the growth from the redifferentiated germination body to the purified seedling of the plant is not performed well when the environment is acclimatized in artificial soil. 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 afforestation in mountainous areas. There is an urgent need for development.

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 Japanese larch tissue culture seedlings 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 fertilizing and culturing the Japanese Larch somatic embryo;

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, including 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 embryonic 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, but is not limited to, a cotyledon stage somatic embryo.

Most preferably, in the present invention, the embryogenic cells derived from the immature seed of the Japanese L. L. japonica are LM (Litvay Medium) medium to which 16.0 mg/L ABA, 54 g/L maltose, and 0.8% gelite are added. 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.

In this step, 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.

At this stage, 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 illumination 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 described above, the germination rate of Japanese larch somatic embryos was significantly increased (FIG. 1b).

Step iii) Redifferentiation

The germinal 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.

At this stage, 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 illumination 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 acclimatization 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 stage, the culture is carried out at 25±1°C for 15-17 hours a day, and 50-70 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, resulting in 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 culture seedlings according to the present invention, the germination rate of Japanese maple somatic embryos is significantly increased, redifferentiation proceeds well, and stems and roots are properly developed to grow into seedlings, and the environment It can be efficiently applied as a system for mass production of Japanese oak tissue cultured seedlings for plantation in mountainous areas, as it is well acclimatized and it is possible to obtain healthy domesticated seedlings with good growth.

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

1A is a photograph showing the germination state of Japanese larch somatic embryos cultured in the dry-treated 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 water content according to the culture period of Japanese larch somatic embryos cultured in the dried conditions 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.
3 is a photograph showing a 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 the acclimatized seedlings transplanted into artificial soil in the acclimatization container.
5A and 5B are photographs showing domesticated seedlings (Example 2) cultured according to the present invention.

Hereinafter, the configuration and effects of the present invention will be described in more detail through specific examples 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 oak, two lines (LK-1, LK-2; provided by the Clone Development Lab of the National Institute of Forest Science) were prepared as test materials, and these somatic cell embryo lines were cotyledon-developed somatic embryos ( cotyledon stage somatic embryo).

After preparing a solid LM medium (Duchefa, Netherlands) with 3% sucrose and 0.3% gelite as a germination medium, a filter paper (quantitative filter paper, Advantec 55mm) was placed on it, and the cotyledon stage somatic cell culture medium prepared above was placed thereon. After implantation, 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. After that, it was transferred to the same new germination medium without filter paper and cultured for a total incubation period of 9 weeks (Example 1), and the results are shown in FIG. 1A .

For comparison, as in the prior art, 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, 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 germinated. Like this callus, it was dedifferentiated and germination did not work properly, and the 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, showing a significant difference according to the drying treatment period.

Specifically, the germination rates of Japanese larch somatic embryo lines LK-1 and LK-2 cultured without drying treatment of Comparative Example 1 were only 7.69% and 14.25%, respectively, but according to the present invention, 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, a filter paper was placed on a 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, without filter paper, 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 in the same manner as above. The water content per 1 g of fresh weight was measured, and the results are shown in FIG. 2A.

In addition, anatomical photographs 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 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 water content of the somatic cell line LK-2 in 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, according to the present invention, it can be confirmed that Japanese larch somatic embryos cultured for 2 to 4 weeks dry treatment have a moisture content suitable for germination.

Conversely, in Comparative Example 1, the water content of the somatic embryos of the somatic embryo 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 is not well germinated due to excessive moisture content.

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 papillae did not develop properly. 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 the 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} The resulting plant photograph is shown in FIG. 3 .

As shown in FIG. 3, when cultured by 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

Japanese larch seedlings (3 to 4 cm) from Example 3 were transplanted into artificial soil [vermiculite: peat moss: perlite = 1: 1: 1 (v: v: v)] contained in a 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% to 90%, and the acclimatization chamber culture environment is illuminated for 16 hours a day at a temperature of 25±1℃ , 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 watering and foliar spraying once a week, and the results are shown in Table 1 and FIGS. 5a and 5b (implemented). Example 2).

For comparison, as in the prior art, the Japanese larch seedlings (3 to 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 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% for somatic embryo lines LK-1 and LK-2, respectively. , it can be confirmed that the significant improvement compared to the case of Comparative Example 2 (85.7% and 61.3%). In addition, the stem and root lengths of the domesticated seedlings of Example 2 according to the present invention were measured to be 2.4 cm, 1.5 cm, 12.6 cm, and 9.5 cm, respectively, respectively, 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 and culturing the Japanese Larch somatic embryo;
iii) transferring the germination body onto a germination medium without filter paper, culturing and re-differentiation to obtain a seedling; and
iv) transplanting the seedlings into artificial soil in an acclimatization container maintained at a humidity of 80 to 90%, and culturing them with Hyponex to obtain purified seedlings; a method of producing Japanese larch tissue culture seedlings, including.
The method according to claim 1, wherein the culturing in step ii) is performed for 2 to 4 weeks.
The method according to claim 2, wherein the culturing in step ii) is performed for 3 weeks.
The method according to claim 1, wherein the germination medium is a solid LM medium to which 2-4% sucrose and 0.2-0.4% gelite are added.
The method according to claim 1, wherein the filter paper is a quantitative filter paper.
The Japanese larch tissue according to claim 1, wherein the culturing is carried out under conditions in which illumination is maintained within the range of ^{50-70 μmol·m -2} ·s ^{-1 for} 15 to 17 hours a day at 24 to 26°C. A method for producing cultured seedlings.
The method according to claim 1, wherein the culturing in step iii) is performed for 4 to 8 weeks.
The method according to claim 1, wherein the somatic embryo is a cotyledon stage somatic 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).
The method according to claim 1, wherein the Hyponex treatment is performed by watering or foliar spraying once a week by diluting the Hyponex 1,000 times.
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 any one of claims 1 to 11.

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